The Unique Acyl Chain Specificity of Biliary Phosphatidylcholines in Mice Is Independent of Their Biosynthetic Origin in the Liver

Transcription

1 The Unique Acyl Chain Specificity of Biliary Phosphatidylcholines in Mice Is Independent of Their Biosynthetic Origin in the Liver LUIS B. AGELLON, 1 CHRISTOPHER J. WALKEY, 1 DENNIS E. VANCE, 1 FOLKERT KUIPERS, 2 AND HENKJAN J. VERKADE 2 The liver synthesizes phosphatidylcholine (PC) de novo from choline via the CDP-choline pathway, and from phosphatidylethanolamine (PE) via the phosphatidylethanolamine N-methyltransferase (PEMT) pathway. Significant amounts of PC, which are highly specific in their acyl chain composition, are secreted into bile by the liver. To determine whether either of the 2 PC biosynthetic routes is sufficient to provide physiological PC concentrations in bile, or is responsible for the unique acyl chain composition of bile PC, we analyzed gallbladder bile composition in mice that synthesized PC either via the PEMT pathway (induced by feeding a choline-deficient diet) or the CDPcholine pathway (based on genetic PEMT-deficiency). The PC concentration in gallbladder bile of mice that synthesize PC mainly via the CDP-choline pathway was comparable with control mice that synthesize PC via both pathways, whereas it was reduced by 40% in mice that synthesize PC via the PEMT pathway. The acyl chain composition of bile PC was similar irrespective of the active PC biosynthetic pathway in the liver. These data demonstrate that the CDP-choline pathway alone, but not the PEMT pathway alone, can account for physiological concentrations of PC in gallbladder bile. Moreover, the specificity of biliary PC fatty acyl composition is determined independently from the synthetic origin of PC. (HEPATOLOGY 1999;30: ) The liver secretes significant amounts of phospholipids, both into plasma as a surface component of lipoproteins, and Abbreviations: PC, phosphatidylcholine; PE, phosphatidylethanolamine; PEMT, phosphatidylethanolamine N-methyltransferase; CD, choline-deficient; CS, cholinesupplemented; TLC, thin-layer chromatography. From the 1 Department of Biochemistry, University of Alberta, Edmonton, Alberta, Canada; and 2 Center for Liver, Digestive and Metabolic Diseases, University Hospital Groningen, the Netherlands. Received March 18, 1999; accepted June 8, L. B. Agellon is a Senior Scholar of The Alberta Heritage Foundation for Medical Research. D. E. Vance is a Medical Scientist of The Alberta Heritage Foundation for Medical Research. H. J. Verkade is a Clinical Research Fellow of the Netherlands Organization for Scientific Research. C. J. Walkey s present address is: Department of Cancer Biology, Dana Farber Cancer Institute and Harvard Medical School, Boston, MA. Supported by grants MT (to L.B.A.) and MT-5182 (to D.E.V.) from the Medical Research Council of Canada, and a NATO collaborative grant (to H.J.V., F.K., L.B.A., and D.E.V.). C. J. Walkey was supported by Studentships from the Medical Research Council of Canada and The Alberta Heritage Foundation for Medical Research. Address reprint requests to: L. B. Agellon, 303 Heritage Medical Research Centre, Department of Biochemistry, University of Alberta, Edmonton, AB T6G 2S2 Canada. luis.agellon@ualberta.ca; fax: (780) Copyright 1999 by the American Association for the Study of Liver Diseases /99/ $3.00/0 725 across the apical membrane into the bile. Bile phospholipids are highly specific in class (90%-95% phosphatidylcholine [PC]), 1,2 as well as acyl chain composition (predominantly C16:0 fatty acid at the sn-1 position and either C18:1 or C18:2 fatty acid at the sn-2 position). 2,3 In mice, the total amount of PC secreted by the liver into the bile in a single day is equivalent to the amount of PC present in the liver. 4 Studies using targeted gene disruption technology demonstrated that the product of the mdr2 gene is essential for biliary PC secretion in mice. 5 It is now firmly established that the mdr2 gene encodes a protein that facilitates the translocation of PC from the inner to the outer leaflet of the canalicular membrane. 