MBBS BIOCHEMISTRY

Practical File
Renal Function Tests & Related Experiments

Experiments 04 · 05 · 06

Exp. 04 Renal Function Test – Blood Urea Estimation

Exp. 05 Estimation of Serum Creatinine (Jaffe's Method)

Exp. 06 Uric Acid Estimation (Uricase Method)

Department of Biochemistry

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 EXPERIMENT No. 04

Renal Function Test – Blood Urea Estimation

Introduction

Urea is the major nitrogenous end-product of protein catabolism in mammals. It is

synthesised in the liver via the urea cycle from ammonia (derived from amino acid
deamination) and excreted by the kidneys. Elevated blood urea is a key marker of
impaired renal function.

Methods of Estimation
A. Enzymatic Methods
• (i) Urease – Berthelot Method
• (ii) Urease – GLDH (Glutamate Dehydrogenase) Method
• (iii) Glutamate Dehydrogenase (GLDH) coupled assay

B. Chemical Methods
• (i) DAM (Diacetyl Monoxime) Method
• (ii) Fearon Method

C. Automated / Instrument-Based Methods

• (i) Autoanalyzer (continuous-flow)
• (ii) Dry Chemistry Analyzers

★ Urease – Berthelot Method (Detailed)

Reagents

Reagent Details

Phosphate Buffer pH 8.0 – 10.0

Urease Enzyme Hydrolyses urea → NH■ + CO■

Sodium Hypochlorite Oxidising agent for indophenol reaction

Sodium Salicylate Chromogenic coupling reagent

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 Standard Urea Solution 50 mg/dL (working standard)

Sample
For urea testing the sample type is typically serum, collected in a Serum Separator Tube
(SST) – often a gold or yellow-top tube – after blood clotting, or lithium-heparinised
plasma in a green-top tube.

Principle

Step 1 – Hydrolysis: Urea present in the serum is hydrolysed by the enzyme urease
into ammonia (NH■) and carbon dioxide (CO■).
Urea + H■O →urease 2 NH■ + CO■

Step 2 – Colour Reaction (Berthelot / Indophenol): The ammonium ions react with
sodium salicylate and sodium hypochlorite in an alkaline medium to form a
blue–green indophenol dye.
NH■ + Salicylate + Hypochlorite → Indophenol (blue–green dye)

Step 3 – Measurement: The intensity of colour is directly proportional to the urea

concentration and is measured spectrophotometrically at 578 nm.

Procedure
Step Blank (B) Standard (S) Test (T)

Label Blank (B) Standard (S) Test (T)

Distilled Water 10 µL — —

Standard Solution — 10 µL —

Serum Sample — — 10 µL

Reagent I (Urease buffer) 1000 µL 1000 µL 1000 µL

Reagent II (Colour reagent)1000 µL 1000 µL 1000 µL

Mix contents and incubate for 10 minutes at 20–25 °C, then for 5 minutes at 37 °C. Adjust
zero of spectrophotometer with Blank at 578 nm. Read absorbance of Standard and Test.

Calculation

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 Urea Concentration (mg/dL) = (AbsTest / AbsStandard) × Concentration of
Standard (50 mg/dL)

Reference Values

Group Reference Range

Normal Adult 10 – 50 mg/dL

Young Adult (mean) ~40 mg/dL

Pregnancy 15 – 20 mg/dL (lower due to haemodilution)

Average (general) Up to 45 mg/dL

Urinary Urea Excretion ~13 g/day

Interpretation of Serum Urea

Serum urea must always be interpreted alongside serum creatinine, eGFR, and
clinical context.

(i) Pre-Renal Causes

Due to reduced blood flow to kidneys or increased protein catabolism.
• High protein diet
• Gastrointestinal bleeding
• Dehydration
• Shock or hypotension
• Severe trauma, major surgery, or starvation

(ii) Renal (Intrinsic) Causes

Due to direct damage to kidney parenchyma.
• Acute tubular necrosis (ATN)
• Glomerulonephritis
• Polycystic kidney disease
• Nephrotoxic drugs

(iii) Post-Renal Causes

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Due to obstruction in the urinary tract.
• Kidney stones (nephrolithiasis)
• Enlarged prostate – BPH (Benign Prostatic Hyperplasia)
• Urethral obstruction
• Bladder dysfunction

Uraemia: A clinical syndrome resulting from advanced chronic kidney disease

(CKD), often worsened by dehydration, characterised by toxic accumulation of
nitrogenous waste products in blood.

