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ESTIMATION OF SERUM UREA

INTRODUCTION:

Urea is the end product of amino acid metabolism in our body. It helps in the
detoxification of ammonia in our body. It is synthesized in liver cells from ammonia
and CO2.

Methods of estimation:

1. Non - Enzymatic method : Diacetyl Monoxime method.

2. Enzymatic method : Nessler’s method, Berthelot method, Glutamate
dehydrogenase method (GLDH).

AIM:

To estimate the amount of urea in the given sample by Diacetyl Monoxime Method
(DAM).

PRINCIPLE:

Proteins present in the serum are precipitated by tungstic acid. In the presence of strong
acid medium Diacetyl monoxime is broken down into Diacetyl and hydroxylamine. Urea
present in the protein free filtrate reacts with diacetyl in acid medium in the presence of
thiosemicarbazide to give pink colour complex. The intensity of the colour is directly
proportional to the concentration of urea in the sample at 540 nm.

REAGENTS:

1. 10% tungstic acid (TCA)

2. 2% Diacetyl monoxime solution.
3. 40% thiosemicarbazide.
 4. Acid reagent ( FeCl3 in orthophosphoric acid and sulphuric acid)
5. Urea standard-1 mg/dL.

PROCEDURE:
Step 1: Preparation of PFF:
In a centrifuge tube, take 3.4mL of DW, 0.1mL of serum, 1.5 mL of 10% TCA.
Mix after each addition and allow to stand for 10 mins. Centrifuge at 2000 rpm
for 5 mins. Use the supernatant as PFF.
Step 2: Take 3 test tubes, name it as Blank (B), Standard (S), and Test (T). add the
following:

Reagents Blank Standard Test

PFF - - 1 ml

DW 1 ml - -

Working standard - 1 ml -

Diacetyl monoxime 1 ml 1 ml 1 ml

Acid reagent 3 ml 3 ml 3 ml

thiosemicarbazide 1 ml 1 ml 1 ml

Total 6 ml 6 ml 6 ml

Mix well and keep in waterbath for 15 mins and cool the tubes. Read at 540 nm
wavelength.
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Data collection and calculation:

O.D of test (T): ……………………
O.D of standard (S): ………………
Conc of standard: 0.01 mg

CALCULATION:
O.D of Test - O.D of Blank X Conc of Std X 100
Urea concentration =
O.D of Std – O.D of Blank Vol of sample
(mg/dL)
O.D of Test X 0.01 X 100

O.D of Std 0.02

= O.D of Test X 50

O.D of Std

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RESULTS:

The concentration of urea in the given sample is ………….. mg/dL

Normal range
Blood Urea = 15 – 40 mg/dL
Blood Urea Nitrogen = 6 – 20 mg/dL

Clinical Interpretation