Biliary Lipid Secretion Rates Research Articles

Hyperhomocysteinemia from trimethylation of hepatic phosphatidylethanolamine during cholesterol cholelithogenesis in inbred mice

Because hyperhomocysteinemia can occur in cholesterol gallstone disease, we hypothesized that this may result from trimethylation of phosphatidylethanolamine (PE), which partakes in biliary phosphatidylcholine (PC) hypersecretion during cholesterol cholelithogenesis. We fed murine strains C57L/J, C57BL/6J, SWR/J, AKR/J, PE N-methyltransferase (PEMT) knockout (KO), PEMT heterozygous (HET), and wildtype (WT) mice a cholesterol/cholic acid lithogenic diet (LD) for up to 56 days and documented biliary lipid phase transitions and secretion rates. We quantified plasma total homocysteine (tHcy), folate, and vitamin B12 in plasma and liver, as well as biliary tHcy and cysteine secretion rates. Rate-limiting enzyme activities of PC synthesis, PEMT and cytidine triphosphate: phosphocholine cytidylyltransferase (PCT), S-adenosylmethionine (SAM), and S-adenosylhomocysteine (SAH) were measured in liver homogenates. Other potential sources of plasma tHcy, glycine N-methyltransferase (GNMT) and guanidinoacetate N-methyltransferase (GAMT), were assayed by gene expression. Plasma tHcy and PEMT activities became elevated during cholelithogenesis in gallstone-susceptible C57L, C57BL/6, and SWR mice but not in the gallstone-resistant AKR mice. Persisting in C57L mice, which exhibit the greatest Lith gene burden, these increases were accompanied by elevated hepatic SAM/SAH ratios and augmented biliary tHcy secretion rates. Counter-regulation included remethylation of Hcy to methionine concurrent with decreased folate and vitamin B12 levels and Hcy transsulfuration to cysteine. Concomitantly, methylenetetrahydrofolate reductase (Mthfr), betaine-homocysteine methyltransferase (Bhmt), and cystathionine-β-synthase (Cbs) were up-regulated, but Gnmt and Gamt genes were down-regulated. PEMT KO and HET mice displayed biliary lipid secretion rates and high gallstone prevalence rates similar to WT mice without any elevation in plasma tHcy levels. This work implicates up-regulation of PC synthesis by the PEMT pathway as a source of elevated plasma and bile tHcy during cholesterol cholelithogenesis.

Abnormal hepatobiliary and circulating lipid metabolism in the Long-Evans Cinnamon rat model of Wilson's disease

Long-Evans Cinnamon (LEC) rats exhibit a genetic defect in Atp7b gene, which is homologous to the human Wilson's disease gene, resulting in an inability to mobilize copper from the liver. This study was undertaken to gain insight into the relationship between liver copper accumulation and plasma lipid profile, circulating lipoprotein composition, hepatic sterol metabolism and biliary lipid secretion rates in 12-week-old LEC rats compared to control Long-Evans rats. Concomitant with hepatic copper deposition, LEC rats displayed increased content of triglycerides (TGs), free cholesterol (FC) and cholesteryl ester (CE) in the liver. Hepatic concentrations of malondialdehyde (MDA), an index of lipid peroxidation were also significantly elevated in LEC rats (50%). This steatosis was associated with aberrant microsomal apolipoprotein (apo) B-100 and microsomal triglyceride transfer protein (MTP) content, hypotriglyceridemia, hypocholesterolemia and abnormalities in both circulating lipoprotein composition and size. Atypical hepatobiliary sterol metabolism was established by the assessment of the activity of key intracellular enzymes for cholesterol homeostasis, which demonstrated, with respect to controls, a 40% reduction in 3-hydroxy-3-methylglutaryl coenzyme A reductase, a 30% reduction in cholesterol 7α-hydroxylase, and a 54% reduction in acyl CoA:cholesterol acyltransferase. During a 6-h biliary drainage, a decline in the bile acid output was recorded and might be linked to the low protein expression of the bile salt export pump (BSEP or ABCB11). Our data emphasize the crucial role of copper balance in hepatic sterol homeostasis and lipoprotein metabolism in LEC rats. Additional studies are needed to delineate the mechanisms of these disorders.

