31P NMR and enzymatic analysis of cytosolic phosphocreatine, ATP, Pi and intracellular pH in the isolated working perfused rat heart

Hearts from fed male Wistar rats (200–350 g) were perfused at low and high workloads with Pi‐free Krebs‐Henseleit medium containing either 10 mM glucose or 10 mM glucose plus 15mU/mL insulin. The intracellular pH by 31P NMR ranged between 6.99 and 7.02 and agreed to within 0.1 pH unit of estimates calculated using enzymatically determined total tissue HCO3−/CO2 contents. At high work, where the tissue contents of phosphocreatine (PCr) and ATP were determined on the same heart as NMR areas (n = 16), the proportionality factors, defined as the 31P NMR area units divided by the total enzymatically determined tissue content (area units/μmol/g dry wt), were 112±8 for PCr, 99±4 for γ‐ATP, 138±9 for α‐ATP and 100±4 for β‐ATP. These values were normalized by taking β‐ATP as 100 area units/μmol/g dry wt. Since the proportionality factor for PCr and γ‐ and β‐ATP were not statistically different (p < 0.05), it was concluded that each was equally visible by 31P NMR and that no significant breakdown of PCr occurred during freezing or tissue acid extraction procedures. The cytosolic Pi estimated from NMR in glucose plus insulin perfused hearts at low and high work was 4.92±0.67 and 6.33±0.42 μmol/g dry wt. Using the near‐equilibrium expression of KCK/KG+G and the metabolite levels in heart extracts, the calculated cytosolic Pi was 13.08±1.83 and 16.17±3.08 μmol/g dry wt, respectively. The cytosolic NMR Pi in the glucose hearts was 8.42±1.0 and 8.42±0.75 μmol/g dry wt at low and high work and 12.08±1.58 and 27.20±4.20±μmol/g dry wt from near‐equilibrium estimates. The total tissue Pi measured enzymatically on freeze‐clamped hearts ranged from 18.0 to 26.42 μmol/g dry wt. The validity of using both the 31P NMR and the near‐equilibrium method for estimating cytosolic Pi in the heart was discussed.