Biliary Phospholipid Research Articles

Management of the biliary complications associated with liver transplantation: 13 years of experience

Nonanastomotic biliary strictures are troublesome complications after liver transplantation. The pathogenesis of NAS is not completely clear, but experimental studies suggest that bile salt toxicity is involved.In one hundred and eleven adult liver transplants, bile samples were collected daily posttransplantation for determination of bile composition. Expression of bile transporters was studied perioperatively.Nonanastomotic biliary strictures were detected in 14 patients (13%) within one year after transplantation. Patient and donor characteristics and postoperative serum liver enzymes were similar between patients who developed nonanastomotic biliary strictures and those who did not. Secretions of bile salts, phospholipids and cholesterol were significantly lower in patients who developed strictures. In parallel, biliary phospholipids/bile salt ratio was lower in patients developing strictures, suggestive for increased bile cytotoxicity. There were no differences in bile salt pool composition or in hepatobiliary transporter expression.Although patients who develop nonanastomotic biliary strictures are initially clinically indiscernible from patients who do not develop nonanastomotic biliary strictures, the biliary bile salts and phospholipids secretion, as well as biliary phospholipids/bile salt ratio in the first week after transplantation, was significantly lower in the former group. This supports the concept that bile cytotoxicity is involved in the pathogenesis of nonanastomotic biliary strictures.

The effects of short term lipid infusion on plasma and hepatic bile lipids in humans

BACKGROUNDPatients on parenteral nutrition have an increased incidence of gall bladder sludge and gallstone disease, thought to be related to bile stasis. Intravenous lipid emulsions, especially those containing medium chain...

Dietary beef tallow alters the fatty acid profiles of bile and liver membranes, but not secretion of cholesterol in bile

To test the hypothesis that type and amount of dietary fat has a regulatory effect on secretion of cholesterol in bile, young growing pigs were fed diets containing either 20% or 40% of energy as primarily either beef tallow or soybean oil or as a 50:50 (wt:wt) blend of tallow and soybean oil. After week 6, pigs were injected with autologous low-density lipoproteins containing 3H-cholesterol. Serial bile samples were collected for 24 hours; then tissues were collected. Amount of tallow in dietary fat correlated positively (P < 0.05) with amounts of palmitoleic and oleic and negatively with linoleic and linolenic fatty acids in biliary phospholipids. In addition, amount of tallow in dietary fat correlated positively with amounts of myristic, palmitoleic and oleic acids but negatively with stearic acid in membranes of liver. Composition of dietary fat, however, did not affect concentration or daily secretion of cholesterol, bile acids or phospholipids in bile. Furthermore, secretion of 3H-cholesterol as biliary cholesterol or bile acids was not affected by type or amount of dietary fat. Concentration of cholesterol in liver tended to be greater in pigs fed diets containing tallow (P<0.10); no differences in cholesterol concentrations, however, were seen in aorta, heart, skeletal muscle, or gastrointestinal tract. Percentage of injected 3H-cholesterol was greater (P<0.001) in perirenal adipose tissue from pigs fed diets containing soy oil than from pigs fed diets containing tallow. There were no significant differences (P<0.05) seen in percentage of injected 3H-cholesterol deposition in several other organs. In summary, type of dietary fat altered fatty acid composition of biliary phospholipids and liver membranes but had no effect on cholesterol secretion into bile and no major effects on cholesterol distribution in major organs of young pigs.

