The thymus-adrenal connection: thymosin has corticotropin-releasing activity in primates.

Endotoxin-free thymosin fraction 5 elevated corticotropin, beta-endorphin, and cortisol in a dose- and time-dependent fashion when administered intravenously to prepubertal cynomolgus monkeys. Two synthetic component peptides of thymosin fraction 5 had no acute effects on pituitary function, suggesting that some other peptides in thymosin fraction 5 were responsible for its corticotropin-releasing activity. In agreement with these observations, total thymectomy of juvenile macaques was associated with decreases in plasma cortisol, corticotropin, and beta-endorphin. These findings indicate that the prepubertal primate thymus contains corticotropin-releasing activity that may contribute to a physiological immunoregulatory circuit between the developing immunological and pituitary-adrenal systems.

Androgen binding proteins of testis, epididymis, and plasma in man and monkey.

Androgen-binding protein (ABP) has been found in the cytosol of testicular and epididymal homogenates of several sub-primate species. In those species which had the plasma androgen binding protein, testosterone-estradiol-binding globulin (TeBG), ABP and TeBG were found to be physically similar. We investigated the possibility that ABP might exist in monkey and man using the cytosol of testicular and epididymal homogenates and aspirates obtained by direct micropuncture of the rete testis. In polyacrylamide gel electrophoresis, pH 7.8, testicular and epididymal cytosols of monkey and man were found to contain several binding proteins of different size and net charge that bind dihydrotestosterone. These binding proteins were either indistinguishable from TeBG or could be related to TeBG as size and/or charge isomers. No ABP was detectable in up to 200 mul of monkey rete testis fluid obtained by direct micropuncture, though ABP is detectable in as little as 5 mul of rat rete testis fluid. The data suggest that the ABP's detected in the testicular and epididymal cytosols in monkey and man represent isomeric forms of plasma TeBG, and their presence in testicular cytosol most likely derives from blood contamination.

Primary cortisol resistance in man. A glucocorticoid receptor-mediated disease.

We have studied a man suspected of having primary cortisol resistance on the basis of high 24-h mean plasma cortisol levels (27.4 micrograms/dl) and no stigmata of Cushing's syndrome. His son had slightly elevated 24-h mean plasma cortisol levels (9.9 micrograms/dl; normal 7.52 micrograms/dl). Both had high plasma protein unbound cortisol and increased urinary free cortisol. Plasma ACTH concentration was high, and both were resistant to adrenal suppression by dexamethasone. The father appeared to have mineralocorticoid excess resulting in hypertension, hypokalemia, and metabolic alkalosis. This was found to be due to markedly elevated plasma levels of deoxycorticosterone and corticosterone. The son, who was normotensive, had mildly increased plasma corticosterone and normal deoxycorticosterone levels. To study the apparent end-organ resistance to cortisol, we examined the glucocorticoid receptor in the white cells and fibroblasts of these patients. In both tissues, using both whole cell and cytosol assays, the glucocorticoid receptor was found to have reduced affinity for dexamethasone. In the cytoxol assays, a reduced receptor number was found as well. We conclude that cortisol resistance is a rare familial syndrome owing to an abnormal glucocorticoid receptor with a decreased affinity for cortisol.

Adaptation during surgical stress. A reevaluation of the role of glucocorticoids.