Evidence That Gallbladder Epithelial Mucin Enhances Cholesterol Cholelithogenesis in MUC1 Transgenic Mice

The gel-forming mucins play an important role in the early stage of cholesterol gallstone formation. We investigated whether the gallbladder epithelial mucin encoded by mucin gene 1 (MUC1) influences susceptibility to gallstones. Gallbladder motility and cholesterol absorption, gallstones and expression of the mucin genes in gallbladders, and secretion rates and compositions of biliary lipids were determined in male C57BL/6J mice transgenic for the human MUC1 gene (MUC1.Tg) and wild-type mice before (day 0, on chow) and at 4 weeks on a lithogenic diet containing 1% cholesterol and 0.5% cholic acid. On chow, expression levels of the gallbladder mucin genes were essentially similar between MUC1.Tg and wild-type mice. The lithogenic diet induced 3-fold higher expression levels of Muc1, Muc3, Muc4, Muc5ac, and Muc5b messenger RNA in MUC1.Tg mice compared with wild-type mice. Gallbladder cholesterol absorption and size were significantly greater in MUC1.Tg mice than in wild-type mice regardless of whether the chow or the lithogenic diet was fed. Gallbladder emptying in response to exogenously administered cholecystokinin-8 was significantly reduced in MUC1.Tg mice but not in wild-type mice. At 4 weeks on the lithogenic diet, mucin accumulation was found in all MUC1Tg mice and in 60% of wild-type mice. Consequently, these alterations greatly accelerated cholesterol crystallization and gallstone formation in MUC1.Tg mice. However, biliary lipid secretion rates and cholesterol saturation indices of gallbladder biles were comparable in MUC1.Tg and wild-type mice. Increased gallbladder epithelial MUC1 mucin enhances cholelithogenesis by promoting gallbladder cholesterol absorption and impairing gallbladder motility in mice.

Impaired Hepatobiliary Lipid Metabolism in Liver Fatty Acid Binding Protein 1 (FABP1) Knockout Mice

Recent evidence from studies with sterol carrier protein 2 deficient and diosgenin fed mice support a role of FABP1 in biliary lipid secretion and gallstone formation. We explored whether alterations in biliary lipid secretion and metabolism occur in mice with FABP1 deficiency Male Fabp1 (-/-) and wild-type mice (n=6–9) received either a standard (SD) or a lithogenic diet (LD) for 4 weeks. Following collection of hepatic bile, livers were harvested for isolation of total RNA, homogenate and plasma membranes. Biliary lipids were analyzed by standard methods and biliary lipid secretion rates were calculated. Gene expression of lipid transporters and enzymes of cholesterol/bile acid synthesis was studied with RT-PCR and Western Blotting. Body weight of wild-type and knockout mice fed a SD was similar. Surprisingly, body weight of Fabp1 (-/-) mice did not increase in response to the LD. Contrary, liver weights significantly (p<0.01) increased to 1.4±0.3g and showed macroscopic steatosis. Bile flow rates of chow fed Fabp1 (-/-) mice were slightly elevated and increased 4.2-fold (p<0.001) in response to the LD. Biliary cholesterol and phospholipid secretion rates were comparable in both groups of mice fed SD, whereas bile acid secretion rates were almost doubled in Fabp1 (-/-) mice. In response to the LD cholesterol, phospholipid and bile acid secretion rates increased 24-, 15- and 9-fold, respectively. The increased bile acid secretion rates corresponded with a 53% increase of BSEP expression (p<0.01). In contrast to a dramatic downregulation of NTCP by 86% (p0.001), Oatp1 gene expression was not significantly altered. Surprisingly, expression of CYP7A1, CYP27A and CYP7B1, key enzymes of the neutral and acidic bile acid synthesis pathway were not suppressed. Based on our findings we propose that i) sterol carrier protein 2 is more important for biliary cholesterol secretion than FABP1, ii) FABP1 may be involved in targeting bile acids in hepatocytes and iii) suppression of FABP1 expression does not prevent fatty liver disease.

The Abcc2-Lith2 locus induces hypersensitivity to the biliary lithogenic effect of oestrogens by precipitating the onset of lithogenic bile

