ESTIMATION OF UREA IN BLOOD
DIACETYL MONOXIME OR DAM METHOD

PRINCIPLE:

urea reacts with diacetyl monoxime under strongly acidic conditions in the presence of ferric ions and thiosemicarbazide to give pink colored complex.

REAGENTS REQUIRED:

1.diacetyl monoxime solution
ferric chloride solution
thiosemicarbazide solution
sulphuric acid 20%
 acid reagent (1000 ml of 20 % H2SO4 + 1 ml of ferric chloride solution)
trichloroacetic acid (10 %)
standard urea solution (0.1mg/ml)

PROCEDURE:

take 3.4 ml of distilled water in a “tt” and add 0.1 ml of blood and mix. Add 1.5ml of 10% trichloroacetic acid. Mix well and allow it to standard for 5 min centrifuge at 2000 rpm for 5 min. take 1ml of the supernatant and pipette into a “tt” labeled as “T”. take 1ml of the STD urea in a “tt” and label it as “S” take 1ml of water in a “tt” and label it as “B”. add 1ml of diacetyl monoxime to “B”, “S”, and “T”.
add 1 ml of thiosemicarbazide, followed by 3ml of acid reagent “B”, “S”, and “T” and mix well. Keep the 3 tubes in a boiling water bath for exactly 15min and cool. Read the absorbance at 540nm using a spectrophotometer. Set the reading to 0 with water

concentration of urea in blood (mg/100ml)=absbnce of T-abmce of B/ absence of S-absence of B X 0.01 X 100/0.2.

CLINICAL SIGNIFICANCE:

normal blood urea ranges from 15-45 mg/100ml.
The level is influenced by the proteins in the diet.
Urea level is decreased on a low protein diet and increased on a high protein diet.
    
    IN general increase in blood  urea and seen in:
acute and chronic nephritis and renal failure
renal tuberculosis
renal cancer
haemo concentration due to severe vomiting and diarrhea and burns and fever.

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