A SIMPLE ULTRAMICRO METHOD FOR DETERMINATION OF PYRIDINE NUCLEOTIDES IN TISSUES

Several methods are available at present for the quantitative estimation of tissue pyridine nucleotide levels. Those that are dependent on the measurement of the absorption of the reduced coenzyme at 340 mμ (1) or on the conversion of the oxidized nucleotide to a fluorescent derivative (2) are capable of detecting reliably concentrations of not less than 5 × 10-6 M or 1 × 10-8 M, respectively. Methods which utilize enzyme cycling (3, 4) can detect concentrations as low as 1 × 10-9 M. In general, however, these latter methods utilize expensive photofluorometric equipment or require several enzymes as reagents which are not usually available commercially in the purity necessary for the determination. A colorimetric method for the determination of the nucleotide coenzymes has been described by Villee (5) and by Slater and Sawyer (6). This method utilizes enzyme cycling with dichlorophenolindophenol as the terminal electron acceptor, requires titration of the sample with dichlorophenolindophenol reagent before analysis in order to correct for some unknown substance (s) that reacts with this dye, and can detect amounts of coenzyme down to 0.5 × 10-9 mole per cuvet. The present paper submits a method which is relatively simple to perform, yet is precise and sensitive, and is capable for determining reduced or oxidized NAD+ or NADP+ in amounts as low as 1 × 10-12 mole per cuvet. © 1969.