TEMPERATURE-DEPENDENCE OF NMR RELAXATION-TIMES OF NUCLEOSIDE TRIPHOSPHATES AND INORGANIC-PHOSPHATE IN THE ISOLATED-PERFUSED RAT-LIVER - EFFECT ON PI COMPARTMENTATION

The effect of temperature on P-31 NMR spectra from isolated perfused r at livers was studied at 9.4 T. Relaxation times (T-1 and T-2) of nucl eoside triphosphates (NTP) and inorganic phosphate (Pi) were determine d at 37, 25, 15, and 4 degrees C. Under hypothermic conditions, an une xpected apparent line sharpening in the Pi spectral region and a clear emergence of an additional Pi resonance were observed. This additiona l signal was assigned to mitochondrial Pi. T-1 values obtained for cyt osolic and mitochondrial Pi at 4 degrees C were 1.14 +/- 0.24 s (n = 5 ) and 0.71 +/- 0.18 s (n = 5), respectively. No significant mitochondr ial contribution to the Pi resonance was observed at 37 degrees C. Qua ntification of Pi and NTP liver contents at 37 and 4 degrees C was per formed by comparing the perfused liver spectrum and the corresponding perchloric acid extract spectrum. Under experimental conditions of low external Pi (0.12 mM), it was concluded that intracellular Pi was com pletely NMR-visible at 4 and 37 degrees C. The observation of the mito chondrial Pi signal at 4 degrees C was well explained by an increase i n the Pi level within the matrix, in response to the mitochondrial swe lling induced by hypothermia, as observed by electron microscopy. T-2 values for the cytosolic Pi at 37 and 4 degrees C were 17 +/- 4 ms (n = 8) and 22 +/- 4 ms (n = 10), respectively. Comparison with measured linewidths indicated that line broadening for the main phosphorylated metabolites-including matrix Pi-was the result of Bo field inhomogenei ty. The additional broadening of the cytosolic Pi resonance at 4 and 3 7 degrees C was attributed to pH heterogeneity within the liver. (C) 1 996 Academic Press, Inc.