Steroid Biochemistry

Technological Advances

Hiort O, Ahmed SF (eds): Understanding Differences and Disorders of Sex Development (DSD). Endocr Dev. Basel, Karger 2014, vol 27, pp 41–52 (DOI: 10.1159/000363612)

a a b Clemens Kamrath · Stefan A. Wudy · Nils Krone a Division of Pediatric Endocrinology and Diabetology, Laboratory for Translational Hormone Analytics in Pediatric Endocrinology, Steroid Research and Mass Spectrometry Unit, Centre of Child and Adolescent b Medicine, Justus Liebig University, Giessen, Germany; Centre for Endocrinology, Diabetes and Metabolism, School of Clinical and Experimental Medicine, University of Birmingham, Birmingham, UK

Abstract Accurate analysis of steroid hormones represents an essential part in the evaluation of a patient with disorders or differences in sex development. Analytical methods based on mass spectrometry (MS) have become the state-of-the-art methodology allowing for the most specific qualitative and quantitative determination of steroid hormones and their metabolites. Liquid chromatography linked with tandem MS (LC-MS/MS) allows for rapid as well as highly specific and sensitive targeted steroid hormone analysis of multiple analytes from a single sample. Urinary steroid proﬁle analysis by gas chromatography (GC)-MS is a non-invasive diagnostic approach and provides qualitative and quantitative data on the global excretion of steroid hormone metabolites. GC-MS remains the most powerful discovery tool for defining inborn errors of steroidogenesis, whereas LC-MS/MS represents a highly sensitive and specific method for targeted steroid hormone analysis. © 2014 S. Karger AG, Basel

Steroidogenesis

The majority of steroidogenic enzymes are cytochrome P450 (CYP) enzymes catalys- ing redox reactions. These biochemical conversions crucially rely on electron supply via specific electron transfer chains. CYP type 1 enzymes are localised to the mito- chondrion and receive their electrons via adrenodoxin reductase and adrenodoxin. P450 side-chain cleavage enzyme (CYP11A1), 11β-hydroxylase (CYP11B1) and aldosterone synthase (CYP11B2) are all CYP type 1 enzymes. In contrast, 17α-hydroxylase (CYP17A1), 21-hydroxylase (CYP21A2) and aromatase (CYP19A1) are CYP type 2 enzymes localised to the endoplasmic reticulum. These microsomal CYP enzymes rely on electron transfer from P450 oxidoreductase (POR) to facilitate the hydroxylation reactions [1]. The acute stimulation of steroidogenesis is mediated at the level of cholesterol im- port into mitochondria, which is facilitated by the steroidogenic acute regulatory Downloaded by: Siriraj Medical Library, Mahidol University 202.28.191.34 - 3/1/2015 6:35:26 PM Cholesterol outer StAR mitochondrial membrane inner Cholesterol ADR/Adx CYP11A1 PAPSS2 POR POR b5 Dehydroepi SULT2A1 Pregnenolone CYP17A1 17OH-Pregnenolone CYP17A1 androsterone DHEA-S Pregnenediol Pregnenetriol DHEA, 16αOH-DHEA HSD3B2 HSD3B2 HSD3B2 Androsterone, Etiocholanolone

POR POR b5 POR Progesterone CYP17A1 17OH-Progesterone CYP17A1 Androstenedione CYP19A1 Oestrone POR Pregnanediol POR Pregnanetriol Androsterone CYP21A2 CYP21A2 17OH-Pregnanolone HSD17B Etiocholanolone HSD17B * POR 11-deoxycorticosterone 11-deoxycortisol Testosterone CYP19A1 OESTRADIOL

ADR/Adx THDOC, 5αTHDOC ADR/Adx THS Androsterone Oestriol CYP11B2 CYP11B1 21-deoxycortisol SRD5A2 Etiocholanolone Pregnanetriolone

Corticosterone CORTISOL DIHYDROTESTOSTERONE THA, THB THF, 5αTHF Androsterone ADR/Adx 5αTHA, 5αTHB H6PDH CYP11B2 HSD11B1 HSD11B2

