Metabolic profiling by 13C-NMR spectroscopy: [1,2-13C2]glucose reveals a heterogeneous metabolism in human leukemia T cells.
Metabolic profiling is defined as the simultaneous assessment of substrate fluxes within and among the different pathways of metabolite synthesis and energy production under various physiological conditions. The use of stable-isotope tracers and the analysis of the distribution of labeled carbons in various intermediates, by both mass spectrometry and NMR spectroscopy, allow the role of several metabolic processes in cell growth and death to be defined. In the present paper we describe the metabolic profiling of Jurkat cells by isotopomer analysis using (13)C-NMR spectroscopy and [1,2-(13)C(2)]glucose as the stable-isotope tracer. The isotopomer analysis of the lactate, alanine, glutamate, proline, serine, glycine, malate and ribose-5-phosphate moiety of nucleotides has allowed original integrated information regarding the pentose phosphate pathway, TCA cycle, and amino acid metabolism in proliferating human leukemia T cells to be obtained. In particular, the contribution of the glucose-6-phosphate dehydrogenase and transketolase activities to phosphoribosyl-pyrophosphate synthesis was evaluated directly by the determination of isotopomers of the [1'-(13)C], [4',5'-(13)C(2)]ribosyl moiety of nucleotides. Furthermore, the relative contribution of the glycolysis and pentose cycle to lactate production was estimated via analysis of lactate isotopomers. Interestingly, pyruvate carboxylase and pyruvate dehydrogenase flux ratios measured by glutamate isotopomers and the production of isotopomers of several metabolites showed that the metabolic processes described could not take place simultaneously in the same macrocompartments (cells). Results revealed a heterogeneous metabolism in an asynchronous cell population that may be interpreted on the basis of different metabolic phenotypes of subpopulations in relation to different cell cycle phases.
Miccheli
A
A
Department of Chemistry, University of Rome La Sapienza, P.le Aldo Moro 5, 00185 Rome, Italy.
Tomassini
A
A
Puccetti
C
C
Valerio
M
M
Peluso
G
G
Tuccillo
F
F
Calvani
M
M
Manetti
C
C
Conti
F
F
D002247:Carbon Isotopes; D002453:Cell Cycle / Q000502:physiology; D049109:Cell Proliferation; D002952:Citric Acid Cycle / Q000502:physiology; D005947:Glucose / Q000378:metabolism*; D005973:Glutamine / Q000378:metabolism; D005998:Glycine / Q000378:metabolism; D006801:Humans; D019169:Jurkat Cells; D019344:Lactic Acid / Q000378:metabolism; D015458:Leukemia, T-Cell / Q000378:metabolism / Q000473:pathology / Q000503:physiopathology; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D008954:Models, Biological; D009711:Nucleotides / Q000378:metabolism; D010427:Pentose Phosphate Pathway / Q000502:physiology; D010429:Pentoses / Q000378:metabolism; D012694:Serine / Q000378:metabolism
D002247:Carbon Isotopes; D009711:Nucleotides; D010429:Pentoses; D005973:Glutamine; D019344:Lactic Acid; D012694:Serine; D005947:Glucose; D005998:Glycine