RESUMEN
Data on vitamin D status of patients with inherited ichthyosis in Europe is scarce and unspecific concerning the genetic subtype. This study determined serum levels of 25-hydroxyvitamin D3 (25(OH)D3) in 87 patients with ichthyosis; 69 patients were additionally analysed for parathyroid hormone. Vitamin D deficiency was pronounced in keratinopathic ichthyosis (n = 17; median 25(OH)D3: 10.5 ng/ml), harlequin ichthyosis (n = 2;7.0 ng/ml) and rare syndromic subtypes (n = 3; 7.0 ng/ml). Vitamin D levels were reduced in TG1-proficient lamellar ichthyosis (n = 15; 8.9 ng/ml), TG1-deficient lamellar ichthyosis (n = 12; 11.7 ng/ml), congenital ichthyosiform erythroderma (n = 13; 12.4 ng/ml), Netherton syndrome (n = 7; 10.7 ng/ml) and X-linked ichthyosis (n = 8; 13.9 ng/ml). In ichthyosis vulgaris 25(OH)D3 levels were higher (n = 10; 19.7 ng/ml). Parathyroid hormone was elevated in 12 patients. Low 25(OH)D3 levels were associated with high severity of scaling (p = 0.03) implicating scaling as a risk factor for vitamin D deficiency. Thus, this study supports our recent guidelines for ichthyoses, which recommend screening for and substituting of vitamin D deficiency.
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Ictiosis Lamelar , Ictiosis , Deficiencia de Vitamina D , Humanos , Ictiosis/diagnóstico , Ictiosis/genética , Ictiosis Lamelar/diagnóstico , Ictiosis Lamelar/genética , Hormona Paratiroidea , Vitamina D , Deficiencia de Vitamina D/diagnóstico , Deficiencia de Vitamina D/epidemiología , Deficiencia de Vitamina D/genéticaRESUMEN
Introduction: An increase of serum dehydroepiandrosterone (DHEA) sulfate (DHEAS) is observed in premature adrenarche and congenital adrenal hyperplasia. Very high DHEAS levels are typical for adrenal tumors. Approximately 74% of DHEAS is hydrolyzed to DHEA by the steroid sulfatase (STS). The reverse reaction is DHEA sulfation. Besides these two enzyme reactions, the DHEAS transported through the cell membrane is important for its distribution and excretion. Case Presentation: We present a female adolescent with overweight and a very high DHEAS. The presence of a DHEAS-producing tumor was rejected using ultrasonography, Magnetic Resonance Tomography (MRT), and dexamethasone suppression. STS deficiency was suspected. Sequence analysis revealed a heterozygous nonsense mutation which predicts a truncation of the carboxyl region of the STS that is implicated in substrate binding. No partial gene deletion outside exon 5 was detected by multiplex ligation-dependent probe amplification. The bioassay revealed normal enzyme activity in the patient's leukocytes. A defect of transporter proteins was suggested. Both efflux [multidrug-resistance protein (MRP)2 and breast cancer-resistance protein (BCRP)] and uptake [organic anion-transporting polypeptide (OATP) and organic anion transporter (OAT) carriers] transporters were studied. Sequence analysis of exons revealed a heterozygous Q141K variant for BCRP. Conclusions: A novel heterozygous nonsense mutation in the STS gene and a known heterozygous missense variant in the BCRP gene were found. The heterozygous nonsense mutation in the STS gene is not supposed to be responsible for STS deficiency. The BCRP variant is associated with reduced efflux transport activity only in its homozygous state. The combination of the two heterozygous mutations could possibly explain the observed high levels of DHEAS and other sulfated steroids.
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Codón sin Sentido , Sulfato de Deshidroepiandrosterona/sangre , Obesidad Infantil/patología , Esteril-Sulfatasa/genética , Adolescente , Estudios de Casos y Controles , Femenino , Humanos , Proteína 2 Asociada a Resistencia a Múltiples Medicamentos , Obesidad Infantil/sangre , Obesidad Infantil/genética , Pronóstico , Adulto JovenRESUMEN
The composition of follicular fluid (FF) has an impact on the developmental capacity of the oocyte and the resulting embryo. FF is composed of blood plasma constituents which cross the blood follicular barrier and the secretory components of granulosa and theca cells. Moreover, it has been shown recently that follicular cells have the ability to synthesize bile acids (BAs). BAs are present in several fluids of mammals especially in bile, blood and urine. FF is an essential impacting factor on the oocyte quality and therefore resulting embryos. To achieve a better understanding of this subject, the presence and concentration of BAs were measured in fluid collected from bovine follicles, categorized according to their size, throughout two entire oestrus cycles and compared to those in blood and urine. The body fluids were collected during the same examination procedure and in total samples from four heifers were obtained. A broad spectrum of 11 BA derivatives was measured applying liquid chromatography-tandem mass spectrometry (LC-MS/MS). The simultaneous and direct quantification of BAs in different body fluids of cattle are reported. Within the follicular fluid, blood and urine, cholic acid and glycocholic acid are the dominant BA subspecies irrespective of the oestrus cycle stage. Moreover, BA concentrations in blood compared to those in the FF were similar. For the first time these results clearly highlight the presence of different BA subspecies in FF, blood and urine during the oestrus cycle in cattle.