Pharmacologic doses of glucocorticoids are administered to patients with adrenal insufficiency during operative procedures to prevent hemodynamic instability, cardiovascular collapse, and death. Since these supraphysiologic doses might not be necessary and might have adverse effects, we examined the effects of different doses of glucocorticoids on hemodynamic adaptation during surgical stress in adrenalectomized primates. Sham-adrenalectomized placebo-treated animals served as controls. Adrenalectomized monkeys were maintained for 4 mo on physiologic glucocorticoid and mineralocorticoid replacement. The adrenalectomized monkeys were then stratified into three groups receiving, respectively, subphysiological (one-tenth the normal cortisol production rate), physiological, or supraphysiological (10 times the normal cortisol production rate) cortisol (hydrocortisone) treatment. 4 d later a cholecystectomy was performed. The intraoperative hemodynamic and metabolic parameters, perioperative survival rates, and postoperative wound healing were compared. The subphysiologically treated group was hemodynamically unstable before, during, and after surgery and had a significantly higher mortality rate than control. In this group, arterial blood pressure was low, and the cardiac index, systemic vascular resistance index, and left ventricular stroke work index were all reduced, suggesting decreased cardiac contractility and blood vessel tone. In contrast, the physiologically replaced group was indistinguishable from either supraphysiologically treated animals or sham-operated controls. All groups had similar metabolic profiles and normal wound healing. These findings suggest that the permissive actions of physiologic glucocorticoid replacement are both necessary and sufficient for primates to tolerate surgical stress. Supraphysiological glucocorticoid treatment has no apparent advantage during this form of stress in the primate.

Continuous administration of synthetic ovine corticotropin-releasing factor in man. Physiological and pathophysiological implications.

The continuous 24-h infusion of a maximally stimulating dose (1 micrograms/kg per h) of ovine corticotropin-releasing factor (CRF) in man caused a modest elevation of plasma cortisol (17.2 +/- 1.4 micrograms/dl) and urinary-free cortisol (173 +/- 43 micrograms/24 h) concentrations, which was far less than that seen with a maximally stimulating dose of ACTH (50.4 +/- 2.2 micrograms/dl and 1,200 +/- 94 micrograms/24 h, respectively). The circadian rhythms of plasma ACTH and cortisol were preserved during CRF administration. An intravenous bolus injection of 1 microgram/kg of ovine CRF given to normal volunteers under basal conditions resulted in elevated plasma ACTH and cortisol peak levels (28 +/- 6 pg/ml and 15.0 +/- 1.0 micrograms/dl, respectively). However, no plasma ACTH and cortisol responses were observed when an identical CRF stimulation test was given at the end of the continuous infusion. These findings suggest that the stimulatory activity of exogenous CRF on the ACTH-secreting cells of the pituitary gland is restrained by the negative feedback of cortisol. The persistent circadian rhythm of ACTH, despite a constant level of plasma CRF during the infusion, suggests that the circadian variation in the activity of the hypothalamic-pituitary-adrenal axis cannot be explained solely by circadian periodicity of the endogenous CRF stimulus.

The developmental changes in plasma adrenal androgens during infancy and adrenarche are associated with changing activities of adrenal microsomal 17-hydroxylase and 17,20-desmolase.

The plasma concentrations of dehydroepiandrosterone, androstenedione, and dehydroepiandrosterone sulfate decrease during the first year of life, remain low during childhood, and then increase during adrenarche. To determine whether alterations in adrenal enzyme activity might explain the changing secretory pattern of the adrenal androgens, we measured human adrenal microsomal 3 beta-hydroxysteroid dehydrogenase-isomerase, 17,20-desmolase, 17-hydroxylase, and 21-hydroxylase activities. 12 adrenals from individuals aged 3 mo to 60 yr were studied. The patients were divided into three groups based upon the age of the patient when the adrenal glands were obtained: group 1, infants aged 3--8 mo (n = 3); group 2, preadrenarchal or early adrenarchal children aged 2--9 yr (n = 4); and group 3, adults aged 20--60 yr (n = 5). The mean activity of the 17,20-desmolase, 17-hydroxylase, and 21-hydroxylase fell by 50% and that of 3 beta-hydroxysteroid dehydrogenase-isomerase activity rose 80% from group 1 to 2. A fourfold increase in 17,20-desmolase (P less than 0.002) and 17-hydroxylase (P less than 0.001) activity and a doubling in 21-hydroxylase activity (P less than 0.005) occurred between groups 2 and 3. We conclude that the decline in plasma adrenal androgens after birth appears to be associated with a rise in 3 beta-hydroxysteroid dehydrogenase-isomerase and a fall in 17,20-desmolase and 17-hydroxylase activity. The subsequent increase in plasma adrenal androgen concentration during adrenarche is coincident with a rise in 17,20-desmolase and 17-hydroxylase activity.

