Urinary Steroid Metabolome Research Articles

SULFATION PATHWAYS: Alternate steroid sulfation pathways targeted by LC-MS/MS analysis of disulfates: application to prenatal diagnosis of steroid synthesis disorders.

The steroid disulfates (aka bis-sulfates) are a significant but minor fraction of the urinary steroid metabolome that have not been widely studied because major components are not hydrolyzed by the commercial sulfatases commonly used in steroid metabolomics. In early studies, conjugate fractionation followed by hydrolysis using acidified solvent (solvolysis) was used for the indirect detection of this fraction by GC-MS. This paper describes the application of a specific LC-MS/MS method for the direct identification of disulfates in urine, and their use as markers for the prenatal diagnosis of disorders causing reduced estriol production: STSD (steroid sulfatase deficiency), SLOS (Smith-Lemli-Opitz syndrome) and PORD (P450 oxidoreductase deficiency). Disulfates were detected by monitoring a constant ion loss (CIL) from the molecular di-anion. While focused on disulfates, our methodology included an analysis of intact steroid glucuronides and monosulfates because steroidogenic disorder diagnosis usually requires an examination of the complete steroid profile. In the disorders studied, a few individual steroids (as disulfates) were found particularly informative: pregn-5-ene-3β,20S-diol, pregn-5-ene-3β,21-diol (STSD, neonatal PORD) and 5α-pregnane-3β,20S-diol (pregnancy PORD). Authentic steroid disulfates were synthesized for use in this study as aid to characterization. Tentative identification of 5ξ-pregn-7-ene-3ξ,20S-diol and 5ξ-pregn-7-ene-3ξ,17,20S-triol disulfates was also obtained in samples from SLOS affected pregnancies. Seven ratios between the detected metabolites were applied to distinguish the three selected disorders from control samples. Our results show the potential of the direct detection of steroid conjugates in the diagnosis of pathologies related with steroid biosynthesis.

11β-Hydroxylase deficiency detected by urine steroid metabolome profiling using gas chromatography-mass spectrometry

Introduction11β-hydroxylase deficiency is the second most common form of congenital adrenal hyperplasia (CAH), accounting for 5–8% of all cases. It is an autosomal recessive enzyme defect that impairs the biosynthesis of cortisol and aldosterone. Mutation of the CYP11B1 gene on chromosome 8q22 causes partial or total reduction of enzyme activity. Clinical manifestations of 11β-hydroxylase deficiency include hypertension, and other signs related to overproduction of mineralocorticoids, and virilisation. Here, we report on a case of 11β-hydroxylase deficiency detected by urine steroid metabolome profiling. Case SubjectThe patient, a 3-month-old male, suffered from truncus arteriosus type I (congenital cardiovascular anomaly) and also presented with hyperpigmentation. An endocrinology consultation was sought and biochemical and molecular testing was conducted. ResultsThe patient’s urine steroid metabolome, as analysed by GC-MS, showed high excretion of tetrahydrodeoxycortisol (THS) and a THS/(THE + THF + 5αTHF) ratio of 2.3, which was higher than normal. Diagnosis of 11β-hydroxylase deficiency was confirmed by mutation analysis of the CYP11B1 gene. ConclusionAnalysis of the urine steroid metabolome by GC-MS can be used to assist in diagnosis of 11β-hydroxylase deficiency. We recommend consideration of urine steroid analysis as a first-line test in the diagnosis of CAH.

Mild autonomous cortisol excess in adrenal incidentalomas - metabolic disease burden and urinary steroid metabolome in 1201 prospectively recruited patients

Mild autonomous cortisol excess in adrenal incidentalomas - metabolic disease burden and urinary steroid metabolome in 1201 prospectively recruited patients

Diagnosis of a malignant adrenal mass: the role of urinary steroid metabolite profiling.

