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after the treatment, while THE levels decreased during the experiment, but rebounded substantially after the DX was withdrawn.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.

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.

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.

Tetrahydro-metabolites of cortisol and cortisone in bovine urine evaluated by HPLC-ESI-mass spectrometry.

Interconversion of hormonally active cortisol (F) into the corresponding inactive 11-keto form, cortisone (E), is catalyzed by 11beta-hydroxysteroid dehydrogenases (11ß-HSDs). With a view to estimating in vivo activities of some 11ß-HSD isoforms, the measurement of urinary F and E and their tetrahydro metabolites (tetrahydrocortisol, THF, allotetrahydrocortisol, ATHF, tetrahydrocortisone, THE) has been suggested. The basic knowledge of THF, ATHF and THE levels in farm cattle is limited. Therefore the aim of this study was first to optimize a simple and quick method to determine F and E tetrahydro-metabolites in bovine urine by HPLC-mass spectrometry with electrospray ionization (HPLC-ESI-MS) and then to apply the method to real urine of bovines treated with prednisolone. The samples underwent filtration, deconjugation, solid-phase extraction (SPE) and the relevant analytes were measured by HPLC-ESI-MS. The method described in this paper is simple and efficient, featuring good linearity (up to 0.996) and reproducibility (6.8-12.5%, CV). Especially, good LODs were obtained, from 1.63 to 2.67 ppb, depending on the analyte. The chromatographic conditions were optimized in order to obtain a resolution which would allow to simultaneously measure two diastereoisomers, i.e. THF and ATHF. In our study, ATHF turns out to be below the detection limit, while for 18 samples tested the contents of examinated metabolites were as followed: THF (12.5±4.8 ppb), THE (10.9±5.5 ppb), F (11.6±3.3 ppb) and E (5.0±2.2 ppb). When the method was applied to the subject treated with prednisolone a major increase in the concentration of tetrahydro metabolites was observed before the slaughter, mainly due to stress conditions; prednisolone treatment, most presumably, influenced the 11ß-HSD activity, as indicated by the decrease in the F/E ratio. This work may provide a useful methodological contribution to the future definition of F, E, THF, ATHF and THE urinary baseline values in order to obtain indirect evaluations of HSDs activity in farm cattle and possible applications in screenings for suspected abuse of synthetic corticosteroids in bovines.

Role of 11ßHSD type 2 enzyme activity in essential hypertension and children with chronic kidney disease (CKD).

BACKGROUND: The mineralocorticoid receptor is protected from excess of glucocorticoids by conversion of active cortisol to inactive cortisone by enzyme 11ß-hydroxysteroid dehydrogenase type 2 present in the kidney. The metabolites of cortisol and cortisone are excreted in the urine as tetrahydrocortisol (5αTHF+5ßTHF) and tetrahydrocortisone (THE), respectively. HYPOTHESIS: Patients with chronic kidney disease (CKD) and essential hypertension have a functional defect in their ability to convert cortisol to cortisone, thus leading to the activation of mineralocorticoid receptor. OBJECTIVE: The objective of the investigation was to study the ratio of urinary steroids (5αTHF+5ßTHF) to THE in patients with CKD, postrenal transplant, and essential hypertension and to compare the ratio with controls. DESIGN/METHODS: We enrolled 44 patients (17 with CKD, eight postrenal transplant, 19 with essential hypertension) and 12 controls. We measured spot urinary 5α-THF, 5ß-THF, THE, free active cortisol and inactive cortisone by gas chromatography/mass spectrometry. We collected data on age, sex, cause of kidney disease, height, weight, body mass index, blood pressure, serum electrolytes, aldosterone, and plasma renin activity. Blood pressure percentiles and z-scores were calculated. The glomerular filtration rate was calculated using the modified Schwartz formula. RESULTS: The ratios of 5αTHF+5ßTHF to THE were significantly higher in patients with CKD [mean±sd score (SDS)=1.31±1.07] as compared with essential hypertension (mean±SDS=0.59±0.23; P=0.02) and controls (mean±SDS=0.52±0.25; P=0.01). In the postrenal transplant group, the ratio was not significantly different (mean±SDS=0.71±0.55). The urinary free cortisol to free cortisone ratios were significantly higher in the hypertension and CKD groups as compared with the controls. The 5αTHF+5ßTHF to THE ratio negatively correlated with the glomerular filtration rate and positively correlated with systolic and diastolic blood pressure z-scores. The correlation of the blood pressure z-scores with ratios was stronger in the CKD group than the essential hypertension and posttransplant groups. CONCLUSIONS: We have elucidated a functional deficiency of 11ß-hydroxysteroid dehydrogenase type 2 in children with CKD and a subset of essential hypertension. Urinary 5α-THF, 5ß-THF, and THE analysis by gas chromatography/mass spectrometry should be a part of routine work-up of CKD and hypertensive patients.