Glucocorticoid resistance in humans and nonhuman primates.

In humans, the syndrome of cortisol resistance is characterized by the absence of signs and symptoms of Cushing's syndrome, elevated total and unbound plasma cortisol concentrations, and increases in urinary free cortisol excretion and plasma adrenocorticotropic hormone. In one family, a severely affected member had hypertension and hypokalemic alkalosis associated with increased plasma concentrations of corticosterone and deoxycorticosterone. These patients are resistant to suppression of the pituitary-adrenal axis by dexamethasone. Dexamethasone therapy, however, effectively corrected hypertension and hypokalemic alkalosis in the severely affected patient, without causing signs of glucocorticoid excess. The glucocorticoid receptor from these patients has a low affinity for glucocorticoids and is unstable during thermal activation. Both the molecular weight of the glucocorticoid receptor and the size of the corresponding mRNA are similar to those of normal controls. Transformation of B-lymphocytes with Epstein-Barr virus leads to induction of glucocorticoid receptors. Receptor induction, however, is lower in patient cells than those obtained from normal controls. This decreased induction parallels decreased expression of glucocorticoid receptor mRNA. Thus, in this form of glucocorticoid resistance the glucocorticoid receptor is abnormal and leads to diminished target organ responsiveness. Many New World primates exhibit glucocorticoid "resistance," without apparent pathology. These species have markedly elevated plasma cortisol, both total and unbound concentrations, increased urinary free cortisol excretion, and marked increases in plasma adrenocorticotropic hormone and beta-endorphin. The glucocorticoid receptors of these primates have decreased affinity for glucocorticoids, are thermolabile, and are not induced by Epstein-Barr virus transformation as indicated by specific binding and mRNA expression. Both the molecular weight of the glucocorticoid receptor and the size of the corresponding mRNA are similar to those of normal controls. Despite the high plasma cortisol concentrations in these primates, there is no sodium retention and aldosterone levels are actually increased. The kidney aldosterone receptor cross-reacts poorly with cortisol, explaining the absence of sodium retention. New World primates also have progesterone, estrogen, aldosterone, and vitamin D insensitivity, suggesting a common factor linking steroid hormone receptors.

Glucocorticoid receptor-mediated effects on rat fibrosarcoma growth.

Glucocorticoid receptors are present in most normal and malignant mammalian cells. To examine the hypothesis that the growth of methylcholanthrene-induced malignant sarcoma is glucocorticoid dependent, we evaluated the behavior of malignant fibrosarcoma (MCA) in adrenalectomized rats treated with either normal saline or deoxycorticosterone acetate and in intact rats treated with placebo or with the glucocorticoid receptor antagonist RU 486. Survival, tumor weight, and loss of body weight (an index of cachexia) were measured. In MCA-bearing rats, neither survival nor loss of body weight was affected by bilateral adrenalectomy or by treatment with RU 486. Tumor weight and time-integrated tumor volume, however, were significantly less in bilaterally adrenalectomized rats without deoxycorticosterone acetate replacement than in animals treated with deoxycorticosterone acetate. Similarly, tumor weight and time-integrated tumor volume were less in intact animals treated with RU 486 than in intact animals treated with placebo. The glucocorticoid receptors in the tumor cells had similar binding capacity (Ro) and equilibrium dissociation constant (Kd) as in control rat fibroblasts. These results suggest that the growth of MCA sarcoma cells is partially dependent upon glucocorticoids. This effect of glucocorticoids, however, was not of sufficient magnitude to improve survival and prevent cachexia. We conclude that glucocorticoids appear to influence MCA sarcoma growth in the rat, and that glucocorticoid receptor blockade, perhaps in combination with other antitumor agents, merits future study in the treatment of malignant tumors.

