Beverly Hughes

Gas chromatography/mass spectrometry (GC/MS) remains a pre-eminent discovery tool in clinical steroid investigations even in the era of fast liquid chromatography tandem mass spectrometry (LC/MS/MS)

Liquid chromatography tandem mass spectrometry (LC/MS/MS) is replacing classical methods for steroid hormone analysis. It requires small sample volumes and has given rise to improved specificity and short analysis times. Its growth has been fueled by criticism of the validity of steroid analysis by older techniques, testosterone measurements being a prime example. While this approach is the gold-standard for measurement of individual steroids, and panels of such compounds, LC/MS/MS is of limited use in defining novel metabolomes. GC/MS, in contrast, is unsuited to rapid high-sensitivity analysis of specific compounds, but remains the most powerful discovery tool for defining steroid disorder metabolomes. Since the 1930s almost all inborn errors in steroidogenesis have been first defined through their urinary steroid excretion. In the last 30 years, this has been exclusively carried out by GC/MS and has defined conditions such as AME syndrome, glucocorticoid remediable aldosteronism (GRA) and Smith–Lemli–Opitz syndrome. Our recent foci have been on P450 oxidoreductase deficiency (ORD) and apparent cortisone reductase deficiency (ACRD). In contrast to LC/MS/MS methodology, a particular benefit of GC/MS is its non-selective nature; a scanned run will contain every steroid excreted, providing an integrated picture of an individuals metabolome. The “Achilles heel” of clinical GC/MS profiling may be data presentation. There is lack of familiarity with the multiple hormone metabolites excreted and diagnostic data are difficult for endocrinologists to comprehend. While several conditions are defined by the absolute concentration of steroid metabolites, many are readily diagnosed by ratios between steroid metabolites (precursor metabolite/product metabolite). Our work has led us to develop a simplified graphical representation of quantitative urinary steroid hormone profiles and diagnostic ratios.

Urine Steroid Metabolomics as a Biomarker Tool for Detecting Malignancy in Adrenal Tumors

Context: Adrenal tumors have a prevalence of around 2% in the general population. Adrenocortical carcinoma (ACC) is rare but accounts for 2–11% of incidentally discovered adrenal masses. Differentiating ACC from adrenocortical adenoma (ACA) represents a diagnostic challenge in patients with adrenal incidentalomas, with tumor size, imaging, and even histology all providing unsatisfactory predictive values. Objective: Here we developed a novel steroid metabolomic approach, mass spectrometry-based steroid profiling followed by machine learning analysis, and examined its diagnostic value for the detection of adrenal malignancy. Design: Quantification of 32 distinct adrenal derived steroids was carried out by gas chromatography/mass spectrometry in 24-h urine samples from 102 ACA patients (age range 19–84 yr) and 45 ACC patients (20–80 yr). Underlying diagnosis was ascertained by histology and metastasis in ACC and by clinical follow-up [median duration 52 (range 26–201) months] without evidence of metastasis in ACA. Steroid excretion data were subjected to generalized matrix learning vector quantization (GMLVQ) to identify the most discriminative steroids. Results: Steroid profiling revealed a pattern of predominantly immature, early-stage steroidogenesis in ACC. GMLVQ analysis identified a subset of nine steroids that performed best in differentiating ACA from ACC. Receiver-operating characteristics analysis of GMLVQ results demonstrated sensitivity = specificity = 90% (area under the curve = 0.97) employing all 32 steroids and sensitivity = specificity = 88% (area under the curve = 0.96) when using only the nine most differentiating markers. Conclusions: Urine steroid metabolomics is a novel, highly sensitive, and specific biomarker tool for discriminating benign from malignant adrenal tumors, with obvious promise for the diagnostic work-up of patients with adrenal incidentalomas.

Clinical and Biochemical Consequences of CYP17A1 Inhibition with Abiraterone Given with and without Exogenous Glucocorticoids in Castrate Men with Advanced Prostate Cancer

