Identification of a human blood biomarker of pharmacological 11ß-hydroxysteroid dehydrogenase 1 inhibition.

BACKGROUND AND PURPOSE: 11ß-Hydroxysteroid dehydrogenase-1 (11ß-HSD1) catalyses the oxoreduction of cortisone to cortisol, amplifying levels of active glucocorticoids. It is a pharmaceutical target in metabolic disease and cognitive impairments. 11ß-HSD1 also converts some 7oxo-steroids to their 7ß-hydroxy forms. A recent study in mice described the ratio of tauroursodeoxycholic acid (TUDCA)/tauro-7oxolithocholic acid (T7oxoLCA) as a biomarker for decreased 11ß-HSD1 activity. The present study evaluates the equivalent bile acid ratio of glycoursodeoxycholic acid (GUDCA)/glyco-7oxolithocholic acid (G7oxoLCA) as a biomarker for pharmacological 11ß-HSD1 inhibition in humans and compares it with the currently applied urinary (5α-tetrahydrocortisol + tetrahydrocortisol)/tetrahydrocortisone ((5αTHF + THF)/THE) ratio. EXPERIMENTAL APPROACH: Bile acid profiles were analysed by ultra-HPLC tandem-MS in blood samples from two independent, double-blind placebo-controlled clinical studies of the orally administered selective 11ß-HSD1 inhibitor AZD4017. The blood GUDCA/G7oxoLCA ratio was compared with the urinary tetrahydro-glucocorticoid ratio for ability to detect 11ß-HSD1 inhibition. KEY RESULTS: No significant alterations were observed in bile acid profiles following 11ß-HSD1 inhibition by AZD4017, except for an increase of the secondary bile acid G7oxoLCA. The enzyme product/substrate ratio GUDCA/G7oxoLCA was found to be more reliable to detect 11ß-HSD1 inhibition than the absolute G7oxoLCA concentration in both cohorts. Comparison of the blood GUDCA/G7oxoLCA ratio with the urinary (5αTHF + THF)/THE ratio revealed that both successfully detect 11ß-HSD1 inhibition. CONCLUSIONS AND IMPLICATIONS: 11ß-HSD1 inhibition does not cause major alterations in bile acid homeostasis. The GUDCA/G7oxoLCA ratio represents the first blood biomarker of pharmacological 11ß-HSD1 inhibition and may replace or complement the urinary (5αTHF + THF)/THE ratio biomarker.

High-resolution daily profiles of tissue adrenal steroids by portable automated collection.

Rhythms are intrinsic to endocrine systems, and disruption of these hormone oscillations occurs at very early stages of the disease. Because adrenal hormones are secreted with both circadian and ultradian periods, conventional single-time point measurements provide limited information about rhythmicity and, crucially, do not provide information during sleep, when many hormones fluctuate from nadir to peak concentrations. If blood sampling is attempted overnight, then this necessitates admission to a clinical research unit, can be stressful, and disturbs sleep. To overcome this problem and to measure free hormones within their target tissues, we used microdialysis, an ambulatory fraction collector, and liquid chromatography-tandem mass spectrometry to obtain high-resolution profiles of tissue adrenal steroids over 24 hours in 214 healthy volunteers. For validation, we compared tissue against plasma measurements in a further seven healthy volunteers. Sample collection from subcutaneous tissue was safe, well tolerated, and allowed most normal activities to continue. In addition to cortisol, we identified daily and ultradian variation in free cortisone, corticosterone, 18-hydroxycortisol, aldosterone, tetrahydrocortisol and allo-tetrahydrocortisol, and the presence of dehydroepiandrosterone sulfate. We used mathematical and computational methods to quantify the interindividual variability of hormones at different times of the day and develop "dynamic markers" of normality in healthy individuals stratified by sex, age, and body mass index. Our results provide insight into the dynamics of adrenal steroids in tissue in real-world settings and may serve as a normative reference for biomarkers of endocrine disorders (ULTRADIAN, NCT02934399).

Is the 1mg-dexamethasone suppression test a precise marker of glucocorticoid excess and cardiometabolic risk in patients with adrenal incidentalomas?

