Determination of the reduced and oxidized pyridine nucleotides in animal tissues

Abstract

A fluorimetric method for the estimation of oxidized and reduced di- and tri-phosphopyridine nucleotide in animal tissues is described. Acid and alkaline extractions of the tissues are used for the oxidized and reduced coenzymes respectively. After extraction, the 4 species of coenzymes are separated by treatment with specific enzymes and selective destruction of oxidized and reduced forms with acid and alkali. The fluorescence of the separated coenzymes is developed by heating with 6 [image] NaOH. The method can be used for the estimation of pyridine nucleotides in concentrations as low as 1.5 /[mu]M, and quantities as small as 0.1 x 10-9 mole have been determined. Conditions affecting the accuracy and specificity of the method have been investigated. Values for the concentrations of the coenzymes in various tissues immediately after death are presented. The suitability of a number of tissue preparations for use in metabolic experiments involving incubation and the subsequent estimation of the coenzymes has been investigated. There is a rapid destruction of coenzymes on incubation of homogenates and minced or chopped tissues. Pyruvate and lactate usually decreased the rate of destruction and in some experiments maintained a high level of reduced coenzymes.