Quantitative Palmitic Acid Synthesis Assay

DNL was measured as previously described (49 (link)). Total palmitic acid labeling assay in the liver was assayed by GC–MS. Briefly, 20 mg liver tissue was homogenized in 1 ml KOH/EtOH (EtOH 75%) and incubated at 85 °C for 3 h, and 200 μl of 1 mM [¹³C₁₆]palmitate was added to samples as IS after cool down. Extracted palmitate acid was mixed with 50 μl N-t-butyldimethylsilyl-N-methyltrifluoroacetamide (TBDMS) at 70 °C for 30 min, and the TBDMS-derivatized samples were analyzed with an Agilent 5973N-MSD equipped with an Agilent 6890 GC system, and a DB-17MS capillary column (30 m × 0.25 mm × 0.25 μm). The mass spectrometer was operated in the electron impact mode (70 eV). The temperature program was as follows: 100 °C initial, increase by 15 °C/min to 295 °C, and hold for 8 min. The sample was injected at a split ratio of 10:1 with a helium flow of 1 ml/min. Palmitate–TBDMS derivative eluted at 9.7 min, and the m/z at 313, 314, and 319 were extracted for M0, M1, and M16 palmitate quantification.
Stable isotope labeling was corrected for natural isotope abundance (96 (link)). Newly synthesized palmitic acid was calculated as: percent of newly synthesized palmitic acid labeling = total palmitic acid labeling/(plasma ²H₂O labeling × 22) × 100.