AIM: To analyze if the 1mg-dexamethasone suppression test (DST) is a reliable marker of glucocorticoid excess and cardiometabolic risk in patients with adrenal incidentalomas (AIs). METHODS: Cross-sectional study of patients with nonfunctioning adrenal incidentalomas (NFAIs, defined by cortisol post-DST ≤ 1.8 µg/dL) and patients with autonomous cortisol secretion (ACS, defined by cortisol post-DST > 1.8 µg/Dl). The urinary steroid profile (USP) was determined by gas chromatography coupled to mass spectrometry. Both groups were matched by sex, age and body mass index. RESULTS: Forty-nine patients with AIs (25 with ACS and 24 with NFAI) were included. As a whole, AIs showed a high excretion of ß-cortolone, tetrahydro-11-deoxycortisol (THS), α-cortolone, α-cortol, tetrahydrocortisol (THF) and tetrahydrocortisone (THE). A positive yet modest correlation between post-DST cortisol and total excretion of glucocorticoid metabolites (r = 0.401, P = 0.004) was observed, with the stronger being observed with total THS (r = 0.548, P < 0.001) and THF (r = 0.441, P = 0.002). Some of the metabolites that were elevated in patients with AIs, were higher in patients with ACS-related comorbidities than in those without comorbidities. Post-DST cortisol showed a fair diagnostic accuracy for the prediction of ACS-related comorbidities (AUC 0.767 [95% CI 0.634-0.882]). However, post-DST diagnostic accuracy improved when combined with urinary cortisone, α-cortol, THS and serum DHEAS (0.853 [0.712‒0.954]). CONCLUSION: The DST has a positive, but modest, correlation with urinary glucocorticoid excretion. Similarly, the diagnostic accuracy of the DST for the prediction of ACS-related comorbidities is only fair, but it may be improved if combined with the results of the USP and serum DHEAS. SIGNIFICANCE STATEMENT: This is the first study aimed to evaluate if 1mg-dexamethasone suppression test (DST) is a reliable marker of glucocorticoid excess and cardiometabolic risk in patients with adrenal incidentalomas (AIs) and if urinary steroid profile was measured by GS-MS could improve such a prediction. We found a positive yet modest correlation between post-DST cortisol and total excretion of glucocorticoid metabolites, with the stronger being observed with total tetrahydro-11-deoxycortisol (THS) and tetrahydrocortisol. Post-DST cortisol showed a fair diagnostic accuracy for the prediction of ACS-related comorbidities (AUC 0.767). However, post-DST diagnostic accuracy improved when combined with urinary cortisone, α-cortol, THS and serum DHEAS (0.853).

URINARY METABOLOMICS OF CENTRAL SEROUS CHORIORETINOPATHY.

PURPOSE: To analyze the urinary metabolomic profile of central serous chorioretinopathy cases. METHODS: In a cross-sectional study, 80 participants with central serous chorioretinopathy were compared with 80 age-matched and sex-matched controls. Urinary metabolites were measured using Metabolon's Discovery HD4 platform. RESULTS: Of 1,031 metabolites total that were measured in urine samples, 53 were upregulated and 27 downregulated in central serous chorioretinopathy participants compared with controls. After exclusion of potentially confounding xenobiotics and bile compounds that could represent digestive processes, 14 metabolites were significantly higher and 12 metabolites were significantly lower in cases compared with controls. One upregulated metabolite (tetrahydrocortisol sulfate) is involved in the corticosteroid subpathway. The downregulated metabolites are unrelated to the identified corticosteroid subpathway. CONCLUSION: The upregulation of urinary tetrahydrocortisol sulfate in central serous chorioretinopathy cases provides a precise molecular basis to further study the role of corticosteroids in producing choroidal venous congestion.

Safety, tolerability, pharmacodynamics and pharmacokinetics following once-daily doses of BI 187004, an inhibitor of 11 beta-hydroxysteroid dehydrogenase-1, over 28 days in patients with type 2 diabetes mellitus and overweight or obesity.

