Single Extraction Method for the Spectrophotometric Quantification of Oxidized and Reduced Pyridine Nucleotides in Erythrocytes

A simplified and reliable assay for the determination of erythrocyte pyridine 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 pyridine nucleotides are inappropriate for RBC, difficult to use, or inaccurate. The method described within this paper provides for both improved reliability and ease of use. In addition, we have documented that significant pools of NADPH and NADH are tightly bound to proteins (e.g., catalase) and not detectable by many of the assay systems previously used. This results in a significant change in not only total RBC pyridine nucleotide content but also in the ratio of reduced to nonreduced nucleotide.