Bile acid synthesis in the Smith-Lemli-Opitz syndrome: effects of dehydrocholesterols on cholesterol 7alpha-hydroxylase and 27-hydroxylase activities in rat liver.
(49/3564)
The Smith-Lemli-Opitz syndrome (SLOS) is a congenital birth defect syndrome caused by a deficiency of 3beta-hydroxysterol Delta(7)-reductase, the final enzyme in the cholesterol biosynthetic pathway. The patients have reduced plasma and tissue cholesterol concentrations with the accumulation of 7-dehydrocholesterol and 8-dehydrocholesterol. Bile acid synthesis is reduced and unnatural cholenoic and cholestenoic acids have been identified in some SLOS patients. To explore the mechanism of the abnormal bile acid production, the activities of key enzymes in classic and alternative bile acid biosynthetic pathways (microsomal cholesterol 7alpha-hydroxylase and mitochondrial sterol 27-hydroxylase) were measured in liver biopsy specimens from two mildly affected SLOS patients. The effects of 7- and 8-dehydrocholesterols on these two enzyme activities were studied by using liver from SLOS model rats that were treated with the Delta(7)-reductase inhibitor (BM15.766) for 4 months and were comparable with more severe SLOS phenotype in plasma and hepatic sterol compositions. In the SLOS patients, cholesterol 7alpha-hydroxylase and sterol 27-hydroxylase were not defective. In BM15.766-treated rats, both enzyme activities were lower than those in control rats and they were competitively inhibited by 7- and 8-dehydrocholesterols. Rat microsomal cholesterol 7alpha-hydroxylase did not transform 7-dehydrocholesterol or 8-dehydrocholesterol into 7alpha-hydroxylated sterols. In contrast, rat mitochondrial sterol 27-hydroxylase catalyzed 27-hydroxylation of 7- and 8-dehydrocholesterols, which were partially converted to 3beta-hydroxycholestadienoic acids. Addition of microsomes to the mitochondrial 27-hydroxylase assay mixture reduced 27-hydroxydehydrocholesterol concentrations, which suggested that 27-hydroxydehydrocholesterols were further metabolized by microsomal enzymes. These results suggest that reduced normal bile acid production is characteristic of severe SLOS phenotype and is caused not only by depletion of hepatic cholesterol but also by competitive inhibition of cholesterol 7alpha-hydroxylase and sterol 27-hydroxylase activities by accumulated 7- and 8-dehydrocholesterols. Unnatural bile acids are synthesized mainly by the alternative pathway via mitochondrial sterol 27-hydroxylase in SLOS. (+info)
Immunodetection and molecular cloning of a bile-salt-dependent lipase isoform in HepG2 cells.
(50/3564)
In this article, we report the nucleotide sequence of the cDNA encoding an isoform of bile-salt-dependent lipase (BSDL) expressed by human hepatoma cells. The BSDL is a 100-kDa glycoprotein normally expressed by the human pancreas. Using a polyclonal antibody raised against an internal peptide located between Ile(327) and Glu(350) of the human pancreatic BSDL, we have immunodetected an isoform of human pancreatic BSDL, with an apparent molecular mass of about 62 kDa. This isoform of BSDL was mainly associated with the cytosol of a human hepatoma cell line (HepG2), the remaining protein being found in the microsome fraction. In addition, esterolytic activity on p-nitrophenyl hexanoate measured in microsomes and cytosol appeared very low and was weakly stimulated by bile salts, such as taurocholate. In contrast to human pancreatic BSDL, which is secreted as a component of pancreatic juice, this isoform appeared to be retained in the HepG2 cells. Reverse transcription, followed by PCR and amplification, performed on RNA extracted from HepG2 cells using specific primers hybridizing to the sequence coding for the entire normal human pancreatic BSDL, allowed us to amplify a 1. 7-kb transcript that appeared to be 0.5 kb shorter than the transcript of the pancreatic enzyme (2.2 kb). From the sequence of the transcript thus obtained, a protein with a molecular mass of 62 kDa might be predicted, which is in good agreement with the size of the isoform of BSDL immunodetected in HepG2 cells. The N-terminal amino-acid sequence, deduced from the 1.7-kb transcript sequence, matched that of the pancreatic BSDL. However, the C-terminal domain appeared truncated, bearing only a single mucin-like sequence compared with sixteen for the human pancreatic BSDL. The actual intracellular function of this human BSDL hepatoma isoform remains to be elucidated. (+info)
Involvement of serotonin and calcium channels in the intestinal fluid secretion evoked by bile salt and cholera toxin.