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 EXPERIMENT No. 05

Estimation of Serum Creatinine – Jaffe's Method

Introduction – Creatinine

Creatinine is a waste product formed by the non-enzymatic, irreversible

breakdown of phosphocreatine in muscle tissue. It is produced at a relatively
constant rate that depends primarily on a person's muscle mass and tone. Creatinine
is freely filtered at the glomerulus and is neither significantly reabsorbed nor secreted,
making it the most reliable endogenous marker of glomerular filtration rate (GFR).

Biosynthesis Pathway
• 1. Creatine is synthesised in the liver and kidney from the amino acids arginine
and glycine.
• 2. It is transported via the bloodstream to muscle cells, where it is phosphorylated
by creatine kinase to form phosphocreatine (energy store).
• 3. During muscle contraction, phosphocreatine is spontaneously and irreversibly
broken down to creatinine, which enters the bloodstream.
• 4. Creatinine is filtered out by the kidneys mainly through glomerular filtration
and excreted in urine.

Chemical Reaction (Creatine → Phosphocreatine)

Creatine + ATP →Creatine Kinase Phosphocreatine + ADP + H+

Phosphocreatine → Creatinine (non-enzymatic, spontaneous)

Clinical Relevance
• Primary indicator of kidney (renal) function
• Diagnosis and monitoring of Acute Kidney Injury (AKI) and Chronic Kidney
Disease (CKD)
• Calculation of eGFR (estimated Glomerular Filtration Rate) using CKD-EPI or
MDRD equations
• Guide drug dosing for renally-cleared medications
• Monitoring in critical care / ICU patients

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Methods of Estimation
• Chemical method – Jaffe's (alkaline picrate) method
• Enzymatic method – Creatininase-coupled enzyme method
• Clearance-based method – 24-hour urinary creatinine clearance
• Alternative biomarker-based – Cystatin C

★ Jaffe's Method (Alkaline Picrate / Colorimetric)

Reagents

Reagent Details

Picric Acid (saturated) Reacts with creatinine in alkaline medium

Sodium Hydroxide (NaOH) 16% solution – provides alkaline environment

Standard Creatinine Solution 2 mg/dL working standard

Serum Sample Venous blood, preferably serum

Distilled Water For blank preparation

Sample Collection
• Specimen: Venous blood, preferably serum
• Anticoagulant: No additive; serum separator gel preferred
• Vacutainer: Red or yellow top (SST)

Principle (Jaffe's Reaction)

Creatinine reacts with sodium picrate in an alkaline medium to form a red-orange

coloured complex called the Janovsky complex (Tautomer complex).

Creatinine + Picric Acid →NaOH Red-Orange Janovsky Complex

The colour formed is directly proportional to creatinine concentration.

Absorbance is measured spectrophotometrically at 490–510 nm.

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 Procedure

Step Blank (B) Standard (S) Test (T)

Label Blank (B) Standard (S) Test (T)

Picric Acid 500 µL 500 µL 500 µL

NaOH (10%) 500 µL 500 µL 500 µL

Distilled Water 100 µL — —

Standard Solution — 100 µL —

Serum Sample — — 100 µL

Allow all tubes to stand for 10 minutes at room temperature. Read absorbance against Blank
at 490–510 nm (500 nm recommended).

Calculation
Creatinine (mg/dL) = (AbsTest / AbsStandard) × Concentration of Standard (2
mg/dL)

Reference Values

Group Reference Range

Adult Female 0.5 – 1.1 mg/dL

Adult Male 0.6 – 1.2 mg/dL

Elderly (reduced muscle mass) May be lower than adult range

Child (0–12 yr) 0.3 – 0.7 mg/dL

Adolescent 0.5 – 1.0 mg/dL

Infant 0.2 – 0.4 mg/dL

Newborn 0.3 – 1.2 mg/dL

Critical value (any age)

> 4.0 mg/dL – indicates seriously impaired renal function

Causes of Increased Serum Creatinine

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 Kidney-Related:
• Chronic Kidney Disease (CKD)
• Acute Kidney Injury (AKI)
• Glomerulonephritis
• Kidney infections (e.g. pyelonephritis)
Lifestyle & Dietary Factors:
• High protein intake
• Creatine supplementation
• Intense physical activity / rhabdomyolysis
Other Medical Conditions:
• Dehydration
• High blood pressure (hypertension)
• Diabetic ketoacidosis
• Pregnancy complications (e.g. pre-eclampsia)

