Lin Y Y, Cheng W B, Wright C E
NYS Institute for Basic Research in Developmental Disabilities, Staten Island.
Anal Biochem. 1993 Mar;209(2):267-73. doi: 10.1006/abio.1993.1118.
A method using 13C isotopes and GCMS/SIM was developed and was used to study glucose metabolism in several mammalian cell lines. Using [U-13C6]glucose, the 13C isotopomer distributions in glucose, lactic acid, glutamic acid, aspartic acid, and gamma-aminobutyric acid produced by the cells were determined, and glycolysis, gluconeogenesis, and the flux into the amino acid pools were calculated. A 1:1 mixture of [U-13C6]glucose and [1-13C]glucose was used to examine the loss of 1-13C carbon from [1-13C]glucose via the pentose monophosphate pathway in the cells; this new method measures the production of [3-13C]lactic acid and [U-13C3]lactic acid, and thus avoids most systematic errors that result from the endogenous production of 12C-lactic acid during metabolic conversion; in addition, glycolysis and the recycling of glucose and lactic acid can be examined at the same time. The mass isotopomer analysis method is reproducible and sensitive, and can be used to analyze metabolites in 10(-7) M concentration with less than 10(5) cultured human cells. This method was used to examine metabolic regulation of glucose in fibroblasts, lymphoblasts, and PC12 cells under various conditions of metabolic disturbance. The method appears to have wide applicability for the study of metabolic regulation of glucose and metabolic defects in cells.
开发了一种使用13C同位素和气相色谱 - 质谱联用/选择离子监测（GCMS/SIM）的方法，并用于研究几种哺乳动物细胞系中的葡萄糖代谢。使用[U - 13C6]葡萄糖，测定细胞产生的葡萄糖、乳酸、谷氨酸、天冬氨酸和γ-氨基丁酸中的13C同位素异构体分布，并计算糖酵解、糖异生以及进入氨基酸池的通量。使用[U - 13C6]葡萄糖和[1 - 13C]葡萄糖的1:1混合物来检测细胞中[1 - 13C]葡萄糖通过磷酸戊糖途径损失的1 - 13C碳；这种新方法测量[3 - 13C]乳酸和[U - 13C3]乳酸的产生，从而避免了代谢转化过程中内源性产生12C - 乳酸导致的大多数系统误差；此外，可同时检测糖酵解以及葡萄糖和乳酸的循环利用。质量同位素异构体分析方法具有可重复性和灵敏性，可用于分析浓度为10(-7) M且细胞数量少于10(5)个的培养人细胞中的代谢物。该方法用于检测在各种代谢紊乱条件下成纤维细胞、淋巴母细胞和PC12细胞中葡萄糖的代谢调节。该方法似乎在研究葡萄糖代谢调节和细胞代谢缺陷方面具有广泛的适用性。
