Effect of portacaval shunt on biliary cholesterol and bile salt secretion in normal rats

In order to assess the role of bile secretion in the serum cholestrol lowering effect of a portacaval anastomosis in rats, bile flow rate, bile salt and cholesterol secretion in bile were determined. The study comprised five groups of animals: (1) rats with protacaval shunt cholesterol free food ad libitum. (2) sham-operated and pair-fed controls; (3) unoperated, pair-fed controls; (4) unoperated controls on cholesterol free diet ad libitum; and (5) unoperated controls on normal laboratory rat chow ad libitum. Bile flow rate showed no difference in any of the groups on cholesterol free diet. Bile salt secretion was significantly elevated in shunted animals (87.1 +/- 7.0 nmol/min) as compared with pair-fed sham-operated (64.7 +/- 20.0 nmol/min) and unoperated controls (56.0 +/- 22.3 nmol/min). Biliary cholesterol output tended to be higher in shunted animals but this was not statistically significant. The data indicate that the atrophied rat liver in shunted animals is able to maintain a normal bile flow and to increase bile salt secretion. Thus, the serum cholesterol lowering action of a portocaval anastomosis may be partly due to an increased cholesterol catabolism to bile salts.     

Growth hormone and bile acid synthesis. Key role for the activity of hepatic microsomal cholesterol 7alpha-hydroxylase in the rat.

Growth hormone (GH) has an important role in the regulation of hepatic LDL receptor expression and plasma lipoprotein levels. This investigation was undertaken to characterize the effects of GH on hepatic cholesterol and bile acid metabolism in the rat. In hypophysectomized (Hx) rats, the activities of the rate-limiting enzymes in cholesterol and bile acid biosynthesis, 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG CoA reductase) and cholesterol 7alpha-hydroxylase (C7alphaOH), were reduced by 71 and 64%, respectively. HMG CoA reductase mRNA levels were reduced by 37%, whereas C7alphaOH mRNA was increased by 81%. LDL receptor expression was reduced by 18% in Hx rats, without any change in the LDL receptor mRNA levels. Although the normal diurnal variation of C7alphaOH activity was preserved in Hx rats, the activity of C7alphaOH was much reduced both at midday and midnight. Total hepatic cholesterol was increased by 14% in Hx animals whereas microsomal cholesterol was unchanged. The rate of cholesterol esterification was enhanced (by 38%) in liver microsomes from Hx rats. Stepwise hormonal substitution of Hx rats showed that GH, but not thyroid hormone or cortisone, was essential to normalize the enzymatic activity of C7alphaOH. GH also normalized the altered plasma lipoprotein pattern in Hx rats, and increased the fecal output of bile acids. The latter effect was particularly evident when GH was combined with cortisone and thyroid hormone. Also in normal rats, GH stimulated C7alphaOH activity. In conclusion, GH has an essential role to maintain a normal enzymatic activity of C7alphaOH, and this, at least in part, explains the effects of GH on hepatic cholesterol metabolism. GH is also of critical importance to normalize the altered plasma lipoprotein pattern in Hx rats.     

Ketoconazole blocks bile acid synthesis in hepatocyte monolayer cultures and in vivo in rat by inhibiting cholesterol 7 alpha-hydroxylase.

In cultured hepatocytes conversion of [4-14C]cholesterol into bile acids was dose dependently reduced by the antimycotic drug ketoconazole, giving half-maximal inhibition at 10 microM ketoconazole in rat hepatocytes and at 1 microM in human hepatocytes. No change was observed in the ratio of produced cholic, beta-muricholic, and chenodeoxycholic acid with increasing amounts of the drug. Conversion of [4-14C]7 alpha-hydroxycholesterol, an intermediate of bile acid pathway, to bile acids was not affected by ketoconazole. These results together with kinetic studies with rat liver microsomes, demonstrating noncompetitive inhibition (Ki = 0.4 microM), indicate that cholesterol 7 alpha-hydroxylase is the main site of inhibition. In bile-diverted rats a single dose of ketoconazole (50 mg/kg) dramatically impaired bile flow and biliary bile acid output (92% inhibition). A similar blockade was observed using [4-14C]cholesterol as precursor for bile acid synthesis. Therefore, treatment of patients with this drug may inhibit bile acid synthesis, resulting in a reduction of the bile acid pool size after long-term ketoconazole therapy.     

