RESUMEN
NSDHL is a 3ß-hydroxysterol dehydrogenase that is involved in the removal of two C-4 methyl groups in one of the later steps of cholesterol biosynthesis. Mutations in the gene encoding the enzyme are responsible for the X-linked, male lethal mouse mutations bare patches and striated, as well as most cases of human CHILD syndrome. Rare, hypomorphic NSDHL mutations are also associated with X-linked intellectual disability in males with CK syndrome. Since hemizygous male mice with Nsdhl mutations die by midgestation, we generated a conditional targeted Nsdhl mutation (Nsdhl(tm1.1Hrm)) to investigate the essential role of cholesterol in the early postnatal CNS. Ablation of Nsdhl in radial glia using GFAP-cre resulted in live-born, normal appearing affected male pups. However, the pups develop overt ataxia by postnatal day 8-10 and die shortly thereafter. Histological abnormalities include progressive loss of cortical and hippocampal neurons, as well as deficits in the proliferation and migration of cerebellar granule precursors and subsequent massive apoptosis of the cerebellar cortex. We replicated the granule cell precursor proliferation defect in vitro and demonstrate that it results from defective signaling by SHH. Furthermore, this defect is almost completely rescued by supplementation of the culture media with exogenous cholesterol, while methylsterol accumulation above the enzymatic block appears to be associated with increased cell death. These data support the absolute requirement for cholesterol synthesis in situ once the blood-brain-barrier forms and cholesterol transport to the fetus is abolished. They further emphasize the complex ramifications of cholesterogenic enzyme deficiency on cellular metabolism.
Asunto(s)
3-Hidroxiesteroide Deshidrogenasas/genética , Corteza Cerebelosa/crecimiento & desarrollo , Colesterol/fisiología , Proteínas Hedgehog/fisiología , Transducción de Señal , Alelos , Animales , Corteza Cerebelosa/fisiopatología , Colesterol/biosíntesis , Femenino , Masculino , Ratones , Ratones Transgénicos , Mutación , Células-Madre Neurales , Neuronas/fisiologíaAsunto(s)
Tendón Calcáneo/efectos de los fármacos , Ácido Quenodesoxicólico/uso terapéutico , Paraplejía/tratamiento farmacológico , Xantomatosis Cerebrotendinosa/tratamiento farmacológico , Tendón Calcáneo/patología , Adulto , Colestanotriol 26-Monooxigenasa/genética , Predisposición Genética a la Enfermedad , Humanos , Imagen por Resonancia Magnética , Masculino , Mutación Missense , Paraplejía/diagnóstico , Paraplejía/genética , Fenotipo , Resultado del Tratamiento , Xantomatosis Cerebrotendinosa/complicaciones , Xantomatosis Cerebrotendinosa/diagnóstico , Xantomatosis Cerebrotendinosa/genéticaRESUMEN
OBJECTIVE: To assess if ezetimibe (EZE), a sterol-absorption inhibitor, improves platelet (PLT) count and size relative to its effect on plasma plant sterol (PS) in patients with sitosterolemia (STSL). STUDY DESIGN: Patients with STSL (5 males, 3 females, 16-56 years of age) receiving EZE intervention as part of their routine care participated in this study. EZE was discontinued for 14 weeks (off) and then resumed for another 14 weeks (on). Hematology variables along with plasma and red blood cells (RBC) PS and total cholesterol (TC) levels were measured at the end of each phase. RESULTS: EZE increased PLT count (23% ± 9%) and decreased mean PLT volume (MPV; 10% ± 3%, all P < .05). In patients off EZE, PLT counts inversely correlated (r = -0.96 and r = -0.91, all P < .01) with plasma and RBC PS to TC ratio (PS/TC), and MPV positively correlated (r = 0.91, P = .03 and r = 0.93, P = .02) with plasma and RBC PS/TC. EZE reduced plasma and RBC sitosterol (-35% ± 4% and -28% ± 3%), total PS (-37% ± 4% and -28% ± 3%, all P < .0001) levels, and PS/TC (-27% ± 4% and -28% ± 4%, P < .01). CONCLUSIONS: EZE reduces plasma and RBC PS levels, while increasing PLT count and decreasing MPV, and thereby may reduce the risk for bleeding in STSL. Plasma PS levels and ABCG5/ABCG8 genes should be analyzed in patients with unexplained hematologic abnormalities.