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Ácidos y Sales Biliares/análisis , Bovinos/fisiología , Estro/fisiología , Líquido Folicular/química , Animales , Ácidos y Sales Biliares/sangre , Ácidos y Sales Biliares/orina , Análisis Químico de la Sangre/veterinaria , Bovinos/sangre , Bovinos/orina , Estro/sangre , Estro/orina , FemeninoRESUMEN
Steroid hormones are regulators in the fine-tuned process of follicular development. During final maturation in vivo a switch from oestradiol (E2) to progesterone (P4) dominance within the follicle is well-described. This change is accompanied by the resumption of meiosis and results in the maturation of the oocyte. It also suggests the important role of these hormones. However, present in vitro maturation (IVM) systems do not completely mimic the in vivo situation, resulting in oocytes of reduced quality. Aim of the study was to determine the temporal pattern of steroid hormone concentrations in the IVM medium of bovine cumulus-oocyte-complexes (COC) at defined time points. The influence of different gonadotropin supplementations during IVM on oocyte maturation, as well as the molecular quality of the oocytes and their corresponding cumulus cells was investigated. COCs were obtained from abattoir-derived ovaries and matured in medium added with different compounds of gonadotropins (eCG/hCG; FSH/LH, each at 0.05 IU or 0.01 IU; only FSH; without gonadotropins) employing a standard protocol without oil overlay. In experiment 1, medium, oocytes and cumulus cells were collected at different time points (0â¯h [control], 4â¯h, 8â¯h, 12â¯h, 16â¯h, 20â¯h, 24â¯h) after IVM in just eCG/hCG-supplemented medium. In experiment 2, medium, oocytes and cumulus cells were collected at 0â¯h (control) and after 24â¯h of IVM with all above-named supplements. The E2 concentration remained similar during IVM whereas P4 concentration increased during experiment 1. No significant changes could be determined after the addition of different gonadotropins (experiment 2). These results suggest that during IVM the temporal pattern of E2 and P4 did not correspond with the pattern during final maturation in vivo. RT-qPCR was used to assess the relative abundance of developmentally important genes in oocytes (BMP15; GDF9; ZAR1; PGR; PGRMC1/2; G6PD; StAR; ESR1/2; SULT1E1; STS; SOAT) and cumulus cells (ESR1/2; FSHR; LHCGR; CYP19A1; HSD3B1; PGR; PGRMC1/2; SULT1E1; STS; SOAT) at all collection points in both experiments. Most transcripts follow a time-regulated mRNA expression pattern during the entire in vitro maturation period. In addition, the expression of the analyzed transcripts was not influenced by the different gonadotropin supplementations during the IVM period. In all, this underlines that present conditions of IVM do not reflect the in vivo situation and require further optimisation.
