23 results on '"Michaela F. Hartmann"'
Search Results
2. Excretion of oxidated cortisol metabolites is markedly lower than previously assumed: An analysis of urinary cortoic acids in healthy children by GC-MS
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Marcel Schauermann, Ulrich A. Wachter, Yifan Hua, Michaela F. Hartmann, Thomas Remer, and Stefan A. Wudy
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Endocrinology ,Endocrinology, Diabetes and Metabolism ,Clinical Biochemistry ,Molecular Medicine ,Cell Biology ,Molecular Biology ,Biochemistry - Abstract
Discovered about 50 years ago, the four C
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- 2022
3. Performance of LC–MS/MS and immunoassay based 24-h urine free cortisol in the diagnosis of Cushing's syndrome
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Felix Beuschlein, Stephanie Zopp, Rong Wang, Martin Reincke, Michaela F. Hartmann, Katrin Ritzel, Andrea Oßwald, Martin Bidlingmaier, Stefan A. Wudy, University of Zurich, and Reincke, Martin
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Adult ,Male ,0301 basic medicine ,Cortisol secretion ,medicine.medical_specialty ,1303 Biochemistry ,Endocrinology, Diabetes and Metabolism ,Clinical Biochemistry ,10265 Clinic for Endocrinology and Diabetology ,610 Medicine & health ,Urine ,1308 Clinical Biochemistry ,Urine free cortisol ,Biochemistry ,Gastroenterology ,1307 Cell Biology ,03 medical and health sciences ,0302 clinical medicine ,Endocrinology ,Tandem Mass Spectrometry ,Internal medicine ,Lc ms ms ,1312 Molecular Biology ,Humans ,Medicine ,ADVIA Centaur ,Cushing Syndrome ,Molecular Biology ,24 h urine ,Immunoassay ,medicine.diagnostic_test ,Receiver operating characteristic ,business.industry ,Cell Biology ,Middle Aged ,1310 Endocrinology ,Cortisone ,2712 Endocrinology, Diabetes and Metabolism ,030104 developmental biology ,1313 Molecular Medicine ,030220 oncology & carcinogenesis ,Molecular Medicine ,Female ,business ,Chromatography, Liquid - Abstract
24-h urine free cortisol (UFC) is an indicator of integrated cortisol secretion and established screening tool for Cushing's syndrome (CS). Doubts have been raised regarding specificity of immunoassays, and mass spectrometric techniques have been proposed as an alternative. In the present study we compared diagnostic accuracy of UFC measured with LC-MS/MS vs. immunoassay in patient with CS and patients where CS has been excluded. We examined 24-h urine samples from patients with surgically confirmed CS (n = 77; Cushing's disease (n = 44), ectopic CS (n = 5), adrenal CS (n = 28)) and patients in whom Cushing's syndrome was excluded (n = 97) by long-term follow up. UFC was first measured by automated chemiluminescence immunoassays (ADVIA Centaur, Siemens; LIAISON, DiaSorin). Aliquots of all samples were also analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Statistics: Passing-Bablok Regression, Receiver operating characteristic (ROC) analysis with Youden's index calculation. UFC of CS patients were higher with both immunoassays compared to LC-MS/MS (913 +/- 235 vs. 303 +/- 155 μg/24 h (ADVIA) and 898 +/-216 vs. 399 +/- 196 μg/24 h (LIAISON)). Similarly, UFC were higher with immunoassays than with LC-MS/MS in the control group (223 +/- 10 vs. 23 +/- 2 μg/24 h (ADVIA) and 105 +/- 6 vs. 27 +/- 4 ug/24 h for (LIAISON)). Passing-Bablok regression showed good correlation between LC-MS/MS and ADVIA as well as between LCMS/MS and LIAISON (r = 0.96 and r = 0.99, p 000.1) but less correlation in controls (r = 0.83 and r = 0.74, respectively, p 000.1). ROC calculation revealed the highest ROC AUC (0.89) for the LIAISON immunoassay, followed by LC-MS/MS (0.82) and the ADVIA (0.80). In direct comparison, AUCs from LC-MS/MS and immunoassays in the same patient were not statistically different (p 0,001). Best cut-off concentration to identify patients with CS was 234 μg/24 h (LIAISON), 51 μg/24 h for LC-MS/MS and 330 μg/24 h (ADVIA Centaur). In summary, UFC values were measured substantially higher by both immunoassays compared to LC-MS/MS. This is most likely due to cross-reactivity from interfering glucocorticoid metabolites. Nevertheless, all three methods correlated well. ROC analysis revealed the highest AUC for one of the immunoassays, although differences between the three methods were not significant. Direct comparison with LC-MS/MS indicates that high diagnostic accuracy can be obtained with suitable immunoassays.
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- 2019
4. Sodium-dependent organic anion transporter ( Slc10a6−/− ) knockout mice show normal spermatogenesis and reproduction, but elevated serum levels for cholesterol sulfate
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Charles C. Love, Stefan A. Wudy, Alberto Sánchez-Guijo, Daniela Fietz, Andrei Golovko, Josefine Bennien, Joachim Geyer, R. Serafini, Martin Bergmann, Michaela F. Hartmann, and Katharina Bakhaus
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0301 basic medicine ,medicine.medical_specialty ,Organic anion transporter 1 ,Endocrinology, Diabetes and Metabolism ,medicine.medical_treatment ,Clinical Biochemistry ,030209 endocrinology & metabolism ,Biology ,Biochemistry ,Steroid ,03 medical and health sciences ,0302 clinical medicine ,Endocrinology ,Internal medicine ,Steroid sulfatase ,medicine ,Molecular Biology ,Testosterone ,Wild type ,Transporter ,Cell Biology ,030104 developmental biology ,Knockout mouse ,biology.protein ,Molecular Medicine ,Spermatogenesis - Abstract
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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- 2018
5. The art of measuring steroids
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Alberto Sánchez-Guijo, Stefan A. Wudy, Michaela F. Hartmann, and Gerhard Schuler
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0301 basic medicine ,Analyte ,Chromatography ,medicine.diagnostic_test ,Chemistry ,Endocrinology, Diabetes and Metabolism ,medicine.medical_treatment ,Clinical Biochemistry ,Cell Biology ,Computational biology ,Biochemistry ,Steroid ,Steroid Biochemistry ,03 medical and health sciences ,Steroid hormone ,030104 developmental biology ,Endocrinology ,Metabolomics ,Immunoassay ,medicine ,Molecular Medicine ,Hormone Measurement ,Molecular Biology ,Hormone - Abstract
Steroids are small and highly important structural or signalling molecules in living organisms and their metabolism is complex. Due to the multiplicity of enzymes involved there are many different steroid related disorders. E.g., an individual enzyme defect is rather rare but can share various clinical symptoms and can thus be hardly diagnosed clinically. Therefore, reliable hormonal determination still presents the most reasonable initial diagnostic approach and helps to avoid uncritical and expensive attempts at molecular diagnostic testing. It also presents a backbone of monitoring these complex patients. In science, reliable hormone measurement is indispensable for the elucidation of new mechanisms of steroid hormone actions. Steroid analytics is highly challenging and should never be considered trivial. Most common methods for steroid determination comprise traditionally immunoassay, or more recently, mass spectrometry based methods. It is absolutely necessary that clinicians and scientists know the methods they are applying by heart. With the introduction of automated direct assays, a loss of quality could be observed over the last two decades in the field of steroid immunoassays. This review wants to meet the need for profound information and orientation in the field of steroid analysis. The pros and cons of the most important methods, such as immunoassays and mass spectrometry based methods will be discussed. The focus of the latter will lie on gas chromatography-mass spectrometry (GC-MS) as well as liquid chromatography-mass spectrometry (LC-MS). Selected analytical applications from our Deutsche Forschungsgemeinschaft Research Group FOR 1369 "Sulfated Steroids in Reproduction" will illustrate the contents. In brief, immunoassays have for long presented the traditional technique for steroid analysis. They are easy to set up. Only one analyte can be measured per immunoassay. Specificity problems can arise and caution has to be exerted especially regarding direct assays lacking purification steps. Mass spectrometry based methods provide structural information on the analyte and thus higher specificity. In combination with chromatographic techniques, they permit the simultaneous determination of a multitude of analytes. Highest specificity can be obtained using GC-MS, a sophisticated but most powerful tool for characterizing steroid metabolomes. LC-MS is a true high throughput technique and highly suited for detecting complex steroids. GC-MS and LC-MS are not competing but complementary techniques. Since reliable steroid determination requires extremely high expertise in the field of analytics as well as steroid biochemistry, it is recommended that collaborations and networking with highly specialized centers of expertise are developed.
