Galactose metabolism in normal human lymphoblasts studied by (1)H, (13)C and (31)P NMR spectroscopy of extracts.
The development of tools to follow and quantitate the fate of galactose in mammalian cells is crucial to the study and understanding of the inherited disorders of galactose metabolism. In this study we incubated normal human lymphoblasts with 1- or 2-(13)C galactose for 2.5 or 5 h and prepared TCA extracts of the cells. The various galactose metabolites were identified and quantified using a combination of proton, carbon and phosphorus NMR spectra. Galactose-1-phosphate (gal-1P), uridine diphosphogalactose, uridine diphosphoglucose and galactitol were present in the extracts. Average levels for gal-1P were around 10 nmol/mg protein and for uridine diphosphoglucose, uridine diphosphogalactose and galactitol in the range of 0.5-2 nmol/mg protein. Galactonate was never found in any conditions. Percentage labeling could be estimated for gal-1P and for the ribose carbons of AMP. The labeling agrees with a conversion of galactose to glucose through the Leloir pathway.
Wehrli
S L
SL
NMR Core Facility, Joseph Stokes Jr Research Institute, The Children's Hospital of Philadelphia, 34th Street and Civic Center Boulevard, Philadelphia, PA 19104, USA. wehrli@email.chop.edu
Reynolds
R
R
Chen
J
J
Yager
C
C
Segal
S
S
D002247:Carbon Isotopes; D002457:Cell Extracts / Q000737:chemistry; D002478:Cells, Cultured; D004376:Galactitol / Q000032:analysis / Q000378:metabolism; D005690:Galactose / Q000032:analysis / Q000378:metabolism*; D005695:Galactosephosphates / Q000032:analysis / Q000378:metabolism; D006801:Humans; D006859:Hydrogen; D008214:Lymphocytes / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D010759:Phosphorus Isotopes; D014531:Uridine Diphosphate Galactose / Q000032:analysis / Q000378:metabolism; D014532:Uridine Diphosphate Glucose / Q000032:analysis / Q000378:metabolism
D002247:Carbon Isotopes; D002457:Cell Extracts; D005695:Galactosephosphates; D010759:Phosphorus Isotopes; D004376:Galactitol; C029973:galactose-1-phosphate; D014531:Uridine Diphosphate Galactose; D006859:Hydrogen; D014532:Uridine Diphosphate Glucose; D005690:Galactose