EXTRACTION AND ASSAY OF CREATINE-PHOSPHATE, PURINE, AND PYRIDINE-NUCLEOTIDES IN CARDIAC TISSUE BY REVERSED-PHASE HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY

The levels of creatine phosphate, purine, and pyridine nucleotides in tissues provide important information on energetic and oxidative cellu lar states. Nevertheless, technical, theoretical, and methodological d ifficulties in extraction and quantification procedures have so far li mited our understanding of the exact role that these substances play i n metabolic processes which take place in cells. The objective of our study was to find an easy and rapid method for extracting, separating, and quantifying creatine phosphate, purine, and pyridine nucleotides in solid tissues. We adapted the classic acid-extraction procedure wit h HClO4 for purine and oxidized pyridine nucleotides and then develope d a new alkaline extraction with phenol in a phosphate buffer solution (pH 7.8) for reduced pyridine nucleotides. Biopsies of myocardial tis sue were frozen and ground at -180 degrees C using the appropriate ext raction procedure. The separation and quantification of the metabolite s were performed using a reversed-phase 3-mu m Supelchem C18 column, w ith the addition of tetrabutylammonium as an ion-pair agent to the buf fer solution, by ultraviolet detection. The recovery of the external a nd internal standards always exceeded 90%, The autooxidation or interc onversion processes were almost insignificant for each reduced form. T his technique allowed us to avoid complex enzymatic procedures and dif ficulties in the selective assay of pyridine nucleotides with chemilum inescence and surface spectroscopy. (C) 1991 Academic Press, Inc.