Linkage between cholesterol 7alpha-hydroxylase and high plasma low-density lipoprotein cholesterol concentrations.

Interindividual differences in plasma low-density lipoprotein cholesterol (LDL-C) levels reflect both environmental variation and genetic polymorphism, but the specific genes involved and their relative contributions to the variance in LDL-C are not known. In this study we investigated the relationship between plasma LDL-C concentrations and three genes with pivotal roles in LDL metabolism: the low-density lipoprotein receptor (LDLR), apolipoprotein B (APOB), and cholesterol 7alpha-hydroxylase (CYP7). Analysis of 150 nuclear families indicated statistically significant linkage between plasma LDL-C concentrations and CYP7, but not LDLR or APOB. Further sibling pair analyses using individuals with high plasma LDL-C concentrations as probands indicated that the CYP7 locus was linked to high plasma LDL-C, but not to low plasma LDL-C concentrations. This finding was replicated in an independent sample. DNA sequencing revealed two linked polymorphisms in the 5' flanking region of CYP7. The allele defined by these polymorphisms was associated with increased plasma LDL-C concentrations, both in sibling pairs and in unrelated individuals. Taken together, these findings indicate that polymorphism in CYP7 contributes to heritable variation in plasma LDL-C concentrations. Common polymorphisms in LDLR and APOB account for little of the heritable variation in plasma LDL-C concentrations in the general population.     

Competitive inhibition of bile acid synthesis by endogenous cholestanol and sitosterol in sitosterolemia with xanthomatosis. Effect on cholesterol 7 alpha-hydroxylase.

The 7 alpha-hydroxylation of two cholesterol analogues, sitosterol and cholestanol, and their effect on the 7 alpha-hydroxylation of cholesterol were measured in rat and human hepatic microsomes. In untreated rat liver microsomes, the 7 alpha-hydroxylation of cholesterol was higher than that of cholestanol (1.4-fold) and sitosterol (30-fold). After removal of endogenous sterols from the microsomes by acetone treatment, the 7 alpha-hydroxylation of cholesterol was similar to that of cholestanol and only fourfold higher than that of sitosterol. Cholestanol and sitosterol competitively inhibited cholesterol 7 alpha-hydroxylase in both rat and human liver microsomes, with cholestanol the more potent inhibitor. Patients with sitosterolemia with xanthomatosis, who have elevated microsomal cholestanol and sitosterol, showed reduced cholesterol 7 alpha-hydroxylase activity relative to the activity in control subjects (13.9 and 14.7 vs. 20.3 +/- 0.9 pmol/nmol P-450 per min, P less than 0.01). Enzyme activity in these patients was 40% higher when measured in microsomes from which competing sterols had been removed. Ileal bypass surgery in one sitosterolemic patient decreased plasma cholestanol and sitosterol concentrations and resulted in a 30% increase in hepatic microsomal cholesterol 7 alpha-hydroxylase activity. Cholesterol 7 alpha-hydroxylase appears to have a specific apolar binding site for the side chain of cholesterol and is affected by the presence of cholestanol and sitosterol in the microsomal substrate pool. Reduced bile acid synthesis in sitosterolemia with xanthomatosis may be related to the inhibition of cholesterol 7 alpha-hydroxylase activity by endogenous cholesterol analogues.     

