Estimation of urea in blood by dicetyl method

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BACKGROUND

In protein metabolism the end product is urea. It is synthesized from ammonia in liver. Through blood it is transferred to kidneys finally from where it is excreted out. In the blood the urea level increases when there is any renal disease, urinary blockages or CHD. In case of pregnancy and liver failure urea level decreases. Through dicetyl method it is very easy to estimate the amount of urea in the blood. In this method, with the help of an dicetyl agent urea is first condensed and then the resulting product is subjected to oxidation with phosphoric nitric acid mixture that gives out yellow colour. So, the basic objective of this experiment is to calculate the amount of urea in the blood by dicetyl method.

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REQUIREMENTS

Reagents:       10% zinc sulphate solution

            Isotonic sodium sulphate solution-30 gm Na₂SO₄+1litre water

Isotonic diluent: 43 ml of 10% ZnSO₄ is mixed with isotonic sodium sulphate to make it total 1 litre.

NaOH (0.5N): Mix 20 gm NaOH in dist. water and make final solution of 1 litre with water

Stock solution of Diacetyl reagent: Dissolve 2.5 gm diacetyl monoxime with small quantity of 5% acetic acid and then make it to 100 ml with same.

Working solution of Diacetyl reagent: 100 ml of stock solution of Diacetyl reagent +working area solution +75 ml DW.

Phosphoric-nitric acid: 300 ml of Syrupy phosphoric acid + 200 ml dist. water + 5 ml concentration nitric acid.

Stock solution of Standard urea (1 mg/ml): 100 mg of dried urea in small quantity of distilled water and then make it to 100 ml with distilled water.

Working urea solution (50 mcg/ml): Take 5 ml of urea stock solution and make it to 100 ml with distill water

Apparatus:    Centrifuge tube

   Centrifuge machine

   Water bath

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PROCEDURE

In a centrifuge tube take 4.6 ml of isotonic diluent. Then to this tube add about 0.2 ml of (0.5 N NaOH solution). Mix this properly and then centrifuge it. For further test take out the supernatant. Now take three test tubes and label it as Blank, test and standard.¹ And on each test tube add as follows:

• Blank: 2 ml water + 2 ml working dicetyl reagent +1 ml phosphoric nitric acid
  
• Test: 2 ml supernatant + 2 ml working dicetyl reagent + 1 ml phosphoric nitric acid
  
• Standard: 2 ml working urea standard + 2 ml working dicetyl reagent + 1 ml phosphoric nitric acid
  

For about 30 mins keep the tube in water bath for properly mixing the ingredients and then cool it down in tap water. Then in colorimeter detect the absorbance at 578 nm.²

Standard values of urea= 5-20 mg/dl

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CONCLUSION

This is a method in which urea can be directly determined without depending on any urease enzyme action

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REFERENCES

1. Marsh, W. H., Fingerhut, B., and Kirsch, E., Determination of urea nitrogen with the diacetyl method and an automatic dialyzing apparatus. Am. J. Clin. Pathol. 28, 681 (1957).
   
2. Fearon, W. R., The carbainido diacetyl reaction; A test for citrulhine. Biochein. J. 33,902 (1939).