28/11/2022

NITROGEN CONTAINING COMPOUNDS

NONPROTEIN NITROGEN COMPOUNDS

UREA

METHODS
CLINICAL APPLICATION ENZYMATIC METHOD PRINCIPLE
FIRST STEP

i. GLDH coupled enzymatic-disappearance of a. Nessler’s reaction

absorption of NADH is measured at 340 nmn
b. Berthelot reaction

ii. Indicator dye

iii. Conductimetric

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METHOD OF ANALYSIS SPECIMEN REQUIREMENTS

Chemical Method Principle 1. Use fasting/non fasting blood sample
2. Avoid fluoride or citrate anticoagulants
i. Fearon’s reaction 3. Refrigerate samples

PATHOPHYSIOLOGY NONPROTEIN NITROGEN COMPOUND

URIC ACID CLINICAL APPLICATION

Asses inherited disorders of purine metabolism
Confirm diagnosis and monitor treatment of gout
Diagnosis of renal calculi
Prevent uric acid nephropathy during chemotherapy
Detect kidney dysfunction

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METHOD METHOD
Chemical Method Principle Enzymatic Methods Principle

First step
Phosphotungstic acid
(Caraway method)
Decrease in absorbance at 293 nm is measured
Spectrophotometric (uric acid v. allantoin)
(Blauch and Koch)

Coupled enzyme
(I) Catalase

Coupled enzyme
(II) Peroxidase

SPECIMEN CONSIDERATIONS PATHOPHYSIOLOGY

1. May be measured using heparinized plasma, serum or urine
2. Avoid gross lipemia, high bilirubin concentration and hemolysis
3. Avoid EDTA or flouride additives (affects uricase method)
4. Salicylates and thiazides ↑ values for uric acid
Increased Concentration (Hyperuricemia)
Enzyme deficiencies
1. Lesch-Nyhan syndrome
2. Phosphoribosylpyrophosphate synthetase deficiency
3. Glycogen storage disease type 1 (Glucose-6-phosphatase deficiency)
4. Frcutose Intolerance (fructose-1-phosphate aldolase deficiency)

PATHOPHYSIOLOGY PATHOPHYSIOLOGY
Increased Concentration (Hyperuricemia) Decreased Concentration (Hypouricemia)
Liver disease
1. Hemolytic and proliferative process (e.g. leukemia, lymphoma, etc.) Defective tubular reabsorption (Fanconisydrome)
2. Treatment of myeloproliferative disease w/ cytotoxic drugs Chemotherapy with azathioprine or 6-mercaptopurine
Overtreatment with allopurinol
3. Chronic renal disease ↓ de novo purine synthesis
4. Toxemia of pregnancy and lactic acidosis
5. Drugs and poisons
6. Purine-rich diet (e.g. liver, kidney, shellfish)
7. Increase tissue catabolism or starvation

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NONPROTEIN NITROGEN COMPOUNDS PHYSIOLOGY

CLINICAL APPLICATION CLINICAL APPLICATION

Determine sufficiency of kidney function Glomerular Filtration Rate (Creatinine clearance)
Determine severity of kidney damage Volume of plasma filtered (V) by the glomeruli per unit of time (ml/minute)
Monitor the progression of kidney disease
Measure completeness of 24-hour urine

CLINICAL APPLICATION METHOD OF ANALYSIS

Given the following data, calculate the creatinine clearance Chemical Method Principle
Serum creatinine = 1.2 mg/dL Direct Jaffe Reaction Creatinine + picrate → red-orange complex
Urine creatinine = 120 mg/dL Jaffe-kinetic (rate of change of absorbance) Rate of change of absorbance (color formation) is
Urine volume = 1.75 L/day detected to avoid interference of noncreatinine
Surface Area = 1.80 m2 chromogens
Jaffe with adsorbent Creatinine in protein-free filtrate is adsorbed onto
Fuller’s earth (aluminum magnesium silicate) or
Lloyd’s reagent (sodium aluminum silicate); then
eluted and reacted with alkaline picrate
Jaffe without adsorbent Creatine in protein-free filtrate reacts with alkaline
picrate to form colored complex

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METHODS OF ANALYSIS SPECIMEN REQUIREMENTS

Enzymatic Method Principle Specimen Considerations Specimen Considerations
Creatininase-CK Creatinine + H2O —Creatininase →Creatine A. Falsely increase due to 1. Glucose
2. α-ketoacids
3. Ascorbate
4. Uric Acid
5. Cephalosporins
6. Dopamine
Creatininase- H2O2 Creatinine + H2O —Creatininase→ Creatine
Creatine H2O —Creatininase→ Sarcosine + ADP B. Falsely decrease results due to 1. Bilirubin
Sarcosine + O2 + H2O ADP —sarcosine oxidase→ glycine + CH2O + H2O2 2. Hemoglobin
H2O2+ colorless substrate—Peroxidase → Colored product + H2O 3. Lipemic specimens

PATHOPHYSIOLOGY
Increased Concentration
Renal failure (glomerular function)
↑ Plasma Concentration = ↓ GFR

NONPROTEIN NITROGEN COMPOUNDS PHYSIOLOGY

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METHOD SPECIMEN CONSIDERATIONS

1. May be measured using heparinized and EDTA tubes
2. Samples should be centrifuged at 0°C to 4°C within 20 minutes of collection and
the plasma or serum removed
3. Avoid cigarette smoking for several hours