AIMS: To study the oral 11 beta-hydroxysteroid dehydrogenase-1 (11ß-HSD1) inhibitor BI 187004 (NCT02150824), as monotherapy and in combination with metformin, versus placebo in patients with type 2 diabetes mellitus (T2DM) affected by overweight or obesity. MATERIALS AND METHODS: This Phase II, randomized controlled trial investigated multiple rising doses of BI 187004 as monotherapy (Arm 1: 20, 80 or 240 mg) and in combination with metformin (Arm 2: 240 mg), in adults with T2DM and a body mass index of 28-40 kg/m2 . RESULTS: In total, 103 patients (Arm 1: n = 62, Arm 2: n = 41) were included in this study. BI 187004 was rapidly absorbed and exposure increased approximately dose-dependently. Target engagement of 11ß-HSD1 was observed with near-full inhibition of 11ß-HSD1 in the liver [decreased (5α-tetrahydrocortisol + 5ß-tetrahydrocortisol)/tetrahydrocortisone ratio]; hypothalamic-pituitary-adrenal axis activation was also seen (increased total urinary corticosteroids). No clinically relevant changes from baseline with BI 187004 treatment were observed for bodyweight or meal tolerance test parameters, or in most efficacy endpoints testing glucose and lipid metabolism; a significant increase was observed in weighted mean plasma glucose (p < .05 for 80 and 240 mg BI 187004) but not fasting plasma glucose. Drug-related adverse events were reported for 14 patients (22.6%) in Arm 1 and 10 patients (24.4%) in Arm 2, most frequently headache, diarrhoea, flushing and dizziness. A dose-dependent increase in heart rate was seen with BI 187004 treatment. CONCLUSIONS: BI 187004 was generally well tolerated in patients with T2DM. Despite complete 11ß-HSD1 inhibition, no clinically relevant effects were observed with BI 187004.

Validation of steroid ratios for random urine by mass spectrometry to detect 5α-reductase deficiency in Vietnamese children.

OBJECTIVES: The 5α-reductase-type-2 deficiency (5ARD2) is a rare autosomal recessive 46,XY disorder of sex development caused by the mutated 5α-reductase type 2 (SRD5A2) gene. In this disease, defective conversion of testosterone to dihydrotestosterone leads to variable presentations of male ambiguous genitalia during fetal development. We aimed to examine characteristics of patients presenting with 5ARD2 over a 4 year period. METHODS: Random urine samples of control and patients with suspected 5ARD2 were collected and urine steroidomic metabolites were measured by Gas chromatography-mass spectrometry (GC-MS) in the period from 2017 to 2021 at National Children's Hospital, Hanoi Vietnam. 5α- to 5ß-reduced steroid metabolite ratio, 5a-tetrahydrocortisol to tetrahydrocortisol (5α-THF/THF), was reviewed by receive operator characteristics (ROC) curve analysis. Molecular testing was offered to 25 patients who were diagnosed with 5ARD2 by GC-MS urinary steroid analysis. RESULTS: Urine steroidomic profiling was conducted for 104 male controls and 25 patients between the ages of 6 months and 13 years old. Twelve of the twenty-five 5ARD2 patients agreed to undertake genetic analysis, and two mutations of the SRD5A2 gene were detected in each patient, confirming the diagnosis. All patients showed a characteristically low ratio of 5α-THF/THF. There was no overlap of 5α-THF/THF ratio values between control and 5ARD2 groups. The ROC of 5α-THF/THF ratio at 0.19 showed 100% sensitivity and 100% specificity for boys between 6 months and 13 years of age. CONCLUSIONS: Analysis of the urine steroid metabolome by GC-MS can be used to assist in the diagnosis of 5ARD2. We recommend consideration of random urine steroid analysis as a first-line test in the diagnosis of 5ARD2.

Maternal Signatures of Cortisol in First Trimester Small-for-Gestational Age.

The objective of this study was to identify predictable maternal serum signatures of cortisol metabolism during the first trimester of women who are expected to deliver small-for-gestational-age (SGA) neonates. This prospective cohort study included 112 pregnant women (with and without SGA, n = 56 each). Maternal serum samples were collected at 10-14 gestational weeks to quantify the levels of cortisol and its precursors and metabolites by liquid chromatography-mass spectrometry. Increased maternal serum levels of tetrahydrocortisol (11.82 ± 8.16 ng/mL vs. 7.51 ± 2.90 ng/mL, P < 0.005) and decreased 21-deoxycortisol (2.98 ± 1.36 ng/mL vs. 4.33 ± 2.06 ng/mL, P < 0.0001) were observed in pregnant women carrying SGA fetus. In conjunction with individual steroid levels, metabolic ratios corresponding to the activity of related enzymes were calculated. In addition to increased tetrahydrocortisol/cortisol ratio (P < 0.006), the SGA group showed a significant increase in the two metabolic ratios including cortisol/11-deoxycortisol (P < 0.03) and cortisol/21-deoxycortisol (P < 0.0003). The receiver operating characteristic (ROC) curve generated in combination with three variables of 21-deoxycortisol concentration and two metabolic ratios of cortisol/21-deoxycortisol and tetrahydrocortisol/cortisol resulted in an area under the ROC curve = 0.824 (95% confidence interval, 0.713-0.918). A significant decrease in maternal serum levels of 21-deoxycortisol and an increase in two metabolic ratios of cortisol/21-deoxycortisol and tetrahydrocortisol/cortisol, indicating cortisol biosynthetic rate, represent potential biomarkers for the prediction of SGA in the first trimester.