18OH-corticosterone Cortisone ADR/Adx 18OH-THA THE CYP11B2

ALDOSTERONE THALDO

Fig. 1. Steroidogenesis. After the StAR protein-mediated uptake of cholesterol into mitochondria, aldosterone, cortisol and androgens are synthesized through the co-ordinated action of a series of steroidogenic enzymes in a zone-specific fashion. The mitochondrial CYP type I enzymes (CYP11A1, CYP11B1, CYP11B2) requiring electron transfer via adrenodoxin reductase (ADR) and adrenodoxin (Adx) are marked (box la- belled ‘ADR/Adx’). The microsomal CYP II enzymes (CYP17A1, CYP21A2, CYP19A1) receive electrons from POR (indicated by circled POR). In addition to POR, the 17,20-lyase reaction catalysed by CYP17A1 also requires CYB5 (indicated by circled b5). Urinary steroid hormone metabolites are given in italics below the plasma hormones. The asterisk (*) indicates the 11β-hydroxylation of 17OHP to 21-deoxycortisol in 21OHD. The adrenal conversion of androstenedione to testosterone is catalysed by AKR1C3 (HSD17B5), whereas the testicular conversion is facilitated by HSD17B3. SULT2A1 = Sulfotransferase 2A1; PAPPS2 = 3 -phos- phoadenosine 5 -phosphosulfate synthase 2; DHEA-S = dehydroepiandrosterone sulphate. ′ ′

(StAR) protein [2]. Thereafter, the first step of steroid hormone biosynthesis is the conversion of cholesterol to pregnenolone catalysed by the mitochondrial P450 side- chain cleavage enzyme (P450scc, CYP11A1; fig. 1). The StAR/CYP11A1 system is the quantitative regulator of steroidogenesis, whereas the CYP17A1 enzyme represents the qualitative regulator determining the class of

42 Kamrath · Wudy · Krone

Hiort O, Ahmed SF (eds): Understanding Differences and Disorders of Sex Development (DSD). Endocr Dev. Basel, Karger 2014, vol 27, pp 41–52 (DOI: 10.1159/000363612) Downloaded by: Siriraj Medical Library, Mahidol University 202.28.191.34 - 3/1/2015 6:35:26 PM produced steroid hormone. In the zona glomerulosa, absence of CYP17A1 leads to mineralocorticoid synthesis. In contrast, isolated 17α-hydroxylase activity of CYP17A1 results in the synthesis of glucocorticoids in the zona fasciculata. Combined activities of CYP17A1 catalyse both the 17α-hydroxylation and 17,20-lyase reaction of 21-carbon (C21) steroids to 19-carbon (C19) precursors of sex steroids in the adrenal zona reticularis and gonads. The main pathway to sex steroids facilitated by CYP17A1 is the conversion of 17α-hydroxypregnenolone (17Preg) to dehydroepiandrosterone (DHEA; Δ5 pathway), whereas the conversion of 17α-hydroxyprogesterone (17OHP) to androstenedione (Δ4 pathway) is hardly relevant under physiological circumstances. The significant predominance of the Δ5 pathway via DHEA in humans is explained by substrate preference of CYP17A1 for 17Preg. The catalytic efficiency of this conversion is about 100-fold higher for the Δ5-steroid 17Preg than for the Δ4-steroid 17OHP. For the 17,20-lyase reaction, CYP17A1 crucially relies on cytochrome b5 (CYB5), which is expressed in the gonads and in the adrenal zona reticularis at the onset of adrenarche [2]. CYB5 acts as an allosteric factor that fosters the interactions of POR with CYP17A1, enhancing 17,20-lyase activity without influencing 17-hydroxylase activity. Another major physiological role of CYB5 is the reduction of methaemoglobin. DHEA is further converted by 3β-hydroxysteroid dehydrogenase (HSD) type 2 (HSD3B2) in the gonads and adrenals to androstenedione. In addition, HSD3B2 is also required for the conversion reactions of pregnenolone to progesterone, and 17Preg to 17OHP. Androstenedione is reduced by 17β-HSD type 3 (HSD17B3) in the testes to testosterone. In androgen target tissues, testosterone is further converted to dihydrotestosterone (DHT) by steroid 5α-reductase type 2 (SRD5A2) [2]. It has recently been suggested that 5α-reduction of testosterone is not the only biosynthetic pathway to produce DHT. In addition, androstanediol can be 3α-oxidated in the target tissues to form DHT via the so-called ‘backdoor pathway’ (fig. 2). In this pathway, androstanediol is hypothesised to derive from sequential 5α- and 3α-reduction of 17OHP to 17α-hydroxyallopregnanolone, followed by its conversion to androsterone by 17,20-lyase activity of CYP17A1, and then its reduction by HSD17B activity (fig. 2) [3]. The physiological relevance of this pathway remains unclear; however, it is likely to play a significant role in the pathophysiology of distinct conditions such as POR deficiency (PORD) or 21-hydroxylase deficiency (21OHD) [4, 5]. Adrenal 21-hydroxylase (CYP21A2) converts 17OHP to 11-deoxycortisol and progesterone to 11-deoxycorticosterone (DOC), both precursors of cortisol and aldosterone biosynthesis. The final steps in the synthesis of glucocorticoids and mineralocorticoids are catalysed by two closely related mitochondrial enzymes, 11β-hydroxylase (CYP11B1) and aldosterone synthase (CYP11B2). Steroid 11β-hydroxylase converts 11-deoxycortisol to cortisol and DOC to corticosterone. CYP11B1 is expressed pre- dominantly in the zona fasciculata, and to a lesser extent in the zona reticularis, but not in the zona glomerulosa. Aldosterone synthase is found only in the zona glomer-

