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Competitive inhibitors of 3-hydroxy-3-methylglutaryl-CoA reductase improve hypercholesterolemia. However, reports about the effects of these agents on bile acid synthesis, the metabolic pathway of cholesterol, are conflicting. We studied the short-term effect on one of these agents, pravastatin, on bile acid synthesis. Six male volunteers were given 40 mg of pravastatin. Plasma mevalonate level (which reflects cholesterol synthesis) and 7 alpha-hydroxy-4-cholesten-3-one level (which reflects bile acid synthesis) were measured every 2 h for 8 h. These plasma levels were compared to those of the same volunteers without pravastatin. Plasma mevalonate level after 2 h was lower than control (3.0 +/- 1.1 ng/mL vs. 6.7 +/- 2.5, mean +/- SD; P < 0.05). This decrease continued for 8 h (2.5 +/- 0.8 vs. 5.2 +/- 1.5; P < 0.05). On the other hand, plasma 7 alpha-hydroxy-4-cholesten-3-one level did not change until after 6 h; then at 8 h it was lower than control (15.7 +/- 11.8 ng/mL vs. 24.7 +/- 16.9; P < 0.05). According to three-way layout analysis of variance, mevalonate level was influenced by both pravastatin treatment (P < 0.01) and time-course (P < 0.01). On the other hand, the 7 alpha-hydroxy-4-cholesten-3-one level was affected by both individual difference (P < 0.01) and time course (P < 0.01), but pravastatin treatment did not influence this compound. This indicates that bile acid synthesis was influenced by pravastatin, although cholesterol synthesis was inhibited. The short-term inhibition of cholesterol synthesis did not affect bile acid synthesis.

The high prevalence of cholesterol gallstone disease in hypertriglyceridemic patients may be associated with frequent metabolic defects in cholesterol and bile acid syntheses and in the concomitant formation of bile supersaturated with cholesterol. This study had the two aims: 1) to assess whether the defects as well as the degree of biliary cholesterol supersaturation in patients with hyperlipoproteinemia (HLP) can be estimated by the simultaneous determination of plasma mevalonate (MVL) and 7alpha-hydroxy-4-cholesten-3-one (C4); and 2) to assess the possible application of an estimated cholesterol saturation index ([CSI]E) as a means of evaluating the clinical effects of simvastatin on biliary lipid composition. Biliary cholesterol supersaturation was observed in patients with both IIa and IV HLP types. Consistent with the high activity and steady-state messenger RNA level of 3-hydroxy-3 methylglutaryl coenzyme A (HMG-CoA) reductase, plasma MVL was significantly higher in 86 patients with HLP (38 type IIa, 44.1 +/- 2.4 nmol/L and 48 type IV, 56.7 +/- 2.3; P < .01) than in 41 normolipidemic subjects (34.2 +/- 1.5), closely correlating with the molar percentage of cholesterol in bile (r = .61, P = .0001; n = 86). On the other hand, consistent with the high activity and messenger RNA level of cholesterol 7alpha-hydroxylase, plasma C4 was significantly higher in patients with HLP (type IIa, 28.8 +/- 2.3 nmol/L and type IV, 38.3 +/- 2.7; P < .01) than in normolipidemic subjects (17.4 +/- 1.5). Plasma C4 was closely correlated with plasma MVL (r = .40, P = .0001; n = 86), but was inversely correlated with the molar percentage of bile acids in bile (r = .49, P = .0001; n = 86). Assuming that cholesterol supersaturation in patients with HLP may be governed by both an enhanced cholesterol secretion (closely reflected by plasma MVL) and a decreased secretion of bile acids (closely reflected by plasma C4), the multivariate linear regression-analyses revealed that an index defined as estimated CSI ([CSI]E) (%) in patients with HLP was given by the following equation using plasma MVL and C4 (nmol/L): [CSI]E = 1[MVL] + 0.7[C4] + 44.4. Biliary cholesterol supersaturation in patients treated with simvastatin improved in a manner parallel to the time course of decreases in plasma MVL and C4. The [CSI]E before and at the end of treatment were correlated with biliary CSI. These results indicate that defects of hepatic cholesterogenesis, and bile acid synthesis, and the degree of biliary cholesterol supersaturation in patients with HLP can be estimated exactly by the simultaneous determination of plasma MVL and C4; furthermore [CSI]E may be adopted for clinical use as a convenient index of biliary CSI.