6-8 PC can be synthesized by 2 biosynthetic pathways: de novo from choline via the CDP-choline pathway or from phosphatidylethanolamine (PE) by 3 successive methylation reactions catalyzed by PE N-methyltransferase (PEMT) 9 (Fig. 1). The CDP-choline pathway is used by all nucleated animal cells, but the PEMT pathway is quantitatively significant only in liver parenchymal cells. In rat liver, 20% to 40% of the PC is derived from the methylation of PE. 10 There is evidence suggesting that PC originating from either of the 2 pathways has a distinct utilization. For example, the PC secreted by hepatocytes in vitro as surface component of lipoproteins appears to originate preferentially from a pool supplied by the PEMT pathway. 11,12 It is not known whether each of the 2 biosynthetic pathways serves a specific function to account for the quantity and/or acyl chain composition of PC secreted into the bile. It has been demonstrated that biliary PC originates mainly from preexisting PC 13 rather than from PC newly synthesized by the CDP-choline pathway. 14 Yet the contribution of either of the 2 PC biosynthetic pathways to provide physiological concentrations of PC in the bile has not been determined. Apart from a specific contribution of each of the 2 pathways to the quantity of PC secreted into bile, a particular biosynthetic route may be involved in acyl chain specificity of PC secreted into bile. The PEMT-deficient mice, 15 in combination with the ability to inactivate the CDP-choline pathway by substrate deficiency (choline deficiency), provide the opportunity to study the metabolic fate and function of PC from either of the PC biosynthetic pathways in the intact mammalian liver. Feeding wild-type mice a choline-deficient (CD) diet induces a state in which PC is synthesized mainly by methylation of PE, whereas in PEMT-deficient mice fed a choline-supplemented (CS) diet, PC can only be synthesized from choline via the CDP-choline pathway (Fig. 1). Both PC biosynthetic pathways are inhibited when PEMT-deficient mice are fed a CD diet. In the present study, the quantitative and qualitative

2 726 AGELLON ET AL. HEPATOLOGY September 1999 iodine vapor. The bands corresponding to PC were recovered from the plates and subjected to gas chromatographic analysis. Gas Chromatography and Mass Spectrometry. Bile samples, PC purified from liver homogenates by TLC, and the Bligh and Dyer extract of the basal semisynthetic diet were prepared for gas chromatographic analysis following the method described by Lepage and Roy. 18 The methyl esters of fatty acids were analyzed using a Perkin-Elmer 8420 capillary gas chromatograph. The retention times of the different fatty acids were compared with those of pure fatty acid standards (Avanti Polar Lipids, Inc.). The Bligh and Dyer extracts of the pooled bile samples were also analyzed by electrospray ionization mass spectrometry 19 using a Micromass ZabSpecoaTOF mass spectrometer at the University of Alberta Mass Spectroscopy Laboratory. Detection was performed in the positive-ion mode. Statistics. Statistical analysis was performed using GB-STAT (version 5.0) software (Dynamic Microsystem, Inc., Silver Spring, MD). Differences among the groups were evaluated using ANOVA with Neuman-Keul s test and were considered significant at P.05. FIG. 1. Biosynthesis of PC in the liver. PC can be synthesized de novo from choline via the CDP-choline pathway or by methylation of PE via the PEMT pathway. The 2 pathways can be separated by using dietary choline deficiency in combination with genetic PEMT deficiency. Wild-type mice (Pempt / ) fed a CD diet synthesize PC mainly via the PEMT pathway. PEMT-deficient mice (Pempt / ) fed a CS diet synthesize PC via the CDP-choline pathway. importance of each of the 2 pathways for the secretion of PC into bile were evaluated. MATERIALS AND METHODS Animals and Diets. Experiments involving the use of animals were performed following protocols approved by the University of Alberta Health Sciences Animal Welfare Committee and in accordance with the guidelines established by the Canadian Council on Animal Care. Mice homozygous for the disrupted Pempt allele 15 and normal littermates were given free access to chow and