GLUCOSE METHODOLOGIES

• Fasting glucose in whole blood is 15% lower than

in serum or plasma
• Serum or plasma must be separated from cells
within 30 minutes
•Chemical Methods
• Reduction – Oxidation Methods
• Alkaline Copper Reduction
Methods
• Folin Wu Method
• Nelson Somogyi Method
• Neocuproine method
• Shaffer Hartman Somogyi method
• Benedict’s method
• Alkaline Ferric Reduction
Method
• Hagedorn Jensen Method
- Condensation Methods
• Condensation with phenols
Commonly used reagents for this method are:
Anthrone resorcinol
Thymol alpha naphthol
Phloroglucinol m-aminophenol
• Condensation with aromatic amines
• Ortho-Toluidine method
• Enzymatic Method
• Glucose Oxidase Methods
• Polarographic Glucose Oxidase method
• Saifer Gernstenfield method
• Hexokinase method
• Considered to be the most highly specific
method for glucose determination.
• Dextrostics (cellular strip)
Samples / Tests for Glucose Determination
• RBS
• Random blood sugar
• Requested during insulin shock, hyperglycemic ketonic coma

• FBS
• Fasting blood sugar
• NPO (non-per-orem) at least 8 hours before the test
• Gives the best indication of overall glucose homeostasis

• 2-HR PPBS
• Evaluate hyper- and hypoglycaemia
• Specimens are collected 2-5 hours after taking his regular meal
• Blood glucose conc should be below 120 mg/dL at 2 hours. If hypoglycaemia is suspected,
do up to 4 to 5 hours PPBS.
• Glucose Tolerance Test
• GTT is unnecessary to do for women less than 25 y/o who
have normal body weight, no family history of diabetes and
are not member of an ethnic group with high prevalence of
diabetes.
• Patient should be ambulatory – most important
• Fasting 8-14 hours
• Unrestricted diet of 150 gms CHO/day for 3 days prior to
testing
• The patient should not smoke and drink alcohol prior to testing
• Glucose load
• 75 gms (standard glucose load)
• 100 gms
• 1.75 g of glucose/kg body weight (children)
Procedure for GTT
• The patient should avoid exercise, eating and drinking (except water)
and smoking during the test
• For non-pregnant women and adults, only the fasting and the 2-hour
sample may be measured

• Collect the fasting blood sample. (urine may also be

collected)
• Instruct the patient to drink the glucose load (within
5 mins)
• Collect blood sample after 30 mins, 1-hour, 2-hour, 3
hour, respectively
• Kinds of GTT
• Oral glucose tolerance test
• Janney-Isaacson Method (single dose method) –
most common
• Exton Rose Method (divided oral dose or
double dose method)

• Added plasma glucose after OGTT

• 30 mins  30-60 mg/dL above fasting
• 1-hour  20-50 mg/dL above fasting
• 2-hour  5-15 mg/dL above fasting
• 3-hour  fasting level or below
Intravenous GTT
• Used for diabetes patients with gastrointestinal disorders
• 0.5 g of glucose/kg body weight (given within 3 mins)
administered IV
• Fasting sample is also required
• The first blood collection is after 5 mins of IV glucose
• Indications:
• Those who are unable to tolerate a large CHO load
• Those with altered gastric physiology
• Those who had undergone previous operation or surgery in the
intestine
• Those with chronic malabsorption syndrome
• Criteria of Fasting Plasma Glucose:
• Non-diabetic = <100 mg/dL
• Impaired PG = 100-125 mg/dL
• Diabetes mellitus = > 126 mg/dL

• Categories of OGTT (2-hour PG)

• Normal / non-diabetic = <140 mg/dL
• Impaired GTT = 140-199 mg/dL
• DM = >200 mg/dL

