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Cerebrotendinous xanthomatosis (CTX) is a rare disorder due to defective sterol 27-hydroxylase causing a lack of chenodeoxycholic acid (CDCA) production and high plasma cholestanol levels.Our objective was to review the diagnosis and treatment results in 43 CTX cases.We conducted a careful review of the diagnosis, laboratory values, treatment, and clinical course in 43 CTX cases.The mean age at diagnosis was 32 years; the average follow-up was 8 years. Cases had the following conditions: 53% chronic diarrhea, 74% cognitive impairment, 70% premature cataracts, 77% tendon xanthomas, 81% neurologic disease, and 7% premature cardiovascular disease. The mean serum cholesterol concentration was 190 mg/dL; the mean plasma cholestanol level was 32 mg/L (normal5.0 mg/L), which decreased to 6.0 mg/L (-81%) with CDCA therapy generally given as 250 mg orally 3 times daily. Of those tested on treatment, 63% achieved cholestanol levels of5.0 mg/L; 91% had normal liver enzyme levels; none had significant liver problems after dose adjustment. Treatment improved symptoms in 57% at follow-up, but 20% with advanced disease continued to deteriorate. In the United States, CDCA has been approved for gallstone dissolution, but not for CTX despite long-term efficacy and safety data.Health care providers seeing young patients with tendon xanthomas and relatively normal cholesterol levels, especially those with cataracts and learning problems, should consider the diagnosis of CTX so they can receive treatment. CDCA should receive regulatory approval to facilitate therapy for the prevention of the complications of the disease.

Cerebrotendinous xanthomatosis (CTX) is a rare autosomal recessive disorder of bile acid synthesis caused by mutations in the cytochrome P450 CYP27A1 gene that result in production of a defective sterol 27-hydroxylase enzyme. CTX is associated with abnormally high levels of cholestanol in the blood and accumulation of cholestanol and cholesterol in the brain, tendon xanthomas, and bile. Hallmark clinical manifestations of CTX include chronic diarrhea, bilateral cataracts, tendon xanthomas, and neurologic dysfunction. Although CTX is a rare disorder, it is thought to be underdiagnosed, as presenting signs and symptoms may be nonspecific with significant overlap with other more common conditions. There is marked variability in signs and symptoms, severity, and age of onset between patients. The disease course is progressive and potentially debilitating or fatal, particularly with respect to neurologic presentations that can include intellectual disability, autism, behavioral and psychiatric problems, and dementia, among others. Treatment with chenodeoxycholic acid (CDCA; chenodiol) is the current standard of care. CDCA can help restore normal sterol, bile acid, bile alcohol, and cholestanol levels. CDCA also appears to be generally effective in preventing adverse clinical manifestations of the disease from occurring or progressing if administered early enough. Improved screening and awareness of the condition may help facilitate early diagnosis and treatment.

Background and aims Phytosterolemia is a rare genetic disease caused by mutation of the ABCG5/8 gene. Our aim was to elucidate the natural history and homeostasis of phytosterolemia. Methods We analyzed a Hutterite kindred consisting of 21 homozygotes with phytosterolemia assembled over a period of two decades, all of whom carried the ABCG8 S107X mutation and were treated with ezetimibe. Results Most of these subjects were asymptomatic and devoid of clinical stigmata, and this, since they were ascertained primarily by a process of cascade testing, suggests that, relative to its true prevalence, phytosterolemia is a condition of low morbidity. All subjects have responded well to treatment with ezetimibe. Initial (pre-treatment) and post-ezetimibe levels of cholesterol and sitosterol were measured and percentage changes on ezetimibe were calculated. We found initial levels to be inversely related to subjects' ages as were percentage responses to ezetimibe therapy. There was also a direct correlation between initial levels and percentage responses to ezetimibe. Hence on-treatment levels were very uniform. Conclusions This evidence of a link with age leads us to propose that an age-related change in cholesterol and sterol homeostasis occurs at puberty in phytosterolemia and that the change is due to high sterol and/or stanol levels causing feedback inhibition of sterol regulatory element-binding protein (SREBP-2) processing. This would explain the well-documented phenomenon of depressed cholesterol synthesis in phytosterolemia. It is also well-known that LDL-receptor activity is increased, and this feasibly explains reduced LDL levels and consequent reduction of plasma cholesterol and sitosterol levels. Downregulated SREBP-2 processing would be expected to also lower proprotein convertase subtilisin/kexin type 9 (PCSK9) levels and this would explain high LDL-receptor activity. The above state could be termed disrupted homeostasis and the alternative, seen mostly in children and characterized by hypercholesterolemia and hypersterolemia, simple homeostasis.

It has been proposed that bile acid suppression of CYP7A1 gene expression is mediated through a gut-liver signaling pathway fibroblast growth factor (FGF)15/19-fibroblast growth factor receptor 4 which is initiated by activation of farnesoid X receptor in the ileum but not in the liver. This study evaluated whether FGF15/19 protein levels in the portal blood reflected changes in FGF15/19 mRNA in the ileum. Studies were conducted in Sprague Dawley rats and New Zealand white rabbits fed regular chow (controls), supplemented with cholesterol (Ch) or cholic acid (CA). After feeding CA, ileal FGF15 mRNA increased 8.5-fold in rats and FGF19 rose 16-fold in rabbits associated with 62 and 75% reduction of CYP7A1 mRNA, respectively. Neither FGF15 nor FGF19 protein levels changed in the portal blood to correspond with the marked increase of FGF15/19 mRNA levels in the ileum or inhibited CYP7A1 expression in the liver. Further, in Ch-fed rats, CYP7A1 mRNA increased 1.9-fold (P < 0.001) although FGF15 mRNA levels in the ileum and portal blood FGF15 protein levels were not decreased. In Ch-fed rabbits, although FGF19 mRNA levels in the ileum and liver did not increase significantly, CYP7A1 mRNA declined 49% (P < 0.05). We were unable to find corresponding changes of FGF15/19 protein levels in the portal blood in rats and rabbits where the mRNA levels of FGF15/19 in the ileum and CYP7A1 in the liver change significantly.

Summary Objective: To assess the effect of ezetimibe (EZE) 40 mg/day on non-cholesterol sterol plasma concentrations in patients with homozygous sitosterolaemia (HoS). Methods: This was a multi-centre, randomised, double-blind, placebo-controlled parallel group study. Twenty-seven patients (≥ 18 years) with HoS and plasma sitosterol levels > 5 mg/dl who had been taking EZE 10 mg/day for ≥ 6 months prior to enrolment received open-label EZE 10 mg/day for the duration of the study and were randomised 1 : 1 to blinded EZE 30 mg/day (4 × EZE 10 mg tablets; n = 13) or placebo (1 × EZE 10 mg tablet and 3 × matching placebo tablets; n = 14) for 26 weeks. Patients were permitted to remain on other ongoing treatments (e.g. bile salt-binding resin, statin and/or low sterol diet). End-points included median per cent between-group changes from baseline in plasma sitosterol, campesterol, lathosterol, low-density lipoprotein (LDL) sterols, LDL cholesterol (LDL-C) measured by gas–liquid chromatography, and Achilles tendon thickness size measured radiographically. Results: Ezetimibe 40 mg/day resulted in median per cent changes from baseline in plasma sitosterol levels of 3.3% vs. −10% in the EZE 10 mg/day group, in plasma campesterol of −0.5% vs. −9.7% in the EZE 10 mg/day group, and in plasma lathosterol of 0.8% vs. 1.1% in the EZE 10 mg/day group (p = ns for all between-group differences). Median per cent changes in the EZE 40 mg/day and EZE 10 mg/day groups, respectively, were 1.3% and 0% for LDL sterols and 2.5% and 4.4% for LDL-C (p = ns for both between-group differences). At study end-point, Achilles tendon thickness remained unchanged in the EZE 40 mg/day group and increased slightly in the EZE 10 mg/day group (2.2%), yielding a non-significant between-group difference of −2.2%. EZE 40 mg/day was generally well tolerated. Conclusions: In patients with HoS, treatment with EZE 40 mg/day for 26 weeks was no more effective at reducing plasma plant sterol concentrations vs. EZE 10 mg/day. EZE 40 mg/day had a safety and tolerability profile similar to EZE 10 mg/day.

The aim of this study was to explore why in rabbits activation of farnesoid X receptor (FXR) is dominant over activated liver X receptor-alpha (LXRalpha) in the regulation of CYP7A1. We cloned the rabbit CYP7A1 promoter and found a fetoprotein transcription factor (FTF) binding element embedded within the LXRalpha binding site (LXRE). Gel shift assays demonstrated that FTF competes with LXRalpha for binding to LXRE. Short heterodimer partner (SHP) enhances the competitive ability of FTF. Studies in HepG2 cells showed that SHP combined with FTF had more powerful effect to offset the stimulation of CYP7A1 by LXRalpha. Gel shift and chromatin immunoprecipitation assays demonstrated that SHP with FTF diminished LXRalpha binding to the CYP7A1 promoter. In vivo studies in rabbits fed cholesterol for 10 days showed that hepatic expression of SHP but not FTF rose and LXRalpha-bound LXRE decreased. We propose that the SHP/FTF heterodimer occupies LXRE via the embedded FTF binding element and blocks LXRalpha from recruiting to LXRE. Therefore, activation of FXR, which upregulates SHP expression, will eliminate the stimulatory effect of LXRalpha on the CYP7A1 promoter because increased levels of SHP combined with FTF diminish the recruitment of LXRalpha to CYP7A1 promoter.