HPLC method for determination of fluorescence derivatives of cortisol, cortisone and their tetrahydro- and allo-tetrahydro-metabolites in biological fluids.

11Beta-hydroxysteroid dehydrogenase isoform 2 (11beta-HSD2) is responsible for conversion of cortisol (F) to inactive cortisone (E). Disturbance of its activity can cause hypertension. To estimate 11beta-HSD2 activity, besides F and E, their tetrahydro- (THF, THE) as well allo-tetrahydro- (allo-THF, allo-THE) metabolites should be determined. This study describes HPLC-FLD method for the quantitative determination of endogenous glucocorticoids (GCs) in plasma and urine (total and free) and their metabolites in urine. Following extraction at pH 7.4 using dichloromethane, GCs (F, E, THF, allo-THF, THE, allo-THE and internal standard--prednisolone) were derivatized with 9-anthroyl nitrile and purified by SPE using C(18) cartridges. The enzymatic hydrolysis of conjugated steroids was provided using beta-glucuronidase. The influence of organic bases on 9-AN derivatization of steroids was investigated. The best yield of the derivatization was obtained in presence of the mixture of 10.0% triethylamine (TEA) and 0.1% quinuclidine (Q). Chromatographic separation was accomplished in the Chromolith RP-18e monolithic column. The elaborated method was validated. Calibration curves were linear in the ranges: for F, E and THF 5.0-1000.0 ng mL(-1), for allo-THF and THE + allo-THE 10.0-1000.0 ng mL(-1). LOD (S/N=3:1) for all analytes amounted 3.0 ng mL(-1). Recoveries of GCs exceeded 90%. The method was precise and accurate, intra- and inter-day precision were 3.0-12.1% and 9.2-14.0%, respectively. Accuracy ranged from 0.2 to 15.1%. The method was applied for estimating endogenous GCs in plasma and urine. Plasma levels of F and E were in the ranges: 133.0-174.5 ng mL(-1) and 17.4-35.9 ng mL(-1), respectively. Free urinary steroids were in the ranges: 12.0-54.1 microg/24 h (UFF) and 37.8-76.2 microg/24 h (UFE). The ratio of (THF + allo-THF)/(THE + allo-THE) amounted from 1.01 to 1.23. The obtained results confirmed utility of the elaborated method in the assessment of 11beta-HSD2 activity in man.

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.

Enduring effects of severe developmental adversity, including nutritional deprivation, on cortisol metabolism in aging Holocaust survivors.

OBJECTIVE: In animal models, early life exposure to major environmental challenges such as malnutrition and stress results in persisting cardiometabolic, neuroendocrine and affective effects. While such effects have been associated with pathogenesis, the widespread occurrence of 'developmental programming' suggests it has adaptive function. Glucocorticoids may mediate 'programming' and their metabolism is known to be affected by early life events in rodents. To examine these relationships in humans, cortisol metabolism and cardiometabolic disease manifestations were examined in Holocaust survivors in relation to age at exposure and affective dysfunction, notably lifetime posttraumatic stress disorder (PTSD). METHODS: Fifty-one Holocaust survivors and 22 controls without Axis I disorder collected 24-h urine samples and were evaluated for psychiatric disorders and cardiometabolic diagnoses. Corticosteroids and their metabolites were assayed by gas chromatography-mass spectroscopy (GC-MS); cortisol was also measured by radioimmunoassay (RIA). RESULTS: Holocaust survivors showed reduced cortisol by RIA, and decreased levels of 5alpha-tetrahydrocortisol (5alpha-THF) and total glucocorticoid production by GC-MS. The latter was associated with lower cortisol metabolism by 5alpha-reductase and 11beta-hydroxysteroid dehydrogenase (11beta-HSD) type-2. The greatest decrements were associated with earliest age of Holocaust exposure and less severe PTSD symptomatology. Cardiometabolic manifestations were associated with decreased 11beta-HSD-2 activity. In controls, 5alpha-reductase was positively associated with trauma-related symptoms (i.e., to traumatic exposures unrelated to the Holocaust). CONCLUSION: Extreme malnutrition and related stress during development is associated with long-lived alterations in specific pathways of glucocorticoid metabolism. These effects may be adaptive and link with lower risks of cardiometabolic and stress-related disorders in later life.

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

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

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.

Endogenous corticosteroid biosynthesis in subjects after bilateral adrenalectomy.

