Specificity and rate of human and mouse liver and plasma phosphatidylcholine synthesis analyzed in vivo

Phosphatidylcholine (PC) synthesis by the direct cytidine diphosphate choline (CDP-choline) pathway in rat liver generates predominantly mono- and di-unsaturated molecular species, while polyunsaturated PC species are synthesized largely by the phosphatidylethanolamine-N-methyltransferase (PEMT) pathway. Although altered PC synthesis has been suggested to contribute to development of hepatocarcinoma and nonalcoholic steatohepatitis, analysis of the specificity of hepatic PC metabolism in human patients has been limited by the lack of sensitive and safe methodologies. Here we incorporated a deuterated methyl-d9-labled choline chloride, to quantify biosynthesis fluxes through both of the PC synthetic pathways in vivo in human volunteers and compared these fluxes with those in mice. Rates and molecular specificities of label incorporated into mouse liver and plasma PC were very similar and strongly suggest that label incorporation into human plasma PC can provide a direct measure of hepatic PC synthesis in human subjects. Importantly, we demonstrate for the first time that the PEMT pathway in human liver is selective for polyunsaturated PC species, especially those containing docosahexaenoic acid. Finally, we present a multiple isotopomer distribution analysis approach, based on transfer of deuterated methyl groups to S-adenosylmethionine and subsequent sequential methylations of PE, to quantify absolute flux rates through the PEMT pathway that are applicable to studies of liver dysfunction in clinical studies. Phosphatidylcholine (PC) synthesis by the direct cytidine diphosphate choline (CDP-choline) pathway in rat liver generates predominantly mono- and di-unsaturated molecular species, while polyunsaturated PC species are synthesized largely by the phosphatidylethanolamine-N-methyltransferase (PEMT) pathway. Although altered PC synthesis has been suggested to contribute to development of hepatocarcinoma and nonalcoholic steatohepatitis, analysis of the specificity of hepatic PC metabolism in human patients has been limited by the lack of sensitive and safe methodologies. Here we incorporated a deuterated methyl-d9-labled choline chloride, to quantify biosynthesis fluxes through both of the PC synthetic pathways in vivo in human volunteers and compared these fluxes with those in mice. Rates and molecular specificities of label incorporated into mouse liver and plasma PC were very similar and strongly suggest that label incorporation into human plasma PC can provide a direct measure of hepatic PC synthesis in human subjects. Importantly, we demonstrate for the first time that the PEMT pathway in human liver is selective for polyunsaturated PC species, especially those containing docosahexaenoic acid. Finally, we present a multiple isotopomer distribution analysis approach, based on transfer of deuterated methyl groups to S-adenosylmethionine and subsequent sequential methylations of PE, to quantify absolute flux rates through the PEMT pathway that are applicable to studies of liver dysfunction in clinical studies. Integration of lipid metabolism is one fundamental role of the liver, converting the diversity of lipid delivered from the diet as chylomicron remnants and nonesterified fatty acids into a relatively constant composition of lipoprotein lipid for export to extrahepatic tissues. Phosphatidylcholine (PC) synthesis is essential for VLDL secretion from the liver (1Yao Z.M. Vance D.E. The active synthesis of phosphatidylcholine is required for very low density lipoprotein secretion from rat hepatocytes.J. Biol. Chem. 1988; 263: 2998-3004Abstract Full Text PDF PubMed Google Scholar), and modulation of PC synthesis and metabolism is directly linked to a number of liver diseases, including alcoholic (2Lieber C.S. New concepts of the pathogenesis of alcoholic liver disease lead to novel treatments.Curr. Gastroenterol. Rep. 2004; 6: 60-65Crossref PubMed Scopus (74) Google Scholar) and nonalcoholic fatty liver (NAFL) disease (3Song J. da Costa K.A. Fischer L.M. Kohlmeier M. Kwock L. Wang S. Zeisel S.H. Polymorphism of the PEMT gene and susceptibility to nonalcoholic fatty liver disease (NAFLD).FASEB J. 2005; 19: 1266-1271Crossref PubMed Scopus (188) Google Scholar), liver cancer (4Zeisel S.H. Choline: an important nutrient in brain development, liver function and carcinogenesis.J. Am. Coll. Nutr. 1992; 11: 473-481Crossref PubMed Scopus (110) Google Scholar), and cystic fibrosis (5Van Biervliet S. Van Biervliet G. Van Biervliet J.P. Declercq D. Robberecht E. Christophe A. Relation between fatty acid composition and clinical status or genotype in cystic fibrosis patients.Ann. Nutr. Metab. 