Background and Aim: We recently established Abcc2 to be the molecular basis underlying Lith2, one of the major cholesterol gallstone susceptibility loci identified in inbred mice fed a lithogenic diet. Based on the direct role of ABCC2 in the hepatohiliary transport of different drug and hormone conjugates, many of which are well-recognised to constitute high risk factors for human cholelithiasis, we hypotbetised that the Lith2/Abcc2 locus impacts not only the fithogenic response to dietary intervention but also to drugs and hormones. We tested our hypothesis here, using estrogen as a model compound. Methods: AKi.L/th2 s congenic mice (Lith2) and AKR controls were injected i.p. with ethinyl estradiol (EE) at the dose of 1X or 3X (5 or 15mg/kg-day) for 3 days. Bile flow and biliary lipids were assessed from hepatic bile with standard procedures. Hepatic bile acids were assayed by LC-MS-MS. Results: Body weights were identical between groups, establishing that all mice received an equivalent amount of EE Without EE-treatment, biliary lipid secretion rates, biliary lipid coupling and CSI were undifferentiable between groups. After EE 1X, however, while the controls increased their bile salts (BS) secretion rate and modestly but significantly decreased their biliary secretion of phospholipids (PL) and cholesterol (Ch) (most likely due to the uncoupling effect of EE-conjugates), the Lith2 group exhibited a declining BS secretion rate, a drop 2 times more pronounced in PL secretion rates and a sharp rise in biliary Ch, culminating in a marked elevation of Ch/BS or PL ratios and CSI. Tripling the dose of EE had no additional effect in lath2 mice but led the controls to now develop an hepatobiliary and lithogenic profile undistinguishable from the congenic animals. Percent of hepatic dihydroxylated BS were elevated in all Lith2 mice treated with EE but only in the controls receiving EE 3X, thereby being in direct relationship with biliary lithogenicity. Conclusions: The Lith2 locus, and most likely Abcc2, is not solely involved in the high-lithogeinc risk following a lithogenic diet, but also induces an hypersensitivity to the hthogenic effect of estrogens. Dihydroxylated hepatic bile acids appears central to the development of lithogenic bile, possibly through activation of nuclear receptor/ABCG5-8 expression. We speculate that such gene-estrogen interaction plays a role in the lithogenic risk associated with gender, pregnancy and estrogen-based therapies.

Serum plant sterols and biliary cholesterol secretion in humans

Ratios of cholestanol, campesterol, and sitosterol to cholesterol in serum are known to reflect cholesterol absorption efficiency. Here, a possible link between these ratios and biliary secretion rates of cholesterol was investigated. Biliary lipid secretion rates and serum sterols were determined in 13 patients with gallstones. Seven were treated with ursodeoxycholic acid (UDCA) (1,000 mg/d). Serum cholesterol and non-cholesterol sterols were also measured in a cross over study in 20 healthy volunteers, who received either placebo or UDCA (750 mg/d). Biliary cholesterol secretion was significantly lower, whereas the non-cholesterol sterols and their ratio to cholesterol were higher in patients with gallstones treated with UDCA. A highly significant negative linear correlation between the ratios of non-cholesterol sterols to cholesterol and biliary cholesterol secretion was observed. In volunteers, administration of UDCA for 4 weeks was followed by a significant increase in non-cholesterol sterols and their ratios. Even 4 weeks after discontinuing UDCA administration, campesterol and sitosterol were still significantly higher than pretreatment levels, which was also true for the campesterol-cholesterol ratio after 8 weeks.The results suggest that the ratios of cholestanol, campesterol, and sitosterol to cholesterol can be used as indicators of changes in biliary cholesterol secretion rates.

Expression of liver plasma membrane transporters in gallstone-susceptible and gallstone-resistant mice

We tested the hypothesis that differential expression of liver plasma membrane transporters might account for variations in biliary lipid secretion rates between gallstone-susceptible C57L/J and gallstone-resistant AKR/J mice. Plasma membrane fractions and total RNA isolated from livers of mice fed with a control or lithogenic (15% fat/1.25% cholesterol/0.5% cholic acid) diet were used for measurements of steady-state gene expression of hepatobiliary transport systems for bile salts (Ntcp1/Slc10a1, Oatp1/Slc21a1 and Bsep/Abcb11), phospholipids (Mdr2/Abcb4), organic anions (Mrp2/Abcc2) and organic cations (Oct1/Slc22a1). Irrespective of the diet, the steady-state gene expression of hepatobiliary transporters did not differ significantly between the two strains. Despite a higher basal bile flow and bile-salt secretion in C57L mice, Mrp2 (Abcc2) and Bsep (Abcb11) expression did not differ between the two strains. Elevated biliary phospholipid secretion in response to the lithogenic diet was linked to increased Mdr2 (Abcb4) protein expression, whereas the induction of Oct1 (Slc22a1) might reflect an enhanced uptake of choline for augmented phospholipid synthesis. In response to the lithogenic diet, Bsep (Abcb11) protein expression was up-regulated only marginally and bile salt secretion did not increase. The down-regulation of Ntcp1 (Slc10a1) protein expression might protect hepatocytes from high intracellular bile-salt loads. We conclude that variations in protein function rather than in the gene expression of liver plasma membrane transporters might account for variations in biliary lipid secretion rates. Our findings support the concept that the formation of lithogenic bile is caused by the hypersecretion of bile salts as a result of augmented availability of canalicular membrane cholesterol, possibly amplified by bile-salt—phospholipid uncoupling due to the increased bile flow.