Asunto(s)
Anticolesterolemiantes/uso terapéutico , Azetidinas/uso terapéutico , Colesterol/sangre , Hipercolesterolemia/tratamiento farmacológico , Enfermedades Intestinales/tratamiento farmacológico , Errores Innatos del Metabolismo Lipídico/tratamiento farmacológico , Fitosteroles/efectos adversos , Fitosteroles/sangre , Recuento de Plaquetas , Adolescente , Adulto , Anticolesterolemiantes/administración & dosificación , Azetidinas/administración & dosificación , Canadá , Recuento de Eritrocitos , Ezetimiba , Femenino , Humanos , Hipercolesterolemia/sangre , Enfermedades Intestinales/sangre , Errores Innatos del Metabolismo Lipídico/sangre , Masculino , Persona de Mediana Edad , Proyectos Piloto , Resultado del Tratamiento , Estados Unidos , Adulto JovenRESUMEN
OBJECTIVE: To quantitatively evaluate feeding impairment in children with Smith-Lemli-Opitz syndrome (SLOS) and to correlate feeding impairment with clinical and biochemical indices of disease severity. STUDY DESIGN: The study subjects were 26 children with SLOS ranging in age from 0.4 to 19 years. Clinical severity was measured using an existing scoring system. We created a tool to quantitatively evaluate feeding. Plasma sterol concentrations were measured, and statistical associations (correlations) with feeding scores were calculated. RESULTS: Oral hyposensitivity or hypersensitivity, adverse behaviors, and risk for dysphagia were seen in â¼65% of the children with SLOS. Thirteen of the 26 children experienced failure to thrive, and 10 children required gastrostomy. Plasma concentration of 7-dehydrocholesterol, as a measure of severity, was correlated with total feeding score and oral function subcategory score (P < .001) and less so with oral structure score, adverse behaviors, or dysphagia. Correlations with cholesterol concentrations were less statistically significant. A plasma 7-dehydrocholesterol concentration >0.24 mmol/L or cholesterol concentration <1.95 mmol/L was predictive of gastrostomy tube use. Feeding impairments may improve with age. CONCLUSION: Feeding impairment is common and complex in patients with SLOS. Our findings confirm that oral sensitivities, adverse feeding behaviors, and risk of oral phase dysphagia are amenable to quantitative evaluation and analysis. Feeding difficulties in children with SLOS are correlated with plasma sterol concentrations, suggesting a link between the biochemical severity of SLOS and feeding function. These findings expand the behavioral phenotype of SLOS and begin to provide insight into the biological causes of feeding difficulties.
Asunto(s)
Trastornos de Deglución/sangre , Conducta Alimentaria , Reflujo Gastroesofágico/sangre , Síndrome de Smith-Lemli-Opitz/sangre , Esteroles/sangre , Adolescente , Niño , Preescolar , Estudios de Cohortes , Trastornos de Deglución/complicaciones , Deshidrocolesteroles/sangre , Nutrición Enteral , Insuficiencia de Crecimiento , Femenino , Reflujo Gastroesofágico/complicaciones , Humanos , Lactante , Masculino , Fenotipo , Síndrome de Smith-Lemli-Opitz/fisiopatología , Adulto JovenRESUMEN
OBJECTIVES: Cerebrotendinous xanthomatosis (CTX) is a rare genetic disorder of bile acid (BA) synthesis that can cause progressive neurological damage and premature death. Blood (normally serum or plasma) testing for CTX is performed by a small number of specialized laboratories, routinely by gas chromatography-mass spectrometry (GC-MS) measurement of elevated 5α-cholestanol. We report here on a more sensitive biochemical approach to test for CTX particularly useful for confirmation of CTX in the case of a challenging diagnostic sample with 5α-cholestanol that, although elevated, was below the cut-off used for diagnosis of CTX (10 µg/mL or 1.0 mg/dL). DESIGN AND METHODS: We have previously described liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) methodology utilizing keto derivatization to enable the sensitive quantification of plasma ketosterol BA precursors that accumulate in CTX. We have expanded this methodology to perform isotope dilution LC-ESI-MS/MS quantification of a panel of plasma ketosterol BA precursors, with internal standards readily generated using isotopically-enriched derivatization reagent. RESULTS: Quantification of plasma ketosterol BA precursors (7α-hydroxy-4-cholesten-3-one, 7α,12α-dihydroxy-4-cholesten-3-one and 7α,12α-dihydroxy-5ß-cholestan-3-one) in a single LC-ESI/MS/MS test provided better discrimination between a CTX-positive and negative samples analyzed (n=20) than measurement of 5α-cholestanol alone. CONCLUSIONS: Quantification of plasma ketosterol BA precursors provides a more sensitive biochemical approach to discriminate between CTX negative and positive samples. A multiplexed LC-ESI-MS/MS test quantifying a panel of plasma ketosterols, with simple sample preparation, rapid analysis time and readily available internal standards, can be performed by most clinical laboratories. Wider availability of testing will benefit those affected with CTX.