2007; 51: 541-549Crossref PubMed Scopus (36) Google Scholar). The bulk of chain elongation and desaturation of the dietary essential fatty acids linoleate (18:2n-6) and α-linoleneate (18:2n-3) to the polyunsaturated fatty acids (PUFA) arachidonate (20:4n-6), eiscosapentoneate (20:5n-3), and docosahexaenoate (22:6n-3) takes place in the liver, and lipoprotein PC is the major vehicle for PUFA exportation from the liver (6Scott B.L. Bazan N.G. Membrane docosahexaenoate ia supplied to the developing brain and retina by the liver.Proc. Natl. Acad. U S A. 1989; 86: 2903-2907Crossref PubMed Scopus (383) Google Scholar). PC is synthesized by all cell types directly from choline and diacylglycerol by the CDP-choline pathway (7Weiss S.B. Smith S.W. Kennedy E.P. The enzymatic formation of lecithin from cytidine diphosphate choline and D-1,2-diglyceride.J. Biol. Chem. 1958; 231: 53-64Abstract Full Text PDF PubMed Google Scholar) (Fig. 1A), but it can also be synthesized in hepatocytes by three sequential methylations of phosphatidylethanolamine (PE), catalyzed by PE-N-methyltransferases (PEMT) (Fig. 1B) (8Vance D.E. Walkey C.J. Cui Z. Phosphatidylethanolamine N-methyltransferase from liver.Biochim. Biophys. Acta. 1997; 1348: 142-150Crossref PubMed Scopus (171) Google Scholar). This pathway uses S-adenosylmethionine (SAMe) as the methyl donor and accounts for about 30% of total liver PC synthesis. The molecular specificity of PC synthesis by both pathways, as defined by the combination of fatty acyl moieties esterified at the sn-1 and sn-2 positions of the glycerophosphate backbone, has been established in rat and guinea pig livers and hepatocytes by metabolic labeling studies using radioisotopes (9Samborski R.W. Ridgway N.D. Vance D.E. Metabolism of molecular species of phosphatidylethanolamine and phosphatidylcholine in rat hepatocytes during prolonged inhibition of phosphatidylethanolamine N-methyltransferase.J. Lipid Res. 1993; 34: 125-137Abstract Full Text PDF PubMed Google Scholar, 10Ridgway N.D. Vance D.E. Specificity of rat hepatic phosphatidylethanolamine N-methyltransferase for molecular species of diacyl phosphatidylethanolamine.J. Biol. Chem. 1988; 263: 16856-16863Abstract Full Text PDF PubMed Google Scholar, 11Burdge G.C. Hunt A.N. Postle A.D. Mechanisms of hepatic phosphatidylcholine synthesis in adult rat: effects of pregnancy.Biochem. J. 1994; 303: 941-947Crossref PubMed Scopus (67) Google Scholar, 12Burdge G.C. Kelly F.J. Postle A.D. Mechanisms of hepatic phosphatidylcholine synthesis in the developing guinea pig: contributions of acyl remodelling and of N-methylation of phosphatidylethanolamine.Biochem. J. 1993; 290: 67-73Crossref PubMed Scopus (23) Google Scholar). The CDP-choline pathway forms mainly monounsaturated and di-unsaturated PC species such as palmitoyloleoyl-PC (PC16:0/18:1) and palmitoyl linoleoyl-PC (PC16:0/18:2), whereas the PE-N methylation pathway synthesizes PUFA-containing PC species such as palmitoyldocosahexaenoyl-PC (PC16:0/22:6) and palmitoylarachidonoyl-PC (PC16:0/20:4) (in this molecular species nomenclature, e.g., A:a/B:b, A and B represent the total number of carbon atoms in each fatty acid at the sn-1 and sn-2 positions of the glycerophosphate backbone, respectively, and a and b represent the number of unsaturated double bonds in each fatty acid). The importance of PE-N methylation for the supply of PUFA from the liver to peripheral tissues is highlighted in PEMT−/− mice, which have substantially diminished plasma concentrations of docosahexaenoyl-containing (22:6n-3) and arachidonoyl-containing (20:4n-6) PC species, although fatal liver failure is prevented and total plasma PC is normalized by dietary choline supplementation (13Watkins S.M. Zhu X. Zeisel S.H. Phosphatidylethanolamine-N-methyltransferase activity and dietary choline regulate liver-plasma lipid flux and essential fatty acid metabolism in mice.J. Nutr. 2003; 133: 