Steroid Biochemistry 43

Δ5

CYP17A1 POR Testosterone OH HO HO HO CYB5 Pregnenolone 17α-Hydroxy- 3β-HSD2 O pregnenolone (HSD3B2) 3β-HSD2 (HSD3B2) O 4 OH Δ 17β-HSD O CYP17A1 O (AKR1C3) 5α-Reductase 17α-Hydroxy- POR 4-Androstenedione (SRD5A1,2) progesterone CYB5 O Dihydrotestosterone 5α-Reductase OH (SRD5A1,2)

O 17α-Hydroxy- OH dihydroprogesterone

3α-HSD 3α-HSD O Backdoor (HSD17B6) AKR1C2,4 ( ) O OH

O 17α-Hydroxy- OH allopregnanolone

CYP17A1 POR 17β-HSD CYB5 HO (HSD17B3/AKR1C3) HO HO Androsterone Androstanediol

Fig. 2. Pathways to androgen synthesis. Conventional pathways are shown in black, whereas the backdoor pathway is shown in red. Cofactors are shown in blue. The conversion of 17OHP to 17α-hydroxyallopregnanolone is mediated by sequential 5α- and 3α-reduction. In humans, type 1 3α-HSD (encoded by AKR1C4), which is expressed mainly in the liver, but also in the foetal and adult adrenal, is the major reductive 3α-HSD. Additionally, the type 3 3α-HSD (encoded by AKR1C2) also catalyses the reduction of 17α-hydroxydihydroprogesterone to 17α-hydroxyallopregnanolone. The next step on the backdoor route to DHT is the conversion of the 21-carbon steroid 17α-hydroxyallopregnanolone into the 19-carbon androgen androsterone by the 17,20-lyase activity of CYP17A1. Next, androsterone is reduced to androstanediol. Beside the type 3 17β-HSD (encoded by HSD17B3), which is dominantly expressed in the testes, type 2 3α-HSD (also known as type 5 17β-HSD; encoded by AKR1C3) shows a pattern of activity similar to that of the testicular type 3 17β-HSD and additionally reduces androsterone to androstanediol with high catalytic efficiency in humans. AKR1C3 is expressed in many different tissues, such as liver, fat tissue and adrenal cortex, and is responsible for the formation of androgens in women. The 3-hydroxyepimerase (also known as retinol/sterol dehydrogenase; encoded by HSD17B6), with the highest activity levels found in liver and testis, and lower levels in lung, spleen, brain, kidney and ovary, oxidizes androstanediol to DHT in humans [2, 4].

