SERINE ISOTOPOMER ANALYSIS BY C-13-NMR DEFINES GLYCINE-SERINE INTERCONVERSION IN-SITU IN THE RENAL PROXIMAL TUBULE

[2-C-13]glycine metabolism was studied in freshly isolated rat renal p roximal tubules. Mitochondrial coupling of the glycine cleavage comple x (GC) and serine hydroxymethyltransferase (SHMT) was confirmed by the formation of three serine isotopomers, [2-C-13]-, [3-C-13]- and [2,3- C-13]serine, detected by C-13-NMR. Incubation with different fractions of C-13-labelled glycine altered the labelling pattern of the serine isotopomers predictably and allowed calculation of the C-13-labelled f ractions of total glycine and methylene in N-5,N-10-methylenetetrahydr ofolate (m-THF) available for serine metabolism. Within 20 min there w as a fall in labelled glycine (to 42 +/- 3, 68 +/- 3 and 93 +/- 2%, (n = 4, mean +/- S.D.) from 50%, 75% and 100% C-13-labelled added glycin e respectively), followed by a slow rate of endogenous glycine formati on for up to 80 min incubation. The C2 of glycine was the source of mo re than 90% of the methylene group of m-THF formed. Gas chromatography -mass spectroscopy (GC-MS) showed that greater than 50% of serine form ed was unlabelled. GC and SHMT proceeded in the direction of serine fo rmation. Serine isotopomer analysis by NMR and GC-MS allowed the actio ns of GC and SHMT and de novo contributions to glycine, serine and m-T HF to be monitored in situ in fresh renal proximal tubules.