Biomedical subjects

S Shefer

Publications and source records attributed to S Shefer.

At least 37 records · Page 2Linked to original sources

PubMed2000

Ileal bile acid transport regulates bile acid pool, synthesis, and plasma cholesterol levels differently in cholesterol-fed rats and rabbits.

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 7alpha-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 7alpha-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 7alpha-hydroxylase) was inhibited in rabbits because an enlarged bile acid pool developed from enhanced ileal bile acid transport. In contrast, in rats, cholesterol 7alpha-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 7alpha-hydroxylase in rabbits.

Animals↗

PubMed2000

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.

In classic cholic acid biosynthesis, a series of ring modifications of cholesterol precede side chain cleavage and yield 5beta-cholestane-3alpha, 7alpha, 12alpha-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, 5beta-cholestane-3alpha, 7alpha, 12alpha-triol 25- and 27-hydroxylases and 5beta-cholestane-3alpha, 7alpha, 12alpha, 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-beta-cyclodextrin increased mitochondrial triol 27-hydroxylase activity 132% but decreased activities of the enzymes in microsomal 25-hydroxylation pathway (triol 25-hydroxylase and 5beta-cholestane-3alpha, 7alpha, 12alpha, 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.

Animals↗

PubMed2000

Hyperabsorption and retention of campestanol in a sitosterolemic homozygote: comparison with her mother and three control subjects.

We measured the percent absorption, turnover, and distribution of campestanol (24-methyl-5alpha-cholestan-3beta-ol) in a sitosterolemic homozygote, her obligate heterozygous mother, and three healthy human control subjects. For reasons relating to sterol hyperabsorption, the homozygote consumed a diet low in plant sterols that contained campestanol at about 2 mg/day. The heterozygote and three control subjects were fed a diet supplemented with a spread that contained campestanol at 540 mg/day and sitostanol (24-ethyl-5alpha-cholestan-3beta-ol) at 1.9 g/day as fatty acid esters. Plasma campestanol concentrations determined by capillary gas-liquid chromatography were 0.72 +/- 0.03 mg/dl in the homozygote, 0.09 +/- 0.04 mg/dl in the heterozygote, and 0.05 +/- 0.03 mg/dl for the control mean. After simultaneous pulse labeling with [3alpha-(3)H]campestanol intravenously and [23-(14)C]campestanol orally, the maximum percent 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 +/- 4.3% as the mean for three control subjects. Turnover (pool size) values estimated by mathematical analysis of the specific activity versus time [3alpha-(3)H]campestanol decay curves were as follows: 261 mg in the homozygote, 27.3 mg in the heterozygote, and 12.8 +/- 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 intake in the homozygote, 2.1 mg/day or 0.3% of intake in the heterozygote, and 0.7 +/- 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 homozygote, but was 100 times more rapid in the heterozygote and three control subjects.Thus, campestanol, like other noncholesterol sterols, is hyperabsorbed and retained in sitosterolemic homozygotes. However, campestanol absorption was only slightly increased in the sitosterolemic heterozygote and removal was as rapid as in control subjects.

Adolescent↗

PubMed1999

Thermodynamic prediction of active ingredient loading in polymeric microparticles.

The growing use of microparticles as a controlled-delivery system for pharmaceutical and non-pharmaceutical active ingredients (AIs) has prompted a costly trial-and-error development of new and effective microparticle systems. In order to facilitate a more rational design and optimization of AI loadings in microparticles, we have developed a molecular-thermodynamic theory to predict the loading of liquid AIs in polymeric microparticles that are manufactured by a solvent evaporation process. This process involves the emulsification of a liquid polymer solution (consisting of polymer and AI dissolved in a volatile solvent) in an aqueous surfactant solution. The theory describes the equilibrium distribution of the AI between the aqueous phase and the dispersed polymeric droplets. The universal functional activity coefficient (UNIFAC) and UNIFAC-Free Volume (FV) group-contribution methods are utilized to model the nonidealities in the water and polymeric droplet phases, respectively. The inputs to the theory are: (i) the chemical structures, densities and total masses of the manufacturing ingredients, (ii) the manufacturing temperature and (iii) the glass transition temperature of the polymer. Since surfactant concentrations exceeding the critical micellar concentration (CMC) are often required in order to stabilize the dispersed polymeric droplets during the emulsion manufacturing process, the theory also accounts for AI solubilization in surfactant micelles present in the manufacturing solution. To test the AI loading predictions, we compare theoretical predictions of AI loadings in poly(lactic acid), poly(methyl methacrylate) and polystyrene microparticles to experimentally measured ones for five model AIs with varying degrees of hydrophobicity (benzyl alcohol, n-octanol, geraniol, farnesol and galaxolide). We also demonstrate how the developed theory can be utilized to screen polymers with respect to their abilities to load a given AI, as well as to provide guidelines for manufacturing microparticles having the desired AI loading.

