Clinical Chemistry 1: Non-Protein Nitrogen Substances Biochemical process is:

Ornithine + CO2 + NH3 -----------------> citrulline

 NPN in whole blood – approximately 75% greater or 1.76X higher than that of serum/plasma Citrulline + NH3 -------------> arginine
 Total NPN in plasma – 250-400 mg/L
 NPN – waste product of metabolism Arginine + H2O ------------->
arginase ornithine + urea

Classification of nitrogenous compounds in blood:

- is readily filtered from the plasma by the glomerulus
1. Protein nitrogen - excreted in the urine
2. Non-protein nitrogen - not good index of kidney function

NPN compounds Approx. plasma conc. (of blood NPN; in %) Analytical Approaches:

1. Urea 45 1. Direct measurement of urea

2. Amino acid 20 1.1 diacetyl monoxine (DAM) method
3. Uric acid 20 - also known as the Fearon reaction
4. Creatinine 5 - R1-CO-NHR2 structure (R1-N; R2 is notnot an acyl radical) will react with diacetyl
5. Creatine 1-2 monoxime in the presence of a strong acid and oxidizing agent to produce a
6. Ammonia 0.2 chromogen
- direct condensation assay (urea is condensed with diacetyl
Biochemical functions of the kidney: - urea is not hydrolyzed to ammonia but is measured directly
Function Example - a colorimetric approach

1. Synthesis erythropoietin, rennin, prostaglandins Principle:

2. Metabolism inactivation of vit. D, formation of creatinine
3. Excretion ammonia, urea, uric acid, several minerals, toxic substances Steps
1. diacetyl monoxime + H2O -------------> diacetyl + hydroxylamine
3 major divisions of renal function test inn clinical chemistry:
2. Diacetyl + urea ------------->
+
yellow diazine + H2 O
H
1. Tests measuring glomerular filtration rate (GFR)
1.1 Creatinine clearance
1.2 Urea clearance  yellow diazine – unstable, photosensitive; so add thiosemicarbazide
1.3 Inulin clearance  unknown and QCS – undergo deproteinization to remove proteins
2. Tests measuring renal blood flow  clear and colorless – ideal PFF
2.1 Kjeldah’s digestion method  blank – check stability of the reagent; set spectrophotometer at 100%T (0 A)
2.2 Dam  urea standard – 10 mg%
3. Tests measuring function tests
3.1 Excretory function test other oxidizing agents:
3.1.1 p-aminohipppurate (PA) or diodrast test
3.1.2 phenolsulfonaphthalein (PSP) dye excretion test 1. K sulfate
3.2 Concentration tests 2. Perchloric acid
3.2.1 Specific Gravity 3. Phenazone
3.2.2 Osmolality
3.2.3 Fishberg Concentration test Reagents used to intensify the color and minimize photosensitivity:

