Excretion Of Steroid Metabolites Research Articles

Prognostic markers of recurrence in adrenocortical carcinoma patients after surgery

BACKGROUND: Adrenocortical carcinoma is a rare and aggressive disease. Tumor recurrence prevention is vital for increasing patients survival rate. Therefore, the identification of prognostic markers is of particular importance.
 AIM: To evaluate clinical, laboratory and chromatographic criteria for adrenocortical carcinoma recurrence using gas chromatography-mass spectrometry to optimize patients follow-up.
 MATERIALS AND METHODS: 40 patients [10 (25%) men and 30 (75%) women] with histologically confirmed adrenocortical carcinoma (according to the European Network for the Study of Adrenal Tumors stage I 3, II 29, III 8 patients at presentation) have been recruited. Measurement of 24-hour urinary steroid metabolite excretion has been carried out by gas chromatography-mass spectrometry (GCMS-ТQ8050, Shimadzu) in preoperative period. The survival distribution has been assessed according to the KaplanMeier method. Cox proportional hazards regression methods have been used to determine predictive factors on recurrence-free survival.
 RESULTS: The patients with early stages of disease (III versus III) had a significantly longer recurrence-free survival, overt hypercortisolism was associated with significantly shorter recurrence-free survival. A significant correlation between pregnenediol urinary excretion and tumor diameter, stage has been found. Increased urinary excretion of tetrahydro-11-deoxycortisol, pregnenediol was associated with a decreased recurrence-free survival in the patients with overt hypercortisolism. Increased urinary excretion of 16-OH-dehydroepiandrosterone was a marker of a shorter recurrence-free survival in the patients without Cushing syndrome. Increased urinary excretion of pregnenediol, pregnenetriol, 16-oxo-androstenediol in all the adrenocortical carcinoma patients was associated with a recurrence risk. In the multivariate analysis pregnenediol urinary excretion, stage and overt cortisol excess were significantly and independently associated with a shorter recurrence-free survival.
 CONCLUSIONS: Adrenocortical carcinoma recurrence prognostic markers have been found by gas chromatography-mass spectrometry. The application of these findings may improve patient-centered outcomes.

The Impact of Obesity on the Excretion of Steroid Metabolites in Boys and Girls: A Comparison with Normal-Weight Children.

Obesity in childhood is associated with several steroid changes, which result from excess body mass. The aim of this study was to evaluate steroid metabolism in children with obesity compared with those with normal weight, especially in relation to sex and puberty progress. We analyzed the clinical data of 191 children, aged between 5 and 18 years, with 115 affected (64 girls and 51 boys) and 76 unaffected (35 girls and 41 boys) by obesity. Routine clinical assessment and pubertal stage evaluation based upon Tanner's scale were performed. In addition, to evaluate the impact of puberty, children with pre-adolescence and advanced puberty were divided into separate subgroups. Then, 24 h urine steroid excretion profiles were analyzed by gas chromatography/mass spectrometry. Significant differences in the excretion of steroid metabolites were found between normal weight children and children with obesity, especially in the prepubertal cohort. In this group, we observed enhanced activity in all the pathways of adrenal steroidogenesis. Raised excretion of mineralocorticoid derivatives such as tetrahydro-11-deoxycorticosterone, tetrahydrocorticosterone, and 5α-tetrahydrocorticosterone supported increased activity of this track. No significant differences were detected in the excreted free forms of cortisol and cortisone, while the excretion of their characteristic tetrahydro-derivatives was different. In pre-adolescent children with obesity, α-cortol and especially α-cortolone appeared to be excreted more abundantly than β-cortol or β-cortolone. Furthermore, in children with obesity, we observed elevated androgen excretion with an enhanced backdoor pathway. As puberty progressed, remarkable reduction in the differences between adolescents with and without obesity was demonstrated.

RF21 | PSAT99 Urinary adrenal steroidome modifications by Ketoconazole and Metyrapone alter the urinary free cortisol immunoassay reliability in Cushing Syndrome