Comparison of effects of two different monounsaturated oils on biliary secretion in rats

Effects of two oils, both with the same high content of monounsaturated fatty acids (MUFAs), 80.28% for olive oil (OO) versus 80.54% for high-oleic sunflower oil (HOSO), on biliary bile acid and lipid secretion have been studied in rats. There was a gradual increase in bile flow, concentration and output of bile acids during the administration of the oils, the increase being higher in HOSO-fed animals. The composition of the biliary bile acids showed a decrease in the percentage of chenodeoxycholic acid, accompanied by an increase in the level of muricholic acid after the administration of two oils when compared to control group. There was no difference in the percentage of cholic acid and deoxycholic acid among experimental groups. Biliary phospholipid levels were not modified in any groups. Biliary cholesterol content was decreased at 3 and 5 days in OO-fed animals when compared with the other experimental groups. Consequently, the ratio phospholipid/free cholesterol was enhanced in these animals. There were no statistically significant differences in the individual phospholipid among the experimental groups. The major phospholipid in the bile was phosphatidylcholine. The fatty acid composition of biliary lipids was lightly modified similar to the major fatty acid composition in the oils. These results showed a decrease in biliary cholesterol content after OO administration compared to other experimental groups, and suggest that the MUFAs are not solely responsible for the biliary cholesterollowering effect. There was no a relationship between biliary lipid and bile acid secretion after the administration of oils.

Cloning and gene structure of rat phosphatidylcholine transfer protein, Pctp.

Phosphatidylcholine transfer protein (PC-TP) is a cytosolic lipid transfer protein that promotes intermembrane transfer of phosphatidylcholines but no other phospholipids. Although its physiological function remains unknown, phosphatidylcholine transfer protein is enriched in liver and evidence from model systems suggests a role in hepatocellular selection and transport of biliary phospholipids. To facilitate in vivo studies, a cDNA encoding rat PC-TP was cloned by library screening and 5'-rapid amplification of cDNA ends. Genomic cloning demonstrated the rat Pctp gene spans 10. 8kb and is comprised of six exons. The putative transcription initiation site was identified 50bp upstream of the translation initiation site. Nucleotide sequence analysis of the 5'-flanking region revealed a CAAT- but no TATA-box. Transient transfection of a series of 5'-deleted Pctp-promoter-firefly luciferase constructs into Reuber H35 rat hepatoma cells, which express Pctp mRNA, and Gunn rat fibroblasts, which do not, suggest that cis-acting elements in a 637bp promoter region contribute to enhanced expression of PC-TP in liver.

Enhancement of fatty acid and cholesterol synthesis accompanied by enhanced biliary but not very—low-density lipoprotein lipid secretion following sustained pravastatin blockade of hydroxymethyl glutaryl coenzyme A reductase in rat liver

A 3-week treatment of rats with pravastatin (PV) augmented biliary cholesterol and phospholipid output 3.6- and 2.2-fold over controls, while bile acid (BA) output and kinetics were unchanged. No major changes were detected in hepatic and serum cholesterol concentrations despite the PV inhibitory property on hydroxymethyl glutaryl coenzyme A (HMG CoA) reductase. To evaluate the mechanisms of this adaptive phenomenon, several parameters of hepatic lipid homeostasis were assessed. Biliary cholesterol changes could not be attributed to an increased influx of lipoprotein cholesterol to the liver and bile. Hepatic low-density lipoprotein (LDL) receptor content, as inferred from Western blot analysis, was unchanged, as was the biliary excretion of labeled cholesterol derived from chylomicron remnants. In vivo 3H 2O-incorporation studies showed an 80% increase in hepatic cholesterol synthesis, evidence for bypass of the PV block. Remarkably, fatty acid synthesis was also stimulated twofold, providing substrate for hepatic triglycerides, which were slightly enhanced. However, serum triglycerides decreased 52% associated with a 22% decrease in hepatic very—low-density lipoprotein (VLDL) secretion. Thus, the biochemical adaptation following PV treatment produces complex alterations in hepatic lipid metabolism. An enhanced supply of newly synthesized cholesterol and fatty acids in association with a limited VLDL secretion rate augments the biliary lipid secretion pathway in this experimental model.