Androgen receptor characteristics in skin fibroblasts from hirsute women.

Hormonal measurements in some women with hirsutism often reveal little or no elevation in androgen levels to explain the disorder. Thus, it has been postulated that increased sensitivity of the hair follicle to androgen may contribute to the development of hirsutism in such patients. We, therefore, sought androgen receptor abnormalities in skin fibroblasts cultured from 10 hirsute women (ages 17-43) and normal or mildly elevated plasma testosterone levels (28-82 ng/dl). Androgen receptor content (Ro) and binding affinity (Kd) in cultured pubic skin fibroblasts were measured using a dispersed, whole cell assay. Ten such cell lines from these women were compared with 19 pubic skin cell lines from 9 normal volunteers (6 males and 3 females) and from 10 other subjects (males with gynecomastia or hypospadias). There was no statistically significant difference in the mean androgen receptor content (11,600 +/- 2700 (SE) sites/cell fibroblasts vs 7900 +/- 700 sites/cell or binding affinity (2.0 +/- 0.3 (SE) X 10(-9) M vs 1.5 +/- 0.2 X 10(-9) M, respectively) between the patients' fibroblasts and those of the controls. We conclude that hirsutism cannot be explained by abnormalities in fibroblast androgen receptor number or affinity. These observations do not exclude the possibility that other mechanisms might lead to increased peripheral androgen sensitivity in such patients.

Adrenal function in women with idiopathic acne.

The adrenal secretion of androgens were examined in 9 women (ages 19-39 yr) with postadolescent idiopathic acne and compared to age and sex-matched normal controls. Plasma dehydroepiandrosterone (DHA), dehydroepiandrosterone sulfate (DHAS), androstenedione (delta 4-delta), cortisol, 17-hydroxyprogesterone, 11-deoxycortisol, and testosterone were measured by radioimmunoassay in the basal state and during a 48 hr ACTH infusion. The mean plasma and time-integrated plasma levels of the 3 adrenal androgens in patients with acne were 15-25% higher than normal controls, but the groups were not significantly different (p greater than .05). The plasma testosterone values, on the other hand, were similar in both groups. In addition, cortisol, 11-deoxycortisol and 17-hydroxyprogesterone basal plasma values and responses to ACTH in patients with acne were similar to the normal control values. These findings suggest that adrenal androgen secretion is at most mildly elevated in patients with idiopathic acne and is unlikely to be the sole cause of acne since many patients without acne have similar hormone levels. Increased sensitivity of the sebaceous gland to androgens or increased local metabolism of androgen hormones in the skin to potent androgen metabolites may offer alternative mechanisms for the pathogenesis of this disorder.

Suppression of plasma luteinizing hormone by prolactin in the male rat.

Although a direct effect of PRL on gonadotropins has been previously suggested, it has not been convincingly demonstrated. The secretion of LH and FSH was studied in response to the stimuli of castration and LH releasing hormone administration in adult male rats made hyperprolactinemic with ectopic pituitary glands. Although plasma LH and FSH levels were similar in non-castrate hyperprolactinemic rats vs. controls, LH concentrations 24 h postcastration were less in hyperprolactinemic animals as compared to controls (P less than 0.001). The level of LH achieved was inversely correlated with the PRL concentration generated (r = -0.71; P less than 0.01). LH suppression was evident in hyperprolactinemic rats at 1 and 3 days postcastration but was no longer observable at 7 days postcastration. After LH releasing hormone administration to non-castrate rats the rise in plasma LH was significantly less in the hyperprolactinemic animals (P less than 0.05). These experiments support the hypothesis that PRL directly inhibits LH secretion, presumably at the pituitary level.

A seminiferous tubular factor is not obligatory for regulation of plasma follicle-stimulating hormone in the rat.