Abstract Introduction Twenty-four-hour urinary free cortisol (24h-UFC) is the most used test for follow-up decision-making in patients with Cushing syndrome (CS) under adrenal-directed medical treatment. In CS patients without medical treatment, 24h-UFC determinations by immunoassays (IA) are commonly overestimated because of interference by cross-reacting steroid metabolites. However, it is unknown how induced changes by Ketoconazole (KTZ) or Metyrapone (MTP) on the excretion of urinary steroid metabolites alter the IA cross-reactivity interference of 24h-UFC determinations and what are its clinical implications. Methods 24h-UFC was analyzed by IA and gas chromatography-mass spectrometry (GC-MS) in 193 samples (81 before treatment, 73 during KTZ and 39 during MTP) from 34 CS patients. In addition, 35 urinary steroids were analyzed by GC-MS on each patient before and during treatment. Results Before treatment, 24h-UFC*IA determinations were overestimated by a factor of 1.75: 1 (95% CI 1.60 - 1.94) compared to those by GC-MS. During treatment with KTZ, the cross-reactivity interference of the 24h-UFC results by IA decreased in a dose-dependent manner (p = 0.000). Thus, in patients taking KTZ >400mg/day, 24h-UFC*IA determinations were no longer overestimated, giving similar results to those by GC-MS (factor of 0.98: 1, 95% CI, 0.83–1.20). Baseline 24h-UFC and upper limit normal (ULN) reductions were magnified (>25%) when assessed by IA vs GC-MS leading to higher falsely KTZ efficacy and misleading biochemical classification of 15% of patients. Despite dosage, in patients taking MTP the IA cross-reactivity only decreased 0.55 which resulted in persistence of overestimated 24h-UFC results (factor of 1.33: 1, 95% CI, 1.09 -1.76). When comparing MTP efficacy among IA vs GC-MS results, ULN baseline reductions were >50% different in some patients, while in others, similar reductions were found. Acceptable 24h-UFC method agreement between GC-MS and IA before treatment (R2 = 0.954) declined in patients under KTZ (R2 = 0.632) but not in MTP (R2 = 0.917). Urinary excretion changes of 22 urinary steroid metabolites explained 86% of the 24h-UFC*IA interference. 6β-Hydroxy-cortisol (6β-OH-cortisol) was the metabolite determining most of the 24h-UFC cross-reactivity variability (R2= 48.3%, p = 0.000) followed by 20α-dihydrocortisol (20α-DHF) (R2= 24.4%, p = 0.000). Each treatment induced a unique urinary steroid signature (p <0.001) and larger excretion reductions of 6β-OH-cortisol, 20α-DHF and 18-Hydroxy-cortisol in patients with KTZ elucidated the higher decrement of interference in the 24h-UFC by IA, compared to MTP treated patients (p = 0.001). Conclusion KTZ and MTP alter distinctly the urinary excretion of IA cross-reactive steroid metabolites, thus decreasing the cross-reactive interference of 24h-UFC*IA determinations present before treatment in a unique manner. Consequently, this interference reduction in 24h-UFC*IA leads to loss of method agreement with GC-MS and high risk of overestimating the biochemical impact of KTZ and MTP in controlling CS because of poor reliability of ULN. Presentation: Saturday, June 11, 2022 1:00 p.m. - 3:00 p.m., Sunday, June 12, 2022 12:36 p.m. - 12:41 p.m.

Ketoconazole- and Metyrapone-Induced Reductions on Urinary Steroid Metabolites Alter the Urinary Free Cortisol Immunoassay Reliability in Cushing Syndrome.

IntroductionTwenty-four-hour urinary free cortisol (24h-UFC) is the most used test for follow-up decision-making in patients with Cushing syndrome (CS) under medical treatment. However, 24h-UFC determinations by immunoassays (IA) are commonly overestimated because of steroid metabolites’ cross-reaction. It is still uncertain how ketoconazole (KTZ)- and metyrapone (MTP)-induced changes on the urinary steroid metabolites can alter the 24h-UFC*IA determinations’ reliability.Methods24h-UFC was analyzed by IA and gas chromatography-mass spectrometry (GC-MS) in 193 samples (81 before treatment, 73 during KTZ, and 39 during MTP) from 34 CS patients. In addition, urinary steroidome was analyzed by GC-MS on each patient before and during treatment.ResultsBefore treatment, 24h-UFC*IA determinations were overestimated by a factor of 1.75 (95% CI 1.60–1.94) compared to those by GC-MS. However, during KTZ treatment, 24h-UFC*IA results were similar (0.98:1) to those by GC-MS (95% CI, 0.83–1.20). In patients taking MTP, IA bias only decreased 0.55, resulting in persistence of an overestimation factor of 1.33:1 (95% CI, 1.09–1.76). High method agreement between GC-MS and IA before treatment (R2 = 0.954) declined in patients under KTZ (R2 = 0.632) but not in MTP (R2 = 0.917). Upper limit normal (ULN) reductions in patients taking KTZ were 27% larger when using 24h-UFC*IA compared to 24h-UFC*GC-MS, which resulted in higher false efficacy and misleading biochemical classification of 15% of patients. Urinary excretion changes of 22 urinary steroid metabolites explained 86% of the 24h-UFC*IA interference. Larger urinary excretion reductions of 6β-hydroxy-cortisol, 20α-dihydrocortisol, and 18-hydroxy-cortisol in patients with KTZ elucidated the higher 24h-UFC*IA bias decrement compared to MTP-treated patients.ConclusionKTZ and MTP alter the urinary excretion of IA cross-reactive steroid metabolites, thus decreasing the cross-reactive interference of 24h-UFC*IA determinations present before treatment. Consequently, this interference reduction in 24h-UFC*IA leads to loss of method agreement with GC-MS and high risk of overestimating the biochemical impact of KTZ and MTP in controlling CS because of poor reliability of reference ranges and ULN.

