Diacetyl Monoxime (DAM) Method for Estimation of Urea

Urea is a waste product formed in liver following the breakdown of proteins. It passes into the blood, is filtered out of the kidneys and excreted in urine. Thus determination of blood urea is the most widely used screening test for the evaluation of kidney function. There are various methods used for the estimation of urea in a laboratory. Diacetyl monoxime (DAM) method is an older method.

Principle

Proteins are first precipitated by trichloroacetic acid. The urea present in the protein-free filtrate reacts with diacetyl monoxime in a hot acidic medium in presence of ferric/cadmium ions and thiosemicarbazide to form pink or red colored complex- diazine. The intensity of the color developed is measured photometrically at 530nm, which is directly proportional to the concentration of the urea present in the fluid.

Requirements

• Apparatus:
  Colorimeter
  Conical flasks and test tubes to hold 20ml
  Pipettes: 50ul, 0.1ml, 0.5ml, 5 ml
  Measuring cylinder, 50 ml
  Water bath at 100°C
• Reagents:
  Benzoic acid
  Ferric Chloride
  Diacetyl monoxime
  orthophosphoric acid
  Thiosemicarbazide
  Tricholoroacetic acid
  Urea
• Specimen:
  Serum, heparinized plasma or fluoride plasma.

Preparation of Regaents

1. Reagent 1: Trichloroacetic acid, 50g/l (5%) solution
   Trichloroacetic acid = 10g
   Distilled water = upto 200ml
2. Reagent 2: Diacetyl monoxime (2,3-butanedione monoxime) solution
   Diacetyl Monoxime = 2g
   Distilled water = upto 500ml
3. Reagent 3: Acid reagent
   Concentrated sulfuric acid = 44ml
   Orthophosphoric acid (H3PO4), 85% = 66ml
   Cadmium sulfate = 1.6 g
   Thiosemicarbazide = 50mg
   Distilled water = upto 500ml
4. Reagent 4: Colour reagent
   Acid reagent (Reagent 3) = 50ml
   Diacetyl monoxime reagent = 50ml
5. Reagent 5: Benzoic acid solution 1 g/l
   Benzoic acid = 1 g
   Distilled Water = 1000ml.
6. Reagent 6: Urea stock reference solution, 125mmol/l
   Urea = 750mg
   Benzoic acid, 1 g/l (0.1%) solution = upto 100ml
7. Reagent 7: Urea working reference solution, 10mmol/l
   Urea stock reference solution = 8ml
   Benzoic acid (C7H6O2), 1 g/l (0.1%) solution = upto 100ml

Preparation of Sample

To obtain protein free filtrate, take 50 ul of whole blood/serum/plasma in a centrifuge tube. Add 1 ml of TCA solution and mix. Centrifuge at high speed (3000 g) for 5 minutes to sediment the precipitated proteins and obtain a clear supenatent fluid. Do same for standard/control sample.

Procedure

1. Take three (or more if needed) large test-tubes and label as follows:
   Blank tube (B)
   Standard tube (S)
   Test tube (T)
2. Pipette into each tube as follows:
   

   	Test	Standard	Blank
   Color Reagent (Reagent 4)	3 ml	3 ml	3 ml
   Protein Free Filtrate	0.1 ml	–	–
   Urea Standard 10 mmol/l	–	0.1 ml	–
   Distilled water	–	–	0.1 ml

3. Mix the contents of each tube. Place all the tubes in the water-bath at 100°C for exactly 15 minutes to allow the red color to develop.
4. Remove the tubes and allow them to cool in a beaker of cold water for 5 minutes.
5. Measure the colour produced in a colorimeter at a wavelength of 530nm.

Calculations

Calculate the concentration of urea in the blood specimen using the following formula:

References

1. World Health Organization, 2003. Manual of basic techniques for a health laboratory. World Health Organization.
2. World Health Organization, 1986. Methods recommended for essential clinical chemical and haematological tests for intermediate hospital laboratories/Working Group on Assessment of Clinical Technologies. In Methods recommended for essential clinical chemical and haematological tests for intermediate hospital laboratories/Working Group on Assessment of Clinical Technologies.
3. Godkar, P.B. and Godkar, D.P., 2003. Textbook of medical laboratory technology. Bhalani.
4. Mukherjee, K.L., 2013. Medical Laboratory Technology Volume 3 (Vol. 3). Tata McGraw-Hill Education.