CONTEXT Abiraterone acetate is a small-molecule cytochrome P450 17A1 (CYP17A1) inhibitor that is active in castration-resistant prostate cancer. OBJECTIVE Our objective was to determine the impact of abiraterone with and without dexamethasone treatment on in vivo steroidogenesis. DESIGN AND METHODS We treated 42 castrate, castration-resistant prostate cancer patients with continuous, daily abiraterone acetate and prospectively collected blood and urine before and during abiraterone treatment and after addition of dexamethasone 0.5 mg daily. RESULTS Treatment with single-agent abiraterone acetate was associated with accumulation of steroids with mineralocorticoid properties upstream of CYP17A1. This resulted in side effects, including hypertension, hypokalemia, and fluid overload, in 38 of 42 patients that were generally treated effectively with eplerenone. Importantly, serum and urinary androgens were suppressed by more than 90% from baseline. Urinary metabolites of 17-hydroxypregnenolone and 17-hydroxyprogesterone downstream of 17α-hydroxylase remained unchanged. However, 3α5α-17-hydroxypregnanolone, which can be converted via the backdoor pathway toward 5α-dihydrotestosterone, increased significantly and correlated with levels of the major 5α-dihydrotestosterone metabolite androsterone. In contrast, urinary metabolites of 11-deoxycortisol and active glucocorticoids declined significantly. Addition of dexamethasone to abiraterone acetate significantly suppressed ACTH and endogenous steroids, including 3α5α-17-hydroxypregnanolone. CONCLUSION CYP17A1 inhibition with abiraterone acetate is characterized by significant suppression of androgen and cortisol synthesis. The latter is associated with a rise in ACTH that causes raised mineralocorticoids, leading to side effects and incomplete 17α-hydroxylase inhibition. Concomitant inhibition of 17,20-lyase results in diversion of 17-hydroxyprogesterone metabolites toward androgen synthesis via the backdoor pathway. Addition of dexamethasone reverses toxicity and could further suppress androgens by preventing a rise in substrates of backdoor androgen synthesis.

Hyperandrogenemia Predicts Metabolic Phenotype in Polycystic Ovary Syndrome: The Utility of Serum Androstenedione

Context: Polycystic ovary syndrome (PCOS) is a triad of anovulation, insulin resistance, and hyperandrogenism. Androgen excess may correlate with metabolic risk and PCOS consensus criteria define androgen excess on the basis of serum T. Here we studied the utility of the androgen precursor serum androstenedione (A) in conjunction with serum T for predicting metabolic dysfunction in PCOS. Patients and Methods: Eighty-six PCOS patients fulfilling Rotterdam diagnostic consensus criteria and 43 age- and body mass index-matched controls underwent measurement of serum androgens by tandem mass spectrometry and an oral glucose tolerance test with homeostatic model assessment of insulin resistance and insulin sensitivity index calculation. We analyzed 24-hour urine androgen excretion by gas chromatography/mass spectrometry. Results: PCOS patients had higher levels of serum androgens and urinary androgen metabolites than controls (all P < .001). Within the PCOS cohort, both serum A and T were positively correlated with the free androgen index (T × 100/SHBG) and total androgen metabolite excretion (all P < .001). All subjects with T above the normal reference range [high T (HT)] also had high A (HA/HT group, n = 56). However, the remaining 30 patients had normal T levels, either in the presence of HA (HA/NT; n = 20) or normal A (NA/NT; n = 10). The groups did not differ in age or BMI. The HA/HT and HA/NT groups had higher total androgen excretion than NA/NT (P < .01 and P < .05, respectively). Multiple linear regression showed a strong negative association between serum androstenedione and insulin sensitivity. The incidence of dysglycemia according to an oral glucose tolerance test increased with the severity of androgen phenotype (NA/NT, 0%; HA/NT, 14%; HA/HT, 25%, P = .03). Conclusion: Simultaneous measurement of serum T and A represents a useful tool for predicting metabolic risk in PCOS women. HA levels are a sensitive indicator of PCOS-related androgen excess.

Impaired glucose tolerance and insulin resistance are associated with increased adipose 11β-hydroxysteroid dehydrogenase type 1 expression and elevated hepatic 5α-reductase activity

OBJECTIVE—The precise molecular mechanisms contributing to the development of insulin resistance, impaired glucose tolerance (IGT), and type 2 diabetes are largely unknown. Altered endogenous glucocorticoid metabolism, including 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1), which generates active cortisol from cortisone, and 5α-reductase (5αR), which inactivates cortisol, has been implicated. RESEARCH DESIGN AND METHODS—A total of 101 obese patients (mean age 48 ± 7 years, BMI 34.4 ± 4.3 kg/m2, 66 women, 35 men) underwent 75-g oral glucose tolerance testing (OGTT), body composition analysis (dual-energy X-ray absorptiometry), assessment of glucocorticoid metabolism (24-h urine steroid metabolite analysis by gas chromatography/mass spectrometry), and subcutaneous abdominal adipose tissue biopsies. RESULTS—A total of 22.7% of women had IGT compared with 34.2% of men. Two women and five men were diagnosed with type 2 diabetes. In women, adipose 11β-HSD1 expression was increased in patients with IGT and correlated with glucose levels across the OGTT (R = 0.44, P < 0.001) but was independent of fat mass. Total glucocorticoid secretion was higher in men with and without IGT (normal 13,743 ± 863 vs. 7,453 ± 469 μg/24 h, P < 0.001; IGT 16,871 ± 2,113 vs. 10,133 ± 1,488 μg/24 h, P < 0.05), and in women, it was higher in those with IGT (7,453 ± 469 vs. 10,133 ± 1,488 μg/24 h, P < 0.001). In both sexes, 5αR activity correlated with fasting insulin (men R = 0.53, P = 0.003; women R = 0.33, P = 0.02), insulin secretion across an OGTT (men R = 0.46, P = 0.01; women R = 0.40, P = 0.004), and homeostasis model assessment of insulin resistance (men R = 0.52, P = 0.004; women R = 0.33, P = 0.02). CONCLUSIONS—Increased adipose 11β-HSD1 expression in women may contribute to glucose intolerance. Enhanced 5αR activity in both sexes is associated with insulin resistance but not body composition. Augmented glucocorticoid inactivation may serve as a compensatory, protective mechanism to preserve insulin sensitivity.

