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.

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.

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.

Bile acids modulate glucocorticoid metabolism and the hypothalamic-pituitary-adrenal axis in obstructive jaundice.

BACKGROUND & AIMS: Suppression of the hypothalamic-pituitary-adrenal axis occurs in cirrhosis and cholestasis and is associated with increased concentrations of bile acids. We investigated whether this was mediated through bile acids acting to impair steroid clearance by inhibiting glucocorticoid metabolism by 5beta-reductase. METHODS: The effect of bile acids on glucocorticoid metabolism was studied in vitro in hepatic subcellular fractions and hepatoma cells, allowing quantitation of the kinetics and transcript abundance of 5beta-reductase. Metabolism was subsequently examined in vivo in rats following dietary manipulation or bile duct ligation. Finally, glucocorticoid metabolism was assessed in humans with obstructive jaundice. RESULTS: In rat hepatic cytosol, chenodeoxycholic acid competitively inhibited 5beta-reductase (K(i) 9.19+/-0.40 microM) and reduced its transcript abundance (in H4iiE cells) and promoter activity (reporter system, HepG2 cells). In Wistar rats, dietary chenodeoxycholic acid (1% w/w chow) inhibited hepatic 5beta-reductase activity, reduced urinary excretion of 3alpha,5beta-tetrahydrocorticosterone and reduced adrenal weight. Conversely, a fat-free diet suppressed bile acid levels and increased hepatic 5beta-reductase activity, supplementation of the fat-free diet with CDCA reduced 5beta-reductase activity, and urinary 3alpha,5beta-reduced corticosterone. Cholestasis in rats suppressed hepatic 5beta-reductase activity and transcript abundance. In eight women with obstructive jaundice, relative urinary excretion of 3alpha,5beta-tetrahydrocortisol was significantly lower than in healthy controls. CONCLUSION: These data suggest a novel role for bile acids in inhibiting hepatic glucocorticoid clearance, of sufficient magnitude to suppress hypothalamic-pituitary-adrenal axis activity. Elevated hepatic bile acids may account for adrenal insufficiency in liver disease.

The effect of spironolactone upon corticosteroid hormone metabolism in patients with early stage chronic kidney disease.

CONTEXT: Aldosterone has emerged as an important mediator of disease progression and mortality in patients with chronic heart and kidney disease (CKD). Despite the increasing use of mineralocorticoid receptor antagonists in these patients, little is known about the effects on corticosteroid hormone secretion and metabolism. OBJECTIVE: To assess corticosteroid hormone secretion and metabolism in patients with early stage CKD before and after spironolactone (Spiro). DESIGN: Randomized, double-blind, placebo-controlled interventional study. SETTING: Single tertiary referral centre. PATIENTS: A total of 112 patients with stable stage 2/3 CKD. INTERVENTIONS: Patients were randomized to receive either Spiro 25 mg once daily or placebo for 36 weeks. MAIN OUTCOME MEASURES: Plasma renin activity (PRA), angiotensin II (AngII) and steroid hormones were analysed by standard assays; urinary corticosteroid hormone metabolites (5α+5ß-tetrahydro-cortisol (5α+5ß-THF), TH-cortisone (THE), 3α5ß-TH-aldosterone (TH-Aldo), 5α+5ß-TH-deoxycorticosterone (5α+5ß-TH-DOC), TH-11-desoxycortisol (THS)) were analysed by gas chromatography/mass spectrometry. RESULTS: Plasma aldosterone concentration (PAC) was inversely correlated with eGFR (r = -0·331, P < 0·001). Urinary 24-h excretion of TH-Aldo was correlated with PAC (r = 0·214, P < 0·05) and diastolic blood pressure (BP) (r = 0·212, P = <0·05), whereas total 24-h urinary cortisol metabolite excretion was correlated with systolic BP (r = 0·316, P < 0·01). In addition, 11ß-hydroxysteroid dehydrogenase (11ß-HSD) type 1 activity (urinary 5α+5ß-THF)/THE) ratio) was correlated with PRA (r = 0·277, P < 0·01). Spiro treatment significantly reduced BP (123 ± 11/76 ± 7 vs 119 ± 11/73 ± 8 mmHg, P < 0·01) despite renin-angiotensin-aldosterone system induction, reflected by increased urinary 24-h TH-Aldo excretion (17·6 (12, 86) vs 26 (18, 80) µg/24 h, P < 0·05). By contrast, Spiro had no effect on total urinary cortisol metabolite excretion, 11ß-hydroxylase, 11ß-HSD type 1 and 2 activity. CONCLUSIONS: Aldo and cortisol are positively associated with BP suggesting that adrenal hyperactivity may in part explain the increased cardiovascular risk in patients with early end-stage CKD. Addition of Spiro had no effect on glucocorticoid metabolism or total 24-h corticosteroid production.

