Tran-Dinh S, Hoerter J A, Mateo P, Gyppaz F, Herve M
Département de Biologie Cellulaire et Moléculaire, CEN Saclay, Gif-Sur-Yvette, France.
Biochimie. 1998 Dec;80(12):1013-24. doi: 10.1016/s0300-9084(99)80006-6.
A new mathematical model, based on the observation of 13C-NMR spectra of two principal metabolites (glutamate and aspartate), was constructed to determine the citric acid cycle flux in the case of high aspartate transaminase activity leading to the formation of large amounts of labeled aspartate and glutamate. In this model, the labeling of glutamate and aspartate carbons by chemical and isotopic exchange with the citric acid cycle are considered to be interdependent. With [U-13C]Glc or [1,2-(13)C]acetate as a substrate, all glutamate and aspartate carbons can be labeled. The isotopic transformations of 32 glutamate isotopomers into 16 aspartate isotopomers or vice versa were studied using matrix operations; the results were compiled in two matrices. We showed how the flux constants of the citric acid cycle and the 13C-enrichment of acetyl-CoA can be deduced from 13C-NMR spectra of glutamate and/or aspartate. The citric acid cycle flux in beating Wistar rat hearts, aerobically perfused with [U-13C]glucose in the absence of insulin, was investigated by 13C-NMR spectroscopy. Surprisingly, aspartate instead of glutamate was found to be the most abundantly-labeled metabolite, indicating that aspartate transaminase (which catalyses the reversible reaction: (glutamate + oxaloacetate <--> 2-oxoglutarate + aspartate) is highly active in the absence of insulin. The amount of aspartate was about two times larger than glutamate. The quantities of glutamate (G0) or aspartate (A0) were approximately the same for all hearts and remained constant during perfusion: G0 = (0.74 +/- 0.03) micromol/g; A0 = (1.49 +/- 0.05) micromol/g. The flux constants, i.e., the fraction of glutamate and aspartate in exchange with the citric acid cycle, were about 1.45 min(-1) and 0.72 min(-1), respectively; the flux of this cycle is about (1.07 +/- 0.02) micromol min(-1) g(-1). Excellent agreement between the computed and experimental data was obtained, showing that: i) in the absence of insulin, only 41% of acetyl-CoA is formed from glucose while the rest is derived from endogenous substrates; and ii) the exchange between aspartate and oxaloacetate or between glutamate and 2-oxoglutarate is fast in comparison with the biological transformation of intermediate compounds by the citric acid cycle.
基于对两种主要代谢物（谷氨酸和天冬氨酸）的13C-NMR光谱的观察，构建了一个新的数学模型，以确定在天冬氨酸转氨酶活性高导致大量标记的天冬氨酸和谷氨酸形成的情况下柠檬酸循环通量。在该模型中，通过与柠檬酸循环的化学和同位素交换对谷氨酸和天冬氨酸碳的标记被认为是相互依赖的。以[U-13C]葡萄糖或[1,2-(13)C]乙酸盐为底物时，所有谷氨酸和天冬氨酸的碳都可以被标记。使用矩阵运算研究了32种谷氨酸异构体向16种天冬氨酸异构体的同位素转化，反之亦然；结果被汇编在两个矩阵中。我们展示了如何从谷氨酸和/或天冬氨酸的13C-NMR光谱中推导出柠檬酸循环的通量常数和乙酰辅酶A的13C富集。通过13C-NMR光谱研究了在无胰岛素的情况下用[U-13C]葡萄糖进行有氧灌注的Wistar大鼠跳动心脏中的柠檬酸循环通量。令人惊讶的是，发现天冬氨酸而不是谷氨酸是标记最丰富的代谢物，这表明天冬氨酸转氨酶（催化可逆反应：（谷氨酸+草酰乙酸<-->2-氧代戊二酸+天冬氨酸）在无胰岛素的情况下具有高活性。天冬氨酸的量约为谷氨酸的两倍。所有心脏中谷氨酸（G0）或天冬氨酸（A0）的量大致相同，并且在灌注过程中保持恒定：G0 =（0.74±0.03）微摩尔/克；A0 =（1.49±0.05）微摩尔/克。通量常数，即与柠檬酸循环交换的谷氨酸和天冬氨酸的比例，分别约为1.45分钟-1和0.72分钟-1；该循环的通量约为（1.07±0.02）微摩尔·分钟-1·克-1。计算数据与实验数据之间取得了极好的一致性，表明：i）在无胰岛素的情况下，只有41%的乙酰辅酶A由葡萄糖形成，其余来自内源性底物；ii）与柠檬酸循环对中间化合物的生物转化相比，天冬氨酸与草酰乙酸之间或谷氨酸与2-氧代戊二酸之间的交换很快。