3386-3391Crossref PubMed Scopus (140) Google Scholar, 14Waite K.A. Cabilio N.R. Vance D.E. Choline deficiency-induced liver damage is reversible in PEMT(−/−) mice.J. Nutr. 2002; 132: 68-71Crossref PubMed Scopus (64) Google Scholar). Considerable evidence suggests that PE-N methylation in liver is involved in the regulation of cell division (15Vance D.E. Houweling M. Lee M. Cui Z. Phosphatidylethanolamine methylation and hepatoma cell growth.Anticancer Res. 1996; 16: 1413-1416PubMed Google Scholar). For instance, PEMT expression and activity are low both in human hepatocellular carcinomas (16Tessitore L. Marengo B. Vance D.E. Papotti M. Mussa A. Daidone M.G. Costa A. Expression of phosphatidylethanolamine N-methyltransferase in human hepatocellular carcinomas.Oncology. 2003; 65: 152-158Crossref PubMed Scopus (30) Google Scholar) and during rat liver growth after partial hepatectomy (17Houweling M. Cui Z. Tessitore L. Vance D.E. Induction of hepatocyte proliferation after partial hepatectomy is accompanied by a markedly reduced expression of phosphatidylethanolamine N-methyltransferase-2.Biochim. Biophys. Acta. 1997; 1346: 1-9Crossref PubMed Scopus (56) Google Scholar). PC synthesis in human liver disease has received less attention largely because of the lack of appropriately safe methodologies. Studies using a combination of isotope ratio mass spectrometry (MS) and multiple isotopomer distribution analysis (MIDA) modeling have determined rates of hepatic triacylglycerol synthesis and VLDL secretion by using stable isotopes (reviewed in 18Parks E.J. Hellerstein M.K. Thematic review series: patient-oriented research. Recent advances in liver triacylglycerol and fatty acid metabolism using stable isotope labeling techniques.J. Lipid Res. 2006; 47: 1651-1660Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar) but have not investigated the synthesis and turnover of hepatic or plasma PC. An alternative analytical approach (19DeLong C.J. Shen Y.J. Thomas M.J. Cui Z. Molecular distinction of phosphatidylcholine synthesis between the CDP-choline pathway and phosphatidylethanolamine methylation pathway.J. Biol. Chem. 1999; 274: 29683-29688Abstract Full Text Full Text PDF PubMed Scopus (304) Google Scholar) analyzes incorporation of stable isotope-labeled substrates into intact PC molecules. Incubation of rat hepatocytes with deuterated methyl-d9-labeled choline chloride, followed by electrospray ionization tandem MS (ESI-MS/MS), enabled determination of the synthesis of individual PC molecular species. Moreover, choline is a major methyl donor for synthesis of SAMe, resulting in transfer of a labeled methyl group to form methyl-d3-SAMe (Fig. 1B) (19DeLong C.J. Shen Y.J. Thomas M.J. Cui Z. Molecular distinction of phosphatidylcholine synthesis between the CDP-choline pathway and phosphatidylethanolamine methylation pathway.J. Biol. Chem. 1999; 274: 29683-29688Abstract Full Text Full Text PDF PubMed Scopus (304) Google Scholar). Consequently, this method is also used to monitor the PEMT pathway flux from the incorporation of the methyl-d3 label (Fig. 1B), as well as to direct incorporation of the methyl-d9 label. Importantly, these analyses of PC biosynthesis fluxes provide information about the individual molecular species that are biologically relevant molecules, and, consequently, analyses of their kinetics are essential to determine the contribution of altered PC metabolism to disease mechanisms. We previously applied ESI-MS/MS methodology to quantify the synthesis and turnover of individual molecular species of PC in lung surfactant in human volunteers (20Bernhard W. Pynn C.J. Jaworski A. Rau G.A. Hohlfeld J.M. Freihorst J. Poets C.F. Stoll D. Postle A.D. Mass spectrometric analysis of surfactant metabolism in human volunteers using deuteriated choline.Am. J. Respir. Crit. Care Med. 2004; 170: 54-58Crossref PubMed Google Scholar) and now use it to describe the synthesis of individual molecular species of hepatic PC in vivo. Initial analyses were conducted in a mouse model to establish whether analysis of the molecular specificity and dynamics of label incorporation into plasma PC can provide a direct insight into liver PC metabolism. This methodology was then applied to human volunteers to show for the first time the molecular specificity of PC synthesis by human liver in by the of