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- 2018
6. The role of sulfated steroid hormones in reproductive processes
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Katja Hartmann, Jens Neunzig, Alberto Sánchez-Guijo, Gary Grosser, Daniela Fietz, Joachim Geyer, Michaela F. Hartmann, Carina Blaschka, Rita Bernhardt, Christine Wrenzycki, Katharina Bakhaus, Dimitrios Papadopoulos, Martin Bergmann, Gerhard Schuler, Stefan A. Wudy, Mazen Shihan, Georgios Scheiner-Bobis, and Y Dezhkam
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Male ,0301 basic medicine ,medicine.medical_specialty ,Ichthyosis, X-Linked ,Organic anion transporter 1 ,Estrone ,Swine ,Placenta ,Endocrinology, Diabetes and Metabolism ,medicine.medical_treatment ,Clinical Biochemistry ,Gene Expression ,Steroid biosynthesis ,Biochemistry ,Steroid ,03 medical and health sciences ,chemistry.chemical_compound ,Endocrinology ,Dehydroepiandrosterone sulfate ,Sulfation ,Pregnancy ,Internal medicine ,medicine ,Steroid sulfatase ,Animals ,Humans ,Molecular Biology ,030102 biochemistry & molecular biology ,biology ,Dehydroepiandrosterone Sulfate ,Reproduction ,Sulfatase ,Biological Transport ,Cell Biology ,Hydroxycholesterols ,030104 developmental biology ,chemistry ,Oocytes ,biology.protein ,Molecular Medicine ,Cattle ,Female ,Steryl-Sulfatase ,Sterol O-Acyltransferase ,Hormone - Abstract
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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- 2017
7. Targeted LC–MS/MS analysis of steroid glucuronides in human urine
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Rong Wang, Michaela F. Hartmann, and Stefan A. Wudy
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Male ,0301 basic medicine ,Androsterone glucuronide ,Endocrinology, Diabetes and Metabolism ,Clinical Biochemistry ,Dehydroepiandrosterone ,Epiandrosterone ,Androsterone ,Biochemistry ,Gas Chromatography-Mass Spectrometry ,03 medical and health sciences ,chemistry.chemical_compound ,Glucuronides ,0302 clinical medicine ,Endocrinology ,Tandem Mass Spectrometry ,medicine ,Humans ,Testosterone ,Gonadal Steroid Hormones ,Molecular Biology ,Chromatography, High Pressure Liquid ,Testosterone glucuronide ,Etiocholanolone glucuronide ,Chromatography ,Etiocholanolone ,Solid Phase Extraction ,Cell Biology ,030104 developmental biology ,chemistry ,030220 oncology & carcinogenesis ,Molecular Medicine ,Female ,Steroids ,Glucuronide ,Chromatography, Liquid ,medicine.drug - Abstract
Conjugation with glucuronic acid is one of the major metabolic reactions in human steroid hormone catabolism. Recently, increasing interest has been raised concerning the biological roles of steroid glucuronides. We have therefore developed and validated a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the simultaneous quantification of 15 urinary steroid hormone glucuronides in human urine: androsterone glucuronide (An-G), etiocholanolone glucuronide (Etio-G), epiandrosterone glucuronide (epiAn-G), dihydrotestosterone glucuronide (DHT-G), dehydroepiandrosterone glucuronide (DHEA-G), testosterone glucuronide (T-G), epitestosterone glucuronide (epiT-G), estrone glucuronide (E1-3 G), 17β-estradiol 17-glucuronide (E2-17 G), 17β-estradiol 3-glucuronide (E2-3 G), estriol 16-glucuronide (E3-16 G), pregnenolone glucuronide (Preg-G), tetrahydro-11-deoxycorticosterone 3-glucuronide (THDOC-3 G), cortisol 21-glucuronide (F-G) and pregnanediol glucuronide (PD-G). Sample workup included protein precipitation and solid phase extraction. Internal standards were used to correct for the loss of analytes during sample preparation and analysis. The method showed good linearity (R2≥0.99) and recovery ranged from 89.6 % to 113.8 %. Limit of quantification ranged from 1.9 nmol/L for F-G to 21.4 nmol/L for An-G. Intra-day and inter-day accuracy and precision were below 15 % for all quality controls. The method was successfully applied to 67 urine samples from children and adolescents in whom total concentrations of free and conjugated steroids had been previously determined by GC-MS after enzymatic hydrolysis. Free and sulfated steroids were also measured by LC-MS/MS. In general, the sums of the respective glucuronidated, sulfated and free forms of an analyte corresponded well with its total amount determined after enzymatic hydrolysis by GC-MS. Regarding the most prominent steroid metabolites, the total mean levels of androsterone and etiocholanolone showed an increase up to 5820.0 nmol/L and 4017.8 nmol/L in the group of 15-20 year-old children, respectively. Glucuronide conjugates (4374.3 nmol/L and 3588.5 nmol/L, respectively) dominated. DHEA was excreted mostly as sulfate (0-1 month of age: 184.5 nmol/L; 15-20 years of age: 1618.4 nmol/L) in all age groups. Cortisol was present predominantly as sulfate (mean: 173.8 nmol/L) in newborns. Levels of sulfated cortisol decreased with age, its glucuronidated form increased. The levels of free cortisol were relatively constant throughout childhood. Sex hormones were preferably excreted as glucuronides. In general, steroid hormone metabolites were conjugated to various extents with glucuronic acid or sulfuric acid and their ratio changed over lifetime.