Cerebrotendinous xanthomatosis, (CTX; MIM 213700) is a rare, recessively inherited lipid storage disease caused by defective bile acid synthesis. The hallmark manifestations include tendon and tuberous xanthomas, juvenile cataracts, and nervous system dysfunction. Chronic diarrhea in children, as well as osteoporosis, bone fractures, and premature atherosclerosis in adults may also be present. The underlying cause of CTX is a block in bile acid synthesis resulting in incomplete oxidation of the cholesterol side chain. This is due to inherited mutations of the mitochondrial enzyme sterol 27-hydroxylase (CYP27A1). Diagnosis is established by detection of elevated cholestanol levels in the plasma, and confirmed by elevated bile alcohol levels in the blood and/or urine. The bile alcohols are thought to compromise the blood–brain barrier function; the subsequent accumulation of cholestanol and cholesterol in the central nervous system (CNS) leads to progressive neuronal damage. Definitive diagnosis can also be made by demonstration of mutations affecting the CYP27A1 gene. The standard of care is oral treatment with chenodeoxycholic acid at 750 mg per day to inhibit hepatic bile acid production, stopping bile alcohols from being made, reducing cholestanol synthesis, and thus preventing damage to the CNS. Normalizing the biochemical abnormalities improves neurologic function and is the mainstay of therapy. Initiation of this therapy before symptoms develop has been shown to prevent many of the sequelae and therefore early diagnosis and therapy is of vital importance. Treatment should be monitored for biochemical response and dose adjustments made for optimal response.

Plant sterols compete with cholesterol (cholest-5-en-3beta-ol) for intestinal absorption to limit absorption and lower plasma concentrations of cholesterol. Stigmasterol (24-ethyl-cholesta-5,22-dien-3beta-ol; Delta(22) derivative of sitosterol [24-ethyl-cholest-5-en-3beta-ol]), but not campesterol (24-methyl-cholest-5-en-3beta-ol) and sitosterol, is reported to inhibit cholesterol biosynthesis via inhibition of sterol Delta(24)-reductase in human Caco-2 and HL-60 cell lines. We studied the effect of feeding 0.5% stigmasterol on plasma and liver sterols and intestinal cholesterol and sitosterol absorption in 12 wild-type Kyoto (WKY) and 12 Wistar rats. After 3 weeks of feeding, cholesterol and sitosterol absorption was determined in 6 rats from each group by plasma dual-isotope ratio method. After 3 more weeks, plasma and hepatic sterols and hepatic enzyme activities were determined in all rats. After feeding stigmasterol, baseline plasma cholesterol was 1.3 times and plant sterols 3 times greater in WKY compared with Wistar rats. Stigmasterol feeding lowered plasma cholesterol by approximately 11%, whereas plasma campesterol and sitosterol levels were virtually unchanged in both rat strains, and stigmasterol constituted 3.2% of plasma sterols in WKY rats and 1% in Wistar rats. After 6 weeks of feeding, cholesterol and sitosterol absorption decreased 23% and 30%, respectively, in WKY, and 22% and 16%, respectively, in the Wistar rats as compared with untreated rats. The intestinal bacteria in both rat strains metabolized stigmasterol to mainly the 5beta-H stanol (>40%), with only small amounts of 5alpha-H derivative (approximately 1.5%), whereas the C-22 double bond was resistant to bacterial metabolism. Hepatic stigmasterol levels increased from 11 microg/g liver tissue to 104 mug/g in WKY rats and from 5 microg/g liver tissue to 21 microg/g in Wistar rats. 3-Hydroxy-3-methylglutaryl coenzyme A reductase activity was suppressed 4-fold in the WKY and almost 1.8-fold in Wistar rats, cholesterol 7alpha-hydroxylase activity was suppressed 1.6-fold in the WKY and 3.5-fold in Wistar rats, whereas cholesterol 27-hydroxylase activity was unchanged after feeding. In conclusion, stigmasterol, when fed, lowers plasma cholesterol levels, inhibits intestinal cholesterol and plant sterol absorption, and suppresses hepatic cholesterol and classic bile acid synthesis in Wistar as well as WKY rats. However, plasma and hepatic incorporation of stigmasterol is low.

Hypercholesterolemia is an important paraneoplastic syndrome in patients with hepatoma, but the nature of this defect has not yet been identified. We investigated the molecular mechanisms of hypercholesterolemia in a hepatoma-bearing rat model. Buffalo rats were implanted in both flanks with Morris hepatoma 7777 (McA-RH7777) cells. After 4 weeks, tumor weight was 5.5+/-1.7 g, and serum cholesterol level increased from 60+/-2 to 90+/-2 mg/dL. Protein and mRNA expression of the ATP-binding cassette transporters A1 and G1 (ABCA1 and ABCG1) was markedly higher in tumors than in livers. These increases were associated with activation of liver X receptor alpha (LXRalpha) as a result of the increased tissue oxysterol concentrations. The accumulation of oxysterols in the hepatomas appeared to be caused mainly by the upregulation of cholesterol biosynthesis, despite the increased tissue sterol concentrations. Overexpression of the sterol regulatory element-binding protein (SREBP) processing system relative to sterol concentration contributed to the resistance to sterols in this tumor. In addition, bile acid biosynthesis was inhibited despite the reduced expression of the small heterodimer partner (SHP) and activated LXRalpha, which also appeared to contribute to the accumulation of oxysterols followed by the acceleration of cholesterol efflux. In conclusion, hypercholesterolemia in McA-RH7777 hepatoma-bearing rats was caused by increased cholesterol efflux from tumors as a result of activation of LXRalpha. Overexpression of the SREBP processing system contributed to the activation of LXRalpha by maintaining high oxysterol levels in tissue.

Background: The presence of hypercholesterolemia is currently not considered a selection criteria for performing gastric restrictive or diversionary bariatric surgery. Methods: We prospectively investigated the effects of the bilio-intestinal bypass (BI-bypass) with a wide cholecysto-jejunal anastomosis and of adjustable gastric banding (AGB) on blood lipid concentrations in obese patients. To clarify the mechanism of the hypocholesterolemic effect of the BI-bypass, daily fecal sterol excretion was measured by gas-liquid chromatography (GLC). Results: At 1 year after BI-bypass compared to baseline, the hypercholesterolemic (n=18) and the normocholesterolemic (n=19) patients significantly reduced total (−38% and −27%, respectively), LDL (−47% and −24%, respectively) and HDL (−11% and −13%, respectively) cholesterol and total / HDL cholesterol ratio (−25% and −13%, respectively). At 1 year after AGB, the total / HDL cholesterol ratio was significantly decreased (−11%) compared to baseline in hypercholesterolemic (n=12) but not in normocholesterolemic (n=6) patients, while total and LDL cholesterol were not affected in both groups. At 3 years after BI-bypass compared to baseline, the hypercholesterolemic (n=9) and the normocholesterolemic (n=11) patients significantly reduced total (−43% and −28%, respectively) and LDL (−53% and −29%, respectively) cholesterol and total / HDL cholesterol ratio (−38% and −21%, respectively). The BI-bypass induced a significant (P

The regulation of the rabbit apical sodium-dependent bile acid transporter (ASBT) was studied both in vivo and in vitro. New Zealand White rabbits were fed 0.5% deoxycholic acid (DCA) or SC-435, a competitive ASBT inhibitor, for 1 wk. In DCA-fed rabbits, ASBT expression was repressed, associated with activated FXR, and evidenced by increased ileal short heterodimer partner (SHP) mRNA. Feeding SC-435 to the rabbits blocked bile acid absorption, decreased SHP mRNA, and increased ASBT expression. A 1.9-kb rabbit ASBT 5′-flanking region (promoter) was cloned, and a cis-acting element for α-fetoprotein transcription factor (FTF) was identified (−1166/ −1158). The effects of transcriptional factors and different bile acids on the rabbit ASBT promoter were studied in Caco-2 cells. FTF stimulated the rabbit ASBT promoter activity fourfold but not after the FTF binding site was deleted from the promoter. Increasing the SHP protein notably inhibited FTF-dependent trans-activation of rabbit ASBT. Adding hydrophobic bile acids deoxycholic acid, chenodeoxycholic acid, and cholic acid, activating ligands for FXR, inhibited rabbit ASBT promoter activity in Caco-2 cells, but this inhibitory effect was abolished after the FTF binding site was deleted. Ursodeoxycholic acid and ursocholic acid, nonactivating ligands for FXR, did not repress ASBT promoter activity. Thus the rabbit ASBT promoter is negative-feedback regulated by bile acids via a functional FTF binding site. Only FXR-activating ligands can downregulate rabbit ASBT expression through the regulatory cascade FXR-SHP-FTF.

Cholesterol feeding upregulates CYP7A1 in rats but downregulates CYP7A1 in rabbits. To clarify the mechanism responsible for the upregulation of CYP7A1 in cholesterol-fed rats, the effects of dietary cholesterol (Ch) and cholic acid (CA) on the activation of the nuclear receptors, liver X-receptor (LXR-alpha) and farsenoid X-receptor (FXR), which positively and negatively regulate CYP7A1, were investigated in rats. Studies were carried out in four groups (n = 12/group) of male Sprague-Dawley rats fed regular chow (control), 2% Ch, 2% Ch + 1% CA, and 1% CA alone for 1 wk. Changes in mRNA expression of short heterodimer partner (SHP) and bile salt export pump (BSEP), target genes for FXR, were determined to indicate FXR activation, whereas the expression of ABCA1 and lipoprotein lipase (LPL), target genes for LXR-alpha, reflected activation. CYP7A1 mRNA and activity increased twofold and 70%, respectively, in rats fed Ch alone when the bile acid pool size was stable but decreased 43 and 49%, respectively, after CA was added to the Ch diet, which expanded the bile acid pool 3.4-fold. SHP and BSEP mRNA levels did not change after feeding Ch but increased 88 and 37% in rats fed Ch + CA. This indicated that FXR was activated by the expanded bile acid pool. When Ch or Ch + CA were fed, hepatic concentrations of oxysterols, ligands for LXR-alpha increased to activate LXR-alpha, as evidenced by increased mRNA levels of ABCA1 and LPL. Feeding CA alone enlarged the bile acid pool threefold and increased the expression of both SHP and BSEP. These results suggest that LXR-alpha was activated in rats fed both Ch or Ch + CA, whereas CYP7A1 mRNA and activity were induced only in Ch-fed rats where the bile acid pool was not enlarged such that FXR was not activated. In rats fed Ch + CA, the bile acid pool expanded, which activated FXR to offset the stimulatory effects of LXR-alpha on CYP7A1.