Drug Delivery Systems↗

PubMed1999

Campestanol (24-methyl-5alpha-cholestan-3beta-ol) absorption and distribution in New Zealand White rabbits: effect of dietary sitostanol.

Campestanol (24-methyl-5alpha-cholestan-3beta-ol) is a naturally occurring plant stanol, structurally similar to cholesterol (5-cholesten-3beta-ol) and widely distributed in vegetable oils consumed in human diets. We measured the absorption and turnover of campestanol by the plasma dual-isotope ratio method and mathematical analysis of specific activity versus time decay curves after simultaneous oral and intravenous pulse-labeling with [3alpha-3H]- and [23-14C]-labeled campestanol, respectively, in New Zealand White (NZW) rabbits: six fed chow and six fed chow with 125 mg/d campestanol and 175 mg/d sitostanol (24-ethyl-5alpha-cholestan-3beta-ol). Plasma concentrations increased insignificantly from 0.08+/-0.01 to 0.09+/-0.01 mg/dL with dietary stanols. The percent campestanol absorption measured by the plasma dual-isotope ratio method after the rabbits were fasted for 6 hours yielded the percent absorption in the absence of competing intestinal sterols and stanols and declined insignificantly from 11.6%+/-3.5% in controls to 8.1%+/-3.7% in the treated rabbit groups. In contrast, the turnover, which measured actual absorption averaged over 24 hours, increased from 0.12+/-0.05 to 0.37+/-0.05 mg/d (P < .05) with campestanol and sitostanol added to the diet. However, the actual percent absorption declined from 3% to 0.3% of dietary intake with the campestanol and sitostanol-enriched diet. Campestanol pool sizes, although remaining small, increased slightly from 1.1+/-0.4 to 2.5+/-1.5 mg. The removal constant (KA) from pool A (MA) did not change significantly with added dietary campestanol and sitostanol (KA= -0.040+/-0.005 v -0.037+/-0.007 d(-1)). The results demonstrate small campestanol plasma concentrations and body pools even when the rabbits consumed substantial amounts because (1) intestinal absorption was limited and (2) was further reduced by competing dietary sitostanol, and (3) campestanol was removed rapidly from the body. Thus, campestanol, which shares the same basic structure and intestinal absorption pathway with cholesterol, does not accumulate when fed, and may be incorporated into the diet to block cholesterol absorption.

Administration, Oral↗

PubMed1999

Comparative regulation of hepatic sterol 27-hydroxylase and cholesterol 7alpha-hydroxylase activities in the rat, guinea pig, and rabbit: effects of cholesterol and bile acids.

