Measurement of Glutamine 13C5 Enrichment

Blood samples were processed to measure ¹³C₅ enrichment in glutamine by gas chromatography-mass spectrometry (GC-MS), as previously described [10 (link)]. A 3-point standard curve was prepared by mixing unenriched glutamine with ¹³C₅ glutamine such that 0%, 50%, or 100% of glutamine was ¹³C labeled. GC-MS was performed using an Agilent 6890N Gas Chromatograph coupled to an Agilent 5973 Mass Selective Detector (Agilent Technologies, Santa Clara, CA). One microliter of each standard or sample was injected and analyzed in scan mode. Fragment ions of m/z 258 (unenriched) and 263 (enriched) ¹³C₅ glutamine were quantified for both standard and experimental samples. Linear regression was used to calculate the enrichment of each plasma sample.