RECENT ..~.

ADV ANCES
IN BIOCHEMISTRY
BY

JOHN PRYDE

LONDON

J. & A. CHURCHILL
7, GREAT MARLBOROUGH STREET
1'126
RECBNT J\DVANCES IN BIOCHEMISTRY
THE RECENT ADVANCES SERIES
[lOO"S l'UHLISlffJ
PHYSIOLOGY.
By C LovAn !'.VAl',." 1,5,.., lIf.R.C ~., LR CI'., F.R.S., .J..x!JT't! I'ro,
k~~')T
u{ 1'1)\,.ld.~,. ! 'fI!~cl":I!tr (,!ktw, }."Ila(!(!. S<xa"d l:cUuon.
7" /!!\I"tl;I(1"J1~. 11$.6d.

OBSTETRICS AND GYNiECOLOGY.

fir ;\1,1." \\ n'WR.'1t;, F.R c.,.. _. Oil'iklnr S~rll'('On, 51. Mary's Hospital
a!l~II,!w"_1'1 lh,ld"ttc'~ lk'''lrltal. :511 Hl(J~tr.ltWlls. 12$.6<1.

MEDICINE.
,,~ (, E. H~M.\lfl.H, ~ .. \, n.!!.t, ERC.P., I),p.n, Physician to the
"r... pita! '0' COtNlfllptlr:>!] and l)JSPfLWS of tnt (lw~t, Brf>mpJ:oll, and
'\~'I~IJ:"! I'hl''Illa/, 1" Ill! Mhl,IlI''l Hospltal. lIml E. C. Dorm!>. M.D.,
H.~. 1')'.1 I., B.S, . J'lvf"'~'{)1 nI 'lnI'hf'Jl!l~try, {'''I't'T)'JI}' of 1 "nfl,)"

~,rJ;;';~~)t~~~~.h(';~~;:I!;~,?,l;;,.~:dd1::""S)~~;~!~,~~:~~l>IjJ~~~":d~Utt01l1flSj~iuti
nOOKS iX l'REJ>ARATION
SURGERY.
I\y), L H, R(lD~RT". l'.R.C.S., amI H.. M. VICK, O.JI.E., F.KCS.,
.'\I'.""t"n\ Sut)i;(..m~, S1. B"rth,)k'nww'!, Hr",pltal.

PSYCHIATRY.
Ry HKNRV l)~VI:-;K, {l.B.E., M.D, F.R.C.l'., Mt-dkal Supt., HQlloway
:"anatofilllH, \'Ht,.J.l"Iia \Ya.ter.

ANATOMY.
B\" H. H. \,'001.\.0\1\1>, M.L)., A"si~tallt I'wfcS$Of of Anatomy, UUiVCIslly
\nUq:e.

XRAYS AND RADIUM.

Bv .I. H. Don,L"" \\t.fI~UJl> "1.0., M.Re.p" I-'hysidan in Charge of
H ..diuhl[.W th.'pt., Mlddk",('x UQ5pitat.

PATHOLOGY AND BACTERIOLOGY.

PEDIATRICS.
n~ ''t'. J.. P)'",tIl_'W.v, I>.S.~., M.e., M.D., Ph~'Slcian ill Chargt', Cbildr~n's. i
CI<-pl., Ch'lrIlig Cross HosplUlI. _ _. _ _)
 MY FRIEND
WILLIAM OGILVY KERMACK
M.A . D.Se.
 PREFACE
lllO('uE!"IISTRY is H sciclH'(' (If such rt'c('lIt dt'\,t'}opuwut I1wl
R('tiy(' JH'ogT('SS that the whoit' (If our suhjt_ct might \\,(..,11 1)('
('mhrtH't'd hy the titlr (If thi~ little \'ohnn('. 1\-ly l'hit.'f
prohl{,lH hw_,; therefore 11('cli th(' ('hoi('c of material. Jt lut-loi
been my aim to inelud(' as dh'('T1'Il' us pu~sible a sd('(~ljoH of
toph's, ill which the fC'('('Ilt adVllTl('CS huw'lwf'll most striking.
alld in which tilt' trend of JHmh'rn hi(}('ht'mit'IlJ rt's('ar('h is
well irv.1i(akd. One.: has hut to contemplate til(' i,nlll('n!ic
incr('a~e in hiodll'mical lit('futurc !o.iH('(_' the war (If HH i-, 1M
to appreciate the magnitudl' of ('\'l'll this tn:-.k. I rt'nlisf',
therefore. that to ('crt:lil! hio('}wmh.ts my .~w}(dion of matt(',
may Seem SOffit'what arhitrary, but this is u. oded in!owpaJ'uhh'
from a work of this lIatur(', in which the prt'dikdiolls of the
author arc bound to filld expf{"SSiOfl. \l'hlJe therefor(', in
view of the limited space at my disposal, I mn.k(' no Ilvo1ogy
for the inevitable omission of mallY nspeet!oo of hio(.'hcmistry
both int.crcstiug and illstructiw', there arf' two omissiolls
whicJl arc in rather a djftcfeut (atrgoTY. ] tt'f{'r to the
mechanisms of tissue oxidations and the ('hcmistry of t.he
int{'rnnl secretions. Both of ti1('SC arc' Hdds of investigu1 ion
in which the interests of blodlemists arc very actin., and in
which striking developments have recently oecuff<'d, while
more many are in prospect. They ha\"c, however, been
treate(! so adequately ill Professor Lovatt Evans' volume
.. Recent Advances in Physiology," in the same series as the
prescnt volume, that I have judged it unnecessary and un-
wise to re-traverse these fields. Rather I have preferred to
det'ote the space, which wight have been utilised for t'lis
'PurPose, to certain aspects of two subjects which do not
 PREFACE
usually find in('l11~ion ill t('xt~books of biochemistry_ These
are t hr {"hapt<>r~ 01\ .. The ('hem.ieal Basis of Sp(.'cific Immuno~
logical HCi\ctions " and on H Chemotherapy, H subjt'cts wlJich
have a spe~'hll appeal to medieal reudf'fS and a dlUU,enging.
illt(,f('S\ to hiochemists and chemists alike.
It is with great plcasure that 1 acknowledge my indebted-
lIess to Professor .T. C. Drummond, D.Se., for reading and
('ll(~('kif)g tny mauuscript of the chapter on dtamins. and to
Dr . .1. 11. Shax],y and Dr. W. H. Tytler for similar services
in fegurd to dlaptcrs II. and X. n~sp('dj"ely. I am also
indebted to Dr. W. O. Kermack for much advice and criti-
('ism while the' book was in manuscript foml. My special
thanks I1r(' due to my colleaguc~ Mr. J. M. Peterson, B.Se.,
for much pninstaking labour in the preparation of drawings
for th(' illustrations and for assistanc'c in the prpparation of
tll(' index, My thuoks are likewise due to Professor Leathcs,
F.H.s .. for the loan of the hlocks and photographs from
whil'h tigurt's 11. 12, 13, and 1-" are rcprodm'ed; to Mes..,rs.
Ogih'Y & Co:, 20, Mortimer Street, W. }, for the loan of the
bloek fr,om which figure 25 was made; and to Messrs. The
M,Graw-Hill Book Company, Inc., of New York for per-
m.ission to f(>producc figures 2. 3, and 6 from Loeb's l"ro-.
H

teins and tl", Theory of Colloidal Hehaviour." I am also

ind"bted to Professor Lovatt Evans, J>'.R.S., and to the
publishers, Messrs .1. & A. Churchill for sending me a proof
ropy of .. neeent Advances in Physiology," thus enabling
me to plan my own book with the avoidance of undue
repetition in fields of cognate interest to phYSiology and
biodl<mistry. I feel, too, t.hat a final word of gratitude i.
<lUt to Messrs.l'hurellill, not only for thdr unfailing assistance
and {_"Ourtesy, but ab~o for the admirable manller in ~hich
they have given expression to their art in the form of the
present volume,
J.P.
1':ifirsJOLOGY INSTITUTE,
NEWPORT ROAD, CARDIFF.
 CONTENTS
1. A~H'}-;(,TS OF PtWTElN C\TAUnl,IS;\I. :\""{XU

..\('11):', A"SD lREA

II. COLl,HIllS AXil TnE I'uY:-.tCAL l'nE\tI~TIt\' OF

l)RoTElss 25

III. THE XITU:O-PIWTEI'SS 48

IV. 'TIlE CARBOJlYllIL\.TES 70

V. TilE BJOCIIEMIS1'RY O:F THE FATS 110

VI. TUE HIO('II:KMISTRY 0.1<' l'uOSI'UORl-S l'OM-

POVNUS 145

VII. THE RoLE OF SCLPDUR IN BlOCIIEMISTRY 186

VIII. THE VITAMINS 211

IX. H.MOGLOBIN AND ItELATED NUCRAI. PIG-

ME~'TS 251

x. THE CHEMICAL BASIS OF SPECIFIC IM~fV"O

LOGICAL REA(""TlONS 295

XI. CHEMOTHERAPY 314

!NDEX 341
nut nat.ul't', who lovps to y{'il }wrl>df at Ollr apprOl:tt'h, wnrkR pl'r tran!';-
formations in my8t~'rinus, bid(kn iaborntorit'>!: and it beinl! ~rnnt.('d thn.t
tilt' human body contJljns {'}wfk, flllJphur. phn.~phorus. iron and h..'ll other
8uhRUlnl'f~ lx~id('$, it is bard indf'{'d to find thl" explanation of hoW' all
this l'an be su~t-ainetl and n'new{'d during a period of .refirs by brend and
watt~r.

BrillatSavarin (Physiology of Tastf. 1825),

 RECENT ADYANCES IN
BIOCHE~IISTHY

CIL\pn:n I

ASPECTS OF PlWTEIN CATABOLIBIL AllmO

'filE
. ACIDS AND UREA
biodwmieal importance of the aamino acids needs no
empha&ising. Thc&c acids arc the ultimate products of tho
hydrolysis of proteins. and therefore represent the oilier
source of nitrogen 8yailable to the animal body. They are
readily oxidised in liying tissu{'s, their nitrogen hdn~ cxcret(>d
in the form of urea, and to a less extent As ammonht. The
relative extent and caM' of occurrence of this pr(){'(>ss varies
with tbe amino acid. In the case of the essential 8.nlino
acids which the animal cannot synthe~ise for ihdf, and which
are required for the e1ahoration of specific proteins or other
products of cellular activity, it probably occurs in the normal
course of eycnts to a much less extent t han in the case of
the simpler amino acids. Thus cystine, phenylalanine, ali<I
tyrosine arc induded in those arllino acids which. when
injected into the animal, do not immediately produce an
in~ase in urea formation such as is observed to occur in
the case of glycine and lencine. The nitrogen of the latter
acids is exaeted readily and completely in the form of ures.
Traces of amino acids are nonnaJly to he fonnd excreted in
the urine, but in general the utilisation of most amill'O acids
i. practically complete.
....
2 ASPE('TS OF' PROTEIN CAT ABOLIS!lf

(4)eLOOD

FIG. 1.-- '''ariations in amino nitrogen of li,er. kidney. muscles. Illld bh~
and in utea of bJood after the injection of amino acids. DraWl) from
data. of van SJyke and Meyer.

The r r - of Deaminlsation
The prwess by -which the amino acids are utilised after
they '!lave been absorbed from the intestine into the blood _
stream bas been studied in recent times by many schools of
 3
bio<:hemists, notably thos< of van Slyk(' nnti ~(llin. As ..
result of UK' injection of amino ados iuto tht \,<'I\OU,<\ circ\lla~
tion, thprt' js a rapid in(,fclks" hI tht" amiHo njttt)",1t'1l of Uu'
}iver~ this orlran takinjE up in th(' :-'P6{,(, of a f('w.rfl;imths S(lfll~
150 to 160 mg. of a.mino nitrogen pt'r 100 J.,'f'tutls.of fresh
tissue. 'fhc amino nitrugen of Ul{' Rctlt'tul hlood ('ir('ulatioJ1
js likewise temporarily jn{,Tcn~('d, Rud n hU'g(' prpportion of
this increas(' is rapidly takell up by oth('r organs, su('h as the
kidm'y and mnsl"lcs. In tht' ("n~c of t1lt' l1\'('r the mnxhrinl
uptake of amino nitrohl'Cu i'i ~o()n fon(lw(~d hy It st(o.dy
diminution, Bnd ;It Uw .'mnll' tiuH' t}u: h}ood urt'n inC'rrnSf'!'i.
Thc' post-ahsorptive fall in HllIiflO nitrogt'tl is not fWa.rly- so
well marked in the ('n~e of the kidncy and m\J.,d{'~. Sonw of
the l'('sults of van Slyko ar,' ,hown in tlw charts r('produeed
in Fli. ]. A point of inb'rest which has l'm('r~t'(l in thts('
investigations i.. the great rapidity with whi~h a.mino acids
temporarily accumulating in the blood stream arc transft'frcd
to the tissues.

The lIIechaniJm of l)eamjnjdtioo

Durin~ the abwrption of amino acid!', from the intestin(',
the blood, in its pass~. through the liver, lose, more amino
nitrogen than it dO<'s in passing through all the other organs
of the body. From these obsermtions w,' eOlldude that the
liver plays an important part in the general meta holism of the
nmino acids. As a result of their passage through this organ
they undergo cxtensi\'(~ deaminisation, and the liberated
nitrogen is converted to urea. But it mu!',t not he as!o.umed
that this function of deaminisation and urca formation is
purelY hepatic. It may occur in animals ill which the liver
has &en cut off from the general circulation, but in these
cases it is well known that t.he amount of urea formed
diminishes, whilst the ammonia is increased. We may,
however, safely conclude that the liypr is the chief seat of
the functions which we are discussing. It has been ","wned
that the amino nitrogen is liberated i~ the form of ammonia,
4 ASPECTS OF PROTEIN CATABOLISM
and that th!' latter is then (OIl\('rt:{~d to urt'n by combination
,,;th carhon dioxidf') pas'ting through tht" internwdiatc stag('~
of lunmonhm ("urbonute aud carbamate. 'Ve ~hall return
later to a dh.('ussion of the mechanism by means of which
amino J:itrogcn is cOllverted into urea, but meanwhile it may
be notf'o that th(' direct formation of ammonia from amino
acids is ycry doubtful. :Folin and Denis found no increase
in the ammonia of the portal blood after injecting the amino
seidl, gly~inc and asparagine, and they haye shown that much
of th(' ammonia present in the portal blood originates from
hl1eterial d('('ompo... ition in the inte,tinc.
\Vhateyer the change' may he hy m('all!-. of which the
f<>nlovnI of the nitrogc'n from the amino acid mol{'{'ulc is
('[(('etrcl, therc is general u,!...rTc{'ment that it involves 11 pl"{)('{'s~
of a-oxidation. By a-oxidation we mc-an that the a""f:'arbon
atom of the original amino a('id to which the amino gToup
was uttaehed ha. . be('orne oxidi::.cd during the proce~s of
d('aminisation. O. N('ubaul~r was the fin,t inn~tigator to
bring forward ('xp<'rimeutal t'yidenc{' in support of the view
that this process involn'd the production of an a-ketonic
acid. In 1883 Schotten had found that after admini,iering
Jlhenylgly~ine (phenylaminoace1ic acid) to dogs he recovered
in tht, urine, in additiotl to some unchanged twid, the corre~
sponding hydroxy a('id, namely, mandelic acid. This latter
acid must haw originated from the phcnylglycine giYen to
the animal at tIl(, commenC'ement of the experiment, and it
would s('{'m to have ori~nated by a proc-ess of hydrolytic
deaminisation, thus : -

C,R, .CRNR . CO OR
(l'booylglydnc.)
+ R.O --> C.R,.lMandeUc
CROll .COOR + NR ..
add.) f'O

'l"hat this was not the case wa." shown in Neubauer~s inyesti~
gations on the fate of optiCAlly inactive phenyl a-aminoacetic
acid in the dog, and in the isolated perfused liver of the dog.
In both inst.ances he found the following products: (1) un- (
changed phenyl aminoacetic acid containing excess of the
 OXlDATH'E DEAMINISATION Ii

Im'o-rotatory compound; (2) Iwvo-mll"odi" acid; (3) pll<'"yl-

glyoxyli,' ""i,1, C,ll,. CO. COOH; 0) IX'llz"ic 'I(-i.1. Tb~
last~nlt'ntioncd {l(id i!- t'ound in the uriJw as hiwmrit- tlt'id.
C.U s . CO . Nil , CII., COOII. On admilli,kril1t the tk.rt",.
'rotatory i~onl(rid( of plH:nylnminom."l,tic add to th~ dog he
also found in the urine phenylgly()xyli(' "Md and Ian,.
manddic at'id. It se('IllS ("\'jdt'l1t from t h('!oo(' ('xpf'rim('nts thnt
the dc.tfl'o.rotatory acid is mo1'(o n'u.diiy Ul{,tt\bolisrd than the
10,1'0 acid, that the ketonic acid is the- main product of the
deamillisatiou, and thttt tht:' manddic aeid mu~t arise by au
asymmetric TetiuefioH uf the ilw('tin' kdoni(' 8fid fo,O fornwd,
<.;inc(' lll'l'O-mIlndeJj" a(,id ('(lull) not be fonm'd dirf'(.'Uy from
the de.rtroalHiuo twirl. The !o.t'rit,s of ('h"llg(_'~ may bt reprc~
~('nt('d us follows :-

C.H, ~HXH, COOH ~ C,H, . (Xl. COOU ..... C,H, . CHon. coon
t
C.H,. COOH.

Many other instancc~ of n sirnilar seric,", of ('han~s M\'C

since been hrought to light. Spt'cial importance .tta,he. to
Ku()()p's invc.ligations. In these )'-plwnyl a-aminobntyric
acid was administered to dogs in the form of the optically
ina('tive acid. There Were reco"ered in the urin(' the {celiO
and tkJ'lrQ isomers of til,' unchanged add, de.ltrll-phenyl a-
hydroxy-butyric acid, the corresponding (.l-k(:toni(" acid, and
hippuric add. III this case, a~ain, the hydroxy acid was
proved to han originated from the k~tonic acid by a prOCfss
of asymmetric rt~duction, sjnce direet administrution of the
sodiu~ salt of the ketonic acid led to the excretion of the
deztro-hydroxy acid_ Knoop made a further very important
disco~ery in this last .tagc of the ex!",riment. He also found
in the urine the acetyl derivative of dCT-lm-phenyl a-amino-
hutyric acid, proving conclusively that the keto"ic acid hod
been utilised in synthesising this amino acid, Dakin has
.represented this important series of inve&tigations It the
following schematic way : -
6 ASPECTS OF PROTEIN CATABOLlS
dJ-C.H .CHt CH 1oC1UIH I .COOH

----------- ------------___ClI.
,J-C.H"CH"CH:.{1iNH.,COOH I-CJj,.CH . CHNHCOUl

. J i
CJ1,.CH,CfJ,.C().C()()1I - - - . "CJl,.CH,.CH CHOH.COOH

J
C'Jl,.CII,.CH,.COOH

I
C.H,.CoolI - . C,H,.CO.NH.CH,.COOH

It will he ,een th.t the stal' represented above, in which

pil('nylpropiolli(' acid, resulting from the oxidation of phenyl
('(k(tohutyrie acid, is eOllvt'rted to henzoie acid, in\"olY.~s the
rt'movul of two ('arbon atoms. Thi~ proces, which we shall
d.js(_'u~s in Chapter Y., i.. known as ,B.oxidation. Dakin
summarises the results of thest~ and many other t'xperiments
on the falt of amino acids in the animal body in the following
terms: .. In general the fate in the body of aamino and
aketonic acids is identicaL wh"reas the a-hydroxy acid.
being presumably secondary reduction products may behave
differently. It is therefore assumed that a-ketonic acids
tll't' ohligate products of th(' direct oxidation of amino acids,
while the hydroxy acids are not directly deri,'ed from the
amino acids. .A ketonic acid may th('refore undergo three
tyJX's of "hanl': (I) it may be oxidised to a lower fatty
acid; (2) it may he reduced to a hydroxy acid; (8) its
ammonium salt may be reduced to an amino acid."
Knoop's demonstration of the synthesis of an amino, acid
from the corresponding ketonic add is of fundamental
importance, and soon Embden and his school showed that
the reaction is a general one. They obtained alanine, phenyl.
alanin~ and tyrosine on JX'rfnsing the ammonium salts of
the corresponding ketonic acids through the isolated liver. I
In the experiments of Neubauer and of Knoop there is no
 YEAST AND A211JNO .4ClDS ..
doubt that the hydroxy acids were sC('ondnry products
arising from tht, T('t]u{'i.ion of the krtanie Odfb, but tht"
rcv{'t'St: changl' of hydroxy add to krtoni(' R('ld is_.one whiC'h
.thr tis...o;ut'.o; art' also capnblt' of ('ffl'(ting. Thus ..1ot only did
EmhrlNI find that }lynlvic ucid yiddrd alanine Oil being
Iwrfused throup-h 111<' liwr, hut thnt tI... <'OTI'C"l'oll(linll
hydroxy u-C'id, b\('ti(' }I('id, likrwis(' ~ft_y(' tit.;c tn th(' amino
ucid. "'p
hav(" Uwrt'i'nrf'. 11." a normal feature of metnlx>Hsm
the rlose~t ('oT'relation \)('t w('t"n tiwsc thrf'e tyP('S of acids,
and their intercollYt'rsion is strictly Tf'vcrsiblc. thus :-
CII" ('HXII,. COOII ~ CH,. (XI , (X'OIi~ eH, ,CHO!! ,coon

As we shall see in u Intcr chapter, both lactic and pyru\'ic

Reid, at<' of the ~..,.enle't importance ill corbohyclrnte
nlcta\-,.,lism, and thc:>sc observations provide a sowld theo-
retical basis for the weUknowll conversion of carbohydrate
into amino acids, and ultimately into prott.>in~ and also for
the reverse chang(', which is readily d('monstrl1ted in the
diabetic subj",t and in the phlorhizinised animal.
The type of biochemieal reaction which We have discussed
in the fOI'Cl(Oing parawaphs, nam('ly, deamini."tion accom-
panied by production of the a-ketonic acid, is by no means
confined to the mammalian organiJi.m, or even to the animal
kingdom. Neubauer included in his investigations a study
of'the action of yeast on di-pbenylamino8('('tic acid, and found
that a series of compounds very similar to tbose produced
in the dog could be isolated as a result of the activity of
this simple organism. }<'rom the culture medium contain
ing yeast and the amino acid the following producta were
isolal&d: (1) unchanged I-phenylaminoacetic acid and its
acetyl derh'ati\'e, phenylglyoxylic acid, I-mandelic acid. and
benzyl alcohol. It was also sho,,", that, as in the animal,
the Imandelic acid arose from phenylglyoxylic acid by
reduction, and that the reverse change could also take place
but much less vigorously than the former ehange~ The
importance of the kctonic acid as a precursor or- the benzyl
,
 S ASPECTS OF PROTEIN CATABOLl~M

alcohol, whieh in thi., instanff' represented the end product

of til{' s('rj{'~ of f('~ctj{)ns, WH!-, shown by the cOllversion in a
:"imilar ('xperiment with yea.,t of RnoUwT kctonic acid,
p-hydroxy].1i1/'nylpyrU\'ic add, into the corresponding alcohol
with on,c carbon atom less, p-hydroxyph<'nylethyl alcohol; ,
110. C ,H . CII, . CO, COOH____. HO , C ,H, ' CH" cn,on.
As we shall ,ec in a lutt'r chapter (1'. 108), such a change
probably iuvolves the formation of the aldehyde, in this
('as(' benzaldehyde, as an intermediate step. Th(' ess{'ntial
similarity of til(' two series of trans.formations in which
pheny\arninoaedl{' a('1.<1 flgurc-'S in th(' dog and in yea~t
cultuff's is shown in the following scheme. The continuous
liue represents ehang'{'s taking place in the mammaJ, whilst
the broken line reprt"scnts those oc('urring in yeast : ~

d/C.HCHNH,.COOll

"'C.H"CII(NH.co'CH")'cOOH/,'/~\
""'- ~ ,"
J-C.H._CHtIH.CO.CH,).COOH
\ /",
,~

d-C.H CHNH,.COOH I-C.H..CHNHCOOH

11
C.H CO.COOH
__.-::;-- \-------------
~-.... ---~ -
I-CJfCHOH.COQH C,.H..COOH B::'.H,.CHCj
1
C.H,.Co.NH.CH,.COOH
1
C.H,.CH.OH

In both cases it should be observed that the de:dro isomer

is preferentially metabolised, and that it is the kwo f";'" of
mandelic acid which is formed by asymmetric reduction of
the ketonic acid. On tile other hand, an interesting difference
between the two processes is the production of the dextro
form t't pm-nylacetylaminoacetic acid in the dog and of its ,
optical antipode, the lrevG form, in the yea.1: fermentation.
 RTRUCTVRE OF UREA
TIle 8trucIure of U_
Ht~for(' passing on to u d.is('ll~... ion of Hl(' nw('llIUli~m of UN'S
formation, Wf' must tirst considt'r Ul(' ~tru('tlll'(' ai thi~ ('OlTlw
potmd. In r{"cent years our idt'ns concerning \r1(' structu}'('
of this fundamental product of amino acid (nbboli'llfrn luwf'
Ilodcrgollt tl consid"rnhl,' ehnn~.... ]n HJllI .J-:. A. \Y('rm'l
SUrh1('sted that Ur('8, in its :-,tabl(' form had til(' ('vdir struc-t.ur('
shown below instead of thf' fumiHnr carbnn'lidf'" stnH'tuff:
u<ually attrihutl'd to it ;-.
/~H, ),\11,
n!'< ~ C(l o ('(
\''''XII 2
o
(Urea.-CycHc Formula..) (Carbamide.)

Thi. ~~'I(estion is of ~".,.at biologkal signiiic.ll(,(" and w..

shall first briefly revi,'w the chemical evidence whi<>h has
been ciiscu",'d and amplifiNI by Werner in support of his
views.
These views are based very largely on a reinvestigation
of the close relationship which ehlsts '",tween urea and its
derivati"es on the one hand and cyanic acid on the other.
This relatiollship has been known ewr since Wtihler's classical
synthesis of urea, which, it will be remembered, involved the
!ntramoJN'ular rt"arran~wment of ammonium cyanate. Thi.s
change, which proreeds, be it Hoted~ as II bimolecular reaction,
may be represented as follows :-
NH,O, C = N or l\'H, . N = C = 0 ~ 0 = C = (XlI.)t.
The change takes place even at ordinary temperatures on
exposi..'1g ammooium cyanate to tbe air, and i. complete
after two days. While the conversion is proceeding ammonia
i. heing constantly given off. On the other hand, if the
ammonium cyanate he enclosed in an atmosphere of
ammonia, there is no conversion to urea even after it bas
.remained at room temperature for eight days. ~mer'.
expl8llation of these observations is that ammonium cyanate
10 ASPECTS OF PROTEIN CATABOLI$M
dissociates, likc> mo~t ammonium salts. with considerable
east~. It thus hr1H.'S ri!'lC to ammonia and (yanic acid. The
latter is lih~'nltt:d ill the enolic form, and in an effort to attain
R rnOT(' ~tat)lf (>onfig'uration it is transformed into the keto
form, i.Jeyanic l\{id. The i.9(J(yanie acid then rc('ombint-'s
with ammonia to form urea. The change is simply rf'Jlre~
scnt{'d in the followillg way : -

NH.O.CN-- NH.' HO.CN=HN'C,O NH,~ m,.c(~H,

~~~~~~; \ ENOL K.f.iO ) ultUl 0

TIlls "dew of the lfIcdHmism of the COIl,"crsiOI1 of ammoniunl

eyanatc into urea is based ('ssentially on tht' supposition that
(!yuni(' acid j" ('omprised of two diffcrf"nt molt~('uJar forms in
(quilihrium. It oup:ht, thcrt'fore, to be l){)ssiblc to ,devise
conditions which lrad to the reversal of the above trans-
formation. Thi~ i!<. c}.cmplified in the partial re('onYersion
of urea to amnWniUll.l {'yanntl:' which takes place in aqueous
or alcoholic solutions at a temp{>ratllre much helow that at
which urea in bulk is known to change.
On being- subjected to heat urea exhibits a series of changes
the mechanism of which haye an important bearing on its
strurtuI'C. These rhanges are correlated by ',"erner with the
('quilibrimll system, which may be r('prcsellted as follows : -
riSt'oftemp.
&~~:~l~ium +- NH, + HOC~;=_~ HNCO + NBs ---+ urea.
. fan of temp.
lEnol IJUlb1e (Ket<> stable
at low temp.) at high temp.)

If ureR is heated in a vacuum at 160 a sublimate is formed

which is found to consist of ammonium cyanate. The. same
sublimate is also formed when urea is heated in the ordinary
way, and further heating leads to the familiar formation of
biuret, which is accompanied by the evolution of ammonia.
Wbel\.the beating i~ continued above 100' cyanuric acid and
ammelide are formed. The first stage of these ~hanges is the
dissociation of tbe urea into ammonia and cyanic acid and
.
 UREA CONDENSATIONS II

the recombination of some of these ('ollstituenh ill tht l~oolt'r

parts of the apparatus to f(lrnl 811lUlOuium CYllIUit(, Tht
bulk of th" cyunic arid formed in this higl;-telllprraturt
\Jis~ocitltion will he pr('!l;ent in tll(' keto form, "ild this on
further heating ('omhim." with th(' Tl'ut'livl' form 0' urrH.
whieh \\~ern('r tiwrres as an open chain ~Hllpound, to Kin.~
the cnolie form of biuret. Tlw lutter then ('hanj,~" to th"
more stabl' k("to form. The formation of nmmdide rt'prcst'nts
a further (,Dnden~tjon of is()('yani(~ acid with hiurtt. '1'11('$c
('hang('s may Ix repr{ ... cntrd in the following way :-

HN~.OH O~.NH,
INH z 135' to ISO'
HN'C + "NCO _, __ \QO. NH --+ NR
'OR ~ ~
IWICTM. FIlIU4
HN-C.OH oC.NH1
at OI\t.A IIIU~

ICO.NHt
lIN, +IINCO
CO.NHt

Biuret, on being heated to stjIJ hj~hcr tcmpcrnturrs, df'('Otl

poses to form ammotlia and cYlluic [l(id, which \'olutilis
considerable quantiti('s of urea and (,'yanuric add, and sm.
quantities of ammelide. Cyanttrie acid is also formed t
polymerisation of cyanic acid at high temperatures.
has tItt constitution shown below. The polymerisation
cyanic add at low temperatures leads to the fonnation
cyameJide.
co . l'<'1I" _

~
0 . qI'ill)~
liN
O.NH/
x;O IlN=C
< O.C(NH)
/
{Cyanuric Acid.)
,
(Cyamelide (Uan_'. F_uJ.).
12 ASPECTS OF PROTEIN CATABOLI/iM
Werner has (!xplained how the formation of thesc two
polytn('r~ of cyani(~ ueid may be (~rrelated with the stability
of t h{' ketPIlic anel (,Holie forms of the paI'{"nt acid at high
lInd low t{'r"'.l(r;dUTf'S T(~}X'('tj\'dv. It wjlJ be Sf'en from the
ubov(' (~quations that the fOfm.ation of biul't"t is a reversible-
reactioll, ~ill(~(' at h'mperaturcs above ]90 it is dissociated
with th(' form:ttion, amongst other produets, of the com~
IKllmds from whieh it originated, namdy~ cyanic acid and
urt'H.. :MuIIY y<'urs ago FinC'kh demonstrated thf" formation
of hiuret by pas!odng th(' vapour of c:vanie Rcid into urea at
it.... lIu,ltiJlg point (132'). "'erner has dcscrilK'd it u]('thod for
dt'moll~tratin~ a l-,.imilar formation of biuret in aqueous
'Solution at low tenlperature.
To '2 !-,rrams of urea dissoly(.~d in 2 C.c. of strong hydrocbloric
.:\('id, diluted with 4, (',C, of water, 2 I-!'rnm~ of powdered
potassium ryHIHltf' ar(~ gradually addf'd WjtJl constant
stirring; after a few moments the solution is filtered to
separate SOUle cyamelide which has been formed. The
filtrate, when tested in the usual manner with copper sulphate
and f'X('('SS of alkali, will be found to give a !)trong biuret
reaction.
}"urtlwr evidence in favour of the new mode of formulating
urea is forthcoming from a consideration of the interaction
of urea and nitrous acid. One Inight expect that if the
C'arbamid(' formula correctly represented urea the reaction
with nitrous acid would be a simple decomposition entirely
analogous to the wdl-known mode of decomposition exhibited
by substances containinp an amino bl1'OUP, thus ; -

OC(NH.) + 2 HNO. ----+ CO, + 2 N. + 8 H.o,

and the reaction ought to be available for the quantitative
determination of urea_ Sueh is not the case, and Werner, in
investigating the behaviour of urea towards nitrous acid, has
established the following points: (1) urea and pure nitrous
acid do not interact in aqueou. solution, but tbe presence
of a mineral acid quickly initiates a brisk reaction; (2) a
\
 NITROUS A.ClD AND l'REA 13
W('sk add, st]<'h 8~ n(~('ti(', dO<'s not promott tilt' intern(tioll
unless tilt' con('entration of th(' ut'id is "l'ry high, nnd (','('n
thrn tht' v('IO<'ity orthe rf'tl(,tioll i~ "t'ry :-.tow; (:illht ,'olnn\('
of nitrogt'"ll ('\oInrl dO('~ Hot ('orrl'spond to thi,f C'tllrulut('d
on the basis of th(' equation gi\'(>1l nl"")\'(" th,' (IUn~ltity of
U1'('a d{'('ompost'd h<-inj.( nlUt'h gr<'ukr tlum thut itldi(uhfl
by the cvolyed nitrogen; (1,) only W}WIl th(' HITn is pr{'~{'l\t
in cOllsidcrabll' exc("s~ i~ the volumc' of nitrogt'l) ohtlliuNI U,l\
approximate estimate of th(' amount of nitruus u('id c\{'('om
posed; (5) the ratio of carhon dioxid{' to nitrogrll in the gaO)
evolved i~ always higlwf thull that indieahd in tht <'(jl1ution.
ThC' Rb~(_>ll(,(' of R('tion of pure' nitrou:"\ M,('id Uti urea indi('utcs
that an amino waul', a~ such, i~ not pn'~rnt. It i"'lntC'r('stil\~
to note that thiourea, which 8ccordinR to Werner may exist
NU.
in solution as UN = C( J is rnpidly de('ompo~cd oy PUf('
1m
nitrou~ acid, giving rbe: to lhiocyaniC' ndd, llitrt)1.1'('U, and
water. \VhCIl, ho'V('n'T, in the ('a!o:le of urra a ~tron,g aeid s\J('h
as hydrochJoric js pr("f,('ut. a !'>a)t i, forJHf'd which r('at"ts
aC"('ording to the eqllatioHS ;--.
NIl, /XH, . Hel
UN = C<I +- HCI -~ IIX = C,
o -OIl
followed by
XII. He]
UN = c) - + IlXO ,~N. -+- IIXCO + !lU/) +- He!.
. II
The cyanic acid fonned according to the second equation i.
.

then decomposed in two ways: (1) by hydrolysis; (2) hy

interaction with nitrous acid, as follows :-
(1) HNCO + lI.O + H(l -----+ :'>.'H,Cl + CO,,.
(2) lINCO + RNO, -----+ CO, + N. t H,O.
14 ASPECTS OF PROTEIN CATABOLlS"lf
Thr formation of ryanjc HC'id during Ul{' ('01lrs(' of tJlt'~e
reactions ('an hc' df'mollstrat(d hy it" precipitation as the
SilVt'T salt on addiIlg ~il\'{'r nitratC'. \Yerm'l' ha!oo confirnU'd
the aho\'/ ~'iplanation of the cours{' of the lut('radion between
ureH apc1 nitrous add hy quantitatiw' nl('a:-'Uf{'menb.

The lIydro)ysis 01 Urea

ttrcu may undf'Tgo hydroly~b. in aquNm:-. solution , . .it h or
without til(' addition of acids or alkali:-.. FawsiU first demon-
strated that the hydrolysis of UT('a hy wateT alone at ]00 0
invo)ycd the formation of (yanni<'. BUfI the prr-scHc(" of the
{'yanate rarli('lc i:-. ('lu.ily :-,hown hy heating a :-.olntion of urea
at the boiling point for a few minutes. cooling. and adding
silver nitrate, In the pTf'senC'{' of neids dissodation into
ammonia and ('vaniC' R{'id jr, Iikt:>wise the first phas(> of the
hydrolysis, th(' e~ourse of the whole> reaction being l'(>P~(>S('Bt('d
jn tIl(" foBowing rquntjoll~, propo~cd by 'Y('rnlT;-
Phasel.
/1\'113
11"" = 'c< I + Hel -> XII ,CI + (II,,"CO ~ nOD').
"0
PI"" .. 11.
(IINCO ~ HOCX) + H 20 + HCl ---> XH,Cl + co,.
SiJH..'t' cyanic acid is hydrolysed very rapidly~ the vdocity
af the w/rate {Wlcti<1ii wiN he ddeiTu!c""7 "5' the rote of
di,ssociation of the urea, both IlToduC'ts of this dissociation
being femOY('o. practically as rapidly as they ar!' formed, In
the presence of alkali the hydrolysis may be pictured as
oc('urring also in two phases, a~ follows : -
Pha ... l.
NH 3
/
HN = c~ , + NaOH --_ NII. + NaOCN + U,O.
pw, II.
 UREASE 15
The Ensymio BJdroInia of Urea
Tht, ammoniac's} or nlkalint, fenut'ntution of urilU~ is a
change which has lx-'{'n inn'stigatc-d by biOl'IK'mi~ts for mort'
than n century. }'ourC'roy ami Yttuqudin ill 17f!HrO ~hOWtd
thut tht' ammonia formt'd during' this fl'rnlt'ntntion pl'(~N
had its origin in the urea. In 1~6~ van 'fi"~h<m i';"laled
the organism, whieh hu., sill(,(, b('cn calkd .'/iffOCm.'NIR urttt't
re!o,poll~ibh- for th(' d('comp()~ition of t}uo unfl. Many oth('f
orgullisnls are also capahle of dt.'(()mpo~iflg: un'n in thr sam('
wily as Jlicrococtfl urea', and in lHIlO Miqud proposed the
UM' of tht' namC' urease to illdi('utl' the' enzyme common to
th{'se organisms which brought nhout the hydrolysis of ur~n
to ammoniulll carbonate. III 190n Tak('u{'hi found urt'a~e
in the aqueous e)"i"rad of ('rushed soya beans, which art'!' the
~('cd!i 0' the plant Glyrine hispida. Th(' cnzylUe has siner
lx">("n found in other leguminous plants, and is said to be
pr('M'nt in the ,\worcl b('un {Canm'(lUa glmJiata} and in the
jack }:wan (C. ensiformis) in ('Vt'll lnr~f'r nnwuJ)ts th/Ul in the
soya bean. The pr!'sence of urC'llSC ju mnny OilH'f plant
sourres has also bet.'ll rceorded. Vr(,flst' hllS abo be'en dttc~cted
ill ,mall amounts ill the Jiver of the horse and the dog. It.
O("currcnc(' in this animal SOUfC(' is not regarded as having
any physiological signifiC'ancr, and. it is sugg(~sted that it is
derived from ingested vegetable matter (Steppuhn and Utkin-
Ljubo,zov).
The enzyme urease app<"n; to be remark. bly sp""ilie in
its action, since it will not promoh' the hydrolysis of the
following substituted ureas at temperatures between 45
and 50': methyl, ethyl, sytn-dimethyl, sym-diethyl. 4$JfI'"
dimethyl, "-propyl, phenyl. pip<ridyl, nor of thiourea,
urethal'le, and biuret. It is stated that pure n-butyl Urea is
decomposed by urease at 45 to 50, and it is suggested that
this i. due to the relative instability of this urea as compared,
for instance, with ethy! urea, leading to dissociation at a
lower temperature. The optimum temperature for the tk!tion
or urease has been pllWed at the remarkably high figure of
16 ASPECTS OF PROTEIN CA7'ABOLl~M

60'\ and this may llceount for the fact that eY('fl ('thyl urea
is slowJy atts('ked hy t h(' (,lIzymc at 70<J, whiC'h must he just
bt:-Iow tht, tt'mperaturc limit of its acti\ity. \V(:rner sugg{'sts
that it is rqJbuble thHt if tho onzyme dcws not promote tho
diss(wiption of a !ooubstitutt-d urea lx-low RO", it cannot
hydrolyse' thnt ur<'a in the ordinary sensc. The spt:'('ificity
of UrNl:~C' would, then,fort', S('('m to lx larJ.,.1('iy d{'tcrmiu('o by
tht, temperature at whieh tilt: urea forming th{' ~l.lb~tra.tc
bf>~jns to bt' dissociatf'd.
It is ~"'nerllny agreed that hefore urease hydrolyses its
substratt' thc' laU('r is ncisorl)f'd by th(' enzynu-. Son1l" surh
theory is Il{'{'('ssitaterl hy Bayliss~ oh<;f'rYstion thnt ur!'a:-;c
still produ('{'s all apprec'iabl{' hydroly!:oois of urC'R in solutions
containing 80 to 90 l~r ('{nt. of akohol, in which m('(lia th(_'
{'llzyUl(' is quite insoluhle. }'oHowing the arisorptio\l phase,
it has bcen assunwd that the ('ol1venion of U1"('ft to ammonium
carhonate in\'o}y('d the simple addition of water, with the
pos:-:iible intermediatf' formation of ammonium carbamate,
thus :-..
CO: (NH,) - . CO: (NH,)(ONH,)--> CO: (ONH.)..
It se,'nlS uatural t hat such a view should he held so long as
un'a is regarded as a rliamino compound such a_" carbamide.
But, 8.s we have sef'll, there arc Y('ry good grounds for formu-
lating urea in an t'nti:rcly difft'rent. mann('r, and .Fearon has
rt'~en't)y inycsti,:rateo 'the m('e~llan'tsm ot 'toe action ot ureusc
from the standpoint of the cyclic formula proposed by
WeMlcr. He comes to the conclusion that the course of the
reaction is not that outlined above. Fearon has shown that
it is possible to isolate cyanic acid in the form of its silver
salt by adding silver nitrate to solutions of urea un&:,rgoing
decomposition by urease. He was able to obtain no evidenee
of the formation of ammonium carbamate during this process.
He therefore formulates tne theory that ammonia and cyanic
acid 'tore intennediate produets of the action of the enzym;
on urea, these products arising by the simple process of
 MlfCHAN18M OF ACTION OF FREASE 17

dissociation which we ha"e already discussed. Ul>'''''' ;',

"",cording to this "iew, n dis.ocinting en~yme. .'oU"wing
the stage of diss()('iation th<' <'yani{" acid Hb("ratt'!i is hydro~
lysed by the water pl'{'scnt, and gin's riS<' to "ltlnJouiu. nml
\'arhon d.ioxid('. Tht-Iatif'f thf'l1 ('Ombinr to form ammonium
hydro~n carbonatt', which is converhd to tht nurmal
carbonHt{~ hy n furUlt'r ('omhination with the amrnontu
libcorated durin~ tht {il'!-,t or dis~o('i"ti()n st.l\I((\ whl('h ammonia
J'(mains ad~orlwu by the {'nzymf' unt.il it is T{mo\,(_d to furm
ammonium carbonatc'. The enzyme IS, t h(rt'fol\.', f-' to
comhirl(' with mor' IIr('n, and the ('),,('1<' filly tw~in afrt"sh.
}'t'IU'Oll addu('(->s ('xperimt'Ht.aJ ,'yideu('(' in support of the> vi('w
that urea.'iC call adl'iorb both un'a find ammonia.
The oydic structure of urea h,,, het'lI acct'pl",1 by M..~k
and Villars in their studieN of tiM' ,arne reaction, hut the",'
workers still attadt importane(' t(l ammonium (nrb8mat.t~
and carbonate at; intt:'rnu,_'diatt" pToducto; ill th(" ('nzymie
decomposition of urea. Mack and Villars, and also Kay, huw
lX'en ahle to df'tnonstrate a slight amount bf urca formation
hy R(ting on strong solutionx of ammonium (~arhaJnate- and
carbonate with urease. This it is cJainJ(~d establish{~,. the
reversible nature of th(" action of urea.w, a claim which is
probahly justified. Hut it would seem to be in opposition to
the \~iews which we have just dis(:usseu. Th(' Ja"Ot-mrntioncd
authors suggt'st that the re8.('tion whi('" is ('utalysNl hy ttw
enzyme is the ('ouvt'rsion
UJ't'a ~ Ammonium carhamate.
Mnek and Villars admit that there i. simultaneous fom",
tion of ammonium cy81lare along with tlw pl'tJduction of
carbaIDare, but they regard the production of the former
compound as due to the spontaneous dissociation of the
urea uninfluenced by the enzyme. As we have stared.
however, }'earon has failed to detect any fonnation of
...mmouiwn carbamate during the action of urease ~ urea,
and be argues that the produet obtained by Mlwk and Villars
....
18 ASPECTS OF PROTEIN (,ATABOLl.JYM
ron.sists of cyanate onJy. Motco,'er, in the absence of the
enzyme, cYl.lnut(" i" not formC"d spont.aneously from urca under
condition~ l{'udlllg: to if.<.; formation when til(' f'l1zyme is
Pr<'sP"t. ft.;s th(,r<'fort difficult to believe that the pl'('Sen"t
of th<".enzymc is not definitely related with the appearance
of (yanutt'. A furth(~r point of importanr.e which is empha-
~i~('(l
by I"earon is that url'Qs(' d(X~s not catalyst' the rab> of
formation of am.monium cyanate from urea, but that its uction
eonsi,b in tlle liberation of the unstable form of cyanic acid,
ill this easc the hto acid. The lath'r is then hydrolysed until
the progress of the reaction results in the accumulation of
sufficient ammonia to retard the action of the enzyme
through til(' formation of an adsorption compound to the
exclusion of the substrate, and to reta.rd the hydrolysis of
the k,to-cyanic acid. The argument that Ur<'8se does not
('utnJyse t.h( ]'t>n'rs(' fe-action of tlw con""l'Sion of ammonium
cyanate into urea, which is used hy both Mack and Villars
and by Kay, is thcrt'fore not r<'garded hy .Fearon as ,-..lid.
Grsn!<t! that such is the case, the production of urea by the
action of urease on mixtuJ"{'s of ammonium carbamate and
carbonate still awaits an explanation. It should, however,
be noted that only very small amounts of urea have been
obtaincd by this PrO<'<'ss, and it seems possible that such
lImounts might be produced through tbe intermediate forma-
tion of cvanic acid. Indeed, Werner has demonstrated that
the fOfm;'tion of urea by thC" action of heat on ammonium
carhamate dOC's inyo]w' this intermediate step.

Urea FormatIon in Living ~

It is obvious tbat tM foreb'Oing observations hllve an
important bearing on tM mechanism of tM formation of
urea as Ii sequd to the deaminisation of amino acids which
occurs in living tisslles,and we must now turn to a considera-
tion of this question. We have already seen that the first
step in the "atabolism of amino acids consists of a p _
of a-oxidation and simultBneous deatninisation. and that tM
 UREA AND DEAM1N1SlITION III
amino group so relnond i!'o ("\tlltmuly (_'xl'rc\{'d as Uf('R. rrhi~
prON.~SS has g't')\('rally hel'll T('prt>M'Ilh-d in tht, {OUOWiH~
wuy!~

R . eo . COOll
.
R . cIl~m . coon / "
- --"XlI, ----> (X11,),('O, .. --->
-_... (:\'H,O). CO. XH, - - . CO(NH,)..
There is no douht that ammotlium sult:-;., l'.uch AS t I\(.~ t'urbonllt('
and chloTidt, l'ithn in l)(.rfu~ioH ~:xp(,Tin\('nb. or wh{'I\
udminbtC-fcd dir('{'tly to fUl.imals, lead to an incret\St~
produC'tivH of un'a. This oh.~('T,ation would .'>{"t'm to )oupport
th(> supposition that tlft'a fornwtion takl'!'. ptuN' from t'rtX'"
ammonia or from an ammonium suIt. }'rom time to time
various. objections huw l.)('{'n rab-Nt to this Vlt'W, and the
pos,ibility of dt'uminisation ()('('lIning without th" di ....ct
liberation of free ammonia or the direct fonnl1t.1oJl of nn
ammonium hoa\t has ht'{'n (on~iclcred. Th('Te art' mauy
difficulties that !lIay he urgNI again,t th" vit-w that free
ammonia hi formed ('\'en temporarily by the oxidative
dcaminisation of amino llcids, and it is certllinly ditftcuJt to
see how this ean occur without disrupting the carbon chain.
Knoop's sugg"sti()n of the illhm.cdiate formation of hydrattd
imillO acids may m' (jted ns an attempt to ('jreutn\'ent some
of these difficulties : -
OH
R,(/Coon ---> R. (..COOH.
'\..NH, XU.
One may here ....fer to the obselvati')lls made hy Fosse and
his co-'\vorkers, which demonstrate the Cll-'" with which
ammonia beeomcs oxidised in alkaline solutions in the
presence of organic compound,. Thes<' observations seem
to render improbable the liberation of free ammonia in an
oxidative change such as we have seen dcaminisation'lo be.
hn the other band, when mixed solutions of ammonium salt.
20 ASPECTS OF PROTEIN CATABOqSM
Ulld gluc()!'>t or otht'r or~ani(' substau('{'s are subjected to
oxidation, both ('yani(_, aci(i and urca un' frequently produced.
Sp('('ial iJuportanc(, attut"h<:'!. to tll(' r('~ults which j1'osse
obtaim,d "'kwn It(' :o.ubjtckd casein and nmino acids, such as
a ... par~i})( und glycinc, to ox;dHtion. In these instan{.'es tJie
nmOUlJt of UN'S formed was Verv mu(:h increas(>tl on heating
the' produC't from tht oxidatio;, with ammonium chloride.
C~anll.te was uouhtl<-s:-.. formt'd durinJ:( the oxidation, but
insum('i(~nt Rmmonia was I>n:sent to enable it to form urea.
Thi~ ddki('Jl('Y was r<~111edi('d by tll(' nddition of ammonium
C'hJorid(' and urt'U wus produced in quantity.
III a di"telt't..,ion of thc-s(' Bnd other obM:rvations \Vernf'r
..ntirdy r"j('cts the ide" that urea is formed from earbon
dioxide and ammonia during the oxidation of nitrogenous'
compounds. HaYing in view the important part pl/ly..d by
<'yani" add in the dlt'mical transformations of urea, Fearon
and M{)nt~m('ry have re(~ntly inve~tigated the possible
relationship of {'yanic acid formation to oxidative deaminiM\-
t ion. Oil oxidising glycine ami alanine with hydr"ll"n
peroxide in alkaline solution the formation of <'yanic acid
nnd of ureH was detectt.d. Much more decisive results were
obtained in the case of glycine on using potassium per-
manganate in an alkaline medium a_.;;. the oxidising agent.
Cynnlc (wid formation wa.or.; specially marked when glucose
or formaldehyde was present along with the amillo acid.
}l"Il'O'" a!'t' advanced for regarding this cyanic acid as
aFl.'tiUR ailY'ctJy in the process of oxidative aean11nisation.
It is, of course, possible that it might be formed as a secondary
oxidation product of ammonia liherated directly from the
amino acid and subsequently oxidised to cyanic acid in tbe
prt'sence of other organic compounds, just as Fosse d'bserved
in his eXJl"riments. Cyanate was, however, detected during
th.. mild oxidation of glycine, when the ammonia liberated
was only 15 !'('f cent~ of the theoreticaJ "alue for complete
dearllinisation. Moreover, in a solution of glycine slowly
<>xidised at -is" during a !'('riod of forty-eight hours the

)
 UREA AND DE.4MINISATION 21
iltC1'eBSe in ammonia WID. not ft("C()01pani('d by n. ('()t1'("s)londihg
ri"" in tl>.. cyani.. acid (ormed. This su~sh that tlK"
ammonia. is formed bv the rontimwu.!O hvdrolv.. is ()f Ul(" (tvaui('
'\Cid at a rate which dCIX'uds on the ('xixrilJl~nta"':>Qnditi()n)o".
Sa,..,,,,,ine (m,thyl glyde) gave grcll!<'r yidd., or "Y"Il'< add
than glycjne under similar rondition't. Thl'S(' obseryatious
suggrst to Fearon and Montgomery th" I)<,,,ihility I)f" union
h<-tween th" amino lITolI" and an nld<;hydi< or simila. suh-
stance resulting from th(' tnmsformatitm of oth('r or;ranh~
fragments undergoing oxidation_ Subsequent uxidation of
the amino 8.("id following un this lIIlion , ...ou)<I rt'luiily ~('()\lnt.
for the production of the ('orre!'.pouding a-ketoHl<' acid,
thus : -
R.CH.NH, R.CH.N CH, It-CO
I .+ H.CHO_ I + 30-+ J +HSeO+H,O.
(:OOH COOH COOR

How well fitted <yanic Reid is to play the part of an inter-

mt'diate suhstan.(c in ur(>a formation will lx M"{'ll from th{!
following consideratj()ns~ advan{'('(l hy ...(aron altd Mont~
gomeTy: (l) in aoid solution cyanic ad,l is ('ompleldy
hydrolysed to ammonia ; -
HOCN + H.O + lICl ----> NH.Cl + CO,;
(2) in neutral aqueous solution it is partly hydrolyS<'d to
ammonia and carbon dioxide, and partly cOlln'rted to ur(~a
by combination with ammonia ;.-

(8) in the presence of cxress ammonia cyanic acid is com-

pletely COll\'erted into urea ; -
NB.

,.
lftI.OCN ~ NR, + HOCN ~ HNCO + NH, ----+ HN : C(J
,
22 ASPECTS OF PROTEIN CATABOLLllM
The behaviour of <'Yunic acid in theS(> resIl<'cts is, therefore,
in tlC'cord with till' oh!oo{'rv('d phrtlomena of Qridosis, in whi('h
ammonia. ('xrrdion i~ incrt'asc'd at the ('xpens(" of urea
formation.' -CyaniC' acid, r('~istant a ... it is to furtiwf oxidatiol1.
is th"JI('fore well fitted to play an important part in the
nl{'chani~ms which rcgulate Ul(' optimal hydrog('n ion
(~ol\centrution of thv tissues of the organism.
If th{'~{' yiC'\\':-, nrc' substantiat-('d, it is d(>ar that the sirnul-
tan('ous formation of the ('o~sponding a-ketonic acid on
the olle hnurl. mal cyanic acid OJ} the other, n('('{~s.sitntf's a
~'()urIT of carbon other than the amino Ildd mo}<'Cuk which
is itsdf be-iug deamini,.,cd. lIt'nee the SiJ.,7Ui/icanc( of tIle
in('reft~{'d yields of cyanic o(id OhS(,fY("d in the ('xperiments
d('scribcd above whell l>.uch an additiOllal S01IrC'(' of carbon
wa..... present in til(' forOl of A'lu('os(' or formaldchy(\(',. It may
1)(" that sud. a prdirninury combination of the amino group
with a cnrbon-containing frngmcnt, normally prcffdl""S the
df'nminisntion ill rillo. Cas('~ in whi('h ~uch a type of combina-
tion have been known to occur arc, therefore, highly
signilirant. We haw already scen (pp. 5, 8) that optically
active a('('tyl derivatiws of phcnylaminnllCctic and 'Y-phenyl
a-anunobutyric acids have been detected in tht' products
]"(>suJting from the deaminisatiol1 of these acids. Dakin ha._'{
observed a similar formation of the o.eetyl derivative of
p-lIwthylphenylalallinc ill a metabolic invcstigation. One
wonld not care to suggest that acetylation normally preeed..
d{'aminisation; but may it not be a sIX'cial aspect of a more
general type of change? It at lca.,t serves to indicate that
Ii-'ing tissues cun effect a substitution in the amino group
similar to that required by the tbeory which we h'l.ve just
discussed.

TIle SjgnifIoaDce III U _ in PiaDVI

The occunence of the enzyme urease in the vegetable
sour&s which have already been mentioned raises t~
question of the presence of urea in the plant world, and of
 UREASE IN PLANTS 23
the significance of tll<' l'llzyml' "lid Its sub>.trutc in plant
physiology. In t h(" light of r('('("nt work UJ'{'ti (,'{Ul no lut1Ilt'r
be 1't~gard('d us a produ(t P('('uUar to animal rud 1\h<)l1 ... m.
During the ('arly part of HI(' )IN.'scnt {'('ntury Ul(.p,r{~t~H('t' of
lirea WAS dt,tc'('trd in man\' fungi. and moTf' n{.... uth it has
been shown to })(' produ(';'d iu many of t}1{' highl'; phwh
when thcS(' aN' aHowed to germinate in media clItif'('Jy ff'('('
fruln ur('Il, It h hut natural, thf'rt'fort\ that ur{"n and \ireUM'
should be found in u.s~oeintion with Ollt' Ruoth{'r, and thj ..
ha.(j, ix.-en shown to he till' ('aM.' in young ~r(>t'll IWU plu1ll~ and
in soya plants. Th(~ pI'('~('t1C(~ of Urt'tl ill pla.nh i:-. }larti('ulurly
a~social('d with the hr('l\kdowli of jltott-in... whieh O('('urs
during germination of the M'(~(i. So frt'quent h. thi~ ph<'-
nomenon that \Yernrr sU~J..rcsts thut urea is a ("on,..tant
producte of th(' disintegration of proh"in iu th(' plant world
just ft ., it h ill the animal world. The hrt'akdowl1 ()fth(' ,tofC'd
protf'in of the s('('d may tht'reforc b" r(,Wlf'd(d U!i tl
phenorncnon which involvt,S the proc.iu<,tion of Uf(,R,
The plant, as we know, syntiu-sises its prntdll frmn the
simplest ('h('mi('al compounds, amnumilt Of nitrah:!; (lilt!
carbon dioxide, III or<i<'f that th" materi"l, of the di,
integrated protein shollill hft'ome available for tJu> building
up of new eell prot.t~ins for the !,'Towing plaut, the trans-
formation of such urea liS is fomleil into cyani(' aeid, and
ultimately into ammonia and carbon dioxicl~ by th~ ag{'nry
of urease, seems a natural step, "~crncr sug~ests that the
synthesis of new prot{'"in may commence at tlw eyanic acid
stage and that the ultimate hydroly,is to ammonia and
carbon dioxidt>, fOT which} in his view, the {'uzymc b not
directly "",ponsible, is not always necessary. We do know,
howev~r, that the plant does utili,e these two simple
substances, but it is, of course, possible that cyanic add
may be formed from tJu>m : -

, co. + NR. __,. HNCO + R.O.

As a synthetic process this seems no more remarkable than
 24 ASPECT.'! OF PROTEIN CATABOLlIjM
the well-known phyto'Yllthc.j, of carbohydrate from earhon
dioxidt' and water. ,,~(! ('annot a.'i yet say what the next
st{p JII t.he- J)t(X'('I.,Oj may he. \V('rner su~gests that the cyani('
u<>id fonu('(t {roOl urea or froln (It llC'r nitrogenous rompounds
j,o; ('ompiurd with H nas(,f'nt" cRrbf)hydrate. The whole*"
,.,'TOllP might th('n lx'('omt" attached. to compound:-; of the tyIX'
of pyruvic 8(id. a prOf'{,ss. more or ){,5S the rcVeTst' of thnt
~hown 01\ p. 21.
Th('<.;r (onsidefHtions ('nab}(' \!!o, to understand why urease
is not founoitl tfl(' higher animals. The animal C'annot utilise
(itht'r ammonia or carhon dioxide; its defkicnt syntlll'tic
ahilitie~ I"('nder these simple compounds useless to it; they
are deflnih' ('tld-products in the metabolic cycle, and there is
there-for(' no rt'Rson for their formation from urea, itself an
t'nd-pro<iuet.
References
I, llAYLlsR. Jmlrn. Physiol., 19J5, 5{), 8i5.
2. VAKIS. ., ()xidations and R(lduc:tioJls in the Animal Body."
l.nudon: Longruaus, (~roen & Co., 2nd Edition, 1922. .
3. EMBHF.N arid SeJUUTZ. Biochem. ZMt., 1912. 88, 393.
4. f"""TT. Zeit. phyukal. Ch.en. , 1902, 41, 6(H.
5. PEAltON. Bioohem.. JoU",., 192:-t. 17. B4, SOO.
6. FHAROS and MONTGOMEkY. Bioohem.. Jou",., 1924, 18, 576.
7. f'OUN and DENIS. JourB. Bioi. (,hem., 1912, 12, 141.
8. Foss};. (''''pt. Rtnd., 1919.168. 008. 1164; 169. 91. 320.
U. K ....:t. Ri.ocJurm. Journ., 1923.17.277.
10. KNOOP and co-worke",. Zeit. phy.iol. Ch<m . 1911. 71.252 ;
lIl!!. 89, 151.
11. MACK a.nd VILLARS. Jount. Amer. C1u:m. Soc.~ 1923,45.501,
505.
12. NEUBAUER and co",orkeno. lJeutsil&. Arch. kUn. Mea., 1909,
95.211 ; Zeit. phylWl. ('Mm . 1910.67.230; 70. 1.326.
:t ~~~O~~~~ .!,t'i.r;i;;: S=~.I;!1: ~~..
213.231.
1913.16.197,
15. ~T""PUllN and UTKlNLJUBOVZ()V. Bioohem. ZtiI., 19ft, 146.
115.
16- WE""ER. Journ. C~. S .... 1913. 103, 1010, 227S.
17. WEnNER. "The C]jemislry of Urea." London: Longt_.
Groon &- Co_, 1923.

~;';_--:'~(!l;
,;;~;':-1-4 g:a . -<<--
 CH.\PTEH II

COLLOIDS AND THE PHYSICAL CBE1IIB'l'RY

OF PROTEI1i8
'''lIES we call to mind the fact tlUlt living maHt' ilii (~om~
prised almost wholly of suhstan('{'s in a (,oHoidul ~tut(", and
that th(> bulk of these (,oBoids urC' proteins, it is ot)\'ious that
coUoid ('hf'mi ... try. more e!\p('(.'jaJly that brRnch of (~(}noid
chemistry whi('h hus heen built up round the pruh'in~f Ulu~t
he iJ)tj~"Jatc1y rf'luh-d to thl' more physjrul 8!o)}>('C"ts of hio ..
ch('mistry. Graham in his C'ariif'st paptn; f((ognist.d this
when he indu<if:>d alhumin~ gdatin, v{'g'('tuhlc unti Iwimul
('xtracti\'C' matt('r~ in 'Ii~ du!'!.s of colloids.
In r('('('nt year~ it hflS hccolTI(' incT("a:-.ingly deur that the
(lucidation of tho."e a.'ipt'C'ts of the (oJ)oirlal reactions of
proteins which arc of importanc{' to th(' hiociwmlst is to be
achieved from two main ,wndpoints- -firstly, hy regarding
the proteins, like the amino acid, of which they arc (!Om-
posed, as amphoteric deetrolytC1i capable of participatinl( in
reaction5. in a strictly stoichiometri( sense; and s(""(~ondJy,
by applying to their study the principles of Donnan's mem-
brane equi1ibriuw since, although they arc t_s.'icntiaJJy
1

electrolytes, their ions arc large and non-diffusihle. More

than to any other individual worker, WI, owe to the late
.Jacques Loeb the foundation of the"" modern vi,'ws of the
coUoida-t behaviour of proteins on a rigid experimental basis.

'l'be BelJavioar of ProteiDI Amphoteric ~

Tbe amphoteric nature of the proteins-by which we mean
~ir capacity to reaet either as bases or acids-is due"to the

..
presence of both amino and carboxyl groups, 8 fact which
26 COI,LOIDS AND CllEMISTRY OF PRC}TEINS

may hecxpress{'d hy the ,ymboi [ P -NIl

_ COOII] ' Propr{'sont-
;"11 the re"lainder of the l>lfgo protein molecule_ It is cleaT
that ~u('h U "ompkx may form sHlts in two ways: (1) th~
hasi(, amino g-roup may ('omhillc with an acid sHe'h as hydro~
('hlori(' acid to form 11 protein hydroC'hlorid{', or (2) the 8{'idic
('arhoxyl group may comhine with a ha."i(" su('h as sodium
hydr()xid(~ to form n sodium pr'Otdnnte. These two types
of protein salts, in whiC'h thc protein part of the compound
form!'J the kutioH in (l) and the auioH ill {2}, may unnergo
dedrolytic (lis~()(i/ltjOH or ionisntion in the followjn~ ways,
that )lurt of the mo}e<,uIc- whi(')\ is capuble of narting with
oti1{'r !-.uh,tan(.'('s heing kept outside the hrackets in this
notation :-

(I)
p -- XII: _lI{'I] _--> r p - N1I, + (1:
[ --coon <---L ---COOlIJ

(2)
[
1' ---- XJI,
-- COON..
;----->[1' --X1I.l +
<------- -- COO Xli.
_j --
Whut are til(' conditions which detemline the bellin-jour of
a protein in onc or the other of these two ways? Loeb has
shown thut the hydrogen ion eoncentration of the solution
in which the protein is present is the deciding factor.
l-Iydrogeu iOIl ('ofleClltration is usually expre.ssed as the
number of grams of ionic hydrogen (i.e., hydrogen carrying
a positive charge) in 8 litre of the solution under consideration
at a stated temperature. In solutions of physiological im-
portance this number is usually small, and for COIlv(II1ience
it may be written as 8 negative power of tbe base 10; thus
a concentration of 0000001 gram of ionie hydrogen per litre
may be written (H] =-10-, It is, bowever, customal'y in
bioeheinistry to use the notation proposed by Sj1Irensen, in.
which a particular ionic concentration is denoted by the
..
 H.l'DROGEN ION CON(,ENTRATION 27
symbol/I., followed by the Iwgat;V(' ('xl)(}ll('nt (lilly, omittiuA'
+
the negative sign. Thus a ,o}ufirm IU"'jng IIII 10" i,
said to have l'H = 6'0. Similarly u ~oJutj(ln ,t"ontnininJ(
O'{)(){)()o2 warn ofionir hytlro,.!.!'{Jl (>(>T litrt' Jws III ,. :!;<. lU~ 6,
This is 103 X 10- 6 or 10'-::;'7, so that Pil 5-7. SimilurJy
'.
[III = '~'5 X 10-' = 101)'" X 10' 10- 73:., ," thnt I'll ...
1'35. t'or a further dis{u!-'!'.ion of hytlrng('1l ion ('Oti("{'nt rut iOB
the reader is t't'f('rrNi to Prof(' ... ~or Lovatt "'~\"mb' hook on
H R{'('ent Ad\'an('('~ in Phy ... iology:'

The- import.ant' of tJw hydrn,U'f'1I iOl, ('ofl{'('ntrntiou in

controlling the })(,ha"iour of th(' proteiu, It .. iUliphoh'rlc
substances was iUu~trat{'d hy IJ(){"b u~ fonow.... )JortioHS uf
nnely powd('rcd gr\atin {'neh '''Tighing 1 ~ralll \\'('r(' hrought
to diff{'rt'nt hydrog'('n ion ('on('pntrntions hy immcf'!,ill,U' ('Il<'h
for one hour at 15 in ]00 ('.c. of nitric acid .. oJutiolJ." of
{'oncentrations varying from M/s down to M;8192. The u(id
was then filtC'red off, Ilnd ('nc,h port inn of ~w1atill WlI'" wnslwd
onc(' ortwic(' at 5:' ,,,,jth 25 (',C', of wah'T to r<'mon' tht ~'1'c'ut<'r
part of the acid hf'hl'~cn thl' partif'1f"s of ~('latjn. Tht portioJl~
of gelatin, which now each posSf'fo.sed a dif(crellt flut wet<> put
for one hour into separate beakers, eH('h containinR l\J/6.'
Jo;ilver nitrate at 15'). Thf'Y were tlwn Tf'moved and washed
six or eight tim('s with 25 C.C'. of i{'('-cold wawr tn T('movc
excess silver nitrate not in combination with the ttdatin.
The latter was then melted by heatin$( to 40", and di,tilled
water was added to each sample to bring the volume to
100 c.e. All the operations so rar d,.,,~rihed w~re (arried nut
in the dark. A portion of each solution wa.s ""cd to d"t('rmi"e
the P.. whilst another portion of each solution was exposed
in a test tuhe to strong light. The results obtained IU'C shown
in Fig. 2, the P. of each sample being marked on the tubes.
It will be seen that up to .. P. of 4'6 no darkening of the
aqueous solutions has occurred, but alxwc this Pa sharp '*'
'change is observed and aU the solutions show darkening.
28 rOLWf[)8 AND CHEMISTRY OF PRQTEINS
'fhe inferrnC'{' drawn from this M'rirs of expcrimt"nts is that
at p" h'l'N\h'r than 4'7 {i.e., lower hydrogt'll ion ('onccntra-
ti()I1~) th(' _gciatin, whi('h to a ('('rtain extent is present as
~ndium or f.hjum g-eiutinate, forms a siIv('r g'('latinate by
double de<-ompo!oiition with the silver nitrate, i.e., the protein
c -NH,J
ion hc.'huve"i H" _ COO + Ago This silver
an anion. thus [
- +
protein salt i, not readily \Va,hed out of the gelatin, and on
exposure to light it f"xhibits the dJarBf'teristk darkening
!i.hown by silY('r salts. At}JlJ less than 4i the g-elatin
fornwd thl' kntion of a I'tutt, in this ('a~c grlatin nitratl',

[ G =~~OIt NO" which could not react with silYer ~itrate.

Ikllce aU th(' ~il\'er nitrate was easily wRsh('d out and no
durkC'uing ('Quld Q('cur on exposurt" to light. A similar series
of ('xperimt'nts is illustratrd in Fig. 3, in which the powdered
!!e(atin, afkr being brought to different PH' was treated for
one hour with Mil2R potassium ferroeyanide. After washing
as before, the gelatin was meltcd and diluted to 100 c.c.
After Ii few days the tube. in which the PH was less than 47
turned blue', whilst those with higher PH remained colourless.
Hence Ilt P. less than 47 the gelatin forols the kation of a
.alt in which Fe(Cl\), is the anion, and the latter betrays its
presence by forming" blue ferric salt in the course of a few
days. But ahove thc PH of 4-7 the gelatin is itself the anion,
and therefore cannot combine with Fe(CN)., which is conse-
quently washed out and no colour can develop.
We sec, therefore, that in the ca..,. of gelatin the fa 4-7
cOJT("sponds to a critical hydrogt"D iQn concentration Dear
which the protein shows a mlU"ked change in
its behaviour.
As wc shall see later, many other properties of gelatin exhibit
a characteristic changc at this PH> which is known as the
isoelecfric point of gclatin, since here it behaves neither as
an acid nor as a base, or more strictly its ionisation in one
 180ELECTBIC POINT 211

direction or in the other is minimal. It must, how("\'ef. he

I",inkd out that L. J. Harri., hll., """'lltly ",Il'an('('<i "rgu-
nwnts for regarding the is()(')ectric protein OIol('("ul.., nut Us
a practically nOll-ionis{"d mule<'ule a.., w(' hay"- SUPl)(~d
~bon', hut as a rno)("('uk ionised equally It~ Em add alld tt.~ it
008(>, that is, as a type of internal ammonium snlt. 'In thi~
cast' tht, total iOl1isation li.t., hoth a._~ add and ha.") i. . tua.rink1l
at the is(wic('tric point. At hyM>J..f(n ion ('on(>(~ntro.tions
aboy<, or twlo\\' th( is{)d(~ctri(' point W(' then hu\'(' in pia<'t'
of what may lx calkd tht .. ('omhillution ., rt'uC'tiul\s ,..huwl1
on p. 26 thc' " Tl'}>lu('('Il\t'llt ,. f(,U(tion!'! shown hdow :"~

\\'hethE:'r Locb's original th('ory or tilt' U('W 011(' gi\"('~ n

c'orn'('t ft'prcM'ntation of the prot rill fIl(I}t'('UI<- ut ih iso-
electric point, both agre(' in regarding tlw latteT Jx)int us
being that PH at which til{' protein i~ ioni<.;e<1 (>qually as an
aeid and as a hase, and ('onM'qU('ntly all ('ss(_'ntiul purt of hoth
th{'ori('~ i!-. the eIcctrieal Jl('utralit y of till' Uloi<'C'we at the
jsodeC'tric point. ..
The position of this luttef point was as('('rtainf'd (~Xlx'ri
mentally by Michaeli, pn"iou, to th~ invrstigations of I",,h
just cited. Michadi,o.; showed that in eatapiIOTesis t'xperi w

menU, gelatin partidct.. did not migrate to one poi(' or to tilt"

other when the P. was 4'7, that is, thpy were dectrieaUy
neutral, whereas at P. ,ith,.r abo,'" this point or IJdow it
they were definitdy charged and migrat('d, to the anode in
the former case and to the kathod,. in the latter. This
meeholl of observing the hydrogen ion concentration at which
protein particles do not migrate in an electrical field consti-
tutes one means of determining the isoelectrie point. We
shaH see that other methods which may be used for the same
purpose are based on the fact that certain physical pr~perties
of the proteins, such as viscosity and osmotic prewUff, are
30 COLLOIDS AND CHEMISTRY OF PR.PTEIN8
minimal at the i~()d(>(tri( point. Mi('hl1rlis and Nakashima
han> r('('(~ntJy d(_'~('rih('d H (;oll\'l'ni(~ut method of ddcrmil1ing
this point for prokin~, whieh i~ basl,.u Oll the oh~cn'atjoll that
tIll' poiut .-v- optimuOl pr(,(Oipitation of n mixtuN' of gum
mastic'. alld gdatin in the pr('se})('f' of an :i('{'tatc buffer
HpprOiH'h{~ tht i"i()(,}("C'tri(' point of th(' gelatin us its relativ('
amount i~ in('rc'a~('{l. In practice, therefor(" n ~eriC's of tubes
('ontaining rnn!.t i(' sol and an UN"tate buffer is made up for
(twh PH which is to lx iuv('~tigat('d. Incl'{'llsing amounts of
t.ht' protein who~(' i~()('k('trie point is to be determined are
ndd('d tu ('fl('h s('ri('s oftu~s. The PH of the ZOIl{' of optimum
pr('('ipitation ris('~ to ft con .. tant value iX'yond whi(~h furthc'r
nddition of the prokin produ('('s no further shift of the zon('.
Thi~ maximum ('onstant yuluc is the PH of the d(>sired
i,;()('lpdrit point.
Tht, folJowill.!.{ ure the iso('}p('trir poinh of ~om(' common
proteins :.-
p,
GlutC'nin }~j:(g
albumin 4'8
{~dHtiH St'rulll globulin 5~
<:ascin . . .destin . 57
S<'rum llihumin Oxyhremoglobin (J'75

The isodedrie point of a protein 'or !'JimiJar eolloidal

amphoteriC' ~ubstlln{'{' is of th(' gl'c'atest importanc(' in T(>lation
to the biophysics of ",,h compounds. We haw stated that
many of the pror.l<'rties of proteins exhibit a charnd:eristie
(>IUUlJ!(' nt this point. and we hay(' already M~n that here the
ionisn.tion of the protein, either as an acid or a._,,- a base, is
\quul (st'e p. 29). u:rtain of these other characteristic
prop,'rties which show a similar heha'1our are the total
swelling of the protein produced by the illlbibition of _er,
the viscosiJ.r, the osmotic pressure, and, .as one might expect,
the eonducth-ity. The" membrane potential," which we
shall discuss later, also has a minimal value at the isoeledric
point.- The CUl\'es gi"en in Fig. " illustrate the..., various,
pbenomena, the experiments on whieh they are based being
 ISOELErTRlr PROTEIN :II

dt.,.~ribed a, follow, hy _ .. ...;t.. ...... -:.::::..." ......

l.Ot": "A IIUI1ll><'r of
rlOM'S, each ('ontaining- 1
,.... L_~ ~" H.: I t-
r~---t:.-I~'tt-f--
+ ..
-t-~'
m !int'ly
g.. of powtkrt'd
Cooper's gelntin whidl
eoP=tH~ 1.'Il ,L
: ~-r+-:tJ'!lr,--r'"'+-+-H'+_1
H: .LJ. . .

hud a PH of 11 little o\'or so ~-'m

7'0 and (,oJI,i~t('({ pnrtly :~1-i~~ I_;~
nf ralcium g'<'iatinatf", z.o~~t::::,,-'1-' ~
W{'T!' put for thirty ~1-9t-'-I-t-i "+~-tr~
nlinuh~s at ]5;' into ,:_, I !-+--H',--t+.+

".('. of lIBr of <liffennl, \ r' .J-+++

h('akt,rs {'ontnlninq 100 1..10 t~~::~~::t~",-"'+'_"f--++-,+~,-+-+-J-.

concentration ... , yurying ':~ J\ _+_ _rH-__t

from M:8 to M S192; eo --l-~..14,,"''''''~-+-+-~1-l
Ilud, a~ n (outrol. I l'Of-:;""l':i'-+-H+...FII~-l-~=ttE
grum of g-d:ltin W8" put '~H-+++--H++ti r
for thirty minute, at ]5' " 0 r
+i-f-.f.
into 100 0.('. of distilled ,uH-++f'oH-++t+-H-l--\-H
water. The powdered '~~ -+ L F"" ...
gelatin was then put so~~~~~lj>ottt:'l::t::ttti-
into a cylindrical funn<"i "'-r,"
and the acid allowed to oH+*H,+++l-H-+++-H
drain otf. The powdered :~~:::t~~+~~J:::t:ttt~:::+-+f-'+-H
gdatin ill th(~ funnd
was t hen perfused !o.ix or
eight tinw~ with constant
stirring, each time with
25 c.c. of cold wakr--
i.e., water not above 5 o "'" i HAUl ........0
-to J!'fllove the exeess tit 4.1 41 "' .. +~ ...4If.. K K H K" Sf
of 8Cld and the salts. FIG, 4.-Curvea $bowing that the total
swelling, vi8cosity. oamotit': preuum,
The water mw.t be rold and conductivity c.l gelatin ate
to pre"ent the powdered minimal at the ieoelectric point,
1'.4'7. (Altoi' Loeb.)
granules from coalescing,
lIInce otherwise the Wa> hing would be incomplete. After
the liquid was drained of( from the filter, the vplume
 32 COLLOIDS AND CHEMISTRY OF P_ROTEINS
(i.e., th(_> rdativ(' !.w{'llitl~ of the gelatin) wat. m(_'a~ured;
thcn the gdutin was m{'lt(~d hy heating to 45, and
(nough watt'r was add('d to bring th(_~ volume in ('ad} case
to lOU ;!r. Then the ('ondu('ti\'ity, o~Jl)oti( 1)J'('ssnrC", and
YiM'O~it Y Wf'f(' ID('U\iUTed and t he PH was also dett-f-
mjnt~d, (jt}r('T ('olorimdricaJly (which gin's fairly 8N'Urate
results with ~dntin. but not with th~~ other proteins) or,
l'rt'ferllhJy, with the hydrogen <:,}ec'trode. In tlu-> experiment
r('pr(''-.(~nt.ed in :Fig. ,~ the Pit was measured coJorimltri('ully.
A glance at the fig-tiff' sho\,..s that i)w {)rdinat(~ of the
('UnT'" f('pr('i'lenting thf' valu(':-' for o~moti(' pr('ssure,
t'ouductivity, swdling, ('hoo, drop very shurply at Pu 47,
i.l'., the i~()('l(,('tfic puint of Rclatill. By this mdhod
the approximate- ]o('ation of the iSOf"kC'tric point ('an'
1)(" f('('ognis{'d at a glance from the osmoti(. prt'ssure
mcasurC'ments, th{' ('oncim'tivity measuf<>ments, etc. The
lowest cur\'l~ in Fig. 4 rep~sl."nts titration for Hr.
Gc..-\atin should ('xbt in t\lC form of ~elatin brOlnide only
on tll{' R('i.d side' of tht' isoclcctri(' point. and titration
for Ilr should be lwgati\'' wht'n the PH is above 47. Tilt:
<,urve shows no Hr wali found when the PH was equal to or
~rcntt'r than 4'7; while it was found on the acid side in ..
cN'nsing in quantity thr lower the P.. On the alkaline side
of th<' isodc(tric point the gclatin <,'xistcd still in the stutf'
of calcium gdatjnat<~. In this f'xperiment the mass of the
gdntiu was diminished by solution and washing to 08 gram,
or possibly a Iitti(' less. ~r
Yet anot.her property of the protdm. which is minimal
at the iso<'lectriC' point i~ t}wir stability 8S emulsoid colloids.
That is, their tendency to coa!(ulate or precipitate is greatest
at the point of electrical neutrality. This is probabl1beoause
the attractive force hetween the dispt'rsed protein and the
surroundin~ water molecules is minitnal at this point. Thus
if 1 pt'r cent. isoelectric gelatin in water be allowed to ,und,
and ~specially on cooling, a doudy suspt'nsion will fonn anQ.
eventually precipitate, but no such separation of gelatin
 I80ELEC,]'RH' PROTEIN 33

O<'('u~ if t1w PH j~ ('\'t.'n ~hllhtly nho\'(' or h('}nw thf.' iMwh-dri('

p('fint. Simijnrly. tilt, ndditioll of 1\ littk ule-ohol to i~t}('kdri<'
gelatill pr(}du('('~ Tnpid pr('(!ipitutinn, hut at n JIM f4dow ~ ..
or ahov(' 5-0 the protein mu\" with:-.tand ttl{' nd~i\ioll or n
(';nsid('rahle ('X('('~~ of al('oh<:l without pr('(il'itntinq. if t Iw
union of the ueid alld the kntiou of tl\f' ulkuli prt~t.llt \w
monovalent. Ifth{,~l" are divHi{'ftt, tht ilwTt'used pl'('('ipitatiHg:
8.(tion of t h('s{' iotl!) will C'UUst' prccipitnt 10n to {)('('\If f'V('1\ At
PH .}',j. or 5'0. "fh{.>. facility with whj('h Ik'at ('Ollgulntioll of It
)1n)t<'in is eff(cted at the i1'l()('I{,(,tnc point. must be fnmilinr
to most rt'adkT:o,. I~o{'k('trir quo( ulhumin ill nqU(,Ol1S sHlu
tion~ will remain clear for Inollth~ in (on~idl._'rlthk t'OIW('ntrH-
tions if thl" te1ll)ll'ratur(' tK' kept at about 2'>; hut if tht'
tf'D1peratuT(' i" raise-d. pT('('ipitatioH of I per ('(nt. isoc'kctric
albumin '(I'M 4-'S} \l"iIJ occur at no". Thi1\ tcmp('rllt urt' is)
th(~refort', that at whidl heat coa~ulation o('C'ur!> ot UK'
is(xl<.'ctrie point. On alterinJ,! th{' PlI to 4" hy "'tiding
hydr<X'hloric s('id the heat coagulation t<'mperaturc rists
to 80,\ and at Pa ""25 or lower coagulatiun i~ in{'otn-
pletc cY(>n at 05-', Kil opalcsC'('B(,c only h(:iuJ:t obtnin('d.
The~e obser,ation.'j demonstrate thnt th(' t{'udtuf'Y of a
protein to separate from its !'Jolution undt-'r the intluenfc
of precipitants or coa!,'lllants i, maximal at it> isOt'I~etrie
point.
Another ('on~ideration of gn'at practical importance III
relation to the isoelectric point of proteins (ollow" from the
fact that at thi, point of minimal iOllis>ltion the protein rou,t
be associated with the minimum quantity of inorgani(:
matter, and Loeb and othe,." have shown that the ash ('ontent
of commercial gelatin and other proteins is rapidly and COIl-
ven~l; brought to a very low value by adjusting the
hydrogen ion concentration of the protein to that of the
isoelectric point, using dilute acetic acid, and then by washing
with cold water. Dialysis of a protein at its isoeleclric ~nt
apinst dilute acid of the same Pa is, therefore, an important
method of purification,
....
34 COLLOIDS AND CHEMISTRY OF PIWTEINS

InlIuence oJ JOII8 on the Behavio1lr oJ ProteiDs JIIII1

the supposed Hofmeister Series
Th,' '",,'lling, osmotic pres>urt>, and viseO>.ity of protein
NoiutiollS are markedly influ('n('t~d by th!' pr{'sence of salls.
and n()fmi'i~t('r. who fir!o;t invrstig,a!t'd these phenomena,
arcw up ft M'rI('" of salt ttnjon~ in the or<icr in which they ,,'ere
!o.uppohl'd to infh.I{'l1(,(, the propertirs n\('nti01wd. Such a
!->('rl('s lHily h{' illu~trah'd by the follo''''ing sr-qur-ncc of ions,
"hi!'}l shows the- order of their adjviti{'s in dimini"hing tile
osmotiC' pr('ssure of gelatin;--
CI>SO,>XOs>Br>I>(,;'\'S.
l.o(,h lIu", howcn'r, pOlut('d out that thes(' earlier iu\cstiga- ..
tions (iid not take into ('onsidrfation thC' varyin~ ('ffeet of
the salts in which th('~e ions wt'r{' pre-sent on the hydrogen
ion ('oll('('ntration of tIl(' :o.olution. 1I(" has shown that if ('arc
bl" tak('H to adju,..,t the hydrogen ion ('Oll('C'utration to a
common ,<ahH' hefore the (~()mparison bt' mmle, the numf'rous
complex llofm{'ister Nerie:-,. may lK' repls("('d by n simple
"alen('Y rule. It ('an, for instance, be ShO"..ll that the acetates
influ('ncc the swelling, osmotic pressure, and viscosity of
protein solutions in the same wny. in a quantitative ~ense~
us the ('hlorides nnd nitruh's. hut not in the same way as the
!'ulphatf"s and salts containing other divalent ions. These
con,idcrations apply also to kations, and of the properties
of ions in gen('ral only the ,'slcn('y and ~ign of ("harb't' influence
the colloidal behaviour of .. protein, provided no consti-
tutional chan{(C in the latter is prodllCCd. The Hofmeister
series t\l'{\ therefoN', meaningless.

'!'be II&oieIJiometri Jlebavioar or ProteiDs

....
Recognition of th" fact that the proteins combine with
acids and bases accorrung to simple stoichiometric relations
follo*OO from the proof of their amphoteric nature. TbP.
most convenient method of demonstrating that these eom-
 TITRATlON (,URn; 35
hinat.ions take- pl8.C"C in ~impl(' molf'Cular })roportiom. i . . by
n study of the titration ('urv('~ of th(' protein" Tht's(' ('Ur\'{'1i
art' ohtained by making up solutions of ('(lUftl N)(l\ntrutions
.'
I. ,\
'"
.7 \
~
'6
'i.
"'
.4
\
13
1\
.%
1\
1\'
"
'0 'r\
\ ~\
" ~\ \'
~
~
~
e
T

..
""'i'-..
~
;",
... ~ ,',,::-., ...
~
'\

~"'"l'\ ~
~
5
... '" ""~
3
't\ '" ,..,-='r&
:it
""~ N ~,..._
c-~ ~

'0 I ....
PH'" 1-0 11 t.4 Z6 1" )00 H ,.4 ~ ,. 400 4-t 44 406 +8
FIG. 5.-Titration curve and PI!' curve of egg albumin IIhQwing
..- ; r;!::~:~ ~~e:n~~!;~~ It::n~:b~ioo
of the isoeleetric protein containing varying concentrations
of acids <Jt alkali., and then determining the PH of the
{"SuIting solution, preferably with the hydrogen dee!rode.
The titration curve is then obtained by plotting the con-
3n ('()LLO[J)S AND CHEJfJBTRY OF Pli07'EINS
c('utratjoH of acid or HlkaH against the I'll" From th<. titration
curv(" (}n(' Hu'n obtHin.", th(' amount of 8('id or a1kali which is
ill ('ombillation with the protein at all)' givC'n Pn by ~ub~
trlU,tillg frt},JH th{' total amount uddtd to bring th(' ~o)ution
to that particular PH' HI(' amoullt of ueil! or alkali whi('h i~
fT('(', This luUef amoullt i~ found hy d('t{'rminin~ that
('oll('('ntration of add or ha~{' )vhi{'h would lX' f(~quir('d to
giv(' tht' silnH' PH to an equal "olullw of pure water. Suppose
thnt ill 100 C,t'. of orig-inaBy i:-.o('}('('trl(' 1 per (('nt. t'g_g- albumin
th('u' i~ present G (',t', of njl0 11('1, uncI that th(' resulting PH
of the solution i~ found to he 3"0. To give this latter Po to
au equal volume (IOn (',('.) of' pure water one must add 1 <'.c.
of n(10 H(,I, l-oin('c tht ('oncf'nfration of the r('~ulting solution
would lx n/I.OOO with r('~pect to the acid, and assuming
(olllplcte dissociation at thi~ dilution, there would l.>e-prcsent
+
0001 ~rram of ionic' hydro~('n in H litre, i.e., IH I would he
10- 3 or l)H !l0. Therefore nt Pu SO 5 C.f'. of nil0 HCI are in
{'om\)\nahon with 1 wanlof originally isoe\cctric cll:g a\buluin.
In l\ s\lni\ar way C1\n he de\('rmined the amount 01 u('id in
('ombination with the ('gg albumin at auy gin>n PH' and the
{'un'{'~ ~iY('n in fig. 5 illustrate an u(-tual series of experiments
('arri('d out by Loeb in this way,
On p. 87 aT(' gin'n the results obtained for th(' two proteins
{'g~ albumin nnd gelatin, u~ing hydrochlori(', sulphuric,
oxalic and phm.phoric Hcid .., in four series of (>xperiments
OB cal'll protein.
In the ('8S(' of both experiments detailed in the,. tahks it
will "" seen that at any gin'n PH hydrochloric and sulphuric
n('id~ ('ombine with the proteins in equiyalent proportions,
both acting as monobasic ""ids, Oxalic acid combilf\,_';.th
the proteins in practically twice the amount of the hydro-
chloric or sulphuric acid at the sam(' p", more especially
""'ow 8,2, whilst the' amounts of phosphoric acid whicb
combiele with tbe proteitL' is, within experimental limits,
three times that of tbe monobasic acids at tbe corresponding'
 fj,T01ClIlO.lfETRY OF PROTEINS 37
T_\llLE I
Cubic Ct'ntimetrt',o.: rd' .';,]0 Acid in rombiuutiotl tcith 1 gmm of
Origillal/y /-,neleclric Crys/al/ille Egg Albumin '" lOO r_e_
(~r S"luti(m (l..Uf'b.)

Pu no HzSO, OulkAdd Ifj~

- ~---"~-'- - .-~"---~ --~----~-

42 H5 1-15 I-N :I-H

HI 17 17 2-6 _~-3
a-s 23 ~a :17 Ii-g
3-6 !Hl 2H 5,0 s-u
IH !}5 :h~ fHI JlHI
3-2 {2 l-a s-u 111-1
I}-O 5,0 51 H'5 16-1

.
2-H 5'S :JB 11-1 19-3
2-0 {)7 1)'.1 IIl-:1 229
2j. 70 7'0 W-u

T_"'LJ,; II
Cu./Ji" Centimetres of n/l0 Acid in combination ,oit" 1 gram of
Originally J.,oelectric Gelatin ill 1(1) t.<'_ 'if Solution (Loeb_)

p. Hel H1ttO, Olt4lkA!'kt H~I'Of

HI 2'7 3-9 6'115

3-H /1-9 1l-75 55 !H
3-6 4-S 4-H 7:j 12-3
84 5-0 :;75 !I-! 15':'>-
32 6.j. 6-15 IHI llH)
3-0 7,2 7'5 IH15 207
28 1-9 8-25 15a 23-6

..
26
.~: 1
S-85
85
S/I
9-3
17-1
180
26-2

PII' _From the cur.-e of combined Hel given in :1<';11_ 5. and

from the fJgllTes quoted above. it will he seen that a flattening
tends to occur at about P. 2. This would seem to in3icaw
that the protein bas 11 limited maximal eapaeity to combine

:lH ('OU,Oll)S AND (,HEJ!l8TRr OF PQOTEINS
with tH'id. and thnt thi~ maximum d('~rrec of eombination is
reach(,d nt til(' PH quoted. TflC',r rf.'SuIts are only to be
('xpfal1\(:(' on the ha~i ... of a simpl(' stoichiometric combination
lid Wf't'll f'lv' protein and tlu' add, su('h as we encounter in
the (r.)::.,talloids. IHt{'re~t.illg confirmation of such a view 'is
fort lwomillg' from n study of the titration curves of deaminised
g('lntin (_'urri('d out bv Hit<heoek. It can he shown that the
mnximum (mnbining: capacity of thl~ drsmiuis('d gelatin for
hydroehlorir add is 1ts~ than that of ~rdntjn by nn amount
Hearly (,tJuint1rllt to the ff{'(> amino gr(}up~ pn'sent in the
origilwJ prot(>in. and whidi ('an be !-.hown to he removed by
tIlt' process of dC8llliHlsation. Thp residual capa("ity for ("orn-
hinin~ with u('id shown by the df'amilli~('d ,!!eIatin must,
thort'fof(', be due to tllt' ll{'ptid<> and other groups unaffected'
by the nitrou~ acid usc'd in thc~ deaminising process. Similar
stoichiometric relationships are found to hold hetween alkalis'
find proteins at l>y above the isodcctric point. Thus in
titration CUfVC'_S ('onstru('tt>d for the fiye proteins fgg albumln,
l.J'datill, (ilb(,jU. serum globulin, alld cdestin, with the strong
ha~es potassium, sodium, calcium, and barium hydroxides,
t Iw poiHt~ obtained for anyone protein with all of the bases
faU on it si ngic' ("urn'.
Thf' ac('eptnnce of the view that proteins fonn compounds
with lwids and alkalis, Ii",! dearly established by the re-
s('ar('hf'~ of L<x-b and his 8...'is(K'iates, is now general. L. J.
Harris, in a l'('('t'llt series of studit's on the titration ("Urves
of amphoteric ..Jeetrolytes, gives the following method for
determining the combining or equivalent weights of proteillS
towards acids and alkalis. Increasing amounts of acid or
alkali are added to a protein solution, and after each addition
a measurement is made of a physical constant ~ther
viscosity, total swelling, osmotic pressure, or conductivity,
etc.), The latter is then plotted against the amount of acid
or alkali added, not Fa. The curve is marked by a very sharp
maXI"Vnum or kink, which, in cases where the equivalent weight
of the protein is already known from the combination curve
 !QUIL4LE:VTS OF PROTEINS :m
(or from conductl\'ity), j,!, fOUlld tC){'uiru'ick' with the addition
of (,X3('tiy 011(' (quinl.lent of ucid or nlkali. Tiw following-
remIt)'. ore from Burris' pnp{'f :---
,
.'
.:;lppro,rimaie f~quit'alr."t ,rripltt.'i (~r Pro/rills t'J1Jrr:v('(/
in Terms (~( 1I,1}0 Acid or Alkali

Gdatiu. jJu-('ornlJinatioJl ('un'(' 1I

Swelling. . . ",.;"j
. Vis{'o~ity . . !l
Slx'ciUe condudivity !)
Osmoti(' pressure II

Edestin . : PIl-Comhilllltion t'UT\'(' 5-0

Osmotic pn,...... uTC !j-(;

Serum globulin Osmoti{' pr(>s.~ure

Casein . PIt-Comhination ('urn~ : >1'5

Solubilitv . . 15
Viscosit,: 15
. Sw,Ilin!i . 15
Egg albumin . p,,-Combination eur\'(~
Osmotic pressurt

Harris concludes that a protein in solution may be de!"r-

mined volumetrically by titrating with standard add or
alP ~tween two given PH points, the amount of titrant
required being strictly proportional to the amount of protein
present, and the estimation is as 6CCllrate as an ordinary
acidbase titration. The practical ,application of such methods
to the determination of tbe protein, of milk i. illustl1ftcd a.
follows. Suceessive small amount. of njlO hydrochloric acid
40 COLLOIDS AND CHEMIBTRr 0); r.t,rulJJillU'

an' run from 8 h1lT1:1 h' into a giv('n volume of milk, and t.he
PH j~ detemJiu{'d aftt'r (at~h addition. Thl"f'c or four such
r{'adjng~ urc !otutTici(,Bt, u:-. th(, result when PH b plotted against
('ubie cNi\imdrt:'!'> of H('id ndd(d is I'radicnlly a stra~ght line,
alld readings at stuhd PI! valu<,,, may be takt'n hy intra-
polati<m. Th(' amount of urid required to takf' the milk from
Oil' UiW'1l PH "HIm' to another is diredly proportional to tht'
nmount of proh'in prt'~ent. Thus the numlwr of ('uhic
('('ntiuwtr('s of n/IO hydrochlorie aetd required to titrate
]0 ('.c. of milk from P" 6,(;5 to 5'2 is numeri('ally {'qual to the
P('Tc'{'utagf' of prott"ln in thf' milk. Readings :;,hould h{' tuken
Oldy lrithin th( range of PH 67 to .J.. .<\t \"H.Jut~ mort' acid
than 1'8 .f. the> hlank correction h<'gins to assume large magni-
tndts. 11y this m(thod the pcrc('ntag<' of protein in normal'
('ollnll(-l'('iaI ('ow. . milk or modifitd milk mixtures ,may be
d('t('rmilwd to within 01 I)(>r ('ent. Tht, hlank ('OTrl>ction'
IHt'lItiolll'd l'eprt'~(,llt . . the amount of 3("id r('qui1'f'd to take a
hypotheti('Hl blank 1'1'(>(' from proh'in to the same final PH
ynilw at the sarne fmal yolnnll'.

The Donnan lIIIembrane Equilibrium and Proteins

If w(' imagine two soiutions of el('cttolyt('~ to he sel)arated
hy H l1\embran(' whi('h is permeable to all thp ions sav<, one
pTl'sent on one ~id(' of the membrane only, it can l:x- shown
1hat un ul1cqual distribution of the difiu..,ible ions must r{'~ult.
Thus if We !-otart with a solution of' NaR, R being a non~
difiu. . ihle ion, on Oil{> side (1) of the membrane represented
by the diyidiuj( lint', and Nan on thp otMr (2), and consider
th.. state of equilibrium attained by diffusion of NaCI from
(2) to (1). we haw first:-
+ + + ~ "'.
Na Na Na Na

n CI and at equilibrium R
"il) (2) CI CI
(I) (2)
 DONNAN EQUIUBRH-;lt 41

In ronsid('rill~ !<.uch u ~ysh'm 1)onn1\n stnks: . '"hl''' 'hiloo

t'quilibrium i~ t'!':Itahlished t}l(' ('nt~rJ,.'Y T('qnin'd to tr1U1S)Xlrt
t('v{'rsibly alld isothermally 1 grRHl m.ol(>(uk Nil (,om (2) to
(.I.) ~qua1s the cnefI(Y which "1m be gailled hy th,','om"pollo-
ing r'v{'rsibh' and isotiH'rlllAl trnn..,port of n ~'l'l\lJI rrfbkcult,

of CL In other words, W(' considt'r the' folJowing inl1nit('b'

smaH isothermal and T('\'('rsibh ('}u\ngC' of Hw looy ...tcm !

(.snmol::O\. (2)-_(1)1
1~" mol ('j (2)----4(1);

Tht, energy wiJiC'h <'an be gaincd in thi ... WHy (i.l'., t hC' dimiHu~
tiou of free energy) i!CoO z(>ro, hCHN' :--
;.

611 UT log I )\,a], -I- ~II RT Jog 1(; j. ,;- ()

INa], lei],
or
, 4

l!'<a1., felj, = 11'0], ,leI), . (Al

where th(' brackt't"i signify molar {on('(>ntrations. H Thus the

product of the (>oncentrations of a pair of diffusihl(' kations
and anions OlU!o.t l)(> equal on both sid{'~ of th(' memhrane.
But since on (1) [Na] is the sum of the kation, in combiuatiofl
with both CI and the nOll-diffusible ion R, and sinoe on (2)
[Na] represents only those kation, oombincd with and ('quell
+ -J_

>
to [Cl], it follows that [Na], [Na1, and [ClJ, <[el] .
~ tls suppose that we have two systems, (A) gelatin
.chloride in equilibrium with hydrochloric acid, and (B) gelatin
sulphate in equilibrium with sulphuric acid, in both of which
the protein salt is separated from the acid by a membrane
premeable to all the ions save that of the gelatin. Til" two
systems may be represented as follows : -
42 ('OLLOIDS AND OHEMISTRY OF PR.oTEINS
,I
zG ;;G

.
;
y'H ,rll yH xII

y+ ocr !I+ 0 - x -
.1{'J
2
SO, 2 so . .
(1 ) (~) (1) (2)
A U

Then using the symbols x, y, and z to denote the various

ioni(> ('onccntrations, or more strictly ioni<.> activities, as
indicated a\){)\T, (lUl' can show thrrmodynamically that in
systt'lYI (A) ",' ~~ y(y +
z), and in system (B) x' = y'(y z), +
assumin~ for purposes of simplicity cou\plete dis~ociation of
the gelatin snIts anti tht' a('ids. \Ye &('(', then, that it is
possible to show mathematically that the valency of th~
diffusible ions will affect the degree of the equation. An'
unequal distribution of diffusible ions at equilibrium on two
sides of a m('mbrarw~ sUf'h as we haye above, must result in
a diffetcnc(' of electromotive forc(, between the two solutions
scpHralt'd by the membrane. Now this electrical potential
different", between the two solutions at equilibrium should

hc g-iwn by the Norust formula and is equal to R : log, :,

F y
where R = the g-as constant in electrical units, }' = the
number of coulombs in a farad of eledricity, and T =
the absolute temperature. In the ease of the system (A) the
formula beconles

~T log ./ij-(y + z) = R! log.

F ,8 Y 2F
(1 -L!)
Y I .......

and in the ease of sys~m (B),

-RT log,
...'
:/y'
- -(y-+
--
11
z)
=
RT
-
8F
log., ( I +_11Z)
 MEMBRANE POTENTIAL
Thus we see that the ratjo of th,' ,'atuts of th, 1'0t('nli..1
difference in systt'm (A) to that in syst<'lll (ll) is 8 : 2.
This potential differt'llc(', dedu('ihk llmthf'mtlticallv us
g!"cn above, was. OC'lllollstratt'd by L<)f'h in \~ ('tl~( of
protpins by 1m' usc of th.. method shown ill ,Fill,. O. A

FlO. 6, - Mt'thod of IDf'&8Uring the potential diifet('nr,e

between gela.tin chloride solution in & collodion hag
and the outaide hydrochloric Mid solution in th~
beaker. (ReproduCed from uwb, .. Proteins and tilt!
'fhoory of Colloidal BehaviouT:' hy permiJt!!.ion of
ltlesare. lolcGraw-Hill.)

~cr l:taJf }f(lk.\iili}f d,,}x;mt J{) ~:C. ol Nie" P~;,y !."t'l'h,eV:t\7t"i,' J~'
immersed in the dilute acid solution, and a!'ter e'luilibrium
has been attained (some eighteen houl'S at ;14') the membrane
~lIti&l is obtained by measuring the E.M.I<'. of the cell : -

Hg
HgCl I
Gelatin
Acid I
i
Acid .\
C"Uodion , Outaide Sat. KCl I I
\ Sat. KCI Solution Membrane Solution! IJgGI IHg.
00 plotting the observed membrane potentials in ml'llivolts
against the P. of the protein-acid solution it is found that
44 rOLLOTlJS A],,;]) (,llEltIISTRr OF PIJOTEINS
the \'ahw~ ,._riV{,1l hy a<"id~ with monovalent ions, sU('h as
hydrm'hlori(', hydrohrotni(\ hydriodic t nitrle, a{'eti(', pro-
piOBi(', and 1udi" Jl(.j<f,.;, nil lj(_ on om' ('urn', whilst th(.~
pf)ttJ)tja)~.~htuiJl{'(J \vith ~uJphurk and sulphosaJicylil." acids
lie on Ii ('Oll'HllOll hut differeut ('urn', lforeovcr, th(' curv~s
all slit,,,, n minimal Ill('mhrafl(, potential at th(-' iso<'lectric
point of t h(' protein, and aU ris(' rapidJy with diminishing
PH untif n maximum i~ fNlf'htd nt about PH 4'0, and fall ng-ain
I{''is rapidly with a further diminut.ion in th(' 1'0' The
following tnhk illu~trat('., tll{'s(' points : -

TAIlLE IV
.lfl'mhrtlll' PIl/ellt;fl/S for Dibatric alld .lfonohasic Acids

P"
Hlbtl.sit' ;\dd~. MonobMk Ac\(k liatio
.
ify~:jc'
M.1\l!\I)ILl
24 7() 067
2(i !Hl 0'6.';
11-1, 11(; 064
:l() J:JO (lGS
a2 l5-S 064
;1.+ lK(l 062
a'li 11l'K 0'64
:l'X 21'2 0'62
HI 21,n 0'61
,.. 2 20H 060
4'4 192 0'62

I{el"{~,again, we oh~t"n'e the phenomenon of a minimum

point in n physical property of the protein corresponding
with the isoelectric point. Further, just as we foftnol.t.he
simple valency rule t.o be "alid in regard to the action of ions
on the osmotic pres.1iut:_e, ,;scosity, and other properties of
the proteins, so we see that this simple rule. applies to the
actiorf of anions on the membrane potential. It was shown
that on mathematical grounds the ratio of the effect of a
 .BIOCHEMICAL Al'PL/('ATJOS 4;;
m0l10n:uent ion to that of u din1knt iOIl ill iutlu('ncing- the
mcmbrane potcntiH1 difft,rt'J)cc should ht, :~ : 2, ~tJ}d the ubo\'(
tabk show!oi how nearly HI{' ('x}wriuwHtul r('~ult!- u('t'ont with
this theoretical ft'quir(:nwnt. an olV,o,('f\"ation whid~ "trjkin~ly
confirms tlK- appli('ability of J)OHlHlll \ tl1('ory of tht, prigill
of this potential dirrettnel'.

Biochemical Application of the Donnan Membrane

Equilibrium
Donnan himself was th( ftr~t to point out that hi .. th('OTY
had obvious important appli('ution~ in hio(lwmh.try tmd
physiology, and, foUowing on LOt'i)h ~tl('('('~h in utHi)or.ltlll the
theory to ('xplain the colloidal behnviour of tht, ptohint-., it
-has bef'll applied to ~uch phy~iol()l!i('uJ piwllomt'llU a~ til('
distributloll of salt ion!'; hetw(,(~lI media separated hy M'mi~
J)Crmf'abk Dll'mbranes. lm!t'{'d it i~ oh\'jous 1hnt IJU"t ('{'U
membranes arc ju~t S\H.'h ~{'l\li-pern){'abk Jl1l'mhrallf'S Ils un'
required by Donnan\; tlll'ory, So fur 11l0-;t of tiWM' applieu ..
tion~ hay<.' b{'{~11 mad!' in !o.tudh,!'o. of tlu.' (quilihrillm ('xi .. tillJ;{
between red blood {'db Hnd pia!'omn. rllld an illv(,!'otigatinll
made by Barcroft and..l.. Y. Bill and their ("ollaboratoN Illay
serve as an illustration of the u~('fuhw~ ... of th('!'o.(' ,<i('w, on
membrane equilibria. Hen the tlwory wu;" uMd to ('xpluin
why at a giYN! hyrlrogeu ion ('oJ}('('}ltration of the hJuod
plao.;maJ tW(I individuals may .. how diff{'J"('flt ox~:.wn-h1(m()
globin diShociatioIl Pun'('!'o. The ~llrpo;-.itioll j:-. mad( that of
the ions prest,nt in the plasma and corpu,,!t-s, 0, nro", II,
Oil, phosphates (P), und hu:-.i(' ion~ (Il), all nw.y pU!o.:-' throu~h
the membrane ~urrounditlg t he ('()rpu~d(' eX{'('pt th{' l"~t t'\\'o
~entioned. Then from thl' consideratiun:-_,. aln~ady advanced
(eq~oa A. p. H),
{H], [01. I HeO.]. IOHI.
[HI. = [a1 = [HC'O;r; = [OH],
or
[Hl, _ fCll. + IHCO.]. + IOHI, (il)
[lIl. - [af+tnco.j,-+ loiiJ.
46 COLWlDS AND CHEMISTRY OF P.{IOTEINS
wheT(> t}){' squarf' hrackets represent iOllic con('{'ntrations, and
th, smtdl numtK'n.. J and 2 the two sides of the corpuscular
memhranC'. It i~ thC'T1 nrgued that of th(" total {'harge carned
h" the i~~ within the corpu~cle h,t'Inoglohin ('an provide
h~Jt a .~mall part, owing to its gTf'at nlOlc('ular weight, and,
Uwrefore, ('ompart"d with the ('Oncentrations of chlorine,
phosphate, bi(,Rrhonate. and basic ions, titt' concentrations
of tho hydro~"'n and hR'moglobin ions aro no!(ligibly small,
Hence the negah"e ions must huye their charges bulanced
almo~t wholly by positin> hatiic ions, and one may write for
the two ~id('s of th(~ tr).{'mhrane

(1) LUll = lO]. + [HeO,), + lOll], + [Ph

nnd
(~) [BJ,'= [('IJ, -+ [HCO,I, + [OUJ, ,t [P],

and ('ombining these with the equation (B) given above, we

huve
\lIh IUl.-I!'I,
111], lBll --[I'J,'
It tiWl'f'fOfe follows that any diff(>fellce in thE.> concentrations
of the \,asio and phosphate ions on tho two sides of the
corpusrular membrane must lead to a proportional but
inverse inequality in the distribution of the hydrogen ions,
Thus we may haw all internal corpuscular hydrogen ion
concentration different from that of the surrounding plasma,
and in two indh'iduals, even if the latter is identical in both,
the former need not be so if the basic ion-pbosphate.i.on
balance is different, Hence the dissociation curves of the
oxyhremoglobin present in the intact corpuscles of the two
individuals may also be different, ,,'en ifthe plasma hydrogen
ion """centrations arc the same, as Barcroft and Hill found
experimentally,
 REFERENCE'" 47
JiIef8renIleII
I. This chapter is based largely on th~ work of Vleh and hit\
aMOdates. for a dIP.taitl'd a,tXl,ount of whiC',h tht' J't,ad('.r l _o\
refette.d to "Prot(~ins and the Th(l,on' of ('oUoidal ~~
havlour," J.l.JOF.B, N~w York and LOftdon! ..ft.(~raw.llm
Book C{)mpany. 2nd Edition, 192t
The following mav aJ~) \)(>. c{Hlt1\lIt,(>,{\ : ..-,
2. BARCROFT, Hn.L and l'OUaborators. ,Journ. PhYlfitil . 1922, H.
157.
3. COliN. Physioa.l CllemliC.f,ry of Prot,(\illtl,." Phy,un. ~oit!W8.
1920. 6, 349.
4. DONNAN. ('ktm. R ..,kw 1924. I. 73.
5. HAlI.Rls. PTOO. Roy. Soe .. 1925. B. 97. 3M.
6. HITCHCOCK. .. Prowin~ and the Donnan Equilibrium."
PhysifJl. llet~iew8. 1924, 4, M,l.
i. i\frcHAEJ.S and ~AKASHn(A. Bi{}('lirm. Z,.it,. 1923.148.484.
8. Tlu: applica.tion of tbt, Donnan ('quihbriurn to gaB and e!tro
~~ ~~~~~~t:Q~!II:r~~On(~;~ t~l~~~~2~~' &6~N76~:YKE, Wu
 CHAPTER III

THE NUCLEO-PB.O'tEDl'S
J:'\ IHtik the !-.ubstanec which w{' !low eall nucleo-protein
Wll!oo i!:ooolated hy Mi{'~('hcr from tht nu('ki of pus C(lls.
Mics('h('r ('aBed hi~ pr('paratioll . Illldein,') and dc~('rib(d it
H!:oo contail1in~ phosphorus lnd r{'sponding to the ordinAry
colour tl'sts for prot(ins. It wa~ soon dc'monstrated by
lJoppe-S<'ylcr sUlci by Ko~sd r('~pc(tivcly t.hat a similar
suhstan{'(' couJd bc' obtnin!"d from the nudej of yeast ('dJs
und from llvian red blond cells. Latc'r :\fic~chcr extended his
ifl\'('~tiA'atiom, to the sp<>rm (,(,JJs of tht> salmon, material well
~uit('d to his Ohi{'ct heeausc of the {'aSl' with which thl' nuclei
may he ""parat<'ll from the relatively ,mall tail and middle
portion of the spermato1.()lm. Jo'rom th{' nuelcithus ohtained.
}w isolat{'d a substance which he rt"gal'ded as a chemical
individual, a salt of the basic protein protamine, and an acidic
substance which he called !lucleic add. The discovery of
tht'Sf' two important ("onstituents of ('(~ll nuclei may, thereforE',
he ",crib<-d to lIIiesdwr. Since that time the chemical nature
of thr- nUdeo-proteins and their oeCUrrenc(' in such funda-
mentaHy important and charactt'ristic structural components
of li"ing mat(cr as the cell nudei ha, sufficed to keep them
in the forefront of the att("fition of biochemists.
The study of the constitutional aspect of this group of
proteins has centred round two main problems--thl ""ture
of the association of nucleic acid with protein, and the
constitution of nucleic. acid itself. With regard to the former
problem, it had been supposed that in the cell nuclei the
nucleie acid was combined with a basic )Kotein to form a

..
substance called, somewhat vaguely, " nuclein," which latter
 NUCLEO-PROTEIN 49
had further ('omplex ru.~odnti()1ls with pn)tcins of tht, histnl\('
typC'. and claims W('TV lIlurie from tim(' to time on bPhtt-lf of
the ('hemical individuality of !o>u('h nU('h'iu-prntcin ('om
pkxf.'~. The positiou at present is much (')('8"": and W('
ru1iy now Tf'f,!ard the nu('k()~prot~.'ins of the li\'lug ~'('JI as {'oll-
sisting of variot}', ~alh of basi( prott'iH~ with lHll'lt'i(' fiNd, in
whieh the form("f eOlnp011('nt is in ('xen.!',. Any md hOft of
('xtrlU'tion of th(_. Ilud{'o-proh'ins, or n pr()c.('~", of pnrtinl
hydrolysis. iuyoln ... n Tl'llIoYal of part or tlH' protein, 1<'Hvin1!
8 mi>..iurt' {)f morf' u('itiic suh~ttm('{':-' of illde'finit{, ('ompo:o-itioH~
Hnd we may Tt'garcl as ~u('h tht . nUC'\f,in" of tilt' c .. rlif'r
ill\'('stigator~ in this field. The prt.s('nt-dny po~itioJl i~ \w1\
('xpress('d hy .}olle!-. a:-. follow~: . Thc' terlH~ nud('o~pruh'in,
'mu'h'ill, and llueh'l(' atid ('xpress Ii nlatioll whi('h tn('tm:-;.
little mqn' than thut ('oB\,(,Yt'Cl hy thc' {(rm . . \H\!->ic' kad
.aeetate, )(>a<1 u('('tatt'. und ~u,(,ti( Heid," and in T('ality
~ JIUC'\(>o-protf'in' m('Ulbi rather a nwthod of pn'pnTation'
than a ('h('mieal suhshuw('." Su(h '"kw ... art' funy hOTIl(,
out hy ttu: r('c('nt ob~ct\'ation:-. of E. lIammnfl<otplI, who finds
that pure nudt'ir u(id prepared from the' thymus ~\RHd ('(\\n
hin('~ 'With pTot('in~ in varying proportions. The nC"id hu~
four dissoeiablc hydrogen atom ... throug-h whi('h it may dft(t
!o.Ufh (omhinatioJ),.,. and proteins wit h an i.!lood(rtric point
of PH 47 eombin{' with the })uc)('ie ucid mort' N'HdiJy during
UJ(' stage' of dissociation of the Hr,o,t two of th('~w atom.It Hum
during tilt, later ...,tagcs of the di.'is()(,jation. Th(' bthu"jour
of nucleic 8!'id in this respect, thef('foTc, conform" to {'XIU'dHk
tions if on(~ regard" it a:f.o a polyhw.;ic Reid.

Nuc1eic Acids
I!. 'lOilIoiJe apparent from the foregoing that the chemistry
of the nudco.protcins centres largely round the nature of
thdr specific acidic component, nucleic acid. Modern views
tend to the recognition of the exi,'tence of but two nucleic
acids_ One of thcse is obtainable from the nuclei of ",I ant
cells, the other from the nuclei of animal cells. Ye.st-
15.
"
50 THE NUCLEO-PROTEINS
"uclcio acid IItid thymo-nudeic acid (from the thymus gland)
will s('r\'(' a~ types of these two acids. The terms yeast-
nnd thytllo-llueJeic arid hav(, been, and ut'{' !o.tiH, widely used
us un illtHcution of til!' source of the lluc)eje adds, but
inssllIuch as all plant lIucleic aci,h may be 1'('garded as
identical with thllt of yelL,t, and all animal acid, with that
of the' thymus cdl nuclei, the application of the terms need
not be f('!-;tri('ted to nucleie acids derived from yeast ('{'Us
and thymus glands respectively. It is legitimate to refer
to the thymo-nuc1l"ic acid of the pancreas, but it is perhaps
prt'ft'rnblt to use the !!('n('ral term .. anima) nuclei(' neid."
Th(" two nueiell' acids closely fes'('mble one another in yielding
on ultimate hydrolysis (1) phosphoric acid, (2) a sugar or
d('ri\'"tin'~ of a sugar, (3) hast's of t}l(' purine and pyrinlidinc
type. Tll<'y dilf(-ll", howen'r, in the nature of tilt-ir ~ugar
r('sidue~ Hnd in the yuricty of their ba~(_,st as shown in tht'
following tu ble :-
TABLE Y
Components of },'uc/eic Add.,
Of Plant. Origin, Of Animal Origin.
Phosphoric acid. Phosphoric acid.
Pentos(~ sugar (d.ribos('). Lrevulinic and fornlic acids
derived from a hexose.
Guanine \ Puri lle bases (Guanine.
Adell;Il(' J \ Adeuine.
Cytosine \ Pvrimidine .fCytosinc.
eracil J bases \Thymine.
Th(>re can be no question concerning the occurrence of one
principal nucleic acid in plant and another in animal cell
nudei, and that these two acids hR,-e respeebvell" the
('{)mpositions given above. But the "whole question is some~
what mote compleK than this would indicate. One can
isolate from the glandular tissues of animals two substances
called inosinic acid and guanylic acid. The latter is obtained
by removing the protein from the so-called ,B-nucleo-protein
 NUCLEIC ACID III
of Hammarsten. These snhstl\nc<:s, although simpler th"u
the nucleic acids proper, U]'{~ V('ry dOSl>ly n'luted to them And
belong to the cla!o,~ of ~ul)'itl\n('{'~ t'llllt'd IHU'lt'otidt's, t ht:
nucleic adds being: formed, as Wr ,hall S('(', by th(.A~"'(Kit\ti()n
of four nudeotidc~ similar to inosinie and gunnyli,' lH1ds.
The study of the ~imflle mono .. nudl'otid(~. tjwrt'fuf(.: M'rn';'.
to elucidate the nature of the nl<lre ('(lllIpl(:x uud('ie uddl-o..
Ino5:inic acid yidds, Oil hydrolysi.'i, phosphoric m:id. tIJ{'
pc:'ntose ribose, and the' purine' bu,'tc hypoxanthiJlt in ('qui ..
molar proportions, whil!o,t guanylic aC'id diff("~ from it only
in eontaining guaniIw ill plac(' of hypoxanthin(', TIt(' poiut
of interest is that, although t}les(' mono~nlH'l('ot.id(s un' uf
animal origin aud ar(' clo~dy ITlnhd to tht._ Ilu('\eiC' add .... th{'Y
eonlain the pt.>nto~(' whi('h i", t ypi(~ul of plnnt~\.'tll nUll{'i,
Thert;' \to, however) no r('ason to a!lo~()('iah' the':>(' two 't.implt
tlueiNJtid(')', with til(' ('('il nudei. Tlwy appeur t.o iH' ('xtrt1-
llueiear l't>H (_onstitu(,llt:-.. A.., a ('ounteTpart to thh. (l(>('UlTf'Jl('C'
of p('nto~ nudeotld('s in animal tj~!o.u(,s. Ilil u<i('niu(" h('xo~id('
has been r{'port(~d from yCll!'.t. It {,,:Hmot he II dt'{'OlUpu",ition
product of y('a.\)t~IHJ('}d(: acid, sinee thi~ ('ontain .... a~ W(, Itave'
seen, a pt'ntos(' only, and theN'forf' it rnUlo.t han' a .!oJ(parah
existeu('e in th{' yea..t ('Pll. Th(' vi('w that an ah~()llltdy
sharp distindioll ~ betw{'t'n plant aud animal nud(~ic uei(h.
cannot be drawn with rt'gard to their sugar rt'sidul' i~ ~aiHing
ground amonw.t biochemi!o.ts. The pr('~{'n("(~ ill animal tis",u<'~
of the two pent~ llu('l('oti(les nwntioflt'd nbov(' h~ heen
re-eogtli~{x1 for ."orne time. More Tel'cntly tJu' bruduc .',aJt.'i
of adenine, ('ytosinc (Jone, and Perkins). and uracil (Jorpes)
pentose nucleotides have beNt isolated,. in addition to guallylic
acid, from the mixed nncleotides obtained by hydrolysis of
t~ .,s..ftucleo-protein of panerea." whilst adenine pentose
nucleotide identical with that obtained from yeast-nucleic
acid has been separated in a erystalline oondition from
deproteinised pig blood filtrates (Hoffman). It has also been
suggested that tbe poly-nucleotide of the" coupled" !W'nyl.
nucleic acid, first described by Feulgen and isolated by him
52 TIlE NFCLEO-PROTEINB
from pancreas, is none' other than y("ast~nudeic acid, which
would accOIwt for the isoJutioll of the individual Huc}eotides
mentioned ahoq'. It i~ at l('H~t dear from th<,s(' ollservations
that tHuI'<ltides ('ontaining: pf'ntosC's have a considerable
rli,'ittrihutioll in animul tissuc~. .Fr-ulJ,!'en and his COw workers
ha\'(' r;'('('ntly d('~('rif)(>d H t('~t. which tlwy cali the" nu('lcal .,
l'f>adioJl, and which may 1)(' app)i(>d to micro!:ocopic prepHrR~
tions of tisslI(,s for Ow purpo!o.c of dC'trding thft- pr('sence of
animal-nucldc R('id. The tissue is subjc("ted to mild partial
hydroly~j~ with n!lO hydrochloric acid at 60 for four
minut(s, wit h the objcet of liocTating the aldehydic groups
in the h(>xos(' units responsibk for the l"{'uction, and tht' tis.'')jue
is tlwn tn"ted with fuchsin-sulphurous acid. A positiv~
1'C'fl('tion, whic'h consists in the development of a red-vio1et
~olour, i.It given only by nucleic acids pontailling hex9Se units
sUe'h as we Imv(' S('CI1 rharaetf'ris(' animal nuelt-i. Nf'vf'rthe
lc'is, tis~uc preparations from ('('rtnin of the higher plttnts,
sUe'h as whent rmhryos and hulbous plantst give' a positive
f(,8('tlon, and therefore presumably c()ntain a nucleic acid of
th,' hexose typ" in addition to the well-recognised pentosc
arid. Bndcria, like yeast ("{'Us, give a negative result, as also
do simple protozoa such a._., try-panosomes, whilst ciliate
protozoa (infusoria) give n positive response to the test.
From these observations Fcul~n COllcludes that the yeast-
nucleic add is the primitiw type, that it is partly replaced
in t lw nu,lei of the higher plants by the hexose (animal type)
acid, aud that it practically disappears in the higher animals,
where it sur\'iws only as tbe extra-nuoJear pentose mono-
nucleotides which we have already mentioned. It is possible,
however, that the coup)"d guanyl-nucleic acid, which contains
pentosc, and probably consists of yeast-nucleic aeid'coopled
with guanylic acid, is a true nuclear acid. If so, we have a
dear case of a plant nucleic add, and not mt'rdy an isolaW
mononucleotide QC('umng in animal tissues.
TM Sugar of Nucleic Adds.-The pentose sugar of plant
nucleic add Ilnd of inosinic and guanylic acids has been
 SUGAR OF NUCLEIC AClD 113

detinitt'ly idt~ntin('d a~ drihost, hut in UK' c'aSt' of tiN' "IUR'UT

of the Hllimal acid no ~uc-h ('(_'rtnint y has yrt h('{'l1 nttaiuNI.
On u}Umat' hyiJroJysi:-. of HlllUW) l1ud('i(' acid, JaTuHnlt- mul
f~)rmi(' u('ids ~"r(' ohtuiJl(',1 in pI8(0(' of 1\ fref' IJ,tt~.HM" 011
oxidation with nitric' arid, Steudt'l hn~ duinU'd tlLl!t tllt'
sugnr residue i~ ohtllJrt('d ns H di('urhoxyJic ll(id of HI<' typ('
of ~a("('harie acid. F('ulg('n hu ..., ~ugJ,{(.~t('cl t hat no )wxo'o{' us
surh is prt"s('nt, hut un Ull!o.lltUfUttd l'Iugnr of tiw llntun' (If
glu(at Steudd and Pf'is('r nscrii)(' ttl(' t(aetioll!o. 011 whit.'h
Frulgf'n hm.(_~d this vi('w, induding the fmh ... in ... ulphurnu'
acid test, to furfural. Hut Ft'u)W'n hul'. p~)iJlt('d ont that the
fuchsin te'st is not ginn hy furfural and is uh..,olutdy ''''IK'('i(it-
for thr partly hydrolysf'{ll1ninmJ llU()(j(, Jwid. Jlldt"rd. jf ttl('
fu('hsin t('st WH~ duc: to furfural, it is diflkuH to S('(' why it
~hou1rl f10t be gi\"(,1t by phmt nudd{' arid, lWUJing' in mimt
the rcadine..,s with which til(' nJdo.'ic f>('nto~('s yidd furfural
011 treatment with hydrochloric U(jd. }'('ulg<'f1".., mo'\t r('C'('nt
eondusion is that the (arbohydrat<' r("sidu(' is not u hl'xos(',
but a related sumtan("(~ of unknown (onstitution. M('IHlwhiJe
a possible nt'w lin(' of iOY{'stigation s('cm!>' to he- opening out.
Mention has hc{'n made of 8n ad('uim' h('xosid{' whi('h was
isolated from yeast (p. 51). This ",I"tam',' 011 hydrolysis
yields lrevulinic add. Suzuki recently mad(" thf' interesting
discovery that the sugar residuC' {'ontained sulphur, and more'
recently still Levene and Sobotka haw identified the "'gar
as a methyl ketopcntose containing sulphur. The sillphur
is not present as a sulphydryl group, but the pr..>;enee of
either an OCH. or " SCH, group has beell demonstrated.
Levene and Sobotka sugwst one of the following structural
formula! for this inh'Testing huh..tanN\ the position
aSlligtle.1 to the methoxyl or thiomdhoxyl group being
arbitrary ; -

CH.OH . C . CHSCH CHOH . CHi

i -. . _ --__ ~ _~.- .---~--
OH 0
 THE NUCLEOPROTEIN8
CH.OU _c _tHOtH, _cnOH _CH,

on
The possibility that some ,ueh .imilar substituted keto"
pt'ntos(l sugar, not n('('('~:-;ari1y ('ontnining !-.ulphur, may
constitut' the sugnr of' the anima] nuckic acid SC('llIS worthy
of l'onsid{'Tlltion, c.-ipecial}r in ,-j('W of the Tl'p('atf'Q failures
to e-haractcris(' this ddinitdy us a hcxo:.,c llut it is ob\'ioU8
that nmC'lt furth('r inv('~tjgation is requln'd to d('ar up this
difficult quc:..tion.

The Structure of Inosinic and Gnanylic Acids and

their Relation to Plant Nucleic Acid
On hydrolysis \\;th ft<'ids inosinic acid d("{'omposes..a('('ord~
ing to the ('qulttioJl :--
(\oH 1 :,!O)<N 4 P -+- 2 H~/) ----+ C\II1110r. + H 3PO" ~ (\Hl0~4
(JuOOute add.} {Rjb~.J (HnlOxantlJine.)

It is, how"",-r, possible to adjust the conditions of hydrolysis

sO t hat intermediate products may be isolated, Thus mild
aeid hydrolysis yields a pcntosc phosphate and hypoxanthine,
whilst neutral hydrolysis under pressure yields pbosphoric
Ht'id and t he mld{'o~ide inosine! a ('om pound of ribose and
hypoxanthine of the formula C,.lI120,N., These results can
only be s('('ommodatrd in a structural formula of inosinic
Rei;1 which places the ribose unit in tht' central position
het w('Cn the IJhosl'horic aeid and the h}'J)()x"nthine, thus : -
INOSINE
n.utml hydrolysis
PHOSPHORIC
acidhY~--~
ACID--RIBO::~~HYPOXANTHINE
_ ...
PENTOSE PHOSPHATE
The constitution of inosinic acid is therefore
HO"
Hg/P-O ,C,H,O" C,H,ON"
 S'!RUCTURE OF NUCLEIC ACW 5r.

Sirnilar observations M'rv(' to ("stahUsh t 11(' ('on~t it uti<m of

1-,1lJnnyJi(' add a ... a mOBo~uud{'otid(' in which pho.'tphoric
Reid and the purine ha!ol{, ~llat1inc are' huk('d through ril~JM',
thus:-
(;rA~O~I::\E
l1f'utral h:rdrlll":li~
..
HO', _ - - __ :--:_ ,
ni:/l'--O-C,H,O" C,H,(J~"

On aplllying to yca~t.nu('ki(' add the' nwthod of lI('utrni

hydrolysis und('f p1"<'s~ur(' th{' four nud('()~idn giv('11 h<-low
arc ohtRinro tog<'th(,t with four ll1oic('ulnr proportion!' of
phosphoric a{'id. Thc ('Darst' of furlhf'T hytlrnlysis of t}u,,'t(,
nud('osidt,s is also included in tht' s(lwIHc.
(I) Guanosine -----+ driho~(' + gunninr \ l'uriH('
(2) A~eno,ilw ---------> d-ribosI' + a.\cninl' ) bfl."",
(8) Cytidine _----, d-ribo,c + "yto,inl' I l',vrimidinl'
(4) rrici.in(' --... -------+ d-rihos(' 'f ura(,il f hnM'~.
On the hasis of theS(' t('sults U:-V{,IW and lac'ohs ~u~gc~t('d
that y('t\st.nuc}>ic acid i~ ('omr)O'icd of four mOIH)nul'I(()ti<i(':-',
eaeh form"d by tbe addition of phosphor;.' Il('id to till'
nu('lt'osides obtained as above. In ("onfinnution of t},il'>
hypotb('si, th('y prepared from yrast-nuclpic add the four
expected nuc}eotldes of the constitution :--
flO,
o_/:Op_o. C,II.O" n.
HO
B representing the bas("s guanine. ad~nin{', cytosine, and
uracil. This hydrolysis of yeast-nucleic acid into its com-
ponent nucleotide. is elfC<'tcd by the action of a thermostable
agent present in a boiled aqueous extract of t} pancre,,, of
tbe pi!!,.' or by the action of ammonia at 105' to 115'. Tilt,
relative ease with which the further hydrolysis of the..,
individuaillucleotides is elf.cted is different in the two type.
of nucleotide. Thus the phosphoric acid is liberated from
1 There now appe&f'! to be SOUle douht.u to the pn....er of the tbefmofJt&bte
agent to libefa.~ ..u the component Dtlc'k,otidaa of yeaat-nueleic add.
 THE NUCLEO-PROTEINS
th{' pyrirnidim' nucl('otidcs at a much slower ratt.' t han is
thnt of til(' purim.' nuclt'otidcs. Thi ... dir('renC't' in tlw
hdUt\'iour of the llu('}cotid{s i!\ illustrated hv .lone" U'i
follow~ :-_'... .
Tt'n. portions of rornmcf{jal y('a~t-ntI('Jei(' neid from tht
!!oaHU' ~p('c.inll'n nT(' weighed into appTopriatf Y{'s!\.t']s, alld in
on(' of the!o.e tllt' total phosphorie add j!j df'teTllliJ)t'd. Tht,
othr-T nine portion.... arc submitted to hydTO)y~jS u'jth 5 per
('put. ~uJphuric acid nt 1(){Y' for various ppriods from 011C-
quart('r to !'t('veH and .a half hours, and after rcnwral of tl1('
guanine with ammonia, th( phosphoric acid is prccipitat{'d
with magnf''!'ia mixture and lVt'ighcu a .., lIlagll('..,ium mIl
monium phosphate, The weight of flIagH{'siulll ammonium.
phosphate obtained ill each experiment is dhided hy the
weight of Ilucleic acid from which it wat;; produC('d, and from
th" compurable results so obtained the upper curl'e (A) ill
Fig'. 7 i5i ('ow,truc'ted. The rapid initial rise in the eUf\'c. its
sharp turn. Hnd tC'rmination ill a ~traig'ht. line sJi,ghUy indined
tn tm- abscissa show dearly that in the acid hydrolysis of
yt'R!-ttRIlucleic acid phosphoric aeif! i:-. pro~-rressiyely liberated
from two sour(,(>s. f'rotn the on(' the produ(~tion is rapid and
('ompktf' in l{'ss t han two hours j from the ot her it is much
~I()w{'r, ('ontinucs beyond the time consume-d in these ex}X'ri-
ments, and is ~o rq~ular that its amount is definitely seen to
be equivulent to 10 nlg. of nlagne~ium ammonium phosphate
per hour. If We make a deduction for this amount from the
values recorded in the fir~t curve, we obtain the lower
curve (B) in :Fig. 7. This curve ends in a straight line parallel
to the abscissa whose ordinate corresponds to half the total
phosphorus ohtainable from thc nucleic acid; or, in other
words, the phosphoric acid which is rapidly and complHely
liberated by acid hydrolysis of yeast-nucleic acid is half the
total. It is possible to. show that at the point where the
curve A makes its sharp turn the purines have been com-
pletely' set free whilst the pyrimidines are still combined.
It is obvious that the two sources of phosphorus are the
\
 s:rRUCTURE OF NIX'LEW ACID 57
purine nudcotidcs and the pyrilnidin{' nlldt'()Ud('-~, nut! a. . .
thf"s(" sources arc equal in amount the purin(' J,trtlUp" of
YNlstMlluc}eie acid must lJt, ('qual in Humber to tll(' Pl'1"imirlil1{'
,t,rt;,oups. In ('onfirmutioll of th('~e views it i:-. fOlltl thHt~ ill
tht' ("liSt'S of the fr('(' lludt'otid(',,; of gURnin<' and udl'nil~(', tht,
H ('a~il.v split " phosphorus is (qual to tilt> tutu). nut! lK)th
gin~ a hydrolysis curn"' practif'ally ('oindd('llt with ('Iun' n
of :Fig. 7. }"urtJwr, 8 mixtuff' of f'qui\'uknt qunntitit's of
adenine HUl'kotide .and UT:wi) lHl(')('otirie givt"s off hulf of it-.
phosphoric acid ('asily "nd holds the oth"T half myeh moTt'

firmly, whilst a mixture of cyto~in(' and uracil nudcotid(s

under similar conditions gives off an amount of ph~phori('
acid which corre'ponds to only 10 mg. of magm'sium
ammonium phosphate per hour, as !;hown in the indiIlt'd
straight line in Fig. 7. The most obvious explanation of
the foregoing observations is that yeast-nucleic acid is com~
flOti'Od OC\wo purine nucleotide. and two pyrimidine nuelcH-
)tides, all of which contain phosphoric acid and ribose, whilst
, each contains a different base. The method by whi"h theS('
. four nucleotide. arc linked together still remains to be
discussed. In their earlier obs.-rvations Jones an3 his
associates failed to demonstrate any increase in the a~dity
 ;38 THE NUGLEO-PROTEINS
of a digest of yeast-nudeic acid with the thermostable agent
in a hoilrd extract of pig's pancrea.<i, which, as we have s('en~
splits the nucleic acid into its component nlldeotides. If the
hydroxylg;Ollps of tlw ph",.phoric acid re,idlles were invoh'ed
ill the internueieotid{' linka_g(:', one might expect a markld
iner(';,. ill I he acidity of the digest during th.. progress of
Hu_' hydroly~is. But rnOft_' 1'('C{'nt ohservlltions by Jones and
Pl'rkins. in whi~h a quantitative {'oncrsion of yeast.nudeic
H(,jd into its ll1ononuC'}eoi.ide!<l wa~ achieved by the action of
1 per c('nt. sodium hydroxide at room telll}X'rature, show a

no,
OP-O-C,li,O, C,H.ON, (GUAHI~l)
HO' I
HOS
0- P-O-C,H,O,. C.H.ON,
b (CYT05INE)
I
o-.t-O-C,H,O,. C.H,(\N,
HO 0 (URJ'Cll)
HO, I
H8;p..O-C)H,O, C,H.N, (ADENI~E)

JONES' FORMULA

progressivE." diminution in the alkalinity as deeompositon

proceeds, indicating the liberation of weak acids. The carlier
view that the phosphoric acid groups played no part. in the
linking has, therefore, been abandoned, and it is now supposed
that tbere are p",scnt two linkings between phosphoric acid
and ribose of an ester type, and another linking of an ether
type between the other two central ribose molecules . -!??"s
and other workers exclude the possibility of the bases par-
ticipating in the linking, largely beeause the rapid rate of
liberation of the purines and the slow rate of liberation of
the f.yrimidines from tile parent acid are tile same as those
observed in the cases of the free nucleotides. Levene, on the
 SUGGESTED STRUCTURES

other hand, suppos~s that all th ....c linkill~" nfl' d(,'('\c,1

through a pc-ntose and a phosphoric' add group. TIl<' wholt,
question of th(~ intt'rna] linkinw- of ven",t ~Jiuc1t,it ndd )Ill)'.t
,.,ti!1 he r{"garded as open, hut tht, tW(~ pos..,ihiliti(, ... u,t'ntiotl('d
aT<" given on p. 58,
In a J"f'eent study of the rlisso('iation ('olistullh of til('
individual nuclPotides and IHH"\l'o\ll1('s of plant ntl<'it"ir' tH'i(l,
Le\'t~ne and Simms addurc import.''Ht {'videHc'(' ill f~n'lJur of
the three ('strr linkings as oppo"l('d to thf' \"ugW'~tinn!oo of .Jnl\(,~.
Nom~ ofth(' formula- y(t proposNI giv('s a r<"nlly !\ath,fndory
explanation of the markl'd dirrerenc(' in the Tntc of furHwT
d('composition of the pyrinli<iine a~ ((Hitrll...ted with thf'
;mrine nudeotid('s.
The formul~ :.,riven abov(' as repn'scntillg' plaut nud(ic
acid may stand in n('('<1 of further ('orTt'dioll. l\.ing til('
method of ~()ld hydrolysis with dilute sodium hydroxi<i('
ITer-nUy dc\'jst'() by them, Jones and Perkin... hun' obtiH('d
from this acid guaninf' nudeotid(', ad('nin(; fl!1()(otid('. latf,("
quantities of cytosine nucl('otid{', hut no urat'il nu<"i('c.1itie.
They likewise obtain very small yidds of the 1atl", on
hydrolysing the nucleic acid with thp tlwrmo'tnhk com-
ponent in an extract of pig's panerf'a.,. They t}wtcfore
eonclude that ura(~il dot's not cxi~t pn:-formf"d in th(' nuddc
bcid molerule, but aris('<;. during hydrolyti{' d{lt\'ag"r hy
secondary de(>()mposition of cytosine This ('xplunat ion of
the occasional presencE' of uracil in ttl(' hydroTY!,j!) produet...
of animal nucleic acid has now he("n held for ;!'ome tim('.
Thus it would seem that uracil is not Pf{,,,,,.t ..., suoh in either
of the two nucleic acids,
..
The ea.", with which plant nucleic acid is decomposed into
its four component nucleotides by the action of ammonia at
1050 to 1150 is not paralleled by the behaviour of the animal
acid, nor does the latter yield it, purines in an identlfmble
form as readily as does the plant acid on hydrolysL' with
,
60 1'lfE NFCLEO-PROTEIN8
dilute miJu'rnJ a(,iel. Theft' is, however, no doubt Tcltar<iing
HI(' ('Jo!-.(' ('oftstitutioual :-.imilanty of th(' two nuC'i('i(' a(~(ds
apart fr.om til(' difkr('l1('{'s in ttl(' natur(' of thdr ultimatt'
h)'tirolyt1a products a~ !-.rlvcn on p. 50. Four of thf~C
prod~l<'ts nre identicu} {pho... phoric Reid, ~uanille, R{ienin(',
and ('yto~in{'), whil . . t the other two are closely related. Tfwsc
oh!-'(rvationo.; nnd the fae-ts that a thymine nucleotidt.", a
guanine nuc1t.'{)..,ide, Itnd various other int('rmediatc decompo-
t'>ition produds .. imilur to those ohtained from the plant acid
ho\'(' l)('en prepar('ci from the animal nuc'kt(, Heid, support the
,-jew that the 11lttt'T is ('oll'itituted on Jiue" f')o'wly ,.,imilar to
those of ttl(' plant Heid, i.f'., frolH four l\udeotidt'~. Thi~ is
horne out by the rect'nt analys(!-' of pun' preparations o~
JHH']"ic ncid obtahwd hy Levene from the thymus gJalld,
spl(,(,fl, kidney, panc'r('a~, and lin'f. All of these pl'l*parations
huw the d(m(.~ntnry (;omp()~ition of a tctra-(h('x()se)-l1ucleo'!.
tid('. \Vith 1'('ga1'd to th(' modt, of int{'rnal linking of the
{'olHIKlllc.'nt nu('}eotiu<'s, discus.,ion ha.... centred round the
(l1.u>stion of the pr(~s('net~ of an c-thcr or of an ('sh'r type of
oxygen linking. .Jon('s and Tannhau~('r hav{' favoured the
!'lTmcr vicw, whilst Lc"efl{' supports t1,,: lattcr_ On hydro-
lysing animal nucleic acid with 2 per ('ent. sulphuri{" add
l.kv{'Jl(~ and .Jacobs obtained two compounds, to which th{'Y
a.'icribcd the eonstitutions of l'ytosine and thymine nucleo-
tides, each carrying one additional phosphoric acid group as
shown in tht' accompanying fonnul!I' : -

They' also daime<! to have isolate<! a dinucleotide containing

the \,,-0 pyrimidine residues_ In later work Levene could
 CLASSIFICATION 61

obtain only the two former ('ompomuls, tlw hariutu snIt of

thymidiIH:'dipho:-;phoric n(~id \)('ing }It'f'julrrd in n (ry!<.tftllim.'
form. I.A:'uying out of consid('ratlon tlw Ulldl'('iu('d q\l('~ti{)n
of_thr po.<,.,')ibJe ('xilsteJl('(' of n dinud('otide in the l..;-drnly. . ;,.;
products of the tUlima} Reid, U,\"cu(, explnlus hi~ r('!>uits (In
the basis of tinct' ('!>;t('r Ililkings cadl iwtw('cil a suJ,tnr ullit
nnd a phosphori(, "wid f('!->idll<'. Th(' strudutt' propos('(l is,
tht'r('fur(_'. ~imilar to thut sUIlg-rst('d for th(' pluut lU'id, hut;
[l" in til(' instan('(~ ofth(" latt<'r add~ it canJlot fX' ...aid thu.t th('

qU{'slio}) is dl'finjtcly s(_ttkd. SUf'h difff'r('JIl'('S of bthuv;our

a.. do exii-tt ~J('hH'('n the two u<'id~ mu. . t oft Hws(' "jew... l)(\
nseriix'd to th(' :-.ugar ('Otn)XlIl(>Ht!<..
To :-.ulll.marist', \\T may dlb:-.ify th(' Ilu('\e\(' l\('\(\-. and
rdated {~om}XHmtl!<. a!-. follow,.., :.-._
1. J.Vu.cJ,eic Add". conla;,ning PrnloflCi.
(1) }folJo-nud('otidt).~. (11) Iuo,.. ini(,l1cid.
(II) (;unnyli(' acid.
(2) Poly-nu('kotidt'!o,. Plant Ilu<,{(j(' HC'id.
II. Xudeic .AM!8 which, afhr !-.piittillg of[ pt1rllU' .... g1v(' n
violet reaction with fu(h~irH,ulphurou ... j-wid 611d It
gI'{'t"1) pil)C' ~havjn.!! reaction, and which prohably
('ontain a readily dc{'onrposnhlc Jl('XO~t' or oUwr
unidentified sligar r(,sidue,
Animal nudde 8t'id.
III. A elm;" of "'Ii,fed Xucu'ic Arid r-.howing til(' rea('tioll~
of both groups I. and n., alld po~ ...ibly (,{lHsi,tlng of n
nwehani('al mixture or JOOM' (oITlhination of the
members of these groups.

Nucleic Acid JIeIaboIism

The ~tabolic transformations of the nudcoprotdns, as
re;;"'ed both hy those ingested in the foodstuffs and by
those arising from the endogenous processes of ti"ue break-
down. provide an exquisite example of a co~ordioated
sequence of specific enzymic activities. It is perhaps r"ll'""t-
table that the hioehemi,t OIIl,t give exp,",,,sion to the""
62 THE NUGLEO-PROTEINS
pht'n'>lnell" hy p",tulatillg the "xistem'C of an elaborate
profn~i()n of di ...1inct, but as yet uHisolated, C'nzymcs. At
thr pn ... C'llt dav our kHowkdtW of hio{'hemical processes
ufJP(ar~t(} ofk~ flO (Itlwr ulternativc. In the meanwhile it
may.. JX'rhaps hdp toward~ un undtrstanding of the realities
hdJind 111(' llUm('~ gin'n to tht,:o.(' hypothetic-al f'Jlzymes jf
Wt' ngnrd Hu' lHnn('~ theml:iel\,("s a~ summarising in a definite
('xpres~ioll th(' ('upaeity of tilt' tissues ('oncerned to bring
nhout. tilt' ~rj\'{'n ('hcmical transformations, without uc(:'Cs~
~arity udopting the vi('w that a distinct enzyme is essential
for Nl('h dint'flout chemical n'aetion or committing ourselves
to ddinitl \'i(_w~ coneerning the m('~hanisms of the tram;
JornHl.tioJl ....
J)eaJing first with the ('xog<-'nous aspect of the lll(>tabolisnl
of thest suh.. tan('('~, one finds that the protein portion of the
nudt'oprot{'in~ in~(~hd ill tht alimentary tract is Tf>movtd
in t ht: ('our~(.~ of gastric' digestion, and by the action of the
pnncfcati{' jui('{', trypsin being mud} Illore eftectiyc t.han
Jx:psin in this I'(>t.pcct. Th('l'C seem., liO reason to doubt that
ttl(' alimentary cnzymes afe capable of completely digesting
tht protein moiety. There is, hOll"e\'cr, no ('ollvincing
",iamce to show that the nucleic acid residue is attacked by
eitlwr the gru,trie or pancreatic enzymes. The exclusion of
pallef('atie juiec fr(J[n the intestine d()(>s not affect the course
of nucleic acid digestion. On the other hand, the succus
l'llierirus and the intt:stinaJ mucosa do contajn enzymes which
effeet a ('ollsiderable disintegration of the nucleic acid mole . .
cuk The results of Levene, of London, and of Jones and
their eo-workers, CQncerning this question may be summarised
as follows: (a) "So digestion or absorption of nucleic acid
occurs in the stomach; (h) Extirpation of the stoma.h Dr the
pancreas does not infiuence nucleic acid digestion; (c) In-
testinal ju;ce (succus. cntmclls) contains a nuckinase which
hydrolyses tetra-nucleotides into 1Il0no-nucleotides, and
nuc1eolidase.. spedfic for the purine nucleotides, which latter
ma~ be hydroJysed ;"to phosphoric acid and Dlwleosides, or
 N.lJOLEO-PROTEIN M1tTAlJOLlS,V 6!1

into purine baS(' Ilnd sugar l'hosphatt'. The pyrimidllw

nudeotides do not appear to h,' aU,,'teti by th" illlt,.tinal
jui('{"; (d) }:xtraeh of the intt..1ilW) mu('o,'ta ('ontain a . . till
wider range of t'nzymc.-;. There is prt..,tut iJf tiWS(' II,
nul'\t'inast', nudeotidases fur hoth purint., and pyrilJlitlint
flue)cotidrs and nurieositio,V/'s ..,:pt'(iH{' for tht' puriH(~ :nud~n
j

.. idas('s which at'(' hydroly~{'d into !-.ugar and baM', Th('

pyrimidine nuckosides appear to rt~~h.t hydrolysb \)oth in the

TAIILE VI
1'h(' Fale of Purincs iujerted Subcuttllft'OUNly ill the .llmda'!1

HP:'i.NNNI
Plaint' Sltf(':!"t'U
X. Tol.a\. J'I'f t '.~ 11 t.
B"~tl\n~. Admin'
H;I~"",.
&(,<'\',
Il!tcreu. AllAn rrlr
win. ;\cid

mil l'JJj! JII': 1II;l

Uric: acid :1" H H 2~ 61
Urit' acid 4H I:! 2:\ 3I', i I
Guanine * 71 3S 2U ,~, 99}I)1')
Xanthine 56 :{;, 2 Ii 54 Ui
Adenint; * 64 Iii :1 :!l :lU 09-76

