Changes in hepatocyte NADH fluorescence during prolonged hypoxia

Abstract

Deprivation of oxygen reduces oxidative phosphorylation and rapidly causes an increase in cellular NADH which can be monitored by fluorimetry. Previous studies have established that increases in NADH fluorescence accurately reflect the impairment in oxidative phosphorylation which occurs during brief periods of acute hypoxia. However, the potential usefulness of fluorimetry for following longer, clinically relevant periods of ischemia has not been explored. We studied changes in NADH fluorescence in rat hepatocyte suspensions and in isolated-buffer-perfused rat livers during hypoxia (pO2 < 50 mm Hg) for periods as long as 180 min. NADH fluorescence of hepatocyte suspensions consistently increased by about 15% after 13 to 15 min of hypoxia and coincided with a marked decrease in tissue ATP levels. Reoxygenation after 15 or 30 min of hypoxia resulted in recovery of ATP and NADH with minimal loss of viability, as measured by trypan blue exclusion. After 60 to 180 min hypoxia, the initial increase in NADH fluorescence was followed by a progressive, irreversible decline which correlated with decreased cell viability. Similar changes in NADH fluorescence were observed in isolated-perfused rat livers in which NADH fluorescence was monitored at the liver surface with a fiberoptic probe. Hypoxia for 30 min had no effect on NADH fluorescence, but hypoxia for longer periods caused a steady increase in fluorescence after 45-60 min. When hypoxia was prolonged (120 or 180 min), fluorescence peaked after 60-75 min and then declined progressively to levels below baseline. The greatest decrease in fluorescence was seen after 180 min of hypoxia.(ABSTRACT TRUNCATED AT 250 WORDS)