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

CONTEXT 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. OBJECTIVE Our objective was to define the impact of mitotane treatment on in vivo steroidogenesis in patients with ACC. SETTING AND DESIGN 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). RESULTS 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. CONCLUSIONS 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.

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

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 The aim of the study was to describe cortisol metabolism and metabolic characteristics of a large PCOS cohort and to delineate the effect of obesity by comparison to body mass index (BMI)-matched controls. DESIGN AND SETTING We conducted an observational, cross-sectional study at outpatient clinics of two secondary/tertiary care centers. PATIENTS OR OTHER PARTICIPANTS A total of 178 PCOS patients fulfilling Rotterdam criteria and 100 BMI-matched controls participated in the study. INTERVENTION The study included 24-h urine collection for steroid metabolite excretion and fasting blood samples, followed by an oral glucose tolerance test. MAIN OUTCOME MEASURES We measured urinary steroid metabolites including glucocorticoids and androgens and the ratios reflecting enzymatic activities involved in peripheral cortisol and androgen metabolism, 5 alpha-reductase, and 11 beta-hydroxysteroid dehydrogenase types 1 and 2. We also measured circulating levels of glucose, insulin, dehydroepiandrosterone, dehydroepiandrosterone sulfate, and testosterone and calculated homeostasis model assessment. RESULTS Total androgen metabolites were higher in PCOS patients compared to BMI-matched controls (4,105 +/- 2,047 vs. 2,532 +/- 1,610 microg/24 h for the nonobese; 5,547 +/- 2,911 vs. 2,468 +/- 1,794 microg/24 h for the obese; both P < 0.001). Total glucocorticoid metabolites were higher in obese PCOS vs. controls (10,786 +/- 3,852 vs. 8,834 +/- 4,487 microg/24 h; P = 0.001). 5 alpha-Reductase activity correlated with BMI, insulin levels, and homeostasis model assessment. Both obese and nonobese PCOS patients had higher 5 alpha-reductase activity than controls (all P < 0.05). 11 beta-Hydroxysteroid dehydrogenase activities did not differ between PCOS and controls. CONCLUSIONS PCOS is associated with enhanced androgen and cortisol metabolite excretion and increased 5 alpha-reductase activity that cannot be explained by obesity alone. Increased adrenal corticosteroid production represents an important pathogenic pathway in PCOS.

Reduced Glucocorticoid Production Rate, Decreased 5α-Reductase Activity, and Adipose Tissue Insulin Sensitization After Weight Loss

OBJECTIVE—The epidemics of obesity, insulin resistance, and type 2 diabetes have heightened the need to understand mechanisms that contribute to their pathogenesis. Increased endogenous glucocorticoid production has been implicated based on parallels with Cushings syndrome. We have assessed the impact of weight loss on glucocorticoid secretion and metabolism (notably 11β-hydroxysteroid dehydrogenase type 1 and 5α-reductase [5αR] activity) and insulin sensitivity. RESEARCH DESIGN AND METHODS—Twenty obese volunteers were investigated before and after weight loss. Patients underwent hyperinsulinemic-euglycemic clamps with simultaneous adipose microdialysis and oral cortisone acetate administration. Changes in glucocorticoid secretion and metabolism were assessed using 24-h urine collections. RESULTS—Before weight loss, fat mass correlated with glucocorticoid secretion rate (total fat, r = 0.46, P < 0.05; trunk fat, r = 0.52, P < 0.05); however, glucocorticoid secretion rate was inversely related to insulin sensitivity (r = −0.51, P < 0.05). Hyperinsulinemia failed to suppress adipose tissue interstitial fluid glycerol release (180 ± 50 μmol [basal] vs. 153 ± 10 μmol [steady state], NS). After oral cortisone (25 mg), cortisol concentrations within adipose interstitial fluid increased (4.3 ± 1.1 vs. 14.2 ± 2.6 nmol/l, P < 0.01), but glycerol concentrations did not change. After weight loss, insulin sensitivity increased. Consistent with insulin sensitization, adipose tissue interstitial fluid glycerol concentrations fell under hyperinsulinemic conditions (186 ± 16 vs. 117 ± 9 μmol, P < 0.05). Glucocorticoid secretion decreased (11,751 ± 1,520 vs. 7,464 ± 937 μg/24 h, P < 0.05) as did 5αR activity (5α-tetrahydrocortisol–to–tetrahydrocortisol ratio 1.41 ± 0.16 vs. 1.12 ± 0.17, P < 0.005). CONCLUSIONS—Obesity is associated with insulin resistance within adipose tissue and increased cortisol secretion rates; both are reversed with weight loss. Reduced 5αR activity after weight loss may decrease hypothalamo-pituitary-adrenal axis activation and reduce glucocorticoid metabolite production.