Asunto(s)
Xantomatosis Cerebrotendinosa/sangre , Xantomatosis Cerebrotendinosa/diagnóstico , Colestanoles/sangre , Femenino , Humanos , Cetosteroides/sangre , Estándares de Referencia , Espectrometría de Masa por Ionización de Electrospray/normas , Espectrometría de Masas en Tándem/normas , Adulto JovenRESUMEN
Human apolipoprotein E (apoE) exists in three isoforms: apoE2, apoE3 and apoE4. APOE ε4 is a major genetic risk factor for cardiovascular disease (CVD) and Alzheimer's disease (AD). ApoE mediates cholesterol metabolism by binding various receptors. The low-density lipoprotein receptor (LDLR) has a high affinity for apoE, and is the only member of its receptor family to demonstrate an apoE isoform specific binding affinity (E4>E3>>E2). Evidence suggests that a functional interaction between apoE and LDLR influences the risk of CVD and AD. We hypothesize that the differential cognitive effects of the apoE isoforms are a direct result of their varying interactions with LDLR. To test this hypothesis, we have employed transgenic mice that express human apoE2, apoE3, or apoE4, and either human LDLR (hLDLR) or no LDLR (LDLR(-/-)). Our results show that plasma and brain apoE levels, cortical cholesterol, and spatial memory are all regulated by isoform-dependent interactions between apoE and LDLR. Conversely, both anxiety-like behavior and cued associative memory are strongly influenced by APOE genotype, but these processes appear to occur via an LDLR-independent mechanism. Both the lack of LDLR and the interaction between E4 and the LDLR were associated with significant impairments in the retention of long term spatial memory. Finally, levels of hippocampal apoE correlate with long term spatial memory retention in mice with human LDLR. In summary, we demonstrate that the apoE-LDLR interaction affects regional brain apoE levels, brain cholesterol, and cognitive function in an apoE isoform-dependent manner.
Asunto(s)
Apolipoproteínas E/metabolismo , Encéfalo/metabolismo , Memoria a Largo Plazo/fisiología , Receptores de LDL/metabolismo , Percepción Espacial/fisiología , Animales , Ansiedad/metabolismo , Apolipoproteína E2/sangre , Apolipoproteína E2/genética , Apolipoproteína E2/metabolismo , Apolipoproteína E3/sangre , Apolipoproteína E3/genética , Apolipoproteína E3/metabolismo , Apolipoproteína E4/sangre , Apolipoproteína E4/genética , Apolipoproteína E4/metabolismo , Apolipoproteínas E/sangre , Apolipoproteínas E/genética , Colesterol/sangre , Colesterol/metabolismo , Condicionamiento Psicológico/fisiología , Humanos , Memoria/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Receptores de LDL/genéticaRESUMEN
Cerebrotendinous xanthomatosis (CTX) is a rare, difficult-to-diagnose genetic disorder of bile acid (BA) synthesis that can cause progressive neurological damage and premature death. Detection of CTX in the newborn period would be beneficial because an effective oral therapy for CTX is available to prevent disease progression. There is no suitable test to screen newborn dried bloodspots (DBS) for CTX. Blood screening for CTX is currently performed by GC-MS measurement of elevated 5α-cholestanol. We present here LC-ESI/MS/MS methodology utilizing keto derivatization with (O-(3-trimethylammonium-propyl) hydroxylamine) reagent to enable sensitive detection of ketosterol BA precursors that accumulate in CTX. The availability of isotopically enriched derivatization reagent allowed ready tagging of ketosterols to generate internal standards for isotope dilution quantification. Ketosterols were quantified and their utility as markers for CTX was compared with 5α-cholestanol. 7α,12α-Dihydroxy-4-cholesten-3-one provided the best discrimination between CTX and unaffected samples. In two CTX, newborn DBS concentrations of this ketosterol (120-214 ng/ml) were â¼10-fold higher than in unaffected newborn DBS (16.4 ± 6.0 ng/ml), such that quantification of this ketosterol provides a test with potential to screen newborn DBS for CTX. Early detection and intervention through newborn screening would greatly benefit those affected with CTX by preventing morbidity and mortality.