We investigated how cholesterol feeding regulates cholesterol 7alpha-hydroxylase (CYP7A1) via the nuclear receptors farnesoid X receptor (FXR) and liver X receptor alpha (LXRalpha) in New Zealand white rabbits. After 1 day of 2% cholesterol feeding, when the bile acid pool size had not expanded, mRNA levels of the FXR target genes short-heterodimer partner (SHP) and sterol 12alpha-hydroxylase (CYP8B) were unchanged, indicating that FXR activation remained constant. In contrast, the mRNA levels of the LXRalpha target genes ATP binding cassette transporter A1 (ABCA1) and cholesteryl ester transfer protein (CETP) increased 5-fold and 2.3-fold, respectively, associated with significant increases in hepatic concentrations of oxysterols. Activity and mRNA levels of CYP7A1 increased 2.4 times and 2.2 times, respectively. After 10 days of cholesterol feeding, the bile acid pool size increased nearly 2-fold. SHP mRNA levels increased 4.1-fold while CYP8B declined 64%. ABCA1 mRNA rose 8-fold and CETP mRNA remained elevated. Activity and mRNA of CYP7A1 decreased 60% and 90%, respectively. Feeding cholesterol for 1 day did not enlarge the ligand pool size or change FXR activation, while LXRalpha was activated highly secondary to increased hepatic oxysterols. As a result, CYP7A1 was up-regulated. After 10 days of cholesterol feeding, the bile acid (FXR ligand) pool size increased, which activated FXR and inhibited CYP7A1 despite continued activation of LXRalpha. Thus, in rabbits, when FXR and LXRalpha are activated simultaneously, the inhibitory effect of FXR overrides the stimulatory effect of LXRalpha to suppress CYP7A1 mRNA expression.

Cholesterol 7alpha-hydroxylase, a rate-limiting enzyme for bile acid synthesis, has been implicated in genetic susceptibility to atherosclerosis. The gene, CYP7A1, encoding a protein with this activity, is expressed normally only in hepatocytes and is highly regulated. Our cyp7A1 gene knockout mouse colony, as young adults on a chow diet, is hypercholesterolemic. These mice were characterized extensively to understand how cyp7A1 affects lipid and bile acid homeostasis in different tissue compartments and whether gender plays a modifying role. Both male and female cyp7A1-deficient mice had decreased hepatic LDL receptors, unchanged hepatic cholesterol synthesis, increased intestinal cholesterol synthesis and bile acid transporters, and decreased fecal bile acids but increased fecal sterols. In females, cyp7A1 deficiency also caused changes in hepatic fatty acid metabolism, decreased hepatic canalicular bile acid transporter, Bsep, and gallbladder bile composition altered to a lithogenic profile. Taken together, the data suggest that cyp7A1 deficiency results in a proatherogenic phenotype in both genders and leads to a prolithogenic phenotype in females.

Familial hypercholanemia (FHC) is characterized by elevated serum bile acid concentrations, itching, and fat malabsorption1,2. We show here that FHC in Amish individuals is associated with mutations in tight junction protein 2 (encoded by TJP2, also known as ZO-2) and bile acid Coenzyme A: amino acid N-acyltransferase (encoded by BAAT). The mutation of TJP2, which occurs in the first PDZ domain, reduces domain stability and ligand binding in vitro. We noted a morphological change in hepatic tight junctions. The mutation of BAAT, a bile acid–conjugating enzyme3, abrogates enzyme activity; serum of individuals homozygous with respect to this mutation contains only unconjugated bile acids. Mutations in both TJP2 and BAAT may disrupt bile acid transport and circulation. Inheritance seems to be oligogenic, with genotype at BAAT modifying penetrance in individuals homozygous with respect to the mutation in TJP2.

Dehydroepiandrosterone (DHEA) is a naturally occurring steroid synthesized in the adrenal cortex, gonads, brain, and gastrointestinal tract, and it is known to have chemopreventive and anti-proliferative actions on tumors. These effects are considered to be induced by the inhibition of glucose-6-phosphate dehydrogenase (G6PD) and/or HMG-CoA reductase (HMGR) activities. The present study was undertaken to investigate whether endogenous DHEA metabolites, i.e. DHEA-sulfate, 7-oxygenated DHEA derivatives, androsterone, epiandrosterone, and etiocholanolone, have anti-proliferative effects on cancer cells and to clarify which enzyme, G6PD or HMGR, is responsible for growth inhibition. Growth of Hep G2, Caco-2, and HT-29 cells, evaluated by 3-[4,5-dimethylthiazol]-2yl-2,5-diphenyl tetrazolium bromide (MTT) and bromodeoxyuridine incorporation assays, was time- and dose-dependently inhibited by addition of all DHEA-related steroids we tested. In particular, the growth inhibition due to etiocholanolone was considerably greater than that caused by DHEA in all cell lines. The suppression of growth of the incubated steroids was not correlated with the inhibition of G6PD (r=-0.031, n=9, NS) or HMGR (r=0.219, n=9, NS) activities. The addition of deoxyribonucleosides or mevalonolactone to the medium did not overcome the inhibition of growth induced by DHEA or etiocholanolone, while growth suppression by DHEA was partially prevented by the addition of ribonucleosides. These results demonstrate that endogenous DHEA metabolites also have an anti-proliferative action that is not induced by inhibiting G6PD or HMGR activity alone. These non-androgenic DHEA metabolites may serve as chemopreventive or anti-proliferative therapies.

A simple method for the simultaneous gas–liquid chromatographic quantitation of fatty acids, sterols and bile acids from human fecal samples is described. The various compounds are directly converted into the n-butyl ester-trimethylsilyl ether derivatives, without prior isolation from the stool. Under these conditions, fecal bile acid derivatives are well resolved from each other and from those of fecal fatty acids and sterols without overlaps. The method was found to be reproducible and recoveries were similar to those obtained after exhaustive solvent extraction of fecal sterols, fatty acids and bile acids. Optimum derivatization conditions that allowed maximum recovery of fecal components with minimal destruction and application of the method for simultaneous bile acid, fatty acid and sterol analysis in human stool are described.

We investigated the role of the orphan nuclear receptor farnesoid X receptor (FXR) in the regulation of cholesterol 7α-hydroxylase (CYP7A1), using an in vivo rabbit model, in which the bile acid pool, which includes high affinity ligands for FXR, was eliminated. After 7 days of bile drainage, the enterohepatic bile acid pool, in both New Zealand White and Watanabe heritable hyperlipidemic rabbits, was depleted. CYP7A1 activity and mRNA levels increased while FXR was deactivated as indicated by reduced FXR protein and changes in the expression of target genes that served as surrogate markers of FXR activation in the liver and ileum, respectively. Hepatic bile salt export pump mRNA levels and ileal bile acid-binding protein decreased while sterol 12α-hydroxylase and sodium/taurocholate cotransporting polypeptide mRNA levels increased in the liver. In addition, hepatic FXR mRNA levels decreased significantly. The data, taken together, indicate that FXR was deactivated when the bile acid pool was depleted such that CYP7A1 was upregulated. Further, lack of the high affinity ligand supply was associated with downregulation of hepatic FXR mRNA levels. —Xu, G., L-x. Pan, S. K. Erickson, B. M. Forman, B. L. Shneider, M. Ananthanarayanan, X. Li, S. Shefer, N. Balasubramanian, L. Ma, H. Asaoka, S. R. Lear, L. B. Nguyen, I. Dussault, F. J. Suchy, G. S. Tint, and G. Salen. Removal of the bile acid pool upregulates cholesterol 7α-hydroxylase by deactivating FXR in rabbits. J. Lipid Res. 2002. 43: 45–50.

The accumulation of various 25-hydroxylated C(27)-bile alcohols in blood and their excretion in urine are characteristic features of cerebrotendinous xanthomatosis (CTX) a recessively inherited inborn error of bile acid synthesis caused by mutations in the mitochondrial sterol 27-hydroxylase (CYP27) gene. These bile alcohols may be intermediates in the alternative cholic acid side chain cleavage pathway. The present study was undertaken to identify enzymes and reactions responsible for the formation of these bile alcohols and to explain why Cyp27(-/-) mice do not show CTX-related abnormalities. Microsomal activities of 5beta-cholestane-3alpha,7alpha,12alpha-triol 25- and 26-hydroxylases, 5beta-cholestane-3alpha,7alpha,12alpha,25-tetrol 23R-, 24S-, and 27-hydroxylases and testosterone 6beta-hydroxylase, a marker enzyme for CYP3A, in Cyp27(-/-) mice livers were markedly up-regulated (5.5-, 3.5-, 6.5-, 7.5-, 2.9-, and 5.4-fold, respectively). In contrast, these enzyme activities were not increased in CTX. The activities of 5beta-cholestane-3alpha,7alpha,12alpha-triol 25- and 26-hydroxylases and 5beta-cholestane-3alpha,7alpha,12alpha,25-tetrol 23R-, 24R-, 24S-, and 27-hydroxylases were strongly correlated with the activities of testosterone 6beta-hydroxylase in control human liver microsomes from eight unrelated donors. Troleandomycin, a specific inhibitor of CYP3A, markedly suppressed these microsomal side chain hydroxylations in both mouse and human livers in a dose-dependent manner. In addition, experiments using recombinant overexpressed human CYP3A4 confirmed that these microsomal side chain hydroxylations were catalyzed by a single enzyme, CYP3A4. The results demonstrate that microsomal 25- and 26-hydroxylations of 5beta-cholestane-3alpha,7alpha,12alpha-triol and microsomal 23R-, 24R-, 24S-, and 27-hydroxylations of 5beta-cholestane-3alpha,7alpha,12alpha,25-tetrol are mainly catalyzed by CYP3A in both mice and humans. Unlike Cyp27(-/-) mice, CYP3A activity was not up-regulated despite marked accumulation of 5beta-cholestane-3alpha,7alpha,12alpha-triol in CTX.

