Molecular Distinction of Phosphatidylcholine Synthesis between the CDP-Choline Pathway and Phosphatidylethanolamine Methylation Pathway

In addition to the CDP-choline pathway for phosphatidylcholine (PC) synthesis, the liver has a unique phosphatidylethanolamine (PE) methyltransferase activity for PC synthesis via three methylations of the ethanolamine moiety of PE. Previous studies indicate that the two pathways are functionally different and not interchangeable even though PC is the common product of both pathways. This study was designed to test the hypothesis that these two pathways produce different profiles of PC species. The PC species from these two pathways were labeled with specific stable isotope precursors, D9-choline and D4-ethanolamine, and analyzed by electrospray tandem mass spectrometry. Our studies revealed a profound distinction in PC profiles between the CDP-choline pathway and the PE methylation pathway. PC molecules produced from the CDP-choline pathway were mainly comprised of medium chain, saturated (e.g.16:0/18:0) species. On the other hand, PC molecules from the PE methylation pathway were much more diverse and were comprised of significantly more long chain, polyunsaturated (e.g.18:0/20:4) species. PC species from the methylation pathway contained a higher percentage of arachidonate and were more diverse than those from the CDP-choline pathway. This profound distinction of PC profiles may contribute to the different functions of these two pathways in the liver. In addition to the CDP-choline pathway for phosphatidylcholine (PC) synthesis, the liver has a unique phosphatidylethanolamine (PE) methyltransferase activity for PC synthesis via three methylations of the ethanolamine moiety of PE. Previous studies indicate that the two pathways are functionally different and not interchangeable even though PC is the common product of both pathways. This study was designed to test the hypothesis that these two pathways produce different profiles of PC species. The PC species from these two pathways were labeled with specific stable isotope precursors, D9-choline and D4-ethanolamine, and analyzed by electrospray tandem mass spectrometry. Our studies revealed a profound distinction in PC profiles between the CDP-choline pathway and the PE methylation pathway. PC molecules produced from the CDP-choline pathway were mainly comprised of medium chain, saturated (e.g.16:0/18:0) species. On the other hand, PC molecules from the PE methylation pathway were much more diverse and were comprised of significantly more long chain, polyunsaturated (e.g.18:0/20:4) species. PC species from the methylation pathway contained a higher percentage of arachidonate and were more diverse than those from the CDP-choline pathway. This profound distinction of PC profiles may contribute to the different functions of these two pathways in the liver. phosphatidylcholine phosphatidylethanolamine CTP:phosphocholine cytidylyltransferase cytidine diphosphate phosphatidylethanolamine methyltransferase polyacrylamide gel electrophoresis modified essential medium Phosphatidylcholine (PC)1 is a major group of phospholipid in all mammalian cells (1Vance D.E. Biochem. Cell Biol. 1990; 68: 1151-1165Crossref PubMed Scopus (147) Google Scholar). PC is comprised of hydrocarbon chains attached to glycerophosphocholine via acyl, alkyl, or alkenyl linkages. The molecular diversity of PC and other phospholipids is dictated by the combination of different lengths, number of double bonds, and types of linkages of hydrocarbon chains. As a result, a single mammalian cell contains at least a thousand species of phospholipids (2Raetz C.R.H. Hawthorone J.N. Ansell G.B. Phospholipids. Elsevier Biomedical Press, Amsterdam1982: 435-477Google Scholar). In most mammalian cells, PC is synthesized mainly via the CDP-choline pathway (3Cui Z. Vance D.E. J. Biol. Chem. 1996; 271: 2839-2843Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). This pathway uses choline as an initial substrate and is catalyzed by three enzymes: choline kinase, CTP:phosphocholine cytidylyltransferase (CT), and cholinephosphate transferase, with CT as the rate-limiting enzyme (1Vance D.E. Biochem. Cell Biol. 1990; 68: 1151-1165Crossref PubMed Scopus (147) Google Scholar). Hepatocytes are unique because they also possess a high activity of phosphatidylethanolamine methyltransferase (PEMT) that converts PE to PC via three sequential steps of methylation (4Vance D.E. Ridgway N.D. Prog. Lipid Res. 1988; 27: 61-79Crossref PubMed Scopus (193) Google Scholar) in addition to a high level of CDP-choline pathway activity. The significance of the PEMT pathway is not completely understood (5Vance D.E. Walkey C.J. Curr. Opin. Lipidol. 