Asunto(s)
Colestenonas/sangre , Xantomatosis Cerebrotendinosa/diagnóstico , Adulto , Calibración , Estudios de Casos y Controles , Pruebas con Sangre Seca , Humanos , Recién Nacido , Tamizaje Neonatal , Estándares de Referencia , Espectrometría de Masa por Ionización de Electrospray/normas , Espectrometría de Masas en Tándem/normas , Xantomatosis Cerebrotendinosa/sangreRESUMEN
OBJECTIVE: To study challenging behavior (destruction, aggression, self-injury, stereotypy) in children with Smith-Lemli-Opitz syndrome (SLOS) using a biobehavioral model that helps distinguish biological from socially mediated variables influencing the behavior. BACKGROUND: SLOS is an autosomal-recessive syndrome of multiple malformations and intellectual disability resulting from a genetic error in cholesterol synthesis in all cells and tissues, including brain. The exact cause of the challenging behavior in SLOS is unclear, but defective brain cholesterol synthesis may contribute. Because the precise genetic and biochemical etiology of SLOS is known, this disorder is a good model for studying biological causes of challenging behavior. METHOD: In a preliminary application of a biobehavioral model, we studied the association between cholesterol levels (as a biochemical indicator of disease severity) and behavior subtype ("biological" vs "learned") in 13 children with SLOS. Parents completed a questionnaire that categorized challenging behavior as influenced primarily by social or nonsocial (thus, presumably biological) factors. RESULTS: The severity of the cholesterol synthesis defect correlated significantly with behavior subtype classification for 1 of 2 challenging behaviors. Greater severity of the cholesterol synthesis defect was associated with behavior being classified as primarily influenced by biological factors. CONCLUSION: The interplay between challenging behavior and defective cholesterol synthesis in SLOS may help explain biological influences on the behavior. Our findings have implications for research on the effectiveness of behavioral and medical treatments for behavioral difficulties in SLOS and other neurodevelopmental disorders.
Asunto(s)
Agresión/psicología , Conducta Infantil/psicología , Discapacidad Intelectual/psicología , Conducta Autodestructiva/psicología , Síndrome de Smith-Lemli-Opitz/psicología , Adolescente , Niño , Preescolar , Colesterol/sangre , Femenino , Humanos , Discapacidad Intelectual/sangre , Masculino , Índice de Severidad de la Enfermedad , Síndrome de Smith-Lemli-Opitz/sangreRESUMEN
Smith-Lemli-Opitz syndrome (SLOS) is caused by mutations in the gene encoding 3ß-hydroxysterol-Δ(7)-reductase and as a result of this defect, 7-dehydrocholesterol (7-DHC) and 8-dehydrocholesterol (8-DHC) accumulate in the fluids and tissues of patients with this syndrome. Both 7- and 8-DHC are susceptible to peroxidation reactions, and several biologically active DHC oxysterols are found in cell and animal models of SLOS. Ex vivo oxidation of DHCs can be a confounding factor in the analysis of these sterols and their esters, and we developed HPLC/MS methods that permit the direct analysis of cholesterol, 7-DHC, 8-DHC, and their esters in human plasma, thus avoiding ex vivo oxidation. In addition, three oxysterols were classified as endogenously formed products by the use of an isotopically-labeled 7-DHC (d(7)-7-DHC) added to the sample before workup, followed by MS analysis of products formed. Analysis of 17 SLOS plasma samples shows that 8-DHC linoleate correlates better with the SLOS severity score of the patients than other sterols or metabolites, including cholesterol and 7-DHC. Levels of 7-ketocholesterol also correlate with the SLOS severity score. 8-DHC esters should have utility as surrogate markers of severity in SLOS for prognostication and as endpoints in clinical trials.
Asunto(s)
Análisis Químico de la Sangre/métodos , Deshidrocolesteroles/sangre , Deshidrocolesteroles/química , Síndrome de Smith-Lemli-Opitz/sangre , Adolescente , Adulto , Recolección de Muestras de Sangre , Niño , Preescolar , Cromatografía Líquida de Alta Presión , Ésteres , Femenino , Humanos , Lactante , Masculino , Oxidación-Reducción , Síndrome de Smith-Lemli-Opitz/diagnóstico , Espectrometría de Masas en Tándem , Adulto JovenRESUMEN
Smith-Lemli-Opitz syndrome (SLOS) is caused by a genetic deficiency in 7-dehydrocholesterol (7-DHC) reductase (EC 1.3.1.21), the last enzyme of the cholesterol synthetic pathway. In SLOS, plasma cholesterol concentration is reduced and immediate precursor concentration (7-DHC) is elevated. Surprisingly, total sterol synthesis is reduced but HMG-CoA reductase activity, a rate-limiting enzyme in cholesterol synthesis is unaltered as judged by normal urinary excretion of mevalonic acid (MVA) (Pappu et al. J Lipid Res 43:1661-1669, 2002). These findings raise the possibility of increased diversion of MVA into the MVA shunt pathway away from sterol synthesis, by activation of the shunt pathway enzymes. To test this hypothesis, we measured the urinary excretion of 3-methylglutaconic acid (U-3MGC), a by-product of the shunt pathway, in 19 mildly to moderately severely affected SLOS subjects (ten males, nine females) receiving either a cholesterol-free or a high cholesterol diet, and in 20 age- and sex-matched controls. U-3MGC was similar in SLOS and controls, and was unaffected by dietary cholesterol intake. Further, no change in U-3MGC was observed in a subset of SLOS subjects (n = 9) receiving simvastatin. In contrast, U-MVA was reduced by cholesterol supplementation (~54%, p < 0.05) and by simvastatin (~50%, p < 0.04). There was no correlation between U-3MGC and either plasma sterol concentrations, urinary isoprenoids, or the subjects' clinical severity score. However U-3MGC was inversely correlated with age (p < 0.04) and body weight (p < 0.02), and higher in females than in males (~65%, p < 0.025). The data show that DHCR7 deficiency does not result in 3MGC accumulation in SLOS and suggest that the MVA shunt pathway is not activated in patients with the condition.