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- 2021
8. Diagnosis of 21-hydroxylase deficiency by urinary metabolite ratios using gas chromatography–mass spectrometry analysis: Reference values for neonates and infants
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Claudia Boettcher, Stefan A. Wudy, Klaus-Peter Zimmer, Michaela F. Hartmann, and Clemens Kamrath
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Male ,0301 basic medicine ,Pregnanetriol ,medicine.medical_specialty ,Endocrinology, Diabetes and Metabolism ,Urinary system ,medicine.medical_treatment ,Metabolite ,Clinical Biochemistry ,Physiology ,030209 endocrinology & metabolism ,Biology ,Biochemistry ,Gas Chromatography-Mass Spectrometry ,Cohort Studies ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Endocrinology ,Reference Values ,Internal medicine ,medicine ,Humans ,Metabolomics ,Tetrahydrocortisone ,Congenital adrenal hyperplasia ,Molecular Biology ,Newborn screening ,Adrenal Hyperplasia, Congenital ,Infant, Newborn ,21-Hydroxylase ,Infant ,Cell Biology ,medicine.disease ,Steroid hormone ,030104 developmental biology ,chemistry ,biology.protein ,Molecular Medicine ,Female ,Steroids - Abstract
One major issue of newborn screening programs for 21-hydroxylase deficiency (21OHD) is the high rate of false-positive results, especially in preterm neonates. Urinary steroid metabolite analysis using gas chromatography-mass spectrometry (GC-MS) is suitable as a confirmatory diagnostic tool. The objective of this study was to analyze retrospectively diagnostic metabolite ratios in neonates and infants with and without 21OHD using GC-MS with emphasis on glucocorticoid metabolism, and to develop reference values for the steroid metabolite ratios for the diagnosis of 21OHD. We retrospectively analyzed urinary steroid hormone metabolites determined by GC-MS of 95 untreated neonates and infants with 21OHD (1-148 days), and 261 neonates and infants (100 preterms) without 21OHD (0-217 days). Metabolites of 17α-hydroxyprogesterone showed specificities below 98%, whereas the 21-deoxycortisol metabolite pregnanetriolone clearly separated 21OHD from non-21OHD subjects. The best diagnostic ratio for 21OHD was pregnanetriolone to 6α-hydroxy-tetrahydrocortisone. The lowest value of this ratio in the 21OHD group (0.47) was at least eight times higher than the highest values in the non-21OHD group (0.055). We have given appropriate reference values for steroid metabolite ratios in the largest 21OHD cohort so far described. Consideration of glucocorticoid metabolism, especially the use of typical neonatal 6α-hydroxylates metabolites, leads to improvement of diagnostic metabolite ratios.
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- 2016
9. Characterizing the steroidal milieu in amniotic fluid of mid-gestation: A GC-MS study
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Stefan A. Wudy, Dov Tiosano, Michaela F. Hartmann, and Rong Wang
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Adult ,Male ,medicine.medical_specialty ,Amniotic fluid ,Endocrinology, Diabetes and Metabolism ,medicine.medical_treatment ,Clinical Biochemistry ,Dehydroepiandrosterone ,Gestational Age ,Biochemistry ,Gas Chromatography-Mass Spectrometry ,Steroid ,Young Adult ,chemistry.chemical_compound ,Endocrinology ,Pregnancy ,Corticosterone ,Internal medicine ,medicine ,Humans ,Molecular Biology ,Testosterone ,Chemistry ,Cell Biology ,Amniotic Fluid ,Steroid hormone ,Pregnenolone ,Molecular Medicine ,Female ,Steroids ,Hormone ,medicine.drug - Abstract
Intact steroid hormone biosynthesis is essential for growth and development of the human fetus and embryo. In the present study, gas chromatography-mass spectrometry was employed to characterize the steroidal milieu in amniotic fluid (n = 65; male: female = 35: 30) of mid-gestation (median: 18.8th week, range: 16.0th - 24.6th week) by a comprehensive targeted steroid hormone metabolomics approach. The levels of 52 steroids including pregnenolone and 17-OH-pregnenolone metabolites, dehydroepiandrosterone (DHEA) and its metabolites, progesterone and 17-OH-progesterone metabolites, sex hormones as well as corticosterone and cortisol metabolites were measured. The dominating steroids were the group of pregnenolone and 17-OH-pregnenolone metabolites (mean ± SD: 138.0 ± 59.3 ng/mL), followed by the group of progesterone and 17-OH-progesterone metabolites (107.3 ± 44.3 ng/mL), and thereafter DHEA and its metabolites (97.1 ± 56.5 ng/mL). With respect to sex steroids, only testosterone showed a significantly higher value in male fetuses (p 0.0001). Of all estrogen metabolites, estriol showed by far the highest concentrations (33.2 ± 26.1 ng/mL). Interestingly, cortisol metabolites were clearly present (59.6 ± 13.6 ng/mL) though fetal de novo synthesis of cortisol is assumed to start from gestational 28th week onwards. Our comprehensive characterization of the steroidal milieu in amniotic fluid of mid-gestation shows presence of all relevant classes of steroid hormones and provides reference data. We conclude that the steroidal milieu in amniotic fluid mirrors the steroidome of the feto-placental unit.
- Published
- 2019
10. Androgen excess is due to elevated 11-oxygenated androgens in treated children with congenital adrenal hyperplasia
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Lisa Wettstaedt, Claudia Boettcher, Michaela F. Hartmann, Stefan A. Wudy, and Clemens Kamrath
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0301 basic medicine ,Male ,medicine.medical_specialty ,medicine.drug_class ,Endocrinology, Diabetes and Metabolism ,Metabolite ,Clinical Biochemistry ,Dehydroepiandrosterone ,030209 endocrinology & metabolism ,urologic and male genital diseases ,Androgen Excess ,Androsterone ,Biochemistry ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Endocrinology ,Internal medicine ,Medicine ,Humans ,Congenital adrenal hyperplasia ,Child ,Molecular Biology ,Testosterone ,Hydrocortisone ,Retrospective Studies ,Adrenal Hyperplasia, Congenital ,business.industry ,Cell Biology ,Androgen ,medicine.disease ,Prognosis ,030104 developmental biology ,chemistry ,Case-Control Studies ,Child, Preschool ,Androgens ,Molecular Medicine ,Female ,business ,Biomarkers ,medicine.drug - Abstract
Adrenal androgen excess is the hallmark of classic congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency. Recently, 11-oxygenated C19 steroids, a class of highly active adrenal-derived androgens, have been described in patients with CAH. The aim of our study was to elucidate the significance of 11-oxygenated androgens in children with CAH. We retrospectively analysed 190 daily urinary excretion rates of glucocorticoid-, 17α-hydroxyprogesterone (17OHP)-, and androgen metabolites determined by gas chromatography-mass spectrometry of 99 children aged 3.0–10.9 years with classic CAH on hydrocortisone and fludrocortisone treatment. Daily urinary steroid metabolite excretions were transformed into z-scores using references of healthy children. Androgen metabolite z-scores were separately calculated for androsterone (AN), the major urinary metabolite of androstenedione (A4), testosterone and 5α-dihydrotestosterone, for urinary metabolites of dehydroepiandrosterone (DHEA), and for 11β-hydroxyandrosterone (11OHAN), the major urinary metabolite of adrenal-derived 11-oxygenated androgens. Multivariate regression analysis was applied to analyse the precursors of 11OHAN synthesis. 11OHAN, cortisol-, and 17OHP metabolite z-scores were elevated in treated children with CAH, whereas AN- and DHEA metabolite z-scores were normalized or suppressed. Multivariate regression analysis revealed that 11OHAN excretion was strongest associated with 21-deoxycortisol (β = 0.379; P =.0006), followed by A4 (β = 0.280; P = .0008)) and 17OHP (β = 0.243; P = .04) metabolite excretion. Androgen excess in treated children with CAH is solely due to elevated 11-oxygenated androgens that derive in addition to the known conversion from A4 also by direct conversion from 21-deoxycortisol. 11-Oxygenated androgens may represent better biomarkers of adrenal androgen status and treatment response than conventional androgens.