Expression of liver plasma membrane transporters in gallstone-susceptible and gallstone-resistant mice.

We tested the hypothesis that differential expression of liver plasma membrane transporters might account for variations in biliary lipid secretion rates between gallstone-susceptible C57L/J and gallstone-resistant AKR/J mice. Plasma membrane fractions and total RNA isolated from livers of mice fed with a control or lithogenic (15% fat/1.25% cholesterol/0.5% cholic acid) diet were used for measurements of steady-state gene expression of hepatobiliary transport systems for bile salts (Ntcp1/Slc10a1, Oatp1/Slc21a1 and Bsep/Abcb11), phospholipids (Mdr2/Abcb4), organic anions (Mrp2/Abcc2) and organic cations (Oct1/Slc22a1). Irrespective of the diet, the steady-state gene expression of hepatobiliary transporters did not differ significantly between the two strains. Despite a higher basal bile flow and bile-salt secretion in C57L mice, Mrp2 (Abcc2) and Bsep (Abcb11) expression did not differ between the two strains. Elevated biliary phospholipid secretion in response to the lithogenic diet was linked to increased Mdr2 (Abcb4) protein expression, whereas the induction of Oct1 (Slc22a1) might reflect an enhanced uptake of choline for augmented phospholipid synthesis. In response to the lithogenic diet, Bsep (Abcb11) protein expression was up-regulated only marginally and bile salt secretion did not increase. The down-regulation of Ntcp1 (Slc10a1) protein expression might protect hepatocytes from high intracellular bile-salt loads. We conclude that variations in protein function rather than in the gene expression of liver plasma membrane transporters might account for variations in biliary lipid secretion rates. Our findings support the concept that the formation of lithogenic bile is caused by the hypersecretion of bile salts as a result of augmented availability of canalicular membrane cholesterol, possibly amplified by bile-salt-phospholipid uncoupling due to the increased bile flow.

Disruption of the Sterol Carrier Protein 2 Gene in Mice Impairs Biliary Lipid and Hepatic Cholesterol Metabolism

Hepatic up-regulation of sterol carrier protein 2 (Scp2) in mice promotes hypersecretion of cholesterol into bile and gallstone formation in response to a lithogenic diet. We hypothesized that Scp2 deficiency may alter biliary lipid secretion and hepatic cholesterol metabolism. Male gallstone-susceptible C57BL/6 and C57BL/6(Scp2(-/-)) knockout mice were fed a standard chow or lithogenic diet. Hepatic biles were collected to determine biliary lipid secretion rates, bile flow, and bile salt pool size. Plasma lipoprotein distribution was investigated, and gene expression of cytosolic lipid-binding proteins, lipoprotein receptors, hepatic regulatory enzymes, and intestinal cholesterol absorption was measured. Compared with chow-fed wild-type animals, C57BL/6(Scp2(-/-)) mice had higher bile flow and lower bile salt secretion rates, decreased hepatic apolipoprotein expression, increased hepatic cholesterol synthesis, and up-regulation of liver fatty acid-binding protein. In addition, the bile salt pool size was reduced and intestinal cholesterol absorption was unaltered in C57BL/6(Scp2(-/-)) mice. When C57BL/6(Scp2(-/-)) mice were challenged with a lithogenic diet, a smaller increase of hepatic free cholesterol failed to suppress cholesterol synthesis and biliary cholesterol secretion increased to a much smaller extent than phospholipid and bile salt secretion. Scp2 deficiency did not prevent gallstone formation and may be compensated in part by hepatic up-regulation of liver fatty acid-binding protein. These results support a role of Scp2 in hepatic cholesterol metabolism, biliary lipid secretion, and intracellular cholesterol distribution.

