Specific Enzymatic Determination of Glucose in Blood and Urine Using Glucose Oxidase

Abstract

Adequate methods for the determination of glucose in biologic fluids based upon its reducing ability are well established. These methods, however, lack specificity since they will detect reducing substances other than glucose. Improved specificity has resulted from procedures designed to remove interfering reducing substances. These measures have been applied most successfully to blood and plasma. The procedure for the precipitation of proteins introduced by Somogyi eliminates the great majority of interfering reducing substances likely to be present in blood, such as proteins, amino acids, glutathione, phosphorylated hexoses, and others. This, however, does not make it possible to distinguish glucose from other hexoses (such as fructose or galactose) or from pentoses and from such substances as glyceraldehyde. For fluids such as urine where many of the interfering reducing substances cannot be removed by available technics the need for a specific determination is obvious. Fermentation, formation of osazones, and chromatography have proved their usefulness, but being rather cumbersome and difficult to quantitate, have not found access to routine laboratories. Muller in 1928 and Coulthard and co-workers in 1942 isolated an enzyme from Aspergillus nlger and Penicillum notatum which had the unique property of specifically catalyzing the conversion of beta-glucose to gluconic acid. This enzyme, which was given the name of glucose oxidase, has been studied extensively by Bentley and Neuberger5 and by Keilin and Hartree. Glucose oxidase was shown to be a flavoprotcin with a molecular weight of 152,000 and an alloxazine-adenine dinucleotide as a prosthetic group.