Exogenous ethylpyruvate vs pyruvate during metabolic recovery after oxidative stress in neonatal rat cerebrocortical slices

Exogenous ethylpyruvate (EP) is known to provide better outcomes in ischemia and oxidative stress than equimolar doses of exogenous pyruvate (pyr). In solution pyr spontaneously forms parapyruvate, a very potent metabolic inhibitor. Exogenous EP's superiority has been attributed to the absence of parapyruvate, better radical scavenging, and improved boosting of TCA Cycle metabolism via pyr delivery after hydrolysis of the ester linkage. Recent in vivo studies found a wide neuroprotection window for EP in cerebral ischemic injury and H2O2 challenges. We wanted to learn the relative importance of EP's additional radical scavenging compared to its metabolic assistance via provision of pyr as a TCA substrate. Oxygenated ex vivo neonatal rat cerebrocortical slices, 350μ thick from P7 Sprague-Dawley rats, were acutely exposed for 1 hr to a 2 mM H2O2 stress, and then recovered during superfusion with oxygenated artificial ACSF preparations having 2 mM glc and one of three unique ACSF components: a) 20 mM exogenous EP; b) 20 mM exogenous pyr; and, c) 1 mM of the non-metabolizable radical scavenger N-tertbutyl-α-phenylnitrone (PBN). Each study was repeated three times along with control runs. Slices were taken and frozen at t=0h (just before the H2O2 stress), and again at t=5h (four hrs after the H2O2 stress.) PCA extracts for the two time points were studied with 14.1 Tesla 31P/1H NMR spectroscopy. In a special study 13C-EP was administered, with the label being at the methyl group (C3) of the pyruvate moiety. 2D[1H-13C] HSQC NMR spectra were acquired to look for intracellular metabolism of EP's pyr, and also for a proposed radical scavenging pathway that has EP being turned into formate and acetate. Acute cell injury was assessed by counting TUNEL stained cells. RESULTS: For each rescue solution 1H NMR spectroscopy provided accurate changes for the following metabolites: lactate, alanine, NAA, GABA, glutamate, glutamine, succinate, PCr/Cr, taurine, and myoinositol. NAA and alanine, measured at t=5h for the EP group were significantly greater than values for the no treatment group. EP of 20 mM was more effective than PBN or pyr, achieving higher levels of Cr/PCr and NAA, the neuron marker. 31P data showed that, compared to other rescue regimens, ethylpyruvate also provided the best ATP preservation, but levels were higher by only 12%-29%, although ADP/ATP ratios were lowered by 30%-45% (p < 0.05). Overlaid HSQC spectra for 13C-pyr and 13C-(ethylpyruvate) showed the same TCA metabolites being generated in both groups. The acetate/formate pathway was not seen. TUNEL positive cell percentage in the ethylpyruvate group was significantly less than half of that in the PBN or pyruvate rescue groups (p<0.05), and less than a third of that in the "no treatment" group. CONCLUSION: Ethylpyruvate easily entered cells and provided pyr as a TCA substrate. It was more protective than pure pyruvate or a pure radical scavenger, and thus appears to work via both mechanisms: substrate addition and antioxidant augmentation. Supported by NIH grant R01 GM034767.