Biliary cholesterol hypersecretion in gallstone-susceptible mice is associated with hepatic up-regulation of the high-density lipoprotein receptor SRBI

Enhanced hepatocellular trafficking of cholesterol to the bile canaliculus and cholesterol hypersecretion appears critical for gallstone formation. Therefore, we studied in more detail the hepatic cholesterol transport pathways in a mouse model of cholesterol gallstone disease. Biliary lipid secretion rates, plasma lipoprotein levels, hepatic expression of lipoprotein receptors, lipid regulatory enzymes, and putative cholesterol transporting proteins were analyzed in gallstone-susceptible C57L/J and gallstone-resistant AKR/J mice, which were fed a lithogenic diet. Biliary cholesterol hypersecretion in C57L mice was associated with decreased plasma high-density lipoprotein (HDL) cholesterol levels and significant hepatic induction of the HDL receptor (SRBI) and cholesteryl ester hydrolase. In response to the lithogenic diet, fatty-acid binding protein of liver (FABPL) was markedly induced in both mouse strains. Caveolin 1 was elevated only in plasma membranes of gallstone-susceptible C57L mice, which also failed to down-regulate cholesterol synthesis. These data suggest a role of the reverse cholesterol transport pathway for genetically determined gallstone susceptibility in the mouse. (HEPATOLOGY 2001;33:1451-1459.)

Phenotypic characterization of Lith genes that determine susceptibility to cholesterol cholelithiasis in inbred mice: pathophysiology of biliary lipid secretion

The inbred C57L strain but not the AKR strain of mice carry Lith genes that determine cholesterol gallstone susceptibility. When C57L mice are fed a lithogenic diet containing 15% fat, 1% cholesterol, and 0.5% cholic acid, gallbladder bile displays rapid cholesterol supersaturation, mucin gel accumulation, increases in hydrophobic bile salts, and rapid phase separation of solid and liquid crystals, all of which contribute to the high cholesterol gallstone prevalence rates (D. Q-H. Wang, B. Paigen, and M. C. Carey. J. Lipid Res. 1997. 38: 1395;-1411). We have now determined the hepatic secretion rates of biliary lipids in fasting male and female C57L and AKR mice and the intercross (C57L x AKR)F(1) before and at frequent intervals during feeding the lithogenic diet for 56 days. Bile flow and biliary lipid secretion rates were measured in the first hour of an acute bile fistula and circulating bile salt pool sizes were determined by the "washout" technique after cholecystectomy. Compared with AKR mice, we found that i) C57L and F(1) mice on chow displayed significantly higher secretion rates of all biliary lipids, and larger bile salt pool sizes, as well as higher bile salt-dependent and bile salt-independent flow rates; ii) the lithogenic diet further increased biliary cholesterol and lecithin outputs, but bile salt outputs remained constant. Biliary coupling of cholesterol to lecithin increased approximately 30%, setting the biophysical conditions necessary for cholesterol phase separation in the gallbladder; and iii) no gender differences in lipid secretion rates were noted but male mice exhibited significantly more hydrophobic bile salt pools than females. We conclude that in gallstone-susceptible mice, Lith genes determine increased outputs of all biliary lipids but promote cholesterol hypersecretion disproportionately to lecithin and bile salt outputs thereby inducing lithogenic bile formation.

Cholic acid aids absorption, biliary secretion, and phase transitions of cholesterol in murine cholelithogenesis.

Cholic acid is a critical component of the lithogenic diet in mice. To determine its pathogenetic roles, we fed chow or 1% cholesterol with or without 0.5% cholic acid to C57L/J male mice, which because of lith genes have 100% gallstone prevalence rates. After 1 yr on the diets, we measured bile flow, biliary lipid secretion rates, hepatic cholesterol and bile salt synthesis, and intestinal cholesterol absorption. After hepatic conjugation with taurine, cholate replaced most tauro-beta-muricholate in bile. Dietary cholic acid plus cholesterol increased bile flow and biliary lipid secretion rates and reduced cholesterol 7alpha-hydroxylase activity significantly mostly via deoxycholic acid, cholate's bacterial 7alpha-dehydroxylation product but did not downregulate cholesterol biosynthesis. Intestinal cholesterol absorption doubled, and biliary cholesterol crystallized as phase boundaries shifted. Feeding mice 1% cholesterol alone produced no lithogenic or homeostatic effects. We conclude that in mice cholic acid promotes biliary cholesterol hypersecretion and cholelithogenesis by enhancing intestinal absorption, hepatic bioavailability, and phase separation of cholesterol in bile.