Asunto(s)
Colesterol/sangre , Colesterol/metabolismo , Ácido Mevalónico/metabolismo , Síndrome de Smith-Lemli-Opitz/metabolismo , Niño , Colesterol en la Dieta/metabolismo , Deshidrocolesteroles/sangre , Deshidrocolesteroles/metabolismo , Dieta Alta en Grasa , Suplementos Dietéticos , Femenino , Glutaratos/metabolismo , Glutaratos/orina , Humanos , Hidroximetilglutaril-CoA Reductasas/metabolismo , Masculino , Ácido Mevalónico/orina , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/metabolismo , Simvastatina/farmacología , Síndrome de Smith-Lemli-Opitz/sangre , Síndrome de Smith-Lemli-Opitz/orina , Terpenos/metabolismo , Terpenos/orinaAsunto(s)
Catarata/diagnóstico , Xantomatosis Cerebrotendinosa/diagnóstico , Catarata/sangre , Catarata/terapia , Extracción de Catarata , Ácido Quenodesoxicólico/uso terapéutico , Niño , Colestanol/sangre , Diarrea/tratamiento farmacológico , Diarrea/etiología , Femenino , Humanos , Implantación de Lentes Intraoculares , Masculino , Agudeza Visual/fisiología , Xantomatosis Cerebrotendinosa/sangre , Xantomatosis Cerebrotendinosa/tratamiento farmacológicoRESUMEN
Smith-Lemli-Opitz syndrome (SLOS) is an autosomal recessive, multiple congenital malformation and intellectual disability syndrome, with clinical characteristics that encompass a wide spectrum and great variability. Elucidation of the biochemical and genetic basis for SLOS, specifically understanding SLOS as a cholesterol deficiency syndrome caused by mutation in DHCR7, opened up enormous possibilities for therapeutic intervention. When cholesterol was discovered to be the activator of sonic hedgehog, cholesterol deficiency with inactivation of this developmental patterning gene was thought to be the cause of SLOS malformations, yet this explanation is overly simplistic. Despite these important research breakthroughs, there is no proven treatment for SLOS. Better animal models are needed to allow potential treatment testing and the study of disease pathophysiology, which is incompletely understood. Creation of human cellular models, especially models of brain cells, would be useful, and in vivo human studies are also essential. Biomarker development will be crucial in facilitating clinical trials in this rare condition, because the clinical phenotype can change over many years. Additional research in these and other areas is critical if we are to make headway towards ameliorating the effects of this devastating condition.