• Diagnostic Criteria for DM

• RBS = >200 mg/dL
• FBS = >126 mg/dL
• 2-hour post CHO = >200 mg/dL
• Glycosylated haemoglobin (HbA1c)
• It is the largest subfraction of normal haemoglobin A in both diabetic
and non-diabetic individuals
• It is a glucose molecule attached to one or both N-terminus valines of
the beta-polypeptide chains of normal adult haemoglobin
• A reliable method in the monitoring of long-term glucose control
• It reflects the average blood glucose level over the previous 2-3 months
• 3-6% of Hgb is glycosylated; 18-20% is prolonged hyperglycemia
• For every 1% change in the HbA1c value, 35 mg/dL is added to plasma
glucose
• Older red blood cells have higher HbA1c, iron deficiency anemia – high
levels
• Not suitable for patients with shortened RBC lifespan disorders – low
HbA1c value
• Specimen for testing: EDTA Whole blood
• Fructosamine
• Also called glycosylated albumin / plasma protein
ketoamine
• It is a reflection of short term glucose control (2-3 wks)
• May be useful for monitoring diabetic individuals with
chronic haemolytic anemias and haemoglobin (Hb S or Hb
C) – decreased RBC lifespan
• It should not be measured in cases of low plasma albumin
(<30 g/L) – low fructosamine
NPN
• Blood Urea Nitrogen
• Major end product of protein (dietary) and amino acids catabolism
• Approx. 80 % of nitrogen excreted
• 10:1 – 20:1 (BUN:crea)
• Synthesized in the liver from CO2 and the ammonia from the deamination
of amino acids (urea cycle)
• Easily removed by dialysis

• Methods:
• Fluoride or citrate will both inhibit urease
• Fasting sample is usually not required
• Thiosemicarbazide and ferric ions are added to enhance color development
• Isotope Dilution Mass Spectrometry – reference method
• Principle:
• Hydrolysis of urea by urease.
• The urea in the sample is hydrolysed by a specific enzyme,
urease, to form ammonia and ammonium carbonate. The
amount of ammonia formed can be measured by:
• Nesslerization
• Urease
• Modified Kaar
• Gentzkow Masen
• Folin Svedberg
• Berthelot’’s reaction
• Acid titration
• Van Slyke Cullen
• Urograph
UROSTRAT CHROMATOGRAPHY PAPER STRIP

When urea in serum comes in contact with

urease by diffusion, ammonium carbonate is
Indicator system ( BCG + tartaric acid) formed which reacts with potassium
carbonate to released ammonia. The
Plastic barrier ammonia released reacts with the dye and
titrated by the acid to form a blue green
color. The amount of nitrogen present is
Potassium carbonate
determined by the height of the color which
is measured after 30 minutes standing at
urease room temperature. It is measured by:

Ruler height in mm x 5 + 10 = BUN in mg%

x 0.357= mmol/L
Caliper – direct reading in mg% x 0.357 =
mmol/L
To obtain the concentration of urea from
BUN – 2.14 x BUN = urea (mg %)
• Direct measurement of urea
• Urea is hydrolysed to ammonia but it is
measured directly.
• Diacetyl Monoxime Method (DAM)

• Principle:
• Urea is made to react with DAM to produce yellow diazine
derivatives (Fearon’s reaction). Arsenic thiosemicarbazide is
added to enhance color formation and to exclude protein
interference.
• Disadvantage:
• Lacks specificity
• Instability of the color produced
• Toxicity and irritating odor of reagent
• Creatinine
• End product of muscle metabolism derived from creatine (a-methyl
guanidoacetic acid)
• It is not affected by protein diet; not easily removed by dialysis
• Most commonly used to monitor renal function

• Methods:
• Both serum and urine creatinine have been used as indices of renal
function because of the constancy of creatinine formation
• Fasting is not required
• Hemolyzed and icteric samples should be avoided.
• ENZYMATIC METHOD
• Creatinase method

Creatinase
Creatinine è N-methyldantoin + NH3

GDH
NH3 + alpha-KG + NADH è glutamate + NAD
• Creatinine Aminohydrolase method
• Principle:
• Creatinine is hydrolysed to creatine by creatinine
aminohydrolase, followed by a series of coupled
enzyme reactions in which creatine reacts with
creatinine kinase, pyruvate kinase, and lactate
dehydrogenase, culminating in the oxidation of the
NADH.
• DIRECT JAFFE REACTION METHODS
• Principle:
• It is the formation of red tautomer of creatinine picrate when
creatinine in serum is made to react with a freshly prepared
alkaline sodium picrate solution (alkaline picrate – Jaffe reagent)

• Composition of the Jaffe reagent:

• 1 part of 10% NaOH
• 6 parts of saturated picric acid (2,4,6, trinitrophenol)
• *The solution must be freshly prepared to prevent its conversion
to picramic acid and methylguanidine.
• Folin Wu method
• A sensitive method but not specific since it measures other chromogenic
substances like ascorbic acid, ketones and barbiturates.