The regulation of the classic and alternative bile acid synthetic pathways by key hepatic enzyme activities (microsomal cholesterol 7alpha-hydroxylase and mitochondrial sterol 27-hydroxylase, respectively) was examined in bile acid depletion and replacement and cholesterol-feeding experiments with rats, guinea pigs, and rabbits. The bile acid pool was depleted by creating a bile fistula (BF) and collecting bile for 2 to 5 days, and it was replaced by intraduodenal infusion of the major biliary bile acids (taurocholic acid [TCA], glycochenodeoxycholic acid [GCDCA], and glycocholic acid [GCA] in the rat, guinea pig, and rabbit, respectively) at rates equivalent to the measured hepatic flux of the bile acids. To study the effects of cholesterol, the animals were fed for 7 days on a basal diet with and without 2% cholesterol. Cholesterol 7alpha-hydroxylase and sterol 27-hydroxylase activities, measured by isotope incorporation assays, were related to bile acid output and composition and hepatic cholesterol concentrations. Intraduodenal infusion of bile acids increased the output of the tested bile acids, but did not significantly change hepatic cholesterol concentrations and had no effect on sterol 27-hydroxylase activity. Neither bile acid depletion nor replacement affected sterol 27-hydroxylase activity when three different substrates (cholesterol, 5beta-cholestane-3alpha,7alpha-diol, and 5beta-cholestane-3alpha,7alpha,12alpha-triol) were tested. In contrast, feeding 2% cholesterol increased hepatic cholesterol concentrations in rats, guinea pigs, and rabbits threefold, twofold, and eightfold, respectively, and increased hepatic mitochondrial sterol 27-hydroxylase activity (conversion of cholesterol to 27-hydroxycholesterol) in all three animal models. The stimulation and feedback inhibition of cholesterol 7alpha-hydroxylase activity by bile acid depletion and replacement were observed in all three animal models, whereas the effect of cholesterol feeding was species-dependent (cholesterol 7alpha-hydroxylase activity increased in the rat, did not change in the guinea pig, and was inhibited in the rabbit). Thus, in contrast to sterol 27-hydroxylase, which was upregulated by cholesterol but not affected by bile acid depletion and replacement in all three animal models, cholesterol 7alpha-hydroxylase activity was controlled consistently and inversely by the hepatic flux of bile acids, but was species-dependent in its response to a 1-week feeding with 2% cholesterol.

Animals↗

PubMed1999

Increasing dietary cholesterol induces different regulation of classic and alternative bile acid synthesis.

We investigated the effect of increasing dietary cholesterol on bile acid pool sizes and the regulation of the two bile acid synthetic pathways (classic, via cholesterol 7alpha-hydroxylase, and alternative, via sterol 27-hydroxylase) in New Zealand white rabbits fed 3 g cholesterol/per day for up to 15 days. Feeding cholesterol for one day increased hepatic cholesterol 75% and cholesterol 7alpha-hydroxylase activity 1.6 times without significant change of bile acid pool size or sterol 27-hydroxylase activity. After three days of cholesterol feeding, the bile acid pool size increased 83% (P < 0.01), and further feeding produced 10%-20% increments, whereas cholesterol 7alpha-hydroxylase activity declined progressively to 60% below baseline. In contrast, sterol 27-hydroxylase activity rose 58% after three days of cholesterol feeding and remained elevated with continued intake. Bile drainage depleted the bile acid pool and stimulated downregulated cholesterol 7alpha-hydroxylase activity but did not affect sterol 27-hydroxylase activity. Thus, increasing hepatic cholesterol does not directly inhibit cholesterol 7alpha-hydroxylase and initially favors enzyme induction, whereas increased bile acid pool is the most powerful inhibitor of cholesterol 7alpha-hydroxylase. Sterol 27-hydroxylase is insensitive to the bile acid flux but is upregulated by increasing hepatic cholesterol.

Animals↗

PubMed1999

Histologic, hematologic, and biochemical characteristics of apo E-deficient mice: effects of dietary cholesterol and phytosterols.

