Fructose-16-Bisphospate does not preserve ATP in hypoxic-ischemic neonatal cerebrocortical slices

摘要

Fructose-1,6-bisphosphate (FBP), an endogenous intracellular metabolite in glycolysis, was found in many preclinical studies to be neuroprotective during hypoxia-ischemia (HI) when administered exogenously. We looked for HI neuroprotection from FBP in a neonatal rat brain slice model, using 14.1 Tesla ¹H /³¹P/¹³C NMR spectroscopy of perchloric acid slice extracts to ask: 1) if FBP preserves high energy phosphates during HI; and 2) if exogenous [1-¹³C]FBP enters cells and is glycolytically metabolized to [3-¹³C]lactate. We also asked: 3) if substantial superoxide production occurs during and after HI, thinking such might be treatable by exogenous FBP's antioxidant effects. Superfused P7 rat cerebrocortical slices (350μm) were treated with 2 mM FBP before and during 30 min of HI, and then given four hours of recovery with an FBP-free oxygenated superfusate. Slices were removed before HI, at the end of HI, and at 1 and 4 hours after HI. FBP did not improve high energy phosphate levels or change ¹H metabolite profiles. Large increases in [3-¹³C]lactate were seen with ¹³C NMR, but the lactate fractional enrichment was always (1.1±0.5)%, implying that all of lactate's ¹³C was natural abundance ¹³C, that none was from metabolism of ¹³C-FBP. FBP had no effect on the fluorescence of ethidium produced from superoxide oxidation of hydroethidine. Compared to control slices, ethidium fluorescence was 25% higher during HI and 50% higher at the end of recovery. Exogenous FBP did not provide protection or enter glycolysis. Its use as an antioxidant might be worth studying at higher FBP concentrations.