Intense physical exercise increases systemic 11beta-hydroxysteroid dehydrogenase type 1 activity in healthy adult subjects.

Intense physical exercise activates the hypothalamic-pituitary-adrenocortical axis but little is known about changes in glucocorticoid sensitivity at the target cell level. No data are available on the acute effects of exercise on 11beta-hydroxysteroid dehydrogenase (11beta-HSD) type 1 activity, which generates biologically active cortisol from inactive cortisone and is expressed also in skeletal muscle. Fifteen healthy, trained males (age mean +/- SE 28 +/- 1) were assessed on three non-consecutive days: at rest, during an endurance and strength sessions. During each session, between 1000 and 1600 hours, 6-h urine and four salivary samples were collected. Urinary total tetrahydrocortisol (THF) + alloTHF, tetrahydrocortisone (THE), cortisol (F) and cortisone (E) were measured with HPLC-tandem mass spectrometry; urinary-unconjugated F and E were measured by HPLC-UV. Salivary cortisol and interleukin (IL)-6 were measured by RIA and ELISA, respectively. Both endurance and strength exercises caused an increase in (THF + alloTHF)/THE ratio (mean +/- SE 1.90 +/- 0.07 and 1.82 +/- 0.05 vs. 1.63 +/- 0.06, P < 0.01 and P = 0.03, respectively), consistent with increased systemic 11beta-HSD type 1 activity. No relationship was found with age, BMI, VO(2max) maximal power load or perceived exertion. No significant change was apparent in F/E ratio, an index of 11beta-HSD type 2 activity. No effect of exercise on salivary cortisol and IL-6 was observed, whereas a significant effect of sampling time was found. Intense physical exercise acutely increases systemic 11beta-HSD type 1 activity in humans. Such an increase may lead to higher cortisol concentration in target tissues, notably in skeletal muscle where it could contribute to limit exercise-induced muscle inflammatory response.

Simultaneous determination of twelve tetrahydrocorticosteroid glucuronides in human urine by liquid chromatography/electrospray ionization-linear ion trap mass spectrometry.

A liquid chromatography/electrospray ionization (ESI)-mass spectrometry (MS) method for the direct determination of 12 tetrahydrocorticosteroid glucuronides in human urine has been developed. The analytes were 3- and 21-monoglucuronides of tetrahydrocortisol, tetrahydrocortisone, tetrahydro-11-deoxycortisol, and their 5alpha-stereoisomers. The mass spectrometric behaviors of these glucuronides in negative-ion ESI-MS/MS revealed the production of intense, structure-specific product ions within the same group of glucuronides. Regioisomeric glucuronides could be distinguished by collision-induced dissociation and tandem mass spectrometry. Using a linear ion trap instrument operating in the negative-ion mode and by monitoring the transition ions of [M - H](-) --> [M - H - CH(2)O](-) for 3-monoglucuronides and [M - H](-) --> [M - H - CH(2)OG](-) for 21-monoglucuronides, a sensitive and specific assay was developed. Initial steps in the assay were a simple solid-phase extraction and the addition of [9,12,12,21,21-d(5)]-tetrahydrocortisone-3-glucuronide (prepared by enzyme-assisted synthesis) as an internal standard. The method was applied to determine the 12 tetrahydrocoticosteroid glucuronides in urine from healthy subjects and from patients with excessive cortisol production. The method described here appears to be useful for clinical and biochemical studies.