Benefits & Limitations of Jaffe's Method

Details

✔ Benefits Cost-effective; easy to perform; rapid results; widely available

✘ Limitations
ketones, bilirubin; limited accuracy at low concentrations; pseudo-creatinine chromogens cause positive bias; considered o

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 EXPERIMENT No. 06

Uric Acid Estimation – Uricase (Enzymatic) Method

Introduction – Uric Acid

Uric acid (C5H4N4O3) is a heterocyclic compound of carbon, nitrogen, hydrogen,

and oxygen. It is the final breakdown product of purine metabolism in humans
(unlike most mammals, which further oxidise it to allantoin). The liver and intestinal
mucosa produce most of the body's uric acid, while the kidneys eliminate
approximately 2/3 of it. Uric acid (as urates) is present in synovial fluid and plasma.

Key Biochemical Points

• Purines (adenine, guanine) are catabolised → hypoxanthine → xanthine → uric
acid (via xanthine oxidase)
• Normal serum urate is close to its saturation point; supersaturation →
monosodium urate crystal deposition → gout
• Kidneys eliminate ~2/3; gut eliminates ~1/3 via uricolysis by gut bacteria

Methods of Estimation
# Method Category Examples

1 Enzymatic
Uricase
Methods
method; Dry chemistry uricase method; Biosensor-based uricase method

2 Chemical Methods
Phosphotungstic acid method; Caraway method; Silver oxide method

3 Spectrophotometric Based on UV absorbance at 293 nm

4 Chromatographic HPLC – most accurate reference method

5 Electrochemical Biosensor-based (point-of-care)

★ Uricase (Enzymatic) Method – Detailed

Reagents

Reagent Role

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 Uricase enzyme Oxidises uric acid → allantoin + CO■ + H■O■

Peroxidase (POD) Catalyses H■O■ reaction with chromogen

4-Aminoantipyrine (4-AAP) Chromogenic coupling reagent

Chromogen (e.g. ESPAS or TOOS) Forms coloured quinoneimine dye

Phosphate or Tris Buffer Maintains optimal pH (~7.0–8.0)

Uric Acid Standard Solution Known concentration for calibration

Principle

Step 1 – Enzymatic Oxidation:

Uric acid is oxidised by the enzyme uricase (urate oxidase) to produce:
• Allantoin
• Carbon dioxide (CO■)
• Hydrogen peroxide (H■O■)

Uric Acid + O■ + H■O →Uricase Allantoin + CO■ + H■O■

Step 2 – Chromogenic Reaction (Trinder Reaction):

The generated H■O■ reacts with 4-Aminoantipyrine (4-AAP) and a chromogen in
the presence of Peroxidase (POD) to form a red-coloured Quinoneimine dye.

H■O■ + 4-AAP + Chromogen →POD Quinoneimine (red dye) + H■O

Step 3 – Measurement:
The intensity of the red colour is directly proportional to uric acid concentration
and is measured spectrophotometrically (typically at 500–550 nm).

Chemical Methods – Overview

Phosphotungstic Acid Method (Folin-Wu): Uric acid reduces phosphotungstic acid in
alkaline medium to form a blue compound (tungsten blue) measured at 700 nm. Less
specific than enzymatic methods due to interference from other reducing substances.

Reference Values

Group Reference Range

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 Adult Male 3.5 – 7.2 mg/dL (208 – 428 µmol/L)

Adult Female 2.6 – 6.0 mg/dL (155 – 357 µmol/L)

Children 2.0 – 5.5 mg/dL

Gout threshold (male) > 7.0 mg/dL (hyperuricaemia)

Gout threshold (female) > 6.0 mg/dL (hyperuricaemia)

Clinical Significance of Uric Acid

Causes of Hyperuricaemia (↑ Uric Acid):
• Gout – monosodium urate crystal deposition in joints
• Increased purine turnover: haematological malignancies, cytotoxic therapy (tumour
lysis syndrome)
• Decreased renal excretion: CKD, lead nephropathy
• High purine diet (red meat, organ meats, shellfish)
• Alcohol consumption (especially beer)
• Drugs: diuretics (thiazides, furosemide), low-dose aspirin, cyclosporine

Causes of Hypouricaemia (↓ Uric Acid):

• Xanthine oxidase deficiency (xanthinuria)
• Wilson's disease
• SIADH
• Uricosuric drugs: probenecid, allopurinol (xanthine oxidase inhibitor – used in gout
treatment)

Advantages of Uricase Method over Chemical Methods:

• High specificity – uricase acts only on uric acid
• Minimal interference from other serum components
• Accurate at low concentrations
• Suitable for automated analysers

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