To study the effect of cholecystectomy on the regu- lation of classic and alternative bile acid syntheses, gallbladder- intact (n � 20) and cholecystectomized (n � 20) New Zealand White rabbits were fed either chow or chow with 2% choles- terol (3 g/day). After 10 days, bile fistulas were constructed in half of each rabbit group to recover and measure the bile acid pool and biliary bile acid flux. After cholesterol feeding, the bile acid pool size increased from 268 � 55 to 444 � 77 mg ( P � 0.01) with a 2-fold rise in the biliary bile acid flux in intact rabbits but did not expand the bile acid pool (270 � 77 vs. 276 � 62 mg), nor did the biliary bile acid flux increase in cholecystectomized rabbits. Ileal apical sodium-dependent bile acid transporter protein increased 46% from 93 � 6 to 136 � 23 units/mg ( P � 0.01) in the intact rabbits but did not change in cholecystectomized rabbits (104 � 14 vs. 99 � 19 units/mg) after cholesterol feeding. Cholesterol 7 � -hydroxylase activity was inhibited 59% ( P � 0.001) while cholesterol 27-hydroxylase activity rose 83% ( P � 0.05) after cholesterol feeding in the in- tact rabbits but neither enzyme activity changed significantly in cholesterol-fed cholecystectomized rabbits. Fecal bile acid out- puts reflecting bile acid synthesis increased significantly in the intact but not in the cholecystectomized rabbits fed choles- terol. Removal of the gallbladder prevented expansion of the bile acid pool after cholesterol feeding as seen in intact rabbits because ileal bile acid transport did not increase. As a result, cholesterol 7 � -hydroxylase was not inhibited. —Xu, G., G. Salen, B. L. Shneider, M. Ananthanarayanan, S. Shefer, L. Ma, A. Batta, L. B. Nguyen, J. J. Lingutla, G. S. Tint, M. Pcolinsky, and F. J. Suchy. Cholecystectomy prevents expansion of the bile acid pool and inhibition of cholesterol 7 � -hydroxylase in rab- bits fed cholesterol. J. Lipid Res. 2001. 42: 1438-1443.

The aim of this study was to study the inhibitory effect of dietary stanols (campestanol and sitostanol) fatty acid esters (SE) on intestinal cholesterol absorption. New Zealand white rabbits were fed regular chow alone or enriched with 0.2% cholesterol, 0.33% SE + cholesterol, 0.66% SE + cholesterol, 1.2% SE + cholesterol, 2.4% SE + cholesterol, and 1.2% SE alone. After 2 weeks, plasma cholesterol levels increased 3.6 times in the cholesterol group and did not decrease after addition of 0.33% or 0.66% SE to the cholesterol-enriched diets. However, after addition of 1.2% SE to the cholesterol diet, plasma cholesterol concentration decreased 50% (P

We examined the effect of hyodeoxycholic acid (HDCA) on plasma cholesterol levels and atherosclerosis in mice. In wild-type C57BL/6 mice, feeding increasing amounts of HDCA resulted in i) progressive decrease in dietary cholesterol absorption, ii) increased concentrations of HDCA in the gallbladder bile, iii) decreased liver cholesterol content, iv) increased liver cholesterol synthesis, and v) increased plasma concentrations of HDCA. In C57BL/6 LDL-receptor knockouts (LDLR-KO) the addition of HDCA to chow and a 0.5% cholesterol diet decreased their total plasma cholesterol levels by 21% and 62%, respectively, because of a decrease in VLDL and LDL cholesterol. Turnover studies showed that HDCA has no effect on VLDL removal from plasma. Furthermore, the addition of HDCA to chow- and 0.5% cholesterol-fed LDLR-KO mice decreased the aortic root atherosclerosis lesion area by 50% and 80%, respectively. Finally, we tested the effect of HDCA on intestinal tumor formation. Feeding C57BL/6 ApcMin mice with HDCA did not affect the number of tumors but decreased the tumor volume in these animals. These results suggest that HDCA might have beneficial effects in the treatment of increased plasma cholesterol levels and atherosclerosis. —Sehayek, E., J. G. Ono, E. M. Duncan, A. K. Batta, G. Salen, S. Shefer, L. B. Neguyen, K. Yang, M. Lipkin, and J. L. Breslow. Hyodeoxycholic acid efficiently suppresses atherosclerosis formation and plasma cholesterol levels in mice.

The effects of feeding cholesterol, sitosterol, and lovastatin on cholesterol absorption, biosynthesis, es- terification, and LDL receptor function were examined in the rat jejunal mucosa. Cholesterol absorption was mea- sured by the dual-isotope plasma ratio method; the rate- limiting enzyme of cholesterol biosynthesis, 3-hydroxy-3- methylglutaryl-coenzyme A (HMG-CoA) reductase, was measured as total and expressed enzyme activities (in the absence and presence of a phosphatase inhibitor, NaF, re- spectively); mucosal total and esterified cholesterol concen- trations were determined by gas-liquid chromatography; LDL receptor function was assayed as receptor-mediated binding of 125 I-labeled LDL to mucosal membranes. Feed- ing 2% sitosterol or 0.04% lovastatin for 1 week significantly ( P � 0.01) decreased the amounts of cholesterol absorbed per day ( � 85% and � 63%, respectively). In contrast, feed- ing 2% cholesterol for 1 week increased the amounts of ab- sorbed cholesterol 27-fold, even though the percent absorp- tion significantly decreased. With all three treatments, there was a coordinate regulation of total HMG-CoA reductase activity and receptor-mediated LDL binding. Cholesterol feeding downregulated both total jejunal HMG-CoA reduc- tase activity ( P � 0.05) and receptor-mediated LDL binding ( P � 0.01), whereas lovastatin- and sitosterol-supplemented diets significantly upregulated both of these parameters. In the control, cholesterol-fed, and sitosterol-fed animals, about half of the total jejunal HMG-CoA reductase activity was expressed (in functional dephosphorylated form). However, in the lovastatin-treated rats with 4-fold stimula- tion of HMG-CoA reductase, only 23% of the total enzyme activity was expressed. Changes in total HMG-CoA reduc- tase activity and receptor-mediated LDL binding in all tested groups occurred with no change in total concentra- tions of mucosal cholesterol, and only cholesterol-fed ani- mals had increased mucosal esterified cholesterol concen- trations. Thus, in response to various fluxes of dietary or newly formed cholesterol, HMG-CoA reductase and recep- tor-mediated LDL binding are coordinately regulated to maintain constant cellular cholesterol concentrations in the jejunum. —Nguyen, L. B., S. Shefer, G. Salen, G. S. Tint, F. Ruiz, and J. Bullock. Mechanisms for cholesterol homeosta- sis in rat jejunal mucosa: effects of cholesterol, sitosterol, and lovastatin. J. Lipid Res. 2001. 42: 195-200.

Cerebrotendinous xanthomatosis (CTX) is a rare, recessively inherited lipid storage disease characterized by a markedly reduced production of chenodeoxycholic acid and an increased formation of 25-hydroxylated bile alco- hols and cholestanol. Patients with this disease are known to have mutations in the sterol 27-hydroxylase ( Cyp27 ) gene. However, one study showed that mice with a disrupted Cyp27 gene did not have any CTX-related clinical or bio- chemical abnormalities. To explore the reason, hepatic cho- lesterol, cholestanol, and 12 intermediates in bile acid bio- synthetic pathways were quantified in 10 Cyp27 � / � and 7 Cyp27 � / � mice, two CTX patients (untreated and treated with chenodeoxycholic acid), and four human control sub- jects by high resolution gas chromatography-mass spectrom- etry. Mitochondrial 27-hydroxycholesterol and 5 � -choles- tane-3 � ,7 � ,12 � ,27-tetrol were virtually absent in both Cyp27 � / � mice and CTX patients. In Cyp27 � / � mice, mi- crosomal concentrations of intermediates in the early bile acid biosynthetic pathway (7 � -hydroxycholesterol, 7 � - hydroxy-4-cholesten-3-one, 7 � ,12 � -dihydroxy-4-cholesten-3- one, and 5 � -cholestane-3 � ,7 � ,12 � -triol), 25-hydroxylated bile alcohols (5 � -cholestane-3 � ,7 � ,12 � ,25-tetrol, 5 � -cholestane- 3 � ,7 � ,12 � ,23 R ,25-pentol, and 5 � -cholestane-3 � ,7 � ,12 � ,24 R , 25-pentol), and cholestanol were all significantly elevated compared with those in Cyp27 � / � mice, although the levels were lower than those in untreated CTX patients. The inter- mediate levels in early bile acid biosynthesis were more ele- vated in male (16-86% of CTX) than in female Cyp27 � / � mice (7-30% of CTX). In contrast, 25-hydroxylated bile alco- hol concentrations were not significantly different between male and female Cyp27 � / � mice and were considerably lower (less than 14%) than those in CTX patients. These results suggest that 1 ) in Cyp27 � / � mice, especially in fe- males, classic bile acid biosynthesis via 7 � -hydroxycholesterol is not stimulated as much as in CTX patients; and 2 ) formed 25-hydroxylated bile alcohols are more efficiently metabo- lized in Cyp27 � / � mice than in CTX patients. —Honda, A., G. Salen, Y. Matsuzaki, A. K. Batta, G. Xu, E. Leitersdorf, G. S. Tint, S. K. Erickson, N. Tanaka, and S. Shefer. Differences in hepatic levels of intermediates in bile acid biosynthesis between Cyp27 � / � mice and CTX. J. Lipid Res. 2001. 42: 291-300.

