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.

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

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

Chemical communication in tilapia: a comparison of Oreochromis mossambicus with O. niloticus.

In allopatric speciation species differentiation generally results from different selective pressures in different environments, and identifying the traits responsible helps to understand the isolation mechanism(s) involved. Male Mozambique tilapia (Oreochromis mossambicus) use urine to signal dominance; furthermore, 5ß-pregnane-3α,17,20ß-triol-3α-glucuronide (and its α-epimer, 5ß-pregnane-3α,17,20α-triol-3α-glucuronide), in their urine is a potent pheromone, the concentration of which is correlated with social status. The Nile tilapia (Oreochromisniloticus) is a close relative; species divergence probably resulted from geographical separation around 6 million years ago. This raises the question of whether the two species use similar urinary chemical cues during reproduction. The olfactory potency of urine, and crude extracts, from either species was assessed by the electro-olfactogram and the presence of the steroid glucuronides in urine from the Nile tilapia by liquid-chromatography/mass-spectrometry. Both species showed similar olfactory sensitivity to urine and respective extracts from either species, and similar sensitivity to the steroid glucuronides. 5ß-Pregnan-3α,17α,20ß-triol-3α-glucuronide was present at high concentrations (approaching 0.5mM) in urine from Nile tilapia, with 5ß-pregnan-3α,17α,20α-triol-3α-glucuronide present at lower concentrations, similar to the Mozambique tilapia. Both species also had similar olfactory sensitivity to estradiol-3-glucuronide, a putative urinary cue from females. Together, these results support the idea that reproductive chemical cues have not been subjected to differing selective pressure. Whether these chemical cues have the same physiological and behavioural roles in O. niloticus as O. mossambicus remains to be investigated.

Steroids excreted in urine by neonates with 21-hydroxylase deficiency. 4. Characterization, using GC-MS and GC-MS/MS, of 11oxo-pregnanes and 11oxo-pregnenes.

In 21-hydroxylase deficiency, urinary metabolites of 21-deoxycortisol, mainly derived from its 11oxo form 21-deoxycortisone, are indicators of intra-adrenal overproduction of 17-hydroxyprogesterone. In affected neonates these metabolites are numerous and most have not been previously described. This work forms the concluding part of a comprehensive study of urinary steroids, aiming to enhance the diagnosis of this disorder and to further elucidate steroid metabolism in neonates. Cortisol metabolites found in untreated patients, similarly almost exclusively present in their 11oxo form in neonates, have been included for completeness. Steroids were analyzed, after extraction and enzymatic conjugate hydrolysis, as methyloxime-trimethylsilyl ether derivatives on gas-chromatographs coupled to quadrupole and ion-trap mass-spectrometers. GC-MS and GC-MS/MS spectra were used together to determine the structure of hitherto undescribed 11oxo-pregnane(enes). Few GC-MS features were associated with the presence of the non-derivatizeable 11oxo group in pregnane(ene)s. GC-MS/MS contributed only to the characterization of structures outside the C-ring, as described in the preceding parts of this study. Parallels were found between the metabolism of 21-deoxycortisone and cortisone. The major metabolic pathway was that of classical 3α,5ß-reduction with a prominent further hydroxylation, predominantly at C6. Oxidation of the 6-hydroxyl was also common. We conclude that further oxygenated metabolites of 21-deoxycortisone have potential as more reliable markers of 21-hydroxylase deficiency in the early neonatal period, because their levels are higher during that period of life than for the classical marker 11oxo-pregnanetriol.

Non-targeted metabolomic approach reveals urinary metabolites linked to steroid biosynthesis pathway after ingestion of citrus juice.