Adrenal masses are highly prevalent, found in 5% of the population. Differentiation of benign adrenocortical adenoma from adrenocortical carcinoma is currently hampered by the poor specificity and limited evidence base of imaging tests. This review summarizes the results of studies published to date on urine steroid metabolite profiling for distinguishing benign from malignant adrenal masses. Three studies have described cohorts of at least 100 patients with adrenal tumors showing significant differences between urinary steroid metabolite excretions according to the nature of the underlying lesion, suggesting significant value of steroid metabolite profiling as a highly accurate diagnostic test. Steroid profiling is emerging as a powerful novel diagnostic tool with a significant potential for improving the management for patients with adrenal tumors. Although the current studies use gas chromatography-mass spectrometry for proof of concept, widespread use of the method in routine clinical care will depend on transferring the approach to high-throughput tandem mass spectrometry platforms. The use of computational data analysis in conjunction with urine steroid metabolite profiling, that is, steroid metabolomics, adds accuracy and precision.

Accurate staging of non-alcoholic fatty liver disease through analysis of the urinary steroid metabolome

Accurate staging of non-alcoholic fatty liver disease through analysis of the urinary steroid metabolome

Steroid metabolomics for accurate and rapid diagnosis of inborn steroidogenic disorders

Background Urinary steroid metabolite profiling is an accurate reflection of adrenal and gonadal steroid output and metabolism in peripheral target cells of steroid action. Measurement of steroid metabolite excretion by gas chromatography-massspectrometry (GC–MS) is considered reference standard for biochemical diagnosis of steroidogenic disorders. However, performance of GC–MS analysis and interpretation of the resulting data requires significant expertise and age- and sex-specific reference ranges. Here we developed novel computational approaches for rapid interpretation of GC–MS data for diagnosis of inborn steroidogenic disorders Methods We analysed the urinary steroid metabolome by GC–MS in 829 healthy controls(302 neonates and infants, 167 children and 360 adults) and 118 untreated patients with genetically confirmed inborn disorders (21-hydroxylase deficiency,17-hydroxylase deficiency, POR deficiency, 11b-hydroxylase deficiency, 3b-HSD2 deficiency, 17b-HSD3 deficiency, 5a-reductase type 2 deficiency, cytochrome b5 deficiency). We calculated age-related normative values for established metabolite ratios representing distinct enzymatic functions. We developed a novel interpretable machine learning technique, Angle Learning Vector Quantisation (ALVQ), which looks at all possible metabolite ratios, computationally reduces these to the most relevant for discrimination, and differentiates disease states by comparison to a representative prototype. The method runs independent of sex and age information, units of measurement and method of urine collection. Results Conventional biochemical ratios had 100% sensitivity but only very poor specificity. By contrast, ALVQ predicted 'affected urine' vs 'healthy urine' with 100% sensitivity and 97% specificity. For our three most prevalent conditions(PORD, SRD5A2 and CYP21A2), the specific condition was identified correctly in 96% of cases. Conclusion We developed a novel Steroid Metabolomics approach to automatically diagnose inborn steroidogenic disorders with very high sensitivity and specificity, superior to current methods, and with high potential for implementation in routine clinical care.

Steroid metabolome analysis reveals prevalent glucocorticoid excess in primary aldosteronism.

BACKGROUND. Adrenal aldosterone excess is the most common cause of secondary hypertension and is associated with increased cardiovascular morbidity. However, adverse metabolic risk in primary aldosteronism extends beyond hypertension, with increased rates of insulin resistance, type 2 diabetes, and osteoporosis, which cannot be easily explained by aldosterone excess.METHODS. We performed mass spectrometry–based analysis of a 24-hour urine steroid metabolome in 174 newly diagnosed patients with primary aldosteronism (103 unilateral adenomas, 71 bilateral adrenal hyperplasias) in comparison to 162 healthy controls, 56 patients with endocrine inactive adrenal adenoma, 104 patients with mild subclinical, and 47 with clinically overt adrenal cortisol excess. We also analyzed the expression of cortisol-producing CYP11B1 and aldosterone-producing CYP11B2 enzymes in adenoma tissue from 57 patients with aldosterone-producing adenoma, employing immunohistochemistry with digital image analysis.RESULTS. Primary aldosteronism patients had significantly increased cortisol and total glucocorticoid metabolite excretion (all P < 0.001), only exceeded by glucocorticoid output in patients with clinically overt adrenal Cushing syndrome. Several surrogate parameters of metabolic risk correlated significantly with glucocorticoid but not mineralocorticoid output. Intratumoral CYP11B1 expression was significantly associated with the corresponding in vivo glucocorticoid excretion. Unilateral adrenalectomy resolved both mineralocorticoid and glucocorticoid excess. Postoperative evidence of adrenal insufficiency was found in 13 (29%) of 45 consecutively tested patients.CONCLUSION. Our data indicate that glucocorticoid cosecretion is frequently found in primary aldosteronism and contributes to associated metabolic risk. Mineralocorticoid receptor antagonist therapy alone may not be sufficient to counteract adverse metabolic risk in medically treated patients with primary aldosteronism.FUNDING. Medical Research Council UK, Wellcome Trust, European Commission.

