Normal Bile Acid Research Articles

Urinary bile acids and peroxisomal bifunctional enzyme deficiency

The biosynthesis of normal bile acids involves beta-oxidation of the 8-carbon side-chain of cholesterol, in addition to numerous modifications of the sterol nucleus. Because beta-oxidation of the sterol side-chain has been localized to the peroxisome, bile acid analysis has been suggested to be useful in the diagnostic evaluation of individuals suspected of having peroxisomal disorders. Although data from subjects with generalized peroxisomal disorders support this, few data exist regarding the bile acids in individuals having single peroxisomal beta-oxidation enzyme disorders. In this study, we analyzed the urinary bile acids from 12 patients with peroxisomal bifunctional protein deficiency using continuous flow fast atom bombardment mass spectrometry. All 12 patients had abnormal spectra, although their ion profiles and rank order of intensity of ions varied considerably. Ten of 12 individuals had abnormal spectra with presence of taurine-conjugated tetrahydroxycholestenoates, allowing a definite diagnosis of a peroxisomal beta-oxidation defect and a presumptive diagnosis of bifunctional protein deficiency; the other two cases were nondiagnostically abnormal. The strengths and limitations of urinary bile acid analysis for the diagnosis of peroxisomal beta-oxidation disorders are discussed.

Urinary bile acids and peroxisomal bifunctional enzyme deficiency

The biosynthesis of normal bile acids involves beta-oxidation of the 8-carbon side-chain of cholesterol, in addition to numerous modifications of the sterol nucleus. Because beta-oxidation of the sterol side-chain has been localized to the peroxisome, bile acid analysis has been suggested to be useful in the diagnostic evaluation of individuals suspected of having peroxisomal disorders. Although data from subjects with generalized peroxisomal disorders support this, few data exist regarding the bile acids in individuals having single peroxisomal beta-oxidation enzyme disorders. In this study, we analyzed the urinary bile acids from 12 patients with peroxisomal bifunctional protein deficiency using continuous flow fast atom bombardment mass spectrometry. All 12 patients had abnormal spectral, although their iron profiles and rank order of intensity of ions varied considerably. Ten of 12 individuals had abnormal spectra with presence of taurine-conjugated tetrahydroxycholestenoates, allowing a definite diagnosis of a peroxisomal beta-oxidation defect and a presumptive diagnosis of bifunctional protein deficiency; the other two cases were nondiagnostically abnormal. The strengths and limitations of urinary bile acid analysis for the diagnosis of peroxisomal beta-oxidation disorders are discussed. © 1996 Wiley-Liss, Inc.

Radiological bile acid absorption test 75SeHCAT in patients with diarrhoea of unknown cause.

The purpose with this study was to investigate the usefulness of the 75SeHCAT test in patients suffering from "functional" diarrhoea and to disclose earlier radiological investigations performed in the course of disease. Twenty patients with chronic diarrhoea of unknown cause were investigated with the 75SeHCAT test. The cholestyramine test and an estimation of oro-caecal transit time (OCTT) were also performed in 13 and 10 patients respectively. Ten patients had low 7-day retention (< or = 15%). Nine of these were tested with cholestyramine and 8 showed complete relief from the diarrhoea. The cholestyramine test had no effect on the 4 patients with normal bile acid retention. No correlation was found between the retention of 75SeHCAT and OCCT. A total of 80 inconclusive gastrointestinal barium examinations were performed during the course of disease. Pathological bile acid retention, studied with the radiological 75SeHCAT test, is common in patients with diarrhoea of unknown cause. The test should be performed earlier in the course of disease in order to prevent unnecessary radiological investigations and to reduce patient suffering and distress.