~~~~~~l~~:I~:t~. : 50
90
2~
:18
3
3 2U
!) 40
(j\l
80
88 96

In tbeso ca.ses t,he urh: add and allantoin IJrodu('t"<i f'aJlta.in but f(1ur~
fiftblf of the orilrinal amino-purllU' nitrc'W'J), antl tht' low('r jj~un' f(lr fH''''
l'~Jltagt! r{"Cov{>ry is based on the a.~ull1ption that thf' I'xtra bat~ an' ammu
p~rines. whih;t the high~r takf'I' 1hpln as IJxy.putinf>lO..

intestine and in the intt~tjnal waU, hehaviour which i~ para-lid

to their resistance to hydroly~is in vitro. It h., thctcfor(",
probable that, as a result of the progressi,," removal of the
prlltein "rom the nucleoprotein, and the subNequent action
of the extra- and intra-ccllul..,. enzymes of the intestinal
mucosa, the products arising from the disintegration of the
nucleic aeid enter the portal circulation in the form of
phosphoric acid, nucleosides (principally pyrimidine Yf &ome
sugar, and guanine and adenine.
 64 THE NUCLEOPROTEINS
Th(' ~uhs('(IU(,lit
fate of these fradions ---and investiga.tiolls
hu,v(' bN'h iurg('Iy (,oIlIiB(~d to th(' purim' ba~('~-~-is W(11 shown
in a M'rit,~ of experiments hy Hunter and Gin'lls, in which
\'uriuus 'rurinc.\ Wert' admini,<o;t('f('d ... uh('utancou. . ly to the
monkey. 'SOnl(, of their results aft' ginn ill Table VI.
on 1'.'6:3.
From thest' rc.\ults we -">('(' that the free purines arp. (,Htn~
holised, ill ~('u(.'raJ, in the' ~am(' way a~ the combincd purines
of lludt'i(' add, giving risc to uric acid and allantoin as final
produets. The T(')fttivdy l!>rnaU{'r amount of nitro,(!cn
recovereo lifter uric Hf'id HdruinistrBtion is interesting, aud
will he J'{f('rrt'd to la-tt'f. The ~('ncraI vil'w ndopt('d at thf'
pr('s{'llt time j_..., that tlw catabolism of hoth ('X0g'<'IlOUS and
f'Hdog'r-!Iou'J nudl'i(' acid produet,., follows idpoti('aJ (,oHrs('&.
TIl(' :.uce('~~ion of cnzymcs r~sponsihk for thrsc fi'lt'taholic
tran~formatlons and th(' changes which ea<.'h on(' brings.
about huy" be'en :-;ummnrisf'd in the schrmr given (Fig. 8)
on p, 65, to whi('h t h(~ reader is referred. Most of the
cnzym('s appear to han' wide di~tributions throughout
allimal tissu(':-t. but sp(~('ics are known in which Ont' or other
of th{' enzymes mf'lltion{'d arC'" larking ill an organ in which
it is known to occur in anotht'r spt'cies. Thus xanthine
oxidas{' appears to be entirely absent fron) t he tissue!; of the
THt., nnd whilst ox livcr will form uric Reid from both adenine
and gun.nint\ pig's Jh'cr wil1 do );,0 only from adenine, rabbit's
liver ollly from guanine, and dog's liv('r froDl neither. On'
of thf' most int~r('sting- caSf'S of diff('.r(>ntial distribution of the
JlUrill<' ellzyme:o; is pro\'jded by the la!'lt stage of the oxidations
shown on p. 65, namely t the conversion of uric acid to
allant.oin which O<'eurs in most. animals prior to excretion.
This further breakdQ\m of uric acid occurs iu all ft1""'W~ls
with the striking exceptions of man and the higher apes.
The oxidation is bro.ught about by the presenre of the
enzyme uricase, found predominantly in the liver, but
""""",onally in the spleen Bnd kidney. The extent to which
uric aeid undergo<-s this final oxidation in various animals
 E,VZYME8 ANII ",r('LE/(' A('IlJ II,.

G4J1'rrf()IIN!.

~.

-r~
_:_$E HY:~:~N
L-----:l lr---J
lUtN~JVI:

UR';1I(,J"..(iJ)+~WWTo'"
FlO. 8.-SchemRtil;' n>prl'.'I(>otaiirl{t flf tilt' {'lltahdillm of lwdl'i(' 4f~}".
The pyrimidine b&fit'.~ art nnt iUl'llJdl-d. 'Ow 'Iw.Vfm'~ whith liN'
re.sponsible fOf the \'Ilri(lu~ Cb31l~f>'i anJ w y, hie}l thf' fll1o.f)f'f>l
refer aTP given in the fHUOu-illg hllt;--
(I) Nucleinase.
(2) Nucleotid8dt' or pholtvhu-nud{:'/t;Je. active in atid 1\t)lllti{,u",
(3) Nw:;lf'otj<1&s~ or purlol'-nuclelu". ad;vt;' in a'ka'ilU'J9i,lutjN~~.
(4) Guanosine-de&roinase
!5~ Adenosine-deamjnase.f
l ~t:r:r:~:.r~;n~~lf
i;:r
n~cC:St:
tbl'Y atw\' cmnpJetely hydrv.
(6) Sugar-phosph.
(7) Nucleosida&es (Guanosine-
__ and A1eno!linf!.bydrolase).