(51/3564)
1. The enteric nervous system (ENS) is activated when exposing the intestinal mucosa to cholera toxin or certain bile salts. Cholera toxin stimulates ENS, at least in part, by the release of 5-hydroxytryptamine (5-HT) from the enterochromaffin cells. Calcium channel blockers of the L-type markedly attenuate the fluid secretion and the luminal release of 5-HT caused by cholera toxin. 2. The objective of the present study was to elucidate if sodium deoxycholate activated ENS in a similar manner as cholera toxin. Furthermore, the effect of several calcium channel blockers was tested on the fluid secretion caused by cholera toxin or bile salt. 3. Sodium deoxycholate (4 mM) caused a release of 5-HT into the intestinal lumen, which was inhibited by calcium channel blockade. Granisetron, a 5-HT3 receptor blocker, partly inhibited the fluid secretion caused by bile salt. 4. The effects of nifedipine, felodipine, R-felodipine, H186/86 (t-butyl analogue of felodipine) on the fluid secretion caused by cholera toxin or sodium deoxycholate were studied. Both secretory states were markedly attenuated in a dose dependent manner by all calcium channel blockers tested regardless of their effects on arterial pressure. 5. It is concluded that both cholera toxin and bile salt activate ENS, at least in part, via a release of 5-HT from the enterochromaffin cells. The antisecretory effect calcium channel blockers is partly explained by an inhibition of this release of 5-HT. (+info)
Stimulation of fecal steroid excretion after infusion of recombinant proapolipoprotein A-I. Potential reverse cholesterol transport in humans.
(52/3564)
BACKGROUND: Apolipoprotein (apo) A-I is the major protein component of HDL, a cholesterol transport particle that protects against atherosclerosis. Apo A-I is believed to promote reverse cholesterol transport, transferring cholesterol from peripheral cells to the liver for subsequent elimination. To test this hypothesis in humans, we measured fecal steroid excretion before and after the intravenous infusion of human proapo A-I (precursor of apo A-I) liposome complexes. METHODS AND RESULTS: Four subjects with heterozygous familial hypercholesterolemia w re studied under standardized conditions. The fecal excretion of bile acids and neutral sterols was determined for 9 days before and 9 days after an intravenous infusion of recombinant human proapo A-I (4 g protein) liposome complexes. Plasma apoA-I and HDL cholesterol levels increased transiently (mean peak concentrations were 64% and 35% above baseline, respectively) during the first 24 hours. Mean lipoprotein lipid and apolipoprotein levels were not different during the 2 collecting periods, however. Serum lathosterol, a precursor of cholesterol whose concentration reflects the rate of cholesterol synthesis in vivo, was also unchanged. The fecal excretion of cholesterol (neutral sterols and bile acids) increased in all subjects (mean increase, +39% and +30%, respectively), corresponding to the removal of approximately 500 mg/d excess cholesterol after infusion. Control infusions with only liposomes in 2 of the patients did not influence lipoprotein pattern or cholesterol excretion. CONCLUSIONS: Infusion of proapoA-I liposomes in humans promotes net cholesterol excretion from the body, implying a stimulation of reverse cholesterol transport. This mechanism may prove useful in the treatment of atherosclerosis. (+info)
Bile acid patterns in meconium are influenced by cholestasis of pregnancy and not altered by ursodeoxycholic acid treatment.
(53/3564)
BACKGROUND: Data on meconium bile acid composition in newborn babies of patients with intrahepatic cholestasis of pregnancy (ICP) are relatively scant, and changes that occur on ursodeoxycholic acid (UDCA) administration have not been evaluated. AIMS: To investigate bile acid profiles in meconium of neonates from untreated and UDCA treated patients with ICP. Maternal serum bile acid composition was also analysed both at diagnosis and delivery to determine whether this influences the concentration and proportion of bile acids in the meconium. PATIENTS/METHODS: The population included eight healthy pregnant women and 16 patients with ICP, nine of which received UDCA (12.5-15.0 mg/kg body weight/day) for 15+/-4 days until parturition. Bile acids were assessed in the meconium by gas chromatography-mass spectrometry and in maternal serum by high performance liquid chromatography. RESULTS: Total bile acid and cholic acid concentrations in the meconium were increased (p<0.01) in newborns from patients with ICP (13.5 (5.1) and 8.4 (4.1) micromol/g respectively; mean (SEM)) as compared with controls (2.0 (0.5) and 0.8 (0.3) micromol/g respectively), reflecting the total bile acid and cholic acid levels in the maternal serum (r = 0.85 and r = 0.84, p<0.01). After UDCA administration, total bile acid concentrations decreased in the mother ( approximately 3-fold, p<0. 05) but not in the meconium. UDCA concentration in the meconium showed only a 2-fold increase after treatment, despite the much greater increase in the maternal serum (p<0.01). Lithocholic acid concentration in the meconium was not increased by UDCA treatment. CONCLUSIONS: UDCA administration does not influence the concentration and proportion of bile acids in the meconium, which in turn are altered by ICP. Moreover, this beneficial treatment for the mother does not increase meconium levels of potentially toxic metabolites of UDCA such as lithocholic acid. (+info)
Screening of newborn infants for cholestatic hepatobiliary disease with tandem mass spectrometry.