Formation of delta 22-bile acids in rats is not gender specific and occurs in the peroxisome

We recently demonstrated that the formation of delta 22-bile acids is a quantitatively major pathway for normal bile acid synthesis in the adult male Sprague-Dawley rat. This pathway is specific for 7 beta-hydroxy bile acids and, when ursodeoxycholic acid is administered, delta 22-ursodeoxycholic acid appears as a major metabolite in the liver tissue, bile, intestinal contents, and plasma. The aims of this study were, therefore, to determine whether this metabolic pathway was gender specific, and to establish that the peroxisome is a site of formation of delta 22-bile acids. Bile acids were determined by gas chromatography-mass spectrometry in liver tissue, jejunum, and plasma of adult female rats and in animals fed a diet containing 0.4% and 1% ursodeoxycholic acid. Bile acid metabolism in female rats was found to be similar to that of male rats, and delta 22-beta-muricholic acid, rather than beta-muricholate, was likewise confirmed as the major muricholic acid synthesized. Ursodeoxycholic acid administration resulted in the appearance of delta 22-ursodeoxycholic acid as a major metabolite. When adult male Sprague-Dawley rats were treated with clofibrate, a drug that induces peroxisomal proliferation, liver weight increased 40-60% and total bile acid synthesis decreased markedly, but the relative composition of individual bile acids was unchanged. When ursodeoxycholic acid was added to the diet, the proportion of delta 22-bile acids relative to the corresponding saturated analogues increased significantly compared with untreated rats, indicating that clofibrate had "amplified" the pathway for formation of delta 22-bile acids. When UDCA was incubated in vitro with a peroxisomal-enriched fraction from normal adult male rat liver, delta 22-ursodeoxycholic acid was formed in proportions comparable to that observed in vivo when this bile acid was given orally. These studies establish that the pathway for the formation of delta 22-bile acids is not gender specific and mainly occurs in hepatic peroxisomes.

A new cause of progressive intrahepatic cholestasis: 3 β-Hydroxy-C 27-steroid dehydrogenase/isomerase deficiency

There have been a few reports of infants with severe neonatal cholestasis related to a defect in primary bile acid synthesis. To assess the importance of such deficiency among children with progressive intrahepatic cholestasis (Byler disease), screening for inborn errors in bile acid synthesis was performed by fast atom bombardment ionization-mass spectrometry of urine samples from 30 affected children. Bile acid analysis revealed a specific fast atom bombardment ionization-mass spectrometry profile for 3 beta-hydroxy-C27 steroid dehydrogenase/isomerase deficiency in five children who had jaundice, hepatosplenomegaly, and fatty stools beginning at ages ranging from 4 to 46 months. None of them had pruritus. Liver function tests showed persistently normal serum gamma-glutamyltransferase activity, low serum cholesterol and vitamin E levels, normal serum bile acid concentrations despite raised serum bilirubin levels, and decreased prothrombin time and clotting factor V. In four of the cases a similar disease was observed in siblings. Liver function returned to normal after oral ursodeoxycholic acid therapy. We conclude that 3 beta-hydroxy-C27-steroid dehydrogenase/isomerase deficiency should be considered when idiopathic cholestatic liver disease with clinical features akin to Byler disease is characterized by the association of normal serum gamma-glutamyltransferase activity, normal serum bile acid concentration, absence of pruritus, and a return to normal liver function during ursodeoxycholic acid therapy. Early identification of these children is essential because they benefit from bile acid therapy and might thus avoid the need for liver transplantation.

Abnormal bile acids in the Smith-Lemli-Opitz syndrome.

The urinary bile acids from four patients with Smith-Lemli-Opitz (SLO) syndrome were analyzed by continuous flow fast atom bombardment mass spectrometry. Two types of abnormalities were noted: (1) a deficiency of normal bile acids (cholenoates) and (2) the presence of abnormal species postulated to be cholenoates and cholestenoates. The finding of abnormal urinary bile acids in children with SLO syndrome led to further investigation of the cholesterol metabolic pathway and to the delineation of a new inborn error of metabolism, deficient conversion of 7-dehydrocholesterol to cholesterol [Irons et al., 1993]. The abnormalities of urinary bile acids, if confirmed by further structural analyses and studies of additional patients, provide an explanation for various aspects of the gastro-intestinal abnormalities and growth retardation noted in SLO syndrome and suggest that exogenous bile acid replacement may play an important role in the therapy of patients with this syndrome.