water, and maintained in a temperature-controlled environment under a reverse 12-hour light/dark cycle. Access to chow was withdrawn beginning 12 hours before the onset of the controlled diet study. The semisynthetic diet, which either lacked or contained 0.4% (by weight) choline chloride, was given for 60 hours. The major species of fatty acids in the semisynthetic diet (#901387; ICN Biomedicals, Montreal, Quebec, Canada) were represented by C16:0 (24%), C18:0 (16%), C18:1 (39%), C18:2 (9%), and C20:4 (0.5%). The mice were fasted for 12 hours before termination. Bile was collected from the gallbladders by aspiration, and the volume was estimated using an automatic micropipettor. Livers were stored at 70 C until analysis. Biochemical Analyses. The total bile acid, cholesterol, and PC concentrations in bile were determined using commercial diagnostic kits based on 3 -hydroxysteroid dehydrogenase (for total bile acids, from Sigma-Aldrich Canada Ltd., Oakville, Ontario, Canada), cholesterol oxidase (for cholesterol, from Boehringer Mannheim Canada, Laval, Quebec, Canada), and choline oxidase (for PC, Wako Chemical USA Inc., Richmond, VA). The inorganic phosphorous and choline concentrations in the bile samples, as determined by the method of Bartlett 16 and the choline oxidase based assay, respectively, were similar. Equal volumes of bile from each mouse within each of the experimental and control groups were combined. The lipids were then extracted by the method of Bligh and Dyer 17 and analyzed by thin-layer chromatography (TLC). The solvent used to develop the plate was chloroform/methanol/acetic acid/water (50:25: 8:3). The separated lipids were visualized by charring the TLC plate, and the migration of the separated phospholipids was compared with those of pure PC and PE standards (Avanti Polar Lipids, Inc., Alabaster, AL). Homogenates of liver from each mouse were prepared as described previously. 15 The lipids were extracted and separated by TLC as described above, and then visualized with RESULTS The CS and CD diets were given to wild-type and Pempt / mice for 60 hours to determine if liver PC synthesized via either of the PC biosynthetic pathways has specific significance for the amount and composition of PC secreted into the bile. The lipid composition of bile collected from the gallbladders of the mice is shown in Table 1. There were no differences in total bile acids, cholesterol, and phospholipid concentrations in the bile of wild-type and Pempt / mice fed a CS diet (hereafter referred to as control mice and CDP-choline mice, respectively). By comparison, the phospholipid and cholesterol concentrations were reduced in the bile of Pempt / mice fed a CD diet (hereafter referred to as PEMT mice ). Inactivation of both PC biosynthetic pathways by feeding Pempt / mice a CD diet (hereafter referred to as PC-null mice ) resulted in the substantial reduction of biliary PC and cholesterol levels (Table 1). The PC-to-bile acid ratio in bile has been related to the efficacy by which bile acids induce the secretion of biliary PC. 1,20 As evident in Table 1, the PC-to-bile acid ratio of PEMT mice was decreased by 39% to 47% when compared with either control or CDP-choline mice, thus illustrating the decreased efficacy of bile acid induced phospholipid secretion when PC synthesis occurs solely via the PEMT pathway. Analysis of gallbladder phospholipids by TLC (Fig. 2) indicated that PC remained the dominant biliary phospholipid regardless of whether the sole biosynthetic source of PC was the CDP-choline pathway or the PEMT pathway. The results from Table 1 and Fig. 2 clearly demonstrate that either of the PC biosynthetic pathways is capable of supplying the TABLE 1. Gallbladder Bile Lipid Composition PC Biosynthetic Pathway (mmol/l) Component Both (control) CDP-choline PEMT None (PC-null) Bile acids Cholesterol * PC NOTE. Bile collected from mice in each of the indicated groups (n 3-5 per group) were analyzed individually for composition. The values shown are mean SD. *P.05, vs. control and CDP-choline. P.01, vs. control and CDP-choline. P.05, vs. control and CDP-choline. P.01, vs. control, CDP-choline and PEMT.