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Measurement of Steroids in Body Fluids

The accurate analysis of steroid hormones and their metabolites represents an important part in the diagnostic work-up in patients with disorders of sex development (DSD). Direct immunoassays without preanalytic steps of extraction and chromatography are obsolete in paediatric endocrinology due to their low accuracy and specificity resulting from cross-reactivity with steroid precursors, metabolites and/or conju- gates, as well as matrix effects. In addition, immuno-based methods in particular are challenging in low concentration ranges. Significant interassay variability has been reported for some steroids, such as testosterone, oestradiol and progesterone [6]. Thus, in cases of DSD, plasma or serum steroids should be measured by either immunoassays after organic solvent extraction and chromatographic purification in specialized paediatric laboratories, or preferably by mass spectrometry (MS)-based methods. Analytical methods based on MS currently present the most specific qualitative and quantitative methods for the measurement of steroid hormones and steroid hormone metabolites. Combination with a chromatographic technique such as gas chromatography (GC) or liquid chromatography (LC) allows for the simultaneous determination (‘profiling’) of analytes. Of all the separating techniques, GC bears the great- est potential for separating steroids and MS allows for the highest specificity in determining steroid metabolites. It is likely that MS methods will improve the diagnosis and management of DSD; however, systematic data such as in the evaluation of the HPA axis [7] are lacking. Urinary steroid proﬁle analysis by GC-MS provides qualitative and quantitative data on the excretion of steroid metabolites. It remains the most powerful discovery tool for defining inborn steroid disorders. Almost all disorders of steroid hormone biosynthesis and metabolism have been characterized and first defined following urine steroid analysis using GC-MS. GC-MS urinary steroid profiling allows the diagnosis of most inborn errors of steroid biosynthesis by identifying characteristic steroid metabolites and by calculating ratios between precursor metabolites and product metabolites (tables 1, 2) [8]. Usually, samples from spot urine are sufficient. A particular benefit of GC-MS is its potential for non-targeted approaches, providing a most comprehensive metabolic ‘fingerprint’ of a biological sample. In general, urinary steroids are extracted, hydrolysed and derivatised, and thereafter subjected to GC- MS. Urinary steroid profiling by GC-MS is used to analyse the metabolites of steroid hormones and their precursors. The catabolism of steroid hormones consists of a series of reduction, hydroxylation and conjugation reactions (fig. 1).

Steroid Biochemistry 45

Full trivial name Abbreviation Metabolite of

Androsterone An Androstenedione testosterone, 5α-dihydrotestosterone, DHEA Etiocholanolone Et Testosterone, DHEA Dehydroepiandrosterone DHEA (and sulphate) Dehydroepiandrosterone 11β-hydroxy-androsterone 11β-OH-An 11β-OH-androstenedione, cortisol 11β-hydroxy-etiocholanolone 11β-OH-Et Cortisol 11-oxo-etiocholanolone 11-OXO-Et Cortisol 16α-OH-DHEA 16α-DHEA Dehydroepiandrosterone, dehydroepiandrosterone-sulphate Pregnanediola PDa Progesterone, 11-deoxycorticosterone Pregnenediol 5PD Pregnenolone Pregnadienol Pregnadienolb Pregnenolone (pregnenediol) 17-OH-pregnanolone 17HP 17-OH-progesterone 3α5α-17-OH-pregnanolone 3α5α17HP 17-OH-progesterone and other foetal origin Pregnanetriol PT 17-OH-progesterone 5-Pregnenetriol 5PT 17-OH-pregnenolone Pregnanetriolone PTONE 21-Desoxycortisol Tetrahydrodeoxycorticosterone THDOC 11-Deoxycorticosterone 5α-Tetrahydrodeoxycorticosterone 5αTHDOC 11-Deoxycorticosterone Tetrahydro-11-desoxycortisol THS 11-Desoxycortisol Tetra-11-dehydrocorticosterone THA Corticosterone 5α-Tetra-11-dehydrocorticosterone 5αTHA Corticosterone Tetrahydrocorticosterone THB Corticosterone 5α-Tetrahydrocorticosterone 5αTHB Corticosterone Tetrahydroaldosterone THALDO Aldosterone Tetrahydrocortisone THE Cortisol, cortisone Tetrahydrocortisol THF Cortisol 5α-Tetrahydrocortisol 5αTHF Cortisol α-Cortolone α-Cortolone Cortisol, cortisone β-Cortolone β-Cortolone Cortisol, cortisone α-Cortol α-Cortol Cortisol β-Cortol β-Cortol Cortisol 6β-OH-cortisol 6βOHcortisol Cortisol a When not specified the A-ring configuration is 3α,5β in all abbreviations. b A chemical artefact formed exclusively from 5PD (pregnenediol) disulphate.