Influence of Pain Killers on the Urinary Anabolic Steroid Profile.

Anabolic androgenic steroids (AAS) are prohibited as performance-enhancing drugs in sports. Among them, testosterone and its precursors are often referred to as "pseudoendogenous" AAS, that is, endogenous steroids that are prohibited when administered exogenously. To detect their misuse, among other methods, the World Anti-Doping Agency-accredited laboratories monitor the steroid profile (concentrations and concentration ratios of endogenous steroids, precursors and metabolites) in urine samples collected from athletes in and out of competition. Alterations in steroid profile markers are used as indicators for misuse of anabolic steroids in sports. Therefore, especially their metabolic pathways with possible interactions are crucial to elucidate. As steroid metabolism is very complex, and many enzymes are involved, certain non-prohibited drugs may influence steroid metabolite excretion. One important group of steroid-metabolizing enzymes is aldo-keto reductases (AKRs). An inhibition of them by non-steroidal anti-inflammatory drugs (NSAIDs), which are neither prohibited nor monitored, but frequently used drugs in sports, was demonstrated in vitro. Thus, this work aims to investigate the influence of NSAID intake on the urinary steroid profile. Kinetic and inhibitory studies were performed using 5α-dihydrotestosterone as substrate. The results obtained from in vitro experiments show that ibuprofen inhibits AKR1C2 and thus influences steroid biotransformation. For in vivo investigations, urine samples prior, during and postadministration of ibuprofen were analyzed using routine methods to monitor the steroid profile. Changes in markers of the steroid profile of volunteers were observed. The combination of in vitro and in vivo results suggests that monitoring of ibuprofen may be useful in doping control analysis. The presented work illustrates the importance to consider co-administration of (non-prohibited) drugs during antidoping analysis. Intake of multiple substances is likely leading to interfering effects. Divergent results in antidoping analysis may therefore be observed and misinterpretation of analytical data may occur. Similar considerations may be appropriate for other fields of forensic applications.

Steroid metabolomics: a rapid computational approach for accurate differentiation of inborn disorders of steroidogenesis

Background: Measurement of steroid metabolite excretion in urine by gas chromatography-mass spectrometry (GC-MS) provides a comprehensive profile of an individual’s adrenal and gonadal steroid production. It has long been acknowledged as a useful tool for diagnosis of inborn disorders of steroidogenesis leading to congenital adrenal hyperplasia and disorders of sex development. Ratios of steroid metabolites can be employed as surrogates for enzymatic activities of distinct steroidogenic enzymes and can also be applied to single random urine samples, making this a more feasible approach for use with paediatric patients. However, widespread use in the acute setting for diagnosis of these disorders is hampered by the considerable expertise required for interpretation. Here we developed a novel steroid metabolomics approach for the detection and differential diagnosis of inborn steroidogenic disorders, combining mass spectrometry-based steroid profiling with machine learning-based data analysis, suitable for automation and interpretation without specialist expertise. Methods: We performed multi-steroid profiling by GC-MS, quantifying 34 steroid metabolites, in urine samples from 829 healthy controls and 178 untreated patients with inborn steroidogenic disorders. This cohort included patients with inborn deficiencies in the following enzymes: CYP21A2 (n=26), CYP11B1 (n=12), CYP17A1 (n=30), POR (n=37), HSD3B2 (n=22), and SRD5A2 (n=51). We assessed the diagnostic performance of conventional biochemical assessment employing 15 steroid precursor-to-product ratios, each historically established as indicative of a distinct steroidogenesis disorder. We compared this to the performance of our novel steroid metabolomics approach, which involved analysis of the GC-MS multi-steroid profiles by a custom-designed approach, Angle Learning Vector Quantization (ALVQ), which classifies samples by comparing similarity of their steroid metabolome to representative steroid metabolome prototypes for each enzyme deficiency. Results: The conventional biochemical steroid ratio approach demonstrated acceptable sensitivity and specificity. However, the automated steroid metabolomics approach (ALVQ) performed significantly superior to this, particularly with regards to specificity. For differentiating patients from healthy controls, sensitivity and specificity of ALVQ were 97% and 98%, respectively. For differentiation of each pathogenic enzymatic defect, ALVQ performed superiorly, with sensitivity and specificity ranging between 95 and 100%. Conclusion: We present a novel steroid metabolomics approach, able to automatically detect and differentiate six different inborn disorders of steroidogenesis, with improved performance when compared to reference standard metabolite ratios. Steroid metabolomics can expedite and standardise interpretation of complex urinary steroid metabolome data, making this technique more accessible to clinicians, and has excellent potential for implementation in routine clinical practice.

