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.

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.

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.

Metabolism of adrenal cholesterol in man. I. In vivo studies.

The kinetics of plasma and adrenal cholesteral equilibration were analyzed in patients undergoing bilateral adrenalectomy for generalized mammary carcinoma. A biological model is proposed to help in the understanding of adrenal cholesterol physiology. It comprises two intracellular compartments: (1) A compartment of free adrenal cholesterol which is small (of the order of 17 mg) but turns over very fast; it is renewed approximately 8 times per day: 3 times by the inflow of free plasma cholesterol, and 5 times by the hydrolysis of esterified adrenal cholesterol, the contribution of adrenal cholesterol synthesis appearing to be relatively small. (2) A compartment of esterified adrenal cholesterol which is 20 times larger; it is constantly renewed by in situ esterification and hydrolysis with a daily fractional turnover rate of the order of 0.25. The direct and selective accumulation of plasma cholesteryl esters is practically absent. Only free adrenal cholesterol returns to plasma, mostly after conversion into steroid "hormones."However small the synthesis of adrenal cholesterol may be, it seems more important in the zona "reticularis." On the other hand, the inflow of plasma cholesterol and the turnover of the free adrenal compartment tend to be faster in the zona "fasciculata." The equilibration of plasma and adrenal cholesterol can proceed unmodified under conditions of ACTH suppression. In one patient with Cushing's disease the size of the two adrenal compartments was clearly increased but their equilibration with plasma cholesterol proceeded normally. In another patient the kinetics of hydrocortisone corresponded to those of free adrenal cholesterol in the control studies.

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.

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.

Formation of steroids by the pregnant mare. V. Metabolism of 14C-isopentenylpyrophosphate and 3H-dehydroisoandrosterone injected into the fetus.

A mixture of 1-14C-isopentenylpyrophosphate and 3H-dehydroisoandrosterone was injected into a horse fetus intramuscularly during laparotomy, after which maternal urine was collected for 4 days. Steroid conjugates in the urine were extracted with Amberlite XAD-2 resin, hydrolysed and separated into phenolic and neutral fractions. From the phenolic fraction estrone, 17alpha-estradiol, equilin and equilenin were isolated. Only estrone and 17alpha-estradiol contained both 3H and 14C, while the ring B unsaturated estrogens contained only 14C. From the neutral fraction 14C-labeled 3beta-hydroxy-5alpha-pregnan-20-one, 5alpha-pregnane-3beta,20beta-diol and 5alpha-pregnan-3beta, 20alpha-diol were isolated. These results demonstrate that the route of biosynthesis of both the ring B saturated and unsaturated estrogens is the same up to the stage of isopentenylpyrophosphate. Thus, the bifurcation in the classical pathway of steroid biosynthesis reported previously by us is occurring at a point after the formation of isopentenylpyrophosphate and prior to the formation of squalene.

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.

Urinary steroid metabolites and the overgrowth of lean and fat tissues in obese girls.

Studies were made of steroid metabolites excreted in the urine of 17 obese girls 11.4 to 16.8 yr and 17 normal girls 11 to 17 yr. Creatinine excretion (muscle mass), total body water (or deuterium space), lean body mass and body fat were determined in the obese girls. Extracellular volume (corrected bromide space) was also measured and by difference with body water, intracellular water or soft tissue cell mass was calculated. In normal girls 24-h creatinine excretion was determined, but body water was predicted from height and weight. It was found, as in previous studies, that the obese girls had excess muscle mass and soft tissue cell mass for height. The excess growth of muscle, lean tissue, and body length in obese girls correlated with increments in oxosteroid (17 ketosteroid) excretion. The overall weight increase correlated with increased excretion of corticosteroid metabolites--a finding of interest since a physiological Cushing's syndrome was postulated for fat girls many years ago. When the normal and obese girls were divided by age at 14 yr and the subgroups compared (normal obese) the younger girls showed differences with respect to height, weight, total body water, fat and percentage fat. Differences in steroid metabolites were not found. In older girls the same findings were made again, but here it was clear that the increments in body size, particularly muscle mass, correlated with augmented oxosteroid excretion. Evidence is cited that these findings are not just related to a larger steroid pool in obese girls.