The urinary steroid metabolome as a non-invasive tool to stage non-alcoholic fatty liver disease

The urinary steroid metabolome as a non-invasive tool to stage non-alcoholic fatty liver disease

Androgen biosynthesis during minipuberty favors the backdoor pathway over the classic pathway: Insights into enzyme activities and steroid fluxes in healthy infants during the first year of life from the urinary steroid metabolome

The steroid profile changes dramatically from prenatal to postnatal life. Recently, a novel backdoor pathway for androgen biosynthesis has been discovered. However, its role remains elusive. Therefore, we investigated androgen production from birth to one year of life with a focus on minipuberty and on production of androgens through the backdoor pathway. Additionally, we assessed the development of the specific steroid enzyme activities in early life. To do so, we collected urine specimens from diapers in 43 healthy newborns (22 females) at 13 time points from birth to one year of age in an ambulatory setting, and performed in house GC–MS steroid profiling for 67 steroid metabolites. Data were analyzed for androgen production through the classic and backdoor pathway and calculations of diagnostic ratios for steroid enzyme activities were performed. Analysis revealed that during minipuberty androgen production is much higher in boys than in girls (e.g. androsterone (An)), originates largely from the testis (Anboys-Angirls), and uses predominantly the alternative backdoor pathway (An/Et; Δ5<Δ4 lyase activity). Modelling of steroid enzyme activities showed age-related effects for 21-, 11-, 17-hydroxylase and P450 oxidoreductase activities as well as 3β-hydroxysteroid dehydrogenase, 11β-hydroxylase type 1/2 and 5α-reductase activities. Sex-related characteristics were found for 21-hydroxylase and 5α-reductase activities. Overall, our study shows that androgen biosynthesis during minipuberty favors the backdoor pathway over the classic pathway. Calculations of specific diagnostic ratios for enzyme activities seem to allow the diagnosis of specific steroid disorders from the urinary steroid metabolome.

Urine steroid metabolomics is a highly sensitive tool for post-operative recurrence detection in adrenocortical carcinoma

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

Steroid Sulfatase Deficiency and Androgen Activation Before and After Puberty.

Context:Steroid sulfatase (STS) cleaves the sulfate moiety off steroid sulfates, including dehydroepiandrosterone (DHEA) sulfate (DHEAS), the inactive sulfate ester of the adrenal androgen precursor DHEA. Deficient DHEA sulfation, the opposite enzymatic reaction to that catalyzed by STS, results in androgen excess by increased conversion of DHEA to active androgens. STS deficiency (STSD) due to deletions or inactivating mutations in the X-linked STS gene manifests with ichthyosis, but androgen synthesis and metabolism in STSD have not been studied in detail yet.Patients and Methods:We carried out a cross-sectional study in 30 males with STSD (age 6–27 y; 13 prepubertal, 5 peripubertal, and 12 postpubertal) and 38 age-, sex-, and Tanner stage-matched healthy controls. Serum and 24-hour urine steroid metabolome analysis was performed by mass spectrometry and genetic analysis of the STS gene by multiplex ligation-dependent probe amplification and Sanger sequencing.Results:Genetic analysis showed STS mutations in all patients, comprising 27 complete gene deletions, 1 intragenic deletion and 2 missense mutations. STSD patients had apparently normal pubertal development. Serum and 24-hour urinary DHEAS were increased in STSD, whereas serum DHEA and testosterone were decreased. However, total 24-hour urinary androgen excretion was similar to controls, with evidence of increased 5α-reductase activity in STSD. Prepubertal healthy controls showed a marked increase in the serum DHEA to DHEAS ratio that was absent in postpubertal controls and in STSD patients of any pubertal stage.Conclusions:In STSD patients, an increased 5α-reductase activity appears to compensate for a reduced rate of androgen generation by enhancing peripheral androgen activation in affected patients. In healthy controls, we discovered a prepubertal surge in the serum DHEA to DHEAS ratio that was absent in STSD, indicative of physiologically up-regulated STS activity before puberty. This may represent a fine tuning mechanism for tissue-specific androgen activation preparing for the major changes in androgen production during puberty.