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Bovinos , Células del Cúmulo/enzimología , Gonadotropinas/farmacología , Oocitos/enzimología , Animales , Estradiol/metabolismo , Femenino , Perfilación de la Expresión Génica/veterinaria , Hormonas Esteroides Gonadales/metabolismo , Técnicas de Maduración In Vitro de los Oocitos/veterinaria , Oocitos/crecimiento & desarrollo , Progesterona/metabolismoRESUMEN
Objective Little information is available on the steroid sulfates profile in obese children. Therefore, we examined whether sulfated steroids are linked with weight status and associated comorbidities in obese children. Methods We analyzed 66 obese children (mean age 10.5 ± 2.5 years, 57.6% female, 53.9% prepubertal, mean BMI 27.0 ± 4.6 kg/m2, 50% with BMI-SDS reduction >0.5, 50% without BMI-SDS reduction) who participated in an outpatient 1-year intervention program based on exercise, behavior and nutrition therapy. We measured intact sulfated steroids (cholesterol sulfate (CS), pregnenolone sulfate (PregS), 17αOH pregnenolone sulfate (17OH-PregS), 16αOH dehydroepiandrosterone sulfate (16OH-DHEAS), DHEAS, androstenediol-3-sulfate, androsterone sulfate and epiandrosterone sulfate) by LC-MS/MS, and insulin resistance index HOMA, lipids, blood pressure at baseline and 1 year later. Results All sulfated steroids except 17OH-PregS, 16OH-DHEAS, androsterone sulfate and epiandrosterone sulfate were higher in boys compared to girls. Concentrations of CS before intervention were higher in children who lost weight. After 1 year of treatment, both groups showed increased levels of DHEAS, 16OH-DHEAS and androstenediol-3-sulfate, but PregS was only increased in children with weight loss. None of the steroid sulfates was significantly related to cardiovascular risk factors or HOMA except 17OH-PregS, which was associated with systolic blood pressure both in cross-sectional (ß-coefficient: 0.09 ± 0.07, P = 0.020) and longitudinal analyses (ß-coefficient: 0.06 ± 0.04, P = 0.013) in multiple linear regression analyses. Conclusions Since higher steroid sulfation capacity was associated with successful weight intervention in children disruption of sulfation may be associated with difficulties to lose weight. Future studies are necessary to prove this hypothesis.
RESUMEN
Estrogens play a pivotal role in the development and proliferation of hormone-dependent breast cancer. Apart from free estrogens, which can directly activate the estrogen receptor (ER) of tumor cells, sulfo-conjugated steroids, which maintain high plasma concentrations even after menopause, first have to be imported into tumor cells by carrier-mediated uptake and then can be cleaved by the steroid sulfatase to finally activate ERs and cell proliferation. In the present study, expression of the sodium-dependent organic anion transporter SOAT was analyzed in breast cancer and its role for hormone-dependent proliferation of T47D breast cancer cells was elucidated. The SOAT protein was localized to the ductal epithelium of the mammary gland by immunohistochemistry. SOAT showed high expression in different pathologies of the breast with a clear ductal localization, including ductal hyperplasia, intraductal papilloma, and intraductal carcinoma. In a larger breast cancer cDNA array, SOAT mRNA expression was high in almost all adenocarcinoma specimen, but expression did not correlate with either the ER, progesterone receptor, or human epidermal growth factor receptor 2 status. Furthermore, SOAT expression did not correlate with tumor stage or grade, indicating widespread SOAT expression in breast cancer. To analyze the role of SOAT for breast cancer cell proliferation, T47D cells were stably transfected with SOAT and incubated under increasing concentrations of estrone-3-sulfate (E1S) and estradiol at physiologically relevant concentrations. Cell proliferation was significantly increased by 10-9 M estradiol as well as by E1S with EC50 of 2.2 nM. In contrast, T47D control cells showed 10-fold lower sensitivity to E1S stimulation with EC50 of 21.7 nM. The E1S-stimulated proliferation of SOAT-T47D cells was blocked by the SOAT inhibitor 4-sulfooxymethylpyrene. IN CONCLUSION: The present study clearly demonstrates expression of SOAT in breast cancer tissue with ductal localization. SOAT inhibition can block the E1S-stimulated proliferation of T47D breast cancer cells, demonstrating that SOAT is an interesting novel drug target from the group of E1S uptake carriers for anti-proliferative breast cancer therapy.
RESUMEN
The prenatal environment shapes the offspring's phenotype; moreover, transgenerational stress and stress during pregnancy may play a role. Brain-derived neurotrophic factor (BDNF) and glucocorticoids influence neurodevelopment during pregnancy, and there is evidence that BDNF in amniotic fluid is mainly of fetal origin, while the source of glucocorticoids is maternal. We tested the hypothesis that maternal early life stress, psychiatric diagnoses, anxiety, perceived stress, and socioeconomic status influence BDNF and glucocorticoid concentrations in amniotic fluid in the second trimester. We studied 79 pregnant women who underwent amniocentesis in the early second trimester and analyzed BDNF, cortisol, and cortisone concentrations in amniotic fluid. The endocrine data were related to maternal early life adversities (Childhood Trauma Questionaire), perceived stress (Perceived Stress Scale), anxiety, socioeconomic status (family income), and the presence of psychiatric diseases. We found BDNF in amniotic fluid to be positively related to maternal early adversity (Childhood Trauma Questionaire). Low family income (socioeconomic status) was related to high amniotic fluid glucocorticoid concentrations. Neither glucocorticoid concentrations nor hydroxy steroid dehydrogenase (HSD2) activity could be related to BDNF concentrations in amniotic fluid. Early maternal adverse events may be reflected in the fetal BDNF regulation, and it should be tested whether this relates to differences in neurodevelopment.