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.

Evidence for Increased 5α-Reductase Activity During Early Childhood in Daughters of Women With Polycystic Ovary Syndrome.

Context:Polycystic ovary syndrome (PCOS) is a heritable, complex genetic disease. Animal models suggest that androgen exposure at critical developmental stages contributes to disease pathogenesis. We hypothesized that genetic variation resulting in increased androgen production produces the phenotypic features of PCOS by programming during critical developmental periods. Although we have not found evidence for increased in utero androgen levels in cord blood in the daughters of women with PCOS (PCOS-d), target tissue androgen production may be amplified by increased 5α-reductase activity analogous to findings in adult affected women. It is possible to noninvasively test this hypothesis by examining urinary steroid metabolites.Objective:We performed this study to investigate whether PCOS-d have altered androgen metabolism during early childhood.Design, Setting, and Participants:Twenty-one PCOS-d, 1–3 years old, and 36 control girls of comparable age were studied at an academic medical center.Main Outcome Measures:Urinary steroid metabolites were measured by gas chromatography/mass spectrometry. Twenty-four hour steroid excretion rates and precursor to product ratios suggestive of 5α-reductase and 11β-hydroxysteroid dehydrogenase activities were calculated.Results:Age did not differ but weight for length Z-scores were higher in PCOS-d compared to control girls (P = .02). PCOS-d had increased 5α-tetrahydrocortisol:tetrahydrocortisol ratios (P = .04), suggesting increased global 5α-reductase activity. There was no evidence for differences in 11β-hydroxysteroid dehydrogenase activity. Steroid metabolite excretion was not correlated with weight.Conclusions:Our findings suggest that differences in androgen metabolism are present in early childhood in PCOS-d. Increased 5α-reductase activity could contribute to the development of PCOS by amplifying target tissue androgen action.

Increased Steroid Excretion in Children with Extremely Low Birth Weight at a Median Age of 9.8 years

Background: Events during foetal or early extrauterine life may affect bodily structure and/or functions and even pave the way for adult diseases. Aims: To find whether extremely low birth weight (ELBW) infants differ from healthy controls regarding the excretion of steroid metabolites. Methods: The study compared 17 female and 10 male ELBW infants, all prepubertal, aged 8-11 years, birth weight <1,000 g, with 27 age- and sex-matched controls. All were healthy at the time of the study. Height, weight and BMI did not differ between the groups. Results were adjusted according to body surface area. 36 urinary steroid metabolites were quantified by gas chromatography-mass spectrometry. Results: In the ELBW girls 33/36 steroid metabolites were higher (19 significantly) than in the controls. All 36 steroid metabolites were higher in the ELBW boys (9 significantly) than in the controls. Sums of mineralocorticoid precursors, metabolites descriptive for cortisol and parameters of adrenal androgen production were significantly higher in ELBW infants (both sexes). Only the sum of the metabolites known to be illustrative for adrenal 11β-hydroxysteroid dehydrogenase activity was not different. Conclusion: Prepubertal ELBW children have an augmented urinary excretion of adrenal androgens, cortisol and mineralocorticoid precursors. These findings corroborate and help to explain the link between early-life adversity and subsequent adrenocortical function.

