GLYCEROL 3-PHOSPHATE AND LACTATE AS INDICATORS OF THE CEREBRAL CYTOPLASMIC REDOX STATE IN SEVERE AND MILD HYPOXIA RESPECTIVELY - A C-13-NMR AND P-31-NMR STUDY

The incorporation of C-13 from [1-C-13]glucose and [2-C-13]acetate into selected intermediary metabolites in extracts prepared from incubated cerebral-cortex slices was monitored by using C-13-n.m.r. spectroscopy under conditions of mild and severe hypoxia. Mild hypoxia had little effect on labelling of tricarboxylic-acid-cycle-related amino acids [glutamate, glutamine and gamma-aminobutyrate (GABA)], although the pool sizes of glutamate and glutamine decreased. There were large increases in the labelling of lactate and of alanine, and an increase in the pool size of lactate. In severe hypoxia, the resonances of lactate and alanine remained high, whereas those of the other intermediates decreased greatly. The pool size of GABA increased. Calculation of percentage C-13 enrichments and total label incorporated showed that lactate was not further affected by severe hypoxia, but the total label in alanine and its pool size were further increased. A new resonance appeared in the phosphomonoester region of the C-13-n.m.r. spectrum only in severe hypoxia. This was unambiguously assigned to glycerol 3-phosphate from a combination of P-31 and C-13-n.m.r. spectroscopy. The percentage C-13-enrichment was calculated from the C-13-n.m.r. spectrum, and the total label incorporated was measured by g.l.c./m.s. The results are discussed in terms of the ability of lactate dehydrogenase to maintain normal levels of NADH in mild hypoxia, but not in severe hypoxia. The pyruvate which accumulates under the latter condition is channelled into alanine, and the increased NADH is reflected by the increase in glycerol 3-phosphate. We conclude that glycerol 3-phosphate and alanine may provide novel means of monitoring severe hypoxia, whereas lactate is a reliable indicator only of mild hypoxia.