t:l
~~~d~?t=lase.} TypeR of nud~id8.JJt.B.
gf~ ~d::: IDe.mini.ing enzym~.
(12) Xanthine--oxidase.
(3) Uriease, not loond in m.lUl and tbf' high?-f' .apt'8.
6H

j" iudit'akd hy the" uri{'oJyti(' index:' whit'h rtprtwJlh. til('

P<T((Hta,l..,I'{ ratio of allulltol11 llitro,U'en to the ~U1H of alhmtoitl
auti uric' aeid llitro~'t'lI ("x(rt:ted. Hunter alld hi" lo~worktrs
hInt dd.'rmin<'d thi", ifl(kx for !HiW'- aninwl .. , Hlal ~om(' of
uit . .
tluit T{ .. {(plOtt'd h\' Ho.... ,) ar(' ('l);hodled in till' foHowi'ng
table': .

T.'Ill.E YII

l'rh'alytic ludict'S of rarioN,' .lni1lla/x.

,tuillW! Onkr j'rl<'o/\f'(, 111(11':..

()pO"'~U1H MHr~upialiH in
Hut Uodeutia 90
~t(lu..,e ,UK
Cuilwa-pig 94
H"hhit !)ti
(..'ut CarlliY()fH nr
Boa: . . m~
HuJmatian ('oach dog :l2
row {Tngulatu na
Jlor;'.<' liS
Sheep HO
(;out H2
].)i~ !I~
J<:kphllllt Pr{)~~<'idf'a 72
)Ionk(y . Primates 89
Chimpimz('(' ()
)It'Il 0

~\pnrt from t he unique position of man and the chimpanzc,

ill the abore list, tb~ very low index of the Dalmatian road
dog- i~ not'\vorthy. This curious metabolic dis('tlv&TY.. ~Y8
lirst lllade by Benedict, wbo showed that on a purine-fre
diet this h""'d of dOjr excl'('tes about as much uric acid.
man. l?ricasc is pri>'sent in the animal\; liYt'r. Onslow. in a
int,,;,osting ",ries of breeding experiments in which D.
matians were crossed 'with other dogs, found that the cross
 l'RI(,OLrsiR 67
~)uH\'('d Uw same high urit'olytit' indt,x as t lit' nt ht.r dog....
TIH.' l)('('uliur I)uimnti1tlt ellHl'adt'ri",tl(' wu.., lo ...t. UIHt tlwn
{Ol"('th(' {H(...II("(' of ur1('a ...(' ill ... tlt'h IUHOUtlb fwd udl\'ity H~
to ('onH'r1 tHo... t of til(' mil' It(,lll h. ulluntoin i, It }It'IHh'hHH
dtllltimmt ('harada. In a ...ln( ... of in\ ('... tig'atHI . . of tht,
purinc' nataholi . . m nftht, Dalmutwll dot.!. 1-lt'lwdil,t fOlllM thut
thYlIlu ... Ji:.'('dillg' pnx.hu'(d 11 Illuch grraft'r ilwn'HM' in thl'
lWIOlwt of Jllhllltoill {'xndt'~1 thHlIl)f uric' nelt!. i.t'. th(' ,'nlue
of tIl(' urieolytic illlh'x wa.., (oll'.. idt'rahly in('rt'u"t'i! nho\'l' it...
nnrmal \'aitH'. On ndrllitlj,t('till~ a )"ulwutlt1lt'OUl'oo llljntioll
of uric lwid lIJ.!nill. u mark{'d illl'f('a't' ill til(' ('x('1'dion of
allantoin w<I"'lIokd d<'."pik the flid that the uric acid tfIj('C'h'd
WI\'.. r('('0\'4'1'('(\ ulI('hun),!NI ill pructieHUy qunnlilatin' mH(lUnt.
Hett'fe'flee til til<' r(,,,"It.. of Huuter and Gh"l'th 01\ p. (i!I
will shOi\' thut til(' illj(_'dioll or uri<' a('irl produ('ed ill tlw
.monk(y hut a ~light inert'liS(' in ulfantoin and II murk(d
iU(,T"U ...C ill IlrJc twid t'x('rf'tioll, ]tJHlugh flU' HHmkf')' Iw ... a
hi~h lIri('olyti(' ilHhx.T1l(' (xp<,dati(lll would nntllwl1y he
that mn..t of tlw urit It{'id :-.hould h( oxidi~l,d to alhmtuil1.
'l'hl'''>e anoma)ou.., r('~ulb IllHy lM.: iutC'rprt!td f'ith('r in til('
M:n~(' tlHJt different mdaholic pltth~ {'xj~t for ('udo..,l'(uou", 1111<1
('xo,i!{'nou"i purine ...> or that the protfudion of uri(' (wid or
allantoin r(' intt'r-felat{'ti in unknown wuy.... The huJanC'{'
of ('videll(,c i ... again~t the former alternatin', a, "'t' hun' !'r('PII,
aHd at pr<. ...cut onc ('IUl hut rt'eord t 1\('",( oh~ernt.tions "'"
inciieating fldd ..., in which furt!}('r jnn-!'>t;g,Utiow. aTC r('{!uir('d.

The SJnthesis of Purines in Animal Orgaoiols

Abundant evidel1ce (-'xbt:-, to ,how that t h(~ animal urgani",m
is cRpabk of ~ynthc!-;j;ing' purines, and ultimab:'}Y llud(>jc
I1<'id apdonudeo-proteins_ We .hall see in a lat .. r chapter that
the-disappearance of part of the ph""pho-protdn phosphoru,
in the incubating hen's egg i~ a.\j~()ci3ted with a formation of
nucleo-protein_ The n"",,"sary purines do not ,-xist preformt-d
in the unineubated egg, but they appear so<m after illcullatioll
has eommenced and progrcSbh-e1y increase during the period
,.!
fl;; 1'lJE X['('LEO-/,R01'EINS
of in('uhatioJl. During till' rnigratioH of tht' ~ahnoll to ih
... pnwlIitlg ground.". a p(riotl in it-; lift, cyd(' wlwil it uhst.Hin.",
from food, f('}atin'ly f"J)orm01I<" HIlJOunt..., of nul'l.j(> llc:itl }If('
j'ornlt'd ii...th(' !-.permat07.nH, til(' material for thi .. "ynt}w..,i ...
Hrilo.inf,( frotH til(' di!-.intt'L!ratioH of otlwr ti!o.~lH.'~. notahlv ttl("
tllu~('l~' fi ....... \U. That thi~ d(}e~ IIH'OI\(' a s~nth('~i ... of p\~ritw:-.
i .. pro\,('({ hy t}H' r<'intivdy !->mali amounts of prefornud
purim'''' prl'~('flt in the Itlll"'(,)("
{'an'fully ('ontroll(>d ('xpcrimcnb ott rab, rabhit..., pllP~,
dog's, and ntl nduH llIan (onelusivdy pro\'(' that these
titlinwl ... nrc iw.kpt'ntknt of (I .... nufct' of prdornud pllrill('~.
III one ('xf)t'rimrut Benedict k{'pt R l>nlmatian dog (1) .a
purint'-frc>{' diet for H yc'ar1 and during' thi~ period it ['xc'TC'h'd
more than 100 gram~ of uri(' a('id, of whic'h it wa~ t'!<.tinlated .
t hat not lnore than 1() })(,T ('(nt. ('QuId ari:-w from .til(' prc-
formcd purin('s of the anlmar~ tis~u{'~. An int('re~ting'
illw..tration of th(' ahility of tht' animal to !".,ynthcsis(, purilll'~
is provided hy tlw bird!<. and f('ptil('s. In these animab the
lar~r('r ptui of th(" une a.dd ex-en'ted f{'prc:.,ellt~ not purin('
mctuholh';lH t but the normal {'xo::..rc110U.., protf'in metaholi",m.
l ~ri(' aeid tak('."i the place of urea as the end-product of
exogel)ou!'., nitrogrn mctahoJi~m. T}w fart that the uric acid
molt'pull' may \X' rt'g8rdt~d Ii!'; l)('ing ('omposcrl of two ureA
mol('('uit-s )inked to_g('thC'r hy a chain of thrt'e (arhon ntorrlS
It,tHis support to the vi('w that in the hird and reptile uric
u('id may be syntheslscd from Ur("a. In any (~ase the ability
of tl\('S( Hnimals to sehi('yc thc r;;yntll{'~is from non~purinc
llitro~en ('ompounds is undoubted. Sugg{'sth"(': experiment!:;
hy Aekroyd and Hopkins point to both histidine and arginine
being possible and indcpt'ndent sources of purines in the
mmnmal. A I'ff'('nt reinvestigation of this ques.tiollby R~'re
Rnd Cox gay!' re~ults in support of this ,"jew so far as histidine
was roncerned, but they were unable to find that normal
I!rowth in mts followed the administration of arginine alone
to n tliet otherwise sufficient, but devoid of histidine, The
failure of gro\\1h on 8 diet lacking histidine might not have
t
 peRINE 8D'TllES1S 61l

h('{'u dUf' to the ab~('twt of n >;uitahl(' ,nlll'{'{' of purim, for

Ilu(Jde ~I('id :-.ynthf ... i .... Iftdtf(l, the tllH' ... tiofl oft", f(lrnutthm
of purin('~ from ('ith('r of thh,C two Mniuo uehf... ruu~t "tiIJ tw
rt~~lTd(d a .. 0lwtl. Jkc('ut t"xpl"riHleuh hy Skun.~ iu w)Ji,.h
",uTvi\'ing ('x('i"l,d ('ah' Jivt,f'> \\'('n' I)f'rfu~ II ",,'HII u"rntt'd
HiH:!er'!'o ,olutioll ('()llhlininJ! hi"tichH(' or ar~nitii'. fnik(1 to
dt'IHollstrate llny formatiotl of ullulltoin, th., lIormai (,lid
prOtlHf't of purine oxidation til tlil' (ut.

I. A('RHO)"l) alltl UnpKt!-.~. Hi(if'hnn . .!(.urn . IOHI, to. ;1:;1.

0' ~'}o;FLm.:'S IUIlI ('o.workf'l'lo\. Zri(. /Jh_(/Ilio/, f'hfm. IU22. 123.
1117; 1{124, 135. 2a:~. 240; 186. 57.
3. HAM:';IAR;.i'f.t~~. nWl'html. Zeft., 1024. 144. !lM3.
4. II('}:f'MA~!\, .fount. ];iol. t 'hem .. IU25. 63. 075,
5. lltsflm Ilnd (;1\1~.'\t'. .}(Hlm, Jlinl, t 'h~ . W14. 17. :li.
6, .T()x.E~. "~ut'l(>i(: Acids:' J,o()luiulI: Lon~nui.u!ii. ('rt.,.,rt & CII"
2nu Edition. I02n.
7. last-;k ml(l }'f;RKI~~ . !flIJrn. Bioi. ('kft1t" lllt:l, 55, !i!J";, ,it}';;
Hl24, 62. 291: IH2:i, 62. 5;"7.
~,.lnRl};~. Bio('"hrm, Zeit,. 1024.151.227.
H. LK\"E~};' .JoUTfL lJifll. (:kern., IO!!I. 48, J ttl. In; JU2:? 53.
HI.
10. L'EVE!liE and .1M'oMs. Journ. Biol. ('h~. Hfl2. 12. :\77. 411.
11. l..:EVEXB and ~HUfS. Journ. BM. {'hem., 1925. 65. M~I.
12. Ll?VEN}~ and SOBOTKA. Jowrn. Hiol, ('hNn . 102.1. 65, iiiH.
13. ONSLOW. Bio('hem ./OtJffl., 1"23. 17, 334, 564.
14. nOSE. ., }'uriIw lJetahoJism.' PhytlilJl, Rt.'l'1.tU'S, t9t;l. 3 .544.
15. Ros}~ atHl Cox . Joum. BioI. ('hem,. 11124,61. i47; lH26.68,
217.
J6. SXEIInEL. Zeit. ph,tp,iol, ('JUnt., }1)08, .55. 407; 56, 2J2.
17. STE].;})}':1. and P.El~ER. Zeit. physWl. ('hl'm., IU24, 132. 207.
18. ~TliWAH.T. lliochem. ,IOtan. 1925~ 19. 266.
19. TASNllAVS};n alld PoADffo!. ZlJ.it. phYNiol. t'Jt-em . J!:J20,21. tOO.
lSi.
 THE CARBOHYDRATES

Tm;>; (\\"crngf' mixed did of man ('on:-'lsts of ..,OnIC' .')0 to

GO JWf ('('lit. of ... tnrefwf. and <;ngnrs, and it i~ C':-..tinwtt"d that
of tlw pro1(.im. sotn(' GO per rent., um! of til{' fat ... approxi-
lHatd~' 10 P('f (,('lit. (tlw glY('(,fol), lt1'{' tran"fol'tn('d intn sugar
ill HI{' ('O\lr~t of normai lH(\taholism. It i-.; tlwfefofr tiot'
~ltrprisillg to fmrl that th( hulk of man \. ('n('rgy f('(luireln{'uts
afl' llwt hy tJw oxidHtion of {'arbohyrlrate. Thi~ oxidatiof)f

to it great e:\.teut in musl'le tissues, where it is probable

{)('('UN
thnt th(' onlv SU~Hr directh' oxidist:'d is d-ghICOS{'. The
metabolism ,;f
tl;~ carbohydrat~ b tlwreforc p;aCtiectlly
that of gluco.s<:>, and the eas{_' and extent of transformation
of th.. other sugars il;to glucose is a measul't' of the extent
to wQich they arc likely to be utilised in the course of nOl'lllal
lUetaboll,ru. Thus sugars which most dosely resemble
gluc,,"se in their stereochemieal configuration are ruore readily.
10
 Sl'ReC7TRE ANV F7'/U};ATlOX 71
ntjlis<.,d hy the' JlUIBUHHlinH nfJ!uni .. m Uum art' ,uM'urs lvhwll
d(,PMrt mort' markedly from t hi ... (ollfij!uratloH. 11\1 lu.' thrnwt
ehh'" we han frudo,('. the kdO,t, nnulogtu uf ,:lltH'HM'; Hml
u:tlnno,('. HIl: cpilw'r, (If _!!h'('-Oo,t\ ill lrhi(h til(' !'otc~.J("h('ftli(,lil
cOHJiguratioll of tlw :'C1l011 :.rr(mp mijU{x).llt to Uw h'l'Juilutl
('arhon atom i . . Tt'\'erM'd as !\hown in t }u uC'(,{lmpoHyilll!
i;>rmllla' (I', 70),
In t he ('aM'~ or gnlndoM' al1d ...orho,(' wr hu\'(' "UJ,fBf"i whidl
differ from g-lu('(~(' in a ~tcr('o("ll(lllli('uJ :',('11-.(' HJore nlt\rk{(n~
than do mtltH\o..;(' Hnd frudo ...t'. Tht tt'\'('r...nl of the ... t(l'f'I)~
('}U"JUj('~ll conliguTiltion of ('arhvJI aft/II) .. ..J. ;wd ;i ;:0, :1 mUl'h
mort' (iiifi('uft ('hellll('at nnd hi()(twmieaI prohklll tfllm I!'o, t iw
('orf('~polldillg f('\'('r-..al {)f ('urbull atoll:\ 2, t 111' proximity of
the latt.r to HI(' rcdtwiflg' C'Hrhon atofH douhtk, .. }wing Ow
ra(,tor deh'Tmining Ow clifrrr{'tl('f._" ill tilt ald(lM~. Thu~ it i . .
douhtful jf galacto...e i .... utili ..,.ed at nB in ttl{' ",,"nSf' of ))f'jug'
oxidiscd, alth{)u~h it i" OIl(' of the ('on..;tihWllt h('xuws of t ht,
mammalihll mi1k.-~ugar hwtos{' and i" pt't~~('nt in tlw {'('f<'"
hro:,:idcs, whi('h O('('ur in }urg-<' ..lmuunt itt hrrull ti:'l."u('. It j,
lle\('rthcles~ probable that the galatto'i( in the~(' liour('("'i i~
formed .at }ca..,.t in part by a syntiwti{! pr(l(~(>1'o~, and i ... hot
deriycd wholly from pnfornwd inges1:ed gaia<'tost'. It i",
furth("r stah'd that la(to"'t', when ftd to diahetit"l'o. may lw
r{'(>(}l-ered ill the urine 81\ 2 mO}f'cu}('S of tl-glm:o"t(-' for ('l}eh
molecule of lactose f('d. Thl'se obsu\'atinn!-. would l'wnn to
jndieait" that the ti.<.;.... ut\.. can Undf."f (.'CrtalB (,onditiou ... traw'l'
form ~Iucose into galaeh)hc, and galactoM' iflto uht(o~('.
Such a change is very interesting from n st{'J'{'o('lwmi('al poiHt
of view and, so far as one can judgt at pr('s(~J)t, it mu.,t
i'l..rnYe" s('i",ion of the oril,rynal hexose int<> two threeearb,,,,
nnits. La'!uer and )leyer have demonstrated in thdr ,llldic'
on the metabolism of carbohydrate in musel,' that, wbiM
llWtic acid is readily formed from glycogen, starch, glu""",'.
fructose. and man"""". it i. not formed in significant .,,"uunts
$rom galactose and sorbose. The following tahle. taken from

72 THE CARBOHYDRATES
tllt'ir pap!'r, ,hows th,>,, n>,ults together with ,orne others of
intert'st :-,-
TAD!.E YII!
Forumlim> '~f La('/il' Arid from l'flrimlN ('(Jrb()h!1dral{',~' by' a
1"!llp '!f Prof.! Skeldal .\Iu},('/t' at iJl). SIl,[!ar Cmt('('lltrati(HI
~ }ier (,,,,If, p~ 7':$.

Cjl.rrn)hydn~u' !\f), of EXI~('''s Formation of Ladie Acid on'r

Addt-tl. I':xpb. ('ontrol!'l. (~1D. ~'r 100 ~Ill. nm ..(')t'.

LoWt,,,C Hi!o!he~t. Av('ragt'.

(;tm'os(' 25 noos -, O'S2H + O':-JH5
Fruetos(' () O-:l~2 -+- O'fi~7 +(J-.lM
~t:l.tuIOS(' -I + ()'17.~ -+ (H195 .,..0827
Galaeto~t + --(HIOl TO'030 +OOH
Sorbo~e 2 ~ O(n:l -+ 0-062 + 0,024
Sucrose. ~ ~ 0080 t ()-(J64 + 0017
~Ili.ltos(' X "_ (H)55 -'- (I-lOS ... O'O2{1
DillHlV}O.,\t' fl 0'(0); ,(HIIlS - 0'004
Tl'tralllylo. . c 8 c (l-()()(> 0'077 -+- 0'035
Inulin 2 0-095 t (Hllll -~ 0'032
Stllrch + (l'1l5 .j 0772 .j O.J,fl11
GlycUg:Cll 23 + U'S50 +- ]'(HS + 0-707

The PentoseB
The lX-lllo,,'s ami methyl I)(>nto.,,,s do not appear to he
mt~hlb()ljsed in tht' majority of mammals in appreciable
amonnts. In the lwrbivorous nnimals) however~ which ingest
a larw amount of ponto'"IIS (p"ntose polysaccharides) in
their foodstuff. a considerable absorption of pentose takes
place, The ,ubsequent fate of this pe)ltose is obscure. It
has been claimed that arubinose, xylose, and rharuno'se; when
fed to star\ing rabbits, are transformed into glycogen.
Substantiation of these results in recent years appears to be
lacking, and it is obvious from tbe formul., given below that
such I. transformation ill the ill"tanCt'S of xylose and rham-
nose inyo!ves a considerable stert'OChe'mical te8lTangement
 PENT08ES 73
of the molecule. If such gly~own forlllutioll dOt, hIke "In"...
onr would thf'refoN' .,,>uggf'st thnt it h. hy wuy of dhdntr#.."f's"
tion product,; of two ot thrt'f' ('arbon utmu., ruUwr thall hy
II direct ronw'rsioH of Uw iK'lIt,o'\(' to l\('x(J~(~. l"-has ulso
}x;'n stated that diabeti( ... ('Xf'rt't(' Uhll'OM' wtwfl I)('ntn)o( ... "n~
administ<-I'<'d to th<'Hl, hut in relution to tht"..t, duim", itilllllll!o.t
Ix hOfl1(' in mind that tilt' admini!-.trutiu)I or Inr_u:p quulltitie'i
of }WlltoS(' to dialwtiC'!-. ha~ }\ ~tiBlulutill).! dfN,t on tlwir
protein {'ataholisrn, Hnd the suppo~{'d formation or ghH'OS('
might he dm> to this indir('('t prOf'('S",.

CHOH CROH CHOH CHOH

HOf~ H~~ Hf~ HO~~

Bcoll 0 RCOH 0
HO~~
HOC H 0

H{~ B~~ a{o/ RCO

I
eHz eH z eHt He
I
eH,
I\IWI'~ 1U1!05. ~ _ND3I:.

In general, it may lx" said that thf.' r)('utos('~ Hfl' far Its~
readily utilised in the animal or~rani~m than arC' the h('XOMS.
and after administration to the animal th,'y ate lurgdy
excreted unchanwd. One might haVl' anticipat<'d that th,
conversion of a pentose ~uch as arahillO~(' into a hc'xtv...(, ;Io,u(~h
as glucose would be " comparativdy simpi, bi(lloJ..~c.1
problem. The stereochemical configuration of thf' two
sugj.l'S ts'\>"ery similar, and th!: change is ('ofnparutivf'ly easy
to achieve in vitro. But the form of arabillOM' wbieh dot',
oceur in nature is de.rtrO~rot8tory, whereas tht: arllhino\lt'-
derived from glucose ofthe natural or deJ1rorotntory ,'ariet)',
by a simple degradation process such Ill< Weerman \, plov,"
to be t_o-rolatory. It is, therefore, the optical enantion~orph
74 7'HE CARBOHY.DRA.1'E8
Clf th(' lll\turul form of nrHhiTH)S('. Thi< faf't s('rn's to
('mph.a;o.,is(' the llIl('Xpc,(,tt-d dim(>lllti(;~ OIlt' N\('()Unt('~ ill
attempting- to \'iew the biological orihrin [Ifld twhHdour of
Uw P('ui'~i(':\ and Iwxo..,(':\ from a ('Ofwnoll .. tandpoiat. On
the OOl('f hand, one f('eis fRirh' ('onfident in tilt pT{.di('ti~n
Uwt ill HI(' phlnt the {)(nto.... e 'i .. d(~ri'('d from a hf'x()s{' hy
!.U1J)(' pro('(' ..... of fiq..,l'J"a<1ation. In tht, animal. jf the p('ntoses
11rt' m(tholi';ed at HIl, it would ~t'('m to he hy m('thods qllit('
difff'Tl'nt from thO. . i 'whiph app1y to thf' }tc'XOSf'S. ]t is indc('d
. . . urpri"iJlg' that th,' onl~' pf'nto\c normally oN'ufring in fl1limnl
tiS"lH', is d-riho~(... which i:-. found in the so-(,:-tJkd ,B-IllH'lNI-
protrhl'>, nJrparfy referred to in ChHptcr III. Th(' {onfigura
tion of riho-.;( rt'lutf's it to the rat(, hexose ~ugars dsUnsl' and
dultrn<.:;(", whi('h so far haw' only \)('('11 ohtaincd hy artifiC'iul
m('nll~ UHd aft' not knOWh to ()('C'\lr ill nature, '!'hey were
ohtnilled by Lc\'('l\c and .ra(ohs on applying KiliaHi'<
t'YHuohydrili ~YIlt'}('~is to rihos('.
In the farl: c()udition known as pentosuria tlwrc is a ('Orl-
tillllt'd l'x('rdion of a pento~e in the urin(>, Thj~ patholugical
~tl:.lt(' hu')<" not bC('tl ('orrelat('d \rith any other ~ignifieant
mduholi(' disturbanc(" and is not ass.ociated with diabet{'s,
ltlthoug-h the ('onditiotl ha", usunUy heen brought to light
through 8 faulty dwgnosis of diahctf's. The '[X'ntose ha~
Jiot yf't hN'l! satisfactoriJy identified. The foHvwin,g- li ... t
~unl111ari ...es the opinions of \'uriou~ investigators of t hi ...
qlll'stioH :----
S.lkowski (1899) . dl~arahiJ}ose.
:-;ellbt'rg (19Q()) . dl-arabinose~
Blumenthal (1908) drotatory pentose.
Adler (1905) Irotatory pentase.
LlIzatto (1905) I-arabinose. t
lllum (UlO6) i-rotatory pento,'e.
Ro."nfeld (1906) . d-rotatory pent",.
Schul.. (1910) . d-rotatory pent""".
Elliot lind Raper (1912) dl-ribose.
f\ro" (1913) . . dl-arabinost'.
Zemel' and WaltlK'h (1913) . d-xyloketost'.
].evenc and La Forge (1918) . i-ribose.
 STARI'Il 70
lA-veil, and La FOf.!:!( (l m ~) . XV\Ukt'tUSf'.
Hillier (l!lJ7) d:;s;v)i)kttww.
\\rz(,~lww ... ki (lH:!~) ,ll~t;ra)}illl)':of"

It would from t lw nhuH' Ii ... t t hut it i .. hy ';0

:,,('('J)} hU'tHJ,,"
in~ptohHhl(' that cliUerl'ut typt'" of Ik'l1to..;urin t'xi~~.

The Relation of the Structure of CarbohYdrates to

their Biochemical Transformations
Th(' hUlk of m.w:-, (,aroohydrtttr intake i~ in the forln of
stardl, t}l(' amount of fT(,(' mono- HIHI di~~u('('hurid('s inU...,ktl
til fond ... turt-... h<'illt,!' r(>lativd.r lll'.. iglliti('ant. IkfoN' lih .. "rp ..
tinn ~tur('h j.., eOlllpktcfy hy<irl1ly,,(d to gl\l(~o,(> hy 1 lit
!-.lI((( ....... i\'( adioJh of JlllJylw'I{'" (dja':>ta"i( ... ) Hilt] HJ:J)1n",', and
that port_ioll of the I!liH'o,,(' whivh i ... Hot jhITll(diat(I~ utili..,N{
l\ stor(d a ... 1,!1ycog'cH. prilldpnl1y ill til<' li\('r alld fHtl'..d(",
Tht' serie" of rlaf'tiOll . . r('j)r(''tcntinl{ thi ... (OJIHr . . ioH, mum')Y,
Starch ~---. (;}U('o'-)(' ~- ~ Glyt'ogI'Jl.
".. hi('h play:-. all important purt if! til(' utili ... atioll of ('arho-
hydrates ill the {Jr~anj'lm, l"Hi . . .t" th(' que.,tiOfJ of Hit' rdatiun-
!o.hip of !-.tarch to the animal lx,ly:-.att'hllril<' glyc()$.t\'n.
R('('('nt il\\'{':-.tigation:-. of tlw f'on!>.titutioll of :-.budl indit'atc
that it i~ prohubly {t polYl1lcri ...ed lwhydl"o~tri- or ll('xu-
saf'charidt'. The vip\\, that t h(' {,~""('I\tiaf unit of stnf(,h j", all
anhydTo-trj~H('rharjd(' or triamyln...,('. in which two of tht"
nulty-uro Jilikings ar(' of the c"aIT!(' type and th< third (If
another typ('. is favoured by lrvio<' and hy Prinj!sht'ftn,
Ling amI Xanji, taking (oglli..,aJH.'t" of the (':\_;",h'Jif'{' within tIl('
starch granule of amylop(",tin and polyn)("ri~td amylol'i(" two
di~tind ~lySA{'('haride!-; Of'l'urring in the ratio 1 : 2, qu("~tio1\
coftaiIf of Ir\'in<''''' rondusions, Amyloptctin. \\"h;(')1 ('ontain't
a !>.mall amount of I)robubly eombilwd phosphuric a{'id. fortu ...
the outer layers of the starch ,{.,rranul(>s, whibt amylo~t j"
found in their interior. Both polys8L<charidet; ~in' ri-.c to
the same chief constituent, glu('ose, on acid hydroly!>;;~. hut
they re,,{'al difr"re,w.'S when they are subjected 10 enly",ic
 78 THE CARBOHYDRATES
hydroly,i,. Thu" wll("n ,t.nh pastp is t,,ated at 50' with
p,r('('ipitatc'd bath'Y rlia:-:.tn~(' whic'h has hetn driNI by treat
nlellt with strohg: alcohol, it is ('ou\'('rhd pru(tically quauti~
tath'(ly.,s> maltose (g)uco.'oc a-g}u('o,',id(') jn uhout tW('!YC'
ht,Uf"l. whibt t hl' HTllylolwetill i~ merely d('polynwrised to an
unhy;-lro.lwxaS8C'(haridt. TIl(' maltosc< may lx' f(>mov(:d hy
dialy... i. . nad t}lf' unclulIJj..rt'd SlmyloJX'ttin tlwil pl't'cipitntf'ti hy
nkohol. ~\Hlylop{'etill on ).,uitahle {'nzymir hydroIY!,j)., yif'Id~
mHlto~(' and iso-filultosc' (g-lucose /3-glu('()side). Ling and
!\'Huji su~g(..,t thnt the poJynwris.('{1 unit of the amylose of
~tnr('h i~ a-iwxa-amylo!->(,. i.e., an ullhydro-Iwxasatcharide in

CO~TliuE.NTS
UNIT 0" ..... MyL.O.$~ U,,",'T OF AM'fl.OPf.C.T11-.1
OF
(a - Hf!KC.-a.myto3e) (a.p~ ~.w.o..-o.rnyIO$e)
5TARtC ....
Fro. 9.

whit'h the glucosidic linkings art' of the a typt" Thi~

hypothesis rPadily explains the occurrence of tetra and di-
amyloses in starPh hydrolysates by a process of further
Oe{m.,meri5l<tian. .For amy}opeetin the con,titution SWf(-
gested hy Ling and Xanji for the polymerised unit is an
ap-hexa-amylose, in which two carbinol hydroxyl groups
.dia",nt to f3 Iinkings arc esterified with phosphoric acid.
The necessity for supposing the prespnce of the /llinJ.;ng
depends on the production of iso-maltose on hydrolysis.
Four of the linkings ))(,tween the constituent hexose units
are suppo,.oo to be of thi, type, whilst the otber twu are
presuri,ed to be of the a tn"'. This latter hypothesis con-
.
cerlling the number of each type of linking accommodates
 A.MYI.JJSE AND .UfrLOl'E("l'IS "
the fa('t that. hv tht., RC'tion of mnlt cHa:-.tll)o,(' nt 70" on Hl1wl\1-
f)(~('tjn. the ~ol;' product ohtaillCO Ulul('r ((rtt.il1 ('onditiuu;\
i~ the triS8{'charid(' ,B-~Iu(o:..ido-Illuftos('. Til('~(' )oul:!J{(~t\d
... tr.udu.t'('~ of til(' lluih of ulllylov ami amyloJ)({'til;.M' sturth
arc .. hOWH in the' ditl.:,!rnms in Fig. 9.
Th(' lin{'~ forming the' ~jd('~ of tht' Il('xt\g-on~ rqn'('sf'n' tlw
illdh'idual hexos('s, the Idtt't It illtil('ating: tht, rc:'{\\1('I,,#o(
end of tht' molt'('uIe, The type nf linkaw' bet W(','n NH'h
Iwxo:-.e uBit is ~hown hy tilt' kt h'r~ f! or fl. TIt(' dotttd lint's
rUllning a('ros'i tlw fi~urc I'(pr(' ..t_utin;.! Ow Hmylop<{tln unit
... how hm,'\. ... ('j.,sion ofthi ... unit may ()('('ur withthp pN)(hution
of ,B-glu('o!-.i<io-maito't( only. The followjn~ 'i('tWIIU' j ... gi\'t'li
hy l.inA' aud !\unji a~ rq>re ...ent in_!.! tilt' ('OU~(' of t'H1Ylllit.~
hydToly~is of amylop{'('tin uJlder variou ... (ondition ... :-.
 7s TUR ('ARBOIlYDRATRS
III \'ariolt~ :-.ttlr('h( .... notably tho~c of hariey. wht.at, ril.("
:-.ago, fliui{.(.', and t;lpio(,ll. Sdlryn'r hth <li",(,o\"('1'(,<1 a IWTIlj
('dlulo~(' ill which n certain nl1HHwt of .,ilicH i~ prc..,('llt in
org'lluj(.~'omhjJwtjon. Thi ... di"'('o\'('r~' further f"ompJi{ut( ...
tilt' c}\('tlfi"try of ... tar('h. The iwmi('('llulo,",(' gi\(: ... a h1ul'
iorlilf(' r('lwtioll, aw.l InH~' th('f('forl' h(, ('ollfuv'd with ...tan'h.
ft b nut, hO\\"l'YI'r, Iwh,d 011 by tlu' din:-.t:'ht' of ung('t'tnillah'd
hurley ("'('ll on I'ro]onW'd incuhatioll at 50", "\'}Wf('::l!-' tll(,
othf'r two ('on ... tituf'nh of ~htr('h~ tht' amy}o\(' emil flu' amy]o~
JwdilJ, Hn' hyilro),\-M'd or lltpolynlt'ri . . {d hy thi~ ('nz~'m{' ullder
thest' ('onditioll:-'. The Il('llIiedlulo..,(' i .. hy<lroly:-.pd hy the
din",tn:-.C' of '!'{('rmillated hari<'y.
TIll' forqroiIlg- (lh~(,f\':tti()n~ srrw' to ~h(lw how ('o01pks the
bioch('mi('ul r('actj(lll~ of ~tur(>h ma.y he, alld Ul('r(' i~ 110
T('a~OIl to SUppO,'i{~ thut the hdwriour of g)y(,o~'n jn the
lluimaJ body i . . llHlit'rially ..,jmp}pr, Hlthollgh IIp to tll!' prc!-.elV"
t ilH(, the d(tHikd stnl('tllre of ,l!1.\'(og(,H hIt-. not hC{~1l :-;0
l'xtellsin'ly illYt. ...tigHh,d as ha ... that of :-.tHf('h. GlyeoW'1\
dos('Jy fl"semhle.., ~tan'h ill heiJl~ a p(_)ly~a('('harid(' of the
i'{,rmula (C 6 H 10 0:;),., in whieh {,H.('h eEl uuit (,Hrrl(,," thrce
hydroxyl ~lTOUpS. Marbdh fHld :'\hwkay han' su('c,(,t'dcd in
prt'pflring- a mrthylo-.uly(o.~n in which the m('t.hoxyl ('ontent
i ... within 5 pt'r (ellt. of thC' th{'oreticui value for th(, t~om
plt'tdy lllethylated suhstal\((. The ditli{'ulty in dfecting
(ompkt<' ~ubstitution (If the hydro:xyl~ hy methoxyl is
probahly phy,iml. 011 hydrolysis of this partly Illf'thybted
f.!JYl{'\~lf')}, m)[' .pf t);w pl'oduct_~ .vbiRjnf'd pJ'J.n:l'o tD bt~ jJ t..rj
methyl glue",,, ideuti"al with that obtained from methylated
{'('Ihll(),..,c, stardl, lactose, and N"IIobiosc, a disaccharide
pr"lmred from eellulose. The residue consist",i d,;.fly of a
dimethyl glucose simillll' to that obtained from .t'irch by
In-iuc and his co-workers. Tht're are, Uwrt'fore, good
grounds for lX"licYing -that starc.'h and glycogf'll arc built up
on closely similar lines. There is as yet no evidence that the
poly~haride giycog"11 is related to or built up from glucose
units of the abnormal or reactiw type, the so-called y type.
 7H

In Tet'('nt yt'ar~ a g'OOt.:i d(,1l1 of SIK't'uh,tioll ha., 1M:,t'1\ (kvotnf

to the possihl(' rtJle of r('tl('tiH' stl!!Hr~ ill 1)iol'llt'lHi ...1rv rut it
~hort rbra/tc; of the po~itioll j" gin'lI h(,tt" ' l
lkadin or y ... ng:aN were lif)..t tli"'('O\'('rt,d ill t I~' fortH oi
tlir g-hl{'o,,,idi{' dN;vati,'c~ hy Fj..,('h('r. ntld ... Jlc:rth' Hfter~
ward ... hy Irvin{' mul hi ... eo~work('n.., in IHIl. TIll' tlif(~rtIH't
bc-twc('U t}J(,,'l' f('::I(,tin' ~ug(lr." and Un' normal "'U_t.."llr-. i ..
n",(ribtd to fl di!'>pla(,(,llll'nt oftht' oxygell hrid)!t,. , .... hid, link,
the tt'rlninn] TnitU'lng parholl ntOJn to lllJotllt'r f'urhon ntom.
from it~ position in th(' nornud :-.tublt,. 'i\!_gar.... {'p fill "~tory
f({'(ntly it has h{'('n a~~unH'<l t hut t lw llXY,l.tt'lI hrid_L.ft' pr('';{'nt
ill til(' stahle ..,ugal'. wa~ of the hut,dell(' oxidi,' t~,)(> Hwt '''',
that it link(_'(l {'urboll <ltollh 1 Hlul ,l a .. ",howl) hd(Jw l:kHnih

1... t:HOH
I ........._
CHOB co
l ........... CHOHl
f~ b~
I 0
I 0
3 .......... ~~
~~
CHOH 0
4. .. ..CH
I
{H~ CH
I
.5 .... -CHOH CH CHOH
I I I
6 ......... CH'OH elItOR ClltOH
BUTVLNE OXIDIC AMYLENf. 0)\11)1(. 5UTVLHf. OXlDIC
SU6AR -WGAJt HOIlOCi\lUlOlIYUC AClD
LACTONE.

evidence in fav(}ur of thi~ type of linkillg ww" la("kin~. and the

as..~umptjQn was made bec8U~{, it '\'as thought to }w u \'('ry
stahle .aWomic j.!rouping, and beeR'''' IIud.on ha.d brought
f";'~d unquestioned e,idenc. that th,. lad(>1I(" of tb. mon-
carboxylic acids derived from the sugars on oxidation ('xi~ted
in the 1 : 4 form, i.e., as rlactou(~~ ~\.., a l'C!o.ult of r('{'c'nt
work carried out bv Hirst, Haworth, L('v{'uc, the a.thor.
and their co~worke;s~ it ha~ l.x--cn possibl(' to demonstrate

 so TIll<; ('A RIIOlI Y [)Ro4 1'ES
thut in aH tilt' ald()~Il('xoM'''' ami ahlo-p<'lIto")('S so far
(~Xalllillt'd -and th"'t, in('lud(' t,.riuco:-;('. mannose, galactosf",
xylos{', Hraoino...,e, and rharnnos(' "-tIl(' stnhJ(' ...tnJ<_1:urt' is that
in whidi. tilt' OX\'W'I\ hridgf' links ('arhon atom" 1 and .f), so
tlmt ttw l;lIt~'kl1~(' (Ixidi(' 'itru('tuff' hith{'rto ltssig1H'd to th;sc
... uunr" Hl1ht HOW he f('plu('f'd hy an aruylelw oXldi(' :-.trurturc.

It has further beeu shown in these reeent studies that the

y or rendive form of the a1doses is none other \oon .t.he
hutylelle oxidit form whieh has for years been ascribed to
the stahle sugar. We see, therefore, that the type of oxygen
hridge which is most stahle in the acid laetones, whell present
ill th.-slIgars, ronf"", on them highly reactive properties, and
the ,!riginaJ selretion of the title yosugar for the reactive
 N:l7'CRE Of' "G,UfJlA" sraARS sl

type ha~ h('PH fortllitou"ly ju ...tilied hy tJI(' fad thnt thi .. type
of ",uj..rar givl':-O ri . . (' ofl oxidation to n y-hwtou{' without
('flange in the po.. itioll ofif:.. OX\'J.{('f1 hrid,lll". :\, iIlu .. trath,'
of ~<H1I(' of the ddJ1s of thi ... ~'('('nt work. a s<'}){',.tw ~UIfI'
nu:ri .. iufl ill{' ("omhin('d r(_:-.nlh of URwurth luu,l of tt\{' R.uthur
on gHlad(}~(' j.., appt'II{)(d (p. SU),
If. tiS showlI ill th(' uhovl' :-,(rit~ of r(l'dion~.
the f)(, .. itiull of
ttl' oxygen bridge i'i pr(,H'nt('d froll! ult(riHf.{ hy ,",ul)!o,titutill,!.C
all the other hydroxyl .l(fOUp'" by nwthoxyl. OH,' ohtains from
tlw nornu\l M'rl('~ nf d('fivativ~.., 1\ ftr,rf-rfH'otlltury 1adm\{',
whereas ill tilt" Y . . (fils. ulld on dir('ct oxidation of HI(' fr('('
'iujtHramI on :-'Uh~('qlH'llt Illdhy)ntioll~ itif'llti('uJ /'(l't'o-rututory
Jadones m't' ohtairwd, The oppo~ition in t II(' dir('diofl of th(
.. rotation ~hown hy tilt"' two luctones j~ iu H('{'ord with tl1<: fud
thnt in grtiaetos(' tlll' hytlToxyl jlT1JUp-; uUa('hf'ti to tilt' {'nrhotl
tttorns 4 Hnd 5 Ii(' on opposih' SiUf'S of th(" ('urhon ehain. In
the ('U"'("'i of glucose nlld nHtmlO~t. ~ill('t Hl(' ('orr(spolldin~
hydroxyl group.. li(, on the :,(lrn(' :-.idf'S of HI(' ('urhon ('hnin~
Ofl(' (:'xpeds the lactoncs prc'pared in the ~aJH(' way 11<,; is

d('.-;erih('d ahove to hav( Hl(' sanlt' dirt'dioll of rotation, hut

. to differ quantitatiYdy in this constant, and such is in fu(t
oh. . ervcd.
The ea,,' with which a sugar in which the oxygen hridge is
displaced from its normal position uu<ierg()(>s oxidation has
ex(~rciscd a strong fascination on biochemists. The hypothc!\i . .
that the reactive form of glucose may play all important purt
in the metabolism of carbohydrates is certainly attracth'c,
but it mu,t he confessed that no convincing evidence i, forth-
coming in ;upport of this supposition, nor, on the other hand,
is the hypothesis disproved. No single instance of an a1uo-
hexose ""~urring in nature in its reactive form, either in tb,'
comhined or in the free state, has yet been hrought to light,
On the other hand, the ketohexose fructose, a.. it is fow,d in
the polysaccharide inulin and in the disaccharide sucrose,
whiht it seems to he of the amylene oxide type and is, ttK:re-
fore, similar to the aldose., is nevertheless much more reactive
. I
 R2 'I'll E l!A RBOll Y DRA TES
in Hti ... form thull it jl'o in !>.tuhlt' cry...,taJIin( fructo ...c. Thl'
latter Uu'rd'nre pft'"muahly pOl'o ...( ...MS a ciifft'rf'nf tnw of
HXyU'f'll hridt[t', A "I('W which i ...... upporfcd h~' poJarinwtri(' find
other (.~.~IH1{'e.
From po1flrirmtrie ob"-t'rnltion of' fluid!o! of pln..,jolog1('aJ
origtll, ~u('h n~ d{"pr(lt('ini~('d hlood Hltr,lh-.... whi(:h ('ontain
f'Hrhohydrat< :lmoll.1!l'ot HlIIU('rOU"i ot her ('OnlpOlients, it ha!'-
iK't'1i Hrg-lH'd that the ~ugar prt ... tnf is not the llormaJ
I'quilihrium lHixtllr(' of t IH' (I.. and ,B)'o,tcrt'oi ... omcridcs of !o.tllhlt'
~Iu(os(. Tilih it i ... found thllt th(' 6pti!'al rotatory ()Ow('r of
~u('h fluid ... i ... rr('q\l(,lItl~' helow that rl'quircd for H ~olution
('olltaillilllr! th(' (qnivakHt nlllount of glm(l..,e a.... ('akulntt'd
from the ('opper r('(iueing power. ~lortoY(r. thl~ ob!'>i('rved
rotation frt'qul'ntJy ('hangt~ wit h lap"i(' of tinw, nmi H C'Io..,tT
('orrl''''}lOndr'Il(,(' hf'twt'(,11 t})(' two valu{'s may hOi nttlliJl('d.~
SotHdinH's til(' l'tTeTS(' J~ the {'d~(', Deductiom. from suoh
oh:-wT\'ation ... mu~t he UC'('f'pted with ...rr('at (,Rutioll, )',in{'(' 011('
j)', {kuling with :-.oJution.., of ,o.,.UR'HT {'(lJItnmiJUltt'd \'\.'itjl many
other ~ub~hHl('('''' of whiC"h the intlw.'H(,(' on tht' rotatioll of the
~ugHr i!-. unknown, Hnd whi(,h may form mon or Ie~.:-. stahle
(oIHpnunds with thl' sugar. The infel't:'ll('t that !>ouch di~
pariti{'~ b(,twt~('n polarimetriC' unO. eoplX'r-redueing' values for
til{' ~ug8r prc..'s(nt in the~t' solution~ indicatt' the presel1(,('
of reudiv(' forms ~e('ms unwarranted. Similar ubnormal
polnrjnw'tri(' ohio,('r\'ation.~ hav( lx'en obtained by Sta~iak in
working with blood Slrum. and have h('en imitated in t'itro
witt. pure mixed solutions of gJucose and globulin, and of
ghu'ose and ('ho)c>stero1. The tYl){' of complication that may
arise, (>\'tU in blood tiltratcs from which tht protein bas been
r<'mov(>d by til(' usual methods, is iJldi('uted by the presence
in pig'. blood of a" adenine pO'nto... nucleotide (Cb!ll.ter,IIt).
and the intt'rt'stillR discov(~ry by Benediet and hi~ associates
of a uric acid-d-ribose compound in the blood of various
animals. mol'(' parti('ularly hl human and in ox blood. The
su~stion of Neuberg that in pentosuria the excreted
pe~tose i. comhined at least in part with urea is of interest
 ALKALlNE DEGRADATlOX 1\:\

iu a ~imilur din'ptioll. SOlll(> yt'Ul' UgH lforrdl alul Bcl1ur..

rt'ported llw forillation of ddillit(, compound .. of Iwxo,t'''' !llJd
gUIlTlidilW, alld morl' rt'('('utly "'if:l~'llInlHl hth jH\,(,~tiglltt'd
Uw intltH'IU'(' of guanidille 011 til(' ('h('mi('nl and p(lil1nillwtri('
h(~lut \'jour of I!Jueo~('. It-. g(ntral df((t i, to product. n
h)\\"{'riH!,!' of t lw rotation of th(' sug-Hr in a lUnnllf'r similnr to
that (,f dilutt' alkali, "'0 that tlil' alllount (If ~ugtlr a .. ('u1<-ulat('d
from tht' rotation of the ,,)Iutioll i, lx'iow thut dt't.(rmitl{tI
hy ('opper reductiol! llwthod... Compounds of which wumi
dint' forms a part art' . . known to lw prt,,>(nt in hlood, wh(~f('
thp,Y may exert all MC'tiotl .. illlilnr Lo thut ohtnin('(1 tOn 1'ilrH

CHOH CHO CHOH CH,OH CH,OH

H{~
I ----I~ I I
R COR C OH COR HCOH
-----11-_--- I

-
I I
'HOC R 0 ROCR HO C H ROC ROC
I ~-""II-'---
'7~
I
H C OR H COR
I - H COH RCOH COR
I I I I I
HC H COH H C OH HCOH HCOH
I I I I I
CH,OH CR,oH CH,OH CR,OH CH,OR
t1LUCO~1'. ALDEHYDIC ,. Z !lIENOL Z:3DIENOl- 3:4DltHOL
FORM

with fl'('(' g-uanidine. In Hlly ('a:,e, the kHown in~tanc{~!o. oj

association!'> bet wet'u ~ugar and other ('ompont'nts of hlood
are suffickntly sugg(:stiv(' of other possible eombinatioTls, the
presence of which would c('rtairily lead to dl~{~r('pIUl(:i('!oj
between sugar determin..d by tht two "uthods which w,' haw
mentioned_ The further fact that c-ompounds of th,' t.vIM of
Jk:l}ediet's uric acid-ribos, derivati"e would yi"I,1 Oil hydro-
Jysis a sugar distinet from glucose provi<i{'s anothf'r pos!'>;bl('
complication.
",,'hen pr('scnt in an alkaline solution. the feadivity of 11
sugar is greatly increw;ed. It has been known sill~ th,'
classical work of Lobry de Bruyn and van Ekenstcifl~ and
,,-,
114 THE CARBOHYDRATES
till' mor(' T('fTut work. or :\'('f, t hat in til{' prl'-.tlI('l' of alkali of

"Hfyiu,L! ~t r('lIgt h .trIlH'o ... {' IllUkl'g'(I(':o-. all f'xh'n ...in ... ('ri('~ (Jf
trall ... for~Hllti(JII"', ill the ('our,(' of whi('h oth('r IH'xo..,r,>.
iJl('luding";j.maflfl()'.(' alld t!frudm,(, art' fornwd, and the'i(,.
tog(~t 1!I'f wit It tIll' 1I1H'hllllgt'd gIUt'OM', thcH und("r~'l) further
c]t'I..!'rl;dation. If O:xygl'Il he ndmitted, oxidation O('e'nrs mud1
BlUff' f{'adiJy ill :--w'h nlkaJiu(' M)]utioll .... than in n{'utraJ or
:H,jd ... oluti(tll.... ::\d H;o.erib( ... th('..,(' resulh- to tlw initial
formatioll from th{' mixture of lwxos(>.<, of 1 : 2, 2 : 3, nnd 3 : 4-
Ii-('ool fortll ... , t h(> op('u-('huin aJd(>hydi(' i"iOnWT probably
h('illg' Ull illkrnl( <lillte product, n:-. ... hOWH 011 p. s:~.
JIl 01(' prl',<"('IW{' of ..trollJ.! <lJkHli Hw....( form ... llrt' ,'xt('n ... jydy
disfUph'd at the poillb. lmlit'uh,d by t h(' <iottrt.i liB<~l'I. and
mmh'fOu .... de:.rmdation produl'ts,. ~ueh a~ formaldehyde and,
uldOpt'll.to ...t'. di()!-'t'~ und nldotctro:o.f'!-', and triose's n~ formed.
Tht' tTio-...e:'~ 11ft' quantitati\'dy til(' most important of th{'s~
prm:!ud..; :md as Wt' .. hall ~('C, \1U'h thrt'('-('Hrhol1 ('ompound ....
are the tir ...t T('('ognisahk (kgradatioll produeb whieh ar('
know]) to H('('ur ",h(,11 glu('os(' is Hlf'taho}i~{'d 'in t'ii:'o. The
bt'st known of thl:'M' produrts form('d in the:' living tissu{'s is
hl('ti(' u('id. Thi:o. HPid is also found when glu('os(' is <ipg:ratkd
ill t'itr() in th(' pn'<.)t'nc(" of alkali, if oxygen is excluded from
tilt, -"yskm.
Another ~('t of conditions under which a hexos{' is Tt'adiJy
oxidi~('(l hy mol~"('ular oxygen is brought about by the
prest'llcc of tht, phosphates of the alkali metals. Thus
lrm'})urg ami l".!ilmsOl' halT' ....}wn-n that fructDse, but not
glucose, is oxiciised in thi!':i way with the formation of carbon
dioxide. As we shall SCt' in a later chapter, the metabolic
transfornlatiolls which gJu('ose undergoes in yeast ferments
tiou and in muscle contraction arc eharaeterisetl by .. the
formation, as all intermediate product, of a hexose phosphate,
which is prohably a fructose diphosphate. It appe!ll'S, how-
('ye-t~ from )J[eyerhof's and from Neuberg's observations that
the 6xidation of fructose ill the presence of phosphate is a
cata}ytic process and does not involve the formation of
 LACTIC ACID
h('xo\oj(' pho~phat(' as nn jnt'rmt:'diuk ~t('p. Xt'\'{'rtJu')c'''''' lhi,
:-,('rn's us n ~ugg{"sti \"(> ntwlog-y to t hl' hifwhemh'al pro('('"
in which pho""phak play~ H fuudalll('lItui illtt'flll(dilltt role
bt'!.\\('('tl tht' sugar and nl('ohol awl ('urhnu ciioxi.it ill tIlt'
in"italll'c of yt'ast fcrm{'ntatioll, Hnd hetwf't'li tltt ~ugnr alHl
lactic u('id in the Jl11b('h' pro('(,s.
In the' mu<.,l'}es, wh<:r(' t lw hulk of ('arhohydratt oxidation
o{'('ur~. la('lie add is quantitutin'ly the lIlo..t Importunt nnd
most (,8,..,ilv demonstrated known int('rnwdinrv. But Tl'ccnt
work mak~'s it prohable thnt tht hH'tjp Ilf'id' j;,; 1I0t furth('r
oxidi .. t'd. but i ... ri'tran . . fornH'd throug-h Ilf'Xo..,p pho .. phHk
Hlld t!hl('os{' to gIY('o_t!'t'Il, oxygcll ht'ing Il('(c .... nrr for til('
prol'f'Ss. II' the oxygton i!'i ddi('iellt. or Hh~l'nt, H)(' lllU ...f'}t
." fati~u(.'~J through the U{,(,l1flllllation of tilt' Indi{~ Il('ill
under th~ t-maerohie l'onditioll"1. It j" during til(' phn<.,t. of
hhcration of lactiC' arid tlmt the ('Ontruetion of th(' mUl'l<'ie
oc'curs, and during til(' rt',"C'rsnI or "Tf'c'o"cry" phnM' U
further ~l1pply of ~ugar j., oxidi~('d to ~upply til!' lJ('('('~"'llry
ener~y for the buiJding.up pro(,,:-'!-o. It j~ thi" lutter portion
of the total :-.u~ar only whieh lS dinctty oxidi~{d, and from
it is formed the carbon dj()xidt~ whieh is tJl(o final pro<illl"t uf
carhohydrate mdahoJi . . m. Although the produ('tion of ladie
acid I>; a fundanwlItaJ pl.lrt of earhohydratf' llI('iaboii!-.1H Htul
is intimately as~o('iated with the eontradion of muscle, W('
eanno~ from this point of vicw T(>gard it a~ all int{'rmeciiut(,
in the direet oxidation of g)UCOS(', It h.. of ('OU~('. po<.;sihk
that the gJu('ost' whk'h i~ dirf'dly oxidj~('{1 is first ('ol)\'erkd,
like the rest of the gJu('o~(', to Jadi(' a<id, but th(~rc is no
cviden<'t' that this is actually the eaS('. nanrce and Hill
have shown in a study of the heat (~\'olved during t.he various
pht!.'iCs"rmuscular contraction that only somcI/47 to 1/6
of the total lactic acid produced during the contraction of the
muscle (or the equivalent of this amount of acid in the form
of carbohydrate) is directly oxidised afkr the contraction.
Meyerhof, Lohmann, snd Meier havt recpntly shown, 'n an
instructi ... series of exp,riments in whi"h they in\'Csti~t('d
 THE CARBOHrI)RATES
the ... yntJU' .. j~ of rarhohydrat<, from lac,tie 3eid ill ttl(' mnsek,
thnt added hl('tif' Heid parti{'ipat{ .. in the" T('('OY{,TY" prof'(''.;';;
in the .....~!lIt' quantitative way a ... til(' HC'id formed d1lring (,OH-
tradiofl. .. Th('y han' ... howll that the glY{'ogf'JI ('olltcnt ,of
frog\ lHlI",('li' iJH'TI.h( ... wh('n it i~ p('rfu..,ed with oxyg('llnted
Hill.!.!(r ~oJlltjOIl ('ontuinlng ladl(' <1(i<1, hut they havt'
ohtuirH'd Ttlu('h 1Il0T(' dt'ei)"iv{' f(:-.ulh hy the ... impJ{ ('xp{'cii(>nt
of' imnH'r... ing j ... olut('d Hlll ...d{'". ""wl! a~ the sartoriu"i or
~1I ... tro('n('tHjll"', ill the ludie Iwid-HingeT sollltioll. Th('~'
T('('ord('d tIl(' oxygen uptake of the' lHlhde during the pro('(\.,!o,
of !'oynt I\(' ... i ... , and han' 1)('('11 ahle t(1 deIllOll'.. trat(' that :-.ynt h('~j\
and oxygen ('on~utllpti{/n Tun parallel to one another. If til('
ratio })('t\w'('n the total amollnt ofladi" R(id whieh di~arp{"ar"i
IIlld t IH' eqni\'aknt allWlIl1t of laetit, H('id (or sligar) whi('h.
i ... Ilxidi:-.('d, It... judged hy t 11(' oxygell (on,umption. "he df't(>r~
mined, 011(' ohtnins wlwt i ... ('all('<1 the (I,ridatt'rm qllofirnt, a~
. . hown in the follo'\\ing tahle taken from the pllpt'f of MC'y<'fhof
Hnd hi:-. ('oIlHhorator.... In all tlll' ('a~(':-' T((orrlt'd tht' ('OTtT-
,..,pondillg llilisele from the oppo:-.ih kg wa:-. H<"('d in a ('ontrol
('x})('rilHf'nt ill whi('h 110 lactic add wa:-. added.

'1'.\ ilL>: IX
('(Ir/mh.'ltiralc _",1IlIllIfSi", {rom LafUe _/eill mifll'd to .tltl.w'it,.
A. O,t'YJ-!t'n Con,lo(umpfirm.

i I I'm.... 02 ronMmwu in .-
l-;xtra
E~I'I 1
WI'ti(ht
of
I l)uratinn
dlh:l)t.
Oxntl'n
t'olllmmptif:m
Mll"dl' ('ollttul. I.artkAf'id
, in Mgmll.

-=1
MIlsc!e,
i
1'-~ .;~j' --~-,~--

!!
3
1-:10
HlO
ZOS
I l~
18
30
20
943
783
1,212
1.385
1,025
1,686
i, (HI.'
'1)'315
0678
1
5
149
128
I 6
16
SO
0
514
515
1,200
977
I
! 098
0661
6
7
105
1-.50
I
I
15
U
30
:In
479
786
(128
1.133
0213
n'l97
I
 O.'(/[)ATION QCO'J'IE.V7'S S7

j'ub"hrllfll!"
S'lllh,.... I., .. ,l t<;'1ujn
l:xpt. ~:!!~.f ; d~ii~~'
~o. Atill ~ (1m,
J,wtl,
t',mltIJl ,\"1'\ (h:kitM'<l tJornt.
!\l,...d ... )1111111< '.

J-1tl 1':3!! 0'21 27 (Hi!) 4,7

~ 15'!l 17':l I 'Hi5 Ia~ (IIS 2a.1, ()';J2/1 7'2
;j lU7 21/1 (HIJ J 011 O'I~ 2-~a , (HlllJl 85
~ Inj. i 175 J.J() 1-17 (I'J:I 2'O:! O'H!! 'J."
:W3 2~t) l';'}S,) 172 O'lS,1 2';i:,! Oti2 a75
"
()* 7-j. N:J;; n 71
o
oso (I-II
I

).1;; , o!W :;'7

7" J:l-! 11-15 ()S7 (HH O'10~1 1';)~ 1 o ~H5 ~ a'2:;

.\nragr oxidntiOlI qllotil'nt Ia:l

In the lu:-.t two (xp<'rinwlIt ... quoted, 'So..... (i nlH1 7, lht'

gly('og-cn aloHe wa~ det(rmilwd. '1'11(' fe:-.lIlt ... art' \'C'ry do~e
to tho~(' ohtalHt'"d in th(_ oth('f (xpcrillJent-... ill whiC'h til(, total
('arbohydrate a ... wdJ a!o, the gly('oW'u \\'a'i (if.krmiu('d. \\'(.
may infer that the ladie acid whi{~h i~ u...ed in th{' :'o>yuthdieal
process is built up not ITl('rel~- into g-itH"OS{', hut. into th.:
polysaccharidf' gty(ogen. It will ht "'('(>-11 that til(> ay(,rage
\'8lut' of the oxidation quotie1lt i~ -l:m. which is in good
a~ecment with the: ~am(' quotient 8!oi detC'rmirwd (1) durin~
r('('ow'ry of the II1u~d(' afh'f ('ontradinn, (:t) aft('r a p(riod
of rest under ana<>robic ('onditiolls, (8) during rest undl'r
aerobic ('onditions, (4) in mhlCed muscle under aerohic sud
anaerobic' ('onditions, and (5) in thc' fati,gucd entire animal.
Il\.all<tl!ese caSes values between 3 and 55 are obtained. In
the experiments of Hartree and Hill already mentioned the
oxidation quotient which would correspond to the ob.erved
ratio between total lactic acid rt"IDoved and ~quivaJent of
lactic acid oxidised would lie between 47 and 6, the .>verage
value beinl! about 5. There is therefore eXcellent correspolld-
.
 8S THE ('ARBOHYDRATES

t'ilet' hdW('('1l the ph~'!'Ii('al m('th()d~... of th(lattct inYl'~ti~ators

and 11l(' dWflIieal rtI('thod~ employed hy .Mey('rlwf.
\Ve JHl\T (I1rtady statNI that tll(' hulk of carbohydrate
(lxiliatith.\ tak('" pla('(' in th(' lllU ... ('i<.. ,"" and it would appear
that (urlx)hydl'at( i . . oxidi~(d hef(' in pr('f('n'T\('(' to otFU:'f
f'ood ...'tII ff..,. X('y('rth('lf~"", tfw faC't that ()tht:'f >.;nh..,talll'f'''' may
}1(' oxidi~(d ;11 the mu ...dc . . i ... shown by a . . tric" of experiment ...
l'lIrrit'd (lut hy Kro.(!h and Linharrl on the J)('rfornHHl('(' of
!ilu!'!.{'uhtr work 011 low t'orbohydrah' did.... Th(y hun' ~hOWJl
that' tht' T('!-,piratory quoticnt! or 0)(' ratio of t)1(' volume of
('arholl dioxide {'xpirf'd tn the \'olullI(' of oxygefl retained..
I1lld"r t}}i'~e ('onditjon~ i ... \\-'ell below th(' T('spiratory qllotjf'lIt
or Ullity whi(h i:-. ObS(,Tvcd when (arhohydrah' material alone
i . . heing oxidi.,('(l. Th('Y ('otlclude that fut IIlU'it be o:xidi~('d
in ~lI('h ('H~(':-. wIwr(' thrre i" a deficiell{'Y of (:uf)Qhydrate.
It is indeed dCBr that h~r(', although lactic 8.('id must bo.
forlnC'd in tlw ('ontracting mus('}('''i, where it i!-. r('uHy part of
Ow nwdlU.1\i~Tl\, it ('annot be the ::-.uh!-.bm(( whi('h i", hurtwd
to )o,upply the t'lwrgy Il('('('ssary for the N'COY('ry pro(t~s.
{~tld('r the eondihpns of ('arbohydrnte deficien(,y whieh
J\rngh and I ..jnhard jJ)v(':-;tigatf'd, nnd ass.umiug th.at the Jadic
~wid Ilt"{'cssary for th(' ('ontra('til(' proccss ean be- dt'riv('d only
from glu(osc'. it is ohyiou~ly important~-ind('ed ('ss('ntiaJ---
tlmt thh. ~ourt'{' of til(' add ~hould be couse-Tn'd. Hen('c the
tj~~u('s turu to some- other sub~hm<'e for th(' energy required
to kt'('p t}l{' contractile m('('hani~m in working ordtr.
It is from nb!-.t<f\ations of the typt ofthos(' whi(~h we lluve
ju . . t disNlssrd that the biochrmist ha._~s the eondu~ion that
in the muscle the pl't"dominating r('nctioH, probably th(' only
rt8('tioll. taking plnCi" in the preseuC!' of oXy~J'('n jn 'whj('h
laeti(~ acid is conct"rncd is e e

Lactic acid - - > Glucose.

Np\'('rthelr-ssf in ('xcj~'('d musplt" and in the JiV<."f the reaction
pro<~s in both direetions,

Lactie a('id ~ Glurose,

 INTERMEDIATE PRODFCTS 811

nc('ording to t'ircnmstall<'t's. Thu~ J)(>rfu ...ion of a lin'r ridl

ill glycogen, or in the {','ent of til(' nhN('Jl('(' of g-JYf'u,!.,ttn ftoUl
the liw'f, th(' prt'M'Il('f' in ttt(' I)('rru~i()n !tuid of ghu'o"{' or
fruetos('. alanine, gIY(,,(,ToJ or otlWT ~uh~b\n("{!\. rt"lt/t!- in tht'
formntion ()f ludjc Itcid. Ttl(' t'('W'rs~' ('(lnn>J",iOf~ of filet if'
acid to ghl('()~(' hn~ 1)('('11 d(mnn . . truh'd i1\ til<' Ji\'(!rs (If
d<'psmT(ati'i('d. and. therefore diul)('ti{', dn,L,rs. III tllt' pn'M'tH'('
of u1knli in 11itro. w(' have "~'('II that ghl('OM' j.!in'~ ri1'>(' UIHOflW't
OtiWT produeb. to laetie aeid; hut if nir or OXYJ.!t'll 1)('
admitted no lactie Hcid formution i ... oh. . <'rn,d. oxidution
heing Hehi{'vrd along !'.omf' othc'r route Hml appHT('ntJr
occurring lx-fort th(' la{'1ie ll('id ha..., lUI opport 11IIit y of f(lrfllin~.
Thw; under anat'Toni(' ('omlition:-. lad;" acid i, forlll{'(1 in lUI
lllkalinf' solutioll of I!lu('os(' ju~t a!oo it .!U'("IHllUlut(' ... ill mu ... rlc'
under th.. same eondjtion~. {~JHlt'r H('robi(' ('ot}(litiOlI!oo; in
lIohe former instance, lactic acid does not HPfwtlr, oxidation
taking somt' other rout(' j in the JnttC"T ('ilS.(' it i~ tran~
formed baek into sugar, whibt that portiOll of the ,",ugaT
whj('h i'i oxidiM'd dire-etly i~ brokt'li own hy nn ("';!o.('utial1y
different Int,thod. w, 'hall ,,'to later (('hlll't,"r \"1.) thnt th"
fermentation of sugar hy yeast, whieh pr()('('... ~ i!o. fr('qu(,fltly
called upon to supply analogi('~ to til(' nwtuholic pr(){'('~s('s of
complex tissues, show~ many ~triking ~imiJariti{'~ to this dual
serif's of J"f'uctions.
The resemblan('c5 ju...t discu~s("(ll)('tw('('n tht, two pr(w('v.;('~
of glu('ose degradation in vir/} and i'l 'ltilro sllgg(~t that oUH'r
possible intermf'diates may be common to hoth, Thus mdhyl
glyoxal, CH . CO . CHO, ha, l)(,"n dt"t,chd in the products
of the action of alkali on glucose in it'itro, and thil'> ~ubstaB('('
and the closely similar dihydroxyaceto",.,CH.OH.CO.CH,OH,
an<!. gl)'Ct'ric aldehyde, CH,OIl. CHOll. ClIO, when sub-
jected directly to tbe at"tion of alkali, gh'e very much the
same results as glucose in regard to lactic acid formation.
'These and similar indications lead to the view that
methyl glyoxal, dihydroxy acetone, gly,:eric aldehyde; and
pyruvic acid, CH . CO . coon, may be all n"L,onahly ~on.
Ilft TIlE CARBOHYDRATES
~i{krt'd a ... intermediate pr<,du('t!o. on the path of j!ht('osl'
(JxiclHtioCi. .\ ~rtltt (It'a) of c:q){>riment.nl ('\'id{,Tlc(' has hccn
pllhli .. hed ill whi('h the <'Hpority of these substan('(.~~ to tit
into tlw',,!htlltt' of gitW(}S(' nl('t"boli~m has he('ll inn'o.;ti,Qat('d
ltv t(' .. tjn~, ill p('rfu ... iof1 and otlier ('xp('rjrncnt!'l, their lKH\'f'r
tt~ g'i~'(' ri . . c to lactic Hcid or to :-.ugar. It \vill he noted that
t he production of Jm'tif' acid has ht'f'H u<;l'd m~ a erih'rioll,
ilnd \w haH' ~('f'H that HWfe is ]lOW !'ct!son to ooubt th('
furtlwr dir('('t oxidation of thi~ u('iti ill the mu~dt". X('n'rth('~
k~ .. \n' IrHI';t regard th(' formntioll or otherwise of ladic
u('id ill a p<'rfll">ed organ or ('x('i..,rd ti"'~ll(' ~!('n('rally a~ a
kgjtiHlutt imfi('H1ion of t1l(, <'Hparity of U)(' ~ubstan("(' h('ing
tt- ... tt-d to pIny n jlHrt ill g-liH'o!-.(' meta holism, hut it is pos-.ihlc
t hnt ill l1\any ('a:-{', the lH'id mHV Hri~;(' Bot direetlv from tIl{'
~nh'ltllfl('t' l1~(d ill th(' t'xp('rinH'~t, hut from Hl(' giflco. . .r into"
whieh thi~ latter may he hansfornwd. It \1-. {'x(_'(>(_dingly
ditli(ult to make a ("hoi(,t, fll\"ouring H11~' OIW of the ~uhstan('('!-.
whidl W(' han: m{'tltiolled ill preft'fCllC(' to the ot}wr.,\, hut Ull
attempt i . . Jlludt, in what follow ... to ... umlllari~(' tlw type of
t'\'id"lll'(' whieh is available,
Glyceric Aldehyde and Related Compounds.~Glyc(ric
"ldl'hydt, 1:-. r{'adil~' oxidb('d in thf' hody, It is dt'stroyed by
lin'r ti ...... ue in dtro. nnd h ('onH'rted to :tJu(_'o~c and ex('rett'd
a:-. :-.lHh hy do~s f(_'ndert'd sugar into)C'rant with phlorhizin,
It in{'f{'aM'S tll(' glY('o,I!('1\ conh'nt of p('rfn~('cl tortoise liver I
und ill (>xfwriruents on the fK'rfu~(>d lin:>r of the dOl! it ha.<;
~in'n ris(' to tl~gIu(>o~(' and d-l'-orho~e. It. is rollverteri into
dl- Hnd }-ltwtj(' ncit}..; in th' perfused livf'r, nnd by the washed
ft'd blood (.,.l1s of the fasting dOl(. We may condude from
th('st~ obs{'rvation~ that sufficient biochr:mic-al evidence is
fortheoming to ~upport thi' view that glyceric aldt!h,'de .J~nay
participate in the intermediate sIn!!", of glucose metabolism.
In a similar way we may consider glycerol and glyceric acid l
two compounds ft'prescnting simple reduction and oxidatio"
prod'ucb of the aldehyde. Glycerol red to fasting rabbit!
prqlUl't'S a marked increase in the blood sugar k-vel (increase,
 DlRfVROXl'ACETONJi: III
up to 035 l)('r ('{'nt. hav(' l)('{'n l't'<'uro('d), whi('11 In:ooh for ..,OIlW
time, TIl(' {'on\'('r~ion of gIYf't'rnl to Itt(>t1(' :1('1(\ and to gJu('()!.(
ha ... heel! ohsrn'{'d in t ht' gly(og{'l}fr('(' liv('r of tlw dog.
GI),('('ri(' acid lik<'wi,e form ... ghl('o"'e in phlorhiy.in~"':d (iog\
liver.
Methyl Glyoxal (Pyruvic Aldehyde). ~I('thyl I:ly()xIII giVt'
It similar ...,('ri('o,; (if r('..,u}ts. It is ('onn~rt('d to J.rlu('(Js(' in
dial)('tic' (phlorhizini ...ecl) dO,!..,f>;;, and to tll-hU'ti(' u(id ll\'
Jx'rfu..;ion through Ul(' lin'r, hy kidrwy ti~~u('. by !'.t(ril~'
1('llc()(_'ytes, llnd hy many ha!'o!h('d ti",!o.ut.'s. Etlzynu.~ ('allf'd
g-Iyo:xaln~('s art' wid('ly di",trihuh'd in animal ti~~ne". '1'11<')'
ar!' capahle of ('onvf'rtillg rtwthyl .l.{lyoxal to Judl(' lit'iil with
gN'at ",p('t~d Hnd in C"on"iderahlt quantity, if it i"t lIot ullow('d
.. to a('('uUluJate as frN> ;ll'ld, hut j,., m'utraJj . . .ed u.s it i .. formed.
It is stat~d that an nqUt'oUs lin>f ('xtrll('t containillj.{ kl-."t than
It gram of organic' matb.'r ('an form in thi" way sl'V('rnl g-rnms
of ladi(' acid in til(' ('OnfSt' of u fl'W minutes. Th(' a('id
produ('{"cl by f!lyoxalas('''i i"i prrdominantly of till' iu:%'fI'
rotatory "andy,
Dihydroxyacetone.--Dihydroxyal'l'ttllll' i~ Hfloth{'r ('{Hll~
pound which gi"f'S risr to dgJ1J(.Ost wheJl Jlchuiui.,t<red to
phlorhizinised doS-,rs, and in Uw ('xcls('{i perfused liver it is
('onv('rtC'd hoth to glucose and to dl-Ia('tic acid. whil ... t by th('
a(tioll of pig's blood ('orpu~cJl'S dlR(1j(~ acid i"i formed from
it. Dihydroxyacetone is ('hara(~terised by a ft'markahk ('ase
of oxidation. Thus it reduces Fehling', solution ill the cold
,,;th great rapidity, and it absorbs molecular oXYgf'fl in th('
presence of phosphates ("ven more rapidly than d()(,s fructost.
f"urthermore, dihydroxyact"tonc has a well-marked anti-
ketogenic action (sec p. 135), and its recent production on a
roa}m~al scale has gi"en a considerable impetus to it", Ul'tC
in the treatment of diabetic coma where glucose is ineflicient
for the purpose. Drs. Kermack and Lambie have brought to
my notice experinwnts in which tbey are engaged, and in the
course of which they have shown that dihydroxyacelone i.
removed more rapidly from the hlood than is glu('Q/iC or
 !l2 THE CARBOHYJ)RATES
frut1o~('. alld. when illj<>d<d into rabhits and miet' sufftring
from th(' f'rkd .. ofillsulitl hypogl.vC'rcmia (ron,'ubions follow(d
hy ('omit). it i." ('apahll' of dfteting a r('('on'f.v in til(' same
WH,V as 4J~t('os(', Dihydroxyac('toll( is also readily IItili~('d hy
diaht,ti{' jJHJi"jdua}s. a1ut this without pfl.<iucing any in('f('HM'
jJJ t lIf' hlood SUg'IlT. Thus a dillhetie ~uhj("('t. vdu) waf. alrf'Rdy
rccciviuJ! SUf.!liT to till' Jimit of Ill'r tol.:-nm{'(' and exefttillg
Ilbout 1 grum of :-.ug:tr ,wr day, did not sho", any incrf"Rst'l
!'>Ilgnr elimination after the addition of 50 ~-'1'ams of dihydroxy-
lfC,(tOll(' to ttl(' diet. That such a result canIlot be explained
hy stomge iII tht:' lin'f i ... n~lIder('d prohahle hy thf' followillg'
{)hs('rYHtion~. .<\. l'ut was d({'("f(hrat(d. ('\'is('fLrated, 111(' livct'
wa.s ('ut off from the rirculation, and the renal ycssds WeTe
Ijgnft'(L TIll' 1>1000 sugar lenl was kept con.st.Ullt by means
of glucose transfusion ut the ratc of 015 gram 1)(1" kilo p<'r
minute. On stopping the glucose trun~fusioll and. gh'ill~
dihyrlroxYfH(tollc in its plarc at the same- TatC', the hlood
sugar }{.,'t>l heg'HH to full. and thc dihydroxya('eto1l(' dis~
appf.'flf('d rapidly and ('omplddy from the C'irrulation. This
ohsern\tion abo eX{'\udes \11(' possibility that <lihydroxy-
u('{'hme is rapidl~' l'()llYcrt('d illto J!lu{'ose in the pTeparation
ust'd. and Kermaek. l ..ambi(', and Sclater conclude that this
~ubstnn('(' is utilised dirc('tJy. In parallel expcrim{~llts sodium
IHl'tat('. sQtJiUJH pyruyate, and methyl glyoxaJ proved to he
jllcnpahle of rdi(,),jug an nnimaJ from the effC'et... of iusuJin
hypoglyc:t'mia. In It prin!te communication. Dr. 1-1. G.
l-l('(>n',s iuforms me tllat ('arefully purified glYff'ric aldehyde
is also quite unable to produ('e any reco\rery in rabhits or
mi('(' su{ft'ring from hypoglyca.ll1ic convulsions or coma. The
foregoing observations may haye considt'rable significance if
one admits the assumption that an intermediate pfoouct of
glucose degradation itt ,';VQ should, under the conditions
mentioned, produce the same ameliorative results as glucose
itself.
Th~ lactic acid which normally ()('Curs in the body is the
de.rtrr-ro4tory isomeride, and the formatioll of the inacli ve
 ABNORMAl. REA("7'lOSS

Of cll-add hOIlJ dl-::!I~'('('ri{' aldehytlt't hut mor(' ( . . pt(ially

froll! Itldhyl ~Iyox,ll, <Iud dihydrpxY:H'dnw'. nud tiw prrwluc"
tion of H [H'C'ponci('r:u!('(' of the la'1'O-afid 1)\' f ht' ,g''':oxnl~...(l'I.
i~ ~Om('whHt lIJlexp('('ted. and rai .. (,,,, tl1(' (P~t"tJ(m (i'~'<)\\' fur
OIl{' {"an rq.tHrd tlw'-.p readiolh whit'h We hun' di~('lI~st'd u ..
f('pr{'~('llting' normal uwtaholi(' pnJ((,~"tt:-.. 'I'll{' ('oll\"{!'rsion
of /-hwtic lwicl illto d-:,.!ilH'u ...r, H nltc,holl whit'h muy he
earried out in rh'u, i~ a~lOth('r ('US(' ill POil1t, Jt would
({rtainl,v ""('('rn to in\"oln'. as Hukill hm. )o,ugJ,t{'!-.tt'd. the iutt1'
lll{'diatt" formation of son\(' inudi\'{' tYJx' of ('ompound,
ll(h")ihJy of tilt lIahu(' of mdhyl glyoxal or dihydroxYR((tolu,
Anoth('f Teaetioll, whieh hu." I>(>('IJ ref'or.hd in thc' prt'('eding
parag-raph.., and whleh probably helong" to thi~ ('ntegory of
.abnormal rt'adiolh, j" the formatioll of cl ... orho ...t along' with
dglu{'o ...('frorn gIY('(>ric uldehy<i(', when Ow la"it-mclltiOlR'U
~ub ... tan((' i~ perfu ... ('d through til(' Jin'r of the dOJ!, Emhtkn
a.nd hi . . (,o-worker... have .sugJ,!{'sted that this Teat'tion lOuy
tukt' IJ1u('(' with t h(, lnh'rtllt,diutc formation of dihydroxy-
a(-,(tollf", whi('h thl'JI r('ad..; with tht I-form of t h(' gIY('{Tie
altldlyde ill the following llHlllIWt :--.
CII,<lll CILOIi
I .
I
('I (' 0

('lI,oll J[O .. ) .. II
-f i
CliO 11---('- OH
I
JIO-C--H llO .. C-I/
I
ell,on ('n,On
I-glyceric ahldlyde dl.orbo!re (k(~to tormula).
TlW !oiug~stioJl is very intt'resting, and Embden intf'rpreh
it as lending strong support to the view that dihydroxy.
acetone is an intermediate in carbohydrate metabolism. But
we have seen that there is good rea....on for supposing that
sorbose is " carbohydrate which is normally not met"bl)liscd
to any extent, and that it does not gh-e rise to la<-1;ic acid
114 TlfE rARBOHYDRATES
ill a pulp of frog\. ... k('ktal nlU",('If'~. On(' ('an but f('('orrl
tlw~(' MHW'u"hut puzzling in:-.tafl(,,{'''o of the fOJ'mation of ''''hut
Wi' llIU ..,t tf'gard a ... u}mormuJ metaboJj{' produet ... without

nth>mptill~ nil ('xplallati(.H1.

The r;,l1owillj.! !'>dwn\(', slightly modifkd from Dakill,
!o>l.lHfuari ...(, ... tht' l'xperinwnt.al cvidf'ti{'(' whi('h we han' ju .... t
di ...'u ..... ed. and i", ha..,ed OIl the f(".;ult"i of trllll ..forrrwtioIlS
c'arrit'd out in 'cir'o, Hnd ,,\'it h ti"'su( ... lind ti ...... lw pulps ill pifro.

All the pos."iihiliti(~ just outlined nlu ...t clearly 1)(' considered
in a full inV(~,.,tig"litioll of thl' intermediate metabolism of
g-hl(o..,(', At pres('nt it d(x's not s{'em possib)(' to arrive at
a delinik decision fC'garding tht~ ff>lati\"e parts whieh these
("om pounds may play in the- j)r<K't'ss.
An t'xt('n..;ion of the results of Meyerhof and his co workers
on the ~ynth(~sis of ('arbohydrate from lactic acid in the
intact fro~'s mus-cIt- gives a new im}>ortan~ to the possible
roll" of pyrm1c aeid ill f..<Iuco~ metabolism. In th_ further
ill\"('Stigat.iolls )[eyerlwf and his collaborators han' t';';W
mnny other suhstances with the view of dis('overing if
altcrnatiw modes of synthesising carbohydrate in the
mUS&!e are 8\ailable. As before, they recorded (1) oxygen
uptake, llJ1d (2) llJllount of carbohydrate present, both in the
 fARBOllYDRA1'E SrNTIIE81S 9i1

{'ontrol Ilm\clt, Ullti in ttl(' lHu ... ('k tl't'Htt-d with flw t(... t ... uh~
!-.tUft('('. Tfl<' nlriOl .... ","h:o.,hlll(('~ whit1t wl're (( ..... (ltI !lU\\, he
da~si{i{rl. from the rt'~ulh whidl tlwy tilt II tllft,to J.{1';IIIP'"

..
,!!HVI"
as folJow~ :
I. JI. lIf.
{ .IIU~Hl~ hIPrl'~_t'll
W.th"ut h,fl.U\'ll\,'fUl j\"'JliUC: lllq":I~I',1 Ihyl(1.lIl11~'lkt.11"
0)(.\'#,')) rI'IJI1o". 1I)O.,~,n 1I"<lk.'. I '~I)"*~~"f1tf<'
1'i\lltht~ll<.
(;IUt'o,\t" EthvkTW 1'1\'('01. )lytu\,j(, lH'iti.
FruC'to!<>(', (;1\,(:01 ai(hj\nl('. IAtc,tJ('U'itl.
DihvdtoXYH('etOII('. I)iil\"(lr()x\,ll;uJ('i(~ ~l('id.
Glycl'r(l-I,jt{~phori(' GIY~('ri(' t;ldthvdt,.
aeio. (;I,'(>cri<' uloid ..
Glv('oHic ,wid. (~l\'((r(~l.
AI;mine. M,:th\'1 g1\'OXl11.
AsprHgiUf'. 1.'\((t~~JdtilY'k )O\wrl'd
till' uptake or O,i
~jflt_t' the \uh~ttlll('('~ iududcd ill ~rouJl I. did 1Iot tflt'f{'UM'
the oxy~rell uptnk(~ Hf th(' lIlu",('k, no ~Yllthl'si!\ of glyc'og('11
or otheI' ('nrh(Jhydrat(' l'ould ha\"(' o('('tlrfed a('('ording to our
pr('...,{'ntday yit'w"", and thi" WH" ('onfirHlld ill th(' dd('rmiBH-
tioll~ ('arri(d out in the"'e pxperilllt"llb, The ~llh~tanc(' ... in
group II. {'au~('(J u'jddy diH'rgt:'nt iJlf'flll.'tc(,,;;'" ill the OXY,LWll
consutHptioU. Thus gIY(~('ri(' u1<Jt.hy<i(' ilH"f{'us('d it by lh
much as 30 Ix'r (('nt., whiht th(' oth('r ~ub~tuJIC(" produC'{'(i
in('r('asc~ of all irregular ami Illuch Jo\\'('r ordtr. In 110 ('Il-SC',
with the ('xC'l'ption of mdhyl glyoxal, did any !-.ynt.h(':-,j, of'
('arbohydrate take pla('C. Ml'thyl glyoXlll produc,'" a ,lillht
increas{' in the musC'le carhohydrate, U f('!o.ult which is
a..'Ocri}){d to 8 partial {'ollver!o.ion to Jal'tic acid hy the' llction
of the glyoxalaM' of the mu~d(', Hence th(' smaU and
variable iu<:'rea!ot(" in the oxyg('u uptake obs(rv(.'d in most of
thes<' .,... "" cannot he due to utili,ation of the u.-,t ,ul"tancc
in tilt strk-t sells(-' of participation in the recov('ry pro(.c"",
since it was not aCCQrnpanied by synthetical activity on the
part of the muscle. g\'en ,light chemical stimulation of the
resting intact muscle often has a marked efff'ct in incllta.'ling
its oXygell consumption, and evell if the substanCt.'S causing
 !ill 7'l1E' ('ARBOlIYDRATES
thi ... itWrta..,l '\TTI' oxidi,(d. tfwy of)\-iou ... ly wef(' lIot ufifi ...(d
ill tJlI' ..,auJI' way ;l.., i .. Jtldif' mid undf'r ~jmjhJr ('()J)ditioHs~
The olll\' . . ub ... lltlU'( of tho~(' ('xumilwd, in addition to lartic
1\('ill, wn~'h fall ... in ~7J'OUp HI. is pyruvj( (I('i<1. Thi~ m'id,
like' ladie H(id. incfN,,,C'd the OXYhr(,H (>OlhUrHl'tiou of the
lHl"(~(' h,\' liO til lfiO pt-'T ((nt.. nnd ut tht' !-.ame time ga\'e
r,,,{' to H .... yutlU' .. i!\ of ('urhohydratt'. ih ill. tlU' JII"tall('(' of
til(' ladi(' Reid ('xl~rimt'nb, the hulk of tlie synthe"i . . f'd
f'urhohrdratt' pr()\"{'d to be J;!lycogeB. On dctf'rmilliug' the
oxidation (lIwtit'nt u!'> lwfore (Hit' ratio of total pyruyjr H('id
which di"aplx'llr... to the equin1.hnt amount oxidisl'd as
ddcrfllfrH'd frotH tli(' ill('reHScd OXY.(.,r(?ll consumption of the
t{'~t nw",C'k (l\"(T the ('olltrol), a.1l uwrage vahl(, of 47 was
()btailH'ci. whieh i-; strikingly <'lo~{' to that gin>n h,' luctic
Q('id. \\"(' ('01}('iuuc from tilt'M' yaluable (,xpi.'rin~r;ts that
PYTtl'ri(' 1H'id, nnd this sub!oJtan('(' alone out of all thf:' :-.ut>--
stai1('CS ill\'('stigat('{i t ('un reproduce the phenomena shown
hy la('ti(' (wid in the n'{'overy Pl'{)('f'SS ill muscle. Th(!o,t
oh,,<'rnltion<..; pla('(' pyrlJ\'lt' acid in an important po~ition
among:..t the intc'rmt'diate products of sugar metabolism.
,y~~ havf' alN'fidy M't'n that it ('an lay claim to a somewhat
!-'imiIar importance in IT'latioll to the deaminisation and
... yntJ}(' . . i)', of ((rlain amino ndds. Tl}f' fact that. the substau('E's
illdud,'d ill !!"oups I. and II. in the table giwn on p. 95 do
110t parti('ipute in th(' rt'covery PfO(>CSS in frog's muscle does
not, of course. mean that they are entil'(>ly excluded from
parti"ipation in I(lucose metabolism. :Many of them may
play some part in the oxidative utilisation of glucose in other
ways, and in other tissuf's or organisms. A noteworthy
instauct> js furnished hy acetaldehyde, which, as we shall sec,
is an important intennediat(' substance in the utiKsatio!l. of
glucose by many micro..organisms.

The Formation of CarboQdrate hom AmiDo Acids

W/have made ,..,{erenee in Chapter I. to the close relation-
ship .whicll exists bet ween a-amino, a-ketonic, aud a-hydroxy
 A.lf/fO AC]J)S ASI) !.'ARBOll)'J)RATA' 97

H(,id:-;;. Th('~(' at'id~ art' lllutuuJly inh'1\'ollnrfihk. and. !<oitlt.T

pynl\"j(' and lactic a('id~ hoth f('artily J:in ri~' to (urhuhydrnt(
ill t h(' animal oTg-unism, it folio\\'\o that (Hl1nino ucid", from
~l-hj.("h thc'tt, two add .. lHay ari"'t, ill t)w ('011,...(' of '!i"tnhnlir
{'hl~n1X('~ mu~' tht>1l1",{'h'f'''i })(' {'onVl'rted into (urhohydruh.
{tnt! 0lH' may ('xp('('t the I'(vt'r...(' ('hutlA't tn lx' a('('Hmp1i~h('lt
with (:qual flH'ility. Emhde)\ show(tI thnt tilt' p<rfu ... inu of
un i:-.,olatcd ,",uT\jyillg-li\,('r rieh ill ,l(J~'('og<'1l did, in fud. yidd
aluniIH". w1l11 ..t H ,.,imilar perfn ...ion of H !i\Tr rTf'(' from
glY('og('n did not produ('(' the nmino R('id. Thc' re\,('J"S(' H!'opP(t
of this plwnoftwflon j~ S('('11 ill Hlt' ('apll{'it~- of the diahdlt"
or phlorhizilliM'd Hnimal to {'onn'rt nWJl~' alllino t\(-id~ into
\u.g'ur, and '\'{' IlHht PTf,.,lHlW that t hi~ pro('t'~;" i .., Ollt' ",hich
plays nn important part in normal fnrtalHlli ... rn .
l)akin I~.~ . . ummarist'd th('l'>(' oh . . ervntiow, on til(' ('apa('ity
of. varion. . amino acids to form ~1U('o",f' in t hfo diaht>tie
orgnni ... m or in the p('rfu"NI Iin'T. and Iw clivid(s the hiolos.,ri-
("aHy importunt amino add.., into thrf'(~ wonp... m. follow ... :
(l) tho..,(' ('apahl(' of furni:-.hing- glu(o...C': arnonu... t tlW"f' an"
.[tlyeim'. uluninc, :'.criflc, ('y~tdtl(', ll"purti(' ndd, ~Jutamie
acid, hydroxYl!lutamie at'id. prolim~, ornithltH', and argininl' ;
(2) those which do not form gluro~(', but which giv(' riM' to
u(rtoa('~ti(' u('id; in this ~rroup are l('ucim', phenylaJunllw,
and tyrosine; (3) a f(w amino aeid~ of the ... iIHli~})('n~ahl(' -,
type, such as lysint' find tryptophant\ whi('h yield m'iHwr
gluco~f' Hor ae('toaedic acid, Dakin sugg(~ts that th(' f('ason
why these two Jast-mf"ntioned aMd~ ('an not be ~ynth('sis('(i,
and at'{> therefore indispensabh~, lies in th(' fact that they
do not occur in thf' ("OlDmon mf'tabolir paths leading to and
from glucose, lactic add, and acetoacetic acid.
In ~n.".aI, in ca<;e. in which glucose is formro, the yidd is
approximately equal to that which would be expectro if
three carbon atoms of the glucogenetic amino acids wer"
transformed into sugar. Thus alanine gives B practically
quantitative yield of glucose, aspartic acid gives the e.fui.
valent of three-fourths, whiM; glutamic acid, proline,. a,!d
 THE CARBOHYDRATES
ornithilw ''It'Iti ")OI)W thr('('~ntth~ of their ('arbon atom:.. M"
J.lhl<~OM. 'i'h{'~(' oh~t'f\'atjon~, Rg'Ultl. "'('rn~ to ('rnphtt~i:-'l' the
I-,Il't'at importKIlC,{' which nne must attll('h to th{' thT{'('~carhon
('ompor.suh H'-, lut(,fllwdintl's in '''U~l1lT )l)ttnbolbm. That thi!'.
" not, l;ow(\"(r, tht' only pos)'oihk nwalh Rvailahlt for'the
~ynfl\(':-,i . . of earhohydrute i ....... ho\\'1\ hy the fnet that the
two-<'Hrhou uruiuo add gJycine' yield . . glueo...!' in Il1mm.t
qwmtitati\'f' amount in the diahf'ti(' (lrgani~nL It i ... th('f('M
fUff' son)('whut IwrpI('xlng to fiud that ll('it h(,T gtY('ollie o(id.
gIY('ol Id{'hyur, glyoxal. nor glyoxyli(' Rcid will yidd gIU('OM'
IHl(kr ('()nditjo!l~ whieh INld to it:, formation from IZlyriIl{',
1'1I,:\1l, 1'11,011 t'll,OH CliO ('110

!'OOIl (,OOll ('llO "110 ('OO\{

(H1~('1I~'1 (llhMm, (Wn"n] 1(,.!rf':'(nJ.) (l>lrf):~ylll'
Acid.) Aldt'hyde-) .-\,Id.)

Wider Aspects 01 the Biological Utillsation of Carbohydraies

1t mu:o.t !lot b(' for~ott('n that mo~t of tilt' {xpi.~rim('nts on
whi!'h Hw moti('nl ,j{'ws of carbohydrate mdaholism an'
foundt~d hUH' l)('('n carned out on musch- hssu(' rrom ('ohl-
blMoed animnb, It is possible that in tho warmblooo ...j
OImnmul mauy of the pr()("("sse~ which \\'(' haw' ju~t ('on
,idt'f('cl may he modified, perhaps both quantitati"dy and
qualitatinly. But thf'bt> :-.tudif-!-., so far a~ they hay('
prOgTPSM'O, ]('avc us ,'rith two fundam{"ntai ob:o.('r\"ation~
{'(Hl('('rnin$! what is probably the most important transforma-
tion whi('h carbohydrate undergoes in the animal hody:
fi...,Uy, that hex""c phosphate is the only intermediate
proouct definitely known to "" formed between glu('()Sc and
htct}c Held in tht" muscles; and ~'CrondlYt that most, if not
all, of tht, laf'tie 8{'id so fOMnNl is built up again infO ~ycogen,
and i, not diI'{'~tly oxidised. If, then, lactic acid is Tt'jccle<
as an intt'rmcdiate prod~('t in the diret't oxidation of UCOS(
--and modern vie\\"S ("('rtain}\" more than tend in this directiol
---.,~ must look elsewheTt' ior t""
solution of our Jlrohlem
It C911nOt 1)(, claimed that any confident answer is forth
 1If!

('oming. X('\\' ~ugg'f'!o.tiou~ hun' rt,(,(,uUy lW'f"ll tidntHf'('(1 in

relation to th( wdl-knowH n.....o('ltltion of tht, la~t !<.tn,llt;.. of
fat oxidation with ('urhohv{lrat(' oxidation. ill which it "
... ug;-.'t . . t<-d that tllt' ,ix-('t:rhon glUt'O,",f' Hut)' he fxidj",(d
dirt:('tly to a ~iHgI{' mol('('ul(, of a " kdolytip" ~\I'}',hu\l'('
withuut pn\'iou!-. sei:-.:-.ioH to n thr('('-('tlftxHI ('ompound . \\'('
)'.haU di"'('\I~s tf\('~(' :-.ug!!( ...hmb wflt'll \\T dt'uI wit h t ht'
oxidation of tht" fah.
Insulin and Carbohydrates. ~I{'anwhill" one ma~' rt'it'r to
th(' h()I)t~ that flUd tx'('n ('ntt'rtain('(f in t'('JutiOll t(, thC'
di"'('O\'f'ry of insulin. It WH:-. thoug-ht that thi ... int.(n).till,i:f
",uh,tmw(' would :o.('f\(' to duc-idul(' iust prt'('i ...d~ how ~lI~ur
i:-. utiJi:-.('d ill ttl(' lIormal :-.uhj(,(t, Hnd \'.;h)' ib utilh,ution fnil ...
in tht' diul:wti('. .F....x(((dill,!!lv inh'r('"tin~ a.., tlw di"'('O\Tn' of
;n"'111in i~,'al\ll widt'~pr('ad a~ ur(' the lx:ll('tih u('('fllinJ,t f~um
j""" thrrapeutieal and pl'f'vrntin' U~ ill diah('tf""\, it ('IHmot
.vet he daiJ)l('d t h:it it JUb -;}ud uny fUJldanwntnJJ_\' Jlf"W Jisrht
on thr Jllt'(hnni..,lJ\!ot. of curhohy<irut(, m('"tahnli. . m. Thi!o.
~uhj(. (-t i~ dealt with ill IJroft~s"'or Lovatt r:\,alh' YOllln\('
. Ilr('('nt Adnul{'('s in Physiology," mul it i.. not pro~
IM):'o.(>d to clahorat(' the question here. It. ha.., beNt stat.Nl
that tll(' glu(ose. which rapidly di,saplK'ar> from th" blood (lr
normal .fmd diaht'tic- .'iUbjNt."i after the injc('tioll of jn,'IouHn,
i~ not rony('rtcd to fat, nor i~ it oxidj,o.{d, nor i~ either hex(Th('
phosphah' or glycogt'n fornlt'd from it. In flU't, the Uly('()~("n
{'ontcnt of UK' hn'r very frequ('ntJy diminh,lu's after IJ(J~
luinio;tration of insulin. lk!'.pit(' mueh .. frantic s.('arch," one
cannot yet ~y what has bc('orll.e of it. H{'(>t'nt f'xP<'ritnenh.
by lIawle-y and )furlin, in ,",",hieh oontinuouf, and prolongHi
r"..'ord. were kept of the respiratory exchange of rahhih
whiSh bed rec";wd insulin, show that more than one hour
after the injection of insulin there is a mJU'ked rise in the
respiratory quotient. So well marhd is this rise that these
workers state: . Sugar burned under the influence of insulin
is far greater than can be accounted r<lr by the disappeJsn""
of sugar from tbe blood or from tilt blood, lymph, and t~sue
7-.
 100 THE CAR/IOElYDRA1'ES
fluid Hi t ht, IHO~t lih('TUl (""tlum.te, nt'1l( e tht' rli~app('arnn('("
o

of j.tfy<,o,ltnt from tilt' fj\'('T and ot h('f or~alh of lIofmal

llilimul". a~ f('potic'd h~' mUll)' author...... It may lw that
uftl'r an... n, lIawhy 1-111(1 :;\Iurlill "ll'!"!,!.W"t, thl' glu('o~t, i ...
"iulply oxidiMtl. alHI that tht' dday whidl n((ur . . before t'lus
takf'~ pla('(' IHO(IHUIt--. f(IT t ht failure' ttl odt'd thi~ II}WI10TlH'non.
It i~ to he Holed t hat during the ilrst hour following tht'
injt,(tioH of in1-.lIlll1 OWT!' i!'o fr('qm'ntly a fall in tlw f('''pirutory
'}llOti{nt.

Metabolic Transformations of Carbohydrate in

Simple OrganislllS
It/ It wid~'r ,"UTn'y of the lIIctllholif' tr.'llJ .... fornwtiun ... of
g-Iwo . . (' tlie hud('riM and rdated ,impk (lrg'Hli~nb. :-.uch a~
yf.'a~t, prc)\-idt admirable lahoratorit,:-, for OW'" P1U1K)~(':
~hmy ~i11lpI(' organi<;Dl\o. flre capahl(' of oxidj~ing glu(_,{):-t~
('olllplddy with the prud\l(tioll of ('arhon dioxide and
watt'f. hut it i~ t't'rtaili thut "'l1<'h pr()('('~~t-':o, Urt' ju ... t HS (omplcx
a~ tIll' ... imi1n1' oxidation which 0<'('111'.., 1n the hlJ,!heT animab,
and ttl(' . . impie ('uo. r(-'~ult prohahly {'OlH'('ah a ~\l('('e..,~ioll of
('('mplex rNwtions. In !-.om_t' ('lb('" bacteria] oxidation of
i;.; Hf'r( ... tt'd at definite ~tUgh, and it j:-, pos~ihl" to
!,!h1('(h('
ilkuhfy in\('rnwrliate prmiu('ts, sHeh u\ ghH'oni(' and
gly('uroHil' 1l(id~.. Ii is douhtful if either of th{,:-,(, :-,uhstanc('&.
piny.... any appn'('iabk pan in thp IlUrmHJ utilisation of
Jrlu{'o... t' in the hif!ht"T animal, ~in('t~ both tlwse ::-.ub....bm(~:-,. arc
('xercif'd ullehanl.wd Wht~1I they art' admini",h'T(d to dogs and
ftibbits. It is true tlIHt J.dycuroui(~ Jl{'id h, frequently en
('ounh'rt:'d as ali ('xct'!'tQry product of animal metabolh.m,
but its proouction in such caS("~ dcp<>nd ... ou th(' presenee of
ot h('t ('ompound[o,. mostly of a toxie nature. and L"i t<? be
l't"garded as u d(.>fen~ivt medlanbm rather than a nomlw
feutu['{' of metabolism.
TIl<' ability to split the gluc<"" mole~u1e into two fraj!ments
and to pr()duCt' lactic acid, by a Might atomic rearrangement,
is {'~\C'Ount(>-rcd in nature in aU types of organisms, from
 FERJfENTATIONS tot
til(' lo\\,("t to the higlw!>t. hi g('IH'rnl till:' pro<ilH'tion of
hH'tir arid hy hu('t('ria, II'" for ill ... tUIWt' lladllll.4; iacti", Ot'rt'R('IU'S
and Em'il/us coli, i ... H{f'oJllpuifd hy t}u' fornmtion of dr-
g-rndat ion produef... '!leI. a .. H}(ohol. nnd ('(IJ11polV'~I' ('Oll~
tni~llnJ.! four Nuholl atom~ ~ueh us hutvit'rw g--lyt'ol. \\'('
haVt' thf'n. n ... a widely di,tribuhd n". u)t of tilt" ndi<:11 of
livi1lg ("elL,_ t h(' ~("i"..)i()11 of the ~hl(,O"'(' IHflJc(td, ihto two
thrt"I'('Hrholl portion .... and tile ,tlh",,'qurnt 1rnn ... formntioll 1.(
the:-o.l' fragml'llt ... , hy <itgradation amI hy ..,Yllthl'iooi.." into hotll
..,imp}(r oIHI lJior(' (ompkx. ... uh:-.tanc(':-,. Thrs(' reudioll)oo UT"
th('rcfoIT of ~rcat iut('f('st ill hiodl(rni~tr.\"" Th("y lJiO,\' f)('
da:-.~itit'(l into two group-.;: (1) tho . . t which illvolv(' It ,ri ... ~iOIl
of tt\(' glu('ost, mokcui<' and :-.uh<.;t'<tw'nt In,s of Ollt' rurhoH
:JtOlll ~ (2) tho")!' i.n which ... ('i . . ",ioH i . . fuJ]owed hy de"rrmiatiHH
and !-.uh . . (.,pwnt syntllf:,i.... )11 tht' Uf'oot group is illdlldfd till'
fAmiliar pro<'('ss of y{'a.."t fl'rm{'ntntion of ~1U('os{'~ with the'
pro<iu('tjoJl of (thy} aJf-ohnj and f'Hrhon dioxid,'. ]n thf'
pro('{':-'.., of ulcoholi{' fermentation, hy.produ{"b !'tueh n...
g-IY('c'roi, !-.U('(ini( tl('id, and ~Hllyl akohol an' fornwci in ~lUall
amount and reprfM'nt 'iOIHe teaction oUl('r than th{' main
O1H'. \\'e ~hall rdurn to t}\(' qu('~tion of origin ofthC:" glyC(rol.
hut meanwhile it should be not(d that the amyl akohol und
>.,u(;eini( IH'id arise Hot from {'arbohydrntc', hut from amino
a('ids Hix:rated by Uw autolysis of y(a:-.t prot<'ins, It is now
known that, a~ in tht, cas(' of the formation of Jad;' aeid in
the lUlls('le, ht'xo.,c dipho~phoric acid play ... a fundanlt'ntal
part in the prO<'cs~ of alcoholic f('rm(,lIt:~tionJ a part which
U'(' shaH djM'u ..,s more fully jIJ Chaptf'T \'1, Arguing from
these premi~('s, and from the fRet that tral'{'~ (If laclie twid
(_'tm be dt'tected ill akohoJic fermentations, a t heory ha~ heefl
,,",uggrstt>ft 1n whi(,h tht n}('chanism of tID!. proCf'~s ha.\:O lx,t'n
formulated as follows ,--
C,lI u O.___, 2 ('H,. CHOH. COOH ----> 2 CH,. CHiHf
!- 2 CO,.
(lA<..-tIrArid., {li:th}'JAI~/

A"cordinll to this view yeast, by a ,imple prO'eSb of de-

carboxylation, would convert lactic acid into aloolool.
102 'THE CARBOHYDRATES
So far 110 cOIl\'im'lll,L: .... upJlort ha", b(>t'll ftJlllld fur thi~
tiuory. amI ~,(a_:-.1 do(.':-' flot ('onvt'rt ad(led larti(' acid
to nleohoi ii,' dirt'('f dc('arhox"JntiOll. If the formation of'
('{hyJ .~'ohoi from ladle nc'ii hy tht' lwtiull of yCH:-.t (!oes
(l1't'UT at alt, it i:-. by much more' ('ompkx paths. Similar
(ll;j(~dioll~ npply to thporit:-. involving thl> fortnatioll of
gIY('cric ald{'hy<k. methyl glyoxal, and dihydroxya<'{hHle.
Ot'('a",ional ).,1J('(( ...... ful f('rnu'ntatiolls ha\'c I)(,(;,H J'('('or<i(d with
gJY('('mhlehydf' and. tiihydroxya('l'hHW, hut Xeuht'rg and
(;oft:-'('lwtlk have fl'('l'ntly Tt'porttt1 that, with tht' ('x(t'ptiotl
o1'oJle . . traill, no yen ....! l'XHInitl{'d hy th('m f('rnl(,Hf:.. dihydroxy-
u('('fOII(' u1 H rat<' r('nlUlei~' approachillg' that of glll{'OM'.
A t Iwory of yt'a~t ft'rnH'1Itation which i~ mOf(' in ar('ord
with ('xpf'rinwlltal fn('t~ i~ that whiell po~tulat('s p~'ruvi(' Il('itl
as an interllll'tiiat( product. ~('llh('rg and hi::. t_.o~work{"r~
hav(, d('mon~trut('d thi' prt'S('hC{' of a carboxylase in y('a<.t
whit>h rapidly ('on\'{'rts pyruvic acid to a('etal(lt'hyd('~ and
pyruvi(' ueid ('lUI b(' isolatt'd from yea~t f('rnwlltHtion~ ('on
dtlttt'ti ill th(' pr(,M.~ll('( of (;akiurn ('arbonat(~. A('duld('hydt,
fotlllntilHl can hkt'wise- he demon~trated in y('a~t kt'pt umit'r
nnuerobi{' {'(mditions. which ('xciudt' its pos:-.ihk formation
from ait-olIol by oxidation. ~tor("ov('r, a l1wC'hanism exi ...ts
ill y(tlst wht'rt'by aCt'taldehydt' and many otht"r ald<:hydt"
aft' l'edU(,N{ to the ('orrt'~p()llding u.I('ohol, and it has hl't'H
shown that thl' produ('tion of alcohol from gIu('o~(> in a
,'oteriit' pulp of JX'8S also prc>'{,f'ds with the int('rmediatt'
formation of 3(,(taldC'hy{k'. This vi(~w of the nlf'chanism of
ukoholic fernll'ntation in\"olH'~ a coupled oxidation and
rt"dU(,tion prQ{'~s, whidl may be formulated as follows : -
/O,.c C,H"O, - - > 2 CII,. CO. COOH .;. 2 H,O---->
2 H,O '" CH,. CHO ..,. 2 CUr .,. 2 H,O
2H,+2I'H,.("1I0 -------> 2CH,. CH,Oll.
Xeubel'1! and his s~hool have Tf.'celltly given eonsiderable
attention ~o the deH'jopnlcnt of these ideas, and han shown
ho,,! t h,'y call he correlated with the formation 'If glycerol
by. wast. If the reduction of the acetaldehyde is bl""ked.
 ~4U'OlJ(iLl(, FElIMENTA1'IOS HI:I

n" it i" throngh til(_' formation of udtlitiq' hi . . ulphih, ('OUl~

poulld~ wh(n "iodium ... ulphih i ... nddt..d ttl til(' ft'ruwntHtioll
mixture. gly(,t'fOi 1:-. found 10 U('('\HHU!ath' ill ('Hu_."idt'ruhk
(pmut it Ih. .\ ""milar rt'!\ult i~ ohtnilwd h,' tIlt' adJit ion HI'
"(fifIlCdon .. (tlillldhyl C!lf'lO-h('X3U('.tliolH'): 'I'll(' Ikt'UIHtdu-
tion of ~Iy('{'rol i .. a:-.('rilwd to tht ri,duetion of looOlIl\thiHI( d:o.('
in pla(T of tht' Ht'dnlti('hydt, in onkr that pyruvi(' add and
{"arhon ,iioxid(' may ('ontillut, to 1)(' formtd R('('ortiillg to till'
('oupled Tt'adiml pidur{'d Jihon', Thi,\ PTl)(>('!\... i ... ('ulJl'd Ow
., S<'('IIHd Form of ..-\I<'ollulic F<'rlHclltHtion," Mnd fir(J('t~'(I~
qll8ntitntin'ly ae(.ording ttl tilt' {'{llll\tioll :
t'atl];!0s ---_ .. t'o: t'IIJ.i'llo (H})H.l'llt}H.~HlIoll.

It Wtl ... 'J-;:<'d in (;('rmany for tit(' Jurge .... euk I'rodudion of
.j..!f\"{'(:ruI during the war of IH14-1H.
pr:.. .
-TIl(> (Il{~t' of alkali . . nh,o moditic~ thl' ('(mr!'t( of akoholi(
rtrlH(utation. A1!aill gly{(rol at'('utllulat(,h and tht yidd of
ukohoJ djmjni ... h('~. In til(' PTf\"('fWt' of nlkuJint, ~ult:,;, slU'n HI<,
(urhouutc)., phosplJut(:-., pyrol'h<"phatc ... etc., tllf" tl(,(tuld{
hydC' i., cOllverted hy a Caunl1.zaro reudion into aleohol and
aec;ti(' aeid and. tb hdoTe, tll(' withdrawal of the aldt'hyde
from t ht, r('adion 1:-. a('('ompanied by the produ('tiol\ of tlu.'
mokC"uJar (~quivalf'nt of gJYC'{'TnL This represf'utf.. !\t'utx'rg-'s
,. Third }'orm of .\koholic F('rnwntatiol1/' and h formulated
a ... ii:Alow ... :-
H 2 0 .~- :!(\HttOs'---)o 2(,0:! ''1"- ella.ClI,iHI t t'Ha.('OO}{
2 ('H,OB . ('BOB. ell,OIl.
'''''(. may ('ondude from thi-; work of .sculwrg und hi,
a .......odat(s that th('re h. very good real-ton for ~uppol>!.ing that
ak'ohofic f('rmentation involv(,s the' following ~tt"P:-, : --
Gln~e -+- Pyruvic 3t'id - ? Acetaldehyde ~ Etl.1yl al"ono!'
It -i, probable that the tra""" of I.ctic acid detectable ill
y('ast ferm('ntations are formf'd by reduction of pyruvic twid,
a mechanism whicb is probahly tbat of lactic acid fermellta-
tion itsc)[ '*
Our second group of degradation readiollB in which glurose

104 TilE ('ARBOHYI>RATES
figure.." i!o. thnt ill whid, the' ~ug'ar i.., "iplit Hnd the fl'Mdtiull
frnguwlit'" UTe dcgl'udtd lind then COlJH'rted into more cnrJ}-
pkx prmlud" Thi ... ~roup of fermentation n'1t('tim)'" l-('swh
in Hw ll~rodlldi()u of {'ompound .. (ont.:1illing fOUT ('ar~m
:Itmu ... "m'h a ... hutyl n}c-oho}, hutyri(' Il(id. ft-hydroxyhutyric
a{'id: hutyli'll!' ~ly('ol, !\('dyJ mt,thyl (;urhillol, Hnd diu(,t.'tyl.
At tht' .. anu.' tiltu' IlHllwr(_)u~ ... illlpler ('otllpound~ Illay lM'
formed, for ill~tan('(. a('('ton('~ iadi(' alHl lU.'('tit' lH'id~, ethyl
Hkohol. carboH dioxide, nwtium(', and hydrogen. Fermenta-
tions of thi ... type, whi(,h ill\"oh-t til(' formation of butyl
lIieoht1i. htlt yrie a(,id, {3-hydroxyhutyrit: r!cid, and He('iollt'- -
and t ht,..,{ ('l)ml)()\lIl(I~ arc' frct.pl('llt}y l'rodm(d to)..{d 11<"r in
ttl(' salllt' fefllwntntioll rnixturt'-Hre tl~rrih('d to tIl(' int{'r-
JHt'diah' forlllutioll of H('ctaIdehyde, alit! lHay then'fon' ht"
l'(:',I.!ard{d m, modifications of tho~e whi('h we hu,'(' uJrt'udy
(,(Hlsidt'l"(u. In till' pr(,~('lIt instulI(,(,s aldol i~ formed by thl
(,{lB(}('fl~atioll (If 2 rHo}c<'ul( ... of the :tld('hyd.e. nuc! thi~ four-
{'arhUH {'ompound i ... tlWli tnl.llsformed by oxiduti\'(' and
rt'dudin' eluHlgc'>, iJitu tlu ('Hd~produi't'i mt'Htimlf'rl noon',
TIlt' Mri('~ of rt'aC'tiou ..., ma,v tw writtel) a~ j'oUow~ >--

CH,CHO
ACE.TALDE.HYDE.

1
CH,. CHOH CH, CHO
.A..L.DOl

cH"cH,.c~FH3,cH,1::::;-~,CH"cooH
&JTYL AlCOHOL BUTYRlC. ACID ,8HYOf(,()XV BIJTYRIC. AC.lD

" .
CH.. CO. CH,.COOH - CH.. CO. CH,
AC(TOACETIC N;I[) ACETONE

In t ht- schell'. proposed by Smedley and Lubrzinska pyruvic

acid,is regarded as being tbe immediate preeu,",or of acetalde
 BCTrR/(' PER:l1ENTA'I'lON 10;,

hyd(" and OIH' llIo)t>euk of t'llC'h of tll('"e t'OtllPOHIHt-. is

""Uppo~(d to ('Ondt'lbt' with til(> other to yield til(' l111~ilturut<-d
keton;,' "('ill CII" ell ell, ('0, ('0011, frollj "hid,
u-~ro1oni(' uld{'hyd<' i ... formed. '1'hi~ t'OIllJKI\lnc( i .. th(,11
()xidi..,(d to the ('orn . . ptHldillg a<'id, tllld front t hi ... fl-P UII-
"'Ht urait'd ('(lmpound t ht' ('lIci-protiud ... llN' ft'1whn\. .\1ttliI11gh
~('ulK'rg ha!-. lK'('1l ahk to dt'IHolI ...trnh' till' fOflllalioll or
lu(tuld('hyci( and of aldol hy Uw nl'tion of yt'Il ..,t on potU .. :-.iUlH
PP'u\,ah', h(' wu!-, not ublt to <'ttU ...(' nny of these ('olll}Jound ..
to u1HI('r~o hutyJi(' or hutyri(' feruwututioti. 011 tht' OUWf
hum1, ht WH~ able to ft'rHlcut t1l(' Jat'ioll<' fornud 1'I'1)111 ~
ulOl{'('uks (If pyr\lvi(' ueid, to ~'idd butyri(' ucid, and on thi ..
rt'sult ~t'uh('rg and .\rin ... t('ill -,uj!gt' ..t til(' foHowiHj.! ltH'('hnlli~1II
~for the PI0t't' .... :

<.;H U.H~ CHJ; (.'H~

I' i I !
CO.COt)}l - . C.OH.C()OH -~ i'H-_ -0 ._.... CH~ - Htlt~'ril'
I I CO:, A('ui, di
Cllz.CO.Ci.IOH CHI. -i,.'u/ l'ft ('0

The ft'rnu:ntntiv(> prodlu'tion of th,' thn'(- (ompouml.....

!!::l hutylt'll" gly('ol, (,H". ('HOB, CHOIl "'II,,, IIl't"tyJ
lIlt'thyl c~rhinol, Of" CO, CHOH ,(,H" a",1 dil...d~l,
fH:}. CO. CO. CH a. const;iutf.. . :woth(r . . 'r;c.'10 of dO"Jjly
Tt'Jatt'd reaction.. whieh 1){'eUT throug-h the adiviti(... of
HUDurou." unj('t"Jlular orgunism.... 1"-.I('t;(- 1H'id j}, frl'c!uentJy
t'nCOUJlteTf'd along with th(s( produrh, :wd it i!'. ,X)s:'-.ibh t(1
P8U'-'(' aectaJd('}lyde to ltc('umulHte in .. mh feTJrl('uiation'i hy
the addition of sodium ~uJphjte. Lacb{' neid ihdf f'U1HJOt ht,
fermented by yt'a~t to f.,rive any of t)l('~t' four-<'urhon com-
pounds. wht'rt'as Neuh'rg and Rcinfurth hav(' shOWli that
acclald~hlde added to y"l.st adh..ly f"rlm'ntfng ,ugar i,
eOllYerted into opticaJJy Bctive acetyl n)('thyl ('arhinol. J'art
of the aldehyde which und('rgoes the t'ond{'l}!)stjon ari.,.(s,
ho-wen-'r, from the sugar. NeuberA' Dscribes. t!Jis tyfC of
synthetie reaction to the pr('bent-'(" of an enzyme, or group
of enzymes,. which he caIJs .. carboJiga.sc: As we have yl."H,
Jnn 'I'HE ('ARBOJ/YIJRATES
y<'uo"t fc'rm('nt at ion of pyruvic aeid pr()('('('d~ Hc{"orciiug to til('
f~llntiOll :

,ell, . ("0. ("0011 ("H,. 010 ,. ("0 ..

Thi, fori1-lUt ion of uc(_tald(hyd(_ luke.., plaC'e< hoth in tlw
prC"(WI('P of t}w inhut y(a . . t (dh. I.Hld on the addition of all
(,);1 met ('ontuillill_ll t hi' (arhox),lus<'. If a "iulphitt, j" }'rt... (llt

GLUCOSE

..~j
LACTIC ADO PR.UVf ACID

ETHYL ALC:OHO/~IC ""'-10

/7~ U,,~"=~waN"
auT't'L"'_ 1WTYR.lC~,1~~
ALCOHOL _,. ACID ~ AClb

AC.f.TOWI!. _
It
ACETOACLTIC. ACI~

ill t}l(' ft'rmehtution mixturt', th(' reaction proc{'eds alnlo...t

wholly a('('ortiing to t hf' ahoyc equation; otherwi~e ac{'t y1
Hwthv} ('arhinol is form.ed alung with the uet"taldehvde and
('ar\~n dioxidt" thus:- t.~ "
:? CN,. CO. coon ---> 2 co, + CR. CO. CHOH . CN,
This latt~r typ" of eondell~ation reaction resembles tbe aldol
roncfensation, and, indC('d, acet yl methyJ carbinol is all isomer
of ~Idol. From t he carbinol 2: II butylene glycol would ""
 ACE1'AU)ElIrliE lui
fornwd 01) tf'<hwtioJl, whil.... t diu('dyf would I'('sult frnm
oxidntion.
Th.' fermentatjon Tt'Hdiow.. whieh Wt' Jw\'(> di:,\.(u"i,'otd JUay
l)(~ hrought to~{'th('r in 011(" (ompr{IU'u:-.i\'f' !o.(I)t'm(~4!j\'('n hy
IJ'fHnilo!lIf'l a!o. ~howll on p. 106.
1n gt'llf'rnl, it may l){' ~Hid that mix('d f('rnwnt nt ion ... HTt~ tilt
ruh; thu~ Hit' n. l'oli J.,troup gi\'(' iaet1e aeid tlnd u){'ohol,
t h(' B. Im'tis ael'o~entw group Jadi(' arid, IlJ('ohvl, ami
hlltyl('IH' gly(~ol, the ll. 81lbtilis group butylcl1l' gly(ol. ut'f'tyJ
mdhyl ('urbilloJ, Hnd tra('{~s of a('dolle. whilst ('\'l'fI !'ouch
typical pr(){'Pss(','o, n,o. u('{'tic add nnd p'n... t feruwutlltiolls nmy
gh-(' rj ...,( to the formation of :H'l'tyJ mt'thyl curl)iuo).
The g'('lwral infer(,tl((O which OIl(> m!ly draw from tl1i:-.
":urn'y of glu('o~( utilb,atiou in the lower form .. of life i~ that
B(,ftI.lJdd\yd(' would "'("('Ill to ()('('upy a position of ('oll .. idernhk
1mpnrtau(,l' 3~ an illt('rln(:'(liut<~ product figuring in n larJ..,"
alld diversifi(,d ~t'rits of r(adion~. Xeu\)('rJ,t and hi~ s('hool.
ll'..iTlj! the :-,ulphite uno din1l'clon .. fixation ., method.." Jlu,('

rN't'ntly en<i(,ltvonr{'d to deOlon.strat< the formatiou of

ut"{ta]lJf"hydt' in tll(' tjs!o.ue~ of hjgher animuh. and }UiV{' mt't
with (otlsid(rahlt :',ueC'('s~. Thl1~ N('uhcrg and Gotts{'halk
have :<.hown. that the sur\'iving (,(lJs of a pulp of J,(uioeR-pi,,( ...
liver ('ontain uc('taJd{;hyd{' which call he .. fix(~d ,. hy the'
addition of eulcium hydrogen sulphite. A pr(;paratioH of
rabbit"s liver. after dryin~ with (1('CtOl1(' and ether, will also
fnrm :u:~C'talddlyde from pyruvic acid. A reaction whieh
u('('taldehyde, in common with many other Bld('hyd('''', under-
goes in pulped ti....,ws is a conn~rsion to the ('orresponding
acid and aJcoboJ, a,_') in t hc Cannizzaro reudion. Parnas halo.
suggested the name H aldehydemutase'~ for the enzynw
su,ppo~1\ to Iw responsible, and the freqm'ncy with whi"h
s!leh conversions of aldehyde occur in the animal hody
suggests that they are of some biological importanc(. Other
investigators have shown that a,'<>taldehyde can be dett'd,'d
iu blood and in sun-i\'ing- muscle tis..'Oue. It seems ptobable
that in these obsen'atiOIls the acetaldehyde has arisen. from
lo~ THE ('ARBOHYDRATE8
pyruvic' fH'id, or from "'Oll\(' (')o ...<'ly rdat<'d ((Hnpoulld. Thi:-;,
of ('OUT,,,,t', dot, ... not lH('ltll tiJ(1t nne ('nn <l~... trt l;l('dalcl('hydr to
lw lIl1 in1l'TUH'diuk produrt of ('arhohydrat(' ()xjciation. :-.in('('
it llIu . . t~' n'rlu'm}wred that pyru\'ic :lejd ('1W ht, fornwd_,in
;';,'(/ ft(lrll Hmin(, iwid ....
Ih:rt \\t' mu ...t It'an' t hi ... pnrt of our .. UbjNt at the' Ill(HIl(,llt,
lwd from t lu' tllav. . of un-lJlahh' p\'id('ll('(' the rt'lHi<-r mu""f
. . (It'c't foJ' hiltl,,(,lfthat tlH'ory whi('h \)('..;t H((ord ... witll hi", t)\\'lI

l)rl'dihdiOll~.

References
I. lh;, ElilCT and C'o-w4,rken-. ./Ollrtl. I\iol. ('''Ht~" Hli t). 20. 6:~:1 :
J H2!!, 54. ,j!),'1. til) I. OO:J.
.} IIARI", .. thitiatiolll' aud Hedudioll!<. ill t}w AllillHl..l Bouy,"
LU1Idnn: Longmanl't. (~l'{'ell. & ('0 . 2nd Edition. 1022.
:t. EltlHH~!'I and ('owork(>n;. Zeit. phl/~iol. ('h.M'n . IHHi, 88. 210.
4. PlS('JU-:rt. Her, dt!r dt'utsrh, ehf'"ffl.. Ges .. IH'4. 47. 19S0. "
;'), If ,lIW};\". .. AI('lJ}lt}lit, }'truwntatioll. ,. I.ondlln: )..ougmantol.
(,rt'f'n ~'\: Co .. ani Editioll. 192:t
ti, H.HVJ.EY auu ~1l'IU,I!\. An~t'r. ./OItTU. 1'Itllliiol .. 102;;.7&. 107.
i, HAUTltEi'~ and HILL. 'oum, }'hyst(ll.. lU'22. 56. 3U7,
N HAWOR1'1\ and tn~work('fl'._, .!oum. ('I,e-m, So,' .. IH:!4. 125.
UfIS; 1925. 127. :165; 11126. t29. H9.
H. Hllt~T awl ''A.\\()rk"n., JOlL'nL ('liNn, ,~(lr . Ht2:1. 123. J:l;,)2;
IH2~1. 127. :I[.~: 1926.129, 22. ~lf")(t.
W, Jun:-a:. Pr~, Addre~... ~{\ti(ln n, Brit. ASSUl'., 1~22,
I I. htYlX\i, }<lyt'FE, and HOGG. JO/un. ('kern. 80(;.. HJJ;"i,
t07 ..524.
)]a 1\ f:RMM'J\. LUUUJ:. aud SCLAn:R. Bwchem.J t))lr'n.. Ht26,
20.
12. KnOGH a.nd LlsHARn. Bitx>Jt,..m. Journ . 1920. 15. 21)0.
1:1. LAQCY.U "lid ~IEYER. Zrit. phy.iol. eMm .. 1923. 124. 211.
Ii, l..1f:Mow!\'E. Hull. Sor.. f~him. d~ Fra'fWc. 192~). 37-88, 1089.
H . J. . EVE~}; aItd JACOBS. BeT. der deutaih. chern. GeJl., 1910. 43,
3141.
Ht LEVENE and ~IEYER. JQUrH. BioI. ('hem .. 1924.60. 167.
17. LiNG and XANJI. JOII/'''N. ('htm. Soc., 1923. 128. 2666; 1925,
127. 629. 636. 652. ,
18. MACBETH "nd M.'CKH. Jau,.... ('hem. Soc., 1924. 121>. 1513.
19. MEY1<mnO}" LOUMANN~ a.u<lltEIER, Bit>enem. Zeit., 1925~ 157.
4511.
20. Ky.!,. .1"HaI." d<r ('h_.., 1910,376. I; 1914.403,204.
21. NEUBERO and oo-work6N. Xumerous papers in B~uc1te
'Z"t.wJorifl f ....m 1911.
22. PltYl>E and ooworkf"J'iI. Journ. (lhem. Sl)C.~ 1923. 123. 1808;
l 1925, 127. 348.
 REFERESCES I nil

2:~. SCIfRYVEU and t"n.wHTk~n;. UIQCJH'fU, ,/1)1(1'''., 192:t t7. W3,

Hl7.
24. ~IU."'}'.:R. "Jut(,flHt>diarv ~htaJw)liilrll of "arhohydra\f!'I.
Ph,1J8u)l, RN'Uu,S. J 923. '3. :HH. .
25. ;-1.r.u:nu;y and L(BRZI~SKA. liiMhNJt. ./(Hrrll., 11%13.,7. :r7;!.
2ft. STA~"\K. l1i.odumt. Zeit., Hl23. 140. 120.
27. "'ARReRt; :tnd 1 ABt',';OE. BtfX"Il"m. If'lt . Hl2.t. 146. :JSU.
 THE BIOCHEM.ISTRY OF THE FATS

}'f\T i~ fmuiJinr to the hjologi~t ll~ tht, chi(.f form in which

\'X.\'~A"",", \om\....tu({ i .......tu\"t~\ in tnt' ti~~U\'~. In Hw anhnal ih
rnlt' ill thi . . ft, ... pet'1 i~ m\wh murf' import;:mt than that of t)}('
(Hrhohydrah~. Thu~ ill mi<'c tIl(' r(_'!-o('nT fat may h:1\'('
hn'nty tinH ..... and in ehif'k:-. fifty tim{'s, the (,tI('rgy "';-tlue of
the TC'ICrn' (arhohydTl1tf'. In tim( ... of ... tre ........ ...,uch R.., ... tan'H-
tion, the rt':o.t'fV(, ... upph(>~ of sugar art' soon ('xhuu,{(,lf. HBd t Jw
organism i~ thell dqw1l(it-nt on its re~{'f"'(' fund .. of fat for tlh'"'
suppl~' of ('uergy f1((,(s:-.ary for it ... maintcllf{tl<'C'. Thi'i Nj/e
of tht' fats as tlw ehit'.f form of f(' ..,t'rY(' fuel in the animal i ..
sutlki('ntly important. hut at the pr('~('nt time the hhxht'mi . . t
is tt_'nding rtloT'{' and mOrt' to ascribe to ttl(' fats l\ fUl\('tional
imporial\('(' ill "itnI pheT10Ilwna of mlleh wider ~ignif1(an('\>.
If we r('g'urd Hs futs not only tho~(' neutral (omwmnd. . of
glyc{'rol and the' fatty acids to which the nam(' i.., familiarly
(h,... igncd, but aJ..,;o aU typf'S of compound ... in ''''hich fatty Hcid")
(x~(tlr or with which fattv adds nuw ht:. asso{"iated in th( cdt,
the' ,ltroup a~sunl{'S an it~lportnnt'{_' in biology which it would
flot otherwis(> po~s('ss, Adopting this wirl{~r vicw, w<' rcquir('
a t('rm which will includ,(., all forms of " fat ., of phy~iolog:ic3l
,ignitknn"". Tho Inh'rnatiollal COllgr." of Applied
('hemi",try has s\l~g('sted th(' ternl H lipidf.'," and Bloor ha~
f('('('ntly f('oommended its adoption, giving- the f01hnying
das.~jJi(,Htjon of tbe compowld.., includ("d in this 1!I'OUp : -

CIassi1hlatidn of the Lipjdea

Li~des al'f' ~ubstanCt's huying the following ('haracter~
ist\t'S :--
110
 L!l'/J)ES III
(a) Tlwy a1'(' in~oltlhl(' in wah-r, alld "rt~ \nluhlt' ill htt
.. oh'c'ot:-. ~ll('h It!>. ('tlwr. dllornform, and ht.H1.t'Ut'.
(h) Their r('Jution"hip to the' fatty Jwid .. i .. that of nil t' .. "'r,
('it Jwr uctual or pokHtinL
(c) They ure uttli.,,(,d h~' Jh"iJlJl or:.r:Uli .. n' .....
I .'Ump/r Lil';fi.f'.\,--,,}<:~te~ of the fatty a(,ld~ with \,ltt'iou ..
alcohoh.
(I) Fah-- -i~!o.kr~ of tlw fatty a(id ... with giY<'tf(tl.
(~) \"nx('~-'--~'!'o,tt'r~ of the fatty ueid ... with nl<.:ohol ... oth('r
t han ~I y('(rol.
n. ('mupmOld lAiJ1itl.('!I.-Jo:...t('r~ of tht' futty 11('ldo.. (,0I1tH11\*
info! group... in ftddition to un alt-ohol and 1'1-\tt Y Heid.
(1) Phospholipidt:!o, or Pho~ph3tiOt'~ ... uh... titutNl fnh
(tHltaiHing- pho~phori(' H('id and nitruf,{<'II; ItTilhin
k('phalin, and ... phiH~Oll1y('lill.
(2) GlyroJipiti('''t-,-(,'ompoHod!<l of tllt" fatty add ... with
a (arhnhydrat< und (,()lltailling- nitro.lltll hut no
pho:-.phoric il('jd ; (tr('hr(,..,jd('~.
Fl) .,\minoJipidc.... SuJpholipidt'!'>, dr'. "~f')'OUp~ n'hic)' JUt'
at pr{~('1lt not .'\ulfi('if'utly well charnd(ri ."ed for
da!io~if}('ation.
III. IJt'ri'l.'rd ]"ipifleS. - Sub~tan(,t":-. d('rin'd from the tthow'
).,'rollp' hy hydroly,is.
(I) Fatty acids of vurious ))('ri(~s.
(2) Stt'rols---mo~tI~' ukohoh. of hil-{h TUoI('(:ular weight~
found jn natll1f~ ('Ombjncd with tllf {'aH.y acid\. and
which are soluble in the fat ,olwllt.; ehol~,l<-r()1
({'"H,.OH). my""yl ak"hol (C,.Il",OlI). ('('\yl
alcohol (ClOH'30H). etc.
The mOl,t I!enera) physical property of the h>TOUP or the
lipides ~"'their ,olubility in th~ so-called fat solvents. But
n;n this property is not an absolute on.'. Thus lecithin will
form ("olloidaJ dispersions jn watf'f wbieh approximate to a
true solution. Other members of the /(I'oup are much Ie..
""'uhle in certain rat soh-ellts than in others. kci~ill i,
insoluble in aceton". kephalin is but slightly solub~e ill
 112 TifF; lIl(l('HEJfl8TRf Of' THE fATS

al(ohol, Hnd ... ph11l,Uom_yefin and t he (t'l'('hro~id(~ nr(' ~olubl{'

in dllt'r H fypi(':!} fat ..,ofn'llt with di{Ji('ulty. Th(' mdtin~
p(lint of 01(' Jipide' i... a phy~jcal property (If ~onl(' interest.
In .L!cllf'1\!1 it i ... fOlHHI that thf' melting point of a gJY(,f'rid('
or " mi,t 11ft' of gly("('ridt'~ b. higlwr than that at , . .hich
it . . oridilit . . (In ('oolinJ,!. Thu ... h('('f fat ha<;. h(>(,11 ohtninrd
flIdtiu,l.! ut t!h,)-' and ~olidifyiHJ.! at 3(),'; a huttPf fat Illay
nwH :It :J.j.. lj mal .\oJidif~ lit 22,7'. It i.~ found, how(>w~r.
Umt jf t}H' Jjquid fBt i., aJlllwr-d In ('hnnt!(' to the <,olid form
n'r~' :"I(lwl~' the lH(ltin,c Hno ..;olidifyillj! points Ilgr('(' to within
Oj to ().~', The ('Xt(,lIt to whk'h til(> liquid fat i" ht'ah"d
al,o\'(' it~ ni('ltih~ point al ...o :o-(,(,fli'. to play '-(Jill(' purt in
(htct'llliuIlll! HII' tt'1lI1wr';lturc at whidl it wiJ} ~olidify EI:;tain.
Tht' ri'('{'nt work of ~\dalH. in whj(}J a study \VUs mndr of tll('
twh}wlonr of thin tlhlls of fatty R('ict..; and thrir ('st('r~ on tht'
. . llrfu('(' (If WU1{,f! ~how... i hat bo1 h Iiqujd nnd solid film.s may~.
he ohtnitl('rl Il!-,ttl~ one and th(' ">n1(' ('ompoulid. Thus
puhllitie arid. normany . . nlid up to a h'mpt'rahlT(, of H2:6".
mm
forrll'. "' litluirl on wa\(~r at ordinary room tempt'future
iftl\(' Pn ofthl' water i~~) or I( ....s. hut the film may lw ('aul.;('d
to ~olidify hy the llppiiellfioll of H latera) prc . . surf' of sufficient
UlUllnitmj(,. TheM' nh. . crnltion . . :trf> of ,ar(>at inteT{A .... t whf'TJ
Wt ('a1] 10 mind thr fact that many form!"> of fat oc(,ur in the
liyil\~ ('dl... a~ sHIHHi(.ro~(opi(' ag_1..Tf('gut('!>,. and ar(' present
in llH'tiia whil'h are prl'dmuinantly aqu{ou"i. Su('h ph('~
nonwna, tog('tlwr with the ddayed ~olidifi('"ation already
rcft'rrrd to. may hdp to explain why many fats in the 1i\-ing
HnimAI havt' mdting poinh. ('oJlsidf'rflhly aboyf' hody
t('mpc:ruturt.' .

Th~ fatty acids which occur in nature may be grouper

..
under the follo,,;ng series : -
(1) The saturated straight-ehain series of the genera
fannltl'd C.HO .. in which are indud.,.J all the fatty acid,
rentai,,;ng 811 eYeIl number of carbon atoms from 2 to 80
 PATTY A{'Il)S 113

TJw arid ... with 1tI) U1)f'H'n JIlltllht't of f'Hrhou HfuHI... do not
o('{'ur ill natutf', Pulmiti(' aC'it! with 10 ('arhnn litoru~. and
... tenril' lH'id with 1~. art' tht' 1lI"..t ilHportunt llH'lIIi)('r, uf thi"
~~ I
(~) '1'11(, tllI ... atuTat('d '\'ri~'''', wiih the' ).!t.lwfal formula
('nIl:!,- _20:!, i.t, . with OIW un ... nturakd linkttJl(" llypoJ!t.j(,
lwid with Hi,okie u{id with lS. s.radolt'i{' Held with 20, :lIId
('TUcI(' u('id with :l2 ("ttr),oIl atom .... aT(' Itwlllll('r~ of tlli"
"'l>ri('~. I...omt'r... of t hf's(" Hcidi' wit h t h(' douhlt hOl"t in
differeut po:-.itioJl'" llIuy o{'('ur. Thu . . oleie u('iti, whi('h j",
th(' mo~t wi(h,:'Jy dii'trihllhd fatty ndel ()(*{'urriJl~ in natural
,oure{'s, Ilormally ha~ tht douhl( bond f)t'tW('('H the nillth
HlHl teuth ('UThOIl atorH, that i~. in tht' middh- of the ('arholl
~'hujn. '1''''(1 otlW-T 11Htural1y {l{'('UrriHJ.{ i~om(r:-. hu\'(' lK'('11
rt'poM('d: OlH' obtaill(d fronl pi/-!""'; fi\'('"f, with tht' 11Il,uturah'd
)"lkll).W bet\\'('('u tIlt' twdfth ~\1Id thirteenth {"arbun ntmul.,
from the (arhoxyI (,lid of HI(' ('hain, alld tht oth('r frolll rUJH:
oil. Tht' ((lIi ....titutioll of tilt iattt'r a{'jd, {l:!.li<.'d rupi(' a('ill.
i:-. l1nkliowll. S('\"{'ral otlwr i:-..t:m\C\'~ huve \)('('n pr('partt\
artilkially. Another typt. of isol'lIl'ri ... m whieh thb. lo,{~ri("", of
aC'id", nHl~' exhibit i~ of the sam!' natun;' a::. thl' ci~~trull' ..
i,..,onl('ri~nl of fumaric' anu nuth.. ie a(id~. TIl(' bOHler of thi ..
t~T)(' whi('h (:orr('spollds to ttl(" normal form of oici(' ucid i:-.
elaidic add. The (,()IlV('rsion of oleic acid into dajdl(~ tLcid
i:o. (ff(,(,ted bv trt:'utmt'llt with the fumes of nitrou:-. acid, or
with :-,ulphu;ous acid, phosphorou:.. a<:-id, or pho~phori(' add.
Jjra~sidic add is the ci",~tran~ isomeride of ('rue;.' acid, and i!o
obtained in a !ooimilar way.
(3) The doubly unsaturated acid, of the ,eries C.H,._ ,0 .
This is known as the lillolic or linokic acid series. The kIlOWII
aci<.b a~1U isomers of an acid with 18 carbon atoms, and are
found ill lin",,-><l oil and in the phosphatides.
(4) The trebly unsaturated ,cries C.H _.O. These acid,
are again found in linseed oil and the phosphatide., and
likc\\;se are isomers of a ell acid. This series is kno:U as
the linolenic acid series,
114 'I'H~: Hf()('HEJfTSTRr Of' THR Plt1'S
(5) The Ull~}lturHtl'll ~('ri(' . . l'flH au ._ 80::. Th... atid ill thi ...
...('rit"", with l~ earhoH ntom~, dupadonie urid. ha~ heen
i~(}l;{t{-d from lJ .. h oi1s, \,'hil ..t that with 20 cluhon HtoHt.."
arn('hidt~jc add, ha" h('('1\ i . . olatcd from )j\'t'r and hn!iH
pho~\)hatid(.'~.
(6) Tht' :-.utufuted hydroxy H('id ~('ri('s (If tht: furmuht
(,,,liz .. 1()2(OU). In<'iuded ill thi ... ~f_'ri{'~ i .. t}l(' .aeiti of tilt'
I't'rthro:-.ide phrt'llo\ill, namely, phnllo... itli(' J.H'id, to whidl
t h(' ('onstitutioH ('211HllOO~ ha~ i)('('n Hs ... ign('d. It' this formula
i ... {'Ofn'('t, it would appear that the ful{, whieh hold~ in tht
jll~tnll('('~ of 111(' Hotl-hydroxyJutcd fatty llcid ... ('oll('rrning
the c'ven munl)!'f of U}f'ir ('arbon atom .... doe,> Hot nppJy ill
tht' ~alll(' :-.trid way to th(' hydroxy a('id:-..
(i) The Ml.turati'd dihydroxy H(ld::-. of the st'rial formula
(',II,,, /l,(OH),. In this ,eries we ha\'c dihydr<lxyskaric
~H'i(l. which f)(('UT .... in l'u-;tor oil. ~Uld til(> lan(){'('ri(' a('jc~.
(' 3011600 4' of wool fut.
(HI TJl(' un:-.atufHted hydroxy adds of the "'erie:-
l'H1l211. i)i1(OU). Rieinok'i(' acid, with Hi ('urbol1 atoHl~,
helOTlh~ to nIb series, Its gly(,,(ridt is the main (_'onstitut'llt
of l'H.:-'\Ol' oil. homer!-. of thi~ add art' known,
(0) The t'ydic fatty add ... , Y{'ry little is known of tht.s('
H(id:-; H' yet. Chtlulmoogri(' Heid and hydno(>arpic add,
C18I-I320t> lutn' tlU' sumt' composition as jinol1c acid. They
oc{~ur in Chaulmoogra oil. whi('h has rC('('ntly been used a~ a
thf'rt\peuti(' agent for l('pI't~y. The constitution as~igned to
tlws{" ('ydic ti('ids i~-

ff('II'
eli' '('H-(CH,). eOOlI.
I I
f"H'!I:-__...._..,CH:f_

In the animal most of 1h" 010'" highly unsaturated fatt

ueids o('Cur, not as triglycerides, but in the phosphatides, <
as esters of cholesterol. Acids of a higher degree of lUlsatUFl
lion than thllt of oleic acid are not usually found in the f.
 T RTaL JTKRI m:s 11."1

~tort.'" of the allilnal hody, with tlw pO'i,ihlf' ('xt"'ptioll of Hi('

lin'T. In th(" plant mallY hig:hlv lltl"aturnh'd fatty u('id,
(){'('IIr 1"1'- tri.!!ly(t'ridt ... ill ~'('~dnhi(' nil .... 'lIcll n~ lill~~rd nil.
By.tll{' pro(( ...... of ('utnlytie h~'dr()gt'll11tioll it i~ J'H!...ihk to
('ou\('rl liquid UHi'luturat(d iJU'tiihk oil ... of pl:lIIt origi1i~
{'.e., ('otton-s('{'d ojl, iuto ~olid fat . . , \\'hiC'h nft' utiliM'd in tht,
animal hody R.~ well a.... the tlatllrall~' hurd futs. The d(t('r~
milling i'aetor ill rdatioll to tll<' utili~atioll or otlu'rwis(' of u
fat i ... ib melting point; tht' higl}('t tll(' I1wltinJ! point t he k ... ~
likely i~ til{' fat tfl l)t, metuboli~('(l.
A tnglY('('riu{' may ('ontain Ulf('.' difrt'rt'nt futty add
r(' ... idlH~, (lr two of t h(".,(, IBny lX' the snnu' whilst til('
third i.., different, or, Ilhtly, uH thre(, fatty 1H'iris ma,v tK'
Ule ';'8nw,. TIlt' natllnilly o((_urriJtj.! fab. ...('('m to ('oH~i ... t
pn'<iuminuntly of mixed trigly(.'(;'ridl'''l, that is to !'>uy. til('
hf:"t two po~:,ihjljtics arf' more frl'qU{'UtJy f'Ilcountt'Tf,d Huw
is the latt(,T. "'(' shall st't thnt ti)(' pho!<.plilltidts, whieh "'.'
. . hall di . . (lh<; in a later dtaptt'r, art' (om.titutNt on ... imil.tr
liJlt'~. in that OlW of tJlt'lr futty add r(',"Juu(,~ i . . "!lfurah-d
whi!::.t the other is unsaturat('d. It i~ appar{'nt thal in
mix('d triglyc(~rid(':.. of the typ('~--

ClI,OR,
, I
CHOIt, <lr (;HOIt,
i I
CH,OIt, !':H,OR,

the central carbon atom is asymmetric, and therefor(

,uch glycerides might he expect<c! to exist in optically
active forms. Mall), attempts hayc l)('en made to obtain
optic.ally -active fats, hut without SUCCC~&, Wt}(>'iS on('
of the constituent fatty acids is itself optically acti\'(',
as in ea'ttor oil and Chaulmoogra oil. A rt'(!ent inn,-
tigation of this qllc,tion is that of Bergmann and Sabeliay.
These workers had difficulty in resoh'ing eye" such markedly
asymmetric compounds as glyceryl monolaurate and glyct':oyJ
8-.
1 J(l THE UJ()(,IlEJflSTRY OF TIlE F.4TS
IllOlII).,t('aratl', ('nlJ"CO, 0('11, ' ('BOll. ('II,Oll Mild
C"JI",.l'O (JClI,. ('HOII . CII,OH ft'.'IX'(,ti\'dy, '1'1",
))OJ)~"IH'('(""" ill tlw j'a:-.f' of Ow IHw,h le ... " IWlrk('(Hy
m.yulIw\rif' trigJyi'l"rid{ ... i ... , tlu'rt'i'ol't', not . . . urpri ... ing.

The Cerebrosides
Two l'q)T(',"'ClIiat ivf' ... of tlH'''{' ('olllpound ..., whic'h art' da~s('tl
H" gula{to]ipidt, .... art' kno\\-'Il, nall)!,}Y. phrpno. . iJl alii] kt'ra~ill.
T)wy eontlliJl thc hexo-.t' ~ugar g)tlado~('. tht: hase sphingo-
"in!". atul 11 fHtt Y l'wid. The fait y lwid found in phreHO.,in i~
phrcllo. . ini{, m'id, which we }P! H' already 1)WntiOIwd. In
k('ra~in if..; phH'(' j." tilkt'n hy lignm:t'ri{' acid, , ... hich bdon~s
to thr nornwl ,...iuratui fHt1y ;H'id ...{'rip,... awllm:-.. 24- .'arhon
utom..... The two ('{'TPhro:-.ilies art' of con...ideTahJ( intpl'c' ..,.t
011 ac{'nunt of their o('('urn'nce in lar,!.!t" amounts in br~l~n
li:\sut'. ~lor('oy('t. tJw ('omhination of faUy acid ,and ('arbo-
hydratt' i . . Hot a tommon Out' in nutuTe, }lor b thl' hexoM'
llu1nt'io,..,t' known to lI{'('ur in any other t'ombinutioJl ill tht,
tis~Ul':o,. The galaC'to. . e of t)l(,,~(' eorupounds i~ linked through
ib. J"{'lhu:ing group to th( ha~t:' sphingosint\ aud thj:-, latter t. .
ill turu linked to thi' fatty acid. TIU' t1uthor and ~lr. }IUlu~
pIney . . han' Tcc(,Htly bet'H able to demou:o,trat(' that the
gahwto.\t h pr(.... ('nt in ih umy1i'ne oxidi(, form, that i ... , in
the ~mne form RS i!-J found in the di:,.aceharidc lactose and in
~tab1c ('ry,.,tnlliHt~ galactose, The significance of the eercbro
sjde~ is as yet unknown.
Th,' phosphatide, al't' dealt with in Chapter \'1,

rndt'r (>f'rtain eondltions ingested fat may })(' hydro)yt.e

tu !)om(' exteut in the !.-;to.mach, a'> was first d('roonstrated b
Yol!>arcl ill 1900, Subsequent observation tend. to show tl"
a ('on>iderabl.. part of the lipolytic activity of the gash
cor tents is due to regurgitation of pancreatic juice from t
 LIPASE 117

inh,tiur. But th(' whok of it cannot 1)(' fi,;('rihcd to It t't'ltux

of duodenal ("ontent.... Of til(' T<'"iduuf 1i1l:IS(' Hctivity. "OIft('
may he <iUt: to un { .. t,t'ra.;,(' rath('r t hun to a trll(' lipUM' (st'4,_'
~) . 121).hn,t ~t .. mall amount of th{' lHtter df)('~ ilP~)(~"ltn o('rt~r
IB g-u ... tn( JUI('{', Thu"o t Iw f('('('nt work of \\ lll ... hith:'r awi hl"-
{'o~work{'n. f' ... tabli~h('s th(' prt':o.('n('(' in gn ... tri(' lipt\.,~ ., Hf
a tru(' Jiplhf' n"i di"tiJl(t from nil f"tt'ra"('< But OJ{' ..,uppo.;.td
clirrCTt'Il('(_' in the: optimal hytlro~{'n iOll {'olwentratioll for tIll'
aetion of ga"tri(' and of pall(,Tl'ati(' lipn:o.( j ... not ... uh...tulltinhd
whell the pr('paratioll~ UTe ('ar('fl1l1y purifittL Th(' only dir('r~
j'Il('(' lW't\"Wt'H tIl(' two lipast's whieh \\"iH.,tatkr wa ... uhh-
to delllon ... tratc j, in tlH'ir action on inadi\'(' manddi(' and
phr-Hykhloracdi<' 8(id ('!-.\ns. Pall('r('atic lil'u~(' yit'ld .. th('
JaTo-rotatory and g-a:-.triC' lipa-.(' tht, (lo'lro-rotHtory fwi(l (1)
h."(1roJy"p.-, of th('~(' ('''iter", and the two Iipu ..{'~ would tll('rt'~
jJlf{' ~('('m to pn.,s-t:'''.., H eertain d{,gJ'('(' of 'itt'r('OC'h{'mical
"'p(('ifil'ity. Gastri(lipa ...C' is d('~troyt'd in Hfkfll mlllut(S hy
(1-02 per ('(,Ilt. hydro('hJon(' H('id <lnd it ,,\'CHild upp<'ut that
t

ih adiol1 as 11 lipolyti(' agf'nt (tlnnot therefore lw \'(ry

importallt. If, how('ver. til(' fat is ftc) in flll ('JJ)ul",ifkd form,
a ... in milk, and if "'lIt1ki('nt pro1('in i" prc ...('llt in tl11' gn.,t.ril'
('ontt'nts to low('r tlU' hydro~'t'n ion ('OII('(:lltratiotJ ('on!-tifi(r
ably, fat hydrolysis in the ~t{)ma('h may then b(' fairly
extensi\'e. Thi~ is prohahly the caM' in young mtunmab
ingesting ('on~iderahlc quantities of milk, and may aho O('-<'UT
in pathological conditions in which hydrodilorie acid !.t:(>r('tion
i~ dtf{'ctiV(~.
Th(' hydroly~is of inge...ted fatty material is carrivd out
chiefly in the alkaline- medium ofth(' intestiIl<'. By the action
of the pancreatic lipasf> in conjunction with f'muJsifying
agents, ,principallv bile solts and the ,oaps forined during
hYdrolysis, the ;cutral fats, whi(,h comprise the bulk of
ingested fat, are completely hydroly,ed to their (:omponent
fatty acids and glycerol. These hydrolysis products are then
absorbed and resynthesised to foml neutral fat in the >11111 of
the intestine. The resynth""ised fat d"", not enter, the
 IIH TilE BI(I('llEJflSTRr OF TilE FATS
hlHOd ~tr('um dir<'dly, a', do ot her food ('omporwub d{'riy(>d
from protein ... anti (nrhohydrat{s, but pa . . M~ into tlw lym M

l'hati<' \'(:-. ...ds and ('\Tntually T<.'a<'iw . . tht blood ...trtam

rili UH' \!lOral'i(' duet. Thi .. met hod of absorption proh'l~)ly
(kpcnd!'> 1f! ~onw nu'n",ure, R"i Blour ha~ ~Ilgg{'~kd, Oll the
iu<.;ol'uhiJity in wakr of fIIO'.. t fllrlll~ (If fut. 'l'twrc i ... no
{'\'id<'lw(' that unhydrol~'~(>d fat i~ nh... orhcd in appr('('iabk
;unmmt. Sub. .tnne('!', of all oily nature wllieh ('annot t){'
hydroly ... tt\ hy lipase, awl ..,0 fendt,ft,tl ~oluhl{' in wateT for

.flU. In. Rf''-'f'noibility of &('tjOD of RipinuJ<.lil'lUl(' fm gJY<'t'ryl

triol('att'. (AJu.'r Artulltl'VnJ!: atld Gmmey.)

ttwir pa~sHg{' throug-h til(' ('pitilt'linl celis of tlw il\tC'~tinal

wan; art' not absorbt~d t~v{'n though t hey arc emulsi n(_d. The
immediate re~ynth('si ... of the neutral fat in the wall of the
intestine is brou~ht about by the sanlt' enzyme, lipase, which
proouc{d hydrolytic clea\'",!!" of the original fat in tile lumen
of the intestine. The romplete re\'Crsibility of the action of
lipase in respect of its hyqrolytit' and synthetic functioll.' has
m'en demonstrated by Armstrong and Gosney, from whose
pa~, the eurws shown in }'ig. 10 are taken, The lip"'''
'''<1 was that of the castor bean (ricinus lip""'). It w."
 REl'ERSJRDE REACTJOKS Ill!

,('{- to nd on the following ~y ... klll'" unOt>t ('onditi-<m .. (ltht'rwl"'~'

('olllparahle :
(II I ~Iol. Oliw Oil Trigl~"'('ri(I,' ,,9 ~Iuk \ruIn.
(2) 1 ~lo1. Gt~-~'('rol ~- H Mol~. Olt-if' .-\('iti () :\11.t, 'Vutt'r .
(:l) 1 ~Iol. Trig-I~'('{'rid(" ~ (i ~lol ..... \\'nkr.
(~) I )101. (;1.\'('('1'01 ',:l )Iu/,. Oleie Acid .i- :1~I"k \,I'ukr.
j;'i) ] ~IoJ. Trigly(,pridc L 3 ~Inl .... \\"utfr.
(0) 1 :\101. (;lY('croi -"-:J )101..;, Oki!' Acid.

In t'aeh of tht"", "y~t(,llh the P('fl"l'lltng-(' of frt'(' lU'id Wa'

'ldermilH'd and plotted ngain ...1 tllm', l\~ ~hown in Hu: ("rH' ....
whiph h{'~}f number .. ('OrTc"'polldifl,l! to tht . . y~t('m ... M'in'H
nhO\T. In ~y ... t{1tl ... (1). (:J) and (1;) the fr('t' :H'idity to.,.,' to
all ('quilihriulH YHlm.' icil'llti('al with that likt'wi..,t, rearilt'd ill
~ the ,..,y .. a,U!s (2). (.~, nnrl {OJ rc">p(eti'\."e)y, whj('h Ht Ow
[OlHtH('lu'(,1Hent of Uw {'xperim('nt eontuillt'd fret U'ly((rol
and acid. Th('~(' ('xpt'rlmrllt-. demoll~trnh' "pry (~l('arly thnt
tIl(' rc]ahn' amount of "",,(ltt-'f PTt'<"('Ht jJl t'Reh sy\hm ddi'r
mined th<' filWI po..,itioli (If equilihrium Tf'R('lwd under ttl('
action of the Jipas(', irrhllj.(ti\'(' of whdhc'f til(' initial com
l)()nf'lJb "n-'rf' g-JYl'{'roJ ~ltld o)ej(' H{'it] or trioJf'ide, Curve .. (7)
and P~) demon ... trAh:- Uw dr('ct of 8n additional mol('cult- of
glyC"t'TOI in altering tht ('quilihriulli position hy fa\'ollrlllj.!
:-.ynthe.sj.... The sy~tf'm!-> in tht'M' jn_.,tan('(~ ... Wer(' :
(7) 1 Mol. Triglyr('rid~ + I Mol. Glycprol + II Mob. "'alt'r.
(X) 2 Mols. GlYCt'rol + 3 Mois. Ol"ic Acid .
. \lthou,ah tht, e\'ide)w(' i~ again!'.t the' dir('pt absorption of
fat Of of its hydrolysi~ product,., into the portal blood nM.t'I!<"
it is ",!'II known thnt only ~orlle 00 llt'f (~('nt. of thf' total fat
wltlch disappean. from the intestilw can he- f('{'ovcrNI from
the thor<'ic duct, where it f"mptJ('s into the venom; cireula
tiot-t. The fute of the remaining 40 JX'r cent. remainfot ~onw~
what of a mystery. Lee (quo\<'d by Bloor) has rcCt'ntly
shown t hat after ligation of the thora("ic duct connections
were established between the duct and the azygof vein
or it, branches, and also to the right thoracic ~Iuct.
 12H TIl}!; HlO(,HEJflSTRr OF TIlE FATS

Tfu' f<.;tIlbJi .. hnwntof . . ut'h additirHluJ pathway:.. iwt W('f'll

the }ymphatj('.~ of' the int(".'1titl(' ami the \"{'unu, {irtnla-
