Urea nitrogen

Urea is synthesised in the liver from ammonia produced as a result of deamination of amino acids and is the chief means of excreting nitrogen from the animal. It is customary in most laboratories to express urea as urea nitrogen. This came about from the desire to compare the quantity of nitrogen excreted in the form of urea with other nitrogenous compounds. Hence, the standardisation of the method is in units of urea nitrogen. Since the molecular mass of urea is 60 Da and it contains two nitrogen atoms, with a combined mass of 28 Da, an urea nitrogen value can be converted to urea by multiplying by 60/28 or 2.14.

Method principle

Urea is hydrolysed to ammonium carbonate by urease, and the ammonia that is released from the carbonate by alkali reacts with phenol and sodium hypochlorite in an alkaline medium to form a blue indophenol. Sodium nitroprusside serves as a catalyst. The intensity of the blue colour is proportional to the quantity of urea in the specimen.

Reagents

Phenol nitroprusside solution

Dissolve 5 g phenol and 25 mg sodium nitroprusside in 500 ml double-distilled (d-d) water. Store at 4 °C. Stable for two months.

Alkaline hypochlorite

Dissolve 2.5 g NaOH in 250 ml d-d water in 500 ml volumetric flask. Cool, add 0.21 g sodium hypochlorite. Mix well and make up to 500 ml with d-d water. Store in an amber bottle at 4 °C. Stable for 2 months.