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.

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.

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.

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.

The early recognition of the 21-hydroxylase deficiency variety of congenital adrenal hyperplasia.

The urinary steroids excreted by three newborn infants with 21-hydroxylase deficiency and by 15 healthy newborns aged two days have been compared after analysis by gas liquid chromatography (GLC). The identity of each steroid was carefully checked by gas chromatography/mass spectroscopy (GC-MS). The enzyme deficiency leads to the elevated excretion of urinary precursor metabolites, mainly 3alpha,17alpha,20alpha-trihydroxy-5beta-pregnan, 3alpha,17alpha,20alpha-trihydroxy-5beta-pregnan-11-one and 3alpha,17alpha-dihydroxy-5beta-pregnan-20-one. In the search for a quick and firm confirmation of suspected 21-hydroxylase deficiency in a newborn baby by means of a GLC-profile of urinary steroids, most attention has up to now been paid to 3alpha,17alpha,20alpha-trihydroxy-5beta-pregnan. However, 3alpha,17alpha-dihydroxy-5beta-pregnan-20-one is a better indicator, as it enables one to confirm the existence of this disease soon after birth directly from the GLC-profile without further analyses by GC-MS.

The diagnosis of 21-hydroxylase deficiency in a prematurely born infant on the basis of the urinary steroid excretion pattern.

The urine of a 6-day-old prematurely born female infant (birth weight 1060 g) suspected of having a 21-OH-deficiency showed no steroid abnormalities on capillary GLC analysis. Using GC-MS tetrahydrocortisone (THE) and also 3 alpha, 17 alpha-dihydroxy-5 beta-pregnane-20-one (17-OH-Polone) were absent, but two androstanetriolone peaks were observed. In the urine collected on day 9 THE was absent, but a large amount of 3 alpha, 11 beta-dihydroxy-5-alpha-androstane-17-one (11-HA) was found by GC-MS to be contaminated by a small amount of 17-OH-Polone. The next urine specimen collected on the 22nd day while the child received cortisol therapeutically showed the characteristic steroid profile for the diagnosis 21-OH deficiency, large peaks of 17-OH-Polone, pregnanetriol (P3) and 11-keto-pregnanetriol (11-keto-P3). Over the next few weeks two other compounds were found to have been excreted in relatively large amounts, 3 xi, 16 xi, 17 xi, 20 xi-pregnanetetrol (16-OH-P3) and surprisingly also a 21-hydroxylated compound, namely 3 xi, 20 alpha, 21-trihydroxy-5-pregnene. These same two compounds were also found in the urine of another infant with suspected 21-OH deficiency. The urinary steroid excretion patterns characteristic for 21-OH deficiency are dependent on the maturity and age of the infant. In the prematurely born infant androstanetriolones appear in the urine before 17-OH-Polone. The occurrence of these different steroid excretion patterns is tentatively explained.

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).

The identification and quantification of three new 6alpha-hydroxylated corticosteroids in human neonatal urine.

The 24 hours urines of six two days old fullterm newborn infants were investigated for polar corticosteroids. 6alpha-hydroxy-tetrahydrocortisone, 6alpha-hydroxy-20alpha-cortolone and 6alpha-hydroxy-20beta-cortolone were identified by gas chromatographic-mass spectrometric comparison of the urinary steroids to compounds synthesized previously. These 6alpha-hydroxylated corticosteroids as well as seven other polar corticosteroids were quantified by gas chromatography or mass fragmentography. It was shown that the newly identified steroids constituted a quantitatively important part of the neonatal urinary corticosteroids. The unconjugated- and glucuronic acid conjugated steroids were quantified separately. It was found that the extent of glucuronoconjugation decreased with increasing polarity of the steroid moiety.

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.

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.