ESTIMATION OF URINARY CREATININE BY JAFFE’S METHOD

Aim:To estimate amount of urinary Creatinine by Jaffe’s method (Alkaline picrate

method).

Principle: Creatinine present in urine reacts with picric acid in alkaline medium to
form orange yellow colored Creatinine picrate, the intensity of which is measured
at 540nm. Intensity of color is directly proportional to the concentration of
Creatinine in urine.

Procedure

Preparation of diluted urine sample: Urine is diluted as concentration of

Creatinine in urine is high. Take 1ml of urine in 100ml volumetric flask & make
up to 100ml by adding distilled water.

Take 3 test tubes and name them as Blank ‘B’, Standard ‘S’ and Test‘T’. Add the
reagents as mentioned below,

Reagent Blank Standard Test

Distilled Water 3ml - -
Creatinine - 3ml -
Standard
Diluted Urine - - 3ml
0.04M Picric acid 1 ml 1 ml 1 ml
0.75N NaOH 1 ml 1 ml 1ml

Mix after each addition and stand for 10 minutes for color development. Read the
OD value at 540nm.
 O.D values Blank Standard Test

Calculation:

OD of T - B Conc. of Std.
Urinary Creatinine conc.= X X100
OD of S - B Vol. of sample

Conc. of Standard:

Conc. of std.is 1mg/100ml (1ml contains 0.01mg)

So, 3 ml contains 0.03mg

Volume of sample:

100 ml contains 1 ml

So, 3 ml contains 0.03 ml

OD of T - B 0.03
= X X100
OD of S - B 0.03

OD of T - B
= X 100 mg%
OD of S - B

Considering urine output as 1500 ml per day,

Urinary Creatinine excretion in a day

OD of T - B
= X 100
OD of S - B
 OD of T - B 15
= X X 100 g / day
OD of S - B 1000

= ______________________ g/day

Report: The given urine sample contains _______________ g/day of Creatinine.

1. What is the normal urinary Creatinine excretion?

A. The amount of Creatinine produced is fairly constant & is primarily a function

of muscle mass. It is not greatly affected by diet, age, sex, exercise.

Males : 1 - 2 g/day

Females: 1 - 1.8 g/day

2. What are the conditions in which Urinary Creatinine levels will rise?

A. Rhabdomyolysis, Muscular dystrophies.

3. What are the conditions in which Urinary Creatinine levels will fall?

A. Obstructions of urinary tract, chronic kidney disease

4. What is Creatinine? It’s significance?

A. Creatinine is endogenous non-protein nitrogenous waste product formed in

muscle from the high-energy compound Creatine phosphate and is excreted in
urine. It is an anhydride of Creatine. The reaction is non-enzymatic and
irreversible.

Creatinine is removed from plasma by glomerular filtration & then excreted in

urine.

So, Creatinine clearance value is a good indicator of GFR. It is decreased in renal

failure.
5. What are the other methods of estimation?

A. Jaffe’s method using llyod’s reagent, enzymatic methods using creatine kinase

6. Is Jaffe’s method is a specific to creatinine?

A. It is not a specific method. Only 80% of color developed is due to creatinine.

20% is due to non specific chromogens like Glucose, uric acid, urea, Proteins etc.

So the Creatinine estimated by Jaffe’s method gives higher values than true values.