In this study, we examined the effects of a "Western-type" diet containing 9% (w/w) fat and 0.15% (w/w) cholesterol, in the presence or absence of 2% (w/w) phytosterol mixture over an 18-week period in apolipoprotein E-deficient mice. Addition of phytosterols to the high cholesterol diet was associated with normalization of the depressed hepatic 3-hydroxy-3-methylglutaryl-coenzyme A reductase activity (from 22.3+/-6.3 to 55.4+/-19.9 pmol/mg protein/minutes, p < 0.05). This finding was associated with a significant decrease in plasma and hepatic cholesterol concentrations compared with animals fed the high cholesterol diet without phytosterols (33.3+/-5.0 versus 19.2+/-6.2 pmol/mg protein, p < 0.05). The activities of cholesterol 7alpha-hydroxylase and sterol 27-hydroxylase were comparable between the two groups of mice. Urinalyses and hematologic data were comparable between the two groups except for significantly lower platelet counts in the phytosterol-treated animals (681.6+/-118.9 versus 857.1+/-185.4 x10(9)/L, p < 0.05). The phytosterol-treated animals had significantly (p < 0.05) less fragile erythrocytes when exposed to 0.08, 0.07, or 0.05 M NaCl compared with cholesterol-fed mice. The consumption of the Western-type diet was associated with the development of xanthomatous skin lesions in 33% of the cholesterol-fed animals, but in none of the phytosterol-treated animals. Histologic examination revealed oil red O-negative vacuolation in liver and kidney parenchymal cells of the cholesterol-fed group, but not in the phytosterol-treated mice. Arrested spermatogenesis and atrophy of seminiferous tubules were observed, to a variable extent, in both groups of animals. We conclude that addition of the phytosterol mixture (2% w/w) to a Western-type diet in apolipoprotein E-deficient mice significantly decreases plasma and hepatic cholesterol concentrations, increases hepatic 3-hydroxy-3-methylglutaryl-coenzyme A reductase activity, and prevents cutaneous xanthomatosis and vacuolation in the parenchymal cells of kidneys and livers.

Animals↗

PubMed1999

Bile acid synthesis in the Smith-Lemli-Opitz syndrome: effects of dehydrocholesterols on cholesterol 7alpha-hydroxylase and 27-hydroxylase activities in rat liver.

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.

Animals↗

PubMed1998

Biliary cholesterol excretion: a novel mechanism that regulates dietary cholesterol absorption.

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 Km of 0.4% (wt/wt) and a Vmax of 0.65 mg cholesterol/g body weight per day. Dietary cholesterol suppressed hepatic 3-hydroxy-3-methylglutaryl CoA reductase activity, stimulated cholesterol 7alpha-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.

Animals↗

PubMed1998

Regulation of early cholesterol biosynthesis in rat liver: effects of sterols, bile acids, lovastatin, and BM 15.766 on 3-hydroxy-3-methylglutaryl coenzyme A synthase and acetoacetyl coenzyme A thiolase activities.

Cytosolic 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase catalyzes the formation of HMG-CoA, the substrate for the rate-controlling enzyme in the cholesterol biosynthetic pathway. To explore the regulation in liver, we developed a new, accurate, and reliable reversed-phase ion-pair chromatographic assay that uses nonradioactive substrates and n-propionyl coenzyme A as an internal recovery standard. The hepatic activities were measured in rats treated with cholesterol, sitosterol, cholic acid, deoxycholic acid, ursodeoxycholic acid, cholestyramine, bile fistula, lovastatin, and BM 15.766, an inhibitor of 7-dehydrocholesterol delta7-reductase, and were compared with microsomal HMG-CoA reductase and cytosolic acetoacetyl coenzyme A (AcAc-CoA) thiolase activities. HMG-CoA synthase activity was effectively suppressed in synchrony with HMG-CoA reductase activity by treatments with cholesterol (-41%, P < .05), cholic acid (-72%, P < .005), and deoxycholic acid (-62%, P < .05). However, ursodeoxycholic acid increased activity 84% (P < .05) and intravenous sitosterol did not change activity. AcAc-CoA thiolase activities also paralleled HMG-CoA reductase and HMG-CoA synthase activities, but differences were not statistically significant. In contrast to inhibition, up-regulation of hepatic HMG-CoA synthase activities by cholestyramine, bile fistula, and lovastatin was much less than HMG-CoA reductase activities. In addition, BM 15.766 did not stimulate synthase activity, whereas lovastatin increased activity 2.4-fold. Thus, hepatic HMG-CoA synthase activity was regulated coordinately with HMG-CoA reductase, and responded more forcefully to regulatory stimuli than acetoacetyl-CoA thiolase activity but usually less than HMG-CoA reductase.