Urine steroid metabolomics as a diagnostic tool in primary aldosteronism

Introduction: The regular diagnostic workup for primary aldosteronism (PA) can be very demanding and involves multiple invasive as well as time and cost intensive diagnostic tests. Here we have explored the value of urinary steroid metabolome analysis in the diagnosis and differential diagnosis of PA. Previously, urinary 3α,5β-tetrahydroaldosterone (THAldo) has been suggested as a reliable screening test for PA and serum 18-oxocortisol and 18-hydroxycortisol have been reported as diagnostic markers with the potential to distinguish unilateral aldosterone-producing adenomas (APA) from bilateral adrenal hyperplasia (BAH). Patients and methods: We studied 180 PA patients (103 APA, 71 BAH) in whom PA had been confirmed by saline infusion test followed by adrenal vein sampling for subtype differentiation. We carried out targeted sequencing for disease-causing somatic mutations in the KCNJ5, CACNA1D, ATP1A1 and ATP2B3 genes in tumour tissue obtained by unilateral adrenalectomy, which was available in 75/103 APA patients. The urine steroid metabolome was analysed by gas chromatography-mass spectrometry comprising the quantification of 38 distinct steroids including metabolites of aldosterone, deoxycorticosterone, corticosterone as well as 18OH-cortisol (18OH-F) and 18-oxo-tetrahydrocortisol (18oxo-THF). Results: As expected, urinary excretion of mineralocorticoids (p 69 μg/24h, with significantly higher THAldo in APAs compared to BAH (p=0.0082). Similarly, APAs had significantly higher excretion of 18OH-F (p=0.0171) and 18oxo-THF (p=0.0005). Genetic analysis of APA tissue revealed mutations in KCNJ5 (n=29), ATPases (n=12) and CACNA1D (n=6) while in 28 patients no known mutation was identified. Patients with KCNJ5 mutations had significantly increased excretion of both 18OH-F (p=0.0002) and 18oxo-THF (p<0.0001) as compared to the other mutation groups. Conclusion: Urine steroid metabolomics is a promising approach for the diagnosis and differential diagnosis of PA.

Urine steroid metabolomics as a novel diagnostic tool for early detection of recurrence in adrenocortical carcinoma

Introduction: Adrenocortical carcinoma (ACC) is an aggressive malignancy with a high rate of recurrence. Regular post-operative follow-up imaging is necessary, but associated with high radiation exposure and frequent diagnostic ambiguity. Urine steroid metabolomics has recently been introduced as a novel diagnostic tool for the detection of adrenocortical malignancy in patients with adrenal incidentalomas. Here we present the first clinical study assessing the performance of this innovative approach in the context of follow-up after complete (R0) ACC resection. Patients and methods: We included 166 patients from 13 centres registered with the European Network for the Study of Adrenal Tumours (ENSAT). We selected all patients recorded between 2008 and 2015 fulfilling the following criteria: i) recorded on the ENSAT registry as confirmed adrenocortical carcinoma with R0 primary tumour resection and ii) availability of at least two postoperative 24-h urines, one whilst disease-free and the other after recurrence. Twenty-four-hour urines were analysed by gas chromatography–mass spectrometry, with quantification of 38 distinct steroid metabolites. A machine learning-based computational algorithm was employed to detect ACC recurrence. Results: Twenty-one patients developed 22 ACC recurrences during the study period as documented by serial cross-sectional imaging and biopsy where appropriate. Steroid metabolomics predicted disease recurrence at the time of first abnormal imaging with a sensitivity of 84% and specificity of 95%. Adjuvant mitotane in 12/21 patients did not affect accuracy. In the subgroup of patients for whom a diagnostic pre-operative 24-h urine sample was available (n=7), we were able to accurately detect all cases of recurrence (sensitivity and specificity 100%). In seven cases, biochemical evidence of disease recurrence pre-dated the first radiological detection by more than 2 months (range 2–11 months). Conclusion: Our study provides proof-of-principle evidence suggesting a role for urine steroid metabolomics as a potent diagnostic tool in the follow-up monitoring of ACC.

