Purine nucleotides and phospholipids in ischemic and reperfused rat skeletal muscle: effect of ascorbate.

The effect of intravenously administered ascorbate on the ischemic and reperfused rat skeletal muscle was investigated. Purine nucleotides and phospholipids in skeletal muscle from rats subjected to 4 h of ischemia followed by 1-h reperfusion were analyzed by high-performance liquid chromatography. In addition, ATP, phosphocreatine (PCr), Pi, and phosphomonoesters (PME) were analyzed by 31P-nuclear magnetic resonance at 202.4 MHz, and individual PME such as glucose-6-phosphate and IMP were quantified. PCr and ATP were exhausted after 4 h of ischemia and recovered poorly upon reperfusion in the soleus and tibialis muscle of untreated rats. Postischemic reperfusion resulted in significant loss of cardiolipin. Treatment with 55 mM ascorbate resulted in total restoration of PCr during reperfusion, and ATP recovered to 42% of control in the soleus. Recovery was improved in the tibialis as well, and the cardiolipin decrease was limited. A lower ascorbate concentration (5 mM) did not enhance postischemic recovery. Our findings show that a high dose of ascorbate improves the energetic state of rat skeletal muscle during postischemic reperfusion, probably due to its antioxidant function.

Phosphorus metabolites in different muscles of the rat leg by 31P image-selected in vivo spectroscopy.

The difference in concentration of phosphorylated metabolites in muscles with different fiber composition was studied in vivo by localized 31P nuclear magnetic resonance spectroscopy in the rat hindlimb 120-160 microliters volumes were selected in regions containing the soleus and gastrocnemius muscles. Concentrations of phosphocreatine (PCr), adenosine triphosphate and inorganic phosphate (Pi) were determined and intracellular pH was calculated in the respective muscle groups. The highest level of PCr was found in the gastrocnemius muscle, containing 30.7 mmoles/dm3 tissue compared to 22.3 mmoles/dm3 in the soleus muscle. Pi was significantly lower in gastrocnemius (1.9 mmoles/dm3) than in soleus (3.2 mmoles/dm3). The ATP concentration was 6.7 and 6.4 mmoles/dm3 and pH was determined to 7.11 and 7.09 in the gastrocnemius and soleus muscle, respectively. Our NMR data show that it is possible to measure high-energy phosphates with precision in small localized volumes with the ISIS method using a Helmholtz coil. Earlier biochemical data are confirmed by these in vivo NMR results. Localized in vivo 31P NMR spectroscopy can contribute to the understanding of the underlying mechanisms of several metabolic events in different regions of the tissue. The method can be used for future studies of varying ischemia tolerance, varying degrees of adaptation to exercise with regard to oxidative capacity, and pH compartmentation in muscles with different fiber composition.

In vivo 31P nuclear magnetic resonance evidence of the salvage effect of ascorbate on the postischemic reperfused rat skeletal muscle.

The effect of 32 mM ascorbate on the time courses of phosphocreatine (PCr), inorganic phosphate (Pi), adenosine triphosphate (ATP) and intracellular pH in rat skeletal muscle during ischemia and reperfusion was investigated in vivo using 31P nuclear magnetic resonance (NMR) spectroscopy. Ascorbate was administered intravenously prior to induction of ischemia and at the time of reperfusion. The changes in PCr/(PCr+Pi), ATP and pH were similar in the non-treated and in the treated groups during ischemia. PCr/(PCr+Pi) fell to < 10% and ATP to approximately 30% of the preischemic values after 4 hours of arrested circulation, and pH decreased considerably. Postischemic reperfusion was followed continuously for 150 minutes. At the time of reflow, treatment with ascorbate had an immediate, positive effect on the recovery of high energy phosphates and pH. The level of PCr/(PCr+Pi) was 86% higher (p < 0.001) and the ATP level was 40% higher (p < 0.001) in the treated group than in the control group by the end of the reperfusion period. The results provide in vivo evidence for a salvaging effect of ascorbate on ischemia-reperfusion injury in skeletal muscle, probably owing to its antioxidant function and other ancillary effects, mainly its provision of additional buffer capacity.

Purine metabolic pathways in rat hindlimb perfusion model during ischemia and reperfusion.

The perfused rat hindlimb preparation was used with a blood cell-free perfusate to investigate alterations in the purine nucleotide metabolism, flow rate, perfusion pressure, and venous excretion in response to ischemia and ischemia followed by reperfusion in skeletal muscle. The development of a physical hindrance during postischemic reperfusion, indicated by an increase in reperfusion pressure and a decrease in flow rate, coincided with a 90% decrease in phosphocreatine and a 50-70% reduction in total adenine nucleotide pool. The reflow impairment could not be explained by blood cell plugging of the capillaries. Washout of several metabolites was demonstrated during reperfusion. Hypoxanthine accumulated intracellularly during ischemia, and a substantial amount of uric acid was excreted into the venous effluent during reperfusion. The experimental data were fitted into a computer simulation model of the purine pathways. The model indicated that AMP deaminase was the predominant enzymatic pathway for the AMP degradation. It was demonstrated that ATP preferably accumulated as inosine-5'-monophosphate during ischemia and that xanthine oxidase was undetectable in skeletal muscle tissue homogenates. However, vascular endothelial cell xanthine oxidase activity responsible for a free radical-induced reperfusion injury could not be excluded.