Cortisol metabolism in depressed patients and healthy controls.

BACKGROUND: Chronic stress as well as major depressive disorders are associated with hypercortisolemia and impaired hypothalamic-pituitary-adrenocortical axis functioning. The aim of this study was to determine whether in major depression changes in the activity patterns of local modulators of glucocorticoid action might contribute to an increase in cortisol bioavailability and if they change during antidepressant treatment and clinical response. METHODS: Concentrations of urinary total cortisol (UFF), urinary total cortisone (UFE), tetrahydrocortisone (THE), tetrahydrocortisol (THF) and allo-THF (5alpha-THF) were measured in 10-hour nocturnal urine samples of 19 depressed patients and 15 healthy controls. The activity of 11beta-hydroxysteroid dehydrogenases (11beta-HSD) as well as 5alpha- and 5beta-reductases was assessed by calculating the ratios of glucocorticoid metabolites. Patients were treated for 28 days with either mirtazapine or venlafaxine. Enzyme activity was observed during the course of treatment and compared to healthy controls. Responders to treatment were selected for this analysis. RESULTS: Depressed patients showed reduced 5alpha-reductase activity manifested as a significantly lower amount of 5alpha-THF (102.8 +/- 167.2 vs. 194.6 +/- 165.8 microg, p = 0.019). The increase in the UFF/UFE ratio (0.73 +/- 0.32 vs. 0.29 +/- 0.13, p < 0.0001) indicates reduced activity of renal 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2). During pharmacological treatment, 5alpha-reductase activity in patients returned to the level of the control group, while the decrease in 11beta-HSD2 activity persisted until day 28. CONCLUSIONS: Our results show changes in activity of intracellular modulators of steroid action in major depressive disorders, particularly a reduced activity of the intracellular cortisol-deactivating enzymes 5alpha-reductase and 11beta-HSD2. These changes suggest an increase in cortisol bioavailability within tissues.

Application of hyphenated mass spectrometry techniques for the analysis of urinary free glucocorticoids.

Alteration of levels of glucocorticoids in plasma and urine can be related to several diseases. In particular, the determination of endogenous glucocorticoids in urine has been reported to provide information on cortisol and cortisone status, on the activities of steroid hormone enzymes and on glucocorticoid metabolism. In this study, the application of hyphenated mass spectrometry techniques (GC/MS without derivatization and LC/MS) for the simultaneous analysis of free urinary cortisol (F), cortisone (E), tetrahydrocortisol (THF), allo-tetrahydrocortisol (A-THF) and tetrahydrocortisone (THE) was evaluated. A sample preparation protocol by solid-phase extraction, mass spectrometry parameters and chromatographic conditions for both techniques were carefully optimized in terms of extracting phase and solvents, matrix effects, recovery, sensitivity and compound resolution. Baseline separation was achieved for the five underivatized analytes both in GC and LC. The LC/MS/MS technique was more suitable for the analysis of urine samples, being less influenced by matrix effects and showing excellent sensitivity and selectivity. A preliminary application of the reported method for the diagnosis of metabolic diseases was also described. The determination of each analyte in its free form, described for the first time in the paper, offers new perspectives in the application of glucocorticoid analysis for diagnostic purposes.

Simultaneous determination of tetrahydrocortisol, allotetrahydrocortisol and tetrahydrocortisone in human urine by liquid chromatography-electrospray ionization tandem mass spectrometry.

