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The incorporation of 13C from [1-13C]glucose and [2-13C]acetate into selected intermediary metabolites in extracts prepared from incubated cerebral-cortex slices was monitored by using 13C-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 13C 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 13C-n.m.r. spectrum only in severe hypoxia. This was unambiguously assigned to glycerol 3-phosphate from a combination of 31P- and 13C-n.m.r. spectroscopy. The percentage 13C-enrichment was calculated from the 13C-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.

biochemical journal

Glycerol 3-phosphate and lactate as indicators of the cerebral cytoplasmic redox state in severe and mild hypoxia respectively: a 13C- and 31P-n.m.r. study