1998; 9: 125-130Crossref PubMed Scopus (14) Google Scholar). The PEMT pathway seems redundant because its product, PC, is also synthesized by the CDP-choline pathway in hepatocytes. Therefore, the PEMT pathway is traditionally considered a backup pathway for PC synthesis in hepatocytes (3Cui Z. Vance D.E. J. Biol. Chem. 1996; 271: 2839-2843Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). However, recent studies indicate that these two pathways apparently have opposite effects on proliferative characteristics of the liver and liver-derived cell lines (6Cui Z. Houweling M. Vance D.E. J. Biol. Chem. 1994; 269: 24531-24533Abstract Full Text PDF PubMed Google Scholar, 7Cui Z. Vance D.E. Biochem. J. 1995; 312: 939-945Crossref PubMed Scopus (43) Google Scholar, 8Tessitore L. Cui Z. Vance D.E. Biochem. J. 1997; 322: 151-154Crossref PubMed Scopus (25) Google Scholar, 9Houweling M. Cui Z. Vance D.E. Biochim. Biophys. Acta. 1997; 1346: 1-9Crossref PubMed Scopus (56) Google Scholar, 10Cui Z. Shen Y.J. Vance D.E. Biochim. Biophys. Acta. 1997; 1346: 10-16Crossref PubMed Scopus (43) Google Scholar). These studies suggest that the higher activity of the CDP-choline pathway favors the faster proliferation of hepatocytes. Conversely, expression of PEMT strongly inhibits the growth of hepatoma cell lines and is negatively associated with the developmental growth of liver and with neoplastic growth of liver tumor induced by chemical carcinogens (6Cui Z. Houweling M. Vance D.E. J. Biol. Chem. 1994; 269: 24531-24533Abstract Full Text PDF PubMed Google Scholar, 7Cui Z. Vance D.E. Biochem. J. 1995; 312: 939-945Crossref PubMed Scopus (43) Google Scholar, 8Tessitore L. Cui Z. Vance D.E. Biochem. J. 1997; 322: 151-154Crossref PubMed Scopus (25) Google Scholar, 9Houweling M. Cui Z. Vance D.E. Biochim. Biophys. Acta. 1997; 1346: 1-9Crossref PubMed Scopus (56) Google Scholar, 10Cui Z. Shen Y.J. Vance D.E. Biochim. Biophys. Acta. 1997; 1346: 10-16Crossref PubMed Scopus (43) Google Scholar). Recombinant expression of PEMT in cultured cell lines leads to an active synthesis of PC via PE methylation (11Cui Z. Vance J.E. Chen M.H. Voelker D.R. Vance D.E. J. Biol. Chem. 1993; 268: 16655-16663Abstract Full Text PDF PubMed Google Scholar). However, the PC synthesized via this methylation pathway does not substitute for the role of PC synthesized from the CDP-choline pathway (12Houweling M. Cui Z. Vance D.E. J. Biol. Chem. 1995; 270: 16277-16282Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). MT58 is a cell line derived from Chinese hamster ovary (CHO) K1 cells by chemical mutagenesis and carries a temperature-sensitive mutation in the rate-limiting enzyme, CT, of the CDP-choline pathway (13Esko J.D. Wermuth M.M. Raetz C.R. J. Biol. Chem. 1981; 256: 7388-9325Abstract Full Text PDF PubMed Google Scholar). At the non-permissive temperature, CT becomes inactive, PC synthesis shuts down, and the mutant dies via apoptosis. Recombinant expression of rat liver CT rescues the mutant effectively, whereas recombinant expression of rat liver PEMT fails to rescue MT58 cells at the non-permissive temperature (12Houweling M. Cui Z. Vance D.E. J. Biol. Chem. 1995; 270: 16277-16282Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). Apparently the roles of the CDP-choline pathway and the PEMT pathway are distinct and not interchangeable. One hypothesis for this distinction is that PC synthesized from different pathways may have different subcellular locations. The ability of exogenous PC to rescue the mutant phenotype (14Esko J.D. Nishijima M. Raetz C.R. Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 1698-1702Crossref PubMed Scopus (58) Google Scholar) suggests, however, that PC synthesized at one subcellular location is unlikely to be restricted from moving freely to another subcellular location. Thus, the hypothesis of distinctive localization of PC is an unlikely explanation for why PEMT fails to rescue the mutant. Another hypothesis is that the two pathways synthesize different pools of PC molecular species. To test this hypothesis, we devised a novel strategy capable of specifically displaying the PC species derived from the CDP-choline pathway and the PEMT pathway. We report here that the PC species synthesized from the CDP-choline pathway were mainly comprised of medium chain and saturated species, whereas the PC species derived from the methylation pathway are mainly comprised of long chain and highly unsaturated species. Our study suggests that the difference in PC composition contributes to the functional distinction between the two pathways. D4-ethanolamine and D9-choline chloride were from Isotec, Inc. 3Hethanolamine was from American Radiolabeled Chemicals, Inc. Dulbecco's modified essential medium (MEM) and fetal