A highly sensitive method for analysis of 7-dehydrocholesterol for the study of Smith-Lemli-Opitz syndrome.
ABSTRACT: We describe a highly sensitive method for the detection of 7-dehydrocholesterol (7-DHC), the biosynthetic precursor of cholesterol, based on its reactivity with 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD) in a Diels-Alder cycloaddition reaction. Samples of biological tissues and fluids with added deuterium-labeled internal standards were derivatized with PTAD and analyzed by LC-MS. This protocol permits fast processing of samples, short chromatography times, and high sensitivity. We applied this method to the analysis of cells, blood, and tissues from several sources, including human plasma. Another innovative aspect of this study is that it provides a reliable and highly reproducible measurement of 7-DHC in 7-dehydrocholesterol reductase (Dhcr7)-HET mouse (a model for Smith-Lemli-Opitz syndrome) samples, showing regional differences in the brain tissue. We found that the levels of 7-DHC are consistently higher in Dhcr7-HET mice than in controls, with the spinal cord and peripheral nerve showing the biggest differences. In addition to 7-DHC, sensitive analysis of desmosterol in tissues and blood was also accomplished with this PTAD method by assaying adducts formed from the PTAD "ene" reaction. The method reported here may provide a highly sensitive and high throughput way to identify at-risk populations having errors in cholesterol biosynthesis.
Project description:Mutations in both copies in the gene encoding 7-dehydrocholesterol reductase (DHCR7) cause Smith-Lemli-Opitz Syndrome (SLOS), which is characterized by a toxic elevation in 7-dehydrocholesterol (7-DHC). Aripiprazole (ARI) exposure, independent of genetic mutations, also leads to elevation of 7-DHC. We investigated the combined effect of a single-copy Dhcr7+/- mutation and maternal ARI exposure on the developing offspring brain. We generated a time-pregnant mouse model where WT and Dhcr7+/- embryos were maternally exposed to ARI or vehicle (VEH) from E12 to E19 (5 mg/kg). Levels of cholesterol, its precursors, ARI and its metabolites were measured at P0. We found that ARI and its metabolites were transported across the placenta and reached the brain of offspring. Maternal ARI exposure led to decreased viability of embryos and increased 7-DHC levels, regardless of maternal or offspring Dhcr7 genotype. In addition, Dhcr7+/- pups were more vulnerable to maternal ARI exposure than their WT littermates, and maternal Dhcr7+/- genotype also exacerbated offspring response to ARI treatment. Finally, both 7-DHC levels and 7-DHC/cholesterol ratio is the highest in Dhcr7+/- pups from Dhcr7+/- mothers exposed to ARI, underscoring a potentially dangerous interaction between maternal genotype×embryonic genotype×treatment. Our findings have important clinical implications. SLOS patients should avoid drugs that increase 7-DHC levels such as ARI, trazodone and haloperidol. In addition, treatment with 7-DHC elevating substances might be potentially unsafe for the 1-1.5% of population with single-allele disruptions of the DHCR7 gene. Finally, prenatal and parental genetic testing for DHCR7 should be considered before prescribing sterol-interfering medications during pregnancy.
Project description:Smith-Lemli-Opitz syndrome (SLOS) is a metabolic and developmental disorder caused by mutations in the gene encoding the enzyme 7-dehydrocholesterol reductase (Dhcr7). This reductase catalyzes the last step in cholesterol biosynthesis, and levels of 7-dehydrocholesterol (7-DHC), the substrate for this enzyme, are elevated in SLOS patients as a result of this defect. Our group has previously shown that 7-DHC is extremely prone to free radical autoxidation, and we identified about a dozen different oxysterols formed from oxidation of 7-DHC. We report here that 7-DHC-derived oxysterols reduce cell viability in a dose- and time-dependent manner, some of the compounds showing activity at sub-micromolar concentrations. The reduction of cell survival is caused by a combination of reduced proliferation and induced differentiation of the Neuro2a cells. The complex 7-DHC oxysterol mixture added to control Neuro2a cells also triggers the gene expression changes that were previously identified in Dhcr7-deficient Neuro2a cells. Based on the identification of overlapping gene expression changes in Dhcr7-deficient and 7-DHC oxysterol-treated Neuro2a cells, we hypothesize that some of the pathophysiological findings in the mouse SLOS model and SLOS patients might be due to accumulated 7-DHC oxysterols.
Project description:Smith-Lemli-Opitz syndrome is a recessive disorder caused by mutations in 7-dehydrocholesterol reductase (DHCR)7 with a heterozygous (HET) carrier frequency of 1-3%. A defective DHCR7 causes accumulation of 7-dehydrocholesterol (DHC), which is a highly oxidizable and toxic compound. Recent studies suggest that several antipsychotics, including the highly prescribed pharmaceuticals, aripiprazole (ARI) and trazodone (TRZ), increase 7-DHC levels in vitro and in humans. Our investigation was designed to compare the effects of ARI and TRZ on cholesterol (Chol) synthesis in fibroblasts from DHCR7+/- human carriers and controls (CTRs). Six matched pairs of fibroblasts were treated and their sterol profile analyzed by LC-MS. Significantly, upon treatment with ARI and TRZ, the total accumulation of 7-DHC was higher in DHCR7-HET cells than in CTR fibroblasts. The same set of experiments was repeated in the presence of 13C-lanosterol to determine residual Chol synthesis, revealing that ARI and TRZ strongly inhibit de novo Chol biosynthesis. The results suggest that DHCR7 carriers have increased vulnerability to both ARI and TRZ exposure compared with CTRs. Thus, the 1-3% of the population who are DHCR7 carriers may be more likely to sustain deleterious health consequences on exposure to compounds like ARI and TRZ that increase levels of 7-DHC, especially during brain development.