Improved Urinary Cortisol Metabolome in Addison Disease: A Prospective Trial of Dual-Release Hydrocortisone.

CONTEXT: Oral once-daily dual-release hydrocortisone (DR-HC) replacement therapy has demonstrated an improved metabolic profile compared to conventional 3-times-daily (TID-HC) therapy among patients with primary adrenal insufficiency. This effect might be related to a more physiological cortisol profile, but also to a modified pattern of cortisol metabolism. OBJECTIVE: This work aimed to study cortisol metabolism during DR-HC and TID-HC. DESIGN: A randomized, 12-week, crossover study was conducted. INTERVENTION AND PARTICIPANTS: DC-HC and same daily dose of TID-HC were administered to patients with primary adrenal insufficiency (n = 50) vs healthy individuals (n = 124) as controls. MAIN OUTCOME MEASURES: Urinary corticosteroid metabolites were measured by gas chromatography/mass spectrometry at 24-hour urinary collections. RESULTS: Total cortisol metabolites decreased during DR-HC compared to TID-HC (P < .001) and reached control values (P = .089). During DR-HC, 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1) activity measured by tetrahydrocortisol + 5α-tetrahydrocortisol/tetrahydrocortisone ratio was reduced compared to TID-HC (P < .05), but remained increased vs controls (P < .001). 11ß-HSD2 activity measured by urinary free cortisone/free cortisol ratio was decreased with TID-HC vs controls (P < .01) but normalized with DR-HC (P = .358). 5α- and 5ß-reduced metabolites were decreased with DR-HC compared to TID-HC. Tetrahydrocortisol/5α-tetrahydrocortisol ratio was increased during both treatments, suggesting increased 5ß-reductase activity. CONCLUSIONS: The urinary cortisol metabolome shows striking abnormalities in patients receiving conventional TID-HC replacement therapy, with increased 11ß-HSD1 activity that may account for the unfavorable metabolic phenotype in primary adrenal insufficiency. Its change toward normalization with DR-HC may mediate beneficial metabolic effects. The urinary cortisol metabolome may serve as a tool to assess optimal cortisol replacement therapy.

Detailed analysis of cortisol, cortisone and their tetrahydro- and allo-tetrahydrometabolites in human urine by LC-MS/MS.

Cortisol (F) and cortisone (E) are metabolized to A-ring reduced metabolites in the reactions catalyzed by 5α- and 5ß-reductase. 5α-tetrahydrocortisol (alloTHF) and 5ß-tetrahydrocortisol (THF) are produced from F. The metabolism of E takes place in analogy to form alloTHE and THE. Up to now, the analysis of endogenous glucocorticoids did not consider alloTHE, limiting the metabolism of E to THE only. Nevertheless, such simplification can generate inaccuracy in the assessment of the function of enzymes crucial for glucocorticoids metabolism: 11ß-hydroxysteroid dehydrogenase type 1 and type 2 (11ß-HSD1 and 11ß-HSD2), as well as 5α- and 5ß-reductase. The paper presents the new LC-MS/MS method for the simultaneous analysis of F and E with their tetrahydro- (THF and THE) and allo-tetrahydrometabolites (alloTHF and alloTHE) in urine. The method was fully validated and allows determining both the unconjugated and total concentrations of urinary glucocorticoids. The method meets the EMA's recommendations and was proved to be useful in the analysis of clinical samples. The LLOQ of 1ng/mL allows the determination of free urinary F, E, THF and THE, but not alloTHF and alloTHE, in samples obtained from pregnant women. The range of concentrations is wide enough for the analysis of total levels of F, E, THF, alloTHF, THE and alloTHE. The undisputed advantage of the method, distinguishing it among others, is the ability to determine F and E and their both 5α- and 5ß-metabolites. Taking alloTHE into consideration enables the thorough analysis of the glucocorticoid equilibrium in human.

