Rapid determination of creatine, phosphocreatine, purine bases and nucleotides (ATP, ADP, AMP, GTP, GDP) in heart biopsies by gradient ion-pair reversed-phase liquid chromatography

Abstract

A simple binary solvent method has been developed for the simultaneous determination of creatine (Cr), phosphocreatine (PCr), ATP, ADP, AMP, GTP, GDP, IMP, NAD, inosine, adenosine, hypoxanthine and xanthine. This allows separation of the most important nucleotides present in myocardial biopsies as, for example, in studies using 31P NMR spectroscopy. In NMR spectra ATP and PCr are the only visible high-energy phosphates, therefore the status of other nucleotides and bases cannot be determined. The nucleotides, AMP degradation products, PCr and Cr in pig and rat heart muscle were resolved with 35 m M K 2HPO 4, 6 m M tetrabutyl-ammonium hydrogensulfate buffer, pH 6.0, and a binary acetonitrile gradient on medium-bore, 250 mm or 125 mm × 3.9–4.6 mm I.D. steel octadecyl-bonded (C 18) columns at a flow-rate of 1.5 or 1.0 ml/min. This method, optimized for use with older high-performance liquid chromatography pumps (100 μl displacement heads), resolves the major porcine and rat myocardial nucleotides and degradation products within 22 min. The amounts found in normoxic porcine muscle are: Cr 9.21 ± 0.75; hypoxanthine 1.40 ± 0.14; PCr 7.20 ± 1.2; IMP 1.34 ± 0.13; βNAD 1.82 ± 0.23; AMP 0.10 ± 0.04; GDP 0.05 ± 0.02; ADP 1.23 ± 0.09; GTP 0.19 ± 0.01; ATP 4.45 ± 0.32 μmol/g wet weight. The method, incorporating adenosine tetraphosphate as an internal standard, allows the documentation of changes in both the high-energy phosphates and their degradation products in a single analysis of myocardial samples as small as 200 μg (wet weight).