LC tandem MS (LC-MS/MS) is increasingly used in clinical laboratories; however, it is still not widely available. Employing LC-MS/MS technology is likely to lead to superior quality of steroid measurements due to increased analytical specificity and sensitivity. Due to the high sensitivity it is ideally suited for hormone measurements in low concentration ranges, which is often a problem in paediatric practice. LC-MS/ MS is ideally suited for high-throughput settings and the methodology requires min- imal sample preparation. Steroid profiling by LC-MS/MS represents a very effective method for distinguishing almost all steroid-related disorders in targeted approaches. Despite methodological improvements, the measurement of some steroid hormones,

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Enzyme defect (gene) Diagnostic ratio

21-Hydroxylase deficiency (CYP21A2) PTONE/THE 11β-Hydroxylase deficiency (CYP11B1) THS/(THE+THF+5αTHF) 17α-Hydroxylase deficiency (CYP17A1) (THA+5αTHA+THB+5αTHB)/(THE+THF+5αTHF) (THA+5αTHA+THB+5αTHB)/(An+Et) 3β-Hydroxysteroid dehydrogenase deficiency (HSD3B2) DHEA/(THE+THF+5αTHF) 5PT/(THE+THF+5αTHF) POR deficiency (POR) (17HP+PT)/(An+Et) (17HP+PT)/(THE+THF+5αTHF) PD/(THE+THF+5αTHF) 5PD/(THE+THF+5αTHF) 5α-Reductase deficiency (SRD5A2) Et/An THB/5αTHB THF/5αTHF 17β-Hydroxysteroid dehydrogenase deficiency (HSD17B3)a (An+Et)/(THE+THF+5αTHF) a Only a small number of patients were studied, further evidence is required.

such as aldosterone and DHT, can be challenging. The wider use of LC-MS/MS will require efforts of appropriately standardized and calibrated methods, as well as the establishment of new reference intervals. Overall, the employed analytical strategy is often driven by the underlying clinical or academic question, as well as by the fact of accessibility to the respective analytical methodology.

Defining of Specific Conditions

Steroid Analysis in 46,XX DSD The majority of cases of 46,XX DSD in newborns or infants are caused by different forms of congenital adrenal hyperplasia (CAH). The specific profile of urinary steroid hormone metabolites can identify the enzyme defects, including deficiency of: • CYP21A2, • CYP11B1, • HSD3B2, and • POR

21-Hydroxylase Deficiency The diagnosis of 21OHD can be established by detecting high concentrations of 17OHP and 21-deoxycortisol or their urinary metabolites (table 1). Despite 17OHP

Steroid Biochemistry 47

11β-Hydroxylase Deficiency The plasma steroid analysis by LC-MS/MS shows increased concentrations of 11-deoxycortisol and DOC, as well as increased androstenedione and testosterone concentrations. 17OHP concentrations are often not significantly increased or can even be normal. Typical indirect signs are significantly increased androstenedione concentrations in combination with relatively mildly elevated 17OHP concentrations, which are commonly found in milder forms of 11OHD [11]. The major advancement of employing LC-MS/MS as a second tier test in neonatal screening has been recently demonstrated by establishing the differential diagnosis of 11OHD during the process of neonatal screening [12]. The diagnosis of 11OHD is straightforward employing urinary steroid profiling. Tetrahydro-11-deoxycortisol is typically elevated. The diagnostic process is simplified by elevated ratios of urinary 11-deoxycortisol metabolites over cortisol metabolites (table 2).

3β-HSD Type 2 Deficiency This CAH form is characterised by high plasma concentrations of the Δ5-steroids pregnenolone, 17Preg and DHEA. However, the interpretation of plasma steroid profiles can be complicated by potentially increased concentrations of 17OHP, which is synthesised from 17Preg via the activity of the isozyme HSD3B1. The urinary steroid metabolite analysis shows a profile dominated by metabolites of DHEA and pregnenolone. The pregnenolone metabolite 5-pregnenetriol is also markedly elevated in children and adults. The excretion of cortisol metabolites is particularly low in the salt-wasting form. In addition, patients with 3βHSD deficiency excrete relevant amounts of 17OHP metabolites such as pregnanetriol and 17α-hydroxypregnanolone. The diagnosis can be established by elevated ratios of urinary DHEA and pregnenolone metabolites over cortisol metabolites [13, 14] (table 2).