The Hidden Liquorice: Apparent Mineralocorticoid Excess Caused by Inadvertent Exposure to Liquorice Root Extract

ABSTRACT Objective: Excessive consumption of liquorice can cause endocrine symptoms of apparent mineralocorticoid excess (AME). This is usually caused by excessive consumption of liquorice-containing sweets and native liquorice root, but various chemical compounds in liquorice may also be used in many other products where they are considered additives and are therefore not explicitly declared. Methods: We here report a 21-year-old patient who presented with severe edema; spells of dizziness, headaches, and peripheral paraesthesia; and general and muscular fatigue. Results: Blood samples showed low serum potassium (2.3–3.1 mmol/L [ref 3.5–4.6]) requiring potassium supplementation (40 mmol × 2 daily) and high normal sodium (142–147 mmol/L [ref 137–145]). Measurement of 24-hour urinary steroid metabolite excretion raised the suspicion of AME. Additional blood tests showed undetectable serum concentrations of aldosterone (<38 pmol/L [ref 50–360]) and renin (<2 × 10−3 IU/L [ref 6–60]). Pituitary function (thyr...

Characterization of trenbolone acetate and estradiol metabolite excretion profiles in implanted steers.

Exogenous growth promoters have been used in US beef cattle production for over 50 yr. The environmental fate and transport of steroid growth promoters suggest potential for endocrine-disrupting effects among ecological receptors; however, the initial excretion of steroid metabolites from cattle administered growth promoters has not been well characterized. To better characterize excretion of trenbolone acetate and estrogen metabolites, steers were assigned to 1 of the following treatment groups: control, given no implant, or treatment, administered a combination implant (200 mg trenbolone acetate, 40 mg estradiol). Blood, urine, and fecal samples were collected over the course of 112 d following implantation. Samples were extracted and analyzed by liquid chromatography tandem mass spectrometry for trenbolone acetate and estrogen metabolites. In both urine and feces, 17α-trenbolone and 17α-estradiol were the predominant metabolites following implantation. Mean concentrations of 17α-trenbolone and 17α-estradiol in feces of implanted steers were 5.9 ± 0.37 ng/g and 2.7 ± 0.22 ng/g, respectively. A best-fit model is presented to predict 17α-trenbolone and 17α-estradiol excretion from steers receiving implants. The present study provides the first characterization of both trenbolone and estrogen metabolites in excreta from implanted cattle and will help provide estimates of steroid production from feedyards in the United States.

Prenatal Diagnosis of Congenital Adrenal Hyperplasia Caused by P450 Oxidoreductase Deficiency

Context:Mutations in the electron donor enzyme P450 oxidoreductase (POR) result in congenital adrenal hyperplasia with apparent combined 17α-hydroxylase/17,20 lyase and 21-hydroxylase deficiencies, also termed P450 oxidoreductase deficiency (PORD). Major clinical features present in PORD are disordered sex development in affected individuals of both sexes, glucocorticoid deficiency, and multiple skeletal malformations.Objective:The objective of the study was to establish a noninvasive approach to prenatal diagnosis of PORD including assessment of malformation severity to facilitate optimized prenatal diagnosis and timely treatment.Design:We analyzed 20 pregnancies with children homozygous or compound heterozygous for disease-causing POR mutations and 1 pregnancy with a child carrying a heterozygous POR mutation by recording clinical and biochemical presentations and fetal ultrasound findings. In 4 of the pregnancies (3 homozygous and 1 heterozygous for disease-causing POR mutations), prenatal analysis of steroid metabolite excretion in maternal urine was carried out by gas chromatography/mass spectrometry during gestational weeks 11–23.Results:Pregnancy complications in our cohort included maternal virilization (6 of 20) with onset in the second trimester. Seven pregnant women presented with low unconjugated estriol at prenatal screening (triple or quadruple antenatal screening test). Overt dysmorphic features were noted in 19 of the 20 babies at birth but observed in only 5 by prenatal ultrasound. These 5 had the most severe malformation phenotypes and poor outcome, whereas the other babies showed normal development. Steroid profiling of maternal urine revealed significantly increased steroids of fetal origin, namely the pregnenolone metabolite epiallopregnanediol and the androgen metabolite androsterone, with concomitant low values for estriol. Diagnostic steroid ratios conclusively indicated PORD as early as gestational week 12. In the heterozygous pregnancy, steroid ratios were only slightly elevated and estriol excretion was normal.Conclusion:Prenatal diagnosis in PORD is readily established via urinary steroid metabolite analysis of maternal urine. Visible malformations at prenatal ultrasound predict a severe malformation phenotype.