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Líquido Amniótico/química , Factor Neurotrófico Derivado del Encéfalo/análisis , Cortisona/análisis , Hidrocortisona/análisis , Segundo Trimestre del Embarazo/metabolismo , Estrés Psicológico/metabolismo , Adulto , Amniocentesis , Femenino , Humanos , Persona de Mediana Edad , Embarazo , Clase Social , Factores Socioeconómicos , Estrés Psicológico/psicología , Adulto JovenRESUMEN
The sodium-dependent organic anion transporter SOAT (gene name SLC10A6 in man and Slc10a6 in mice) is a plasma membrane transporter for sulfated steroids, which is highly expressed in germ cells of the testis. SOAT can transport biologically inactive sulfated steroids into specific target cells, where they can be reactivated by the steroid sulfatase (STS) to biologically active, unconjugated steroids known to regulate spermatogenesis. Significantly reduced SOAT mRNA expression was previously found in different forms of impaired spermatogenesis in man. It was supposed that SOAT plays a role for the local supply of steroids in the testis and consequently for spermatogenesis and fertility. Thus, an Slc10a6-/- Soat knockout mouse model was established by recombination-based target deletion of the Slc10a6 gene to elucidate the role of Soat in reproduction. However, the Slc10a6-/- knockout mice were fertile, produced normal litter sizes, and had normal spermatogenesis and sperm vitality. This phenotype suggests that the loss of Soat can be compensated in the knockout mice or that Soat function is not essential for reproduction. In addition to reproductive phenotyping, a comprehensive targeted steroid analysis including a set of 9 un-conjugated and 12 sulfo-conjugated steroids was performed in serum of Slc10a6-/- knockout and Slc10a6+/+ wildtype mice. Only cholesterol sulfate, corticosterone, and testosterone (only in the males) could be detected in considerable amounts. Interestingly, male Slc10a6-/- knockout mice showed significantly higher serum levels for cholesterol sulfate compared to their wildtype controls. As cholesterol sulfate has a broader impact apart from the testis, further analysis of this phenotype will include other organs such as skin and lung, which also show high Soat expression in the mouse.
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Ésteres del Colesterol/sangre , Fertilidad/fisiología , Transportadores de Anión Orgánico/genética , Espermatogénesis/genética , Animales , Ésteres del Colesterol/genética , Femenino , Fertilidad/genética , Tamaño de la Camada , Masculino , Ratones Noqueados , Transportadores de Anión Orgánico/metabolismo , Espermatogénesis/fisiología , Esteroides/sangre , Esteroides/metabolismo , Testículo/fisiologíaAsunto(s)
Ictiosis Ligada al Cromosoma X/genética , Proteínas de Filamentos Intermediarios/genética , Mutación , Adolescente , Adulto , Anciano , Niño , Preescolar , Proteínas Filagrina , Humanos , Proteínas de Filamentos Intermediarios/deficiencia , Persona de Mediana Edad , Prevalencia , Estudios Prospectivos , Adulto JovenRESUMEN
Historically sulfonated steroids were primarily considered as inactive metabolites destined for elimination. However, more recently they have been increasingly recognized as precursors for the production of bioactive steroids in target tissues and as functional molecules without preceding hydrolysis. In order to comprehensively characterize their occurrence in cyclic cows and their formation and hydrolysis in bovine ovarian steroidogenesis, ovaries from cyclic cows were screened for the expression of oestrogen sulfotransferase (SULTE1) and steroid sulfatase (STS) by Western blot and immunohistochemistry. Moreover, a broad spectrum of 13 sulfonated steroids was measured applying liquid chromatography-tandem mass spectrometry (LC-MS/MS) in blood samples collected from three cycling heifers during defined stages of the ovarian cycle and in fluid obtained from ovarian follicles of different size. SULT1E1 was undetectable in ovarian tissues. For STS only a weak immunostaining was found predominantly in granulosa cells of larger follicles. However, no specific band occurred in Western blot. In blood, concentrations of all sulfonated steroids investigated were below the limit of quantification (LOQ). In follicular fluid, only cholesterol sulfate was measured in considerable concentrations (328.3⯱â¯63.8â¯ng/ml). However, the role of cholesterol sulfate in bovine follicular steroidogenesis remains unclear as concentrations were obviously unrelated to follicular size. The remaining sulfonated steroids investigated were undetectable or only slightly exceeded LOQ in a minor proportion of samples. The results are clearly contrary to a role of sulfonated steroids as important precursors, intermediates or products of bovine ovarian steroidogenesis.