tiOfl I1Hl~' ('xplaill nw,t. if not ull. of Hw di",(TqKlIl('.v aln-'<udy
llwllti()~i.
Fat if/!t'dcd ~lIh(utneoH ...ly or intp tht' pt'Titolll'al t'/I'!lty
i, uh,orlwd ill ('on:-.id<'rnhlc 'IttlOUlit. Thi... ah... orJltioll
tak('" piH(,(' thrlluj.!h th<' Iymphati(' "'y",tt'lli Hlili the thOfIWi('
flud.
After ah~orpti(ll) HI(~ fut i!o. found in the hlood ill Hw form
of I_l fille (,tH\tl ... ion. th(> partiele ... being ahout 1 JA in diameter.
])l1rjJl~ tilt' po:-.t-nh;",orptiv(' ill('r{'n~e in hlood fat the pho'iph.l-
tide!'> of UI(' hlood sl ... o ... how an inert'asc, which nUl .." roughly
)luraBd to th{, iB(,f(,UM' in hlood fnt. ~I()~t of the' in('f"{'a,,('
in the phosphutidt'S tnk('~ phi('{' wlthil! the ('()rp't'('h'~. It
would apP('HT thnt thi", ll('w)r~formt'd phosphntitk j", pTO-
due(,d from tIl<' fatty a('id~ ah~orlwd. If thi:-. i~ the ('Ih(', it
h'nd. . ~trong :-.nppmi 1:0 the view that tl\l' pho:-.phatitk"i pla~'
an important part in Uw metaholi .. m of the t'Ht . . a ... H whole
(M'C p. 12!l). The amount and distrihution oftll<' totul hpid(\
of dog':-, hlood h{,for{' and after f('('dln.~ on H meal eontainin,!!
f~lt j.., ",hOWl) in thc' foIlo\\'inu figures ohtMj)wd by niOOf nnd
Knud ..on {quoted by Ikathcs and Raper) :-

TAIlL>: X
_lm,otwt lind Distrib-tlfioll ,if Blood LipilifS
}kfol'(, Feed.in_g. four hours a.ftt'
Fetlding.
J n Ilu' Corp'ltscit'S. Gr.m~.
Phosphatides (1,1-10
Cholesterol. . 0'012 0-097
Glycerides, soaps, etc. 1-598
III tM Pla.."ta.
Phosphatide, 0-601 0-647
Cholesterol . (1,216 0248
Gly,-"rides, soaps, ete. 1-1)13 1'75!l
nur.ing absorption, in addition to increasing in til<, bloo
 :"'TOREn AXlJ 1'JSSrE l'AT 121
fat i!-. also found to C\cC'tunulat< ill trn' li\'('r, ulld to a It-.. ~
('xtent in othf"t organs, ..,n('il a . . hOIlt' mntrow. !o-pie"H. und
mu:->d('~.
The PEt!>.SH#!f of tilt from Hl(' blo(l() into thc...(> n~1 oHwr
of.!lan ... is not pr('('('(iefl hv hvdroh... i ... , ~u('h n. . i . . '('(( ... ~HrY
lwfore th(' fat ('an h(' ah~~)rh;d fr(~Jn tlt(' illh'stillt'. If .... ll1'i,
Ii prol'{':-'~ did (K'('Ur. th{' }In'wlw(' {If lipn..,(':-, in tlw 11100.1 and

ill th(' ti"stl(> ('db would be 1H{('..,.~ary. The u\'Hiluhh ('"hku('('

j, RJ,(aillo;t thi ... po... o,;ihility. It i . . true thllt (U7.)'lW'S ('alkd
lipas('s aft' widdy distrihuh'd in the 11s<o;\I(... J,f(,lwrnHy. hut
"'iI1stntt("T Ilnd hi~ ",(~h()()l difkr.:ntiuh' th(s(' from trtH'
lipa!o,('..... TIl(' )"'o-('aB("d ti!-;sll(' lipn ..t:s. or, hd.kr... t~t{'ra ...(~."
:-.u('h a ... liw'r Iipa~e, readily hydroly ...e (, .. ter\o, l':olU'h it ... d hy!
(lud amyl hutyratf'. and tht, gJy(,l'rid(>~ of .. hort(,lmill futty
a('id~. hut-do not hydrolys(' the ,aly('(.'ri<ir", (If lOllg~{'huin rutty
~~jds with anything like the ~all\e fn('ility, Lipa,.,(', in th('
",trict s('ns{', s('t'ms to ()('cur only in th(' g'a~tro.illtestilull
nw('hanism.

Stored Fat and Tissue Fat

Fnt (J{(ur,., in an organ,., and tis~urs. hut frOIu t li(' poi 11 t
of "jew of ~torag(' til(> m()~t im}>ortnflt "dt'pbh.' art':
(I) int('rmu~('ular eOlllwctiv(' ti~\Uf'; (t) til(' al}(loruinul
C8,-jty; (3) suhcutan{>ou... conned i n' tj~SUl. The fat prf',"'f'ut
in tht's(' deposih eonsi~b in gnat part of tlw nt'utral mixed
triglycerides of palmitic, stearic, and o}eic acid..,. It il'> forrnpd
mor' or les~ dirt"(tly from the fat inge ...tt-d in the {'c)(,d or
from transformed carbohydrat<- or prot{'in. It is pOl-."ihle
to modify the nature of weh dl'pot fa! by altering til(" food
stuff. In general, th,' fat ,tored is diff, ....nt from that fNI
and is l1lQI'e or less characteristic of the animal in which it
OCCUI'SI the differences being accountable for hy alteration~
in the reiatiw" amounh. of saturated and un."aturated fatty
acids present. By feeding t'xccs.....h'e quantitip~ of fat it j~
possible to lessen the extent of the moditkation whicl't the
fat normally Undf'lgo<'!'1 on it!-. WHy from t.h(' il1h'stitw to ,the
 122 THE BIOCHEMISTRY OF THE FATS

,tilrc,. 1.,(1 that the tyW' of flIt d('pOf-llteo in the Jatt('r Hpproxi-
fJlutf'''' to that f('d. A,!!aitl, when fat is ht'in~ laid dO"ll on
a ri('h ('arhohydrfl.h' did, it i ... u,;uaHy (If the ., hard" yaridy_,.
that i\ it (,olltHin~ II f{'iati\'dy high proportion of fully
. . atnratr'-i flltt~ IIdd.,. '
)n 1 he t j"',"'1H'~ g~'lH'r:ll)~- t hl'Tf' is a largf' amount of fat
whidl CllIIIH>t h(, t'xtrad('d hy mU1U:,\ or the u ... wtl fat ... Oh'Cllts
{('t herl ('h}oroform, fwd h(mr.CtlC). hut which may he t('mov('d
hy hot nkoho!. Such fat ('annot 1K' ~(,(_'I1 v.ith the mi(ros{'oJw.
nor ~'an i1 Itc "tnlm'd with thr usual fat staill~ used hy the
hi ...tolo1.6~t. Thc>.;(' forms of fat ('onsist ill largt> part of
ph()~pha1 ides Hnd t'holt"... tcTO] and it-. lSOIllf'rS and {' ..tf'r~,
Jt j~ pos~ib]l by \.>ir;ou"i }l}t'mb. ~uch us the inje('tion of
diphtheriu toxin, to lHUHHSk ~omc of this hi(hkn fat lind
{,RUM' it to <,oll{'('t ill dis('ri'tr and \'isible droplets. In th('
prO(>{'s~ or fatty dt'g('lwrntion we hAvC' i\ similar unmasking:
{)(.(nrring no,;, a pat hoiogi('al pro('{':;;'~, Slll(,(, it is w(l1 kno'wn
t hllt the' dCj.!t'uf>rntcd organ frc'qucnt1y ('ontuin ... no more
Int.)1 fat th:m the lIornMl organ. A f'haruC'teristi(' differell('c
hd W('('n the flit ~tor('d in connc('tin' tis~u(> or adipose tissue
~H\d tllnt of ti~,:-u('" :;,uc'h a~ the li\"~r, mu~d('s, or gland" is
tht, higllt'f degrn: of un~atu.Tution of til(' fatty acids of th('
Jatter. ~lor~' ('~I)('('ialIy is this the c'ast' with Iiv{'f fat. and
the prescJw(' in this ort!un of a rrlatiwJy larg't' amount of
unsaturut("d acid~ i ... a.<;crii){'d by uathC"s to the fact' that the
tir~t stttg'(> of fut ('atubolism. namely, d{'saturntion of the
fatty m:'id:... tnk(s pl(\('(' prC'dominantly in th{~ liver, 11
. . tarvation und ill phosphorus poi~oning the amount of ffl'
ill th(' liver und('r~s an increase. and the fatty acids the1
founo art mo1'l' saturateo than those normally present. Ther
iii; a mobilisation of fat in the liver at the expense iff the mor
s.turtlted o<pot fRt.
It is fr<'<juent1y stateo that the fat stores of the body'"
t'Xhalbtl'<l ourin!! prolonged starvation. It is true th,
artd- t he exhaustion of the carbohydrate stores, which tak
pl~{'f' durjn~ the first few days of romplett' starvation, tl
body dra'\'~
.UPOCYTIC eOEFFlrlEXTS
ih. !o.upplyn('{'t> . . ~ary ('nf'ruy frmn tlw' oxid.atiol\
t)f
123

of fats, and this pro(-'rs .. muy ("ontimlt' until tlU' :-.tortd ft\1 i ...
{'xhau~t('d. But th<' T(>('cnt work of )lay<'r ~'w<l (If 'l('rrolfw
proves that organs, such a." the kidney, lung'. ,.,.ple1'Y. he}lrt,
d(~. of animal... whieh have h{'('n ~tarn'cl to <h.-nUf {'ontain
pradi('all:' the sanl(' llmount of fat a., tll<' ('(tl'fl"!'o}lOlMHHJ,!:
On.~all'" of normal animub. It i" Ul{' ... t()T('d fut of t 1)(' ndipn ... t'
ti ... Sllf', anci to n h':-,~ {'xtent thut of ttl(' Hluscl(' ... , whidl i.., 1I"{'"
lip at IIt'pd, nnd lIot tht' fat of till' U('lll'ml ti . . 'm' (Til.... The

Animal.
. LUTl.~. SpIN'n. Kidnf .... Uvpr, Hpart llu~dj.

DOI/ 20-0 15';j 10-5 (j-H 11<0 '-',"

Huhhi! 17-1 l:HI S--I 7':i
Guinea-pig 155 nn 7H f)./j Hi
J_)jg-ton ~H'J !H jn 3'(\ lti
Ed ]],2 (gilb) 71 :I'X oj

modern tend{'ncy i!'> to rt',U'ard the fatty nwtt'ri}JJ of Uw )uttef

a~ an e~M'ntial {'onslltu(_'nt of the ('ell ~tf\J('tur(' at h'ast as
important as the proh'im,. It ('umlot h(' di"',)('llsed with
(lltirt'ly. and tbert'fof{' eannot he utili ...td in the ~~ml(' way
as the ~torcd fat if tht animal i ... to sllrvin: at all. Tll<"
J'f(nch school refers to th( fatty u"id .. whith ('ann(,t 1)('
diminished in amount without (>au~ing death "J_' th(' " ('{m~bmt
dement," whilst. the fatty 8citl<,. of the flr-utral fats whith
compri~. the true fat rt:'~eTV(" constitute the .. variabl('
elemeut.'~ One naturally nssociates the ('om.tant element
with the cell phosphat ides and with cholt-sterol. It i,
certainly rich in phosphoru"i, and theN' apllf"OfS to bE a
definite ratio between the constant element alld' the
ch()]~t('rol of tht' tissues. The ratio is expressed numeri~aJJ~'
 124 TIlR lUO(,IIRJ1l8TRr OF 7'HE FA7'S
('hol<, .. t(,t'lJ!
n ... fatt~. lwid ... "; lOti, and i ... ('affi'd the lilkl('~,ti(' ('ndliciellt.

It i . . eliaraf"t!'r, ... ti, for HJl:-' gin'l! nr::aH, hut \arif' .... ill dirrt'Trnt
... ptC;I' ... \ The tahle :-.11(1""1) 011 p. l~:l ::j\'( .... "onw of !\'Iayn
Hlld Seh(wrfcr\ re.,'11t1., for ('orf('~I~mdillQ org:1lb from
nlriou ... 'lIlim;:d~"
11 will he oh..,('rv('d that the lipfWytic ('(wflieirllt ... alway..,
fall ill the MlHH' {mkr in 1 iw or.!.!Hlh of t h(' diikrent "'p("(ie..,
j1\v(' ...tigatcd.
The diff<>re)H'(' het W('('I} ... tof(d fnt and (' ...... (ntifll fat is
",trikil!gl~' iBu.,tralcd III th~' (';1-.(, of the hrnill. Thi ... org1-lIJ,
:!~ i ... well kHOWH, does Hot lo~e weight dllrinu ~t.trYl~tion,
(wd a ... LCllt'w~ hw~ pointed out, ,. that JHcnn ... that the (lr~au
whi('h ('ontnins thc larg('~t amount of whnt HW~' he ('aUed
OTJ.!:llIi"cd fut ('I.\Il di:-.}){_t)'.;(' WIth none of it if it i; to lh'e:'

Synthesis of Fat in Living Tissues

From t he oh~('T\'ati()n~ 'which we han' ju ... t di.,{'u, .. ('d In'
fIIU.,t fcgnrd the lipide.., a~ jHdi~p{'n~ahk c(ln ... titH(,lIt~ of the
li\,jll~ ('cH, The qu('slion which Ht Oll(_'(' ari:-.(>~ i ... the ahilit~
01' otherwi"l' of the animal nrganl:-.m to "ynthe... i:-.e the n('('(~
:-.ary lipide", for it . . (lf from other food (on"tiht{nh.. This
I\hilit~, it "P1X';\N\ to pO'St:-.~. GjYCll an u{it'quah' :-.uppJy of
prott'ins Jlnd (,arhohydratr- t()~{th{'r with the IWf'p"..ary
hwr~alli(' ('on ... tituf'nts anrl yitamin .... young animal .;; grow in
a norma} manuer. It b, huwcn>r, important, as we shall
~(.'(' ill a Jater ehaphr. to (,lISUI'(' that the \"itnmin~ normally
pr{'sent in fat... are supplied from some tlltenlati\'{' souret',
Cnder ~\ll'h ('ondition~ fat may he entirdy eliminated fronl
tht:' didary, yet no harnlful resulh. ('n!"tUt'. One mu~.('()ndude
that tht n('C'('~ar~' fat is bt;ojn~ !-'yntlwsiM"d from tilt' other
('arbon..('ontainin~ foodstuJfs, namely. from the carbohydrat.e
or from the protein, or from both. Th,' evidence for the
Imrtkipation of thesc separate rompoll('ut> of the foodstuff
in the .
, synthesis of fat will now Ix- ronsidore<i.
 FAT Sl"NTlIF:S1S

Synthesis of Fat from Carbohydrate. Klllil Fi'ch .. r ""li,,1

atb-Iltiofl to til(' fad flint ttw natllrHJ fatty m'ld ... \\hidt ill
It quafltitatin' ",("l',p utt hy far thl' mo.... t importullt are tho..,.'

('ollJaillilig tt-! ('arboll atolll"', StH'h uI'id ... mi~ht ,i)o.t' hy

cond.ellsatioll (If thre(' IIt'xu..,{_' ullib. Itml ... uh...t'qm'lIt tfxitiutinl\
atld reduction. Sugl.!e ... tiv(' a ... thi~ Hnnlogy i:-., th<'fP i~ llo
l'x}writllcntul t'yj<klH't' which {'all ht' quoh'd in ~upport of
... ueh a ~YHth(' ... j..,.. It . . {tJlh mort' prohahk from the
ohsernttivII that only u('id ... with un ('\"('Ii BUIlth.." of ('urbHH
atoms occur ill twturr that til(> ... yntlw .. i:.. i~ a('hi('v('d hy tlt('
~ll((c,,, .. i\( ('olHi('n!o,ution of two~('urhon unit... Incit'(ti, it is
~tat('d that nll the (.'\'{'IHHlmbt'f((l fatty atjcl ... from hlltyri('
to ... t(,Rrie Hcid\ art' found ill milk. nir~:d proof (If tht'
\:oll\'('r... ion of ('arhohydrat(' illto fHt in liYillg animnl~ ',"'U'"
liNt furn:shed in thl' (']J-I ..,.,i('lil ('xperiJJIf'Uh of LU\\T~ anti
irillwrt. The. . e :wed Hot }:)(' diMUsS{d hC'f(', Sutfiee it to ..,tatt
tJalt sub:-'NIUl'ut oh~('r\'ati(Jns han' unll,ly ('(Jfltirmcd til{'
urij.!inal work. TIl(' dwmi{'aI 111('(huni ... m which untilrlit ...
the transformation i.., lIot yl't ~ati ... f~u,torily chl<'idatt.'d, hut
that whi('h is mu . . t favOUft'd hy bj(Jc:h('tlli .. t--, ill\'oh( ... "'UC_'('("'I-
:-oiv(_> aldol (ondcn ...ation::-. of 'l('('taldeh_ydt" with nppropri~.te
rf'ducti\'t, and oxidativ(' (:hang<'s. Th(' produ<1ion of butyri('
acid hy ,-anou . . bach'ria acting' on ghw(J.lw and 011 1m'tit' a(jfi
i:-. wdl knO\\"fl, and the dumgc may h( fOfll1ldut<-d u.. . follow ..
(,cc also p. 106) :--

OI(I('<HK' ---to ~ or ~~~'i~,(:~~id} ~_,. ~\(>dltMd~J"dt,

(aluol ('(Jud(.'nfl1Ltioll) fj.Hydroxj'butyri(~ aldphyd. .~-. Butyrj('
add.
In such bactt'"rial fermentation~ caproic add and. small
nmpunt" 'bf aciDic and decoic acids "rt' also found. Th"ir
pI't-'S('ne(> would se<~m to point to further aJdol condensation ... ,
,tarting perhaps with butyraldehyde. Such a seTi," of
reactions may well form the basis of the synthesis which We
are discus-sing:, and there are no serious experim(~ntal 8bjec~
tiOl" to it. There is as yet, it is true, little biological c\'idfnce
 12f, THE BTO('HEM/8TRY Oli' THE 1"..1'1'1'1
in favour of 1Ill' vi('w 1 hat Judie <wid i" trnn ... fornwd into
;wdald<'hyd(', hut no sH('h ohj('('tion ('xi . . h in t Iw ('t\""(' tlf
pyruv)<' atid. whieh i~, a.., W(' hlt\,(, . . ('t'll. a p()~ ..ihl< intt'f-
tllN{iar~\produd ()f (,arllOhydratt llldaholi~m. Alt('rllatj\"('
foutt'!-. fOl~ tiJt' hiolog-i<'ui :-.ynHwsi . . of ..,imp!< fatty R('id~~ sueh
th h"ltyri(' Held., from ('Il.rhohydrnt('~ hay(> already h(>('1)
tli..,(u ......('d in the pr('viou~ ('haptet, Wilt'fe, to0 1 (,(rtain asp<.'t'ts
of th{' hiolo),!it'lli ('OIl\'efSl()l\ of ('arbohydrate into gIYf?erol,
whidl i ... ll(('l'~ ... ary for th('> ('omph'h- syntht'",is of u neutral
fat. lin H' tW('l\ <kaIt with.
Synthesis of Fat from Protein.~-l\lallY of tiw amino acid ...
d~ri\(d ('rOfn plot('in . . arc' f('aciiJy transfornH'd into c'arho~
hydrate, and sinc'(' tlw luH('r mny be utilised in forming fat,
it i!'o tlwrefort' natural to prCSllnlt' that a dir('(~t tran!'oform.atiol\,
of pr()t<~iH into fat may tuk(' pln('e. .Eyen UUd('f optimal
('ollditiol\s for thi~ trallsformation, it is (']<'ar that it ('an lWVt"r
he moJ'(' than pnrtinL Silll'(-, only u limitc,d n\l1nh('r of tlw
amino )with comprising tJw prot pin mole<'ul(' CHI) po,,~ihT
gin ri..,( to ~llg-ar. DirN't ['vidf'nc'(' }.., 1ht'rt'fore diflkuH to
at'l}llirt'. Therr- i~ no doubt that much of tlw t:arli('r work,
Hotub)y thnt of Yoit. on the suppost'd formation of fat from
proteiu ('annot now be UC('f'pted, and in the lJormru {'ours('
of nwtabo1ism this transformation probably dOf'~ not take
place to nny important extent. !'\ev('rthelt"ss the 1Ulima)
organism em) under ('crtuin ('onditions synthesise fat from
protpin. '''{'inland's demonstration of this sYllth('sis in til{'
case of the Jarv,e of tbe Ily Calliphora still remains un-
challenged. A l"("cent invostigation by Atkinsou, Rapport,
llnd Lusk elucidates the conditions under which protein is
transformed into fat in the dog. Their method of inter-
preting the ,...su)ts is lx'st sbown by a quotation from their
pap<'r :--
.. If protein is ronverted -into rat two conditions must be
fuJfi)J':9: (a) the R.Q. must be higber than tbat of protein
itself and (1)) there must be a retention of protein carbon.
The urinary nitrogen is usually higher in the dog when the
 FAT SYNTHESIS 1:!'i

animal is ('uthekri,,"d hourly thun it j, when it lU'('Ulllldutt"

ill tht' hlmhler dllrin~ a Cu.Jorilllt'icr period. alltl tlH' trUt' twh'"
of prot('in md1iho)i ... m j.,. 11l'('Oun!rd to 1", tiu' J,>vd to whid)
thl' .urinHry Hitro~l'l\ ri...,e.., WIWll the nrilH' is. frndilOutt'd
hourly on a day that tilt.' dog i ... gi\'t'll tlu' sHirl(' di~t hut i ..
not in th(' ('aJonml't('r.
"Tht method of ('akulation of the lifth hour ufti'r J,!'i\ iu~
1,000 :,!ram ... of Ill('at to f)o,~ X\'IIL will 'Uffiet':h n guitit, to
all the l'xpnim('lIt-..
" CriHary llitrogf'H 1"~ ~ ~ram"'.

]:<.~ql1intlcnt.of 1'..J.4 gram.., orx , In'u) !iH'l i

F(:und in rh.piratiOJ! 10]0

akj. Jl':l~

Caloric.., indir{'('t
RQ. of depo,it c U'7()~.
Valu( of fat depo,ih'cl 1] '32 ('ulori{ ...
('Hlori,'s (indin'd) . 2(Jt(5.
Calories (diro('t) 275~!'
I -_-:-. : 1,2 gram ... of faf.
C retained :. :;.-; 092 I-{ram ~= 28 ,l!ram~ of ghl('o~('
i c<ll()rit~).
Calories if C had been retained a~ glycog(,1l '"'_- :.W;jt

.. Th(' f('l')piratory quotients and th<: heat directly m{a~ur(d

confirm th{' conception that fat and not lllyt'"gcJI wa., the
form in which the carbon was depo.,ited.
" 1f one makes ""e of this method of cal,'ulatioll for the
aver~ge ~rly heat production for th,' ,,'rie, of three t'xp{'ri
ments, one mav construct Table Ill.
H It is e",de;lt tbat, computed 011 the oxygen aru.orption

by the method of indirect {'alorimctry aln'ady outlined, tilt

('alcuJated heat production is almo.t exactly the same it t,
is when the computation is based upon the hypothesis tqat
 12!> THE 1iI0('JlEJfJSTR r OP TilE PATS

t 1)(' ('arhol) rf't }lilll'li i, l:titl down i" t JH' form of fat (1 Stram of
t' retained it ... fa! }!!,:n ('illori('~),

"T,'"I.I: III
~
" /)irt'l'/lIlJtJ illl/ir/'f'/ ('fl!orimrlr,ll in lIour1.ll Pl'rimls (~Ii(')'
tht' I fl{!('~'I;(IIl (d' J ,not) l!rams (If J/t'l~!
('IIJ"rit-.,., ('
J.;Xpt'rJ-
WI'll!
Xl)
,
{"rin"
I'rotl!lh
('to
b\)\J~
DirN'! },KlIN'I'1
{;
d~il(1l1itl'd
:lJ<fat.
Ut'llO!!itK-J
:l1l('lUI,W-
h;o,orah'.

Dol' XVlll. t)l'\ I'H U'S~ ~H'H 27']).~ 27,S:) :30,30

I)og XIX, Ii) I ,HI) ) ,~() a2'1H :3U'ln aU'51 :34-;')7
77 I'HII HI~ :36'1(1 al'Ha :15'25 :lX29

Tota! 97'06 0335 H:l.I)9 10:{'1,;

-. Tlw hetwt'('n dirt'd and indirect eHlorimdry

agN.'(lllClIi
i ... Hot a~ ('lOSt, a~ on('
would wi~h . . . . Tht.' (ulori( ... a~
{'Hh-uht11'd aft' as pl'r {'tnt, h~~ than the ('nlori{'~ found.
Howt'ver, if t lit, eurhon retention is H!'.~um('d to take the form
of gly('og(,ll, then til(> t'Hkulatrd Iwat 'would lw 63 })(~r C{'nt.
higher 1han the anlOunt directly meH~ur{'(l. The tinding~.
th('refort" favour the idea of the ret('utiun of a pabulum ill
t itt form of fat. . . . Tlw::-o(:' experiments, by a H('W method,
mid another link to tht- chain .of (\ide-nee that protein may
lw conn'rterl into fat. It must be remembered, however.
that the ('ontlition.s w{'l'e f'xccptionaJ. in that the animals
w{>n~ mnintaint'd upon H nutritive plane which would fiU the-
glycogen resen'oirs, wen" giw'n meat up to the limit of their
willing-nes.... to ("onsumf:' it, and were kept in absolute rest ill
8 calorim<>ter at an environmental temperature- "f 2iio C
l;ntlct th(,M~ clfl'ulllstanecs fragm<'utors of protein nlt"tabolbmi
whieh would ordinarily have been oxidised or conv~rtrd int,
gluco;;e and laid down as glycol!"", found no other path",,,:
ol",n than eonv<:'rsion into fat. Under a natural diet the,
('ouditions would not exist.'
 PHOSPHATIDES 1211

,,',e muy ('onclud{' from tilt' ('xpl'rinU'nh jU!'tt dt~tnil('(1

that til{' in h1('stion of :.t larg-e (l'umtity of 1H('at prott-in
brins..~ about n deposition of hoth ~lY{'oge1\ nnd rut. Only
when n w'ry lar~w ('X{'C':"oS of I1WUt is f~d will fnt."loHt lx'
retained.
The Catabolilm of the Fats
\Yhen reM'n'(' fat i~ n'quin'd fot w ..(~ in th(' 1i~~u(", it ilo.
first dealt wit h prc'dominantl), by thl' liv('r. Th(' transfert'n('(>
of fat from th(' adipos(' ti~sn(' and ih mohilisation in the
}in'r is s{'en in un t'xaggt'rukd form in pf\tholo~("ul ('oil
rlitions involving H('ut(' poisonin.f!! and to n IC'ji;s rnnrk('(l
dt,J,.,'T(,(, in partial :-.turnllion. '\.(" hun' ulr'Nuty ~('('n that
much ('\'idt'u('(' is ft('('umulating whirh point!'> to th( }wput i("
(unction lwina eXt:t'{'ist'd in hm dir('dions~ nnrndy, the
('onn'r~ior), of til(' neutral fats into phosphntid('s and titf'
.""ultaneous desaturntion {lr the fatty adds. With I'<'gard
to the first point, ('ollvC'n-ion to phosphati<i('s, lht, ('\'ici<'ne('
for thr participation of th('s(_' {'ompounds in fut metal)(JlisJIl,
whih.t it is !'Iug_g(~stiV(', i... admittedly cirvum!oitantial. In('i~
dental ref{'r('n('(~ to ~om(' of lhi~ cvi<i('n('(' has already hel'n
made in this chapter, and it will lx furtht.'r (~x('mplified in
Chapter YI. HeJ'(' it may be summarised as follows:
(1) phosphatide. al'<' univ,>",al constituents of all tissues and
organs; (2) phosphatides a1'<' much more readily oxidised
and hydrolysed than the neutral fa!.,. and they mix more
readily with the blood plasma and the tissue fluids; (8) the
phosphatides of the blood inerease in alimentary lipremia, and
a similar increase in phosphatides is observed during liplCmia
in individual organs; (4) the character and amolint of fat
in the diet of hern; influences the degree of saturation of the
fats .lmd 'I:If tbe phosphat ides of the eggs (see Chapter VI.) ;
(5) there is e..Mence that the fat of milk i. derived mainly.
if not entirely. from blood phosphatides; (6) the bulk of the
fatty acids of the liver are present in pho&phatides, andJ>otb
these latter and the neutral triglycerides of the liver (which
are probably present in small amounts only) are nt>re
....
 1:111 TilE BJOC'HEldlSTRY OF TilE F:ATS
un~nturat(;'d than un' those of tht' fat !'.tOf(>S. This last pi('('c
or ('vidl'nC'(' hrin,:.,rs U!lo to the M'('(}Jul point mentioncd ahon-,
namely, thl:' dr-saturation of tht' fatty a('id!';.~ whirh takt's
pla('(' llI~inJy, but not n(,ffssarily ('xdusi\'dy~ in the lin'r.
Tlw 9\!emge iodin(' Humb{,t of the fntty a(ids of tiki' Hn>T,
kidll~Y. lind heart vllrirs from 115 to 185, whibt that of the
fut !o.tOft'''" varies from as to 65. Direct {'xpcrimcntal ('\'io<-'n('{'
of tilt" df'saturating a{'tion of the lin'r OJ) inge ..hd fatty adds
hns h('('n furuis.hed hy L<-ath('t\ and hi~ ass(}(,late'S in the
folJowill~ way. {'nd}ivt>r oil or h('rring tk'sh--rich ~our(':cs
of fAt1y acids with a vtry high iodine llumhC'f-,wCr(' fed to
rats or ('ats, 11lld it WQ!oo found that tht, fatty "('ids rCl'ovC'rt"d
from tIl{' nnirnar1'i liver had au iodin{' number still higher
than that of the mixed add, of the oil, oril(in"lIy fed. Thi,
result might possibly be due to a selective retention 011 the
part of the 1ive'f of th(' morc unsaturat('d acids, hut furth""r
l,\"jdt"Jw{' ad,"an('{'(l by Hartley shows that this {'xplunation
is most improbahle. Tim; the reserre fat of the pig (lard)
('olltains both stearic add and the normal form of oleic add.
wilh til" unsaturated linkal(e between tbe ninth and tenth
('arbon atoms. In the lin'r of the pig is found an oleic acid
with the double bond between the twdfth and thirteenth
{'arbon stonlS, an.d also a linQlic. acid with its double hond!;
between the ninth and tenth and the twelfth and thirteenth
carbon atoms. Neither of these acids is present in lard, but
hoth would te fonu"d from the acids which are present,
namely stearic and normal olej{'"~ by the samt" change, the
inscrtion of a double bond between the twelfth and thirteentl,
('arbon atoms in each of these aeid~. A similar chang<"
occurring in the fish oils fed to animals in the eX}lerimen'
previously mentioned would serve to account fiX tbe in
creased iodine number of the fatty acid., found in the Iivet
In addition to the foregoing evidence, the occurrt>nce in H
liver phosphatides of unsaturated acids containing up I
fOUT double bonds affords collateral support for this ps:
of 'Leat!"",' tb<-ory.
 .. BETA" OX/JIAT[(JS 1:11

\V(' ;"('(" tht'n. thnt thcrt' i!oo good t'vidt'lu'(' for til(' \'i('w thnt
the Hr~t ...tal-,'(' in tll(' catuholi"IH of tht' t'nb. i ... tI 1)f(.)((' ...... of
de!'.aturation. whi('h fHny or may Hot ht, a~ ..odahd with tl
~imliltal}(,ou~ ('oH"('r~ion to pho~phn.tidt,,,,,, and ttu~t ttw linr
ads in n ',X'('iul w\- a!'. n rt('(i\ill~ nrj.tull for the r:t whkh
Ilfld('rgo('~ thi~ trlw~f()rmatiofl. The ~uh"'('(IUt'ut utW .. atim1
of the rub. illyol\'('!\ tht'ir oxidatioll ill ttl(" ti'i"'Ut,~, Illld Ollr
illfornHltioli a ... to whal hapP;"II'" hdw(,(,11 thl' (It-~:dllrl\ti(ln
:-.tag't' and the oxidation PTI)(t~~ is Hot ltioo compid(' a ... 01lt'
might wi",h. "TheIl We pl('k up th(' thr('tHl of ('yiti('IH'f' Itg-nln,
a ... tepre"enh'd hy th(" frngnlC'nt ... ofthr original fut t'('(onTuhlt
from normal Jlwtuho)ie pr<K"(,~~{'~, \w find thut tlw lonu (JUlin
of the futty tu,irl rtlol('('ul(' hu~ ht'('ol1l(' r('du('t'd to ('ollllmrn-
ti.\'{Jy ~irnpl(' rlirrH'll<.;ion.... Th(' da ... ~i('al work of .Knoop. of
Dakin, aIH' of Emhd(~Jl arrord ... A ~ound (>xp(.'rim(,lltul ha~i."
fo~ our theorie"" 8!-. to what ha.~ h{'(~n haPIK'ning to the! fatty
:.I<'io bdw('('n these t\\"o ~tag(''''' The work of th{'!oJ(' inv.... ti
g-ator. . in relation to the pro('('s:-. of ,B.oxidRtion. whi<'h the
... h()rt('hailJ fragmf'nh of the original fatty uC'ld Undt'TJlo in
tilt' ti~~ll('~, i~ too w('l1 kJlown to tfquirt, ddaikd repetition
h(>l"(. The pro('(>ss of ,Boxidatioll iJl\'oln'!\ the oxidati\'c
di:-.ruption of the fatty acid moJ('(~uk by two ,('arhon atoms at
a tinw, and i ... tl)("r('fore analogous to the OJechanism b~' whi('h
it is synthesised. Bridly, the (,yjdt'nc'{' in fa\'our of th{'
prQ('css of p-oxidatiol1 as repn'senting the normal ('()ur!ol(~ of
t he catabolism of at least t he shorter chain fatty acids is ns
follows : -
(I) Knoop found that, on administering the ,odium slllts
of the phenyl derivatives of the lower fatty acids to dogs, he
eould recover from the urine either hippuric acid (henzoyl-
glycive) dr' phenaceturie acid (phenylocetylglyeine),
C.H . CO. lI.'H . CH,. CooH
and C.H . CHI . CO . NH . CH, . CooH
respectively. The interpretation is that derivative" of
fatty acids with an odd number of carbon atoms, which
yielded hippuric acid, were oxidised to benwie acid a:1td
11-,
 13t TIi/<: BIO('IIf:JIl87'Rr OF' 7'H1:: fA7'8

!:'>Uh-..tCPll'lltly {,(Illden"f'd wi1 h glycilH' lX'i'orr ('xcrdion,

whit.. t tho.,(' derinltiv('>., of Heid>.; ,yith an ('\'cH Humht'r of
('arbon atom ... , which Wef(' (xtrdNI a~ pht.'nu('dnri{ a(id,
W('ft' o:\ldi ...ed to p}H'll~'l:H'eti(' H(jd, Hnd W('Te abo ('(md('~lsed

Wiq, gl~'dtH'. The ~trid ulh'rnation of tht'st' hHl ('xerdory

Jm~lu('h in Hi{' (':t'w of tht' homo}ogmh ""('ri{'", of acids u~('d
pointcd_ to tilf' f('lHOYHI of til(' ('orhon "toni" two at a time,
(2) Dakin admilli'ttcfi'd pht'nylpropivlIl(, Heid ~ul)('utan{~~
ou;.;ly to dogs and rats, and found ill til(' uril\(\ in addition to
hippuric :wid, f3.phtnyl {3.hydroxypropionir m'id, benzoyl-
u('di(' (-wid. (lnd H(,(toptwIlOIlt', Ii ~('rit,~ of ~uhstan('('s
HlHtlf)~OI1" to the " Jl{'{tOI1f' bodies." He al.... n found ('innamic
.wid, the <'ofn''''pollding- (l~,B~urt~atuI"nt('d aeid. Dakin dcntOIl-
:-.tratNi ahout Uw ~llJl)(' time that a simijar oxjdation of the
fl~('ttrhon ntom ('(mid be ('fkctcd hy the a('tion ()f hydrogen
pt'roxid(' un ammonium hntyratf' i)j dfm at 37',
UJ) In lln'Ts pcrfw.,(>(i with oxyg('lwt('d blood ('ontaining
nH'mtl('l'~ of the saturated fatty acid !-.('ri('~. Elllhden alld hi!-.
('o-workers found thtl.t mu('h larg('r amounts of acetone- were
fornwd (from a('t'toac(ti{' mid} wl1('11 the fatty 1l('id pr(,~(,l\t
in the hlood ('ontaiu('d atl (,Yell Humm'f of carhon atoms than
wht'n one with an odd number wa ... pr('sent.
These ohs('f\'ations uU.-.upport }i.lloOP!'> original hypothesis
thot the fatty acids arc oxidiscd through their ,8-carbon
atnm,'i. It is usuaUy assumed that this type of oxidation
Pf()('{'('d<.; with t hr int('rmediatt' formation of the ,S-ketoni(
,,,,id, hut we han' seen that J)akin was ahle to detect in hi,
('xpt'rim('nts the formation of an a-fj'-unsaturated acid, 3W
the e"id"llr" available up to the present time docs no
enable us to dra w a di,tinction l:J<.tween these two possibJ
types of illt~rmediate rompoullds. In a re!'ent Sindy of tt
e,atabolism of capro;<- acid and its derivatives Dakin ht
shown that on perfusing through a survhing liver tl
folJ,nwing derivatives of the .ix-Cal'bon fatty acid, namel
<,proie acid itself, ,8-hydroxyeaproic acid, ,8-ketocaproic S(
(IJutyrylacetic acid), and ,,-,8-bc-Xenic acid (i.e., the cor
 .. BETA ., OXI/JATIOA'
!'opowiing UlhHtutakd o'l('id). the uufltitnth'c' dif('n'nffl
hetween th(' amount:-. of til(' "a{'dOll(' hodit::-. ., furnU'tl ill
eU('h CHS{' w{~r(' iu)"um"icnt to indieuh' whit'll, jf allYl of thes,('
adds wa ... pn'{"f'Tt'lltiulJy lIU'tuho)i:o.l'(l. Dakin ~U~,L.I'(_".... h thnt
tht H<,id . . aft' all readily iukr(oll\'(.'rtihlt' and urf' l..)rmal1~
pr(,,('ut tU equilibrium. Xor j~ it fK,>:o,ibll' It ... yf't to ('(lrr,.{a1c
<.,ati ... fadllrily the pro('(':-':-' of J3-oxidation with the ('nrhl'r
Pf()('P:-'s of d( ... aturatioll. It i~ po..... ihJt. that til(' longdulin
fatty add ... are hrokcll dowll at tlwir douhle howl .. iuto II
..,erif!-> of .... hort~(hain H('ids. and that tht'~( aft' tht'n ~uhjt("trtl
to oxidation, whf'r('by two atoJU! of l'uroon are rf'tllo\'('d l\t
1)11(' tinu.', Or it may be that th(' )on~ ehaill i~ oxidi~'d iu
situ, thl' prp:-'eJle(' of t}l(> douhle bond . . ('onf('rring ~onl{' sort
of :.. timulu,) OJ) tilt, degradatjon PN)[,('''S,
The for~oin~ ObSefyations aft' ~trougly in favour of the
\'i~w that {l*oxidatioll is 1ht, predmninallt nwdt of utiiihlltion
of the fatty adds ill the li\'illg tiSSll(S. hut tilt.' possihility thu.t
other types of oxidation may play ~otH{' part in tht~ pr()('(~s"
i... by IW means excluded. It wlH he S{~{'n Hmt our {'vidt'H('('
for tht> fi*oxidation process is haS{,d lur~dy on tl1<.' b("haviour
of short-ehain fatty add~ alld their deri\'ati\'ps in th( animal
or 111 surviving tissues. A (~onsidcrahl(' part of the (\'i<1en('(.'
is founded on. the formation of thf' . acctotle h()di(~s." \Vt
know nothing of the intermediate stages which must O<:1'!ur
between the desaturation of the long~chain fatty a('ids and
the produdion of acetoacetic acid in the ('our~ of normal
nutabolic proccssl>S. Dakin's ohSt':fvations on the action of
hydrogen peroxide on the fatty acids lends welcome support
lor the w!wle theory in showing that f/-oxidatioll may ""'eur
as a perfectly nomUlI chemical proces., iTt vitro. It is all the
more sig.\i.licant when we bear in mind the close analogy
which has generally been observed between oxidations carried
out with this reagent and biological processes, It is precisely
for this reason that recent observations by Clntterbuck and
Raper on the oxidation of the normal saturated fatty tl!ids
by means of hydrogen peroxide are of considerable intelCft,
1:14 T1U; RIO(,HEMI8TRJ' OF THE fATS
Th('M' irl\('~tigHtor~ lIan' shown that the ammonium salt..:
of l1('xnic'. }I{'ptoi('. (){toi(', myrilo!ti(' (('14). palmiti('~ and
sh'arie a{ id ... yidd Hpproxilllah'ly equnl amouflts of fJ~, y-,
owl O-\xi<iation produC't~. Sp(,f'htl sig'lliiieatl('(" nttul'hes to
the fndthat thcy find sU('('inic ueid (Col) nnlOll!.,J'!it tht, further
oxitlutioll prodllets of the y- anct 6-kdo-aeid!-l so formed.
1'lu' pro{'ess ('nn ht represented somewhat as follo\'t,~ :----
It. ('H, ('0. eH,. ('II,. ('0011 R. COOH +
IIOOC. ('II,. ('H,. COOl!
1(. to. rH, .';'H, :'.-11,. ('o'm! R. COOH +
1I00e. CIl,. CIl,. coon.
The molt,pule undeTJ.,F()('l; disruption betwe('n the '1- and til('
b-('arhon atoms if t'ithf'T of these atoms should become
oxidisr-d. If such a pro{'('ss o('C"urrt'{l in th( hody the ultimate
Tt'sults of sUt'h a seissioll would he iu aWt'('llWI1't: with tilL
observation~ of Klloop and of Emhd('lI, for the: fatty a0d.
in plaN' of h('ing degraded two atoms at a time, would Jose
four atoms, aud the rt"sidual ('hain would still {'ontain an ('ven
Hum her of ('arboH atoms. Incideutally. as Clutterbuck and
Hapt'r point out, su('cinic aeid is readily transfonncd into
carbohydrate (ilia malic acid), and its production in such a
way as we have outlined would help to explain the bio-
dwmieal COIl\'r-rsion of fat into carbohydrate. Succinic acid
is also dosely r~l!lted to aspartic acid, the latter being tbe
oc-tlmino d('riYative of the former.
Wbatever t.he process may be by which the tissues hridge
the intermediate stages between the long- and the short-chain
fatty acids. we may presume that oxidation proceeds until
the simple compounds which we encounter from time to time
in the course of normal metabolism are reached, These are
the acids c.ontaining four carbon atoms, acet.qcetic and
fl-hydroxybutyric acid. It may be .'tRted in passing that
it is much more prohable that the latter acid arises from
the former than that the hydroxy acid is an intermediate
in lite formation of the keto acid. At this stage a new type
at oxidation is necessary to ensure the disruption of the
 ANTI KETOGENESIS 131\

four~('arbOil add. altd for its ('omp)(>tion til(> )t('M'lH,(, or('urhn~

hydrate i~ nNf':&o.~ary. It i!oo for thi" rt'aMlH that fu.tty add
oxidation i.., arr('~t('d ill th{' U(halU"('(1 dinbdie fit th(' nc(~t(l
HPeti(' add stag"(' and this 8t'id. tos,.'f't)wr with Hit' oth('r
. "('(tolle hodi(",." i!oo rX(1'ehd in thr urine. III thl dlalH'ti{'
('ou<iitioll. as in ~tar\'atioll and afh.r administratiuh of
phlorhizin. thert' is in t(f(>('f a stnk (Jf (~arh(}hvdrnt starva-
tion. and in th(' instan('(' of simple starvation fI("(~umrt\ny
tl,('
ing ('x{'r(,tion of 8('(,t01\(' hodies ,. j", promptly nrrc-stt'(i hy
th(> udminifootration uf 1l1u('o;.;.r. Hukill has shoWII that ttl('
oxidation of K('rto8cetic lu~id may he aC'rolllpHshcd ill l.'itro
hy the action of hydrogen pC'roxid(', R(>(tic, ~lyoxyli(" HHd
formi(: adds Rild carboll-dioxidt, heing pro<iu('('d. ShaffrT
has demonstrated that this disapP"aran,'(' of the k.to .<id
i~ great!, accelerated hy thl' pn's('nc'{' of ghl(,Os('. Ttt<'
~)rmatioll of .. a('t:toJU..' bodi('~ .. luu. he('u ('ulJ{'(l k"t(lp.nlCl'ii~
.and Shafft'r a5tTiocs to ('om pounds su.ch as carhohydrute~
anliketogl'uic or ktt(}iylic functious, in that tlw)' oppose th('
formation of' .. acetone bodies ,. or an' nC(tssary for tlwir
oxidation. Various explanations of this ("Orrdation of fut
and C"arbohydrate oxidation have h(~>n ad\'all('ed from tim'
to time, most of which are based on the SUPPOM-d fonnatiolJ
of hypothetical intermediate products composed of fatly
acid and sugar or of bodies resulting from their d('('ompo-
sition. There is as yet no experimenta' evidt'nee which
supports these suggestions. In a recent detailed investigation
of tilis problem Shaffer suggests that there is a quantitat;'"
relationship between the amount of acetoacetic acid and of
carbohydrate being oxidised, whereby two molecul<.. of aceto-
a(~etic acid require the simultaneous combustion of one mole
euJe of f!Ilucose to ensure their complete oxidation. If the
rat10 of ketogenic to ketolytic molecules undergoing oxidation
in the bodvexceeds two, .. acetone bodies H will he excreted.
Much e~mental support for this supposition has been
obtained and reasonably accurate prediction. have" oc'Cn
made of the amount of acetone exereted by stan,jng ,and
136 THE BIOOHEMISTRY OF THE FATS
diabetie !Suhjects on diets of known composition. It must,
however. he rC'Illcmhered that a certain proportion of the
k(togenic and of the ketolytic suhstancf's is deriycd from
proteins, und the Ilecessary ('alculation itl\'oln,d does not
yt'l, rl'st ~I all assured hasis. Moreover, tht'St' eoruputations
1tssnu!lt' thnt Oil(" moi(,(.,ttle of fatty acid gino'S rist, to but
one lllo1t(ult of' Ul('t01t('{tic u(,id. Onto ('annot state dctinite1)t
that SUdl is tht' casco 1'lwse \jews have led Shaffer to make
an inic'T{'sting- suggestioll regarding the oxidatioll of ('urho-
hydrat(, (s('(' p. 09). and We may give his \'i{'ws in his own
word.'): .. JJI U}C human subjt'ct th{' maximum ButiketogC'nic
dfeet of glueosc {'oTre-sponds to approximately two Inolet'uIcs
of U.Ct'toHcctie s(id for onf> of glucose, which is also the ratio of
glu{'ost, to kf'to acid in the ,in vitro reaction. But in vitru,
t'lu.'h mole('ule of glyceric aldehyde. gIY('ol aldehydt!, g]yoxal'
or their oxidation products likewise feucts with two oj
8('ctoacetic add. 11' ill the body {or ill l'itl'o} glucose were
first dissociated into two molecules of glyceric aldehyde, we
~hould CXP('ct that under favourable conditions it would
u('complish the oxidation of (2 X 2) or four molecule, of keto
U(id. But this would be twice the maximum antiketogf'nic
effed of glucose which has so far be('n obsen'ed, According to
this Jille of argument it must therefore be concluded that the
six-carhon glucose is probably oxidised to a single molecule
of ketolytic subsb.nL'e without previous splittinq to a triose."
Ueturning to our main theme to summari<ie what is known
concerning the oxidation of the fatty acids, we may say
that the stable fatty acids present in the food and in the fat
stores are rendered less stable by a process of desaturation
whl~h takes place mainly in the liver. A pos!>ible sou1'('e of
further reactivity is their com'ersion into phosphati<ies. The
long chains of the fatty acid moleenles are then shortclled
two carbon atoms at a time until they are reduced to the
dimensions of a four-carbon compound. }!'urther oxidation
is onI:v achieved in most animals by the simultaneous
oxidttion of carbohydrate. It should be noted that
 BIOPHYSICS OF FAT 137
carnivorous animals are not so dqx'ndent on t ht' Sim\lltuUt..OU,\
oxidation of ('arbohydrate in order to eIlSllr(' (olI1pht(, fut
oxidation as are animals prt.~dominant Iy h('rhin}rOUh ill hnhit.

Bioilh1Bical Aspects of the Lipides and thrJ

Signjflcance in Biology
rntil comparatiyt'ly ree'eutly most bi(w)lI'IHi ... ts hun lw('u
content to regard the fats and tlwir fatty "('ids simply ItS
stores of potential energy. They C'ertainly i\f(' of \'('rr gr('J~t
importaul'(_' in this f(sIX'ct., hut th(>ir ntility as fo()(h.tuffloJ l)y
no rncans exhausts their bio{'hC'mif'ul importulHt 'I'}w l'it'ur
o

demonstration of the n{'('('ssity for lipide lI1utninl in the edl

so long as it continues to live c('rtainly points to some
intimate ('orreiation with c('Uuiar prO('l'sses. As Wt' hun'
"ct'n, muck, if not aU, of this ('Ss('Htiul Jipide matl'ria.l j~
vrrsent ill the cell in th(' form of phosphatidt's or ill a:o.soeintioll
with cholesterol. For a proper uppr('('iution of tt.l(' roil' thut
phosphatides may play ill }i\'il.lg matter, it is m:lc:>:o.:o.ary that
one should understand their (~hcl1lical stru('tur(~. Tllis ~Uhjl'('t
will be takeu up in detail in tll(~ next chnpt.u, hut for our
prt'Sent purpose it is sufficient to bear iIi mind that. in additioll
to possessing great structural similarit y to th(~ neutral
triglycerides, they also possess one free addi(: group nttadwd
to the phosphoric acid residue, and also a hll,',ic nitrog('u
group. They are therefore amphoteric, they have an i",
electric point at which they may be expech'd to show "
greater physical instability than at other hydrogen ion
concentrations, and they can form saltlikc ('omhinations
with acids at a P. below this point, and ,vith bases ahow
it. The constant occurrence of small amounts of inorganic
matter (INtassium, calcium, magnesiulIl) in 1I10st phospha.
tides'suggests that such combinations do have some biologiCBI
significance. In these respects, then, the I'hosphatides arc
analogous to the proteins, and lecithin shows many of the
precipitation reactions typical of the latter, more espedally
in regard to electrolytes. Thus salts with lIlGnova1<;nt
1:IS TIlE f$/()('HEMISTRY OF THE fATS
kat iOIl~ tio not pT('('ipitat< all IH)U{-"{)l1S eOluh,ion of' It'cithin,
wlH'Tea:-., within ddinitc TIlOgl'S of eUll('(,l1trntioll, salts with
<li- and tri-\'alent kntions do, trIYnl('nt katioBs heing ('rr('('ti'n'
in IOWf},T ('otlC'clitratioll"i than tIl(' dinllt'nt ions. It has
furUH'l' ~{'(,B eininwd that tht precipitating 8('tiol1 of anions
011 1lho:o.phntid('", fotlow~ the Hofmeister st'rie.., orihrinally
oh'wrn'd in the ('(-IV' of pratt-ins, hut tlli.., work prHbahJ~'
rt'uirl'''i rt_'\'isioll in the light of m.OTe re{'ent ideas on t1l("
!'.itHpl(' \'Ull'lH'y Tille. and on tlw ('fre<,t of hydrogen ion
('oll('C"lltrntiolt in :-.u(h phlIJOBH'na. ideas hasC'd largely on tll('
work of Loch on prot.lns.
MallY plWIlOllWlHl exhihitt'd by Jidng ('dIs, 'Sueh as sp('C"ifk
Il'TTTH'ahilily to e(~rtain molN:"ules and ions and impermea~
hihty to others, point to the presence of some retaining
J1}l'mlirnuc surrollHdin~ tht- surfaee of th~ ('ell irT the for,;}
of a deHnih' htY\>f. And UW1't~ is a TeRsoHnble presumpti"JIl
that this layer is t'omposcd es""entially of lipid(' material, juto
the nHlk{'~up or which the pho:,phRtide" probably enter.
Sueh a layer in allY caSe has heen the basis of a great many
spe('ulations ('onL'erning ('eH pt'rme-ahiJity. Lipides, when
pre-sent in an aqueous solution containing electrolytes, such
as we hayc in the (ell. will form a two~phase system and,
more especiaHy in the ('R.se of the phosphatides, the ionic
equilihria at the intersurfaces of sueh heterogeneous systems
arc likely to be ('ontrolled by the Donnan membrane equi-
librium. The phosphatides assist us therefore in our efforts
to gin a physical basis to theories of cellular activity which
demand differf>nc-es in ionic concentrations on the opposite
sirles of a limiting mf:"mbrane, with ronsequent differences in
f"iectrical potential, osnlOtic and diffusion effects.
!lut a mere ('rude fatty pellide, wh..ther a.imi""d or not
with proteins and other substances, is quite inadequate to
account for the ever-varying Ill-tivities of the living cell. It
seeJns certain that the limiting nlembranes sutTounding the
""II 'and also within it are much more subtly constituted.
Lthes in his rec<'nt suggestive Croooian - Lectures bas
 srRFACE FILMS 130

('alied alb'lItiol) to ('('rhlin M~P('tb. of tilt" Iwlul\ i~lllr of ttll'

lipid('~ which hd)' u), to UII IlHti('rstllll<iinJ,:' of wlutt the
phy!O.i(al stah' of th('!<,.(' 1l1C'lIlhrHnt'., nUH' 1)(',
\"re han' madt a reff'tf'Jt(,c ('ar1ier i~1 thi" dmpt4'r to tlu'
work of Adam on the ..,tructurt' of \"('rv thin tihwf of l:lltt"
a('ids and similar slIh"hu)('('s on tht StJ~fH('f' of ""}lh'r, 'fh~'
lon~ chain of a fatty aeid 1l1Oi<'t'ule i!-> \Try like t ht' "Hlill'lr
(hnin of a paraffin' hydro('srb()u ('x('t'pt . that tht' form('r
terminates in fl (~arhoxyl l-,7fonp whi{'h t"onf<'rs Oil t hnt ('lui
of the' tuole('ule proJwrti('s whi('h uft' not nppar('lIl ill Hn~'
other part of thp molc('uk or ill tlw purn.f1in hydffH'arhoH.
The physical df('('t of this constitutio11ul dirkrt'IH'(, is nt 011('('
shown by the bf"ha\'iour of tht t w() tY'H'~ of .'iuhsbuw("_ wheu
placed in ('Olltact with water. Ttl(' hydrocarbon mo}((ttl(!-o
tend to <!ling togt"thcr out of ('outad as fur u.s lK)ssiblc' with
tJ\( water. They do not i()rm a film. WhtfelH. ttl(' futty H(id
molecules, although thf'Y are, like th(' para.nins, ill~oluhl( in
watcr, spr('ad themselves out over the surfu{'(' of th(_' wut('r
in the form of a vcry thin film which. if frN' to ('''paud,
assumes the thi('kness of hut ol1e mol('(.'ui(' of fatty ueid. If
a small volume of a benzene solution eOHtaining a known
arnount of ratty add be dropped upon the Mlrl'U('(' of tll('
water. the bcnzene soon ('vaporat.(.~ and le'an-"s f:t. kno\\'n
number of fatty add molt'cules in t.h(' tUm ",huSt" anon. ('UII
be determined ~by simple devices. It has lwt'll ~hown that
the area of the film, when the molecule. ha,'. been " ..eked
close together by a lateral compres&ing- fon('. ("OTrt'\J.tOltds
to a given area per unit mole('ule. which area is til(' salU('
for all saturated fatty acids that are <"pable of forming
films. The vertical dimension or "depth ., of til<' film call
also be _Iculated, and it varies with the lell~>"lh of tbe futty
acid chain. In such fashion it can be shoWI! that ill tllli-
molecular films the molecules are definitely oriented with
their carboxyl groups, in which the affinity for water resides,
anchored, so to speak, on the surface of the wate! from
which the rest of the molecule projects upwards. .Surn.-tJyl_
 140 THE BIO 'HEAll, 'l'RY Oft' 'J'HE F~T
present in film, of tltil. nature exhibil properties quite
distinct from those which they possess in bulk. Pa.lmitic

f't(l.Il._..-:uOOt"!\8i\c phueA in the de:velopmcnt of myelin

outgrowths from R. .treak of leci thin in 11/100 hydro.
ch l Oli~ al"ia. (CourlCllYof ProfcSSQr Leadru.)

FJG. l2. -Mychu out~lh.s from ~thin &fier"!!. hours in

E'9"Ual parts of n.' lOO calcium and sodium hydroxides.
(Courtt'iy of Professor ~tbes.)

acid limnins liquid in sucb films even at room temperature,

and "t sOllie 30' below its true IlIl'lting point of 63 it passe
 QASEOOS AND MQe lD FlLJJ, 14 I

into a physi "I state resembling that r n glls \\',th ,(, mol _
culcs ll1l anchored Ilt one end. as l.entho call< it. ". go"
in two dimensions insteud of thrt.~." Lceilhin n)~ form"
c,."<J)llnded H gaseous" mlll~ at. tCJl'l1 .ralures 8~ low I~ !) .
Cholesterol. which as a film is liqujd up to ~O. I "'e t h.
molecules of ti,e cxpu"ded film. of fAlly acid Rnd or lceithin
to drAw closer together, although tbcy do not pack >ulllci("tl~'

Jo~IO. 13.-Myelin form, of i('(!_ilbin C(lninininp: clll}letlb_

'1'I,1.
(Courl~y IJI ProfeS8l1r Leathc J

closely together to foml a liquid lilm. Liquid unin,ol .. I.,

films of fatty acids can be caused to sc>lidify by th~ appli-
cation of a lateral romprcssing force. The solidification "
demonskated by small particles which were mo"iug freely
on the surface of the film suddenly becoming stationary.
E,.cn in a condensed or solid film, witb the illdkidual
molecules of fatty acid packed as closely together a' they are
in the solid in bulk, it is of great interest to note lhat"'Rtcr
molecules may readily pass through the fUoo. ,\.n cxpaJ'dcd
 142 'PilE BlOCHE~Il 'I' RI" OF THE F.A1'S
or gaseous ftlOi will oller even greater facilities for such
."'poration of water through Ute mm. lnlceithin films this
may be still more marked. since here the lorl,.'" polar group
compri irg the glycrryl cbolyl pho'phoric acid, by means, of
which th~ whole molecule i:,. ancbored, I rcvcnts clo e packing
of thl: fatty acid chains.
Th import"nce of these obscn'ations for the biochemist

}"'l:o. 14.-~Jye'in forms of )<'Cithiu eoutaining cholesterol.

(Courtesy of 'Professo! Leathus.)

lies in the fact th.t they direct attention to wbat is practi-

onll), a new ph~-si<:al state of matter at an intersurfaoe-and
lh'jng matter is one mas ' of intcl'Surfsecs-and particularly
do tbey stress tbe fact that at these intersurfaees matteT is
not arranged in 8 random manner; the molecules assume
some de6nite polar configuration with regard to each other
whichC obdously nUlst profoundly affect their interactions.
Thi , concept of ordered array ill place of ra.rt<lom mixture
 .M YELlS FOR.lIS

mil)' ob,';ou Iy 1)(> extended Ihroughout II. II hoi., of th~ ( "

protoplasm. and ~ill1ple enlculntion, f\'(" to 'John\\' lhut tt\( ,t
animal cells :ontain ."mdcllt Icdlhin 811d (Jlb~r r 1,,1,'<1
substances to pro\ ide for u Ilt'lwork or himol\~ulllr mill
eBen "'ith their polnr group, di=ted 10llurd Ih, C\t'I"' N'cl
."ucow. "h8"" Iyilll( 011 cach ide of the IlIno.
A further indication of thi, oOIl.pl it)", it llIn~ dC' \clol'
within thc cell is alf rdl'CI by a. phenomenon cl "y rei. led
to those which we ho\'c jUht d_il!lclI%(.'d llJ,d \\llirh i.. 'l' t\
when t\ streak of solid l(,(,jlliin in contact with )\ulc-r b
observed under the micT'thcope. Thi~ coll.!'.ist" in thr devclop.
ment of what have been cuUed n)~ elin forlll' of Icdt hill
cOlllplicated outgrowths gh;lIg ri.e to many compl forl1l8 '
Uons. omc of lhese arc illustntlcd in th criall'lic)tograph<
t;'ken by P rofe,.or unlh nnd r.produed ill 'Figs, 1110 t k
lAnlhes ascribes I he formation of Ihese outgrowths to thr
tendency of n.olccules of lecithin ill ('ontact wilh waltr 10
increase ill number; the behaviour or leeiUlin in buJk j~
therefore the Ol>posit of that of oil, wher Ow tendcel 'y i,
for the surface area to be kept .. 'mall lIS lXl!>Siblt. The
attract;,'e force ill the CIC>C of lecithin ;, dirt'Ckd from the
water towards the polar glycer)'l cholyl phosphoric acid
group, and thi attrnction exerts itself beyond the surf"""
layer of molecules initiaUy in conta~t with the ...uter, ."tI
which are probably in an expanded form. Water mokCl,I,...
may there.fore penetrate into the deeper layers of the lecithin
with ease and there giw rise to further complicated patlorns.
The net result ;, that fIlm formation take,,> plact' ill nil
directions, and not merely in one. The ('xocul and form
of these myelin outgrowths i. modified by lbe pr... 'II('C
of ions 8lld of proteins and other sub tances. Some of
these effects are illustrated in the photographs rcprodu,-ed
aoo,c.
It is clear that tb= phenomena of surface film. and myelin
formations must have some hearing on the distributi"n of
lipides throughout liying cells, and they at Iew;l !>erl'e, to
144 TilE BlOCllElIfJ8TRY OF THE fATS
ilHiicak how our older notions of the " lipoid memhrane ,.
nlll~' be ('xpa1l(ied in a manlier whi('h is probahly more neurly
III :l{'('ord with hiologieal Tculity.

'. Beferences
L tI\I>A!l1. /'/'0(', HO,t/. 8oc . 1922, lOt. A. 452, .5J6.
t. ;\H\{:-iifttlSn awl l;o~,t:r. Pro('. {("yo Soc. 1014 15. 88, H,
1 ill.
3. ATKI~~ON. R.\PPORT, and IJusK . Iourn. Bioi. ('hem., 1922,53.
1:1.5,
4. HElW~L\~ and ;';::ABJn'AY. Zf'il. phYRiol. ('hfflt., 1924.187,47.
n. BI.OOH. l'h,IIHi,ui. U"l'W'IA'S, 1922, 2. 92.
0. HI,(lOR, ('/u'nl. Rct'iew8, 1925.2.243.
j, i'l.l'l"U:HllU(,K aud RAPER. JJwcht',tn .IQurll .. 192.5,19. 38a.
!o\, )hKIl-i . . Oxidati(Jh~ and Reductioml; in thl'- Animal Body:'
I~n~don: LHngl~HuI8~ Green & Co .. 2!~d Edition, 1922.' .
n. ]lAKIX, .]ourn. B'l.oi. (lU'm . 1908,4, I I , 419; 5. 173, 303,
l!)O9. 6.203.221; 1923.56.43. . '
W, EMBIJF.'S and ("owoTk(>I'f\. Beifr, chem, Ph.llsinl. Path., 1006,8,
129; I!KI8. 11. 318.
] I. Jh.Hn..Ey. JOttl'rl. Physiol . 1907.36. 17; 1909.38,353.
12. KNOOP. UeiJ.r. rUm. Phys;'ol. P(f,th., H)05,6, 150.
I~. Lr:.\nn~ft ... The- RoleofFat~in Vital Phenomena." C'roonian
Leetnrelo<, 1923. Lannt. 1925.
14. LEATIIES and RAPER. .. The Fats." London: Longmans.
(;'roon & Co . 2ml Edition, 1925.
15. ~lA YER and ~cnAEFJo'ER. JOlun. de Ph.yswl. t;i de Path. Gen.,
l!)] 3. 15. 534.
16. PR)'j)I:anti I1l'MPHREYS. Bior-hNn. Journ., 1923,.18.661, and
llnpublishfld work.
17. RHAFI-~ER. Phys1ol. Reviews.. 1923.3,394.
IR. I'IIAFt'ER. .lollrn. BU.!. Chern . 1921. 47. 433. 449; 49. 143;
1922. 54. 399.
19. SHAFFER and }'RJ.EDEMANX. .Toum. Bioi. Chem.. 1924, 61,
585.
20. TERROINE. "l;)hysiologie des su~tanOO8 grasses." Paris}
1919.
21. WE[NLAND. Zeit. Riol., 1908, 51, 197.
22. WILLSTATTER. HAUROWITZ. and lIIX1...EN. Zeit. pftllM.
,_ .,; Ch ...... 1924. 140. 203.
 J

THE mOCHEJOSTRY OF PHOSPHORUS OOJlPOtniOS

1:--.' th(' two pn.('(>ding ('haptef:'. f('ferNI('(' has th'iflH'utly

h('ell Illudt, to tht, O('Purr('Il('{' of organil'nJly (,Ofuhiut'd
phosphori(, H('id in rt'iation to earbohvdrah' lUll) fut C'Ofn~
pounds und tlwir mdabolism. In tit; prf's('nt dmptt'T We'
shall pa~s on to a more detailed dis{'ussioll of' this type of'
(~mpoun{L amI of its bioloJ.,ri(al significan('e.
Th('rt, UTP tlir('t' ehiC'f ~Ollf('('S of phosphorus uvttHahl( ill
lH~rmal eireumstall('('s to the animal hodv. 1'h('",( UTe:
(1) phosphoprotdlls su('h a~ (,n:-.einogen of milk or vikllin of
('gg-yolk; (:!) Ilucl('oproteins, wherr the phosphorus is Pl'f's(>ut
in the prosthetiC' group in association with a (arhohydrah'
and a purine or pyrimidilw base; and (8) phosphatides. i.t"
Jipides or fat-like bodies (~ontnilling phosphorus. To a les!ooer
extent inorganic phosphates may cOlltriiJutf' to til{' hodily
store of phosphorus. It is possible that tltis list may 1111""
to be extended ill "iew of the discovery, reported by Kay,
in {_>Qw's, goat's, and human milk of at It'ast two, and
probably three, acid-soluble organic phosphorus compouud.,
which are non-protein and readily diffusible. Kay gin'S a
useful table of the fol'lllll in which phosphorus occurs in
milk, thus emphasising the importance of this fSOurce, which
serves as the sole foodstuff of most young growing mammals
during their earliest days. These are :-
(1) Free Inorganic Phospbate.
(2) Phosphate loosely associated with caseinogen alld lint
diffusible. 0
(8) Caseinogen phosphorus.
LB.
,,,
14(j PllOSI'IJOR1I8 OOMPOUNDS

(') I.edt hin, a morwminoInol1ophosphBtide.

(5) lliamillOmOJlophosphatide,
(G) A(id-soluhl, l'hosphori(' Ester hydrolysed by Muscle
})llO!'lp}utasc.
(\) A('id-soluhle I'hosphorie Ester hydrolysed hy Bone
Phos})hHtasc.
(S) A('id-soluhlc orgBuie {'ompound ul1af(cded by thf'se
enzymes and stahle to acids.
:'I:os. (ti) "nd (7) are probably of the nature of hexose
phosphute's. It wiH be s{'{~n that miJk ('ontains aU the forms
of' phosphorul-. of bio('hcmic-al importance with the C'xeeption
of Ilueleoproh'in phosphorus. .AU the organic phosphorus
eompouwls ml.'ntion('d contain this element ill the form of
phosphoric acid linked to various organic rt'sidu{'s.
The high cont('ut of lipide phosphorus in brain and ner\'('
tissue and ib ImiY{'rsa1 distrihution throughout all living
((lIs has in the past been a marked stimulus to hoth investi-
gation and speculation as to the biochemical functions of'
phosphorus. 1n reeent years the interest in this elemf'nt
has tended to widen. and another series of phosphorus
('ompounds, eomprising the arid-soluble organic phosphorus
of the tissues, lIas :received 8 great deal of attention from
LiO('hemists. Included ill this series arc the hexose and
related phosphates, and we now know that compounds of
this type play a fundamental part in apparently widely
different vital processes such as yeast fennentation, muscle
eontractioll, and the ossification of cartilage. The phos-
phatases are enzymes which have a wide distribution
in tissues and which are capable of hydrolysing aud
s ynthesisiug such hexose and other phosphates.

The u - PhoIphat,es
The hexose phosphates are phosphoric acid esters of the
hexose sugars, but we do not yet know whether they are to
be regarded as derivatives of an aldose or of a kM:ose or of-
Ix-th. Assumiug that both types of hexose phosphate may
 lIBXOS1<; I'llOSPIIATB8 147
(xist. illu'itrntc hplow th(' l)()~,... iblt ,.;trlu'turt'''I of an uldo~t'
W('
mOHopl](~'phHtt' (1.) H!Hl a kdo~(' tfip1.n'I)lmt( (II.). whid,
will s('rH' II'> type" of I lItw ('otupOHn(J.., :

r-------O------~
CHOH-CHOH-CHOH-CHOH-CH-CH,
I
o
I HO-~-OH
o

CH - COH -
I z
CHO~ - CHOH-JH-CH.
I
o 0
I I
HO-P-OH II HO-P-OH
I I
o 0
Th(~ phosphorie groups ('tnmot a ... ~(t 1)(' a.~signcd dt'ljnitdy
to any gin1i. hydrox,vl of tht' pun-nt Jl('XOM:~ und tht pOl'litions
sf>lf'<.>tt'd in the abon' illustrativ(' cxaUlpl~ are arhitrary. ft..ij,
are also the positions of the oxygen bridges. Three naturltlly
occurring hexose phosphaks have been delinitely chltrac
terised, but it is 11<lSsiblt-, indet'd prohable, that others as
well as pentose and triose phosphates will be found to Illay
a .part in biochemistry as important as that assigned to the
known phosphates. These are; (1) hexose diphosphoric
acid, C,HlOO.(PO .H.).. discovered by Harden and Young
in the produets resulting from the fcmlentation of suga...
by Y<:BSt id"the presence of inorganic phosphates; (2) hexose
monophosphoric acid, C.HuO.(I'().H.), obtained by Xeuberg
from the diphosphoric acid on partial hydrolysis; (8) "
second hexose monophosphoric acid obtained by Harden 4Uld
Robison, like the diphosphorie acid, by the yeast fermentation
oCsugars.
......
14R PHOSPHORFS COMPOUNDS
Preparation of Hexose Djphosphoric and Monophosphoric
Acids by Yeast Fermentation (Hohi:"'t)Jl':-, !\Iethod}.---.Julcc
)It('}lIlf{'d from fr('~h l)rt':-.~('d yeast i!':o mixcd with IO pf'T ('{nt. of
its W('iM:ht or fr\Hto~l' or glu('o:-,c (umnllo~c or suprosc may also
Iw us(l) alld warnwd to 26 in a wat{'T bath. ''''Iwn fcrn;{:nta~
tio;1 ('OIHIll('f1C'('S a:!O per ((nt. flqU('OUS solution of Na 2 HPO" .
12 Itz() i ... adl}(d from time to tinw in such quantity a~ to
produc(' the maximum fat{' of evolution of carbon dioxide.
\\'111'11 the total volume of phosphate solution added {'xcecds
half the original volume of juice a further quantity of sugar
is dissoln~d in thl' fl"rm('ntation mixture. "'hen the further
addition of pho~phate no longer causes any considerable
rise in the rate of p"olution of carhon diox.id{, solid barium
acetal<' in amount equal to the weight of the crystalline
sodium phosphat(' added during the f('rmentation' is dissoh'ed
in the f('action mixture. which is then r('nd('rcd just alkalin('
to phenolphthal~in with baryta, and an equal volume of
IIloohol is added. This pn.cipitates the barium salts of any
(-'XCtss phosphate and of the two hcxos(' phosphah's together
with the yeast juice proteins. After standing overnight the
pre<'ipitatc is filtered, washed with 70 per cent. alcohol,
treated with boiling absolute alcohol, and allowed to stand in
contact with the latter onrnight. This renders the precipi-
tated protein insoluble. The crude dry harium salts are
ground with 10 parts of cold water, which dissolves the
barium hexose monophospbate, but scarcely any of the
diphospbate. The residue is ground twice with small amounts
of water, filt<!ring each time, and is then extracted with
200 parts of water, which dissolves the barium hexose
diphosphate, lea\1ng the insoluble barium phosphate and
protein. Tbe filtrate is treated with lead aeel!llte a~d the
precipitated lead hexose diphosphate is separated, washed,.
suspended in water, and deromposed by passing hydrogen
su,lphide. The clear filtrate is heed from sulphide by
aeration, and is filially neutralised to phenolpht,halein witk
(!au.tie soda. Traces of free phosphate may be remond by
 PREPARATION 149

predpitation with mag-n('siulll 8('('tnt('. Thf' prf'('ipitfttiou

of the It'ad salt is rq)('tt'd at It'ast twil"l" Mut th(' 11n81
product is found to f){' (rN'" from nitroJ._'t"u and to hfl\'(' tlw
composition C.H,oO,(PO,Ph),. By t",atinl! thr It'ud 'nIt
jn S!]Uf"OtlS solution with hydrogt'Jl ~ulphjdt\ mh~g. ntul
{'onccntrating the I.;olution in flfl("UfI, the f1''(' hrxose
phosphorit ft(id may hf' ohtaiHed .. " :-.yrup,
The Hrst aqueous cxtrnd uf the C'tudt' harium salts i,
treated with basi<- h's(l aCt'tatC', and thl' insoluhle hu~h" It''8d
lwxos(: monophospnate is tHtcrNi and wa..\ihed. Jt is d('~'urn~
pos('{l with hydrogen sulphide as d('s(Tilwd tthon'. and tlw
add solution is nt'utraliS{'d to pIWllolphthnl('ill wit.h hot
saturated baryta. After IiItration the prN'ipitation with
hasic lead acctatc is npl'atNI. TIlt' produ("t is ('ontamiunt('d
With saltsof sucC'illi(' aTld othrr fH'lds. hy~prodtJ('ts of tht
frQllcntutiol\. and with" little of the diphosphate. Further
purification is eff('cted hy forming tht> !o.oluhlc hurium salt,
precipitating thi~ with a1<ohol. and adding m{'r('uri{~ O((tJltt-
to the filtered solution of tht" hnrium '>nlt in 10 TX'r (,(nt.
alcohol. ,From the filtrah' the lead h('xoS(~ fHonophosphnt(
may again be predpitated. R(,COHY("TSiott to thf' harium
salt and repeated p...,eipitation of the fatt"r di ..""f",d in
10 per cent. alcohol by the addition or an equal yolmn<' of
absolulr alcohol completes the puriiieation. Aft..r dryillg at
78 12 mm. over phosphorus pentoxidc it has thf" composition
C.HllO, (pO,Ba).
Preparation of Hexose Monophosphoric Acid by Hydrolysis
of the Diphosphoric Acid (Neuberg's J\ffthod).-Th( hydro-
lysis is best effected by means of oxalic add, but mi'l<'rl11
acids may be used. Calcium hexose diphosphaw (13'7
grams) is ,.;Iisso]wd ill J.50 c.c. of hot N-oxalic acid. Aflrr
boiling for thirty miflulrs the solution is cooled and fillrred,
warmed again and shaken with an eXceas of barium carhonnw
until the reaction to litmus is no longer acid. AfteT standing
in tbe ice-chest the clear filtrnte i. ooncentralrd in .-3cuo
at 40. After filtering It further tmce of pf't'cipitalr, tfte
J50 PHOSPHORUS COJIPOUNDS
('rude harium hexose monophosphat(' is precipitated hy the
uddition of alcohol. It is purified hy ft'dissolving in wah'r
and again precipitating. l"hf'u aHow{'d to ("it'posit slowly it
is ohtained in It mjcr()~{'ry!o;tuUill{, form. After dryiug ut 10.'1
Q

it has tlw composition (',HuO,(PO,Ba) + Bp.

Properties of the Hexose Phosphates

Hex{).s!' diphosphorii' acid is feebly tle,ltro-rotatory. hadllg
lal il + :\'4'". it reduc('s }'(hling's solution after soru(' hours
in the ('old, and rapidly when hoiled, hut its rr-du('ing- power
is only ot}('4third that of an cqui"alent amount of glu("os('.
"'hen warmed with phenylhydrazine hydrochloride and
sodium acetate it yields a phenylhYdrazine hexose mono-
phosphate 0S87,onc ; -
('ll: IN . XII. (',H,). C: IX . ~H . C,H,). C,lI.(OH).,. pO,H,.
H,X. NIl . (',fl"
whicb forms yellow needles melting at 151 0 to ];;2 (HR'
according to Embden). In the cold the product formed is
diphcnylhydrazine hexose dipl.osphate phcnylhydrazone :---
(,H: (N. NH. C,Il,). C,Il,IOH),. (l'O,H,. H,N. NH . (",H,),
The reducing power of the parent diphosphate, the presence
of hoth phosphoric acid groups in the hydrazone. and the
loss of one of these in forming the osazone~ point to the lost
group occupying a position adjacent to the reducing group,
i.e .. ill tbe 2 position if the diphosphate is. glucose derivative,
in the I position if it is deri,'cd from fructose, which is more
probable. The position of the other phospllOric acid group
remaills undetermined. When the diphosphoric acid is
boiled with acid or with water, free phosphoric add is
produred, and from the resulting Solution, which beeornes
lavo-rotatory, crystalline fructose can be isolated:' The cold
{""'o-rotatory solution reduces J'ehling's solution in a few
minutes in the cold-a property whi('h is also shown by the
re8l!tive so-called " y ., sugars, but which here may be due
t~ triOS<'-like deeomposition products, The diphosphoric acid'
 PROPERTIES 11\1
is also hydrolys(>d by emulsin. and by th( l)ho~phorj(' ('Sh'TA."l'
pI't'sent in yeast juiC(' whi('h has lw('n f'rC'('ct front CO~("HZVIU{'.
In hoth C-US{'S a IWt'(}*Totntory produet whj('h is appar,:ntly
fructose is form('d. Tlw hvdrolvti(' ndion of R('ids "nd
('1l1:YIOC'S 011 tllt' diphosphate ~\'ouhi M'O)), tht'Tt'foTC', ') ht' the
same.
Hexos(' Jllonophosphorie a.dd fomlcd along with the di-ndd
during yeu-.'4 fernwutatioll i~ ulso fu',tlro*rottttory, hU\'lug'
I:xl v t 25'0"', It. forms a p}u~nylhydr8r.it\( h(,xoM~mono
phosphate OS6.Z'.orl(' which melts nt 18H'? with d('{'()mpositiou.
A phcnylhydral..orH:' has not yet he-l'n oht.ain~d. 011 hydro...
lysing the monophosphori(' a('id with acids or with ('mu'~in
the rotation dirninishts. hut remains d.f'.rtr(l. Tht futio of
the rotatory power of the resuitinJ! solution to its feduring
power is ItlU('h lower than that of' solutions of pure glU('()~,
bl,t th{' ratio reducing power to phosphate lilwrnhd indirnh'!\
a {'onsiderable dcstru(tion of sugar. From the hydn:,))"wd
solution glucosa1.one may be obtained. It i!\ deAf from til('
foregoing e,"idence that the hexose monophosphori<- acid
formed along with the diphosphoric add during yeast
fermentation is quitt" a different product and drn:s not arise
from the latter by hydrolysis. nor would it appear to he all
intermediat, between the sugar and tll<" diphosphate.
The hexose monophosphoric acid obtained by hydrolysis
of the diphosphoric add is much le'Ss strongly dextra-rotatory
than the monophosphoric add described ahove, having
+
1ID 1'5. It is interellting to note that, unlike the
diphosphoric acid from which it is derived, and like the other
mono-acid, it is fermented by lhing yeast and by dried yeast
preparations such as zymin. It seems not improbable that
tbis monQPhospboric acid is It derivative of fructose, whereas
tbe "mono-acid formed directly during the fermentation of
sugar hy yeast may he a glucose monopbosphoric aeid.
These interesting questions of structural chemistry require
further elucidation. 0
The Signiiican~ of Phosphates in Yeast FerDll'Dtatioq.---
1;i2 PHOSPHORUS ('OMPOUNlJI:;.

ObM'f\ ati()l1~ of the drt.'d of ~odium phn~phat(' in inc.reasing

tilt' hItal f('rnH'lltatioll pro<iuN'd by yt'ast jui('t' date from
HlO1. but it WHS not UlItiJ Hardeu lwd hi~ H;'\sociat<"s took up
th(' illH"tigntioll of this prohlem in 1905 that til(' nw{'hanjsm
or this (,(tN't Hnrl tilt' fundamental rolf' of ph(hphat('~ ill y("ast
fnniE'lltatioIl bt't'unlP dear. TIH' A"('tH'ral dfe('t ofth(' adctition
of' ~odium phosphuh' to "iugur lmo('rgoing fermentation hy
yt'ost jui('(' is shown hy tht' ('urn>s repro(h1<'cd in }'ig-. 15.

10
melH~
10 .10 040 50 .0 10 eo 10 lOll III: 120 IJO * ISO 110

FIG. 15.-Influence of phosphate' on yeast fermentation

of sugar. (Alter Hard{"n.)

('urv' A frpresents the rate of erolution of carbon dioxide

from glurose undergoing fermentation by yeast julee, whilst
cur,'c B shows the effect produced Oil tbe same amount of
g!U('ose acted on by th~ same amount of y~ast juice under
parallel conditions, but with the preliminary addition of a
mix{ure of primary and secondary sodium phosphat""_ The
lit~ar porti911 of ('urye A shows a carbon dio:-<ide production
 YEAST FER,t[ENTA7'IOS
of about 1-4 {".c. IM'r fiv{' minutes. 10 ('unT B it will ht ~f'l'li
that the presetlc(' of phosphate rAise", this to somc' H5 (',t',
per 1iv{' minutes. hut this rut<> fnlls off #..fTlHiunJiy Hlllil uft('t
an hour a uniform rate pro('ti('uil.v tilt' snnl(' n.,; t hat of
{'urw' A is nttainf'd. ('un'(' (' shows that tht nddifton of II
fresh supply of phosphat<\ f.qual in amount to tlw 1i,.,..t.
('nus!'_'" th(' whoJe phPflUHIt')Wf) to f,fUT. H in tJ}('.st~ ('urn',
one extrapolates the line of steady rnh' hll('k to t h(' :fA'ro
tinw axi~ as shown hy ttw dott ..d lines it will 1)(" S('ell tlmt
the first Hddition of pho'iphntc CllUS('(f an ndditioual ('urhfHl
dioxide productioll of some :JS ('.('. whell ('ompare1I wit h
('urw' A. and that the' S("('ond additioll prndm'('(i tlH ahuo,t
identical increase in the total carhon tiioxid('. The (,Ilkufntc'd
ill('f('sse in the carhon dioxide, assuming thnt ttlf' {,fiN" j",
l;roportional to th(' phosphate addt~d as <'xpr('..s(><t hy 111(,
ratio (,0 2 : H 2 HPO", is~ in th('~i(' cXI)('rlmc-nts, 3~HJ ('.t', By
SIH'h experiments it is possiblt to show that til(: "dditinH of n
soluble phosphate to a Y('a!oit jtli('('~h{'xos{' ff'rmcl1tntion
mixtur(;' produ(>e'S an equi\'alt'nt amount of ('arhon dioxide
and all'ohoJ, the efff>i't heiug fluantitatin'Jy t'xpr~~~(d h.v the
equation ;.-
2 C.HI~08 '" 2 R 2 HPO" --.. t COz ': 2 (':Hr,Oll t 2 H:O
+ C,H"O,(pO,n,), . . . . . . . . II.)
It will he seen that according to the aho\'(' ('xpr('~sion two
molecules of hexose sugar are involved, OIl(' lIt-jng ('oJ\vt.'rt('d
to he~t)se diphosphate. whilst the' OUl{'f is brokrll duwlI to
carbon dioxide and alcohol. It is also apparent that fh, frt'f'
phosphate is used up in formin/( the dipho!;phate, and sill''''.
when the amount of phosphate present in the materia" used
in a fermentation is redu('cd to as low a hmit as possibl(',
the. rate.,,; ft~rm('ntation is proportionately diminishrd. it
is reasonable to sUpJlO"-e that a supply of phOliphate is
indispensahle for the continuation of the rcadion. Whence
is this to be derived? The answer is U, be found ill all
probability in a study of the distribution of inorll:ftnif! aud
orRanie phosphate in a (em.t'nting mixture to whid. 1'''''''
1.54 PHOSPHORUB COMPOUNDS
pMt,. has bc,'n added. Such a study reHRls th,. following
thr(,t' stag'('s in the process :----
(l) A period ('oineident with the in('reasrd fermentation
durin~ whivh the fret' phosphate rapidly dirninhh('s.
(2) Ths' uniform period following the Q('ce1erated f('rmettta~
tion .dllriug' whi('h theft' occurs no alteration in the small
amount of (rce phosphntt' pres('nt.
(II) The J)('riod durilll( which fermentation lessens and
tinully ('cast'Sin whi{'h there is a rapid inCfeRSt' in free
pho~phu.k and a ('orTC'sponding diminution of hexose
phosphat...
All enzymiC' hydrolysis with proriu('tion of free phosphate,
similar to that oc('urring in stagc (3), is also effected hy
tn'u1ing hexose' tliphosphatp with yeast juice from whieh
tht' diffusibk eo-enzyme has hecn removcd hy dialysis, so
dqlrh-ing the juipe of its ferment.ing power. The su~r
formed at the sume tim(' is found to hr readily fermt:'ntahie
hy living yeast Hnd to possess the reactions of fruetose.
Assumiug that this last r)hasc mentioned above can be
represe-ntcd by the equation :~
(',RIOO,WO ,R,), -, 2 H,o ---> C.1l ,,0, + 2R,HPO.. . (11.)
H"rd~n gins the following simple explanation of the
~('f}lIenc(> of ('vents during fermen.tation. .. The rapid
diminution in the amount of free phosphate during stage (1)
('Orresponds with the occurrence of reaction I. During the
whole period of fermentation the enzymic hydrolysis of the
hexose phosphate is proceeding according to equation (11.).
Up to the end of stage (2) the ph""phate thus produced enters
into ",action. according to equation (1.), with the sugar
whi('h is present in excess and is thus reconverted into
hexose phosphate so thB.t as long as alcoholic fer;;;'entation
is proceeding freely no aeeumulation of free phosphate can
occur. As soon as alooholle fennentation ceases, however,
it is 1'0 longer possible {or the phosphate to pa$ baek into
hexose phosphate, and hence it accumulates in the free state."

 LAC'TACIDOGEN
The following diagrammatic ,.;;('}u'mf' "prf'M'nts tlli . . "i{"(IU('lU'f'
of events:-

The part played hy the hexoS(" llIonophosphatc wili('h is

formed along with the diphosphll\(' in this <wl,' j, not wt
clear. It may possihly rt'pt{'~nt all iut('TUU'(jiuti' ('ornpm;ud
in the- reaction serie:-- leading frorn the sugar to a(l'ohol Hnd
carbon dioxide, in which case it might l)t iBst'rted in tIlt'
8.bOre sc~me at the pG-int,o; marked X. In this eOllflNtion
it.is 5uggtstivc that its init.ial ratc of {<'rnwntJ\tioll by ).'entot
juice and zymin is approximately the sam.(~ as that showH
by a mixture of phosphate and gluC()sc,

Lactaeidogen and the Chemistry oJ Muscle Contraction

Physiologists have recognised for many y,'ars that t hl'
contraction of a muscle is associated. with a utiJi.,ution (If
carbohydrate, which in disappearing gives rise to ladic add,
and which is finally oxidiscd to carbon dioxid~ and wah'f,
The actual state of contraction of the nlUsde i. closely bound
up with the liberation of lactic acid from the muscle sugar,
whilst tbe subsequent oxidative removal of the lactic acid
occurs during the period of rec",'cry following on the acti\"<'
contractile state, In the absence of an adequate supply
of oxygen during this second phase of recovery, the Iactit:
acicj forn'l'ed during the first phase acCumulates in the tnllsclc
tissue, which becomes H fatigued." or contrs<-'{..., with less
vigour or not at all. In 8 long series of researchL'S Embden,
Meyerhor and their associates have established an inti_I.e
"","",(ation between the conversion of carbohydrate int<>
 J;}11 PHOSPHORrS COMPOl'NDS
lactit add in t hc ('ontract ing tnusck and t}w presf"nce of
Jlhosphatt'~ in Hit' latter. Thu!\ during ('ontraction phosphate
and la('ti<- ucid Urt' formed in t:quimolar proportions. By
IHiding ex('ess phosphnt< to the muscl(_. ill an atmosphere of
hy<i'ro,l!et. all its stofC'd carhohydrah' (glycogen) may -be
('on\'('rh'd to ladie u(jd. and ns long as supplies of phosphate
and gl:n'ogcll or other eurhohydrate arc kept up. further
formation of laC'tif' Reid may tak('- place. It ean also be shown
thnt OJ{' lwxos{' diphosphatt-. whirh j.., fornlf'd during the
f('rlllt~ntation of su,ltar by 'yeast. is ('onn'rted to lactic' acid
and phosphoric' aeid hy thf' A-f'tion of thc' juice (mus('}(' press
jUjl"t') f'xtruf'ted under high pressure from fresh pulped
muscle tissues. The addition of hexose diphosphate to the
t'ontracting musl'lt produces the same increased formation
of IH('tic' acid vh!oOen'f'd when glycogen and phvsl>hate 8re
",lned. These and similar observations Jed Embden and hjs
a~sO('illt{"S to postulnte the existence of an intermediate
(,Hrhohydr[ltt~~phosphah' compound, to which they gan' the
mlHH' /nclo(idogf'lI. since in their "jew the formation of this
compound preceded the appearanl'<' of the laetic acid in the
muscle. and the add originated from it. It was found later
that lHC'tnddogeu, isolated from muse}(', i~ eonnrted into
ladi(' add and phosphoric acid by musde press juice just as
})(xos.e diphosphate undergoes the same COll\rersioll. There
is now (',,"ery reason to belie\-e that the lactacidogen is
identical with the hexose diphosphate concerned in the pro-
cesses of yt'"ast ft'"nnentation. Embrlen and his co-workers
han' isolated from a fresh pulp of striated muscles a substance
(their lactacidogen) which giYes a phenylhydrazine salt of
a hexosazone monophosphori(, acid of the composition.
C..HUO,X,P, identieal.with that obtained from t~ hexose
diphosphoric acid prepared from a yeast-sugar fermentation.
Method of Isolating Lactacidogen OsazoDe.--Immediately
after death the ice-cooled muscle tissue is finely minced,
stirre/! for one hour with its own weight of ice--cold " per
cen\. hydrochlori(' add to desnoy the '''''tacidogen-splitting
 LAr'T Ar' T[)(J(I E X ORA Z() N E liOi

t'llzymr. a VO]UlUt' of S per lIIen,uri(' f'h}uridc' ('quHl to

('{'lit.
that of the hydf(whlorie tH.'id i"" add(,tL Hnd t lu' \, hull' j, kt'pt
owrnight ill the i('(' dlt'st. ~h'rc'lIrv i ... rt'IlHI\{',l froIH ttl('
m~ratt~ hy lH('nus of hyclrogcu ~ul,,hid(.. "II(} tlt-c l.tltc'r , ...
removcd by u{'ration. !o.odiulH hydroxide i . . 1i1f'lI IHit\cd, with
('xt('rnal {ooling. until the' rf'adiou j" Jo.hH just ntilJ, UHf)
tinaH~~ harium. hydroxiti< (fh'(' frotH {'nrhonut("j uutil ifw
rt"aetion is strongly alkalint'. The t('SllltillJ,.: !If('{'ipitate i ..
{'oUccted, washed with i('('~{'()ld Wllt<-r uutH (1'('(' from uJkJi,
ground in a mortar with 25 p{>r {'(Int. ~ufphuri(' It(id. ltguill
cooling. and from tht' filtrate' JnllglH'sium is prt(jpitntNi ,,~,
tIl(' addition of a slight ('X('('S" of IIt'utrul I('ad u('dftft'. Thi,..
precipitate is ('xtrart('d as hdort' wit h ~r) Pl'f t.'~>I\t. hu\plmri('
!lcid and thoroughly washed. TJu' mtc'tN} solution ('mJtHjfl~
iactacidogen. phosphoric add. and 1lU(leie a('id. Til(' lath'r
ili precipitat<'d hy adding .j. to 5 yohltll('~ of' purt IIldhyl
ak-ohol, aud the filtrate is ('oll('('ntrakd ill ractltJ at ttl. "'0
that from 5 kg. of musd{' ('xtru('t('(i !'oOlll(' 100 t () 150 (.. to. of
~o1utioll are ohtaiJled. This SO}UtiOll r('dtH(,~ }'t'hHIlU'..,.
solution. To it are adde(.l powdered sodium a('date, until it
no longer reacts add to Congo papcr. anti plW'tylhydraziIH'
hydrochloride. It is warmed at 80' and filt"f('(i if nC'",,,'"ry.
After thirty to forty minutt~s the OSa7.()IU '"'('purahs <'itl1('t
in the warm solution or on (ooling. It may hf' purili(otl hy
recrvstallisation frolll hot methvl alcohol to whi('h hot ..!tloro-
fo";' is added, It melt., at i4l!'.
Emhd<:" h",ohtain"'l
from 7 kg, of dry muscle 8-52 gram" of Ih" T('CrystaUi"",1
osazone.
The isolation of the same osa7.0ne frmil Jaetacidog<'ll and
from the hexose diphosphorie acid obtained from yeast doc..
not Dea8SS8.rily nlean that the two parent l'ompound~ are
identical, since the formation of the osazone inl"oh:("S t.he
loss of one phosphoric acid residue from the yea.,t acid, and
lactacidogen might be a hexose monophospboric acid, or
if it were a diphosphoric acid the lost group need not &ceul>Y
the same position in both compounds, The foregoing
 liiR l'lIOSPlIORUS COMPOUNDS.
obs(rnl-tiuw. do, how('vC'r, estahlish tll(~ identity of la('tacido'
W'1l n~ H hex()st' pho:-.phut(, It!-o tinal id(\ntifi<'ation had tc
aWliit tht' isolatioll of thf' utwhangeo lacta('idogen. and thi~
Etnbdt'l\ and ZimtHl'ftnallll ha\'{\ successfully u((omplisl\NI.
Tlw diilihtlty of isolating the musl,le laC'ta('idol!('ll unchanged
b largely du(' to it~ ('ontamiTlutioll with free phosphori(' acid.
This diflkulty has he('n o\'crCOflU' by COH\Trtilig the excess
pho!oophori(, Iwid into lactaeidogen by a process of fermenta-
tive ).;ynth('~i~. Emhd{'n rnade us(' of tIl(' property of cC'rtain
iOllS, su('h HS tlw fluoride iOB. of promoting the fornlution 01
laduddo,l{('11 from phosphates and eurhohydratc' by th{'
8ctioH of fresh ruww}(' pulp or muscle pre-s5 ju1C'('. The method
is as follows ;----
The' mus(1t- tissuf' is findv ITlinc(d. ground in a mortar
with sand and ki{'sellZuhr, au;} the juiee is extrad("(i from the
pulp, using the l1uchner apparatus up to 320 atnw..,pheres
pressure. The.se operations must be carried out rapidly with
effkicnt icc-cooling. The ice-cold juice is treated with an
equal "olume of n/8 sodium fluoride and 0'4 per {(nt.
glycogen. Sodiulll bicarbonate (2'0 per (oent.) is added and
tl", mixture is allowed to stand for four hours at about 10.
the free phosphate being converted to laetaeidogen. It is
then treated with 4 per cent. hydrochloric acid and 5 per
cent. mercuric chloride to precipitate the proteins, and after
standing it is filtered, treated with hydrogeu sulphide to
remove soluble mercury salts, agaiu filtered, aud the excess
of hydrogeu sulphide is removed by aeratiou. The laetacido-
gen is preeipitated from the filtrate by the addition of onc-
tenth volume of 10 per cent. copper sulphate and sufficient
slaked lime to precipitate all the copper as the hydroxide.
The precipitate is separated, ground in a mortar with exc;t'ss
of !l5 per cent. sulphuric acid, filtered and washed, and the
IiItl'8te is again treated with lime and copper sulphate. Th~
precivitate is ex'tracted with sulphuric acid as before, this
time avoiding excess of the' latter. Dissolved copper is
~ved f\'OUI the "leu filtrate in the usual way with
 lJRI'('INE SALT l.tH

hydrnW'Jl sulphide. Thf' acjd solutio!) t!lu .. ohtntlWd gl\'('. .

R strong l\fo}is(>h r(,Clctiofl HUfl redu('t's )<'t'ltJinq\ !-tolutioll
vigorously. It is treflt('(i with the f'XH.<'t IUHOHl1t of hur\'tn
n("C1'Ssar~: to prN'ipitatc all tilt' snlpllllrit' tH'id. utHl tll\
fHtrate. still a<'id to litmus. b pre<"ipitatl'd hy Hddil'~ 10 p.. 'r
('('lit. If'ad ft('(>tate. The \'ohuninous pr("('ipitatt- is nllr,wt'd
to settle and is U}{'n separuted and pomplttj)Y def'umlK .... (,d
by means of hydrogen sulphide. ..1ftt'r lwiug- worked up iu
the usual way the filtrat<' from the dC'{'ompo!-.('d it'ad )In'('ipi~
tatc contains all Hu' lact3{'idogeH. smitH anHJIIHh of IlU<"lf'tt'
R(id ('ontaining l){~l1tose. and hut trf\('('s of ph()~phori{' o('id.
It is treat('d with 11. ~aturated tnrthyl ui{'olloJie ~()Iutiull (,f
brueim~ until it is Jwarly neutral to lit;llUS. Pr<'{'ipitntioJ) HIHI
C',fvstalJisation of the hrueine salt soon ('omm(O',('f'S HUrl j,..
h~stened l)y (()oling. The crY1oital!ii an> separatefl, dr;(d in a
VAt'Hum, and recrystaHis('d from nwthyl altohol (olltainillJ!
50 Iwr <"ent. wahr. 15 grams of pun' r(~ry~taniS{tI ~Hlt IUAn'
bet'll obtained from the muscles of one tnhhit.
The brucine salt ohtainf:'d as described aboH' gin'!'> f\f1u~
lyticaJ fiJ!Ures which identify it as th!' B('utral sn1t of ilt'xost,
diphosphorie acid. It. physic'al properties (,oIl/hili,}", "l'ti('111
rotation. and mclting point) are identical with thn~{" of til('
eorresponding derivntin' of the hexl)~w diphosphorie uC'id
from yeast. We may the-reforc cOlwludt' that on~ and th('
same hexose diphosphate is primarily cOllc"'rned in the'
processes of yt"ast fennentation and of the C'ontra{,tinn of
,,,,,mll1lllian muscle. ]t is to be noted that Embo"IJ dot's not
exclude the possibility of a hexose monophosphate playing
some part in the latter process just as it does in the fomler.

'!'he~ of Chemical Eventa in JIucle Contraetion

iccording to present-day ideas the succession of events
as they occur in a muscle contraction are as follows: The
muscle glycogen combines with phosphate (prestu;'ahly
passing through some intermediate simpler form of carbo-
hydrate j, the resulting hexose phosphate is hroken down'by
 HiO J'[IOSI'IJORCS COMPOUNDS

til<' lllWWi<" t'!\'I.YIlH' or ('l\1.ylll(... with til(' iibf'ration of phos~

"hur-if' H(id Hnd la~tic M'id. Hnd it j, during thi.., phase of
:wid produdion thut tilt.' lllUS(I(, Hl,r('!-' (ontr.:wt. ('olu('i<it'nt
with'the fOfllHttioll (If ludi(' i{(id. fr('(' g-lm'oM' is ab,o formed
from t ilc' ~ly('ng('IL It i!'oo thi!-. latb:r portion of the original
gl~t-.:)gt'H ynoit-{'ule whieh i:-. dired ly {lxidised 10 ('arboll
di4lxide and water. t ht Jad if' acid pa . . silll! through a phase
of tc(omhinatioH. Tht' modern tend(ncy is to ascribe til('
u(twd (_'ontrlll'tilc effed to li ('hange in the surfa(~(' energy of
til(' Hhrl'~ ('QutlitJoH<d uy tht-' ~ud<len. pra(tieally t'xplosin.
f(~kH~(, of ~H'id. hut a diffcr('O('(' of opinion ('xists a.s to tilt,
prh. phtYfd by tlw two 3(jd),. Elllhc}('n nthwhes importanee
to tht, phosphoric u('id ill this respect, whilst ohservations on
tlw }wat pro~hl('tion of the> ('ontrllC'ting muselt' point to lactic
udu as tlH_' a(tin agent. TIl(' whole ('ontra('til(' pt.Ulo.'{,. whieh
includc's Ul(' (.)(l~('t and maintenance of tilt, ('outraction. and
til(' relaxation to tll(' norrnal resting condition. proet~cds \\:ith
a relatiYl~Jy ~nulll heat output and cun take place ill the
('ulite abseJlce of oxygen. It i:'i therefol't" {'a1Jf'rl the nnIJt'Yobh'
pha~e. During the r('t'overy or aerobi(' pha..')c whi(~h follows
the rdaxation. and which is onJy completed in the prf'sen('{'
of oxygen, there is a prolonged heat produrtion. The uptake
of oXYb-'t:"n during this l>hase (.'Orr('sponds to the oxidath'('
n'lllo,"al of the lactic acid formed durillg the ftrst phase.
Hut this acid is not directly oxidised to carhon dioxide and
water; illdrt'd the OXYII"" ahsorbed i. only one-fourth of
that re,!ui,ed to a""oullt for the oxidation of the lactic acid.
It is resynthesised to hexose phosphate and ultimately, as
]lleyorhor and others have shown. to glycogen, whilst the
phosphoric add is Iiherated durillg the last stage of this
synthetic process_ and tberefore becomes free to R'lrticipate
in the chemical events of the next contractile cycle. The
production of carbon dioxide during the aerobic or recovery
phase proCeeds from the oxidation of the free glucose fonned
along with the hexose phosphate. and whieh represents one-
tlfth part (approximately) of the glycogen molecules taking
 MCSCLE COSTR.4('TlON 161

purt in the eyd,'. We haw tho" in the ('olltrooting mu""l.

a suc(!('ssiolt of ('\'cuts of {l (Tdi(" flU tun'. in the roUt'NC' ()f
which one-fifth of the glyCOW'", is (~.mpl<-t('ly oxidised to
t'nrVofl dioxide nnd wattr, whil" th" oth"r four-lifth'1pns.in g
through a Tf;>Y('rsihle proces." whf'rt'"hy tht'y nr(> f'Onn'rtNl to
hexosl~ phosphat< nnd lu(,tic a(id. arE" {'\'f'ntunlly l'ffon!lOtit~'trd
as glycogen and pass{'rl on to suhs('qutnt ryd(s. The'S(>
su{'cessive and reversible rt'ftctions have h.cn ('xpt'('"fJsed by
lkyf'rhof in a sl'ri('s of ch('mi(~ul c-quaticlHs whi<:'h. althouJl'h
they ('annot bc' U('reptt'd too litt'r8-U~\ llev{'rtheic's,,, fWr\'e a)lt
Auseful btlsis for the theorrti,'al aspeets of the chemistry of
muscle ('ontru('tion. These ("quations arp :
(I) The A,ueroiJic or Contractile P//(/<ie.
5in(C.H100,). + 5H,O 8 K,IH'O.
~CC .~ l',HIOO,(pO,K,), t (',IJ"O, f H H,O
= 8 C,H,O, 8 K,HPO, + (',U,.O,.
(2) Tit, iErobi< 'ff RecoveTY Pita...
R t aH,03 +" K.HPO. + <",H"O, 6 0,
= 4 C.H.oO,(l'O,K,), + 6 CO, ,- 14 H,O
= 4/,,(C,H. oO,). + 8 K,HPO. + 6 ('0, + 10 II,O.
A schematic r('presentation of tht" events in th{" anovc series
of equations may be drawn up as follows :-

Such " scheme, when compared with that given on p. 155

.
for the processes of yeast fermentation, serves to em!:ha..ik
....
11,2 PIl08PI{(JBF8 COJfPOl'N08.

th(' t~sS('ntinl similarities of the two phenomena. "'(. han'

in hoth a T{'v('rsihlr (yelt." in which the hexose phosphate
plnys an indisp('ns"bl~ part. althou!(h in neith,'r c"s~ is it
dir(ctly transforul'd into the (nd-produ<.ts. In yeast
fC"rnwntation the ('_arhon dioxide and alcohol art' fonned from
the fcrnwutahlt ~ugar produced along with the h{'xost'
phosphak. whilst in the contracting muscle the carbon
dioxide also arises from the sugar simultaneously produc{'d
with tht fl('xos(' phosphate. It is an impressi\'(' fn{'t that
l\:'nture should have devised sllch fundam(,l1,tally similar
nw('hanisms f()r two apparf"ntly dissimilar prt){'('SS(,S. the onc
rc\"eal~d hy one of the lowest unicellular plants, the other by
the highly organised muscles of the highest animals.

The Chemistr7 of Ossification

Dry nlsuoY{\ess booe consists of some 60 per (cnt. of
t

calcium and magne-sium phosphates and carbonates deposited

on a matrix of mucoids, collagen, and a small amount of
other proteins. This matrix thcr("fore is ('losely similar to
the ~artjJage from which the bone is derh-cd by the prO<'esses
of ossification. Of the inorganic constituents of bone, calcium
phosphate is the most important, comprising some 85 per
'nt, of the total inorganic matter, and therefore im'estiga-
tions of ossification. processes haye largely centred round
tll<' problem of the deposition of this insoluble salt. In hone
it is probably prt'sent as a romplex phosphate of the type
of hydroJ:Y<lpalite. (Ca,P,O.). . Ca(OH) but for our purposes
it will be sufficient to consider the tribasic phosphate,
Ca.(PO.l ..
The problem of the <ieposition of calcium pbospl1iite du,ring
os..ification has always been a difficult one, and it is natural
that the capacity which proteins possess of fixing both
calchlID and phosphate in a non-diffusible fonn has beeu
roade the basis of theories of ossification. Freudenberg and
GyUl'gy are present-day exponents of such a theory, tbeir
 nSSl FICA TWX HI:!
Vlt'w }){jug that n f<'\'crsihk fixutioH uf l'aldultl ltv til(' H~",Ut
proh'iHs is tht' t':.s{,Htiul m('t'ill.miMH. The (ftp:;eity of the
proteins to fix el.lI(imH find phosphah' is ilhtstruh'(1 hy th(
r(,s1flt~ of {;yi;rgy. wlto found that of til{' totfll t'ftlt:'jm1\ 8ud
iuorgaui(' phosphat<- of ('ow's milk some :m to .J.O pc..I- ('(ut. ;l'I
jndifru~ihlc. and that this st('adil~' dimil1islw~ "dth itwr(il!ooillg
hydrogel) ion {'om't'ntrntioll until at til<' iso<'iedri(' perint of
(_~as(>in all til(> (,flieium und inorg-uni{' phosphat(' nfe dinJysnhl('.
Th(' sault' itH'Tf'llSt' in th( diffusihlf' eal(';nm nnd phusphatt"
wa~ OhS{'Ty('d during the' tryptic <iigt'stioH of milk at u.
c<tn.o,;tant 1Ja' After th(' thsu(' protein!> h('giu to fix ('uldum
thp mduholit' activity of tht' tissuf> rlimini ...lu's nmt UU" 1hld
caidmn reads with phosphate and ('nrhonate to gin comph'x
Hrotein-(alciunl-phosphatt~-(ltrbonat(' rom pounds. from whi(~h
the carbonate is ~lowly eliminated by 1o<,al tissue addity.
T!u' exc(;"ss calcium phosphnt<' is thf'1I split orr, l('avillg' the
original ft(,tin> group of the protein fr('(' to combine
with more caJcium. Such theories Jark tht "upport of
t.~xperjmental evidence obtainrd at tht' 3(,t1181 S(,ftt (Jf ossiti('a-
tion, hut they ma.y po~sjhly play an important part in the
slow cakification of tissu("s! such a5. artcri('s, with advanc>jng
81l".
It appt'ars that the concentration of diffl1,ihl, eal"ium.
phosphate, and ("Brhonate in the bJood plasma, front which
the hone constituents must ultimately be denvt:d, is above
that corrcspondinj! to the solubility of calcium phosphate and
(~arbonate, and it has ht-en suggest.('d that th(;<o;c ar(' present.
in the plasma in the form of a metastable supersaturatl'{l
solution which might deposit the solid phase of these ""Its
Oil coming in ~ontact wit h them in the bone. nut as Rohison
points o~, it is not justifiable to regard plasma as sueh It
sup;rsaturated solution until we kno;" more concerning the
factors which control the degree of ionisation and solubility
products of the various components of such complex sysVms.
Regarding the plasma or the tissue fluid. "" simple saturat.~
solutions in which we have the system solid phosphate' in
U-l
1M I'flOSPf{ORFS COJfPor'NllS

NJujlibrium ll'ith the dissoln>d salt. the conditions aTC met

by the ('quation : .-
3 Cu" -j 21'0,'" ~:::_-::~ ('.,(PO,), ;::~~::::' (''',,(1'0,12.
ponillNi.) (In solution (t-I.ohd.)
unioniS6(l. }
An ihcr(,~lsl' jn the C'O!lct'utration of dtht'T ('a" or PO/"
wouJd PRuse tht' Tcuf'tion to pro('{'eo tmvurd ... thf' right and
result in the deposition of solid calcium phosphate, and
('oJl,'erseJy II d('crellse ill the COllc{:,l1trlltion of t'ither of thl'sC
iOlls would he c01mterhftlunced by some of the solid phosphnh'
pmi:SluR' into solution and so causing the rcactiofl to mon:'
towards the left. Accordillg to the mass action law,
[Ca"J' [PO,"']" = KlCa 3(PO.),J
and siIwf' we are dealing with It salt of minimum solubility,
[Ca"l' X [1'0,")' = K. or the solubility product.
Hence solid eai<-ium phosphate will be deposited when the
produet of the concentrations of tht calcium and phosphate
ions h("comes greltter than the va1ue of thf' soluhility product
of calcium phosphate. There is no reason to suppose that
the deposition of calcium phosphate in hones can he expressed
in quit(' SHe'h simplt' trrms, sinee, as ha..... bC<:'11 mention('d l

hydroxyapatite and not tribasic calcium phosphate is laid

down in tbe hone; but thesc considerations illustrate the
fundamental point. A shift of equilibrium towards the right
in the ..bow equation followed by a deposition of solid
l"udum phosphate would result from a local increase in th"
phosphate ion concentration, and a mechanism whereby such
an increase can he brought ahout locally in ossif}~ng cartilage,
holle, and teeth has been revealed in a series of investigations
"arried out by Robison and his associates.

The B6Ie of Orpaie l'hospbates in 0ssillcati0D

In an examination of the action of the enzymes present in
"sri"". tissue extracts on the soluble calcium an(i barium
salts of hexose mOllopbosphoric acid from yeast fermenta-
tions it was found that a preeipitation of the sparingly
 OROAN/(' PHOSPHATES 165

so1uhle ('akium or haJ'ium phosphatt o(!rufrl'd as tht suh ..

strate lwcanw hydrolys(d. This ohservation suggrsted to
HohisOH t ht, qUCTY wh("t}\t~r SOI1)(' stl('h J'{"RC"tioH might bt,
('o))('f'rned in the deposition of ('a)chuu phosphHh' during th('
os,';ifieation of ('artilagc, It was found Hud un f"nz)'J't'. whidl
Wt~ ('Hn (,HII Ii phosphorie cst('rnse, ('apah!{' of hydrolY''';llIfl til('
~llt.", of }I{'XOS{' nlOnopho!\phoric arkl {ohtaiuC"o frotlt yeast)
was pres('ut in many tissues, but mon especially docs it o(\Cur
in o~sifying bone and teeth. The dtstribution of thr enzyme
is g--iven hy the following tahIr, whieh show.,: thr lJ{'recntagc
of (a) potassium hexose monophosphat-(, and (iI) llota'isiuIll
glYNromonophosphatc. hydrolySt.'d hy a .5 l)t~r ((>nt. aqueous
extract of rat tilisu{~s in the ('OUfS(' of ('ightf'{'u hours at 87')
and 1'. 9-92, and the percentage or
(e) potassium 11I,xoll('
lilOnophOllphatc, and (d) potassiullI hex"". diphosphate
h~'drolys("d in another scri('s of C'xperiments hy aqurouf<O
{~xtracts of rabbit tissues in the COl1l"S(' of forty hours u.t 37-',
til(' PH b('ing uncontrolled :--

l'AIIl.E XII
Hyciro/y.;i.; "J {Jr/!.((Ilic Phosphates "Y TiNA"''; R.rlm<1'
(After Robi.,m.)

I'er(~ntage of PhofiphoritJ
FAtr.r& HydroliMed.
Tis.me.

(/1 (/;I (r;

Bone cartilage (epiphyses) 92 95

Rib cartilage (unossifwd) 2 8
Kidney _ 71 S8 41
Lh",r . 10 8 22
Spleen 17 20 22
Pancreas 1 3 6
Muscle 9 1
Blood . 0
](If> 1'1lOSPHORFR (YfMPOUND/'i ,

The range of lH'tj,'ay of the f'JJzynw or enzym("~ at yurious

hydrogen 1011 (otH'(,Htratiotih j", shown in the following tahle:--

T"m.E XIII
Adirif.!1 (~( 1'il's(((' Phosphoric R~tf'ras('.., at rflr!lluj?, piI
(Ajirr Robisltll.)

Uutil' lIf Amollnttl of E~t~'r

H.\'droJYi'U~1 at p".
'fi'l8\]1'.

7':J R':l !1':J

Boni' a.3 55
Tc,th :~'7 5S
Kidllt'y 3'() 51
Liver on HI
Spl('C'Il . :lZ
Pallen'as 1,,)

}'rom these results one may con<'imh.' that there is prpscnt

in these t.issues an enzyme ('apable of hydrolyslng hath
hexose and glYN!rornonophosphoric ft('ids, that it is pf{'sent
notably in ossifying hone, teeth. :md kidner, that it is
present only in traces in unossiiied cartila~e. and that the
C'l1zymC? rc!)ponsible for the hydrolysis shown by the Ih'cr,
spleen, pancreas, and must'le extracts is probably different
from the bone enzynlc~ sint'(' the latter shows a steadily in
creasing activity as the PH rises front 78 to g-s. whereas the
enzymes present ill the other four extracts do not. In these
experiments the enzyme solutions Were kept sterile by the
addition of chloroform, and the slight amount of spontaneous
hydrolysis shown by t,he phosphoric esters was cMckcd by
controls and has been deducted, A further conclusion which
one may make from these results is that the C1lpacity of a
given tissue extract to hydrolyse the monoph()Sphoric ""1er
does 1)ot Yary in the same way as its C1lpacit)' to ou.'t on the
dip'lOsphori~ ester. After Robison'. discovery of this bone
 BONE ENZYMJ<:
l'J)zynJt' Taknhashj 'V8.," ahle to .'~,how tlwt uqUt'()u!!o t'xtmd.,;
of th<' f('mnr nJ~o hydrolysed X('lIlJ('rlit"~ potassium h("xuse
monophosphatt' obtnilwd hy partiRI hydrolysis of the
diphosphatt> from yt"flSt. and whi(~h 'n' hu\'(' S(,{"H to din\'r
frotn ttl(' monophosphatt' ohtailH.'d dirt'ctl,' frolll V(~rllt. 'flw
bOJle {_nzynw Uwnfort' fippea~ to act indirrt'r('lItl;' un nllHlo.
pho:sphoril' ('stl"fS, hut it is iutHt'stiHg to .now thnt it ha,,"
no udio" on I('~ithin or Oil the hlood phosphatid,'s, although
the fornwr is a d(>rivativt, of gIY('e-fomooophosphotie M(id.
"'hilt thf' ("vitit'l\('(' gi\'C'1\ "hon' is highly su~g(~st.h'( of Uu'
part played by the Jl\onophosphorie ('steTnse iii hrillgilt~
about a )o('al inerf'sst' of }Jhosl1hat{ ions and ,~) causing a
depo~ition of ~oJid pno.liphate in artjvely ()s!I;ifyiuj( e{otrt's. it
i~ not (:onclusivf' until it ('u.n he sho\\'rt that there i~ a delinitr
(~rr((atiot1. betw('cn tht, initiatioH of ossification proct'.s~ and
thj, appearance of the active enzyme. This has b<>e1l dOll<'
by investigating the distribution of the phosphoric' esterase'
in humall fu::t.ust'S and young infant" with the result that. ill
purely cartilaginous structures the enzymt' i!t found to be
absent, and in partially ossified bones it is found only at Ul
ossification centres. Robison therefore concludes that th(" h

production of the cozynl, is a part of those (!(>llular activities

which result ill the fonuation of bone."
The range of optimum act.ivity of the (,nt.yme is an interest
ing one. It shows a broad optimum from PH S'4 to 94.
This is usually regarded as being above the normal rang.' of
hydrogen ion eoncentration of tho tis'u", generally. but Ii
localised increase of PM to this range is not improbahle. and
in any case instances art" not lacking of enzymes with '>ptima
considerably out with the small range of the blood P bllt
whi;h ne...ertheless play important part. in tissue proce....s.

The ProIIlem of BickeIos in JWation to the BoDe EnB.Jme

As we shall see in Chapter VIll., wben the growing animal
i. deprived of the antirachitic vitamin D, present in coJ..li\er
oil and other natural fats, ossification becomes defect"'e.
 HI!! PHOSPHORUS COMPOUNDS
Attempts have hern made to ('orrdatt~ this metaboli(' disturb
ancc with u defect in the (,fllcium or phosphate balanc(....
Theft; is. howe\'cr; no frasan to suppose thnt the tot.aJ c:alcium
of th" blood is "pp"'"iably altered ill rachitic children or
unimal"", Although rickets is sometimes nssO<'iated with a
iow __ ('ontellt of inorgat\i{~ phosphate in the hlood the associa-
tion is not i'r<_'qul>nt enough to he ft'gar<ied as specific', TIlt.'
prohl'1l1 of ricktts would s{'('m to lie dl'cper than a mef('
quantitative deficiency of the inorganic ('onstjtuents of bOlle.
u view which is supported by the Jlistoiogieul pit-tun> of the
ru('hiti(' bone. It is ob,'iously a matter of interest to examine
the enzymic activity of bmw extracts obtained from animals
affected with rickets. Such an l>xamination fc\-eah;. the
prl~s('nce of the active enzyme in amounts no less than those
oecurrillg in normal animals, and iu some cases If.Ort' aeti\.'{'
preparations ,,,"'ere obtained frorn the rarhitie animals than
from the noruUlls. Rickets therefort~ cannot lw aseribcd t~ a
deficient'Y or the monophosphoriC' esterase. It caB. however,
be shown that imperfectly ('alcified bones from young rats
show extensive deposition of call'iurn l)hosphate if they are
immersed in solutions of calcium hexose or glyceromono~
phosphates. whereas no su(h deposition occurs in a solution
of disodiulll phosphate under similar conditions. It appears
therefore that the causation of rickets might be found in a
deficiency of the phosphoric esters from which the phosphate
of ossifying cartilage is derived. It is oh,iously difficult in
these experinlCuts, necessarily carried out in vitro on bones
out of contat't with their normal envirollnlent~ to be quite
~..".tain how far these methods reproduce L'Onditions in the
living animal. In any case attempts to demonstrate in
rachitic animals a deficiency of the phosphoric es~rs of the
hlood which are hydrolysed by the hone enzyme have 'liot
yet led to conclusive results. Korenehevsky and Qar have,
however, been able to show that sodium glycerophosphate,
but l1.ore especially calcium glycerophosphate, when injected
su~utaneollSly into rats fed on a rachitic diet, produces 11
 BLOOD PH()SP}JORT'B IIllI

marked iHere8Se in Ul(> t"xt('nt of ('u){'if\cation of thf:ir !o.kth._

tons. The superiority of tht' ealrium over tht~ sodium salt
in this respect suggt'sts that a d(>jjcit~f1('Y of ~ou)(' fortH of
eaicium is at Ie-8st a contributory CIIUJj.(' of Ti{~k('b. Jt S(~t'n1'>
higidy probahle that s('parutt' m~chRnisl1ls ('XiJ.,t for ~ypplyi1i~
the ('l\)riuIn and t}l(> phosphate required for ossiUl"ati(llI .. and
it may be that tll(> pathologieal disturhanl't'S nsS(wintt"(\ with
rit'kets do not afft,!'t both t h('s(' Im.'('hanistHs in Hw !">fUtll' wny
or to t1t(' same extf'ut. A furHwT f)Oint worthy of lIott' h(-'n~
;s that jng{~sh~tl caJC'ium gl)'ceropho."tphutt' has UQ elff-'d on
tht:> ('aldum retention of radlitl{, animals, a result whit1I
a('C'ords with Robison's demollstratioJl of ttl(' pr(st_nc(' in tht'
intf:stinal mueosa of an actin> mOl1ophosphori(' ('steras(' in
amount similar to that of the kidney. It would ",--em that
hoth the absurption from the intestiI;e aud the- l"xC'retinn t'~id
the kidney of the ullhvdrolV1'i{'d J1h(J~phori(' ester aTe saf('-
gu:'rdt'd against, so that the ;)C('{'ssary ~st<'r or ('Sh'TS UlU!!ot Iw
synthesised hy the tissues. possihly by the liver.
The Acid-soluble Organic Phosphorus of the Blood. fI,,
problems whith Wf' have considered ra.lse til(' further cIU('stioll
of til(' supply of the necessary rnonophosphork (~stcrs to tht'
ossifying tissues. This must be through the blood supply.
and it is an obvious step to investigate the oc('urrencf.." Dn(i
distribution of such esters in the blood. The addsoluble
or!fanie phosphate of blood ocrurs almost wholly in I he
corpuscles. The following details an' given hy nloor of I ht
phosphoru):; distribution in human blood >'~
TABI.E XIV
Plwsphoric Acid oj Blood ill Milligram. Per Cell'.
Add~..}Juble
To,,". Lipide. Inorganic. (hganic.
1----_. _-_. _--
Corpuscles J72 160'0
Plasma 99 '1'5
170 PlIORPHORF8 r:OMPOFNDS

11I(luded in the lipidt. Vho:.phoric arid frat'tion are IN."ithin

and kephalin. hllt although these eontain a glyceromono-
phosphoriC' add tf"sieiu(' they RTf> not acted on hy the phos-
phoriC' f'stt'ras{' of hOIH'. as has h(,{,11 IHrntiollt'd alr(ady.. aud
~

s ..
"'IIIJ!.IH~ I"(#.~ .... MID,e
,FlO. Itt-Hydrolysis of the orga.nic acid.soluble phosphorus
compmmds of the bl()()(l by the boll~ enzyme a.nd by
sulphuTie acid. (After Kay a.nd Robison.)

therefore these Iipides presumably can play no dired part in

t.he ossification ptucesses which we are considering~' although
they lllay form a source of phosphoric acid for the hexose
phosphate and so participate indirectly, as indeed we shall

1
.
see is probably the case in the embryo chick (see p. 184).
,

1'bt" g)yoorophoapbatase pre8e:Dt in ~ appe&..nl. bO'Wt'"\.'er. tn

.cc ()u lecithin. (Ak.maDU, ~ 'z'f 1923~ 141, 186.)
 ORr;.~NI(, PfI08PlIA7'E OF BLOOD IiI

'fhe fraC'tion of til<' blood phosphorus whirh i", of import"JUH...

in relation to the prpsent tiis('ussion is tht' a{'id~soluhk organiC"
phosphorus. Th(, (_'UTY('S l'('produ('ct\ in f'ig. 16 Tf'prl's('nt t h('
results obtained by Kay anci Uohisol\ on hydrolysing tht'
neid-~olublf' organiC' phosphorus C'ompounrl:o. prt'S('nt In d('~
prot('inised filtrntt"S from rabhit's hlood. ('urn' A !oOhUA\'s
the ratt> and extent of hydrolysis nhtain('(t with tllt' hml('
.. enzyme at 37'5(., aHd at PH 9'3. ,,,,hilst ('UT\'l' (' ~ho\\'s Uw
hydrnlyti(' action of ni5 sulphuric .wid at 100". A ('urn' (II)
~howing the hydrolytiC' a(tion of tilt, phosphorit- t'sf('Tas.c.' of
bOBe on sodium lwxos{'mol\ophosphate is alsu inducltd in tlw
figure. It i~ clear that whilst t.h( sulphuric ul'id (>\'('ntuaJl.\'
hydrolyses all th~ acidsoluhl, phosphoric ""tn, (If blood, till'
action of th(" enzyme ceases when some 25 per ('('nt. of
the esters h!we been hyrlro1ystd. In other f."XIK'Timents th('
rt'sults vary between 1 ~ Rnd 36 I)(,f l'cnt. of th(, total I\.(id~
soluble organic phosl)horus. One might t}wrefol'(> ('()H('lutit,
that the phosphoric ester> of the blood arc divisible inlo two
fractions, one of whirh represents that portion of tht
phosphorus compounds hydrolysablc by the bom' ('sl<r,,-'<{',
whilst the other portion is resistant to the action of this
enzyme. Following up this information, Goodwin and
Robison M,'e prepared from deprotcinisrd blood filtrat"s
two fractions of the barium salts of the phosphoric elih'rJoi
with the following properties : -
Fraction A.- Practieallv insoluble in water, no J'(ducillg
action on Fehling's solution, fom.. the gre"tn part of tht'
total organic phosphoric acid, and contains the esters not
hydrolysed by the bone enzyme.
Fraction B.-Readily soluble in wah.., reduces Fehling'.
solution, alid is hydrolysed by the bone enzyme to th" extent
0
of 90 per c('nt. in six hours, The ratio of the reducing power
calculated as glucose to the phosphorus ('on tent is very
similar to that of hexosemonophosphoric ester. Fraction B
is optically active, heing /,n'D-rotatory. t
One may therefore conclude that a portion of th" ph",,-
 172 PHOSPHORUS COMPOFNDS
phorie (ijtt'f!-. pn's('ut in blood it-. rapidly hydrolysed ny the
bOIlt' tHzynw. whilst the other portion is resistant to its
action, Kay alld Hobi,oll han' been able to show that the
wenter part of tht, blood esters ... hich are altac'ked by the
hone tl'J)zyme js not hydrolysed by the phosphoric f'su-rase
oi')lw 1ll11"t('1e. and h<'Il('c this portion of the estC"fS constitutes
a sp('('ifi<- substrate for th(~ bone enzyme stl(.'h as is required
for the .d'eme of ossillcalion suggested hy Robison and his
'o ... orkers. It ...iII he seen from the table given on p. 169
tlu.1 most of the aeidsoluble organic phosphorus of tht blood
is pres('nt in the corpuscles. On the other hand it serms
probable that ossilication must be determined by the con
centrations of calcium and phosphate ions in the plasma,
Uobison points out, howe,'cr, that a ,'ery small amount of
('ster pre-sent in plasma saturated 'with calciunt phosphate
would he sufficient to bring about deposition of the solid
phase by increasing the ,'oncentration of phosphate ions
when hydrolysis occurred. On this view, if one regards the
con('cntrations of ester in the corpuscles and plasma as being
in equilibrium. the corpuscles would act as reservoirs from
which the supply in the plasma would constantly be re
pl~nished,

The Significance of Phosphoric Esterue in the KidDey

It will be observed from the foregoing that a mono
phosphoriC' esterase is present in the kidney in amount and
activity scareely inferior to that of ossifying tissues, It is
possible that its presence there may have some special
significance iI, relation to the excretion of phosphates,
Eichholtz and Starling have shown that the isolated kidney
perfuS<'d by means <;>f the heart lung preparatipn will not
concentrate the normal inorganic phosphate of the Mood
serUIll. On the other hwd, Eichholtz, Robison, and Brull
hal'e shown that such a preparation is able to hydrolyse and
to "oneen_te added organic phosphates and to excrete'
t/leir phosphorus as inorganic phosphate in a mueb higher
 f{.lDNEY AND PHOSPHATES 173

COHl'C'ntration than that of the' inurgnni(' phosphatrs of tht'

serum. In the intn('t animal it is po!o<sihl(' hy rC:"movai of t ht'
pituitary body or by punt'turt' of the tubrr riuerrum to ('.nUS('
the lVinary inorganic phosphates to sink to a minimum. If
now orgnni{' phosphate be injC"('t("cl. phorsphorus is ('xlrrh'd .
.after .all initia.l period. eutiN"ly in the inorganiC' form. .,h('
.. sllgg<'stion is put forward that normally n {'olls,tit'rahi(' pn:rt.
ir not all, of th~ urinary "hosphal;" is drriv,,1 rrom til('
organic "hosphate of the blood serum, The hydrolysis "ftll..
organic blood esters is hrought anoul in th" e"lIs or tll<' kidney
hy the phosphoric <,ster'st' presrnt tlwre.

The PhoIpbatidell
The ph~phatides arc organic phosphorus compounds
(frequently referrrd to 11. lipins or lipoid.) with a widt
distt'iblltion in both animal and plnnt. tissues. Tht'Y nrr
('sters of phosphoric acid and possess cht'tni('nl strUl'tuf(':-',
and certain physical properties, analogous to thm,,> of the
fats, They may he extracted along with the latter by tb.
usual fat solvents. Their isolation and separation from onr
another in a pure condition is a matter of r.onsidcrablt'
diffi<;ulty, and it is probabl,> that nO other similar fidel of
biochemistry contains a more extensive gra\-cyard of d(,CCHSc>d
compounds, with accompanying theories of their biological
significance, than does this fidd. Of the phosphatid,.,. one
may sing with the poet :
H The dead are more in mDAtcr
. . . than the quick."
for at the present time the number of definitely chara,1erised
phosphatides may be limited to three, although these three
may .exist "'In numerous isomeric or closely related fonns.
They are the tnOnoaminomonophosphatides leeithin and
kephalin, and the diaminomonophosphatide sphingomyelin,
The Isolation of Pbosphatides.-For the separation, of
phosphatides (rom ti""""" it is best to start with material
which has been freed from visible fat and dried as rapidly ...
IU PHOSPHORUS UOllfPOCSI>1i

pos!-r.ihl<' at a low tcmp('raturt' out of contact with air. other-

wist then: is no ('('rtainty that ol\e will isolah' thc Ulwhallgcd
phosphatidt~. Drying th(' tissu{' with ('old Redone j:-. a \'('ry
wwl'ul Int,thod ....im(' most of th(' J~lt and (hokhh'rol is
r('mo\~d along with th(~ wutt'T, and at the same time \Try
litUc pho:o.plultitit' is lost. Dryillg' at low trllq)('ratllT(, in a
vacuum is also an t'rfrdin~ nU'thod. Tht'N'after the' sd('('tion
of Ii ~uitahh' solvent must he dett'"Trnillcd by the particular
phosphutidr rt:'quired. I.A'YC'llc gives tIl(' following ~r('n('ralisa
tion~: \\,hf>T1 it is desired to obtain n"'IT pure 1{'C'ithin or
pure kephalin the' aectofl{", extract is (>mployed. '''11('11 the
objec>t of the extraction is the> preparation of all uHsaturat('d
phosphatiiles. then {>thef is chosen as the fcxgcnt. .FilluHy,
wht:'Jl it is desired to prepare ~phjngomyelin, the dried tissues
21rt~ ('xtrncif'd dircctly hy means vf aJ(ohoJ. As a mnttf'T of
routine. all thrp{, reagents aTc ('mp)o.)Ted (ons,t'(uti\'f'ly. the
pr{)(,t.~dure bf'ing as follows. The dry material is pulY('rised
and extracted ('ons{'cutively with acetone, ('ther. and a1eohol.
:F.aeh of th.f' (_'xtrll('ts is worked oyer separately. Th(' R(etOlW
extract ('ontains lecithin and kephalin and only a small
proportion of their decomposition products. This fraction
is til' olle best suited for the prt'paration of pUrt~ ic('ithin.
The crude etherral extract contains unchanged lcdthin and
kephalin RssO<'iated with the products of their partial d.'
composition. This fraction ('an he. separated into two sub-
fractions. One is soluhle in cold as well as in hot alcohol,
Rnd is commonly nanwd "lecithin!'; and the other is
soluble in ether but insoluble in alcohol, and is generally
rt'ferred to as . kephalin."
.Macleans general principles for separating phosphatide>;
from assoeiated substances are as follows: Afte~ thorough
.xtraction of the tissue. the extract is concentrated and the
residue treated with ether. when a thick opalescent mixture
is obtained. The insoluble fraction consists partly of im-
puri'iies. such as inorganic salts and nitrogt'Jlou. substances,
ard partly of sphingomyelin and cerebrosides. This ether-
 PHOSPHATIDES 175

insolub)(' part is Iooeparattd dthrt hy tiltrlltion or h~' ("t'litri-

fuging. ((,living a dC"ar yellowish ethrt solution. This ~orutilJn
is ('oJ)('('ntratt'd and tr{'att'd with t'X('('ss of lu('tOl1e, whi('h
precipitates the phosphatides, leaving "'l<)lesterol IIIHI fats
in solution. 011 redissolving the prt'('ipituh' in (t}WT. a :lightly