Effects of ursodeoxycholic acid on plasma lipids.

Interference with the enterohepatic circulation leads to changes in plasma lipoprotein metabolism. Thus, increased bile acid synthesis (such as after cholestyramine therapy or biliary diversion) stimulates hepatic triglyceride production and increases the number of low density lipoprotein (LDL) receptors in the liver. However, treatment with chenodeoxycholic acid reduces triglyceride production and appears to reduce LDL catabolism. Ursodeoxycholic acid therapy, which has minor effects on bile acid synthesis, results in relatively minor changes in lipoprotein metabolism. A tendency for lowered LDL cholesterol levels may be related to the fact that ursodeoxycholic acid interferes with the enterohepatic circulation of the normal bile acids, particularly when administered at a high dosage.

Effect of epomediol on taurocholate uptake in hepatocytes isolated from normal rats or rats with ethinyloestradiol-induced cholestasis.

In this study the effect of epomediol (1,3,3-trimethyl-2-oxabicyclo 2.2.2 ocatan-6,7-endo, endo-diol) (Clesidren) on rat hepatocyte bile acid transport was evaluated using [3H]-taurocholate as a probe, in order to clarify the mechanism of action of this drug. To this purpose, primary cultures were prepared with hepatocytes obtained from normal rats (Group I), and rats receiving a 4-day treatment with either epomediol (100 mg/kg) (Group II), or ethinyloestradiol (5 mg/kg) (Group III), or ethinyloestradiol plus epomediol (5 mg and 100 mg/kg respectively) (Group IV) or vehicle DMSO 50 microliters/kg) (Group V). All hepatocytes were isolated 10 days after the end of treatment. Hepatocyte [3H]-taurocholate uptake was evaluated in vitro after 48 hr of incubation in the presence or absence of epomediol. In both cases no difference was found when evaluating the uptake of hepatocytes from Group I, II and V. In the absence of epomediol [3H]-taurocholate uptake in hepatocytes from rats of Group IV was significantly higher than that observed in hepatocytes from rats of Group III. On the other hand, the presence of epomediol did not influence [3H]-taurocholate uptake in hepatocytes from rats of Group III, which remained significantly lower compared to that of control hepatocytes (Group V). The protective effect obtained when administering epomediol simultaneously with ethinyloestradiol (Group IV) was not due to its ability to compete with ethynyloestradiol for the binding to oestrogen receptors. Our results indicate that epomediol is able to restore a normal hepatocyte bile acid uptake when given in vivo simultaneously with ethinyloestradiol but does not influence bile acid transport in cultured hepatocytes. Further studies are required to better define the choleretic activity of this drug.

Early and Long-Term Effects of Colectomy and Endorectal Pullthrough on Bile Acid Profile