- Published
- 2017
11. Transport of steroid 3-sulfates and steroid 17-sulfates by the sodium-dependent organic anion transporter SOAT (SLC10A6)
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Gary Grosser, Joachim Geyer, Josefine Bennien, Barbara Döring, Katharina Bakhaus, Stefan A. Wudy, Alberto Sánchez-Guijo, and Michaela F. Hartmann
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0301 basic medicine ,Organic anion transporter 1 ,Endocrinology, Diabetes and Metabolism ,medicine.medical_treatment ,Clinical Biochemistry ,Androstenediol ,Organic Anion Transporters ,Androsterone ,Hydroxylation ,Biochemistry ,Steroid ,03 medical and health sciences ,chemistry.chemical_compound ,Structure-Activity Relationship ,0302 clinical medicine ,Endocrinology ,Sulfation ,medicine ,Humans ,Testosterone ,Sulfate ,Molecular Biology ,Testosterone Sulfate ,biology ,Estradiol ,Biological Transport ,Dihydrotestosterone ,Cell Biology ,Androsterone Sulfate ,030104 developmental biology ,HEK293 Cells ,chemistry ,030220 oncology & carcinogenesis ,biology.protein ,Molecular Medicine ,Steroids ,Pregnenolone sulfate - Abstract
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, 17β-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/3β-sulfate, 17α/17β-sulfate, mono-sulfate/di-sulfate, and 17α-hydroxylation. We found significant and sodium-dependent SOAT-mediated transport of 17α-hydroxypregnenolone sulfate, 17β-estradiol-17-sulfate, androsterone sulfate, epiandrosterone sulfate, testosterone sulfate, epitestosterone sulfate, and 5α-dihydrotestosterone sulfate. However, 17β-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 β- 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 17β-estradiol-3,17-disulfate is not transported by SOAT.
- Published
- 2017
12. Simultaneous profiles of sulfonated androgens, sulfonated estrogens and sulfonated progestogens in postpubertal boars (sus scrofa domestica) measured by LC-MS/MS
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Gerhard Schuler, Michaela F. Hartmann, Alberto Sánchez-Guijo, and Stefan A. Wudy
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0301 basic medicine ,Male ,medicine.medical_specialty ,BOAR ,Endocrinology, Diabetes and Metabolism ,Clinical Biochemistry ,Sus scrofa ,Dehydroepiandrosterone ,030209 endocrinology & metabolism ,Estrone ,Epiandrosterone ,Androsterone ,Biochemistry ,Chorionic Gonadotropin ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Endocrinology ,Tandem Mass Spectrometry ,Internal medicine ,Testis ,medicine ,Animals ,Molecular Biology ,Testosterone ,Chromatography ,Chemistry ,Dehydroepiandrosterone Sulfate ,Epitestosterone ,Puberty ,Estrogens ,Cell Biology ,030104 developmental biology ,Pregnenolone ,Androgens ,Molecular Medicine ,Female ,Progestins ,Sulfatases ,medicine.drug ,Chromatography, Liquid - Abstract
Sulfonated steroids (s-St) have been usually regarded as inactive metabolites but are progressively considered as precursors for the intra-tissue formation of bioactive steroids. Moreover, independent effects without preceding removal of the sulfate group have been observed. We use the porcine testicular-epididymal compartment as a model to investigate the still largely unknown s-St physiology as the boar exhibits an intriguingly broad s-St spectrum predominantly originating from the testis. The application of LC-MS/MS in steroidomics enables the determination of unconjugated and intact sulfonated steroids with currently highest specificity and good sensitivity, allowing the concurrent measuring of numerous analytes in larger quantities of samples. Profiles (6h, 20min intervals) were generated for sulfonated 5-androstene-3s,17s-diol (Adiol-S), androsterone (A-S), dehydroepiandrosterone (DHEA-S), epiandrosterone (EA-S), epitestosterone (ET-S), estrone (E1-S), estradiol-17β (E2-S), pregnenolone (P5-S), 17αOH-pregnenolone (OHP5-S) and unconjugated testosterone (T) in four unstimulated and four hCG-stimulated boars. Moreover, concentrations were measured in individual samples collected from testicular afferent and efferent blood to differentiate between testicular vs. extratesticular origin. Highest concentrations were found for EA-S, followed by ET-S, Adiol-S and DHEA-S, which mostly exceeded the levels of E1-S and A-S. Lowest concentrations were obtained for E2-S, P5-S and OHP5-S. The analytical profile also included sulfonated T, 5α-dihydrotestosterone and cholesterol. However, their concentrations were below the limit of quantification. Profiles of quantifiable s-St were consistent with a wave-like pattern associated with T pulses. In postpartal females (n=5) concentrations of all analytes assessed were undetectable, suggesting that in pigs the adrenals are not a quantitatively significant source of s-St.
- Published
- 2017
13. Efficiency of the sulfate pathway in comparison to the Δ4- and Δ5-pathway of steroidogenesis in the porcine testis
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Alberto Sánchez-Guijo, M.C. Klymiuk, Stefan A. Wudy, Michaela F. Hartmann, Rita Bernhardt, Jens Neunzig, and Gerhard Schuler
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0301 basic medicine ,Male ,endocrine system ,Swine ,Endocrinology, Diabetes and Metabolism ,Clinical Biochemistry ,Hydroxylation ,Biochemistry ,03 medical and health sciences ,chemistry.chemical_compound ,Endocrinology ,Dehydroepiandrosterone sulfate ,Microsomes ,Cytochrome b5 ,Testis ,medicine ,Animals ,Molecular Biology ,Progesterone ,NADPH-Ferrihemoprotein Reductase ,chemistry.chemical_classification ,030102 biochemistry & molecular biology ,Chemistry ,Sulfates ,Steroid 17-alpha-Hydroxylase ,Cell Biology ,Metabolism ,Recombinant Proteins ,030104 developmental biology ,Enzyme ,Cytochromes b5 ,CYP17A1 ,Pregnenolone ,Microsome ,Molecular Medicine ,Pregnenolone sulfate ,Metabolic Networks and Pathways ,medicine.drug - Abstract
Sulfonated steroids are increasingly recognized as a circulating reservoir of precursors for the local production of active steroids in certain target tissues. As an alternative to sulfonation of unconjugated steroids by cytosolic sulfotransferases, their direct formation from sulfonated precursors has been described. However, productivity and physiological relevance of this sulfate pathway of steroidogenesis are still widely unclear. Applying the porcine testis as a model, conversion of pregnenolone sulfate (P5S, sulfate pathway) by CYP17A1 was assessed in comparison to the parallel conversions of pregnenolone (P5, Δ5-pathway) and progesterone (P4, Δ4-pathway). To characterize conversions in the virtual absence of competing enzyme activities, in a first series of experiments porcine recombinant CYP17A1 was incubated with the respective substrate in the presence of bovine recombinant cytochrome P450 oxidoreductase (CPR) and cytochrome b5 (b5). Moreover, porcine testicular microsomal fractions were used as a source of homologous CYP17A1, CPR and b5. Invariably 17α-hydroxylation of P5S was, if at all, only minimal and no formation of dehydroepiandrosterone sulfate from P5S was detectable. Consistent with earlier studies porcine CYP17A1 efficiently metabolized P4 and P5 in both assay systems. Metabolism of P4 and P5 by testicular microsomal protein varied substantially between the five animals tested. In conclusion, a physiologically relevant sulfate pathway for the production of C19-steroids from P5S via CYP17A1 is very unlikely in the porcine testis.