opa}('s('('nt solution i.,; again ohtninf'c): tht opah'S{'('ue!' is liuf'
to the pr(>scu{'(' of small UlJIOlJut" of oR mixture of SphlH~r('"
" myelin and c(>rehrosidcs. On l'(,lltrifu~inJt~ the liquid yidd!>;
a (~lcar solution. which is ('oH('('ntratc'ci and precipitate.-_{ with
aedone as before. The prf'cipitah'd phosphatide's art now
C'omparativ('ly frC'f' from (holesh'rol. fat. fatty fU'iti t awl
~phjl)gomyclill; hy S('\'f'TaJ Tepf'tjtjorh of th(' ahnn' pTf)(t~:'s
of dr!lsoJving in ether and pre<>ipitutinf,! with 8,('i'tO[W ('\'('ry
trt\{'e of thes(' impurities is t~vcutul1Hy {'liminnft"tL The
phosphatid~ mixture isolated in t.his way is di'isolvt'ti in tJ.
smaJ,1 amount of ether and ('xcess of ahwluh.' a1cohol add(d.
This addition of al('ohol caus('"s ~om(' of t he phosplu~t ide
to separate; thir;. alcoho1-jnso1uble part ('onstitutt's t:rude
kephalin. The alcohol-soluble part, when further purififtl,
yields a substan("c with a nitrogen allt! phosphorus l)('f('(,lltage
('orresponding to that of ieC"ithin and is ('onsidcred to ht flUte.
These IDf"t.hods rcpl'(>sent the essential manipulations. but
rnany modifications may be introduced to suit parti('ular
cases. The cadmium chloride double compound (If lecithin,
which is crystalline and may he recrystallised from a mixture
of alcohol and ethyl acetate. is very useful for the preparation
of this specific phosphatide. In identifying phosphatidf'S
Le"ene has used extensively the catalytic hydrogenation
method. The resulting saturated phospluitides arc '}llite
stable in air, are crystalline, readily recrystalJised, and arc
ronsequelMly ohtainable in " high state of purity.'
Stiucture.-The structure of each of the three phllbphatides
will be considered in tum.
(1) Lecithin, which on complete hydrolysis gives rio.: to

1 Fnr deWkd tnethodll of purifying pho8phAtidefi th~ origina.) Jlaper~ (if

Levene and of Maclean. or Macleans UlODOJrn'ph. ahoWd be CODsultOO.
 176 PHOSPHORUS CO"l1POUNIJ$
tho base ",,,,line. gIY'rol, phosphorie acid and fatty acids,
is rrprc-scntt'd by the' formula given, in which Rl and R2
(H,O. ('CR,
,~ /no. (leI!,
/llH on
CH,O. (I.,], (l
,",,0. eH,. Cll,. ~ == (CH,1,.
rt'pr('sent the fatty Heyl radi('les. 'l'h(' le-C'ithin obtaineo from
nlrious S()UfC'(>S yi-drls l){)th saturated and unsaturated fatty
u('ids, hut the proportion of highly unsaturated R('id is much
higher in li\'Cr lecithin than in that obtained from egg-yolk
or hrnin. The saturated acids arc palmitic and stearic, and
tho unsaturated adds arc old" (',.H ..O,) with ooe douhle
bond, !inoHc (',.11,,0,) with two double honds. linolenic
(C 18 H.,.O,) with three double bonds. and finally arachidc>nic
(l',.H"O,) with four double bonds. As yet all these un-
saturated acids have not brcn demonstrated in all thf.>
lecithins obtained from individual tissues. but they are
probably all present nevertheless. Thus it appears that no
Ic~s than six fatty acids hare to be a<'COmmodated in the
stru<"lul'(' of lecithin. This, in the opinion of Levene (who
with Maclean is responsible for most of the recent detailed
structural illl'estigatiolls on phosphatides), can best be
achieved by the assumption that different lecithins exist.
the variations being in the nature of the fatty acid residues,
Thus stearyl-oleyl. palmityl-oleyl, stearyl-linolenyl, and
stearyl-aTachidonylledthins are all possible and prohahly do
()('CUf.
Leeithin from a vegetable source (soya-bean) contains less
saturated fatty acid than lecithin from animal ti..sues, but
again palmitic and stearic acids are found in the satUTated
fraction, and oleic, linolic, and linolenic acids occur in the
unsaturated fraction. In tbe latter U1lS8turated hydroxy
fatty acids arc probably also present.
!'here is a fu:tber possibility which must be considered in
 .U:ClTHTN AN/) KEPfI.I/,/.\' 177

relation to tht, prohlem of th<> stru('hlr(' uf h. dthiu. It j ...

po~sihk hy lnild nlkalhw hydtoiysi!oo to i:'iOfak tht~ .L:Iy(~rn.
phosphori(' add of I(>cithin. and t hi .. pro\'('\ to 1)( optit'llily
ucti\'('. Hell(,(, of Hi(' two possihle ilWHlt,n. of gly(.(rv~
phosphoril' ncid. th" ~f()rlll. (,B,OB ' ('1I0Il . (,H,"P .u,,1
tht jl.forrn. ('B,OIl ,0101' . ('II,Oll (in ",hid. I' f\!lI'< ..nls
the phosphoric a(~id residue). til<' )aUt'T would nppt:'ar to h(
t'Xdlldcd, Sill('C it do('''; Hot possr~s an tl."iymuwtrh.' N\rhull
atom. The existt'1\C"(, of a lecithin d('ri\'Nl from th( iirst of
tht,S(, isomers is undispuh'd. 1H'\'(>rt ht'i('ss it hal, ht't'll dRil1w<1
hy Bailly thlit it is possible to bolRh' from t Iw gly(tru-
pho!-.phorie add of ]('('ithin two salts or difkrf'llt ('ry!'!tnHitw
form. on(' of whi(,h yit'lds dihydroxyu<'(tmw lllOHOphosphutt:
on oxidation with bromine, whilst t.h{ oth{'r faa." to do .~o.
1';, th" laHer tIlt' ,8.stru"tuJ'('. CH,OH, {'HOP , ClI,OH. is
a\-~rihed. This obs('rvation furth('r multiplits UIt' po",sihUiti(;~
of i)){)merism in the fc('ithills. I...e'ithin itSRii' ito. opt1<-aHy
active in a deximrotator}' sense, but both typt'''i of glyet'ro-
phosphoric' seid can give riS(' to tl"ymmetri(' fto{'ithin ....
CH,OR, ' CHOR, ' CH,OI' (I)
CH,On, . CHOR, . CH,OP (2)
CHpn, . ('HOP. ('H 20}!, (II)
CH,OU, ' CHOP, CH,OR, (l)
Of the formulre given ahove (1) and (2), derived from the
...glycerophosphoric acid, and (8). derived from the ,8-acid.
would be optically active. whilst only (,I) would Iw illad.in',
(R, and R. represent the ratty acid residues,)
(2) Kepho!itl yields on hydrolysis a series of <'Ompounds
very similar to those obtained from lecithin; in fact the only
essential <iillerence is the presence of th.. much weaker base
amilioethyi alcohol, CH.OH. L'H"NH., in place of choline,
so that with this dillerence the formula already gi"en for
lecithin represents also that of kephalin. Question. already
discussed relating to the structure of lecithin therefote :pply
to the constitution of kephalin. It must, however, be poin1led
17S l'lJ08PllORC8 COMPOFNDS.
out that it is much more diflkult to obtain the fatty acids
of kephalin free from phosphori,' acid than it is in the cas"
of lodthin. On this account Maclean has considered the
possihility of Thudi('um's original sugg(stion being correct,
namely:' that th,' arrangement: F'atty A(,ids--Glyeerol---
l.)hoophori( A(icl found in l(>Cithin is moditit'd ill kt'phalin
to: Glyc(rol --Phosphoric Acid -- Fatty Acids_ The isolation
from hphalin of definite intermediate compounds of fatty
a(id and phosphoric acid has been daimcd by Thudi('um and
hy lluruRs. The bulk of the evidence seems neverthdt'ss to
favour the yiew that. lecithin and kephalin an' eonstitutc-d
on Ilnalogous lines. The fatty acids of kephalin hn V(' not
lwen so extensively investigated as have thos(~ of lecithin,
hut they appear to be the same. Thus the unsaturahd aeitIs
de-finitely known to occur ill kephalin urc oleic, ara('hidonic
(in brain kephalin), and linolic acids. Of the saturat('d aqids
stearic only has so far IM'en detectrd. Kephalin of wj.!dablc
origin (soya-ht"an) shows no nutrked differclw{' from that of
animal origin.
Lysolecithin (Lysocithin) and Lysakephalin.--])eleZt'llnc
and FOUnleRU showed that by acting on lecithin of egg-yolk
with snake (cobra) Honom a partial hydrolysis ()('('urred,
in\'olving the removal of unsaturated fatty a.cids~ and a
crystalline compound was obtained which had the compo-
sition of a choline monopalmitylglyeerophosphate. They
c.alled this compound lysocithin. The action of tIM' venom
is due to the presence of an enzyme which has the remarkably
specific property of hydrolysing only the unsaturated fatty
acid groups. Levene and his ,,-,sociates have more recently
shown that kephalin undergoes a similar loss of its un-
saturated fatty acids .when acted on by cobra ven..;m. They
suggest the names lysolecithin and lysokephalin for these
L'Ompounds, and in as far as lecithins differing in the nature
of ~ir saturated fatty acids are known to oceur, it is
pI>Obable that at least two of these Iysolecithins exist. These'
imeresting oompounds shed wnsiderable light on the question
 LYSOLEf'ITJlINS 179

of till' :-.trlH'turt' of th(' part'ut phosphat.idt'!-. I'-rotH til('

lIIixtUT(' of lysoh'<'ithin nnd Iy:-.ok('phulin pn'tmred ftol1\ ('~JJ!
yolk two fnwtiolls nrt' ohtnilH....t. OBt' yirldil1~ palmitiC' uHd
stearic adds (from Iysol('('ithin), and th(_'> other !'oh.~It.ri( at'id
only (from lysokt:"phaiin). on hydrolysis. Thus. t.1t!' nllting of
stearic only in the sltturated acids of k('phaliu is l)araHeJt'fi hy
its pres(,Jw(' alonc in Jysokephuhn. whi)!..t both tht, saturall*tt
adds of lecithin turn up in the lyso t'Ompotlud. l'urtlw"r. if
egg-yolk ('ontainr<\ INithins df.>rind frolll saturnt(>d adds
ooly. these would sun-in' til(' adion of th{' venom, and if
dioi("yl lecithins Were pr("sent (me would expect to find an
oleyl I~'solcoithin (or ('holine glycerophosphate) in the
product eOlltaining the Iyso compounds. Hut 110 unclUlng"d
1N~jthin is found to he present. nor arc unsaturated Iyso-
ie~'ithins llnowl1, so that the conclusion that only nlixcd
lecithins oceur in nature, that is, those- containing a saturab-d
and an unsaturat('d fatty acid residu(', would sft"JU to h(.'
justified. These partially hydrolysed phosphatides exert a
powerful hremolytic action on red blood eells. lysolecithin
being three times as active as Jysokephalin in this r~fW"<"t.
It is probable that the tllemolytic properties ""'Iuired by
blood .(,rum to which a small "usntity of sunke venom hall
been added are due to the conversion of the serum phospha-
tides to the corresponding ly80 compounds, which are also
toxic in themselves. Tht lyse phosphatides have a matked
affinity for cholesterol, the loose compound formed consisting
of monomolecular proportions of each -component. It is
dC"oid of hremolytic properties. Lysolecithin. have been
used for the preparation of synthctic lecithin.; thus acetyl-,
benzoyl-, oleyl-, and elaidyl-Iecithins ha,'e been obtained by
Levene aM Rolf in a pure state by acting on lysolecithin
witti the appropriate acid chloride. They have no ba:molytic
action, &Ild most of them form waxy masses; oleyl-lecithin
so prepared is, as one might expect, closely similar to natural
lecithin.
(3) SpmnllomgelVn yields on hyd1'olysis four oomponedt$
12-2
ISO PHOSPHORUS (fOMPOT:NDR
in ('(lui, alt'nl Im)portiOJls. Th('s(> aft' phosphori<' add, two
hu,('!'., choline alld ~phiH~()sill(" Hilt! fatt,v a(Oio!'>. It b tlwf('-
fon' a diulllillomollopliol'-phutidt', or the haM'~ dlOlill(, is
much Hwn' ca . . il,\' ..qmrakd h~' h~droJ.n.:il't than is ~phjHgosin('.
'I'll(' occurrt'tH'e of sphingo:-.jl)(' (awl therefofe of sphingo-
1I1~'('iili) is limited to tht' HlliTlial "hosphatide~. Tht'struduff'
of sphing'osill(, is
('II, . (CH,ln . ('II = CH. ('HOH . CHOH . nf,XH"
hut til(' nHo(,lttioti of the HIIlino group and the two
hydroxyl groups h('tW('t'll carbon atoms L 2 or 3 i ... Hot yet
{'stahlished. lIor i:-; tht ~h'r('o('h(,llli('al ('ottfigut'utioll of tht'

'"
/
CHOll g-roups. Two futty adds have h{'{'}l ohtaiBNl up
.
to the pr('St~nt time from sphiHg'omyelin. and these are both
suturat('{i- -liguO<'cri(' acid, ('2..tH4/jO,. and a hydroxy d('ri,a~
tin: of an fH.'id of lower ('arhou content. whidl is possihly
hydroxystt'nril' acid. On the analogy of tht:" other phospha~
tidt's it is r<'aMmahl(' to atisigu the ecntral positiofl in the
structure of this phosphatide t.o t he hydroxy compound, in
this case the base sphingosine. Ncither sphingomyelin Hor
ligno<'crylsphingosine obtained from it on partial hydrolysis
contain it free prinlary amino group. These facts together
with tlw case of deta('hment of the choline residue are best
accommodated by assigning to the phosphatide the
stru{'turc~
/ll-l'" H,,(OH)XH-R
o =l'---OH .
"'O-eH,CH,. N == ~H,),
JH
in which R represents the fatty acid. As in tlte ease of
Ie<'ithin and kephalill, one must suppose that at least' two
sphingomyelins exist differing from one another in the nature
of ~eir fatty acids.
Tilt' numerous phosphatide. whose existence has been
~ from time to time. such lIS heparpbosphatide, "uOOn,
 PHOSPHATIDES IN METABOI.lSM IRI
protagon, tf'tlw1in. carnauhon, \"('salthin, lH"t)ttin. ~ahidin,
jt>('orin, ("t(',. in ,,,'hi('h \'urious X ; P rnti{)~ nfl' tt'})f)rtf'ti. fUn".t
he r(garcicd ns mixtures of tIl(' thr('(' khOWH ph()~phatid(',.
with each otlwr. or with tIlt'ir d('~rradntion prr)(ilwts. or with
thC" ('crehr()sidt's whi{'h hUH' snlubiHti('~ und di!\trjHltions
simiJar to t hosf' of th(, pho,.,phntidt,!oJ.
Phosphatides in Metabolism. _.. "'e han' aln'ndy di:-'C'HMWd
in C'haph'r , .. , those aspects of tht tlldnhoti~m of pho!'tpha.tlc(("
which are ('orrclaten with the ut.ilisation of fat in t}\t~
organism. Turning HOW to the mOTe spf'f'iulis('d aspects of
thi!' prohlem. Wt han' to Ilot{ tirst of all that th('rt i~ little
information rdating to the lIl(xlt' of orig-iu of phosphntidt's
in either animal or plant tissur!i. TJwrp is jlJ t h' inh<.jtinul
muCQs,,"l an enzyme capahle of hydroJysiull (and pr('sufOnhJy
therefore dfsynthcsising) lecithin. llno the {X'('U1T('t\('(, OfhU(h
an enzYI1lC' in takadiastust'~-~ that :'OtordHH1S(' of lnzymt~!'-
ha.: also rn't'J} rteorded. Again, it b (')nimt'c) thnt ill'us or
duC'"ks kept on a diet with a miuiOlal lecithin ('ollh'nt Jay
eg~J1o; with a normal content of th1' phosphatidt, !'>(, that til('
parent bird appears to lw ahle to syn1 hestS<' th{ Ia.Ut'r. 'fhi!>.
js supported by the ohservRtion of M...CollUlJl and hi" ('0"
workers that thf' nature of til{' fatty at'ids of It'('ithill in UI('
("gg~yolk changes with the nahUl' of Hlt' di('t. as is shown III
the foIlO',;ng table (from )la<:iean, .' L",ithill alld AliiNI
Sub,tRuces," p. 172);-
T.Ull.F. XV

I.ldinf' numlJf'r of;

Diet.

. (II
(a) Fats.

(21 (31
(b) PbMphatltW!\.

ilJ m (3)
nation nearly free
from fats and
phosrhatides 51)0 54-86 5H 3522 3407 3Hl
Norma ration
~--.. --_ ~
632 655 63'7 63'1
.
182 PHOSPHORUS (,OMPOlJNDS
It is ther,fore clear that the degree of saturation of the fatty
a.cids of the phosphatid('s is ('VI."'Tl mor!' affected than is that
of the fats by a "ariation in the diet. Eichholtz has r"'fltly
reportcd, howen'T, that Jei'ithin. Wht'll fed to young dogs.
;flcreast;.'l the ether-soluhle phosphorus of the blood. hut' not
tht~ t.otal fat. and he (,OIlciudes that le("ithin is aSlooimilah>tl as
such and that its fatty acid residues arc uot built up into
neutral fats. which p"""ess would necessitate a breakdown
of the l('f'ithin fed. J4~{'kst('jn has. however, arriv'd at a
diametrically opposite cOlU'iusion. He reports that th,
ingestion of Iccithin inereases tlw total fatty add contcnt of
the lymph. and concludes that. like simple fats, lecithin is
transformed into neutral fat during its transport through the
intest.inal mucosa.
The a..sociation of fats with phosphoric acid which we
encounter in the phosphatides is highly suggestive now tpat
w(> realise the importance of phosphoric acid compounds in
the intermediary metabolism of the carbohydrates. Indeed,
as w(, have seen in an earlier Cha}1ter. where the e\'idence is
reviewed, theories have been advanced which postulate that
fatty acids, before oxidation. are combined with glyt'cro-
phosphoric acid. It is known that the neutral fats of the
Ih'er are more highly unsaturated than the fats of other
tissul'S, and Levelle's demonstrlltion that the fatty acids of
the liver phosphatides are likewise more unsaturated than
those of brain and egg-yolk phosphatides is in harmony with
the general theory nlentioned.
A study of the phosphorus metabolism of the de"e1oping
egg during incubation e1ucidatl'S the catabolism of the
phosphatide. rather than their anabolism. Thus it will he
seen from the aeeompa~yingcurYcs in Fig. 17, draWl! from the
data of Plimmer and Scott, that the steady drop in phospha-
tide phosphorus, which represents some OS per ""nt. of the
total phosphorus in the unineuhatcd egg, and in the phospho-
proMin phosphorus during incubation, is accompanied by an
equally st..ady increase in inorganic phosphate and in nucleo-
 PHm/PHOBl'S OF EGr. IS3
protf"in phosphor\l~; the iargt' amount~ of phoNphntidt in
tht.~ hen's f'gg nppl'ar to fUTUish a !;tOfe or phosphoric lwid.
'hidl is rrl('us{'d tt!-o it is requirNi (or otiwr purpoS("l'o. ~'.~ .. th('
!o,ynthesis of thf' important HlH'l(oproteins. and for f)SSin(,li~
tiOL'. In regard to this Inst qu{'stion it is \,t~ry int{'l'~tillg tu

00

.14ICI.UJ~H~.'~IC~

.At:m~~~
IN ~.If~1fYO CH~

FlO. 17.-~.Altera.t.jvntl in the diittJ'ibutlon of p1Hl6Jlhoru~ in thf'

incubating tgg. (Drawn from da.ta. Qf Phmm(,f and ~;ott.)

note that Kay (quoted by Needham) reports the ab... nc( in

the uninJ'uhated egg of both the bone enzyme of }lobi,on
and of a suitable substrate, a finding which i. in agreement
with the low content of acid-soluble organic phosphorus
found in the early stages of incubation of tbe egg by Plimmer
and Scott. By the twelfth day of incubation Kay finds both
tbe enzyme and substrate in considerable amount. f'ro,,\thi.
 1~4 PHOSPHORUS COMP01'NDR
tinU' onwards. howcycr. as wiU h{~ seen from the ('l!rYC, the
add-soluhle orgflllie phosphorus of tl1<' embryo chick only
stcadily and rapjdly diminishes (a ('}13Jlgc' which is al,,() shown.
hut I{'ss strikingly. hy the who1t~ egg). and this period ('oincidcs
with \"iaonm~ I:mw d(ydopmtnt. Thrs(' fa('b lend further
strik,jllg support to the yi<'w~ of Hobison (1) ossifieation, and
111 thi:-o pnrti('ular CUS(' it is probahl{' that a cOllsidt'rabl('
proportion of thr phosphori(' acid released from the phospha-
tidt's g'()('!>, to form Iwxos(' or similar l)hosphah's. whi('h afe
thl'n hrokpll down and their phosphoric' a(,id ('\"C'ntunH,:'
dq)O~it(_'d in bone R!:o calcium phosphat{'.

llaolU' Phosphatf.1f in YtiMt FentH'lIiaiultl lind .lilli/rit'

('ontrarlion
1.EMBn):~ and LAQvlm. Zt"it. physiol. ('hem., IH21. 113. 1.
2. EMBI)"F.:!I; and ZIMM1:RMANN. Zeit. plll/iliol. ('hetn., 1924, 141,
22;,).
:-I. HARnEN . , Alcoholic Permentation:' London: Longmans.
nroon & eo., 3rd Edition. 1923.
4. HARDE'S I1wl Yovs<... l'rfH'. ('hem. Soc., 1905.21. IS9.
;). HJLL .. .Mrl"hanism of MmwuJar f'ontr3-('fion:' rh!l~iol.
RfWiffll8, 1922,2. 310.
ti. KAT. WO('h~ .Iourn., 1925. 19. 433.
7. X}:l1BERG. Bu)('/um. Z{'il .. HHS, 88. 4:l2.
S, RORI~ON. RWtJH~m. Jour"" 1922.16.809.

II e.xoJl,{' Photlplmtf-ll in Ossi_fi{'atwn and Related Phencnnena

L EICItUOLTZ, ROBISON, and BRULL. Proc. Roy. Soc . 1925, 99,
B,DI.
2. EICHU01.TZ and STARLING. ['roe. Roy. Soc . 1925.98. B. 93.
3. FREl'DE"BERG and (lYORGY. BWch_. Zftt., 1923, 142, 4117.
4. (:oonwIN and ROBISON. Biochem. Journ., 1924, 18. lUH.
~: i::~R::d R~: Z~A~3;,!..~.:' 1924, 18, 7s!. J 139
7. K(}REN~UEVSKY and CARR. Biodum. Jot<rn., 1925,19, 101.
8. Y,,"RTLAND Md ROBISON. B""'''-. Jour. , 1924, 18, 1354.
9. ROBISON, Bioe1_. Jour . , 1923, 17. 286.
10. R(\_",solr 81ld SOAK"" Bio<Jum,. J"",rn,. 1924, 18, 7411; 19U,
19,153.
11. 1.'AKAHA,m, ~. Z<!it., 1924,146,16),
 REFEREN('NS
PkosplMtidt'1t
I. BAILLY. f'ompt. Rnul . Hllf).160. 39.1),
t. lJELEZNSN): awl ,FOuRNIi:Ae. Rll. 8cJr. t'.hihl. ,If' }<'ru,H'f'. H1t4.
15.421.
:J. ECKsu:n.. .1(Jurn. Riol. (,hl"m .. 1$125. 62, j 43.
4. l<:KIIHOLTZ. Bim~htm... Zt"it . li124. 144. 06. A
."i. LEVENE. " Stru('turo and :';ignificam.f, nf tll(1 l'hujll'ha1itl~."
PIt.y.iol. ](tt~ewJJ. 1921, 1. 327.
6. I.F.VENE and Rou', .Ioluu. Riol. f'lu:m., 1022. 51. ;)4~7 ~ 54-.
91, 99; j 92:J, 55. 743.
7. LEVENE, ROLf' and ~IMM_S, .Iou.rn. Hwl. ('htm .. 1024,58. S.:,{i,
8, LEVENE and ~[MMS. ,/ourn. BioI. ('hem .. 1922, 5t. 21\:).
n. MACI.EAloi. . Lecithin and AWed Suf.lf<ta.1l("~." Lnrttinn:
Lmlgrnaull. (;ref!Tt &.: (A., . lOIS.
JO. ~EEPJlAM. "T1w :\Jotfi.hoJil'lm 01 thf>- Pf'VlIJnpjJlg 1:,i:,I.{,"
Ph-1/8wl. /levi.MAI", Ifl2.'l, 6. I.
11. }'LUUdF.R aud ;o:.('I)TT. .JOU1'''. j'hYilil)l., IltOP, 38, ~4-;
 I,
(,HAPTER YII

THE ROLE OF stILl'BUB. IN BIOCBEMIS'l'RY

S{'l.PHl'R is a uni\"ersal but quantitath'cJy minuk {'om ..

ponellt of practically aJl animal ttssu-t'"S. It finds its way
into the aninml body f'hiefly in the form of the amino acids
eystcine and (ystine. dt'rivcd from proteins~ and to a lUueh
less ('xtent in phosphatides containing sulphur. and in
inorganic sulphates, etc.
There is a eonsid(,l'abl(' amount of ('vidence pointing to
the presence in the protein molecule of a source of organic
sulphur additional to ('ystcinc or cystine. Thus east'in which
yields only tra~es of cystine loses but HI per <,<nt. of its total
sulphur on boiling with alkali, and ovalbumin gh'es a very
similar result. The amount of methyl sulphonic acid obtained
from proteins on oxidation with nitric acid bears no relation
to their "ystine content, nor is cystine regarded as a possible
source of m{'thyl sulphonic acid in such oxidations. }{ueller,
indeed. has recently isolated a compound of the formula
C,HuO;NS. isomeric with ethyl eysteine. from "arious
proteins, including casein. It is regarded as an a-amino acid
and a primary product of protein deal'age. It gives no
nitroprusside reaction, does not blacken lead, and on being
fed to man its sulphur, which is more firmly bound than that
of cysteine, is readily oxidised and excreted as inorganic
sulphate. Another observation of importance in .relation to
this question has been
made by Hoffman and GoTtne.. These
workers prepared from pure erystalline cystine (bexagonal
plates) an isomeric cystine (microscopic prisms) obtained by
p~ boiling of the former with hydrochloric acid. The'

. '
,..
n~w form of cystine is optieally inaHive and is more soluble
 .CYSTEINE AND CYSTINE 187

in watc:'r than .. plate" cystint'. as also is its r)lusphn-

tungstate more soluhle than that of the natural "plft!.. "
cystine. It is suggC$ted that this isomeric' {"ystint" is t h(' t nw-
ohtain~d in artificial syntheses. Tht" oJlti~81 insptivity muy
be riue to ra(cmisation. It would be of intrrf'St to WlOW if
the n<>w form of cystinc is capable of rt-"SoJution into ("lHu:~Ho
morphic forms.
The fact that cysteine. l'H,SH . l'H~H, . ('OOll. i, onto of
the indispensahle amino acids whidl the anim..1 is una hi, to
~ynth(>Sjse. and which must lw supplif'd to it in some furm or
other with the protein portion of" di"!ary in onk. that tit('
Hfr processes may proceed l1ormally, has led hi()d\(>lfIi~b to
attach to this sulphur-containing acid a J>e('uliarly important
r6i in animal metabolism.
l 'ysteine itself is a typical (,ryswUine amino add whiC'h
may he obtained from -all H complete 11 proteins on hydn)
lysi;' and which is characterised by t he ease with wlti"'. it i.
transformed into its bimolecular form. C"ystint. with til(' los~
of one atom of hydrogen from each of th, two cysteine mol,,
cules. This conversion takes place in the pt{"!\cIH.(~ of
molecular oxygen and is greatly facilitated by slight <iegr.'t'
of alkalinity in the solution. Again, with a suitshle choi ....
of conditions it is possible to rc,'erse this process and eonvt'rl
the oxidised cystine back again to the reduced cysteine. mild
reducing agents effecting this change slowly. more \'igorou,
reagents, such as tin and hydrochloric acid. rapidly.
+0
0+ CooIl. ClINIl. CIl,8Il___. COOIl. cm!Il . CII,-S I ., H,O
CooH. CHNB . CH,8B .,._._... COOH. CHNH,. CH,-l:!
+21{
It is ob;rious that such a rHersible autoxidillahle system
as tlmt pictured above i. of great interest to biochemists. and
Hefl'ter in 1908 and Mathews and Walker in 1900 Were
amongst the first to suggest that cysteine or cystine, or a
complex of one or both of these bodies with a proteib or
similar residue, i. related in an intimate way to ti........,
 lR8 ROLE OF' SULPHUR IN BIOCHEMISTRY
Tt"piration, that i... to ,"tHy. with the Dxidation and reduction
pr()(~('ss{'''' whkh go on within til(' Jiving cel}. ('ombined
<,y.'it(jue Nmst:tuting no; it were a point (fappui ill the molecule
nround which th{'~;(> reactions nre initinted. If such dews
('ould be ;'ouhstantiatc'd the ne('("Ssity for the indusiofl of
t Itt.'$"<:' sulphur.. (,outaining amino acids in any die-tary- would
n~c(in' a ready {'xpJ.:inntion.
The Autoxidisability of Cysteine.-Th,' probkml of t hr
alltoxidisability of ('ysteip{' has thr-f('foTf> an ob,"iously im-
portant hearing on t he whole question of tissuE." oxid.ations,
and has t('('('-ntiy b{'{'1\ tht> objE."{'t ofmuth investigation. Like
most reaetions of biochemical intel'('St, it i:-; found to he
exC'ccdingly suSC't'ptible to the influence of small amounts of
foreign substanct's. and indeed "rarburg and Sakuma regard
the autoxidation of t'ysteill(, to cystine as a catalytic phe-
nomenon d<'pt'nd("nt on the prt'senct' of traces of heavy
metals. more especially of iron. This hypothesis affords ~
i:>imple and eonvenient explanation of the marked inhibitory
~\{.-tion of hydrocyani{' at'id and the eyanides generally on the
Qutoxidation of ('yst('ine~ a phenomenon first recorded by
~{nthews and Walhr, espeeially when one ralls to mind the
t('fld('n<'y of iron and the ex radicle to fonn ('omp)ex ions in
whi('h the iron no longer carries a pos:itive charge. In sub-
stantiation of these "iews Sakuma has prepared a specially
pure cysteine which be l1'Cry,"\allised from ethyl or propyl
alrohol. using quartz vt'SSels for all operations. including the
preparation and purification of reagents and solvents, and
tbu. reducing possible contamination by iron and other
heary metals Ie a minimum. Such a preparation of cysteine
shows a rate of autoxidation measured in quartz vessels,
some 100 to 250 time~ less tl>all that of previou~ "pure"
preparations of the amino acid. and it is estimated that sbn,e
09,1 per cent. of the so-called autoxidisability of cysteine
depends on a catalysis resulting from impurities, Working
with 'Ilighly purified preparations of this kind it is founel that
tM merest trace of iron htlS a marked efleet in eatalysing tile
 A L'TOX I ))A 'flO); IS!)

rate or oxidtioJl to ('.\'stiJH' (Sf'(' }<'jg. lM}. HUi! \Varhurg statt~

that 1/10(},000 mg. of iron ill }() {',{', i:-.. (h.~tt'{tnbh' hv ih {fft-et
ill pronl0tillg this {lltn.lysis. Ahd('rllRldt'll N1Hi hi!'! (~).workl'l'~,
howc\'er, adhcf'(' to the dew that cvst('ftl( ,~ uuto}.idisahl(, 1(\
the complt'te nbsene(' of iron. alth~)Ugh Hwir pt{'('nuJIon', tn
('xc\lld{' possible tr~w{'s of the nwtal do not seeHl to }'Ul\(.bN~h
s.o rigorous a . . those of \\"arlHlr~ and Sakuma, III allY tms('
Abcicrhalde,n attaf'hes importan("(O to tltt, slight T(>1O;iilltul
(}xygf'tI uptake shown h~' the PU[('st a .... ailahk IJI'('Jutn,ti(Jtts

/e-

/v
TO

/
'/ . ..
"'0 '"
]<~JO, 18.-Eflif'd of inr.t'(>1l81ng eoncentIlltiolls of iron {Ill tht! ralt'
vi Hxygen uptak~ by pure (:yIJtRim' hydn:whhJridf' (12 mp.).
Iron added as Ft.'C"I1.; p" =. kO; temp. 2(rO~. (AiU>r
Htm'ison.)

of the a.mino acid. Harrison, reinvestigating the quc!!.tion,

has confirmed most of the observation. of Warburg, but
nevertheless linds that the slight residual autoxidisabillty i.
rather more than caD be RccoWlted for by the trace of iroll
still present in his preparatiollS. Thus by extrapolation of
his curv ... showing the linear relationship between oxygen
uptake and amount oCiron present Wig. 18), the distance AO,
which corresponds to .. possible residual trace of iron nee"'"
sary to produce the effect observed, is found to ...,present
000005 mg. iron in the 12 mg. of cysteine used. In the Jlctual
preparation the amount of iron found was only 0000001 J'$g"
!!II) ROLE OF SULPHUR IN BIOCHEMISTRY
froll! whieh lIarri~nll (_'ondud('s that the residual uptakt, of
ux_y,gt'tJ is IIot entirdy dm' to tlU' prpsen('{' of iron. Htncr
('ith<-r tra('_('s of ('atalysts other than iron are present in the
pliriHed ('ysteinc. or cysteine is to n minute extent stri(--tiy
Rutoxiliisabh'. The latter conclusion would seem to correlatf
w(lL with th(' ('iassi('al definition of a eatalyst lL'O a substan('t_'
which llloditl("!!t tilt' "clocity of a reaction, but which cannot
of' itsf'lf initiatf' that reaction. The ['eaction must therefort:..
Ill' already in progress. howevcr slowly, hefore it may be
(~atnlys(d.

The Autoxidisable Sulphur Constituent of tbe Cell-- Glutathione

\\'hil...,:t the cysteinc-eystinf' system (with appropriate
l'utalysts) might. as We have seen, play an important part. in
ti!o.sut' respiration, thefe is ill actual fact no eviderice to show
that either fr{'(' eysteinc or cystine is present in normal ti.ss,ues,
alld tht' low solubility of cystine at reactions in the vieinity
oj' ll('utrality uO<'s not accord with the supposition that it is
litted to play such a part. ~e,"ertheless the fact that most
tissues which aft' the seat of active metabolic proeesses give
'the nitroprusside reaction, which we shall discuss later, has
Ut'Cll ae<:{~pkd for some tinle as e"idence of the occurrenec ill
these tissues of some compound wry closely related to
cysteine. An examination of the constituents of extracts
from such tissues initiated hy Hopkins has led in recent
years to the isolation of the compound responsible for the
reaetion, and to a very noteworthy extension of our know-
ledge of the autoxidisable systems of the living cell and of
the oxidative and reductive changes associated with tissue
respiration. Hopkins gave to his new substance the name
glutalhiOl',e, since he <.lemonstrated it to he a dipeptide of
(}'Steine and glutaminic acid. It is of very wide oecurience
and Was first isolated from yeast cells and from mammalian
liver and muscle, in which it is found in a free uncombined
stab!. A description of the method by which this substance
_,y he isolated is given.
 GLl:TA '1'H ION E 1111

lsolabon of Glutathione.-Yr".t (+5 kg.) j, "oil ...1 with

three suC'cessiv(' quantities ('a,(h 10 litns) of tnp w"h'r~
tHtered hot t"ti(>h tim<>, and th(' combined extru('ts 8r(' pnrtiaUy
neutralis('(\ with dilute sotlium hvdtoxid,', 1'0 Illf' still
slightly acid (>xtrat'ts n~utral1('ud n(~('tn.tt, is ud(\t'd. tYlHf tlw
resultiltg pr(>('ipitate ifo. allowed to ~cttk and s('JJ4lrah'd fcum
the sup('rnatant solution hy siphonilll{. ~rh(' pn{ipitntl ..
after being thoroughly washed on Isrw' lIi\<'lmcr nlhrs. is
~11"ound in mortars and ('xtrntt<d with rold n/2 sulphuric
add until fresh t'xtraets {,('RSt" to gh'(' the })itr()J>ru~sirl('
reaction. Pranium acetate is then add('d to the folulphuri(
add extracts, which amount to about ] 2 Iitr('!O. until it. i ...
present in slight excess. Hot saturated barium hydroxidt'
sol.,ut.iou is now added until pl'ttipitation C('UstS\ and tlw
(lroripitate is IiItered under p",",sure and washed with cold
watfT. ThE'S(' treatments Tcnwn' various JX>ptides allti nJi
the phosphoric ..cid from the ytast. From th,' fIItrstt harium
is rl"moved as the sulphate and acid ntcrc-urie sulphatt> i~
added. The "redpitate is washed, susp"nded ill water.
treated with H.s. lind the filtrate and w".hin\,,,, aftor hdnl:
freed from Hri.) by aeration. art' made half normal ill u('idity
with sulphuric a(id. The solution (1,()()()-1.500 ".('.) i. then
treated with phosphotungstic acid until the pre<ipitatt is
maximal. the latter is filtered. and from the filtrate the ex",""
precipitant is Temo"ed hy barium hydroxide. ami the latu'r
by sulphuric acid. The solution is once again treated with
acid mereuric sulphate. the susl"'nded metcury predpitate
is decomposed with H.s. and the sulphide precipitate washed
with small volumes of water to keep the concentration of the
product as high as possible. From this solution, now ridl
in glutathione, the dipeptide may he separated either ,.,. the
copper or as the lead compound.
Copper Compound.-To the solut;on obtained froOl the
second mercury separation, moist, freshly precipitated
copper hydroxide is added, followed by suftlcient soAium
hydroxide to render the sUl"'matant blue liquid nea.ly
 Ifl2 R(JLE (Jj' SULPHUR n, BIOCHEJflSTRr
lIf'utral to litnlu" The eOppf'f {'ompouno so obtaiuC'd i~
d(,(,OIlIPOSfd. uft<'r <"'('}laration, with H 2S. and aftef remoYltl
(If t'.W'("'oj1o of th~' Jatter the tiJtrate is marl<' alkaline with
hariulll hydroxide and a(~rah'd to ('n!olurc' oxidation of the
dip<'pli<ie to til(' bimol{'('tllar form. using the nitroprusside
ksf~ whieh is unly gi\'(~n hy th(' reduccd form. as a cor.troI.
Tht" solutiu)} is then earefuHy f'ret~d from both barium and
slllphuri(' 8(..'id, ('oll('(-'ntrated under reduced pressure to about
10 (',t'" and pouf('d into 100 ('.c. of absolute alcohol. The
pr('('jpitated ~lutathion{' should tfwll stand undt'r the alc'ohol
until it is quite dry and friablt',
Lead Compound.---l..ead acetate i~ added to the solution
resulting from the deeomposition of the s('cond mercury
precipitate, and the resulting lead salt is deoompused with
H~, TIU' suhSC'({uent prcccdure i.s then the same as that
d('scrih('d for thl' copper compounrl.
\Vorking on a small seale the silver rompound. obtained
by trC'atment with sih'er sulphat(" and barium hydroxide,
may ('onvtmiently he used for purposes of purification, It
is soluble in hot water and separates again on cooling.
In extracting animal tissues the finely minced organ is
allowed to extract twice for two to three hours with its own
wdght of water at 40", and the procedure is then as described
for yeast.
The oxidised form of glutathione is a snow-white, non-
hygroscopic puwder, which softens at 165 to 161 and melts
with decomposition at 182 0 to 185, It is readily reduced to
the mono-molecular compound, which may be obtained in a
crystalline fonn,
The Constitution of Glutathione,-The elucidation of the
t'Onstitution of glutathione provides an admirable jllustration
of the application of analytical and synthetical methods to the
complete solution of a none too easy problem, rendered much
more diffieult by the !!maIJ scale nn which it was necessary to
work owing to the difficulty of securing adequate supplies of -
the substance, It is worthy of consideration in some detail.
 CON8T1Tl'TION 193

As has }wen nwntionCt), Hopkill" showed 111utathionc to llt'

a dip<>ptirk of giutamini(' ndd nnd ('Ysteillt' of til(' fotu\ula
r8Hu06N~'i. Such 11 dipeptide in it~ monO-ll1ok('ulllr form
may poss('ss anyone of til(' following three fonnuli(' :
, J. 11. Ill. "
CU."'U (,Il,Sli ('Il,~ll
i I
(!~HXH
CHXll, COOl! ('HXlI co ('0

/0- -XH. LH (lOOI! {J'IIXH, (l'OOH

IlH (\n,
I '
ClI, JH, hl:>II,
I I
COOR Cool{ (loOH.
1'he facts ~m which a decision may bf> made art' :--_.-.
(1) Glututhion .., afwr lJt'ing condensed with 2: II: i tri-
nitJotoluene. which rea('ts with primary amino groups, and
then suhjected to hydrolysis, yields frc(' <'yst(ine.
(2) On ",placing the free amino group of glutathione with
h~'droxyl,using ice~cold hydrochlori(' add and silver nitrit("1
and 011 hydrolysing til(> product. o:.hydroxyglutari(' arid is
ohtained.
(8) On oxid;,ing glutathione with hydrogen peroxide in
th(" presence of a trace of frrrOllS sulphate, a reaction which
(.'Onvcrts oc-amino acids into carboxylic acids with one carbon
atom less, and on hydrolysing the oxidation product, fTee
succinic acid is obtained.
Results (J) and (2) exclude (onnula I. 11-' heing that of
glutathione. since in it the free amino group which would
condense with trinitrotoluene is in the cysteine residue, and
hence free cysteine would not be obtained on hydroly.is;
whilst likewise o:.hydroxyglutaric acid could not be formed
on deaminising and hydrolysing a peptide of this constitution.
Of the remaining two formulte only a compound of the
constitution III. could give the third result obtained.,since
e,-en if the )en. NIl, group of compollnd U. were .oxidi",d.
~ u
 1(14 R(jLE OF SPLPHUR IN BIOCHE,lIl8TRr
this would of llc('('s .. ity involvc u hrt'ak in tht' ('hain and
su('C'inie acid would aplwHf b(,fore hydrolysi~ if it w('r(' to hf'
lornwd at all. Thus the analytical findings point to gluta
thiouc hadng the constitutioll III .. lind more r('(Y'ntly this
has befl;,l ('onfirrw'd by the' synthf'si ... of this compound and
Ihe il~tahli~hnl('nt of its idcntit.\' with natural glutathione.
The st'rk~ of nR(tions employed wa!.: the ('on\"l'r~ioll of
giutaIllini(' Hcid into hydantoinpropionic add, the preparation
of tll(' ueld hromide- of this Inttc'r add) and its (oupling with
('ystillc dimdhyl ('steT hydrO<'hloridf', wher('hy ttl{' (,Olll-
POlllld:

(U:-\ll---('O. ('H,

'\0011 tn2
.I'H_.:-\Il"
I ('0
('O_--:-\lJ/
dihydnntoinpropionyl c),stinf' was obtained. By boiling
with ealdlll1l hynroxide solution. followed by tr('atnwnt with
nitrous add. thc hydantoin ring was o}X"ned. and the resulting
Hramino acid wus cOllYerted to tlw corresponding amino
Reid:
CHt~----- CH28--~

(lll:-\II ___ CO.CR, (IHXR-_-CO, ('II,

(~Hj! dOOR ~n:!
(lU,NH,CO,XII, ([HXU,
(lOOR (,OOR
This {'om pound pr<wed ,to be identical with naturoJly occur-
ring glutathione "xcept for the fact that, "hilst the Ilttter
has in aqueous solution the specific optical rotation [<xl .."
- 98'8, th" synthetic product showed only {"'l'lft - 80'6,
the <liS{'r<'I'"ney being ascribed to racemisation during the
fONnation and subsequent opening of the hydantoin ring.
 ])J8TlIlIW1'lON !!I5
Ho\,'('\"er. by an alh,ttHltiq> s.\'nt}lt'~js affording furi)wr {'f)n~
tirmatoTY t'vi(h'I\{'l' of ((Hlstitution. a pr('l)tu'utiul\ has hC-'<'H
obtained possessing til(' ..,HllIe optical ndirity as the:
natural dipt'ptid('. On tn'ntillJ,! glutaruinl{' twirl with phtls~
phorus trihr{}mid(~ in dry tolu('II(, tll(' tllotl(Hldd ~Hl1Iid.( ..
IIrO(,. ell . l'H . CH:-;H . COOlJ. was ohtni.wd. nlJd I !lis Oil
('ondeHsntion with <,ysthw dimethyl cosh'r yit'hl(~d grutathiouf'
pOS-"lcssing thi' ~l){'('ifie rotatjon {~lsul 97,j., in H'ry l'ln"ie
:1WN"lln-nt with thf' ('onstunt fur til(' natural pro<iut't. \V~
may thf'ref(}tc {lcet'pt this work of th( CRrnhrid~Y(> laboft4tory
as ('stabH"hing tIw constitution of this intert'!.till,Q dip<ptid('.
It !'Ihould be noted that th('~(' s~'nth(''i(,'" yiddNI tht oxidi~t'd
or himolecular form of the dipt'ptide.
Occurrence, Distribution, and Determination of Gtutathion~.
~In his cttrly work on Y<'llst Hopkins ohtuifl('d from 0-1 to
0-15 J-rram of glutnt hiotw in (>u('h kilo,!..~am of makriul
('xtraetf'd, a yi('ld whidl was in('T('a~{'d ill latlor (x()t'rill1t'lIh
)]OTC TN'eutJy Tunuidiffc, working jJl tllt" ('Bmhridgt' JahorH~
torif'S. has dcvis('({ a nwthod of d('tC'rtuining: glutathimH' and
ha~ made a systematic inn~sti~ation of th( distrihution of
the suhstau('c in various ('ells and t.issues, Tlu' quantit.uth'(,
det(~rll1ination of glutathioJle is carried out as follows, . _.\.
knm,vJl weight of the tissu(' is grollml in a mortar with sand
and ]() per cent. tri(hlora(,(tie add, and nft.cl' filtration under
pressure it is re-extracted twice with the 10 [>('r ('ent. arid.
Th(' clear extracts are- combined and titrated with n:l()()
iodinv, using sodium J1itn.lflrus~i(l~'" Jl:i" aN f:,xb._"fYtitl (<<diNtt<ft'.
This substance gin's a purpl, rolollt with the reduced. hut
not with the oxidised. form of the dipeptide. From tit,
equation:
! G . SH + I. c--+ G. 5S . G + 2 HI,
1 c.c. of njl00 iodine corresponds to 25 mg. of red"ce<l
glutathione. The method gj,es satisfactory result, when
known amounts of glutathione are added to tissue pulp; and
no abnormal results are obtaine<l when urea, uric aqjd,
J3- .:
!PO R()LE OF Sl'l.PHFR IN RlOCHEJllS1'RY
rfl'atinim'. glu('osf". or frucios(' afC prf's('nt. l)>tefminations
ill tisstH.' t'xtru<'is of the soluhfc or~"Rni(' sulphur hy difer(,H('('
(Iotlll sulphur millll' sulphate sulphur) gi\'~ result, ill close
Uf'('orclan('(' with tht iotliu(' titration figure, from whi('h it is
conelUlisd that ill the latter method the ,ulphytlryl groups
alou,,' nrC' twin#! ('stimat{'d. and that by fur the greater part
of th(, ~Iutathion(' is Pt('S('ut in the tissu('s in the redul'eo
form. rsing the iodine titration method. awl assuming that
tilt' solubl(' sulphydryl l-,'Tou}>!'1 repres('llt glutathinn(' only, th("
following- nY('ra~t_' peT(,(,lltll,K'<' distributions of th(' dipeptide
nr(' giv(,ll :--
Fresh yeast IH K
Hat liv(,r (gIY"ogcn frce) . (HX
Hahbit lin'r (glycoJ.(l" fTCC 1 U'2~
Rat skeldal mils"',. ,O'U:U
Hilhbit. ,kektnl mll.s..!" O'()J5
Kidney 0-]7
1lI00 . Ahsc'lI\.
Hrn 's {'~11 ..:\ hsent.
The assumption that one is dealing with gJutathionf' only in
th(' abo\'(' drterminntiolls is justifiahle until it can be shown
that othrr pf'ptid('s {'ontaining C'ysteiu{' of the type of
g[utnthiont~ do occur in tissues.

The AutoxidiBability 01 Glutathione

Wo hn,'. seen that the autoxidation of cysteine is a reaction
which proceeds of itself wry slowly. but that it may he
catalysed by the presence of traces of iron. Harrison has
shown that glutathione is I'SS<'lltially analogous in its be-
haviour, When it is purified with the same precautions as
were used in the case of cysteine. it is found that the rate
of uptake of oxygen is greatly lowered, that thS presence
of minute amounts of' iron markedly increase the rate of
oxidation, and that this process is inhibited by the addition
of hydrocyanic acid, the relative amount of inhibition being
less'in samples rontaining small amounts of iron than in
those rontaining more iron. These results are shown in
 AUTOXIDATION Ini
}'jg.]fl, from whi('h it wiJ) h{ ~('('n that tilt' rak Hf OX\'gt'u
uptakt~ hy impure gfutathioll(' i!'o; \'('ry fn)litl. hdn~ prlwli~'n1ty
eomplt,tc in huH an hour {('Uf\"(' D). WhVr{lL."i with purified
glu~athiOll(' tlu" proc('ss is s('ar('{'ly ('ompJd(' in ,~IX hours
'urve B). ('urn' A shows the ('llormous a('('('lt~ratir'" dfl'd

}'JO. 19. -('omparil}on of tht" ratfil. of o"yp:en upta.kt in thf' f(lJl1)wjn~ ~.v!,;uml4.:

A. +
16 mg. pure (_{r-;H r!,M)
ReS.
H. 16 mg. pure aSH.
+
c. 16 mg. p1lJ1:" GSH (j.(1(Hi4 mil. Ff' (MI FeCJ,).
D. 16 mg. crudf' (iSH.
Pl' = 8-{); temp. -" 20".

of a minute trace' (0004 mg.) of iron on a sample of pure

glutathione which was oxidising very slowly owing to the
presence of cyanide. An inhibiting effeet very similar to that
produced <by cyanide may be brought about by the addition
of sOdium pyrophosphate, which, like cyanide, is known to
form complexes with iron. The difference between the
autoxidation of cysteine and that of glutathione is therefore
one of degree rather than kind, alld whilst ill the ~ at
aerobic oxidatinn which we have been discussing .here the
HIH R(JLE OF SULPHUR IN B[()(,HJ<:J1l8TRr
tiip(.'pti(_h oxidi~('!', Ilion' rapidly thut! thp amino acid, the
r('Y('r~(' i!- the ('HM' ill HIllWrohi(' ()xieintioll, wh('1\ jllsh'uo of
oxygc'll the dy('~tllf{ IIwthylen(' hlu{" is u~l'd U~ the hydro~rcJl
a(((ptor. Th~s(' fa<'t~ all point to the nf'{'C'ssity for symc
interacltoJJ between iron, hT some !o;imilar metallic catalyst,
and giut.athioHt' lwfore thl' Jattef ('an exhihit 1he eharaderisti('
autoxidisahiIity whieh it poss(>ss{'!o, iu the tbsu{'s, and the
t.Ulin'rsaJ o('('urrnl('C of iron ill the mammalian and most
other tlSSW',,,, ClISUT('!t t}mt all adequate supply of this ('utaJr,-;t
w iJi h(' j()rtl1<'ollling.
The Nitroprusside Reaction.-- ~\~ ha . . be(,11 1lH.'lltiOlwd, tl)('
nitroprusside f('u('tioIl is used by biochemists as a sp(>cifi(' test
for organic sulphydryl groups in tissues and tissue extrads.
As applied to these latter, it was first used by Arno"~. The t",t
is best ('urried out as follows: To the tissue ('xtrat't. or to
the ffuid in whj('h a pi{t'e of th{' fresh tissue is slispendcd,.are
added a 1(w ('ubi(' (,{>lltim!'trc:, of a saturah'd solution of
ummOnlUl!l ~ulphate followed by two or three drops of n
.S pel' cent. solutiOll of sodium llitropru~sidt' and th{~n {'x('ess
of ammonium hydroxide. A positjyc rt>adion is jndicated
hy the d{'\'e1opmel1t of an intense purple- colour very like that
of permallganate. The colour is intensified in the case of
fr('sh tissu{'s by a preliminary heating with vcry dilute acetic
aeid, or by !,'rinding the tissue with sand. It will be seen that
the meehanisrll of the reaction must be vt'ry similar to that
of the Rothera test for acetone.
The suhstance which gives the nitroprusside reaction, and
which we may assume with considerable confidence to be
glutathiolle, may be extracted from the tissues by hot water,
by washing wjth isotonic soJutions containing acetic acid,
or even by prolonged washing with tap water. THe applica-
bility of the reaction is illustrated by the following cases.
We haYe seen that the method available for the quantitative
dete>;mination of glutathione re"eals the absence of the latter
from the hen's egg, and this is confirmed by the negative
nitroprusside test. But after incuhating the egg for thirty
 .YITROPRUS8lDE TEST
hours. hy whi('h tinl<' d('vdopm('nt of tfU' ('mltryo with
in('ipieut oxidation pro('('ssC's hu." hc:_gull. n stru1\J,{ I\itro~
prus!oiid(' rNlctiou is ohs{r\'('(l. Again. Sh(,ftN'r ftH ... d('nlOu~
. . t~att'd that in thf' immaturt~ eggs and "'p{'rm of ('C'hiuo<irrru ..
tht, mtroprusside reaction is }}tgatin'. It is po~iti~' ill Iht'
ripe eggs and sperm. and one minute nrh')" f{~rtilisatjf'" with
un ill(,Tl'ftSl' in oxygl'Il l'onsumption estimut\'d at >t.ouo pt~r
('('nt. of that of tht' un.ft..rtilised ('gg it has hC('otne \'("ry
intense. Abderhalden and \Vt>rthC'in:wr luwt' shown thut 11\
ttl{' tissues of pig('ons krpt on Ii d(,fif'icnt did of polisJwd ril't
th{ nitroprw,:side I'('adion is \'C'ry weak Rlthou1!h (ystim j!\
apparently present ill normal amount.s. The pigeons t>.dlihit
Ii n>ry llnl('h impaired gaseous nwt.aholism. with dimiui~h{'d

~elJ rf"s-pjratioll nnd a low body tt'ml)("ratuJ"c, Tht' \'itllmin.

ddi<'ient dif"t appears here to havt' prodnr(~d all inahility tn
{'(lnvt'rt cystim,' into (>vsttinc', in which form it gi,,{s the
nitroprusside test, with a t:OIlst'qUf'ut lovn'ring of r('spirator~'
dlieiellC-Y. A similar ahscll(,(, of nitroprusside reuet.iou W""
demonstrated ill the tissues of' rats in whidl a tailorl' of
nutrition was produced by a ddiciem."y of ('Y~tinc iii the dirt.
Ahderhalden attempts to correlak tht'se obJ.ti'rvntion,to;: by
suggesting that in alimentary dystrophia <"jtamin dditjtmy)
the normallv reversible reaction, R--SH + HS---R ...::-_:~
R-S ,S-l~ can proc'eed only towards the right with the
formation of the oxidised form of glutathione, so that the cell
loses its powers of reduction,
It is not proposed to discu.., here the part play(,d hy
glutathione in promoting tis.me respimt(on, as the f"(~lld<'r
will find an admirable account of the subject in the (-Oln-
panion volume of the present work, ProfC!;sor IA)vatt }'~ya),,'
.. Uecent Advances in Physiology:' Suffice it to state here
tl>3t the views now held concerning the role of glutathiolle
as Il co-enzyme of the oxidath'e-reductivc systems of the
tiss'Jes are firmly grounded on numerous obser\'atio1L~, and
these views atrord an explanation of the reason why the
amino acid cysteine is essential for the growth of y&ung
 200 ROLE OF SULPH[;R IN BIOCHEl(fISTRY
animals and for the pres(~rvation of nitrogenous equilibrium
in th(~ adult, lUI explanation which is at least as satisfactory
ns our "jews concerning the l}{'cessity of tyrosine for the
biosynthesis of adrenalint'. Nevertheless this is not the only
irnportpnt role which has been assib,nlcd to cystPine and'its
dt"riU(..ti\'(;s, The high sulphur ronu'nt of epithelial strut'turt's
has frt,<tuently attract<'d the attention of him'h(mists. Thus
hair and woof contain some 5 to 10 peT ('ent. of cystill(' in
combination in th(' prutein keratin. ,,"'lIra has rt>('(ntly
shown that of numerous aUlino adds ('xamiu('d cystin(' is
til(' only one showing any nl.arkt>d pow('r to ahsorh radiations
in til{' r('glon of the solar ultra-violet. Its hC'huyionr in this
t<-'Spt"'<'t diefers from that of aIip:M.tic amino acids. and its
general ahsorption is as strong as that of p}lcnylalauiue. !t
is suggt.'stcu that it may therefore possess a ey('lie strU('tuTt'.
and that its sp("('iul fUfletion in epithelial tiS3Ut'S is to prot1,,{t
underlying structures from the lethal effects of an ('xcess
of the ultra-yiolet radiations. It is noteworthy that ultra-
violet irradiation is 8.('{'ompanie-d by the d('velopmt'nt of pig-
mentation (sunburn). and that the pigments so formed
are probably sulphur-oontaining melanins. It would be of
intt~Test to know what efrcct ultra-yiolet irradiation had on
the quantitative and quaIitath'e aspects of cysteine meta-
boli,m in e],ithelial tissues. It may be mentioned that the
nitroprusside reaction. is given by the cellular structures of
th~ skin, for example, the epidermis, the hair follicles, the
lining of sebaceous and sweat glands) and is stronger in the
roots of healthy pigmented hairs than in those of healthy
white hsirs. In collagen and elastin fibres, hair shafts. and
stratum corneum the reaction is negath'e (Kaye).

The IWaboIic Traudtirmatious of Sulplmr Com~

in the Organism
The action of the l'roteoiytic enzymes on proteins con-
tainil~ sulphur is conveniently followed by meaus of the
nitn.pl'usside reaction. Egg white and frog muscle proteins,
 RULPHliR IN PROTEIS8
which initially give ft J)l'gati\'(' rendion to thi!\ tt,,,,t. SOon
develop a positive f('uction WhN} suhj<'Ct('ti to th<' a('t1on of
pepsin at appropriate hydrogen ion cOIH'rntrntiuu ... ill<ii<'uting
a gr!ldual liberation of sulphydryJ hrt'OUpS fnHlI SOIn(' (,OIuhi~
Bation with other group};. It mn.)' lw that ('ystjn('~uJ~' is
prt'scnt in the native prl)tc'ill~. and no l'oH\'itwin)! ('\'id(~(' i:o.
y(lt a\'ailahk' to indicate whil'll of the two R('id!. i!'oo pres('nt.
,. In a.ny case cystine is readily trunsformcd itlto cyshiu( hy
such reactions us are involved if] th{' lwut nnd a.)fohul
('oaguiation of proteins, and in til(' formation of atid or
alkali metaprotcins. The obst"rvation that pfl('nyl('flfhllfllido-
cystille, administ<~r(>d orally or suhcutarwously. ilO ('xcret{d ll"i
ph('ny1['nrbamid{)~cystejne. supports thi' yj(,w that the Hr~t
sta,.ge in the catabolisln of ('ystinc is (~{)n\"('rl';i()n to ('yslt'illt'.
Th' two arnino adds would therefore appc.~ar to ht, nu-tn.-
bolif'ally {'quivalt"nt. Another .'mggestion hn..,: lJ('crI ndvurH(d
by Harris to a{'(~ount for the graduaJ liheration of sllJphplryl
~J1'OUpS during protein hydroly!-ois, 1Il' sllggt:sts thai t ht' SH
groups rnay play some part in linking tOgt'tJWf individual
:unino acids, the possible tYfK'S of linkage bdug flu'll of It
thiopeptidc, R--CS . NH-U, or R . coon I- liS . H
H--CO. S--R + H.O, instead of H. COOl[ " H.N. It
~-~ R-CO. XH--R, or, abJ'8.in, un iuternal dug' forma-
tion between {I-sulphydryl groups and clldwxyl ~".oups. tim' ;
CH CHNH . CO
!
S--------
The action of trypsin on proteins ('Oubuning !Sulphur. on the
other hand, i. not accompanied by the lilK'ratioll of
sulphydryl groups, as shown by the ncgatin' llitroprusside
...,,,,dioll gi"ell throughout. This may be due, however, 1o
the ready conversion of cysteine to cystine in tbe alkaline
media necessary for the action of try!",in.
An inte"",ting by-path in ,;dphur metabolism is re""aled
by tbe occurrence in the bile acids ot taurine, aminoetllyl
2(12 ROLE OF SULPHUR IN BIOC'HEMI8TRl'
",Ip}w"j,' Iwid, ('H,NH" ('H,SO,H. in ""njugulion with
{,hoi it lujd. Thef(' . . (I'ms to hr- Htt}(, doubt U,S to th(' origin
of taur;,,!' froIH (',\'sh;ut hr n prfK'f.",.,s of d((arhoxylation and
oxililitiull of Uw !'tI1Jph~'rlryl group. but the mechanism o,f thi,.,
trmls".,rmatioll i!; not y(~t ('kaT. Taurine iV,('lf appcars to be
1'(\~1:ltllt to furf her oxidation, Clnd if administf-red to man or
to the dog' it is {'xer{'ted as lleutral unoxidised sulphur (set'
p. 20:;). TIU'f'(' appt"Hrs to he a taurine rt'ser\'c in the animal
hod,\" sin('(' the arllount of taufQ(>hoJic add s('creted in th('
bil(' is not irH'rv8.serl hy administering taurine. whereas it is
IIlC'f('H<.;(d hy th(' administration of dwli(' acid (Fost<r.
1I001','r, and Whippl<'), TIll' output of taurocholie aeid ill
th(' biJr is also. as one might expt"et. increased hy the f(,eding
of ('~'steillf' und sodium eholat(. It is possible that .th(
oxidation -of thl' thriller to tauriIw may takt' plu'cl' after tlu.'
('omhina1ioll with cholic add. although the d(>InOllStlahlC'
ot'{'Urr('Il('{, of fr('(' taurine in certain tissues and the tauritw
l"t'S('T\'(' already lllentioll.('d would s('{'m to f('nd(,T sHeh a
hypothesis at least unnet'cssary. Thc hulk of the {'yidet)(C'
wOllid appear to b{' in favour of tllf~ "jew that taurine dot,S
110t r('preSt'Ht 011(' of tht> normal intermediates on the path of
(y!'.t(>illC' metaholism.

The Escretion of Sulphur

In as fur as th(' excretion of nitrogen and of sulphur com ..
pounds in the urine repTf'sents the protein catabolism of the
hody, one expeds to find a quantitati,-c parallelism hetween
the,,' two types of excretory cnd-products, This is shown by
a more or less constant nitrogen to sulphur ratio (:S : S), the
fasting value of which is placed by various workers betWl"t"ll
10 and 10, The ratio naturally varies according'to the diet
and metabolic activity, and thus a much higher N : S'ratio
than those quoted, indicating a conservation of sulphur, has
hee\, obtained on feeding meat to a dog after a prolonged
fast, whilst lower ratios are orn.erved during the period of
f~Sting, Low ratios h",-e also been reported after severe
 N: S RATIOS
muscular {'xert'is(', ~UPP()s('(tI.v tim' to tht> illl'r('n"t' in tissue
respiratory proe(,SM'S and the illtimate rdation-:.hip of t h( ...(_'

TAlIl.>: An
"'{JriutifHls ill S : S llatio protiu4.;{'(l by rariatiullJ..' ;tI'<!!.t~1
(.Iftrr Wil."",.)

Dny. Dif't. Totui S. 'r'llal S, ~:S

Ba",J, 1\ {rt'C -!7KS (H1022 15S

1 ZfJIIIl 0-2111i 12'.!'
5 "
Staryil\~ 2-W2 0-211'1 11(1
{; I-H72 lQ2HU lHI
7 Ba'~I, l\ f"'r (H~H (HOI~ 17,a
'K I-ISO 0-21111K HHt
15 ,~1J.~2 O':]!!.'18 H"
IG. na~al +('g~-an;UllliIl'1 a-g:w (J. ~H7(j K(I
17 StarYing 51l2S (l'5tl1~1 Ht)
III "llll IH5(1{i 1.1-7
1 ~) Ba!-'~l. l'; fr('(' 7'5:~2 O'5(}50 H-K
20 a-!)()() 1l-2m):1 167
21 2'{Hu) O'25H3 11-5
_..-- -~----------.--

I Baf;al, cOHtatlting 6,1 gm. X 5--IGiI (j:n:17 14011

10 (i-(170 O'45fH I:J'H
11 na.~a} (6'4 g~). ~) +;gg-AJbumin .~ 7-252 ()'7H12 91
12 Basal (6'4 gm. X) R-M4 0'7178 11-1
18 6076 (l-H67 ):10
14 6-272 ()'4iiOfI 1:3-1..
----- ------------.-----
1 Basal, containing) 1 gm. N Il-1l0 0'610 IH-2
9 11-680 lH,iH2 20'{)
10 Ba;al (11 in.
N)~gg-alhumin; 13'8R~ H9R 11-6
11 Basal (II gm. X) 15'282 HH7 12'8
12 ! !-Ill:! 0-7864 15-3
18', 11-704 (1,747 156

1 Containing 887 gtaJhS Nand 0-96 gram Ii; N: S = 92.

:I; Containing 10206 grams N And 1-059 grana S; N! S = g-6..

._-
Containing 9-8 p"" N and 1-2(\4 grams S; N: S = 11-6_
Baeal diet CQD8iated of 100 grams olive oil, 300 gra.m.s tapiQC3, 100 gtJltnfl
204 ROLE OF SULPlfUR IN BJOClfE,tfISTRY
to ('y~tdJH' (,I!lllt~lthioJJe) HH'tnboJh.m. The IJf"haviour of tht,
!'\ : S ratio utHI('f variolls ('onciitions of dieting is well
I-Ihown ill the u('companying table, p. 20::1. taken from some
J'{'('('lIt ('xpprilIl('nts on the human ~\lbj{'('t reported from
l'ath(~ts luboratory. '
TkCs(' {i.cUN'S iHustrnh' Uu.' gradual diminution in tht' ~ : S
futio during a period of nitrogt'n~free dieting or during
~tnr\'ation. TheTl' would appear to be no att('mpt on the
pnrt of the' body to Conserve sulphur in prefercnc(. to nitrogen,
despite the important role 'which tht' former plays in tissue
mduholism. but rather th{' ren:rs<'. Apparently the nitrogen
('ompounds art' as important, possibly more so, than the
sulphur ('ompounc!s during a crisis such as starvation. Hut
the ,udden rise of the ratio to 17'3 on again laking fO?d
aft<r H fast is intt'resting, and indlC'ates a much m.ore marked
r('t('lltiOIl of tissu(' sulphur than of nitrogen during Ul{' pt.pod
of n'(overy. All. inspection of theS{' figur(s also revt'Rb the
raet. that ill clwh of tht thrt-e experimenh in which egg
.. (humin was fed in addition to the basal diot there followed
nil immediate drop in the X: S ratio, as 011(' would expect,
and the excretion of exceSS sulphur was more rapid than that
of ('x('es~ nitrogen. On these grounds it would seem justifi
able to assume that the sulphur portion of the protein is
metabolised in advance of the nitrogen both in the c.atabolic
and anabolic phases. In the second experiment, in which
w""
el!:\!:-..Ibumin "ul"'rimp<>se<l on the bo.sal diet, the mate<W
ston'd and broken down again is .\;dently rich in sulphur,
sinee the N ; S ratio is low for the ele\'enth and twelfth days,
whilst on the thirteenth day, when the nitrogen output has
returned to its hasaI figure, the N ; S ratio is la6, a little
below the average for the whole experiment. The balance
may be analysed as follows ; -
GraQlg.
Sitrogen excreted above the basal level for
the three days . 3'UO
Nitrogen ingested on first day 10206
 EXCRETE/) fWLPHUR
(~ram~.
r\itt-og{,ll rdaincd i:-, t herr,fOft'. . . n,7Hn
Sultlhur ('xcr<'ted abov(' th(' hn!'>nJ knt for
the thr<"t> d<\v~ . . O'62H:!
Sulphur in~~ted*' on first day. . . t,o;,)!!
-Sulphur Tctui))cd is thrrefoTc. . . f)'4im~
The ~: S ratio of the ,fltorrd tlllIte-rinl i., thtrd<lrr\;t"(I!
o~a2 =~ J.I)'2. This is \'err ,war thf> X ~!oi rnt;" of mllsd('
., protein. and it would seenl that t}ll~ hody ha. . M)l'(ti\'t~I~
T(tained a material which ('Orrf'spollds in jt~ lIitrogt'U amI
sulphur ('ontent to that of Ulf' musl'l(', A similar lUlUI.\'si ....
of the third ('xpcriulf'nt in whi,'h (gg-a1immitl \\'Jl!'. ... up('r~
1

impos('rl on a hasal rliet much ri('ht'T in llitrogf'n. gi\'('~ Hit'

following fi!-,1Ur('s: nitrogt'tl stOf{'<l. 2'}i grams: sulphur lo!'.t
{since there is a negatiY(' halan('(' in this (,US('). (NnSl /,fruln.
Th'us there-was a consid'Tabit.' loss in sulphur and hut. n slight
r('t('ntion of nitrogen. At the high iC'vd of protein jlltuk(' it
s{'en\s that the body's power to ~tore (J.!galbmnin. hoth as
rf'gards the nitrogen and the ~ulphur, is nnl('h h('low tlmt
shown in the prcyious experiment 011 a Jow('r husal nitro~(,11
iutak('.
The' total sulphur excreted, which w(' han~ })('('1I ('(msidcrillJ.,:'
in the abon::: experiments, is usually dj,id{o iuto three
fra<'tions: (0) inorganic sulfJhat~s. (h) organi(' or ...th~r",,1 ..
.,ulphates, (e) unoxidised or" neutral" sulphur. :Fradjo" (II)
represents practically wholly exogenous suJphur Hldaholisrn,
just as urea docs that of nitrogen. and it COIli<;titutf'S on tht'
aYeIage some 85 to 90 p<>r cent. of the total sulphur ,x('J'(t<d.
In ~neral the fluctuations of this fra{tion follow (:l()~;('ly
those of urea. J1raction (b), the organic sulphates, consists
of bodies of the type of phenol and indoxyl conjugated with
sodium 0t potassium "'lliphate, and their origin is IUKTihed
in general to the detoxication of cyclic hydroxyl comp()\lnds
arising from the bacterial degradation of l,roteins or amino
acids in the intestine. The experimental method which most
markdly increases the excretion of organic snlphates is the
joint oral administration of cysteine and phenol. It is stated
:!O/i R()U; OP 8ULPIIl'R IN BI()('HEJlfI8TRY
hy Hhod('. und {'onfirnwcl hy OtlWfS, that the substitution of
~orliulll sulphate or thiosulphatt' for til(' eystein(' leads. in
th(' rahhit. to 110 ill('f('tlsrO ('xcrt,tion of organic sulphates, in
addition to thnt produ('('d hy phenol aIOl1(', hut that til('
~ulfJ}u>!(, is <fluwtitatin"Y ('xc)"(_~t('(1 flO; sueh. Thi~ might
'u,q'~f that {'onjugHtjon of the sulphur ('ompound with the
phrllol pr{'('cdcd the oxidation of the former. hut on the
othl'f hnnrl. til{' administration of sodium ~ulphit(' did
prnlu('{' an incrcas(d_ ('x(,fetion of ph{"tlol-sulphah'. MOT(_'
t'l'{t'Jltly IIde has ~hown that sodium sulphate, sodium
hydro~(,11 sulphite. Bnd ('ysteiUt', administ('Teo orally togethf'T
with g'uaia('oJ ('Hrhouatc to the dog, are utilised with equal
r('1lfJin{'!oi~ in th( synthesis of organi(' sulphah-'. TheTC h,
how('Y('r. the possihility that guaiacol is a substance too far
fCmOYNi from the Ilormn) metabolic paths to warrant ('dl1l~
parison with phenol in this rr-spcd. hut ill any eRse the
diser('pal1cy is intef(~sting. Xcuherg's diseonry in t1.tk(l~
diastasr. and late-f in the kidney and in othef organs of man.
of all cJlzym{' which he has called . . .ulplwtase. and which
hydrolyses organic sulphates, suggests a possihJe widening
of OUf yitws on th(' biologiC's] role of organie sulphates. It
may Ile that the attaehIuent of the SUlphuric acid groujJ
to certain eompounds repfesents a definite stage in their
metabolism. As an analogy one can point to the part played
hy the phosphori(' acid l"esidue in relation to the utilisation
of ~arbohydrat~s, and possibly also of the fats. In any e,'cnt
th' suggestions made from time to tilue by Sherwin and his
associates r.ollccrHing the possible endogenous origin of
,onjugated organic sulphates are worthy of note in this
(onnection.
The sulphur fra~tion (c) of the urine, consisting of un-
oxidise<! sulphur. is generally assumed to be of e~dogenous
origin, but convincing evidence on this point and concerning
the question of its exact com~ition is lacking. Cystine '"
peptides contBining <'ystine hal'e bet'n stated to be present,
bu.t rerent quantitative determinations show that cystine is
 OROAN/(' I'WLl'JlA'I'ES 207

but a smaU ('OlHIK)JI{'nt of this fraction in nnrnud urine, In

tht> puzzling condition of eystinurilt. howcnr, Wt.' ('Il('hHnh'r
U ('ontinu('d ('xeretion in the' urine of t'oH~ith'rl\hl~' ~lunntiti{''''
of cystine. A small portion of this <,y!'\tilH' st't'm .. tu h(' of
('U(io)Zrnotis origin. whilst tilt' mnjor portion vuries with tlU'
protpin intak(' (LOOHf'Y. Bt'rghmd. and (;ruy('s j. ;\('(\"1 II('
less ('a!.;{'s of this pathuloJ,?i('ul ('onditi<m art' Of! r{>('o;:t in
whic'h illl-,J'{'st(>d ('\'~tillc WitS readih' oxidis('d; iu<'iudnf in
these r'fiSt'S is one 'in whid. -t..(J grlln;s of ("Yfo,.til1t' isolakd frull)
tilt' patient's own urine was {'ompkt('iy o~hii,,,,('d wiwll it wus
fi.d to him (Thide). In oth{,T ('n~('s Hwrt' 1llltY lw- n ('ol1\ph'h'
T(,fwm] to oxj(};!!it' ('X0R'f'lWIJ."- cY,'i:tiUf'. IUlt) thi.. ('HudHion is
~()m('timt~ associakd with an ('x('rdioll of diawhll",' (tdra
and p<>nta-methyJellc diamiu('s), whi('h is indi(atirc of M,IHC
imi~airmen~ of' protein m{taholism other than that nfr('(,tirl~
thf' sulphur~('ontaining amino a(,ids. In g<"ueral. ho\\,('\'(r.
the mt1C'f only exhibit a faulty eatnholbl1l. whidt lnll"it lw
of it rughJr spee1llJiseo naturf', sin('(' othtr t~'r)(.'" of io.ulphnr
('om pounds are oxidised and considerable qwmlitif''' of
inorganic sulphates appear in the urine.
Organic Sulphate. Pretent in Tissues
Organically ('omhjncrl sulphuric add is knowJI to ()('('ur in
the tissues in two form ... : (l) in ass()('iatioh with a ,'srho-
hydrate or derivative of a earhohvdratf' in th{' mucin .. and
similar proteins; (2) in association with phosphat ides or
similar (,ompounds not necessarily ('ontaining phosphorus.
The existence of thes(' contpounds. imperfectly ('harfwtc-ris{'(i
as we must admit them to be, yet s<:-rves to show that sui ..
phuric acid may be found in organic comhinat.ions apparently
very similar in nature to those in whieh phosphoric acid
pam"ipate's.
The Conjugated Organic Sulphate Residues present in Muco-
proteins. '_The presence of conjugated sulphuric acid in
l The muooproteio include the typical mucih8 and the mueojds \I-'hkh
dUfer from arm another in the phylri.e.1 clte:taeter of their 8f)JutioJ18, and of the
precipitate when they .are foried fmfJ! aD aqUeo'ld JJOlution.
2\1H R()"E OP SFLPH(,R IN BlOCHEJllSTRY
mueins Wus Hrst disco\,c'red by Morn('r in lR89. and he later
isolated a suhst~lI1(,(, whirh he c'aned ('hondroitin sulphuric
liPid. Different nmdns and lllu('oiris showed a nry variahlt'
h<>haviour in T('sped of the ('ase with \\'hi{'h tht'y yielded this
u(id. _ij!H-I it WitS Hot until I~\'{'n(' began his inv('stigations in
thisjl{'ld thut n df'flnih' ehf'mical hasis for tIl(' differentiation
tm('r~r('(1. It is HOW draT that from ('artilag<>. t('ndon r aorta,
and s(']cra mu('oii1s a (hondroitin sulphuriC' acid may be
j."ioluted whi{'h is ('onstitut{'(1 of two molecules of an acctyl~
aminotlllos(' (chondrosaminc). in whi(>h one hydroxyl is
replae('d hy th" sulphate grouping, O. SO,oU, linked
togt'tfwr through t.wo mulcc'uies of glyc'uronic acid. thus ;---
~lIh~tit\lh'd . - 0 - (T - -- -0 --G--O---Suhstituted
iwxO~alllin('! i hexoAAminf',
{,OOH (,DOli
th'~f(' h('ing present a giueosidic Linking hd,wt'en the glyeu-
roni(' Reid r('sidu('s. both of which have their carhoxyls
fret'. The' pO~;tiOl1 of the sulphuric acid and arllino nitrogen
Uroups in the substituted hexosamim>. and the point of
nHal'hment of the latter to the gly('uronie acid, han." not yet
heton satisfnt'torily d{'ternul1~d. A very similar acid filay he
j~olntt"d from the yitr{'ons humour and cornea of the eye,
thf' mud)) of the gastriC' mucosa. serum mucoid. ovomuf'oi<L
oyarian cysts. IJnd }'unis murin. which, however. differs from
('hondroitin sulphuril' a(,id in being much more lahile, and
('onsl''luentJy more dimeult to isolate without degradation.
This aeid, named muroitin sulphuric acid, consists of two mole-
('ul,s of acetylaminomannose (chitosamine) sulphuric acid
linked together as before by two glycuronic acid residues.
It differs. therefore, from the acid already described in the
nature of its hexosamine, and probahly also in the position
of t he sulphuric acid group attached to the latu,r. These
two compounds, chondroitin and mucoitin sulphuric acids,
constitute the prosthetic groups of the conjugated proteins
(glucoproteins) classed as the mucoproteins, but as yet one
ca.n but speculate lIS to what special metabolic significance
 srLPffATIDEi, 2{)!l

atbu}w.... to ~Udl (omp}{x Hmillo~\tlgHr ",ulphuril' {t('id

C(JlIII)('WlCh. It i,,,, pm"ihle that tlH'Y might rtpn."tut trnt\ .....
I<:Jrmatiofl !-.ta~I'{'~ ht'tw('('n ~lIh'1l~ nfld prot{'in~, Hltd tltt,
prt'M'fl(,(' of flrt :X-Amino J!roup ill 1IH\11~' of tlu-"t :'<oH~H.r.. i~
~lIgl!tstiq in this l'p:o,pp,,'t. a . . is ul ..o tht, pr('M'tH'(' .t.ar tlu'
sulphuri(' ~l('id group on tlw pos"'Iihle analogy. lllrt'lldy IlWll
tioll(>d. lwtw('l'U phosphori(' Rnd sulphuric l;whh.: Imt nt th('
~an\(' time the larg(' part whil'il such ('Olnpountis play in tht'
('(Hllp01)ition of sU('h mdaboli('ally itwrt tissue',", t\,. t('!Hloh.,
Ilnd supporting tissu('s g('IIC'raUy dQ{'~ not rt'adity ti("('ord with
! his hypo! h,sis.
The Sulphatides. Vcry little j ... kllonll ('oIH>(TJling tlw ... c
('ompollnds. Thry havt' already hN'll nft"rred to in thl'
general clru;siflcation of thf> lipiues gjY('ll OJ) p. 11]. 'I'lli'
_",ulphatiues ('omprise an obs('urc type of sulphur (:ompouud
with properti('s somewhat analogous to tho~(' of the phu~phn~
tjd('~" Their t~xisten('(' was tirst r('cnrd{~d by TlmdiduHH.
who reported the preseIlc(" of both phosJihorw~ and ~wJphur.
Kodl deteeted the prcsen(:e of phm.phoru!oi. ~ulr)hur. "lId u
I"(ducing sugar, and suggested. that the ('ompoulid wm. II
~ulphah' of a pho~phatide and ecrcbrosidf" of th(' t~'lw :

o
Ii
J)ho8phatidt.------- ()--s _O -.o------('j'nhfl>fiidt,.
<)
In 1912 Levene reported the preparation from hrain of "
sufphahdr containing no pnospnorus ana' iUlSing tilt" pt?r~
ccntage composition C = 00'9; H = 10r.r; S'~ 2'31 ;
S = 2'66; 0 = 28'46 per cent. This illkrestill.1f compound
does not ,!pm to have beeu further invcstigated u}' 10 1h"
prese;:!t time.
BefIIrenoes
J. Al>D1UUULD"" and W"RTllEllIElL Pfluger', A",}i.. 1923. 198,
169 ; 200. 649.
%. ARNOLD. Zeil. pky.iol. Clt_.. 19)(J, 70. 314.
3. H.Ltuu P"",. R4y. 8"... !l11l3. IN. B. 426. 441.
... "
210 R(i/,E OF ST'LPliCR n~ BIOCHEJ!lSTR1'
.t. 1I,\RIWolO.\, Hiod/I'tU. ,fmun., HJ24. 18. lOU!!.
5, lh:u:. Uim'Jum. "minI., IH:!-l. 18. I HI, flrW.
0. JJ(lFVMA~ :-till! (;(lln"Nt:n. ,ffmrn . .. lnlf'f. f'hn;/. 8(w.o 1922.44
:141.
7. HOI'KINS. Bio('henl-. JOllnt .. H12J. 15. 2~6. :-:'Pt' all(o Bio('hnu,
r!_0urn 1923,17. 5M6; 1925.19,207.
R. ~l"E. Uiorhem .!our1L, Hlt4. 18, 12tW.
I;:: L}~~,;;I~E. Zt'i~: {~:'i~::Ln(i:':;:" a!;:lH}i~;'(;~~;nt(;lll~." iJtlllilul!:
lJ()Ill.!;llIalll'l, Gret"ll & en., In2{;.
11. L.EVJ..:'Sl'; . 1ourn. Bi(JI. ('hNfl . IH12. 13. 46:l.
12. LEWIS. .. ~ulp}lllr 11t,taboli8m:' J'hYliiol. lierieu'x. I !l24. 4.
:194.
l:l. LooxJ.;Y. B}~nGM}X-J), and t;nAVJ:s. Jolln;. BioI. ('Jum . JH23,
57, 515.
14. Bioi. ('hem., IH23, 56. 1;")7; 58. :li:t
~h'};l.l.lm . JfHJTn,
15. Xl~(;BEIW autI cn-workt"J'8, Bi(}('kem, Zeit., 192:l. 140. 2fJi;:
142, 191; 143. 186; 11l24,144, 138.
~;: A~~~~;~~, ZR1~~!!~~i~~i~"~;~~2':J.H~~., 1~' 11).
18. ~IfEAR}:R. 1'I'OC, ROll, Sm',. 1922. 93. B. 213.
lU, SIIERWI!..: ruld cQ.workct'8 . fount. UitJl. Clwm., Ht2..t. 59. 675 ;
60. fiR ,.
20. TiliELE, .lotH'''. ]Jh.lJHio1 . 1nOi. 36. fi8.
:.!J. TrXSICLJFl'E. Biochem . .JOInn., IH20, 19. lil4.
22. \fUtBURG and ~AK{'llA. Pfluger'S Anh .. IH23. 200, :?U:t
2:1. 'VAltLI. Ri..odHm~ . Tour" .. 1\\23.17,898.
24, "'ILSOl\. Rio('iwln .'ount .. J92!1. 19, :\22.
 ('JHI'TEH nil

THE VITAMINS

E'U'IHI(',\L oh~f'rvatjon ... on the nutritional di ... turuan('(,,;

which w(' now aseribe to vitamin (i<'ficit'Il('v date from the
experiC'ne(>S of the early eightt't'nth-~lItury "I\IlYiJ:Hturs. who
not{'d tht euratin' dft'ets of thf' juic('s of (mmgrs. lefHolls,
and t'itrons on t))(' diseuse knowH U~ s{'urv", whj('h IJH'\iitahl"
followed prolonged omission of fresh veg.etahle foods rroJ~
Hi{' diet. ~l(, curative effect of fruit juiees was so dt'arly
cstublishC'd even then that in ] SO''" the j~':tu(, of 8 rution vf
1(,lllOn- juic( ''''a~ nutde ('ompulsory in tht British Say)'. and
thr-rC'aft('r seurv)' bc('amc a ran' dist'n.sc. During th(" latter
half of th(' nineteenth century a problem similar to that
presented by S(Ourv)" aros(' ill th{~ Japanes(' Navy, wh('u Ul("
dis{'nsf of ben-beri, which had b(,(,11 promin(>nt for some tinw,
was ascribed t.o a dietary deficiency. Empiri(>-Hl ertorttt to
modify the then existing diet, composed largely of polished
rice, were successful in suppressing the disease WhCh a ration
of barley was substituted for a portion of the polished rice.
In relation to the problem of beri-beri Eijkmall, then medical
officer of a prison in Java, made a series of obst'rvations,
dating from lS9() to 1896, in which he noted the effect on
fowls of a diet of polished rice. He found that they de"eloped
a definite patholog;cal condition of the nature of neuritis, and
Was much -itupressed by the resemblance of this disease to
huma.; beri-beri. He was able to show that his birds could
be cured if an extract of the outer husk of the rice, which bad
been removed in the process of polishing. was added to the
diet. Eijkman clearly excluded the possibility of the beri-
beri of his birds being due to insufficiency of any. of the'
211
 212 THE VITAMINS
k1l0WlI food ('ornpol1{'nts or to the prt>~l'I\t'C of pathogellie
orgunisllls. 111 lHOr, he tkfinitt'ly ~tai('d that til(' Hutritional
pof\'llt'uritj" OJ' J)('rj~l)f'ri \VBS rill!' 10 thl' uhs('JH'c from the
didury of some sHhstan('(' preSt'lit in ri('(' poIishings l and
w}l],JA wa ... liot 1) prott'in. fat! C'urhoh:vdrat<" or mineral salt.
lIt 81:0,0 ('otwluded that the prt'SPll('{' of tIlt' unknown suh-
stUtl('(' WU"'i indisp(nsuhl(, to health. So long- ago as l~R]
LUIIin tt'(oglti'i.t'rl that nrtiti('ial mixturt,s of proteins. fats."

~
to~=-_------};c
o ~
___ --;!
..,

}'w. ~~~~:~~~l~:~l:~n~):htb~~~l; dt.i'~i~ r:~:~~la~G~

a)lowaIll't' of milk. {_.\ft,eT Hopkins.}

and {'arhohydrates, together with salts and water, cannot

be used for rearing experimental animals, whereas milk.
"'hicp <'Ontain. these food <'Omponents, is adequate for the
purpose, He therefore argued that milk nrust ~ntaill
substances, other than these five well-recognised components,
whi"h are indispensable to the growing organism. It was
not, however, until 1906-7 that this important fact waS
, definitely establiahed by Gowland Hopkins in a senes of"
experiments ~hich w~e not published until 1912, The
 UROJrTH (TRrE8 213

ac('()mpunying 1ljg. 20 show~ some of Hopkins' rt.'!!<uHs.

The lower ('urn' (up to tht' ('iJ,'!htc'enth dny) show", tlw 1\\"t'rnJ.,J('
wf'ig-ht in w-ams from day to day of ('ig'ht Hl1l1{, rn.h Up-Oil n
pure dittlH,Y ('Otn})('ti('d of puritictl ('n~eiH ..,ta~h, lard,
inorganie salts and wut('r. whilst tht, UP1K't ('\In'(' sh0l!'~ tiw
w(ight of (jght similar rut.... takillg .'1 C.l', (If milk Iwr \.iem
in additioll to th(' pUfe hasH] did mc'nfjOlwcL On til{"
('ight('(,llth day. marked by u yt'ftiC'Hi dotted lint', til<' milk
W8 .... transferred from out:> sd of rat~ to the oth('r. At th(' heM
gjylJJjn~ of t}l(' l'xpf'riuw))t tht' flit-; fr't'ciyhlg thi' milk showt'd
a nnl<'h h'1'('ah~T ralt' of growth than ttl!' ruts kept on tlu.>
artificial diet. an iucrC'as(' aitog('tner out of ptoportion to the'
small a.mount of h-olid math'r in til(' :l ('.('. of milk whi('h tht,y
r('(eiw'd. and which th(' otlwr rats did HOt. Th(' fornt('f sf't
of ;ats. ho~('vt'T. when cit'prh'ed of th(' milk. soon ('rasf'd to
gron-, and in fa(t lost wright. whi}r."t, the tm<}er-dN'{-lop<'{} rah
at 01;('(' h(>gan to s.,rrow rapidly whrJ\ thf' milk was nd,dt'd to
their d('fil'irnt dietary. .Jll~t hd'on' the plihli{'atioll of
Hopkins' results! Oshorne am} lft'ndcl and at HII' salll<' time
~J('CoJ1um and ll8yjS. had hN'B workillg on similar dirt(>ti('
probI('ms and had arri\'('d at ('ondnslohs very similar to
those of Hopkins. The Anl<'riean work,", ,howed that. tlw
milk fat. aHd also the Tf','tieiut' oht.allJM after Tt'movRI of t))t'
fat~ eRsein, and albumin from the milk. ('ontnin('d !-,.'l"owth
promoting substances similar to thos(' whidl Hopkin~ had
also been able to extract from the milk h~r Ttleall~ of
alcohoL
Thus we se(' how thtsc t'arJv observations 011 til{' failurv
of normal growth of animals kept on artificial diet. l,'d to
the recognition of thr- ("xisten{'(' of a growth~promoting 1'ouh~
stance, wllst the still earlier work on speeilk di-..... , j,
responsihle for the con<.-ept of two other essential sunstane<:s,
one having antj~neurjti('. the other having anti~SC<)J'buti(~
functions. TheS<' substances, present in the natural food-
stuffs in variable and small amounts. have been u-rm('d
accessory {aC'tors, food hOTm01J(~s. and vitamines. Th(' la,<;t
214 THE J'ITAJ!INS
trnn, sugg<'Sted by Funk, was used to indi('at(' a supposed
rf'1ationship to organiC' nitrogen compounds which furth('r
investigation has not substantiated. At prr8f'flt thj~ last
t('rm is retained. hut tht' terminal .. (' " is dr(lpp(~d, and "\,'('
now ~}assify the thr('(> suhstances nJ{'ntiOBtd aoo\Jc as
vitatf.ins A. H. and (' respeetivdy, and differentiate them hy
their solubilities, A being fa! soluble, and Band C watt'r
soluble, This system of terminology is pref('rablc to a loose
USf' of the o\"'r-workrd expression fa('tor;' anel its nou-
H

('ommittal significance is to he reeornmended until clearer

(,\"1dcl1<'C of tht' chemical nature of these suhstanc(>s is
a,'ailable. The system also has the advantage of heing
readily expanded to accommodate tbe discovery of neW
\'itamins. Thus recent work clearly establishes the occur
rrnce in tht' fat~soluble group of vitamins of a grov..-th-
promoting substance. an antl-r.arhitie suhstaJ)("{'. and an
nnti-stcrilit),T substance) the two latter being now dasscd as
yitamins D and E. Further, vitamin B has growth promoting
as; well as anti-neuritic properties. whieh mayor lnay not
I", due to distinct and separate vitamins. It seems highly
probable that the number of yitami"s will increase with th,-
prosecution of furthf'r reseal"Ches of a more detailed nature,
but at present the vitamins whose individuality has been
establish,'d arc as follows : -

Solubility. Etl~ts by whi.ch vitamin iR recoguiiWd.

A lo'nt soluble. Growth-promoting, essential to

young animals.
B Water soluble, Anti-neuritic~ growth-promoting,
essential to normal lMltrition at
all ages.
C Water soluble, Anti.scorbutic.
D Fat soluble, Anti-rachitic, essential to young
animals.
E Fat soluble, Anti-sterility in both sexes,
 .. BI08" 21!i

~l(.ntion must h{, mad' of S\notiWf sf'rif's of ~uh"hmf't...

the ('Xistt'Ul't' of whi<'il has h('('n p(l~tulat('d. and whit,)' nut\' ht'
rellltf'd to the ,-itamiu ... mort' (sptriuHy to vitamiu B. T'h('M'
!o.uhstan<'l'l'>. lift' ~lIppm..('d to lWH-r n f('luticm to the growth of
~~Ill~)[(' orguni~lIls, l\\I('h .n!. )':tast. bH.(,t<~ia. nnd. p\'!to/.un.
'imular to that of the ntaItUll!-. to th(' lugh(>r UllIlHa~, III
til(' ('ast' of ~Tst thf' minH' .. hios .. h.a~ hel-of! ~i\'('n to this
hypothdi('aI suh~hHHt. Hit),.., has n widt di ...trihtltion til
H't,,<'tahlc- ('xtrad ..... and it i\ l,upp0S{'d to he f'harn<'i{,l'i"t'd
hy its property of ... timulatiuR the ~rrowth of ycm;t t,t,lh
in artlfi('ial ('uIture llwdia. Thus it hus lW(,Tl duifllf,tl
that normal growth of yeast ('('lis in a ~uitahl(' lIutril'Ht
JlHdium will ollly ('H"'lU'. ill the Hh'\{'lH'(~ of un ouf!\idc ~OUrf'f'
of bios. if tilt, original inOt.'uiuTll is rompoSt'd of Hot It's." th"H
a cl'rtain l .... iuimalnumher of ('dIs. Tlw th{'OfY is that j.{Towth
i,<; ('nta)y~(>d hy the pr(~.'i{~mc of 1>io.., in suflidt'nf IlIfWUht.
('udf ('{'1I ('ontrihuting it, quota: if til(' Humb('r of ('t'I1!>' in
the illO('uiulH ih in~um('iel\t to make up til(> fl('('{,,"SHry supply
of bin... ~1T(}wth is r.ctardNi or suppre.s,"Ud. )fw'h l'untro\'ct'Oy
hah ragt,(l round tilt' question of th(, Tf't\lit_v or otherwiS{' klHl
possible fun(tions of hios in relation 10 tht' ~rrowth of )'l'n'st.
and ~onJf'what l'iimilar cJaims han' ht'clI made co-mt-rning-
th~ dependeIl('(' of the ~I'fowth of hacteria. and t'n'n of gTt'('n
plants. on the pr{'s(~ne{' of tra('('s of prd'ormed organl{'
substaneffl. l util more i~ known (>onC'{>rning Uw l:,rr{~d,;o; of
T

!-.ima}} nUTtltiows' in the rdath'{' ('<)/l{Ywtr8liol/~' qj' kmnf'H

components of culture media on the deve-iopment of ),,{'ust
(!ells and bacteria, these claims must f)( 8.C'ct'pt<'d \\'ith
eaution. )lany of the f"xperiments in\o)\"(d S("("m to ba\'{'
been very inadequately controlled. In relation to the
question of the existt"nce of plant . auximones," a.~ thf'
5uhstanc~ said to stimulate plant growth ha ve 'hren called,
Drummond and his co-workers have shown that a diat<>rn
U'iii:U1chia closftrium) can grow nonnally in sterilised or
artificial sea water, and tbe fresb-water alga, ChlorelLl, like-
wise- flourishes in a solution contflining inorganic salts only.
 THE rIT.HflSS
FuinH'r. ~t'J"'O!l. and ""hitt> haw' df'mon~trah'd that yeast
will g-row ill n synt Iwti(' artifh:'ial medium prepared from
inorglmic . . nlts llnd PUff' SU~clr. ('Yeli when thp latter is n
~ynU\('ti(' o;ugar. Drummond points out that it would he
.. Hn <:\frnorninury fad if in the ('ourSt' of (\'ulution. erell
~il1lp' g'Tt'('n plants had l{)~t tlH.'ir u.utCltrophi{ natur(' and
11('('0111(" to R ('('Main ext('nt d('pen<ient on preic)rmed orgallie
Illatter." lit' al5.o draws ft di!-;tin<'fion between suhstall(,cs
which ux('('Jt~rat(' or stimulatr g'n)\\,th and ""uhstances whi(,h
HT(' t'~Sf'HtiRl for growth. He r{'sct'\'{'S tIl{' term" vitamin "
for t iws(' luttC'T. and regards .. a suhstan('(' H~ a vitamin if it
j,,", essential to the life Mnd wc11~bci1JR' of an organism which
dnt's not possess the })QWt'T to syntlwsi!o.c that substan('(', and
aJ.sn jf it is or~ranj(' in nature, and does not belong to any
Ollt' of the thr('(' great ciass("s of foodsturrJ... protrins, fat!-,.

a1l(1 (,HrhohnlrRh~s," Bios. which i~ not {'~.o;{'ntjLtI for the

MTowth of ;'e8st, but at the most l1\('re1y stimulates it. is
t h('refor(' not a \'itamiH.
Certain of the simplest living animals, r.J!., the {'ilinte
protozo()n Colpidium ('oipoda, appear to rt"semble the plant
ill being iIHiepf'ndf'nt of a SOUT('f' of preJormt'd ,-itamins,
although their growth is stimuJatt'd by substa.n('~ of th('
JUltUrt' of the hypothetiNll bios. As ont' asee-nds the animal
sPate d('l)(>}uiNl{,{, on OOt or nlOr(> vitam.ins bee-omt's mof(' and
mor.f' marked, anrl normal Hfe processes become impossihJt
in the absen('{' of these definite substance~ synthesised by
plant agen<'y. As yet our information roncerning the
Ilt'('essity of vitamins for the lower animals iii very frag~
"",ntary. but the following table attempts to summarise
the n vailahle data : -

Type of Animal. Vitamins n~ry.

J<1y (Dro80phi/O). B
Tadpole and f:to!.'. B A?
Fish. A II
Bird. A B ('1
MalnJllaJ. A B nDE.
 80f'RCE OF rTTAJIJ.VS
The animal world i< dependent Oil the plaJlt world for it,
~upply of vitamins. f'uT('ful ('xJlf'rimruh haH' shown that
th(> "itamins present in animal tisSUt's unci 111. th( pf1Jdnd-.;
of animal adi\'ity ha\"(~ not oriJ.,.r111ohd tiwrt'. hut havl' ht"t,})
trahsferrt'u from H n'gt'hlhh, svur('('. Thus tIlt', vit~lin~ of
(ow\; milk fat have b(,(,fl trul,('d to their origin In tIH\t.trrt'li
foods on which th( ('OW f((rl_.",. 01)(' of tht rlrh('st SOlJt('('S of
thl' fat-soluhle vitamins A and n is Ul(' liver oil of thl' ('ltd,
and Drummond and Zih'a ha \'(' followttJ t lw sti1.IX('S in t 1\(.'
trallsf{'rrn('(' of the vitnmins from th('jr V('gC'hlhlc soum' to
their final place of storage in tht, cod'" Ih(r. Th{' ('od i~
('arnh'orous alld f((ds on small('r fish. slI<"h n" h('rriHg'l'i.
(aplins. C'rusta('t'Hns. and squids. whic'h latter in turn f('('(l
on the small organisms floating on the /!Iurfu{'(' of tJu' s{'a.
all~i class(otd as plankton. Thr plankton ('ompri!'lt's milluk
('rllsta(>('IlJ}s. "torms. dfrapods. {'op(podJo>. ann Hl)nll'rnUS
llln~a} forms, which nOllrish th{,lHSt'!n's nt th( t'Xlwns(' of
th{' minute chlofophylla('<'ous diatoms which ahotwd in the
surface fayers of thf' sea. The vitamins of the {od'.,,> Jj\"f'f c'nll
he traced hR.ckwards through this ('hail! of animal"" f'l:wh
f{'-('ding on smaH{'r animals. to tJl<'lr SOltrt'f' in tilc' minllt(
plants. the djatoms. Djatom~ han' h"{'n J.tTOW!) ill tll('
lahoratory, as we han' s{'~_'n. in st<>rile Sea watrr or salt
solutions. and in the' pre~(,ll('e of dilfus{' sunliJ:ht tl1("), nrc
capable of forming vitamjns from the purdy inorgani(.
constituents of the Illedium in which they grow. Fre>h-
water alga' likt'wise flourish und.er thcs{' {'01lClitioIlS, and
at the same time form vitamins l'apahlr of tf'stoTins,r
to rats kept on a vitamin-free diet their power to grow
nOflnally.

In the animal sources in which this vitamin was fin1

found, namely in butter fat, egg-yolk fat, cod-Ih'er oil, and
ox fat, it occurs dissolved in the fat, alollJ{ with which it i.
_18 THE VITAJllNS
fLxtra(ttd hy means of the usual fat soh'ents. In the plant
wher(~ tht, vitamin is synth(~siscd its association with fats is
hy no m(,RHS so uHiversal as it is ill the animal. and. indt"ed.
as it O('curs ill t hc' plaut it secms to he much I('.\is freely
soluhle ~in fat solvents than wht"n it occurs in animal fat.
This IJay he (hH~ to its n~so<'iation in the plant with other
nlO)('cuhs which arc not thcmsclvf's fat-soluble. 'I'lli' ahsenee
of the vitamin from practically all vegetable- oils is note-
worthy in this ('oJUw'dion. In 1914 !ltd.'oHum and Du\'is
show('d that if uutter i~ saponified and tht'n shaken with
olin' oil the lattf"r at'quires growth-promoting properties.
hut Steonb(){'k. St,ll. and Buell first showed elmrly that if
prt'('IJUtioJ)!; art' tRkf'U to prevent oxidation the ,-itamin j:..,
Hot n.fre('icd hy the saponification by means of boiling- ako-
holi(' potash of the ruts in whi('h it is pr('&cnt, The yitanim
may th{'refor(' lw r('(~oyert'd in undimini~ht'd adivity and
(oJls.idfrabJy {'onf't~ntrated in thf' non-snponiJlahk frat'Uon.
rt'prc:>st'flting in most futs a little less than] per ('cnt. of the
original mnttrial. r~;t' has I)('(>n made of thi~ rcsistuH(,{, to
al<'oholi(, potash in attempts to isolate th!' vitamin. The
t('C'hni<'u.l diftkulti('s aTt' grNlt. in part du{' to the minute
amount of the ritamlll angina})y pr('!~eut even in its richest
SOUfIT, (od1inr oil, and in part du(, to its liability to undergo
oxidntivc drstruction during the prolonged manipulations
i,wol\'ed. It has not yet been found possible to identify the
"jtalniJ) as a distinl.'t cheD}ica) substance. but Drumu)ond
and his co-workers have accumulated much interesting
information concerning th~ chemical composition of the
nnsaponifiable suhs1:anres with which the "itanlln is asso-
('jated. in the course of which work highly roncentratt'd
preparations of the \jtamin have been obtained. Clajms have
heen made bv Takahashi to haye isolated vitamin A in a
state of purity, but on the basis of Drummond's work there
is no doubt that these preparations represent snbstances
which are themse),,"s ;,,,,ctive, but with which the vitamin
i. closely associated. These substances, constituents of the
 ,rITA;IfIN A CONCENTRATES 21l!

non-saponifiable fraction of natural oils and fat~. Wi' nttl~t

now ('onsider.
The sapoJlifi('ation of the oil or fat iloi H('hi('\'('d hv ntNHl" (If
hoiling aleoholic' potussiUOl hydroxid<" foHoWld t;y 1\ .. "II)'w-
q\tellt treatnu~nt with sodium ('thoXldt,. Th(_'s(' hVl-iroivst,!o,
nrf' tarried out in an atmo."phCT{ of nitrORt'tl. to" ?l't;~'t~Ht
oxidath~> loss of thf' yjtamin. Th ... non-""aponiHahl{' Tt's.ifhw
of the ori~ina) oj) ('ontains considrrablt amoullts of {holt
st('roJ. and when carefully prepared it is fIT'" {rom all trlt{,(
of fatty acids. iodinc, and uitrogtn. ]t contAins the whole
of tht' ,"itaruin present in the original oil. The (hoh,,stefol
is r(,lnov('d from this fra(tion without imrniring til' adivits
of tIl(' vitamin. by crystallisation from mdhyi nkohot at low
f('mperatuT(,s (not below 1..ero), ami by prf'dpitatioJ) of tht
"hole~tf'T61 whil'h stiH remains h.Y m(>lw ... of digit.onin. Tht:'
Tcsidu(' from these trt'atJncnts {'onsi,ts of It T('d)>rHWH nil
whkh still retains the full activity of the vitamin . 11 Illt'
otht'f products separatt>d so far lJ('illg- quit<- iruutin. A
further purifi(',ation of th(' vitamin~hearinll frndion Uluy tK'
a<:'hi(,YM by distilling it in supcrheat('d ~tcam in nn Htmn~
sphf"rc of nitrogen. l:ndtr th(>s(' ronditions the vitamill is
".Brri{d o,'cr with other vo1ntiJe produf'ts. Hnd the< rtosidut'
of resins left in the still is found to ht~ inactin'. It i ... pr{'fr'r.
able, however, to ~uhjeet the c-holestcrol-frN' p!Jrtiml to
distillation in a high vacuum. In th(' ('Qursc (.If their r>('('nt
work on cod .. liver oil. Drummond~ l'hannon, and ('oward
obtained from a large-scale preparation some 750 grams of
unsaponifiable matter, from which they separated 370 gram,
of cholesteroL They theu fractionally distilled part of the
residue in a high ,'aeuum, and obtained the following series
of frnctipns. Distillation was more or les~ coutinuou.li o,er
tilt! whole temperature rang!! and there was no well-mark,,1
separation. The first fraction to Ix; collected consi,~-d of
impurities which had accumulated from the variolL' solWllts
which had to be lL..ro in very iarg!! quantities. This fraction
is neglected,
220 THE VITAMINS
TARl.E XVII.

A~('J.!
lradloll,
Tetl\JX"ratll~
Quantity. lOOlm' C'olol\f
.Rangt', ~IlJllhrL
lWa;:1ioll.
"
rr 'trry pall' yrf{(}W oit w(tn Its -I."i5'>.':~ mill. tJgrlimll, ,,,
IlllukNl krJ ...lwllkt> ndOl\r.
III l',,!t, yrllollr nil, wq>t'lle tM,O-1~~i2 mm. 7gtaffil\, 120 ++
1\',
(Ki(1lit.
Y~Uow 0\1, ~Imilf\.r ",men. lR4"lOO"::!mm. 6 gtaDlll. 12:, ,
,.,.-1.,-
\'. \'('lIow oil. plmll.. nt t-t't-
f.('III'-llkf1-!1mdl.
HIO"- :!:"'()~ll--~ nun. 1(, gl'aIru.. 12t1 +
\'f. Oranlc yell(lw 011, pl1np:t'nt :!:~W-270',1-2 mID. 27'~' Irfll.ms., 14.\)
lItnelt.
VII llfotldl~h,hrown
r,,,,,ln
11.l1lbt>r-lIk(' Jlcsldu(' 270' - :"'O;,graffilj,

In tlw last column thef{' is recorded the intensity ot' the

('olour rt's('tion givcn with arsenic (_'hloride, Thi~~ ddie-ate
('olour test, which we shan discuss shortly. scrYes as an
indication of the prrscnce of vitamin A ~ in the Yari{)us
frnctions. It will m> s{"('n that the actin> ritamin is definitely
associated with fractions IV. and ,~ .. and the further (>xam.ina-
tion of these fructions is therefore a matter of grl~at interest.
There is found in these fractions. in addition to a small
amount of what is possibly lipochrome pigment. the un-
saturated complex hydrocarbon spinacenc. with the formula
(, H, .. and u saturated solid alcohol with a melting point
of about 6(r', which is probably batyl alrohol, r ,,,H,,O,. The
bulk of the neti"e ftactions consists of an unidentified
unsatura,d alrohol or alcohols with a molecular weight of
about 300, and an iodine number of about 103. From other
fish liver oils oleyl alcohol, C,.H O, selachyl alcohol,
C,,,H O,. and batyl alcohol, C,,,HuO,, have been isolated
in the non-saponifiable .fraction, and these, togetber with
the alcohol phytol, C,,,H O, which is present in the chloro-
phyll molecule. prove to be quire inactive in re-establishing
gro,,'lh when red to rats maintained on a deficient diet.
"'_;nacene and the satnrated solid alcobol isolated from cod-
is cl",U are likewise inactiv.., and th..re is no reason to believe
 .. BIOSTE'RIN '. 221

rHat IIP()('lltUJHl'pignwHts. when ('Rtt'fuliy pnritkd. hnn' au)'

brrowth~pr()JllOtin,t! I)to})(,rtie."', Tht' Mlh",tHIW(' whi{'h TnkH'
htlshi hH~ clninH'd to he \'it.uuin .\. whidl ht Itt.... ('uUed
.. Hiost('rin."and to whidl he 1t!-.~ig1\'" tltl' fOTHHlla Cn ll""Ol'
prohably rcpr('Sl'Hts mixtufes of l1w protilH'ts ~i"i(US"ld
ab()\'(. jn whidl the yjtamin is pr('s('nt iu a hj~hlr (01Hen~
tmted statf'. just as it is in DruuulIond'!'> pr(plt.rRti()Jls~ \V,,;,
must ther(fore ('oncludl' Hutt nOnt~ of t Iw ('()n~titUt'uts
prest'nt in the non~saponifiabh. portion of c()d~ti\'('r. and NO
far identified, really Tepr(.'S(nt thr IHU'{' \'jbunin A. 'fill'
vitamin itself is Ul('refote prohahly pr('sent ('\'('Il in t}1('se
concentrated ]lrt'parations as an .. jrllpurity " iff th(' rnntll
bulk of ullsaturatc'd a}<'ohols and other :-'UiJstHIH'l'!o.. Tlw!'ot
filets illustrate weB the gr(a.t djffi('ulti~,o; attendant 011
isolation- and the exe("edingly small amouut of the dtamin
which is phvsiologically df,,(tive. Tim; a I'I\t weighinJ(
about 1on gr~ms will c~t about H grams of prot.rin. ~ gram!.
of fat, and )() grams of carhohydrah' per di{'m, and to
supply the nec('ssary fat--soluhlc \'itamins A and n it i.Oj,
~ufflcient to add to this daily ration sotn(' 20 mg. of ('od-lin"r
oil. This represents a.bout 02 mg. of non~sapotlHiahl('
matter and about 002 mg. of the fraetions in which th!'
bulk of the vitamin is found after vacuum distillation. TIl{'
actual amount of vitamin which is effective therefor~ be~on1('s
yarushingly small.
Some information concerning the possible chemical l11Itu",
of ,itamin A may be gained from the observation that it is
not affected in any deleterious way by the substitution of
the hydroxyl group of the alcohols with which it i. associated
by the acetyl or benzoyl groups, or hy treatment with
phthallic anhydride. On the other hand, the vitamin is
,,ompletely destroyed by changes which affeet tbe unsatu-
rated linkages, for example by bromination, by hydrogcll~
tion, or hy oxidation of the unsaturated compound. of the
active fractions.
The presence of vitamin A in natural fats and other
 222 THE VITAJ\{lNS
Mlhstl:HI('t'S i~ d('h'rmiJwt\ hy f('eding th(' !-.uhstan('c und('r
inn'!-.tigatinn itl vHriol)S amounts to rah or oth('r anllllnis,--
rnb. IITt' "'if'){>dt'd lar~Jf'h' h({uus(' of thC'ir {'nnn'nient sizto and
relatively rap-id mctal~li(' pr(){'c,Sses-- which tUH'C b{'('lJ kf~pt
thr H I~t(iod on a di(,t deficient in vitamill A. A suitable
bllsnJ did, whi(h ('ontains ('\"l'ry JI('{'essary food ('OJllllOJlf'nt
with the exc('ption of vitamin A, is composed a~ follows:--
})uritied cus('in ZO part.,.
l'urili{'d ~tarch . . . . ,35
Flit (lare! or h"re!<'n~d cotton,eed oil) 15
Mixturi' of' ('ry ... taUim' InorJ,!anie ..,11lts . ,?
Yen.,t extract . 5 ..
lA"'nloll or orangr juj('(> A few ('uhie
('(,t\timctrc~.

The mixture of inorganiC' salts is ('om posed as' follows

(McCollum and Davis) ;._-
.:'linn s]!) part~.
Mf[S04' . 7'!)H
"aHjPO" H,O 10'41
K.Ill'O, ' 2Sti2
CnHil)O,b Il 20 16'20
fa lactah' 39'IM)
IrOll citrah' a'54
Iodine . Trae(".
In pl't'fmring this diet tll(> casein is fin(>ly powdered, and is
puri{i{'d by heating, with frequent stirring, for thirty-six
hOUT> at 120 0 on shall()w trays exposed to a current of air,
Any vitamin A which is normally present in crude casein is
thus oxidiS('d and destroyed, Extraction of the casein with
warm alcohol followed by light petroleum is an alternative
procedure, Rice starch, which is generally employed,
requi1't'S no purification. As a vitamin-free fat, krd, or
preferably refined hardened (i.e hydrogenated) cotton-seed
oil, is used, Young rats weighing about 50 grams are kept
on this diet for a period of three to four weeks. and at the end
of this period they have usually ceased to grow, their weight
not being in general above 90 grams. The substance to be
 COLOC'R REA(,7'IOS
testNI for th<' prc...(~,H'( of vitumin :\ t~ HWfI :Hl(kd to the
dif'hlry in "arying quuntiti<'''1 until tht' fflflliuHUi-I tifliOH1l1
whkh will inou('p d~'fiuitc and ('OlltimlOtiS I!f'nwth f(lr n f",ri~Jil
of four w('cks is as{"{rtuinc-d. ThiN hiologi('1 HU'thnd of n"''''iI~'
i~ ;lccessariJy trdiolls. but it is til(' Huly rdilllJlq..rtH'ltfl!-
8\'ailahl(> up to Ul{> present for deterrHinin~ the pr('S('Il(,{' of
the "jtamin ill allY substnnee. A sfiti~radory dl('mi(,tti h'st
t(Jr the \'itamiu would ,:{rently fa(~ilitl{t( the w(Jrk of hio~
dwmists in this Held, and there- is r('a~on to b('lit'\'(' thut ,tl('i!
a test nHly he forthcomiuj!. A parallelism has lit'eli oh!'!('n'('d
hetween th{' purpk ('o}our t(tte-tion ginl) by li\'('r oil... W}H'll
they nrc dissolved in an (lrgani{' ~o)\,(,llt and trt'utNl with It
drop of sulphuric ucid, 811d the preserH'(' of dturnin .\ iu tht'
oil.. But so far this kst catlnot 1)(' used a .. all uufitiliut{
illdie{ltior~ of the prcsen<'C of the t'itamin. Thu~ th4' oil
{'xtract('d from marine diatoms (Xit:;xC/da). which ('oll1priv'
the'synthetic sour('(' from whi('h til(' {,H(} derj\'(~ it;. 'iupply
of the "itamin, dOt'~ not give thi!'> ('olour reactioll. Allothf't
simple colour t('st of a nor}' promising Hutufe ha.' just hel'lI
descrihed by Roscnhdm and Drummond, Thi", dc1iptlt(,
colour reaction is ('"arried out by adding 1 c.c. of pnrl' ark('u;('
trichloride to 1 drop of codli,-er oil and shaking th,. t~,ttul)('
at one(>, The oil dissolv{'s immooiately to form a soJutiolJ
of a clear ultramarine blue colour. which in du' ('our~w of 8
few seconds assumes a purple tint lind gradually fad,._ Th,.
coloured product formed bas a wellddiTl(~1 absorptioll hand
extending (rom A 55<1---511<1, whicn persists ,r .. 1"lUl ,;"'>
minute..~. This reaction is twenty times as delicalc a.~ tbt>
sulphuric acid test already m~ntioned, the limit ""iug
reached with 0.05 mg. of Xewfoundla"d codli\'{," oil. After
saponifieation of tbe oil the cholesterolfree fraction of Ih,
unsaponifiable portion gave the reaction in a dilution of J in
2,000,000. A complete parallelism has been foun<! hetweN'
the chromogenic substance and the presence of ,-itamin A
so far as the in"cstigations have been pursued. and reactions
which are known to destroy the vitamin also destroy the
 224 1'HE rITA-WINS
~UhI4alH'(' "'hie}! th{ ('olour ftIl(tiOll. An inh'resting
gin ....
point is thllt chol<, ...t(,fol whkh ha!o, tH'quirC'ti anti-ra{_'hitie
llrtll)erti('s thrnlJj!'h irradiHtion with ultra-violet light (st'{_~
p, :H:1) dol's 'lOt gin' thb ('olour Tf'adion. .\ <'olour is
ohtaill~1. but it rliff{ts from that gin'I1 hv eod-iin'r oiL
Ho,,,('u}l('irn and Drlmmwlld regard the IH''': ('o)our test as
IwillJ[ ~l)('('iti" for \'ltaJuin A, and it may theTi,fort, afford a
rurtiH'r IlIt'aJI!o. of distinguishing tht, growth~prontotiflg
"Humin from tlw ;Jnti-rachitie vitamill. Colour rea('tions
very similar to that f,!lH'H b:v ars('uie chloride af{' also given
by ('{ld-Hn'r oil iii th{' pr('s{'ne(' of diowthyl sulphllte, trichlor~
a(t'jl(' ut'id~ acetyl chlorid('. and l.){'ozoyl chloride, the last
two only in tllt' pr('sC't\ct' of zinc ('hloridt~. Th(' rcaetion may
he utilised for t}1(' ('olorimetric deterrnination of vitamin, A
in the following way ;--
.:l suitahle colour standard for comparison is provided hy
mixing 100 {',(" of cryst.al "iolet solution (1; 10.000) with
,')H ('.C. of methylene hJue of the same ('on('('utration, ooth
dyestuffs heing dissoht'd in aleoho1. Tlnnty mi1ligTam~ of
's('wfuulldland ('od~liver oil (which contains more yitamill A
tluUl the' Norwegian oil), equivalent to 1 drop from a
pointed glass rod 3 nUll. in diameter, when dissolvcd ill 1 c.C,
of arsenic chloride, matches the standard dye solution
diluted in the proportion 3: 2. The comparison must be
made within a rew seconds aftor the addition of the reagent.
After arraIll1ing " set of suitably diluted standard solutiOlIS
in test-tubes, all the oils so far examined were ranged by
Hos(>nheim and Drummond in a series which agreed with
their growth-promoting ad;,ity.
In view of the somewhat similar colour reactions given by
cholesterol, and the flO'>'Sible relationship between sterols and
lipochromes and thrjr general association with vitamin A in
plant tissues. Rosenheim and Drummond suggest that the
arsenic chloride and similar reactions are concerned with
a substance derived from thrse types of synthetlc plant
products under t~e influence of sunlight.
 1"l1'AMIN A STORAGE
Physiological Effects 01 Lack 01 Vitamin A... Th,. ""'<1 for
\'itamin A jn the rlietary of tIlt' Hflult j .., not so m~lrk('d its it
is in thnt of th(' yOlll1g ~fJ"owing .win);!!. Tim ... f1JII~",:..l1'nwu
rats.haye been maintalJH'rl for sen'rn) J)11.mt)p\ on u di~,t frr('
from vitamin A and han' shown no appr{'ciuhJ(' }o..... oJ'4tight
or otlwr siJ,~n~ of iU-twalth. In pnrt this (th(1. lJlllV \t(' dw' to
the ('otlsidt'rnbl(, ('apaeity for storn,!w of t he "itaflli~\ pO ..M ...MII
hy tht' adult animal. a
~torag(' rapadty mUf'h
J,{f('at(,T than thnt shown
fhr vitamins H or C.
Adult allituals kt'pt 01\ a
diet ddieient in \'itfmlin A
do.- ho\\r('\~er. show sonl('
impairment of 1lutrition
in 'he form of an i1l~
('reased liability to contract
had~rinl infections and a i40~C-:'1--+'-+" 1 .
diminish(>d power of' J'{'-
~
production. In th, young
animal d('pri)"atioll of the
vitamin results in a ('essa-
tion of growth which is
r
oL-~~b-~40b-~h-~~h-~
TI"! IN DAV,
}o"]"o. 21.--('ur\'{'~ tl.h!Jwin,!l the infiu(m('t'
eve II t U a Ily fatal. No upon gl"o.'th of pt"t>t'ivul'I iNV"lillg
satisfactory physiological un diets deficient in vitAmin A.
(After Hbt'!'m.a.n.)
basis for this failure of
prowth has yet. be~n discover~d. Ther. is no mark.d
sign of a disturbed fat metaholism. The definite disturhanc<%
in calcium and phosphorus metabolism resulting in defective
ossification (rickets). and formerly ascribed to vitamin A,
are I\OW r~garded as being due to lack of another fat-KOluble
vitamin, the anti-rachitic or D vitamin, whieh is usually
associated with A. The oommencement of failure of nutrition
produced by absence of the ,itamin from tbe dietary is not
immediate, but begins to be apparent after the lapse of
some time, which varies with th.. previQus Ceedi~ of the
 .THE rITAMLVS
allinm1. and presHmahly clt'p<'lHls on thC' ('xhaHstion of thl~
iJodil~' st{)T('t" of the ,'itamin. This is ioohoWH in Fi~. 21. wh'('h
r('pr('srnts t h(' inRucn('{' of previous f('('din~ on tlw growth
of rIfts p11lccd all ll. diet lnl'killg dtnmin ~4. Cl1r\,(' 1. r('prc~
.~C!lts {ht' growth of It rnt whos(> prc\'iows did had ('ontajni'd
titth vitamin A (five-sixths whole wh(nt. Olw-~ixth drit'd
whole milk), ruryc II. is that of 8 rat whos(' previous diet

T.IlIl'}; XVIII.
incidellcf (!f ENe lJiscast' in Rats.
TotRL NIl.ll.fit't'{('d
with I<:,Vf'
J)il'tf'a.'1('.
On dids dditjent in vitamin A . la(i uS)
On diets ddi('i('nt in ,-itamin 11 . 22.5 .,
On dil"ts ot h('rwi~{' d('Ht'lC'ot . . 90 0
OJ) diets vXpt'rimclItal hut pTf'slUHably
ud,qua1<. . .. 20 I (l
On mix"d food (sto"k .nimab) . 34K 0
1,000 60

had he{,Jl richf'r in vitamin A (two-thirds whole wheaL 0l1('-

third dried whole milk). whilst curve In. is that of a rat
whirh had r<'cd\"ed "diet richer stili in the vitamin (onethird
whole meat, two-thirds dried whole milk). AU three rats on
the twenty-eighth day of their lives were placed on a diet
(ntirely de"oid of vitamin A. The rats died at the points
lit which the curVeS end. It will be seen that growth and
duration of life Yaried with the amount of vitamin A in the
previous diet, alld therefore presumably with the amount
of yitamin which the rats had been able to store during the
preliminary dietetic regime.
In addition to the effects alrt'ady mentioned, ill rats and
ill other experimental animals. lack of ,itamin A is frequently
lI$liOclated with _a characteristic eye infection ""lied xeroph-
 X ER(}l'lITIIALff fA
thaililia. whit-h. if untnnkd. aff('1,t ... the l'UfIWl\ anti inn\"
resuh ill tot,II hlindnf's,... TtmJ t hi .. di ...ens(' j, !'opt'('itknn~
n"s(){'inh'd 'with ddieirllf'Y of yitft.min A j ... ~hllWll h~' th~'
UhO\T Tf'Sllits of (Nl~lrlw nlld :\!t'lldd. uhtahwll on their
pf'dij!r('(' rnt!-i tltlwlW(h :;,000 to S.H!J!i (,"'(' Tullte Xfl\I I.
Ttw f'Y<' diM'n<.;C' npp<'ar"i tu 111\'01\'( inf(,(tioH. and tIluy 1)(,
{'(Irn'lntf'd wit h t iH' W'l1f'rnJ )t.,.\'j'riflg of Tf.... i .. tHllrt' )'O)U)Wll I)v
the unirnal kept Oil a ddi('ient diet. But flu_- )"p...dtit' nut\l~'
or tlH' a~s(_l('i:1tiOI1 of the dbea ..c with !1IC'k of \'itafuin. A
,.,hown in t)}(' uh()\'( tuble is al~o ..,}wwu hy the fud thut., jf
I\ot too far advaJwed. fhe di)'('a,,(' i ... u!>.ually ('tU'1.'" without
auy otht'r treutmellt hy lllhnilli.."trutioll of \'ih,llIill A.

Vit&mlh B, the W&ter-aoluble Growth-promoting and

. Anti-neuritic Vitamin
Thf' phy~iologicn[ ('ffed ... of "itllmin Hun' f('('oA'ni<;(,d in
two ways: (1) it .. (,llrati\'(' drt(,t 011. or prdtrnbly it~ pr('-
,"cntin' action in relation tn, ttdull po}ytl(-uritis. alHi (t) its
(~apaeity to enahle young rah to mn.iutuIII a normal rah> (If
R'rowth. Thps(' two eff!,pt'-i mll): be dUi' to the SaJTH' vitami",
and the hulk of present~duy opinion fanHlrs this ykw, hut
it ('annot he said to h(' ahsol\ltf'ly (stahlislud. TIl(' tlistrihu-
hon of wat:-rsoluhJ( H as dt'tt'rminNJ hy growth-prom()tln~
properties is in tht' main ('oiueid('nt with that deh'rmllwd
from its anti-nt'uritic 8ttioll. Xeyerthdess the plurality of
function shown by vitamin 11 is \'('ry similar to that of t.he
fat-soluble vitamins. now diffel't"ntiated into vitamin A and
yjtarnin D.
In ex-peritnental work on mammals dcsif:.,TJl('d to in\,f~tigate
the distribution of vitilmin B the bar;al diet used is that
given on p. 222, with the substitution of butter fat for the
lard or hardened cotton-seed oil, thus ensuring a supply of
vitamin At and the omission of the yeast extract oontaininu
B. The casein U1led must he wry earefully puritled either by
exhaustive extraction with wann 95 per cent. alcohol or by
,......
22~ TilE rITAJlIS8
r('}l<'ntcd pre('ipit;ltiotl with dilutl' (l('di(' H(id. ""he'l! nils
aN' k('pt (If! ~lIdl It did the failurf> of g-ro\\'th whi('h rf' .. ults
dirkrs from that pr()(tu{'cd by ahscJlf't' of .-\ in o('cOurring'
almo:-:.t illlJlH'c1iatr-iy. thus indi('ating nn iuability 10 ..,tore
yitulH.;' H. which i..; iii lHnrkf'd ('ontra~t to the ('onsidrrahk
res('rn' of' A u~unlfy lbund in t h(' hody. Tht, immediatt.'
(''ssHtioll of growth is follow('(l hy a short period during-
which the hody wdght rf"B1Uim; mor(' or ){'.'>s {'on!'!tant: then
n }O~~ of wright ('omJT)cncf'S and h('{'on-ws progn:s~in'ly mort'
rapid up to tlw (h'ath of the animal. DeprivHtion of yitnmin
H aff('(,ts hoth young and adufi animals in the sa1n(' way. and
nit hough th( adult may at first ~how a \Trr g-radual los~ of

, ,
"'..t '''I'IQM''T~

F{( . 22.-Curn-~ ioi!JI\winp: fl~iluJ'('

of j'zyowth on a ditt dt'tkicut in "\itall1il1
Rand f('<-ovt"ryon u.dditioll of tht' 'Iitarnin, (Aft('r ~L H. C. Rf:'purt,
lUN).

weight. a rapid decJine f'\-entual1y appears, and soon T<'Bch('s

n fat-al termination. \Ye haw seen. on the other hand. that
the adult is able to withstand deprivation of vitamin A with
apparently hut minor ill effects. The eur\'es gi\'en in Fill. 2'.l
illustrate the effeets of withholding the water-soluhl. B
\jtarnin from the dietary of rats. Cur,Te I. is a normal
control, ouryes II. and HI. show the effect.. of suecess;yc
I"'riods of adequate meting and B-deficient dft-ting: on
growth, whilst curves IV., V., and VI. show that the more
mature the animal is the longer it is able to withstand the
damaging effects of a deficiency of B in its diet. The animals
representNl in the thn>e last curves died at H.e points where
~ curyes end. /I. certain degree of muscular inco-ordination
 BERl-}iJ<:Rl

is usunHy ObS,Cfqd a~ .a ,""l'quel to Jllek of \'itaruill H ~ thts i~

g'l'llt'rally aserilwd to a fUlwtlunal llHpnirm('Ht or Ilt'r\';' {dh.
llt~('n('ratj\'t "'YlllptOtH~ ('haradt'ristk or htrilwri nplwar
later. aud ('uth ~(Jon follows.
I:seri-Beri. If [}/!!{'Vll ... or uthe!" hird . . Hn' kept fH~.n (ltd
llleking \itnmin B. !'-t'\"{'Tt' ~ymptotlJs of adah polyru!lIritis.
\vhi('h i:. U(,(,t'ptl'd a .. the t'qui\'IlI{'nt of hmuull h{~r'htri.
d('\ dop if! from fift~t.'11 to tWt'HtyHvt' dnys. Thi:-. i:-. HM!o{)('int(',l
wit h loss of weight. hut ill birds tJJ(" Hernm~ ,yrnptnm"
pr('(iomillatt'. awl nppt'Hf nllwh 'iOOHt'T thun 11}('), do ill tlu'
mammal. Till' l{'g~ u,HlI winJ.,.'S h{'('oIHc pnraIYM'i.i, n.ml ('Oln w

pick hdph'!oi~I\(;Sl, and dt"u1 It i(tHo\\" 111 from t w(ntyfollf to

l()rtY-<'ight hours. The udmiurstratiotl of vittuuiu H in
!oIuHicient amount to birds in 11 eomplttt-Iy Jlt'Jph'S\;. {'owlition
is u!-.ualIyfoUowcd hy !'otrikinJlly rapid cur(~s; pi,g('ulIs han'
hel'n knowll to walk and fly within a {(w lU')lm. ur ('ven I(~s.
of rcc'{.'iving the dtumitl. A met.hod fN"tlU('utly ustd for
inducing poI)'Jleuritis in bird~ is to feed them OH poHs}wd
tlC'(", but Simonuet hl:l~ pointed out that many of tht: rt'sults
onservl'd under this regime may not b(' due to lack of vitamin
B only, sirwe polishC'd rice is dclidt'nt in proteins. fat!
"itamins A and C, and salts in additioH t4) Jut'king B entirely.
r!)iHg a diet of the fol1owing (ompol:>itiou : -._

Mcat re'idue (extracted hy builinl{ alN,hol

and ether) 11 part '.
Salt mi'--turc I
('cUllio," 5
Agar-agar . ... 5
Pell-nut oil (heated at ):JO - fbr tin,",' h('lIrs) 5
Butter (clarified) . ](I
Potato ,ta~h 60

giveh to pigeons in the form of pellets. SimonI..t has shown

that the birds do not lose weight, but avian polyneuritis
dewlaps in about thirty days. and is usuaUy fatal ill four
days ill the absence of treatment. It is stated that th.
polyneuritis developed on this deficient diet i. not ",,-com-
230 THE rJTAJIINS
pHui('d hy paralytic' or at rophi(' symptoJ))t;, and although
there i!-; YOmitiHg. inahility to swallow. diarrh(l'u, ('xrrt"tioll
of hih ~alt~. alld an iu('r('SH;('O output of uri{' add. th<.'r(' is no
marked intcrkr{'II('(' nith till' 1Hf('haHj~m of (Hg{'stioH.
SinlOJ~t't (()Il~id(."r.- thnt many of the symptoms us'tally
iuelucf(d in polynt'uriti!-. arc really du(; to inanition and
muJtipJe ddi(it'n('ie.l;. By t}u" addition to tht, abo\'e di(,t of
()'5 .cram of elfiNl yeast pt'r dinfl pigeon!'; ('rm be m.aintained
ill ('x('piJent il('alth for many months.
I '\"nt('r-~oluhJ(' "itamin B Q('C'urs jll a. wide varidy of
animal Rnd \'(~gd.ahk substances, heing pf{'~ent in vuriahl(
alHOtlilb. in praetiralty all natural footistuffj:" Its prinripal
sourc('s un.> plant seeds, bird\; Cg'f..,ts, uIHl YCRst. The In.st
nwntiOht,ci S\lb~tnH(,(' provides a vcry ('onVt'uient souref'. of
the' ,"itamil\\ since it is not Ilffc{'ted hy autolysis of the yeast
('el1s (('.~., in tilf' preparation of ('rtain ('omrll(.'reial yeast
extrn{'h.), nor by t~xtTJletion or drying. A convenient method
(If ('ol)C'el)trating tht, "itamin fTom yeast has been des('ribed
by Oshorn(' and \Vakeman: :!\Ioist washed yeast is extracted
OJU'f' with nhout 2 parts of boiling 001 per ceut. aectie add,
and again with about half this amount. It is then centrifuged
and {'\upora.ted to 0" part. This is made up to 52 per cent.
hy wt'ight of alcohol, and the precipitate is separated and
washed. The filtrate and washings arc evaporated to ahout
6 }:Ier (('nt. of the volume of the original concentrated extract,
and til(' ak'O}lOJ ('oll{'cutration is brought up to 79 per cent.
hy weight. Th. preoipitate is washed and twice reprecipi-
tated by adding al""hol to its solution in a small volume of
water. It is finally digested under alrohol. and on drying
forms a friahle mass which coITel\ponds to 1),2 per C("nt. of