Although total colectomy with mucosal proctectomy and endorectal pullthrough affects two sites critical to the enterohepatic circulation of bile acids, little information is available regarding the manner in which normal digestive physiology is altered by these procedures. This study defines the early and long-term effects of colectomy and endorectal pullthrough on bile acid profile and the long-term effects on biliary lipid metabolism. Specific changes in bile acid absorption have been reported in patients after ileal resection. Recent studies from our laboratory indicate that in the early postoperative period, colectomy with endorectal pullthrough causes a significant decrease in gallbladder bile concentrations of total bile acids, cholesterol, phospholipids, and calcium. The observation by several authors that the pouch undergoes morphologic and perhaps functional adaptation suggest that these changes may be transient and perhaps reversible. These studies were done in an awake, unanesthetized canine model that allows periodic sampling of gallbladder bile without creation of an external biliary fistula and its associated sequelae. Animals were ultimately randomly assigned to either laparotomy and gallbladder cannulation (N = 6), or gallbladder cannulation with total colectomy and ileorectal anastomosis (N = 7), or biliary cannulation, colectomy, mucosal proctectomy and endorectal pullthrough with ileal reservoir (N = 5). Six weeks after operation, colectomy and ileorectal anastomosis were associated with a significant alteration in the relative composition of bile acids in gallbladder bile. These early changes were manifested by a significant (p < 0.05) increase in taurocholic acid and a concomitant decrease in taurodeoxycholic acid. These changes became even more pronounced in the ileorectal anastomosis group 12 weeks after colectomy and ileorectostomy. Although similar changes in the relative concentrations of individual bile acids occurred in the 6-week endorectal animals, bile acid profile was restored to normal by 12 weeks. Colectomy with ileorectal anastomosis leads to early and significant changes in bile acid profile, which persist and become even more pronounced with time. In contrast, the construction of an ileal reservoir after colectomy facilitates restoration of a normal bile acid profile. We propose that these alterations in bile acid metabolism result from adaptation of the ileal reservoir as its mucosa assumes functional characteristics of normal colon.

Disorders of bile acid metabolism in cholesterol gallstone disease.

The aim of the study was to evaluate the metabolism of individual bile acids in patients with cholesterol gallstone disease. Therefore, we determined pool size and turnover of deoxycholic (DCA), cholic (CA), and chenodeoxycholic acid (CDCA) in 23 female gallstone patients classified according to their gallbladder function and in 15 healthy female controls. Gallstone patients had normal hepatic bile acid synthesis, but, depending on gallbladder function, differed with respect to turnover and size of the bile acid pools: Patients with well-emptying gallbladder (group A, n = 9) had enhanced turnover and reduced pools of CA (-46%; P less than 0.01 vs. controls) and CDCA (-24%; P less than 0.05), but normal input and size of the DCA pool. With reduced gallbladder emptying (less than 50% of volume; group B, n = 6), turnover and pools of CA, CDCA, and DCA were similar as in controls. Patients with loss of gallbladder reservoir (group C, n = 8) had increased input (+100%; P less than 0.01) and pool size of DCA (+45%; P = 0.07) caused by rapid conversion of CA to DCA, while the pools of CA (-71%; P less than 0.001 vs. controls) and CDCA (-36%; P less than 0.05) were reduced by enhanced turnover. Thus, in patients with cholesterol gallstones, the pools of primary bile acids are diminished, unless gallbladder emptying is reduced. Furthermore, in a subgroup of gallstone patients, who had completely lost gallbladder function, the CA pool is largely replaced by DCA owing to rapid transfer of CA to the DCA pool. This probably contributes to supersaturation of bile with cholesterol.

Structure-activity relationship of the cholestatic activity of dihydrotestosterone glucuronide, allo bile acids and lithocholate

Dihydrotestosterone glucuronide (DHTG), a series of 5α-bile acids, or allo-bile acids (3α-hydroxy-5α-cholanic acid, 3-keto-5α-cholanic acid and 3β-hydroxy-5α-cholanic acid) and their normal bile acid analogues (3α-hydroxy-5β-cholanic acid or lithocholate, 3-keto-5β-cholanic acid and 3β-hydroxy-5β-cholanic acid) were administered intravenously to female rats in order to determine their effects on bile flow. All agents caused a rapid and profound inhibition of bile flow which was dose-dependent. The logarithm of the dose vs the cholestatic response curve for DHTG, the allo-bile acids and lithocholate were all parallel. DHTG was the most potent congener and was two times more potent than 3-keto-5α-cholanic acid and 5 times more potent than lithocholate. These data indicate that the glucuronic acid moiety and the trans configuration of the A and B rings of the steroid nucleus confer the greatest cholestatic potency.