3 HEPATOLOGY Vol. 30, No. 3, 1999 AGELLON ET AL. 727 homogenates revealed a large reduction in the contribution of C18:1 to bulk hepatic PC when the main source of PC was the PEMT pathway, or when both PC biosynthetic pathways were inhibited (Table 3). On the other hand, the C20:4 fatty acid content of hepatic PC was not affected even in PC-null mice. Because gas chromatographic analysis of fatty acids includes their hydrolysis from their parent compounds, we also analyzed bile PC composition by electrospray ionization mass spectrometry 19 to determine the molecular masses of intact PC molecules. The mass spectra (Fig. 3) of bile from Pempt / and Pempt / mice on the CD diet were similar, indicating that the unique acyl chain specificity of biliary PC largely remained unchanged even under an extreme metabolic condition. FIG. 2. TLC analysis of bile lipids. Bile was collected from mice (n 3-4 per group) representing the 3 Pempt genotypes ( /, wild-type; /, heterozygotes; /, deficient) that were given CS or CD diets. Biliary lipids were extracted from pooled bile samples (equal volumes from each mouse) and analyzed by TLC. The migration of pure PC and PE are indicated on the left. BA, bile acids. pool of PC for bile secretion under conditions in which one pathway predominates. However, the hepatic capacity for providing normal levels of biliary PC in PEMT mice appears to be insufficient. The fatty acid composition of bile was analyzed by gas chromatography to assess if either of the PC biosynthetic pathways influenced the unique acyl chain specificity of biliary PC. As evident in Table 2, the C16:0, C18:0, C18:1, and C18:2 fatty acids comprised 84.8% of the total fatty acids in the bile of control mice. The total contribution of these fatty acids to the bile of CDP-choline mice and PEMT mice was not significantly altered (84.9% and 84%, respectively). In PC-null mice, which are under the extreme condition of dietary choline deficiency and genetic PEMT deficiency, the contribution of these 4 major fatty acids in bile was reduced. In contrast, the percentage of C20:4 increased and this was correlated with Pempt gene dosage as the amount of C20:4 fatty acid in the bile of Pempt / mice fed the CD diet was intermediate that of Pempt / and Pempt / mice fed the same diet (data not shown). Analysis of PC isolated from liver TABLE 2. Fatty Acid Composition of Bile PC Biosynthetic Pathway (mol%) Fatty acid Both (control) CDP-choline PEMT None (PC-null) 16: * : : : : Others NOTE. Gallbladder bile collected from mice (n 3 per group) were analyzed individually for fatty acid composition. The values shown are mean SD. *P.05, vs. control, and PEMT. P.01, vs. CDP-choline. P.01, vs. control. P.01, vs. control, CDP-choline and PEMT. DISCUSSION In the present study, we investigated the importance of the PEMT and the CDP-choline biosynthetic pathways for the quantity and acyl chain composition of PC in mouse gallbladder bile. The results demonstrate that physiological gallbladder bile concentrations of PC are present in mice with only the CDP-choline pathway, but that PC concentrations are decreased by 40% in mice with mainly the PEMT pathway. Interestingly, the unique acyl chain composition of gallbladder PC was independent of the activity of either or both of the PC biosynthetic pathways. The existence of PEMT in the liver is compatible with the hypothesis that some specific, PC-requiring function of the liver has an obligate need for PC from one specific biosynthetic pathway. For the secretion of PC in the form of very-lowdensity lipoproteins, a preferential contribution of the PEMT pathway over the CDP-choline pathway has been demonstrated in cell cultures. 11,12 Alternatively, the synthesis of PC from PE may simply represent a general mechanism that is used to provide the large quantity of PC required by some other liver-specific function, such as bile formation. Another potential benefit of the PEMT pathway involves its ability to guarantee choline homeostasis, via subsequent catabolism of PE-derived PC, in instances of nutritional choline insufficiency. 4 The contribution of the 2 biosynthetic pathways for bile PC formation has not been clearly resolved, because it was previously not possible to consider each of the 2 PC biosynthetic pathways separately and independently. Available data from past studies do suggest that PC from the CDP-choline pathway, as well as PC originating from PE-methylation, are secreted into bile. 21,22 The specific activity of methylation- TABLE 3. Fatty Acid Composition of Hepatic PC PC Biosynthetic Pathway (mol%) Fatty acid Both (control) CDP-choline PEMT None (PC-null) 16: : : * * 18: : Others NOTE. PC was isolated from liver (n 3-5 per group) homogenates and analyzed for fatty acid composition. The values shown are mean SD. *P.01, vs. control, and CDP-choline mice. P.05, vs. control, and CDP-choline mice.