- Published
- 2017
14. Transport of the placental estriol precursor 16α-hydroxy-dehydroepiandrosterone sulfate (16α-OH-DHEAS) by stably transfected OAT4-, SOAT-, and NTCP-HEK293 cells
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Ernst Petzinger, Bernhard Ugele, H. Schweigmann, Barbara Döring, Stefan A. Wudy, Gary Grosser, Michaela F. Hartmann, Joachim Geyer, Katja Hartmann, Alberto Sánchez-Guijo, Martin Bergmann, and C. Pfarrer
- Subjects
Organic anion transporter 1 ,Placenta ,Endocrinology, Diabetes and Metabolism ,Blotting, Western ,Clinical Biochemistry ,Organic Anion Transporters ,Organic Anion Transporters, Sodium-Dependent ,Transfection ,Biochemistry ,Immunoenzyme Techniques ,Endocrinology ,Syncytiotrophoblast ,Pregnancy ,Tandem Mass Spectrometry ,medicine ,Humans ,Steroid sulfate ,RNA, Messenger ,Molecular Biology ,In Situ Hybridization ,Symporters ,biology ,Chemistry ,Membrane transport protein ,Biological Transport ,Estriol ,Dehydroepiandrosterone ,Cell Biology ,Apical membrane ,Trophoblasts ,Organic anion-transporting polypeptide ,HEK293 Cells ,medicine.anatomical_structure ,biology.protein ,Molecular Medicine ,Female ,hormones, hormone substitutes, and hormone antagonists ,Chromatography, Liquid ,Sterol O-Acyltransferase - Abstract
16α-Hydroxy-dehydroepiandrosterone sulfate (16α-OH-DHEAS) mainly originates from the fetus and serves as precursor for placental estriol biosynthesis. For conversion of 16α-OH-DHEAS to estriol several intracellular enzymes are required. However, prior to enzymatic conversion, 16α-OH-DHEAS must enter the cells by carrier mediated transport. To identify these carriers, uptake of 16α-OH-DHEAS by the candidate carriers organic anion transporter OAT4, sodium-dependent organic anion transporter SOAT, Na(+)-taurocholate cotransporting polypeptide NTCP, and organic anion transporting polypeptide OATP2B1 was measured in stably transfected HEK293 cells by LC-MS-MS. Furthermore, the study aimed to localize SOAT in the human placenta. Stably transfected OAT4-HEK293 cells revealed a partly sodium-dependent transport for 16α-OH-DHEAS with an apparent Km of 23.1 ± 5.1 μM and Vmax of 485.0 ± 39.1 pmol/mg protein/min, while stably transfected SOAT- and NTCP-HEK293 cells showed uptake only under sodium conditions with Km of 319.0 ± 59.5 μM and Vmax of 1465.8 ± 118.8 pmol/mg protein/min for SOAT and Km of 51.4 ± 9.9 μM and Vmax of 1423.3 ± 109.6 pmol/mg protein/min for NTCP. In contrast, stably transfected OATP2B1-HEK293 cells did not transport 16α-OH-DHEAS at all. Immunohistochemical studies and in situ hybridization of formalin fixed and paraffin embedded sections of human late term placenta showed expression of SOAT in syncytiotrophoblasts, predominantly at the apical membrane as well as in the vessel endothelium. In conclusion, OAT4, SOAT, and NTCP were identified as carriers for the estriol precursor 16α-OH-DHEAS. At least SOAT and OAT4 seem to play a functional role for the placental estriol synthesis as both are expressed in the syncytiotrophoblast of human placenta.
- Published
- 2014
15. The steroid metabolite 16(β)-OH-androstenedione generated by CYP21A2 serves as a substrate for CYP19A1
- Author
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Lina Schiffer, Yogan Khatri, Jens Neunzig, Alberto Sánchez-Guijo, Michaela F. Hartmann, Mohammed Milhim, Rita Bernhardt, Josef Zapp, and Stefan A. Wudy
- Subjects
0301 basic medicine ,Magnetic Resonance Spectroscopy ,Stereochemistry ,Endocrinology, Diabetes and Metabolism ,medicine.medical_treatment ,Metabolite ,Clinical Biochemistry ,Estrone ,Endocrine System ,Reductase ,Crystallography, X-Ray ,Endocrine System Diseases ,Biochemistry ,Catalysis ,Steroid ,Hydroxylation ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Endocrinology ,Aromatase ,medicine ,Escherichia coli ,Humans ,Androstenedione ,Molecular Biology ,biology ,Dose-Response Relationship, Drug ,Chemistry ,Aromatase Inhibitors ,Estrogen Receptor alpha ,Cell Biology ,Recombinant Proteins ,Androgen receptor ,Kinetics ,030104 developmental biology ,Receptors, Androgen ,030220 oncology & carcinogenesis ,Child, Preschool ,biology.protein ,Androgens ,Molecular Medicine ,Female ,Spectrophotometry, Ultraviolet ,Steroids ,Steroid 21-Hydroxylase - Abstract
The 21-hydroxylase (CYP21A2) is a steroidogenic enzyme crucial for the synthesis of mineralo- and glucocorticoids. It is described to convert progesterone as well as 17-OH-progesterone, through a hydroxylation at position C21, into 11-deoxycorticosterone (DOC) and 11-deoxycortisol (RSS), respectively. In this study we unraveled CYP21A2 to have a broader steroid substrate spectrum than assumed. Utilizing a reconstituted in vitro system, consisting of purified human CYP21A2 and human cytochrome P450 reductase (CPR) we demonstrated that CYP21A2 is capable to metabolize DOC, RSS, androstenedione (A4) and testosterone (T). In addition, the conversion of A4 rendered a product whose structure was elucidated through NMR spectroscopy, showing a hydroxylation at position C16-beta. The androgenic properties of this steroid metabolite, 16(β)-OH-androstenedione (16bOHA4), were investigated and compared with A4. Both steroid metabolites were shown to be weak agonists for the human androgen receptor. Moreover, the interaction of 16bOHA4 with the aromatase (CYP19A1) was compared to that of A4, indicating that the C16 hydroxyl group does not influence the binding with CYP19A1. In contrast, the elucidation of the kinetic parameters showed an increased Km and decreased kcat value resulting in a 2-fold decreased catalytic efficiency compared to A4. These findings were in accordance with our docking studies, revealing a similar binding conformation and distance to the heme iron of both steroids. Furthermore, the product of 16bOHA4, presumably 16-hydroxy-estrone (16bOHE1), was investigated with regard to its estrogenic activity, which was negligible compared to estradiol and estrone. Finally, 16bOHA4 was found to be present in a patient with 11-hydroxylase deficiency and in a patient with an endocrine tumor. Taken together, this study provides novel information on the steroid hormone biosynthesis and presents a new method to detect further potential relevant novel steroid metabolites.