Selection and early clinical evaluation of the brain-penetrant 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1) inhibitor UE2343 (Xanamem™).

BACKGROUND AND PURPOSE: Reducing glucocorticoid exposure in the brain via intracellular inhibition of the cortisol-regenerating enzyme 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1) has emerged as a therapeutic strategy to treat cognitive impairment in early Alzheimer's disease (AD). We sought to discover novel, brain-penetrant 11ß-HSD1 inhibitors as potential medicines for the treatment of AD. EXPERIMENTAL APPROACH: Medicinal chemistry optimization of a series of amido-thiophene analogues was performed to identify potent and selective 11ß-HSD1 inhibitors with optimized oral pharmacokinetics able to access the brain. Single and multiple ascending dose studies were conducted in healthy human subjects to determine the safety, pharmacokinetic and pharmacodynamic characteristics of the candidate compound. RESULTS: UE2343 was identified as a potent, orally bioavailable, brain-penetrant 11ß-HSD1 inhibitor and selected for clinical studies. No major safety issues occurred in human subjects. Plasma adrenocorticotropic hormone was elevated (a marker of systemic enzyme inhibition) at doses of 10 mg and above, but plasma cortisol levels were unchanged. Following multiple doses of UE2343, plasma levels were approximately dose proportional and the terminal t1/2 ranged from 10 to 14 h. The urinary tetrahydrocortisols/tetrahydrocortisone ratio was reduced at doses of 10 mg and above, indicating maximal 11ß-HSD1 inhibition in the liver. Concentrations of UE2343 in the CSF were 33% of free plasma levels, and the peak concentration in CSF was ninefold greater than the UE2343 IC50 . CONCLUSIONS AND IMPLICATIONS: UE2343 is safe, well tolerated and reaches the brain at concentrations predicted to inhibit 11ß-HSD1. UE2343 is therefore a suitable candidate to test the hypothesis that 11ß-HSD1 inhibition in brain improves memory in patients with AD.

HPLC-ESI-MS/MS assessment of the tetrahydro-metabolites of cortisol and cortisone in bovine urine: promising markers of dexamethasone and prednisolone treatment.

The effects of long-term administration of low doses of dexamethasone (DX) and prednisolone (PL) on the metabolism of endogenous corticosteroids were investigated in veal calves. In addition to cortisol (F) and cortisone (E), whose interconversion is regulated by 11ß-hydroxysteroid dehydrogenases (11ßHSDs), special attention was paid to tetrahydrocortisol (THF), allo-tetrahydrocortisol (aTHF), tetrahydrocortisone (THE) and allo-tetrahydrocortisone (aTHE), which are produced from F and E by catalytic activity of 5α and 5ß-reductases. A specifically developed HPLC-ESI-MS/MS method achieved the complete chromatographic separation of two pairs of diastereoisomers (THF/aTHF and THE/aTHE), which, with appropriate mass fragmentation patterns, provided an unambiguous conformation. The method was linear (r(2) > 0.9905; 0.5-25 ng ml(-1)), with LOQQ of 0.5 ng ml(-1). Recoveries were in range 75-114%, while matrix effects were minimal. The experimental study was carried out on three groups of male Friesian veal calves: group PL (n = 6, PL acetate 15 mg day(-1) p.o. for 31 days); group DX (n = 5, 5 mg of estradiol (E2) i.m., weekly, and 0.4 mg day(-1) of DX p.o. for 31 days) and a control group (n = 8). Urine was collected before, during (twice) and at the end of treatment. During PL administration, the tetrahydro-metabolite levels decreased gradually and remained low after the suspension of treatment. DX reduced urinary THF that persisted after the treatment, while THE levels decreased during the experiment, but rebounded substantially after the DX was withdrawn. Both DX and PL significantly interfered with the production of F and E, leading to their complete depletion. Taken together, the results demonstrate the influence of DX and PL administration on 11ßHSD activity and their impact on dysfunction of the 5-reductase pathway. In conclusion, profiling tetrahydro-metabolites of F and E might serve as an alternative, indirect but reliable, non-invasive procedure for assessing the impact of synthetic glucocorticosteroids administration.

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 11ß-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 11ß-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.

Glucocorticoid activity and metabolism with NaCl-induced low-grade metabolic acidosis and oral alkalization: results of two randomized controlled trials.