Background/aims: Intrahepatic calculi, which are characterized by cholesterol-rich pigment stones, are highly prevalent in East Asia. Their pathogenesis remains unknown. To elucidate the etiological factors underlying the formation of cholesterol-supersaturated bile, which leads to the formation of cholesterol-rich pigment stones cholesterol and bile acid de novo syntheses in the liver were studied., Methods: Liver specimens were assayed for the catalytic activities and steady-state messenger RNA levels of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase and cholesterol 7 alpha-hydroxylase., Results: The activity of HMG-CoA reductase, consistent with the messenger RNA level, was significantly higher in 13 patients with intrahepatic grown pigment stones (11.2 +/- 1.3 pmol.min-1.mg protein-1 [mean +/- SEM; P < 0.0001] for affected hepatic lobes and 13.4 +/- 1.7 [P < 0.0001] for unaffected ones [P < 0.0001]) than in 19 control subjects (6.4 +/- 0.4) and in 29 patients with gallbladder cholesterol stones (2.1 +/- 0.1). On the other hand, the activity of 7 alpha-hydroxylase, consistent with the messenger RNA level, was significantly lower in patients with intrahepatic brown pigment stones (2.8 +/- 0.5 pmol.min-1.mg protein-1 [P < 0.0001] for affected lobes and 2.6 +/- 0.5 [P < 0.0001] for unaffected ones) than in control subjects (6.0 +/- 0.6) and in patients with cholesterol stones (5.1 +/- 0.5)., Conclusions: In intrahepatic calculi, the formation of supersaturated bile and cholesterol-rich pigment stones may be attributed to the primary dual defect of up-regulated cholesterogenesis and down-regulated bile acid synthesis in the liver.

Patients with cholesterol gallstone disease have a reduced pool of bile acids. Overly sensitive feedback inhibition of bile acid synthesis has been postulated to explain this size reduction. To test this hypothesis, hepatic bile acid concentration and the activity of cholesterol 7 alpha-hydroxylase, the rate-limiting enzyme for bile acid biosynthesis, were determined in ten patients with cholesterol gallstones and ten patients without gallstones. The bile acids present in liver tissue are the sum of those returning to liver and those newly synthesized in liver. If an overly sensitive feedback inhibition truly existed in our gallstone patients, a decreased concentration of hepatic bile acids would have been expected. However, patients with cholesterol gallstones had significantly higher total (143.3 +/- 25.5 vs 64.5 +/- 10.8 nmol/g liver, P < 0.01), chenodeoxycholic (64.1 +/- 9.9 vs 29.8 +/- 5.4, P < 0.01), deoxycholic (22.8 +/- 10.9 vs 2.0 +/- 0.7, P < 0.05), and ursodeoxycholic acid (6.2 +/- 1.4 vs 1.5 +/- 0.6, P < 0.01) concentrations than patients without gallstones. The activity of cholesterol 7 alpha-hydroxylase did not differ significantly between the two groups. Impaired hepatic transport or secretion of bile acids is strongly suspected in cholesterol gallstone patients. The findings of the present study showed no evidence of overly sensitive feedback inhibition of bile acid synthesis in cholesterol gallstone patients. Bile acid pool size may be affected by the inappropriate increase of hepatic bile acids rather than by overly sensitive feedback inhibition.

A highly sensitive and specific method has been developed for determination of the level of 7 alpha-hydroxy-4-cholesten-3-one in plasma. This method is based on a stable isotope-dilution technique by gas chromatography-selected-ion monitoring mass spectrometry. 7 alpha-Hydroxy-4-cholesten-3-one was extracted from plasma by saltingout extraction, and then purified by serial solid-phase extractions. The extract was treated with O-methylhydroxyl-amine hydrochloride and then dimethylethylsilylated. The resulting methyloxime-dimethylethylsilyl ether derivative was quantified by gas chromatography-selected-ion monitoring mass spectrometry with a high-resolution mode. The plasma levels of 7 alpha-hydroxy-4-cholesten-3-one were correlated with the cholesterol 7 alpha-hydroxylase activity to a higher degree than those of any other form of 7 alpha-hydroxycholesterol (r = 0.84, n = 16, p < 0.0001). The present method was applied to monitor the circadian rhythm of 7 alpha-hydroxy-4-cholesten-3-one levels in human plasma. It was concluded that the plasma level of 7 alpha-hydroxy-4-cholesten-3-one is a useful index for the monitoring of bile acid biosynthesis in the human liver.