Citrus juice intake has been highlighted because of its health-promoting effects. LC-MS based metabolomics approaches are applied to obtain a better knowledge on changes in the concentration of metabolites due to its dietary intake and allow a better understanding of involved metabolic pathways. Eight volunteers daily consumed 400 mL of juice for four consecutive days and urine samples were collected before intake and 24h after each citrus juice intake. Urine samples were analysed by nanoHPLC-q-TOF, followed by principal component analysis (PCA) and Student's t-test (p<0.05). PCA showed a separation between two groups (before and after citrus juice consumption). This approach allowed the identification of four endocrine compounds (tetrahydroaldosterone-3-glucuronide, cortolone-3-glucuronide, testosterone-glucuronide and 17-hydroxyprogesterone), which belonged to the steroid biosynthesis pathway as significant metabolites upregulated by citrus juice intake. Additionally, these results confirmed the importance of using the non-targeted metabolomics technique to identify new endogenous metabolites, up- or down-regulated as a consequence of food intake.

Steroids excreted in urine by neonates with 21-hydroxylase deficiency. 2. Characterization, using GC-MS and GC-MS/MS, of pregnanes and pregnenes with an oxo- group on the A- or B-ring.

Urine from neonates with 21-hydroxylase deficiency contains a large range of metabolites of 17-hydroxyprogesterone, 21-deoxycortisol and androgens but few have been previously described. We present the second part of a comprehensive project to characterize and identify these in order to enhance diagnosis and to further elucidate neonatal steroid metabolism. Steroids were analyzed, after extraction and enzymatic conjugate hydrolysis, as methyloxime-trimethylsilyl ether derivatives on gas-chromatographs coupled to quadrupole and ion-trap mass-spectrometers. GC-MS and GC-MS/MS spectra were used together to determine the structure of the A- and B-rings containing an oxo group. Fragmentations indicating presence of 3-, 6-, and 7-oxo groups and also 1ß-, 2α-, 4ß-, and 6ß-hydroxyls are presented and discussed for the first time. Interpretation was aided by comparison with spectra of available relevant standards, of oxidation products of standards and urinary metabolites and of deuterated derivatives. Endogenous 1-enes and 2(3)-ene artifacts of non-hydrolyzed 3α-sulfates are also reported. D-ring and side chain structure was determined according to our previously published criteria. Likely metabolic relationships were also explored. We conclude that GC-MS combined with GC-MS/MS allows identification of the A- and B-ring structure of pregnane and pregnenes in the presence of an oxo group on one of these rings. Major oxygenations are 1ß, 15ß, 16α and 21-hydroxy and 6- and 7-oxo groups. Minor positions of hydroxylation are those at 2α, 4ß and 6ß. Three major metabolic streams exist in affected neonates in addition to the classical 3α-hydroxy-5ß-pregnane pathway, i.e. these of the 3-oxo-4-enes as well as 3α- and 3ß-hydroxy-5α-anes.

Steroids excreted in urine by neonates with 21-hydroxylase deficiency: characterization, using GC-MS and GC-MS/MS, of the D-ring and side chain structure of pregnanes and pregnenes.

Steroid metabolites in urine from neonates with 21-hydroxylase deficiency are predominantly polyhydroxylated 17-hydroxyprogesterone and androgen metabolites, and most have incompletely defined structure. This study forms part of a comprehensive project to characterize and identify these in order to enhance diagnosis and to further elucidate neonatal types of steroid metabolism. Steroids were analyzed, after extraction and enzymatic conjugate hydrolysis, as methyloxime-trimethylsilyl ether derivatives on gas-chromatographs coupled to quadrupole and ion-trap mass-spectrometers. GC-MS and GC-MS/MS spectra, obtained with constant excitation conditions, were used together to determine the structure of the D-ring and the side chain of 20-oxo and 20-hydroxy pregnane(ene)s without oxo groups on the A-, B-, and C-ring. All possible combinations of D-ring and side chain configuration were considered. Most fragmentations could be interpreted as partial or complete D-ring cleavages with loss of the side chain, aided by comparison with spectra of deuterated derivatives and of borohydride reduced metabolites. Possible rearrangement ions are also discussed. More than 140 endogenous metabolites were characterized. GC-MS/MS was especially beneficial for characterization of compounds with 16,17-dihydroxy-20-oxo structure, interpreted as markers of intra-uterine enzyme induction. It also assisted the differentiation of 16-hydroxy-20-oxo metabolites, present in urine of non-affected neonates, from the diagnostic 17-hydroxy-20-oxosteroids and enabled the detection of 15,17-dihydroxy-20-oxo compounds in low concentrations. The presence of 17,21-dihydroxylated pregnane(ene)s despite the deficit in CYP21A2 is discussed. We conclude that GC-MS combined with GC-MS/MS allows reliable identification of the structure of the D-ring and side chain of pregnane(ene)s without prior isolation, even when in low concentrations in urine.