• Lloyd Method
• A sensitive and specific method since it measures true creatinine. It is the
reference method for creatinine. Its advantage is that creatinine adsorbed by
the hydrated aluminium silicate or oxalic acid (Lloyd’s reagent) which isolates
creatinine from other interfering substances. Elution techniques are then
utilized to separate the creatinine form the adsorbent which is then made to
react specifically with the freshly prepared Jaffe reagent.
• Blood Uric Acid
• Major product of purine metabolism
• Formed from xanthine by the action of xanthine oxidase in the liver and
intestine
• Exists as monosodium urates

• Methods:
• Uric acid is stable in both serum and urine for 3 days at room
temperature
• Potassium oxalate anticoagulant cannot be used (forms potassium
phosphotungstate – promotes turbidity)
• Ascorbic acid and bilirubin are the major interferences.
• DIRECT REDOX METHODS

• Methods based on their alkalinizing agent:

Uric acid + phosphotungstic acid
• Sodium cyanide (NaCN)
• Folin
• Brown Sodium cyanide or sodium carbonate

• Newton
• Benedict Tungsten blue + allantoin + CO2

• Sodium carbonate (Na2CO3)

• Henry
• Caraway
• Archibald
• ENZYMATIC METHOD
• Blaunch and Kock (uricase method)
• Simplest and most specific method (reference method)

uricase

• Uric acid + O2  allantoin + CO2 + H20

Protein Methodology
• General Methods:
• Salt Fractionation Methods
• Turbidimetric Methods
• Flocculation / Turbidity Test
• Electrophoresis
• Ultracentrifugation
1. SALT FRACTIONATION
• will differentiate proteins according to their
solubility using different concentration of salt.
• Ammonium sulphate – most frequently used salt.
SOLUBILITY PROPERTIES:
• Albumin
• Soluble in: water
• Dilute and moderately concentrated salt solution
• Insoluble in: hydrocarbon solvents
• Saturated salt solution
• Highly concentrated salt solution
• Globulin
• Soluble in: hydrocarbon solvents
• Weak salt solution
• Insoluble in: water
• Saturated salt solution
• concentrated salt solution
• Albuminoids
• Characterized by being insoluble in most common reagents
Methods of protein determination under salt fractionation:
1. Kjeldahl – a standard reference method based on nitrogen determination
1 gram nitrogen = 6.54 grams of protein

2. Biuret method Measure of TP & ALB using the Biuret

3. Kingsley biuret reaction
4. Weischelbaum

5. Greenberg Measure of TP & ALB using the Redox

6. Folin & Ciocalteau reaction
BIURET REACTION
• The formation of a blue violet complex when
total protein is serum reacts with alkaline copper
sulphate reagent. To determine the albumin
fraction, the globulins are precipitated with 22-
26% sodium sulphate reagent.
• Compositions of the Biuret reagent:
• Alkaline copper sulphate
• Rochelle salt (NaK tartrate)
• Sodium hydroxide
• Potassium iodide
Interference with the Biuret method:
1) Lipemia
2) Hyperbilirubinemia TP is falsely elevated
3) Hemolysis

4) High blood ammonia – falsely low total protein level

 II. TURBIDIMETRIC METHODS

1. Looney Walsch method Measures of TP and globulin using ammonium

2. TCA method sulphate and sulfosalicylic acid (SSA)

Interference with the Turbidimetric methods:

• Bacteriuria
• Cephalosporin
• X-ray contrast media
• Sulfa drugs
• Penicillin
• Tolbutamide
FLOCCULATION / TURBIDITY TEST
• Also referred to as Globulin Reaction test, serum colloidal
stability test
• Cephalin-Cholesterol Flocculation Test (CCFT)
• Thymol Turbidity Test (TTT)
• Methods: a) Mac Lagan

b) Shank Hoagland
• Colloidal gold
• Zinc Sulfate Turbidity Test
• Method: Kuakell Test
• Takata – Ara  using mercuric chloride
• Cadmium sulphate
III. ELECTROPHORESIS
• Provides quick and useful information regarding altered levels of
protein groups within the circulation.

