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Both acute and chronic stress can impair maternal behavior and increase rates of infant abuse in several species. The mechanisms inducing these effects are unknown, but experimental manipulation of circulating corticosterone levels alters maternal behavior in rats, and circulating or excreted cortisol concentrations have been found to correlate either positively or negatively with maternal behavior in humans and nonhuman primates. In this study, therefore, we experimentally tested the hypothesis that both acute and chronic treatment with exogenous glucocorticoids would alter maternal behavior in a primate, the common marmoset (Callithrix jacchus). Multiparous females, approximately 3-5 weeks postpartum, received daily injections of either cortisol (hydrocortisone sodium succinate and hydrocortisone acetate; N=7) or vehicle (N=7) for 8 days, and maternal behavior was characterized under baseline conditions as well as during exposure to a noise stressor. Cortisol treatment successfully elevated both morning and afternoon plasma cortisol concentrations and suppressed circulating levels of adrenocorticotropic hormone. In home-cage observations, cortisol-treated females carried their infants significantly less than control mothers, and in noise-stressor tests, several hours after the first cortisol or vehicle treatment, cortisol-treated mothers inspected their infants significantly more often than controls. Aggression towards infants was infrequent and mild, and did not differ between treatment groups. These findings provide the first experimental evidence that cortisol elevations can alter maternal behavior in primates. As these effects were limited in scope, however, they suggest that other stress-responsive hormones or neuropeptides may additionally play a role in mediating the effects of stress on maternal behavior.

An abnormality of rapid cortisol feedback on activity of the hypothalamic-pituitary-adrenal axis has been reported in depression. However, there is controversy regarding the existence of rapid cortisol feedback on corticotropin (ACTH) secretion in humans. We investigated the effects of cortisol on ACTH levels in healthy subjects using a placebo-controlled, double blind, random assignment, cross-over design. Ten medication-free volunteers with no psychiatric history and no active medical problems underwent a standard protocol on two occasions separated by at least 2 weeks. Each time, subjects were admitted to a General Clinical Research Center and had infusion of 15 mg cortisol (hydrocortisone sodium succinate) over 120 min or placebo. Serum levels of cortisol and plasma ACTH levels were determined at baseline and over the 4 h after the start of the infusion. Over the two GCRC admissions subjects received both cortisol and placebo infusions, and the order of the two infusions was randomized. Compared to placebo, cortisol infusion produced a significant decrease in plasma ACTH levels beginning within 60 min from the start of the infusion. We conclude that cortisol infusion produces early inhibition of ACTH secretion in normal humans.

Electrophysiological responses to glycine (0.25-5 mM) were obtained on preparations of rat optic nerve. The log dose-response curve for glycine was shifted to the left by a factor of 2 by 1 microM 20 alpha-dihydrocortisol and by a factor of about 1.5 by 1 microM alpha-cortol and 10 microM hydrocortisone. A similar effect was obtained with 100 microM chlormethiazole but the GABA-potentiating steroid alphaxalone (1 microM) was ineffective on responses to glycine. 20 alpha-Dihydrocortisol and chlormethiazole also appeared to increase the antagonistic potency of strychnine against glycine. These observations suggest that the active steroids and chlormethiazole increase the functional interaction of both glycine and strychnine with the glycine-gated chloride channel.

Binding of tracer or competing steroids to transcortin can compromise specificity studies on receptors for adrenal steroids. Recently Alexis et al. have used cortisol 17 beta acid at high concentrations to prevent steroid binding to any transcortin possibly contaminating rat brain cytosol preparations. On the basis of limited specificity studies of [3H]dexamethasone and [3H]corticosterone binding under such conditions, it was claimed that binding sites for the two steroids are indistinguishable, and it is thus unnecessary to invoke distinct binding sites for each glucocorticoid. We have extended these competition studies in the presence of cortisol 17 beta acid, and shown that in rat hippocampus Type I, corticosterone-preferring glucocorticoid receptors can be clearly distinguished both from transcortin and from Type II, dexamethasone-binding glucocorticoid receptors.