Simultaneous quantification method of three major metabolites of cortisone and cortisol, tetrahydrocortisol, allotetrahydrocortisol and tetrahydrocortisone by liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) was investigated in a positive mode using a recently developed picolinyl derivatization. Conversion of each steroid into the corresponding picolinyl derivatives (1b, 2b or 3b) was performed by mixed anhydride method using picolinic acids and 2-methyl-6-nitrobenzoic anhydride. Derivatization proceeded smoothly to afford the corresponding 3, 21-dipicolinyl derivatives. Positive ion-ESI mass spectra of the picolinyl derivatives were dominated by an appearance of [M+H](+) as base peaks in all cases. The picolinyl derivatives provided 15 to 80-fold higher ESI response in the LC-ESI-MS/MS (selected reaction monitoring: SRM) when compared to those of underivatized molecules in a positive LC-ESI mode. The use of the picolinyl ester, solid-phase extraction, and deuterium labeled internal standards enabled the concentrations of these metabolites in human urine to be determined simultaneously by LC-ESI-MS/MS (SRM) with a small sample volume of less than 1microl urine.

Reduced activity of 11 beta-hydroxysteroid dehydrogenase in patients with cholestasis.

Enhanced renal sodium retention and potassium loss in patients with cirrhosis is due to activation of mineralocorticoid receptors (MRs). Increased aldosterone concentrations, however, do not entirely explain the activation of MR in cirrhosis. Here, we hypothesize that cortisol activates MRs in patients with cholestasis. We present evidence that access of cortisol to MRs is a result of bile acid-mediated inhibition of 11 beta-hydroxysteroid dehydrogenase type 2 (11 beta-HSD2), an MR-protecting enzyme that converts cortisol to cortisone. Twelve patients with biliary obstruction and high plasma bile acid levels were studied before and after removal of the obstruction. The urinary ratio of (tetrahydrocortisol + 5 alpha-tetrahydrocortisol)/tetrahydrocortisone, a measure of 11 beta-HSD2 activity, decreased from a median of 1.91 during biliary obstruction to 0.78 at 4 and 8 weeks after removal of the obstruction and normalization of plasma bile acid concentrations. In order to demonstrate that bile acids facilitate access of cortisol to the MR by inhibiting 11 beta-HSD2, an MR translocation assay was performed in HEK-293 cells transfected with human 11 beta-HSD2 and tagged MR. Increasing concentrations of chenodeoxycholic acid led to cortisol-induced nuclear translocation of MR. In conclusion, 11 beta-HSD2 activity is reduced in cholestasis, which results in MR activation by cortisol.

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.

Urinary cortisol and cortisol metabolite excretion in chronic fatigue syndrome.

OBJECTIVES: Reduced basal hypothalamic-pituitary-adrenal (HPA) axis output in chronic fatigue syndrome (CFS) has been inferred from low cortisol levels in blood, saliva, and urine in some studies. Because > 95% of cortisol is metabolized before excretion, we assessed cortisol output by assay of both cortisol metabolites and free cortisol in 24-hour urine collections and also investigated sex differences in these between CFS and control groups. METHOD: We calculated total urinary cortisol metabolites (TCM) and cortisol metabolite ratios from individual steroid data in 40 patients (20 males and 20 females) with CFS who were free of medication or comorbid psychiatric disorder likely to influence the HPA axis. Results were compared with those of 40 healthy volunteers (20 males and 20 females) well matched for age and body mass index. Data for free cortisol was obtained on 28 of the patients and 27 of the controls. RESULTS: The mean of TCM and cortisol metabolite ratios was not significantly different between patients and controls for either sex (p > .05 for all parameters). Previously established sex differences were confirmed in our controls and were found to be similar in CFS for TCM and the ratios 11OH/11OXO, 5alpha/5beta THF, and 20OH/20OXO (see text) (p < .005, p < .05, p < .05, and p < .005, respectively). Urinary free cortisol values were numerically (but not statistically) lower in patients with CFS than controls, and correlated inversely with fatigue levels in patients. CONCLUSION: The finding of normal urinary cortisol metabolite excretion in patients with CFS is at variance with earlier reports that CFS is a hypocortisolemic state. If serum and saliva cortisol levels are lower in CFS, this would suggest that metabolic clearance of cortisol is faster in patients with CFS than controls. This study also demonstrates that sex differences must be taken into account when interpreting results in patients with CFS.