A detailed comparison was made of the bile acid composition in gallstones (brown pigment stones) and paired bile and liver from both affected and unaffected lobes by gallstones, which were taken at operation from 16 patients with hepatolithiasis, with the aim of elucidating whether stone formation is derived from possible local disturbances limited to intrahepatic bile ducts. Brown pigment stones in the intrahepatic bile ducts, most of which were accompanied by bile with high cholesterol saturation, had significantly more cholesterol, and less calcium bilirubinate and bile acid than those found in the extrahepatic bile ducts. Intrahepatic gallstones had significantly lower amounts of secondary and unconjugated bile acids, the bile acids modified by bacterial intervention, than extrahepatic stones. Bile specimens from both affected and unaffected lobes showed significantly increased molar percentages of cholesterol and decreased percentages of bile acids than bile from controls. In contrast, liver specimens from both lobes showed significantly higher concentrations of total bile acids. Secondary bile acids were present in a much lower proportion in bile and liver from both lobes than in bile and liver from controls. On the other hand, unconjugated bile acids were present in a much higher proportion in bile and liver from patients and only in negligible amounts in bile from controls. Furthermore, the plasma levels of mevalonate and those of 7 alpha-hydroxy-4-cholestene-3-one were found to be significantly higher and lower in patients than in controls, respectively, indicating that in hepatolithiasis cholesterol synthesis might increase and bile acid synthesis might decrease in the liver. These findings suggested that alterations of bile acid composition in gallstones, bile, and liver of patients with hepatolithiasis may be attributed to not only secondary changes resulting from local disturbances limited to intrahepatic bile ducts but also possible primary alterations of hepatocyte metabolism, such as bile acid conjugation and primary defects in cholesterol and bile acid synthesis.

In Japan the composition of gallstones is changing rapidly from the once-predominant brown-pigment stones to cholesterol ones. The present work was undertaken to clarify the mechanism of cholesterol supersaturated bile production in Japanese patients with cholesterol gallstones. In 26 non-obese and normolipidemic patients (11 with cholesterol gallstones, 8 with black- or brown-pigment gallstones, 7 without gallstones) a liver biopsy and hepatic bile were surgically obtained under standardized conditions. The cholesterol saturation of hepatic bile was significantly higher in cholesterol gallstone patients than in gallstone-free controls (195 +/- 10 vs. 146 +/- 8%, respectively; P < 0.01). The microsomal activities of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, the rate-limiting enzyme for cholesterol synthesis, cholesterol 7 alpha-hydroxylase, the rate-limiting enzyme for bile acid synthesis, and 7 alpha-hydroxy-4-cholesten-3-one 12 alpha-hydroxylase (12 alpha-hydroxylase), the rate-limiting enzyme for cholic acid synthesis, were assayed simultaneously in the same subjects. There were positive correlations between HMG-CoA reductase and cholesterol 7 alpha-hydroxylase activities (Rs = 0.62, P < 0.005), and between cholesterol 7 alpha-hydroxylase and 12 alpha-hydroxylase activities (Rs = 0.44, P < 0.05) in all subjects, irrespective of the existence of gallstones. The activities of the three rate-limiting enzymes did not differ significantly among the three groups (cholesterol stone, pigment stone and stone-free). In conclusion, the cholesterol supersaturation of hepatic bile in nonobese and normolipidemic Japanese patients with cholesterol gallstones does not result from an increased hepatic cholesterol synthesis or a decreased bile acid synthesis.

