Abstract
Despite being widely hypothesized, the actual contribution of choline as a methyl source for phosphatidylethanolamine (PE) methylation has never been demonstrated, mainly due to the inability of conventional methods to distinguish the products from that of the CDP-choline pathway. Using a novel combination of stable-isotope labeling and tandem mass spectrometry, we demonstrated for the first time that choline contributed to phosphatidylcholine (PC) synthesis both as an intact choline moiety via the CDP-choline pathway and as a methyl donor via PE methylation pathway. When hepatocytes were labeled with d(9)-choline containing three deuterium atoms on each of the three methyl groups, d(3)-PC and d(6)-PC were detected, indicating that newly synthesized PC contained one or more individually mobilized methyl groups from d(9)-choline. The synthesis of d(3)-PC and d(6)-PC was sensitive to the general methylation inhibitor 3-deazaadenosine and were specific products of PE methylation using choline as a one-carbon donor. While the contribution to the CDP-choline pathway remained intact in hepatocarcinoma cells, contribution of choline to PE methylation was completely disrupted. In addition to a previously identified lack of PE methyltransferase, hepatocarcinoma cells were found to lack the abilities to oxidize choline to betaine and to donate the methyl group from betaine to homocysteine, whereas the usage of exogenous methionine as a methyl group donor was normal. The failure to use choline as a methyl source in hepatocarcinoma cells may contribute to methionine dependence, a widely observed aberration of one-carbon metabolism in malignancy.
Highlights
In hepatocytes, PC1 is synthesized via two pathways
Using a novel combination of stable-isotope labeling and tandem mass spectrometry, we demonstrated for the first time that choline contributed to phosphatidylcholine (PC) synthesis both as an intact choline moiety via the CDP-choline pathway and as a methyl donor via PE methylation pathway
If choline is used as a methyl group donor, we expected to detect PC with one deuterated-methyl group (d3PC) or two deuterated-methyl groups (d6-PC) when hepatocytes were labeled with d9-choline containing three deuterated methyl groups (HO-CH2-CH2-NQ[CD3]3)
Summary
Materials—d9-Choline chloride, d4-ethanolamine, and d9-betaine were purchased from Isotec, Inc. d3-Methionine was purchased from Medical Isotopes, Inc. [3H]Ethanolamine was from American Radiolabeled Chemicals, Inc. [methyl-3H]S-Adenosylmethionine was purchased from PerkinElmer Life Sciences. Deuterium Labeling—Cells (3.5 ϫ 105) were incubated in serum-free DMEM containing 2 mg/ml bovine serum albumin and 500 M unlabeled or deuterium-labeled choline, betaine, methionine, and/or ethanolamine, as described under “Results” and in the figure legends. Detection of Water-soluble metabolites by ESI-MS—For the detection of choline and betaine, the aqueous portion of the Bligh and Dyer [17] extract from 3.5 ϫ 105 cells containing 1% formic acid was analyzed directly by ESI-MS in the positive ion mode at a cone voltage of 35 eV. Reaction mixtures for enzymatic assays contained 40 mM glycine (pH 8.5), 130 M d9-choline, 50 g of protein extracts, and homogenization buffer to a final volume of 100 l and were incubated at 37 °C. The concentrations of d9-betaine and d3-methionine in each sample were determined by comparing the intensity at each time point with a standard of known concentration
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