the dry weight of the original yeast. It possesses all the
potency of the original yeast. Vitamin B is more resistant
to heat thll1l the other \"itamins. and is also least susceptible
to inactivation by oxidation processes, with the possible
ex('Cption of the recently discovered ,itamin E. Despite
numerous atteml?ts to jsolate vitamin D in amounts sufficient
 J'17'A2IflN R rON('/i:N7'JiA7'ES lilt

to permit of the ddermiuation of ih dt('mi('allHttuN\ tt'linhk

information is .... til1 hwkiug. Sum('rf)u~ suhstuf}('('*lo rHlrp()rhl1~
it, iw yit.amill B han' h(,(,lllso1nh:d. Il.I1d hA\'(' ("Til lX'('1\ ,(C1H'h
names. But tht'tl' is 110 reason to ~lIPpUS(\ thut any hf thf's{'
pr<'parat.iolls frally rt'pres('nts th( \ italllttl. sin~(' ot.) twinJ,l
furth,T puritiNf their adi\,ity is ~"Nl('raHy Ju~t. ~c\'(;"'hdt's"
the frt'qUl'Jwy with whid} purilH' and pyrimidim' {'ollllxHmd~
art' assO('iakd with tlw~(' n('ttn' prt'parptiOlI-' i ... of MUlW
intt'f(~!'.t. morf' ('sIWC'iuHy in "i('w of Fiudlay'~ S,UIlJ,,'l'stiun
that the phY!o.iologieal Lasi!-i of HI(' adi<HI of dttlHttu II i~
{'orrp!a1(d with JHwl("ur 1lwtahoJisllJ. Thus}w fluds that tiw
('hii.f (hangt in the edl~ of the ('('Htral llCrYHllS 'y!<.tNIl of
hird~ sukring from polYIl('urit is i .. It d,~uPJ)('arn.ll('(> of till'
~issl grnllules, whirh urt' {ompos(d ofnm'!{nprottin. Dt"'ipitl'
th('!.;{, indif'atiolls, lIO pur{' pyrirnidiuc or purilw (ompouud.
or JJJ flU.,t any othf'T PUN' -suhstanel', has yt't h('eJJ fouuci
wh~eh POSS{'ss('s Hl(' Cl1rat.iY(' prop{>rti('s of y('a~t ('xtrat'i or
otlwr sources of vitamin B. P(,tets ha!-. shnwlI that HltroH'..
acid do('!) not (h'stroy Hl{' antju('uritic' profX'rtk~ of Y('u~t
('xtrBct, whi('h \l'ould !'Jee1H to C'xeiude yHamin H from th,
group of primary amines. HeN'nt attempts to ohtain ('011-
('cntrated preparations of vitamin H earried out hy 1.(\'t'11(,
and his co-workers hav(' resfdtcd in considc'l;uhle adnth('t'!>,.
Thus, sturting from a. COllccnt ratfd prqmrat ion ohtai1J('d
a(,cording to Osborne Hud Vl'akem3n's nwlhod, th('y ('arry
out a furth('r series of pr(~('ipitations with bu'S;c' I('ad ac:d.at(:
followed by barium hydroxide, and eventually ohtain an
fl."Jh-free substance containing 4-0 per ('ent. of nitrogen,
potent in doses of 1'23 mg. per diem, ha\'ing an elementary
eompositioJl approximating to that of a ('arhohydrate, and
yieldiQ~ reducing substances on hydrolysis, By adsorption
ot( silica, preparations are obtained containing 15 per eent.
of nitrogen and potent in daily doses of {H mg, Kiunersley
Bud Peters b,we also recently describt-d the preparation of
a yeast concentrate whleh cures and proteets pigeons (I'oly-
neuritis) in d<>Se$ of {HIM mg, per dirm.
 2:12 THE VITAMINS
Vitamin C, the Water-soluble Anti...scorbutic Vitamin
TIl(' (xperimental animals whi<:h aTe HOW most bJ('nt~rally
{~mploy('d in 'ltudies of ,-itamiu (_' arc hmin('a~pi~J'S. It is an
intcTl'sting fact that the~(' animahi, together with man and
ttl(' UPfS and monkt'ys. arc yery sus('eptihle to S('lln~. whil ... t
rat~. "~('t" and rahhits aft' "t'ry nnt(')\ HlOT(' resi . dallt. with~

.... ,_.=-~.
..... -:'-!"~":--:'.:-:":-::":-:lT:;-;",~"~";'-=":-:,:,,:-:,:,,,:-::,,:;-;,,c---:,,O-:5T~
f'lG. 23.-W('ight cbarts of ~uinea.pig:s sh(Jwing tht' anti'fI('urbutk vaiuf' of
\)l'al~ juic(' a.nd iresh ('6bbagt-lea.v('iI. (Afte- M. R. ('. RE"port. 1924,)

stnnding deprin.tion of the yitnmin for prolonged periods

with relatively little sign of disturhed health. If gu\itfa-pigs
are kept on a diet of oats. bran, and a limited supply (about
40 c.c. daily) of milk autoclaved at 120 for one hour, they
develop the typical effects of scurvy. The autoclaved milk
provides the necessary supply of vitamin A. Normal growth
takes place for a period of from fifteen to twenty days;
 SITHrr
s(''Orbuti(' symptoms then appear. l()s~ of wd~ht follow,", and
1('ath results in about four to tin' Wt'('k.ii: from the Ntmmt'lH'('-
ment of the period of dC'fit'if'llt dit'lill~. Typl('al r('~ult~ of
f'xJ>erimenbr earrit'd out by (,hi{'k and Huult' (m Muiut'lI~pi~
Te<t-iving n s('orhutie di(,t. with flnd without. th(' add.;cioll qf
autocJ.:wed milk. art' .,hOWll in Fig. 2:l ('urn' 1. ~'hflrt'"
th<. wdJ!ht los!'> on a (lit,t of oaho. hran, and watt''!' : ('Uf"" 1 L
!oihuw. . a similar t'xI)(_'rill1('lIt jll whidl the did (milHIII(d (tllto
eiuv(,d milk; whib"t curv(' Ill. shows th(' ('ur(' dfr-ttld hy
adding orallf.,J'(' juic(' nud uutodlt\'ed milk to the did of (lob-.
hran, and wah'T. In ('UTV(' 1\', 5 ('.(', of rr('~h orall.t!t' jui('t.
wer(' add('(J daily to the did from th('(X1Huu(>tI('('tIICHt (If tIl('
t'x}){-'ritncnt. ('urn'!oo Y. alld Yl. art' lIormal grow\ h ('\If\T''i
obtaiuc'd Oil di<'b. of oats, bran, nrld ('llhhnf.,J'(' kll\';'., nwl (If
oats, hrah~ autoeian:d milk. and (',t', of fre..,h Ufallj.!t' juke
daily. .-\n alternative seorbutie diet Pf'{JIKlSNI h~' S~wrmnn
and'tls asso('iat{'!o, is (omI)(}..~('d as follows: (lab, 5~1 pl'r ('('tit, j
skimmed milk powd{'r heakd in oI)(>{J tray~ for two hour!. ut
110\) to destroy vitamin t. 30 per ('("nt.; hutter fut rr(shly
[lr-cparcd, 10 per cent,; sodium ('hloridt'. 1 per ('('lIt, Tlti:'oo
diet ha.s the advantage of avoidjng possihle lra!'('\') of \'ihunin
C which might be prescnt in auto<'la"cd !flilk.
Vitamin l' i~ found in nature in fresh fruib and \'('g"t"t.a"lc~
in which active metabolic proc-esses arc still Jiroceeding,
Thus in resting tissues such as st'eds it is usually abscllt, but
reappears when the seed begins to germinate. The al""',,ec
of vitamin C from drit'd seeds is 111 striking (~OJltrast to tht
oceulTence of vitamin B in this SQuree. Of fresh fruit, th~
orange and lemon take first place as anti-scorhuti<- agents,
whilst the juice of fresh liml'S is distinctly inferior. Fn."b
greell lea:..es and the tomato are rich SOUl'<~ of the \'itamin.
Driea vegetables are poor in the vitamin; meat and milk
are variable, but generally poor. Vitamin C i. highly
su$Ceptible to destruction by oxidation, more so than
vitamin A. It does not thM"efore sUr\'ive the drying (Jf
fruits or vegetables at all well. Zilva has shown that ded-
234 THE VITAMINS
trai('d lemoll jlli('(' of Pfl noR (.'an 0(' iloilt'd in an atmospher('
of ('arhon dioxide for two hours without los~ of poterl('Y.
wh~>r('a.'i ,..,imilaT tr('utlHt'nt fot OIl{' hour in II ('urrt'llt of air
destroys moT(' th:m 75 Jwr "rnt. of the vitamin. In more
~l('id seJuti()ns of P'I 22 to 2'4 the loss in the prl'sem'c of air
j<; I111l~'h Ie~s. hut on tIlt' otlu'r hund ill alkaline solutions of
PH J:t tht ,-itnmiu lo. . e~ NU 1H:'r ('('ut. of its lwti\'ity in thirt~

"J
300

Fw. 24.~-Etl{'d of {"xpos,lt(> t(l air on d{'(.'it.rat-ro It"mon jui~t> kept at PII 12.
Ttl(' anti'!II'nrbutil' propt>rties of the portion exposed to air hl\v{' ~11 ICIt.
whilst tht" jUil'f' lu'pt out of {'-oulad with ail' 18 l!-tjil strongly anti
~'ilrrhnI(. {~.-tl~r ;.1:1'n. ,-

minutes at room temperature, and is completely de5Ji.royed

at room temperature in twenty~four hours at this reaction.
The practice of boiling vegetables in the presence of a little
sodium carbonate or bicarbonate would ,tberefol"f seem to
be strongly contra-indicated. In the absence of air or
oXYg<'n no appredabJe loss of vitamin C occurs at ordinary
temperatures even in alkaline solutions. At higher tempera-
tures a slow beat inactivation takes place even in the absence
of oxygen. :Fig. 24 gives the weight curves of two of ..,t.
 .rI'l'A;!lIN (' ('O,Y('F),VTRATf:$ 23,;
,Q'uinca-pisr.;. hoth of whif'h Weft' rr-f(,j\-jug 11 s(,hrbuti~' did.
:Frorn thf' points rnarkl'd h\' thr dothd lin('~ 011(' Slt TN'('i\'('d
daily doses of 7 (',c. of d'eeitrat('d lemmt jui(O(' whi('h hlilf
h('~n standing at rooll) t('lHp(tattlf(' fur tw(nty-fouT hours
at PI{ 12 and fr{'('ly cxpo!><t~d to th(' nir. It wili Iw !\,,'\.'1\ thRt
this pr('[mration had 1)/) anti-''"('orhutic prop~rth's. ~jl~('t' flit'
animab rapidly lo~t wt'ight and tliC'd of sNlt\)' nbOllt til('

1 litn' It'JI)on juktl ('outaiuiug no j,!raJu,. ~nli!l WaUI'f.

Fn'('ipillttioll will!
huH'.
I

I
~() gram~ t'itrit and nflll'!' )0 gram~ a(!ti\'~\ fNljtitW.
.~lI~jtl ... , prnttin,
(h~wtin;).
+,t{;.
i
Fi'rJlWflfali"ll.

7 -,1 eraInS Hugar

(inlwti\'e).

(}8 ~ranl prpc'ipitaftl 1,7 gralUfoI u('.tiVf' f(,,,idtw.

(in~H'tivc).
Pn)eiJlitatiH~l with ImRil' }I':1(1
fll'('!tait,.

14 gnu.h~ nunpr.e(ipjtah~l
residue
(inacti,,e).

thirtieth day. On the other hand, in the ",,"ond set (If

animals the administratiDn of 7 ('.f', of similar dt>{_jtratfd
juice which had been fttanuing at room tNnpt"l'atuf(' under
anaerohic conditions, hut at the same JIM ali the- 8C-TObi('
sample, effected a rapid cure of scurvy, and the weight of
the animal. rose almOlit at onee. It will be ",-en from these
"",ults that vitamin C is gradually destroyed by risc of
temperature in the PftSence of air at a rate whi~h increast's
 THE YlTAJfJNS
with dilllillbhi!Jg hydl'ogrll ion ('oHeentratioJ). whilst in the
ahwlHT of nil' ttl(' ruh' of imt(tivation J!'. gT{'atly de(_rtased.
In n :-,t'ri('s of investigations whidl an' iwing earried out at
tlH.' Li~t('r Inst1tut<~ in London hy Zih-a, eOIll,id<.>rabk in1brma~
tioll i;". hdllg f.,'ltiIlt'd it! relntion to th(' dl('mieuJ nature of
,"jtnmin 1'. ('onc(ntrnt(d preparations of the \'italliln h~n'('
h('('J) ohtained from lemon juic(' by fC'moving the eitri(' und
ulhn a('j(b hy pr('C'ipitation. (Uld til{' sugar by fernwutation.
without P('Tt'('ptihly afft'cting' the pottfi(Y of the jui('C'. Such
('U{H'('lltratcd jui('(' ('ontalns only olH-thirti(th part of the
total ).ulids of the' ori~infll juice. and 011 ('onc(4Iltrating
the jui('(' still further and prt'('ipitating it with akoItol the
\'itamiu is found in the sup{>rnatant liquid. from whi('h it
IlIUY he prt'('ipitah'd hy the addition of hasic leaa a{'dute.
On It'moying th(> lead' from this precipitate a ve.ry }l()t~'Ht
preparation of th(' \.'itarnin is obtained. This proecss of
('oHc('ntrntioH call be summarised as showtl 011 p. 285.
A fur1:hcr con{'t~ntratioH of the vitaJuin mav be crreet('d b,
dis~oh'ing the basic lead ace-tail' prNjpitate: eontaining th~
lIt'tin' slIhstan('c from 1 litr{' of lemon juic(", in the mjnimal
amount of dilute accti(' acid and adding lleutrallead acetate.
The latter reagent does not precipitate t.he vitamin, but
rt'(noY{,s consid('rable quantities of non.acti\'e matter. The
PUI'('st concentrated prqJUrations of vitamin C obtained in
this way ('ontain n~ry little nitrogen, some 1 to 2 mg. per
('(nt.. somt' of which is in the forIn of amide and aInhlO
nitrogen. The amide nitrogen is not related to the anti-
scorbutic acti~ity. Phosphorus is present in traces oIlly.
The active preparations reduce anunoniacal sih'er nitrate
and potassium permanganate, but the reducing properties
are lost on oxidation Ilt a much slower rate than ,+he anti-
scorbutic properties. Swed.. turnip juice and cabbage "leaf
juice may I)e conct'utrated by a t ..."tment similar to that
outlined above, and the vitamin obtained from these souret's
shows physico-chemical properties similar to those of the
preparations ~ lemon. From a study of the dialysis of
 rITAJllN ])
a('tiH' ('oll('(-'ntratt'd jui('l'~ Zil"n ('olw)ude'" t1mt tll!' a"',Ye'
suhstauce appl'oxinlnft' ... In mnJe('ular magllitlHk ttl tiw
rlim('nsion."i of a h('xo~(' nwh(,IIJc. hut j ... tlh.tiltd frum tIll'
r(~d~l('inJ.! unrl nitro,l.,.'tnou!o. slIhstall('(" prt'~{'nt in the ndiy{'
prepRrations. _.

Vitamin D. the Anti-rachitic Vitamin

This name was first gin'lI hy Funk twd J)flhiu to tIll'
substan('(' nlrendy )Hrntiont'd, whid} ""n, ",upPoM,d to hl'
fl('('('ssary for thl' growth of yeast, hut it i ... now dt'finitefy
IJsf'd to indi('Jlt-(' the anti.rachitic vitamiu prf':'ot'lit ill ('f)d~
liver oil and in ntiwT natural animal fats. UI\({ sllti,flldnrilv
dirfef<'lltiated from th(' wowth-pronJoting ,'itallliu .-\. nl~'
prOf'ient in.these fats. Yitnmin J) ac(omplwits "itamin A ill
the Bon-saponifiable residue ohtainf'd in fradionnting ('ocf
Ihct oil, and is found in the .'.,am~ fiual fraction of (t1mpll'x
unsaturated alc'ohols as vitamin A aftcr \'a('\1um .list illntion
of the'? actin'~ rcsidu{'s. It is tru(' that Hi(' #!n)wthpr(lnlOtjn~
and anti~xerophtha)mje at"tion of nAtural oiJs and fats on thc'
one hand. and their anti~racbiti(' adion Oil the oth{'r hllUd,
run closely parallel. N",'crthcless the more d(,tailt-ll "ml
more carefully ("ontrollec] the' inv('stigation'3 of th(,S4' prop(r
ties bc('ome, the more ar(" diff('f(-'nc('s }wtw('('u th<' tW(J
properties apparent. rntil nory TN'{'nt yC'ars, "'.1('1) tlw
possibility of these differences became ",,ognised. it had not
been found possible to draw a simple and tlir('ct rt'lntiHu
hetween rick('ts and the fat~s(}lu\)le vitamin A as SH{h. a~ '"
done in the CAS(>S of tht' vitamins Hand (' and the spN'ific
dist'ast's of heri-ber; and !""""y respeMh-ely_
Vitamin D is less liable to undergo oxidative d(~trtl("'liotl
when hettted in the presence of air than is dumi" A. Thus
cod-iiver oil loses ita curative action on xerophthalmia
induced by lack of fat-soluble vitamin after being subjected
to the action of oxygen at 101) for period. ,-arying from
twelve to twenty..,ight hours. On the other hanrl, the anti-
rachitic action of the oil i. still apparent after this treatment.
 TilE VITAMJSS
A_gllin, 0-1 of spinach l('an's b suffkif'Ht to r{'stot'(' tilt'
g'TUHl
po,\"ef of' _growth to unimal~ kept on a udic'ic'nt did. whf'rt'us
a(j grams wlll not ('UTe ri(.'kds. Furtlwf. ti1(' allti-fUChHi('
lWtiOIl of huHn is Illtlrh less than that of eod-Jiypf oil,
where!!'" tll(> ('uratiYe f1C'tioli of HI(' form('r t~lt (Ill x('ro'l'h~
tJulimia i~ I-,omew}wt g'N'atcr t hnn thM of the latter. I~u('('
ha!. N'c'('ntiy found that. when a {'ow f('('('il'ing a diet ddicicnt
ill t[tt-~olHhl(' "itllmins i ... ('x posed to summc'r sunlight ill the
open nlr. the' growth-promoting prop{rtit'.., of the milk which
she ~'j{'lds :IT(' lIot ~ig}}itkllntly afftO(,ted; on the otht'r hand.
there is II distinct iueTt'aSf' ill tht anti~ra('hiti(' nl}ue of the
milk Tht fuet that thf' Ilt'W ars{'nic ehloride test for vitamin
A Kpl'eifica1ly distinguishf"s it from vitamin D has aJready
hc'cn weutiOJIl'(1. Such is the type of e\-iden('c on wbj{'h
yitamiu :\ is difft-r{'ntiat('(l fTom vitamin D.
In the' ('ontlition known as rick('h; the' l'lini('ai app('aran('('t;
YSTY with th{' animal in which the diseas{' is <{('v{'loped.' inlt
ill(_' most (~hara(t('risti{' ~ymptom is Ii rlisturhan('c of ('aJeiuIH
aud phosphate' m('taholislll whieh r('~ulb. in a ddicif'nt
d('po:sition of C'ulcium salts, C'hiefly ('al(~ium phosphatC', so
that th(_> lJOH{,S do not hecome sufficiently rigid to support
t heir normal stresses. and defonnation results, The histo~
logi(Oal examination r<'Ycals an imperfect caldfication of the
lIlt'mhranous and ('artilaginous matrix of the boue. The
oskomalaeia of adults is essentiaHy similar to rickets in the
l1r<lwing animal. Experimentally the condition of rickets
is readily produced in puppies and in rats. In the former
animals the disease is produced by cutting down the supply
of fat-soluble "itamin in a diet otherwise adequate. Growth
pTOCf't'ds more or less normally. a feature of rickets in the
humsn. hut ossification becomes defective. In the ra! it
has been supposed that the rachitic condition could "not
generaUy be induced by cutting down the fat-soluble vitamin
alolle; the calcium and phosphate intake had also to he
reduced or thrown out of balance hefore obvious r""hitie
conditions develpped, a.nd these were generally associated
 RICKETS
with l\ I()'\~ of weight. ~fany other animals ~h()w ('on~id('fnhk
variations in resp{'(" fir tht~ir liahility to d('\'dop rkk('t!'o UlHit'r
deficient dieting. But Wi' haw' S('('lJ that ... iruHnf nlriutioH!'<
in (!iff(,Tcnt animal sped{'~ ar(' t'n~o\lnh'r('d in t la' re(o~l\i!'o(d
drHcicn('y discas('s prodH('{'(1 h~' hwk of vi1HHlins -,\1111 l'. n
~for<> ft'('ently it has h('('fI f01Wd that with strid ('ontrol (If
til(> ditts Bnd aU('lttion to the ng(' of til{' animal ...dl,t't('(l for
th" ('xIK'rinlt'lJt it is po~~jh)t to indm'(' typi('nJ rir'keh ill
youltg rats. M('CoHutn und hi~ ('o~worh'''''' hll\'(' rt'('('t1t1y
J.!i\'t"ll l-fonlf' ath'ntiol1 to this qu('stion. nnd hun' ('"oln'li tht'
foJlowinU' two djtts. on whi{'h young rats \\(jghing from ,lot)
10 45 J..Yfams tit-velnp S('\"('f{' ri('k{'t~ aHd ar(~ rt'udy for liM'
aftt'r ha\jng }x>('tl eonnnf>ct to th('~c dids for ahout tw('nt)-
JiYl~ days . The diet.s are:--

. lJirt 4025.

Wheat f!erm .
UranHI
500
/Jil" J.H:,!'H.

\\'11('" t hq'rm
';"<11/H1.
,I'iO
Salt mixture 31 ,),1.) Salt mixtuf(' :~~ ~':l
Calcium earhonaf( I50 Caleium (~arhonat(' 15
C'.datin 1 (HK' (,,,,,,in (purifi('d) 20n
Ef!g albumin l(HK) Gelatin .~.(l

Wheat. f!lut.n 12()() \\'I",at glu\('n ,,0

Agar-agar 2m. Agar-agar 2'()
Dextrin W3J Dext.rin .12'2
Butter rat ~)'OO Hutt,'r r"t 3'0

In both til(> aho\'(' die-ts the' wheat gf'rm may he n'phu'('d

hv the same amount of yeast.
~ The salt mixtures u~d in the aho\'(' dids are ('otllJ)()M:d
as follows :._
Salt .\Ji.rturr 37. Sail .\li:flurr as.
CaCO. '. 1'50 IdellticaJ with salt mixtlJl'<~
Kef . 1'00 87, except that no KH.I'O,
.saCl 1'00 is added. The ca.'!Cin in tl.e
NaHCO,. 040 diet furnish.s an adequate
)[gO . 0'20 amount of phosphorus.
FeSO .. 7H.0 020
KH'po . 085
 7'/JE rITAMIA'S
It j ..., HOW t:!'('llt'm"~' admitt('(} that it is po.'i~iblt_. so toaojust
difh. thnt the d('('fdill~ fador at.. to whdher or not any
animo I (h\"dops ri('kt,h, 1)t'C'om('s th( pr('!';:('1}('{' or nhs(,llC'c
from tiw di('t of Ow nnti-ffH'hiti(' yitamin of whi('h ('od-Liypr
oil is tJt most potc'nt SOllT(P. This aSSllm('s that Iln Rctf'qnat('
and hnlac){'('d .'1upply of the inorganic dements of the houe is
fort}worllin~. this f(>quirtment being paraHe) to the necessity
for nn nd('quut{' supply of the speC'ific amino 1wids essential
fOf normal ,LITo\vth. apa.rt fron) the q\l($tion of the presence
or ntlwrwist' of ,L,"fJ"owt h-promoting yitahlins. It is th('fcforc
ohyiolJR thut a ra(hitie ('ondition wi)} hr mort' speedily
produ('('d hy simultaJl{'ous dcprinltion of hoth thi' anti~
rachitic \-itHOIin and supplies of calcimu and phosphorus,
mOf(' ('sJ){'(,ially if tht' ddkil'ncy hegins at an earJ~' age. -In
11l(' Hqmrt of the ~kdi{'al HeS('ar{'h Coun{'i[ of Great Britain
(l92~) the points of l1en('ral agreement regarding the relative
importamoc of a deficif"ncy of caleium, phosphatt~. and anti-
rfl('hitl(, vitnmin in th( causation of n('kf'ts tlrt' given as
(c.)ilowl'o ;---
(1) An anti-ra('hiti(' yitamin in the diet (orf('<.is itnprop(,r
halalw(' in th(' {"atrium and. phosphorus intakt,. and tlw greater
t hi' disproportion or drfcct in these elements. the more
important is the role of thf' vitamin in the prevention of
rickets.
(2) E\'Otl when the {'alcium and pho"phorus balance is
good and the supply of eaeh adequate, the absen"" of anti-
rachitic vitamin from the dirt will result in the production
01' imperfectly calcified bone.
The Effect 01 Light in Preventing Ricl<ets.-Mllch recent
work in relation to rickets has centred round the curative
influence of direct sunlight on the disea.o;e. These IleneficiaI
results, which have been sbnwn to be due to the ultra-violet
light radiations of about 800 Pi" or less in wavelength, and
which are cut off by ordinary glass, haye giYen rise to an
interesting d('ve\opment in vitamin chemistry. Hess, Unger,
at)d Pappenhej.lleT ha \'e shown that six young rats given a
 U(Nlr AST) R{('KJJ:7'S 241

diet of whit( flour (n,) per ((Ilt.), ('nlr-ium ludah (2:'H7 prr
('<'fit. 1. ~odillnl
f'hlori<it' (~ I}('r ('f'u1. J, f('rri(' <'itrah' (H'1 pt:'t
('O)t.). which lIormaU," prodw't,s ri('k(t'i in tilt' lahorntory.
did ill fnet d('\Tiop the dis(,R!'o{' ill h\'('l1ty~two to thirhfour
day~ wlH'1I kq)t in ('olllpl{'h' durkll{,~!o, .. Srn'lI l'oimilai,rats,
takjll~ til(> SHIH!' diet, Wrn- k('pt tl1ld(~r th(' stUll(' (oHditions.
('x{'('pt tlint for It period of fifteen to twenty mifluks {,ad,
dl:l~' t hey wert' ('xpo"ed to sunlight ill til(' opc."n nir. TIl('~('
nIl failed to tln'plop M('kch in the snml' ~ri()d RS tht"ir ft'llow'l
kept ill tilt' dnrk. TIlt" pre\'('llti\,(, twtlon ~hnwll hy rlirt(t.
~lIlllight was oh1ailll'd {'qualfy W{,jj fly suhj('('ting th, ruts to
th{, ultra-\';old radiations of a qUHrtz mCITury nlpouT hlll!f)
or of a ('arhon are lamp. Th(' !-.unlight lost its ('uratin' ad ion
Whql it was first passed through 1l flint glass window h(foT('
reaching t~e rats. It has also hl'en ohs(rv(d t hut ruts k<,pt
(Ill a ruehitk diet ('ontining t wi('(' the optimal aruouul of
C'alcium. hut ddkient in phosphorus and fat~soluhl(' yitnrnin
D, do not d('vclop rickets if they an' (xpost>d to sunlight in
ttl(' open air. Thdr h01U'S are thill, hut firm and compl(hly
oS!o,ified. It is till'refor(' apparent, as we might exped. that
uJtra-"iold irradiatiun wm not t'Htirt>ly ('ompl>u.5Iltf f_)r 11
dietetic' phosphorus dcti<'i("nty, hut it do('s exert a very
favourahle action on the utilisation of the ~uppli("S uf
phosphorus which are ayailable. Similar hcmficial result.
on growth are obtained when rats kept on a vitamin A
defident diet are irradiated. In couJ's{' of time. howe\"er~
growth ceases, presuma.bly on exhaustion of the stores of
the gro"th-promoting vitamin in the anima!". own body,
and the rat. decline in weight and die, despite continued
ultra-,~olet irradiation. Since rats kept Oll all adeqUAte
diet, but :in absolute darkness, develop perfect bones, it i.
appai-ent that the anti-rachitic ,;tamill is prepotent in
preventing rickets. and is capable of doing !IO in the entire
absence of light. At the same time it is ..Iso apparent that
the ulm.-y;olet region of solar and other radiations is of
great importance in ~ifying partial, but not absolute, lack
.... ,6
242 THE VITAMINS
of the di('{('tit' ('OIIl}>OIH'llts n{'('essar~' for ossifirution. At
ttw present tinw thc' aeN'pted dew of tht at"t1oB of ultra ..
violet irradiation 1n this r('~p('ct is that it mohiiis('s th{'
reN{'Tn"s of th" Hntj~rachitje vitamin. In t}l(' 1ight of rN'{'nt
work" whi('h wc' shaH HOW db(,tlss, llltra-vioid irrarlja~iol1
may nlso gjn' rl...f' to additional supplk... of yjt:llnin ]) from
the hodily stores of ('hahst.erol.

Anti-Rachitic Properties induced in Non-acti.-e

Substances by Ultra-Violet lrradiation
A rt~mnrkahlt> and intcr('~ting ('xtcllsion of t h('::;(' ohs('rnl-
tiom~ 011 the ('uratiq, adion of ultrll-"jold light on rkktts.
whkh we have just diseusst'd. was made duriug H)24 by
St('c'nhoek and his co-workers, when the\' demonstrated
that it is possihl(> to indu{'{_~ anti~ra('hjti(' pr~)pertL's in food ..
stuffs ",hidl arc initially quitf' iuudin> in this rf'sp('ct hy
subje('ting them to irrudiation with ultra-yjolet light. Thi!o.
original observation of S{{'cnhO('k has been ('onnrn1l'd and
f'xtendrd hy many other workf'rs~ The irradiated foodstuff:-.
a('quire not only anti-rachitic properties, but ah;o (:>xhiIJit
gto\o\th-promoting iHtj"ity. \Ye shaH return to a discussion
of this duality of funotion later. It is 1IOW apparent that
whilst it is possjbJe to ('onfcr anti-rachitic prop{'rti('s on mnny
natural foodstuffs and vegetable oils which are lacking or
ddicit'f}t in ,-itamin D, such as {.'ommereial casein, dried Ini1k~
meals, flour. lanoline, oliye oil. bean oil, linseed oil, cotton-
SCt>d oil, lettuce. spinach. etc" no such results are obtained
when the following carefully purified substances are irradi-
ated: glycerol, fatty acids, hardened ,egetable oils, fats,
mineral oils. terpenes, proteins, carbohydrates. salts, water.
ether, hydroquil!one. phloroglucinol, paraffin hydrocarbons,
ete. It is therefore apparent that it is some constituent of
the natural product absent from these pure products which
is responsible for the formation of the anti-raehitic \;tamin
under the intluence of ultra-\;olet light.
In a vegetable oil which has acquired anti-rachitic
 lRRAlJlA1'lW ('/I0LESTEROJ. 24:1
propt'rtit,,,, lilt, uctin' ~1t1~tatH't is fOlllnl ill H,sudntioJ) \\'ith
Uw lIon,~:lpolJiJl.ahk frndiolJ. ill whi('h it mn,' J}(. ~HlHltI
trakd ill tfu> lWlltll('r :drnuly tic\{'rih('d 11-' h/lvill~ lJ('('u
utili:-.cd in HUt'mph to i~oJnk \'itamili A. The Hwt hH~
(_IIH.'rgt'ti that tht' {'apa('ity of any lint ural produd to l~..quir{
sudl allti~\n('hiti( propertit,s i:-. ("oIT(lnkd with ih. ~h-rol
('Ollt('nt. more parti('uluriy with (holtstno} HIUl phytosttrnl.
Both of t1w~(' ~terol:-'l 011 hdng j!o.oluh>d from naturul oils awl
i~lts or frorn animal Of plaut ti:-tsue ... and on ht,jng purilitd,
an' th('JUsf'ln'~ {)Pyoid of OilY anti-rarhitie uction aud ("ontuill
no ,itRmin D. Hilt hoth lif(' t{'ltdilr ud;nltcd Ofl heing
!-'ubjeded to ultm\'iold irradiatioll. The pur('!<.:t ~(J('('iflI('IIS
of (hoi<'sterhI yet obtailH'd exhibit this prol)4:rty. Thu\
lJrunuliond Hlld hi.., eo-work(r!o. ha n' suhj('(t'd (holt'\\(rol
to thret s\f("(.'{'ssin' trt"atmcllts with hoiling ai('oholic' pota!-oh
to remon' ail tra(~('s of fatlot. and IUl.\'{' r('{'rystal1ist'd tlw
(.'hol~tt'rol twentY-Mnn tinH.'~ frorn akohol. dlwr. und h:.rht
petroleum. It then had a JIIdtiug point of 1 4}P\" , {ulll'urr.),
Hud spel'irnens have h-cn obtaiJH'd mdting as high as Ljn~',
Th('~e rif,Tid prccaution~ to eliSllfe purity. and other similar
methods in which th{, dlOkst.erol was finally f(r.fystaJliMd
as the henzoate and al'date, havl' ail rcsulttd in pnparu.tions
which still rttain the power of forminJ,! the anti-raehitie
\itamill. IH!o.istcnc(' Oil the rigid purittcation of choit"stcfO)
is very important. as this substance <.'lings to impuriti{"s with
great tenacity t and the al,tivution results might he dut' to
these impuriti('s. It seems, howev(..'J', that we must aC('('pt
the fact that it is from the cholesterol it-relf that the anti-
rachitic vitamin is formed under the influence of ultra~violet
radiations, and not from an adventitious impurity.
After irradiation ('holestcrol acquires a yellow colour, its
melting point falL. some 20 or 40", and its sJX'Ctral absorption
in the ultra-violet region is altered (Hess and Weillstock).
It appears, however, that a very small amount of the
cholesterol is transformed, for it is possible to obtain an
almost quantitative ,recovery of the original chol.,..terol by
,....,
244 THE rITAM1SS
prc('ipitatioll with dif,(itOflill ufter th(' '>tirllple hn... he('n
irradiatc,1( alld th(' \ Hamill hn~ h('t'h forllled. TIl{' quantity
trJlIl!'SoTHH'd )JIB..,t tiWTcfoTC he If'...... tJlHfl thl' {'rror ill the
digitonin Iftdhod for det('rJllinillg' dlolest('rol. whi('h i!-> \'cry
!-.11lnll'jHd("('d. 1 It ha .. not h(,(,ll found po~sihle to a(ti~atl'
dihyClroehoiestf'To] or dihydrophyto~h'roL nor do th(' absorp~
tiofl Sp(~(ltra of the~(' saturated ('ompounds show any chuJif,.f(
uncr irradiatioll. This dt'monstrates furtlwf the eorrelation
which t'xists, aud \vhieh has a)Tf'ady hcel) nwntiol)(d, hdwN?Jl
the activity of tIl(' fat-soluble A and U \'itu1Hin~ and un-
saturated linkag(>s. Long-('ontinUt'tl irradinti()tl of eho\('stC'TO\
does not dimiHish the 8('tivity whjrh has dCH,joped initially.
pro\'idNi t hat during the> pr()(_'('~s the suhsh\1\('(_' is adequately
prot('('h'd frotH oxidatioll. t'.;! . hy 07.one, whieh i!-. form<.:d in
cOlisidf'rahk quantity ncar un ultra-"jo)et lamp. Owing to
tJ1(~ df'struC'tiw' al'tion of oxygen or ozone on tht> vitamin, it
is. preflrahk that when dlOlest{'rol i:-. being irradiatrd it
should lx' enclosNi in (,"acuuted quart!. vcssels. These
ohservations ail s(_>('m to point to the supposition that
vitamill D is a product relatf'tl in a ('omparativeiy simple way
to tl ... sterols. We l""'e "In'ady seen (1'. :124 rthat the clostJy
reiatf'd vitamin A may also stand in SOllW such similar
..lationship to these products. If these suppositions are
borne out br further ir"'"stiglltions Wi' may at last be able
to assign an indispensable physiological function to these
curious and widely distributed ('ompounds,
\Vhilst irradiated cholesterol does gi.'e a colour reaction
with arseni,' chloride. this is not the typical reaction regarded
by llrununond and his associates as being specific for vitamin
A. One concludes that vitamin A is not formed when choles,
tero! is irradiated. -Nevertheless irradiated choltbtero! does
produce 8 resumption of growth in rats kept 011 a deficient

1 ~tly reported experhn~nt,s seem to indit.'ate that the recovery of

unch~ cholesterol after irradiation is not ao nN.t A quanHtative ()p(! aft
had. been a.t fi.n.t sup-poaed. ~ of 8 to 9 pel' oont. n..ve been reported.
(H_ Weiostoot. ODd SIM......... J. Bioi. C"-..I926, 6'1. 413.)
 rITAJflS j) AS /) OROJrTll 2411
di(~tRry. H{'C'ent work of St('('uh()('k in ,\I1I('ri('1\ and ('{
Drummond in this ('(luntrv ~Hg'f.."t:<.ts thnt dtntniu 1) Olav
it"i('U' hllH' the property ofprmnotillj!' t.,1'THWth ill ltdditiofl t~
jt,,,, w('H~r{"cogllhwd nut jriwhitic ftw(tion:o.. Thu~ it hl\~ hi'cn
showlI tJmt although th( t.,'l'O\l:th U'Sf)(/H,'W .,\howlt h.\~ome
~mlmnh kC'pt on a ddiej{'nt did nmy l}(' rapid "'IWll they nfC
given irrmJiJlt('d cho)c-;ft'roJ. ill other., it i .. irregular or t'lJtird~'
lurking. aud j" not correlated ill n q!U1Utitutin' W21Y with till'
muouut of the supph'Jl)('nt 'whidl is firI. )lurt,(H'f'r. in animals
l\'hif'h do gin' 11 h'l'owth Te:-,pOllM' to jrradjnt~'fl ehol(',itcTO). n
rdHrdtltiofl foJ]O\vt'd hy H c'{'."!<IutjOH of growth may :...t't in IIfkr
11b01lt fOUT l'lf'f'ks. A lnrgt' iller('a"'t' in1)w MliOImt oft ht' .\OIlPpJ,.
nwnt of irradjakd ('hoJ('~t('rol dot's Hot prOdlJ(~f' a r{.sullJptiun
or J.,1Ofowth pfter this ";('('olld ('C'sMttion; (In til(' othf'r hHluJ,
lIorma) J(To'\\"th is reslIlHNl OIl administ.crin,t vitnmiu ;\, It
is Slliig'('steci that in ~u(h ('aM'S th(~ first ('('S~atioH i~ due to
rxhaustion of the bodily stem's of \'itamill D at a tillw wlwn
t h(' unimal has Hot yet (xpendcd All its ~tor('~ of \'italllio A.
and that the tinai ('('ssatloll of _L.rrowth, w}Wt1 1)0 further f<'('(ling
of D produ('ed any crC{,(t, ("orrespouds to Ul<' ('ompit"k
cxhau>ition of A from the nnimal's OWl! tis!o;ut'~. This S(,{'III"i
to imply that vitamill D ('xerts ih. full growthprollioting
influence' only in the prC~l'Hc{, of vitamin A.
Thf"sC obs('rvation~ mak(' it ('vident that many f'onlpli('akd
relationships exist l)('twc('Jl the numerous influ(>Ht'C'S whi'h
determine growth in the young animal. Th(' diseo\'('ry that
vitamin D is formed when ehoiestt'Tol is irradiat{'d has be!'11
utilised by Drummond to control more strictly what ron,ti
tutes a vitamin A frcC' diet, and so to assist in dirrcrc-ntiating
more dearly the rdatiw parts played hy these two vitamins
in regulatIng growth process"s. The new basal rntion used
hy Drummond, and believed to be ad"'luate in all res peets
except in vitamin A, is cnmp,)jjed as fnUnw. : -
Purified casein . 15 parts.
Pure ric<> stan-h 70
Ye...t extract 5
246 THE VITAMINS
Snit lnixtuf(' ~ part~
lA'Dlon jui('{' . . ~) "
Irrndinh'd ('hol('st('r(ll . 2 mg-. daily (ndmini-.;h'rl'd
in liquid paraffin).
Cnnt;~,1 (>xpcrim('nts ,<;;how that the (ompkt( ('xdw.ioll of
fltt docs not disturh the norm.) J)wtuho1i..,m of tht' animal.
ft is pr~sumahIy able to synthesisl' all thC' fat whic'h It
requires from the other ('omponents of th(> dietary. Fat i ...
th('rt'foTr t'xeiudcd ('ntirelY}ls an unrw{'f'ssary potcHtial sourec
uf errOT.

Vitamin E, the Anti-sterilitJ' Vitamin

In th{' ('ourse of investigations on the eff(,(ts ()f vitamin
ddieien<'y 011 experimental animals it has freql}('utiy {)eel\
oh,'R'ncd that. in addition to deft'ctin' t:,'l"owth and other
~.vmptoms whi('h han' air('ady [wen dis('uss('d, the fl'pro-
dUdive powers of the animals were impuired or eycn cntir{'ly
lost. Thus ddiej{mC'y of \'itamin B results in marked atrophy
of tIl(' h'ste's in mnk animals, hut f('males arc. in gt't\("ral~
Illll('h less nffrcted than thr- males. Hats, whi(~h. as we han'
~c('n. nre not very sHs<*ptihle to s{'"un'y when kept on a diet
ht<'king in vitamin (', n{'Yr-rthe1ess produ{'{' a larger number
of litters of young when the anti-scorbutic yitamin. is added
to their diet. More especially has a rorrelation between
reproouetiye power and the fat -soluble vitamins been
established. EYans and his associates haw shown that rats
may be rearoo on a diet somewhat deficient in these ,;Jamins,
thai they may grow normally under these conditions and
1I0t suffer from xerophthalmia. and yet they show a marked
diminution in th(>ir reproducti-n' powers. In the female tillS
is associated with" 'prolongation of the restrous {yele and a
failure of ovulation, as revealed by the vaginal smear test.
Administration of the dry powdered leaves of young alfalfa
plants or of small qUlUltities of butter fat has ""red
these
abnormalities. It is on the whole not surprising that a lack
of vitamins "hich eventu"Uy leads t., such profound dis-
 247

turhan{'('~ in mf'tahoiism as Wt' hUH' St't'll to Iw th(~ ('1\"-(',

should first afft.ct tIl(' rt'pY'mhU'tiv(' ('apa('iti(',!o, of tit(' l\lIimnls
Whf'H the la(k i~ Hilt ahsolute, Hut hI mort r"('l'nt tinws
disturlml1('('s of f!'prmluctin' fwwtiotls of u dutrllC'kristi{'
fypf' han' h{'('n tra('('d to hH'k of II fnt~oluhh \'itlLtlin to
whi{'h the lettpT E has h('(,11 nssigtwd. If rnt:.; art' kt'pt 011
t ht, following di("t >-,-

Skimnwd milk powdC'T 50'0 per ('('lit.

Dextrin. ' , ~5-:1
Agar~agar ~.(l

('odiiycr oil. . 2'()

yea. . t vitamin )KI\\'<ier 0'5
Ferrie (itrat f' n!!
or. on u similar diet cOHtuining fat. prohiu. (nrbohydrattt.
saIh.. Hn~ vitamin . . A and H. ~Tf()wth i ... lIormnl. hut u.fh.r
a transitory J){'riod or f(rtility ('()rnpl('t~ !>ih'rilit)' d(\'(lop~.
};~\":ill~ and Hurr han' ~hOWJJ that till' Jo.krility j~ a"Mwinh,d
with characteristic ('hang~ in hoth fUuk lind [{>!lIltle rub.
In the formcr therl' is destru{,tion of flU' germ ('"lis, and
eventually of Hl(' ('utirc !iwminifc'rous epitheliuHI. In th('
jt-male the ovary and the proc('ss of ovulation rf:mnin normal,
but during gestation the emhryo, which at fi~t. develop...
normally, begins to show a rC'tardation of development al)(Hl1
the eil?hth day, and betwecll the twelfth and twentieth uay
th" frctus dies and is ~ompldely resorbed. The mah'rnal
plac'Cnta continues to li,'e for some time after ferlal death ha..
occurred. These ..terility effects Me prevented in both male
and feluale by administration or \itamin E. The distribution
of the vitamin has heen studied by observing its curative
effects on these reproductive abnormaliti"$. The rich""!
source M) far discovered is wheat germ oil, but the vitamin is
alsO present in high concentratIOn in other cer",,1 embryos.
the green leaves of plants (lettuce, alfalfa. pea, ete.). and in
lower concentrations in various other vegetable oils. It is
present in 10.'" concentration in animal musculature alld
other tissues, and, is present in extremely smaU amount in
 24H THE rITAMIS8
milk fat und ('od~lin'r oil. a fact in striking contrast to the
importance of tht:'~(' two sour('es of th(' other fat-solublf'
\itarnins. Allimal ano Y(~_g('h\hl(' oils th"'~T{'forr h(>(>Tll to play
nil illn'rsc' rii/,' to OIl{' tluothf"r in rdution to the rlist.rihutioll
of th(' /Ilt-soluhle A and D vitamins. and. th(, IWW fat-soluble
E \ihuuin.
III rah suift:'ring from :-.t('rility duf.' to iaC'k of vitamin f: a
f'urntiv(' dnily dose of \\t}wat germ oj) is 2.lj mg.. hut fl ~jnglr
llln~siv(' do~c of 550 mg., rC'pres{'ntinR twent;.-'-two daily do~('s.
is also ('llratiYt' (>y('f} when g-in'lI on ttH' fifth or ~ixth day of u
prt'gtwl\('Y whi(h would otlwrwise fail throug-h fo.'tal r{'sorp-
tion.
Starting from 6 kg'. of wheat g'(,flll .E\"an~ and hb ('O~
workefloi have ('xtra('t{'d the fats. Rnd oib with ctiwr. and ha.n'
~n pOllifi!'d t h(> extr8("t so ohtnillcd. They ha '"(' ~}{own that
th{" Yitnmin pass('s into th(' llon~saponinahl(' fraetion. and.
makin~ HS(; of a method similar to that de~crih('d micler
vitnm.in A, A. ('onc('ntrai('d pn'paration has been ohtal1wd hy
T('m()dn~ from th(' non~saponifi8.ble Tt'sidUt, the sterols and
othf'r impuriti,(,s by cryst.allisation and by digitonin pr('t'ipita-
tton. ThC'Tt" is finally obtainf'rl 8 stero1-fTf't' oB active in singlr
doses of from 5 to 10 mg. This has b(;en purifiNI by vacuum
distillation. and a fraction boiling at 200 and upwards
at (l'5 mm. is found to contain the bulk of t.he active
U,1{<'l1t. These r(~sults arc.' thert'fore very similar to those
obtained on purifying and fraetionating cod*lh-cT oil. its in
the MS" of the "itamins of rod-liwr oil. it is probable that
th<> active fract.ion does not eonsist of the pure vitamin. but
is simply 8 fraction of the compounds originally present in
the wheat germ with which the \'itamin has heCOlW' associ-
ated during isolation in virtue of a similarity of fJhysico-
chemical properti.... The close similarity of the r...ults
obtained all fractiollating rod-liver oil and wheat germ oil
in the attempted isolation of their fesped;ve vitamins is
of great interest.
Vitamin E is _remarkably stable to hellt, light, air, and
 S7'ORAOS OF rn'.HfLI; E; 24U
('ht'mi('sl Tf'IH1ions ~n(,Tal)y. It is not dt'stro!'f'(l hy hf"ntinS!
at 170". by steam distiBation for s('v('rnl hour., nt 1totti'. nur
hy ,RC1Hl1n distillation up to 2aa, "'htH k{~pt fN' tWt'h,t,
hfH~rs fit 97 in a ('HrT{'nt of nfr. or trent cd with pRf1ndiuHI iu
a ('1IrTent of hvrlrog't'll at 7'> .'. I}O d('!oItru(ti01J or
the "ttlltllill
was d{"h'('tld. ~ Thr- "jtamill S{,{'HJS UWNfoTC' to lw r('u(1,uln"'~'
r('~istant to both oxidation and n'(itH'tiOH. H furtlwr di~tih('~
tion from the otlwr fat-solubk vitamin ...
Hats have U ('clHsicit'fllhh' (apadty 1(\ !ot10ft' vitnmtu t:.
Thus uftpr ht'lng put on an l:fr('(' dif't f(rtilit~, p('fsi!o.ts for
thr{'(' or four fIIotlth:'o.. or for two or thr('(' w'statioll:'<., tt:o.. T11h
of dj~appenrun{'(' beiJlg' th{' 'wmf' whdlwr (lr not tIl<' fwimnl
is .,uhjectt'd to the drain of ~J'{'.'tntj()Jl, TIlt' dtawill <10<"
not. ill('l'ea~(' f(~rtility h('volld 1lormut Jiwit!\ or 01 herwi ...("
improve ti~(' pf"rforn;umt.' of til(' r('pro{Jut'ti\,t, fW'()uwi.,1I1 in
Hllin!ais alrpany adt'quatdy !'>Hppli{'d.

Refel'el1ces
1. Ilut>MMOSl.I. :SOUl(' ~l()d(,f1I TtIUI(~II('it{o, of \'ltalJlin J{( .
"cllfc.b." ..fOlun, Sf)('. ('hem. ]nd . Itt:!4. 43. HUH .
) ])RU\JMOSJ), .. ('od-Li\'f~f OiL" JlIlun . .'101'. i 'hNlI. huT . H124.
43, 928.
:1. DRIT_\fMOSD, t'HAS:SO!\. and COWAJUI, /fio('hf'rrl. .100an., 1025.
19, 1047,
4. DRFMlIOND. COWARD, and HA}I;JI\". JIJ{K'h('m.lourn . H'~5.
19, 1068,
t,. l)RUMMOXD, Rf)SE~Jl}:lM, ami Cow..uw. .Jmtf-n. SOt'. ('hn't.
1 M . 1925. 44. 123.
6. EVA1tr.S and BURR. Proe. Sat, Arod. 8cienceh, JlI2;,), H. a;u.
7. HESS and WEINSTOCK. Journ. BiNI. ('],un., J925, 64, Hn, HI;\'
R. HoPI""'. Jo" ..... Phy'WI" 1912,44, 42.S,
H. KINXEBSl,ET" and PETERS, BWchem. rJWnJ., 1925. 19, ~20.
ltJ. LEVENE alld 'VAX VER HOEVEN . Jmlrn. IJitA. ('hn.., 1925.65,
483.
II. L!TCE:~ BWcMm. ,loum,. 1924, 18, 1279,
12. McCOLLUM, SnUlmws. BECKER and H-lJJPLEf. .JQurn. BioI.
0,."".., 1925, 85. 97.
13. MCCOLLUM &lid DAVIS. ,l"urn. BWl. f'k .... , 1914, 19, 24.~,
14. NELSON" and STRENBOCK. Journ. Bwl, (.'hem., 1925,64. 2HU.
15. OSBOR..'fE and llTAl[JfAl'.i. JOfl.rR. Rial. Clum . HH9, 40. 383.
16. OSBORNE and MENDEl.. .TouNt. A1n6" lfM. A.lJKor.~ 1#2J. 76.
OO~.
 THE VITAMINS
I;. PARK. "Tht, Etiology of Ri('.kpt,e:' Ph",lIwl. It",iflNI. 192:l.
3,1(16,
Hi. HO/'{EXJn;J.M MHI DHl'MMf)Sn. lJi{)cknn .7()ftTn .. 1925,19, i;,)3.
Hi, ~JJ)l.RMAX ami SMlTJl. . Tht' Vitamin!'." N~w York: f'hemi<'al
CatllJo~ut r'o. Inc., 2';ew York. J V22.
~(:: ~~\;~'!>;I~~~~T()~~t~;, 'i;~:~'I(:::~m ... I~~;~)::~;: t8bp5~:;~t.nt ~ta1'~ of
, Kunwltdgc of A('('~r<H()ry Pood Fa.c-tofi; (Vitamin,.)."'
Loudnn, J H24.
22. ~T:KESBOCK and Ih.AcK. .1oItTu. Biol. ('hem . ) 024. 61. 40fi ;
11)2:;, 64, 263.
2a. ~n;}':~l_\(O('K. ~E1-1., and Btna.L. ,/ourn. niol. ('hem., 11)21,47.
RH.
24. TCKslm. "Th{' l~im. QU('StiOll:' Chemic-al Rf'It1NCii, 1025, 1.
:IH7.
2~j. 7:n,vA. IHoehf'm .IOlf-TtI .. 1923.17,410,416: ibid., 1924,18.
182, 186, 1132, 6:18. 641; i~id . 1925.19. fi89,
 l'HAPTEH IX

HlEMOGLOBlN AND RELATED NATURAL PIGlI1ENTS

TH1'~ pignwntNI ('ornpouuds of which ha:m(J~l()lIin il\ U(,
most familiar repn's{'ntatin' ar.' of "I:idesprt'nd O{'j'1)rrrlH't'
hoth in the animal and in til(' plllnt worid. In tht' flll'IIH.'T
th('y arc intimatdy assoC'lated. eit}wr lIS oxygeu Ntrrlf'tf- or
as oxirlutiv(' ratalysts. with rf:spiratory pro(('!'\!"t's. unci In tt)f'
latter with the photosYllthcti('af al'fi\'iti{'~ of til(' piallt ('('il.
Both the animal and the plant cOllipoWHh. PO~S{'~" u ('OUHlHHI
~tr.ucturfil unit known as a porphyrill. whif')' I!o. ('oll!'ltitllt("
of pyrrol groupl'i. In the naturally (I{'{'urrill~ pignwlIt tlw
porphyrin is associated with a mdal, and tlwre j!oo in tt('Il('rnl
another group. usually hut not ne<'('!o;saril~' of a proteiH
nature. in eomhinatioll with tht' m{'tuH()~porphyriJl, Till'
metals whi<,h Nature has appart'utJy found mo~t suited for
the elaboration of compounds of thi~ ('laM; art' iron nnd
magnesium. Thus iron is found in hlctTIogfohin, ,'hlnro~
cruorin. and helicorubin. aU of whi{'h aT{' of "ujlllul ori~itl.
whilst magnesium is present in the chlorophyll of #,,""'('11
plant..'\. In addition to thes(' compounds. otfwts nrc kUUWH
;to occur in the lower animals in which {"opper all(} nuwg:uw.\l'
are present, the former in hremo('yanill, thC' latl<'r iu pinna~
globulin. Whilst these two pigments arc funetionally rdat'~!
to the hremoglohin series. it is not yet pron'd that HJ(y
co.ntailt a porphyrin unit within thdr molecule. although thi'
seems ve1')' probable at teast in the instance of' h1CmO'yanil1.
The occurrence of hremocyanin and pinnaglohulin is (""tlned
to limited groups of invertebrates. One might therefore infer
that in the animal the copper and manganese pigments
proved to have but a restricted ranllf" of usefulness afJ respira-
2l)1
 252 ll ..EMOGLOBLV
tor~' or cHtnlyti(' ('ompollncis. Imd that XatuT{- in thf' ('OllN'('
of JWf ('volutionary trinl and error IHf'thods soon abandoned
their ns(' in favour ortll(' nmeh more suitable iron t'ompounds.

The Occurrence and PhJ'llico-Cbemical Properties of '

Hremoglobin
lIa'llwgJohiu b mo. . t familiur to u." a~ th(' proti'in-piglll{'ut
('omp!('x preSt'Ht in mnmmnlian r<"d hlood cf.'Il!-.. Its function
tlH'T(' is to ad a~ It ('arrier of IllOieeulHT oxygen frorn the lungs
to th(' tissu('''1 where the oxidatiw' r('adions o('('ur. But ill
the nnimal world the association of hrcmoglobin and rdatcd
suhstaJH'('s wit h oxidative pro('('ssc!-i is muc'h "'ider Hum this
would. imply, I1.\~moglohill itself is by no means confincd
to the l'T~thro('ytts of \"('rlcbrah' blood. as the foHowiVg
tl'lOtatiOiI from a rccent paper hy Anson and Mirsky wHI
show. ,. Ha'mogiohin i.s found in t he bloods of all vrrtebrates.
Thi!o. h. ahout the. only generalisation that ('an be made ahlmt
ib distrihution. Its o('('urrcu('"c is \'(~ry wide~pr('ad through-
out tlw anim.al kingdom~ but it is absolutely haphazard, not
fo)Jowing at all tht, Willa) {'yolutiomuy {'lalisifications. 11:Vnlo-
glohin has lW('n found in the starfish (Opltiadis vireRcen8), and
ill no related forms. It has be-en reported in the body fluids
of the Jarnt.' of two or three insects, although not in any
other inse{'t lan'a, Of in the body fluids of any adult inserts,
Tht' audent and often-repeated staternent that the blow-fly
Jlu ..~ca <'Outains hl:(~Uloglobin has no basis in fact. AU the
pulmonate snails. with one exception t contain hOC'JUO('yanin.
That on . .' t'x("('ptiOll 1 J>lallorbi~v, contains hRmoglobin. Some
wornlS have ha'.moglohin, sonle ('hlorocruorin, some neither
pigment, A hll'rnoglobin oeeurs occasionally, though on the
whol" "ery rarely, in nmsde; for instance, in the striJi'-..->d and
heart muscle of many vertebrates, and in the pharyngeal
rnuscles of cert.ain gastropods, Frog rnusc)e contains none.
In the parasitic worm Ascaris, not only the musdes, hut also
aU the tissues except those of the generative organs, are
impregnated with hremoglohin, A"parent~y a "ery compli-
 M!('R(U,PE('TROS('(II'I<;

['att'cl ~l1h ... tnn(t' \\ it Ii Y~'r~' pe\'ldiltr propt'-rtit":o. hn... )~~'Il

{'\'oln'd illdt'p{"l(k/l'I~' lI,I!l~ifl mlfl Hg'uill in :\aturt',"
Out' of the 111()~t ohviou ... propl'rtic''_ of hwtll()J,!lnhill nml of
similnr natuI'ai pij!JlII'Jlb i . . Uw dwnwtt'ri .. tie ~f'rj~... of nh...orp.
tio!1 hands which tht", ~h{)w in thc' ,i .. ihk ft'cilu\.nf fhc'
('ontinnotls spe(trunJ oj whitt- light. Tht"oW hauds ... t.;,\. us It
('oll\THiellt rt}('lltlS of id("ntif~'IIlg' the piJ,!'uu'uts find for HI('
in\'('stigation of their l)hy~i{,(H'twmil"ul Im~lwrtit.... Thl'
approxinlHtp p():o.itions of tht, uh:-.orpti"I! )mud ...... howli ",\'
such a pigl1l<'lIt a" h'('HlOglohfll
will lw familiar to Tenders. simf
tht'sC band . . arc TcadiJy Ob"'t'T\ (,II
hy mean!o. of tilt' simple ).,pedro
Sf'OP<' or the waytk1ll-,rth spt'drn*
mete'r, -Ill n:('('flt :'('an, the
int I' 0d u (' t ion of t 1)(' micro
~pt'~'tro~('oJU' has cllabler! l,jo*
e}wmists to oh~('ryt' the ubsorp*
tion Sp('('tTa of pignwHt!) pT('M'nt
in very srnaH (luaHtiti{'~ awl
permits of til(> ('xnminatioll of'
miera-preparations of ) i \' j 11 g
tisfooues and ('ells, This jn~trl1
ment. which can he' llst'd aft'r
the manner of the o('ular of IW
ordinary mieroscolw. is shuwll in Fig, 25, It has pro\ (d
specially u.'tt:'ful in the examination of tht, pjgm('nts pr,'st'Jlt
in the tissu{'s of small animaL~ su('h as ins("cts.
It is frequently essential that the prt'cbt, positions of til<'
absorption bands should be determined with a{'curscy, t,in('(',
apart f1rom the well-marked diffcl't'n('('s hetween ohviously
disHnrt pigments, in many ('a.&f>S th{' charactt'riliiation of
closely related and at first sight identical pigments derwnds
on the observation that the mean wavelengths of corrt'Spond-
rug bands are not coincident.The determination of the mean
wavelength of the, absorption bands of pigments by means
2;'4 H.tEMOGWBIN
hi' thc' ordiHary spcdroseop(' is ina('('urate h{'('HU~(' of tfw
lm'Hcitl! of Hw hRlld ... and th{' in(h-tinih'ncss ()f tlH'ir margin!>.,
ami for till' purpo...c of making thi ... IJJrl}SUr('IU('IIt with the
1H'('{'SSHr~' tU'CUTa('Y IIartridg( has c{{'\'is(d a ItlOditied instru-
1I1t'lit ~'hi('h h(' ('ulls a r('\'(>rsion speetro<';(olw. The pl.IB' of
til{' ill~.trlltll(.'llt i~ ~hown in }-"ig. 26. III phU'e of tilt ordinary

('ross-wire, it nlakes use of an absorption hand of the same

width, mean density, and distribution of absorption.,!'. that
to be measured, but re,ersed. The absorption band pro,;ded
as an iude-x is fornloo in a second spectrum reversed in
direetion to the first, as shown in the figure, both spectra
luwing tpe same brightness and dispe....ion, and both being
formed, the one above the other, from light that bas passed
 RBI'J<JR8J(1N Nl'E(,1'RO/:i('(J/'f; ~,i;;

throug'h the pignwnt undt'r (xJt.mifudioll. 'I'h\' halld~ III tht

in<iex speetrum art' thctefnt(' ... imilnr to thOM' ill llH' other.
no mattrr what may h(' tiw thil'klH'~!oJ Hnd ('otU'('lltr"tintl of
th( pignH'nt or tht~ propertil's of HI(' .'tour('(' of Jj~ht. Th!'
im.t"rum('nt is so ('otlstnlctNl that th( ;IHhx t;jledrullt IVlt~' 'I("
shifted paraHt'1 to its lOUR nxis hy the nlllnipuflltiollct( UU'
mi(,foHlett-r tlll'l'hnllism so thut the two ... illlitar hUlids Inuy Ilt,
moved until they app{'ar to tIl(' obsernr to II(" in lint', Til('
mieruflleter index tht'll points on til(' :-,(nlt to ttl(' !Hean Wll\ ('-
ICJlgth of the band whi{'h has htt~1l tilt' suhj,,(t (If IHt'a!oolJTt'-
I1I('nt. Suppose that two uhsorptioll hand ... he ,..,d "'0 thnt til{'
left-hand edg(' ofOlle haHd b ill ('OiIU'id('II('(' with the Itft-hulId
(~dgt. of the other hand abo\'' it. thelJ the rl!!ht-Iumd (-dloW'"
must also \)f' ill eoincidt'u('(', silwt> tilt' two hand ... arj' 11Iirror
irn:ges ofollc another, aml if ).' i~ tht' WR\Tlt"llgth (if (11\('
edge of the hand and A" that of tht' utlH'T (dgt. th('11 tilt'
point midw.ny hf'hn'clI the edg{'~ wil) )wn' a WtHTleng-th uf
t( i: -+ ,1"). Since when the ('dhl'(>s (If the lutnd .. ur(' in
roil)('idt'nc{' th(' midway points must aJso tw ill ('OiIWidl'll('(',
it is clear that th(' wavdtngth of !( i: f' i:') of 01)(' !o.pedrum
is in ('oin('jdt'lH~ with that of the' otha. and thtrt.'f(lrt' the
reading giv('n by tlw mi('rom{~t('r indtx will h(' t hut of
it).' + X'). In tho instrunl('nt (s,. }'ig. 26) light fr(J1lI 1L
sour('e L is {'oll('('ntrated, hy menus of .u simpk (,-()Hdl'H~jng
lens C, on to the adju,tahle slit of the sp<ctrosrol'(' S, through
which some of it P"""'"' to the lens P, by whid, it ih rClld.,rcd
parallel. Half of the light then pass<'s through the prism of
small angle D, which deflects it slightly upward,. and reach,,
tbe surfact' of tlte grating G, by whicb a '1"','trum is form"d
in whi('h the violet end is to the left and the red end to till'
right. 'J;he otlter half of tlte light from tlte L'Ollimating le,,,
is deflected slightly downwards by the prism 11', and th,'l1
reaches the surface of the steel mirror M, by which it also
is reflected on to the grating, but from the opP""iw side of
the normal N as compared "ith the light reaching the grating
directly. In this ~r a second spectrum i. formed which
 llJEMOOLOBIN
'Ie . . with OJ(' \ '(lid clld to the right and the fed (,Ild to the
I<.j't. i.I .. ill t hi' f('n'r",l' dirl'ctinn to til(' ~p('('trlIlll preyiOlI'.ly
('ofl ... ider('(1. If it w('re (lot for th(' interposition (If th(' weak
)lri"'lH~ l> Hild 1),. t hest' two s}){'dra would lie over one
Htlotlwr. hut Ute prjslll~ art' ~ri\'(,11 su('h all angle that they lie
~id(' l~' ~id(' a ... :-.hOWH ill tht' !-ilnall dia~"l"am. Tlw Spt(tra are
~hifkd Ily rotatiflg tile gfatiug nnd mirror ahout tll(' fulcrufIl
J.' hy lllt'all~ of the llli(,TOnlt'tt'r SeT('W shown. '''heu a ~mall
rUflgr of \\'U\'pll'ngths lin ... to h(, (on'rt.. d with gr('atn a('('uraC'y
tht' plnh'-.!!lu",~ mi(,fOlll{'h'T l;P is ust'(L This ('onsists ()f a
thill gla".., plnt(' aHadwd to H pointer. \\'twn th(' plah- is
~d Ht an anu1( to tlit, lH.'am passing h('twt'rJl tIl(" slit Sand
1l'1I~ 1'. till' hearn is d{'\"iah'd to om: sidt' jUl'iot a!o; if the slit
itst'lf had }Wf'1l gin'lI a slight side shift. and this ('BUSt'S tilt.'
two !-.pl'<'tra to undergo 8 small side shift rdati, e to onc'
;lHot lwr. Both m!eromt'it'J'S at(' cahhratt:'d a~aill.'>t standard
lilH'~ of kllown wanlength. so that the final f(ading g;yl'!oo
dirt'{'tJy t}w mean wanh:n,.,rth of the coincident bands.
All iJlt('rtl>.tin~ appli('ation of thi!>. instrument has hef'll
IlHldt hy Hartridge and his ('o~W()rk('TS jJl dett'rmining the
rtiuti\"t' amouut!:o. of two different pigrn.cnts present in a
solution. f'.~". in mixtures of oxyhamlOglohin with carhoxy-
Of reduced h:l'moglohin. The principle upon which th.e
nppljeution i!o\ based is as follows: If the absorption bands
of two pi~n1t"llts ha\"c difft'rent mean wavelengths, then, if
hoth pigme-nts are present in the samt_ solution and are
tog(t1wr "ausing absorption of light, the measurement of the
mean wa\'denl,.>th of the absorption bands will he found to
ya.ry with the relativf> coueentrations of the pigments if
(1) the hand of one pigment is approximately similar in the
form of its density curve (i.e" absorption coefficient. plotted
,,!!ainst wa"elength) to a band of the other pigment, and if
(2) at no l'Oncentration do the bands appear separated, tbat
is, that t-heir mean wavelengths must not differ by more than
the apparent mean width ofthe hands. One or both pigments
lllay vary ill l'Oncentratioll, and the pigments lllay he ill the
 OXYOKV 8ATI'RATJ(lS
"-3tnf'glass v('sseJ or in differt'nt #lln.s~ \,(',..,e)s through which
th(' light passe~ in ttlnl. It i!-i WI'1l known fhnt tfw two
ahsorption hands of (,xyha!mogtohin HT'{> ... hiftt-<l townrd~ th~'
violft if til(' oxygt'Jl 1)(' 1'(pla~t'd hy ('arhon luonuxidt" U ~hJrt
whid) may tw dcmoH.'ltrnted with HI(' ordinury s()(>ctrn:'\'(j!w.
{'sing tht, Tt'Y('rsioll sPt.C'trOS(oPt'. Hw mt'Ufo.lIfr'owllt of t 1'1('
extent (If thb. shift ht\s heru madl' Ow hn~i, of nwthod ... nf
rJttf'Tmjniu,!:' fJuantitatin,ly 1hf' ff'Jutin' nulOunt . . ofox.\'~ ami
r('(hwf>d hICrtJog"lohin. or of oxy- and (nrt,oxy'ut~fHoRlohih.
ptt'sent ill mixtuf('<" of tht's(" pi,rlllents. Tht' mdhue.i. "...
d('s{_'ri~wrl hy llartridg{' nnd Houghton. 1!-. n~ fl)lIo\\'~: (hw
compartment of a douhh wrdgp-.. hapcd trough /'...r't' Fig, 27)
i~ tiHt'd with a solutioll of oxyhafl1()~lohin. and. th(' other
with: a r-.ohition of th(' sume ~tf{'n~rth of r((hlf'(d hl{'nU}~lohin.
Examination'in t he ~pe('tros('opt' ()f the li.i{ht whi('h hn~
pU.!oo.sed through on!' ('nd of thC' trough show!\ tht: bauth' of
oxyh;\>moglohin (1\). whilst tht' other end shows tilt, bauds
of tNhwcd hn~moglohin (B). interuwdiatc positions showing
tht, hand, of variolls mixturrs of tht'se suh8t.an('('!ot I:l('(~orlhng
to their rdatin' thicknHis. An additional trough is now
filled with a solution of ,'arhoxyh!Cmof(lohin and plar.'"d
l"tween tho douhle w,.dg<.-shapcd trough and the "Ou',' of
light so that th .. h,'uID of light first I""""s through this 'iug1<'
trough, and finally through the spectroscope. Sup!,o", the
thi"kness of the single trough multiplied hy the ,'onccntratioll
of earhoxyhremoglobin ('ontain('d ill it, is equal to the maxi-
mum thkkness of tlli oxyhremoglohin compartrlJ{'nt of the
doubl,' wedge-shapro trough multiplied by the ",,,wcntratioll
of pigment ill it, i.I'., T[Hbt'Oj Cc TmuIHhO.I. Then when
the douhle wedge-shaped trough is shifted as far to the left
as possib/t, (A), the position of the ",-band in the spectrum
is the same as that of a sillgle solution in which 5Q per cent.
of the ha:moglobin is comhined with 0 and the remaining
5Q per cent. with CO, If the trough is shifted as far as
possible in the opposite direction (B) the ",-hand appears
more diffuse and slightly to the \;olet side of the correspond-
....
 ll.EMO(JLOBIS
iug Iwnd of /! ... ingh' ~()lutioJ) full,\' :-.atufnh'd with ('0. If
ttw troul!h j,., shifted to an iui<'rnlPdiRtt> position. t}l(' ah~orp
tiOlI Imnd;., a},.,o ~hift to A position internl(>ciiate hetween tlwir
t~nl cxtrNIW f1o~itjow. ill .\ 8nd H. their t'X!lct p()Si~ioH

,,

lE9
1~1
: 8

l~lH. 2.,- P08ititlU nf troughs and f'aUbratiull CUI'Vt' as uM'(i in Hartridgt> and
Roughton '8 tn(>thod of deu>rmining ~rcf'nt~f." saturation oj bluod
pi~ml"m-s. (After HtLTtridgf" and R(nlghton.)

depending on the ratio HbO. : Hb trawrsed by tll~ beam of

light, F'igs, 27 and 26 make this point dear. Br leaving the
HbCO trough in situ, and by a suitable adjustment of the
double wedgeshaped trough. the position of the ".band o..f
t he spectrum ean be measured for 8 series of different valores
or the ratio 'l;'[HbO,J to T[HhJ. A calii,ration cur,'" of this
 oxrOEN SA7TRA7'IOS
kind iun'illJ,! I)('en ohtailwd ht'(' C, ....ig. :!7'). t ht, IX>ft't'nt.ultt' ttl'
oxyh:ctlloglohin in an~' sing-If' ~olutiOI\ or hll~lH()globitl in
whit'h tht'f(, i~ a partinl ('omhillafinn \",'itlt O';} ('nn tw littn
mined. Thl" douhlt' \..,(d,L.w ... lUlptiI trough i . . npll\l'('tl h~ 1\
Y(,SS(j of sHell dimf'nsiml~ tlmt whell til(" Illlknowll.1H\'H\o-
glohin is pia('('(l ill thi!'\. \"{'sM,1 tht totul ('ow'futril,iutl of
h:'l~llloJllohill (Hh Hml llhO::) ill tilt' IIltkllllWII ~olutiotl IJItlltl~

.. 8

",0,
I I
:0. /I

I I
-=-1;,
:::::~~~ :;' ,
,., llPC.
OWl

FIt.. :l~.-~ Dialr~UlIn,~til rt'"pr"~'nta'iOl1 of lh(' 1'I'latl\\' PO>lltj()Hk 'If Hu

aXffl (mInimum ahtolurptir,n) ot th{' Il bandl< I,f: A. {)X;VhlkmO}(;lohin ;
B. ('Q.rb(jJ:;~illl'mof(l(Jhlll: C. f"tducf',j ha:'mit,cl"bil1; l~. it mixrurf' ot
f)() ptr eenC rt'duced haomuglHbin and ;~I pt'r Nnt. rarbox.)"hslJll1tgl'Jbin.

plied hy the thickness of the vcs;d is ('qual to T,,,,.IHhO,)

or !HhJ in the double wedge-shaped trough, The position
of the ~-band is thell determined, a11l1 this with the aiel of
the caiilJration <'u"'c gives the value H of the ratio TfHhO,j
TIHb]
of the unknown hremoglobin solution. But since T HbO =
T Hb = thickness of vessel containing the unknown solution
then R = ~I_IbO.1 in the latter solution.
tHb]
17-1
200 fI_EMOGLOBIN

The Existence of Different Hemoglobins in Different

Animals.-Th(' nwthod ... whi('h We flU.H' ju!o,t. d('scritx-d
)u\\'(' h('t'll nppli"d to H study of the problem of tht' possible
('xistnU'(' of difft'Tent hremoglohill'" in diff('rent spee1cs. The
npp1ic;Mion has heen in two main oirf'ctinn.s: () a :-.tudy
of tht: affinity for oxygen of the h~pmogJ(}hins from diff(,Tent
unimal MHlt('('S, and (2) an (:XRct determination of the mean
wuyclt'ngths of the ahsorption bands of tht hR~m()glohins.
It hns \)('en T('{'ognist'd for many y(~ars that the h.crnoglohin
frotH the hlood of various mammals \'ari('s g'l'f'atly in the' f'RSC
with whieh it may he (.rystalliS<'d, and also in tht' form of
the C'rystnls obt.ained. Hf'h'hert and Brown. in their pain~
tHkitlg' and elaborate investigations. harf' dcmon"itrat(>d thi~
ph(,1I0fH{'Hon in a ,'ery convincing manlier. They h"'ve
l'xnmined th(' crystals of oxyhremoglohin pr(:'part:'d from th('
hlood of over 100 speeit"s of rnarnmals, and the mict'o-photo-
graphs rcpl'odu('{'d in Figs. 29 and 30 illustrate some of the
~triking difh:'rt"I1<'t's in ('rystaHin(' form whiC'h they han'
recorded. Thrs(" observations lend cOllsid('rahle ~upport to
HH' "jew that actual chemical rlifferen("e.~ exist betwt'en the
,'anous hremoglobins.
The question of the identity or otherwise of the gas
combining power of hremoglobins froul various animals has
been examined by Barerdft and Barcroft, and by Maeda and
Seli~kar. The former investigators have shown that the
hremoglobin of ArNJicolo has an oxygen dissociation curve
which shows 8 higher affinity for oxygen than that of huma"
luemoglobin, so that the gaseous exchange takes place at
v'ry low pressures. They also showt'd that carbon monoxide:
instead of having an affinity 250 times that of oxygen, as in
the case of human' hremoglobin, has only about' fifty to
seventy times the affinity of oxygen for the hrernoglobin of
Arrn;cola. MattIa and SeliSkar have investigated the
inlluence of tempt'ratllre. on the equilibrium between oxygen.
and hrernoglobin from widely different types of animals.
They used dilute solutions of hremoglobin "arying ill colleen-
 A._! , . , \.

I .~~~
(.~...",~
~#-
.. .
(I)

(3)

FIG. :!!:L--Oxyhlemoglobin crysta.l~ (rom : -

(1) The Htuf!,!{"01l (A.cipt!ftMr stllr;u). showing hr&t'hypina,roid aspt"ft.
(2) XeclurlU ,HwulallVt. showing I'loml' twinnt"d ('r-Y!ltalt'l.
(3) The' Trulnpt>ulr Swan (Olor bN~-cinntor).
(4) The Guint>-8 Fowl ( ..V",mida. me/Mfl"i8).
(Rto-produced from Reichert and Brown.)
 (I) (2)

FIG. M.-(h:yb.. m~lohill c1)'Ktal>l from : -

(I) Guinea-pig ('aria ('utteri), Ititowim.:: lIom(' twin cry~tal".
(2) Grey &j:uirrel (8rjuTu.IIt"arolillen.tli.IJ), Id,O~ in$! ptK'udQbcxajlculAllUuak>
cryatals and group" ill: par.'llJd l-"oMth.
(3) Ka.ngaroo (J/fJrroplllJ Vigtlnlt:tM), ~how~ Jath.1!Iha.pet.I "r_VlltaJJil
growing loIin~ly ami a.gS{n~ated int~) Hhcfl.flik", bundle".
(4-) Horst' (l';gUIUI r-ahalht.'f). sho..... ing long priMmatk t'r~.rt.alA.
(keprodtlced (rob) Reichert and RrO\nl.)

 j)ISSOClATION CrRl'E8 261
tration from 1 in 2RO to 1 ill 1.5tlO. nt whidl dilutioH' til('
dissot'iatioll ('nrn>~ arc T((tallJ!ulnr hype'rhollt' and nrC' not
inftul'Il('NI hy th(' snlt~ pr(,M'nt. th(' {'quilihriul\1 h.. tw('('u
(lx.\'1!cn and h~l'nH)glohill d('{>('utiiJlg ouly ()Ji tht }lydroW'n juu
('o~H'(,lltratl0n and tf'fllJWTutUf(', TIlt' I'll of Ul(' ... olutigns was
kpIlt at 7'4 with Ii pho~phat(' mixturt'. TIl(' dd('rmiH~ltim)s
of JWN'('lltag(' suturation W('ft' mn<ie with tht rl'\'('r~joH
spt'dros('opt' by til(> ou.'thod already dl's('rilxd. Fig.:n

s ~ IS ~ ~ ~ ~
o.~ )II.~~"""

FIG, 31.--1~:;;~~;it;~;;;;:~~~:<nn(.Mt!1ia~:taI:~:r)~~:;.lk::':tjlll'hinB at
shows some of their dissociation CUT\"CS of thr dHutc ha:mo-
globin obtained from man, Planorbis,. and the frog at
various temperatures. It will be seen that the percent
age saturation \\;th oxygen at the same ten~ion.s varie1i
greatly in these different hremoglobins under identical
physical conditions, and also in the same hremoglobin if the
temperature is altered. Not only is this the case, but it is
also found that not all hremoglobins respond in the sanl('
degree to a temperature change, that is, their temperature
coefficients arc not the same. J'ig. 82 shows the relation
2112 II.EMOGLOBIN
ht~t \'H'ell hmpCr;lture nml {'(luilihrium constant of th("
Tc'action.
Hb I 0, , - -:' HhO,.

for lH1/wUl. frog. tortois(', and Planorbis Jnemoglohin. The

log-ari\fllll of til(' f{'actioll ('onstant I\: i ... plott(d IH!nillst tilt:'

Tt'ciprof'HI of th' absoluh' tt'llIp{'TntUft" i.t'. 1. 'flit' rl'latin'

T
nHinit., of a '-"P('('illl h,H:'llJoglohiu for oxyg('11 ('un ht' l'('ad from

+o~-------------------.

"0

t
"~
....T
Q'''Cl
}i'm. :l2.-Rf"1&t.ion hetW('(l1l temperature and the
("t{uilihriuUl ('onstant for human, turtoise>, fruR.
and PWllorbioil luemoglobiful. (Afu-r Ma<''la and
~liiJkl\r.)

this figure by comparing the values of log K at a given

temperature, this vahw being greatest in forms with the
lo"",,st affinity for 0" Til" "alue of til" temperature eo-
efficient is representtd graphically by the slope of the lines.
These eoeflleients vary greatly, tbe largest heing the human
onc, which is O\'er 5, whilst that of the frog is under 3, and
that of Plmwrms probahly less, These disparities have, as
nar.,roft has pointed out, great biologi<'al interest, H;
expresses this as follows: "The temperl'ture of the higber
 "Sl'AN" J)fAGRAM
mammals (apart from hibernating anima)!..) unchrg'Ht... \ ('r.,
... light '-ariHtiol)s: if ~u('h variations art' to produ('(' nl1~' dft(t
of signiti('an('C' on tht' diss(}{.'iatioH ('urn', (itanlv 01(' ,'urH'
must ht H'ry ,."in . . 'tin'. On the other Juwd, Uw t:roj.(', in "OftI('
('olmtrh's, nm~' change in l<mpc>rn.t\irt~ Iwrhllps R(r- .:l~ he-
tWf'{'1) the willter nud the Sl111Hll('T, ill whidl ('a ... ,'tlnwr-
..,ensiti\'{IW."i' of if!.. dl"Sodlttion <,urw' rnil!ht tw (,Htird~' (lit I

J .. , - - - - - - - - - - - - "

~e..- .",~e:

2,. ..= ....... _.;\-: __ . __ <..,

"".
2-6
P'IGIEDK - " ~ - ~ - - - - _.
"'MOV~
""",.

'J/. Z'I

g
t
Fw. a:J. - .. ~p(l.n .. di&f(fam (If v)tril,u!'l mtl.fllml!dian a.nd ,,"!J.1htl.fII
malia.n hwnu',Itiobiru. (Aft.t'T R.U'(~rf)f{.1

of place. Clearly it is a hioiogicai neN:~\lty that the oXYJ.!(n

diss(.){'iatiou in forms whi{'h exhihit a high dt'gre(' of p()ikilo.
themtidty should }J<~ adapted to the ge-Tleral condition.. of tJw
animal, and therefore. from the hiological standpoint, th,.
factors to which the temperature coefficient of K must
conform are quite different from those ill hon}(>jothermi,'
animals."
An exact dete,:,ni1l8tion of the mean wa\'~leugtl", of the
264 HLEMOGLOBIN
ub.sorption hands of the' oxy- or earboxy-derivatives of
various lurrtloglohins n~n'als the tad that slight but ~ignjti
('alit diff'reIlC('!Ij exist. (~uantitativl' IJWUSUTt'mcnts of this
ph('Il(_mH'llOn art' usually mati(> on tlw :x-hand, sin('(' it is the
!->hurpr;r und narrower of the two hands.. 1t is possible"to
('orr(,kl\(, these sJwetral ciiffrren('(>!o> with the \'urying affinity
ror gnst's shown by \'ariou~ }llcm()glohill~. aJl(i which We hun'
dis(us~(d in tilt' prt'cNling parugraph. In the followin~ tahl('
the results of Barrroft and his associates aTe given. It
t('('ords tIlt" r(lativt, position of maximum density of the oxy-
and ('urhoxyhll'Inogiohill hands in the spedra of humall
IUl'moglohin and that of s{'vf'ral ~ubmammalian forms of
Ii 1,-.
TABLE XIX.

Po.!ition of .Hand in Span (S),in

Angstrom Cnitt\. Angstrom l'uit.<;.
Animal.

A in HhO: Bin Hbf'O. S A-B

.\tan 5,7fH 5.ilO ;;..J.

.4 renico/ 5.746 5,fi9H ~S
Lumbrl.l14.-.; 5,755 5,720 :15
PlanorbiR 5.7.!t) 5,708 as
Chiranomw. 5.777 5.727 50
Pi~on 5,762 5,710 52
Carp 5,762 5,710 46
Tortoise 5,766 5,7]7 49
Fowl 5,769 5,1IH 51
Lizard 5,762 5,715 47

If. as supposed above. the position of maximum absorption

of the ",band of oxyhremoglobin ('orresponds to A Angstrom
units, and of carbox~'ha!Illoglobin to B Angstrom units, then
the \'a)ue of A ~ 11 represents the number of AngstrOm units'
throug!l which the ,,band moves when t!'e oxygen is com
 " SPANS"
plt'tdy uispla<'f'"d by ('url)f)) moumddf' H("('otlJiHJ,! to HU'
equatiOll
HhO, + CO ,~.. ' 111>('0 ; 0,.
T1vs djsbul('(' A to H i.-, {'allt,d the" span." uwl. U'I' Wt' 'it'p. if
diff('rs ('ollsid('rahl,Y in dirrer(nt. ""tttbratt' :ttullowt'r ~;wmal-;,
TIlt' VU.llH' of the rt'actiofl {'Oll!o.tllflt I\" for tilt, aIIO\"{' t'tllIHti('II.
Ilam"h', IIlbO,1 ,1(:01, abo (liff('''' ft"'" '1.'l'i,'., 1<> '1"";'"
, 1111>(:01,10,1 '
If the i<'ngth of the span in Ang!'.triilll unit ... is plotted ag'ninst
thr logarithm of this rcu('tion constunt at Ii g-iv('ll ttlHp<'rahlrt'.
the poillts obtairwd lie mort' or ks ... on f{ !o.traight lim', a1. ",IJOWh
in t.he acc(}mpanying }~jg. 33. SpU{'(' doe... lwt JW.'Tmit vI' H
discussion of the intriguing question of tht' lwuring of tIH'S{'
oh~er\'atwns all a possihlt' ('hemi{'ul hasis of nut ural ~(l('(tioll
and {'\'olution, but it is obvious that" hU'fliog-lohili .. i~ IUlt u.
!o.ingJt substau('f' common to allllw spt(i(s ill whieh it ()(;('ur.".
It is a complex slightly but defillitt'ly variahle from "pc:{i('i
to sp(_~ci('s, th{' variations heing atiaptatiOllloo of til(' pig--IHC'llt
to suit th<~ endronment and IH'Cds of the ;ndkidullJ aninwl.

The Chemical Basis of the Differentiation

of BlBmoglobins
It is reasonahl(, to suppose that th~ physical diffefrll('('s
between individual hremoglohillS. which filld~ ('xpr('ssioH in
varying crystallographi(' fonns, absorption sJJCctru, and
diasociBtian cun'",', .'/wukl ),.,. o"wciated with &,lilld dllmi
~al differences, But before we can even suggest aclequah'
ehentical grounds for this differentiation we mu..~t know 1iOtl}f'-
thing more of the chemical ronstitutiorl of htcmoglohjt) l1t)d
of its related pigments,
Readers are familiar with the view that hremoglohin i8 U
conjugated chromoprotein in which the oxyg'(~n-('I:l1Tying
pigment, hrematin, a metallo-porphyrin ~omposcd of iron ill
association with substituted pyrrol groups, is combined with
a protein of the iJ;istone class called globin. UreOloglohill
26H H.'E.}'[OOLOBI N
ouly exi~t ... til' such llear Uu' JlC'utrul poiJlt. and it has hecn
suppoMd utltil rt'{'{'lltl~' that th(" add.ition of diluh- aeid or
nlknli (ffet'1td a disruptioll of' th(' ('hl'omopro!('in into its
prot('in portiou alld hwmatifl. By the addition of ~tlitable
r(dll()ng r('ag<'llts Ow lattt'r could. the1\ he ('oll,'erh,d iHto
ha'tll(u'hroIlU)gf'n. Further. it has hC(,1l aSSllllwd that ha:matin
and hWnlochrorllog<'n reprcs('ut resp<,din:ly the oxidised and
ft'Ciu('{'d forms of til(> iron-eontnininpr pigment I-,.YfOUp of ha:mo-
glnhiu. and that t}H'~{" two pigmpnts ('orr(''spond to (Ix_\'-
htcnrog}ohin and r{'du('('d ha'mog-lobin miuus their protciu
Illoidj(,s. That tl1{'st~ ,'iews do not givl' a tru(' reprcs{'Iltation
of tlU' fads has h('('n shown hy the rc('('nt wt;rk of llareroft
nnd his 8sso('iates. Thus Alison and )Iirsky hav(' h('Cll able
to dt'lHonstrnit' t hut ha~matin and hR~m()chromogcn are not
prott"iTl~fr('(' pigm(nb.. hut that thl' globin of thf' original
hWBwglobin i~ stilJ pres('nt in these compounds.
\,"hen dritd hlood ill th(' pr('scll('c of sodium chloride is
tr{'atNi with giaeial acctic acid or with strong sulphuric acid
in aleohoJ it crystalHne preparatioll ealled ha::~min is obtaiued.
This substance has long been familiar to biochemists and is
T('('ognis('d to he th(' hydro('hloride of the pigment portion of
h:CHlogJobin freed from protein. The empirical formula and
(,OllStitlltioll of this substance cannot yet be regarded as
....Wed. hut the approximate ('om position is indicated by the
suggestions of Ktister (C ..H O.N.Fe-('I). of H. }'iseher
(C"H,.O,N,FeCI). and of WilisUitter and M. Fischer
(C ,.Il"O ,l\ ,FeCI). The following constitutional fonnulre are
t/"",e suggt'sted by Kiister and Willstlitter (see p. 267.)
When h"'min is treated with sodium hydroxide in the
pr('sell{,(> of ft reducing agent such as sodium hydrmmlphite,
.sQr-"i20 4' tJIC ('orrc:sponding base in its reduced form js
liherated from the hydrochloride. Until recently this base
would haYe h .... n regarded as hremochromogen. But, as we
rullrC seen. tbe latter contains protein, whereas hremin does
not. Anson and Mirsky have suggested the name Iunn for
tht base of which hamlin is the hvdrochloride. Hrem may
 2(17

hC' ohtain<>d from hremortlohin in ill(' fol1owinf,t wny. whi('h i ...

d(As('ribt'd hy the Inst~rl)('ntioIH'd authors. ulld i... h'ht'd 01\ th'

method of Schulz. A few drops of dilute hydro<,b1oric acid

are added to a dilute aqueous solution of Iucmoglobin to
convert the latter !nto acid b",,,,.atiJl. Ether and ai"ollUl arc
 268 HAtMOOLOBIN
tll{~n ndd~d in appropriate proportions and the mixture is
shak(Il. Th(' al{'ohol-water phas(' Ins(~s its ('OIOUT. It ('ontains
glohiu only. Th(' ai('ohol~eth{'r phas{' ('ontains the eolouTt'd
('olHpOllcnt of ha;moglohin. It is to h(' notf'd that tht hydro-
('hlori(' 1wid is Hot Trsponsible for the hr<'akdowll of the htemo-
globi'u' into Imml and glohill. lI,cm is in~olubl(' ill all acid
aqueous solution. and silw(' lJO preeipitah' appears when aeid
i!'o. ndd('d to lucrt1ogiohin. no appreciable amount of frec h,cm
PHil he formed. Hydro('hJoric acid ('hanges hremoglobin into
ueid htcruatiu,-u ('ompouHd of lucm and globin-and thj~
proh'ill is readily soluhle in u('id, "'hen alcohol alld dher aft'
added to the solution of add lucmatin the equilihrium,
}uno + glohiu .;=_::'-~ add hrematin, is distutbed. The
h:em P8,s,\j{,S illto Hu al('ohol-l'th{~r phtlSf' (in which it is rery
solublt. and in whieh the globin is insoluhlt,). thereby colour-
illg it. whjJs1 f}w globin rf'JlwjJl!'. in thf.> now only slightly
{'olourNI aqUt~ous phase. The hrem pr<'pared in this t way
will show the ('haracteristic absorption sp('ctra of hrernatin
or luclllochrolllogen only after globitl or a globin substitute
( ... t'{' p. 270) has het'll added to it.
On the basis of Willstiitters formula for hromill the
composition of hR~m will he expressed by some such formula
~s C 331I 320.::\'.]<'eOH. H:r;m is insoluble in water and in
add solutions, hut it dissoh'cs in the presence of alkali. It
shows a diffuse absorption spt"drum in the yellow region, and
with carbon monoxide it gives a spectrum of a pattern siniilar
to that of carboxyhremoglobin. The relationship of hrem to
hremochromogen and to reduced hremoglohin is shown in the
schem(' gh'cn below. In the same scheme is shown the
rdationship of the oxidised form of hrem to hrematin and to
methromoglobin. Ml'thremoglohin is the true oxide of hremo-
globin, oxyhremoglobin heing an 03'!Jgenated, as opposed to
an o..l'idised, deri\ati\Fe.
The oxygen cannot be puml""l off from methamtoglobin as
it call from oxyhremoglobill, and. conversely, the formatio,,'
of metluemoglobin therefore requires ~ higher oxidation
 II.BJfO('f{ R()MOaEN
111'1 Xtl,S:O.
Hwmill 4.'- 111\'J1I -_- -+ R"duN.t! hll'W
OJ t
(;Iullin

1:\Ui~2(_)~
IJ:~m.ati1\ --- I
(I - II tf>III(W}lrOtlH'~NI
t ~
...
\), I ~.Ha'm~'('hnII1H}"I'11
r PIII\'HH'flKutiutl
. h~' 1)"
a,djuJol lllH'nl
~
~h>i hft'U\op;Iobin [{t:du,:.tI j )'l { h,\'h.tt'tUtI~
iUl.'lllllg1ohin <Ii :,:.llIlIin.

potf'utiul thaH that iH'{'{' ... sary J(tr tl1{' prodlldioB of til('
oxygt'lIutt-rl d{'rh'ntin:, oxyh:cnlOgfohill. But whilst moli'-
("ular ox:rg(n.i~ unahJ(' to ('()ll\'('rt hWfUogJohin intn mt" hH;mn~
gloi1tn. it will ('onn~rt both rt'dtH't'd h{l~lll alld ha:Hl(whrhtllOl:f'U
into UU-'ir truc' oxides, oxidise-a ha:rn and hwnwtill Tr..,.pC(-
t1,,<:'Iy. It is therefore apparent that l\eith('r tHem Hor ha:!fUO-
ehromog'el} is ~ujted for tilt {~arrjaR'{' of ()xy~efl in th(' bJoOtI.
One r('quire~ a compound whi('h forms u 100M' ('omhiliutioH
with oxygen, and from whieh the latter is T('aclily Jj}wrat('d
at the low oxygen h'nsions of the ti!'t!o.u(~. NntuTf' tis'"
obtained such a suhstancc~ by fir~t (,{)tnhinill~ ha:m wjt II t lw
protein globin, so forming a-ha:mo('hrotnogeu, which OJ!
polrmerisation finally yields h"'lllog1obin, It is proh~"l<'
that two fomls of ha:,"ochromogen cxist. Those han' "':ell
called the a and f3 forms, and it seems likdy that the f3
compound is an intermediate product of th(' p()I~'merisati(Jn
process which leads to the formation of h",",oglobin, Ha:1lI
is completely insoluble in water at about P. 7,0, whiM
a-hremochromogen, ,B-hremochro",ogen, and hremoglobin
constitute a series of compounds with increasing solubilities
near the neutral point, and therefore with increasing gas
carrying capacities. They represent successive advances in
the produdion of pn ideal respiratory pigment. Accorrung >
270 H,EMOGLOBIN
to tilt )'('st ~lUth('nti('Rtt~d determinations. the JlJOI('('ular
weight of acid Immatin. and th('refoTe of a~}m:m()('hromog'{'ll,
is about 17.0()O, whilst that of ha~moglohin approximat{s to
t)S.ooo, \\'e may thl'rt~forc assume that four fHl'mochroJ1logcB
units v,o to fortH one hn.:moglobin molt'(uIe.
:'\ltt'li~ IIjtr(lg<'llou~ s\lh~tall('('s other than globin may unite
wit h f('dll('('d tuem to form lucnu)('hromogeIls. Amongst thc.!o.c
aTt ot}}('r prott'ins. amino adds. amint's. ammonia. hydrazine
hydrate, pyridiw:, ni('(ltim', pyrroJ. etc. The 111('mo(.'hromo-
gl'll~ ohtained in th(~s{' install(,-{:,s. whilst they all gh'(' sp<'ctra
'Try similar to the familinr luclllochromog(_~n spectrum. differ
from onC' ullothn in rt'spcct of the mean wa\'cIell!.rths of the
maxima of th('ir a-ahsorption hands. Thus in ammonia-
hll'llloC'ilrolnogt:'Il (i.t' .. the ('oillpound fornwd by ammonil;1 -+
hwm) the position of maximum abh.orptioll of tlie a-hand
lie!. 26 Angstri:nll unit.... more towards the fed than the
('(~rf(!-.J)(mding maxizuunl of glvhin-h:z:'mex'hromogt'H ,{i.!'.,
~lohin -~ htl'HI}, and th(' other hll'mo('hromogen~ show
(lifkrenc{'s of a similar nature. G1obin. howt'H'r. has a nnl('h
gr('at~r affinity for hrem than any other nitrogenous sub-
stan('e so far ~xamined. Of those mentioned above only
pyridine Hnd ni('otine possess an affinity at all ~omparnblc
with that of globin. It will be observed that the differe,wes
hetWf"f'B these artificial h:cmoehromogens aft' akin to those
which ln~ harc Sft.'l1 distinguish thf- hlf:mogJohin of one species
from that of another.
Mnny of the specific differenc<'S between individual hrcmo-
globins are either lost or are much less marked in the
hR'lllo('hromogens prepared from them. Thus. whilst the
illl'llloglobins of rabbit and sheep blood haye very different
atlinities for carbon monoxide, the corresponding bremo~
~hromogens are not detectably different, eyen though the
two globins present are known to be slightly different. More-
over. it would appear that slight ysriations in the gIobins".
which produce measurable shifts in the ab'lOrption bands of
the bre:moglobills, do not give shifts in. the corresponding
 ('ON PfW.v JlS OF IJ .EM :!71

JInJlI{l{'hr(Jrno#-.1(~n hand~ wh;eh ~tr(' gt"{'at ('lIoug'h to fl(' d('kdt'd

hy til(' HaT'trid.~ J'('\'('rsion Sl)(,(troMolw.'. On the nthtr hlll1<l,
til<' (orrespondillg hlt'Tn<-; ohtainahh' from tilt' fllhlJit mitt
~h~('r hwltloglobins mentioned nhoH' are ahsolr,h'ly idi.'flti('ul.
Rnd suC'h i", ah~<\ thl;> ('US(' in all ha'IlIOI.!'iniJiu!'> ~o far ('xnl',itwd .
\Ve m3:. tlwT'('fore infer thnt t Jw dwmlc'al hn"lis 01' 11w
... p('(itj('it~ of the hll'nwglohin.... rc~id<~ ill tlw t,(Johiu", and thut
!ootiH W't'nt('r moditkatioH' in the prop(."rtit's of tfw t't"piratnl'Y
pigment mntc('u](' w()uld lw df{'ct('d h~' ~uh~titl1tion of otlwr
IlitrogcllOl'" . . uh.... tNIJ(('~ for tIlt gJohill.

The Oecurrence of Compounds of HaIIll in Nature

That ~atlJr(' h",." utilised .'oOJllC of tht po..,; .. ihilitit" lIH'fI~
ti(l')t"d in th{' for('goiHg paragraphs i~ ~h{)wn hy t 1)(" ()('('UrTl'II('(.'
of s{'verat types of pigmented {'ompounds other Hum hlt'1I10~
globin. hut. Jikp it. R~-~'J-()('iatNj 'with r{'~pjTfitory or oxidati, f'
pr<J'esst>s, Certain of the,t' pign.cllh. hear a ('Io~(' filTllily
tescmblanec to ha~moglohin in that th('y ('olltnill hn'Ill. hilt,
Oil the> oth(~T hand. the gJohill i~ N'plac'f'd hy quite difff'T~'n1
J!roups. Htcmoglobin is really 8 highl~' sfJ("("iHlis('d d(\'(')op
llwnt of a partiC'ular type of pigmtnt. and it is tmturul thnt
in the lower animals and in plants. Wh{'T(' tlH' pT('\'aj1inp'
ef)\"ironrncntal and oth('r cOlldition"" Rr(~ \'(ry dil1crf'ut from
those- of the higher animals. much mor" fllUdanH'HtaJ v8ria~
tions aTt' euC'onnterpd than t h(l.~f' a~S(){jatf'(j with ttl(' ",Jig-ht
yet signifi{"ant differences in the protcin resiciu('s of the
yariolls hremoglohins. Amongst. naturally O({1.uriuJ! pigw
ments wrueh COIDt' within the SMfK" of tht>se obs('r"n1'iuns,
and which possess the same h(Cm as is found in h~ magJobin.
are helicorubin. actiniohrematin, al1Q cytochrome.
BeIioorubin.-Hclicorubin is really a naturally (",curring
hremoehromogen. It is found in the li"er alld gut of the
snail (Helix pomatia) and in other puJmcnate molfu"cs. It
i. also found in the liYer of the crayfish. Helicoruhin has a
spectrum of the same pattern as hremochromogen (globin _j.-
ha:m), hut its ba..d. are shifted somewhat into the red as
272 HJE.tlOGU)BIN
(omparr-d "'ith tht, iattf.'r pig-ment. A shift in thf' same
dir<,('tion is fr<,qlwntiy m"t with in mHny of tht> artiflcial
h.{;mochromogclls wmeh may he prepared from hR~m and
th' "llriOIlS nitrog-ellolls compounds !lIre-ady Il:lt'ntioned. On
r('mo\~n~ the C'ompound with whieh the hrem of he1i('ofuhin
is ('omllint'o, and on repJa('ing it with ammonia, as An~oJl and
~firsky have done, tht're is obtained an artifirial hremo-
chromogen (ammonia + hrem) whioh is identical with the
('orresponding ammonia-luemo<~hromogen prepared from
ha:moglohin or from any other ('ompoulld in which hR!nl is
known to {)('('ur. Ont' must ('oneiude that ht'lieorubin ('on-
tain!>. ttl(' SaUle ha~m as hremoglohin. It is noteworthy that
ttl(' influent'( of altered PH on the affinity of helicorubin for
oxn~('n is just the' opposite of that of a similar alteratior of
IJ n on the amnity of hremoglobin for oxygen. On the- other
hand. artificial pyridine-hremochromogcn (pyridine -+ h",m)
shows a lwhavionr yery sinliJar to h('licorubin in this respC'et.
Heli('ofllbin in a sHghtly acid medium. such as the gut of th("
snajJ jn which it occurs. can combine loosely \\-lth oxygen,
t hat is to say. under these conditions it forms not a stahle
{)xid('. hut an oxygenatf'd compound like oxyhremoglohin.
In ('ontrflst to its beha\'iour in acid solution. its affinity for
oxygen in an alkaline solution is very small. Although we
cannot regard the problem of the function of helicorubin in
the pulmonate mollusL'S as definitely settled, its close physico-
"hen.ioal similarity to hremoglobin certainly suggests that it
is corrdated with respiration, even although it cannot he.
shown to C'irculate in .R nuumer similar to the respiratory
pigments of the higher animals.
ActiniohlBJD8tin.-Actiniohrernatin is another pigment of
the same type a.~ h~1ieorubin. It occurs in eertain aetini8!,
and from it there can he prepared all anunonia-h...mo-
chromogen identical with that obtained from hwmoglobin
and from helicorubin.
C)1oeIu:ome.-ln 1884-86 lIIacMunn described a pigment
which he called myoh""""tin or histohW'l1Btin, and which
 (' r T(W 11 flO JI ;:
lutd :1 wide di ... trihHtioH ill th(' mu/\('/e...Hld other ti .... fj(' .. of
.dlllo~t nIl orders of animals. Ht wa~ not ahl( to h.olntt- tl~i ...
pigmt'nt ill an ulicluHI:,!eci ('Ollditioll. hilt IH' l<>how('(1 thnt it
('(ntld ('xi~t in a r\,du("{'d and in nil oxidised fotlll. til!' forult'r
hn' inR 811 alnjorptilm ,.."n(trum "'ith four humt.;. whi,.t.;t Hi('
iattt'l' showed no ah!,>(lrption. )lue-:\lullll ",us nl~tI dt,lt to
prc'par(, from hi!-. pigmellt compounds whi('h Ill" rC'ferrt,d to
as acid hll'matin and ha'matoporphyrin, lIt, t}u'rt't'oft'
c'ttabJishcd its Jiualogy to Juvrnog}ohin, but daimed thut
it was distinct from the latter pigment.. Mtu"llIlI1t ... result!'.
\H'n' ehnll(nged. and other work('ni !'!tah'ci that his " rmJ{lifit-d

myohH:'IIIHtin." froIlI \\'hieh he I)Tepart'd Iti ... tleid ha'matill

alld }ucllwtoporph)'rill, IHl,.,' .~imply hl{'/H(J('hromo~f('n d('rin'd

Fw. :~4. Ah~orption "pf'Ctnllll of (',ylo{;hrOflW III thota.. ir mw.d.." hf II b" .. ,.

(Aftt'r Kt'illn.)

from the ht"Ctuoglobin present in tilt.' tissu('s whi('h II(' ('X-

amin('{1. Keilin ha~ reef'ntly r(-(xamimd the qu('~tiol1 of the
('xish'nc{' of a suhstan('(' of the typ(' dt'srril-wd hy ~11l('~fl1nn,
making use of the rnicl"ospeetros(>opl. and the HUl'triilg('
rC\'crsiOll spectroscope for the examination of the pignunt
as it occurs ill fresh tis')ues. H(' tind~ that the pignwHt do<:s
t~xist and has a very much wider distribution and !Jignificallee
than e\-'en ~fac}{unn antidpated. KciHn calls his piglJwnt
cytochrome.
In the animal kingdom cytochrome is found in all typts
of animals-Turbellaria. Oligochretes, Nematod,s. Mollusc'S,
Crustacea, M,rriapo<is, Ara('hnids. Insecta, and vertebrates
.ueh as the frog. pigeon. guinea-pig. and rabbit. It is pres~llt
in the muscles of the last-mentioned animals together with
the muscle Iucmog1l'bin. But its di.1:rihution is not limited
J.
274 H.iEMOGLOBIN
10 tilt lIHinml world. HfI(l tht'r('ill lies its !-.p<'('inl ~ignifi('alH(.
It is found in had('riH. ill ~'('a!\t. find ill Hw higher plants.
The typi(al appeurau('(' of tI)(' ahsorption sppdrum of C'yto-
ehrOIlI(' is M'('1l it! Fig. ;-u.. whidl show!i the positions and
appr0Xcimu.t{ int('n~itif'~ of til(' hands as s('en ill the thoradc
Illuftdd of a bcl' ('XUJllllH'd in the mi(ro(o,p(dro~(,()pt:. It will
b(' Uh.lOCT1:l'd tlVlf 011(' of tlJl's(' }mJJds really (oIJsi.st.~ of tJlrf'('
lit'li("utl' hands Ilt-arly f'usl'd together. TIl(' bands Yury
.. lightly in thdr f('lativ{' densities and in tlwir t'xad positions.
('ytoehrouw is re-adily ('OuH'rted hy Uw oxygcu of the air,
or h~' nwans of potHb~ll1m ferri('yanide or hydro~wn pt'roxidt'.

t'w. 35.-1hagram "howing the tllrt'C' hll_lfll~l{'hr"Hm()gen l'('tuPOUfld"l rt'. '/.

and elf of C'ytocbrome. (After Kt'ilin.!

into its oxygC'nated form. which shows no distin('t absorption.

It is reduct'd by the tissues thetnsclvcs if air be excluded, or
by the addition of sodium hydrosulphiie.
On adding alkali to cytochrome the appearance of the
absorption spectrum is that of a typical hremochromogen.
Fischer and Schneller have obtained porphyTins frolll fresh
yeast, autolysed yeast, and putrefying yea.-.,:t, and, on tre-ating
with pyridine the re!<idue obtained on evaporating an extract
of such yeasts, a definite hremochromogen spectrum was
observed. Tl,e spectra of prophyrins b....e also been det""ted
by }'iseher and his co-workers in grasses and in coconut milk
free from chlorophyll. It seems highly prohahle, as Keilhi
suggests, that the hremochromogen and porphyrins which
 OY"()(,IJROME 271;
have \)('('f1 ddt(ted ill tht'!o.( ~o\lf('('s aft' dtri\ (>.(1 from (\'t.{)~
(hron~{. .
Kdlin ha~ !o.hown thnt feuHv tOIl~I:-.t~ of tllt",
('vtot'hrOlllt'
h.t:mo(hrolllogr-IIS. or of tw'() IHeln()('hronlO~'lls and of 1\ third
simllar substan<'{'. ~:u('h 011(' of thes(' (()lIlpOlltuts j~ n
spolI!'tihl(' for one cac,h of tht. 0. b. and (' hanos. nlHl.~~o for
Oil(' of Ul(' constitll('nt bands of th.., ('omposite d band. n~
,.,hown ill Fig. 35. Two of thes(' ('ompOIwntl'o, nOI11('ly. theSt'
""hi('h gin' the band d'l hands and tlu' (' and d'J h.mds rt~:-.P((
tivcly: yield the same pyridine lucmo('hromol-.Pf'tl (pyridint -+
hrem), and thenfore Ulust ('ontain tlw same ha:-Ill. Thf' ('xud
uatur{' of the third component has (lot yet h('C'fI (stahJi&hcd,
hut it, appears to ('ontain a dHfefeut ha'm from tJl(' other two
(>'omponents. "'' 'e
can now see tha.t va.riations in the- pro ..
fXlrtionate amounts of these t hre(' eompoltcnts. and in UI('
extents to which t'Bch is oxygclIated, wiH llccount for tht
diUerClH>("s OhSCfl'cd in the relative iutellsities of Uw haud!,'
of cytochrome oc('urring in diffcT(.'nt tis~ut,S or ("f'US. ''''("
therefore have in eytochromr a. compound of }H.cm-~ tht same
hrem as is found in hremogJobin --with two diff('t('lIt nitrogt>ll
compounds. resulting in the formation of two diff('rcnt
h:emochromogens, whilst a third component ('outains a
substance different from. but possibly related to, hrem.
It is therefore clear that most, if not all, living animals,
and also nlany plants~ posses!; the constituents from which
bremoglobin may arise, and from which it would certainly
seem to Ita,'e developed in tlK' course of the <'voJution of tbe
bigher animals, And the widespread distribution of hrom as
a constituent part of respiratory pigments both in the animal
and plant worlds renders unnecessary the assumption.
advanced in order to e"plain the haphazard distribution of
hremoglobin in Nature (if, p. 252). that the power to synthe-
sise the latter pigment bas been developed again and again
by independent steps in successive types of animal.. The
question arises as to whether this primitive form of respiratory
substance really ~ have .. functional signillca.nce similar to
18-.
276 lLEJWOLOBlS
t hat of ha:III(1~lohili in the higher HuiflHtI .. , 'I'll(' r('ndjnc~s
with whi(,h ('yttwhrOIlH' i.s oXYI!('lwt,'d h~' air. thl' equal rt'Hdj~
lI{' ..... H'ith \l'hi(l! it i .. rcdw,('d h.\' the Itormal adi"itics of the

{'ell. aIHI th(' filet that. like 11a:JlIoglohill. it exhibits the

prOr){'rti/'s of H th{>rlll()~tlt"l(' p('roxida ..,c, alJ j('nd support tt?
tilt' \'ifll\\ thnt <,.\hwhrol1lt" is H~"o{'iakd with re"piratol'Y
prO(fsS(~. In th( IJlw~dc."i of the hi1-(}I('r lIlJim:.ds. where it
o('('urs along with tlllls('i{' h:elHoglohin. it lIluy. as KriJin
.. u~gf'.',t... , r{'pft'Scllt :Hl iutenlH,diHt(' step in the {'t,rriNge of
oxygc.l) from thi,lungs to the' oxidatin' Jo..\':-.tf'Hl:-' ofth(_' tissu(,s,
t Jw su('{'('s .. in' <, ('arriers" heing hlood }ucnwglobill. muscle
hlt'mogJohin, and {'ytochrome. It would therefore seem that
iu cytot'hromc there ar!' ren'alpd the ijrst step~ made hy
~l\tUT(' in dt,yrloping tIlt' typr or respiratory pigment which
has uttllinf'd its hight''>t ('xprf'!.~j()n ill thf' ha'OH)gJohi;) of tile
\'("rtehrat< unimals.

Respiratory and Other Pigments which do not

contain Brem.
]1 must not }J(' supposed that ill NatuTe the {'{forts of
nHi()u~ groups and spc('it's of living or~anisms to obtain a
r('spiratory pigment with the requisite propC'rti('s for their
indi"idllal n('eds have been contined to the type of compound
whidl we han>' just discussed., namdy, those resulting from
the rombirwtion of htcm with a nitrogen cOlnpound. Indeed.
we have just S{,,("J\ that the third constituent of cytochronle is
not a compound of ha::m. The best-known instances of
natural pigments with respiratory or sinlilar properties, which
pigments. although they do not {'Ontain hrem, are in other
respects constituted C!1l lines very similar to hremoglobin, are
furnished by the green pigments ehlorocruorin and chloro-
phyll. The former is an animal pigment, whilst the latter is
familiar as the green colouring matter of the plant world.
Chlarocruorin.-.Chlorocruorin, which i, red in concentrated
and green in dilute solutions, is a pigment found dissolved
 ('HWRO('Rf'OR/S ...... ,...
_j<

in the plnsmn of ('('Ttnin mnrjJJ(, worms h .. Jon",riuJ: to tJu' g-ruup

of the poly('ha~ks. ~1unro Fox hils :-.ho\\,11 tlmt. ulthoHllh
ehloro('ruorin dO('~ 1I0t (>ontnin ha:1I1. it", piglllt"lIt j.!roup I~
<;.ompo\C'd of a porph~rill ('om hi ned wit h iroll. lind mlt,t
therdorf' he \"('ry ('Iosel." ullied to hit'UL It h tli('N.jt~'rc tint
~urprisillg to find thnt chloroC'ruorill .l!1\'(,,, ri,c to u ... ('rh~
of oeri\'atin's (,OfT{'spondillg tl) lllt'tha:II\{lglc)hin, ha'lImtin.
hR.:mOt'hromogl'lI. and ha~matoporphyritl. TIl('s(' emupound!oo
luwC' their ahsorption halld~ !-.hiftt'ti mnrC' into th. TNI thnn
those of the "orr('sponding df'Tinltin ... I)f Jlwrnog)ohin. Hf
stri('ted as is the distribution of (hloro(,'rtlOrin. it t\(\'('rt}\{j{'!oo"
shows a striking parallelism to hll.::rnoglohin in thnt spec-Hit'
(_'hlorocruorinb {'xist. with diffcr{'J1t (lXYg'C'1l nmnith~. different
H,can )\~a\'elcJlgths of the pw-,itiou.". of maximal nh!ooorption of
t fair a-ba.nds, and with different" "'pans. Thus :ll.1j,I';colt"
Sl~ir()l!.raJ'his.
Sabella. and lJral1('li;omma ('hloroeruorilJ' ft)rm
a series with oxyg(,11 affll1iti('s diminj~hiHg in tltt' ord.'r
quoted, SineC' ammol1ia~<:hl()roeruor()('hromog(n has it ..
a-band ill It difft'rpnt positiOfI from that of lIH1mollj;)~Jm~lJ)o
ehromog('I), it is eondudrd thnt the prophyrin of (,hloro-
(,fllorin is not h~cmatoporphyril1. nit hough it j" ill HH' nat ural
pigment. linked to iron jU'it as ha::matop()rph~rrin i .. ill lm:JIlo-
globin.
Chloroerl1orin its{'lf exists in an oxidi"ed and a. rc<iu('('d
('ondition, and in these !)tatts show" absorption sp("('tra
analogous to those of oxy- and reduced hllOlTloglohin. hut
with the bands shifted considerably into the red. Oxy-
chloTOCroorin can be TNJuced by exposure to U \'Sf'uum or
on contact with living tissues. Sp;rfJf!.Taphi... ()xy-('hloro~
cruorin is 90 per cent. saturated at the oxygen tension of the
atmosphere. The pigment is Ii much I,,,,, emdent carrier of
oxygen than is Iurmoglohin. and although it is found itt the
blood plasma, the lotrer does not in the polychretL.. undergo
a eomplek circulation. Nevertheless it ..,.,ms lrighly probable
that the functions of chloroeruorin are more or .less closely
associated with dxidativc proceJ;s<"s. and it therdore repre-
278 H..EMOGLOBIN
s('nts th(" pHralkl evolution of it substancT whir-h has a <'lobe
phrsj('o~('h{'mic'al similarity to hremol!lohin.
Chlorophyll.-l'hlorophyll i, a m('tall()porph~'rill derivR
thoi' whirh has 11 widespread distributioll in grt'cll plant~.
uml hl}~ functions which lit, at the Ycry hasis of life itself.

CHLOROPHYLl.. A

CHI..OROPliVLL B

There are present in'most green leaves two chlorophylls and

a number of other pigments differing fundamentally from
chlorophyll and ha,inj! a signilkance wbich is not yet
understood. Included ill these latter are carotin, whicfr.
is a coloured hydrocarbon of the formula C.II..,
and a nnmber of xanthol>hylls having' the constitntion
 ('llLOROPlfYU 2711

C,oH Ge01)' The-sc ar~ {'lassed to~ctllt'r as rnrntittoith or. Otl

a(~c()unt of their soluhility in fnt !'ooolqnb. n~ lip(whrollle",
Th('Y do not. )loW{'\'('f. ('ornt' wit hill flu' .,,(ol~ of thi ..
c~npter.
l'rr OW(' most of' our infc.,rmllt Ion ('ol\('('rH'II~ tilt' l~tt"tl\it'n'
llRtur{' of chlorophyll to 'ViH-..;tiltt{'T awl his ,('hoor. Two
('hloroph.vll~ arr known, alld tlws(' aTe di!'ttingui ... hl,d by tIlt
letters a lind b. "~mstiitt('r lis('rib(')o, to t}I('HI OW n",H(I"'iug
f()rmuitt' :.---

(~lgN ,C"H" I('ON II )(1'()(It'H ,l(I'OfH""H ,,) t<hlotul,hytl a

(~!~N ,C"H"O)(f.'ON Uj(I'On( 'II ,)(I'O(W,,,II ,,) ('hlllfnphyll II

Tbes(. two ('ol11pon(>nts of (,hlorophyll ('xhihit, lik(, thl' hlood

pignH_"u(, charaetrristi(' ahsorptinn ~pe(tru. whidl ar" shown
in Fig. 36. It will lU' S{'CIl that hoth pignH"llts show ~troltg
{'un ahsorption jn the vio)ct region. an ohst'f\'atioll whh,h hn ..
an important Jwaring on th('ir fUflction~ ill HI<" gre(,H pltud
cell.
On actinJ,t on dllorophyll (J with cold alknli (and ('hloro-
phyl1 b gh-cs a similar st'ries of n'MJlts) th(' (ster group'" arc
hydrolysed, mcthyl al("ohol and phytol. C"II"On, urI'
Hberated, and ehlorophyllin a i!'t ohtaillC'd. Thc' hltt('(' ha:'.
the composition (MgN.('.,H"O)(COOH),. a"d on furll ... r
treatment with hot alkali it vil'lds ,"anous intC'rm('diafl'
phyllins, and finally rliophylli;'. C,.H"N,Mg. Treatnwllt
with acid remo\"es the magnesium aud tCtinporphyriJl.
C31 H,.N., is obtained. If, on the other hand, chlorophyll "
is treated with acid the magncNium is rt'nloV{'d hefof(' the
",,'ter groups are hydrolysed. and the produd obtai ....d in thi,
ease is phreophytin n, (N,r.,H.,O)(COOCH.)((,OOCIJ..).
Subsequent treatment with cold alkali yields metbyl al"Qhol,
phytol, and phytochlorins of the fomml", (S.C"H O)
(COOH)(COOH), whilst further treatment with hot alkali
gives rise to a series of intermediate porphyrin derivath'es,
and finally rlio)lOrphyri'l idt-ntieal with that obtained from
 1i..'EltfOr:LOBIS

chloropll.dJin (f. Tn :t'tioplJyHin 'rill~tatt('r and .Fi~('her

hn\"{~ as('rilwd thf' ('ollstitution :

CH=C'H
I I
CH,-C-C"" C-C
"
II /N N;'- II
C.H.-C-C ,. "C-CH
II II
C ,C
I .. : ,
C,H,-C=C ',; C=C-C,H.
i
CHc('=C
>-Mg-r< C=C-CH~
I
'I I
CH, CH.
,l:tiophyllin {Will~4ntt('r and l"Hwher). F

One hm. only to ('ompart' the nhon"'> formul:l ",.. ith that of
hwmin ~iY('n on p. 2(17 io seC' tht' elos(' similarity of the
hasal porphyrin:.. of h~cmoJ!lobjn and chlorophyll. Onr of
tlw ('Sst'ntial diffcrt'll(,('s is the O('curr{'ll('(' of iron in the
former pigllwnt, and ma~'1l('sium ill the lattpT.
llhytol. the nl('ohol whi('h f'st('rifi("s OH{" of the ('arboxyl
J.,TfOllPS of ('hloroph~ll. is an rt.fJ unsaturated primary alcohol,
and this interesting and ('uTions substance is supposed to
han' tilt' constitution :~
CH,-CH-CH-CH--CH--CH--CH-CH--t'=C--Z'H,OH
I I I I I I I
<'H, CH, CH, CH, CH, eH, rH, CH,CH,

Theord.i('&lly one should obtain from a glyen amount of

chlorophyll onethird of the weight of the latter as phytol.
but in actual praetiC<' the yield is usually lower. This is
probaLJy due to the adion of a hydrolytic enzyme, ., chloro-
phyHasc," which OCCUl'S ill green lctl"es 8ud which is Bcth'e
in alcoholic solution, ("Ollvcrting the chlorophylls into"
chlorophyllides; that ohtainoo from chlorophyll has the
 PHOT08YNTH~;8[S 2!l1

"omp",ition (MgX,C"H 3 .O)(COO(,)I.l(CO()(,1l,). WIlI'1I

the dllorophyllides aT(' treut('d with n('jd~ tht~, ll)~t' nUl!.!-
I)r!-.iulH and form ph:l'ophorhidt's of tht' gt'II("rnl j()rlHllln
(X,C"Jl"O)(COOH),. in whi"h It l'l'pr<',mh til<' aknho\i,'
group. Dir<'<'t tn'atnJ{'nt of phR'Ophytili (/ with hydro.hlorh
.. ueid would also yidel a pfw~ophorhid('.
Chlorophyll and Photosynthesis.- Alt hongh tlu' {'Ull('ti"n
of ehlorophyH in hYf('rll plants has 1)('('11 n'('{lglliM~d for IHiHly
yl'ar~,the m('chanism of the photosyntlll'ti('ul pr()('{'s" ill
whieh it participates is stjJl a matft'r for diM'IN'l-ion. It"
prest'H(,(' (,HabIt,!' the ween plant to utilisr the rnriiHnt {'It('r~y
of sunlight. prineipally that of thf ~horkr wu \'t'lt'Ilj...1\ iiI'., to
"Yllthesisc complex orgalJir l'onJpoumh from ('arbon dioxifh'
and. water. Monosue(harides Rnd Slll'ros(' afC' tunun~""i lh('
tirst Tc('6~Hisahl(' produrts resulting' from thi... pltot()~
syuthf'ti.('aJ adj, ity. whilst stareh UppNIT.... lllt('f. A('('ording
to Bh(ycrs original hypothesi~, the nr~t sit'" ill thi", pr()('(''''~
i .. the reduction of earbon dioxidt: to fortlluldt'hydt'. alll1 tilt'
subsequent sh'ps in\'okc the polymeri!toRtion ofthi ... ",uhstall(,(,
to a hexose, and so to di-, trl-, and poly~sa('('harhlt,~, Thl'
f{'duction of carholl dioxid(' to formaldfhydc i... a markedly
f'lJaothermic rcu.etion; lu:nc(' the Il('('('ssity for H large input
of enC'rgy in the form of light radiatiolls. \\0(. ('an ther('for('
regard chlorophyll 88 a phot()('atalyti(' {,lIergy trall!'lfornwr
(Ba1y and assodates). or we may T(gard it a!', participAting'
directly ill the chemical transformations by which formald,"
hyde is formed from ('arbon dioxide (l\'iIlstatter and Stull j
Maquenne). The process of photosynt hes;s i""ol\ os t he up
take of carbon dioxid(~ and the cV'Jlutioll of oXYJ!(o. hut at
the same time til< plant cell is also respiring. that i; to say,
it is absorbing oxygen and gi \-'ing off earoou dioxiu('----il
process ill which cytochrome may play an important part.
If a correction be made for the respiratory proc,'ss, the ratio
of the carbon dioxid.,. absorbed to the oxygen ""olved
l'f'presents that portion of the ga.<;;cous ex('hangc of th(> })Jant
which is due to photosynthesis. This is known a, the
282 HIEMOGLOBIN
,. ttssimilalory codftcicHt," and its valu(' may he obtained by
gasomrtri(' rndhods. A st-ri('s of ('arC'ful mf"RSUrcments made'
hy \YilIstiitter shows that this ('o<'ili('ient has a \'alue of unity
\1I1<1('r wicit'ly ,"ar_villg ('onditions of tcnt}wrahu{\ streng1h of
illumination. contellt of ('arhon dioxidf' in the atmosphere.
and'i:\ wielely dif(rrt'nt plant spe(jps. FrOln this important'
ohsrrnltion \VilJstiitt('r 1Hld Stoll (onclud( that tJif' oxygen
of the uh!'orlKd carhon dioxide is entirely ghTen of! as
Hlolc("ular oXYg'{'fI. whilst the dcoxidiscd ('arbon fi>!i:iduc
r(>tnains as slIC"h. or in a hydrated form (formaldehyde), in
t h, plant ""11. r.... avcs which do not normally contain chloro-
phyll han" no assimilatory power, but, on the other hand,
('tiolat{'d lean"s of grecn plants and yellow leaves show an
lJH('xpedf'dly marked assimilatory power. The further
ohset,"ations t hnt neither the isolated C'hJoroplasts nor
('hloroph:vll in eolloidal solution assimilate carbon dioxide
nil point to the presencr in thf' 1eaf of some agent otherthan
chJorophyll whieh regulntcs photosynthesis. This agent is
g<'Jlerally suppo.'\ied to be of a protein nature. Here we must
1co\'(' these fascinating questions. suffi('ient having been said
to indicate thf' nature of the proc(>ss("S in which chlorophyll
pnrti(ipates. It will be seen that the functions of this
magnesium~porphyrin compound differ considerably from
those of the iron-porphyrin compounds of the hlCmoglobin
type.
Heemoeyanin.-"c have not v('t exhausted our informa-
tion cont"'('rning t he efforts which-liVing organisms have made
to s{'{'ur(' substances with respiratory or similar properties.
HR!"moryanin furnishes an instance of a pigment with proper-
ties al1alogous to those of the pigments to which we have
alff'ady ff'ferred. and into whose composition a heavy metal
('ntcrs. This metal is copper. and its occurrence in a com-
pound ofthi. type is ofpanicular interest in view of the well-
known toxic action of compounds of copper on many liviQg
organisms.
Hremoeya?in is fonnd only in invertebrates, in which its
 HJEMOCYANIN
distribution. lik<.' that of hR'moglohill. is TfI,1h('r ~rrnti(. It
is found in the hloods of ('crtain arthropod . . (t'ru... ta(('u Imd
arachnids). and in certain mollusl's (gnsh>ropm\-; nlHi (,l'phlllo~
})()(i,'i). It ('an form )008(' ('omhiuntin)}s with ox~~('n. Jlull III
thr form of oxyiuemoryanin jt has a blue (,,,Jour. whi',..,t lit
'ht rnlu('{'d condition it i.'1' (olourlcs.... It ~(>('m~ highly
prohahle that here again W(~ hun' u pignH'lIt whidl diUl'r. .
slightly but definitely from spedes to s!',,,i,s. '1'1"" :\I,lwrl(
and Clark diCf('rt'ntiatt" the tucmocyunin of IAmuiw; from thut
of Octap-u~+; on the grounds of a diftcrf'lW{' in p(f{entuf..'{'
composition. anel in prc('ipitation rCR('tions with suturntrd
magnesium sulphate, with half-saturat<>(J ammoniulll sul~
phate, and on dialysis. The two hre1ll{)('Ylluins also ('xhihil
dif!('ten{'f1 in their tendencies to ('rystaBis(' amI in t h('jr
!oIcnsith:ity towards acid. Rc>dfield ami Hurd also ObSf'Tn'd
differene("s between the hremoc,'llOins of Limulu.,< alld [,oli{.!fI.
Sted:;"an and Stedman ha\,('" ;c('cntly in\'cstigatt~d th(' dis~
s-o('iation curves of hfCnlocyanin from th{' hlood.", of {<IlIT
f.>J~cies of crustacere. namely. lIomarus, Palinur!ls, Jlolo. Rnd
Cancer. They hav(' shown that, whilst the oxyg<'11 cnpn('iti(>~
of the four hloods differ widely, the l':ihap('s oftht' diss()(iation
CUf\"("S are \'cry similar, and when thc' }x'T('entag<' liat uration
is plotted against the oxygen tension thr points ohtaiJwd ali
he on one curve. ender tempt~ratur(' conditions 'whif'h
approximate to the normal for these animals the hremocyaHiu
is about 85 per cent. saturated when it i8 in equilibrium with
oxygen tensions of 20 mm. At lower ten~ions dissol'iatiolt
would occur, and thus oxygen would be available for the
tissues. It is concluded that hremocyanin is capalM "I'
exercising real respiratory function,. According to I)h'rl.
the amount of oopper in the blood of animals in which
hremocyanin occurs varies from 3'5 to 23'7 mg. per lOll c.c.
of blood, B variation which amply a"oounts for the difference.
in oxygen capacities observed by Stedman and St.edman,
Hremoeyanin has about one-Courth of the ('()mbin;ng eaplK'ity
oc ruemoglobin for oxygen, The ease of th~ snail (/leUr
284 I1.ElIfOGLo"BIN
pOlilalia) i~ ofilltt'r{'~t. SilH'fboth iuern(l('Y8IliJl and heli(,oruhin
art-' found in iti- ti!o.su('s.
J ra~l1lo(,y8njn and inclllOgiohin show a very ('Io~{' ('orre-
1'>polldellC'{' in their J){>f{"'ntag(' ('ompohitiollS. as i~ shown by
111(', ~)lIowing Hnal~ti('al tigur!', :

I )"ylul'lllOl-{lohill

(hork(" ii4'j;j 6ft8 2012 I 'i .:~.) (l42 O;JS!F,)
Oxyhff"1Ho('\'allill
. inrtHpllJ'.) :;;H.i6 7:t1 2167 Ji)on (186 (l:JI<(('u)

This ('orrespondt'l\ct.: certainly suggests a dos(' chemi('al

similarity hrtw{,(,1l the two (ompounds. and I'hilippi, has,
jud('ed. i~olatt'd an amorphous pigment group frnm lucmo-
(,YHllin whi('h would ('orr('spond to hit'matoporphyrin. and
whieh gives pyrrol 1"eactions. It is ('('ruinly all attr('tive
hypotlwsis that in hremo('yanill we s('(' "Xaturr's experiments
'with a {'oppt'r-porphyrin deri\"ativ('. ('xpcrill}t'nts wweh werr
appart'ntly not ~uffi('it'llt)y ~n('('('ssful to enablf:' ha~mo('yanin
to compete with the iron~porphyriHs us r{'spiratory pigrnents
::.uihd to the IH'eds of the higher animals.
Pinnaglobulin.--Jliullaglolmlin is a brown pig[m_'nt whi('h
is pr('se-ut In the blood of the lam('ilihram'h Pinna .'iqtIQJ1WRll.
It is stated to be' Yer)' similar to hff!mo('yanio 1 but {'ontains
man}!anf's(' in place of ('opper. So far as is known at pr("s(.~nt.
this pigment is the result of 811 isolatcd experiment on the
part of ~atur('. sinc{" the presen('{' of manganese has not been
f'portcd in any other animal species. It cannot yet be stated
how the manganese of pinnaglohulin is ~omhined with the
protein part of the mol('cuJe. nor is it known whether or not
the metal is associated with eompounds of the nature of
porphyrins.
The PorphyriDs
The feature which is ronUJlOIl to tile luemoglobjns~ the
chlorophylls: alld similar substallC('s is< the presence of a
 porphyritl, and tht'rt'fort, ('om,i(itrnhlt int('rt'~t aitudlt,,,, to
the Ot'('Ufl'('!H't' of' frec porpil~'ril\'" ill :\lllllr('. \\'1' HlU"'! flr ... t,
howen'r. Tl'f't'r to ('crtaill tlf tht, nUIIH'rou'" porph~-rill.., whit'lI
han he{,1I prqlHT('d artilieiHlly,
The term" hWlllatoporphyrin "i ... Mllilt'wliut Jon..,d.", npwii(d
tp the irOfl-fre(' pig'Hl('lIts whieh are obtHilH'd (r01l1 Juf!f11I(lk
globin. ha~m()('hr()rn(lf!(,II. lw:matill. or hre-min h~' flU' lH'fifitl
of strong acids. Thc' t'XtH't ('olllpl>''Iitioli (If thes{' porph,\'rins
varies according to tht> Hlcthod of prrlmrnt;oll. Oil the hu_.. i",
of tht' formulu preyjou .... ly a,...... j~llcd to ha:wjn. it j~ IJslJnl to
a~sigll to hremntoporphyrin prol'('r S{)lll(' slleh ('ofllpositioJ) II~
t':J.1 or 3"H 360 8 X... By redut'tion with hydriodi(' 1111<1 a('dl('
adds it is {,OTI\'ert('d into )Jl('soporphyrill, (' ...... 1I 3H0 4 :'\". awl
with !llt'thyl al('oholic potassium hydroxidt, it yidd~ IUl'rJIO-
porphyrin. whi{'h is either an isomer of Jlwsoporphyrin or
(:outains two hydrogen atoms l(~s. The laU('r !'tt'('I1I!>. more
probaai(', sinc{' hrernoporphyriu i . . (onnrtt'd into 1Il(',,()~
porphyrin wheH it is treated with hydriodit' acid ill glut'inl
aeetic acid. It would ~Cl'm that lwither ha:!lIIutoporphyrill
nor mcsoporphyrin is forml"<l in the human hotly. All;
important artificial porphyrin lS retioporphyrin. whi('lt. it ...
We have seen, is the proouet t'\-t'ntualI!' obtaineo from
t'hlorophyli on hydrolysing the ('sh>r group:-. and on renHH'ing
the nlagnesinm.. But retioporphyrin is abo ohtailwd from
hremoporphyrin when the laUer is heated with Jo.ocla lirtlt.' ,
fronl mesoporphyrin hy ('ofl\'ersion iuto it.s ph:'lIin folJow(,d
by deearboxylation, anti by the action of magn{~sium oxid(>
and potassium methylate on uroporl,hyrin. a naturally
occurring porphyrin to which we shall make further rei,renCl'
shortly. These observations serve to link tog('tht~ hy dJTect
experimental methods the porphyrins of the hremoglobins
and chlorophyll.. Hremoporphyrin. its methyl ester. and
!etioporphyrin are scareely distinguishable spectros<opieally.
and their spectra differ ollly slightly from those of meso-
porphyrin and its ester. It is of interest to note that
hrematoporphyrin ha!: B ,"ery powerful light M"nsitjsing action
286 IVEMOGLOBIN
TIJH!oo. WIWB ifljt(ted into animals, it r('nd('TS thf'1lI JK'(,uliurly
"'C'llsitin' to th( noxious d{ed~ of ultra-violet irradiation aHd
ill oth('r way~ iJl('f('Nsl'~ the f{'SpOnSl' of HUWY ti\:-'lWS to light
(ultra\"iokt) ,tirnuli.
N4turally Occurring Porphyrins. ~ III not'cut years H.
Fisd'l('T find S('huOlm and tlu'ir rpSpt'('tiv<' schools han;,
dt'\'oted (ollsid(rahle att(>ntioll to the relationships and
('ollstitutions of the naturally o{'('urring porphyrins. 'l'ht's(' 4
may b(' diyided into two groups. u('('ording to their soluhilities
in ~'hlor()form ;._
(,'mull I. Insoluble in chloroform.
(1) Coproporphyrin. found iu fa.x'f's and in y('ast a.'t a.
normal constituent.
(2) {'roporphyrin, found in pathological urine .(con.
gf"uitul porphyrinuria).
(:3) Turacin, the copper salt of a porphyrin found as a
pigment ill the feathers of certain hirds.
(;nm)J ll.-~Solubl(' ill chloroform.
{I) Kiimlllt'rer's porphyrin, extracted from putrefying
bloo<j by dher Bnd acetic acid.
(2) Pap('J}dieck's porphyrin, found in freces after a meat
mea}.
(3) Ooporphyrin, occurring as 11 pigment in tl.e egg-sheUs
of certain birds, from whieh it may be e>:tracted hy
hydrOl'Woric acid.

Xo doubt other porphyrins eJcist, either in the free state

or in such a form as permits of their extraction by means of
acid reagents. But there is considerable doubt as to the
"h<'mica} individuality of the numerous members of the class
described. from time to time. Recently Fischer has claimed
that Kammerer's porphyrin, ooporphyrin, and the porphyrin
obtained. by Papendieek by the action of hydrogen sulphide
on blood, are identical, and he suggests the gene-ral name
prol.tJparphyrin for this derivative. Since ooporphyrin may
he prepared from hrematoporphyrin on heating the latter in
 PORPHYRINS 287

a yaeuum, and siuC't' in th(' pro('('ss t Wt. ntn1t't'ul("s of wutl'r UTt'

lost. it is snggcsh'd that tit(' most prohahle l'ompo"ition of
protoporphyrin is ('34H:J4()4~C' If thes(' ~tI.f.J!{':-.tiolls ~h(lliitl
pro\'{" to be well fOllnded, a {'ollsidt'Tllbl,' Hnd W(Ir'OHIf'
sinll)1ification in the dassitiratioJJ of thj' porphyrjns w(}IJJd Iw
.fff((,tfd. ,
Apart from h:l'mnto- aud m('so-pt)rphyrin~, mil:, {'l)prH~
, porphyrin and uropurphyrin han' been isol:lkd. uWtJ,\''>t,d.
alld deJiniteJy ('haraeteris('d as ch<'micul im.Hddu-ls. 'I'll('
identity of turaC"i1i as the ('oPpt't' snit of uroporphyrin iN nl;\o
fairly wd} estubHf;hed. eoprofJorphyrin is t(,rrm,if in the
intestine uuder normal (onditiotl~. It j~ also found liS MH'h
ill normal urillt'. ~:Y(,II in th(' majority of path(Jlo~i('111
con<}itions whirh arc ass(){'iated wif h porplJyrilluria it would
appear drat coproporphyrin. is cxcrt't('d, and onl~ ill ('as('~
of ('ongenital porphyrinuria is uroporphyrin found. TheM'
two .,porphyrins have very similar spet'traf prop('rti(.... und
with the heavy metals, especially with iron. tfwy f01'1I1 f'orn~
piex('s, ha,ring !,lx~ctros('opi(' propcrti('s H'ry ... i1l1ilar to UfO,e
of hremin, and forming hremochromogens with pyridim' alHl
other nitrogen conlpounds. TIU' similarity of ttl(' two pOT
phyrins is further borne out by tht' fad that uropnrphyrill
is converted into coproporphyrin on being h('atrd \\ it h 1 1)('1'
(cnt. hydrochloric acid for three hours at IS0 0 to ]x[J'>.
<':oproporphyrin has been d(tected as a normal ('OIhtitm-nt
of the sera of human subjt'ds, and of hors~s_ oxen, and fowl,_
It is also found in the freet's of individuals C'xisting on n.
purely vegetarian diet, and together with KilmOl('n~r's
porphyrin it is found in yeast and in l)('{>f durin~ sh-ril(-
autolysis or putrefaction. The presence of porphyrin' ill
yeast, which at first seemed a very puzzling fad. i't BOW
