Evaluation of carbon flux and substrate selection through alternate pathways involving the citric acid cycle of the heart by 13C NMR spectroscopy.
Evaluation of carbon flux and substrate selection through alternate pathways involving the citric acid cycle of the heart by 13C NMR spectroscopy.
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DOI：
10.1016/s0021-9258(18)68590-4
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发表时间：
1988-05
期刊：
影响因子：
--
通讯作者：
Craig R. MalloyS;A. D. Sherrysll;Mark;Jeffrey
中科院分区：
文献类型：
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作者：
Craig R. MalloyS;A. D. Sherrysll;Mark;Jeffrey
先前的13 C NMR技术（Malloy，C. R.，雪莉，AD，和杰弗里，F.M.H. 04 The Dog（1987）212，58-62）用于测量分子通过涉及大鼠心脏的柠檬酸循环的氧化途径与回补途径的相对通量，已经扩展到包括整个谷氨酸13 C谱的完整分析。虽然在实践中仍然简单，这个更复杂的模型允许13 C分数富集的分子进入氧化和回补途径在稳态条件下的评价。该方法用于分析在利用13 C富集的丙酮酸盐、丙酸盐、乙酸盐或其各种组合期间完整心脏或其酸提取物的13 C NMR光谱。[2- 13 C]丙酮酸被用来证明丙酮酸通过丙酮酸羧化酶的稳态流量在丙酮酸和乙酸的共灌注期间是显著的，并且我们首次证明了在完整的跳动的心脏中可以检测到九线13 C多重峰。乙酸或丙酮酸单独提供约86%的乙酰辅酶A;组合时，约65%的乙酰辅酶A来源于乙酸，约30%来源于丙酮酸，其余来源于内源性来源。丙酸盐减少了外源性乙酸对乙酰辅酶A的贡献至77%，并且还减少了内源性底物的氧化。方程，允许这种相同的分析多标记的底物，使这种技术非常强大的评价底物的选择和相对代谢通量通过回补和氧化途径在完整的心脏。
A previous 13C NMR technique (Malloy, C. R., Sherry, A.D., and Jeffrey, F.M.H. (1987) FEBS Lett. 212, 58-62) for measuring the relative flux of molecules through the oxidative versus anaplerotic pathways involving the citric acid cycle of the rat heart has been extended to include a complete analysis of the entire glutamate 13C spectrum. Although still simple in practice, this more sophisticated model allows an evaluation of 13C fractional enrichment of molecules entering both the oxidative and anaplerotic pathways under steady-state conditions. The method was used to analyze 13C NMR spectra of intact hearts or their acid extracts during utilization of 13C-enriched pyruvate, propionate, acetate, or various combinations thereof. [2-13C]Pyruvate was used to prove that steady-state flux of pyruvate through pyruvate carboxylase is significant during co-perfusion of pyruvate and acetate, and we demonstrate for the first time that a nine-line 13C multiplet may be detected in an intact, beating heart. Acetate or pyruvate alone provided about 86% of the acetyl-CoA; in combination, about 65% of the acetyl-CoA was derived from acetate, about 30% was derived from pyruvate, and the remainder from endogenous sources. Propionate reduced the contribution of exogenous acetate to acetyl-CoA to 77% and also reduced the oxidation of endogenous substrates. Equations are presented which allow this same analysis on multiply labeled substrates, making this technique extremely powerful for the evaluation of substrate selection and relative metabolic flux through anaplerotic and oxidative pathways in the intact heart.