Phospholipid changes in children with pancreatic sufficiency and insufficiency

The phospholipid secretion rates and phospholipase A 1 and phospholipase A 2 activities in biliary-pancreatic secretions of patients with pancreatic sufficiency and insufficiency were measured using 31P-NMR spectroscopy. It was possible to quantify conveniently the individual phospholipids without prior extraction of lipids or treatment of the samples with detergent. The reciprocal nature of the decrease in phosphatidylcholine concentration, compared with the increase in the concentration of 1-lysophosphatidylcholine and 2-lysophosphatidylcholine, suggested a substrate/product relationship consistent with the activities of phospholipase A 1 and phospholipase A 2, respectively. Although the secretion rates of total biliary phospholipids among the patients with pancreatic sufficiency were similar, the phospholipase A 1 and phospholipase A 2 activities varied considerably. The latter differences were similar to their pancreatic lipase and colipase secretion levels and hence their degree of pancreatic dysfunction. The biliary-pancreatic secretions from patients with pancreatic insufficiency showed no enzyme activities. Total biliary phospholipid secretions in patients with pancreatic insufficiency with common bile duct stenosis were significantly lower than those in patients with pancreatic sufficiency, and pancreatic insufficiency without common bile duct stenosis.

Sphingomyelins of rat liver: biliary enrichment with molecular species containing 16:0 fatty acids as compared to canalicular-enriched plasma membranes.

We harvested canalicular-enriched plasma membranes of hepatocytes and collected fistula bile from male rats and isolated the sphingomyelins. Following sphingomyelinase hydrolysis, we identified the sphingomyelin molecular species on the basis of their benzoylated ceramide derivatives employing high performance liquid chromatography. Sphingomyelin constitutes </=3% of total biliary phospholipids (which are mostly sn-1 16:0 long-chain phosphatidylcholines) and approximately 30% of canalicular-enriched membranes. In both cases, the principal molecular species were composed of 16:0, 18:0, 20:0, 22:0, 23:0, 24:0, 24:1 and 24:2 fatty acid classes. However, the 16:0 fatty acid species was enriched in biliary sphingomyelin to a significantly greater degree than in sphingomyelins of canalicular-enriched plasma membranes (46% vs. 25% of total). We argue a physical-chemical case for laterally separated domains of very long chain sphingomyelins on the exoplasmic leaflet of the canalicular membrane. We bolster our hypothesis by the likelihood that the least hydrophobic, e.g., 16:0 sphingomyelin molecular species, are miscible with biliary phosphatidylcholines, and are secreted into bile. Laterally separated domains of very long chain sphingomyelins on the exoplasmic leaflet of the canalicular membrane could provide a means of sequestering cholesterol molecules prior to secretion into bile.

Effect of Intravenous Bilirubin Infusion on Biliary Phospholipid Secretion, Hepatic P-Glycoprotein Expression, and Biliary Cytotoxicity in Pigs

Background: Infusion of large intravenous bilirubin loads in bile acid-depleted pigs reduces P-glycoprotein-dependent biliary phospholipid secretion and increases the cytotoxicity of bile. The reasons for the diminution of biliary phospholipid secretion and the increase in biliary cytotoxicity are not known. This study was undertaken to determine whether the bilirubin-induced lowering of biliary phospholipid secretion is associated with alterations in hepatic P-glycoprotein (P-gp) expression and to determine why bilirubin infusions increase biliary cytotoxicity. Methods: Hepatic bile was collected from bile acid-depleted pigs before and during intravenous bilirubin infusion. Hepatic P-gp expression was measured with protein blot analysis, using the P-gp-specific antibody C219. Biliary cytotoxicity was assayed against erythrocytes. The biliary phospholipid fatty acid profile was determined by means of gas chromatography. Results: Bilirubin infusions lowered biliary phospholipid secretion by 69% without changing hepatic P-gp expression, suggesting that bilirubin infusions have an inhibitory effect on hepatic P-gp activity. Bilirubin infusions did not cause P-gp losses into bile. An unequivocal, proportional relationship (r2 = 0.80) pertained between cytotoxicity and the bile acid to phospholipid ratio in bile secreted before and during bilirubin infusion and in phosphatidylcholine-supplemented bile. Unconjugated bilirubin in bile did not contribute to biliary cytotoxicity. Biliary phospholipids were always phosphatidylcholine > phosphatidylethanolamine, mainly of C16:0, 18:2 and C16:0, 18:1 fatty acid configuration. Conclusions: Intravenous bilirubin loads reduce biliary phospholipid secretion without changing hepatic P-gp expression. Bilirubin infusions increase biliary cytotoxicity by augmenting the biliary bile acid to phospholipid ratio.

