Fructose-1,6-bisphosphate does not preserve ATP in hypoxic–ischemic neonatal cerebrocortical slices

Abstract

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 T 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 (350 μm) were treated with 2 mM FBP before and during 30 min of HI, and then given 4 h of recovery with an FBP-free oxygenated superfusate. Slices were removed before HI, at the end of HI, and at 1 and 4 h 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.