DETERMINATION OF FREE CREATINE AND PHOSPHOCREATINE CONCENTRATIONS IN THE ISOLATED PERFUSED RAT-HEART BY H-1-NMR AND P-31-NMR

To measure free creatine in the isolated perfused rat heart, the concentration of phosphocreatine, and phosphocreatine plus creatine (SIGMA-Cr) were measured by P-31- and H-1-NMR, respectively. Quantification was performed in the presence and absence of an intraventricular balloon filled with a known amount of PCr, which acted as an external standard. Total (free plus bound) phosphocreatine and creatine were measured by HPLC analysis of extracts from the same hearts, freeze-clamped at the end of the perfusions. A greater concentration of creatine (mu-mol/g dry wt.) in the perfused rat heart was measured by HPLC analysis (40.3 +/- 2.38 (11)) as compared to NMR (34.6 +/- 1.95 (11)), whilst no significant difference was observed in the measurement of phosphocreatine between the two assay methods. Consequently, a greater SIGMA-Cr was measured by HPLC. This work suggests that the majority of Cr in the heart is NMR visible and unbound, so available to interact with creatine kinase. The lower free ADP concentration calculated from NMR measurements (53.3 +/- 3.80-mu-M (9)) was not significantly different from that determined by HPLC analysis (56.9 +/- 5.90-mu-M (9)). This suggests that the concentration of free ADP in the heart is higher than values where it can regulate oxidative phosphorylation most effectively.