Acetyl-CoA C-Acetyltransferase↗

PubMed1998

Relationship between abnormal cholesterol synthesis and retarded learning in rats.

We examined the relationship between brain sterol composition and associative learning (classical conditioning of the eyeblink response) in newly weaned rats fed BM 15.766 (BM) for 4 months. This compound inhibits 7-dehydrocholesterol-delta7-reductase, which catalyzes the conversion of 7-dehydrocholesterol to cholesterol, the last step in the synthetic pathway. As countertreatment, half of the BM-treated rats were fed 2% cholesterol during the last 2 months. With BM, cholesterol concentrations declined 91% in plasma, but with cholesterol feeding, the levels increased 50% compared with baseline values. 7-Dehydrocholesterol, which was not detected at baseline, increased to 55% of plasma sterols with BM but decreased to 5% of total plasma sterols when cholesterol was added. With BM, brain cholesterol levels decreased 60% and did not increase after cholesterol was added. However, 7-dehydrocholesterol, which comprised 39% of brain sterols with BM, decreased to 31% (P < .05) when cholesterol was fed. Hydroxymethyl glutaryl coenzyme A (HMG-CoA) reductase activity in the liver increased 2.2-fold with BM and declined 95% after adding cholesterol, but did not change in the brain. BM treatment for 4 months prevented learning of the conditioned eyeblink response as compared with controls. In contrast, BM-treated rats supplemented with cholesterol acquired the conditioned eyeblink response. Chronic inhibition of 7-dehydrocholesterol-delta7-reductase reduced cholesterol and increased 7-dehydrocholesterol levels in plasma and brain, and was associated with impaired learning. Cholesterol feeding corrected plasma and hepatic sterol levels and reduced brain 7-dehydrocholesterol concentrations to reestablish normal learning.

Administration, Oral↗

PubMed1998

Accurate detection of Smith-Lemli-Opitz syndrome carriers by measurement of the rate of reduction of the ergosterol C-7 double bond in cultured skin fibroblasts.

The activity of ergosterol delta 7-reductase (3 beta-hydroxysteroid delta 7-reductase) was measured in cultured skin fibroblasts from 7 controls, 10 Smith-Lemli-Opitz syndrome (SLOS) patients, and 10 parents (obligate carriers). The fibroblasts were exposed to delipidated medium supplemented with lovastatin for 24 h and the enzyme activity was determined by incubating cell-free homogenate with ergosterol (ergosta-5,7,22-trien-3 beta-ol) and measuring the mass of brassicasterol (ergosta-5,22-dien-3 beta-ol) formed by gas chromatography-mass spectrometry with selected-ion monitoring. In carriers, the activity was significantly lower than in controls (22 +/- 2 vs 65 +/- 10 pmol/min per mg protein, p < 0.0005), and no overlap was observed. The mean activity in carriers' fibroblasts was more than 100 times higher than in patients' cells (0.2 pmol/min per mg protein). The use of ergosterol avoids the many problems caused by the instability and lack of availability of radiolabelled 7-dehydrocholesterol. The present method makes it possible to discriminate SLOS carriers from both controls and patients using a commercially available substrate and common analytical equipment.

Cells, Cultured↗

PubMed1998

Cholesterol and oxygenated cholesterol concentrations are markedly elevated in peripheral tissue but not in brain from mice with the Niemann-Pick type C phenotype.