Pediatric renal allograft transplantation does not normalize the increased cortisol/cortisone ratios of chronic renal failure.

OBJECTIVE: The conversion of cortisol (F) to cortisone (E) is catalyzed by 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2). Children suffering from chronic renal failure (CRF) have a decreased activity of 11beta-HSD2 contributing to increased arterial blood pressure. The objective was to investigate whether a normal conversion of F to E is achieved after renal transplantation (TX) in children. METHODS: Fifteen children with CRF, 17 children with steroid-free immunosuppression after TX, and 18 healthy controls (CO) were enrolled. The activity of 11beta-HSD2 in plasma was calculated using the ratio of F/E determined by tandem mass spectrometry, the ratio of tetrahydrocortisol (THF) +5alpha-tetrahydrocortisol (5alphaTHF) in urine determined by gas chromatography/mass spectrometry, and the ratio of (THF +5alphaTHF)/tetrahydrocortisone (THE) in urine determined by tandem mass spectrometry. RESULTS: The F/E ratio (mean +/- S.D./S.E.M.) was significantly higher in CRF and TX (5.6 +/- 1.9/0.6, 7.12 +/- 3.1/0.9) than in CO (1.18 +/- 0.2/0.03, P < 0.0001) groups. The (THF + 5alphaTHF)/THE ratio in CRF (1.19 +/- 1.1/0.5) and TX (1.19 +/- 0.1/0.5) groups was significantly higher than in controls (0.21 +/- 0.05/0.18, P < 0.0001). Positive correlations between plasma and urinary ratios (P = 0.0004. R(2) = 0.73 in CRF, P = 0.0013, R(2) = 0.56 in TX, P < 0.0001, R(2) = 0.66 in CO) were found, whereas significant correlations between F/E or (THF + 5alphaTHF)/THE ratios and blood pressure, the number of antihypertensive drugs taken or creatinine clearance could not be found. CONCLUSIONS: In all children with chronic renal failure plasma and urinary cortisol/cortisone ratios are elevated and do not return to normal levels after renal allograft transplantation. This suggests that renal transplantation does not normalize 11beta-HSD2 activity.

Direct determination of the ratio of tetrahydrocortisol+allo-tetrahydrocortisol to tetrahydrocortisone in urine by LC-MS-MS.

The 11beta-hydroxysteroid dehydrogenase (11beta-HSD) is responsible for the interconversion of both the hormonally inactive cortisone and the active cortisol. This enzyme activity, which has implications in the pathogenesis of numerous diseases, is reflected in the ratio of tetrahydrometabolites of cortisol (allo-tetrahydrocortisol and tetrahydrocortisol) to those of cortisone (tetrahydrocortisone). Several methods have been proposed in the literature to determine such a ratio in urine. Most of them require tedious and extensive extraction and derivatization steps and make use of gas-chromatographic techniques, including gas chromatography coupled to mass spectrometry (GC-MS). We present here an alternative approach for the direct determination of such a ratio in urine by using liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS-MS), based on a minimal sample treatment. Actually, the limit of detections (LODs) for pure standards in water permitted a simple dilution of the urine samples prior to the analysis, hence, an accurate optimization of the high performance liquid chromatography (HPLC) separation was needed in order to get rid of the severe influence of the urine matrix on the ionization efficiency. Besides, the nature of some interfering species was deeply investigated, as well as the suitability of some commercial deuterated steroids as internal standards. All these led to the final method, which was based on a HPLC separation on a C8 column and a ternary gradient water/methanol/acetonitrile. In parallel, an appropriate sample preparation was set up, which consisted of an enzymatic hydrolysis of the conjugated species and a followed 1:20 dilution. Preliminary measurements on real urine samples were performed as well.

