Quantification of cortisol and its metabolites in human urine by LC-MSn: applications in clinical diagnosis and anti-doping control.

The objective of the current research was to develop a liquid chromatography-MSn (LC-MSn) methodology for the determination of free cortisol and its 15 endogenous metabolites (6ß-hydroxycortisol, 20α-dihydrocortisol, 20α-dihydrocortisone, 20-ß-dihydrocortisol, 20ß-dihydrocortisone, prednisolone, cortisone, α-cortolone, ß-cortolone, allotetrahydrocortisol, 5α-dihydrocortisol, tetrahydrocortisol, allotetrahydrocortisone, 5ß-dihydrocortisol, tetrahydrocortisone) in human urine. Due to its optimal performance, a linear ion trap operating in ESI negative ion mode was chosen for the spectrometric analysis, performing MS3 and MS4 experiments. The method was validated for limit of detection (LOD) and limit of quantification (LOQ) (0.01 ng mL-1 and 0.05 ng mL-1, for all compounds, respectively), intra- and inter-day precision (CV = 1.4-9.2% and CV = 3.6-10.4%, respectively), intra- and inter-day accuracy (95-110%), extraction recovery (65-95%), linearity (R2 > 0.995), and matrix effect that was absent for all molecules. Additionally, for each compound, the percentage of glucuronated conjugates was estimated. The method was successfully applied to the urine (2 mL) of 50 healthy subjects (25 males, 25 females). It was also successfully employed on urine samples of two patients with Cushing syndrome and one with Addison's disease. This analytical approach could be more appropriate than commonly used determination of urinary free cortisol collected in 24-h urine. The possibility of considering the differences and relationship between cortisol and its metabolites allows analytical problems related to quantitative analysis of cortisol alone to be overcome. Furthermore, the developed method has been demonstrated as efficient for antidoping control regarding the potential abuse of corticosteroids, which could interfere with the cortisol metabolism, due to negative feedback on the hypothalamus-hypophysis-adrenal axis. Lastly, this method was found to be suitable for the follow-up of prednisolone that was particularly important considering its pseudo-endogenous origin and correlation with cortisol metabolism.

Two-step biochemical differential diagnosis of classic 21-hydroxylase deficiency and cytochrome P450 oxidoreductase deficiency in Japanese infants by GC-MS measurement of urinary pregnanetriolone/ tetrahydroxycortisone ratio and 11ß-hydroxyandrosterone.

BACKGROUND: The clinical differential diagnosis of classic 21-hydroxylase deficiency (C21OHD) and cytochrome P450 oxidoreductase deficiency (PORD) is sometimes difficult, because both deficiencies can have similar phenotypes and high blood concentrations of 17α-hydroxyprogesterone (17OHP). The objective of this study was to identify biochemical markers for the differential diagnosis of C21OHD, PORD, and transient hyper 17α-hydroxyprogesteronemia (TH17OHP) in Japanese newborns. We established a 2-step biochemical differential diagnosis of C21OHD and PORD. METHODS: We recruited 29 infants with C21OHD, 9 with PORD, and 67 with TH17OHP, and 1341 control infants. All were Japanese and between 0 and 180 days old; none received glucocorticoid treatment before urine sampling. We measured urinary pregnanetriolone (Ptl), the cortisol metabolites 5α- and 5ß-tetrahydrocortisone (sum of these metabolites termed THEs), and metabolites of 3 steroids, namely dehydroepiandrosterone, androstenedione (AD4), and 11ß-hydroxyandrostenedione (11OHAD4) by GC-MS. RESULTS: At a cutoff of 0.020, the ratio of Ptl to THEs differentiated C21OHD and PORD from TH17OHP and controls with no overlap. Among metabolites of DHEA, AD4, and 11OHAD4, only 11ß-hydroxyandrosterone (11HA), a metabolite of 11OHAD4, showed no overlap between C21OHD and PORD at a cutoff of 0.35 mg/g creatinine. CONCLUSIONS: A specific cutoff for the ratio of Ptl to THEs can differentiate C21OHD and PORD from TH17OHP and controls. Additionally, the use of a specific cutoff of 11HA can distinguish between C21OHD and PORD.