bovine serum were from Life Technologies, Inc. Silica gel H plates were from Analtech, Inc. Phospholipid standards were from Avanti Polar Lipids. All other chemicals and materials were from Fisher Scientific. Rat primary hepatocytes were obtained by collagenase perfusion (15Davis R.A. Engelhorn S.C. Pangburn S.H. Weinstein D.B. Steinberg D. J. Biol. Chem. 1979; 254: 2010-2016Abstract Full Text PDF PubMed Google Scholar). Hepatocytes were cultured on collagen-coated culture dishes overnight prior to experiments in Dulbecco's MEM with 10% fetal bovine serum, 10 μg/ml insulin, and 10 mm Hepes buffer. McArdle RH7777 cells were dually transfected with 10 μg of pCMV5/PEMT2 (11Cui Z. Vance J.E. Chen M.H. Voelker D.R. Vance D.E. J. Biol. Chem. 1993; 268: 16655-16663Abstract Full Text PDF PubMed Google Scholar) and 1 μg of pSV2neo plasmid by calcium phosphate precipitation as described (16Chen C. Okayama H. Mol. Cell. Biol. 1987; 7: 2745-2752Crossref PubMed Scopus (4799) Google Scholar). G418-resistent colonies were selected with 500 μg/ml G418 and maintained in 200 μg/ml G418. 3.5 × 105 cells were labeled with 1 μCi/ml each, 3Hcholine chloride, or 3Hethanolamine HCl for 24 h in low choline medium (choline-free MEM + 2% fetal bovine serum). Cells were scraped into methanol, and lipids were extracted according to Bligh and Dyer (17Bligh E.G. Dyer W.J. Can. J. Biochem. Physiol. 1959; 37: 911-991Crossref PubMed Scopus (41848) Google Scholar). The lipid extracts were separated on silica gel H plates in a solvent system of 65/35/8 chloroform/methanol/ammonium hydroxide. The phospholipid bands were visualized by iodine vapor and the PC and PE bands were scraped and counted by scintillation counter. 3.5 × 105 cells were incubated in low choline medium containing 50 μg/ml D9-choline chloride and 50 μg/ml D4-ethanolamine for 24 h or otherwise noted. Cells were scraped into methanol and lipids were extracted. Extracts were measured for lipid phosphorus content (18Rouser G. Siakotas A.N. Fleisher S. Lipids. 1966; 1: 85-86Crossref PubMed Scopus (1312) Google Scholar). Lipid extract equaling 500 pmol total lipid phosphorus in 100 μl of 2:1 methylene chloride:methanol was analyzed on a Micromass Quattro II triple quadruple mass spectrometer (Micromass, United Kingdom) in a solvent system of 45/45/10 methylene chloride/methanol/H2O. Data were acquired using MassLynx NT software (Micromass Limited, United Kingdom). A mixture of di-14:0, di-16:0, 16:0–18:1, and di-20:4 PC standards of equal concentration was used to determine instrument settings and concentration for optimal signal intensities. Standards and samples contained 1% formic acid for positive ion analysis, and 1% ammonium hydroxide for negative ion analysis. PC molecular species were detected by precursor ion scanning for m/z +184, +188, and +193 in the positive ion mode. PE molecular species were detected by neutral loss scanning for m/z 141 or 145 in the positive ion mode. The fatty acid composition of each molecular specie was determined by daughter ion analysis in the negative ion mode. The intensities of PC species with equal concentration decreased significantly as mass increased. To correct spectra for mass discrimination, PC standards were analyzed at equal concentrations. The equation derived by plotting intensity versus mass was used to correct raw results: y = y′ · b/([−4963.4 · (x − 678) +b)] (y = corrected intensity of each peak,y′ = actual intensity of each peak, b = intensity of the internal di-14:0 PC standard, and x =m/z of each peak). Fifty μg of total protein from each cell line was separated by SDS-polyacrylamide gel electrophoresis (19Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (205531) Google Scholar) and transferred to nitrocellulose membrane (20Towbin H. Staehelin T. Gordon J. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 4350-4354Crossref PubMed Scopus (44644) Google Scholar). The membranes were probed with anti-PEMT2 (11Cui Z. Vance J.E. Chen M.H. Voelker D.R. Vance D.E. J. Biol. Chem. 1993; 268: 16655-16663Abstract Full Text PDF PubMed Google Scholar) and goat anti-rabbit IgG horseradish peroxidase conjugate. PEMT protein was displayed by a reaction with Supersignal Chemiluminescent Substrate (Pierce) and exposure to x-ray films. Electrospray ionization tandem mass spectrometry analysis (18Rouser G. Siakotas A.N. Fleisher S. Lipids. 1966; 1: 85-86Crossref PubMed Scopus (1312) Google Scholar, 21Jensen N.J. Tomer K.B. Gross R.W. Lipids. 1986; 21: 580-588Crossref PubMed Scopus (164) Google Scholar, 22Bryant D.K. Orlando R.C. Fenselau C. Sowder R.C. Henderson L.E. Anal. Chem. 1991; 63: 1110-1114Crossref PubMed Scopus (47) Google Scholar, 23Kerwin J.L. Tuininga A.R. Ericsson L.H. J. Lipid Res. 1994; 35: 1102-1114Abstract Full Text PDF PubMed Google Scholar, 24Brügger B. Erben G. Sandhoff R. Wieland F.T. Lehmann W.D. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2339-2344Crossref PubMed Scopus (719) Google Scholar, 25Bernstrom K. Kayganich K. Murphy R.C. Anal. Biochem. 1991; 198: 203-211Crossref PubMed Scopus (44) Google Scholar) of PC species labeled with specific deuterated precursors allowed us to distinguish the PC species synthesized from the CDP-choline pathway and the PE methylation pathway (Fig.1 A). Different phospholipid classes were specifically detected by the unique mass/charge (m/z) ratio of the molecular fragments produced upon argon-induced collision. All PC molecules produced a fragment withm/z of +184 corresponding to the protonated phosphocholine head group. All PE molecules were detected by the loss of a neutral fragment of m/z 141, corresponding to the uncharged phosphoethanolamine head group. D4-ethanolamine-labeled PC species with m/z +188 head group were exclusively derived from the PE methylation pathway, and D9-choline-labeled species with m/z +193 head group were specifically synthesized from the CDP-choline pathway. The major advantage of this strategy is that the metabolism of major phospholipid groups can be analyzed from total lipid extracts without any chemical modification. Thus, the peak intensity of each lipid reflects a physiological concentration. To determine whether the incorporation of the D4-labeled choline into PC is a reflection of true PC synthesis from natural choline, we compared the profile of unlabeled PC to that of deuterium-labeled PC in RH7777 cells. RH7777 is a hepatoma-derived cell line in which PEMT activity is absent (7Cui Z. Vance D.E. Biochem. J. 1995; 312: 939-945Crossref PubMed Scopus (43) Google Scholar). Thus PC is synthesized exclusively from the CDP-choline pathway in RH7777. The profile of labeled PC species was nearly identical to the profile of endogenous PC species (Fig.1 B). PC species with m/z +193 head group were not detected in the absence of D9-choline (data not shown). Daughter ion analyses of the molecular each peak 1 and were capable of lipids from lipids with are of in addition to the PC species, lipids with any of or The of these novel lipids are because of most species were in the lipid according to the of Bligh and Dyer (17Bligh E.G. Dyer W.J. Can. J. Biochem. Physiol. 1959; 37: 911-991Crossref PubMed Scopus (41848) Google Scholar). This can be by of the lipid and species be a of To determine whether was a precursor of PC synthesis, we incubated the RH7777 cells with the labeled choline for A of the labeled PC was 1 that deuterium-labeled choline was a precursor for PC To determine the PC profile of the PEMT pathway, we the rat liver PEMT into the rat hepatoma cell line in which PEMT is Thus, in PC synthesis as a specific of PEMT expression were The PEMT was by analysis using a rat liver M. Cui Z. Vance D.E. Biochim. Biophys. Acta. 1997; 1346: 1-9Crossref PubMed Scopus (56) Google Scholar) A). The PEMT pathway was by the incorporation of 3Hethanolamine into PC by B). The cells were labeled with D9-choline and D4-ethanolamine for 24 h at lipids were extracted and analyzed by tandem mass spectrometry. The PC species with head group mass of were derived exclusively from the PEMT pathway and were detected in the RH7777 cells that PEMT and not in the cells, in which PEMT activity was absent The PC species with head group mass of from the CDP-choline pathway were in both and cell lines and were not by the expression of PEMT In the RH7777 cells, between PC species synthesized from the CDP-choline pathway and that from the PE methylation revealed a The major species of the PC derived from the CDP-choline pathway were and which a of the On the other hand, PC derived from the methylation pathway contained significantly more long chain, polyunsaturated PC species These were in in with the report that PEMT the long chain polyunsaturated PE as substrate N.D. Vance D.E. J. Biol. Chem. 1988; Full Text PDF PubMed Google Scholar) for PC To determine whether these distinct profiles of PC in RH7777 cells the physiological of PC synthesis in the liver in which the CDP-choline pathway and PEMT pathway are we studies in hepatocytes. The primary hepatocytes were labeled with the D4-labeled ethanolamine and D9-choline for 24 The total lipids were extracted and The distinction of the PC species from the two pathways was much more profound in primary hepatocytes than that of cells and The PEMT pathway in primary hepatocytes produced more PC species of and than that in cells. The between and primary hepatocytes may the that RH7777 cells are cells in which PC species are faster than those in the of PC species in