- Published
- 2016
16. The urinary steroidome of treated children with classic 21-hydroxylase deficiency
- Author
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Michaela F. Hartmann, Clemens Kamrath, Stefan A. Wudy, Claudia Boettcher, and Lisa Wettstaedt
- Subjects
0301 basic medicine ,Male ,endocrine system diseases ,Hydrocortisone ,Endocrinology, Diabetes and Metabolism ,Clinical Biochemistry ,urologic and male genital diseases ,Biochemistry ,Cohort Studies ,0302 clinical medicine ,Endocrinology ,Reference Values ,Child ,biology ,Chemistry ,21-Hydroxylase ,Child, Preschool ,Fludrocortisone ,Androgens ,Molecular Medicine ,Female ,Steroids ,Glucocorticoid ,medicine.drug ,medicine.medical_specialty ,Adolescent ,medicine.drug_class ,Urinary system ,030209 endocrinology & metabolism ,Urinalysis ,Androsterone ,Gas Chromatography-Mass Spectrometry ,03 medical and health sciences ,Internal medicine ,medicine ,Humans ,Congenital adrenal hyperplasia ,Adrenarche ,Molecular Biology ,Glucocorticoids ,Retrospective Studies ,Adrenal Hyperplasia, Congenital ,Body Weight ,Cell Biology ,Androgen ,medicine.disease ,Body Height ,030104 developmental biology ,biology.protein ,Steroid 21-Hydroxylase - Abstract
Monitoring treatment of children with classic congenital adrenal hyperplasia (CAH) is difficult and biochemical targets are not well defined. We retrospectively analysed 576 daily urinary steroid hormone metabolite profiles determined by gas chromatography-mass spectrometry of 150 children aged 3.0-17.9 years with classic 21-hydroxylase deficiency (21-OHD) on hydrocortisone and fludrocortisone treatment. Daily urinary excretion of glucocorticoid-, 17α-hydroxyprogesterone (17-OHP)-, and androgen metabolites as well as growth and weight gain are presented. Children with classic CAH exhibited increased height velocity during prepubertal age, which was then followed by diminished growth velocity during pubertal age until final height was reached. Final height was clearly below the population mean. 11β-Hydroxyandrosterone was the dominant urinary adrenal-derived androgen metabolite in CAH children. Adrenarche is blunted in children with CAH under hydrocortisone treatment and androgen metabolites except 11β-hydroxyandrosterone were suppressed. Cortisol metabolite excretion reflected supraphysiological hydrocortisone treatment dosage, which resulted in higher body-mass-indices in children with CAH. Reference values of daily urinary steroid metabolite excretions of treated children with CAH allow the clinician to adequately classify the individual patient regarding the androgen-, 17-OHP-, and glucocorticoid status in the context of the underlying disorder. Additionally, urinary 21-OHD-specific reference ranges will be important for research studies in children with CAH.
- Published
- 2016
17. Profiling of bile acids in bovine follicular fluid by fused-core-LC-MS/MS
- Author
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Stefan A. Wudy, Carina Blaschka, Alberto Sánchez-Guijo, Michaela F. Hartmann, and Christine Wrenzycki
- Subjects
0301 basic medicine ,Taurine ,Spectrometry, Mass, Electrospray Ionization ,Lithocholic acid ,Endocrinology, Diabetes and Metabolism ,education ,Clinical Biochemistry ,Glycocholic acid ,Cholic Acid ,Chenodeoxycholic Acid ,01 natural sciences ,Biochemistry ,Bile Acids and Salts ,03 medical and health sciences ,chemistry.chemical_compound ,Endocrinology ,Limit of Detection ,Tandem Mass Spectrometry ,Chenodeoxycholic acid ,Animals ,Molecular Biology ,Chromatography, High Pressure Liquid ,Chromatography ,010401 analytical chemistry ,Deoxycholic acid ,Cholic acid ,Cell Biology ,Follicular fluid ,0104 chemical sciences ,Triple quadrupole mass spectrometer ,Follicular Fluid ,030104 developmental biology ,chemistry ,Molecular Medicine ,Cattle ,Female ,Lithocholic Acid ,Deoxycholic Acid - Abstract
Bile acids (BAs) are present in follicular fluid (FF) from humans and cattle. This fact has triggered an interest on the role BAs might play in folliculogenesis and their possible association with fertility. To achieve a better understanding about this subject, new methods are needed to provide reliable information about concentrations of the most important BAs in FF. In this context, liquid chromatography-tandem mass spectrometry (LC-MS/MS) offers high specificity with a relatively simple sample workup. We developed and validated a new assay for the quick profiling of the 9 most abundant BAs in follicular fluid from cattle. The method uses 200μl of FF and can quantify cholic acid (CA), chenodeoxycholic acid (CDCA), deoxycholic acid (DCA) and their glycine (G) and taurine (T) conjugates. Lithocholic acid (LCA), its conjugates GLCA and TLCA, and sulfated forms, were present in some samples, but their concentration was low compared to other BAs (in average, below 60ng/ml for LCA, GLCA or TLCA and below 20ng/ml for their corresponding sulfates). Method performance was studied at three quality controls for each compound in consonance with their physiological concentration. Excellent linearity and recovery were found for all compounds at every control level. Intra-day and between-day precisions (%CV) and accuracies (relative errors) were below 15% for all the compounds. Matrix effects were negligible for most of the analytes. Samples undergoing freeze-thaw showed no degradation of their BAs. The method makes use of a fused-core phenyl column coupled to a triple quadrupole tandem mass spectrometer to achieve chromatographic separation within 5min. We quantified BAs grouped in four different follicle sizes (3-5mm, 6-8mm, 9-14mm, >15mm), obtaining a similar relative BA profile for all the sizes, with CA always in higher concentration, ranging between 1600 and 18000ng/ml, approximately, followed by its conjugate glycocholic acid, GCA, which ranged between 800 and 9000ng/ml. The highest concentration in CA, DCA or CDCA was always detected in FF stemming from follicles of 6-8mm. To our knowledge, this is the first report in which BAs subspecies have been detected and quantified in bovine follicular fluid.
