Corticosterone Metabolites Research Articles

Separation and identification of corticosterone metabolites by liquid chromatography–electrospray ionization mass spectrometry

High-performance liquid chromatography coupled to atmospheric pressure ionization–electrospray ionization mass spectrometry (API–ESI–MS) was investigated for the analysis of corticosterone metabolites; their characterization was obtained by combining the separation on Zorbax Eclipse XDB C 18 column (eluted with a methanol–water–acetic acid gradient) with identification using positive ion mode API–ESI–MS and selected ion analysis. The applicability of this method was verified by monitoring the activity of steroid converting enzymes (20β-hydroxysteroid dehydrogenase and 11β-hydroxysteroid dehydrogenase) in avian intestines.

Alteration of endogenous corticosteroids and catecholamines in allergen-induced eosinophilic inflammation in Brown Norway rats

Although various types of stress activate a pituitary adrenal response, the alteration of endogenous corticosteroids and catecholamines during asthma remains unclear. The aim of this study was to assess changes in endogenous corticosteroid and catecholamine levels in allergic eosinophilic inflammation in rats, using metabolic cages. Brown Norway rats (female, 6 weeks old) were sensitized with intraperitoneal injections of ovalbumin on days 0 and 2 and challenged with either an aerosol of ovalbumin or saline for 30 min on day 21. Levels of urinary 11- hydroxycorticosteroid (OHCS), a primary metabolite of corticosterone; epinephrine and norepinephrine were determined in pooled samples taken 0–24 h before and 8–32 h after the challenge. Serum adrenocorticotropic hormone, corticosterone levels and cell counts in bronchoalveolar lavage fluid were assessed 32–36 h after the challenge, as well as lung eosinophil peroxidase activity, an indirect index of eosinophil infiltration. The numbers of total cells and eosinophils in bronchoalveolar lavage fluid and lung eosinophil peroxidase activity were significantly increased in the ovalbumin-challenged rats compared with the saline-challenged rats. While urinary OHCS and serum corticosterone levels were significantly increased after challenge in the ovalbumin-challenged rats, compared with the saline-challenged rats (2.1 ± 0.1 × 10 − 1 vs 1.7 ± 0.1 x 10 − 1 mg/g creatinine, P < 0.05 and 482 ± 49 vs 348 ± 19 ng/mL, P < 0.02, respectively), serum adrenocorticotropic hormone levels did not differ between the two groups. Urinary epinephrine and norepinephrine excretion also did not differ between the two groups. It is concluded that endogenous corticosterone, but not catecholamine, increases as a pathophysiologic adrenal response, possibly to protect lung during allergic eosinophilic inflammation.

Furosemide Inhibits 11 -Hydroxysteroid Dehydrogenase Type 2

11β-Hydroxsteroid dehydrogenase 2 (11β-OHSD2) protects the nonselective renal mineralocorticoid receptor from the endogenous glucocorticoid cortisol. Thus, drugs inhibiting 11β-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 11β-OHSD2. Furosemide was the only inhibitor. Its inhibition constant (Ki) was 30 μmol when extracts from COS-1 cells transfected with human 11β-OHSD2 were used as an enzyme source. The type of inhibition was competitive. To establish whether furosemide inhibits 11β-OHSD2 and 11β-OHSD1 in the renal target tissue, isolated tubular segments from rats were analyzed. Furosemide decreased the oxidative activity of 11β-OHSD2 in intact distal tubules and 11β-OHSD1 in proximal convoluted tubules. For the assessment of furosemide on the excretion of corticosterone metabolites in vivo, rats were given furosemide ip, and the ratio of tetrahydrocorticosterone plus 5α-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 11β-OHSD. Thus, furosemide inhibits the 11β-OHSD2 enzyme in the target tissue and might by that mechanism enhance the mineralocorticoid effect of 11β-hydroxyglucocorticoids.

Furosemide inhibits 11beta-hydroxysteroid dehydrogenase type 2.

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.

The relationship between social stress and dominance is seasonal in greylag geese

Studies of captive animals have suggested that social stress affects subdominants, whereas recent data from the wild have revealed that stress mainly affects dominants. We used a non-invasive approach to investigate, for the first time in a social bird, the circannual stress–dominance relationships between low-ranking single males, intermediately positioned paired ganders without offspring and high-ranking paired males with offspring from a flock of semi-tame, free-ranging greylag geese,Anser anser. We collected 933 faecal samples from 43 individuals, 12 singletons, 18 paired males without offspring and 13 paired males with offspring over an entire year and analysed them for corticosterone metabolites by enzyme immunoassay. During the mating season (February–April), singletons had marginally higher corticosterone than paired males (P<0.1), whereas during the parental season (May–January), the paired males with offspring had significantly higher corticosterone than both paired males without offspring and singletons. All three male categories had significantly higher corticosterone during the mating season than during the rest of the year. These results suggest that social stress in ganders is caused mainly by competition between males and by constrained access to females during the mating season, but by parental commitment during the rest of the year. We suggest that dominance per se may not be a direct cause of stress. Rather, the amount of social stress may co-vary with the behavioural investment individuals need to make to optimize their fitness and with the relationship between such demands and the individuals' rank positions. This relationship seems to be seasonal in geese.

Evidence of coexisting changes in 11 beta-hydroxysteroid dehydrogenase and 5 beta-reductase activity in subjects with untreated essential hypertension.

We compared corticosteroid metabolite excretion rates and patterns in a group of 68 subjects with untreated essential hypertension and a matched group of 48 normotensive control subjects. The ratio of tetrahydrocortisol plus allotetrahydrocortisol to tetrahydrocortisone and the ratio of allotetrahydrocortisol to tetrahydrocortisol were significantly higher in the hypertensive group. This is qualitatively similar to the situation found in patients with the syndrome of apparent mineralocorticoid excess or subjects treated with licorice or carbenoxolone where hypertension is known to arise from deficiencies of 11 beta-hydroxysteroid dehydrogenase and 5 beta-reductase activities. The equivalent ratios for corticosterone metabolites were not different between groups, but total corticosterone metabolite excretion was higher in the hypertensive group. Plasma cortisol levels were lower in hypertensive than in control subjects, but corticosterone levels were higher. This evidence supports a previous suggestion that the activities of these two enzymes may be reduced in essential hypertension, but the contribution of these changes to hypertension is not known.

Hyperandrogenism in polycystic ovary syndrome. Evidence of dysregulation of 11 beta-hydroxysteroid dehydrogenase.

Hyperandrogenemia is the hallmark of the polycystic ovary syndrome, yet the relative contributions of the adrenal cortex and ovary to the overproduction of androgen remain unclear. To identify possible causes of adrenocortical overactivity, we studied the metabolism of adrenal and ovarian steroid hormones in women with this disorder. We measured 24-hour urinary excretion of steroid hormone metabolites by high-resolution capillary gas chromatography in 65 women with the polycystic ovary syndrome and 45 normal women matched for body-mass index. After adjustment for body-mass index, the urinary excretion of testosterone and androstenedione metabolites was 1.9 times higher in the women with the syndrome than in the normal women, and the excretion of dehydroepiandosterone metabolites (C19 steroid sulfates) and cortisol metabolites was 1.5 and 1.3 times higher, respectively (P < 0.01 for all comparisons). The affected women also had significantly higher ratios of 11-oxo (oxygenated) metabolites to 11-hydroxy metabolites of cortisol (1.4 times higher, P < 0.001) and of 11-oxo to 11-hydroxy metabolites of corticosterone (1.8 times higher, P < 0.001). In the group with the polycystic ovary syndrome, 55 percent of the nonobese women and 24 percent of the obese women had ratios above the upper limit of normal; the ratios in the obese women did not differ significantly from those in the nonobese women. Adrenal secretion of cortisol and androgens is increased in women with the polycystic ovary syndrome. The increases may be explained by dysregulation of 11 beta-hydroxysteroid dehydrogenase causing increased oxidation of cortisol to cortisone, which cannot be accounted for by obesity.

Effects of adrenocortical steroids on long-term potentiation in the limbic system: basic mechanisms and behavioral consequences.

Hippocampal structures are a major target for adrenal steroid hormones, and hence these neural regions are some of the most likely mediators of the effects of adrenocortical steroids on behavior. Memory disturbance, in particular biasing toward negative contents, are part of the symptomatology presented by depressive patients. In turn, a sizeable subset of depression also presents with hypercortisolemia. Adrenocortical hormones are also known to affect memory processes. Hippocampal formation is essential for declarative memory. We thought it appropriate then to study the effects of adrenal steroids on long-term potentiation, a putative memory mechanism in the hippocampus. Two clearly distinguished components of the evoked response to perforant path stimulation can be studied in the hippocampus: the excitatory postsynaptic potential (EPSP) which denotes the graded depolarization of the somatodendritic region of the neuron and the population spike (PS), a manifestation of the all-or-none-discharge of the cell action potential. Corticosterone had a significant depressant effect on the EPSP component of the evoked response immediately and 15 min after injection. Thereafter EPSP amplitudes were within normal values. Corticosterone significantly decreased the PS immediately after the train, the component remaining low 30 min after the train. 5 alpha-Dihydrocorticosterone (a ring A-reduced metabolite of corticosterone) significantly reduced the PS component of the response at all times after injection. 18-Hydroxydeoxycorticosterone and deoxycorticosterone significantly decreased both EPSP and PS components of the evoked response from the time of infusion. Contrary to expectation, tetrahydrodeoxycorticosterone was ineffective in decreasing and if anything, enhanced the development of long-term potentiation. 18-Hydroxydeoxycorticosterone 21-acetate behaved like vehicle, except for the first 30 min after injection when the EPSP was decreased. Allotetrahydroprogesterone decreased all EPSP's values and had no effect in the PS development in comparison with vehicle. The suggestion is made that the study of steroidal effects on hippocampal LTP can serve as a preclinical model of some aspects of depression in a specific subset of the disease.

Importance of hypothalamic function to stressor-induced responsiveness of the GABA A receptor in the cerebral cortex: a non-corticosterone influence

Catecholamine terminals in the paraventricular nucleus (PVN) of the hypothalamus of 60-day-old rats were destroyed by the stereotaxic injection of 6-hydroxydopamine into the PVN (6-OHDA; 9 μg/1.5 μ1 bilaterally), and the rats were tested 2 weeks later. Lesions led to a 70% reduction of norepinephrine in the hypothalamus and a loss of dopamine-β-hydroxylase immunoreactivity in the PVN. Furthermore, 6-OHDA lesions in the hypothalamus disrupted stressor-induced (15 min of restraint) changes in GABA A receptor function in the cerebral cortex (assessed by measuring chloride-facilitated benzodiazepine binding) but did not alter stressor-induced increases in plasma corticosterone levels. Additionally, the lesion did not change the responsiveness of the GABA A receptor to the corticosterone metabolite, allotetrahydrodeoxycorticosterone. These results indicate that stressor-induced changes in cortical GABA A receptor function are not driven by the stressor-induced release of corticosterone. A separate group of animals were tested for behavioral responses to challenge, and while 6-OHDA-induced lesions did not alter total scores in the test of environment-specific social interaction, the lesions did induce a change in composition of the behavior. Lesioned animals demonstrated increased physical (vigorous contact) interactions, similar to behavior previously observed in younger rats. The results of the behavioral study support a role for the GABA A receptor in the cerebral cortex in mediating appropriate behavioral responses to challenge in the adult rat. Thus, a hypothalamic lesion that prevented challenge-induced changes in GABA A receptor function in the cortex (with no change in the corticosterone response to the stressor) also led to altered behavioral responses to challenge.

Measurement of the cortisol production rate in two sisters with 17α-hydroxylase deficiency using [1,2,3,4- 13C]cortisol and isotope dilution mass spectrometry

[1,2,3,4- 13C]cortisol was i.v. administered to two sisters aged 11 yr (patient I) and 3 yr (patient II) who suffer from 17α-hydroxylase deficiency. This is the first time that the cortisol production rate (CPR) in patients with 17α-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 ≈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, 11β-hydroxy progesterone and corticosterone metabolites. Tetrahydrocorticosterone and 5α-tetrahydrocorticosterone were found in urine at elevated excretions of 2.5 and 5.7, 0.9 and 2.0 μmol/24h, 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.

Release of arachidonic acid and the effects of corticosteroids on steroidogenesis in rat testis Leydig cells

The release of arachidonic acid by luteinizing hormone (LH) and the effects of inhibiting phospholipase A 2 (PLA 2) in vivo and in vitro on LH stimulated steroidogenesis in rat testis Leydig cells has been investigated. It was found that arachidonic acid is rapidly incorporated into phospholipids and is released within 1 min after addition of LH. The effects of treating adult rats with dexamethasone and human chrionic gonadotropin (hCG) in vivo on steroidogenesis and prostaglandin synthesis in Leydig cells isolated 6 h later were determined. It was found that hCG caused a marked increase in prostaglandin F 2α formation which was inhibited by treatment with dexamethasone. LH-stimulated testosterone production was inhibited in the hCG treated rats and dexamethasone caused a further decrease. Treatment with dexamethasone alone also caused a decrease in the response to LH. HCG, but not dexamethasone, had similar inhibitory effects on LH-stimulated cyclic AMP production. Similarly, the PLA 2 inhibitors quinacrine, dexamethasone and corticosterone, added to the Leydig cells in vitro, inhibited LH-stimulated testosterone production but not cyclic AMP production. 11-Dehydrocorticosterone also inhibited LH-stimulated testosterone production, but higher concentrations were required to give 50% inhibition compared to corticosteone (50 and 25 μM, respectively). Ring A-reduced metabolites of corticosterone and progesterone were also found to inhibit LH-stimulated steroidogenesis. The results obtained in this and previous studies are consistent with the activation of PLA 2, (either directly by LH and/or via cyclic AMP), which results in the release of arachidonic acid and the formation of leukotrienes, which stimulate steroidogenesis in the Leydig cell. This study also indicates that corticosteroids and their metabolites may exert inhibitory effects at other sites in the steroidogenic pathways, in addition to PLA 2.

A Comparison of the Fatty Acid Esters of Estradiol and Corticosterone Synthesized by tIssues of the Rat

The biosynthesis of the fatty acid esters of the corticoid (corticosterone) and estrogen (estradiol) was compared in parallel incubations of corticosterone and estradiol with several tissues of the rat. The fatty acid composition of the esters of the two steroids was characterized in mammary and uterine tissue. In both of these tissues, the esters of estradiol were extremely heterogeneous. To the contrary, in the same tissues only one predominant ester of corticosterone, corticosterone-21-oleate, was formed. It comprised 70-80% of the total. The oleate ester of estradiol accounted for only 20% of the esters of this estrogen. In addition, fatty acid esters of an A-ring reduced metabolite of corticosterone, 5 beta-dihydrocorticosterone, was also identified. Its fatty acid composition is identical to that of corticosterone. In other experiments the fatty acid esters of both steroids were isolated from several tissues and quantified. When the amount of steroidal ester formed was compared, there was over a 100-fold difference among the various tissues in the ratio of estradiol to corticosterone ester synthesized. Thus, the rate of synthesis of the fatty acid esters of each class of steroid varies dramatically from tissue to tissue, and their fatty acid composition differs markedly as well. If the same enzyme synthesized both the estrogen and corticoid esters, then it would be expected that the relative amount of both esters synthesized in various tissues should be constant and likewise that their composition should be the same. Since neither occurred, these results suggest that the enzyme which produces the C-17 fatty acid esters of the estrogens may be different from the one which synthesizes the C-21 esters of the corticoids. The existence of separate enzyme systems for the synthesis of the fatty acid esters of these steroid hormones opens the possibility of specific physiological controls of each of these unusual steroidal metabolites.

Corticosterone's metabolite is an agonist for Na+ transport stimulation in A6 cells.

This study tests the hypothesis, in A6 epithelia, that 1) corticosterone stimulates active Na+ transport (short-circuit current, Isc) by an additional receptor mechanism to the type I (mineralocorticoid) and type II (glucocorticoid) mechanisms shared with aldosterone (Aldo) and 2) that the agonist may be 6 beta-OH-corticosterone made in the effector cell. The dose-response relationship of corticosterone at 24 h resolves into two components, by curve fitting, with a 50% effective concentration (EC50) for 10% of maximum Isc stimulation of 2 X 10(-9) M and an EC50 for the other 90% of 3 X 10(-7) M. The EC50 of the smaller component correlates with the apparent dissociation constant (K'd) of corticosterone for high affinity (type II) nuclear binding sites shared with Aldo. In unlabeled analogue competition studies Aldo and corticosterone displaced nuclear binding equally below 10(-8) M [3H]corticosterone, indicating only shared sites. However, nonshared saturable sites (displaced by corticosterone but not by Aldo) were found at [3H]-corticosterone concentrations above 10(-8) M. Concentration-binding curves performed with [3H]corticosterone, in presence of 1,000 X Aldo to displace shared sites, revealed a single class of binding sites with a half-maximal saturation of 2 X 10(-7) M, which is quite similar to the EC50 of the lower affinity component of Isc stimulation by corticosterone at 24 h. Reversed phase high-pressure liquid chromatography of nuclear extracts indicates that the saturable component of bound [3H] was 6 beta-OH-[3H]corticosterone derived from [3H]corticosterone. Thus, A6 cells metabolize corticosterone to 6 beta-OH-corticosterone, which in turn occupies lower-affinity receptors not shared with Aldo or corticosterone, to mediate most of the active Na+ transport stimulation by corticosterone.

HPLC separation of corticosterone metabolites and its use in following corticosterone metabolism and transport in the isolated rat hepatocyte

Incubating isolated rat hepatocytes with tritium labelled and unlabelled corticosterone at 37°C resulted in the rapid appearance of at least nine corticosterone metabolites. A quick, relatively easy, and quantitative high performance liquid Chromatographic (HPLC) method was used to separate these metabolites and follow their rate of appearance both intra- and extracellulariy. We found different intraand extracellular amounts of each metabolite at a particular time and this suggested that some metabolites were more available for transport than others.

Effects of 5α-dihydrocorticosterone on evoked responses and long-term potentiation

The effect of a Ring A-reduced metabolite of corticosterone, 5α-dihydrocorticosterone (DHB) on long-term potentiation (LTP) in the dentate gyrus (DG) of the rat were studied in barbiturate-anaesthetised animals. It was observed that DHB significantly impairs the development of LTP, more particularly the population spike (PS) component of the evoked potential (EP) to perforant path (PP) stimulation. Nutralipid, an inert control solvent of the steroid, did not affect LTP development. We argue that both, membrane and intracellular effects of DHB, are involved in the mechanisms responsible for DHB blocking of LTP.

URINARY STEROID METABOLITES IN SUBJECTS WITH MALE PSEUDOHERMAPHRODITISM DUE TO 5α‐REDUCTASE DEFICIENCY

To investigate the enzymatic basis for abnormal steroid metabolism in subjects with male pseudohermaphroditism due to 5 alpha-reductase deficiency, the ring A reduced urinary 5 beta and 5 alpha metabolites of testosterone, androstenedione, 11 beta-hydroxyandrostenedione, cortisol and corticosterone were measured by gas chromatography. Assays of the four pairs of urinary 5 beta and 5 alpha steroid metabolites revealed decreased conversion of the parent steroids to 5 alpha-reduced urinary metabolites, with increased 5 beta to 5 alpha urinary steroid metabolite ratios. These studies establish that increased urinary 5 beta/5 alpha ratios are distinctive for this disorder, and represent the most reliable method for confirming the diagnosis of primary inherited 5 alpha-reductase deficiency. These data also suggest that the conversion on the many delta 4-3 ketosteroids to 5 alpha-reduced steroids may be due to a single enzyme with broad specificity, or multiple enzyme reductases with a common regulator.

Excretion of 5β-cholestane-3α,7α,12α,26-tetrol and corticosterone metabolites in rat bile. Studies on the role of alcohol dehydrogenase in steroid metabolism

The monosulphate of 5β-cholestane-3α,7α,12α,26-tetrol was identified in bile of female and male rats. The excretion in the female rats decreased from 10–20 nmol/h directly after insertion of the bile cannula to very low values after 6 h. It then increased to reach a constant rate after 24 h that was slightly lower than during the first hour. Administration of 4-methylpyrazole or ethanol failed to change the rate of excretion. Thus there was no increase in the rate of excretion that might have resulted from an inhibited oxidation catalyzed by alcohol dehydrogenase (EC 1.1.1.1). Male rats were fed liquid ethanol-containing and control diets for 24 days prior to insertion of the bile cannula. The excretion during the first 3 h was 10–30 nmol/h in both groups, and chronic ethanol treatment did not change this rate. The effect of 4-methylpyrazole on the ratio between the excretion rates of the 3β- and 3α-isomers of 3,11β,21-trihydroxy-5α-pregnan-20-one disulphate was measured by a gas chromatographic-mass spectrometric method. The absence of any significant change indicated that the 3β-hydroxysteroid dehydrogenase activity of alcohol dehydrogenase was of no significance in the metabolism of corticosterone.

Synthesis of mono- and diglucosiduronates of metabolites of deoxycorticosterone and corticosterone and analysis by a new mass spectrometric technique

By condensing 3α, 21-dihydroxy-5β-pregnan-20-one, or its appropriate monoacetate, with methyl 2,3,4-tri-O-acetyl-1-bromo-1-deoxy-α- d-glucuronate in the Koenigs-Knorr reaction β- d-glucosiduronates 10 , 4 , and 7 were obtained as polyacetate methyl esters. Alkaline hydrolysis of these substances cleaved the ester groups and gave the corresponding steroidal glucosiduronic acids 12 , 6 and 8 . Upon treatment with diazomethane, these acids produced the equivalent methyl esters. The C-3, the C-21 and the C-3,21 glucosiduronates of 3α,21-dihydroxy-5β-pregnan-11,20-dione were prepared by previously reported methods and converted into the corresponding C-20 semicarbazones ( 14 , 20 and 26 ). With C-20 stabilized by the semicarbazone group against reduction, it was possible to reduce the 11-oxo function in these substances to an 11β-hydroxyl group; after removal of the semi-carbazone moiety from these products at pH 2.0, glucosiduronic acids 18 , 22 and 28 were obtained. The mass spectra of a representative group of the mono- and diglucosiduronic acids and esters were determined by utilizing fast atom bombardment and monitoring ions in both positive and negative modes of operation.

A mixed longitudinal study of adrenal steroid excretion in childhood and the mechanism of adrenarche.

Twenty four-hour urine samples have been collected 6 monthly for 18 months from 127 normal boys aged between 7.5 and 17 years. Urinary steroid metabolites have been measured by gas-liquid chromatography and ratios of steroid metabolites used to draw conclusions about likely enzymic activities at each step in the adrenal biosynthetic pathway. DHA excretion rose throughout the study, especially between 7.5 and 11 years. Metabolites of cortisol and corticosterone did not increase when related to surface area. During adrenarche there were changes consistent with a decrease in 3 beta-hydroxysteroid dehydrogenase activity, a slight rise in 17 alpha hydroxylase activity, a marked rise in 17,20 lyase activity, and a fall in 11 beta hydroxylase activity. These data are compatible with the hypothesis that a small reduction in the ability to synthesize cortisol requires a marginal increase in the secretion of ACTH and/or other CRF-induced peptides to maintain cortisol production rate. ACTH itself or a dexamethasone-suppressible CRF-induced peptide would in such a case control the growth of the zona reticularis at adrenarche, which thus appears to be primarily an adrenal event.

17 alpha-hydroxylase deficiency: mineralocorticoid hormone profiles in an affected family.

The plasma concentrations of mineralocorticoid hormones, basal and after stimulation and suppression with ACTH, can identify the heterozygotes in a family with two siblings with 17 alpha-hydroxylase deficiency. Both parents and one sibling had elevated levels of plasma deoxycorticosterone, corticosterone, 18-hydroxydeoxycorticosterone, and 18-hydroxycorticosterone, but normal cortisol and aldosterone concentrations. Stimulation with ACTH effected additional increases in the elevated steroid and cortisol levels, but not in aldosterone, further increasing the discrepancy and the ratio between 18-hydroxycorticosterone and aldosterone. One sibling had normal steroid patterns and an 18-hydroxycorticosterone to aldosterone ratio. Suppression of ACTH restored the steroids to low normal levels. In addition, the ratio of the gas chromatographic analysis of the total major urinary metabolites of corticosterone to total metabolites of cortisol was greater, and the sum of urinary androsterone and etiocholanolone to total corticosterone and cortisol metabolites was less in the heterozygotes than in normal subjects. This identifies deficient 17-hydroxylation, which is required for the production of cortisol and C-19 steroids. These criteria appear unique for the 17 alpha-hydroxylase defect in the heterozygote.