Proton magnetic resonance spectroscopy played a crucial role in the identification of a new corticosteroid, 18-hydroxycortisol, recently isolated from the urine of a patient with an aldosterone-producing adenoma. Mass spectrometric analysis and chemical degradative studies demonstrated an empirical formula of C21 H30 O6 corresponding to a diketopregnenetetrol and placed three of the four hydroxyl groups at the 11 beta, 18 and 21 positions. The first suggestion that the locus of the fourth hydroxyl was 17 alpha came from the n.m.r. spectrum in the form of negative evidence for proton linked to the carbon atom bearing this fourth hydroxyl. The n.m.r. spectral features of the 18-hydroxy derivatives of cortisol and corticosterone are compared. Both were found to exist in the 20, 18 cyclic hemiketal form.

The role of the glucocorticoid receptor in the expression of antiglucocorticoid action has been investigated with a chemically-reactive derivative of three glucocorticoid steroids with differing biological potencies, i.e. the C-21 mesylates of cortisol, dexamethasone and deacylcortivazol. Dexamethasone 21-mesylate (Dex-Mes) was the most useful derivative due to its favorable balance of high receptor affinity and predominantly irreversible antiglucocorticoid activity. A number of criteria have been used to conclude that [3H]Dex-Mes covalently labels glucocorticoid receptors in the steroid-binding cavity. The available data indicate that covalent Dex-Mes-labeled receptors (mol. wt approximately equal to 98,000) are responsible for the irreversible antiglucocorticoid activity while the partial agonist activity of Dex-Mes is due to non-covalent Dex-Mes-bound receptors. Further support for this hypothesis comes from the observations that deacylcortivazol 21-mesylate was a full glucocorticoid and did not affinity label receptors (and marginally labeled cytosol proteins) although it was capable of covalently-labeling bovine serum albumin. Several mechanisms for the expression of irreversible antiglucocorticoid activity by covalent Dex-Mes-labeled receptors were examined and can be eliminated. Covalent receptor-Dex-Mes complexes formed in whole HTC cells were found to have a decreased capacity for nuclear binding. This decreased nuclear-binding capacity could be responsible for the whole-cell irreversible antiglucocorticoid activity of Dex-Mes.

ELISA technology using immobilized steroid conjugates has allowed the rapid screening and characterization of hybridoma supernatants. Although cortisol-3CMO-BSA was the immunogen, a monoclonal antibody with exceptionally high cross-reactivity (greater than 1000%) to prednisone was obtained. Characterization of the antigenic determinants shows a requirement for overall glucocorticoid 21-hydroxyl and 17 alpha-hydroxyl groups with additional high specificity for the 1,2-double bond and 11-position. There is potential use for this antibody in the assay of prednisone.

The sulfates of deoxycorticosterone (DOCS), corticosterone (BS), cortisone (ES) and cortisol (FS) were radioimmunoassayed in umbilical vein blood plasma samples collected in 97 fetuses between 19 and 32 weeks of gestation after extraction and chromatography on Sephadex LH 20 columns. While DOCS and BS displayed a decreasing pattern until 27-28 weeks, FS and ES did not show important variations throughout the period considered. All sulfates, excepted BS, increased at 29-30 weeks but this rise was only significant for FS and ES. Thereafter BS significantly increased while no significant difference could be displayed for the three others. In view of the difference between the patterns of 17-deoxy- and 17-hydroxycorticosteroids, one can speculate that, during this period of pregnancy, a shift in steroid biogenesis might occur towards a more important production of cortisol. DOCS was correlated with BS and with FS but not with ES. FS was correlated with the three other sulfates and with unconjugated F. ES was correlated with BS and FS but not with DOCS or unconjugated E. The significance of these correlations are discussed according to the origin and the metabolic interrelationships of the four steroid sulfates and unconjugated F and E.

In cytosol preparations renal mineralocorticoid receptors have equivalent affinity for aldosterone and corticosterone; in vivo, such receptors are clearly aldosterone-selective. To explore this difference, we have compared the binding of both steroids in kidney slices, high speed supernatants and true cytosols in vitro, in the presence of RU28362 to exclude steroids from Type II glucocorticoid receptors, and/or in the presence of cortisol 17 beta-COOH to exclude corticosterone from transcortin. In addition, we have explored the effect of tissue preparation and cortisol 17 beta-COOH on the level of activity of the renal enzyme 11 beta-OH steroid dehydrogenase. The results of the present studies support the hypothesis that the predominant aldosterone specific-conferring mechanism for renal Type I receptors in vivo in the enzyme 11 beta-OH steroid dehydrogenase, and underline the potential difficulties in interpreting studies which include multiple potential ligands to multiple potential binding sites.

