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.

[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.

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.

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.

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.

The diagnosis of 5 alpha-reductase deficiency in infancy.

The diagnosis of 5 alpha-reductase deficiency in infancy is reported for the first time in three male pseudohermaphrodites from the Dominican Republic. Basal plasma testosterone to dihydrotestosterone ratios were significantly elevated in two of the three affected infants, and increased markedly in all three infants after administration of hCG. Since urinary etiocholanolone to androsterone ratios could not be determined accurately in this age group, the diagnosis of 5 alpha-reductase deficiency was confirmed by the finding of elevated urinary tetrahydrocortisol (THF) to 5 alpha-tetrahydrocortisol (5 alpha-THF) ratios, as determined by gas chromatography/mass spectrometry, in the affected male infants compared to those in age-matched normal infants. The THF/5 alpha-THF ratios, however, were lower in both the normal children and the affected infants, suggesting increased 5 alpha-reductase activity in infancy. The affected infants had THF/5 alpha-THF ratios comparable to ratios in adult carrier males and significantly lower than ratios in adult homozygotes. Two of the three affected infants are related to the large Dominican kindred we studied previously and initially were raised as females but, after parental counseling, were changed to a male sex of rearing.

Quantification of polar glucocorticosteroids in the urine of pregnant and nonpregnant women: a comparison with 6 alpha-hydroxylated metabolites of cortisol in neonatal urine and amniotic fluid.

6 alpha-Hydroxy metabolites of cortisol were determined in the urine of pregnant (36-40 weeks of gestation) and nonpregnant women and in amniotic fluid from nearly fullterm pregnant women because relatively large amounts of these compounds are excreted in the urine of 2-day-old infants (> 200 micrograms/day). The corticosteroids analyzed by high pressure liquid chromatography and gas chromatography-mass spectrometry were 6 alpha-hydroxy derivatives of (allo)tetrahydrocortisone (3 alpha, 17 alpha, 21-trihydroxy-5 epsilon-pregnan-11,20-dione), (allo)tetrahydrocortisol (3 alpha, 11 beta, 17 alpha, 21-tetrahydroxy-5 epsilon-pregnan-20-one), and alpha- and beta-cortolone (3 alpha, 17 alpha, 20 epsilon, 21-tetrahydroxy-5 beta-pregnan-11-one). All of these compounds were found in the urine samples from both groups of women and in the amniotic fluid samples in contrast to those found in the urine samples from the neonates where 6 alpha-hydroxy compounds of (allo)tetrahydrocortisol and allotetrahydrocortisone were not positively identified because of insufficient yields. The pregnant women excreted significantly larger amounts of 6 alpha-hydroxy metabolites of cortisol (approximately 600 micrograms/day) than the control women (approximately 90 micrograms/day), and the rate of urinary excretion of these 6 alpha-hydroxy compounds was 7.82 and 1.30 micrograms/kg . day, respectively, for these groups of women compared to 54.3 micrograms/kg . day for the neonates. The precursors of these metabolites within the fetal body originated largely from the maternal circulation, and, therefore, the 6 alpha-hydroxy metabolites of cortisol excreted by the mother refer mainly to fetal metabolism and to a lesser extent, to the fetal secretion of cortisol.

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.

Effect of flutamide on cortisol metabolism.

The effect of flutamide on cortisol metabolism was studied in eight patients with prostate cancer. Flutamide markedly decreased the formation of 3 alpha, 17,21-trihydroxypregnane-11,20-dione (THF), and the 11-oxy-17-ketosteroid metabolites by 72%, 50%, and 46% respectively; however, 3 alpha, 11 beta, 17,21-tetrahydroxy-5 alpha- pregnan-20-one was increased by 46%. The 24-h mean plasma cortisol concentration was not altered. The cortisol production rate decreased by an average of 53% (from 32.7 to 15.5 mg/24 h). The effect of the drug on plasma cortisol kinetics was studied in three patients. This showed that flutamide increased the t1/2 (from 80 to 108 min) but decreased the distribution volume (from 17.8 to 13.8 liters) and the MCR (from 222 to 130 liters/24 h). The changes in THE and THF formation and in the t1/2 and MCR of [C]cortisol are similar to the effects observed in patients with intrahepatic cholestasis. It is suggested that in the case of flutamide these changes were also due to a cholestasis-producing effect of the drug on the liver. As the clinical response to the drug did not correlate with the cortisol metabolic changes, its therapeutic effect was probably not mediated by its effects on cortisol metabolism.