Further advances in diagnosis of adrenal cancer: a high-throughput urinary steroid profiling method using liquid chromatography tandem mass spectrometry (LC-MS/MS)

Context: Differentiating adrenocortical adenoma (ACA) from adrenocortical carcinoma (ACC) represents a continuous challenge in patients with (often incidentally discovered) adrenal masses, with unfavorable sensitivities and specificities provided by tumor size, imaging and even histology. We have previously developed urine steroid metabolomics as a tool for the detection of adrenal malignancy employing gas chromatography mass spectrometry (GC-MS) for the detection of 32 distinct steroid metabolites (JCEM2011; 96 (12): 3775–84). Using the most informative nine steroids, as determined by machine learning analysis, this method can diagnose ACC with superior sensitivity and specificity to currently used imaging modalities. However, GC-MS is a labor-intensive, relatively expensive and low throughput method. Methods: Here we developed a high-throughput liquid chromatography tandem mass spectrometry (LC-MS/MS) method capable of detecting 16 distinct steroid metabolites in a single 5 min run. This method was validated assessing linearity, sensitivity, specificity, reproducibility and matrix effects. We collected 24-h urine samples from 130 healthy controls, 294 ACA and 96 ACC patients and analysed steroid excretion both by GC-MS and the novel LC-MS/MS method. Results: Comparison of steroid analysis results showed very good correlation between the two methods. LC-MS/MS data revealed significant differences in steroid output between ACC and ACA, with 13 of the 16 measured steroids significantly increased in ACC. Steroid data were subjected to Matrix Relevance Learning Vector Quantization, which identified for both methods the same three steroids as most informative in detecting ACC. These were THS, 5-PD and 5-PT, the metabolites of 11-deoxycortisol, pregnenolone and 17OH-pregnenolone, respectively. Importantly, we could demonstrate very similar diagnostic performances of GC-MS and LC-MS/MS when using 16 steroids. Conclusion: This work represents an important step in the implementation of urine steroid metabolomics in the routine work-up of adrenal incidentalomas. We anticipate LC-MS/MS screening in all patients, followed by GC-MS confirmatory analysis of samples with a positive screening result.

Estimation of reference curves for the urinary steroid metabolome in the first year of life in healthy children: Tracing the complexity of human postnatal steroidogenesis

ContextComplex steroid disorders such as P450 oxidoreductase deficiency or apparent cortisone reductase deficiency may be recognized by steroid profiling using chromatographic mass spectrometric methods. These methods are highly specific and sensitive, and provide a complete spectrum of steroid metabolites in a single measurement of one sample which makes them superior to immunoassays. The steroid metabolome during the fetal-neonatal transition is characterized by (a) the metabolites of the fetal-placental unit at birth, (b) the fetal adrenal androgens until its involution 3–6 months postnatally, and (c) the steroid metabolites produced by the developing endocrine organs. All these developmental events change the steroid metabolome in an age- and sex-dependent manner during the first year of life. ObjectiveThe aim of this study was to provide normative values for the urinary steroid metabolome of healthy newborns at short time intervals in the first year of life. MethodsWe conducted a prospective, longitudinal study to measure 67 urinary steroid metabolites in 21 male and 22 female term healthy newborn infants at 13 time-points from week 1 to week 49 of life. Urine samples were collected from newborn infants before discharge from hospital and from healthy infants at home. Steroid metabolites were measured by gas chromatography-mass spectrometry (GC–MS) and steroid concentrations corrected for urinary creatinine excretion were calculated. Results61 steroids showed age and 15 steroids sex specificity. Highest urinary steroid concentrations were found in both sexes for progesterone derivatives, in particular 20α-DH-5α-DH-progesterone, and for highly polar 6α-hydroxylated glucocorticoids. The steroids peaked at week 3 and decreased by ∼80% at week 25 in both sexes. The decline of progestins, androgens and estrogens was more pronounced than of glucocorticoids whereas the excretion of corticosterone and its metabolites and of mineralocorticoids remained constant during the first year of life. ConclusionThe urinary steroid profile changes dramatically during the first year of life and correlates with the physiologic developmental changes during the fetal-neonatal transition. Thus detailed normative data during this time period permit the use of steroid profiling as a powerful diagnostic tool.

