Expt No: Date:

Estimation of serum Creatinine by Jaffe’s Method

Introduction:
Creatinine is a waste product derived from endogenous sources by tissue creatine breakdown
and is excreted by the kidney. Creatinine is the anhydride of creatine. The reaction occurs
nonenzymatically.

Creatine creatinine
H2O

About 98% of the total creatine is in the muscle as creatine phosphate. About 2% of the total
creatine is converted daily to creatinine so that the amount of creatinine produced is related to
the total muscle mass. As muscle mass remains approximately same, creatinine also remains
same in plasma and urine.

Aim : To estimate the serum Creatinine by Jaffe’s Method

Method:
Jaffe’s alkaline picrate method

Principle
Creatinine present in Serum reacts with picric acid in the presence of sodium hydroxide to
form an orange, red color Creatinine picrate. The intensity of the color developed is directly
proportional to the amount of creatinine present, the intensity of the orange red color is
measured at 520 mm (green filter).

Reagent:
1) 0.04m Picric acid
2) 0.75N Sodium hydroxsaid
3) 10% Sodium tungstate.
4) 2/3 H2SO4
5) Creatinine Std solutions 0.01mg/ml in (0.1 N HCl).

Procedure:
Part – 1 : Preparation of Protein free filtrate.
1) In a dry test tube take 3ml distilled water 1ml serum or plasma, 1ml 10% Na
Tungstate and 1ml 2/3 H2SO4 (dilution of Blood = 1 in 6). Mix keep for 10 minutes
and filter in a dry test tube obtain a clear solution of PFF.
2) Part – II : Estimation of Creatinine.
Label three test tubes as test, blank and standard and add PFF and Reagents as follow.

Reagents Test Standard Blank

Protin free Filtrate 3ml - -
Standard Creatinine Solution - 3ml -
Distilled water - - 3ml
Picric acid 1ml 1ml 1ml
NaoH 1ml 1ml 1ml
Mix and keep for 15 minutes read O.D using a colorimeter with green filter of
wavelength 520 nm.

Observations:
O.D of Test
1
O.D of Std
O.D of Blank

Calculation:

O . D of Test – O . D of Blank Concentration of Creatinine

Concentration of Creatinine= X X 100
O . D of Test – O . D of Blank Volume of Sample

O. D of Test – O . D of Blank 0.03

¿ X X 100
O. D of Test – O . D of Blank 0.5

O. D of Test – O . D of Blank
¿ X6
O. D of Test – O . D of Blank

= mg/dl
Report …… mg of creatine is present in 100 ml of serum

Normal Values
Adult Male 0.6 – 1.5 mg/dl
Adult Female 0.6 – 1.1 mg/dl
Children 0.2 – 1.0 mg/dl

Clinical Significance:
The value may increase during kidney diseases like acute or chronic renal disease. Urinary
tract obstarction and impairment of renal function include by some drugs, nephritis and
muscle wasting disease.

2
Expt No: Date:
Estimation of Urinary Creatinine by Jaffe’s Method and to calculate the
Creatinine coeffecient & Creatinine clearance

Aim: To estimate the Urinary Creatinine & creatinine coefficient by Jaffe’s Method and To
calculate the & creatinine clearance on given serum creatinine level.
Procedure:
Take three 100 ml volumetric flasks; mark them as B, S and T. Add 1 ml of distilled water to
B, 1 ml of standard solution to S and 1 ml of 1:10 diluted urine sample to T. Then add 1 ml of
picric acid and 1 ml of 10% NaOH to all the three flasks. Mix well and keep it for 15
minutes. The orange color will develop. Then dilute the solution up to 100 mark using the
distilled water. Read the optical density of the solution at 540 nm or using the green filter.

Reagents Test Standard Blank

Distilled water - - 1ml
Standard solution - 1ml -
1:10 diluted urine sample 1ml - -
Picric acid 1ml 1ml 1ml
10% naoh 1ml 1ml 1ml
Mix well and keep it for 15 minutes. Then dilute the solution up to 100 mark
using the distilled water. Read the optical density of the solution at 540 nm or
using the green filter.
Calculation: - mg of Creatinine present in 100 ml of urine.

O. D of Test – O . D of Blank Concentration of standred

¿ X X 100
O. D of Test – O . D of Blank Volume of Sample

O. D of Test – O . D of Blank 0.1

¿ X X 100
O. D of Test – O . D of Blank 0.1

O. D of Test – O . D of Blank
¿ X 100
O. D of Test – O . D of Blank

= mg/dl

Assuming the 24 hours urine output is about 1500 mL/day the amount of creatinine excreted
in g/day.

O. D of Test – O . D of Blank 15
¿ X X 100
O. D of Test – O . D of Blank 1000

T −B 1500
¿ X g /day
S−B 1000

= ___________ g/day

Report : ….. g/day is excreted creatininie in urine.

Normal Range: 60-120 mg in 100 ml urine sample or 1-2g/day

Creatininie coefficient: Mg of critinine excreted in urine per Kg body weight/day.

3
 gr /dayX 1000
Creatininie Coeffeciant=¿ 70(average weight of adult ) ==…mg/kg/day

Normal Values: Adult male 24 - 26 mg/kg body weight/day,

Adult female 20 -22 mg/kg body weight/day

CREATININE CLEARANCE (Ccr)

Clearance is defined as the amount of plasma in ml which would have to be completely

cleared of the substance, each minute by the kidney, in order to account for its rate of
excretion.

Procedure: For Creatinine clearance a 24 hours urine specimen is collected and blood taken
during the day. Serum and Urine Creatinine are estimated. Urine volume / min is calculated
by dividing urine volume (ml) by time of collection. (min).

It is calculated by
UV
C¿
P
Where U and P are the concentrations of the concerned substance in urine and plasma
respectively and V is the rate of urine flow in mL/min. Clearance varies with body size and is
proportional to the surface area. The clearance is a parameter for assessing the glomerular
function of kidneys.

CALCULATION OF CREATININE CLEARANCE (CCR)

C: Creatinine clearance
U (Concentration of creatininie in urine in mg/dl) = ---------
P (Plasma concentration in serum in mg/dl )= --------
V (Volume of urine in ml excreted per minute (24 hr urine divided by 24X60)) =

24 hr urine output in ml
V= ____________________
24X60

V= ----------------ml/min excretion of urine

UV
Creatinine clearance ¿ =
P
Concentration of creatinine∈urine∈mg/d l X Volume of urine∈ml excreted per minute¿ ¿ =
= ……ml/minute
Results:
Creatinine clearance =
Normal Values:
Male – 95-140 ml/min
Female – 85-125 ml/min
These values are close to GFR