Decreased urinary C19 and C21 steroid 5 alpha-metabolites in parents of male pseudohermaphrodites with 5 alpha-reductase deficiency: detection of carriers.

The urinary 5 beta/5 alpha ring A-reduced metabolites of C19 and C21 steroids from obligate carrier parents of male pseudohermaphrodites with 5 alpha-reductase deficiency were analyzed by gas chromatography. Etiocholanolone/androsterone, 11 beta-hydroxyetiocholanolone/11 beta-hydroxyandrosterone, tetrahydrocortisol/allotetrahydrocortisol, and tetrahydrocorticosterone/allotetrahydrocorticosterone were the paired 5 beta/5 alpha-metabolite ratios measured. Increased mean 5 beta/5 alpha ratios were found for all paired metabolites compared to mean ratios in normal subjects. In men, the highest index of discrimination of the carrier state was the tetrahydrocorticosterone/allotetrahydrocorticosterone ratio, while in women, the etiocholanolone/androsterone ratio was more diagnostic. In obligate carrier men, plasma testosterone, dihydrotestosterone, androstenedione, and 17 alpha-hydroxyprogesterone levels were normal, as were testosterone/dihydrotestosterone ratios. These studies demonstrate a generalized defect in 5 alpha-reductase activity involving C19 and C21 steroid metabolism in obligate carrier parents and provide further confirmation of an autosomal recessive mode of inheritance in this condition. The data from parents of sporadic cases of male pseudohermaphrodites with primary 5 alpha-reductase deficiency suggest that there is a carrier rate within the general population, although the exact frequency remains unknown.

Metabolic profiles of steroids in urine of alcoholics after withdrawal.

The metabolic profiles of steroids in urine were analyzed in 13 male alcoholics during long-term abstinence, in most cases exceeding 3 months. The ratios of 5 beta- to 5 alpha-reduced steroid metabolites (etiocholanolone/androsterone and tetrahydrocortisol/allotetrahydrocortisol) were initially elevated but decreased slowly following withdrawal. The half-life of this normalization exceeded 3 weeks. The change was most marked in patients with signs of liver injury, and may reflect a relative decrease of the activity of hepatic 5 alpha-reductase. The ratio between cortisol metabolites carrying a 11 beta-hydroxy and an 11-oxo group was elevated in the patients and showed no tendency to normalize. This might reflect a decrease in the peripheral inactivation of cortisol.

Polar corticosteroids in human neonatal urine; synthesis and gas chromatography-mass spectrometry of ring A reduced 6-hydroxylated corticosteroids.

This report describes the synthesis of 3alpha,6beta,11beta,17alpha,21-pentahydroxy-5beta-pregnane-20-one, 3alpha,6beta,11beta,17alpha,21-pentahydroxy-5beta-pregnane-20-one, 3alpha,6alpha,11beta,17alpha,21-pentahydroxy-5alpha-pregnane-20-one, 3alpha,6beta,17alpha,21-tetrahydroxy-5beta-pregnane-11,20-dione, 3alpha,6beta,17alpha,21-tetrahydroxy-5alpha-pregnane-11,20-dione, 3alpha,6alpha,17alpha,21-tetrahydroxy-5beta-pregnane-11,20-dione and 3alpha,6alpha,17alpha,21-tetrahydroxy-5alpha-pregnane-11,20-dione. The gas chromatographic-mass spectrometric properties of these compounds are given. Proof of structure was accomplished using gas chromatography-mass spectrometry, microchemical reactions, optical rotatory dispersion and nuclear magnetic resonance spectroscopy.