4 728 AGELLON ET AL. HEPATOLOGY September 1999 FIG. 3. Analysis of PC species in lipid extract of mouse bile by electrospray ionization mass spectrometry. Lipid extracts of pooled bile from Pempt / (n 3) or Pempt / (n 3) mice fed the CD diet were analyzed. Detection was performed in the positive-ion mode. The numbers in the parentheses indicate the total number of carbon and number of double bonds in the fatty acid component of PC. The molecular masses of the specific PC species are also shown. derived PC was higher in bile PC than in cellular PC, suggesting preferential secretion into bile. On the other hand, a reduction in biliary PC secretion has been documented in rats fed a CD diet, compatible with a quantitatively important role of the CDP-choline pathway. 21 Previously, it was demonstrated that 3 days of choline deficiency reduced choline concentration in rat liver by 12-fold, leading to severe impairment of the CDP-choline pathway. 23 Severe liver pathology is evident after 3 days when mice lacking PEMT are fed a CD diet. 4 This finding illustrates the physiological importance of ongoing PC synthesis in normal hepatic function. Surprisingly, neither the CDPcholine mice (i.e., Pempt / mice fed the CS diet), nor PEMT mice (i.e., Pempt / mice fed the CD diet) showed any overt signs of abnormality, suggesting that inactivation of either of the 2 pathways can be compensated for, at least partially, by the remaining pathway. 4 Nevertheless, in the present study, we identified a liver-specific function, namely the supply of physiological amounts of PC to bile, that cannot be fully compensated for by the PEMT pathway. Although the results clearly illustrate that each of the 2 PC biosynthetic pathways can provide PC for secretion into the bile, the reduced concentration of PC and especially the reduced PC-to-bile acid ratio in gallbladder bile of PEMT mice indicates that, under physiological conditions, the CDP-choline pathway contributes a greater proportion of PC to bile than the PEMT pathway. Interestingly, the total level of PC in the liver is not significantly altered in PEMT mice, and these mice appeared phenotypically normal. 4 Apparently, under the condition of reduced PC biosynthetic capacity as a result of the diminished CDP-choline pathway, the liver prefers to retain synthesized PC inside the liver rather than secrete it into the bile. The significantly decreased PC-to-bile acid ratio in mice in which the CDP-choline pathway is impaired reflects the decreased efficacy by which bile acids induce the phospholipid secretion into the bile. Based on the determinants of the efficacy of bile acid induced phospholipid secretion, 1,20 it is tempting to speculate that an alteration in the composition of the bile canalicular membrane is involved in this process. We are presently investigating whether down-regulation of mdr2 P-glycoprotein function contributes to the decreased PC concentration and PC-to-bile acid ratio when the CDPcholine pathway is diminished. It should be noted that the present data on the preservation of hepatic PC concentrations were obtained under a condition in which biliary PC secretion was not stimulated. It remains to be determined whether hepatic PC content is still upheld when a metabolic stress is imposed, such as through administration of bile acids that favor the secretion of PC into bile. The molecular speciation of biliary PC is distinct from that of PC in the bulk liver, 3,24 and therefore, it was of interest to determine if the biosynthetic origin of the PC headgroup has any significance in specifying the ultimate fatty acyl composition of biliary PC. In mice, the C16:0, C18:1, and C18:2 fatty acid species predominated in bile regardless of whether hepatic PC was derived solely from choline or from PE. Remarkably, the fatty acyl composition of biliary PC was not drastically altered when either of the 2 or both PC biosynthetic pathways were inhibited. These findings indicate that the process responsible for the molecular speciation of biliary PC largely operates independently from the origin of hepatic PC in its substrate pool. These observations now allow a reinterpretation of previously reported differences in the proportion of choline-derived and PE-derived PC in bile. 21,22 Based on our present data, it seems highly likely that those results have been due to the specific activities of the respective biosynthetic pathways at the time of sampling. The activities of these 2 pathways are likely adjusted independently according to the overall metabolic demand of the liver for PC. In contrast to bile in which the effect of choline deficiency on the contribution of C18:1 fatty acid to biliary PC was small, a large decrease in the proportion of C18:1 fatty acids in the bulk hepatic PC was apparent. The basis for this change is not clear, but appears to be specific for dietary choline status, because the same effect was evident regardless