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Ovario/metabolismo , Esteroides/sangre , Esteril-Sulfatasa/metabolismo , Sulfotransferasas/metabolismo , Animales , Bovinos , Ésteres del Colesterol/metabolismo , Estradiol/sangre , Estro/metabolismo , Femenino , Líquido Folicular/metabolismo , Progesterona/sangre , Esteroides/análisis , Esteroides/metabolismoRESUMEN
The sodium-dependent organic anion transporter SOAT/Soat shows highly specific transport activity for sulfated steroids. SOAT substrates identified so far include dehydroepiandrosterone sulfate, 16α-hydroxydehydroepiandrosterone sulfate, estrone-3-sulfate, pregnenolone sulfate, 17ß-estradiol-3-sulfate, and androstenediol sulfate. Apart from these compounds, many other sulfated steroids occur in mammals. Therefore, we aimed to expand the substrate spectrum of SOAT and analyzed the SOAT-mediated transport of eight different sulfated steroids by combining in vitro transport experiments in SOAT-transfected HEK293 cells with LC-MS/MS analytics of cell lysates. In addition, we aimed to better understand the structural requirements for SOAT substrates and so selected structural pairs varying only at specific positions: 3α/3ß-sulfate, 17α/17ß-sulfate, mono-sulfate/di-sulfate, and 17α-hydroxylation. We found significant and sodium-dependent SOAT-mediated transport of 17α-hydroxypregnenolone sulfate, 17ß-estradiol-17-sulfate, androsterone sulfate, epiandrosterone sulfate, testosterone sulfate, epitestosterone sulfate, and 5α-dihydrotestosterone sulfate. However, 17ß-estradiol-3,17-disulfate was not transported by SOAT. IN CONCLUSION: SOAT substrates from the group of sulfated steroids are characterized by a planar and lipophilic steroid backbone in trans-trans-trans conformation of the rings and a negatively charged mono-sulfate group at positions 3' or 17' with flexibility for α- or ß- orientation. Furthermore, 5α-reduction, 16α-hydroxylation, and 17α-hydroxylation are acceptable for SOAT substrate recognition, whereas addition of a second negatively charged sulfate group seems to abolish substrate binding to SOAT, and so 17ß-estradiol-3,17-disulfate is not transported by SOAT.
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Transportadores de Anión Orgánico/metabolismo , Esteroides/química , Esteroides/metabolismo , Androsterona/análogos & derivados , Androsterona/química , Androsterona/metabolismo , Transporte Biológico , Dihidrotestosterona/química , Dihidrotestosterona/metabolismo , Estradiol/análogos & derivados , Estradiol/química , Estradiol/metabolismo , Células HEK293 , Humanos , Hidroxilación , Transportadores de Anión Orgánico/química , Relación Estructura-Actividad , Testosterona/química , Testosterona/metabolismoRESUMEN
Sulfated steroid hormones, such as dehydroepiandrosterone sulfate or estrone-3-sulfate, have long been regarded as inactive metabolites as they cannot activate classical steroid receptors. Some of them are present in the blood circulation at quite high concentrations, but generally sulfated steroids exhibit low membrane permeation due to their hydrophilic properties. However, sulfated steroid hormones can actively be imported into specific target cells via uptake carriers, such as the sodium-dependent organic anion transporter SOAT, and, after hydrolysis by the steroid sulfatase (so-called sulfatase pathway), contribute to the overall regulation of steroid responsive organs. To investigate the biological significance of sulfated steroid hormones for reproductive processes in humans and animals, the research group "Sulfated Steroids in Reproduction" was established by the German Research Foundation DFG (FOR1369). Projects of this group deal with transport of sulfated steroids, sulfation of free steroids, desulfation by the steroid sulfatase, effects of sulfated steroids on steroid biosynthesis and membrane receptors as well as MS-based profiling of sulfated steroids in biological samples. This review and concept paper presents key findings from all these projects and provides a broad overview over the current research on sulfated steroid hormones in the field of reproduction.