The molecular mechanisms regulating the amount of dietary cholesterol retained in the body, as well as the body’s ability to exclude selectively other dietary sterols, are poorly understood. An average western diet will contain about 250–500 mg of dietary cholesterol and about 200–400 mg of non-cholesterol sterols. About 50–60% of the dietary cholesterol is absorbed and retained by the normal human body, but less than 1% of the non-cholesterol sterols are retained. Thus, there exists a subtle mechanism that allows the body to distinguish between cholesterol and non-cholesterol sterols. In sitosterolemia, a rare autosomal recessive disorder, affected individuals hyperabsorb not only cholesterol but also all other sterols, including plant and shellfish sterols from the intestine1,2. The major plant sterol species is sitosterol; hence the name of the disorder. Consequently, patients with this disease have very high levels of plant sterols in the plasma and develop tendon and tuberous xanthomas, accelerated atherosclerosis, and premature coronary artery disease3. We previously mapped the STSL locus to human chromosome 2p21 (ref. 4) and further localized it to a region of less than 2 cM bounded by markers D2S2294 and D2S2291 (M.-H.L. et al., manuscript submitted). We now report that a new member of the ABC transporter family, ABCG5, is mutant in nine unrelated sitosterolemia patients.

We measured the percent absorption, turnover, and distribution of campestanol (24-methyl-5 a -cholestan-3 b - ol) in a sitosterolemic homozygote, her obligate heterozygous mother, and three healthy human control subjects. For rea- sons relating to sterol hyperabsorption, the homozygote con- sumed a diet low in plant sterols that contained campestanol at about 2 mg/day. The heterozygote and three control sub- jects were fed a diet supplemented with a spread that con- tained campestanol at 540 mg/day and sitostanol (24-ethyl- 5 a -cholestan-3 b -ol) at 1.9 g/day as fatty acid esters. Plasma campestanol concentrations determined by capillary gas- liquid chromatography were 0.72 6 0.03 mg/dl in the homozygote, 0.09 6 0.04 mg/dl in the heterozygote, and 0.05 6 0.03 mg/dl for the control mean. After simulta- neous pulse labeling with (3 a - 3 H)campestanol intrave- nously and (23- 14 C)campestanol orally, the maximum per- cent absorption measured by the plasma dual-isotope ratio method as a single time point was 80% in the homozygote, 14.3% in the heterozygote, and 5.5 6 4.3% as the mean for three control subjects. Turnover (pool size) values esti- mated by mathematical analysis of the specific activity ver- sus time (3 a - 3 H)campestanol decay curves were as follows: 261 mg in the homozygote, 27.3 mg in the heterozygote, and 12.8 6 7.6 mg in the three control subjects (homogygote vs. controls, P , 0.001). The calculated production rate (mg/24 h) equivalent to actual absorption in the presence of dietary sterols and stanols was 0.67 mg/day or 31% of in- take in the homozygote, 2.1 mg/day or 0.3% of intake in the heterozygote, and 0.7 6 0.3 mg/day or 0.1% of intake in the three control subjects. However, the excretion constant from pool A (K A ) was prolonged markedly in the homozy- gote, but was 100 times more rapid in the heterozygote and three control subjects. Thus, campestanol, like other non- cholesterol sterols, is hyperabsorbed and retained in sitoster- olemic homozygotes. However, campestanol absorption was only slightly increased in the sitosterolemic heterozygote and removal was as rapid as in control subjects. — Salen, G., G. Xu, G. S. Tint, A. K. Batta, and S. Shefer. Hyperabsorp- tion and retention of campestanol in a sitosterolemic homo- zygote: comparison with her mother and three control sub- jects. J. Lipid Res. 2000. 41: 1883-1889.

Background & Aims: The mechanism for abnormal hepatic bile acid transport was investigated in an 18-month-old Amish boy who presented with pruritus, poor growth, and severe bleeding episodes. Serum bilirubin, γ-glutamyltranspeptidase, and cholesterol levels were normal, but prothrombin time and partial thromboplastin time were prolonged and bone alkaline phosphatase level was elevated. Methods and Results: Cholic acid plus chenodeoxycholic acid levels measured by capillary gas-chromatography were 32 times higher than control in serum (34.7 vs. 1.1 ± 0.4 μg/dL) but were not detected in liver and were reduced in gallbladder bile. Treatment with ursodiol, a more hydrophilic bile acid, improved pruritus, produced 37% weight gain, and after 2 years reduced serum primary bile acid concentrations about 85%, while accounting for 71% of serum and 24% of biliary bile acid conjugates. On ursodiol therapy, hepatic bile acid synthesis was enhanced 2-fold compared with controls, and microscopy revealed chronic hepatitis without cholestasis. Three younger sisters with elevated serum bile acids responded positively to ursodiol. Microsatellite markers for the FIC1 (gene for Byler's disease) region in these 4 children were inconsistent with linkage to FIC1 . Conclusions: Conjugated cholic acid and chenodeoxycholic acid were synthesized in the liver and secreted into bile but could not reenter the liver from portal blood and accumulated in serum. In contrast, unconjugated ursodiol entered the liver and was conjugated and secreted into bile. Thus, the enterohepatic circulation of all conjugated bile acids was interrupted at the hepatic sinusoidal basolateral membrane. Unconjugated ursodiol bypassed the hepatic uptake block to enlarge the biliary and intestinal bile acid pools. A mutation in FIC1 recognized among the Amish and linkage of the disorder to FIC1 were excluded. GASTROENTEROLOGY 2000;119:188-195

In classic cholic acid biosynthesis, a series of ring modifications of cholesterol precede side chain cleavage and yield 5β-cholestane-3α, 7α, 12α-triol. Side chain reactions of the triol then proceed either by the mitochondrial 27-hydroxylation pathway or by the microsomal 25-hydroxylation pathway. We have developed specific and precise assay methods to measure the activities of key enzymes in both pathways, 5β-cholestane-3α, 7α, 12α-triol 25- and 27-hydroxylases and 5β-cholestane-3α, 7α, 12α, 25-tetrol 23R-, 24R-, 24S- and 27-hydroxylases. The extracts from either the mitochondrial or microsomal incubation mixtures were purified by means of a disposable silica cartridge column, derivatized into trimethylsilyl ethers, and quantified by gas chromatography–mass spectrometry with selected-ion monitoring in a high resolution mode. Compared with the addition of substrates in acetone, those in 2-hydroxypropyl-β-cyclodextrin increased mitochondrial triol 27-hydroxylase activity 132% but decreased activities of the enzymes in microsomal 25-hydroxylation pathway (triol 25-hydroxylase and 5β-cholestane-3α, 7α, 12α, 25-tetrol 23R-, 24R-, 24S- and 27-hydroxylases) 13–60% in human liver. The enzyme activities in both pathways were generally 2- to 4-times higher in mouse and rabbit livers compared with human liver. In all species, microsomal triol 25-hydroxylase activities were 4- to 11-times larger than mitochondrial triol 27-hydroxylase activities but the activities of tetrol 24S-hydroxylase were similar to triol 27-hydroxylase activities in our assay conditions. The regulation of both pathways in rabbit liver was studied after bile acid synthesis was perturbed. Cholesterol feeding up-regulated enzyme activities involved in both 25- (64–142%) and 27- (77%) hydroxylation pathways, while bile drainage up-regulated only the enzymes in the 25-hydroxylation pathway (178–371%). Using these new assays, we demonstrated that the 25- and 27-hydroxylation pathways for cholic acid biosynthesis are more active in mouse and rabbit than human livers and are separately regulated in rabbit liver. —Honda, A., G. Salen, S. Shefer, Y. Matsuzaki, G. Xu, A. K. Batta, G. S. Tint, and N. Tanaka. Regulation of 25- and 27-hydroxylation side chain cleavage pathways for cholic acid biosynthesis in humans, rabbits, and mice: assay of enzyme activities by high-resolution gas chromatography–mass spectrometry. J. Lipid Res. 2000. 41: 442–451.

Sitosterolemia is an inherited recessive disease characterized by abnormally increased plasma and tissue plant sterol concentrations. Patients hyperabsorb sitosterol. In addition, hepatic, ileal, and mononuclear leukocyte 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, the rate-controlling enzyme in the cholesterol biosynthetic pathway, is markedly suppressed in this disease. It is still controversial whether the down-regulation is due to accumulated sitosterol, but the effect of sitosterol on HMG-CoA reductase activity has not been studied in sitosterolemic tissues. To investigate whether sitosterol inhibits HMG-CoA reductase activity in sitosterolemia, we measured the enzyme activities in liver and cultured skin flbroblasts from patients. Hepatic HMG-CoA reductase activities in patients were decreased 76% (P < .05) as compared with results in control subjects. In contrast, HMG-CoA reductase activities in sitosterolemic fibroblasts were not decreased as compared with results in control fibroblasts, and the activities in all cells were up-regulated similarly when they were exposed to delipidated medium. Because the cultured sitosterolemic fibroblasts contained only trace amounts of plant sterols, we added 20 microg/mL sitosterol directly to the cell medium. Raising the intracellular sitosterol concentration to 7% of cellular cholesterol level increased HMG-CoA reductase activity 23% (P < .05), while the addition of the same amount of cholesterol to the cells reduced the activity 46% (P < .05). Thus, when sitosterolemic skin fibroblasts were used, it was possible to distinguish between the effects of cholesterol and those of sitosterol on the activity of HMG-CoA reductase. These results suggest that reduced HMG-CoA reductase activity in this disease is caused by secondary effects of unknown regulator(s) other than sitosterol.