readily explained in view of Keilin's discovery in this phmt
SOUl'Ce of the various hremochromogells which comprise
cytochrome.
No general agreement has yet been reached regarding the
constitution of the porphyrins, but the following obst-nstioll.
28f1 II.:EMOfiLOBlS
rcpn"wllt tIw l)(,,,,t lillth(,llti('akd \ i{'w~. Th(' pnl'phyrin:-.
('oll .... i ... t of fll11r "'llh!-.titIl1('d JI) rrol gr"lIJl~ lillk{'d tORdhtr
through t wo.or fOllr ('arholl at o Ill:". .'Ftioporphyriu ('ontuins
110 ('Hl'iloxyl '-!'fOUP' and ha~ the' fOrthIdn (':HB3ti:\' .. ("'ill-
.... tiittN) or (';I;!H: 16 ',j 3"':\'4 (Fi..,(hC'r}. Copr(lpurph~'rin and
IIrop~rpll~Tjll are fcgHrd(,d a . . heiug d('rin-lt[, ('''' of (dio-'
p(}rph~'rjll, iu whid, fOllr and ('lght ('arhoxyl !,ffOIlP." rf'!o.pt'c-
tin'ly nre inst'l1f'd ill the suhstitueut. side ('haius of the
p:vrro! r('sidu{'s. The. . c two porphyrill'" therefor!' have
tht (omposition . . l":ItIH31i ,r :l~()fI~J. (eoproporphyrin). and
l\l)l1a6 "f :;[oI01fi~4 (uroporphyrin)1 hoth 011 the hasis of
}'j.'wla-r\ fi>fmllb',

/pC~
R-CH:rC- C, C=C-CH,
II ~N N( I
CHrC- C C=C-CH~ R
I I
CH CH

CH.C-C" II
~-C-CH:rR
II :;N NH, II
R-CH.-C-C C-C-CH.

~/
CH
One (If f lit> sug('\_~t ions of FiSl'her and H iJgel' for the ionnuJJP of
retiol)orphyrin [H 0_- eH 3 1. (opt"<.porphyrin lR "'"_,_ CHt,t'OOH1,
tUid uroporphyrin (R ""'- CH.(COOH)!J.

Th. (l{"('Urrell('{' of turacin as a pigment in the feathers of

ee-rtain birds is of some intele'st, more especially in view of the
faet that it ('Ontains copper, The futl('tions of this compound
as a featller pigment appear to be in marked ('Ontra.'it to
tllOS<' of hlt'mo~yanin, whit'h is also a ropper compound 'If,
presumably. a porphyrin. In 19011 Laidlnw showed that a
compound, syeettoscopically "ery similar to turacin, could
 ORfGlN OF BnE P1(J.IIENT 2RfI
hf' obtained from ha-matoporphyrin b.,' frt'atnH'ul with IW
:mlmonitu'al copper !>ohrtioll. The IIIOr(' rr('t'ut nnn!vst'''' of
J.'isdlf'r and Hill-,"l'>T SlI~!l..,J'(_;.st \'('ry stron~r~ that the pil..'1Ht'llt
is .th( ('oPlwr salt of uroporphyrin, with ttlt' ('()Illposition
f'4o-H ;lj p.f J&OJON"Cu.
"
The Bile Pigments
In ('1050(' ('h(,OllcaJ relationship to t hc' pHrphyrill~ Afl: tilt,
hile pignwI1t'-;, and in "i{'w of thi"i rdntioHship it i ... hut
naturnl that Onto should t(~gard the hilt piJ,!IlI(,lIts as h(~ill.l
deri\"l~d frmu hmmog}ohin or from .'HHlI( rtI:~hd sIJh",tum'(',
There is ampl(' cxperime-utaJ f>orroboratirm of thi'" ('xrwc'hl-
t;Olj so far as the intaet animal is f'otl('('rrwd. hut th(' truH,"~
formatiOIl of hremoglobin or any of its d('rivati\'r~ into f,ilt,
pigment has not yet hel'n cOll\'im'ingly df'tlloflstratrd ill 'l'itrll,
It Itas, hOW("'O., h('rn daimed that whm lak",1 1.1( 1 i,
inC'uhated with adrt'nalinc, hiliverdilJ is formed. Bru/..rs(h
alld PoHak hBxe also ('Iaimed that hy th(' R('tion of p.vrn-
('atechol in vitro h:-cmin and it,!, rlerivatin's are t.ffln~forlfl{d
into bilirubin, These claims may ht. right. hllt !<JO fur th(y
do not. seem to have h('-("I) suflidcntly inv('sti!-,rutC'd. The
('yidence upon whi('h is ha~(>d th(' stat~nwllt that JJilt. piJ;!Hlt'nt
arises in '0",'0 from hx.moglohin InaY' h(' .lHnnmari-;f'd as
follows: (I) Intravascular hlClllolysis of nd hlood ,",'lIs
produced by the injedion of distilled wau,r. hile salts. or
other hromolytic agents. leads to the t'xC"r(>tioI1 of bilirubin in
the urine. (2) The introduction of pure h,emoglohin into
the circulation of an animal with a hiliarv fhtuls in~f('a""
the bile pigment excreted by the linr. (8) Pat Iwlogicn I
conditions in wbich excessive 8mOWlh of hremoglobill arc
liberated in the circulation or in injured ti"ues, lead to
increased excretion of bile pigment or of its deri,ativ~.s, or to
its local fOID.ation at the site of the injury. (4) Lru.'tly, the
close chemical relationship hetWeetl the porphyrins of h",mo-
globin and hile pigment may he <:ited !!l' col1'Oboratin,
..
 lJ .E.MOGLOBI S
(\"jdt:'J1l/, I)wHlof!)uhin is the on]y (If-finitely kuo\\ n ~aur('('
of hilt' pigment, hut the' possibility of the formatioll (If'the
latt('f from th(' dcgradatioll prodmts of l'hl()fophyll inf,!{,,,ted
ill HR(tnhle matt('r, Hnd front thc- nHl\(')(' pignH'Tlt ('yto('}m.Hll('
(myo.k,pm.utin). is h~T no m{'H))~ exl'ludt'd. It was for)1J('rly
nfo!"urh~'d that the tran~t(JrlTlati()n of hamoglohill into bile ~
pigment took piaN' in thr jj\(r. 1I10f(~ ('s{)l"('iall~' in til('
('pithelinl ('dis of thb organ. That ttl{' formation of bile
pigrnC'ut niR.,,". and does normally. tHkt' piu('t" out<;;ide the lin'r
hi HOW WHJU<ljtiOl}('rl. )mt (Olt..;idcrahle difference of opinioH
('xists as to the sit(' of t hf' transformation atHl cOll('('rning the
uw{'hanism by which it is hrought about. \Vhipplt' and his
nssO<"iat('s still atta('h imporhm('(' to the epithelial ('(,lIs of
the lin'r in this r('spect. whilst Rich r('gard~ the hCPfltic
fUJI('tion as twing me-rely an ex(,retory one. IlHrlwly, the
elimination of the hile pigm('nt hronght 1:0 it fly the blood.
In IIddition to the epithelilll cells. the Kupffer endothelial
(,dis of the li\,f'r haw' bern regard en as playing an important
part ill the formatioll of hile pigment, aud in rcC(>nt yt'ur,'\
('onsiderablr importan('(' has bCll atta('hed to the reticulo-
endothelial system generally. to which the Kupller cells and
most phagocytic rells helong. Cells of this type are found in
th( spleen, hone marrow, lymph glands, blood strt'am, con-
ll('('ti\'c tissue, eft',. and any or all of these may be regarot""<l
as tlw locus of the {xtrahepatic for.nation of bile pigment.
Tht']'e is no satisfactory evidence of the presence in any of the
tissues mf'ntioned of an enzyme capable of efft"!'tjng thf'
tl1lllsformation t'; hile pigment outside the cell.
Th. two .most important bile piIVncnts are bilirubin and
hilinrdin. the compositions of which are expressed in the
formul", (' .H ..O.N. and (',.H O",'<. respectively. Theso
fannul", at 0IlCe indicate a dose correspondence with the
porphyrins as regards eiementBl'Y ""lnl'osition, and there is
s mass of chemical eyidence ,,"hieh bears out this relationshjp.
}Iuch of this "vidence is bast'd on the ultimate degradation
products o~taincd from the blood and hile pigments. and a
 8I'B87'ITF7'g}J prR1Wl.S
brit'f rd(ren(( will t)(' mati(' Ilt'rt' to ttl<' nutufe of th",(,
prnduC'f~, !<.in('(' tlwy hu\'(' pJa~..('d nil important purt ill tht
as~igning of {'onstitutional formula' to tilt' parl'lIt substnIH'f' ....
TIlt' porph'\-'rills tlwms~fn.s {'ollsi~t of ttmr pyrrol r("iduc:-.,
niH. it is possihl(' to ohtaill from hiliruhill and from bWlllili.
fly f('du<'fioll with hyciriodif' m'ld in g-Iadul fH'l'tit- ~~id. H
pr(Kiuct in whi('h two ofthcs(' p:\,rrol g'ro\lp~ af" "till uttuc'lwd
to OB{' nnothtr. This i~ knowfl H~ hiliruflini(, udd. rind
FiseheT aserilx's to it the following- ('Olhtitlllilm :

CH.-C-C-C,H. CH.-C-C-CH,-CfkCOOH
II II II II
OH-C,,-.f. _______ ...-------C, ,C-CH~
I'm CH. Mf
lIiJiruhinie add. on Iwing oxidis('ci with lk:rnulIl,f,!':llutk.
yiel(lS xanthobilirubinie a('id, in whi('h tlw h~'drox~'1 Aroup
of the parent add is ('oIln'rted into a ketonic' group. Oil
"lJbj(~eting these derh'atin's to more drn ... ti( elwmi('al tr('nt-
fi)eut various sub~titut(>d :-'IIHl"e pyrrols art' ohtui[u'cI, ~lIld
a large number of ~u('h eompoUlld~ han' h(,('1l i!otu!att,d nuel
identified in the degradation produ(ts of th{' bile pigmcntJ...
and of the porphyrilJ:', obtained fromh:cllloglohin. (,hlorophyll.
and their derh'atj\"t-"'s, Thus hy oxidatin' pnK'('SM'''' thefe
have been obtained from hoth hlood and hik pigwenh,
hrematinic acid and methyl t'thvl mal{'iHill1iu(', tht fOTJUulw
of which arc given below :_:__. .

Hematinic acid. ltetbyl ethyl maleinimidf',

Similarly, by reductive proce"ses the foUowing sub.tituu-d

pyrrol compounds .have been obtained, 3 : cthyl-4 : methyl-,
,,,_,
 11.-EM0{1LOBIN

a: dhyl--l : ti : tiiJHf'thyl-, :t: 4 : dinwt hyl-3 : dhyl-. and:! : .1- :

5; trinll'thyl-a : dhylpyrrob., and t: ,;: dimcthylpyrryl-:J :

CH.C-C-C.H~
II !I
CH~C" /C-CHb
Nfl
: ('thyl-4 ; mt'thyl}lyrrol. :t: (,thyl-4 :."i: dimNhylpYlTol.

CH,C -C-C.Hz
II II
CH.-C" FH
NH
2 : _, : J : triuwth,vl-3 : ~th.rlp;rrrlll

"' : [): diU\ethylpyrryl-3: PrlIpiPllll [wid.

CH~ C -C-CH. CHrCOOII

II I
He" F-CH~
NH
2: l ; diIDrthyJpJ'1TyJ.3 ; prupionjt' add.

C}tr C-C-Cl:h-CH, COOH

Ii \I
CH.~\ /C-Cli~
NH
2 ; 4 ; 5 ; trimetbylpyreyl-3 ; propionic acid.

propionic acid, 2 : " : dimethylpyrryl-3 : propionic acid, a~d

2 : "; 5 : tri~thylpyrrYI-8 : propionic acid. On consulting
 {'ROB/US
the formu];.(' I(in'n oppo. . iff the r('adt'T will h(' uhJe to M-,:' the
relatiollship of t hes{' ('ompolJnd.~ to Hit' more- ('OIlIJ11(Ox
porphyrins lwd their deri,at;v('s. tIte (,OflstitHtionnl fnnuultc
of whiph han: already hc('n gin'n. .For 11 full fU'('Olwt of ttu'
r<~latjollshi[J of {'Heh dq{tadution produd to tlu' pif(nwHt ...
from whi(h it i!:- ohtuirwd tlu' reu<h'r HHI'if ('OlhUIt f.'.~~h('r\
original pa pel's.
On subj(.('ting hilirubin to I<>ss <irasti(' r(du('tioll thull is t('
quired for the scission of the iudi\'iduai pyrroi 1I11('1('i. nH'''U~
hilirubin. C:,;aH"oOtlN". and IlI('sobifirubil\ogtll. (''};lII",Oe:\,'
art' obtained, the form('r by the use of hydr()~'n und
palladium in alkaline solution, the lattrr hy tht, ndion
of sodium amalgam. Mesohiliruhinogcn (urobilinogt'n) is
fovnd in normal bile and urine, Rlld in ('onsid('rahh' qualltitl{",
in ({'mfn pathologlC'ul urincs. It is a (>olo11rl(ss (hromogfl}
which under the aetion of air and light il'll tran~forll1('d into
tht" pigmf'nt " urobilin," supposfd to he r('spollsihl( for t Iw
strong gr(~{"n fluoresct'llce observed wh(,1l zim' (hloridt nnd
ammonia arc added to urines containing llllWh of tIl(
~hromo!,.'en, Urobilin is probably idcnti"al with th" pigm('nt
stereobilin, found in the freees. It would appear that Ill'itll('r
pigment is a simple substance, but a mixture of pih"lU('nts
fonned from the parent chromogen, It is probably th"r('for('
preferable to refer to the urobilin type of pigment rath tluln
to urobilin itsdf. Presumably a reduction prOl'{,ss for which
the intestjnAJ bacteria aN! responsible a(.'Counts for tlw
fonnation of mesobj)jrubjnogen in vipo.
Many other bile pigments are known, including choleprasill.
biliprasin, bilifuscin, and phylJoerythrin, but these ar!' J.'~,
well defined tluln those which ha,'" been mentionetl in th.,
preceding pages. The 1B..t-mentiOl1ed pigment lIB" the
composition eu or 3f,H 3flO,N_.. It occurs in bile not as su('h.
but as a colourless chromogen, from which the pigment is
formed on contact with air. A porphyrin isolated by Ki:lm'ri,
and found in the freees on both meat and wgetable diets, is
probably identical with pbylloerythrin,
 }/.EMOGLOBIN

References
t. ALSBF.HH l1.lId CLAllK . '<Hun. Biol. ('/um., 1910,8, I; 1914,
19, 50:l.
2. AN~()N and :\lIRi4KY. ,Tourfl. I'hYl'iol . 1925,60. :";0, 161,22,1.
:1. H.\W;HOl-'T. "'I'll(' Si~nifk-all(,.e of Hll'lllHgltlhin:' Ph,llsifJI.
]:et'il'w!J. 1H2a._ 412: .. The SiglJifj(~aul,{, of HWlTIoglohili
in Suh\Janllllalian Pnrms of Life," Pltysiol. Ufl'jews, IH2;l,
5, ;W6.
~. H,UC.CHOFT and R4.RCHO}'T. 7'(0(', Noy. SO(' . 1!l2:{, 96H. 28.
:i. Hlwns(,11 and POI.l_AK. Hiorhrm. Zeit., 1924, 147. 253.
n. DU":Rt. JI,>U":.'f. d,. I'h]lHiol. el d(' ruth. Gt?!., H))!), 16. 985.
j. FHWlJlm. .. {'ber Hiut llud (~aJlmlfarh8t,()tr." ft}rgeiJni8!UJ dtl'l
Phllsivl. ]UI6. 15. 1S!), And numerous pape~ published
duriu~ til(' PI1.kt. d('t',adt> in Zeit. pltysiol. ('hem.
K. }<'{sCln:n UlHl llluaaL Zrit. l,h.'l",,i,f)t ('hf'nt . H)24. 138. 49.
~ . J.'l~clllm amI ,.... ('llSELU;R. Zelt. l~ky8ifll. ('hem .. J f24. 135. 253.
10. Fox. Proc. Ro!!. Soc., 192ft. 99B. 109.
11. IIARTRlnGE. Prot. Roy. Moe . 192:~, 102A, 57il.
12. lLu<TRWGE and I~oUGflToN. I'roe. l:.'(}ll. :""0('.,102:1, tMA. :~H5.
1:l. K}O~ILIN. I'rO(:, RQ.lJ. Soc . 1925, 98B. ':H2.
H. KlhIER[. Riochrm. Zeit., 1924, 151, 4:18.
15. Ktj~TER. Z(1it. ph.llsloi. {'hem., 1920. 110, 9:-l.
J 6. !\-JJtCE]~A and ~ELJ~K.4.R. JOlJrn. Physiol .. ] 925, 60. 428.
17. PHlLlPrI. Zeit. physwl. Chem .. UH9. 104, g8.
IS. RIUCllERT and. BROWN. .. Th{'l Cry"t,aUo~'Taphy of Hamto
globins," Pub. No. ]16. Carnegie lnst. of \\'a.shington.
190n.
HI. RICH. . }<'(lrmation of Bile Pigment:' PhYBiol. Re!oiewIJ, 1925,
6, 182.
20. ~TEIlY:A." and STEllMAN. Biochem. JO!lrn., 1925.. 19, 544.
21. 'VIIIPPLE. " The Origin and Rignificance of the Constituents
of the Bile," Phy,wl. R",,,,s, 1922, Z, 440,
22. \VILLSTATTER. Bff_ dn- dtutSCR. f'.Mm. GeBeU8cAaft. ]914,47.
2854.
23. \VILI.8TATTER and F~lgGnER. Zeit. physiol. Cum., 1913, 87,
~23.
24. WILl.,';'TATT1:R. ~TOU., and eTZlSGER. ~4I1n. de-r Chemi.f?, 1911,
385, 156.
 (,HAPTEH X

THE CHEMICAL BASIS OP SPECIFIC

IlOIUNOLOGICAL REACTIOIIS
I;.; t1w prf's{'nt ('hapt('T it is propmwd to Tf'V;f'W ('{'rtnin of
the Inore purely rlwlllif'ul ph{'HOIH(>lUl whi{'h S('('[11 to ull<it'rli('
the sp('('ifi{' lUltUTf' of immnlloiogicall'ett.cotiom., Th(' method~
whi(h hav(' hiUwrt.o beN} utilis{'d for til(' innstigahol1 (If
th~s{' readions hay{' \)('('11 largely those of Hit., ph)'~i('at
chemist't>r of the hiologi,,,t, and it hits not h('('n fowal pos,,,ihl('
to (Oorrdate to allY extent tht' findin~ of' {ith('r of tfw"t
graups of workers with the chcmi('al ('ompositjon or t'OJl~titlJ~
tion. of the r>acting hodies so far as these W('Tf' known. But
within comparatively rf'C'rnt },t'ars an almost purely dwmic'nl
s{'ries of il1vestigations has yielded reiilllt" of tht' grcukst
importance ill contributing to our understanding of til(' hasal
mechanisms of' immunologka.]_ readi()n~, whidl, in \'irtue of
their marked specificity and great sensitivity. art' amongst
the most remarkable of biologi(,al pro('es,,s. llefore dealing
with our main suhject w(' shall ue\'ou' snnw hrif>f (,OH-
sideration to the g'neral aspf'cts of immunology whj(~h ar('
of importance for a proper appreciation of the immediate
problem.
The term " immunological reactions" embrace." thos<'
specific properties which are not possessed by the nom",l
animal, but which are acquired by tbe tissues of an animal
into which have heen introduced intad baderial cells (lr
"foreign" proteilll< of bacterial or other origin. It is well
known that when It suitable suspension of bacteria such as
pneumococci, either alive or dead, is injecled in suitablt,
amount into th~ blood stream of all animal, the latter
 2!1~ IM.l1FNOLOGlOAL REACTIONS
u('qujr(,S aft(,r thf' inocuJntioll a)) inmJUnity to subsf'qut'nt.
infl>(tion hy a lh'ing ('ulture of th( saIne hacteria, and h
('onsl'<)U('ntly more or less immunt' to the specific disease
()('('asiolwd hy their prcsem'(', The blood serum of an animal
whi('h)lllS ;:wquirt'd "Udl an immunity towards a partieulnr
type I(J.' pllC'unlO('(){'('US gives, when added ill 111'Iro to a ~us
p<'HsioH of t h(' saJli(' huettria. ('('rtain highly ('haraet'fistie
r(al'tiolls. In describing tll<'se reactions th!' buet('riologist
has evolved a terminology to which Wt' must now refer.
\\Then the serum of an immu(lis('d animal. whitII is referred
to as .. illlmUt1E' s{'rum, ,. is added iu considerable, and som('~
times iu VCTY high, dilutions to 8: SlJsp<>llsiolf of the S~Hn(' type
of bacteria as WH~ ll.~d in the original .inocuJntion, the latter
may either .. agglutinatl"':' or adht're to one anothcr in
dumps, they may he dissolV(d or H )ys{'d."' or if an extrat't
of tht bacteria is added to the imnulI1e ~i('rum n prc('ipitat('
Illay he produc(d, or the mixture may at'quire tht, prop(;'rty
of inhihiting the action of fresh S('rUJn "." complt'rnent," the
suhstanl'c present in both normal and immune sera which
{'olilplement.... or assists. certain lytic reactions. The-st,
reactions are not ohtained when normal blood serum from
tlw same animal species i~ added to the bacteria or to an
{'xtract prepared from them. In the instances mentioned
.. agglutinins,~' . lysins," or ., precipitins ., respecti"ely afC
said to be present in tbe immune serum. These hypothetical
substance-oS are classjf}{"d tegcther as ". antibotiies," wlulst
the suhstanres which when introduced into the blood stream
of the living animal bring about their formation are called
H antigens." ,\"(' shall pass on to a Rent"'ral consideration of
tht properties of anti!!"ns and antibodies.

AntigeuB
Mention has been made of the fact that both li\'ing anq
d~ad pneumococci behave as antigens. Antigenic power is
ther<>fore not II function pooseosed only by the living cell.
 ANTJOEN8
lIor, as we shall see~ is it evert ('(mtined. to unih 1)(}sS{'~sitlg tt
cellular structure. Most proteins, Wh<'H luj("ded ill the "Hnw
manner as bacteria, jndu{'(' t h(, formntion of ,'11)((jfk suh~
stan('es whirh aTf' found in tht, St'TUHI of tilt' illjf'dt'd uuimal.
ol'd whieh will cau.';;:' th{' mutuul pn'('ipitation of tl)i' ('01)-
stitut'nts of inln1UIlt' st.'rum Hud tilt pnrtieulur prot<'TI' u"ot'd
for the immunisation wll('11 the prot('ill is adtkd to ttl(' ::.t'l'tIln
ill dtro. That is to say, most proh'ills will hehuv(' us UUtia(>IIl\;
inducing the formation of s}){'(~ifi(' pr('(iJlit1n~. 111 order tu
exhihit, this behaviour the injected protein must. hm\'('-I'r.
h( "foreign" to the serum of the> anirual into whi('h it i ..
injected; that is, it must not ht, a normal t'OIfl)KIIl('nt of t h('
serum. Now proteins arc', of eOUNi{', pr(,~(,Ht ill all ha(ttrin,
any' we may ascribe the immullological r(sctiOlU. showli by
the intact bacteria, or by their disitltegratioll Vrodut'fs. to t Iw
presence of thes(' proteins. It is, inde(d. douhtful if lUlY
sumtaflct's other than proteins possess auy Rutig'('n;(' propt.'r-
ties, and it seem~ probable that ,'-melt prop<'rti('s art' po!-tse!Sscd
by aU rroteins with the exception of c'f'rtain mf'm}wN of til('
group. such as gelatin, which arc deficient in nroJnu.ti{' Itminn
acid~. On the other hand. protdns suh as lA,in , iIl<'om-
plete" as regards tryptophane and lysine, but ('ontaining
considerable amounts of tyrosine, do POS!'tCfoIS antigcl,i(~
properties, Proteins which have been irrncrsibly coagulated
by heat or by treatment with alcohol arc in general de\'oid
of antigenic activity, but if heating does not lead to 10" of
solubility, as in the instance of casein, the antigenic action
i. usually unaffected. Proteins do not lose their antigenic
properties 011 being converted into soluhle ",id metaproteins.
but, Oh the other hand, the corresponding alkali mt-ta~
proteins are not antigens. It is known that the peptides and
amino a"ids isolated after alkaline hydrolysis of proteins
han, ill great measu~ lost their optiCAl activity. whereas
those resulting from acid hydrolysis still retain thi, property.
Further, the partly disintegrated products of alkaline
proteolysis are notJurther hydrolysed by suitable proteolytic
zn~ IMMCNOLOCJlCAI. REA('TIO]l;S
(,1i:r.~IJWS. Dakin has explnimd tht>~e frsults as hdng due
,
to ('nolio.;atioll ()((urring h'tw('("t1 til(' to group of til(' Jwptid('

I
linkug;' Hlld 1 he adjoining _.. cn group. wlU:f('b~' TU('('misntion ~,
!
of tht, origiHally optically B('un' amino R('id (){(,UT$. It
would tli('refof(' seem possihle' that Joss of antjg<:ni(' power
Hnd of Hl(, property of undergoing dig:estiolt hy (,llzyOH'S is
l'orreluted in ~on1l' way with rac(misat1ntl. }'rugmcntatioll
of HI(' IHT_q(' C'olloidal protdB molecule into smaller molecul('s
prorttds with rapid }os!o. of antigt'nj( power. :\IOTe-OV{'T. th(
injection of the total produC'ts n'stdting from the ('ornp 1et('
hydrolysi~ of a protf'in do~'''i not product, any 'antibod~'
formation. TIlt' fOfl'!{oing observations would st'('m to
wnrrant. the ('oll('lusion that ill order to fun('tion R!<" an
ulltigcn fi suhstalw(' rnH.~t be a solubJe roUoid of the nature
of H protein. forei,hrfl to the parti('ular animal into whidl it
is injedl"d : apparently it nmst Hot be racemist'd. and it must
('ontaill aromatie amino W:'ids. \Vhilst we must conclude in
the uh'iew'e of dt:'tiuite {vidence to the ('ontrary that nOJl~
protdn substar\('es eannot function as antigens, there is a
possihiJity thnt (_'ertain toxic glu('osides may have antigenie
properties. and the protein nature of the bacterial toxins
whi('h ari' undoubt('d antigens has not yet heen de-fllonstrated.

Antibodies
The parti('ular rt'sponse which the organism makes to any
given type of antigen varies with the nature of thr latter.
Thus bacteria Ilnd illu>ot rells produ"" agglutinins and Iysins,
proteins produt'e precjpitins~ and bacterial toxins give rise
to antitoxins. Like vitamins and enzymes~ antibodies ean
only he recognised by their effects, and not by what they s;:".
They art> p,"?bably coIloid.1. and it is _nmed, but it i. by
 ANTIBODIES
no nwaH~ ('("rtniu. that tIH'Y art' prot ('in", Thf')"' ('Ull })('
s('parated from the hlood in th(' ~lohulill frtwtiOl\ (If tilt' 1-;('Tnm
proteins .
Th(' antihodies Illay Iw diyi<il'd into two elnsM's, ill the tiT"t
of whiC'h art' tll(' ag~lutinills. prt'eipitim., I\' ... in~. find !IIiJuilnT
suhstanet's. TIH'Te is ('oB"iidf'rahk diff('ff';H'f' of op;J~i~m lJ,'"
to w}}{~th{'r th" Tcadious \-"'hj(,h ('hurudt'ri ..-..e the,.,t' ~uhstMU't ..
arc to h(' as('rj)t'd to distjfl(t chemical indhjduals Of tH
difft~rt'nt aspects of th{ re-a('tj\';t~ of a _"i-ingl(' ~uh"hUl('c. }"
many cases. uftpr the injection of a "in~k PUT(' prot<'in
alltig(~n. tll{' inmHHI(, s('rtun gin's most, or ('\'('n all. of the'
wdl-known immunological rC'(u,tions. nut th('rt~ utf' ('('rlujll
discrepandes which do not pt:'rmit of' the 8(({pt.nn(( of tilt'
simple ": unitarian t ' hypoth('sis. Agglutiuntioll. pr('('ipita~
tion. and the lytic Tt'a(tioBS de})('nd on nltcratioll~ in the'
df'l,Tfce of dispersion of the rolloids ('ollstituting tlw suhshtlwt'
athtcked, but little is known cOl1(,("Tning thr 1Il{'('huuislH ()f
these alterations. In the second cla..~lt of tilltibodhs an' the
antitoxins, and these differ from those which W(' hav(' alTtady
mentioned in that they seem to rtf'utrali.,(, the t.oxin for whi{h
they are specific by dirc{t1y (ombining with til(' lath'r in a
quantitative manner. \Vhen animal ... of diffcrt'llt spt:(j(s nrt'
imnmnised with the same' toxin. thc' antitoxin formrd 111
each ease is also the Salnf', hut? on the othr-T hand. a marked
specificity is shown by the fad that Ii given antitoxin will
only neutraHse its own toxin. All lith'mph to prt'pnrc
protein-free antitoxins have so far faHed.

The SpeciticitJ of Immunological Reactions

In recent vears the main interest. of tht biodu"mist in
immunol~1 reactions bas centred round the 'IUl-"tion of
their remarkable specificity_ Thus precipitin. may h"
obtained which throw down one ot the proteins prt...,,,! in
the vitreous humour of the eye. but not the other. Again.
if a sheep or rabbit he inoculated with the proteins of human
 :lOO IMMl']IlOLOOIC'AL REA('TJON8

hlood st~rUIl1 th(" untibodies so produ{"ed will give little or no

rea(!tion with the lo,{'rum of the ape. Of spt'eialiHterest in their
bearing on this question afC results whi(~h have been obtained
with an antiwhuman !-ocrum, and whieh arc quoted by 'VeUs.
This H'rum ga.vc litti(.> or no reaction with the blood' of
Tualll\l'als other than the primat<~s, whilst ('xc('lIt~nt reactions
were obtained with the hJood of primates, a<.; shon'n by the
followillg table :---

TAllLE XX,
Allti-hUIIUW Prt'cipifatillg .\~er1iln.

Test(ld against Pr(>Cipita.te.

84 !->p('ci mf'n~ human blood lOOper {-(i'ut.

8 'p""imtns anthropoid blood , J 00
36 s}-)('cinwils blood frotu common monkey!:. 9~
18 sp('cimells blood from eapuchins and spider
monkeys . 7~
.... !-.pecim(ti!-' blood from marmo~ds 5S
2 specinWl1s blood from lemurs. 0

(Quoted from Wells,)

This spceitkity is peculiarly striking in the instance of the

antibodies produced by bacteria, for not only is the serum.
of a patit'ut sufft'ring from pneumonia specific for pneumo~
coc('us alone and quite inactive to typhoid bacilli, and vice
t>ersa. but within one species of pathogenic organism it would
appear that ('orum.only various types exist which are immuno~
logil'a1ly quitt distinct Bnd characteristic. To find an
adequate chemical has is for the multiplicity of these specific
reactions might well he regarded as heyond the powers of
even tbe most sanguine biochemist. Nevertheless, recent
developments in this field have yielded results of such a
promising nature that considerable advances in the interprt-
tBtion of the mechanism of these phenomena have already
been made,
 RES/DeE AN1'lOfJ.lis :101

t'p till th(' pr(~s('nt time the hlH't('riUUJ whl('11 hm. . 111'<'11
most ('xt{,llsh'cly iH\'cstigakd from the ~tnudpoi1lt with
which we tire (>01l('(,f1l('d i~ th(' PfH'WIICI{'O('('US. I)lIt'ullltM"o('('i
ohtaillcd from dif('rcllt SOUf('('''i frf'qtlC'ntlv prt'sent intli"ldunl
cl:araderisti(s. aIld it i<.; possil)lc' to di"i(h' 1in'nl int,_ t hr('('
wl'-flddifWd strains. whil'h flrt' refcrr(d_ to as Types .. ~. [I..
nnd III. C('rtain suh-typt,S hUH' h(,(,11 r{'(():,,'11i~(d. htlt thl"!.('
Heed not cnh'f into our prt'srnt ('onsid('ratiolls. If we' ntc'
<{eating with Type I.. tht, immune serum or nntiS('rHlll
prodm'(,d OIl ino('ulation of Type L PUCUfllO("O('(j is l'onid to
b(' an "homologous antiserum." whilst S('TUOI prnriu("('d hy
the other typcs is said to hC" . hetc-roiof.,l'())ls:'
In ]917 Doche? and An~ry showed that ill hH('krin~fr('('
filt"ates of young cultures of pneum()('o('{'u~ tlwr(' was
present a specifi{'olly Teu('ting suhstullcr whid. prc(ipitu.ttd
wit h antipnrulllocoeC'al serum of homologous t YIK'. The
S&ITJIf;' substan{'c was also found in til{: hlood and l1rin(' of
animals infechd with pneumo('oeclIs. In a stndy ~f Hit'
urine of 112 cases of lohar pnf'umonia it was found that in
625 per cent. of the eas('s attributahle to pnl"Ufllo('()('C'us of
'fyPf'S I .. Il.~ or III. a substance was cxC'rcted ill the nrim'
which reacted speeifically with homolo,r,.rous llhti})11(~UI}l(I~
coccal serum. Later Zinsser and Parker isolated what they
called " residu{' antigens " from alkaline extracts of pulv(riStid
bad-eria of S{'\'t'Ta} Y.arieties, including pneumococci. ThcS('
residue antigens were resistant to acid and to tuoat, tljd not
appear to contain protein, and were specifieaU)' pr(,(,jpjtabJf'
by homologous antisera. Despite tbi, last-mentiOllPu r.M,
these residue antigens did not produ~e antibodies wben
injected into animals. They were therefore, strictly 'peaking,
Iwt antigens. At the time ofits discovery Doche. and A,-cry
found that their specific substance is not destroyed by boiling.
It is readily soluble in water, is precipitated by acetone,
alcobol, ether, and by dialysed iron, does not diffuse through
pa:tchment, and is not affected by treatment with a proteo-
Iytie enzyme suell as trypsin. In view of these' properties
:1112 01 N I'NOL()OJrAL REAC7'/()S.~

Hdddherg<'f and A \-eTY regarded this substance as ,,'dl

~uited for the {'omTJlel\('CH1cnt of th('ir studies of the T('latioll
hetw('('n hade'rial Sl)('(ifi<ity nlld {'}wmical ('onstitutioH.
III 1H2:l the first n~ulh. of' this in\{'~tigation wen' pub-
l1slwd.,. The Imper. together with suh~('(lucnt puhli(atiol\s.
d('nlt,y;ith two of til<' ('e-Bular constituents of til(> pueunlO-
On(' of these i~ pr('(~ipitated from solution hy dilute
("O('(_'U!-.,

Ht,(otic a('id, auel i~ rei'err{'(l to as bacterial 1l11cleoprotc.;iH,

alt hough it probably eonsists of a mixture of protf'ins. The
other (()JlstitU{,llt. tiJ{' soluhle sped fie suhstau('('. is now
knowll to h(' H ('arhohydrah' of the' polysa('eharid( typ<',
I1eitieHwf.Lwr and A H'ry point out that tht'se two substances
prohahly do not ('omprise thf' whole antigenic mosai(' of thc
(ell. hut. as we shaH sec, tlwy hayC' s("rYcd to ducidatc sqnw
of tlw fundanH'ntal a~pl"ds of immunological reactions.
In\'estigations }lan' hf'f'B made of tilt, behaviour of the
1ludeoprotrin and of tht' soluble speC'iflc earhohydratc, voth
as hHiividlml suh:-.taUl(s and as they exist together in the
intaet c('ll, }::ach type of pneumococcu.'i yields a lluc)eo-
prattin possessing antigenic pr()pertii~S which on inje<-'tiou
into rahhit.s produces an antinucl('oprotein serum. nut
these antinudeoprotein sera are not type-specific, so that the
antiserum produced by ino<.'ulatioH of Type I. pneumococci
will read equally well with the proteins of Type II. and
Type III. as with those of Type I. cocci, The same nucleo-
protein would therefor<' seem to be present in all types.
It should be noted that these antiproteill sera will not
agglut.inate typc-specific strains of pneumococcll..o;;, and will
not prt'Cipitate solutions of the soluble speciflc carbohydrate
(see .Fi!(, 3i). On tbe other hand, the carbohydrate is
different in each type of pneumococcus. The following
quotation from one of the papers of Avery and Heidelberger
\\ill serve to emphasise these points: "Pneumococcus is an
encapsulated organism, and there are grounds for the belitf
that the ectoplasmic layer of the cell i. romposed of carbo-
hydrate material which i. identical in all its biologjcal
 SPECIFIC ('ARB(mY])RATES :1H:1
dWTHdeTS with thc typc'-.'tJX"C'ifl(" suh~hwc(' of InH'umH(tx(H~.
On til<' oth('"r hHud. the t'udoplasfu. or ~olJlllth' ~uh'ttH!(,I'.
('()U!'iist:-. largdr of prott'in whi('h is SPl{j(':oo. and Hot tyPt
sJlE'citk. The protein is posM'sS('d in ('0111111011 hy nil PIH'\llHO.
('o{-ei. while the ('arbohycirnh' i\i ('ht'lUi('nliy di:-,tilJ(.l Hud
M'ro}ogieully sp{'(ilk for ('i\('h of the thft'!' fixed type"'. Tilt
<'<,JI. therrfoTf'. may lx' (OJl(_(j\'(d of H-'> so "Oll~titutf'd tlmt
tht're 1." dj,"'poscd at its l)('riphery II hiJ!hly f('udin' ""Jb... tufH'(>
upon which the typt' ~I)('t'itk'ity dtp(,nds." The fUII({HHH'niuf
distindioIl, thell. Jwtwpen the two suhstHI\('t" i:-. tlUlt tlU'
lluC'i('oproteill is spcC'i('S-SPN:itk ancI nntiQ('lli(', wiu'rt'u", t ht,
('arhohydratei:r, tYP(sj)('(ific, hut TwltuntigtlIi('.

The Chemical Naturtl of the Specific Carbohydrate Substan_

Th(' method adopt<>d for the s('(ntratioll ttl' th{' '-nlubk
specific substan(~es of pn{'ultloco('eu~ is nat ItrllBy JOftg Rnd
tedipus, and th{ yield of finaj produ('t is. as might fw 3Ilti{'i-
pated, smaU, SOIll(' 2 to 3 I-,rrams from 3()O to !l:Z.) litrt's of
{'uiturc. BripHy. til(' s{~paration of the Type 1. "'I)('('ifi('
suhstance invoh'cs the following operations. Th(' pulttlrtS are
('oncentrated in 2~Htrf' Jots amI prt'ripit-Rti'd hy OwarJdit;ou
of alcohol. On ('('ntrifugiltg thre(' layC'f!\ are ohtained, the
middle olle of which ('Outains the !-.pf'('itk "'Uh~tRhC(,. Thi:-.
layer is dissolved in water, and variou!\ impuritit,!\ at'(" pr('{'ipi-
tated and removed by acidifying strongly with hytiro('hlorie
acid. After repeated centrifuging at high spct'd the' "rwIt,
~pceifi{' substance is thnnnl down by the' addition of c:hiH('(l
al<'Ohol. separated by centrifuging. and wash"d with OS "
acetic acid to remove glycogc'n and crytbroclextrjll. The
product is then dissoh'(>d in sodium hydroxide, c("ntrifugtcI)
and precipitated by the addition of alcohol in th" pres""c.
of sodium acetate. Tbe subsequent lengthy purifwstion
processes invoh'e redissolving the substance. repeated
precipitations, filtration through Herkfdd ('3ndles, precipita.
tion at the isoeJectric point. dialysis in collodion sars, and
washing with acet.one. Although no claim is made that the
3(14 INJll'NOLOmCAL REACTIONS
{'arhohydrate!'ot ohtained in thl' aboy(' and. in similar ways afC
pure ('lU'mil'ul individuals. the various preparations show a
markf'd ('onstam':v in their composition and in their physical
ano clwlHi('al propertl('.... and ('very rart' s{'cms to ha,'c bet'll
taken.to guard again!'.t the possihility that the R('tiv(' sub~
!o.ta,}(q.,~' miR"ht h(, Wi'Tl"Y adsorhcn impurities. The chemical
('hRmderistic!i, of the sfweific suhstan{'(>s !Sharply diffeT-
t'ntiatf' th{' thrt't' ('arbohydratf's. Th("se charactt'ristics aft'
gin'n in Tahlt, XXI., whif'h is con...tructcd from the averng-e
t('sult:-; and analyses T('('orcied by Hejdelher~r. Goel1t'l, and
Av(ry.
TABLE XXI.
('''nnira! Characteristics (~l
the Spffijic Carbah.1Jdratu
"'{~f1ar((I{'(1 from 1'IlCUtTWC()Ccw< 1'.lJ[Jf!s I.~ II .. ami Ill.

II.
:~~~. gi~:~:a- k~f~~~;I~. H"Iiudul( Sllgars on
HfdrolYIIIII.
on
Hytlro-
lrs!~

I. 433 [).(\.5 H 25,5 285 t 3(10" Galo.cturoni('. add,

a.mino sugar.
11. 458 6-. ().(I 683 1,175 +66'-1" <;luctlst".
lll. 427 53 0-0 732 343 _ 330" Glucose-.glyt.'uronic-,
acid.

- .. A poly!;3Ccharid(" ~f i.be compositi~n -(C,H-toO:;,.. rt":quire~- C-~-= 444 -~nd

H 0-2 pt'T cent.
_""00

I-imilillg lJi/ul;"". ill whi<'h PrecipiUdum with h(mw/ogo,,<

..lntipneumococcu.~ serum '1:0; obtained.
Type. Dilution.

I. 1 : 4,000,000 to 1 : 8,000,000
II. . 1 : 2,000,000 to 1 : 6,000,000
III. . 1 : 5,000,000 to 1 : 6,000,000

From the above table it will he seen that the Type I.

substanee ~lIers from the other two in C('ntaining nitrogen.
 .,'lPEr'IPlC ('ARBQElYDRA7'/i'", :)0:1

Despitc' thi"t, it gin's Hour of the u,.uaJ protein ('o)uur k~ts.

0" 1){'illM trNltpd with nitrous add it Ins('''' nHI'~hnlf of i...,
nitrogen. )0.0 that this proportioll is pr('\ulllahty amiuo
nitrogt'H. At the same tinlt' f('dut'il1l-!' !oo\lgur~ npptar. alut t.he
... p(.('i1i.{~ adion is lo"tt. Similar trt'atmrnt with lIitroll!) acid
, of the TYI)(~S II. and Ill. ~uh!itall(,{~. whj('h do not (fh\t.ain
lIitrogen. does Hot rfsult in lo~!<' of' th(~ir Spt>{'ifi(' a('ti\'ity.
One WHy therefore infer that at z""st oll(half of til(' nitrog<'n
of substance I. is an intt'W'H) part of tht ('ompound nud
directly .,"ociated with its sp'('ilicit,v. f'rom the fact that
the ('arbon and hydrogf'1I eonh'Ht (')os(,ly approximate to
those required for a tarhohydrah' it. is inferred that thert'
110 present in this :-;uhstauC'c a nitrogt'J)ollS SUg'Rf of thf' tyP('
of gJ_ucosarnine. Oxidation with nitri(' arid yields murir add.
which indicates the preSCHer of galad,ose, or it may f_w thnt
this acid arises from oxidation of the galacturoni(' add whidl
i~als:o thought to be present. Typt" I. substance is amphotf'rk.
The soluble specific substan"e ontailled from Typt' n.
pneUlnO('oc('us is a nitrogen-fret' poiyS!wdmridc lluHlt' up of
glucose units. this hexose lta"ing been identified by th,
isolation of its osazon{' and of sacchari(' a('id. Typt II.
substanee has been acctylated with pyridine and uNfit
anhydride. The resulting seetyl compound can h, hydro-
Jysed, and the original 8jX'cific subst8.nct may he rccon~r('d.
Carbohydrate II. is weakly acidic.
The Type III. substance is similar to Type II., but appeal'!; to
c()ntain in addition glycuronic acid or some deri\'atjVt~ of thi~
acid. It is sharply distinguished from the Type II. sub.tan,,.,
iff being lievo-rotatory. Like substance II., it is also acidit.

Immunological JI.eaetioDa of the 8pee11le 1'IleumoeoecIII

CarIJobydraies
The specific carbohydrates which we have just descrllx:d
are not antigens. They do not evoke any antibody reSp'J1lM'
when they are injected into animals. Thus if a solution of
one of them he injected into a rabbit, the serum of that
.... ..

~{)6 UUIUNOl.OUlCAL R~JAC7'lON8

rahhit d()('~ not acquir(' the prop{rt~ of formill~ a pn{i,.itatt'

",.'hi'l! it is ad(lfd to Ii solution of thl' f'Hrhohydratt' used in
the illjedioll. On the other hand. the carbohydrate, do
Teuct spt.'eificaUy with till' antihody in the hornolo h1Ous: scrl
produ-"Nl hy immunisation with th( {'orresponding intHc1
ha('t{~rla. }I;Spt'ljHlly signiti<'ant from our pf(~St~llt standpoiu1
is the fad that, although t'Hc'h spe('ifie f'arhohydratt pre<jpi
tat<s homologous antisera in ('xtrrm' dilutions ranging up
to 1 ; X.OOO,OOO. th<' Type 1. cnrbohydratf' will not precipitnte
Type Il. or Type III. antisera. hut reaets s"e('ific'ally with
'f.,P(' l. alonr. Similarly. Tyl'" II. and Type III. carbo-
hydrates arc equally specific. At muC'h higher ('on('(>ntratiolls
of th(' carbohydrates slight and indefinite crossprc('ipitations
may tJ<" ohtained. but thCff' is no question of the essential
sp('cifieity of these suhstances. It llH.s bt't'u shown that the
('nrbohycirlltc is present in tht prt'>f'ipitate.s and may he
r{'co,'ered in a practically unchanged condition after trypti~
digestion lind used again.
The obseryation that these carbohydrates are non-antigenir
whe-n they art' diss()("iated from the other ('cllular constituents.
and un' capable of inciting spt'dfic antihody formation only
when they ar!' present in the intact ccII. leads Avery and
H"idelberger to conclude that in the intact pneumococcus
the carbohydrate is probahly present in combination with
sonw other substan('{' whi('h confers upon it antigenic proper
ties. 1n these authors' own words, _, Immunisation with
intact bacteria eontaining this carbohydrate complex elidts
antibodies which not only agglutinate the formed rells, but
preripitate solutions of the carbohydrate isolated from
pnenmo<;()('ci of the homologous type. How the specific
"artohydrate is 'combined in the rell, whether with protein
or some other ("ollstituent~ is not yet clea.r, but it is evident
that the eompound thus formed is the dominant and essential
antigen of the cell, and the one responsible fur type
specificity." Strong support for the riew tbat !lOme definite
combinati9n between carbohydrate and s<>me other cell con-
 BACTERIAL CE[,L ('ON8TITr.'ENTS 307

stituent must exist in tilt' intad N,jJ j~ nnorflf'd hv Uw

obs{"r\'atin[l that on mnrphnloJ!iral di~~oltlti(ln of ttw
hllderiu, either b~ fr('('zillA' HtHI SUhS('fIU('llt thnwing or hy
tpe- Iytit 8('tioll of bile, the solution ohtaiHt'{I, Illthough it
contains all the ('nrbohydrah' and prott'in substan("('!(of tht'
original whole ba("tcrium. does not indu{'t" the formnt1()11 of
tY}lf'-specifir antibodies. Alltiprotein Sf'ra un' ol)taim'd
which are identical in the cases of .U th"'e tyfl""< of "ocr;.
and thes(' will not rea('t with the homologous .orbohydrates.
When the {'ell is ruptured its constituents puss illto solution,
the linkage between the carbohydrate and the other ('on
stiluent is broken, and the free ('arhohydrnte fraction,
although stiU retaining the property of 'Ile('ili<- union with
iL~ type antibody, is no long('"r capable of inciting t he forl1lft~
lion of thi' same immune body. It would app"ar that the
complex, if such it is, is not ("asHy rt'-formNf 00(,(' it is
dest.rorro, since sera prepared from filtered ,')olutions of
disintegrated cells frcc from formed elements rail to show any
of the dominant typespecifi<- profl"rties whit,)} characteri.,.
sera obtained by immunisation with whole hacteria (compare
A and C. .Fig. 37). A"cry and Heidelberger condude that
.. morphological integrity of the bacterial cell, therefore, is
requisite for the expression of its full antigen;" pnwer."
When intact pneumoeocci are injected into a rabbit
specific agglutinins are formed for that tyfl" of pneumo
coccus, and also specific precipitins for the correspnnding
carbohydrate. In addition some varying amounts of
nucleoprotein preeipititrs are formed. but these, os already
mentioned, are not type-specific. There is some doubt as to
whether atry nucleoprotein precipitin is formed os " result
of the injeetion of the intact pneumococci as such. It is the
opinion of Avery and Heidelhergel' that the nucleoprotein
precipitin. whieb usually appear are due to the fact that in

_.
mtJSt suspensions of pneulll(lCocei as used for injection a
certain amount of dissolution of the bacteria cat. scarcely
be avoided, and IIence a IIft'liIIl quantity of free nucleoprotein
308 IMMUNOLOGICAL REACTIONS
is usuaHy prest'flt. Even if this dissolution W('I'(' a,-oided
di!\int('grntion of ~OI!1{' of th(' illjt,(t(rl haderiu would (){'{'ur
ufttr thf' ilH)('ubltiOIl.
The n'udC'f will S('C' from the j()r('going that, whilst it is not
),(t po~ibk to aITin~ at a d('t1nitt C"onl'1usion with regard to
the fohn in whi .. h til{' carhohydrate exists in tho intact cell,
it s('elllS at present justifiable to suppmw that it is associated
ill some way with the bneteriaf nucleoprotein. Ac("ording to
this sUl>Position. th{' scheme briven in :Fig. 37, whi('h is
reproduced from one of Awry and Heidelberger's papers,
and whit'h is purely symholic. will s("rvc to yisualise the
{'orrdation l){>tw('eH the cell constituents and their respeetive
antihodies.

Specific Substances from other Micro-organiscs

The nature of th(' results obtained in the case of the
various typ<>s of pnC'lUlloco('CUs naturally led to the inyesbga~
tion of th(' possible occurrence of soluhle spedfie substances
in oth('T mi(ro~organisms. Following up the T('suits of
Zinsser and Parker on the preparation of the so-caHed residue
llntigens, lIfueilor and Tomcsik were able to show that from
a totally different typt' of micro-organism, namely. yeast,
it was possible to isolate a complex carbohydrate which was
apparently idenbcal with the "yeast gum" described by
earlier workers, and which gaY(' precipitin tests \\~th immune
yeast sera in dilutions u}' to 1 ; 400,000, and negabve results
with the serum of nonnal controls, Like the pneUlllOCOCCUS
carbohydrates, the spceific substance isolated from yesst
completely failed to in voke any antibody response wheu
injected into animals, The spceific rotation was of the order
+ 9Qo, and small' amounts of nitrogen and phosphorus were
still present in the purest preparations obtained,
In extending thei~ work on pneumoeoceus Heidelberger.
Goebel, and Avery decided to investigate li'riedlilnder'.
bacillus, presumably OIl account of its voluminous CApsule,
It will be remembered that the e&PSuJe of pnewnococeus _
 B.ACTERIAL ('ELL .('O.\'STI1'rESTs :100
A.INTACT au. ,.cJ.
...... , ~ 2J AMT1--(' stIW" 1VI"! J ~cmc
...... J1
~ 3J n
TV>f.m
~ !J .II!

15. 10000TtI:> C('' 'TTTIJ!NT$ M au. I'J, (C).

..
PlWl'!.IN [8 a Atm" XlWM. NOT 1'W'!$II".tI"lC

FIG. 37.-lmmunological relathmlfhip of the cell constituent.. of

Pneumococcus of Typea I . It. and Ill. e ('.rbohydrau~.
P = protein. (AI.... Heidelberger and Avery,)
supposed to be composed almost wholly of th~ specific
carbohydrate substance, and on analoJO' it Was therefore
:110 IMMUNOJ.,()G!/}AL REACTION8
rf.'a.{jonable to suppose that a bacillus with a promin('nt
"npsul~ offered suitable material for the isolation of a soluble
slX,cific substance should such be prest'lTt, Fri,'dland,'rs
bacillus is quitf" distinct from pnt'umm'(){'('us both in fOfm
and in.function. although it is sometimes associated with it
in tht' 'production of pneumonia. In one respect the I wo
bacteria resemble on' another, since both exist in the form
of a number of distinct immunological types, but in the
ease of FriedHinder's bacillus these have not been worked
out so completely as have the types of pneumococcus. \\'ork
was ~olllmenred on a strain (E) isolated from a chance
infection of a laboratory gl.inea-pig. Using the methods
aJready described. a carbohydrate was isolated which had the
foHowingo rharaC'teristics. It containeo no nitrogen, was
weakly acidic. having an acid equivalent of 695, anI:! had a
specific rotation of + lOO~. The principal hexose ('ompo!lent
of this new polysaccharide proved to be glucose, which 'l'as
identified by the isolation of the osazone Rnd by the formation
of saccharic acid on oxidation. The presence of glycuronic
acid could not be detect"d. The analytical figures showed
(' = ~~'6 and H = 61 per cent., in very close agreenwnt
with the figures required for a polysaccharide of the compo-
sition (C ,H O,),.
A s('rutiny of the properties of the :Friedlander substance
ren~als a striking resemblance to those of the pneumococcus
Type II. substance. The agreement, while not absolute.
suggt'Sted to Heidelberger and Avery the experiment of cross-
precipitation tests, and the remarkable fact emerged that
the Friedlander SUb,i;allCC gal'e a precipitate with the anti-
pneumoc~us Type II. serum in dilutions up to I : 2.000,000.
Conversely, pneumococcus Type II. substance gave a
precipitate with anti-Friedlander (E) serum. On tbe other
hand. no trace of reaction was observen with the FriedlAnder
substance and Type I. or Type III. antipneumoeoccus ~.
It must not, however, be supposed that the two substances
~ identiCf}; the rotatioru;, acid equi'-Nel}ts, and rertain
 TUBERCLE pACnU
precipitation rf'octions. ('\'('11 grant('d that fwitflf'l' "'Uh~tJ.HU>-(
has t~n obtained pure. art' t.oo din'TJ.,'t:'"Ht to permit of thb.
possihiilty. Xe\,('rtht'icss their mark('d immulloloJ,!h'ui simi-
larity renders reRsonablt> lht, as!>.ulnption t hat both SUh... tItHt.(...
(,(lutaill in a portion of t.heir comple'x lHol('('ule tilt' ~,rnt' or
a dost"'ly similar ('onlig-uration of lttOIliS. This rcmAPknhl(
example of what is ('aUed .. h(tctOg('Hdit: !oIpf'C'iH('ity"
strikingly illustrates t.hp fral importarH't' of the C'}wlHit'"1
natUf{> of the' carhohy<iratt' in the immullologit'ul rt'R("tiollt;.
It may also he mentioned heN' that tlw!oo(' crossf('u(,tioll!o! 81"'0
apply to the intact. organisms. Thu~ rnj((, ('ould he pr(jtt_~(,tt'd
against inoculation with Typ(" I I. pneumO('()('us by ImH.n~
of a previous injfC"tion of anti-}"ri('dliind(,T {}:) st'rm)) just a~
wPJJ as, anrJ possibly even }Jf'tU'T than. hy inje('tjoH of tlut;-
lm('umo~oc('us Type II. ,<;crlnu.
I..aidlaw and Dudl(y haw' made 11 furthrr itfkr(,\tinU
(>(lI\tributioll to the- problem of these Nuh;\tlW(,(>!'J wit" ~p('('itk
imnlUIloiogical reactions. Th('s(~ iu\(stjs.,l1ltor~ han' ('xtru('t,('(l
from tuhercl( bacilli (HurnsH Tyt)(;', Strairl ]OO} n ('tlrho-
hydrate pm.sessing properties closdy analogous to t 110'<' of
the pneumocorcus carbohydrates. After trt~atill~ tht,
pulverised bacilli so as to relTlovr th( hu(tf'rial Tt!>i<it.t<'.
proteins, and tuherculinic acid (a tlluleic acid), a Ilmterial
was obtained which proved to 1)(' largely ('arbohydrak ill
nature. and whieh could he separateu into glY('og('n nud
another carbohydrate isolated in tht form of a gum and
finally as a white powder. Solutions of the latter i.""T ill
high dilutions (up to 1 : 6.400,(00) a precipitate with immune
horse serum, whilst with normal horse serum no prccipitatiun
was obtained in any concentration of the precipitating
substance. This specific carbohydrate from tul)('rcl( bacilli
gives no colour reaction with iodine, doe. not reduce alkaline
copper solutions, nor does it contain nitrogen or sulphul
On hydrolysis pentoses and methyl pentose-s Were detect"c
No proof of the presence of glucose or of other hexose-s wa
obtained. DiS80h'ed in 25 per cellt. sull'hurir add, the 'U~
 :112 IMJr:NOLOWCAL REACTIONS
!o.tan("(' showrrl 8 sfW'C'ific rotation of + 61. On heatitt#! th('
rotation diminhhrd to U'TO. and from the- flcutralis{'o Sohltion
a small quantity of an unidentified cl'ystaHin(' osazone was
ohtained. Mu('lIt'r. n'orking with filtert'd culture media in
whiC'h tuiJcrrit' baC'ilIi had grown, has rf'eentiy reportt>d the
J!Jolation of a .'timilar carhohydratc. ('ontaining at ll'ast 55 per
<:<'nt. of pt'lltoS(' ('onstitllents, whidl gives spedfie precipitin
re8<>tions in high dilution in the preSCJl(,(, of homologous
i01munt._~ sera,
Still another ('as(' in point j~ pr()\>ided hy the nonhamlOlytTc
l"trcpto('(Jccus 1'iridalls, Th(> immunologi('al T('fl.dions of two
of the cd) ronstituf'lJts of thi') mjero"oF1(llnism han" bern
studied by LRn('efirld. At tht" tim.(' of writing this inw'stiga-
tion is incQmpirte. but the results. so far as they have bf'cn
rt(ordf"d. bear out t}lr analogy to thr pnrumoco('C'tis ff'SUJts
ill a striking way. 011(' of til(' (onstitut'nts. the so-called
Hurlfoprotein. is reJntiv('Jy :tIon-~peeiH( and gin"s rise t(l ali
untihody which shows group reaction!.; with nu('leoproteins
of r('latrd species. Thl' other ('onstituent is non-protein. as
indiC'ated hy qualitative chemical h_~sts. and is vcry probably
~arbohydr.te. Alt.hou~h the latter i, highly reactiw with
the spe('ifi(' al~tihodi('s produ('ed by inlnIUnisation with the
int"et. hacterial cell, in the purest ('ondit.ion in which it. has
ht'el)' obtained it is itself non-antigenic,. It will be remetn-
'",red that Heidelberger and A \'Cry and their associates
isolatffi from the three types of pneumococcus one and the
same nucleoprotein. identical in immunological relationships,
and it will he seen that a similar non-spedlk protein is
obtained from Stnptococcus liridan... These proteins consti-
tute a highly interesting group of substances. hut since from
the chemical po;,;t of "icw they haYe not yielded to purifica-
tion to the same extent as the carbohydl'8tes. our information
about them is stili somewhat indefinite, They seem to exiflt
constantly along with the carbohydrate!;, and it wonld appear
that fhey too play an intimate .Ok in immunological
phenomena.
 SUGARS AN j) !)pEel F/Cl1' r 313

From this brief skf'kh of t}\t"St~ Tt'{'('nt df'v('}opnwnt:-; in

immunology it i!' apparent that th('r(' is Illrc-arly a suh'!.tnntiul
bulk of cvidell(,(' to !olhow that th(>s(' })(,(tllint tarhohytlrntt,
arc of funoamt'ntal importancl' in whnt }U\V(' hf'(,ll rf'~nrdr,l
l:P till the prf'sent timp AS oh'teUT{' iruHHHlologil'nl TPa,tioHS ;
more espf'('iaJly are they of import8m'(' in tldt'fmiu1,tg om'
of their most interesting fC:'aturcs, Jlnmdy. their "pt'cilkHy.
That Nature should han' ,deet.,l carbohydrates for thi.
role is perhaps not surprising when Wt' bear in mimi tfu'
numerous possibl(' ways in whi("h two simplt suJt8l' nmh.('ulC:'"
may be linked together. t!a('h on(' rli.-.;tin('t from the oth{'rs
in stereochemical configuration. Thus thf'f(' ur(' at lenst.
twenty-eight ways in which two mol('('uit's of (lJ!lulOS(' may
eQlnhine with one another, and when w(' C'onsidt'r th(' possi~
bilities 'offered by two different sUl!"rs tbl'se I~'('om(' ,till
greater. In comparison the possib)(' modes of comhination
of two amino acids arc very limih'-d, and although numerous
isomeric peptides could result from the' ('ornbinatioJl ur, let
us say. tweh'(' amino acids. thi ... Ilumbt!r h('('onH'~ iu ..igHifiellnt
when compared with the lIUmbl'T of possihJe (,llrhoh)'drntt.~
obtainablt:> from twelv{" molecule.", of glucose sonl(~wht'Ti'
in the order of lOIS!
References
1. AVERY and HEIDELBERGER. ,Journ. F~'fp. M,4 .. HI2:t:. 3S. ~t ;
1925,42. 347. 367.
2. AVERY, HElDELBERGElt, o.nd GOEBEL. JOlin" /<:.cp. JIl!d.,
1925. 42. 709.
3. DooHEZ &Ild AVERY. JQurn. Exp. ~Ved . 1917.26. 47i.
4. HEIDELBERGEk and AVERY. JO'UTn. E;rp. j[ed HJ23, 88. 73 j
1924, 40, :WI.
5. lIEIDEJ...BERGER, GOEBEL, and AVERY. ,louro. Erp. Med.,
.1925,42,701, 727.
6. LAlDLAWand DUDLEY. Brit. Joum. Exp. Path . 192.';,6.191.
7. LANCEFIELJ). Journ. Exp.ffed . 1925.42,377.397.
S. MUELLER and TOlle"",. Jon,.,.. Exp. Jled. 1924,40.343.
9. MUELLER. Journ. Exp. "lfed .. 1926. 43, I. 9.
10. WEU8. .. The Chemical Asped>; of Immunity." Xow York,
Chemical Catalogue Company. Ine . 1925.
11. Zm8llER and PARJO!.R. Jour,.. Exp. Ned.. 1923.37.275.
 rIlAP1']<;n XI
"
CHEMOTHERAPY
('U>OMOTlI>:UAPY is that branch of pharmacology which is
('ollC"t'rlll'd with til(' ('omooting of inle('tiolls diseases of
bat'tf"riaJ or protozOaJ orib,.l'iu by t})(> USe of intrrna1 antiseptirs,
that is to say, of antiseptics which lllUst he introduced iuto
th~ hlood stream of th" li"ing infcd.ed host in order to
produce their spccific e{feets. One may also reil'ard the term
u (~hemotl){~rapy ,. as embraeing the use of external anti-

septics. many of which 8fe identical with or dosely rt'latt'd

to the internal antis<'pties. Tht whole field is oue in which
the <"lIdC'u"ours of the srllthetic orKS-nrC' cht'mist haw'
rf'('cntly led to som(' strikiu,:! sue('f_'Ss('s.

ChemotheraPY of Inlectious DiseUlII

The prartit(' of combating certain maladies of an infectious
Hut un- with spe<'ific organic or inorganic medicaments is a
"t'r}, old one, dating from times long hefore the causation of
t he disease or th(> rationale of its treatment were known,
but. being based purely on empiricism, it necessarily remained
more of an art than 8 science until comparatively recent
years. Modern developments in this branch of biochemistry
have their (}rigin in the early observations (1890) (}f Ehrlich
on the selective staining action of certain organic dy;"tuffs
towards various living cells and pathogenic organisms, and
the attempt to' combine with this property a toxic action
which would kill the parasite without harming the infected
host. The aim has been, then, to obtain a preparation of
maximal toxicity to the parasite (parasitotropic). and of
su
 "RJ<JOEPTOR" THEORIES :1l5

minima} toxiC'ity to tht' tiss-Ut's of the host (orf,{llliotropi{').

so that if C he the "urali,," de"" whieh kill" .11 til<' l"m",itc",
and T tht' maximal dost' whi('h tht> host will tnit'r!.ttl' without
suffering injury, the ratio ('/T should b(' SllUtll if tht prepnrn~
ti()n is to Jll('("t wit.h ~ueees"i. MUf'h of )<;hrli"h's Inkr work
was hasco on the vj(,w that it was possihl(' to ~d('(''' "om~
pounds for which tht" partic'ulllr organism who!<tt' {'ud ttw~\'
W('rt~ designed to encompass hnd H ",pi'('ial affinity. tl'",idt'nt
in certain . rc('<'ptor ., groups which lw('am(' Iink(d to SOIll('
special atomic grouping in the antisrpti{'; ('omhinotion l)f:~illg
established, th(~ \'ital acth'ity of the inyading purasitt' way.,
then so lO\\'('f(,O that it ('cased to produce its toxin'> lind dit'd,
or tht, natural dcf("nsi\'(' m("C'hanisms of tllf' ho~t'~ t i~"u('s
att{lined the upper hand and uC'hi('\'('"d the samc' end. Thi~
simple theory of dif(>et uflinity }wtw('('n parHsit,(' and tlltti~
septic has pro\'("d t'xccfdingly useful in a purely prat'tit'ul
\\'8)&. und has led to the rlaboration of' mUlIY Ill'W and tHore
toxic compounds of hillh('r sf'lectidty for ginn infc(1.iuJts.
hut. as Dale points out, it is Uecoming 11)('Tt>HsingJy nppnrt'lIt
that such a simple hypothesis is no longcr tf'nahlc. Thus.
whilst in eertain ca."ies the mechanism postulated by f;hrli('h
would still seem to furnish the simplest (xplanation. tiwrt'
are mnny examples of th{'raIX!utic ag<'nts which arc toxic to
organisms in ritro or in cultur('s, hut whit,}) arc (Jltird.v
ineffective when introduced into tht' host's hlood stream.
aild conversely many of the most powf'rful antis<'ptic'I ill
"(Flvf., ~t:\.o toe \\IUb""t -w'rin?n l:Ct't qtit\t: ~1.l'l"M!t-:tn:. 'tit Y4'1Jx!t='"t:a1l1ry
SO, in vitro. Evidenct>, too, is 8ysilahJe whirh ~hows that
certain dyestuffs of a powerfully specific antiseptic nat.""
have a much greater affinity for the tissues of the host than
for the isolated parasites in pitro, and, again. at lea."t one case
is known in which the antiseptic agent eonfers on the host a
specific immunity to the infection not unlike that produced
by protein antigens to bacterial toxins, Ohviously a simple
theory of direct affinit.y mllSt he extensively modified to
explain these ~,
 316 CHEM,OTHERAPY
Tilt' diseases which we are ('onsidering fall into one or other
of two main (,lasses ac('ordinJ,! as thf'Y are caused by the
prrs('ne{' in the infcch>d animal of a foreign protozoon. or of
" barteriurn. and it is to be noted that chemotherap<'utical
111f'thvds haY(' pro\'('d very much more effective in eombati~lg
tIl{' t.,I'mer type of infection than the latter,

Chemotherapy of protozOal Diseases

H j.,-; in this group that the most st.riking SUC('C.\:;cs
of ehf"nlOt hcrapy ha \'C' been achieved. 'Four type~. of
antiseptics ha vc proved useful; these arc: (1) Organic
dyestuffs; (2) organic compounds related to dyestuffs. but
not necessarily dyestuffs or .,'en coloured; (8) orga.no
metallic compounds, chiefly those of arsenic, and to a less
('xtl'ut derivatives of antimony. bil\muth. mercury, and
cert;.in rare motals; (4) alkaloids, chiefly those OCCUlTing
in rinchona hark or ipccQ('uanha root, or artificially prepared
d('rivati\'(~s of these. The diseases which have proved most
amenahlf' to treatment by these specifics are trypanosomiasis,
spirochw.tal, malarial, and amcebiC' infections, many character
isti('ally t hose prevalent in, or a('quired in, tropical countries.
(I) Dyestuffs,--Ono of the ('"rli('st observations in this field
is due to R. Koch. who noted the antiseptic action of
Malachite green, and in 1891 Ehrlich and Guttman investi
gated the possibility of using Methylene blue, which has a
strong staining action on the malarial parasite, as a specific
curative agent for that infection. Following on Koch's early
observations on Malachite green, many other dyestuffs of
the triphenylmethane series - Parnfuchsin, Tryparosan,
l\lethyl \;olet, Methyl green. Aniline blue-have been used,
but, o"ing to tlleir dt"Ieterious local actiQD on tissues, tIley
have not been extensively employed. Later investigations
led to the use of dyestuff. obtained by tetrazotising benzidine
ow ~lidine and coupling the product of this reaction with
various substituted naphthylarnines or .mino naphtliol~;
 DYESTUfFS ~17

thus Trypnn f('d and Trypan hh)(~ hun' prond of (01l'iidt'r.

able W;t'. The former dyestuff illu ... trnhs the poiut. nlr('u.dy

So,JId

M?O:~:'
ro.so.W5O,Na. TIM'AN
RED
lIa.lO,WlO,No
mentioned, of an antiseptie whiC'h is innctiv(' ill t'ilro. yd
proving effective against ('('rtaln trypanOSOlllC'S (7'. fquinuw)
in mjee~ and demonstrates the further poillt of Ii ntHrktd

specifieity. heing jnaetjVt~ against T. rqui1lU111 ill Hlt' fnt, Of

against the trypanosomf> of Nagana <1'. bruCi'i) in Uw mOlJ\p.
Trypan blue is effectiv(> against th( Inst-Hlrutioll('(] inf('diofl.
and has been used in ('uring do~ and ('attl(' of' H pnrnsiti('

PR.OfI.AIIllIl lRVPAfLAVIlil

infection of the red blood cells (Piroplu$ma), Of more ro,,('ut

interest is the introduction of dyestuffs of the acridine serif's :
thus Proflavine and Trypaf!a"ine (Acriflavine) have proved
of some use in trypanosome jnfections~ hut are mu~h morc'
useful 011 account .of their anti-hacterial action or, as external
 :JIR CHEMOTHERAPY
nnthwpti("s. Browning hn~. indc(>d. ~ho\v1l them to poss('ss
the lI,',{'ful property of augnlt'nting their hadel'i('idaf action

,
in thl' prc,"it'I)('{' of organic' media such a." blood sent. The
dyt>stuff Afridol "iolet, which is very similar in ('()nstituti~m

H ON NH,

Na.
CD
so,
N-"Ollll-CO-NH-<=)-"_N
so,leG. .o\FIlIOOL VIOLET ~s
o,Wsc,
to Trypan bhl(', but is obtained by u~ing tetrazotised
N4-

dialHiuodipiwnylurea in plaC'{' of tolidine, is of considerable

interest, not. on U('('ount of its own practical value, which is
hut slight, but hecause it is a complex substituted urea, and
as sud~ forms a link between the trypanocidal dyestuffs ~nd
the second group of compounds already mentioned.
(2) Organic Compounds related to Dyestuffs.-Om of, the
most striking adyau{'<'s in the ('h{'motherapy of trypano~
~omjasis hus rt'su1teci from the introduction. not of a neW
dyestuff. hut of a related compound which is d"'oid both
of dydllg properties and of colour. This is known as " Bayer
205," and has been introdu<'ed by the Bayer Company, who
llBxc lh.)t. hmvevcr, disclosed its constitution. It would seem
to possess a much more nlarked action against trypanosomcs
than allY of the numerous dyestuffs preyiously used. It has
f('eentiy bftm extensively studied by Fourneau and his
roliaiJorators. and these workers flu ve succeeded in synthe-
sising a (~ompound to which the serial number 809 is gh'en,
and which has the constitution shown : -
 BAYER.205
This IWW l'ompound :J09 hAS trypafl(widul and otlwf prop('r~
ties Y('ry dost"Jy Tf'semhliug. if not ith'uti('ul wit h. Bnw'r ~O.~.
Moreover. ,Fourlleuu an<1. his (o~\\'ork(t'!oo hnH' ,;rf'pur..ct
num(."rous substan{'("s of a natuf(' dosf'lv similar to that of
309. and Tf'port that a vt'ry slillht ('(~nstituti()llal (ihnnw:'
completely suppr(~ssf's the trypano<'idal ("tion. ()A .('(>ft!.
paring the fonllula of a09 with Africlol vinlf't or Trypnn
blu{' it will be S('{'II that til(" fOTllwr differs frow till' two
latt~r in the absence of diazo linkill!(S (uerollnting for its I.ck
of colour) and ill possessing a ('hailt (If . Nil , ('0 1(1'0111'-
ings ",hid) confers on it a <'f'rtain ('onstitutioHal similurit\' tn
the peptides. Hayer 205 has no actiou on tryptHlOSOm('~ ill
vitro. and one of its most r(~markflb)("' propE'rtie", i~ the- (>npn('ity
of Jl single injection not mrr(j-y to ('lIrt' the' inffdion. hut to
('onfcr on the animal a.n immuuity to suhs('l'{w'lJt tr.Vfmuo~
SOtnf' infections which may last for wcocks. or t'\'(u mf)1Jth~,
:FoJlowing the initial suc('cssfuJ use of Hayt'T 205 in animllls,
it has been applied to the ('Uf(' of' trypullo,..umia\is in Ulan.
and is reported to have ('ured cornpll"'tely Ildvun(>('o ('tUI('~ of
infection with T. gambictlRe (sleeping si!'k'If'ss) nnd 7 1
,

rlwdesiense. It i. just possible that early ,stimat". of the

potency of this new specifi<, applird to human trypul1o.
somiasis are a little too optimistiC', It would aPP('ur that
Bayer 205 is slow in its action and jlersists in t ht" hlood and
tis.~mes for some time. The sera of patients who havt' heen
tr,..ated with the drug several weeks previous to tI,, with-
drawal of the sample of blood under examinatioll 81(' found
to possess a trypanocidal action, which, however, Luengo and
, de Buen ascribe not solely to the drug still present in lh.
blood, but also to tbe products resulting from the destruction
of the trypanosomes. Most investigators report that Haypr
205 has a tendency to produce a more or I""" persi.tent
nephritis, In any ellJle, the evolution of this type of <ok",r-
less, complex, substituted urea-Bayer 205 f1r J.'ourneau',
~from the tetrazotised benzidine dyestuffs is all interest'
ing chapter in therapeutics, and represents a very noteworthy
320 f'llEMqTHERAPY
adVall(T whi('h pr('sf'lIts IHauy possihiliti('!', of future dt'n'iup-
IIwnt.
(:J) Organo-metallic Compounds. The ('hlef of the,..,(' arc
the ar~('nitals. and they haH- attained hJ'}'(_~at importante in
C'omhn.ting trypanOSOllH' and spiro(ha.'tal infcdions. The use
of arfoilf'hlc ckrivativcs in this eOIlIl('C'tion dates from th(' end
of lu.!-.t (,{,Htury. wh{'n arsenious acid wa'i used as a remedy
for ('{'rtail) parasitic iufe('tions in animals. But the first
(omplcx organie arseni('at to (l(hicn> success--and it still
remains in usc to-day---was ntoxyl. which was shown by
Ehrlidl nlld Bf'rth('im in 1907 to be thr sodium sal: of
lifsunilie add (p-amillophclIylarsillie acid). Ataxy' has
pron'd a ll~d'lJl starting 8uhstanC'(' for the prf'paration of
many nr~('ni('a}s and for the gt'llt'ral study of arsenic therapy,
and is itsrlf!'ootill Ollt' of the most effecti\'(' Tt'medit's for ~lt'eping
si(kness. AtoxyI is prmticaiIy devoid of aetion on trypano~
:-;Ol}Jt\\i in 'i'itro, uud Ehrlich form{'d the theory that it became

r('du('t'd iTt rh'() to un uetin'iy toxic modifi(ation. Aeting on

this view. he Jlrepared a numher of reduced arsenicals and
,"as ahle to demonstrate their increased toxicity. Thus he
found that p~hydroxyph("nylarsinic acid. etfective ill vitro
against trrpanosomes in a rlHution of ] /20. on reduction
yi{'lds p~hydroxyphN).ylarsenious oxide, which is effective at
a dilution of 1/1,000.000. A similar relation was found to
hold hetw~~ll atoxyl and its reduction product, p-amino'
phenylarseniolls oxide. but~ unfortunately. the increased
toxidty of the reduced compound to the parasite i. accom
panied by a corresponding increase in toxicity to the host, so
that the tolerated dose diminishes; hence the impracticability
of direct injection of arsenoxides in chemotherapy. Th,'
general fact em.;orged from these researches that derin.tives
in which the Br,>enic is present in a trivalent condition a ....
milch more toxic to spirochretes than compoWlds containing
p<"nta ""lent arsenic. and this led to the preparation of
a<seno derivatives of the general type. R-As = As--R, ot
which arsenopltenylglycine and the well-lrnown salvars..n
 ARSEN/ALS 321
Qr" 606 "(Ehrlich and BcrthcIOl. HH2) and their i.krh"llti\'('s
have proved most usrful. It should he IIt)h'd t hut thc'St~

NIl
J~
~ A>. A5C)NH
f
~Ht ~Ht
COON. COON.
AAIDIOPIIENYt.C.L'f""E !>ALVAJU,O>.N

compounds T('present It furt hef stagl' i 1\ till" r.'dud iOll

process : -

-Oil
R-As ~ 0 -~._. H--A.. 0
OlI

A"rsenoph('nylg)Y('inf' is very erff'div(' H,itllill.,t trypano,"-ullw

infections in animals. hut whell appliNi to t he ('ur(' of ..1(('l'ing
slckness in man r('suits or a I{_'s~ promising- natu}'..- han' 1.('('11
ol;tairl('d. Here the SO}ficwhut ;momaJoIIs. i:ljd mny he flot'(d
that the most efrcctin' arseui('ui yet producTd for tht trc'ut-
In('nt of human sleeping si('km's'lO is trypm''>lUllidt', ill whi('ll
the arsenic is pentuvalput, lin iudielltiofl tllttt. fruitful .as

Ehrlich's reduction theory proved in prouucing: r\~"\l\h of

practical value, it does not represent UI(' whol(.~ truth. In
any case~ tryparsanlide and Bayer 205 ('on~titut,( our f)("St
defences against the attacks of T. gambiellsr.
Salvarsan remains one of the greatest diS(~on'riet; of thi~
btanch of therapeutics, being effective in both spil'<",hlt'tal
and trypanosome infeetions, although its widespread USf' a,
an anti-syphilitic agent in the fonner capacity rather ohs(!ure~
its usefulness in the latter. As in the case of the atoxyl
derivatives, tb_ fullyreduced arseno compounds are in-
~ n
322 CHEMaTHERAPY
effective against trypanosonlCs 01' spirochretcs in nitro. More
recent developmeuts of this aspect of chemotherapeutics have
resulted in the pr{'paration of llcosalvarsan, in which one
substituted amino grouping, -NH. ClI.O. SONa, is present,
and 0 ' sulpharsenol l in whidl two -NH. ('H 20. S02Na
grouptrfgs nrc 111'('S(l)t. These have the advlmtagc o'er
sa]nlTsan of lwing more ponn'nirllt in administration. since
they Ilrc less toxic: in their immediate df{"rts and have
t.he further apparent ad"anlage of being soluble in water at
lleutral f('udioll. Hut, despite this apparent advantage,
equally good results seem to have heen obtained with
sal\arsun, which is insoluhle at the hYdrogtn ion conc('ntra-
tion of the hlood, liS with these soluhle derivatives. Sih'er
salvarsan represents all example of a eombination of a loose
ChClUi{'al nature hch\'c("11 snh"arsan and silver which has
b(,(,11 ('xt(,lIsiH'ly used in treating spirochretal infections.
notably in Gcrrnany.
A r('ct'ut c.{cv(lopnlcnt of a highly promising nature is the
produetioH by }<~ourll('an and his eollaborators (1928) of
stuyarsol, p-hydroxy-m-uc(tylaminophenylarsinie acid:~

(H,.CO.NII OH
HOO~O
'OH
STOVARSOL
whirh, it is claimed, is eff('ctive in curing syphilis when
administered by mouth, thus representing the first compound
for which such a claim has been made. St",urso} has recentl)
been applied, again orally, to the treatment. of amrebi,
dysentery. It appears to have a specific action on th.
intestinal lesions, and highly promising results, wbich hay.
been confinned by other investigators, are recorded_
The non-aeetylated compound treparsol (p-hydroxy
",.aminoph..nylarsini(' acid) has likewise been used. b3
 TRYPA1WCllUL ACTIOS 323
Flandin for the Irl'atnlt'nt of mlltl'iJiliSis in {;onjtuH'tlorl with
emetine gin'Jl inll'a\TIIOusl".
In un intere!'.ting study' of tlU' fl'lHtioll of trypano"idnl
a.C'tiotl to (hclllieal ('on~titlltioll rt'('('utly COmnH'IH"'(ll,y KllIg.
nn attempt has 1,('('11 nHui( to eomhin(' tIlt' w(II.rtt'f.J!niM'11
trypanocidal ad i(11l (If c'oIlIIHHlIHI ... of tile pJIt~llyllll... ildt' tuid
sprics. to which 1rypllr"alllidc Iwloll,!!!-., with tlwt of ,'0111-
pounds f(jutl'd to Bay('r 20;) or FOllfJlt':lu'S :iO~J. Tlw intell'
tion was to attach two <ll'siui(' add r(~idut,S to the emls of n
t'lmin similar to that whkh link~ tht' naphthylnmillc !'Iulphonl"
u('id r(,.,i(hl('~ in :lOll. King llmil\iul't'h hav(', indl't,d, SYhtli('
sisNI ('ompounds of the ty}><,~-

anti noh that, although SOllW of the ~ilJlph'r illt(,l'llli'dint(

compmuHis show{'d trypu,1Hw\dal adi\ity, tlwy \1t('B\~('lv('!o.
arc totally devoid of any a('tioll OJl t ht' t rypaflo~onU''''. a
striking illustration of the imperfection {If (llll' IIl'l'Sf"llt tli('(lri{'s
in ('hemothcrapy. It should, howev('f, Iw rnlll.'mlwl'(d. u;.;
Killg points out, that SH('h ('ompolilicis ari' ('olloilh of ('011-
sidero.blc mokf'ular \'\-'cight (!mO) and HI'(' but ~Ijgiltly ~olll"I('.
This last property is nu~ 1\('('{'s...:uily ill('{)mpatihk ..... ith
ijlerd.pcutieul adh'ity, as we have ~C{'II ill tilt' ea~(_' of ~al
varsan, but ncn'rtli{'ie!'i'" it is noteworthy that BaYf'r 20~; (or
"ruurlwu"{J'~ ~lfm,\" -n;th. ;A'~ '.,h:-.. '.:.u}qht'(}iit<. 'J((;t~. 'P'.'lufh, :ji> "'~"5
soluble in water. In continuation of tlwse iJl\'t'stigutions.
King and Murch have prepared a nUmfK'l" of d("'rinllin's of
3'-aminobeu7..oyl-.!.-am,inophcnylarsinie at'id (Forml11:1 1.),
and lind that when It = OCH. of ('I ill til" formula 1",lnw

NH.
tCJo. NH~A>O,1l,
 324 OHEJfOTHERAP Y
the resulting rompound eHeeted permauent cures jn mice
infc<"ted with Trypanosoma equiperdlltn. The 8' : 8~diamino
acids WCTC' also perma.nently curative in the instances where
R = H. t'H3' or CI (Formula II.). Balaban and King haye
prepaffil(l similar derh"atin's of phenylarsiui(> acid in whi(il
11- J!]y(J~Jjn(' r('sitillC' is prt'Sellt. and they find that glyoxaline-
'l'-('fjrlJ{)x~'%p-mHiHnpheHylal'sini(' Reid (Fommla III.) nnd
glyoxaline - .J.' - ('arb()x~' - p - amino - 3 - aminopheny larsinj( acid
(Formula I\'.) ean hoth eff,'ct permanent cures.

AiO,II,ONHCO.C=CIl
I I
N Nil
ill '(H/

The furth('r Ol1teome of 1hesc investigations, which arc adding

matt'l'iu.lly to our information ('ou('('rning moleeular configura-
tion and trypuuo<'idal u(tivity, will he followed with int('rest.
"'ith reft'ff't\('C' to the: varying toxicities shown by organic
arsf'nj('als jn which the ars(-nie is in rllfff'rent states of oxida-
tion. 111ention may ht.'f'(' he made of an interesting thf'ory
put forward by Yo('gtliu to explain the mechanism of the
toxic aetion of these arsrno compounds on living matter_
He argues that the lirst st,p is all oxidation to the correspond-
ing aJ"Sf'Jloxide, which, as Ehrlkh showed, is much the most
dired ly toxic of the arsenieals. The arsenoxide then react~
with reduced glutatIlione of tlte tissues ; -
H8----G /8----G
R--As ~ 0 \U;--G R--AS\.8----G +H,O
The eyidcnce 011 which this reaction is based is; (1) the
death of aniJnal& given a fatal intravenous injection of an
arseno:xide, such as 3-amino.4hydroxyphenylarsenious oxide,.
is greatly delayed by a simultaneous injection of sodium
, thioglycollate or other sulphydryl compound ; (2) the feeding
with moderately large &.iuvlints of the two constltuent amino
 A.RSEXOX/JJE AS~ (lLf'TATlIIlJ.Yh' 32r,
acids of gIutathiollC"-- glutamillic at'id Hlld ('v-.h'irH' of((,t,
considerable protpdion ngnin."it u ktJutl do!-.(' of ull arM'Hoxitit
gin'll two to thr('(_' hours Illtn. Tlw illf('n:ll<'t' i ... t llUt t h(,
arsenoxide may ht, considered a"i 11 Sl)()('ilil- IHli"iiJll for rt',itu'('d
glutathione, and a toxi(' dose will tlwrt-for(' 1(,IHi to.<'('IIIIIu-1'
asphyxia. Puralld ]'('slilb han' b{,(On ohtniued ill itt,.. .. tigUM
tions on inf('('ted l'at!-.. rarlow. slIlph,vd"yJ ('(lmpouud,
(reduced glut at hioJle.', thiogl~Tolli('. llli()llldi{', tlli(l~iili('ylii'

,ooc -- -- --- -.r~ll-

000

~
f-- L
!'~~
~(:o ,"
000
Cl Ie-- - -- ~
1---
"'4

')

FIG. 38.-TrypanOl,idallVCtion of IUt.lvanllt.n, Iln;f'noxid! "In" n-tl1H't:'!i gluta.

thione, and arsenoxide alont', in ratfoll. (Afte1' VOIgtHn.)

acids, cysteine, and glycykysteine), if illjected illto the

t'nfected rat just before a minimum d({'ctin' dosf' of all
arsenoxide, greatly lower the rate of disapJx>-anw('(' of the
trypanosomes from the bloud. Indeed, the nrS(_,noxidt.
instead of proving immediately lethal .as it is when illj((\td
alone, shows a latent pcri(KI very similar to thnt whieh
salvarsan itself exhibits (see Fig. 38), From the positi\'c
nitroprusside test given by trypanosomes Vo<"gtlin assumes
the presence of sulphydryl compoun,t. in these pamsitt's, and
postulates a mechanism essentially similar to the olle ft
sponsible for the toxic action of arsenoxide on mammalian
tissue. Such v~ws are highly interesting and suggestive,
326 CHE.M((I'HERAPY
but they still do not explain the seledin' netion of the
arsenicals on the parasit('s. This, how('ver, might he a
question of H sulphydryl r('scrv{','~ or, as Yoegtlin sug~:wsts,
might depend on tIl(' tissllf's haying a highC'r oxidation
pot{'n~\l for arseHoxicle than the pnrasit(s, wlll'rehy n more
rnpid"llnvcrsinn to the llon-ntoxie p{'ntava\eut HT'sl'ni{~ ('.om-
pounds wouM re~111t, and (limination take phl{'(~ lwfore
"pprceiahlc crBular asphyxia could O(;('llr in t hf' tissue ('fIls
of the host.
It is n natural stcp from arsf'ni(' dt'rivativ('s to lIlv('stigale
the th('rapcuti(~ action of compoullds of tite dosely-rtlatrtl
metals antimony and hi.sIHuth. The type of compound
wilieh has been used is much simpler thnn the complex
arsenicals which we have been considering. Tartar cn\(~tic
and its sodium n.nalof.,ruc and the corresponding bisnmthyl
tartrates hu\'C heen us(>d, both in "vitro a.nd in clinical pra.cti(~c,
against trypanosomes and spirochretcs. Here it is found thlt
the' rrsults obt.ained 1,'" vitro ~111d in l'it'O arc parallel. and the
action of these compounds seems to ('ollsist in a direct toxic
effect on the parasites which is more or lcf.;s proportional
to the amount of nwtal-antimony or hismuth-present.
Indeed, rnetnHk antimony Sl1sp<'Bd('d in oil and injected
iHtranlU!ScuJady is as cffeethT as tartar emetic in combating
trypanosomiasis. ''''hilst antimony is more toxic than
hismuth for trypnnosomes, the }'f'Yersc 1"c1ationship holds
iu the case of sJ>irocllll.tcS. Mercury in various fom~"f) ha:~
~ u/,.If:\'l i."JJ" ~-in'UYF _H?Rt"5' R!> Ii ~rXY~:HA-:- j.Y &f.i"~' <It sJphl-}is~
and it is natural that considerable attention has been given
to the c1abomtion of eompkx organic mercurials. But here,
again, the toxidty to parasites seems to he proporlional to
the amount of mercury present, since all attempts to alter
the ('IT ratio h;wc failed. The inference is that in these
instances also one is dealing with a direct lethal action on the
parasiu.,. The continu...! activity in the elaboration of new
mercury compounds would seem to he a response to the
clinicians' demand in rountering syphilis f()l: Q shorl inten'Sive
 AI.KAJIIlJ)'" 32i

.. ,,,,,:nent to~dh('r with It prultmgt'd l1W1"1'ury

treatm("nt.
(4) Alkl1lnid,"J.--Th(~ tr('utTlH'lIl of lIIalarial iHt~('tioll!\ with
ttl(' alkaloids of ('itwllOna hark, I1HU'C ('~J)(,"iall~' fflllUilw, 1:-; n
vcry old therap<'Hti(' llwaSHrt" hut, d('~pitc' the prt'liurntiolt
within r('ec'ut :n'ars of TlHllIV artin(tal (i<-ri\'utin's"l( tlll''''('
alkaloid." none shows any' ),{,lIi adnmtllge 0\ t't '1l1illiw'.
\Vith our improving- knowled.!w of the alknloids (x('ufrin,!.!
in this hark-and it is ('-.;timutt'd thnt tIH'r( nr(' mort' 1h:m
tw('ntv--it has been found thnt of tIH'M' nlknloids hvtlrn
quinit~e, quinidine, alltl eincilotlidill(' Hl'{' fit IC'ltst no 1t's,," tu'Xit
to the parasite, alld Hot any more toxit to HUlll, thall i~
quinine. This is of int(>rest, as Dak P(lillt .., out, ill d('fuon~
~trating that thl' toxicity of quinine to ttl(' malnrinl protol.{)UIl
is not" dep('lHicnt on the \'iny] gronp (M'(' til(' formula 011
p~ 334). nor OB tJlC' Olf'thoxy j:!rnllp. Hor (Ill tlw stl'n'o "Oil
f\guration of thi~ alkaloid, siu('{' til(' lirst-nwlltiolltd group
is rffiuc('d to ethyl in hydJ'o(Juinim'. till' J1w1JJOXY ~rHIJP is
ahsent in einchonidiuf', and quinidiut' dirr(,rs frllJ1l qllinjfl{~
jn at ]Cl!st one of its ll'oymnH'trk ('arholl atol1l."'. lJ('rt', again.
it is to'be noted that quinlIlf', although toxic: ill ',f7'O, il) df'\'uid
of parasitiddal action in "jlm.
As in the case' of malaria, ~o hopi{'al (arnu'hi(') dy:-.entf:ry
ha.s been combat.t_d by t)u' 11.<;(' of J-lJku1ojd~ Si,H'C at J{,H,~t Uw
seventeenth century. Of tile alkaloids of jpct'ul'uanlm fuot,
the specific for dysentery iutrodu('{"d hy 1..(' Gra.\ in J 672,
cephR'line. CtSH3S0"Nz' and its ITlf'thyl estet crnf'tiu(',
C 2t H.,oO,N2, luwe been idC'utificd, and hoth pHfo,s(~,'t~ atoxjf~
action on Entama?ba hi'ltolytica l11u('h more markf-d than that
of any artifbal derivat.hL"i prepart,j from them. &.rlier
experimcr.ts carried out in r:itro on the action of ('tll(~titw nil
E. kistolytica were interpreted as showing a .implc dirtct
toxic action of the alkaloid on the parasite, hut more recent
investigations by Dale and Dobel! (1fI17) and Allen (1920)
have shown that emetine iii very much less toxic in ritro.
than wa.< at fil'ljt stated, and, indeed, 80 far 'IS in .,ura experi
 32S CHEMQTHERAPY
ments _go, Quinine is muC'h m()r~ ef{(,cti\'{' than ('met inc.
although th former is quite d('Yoid of any (>ffN't in dro, and
cOClM'quenfJy (If no ('uratin' valm' in this di~('[ls('. Ag-Bin.
Dale ami Hohell found olle of thf' less important alkaloids
of ip<t;u('uanllR root. namely, methyl psychotrinC'. to he
,.,tr()Hpl~ toX)C to the i.mlated .!H1H1'Int, and almost non-toxic
to them in t h(' mammal. Th('s(' illustrationloi demonstrate the
('xtrrl1l(, ('omplcxity of what Was at first an apparenUy simple
qU('stioll in chetnotherapeuti{s. and again cmpha~ise the
romplph- failut'{' of results obtained in ritro to correlate with
thos(' ohtained in vivo.

Chemotherapy of Bacterial ~

In tIl(> SN\TCh for chemicul suhstances spccifkally toxic to

pat hogf'nie hacteria mu('h ]('ss striking l'{'sults are to he
Te('ord('d than in the l'aSt of prot.oz(}al diseases. In\"f>stj~-

TABLE XXII.
Comparatire Actio1l oj Antiscptics in Uatcr
and in Blood Serum

8lopbyJc;rornu IlUTl!/U. BorilJus ro# romm.

8tlhl<tallcf'.

Merturic chloride I , 1.000.000 I, 10.000 I , 1.000,000 I I : 10.000

Phenol. .
Malll('hit,f'~
Brilliant~n
:u~ooo.ooo i :: ~ooo
,1:500
I : 20,000
I : 10,000,000 : I : 30,000 : I : 130,000
.1,500
I : 1,000
I : 3,500
Crvstal violet I : 4,000,000 ,I: 400.000 ; I , 8,000 I : 8,000
Trypafl.a.vinf' . I : 20,000 ! I : 200,000 ' I : 1,300 I : 100,000
:
tions in this field hi",e frequently ended in the discovery that
an antiseptic powerfully bactericidal externally proved quite
( innocent in ttiV(), the most probable cause for such dis~
crepancies being the presence of blood prot<;ins which were
 RACTElllA
dt"moJlstrateti hy B('('hhold in nIllllY ('U'WS to low('r tlu'
toxic'ity of all nut iSf'pti(' ('\"('11 ill rifro hy fOfmillJ,! It (tH't~t'l
('omhimLtioB with til(' lutkr. This d(f'1't j" illu!'ootl'ntt-d ill th('
tablt" shown on p. !l2S (Hefner. \"oJ. i .. l!!,;H). \\)llt'h gh t'!.
the millimum lethal ('OIl('(,lltrntion.'i of tht' \arioll .. ltnti ..... pti(~
(a) in wutel' and (b) in hlood Sf'l'um.

.From time to tim(' nUllH'NlUS dytstnfh. with ~p{'('iJit- ... btlfl~

ing reuctions for giv(,1l hu.deria hun' b('1.'1l im ... ,tigut(d WII h
the view of utilisation as hach'ri('i<ies ill n't'O hut IlUlIl\' of
these are found to inhibit tht' phaJ,.l"(wytic ndicll~ of h1J((w;t(' ...
f.g., on StfJphN/(}('ocruJa', in diJutioll~ helow tllnt Ht whiell thtir
hactf'riciliul action c('as('.", alld thus, in addition to tlU'
inhibiting aetioll of th( blood protdn"oon tJI(, antis('ptics. we
ha\'c to rcC'koH with an inhibitillg a{tion of tilf' Hlltis<'pti ..
on one of tht' natural defl'll('e n1("t'lHlIlism~ of til(' hody. Mort>
sliccessful results have bef.'H f{'('or(kd In tJH' C'a"o{'~ of

",OJxr ilYb:' I
NH,
R.IVANOL FLAV1(.lDE.
p-methoxy-Malachite green allo Ethyl violet of thC" trio
pllenylmethane serit's. ann. notably in th(' ('uses of Profls\'JIIf'
and Tl:vpaflavine (Acrifiavinr), of the llC'ridine s('ries of {Iyt
stuffs. These latter, which nIT more powerfully bad(ri(jdal
in the presence than in the ahsen(~e of senun proh'ins (Sf'('
Table XXI1.), have already been mentioned 8' having ,orne
application in trypanosomiasis (p. 817). ~Iorc r('ccntly
Morgenroth (1921) and his collaborators haw introduct,d
rivanol, which is the hydrochloride of 2-ethoxy-6 : O-diamino-
acridine, and Langer (1922) has utilised flavicide. 2: 7-di-
methyl- 6 - dimethylamino - 8 - amino - 10 -methylacridinium
chloride; but, pl'QIIlising as these substances are, there i. no
330 ClIEJiOTHERAPY
l'ollolu!>.ln' c\'idcncc a.<.; ytt to show that thcy arc capahlc of
(Ouring haderial s('pti('a'mia..... This, howe vcr. has heen more
IH'arly attained hy the usC" of an artifieial (krivativc of the
('ilwholJ:t S('l'iN' of alkaloids. namely. dh:vlhydr()('nprr~ne
(ophv'hju), first u'Jpd hy MOI'gt'nfoth Hnd his collaborators
ill l~fl -]2.
Optochin is to_xic to several straill."i of J}I}{>Hm<)('{l{~('us
hoth ill ,;.'ilro and ill vit'o, and in c(mjundion with spedJic
ftl1ti-pll('umo{'o('('a.! S('ra gives results Illueh greater than
would. he' ohtained hy sUllunation of t}l('SC two individual
df('ets. _From the results obtained with opto('hin and mAny
oth{'r artifiC'ial f'indlOllll alka.loids it wouI(i uppear that to
ohtain tIlt' optirnal ba('t('l'it'itiul dfed in l.'it'O the ('o~operation
of the host's dl'f>l1siv(.' m("('hanisms is 1l('('{'SSRT\\ and that
tlwsc latter are rt'adily impaired or suppr('ss~d 'by ovcr p

administration of the alkaloids. ("ven though the amount

inj('('ktl is' wdl withill tIll" host's tolcranec (Felton ,,"and
Dougherty. IH22).
Chemotherapy of Tuberculosis.-l'uhereulosis is a disease
which has }l}'(),pd to be (,yt'JJ nlOrc resistant to ehemoth('ra-
pelltic'at tr('atm('ut than ot.her ll(terial or baeillary infections,
despite flllBlel"OllS attempts to utilise remedies which gave
promising" results in other cas{'s. This rt:'sistance may be
aserillt't{ to the physical nature of the baC'iHus, which possesses
a fatty (-,Ilydopc probably impermeable to Inost of the
substa.u('(-'s used. or to the Vef)' poor blood supptv whIch
eharaderis('s tubt'fCuiar lesions, rendering uncertain the
a<,cess of the curative agent to the site of the infection.
Newrthdess, from time to time partially successful results.
han b,'t"n claimed, more especially on behalf of metallic
deriYatin's, amongst thenl those of gold. None of the earlier
efforts seem to have stood the test of time, but recently
Mllgaard, a Danish physician, has introduced a gold salt,
sodimu auritbiosu)phate, of the composition;
Na
[S.O.AuJ Na.S.O.,
 EXTERNAL ANTISEPTlrS ~31

whidl hl~ has T'('gistrrN) \lIldl~r tlH~ nanw of Sallo('rv"'in~

and in whiC'h the gold is prest'llt ill a HOIl-iouisllhll> f~mn.
This salt is now Tl'('l'i'"illg' n gTt'at d(~HI of at h'nt ion ns n
specific curatiYe ng('nt ill tull('rcuiosis 011 !tf'('Otiut of tlH'
highly promisillg prrliminul'Y ({'<mlts ol)taiIH'd Ily ~1~"glulr(1
nnd his nssoeintes. It undouhkdly IU1S a SPfTiJi(' fu1i'tm 0),
tissues inf("C'ktl hy t111wrcl(' hacilli. bllt it t('tlHlill'" to h(, 'f'('n
whether or not this ad ion ('un he mmt( til(' hasis of a ('111"('.
On injection into tub('t{,tllOlis patients it prmlllet'l'o. a t ypi('nl
reaction with rise ill temp<'fatun" alhmniuurin, dc. wlli(,h
is not shown hy the normal suhjf'd, and tlw sc'\"('rity of ",hid!
is more or l('ss 1lrnportjonal to th(' f'xt('ut of thr inf('dion.
The treatment may he comhined with a "'pC'dfk seruUl tr{'n.t~
m~nt aimed at ('omhating the toxie T('udion. 1'111' most
promisi;lg' fesults nrc thos(~ obtained in mild illft>diohS;
advanced pulmonary cast'S are 11111('h kss tra(;tnhl('.

External Antiseptics
In the field of {'xtenl1ll aIlH'j('ptics it hits not tK't'fl diflir'ult
to find IllUn('rOllS ('x{'('('dingly t'ffedh'c dlCltlkai ('OITIIW'Ulhd'li
having many useful app1ieutions iu vitro, nnd h.iSf) for til('
local external treatment of infceted wOlllHl ... and nuwous
surfac(s.
In se1eeting an antiseptic for any gin'u pHrpos-p .'fcnraJ
J.lOints Dlllst be considered, as, for ('xmnpl{', the- followillg :
(1) The nature of the m.'dium in which tl ... antiseptic, is
to act is of great importance. Mauy nntisq>ti('s tUl\'e a
nmrked ~"lmnlty for proteins and fats., whh whj,'h t]J('}' J))JJ.J'
fonn more or less stable rompounds which are in gf'Ht'ral
much less reactive than the parent suhstauf'c. The (,X('f'P-
tiona1 case of the acridine dyestuffs may again he mcntiOh('ci
here; these, as Browning hat;; shown, arc more readive if!
the presence of proteins than in their absenl'e.
(2) The nature of the particular organism tn be dealt with
must.be considered. It may have a waxy or chitinous
envelope which Hsists the entrance of It given anti.eptic, or,
 332 CHEMOTHERAPY
again, the spores ma.y be lnu<.'h more resistant than the
parcnt orgnnism.
(;3) MUllY antiscptj(_.s show n highly spc{'ific ad ion. heing
toxic: t.o (lite tq)(' of organism and lJontoxie to another
whi('i. may he (:!os(,ly rdated to the first. This spccifi('ity ~f
tletiotl is utilised in ha.cteriology for the selection of one
spceie . . of hacteria out of a mixture of nutny. un Rntist'ptic
Iwing ('hOS'll which inhihits the growth of the oth('l' organisms
more than that of the one which it is desired to select.
(-J.) T1J!' ('apJl'ity of nwny organisms to dc\'elop a specific
resistance ou p"olongcd treatment with a gi\'en antiseptic
hns: already bt~ell U1elltioIled) and has an otJl. 'ious: importance
here. ~fost bioehcmists cannot fail to have observed the
growth of moulds, etc., in solutions which might be expected
to he' quit(' toxie. e.g., in 10 })('r cent. snlphuri(' acid of sodium
hydroxide, an illustration of the enpucity of lowly organisms
to adapt themselves to ('xc('cdingly unpromising envi:ron-
ments.
(5) An antiseptic lllay inhibit thc growth or reproduction
of haderia, or in other ways interfere with their metabolism.
without killing them. On removal of the antiseptic they
may again flourish.
Rt'arillg in mind the St. OhSl"f\'ations, it is not surprising that
the ideal antis('ptie still awaits discovery, and the task is
ohYiousJy diffieult, since a suhstance toxic to all micro-
organisms will probably be toxic to other lh'ing cells. Ex-
temal antiseptics are of many types-halogen, sulphur,
bismuth, silver, and mercury compounds. phenol and
fonnaldehyde derivatives, dyestuffs and alkaloids-but these
cannot be considered in detail here, many having attained
but a Ilft'ting popularity. Nevertheless recent progress with
alltiseptics of the type of chloramine T, the acridine dye-
stuffs, and the artificial quinine alkaloids is highly promising.
Chlorine, in the fonn of IlypoeWorif;e solutions, h"" a wide
application as an antiseptic, but most hypocWorites !Ire ~
ullstable in dilute aqueous solutions to be really serviceable.
 MODERN AXWISA'PT{(,S 333
Dakin, and latrr Carrel. found that th(" uddition of hOrl(' add
greatly inerrascd th<' stability or hypochloritc' SHlllhol\~. nlHI
such comhinations w('r(' ext('lIsiy('ly i:'ll1l)I(l~'td (1IJrifl~ tht'
war of ]U14-1~. Dakin ('ontiHUl'd hi,"i . . tm'l,h for .. liB lJlI)f('
eifective chlorinc ('ompmmds whidl wOl1ld lilwnlt.' thi ...
elemC'nt ill pontu('t with a wound or SlIft', 11IHI n ... Ii ftlt..tllt Iw
has prodtH'l'd dlloramill(, T and tiit'ld()rnlllill{' T. till' 1Il0"it
notahle dllorinc alltist'pti('~ yd di,,(o\'('f(ll. rhlofllillillt' T
is asoJublc sodium derinlti\'c of p.tolu('w"... uJph,,'H,hJnrIlHlidl"
CH 3 CaB ... SOzNa : NC], whilst. d;('hloramillt T j-; lUI
insoluhle nlOdilieation ('ontainillg t \'\0 C'hlorilw utOllls,
CH 3 (,~1I4 . sO,n : ~("J or (,11 3 ('.11, . SO, . ~n,. Thes!"
antiseptics nrt~ stahl{' in tilt' dark. art' 1'1'('(' frOll1 irritant
prqperties, and arc ,'err lIst'flll in It great ntridy of M:ptir
conditioilS.
Of the dyestuffs many han' h('{'11 used. alld SliitHhilitv i ..
obviously dt~t('nnincd by partic'ular ('HM'S. Thl' 1I'iph{';'~I.
methanes and the B('riditws pro"ide tli(" JlIII."t ll~d\ll ('xh'rnal
antis{ptics. It is inter('stiHI-{ to lwk that OII{' uf the Hr... t
dyestuffs to feveal its Rntisepti(' properties - }Iaiat'hit( g-r('{'n
-still remained in use during the late wnr. TIl{' a(ridill('s.
already considered as internal antis<'pti{s. art~ !lOW J'('('('glli!'!tNI
as the most potent of extcruul dye~turr UlItiM'pti(s. alld ~(t:m
destined to retain that po~iti(Jn for som!' time, WhCf(,U,"j tlw)'
are likely to be superseded as intenud ullti!oo('pti(s.
-The efftciency of quinine as nn internal anti!oot'ptic in
malaria has already been noted. and we han' M'cn t hnt of t lu-
numerous artificial derh~ath'cs of the cinchona alkaloid... IIOIl!'
.. has proved more efficacious against tIle para.~jtc tluUJ tit'
natural compounds. Nevertheless many of these artifi,ial
alkaloids have useful applications as external (and som,tin,,".
as internal, e.g., optochin and vuzin) antis(:pti('s in oth('f
directions. "'''e owe an interesting series of inyC'StigatioIls on
the potency of these substituted hydrocupreine deri,ativcs
.principally to Morgenroth and his collaborators, from whi"h
s'ilme correlation between constitution and antiSC'ptic action
334 CHEM{)THERAP Y
emerges. The relationship of these antiseptics will be seen
from the formula :-

"

In quinine Rl is -CH = CH., the vinyl radicle, whilst in

hydroeuprcine this is I'educed to -CH2-CH" ethyl. In
quinine H2 is methyl, and it is this substituent which has
becn variNl in. the series which we are considering. Some of
the results obtained afe summarised in the table givcn
(p. aS5). It is eIear that the nntise})tic ad ion in 'I.:itro oftlU'sc
derivatives is nlRximaI against diphtheria, tetanus, anthrax
:Ind gas gangrene bacilli, and again....t streptococcus, when R2
is the iso-oetyl radicle. the toxicity gradually increasing as
the substituent becomes larger until this eight-membered
carbon chain is reached, and then diminishing again, with a f

further continued increase in the complexity of the side

ehain. Against other bacilli similar results, generally showing
a different optimal size of side chain, are noted. Thus for
Staphy/ococCIU aUTt'ItS the heptyl radicle is most deadly, and
for pneumococcus and trypanosomes the ethyl radicle, as in
optochin. With regard to the relationship of other parts of,
the quinine structure to toxicity, the fact .that reductiOft ol
 SUBSTITUTED QUI.YLYES 335

6 i C
~ c:
t~
:E t!." ~
c ;;: .,
~" E ~.i : I ~i
"
"
is ~t
~"
~
, :
.:: 8 '"'"
'0

"
'E
~
]
.5
~

~~ i
~ :
.~
.;::" ~ ~i
I

..;;::: . l-

a
X .~
0-
";j
:::: ::. :
z;:: ;:

~=.::'
X t:
"T:
...;
"'"
;r; ~
-<
Eo<
~
--:
."""
h
~
"t;
~ !

.r ,
~
;:s
 336 CHEM.OTHERAPY
the ,"illyl group to ethyl (quinine ----> hydroquinine) docs
not alter th(' therapeutic action in malaria has already been
meutiorwrl, and this point is further demonstrated in the
tubh'. Xevcrthclcss, complete oxidation of the side chain
to {J.lrhoxyI d('stroys all toxicity, as ill quite'nine, Other
ohs~tu.tiolls b('arill~ on this question arc thnt alteration of
til' st'{'ondary aleohol group at (A) does not arced the toxif'ity
of quinil\{' dt'rh"atiH's, Hor does an op(ning of OB{, of th("
rings at (B). as in quinatoxinc. destroy it, hut it is interesting
to note that trypanosome's resistant to the original quinine
111kuJoid '10 longer dispJay this resistaw't_' to the ('orrcsponding
quinatoxiue. whilst tho.se resistant tu the toxiue nre likewise
rt'sistuut to tJI(.' quinine. Morgenroth assumes that quina-
toxines hnvt' a greater amnity for the quinine receptor ." of
H

the parasite, or host's tissnes, than has quinine itself, and so

('orrelat.cs these results, the inference then heing that int.egrity
of the ring at the point (B) is not all essential factor.
It is d('ur from the foregoing survey of the chefllOtherapy
of l)rotozoal and bacterial infections that no one theory,
simplr or otherwise, wiH aecoD1Dlodate all the known facts.
\Ye have. in the Jirst p}acc, the (Brect toxic action of mercury.
u,utimouy, and hismuth derh'ath'cs, both in titro and in v;'vo,
which apparcntly depcuds simply on the introduction of a
highly toxit ul.ctaf in some suitable physical state into the
organism. Sl'C'ondly, we have many cases, including all the
reany effective agents, in which the toxicity in 'l.'itro beifrs
hnk rdatitm.<hip to that sho ..." i" the liring animal. Thl1s
the toxicity of quinine to the malarial and dysenteric parasites
is summarised in the following table ; -
I" dt7O. In ,ivo.
Parasiticidal effl"('f. of quinine on : -
.Malarial Plasmodium +
E'uta mO'ba Jiistolytica +
Again, we have the great lowering in the toxicity of Mala-
chite green ill the presence of serum, and the corresponding
increase ill that of Trypaflavine under th" same conditions,
 RESISTANT .'1TRAT.VS 337
and the powerfully toxic' uetion of ..nlvursuH anti llft\,C'T 20.5,
which is only r(,\"f..~al('d in l'it'll. Thirdly. n point ~Jf "'Tf~>nt
i)}lportal1{,{, is thf' rHark(',' 'P(('jti('it~ of fIIlllly of to("'S('
antist'ptics (M'e Tali]," XXII. IIlId XXIII.). II 'IK"'ifieil\" whieh
sometimes difr('r('ntiat{'~ in a rf'IIHtrkultlt tnHIlJI('f t~t W('(')1
two dOM"h'-Tf'Jati'll organj,'HHs. Awl JWitJ" nutv h(' nH~jrion('d
the claim~ whi('h han' heen made 011 l;('lmli of BuY.r 205
in relation to ('onfC"rring Oll the ho"t nil imI1111nit,," to tltt'
infediw' parasitf"s in addition to dt'strf)yjn~ tho\(" Ht'tuUJJy
pr('sent in the tissues. All these fft('ts on- ('x('N"(linglv
difficu)t to f(condlt with Ehrlieh's" eheUiOTt'('('ptor" tl1l'or~:,
faithful servant as it has pro\'t'd itself: nor. in<i(,l"d, dm's allV
other singJ( theory siller propoundrd off('r a Iwttf'r ('xphm~w
tiQll. In particular (as(s. {'.g., th(' arst"llieals, mort' <htailffi
('xperitnental inyt>stigation j" h(>gillning to hint at tIl' wider
"\'ision, and, indeed. it is drar that in alleflscs the suhstau('e
Bop-toxic ill vitro must undergo nih'ratioll in ('out Ret with
the host's tissues, or indu('{' som(' ehrmi('I\) or ph~t~i('al ("hnnge
in the tissu('s leading to production of the toxi(' "lIhst.an('( or
('on<lition. The complexity of th(' whol{' qu(,stion is Ktill
further inrreascd by a factor which has not pr('\'ioulloly hf.cn
discussed h{'re, namely, the dt'\'r)opment of strains of para ..
sites Which, after bring suhjeded to tht aetion of th{" a.nti-
septic, acquire a resistance towards it. It is poioisihle to
produce such resistant strains in dim without the co-op("ra w

lion of the host's tissues; thu, pneumoc,oeci ha\'" been

developed to resist the action of opt<x'hin, and certajn ('holera
bacilli have likewise heell modifit'd in serial media containing
increasing amounts of the toxic substance to resist hoth
dvestuffs and arsenic derivati\"es. It is interesting to note
that in the latter case the resistance to antimony was found
to have risen too; and, indeed, Gonder (1912) has recorded
that resistance to tartar emetic in the case of protozOa is
better attained by treatment with arsenious acid than with
an antimony compound! Such resistant strains may also
develop in viva, and the resistance is generally found to he
n
338 CHEMQTHERAPY
markedly spC'{'ifie; tllti~ a straill of t.rypanosomcs resistant
to Trypan fed might still retaiu it~ full i'leH~itiYity to atoxyl.
and two or more r('sistan("(':', may be d('\'('lop('(l in the same
straill ind('1wnd('ntly of one anoth(,f. One of the most
intefes_J-ing ohSf'fvations Tf'cordcd in this field is that in whif'h
8 trYI'rt'losomc strain. resistant to ntoxyl in the mouse, lost
this nlld )w('unl(' normally 5,('n~itin' in the rat durin~ the
cours(' of fort~ passfthr('!!, thruugh the s('('ond host, and
inun(->(liatcly rcgairwd its original r('6i~tan('c 011 ueiug rctrans-
fern'd to its first host (lIlesniJ and Brimon!, J 9(8), Of a
similar JUlture is an ohS('rvatioJ] thnt a strain of dysenter,v
atnu'bw, readily &us('cptihJ' to {'mt'tiu(' ill the human, became
quit!' resi~tant when transferred to th(' kittf'll. In these Cfises,
as 1)al( {'xprcss('s it ... to suggest. as an explanation, ~o
operation of the ho~t 's tissues, in u manner concerning which
tht' knowlC'dgf' yet a\"ailahk affords not evell a hint. is
unsatisfactory; hut it Si'eI11S at present the only alternatjv(>
to a (ompi(tcly agnostic attitude." Although tlw ehemo-
therapy of infectious diseases has not yet realised Ehrlich's
ideal-therapia stcrilh:all ... 1I1aglla-"in which a single large
injcctlon would effect a romplete cure, there is no reason to
douht that nlOre and mOTt' potent Temedit'S "",jJl be forth-
coming as. our jnformation of the physical, chemical, and
phYE-iologieal reae-tions of the internal antiseptics increases,
and meanwhile the succ('ss{'s a1r('ady achi('vcd in what were
previously regarded as prac-tieaUy intractable diseaseS~
partial though many of these successes are, constitute note-
worthyad,-ances.

The author ae].-no,,-ledgt's hi. indebtedness for mucb of the

material elubowed in this cbapter to au article by Dr. H. H. Dale
on "Che-lllotherapy .. (Physiol_ Ret'i~8. 19~3. 3. 359). The reade1'
mav oonsu1t th{> following reff.'!TeDOOS in addition to those which
wili 00 fOlWd in the above-mentioned artiele.
1. BALAIlAN and KUiG, JOlt'''', Ch".. , Soc" 1925, 127, 2701. '
2. FouR~E.t\r, ~. Orga:nie Medieamentf; and their Preparatio~_"
London: Lhurebill, 1925.
 CJlJ<:lllU1'1l IJRAI'}, 33!1
3. and (H.w()rkt~. :tun./lIlll. l'l(i'iinft', Hl2:I. 37. i.:a.
}'OliR!\E.\tl
IH24, 38, KI.
,t-. I"LANHlS. I:ull. SO(', uull. II/'"/Ii(. II!' I'arill, IH:.!>t. 48. III:!!'\.
5. HElTTlm. '" lIantllllu'h d,'r EXP('flflWlltt'iJt'lI Ph:lI'IHOlklllo~i."
H.rUn : .Julillt' ;o".prilLg"t'f, In:!!) :!t.
'6. KrsGaud \h'lH'I1. .J(JUnt.,'hm, .'1'''-.. 1\121,125, :!;l!l:-" HI:!;,.
127, 20:12.
7. LAXO};R. Zf'i.t. yCH. Il.T)1. ,If('~l .. l!l:!:? 27. 171.
~. L1J.E~HW allli H}~ HI't;~. ("am/If, Nt'I/.I. SI/I', f,jol" I\):!-l, 91. ~2;).
U. ~IAn(,l!ot_;x. l)!lll. Sm', Jafh. l!1;!:I. 16. 7ll,
1'.fO(.,
11). ~hl1.. U;.\,\Hn. .. \ '1t('HlotliNaps of Tu!w1'luh,;.J.... " ,'Opt'o.
ha~l'lI : Arnold Hu"d.. , I !I:! L
11. V"')~~UTl,l~
.. Tllt, PtmnH"H'llh'IrY o( .\r",\,htH~lHliHt, (~al\'ar*",llIl
and R(,lat{_d .\rt>t;'uir'al"," J'II.11:<iol, lit'ril~If'''' HI:!;,. 5, ttl.
 INDEX
Absorption of fltt. II';. liS. I HI. Ammomurn ('\'11111111' III UN'II (o!'tll'"
l~o tloll \1, 10:11. ;!n, ;!I
Absorption sp~'d m Ampboterlc dl'I'1 r"ln, ... , :!;,
ehlllT{lphyJj~. !!7S Amyle,.. o,\IIJi,' .. ,/(';,('>1. ill, ~f)
t'yt&hromf>. 2i:t :!i4 Amylol*itu, 7;1, 77
m('OSIIN'm('nt (If hnndtt. :!ii:1 AtDylOM, 7:. iii
Acetaldehyde iu fl'rmf'JJtafilmll. J():.!. AllimAl JJudf'll' lWld, t~1)
10:1. 104, lOti AbtibodlM, :!l;li. :'~.1'4
Acetone hot1it~, l:l:!. 1:1:1, 1:15 Antirla :!'!Mi
AcetylamiDo I.kids, ii. t'i, :!:! Aotiketo,ell8sil, 111, 1:t,j
MetylmethYl earhinnl in f(-rnwnta. Antimony iT11'lumnttw(1t1'),. :1:..'11
tions,"H5 Antiseptic.. IIrtili('i.1i 'luifHlW tI(ri
Acriflavine in l,hf'motiwl'apy. :U7. valin,,>, a;,,~,
3"'8 (',~t.-rrlftj. :l:n
~a:::~:::it':!;~m yt'llllt, ~I. f!:!
int-<'rwll. :HI.:l1ti
Antisera. ;;01
J)('nt-ot' flu('it(ltid, from pi.t::'~ ArabiDOIe. 7:1. !oi.41
hlood.51 Arachidonic add. lit. 17K
.&tioPbJUin. 2ill. :!I'J() Arf'ni.('fJbI. M(I~"I pigult'nt. :!'HlI, ~I~
lEtioporpbyrln. 27!1. 2M. 2AR Arjniae. r"illtil'll 1(1 tIm II}!' ~yJl\lw'
AfridoJ violt,t in <:hemutilt'Tu.PY, :0 S "i".'~.{iH
A"lutinitt.. 2fJ6 A,..,DIC diloril!" I~.,.,t (or, itMtI;rl .-\.
Alanine. 6, 20, tiH, Ofl, H7 :!:!il.2;!4
Alcoholic lennf'ntat;on. ](1] Arsenic d.rival /vI''l.indwOlntiH'T4PY,
and phoal)hah'~' 15:! ;t211
pyru vic arid thl>()ry, 10:! Anenophel'lJlIlYcine. ~t;! I
8("('Ondform. )II:! Aneoozides,. tnxil'jtv. a:.!4
third form, ltt3 ..fIlNn'.... 01('0<.1 pi~m~'Jlt, :.';;:!
Aldehydes. ndudiOl1 t.o t\kohol~, S. Asparagine. 4, ~H, Hi)
]0'2,107 Animil&&orJ. 1'~)(.f6df'nt, :''S:':
AJ4ehydematue, Wi AtoXl'1. ;~:"IO. :C18
Aldol in fat A)'ntht"Si8, J2lj Autotrophic nnturt (If plittltli. 21f1
in ff'.nnentationf~., IH4, 105, 10fi Autosi4atioa. (y~t("irll", ISH
AIkaIiI and aleoholic ft'rrnentAtion, ,rlutathioflf" I Hf\
103 AuimODet,,:!I:,
Alkaloids lUI t'xtemal antwptiell, 3:l5 A vitamin. 217
in chemotberapy. 327 l'fJlour read Ion!!. !t23. ~.!4
AIIantoia, 64 phy.llj(JJo~iU41 (tfM:1ff of l1:u'k,
AminoacidR,.l 225
conversion to t'8.rbohydrate, 7,
00 Bacterial earbohydrat.{_'R, :W:1. 3W.
A.JDiDo-nitrogen.. 2, 3 : 311
AJUtOIlitua earbonate and e:a:r. d~ and cb.mothcrapy. :12H
bamateinurea fortnation.4, 16.1; , nuc:looprouin, :i(J:t
an
 342 I/iDEK.
Bacterial t()XinH. 2t1R C&rbohyc\rate----r-onl;llued.
Bayer 205 in dl('nlntlwm{1Y, 31S of Ulwleie jUjdil. f):!. 53
Benzoic add, 5 of p(>ntoHuria, 74, 7fl
BeD.z-yl alcohol. i spt'eific bad-crin!, 304, ,3ns, 311,
Beri-ben. 2J i, tit, :!:.!!I :H:!
Bill" pj;ll1i'nb" ~KH Carboligase, 105
formation in n'frfl awl in l'ir'o. Carboxylase ill WMt, 102
2!f1.J CarotinQid.. :r;'!i
Bilirobftl,'.?IIO Cerebrmides, J J.... 1Hi
BiUrubinie al:irl, 2tJ 1 Chemotherapy of had_,(>rial tli8ClUles.
Biliverdin, :.WO :e8
Biophysics of lipidt.'\, l:ri protot.oa! di~!(,Ht.f>j;, :J16
})rtJh,jns, ~s tuht'rCllli.,i:>. ;;:itl
Bios, 215 Chloramine T. :l:>:!. :1:15
Biosteriu, 221 C'ldnrelfa. growth, 21G
Bilmutb in ('h(,flIotll(>rllJlY. 32H Chlorocruoril1,. 2lJ 1. 2.S;!. 276
Blunt. 11. I:! Chlorocruoroehromogen, 277
Blood amino nitrogen. 2 Chlorophyll. i7S
and JJonllau's ('(juiiibrillIll. 4fi ChloroPhyUaft; Z&I
Jjpjdt>.'1. ]20 Cbolesterol, j 1}
phosphoru", ll\fl irratliatoo. 243
pignu.'nt$., 251 Cinchona 11lkaloids in mll.latla, 317,
~ugllr, oS:! ::t:t:l
BonE! ('oml~iti(ln. H\2 Cod oil. origin of yitamins in.
IiV('f
fUl'matiotl. In:! t17
BODe t'uzyme. J(li.), Hi() frurtionati(ln,219
alld }lydl"Oly~jg of
ndd soluhle (,oIpidium, growth, 216
orgllnic llhosphorus of blood. Complement, 21M\
170 Constant" cit'ment of fats, 123
ill incnhltting egg. 183 Copper in blood of mol1lt.'K'll and
in kidn(v. 172 (,nlsta.cea, 283
in l'ick('t..~. 167 and poTphyrin:;;, 286. 288
Brucine salt lIf ht'xO!W diphosphoric Coproporphyrin. 2~6, 288
add. )51) Crou J'tactioll.s in immunology, 300,
Butylene ~lyrol, 10,j :lll
But,iene oxidic !!iugat'$., 79. RO C vitamin. 232
ButJTio add ftl'lIlootatiun. 104-, 10.7, Crntallograpby of bremoglobim. 260
106,126 Cy_186
B vitamiD, 227 nutQXhlation. )88
and uudear metllboli.'im. 231 inhibiting action of cya.nides on
aut-oxidation. 18t\ 197
relation to t.oxic action of a.rsen-
Calcium. of milk. 163 oxides, 325
Calcium pboephate in ONJifil.'.ation, Cyloeluo_ 272
162, 238, 24Q functions, 276
Caru:er, blood pigml..'nt, 283
Carkh)'drate and insulin, 92, 90
formation from &mmQ t.cids, 96 Dalmatian dog and purine meta-
formation {lOin lactic a.dd, S6 bolism.66
formation from pyruvic ftf'id. _1l,2
96 in livet'.:J.
fOl'1llation of fat from, 125 m~ha.nistn. 3. 19
forma.tloD of lact.ie aeid from. 72 Duat_ ()/ fatty ..ids. 122. 130,
()/ cereb_des. liS 132,133,
 INDN.X 343
Diacetyl in fl'rmrntAtinns, 105 Fat, of ('Io!g }llw"'plml ith'tl, 1S:I
Diaminea, cxcn>tiou of, :!Oi Ilf j.rlll( U!i(' nftt'r in~uliu IUJI'.'l.ull.
Dichloramiue T, :i3;j HII
Dlh7droJ'l&ciltone, Sil, !II. H:') of Illj\~t.'d \llInw"l!o, Ii:l
nntikt!toJ,!:cni<: Ild,jOll, HI "f 1111"1 It' II, ,.I !tI IlIH~dc. !'II;, ~s
eff~~~l!~~~I~hypocly('!.'mk ((HlYul I>f 1'~'rH\ Ie IV It! III tHll~( 10" 11.'" ~~;
FattYI\~I!I,.., d"l'iatl!mtl"I!. Ii:!. I:iq
" DimedoD" and ai<-uitoilc f"I"
m~ntat ion, 10;\ Film~:'~:~'f(\~ ~.~'1 ~I:;I,
lit
DiuoeiatioD ('urv{'/ol of (l"\)'-hU"llh,. FJavlcide II) dWl!lnliwfJlp.L a:.'ll
globin,4H FOUfneau'. 309 Ul .1H'ltlPll",t.IJlY,
of ha'IlH~lohilll', :W 1 :l!l!I, ;m'
Donnan', lllt'mlmHII' (!ljllilillrilltll, Friedlander', Im'lllHoi, )l.P"I'lti. "at
:.!o.40, J3S hollvdrat."" :1111
uppJkafi(1) to blrxl(),.J.;) FroctOu, 711
D vitamin. :.!:li ofllllllm.1'I1
growth promotin~ action, 24[1 o\it1allol) III J't!"wm'I' of l.hull
Dyutid!s ill Cht'llwthernpy. :lIfi ph:lll', H-I
DYJeDtery. :t!7. :J3G

G&laetose. 7o. ~I
Egg. }}hoflphorou>! tll&trihution. HI:; of ((t!),n .... id.,'l, III;
Emetine. :it7 Gamma J,:iu("}f->, kn
Euymia byuroJyRi.'I (If "tanh. 76 Gastric Ilpa,..,,, J 17
of tdJ!ly('t'ridf'8, ) I ~ Glucoprotetns. !,'OH
of urea, ]5 Glucose. 70. ki)
EQuilibrium ('01181.ant-8 of hremo u.diuu of alkali 1)1l. s:t
J;lohiIl~. 2():! llnolill.!<ulill,!ljI
EQuivalent. of protein.'!. :~H al\lilwtr~,'r!j"l;IctJ'IIl, 1:111
Elterase., 12l /orlUl1t;ml from "wlino 11i il)",. Hi
of kidney. 17t IltJlj"ati'lll hy )'jlll}llr' otj:Jlni:mll>.
phosphoric. Jfj,i, 161) 10(1
Ethereal f;.ulphllu-'S. 21ilj Glutathione. litO
formation. 206 /Lilli tr~'ILuoddr'i'I, :I:.!.J
E vitamin. 246 di.'Itrilwtiafj (Ill" dl'tf'rmitJntiIJff,

.
Extem.al anti!leptie~. 3:n til;" HHi
ifOouluti'IIl,1\ll
fltm('inre. Ill:!
Fat (su aIM) Lipidefl), Glyceric aiJIfhyti.,. S!I !to, !fl
absorptioo. 117. 11~ negativp - tu'fi(oIl in hnll''jlly.
catabolism. 12 ('ll'mk (.OJl\'UiSjHU", ut
constant eJement. ) 23 Glycerol Ilnd hypfrgly,remia. HIJ
hydrolY8:iB: by lipaRC. I JS. J J9 {ormatioll in ra!eolj(,lk (ptnH'uta
nwlting-point. 112 ti(lll.I~l
metabolism, 1{6 Glyeetopboq,boric add'il, Iii
"",...,121 GI,eille, 1, !..'o
synthesis, 125, 126, 246 Nm\'cndon t,1I tar}Juhydrat~', ms
oxidation and carbohydrate meta- Gtydrr,e hi<!lpUla. 15
bolism, 135, 136 GIJCOCeD.. 75
utilisation in muscle, 88 (:hemic&i nature, 1H
variable element, 123 fonn.a.tion from I~wtic ll<1wi in
.... of -amino acids, 6, Oi mWlCre, 86, 8;
of a-hydroxy acids. 7. 97 formation from Ilynl\'i~ ..cid in
of u-ketooic acida. " 97 muscle, 95. 00
 INDEX
Glyeo.en in mmwlf'. tun, WI Insulin, !.l
Glyoxalase.. !II, H:l lind dihvdroxvl\N>tonC'. !l2
Gr~:~:h curn.... :!I.:!. :!::!ti, 2::!~. :!:l.:? Ilnd gh'N'ril' ;Idt.hvdf., II:?
lnteuurface. ph;'i"i"~1 IUttUl'f', 1~2
G~~.h:!}~I.omutiTl!! ,Hamill". 217. Intravascular iUt'nlol\'Bis, :?SIJ
Inulin., HI '
GuanidiJe. ,:dioll 1m )W\IN':'( ...., It>diD81wmJ>{r..... J:JO, JSJ
GnaDYijc.ul~l. [tI), ,~~; , "
IP~l~;cuanba ulklllnids in clYM'tltN,Y.
Guanylnucleic :wi/1. :i1 ..-)~
Iron and autoxidation. JR!I. lHi
Hlem.:!I\1i Zrr;1:f.tted "flak''''kJmJ. (11U(kyt"ie....
Blewatin, :.~l(i.
;?fiH
Hmmatinic a~jd, :!!I) Irradiation aud ril'ktt.s. 2411
Blematoporpbyrin. :!.sri, :!~'; Isoelectric diaiysis, :j:~
Hlemin. :!Gli. :!1l7 1-'gJ:! alhumi,;. a:3
Hmmochromoaenll. ~tl{l, ~()H geirl-till, :lL
nrtitidu.l. :!io points and ('ailtphol'f'F.is, 29
plJini!-l of prl)tdn!',. :1<1
B~::;~~i~).('~~;rjl1g, :!il, :!'i5 Isomaltose from :-;tan'b, 7i), 7i
Hte~~~)lObins, (tltmilul difl(,"('fi('p,;;,
Isomerism of fatty !wid;.;, 11:l

f'ry~t.I\JI()gTaphy. :.!HO
di:-l)"lf){'jation ,'III'V(':-<, 41\. ~f)1 Kammerer's porph!'l"in. ~B.f;. 287
('quilihdutll ('On"tllnts, :.)(j~ Kepb.&lin. 177
(l('/'nrrPIl1'f,,23;! Kerasin. 11(1
"NV/HllI." 26:~ Ketogenesis, I :~5
Ra:emoporphyrin. 2~5 Eetomc /l-!'ids, transformation of
Hea.t ('(Ing-1I11~ti()n (If pl'ot.dn<.;:. 3~~ a.JllmOllium ~alt$. tI
}h~~~~ ji(lfrifdM. ~Ilo'(ld pi).(fll('lil". ;?;r. Kidlley fmwtioll find phoSphori(l
('~t('rMt'. 172
Bemicellulole in "tardu..,.. 71{ Kup.8'e:r {'('lIs, :!HO
Heterogenetic I"Ip(,('ifidr\' :111
Heterologow allti;l{'run~ . 3tH
Bnoumines. :?oS . Lactacidogen, 156
Hexose l,ho!lpnatt';o.. l~i lind JIJu::wk {,ontraf'tiflJl, Ir.n
hI'ueilll' salt. HiS
:~ ::~rt~':,~:t~::~~~t(ln, 1.15 t1,.,\zon(', lr~i

. ,il~ yt'u..... t ft',:melltation. 1.11 Lactic aeid. 7 f

ft'Tlll{"ntations, 101, 103, 105.
~~~:;~Sir~~~~lOn to purim, syn. 107
Bistohmmatin. 27:! forma.tion from (ft.l'bohydrat-{. in
Kotmeister Sf'ri('os, at, 138 ffOll's musde. 72
pig-m{'nt. 2R3
HOIIWYII$. bIOll({
formation in alka.line sugar
Homologous n.nti!'tUlu. :w I solutions, S4
fonnatinfi in muscle. 85. 88. 155
B~~r;,XY:rtite amI honf' forUultion. formatj(m in perfused liver. 89
Lactones and Sl1,1rW structuro~ i9
Luooeric a.cid. 114
Immune !'rum, 296 Lecithin. mytlin fonns, 14.3
ImlDWlolot{"ieaJ reltd ioo.'1, 2tm RtrnC"tUlt', 116
spocifidty. 21)9 svnthctie, 179
Inf3e;!iOua di~. cb{'moU~rapy. Leuum. jui<.-e.. fractionation, 235
Leue..... 1.
lDosin.ic acid, 00. 54 Limn!1u. bJ"1Od pigmt?ut" 283
 IX/i19X
Linoleic (linolid IHid, II:~. 1:111 .ulClt ('(>utmdtnl1, t'hNutt,,1 t'\l'Ur""
Linolenic twid, t ItI lH, )01,"', I ;,~.
Lipemia. I ;!O, J:!!) IIlyelin. f(lrm~, In
Lipue.117 Myocbll!!ruahn. :!'';';!
J'{'Vt'l'!<ihility o{ aciitJll, liS, II!!
t iH~t1l', 1:!1
Lipides. ('lilK'!itkatiuu, It 1 Neutral "'ull'lillr (Of IIrlm . :..\,\
of blond. I:!I Nilropru.sl,de 1"I'lldl"II, 1!liW
Lipochromes. :!7H X,I.;"rIH" '!f~~IIII'f"'. ).':r,,"" nl. :.:15
L!pocytie ("(wffi{'i('nl~, I :!a. 1~t lIon~laponiHablt! h"'I,lIi1' "I ...... 1 In'(r
Liver. rhief R('nt of dlll.lIlini."Itliutl, :I "il, :.!~\)
LoliglJ, blt)()tl pi)!ul\'ltt, :.!S:J N:S.mtu,,:.!n:!
Lylins. :.'1Hi Nuclea' rt',wl WH. :j~
Lysolecithin Itllli Iyt'okpphuliu, I,;"S Nueleic :wid, I!!
~'lll,\IW'" 'Il tiHr' on, n;!, I';,
IIl!"hiloilsm,til
Jllairr, bluod pignll'tlt, :!H~4 (Jf ll.llinud n'!k r,!1
Malachite t!:tt'"{'11, :.I w, :J:~;I (If plllllf (pl! .. , ,Wttl
.IIalaria. :127. ;tjtj l'iJo""'phori(' ilnd of, ,';1', .~,7
:Kalioae {rom Hhm,IJ. 7H, Ii s!flll'!un, ;,)\