primary of total extracts of primary hepatocytes were as described with head group mass of the PE methylation and the CDP-choline were by electrospray tandem mass spectrometry. acid composition was determined by daughter ion analysis. m/z of all major unlabeled PC are in The of all in the total PC species derived from the CDP-choline pathway are in and in the total PC species derived from the PEMT pathway are in species in each m/z peak, are in in of or fatty acid at in a extracts of primary hepatocytes were as described with head group mass of the PE methylation and the CDP-choline were by electrospray tandem mass spectrometry. acid composition was determined by daughter ion analysis. m/z of all major unlabeled PC are in The of all in the total PC species derived from the CDP-choline pathway are in and in the total PC species derived from the PEMT pathway are in species in each m/z peak, are in in of or fatty acid at primary hepatocytes not in we to the endogenous PC was by the synthesized PC D4-ethanolamine and D9-choline were in the culture We labeled the cells for 24 h with both deuterated of unlabeled PC between labeled cells and unlabeled cells revealed that of endogenous PC was by the synthesized PC in 24 h A and B). the labeled PC, was from the CDP-choline pathway and was from the PE methylation pathway. The of PC was active even hepatocytes were in a of the PC species to that in the unlabeled cells suggests that the of and species were much more active than that of and PC species derived from PE may be more than the PC species. PC species from the methylation pathway contained a higher percentage of arachidonate B). the of acid in the PC species derived from the PEMT pathway in the liver may as an for lipids active in The significance of the PEMT pathway in hepatocytes has of because the CDP-choline pathway is in all mammalian cells and is for PC studies that the PEMT pathway contributes significantly to the of of choline (3Cui Z. Vance D.E. J. Biol. Chem. 1996; 271: 2839-2843Abstract Full Text Full Text PDF PubMed Scopus (59) Google that of PEMT pathway at least in substitute for the role of the CDP-choline pathway. At the however, is that PEMT fails completely to for the synthesis of Our study revealed a molecular for distinction between the CDP-choline pathway and the PEMT pathway. This distinction apparently the unique of PC species derived from different pathways. Our studies also that the CDP-choline pathway synthesized two or three major species of PC in primary whereas the PEMT pathway at least major species of PC with Therefore, the substrate of the PE methylation pathway is much than that of the CDP-choline pathway. In MT58 cells, CT activity is temperature (13Esko J.D. Wermuth M.M. Raetz C.R. J. Biol. Chem. 1981; 256: 7388-9325Abstract Full Text PDF PubMed Google Scholar). PC with medium and high of can rescue the mutant cells at the non-permissive temperature (14Esko J.D. Nishijima M. Raetz C.R. Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 1698-1702Crossref PubMed Scopus (58) Google Scholar). However, and highly unsaturated PC species are to the cells. of PEMT in RH7777 displayed a and of cell growth (6Cui Z. Houweling M. Vance D.E. J. Biol. Chem. 1994; 269: 24531-24533Abstract Full Text PDF PubMed Google Scholar). Our suggest that the of PEMT expression in RH7777 cells may be because of the of highly unsaturated PC species that are to the cells. The profound distinction in PC molecular species from the two pathways was the opposite effects of the two pathways on this distinction in PC species may for not of the functional difference of the two pathways. This profound distinction may also a that both the and the PE methyltransferase have for fatty acid in addition to the head group However, be that the distinct of the PEMT pathway may also be because of a of PE species as the initial of PC species at and different even in cells. strategy of and all molecular species of PC can be and can the PC species from different pathways be via with distinctive precursors, the synthesized PC can be compared with the PC species. The of this strategy a unique advantage a of of all and all species of spectrometry a of at least m/z for PC a is capable of of one at head group or mass spectrometry any for phospholipid analysis. Thus, extracts of cells can be This advantage for analysis of phospholipid species because as the loss of species not shown). The of this strategy more than cells for the profiles of phospholipid species. the addition of a lipids from as as 50 cells of materials for analysis. and of tandem mass with stable isotope precursors of lipid metabolism that be by We Vance for to for for mass spectrometry analysis, and and for the primary rat hepatocytes.