(54/3564)
OBJECTIVE: To assess the feasibility of screening for cholestatic hepatobiliary disease and extrahepatic biliary atresia by using tandem mass spectrometry to measure conjugated bile acids in dried blood spots obtained from newborn infants at 7-10 days of age for the Guthrie test. SETTING: Three tertiary referral clinics and regional neonatal screening laboratories. DESIGN: Unused blood spots from the Guthrie test were retrieved for infants presenting with cholestatic hepatobiliary disease and from the two cards stored on either side of each card from an index child. Concentrations of conjugated bile acids measured by tandem mass spectrometry in the two groups were compared. MAIN OUTCOME MEASURES: Concentrations of glycodihydroxycholanoates, glycotrihydroxycholanoates, taurodihydroxycholanoates, and taurotrihydroxycholanoates. Receiver operator curves were plotted to determine which parameter (or combination of parameters) would best predict the cases of cholestatic hepatobiliary disease and extrahepatic biliary atresia. The sensitivity and specificity at a selection of cut off values for each bile acid species and for total bile acid concentrations for the detection of the two conditions were calculated. RESULTS: 218 children with cholestatic hepatobiliary disease were eligible for inclusion in the study. Two children without a final diagnosis and five who presented at <14 days of age were excluded. Usable blood spots were obtained from 177 index children and 708 comparison children. Mean concentrations of all four bile acid species were significantly raised in children with cholestatic hepatobiliary disease and extrahepatic biliary atresia compared with the unaffected children (P<0.0001). Of 177 children with cholestatic hepatobiliary disease, 104 (59%) had a total bile acid concentration >33 micromol/l (97.5th centile value for comparison group). Of the 61 with extrahepatic biliary atresia, 47 (77%) had total bile acid concentrations >33 micromol/l. Taurotrihydroxycholanoate and total bile acid concentrations were the best predictors of both conditions. For all cholestatic hepatobiliary disease, a cut off level of total bile acid concentration of 30 micromol/l gave a sensitivity of 62% and a specificity of 96%, while the corresponding values for extrahepatic biliary atresia were 79% and 96%. CONCLUSION: Most children who present with extrahepatic biliary atresia and other forms of cholestatic hepatobiliary disease have significantly raised concentrations of conjugated bile acids as measured by tandem mass spectrometry at the time when samples are taken for the Guthrie test. Unfortunately the separation between the concentrations in these infants and those in the general population is not sufficient to make mass screening for cholestatic hepatobiliary disease a feasible option with this method alone. (+info)
Effect of dietary taurine on endogenous hypercholesterolemia in rats fed on phenobarbital-containing diets.
(55/3564)
The effect of dietary taurine on endogenous hypercholesterolemia induced by a phenobarbital-containing diet was investigated. Supplemented taurine did not affect the concentrations of serum cholesterol, but further potentiated the accumulation of hepatic cholesterol in the hypercholesterolemic state induced by phenobarbital. It is suggested that taurine might amplify the hepatic cholesterogenesis in phenobarbital-induced hypercholesterolemia. (+info)
Substrate specificity of the ileal and the hepatic Na(+)/bile acid cotransporters of the rabbit. I. Transport studies with membrane vesicles and cell lines expressing the cloned transporters.
(56/3564)
The substrate specificity of the ileal and the hepatic Na(+)/bile acid cotransporters was determined using brush border membrane vesicles and CHO cell lines permanently expressing the Na(+)/bile acid cotransporters from rabbit ileum or rabbit liver. The hepatic transporter showed a remarkably broad specificity for interaction with cholephilic compounds in contrast to the ileal system. The anion transport inhibitor diisothiocyanostilbene disulfonate (DIDS) is a strong inhibitor of the hepatic Na(+)/bile acid cotransporter, but does not show any affinity to its ileal counterpart. Inhibition studies and uptake measurements with about 40 different bile acid analogues differing in the number, position, and stereochemistry of the hydroxyl groups at the steroid nucleus resulted in clear structure;-activity relationships for the ileal and hepatic bile acid transporters. The affinity to the ileal and hepatic Na(+)/bile acid cotransport systems and the uptake rates by cell lines expressing those transporters as well as rabbit ileal brush border membrane vesicles is primarily determined by the substituents on the steroid nucleus. Two hydroxy groups at position 3, 7, or 12 are optimal whereas the presence of three hydroxy groups decreased affinity. Vicinal hydroxy groups at positions 6 and 7 or a shift of the 7-hydroxy group to the 6-position significantly decreased the affinity to the ileal transporter in contrast to the hepatic system. 6-Hydroxylated bile acid derivatives are preferred substrates of the hepatic Na(+)/bile acid cotransporter. Surprisingly, the 3alpha-hydroxy group being present in all natural bile acids is not essential for high affinity interaction with the ileal and the hepatic bile acid transporter. The 3alpha-hydroxy group seems to be necessary for optimal transport of a bile acid across the hepatocyte canalicular membrane. A modification of bile acids at the 3-position therefore conserves the bile acid character thus determining the 3-position of bile acids as the ideal position for drug targeting strategies using bile acid transport pathways. (+info)