Asunto(s)
Colesterol/metabolismo , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/deficiencia , Síndrome de Smith-Lemli-Opitz , Animales , Deshidrocolesteroles/metabolismo , Humanos , Ratones , Mutación , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/genética , Ratas , Síndrome de Smith-Lemli-Opitz/genética , Síndrome de Smith-Lemli-Opitz/fisiopatología , Síndrome de Smith-Lemli-Opitz/terapiaRESUMEN
In this study we profile free 3-oxo sterols present in plasma from patients affected with the neurodegenerative disorder of sterol and bile acid metabolism cerebrotendinous xanthomatosis (CTX), utilizing a combination of charge-tagging and LC-ESI-MS(n) performed with an LTQ-Orbitrap Discovery instrument. In addition, we profile sterols in plasma from 24-month-old cyp27A1 gene knockout mice lacking the enzyme defective in CTX. Charge-tagging was accomplished by reaction with cationic Girard's P (GP) reagent 1-(carboxymethyl) pyridinium chloride hydrazide, an approach uniquely suited to studying the 3-oxo sterols that accumulate in CTX, as Girard's reagent reacts with the sterol oxo moiety to form charged hydrazone derivatives. The ability to selectively generate GP-tagged 3-oxo-4-ene and 3-oxo-5(H) saturated plasma sterols enabled ESI-MS(n) analysis of these sterols in the presence of a large excess (3 orders of magnitude) of cholesterol. Often cholesterol detected in biological samples makes it challenging to quantify minor sterols, with cholesterol frequently removed prior to analysis. We derivatized plasma (10 µl) without SPE removal of cholesterol to ensure detection of all sterols present in plasma. We were able to measure 4-cholesten-3-one in plasma from untreated CTX patients (1207±302 ng/ml, mean±SD, n=4), as well as other intermediates in a proposed pathway to 5α-cholestanol. In addition, a number of bile acid precursors were identified in plasma using this technique. GP-tagged sterols were identified utilizing high resolution exact mass spectra (±5 ppm), as well as MS(2) ([M](+)â) spectra that possessed characteristic neutral loss of 79Da (pyridine) fragment ions, and MS(3) ([M](+)â[M-79](+)â) spectra that provided additional structurally informative fragment ions.
Asunto(s)
Espectrometría de Masas/métodos , Esteroles/sangre , Xantomatosis Cerebrotendinosa/sangre , Animales , Colestanotriol 26-Monooxigenasa/genética , Cromatografía Liquida/métodos , Femenino , Humanos , Masculino , Ratones , Ratones NoqueadosRESUMEN
CK syndrome (CKS) is an X-linked recessive intellectual disability syndrome characterized by dysmorphism, cortical brain malformations, and an asthenic build. Through an X chromosome single-nucleotide variant scan in the first reported family, we identified linkage to a 5 Mb region on Xq28. Sequencing of this region detected a segregating 3 bp deletion (c.696_698del [p.Lys232del]) in exon 7 of NAD(P) dependent steroid dehydrogenase-like (NSDHL), a gene that encodes an enzyme in the cholesterol biosynthesis pathway. We also found that males with intellectual disability in another reported family with an NSDHL mutation (c.1098 dup [p.Arg367SerfsX33]) have CKS. These two mutations, which alter protein folding, show temperature-sensitive protein stability and complementation in Erg26-deficient yeast. As described for the allelic disorder CHILD syndrome, cells and cerebrospinal fluid from CKS patients have increased methyl sterol levels. We hypothesize that methyl sterol accumulation, not only cholesterol deficiency, causes CKS, given that cerebrospinal fluid cholesterol, plasma cholesterol, and plasma 24S-hydroxycholesterol levels are normal in males with CKS. In summary, CKS expands the spectrum of cholesterol-related disorders and insight into the role of cholesterol in human development.
Asunto(s)
3-Hidroxiesteroide Deshidrogenasas/genética , Anomalías Múltiples/genética , Alelos , Enfermedades Genéticas Ligadas al Cromosoma X/genética , Temperatura , Adolescente , Adulto , Secuencia de Aminoácidos , Animales , Exones , Femenino , Humanos , Masculino , Datos de Secuencia Molecular , Mutación , Linaje , Homología de Secuencia de Aminoácido , Adulto JovenRESUMEN
Cholesterol is esterified in mammals by two enzymes: LCAT (lecithin cholesterol acyltransferase) in plasma and ACAT(1) and ACAT(2) (acyl-CoA cholesterol acyltransferases) in the tissues. We hypothesized that the sterol structure may have significant effects on the outcome of esterification by these enzymes. To test this hypothesis, we analyzed sterol esters in plasma and tissues in patients having non-cholesterol sterols (sitosterolemia and Smith-Lemli-Opitz syndrome). The esterification of a given sterol was defined as the sterol ester percentage of total sterols. The esterification of cholesterol in plasma by LCAT was 67% and in tissues by ACAT was 64%. Esterification of nine sterols (cholesterol, cholestanol, campesterol, stigmasterol, sitosterol, campestanol, sitostanol, 7-dehydrocholesterol and 8-dehydrocholesterol) was examined. The relative esterification (cholesterol being 1.0) of these sterols by the plasma LCAT was 1.00, 0.95, 0.89, 0.40, 0.85, 0.82 and 0.80, 0.69 and 0.82, respectively. The esterification by the tissue ACAT was 1.00, 1.29, 0.75, 0.49, 0.45, 1.21 and 0.74, respectively. The predominant fatty acid of the sterol esters was linoleic acid for LCAT and oleic acid for ACAT. We compared the esterification of two sterols differing by only one functional group (a chemical group attached to sterol nucleus) and were able to quantify the effects of individual functional groups on sterol esterification. The saturation of the A ring of cholesterol increased ester formation by ACAT by 29% and decreased the esterification by LCAT by 5.9%. Esterification by ACAT and LCAT was reduced, respectively, by 25 and 11% by the presence of an additional methyl group on the side chain of cholesterol at the C-24 position. This data supports our hypothesis that the structure of the sterol substrate has a significant effect on its esterification by ACAT or LCAT.
