Estimation of Urinary Creatinine by Jaffe's

Method
Aim: To estimate the amount of urinary creatinine in the given sample by Jaffe’s
method.

Principle:
Creatinine present in urine reacts with picric acid in the presence of sodium
hydroxide to give an orange color. The intensity of the color developed is directly
proportional to the amount of creatinine present. The color intensity is compared
with standard and is measured at 540 nm (green filter).

Reagents:
1. Creatinine standard (10mg/dl)
2. Picric acid
3. 0.75 N Sodium hydroxide

Procedure:
Dilution of urine: Take 1ml of urine, add 9ml of distilled water to make it 10 ml.
Label three test tubes as test (T), standard (S) and blank (B). Into T, pipette 5 ml
diluted sample. Into S, pipette 5 ml standard creatinine solution (0.5 mg). Take 5 ml
distilled water into B. To each tube, add 2 ml of saturated picric acid solution and 2
ml of 0.75 N sodium hydroxide. Mix. Read optical density values (OD) after 15
minutes using green filter (540 nm).

Reagents Blank Standard Test

Distilled water 5ml - -

Diluted sample - - 5ml

Saturated picric acid 2ml 2ml 2ml

0.75N sodium hydroxide 2ml 2ml 2ml

Mix. Read the OD values after 15 minutes using green filter

Page 1 of 5
 OD at 540nm

Calculation:
Concentration of standard 10mg/dlthen for 5ml of standard solution the
concentration is 0.5mg/dl
Volume of sample1ml of sample is diluted in 9 ml of distilled water
Therefore, 5ml of diluted sample consists of 0.5ml of sample
Concentration of creatinine in the given sample (mg/dl) =
𝑂𝐷 𝑜𝑓 𝑇𝑒𝑠𝑡−𝑂𝐷 𝑜𝑓 𝐵𝑙𝑎𝑛𝑘 𝐶𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 𝑜𝑓 𝑆𝑡𝑎𝑛𝑑𝑎𝑟𝑑
X X100
𝑂𝐷 𝑜𝑓 𝑆𝑡𝑎𝑛𝑑𝑎𝑟𝑑−𝑂𝐷 𝑜𝑓 𝐵𝑙𝑎𝑛𝑘 𝑉𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 𝑆𝑎𝑚𝑝𝑙𝑒
𝑂𝐷 𝑜𝑓 𝑇𝑒𝑠𝑡−𝑂𝐷 𝑜𝑓 𝐵𝑙𝑎𝑛𝑘 0.5
X X100 mg/dl
𝑂𝐷 𝑜𝑓 𝑆𝑡𝑎𝑛𝑑𝑎𝑟𝑑−𝑂𝐷 𝑜𝑓 𝐵𝑙𝑎𝑛𝑘 0.5
𝑂𝐷 𝑜𝑓 𝑇𝑒𝑠𝑡−𝑂𝐷 𝑜𝑓 𝐵𝑙𝑎𝑛𝑘
X100 mg/dl
𝑂𝐷 𝑜𝑓 𝑆𝑡𝑎𝑛𝑑𝑎𝑟𝑑−𝑂𝐷 𝑜𝑓 𝐵𝑙𝑎𝑛𝑘

Assuming the 24 hours urine output is about 1500ml/day the amount of

Report:
1. The concentration of creatinine present in the given sample is ______ mg/dl.
2. The amount of creatinine excreted is ______ g/day.
Clinical significance:
i. Normal range of urinary creatinine: 1-2g/day
Serum creatinine: 0.5-1.5 mg/dl
ii. Increased urinary creatinine:
Muscular dystrophy
Myasthenia gravis
High catabolic states like hyperthyroidism, fever
Diabetes mellitus

Page 2 of 5
 High dietary intake of meat
iii. Decreased urinary creatinine:
Renal failure
Glomerulonephritis
Reduced blood flow in dehydration
Urinary tract obstruction
Metabolism:
Creatine is synthesised in liver made up of 3 amino acids glycine, arginine,
methionine taken by skeletal muscle and stored as creatine phosphate as storage form
of energy.
About 2% of creatine daily converted to creatinine and is excreted by kidneys. Hence
serum creatinine level is an index for renal function test (RFT) and muscle mass.
Creatinine Clearance Test:
Creatinine clearance is defined as volume of plasma completely cleared of creatinine
per minute by the kidney.
Procedure:
The test is performed in the morning. The patient is given 600 ml glasses of water to
drink. The bladder is emptied completely and the urine is discarded. The time is
noted.
Urine is collected for the next 5 hours in a container, A sample of blood is
drawn for creatinine estimation in serum. Measure the total urine volume.
Estimate the creatinine in the urine.
Calculation:
Creatinine Clearance (ml/min) = U x V x 1.73
PxA
Where U = mg of creatinine/dl in urine
P= mg of creatinine/dl in serum or plasma
V = Volume of urine in ml/min
A = Body surface area of the patient
1.73= Standard average surface area of normal individual

Page 3 of 5
 Clinical Significance:
 Normal value of creatinine clearance in
Males: 95-140 ml/min/1.73 sq. mt mean: 120 ml/min
Females: 85-125 ml/min/1.73 sq. mt mean: 110 ml/min
Creatinine clearance cut off values in assessment of renal function, considering
125ml/min as 100%
70%  Normal
70-50%  Mild renal damage
49-20%  Moderate renal damage
<80%  Severe renal damage
Significance of clearance:
Clearance is affected in very early stages of glomerular damage where serum level is
likely to be normal. It is better than urea clearance as it is not affected by dietary
proteins and not reabsorbed by tubules.
Diagnostic Importance
 A decrease in creatinine clearance value (<75% of normal) serves as
sensitive indicator of a decreased GFR due to renal damage.
Points to Remember
 Creatinine is the end product of creatine metabolism.
 Creatine is found as creatine phosphate in muscle, plays an important role in
muscular contraction.
 Normal excretion of creatinine in urine is in the range of 1-2 g per day.
 Excretion is more in males because of more muscle mass.
 Creatinine excretion is useful to check the reliability of 24 hours urine samples
in assaying other biochemical parameters. Urine creatinine is largely
endogenous and is little influenced by diet. The excretion is therefore
remarkably constant.
 Excretion of other metabolites in random samples of urine may be expressed
in terms of creatinine in the same sample.
 Creatinine coefficient is milligram of creatinine excreted in urine per kg body
weight in 24 hours. Normally it is 20 26 mg/kg/day in men and 14-20
mg/kg/day in women.

Page 4 of 5
 Creatinine coefficient is more precise and is used to assess the functional
muscle mass in the body.
 Very little creatine is normally found in adult urine except in women during
pregnancy and early postpartum.
 The excretion of creatinine increases in fevers and wasting diseases.
 The excretion of creatinine decreases in myopathies and renal failure.
 A variety of compounds, like proteins, glucose, pyruvate, ascorbate and
ketones interfere in Jaffe's method for creatinine estimation.
 In serum the interfering compounds contribute to about 20% of the color,
whereas in urine, the interference is only to the extent of 5% or less.

Page 5 of 5