Effects of bile salt hydrophobicity on crystallization of cholesterol in model bile

Precipitation of cholesterol crystals is an essential step in gallstone formation. In the present study we found much faster and more extensive precipitation of various cholesterol crystal shapes in whole model biles containing the hydrophobic bile salt taurodeoxycholate than in biles containing the relatively hydrophilic taurocholate. Addition of taurodeoxycholate to isolated cholesterol–phospholipid vesicles also induced more crystallization than taurocholate. Crystallization behaviour in whole model biles and in vesicles after addition of corresponding bile salts was very similar. The very hydrophilic bile salts tauroursodeoxycholate and taurohyodeoxycholate never induced crystallization from vesicles, and crystallization in corresponding whole model biles did not occur. These bile salts also reduced crystallization dose dependently after addition of taurodeoxycholate to vesicles. Ultracentrifugation experiments suggested a higher vesicular cholesterol–phospholipid ratio for whole model biles containing more hydrophobic bile salts. These findings indicate that bile salt hydrophobicity influences shape of cholesterol crystals and extent of crystallization, possibly by modulating the vesicular cholesterol–phospholipid ratio.     

Effect of periodic long-term ethanol administration on biliary bile acids and bile secretion in the rat

Hepatic bile flow and its bile acid composition were determined in a group of 23 rats receiving 20% (W/v) ethanol by daily intubations 5 times a week over a 10-12 week period and in a control group of 23 rats. The aim was to elucidate the well known role of alcohol in pancreatitis. Chronic ethanol administration resulted in a significant increase in 2-h bile production and bile secretion rates. Molar concentrations of total and individual bile acids were determined by the hydroxysteroid dehydrogenase method after thin layer separation. No significant differences in molar concentrations of total or separate bile acid were observed. Special attention was paid to free bile acids. A spot with a Rf value corresponding to cholic acid was found in 10 rats in each group. This was examined by mass spectrometry using direct inlet technique, but no free cholic acid could be identified. Thus it seems that the changes in the amounts of bile acids are not decisive for the origin of acute alcoholic pancreatitis but that increased bile flow caused by chronic alcohol ingestion may favour reflux of bile into the pancreas, resulting in pancreatitis.     

Unexpected inhibition of cholesterol 7 alpha-hydroxylase by cholesterol in New Zealand white and Watanabe heritable hyperlipidemic rabbits.

We investigated the effect of cholesterol feeding on plasma cholesterol concentrations, hepatic activities and mRNA levels of HMG-CoA reductase and cholesterol 7 alpha-hydroxylase and hepatic LDL receptor function and mRNA levels in 23 New Zealand White (NZW) and 17 Watanabe heritable hyperlipidemic (WHHL) rabbits. Plasma cholesterol concentrations were 9.9 times greater in WHHL than NZW rabbits and rose significantly in both groups when cholesterol was fed. Baseline liver cholesterol levels were 50% higher but rose only 26% in WHHL as compared with 3.6-fold increase with the cholesterol diet in NZW rabbits. In both rabbit groups, hepatic total HMG-CoA reductase activity was similar and declined > 60% without changing enzyme mRNA levels after cholesterol was fed. In NZW rabbits, cholesterol feeding inhibited LDL receptor function but not mRNA levels. As expected, receptor-mediated LDL binding was reduced in WHHL rabbits. Hepatic cholesterol 7 alpha-hydroxylase activity and mRNA levels were 2.8 and 10.4 times greater in NZW than WHHL rabbits. Unexpectedly, cholesterol 7 alpha-hydroxylase activity was reduced 53% and mRNA levels were reduced 79% in NZW rabbits with 2% cholesterol feeding. These results demonstrate that WHHL as compared with NZW rabbits have markedly elevated plasma and higher liver cholesterol concentrations, less hepatic LDL receptor function, and very low hepatic cholesterol 7 alpha-hydroxylase activity and mRNA levels. Feeding cholesterol to NZW rabbits increased plasma and hepatic concentrations greatly, inhibited LDL receptor-mediated binding, and unexpectedly suppressed cholesterol 7 alpha-hydroxylase activity and mRNA to minimum levels similar to WHHL rabbits. Dietary cholesterol accumulates in the plasma of NZW rabbits, and WHHL rabbits are hypercholesterolemic because reduced LDL receptor function is combined with decreased catabolism of cholesterol to bile acids.     

Effects of dietary cholesterol on cholesterol and bile acid homeostasis in patients with cholesterol gallstones.

