Quantitation of PEMT and Kennedy Pathways in Liver Phosphatidylcholine Biosynthesis Using NMR

Phosphatidylcholine (PtdCho) is a major phospholipid that regulates diverse cellular processes, including lipid metabolism, signal transduction, and cell proliferation. PtdCho is synthesized through two distinct pathways: the Kennedy pathway and the phosphatidylethanolamine N-methyltransferase (PEMT) pathway. Because a quantitative comparison of these pathways is critical for understanding lipid homeostasis, several analytical approaches have been developed to assess their relative activities in biological samples. However, most are limited by their reliance on radiotracers and their low sensitivity. Here, we developed a modified high-sensitivity heteronuclear multiple-bond correlation (HMBC) NMR pulse sequence that distinguishes PtdCho species synthesized via the two pathways within a single experiment using U-13C-choline and U-13C-ethanolamine. By optimizing band-selective polarization transfer and implementing relaxation-based correction, we accurately quantified the ratio of 12C- and 13C-labeled N-methyl carbons, enabling a precise estimation of pathway contributions. Application of this method to murine primary hepatocytes, nontransformed human hepatocyte cell lines (HL-7702 and NKNT-3), and the human hepatoma cell line (Hep3B) revealed species-specific differences: murine hepatocytes exhibited nearly equivalent activities of the PEMT and Kennedy pathways, whereas human liver-derived cells relied predominantly on the Kennedy route. PEMT overexpression in Hep3B cells significantly boosted the PEMT contribution without affecting proliferation, which might suggest species-specific metabolic regulation rather than a conserved tumor-suppressive role. This HMBC-based strategy allows simultaneous and quantitative assessment of PtdCho biosynthetic fluxes without radiotracers, providing a robust platform for elucidating lipid metabolic reprogramming in liver physiology and cancer.