Reduced Activity of 11β-Hydroxylase Accounts for Elevated 17α-Hydroxyprogesterone in Preterms

To characterize the urinary steroid metabolome of neonates and infants born either at term or preterm. We retrospectively analyzed urinary steroid hormone metabolites determined by gas chromatography-mass spectrometry of 78 neonates and infants born at term and 83 neonates and infants born preterm (median 34 weeks of gestational age). The subjects' 11β-hydroxylase and 21-hydroxylase activities were assessed on the basis of urinary metabolite substrate-to-product ratios. Preterm neonates and infants had elevated urinary concentrations of 17α-hydroxyprogesterone (17OHP) metabolites (P<.001) but lower urinary concentrations of the 21-deoxycortisol metabolite pregnanetriolone (PTO) (P<.01). One reason was lower 11β-hydroxylase activity in preterms. We could demonstrate a correlation between low 11β-hydroxylase activity and high urinary concentrations of 17OHP metabolites (r=0.51, P<.001) but low urinary concentrations of the 21-deoxycortisol metabolite PTO (r=-0.24, P=.03) in preterms. Low 11β-hydroxylase activity may explain increased 17OHP but decreased 21-deoxycortisol metabolite excretion in preterms. Our analysis clarifies, first, why preterms have higher 17OHP levels and thus higher rates of false-positive screening results for congenital adrenal hyperplasia than do term infants, and, second, why 21-deoxycortisol or its urinary metabolite PTO is more specific than 17OHP for the diagnosis of 21-hydroxylase deficiency.

Adrenal hormonal imbalance in acute intermittent porphyria patients: results of a case control study.

BackgroundAcute Intermittent Porphyria (AIP) is a rare disease that results from a deficiency of hydroxymethylbilane synthase, the third enzyme of the heme biosynthetic pathway. AIP carriers are at risk of presenting acute life-threatening neurovisceral attacks. The disease induces overproduction of heme precursors in the liver and long-lasting deregulation of metabolic networks. The clinical history of AIP suggests a strong endocrine influence, being neurovisceral attacks more common in women than in men and very rare before puberty. To asses the hypothesis that steroidogenesis may be modified in AIP patients with biochemically active disease, we undertook a comprehensive analysis of the urinary steroid metabolome.MethodsA case–control study was performed by collecting spot morning urine from 24 AIP patients and 24 healthy controls. Steroids in urine were quantified by liquid chromatography-tandem mass spectrometry. Parent steroids (17-hydroxyprogesterone; deoxycorticosterone; corticoesterone; 11-dehydrocorticosterone; cortisol and cortisone) and a large number of metabolites (N = 55) were investigated. Correlations between the different steroids analyzed and biomarkers of porphyria biochemical status (urinary heme precursors) were also evaluated. The Mann–Whitney U test and Spearman’s correlation with a two tailed test were used for statistical analyses.ResultsForty-one steroids were found to be decreased in the urine of AIP patients (P < 0.05), the decrease being more significant for steroids with a high degree of hydroxylation. Remarkably, 13 cortisol metabolites presented lower concentrations among AIP patients (P < 0.01) whereas no significant differences were found in the main metabolites of cortisol precursors. Nine cortisol metabolites showed a significant negative correlation with heme precursors (p < 0.05). Ratios between the main metabolites of 17-hydroxyprogesterone and cortisol showed positive correlations with heme-precursors (correlation coefficient > 0.51, P < 0.01).ConclusionsComprehensive study of the urinary steroid metabolome showed that AIP patients present an imbalance in adrenal steroidogenesis, affecting the biosynthesis of cortisol and resulting in decreased out-put of cortisol and metabolites. This may result from alterations of central origin and/or may originate in specific decreased enzymatic activity in the adrenal gland. An imbalance in steroidogenesis may be related to the maintenance of an active disease state among AIP patients.

