Biochemistry Clinical practice Lecturer of Biochemistry Serum biochemical parameters urea assay Biochemistry Clinical practice CLS 432 Dr. Samah Kotb Lecturer of Biochemistry 2015

principle Serum urea concentration was assayed by using commercial kit, supplied by Biodiagnostics (Egypt), (Fawcett and Scott, 1960). Enzymatic determination of urea according to the following reaction:

principle Urease Urea + H2O 2NH3 + CO2 In an alkaline medium, the ammonium ions react with the phenol and hypochlorite to form a green colored indophenol which absorbs light at 550 nm proportional to initial urea concentration.

Buffer- Enzyme reagent Reagents Reagent 1 Urea Standard 50 mg/dl Reagent 2 Buffer- Enzyme reagent Phosphate buffer Urease > 5000 U/l 50 mmol/l >10000U/l Reagent 3   Color reagent Phenol Sodium -nitroprusside 100 mmol/l 0.2 mmol/l Reagent 4 Alkaline Reagent Sodium hydroxide Sodium hypochlorite 150 mmol/l 15 mmol/l

Procedure Blank Standard Sample ---------- 10 µl Reagent 2 0.2 ml   Blank Standard Sample ---------- 10 µl Reagent 2 0.2 ml All tubes were mixed and incubated for 5 minutes at 37 oC Reagent 3 1.0 ml Reagent 4

All test tubes were mixed and incubated for 10 minutes at 37oC All test tubes were mixed and incubated for 10 minutes at 37oC . The absorbance of the sample and standard were measured against reagent blank at 550 nm.

Calculation: The concentration of serum urea (mg/dl) = = (A sample )/(A standard )x 50 (standard conc.)

Serum urea normally vary depending on Age ( due to change kidney function ) Sex ( conc. are slightly higher in men ) Diet ( protein diet Urea )