48 Kamrath · Wudy · Krone

Steroid Analysis in 46,XY DSD The differential diagnosis of 46,XY DSD can be established using steroid hormone profiles from urine or plasma obtained by GC-MS and LC-MS/MS: • high defects in steroid synthesis, – lipoid CAH due to defects of StAR protein – P450 side-chain cleavage enzyme (P450scc, CYP11A1) defects • CAH variants, – combined 17α-hydroxylase/17,20-lyase (CYP17A1) deficiency – isolated 17,20 lyase deficiency (CYB5) defects, mutations of CYP17A1 and POR – POR deficiency – 3βHSD2 deficiency (HSD3B2) • defects of extra-adrenal androgen synthesis, – 5α-reductase type 2 deficiency (SRD5A2) – 17β-HSD deficiency (after puberty)

Lipoid CAH (StAR) and P450 Side-Chain Cleavage Enzyme Deficiency (CYP11A1) Low concentrations of all steroid hormones are typically found. This is also mirrored by the extremely low excretion of urinary C19 and C21 steroid metabolites. StAR and CYP11A1 are almost impossible to diagnostically dissect [19, 20]. Ultrasound scan- ning of the adrenal can potentially be helpful to distinguish both entities with large adrenals in StAR deficiency and small or absent adrenals in CYP11A1 deficiency [2].

Steroid Biochemistry 49

Isolated 17,20-Lyase Deficiency The urinary steroid profile in patients with isolated 17,20-lyase deficiency is dominated by cortisol metabolites, whereas androgen metabolites are very low or absent. In contrast to combined 17-hydroxylase/17,20-lyase deficiency, corticosterone metabolites are normal [22]. Patients with defects in the CYP17A1 gene also commonly show an impairment of 17α-hydroxylation [23]. Diagnostic ratios in PORD are also influenced by the concomitant impairment of 21-hydroxylase activity. Recent, evidence suggests that ‘true’ isolated 17,20-lyase deficiency is caused by defects in CYB5 [22].

SRD5A2 Deficiency The diagnosis of SRD5A2 deficiency can be established by increased testosterone concentrations and an increased testosterone over DHT ratio after hCG stimulation. A common limitation is the difficulty to reliably analyse DHT. Improvements to this test using MS-based methods have been reported [24]. The analysis of the urinary steroid profile represents a very powerful and elegant alternative to biochemically diagnose this condition. It is, however, important to con- sider that SRD5A2 expression is detected postnatally after about 10–12 weeks. Thus, negative results before the age of 3 months do not rule out the diagnosis of SRD5A2 deficiency. SRD5A2 is not only confided to the testes, prostate and genital skin, but can also be found in other tissues, such as the liver, where it contributes to the metabolism of glucocorticoids and mineralocorticoids. Thus, the diagnosis of SRD5A2 does not only rely on reduction of 5α-reduced androgen metabolites, but is aided by reduced concentrations of 5α-reduced cortisol and corticosterone metabolites. This is particularly helpful in young infants and patients after gonadectomy [25]. Thus, the extremely low excretion of 5α-reduced steroid metabolites indicates 5α-reductase deficiency [25, 26]. Diagnostic ratios using 5β-reduced over 5α-reduced metabolites are significantly increased (table 2).

50 Kamrath · Wudy · Krone

Conclusions and Future Perspective

For more common conditions and well-known analytes, high throughput LC-MS/MS can replace most immunoassay-based steroid analytic techniques for measuring hormones and precursors. However, work is still required on improving reproducibility between laboratories. In addition, systematic evidence is lacking on whether LC-MS/ MS is improving the diagnostic yield in DSD. Despite such advances, GC/MS will continuously play an active role in studying rare and undefined conditions. Furthermore, GC/MS continues to be the pre-eminent discovery tool for defining new and aberrant metabolic pathways and first-time char- acterization of unknown steroids. GC/MS is of particular value in allowing for non- invasive testing in paediatric situations when it is desirable to minimize blood sam- pling. Both mass spectrometric techniques are complementary tools rather than com- peting technologies.

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Stefan A. Wudy, MD Division of Pediatric Endocrinology and Diabetology, Laboratory for Translational Hormone Analytics Steroid Research and Mass Spectrometry Unit, Center of Child and Adolescent Medicine Justus Liebig University, Feulgenstrasse 12 DE–35385 Giessen (Germany) E-Mail [email protected]

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