Absent Adrenarche in Children with Hypopituitarism: A Study Based on Urinary Steroid Metabolomics

Background: Up to now, the regulation of adrenarche remains a myth although ACTH may possibly play an important role. Methods: Urinary steroid profiling by gas chromatography-mass spectrometry was used to study non-invasively the adrenarchal steroid metabolome in 13 children aged 6-16 years with partial or complete hypopituitarism (HP) whose ACTH/cortisol axis was affected and compared it with 24 healthy age-matched controls. The sum of DHEA, 16α-hydroxy-DHEA and 3β,16α,17β-androstenetriol served as markers for adrenarche parameters (AP). The excretion rates of major urinary cortisol metabolites were also determined. Results: The excretion rates for AP were significantly lower for the HP subjects than for the controls (p < 0.001). After dividing the HP group into a subgroup treated with hydrocortisone (HC) and an HC-untreated subgroup, a significant difference for AP remained for each subgroup when compared to the control group (p < 0.001 and p = 0.045, respectively). Treatment with HC had no influence on AP. Conclusion: The data imply indirectly a significant contribution of ACTH to the regulation of adrenarche. Our results also signify important diagnostic information: absent adrenarche can be indicative of ACTH deficiency.

Gas chromatography–mass spectrometry profiling of steroids in urine of patients with acute intermittent porphyria

ObjectiveAcute intermittent porphyria (AIP) is an autosomal dominant disease that results from a deficiency of hydroxymethylbilane synthase, the third enzyme of the heme biosynthetic pathway. AIP carriers may present acute neurovisceral attacks with hepatic overproduction of heme-precursors. In some patients, remission of the acute symptoms leads to long-term hepatic metabolic abnormalities. In this study, gas chromatography–mass spectrometry (GC/MS) was used to investigate urinary steroid metabolome of AIP patients. Design and methodsSteroid profiling in urine was performed in a group of AIP patients with biochemically active disease (n=22) and healthy controls (n=20). Five asymptomatic AIP family carriers were also studied. Commonly used ratios for the evaluation of disturbances in the steroid metabolism were calculated. ResultsWe found that etiocholanolone/androsterone and tetrahydrocortisol/5α-tetrahydrocortisol (THF/5α-THF) metabolic ratios were significantly increased in the urine of AIP patients compared to controls (2.3±0.3 vs 0.8±0.1; p<0.001 and 2.9±0.7 vs 0.9±0.1; p<0.01). The (THF+5α-THF)/tetrahydrocortisone ratio was reduced among the AIP patients (p<0.01). Quantification of the steroid absolute concentrations showed that these variations were due to a decrease of the 5α metabolites. Other ratios, like cortisol/cortisone and 6β-hydroxycortisol/cortisol in the free steroid fraction did not show differences between patients and controls. All ratios were normal among the family carriers. ConclusionA significant number of AIP patients present a basal decrease of steroid 5α-reductase activity in the liver. The deficiency may be related to malnutrition and hepatic energy misbalance associated with active AIP. Urinary steroid profiling by GC/MS may be a valuable tool to assess hepatic metabolome in AIP.

Androgen Synthesis in Patients with Congenital Adrenal Hyperplasia due to 21-Hydroxylase Deficiency

A hallmark of severe congenital adrenal hyperplasia due to 21-hydroxylase deficiency is pre- and postnatal virilization. The most characteristic biochemical abnormality is the elevation of 17α-hydroxyprogesterone, which is metabolized to the most potent androgen receptor agonist dihydrotestosterone. 17α-Hydroxyprogesterone can be metabolized to dihydrotestosterone via 4-androstenedione through the classical Δ⁴-pathway or via 17α-hydroxypregnenolone and dehydroepiandrosterone through the classical Δ⁵-pathway, as well as through an alternative route, called the 'backdoor pathway', that bypasses dehydroepiandrosterone, 4-androstenedione, and testosterone as intermediates. This review article will summarize recent advances in the understanding of the activities of androgen synthesis pathways in patients with 21-hydroxylase deficiency obtained by urinary steroid metabolomics based on gas chromatography-mass spectrometry. Compared with healthy controls, the relative activities of the backdoor and Δ⁴-pathways increase in patients with congenital adrenal hyperplasia during neonatal age and infancy, whereas the activity of the Δ⁵-pathway remains unchanged. Thereafter, the activity of the Δ⁵-pathway dominates, whereas a decreasing 5α-reductase activity leads to a diminished role of the backdoor pathway for androgenic steroid production. Beside the backdoor pathway, the Δ⁴-pathway seems to be responsible for increased androgen generation in patients with 21-hydroxylase deficiency before the onset of adrenarche, whereas the Δ⁵-pathway might contribute to the increased androgen formation in those patients only after the onset of adrenarche.