- Published
- 2015
18. Phenotypic, metabolic, and molecular genetic characterization of six patients with congenital adrenal hyperplasia caused by novel mutations in the CYP11B1 gene
- Author
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Stefan A. Wudy, Frank Hannemann, Michaela F. Hartmann, Huy Hoang Nguyen, Ewa M. Malunowicz, Rita Bernhardt, and Antje Eiden-Plach
- Subjects
0301 basic medicine ,Male ,Endocrinology, Diabetes and Metabolism ,Clinical Biochemistry ,Nonsense mutation ,Mutation, Missense ,030209 endocrinology & metabolism ,Biology ,Compound heterozygosity ,medicine.disease_cause ,Biochemistry ,03 medical and health sciences ,Exon ,0302 clinical medicine ,Endocrinology ,Chlorocebus aethiops ,medicine ,Missense mutation ,Animals ,Humans ,Congenital adrenal hyperplasia ,Molecular Biology ,Genetics ,Mutation ,Adrenal Hyperplasia, Congenital ,Intron ,Cell Biology ,Exons ,medicine.disease ,Molecular biology ,Introns ,Pedigree ,030104 developmental biology ,COS Cells ,Molecular Medicine ,Steroid 11-beta-Hydroxylase ,Female ,Steroids ,RNA Splice Sites ,Minigene - Abstract
Congenital adrenal hyperplasia (CAH) is an autosomal recessive inherited disorder of steroidogenesis. Steroid 11β-hydroxylase deficiency (11β-OHD) due to mutations in the CYP11B1 gene is the second most common form of CAH. In this study, 6 patients suffering from CAH were diagnosed with 11β-OHD using urinary GC-MS steroid metabolomics analysis. The molecular basis of the disorder was investigated by molecular genetic analysis of the CYP11B1 gene, functional characterization of splicing and missense mutations, and analysis of the missense mutations in a computer model of CYP11B1. All patients presented with abnormal clinical signs of hyperandrogenism. Their urinary steroid metabolomes were characterized by excessive excretion rates of metabolites of 11-deoxycortisol as well as metabolites of 11-deoxycorticosterone, and allowed definite diagnosis. Patient 1 carries compound heterozygous mutations consisting of a novel nonsense mutation p.Q102X (c.304C>T) in exon 2 and the known missense mutation p.T318R (c.953C>G) in exon 5. Two siblings (patient 2 and 3) were compound heterozygous carriers of a known splicing mutation c.1200+1G>A in intron 7 and a known missense mutation p.R448H (c.1343G>A) in exon 8. Minigene experiments demonstrated that the c.1200+1G>A mutation caused abnormal pre-mRNA splicing (intron retention). Two further siblings (patient 4 and 5) were compound heterozygous carriers of a novel missense mutation p.R332G (c.994C>G) in exon 6 and the known missense mutation p.R448H (c.1343G>A) in exon 8. A CYP11B1 activity study in COS-1 cells showed that only 11% of the enzyme activity remained in the variant p.R332G. Patient 6 carried a so far not described homozygous deletion g.2470_5320del of 2850 bp corresponding to a loss of the CYP11B1 exons 3-8. The breakpoints of the deletion are embedded into two typical 6 base pair repeats (GCTTCT) upstream and downstream of the gene. Experiments analyzing the influence of mutations on splicing and on enzyme function were applied as complementary procedures to genotyping and provided a rational basis for understanding the clinical phenotype of CAH.
- Published
- 2015
19. A steroidogenic pathway for sulfonated steroids: the metabolism of pregnenolone sulfate
- Author
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Alberto Sánchez-Guijo, Jens Neunzig, Joachim Geyer, Stefan A. Wudy, A. Mosa, Michaela F. Hartmann, and Rita Bernhardt
- Subjects
endocrine system ,medicine.medical_specialty ,Endocrinology, Diabetes and Metabolism ,medicine.medical_treatment ,Clinical Biochemistry ,Hydroxylation ,Biochemistry ,Steroid ,chemistry.chemical_compound ,Endocrinology ,Internal medicine ,Cytochrome b5 ,medicine ,Escherichia coli ,Humans ,Molecular Biology ,Cholesterol side-chain cleavage enzyme ,Steroid 17-alpha-Hydroxylase ,Cell Biology ,Metabolism ,Cytochromes b5 ,HEK293 Cells ,chemistry ,CYP17A1 ,Pregnenolone ,Molecular Medicine ,Pregnenolone sulfate ,medicine.drug ,Plasmids - Abstract
In many tissues sulfonated steroids exceed the concentration of free steroids and recently they were also shown to fulfill important physiological functions. While it was previously demonstrated that cholesterol sulfate (CS) is converted by CYP11A1 to pregnenolone sulfate (PregS), further conversion of PregS has not been studied in detail. To investigate whether a steroidogenic pathway for sulfonated steroids exists similar to the one described for free steroids, we examined the interaction of PregS with CYP17A1 in a reconstituted in-vitro system. Difference spectroscopy revealed a Kd-value of 74.8±4.2μM for the CYP17A1-PregS complex, which is 2.5-fold higher compared to the CYP17A1-pregnenolone (Preg) complex. Mass spectrometry experiments proved for the first time that PregS is hydroxylated by CYP17A1 at position C17, identically to pregnenolone. A higher Km- and a lower kcat-value for CYP17A1 using PregS compared with Preg were observed, indicating a 40% reduced catalytic efficiency when using the sulfonated steroid. Furthermore, we analyzed whether the presence of cytochrome b5 (b5) has an influence on the CYP17A1 dependent conversion of PregS, as was demonstrated for Preg. Interestingly, with 17OH-PregS no scission of the 17,20-carbon-carbon bond occurs, when b5 is added to the reconstituted in-vitro system, while b5 promotes the formation of DHEA from 17OH-Preg. When using human SOAT-HEK293 cells expressing CYP17A1 and CPR, we could confirm that PregS is metabolized to 17OH-PregS, strengthening the potential physiological meaning of a pathway for sulfonated steroids.
- Published
- 2014
20. The balance of cortisol-cortisone interconversion is shifted towards cortisol in neonates with congenital adrenal hyperplasia due to 21-hydroxylase deficiency
- Author
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Stefan A. Wudy, Michaela F. Hartmann, and Clemens Kamrath
- Subjects
medicine.medical_specialty ,Hydrocortisone ,Cortisol/Cortisone ,Endocrinology, Diabetes and Metabolism ,Urinary system ,Clinical Biochemistry ,Biology ,Biochemistry ,Gas Chromatography-Mass Spectrometry ,Endocrinology ,Congenital adrenal hyperplasia due to 21-hydroxylase deficiency ,Internal medicine ,medicine ,Humans ,Congenital adrenal hyperplasia ,Molecular Biology ,Glucocorticoids ,Retrospective Studies ,Adrenal Hyperplasia, Congenital ,Infant, Newborn ,Infant ,Cell Biology ,Metabolism ,medicine.disease ,Prognosis ,Cortisone ,Case-Control Studies ,Molecular Medicine ,hormones, hormone substitutes, and hormone antagonists ,Glucocorticoid ,Homeostasis ,Biomarkers ,medicine.drug ,Follow-Up Studies - Abstract
Patients with congenital adrenal hyperplasia due to 21-hydroxylase deficiency (21OHD) have an impaired cortisol synthesis, but it is unknown whether the metabolism of glucocorticoids differs between neonates and infants with and without 21OHD.The objective of this study was to compare the glucocorticoid metabolism between neonates and infants with and without 21OHD.We analyzed 14 urinary glucocorticoid metabolites, 7 metabolites each of cortisol and cortisone, by gas chromatography-mass spectrometry of 89 untreated 21OHD neonates and infants and 161 neonates and infants without 21OHD.Neonates with 21OHD exhibit elevated relative amounts of cortisol metabolites in total glucocorticoid metabolism and an increased ratio of cortisol to cortisone metabolites (p0.0001). This reflects a shift toward cortisol in the relative balance of the interconversion between cortisol and cortisone. The ratio of cortisol to cortisone metabolites correlated significantly with low urinary glucocorticoid concentrations (p0.03), with low 21-hydroxylase activity (p0.001) and high urinary sodium and chloride concentrations (p0.05) in neonates with 21OHD.Our results demonstrate substantial changes in the relative cortisone to cortisol interconversion in neonates with 21OHD. The shift of glucocorticoid metabolism toward active cortisol in neonates with 21OHD seems to be related to the severity of 21OHD and adrenal dysfunction. Our data provide new insights into the regulation of glucocorticoid homeostasis in 21OHD.