Xenobiotic action on steroid hormone synthesis and sulfonation the example of lead and polychlorinated biphenyls.

OBJECTIVES: In the present study, the metabolism of steroid hormones has been investigated to determine whether and how xenobiotics like lead (Pb) and polychlorinated biphenyls (PCBs) interfere with steroid hormone biotransformation in humans. METHODS: Three groups of subjects were tested for concentration of urinary total steroids, 17-ketosteroids (n = 5), pregnane derivates (n = 6), 17-hydroxycorticosteroids (n = 11) and their sulfonated compounds: 14 workers exposed to lead, with a mean Pb blood concentration (PbB) of 29.21 microg/dl; 15 subjects exposed to PCBs, with a mean PCB blood concentration (PCBB) of 61.69 microg/l; a control group (n = 25). RESULTS: The urinary concentrations of 17-ketosteroids and 17-hydroxycorticosteroids were significantly lower in the PCB-exposed groups. There were significantly fewer sulfonated 17-hydroxycorticosteroids in the subjects exposed to PCBs as compared to the controls, while the percentage of sulfonated steroids was lower for both 17-ketosteroids and 17-hydroxycorticosteroids in the PCB-exposed subjects, but only for the 17-hydroxycorticosteroids in the group of subjects exposed to Pb (P < 0.05). Pregnane derivate urinary concentrations did not differ between the three groups. CONCLUSION: Our results suggest that PCBs and Pb act on steroid hormone metabolism with different effects and only partially using the same hormone pathways; they may cause changes in endogenous hormone homeostasis and interfere with the xenobiotic phase II of detoxification. PCBs interfere on a larger number of steroids and cause more significant effects than Pb. It is likely that different mechanisms are involved in steroid hormone metabolism interference.

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.

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 metabolites and 11beta-hydroxysteroid dehydrogenase activity in patients with unipolar recurrent major depression.

BACKGROUND: The aim of the present study was to obtain comprehensive information on steroid metabolism in depressed patients. METHODS: 24-h urinary steroids were measured by gas chromatography in patients with unipolar recurrent major depression (URMD) compared to controls, and an index of relative activity of the 11beta-hydroxysteroid dehydrogenase (11beta-HSD) enzyme was calculated. RESULTS: The levels of etiocholanolone (E) (p < 0.05), beta-cortolone (beta-CL) (p < 0.01) were significantly decreased, while levels of allo-tetrahydrocorticosterone (aTHB) (p < 0.05) and cortisol (F) (p < 0.01) were elevated in depressed women. The levels of dehydroepiandrosterone (DHEA) (p < 0.01), tetrahydrocorticosterone (THB) (p < 0.01), beta-CL (p < 0.01), and aTHB (p < 0.05) were found significantly decreased in depressed men. The index of 11beta-HSD activity (p < 0.01) was significantly decreased in patients in both sexes. LIMITATIONS: The sample is limited to only urine samples of patient with URMD; the correlation between the severity of depression and F and DHEA was not analyzed. CONCLUSION: Our investigations confirmed that URMD associated with altered steroid metabolism, which shows gender differences, pointing to the different stress sensibility of women. These differences may be the cause as well as the consequence of the major depression (MD).

Endogenous urinary steroids in premenopausal women with uterine leiomyomas.

