7-dehydrocholesterol Delta 7-reductase Research Articles

Ezetimibe normalizes metabolic defects in mice lacking ABCG5 and ABCG8

The ATP binding cassette transporters ABCG5 (G5) and ABCG8 (G8) limit the accumulation of neutral sterols by restricting sterol uptake from the intestine and promoting sterol excretion into bile. Humans and mice lacking G5 and G8 (G5G8-/-) accumulate plant sterols in the blood and tissues. However, despite impaired biliary cholesterol secretion, plasma and liver cholesterol levels are lower in G5G8-/- mice than in wild-type littermates. To determine whether the observed changes in hepatic sterol metabolism were a direct result of decreased biliary sterol secretion or a metabolic consequence of the accumulation of dietary noncholesterol sterols, we treated G5G8-/- mice with ezetimibe, a drug that reduces the absorption of both plant- and animal-derived sterols. Ezetimibe feeding for 1 month sharply decreased sterol absorption and plasma levels of sitosterol and campesterol but increased cholesterol in both the plasma (from 60.4 to 75.2 mg/dl) and the liver (from 1.1 to 1.87 mg/g) of the ezetimibe-treated G5G8-/- mice. Paradoxically, the increase in hepatic cholesterol was associated with an increase in mRNA levels of HMG-CoA reductase and synthase. Together, these results indicate that pharmacological blockade of sterol absorption can ameliorate the deleterious metabolic effects of plant sterols even in the absence of G5 and G8.

Normal cognition and behavior in a Smith-Lemli-Opitz syndrome patient who presented with Hirschsprung disease.

Smith-Lemli-Opitz syndrome (SLOS) is an autosomal recessive disorder of cholesterol biosynthesis. It is caused by mutations in the gene encoding the enzyme 7-dehydrocholesterol Delta7-reductase (DHCR7), which catalyzes the final step in cholesterol biosynthesis, usually resulting in cholesterol deficiency. We report a 3.5-year-old girl who has cognition in the low average range and normal behavior, but in whom molecular studies identified two missense mutations in DHCR7: V326L and F284L. She was born at term following an uncomplicated pregnancy and delivery, and presented at 12 days of age with poor feeding, abdominal distention, and jaundice. Colonic biopsy was consistent with Hirschsprung disease. On physical examination she had mild ptosis, a long philtrum, mild micrognathia, a short, upturned nose, and subtle 2,3 syndactyly. Her 7-dehydrocholesterol (7-DHC) level was markedly elevated at 8.7 mg/dl (normal 0.10 +/- 0.05), and her cholesterol level was normal at 61 mg/dl (normal for newborn period 50-80 mg/dl). Karyotype analysis was normal, 46,XX. Breast milk feeding was initiated and continued for 18 months. Cholesterol supplementation was implemented at 100 mg/kg/day at 3 months, which resulted in increased cholesterol levels and reduced dehydrocholesterol levels. Neuropsychological testing has shown functioning in the low average range, between the 14th and 18th centiles when compared to peers. This is markedly higher than most children with SLOS. She has no behavioral problems. MRI and MRS testing of the brain revealed no structural abnormalities. This is in contrast to a recently reported case by Prasad et al. [2002: Am J Med Genet 108:64-68] with a mild phenotype, behavioral problems, and abnormal MRI, who is compound heterozygote for both a null and missense mutation. Our case suggests that patients with severe feeding disorders with or without Hirschprung disease and postnatal onset microcephaly may warrant screening for SLOS.

Detection of a common mutation in the RSH or Smith-Lemli-Opitz syndrome by a PCR-RFLP assay: IVS8-G-->C is found in over sixty percent of US propositi.

The RSH or Smith-Lemli-Opitz syndrome (SLOS) is a relatively common autosomal recessive disorder of cholesterol biosynthesis resulting from a deficiency of the enzyme 7-dehydrocholesterol delta7-reductase (7-DHCR). Mutations in 7-DHCR gene cause SLOS. Among these, a G-->C transversion in the splice acceptor site of exon 9 (IVS8-1G-->C) was suspected to be a frequent mutation, having been detected in about 18% of SLOS patients so far. This mutation results in the elimination of a AlwN1 restriction endonuclease site. We report a simple PCR-RFLP assay to detect the IVS8-1G-->C mutation. Using this method, we identified the IVS8-1G-->C mutation in 21 of 33 SLOS propositi. This mutation was detected in one of 90 normal adult Caucasian Americans; but not among 121 Africans from Sierra Leone, 120 Caucasians from Finland, 95 Chinese or 103 Japanese adults. The results of this study provide further evidence that IVS8-1G-->C transversion is a very common mutation in SLOS patients from the US and that the carrier rate in US caucasians may be high. The simple PCR-RFLP assay developed makes identification of this mutation convenient for diagnosis and for carrier detection.

A simple PCR-based assay allows detection of a common mutation, IVS8-1G-->C, in DHCR7 in Smith-Lemli-Opitz syndrome.