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.

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.

Multiplex Surface-Enhanced Raman Scattering Identification and Quantification of Urine Metabolites in Patient Samples within 30 min.

Successful translation of laboratory-based surface-enhanced Raman scattering (SERS) platforms to clinical applications requires multiplex and ultratrace detection of small biomarker molecules from a complex biofluid. However, these biomarker molecules generally exhibit low Raman scattering cross sections and do not possess specific affinity to plasmonic nanoparticle surfaces, significantly increasing the challenge of detecting them at low concentrations. Herein, we demonstrate a "confine-and-capture" approach for multiplex detection of two families of urine metabolites correlated with miscarriage risks, 5ß-pregnane-3α,20α-diol-3α-glucuronide and tetrahydrocortisone. To enhance SERS signals by 1012-fold, we use specific nanoscale surface chemistry for targeted metabolite capture from a complex urine matrix prior to confining them on a superhydrophobic SERS platform. We then apply chemometrics, including principal component analysis and partial least-squares regression, to convert molecular fingerprint information into quantifiable readouts. The whole screening procedure requires only 30 min, including urine pretreatment, sample drying on the SERS platform, SERS measurements, and chemometric analyses. These readouts correlate well with the pregnancy outcomes in a case-control study of 40 patients presenting threatened miscarriage symptoms.

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.

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.

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.

Diagnosis of 21-hydroxylase deficiency by urinary metabolite ratios using gas chromatography-mass spectrometry analysis: Reference values for neonates and infants.

One major issue of newborn screening programs for 21-hydroxylase deficiency (21OHD) is the high rate of false-positive results, especially in preterm neonates. Urinary steroid metabolite analysis using gas chromatography-mass spectrometry (GC-MS) is suitable as a confirmatory diagnostic tool. The objective of this study was to analyze retrospectively diagnostic metabolite ratios in neonates and infants with and without 21OHD using GC-MS with emphasis on glucocorticoid metabolism, and to develop reference values for the steroid metabolite ratios for the diagnosis of 21OHD. We retrospectively analyzed urinary steroid hormone metabolites determined by GC-MS of 95 untreated neonates and infants with 21OHD (1-148 days), and 261 neonates and infants (100 preterms) without 21OHD (0-217 days). Metabolites of 17α-hydroxyprogesterone showed specificities below 98%, whereas the 21-deoxycortisol metabolite pregnanetriolone clearly separated 21OHD from non-21OHD subjects. The best diagnostic ratio for 21OHD was pregnanetriolone to 6α-hydroxy-tetrahydrocortisone. The lowest value of this ratio in the 21OHD group (0.47) was at least eight times higher than the highest values in the non-21OHD group (0.055). We have given appropriate reference values for steroid metabolite ratios in the largest 21OHD cohort so far described. Consideration of glucocorticoid metabolism, especially the use of typical neonatal 6α-hydroxylates metabolites, leads to improvement of diagnostic metabolite ratios.

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.

Hypoxia causes down-regulation of 11 beta-hydroxysteroid dehydrogenase type 2 by induction of Egr-1.