IV. ULTRACENTRIFUGATION
• Will differentiate proteins according to their differences in molecular
densities; thus, heavier molecules settle faster than lighter molecules.
• Albumin – measured by direct methods based on its dye-binding property
Dyes:
BCG (Bromcresol green)
HABA ( hydroxyazobenzene – benzoic acid)
MO (Methyl orange)

• Fibrinogen – the only protein found in plasma which is not found in serum
Methods:
Howe’s method
- Fibrinogen is precipitated with calcium chloride
Parfentjev’s method
- Fibrinogen is precipitated with ammonium sulphate and
sodium chloride
 LIPIDS DETERMINATION

• Free Fatty Acid (FFA) / Non-Esterified Fatty Acid

(NEFA)
• Bound to albumin in plasma

• Methods for Laboratory Assays of FFA

• Microtitration method using dilute alkali
• Principle:
• Fatty acids are extracted from the acidified plasma by a mixture of
isopropyl and heptane.
• The extract is then titrated with a dilute alkali using ethanolic
solution of thymol blue or thymolphthalein as indicator.
Colorimetric method: Formation of cupric soaps and free
fatty acids.
• Principle:
• FA in plasma is made to react with a copper solution to form cupric
soaps of fatty acids.
• The soaps are then extracted in a mixture of organic solvents
(Chloroform-heptane-methanol solution)
• The amount of bound cupric ions is determined colorimetrically,
after reaction of copper with diethyldithiocarbamate.
• Practical Considerations in FFA
determination
• Serum or heparinized plasma can be used and rapid
separation of serum from blood clots is suggested
because presence of lipase permits slow in vitro
lipolysis.
• Stress must be avoided, as in those encountered
before and during blood sample collection because
cathecolamines are released which enhances the
activity of hormone sensitive lipase.
• Patient must be under NPO state
Triglycerides / triacylglycerol (TAG)
• Contains 3 molecules of fatty acid and one molecule of glycerol by
ester bond.
• Main storage form of lipids in man
• Stored fats are more efficient in its energy storage than CHO
because of:
• Purity of storage – interaction of lipids and water is minimal
thus can be stored in its pure form (anhydrous)
• Unlimited storage – CHO can be stored only chiefly in the liver
and to a lesser extent in skeletal muscle, whereas adipose
tissue covers different parts and organs of the body.
• Greater ATP yield – the energy yield from complete oxidation
of fatty acids is about 9 kcal/g in contrast to 4 kcal/g for CHO
and CHON.
 LABORATORY METHODS:
• In general, TAG assays involve the hydrolysis of TAG, either
enzymatically or with alkali (saponification) to liberate glycerol which
then can be measured by colorimetric, fluorometric or enzymatic
methods.

• Colorimetric method of Van Handel & Zilversmith

• Principle:
• Serum lipids are extracted with Folch’s reagent (chloroform-
ethanol mixture)
• TAG are then saponified using alcoholic KOH to release glycerol
and fatty acids.
• The glycerol is oxidized with periodic acids to produce
formaldehyde (HCHO)
• A blue colored compound of unknown composition is formed
when formaldehyde is treated with a sulphuric acid solution
(chromotrophic acid).
• Fluorometric method (Hantzch Condensation
method)
• Principle:
• Reactions a, b and c of the colorimetric method is basically
the same.
• HCHO combines with diacetylacetone and ammonium ions
to produce 3,5 diacetyl 1,4 dihydrolutidine, a yellow
fluorescent compound.
• Enzymatic method (Glycerol Kinase)
• Principle:
• Glycerol is released from TAG by the usual saponification step.
• Glycerol is phosphorylated and catalysed by glycerol kinase to
produce glycerol-6-PO4 and ADP.
• The ADP is made to react with phosphoenol pyruvate and
pyruvate kinase catalyses the reaction yielding one molecule of
ATP and pyruvate.
• The pyruvate is converted to lactate by LDH and subsequent
oxidation of the cofactor NADH to NAD.
• The decrease in absorbance at 340 nm is equivalent to the
amount of glycerol present in the specimen.
• Practical Considerations in TAG assays
• The best sample is fresh serum taken from a patient after a 14-hour
fast. Peak of plasma TAG occurs 2-6 hours postprandially.
• Plasma has a slightly higher level than its corresponding serum
because of the trapped chylomicrons and lipoproteins during clotting
and also because of lipolysis during clot formation.
• Heparinized or EDTA treated plasma can be used but fluorides and
oxalate can cause interferences.
• Glucose and phospholipids present interfere in the non-enzymatic
methods for TAG assay because the oxidation step of glycerol to
formaldehyde is not specific for glycerol alone.
• Extraction of TAG plasma using adsorbents like zeolite (magnesium
aluminium silicate) or silicate acid removes interfering substances.
Cholesterol
• 3-hydroxy- 5,6-cholestene
• The presence double bond and a hydroxyl group structure
allows interaction of cholesterol with different reagent