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Sulfato de Deshidroepiandrosterona/metabolismo , Estrona/análogos & derivados , Ictiosis Ligada al Cromosoma X/metabolismo , Reproducción/genética , Esterol O-Aciltransferasa/metabolismo , Esteril-Sulfatasa/metabolismo , Animales , Transporte Biológico , Bovinos , Estrona/metabolismo , Femenino , Expresión Génica , Humanos , Hidroxicolesteroles/metabolismo , Ictiosis Ligada al Cromosoma X/genética , Ictiosis Ligada al Cromosoma X/patología , Masculino , Oocitos/citología , Oocitos/metabolismo , Placenta/citología , Placenta/metabolismo , Embarazo , Esterol O-Aciltransferasa/genética , Esteril-Sulfatasa/genética , PorcinosRESUMEN
The impact of steroid sulfatase (STS) activity in the circulating levels of both sulfated and unconjugated steroids is only partially known. In addition, the sulfated steroid pathway, a parallel pathway to the one for unconjugated steroids, which uses the same enzymes, has never been characterized in detail before. Patients with steroid sulfatase deficiency (STSD) are unable to enzymatically convert sulfated steroids into their unconjugated forms, and are a good model to elucidate how STS affects steroid biosynthesis and to study the metabolism of sulfated steroids. We quantified unconjugated and sulfated steroids in STSD serum, and compared these results with data obtained from serum of healthy controls. Most sulfated steroids were increased in STSD. However, androstenediol-3-sulfate and epiandrosterone sulfate showed similar levels in both groups, and the concentrations of androsterone sulfate were notably lower. Hydroxylated forms of DHEAS and of pregnenolone sulfate were found to be increased in STSD, suggesting a mechanism to improve the excretion of sulfated steroids. STSD testosterone concentrations were normal, but cholesterol and DHEA were significantly decreased. Additionally, serum bile acids were three-fold higher in STSD. Correlations between concentrations of steroids in each group indicate that 17α-hydroxy-pregnenolone-3-sulfate in men is mainly biosynthesized from the precursor pregnenolone sulfate and androstenediol-3-sulfate from DHEAS. These findings confirm the coexistence of two steroidogenic pathways: one for unconjugated steroids and another one for sulfated steroids. Each pathway is responsible for the synthesis of specific steroids. The equal levels of testosterone, and the reduced level of unconjugated precursors in STSD, support that testosterone is primarily synthesized from sulfated steroids. In consequence, testosterone synthesis in STSD relies on an enzyme with sulfatase activity other than STS. This study reveals that STS is a key player of steroid biosynthesis regulating the availability of circulating cholesterol.
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Homeostasis , Ictiosis Ligada al Cromosoma X/metabolismo , Ictiosis Ligada al Cromosoma X/patología , Esteroides/metabolismo , Esteril-Sulfatasa/metabolismo , Sulfatos/metabolismo , Adolescente , Adulto , Anciano , Estudios de Casos y Controles , Niño , Preescolar , Deshidroepiandrosterona , Estradiol Deshidrogenasas/metabolismo , Humanos , Persona de Mediana Edad , Pregnenolona , Esteroide 17-alfa-Hidroxilasa/metabolismo , Adulto JovenRESUMEN
The consequences of ubiquitous exposure to environmental chemicals remain poorly defined. Non-targeted metabolomic profiling is an emerging method to identify biomarkers of the physiological response to such exposures. We investigated the effect of three commonly used ingredients in personal care products, diethyl phthalate (DEP), methylparaben (MPB) and triclosan (TCS), on the blood metabolome of female Sprague-Dawley rats. Animals were treated with low levels of these chemicals comparable to human exposures during prepubertal and pubertal windows as well as chronically from birth to adulthood. Non-targeted metabolomic profiling revealed that most of the variation in the metabolites was associated with developmental stage. The low-dose exposure to DEP, MPB and TCS had a relatively small, but detectable impact on the metabolome. Multiple metabolites that were affected by chemical exposure belonged to the same biochemical pathways including phenol sulfonation and metabolism of pyruvate, lyso-plasmalogens, unsaturated fatty acids and serotonin. Changes in phenol sulfonation and pyruvate metabolism were most pronounced in rats exposed to DEP during the prepubertal period. Our metabolomics analysis demonstrates that human level exposure to personal care product ingredients has detectable effects on the rat metabolome. We highlight specific pathways such as sulfonation that warrant further study.
