Laboratory Examination Of Creatinine

ESTIMATION OF CREATININE

Creatinine is a nitrogenous product that is produced when metabolism of creatine in the skeletal and muscle. They are filter by the kidney and excreted in the urine.

REAGENT

  1. SATURATED PICRIC ACID SOLUTION

COMPOSITION

Picric acid

Distilled water

PREPARATION

Place an excess of picric acid in an amber colour bottle containing distilled water, leave for a few days then discard the suppernant in another container. The suppernant is the saturated solution, store in the bottle.

NB: picric acid is explosive so it must be store always in damp.

  1. 10% W/V SODIUM HYDROXIDE
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COMPOSITION

Sodium hydroxide

Distilled water

PREPARATION

Dissolve sodium hydroxide in a distilled water and store

  1. CREATININE WORKING REAGENT

COMPOSITION

Saturated picric solution                               10ml

10% Sodium hydroxide                                2.0ml

PREPARATION

Mix 10ml of saturated picric acid solution with 2.0ml of 10% sodium hydroxide.

It should be prepared freshly for use.

  1. STOCK CREATININE STANDARD 10MMOL/L REAGENT

COMPOSITION

Pure creatinine                                             0.113g

(0.1N) hydrochloric acid                             100mmol/l

PREPARATION

Dissolved 0.113g of pure creatinine in a quantity of (0.1N) hydrochloric acid and make up the solution to 100ml with (0.1N) hydrochloric acid, the reagent is stable for two month when store at 2-8ºC

  1. WORKING CREATININE STANDARD

COMPOSITION

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Stock creatinine standard                                       1%

Distilled water                                                         200µlmol/l

PREPARATION

Dilute 1:50 of stock creatinine standard and make up to 200µlmol/l

Stable for one month when store at 2-8ºC

METHOD

STEP 1

Set up three tubes as follow;

REAGENT TEST TUBE STD TUBE BLK TUBE
Working reagent 2.0µl 2.0µl 2.0µl
Standard reagent   0.2µl  
Test sample 0.2µl    
     

Mix the tube very well leaves it at room temperature to stand for 1 hours 30 minute

STEP 2

Set the instrument to zero with the blank.

First reading, use 490nm wavelength or use blue green filter.

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Record your reading down

STEP 3

Add 50µl (0.05ml) acetic acid 60% v/v to all tubes.

Set the instrument to zero with the blank and read the absorbance using 490nm, or blue green filter

Record the reading separately.

STEP 4

Subtract second reading from the first reading to give final reading.

CALCULATIION

Second reading – first reading = final reading

Then calculate

                                                       Ab. Test

Creatinine µmol/l       =                   —            x   200

                                                      Ab. Std

 

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