Quantitation of PEMT and Kennedy Pathways in Liver Phosphatidylcholine Biosynthesis Using NMR.
5 affiliations
- 1Natural Products Research Institute, College of Pharmacy, Seoul National University, Seoul08826, Republic of Korea.
- 2Department of Biological Sciences, University of Ulsan, Ulsan44610, Republic of Korea.
- 3Research Institute of Pharmaceutical Sciences, College of Pharmacy, Seoul National University, Seoul08826, Republic of Korea.
- 4Future Medicine Co., Ltd, 54 Changup-ro, Sujeong-gu, Seongnam, Gyeonggi-do13449, Republic of Korea.
- 5KIST Gangneung Institute of Natural Products, Natural Product Drug Development Division, Center for Natural Product Systems Biology, Gangneung25451, Republic of Korea.
Abstract
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.
Related
- Lysophospholipase PNPLA7 is a critical checkpoint of phosphatidylcholine catabolism and turnover for skeletal muscle homeostasis
- Cholesterol Reshapes Electroporation Energetics in Phosphatidylcholine Model Membranes
- Scale degeneracy: the physical basis of calibration and absolute quantitation in molecular binding
- PiProteline: An R Package for Integrated Proteomics Data Analysis, from Label-Free Quantitation to Systems Biology.
- Optimisation and Validation of a UHPLC-MS/MS Assay Using Timed Post-Column Ammonium Fluoride Infusion for Sensitive Serum Steroids Quantitation
BibTeX
@article{lee2026quantitation,
title = {{Quantitation of PEMT and Kennedy Pathways in Liver Phosphatidylcholine Biosynthesis Using NMR.}},
author = {Ji Yun Lee and Tin Tin Manh Nguyen and Thi Ha Nguyen and Eunbyul Cho and Seungjoon Lee and Hyuk Nam Kwon and Lak Shin Jeong and Dnyandev B Jarhad and Jin Wook Cha and Sunghyouk Park},
journal = {Analytical chemistry},
year = {2026},
month = sep,
doi = {10.1021/acs.analchem.6c02190},
url = {https://europepmc.org/article/MED/42915245},
note = {CC BY},
}
RIS
TY - JOUR
TI - Quantitation of PEMT and Kennedy Pathways in Liver Phosphatidylcholine Biosynthesis Using NMR.
AU - Ji Yun Lee
AU - Tin Tin Manh Nguyen
AU - Thi Ha Nguyen
AU - Eunbyul Cho
AU - Seungjoon Lee
AU - Hyuk Nam Kwon
AU - Lak Shin Jeong
AU - Dnyandev B Jarhad
AU - Jin Wook Cha
AU - Sunghyouk Park
PY - 2026
DA - 2026/09/01
JO - Analytical chemistry
DO - 10.1021/acs.analchem.6c02190
UR - https://europepmc.org/article/MED/42915245
AB - 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.
SN - 0003-2700
SN - 1520-6882
ER -