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Cosméticos , Metaboloma , Parabenos/administración & dosificación , Ácidos Ftálicos/administración & dosificación , Triclosán/administración & dosificación , Animales , Relación Dosis-Respuesta a Droga , Femenino , Fenol/metabolismo , Ácido Pirúvico/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
Metabolic acidosis induces elevated glucocorticoid (GC) levels. However, the influence of less strong daily acid loads on GCs is largely unexplored. To investigate this, we studied whether higher acid loads in children, fully within the normal range of habitual diets, associate with endogenous GCs. In a specific quasi-experimental design, we examined 200 6- to 10-year-old healthy participants of the Dortmund Nutritional and Anthropometric Longitudinally Designed (DONALD) Study equally divided to either high or low 24-hour renal net acid excretion. Major urinary GC metabolites were analyzed by gas chromatography-mass spectrometry to assess daily adrenal GC secretion and metabolites of tissue cortisol catabolism (6ß-hydroxycortisol and 20α-dihydrocortisol). Liquid chromatography-mass spectrometry was used to quantify urinary free cortisol and cortisone. After confounder adjustment, significant positive associations were unmasked for urinary potential renal acid load and net acid excretion with adrenal GC secretion, free cortisone, free cortisone plus cortisol, 6ß-hydroxycortisol, and 20α-dihydrocortisol. An inverse association emerged for an enzymatic marker (5ß-reductase) of irreversible GC inactivation. Our data suggest that existing moderate elevations in diet-dependent acid loads suffice to raise GCs and affect cortisol metabolism. Thus, potential detrimental effects of high acid loading appear to be mediated, in part, by increased GC activity via increased GC secretion and/or reduced GC inactivation. Higher cortisone levels, directly available for intracrine activation to cortisol may play a special role.
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Acidosis/metabolismo , Glucocorticoides/metabolismo , Riñón/metabolismo , Eliminación Renal , Niño , Cortisona/metabolismo , Cortisona/orina , Dieta , Femenino , Cromatografía de Gases y Espectrometría de Masas , Glucocorticoides/orina , Voluntarios Sanos , Humanos , Hidrocortisona/análogos & derivados , Hidrocortisona/metabolismo , Hidrocortisona/orina , Masculino , Valores de ReferenciaRESUMEN
Steroids are primarily present in human fluids in their sulfated forms. Profiling of these compounds is important from both diagnostic and physiological points of view. Here, we present a novel method for the quantification of 11 intact steroid sulfates in human serum by LC-MS/MS. The compounds analyzed in our method, some of which are quantified for the first time in blood, include cholesterol sulfate, pregnenolone sulfate, 17-hydroxy-pregnenolone sulfate, 16-α-hydroxy-dehydroepiandrosterone sulfate, dehydroepiandrosterone sulfate, androstenediol sulfate, androsterone sulfate, epiandrosterone sulfate, testosterone sulfate, epitestosterone sulfate, and dihydrotestosterone sulfate. The assay was conceived to quantify sulfated steroids in a broad range of concentrations, requiring only 300 µl of serum. The method has been validated and its performance was studied at three quality controls, selected for each compound according to its physiological concentration. The assay showed good linearity (R(2) > 0.99) and recovery for all the compounds, with limits of quantification ranging between 1 and 80 ng/ml. Averaged intra-day and between-day precisions (coefficient of variation) and accuracies (relative errors) were below 10%. The method has been successfully applied to study the sulfated steroidome in diseases such as steroid sulfatase deficiency, proving its diagnostic value. This is, to our best knowledge, the most comprehensive method available for the quantification of sulfated steroids in human blood.
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Ésteres del Colesterol/sangre , Progestinas/sangre , Sulfatos/sangre , Congéneres de la Testosterona/sangre , Cromatografía Liquida , Humanos , Espectrometría de Masas en TándemRESUMEN
Steroid sulfatase (STS) deficiency is the underlying cause of the skin condition known as recessive X-linked ichthyosis (RXLI). RXLI patients show scales on their skin caused by high concentrations of cholesterol sulfate (CS), as they are not capable of releasing the sulfate group from its structure to obtain free cholesterol. CS has been reported, so far, as the sole sulfated steroid with increased concentrations in the blood of RXLI patients. A non-targeted LC-MS approach in negative mode detection (LC-MS precursor ion scan mode) was applied to serum samples of 12 RXLI patients and 19 healthy males. We found that CS was not the only sulfated compound consistently elevated in RXLI patients, because a group of compounds with a m/z of 481 was found in high concentrations too. Further LC-MS/MS demonstrated that the main contributor to the m/z 481 signal in RXLI serum is 27-hydroxycholesterol-3-sulfate (27OHC3S). Accordingly, a new method for 27OHC3S quantification in the context of RXLI has been developed and validated. Other hydroxycholesterol sulfate compounds were elevated as well in RXLI patients.
