Metabolism of 13C galactose by lymphoblasts from patients with galactosemia determined by NMR spectroscopy

Abstract

In order to assess the pathways by which galactose is metabolized by galactose-1-phosphate uridyltransferase (GALT) deficient cells, lymphoblasts from 10 galactosemic patients with defined genotypes (six Q188R homozygotes, two S153L homozygotes, and two with homozygous deletions) were incubated with 1 mM 1- or 2- 13C galactose for 2.5 and 5 h. The 13C-labeled metabolites were identified and quantified using nuclear magnetic resonance and the results were compared to that obtained with cells from eight normal individuals. Cells from galactosemic patients formed two to three times the galactose-1-phosphate (Gal-1P) in normal cells, no difference being observed between the various genotypes. Galactitol formation was not significantly different from normal cells. No labeled galactonate was detected. Cells with the Q188R and S135L mutations formed both labeled uridine diphosphogalactose (UDPgal) and uridine diphosphoglucose (UDPglu), but to a lesser extent than normals, whereas cells with the GALT deletion did not. The pattern of 13C enrichment of the ribose carbons of adenosine monophosphate upon incubation of the normal cells with 1- 13C galactose paralleled that found for incubations with 1- 13C glucose, which is consistent with galactose disposition through the Leloir pathway to glucose and its subsequent metabolism to ribose. Cells with the GALT deletion formed no detectable labeled ribose, whereas cells from a patient homozygous for Q188R mutation formed labeled ribose in a pattern similar to normal albeit with lower enrichment. The results suggest that there is residual GALT activity and function of the Leloir pathway in the presence of the Q188R as well as S135L mutation.