:==..:e::~'tj:)l::t :,r I'ii/fw, :!b4

~lJl-.:'lIr of, .i;_!
Nllclein.JI'I, 4\1
MaD.DOJe, 70, 80 .ueleoproLein. I~. !:I
lIecbanilm of action of III'pam', I H, of pt/(lllflO("fJ('u.... ;IO:.!
.17, 18 of <'ftrf"p/'lrrIOJl,', ;ll:!
of dcamillhmtioll. 3, J!l Nucleoside.. ;,;;
Membrane poh.ntiai!i. :~I', 4:! NueleotideJ .'",4, r).\ HI
of mono and di.bAAi(~ tu.'idJi, 44
Mercury in chctllotlwrapy, :::!6
IlUObilirllbin. ;..'9a OrtOl),IH. hl!lod pl~rrt!'llt, ~ioI:l, :!"t
Ileloporphyrin. :!~3" 287 Oleic lHid". J 1:1, 1:1
Metabolism of fatlo, 1 Hi Ooporphyria. :!I#i
of nucleic acid, ti I (}pltiw't,,<. IJI,....,.-} pif[IlIl'1I1. :![I~
of pent,c)8f'H, 7t Optically al tin ul.v'"fl,lf,'I. II:;, llli
of phosphatiUes, lSi OptocbiQ, a;PJ, :1:1:1, :~:Wo. a:n
of ph()Hph()ru~ (~()mImunds in Organic pllo!ophate" awl (lIfI'iilil'1"tthin,
incuba.ting- t1/-Ut, 182 H~4
'> of 8Uglll'H. &5, J 50 Orgallic pJJ(lspbo.ru.~. IH'Hi !<-'Jlul,li~ (If
of sulphur ('{)mpounm, 2UtJ blof){!, lli1I. J'In, 171
Metb83moglobin. 26R Organic ,"u1f,h.atf"H iu tiliiOIW;;, tU7
Methyl ethyl maleinimiu{', !!iJ I of urirw, :..~o.'i
glyoxal, 8!J, Ill, 95 Ore.no metallic l~omp{)UndH in
Methylation and sugar ~tructure, 81 ehf'motht'rapy, :1~)
1loIh.1thI....lope.-.53 OsUOIUt of lat't.fl.eiougefl. 1.5fJ
MU:rOtX)(',cUll urm. 15 OuUieation. I tit
~pe.253 protf>in., and, Jua
IIiIk, phosphorus compoul.lds, 145 organi(' ph(~'!phll.k~ in, 16t
pt"Oteins, detennination by titra Osidation. tiAAue, lW
tion, 39. 40 a- 4
proteins~ .6.xa.tion of calcium and ,'.6,131
ph06j)hate. 163 .(~ and ,_ 134

.QI:Oitia sulphuric acid, 2U8 Oxidation ({lmtient, 8fi. ~7. 00

- - . 2 0 7 . OQreu uptake in mU8t')e. 8.5, 86, 9~
M __ blood pigment, 2.'12 160
 346 INDEX
Oxyhromoeyanin. l'ompo.-.;itirm. :!&l heeipitin.. 2tlH
Oxyhremoldobin. l'\lll)ll<noIitilllJ, ~g.t Proflavineiu chemothl'J'apy, 317, 321)
di~ocj1l1 jon ('UrVeM, 40, :!()(I ProteiD&, amphoteric bt>ha;dour. 25
luul Donnan'" lU{'mbrane equili.
brium,4()
P'JliIlUrlM, hlm)!J plj.!IIH'lll, :!H;J cat&ln-JIi..qm,l
Pa.ncreatic lipn:-!{', 117 cnnwrsjOl) to fat, l~ti
Papentliftk's porphyrin, :!i'lH d{tl'rminati(m hv titration, 39, 4()
Peniose 52, 7:! equh'j\.l!'ntfl, 30 "
Pentosuria. 74 twat t'ClBI-!Ula.tion. 3:1
l)(mt_(jjl(, ('omIJill(d with un'a j)l, H:.! jiK)f'kdrk poiou-., 30
Phenaceturie Iwid, l:H f;loiehiouwtry, :H
Phenyl alauiuc. l titration ('UrWR, 35, 38
't,Ph6Dyl a-IH.!lillH!JUtyric Reid, r, ProtopOl'ph:rrin. 280
Phenyl glyfjllf" 4- Purines. fIlii' of f'xogenolls, t;;}
Phenyl ,l!l)'lIxylil' add, r. ~ynt,hO!'.i". ti7, {1M
Phosphate in hol1(, formfltion, ltl:! Pyrroles, 8uhstitut.t'd, :W:!
in yi'l\.... t ff'rtlWntlltiol1. lIt! l'yruvic add, 7
oxi~latioll of frudo;,w in prest'nce ('onvetsion to glycogt'n in muscle,
uf. H4 fiG, mi
Phosphatide.. 1 i3 in hutvric fenllt'ntation~ 105
nmpliott'rk IIltlll\\ 1:~7 in yel~t fl.-'rmentat.ion:', 102
and cl'n J)('rmf'llbility. 1:~'l
and fIlt ffii'taboillim. J 20, 121)
Ilnd mydin form"!, 140. 14:J Quinine dtt-i'vati\"Nl as anti.scpli("-S,
fatty adds of, }7(), Ii!';, ISO 3:H. :l35
in n1('taoolism, 181
Phosphoric add of lludt'ic add, 56
PhOSPhOric {'i'.f('m.'!(>i', W5, H~ &eceptors in ~h('motherapy, 315, 336
of kidnc\', Ii2 Red blood cells and Donnan equili-
Pho,phorua ('Jf blood. Hm brium,45
of ~f!}t, 6';, )S2, lS,'1. 184 Besidue antigens, 301, 308
of milk, ] 45. ] 4(). I t;3 ResiJtant strail18, 337
Photosynthesis. 281 Retieolo-endothelia-I syf!ltem a.nd bile
Phrenosin, ]16 pigment formation. 200
Phylloerythrln. 293 Bnenibility of action of Ricinu.a
Phytol. 220. :.!80 lipase. liS. 119
Phytosterol. irradiat.t.-U. 243 R4!1veraiOll spectroseQ~. 254
Pirmeats of bil(', 2S!I Rhamn.... 73. 81)
P.inaagiobalm. 251, '::84 1Ii_53.S5.73
l'tannrbiP, bJood pigmc-ut. 252. 2m, urjc acjd rorupound, 82
')6') 264- Ricinoleie acid,. 114
P~i'nncl('ic Mid, structure, 58 lticketl and bone enzyme, 167
P,wttmOCOCctM ~11 ronstituents, 309 and calcium-phO$pborus balance,
nuclooprotein. 302 2<10

P_. . -
specific carbohydrat.es, 303, 304 and irradiated cholesterol, 242
types, 301 . and Jight, 24Q
PolJCluates. blood pigment, 277 and vitAmin A, 225
poJ.nMuritil. lll'"ian. 227? 229 and vitamin D, 238
BivaDOl in chemot.her.pYt 329
in vtgetable sourees~ 274, 287
natural. 286
_ure.2S8
Porp _ _ 287
 IND:&t :147
8areOliDe, 21 8a1phatidea, :''l~}
8aturati~D of hi(lorl pigmrnt!'l, dl'h'T' 8u1phur t""h"tj"tl, ~Il~
nunatlOll, :.';}8 mf'taiW:III"'IlI. :.~.,
Soony, :.'J J. :!:,:! 8'l1lbHil of (,t\thnh:nft1l.lt> fWIlI
Sodium !:iUlphitt anti akohoik ft.r. aUlIno (wl.l.. , II(;
tnentl\tiou. lO:l in InIlJ.dl'. Ktl. I.... ItH
~rbose. ';'0 fnt frmn ('l\riMlhvdtl4t<' I:,:;;
8ynth~is in liI~). !to. \I:{ from J!l'Phjll. '):!H
Ioya beRn, Sir (;fYf:inf' hi~pid(l . purim...:. tl';'. ~IS ,.
.. Span H of hwtllof..'iobius. :.'Il:~. :,'t\4. un'a b,v IIJ'1'il"f', 1';'
:.'65 a,.ntbetic ~Iut,dljoll'" 1!'.a. Ht.'i
of dl1oI'twnwrin. 277 !t'('lthiul:I. ]";H
lpeci!c.itr Ummuna/of,!i{,8l). :1.1
309
.pectrolCoPfl. mil'ro. 2;')3 huriDe, :.'01
J'('ve~ion, 2M TaurochlotlC lwid, :!iI:!
.pbingomyelin. ~tnh'tun. I~) ThatJpi(I .t({'rilUlflll" 1W:'11'l1f, :l:lS
Stareb (lonstii lIt'nt~, if). i(i Tbermoa1abll: 1I~"llt (If pig'k JlIln.
stercobilin. ~;:{ at'IUI, ;",>, !)~I
sterility and \'itandl1 E. 2-16 Thiokelopentoae. 5:J
at.icbiometry of pl'ot.f'ini-, 34 Thiopeptide hnk,u[l'. :!ol
Stored (1\1,. 1:.'1 Tbiourea, 1;1
Itovanol, 3:!2 TbymoDtlcleic 1l1'ld. till
Streptoc<xxus, SIk'('itk ('arhohy- Tinue fnt. I:!l
drat.e. 312 lipa.e'('S, 1:!1
Sttboture, IPtipphyllin, 2:8(1 Titration {'llrn..,. of prnt,iw., 3.'}. :,8
tetioporph.rrin, 2SS f'rypdanlMt. :0";, :1:..'1-'
and antiseptic al'tion of sub Trypan hhu' in {'ht'nJottu,mpy, :H7
I!tituted quinines. :1:W. :~3:), 33t1 red in d'('nwtlwrupy, :Hi
bilinlbinie a.dd, 21H Trypanoei4al IU.'t iOB (till! ('oHlltit II
coproporphyrin. 288 tinll, :J23
gamma sugar. 8(1 tht'ury. :J:!4
glutat.hione. 192 Tryparsamide. :t!l
guanyliC' acid. 55 Tubercle I,mdili. !l:lk'<'ifk c'tlrhoby.
hrematinic acid. 2H1 drat._. :)11
hremin.267 Tubereu1otw., (hlnlntht'rapy. :J:Ju
inosinic add. M Turacin. :!xti. :!l(b
:" kephalin, 1ii Tnotine. H, :...'(10
lecithin, 176
met.hyl ethyl maleinimidt", 291
nueJei(' lU'id&. 58 11Ura-vj()let ljht lind i,huJeI'ItNfJ)
porpnyrin~. 2BS !?42
quinine. 334 and porphyrins. ~
aphin$z:omyelin, 180 and rickets, 2"0
8ubetitnted pyrro~, 29".! UDimolecwar filffil:<. 13H
8ug&rs. 70. 73, 79 "UnitariaD n bYP(lthf:'_"lil-l o{ anti
urea,9 bodif'S.2Un
IU'flpo>phyrin, 288 UllUturat" fats. It:!. 13(J
8qu, g&mma. 80 fatty add-~. J J:l
of blood, 82 Ur&ciJ. pn:liferwe in anintallUld pi.!UIt
of nucleic acids. 52, 53 nuclei!' ~idJJ, 59
Fpentosnria, 74. (Su aho car- Urea. a.ctinu of nitrtmJ\ ac-td (}II. 12.
bohydrate). 13
ItaIpIaaIaoe. 206 combination with pentrnw. 82
348 IqDEX
Urea,. ('nzymit, hydrolysis. 15 Vit.mma. origin. :!17. (8et' (1[80 A,
formation in t i~!I\l{'M. 18 B,C,D. E).
Jlyc1rolYllif'l. 14 Vuan. 333, 3:~5
structure, lJ
Urea.e. 15
a diS80da.tin,l2." (nzyrne, 17 WU... lll
i~ ;r~~:~~ii.6:!~
Wheai ..germ oil. fraetionation, 248 .

Dl(.-.cMt.ftiHm (If a-ction. 16. 17, IS

~vnthNli!; of un'a. 17 Xanthine oxidaS('. 64
Uric add. comhined with ri\:x)S(> in Xerophthalmia. 2:!6, 227
blood. S;! XylOM, 73. 80
cnd product of purine metabolism.
63,64,6,
in hird.'! and l'f'ptil(>.Ol, 68 Y.ut. ron{,f"ntr~tion of yjtarnin B
Uricue. 64. 65 from, 231
a.nd Mtmddilln inheritance, 67 f .... rm~lltation and hexose phos-
Uricolytic index, ij() ph.,.., ]v]
Urobilin. 203 ft'rmentations, 101
'Uroporphyrin, 286. 288 gum. 308
copper lUlU. 281) Ye&Jt nut'it'il' !lCid, 5
"Variablet " element of fata. 123
VitaDlinJ. 214 Zymin 151
 Q'~~'v,_~

. -
; NO.2
..

J. & A. CHU~CHILL
APRIL. ,,;a6." "'I 1

INDEX.
I(
..~tlAn.tom)'. Phy.,ofoiY. ;
3 Mooterl. Med",.. ........ (
macy .
HYJ!eae. 811<terIoIOI7. ,
5 Patholov. Plycho-"'
Selected lory. Electro-
therapeutic
PktIon_.
6 Medlclne.
600ks 1 Medlcl ....
S Surret'y. ,
II Surrery. An_thet!<:a. ~
for
100'......toloa. Nea_ (
101Y. Urinary I
Students PI_..... Troplcal.
01_10
I I Midwifery. 0,......,-'
and 100. .
I~ MedkalJurloprudence. .
, Ophthalmolor;y. '
Practitioners
of :I.13 Otoloa. Pe4tatrlc..
Dentlary.
Cheml.try.

. :l--el-
i .s Che,,"try. Phy.lc:..
Medicine. .! .6 Physka. Mlc:roICOPY.
:1 MlM:.eII._.

FREE ON
i I APPLICATION'.
I ,
1 _
- _

1_
1 LONDON i
I "
~ :} 0" ..,.,,_.. Street.!1 .
-..

O~""""'~~__'''''~''''-~''''''''''''''''---4
 Anatomy

TJ1e Principles of Anatomy as Seen in the Hand:

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S)'n=r~A!::!~~;~itr~lLo~~i n~:::U~-:~2..~:~
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~::~:.:~~ ..~1\y:'~~.~8
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