We investigated the effect of ileal bile acid transport on the regulation of classic and alternative bile acid synthesis in cholesterol-fed rats and rabbits. Bile acid pool sizes, fecal bile acid outputs (synthesis rates), and the activities of cholesterol 7α-hydroxylase (classic bile acid synthesis) and cholesterol 27-hydroxylase (alternative bile acid synthesis) were related to ileal bile acid transporter expression (ileal apical sodium-dependent bile acid transporter, ASBT). Plasma cholesterol levels rose 2.1-times in rats (98 ± 19 mg/dl) and 31-times (986 ± 188 mg/dl) in rabbits. The bile acid pool size remained constant (55 ± 17 mg vs. 61 ± 18 mg) in rats but doubled (254 ± 46 to 533 ± 53 mg) in rabbits. ASBT protein expression did not change in rats but rose 31% (P < 0.05) in rabbits. Fecal bile acid outputs that reflected bile acid synthesis increased 2- and 2.4-times (P < 0.05) in cholesterol-fed rats and rabbits, respectively. Cholesterol 7α-hydroxylase activity rose 33% (24 ± 2.4 vs. 18 ± 1.6 pmol/mg/min, P < 0.01) and mRNA levels increased 50% (P < 0.01) in rats but decreased 68% and 79%, respectively, in cholesterol-fed rabbits. Cholesterol 27-hydroxylase activity remained unchanged in rats but rose 62% (P < 0.05) in rabbits. Classic bile acid synthesis (cholesterol 7α-hydroxylase) was inhibited in rabbits because an enlarged bile acid pool developed from enhanced ileal bile acid transport. In contrast, in rats, cholesterol 7α-hydroxylase was stimulated but the bile acid pool did not enlarge because ASBT did not change. Therefore, although bile acid synthesis was increased via different pathways in rats and rabbits, enhanced ileal bile acid transport was critical for enlarging the bile acid pool size that exerted feedback regulation on cholesterol 7α-hydroxylase in rabbits. —Xu, G., B. L. Shneider, S. Shefer, L. B. Nguyen, A. K. Batta, G. S. Tint, M. Arrese, S. Thevananther, L. Ma, S. Stengelin, W. Kramer, D. Greenblatt, M. Pcolinsky, and G. Salen. Ileal bile acid transport regulates bile acid pool, synthesis, and plasma cholesterol levels differently in cholesterol-fed rats and rabbits.

We report a convenient method for the synthesis of dinorbile acids (23,24-dinor-5beta-cholan-22-oic acids, pregnane-20-carboxylic acids) in fair to good yields from norbile acid nitriles in one step by oxidative hydrolysis with oxygen in the presence of potassium-t-butoxide. The method results in stepwise overall removal of two carbon atoms in bile acid side chains in two steps. Dinorbile acids corresponding to several common bile acids have been prepared and their structures confirmed by spectroscopic methods. This simple method for synthesis of dinorbile acids may facilitate their study metabolically.

Recently we described a block in bile acid synthesis in cerebrotendinous xanthomatosis (CTX), a lipid storage disease related to an inborn error of bile acid metabolism. In this disease a defect in hepatic microsomal (24S) hydroxylation blocks the transformation of 5beta-cholestane-3alpha,7alpha,12alpha,25-tetrol into (24S) 5beta-cholestane-3alpha,7alpha,12alpha,24,25- pentol and cholic acid. Mitochondrial cholesterol 27-hydroxylation has also been reported to be abnormal in CTX subjects, but the relative importance of the enzymatic defect in this alternative microsomal pathway (namely, the 24S hydroxylation of 5beta-cholestane-3alpha,7alpha, 12alpha,25-tetrol relative to the abnormality in mitochondrial 27-hydroxylase) has not been established in CTX. To delineate the sequence of side-chain hydroxylations and the enzymatic block in bile acid synthesis, we synthesized the (23R and 23S) 24-nor-5beta-cholestane-3alpha,7alpha, 12alpha,23,25-pentols utilizing a modified Sharpless asymmetric dihydroxylation reaction on 24-nor-5beta-cholest-23-ene-3alpha, 7alpha, 12alpha-triol, a C26 analog of the naturally occurring C27 bile alcohol, 5beta-cholest-24-ene-3alpha,7alpha,12alpha-triol . Stereospecific conversion of the unsaturated 24-nor triol to the corresponding chiral compounds (23R and 23S), 24-nor-5beta-cholestane-3alpha,7alpha,12alpha,23 ,25-pentols, was quantitative. However, conversion of the unsaturated 24-nor triol to the chiral nor-pentols had absolute stereochemistry opposite to the products predicted by the Sharpless steric model. The absolute configurations and enantiomeric excess of the C26 nor-pentols and the C27 pentols (synthesized from 5beta-cholest-24-ene-3alpha,7alpha,12alpha-triol for comparison) were confirmed by nuclear magnetic resonance and lanthanide-induced circular dichroism Cotton effect measurements. These results may contribute to a better understanding of the role of the 24S-hydroxylation vs. 27-hydroxylation step in cholic acid biosynthesis.

Bile acids, the end products of cholesterol metabolism in the liver, are of vital importance in the tissue distribution of cholesterol. Abnormalities in cholesterol biosynthesis or metabolism are often reflected in the proportions, concentrations and conjugation of bile acids in various tissues and determination of bile acids in these tissues is important in the diagnosis of hepatobiliary diseases. Several methods for quantitative determination of bile acids in biological fluids are known and have been reviewed. In this review, we have discussed the gas-chromatographic method for determination of bile acids with special reference to bile acid quantitation in plasma, bile, urine and stool.

The mechanism for the catalytic reduction of the double bond at C-7,8 in 7-dehydrocholesterol by 3β-hydroxysterol Δ7-reductase was investigated by testing structurally related sterols as substrates and potential inhibitors. The hepatic smooth endoplasmic reticulum was identified as the site of enzyme activity. All putative substrates contained 27 carbons, but differed from 7-dehydrocholesterol by the addition of either an ethyl substituent at C-24 (7-dehydrositosterol), a double bond at C-22 with a methyl substituent at C-24 (ergosterol), epimerization of the hydroxyl from the 3β- to 3α-configuration (7-dehydroepicholesterol), or a saturated double bond at C-5,6 (lathosterol). Two non-steroidal compounds that inhibit 3β-hydroxysterol Δ7-reductase in vivo (AY 9944 and BM 15.766) were also tested. Ergosterol, 7-dehydrositosterol, and 7-dehydroepicholesterol were reduced at C-7,8 to form brassicasterol, sitosterol, and epicholesterol, respectively, but 75% less efficiently than 7-dehydrocholesterol. Increasing concentrations of these sterols competitively inhibited 3β-hydroxysterol Δ7-reductase activity. The double bond at C-7,8 in lathosterol was not reduced. AY 9944 and BM 15.766 inhibited 3β-hydroxysterol Δ7-reductase activity non-competitively. 3β-Hydroxysterol-Δ7-reductase activity declined after microsomes were exposed to alkaline phosphatase, and enzyme activity was increased by phosphorylation with Mg2+, and ATP. These results demonstrate that the reduction of the double bond at C-7,8 requires binding of the enzyme protein with the B-ring of the sterol substrate that contains a double bond at C-5,6. The reaction is hindered by substituents located on the apolar side-chain and epimerization of the hydroxyl group in ring A to a 3α-configuration. 3β-Hydroxysterol Δ7-reductase exists in two forms: an active phosphorylated form and an inactive dephosphorylated form. —Shefer, S., G. Salen, A. Honda, A. K. Batta, L. B. Nguyen, G. S. Tint, Y. A. Ioannou, and R. Desnick. Regulation of rat hepatic 3β-hydroxysterol Δ7-reductase: substrate specificity, competitive and non-competitive inhibition, and phosphorylation/dephosphorylation. J. Lipid Res. 1998. 39: 2471–2476.

Objective: Ursodeoxycholic acid (UDCA) improves liver biochemistries and enriches the bile with UDCA in patients with primary biliary cirrhosis. The aim of this study was to determine whether the degree of enrichment of bile correlated with that of serum and whether either of these measures correlated with improvement in measures of liver disease. Methods: In a randomized study, biliary and serum bile acid analyses were performed at entry and after 2 yr of UDCA or placebo. Results: The percentage of ursodeoxycholic acid in bile increased by 42% in the UDCA group (n = 61) compared with 8% in the placebo group (n = 57) (p < 0.0001). Measurement of serum bile acids in 32 patients (18 ursodeoxycholic acid, 14 placebo) indicated that at 2 yr, ursodeoxycholic acid comprised 65% of serum bile acids in the treated group and 7% in the placebo group. Agreement between bile and serum was fair (r = 0.75, p ≤ 0.00002) because in some patients, plasma but not biliary bile acids were enriched with UDCA. Changes in biliary ursodeoxycholic acid correlated significantly but weakly with the changes in serum alkaline phosphatase, AST, bilirubin, and in Mayo risk score. Correlations between changes in serum bile acid composition and biochemical measures of disease activity were even weaker. Conclusion: The measurement of biliary bile acids is superior to that of serum bile acids for assessing the compliance and changes in the circulating bile acids in patients receiving ursodeoxycholic acid for the treatment of primary biliary cirrhosis. Furthermore, measures to further increase the proportion of ursodeoxycholic acid in circulating bile acids should be explored.