Specimen requirements: 1. Thiosemicarbazide

2. Phenylanthranilic acid
1. Serum 3. Glucoronolactone
2. Plasma
3. Whole blood 2. Indirect approaches
4. Other body fluids such as urine - uses urease enzyme to decompose/hydrolyze urea
- oldest and most often used method
Protein Urinalysis – primary and most cost-effective test - initial step: hydrolysis of urea (products: carbonate anion and ammonium cation)
NPN
Urea + H2O ------------>
urease 2NH4+ + HCO3
1. UREA
- Blood urea nitrogen (BUN)  2NH4+ - proportionate to amount of ammonia
- major NPN in blood
- major excretion product in protein metabolism
- synthesized in liver via the ornithine or Krebs-Hensleit urea cycle
- first metabolite
Quantitation of the 2NH4+ 1. Prerenal causes
2.1 NH4+ + Nessler’s reagent -------------> yellow dimercuric ammonium iodide  Congestive heart failure
 Shock
 Yellow dimercuric ammonium iodide – double iodide of mercuric ammonium  Hemorrhage
 Category: enzymatic colorimetry (480-500 nm)  Starvation
 Dehydration
2.2 NH4+ + NAOGL + phenol ------------> indophenol (blue) 2. Renal causes
Na
nitroprusside  Acute and chronic renal failure
 NAOGL – sodium hypochlorite  Glomerular nephritis
 Berthelot method (Chaney Marbach method or Urease-Berthelot reaction) 3. Post renal causes
 Absorbance is read at 630 nm  Urinary tract obstruction
 only in alkaline medium B. Decreased levels BUN
1. Decreased CHON intake
2.3 coupled enzyme assay  Severe liver disease – decreased synthesis
 Normal pregnancy – increased glomerular function
Urea ------------> NH4+ + 2HCO3
urease *azotemia – increased level of urea as well as creatinine in the blood largely related to decreased GFR
 source of urease – soybeans/jackbeans *uremia – very high levels of plasma urea and creatinine
Signs and Symptoms of Renal Failure:
Indicator reaction:
1. Metabolic acidosis due to failure of kidney to eliminate acidic products of metabolism.
GLDH
NH + +
+ alphaketoglutarate + NADH -------------> Glutamic acid + NAD +
+ H2O 2. Hyperkalemia (increased K) due to failure of K excretion.
4
3. Generalized edema due to water retention.
 GLDH – glutamate dehydrogenase
 unique characteristic – use of coenzyme which is NADH+ or nicotinamide adenine Increased CHON diet:
dinucleotide
 coenzyme  Fever
- not an enzyme  Major illness: may increase urea levels because of increased CHON breakdown
- can be altered or reduced
- attached tightly to an enzyme Used for renal function test:
- added as a reagent
 high peak of absorbance at 340 nm  BUN
 uses multiple readings/analysis of absorbance  Creatinine test
 decreased absorbance reading as NAD + is formed from NADH+  Uric acid test
 principle: rate of decrease of absorbance of NADH+ at 340 nm is proportionate to amount of
urea *Creatine and ammonia level determinations are not good renal function tests
 category of estimation/quantitation: enzymatic UV spectrophotometry *ammonia det. is more on liver function test
Creatine and Creatinine
Other enzymatic methods: Physiologic Chemistry/Biochemistry
Creatine:
4.1 acid titration of ammonium – Van Slyke-Cullen method
4.2 urograph – urostrat? (paper chromatography method)  From glycine, arginie and methionine
4.3 Conway micro diffusion method  Synthesized mainly in the liver
 Transported as phosphorylated (muscle cells) creatine kinase/creatine phosphate (high
General considerations in BUN method: energy source in muscle tissue)
1. The determination is affected by increase CHON diet, hydration and other physiologic  Creatine phosphate/creatine, under physiologic conditions -> lose phosphoric acid and H2O
functions.
2. Whole blood should be deproteinized to eliminate interference of hemoglobin. Creatinine:
3. NH4-containing anticoagulants are contraindicated in enzymatic methods.
4. Increase concentration of NaF and sodium citrate must be avoided since they will inhibit the  Anhydride form of creatine as a result of non-enzymatic dehydration of muscle creatine
urease. (degradation product of creatine)
5. Urea is quite susceptible to bacterial decomposition; samples (esp. urine) that can’t be  Excreted into the circulation at a relatively constant rate
analyzed within a few hours should be refrigerated. A few crystals of thymol can be added to  Removed from the plasma almost entirely by GF and excreted in urine, therefore it is used to
the urine sample as preservative. check the completeness of a 24hr urine sample
6. Bilirubin at a concentration of 10mg/100ml increases the urea nitrogen value by about  Measurement of creatinine clearance (CC) is used as a measurement of GF and thus plasma
3mg/100ml of serum. creatinine maybe used as an index of GF/index of renal function
7. Hemolysis and lipemia does not interfere with the coupled enzyme assay but a non-  More reliable than BUN
hemolyzed sample is ideal.  Free creatinine is not reutilized in the body’s metabolism, thus function solely as a waste
product of creatine
Clinical Significance  Increased in the latter part of renal diseases because it increases slowly than BUN but also
decreases more slowly with hemodialysis
A. Increased level BUN  decreased renal blood flow
  Is not routinely affected by diet (CHON intake), however, large quantities of creatine are sarcosine
present in: sterilized canned meat (e.g. sardines), cooked meat Sarcosine + O2 + H2O --------------> glycine + HCHO + H2O2
 It is among the most frequently performed clinical assay ranking second only with glucose
(K+) and BUN oxidase
H2O2 + EHSPT + 4-aminoantipyrine ------> red quinine (quinonimine)
(A@600-700 nm)
Analytical Assays: *EHSPT – n-ethyl-n-2-hydroxy-3-sulfopropyl-n-toluidine