Asunto(s)
Trastornos del Metabolismo de los Lípidos/metabolismo , Fosfatidilcolina-Esterol O-Aciltransferasa/fisiología , Sitoesteroles/metabolismo , Síndrome de Smith-Lemli-Opitz/metabolismo , Esterol O-Aciltransferasa/fisiología , Esteroles/metabolismo , Adolescente , Adulto , Niño , Preescolar , Esterificación , Femenino , Humanos , Lactante , Recién Nacido , Masculino , Adulto Joven , Esterol O-Aciltransferasa 2RESUMEN
BACKGROUND: The genetic disorder cerebrotendinous xanthomatosis (CTX) frequently remains undiagnosed for many years. Left untreated CTX is associated with the development of cataracts, xanthomas and severe neurological dysfunction. The method routinely used to screen for CTX is GC-based measurement of elevated 5alpha-cholestanol from hydrolyzed plasma. A plasma test for CTX utilizing ESI-MS/MS methodology would be beneficial. METHODS: Development of rapid, simple LC-ESI-MS/MS methodology to test plasma for CTX is described. Two hour Girard derivatization allowed for 7alpha-hydroxy-4-cholesten-3-one quantification by isotope dilution LC-ESI-MS/MS within 12 min from un-hydrolyzed affected patient plasma (5 microl). RESULTS: Adequate sensitivity and reproducibility were achieved for quantification of 7alpha-hydroxy-4-cholesten-3-one, which demonstrated improved utility as a diagnostic marker of disease and to monitor treatment compared to 5alpha-cholestanol. The mean plasma concentration of 7alpha-hydroxy-4-cholesten-3-one in untreated CTX-affected patients (n=6) was 107-fold that in unaffected subjects (n=9), with the lowest concentration in affected patients >10-fold the highest concentration in unaffected subjects. CONCLUSION: Quantification of the bile acid precursor 7alpha-hydroxy-4-cholesten-3-one with LC-ESI-MS/MS is a novel approach to improved diagnostic testing of plasma for CTX, amenable to high-throughput analysis and automated sample handling. Development of ESI-MS/MS methodology should make CTX testing more widely available and facilitate easier diagnosis of CTX.
Asunto(s)
Colestenonas/sangre , Espectrometría de Masa por Ionización de Electrospray/métodos , Espectrometría de Masas en Tándem/métodos , Xantomatosis Cerebrotendinosa/diagnóstico , Cromatografía Liquida , Humanos , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Xantomatosis Cerebrotendinosa/sangreRESUMEN
In June 2007, the Smith-Lemli-Opitz/RSH Foundation held a scientific conference hosted jointly by Dr. Robert Steiner from Oregon Health & Science University and Dr. Forbes D. Porter from The Eunice Kennedy Shriver National Institute for Child Health and Human Development, National Institutes of Health. The main goal of this meeting was to promote interaction between scientists with expertise in cholesterol homeostasis, brain cholesterol metabolism, developmental biology, and oxysterol and neurosteroid biochemistry, clinicians researching and treating patients with Smith-Lemli-Opitz syndrome, the patient support organization and families. This report summarizes the presentations and discussions at the conference, represents the conference proceedings, and is intended to foster collaborative research and ultimately improve understanding and treatment of Smith-Lemli-Opitz syndrome and other inborn errors of cholesterol synthesis.