Project description:Well-established cell culture models were combined with new analytical methods to assess the effects of small molecules on the cholesterol biosynthesis pathway. The analytical protocol, which is based on sterol derivation with the dienolphile PTAD, was found to be reliable for the analysis of 7-DHC and desmosterol. The PTAD method was applied to the screening of a small library of pharmacologically active substances, and the effect of compounds on the cholesterol pathway was determined. Of some 727 compounds, over 30 compounds decreased 7-DHC in Dhcr7-deficient Neuro2a cells. The examination of chemical structures of active molecules in the screen grouped the compounds into distinct categories. In addition to statins, our screen found that SERMs, antifungals, and several antipsychotic medications reduced levels of 7-DHC. The activities of selected compounds were verified in human fibroblasts derived from Smith-Lemli-Opitz syndrome (SLOS) patients and linked to specific transformations in the cholesterol biosynthesis pathway.
Project description:Recent studies suggest that 7-dehydrocholesterol (7-DHC)-derived oxysterols play important roles in the pathophysiology of Smith-Lemli-Opitz syndrome (SLOS), a metabolic disorder that is caused by defective 3?-hydroxysterol-?(7)-reductase (DHCR7). Although 14 oxysterols have been identified as the primary products of 7-DHC autoxidation in organic solution, the metabolic fate of these oxysterols in a biological environment has not yet been elucidated. Therefore, we incubated these primary 7-DHC oxysterols in control Neuro2a and control human fibroblast cells and identified metabolites of these oxysterols by HPLC-MS. We also incubated Dhcr7-deficient Neuro2a cells and fibroblasts from SLOS patients with isotopically labeled 7-DHC (d(7)-7-DHC). The observation of matching d(0)- and d(7) peaks in HPLC-MS confirmed the presence of true metabolites of 7-DHC after excluding the possibility of ex vivo oxidation. The metabolites of primary 7-DHC oxysterols were found to contribute to the majority of the metabolic profile of 7-DHC in cells. Furthermore, based on this new data, we identified three new 7-DHC-derived metabolites in the brain of Dhcr7-KO mice. Our studies suggest that 7-DHC peroxidation is a major source of oxysterols observed in cells and in vivo and that the stable metabolites of primary 7-DHC oxysterols can be used as markers of 7-DHC peroxidation in these biological systems.
Project description:A small library of pharmacologically active compounds (the NIH Clinical Collection) was assayed in Neuro2a cells to determine their effect on the last step in the biosynthesis of cholesterol, the transformation of 7-dehydrocholesterol (7-DHC) to cholesterol promoted by 7-dehydrocholesterol reductase, DHCR7. Of some 727 compounds in the NIH Clinical Collection, over 30 compounds significantly increased 7-DHC in Neuro2a cells when assayed at 1 ?M. Active compounds that increased 7-DHC with a Z-score of +3 or greater generally gave rise to modest decreases in desmosterol and increases in lanosterol levels. Among the most active compounds identified in the library were the antipsychotic, antidepressant, and anxiolytic compounds that included perospirone, nefazodone, haloperidol, aripiprazole, trazodone, and buspirone. Fluoxetine and risperidone were also active at 1 ?M, and another 10 compounds in this class of pharmaceuticals were identified in the screen at concentrations of 10 ?M. Increased levels of 7-DHC are associated with Smith-Lemli-Opitz syndrome (SLOS), a human condition that results from a mutation in the gene that encodes DHCR7. The SLOS phenotype includes neurological deficits and congenital malformations, and it is linked to a higher incidence of autism spectrum disorder. The significance of the current study is that it identifies common pharmacological compounds that may induce a biochemical presentation similar to SLOS. Little is known about the side effects of elevated 7-DHC postdevelopmentally, and the elevated 7-DHC that results from exposure to these compounds may also be a confounder in the diagnosis of SLOS.
