Quantitation of erythrocyte pentose pathway flux with [2-13C]glucose and 1H NMR analysis of the lactate methyl signal.
A simple and sensitive NMR method for quantifying excess (13)C-enrichment in positions 2 and 3 of lactate by (1)H NMR spectroscopy of the lactate methyl signal is described. The measurement requires neither signal calibrations nor the addition of a standard and accounts for natural abundance (13)C-contributions. As a demonstration, the measurement was applied to approximately 3 micromol of lactate generated by erythrocyte preparations incubated with [2-(13)C]glucose to determine the fraction of glucose metabolized by the pentose phosphate pathway (PP). PP fluxes were estimated from the ratio of excess (13)C-enrichment in lactate carbon 3 relative to carbon 2 in accordance with established metabolic models. Under baseline conditions, PP flux accounted for 7 +/- 2% of glucose consumption while in the presence of methylene blue, a classical activator of PP activity, its contribution increased to 27 +/- 10% of total glucose consumption (P < 0.01).
Delgado
Teresa C
TC
Center for Neuroscience and Cell Biology, Faculty of Sciences and Technology, University of Coimbra, Coimbra, Portugal.
Castro
M Margarida
MM
Geraldes
Carlos F
CF
Jones
John G
JG
D002247:Carbon Isotopes; D004912:Erythrocytes / Q000378:metabolism*; D005947:Glucose / Q000378:metabolism*; D006801:Humans; D019344:Lactic Acid / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D010427:Pentose Phosphate Pathway
D002247:Carbon Isotopes; D019344:Lactic Acid; D005947:Glucose