Method and reagents for the determination of nicotine adenine dinucleotide phosphate and of glutathione

Abstract

Abstract The level of nicotine adenine dinucleotide phosphate (NADP) or of glutathione in a given sample is determined spectrophotometrically to extremely low levels, the method employed in testing for either chemical being to add an excess of the other together with a dithiobisheterocyclic reagent chemical as represented, for example, by 2,2''-dithiodipyridine, 6,6''dithiodinicotinic acid, 6,6''-dithiobis-(isonicotinic acid) or 2,2''-dithiodipyrimidine. Enzyme catalysts to induce cycling of the glutathione and optionally of the NADP are also employed. In analyzing for either chemical the glutathione is converted from the reduced to the oxidized state by the reagent chemical as the latter is irreversibly converted to a product compound which normally possesses a characteristic ultraviolet spectrum. The rate at which said product compound is formed, or at which the reagent compound disappears, is observed spectrophotometrically and is a function of the concentration of the chemical under test. The oxidized glutathione is continuously converted to the reduced form by reaction with reduced NADP in the presence of glutathione reductase. When testing for NADP ( and optionally when testing for glutathione as well) the oxidized NADP formed on reacting with glutathione is continuously converted to the reduced form in the presence of added substrate and dehydrogenase.

Inventors

• 👤 Davide R Grassetti
• 👤 John F Murray Jr

Bibliographic Details