We examined changes in cholesterol and bile acid metabolism produced by dietary cholesterol in gallstone subjects and matched controls. Healthy women were recruited and, after confirming the presence or absence of radiolucent gallstones, they were studied on regular diets and again on the same diet supplemented with five eggs daily for 15-18 d. Studies included plasma lipids, lipoproteins and apolipoproteins, dietary records, cholesterol absorption, cholesterol synthesis, plasma clearance of chylomicron remnants, biliary lipid composition, and secretion and bile acid kinetics. On low cholesterol, gallstone subjects absorbed a slightly lower fraction of dietary cholesterol, synthesized more cholesterol, and had smaller bile acid pools and faster fractional turnover rate (FTR) of bile acids. On high cholesterol, the fraction of cholesterol absorbed decreased in both groups and cholesterol synthesis decreased, especially in the gallstone group. Biliary cholesterol secretion increased in the gallstone group only. FTR of bile acids did not change in either group. Bile acid synthesis and pool tended to increase (P = NS) in the controls, but in gallstone subjects, synthesis and pool size decreased. We concluded that in gallstone subjects cholesterol and bile acid homeostasis is significantly altered, and that increasing dietary cholesterol increases biliary cholesterol secretion and decreases bile acid synthesis and pool, changes associated with cholesterol gallstone formation.     

Adenovirus-mediated transfer of a gene encoding cholesterol 7 alpha-hydroxylase into hamsters increases hepatic enzyme activity and reduces plasma total and low density lipoprotein cholesterol.

Clinical interventions that accelerate conversion of cholesterol to bile acids reduce circulating low density lipoprotein (LDL) cholesterol concentrations. The initial and rate-limiting step in the bile acid biosynthetic pathway is catalyzed by hepatic cholesterol 7 alpha-hydroxylase. To examine the effects of transient primary overexpression of this enzyme on sterol metabolism and lipoprotein transport, we constructed a recombinant adenovirus in which a cDNA encoding rat 7 alpha-hydroxylase is expressed from the human cytomegalovirus immediate-early promoter (AdCMV7 alpha). Syrian hamsters administered AdCMV7 alpha intravenously accumulated transgene-specific mRNA in the liver and demonstrated a dose-dependent increase in hepatic microsomal 7 alpha-hydroxylase activity. The increased conversion of cholesterol to bile acids resulted in a compensatory increase in hepatic cholesterol synthesis. In addition, overexpression of 7 alpha-hydroxylase reduced the rate of LDL cholesterol entry into the plasma space and, in animals maintained on a Western-type diet, restored hepatic LDL receptor expression. As a consequence, plasma LDL concentrations fell by approximately 60% in animals maintained on control diet and by approximately 75% in animals consuming a Western-type diet. Plasma high density lipoprotein cholesterol levels were reduced to a lesser degree. These results demonstrate that transient upregulation of bile acid synthesis by direct transfer of a 7 alpha-hydroxylase gene favorably alters circulating lipoprotein profiles and suggest one potential molecular target for genetic strategies aimed at reducing cardiovascular risk.     

Effects of cholic acid, 7 beta-hydroxy- and 12 beta-hydroxy-isocholic acid on bile flow, lipid secretion and bile acid synthesis in the rat

The effects of three epimeric trihydroxy-cholanoic acids, cholic acid (C), 7 beta-hydroxy-(7 beta) and 12 beta-hydroxy-(12 beta) isocholic acids on bile flow, lipid secretion, bile synthesis and bile micellar properties were studied in the rat with a bile fistula. The bile salts were infused intraduodenally starting 72 hours after cannulation when endogeneous bile salt synthesis had plateaued after the bile salt pool was drained. The bile salts were infused at two levels approximately 2 and 4 mumol min-1 kg-1. All three bile salts were absorbed and secreted almost quantitatively into the bile. Cholic acid was secreted in the conjugated form, 7 beta conjugated to approximately 60% and 12 beta completely in the unconjugated form. The bile salts did not undergo any significant biotransformations during the one passage from the intestine through the liver. Bile flow increased from the preinfusion level for all three bile salts infused in the order 7 beta greater than 12 beta greater than C. The bile flow increased linearly with bile salt secretion more for 7 beta than for C and 12 beta. Infusion of C increased the secretion into bile of phospholipid (PL) and cholesterol (CH) over the preinfusion values. Infusion of 7 beta as well as 12 beta resulted in a parallel decrease in the secretion of PL as well as CH compared to the preinfusion values. The infusion of C and 7 beta at the two levels used decreased the secretion of newly synthesized bile salt below the control level.(ABSTRACT TRUNCATED AT 250 WORDS)     