- Published
- 2014
21. Human aldosterone synthase: recombinant expression in E. coli and purification enables a detailed biochemical analysis of the protein on the molecular level
- Author
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Michaela F. Hartmann, Michael C. Hutter, Rita Bernhardt, Norio Kagawa, Stefan A. Wudy, Frank Hannemann, and Anna Hobler
- Subjects
Aldosterone synthase ,Models, Molecular ,medicine.drug_class ,Endocrinology, Diabetes and Metabolism ,Clinical Biochemistry ,Biochemistry ,Hydroxylation ,chemistry.chemical_compound ,Endocrinology ,Biosynthesis ,medicine ,Escherichia coli ,Cytochrome P-450 CYP11B2 ,Humans ,Homology modeling ,Enzyme Inhibitors ,Molecular Biology ,chemistry.chemical_classification ,Aldosterone ,biology ,Cell Biology ,Recombinant Proteins ,Enzyme ,Ketoconazole ,chemistry ,Docking (molecular) ,Mineralocorticoid ,biology.protein ,Molecular Medicine ,Corticosterone - Abstract
Aldosterone, the most important human mineralocorticoid, is involved in the regulation of the blood pressure and has been reported to play a key role in the formation of arterial hypertension, heart failure and myocardial fibrosis. Aldosterone synthase (CYP11B2) catalyzes the biosynthesis of aldosterone by successive 11β- and 18-hydroxylation followed by an 18-oxidation of 11-deoxycorticosterone and thus comprises an important drug target. For more than 20 years, all attempts to purify recombinant human CYP11B2 in significant amounts for detailed analysis failed due to its hydrophobic nature as a membrane protein. Here, we present the successful expression of the protein in E. coli yielding approx. 90 nmol/l culture, its purification and detailed enzymatic characterization. Biochemical analyses have been performed using in vitro conversion assays which revelead a Vmax of 238 ± 8 nmol products/nmol hCYP11B2/min and a Km of 103 ± 8 μM 11-deoxycorticosterone. Furthermore, binding analyses indicated a very loose binding of the first intermediate of the reaction, corticosterone with a Kd value of 115 ± 6 μM whereas for 11-deoxycorticosterone a Kd of 1.34 ± 0.13 μM was estimated. Upon substrate conversion of 11-deoxycorticosterone, new intermediates have been identified as 19- and 18-hydroxylated products not described before for the human enzyme. To understand the differences in substrate conversion, we constructed a new homology model based on the 3D structure of CYP11A1, performed docking studies and calculated the activation energy for hydrogen abstraction of the different ligands. The data demonstrated that the 11β-hydroxylation requires much less abstraction energy than hydroxylation at C18 and C19. However, the C18 and C19 hydroxylated products might be of clinical importance. Finally, purified CYP11B2 represents a suitable tool for the investigation of potential inhibitors of this protein for the development of novel drugs against hypertension and heart failure as was shown using ketoconazole.
- Published
- 2011
22. Alu Sx repeat-induced homozygous deletion of the StAR gene causes lipoid congenital adrenal hyperplasia
- Author
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Stefan A. Wudy, Huy-Hoang Nguyen, Frank Hannemann, Rita Bernhardt, Antje Eiden-Plach, Michaela F. Hartmann, and Ursula Schneider
- Subjects
medicine.medical_specialty ,Endocrinology, Diabetes and Metabolism ,Fludrocortisone ,Lipoid congenital adrenal hyperplasia ,Clinical Biochemistry ,Molecular Sequence Data ,Alu element ,Biology ,Biochemistry ,Primary Adrenal Insufficiency ,chemistry.chemical_compound ,Exon ,Endocrinology ,Alu Elements ,Internal medicine ,medicine ,Humans ,Metabolomics ,Cholesterol Side-Chain Cleavage Enzyme ,Allele ,Molecular Biology ,Gene ,Sequence Deletion ,Genetics ,Aldosterone ,Disorder of Sex Development, 46,XY ,Adrenal Hyperplasia, Congenital ,Base Sequence ,Homozygote ,Cell Biology ,medicine.disease ,Phosphoproteins ,Pedigree ,chemistry ,Molecular Medicine ,Female ,medicine.drug - Abstract
Lipoid congenital adrenal hyperplasia (Lipoid CAH) is the most severe form of the autosomal recessive disorder CAH. A general loss of the steroid biosynthetic activity caused by defects in the StAR gene manifests as life-threatening primary adrenal insufficiency. We report a case of Lipoid CAH caused by a so far not described homozygous deletion of the complete StAR gene and provide diagnostic results based on a GC-MS steroid metabolomics and molecular genetic analysis. The patient presented with postnatal hypoglycemia, vomiting, adynamia, increasing pigmentation and hyponatremia. The constellation of urinary steroid metabolites suggested Lipoid CAH and ruled out all other forms of CAH or defects of aldosterone biosynthesis. After treatment with sodium supplementation, hydrocortisone and fludrocortisone the child fully recovered. Molecular genetic analysis demonstrated a homozygous 12.1 kb deletion in the StAR gene locus. The breakpoints of the deletion are embedded into two typical genomic repetitive Alu Sx elements upstream and downstream of the gene leading to the loss of all exons and regulatory elements. We established deletion-specific and intact allele-specific PCR methods and determined the StAR gene status of all available family members over three generations. This analysis revealed that one of the siblings, who died a few weeks after birth, carried the same genetic defect. Since several Alu repeats at the StAR gene locus increase the probability of deletions, patients with typical symptoms of lipoid CAH lacking evidence for the presence of both StAR alleles should be analyzed carefully for this kind of disorder.
- Published
- 2011
23. The human adrenal gland as a drug metabolizer: First in-vivo evidence for the conversion of steroidal drugs
- Author
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Rita Bernhardt, Stefan A. Wudy, Michaela F. Hartmann, and Martin Reincke
- Subjects
0301 basic medicine ,Male ,Endocrinology, Diabetes and Metabolism ,Clinical Biochemistry ,Pharmacology ,Spironolactone ,Biochemistry ,Gas Chromatography-Mass Spectrometry ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Endocrinology ,Mineralocorticoid receptor ,In vivo ,Canrenone ,Adrenal Glands ,medicine ,Cytochrome P-450 CYP11B2 ,Humans ,Steroid 11-beta-hydroxylase ,Molecular Biology ,Active metabolite ,Aged ,Mineralocorticoid Receptor Antagonists ,Chemistry ,Adrenal gland ,Cell Biology ,Middle Aged ,030104 developmental biology ,medicine.anatomical_structure ,030220 oncology & carcinogenesis ,Molecular Medicine ,Steroid 11-beta-Hydroxylase ,Drug metabolism ,medicine.drug - Abstract
The metabolism of drugs in mammals is attributed mainly to the liver and its cytochromes P450 localized in the endoplasmic reticulum. Here, we demonstrate for the first time in humans that there is no strict subdivision between P450 s involved in exogenous and endogenous metabolism. We determined the widely used mineralocorticoid receptor antagonist spironolactone, its active metabolite canrenone and their metabolites in the adrenal venous blood of treated patients with gas chromatography-mass spectrometry. 11- and 18-hydroxylated metabolites of canrenone were found in the efferent right and left adrenal veins, indicating that they were produced by the adrenal mitochondrial cytochromes P450 CYP11B1 and CYP11B2. Thus, the adrenal has to be considered as a new organ for drug metabolism. In future, application of drugs may need further investigations concerning side effects due to interactions with adrenal enzymes.
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