Comte B, Vincent G, Bouchard B, Des Rosiers C
Department of Nutrition, University of Montréal, Montréal, Québec H3C 3J7, Canada.
J Biol Chem. 1997 Oct 17;272(42):26117-24. doi: 10.1074/jbc.272.42.26117.
We present a strategy for simultaneous assessment of the relative contributions of anaplerotic pyruvate carboxylation, pyruvate decarboxylation, and fatty acid oxidation to citrate formation in the perfused rat heart. This requires perfusing with a mix of 13C-substrates and determining the 13C labeling pattern of a single metabolite, citrate, by gas chromatography-mass spectrometry. The mass isotopomer distributions of the oxaloacetate and acetyl moieties of citrate allow calculation of the flux ratios: (pyruvate carboxylation)/(pyruvate decarboxylation), (pyruvate carboxylation)/(citrate synthesis), (pyruvate decarboxylation)/(citrate synthesis) (pyruvate carboxylation)/(fatty acid oxidation), and (pyruvate decarboxylation)/(fatty acid oxidation). Calculations, based on precursor-product relationship, are independent of pool size. The utility of our method was demonstrated for hearts perfused under normoxia with [U-13C3](lactate + pyruvate) and [1-13C]octanoate under steady-state conditions. Under these conditions, effluent and tissue citrate were similarly enriched in all 13C mass isotopomers. The use of effluent citrate instead of tissue citrate allows probing substrate fluxes through the various reactions non-invasively in the intact heart. The methodology should also be applicable to hearts perfused with other 13C-substrates, such as 1-13C-labeled long chain fatty acid, and under various conditions, provided that assumptions on which equations are developed are valid.
我们提出了一种策略，用于同时评估回补性丙酮酸羧化、丙酮酸脱羧和脂肪酸氧化对灌注大鼠心脏中柠檬酸生成的相对贡献。这需要用13C标记的底物混合物进行灌注，并通过气相色谱-质谱法测定单一代谢物柠檬酸的13C标记模式。柠檬酸中草酰乙酸和乙酰部分的质量同位素异构体分布可用于计算通量比：（丙酮酸羧化）/（丙酮酸脱羧）、（丙酮酸羧化）/（柠檬酸合成）、（丙酮酸脱羧）/（柠檬酸合成）、（丙酮酸羧化）/（脂肪酸氧化）以及（丙酮酸脱羧）/（脂肪酸氧化）。基于前体-产物关系的计算与池大小无关。我们的方法在常氧条件下用[U-13C3]（乳酸+丙酮酸）和[1-13C]辛酸对心脏进行稳态灌注时得到了验证。在这些条件下，流出液和组织中的柠檬酸在所有13C质量同位素异构体中都有相似程度的富集。使用流出液中的柠檬酸而非组织中的柠檬酸，能够在完整心脏中以非侵入性方式探测底物通过各种反应的通量。该方法学也应适用于用其他13C标记的底物（如1-13C标记的长链脂肪酸）灌注的心脏，以及在各种条件下，前提是所建立方程的假设是有效的。