• Importance
• Constituent of cell membrane
• Precursor of various steroid hormones
• Bile acid synthesis
• Vitamin D formation
• Laboratory Methodology
• The presence of a double bond and OH group in this sterol’s structure
makes it possible that color reaction be carried out. In general, the
cholesterol is measured by using strong concentrated acids to form colored
product.

• 2 general color reactions used in cholesterol assays:

• Liebermann-Burchard reaction
• Cholesterol is dehydrated and oxidized to form green cholestadienyl
monosulfonic acid.
• Salkowski reaction
• Cholesterol is reacted to a mixture of acids to produce a red
cholestadienyl disulfonic acid.
• In cholesterol assay, it employs a special reagent
called color developer mixture (CDM) which
consists of:
• Glacial acetic acid
• Acetic anhydride
• Concentrated sulphuric acid
• Iron in the form of ferric chloride is sometimes added
which enhances oxidation reaction
CLASSIFICATION OF PROCEDURES FOR
CHOLESTEROL ASSAYS BASED IN THE NATURE
OF PRELIMINARY TREATMENT OF SPECIMEN
• Direct colorimetric methods – these procedures
involve direct addition of color reagent mixture
to the aliquot of specimen.
• Pearson Stern and Mac Gavack
• Wybenga et al
• Interference encountered:
• Presence of plasma proteins may produce a multitude of
colored products
• Non-specific chromogens like bilirubin, carotenoids and
others may react.
• Instability of final colored product
• Extraction and Colorimetric methods (Two step method)
• In these procedures, the lipid in the specimen is extracted first
before treating with the color reagent. The use of petroleum ether
isolates cholesterol from protein.
• Bower’s method

• Saponification, Extraction and colorimetric methods (3 step

method)
• Cholesterol esters (CE) are first converted to free cholesterol (FC)
using an alkali saponifying agent. The FC produced and the FC already
present in the sample is extracted and finally set for color reaction.
• Abell’s method
• Saponification, extracted, precipitation and
colorimetric (4-step method)
• the CE are saponified, the total FC is extracted and further
isolated by precipitation with digitonide before subjecting for
color development. This is a very specific method and is
accepted as reference method.
• Parelch and Jung
• Schoenheimer and Sperry
• Sperry and Webbs
• Enzymatic method for cholesterol assay
• Principle:
• The enzyme cholesterol esterase splits CE to release
cholesterol and fatty acids
• The FC is then oxidized to 4-cholestene -3-one and H202
using cholesterol oxidase
• The peroxide produced is then measured by reaction with
aminoantipyrine and phenol catalysed by the enzyme
peroxidase.
• The final end product is a quinonemine dye with a peak
absorbance at 505 nm.
• PRACTICAL CONSIDERATIONS
• Serum is preferred specimen because anticoagulant cause water shifting from
red cells, dilutes the specimen which lowers the value.
• The incubation period of cholesterol assay is critical; prolonged incubation
increase cholesterol value. For color development, cholesterol is incubated
for 20 minutes at RT and 10 mins at 37oC.
• Jaundiced, lipemic and hemolyzed specimen must all be rejected, a series of
interferences encountered.
• Extreme exposure to light of the specimen must be avoided since it lowers
cholesterol value.
• Introduction of water and metallic contamination cause falsely decreased
cholesterol value.
END