33 DEFECTIVE BILE SECRETION OF BILE ACID GLUCURONIDES IN RATS WITH HEREDITARY CONJUGATED HYPERBILIRUBINEMIA: IMPLICATIONS FOR THE MECHANISM OF CHOLESTAIS

Bile acids glucuronides have been identified in human bile, plasma and urine. We investigated the biliary secretion of lithocholate-3-0-glucuronide (LCG) and of cholate-3-0-glucuronide (CG), as well as the cholestatic potency of LCG in normal Wistar rats and in Wistar rats with hereditary conjugated hyperbilirubinemia. These rats show an impaired biliary secretion of bilirubin conjugates and other organic anions, but have a normal bile acid secretion. Bile secretion of an i.v. administered dose of (3H)LCG was strongly reduced in the mutant rats; 24% recovery in bile at 1h after injection vs. 96% in control rats. Corresponding values for biliary recovery of (3H)CG at 1h were 71% and 98% resp. Bile secretion of (3H)LCG was delayed by i.v, infusion of dibromosulphthalein (DBSP, 1.1 umol/min/kg) in control rats, whereas that of a simultaneously administered tracer dose of (14C)taurocholic acid was slightly accelerated. Low doses of LCG (0.5-2.0 mg) caused a transient reduction of bile flow in control rats, followed by a choleresis. A dose of 4 mg caused an almost complete cessation of the bile flow within 30 min. In contrast, LCG at the same dosages did not affect bile production in the mutants. It is concluded that bile acid-3-0-glucuronides share transport systems for biliary secretion with bilirubin and DBSP. Our data indicate that transport across the canalicular membrane is of importance for the development of LCG-induced cholestasis.

Similarity of unusual bile acids in human umbilical cord blood and amniotic fluid from newborns and in sera and urine from adult patients with cholestatic liver diseases

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.

Separate transport systems for biliary secretion of sulfated and unsulfated bile acids in the rat.

Biliary secretion of 3 alpha-sulfated bile acids has been studied in Wistar rats with an autosomal recessive defect in the hepatic transport of bilirubin. Liver function, established by measurement of various enzymes in plasma, by enzyme histochemical methods, and by electron microscopy, appeared to be normal in these rats. Serum levels of unconjugated, monoglucuronidated, and diglucuronidated bilirubin were 0.62, 1.62, and 6.16 mumol/liter, respectively, compared with 0.17, 0.08, and 0.02 mumol/liter in control rats. Biliary bilirubin secretion was strongly reduced in the mutant animals: 0.21 +/- 0.03 vs. 0.39 +/- 0.03 nmol/min per 100 g body wt in control rats. Despite normal biliary bile acid output, bile flow was markedly impaired in the mutant animals, due to a 53% reduction of the bile acid-independent fraction of bile flow. The transport maximum for biliary secretion of dibromosulphthalein (DBSP) was also drastically reduced (-53%). Biliary secretion of intravenously administered trace amounts of the 3 alpha-sulfate esters of 14C-labeled taurocholic acid (-14%), taurochenodeoxycholic acid (-39%), taurolithocholic acid (-73%), and glycolithocholic acid (-91%) was impaired in the jaundiced rats compared with controls, in contrast to the biliary secretion of the unsulfated parent compounds. Hepatic uptake of sulfated glycolithocholic acid was not affected in the jaundiced animals. Preadministration of DBSP (15 mumol/100 g body wt) to normal Wistar rats significantly impaired the biliary secretion of sulfated glycolithocholic acid, but did not affect taurocholic acid secretion. We conclude that separate transport systems in the rat liver exist for biliary secretion of sulfated and unsulfated bile acids; the sulfates probably share secretory pathways with the organic anions bilirubin and DBSP. The described genetic defect in hepatic transport function is associated with a reduced capacity to secrete sulfated bile acids into bile; this becomes more pronounced with a decreasing number of hydroxyl groups on the sulfated bile acid's molecule.