Niemann-Pick disease type C (NP-C) is a rare genetic disorder characterized by progressive neurodegeneration, frequent developmental delay and early death. Tissues of affected individuals accumulate large quantities of free cholesterol in lysosomes. Because cytotoxic oxygenated derivatives of cholesterol are known to form readily when cholesterol concentrations are elevated, we searched for these compounds in liver, kidney, spleen and brain from mice with the NP-C phenotype. In order of abundance, we identified 7 alpha- and 7 beta-hydroxycholesterol, 5 alpha, 6 alpha-epoxycholestan-3 beta-ol, 4 beta-hydroxycholesterol, cholest-4-en-3 beta, 7 alpha-diol and cholest-4-en-3 beta, 6 beta-diol in most tissue samples. Cholesterol concentrations in affected mice were increased 3-fold in kidney and 7- to 8-fold in spleen and liver compared to controls (all p < 0.001) but were unchanged in brain. Although oxysterol levels were markedly elevated in nonbrain tissue, the oxysterol and cholesterol concentrations increased proportionally so that oxysterols expressed as percentage of total sterols were the same for all animals (0.34 +/- 0.19% averaged over all organs in affected animals vs 0.40 +/- 0.42% in control mice). In contrast to peripheral tissue, we could not detect any increase in either absolute or relative oxysterol levels in the brains of affected and control mice (49 +/- 61 vs 53 +/- 43 micrograms/g wet weight and 0.45 +/- 0.52 vs 0.47 +/- 0.37%, respectively). Thus, brain sterols are normal in NP-C mice and it is unlikely that an accumulation of cytotoxic oxygenated derivatives of cholesterol could account for the progressive neuropathology seen in the disease.

Animals↗

PubMed1998

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 delta7-reductase in liver and mononuclear leukocytes.

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 delta7-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 delta7-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.

Acetyl-CoA C-Acetyltransferase↗

PubMed1998

7-Dehydrocholesterol down-regulates cholesterol biosynthesis in cultured Smith-Lemli-Opitz syndrome skin fibroblasts.

The Smith-Lemli-Opitz syndrome (SLOS) is a common birth defect-mental retardation syndrome caused by a defect in the enzyme that reduces 7-dehydrocholesterol to cholesterol. Because of this block, patients' plasma cholesterol levels are generally low while 7-dehydrocholesterol concentrations are markedly elevated. In addition, plasma total sterols are abnormally low and correlate negatively with the percent of 7-dehydrocholesterol (r = -0.65, P < 0.0001) suggesting that 7-dehydrocholesterol might inhibit the activity of HMG-CoA reductase. Cultured skin fibroblasts from SLOS patients grown in fetal bovine serum or for 1 day in delipidated medium contain little 7-dehydrocholesterol (3 +/- 1% of total sterols) and HMG-CoA reductase activities are indistinguishable from that measured in control cells. However, raising the 7-dehydrocholesterol concentration to 20 +/- 3% of total sterols, equal to the mean proportion in plasma of SLOS patients, by either growing cells for 1 week in delipidated medium or adding 20 microg/ml 7-dehydrocholesterol directly to the cells reduced HMG-CoA reductase activities from 74 +/- 7 to 9 +/- 2 pmol/min per mg protein, or from 92 +/- 22 to 16 +/- 4 pmol/min per mg protein, respectively (P < 0.01). In contrast, adding 20 microg/ml cholesterol evoked a 2- to 4-fold lesser suppression of activity (39 +/- 8 pmol/min per mg protein, P < 0.05, vs. 7-dehydrocholesterol). HMG-CoA synthase and LDL binding were inhibited equally by 7-dehydrocholesterol and cholesterol. Ketaconazole prevented the down-regulation of HMG-CoA reductase by 7-dehydrocholesterol, suggesting that an hydroxylated derivative of 7-dehydrocholesterol may be especially important in suppressing cholesterol synthesis. These results demonstrate that 7-dehydrocholesterol, perhaps as an hydroxylated derivative(s), is a very effective feedback inhibitor of HMG-CoA reductase.

Cells, Cultured↗

PubMed1998

Fetal Smith-Lemli-Opitz syndrome can be detected accurately and reliably by measuring amniotic fluid dehydrocholesterols.

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.

Adult↗

PubMed1998

Regulation of classic and alternative bile acid synthesis in hypercholesterolemic rabbits: effects of cholesterol feeding and bile acid depletion.

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 7alpha-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 7alpha-hydroxylase activity but not sterol 27-hydroxylase activity in all three 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 7alpha-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 7alpha-hydroxylase is controlled by the circulating hepatic bile acid flux.

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