Urinary 6 beta-hydroxycortisol is a useful, non-invasive index of microsomal mixed-function oxidase activity in man (review: [1]). Administration of phenobarbitone (40 mg/kg/day) for 4 days to male guinea-pigs, produced significant increases in hepatic microsomal cytochrome P-450 content and cytochrome P-450 reductase activity but did not increase 6 beta-hydroxycortisol excretion. The lack of relationship between urinary 5 beta-OHC excretion and microsomal mixed-function oxidase activity in the guinea-pig may be accounted for by the finding that 6 beta-hydroxycortisol itself undergoes extensive and variable metabolism in the guinea-pig.

The isolation of 18-hydroxycortisol from the urine of patients with primary aldosteronism was recently described and no synthetic procedure was available for its preparation. The C-13 angular methyl group of prednisolone-17 alpha,21-acetonide-11 beta-nitrite was functionalized by photolysis in the presence of oxygen to give the product 18-hydroxy-prednisolone-17 alpha,21-acetonide-18-nitrate. The 18-nitrate was reduced with zinc and ammonium acetate to the corresponding 18-hydroxy compound, 18-hydroxy-prednisolone-17 alpha,21-acetonide. Homogeneous hydrogenation with Tris(triphenyl-phosphine)rhodium (I) chloride as catalyst resulted in the formation of 18-hydroxy-cortisol-17 alpha,21-acetonide. Acid hydrolysis of the latter compound gave 18-hydroxycortisol. Oxidation of 18-hydroxycortisol-17 alpha,21-acetonide with pyridinium dichromate followed by acid hydrolysis gave 18-hydroxycortisone. The 18-hydroxylated steroids exist as the 18,21-hemiacetals. Catalytic reduction with tritium gas with Tris(triphenyl-phosphine)rhodium (I) chloride of 18-hydroxyprednisolone-17 alpha,21-acetonide and acid hydrolysis gave [1,2(3)H]18-hydroxycortisol.

Affinity for the dexamethasone binding sites of tixocortol pivalate, (the ester of the 21 thiol derivatives of cortisol), a steroid with local anti-inflammatory activity similar to cortisol acetate and with no systemic activity, was investigated in comparison with other steroids: tixocortol (the thiol derivative yielded by esterase hydrolysis), cortisol acetate and cortisol. The rank order of relative affinity for dexamethasone receptor of mouse thymocytes (37 degrees C) was: dexamethasone (1), cortisol (0.20), tixocortol pivalate (0.16), tixocortol (0.065), cortisol acetate (0.05). The corresponding 21 oxygenated ester (cortisol pivalate) was found less potent (0.080). Using rat renomedullary interstitial cells in culture, tixocortol pivalate showed also a higher receptor affinity than tixocortol. Cortisol acetate was as potent as tixocortol pivalate. The biological activity of tixocortol pivalate measured by the inhibition of PGE2 secretion on the same model, was similar to cortisol acetate and cortisol (10(-6) M, 24 h incubation, 38-55% inhibition). Tixocortol was less active (26%). These results with tixocortol pivalate are in good agreement with previously reported in vivo studies and show a good correlation between its binding ability and biological effect.

The urinary excretion of 18-oxocortisol in 37 normal subjects consuming a normal sodium diet was 1.2 +/- 0.9(SD) microgram/24 h. Dexamethasone administration to 5 normal individuals suppressed the excretion of 18-oxocortisol from 1.16 +/- 0.5 micrograms/24 h to 0.6 +/- 0.2 micrograms/24 h. While they still received dexamethasone, ACTH administration raised the 18-oxo-cortisol excretion to 3.82 +/- 1.2 micrograms/24 h. Seven normal subjects were placed on a sodium restricted diet, and the urinary excretion of 18-oxocortisol rose from 1.5 +/- 1.21 micrograms/24 h to 8.54 +/- 5.08 micrograms/24 h and aldosterone from 6.6 +/- 2.0 micrograms/24 h to 39.7 +/- 14.6 micrograms/24 h. Two of the seven individuals showed minimal increases in the excretion of 18-oxocortisol, but in all cases aldosterone increased with sodium restriction. The urinary excretion of 18-oxocortisol correlated significantly with the excretion of aldosterone, 18-hydroxycortisol, cortisol, and 19-nordeoxycorticosterone. These studies indicate that 18-oxocortisol secretion is under ACTH regulation, but since sodium restriction also increases the excretion of 18-oxocortisol, the renin-angiotensin system must also participate in its regulation. However, some individuals do not increase their excretion of 18-oxocortisol with sodium restriction, although aldosterone excretion increases as expected, suggesting that additional factors participate in the regulation of 18-oxocortisol production.

The isolation of 18-hydroxycortisol and 18-oxocortisol was recently described. These steroids have been shown to be excreted in exaggerated quantities in patients with primary aldosteronism, with adrenal adenomas and in glucocorticoid suppressible aldosteronism. We report the measurement of both steroids in the urine of patients with essential hypertension. 18-Oxocortisol excretion did not differ in patients with normal renin essential hypertension (0.7 +/- 0.7 micrograms/24 h), low renin essential hypertension (0.7 +/- 0.5 micrograms/24 h) and normal individuals (1.2 +/- 0.9 micrograms/24 h). Patients with normal renin hypertension excreted 54 +/- 43 micrograms/24 h of 18-hydroxycortisol, those with low renin essential hypertension excreted 58 +/- 54 micrograms/24 h, and normal individuals excreted 63 +/- 36 micrograms/24 h. Three of the low renin and one of the normal renin hypertensive subjects excreted greater quantities of 18-hydroxycortisol than the upper limit of normal, but all excreted normal quantities of 18-oxocortisol. As 18-hydroxycortisol is inactive, the meaning of this elevated excretion is unclear, but it may be a marker of an adrenal enzymatic abnormality which may be playing a more direct role in hypertension.

During in vivo metabolism the addition of six atoms of hydrogen to cortisone at the appropriate location and configuration can lead to formation of either 3 alpha,17,20 alpha,21-tetrahydroxy 5 beta-pregnan-11-one (cortolone) or 3 alpha,17,20 beta,21-tetrahydroxy-5 beta-pregnan-11-one (beta-cortolone). Likewise, metabolic reduction of cortisol can lead to formation of either 5 alpha-pregnane-3 alpha,11 beta,17,20 alpha,21-pentol (cortol) or the 20 beta isomer (beta-cortol). This paper describes the chemical syntheses of the C-3 beta-D-glucosiduronates of cortolone, beta-cortolone, cortol and beta-cortol-conjugates which are normal excretory products of man. The foregoing conjugates are characterized as free acids (or salts), as methyl esters and as polyacetate methyl esters.

The adrenal cortex shows a histological and functional zonation which allows it to function as two distinct glands. Aldosterone is produced only in the outer area, or zona glomerulosa and cortisol is produced in the zona fasciculata. Adrenal slices (200 mu) were prepared from cores of beef adrenals using a McIlwain Tissue Slicer and incubated. Cortisol, corticosterone, aldosterone, 18-hydroxycorticosterone, 18-hydroxycortisol and 18-oxocortisol were measured by radioimmunoassay. Aldosterone and 18-hydroxycorticosterone were produced primarily in the outer slices of the adrenal. Cortisol and corticosterone were produced throughout the adrenal with the production of cortisol in the first layer probably due to contamination with fasciculata. 18-Hydroxycortisol and 18-oxocortisol were produced primarily in the outer slices. The coexistence of cytochrome P-450 corticosterone methyl oxidase I and II and the 17 alpha-hydroxylase in the outer slices leads to the synthesis of these two hybrid steroids.

The naturally occurring mineralocorticoid agonist, 18-oxocortisol, is secreted in increased amounts in two hypertensive syndromes. One is primary aldosteronism and the other a genetic disorder first described by Sutherland and co-workers in which aldosterone secretion is ACTH-dependent and the mode of inheritance is autosomal dominant. 18-Hydroxy and -oxocortisol are the components of the cortisol oxidation pathway which arise when cortisol becomes an alternate substrate for corticosterone methyl oxidase. This enzyme system normally resides in the glomerulosa zone of the mammalian adrenal cortex. In an effort to account for a larger fraction of 18-oxocortisol and provide a reliable index of its secretion and of the expression of the cortisol C-18 oxidation pathway, metabolites were sought in the urine of a patient with the ACTH-dependent autosomal dominant form of aldosteronism. Using a variant of the technique of reverse isotope dilution, a pool of [3H]-labeled urinary metabolites form a normal subject was mixed with the patient's urine and subjected to customary methods of hydrolysis for urinary steroids. The radiolabeled glucuronide fraction was the most abundant and was subjected to repeated HPLC fractionation to yield the predominant component. The evidence from gas chromatography-mass spectrometry indicated that this metabolite was a tetrahydro derivative. The structure of the isolated tetrahydro 18-oxocortisol was confirmed by a biosynthesis of a reference standard from 18-oxocortisol and a 5 beta-pregnane reductase preparation.

Comparative pharmacokinetic studies of [14C]tixocortol pivalate ([14C]TP) and [14C]cortisol were carried out in rats. A 2.5 mg/kg i.v. dose (TP and cortisol) and oral doses of 1 and 25 mg/kg (cortisol), 25-250 and 1500 mg/kg (TP) were given separately to male and female rats. 14C-Radioactivity, [14C]cortisol, [14C]TP and [14C]T were determined in plasma samples, using TLC determinations and HPLC analysis. The results showed that plasma clearance and volume of distribution values of TP were respectively 6 and 10 times larger than those of cortisol (ClC = 4.7 l/h/kg and ClTP = 33.3 l/h/kg; VdC = 1.9 l/kg and VdTP = 21.7 l/kg). TP was rapidly converted into T whose plasma concentrations were close to those of TP. By the oral route, the bioavailability of cortisol was complete, whereas that of TP and T was 0.10-0.20. For the same 25 mg/kg p.o. dose, plasma Cmax values of TP and T were 100 times less than those of cortisol. It is concluded that a faster rate of metabolism combined with a larger volume of distribution and a low oral bioavailability all contribute to the lack of systemic activity of TP compared with cortisol.

A procedure is described for the microsynthesis and purification of the high specific activity tritium labeled cortisol metabolites, 20 alpha- and 20 beta-cortolic acids and 20 alpha- and 20 beta-cortolonic acids.

18-Oxo-cortisol (18-oxo-F) has been isolated from the urine of subjects with primary aldosteronism. This study examines the pressor, mineralocorticoid and glucocorticoid effects of 18-oxo-F in conscious sheep--a well studied species for the assessment of the pressor effect of steroid hormones. 18-oxo-F (24 mg/day i.v. for 5 days, n = 3) increased mean arterial pressure MAP (64 +/- 2 mmHg control and 75 +/- 6 mmHg on day 5 P less than 0.001). There was no change in heart rate. Plasma [K+] decreased from a control of 4.3 +/- 0.1 mmol/l control to 2.9 +/- 0.3 mmol/l on day 5 (P less than 0.001). Urinary Na+ excretion decreased on the first infusion day (233 +/- 18 mmol/day control and 124 +/- 20 mmol/day on infusion day 1 P less than 0.001). Urinary K+ excretion was reduced on days 1, 4 and 5 of the infusion. Thus in sheep, 18-oxo-F increased blood pressure associated with in vivo evidence of mineralocorticoid activity.

7 alpha- and 7 beta-Carboxymethylderivatives of cortisol, corticosterone and deoxycorticosterone have been synthetized. After coupling to bovine serum albumin, they were used to elicit antibodies in rabbits. Highly specific antisera were obtained which may possibly be used for a direct radioimmunoassay of these steroids in human and rodent plasma. In the case of the derivatives of cortisol and corticosterone and stereoisomery of the coupling had an effect on the affinity and the specificity of the antisera. In all immunized rabbits the antisera obtained with the 7 alpha-derivative had a higher affinity and a narrower specificity than the antiserum obtained with the 7 beta-derivative.