Low-grade metabolic acidosis (LGMA), as induced by high dietary acid load or sodium chloride (NaCl) intake, has been shown to increase bone and protein catabolism. Underlying mechanisms are not fully understood, but from clinical metabolic acidosis interactions of acid-base balance with glucocorticoid (GC) metabolism are known. We aimed to investigate GC activity/metabolism under alkaline supplementation and NaCl-induced LGMA. Eight young, healthy, normal-weight men participated in two crossover designed interventional studies. In Study A, two 10-day high NaCl diet (32 g/d) periods were conducted, one supplemented with 90 mmol KHCO3/day. In Study B, participants received a high and a low NaCl diet (31 vs. 3 g/day), each for 14 days. During low NaCl, the diet was moderately acidified by replacement of a bicarbonate-rich mineral water (consumed during high NaCl) with a non-alkalizing drinking water. In repeatedly collected 24-h urine samples, potentially bioactive-free GCs (urinary-free cortisol + free cortisone) were analyzed, as well as tetrahydrocortisol (THF), 5α-THF, and tetrahydrocortisone (THE). With supplementation of 90 mmol KHCO3, the marker of total adrenal GC secretion (THF + 5α-THF + THE) dropped (p = 0.047) and potentially bioactive-free GCs were reduced (p = 0.003). In Study B, however, GC secretion and potentially bioactive-free GCs did not exhibit the expected fall with NaCl-reduction as net acid excretion was raised by 30 mEq/d. Diet-induced acidification/alkalization affects GC activity and metabolism, which in case of long-term ingestion of habitually acidifying western diets may constitute an independent risk factor for bone degradation and cardiometabolic diseases.

Decreased maternal hypothalamic-pituitary-adrenal axis activity in very severely obese pregnancy: Associations with birthweight and gestation at delivery.

BACKGROUND: The maternal hypothalamic-pituitary-adrenal-axis (HPAA) undergoes dramatic activation during pregnancy. Increased cortisol and corticotrophin-releasing-hormone (CRH) associate with low birthweight and preterm labor. In non-pregnant obesity, the HPAA is activated but circulating cortisol levels are normal or lower than in lean women. We hypothesized that maternal cortisol levels would be lower in obese pregnancy, and would associate with increased fetal size and length of gestation. METHOD: Fasting serum cortisol was measured at 16, 28 and 36 weeks gestation and at 3-6 months postpartum in 276 severely obese and 135 lean women. In a subset of obese (n=20) and lean (n=20) we measured CRH, hormones that regulate bioavailable cortisol (corticosteroid-binding-globulin, estradiol, estriol, and progesterone). Urinary glucocorticoid metabolites were measured in pregnant (obese n=6, lean n=5) and non-pregnant (obese n=7, lean n=7) subjects. RESULTS: Maternal cortisol and HPAA hormones were lower in obese pregnancy. Total urinary glucocorticoid metabolites increased significantly in lean pregnancy, but not in obese. Lower maternal cortisol in obese tended to be associated with increased birthweight (r=-0.13, p=0.066). In obese, CRH at 28 weeks correlated inversely with gestational length (r=-0.49, p=0.04), and independently predicted gestational length after adjustment for confounding factors (mean decrease in CRH of -0.25 pmol/L (95% CI -0.45 to -0.043 pmol/L) per/day increase in gestation). CONCLUSION: In obese pregnancy, lower maternal cortisol without an increase in urinary glucocorticoid clearance may indicate a lesser activation of the HPAA than in lean pregnancy. This may offer a novel mechanism underlying increased birthweight and longer gestation in obese pregnancy.

A simple LC-MS/MS method for the determination of cortisol, cortisone and tetrahydro-metabolites in human urine: assay development, validation and application in depression patients.

