Estimation of Blood Urea by Diacetyl Monoxime method, Normal values, calculations, Clinical significance.

 

Estimation of 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 breakdown 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 Diacetyl Monoxime Method:-

blood urea

click here to watch  :-   Estimation of urea Berthelot method

click here to watch :- Blood and its composition

Principle:-

 In this method,  urea is heated with  Diacetyl monoxime in the presence of strong acid and an oxidizing agent to form a yellow diazine derivative.

Thiosemi-carbazide is added to the mixture to enhance and stabilize the colour.

 

Reagents:-

 

      1.     Sodium tungstate (10%)

 
2.     2/3 N Sulphuric acid:-

                Concentrated H₂SO⁴    --- 2 ml

                 Distilled water                -  50 ml

Dilute up to 100ml.

   

 

3.     Colour Reagent :-

 

(i)                 Solution A:-

 

       Diacetyl monoxime    ---   1.0 gm

       Thiosemicarbazide     ---   0.2 gm

       Sodium chloride         ---   9.0 gm        

       Dilute up to one litre.

(ii)               Solution B:-

                         Concentrated H₂SO⁴   ----  60 ml.

                          Phosphoric acid(85%)  --- 10 ml.

                          Distilled water            ---   100 ml.

                          Mix well make the mixurer.

                          Add ferric chloride      --- 0.1 gm in this acid mixture.

                          Cool it and make the volume up to one litre.

 

 

4.          Urea standard solution:-

 

                     Urea                        –    300 gm

                   Distilled water        --     600ml

     Make the volume up to one litre.

 

 

Sample required:-
 

EDTA blood, plasma, Serum, Urine.

 

Material required:-
 

Test tubes

Test tubes stand

 Pipettes

Tissue paper

Reagents and standard

Water bath

Urea procedue

 

Method:-

·        Prepared protein-free filtrate-

 (by mixing)                  Blood               ---     1.0  ml

                                      Distilled wate  ---     7.0  ml

Mix well  and add—

                                       Sodium tungstate   ---   1.0ml

                                                        2/3 N sulphuric acid  ---  1.0ml

        Mix and allow to stand for 5 minutes and centrifuge the mixure.

 

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

·        In the Blank tube  add

       Colour  reagent –  5.0 ml.

        Distilled water--   0.5 ml.

·        In the test tube:-

        Colour reagent  -   5.0 ml

        Specimen             -- 0.5 ml

·        In the standard tube   :-

      Colour reagent -    5.0 ml

      Urea Standard    --  0.5 ml.

·        Heat  all three tubes in water bath at 37^o C for 15 minutes.

·        Cool it and then read the absorbance of test and standard against blank,  at green filter 520 nm.

·         calculate the reading..

 

 

Calculations:-

 

The urea concentration in mmol/l

 

    Absorbance of test       x    conc. of std.  x            100

Absorbance of standard                                     Amount of blood

 

 
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.