The regulation of dietary cholesterol absorption was examined in C57BL/6 and transgenic mice with liver overexpression of the scavenger receptor BI (SR-BI Tg). In C57BL/6 animals, feeding 0.02 to 1% (wt/wt) dietary cholesterol resulted in a dose-dependent decrease in the percentage of dietary cholesterol absorbed. A plot of total daily mass of dietary cholesterol absorbed versus the percentage by weight of cholesterol in the diet yielded a curve suggesting a saturable process with a K m of 0.4% (wt/wt) and a V max of 0.65 mg cholesterol/g body weight per day. Dietary cholesterol suppressed hepatic 3-hydroxy-3-methylglutaryl CoA reductase activity, stimulated cholesterol 7α-hydroxylase activity, and enhanced fecal excretion of bile acids, but none of these changes correlated with the percentage of dietary cholesterol absorption. Dietary cholesterol also caused an increase in biliary cholesterol concentration, and in this case the concentration of biliary cholesterol was strongly and inversely correlated with the percentage dietary cholesterol absorption ( r = −0.63, P < 0.0001). Biliary cholesterol concentration was also directly correlated with daily cholesterol intake, dietary cholesterol mass absorption, and liver cholesterol ester content. Transgene-induced overexpression of SR-BI resulted in a stimulation of excretion of cholesterol into the bile and suppressed percentage dietary cholesterol absorption. Furthermore, biliary cholesterol levels in SR-BI Tg mice were strongly and inversely correlated with the percentage of dietary cholesterol absorbed ( r = −0.99, P < 0.0008). In summary, these results suggest that the excretion of cholesterol into the bile plays an important role in regulating the percentage absorption of dietary cholesterol.

Aminosalicylic acid conjugate of ursodeoxycholic acid was synthesized in above 90% yield by adding a basic solution of 5-aminosalicylic acid into the mixed anhydride formed with ursodeoxycholic acid and ethyl chloroformate. The 5-aminosalicylic acid conjugate of ursodeoxycholic acid was poorly secreted into the bile and was deconjugated with cholylglycine hydrolase and Clostridium perfringens, that deconjugate naturally occurring glycine and taurine conju- gates of bile acids. However, ursodeoxycholic acid 5-amino- salicylic acid conjugate was not absorbed from the duode- num but was concentrated in the colon wher e it was partially hydrolyzed by the intestinal bacteria to ursodeoxy- cholic acid and 5-aminosalicylic acid. We believe that this unique conjugation of ursodeoxycholic acid with 5-ami- nosalicylic acid may facilitate the transpor t of both 5- aminosalicylic acid and ursodeoxycholic acid to the colon and may be useful for the treatment of colonic inflamma- tory bowel diseases, ulcerative colitis and Crohn's dis- ease.— Batta, A. K., G. S. Tint, G. Xu, S. Shefer, and G. Salen. Synthesis and intestinal metabolism of ursodeoxy- cholic acid conjugate with an antiinflammatory agent, 5-ami- nosalicylic acid. J. Lipid. Res. 1998. 39: 1641-1646.

The effect of cholesterol feeding (3 g/day) on bile acid synthesis was examined in 10 New Zealand white rabbits (NZW), 8 Watanabe heterozygous and 10 homozygous rabbits with partial and complete deficiencies of LDL receptors. After 10 days of cholesterol feeding, bile fistulas were constructed and bile acid pool sizes were measured. Cholesterol feeding increased plasma and hepatic cholesterol levels in all rabbit groups. Baseline bile acid pool sizes were smaller (P < 0.01) in heterozygotes (139 ± 3 mg) and homozygotes (124 ± 30 mg) than NZW rabbits (254 ± 44 mg). After feeding cholesterol, bile acid pool sizes doubled with increased cholic acid synthesis in NZW and, to a lesser extent, in Watanabe heterozygous rabbits but not in homozygotes. Baseline cholesterol 7α-hydroxylase activity in NZW and heterozygotes declined 69% and 53% (P < 0.001), respectively, after cholesterol feeding. Sterol 27-hydroxylase activity reflecting alternative bile acid synthesis increased 66% (P < 0.01) in NZW and 37% in Watanabe heterozygotes but not in homozygotes after feeding cholesterol. Bile fistula drainage stimulated cholesterol 7α-hydroxylase activity but not sterol 27-hydroxylase activity in all thr ee rabbit groups. These results demonstrated that dietary cholesterol increased hepatic sterol 27-hydroxylase activity and alternative bile acid synthesis to expand the bile acid pool and inhibited cholesterol 7α-hydroxylase in NZW and in Watanabe heterozygous rabbits but not in homozygotes with absent hepatic LDL receptor function. Thus, in rabbits, sterol 27-hydroxylase is up-regulated by the increased hepatic cholesterol that enters the liver via LDL receptors whereas cholesterol 7α-hydroxylase is controlled by the circulating hepatic bile acid flux.—Xu, G., G. Salen, S. Shefer, G. S. Tint, L. B. Nguyen, T. T. Parker, T. S. Chen, J. Roberts, X. Kong, and D. Greenblatt. Regulation of classic and alternative bile acid synthesis in hypercholesterolemic rabbits: effects of cholesterol feeding and bile acid depletion.

The Smith-Lemli-Opitz syndrome, characterized by limb, face and organ abnormalities, and mental retardation, is caused by an inherited block in the step of cholesterol biosynthesis in which the delta 7 double bond of 7-dehydrocholesterol is reduced. It is diagnosed by the presence of markedly elevated levels of 7-dehydrocholesterol and 8-dehydrocholesterol in plasma and tissue. We measured amniotic fluid sterols in 15 pregnancies in 13 women who had previously carried an affected fetus. Cholesterol, 7-dehydrocholesterol and 8-dehydrocholesterol concentrations averaged 18 +/- 3, 9.8 +/- 2.9 and 5.0 +/- 1.7 micrograms/ml, respectively, in seven pregnancies with an affected fetus or child. In contrast, these levels were 19 +/- 3, 0.05 +/- 0.01 and < 0.005 micrograms/ml, respectively, in eight increased-risk pregnancies with normal outcomes and 16 +/- 2, 0.07 +/- 0.01 and < 0.005 micrograms/ml in normal controls. 7-dehydrocholesterol concentrations, 2.2-26 and 0.05-0.10 micrograms/ml in pregnancies with an affected and unaffected fetus, respectively, did not overlap. Thus, abnormally elevated amniotic fluid dehydrocholesterol concentrations are an accurate predictor of fetal Smith-Lemli-Opitz syndrome. A false-positive or a false-negative result is highly unlikely.

Gas chromatographic separations of n-butyl ester-trimethylsilyl ether derivatives of several common bile acids were compared with those of the corresponding methyl ester-trimethylsilyl ether derivatives on a CP-Sil-5 CB capillary column. Both types of derivatives were similarly resolved from each other. However, the n-butyl ester-trimethylsilyl ether derivatives of the bile acids showed longer retention times than the corresponding methyl ester-trimethylsilyl ethers and unlike the methyl ester-trimethylsilyl ether derivatives, were completely resolved from and eluted later than the trimethylsilyl ethers of common plasma sterols including sitosterol. A simplified method of plasma work-up for quantitation of bile acids and application of the above method in quantification of plasma bile acids in humans is described.

Sitosterolemia is a recessively inherited disorder characterized by abnormally increased plasma and tissue plant sterol concentrations. Patients have markedly reduced whole body cholesterol biosynthesis associated with suppressed hepatic, ileal, and mononuclear leukocyte 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, the rate-controlling enzyme in cholesterol biosynthetic pathway, coupled with significantly increased low density lipoprotein (LDL) receptor expression. To investigate the mechanism of down-regulated cholesterol biosynthesis, we assayed several other key enzymes in the cholesterol biosynthetic pathway including acetoacetyl-CoA thiolase, HMG-CoA synthase, squalene synthase, and 7-dehydrocholesterol Δ7-reductase activities in liver and freshly isolated mononuclear leukocytes from four sitosterolemic patients and 19 controls. Hepatic acetoacetyl-CoA thiolase, HMG-CoA synthase, reductase, and squalene synthase activities were significantly decreased (P < 0.05) –39%, –54%, –76%, and –57%, respectively, and 7-dehydrocholesterol Δ7-reductase activity tended to be lower (–35%) in the sitosterolemic compared with control subjects. The reduced HMG-CoA synthase, reductase, and squalene synthase activities were also found in mononuclear leukocytes from a sitosterolemic patient. Thus, reduced cholesterol synthesis is caused not only by decreased HMG-CoA reductase but also by the coordinate down-regulation of entire pathway of cholesterol biosynthesis. These results suggest that inadequate cholesterol production in sitosterolemia is due to abnormal down-regulation of early, intermediate, and late enzymes in the cholesterol biosynthetic pathway rather than a single inherited defect in the HMG-CoA reductase gene.—Honda, A., G. Salen, L. B. Nguyen, G. S. Tint, A. K. Batta, and S. Shefer. Down-regulation of cholesterol biosynthesis in sitosterolemia: diminished activities of acetoacetyl-CoA thiolase, 3-hydroxy-3-methylglutaryl-CoA synthase, reductase, squalene synthase, and 7-dehydrocholesterol Δ7-reductase in liver and mononuclear leukocytes. J. Lipid Res. 1998. 39: 44–50.