1. Jaffe’s rxn (direct approach) 2.1.2 reagent strip method/Sakaguchi reaction

 Classical assay for serum and urine creatinine Creatinine + DNBA ------> purple chromogens
 Principle:
*DNBA – 3,5-dinitrobenzenoic acid/dinitrobenzene or its derivative
Creatinine (PFF) + alk. Picric acid ----------> amber yellow/bright orange-red
alkaline creatinine picrate complex 2.2 UV-spectrophotometric method

 Composition of alkaline picrate sol’n: 2.2.1 creatinine + H2O ---------------> creatine

creatininase
1 part 10% NaOH
5 parts saturated picric acid (2,4,6-trinitrophenol) Creatine + ATP ------------------------> creatine phosphate + ADP
Creatine kinase

 Major drawbacks: ADP + PEP -----------------------> ATP + pyruvate

phosphokinase
1. Lacks specificity and sensitivity (because of pseudocreatinine like glucose, ascorbic acid,
and alpha-keto acids such as acetone and acetoacetate)
2. Temperature sensitive (should not exceed 30°C) Phosphoenolpyruvate
3. Affected by length of time of incubation (10 min)
4. Affected by incomplete removal of CHON Indicator reaction:
 To improve Jaffe’s method, modifications were made: Pyruvate + NADH + H+ ----------> L-lactate + NAD+
1. Addition of Lloyd’s rgt.
2. Employing ion-exchange resins *the concentration of creatinine is proportionate to the rate of decrease in absorbance at 340nm
*NADH characteristics: reduced form co-enzyme
Lloyd’s reagent high absorbance at 340nm
*NADH oxidized to NAD+
 purified fuller’s earth which is a hydrated aluminum silicate powder as a kaolin is added for *advantages: precise and accurate
the system to improve sensitivity and specificity of the Jaffe’s method not subject to interference

Specimen Requirement:
Purposes of the reagent:
1. Specimen: serum, plasma, urine
1. used to absorb, therefore separate the creatinine from the other chromogens 2. Fasting not required
2. a trapping agent which removes reactive impurities before the alkaline picrate sol’n is added 3. Serum/plasma is preferred over whole blood
4. Hemolyzed sample should be avoided esp. when using Jaffe method
Oxalic acid 5. Bilirubin, elevated amounts in icteric serum samples causes low creatinine result in the
Kinetic Jaffe and enzymatic methods
 Added to PFF which results to the formation of creatinine oxalate which is then absorbed 6. Lipemic samples falsely low results in kinetic jaffe
and held by Lloyd’s reagent in the acid sol’n 7. Fresh specimens are recommended as creatinine and creatine are labile, stable when frozen
 Color of “true” creatinine is less resistant to acidification than color of pseudocreatinine and ph of specimen should be maintained at ph7 during storage to minimize conversion
substances 8. Separation of serum and cells is required
9. Levels of alphaketo acids at levels that maybe found in diabetic patients will give falsely high
Advantages of the use of Lloyd’s reagent: values
10. Ascorbate, pyruvate, acetone and glucose will also form a color in the presence of the
1. Kinetic Jaffe method by Romeo reagent – significant (+) error in Jaffe method using PFF
Principle: 11. Drugs: methyldopa, cefoxitin, dopamine – false high
Serum/plasma + alkaline picrate ------> rate of change in A is measured bet 2 time points Antibiotics: cephalosporin – false high with Jaffe’s reaction
Advantages: inexpensive, rapid and easy to do
Disadvantages: still subject to interferences by acids and cephalosporins Clinical Significance:
Bilirubin and hemoglobin give A(-) bias probably due to their destruction A. High creatinine levels
in strong base used 1. Renal diseases (decrease renal blood flow)
-------------------------------------------------------------------------------------------------------------------------------- 2. Shock
3. Congestive heart failure
2. Indirect Analytical Approaches 4. Muscular dystrophies
2.1 Enzymatic spectrophotometric 5. Decreased urine excretion
B. Low creatinine levels
2.1.1 creatinine + H2O creatininase
---------------> creatine  Not associated with any disease
creatinase  Due to drug interference
Creatine + H2O -------------> sarcosine + urea
Reference values: 2.3 enzymatic UV-spectrophotometric assay
uricase
 Male: 0.6-1.2mg/dl (53-106 umol/L) u.a. + 2H2O <-------------> allantoin + CO2 + H2O2
 Female: 0.5-1.0mg/dl (44-99 umol/L)