Asunto(s)
Encéfalo/metabolismo , Colesterol/biosíntesis , Síndrome de Smith-Lemli-Opitz/diagnóstico , Síndrome de Smith-Lemli-Opitz/patología , Terapia Genética/métodos , Humanos , National Institutes of Health (U.S.) , Síndrome de Smith-Lemli-Opitz/terapia , Estados UnidosRESUMEN
Deficient cholesterol and/or excessive 7-dehydrocholesterol (7-DHC) may be responsible for the pathology of Smith-Lemli-Opitz syndrome (SLOS). Both high-cholesterol diets given to ameliorate cholesterol deficiency while decreasing 7-DHC and cholesterol-enriched diets plus simvastatin to further decrease sterol synthesis have been used as potential therapies. However, the effect of dietary cholesterol and simvastatin on cholesterol synthesis in SLOS has not been reported. Twelve subjects with SLOS enrolled in the study: Nine had received a high cholesterol diet (HI) for 3 y and three were studied after 4 wk on a low cholesterol diet (LO). Cholesterol fractional synthesis rate (FSR) was measured after oral administration of deuterium oxide, using gas chromatography isotope ratio mass spectrometry. FSR was lower in HI compared with LO (HI: 1.46 +/- 0.62%/d; LO: 4.77 +/- 0.95%/d; p < 0.001). Three HI subjects were retested after 0.8 y taking simvastatin (HI + ST). Simvastatin tended to reduce FSR and significantly decreased (p < 0.01) plasma 7-DHC compared with cholesterol supplementation alone. The study demonstrates the utility of the deuterium incorporation method to understand the effect of therapeutic interventions in SLOS. The data suggest that dietary cholesterol supplementation reduces cholesterol synthesis in SLOS and further support the rationale for the combined treatment of SLOS with a cholesterol-enriched diet and simvastatin.
Asunto(s)
Colesterol en la Dieta/metabolismo , Colesterol/biosíntesis , Simvastatina/uso terapéutico , Síndrome de Smith-Lemli-Opitz/metabolismo , Adolescente , Anticolesterolemiantes , Niño , Preescolar , Colesterol en la Dieta/administración & dosificación , Deshidrocolesteroles/metabolismo , Suplementos Dietéticos , Femenino , Humanos , Lactante , Masculino , Síndrome de Smith-Lemli-Opitz/dietoterapia , Síndrome de Smith-Lemli-Opitz/tratamiento farmacológicoRESUMEN
OBJECTIVE: To test the hypothesis that there is a correlation between the ratio of plant sterols to cholesterol in plasma and dietary cholesterol absorption in children with Smith-Lemli-Opitz syndrome (SLOS), a cholesterol synthesis disorder. STUDY DESIGN: We obtained measurements of cholesterol absorption with a direct radioisotope cholesterol absorption method during 9 visits of children with SLOS. We measured plasma sterols in 22 children with SLOS and 16 control children, and we measured dietary intake of cholesterol and sitosterol (n=11 SLOS). RESULTS: The correlations of 2 plasma plant sterol ratios (sitosterol/cholesterol and campesterol/cholesterol) with direct cholesterol absorption measurement were poor (R= -0.33 and R= -0.25, respectively), significantly lower than the published correlation in adults (R=0.73; P< .02). CONCLUSIONS: Although the ratios of plant sterols to cholesterol in plasma has been used as a surrogate for cholesterol absorption in adults and children, these ratios may not accurately reflect cholesterol absorption in children with SLOS. These ratios should not be used as a surrogate for cholesterol absorption in children without further validation.
Asunto(s)
Colesterol en la Dieta/metabolismo , Absorción Intestinal , Fitosteroles/sangre , Síndrome de Smith-Lemli-Opitz/sangre , Síndrome de Smith-Lemli-Opitz/dietoterapia , Adolescente , Adulto , Biomarcadores/sangre , Estudios de Casos y Controles , Niño , Preescolar , Colesterol en la Dieta/sangre , Femenino , Humanos , Lactante , Masculino , Sensibilidad y Especificidad , Sitoesteroles/sangreRESUMEN
Previous studies from this laboratory have shown that maternal-derived cholesterol can be effluxed from trophoblasts to fetal HDL and plasma. We had the opportunity to study for the first time the ability of HDL and plasma from a fetus with the Smith-Lemli-Opitz syndrome (SLOS) to efflux cholesterol from trophoblasts. It was unclear whether cholesterol could be effluxed to fetuses with SLOS since lipoprotein levels are often very low. To answer this question, cord blood was collected from the placentas of an SLOS fetus and unaffected fetuses just after delivery. Plasma cholesterol concentrations were very low in the affected fetus; cholesterol, 7-dehydrocholesterol, and 8-dehydocholesterol concentrations were 14.1, 4.5, and 5.2 mg/dl, respectively. The HDL from the fetal SLOS effluxed approximately 50% more cholesterol from a trophoblast cell line, were smaller in size, and had a lower cholesterol to phospholipid ratio as compared to HDL from unaffected fetuses or adults. Plasma from the SLOS fetus effluxed cholesterol to a similar percentage as unaffected fetal plasma or adult plasma, possibly due to fewer HDL particles as demonstrated in previous SLOS patients. These novel data demonstrate that the cholesterol-deficient SLOS fetus is able to obtain cholesterol from trophoblasts at a time when cholesterol is playing a critical role in development, and has implications for design of treatments for cholesterol deficiency syndromes as well as understanding of prenatal cholesterol transport in humans.