Spectrophotometric Determination of Uric Acid in Urine Based-Enzymatic Method Uricase with 4-Aminodiphenylamine Diazonium Sulfate (Variamine Blue RT Salt)

Abstract

A simple spectrophotometric method based-uricase enzyme for the detection of uric acid in normal urine and gout patient’s urine samples has been developed based on the reaction of hydrogen peroxide (H2O2 ) with yellow color of 4-Aminodiphenylamine Diazonium Sulfate (variamine blue RT salt) to yield a pale yellow-green coloured solution. The absorbance of products as the output of enzymatic reaction and variamine blue RT salt were detected using uv-visible spectrophotometer with maximum absorbance at 269 nm. The response time of this proposed method was 20 minutes. The optimum pH activity on enzymatic and variamine blue RT salt reactions was foud to be pH 9. The relative standard deviation (RSD) of the reproducibility of this method was very good at 0.7%. The calibration plot of different concentrations of uric acid was found to be between 0.5-13 mM with a detection limit of 0.58 mM. The kinetic parameters the Michaelis-Menten constant (KM ) and the maximum abosrbance (Absmax ) in the presence of different concentrations of uric acid were evaluated using the Linewaver-Burk and Hofstee plots respectively. The enzymatic-spectrophotometric method was compared with established clinical method and satisfactory agreement was achieved.