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DAM Method of Urea Estimation

In this DAM method of urea estimation post we have briefly explained about DAM method, principle, requirements, procedure, result and calculation. 

DAM Method of Urea Estimation

Urea is the principal nitrogenous waste product of metabolism and is generated from protein breakdown. It is eliminated from the body almost exclusively by the kidneys in urine, and measurement of its concentration, first in urine and later in blood, has had clinical application in the assessment of kidney (renal) function for well over 150 years.

Principle

Urea in an acidic medium condenses with Diacetyl monoxime at 100°C to form a red coloured complex. Intensity of the colour formed is directly proportional to the amount of urea present in the sample.

Requirements

Reagents

Diacetyl monoxime: 1.56g of diacetylmonoxime in 250ml of distilled water.

Thiosemicarbazie: 41mg of thiosemicarbazied in 250 ml of distilled water.

Ferric chloride reagent: 324mg ferric chloride in 10ml of 50% orthophosphoric acid.

20% sulphuric acid

Preparation

Stock standard: Dissolved 100mg of urea in 100ml of distilled water. This solution is prepared in saturation solution of benzoic acid for long use. 1.0ml of this solution contains 1mg of urea.

Working standard: 2.0ml of stock standard was diluted to 100ml with distilled water. 1.0ml of their solution contains 20 µg of urea.

Urine Sample: 1.0ml of urine was made up to 100ml with distilled water. 1.5ml of diluted urine was taken for the experiment.

Procedure

1. Take 1.8ml of trichloroacetic acid and 0.2ml of blood centrifuge for 10 minutes. 0.5ml of supernatant was taken for experiment into a series of test tubes take 0.5, 1.0, 1.5, 2.0 and 2.5ml of working standard urea solution.

2. Corresponding to the values of 10, 20, 30, 40 and 50 µg respectively. The volume was made-up to 3.0ml with distilled water n all the test tubes.

3. Added 1.0ml of diacetylmonoxime, 1.0 m of thiosemicarbazide and 3.0ml of acid reagent and heated vigorously in boiling water for 20 minutes.

4. Along with this a blank was also conducted. Remove the tubes and cool. Reading was taken against the reagent blank at 540nm in a colorimeter to complete DAM method of urea estimation. A standard graph was drawn by plotting colorimetric reading on y axis and concentration of urea on x axis.

Normal Values - Blood – 19-30 mg/dl

Result

The amount of urea present in the given serum sample is —————- mg/dl

The amount of urea present in the given urine sample is ————— mg/dl

Further Readings

Reference