Chronic stress as well as major depressive disorders is associated with cortisol metabolism. Two enzymes modulate cortisol (F) and cortisone (E) interconversion: 11ß-hydroxysteroid dehydrogenase type 1 and type 2 (11ß-HSD1 and 11ß-HSD2). Furthermore, F and E were inactivated by 5α and 5ß reductases to their tetrahydro-metabolites: tetrahydrocortisol (THF), allo-tetrahydrocortisol (5α-THF) and tetrahydrocortisone (THE). To better understand depression a LC-MS/MS method for simultaneous determination of F, E THF, 5α-THF and THE in human urine has been developed and validated. The quantification range was 0.1-160 ng mL(-1) for F and E, and 0.2-160 ng mL(-1) for the tetrahydro-metabolites, with >86.1% recovery for all analytes. The nocturnal urine concentrations of F, E and tetrahydro-metabolites in 12 apparently healthy male adult volunteers and 12 drug-free male patients (age range, 20-50 years) with a diagnosis of depression were analyzed. A series of significant changes in glucocorticoid metabolism can be detected: F/E ratios and (THF+5α-THF)/THE ratios as well as F and THF concentrations were significantly higher in depression patients than in healthy subjects (p<0.05); 5α-THF/F ratios, 5α-THF/THF ratios as well as 5α-THF concentrations were significantly lower in depression patients (p<0.05). The results pointed to the decreased 11ß-HSD2 activity and a dysfunction in the 5α-reductase pathway in depressed patients. This method allows the assessment of 11ß-HSD1/2 and 5α/ß-reductase activities in a single analytical run providing an innovative tool to explain the potential etiology of depression.

[11beta-hydroxysteroid dehydrogenase type 2 enzyme activity effect after exposures phthalate esters in maternal].

OBJECTIVE: To study the association between phthalate esters (PAEs) metabolites in maternal urine and 11beta-hydroxysteroid dehydrogenase type 2 (11ß-HSD2 ) enzyme activity, explore the possible mechanism of PAEs effect on fetal development. METHODS: All of 33 cases of intrauterine growth retardation (IUGR) newborn were selected by random sampling in 2012. And 33 cases of normal control newborn were enrolled, use high performance liquid chromatography-tandem mass spectrometry method was used to detect 4 kinds of phthalate esters (PAEs) metabolites in maternal urine: mono-n-butyl phthalate ester (MBP), mono (2-ethylhexyl) phthalate (MEHP), mono (2-ethyl-5-hydroxyhexyl) phthalate (MEHHP), mono (2-ethyl-5-oxohexyl) phthalate (MEOHP) and three kinds of cortisol corticosterone metabolites, tetrahydrocortisol (THF), allo-tetrahydrocortisol (allo-THF), tetrahydrocortisone (THE), and analyze the association between phthalate esters (PAEs) metabolites in maternal urine and 11ß-HSD2 enzyme activity. RESULTS: MBP, MEHP, MEHHP, MEOHP metabolites can be detected in 98% (65 cases) , 89% (59 cases), 91% (60 cases), 91% (60 cases) of all 66 maternal urine samples, respectively. The median concentrations of test material in case group were 31.20 ng/ml for MBP, 24.61 ng/ml for MEHHP, 11.72 ng/ml for MEOHP and 48.67 ng/ml for SumDEHP which were significantly higher than those of the control group (were 17.32, 12.03, 5.68 and 28.64 ng/ml); 11ß-HSD2 activity in case group ((THF+allo-THF)/THE = (0.79 ± 0.09) ng/ml) was significantly lower than that of the control group((THF+allo-THF)/THE = (0.58 ± 0.04) ng/ml); PAEs metabolites MBP (ß' = 1.12), MEHHP(ß' = 1.14), MEOHP(ß' = 1.10), SumDEHP(ß' = 1.08) in baby boy mother's urine was reversely correlated to 11ß-HSD2 activity. CONCLUSIONS: PAEs could affect fetal development by inhibit 11ß-HSD2 activity.

Study of urinary steroid hormone disorders: difference between hepatocellular carcinoma in early stage and cirrhosis.

Hepatocellular carcinoma (HCC) is the fifth most common cancer in the world. Discovery of novel biomarkers for early HCC from other liver diseases such as cirrhosis is of great clinical benefit. In this study, a novel steroid hormone metabolomic method based on liquid chromatography-mass spectrometry combined with logistic regression analysis was applied to study the steroid hormone disorders and to screen potential urinary steroid hormone biomarkers of early HCC. Thirty-six urinary steroid hormones were detected and quantified in healthy controls, cirrhotic patients, and early HCC patients. Heat map analysis and multivariate statistical analysis suggested severe disorders of steroid hormone network and holistically decreased urinary steroid hormone pattern in cirrhotic and early HCC patients. Logistic regression analysis reveals that a panel of two urinary steroid hormones (epitestosterone and allotetrahydrocortisol) displayed excellent diagnostic capability for distinguishing early HCC from cirrhosis with area under the curve (AUC) = 0.938 of receiver operating characteristic (ROC) analysis. These results help to overcome the disadvantage of lower sensitivity and specificity of alpha-fetoprotein for distinguishing early HCC from cirrhosis. Our work shows that steroid hormone metabolomics is a promising biomarker tool for biomarker study of early HCC.