OBJECTIVES: To study the effect of endogenous steroids on the presence of uterine leiomyomas. METHODS: Urine samples of 27 premenopausal women with leiomyomas and 25 age-matched healthy premenopausal women were collected. The concentration of estrogens and androgens in the urine samples of the two groups were determined using a gas chromatography mass spectrometer and the two groups were compared. To study metabolic changes in patients indirectly, the concentration ratios of precursor metabolite to product metabolite of the two groups were also compared. RESULTS: Urinary concentrations of 17beta-estradiol, 5-androstene-3beta, 16beta, 17beta, triol, 11-keto-ethiocholanolone, 11beta-hydroxy-androsterone, 11beta-hydroxy-etiocholanolone, THS, THA, THE, alpha-cortol and beta-cortol were significantly higher in patients than in controls. The concentration ratios of 17beta-estradiol/estrone and 11/beta-hydroxy-ethiocholanolone/11beta-hydroxy-androsterone increased in patients. CONCLUSIONS: The presence of uterine leiomyomas correlates with an increase in urinary concentrations of estrogens and androgens, and it appears to be caused by a decrease in patients' metabolism of steroids.

Hormonal diagnosis of 21-hydroxylase deficiency in plasma and urine of neonates using benchtop gas chromatography-mass spectrometry.

We aimed at measuring the first plasma concentrations of 17-hydroxyprogesterone (17OH-P) determined by benchtop isotope dilution/gas chromatography-mass spectrometry (ID/GC-MS) in term neonates with or without 21-hydroxylase deficiency. Plasma samples from normal cord blood specimens (n=30), unaffected neonates (n=38) and neonatal patients with classical 21-hydroxylase deficiency (eight salt-wasters, three simple virilizers) were analyzed. Steroid profiling of random urinary specimens by GC-MS served as a confirmatory test for 21-hydroxylase deficiency. 17OH-P (nmol/l) in cord blood plasma lay between 11.66 and 75.92 (median 24.74). It declined shortly after birth. In the first 8 days of life, the time that screening for 21-hydroxylase deficiency is performed, 17OH-P ranged between undetected levels and an upper limit of 22.87 (median 4.11). Thereafter (days 9-28) its concentrations lay between 2.18 and 20.30 (median 6.22). Except one simple virilizer, all other patients with 21-hydroxylase deficiency had clearly elevated plasma 17OH-P at the time that screening for 21-hydroxylase deficiency would be performed. We suggest ID/GC-MS, which provides the highest specificity in steroid analysis, for checking suspicious concentrations of 17OH-P in neonates and underscore the potential of urinary steroid profiling by GC-MS as a rapid, non-invasive and non-selective confirmatory test for congenital adrenal hyperplasia.

Quantification of cortisol inactivation in cirrhosis of the liver.

Impaired cortisol inactivation in patients with cirrhosis of the liver has been described, but precise data are limited and the pathophysiological significance of this finding has to be elucidated. Therefore, we assessed the main urinary cortisol metabolites using capillary gas chromatography and urinary free cortisol using an enzyme immunoassay in 20 consecutive patients with cirrhosis of the liver and in 28 healthy controls; ratios of cortisol inactivation were calculated (cortisol metabolites/cortisone metabolites, and sum of tetrahydrogenated cortisol metabolites/free urinary cortisol). In patients with cirrhosis free urinary cortisol was normal, whereas the sum of cortisol metabolites was significantly reduced; therefore, cortisol synthesis seems to be adequately adapted to the decreased hepatic inactivation (conjugation, ring A-reduction). A significantly reduced ratio of cortisol metabolites to cortisone metabolites indicating impaired renal 11beta-hydroxysteroid dehydrogenase activity was only found in a subgroup of patients with ascites.

Direct determination of human urinary cortisol metabolites by HPLC/CRIMS.

