Evaluation of a procedure for the simultaneous determination of oxidized and reduced pyridine nucleotides and adenylates in organic phenol extracts from mitochondria.

An extraction procedure using mixtures of phenol, chloroform, and isoamyl alcohol originally applied to quench mitochondria for determining adenylates proved suitable also for the quantification of reduced and oxidized pyridine nucleotides yielding recoveries of more than 90%. In combination with HPLC, this approach allows the simultaneous determination of NAD+, NADP+, NADH, and NADPH as well as of adenylates within one extract. A comparison of this extraction method with fluorimetric measurements of pyridine nucleotide reduction in intact mitochondria revealed that about 30% of the fluorescence signal in the resting state of liver mitochondria is caused by NADPH.