Hypoxia causes several renal tubular dysfunctions, including abnormal handling of potassium and sodium and increased blood pressure. Therefore, we investigated the impact of hypoxia on 11beta-hydroxysteroid dehydrogenase (11beta-HSD2) enzyme, a crucial prereceptor gatekeeper for renal glucocorticosteroid-mediated mineralocorticoid action. The effect of hypoxia was assessed in vitro by incubating LLC-PK1 cells with antimycin A, an inhibitor of mitochondrial oxidative phosphorylation. Antimycin A induced a dose- and time-dependent reduction of 11beta-HSD2 activity. The early growth response gene, Egr-1, a gene known to be stimulated by hypoxia was investigated because of a potential Egr-1 binding site in the promoter region of 11beta-HSD2. Antimycin A induced Egr-1 protein and Egr-1-regulated luciferase gene expression. This induction was prevented with the MAPKK inhibitor PD 98059. Overexpression of Egr-1 reduced endogenous 11beta-HSD2 activity in LLC-PK1 cells, indicating that MAPK ERK is involved in the regulation of 11beta-HSD2 in vitro. In vivo experiments in rats revealed that Egr-1 protein increases, whereas 11beta-HSD2 mRNA decreases, in kidney tissue after unilateral renal ischemia and in humans the renal activity of 11beta-HSD2 as assessed by the urinary ratio of (tetrahydrocortisol+5alpha-tetrahydrocortisol)/tetrahydrocortisone declined when volunteers were exposed to hypoxemia at high altitude up to 7000 m. Thus, hypoxia decreases 11beta-HSD2 transcription and activity by inducing Egr-1 in vivo and in vitro. This mechanism might account for enhanced renal sodium retention and hypertension associated with hypoxic conditions.

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.

Cortisol metabolism in the baboon during pregnancy and the postpartum period.

The metabolism of iv-administered 14C-cortisol (F) by pregnant baboons (107, 124 and 150 days gestation) was compared with that previously reported for nonpregnant animals and with that of animals examined 6-18 h after spontaneous vaginal delivery (178 plus or minus 4 days). Unconjugated, glucuronoside (beta-glucuronidase) and sulfate (H2SO4-ethyl acetate) fractions were extracted with ethyl acetate from urine containing more than 80% of injected 14C. Metabolites of interest were isolated by paper partition chromatography and purified by crystallization and derivative formation. Compared with nonpregnant animals, the following changes (P less than 0.05) were observed in pregnancy: (1) an increase in the percent urinary 14C in the unconjugated fraction and a decrease in the proportion of 14C appearing in the glucuronoside fraction; (2) an increase in excretion of metabolites more polar than the cortols; (3) a decrease in excretion of metabolites less polar than cortisone in the glucuronoside fraction; (4) an increase in unconjugated F excretion. Production rate of F (11.9 plus or minus 0.7 mg/day) estimated by isotope dilution and percent urinary 14C in tetrahydrocortisol and tetrahydrocortisone from the glucuronoside fraction were as in nonpregnant animals. With the exception of an increase in F production (22.7 plus or minus 0.8 mg/day), presumably the result of the stress of labor, F metabolism in the immediate postpartum period was strikingly similar to timals indicates that changes in the mother alone can account for the altered metabolic disposition of F in pregnancy and suggests that the fetus takes little part in metabolism of maternal circulating F.

Furosemide inhibits 11beta-hydroxysteroid dehydrogenase type 2.

11Beta-hydroxsteroid dehydrogenase 2 (11beta-OHSD2) protects the nonselective renal mineralocorticoid receptor from the endogenous glucocorticoid cortisol. Thus, drugs inhibiting 11beta-OHSD2 might enhance urinary loss of potassium. As diuretics influence the renal handling of potassium, we analyzed the impact of 13 commonly used diuretics on 11beta-OHSD2. Furosemide was the only inhibitor. Its inhibition constant (Ki) was 30 micromol when extracts from COS-1 cells transfected with human 11beta-OHSD2 were used as an enzyme source. The type of inhibition was competitive. To establish whether furosemide inhibits 11beta-OHSD2 and 11beta-OHSD1 in the renal target tissue, isolated tubular segments from rats were analyzed. Furosemide decreased the oxidative activity of 11beta-OHSD2 in intact distal tubules and 11beta-OHSD1 in proximal convoluted tubules. For the assessment of furosemide on the excretion of corticosterone metabolites in vivo, rats were given furosemide i.p., and the ratio of tetrahydrocorticosterone plus 5alpha-tetrahydrocorticosterone to 11-dehydrotetrahydrocorticosterone was determined in urine. This ratio increased after the administration of furosemide in all animals, indicating inhibition of the oxidative activity of 11beta-OHSD. Thus, furosemide inhibits the 11beta-OHSD2 enzyme in the target tissue and might by that mechanism enhance the mineralocorticoid effect of 11beta-hydroxyglucocorticoids.