VISIBILITY OF ATP AND ADP IN FREEZE-TRAPPED TISSUE FROM PERFUSED-RAT-LIVER DURING NORMOXIA AND ISCHEMIA USING P-31-CRYO-NMR

The visibility of ATP and ADP to NMR was studied by comparing simultaneous measurements of freeze-trapped tissue sections from perfused rat liver under normoxia and ischemia using a modified P-31-cryo-NMR method and biochemical assay. The P-31-cryo-NMR method provides good time resolution and allows the quantitation of absolute metabolite concentrations. Prior to P-31-cryo-NMR measurements, freeze-trapped tissues were thawed in the presence of cryoprotectant and EDTA. With this sample preparation procedure, the integrity of the plasma and mitochondrial membranes was not maintained, inducing homogeneous microviscosity and chelation of intracellular divalent cations, thereby increasing the visibility of metabolites compared to the in vivo NMR measurement. With ischemic stress, total cellular ATP concentration decreased significantly (P < 0.001). While ADP concentrations measured by cryo-NMR and biochemical analysis were consistent during normoxia and ischemia, ATP concentrations measured by cryo-NMR were significantly lower (P < 0.05) than those obtained by biochemical analysis. The amount of invisible ATP (0.42 +/- 0.10-mu-mol/g wet weight: mean +/- S.E.) did not change after the induction of ischemia. The results of this study suggest that ATP invisibility to cryo-NMR is not due to compartmentation into regions of high paramagnetic ion concentrations or high microviscosity, but is influenced by other factors.