Feasibility of using high performance liquid chromatographic input to the chemical reaction interface mass spectrometry system was assessed by measuring the profile of hydrolyzed urinary metabolites of [9,12,12-2H3] cortisol in six human subjects with no preparation other than hydrolysis and solid phase extraction. Relative amounts of tetrahydrocortisol, tetrahydrocortisone, and cortolones (as the sum of alpha- and beta-) were 0.417 +/- 0.047, 0.523 +/- 0.036 and 0.059 +/- 0.019, respectively. The constant reproducibility of the measurements coupled with a profile consistent with that observed by other workers shows that the technique represents an important tool in the determination of metabolites of endogenous molecules.

Role of 21-deoxyaldosterone in human hypertension.

21-Deoxyaldosterone has been postulated to be a precursor of aldosterone in an alternative biosynthesis pathway and Kelly's-M1 is considered to be its metabolite. In healthy volunteers, the excretion rate of 21-deoxyaldosterone and of Kelly's-M1 are significantly lower than the aldosterone metabolites, aldosterone-18-glucuronide and tetrahydro-aldosterone and than the aldosterone precursor 18-OH-corticosterone. Essential hypertension patients (with low and normal renin) excrete comparable values of 21-deoxyaldosterone and Kelly's-M1 as normotensives. In 66% of aldosterone-producing adenoma cases (APA) and in 60% of idiopathic hyperaldosteronism (IHA) patients, significantly raised values of 21-deoxyaldosterone and Kelly's-M1 were found. The patients with the high excretion rates of both steroids showed only moderately increased values of the aldosterone metabolites, aldosterone-18-glucuronide and tetrahydro-aldosterone, as well as of the aldosterone precursor 18-OH-corticosterone. In contrast, the latter mentioned steroids were excreted in higher amounts in those patients with normal excretion of 21-deoxyaldosterone and Kelly's-M1. Hence, it is suggested that aldosterone is produced alternatively either via 18-OH-corticosterone alone or additionally via 21-deoxyaldosterone. Furthermore, in three cases of "incidentally" discovered adrenal adenomas, 21-deoxyaldosterone and Kelly's-M1 were the only elevated steroids. After adrenalectomy, excretion of 21-deoxyaldosterone and of Kelly's-M1 and blood pressure returned to normal, which proves that these steroids play a role in blood pressure regulation. In essential hypertension, ACTH infusion induced a significant increase of 21-deoxyaldosterone and Kelly's-M1. However, the increase after angiotensin II was 3- to 6-fold higher than after ACTH. IHA patients proved to be more responsive to angiotensin II; and, in contrast, APA cases proved to be more sensitive to ACTH. The data suggest that beside the main route of aldosterone biosynthesis via 11-deoxycorticosterone, corticosterone and 18-OH-corticosterone an alternative pathway exists via 21-deoxyaldosterone in healthy and in hypertensive patients. There are similarities between the regulation of 21-deoxyaldosterone and the regulation of aldosterone. The determination of 21-deoxyaldosterone and its possible metabolite Kelly's-M1 might be appropriate in the diagnosis of mineralocorticoid-induced forms of hypertension, especially when an adrenal adenoma is discovered.

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.

Development of an isotope-selective high-performance liquid chromatography detector using chemical-reaction-interface mass spectrometry: application to deuterated cortisol metabolites in urine.

An isotope-selective detector for HPLC based on the particle-beam-interface and the chemical-reaction-interface mass spectrometer (CRIMS) principle is described. This paper focuses on the selective detection of deuterium-labeled analytes. The CRIMS product HD is detected as has been previously described for GC-CRIMS. The analytical performance was not affected by analyte structure or solvent composition. Deuterium detection was linear from 20 ng to 490 ng using 2H3-labeled cortisol. Based on this method, the fractional abundance of three labeled metabolites of cortisol were determined in the urine of a patient infused with tracer amounts of deuterated cortisol.

New identified 15 beta-hydroxylated 21-deoxy-pregnanes in congenital adrenal hyperplasia due to 21-hydroxylase deficiency.

