Glucose metabolism in mammalian cells as determined by mass isotopomer analysis

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.