SI unit = conventional unit x 88.4 H2O2 + ethanol <------------>

catalase oxidized acetaldehyde + 2 H2O2

Acetaldehyde + NADP+ <---------->

AIDH acetate + NADPH + H+
2. URIC ACID
 Major end product of purine metabolism *NAPD – oxidized form
 Final breakdown product in purine metabolism in humans *kung naay “H” – reduced

*urinary excretion depends upon: purine, metabolism, renal function  acetaldehyde is oxidized to acetate by aldehyde dehydrogenase and the simultaneous
*purine rich foods: legumes, visceral organs, sweet breads, shellfish reduction of NADP+ to NADPH, the increase in A is proportional to the u.a. concentration
(initial A is lower than the 2nd reading) @340nm
 u.a. has a maximum absorption peak at 285-293nm while allantoin and H2O2 has no
 96.8% of uric acid in blood plasma is present as sodium urate or monosodium urate, relatively absorption at this wavelength
insoluble  the disappearance of u.a. is indicated by the decrease in light A and is proportional to the u.a.
 UA level above 6.4mg/dl, the plasma is saturated, urate crystals may form and be deposited in the initially present in the sample
joint-tophi
 Or deposited in the gut as UA stones
 Not a primary evaluation test for kidney function but serves as a confirmatory check on creatinine Specifications:
and BUN analyses
1. diet
Analytical Assays: 2. specimen: serum/heparinized plasma and urine, potassium oxalate can’t be used since it
combines with phosphotungstate promoting turbidity
3. gross lipemia/grossly turbid sample: erroneously high result
1. Caraway method 4. refrigerate serum sample to avoid bacterial contamination
 Direct redox method 5. fluoride and thymol are added as preservatives
 Based on the alkalinizing agents such as Na cyanide, Na carbonate 6. elevated bilirubin (3mg/dk) erroneously low results with the enzymatic assay due to
destruction of peroxidase
Principle: 7. ascorbate (vit. C) usually causes low results due to competition of ascorbate with
u.a. (serum) + phosphotungstic acid <----------->
NaCN (blue) tungsten + allantoin + CO 2 chromogen as a hydrogen donor in the reaction
*competition is eliminated by including K ferricyanide and ascorbic acid oxidase in the
Na2CO3
reagent to oxidize ascorbate. This occurs in the uricase/H2O2 coupled reaction.
 u.a. is oxidized by phosphotungstic acid to allantoin in the presence of sodium carbonate 8. Hemolysis releases nonspecific reducing substance such as ergothionine, glutathione,
 phosphotungstic acid – reduced to tungsten by u.a.; color developer and oxidizing agent tyrosine, ascorbic acid and glucose, which, like u.a., reduce phosphotungstic acid
 sodium carbonate – provides the alkaline (buffer) medium necessary for adequate color
development Reference values:
2. Coupled Enzymatic Assay
2.1 Blaunch and Koch Method  Male: 3.5-7.2mg/dl
 An enzymatic method  Female: 2.6-6.0mg/dl
 Utilizes the uricase enzyme
 Enzymatic-colorimetric analysis
 Aka uricase method *SI unit in mmol/L
 A coupled enzymatic reduction assay
Principle: Causes of increased plasma uric acid:

u.a uricase
--------> allantoin + CO2 + H2O2 1. Increased dietary intake
2. Increased urate production: gout, treatment of myeloproliferative disease with cytotoxic
H2O2 + o-aminobenzenoic acid + MBTH ------> blue chromogen (w/ an A at 520nm) drugs
3. Decreased excretion: lactic acidosis, toxemia of pregnancy, glycogen storage disease type 1 B
*MBTH – 3-methyl-2-benzothiazolinone hydrazone (glucose-6-phosphate deficiency), drug therapy, poisons (lead, alcohol), renal disease
4. Catabolic pathway enzyme defects: Lesch-Nyhan Syndrome
2.2 Coupled Enzymatic-Spectrophotometric Assay
peroxidase
H2O2 + 3,5 DCHBS + 4-aminophenazone ------------------> red dye complex

*3,5- DCHBS – 3,5-dichloro-2-hydroxybenzene sulfonic acid

Indicator compounds:

1. O-aminobenzoic acid
2. MBTH – produces color with absorbance in the visible region