Mitotane Therapy in Adrenocortical Cancer Induces CYP3A4 and Inhibits 5α-Reductase, Explaining the Need for Personalized Glucocorticoid and Androgen Replacement

Mitotane [1-(2-chlorophenyl)-1-(4-chlorophenyl)-2,2-dichloroethane] is the first-line treatment for metastatic adrenocortical carcinoma (ACC) and is also regularly used in the adjuvant setting after presumed complete removal of the primary tumor. Mitotane is considered an adrenolytic substance, but there is limited information on distinct effects on steroidogenesis. However, adrenal insufficiency and male hypogonadism are widely recognized side effects of mitotane treatment. Our objective was to define the impact of mitotane treatment on in vivo steroidogenesis in patients with ACC. At seven European specialist referral centers for adrenal tumors, we analyzed 24-h urine samples (n = 127) collected from patients with ACC before and during mitotane therapy in the adjuvant setting (n = 23) or for metastatic ACC (n = 104). Urinary steroid metabolite excretion was profiled by gas chromatography/mass spectrometry in comparison with healthy controls (n = 88). We found a sharp increase in the excretion of 6β-hydroxycortisol over cortisol (P < 0.001), indicative of a strong induction of the major drug-metabolizing enzyme cytochrome P450 3A4. The contribution of 6β-hydroxycortisol to total glucocorticoid metabolites increased from 2% (median, interquartile range 1-4%) to 56% (39-71%) during mitotane treatment. Furthermore, we documented strong inhibition of systemic 5α-reductase activity, indicated by a significant decrease in 5α-reduced steroids, including 5α-tetrahydrocortisol, 5α-tetrahydrocorticosterone, and androsterone (all P < 0.001). The degree of inhibition was similar to that in patients with inactivating 5α-reductase type 2 mutations (n = 23) and patients receiving finasteride (n = 5), but cluster analysis of steroid data revealed a pattern of inhibition distinct from these two groups. Longitudinal data showed rapid onset and long-lasting duration of the observed effects. Cytochrome P450 3A4 induction by mitotane results in rapid inactivation of more than 50% of administered hydrocortisone, explaining the need for doubling hydrocortisone replacement in mitotane-treated patients. Strong inhibition of 5α-reductase activity is in line with the clinical observation of relative inefficiency of testosterone replacement in mitotane-treated men, calling for replacement by 5α-reduced androgens.

Steroid metabolism and excretion in severe anorexia nervosa: effects of refeeding

To our knowledge, changes in steroid metabolism in subjects with anorexia nervosa (AN) after weight gain have not been elucidated. We characterized urinary steroid excretion and metabolism in AN patients and investigated the effects of refeeding. In an intervention study, we recruited 7 women with life-threatening weight loss upon admission and after a median [interquartile range (IQR)] of 95 d (88-125 d) of intensive refeeding; 15 age-matched women were recruited as control subjects. The major urinary metabolites were quantified in 24-h collections by capillary gas chromatography. A single examiner measured weights, heights, and skinfold thicknesses. The median (IQR) age of patients was 24 y (21-26 y), and the duration of AN was 4.0 y (3.3-8.0 y). Body mass index (BMI; in kg/m(2)) increased from 12.8 (12.7-13.1) to 18.6 (18.0-19.6) after refeeding (P < 0.0001). Steroid values [median pre-, post-refeeding (P value)] were as follows: androgen metabolites [472, 1017 μg/24 h (0.93)], cortisol metabolites [1960, 3912 μg/24 h (0.60)], and ratios of androsterone (5α)/etiocholanolone (5β) [0.28, 0.63 (<0.001)], 5α-/5β-tetrahydrocortisol [0.20, 0.48 (0.02)], tetrahydrocortisols/tetrahydrocortisone [0.87, 0.61 (0.09)], 20-hydroxy-/20-oxocortisol metabolites [0.29, 0.47 (0.01)], and 20α-/20β-reduced cortisol metabolites [1.18, 1.89 (≥1.00)]. BMI change was positively correlated with 5α-/5β-tetrahydrocortisol (r = 0.95, P < 0.001). Before refeeding, the following metabolites were lower in patients than in control subjects: androsterone, 5α-tetrahydrocortisol, α-cortolone and α-cortol, 5α-/5β-tetrahydrocortisol, androsterone/etiocholanolone, and 20-hydroxy/20-oxocortisol (all P < 0.05). After refeeding, all steroid metabolites in patients were at concentrations that were comparable with those in control subjects. Significant changes in urine steroid-metabolite excretion occurred upon starvation, which were reversed upon refeeding. For cortisol, there were decreases in 5α-/5β-tetrahydrocortisol and 20-hydroxy-/20-oxometabolites; for androgen, there was a decrease in androsterone/etiocholanolone.

