2.4. 13C NMR analysis for metabolite quantitation and 13C enrichment

The relative concentration of each metabolite was calculated using a singlet from the metabolite normalized by the signal of DSS. The singlet from natural ¹³C abundance reflects the pool size of a metabolite. Excess ¹³C enrichment in metabolites was calculated using a doublet, the signal due to ¹³C–¹³C spin-spin coupling, based on an assumption that a singlet was natural abundance (1.1%). In addition, the relative levels of ¹³C-labeled metabolites in ¹³C NMR spectra were calculated using the peak areas of specified doublets normalized by the peak area of DSS. Glucose concentration in plasma was measured using glucose oxidase method (YSI 2300 Glucose Analyzer; GMI, Inc).