31P exchange rates through the creatine kinase-catalyzed interconversion of phosphocreatine and γ-ATP were measured in a total of 27 miniature swine ranging in age from 5 days preterm to 5 weeks old. A steep increase in the forward rate constant for 31P exchange from phosphocreatine (PCr) to γ-ATP was observed between 2 days preterm and 3 days postterm, with a more gradual increase for older ages. In contrast, the [PCr]/[NTP] ratio measured by in vivo 31P nuclear magnetic resonance (NMR) remained constant throughout this age interval and close to unity. Forward and reverse rate constants and the rate of flux for 31P exchange were equal to each other for both preterm and 5-week-old animals, suggesting that the creatine kinase reaction is near-equilibrium for this span of age. Multifrequency steady-state saturation of Pi and PCr compared to single-frequency saturation of PCr produced the same extent of saturation transfer to γ-ATP, and the saturation of Pi alone had no effect on the γ-ATP 31P NMR signal. These results suggest that even for immature swine brain, creatine kinase activity should be adequate to buffer against changes in [ATP] when there is a mismatch between energy supply and energy demand, during conditions such as ischemia or hypoxia. The results from the present study indicate the unlikelihood that previously reported discrepancies between forward and reverse 32P flux rates in rat brain (Shoubridge et al., FEBS Lett 140:288–292, 1982) were due to neglect of γ-ATP to Pi exchange. If the contribution of nonadenosine triphosphate to the in vivo rat brain 31P NMR signal is accounted for in the calculation of reverse flux and a literature value for rat brain [PCr] is used in the calculation of forward flux, then forward and reverse flux rates are equal.
Efficient 31
P band inversion transfer approach for measuring creatine kinase activity, ATP synthesis, and molecular dynamics in the human brain at 7 T