Insulin enhances ACTH-stimulated androgen and glucocorticoid metabolism in hyperandrogenic women

In hyperandrogenic women, hyperinsulinaemia amplifies 17 α-hydroxycorticosteroid intermediate response to ACTH, without alterations in serum cortisol or androgen response to stimulation. The aim of the study is to assess whether acute hyperinsulinaemia determines absolute changes in either basal or ACTH-stimulated adrenal steroidogenesis in these subjects. Twelve young hyperandrogenic women were submitted in two separate days to an 8 h hyperinsulinaemic (80 mU/m² × min) euglycaemic clamp, and to an 8 h saline infusion. In the second half of both the protocols, a 4 h ACTH infusion (62.5 μg/h) was carried out. Serum cortisol, progesterone, 17 α-hydroxyprogesterone (17-OHP), 17 α-hydroxypregnenolone (17-OHPREG), DHEA and androstenedione were measured at basal level and during the protocols. Absolute adrenal hormone secretion was quantified by measuring C19 and C21 steroid metabolites in urine collected after the first 4 h of insulin or saline infusion, and subsequently after 4 h of concurrent ACTH infusion. During insulin infusion, ACTH-stimulated 17-OHPREG and 17-OHP were significantly higher than during saline infusion. No significant differences in cortisol and androgens response to ACTH were found between the protocols. Nevertheless, urinary excretion of ACTH-stimulated C19 and C21 steroid metabolites was significantly higher during hyperinsulinaemia than at basal insulin levels (both P < 0.005). Changes in steroid metabolites molar ratios suggested stimulation by insulin of 5 α-reductase activity. These in vivo data support the hypothesis that insulin acutely enhances ACTH effects on both the androgen and glucocorticoid pathways.

Increased 5α-Reductase Activity and Adrenocortical Drive in Women with Polycystic Ovary Syndrome

ABSTRACT Context Polycystic Ovary Syndrome (PCOS) is characterized by hyperandrogenism, anovulation and susceptibility to the metabolic syndrome. Altered peripheral cortisol metabolism has been reported in PCOS but also in simple obesity. Objective To describe cortisol metabolism and metabolic characteristics of a large PCOS cohort and to delineate the effect of obesity by comparison to BMI-matched controls. Design Observational, cross-sectional study. Setting Outpatient clinics of two secondary/tertiary care centres Patients or Other Participants 178 PCOS patients fulfilling Rotterdam criteria and 100 BMI-matched controls. Intervention 24-h urine collection for steroid metabolite excretion, fasting blood samples followed by an OGTT. Main Outcome Measures Urinary steroid metabolites including glucocorticoids and androgens and the ratios reflecting enzymatic activities involved in peripheral cortisol and androgen metabolism, 5α-reductase and 11β-hydroxysteroid dehydrogenase type 1 and 2. Circulating levels of glucose, insulin, DHEA, DHEAS and testosterone, calculation of HOMA. Results Total androgen metabolites were higher in PCOS compared to BMI-matched controls (4105 ± 2047 vs. 2532 ± 1610 μg/24h for the non-obese, 5547 ± 2911 vs. 2468 ± 1794 μg/24hr for the obese, both p &amp;lt; 0.001). Total glucocorticoid metabolites were higher in obese PCOS vs. controls (10786 ± 3852 vs. 8834 ± 4487 μg/24hr, p = 0.001). 5α-reductase activity correlated with BMI, insulin levels and HOMA. Both obese and non-obese PCOS patients had higher 5α-reductase activity than controls (all p &amp;lt; 0.05). 11β-hydroxysteroid dehydrogenase activities did not differ between PCOS and controls. Conclusions PCOS is associated with enhanced androgen and cortisol metabolite excretion and increased 5α-reductase activity that cannot be explained by obesity alone. Increased adrenal corticosteroid production represents an important pathogenic pathway in PCOS.

A randomized placebo-controlled study on the effects of pioglitazone on cortisol metabolism in polycystic ovary syndrome