Decreased Na +-dependent taurocholate uptake and low expression of the sinusoidal Na +-taurocholate cotransporting protein (Ntcp) in livers of mdr2 P-glycoprotein-deficient mice

Background/Aims: Ntcp-mediated uptake of bile salts at the basolateral membrane of hepatocytes is required for maintenance of their enterohepatic circulation. Expression of Ntcp is reduced in various experimental models of cholestasis associated with increased plasma bile salt concentrations. Mdr2 P-glycoprotein-deficient mice lack biliary phospholipids and cholesterol but show unchanged biliary bile salt secretion and increased bile flow. These mice are evidently not cholestatic, but plasma bile salt concentrations are markedly increased. The aim of this study was to investigate the role of Ntcp in the elevated bile salt levels in mdr2 P-glycoprotein-deficient (−/−) mice. Methods: Plasma membranes were isolated from male wild-type (+/+) and mdr2 (−/−) mice for measurement of Na +-dependent taurocholate transport and assessment of Ntcp protein levels by Western blotting. Northern blot analysis and competitive reverse transcription-polymerase chain reaction were used to determine hepatic Ntcp mRNA levels. Results: Kinetic analysis showed a 2-fold decrease in the V max of Na +-dependent taurocholate transport, with an unaffected K m in (−/−) mice compared with (+/+) controls. Ntcp protein levels were 4–6-fold reduced in plasma membranes of (−/−) mice relative to sex-matched controls. Surprisingly, hepatic Ntcp mRNA levels were not significantly affected in the (−/−) mice. Conclusions: Elevated plasma bile salt levels in mdr2 P-glycoprotein-deficient mice in the absence of overt cholestasis are associated with reduced Ntcp expression and transport activity. This is due to posttranscriptional down-regulation of Ntcp.

Taurocholate induces preferential release of phosphatidylcholine from rat liver canalicular vesicles.

Biliary phospholipid secretion involves predominant segregation of canalicular phosphatidylcholine into bile. We tested the hypothesis that micellar concentrations of the major physiologic bile salt taurocholate can preferentially solubilize phosphatidylcholine from the canalicular rat liver plasma membrane. Subcellular fractions from rat liver and kidney were isolated with standardized procedures, incubated in vitro with taurocholate or 3-[(3-cholamidopropyl)dimethylammonio]-propane-1-sulphonate (CHAPS) and released phospholipids determined after centrifugation. After incubation of canalicular (cLPM) and basolateral (blLPM) rat liver plasma membrane vesicles with 6 and 8 mM taurocholate, the proportion of phosphatidylcholine released was about two-fold higher as compared with its relative contribution to the overall lipid composition of the membranes. Quantitatively, this taurocholate-induced preferential phosphatidylcholine release was about four-fold higher in cLPM (117 nmol) as compared with blLPM (28 nmol). Comparison of membranes from different organs showed that increased sphingomyelin content reduced taurocholate-induced phosphatidylcholine release. Furthermore, phosphatidylcholine release from cLPM did not fit an inverse exponential relationship between membrane sphingomyelin content and phosphatidylcholine release from different starting material, indicating that cLPM is especially prone to taurocholate-induced phosphatidylcholine release. In contrast, in rat liver microsomes and kidney brush border membranes, taurocholate released phospholipids in proportion of their membrane contents, indicating an unspecific membrane solubilizing effect only. Similarly, CHAPS had an unselective lipid solubilizing effects in cLPM and blLPM. These results support the concept that the very last step of canalicular phospholipid secretion is mediated in vivo by bile salt-induced vesiculation of phosphatidylcholine-enriched microdomains from the outer leaflet of cLPM.