To extend the enzyme deficiency in Smith-Lemli-Opitz syndrome (SLOS) to extrahepatic tissues, 7-dehydrocholesterol-delta 7-reductase activity was measured in fibroblasts from 10 controls, five SLOS homozygotes, and five obligate heterozygotes. In cells grown almost to confluence in cholesterol-containing medium (4 mg/dL), the conversion of [1,2-3H]7-dehydrocholesterol to cholesterol (7-dehydrocholesterol-delta 7-reductase activity) was 3.8 times higher in control than in homozygote cells and 2.2 times higher than in heterozygote cells. After 24 hours' exposure of the fibroblasts to cholesterol-deficient medium supplemented with lovastatin, 7-dehydrocholesterol-delta 7-reductase activity increased twofold in controls, but did not change significantly in either heterozygous or homozygous cells. In contrast, the activities of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase and lathosterol 5-dehydrogenase, two key enzymes that precede 7-dehydrocholesterol-delta 7-reductase in the cholesterol biosynthetic pathway, and low-density lipoprotein (LDL) receptor-mediated binding were equal in control, homozygote, and heterozygote fibroblasts. Further, HMG-CoA reductase activity and LDL receptor-mediated binding increased after exposure of the cells to cholesterol-deficient medium. Fibroblast cholesterol concentrations were approximately equal, although homozygote cells contained 30 times more 7-dehydrocholesterol. Thus, markedly reduced 7-dehydrocholesterol-delta 7-reductase activity that cannot be upregulated after exposure of the cells to cholesterol-deficient medium is diagnostic for the biochemical defect in SLOS. Significantly reduced enzyme activity between the levels in controls and homozygotes without accumulation of 7-dehydrocholesterol in fibroblasts identified heterozygotes.

A rapid and easily performed procedure for the synthesis of 5β-cholestane-3α,7β,12α,25-tetrol by means of an efficient homologation sequence of the intermediate, 3α,7β,12α-triformyloxy-24-oxo-25-diazo-25-homo-5β-cholane is described. The reaction sequence involved treating the intermediate, α-diazoketone in methanol with 3% AgNO3 or Ag2O, anhydrous Na2CO3, Na 2 S 2 O 3 H 2 O resulting in the formation of homoursocholic acid in high yield. Esterification of the homoursocholic acid in methanol containing a catalytic amount of methanesulfonic acid under microwave irradiation conditions gave methyl homourscholate. The subsequent treatment of methyl homoursocholate with methyl magnesium iodide provided 5β-cholestane-3α,7β,12α,25-tetrol in 88% yield. The products and synthetic intermediates prepared in these studies were fully characterized by the results of 1D and 2D NMR, and high-resolution mass spectral studies. These studies will help in further investigation of the defect of cholic acid biosynthesis in patients with cerebrotendinous xanthomatosis (CTX) as well as other inborn errors of bile acid metabolism.

The Smith-Lemli-Opitz syndrome is a common birth defect syndrome caused by a deficiency of 7-dehydrocholesterol delta 7-reductase, an essential enzyme in the biosynthesis of cholesterol. The syndrome can usually be diagnosed easily from the plasma markers of markedly elevated 7-dehydrocholesterol and reduced cholesterol concentrations. However, atypical cases with normal plasma levels of cholesterol with only moderately elevated 7-dehydrocholesterol have been reported. To establish a sensitive method for the biochemical diagnosis of the atypical cases of the syndrome, we measured sterol concentrations of cultured skin fibroblasts. 7-Dehydrocholesterol concentrations in patients' fibroblasts grown in the presence of 10% fetal bovine serum were significantly higher than those in controls and parents (P < 0.0005), but they were not elevated proportionately as much as in plasma. To re-produce the accumulation of 7-dehydrocholesterol, the cells were exposed to delipidated medium to induce sterol biosynthesis. After 4 weeks, 7-dehydrocholesterol concentrations in patients' fibroblasts increased from 2.8 +/- 0.3% to 34 +/- 3% of total sterols (cholesterol + 7-dehydrocholesterol + 8-dehydrocholesterol). The increase was also observed in fibroblasts from an atypical patient who has a normal plasma cholesterol level and a 7-dehydrocholesterol concentration of only 0.15 mg/dl. In contrast, cells from parents and controls accumulated very little 7-dehydrocholesterol (< 1% of total sterols). These results demonstrate that cultured fibroblasts exhibit abnormally high accumulation of 7-dehydrocholesterol after cells are exposed to delipidated medium not only in typical patients, but also in an atypical case. The present method is a sensitive procedure for the biochemical diagnosis of this syndrome.

We describe the clinical effects of cholesterol supplementation in 6 children with the RSH-"Smith-Lemli-Opitz" syndrome (SLOS). The children ranged in age from birth to 11 years at the onset of therapy, with pretreatment cholesterol levels ranging from 8 to 62 mg/dl. Clinical benefits of therapy were seen in all patients, irrespective of age at onset of treatment, or severity of cholesterol defect. Effects of treatment included improved growth, more rapid developmental progress, and a lessening of problem behaviors. Pubertal progression in older patients, a better tolerance of infection, improvement of gastrointestinal symptoms, and a diminution in photosensitivity and skin rashes were also noted. There were no adverse reactions to treatment with cholesterol. This preliminary study suggests that cholesterol supplementation may be of benefit to patients with the SLOS.

The Smith-Lemli-Opitz syndrome (SLOS) is a common condition caused by deficiency of 7-dehydrocholesterol Δ 7 -reductase. The syndrome can usually be diagnosed by demonstrating markedly increased plasma concentrations of the cholesterol precursor, 7-dehydrocholesterol. We describe a simple and rapid method for detection of plasma 7-dehydrocholesterol by use of ultraviolet (UV) spectrometry. Lipids were extracted from plasma by addition of ethanol and n-hexane, and the n-hexane phase was directly subjected to spectrometry. The absorption maxima characteristics of 7-dehydrocholesterol (λ max 271, 282, and 294 nm) were observed in patients' plasma but not in controls. For quantitative measurements, absorbance at 282 nm was used. Since this absorbance is the sum of the absorbance derived from 7-dehydrocholesterol and background absorbance, the concentrations of 7-dehydrocholesterol in various plasma samples were quantified by subtracting estimated background absorbance at 282 nm from observed absorbance at 282 nm. The results correlated well with total (free plus esterified) 7-dehydrocholesterol concentrations measured by gas-liquid chromatographic method. The UV spectrometric assay was sensitive enough to detect increased 7-dehydrocholesterol in cultured skin fibroblasts from patients grown in delipidated medium. The present method will make it possible to screen plasma or fibroblasts to detect the syndrome rapidly in general clinical laboratories.

Patients with the RSH or Smith-Lemli-Optiz syndrome (SLOS) have an inborn error of cholesterol biosynthesis which results in a deficiency of cholesterol and an elevation of the cholesterol precursor, 7-dehydrocholesterol. A treatment protocol consisting of administration of cholesterol +/- bile acids was initiated in an attempt to correct the biochemical abnormalities seen. Fourteen patients (8 female, 6 male: ages 2 months to 15 years) have now been treated for 6-15 months. Three patients received cholesterol alone, while 11 patients received cholesterol and one or more bile acids. Biochemical improvement in sterol levels and in the ratio of cholesterol to total sterols was noted in all patients. The most marked improvement was noted in patients presenting with initial cholesterol levels < 40 mg/dl. No toxicity was observed. Clinical improvement in growth and neurodevelopmental status was also observed.

A new sensitive and specific method for the evaluation of 3 beta-hydroxysteroid delta 7-reductase activity, the defective enzyme in the Smith-Lemli-Opitz (SLO) syndrome, is described. The assay is based on the use of gas chromatography-mass spectrometry with selected-ion monitoring to measure the mass of brassicasterol (ergosta-5,22-dien-3 beta-ol) produced by the incubation of ergosterol (ergosta-5,7,22-trien-3 beta-ol) with cultured human skin fibroblasts. Although the conversion of ergosterol to brassicasterol was slower than the transformation of [3H]7-dehydrocholesterol to [3H] cholesterol, cells from control subjects produced brassicasterol efficiently. In contrast, cells form SLO patients produced very little brassicasterol (P < 0.0001, patients vs. parents or vs. controls). These results indicate that the reduction of ergosterol can be used as an assay for 3 beta-hydroxysteroid delta 7-reductase in human skin fibroblasts, which avoids the many problems caused by the instability and lack of availability of radiolabeled 7-dehydrocholesterol. The present method made it possible to diagnose the SLO syndrome with high sensitivity and reliability using a commercially available compound.

Selected aryl aldehydes were treated with 17-oxo-5-androsten-3β-yl acetate (I) to give the corresponding 16-arylidene 17-oxo-5-androsten-3β-yl acetates(IIa-e). Condensation of these chalcones with urea revealed the formation of the corresponding substituted pyrimidin-2′-ones (IIIa-e) respectively.

YM 9429 ( cis -1-[4-( p -menthan-8-yloxy)phenyl]piperidine) is a hypolipidemic agent with a potent and specific teratogenicity, inducing cleft palate and skeletal variations in rats. Since cleft palate is generally observed in the Smith-Lemli-Opitz syndrome, a common syndrome of multiple congenital anomalies caused by reduced activity of 7-dehydrocholesterol Δ 7 -reductase (3β-hydroxysteroid Δ 7 -reductase), the final enzyme in the cholesterol biosynthetic pathway, YM 9429 was suspected of being an inhibitor of this enzyme. To prove this hypothesis, YM 9429 was added to cultured human skin fibroblasts and to cultured Morris hepatoma cells and incubated with [5- 3 H]mevalonolactone. After 24 h, radiolabeled 7-dehydrocholesterol accumulated in the cells, whereas the formation of radiolabeled cholesterol was markedly reduced. The results indicate that YM 9429 inhibits the conversion of 7-dehydrocholesterol to cholesterol catalyzed by the microsomal enzyme 7-dehydrocholesterol Δ 7 -reductase. In rat liver microsomes, the mode of inhibition was found to be noncompetitive, with a K i of 40 μM. These results suggest that YM 9429 induced developmental abnormalities in rats by the same mechanism as the Smith-Lemli-Opitz syndrome. This compound might be useful for studying the pathogenesis of anomalies in animal models of the Smith-Lemli-Opitz syndrome.