Smith-Lemli-Opitz syndrome (SLOS) is an autosomal recessive multiple malformation disorder. A deficiency of the enzyme 7-dehydrocholesterol delta 7-reductase (DHCR7) is the primary abnormality in SLOS. The gene encoding DHCR7 has been cloned, and we have identified a mutation affecting the splice acceptor site 5' of exon 9 that occurs frequently in affected individuals. We developed a novel PCR-based assay to detect this common mutation in DHCR7. Using this assay, heterozygosity was detected for this mutation in 18 of 26 and homozygosity in 1 of 26 unrelated affected individuals. The high frequency of this mutation is suggestive of either a founder effect in our group of patients or a mutational hotspot. The simplicity and reliability of this assay will allow it to be used as a clinical test to aid in diagnosis of atypical cases, in carrier testing, in prediction of prognosis based on genotype, and in prenatal molecular genetic diagnostic testing.

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.

Sterol concentrations in cultured Smith-Lemli-Opitz syndrome skin fibroblasts: Diagnosis of a biochemically atypical case of the syndrome

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.

Regulation of the last two enzymatic reactions in cholesterol biosynthesis in rats: Effects of BM 15.766, cholesterol, cholic acid, lovastatin, and their combinations

The Smith-Lemli-Opitz syndrome is a common inherited birth disorder caused by markedly reduced 7-dehydrocholesterol delta 7-reductase activity, the final enzyme in the cholesterol biosynthetic pathway. BM 15.766 (4-[2-[1-(4-chlorocinnamyl)piperazin-4-yl]ethyl]-benzoic acid) inhibits 7-dehydrocholesterol delta 7-reductase activity, reduces plasma cholesterol levels, and increases 7-dehydrocholesterol levels to reproduce the biochemical abnormalities of the syndrome in rats. Cholesterol, cholic acid, and lovastatin, alone or in combinations, were fed to rats given BM 15.766, and hepatic activities of the last two enzymes in the cholesterol biosynthetic pathway, lathosterol 5-dehydrogenase and 7-dehydrocholesterol delta 7-reductase, were measured. After feeding BM 15.766, hepatic 7-dehydrocholesterol delta 7-reductase activity decreased by 77% while lathosterol 5-dehydrogenase activity tended to increase, so that the ratio of 5-dehydrogenase to delta 7-reductase activities increased from 0.33 to 2.8. In BM 15.766-fed rats, treatment with cholesterol suppressed both 5-dehydrogenase and delta 7-reductase activities by 76% and 66%, respectively, and decreased the 5-dehydrogenase: delta 7-reductase activities ratio from 2.8 to 2.2. In contrast, treatment with cholic acid and BM 15.766 further inhibited delta 7-reductase activity by 67% without changing significantly the 5-dehydrogenase activity that had increased the ratio to 5.5. Combining BM 15.766 with lovastatin increased 5-dehydrogenase activity fivefold but did not change delta 7-reductase activity, raising the ratio to 14.3. In BM 15.766-treated rats, the first and last two enzymatic reactions in the cholesterol biosynthetic pathway catalyzed by 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, lathosterol 5-dehydrogenase, and 7-dehydrocholesterol delta 7-reductase are down-regulated by cholesterol. Thus, only cholesterol and not cholic acid or lovastatin could reduce elevated plasma 7-dehydrocholesterol levels induced by BM 15.766.

Regulation of the last two enzymatic reactions in cholesterol biosynthesis in rats: Effects of BM 15.766, cholesterol, cholic acid, lovastatin, and their combinations

The Smith-Lemli-Opitz syndrome is a common inherited birth disorder caused by markedly reduced 7-dehydrocholesterol δ7-reductase activity, the final enzyme in the cholesterol biosynthetic pathway. BM 15.766 (4-[2-[1-(4-chlorocinnamyl)piperazin-4-yl]ethyl]-benzoic acid) inhibits 7-dehydrocholesterol delta 7-reductase activity, reduces plasma cholesterol levels, and increases 7-dehydrocholesterol levels to reproduce the biochemical abnormalities of the syndrome in rats. Cholesterol, cholic acid, and lovastatin, alone or in combinations, were fed to rats given BM 15.766, and hepatic activities of the last two enzymes in the cholesterol biosynthetic pathway, lathosterol 5- dehydrogenase and 7-dehydrocholesterol delta 7-reductase, were measured. After feeding BM 15.766, hepatic 7-dehydrocholesterol delta 7-reductase activity decreased by 77% while lathosterol 5-dehydrogenase activity tended to increase, so that the ratio of 5-dehydrogenase to delta 7-reductase activities increased from 0.33 to 2.8. In BM 15.766- fed rats, treatment with cholesterol suppressed both 5-dehydrogenase and delta 7-reductase activities by 76% and 66%, respectively, and decreased the 5-dehydrogenase: delta 7-reductase activities ratio from 2.8 to 2.2. In contrast, treatment with cholic acid and BM 15.766 further inhibited delta 7-reductase activity by 67% without changing significantly the 5-dehydrogenase activity that had increased the ratio to 5.5. Combining BM 15.766 with lovastatin increased 5-dehydrogenase activity fivefold but did not change delta 7-reductase activity, raising the ratio to 14.3. In BM 15.766-treated rats, the first and last two enzymatic reactions in the cholesterol biosynthetic pathway catalyzed by 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, lathosterol 5-dehydrogenase, and 7-dehydrocholesterol delta 7-reductase are down-regulated by cholesterol. Thus, only cholesterol and not cholic acid or lovastatin could reduce elevated plasma 7- dehydrocholesterol levels induced by BM 15.766. (Hepatology 1996 Aug;24(2):435-9)

Abnormal cholesterol biosynthesis in sitosterolaemia and the Smith-Lemli-Opitz syndrome.