OBJECTIVE: Corticosteroids can be synthesized in extra-adrenal tissues but the contribution of this to circulating levels in humans is not known. Previous in vitro studies suggest that the 'hybrid' corticosteroid 18-oxocortisol (18-oxoF) is produced from cortisol by aldosterone synthase. We looked for evidence of extra-adrenal production of this and other corticosteroids in 10 subjects stable on long-term glucocorticoid replacement following bilateral adrenalectomy. METHODS: In phase 1, patients were maintained on cortisol alone (30 mg/day), in phase 2 dexamethasone (2 mg/day), and in phase 3, both cortisol and dexamethasone. Each phase lasted 3 days. MEASUREMENTS: On the last day of each phase, 24-h urine collection was performed for analysis of steroid metabolite excretion [using gas chromatography-mass spectrometry (GCMS)] and plasma aldosterone and renin were measured (by radioimmunoassay). RESULTS: Cortisol metabolite excretion rate [tetrahydrocortisone (THE) + tetrahydrocortisol (THF) + allotetrahydrocortisol (aTHF)] fell from 9169 nmol/24 h in phase 1 to 22 nmol/24 h in phase 2, rising to 6843 nmol/24 h in phase 3. Tetrahydroaldosterone (THAldo) excretion was readily detectable and did not alter significantly between phases (26.5, 23.5 and 28.5 nmol/24 h, respectively; P = 0.474). 18-Hydroxycortisol (18-OHF) excretion was easily detectable in phases 1 and 3 (252.5 and 212 nmol/24 h), falling in phase 2 (12 nmol/24 h). 18-oxoF excretion rates were lower but followed a similar pattern (1.62, 0.085 and 1.785 nmol/24 h in phases 1, 2 and 3, respectively). CONCLUSIONS: Significant levels of adrenal steroids are found in adrenalectomized subjects. We speculate that this occurs at extra-adrenal sites or in residual adrenal cortex tissue in an ACTH-independent manner. Our data suggest that aldosterone synthase, acting on cortisol, is the source of 18-oxoF and 18-OHF in these subjects. Further studies of corticosteroid production within adrenalectomized subjects, looking for evidence of adrenal regrowth or residual adrenal tissue, are justified.

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.

Urinary steroid excretion rates in acromegaly.

Using gas chromatography/mass spectrometry, urinary excretion rates of cortisol, cortisone and of various steroid metabolites were determined in 35 acromegalic patients (18 men, 17 women) and in 45 age- and weight-matched controls. The ratio of excreted cortisol/cortisone was similar in acromegalics (0.75 +/- 0.20) and in controls (0.75 +/- 0.24). Hence, the preponderance of the main cortisone-derived metabolite, tetrahydrocortisone, over the main metabolites of cortisol (tetrahydrocortisol and allotetrahydrocortisol; p < 0.01), which was seen both in female and in male acromegalics and which was directly correlated with the postglucose concentrations of growth hormone (r = 0.508, p < 0.01), suggests a decreased activity of 11beta-hydroxysteroid dehydrogenase type 1 in acromegaly. Furthermore, the preponderance of etiocholanolone over androsterone (p < 0.01) in men (though not in women) with acromegaly--the ratio androsterone/etiocholanolone being negatively correlated with the serum concentrations of insulin-like growth factor type 1 (r = -0.406, p < 0.05)--suggests a relatively reduced activity of hepatic 5alpha-reductase in male acromegalics.

Hydrolysis of conjugated steroids by the combined use of beta-glucuronidase preparations from helix pomatia and ampullaria: determination of urinary cortisol and its metabolites.

This study describes the enzymatic hydrolysis of urinary conjugates of cortisol, cortisone, tetrahydrocortisol, allotetrahydrocortisol, and tetrahydrocortisone with beta-glucuronidase preparations from Helix pomatia and Ampullaria. The objective of the present studies was to find optimal hydrolysis conditions for these conjugated steroids. Assay of the isolated steroids was carried out by GC-MS using deuterium-labeled compounds as internal standards. The allotetrahydrocortisol conjugate was clearly the hardest to hydrolyze with enzyme from Helix pomatia and required increased enzyme concentration and prolonged incubation. Hydrolysis of a urine sample for 2.0 h with the simultaneous use of 3400 units/ml Ampullaria and 5400 units/ml Helix pomatia enzymes in 0.5 M acetate buffer at 55 degrees C achieved more complete cleavage of the urinary conjugates of the five steroids examined. It is thus advantageous to use the Ampullaria and Helix pomatia enzymes in combination to obtain the highest yield in the urinary corticosteroid assay.

Sexual dimorphism of cortisol metabolism is maintained in elderly subjects and is not oestrogen dependent.

