Serine isotopmer analysis by 13C-NMR defines glycine-serine interconversion in situ in the renal proximal tubule.
[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.
Cowin
G J
GJ
Department of Medicine, University of Queensland, Royal Brisbane Hospital, Australia.
Willgoss
D A
DA
Bartley
J
J
Endre
Z H
ZH
D000818:Animals; D008401:Gas Chromatography-Mass Spectrometry; D005998:Glycine / Q000378:metabolism* / Q000494:pharmacology; D012696:Glycine Hydroxymethyltransferase / Q000378:metabolism; D007553:Isotope Labeling; D007687:Kidney Tubules, Proximal / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D008297:Male; D051381:Rats; D017208:Rats, Wistar; D012694:Serine / Q000032:analysis / Q000096:biosynthesis*; D013763:Tetrahydrofolates / Q000378:metabolism
D013763:Tetrahydrofolates; C013123:5,10-methylenetetrahydrofolic acid; D012694:Serine; D012696:Glycine Hydroxymethyltransferase; D005998:Glycine