A very sensitive and specific method for the simultaneous determination of mevalonate and 7 alpha-hydroxycholesterol in human plasma is described. The assay is based on isotope dilution mass spectrometry: the extracts from plasma were treated with benzylamine followed by dimethylethylsilylimidazole, then the resulting dimethylethylsilyl ether derivatives of mevalonylbenzylamide and 7 alpha-hydroxycholesterol were determined by gas chromatography-mass spectrometry using high-resolution selected-ion monitoring. Simple regression analysis revealed significant correlations between the plasma level of mevalonate and the hepatic activity of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (EC 1.1.1.34) (r = 0.83, P < 0.01) and between the plasma level of free 7 alpha-hydroxycholesterol and the hepatic activity of cholesterol 7 alpha-hydroxylase (EC 1.14.13.7) (r = 0.76, P < 0.05).

Brown pigment stones in the intrahepatic bile ducts were compared with those found in the extrahepatic bile ducts with special reference to the bile acids modified by bacterial intervention, that is, unconjugated, glucuronidated, secondary, and ketonic bile acid fractions. The former showed significantly lower amounts of total bile acids (P less than 0.01) and lower proportions of unconjugated bile acid fraction (P less than 0.01), secondary bile acid fraction (P less than 0.05), and ketonic bile acid fraction (P less than 0.05) to total bile acids than the latter. The discriminant analysis using these bile acid parameters led to complete separation between intrahepatic and extrahepatic stones in the case of brown pigment stones. In contrast, cholesterol stones in the intrahepatic bile ducts showed the bile acid composition close to those found in the extrahepatic ducts and gallbladder. The above data show that the bacterial infection plays a less important role in the formation and ensuing growth of most intrahepatic brown pigment stones than in extrahepatic stones, and that factors other than or in addition to bacterial infection are involved.

C-1 and C-6 hydroxylated bile acid metabolites in various biological specimens from subjects with liver disease (cholestasis, liver cirrhosis, chronic hepatitis, acute hepatitis) were determined by gas-liquid chromatography-mass spectrometry. Five C-1 hydroxylated bile acids and nine C-6 hydroxylated bile acids were identified in the urine studied; 1 beta,3 alpha,12 alpha-trihydroxy-, 1 beta,3 alpha,7 alpha-trihydroxy-, 1 beta,3 alpha,7 alpha,12 alpha-tetrahydroxy-, 3 alpha,6 alpha,7 alpha-trihydroxy-, and 3 alpha,6 alpha,7 alpha,12 alpha-tetrahydroxy-5 beta-cholanoic acids were found as the major components. Most of the 1 beta- and 6 alpha-hydroxylated bile acids were excreted into urine in the nonsulfate-nonglucuronide form. The amounts in the urine were greater than those found in the bile, portal and peripheral venous sera, and liver specimens. The biliary excretion and hepatic extraction of 1 beta-hydroxylated metabolites were more impaired and less efficient than for cholic acid. These findings suggested that hepatic 1 beta- or 6 alpha-hydroxylation of bile acids occurred concurrently in the patients with liver disease and that the resulting hydroxylated metabolites were efficiently excreted in the nonsulfate-nonglucuronide form into urine rather than into bile.

To explore the effect of type of dietary fat, cholesterol and chenodeoxycholic acid on gallstone formation, bile formation, bile composition, bile acid kinetics and plasma lipids in squirrel monkeys, 39 monkeys were studied using seven different diets. Safflower oil, a highly unsaturated fat, added to a diet with cholesterol resulted in at least as high an incidence of cholesterol gallstones as butter added to the same diet. On the other hand, diets with high levels of saturated or unsaturated fat without cholesterol did not result in gallstone formation. Dietary chenodeoxycholic acid (0.1%) did not reduce the incidence of cholesterol gallstones, although the proportion of bile acids as chenodeoxycholic acid increased. Gallbladder biles from monkeys fed semipurified diets with cholesterol had a significantly higher lithogenic index than the comparable groups without cholesterol. Pool sizes of bile acids in all semipurified diet groups were reduced and the lithogenic indices were increased compared with the group fed a commercial feed. Dietary chenodeoxycholic acid caused a decrease in plasma cholesterol in butter groups and an increase in triglyceride concentrations in safflower groups. Diet infuences bile composition and bile acid kinetics, as well as the incidence of gallstones, in squirrel monkeys.