Identification of 7xi-hydroxytetrahydrocortisol and 7xi-hydroxytetrahydrocortisone from urine of newborn baboons (Papio papio).

Two highly polar, unconjugated metabolltes of tetrahydrocortisol and tetrahydrocortisone, which had been shown previously to account for greater than 40% of [14C]cortisol (F) administered to baboon neonates, were isolated from urine of a term newborn animal. Metabolites were extracted with ethyl acetate, purified by sequential paper chromatography and crystallization, and identified by chemical and biochemical procedures. Both metabolites of F are 5beta-pregnanes with a hydroxyl of unknown orientation at C-7, a 3alpha-hydroxyl, and a dihydroxyacetone side chain. The two compounds differ from each other by the presence of either oxo or hydroxyl functions at C-11. The following nomenclature is proposed: unknown 1: 3alpha,7xi,11beta,17,21-pentahydroxy-5beta-pregnan-20-one (7xi-hydroxytetrahydrocortisol); unknown 11: 3alpha,7xi,17,21-tetrahydroxy-5beta-pregnane-11,20-dione (7xi-hydroxytetrahydrocortisone). Production of these compounds compensates quantitatively for the decreased formation of F glucuronoside metabolites by baboon neonates, compared with that in adults. Therefore, we propose that 7-hydroxylation is a major alternative pathway for F metabolism in the perinatal period of the baboon.

C-3 glucosiduronates of metabolites of adrenal steroids.

On treatment with methyl 2,3,4-tri-O-acetyl-1-bromo-1-deoxy-alpha-D-glucuronate and silver carbonate, tetrahydrocortisone 21-acetate gave the corresponding 3-glucosiduronate triacetyl methyl ester. This product was converted into the 20-semicarbazone which, by treatment with alkali to hydrolyze the ester functions and acid to hydrolyze the semicarbazone moiety, gave tetrahydrocortisone 3-glucosiduronic acid. The acid was converted into the crystalline barium salt and into the methyl ester. An analogous series of reactions was carried out on tetrahydrocortexolone 21-acetate. Treatment of the 20-semicarbazone of tetrahydrocortisone 3-glucosiduronic acid with potassium borohydride reduced the 11-oxo function to an 11 beta hydroxyl group; acid-catalyzed removal of the semicarbazone group produced tetrahydrocortisol 3-glucosiduronic acid which also was obtained as the barium salt and the methyl ester.

Late-onset 21-hydroxylase deficiency: reliable diagnosis by steroid analysis of random urine collections.

The feasibility of performing steroid analysis by capillary gas chromatography on random urine samples for the detection of mild late-onset 21-hydroxylase deficiency was evaluated. Comparisons were made of basal excretions of androgen and 17 alpha-hydroxyprogesterone metabolites with plasma levels (basal and stimulated) of 17 alpha-hydroxyprogesterone and testosterone in six patients with the disorder. The following steroid metabolite excretion ratios were determined for normal controls and affected individuals. 1) 17 alpha-hydroxypregnanolone/tetrahydrocortisone + tetrahydrocortisol + 5 alpha-tetrahydrocortisol (cortisol metabolites) (normal 0.017-0.10, affected 0.17-0.42); 2) pregnanetriol/cortisol metabolites (normal 0.03-0.15, affected 0.17-0.99); 3) pregnanetriolone/cortisol metabolites (normal 0.02-0.014, affected 0.08-0.20); 4) androsterone + etiocholanolone/cortisol metabolites (normal 0.26-1.02, affected 0.34-1.47). Among the 21-deoxy steroid ratios, there was no overlap between affected and unaffected individuals. Two of six affected individuals had androsterone + etiocholanolone/cortisol metabolite ratios in the normal range. This method provides excellent discrimination between normal and affected individuals, precluding the need for an ACTH-stimulation test. It is anticipated that it will be increasingly used for diagnosis of the condition.

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.

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.

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.