Cholesterol absorption regulates cholesterol metabolism and plasma lipoprotein levels in patients with gut exclusions

The occurrence of cholesterol malabsorption and its role in the regulation of cholesterol metabolism were studied in 30 patients with an earlier gut resection and 9 patients with a jejunoileal bypass for treatment of obesity. Fractional cholesterol absorption varied from 0.1% to 70%, and was lowest in jejunoileal bypass (8.3%) associated with severe fat and moderate bile acid malabsorptions and in 15 patients with a long small-intestinal resection (20.4%) associated with severe bile acid and moderate fat malabsorption. Seven resected patients with normal fecal fat and bile acids and 8 resected patients with malabsorption of only bile acid had normal cholesterol absorption. Low fractional cholesterol absorption was associated with a short length of the remaining proximal small intestine, high dietary intake of plant sterols, and high fecal fat and neutral sterol excretions, but not with bile acid malabsorption. In the whole study population, plasma levels of total, low-density lipoprotein, and high-density lipoprotein cholesterol were positively correlated with fractional cholesterol absorption and the amount of total, dietary, and biliary absorbed cholesterol and were negatively correlated with fecal cholesterol elimination as neutral sterols (less so as bile acids) and cholesterol synthesis. The results emphasize that, in patients with ileal exclusion, plasma levels of low-density lipoprotein and high-density lipoprotein cholesterol are regulated more effectively by cholesterol than by bile acid malabsorption. Moreover, although the fecal loss of bile acids is the main determinant in cholesterol elimination and stimulation of cholesterol synthesis in patients with intestinal exclusions, intestinal cholesterol absorption also contributes noticeably to the regulation of cholesterol synthesis.

Assessment of taurine deficiency in cystic fibrosis

Bile acid taurine deficiency is common in cystic fibrosis (CF) and is thought to be associated with impaired fat absorption. The relationship between the glycine: taurine bile acid conjugation ratio (bile acid G/T ratio) and taurine concentrations in plasma, urine and leucocytes was examined in 27 CF children aged 4–15 yr. The bile acid G/T ratio was elevated in serum in 14 of the 27 and in duodenal juice in 5 of 6 children. Subgroups of CF children with elevated and normal bile acid G/T ratio and controls ( n = 8) all had similar plasma, urine and leucocyte taurine concentrations. The results suggest that either taurine measurement in plasma, urine and leucocytes does not accurately reflect stores elsewhere in CF, or that taurine deficiency is confined to bile acids.

Bile acid metabolism in heterozygous familial hypercholesterolaemia: a study comparing affected and unaffected siblings of four kindreds.

Previous studies have indicated that quantitative as well as qualitative abnormalities of bile acid metabolism frequently occur in hypercholesterolaemia. In order to determine if this is a feature of familial hypercholesterolaemia, bile acid kinetics and biliary lipid composition were determined in 15 affected (heterozygous) and six unaffected siblings of four kindreds with familial hypercholesterolaemia. Furthermore, serum levels of cholic acid, chenodeoxycholic acid and deoxycholic acid were measured with a mass fragmentographic technique in 15 members of two of the kindreds, and secretion rates of biliary lipids were measured in six members of two kindreds. No differences with regard to these parameters between affected and unaffected siblings could be detected. There was a close resemblance between relatives of a given kindred concerning bile acid pool size and serum bile acid levels. No evidence for a defective bile acid metabolism in familial hypercholesterolaemia could be gained from the present study. It is concluded that the deficient receptor-mediated elimination of low density lipoprotein cholesterol in this disorder does not influence the maintenance of normal bile acid metabolism.

Effects of bile acids on the mutagenicity and recombinogenicity of triethylene melamine in yeast strains MP1 and D61.M.

