A C-NMR study on the influxes into the tricarboxylic acid cycle of a renal epithelial cell line, LLC-PK1/Cl4: the metabolism of [2-C]glycine, l-[3-C]alanine and l-[3-C]aspartic acid in renal epithelial cells

Perchloric acid extracts of LLC-PK₁/Cl₄ cells, a renal epithelial cell line, incubated with either [2-¹³C]glycine l-[3-¹³C]alanine, or d,l-[3-¹³C]aspartic acid were investigated by ¹³C-NMR spectroscopy. All amino acids, except labelled glycine, gave rise to glycolytic products and tricarboxylic acid cycle (TCA) intermediates. For the first time we also observed activity of γ-glutamyltransferase activity and glutathione synthetase activity in LLC-PK₁ cells, as is evident from enrichment of reduced glutathione. Time courseS showed that only 6% of the labelled glycine was utilized in 30 min, whereas 31% of l-alanine and 60% of l-aspartic acid was utilized during the same period. ¹³C-NMR was also shown to be a useful tool for the determination of amino acid uptake in LLC-PK₁ cells. These uptake experiments indicated that glycine alanine and aspartic acid are transported into Cl₄ cells via a sodium-dependent process. From the relative enrichment of the glutamate carbons, we calculated the activity of pyruvate dehydrogenase to be about 61% of when labelled l-alanine was the only carbon source for LLC-PK₁/Cl₄ cells. Experiments with labelled d,l-aspartic, however, showed that about 40% of C-3-enriched oxaloacetate (arising from a de-amination of aspartic acid) reached the pyruvate pool.