Quantification of cholesterol nucleation promoting activity in human gallbladder bile

We have developed a simple method to quantitate cholesterol nucleation promoting activity in bile. The method makes use of the fact that gallbladder bile of cholesterol gallstone patients contains potent nucleation promoting activity. Gallbladder bile samples were serially diluted, routinely from 1/25 to 1/6,400. The diluted samples were mixed with a supersaturated model bile and the nucleation time (NT) of the mixtures was determined. The greatest dilution that resulted in a significant shortening of the NT was called the nucleation promoting activity titre (NPAT). The determination is independent of the original lipid content of the bile sample. The NPAT was measured in 14 gallbladder bile samples derived from patients with cholesterol gallstones and 9 controls. In all samples promoting activity was found. In the samples from the stone patients the NPAT was significantly elevated as compared to the patients without cholesterol stones (p = 0.01). Our results suggest that the cholesterol saturation index and the activity of cholesterol nucleation promoting factors are the most important factors in the pathogenesis of cholesterol gallstone disease. Assessment of the NPAT allows the differentiation of groups of patients with a normal cholesterol saturation index who are at risk for gallstone formation due to a high NPAT.     

Chronic administration of chenodeoxycholic acid increases cholesterol saturation in bile in the dog

Six dogs were given chenodeoxycholic acid, 200 mg/day for 15 days; gallbladder and hepatic bile samples were taken and biliary bile acids, phospholipids and cholesterol were compared to those of fourteen control dogs. It was found that: (1) the cholesterol saturation index of gallbladder bile was higher in treated dogs (0.12 +/- 0.06; m +/- SD) than in controls (0.07 +/- 0.01; P less than 0.05); the proportion of chenodeoxycholic acid in gallbladder bile was higher in treated dogs (27.8 +/- 12.5%) than in controls (4.5 +/- 1.9%; P less than 0.01); (2) in hepatic bile, the saturation index and proportion of chenodeoxycholic acid were significantly higher in treated dogs (respectively 0.13 +/- 0.03 and 21.6 +/- 8.8%) than in control dogs (respectively 0.07 +/- 0.01; P less than 0.01 and 4.6 +/- 2.2%; P less than 0.01). In the dog, chronic administration of chenodeoxycholic acid increases cholesterol saturation in bile. It is concluded that the difference of effect of chenodeoxycholic acid in man and in the dog is not related to the duration of administration, but to a species difference.     

Effect of physical exercise on erythrocyte lipids, biliary cholesterol and bile lithogenicity in rats

Male Wistar rats were exercised (E) by daily 1 h swimming for 24 days. Sedentry controls (S) were caged individually. Bile analysis at the end of the experiment (24 h after the last exercise) showed a decrease in bile cholesterol (CH) (P less than 0.02) and phospholipids (PL) (P less than 0.01) in the exercising animals, but no significant change in the bile acids (BA). These changes in the exercising rats resulted in a decreased CH saturation of bile: in an improvement in the (BA + PL)/CH ratio (P=0.05) and a trend to a decrease in the per cent saturation of bile CH. Exercise did not affect the bile flow or bile acid flux rate. Exercise decreased erythrocyte phospholipids (P less than 0.01). The effect of exercise on bile was associated with a trend to lower CH in the erythrocytes and in the adipose tissue and thus most likely does not represent a shift of cholesterol from the liver to the peripheral tissues. Physical exercise may be a preventive factor in cholesterol gallstone formation.