Blood Urea defination, Estimation of Urea, Berthelot method, Normal values, calculations, Clinical significance

 

 Blood Urea:-

Urea is the major excretory product of protein metabolism. Urea contain almost half the total of the non-protein nitrogenous substance of the blood.

Urea is manufactured in the liver from carbon dioxide and ammonia, from the break  down of the amino acid. Urea is carried by the plasma to the kidney. In the kidney Urea  is filtered from the plasma by the glomerulus. About 40% of the urea in the glomerular filtrate is reabsorb by the renal tubules.  Most of the urea in the filtrate  is excreted in the urine, and small amount of the urea are excreted through the gastro intestinal tract. 

Estimation of Urea By Berthelot. reaction,

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Estimation of Urea by Berthelot Reaction:-

 

Principle:-

 In this method, Ammonia produced by the action of urease, urea is estimated by the measuring the blue colour. The blue colour forms with phenol and hypochlorite.  Sodium nitroprusside used as a crystals.

 

Reagents:-

 

1.     Buffered urease reagent

2.     Phenol colour  reagent

3.     Alkaline hypochlorite reagent

4.     Urea standard solution

 

 Composition of Reagents :-

 

1.        Buffered urease reagent :-

 

Ethylene diamine tetra acetic acid (EDTA)     --           1.0  gm

     Urease                                                                ---       150 mg

     Distilled Water                                                    ---         70  ml

             Mix well and adjust Ph 6.5 and makeup volume 100  ml.                                                      Store in a plastic bottle at 4^o C. This reagent is stable for one month.

 

2.       Phenol colour Regent :-

 

(i)                 Analytical grade Phenol        --    50 gm

              Distilled water                          --   400 ml.

 

            Mix well in the volumetric flask.

 

(ii)              Analytical grade sodium nitroprusside    -   250gm                                     Distilled water                                        -     50 ml.

                          Mix well in volumetric flask.                                                                                              Mix well (i) and (ii) solutions together and make the volume up to 1 litre with distilled water, Store in the dark brown bottle. Stable for two months.

 

3.       Alkaline hypochlorite Reagent :-

 

(i)                  Sodium hydroxide   --   25 gm

             Distilled water       --     400 ml.

 

        Mix well in the volumetric flask.

 

(ii)                 Sodium hypochlorite    ---    2.0 gm

     Distilled water               --     100 ml.

 

 Mix well in the volumetric flask.

Mix well (i) and (ii) solutions together and make the volume up to 1 litre with distilled water.  Store in the dark brown bottle.  Stable for two   months.

4.          Urea standard solution:-

 

                     Urea                        –    600 gm

                   Distilled water        --     600mg

      Add a few drops of chloroform as a preservative.  Store at 4^o C.

 

 

Sample :-

 

EDTA blood, plasma, Serum, Urine.

 

Material required:-

 

Test tubes

Test tubes stand

 Pipettes

Tissue paper

Reagents and standard

Water bath

 

 

Method:-

 

·        Take three test tubes an d label as  blank, test and standard.

·        In the Blank tube  add

       Buffered urease reagent - 200 microlitre.

        Distilled water                --   20 microlitre.

·        In the test tube:-

        Buffered urease reagent - 200 microlitre.

        Test plasma                       -- 20 microlitre.

·        In the standard tube   :-

     Buffered urease reagent -    200 microlitre.

    Standard                             --     20 microlitre.

·        Incube all three tubes in waterbath at 37^o C for 15 minutes.

·        After 15 minutes, Remove all three tubes from waterbath .

·        Add phenol colour reagent in each tube ---   1.0 ml

·        Mix well.

·        Add hypochlorite reagent to each tube  -   1.0ml.

·        Incubate all the tubes at37^o C for 20 minutes.

·         Remove all three tubes from waterbath

·        Add 5 ml distilled water to each tube

·        Then read the absorbance of test and standard against blank, at red  filter 630nm to 650nm.

·         Calculate the reading..

 

 

Calculations:-

 

The urea concentration in mg/dl

 

    Absorbance of test       x    60

Absorbance of standard

 

 

Normal values:-

 

Adults   ----       10-40  mg/dl

 

Clinical Significance:-

 

Blood urea Increase  in kidney disease, Dehydration, Diabetes, Dehydration.

 

Blood urea Decrease  in liver disease, increase urine output.

 

 

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