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.

Diacetyl-monoxime-DAM-urea

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:
DAM-Calculation

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.


About Dhurba Giri 27 Articles
Dhurba Giri is the developer and contributor for LaboratoryTests.org. He is a Medical Laboratory Technologist and Scientific Blogger from Pokhara, Nepal. Connect with him:

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