The identification of 3 new 15 beta-hydroxylated 21-deoxy-pregnanes in the urinary steroid profile of a 4-month-old girl with congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency (21OHD) is reported here. These steroids were identified by gas chromatography and gas chromatography-mass spectrometry as 3 alpha,15 beta,17-trihydroxy-5 alpha-pregnan-20-one (5 alpha II), 3 alpha,15 beta,17,20 alpha-tetrahydroxy-5 alpha-pregnane, and 3 alpha,15 beta,17,20 alpha-tetrahydroxy-5 beta-pregnane (20 alpha DH-II). Two other compounds in the urine, 3 beta,15 beta,17- trihydroxy-5 alpha-pregnan-20-one and 3 beta,15 beta,17-trihydroxy-5 beta-pregnan-20-one were also characterized. The identification of the former 3 steroids was obtained by comparing their methylene unit values and mass spectral data with the corresponding data of the standard steroids synthesized from 15 beta,17-dihydroxy-4-pregnene-3,20-dione. Seven other synthesized and identified 15 beta-hydroxylated steroids were 3 alpha,15 beta,17-trihydroxy-5 beta-pregnan- 20-one (II), 3 alpha,15 beta,17,20 beta-tetrahydroxy-5 beta-pregnane, 15 beta,17-dihydroxy-5 alpha-pregnane-3,20-dione, 15 beta,17-dihydroxy-5 beta-pregnane-3,20-dione, 3 alpha,15 beta-dihydroxy-5 alpha-androstan-17-one (15 beta OH-An), 3 alpha,15 beta-dihydroxy-5 beta-androstan-17-one (15 beta OH-Et) and 3 alpha,15 beta,17,20 beta- tetrahydroxy-5 alpha-pregnane. Of these the latter two have not been reported previously. This study supports the findings that 15 beta-hydroxylated steroids are common in the neonate and could play an important role in the diagnosis of CAH due to 21OHD, where II and the newly identified steroids from this investigation viz., 5 alpha II and 20 alpha DH-II appear the most important 15 beta-hydroxysteroid markers for this disease.

Urinary excretion of 17-hydroxypregnanolones in patients with different forms of congenital adrenal hyperplasia due to steroid 21-hydroxylase deficiency.

To improve diagnostic criteria in different (classical salt-wasting (SW), classical simple virilizing (SV) and non classical late onset (LO)) forms of congenital adrenal hyperplasia (CAH) due to steroid 21-hydroxylase deficiency, we investigated the urinary excretion of 17-hydroxypregnanolones (17OH-PO(5 beta) and (5 alpha)), 15 beta-hydroxypregnanolone(15 beta OH-PO), pregnanetriol(PT) and 11-oxo-pregnanetriol (11-O-PT) compared to hydrocortisone metabolities. During the 1st month of life newborn infants with CAH-SW excreted from barely detectable to very large amounts of 17OH-PO(5 beta), 15 beta OH-PO and PT, and, in 12 of 14 cases, also 11-O-PT in their urines. From the 1st to the 28th day of life, cortisol metabolites were virtually absent in urines of CAH-SW infants. This was in contrast of 36 healthy newborn infants. We measured the excretion of 17OH-PO(5 alpha) in children with CAH of whom 19 patients with CAH-SV had a median 17OH-PO(5 alpha) excretion of 1110 micrograms/day (range: 152-5515). In 21 patients with CAH-LO, median excretion of 17OH-PO(5 alpha) was 294 micrograms/day (range: 66-1273). Besides the conventional metabolites of 17-hydroxyprogesterone (17OH-PO(5 beta), PT and 11-O-PT), no 17OH-PO(5 alpha) was detected in the urines of 14 patients with precocious pubarche, in 14 patients with virilization of unknown origin and in 94 healthy children of comparable age. The ratio of 17OH-PO(5 alpha) to tetrahydrocortisone (THE) discriminated between CAH-SV and CAH-LO from the 1st to the 18th year of age.(ABSTRACT TRUNCATED AT 250 WORDS)