Fasting serum bile acid (SBA) was measured by the enzymic fluorimetric method coupled with the NAD-resazurin system in 23 controls, 35 asymptomatic carriers of HIs antigen including 4 e antigen carriers and 91 patients with various liver diseases. All GHBs and e antigen carriers showed SBA within the normal range. SBA was most significantly correlated with serum bilirubin (gamma=0.74) and was a more sensitive index for impaired liver function than bilirubin or alkaline phosphatase in 164 radomly chosen samples from the liver disease group. In serial determinations of SBA with reference to GOT, GPT, changing patterns of these two parameters were classified into the parallel type and the discrepant type. Thirty two out of 40 cases with chronic liver disease belonged to the parallel type. SBA remained abnormal even after the normalization of transaminase in 12 out of 20 resolving episodes in cases of the parallel type, regardless of diagnosis. Since SBA changes according to the stage of the disease activity, serial and simultaneous estimation of SBA and GOT, GPT was found to be helpful in the observation of liver diseases. Maximum values of SBA elevation in an endogenous bile acid tolerance test after eating two egg yolks were higher than controls in 4 out of 7 cases with liver disease, who were associated with normal fasting SBA.

Bile acids, such as cholic, chenodeoxycholic, deoxycholic, lithocholic and ursodeoxycholic acids, were allowed to react with hexafluoroisopropanol and tri-fluoracetic anhydride at 37 for 30 min. The resulting derivatives were gas chromatographed on QF-1, with flame ionization detection, and were identified by gas chromatography-mass spectrometry. Separation was good. By using this method, these acids were detected in samples of human duodenal fluid; the ratios of each were 24.4, 41.5, 24.9, 2.3 and 6.9%, respectively.

3 beta-Hydroxy-(delta 5-3 beta-ol), 3 beta,12 alpha-dihydroxy-(delta 5-3 beta,12 alpha-ol), 3 beta,7 alpha-dihydroxy-(delta 5-3 beta,7 alpha-ol) and 3 beta,7 beta-dihydroxy-(delta 5-3 beta,7 beta-ol) 5-cholenoic acids were identified in patients with liver diseases by gas-liquid chromatography-mass spectrometry (GLC-MS). Of these unusual 3 beta-hydroxy-5-en-metabolites, delta 5-3 beta-ol and delta 5-3 beta,12 alpha-ol were found as major components in the urine of patients with liver diseases (cholestasis, liver cirrhosis, chronic hepatitis, acute hepatitis). Other 3 beta-dihydroxy-5-en-metabolites, delta 5-3 beta,7 alpha-ol and delta 5-3 beta,7 beta-ol, were found as minor components in the urine. The levels of delta 5-3 beta-ol and delta 5-3 beta,12 alpha-ol in urine were correlated with their levels in serum, with total bile acids in the urine, and with liver function, implying that the degree of their increment correlated well with the severity of liver diseases. The most abundant amounts of delta 5-3 beta-ol and delta 5-3 beta,12 alpha-ol were found in the urine as sulfate conjugates in comparison with bile, portal and hepatic venous sera, and liver tissue of the patients. The biliary excretion and hepatic extraction of these 3 beta-hydroxy-5-en-unsaturated bile acids were more impaired and inefficient than those of cholic and chenodeoxycholic acids.

A simple and sensitive method was developed for the quantification of serum total 3alpha-hydroxy bile acids. 0.1 ml of serum was mixed with tris(hydroxymethyl) aminomethane hydrochloric acid buffer and heated at 67 degrees C for 30 min. To the solution were added 3alpha-hydroxysteroid : oxidoreductase (EC 1.1.1.50; 3alpha-HSD), NAD, diaphorase (EC 1.6.4.3) and resazurin. The mixture was incubated at 20 degrees C for 1 h. The resultant fluorescence of resorfin was measured at 580 nm with the excitation at 560 nm. The blank value was obtained after the same treatment of another 0.1 ml of the same serum without 3alpha-HSD. A linear relationship was obtained between the amount of bile acids and the fluorescence intensities in the range of 1 to 150 mumol/1. The recovery of bile acids added to the serum was 81.4 +/- 2.5 (S.D.)% for cholate, chenodeoxycholate and deoxycholate. The bile acid content in the serum was 48.8 mumol/1 with a standard deviation of +/- 0.42 and a coefficient of variation of +/- 0.87% in 10 replicate determinations. The mean bile acid content of normal fasting male sera was 8.0 mumol/1 (3.6-12.6 mumol/1, n = 12) and of female sera 6.8 mumol/1 (3.2-12.7 mumol/1, n = 13).