5 HEPATOLOGY Vol. 30, No. 3, 1999 AGELLON ET AL. 729 of hepatic PEMT status. A simple explanation is that choline deficiency has independent effects on fatty acid metabolism. Alternatively, the processing of PC that is not secreted into bile, in contrast to PC destined to bile, may actually differ depending on the origin of the PC headgroup. In summary, we provide definitive proof that the PEMT and CDP-choline PC biosynthetic pathways contribute independently to the pool of PC that is secreted into bile. Apparently, the diminution of either of the 2 pathways can be tolerated without overt signs of hepatic failure during the experimental period. In the case of the loss of the CDPcholine pathway, hepatic PC levels appear to be maintained at the expense of PC secretion into bile. The unique fatty acyl composition of biliary PC is determined independently of the biosynthetic origin of the PC headgroup and appears highly conserved, even under the extreme metabolic pressure imposed by dietary choline insufficiency and genetic PEMT deficiency. Acknowledgment: The authors thank L. Yu and R. Waite for technical assistance. REFERENCES 1. Verkade HJ, Vonk RJ, Kuipers F. New insights into the mechanism of bile acid induced biliary lipid secretion. HEPATOLOGY 1995;21: Cohen DE. Hepatocellular transport and secretion of biliary phospholipids. Semin Liver Dis 1996;16: Hay DW, Cahalane MJ, Timofeyeva N, Carey MC. Molecular species of lecithin in human gallbladder bile. J Lipid Res 1993;34: Walkey CJ, Yu L, Agellon LB, Vance DE. Biochemical and evolutionary significance of phospholipid methylation. J Biol Chem 1998;273: Smit JJM, Schinkel AH, Oude Elferink RPJ, Groen AK, Wagenaar E, van Deemter L, Mol CAAM, et al. Homozygous disruption of the murine mdr2 P-glycoprotein gene leads to a complete absence of phospholipid from bile and to liver disease. Cell 1993;75: Ruetz S, Gros S. Phosphatidylcholine translocase: a physiological role for the mdr2 gene. Cell 1994;77: Van Helvoort A, Smith AJ, Sprong H, Fritzche I, Schinkel AH, Borst P, Van Meer G. MDR1-P-glycoprotein is a lipid translocase of broad specificity, while MDR3 P-glycoprotein specifically translocates phosphatidylcholine. Cell 1996;87: Crawford AR, Smith AJ, Hatch VC, Oude Elferink RPJ, Borst P, Crawford JM. Hepatic secretion of phospholipid vesicles in the mouse critically depends on mdr2 or MDR3 P-glycoprotein expression. J Clin Invest 1997;100: Vance DE. Glycerolipid biosynthesis in eukaryotes. In: Vance DE, Vance JE, eds. Biochemistry of Lipids, Lipoproteins and Membranes. 31. Amsterdam: Elsevier, 1996: Sundler R, Akesson B. Regulation of phospholipid biosynthesis in isolated rat hepatocytes. Effect of different substrates. J Biol Chem 1975;250: Vance JE, Nguyen TM, Vance DE. The biosynthesis of phosphatidylcholine by methylation of phosphatidylethanolamine derived from ethanolamine is not required for lipoprotein secretion by cultured rat hepatocytes. Biochim Biophys Acta 1986;875: Vance JE, Vance DE. Specific pools of phospholipids are used for lipoprotein secretion by cultured rat hepatocytes. J Biol Chem 1986;261: Patton GM, Fasulo JM, Robins SJ. Hepatic phosphatidylcholines: evidence for synthesis in the rat by extensive reutilization of endogenous acylglycerides. J Lipid Res 1994;35: Robins SJ, Brunengraber H. Origin of biliary cholesterol and lecithin in the rat: contribution of new synthesis and preformed hepatic stores. J Lipid Res 1982;23: Walkey CJ, Donohue LR, Bronson R, Agellon LB, Vance DE. Disruption of the murine gene encoding phosphatidylethanolamine N-methyltransferase. Proc Natl Acad Sci U S A 1997;94: Bartlett GR. Phosphorous determination in column chromatography. J Biol Chem 1959;234: Bligh EG, Dyer WJ. A rapid method of total lipid extraction and purification. Can J Biochem Physiol 1959;37: Lepage G, Roy CC. Direct transesterification of all classes of lipids in a one-step reaction. J Lipid Res 1986;27: Lehmann WD, Koester M, Erben G, Keppler D. Characterization and quantification of rat bile phosphatidylcholine by electrospray-tandem mass spectrometry. Anal Biochem 1997;246: Verkade HJ, Havinga R, Kuipers F, Vonk RJ. Mechanism of biliary lipid secretion. In: Hofmann AF, Paumgartner G, Stiehl A, eds. Bile Acids in Gastroenterology: Basic and Clinical Advances. Lancaster: Kluwer Academic Publishers, 1995: Balint JA, Beeler DA, Kyriakides EC, Treble DH. The effect of bile salts upon lecithin synthesis. J Lab Clin Med 1971;77: Robins SJ, Armstrong MJ. Biliary lecithin secretion. II. Effects of dietary choline and biliary lecithin synthesis. Gastroenterology 1976;70: Haines DSM, Rose CI. Impaired labelling of liver phosphatidylethanolamine from ethanolamine- 14 C in choline deficiency. Can J Biochem 1970;48: Balint JA, Beeler DA, Treble DH, Spitzer HL. Studies in the biosynthesis of hepatic and biliary lecithins. J Lipid Res 1967;8: