Search

Quantification of glucocorticoids and their metabolites as a central component of the physiological stress response has many potential applications in ornithological research, but require careful species-specific validations. We established a reliable non-invasive method to measure faecal glucocorticoid metabolites (fGCM) in the southern yellow-billed hornbill (Tockus leucomelas). Following an adrenocorticotropic hormone (ACTH) challenge, plasma corticosterone levels increased by 110%, with a tetrahydrocorticosterone enzyme immunoassay (EIA) revealing a ~ fourfold increase in fGCM concentrations approximately 3 h post-injection. These data reveal droppings are a suitable matrix for monitoring stress-associated biomarkers in this species and suggest the tetrahydrocorticosterone EIA may often be appropriate for wild birds.

Steroids that contain a 3-hydroxyl group (3-OH steroids) are widely distributed in nature. During analysis with ESI-MS, they easily become dehydrated while in the protonated form, resulting in the production of several precursor ions and leading to low sensitivity of detection. To address this analytical challenge, here, we developed a method for the quantitation of 3-OH steroids by LC-MS/MS coupled with post-column addition of lithium (Li) ions to the mobile phase. The Li ion has a high affinity for the keto group of steroids, stabilizing their structures during ionization and permitting detection of analytes exclusively as the lithiated form. This not only improved the intensities of the precursor ions, but also promoted the formation of typical lithiated fragment ions. This improvement made the quantitation by multiple reaction monitoring more sensitive and reliable, as evidenced by 1.53–188 times enhanced detection sensitivity of 13 steroids that contained at least one keto and two hydroxyl groups or one keto and one 5-olefinic double bond, among 16 different 3-OH steroids. We deployed our newly developed method for profiling steroids in mouse brain tissue and identified six steroids in one tissue sample. Among these, 16-hydroxyestrone, tetrahydrocorticosterone, and 17α-hydroxypregnenolone were detected for the first time in the mouse brain. In summary, the method described here enables the detection of lithiated steroids by LC-MS/MS, including three 3-OH steroids not previously reported in the mouse brain. We anticipate that this new method may allow the determination of 3-OH steroids in different brain regions.

Simple Summary Faecal cortisol metabolites (FCMs) are a useful, non-invasive tool for the assessment of stress in koalas. However, FCM stability after defecation is a critical issue. Therefore, we exposed faecal pellets of koalas to three different environmental conditions and measured FCMs with three recently evaluated enzyme immunoassays (EIAs). Because water loss over time exerted the greatest influence on FCMs, we strongly recommend collecting freshly defecated pellets in koalas. Abstract Faecal material can be a valuable source of information for a range of animal health aspects and can be used to measure faecal cortisol metabolites (FCMs). FCM values can relate to physiological stress responses. However, freshly defecated pellets are not always available and environmental conditions, such as temperature and humidity, might affect faecal pellet consistency and FCM levels. Therefore, the impact of environmental conditions on FCMs needs to be evaluated. We collected 107 pellets from two female and two male koalas, exposed them to three types of treatments, and analysed FCMs in these samples with three enzyme immunoassays (EIAs). After analysis, the original FCM values were mathematically corrected for water loss. Results show that the FCMs were more stable when measured using tetrahydrocorticosterone (50c) and 5α-pregnane-3β,11β,21-triol-20-one (37e) EIAs, and were less stable when measured with the cortisol EIA. With 50c, the FCM values did not vary significantly over time either before or after the adjustment with water in the environment treatment group. For samples kept under constant low (25 °C) and high (35 °C) temperatures, the 50c FCM values did not vary significantly over time, after adjustments were made for water loss. Thus, this study highlights the importance of considering the suitability of faecal field samples for FCM analysis. Because water loss was the main driver of FCM changes, we strongly recommend collecting koala pellets that are freshly defecated, despite the effort and time it might take to collect such pellets.

The koala (Phascolarctos cinereus) is an arboreal folivorous marsupial endemic to Australia. Anthropogenic activities and climate change are threats to this species’ survival and are potential stressors. A suitable non-invasive method is needed to objectively detect stress in koalas. Under conditions of stress, the concentration of the hormone cortisol in plasma or in saliva is elevated, and this would provide a convenient measure, however, collecting blood or saliva from wild animals is both practically difficult and stressful, and so likely to confound any measurement. In contrast, measurement of cortisol metabolites in faeces provides a practical and non-invasive method to objectively measure stress in koalas. Unfortunately, the identity of the main faecal cortisol metabolites of koalas is unknown. In this study, we have used both untargeted liquid chromatography–mass spectrometry (LC-MS) and enzyme immunoassays (EIAs) to identify several faecal cortisol metabolites in two koalas, one female (18 months old, 4.1 kg) and one male (4 years old, 6.95 kg) upon administration of hydrocortisone (cortisol) sodium succinate. The LC-MS analysis identified tetrahydrocortisol along with several other isomers as cortisol metabolites. After a survey of five enzyme immunoassays, we found that two metabolites, tetrahydrocortisol and 3β-allotetrahydrocortisol, could be detected by EIAs that used antibodies that were raised against their structurally similar corticosterone counterparts, tetrahydrocorticosterone and 3β-allotetrahydrocorticosterone, respectively. While the 3β-allotetrahydrocortisol metabolite was detected in the faeces of only one of the two animals studied, tetrahydrocortisol was detected in both. These results ultimately indicate that tetrahydrocortisol is likely the main faecal cortisol metabolite in koalas, and we demonstrate that it can be measured by an EIA (50c) that was originally developed to measure tetrahydrocorticosterone.

Simple Summary In the present study, we performed an untargeted metabolomic analysis of skeletal muscle of rabbits and found that the skeletal muscle of rabbits fed a high-fat diet is rich in many metabolites, most of which are associated with type 2 diabetes and metabolic syndrome. In this paper, the mechanism of action of these metabolites in skeletal muscle and the metabolic pathways that interfere with the normal operation mechanism of the body are described and presented in the form of charts. Finally, we found that skeletal muscle-rich phospholipids, long-chain carnitine, histidine, carnosine, and tetrahydrocortisone may be potential markers for type 2 diabetes and metabolic syndrome, and may serve as potential therapeutic targets for related diseases in the future. Abstract Type 2 diabetes and metabolic syndrome caused by a high fat diet (HFD) have become public health problems worldwide. These diseases are characterized by the oxidation of skeletal muscle mitochondria and disruption of insulin resistance, but the mechanisms are not well understood. Therefore, this study aims to reveal how high-fat diet causes skeletal muscle metabolic disorders. In total, 16 weaned rabbits were randomly divided into two groups, one group was fed a standard normal diet (SND) and the other group was fed a high fat diet (HFD) for 5 weeks. At the end of the five-week experiment, skeletal muscle tissue samples were taken from each rabbit. Untargeted metabolomic analysis was performed using ultra-performance liquid chromatography combined with mass spectrometry (UHPLC-MS/MS). The results showed that high fat diet significantly altered the expression levels of phospholipids, LCACs, histidine, carnosine, and tetrahydrocorticosterone in skeletal muscle. Principal component analysis (PCA) and least squares discriminant analysis (PLS-DA) showed that, compared with the SND group, skeletal muscle metabolism in HFD group was significantly up-regulated. Among 43 skeletal muscle metabolites in the HFD group, phospholipids, LCACs, histidine, carnosine, and tetrahydrocorticosteroids were identified as biomarkers of skeletal muscle metabolic diseases, and may become potential physiological targets of related diseases in the future. Untargeted metabonomics analysis showed that high-fat diet altered the metabolism of phospholipids, carnitine, amino acids and steroids in skeletal muscle of rabbits. Notably, phospholipids, LCACs, histidine, carnopeptide, and tetrahydrocorticosteroids block the oxidative capacity of mitochondria and disrupt the oxidative capacity of glucose and the fatty acid-glucose cycle in rabbit skeletal muscle.

The article presents the analysis of renin–angiotensin system and content precursor of aldosterone and their metabolites, investigated by methods of high-performance liquid chromatography and gas chromatography – mass-spectrometry in 49 patients with pheochromocytoma (РНЕО) and 28 patients with non-active adenoma of the adrenal cortex and 26 healthy people. In the group of patients with PHEO the increased blood levels оf aldosterone and urine excretion of 18-hydroxycorticosterone, tetrahydrocorticosterone and it 5-α form were obtained. These patients had features of hyporeninemic and hyperreninemic hyperaldosteronism. Positive correlations of urinary excretion of free normetanephrine with blood aldosterone, renin, corticosterone and 18-hydroxycorticosterone suggest the relationship between adrenal cortex and medulla.

Liver fibrosis is a common stage in the majority of chronic liver diseases, regardless of the etiology, and its progression may lead to hepatic cirrhosis or hepatocellular carcinoma. Metabolomics, a powerful approach in systems biology, is a discipline used to qualitatively and quantitatively analyze the small molecule metabolites of cells at specific times and under certain conditions. The present study aimed to investigate serum metabolic changes following Shu Gan Jian Pi formula (SGJPF) treatment of carbon tetrachloride (CCl4)-induced liver fibrosis in rats using gas chromatography-time of flight mass spectrometry (GC-TOFMS). In addition, the potential mechanisms were explored. Rat liver fibrosis was induced by twice-weekly subcutaneous CCl4 injection for 12 continuous weeks. During the same period, the SGJPF group received 16.2 g/kg body weight SGJPF, diluted in water, once a day for 12 weeks. Rats in the control and model groups received oral administration of the same volume of saline solution. Serum samples from the control, model and SGJPF groups were collected after 12 weeks of treatment, and metabolic profile alterations were analyzed by GC-TOF/MS. Metabolic profile analysis indicated that clustering differed between the three groups and the following 12 metabolites were detected in the serum of all three groups: Isoleucine; L-malic acid; D-erythro-sphingosine; putrescine; malonic acid; 3,6-anhydro-D-galactose, α-ketoglutaric acid; ornithine; glucose; hippuric acid; tetrahydrocorticosterone; and fucose. The results demonstrated that SGJPF treatment mitigated the effects of CCl4-induced liver fibrosis on biomarker levels, thus indicating that SGJPF may have a therapeutic effect on CCl4-induced liver fibrosis in rats. The mechanism may involve the regulation of energy, amino acid, sphingolipid, cytochrome P450, glucose and water-electrolyte metabolism.

Developing non-invasive techniques for monitoring physiological stress responses has been conducted in a number of mammal and bird species, revolutionizing field-based endocrinology and conservation practices. However, studies validating and monitoring glucocorticoid concentrations in reptiles are still limited. The aim of the study was to validate a method for monitoring glucocorticoid metabolite concentrations in urine (uGCM) and faeces (fGCM) of the cordylid lizard, the Sungazer (Smaug giganteus). An adrenocorticotropic hormone (ACTH) challenge was conducted on one male and two females with both urine and faecal material being collected during baseline and post-injection periods. Steroid extracts were analysed with four enzyme immunoassays (EIAs)namely: 11-oxoaetiocholanolone, 5α-pregnane-3β-11β-21-triol-20-one, tetrahydrocorticosterone, and corticosterone. A considerable response in fGCM and uGCM concentrations following ACTH administration was observed in all subjects, with the 5α-pregnane-3β-11β-21-triol-20-one and tetrahydrocorticosterone EIAs appearing to be the most suited for monitoring alterations in glucocorticoid metabolite concentrations in S. giganteus using faeces or urine as hormone matrix. Both EIAs showed a significantly higher concentration of glucocorticoid metabolites in faeces compared to urine for both sexes. Collectively, the findings of this study confirmed that both urine and faeces can be used to non-invasively assess adrenocortical function in S. giganteus.

Urinary steroid profiling (USP) was studied using high-performance liquid chromatography (HPLC) and gas chromatography-mass spectrometry (GC-MS) methods in 108 patients with adrenocortical adenoma (ACA) and in 31 patients with adrenocortical carcinoma (ACC). Thirteen ACC and Cushing's syndrome (ACC-CS) patients had two types of USP as well as 18 ACC patients without hypercortisolism. These four types differed by androgen and glucocorticoid secretion of the adrenal cortex. Fifteen main ACC features were observed by GC-MS. Urinary excretion of dehydroepiandrosterone (DHEA) was increased in 67.7 % of ACC patients and tetrahydro-11-deoxycortisol (THS) in 74.2 %. By combination of the following parameters: THS >900 μg/24 h and/or DHEA >1500 μg/24 h with ratios of 3α,16,20-pregnentriol/3β,16,20-pregnentriol (3α,16,20dP3/3β,16,20dP3) less than 6.0 and 3α,17,20dP3/3β,17,20dP3 less than 9.0 and the detection of "non-classical" 5-en-pregnens, not found in ACA and healthy persons, 100 % sensitivity and specificity of ACC and ACA differential diagnosis were achieved. Features of 21-hydroxylase and 11β-hydroxylase deficiency were observed by GC-MS in 32.2 and 61.3 % of the ACC patients, respectively. Additional features for ACC-CS diagnostic were increased urinary excretion of 6β-hydroxycortisol, 18-hydroxycorticosterone, the sum (UFF + UFE) obtained by HPLC, tetrahydrocorticosterone, and the sum (THF + THE + allo-THF) obtained by GC-MS.

The intracellular availability of glucocorticoids is regulated by the enzymes 11β-hydroxysteroid dehydrogenase 1 (HSD11B1) and 11β-hydroxysteroid dehydrogenase 2 (HSD11B2). The activity of HSD11B1 is measured in the urine based on the (tetrahydrocortisol+5α-tetrahydrocortisol)/tetrahydrocortisone ((THF+5α-THF)/THE) ratio in humans and the (tetrahydrocorticosterone+5α-tetrahydrocorticosterone)/tetrahydrodehydrocorticosterone ((THB+5α-THB)/THA) ratio in mice. The cortisol/cortisone (F/E) ratio in humans and the corticosterone/11-dehydrocorticosterone (B/A) ratio in mice are markers of the activity of HSD11B2.In vitroagonist treatment of liver X receptor (LXR) down-regulates the activity of HSD11B1. Sterol 27-hydroxylase (CYP27A1) catalyses the first step in the alternative pathway of bile acid synthesis by hydroxylating cholesterol to 27-hydroxycholesterol (27-OHC). Since 27-OHC is a natural ligand for LXR, we hypothesised thatCYP27A1deficiency may up-regulate the activity of HSD11B1. In a patient with cerebrotendinous xanthomatosis carrying a loss-of-function mutation inCYP27A1, the plasma concentrations of 27-OHC were dramatically reduced (3.8 vs 90–140 ng/ml in healthy controls) and the urinary ratios of (THF+5α-THF)/THE and F/E were increased, demonstrating enhanced HSD11B1 and diminished HSD11B2 activities. Similarly, inCyp27a1knockout (KO) mice, the plasma concentrations of 27-OHC were undetectable (Cyp27a1WT mice). The urinary ratio of (THB+5α-THB)/THA was fourfold and that of B/A was twofold higher in KO mice than in their WT littermates. The (THB+5α-THB)/THA ratio was also significantly increased in the plasma, liver and kidney of KO mice. In the liver of these mice, the increase in the concentrations of active glucocorticoids was due to increased liver weight as a consequence ofCyp27a1deficiency.In vitro, 27-OHC acts as an inhibitor of the activity of HSD11B1. Our studies suggest that the expression of CYP27A1 modulates the concentrations of active glucocorticoids in both humans and mice andin vitro.

This study describes the development of a method suitable for the analysis of nineteen major urinary steroid metabolites in human urine. The analytes of interest were isolated from urine using solid phase extraction, subjected to enzymatic hydrolysis and again extracted applying solid phase extraction. After derivatization, methyloxime-trimethylsilyl ether derivatives of steroid hormones were identified by gas chromatography-mass spectrometry (GC/MS) and quantified by gas chromatography with flame ionization detector (GC/FID). The quantification method was validated for linearity, trueness, precision and selectivity. The limits of detection were between 6.2 and 7.2 ng/mL and limits of quantification were between 12.3 and 14.8 ng/mL. The established method was applied to analyze 28 urine samples from patients diagnosed with non-functioning adrenal incidentalomas (AIs) and 30 healthy subjects. Hierarchical cluster analysis (HCA) and principal component analysis (PCA) were employed to visualize the differences between metabolic profiles of patients and the control group and to determine possible markers of AIs activity. Both multivariate methods separated seven patients from the rest of the examined individuals. Five urinary metabolites including α-cortol, tetrahydrocorticosterone, tetrahydrocortisol, allo-tetrahydrocortisol and etiocholanolone were identified as potential biomarkers of pathological adrenal function. The altered metabolites reflected pathological metabolism mainly of cortisol and cortisone. This research proved that metabolomics is a suitable tool for disease research.

Objective The aim of this study was to investigate the possible urinary markers of hormonal activity in patients with non-functioning adrenal incidentalomas. In order to evaluate the endocrine activity of forementioned tumours, urinary steroid metabolite levels were analyzed in samples from patients and controls. Possible blocks in metabolic pathways of the examined hormones were determined by comparing selected urinary steroid metabolite sums and ratios in both groups of interest. Design Urine samples were collected from 20 patients with non-functioning adrenal incidentalomas and from 25 controls matched in terms of age, sex and BMI. Excretion of 19 major urinary steroid metabolites was analyzed by gas chromatography. The results were subjected to statistical analysis. Results In patients with adrenal incidentalomas sum of total urinary cortisol metabolites was significantly increased in respect to the control group. We also observed a shift towards tetrahydrocorticosterone, cortisol and etiocholanolone production in patients. No significant differences in production of other urinary steroid metabolites were noted in patients with adrenal incidentalomas in respect to control group. Conclusions Our data suggests that not only urinary free cortisol but also its metabolite such as tetrahydrocortisol and other steroids including etiocholanolone and corticosterone tetrahydrometabolite might be urinary markers for the endocrine activity of adrenal incidentalomas. Enhanced levels of these urinary steroid metabolites indicate an impairment of 11β-hydroxysteroid dehydrogenase activity and slightly increased activity of 5β-reductase in patients with adrenal incidentalomas.

Data are presented on the urinary corticosteroid metabolic profile of the mouse strain 129/svJ. Through the use of GC/MS we have characterized, or tentatively identified corticosterone (Kendall's compound B) metabolites of both the 11beta-hydroxy and 11-carbonyl (compound A) series in urine. Full mass spectra of the methyloxime-trimethylether derivatives of 15 metabolites are included in the paper as an aid to other researchers in the field. Metabolites ranged in polarity from tetrahydrocorticosterone (THB) to dihydroxy-corticosterone with dominance of highly polar steroids. We found that prior to excretion corticosterone can undergo oxidation at position 11beta, reduction at position 20 and A-ring reduction. Metabolites retaining the 3-oxo-4-ene structure can be hydroxylated at position 6beta- as well as at an unidentified position, probably 16alpha-. Saturated steroids can be hydroxylated at positions 1beta-, 6alpha-, 15alpha- and 16alpha. A pair of hydroxy-20-dihydro-corticosterone metabolites (OH-DHB) were the most important excretory products accounting for about 40% of the total. One metabolite of this type was identified as 6beta-hydroxy-DHB; the other, of similar quantitative importance was probably 16alpha-hydroxy-DHB. The ratio of metabolites of corticosterone (B) to those of 11-dehydro-corticosterone (A) was greater than 9:1, considerably higher than that for the equivalent "human" ratio of 1:1 for cortisol to cortisone metabolites. Results from this study allowed the evaluation of 11beta-hydroxysteroid dehydrogenase (11beta-HSD) activity in mice with deleted glucose-6-phosphate transporter (G6PT). These mice had attenuated back-conversion of A to B resulting in an increased ratio of A-metabolites to B-metabolites [Walker EA, Ahmed A, Lavery GG, Tomlinson JW, Kim SY, Cooper MS, Stewart PM, 11beta-Hydroxysteroid dehydrogenase type 1 regulation by intracellular glucose-6-phosphate, provides evidence for a novel link between glucose metabolism and HPA axis function. J Biol Chem 2007;282:27030-6]. We believe this study is currently the most comprehensive on the urinary steroid metabolic profile of the mouse. Quantitatively less steroid is excreted in urine than in feces by this species but urine analysis is more straightforward and the hepatic metabolites are less subject to microbial degradation than if feces was analyzed.

The identification and quantification of tetrahydrocorticosterone isomers (THBs; 3alpha,5alpha-, 3beta,5alpha-, 3alpha,5beta- and 3beta,5beta-THB) in rat brains using liquid chromatography (LC)-mass spectrometry (MS) are described. For the identification, the THBs were converted to the atmospheric pressure chemical ionization (APCI)-active derivatives, i.e., the dinitrobezoyl esters and 2-nitro-4-trifluoromethylphenyl hydrazones, and detected in the negative-ion mode. These derivatives showed 60- and 40-fold higher sensitivities, respectively, than intact steroids measured in the positive-APCI-MS. The derivatized THBs were satisfactorily separated from the others during the reversed-phase LC. The THBs were not detected at all in the brains of the unstressed rats. When the rats were exposed to the immobilization for 20 min, 3alpha,5alpha- and 3beta,5alpha-THB were detected as the major metabolites together with small amounts of 3alpha,5beta- and 3beta,5beta-THB in the male rat brain, while only 3alpha,5alpha-THB was detected in the female rats. Thus, the steroid variety found in the brains was different between the sexes. In the next step, 3alpha,5alpha-THB, a major metabolite found in the brains of the stressed rats, was quantified as its dinitrobezoyl ester. This method was accurate and reproducible, and the limit of quantitation was 1.0 ng/g tissue when a 50 mg tissue sample was used. There was also a sex difference in the brain 3alpha,5alpha-THB level; it was significantly higher in the female rats than in the male rats (P0.05), although the brain corticosterone level was not higher in the stressed female rats than in the male rats (no statistical difference).

When resources are limited, life-history theory predicts that long-lived animals should allocate available resources to body maintenance rather than to reproduction in order to maximise their lifetime reproductive success. In the present study, we estimated physiological stress in a small procellariiform seabird, the Wilson's storm-petrel Oceanites oceanicus, as a means of understanding how limited resources are partitioned between provisioning parents and their chicks. We analysed adrenocortical activity of Wilson's storm-petrels during the breeding season by measuring glucocorticoid (GC) excretion, using an enzyme immunoassay measuring tetrahydrocorticosterone concentrations in extracts of faeces and urine of chicks and adults. Faecal GC measures were negatively correlated with chick body condition, suggesting that measures of tetrahydrocorticosterone in faeces and urine can be used to assess adrenal activity characteristic for physiological stress in Wilson's storm-petrels. In the breeding season of 1999, the colony was subject to low food availability, and the faecal and urine GC levels of chicks were elevated during these months of chronic starvation. In contrast, adults did not show elevated GC levels. The data thus suggest that Wilson's storm-petrels respond to unfavourable conditions by maintaining their own body condition and reducing provisioning of food to their chicks.

Metabolomics techniques are the comprehensive assessment of endogenous metabolites in a biological system and may provide additional insight into the molecular mechanisms. Er-Zhi-Wan (EZW) is a traditional Chinese medicine formula, which contains Fructus Ligustri Lucidi (FLL) and Herba Ecliptae (HE). EZW is widely used to prevent and treat various liver injuries through the nourishment of the liver. However, the precise molecular mechanism of hepatoprotective effects has not been comprehensively explored. Here, an integrated metabolomics strategy was designed to assess the effects and possible mechanisms of EZW against carbon tetrachloride-induced liver injury, a commonly used model of both acute and chronic liver intoxication. High-performance chromatography/quadrupole time-of-flight mass spectrometry (HPLC/QTOF-MS) combined with chemometric approaches including principal component analysis (PCA) and partial least squares-discriminant analysis (PLS-DA) were used to discover differentiating metabolites in metabolomics data of rat plasma and urine. Results indicate six differentiating metabolites, tryptophan, sphinganine, tetrahydrocorticosterone, pipecolic acid, l-2-amino-3-oxobutanoic acid and phosphoribosyl pyrophosphate, in the positive mode. Functional pathway analysis revealed that the alterations in these metabolites were associated with tryptophan metabolism, sphingolipid metabolism, steroid hormone biosynthesis, lysine degradation, glycine, serine and threonine metabolism, and pentose phosphate pathway. Of note, EZW has a potential pharmacological effect, which might be through regulating multiple perturbed pathways to the normal state. Our findings also showed that the robust integrated metabolomics techniques are promising for identifying more biomarkers and pathways and helping to clarify the function mechanisms of traditional Chinese medicine.

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

To monitor adrenocortical activity in Adelie penguins (Pygoscelis adeliae), we validated an enzyme immunoassay (EIA) for faecal glucocorticoid (GC) metabolites. An adrenocorticotropin hormone (ACTH) challenge was conducted on a paired female and male. The EIA for tetrahydrocorticosterone showed a clear response to the ACTH challenge in both birds. After high-performance liquid chromatography using pooled samples generated from the ACTH challenge, and analysing each individual fraction, three immunoreactive peaks were detected. Both biological and chemical validations strongly suggest that the EIA can be a useful tool for non-invasively measuring GC metabolites in faeces of Adelie penguins.

Renal allograft donors are at risk of developing hypertension. Here, we hypothesized that this risk is at least in part explained by an enhanced intracellular availability of 11β-hydroxyglucocorticoids due to an increased 11β-hydroxysteroid dehydrogenase type 1 enzyme (11β-HSD1), an intracellular prereceptor activator of biologically inactive 11-ketocorticosteroids in the liver, and/or a diminished 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2), an inactivator of 11β-hydroxyglucocorticoids in the kidney. To test this hypothesis, uninephrectomized (UNX) (n=9) and sham-operated (n=10) adult Sprague–Dawley rats were investigated. Mean arterial blood pressure and heart rate were measured continuously by telemetry for 6 days in week 5 after UNX. The mRNA of11β-Hsd1and11β-Hsd2in liver and kidney tissues were assessed by RT-PCR and the 11β-HSD activities were directly quantified in their corresponding tissues by determining the ratios of (tetrahydrocorticosterone+5α-tetrahydrocorticosterone)/tetrahydrodehydrocorticosterone ((THB+5α-THB)/THA) and of corticosterone/dehydrocorticosterone (B/A) by gas chromatography–mass spectrometry. The apparent total body activities of 11β-HSD1 and 11β-HSD2 were estimated using the urinary and plasma ratios of (THB+5α-THB)/THA and B/A. Mean arterial blood pressure was increased after UNX when compared with sham operation. Hepatic mRNA content of11β-Hsd1and hepatic, plasma, and urinary ratios of (THB+5α-THB)/THA were decreased after UNX, indicating diminished access of glucocorticoids to its receptors. In renal tissue,11β-Hsd2mRNA was reduced and B/A ratios measured in kidney, plasma, and urine were increased, indicating reduced 11β-HSD2 activity and enhanced access of glucocorticoids to mineralocorticoid receptors. Both 11β-HSD1 and 11β-HSD2 are downregulated after UNX in rats, a constellation considered to induce hypertension.

[1,2,3,4-C-13]cortisol was i.v. administered to two sisters aged 11 yr (patient I) and 3 yr (patient II) who suffer from 17-alpha-hydroxylase deficiency. This is the first time that the cortisol production rate (CPR) in patients with 17-alpha-hydroxylase deficiency has been measured with a stable labelled tracer using the urinary method. The urine was collected for 3 days. High-performance liquid chromatography (HPLC) of approximately 100 ml urine extracts was carried out to isolate the small amount of cortisol metabolites excreted. The cortisol metabolites were oxidized to 11-oxo-aetiocholanolone. The isotope dilution in the methyl oxime tert-butyldimethylsilyl ether derivatives was measured by selected ion monitoring gas chromatography/mass spectrometry (GC/MS). The CPR calculated from tetrahydrocortisone (THE) and the cortolones was 765 and 536 nmol/day, respectively in patient I. The CPR in patient II was only calculated from THE and was 62 nmol/day. If radioactive labelled cortisol had been used, much larger quantities of urine would have been needed for isolation of sufficient mass of metabolites, even then purification may have been difficult. Steroid profiling of 1 ml urine samples by GC and identification by GC/MS revealed high concentrations of pregnenolone, progesterone, 11-beta-hydroxy progesterone and corticosterone metabolites. Tetrahydrocorticosterone and 5-alpha-tetrahydrocorticosterone were found in urine at elevated excretions of 2.5 and 5.7, 0.9 and 2.0-mu-mol/24 h, in patients I and II respectively. No cortisol metabolites were detected by routine GC or GC/MS as the low amounts excreted co-eluted with the relatively abundant corticosterone metabolites.

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 11β-hydroxysteroid dehydrogenase (11β-HSD) enzyme was calculated. Results: The levels of etiocholanolone (E) (p

Background. The disease state of the nephrotic syndrome is characterized by abnormal renal sodium retention that cannot be completely explained by a secondary hyperaldosteronism for the following reasons. Firstly, in rats an enhanced sodium retention is observed before proteinuria with intravascular volume depletion occurs. Secondly, in patients with the nephrotic syndrome, volume expansion with hypertension has been reported despite suppression of the renin-aldosterone system. Therefore, another mechanism for sodium retention must be postulated for this disease state. We hypothesize that this mechanism is a reduced 11b-hydroxysteroid dehydrogenase 2 (11b-HSD2) activity, a phenomenon known to cause enhanced access of cortisol or corticosterone to the mineralocorticoid receptor. Methods. We assessed the 11b-HSD activity by measuring the urinary ratio of tetrahydrocorticosterone (THB) plus 5a-tetrahydrocorticosterone (5a-THB) to 11-dehydro-tetrahydrocorticosterone (THA) by gas chromatography–mass spectrometry in rats with puromycin aminonucleoside (PAN)-induced proteinuria and with adriamycin nephrosis. Furthermore, the plasma ratios of corticosterone to 11-dehydrocorticosterone were measured. Results. The urinary ratio of (THBq5a-THB)uTHA increased in all animals following injection of PAN or adriamycin, indicating a reduced activity of 11b-HSD. The reduced activity of 11b-HSD was confirmed by an increased plasma ratio of corticosterone to 11-dehydrocorticosterone. The changes in the glucocorticoid metabolite ratios were already present before significant proteinuria appeared. Conclusion. PAN- or adriamycin-treated rats develop proteinuria with a reduced activity of 11b-HSD, a mechanism contributing to the abnormal sodium retention in nephrotic syndrome.

Renal sodium retention and potassium loss occur early, in many instances in the preascitic state of cirrhosis, an observation that cannot be fully explained by increased aldosterone concentrations. We therefore hypothesize that 11beta-hydroxysteroid dehydrogenase 2 (11beta-HSD2), which protects mineralocorticoid receptors (MR) from glucocorticosteroids, is down-regulated in cirrhosis. Cirrhosis was induced by bile duct ligation in rats. The urinary ratio of (tetrahydrocorticosterone + 5alpha-tetrahydrocorticosterone)/ 11-dehydro-tetrahydrocorticosterone [(THB+5alpha-THB)/THA] was measured by gas chromatography. Cortical collecting tubules (CCT) were isolated by microdissection and used for measurements of the activity of 11beta-HSD2 by assessing the conversion of corticosterone to dehydrocorticosterone. The mRNA content of 11beta-HSD2 was determined by reverse-transcription polymerase chain reaction (RT-PCR) in CCTs. The urinary ratio of (THB+5alpha-THB)/THA increased concomitantly with the urinary excretion of bile acids following bile duct ligation. Chenodeoxycholic acid (CDCA) dose-dependently inhibited 11beta-HSD2 in CCT with a Ki of 19.9 micromol/L. Four weeks after bile duct ligation, 11beta-HSD2 activity was decreased in CCT, an observation preceded by a reduced mRNA content at weeks 2 and 3. In cirrhosis, the MR-protecting effect by 11beta-HSD2 is diminished, and therefore, endogenous glucocorticoids can induce MR-mediated sodium retention and potassium loss.

The changes in the anthropometric data and urine steroid metabolites caused by regular training in children in two age groups (11 and 14 years old) were investigated. The skinfolds of older girls participating in regular athletic, swimming or soccer training were thinner compared with age-matched control groups (P < 0.01) and their body mass and constitution were lower (P < 0.05). In the other groups no significant differences were observable in the anthropometric parameters. The trained children in all groups had significantly higher exercise times on the cycle ergometer (P < 0.01, in young boysP < 0.05). The strength of their hands was lower in three trained groups: in younger boys (P < 0.05), in younger girls (P < 0.01) and in older girls (right handP < 0.01, left handP < 0.05). The urinary excretion of androsterone (P < 0.02), 11-ketopregnanetriol (P < 0.01) and pregnenetriol (P < 0.02) was decreased in the older trained girls; pregnenetriol was increased in younger boys (P < 0.05). Urinary excretion of cortisol metabolites was increased in trained boys [in younger boys: tetrahydrocorticosterone (P < 0.05) and 20α-hydroxycortisol (P < 0.05); in older boys allotetrahydrocortisol (P < 0.02), cortisol (P < 0.05) and 20α-hydroxycortisol (P < 0.05)]. There were no significant differences in the younger girls. In the trained older girls urinary excretion of cortisol metabolites was decreased: tetrahydrocortisone (P < 0.02), allotetrahydrocorticosterone (P < 0.01), tetrahydrocortisol (P < 0.05), α-cortolone (P < 0.01), cortisol (P < 0.02), 6β-hydroxycortisol (P < 0.01) and 20α-hydroxycortisol (P < 0.05). A multivariate analysis of the data from the trained groups and sedentary, age-matched control groups showed that regular training has a significant effect on steroid excretion.

Male subjects administered the 5 alpha-reductase inhibitor finasteride were studied to determine its effect on C19 and C21 5 alpha-metabolism. Plasma testosterone (T) and 5 alpha-dihydrotestosterone (DHT) were measured and T/DHT ratios determined at doses of 0.2-80 mg. Urinary etiocholanolone (5 beta)/androsterone (5 alpha) ratios and 5 beta/5 alpha metabolite ratios of cortisol, 11 beta-hydroxyandrostenedione, and corticosterone were also measured. The steroid profile was compared to male pseudohermaphrodites with inherited 5 alpha-reductase deficiency who have a global defect in C19 and C21 5 alpha-metabolism. The mean plasma DHT levels were decreased at all doses, resulting in elevated T/DHT ratios. The mean urinary etiocholanolone/androsterone, 11 beta-hydroxyetiocholanolone/11 beta-hydroxyandrosterone, tetrahydrocortisol/allotetrahydrocortisol, and tetrahydrocorticosterone/allotetrahydrocorticosterone ratios were elevated compared to pretreatment levels and placebo control values. The mean ratios appeared to be dose dependent for plasma T/DHT, urinary etiocholanolone/androsterone tetrahydrocorticosterone/allotetrahydrocorticosterone ratios. The mean 11 beta-hydroxyetiocholanolone-hydroxyandrosterone ratio was maximally elevated at the lowest doses. The results indicate that finasteride has a broad steroid spectrum inhibiting C19 and C21 5 alpha-steroid metabolism and affecting hepatic and peripheral 5 alpha-metabolism. These results suggest that a single gene codes for a single 5 alpha-reductase enzyme with affinity for multiple steroid substrates. The steroid profile is strikingly similar to that of male pseudohermaphrodites with inherited 5 alpha-reductase deficiency.

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.

Urinary steroids from a patient with 17α-hydroxylase deficiency syndrome have been identified. The steroids were obtained by enzymatic hydrolysis of urine, Amberlite XAD-2 extraction and Sephadex LH-20 column chromatography. Following preparation of volatile derivatives, the steroids were separated and estimated by gas chromatography on open-tubular capillary columns (OV-1, OV-101 and SE-52) and were identified by combined gas chromatography-mass spectrometry. The major steroids excreted were metabolites of pregnenolone (e.g. 5-pregnene-3β,20α-diol and 5-pregnene-3β,16α,20α-triol), progesterone (pregnanediols and 5β-pregnane-3α,16α,20α-triol) and 11β-hydroxy progesterone or corticosterone (11-oxo-pregnanediol, 11β-hydroxypregnanediol, 1,3,20-trihydroxypregnan-11-one, tetrahydrocorticosterone, allo-tetrahydrocorticosterone, and their 20- dihydro metabolites). It is probable that the 11-oxygenated 21-deoxysteroids are principally formed by microbial 21-dehydroxylation of the tetrahydro metabolites of corticosterone secreted into the bile. These metabolites are then reabsorbed in the intestine and excreted in the urine. Tetrahydrodeoxycorticosterone, 18-hydroxytetrahydro-deoxycorticosterone and 3α,18,21-trihydroxy-5β-pregnane-11,20-dione, were also identified. The daily excretion of 17-oxosteroids and cortisol metabolites was extremely small (

The chromatographic, physiologic, and enzymic characteristics of labeled material isolated from cloacal fluid of ducks given a single iv dose of labeled corticosterone suggest that several metabolites of labeled corticosterone are excreted. More than 85% of the labeled material in cloacal fluid was excreted via the kidneys. Approximately one-tenth of the labeled material was soluble in methylene chloride, and chromatographic separation of this extract in a cyclohexane:dioxane:methanol:water (100:75:50:25) system showed three peaks of radioactivity. The least abundant fraction had a mobility (Rf = 0.6) similar to authentic corticosterone and tetrahydrocorticosterone, the most abundant fraction was found in the slowest moving (Rf = 0.14 – 0.29) peak, and the remainder was associated with a peak of intermediate polarity (Rf = 0.38 – 0.46). The n-butanol extract of the aqueous phase contained more than 90% of the total radioactivity collected. Alumina column and paper chromatography of this extract revealed seven peaks of labeled material. Enzymic hydrolysis of these labeled compounds suggested the presence of conjugated labeled corticosterone and conjugated and unconjugated derivatives of labeled corticosterone. The labeled material present in each chromatographic peak was stable when added to urine and separated once more chromatographically. Also, with the exception of the labeled material in one peak, each was stable when recirculated intravenously in intact birds and isolated from the cloacal fluid for a second time. The recirculated labeled material from each chromatographic peak showed a pattern of disappearance from plasma and a distribution in compartmental volumes that permitted it to be distinguished from its chromatographic neighbors. The relative abundances of the chromatographically distinct labeled substances in n-butanol extracts of cloacal fluid from intact birds maintained under normal laboratory conditions and conditions of acute cold-stress and starvation were the same. A significant shift in the relative abundances of these labeled materials was found in cloacal fluid from chronically adenohypophysectomized birds, and the relative abundances were restored to normal after treatment with corticosterone.

In order to study the metabolism of 18-OH-11 deoxycorticosterone, the mineralocorticoid hormone responsible for hypertension in rats and humans, we have synthesized the following dihydrogenated and tetrahydrogenated reference derivatives: 18,21-dihydroxy-5α-pregnane-3,20-dione, 18,21-dihydroxy-5β-pregnance-3,20-dione. 3α,18,21-trihydroxy-5α-pregnan-20-one (I), 3β,18,21-trihydroxy-5α-pregnan-20-one (II), 3α,18,21-trihydroxy-5β-pregnan-20-one (III) and 3β,18,21-trihydroxy-5β-pregnan-20-one (IV). A complete separation of these compounds from each other and from tetrahydrocorticosterone isomers has been realized only by the association of thin-layere chromatography and gas-liquid chromatography on high-efficiency glass capillary columns. Characterization by gas-liquid chromatography-mass spectrometry is described. The stereoisomer distribution in rats is: adrenals of adult males and females (compounds I and II in the ratio 4:1), adrenals of 23-day-old males and females (compounds I, II and III, 11:11:3); liver of females (compound I and traces of III); and the liver of adult male (compounds II, III and IV, 4.5:4.5:1).

Analysis of urinary steroids excreted by a 7-year old girl with low renin hypertension following ACTH treatment revealed several unknown steroids, which have been analysed by gas chromatography-mass spectrometry. It is proposed that these steroids are monohydroxylated derivatives of cortisol, cortisone, either or both tetrahydro and allo-tetrahydrocortisol and either or both tetrahydro and allo-tetrahydro-11-deoxycortisol. Further analysis indicated that there are two likely positions for the additional hydroxyl group, either on the A or B ring.

The microsomal fraction from liver of female rats was found to glucuronidate pregnanediol. Enzyme activity was stimulated by Mg2+, Ca2+ and n-ethylmaleimide; p-chloromercuribenzoate did not affect enzyme activity, while dithioerythritol inhibited the activity 34%. The KM values for pregnanediol and UDP-glucuronic add were 0.84 and 60.8 μM, respectively; the mechanism of enzymatic reaction was sequential. Oestrone, etiocholanolone, pregnanolone and 11-deoxytetrahydrocortisone inhibited pregnanediol glucuronyltransferase competitively; bilirubin and phenolphthalein were noncompetitive inhibitors of the enzyme. Testosterone, tetrahydrocortisone, tetrahydrocorticosterone, p-nitrophenol and o-aminophenol did not affect pregnanediol glucuronidation. The activation energies with pregnanediol, oestrone and testosterone as substrates were 18.0, 11.7 and 12.0 kcal/(K × mol). Glucuronyltransferases conjugating pregnanediol, oestrone, testosterone and bilirubin show different rates of development and reach maximal activity at different times after birth. These results indicate the presence of an enzyme glucuronidating pregnanediol, which is different from those glucuronidating oestrone, testosterone and bilirubin, and provide additional support to the presence of multiple, steroid specific glucuronyltransferases in the liver of the rat.

The effects of melatonin and an aqueous extract of bovine pineal glands on adrenal function were studied in pinealectomized, ovariectomized and hypophysectomized rats. Melatonin (50 microgram i.p., twice daily for 1 week) stimulates adrenal 5 alpha-reductase activity in intact animals and after ovariectomy. A corresponding enhancement in the secretion of the principal 5 alpha-reduced metabolites of corticosterone (B), dihydrocorticosterone (DHB) and tetrahydrocorticosterone (THB), occurs in vivo. As a result, proportional output of B declines. In contrast, an aqueous pineal extract (360 mg bovine pineal tissue/day for 1 week) fails to alter reductase activity or to produce the secretory changes associated with increased reductive metabolism of B. Melatonin also stimulates adrenal reductase activity in hypophysectomized rats. The results suggest that melatonin is a potential contributor to the physiologic regulation of adrenal steroidogenesis.

The excretion of steroids by an infant with pseudohypoaldosteronism is reported. The patient presented with symptoms of hyperkalaemia and hyponatraemia, and was maintained for a period on a high salt intake and mineralocorticoids. However, he collapsed on the 48th day of life and could not be resuscitated.The urinary steroids were identified by combined gas chromatography–mass spectrometry and quantitation was achieved by gas chromatography. The following steroids were identified: tetrahydroaldosterone, tetrahydrocortisone, α- and β-cortolone, 1 β-hydroxytetrahydrocortisone, 1 β-hydroxycortolone, tetrahydrocorticosterone (THB), tetrahydro-11-dehydrocorticosterone (THA), 3α, 20α, 21-trihydroxy-5β-pregnan-ll-one, 5-pregnene-3β, 16α, 20α, 21-tetrol, and 5-androstene-3β, 16αa, 17β-tiiol.The patient evidently secreted an excess of aldosterone so the disorder was probably due to a form of renal-tubule unresponsiveness to this hormone. Some of the urinary steroids normally excreted in large amounts during infancy (16α-hydroxy DHA, 16-oxo-androstenediol, and 16α-hydroxypregnenolone) were not detected in the urine samples, and it is suggested that the precursor of these steroids (pregnenolone) was being fully utilised in the synthesis of aldosterone and cortisol.

SUMMARY The α-ketolic steroid R6, previously described by de Courcy, Bush, Gray & Lunnon [1953], has been identified as tetrahydrocorticosterone. The structures of X6 and some other αβ-unsaturated ketones appearing on chromatograms of urine extracts and giving the Bush soda fluorescence reaction are discussed. Four of these compounds are of exogenous origin and follow the ingestion of citrus fruits.

The heparinoid (RO1-8307) has been administered for from 2 to 5 days to 3 patients with subsequently proven primary aldosteronism. Patients with this disease are uniquely suited for study of the mechanism by which heparin and heparinoid compounds decrease adrenal production of aldosterone because in them the renin-angiotensin system is severely suppressed. This system, therefore, can be eliminated as a possible site of action of heparinoid. In all 3 patients heparinoid induced large decreases in both excretion and secretion of aldosterone, plasma renin activity remaining undetectable before, during and after administration of the compound. Excretion of 18-hydroxytetrahydro-11-dehydrocorticosterone, as an index of 18-hydroxycorticosterone production, was measured on one patient and was sharply suppressed by heparinoid. Excretion of tetrahydrocorticosterone, initially elevated, did not change. It is concluded that heparinoid acts directly upon the adrenal tumor tissue (and presumably normal adrenal tissue a...

Adrenocorticotropin was administered continuously for 3–7 days to normal subjects and to patients with essential hypertension or an aldosterone-producing adenoma. Tetrahydrodesoxycorticosterone and tetrahydrocorticosterone showed sustained increases but aldosterone showed only an initial transient rise. With cessation of adrenocorticotropin, aldosterone fell to

The effects of changes in the dietary content of sodium and potassium in a patient with isolated aldosterone deficiency are described. Poor renal sodium conservation on a low salt intake is demonstrated, as well as an inability to correct the persistent elevation in serum potassium by dietary potassium restriction. Markedly negative sodium and chloride balance occurred with dietary salt restriction, and was associated with hyponatremia and a further increase in serum potassium, as well as more frequent paralytic episodes. Correction of the biochemical and clinical abnormalities was achieved by the administration of ACTH, desoxycorticosterone, 9α-fluorohydrocortisone, and d-aldosterone. Administration of the salt-retaining steroids, however, resulted in sustained systemic arterial hypertension. Chlorothiazide, while able to lower the serum potassium concentration, also enhanced the excretion of sodium and chloride. Paper chromatographic separation of the individual urinary hydroxycorticoids disclosed an excess of tetrahydrocorticosterone, which increased after the administration of ACTH, while aldosterone remained absent in the urine. This strongly suggests the presence of a defect in the 18-aldolase enzyme system. A slight elevation in tetrahydro-S excretion raises the possibility of an associated mild defect in the 11β-hydroxylating system, although confirmatory evidence is lacking, since hydrocortisone metabolites in the urine were present in normal amounts and desoxycorticosterone metabolites were absent. The appearance of aldosterone in two consecutive 24-hour urine specimens toward the end of the study period could not be clearly related to the administration of any exogenous compound, and apparently represented at least a temporary restoration of 18-aldolase activity to normal or, perhaps, the activation of a different biosynthetic pathway for aldosterone formation.

The urinary excretion of 11β, 17α-dihydroxy-3,20-diketopregn-4-ene-21-yl sulfate (cortisol sulfate), 17α-hydroxy-3,11,20-triketopregn-4-ene-21-yl sulfate (cortisone sulfate) and of 11β-hydroxy-3,20-diketopregn-4-ene-21-yl sulfate (corticosterone sulfate) by newborn infants aged 2 and 6 days was quantified by double isotope dilution derivative assays and compared to that of known metabolites of cortisol and corticosterone. In a series of 7 newborns the sum of the urinary concentrations of cortisol and coitisone sulfates on the 2nd day of life represented represented from 43 to 114% of the amount of tetrahydrocortisone glucuronide, and on the 6th day from 8 to 155%. Corticosterone sulfate was excreted in amounts consistently much higher than tetrahydrocorticosterone glucuronide. It is concluded that the presence of these sulfate esters in urine reflects the existence of yet another pathway of corticosteioid biosynthesis and/or metabolism of particular significance during the perinatal period.

To elucidate further our previous findings that patients with essential hypertension produce more sulfate conjugated 17-hydroxycorticosteroids (17-OHCS) and less glucuronide conjugated 17-OHCS than normotensive subjects, individual urinary C21 αketolic steroids were separated by paper chromatography and quantitated in 11 hypertensives and 11 normotensives. The chromatography was performed separately on free, glucuronide and sulfate conjugated steroid fractions. The results obtained revealed the following statistically significant differences between the 2 groups of subjects: 1) in the free steroid fraction, excretion of cortisol and cortisone was higher in hypertensives; 2) in the glucuronide conjugated fraction, excretion of tetrahydrocortisol, tetrahydrocortisone, tetrahydrocorticosterone and tetrahydro-11-dehydrocorticosterone was lower in the hypertensives; 3) in the sulfate fraction, excretion of the most polar steroids, 6α- and 6β-hydroxycortisol, was higher in the hypertensives. The ratio ...

An ultradian rhythm was found in the daily variation of the parameters concerning the peripheral metabolism of corticosterone in sexually mature male Japanese quails. The period of this rhythm was eight hours; three peaks and minima were found in the daily fluctuation of metabolic clearance rata and an antiphysed rhythm in the biological half-life as well as in tetrahydrocorticosterone/corticosterone conversion rata. The findings emphasize the importance of the peripheral metabolism in developing of corticosterone plasma levels; and analyze the circadian rhythm of corticosterone plasma level as a result of secretion rate and peripheral disappearance.

Female rats were ovariectomized and either sham-pinealectomized or pinealectomized on day 24 of age and exposed to a photoperiod of 12 h light: 12 h dark. In vivo and in vitro measures of adrenocortical function were made at 6, 7, and 8 weeks of age (18, 25, and 32 days post-pinealectomy, respectively). Pinealectomy diminished the post-castration rise in adrenal 5alpha-reductase activity in all age groups (P less than .05). The proportionate secretion of corticosterone (compared with the total steroid output) by adrenal slices was likewise enhanced (P less than .05) although the secretion and production of corticosterone in vitro was not altered. Pinealectomy substantially diminished (P less than .05) the in vivo secretion rates of reduced metabolites of corticosterone (dihydrocorticosterone and tetrahydrocorticosterone) without altering corticosterone secretion. Consequently, proportionate secretion of corticosterone in vivo was also enhanced (P less than .05). These findings are consistent with the notion that in ovariectomized rats removal of the pineal gland diminishes adrenal 5alpha-reductase activity without affecting ACTH secretion. However, in rats with ovaries intact, estrogen modified the effects of pinealectomy. Not only was the intra-adrenal metabolism of corticosterone diminished (higher proportionate output), but also resting levels of plasma corticosterone (P less than .01), corticosterone production in vitro (P less than .05), and total adrenal steroidogenesis in vitro (P less than .01) were increased. Thus, ACTH secretion may be enhanced following pineal removal in the presence of estrogen. The data suggest that the pineal gland, together with the ovaries, plays a role in the modulation of adrenal steroidogenesis.

SUMMARY The quantities of nine corticosteroids in 24 h urine samples collected by pregnant women (nine with normal foetuses and nine with anencephalic foetuses) were measured after hydrolysis with β-glucuronidase and separation by paper chromatography. The excretion (μmol/24 h, mean ± s.d.) of pregnanetriol (0·85 ± 0·17), 3α,17α-dihydroxy-5β-pregnan-20-one 17α-hydroxypregnanolone, 0·55 ± 0·17), 3α,17α,21-trihydroxy-5β-pregnan-20-one (tetrahydro-11-deoxycortisol, 0·17 ± 0·14) and tetrahydrocorticosterone (0·65 ± 0·26) by women with an anencephalic foetus was significantly lower (P < 0·01 or No evidence was obtained therefore to indicate the secretion of cortisol or deoxycorticosterone by the foetal zone of the adrenal of the undisturbed human foetus in late gestation.

Gamma-Glutamyltransferase activity was detected in the plasma membrane of the highly differentiated hepatoma cell line Fao, (0.93 mU/mg cell protein). Dexamethasone (1 microM) provoked a 2-3-fold increase in the activity of the enzyme in the presence of fetal calf serum. Maximal induction occurred 48-72 h after addition of the glucocorticoid to the cell culture medium. The hormonal specificity was demonstrated by the relative potencies of several glucocorticoids and sex steroids: hydrocortisone and corticosterone increased gamma-glutamyltransferase activity while tetrahydrocorticosterone and all sex steroids tested were ineffective. The effect of dexamethasone on gamma-glutamyltransferase activity wa specific since the activities of several other plasma membrane enzymes were not modified. The mechanism of the dexamethasone-induced increase in gamma-glutamyltransferase activity was neither by modification of the affinity of the enzyme for its substrates nor by alteration of the subcellular distribution of the enzyme. This increase was prevented by cycloheximide and actinomycin D. The data presented are consistent with a specific glucocorticoid receptor-mediated induction of gamma-glutamyltransferase activity in Fao cells. The kinetic parameters of the induction process by glucocorticoids are very similar to those found in adult rat liver. These results suggest that the Fao cell line is a very convenient system for the study of the molecular mechanisms of glucocorticoid effects on differentiated cells.

In late pregnancy the mean excretions of tetrahydrocorticosterone, tetrahydro-11-deoxycortisol, pregnanetriol and 17-hydroxy-pregnanolone by women pregnant with an anencephalic fetus were significantly lower than the excretion of these steroids by women pregnant with a normal fetus. In contrast, there was no significant difference between these groups of subjects in the excretion of tetrahydrocortisol, tetrahydrocortisone, cortolone (20α + 20β), cortol (20α + 20β) and tetrahydrodeoxycorticosterone. From this evidence it is concluded that the fetal zone of the human fetal adrenal near term contributes to the secretion of corticosterone and 17-hydroxyprogesterone or their precursors, but not to the secretion of cortisol (or its precursors). Radioactive cortisol was formed during incubation of radioactive progesterone and pregnenolone with adrenal tissue (largely definitive zone) from newborn anencephalic infants. In contrast, the incorporation of [ 14 C]-acetate into 4-en-3-oxo steroids by adrenal tissue from fetuses of eighteen weeks gestation could not be demonstrated. The evidence presented suggests that any synthesis of cortisol by the human fetal adrenal occurs largely in the definitive zone.