The role of dietary choline in the beneficial effects of lecithin on the secretion of biliary lipids in rats

Earlier studies showed that dietary soybean lecithin increases biliary lipid secretion, which mainly comes from the contribution of high density lipoprotein (HDL) and hepatic microsomal pools of phosphatidylcholine and cholesterol. In addition, a lecithin diet enhances bile secretion and prevents bile acid-induced cholestasis. This study evaluated the contribution of choline, a component of lecithin, to the observed effect of lecithin on biliary secretory function. Rats were fed either a control diet (CD), a choline diet (ChD) or a lecithin-enriched diet (LD) for 2 weeks. Results showed that like LD, ChD induced an increase in bile flow and bile acid secretion rate when compared with the control diet. However, unlike LD, ChD did not significantly increase biliary phospholipids and cholesterol output. An increase of hydrophilic bile acids (i.e. ursodeoxycholic and muricholic acids) in bile of rats fed choline could explain why the biliary phospholipid and cholesterol secretion was not increased. During taurocholic acid infusion, both experimental diets increased bile flow and the bile acid secretion rate maximum (BASRm). The cholestasis usually observed after the BASRm is reached was inhibited by ChD and LD. Both diets induced a decrease in plasma cholesterol (total and HDL), however, only LD induced statistically significant changes. Analysis of total cholesterol and phospholipid content of microsomes and canalicular membranes indicated no statistically significant difference between control and experimental groups, either under basal conditions or after bile acid infusion. Similarly, the phospholipid classes and fatty acid composition of biliary phosphatidylcholine were not altered by feeding ChD and LD. We conclude that choline contributes to the beneficial effect of a lecithin diet on bile secretion. It is postulated that this effect may be attributed to modulation of HDL and an enhancement of the cholesterol and phospholipid pools destined for biliary secretion.

The mechanism underlying the hypocholesterolemic effect of chronic fish oil feeding in rats is not due to increased excretion of dietary cholesterol

The role of the excretion of dietary cholesterol in the hypocholesterolaemic effect of chronic fish oil feeding in rats was investigated. The hepatic uptake and processing of [ 3H]cholesterol carried in chylomicrons derived from fish oil was studied in vivo in rats fed a low fat diet or a diet supplemented with fish oil for 21 days. In addition, the effects of the fish oil diet on cholesterol esterification, cholesteryl ester hydrolysis, bile acid synthesis and biliary lipid secretion were determined. In rats fed the fish oil as compared to the low fat diet, the uptake of [ 3H]cholesterol from the blood and its secretion into bile as bile acids was significantly slower, and this was entirely due to a decrease in the bile acid fraction. Biliary bile acid mass secretion was unchanged by fish oil feeding, while biliary cholesterol and phospholipid secretion was increased. No significant differences were observed either in the expression of mRNA for cholesterol 7 α hydroxylase or the secretion of bile acids into bile after 20 h biliary drainage between the fish oil and low fat diet groups, suggesting that bile acid synthesis is not affected. These results indicate that the access of chylomicron cholesterol to the hepatic substrate pool for bile acid formation is decreased in the fish oil fed rats, and this, together with its slower uptake from the blood, accounts for the retardation of its excretion via the bile. Thus, the hypocholesterolemic effect of dietary fish oil in rats is not due to more rapid metabolism of cholesterol originating from the diet.