When Saccharomyces cerevisia MP1 was treated with bile acids alone or in combination with triethylene melamine (TEM), cholic acid was found to be comutagenic and antirecombinogenic while lithocholic acid had the opposite effect. Other bile acids enhanced the mutagenic and recombinogenic effects of TEM. Chenodeoxycholic acid, deoxycholic acid and ursodeoxycholic acid had stronger comutagenic than corecombinogenic effects; hyodeoxycholic acid did not show this difference. Ox gall and a mixture of bile acids antagonized the genotoxic effect of TEM, and it therefore seems plausible that in normal composition bile acids neutralize each other, i.e., the comutagenic (corecombinogenic) effect of one substance is antagonized by the antimutagenic (antirecombinogenic) effect of another one. However, when the composition is altered, bile acids may become harmful. Experiments with yeast strain D61.M were performed in order to test for aneuploidy caused by bile acids; no effects were observed.

Plasma bile acids in patients with peroxisomal dysfunction syndromes: analysis by capillary gas chromatography — mass spectrometry

Six patients with disorders of peroxisomal function have been studied. Two presented in the neonatal period with the classical features of the Zellweger syndrome, two had incomplete Zellweger phenotypes, one infantile Refsum's disease and one rhizomelic chondrodysplasia punctata. Plasma bile acid profiles were determined using capillary gas chromatography-mass spectrometry. In all patients, except the case of chondrodysplasia punctata, 27-carbon and 29-carbon bile acids were present. The compounds identified included trihydroxycoprostanic acid (THCA), dihydroxycoprostanic acid (DHCA), C24-, C25- and C26-hydroxylated derivatives of THCA, a 27-carbon acid with four nuclear hydroxy groups and 3 alpha,7 alpha,12 alpha-trihydroxy-27a,27b-dihomo-5 beta-cholestan-26, 27b-dioic acid (C29-dicarboxylic acid). THCA was present at a low concentration in the patient with infantile Refsum's disease; the concentration of DHCA and the C29 dicarboxylic acid were considerably higher. The presence of abnormal bile acids in patients with Zellweger syndrome and infantile Refsum's disease could be explained by the absence of peroxisomes from their hepatocytes. In chondrodysplasia punctata the cause of peroxisomal dysfunction must be different, since normal bile acid synthesis is preserved.

Gas chromatography-mass spectrometry of isobutyl ester trimethylsilyl ether derivatives of bile acids and application to the study of bile sterol and bile acid biosynthesis in rat liver epithelial cell lines

The derivatization of bile acids into trimethylsilyl ether isobutyl ester (IBTMS) and of neutral sterols into trimethylsilyl ether (TMS) allowed the separation on an OV-1 capillary gas chromatography column of 15 bile steroids as follows: cholesterol, 7α-hydroxycholesterol, 6β-hydroxycholesterol, 6α-hydroxycholesterol, 7β-hydroxycholesterol, lithocholate, deoxycholate, 25-hydroxycholesterol, chenodeoxycholate, cholate, murocholate, hyodeoxycholate, ursodeoxycholate, hyocholate, and β-muricholate. Fragmentation data of the coupled gas chromatographic-mass spectrometric (GC-MS) analysis of these nine bile acids as IBTMS derivatives under electron impact and chemical ionizations (methane, isobutane, and ammonia) are given. The ammonia chemical ionization appears to be the best mode for compound identification and quantitation due to fragmentations into high mass ions. The comparison of methylene units of the five sterols as TMS derivatives and of each type of methyl, TMS, or isobutyl ester of the nine bile acids as TMS ethers showed that isobutyl esterification increased dramatically the retention time of the bile acids, allowing their separation after the neutral sterols. Different methods of GC-MS analysis were applied to the study of bile steroid secretion in long-term rat liver epithelial cell lines, either serum-supplemented cell lines or serum-free cell lines, growing in serum-free medium since the primary explantation or after adaptation of serum-supplemented lines to this medium. It is demonstrated for the first time that liver epithelial cell lines maintain the metabolic pathway leading from synthesized cholesterol to dioxygenated sterols and the two normal main primary bile acids of the liver, chenodeoxycholic acid and cholic acid, up to 32–47% of the in vivo daily rate, and in addition the production of α-muricholic acid, the bile acid marker of murine liver.