Effect of ileal autotransplantation on cholesterol, bile acids, and biliary lipids in pigs with proximal small bowel resection

Our major aim was to investigate the consequences of ileal autotransplantation in pigs with proximal small intestinal resection on biliary lipids and metabolism of bile acids. Biliary lipid secretion rates and bile acid absorption were assessed by measuring dietary and biliary lipids, fractional cholesterol absorption, and fecal excretion of cholesterol and bile acids. In addition, serum bile acids and cholesterol, biliary and fecal bile acid species, and ileal villus height were determined after resection of the proximal 75% of the jejunoileum (n = 15) and autotransplantation of the remaining ileum with systemic venous drainage (n = 15) or transection (n = 5). Autotransplantation further increased fecal excretion of neutral and acidic steroids and serum concentration of bile acids after proximal resection (P < .05 for all); autotransplantation significantly decreased serum cholesterol, ileal villus height, fractional bile acid and cholesterol absorption, and biliary molar percentage of total and primary bile acids, whereas biliary secretion of bile acids, enriched by secondary bile acids, and cholesterol remained unchanged. At 14 weeks, ileal villus height, fractional bile acid and cholesterol absorption, biliary molar percentage of bile acids, and proportion of secondary biliary bile acids were altered by transplantation from the respective postresection values of 864 +/- 22 microm, 97.9 +/- 0.6%, 26.9 +/- 3.9%, 91.8 +/- 1.2% and 9.2 +/- 1.3% to 428 +/- 21 microm, 91.1 +/- 1.5%, 9.5 +/- 1.1%, 83.9 +/- 1.4% and 52.5 +/- 3.5% (P < .005 for all). Posttransplantation biliary bile acid secretion correlated positively with fractional reabsorption (r = .70) and biliary molar percentage (r = .73) of bile acids and ileal villus height (r = .65; P < .01 for all). Decreased absorption efficiency and biliary molar percentage of bile acids, increased biliary secondary bile acids, and short ileal villi point to bacterial overgrowth-induced bile acid malabsorption, which with decreased absorptive area may contribute to malabsorption of other lipids after ileal autotransplantation. Compensatory increase in cholesterol synthesis in the pigs with autotransplanted ileum appeared sufficient for constant biliary secretion of cholesterol and bile acids.

Relationship between biliary lipid and protoporphyrin secretion; potential role of mdr2 P-glycoprotein in hepatobiliary organic anion transport

Background/Aims: Erythropoietic protoporphyria, caused by ferrochelatase deficiency, leads to protoporphyrin accumulation in the liver. Therapeutic attempts to increase the secretion of ths hydrophobic organic anion into bile are hampered by a lack of understanding of the secretory mechanism(s) involved. We have investigated biliary secretion of protoporphyrin in rats and mice, primarily targeted on the role of biliary lipids in this process. Methods: Gel permeation chromatography was applied to investigate the association of porphyrins with lipid fractions in bile. Secretion of endogenous porphyrins was studied in (GY mutant) rats and mdr2 P-glycoprotein deficient mice, under conditions of widely varying biliary lipid secretion rates. Results: Gel permeation chromatography revealed that, in native human and rat bile, protoporphyrin associated with cholesterol/phospholipid vesicles upon elution with bile salt-free buffer. In contrast, the more hydrophilic corporpyrin isomers I and III were found only in bile salt/organic anion hybrid particles and smaller aggregates. Interruption of the enterohepatic circulation in normal Wistar rats resulted in a parallel decrease of endogenous protoporphyrin-, lipid-, and bile salt secretion, but did not alter the secretion of coproporphyrin I and III. Uncoupling of lipid- from bile salt secretion by sulfated taurolithocholate resulted in imparied secretion into bile of protoporpyrin only. Conversely, secretion of coproporphyrin I and III, but not that of protoporphyrin, was impaired in mutant Gronigen Yellow rats with defective ATP-dependent hepatobiliary organic anions transport. In mice homozygous for a disruption of the mdr2 P-glycoprotein gene, resulting in complete absence of phospholipids in bile and strongly reduced cholesterol output, secretion of protoporphyrin was reduced by 90%, whereas that of coproporpyrin I and III was affected to a much lesser extent. Conclusions: Our data demonstrate a close association between protoporphyrin and lipid secretion into bile, indicating that these processes are, at least functionally coupled. This finding implicates a role of mdr2 P-glycoprotein activity in hepatobiliary removal of the hydrophobic organic anion protoporphyrin. Hence, it may be speculated that protoporphyrin secretion can be influenced by drugs, diet or other means that affect biliary lipid secretion.