The effects of phospholipid molecular species on cholesterol crystallization in model biles: the influence of phospholipid head groups

Background/Aims: Variations in the molecular species of biliary phospholipids have been shown to exert major effects on cholesterol solubility and carriers in model and human biles. The aim of this study was to explore systematically the effects of various phospholipid head groups on the cholesterol crystallization process in model biles. Methods: Three different control model biles were prepared using varying proportions of egg lecithin, cholesterol and Na taurocholate. In the test biles, 20% of the egg lecithin was replaced with synthetic phosphatidylserine, phosphatidylethanolamine, phosphatidylglycerol or phosphatidylcholine, keeping the phospholipid acyl chains and other biliary lipids constant in each experiment. Results: Phosphatidylserine and phosphatidylglycerol significantly prolonged the crystal observation time, from 2 days to 10 and 6 days, respectively ( p<0.02), while phosphatidylethanolamine had little and phosphatidylcholine no effect. The crystal growth rate was significantly slowed down with 20% phospholipid replacement in the following order: phosphatidylglycerol > phosphatidylserine > phosphatidylethanolamine. The total crystal mass after 14 days, as measured by chemical analysis, was reduced by 59% with phosphatidylserine ( p<0.05), and by 73% with phosphatidylglycerol ( p<0.05); while phosphatidylethanolamine had little effect. The precipitable cholesterol crystal fractions after 14 days were significantly reduced with phosphatidylserine (54%) and phosphatidylglycerol (37%), but not with phosphatidylethanolamine or phosphatidylcholine. Conclusions: Variations in the head groups of biliary phospholipids may markedly slow down the cholesterol crystallization process in model biles.

Reduced plasma cholesterol and increased fecal sterol loss in multidrug resistance gene 2 P-glycoprotein-deficient mice

Background & Aims: mdr2 P-glycoprotein (Pgp) deficiency in mice leads to the absence of biliary phospholipids and cholesterol in the presence of normal bile salt secretion. The aim of this study was to evaluate the importance of the biliary pathway in cholesterol homeostasis by determining the effects of mdr2 Pgp deficiency on hepatic and plasma lipid levels and cholesterol kinetics in chow-fed mice. Methods: Hepatic lipid content, enzyme activities, plasma lipoprotein levels, and fecal sterol excretion were measured in wild-type (+/+) and mdr2 Pgp–deficient (−/−) mice. Cholesterol kinetics were determined using radiotracer techniques. Results: No differences in hepatic lipid content were observed between (−/−) and (+/+) mice. Plasma high-density lipoprotein cholesterol and apolipoprotein A-I levels were strongly reduced in (−/−) mice compared with controls, whereas the apolipoprotein B contents of very-low-density lipoprotein and low-density lipoprotein were increased. Hepatic activity of 3-hydroxy-3-methylglutaryl–coenzyme A reductase was threefold greater in (−/−) mice than in controls; however, compartmental analysis of plasma cholesterol decay showed no differences in cholesterol synthesis between (−/−) and (+/+) mice. A dual isotope approach for estimating cholesterol absorption yielded ~50% lower values in (−/−) mice than in controls. Surprisingly, (−/−) mice showed a fourfold increase in fecal neutral sterol secretion. Conclusions: This study unequivocally establishes the important direct role of biliary lipids in the regulation of plasma lipid levels in mice. GASTROENTEROLOGY 1998;114:1024-1034

Hyposecretion of biliary phospholipids (PL) significantly decreases the intestinal absorption of cholesterol (Ch) in Mdr2(−/−) and (+/−) mice

Hyposecretion of biliary phospholipids (PL) significantly decreases the intestinal absorption of cholesterol (Ch) in Mdr2(−/−) and (+/−) mice

A role of phospholipase A2 in cholesterol gallstone pathogenesis is associated with biliary phospholipid species subselection at a site of hepatic excretion

A role of phospholipase A2 in cholesterol gallstone pathogenesis is associated with biliary phospholipid species subselection at a site of hepatic excretion

Abnormal duodenal bile composition in patients with acalculous chronic cholecystitis.

