1 CREATININE

(Alkaline Picrate Method)

CLINICAL SIGNIFICANCE
The evaluated level in serum/plasma is usually associated with various renal diseases. In the earlier stage
of renal diseases, creatinine clearance, test is sensitive index of impaired renal function. The prerenal
factors which can increase blood urea, have less influence on creatinine concentration. Hence for
diagnosis of renal diseases serum creatinine is preferred over urea estimation. In additional to renal
diseases, elevated levels of serum creatinine and creatinurea may be observed in extensive muscle
destruction.

PRINCIPLE
In alkaline medium Picric Acid reacts with creatinine and produces a red coloured complex, whose
absorbance is proportional to creatinine concentration. Picric Acid reagent has a dual role, as a
deproteinzing agent and as a reactant.

REAGENTS PROVIDED
1. Picric Acid Reagent
2. Sodium Hydroxide 0.2N
3. Creatinine Standard 2mg%

REAGENTS STORAGE & STABILITY

All reagents are stable at Room Temperature till expiry date mentioned on the label.

SPECIMEN
Serum/Urine (24 hours urine is preferred. Dilute 1 ml of urine to 50ml with distilled water).

PROCEDURE

Step 1 :- Deproteinistation of specimen.

Pipette into clean dry test tubes labeled (T):
(T)
Serum/Diluted Urine 750 µL
Picric Acid Reagent (1) 4.5 ml
Mix well and filter/centrifuge at 2000-3000 rpm for 10 minutes to obtain a clear filtrate/supernatant.

Step II :- Colour Development

Label three clean dry test tubes at Blank (B), Standard (S) and Test (T), Pipette into each test tube as
shown below:
Description (B) (S) (T)
1 Supernatant/Filtrate from step 1 -- -- 3.5ml
2 Creatinine standard (3) -- 0.5ml --
3 Distilled water 0.5ml -- --
4 Picric Acid Reagent (1) 3.0ml 3.0ml --
5 Sodium Hydroxide 2.0ml 2.0ml 2.0ml

Mix well and allow to stand at R.T.for exactly 20 minutes and measure the absorbance (A) of standard (S),
Test (T) against Blank (B) against distilled water either on a photocolorimeter with green filter or on a
Spectrophotometer at 520 nm (Hg 546 nm).

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 CREATININE
(Alkaline Picrate Method)

INTERFERENCE:
Drugs causing nephrotoxicity
Acetoacetate, acetohexamide (ASTRA aca) acetone, ascorbic acid, cafamandole, cafazolin, cefoxitin
cephalothin, flucytosine (Ektachem) fructose, glucose ibuprofen (deproteinization), levodopa,
methyldopa,pyruvate, uric acid. Enz. Cephalothin (aca)

CALCULATIONS
A of (T) - A of (B)
(a) Serum Creatinine in mg% = ------------------------ x 2.0
A of (S) – A of (B)
A of (T) - A of (B)
(b) Urine Creatinine in gm/L = ------------------------ x 1.0
A of (S) – A of (B)
(c) Urine Creatnine in gm/24 hrs. Urine collection. = (b) x24 hrs. Urine volume in liters.

QUALITY CONTROL
It is recommended that normal and abnormal controls or a known patient serum should be included in
each set of assays. Factors other than reagents which might affect the performance of the test include
proper instrument function, cleanliness of glassware and accuracy of pipetting.

NOTE
1. Do not pipette Picric Acid Reagent by mouth.
2. During urine creatinine estimation do not fail to dilute urine sample.
3. Adhere to the reaction time of 20 minutes as closely as possible, since if the samples for any analysis
are allowed to stand any longer, there may be increase in absorbance due to pseudochromogens.
4. Hemolysed or lipemic serum should not be used as it may give erroneous results.
5. The filtrate/supernatant obtained in Step 1 should be crystal clear.

NORMAL VALUES
Serum creatinine Urine creatinine in 24hrs Urine Collection
Male 0.9 – 1.5 mg% 1.1 – 3.0 gm
Female 0.8 –1.3 mg% 1.0 – 1.8 gm

Due to assay conditions, instruments and demographic variations it is recommended that each laboratory
should establish its own normal range.

PERFORMANCE
Linearity : Upto 6 mg %.of Creatinine
Accuracy : Accurancy was evaluated using normal and abnormal assayed control
sera and was found satisfactory. The recovery studies showed 95%
recovery.
Precision : The day to day Quality Assurance testing suggests that the method has
good precision. The coefficient of variation determined was 4.4% (n=20)
REFERENCES
1. Bonses, R.W. & Tausskay H.H. (1945) J.Biol.Chem. 158, 581.
2. Slot C. (1965) Scand J.Clin.Lab. Invest. 17.381.
3. Taro G. & Ackermann P. (1975) Practical Clinical Chemistry P. 154.

RECOMBIGEN LABORATORIES PVT LTD: A-2, IInd Floor ,Badli Industrial Estate, New Delhi-42. India
Ph: +91-11-27295102, Fax::+91-11-27295101,Website: www.recombigen.com , email: salesrecombigen@gmail.com