The antitumor activity and toxicity of two new 1-beta-D-arabinofuranosyl-cytosine (ara-C) conjugates of cortisol and corticosterone linked through a phosphodiester bond between the 5' and 21 positions of the respective moieties (cortisol- and corticosterone-p-ara-C) were investigated in L1210 lymphoid leukemia cells in mice. They are highly active against both i.p.- and i.c.-implanted ara-C-sensitive lymphoid leukemia in mice, exceeding the activity produced by the parent drug, ara-C. For example, corticosterone-p-ara-C exhibited the respective ILS values of 306% at 50 mg/kg/day X 9 and 294% at 75 mg/kg/day X 9 on survivals of i.p.- and i.c.-inoculated L1210 leukemic mice. The effectiveness of the conjugates seems to depend on schedules of the treatments. The 9-day continuous treatments showed a better therapeutic effectiveness than those with either a 5-day, a single or a widely spaced (q 4d., 1, 5, 9) treatment. However, they were found to be marginally effective against i.p.-implanted ara-C-resistant L1210 leukemia in mice. They were also inhibitory against proliferation of human leukemia-lymphoid cells in culture. Their superior antitumor activity and resistance to cytidine deaminase suggests that they serve as a prodrug form of ara-C or ara-CMP.

The course of oxidative side chain cleavage of two recently isolated 17 alpha-hydroxy C-18 oxygenated naturally-occurring corticosteroids differed in that 18-hydroxycortisol yielded a 17-ketosteroid (Chu M. D., and Ulick S. (1982) J. biol. Chem. 257, 2218-2224) whereas 18-oxocortisol yielded a gamma-etiolactone (Ulick S., Chu M. D. and Land M. (1983) J. biol. Chem. 258, 5498-5502). In an analytic application of the periodic acid oxidative cleavage reaction to 18-oxocortisol, the finding of a second product of the reaction prompted a reinvestigation of its course. Both the delta 4,3-ketone secreted form of the steroid and its tetrahydro urinary metabolite were cleaved predominantly to 17-ketosteroids along with smaller amounts of gamma-etiolactones, whose proof of structure is herein reported. This anomalous course of oxidative cleavage was considered to reflect an equilibrium between C-20 ketone and cyclic-hemiketal forms in which the glycerol-type side chain structure of the later becomes the precursor of a 17-ketosteroid. Because of the similarities between in vitro oxidative side chain cleavage and in vivo corticosteroid metabolism, these findings suggest that C-18 oxygenated steroids may contribute to the 17-ketosteroid fraction of human urine.

A reliable radioimmunoassay for plasma and urine 6 beta-hydroxycortisol after chromatography on Sephadex LH 20 column has been described and evaluated. The antiserum used was raised in the rabbit injected with 6 beta-hydroxycortisol-3(O-carboxymethyl)oxime-bovine serum albumin. In control subjects, urine 6 beta-hydroxycortisol levels ranged from 137 to 348 micrograms/24 h (mean +/- SD: 238 +/- 66) in adult males (n = 14) and from 80 to 432 micrograms/24 h (mean +/- SD: 210 +/- 93) in adult females (n = 15). In plasma, 6 beta-hydroxycortisol levels ranged from 0.53 to 3.13 ng/ml (mean +/- SD: 1.14 +/- 0.57) in adult males (n = 17) and from 0.53 to 2.69 ng/ml (mean +/- SD: 1.22 +/- 0.53) in adult females (n = 19). In patients with Cushing's syndrome high levels were found in urine as well as in plasma. Finally the higher concentrations of 6 beta-hydroxycortisol found in adrenal effluent in comparison with those of the peripheral vein blood has clearly demonstrated that 6 beta-hydroxycortisol is secreted by the adrenal besides its extra-adrenal formation from cortisol.

The hapten (5 beta-pregnane-17,20 alpha-diol-3 alpha-yl glucuronide) required to develop the radioimmunoassay was synthesized by an unambiguous chemical synthesis. An immunogenic complex was synthesized by coupling bovine serum albumin (BSA) to the glucuronide by mixed acid anhydride reaction. The immunogenic complex was used to induce the formation of specific antibodies in rabbits. In addition, the required radioligand [6,7-3H]5 beta-pregnanetriol 3 alpha-glucuronide was prepared with appropriate specific radioactivity. The characteristics of the antibody were determined with regard to specificity and sensitivity and the precision and accuracy of the assay method established. As applied to urine samples this method is superior to existing procedures in that they avoid the hydrolysis step and can be used directly on diluted urine. Examples have been given of useful application of this technique in clinical practice.