Our goal was to characterize biliary lipid composition in patients with the syndrome of chronic biliary pain, absence of gallstones, and inflammation of the gallbladder mucosa (acalculous chronic cholecystitis). Duodenal bile, obtained from 27 patients with a history of right upper quadrant pain and with negative imaging studies of the biliary tract, was analyzed enzymatically for bile acids, phospholipids, and cholesterol. Fifteen patients were found to have inflammation and/or fibrosis of the gallbladder at cholecystectomy. The 15 patients with abnormal gallbladder histology had more dilute duodenal bile, as indicated by a low bile acid concentration and a lower proportion of phospholipids (p < 0.01) when values were compared with those of duodenal bile samples from postmenopausal women without gallbladder disease or from radiolucent gallstone subjects participating in the National Cooperative Gallstone Study. Cholecystectomy relieved pain in 9 of 14 patients. Some patients with acalculous chronic cholecystitis have duodenal bile samples characterized by a decreased bile acid concentration and a decreased proportion of biliary phospholipids. The low biliary bile acid concentration may result from impaired gallbladder contraction and/or secretion by the biliary tract epithelium. The low proportion of phospholipid may result from posthepatic hydrolysis of luminal phosphatidylcholine followed by absorption of the hydrolysis products. The latter process could be caused by and/or contribute to mucosal inflammation and would also elevate the cholesterol saturation of bile, increasing the risk for cholesterol gallstone formation.

Increased saturation of the fatty acids in the sn-2 position of phospholipids reduces cholesterol crystallization in model biles

Changes in the molecular structure of biliary phospholipids were shown to have major effects on cholesterol solubility, carriers and crystallization in human and model biles. This study investigated systematically the effects of varying saturation of the phosphatidylcholine (PC) sn-2 fatty acid on the cholesterol crystallization process in 3 different model biles. Twenty % of the egg PC (EPC) in these biles were replaced by synthetic PC's with 16:0–18:0, 16:0–18:1, or 16:0–18:2 fatty acyl chains. With 18:0 in the sn-2 position, the crystal observation time (COT) was prolonged from 2 days in the control EPC solution to 14 days ( P<0.05). The crystal growth rate (CGR) was reduced from 0.1 OD/day to unmeasurable levels, and the total crystal mass on day 14 decreased by 86%. The introduction of one (18:1), and two (18:2) double bonds in the sn-2 fatty acid rapidly reversed these effects. Ultracentrifugal analysis showed precipitable cholesterol as monohydrate crystals. In the 16:0–18:0 test solution, most of the precipitable cholesterol remained in the supersaturated multilamellar vesicles. Saturation of the biliary PC sn-2 fatty acyl chain prolongs the COT, slows the CGR, reduces the crystal mass, and extends cholesterol solubility in multilamellar vesicles. Desaturation of the sn-2 fatty acid reverses these effects.

Mutations in the MDR3 gene cause progressive familial intrahepatic cholestasis.

Class III multidrug resistance (MDR) P-glycoproteins (P-gp), mdr2 in mice and MDR3 in man, mediate the translocation of phosphatidylcholine across the canalicular membrane of the hepatocyte. Mice with a disrupted mdr2 gene completely lack biliary phospholipid excretion and develop progressive liver disease, characterized histologically by portal inflammation, proliferation of the bile duct epithelium, and fibrosis. This disease phenotype is very similar to a subtype of progressive familial intrahepatic cholestasis, hallmarked by a high serum gamma-glutamyltransferase (gamma-GT) activity. We report immunohistochemistry for MDR3 P-gp, reverse transcription-coupled PCR sequence analysis, and genomic DNA analysis of MDR3 from two progressive familial intrahepatic cholestasis patients with high serum gamma-GT. Canalicular staining for MDR3 P-gp was negative in liver tissue of both patients. Reverse transcription-coupled PCR sequencing of the first patient's sequence demonstrated a homozygous 7-bp deletion, starting at codon 132, which results in a frameshift and introduces a stop codon 29 codons downstream. The second patient is homozygous for a nonsense mutation in codon 957 (C --> T) that introduces a stop codon (TGA). Our results demonstrate that mutations in the human MDR3 gene lead to progressive familial intrahepatic cholestasis with high serum gamma-GT. The histopathological picture in these patients is very similar to that in the corresponding mdr2(-/-) mouse, in which mdr2 P-gp deficiency induces complete absence of phospholipid in bile.