OBJECTIVE: The net interconversion of inactive cortisone to active cortisol by 11beta-hydroxysteroid dehydrogenase type 1 (11betaHSD1) may determine hepatic and adipose tissue exposure to glucocorticoid action. Cortisol metabolism exhibits a sexual dimorphism with an apparently lower activity of 11betaHSD1 in females that, in an animal model, has been attributed to the effects of oestrogen. The aim of this study is to determine whether the sexual dimorphism of cortisol metabolism persists between post-menopausal, oestrogen-deficient women and elderly men. PATIENTS: Fifteen healthy men, aged 60.8-82.0 years, and 7 healthy women, aged 62.4-87.5 years, were studied. None of the subjects was receiving steroid medication at the time of the study. All the women were post-menopausal and none was receiving sex steroid replacement therapy. MEASUREMENTS: 24-h urine collections were taken from each patient and assayed for steroid metabolites by gas chromatography. Body composition was determined by dual energy X-ray absorptiometry. Blood was drawn, after an overnight fast, for the determination of serum IGF-I and IGFBP1 levels. RESULTS: The ratio of 11-hydroxy cortisol metabolites to 11-oxo cortisol metabolites (Fm/Em) was significantly higher in men than in women, 0.80 (0.55-1.86) vs. 0.67 (0.46-0.98) (P < 0.02), as was the ratio of allo-tetrahydrocortisol (5alpha-THF) + tetrahydrocortisol (THF)/tetrahydrocortisone (THE), 0.74 (0.37-2.08) vs. 0.40 (0.22-1.10) (P < 0.047). In the group as a whole there was a negative correlation between Fm/Em and percent body fat, r = - 0. 43 (P < 0.05), and the negative relationship between cortisol and cortisone metabolite (Fm/Em) and total fat mass approached significance, r = - 0.39 (P = 0.07). These relationships were not apparent in the women when considered alone. Among the men there were negative relationships between Fm/Em and total fat mass, r = - 0.48, and Fm/Em and trunk fat mass, r = - 0.48 which approached significance (both P = 0.07). Serum IGFBP-1 levels were not significantly different between the two sexes. There was a significant correlation between IGFBP-1 and Fm/Em in the men, r = 0. 84 (P < 0.0001) which persisted when total body fat mass, r = 0.85 (P < 0.0001) and trunk fat mass, r = 0.83 (P < 0.0001), were controlled for. This relationship was not evident among the women. CONCLUSION: This study demonstrates that the previously described sexual dimorphism in cortisol metabolism is not dependent on oestrogen, although the possibility that oestrogen exerts a permanent modifying effect on 11beta-HSD1 gene expression during the pre-menopausal period cannot be excluded. The findings confirm the primary importance of body fat as a determinant of cortisol-cortisone conversion.

Molecular basis of human salt sensitivity: the role of the 11beta-hydroxysteroid dehydrogenase type 2.

Salt-sensitive subjects (SS) increase their blood pressure with increasing salt intake. Because steroid hormones modulate renal sodium retention, we hypothesize that the activity of the 11beta-hydroxysteroid dehydrogenase type 2 (11betaHSD2) enzyme is impaired in SS subjects as compared with salt-resistant (SR) subjects. The 11betaHSD2 enzyme inactivates 11-hydroxy steroids in the kidney, thus protecting the nonselective mineralocorticoid receptor from occupation by glucocorticoids. We performed an association study using a recently identified single AluI polymorphism in exon 3 and a polymorphic microsatellite marker of the HSD11B2 gene in 149 normotensive white males (37 SS and 112 SR). The activity of the enzyme 11betaHSD2 was assessed by determining the urinary ratio of cortisol (THF+5alphaTHF) to cortisone (THE) metabolites by gas chromatography in all the 37 SS subjects and in 37 age- and body habitus-matched SR volunteers. Mean (THF+5alphaTHF)/THE ratio was markedly elevated in SS subjects compared with SR subjects (1.51 +/- 0.34 vs. 1.08 +/- 0.26, P < 0.00001), indicating enhanced access of glucocorticoids to the mineralocorticoid receptor in SS subjects. In 58% of SS subjects this ratio was higher than the maximum levels in SR subjects. The salt-induced elevation in arterial pressure increased with increasing (THF+5alphaTHF)/THE ratio (r2 = 0.51, P < 0.0001). A total of 12 alleles of the polymorphic microsatellite marker were detected. Homozygosity for the allele A7 was higher in SS subjects than in SR subjects (41 vs. 28%, P < 0.005), whereas the occurrence of the allele A7 with allele A8 was lower in SS subjects than in SR subjects (8 vs. 15%, P < 0.03). The prevalence of salt sensitivity was 35% in subjects with allele A7/A7, whereas salt sensitivity was present in only 9% of the subjects with allele A7/A8. The (THF+5alphaTHF)/THE ratio was higher in subjects homozygous for the A7 microsatellite allele as compared with the corresponding control subjects. The prevalence of the AluI allele was 8.0% in SR subjects and 5.4% in SS subjects and did not correlate with blood pressure. The decreased activity of the 11betaHSD2 in SS subjects indicates that this enzyme is involved in salt-sensitive blood pressure response in humans. The association of a polymorphic microsatellite marker of the gene with a reduced 11betaHSD2 activity suggests that variants of the HSD 11B2 gene contribute to enhanced blood pressure response to salt in humans.