Asunto(s)
Ésteres del Colesterol/sangre , Ictiosis Ligada al Cromosoma X/enzimología , Esteril-Sulfatasa/metabolismo , Humanos , Ictiosis Ligada al Cromosoma X/sangre , Masculino , Espectrometría de Masas en TándemRESUMEN
Whether higher production of glucocorticoids (GCs) within the physiological range may already be affecting bone status in healthy children is unknown. Because dietary protein intake affects both bone and GCs, we examined the association of urinary measures of glucocorticoid status and cortical bone in healthy non-obese children, after particularly controlling for protein intake. Proximal forearm bone parameters were measured by peripheral quantitative computed tomography (pQCT). Subjects studied (n = 175, 87 males, aged 6 to 18 years) had two 24-hour urine samples collected: the first sample at 1 year before bone measurement, and the second sample at the time of bone measurement. Major urinary GC metabolites were measured by mass spectrometry and summed to assess daily adrenal GC secretion (∑C21). Urinary free cortisol (UFF) and cortisone (UFE) were summed to assess potentially bioactive free GCs (UFF + UFE). After controlling for several covariates and especially urinary nitrogen (the biomarker of protein intake) cortisol secretion ∑C21 was inversely associated with all analyzed pQCT measures of bone quality. ∑C21 also predicted a higher endosteal and lower periosteal circumference, explaining both a smaller cortical area and (together with lower BMD) a lower strength-strain-index (SSI). UFF + UFE, UFE itself, and a urinary metabolite-estimate of 11beta-hydroxysteroid dehydrogenase type1 (11beta-HSD1) activity showed corresponding reciprocal associations (p < 0.05) with BMD and bone mineral content, but not with SSI and bone geometry variables. In conclusion, higher GC levels, even within the physiological range, appear to exert negative influences on bone modeling and remodeling already during growth. Our physiological data also suggest a relevant role of cortisone as the direct source for intracrine-generated cortisol by bone cell 11beta-HSD1.
Asunto(s)
Proteínas en la Dieta/farmacología , Glucocorticoides/metabolismo , Salud , Radio (Anatomía)/fisiología , Adolescente , Biomarcadores/orina , Densidad Ósea/efectos de los fármacos , Niño , Dieta , Femenino , Humanos , Masculino , Radio (Anatomía)/efectos de los fármacos , Valores de ReferenciaRESUMEN
Urinary free cortisol and urinary free cortisone are decisive markers for the diagnosis of syndromes related to the dysfunction of the adrenal gland or to evaluate certain enzymatic disorders. Here, we present a new method, designed for routine laboratory use, which enables quick determination of these analytes with minor sample workup. Turbulent flow chromatography shortens sample preparation, and connection to a fused-core particle-packed column (rugged amide-embedded C18 phase) permits a rapid and effective separation of the analytes, as well as additional separation from other related and isobaric compounds present in urine. Urinary isobaric compounds were successfully identified. The method requires only 100 µl of urine supernatant per sample. The total time between injections is 9.5 min. The solvents used for both turbulent and analytical chromatography are water and methanol, and the relatively low flows needed during the method resulted in an extended life of the columns. Linearity showed a R (2) > 0.994. Limit of detection and limit of quantification are 0.5 and 1.0 ng/ml for cortisone and 1.0 and 2.0 ng/ml for cortisol. Recoveries ranged from 99.7 to 109.1 % for cortisone and from 98.7 to 102.9 % for cortisol. Accuracy values (relative errors) for intra- and inter-assay experiments were always below 8 %, whereas precision (percent CV) ranged from 3.7 to 10.7 %. No matrix effects were detected during the validation process. The reproducibility for each analyte's retention time was excellent, with a coefficient of variation always below 0.2 %. The final validation step included the study of urine samples from healthy children and from children previously diagnosed with corticoidal disorders. The high selectivity achieved enables quick data handling.
Asunto(s)
Cromatografía , Cortisona/orina , Hidrocortisona/orina , Espectrometría de Masas en Tándem , Urinálisis/métodos , Adolescente , Biomarcadores/orina , Niño , Síndrome de Cushing/orina , Femenino , Humanos , Límite de Detección , Masculino , Síndrome de Exceso Aparente de Mineralocorticoides , Estándares de Referencia , Reproducibilidad de los ResultadosRESUMEN
Gas chromatography-mass spectrometry (GC-MS) is a technique of pivotal importance for the analysis of hormones in biological fluids. In consequence, it has gained relevance in clinical and endocrinological laboratories, providing reference analytical methods. This chapter offers a general description of its principles, and a real example for GC-MS profiling of plasma steroids.