SINGLE EXTRACTION METHOD FOR THE SPECTROPHOTOMETRIC QUANTIFICATION OFOXIDIZED AND REDUCED PYRIDINE-NUCLEOTIDES IN ERYTHROCYTES

A simplified and reliable assay for the determination of erythrocyte p yridine nucleotide (NAD and NADP) concentrations, as well as the ratio of the reduced [NADH/NADPH] to oxidized [NAD(+)/NADP(+)] nucleotide, is important in understanding both normal and abnormal red blood cells (RBC). However, previously published methods for quantitating pyridin e nucleotides are inappropriate for RBC, difficult to use, or inaccura te. The method described within this paper provides for both improved reliability and ease of use. In addition, we have documented that sign ificant pools of NADPH and NADH are tightly bound to proteins (e.g., c atalase) and not detectable by many of the assay systems previously us ed. This results in a significant change in not only total RBC pyridin e nucleotide content but also in the ratio of reduced to nonreduced nu cleotide. (C) 1994 Academic Press,Inc.