We describe a highly sensitive and accurate automated continuous-flow method for determining bile acids in serum. The bile acids are first liberated from serum protein by dialysis at alkaline pH and then measured fluorometrically after the following enzymic reaction. Bile acids are converted to 3-oxo bile acids with 3alpha-hydroxysteroid dehydrogenase (EC 1.1.1.50) with concomitant reduction of NAD+ to NADH. The hydrogen in the generated NADH is transferred by diaphorase (EC 1.6.4.3) to resazurin to yield resorfin, the fluorophore. Only 100 microliter of serum is required and 40 determinations can be done per hour. The CV for 20 replicate determinations in serum with a mean bile acid concentration of 9.8 mumol/liter was 2.6%. The CV for day-to-day variation for another serum on 27 successive days was 3.0% (mean concentration, 10.0 mumol/liter). We applied this method to 826 sera from various diseases; 29% exceeded the upper limit of normal, 10 mumol/liter, and abnormally high values (greater than 20 mumol/liter) were almost exclusively limited to sera from hepatobiliary and enteric disorders.

Unusual bile acids in umbilical cord blood and amniotic fluid of term newborns and in sera and urine from adult patients with cholestatic liver diseases were analyzed by use of gas-liquid chromatography-mass spectrometry. These bile acids were compared in order to elucidate possible similarities of bile acid metabolism between fetal and cholestatic liver. In both umbilical cord blood and amniotic fluid, 14 unusual bile acids were found in addition to normal bile acids (cholic, chenodeoxycholic, deoxycholic, and lithocholic acids), and 15, excluding ursodeoxycholic acid, were found in sera and urine from patients with cholestatic liver diseases. Of the unusual bile acids detected, 12 were common to both samples. Six unusual bile acids, 3 beta-hydroxy- and 3 beta,12 alpha-dihydroxy-5-cholenoic acids, 3 alpha,6 alpha,7 alpha-trihydroxy-5 beta-cholanoic acid, 1 beta,3 alpha,12 alpha-trihydroxy-1 beta,3 alpha,7 alpha-trihydroxy-, and 1 beta,3 alpha,7 alpha,12 alpha-tetrahydroxy-5 beta-cholanoic acids were more abundant than others. They could be classified into three groups, i.e., unsaturated, 6-hydroxylated, and 1 beta-hydroxylated bile acids. 1 beta-Hydroxylated bile acids, which were not found in serum specimens, were detected in sera from umbilical cord blood and from patients with cholestatic liver diseases. The presence of these unusual bile acids suggested similarities between the altered metabolic states of the two groups examined.

Trihydroxy and tetrahydroxy bile acid metabolites substituted at the C-1 or C-6 position were studied using the urine, serum and liver tissue from sixteen patients with cholestatic liver diseases. Following extraction, isolation and hydrolysis, bile acids were converted into the dimethylethylsilyl derivatives and assayed by capillary gas chromatography-mass spectrometry. Five 1 beta-hydroxylated bile acids, viz. 1 beta,3 alpha,12 alpha-trihydroxy-, 1 beta,3 alpha,7 alpha-trihydroxy-, 1 beta,3 alpha,7 beta-trihydroxy-, 1 beta,3 alpha,7 alpha,12 alpha-tetrahydroxy-5 beta-cholanoic acids and an epimer of the first compound, and two 6 alpha-hydroxylated bile acids, viz. 3 alpha,6 alpha,7 alpha-trihydroxy-, 3 alpha,6 alpha,7 alpha,12 alpha-tetrahydroxy-5 beta-cholanoic acids, were completely or partially identified. Large amounts of 1 beta-hydroxylated and 6 alpha-hydroxylated bile acids were found in the urine, whereas only trace amounts were detected in the serum and liver tissue. These findings indicate that altered metabolism, such as 1 beta- or 6 alpha-hydroxylation of bile acids, is enhanced in cholestasis, and that the resulting hydroxylated metabolites are eliminated in the urine.