In patients with orthotopic liver transplantation, serum markers of cholestasis are unreliable indicators of biliary secretion

Background/Aims: In patients after orthotopic liver transplantation, treatment with the novel immunosuppressant FK 506 may lead to elevated levels of alkaline phosphatase, gamma glutamyl transferase and bilirubin. Up to now it was unclear whether the excretory capacity of the liver in such patients is impaired. Methods: We measured quantitatively the secretion of bile acids, phospholipids and cholesterol using the duodenal perfusion method, which allows assessment of biliary secretion without interruption of the enterohepatic circulation. Six healthy volunteers served as controls. Results: All patients studied after orthotopic liver transplantation had elevated concentrations of serum alkaline phosphatase and gamma glutamyl transferase, whereas only half of them had slightly abnormal serum bilirubin levels. On average, the FK 506-treated patients excreted 1.23±0.27 mmol/h bile acids, 0.23±0.04 mmol/h phospholipids and 0.11±0.02 mmol/h cholesterol, which was not significantly different from the healthy controls. Conclusions: The normal secretion rates of biliary bile acids and lipids in FK 5-6-treated patients with elevated serum alkaline phosphatase and gamma glutamyl transferase indicate that the excretory capacity of the transplanted liver has completely recovered 2–3 months after surgery. In addition, in the majority of these patients elevated serum levels of alkaline phosphatase, gamma glutamyl transferase and bilirubin do not reflect impaired biliary secretion.

Enterohepatic circulation in hamsters with an extracorporeal bile duct.

The present study describes a novel technique for investigations of the enterohepatic circulation in the hamster with an extracorporeal bile duct that allows long-term bile collection in the free-moving animal. The animals recovered for 7 days after the operation before the external loop was cut and bile was collected over a period of 78 h. Under these optimal conditions, initial bile flow (651 +/- 89 microliters per 100 g.h-1) and the secretion rates of biliary lipids were several-fold higher than reported in an earlier study using the acute fistula hamster. Biliary cholesterol secretion amounted to 369 +/- 32 nmol per 100 g.h-1, phospholipid secretion was 2.6 +/- 0.3 mumol per 100 g.h-1, and total bile acid secretion was 31.9 +/- 2.2 mumol per 100 g.h-1. A clearcut diurnal rhythm was demonstrated for bile flow and all biliary constituents. After 9 h the depletion of the bile acid pool was complete and cholic acid synthesis derepressed 1.4-fold from a basal rate of 818 nmol per 100 g.h-1, whereas the derepression of chenodeoxycholic acid synthesis was even less pronounced. Biliary cholesterol output increased 2.2-fold, but the phospholipid secretion was constant during the full experiment. It may be concluded that the technique of an extracorporeal bile duct in the free-moving animal allows studies of bile secretion under optimal conditions. Most likely the bile secretion rates given above approach the physiological rates in the hamster.

Effect of complete sulfation of bile acids on bile formation: role of conjugation and number of sulfate groups.

The effect of complete sulfation of conjugated cholic, chenodeoxycholic and deoxycholic acids on bile formation was investigated in rats. The sulfated bile acids were infused intravenously in stepwise increasing doses (1, 2, 3 and 4 mumol/min/100 gm body wt) in rats after 90 min of bile acid pool depletion. The effects of these bile acids on bile flow, bile salt, biliary phospholipid and cholesterol secretion rates were determined. In addition, their choleretic activity and their effect on biliary lipid secretion were calculated. Appropriate controls infused with nonsulfated bile were also performed. The sulfated bile acids increased bile flow with increasing the infusion doses, and the maximum bile flow was significantly higher than nonsulfated bile acids. Although cholestasis was developed during the infusion of nonsulfated bile acids, no cholestatic effect was observed for sulfated bile acids. With the exception of cholic acid, sulfation significantly increased the bile acid secretory rate maximum. The sulfates of chenodeoxycholic and deoxycholic acids were further hydroxylated. The choleretic activities for all the sulfated bile acids were significantly higher than the nonsulfated bile acids. All the sulfated bile acids significantly reduced the biliary lipid secretion, and a significant correlation was found between the choleretic activity and the phospholipid-dependent bile acid secretion. The data also showed that infusion of sulfated taurine-conjugated bile acids produced higher bile flow and bile acid secretion rate and was less effective when biliary lipid secretion rates were reduced compared with glycine conjugates. It is concluded that sulfated conjugated bile acids may have a role in protection during cholestasis either by stimulation of bile flow or by reduction of biliary lipid secretion, thus protecting cell membranes from the detergent properties of high concentrations of nonsulfated bile acids.

