ESTIMATION OF PLASMA/SERUM UREA USING BERTHELET METHOD

REAGENT

1. BUFFERED UREASE REAGENT

COMPOSITION

EDTA (Ethylene diaminetetracetic acid)                     1.0g

Urease                                                                           150mg

Distilled water                                                              100ml

PREPARATION

Dissolved 1.0g of EDTA (Ethylene diaminetetracetic acid), and 150mg urease in 70ml of distilled water, adjust the solution to PH 6.5 and make up the solution to 100ml with distilled  water.

The solution is stable for 1 month.

2. PHENOL COLOUR SOLUTION

COMPOSITIION

Analytical grade phenol                                    50g

Analytical grade sodium nitroprusside              250mg

Distilled water                                                   1litre

PREPARATION

STEP 1

SOLUTION 1

Dissolved 50mg of Analytical grade phenol in 400ml of distilled water.

STEP 2

SOLUTION 2

Dissolved 250mg of Analytical grade sodium nitroprusside in 50ml of distilled water.

STEP 3

Mixed solution 1 with solution 2 and make it up to the final volume of 1litre with distilled water.

Store the solution in a dark brown bottle and it should be kept away from light, store at 4ºC it is stable for 2 month.

3. ALKALINE HYPOCHLORITE REAGENT

COMPOSITION

Sodium hydroxide                                        25g

Sodium hypochlorite                                     2.1g

Distilled water                                               1lire

PREPARATION

STEP 1

SOLUTION 1

Dissolved 25g of Sodium hydroxide in 400ml of distilled water

STEP 2

SOLUTION 2

Dissolved 2.1g of Sodium hypochlorite in 100ml of distilled water

STEP 3

Mixed solution 1 with solution 2 and make it up to the final volume of 1litre with distilled water.

Store the solution in a dark brown bottle and it should be kept away from light, store at 4ºC it is stable for 2 month.

4. UREA STANDARD (10mmol/L) REAGENT

COMPOSITION

Urea                                              600mg

Distilled water                              1litre

ChloroformPREPARATION

Dissolved 600mg of Urea in a quantity of distilled water, and make up the solution to 1litre with distilled water, add a few drop of chloroform as a preservative.

METHOD

STEP 1

Set up three tubes as follow;

 

REAGENT	TEST TUBE	STD TUBE	BLK TUBE
Plasma	20µl
Buffer urea reagent	200µl	200µl	200µl
Standard		20µl
Distilled water			20µl

STEP 2

Incubate all the tube using water bath at 37ºC for 15 minute

Remove all the tube from the water bath to room temperature, add 1.0ml phenol colour reagent to each tube, and shake each of the tube gently to mix very well.

Add 1.0ml hypochrite to each tube and shake to mix.

 

STEP 3

Incubate all the tube again at 37ºC for 20 minutes

STEP 4

Remove all the tube from water bath; add 5ml of distilled water to each tube

Read absorbance of the standard and the test, using the blank to set the machine to zero, record your reading.

 

CALCULATION

Ab. Test

Urea plasma Con. In mol/l =  ———————   x 10mmol/l

Ab. Std