label in plasma PC. Finally, we present a mass isotopomer analysis for the of flux through the PEMT pathway that both the and of stable isotope labeling with for the of altered hepatic lipid metabolism in in vivo. received of of in and were then at and time was by and in and plasma was by for and at and were in and at received to of were conducted of was in and human volunteers with were at from to after the of were at at for and plasma was at Initial and those at and were after but volunteers not all were volunteers their and a the were was in this to or during the The was by the at was and of or hepatic A total of of mouse liver or lung was and in of on were from and plasma by using and A and sensitive method of total lipid and J. PubMed Scopus Google Scholar), and by PC and were as PC species were using a mass with an were in and into the mass by direct using group with of to and of for PC synthesized the CDP-choline pathway A.N. Postle A.D. phosphatidylcholine is synthesized in and with CDP-choline pathway Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). of the with or containing one or methyl incorporation of into the PC group the PEMT pathway A.D. Hunt A.N. dynamics by electrospray mass Lipid Res. 2007; PubMed Scopus (64) Google Scholar). PC molecular species were from of the A of analysis of diacyl molecular species. of were the of the mass Importantly, the of lipid this not the PC or molecular species or the incorporation of stable isotope were using and The from was and and of the methyl-d9 label in choline was Choline was using G.C. Kelly F.J. Postle A.D. of phosphatidylcholine in lung acyl remodelling and turnover of individual molecular Biophys. Acta. 1993; PubMed Scopus Google Scholar). and was by ESI-MS/MS in the multiple and of labeled into mouse liver PC by both the CDP-choline and the PEMT pathways after of are in have been mass on the to synthesized and with molecular species show the of molecular specificities of PC synthesis by the PC (Fig. MS was a combination of and polyunsaturated species. PC synthesized directly by the CDP-choline pathway (Fig. was in mono- (PC16:0/18:1) and di-unsaturated species. PC synthesized by the PEMT pathway (Fig. containing one methyl-d3 was predominantly and was the major species This of molecular species was similar but not to that of PE, the for the PEMT pathway (Fig. which was of and species. The of all PC species in with of the in each were for and MS in of mouse liver, and plasma and of human was for as for PC in this The incorporation of methyl-d9 label into mouse liver and lung PC and to total PC is in and the incorporation of methyl-d3 label is in The rates of hepatic and lung PC methyl-d9 labeling were very by at and methyl-d3 labeling was for liver PC at and the lung labeling at PC synthesis by the CDP-choline pathway from and was not with PE-N methylation of label in mouse plasma PC labeling of liver PC by both pathways (Fig. that not was PC synthesized very by mouse liver but that it was into the The of stable isotope labeling of human plasma PC a prolonged time (Fig. that both pathways were active in the synthesis of lipoprotein with less PC synthesized by the PE-N methylation pathway in human in mouse The composition of and synthesized PC is in and in for the PC species. species were to the major that time to the stable isotope incorporation by the CDP-choline (Fig. and PE-N methylation (Fig. pathways into PC from mouse liver (Fig. mouse plasma (Fig. and human plasma (Fig. are although the of the to mouse liver PC synthesis by the CDP-choline pathway is with acyl at both the sn-1 and the sn-2 with synthesis as followed by of label to and and the PC composition by (Fig. The labeling is for mouse plasma (Fig. and, very similar for human plasma (Fig. Initial incorporation into in mouse liver PC by the PE-N methylation pathway (Fig. the of species in liver (Fig. The methyl-d3 label in with accompanied by labeling of and and the PC composition by The distribution of methyl-d3 label in mouse plasma PC the that in liver PC (Fig. The distribution of methyl-d3 label in human plasma PC molecular species followed a very similar with an incorporation into and and a subsequent to species (Fig. This analysis for the first time that the PEMT pathway in human liver is for the synthesis of and PC molecular species for lipoprotein