Dietary fish oil effects on biliary lipid secretion and cholesterol gallstone formation in the African green monkey

We studied two groups of adult male African green monkeys to assess the effects of dietary fish oil on biliary lipid secretion and cholesterol gallstone formation. One group was fed 0.8 mg cholesterol/kcal and 42% of calories as fat with half of the fat calories derived from lard; the other group was fed a similar diet except for the isocaloric substitution of menhaden oil for lard. After 2 1/2 to 3 yr, necropsies were performed and the presence of gallstones was determined. Gallbladder bile specimens were analyzed for cholesterol saturation index, as well as for bile acid species and biliary phospholipid fatty acid composition. Our results showed that 67% of animals fed the lard diet had cholesterol gallstones compared with only 22% of animals in the group fed the fish oil diet (p = 0.08). The cholesterol saturation index of gallbladder bile also tended to be higher in the lard-fed group (1.15 +/- 0.11) compared with the fish oil-fed group (0.86 +/- 0.09, p = 0.06). No differences between the two dietary groups were noted in the percentages of the various types of bile acids. However, a greater percentage of omega-3 fatty acids and a lesser percentage of 18:1, 18:2 and 20:4 fatty acids were found in the biliary phospholipids from the fish oil-fed group compared with the lard-fed group. Biliary lipid secretion rates were then measured during isolated recirculating liver perfusion performed with a constant sodium taurocholate infusion.(ABSTRACT TRUNCATED AT 250 WORDS)

Dietary fish oil effects on biliary lipid secretion and cholesterol gallstone formation in the African green monkey*1

We studied two groups of adult male African green monkeys to assess the effects of dietary fish oil on biliary lipid secretion and cholesterol gallstone formation. One group was fed 0.8 mg cholesterol/kcal and 42% of calories as fat with half of the fat calories derived from lard; the other group was fed a similar diet except for the isocaloric substitution of menhaden oil for lard. After 2 1/2 to 3 yr, necropsies were performed and the presence of gallstones was determined. Gallbladder bile specimens were analyzed for cholesterol saturation index, as well as for bile acid species and biliary phospholipid fatty acid composition. Our results showed that 67% of animals fed the lard diet had cholesterol gallstones compared with only 22% of animals in the group fed the fish oil diet (p = 0.08). The cholesterol saturation index of gallbladder bile also tended to be higher in the lard-fed group (1.15 +/- 0.11) compared with the fish oil-fed group (0.86 +/- 0.09, p = 0.06). No differences between the two dietary groups were noted in the percentages of the various types of bile acids. However, a greater percentage of omega-3 fatty acids and a lesser percentage of 18:1, 18:2 and 20:4 fatty acids were found in the biliary phospholipids from the fish oil-fed group compared with the lard-fed group. Biliary lipid secretion rates were then measured during isolated recirculating liver perfusion performed with a constant sodium taurocholate infusion.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparative evaluation of chenodeoxycholic and ursodeoxycholic acids in obese patients: Effects on biliary lipid metabolism during weight maintenance and weight reduction

Obesity is a condition associated with an increased frequency of gallstone disease. This study attempted to evaluate the comparative effects of two gallstonedissolving agents, chenodeoxycholic acid and ursodeoxycholic acid, on bile acid metabolism and biliary lipid secretion in obese subjects in order to identify the bile acid of choice in preventing and treating gallstone disease in obesity. Twenty obese subjects (> 120% ideal body wt) were randomly treated with ursodeoxycholic acid (10 mg · kg−1 · day−1 · 1 mo−1) and then with chenodeoxycholic acid (15 mg · kg−1 · day−1 · 1 mo−1) or with chenodeoxycholic acid first and then with ursodeoxycholic acid. Patients 1–10 were studied while eating an unrestricted weightmaintenance diet, whereas patients 11–20 were eating a 1080-kcal/d hypocaloric diet. Biliary lipid composition, cholesterol saturation index, and biliary bile acid pattern were evaluated in all subjects before and after each treatment period; in subjects 6–10 and 16–20, biliary lipid secretion rates and bile acid pool size were also evaluated. Both ursodeoxycholic acid and chenodeoxycholic acid decreased cholesterol outputs and cholesterol saturation index. However, during the weight-maintenance period the decrease induced by chenodeoxycholic acid was not significant. Biliary cholesterol outputs and cholesterol saturation index were always lower during ursodeoxycholic acid administration than during chenodeoxycholic acid therapy. Ursodeoxycholic acid levels during ursodeoxycholic acid administration and chenodeoxycholic acid levels during chenodeoxycholic acid administration increased in bile to 50% and 77%, respectively, of total bile acid levels. Bile acid pool size remained unchanged during chenodeoxycholic acid administration and was significantly reduced by ursodeoxycholic acid administration during the weight-reduction period. In conclusion, ursodeoxycholic acid in obese subjects seems more effective than chenodeoxycholic acid, at least during weight maintenance, in reducing cholesterol saturation of bile. This effect is related to a significant decrease of biliary cholesterol output.