To investigate possible effects of insulin-sensitizing treatment on cortisol metabolism in insulin-resistant patients with polycystic ovary syndrome (PCOS). Randomized placebo-controlled study. Academic tertiary care medical center. Thirty insulin-resistant PCOS patients. Sixteen weeks of pioglitazone (30 mg/day) or placebo treatment. Twenty-four-hour 20 min integrated blood sampling for measurement of cortisol and 24 h urinary excretion of steroid metabolites. Relative 5alpha-reductase activity was evaluated by allotetrahydrocortisol (alloTHF)/THF and androsterone/etiocholanolone (A/E) ratios. Delta values denoted changes during the treatment period (16 weeks--basal). Pyridostigmine growth hormone (GH) stimulation tests were performed, and testosterone (T), dihydrotestosterone (DHT), DHEA, DHEAS, adiponectin, and insulin-like growth factor I (IGF-I) were measured before and after intervention. Insulin sensitivity, GH, adiponectin, and IGF-I significantly increased during pioglitazone treatment, whereas alloTHF/THF levels significantly decreased. Delta alloTHF/THF levels inversely correlated with Delta adiponectin levels. Delta A/E ratio inversely correlated with Delta IGF-I and Delta peak GH during GH stimulation tests. No significant changes were measured in T, DHT, DHEA, DHEAS, 24 h mean cortisol, or urinary excretion of steroid metabolites. Pioglitazone decreased relative 5alpha-reductase activity, whereas no significant changes were measured in cortisol levels or urinary cortisol excretion.

Reference intervals of urinary steroid metabolites using gas chromatography–mass spectrometry in Chinese adults

Background Urinary steroid profiling by GC or GC–MS are established clinical tools to complement other biochemical tests in the diagnosis and investigation of a wide range of adrenocortical disorders, but normative data on adults using the more specific GC–MS are lacking. Our objective was to set up the reference intervals of commonly detected urinary steroid metabolites as well as marker metabolites seen in disease states. Method Apparently healthy adult Chinese males and females were recruited by completing health questionnaires. A 24-h urine specimen was collected from all the participants for urinary steroid profiling by GC–MS in cyclic scan mode. The analyzer was calibrated by using authentic steroid standards. Statistical methods recommended by the National Committee for Clinical Laboratory Standards were followed for setting up the reference intervals of various steroid metabolites. After outliers were excluded, the data were tested for the necessity to partition into sex-, menopausal status- and age-specific reference intervals. Results 83 males and 89 females were recruited for the study. Necessity to partition into sex-specific reference intervals was demonstrated for almost all steroid metabolites. Menopausal status and age also had a significant impact on steroid metabolite excretion, making separate reference intervals necessary. Conclusions We have set up the normative data on the levels of urinary steroid metabolite excretion in Chinese adults for future reference in patient management and research in steroid metabolism.

Sex hormones and bile acids in intrahepatic cholestasis of pregnancy

Sex hormones and bile acids in intrahepatic cholestasis of pregnancy

Phenotypic consequences of variation across the aldosterone synthase and 11‐beta hydroxylase locus in a hypertensive cohort: data from the MRC BRIGHT Study

Aldosterone is an important cardiovascular hormone; 15% of hypertensive subjects have alteration in aldosterone regulation, defined by a raised ratio of aldosterone to renin (ARR). Studies of the aldosterone synthase gene (CYP11B2) have focused on a single nucleotide polymorphism in the 5'promoter region (-344 C/T). In normotensive subjects, the T allele associates with raised levels of the 11-deoxysteroids, deoxycorticosterone and 11-deoxycortisol which are substrates for 11beta-hydroxylase, encoded by the adjacent and homologous gene, CYP11B1. We have speculated that this altered 11beta-hydroxylase efficiency leads to increased ACTH drive to the adrenal gland to maintain cortisol production and reported herein the association between the -344 C/T single nucleotide polymorphism (SNP) and adrenal steroid production in subjects with essential hypertension. The CYP11B2-344 C/T polymorphism was genotyped and urinary excretion of adrenal steroid metabolites was measured (by GCMS) in 511 unrelated hypertensives from the Medical Research Council (MRC) British Genetics of Hypertension (BRIGHT) study. Thirty-five per cent of subjects were homozygous for the -344T allele whilst 16% were CC homozygotes. There was no difference in cortisol excretion rate between the two genotype groups but the index of adrenal 11beta-hydroxylation (ratio of tetrahydrodeoxycortisol/total cortisol) was significantly higher in the TT group (P < 0.005) than in the CC group. Excretion rates of the major urinary metabolite of aldosterone (tetrahydroaldosterone) correlated strongly with the ACTH-regulated steroids, cortisol (r = 0.437, P < 0.0001) and total androgen metabolites (r = 0.4, P < 0.0001) in TT but not CC subjects. Hypertensives homozygous for the -344 T allele of CYP11B2 demonstrate altered 11beta-hydroxylase efficiency (CYP11B1); this is consistent with the hypothesis of a genetically determined increase in adrenal ACTH drive in these subjects. The correlation between excretion of aldosterone and cortisol metabolites and suggests that, in TT subjects, ACTH exerts an important common regulatory influence on adrenal corticosteroid production in subjects with hypertension.