We investigated the enzyme defects in two inherited disorders of cholesterol biosynthesis: sitosterolaemia and the Smith-Lemli-Opitz syndrome. In sitosterolaemic homozygotes, plasma plant sterols (sitosterol and campesterol) concentrations are elevated because of enhanced intestinal absorption and diminished removal. Underlying these changes is very low cholesterol biosynthesis to provide extra sterol for cell growth. Extremely reduced activities of HMG-CoA reductase, the rate-controlling enzyme for cholesterol biosynthesis, caused by deficient HMG-CoA reductase mRNA is responsible and is the suspected inherited abnormality. The Smith-Lemli-Opitz syndrome is caused by a block in the last reaction in the cholesterol biosynthetic pathway, the conversion of 7-dehydrocholesterol to cholesterol, which is catalysed by 7-dehydrocholesterol delta 7-reductase. As a result, low plasma and tissue cholesterol with high 7-dehydrocholesterol levels are found in homozygotes, who show characteristic phenotypes of mental retardation, facial dysmorphism, and organ and limb congenital anomalies. Similar biochemical findings are produced in rats fed BM 15,766, an inhibitor of 7-dehydrocholesterol delta 7-reductase. Interestingly, feeding cholesterol can suppress abnormal cholesterol biosynthesis and improve symptoms in homozygotes and rats fed BM 15,766.

Defective conversion of 7-dehydrocholesterol to cholesterol in cultured skin fibroblasts from Smith-Lemli-Opitz syndrome homozygotes

The Smith-Lemli-Opitz syndrome is a common birth defect syndrome characterized biochemically by low plasma cholesterol levels and high concentrations of the cholesterol precursor 7-dehydrocholesterol. The present study was undertaken to prove that the enzyme defect is at the step in which 7-dehydrocholesterol is converted into cholesterol and to establish a new biochemical method for the diagnosis of this disease. We assayed the latter part of the cholesterol biosynthetic pathway by incubating [3H]lathosterol (the immediate precursor of 7-dehydrocholesterol) with cultured skin fibroblasts from 15 homozygous patients, 14 obligate heterozygous parents, and 8 controls, and measuring its conversion to 7-dehydrocholesterol and cholesterol. The formation of cholesterol from lathosterol in parents was not significantly different from that in controls. In contrast, cells from patients made very little cholesterol (P < 0.0001, patients vs. parents or vs. controls) but readily converted lathosterol to 7-dehydrocholesterol. The defect was especially profound in a subgroup of 8 of the most severely clinically affected patients, as virtually no label was detected in the cholesterol fraction. These results provide compelling evidence that 1) this disease is caused by a primary defect in 7-dehydrocholesterol delta 7-reductase, an essential enzyme in the biosynthesis of cholesterol; 2) the most clinically severe form of the syndrome may be associated with the most inhibited enzyme; and 3) the enzyme lathosterol 5-desaturase that converts lathosterol to 7-dehydrocholesterol is fully intact. The present method using fibroblast and amniocyte cultures establishes it as a useful procedure for the biochemical diagnosis of this syndrome.

(2-Piperidine)- and (2-pyrrolidine)ethanones and -ethanols as inhibitors of blood platelet aggregation.

(E)-4-[4-(Methylthio)phenyl]-1-(2-piperidinyl)-3-buten-2-one hydrochloride (44, RMI 14 133A) was found to inhibit ADP-induced aggregation of blood platelets. It was selected from a large series of (2-piperidinyl)- and (2-pyrrolidinyl)ethanones synthesized by a modified Schopf reaction from enolate magnesium salts of beta-keto acids and 2,3,4,5-tetrahydropyridine trimer or 3,4-dihydro-2H-pyrrole trimer, respectively. Evaluation of the compounds was carried out in vitro on human blood platelets. Structure-activity relationships are discussed. 44 also inhibited platelet aggregation ex vivo in guinea pigs. Subacute toxicity evaluation in dogs and guinea pigs showed it to have an unfavorable therapeutic ratio. 1-[4'-Chloro(1,1'-biphenyl)-4-yl-a1-2-(2-piperdinyl)ethanone hydrochloride (18, RMI 12436A) was found to lower serum cholesterol levles in rats with concurrent accumulation of (3beta)-cholesta-5,7-dien-3-ol, suggesting inhibition of 7-dehydrocholesterol delta7-reductase.