Project description:Treatment of Sprague-Dawley rats with AY9944, an inhibitor of 3?-hydroxysterol-?(7)-reductase (Dhcr7), leads to elevated levels of 7-dehydrocholesterol (7-DHC) and reduced levels of cholesterol in all biological tissues, mimicking the key biochemical hallmark of Smith-Lemli-Opitz syndrome (SLOS). Fourteen 7-DHC-derived oxysterols previously have been identified as products of free radical oxidation in vitro; one of these oxysterols, 3?,5?-dihydroxycholest-7-en-6-one (DHCEO), was recently identified in Dhcr7-deficient cells and in brain tissues of Dhcr7-null mouse. We report here the isolation and characterization of three novel 7-DHC-derived oxysterols (4?- and 4?-hydroxy-7-DHC and 24-hydroxy-7-DHC) in addition to DHCEO and 7-ketocholesterol (7-kChol) from the brain tissues of AY9944-treated rats. The identities of these five oxysterols were elucidated by HPLC-ultraviolet (UV), HPLC-MS, and 1D- and 2D-NMR. Quantification of 4?- and 4?-hydroxy-7-DHC, DHCEO, and 7-kChol in rat brain, liver, and serum were carried out by HPLC-MS using d(7)-DHCEO as an internal standard. With the exception of 7-kChol, these oxysterols were present only in tissues of AY9944-treated, but not control rats, and 7-kChol levels were markedly (>10-fold) higher in treated versus control rats. These findings are discussed in the context of the potential involvement of 7-DHC-derived oxysterols in the pathogenesis of SLOS.
Project description:Smith-Lemli-Opitz syndrome (SLOS) is a severe autosomal recessive disorder resulting from defects in the cholesterol synthesising enzyme 7-dehydrocholesterol reductase (?7-sterol reductase, DHCR7, EC 220.127.116.11) leading to a build-up of the cholesterol precursor 7-dehydrocholesterol (7-DHC) in tissues and blood plasma. Although the underling enzyme deficiency associated with SLOS is clear there are likely to be multiple mechanisms responsible for SLOS pathology. In an effort to learn more of the aetiology of SLOS we have analysed plasma from SLOS patients to search for metabolites derived from 7-DHC which may be responsible for some of the pathology. We have identified a novel hydroxy-8-dehydrocholesterol, which is either 24- or 25-hydroxy-8-dehydrocholesterol and also the known metabolites 26-hydroxy-8-dehydrocholesterol, 4-hydroxy-7-dehydrocholesterol, 3?,5?-dihydroxycholest-7-en-6-one and 7?,8?-epoxycholesterol. None of these metabolites are detected in control plasma at quantifiable levels (0.5ng/mL).
Project description:7-Dehydrocholesterol (7-DHC) accumulates in tissues and fluids of patients with Smith-Lemli-Opitz syndrome (SLOS), which is caused by mutations in the gene encoding 3?-hydroxysterol-?(7)-reductase (DHCR7). We recently reported that 7-DHC is the most reactive lipid molecule toward free radical oxidation (lipid peroxidation) and 14 oxysterols have been identified as products of oxidation of 7-DHC in solution. As the high oxidizability of 7-DHC may lead to systemic oxidative stress in SLOS patients, we report here lipid biomarkers of oxidative stress in a Dhcr7-KO mouse model of SLOS, including oxysterols, isoprostanes (IsoPs), and neuroprostanes (NeuroPs) that are formed from the oxidation of 7-DHC, arachidonic acid and docosahexaenoic acid, respectively. In addition to a previously described oxysterol, 3?,5?-dihydroxycholest-7-en-6-one (DHCEO), we provide evidence for the chemical structures of three new oxysterols in the brain and/or liver tissue of Dhcr7-KO mice, two of which were quantified. We find that levels of IsoPs and NeuroPs are also elevated in brain and/or liver tissues of Dhcr7-KO mice relative to matching WT mice. While IsoPs and NeuroPs have been established as a reliable measurement of lipid peroxidation and oxidative stress in vivo, we show that in this genetic SLOS mouse model, 7-DHC-derived oxysterols are present at much higher levels than IsoPs and NeuroPs and thus are better markers of lipid oxidation and related oxidative stress.
Project description:Inhaled ground level ozone (O3) has well described adverse health effects, which may be augmented in susceptible populations. While conditions, such as pre-existing respiratory disease, have been identified as factors enhancing susceptibility to O3-induced health effects, the potential for chemical interactions in the lung to sensitize populations to pollutant-induced responses has not yet been studied. In the airways, inhaled O3 reacts with lipids, such as cholesterol, to generate reactive and electrophilic oxysterol species, capable of causing cellular dysfunction and inflammation. The enzyme regulating the final step of cholesterol biosynthesis, 7-dehydrocholesterol reductase (DHCR7), converts 7-dehydrocholesterol (7-DHC) to cholesterol. Inhibition of DHCR7 increases the levels of 7-DHC, which is much more susceptible to oxidation than cholesterol. Chemical analysis established the capacity for a variety of small molecule antipsychotic drugs, like Aripiprazole (APZ), to inhibit DHCR7 and elevate circulating 7-DHC. Our results show that APZ and the known DHCR7 inhibitor, AY9944, increase 7-DHC levels in airway epithelial cells and potentiate O3-induced IL-6 and IL-8 expression and cytokine release. Targeted immune-related gene array analysis demonstrates that APZ significantly modified O3-induced expression of 16 genes, causing dysregulation in expression of genes associated with leukocyte recruitment and inflammatory response. Additionally, we find that APZ increases O3-induced IL-6 and IL-8 expression in human nasal epithelial cells from male but not female donors. Overall, the evidence we provide describes a novel molecular mechanism by which chemicals, such as APZ, that perturb cholesterol biosynthesis affect O3-induced biological responses.