Genotype-phenotype analysis in congenital adrenal hyperplasia due to P450 oxidoreductase deficiency

Context: P450 oxidoreductase deficiency (PORD) is a unique congenital adrenal hyperplasia variant that manifests with glucocorticoid deficiency, disordered sex development (DSD), and skeletal malformations. No comprehensive data on genotype-phenotype correlations in Caucasian patients are available. Objective: The objective of the study was to establish genotype-phenotype correlations in a large PORD cohort. Design: The design of the study was the clinical, biochemical, and genetic assessment including multiplex ligation-dependent probe amplification (MLPA) in 30 PORD patients from 11 countries. Results: We identified 23 P450 oxidoreductase (POR) mutations (14 novel) including an exonic deletion and a partial duplication detected by MLPA. Only 22% of unrelated patients carried homozygous POR mutations. p.A287P was the most common mutation (43% of unrelated alleles); no other hot spot was identified. Urinary steroid profiling showed characteristic PORD metabolomes with variable impairment of 17α-hydroxylase and 21-hydroxylase. Short cosyntropin testing revealed adrenal insufficiency in 89%. DSD was present in 15 of 18 46,XX and seven of 12 46,XY individuals. Homozygosity for p.A287P was invariably associated with 46,XX DSD but normal genitalia in 46,XY individuals. The majority of patients with mild to moderate skeletal malformations, assessed by a novel scoring system, were compound heterozygous for missense mutations, whereas nearly all patients with severe malformations carried a major loss-of-function defect on one of the affected alleles. Conclusions: We report clinical, biochemical, and genetic findings in a large PORD cohort and show that MLPA is a useful addition to POR mutation analysis. Homozygosity for the most frequent mutation in Caucasians, p.A287P, allows for prediction of genital phenotype and moderate malformations. Adrenal insufficiency is frequent, easily overlooked, but readily detected by cosyntropin testing.

11β-Hydroxysteroid Dehydrogenase Type 1 Regulation by Intracellular Glucose 6-Phosphate Provides Evidence for a Novel Link between Glucose Metabolism and Hypothalamo-Pituitary-Adrenal Axis Function

Microsomal glucose-6-phosphatase-α (G6Pase-α) and glucose 6-phosphate transporter (G6PT) work together to increase blood glucose concentrations by performing the terminal step in both glycogenolysis and gluconeogenesis. Deficiency of the G6PT in liver gives rise to glycogen storage disease type 1b (GSD1b), whereas deficiency of G6Pase-α leads to GSD1a. G6Pase-α shares its substrate (glucose 6-phosphate; G6P) with hexose-6-phosphate-dehydrogenase (H6PDH), a microsomal enzyme that regenerates NADPH within the endoplasmic reticulum lumen, thereby conferring reductase activity upon 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1). 11β-HSD1 interconverts hormonally active C11β-hydroxy steroids (cortisol in humans and corticosterone in rodents) to inactive C11-oxo steroids (cortisone and 11-dehydrocorticosterone, respectively). In vivo reductase activity predominates, generating active glucocorticoid. We hypothesized that substrate (G6P) availability to H6PDH in patients with GSD1b and GSD1a will decrease or increase 11β-HSD1 reductase activity, respectively. We investigated 11β-HSD1 activity in GSD1b and GSD1a mice and in two patients with GSD1b and five patients diagnosed with GSD1a. We confirmed our hypothesis by assessing 11β-HSD1 in vivo and in vitro, revealing a significant decrease in reductase activity in GSD1b animals and patients, whereas GSD1a patients showed a marked increase in activity. The cellular trafficking of G6P therefore directly regulates 11β-HSD1 reductase activity and provides a novel link between glucose metabolism and function of the hypothalamo-pituitary-adrenal axis.