Diurnal excretion of urinary cortisol, cortisone, and cortisol metabolites in chronic fatigue syndrome.

OBJECTIVE: The aim of this study was to obtain comprehensive information on basal hypothalamic-pituitary-adrenal (HPA) axis activity in chronic fatigue syndrome (CFS) patients who were not affected by medication or comorbid psychiatric disorder likely to influence the HPA axis. METHOD: Steroid analysis of urine collections from 0600 to 2100 h at 3-h intervals in CFS patients and in controls. RESULTS: Urinary free cortisol and cortisone concentrations showed a significant normal diurnal rhythm, but levels were lower across the cycle in CFS. In contrast, while urinary cortisol metabolites also showed a normal diurnal rhythm, levels were not significantly different between the CFS and controls at any time. Derived metabolite ratios were similar in both groups. CONCLUSION: This study provides further evidence for reduced basal HPA axis function in patients with CFS, based on lower free cortisol and cortisone levels, but this is not corroborated by cortisol metabolite data. The difference between these measures cannot be explained by an altered timing of the diurnal rhythm.

Hormonal status of breast cancer. III. Further analysis of ovarian-adrenal dysfunction.

Detailed studies of urinary steroids in patients with breast cancer and normal controls have shown that abnormal aging processes and depressed corpus luteum function were associated with the presence of this form of cancer. The ratio of androsterone to tetrahydrocortisol, an index of the aging process, was significantly lower in a breast cancer group than in a normal group at both premenopausal and postmenopausal stages. In premenopausal women, the ratio decreased in the order of rural controls, urban controls, and patients with breast cancer. It was indicated that the ratio may serve as a measure of risk for breast cancer. Aside from the general depression of menstruation-dependent steroids, breast cancer was associated with disproportionately low excretions of prenanediol and pregnanetriol within the menstruation-dependent steroid family. The notion that this finding reflects the delay or complete failure of ovulation was supported by a reduced parturition rate of our patients with breast cancer as compared with that of our normal controls. The physiologic significance of steroidal disturbances was considered in relation to the genesis of breast cancer.

Hormonal status of breast cancer. II. Abnormal urinary steroid excretion.

The urinary excretion of 14 neutral steroids was measured by gas-liquid chromatography in women with early and advanced breast cancer, in women with early uterine cancer, and in healthy women from urban and rural districts. The premenopausal patients with early breast cancer excreted subnormal amounts of five steroids (11-hydroxyandrosterone, 11-hydroxyetiocholanolone, pregnanediol, pregnanetriol, and tetrahydrocorticosterone) and increased amounts of tetrahydrocortisol as compared with the normal subjects of corresponding ages. From our findings, a new parameter was proposed by which a premenopausal breast-cancer patient was separated from the control. Postmenopausal breast-cancer patients excreted greater amounts of five steroids (one steroid from 17-ketosteroids and four from 17-hydroxycorticoids) than the corresponding controls. The discrepancy between premenopausal and postmenopausal breast cancer was tentatively related to ovarian-adrenal dysfunction in the course of aging. Oophorectomy induced a long-lasting tumor regression only in patients with a high value for the ratio of 11-deoxy-17-ketosteroid to 17-hydroxycorticosteroid in urine taken before surgery; the ratio in the responsive patients decreased remarkably after surgery. A constitutional change in 17-ketosteroids, as observed in a postmenopausal breast-cancer patient and a premenopausal healthy woman of urban origin, favored the geographic importance in the genesis of breast malignancy. The steroid abnormalities in uterine cancer were distinguishable from those of breast cancer in both premenopausal and postmenopausal women.

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.

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.