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[2-(13)C]glycine metabolism was studied in freshly isolated rat renal proximal tubules. Mitochondrial coupling of the glycine cleavage complex (GC) and serine hydroxymethyltransferase (SHMT) was confirmed by the formation of three serine isotopomers, [2-(13)C]-, [3-(13)C]- and [2,3-(13)C]serine, detected by 13C-NMR. Incubation with different fractions of 13C-labelled glycine altered the labelling pattern of the serine isotopomers predictably and allowed calculation of the 13C-labelled fractions of total glycine and methylene in N5,N10-methylenetetrahydrofolate (m-THF) available for serine metabolism. Within 20 min there was a fall in labelled glycine (to 42 +/- 3, 68 +/- 3 and 93 +/- 2%, (n = 4, mean +/- S.D.) from 50%, 75% and 100% 13C-labelled added glycine respectively), followed by a slow rate of endogenous glycine formation for up to 80 min incubation. The C2 of glycine was the source of more than 90% of the methylene group of m-THF formed. Gas chromatography-mass spectroscopy (GC-MS) showed that greater than 50% of serine formed was unlabelled. GC and SHMT proceeded in the direction of serine formation. Serine isotopomer analysis by NMR and GC-MS allowed the actions of GC and SHMT and de novo contributions to glycine, serine and m-THF to be monitored in situ in fresh renal proximal tubules.

Glycine-serine interconversion is important to numerous metabolic processes and serine release by the kidney. Incubation of freshly isolated rat renal proximal tubules with 5 mM glycine 75% 13C-labelled in the 2-position resulted in 13C-labelled incorporation into serine of 69 micromol.g protein(-1) (+/- 14, n = 16) at 20 min. Addition of 5 mM glucose, 4 mM lactate, 1 mM alanine, 1 mM butyrate and 1 mM glutamate increased 13C-label incorporation into serine to 173 micromol.g protein(-1) (+/- 32, n = 4) at 60 min, 50% greater than tubules incubated with 5 mM glycine alone (P < 0.05). The increase was prevented by hypoxia. Reoxygenation for 20 min restored the rate of incorporation of 13C-label into serine. The fraction of unlabelled serine remained approximately 47% at 20, 40 and 60 min in each group. The results indicate that in the presence of oxygen, TCA and glycolytic intermediates stimulate serine synthesis via the glycine cleavage complex and serine hydroxymethyltransferase pathways and not the phosphorylated pathway. In addition, significant serine production occurs from an unidentified source, which is also tightly coupled to glycine metabolism. Both in the presence and absence of added TCA and glycolytic intermediates, glycine was the principle source of the methylene group in methylene tetrahydrofolate.