Enzymatic Micmassay of Glucose, Uric Acid and Cholesterol Using 3-(p-Hydroxypheny) Propionic Acid as Fluomgenic Substmte for Horsemdish Peroxidase

Abstract

Highly sensitive fluorimetric methods for the enzymatic assays of glucose, uric acid and free and total cholesterol in biological samples are described.Glucose.uric acid and cholesterol are oxidized by glucose oxidase-, uricase-and cholesterol oxidase-catalyzed reactions to give hydrogen peroxide, respectlvely, which is measured by horseradish peroxidase-mediated reaction with a highly sensitive fluorogenic substrate 3-(p-hydroxyphenyl) propionic acid.Cholesterol ester is hydrolyzed in the presence of cholesterol ester hydrolase.The methods are readily performed with human serum of 1μl or less for glucose, 2μl for uric acid and 1μl or less for total cholesterol and with rat liver of 0.5mg (wet weight) for free cholesterol.The lower limits of determination for glucose, uric acid, total cholesteroi in human serum and free cholesterol in rat liver are 50ng (275 pmol), 60 ng (354 pmol), 110 ng (286 pmo-) and 0.6μg (1.55 nmol), respectively.