Importantly, the similar labeling of mouse liver and plasma PC by both pathways at each time show although synthesized liver PC a of acyl after this is not required for secretion of lipoprotein PC. these suggest that synthesized PC is from the liver into the with to the synthesis liver PC labeling are not the human plasma labeling also strongly suggest a similar for synthesis and secretion of human plasma lipoprotein PC. The methyl-d3 in represent the of all PC species, but the distribution of incorporated label into PC species with and are the PC species respectively, by the PE-N methylation and CDP-choline The methyl-d3 in demonstrate a flux of through the PE-N methylation pathway with synthesis of this The of PC synthesis by the CDP-choline pathway at was based on the that all methyl-d9 label was incorporated during the PC labeled between and was of the choline be and the was at The of (Fig. in liver followed a with a to followed by a to Although these are suggest a with the of lipoprotein by the liver and of labeled choline B. Vance D.E. of low density phosphatidylcholine to triacylglycerol by hepatocytes.J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). that at the of was and that of was between and an of the of synthesis by this pathway was as of total liver A similar for PEMT pathway flux was using to determine the of the methyl-d3-SAMe in the liver M.K. New stable spectrometric for fluxes through intact metabolic pathways in of into and 2004; 6: PubMed Scopus Google Scholar). these can to three methyl-d3 groups during to PC by of one methyl-d3 group be by a MS and that of methyl-d3 groups by a with of incorporation of methyl-d3-SAMe is then with the for clinical very similar can be from the analysis of both mouse plasma and liver PC (Fig. an can then be using human plasma PC to provide a measure of the methyl-d3-SAMe in human liver (Fig. for methyl-d3-SAMe were then used to to provide of total PC synthesis by PE-N methylation at each time for mouse liver and plasma (Fig. and human plasma (Fig. Initial incorporation for each were of flux this pathway. The in synthetic rates for mouse liver and plasma strongly suggests that the of for human plasma can be used as a for human liver PC flux by the PEMT pathway in vivo. The specificity of this synthesis (Fig. for PC species and that synthesis rates of for all three with and was for PC species, for and for and This in synthetic rates suggests that this stable isotope labeling approach can be used to the specificity of hepatic PEMT activity in vivo in human as well as in rates of synthesis by the PEMT pathway of molecular species of liver PC. Rates of PC synthesis were from methyl-d3 into individual PC molecular species for mouse liver mouse plasma and human plasma are as synthetic rates for each individual PC of methyl-d9-labeled choline has previously been used to the molecular specificity of PC synthesis by rat hepatocytes and hepatoma but the are the first of the specificity of human plasma PC synthesis. between the specificities of mouse liver and plasma PC synthesis suggest that incorporation of label into human plasma PC can be used as a for human liver PC synthesis in vivo. This approach is relatively and and the use of substrates labeled with use of for methyl-d3-SAMe absolute rates of synthetic flux through the PEMT pathway to be in a method for liver PEMT activity in vivo in is important to that of methyl-d3-SAMe by this approach is of the of the methyl donor and be for instance, to studies of the incorporation of This to quantify PEMT metabolic flux in vivo has clinical to a of metabolic and hepatic PEMT is a methylation activity in the L.M. Vance D.E. it time to methyl in J. Nutr. 2006; PubMed Scopus Google Scholar) and is a of plasma C.S. Phosphatidylethanolamine N-methyltransferase and regulation of 2006; PubMed Google Scholar, L.M. Vance D.E. is by Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar), an established for disease and Med. PubMed Scopus Google Scholar). of PEMT is in liver from patients with J.M. and are in human 1988; PubMed Scopus Google the pathway is with (3Song J. da Costa K.A. Fischer L.M. Kohlmeier M. Kwock L. Wang S. Zeisel S.H. Polymorphism of the PEMT gene and susceptibility to nonalcoholic fatty liver disease (NAFLD).FASEB J. 2005; 19: 1266-1271Crossref PubMed Scopus (188) Google Scholar, Wang J. A. M. M. S. The phosphatidylethanolamine N-methyltransferase gene the susceptibility to in 2007; Full Text Full Text PDF PubMed Scopus Google PEMT−/− fatal on a diet C.J. L. Vance D.E. and of Biol. Chem. Full Text Full Text PDF PubMed Scopus Google and PEMT pathway flux is an essential of the dietary mouse model of G. L. B. J. Lee S.M. a diet with hepatic of Lipid Res. 2006; 47: Full Text Full Text PDF PubMed Scopus Google Scholar). PEMT−/− on a diet have a liver PC but of plasma PC and PC species containing and (13Watkins S.M. Zhu X. Zeisel S.H. Phosphatidylethanolamine-N-methyltransferase activity and dietary choline regulate liver-plasma lipid flux and essential fatty acid metabolism in mice.J. Nutr. 2003; 133: 3386-3391Crossref PubMed Scopus (140) Google Scholar). in the importance of PEMT activity in hepatic lipoprotein exportation of PUFA-containing PC to extrahepatic tissues and strongly suggest that all synthesized PUFA-containing PC species by mouse liver are from PE-N methylation and not from the CDP-choline pathway. and of methyl-d9 and methyl-d3 in human compared with mouse plasma PC suggest similar for the metabolism and secretion of lipoprotein PC in although this to be in studies using human liver A of VLDL is synthesized by while synthesis from choline is essential for PC secretion into the G. S. W. of phosphatidylcholine secretion in rat Biophys. Acta. 2004; PubMed Scopus Google Scholar), the contribution of PC synthesis to plasma lipoprotein and secretion is not The methodology not directly this The of molecular specificities of synthesized PC synthesis in mouse liver and plasma has for and secretion of plasma studies have that rat liver PC (9Samborski R.W. Ridgway N.D. Vance D.E. Metabolism of molecular species of phosphatidylethanolamine and phosphatidylcholine in rat hepatocytes during prolonged inhibition of phosphatidylethanolamine N-methyltransferase.J. Lipid Res. 1993; 34: 125-137Abstract Full Text PDF PubMed Google Scholar, 11Burdge G.C. Hunt A.N. Postle A.D. Mechanisms of hepatic phosphatidylcholine synthesis in adult rat: effects of pregnancy.Biochem. J. 1994; 303: 941-947Crossref PubMed Scopus (67) Google Scholar) is synthesized by the CDP-choline pathway largely as and PC and is then acyl to the composition by a of and S.B. of rat hepatocyte by selective acyl Biol. Chem. Full Text PDF PubMed Google Scholar, S. D.E. M. G. and of a major liver Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). in demonstrate that liver synthesized by acyl acyl a of (9Samborski R.W. Ridgway N.D. Vance D.E. Metabolism of molecular species of phosphatidylethanolamine and phosphatidylcholine in rat hepatocytes during prolonged inhibition of phosphatidylethanolamine N-methyltransferase.J. Lipid Res. 1993; 34: 125-137Abstract Full Text PDF PubMed Google Scholar, 11Burdge G.C. Hunt A.N. Postle A.D. Mechanisms of hepatic phosphatidylcholine synthesis in adult rat: effects of pregnancy.Biochem. J. 1994; 303: 941-947Crossref PubMed Scopus (67) Google Scholar) and as of the flux through both pathways is for both mouse liver and plasma PC acyl be one of a number of that determine liver PC molecular species this the contributions to the composition of liver and plasma PC of S. D.E. M. G. and of a major liver Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), M. studies on the specificity of the molecular species of phosphatidylcholine in the plasma of Lipid Res. 1996; Full Text PDF PubMed Google Scholar), of extrahepatic PC to the liver B. Vance D.E. of low density phosphatidylcholine to triacylglycerol by hepatocytes.J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), and secretion of PC in S. of the of phosphatidylcholine molecular species for secretion into in the PubMed Scopus Google Scholar) have to be have strongly suggested that modulation of the methylation pathway for the supply of PUFA from the liver to extrahepatic which is largely of PC synthesis by the CDP-choline pathway. of to absolute rates of PC synthesis by PE-N methylation this pathway flux to be in vivo in human patients and has for the of a of important liver diseases, including hepatic alcoholic and nonalcoholic fatty liver and cystic with multiple isotopomer distribution analysis multiple liver disease phosphatidylcholine phosphatidylethanolamine S-adenosylmethionine