Acute effects of physical exercise and phosphodiesterase's type 5 inhibition on serum 11ß-hydroxysteroid dehydrogenases related glucocorticoids metabolites: a pilot study.

Endogenous glucocorticoids (GC) rapidly increase after acute exercise, and the phosphodiesterase's type 5 inhibitor (PDE5i) tadalafil influences this physiological adaptation. No data exist on acute effects of both acute exercise and PDE5i administration on 11ß-hydroxysteroid dehydrogenases (11ß-HSDs)-related GC metabolites. We aimed to investigate the rapid effects of exercise on serum GC metabolites, with and without tadalafil administration. A double blind crossover study was performed in eleven healthy male volunteers. After the volunteers randomly received a short-term administration of placebo or tadalafil (20 mg/die for 2 days), a maximal exercise test to exhaustion on cycle ergometer was performed. Then, after a 2-week washout period, the volunteers were crossed over. Blood samples were collected before starting exercise and at 5 and 30 min of recovery (+5-Rec, +30-Rec). Serum ACTH, corticosterone (Cn), cortisol (F), cortisone (E), tetrahydrocortisol (THF), tetrahydrocortisone (THE), cortols, cortolones and respective ratios were evaluated. Pre-Ex THF was higher after tadalafil. Exercise increased ACTH, Cn, F, E, THE, cortols and cortolones after both placebo and tadalafil, and THF after placebo. The F/E ratio increased at +5-Rec and decreased at +30-Rec after placebo. Compared to placebo, after tadalafil lower ACTH, F and Cn, higher THF/F and THE/E, and not E (at +5-Rec) and F/E modifications were observed. Acute exercise rapidly influences serum GC metabolites concentrations. Tadalafil influences both GC adaptation and 11ß-HSDs activity during acute exercise. Additional researches on the effects of both exercise and PDE5i on tissue-specific 11ß-HSDs activity at rest and during physiological adaptation are warranted.

Altered systemic cortisol metabolism in bipolar disorder and schizophrenia spectrum disorders.

Dysfunction of the hypothalamic-pituitary-adrenal (HPA) axis is suggested as a pathophysiological factor in bipolar disorder and schizophrenia. Increased clearance of cortisol was recently indicated as a component in the HPA axis hyperdrive. The aim of the present study was to test the model of increased cortisol metabolism in a new replication sample separately and combined with a previously published sample of bipolar disorder and schizophrenia. Spot urine was sampled from 212 healthy controls (HC) and 221 patients with a schizophrenia spectrum disorder (SCZ, n = 115) and bipolar disorder (BD, n = 106). Of these, a subsample of 169 HC and 155 patients was included in a previous report. Urinary free cortisol, cortisone and their metabolites were measured, and the activities of 5α-reductase, 5ß-reductase and 11ß-HSD were estimated and analyzed for differences between groups. In the new sample, there was increased enzyme activity in SCZ for 5ß-reductase (p = 0.024 vs HC; p = 0.027 vs BD) and 11ß-HSD2 (p = 0.014 vs HC; p = 0.004 vs BD). In the combined sample, there was increased activity in SCZ for 5α-reductase (p < 0.001 vs HC; p = 0.020 vs BD), 5ß-reductase (p < 0.001 vs HC; p = 0.045 vs BD) and 11ß-HSD2 (p < 0.001 vs HC; p = 0.043 vs BD), and in BD for 5ß-reductase (p = 0.002), 11ß-HSD2 (p = 0.039) and 5α-reductase (trend, p = 0.084) (all vs HC). The findings confirm increased systemic cortisol metabolism in BD and SCZ. This is most consistent in SCZ, with BD taking an intermediate position. The design makes it impossible to determine the direction of the effect. However, the findings merit further study of cortisol metabolism as a possible component in the HPA axis dysfunction and pathophysiology of BD and SCZ.

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

OBJECTIVE: Acute 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 METHODS: Steroid 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. RESULTS: We 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 6ß-hydroxycortisol/cortisol in the free steroid fraction did not show differences between patients and controls. All ratios were normal among the family carriers. CONCLUSION: A 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.