Plasma testosterone, FSH, and LH levels were measured by RIA in sham-castrate control rats and in castrate male rats with testosterone-containing Silastic capsule implants to evaluate the relative contribution of testosterone and inhibin in maintaining normal FSH secretion in vivo. Silastic capsules maintaining normal testosterone levels maintained normal levels of both FSH and LH over the 5-day course of these studies. The data suggest that testosterone or its metabolites can account for at least 78%, and possibly all, of the FSH-suppressing activity of the testis. The data do not support an obligatory role for other testicular factors, such as inhibin, in the regulation of plasma FSH when normal testosterone levels are maintained.

Mechanism of interaction of digitalis with estradiol binding sites in rat uteri.

Digitalis preparations have a weak estrogenic effect in man. The data in animals are equivocal. We have studied.the biologic effect of both digitoxin and digoxin on the rat uterus in vivo and the interaction of these drugs with the rat uterus estrogen receptor in vitro. Digitoxin and estradiol significantly increased the uterine weight of immature rats, while digoxin did not. The interaction of digitoxin and digoxin with the rat uterus estrogen cytosol receptor was studied using protamine sulfate precipitation and dextran-coated charcoal (DCC) assays. Both methods gave a Kd for the estradiol-receptor interaction between 0.8-3.1 X 10(-9) M (n = 20). Digitoxin at concentrations of 0.5-2.0 X 10(-6) M significantly inhibited the binding of estradiol to the specific or saturable binding sites with minimal inhibition of hormonal binding to nonspecific sites. The binding was competitive with a calculated Ki for digitoxin of 5.2-7.8 X 10(-7) M (n = 18). Digoxin failed to inhibit estradiol binding to the receptor protein in vitro. We conclude that digitoxin probably acts directly as a weak estrogen and that this effect probably explains the estrogen-like side effects seen with digitoxin therapy in man.

Central kappa- and mu-opiate receptors mediate ACTH-release in rats.

The control of ACTH secretion by opiates seems to involve stimulatory and inhibitory pathways, since opiate agonists and antagonists are capable of releasing ACTH in conscious rats. To elucidate the role of different opiate receptors in the control of ACTH release, rats were treated with receptor-selective opiate agonists and antagonists. The mu-opiate agonists, morphine and (D-Ala2, MePhe4, Gly5-ol)enkephalin, and the benzomorphan kappa-opiate agonists, MR 2034 and MRZ 2549, both stimulated ACTH release after central or peripheral injection. The effects of morphine, but not those of MR 2034, were blocked by a low dose of naloxone (50 micrograms/kg) and by the mu-receptor antagonist, beta-funaltrexamine. A 20 times higher dose of naloxone also blocked the effects of the kappa-agonist. Our data suggest that both mu- and kappa-opiate receptors are involved in the stimulation of ACTH release in rats.

Adrenarche: a survey of rodents, domestic animals, and primates.

The concentrations of the adrenal steroids dehydroepiandrosterone (DHA), dehydroepiandrosterone sulfate (DHAS), and delta 4-androstenedione (delta 4-A) have been measured by RIA before and after sexual maturation in plasma of rodents, domestic animals, and primates to determine whether these species exhibit and adrenarchal process comparable to man. The average concentrations of DHA and DHAS were less than 60 ng/dl and 5 microgram/dl, respectively, in plasma of sexually mature rodents and domestic animals, and a significant increase in the plasma DHA level after sexual maturation was seen only in the rabbit and dog. The concentrations of DHA, DHAS, and delta 4-A in 21 rhesus monekeys from 0-3 yr of age were 2021 +/- 235 ng/dl (mean +/- SE), 357 +/- 60 microgram/dl, and 107 +/- 9 ng/dl, respectively, and did not increase during sexual maturation. By contrast, DHA, DHAS, and delta 4-A levels in plasma of chimpanzees were 5.9-fold, 3.3-fold, and 4.8-fold greater, respectively, in 7- to 22-compared to 0- to 3-yr-old animals. Temporally, the increase in DHA levels in the chimpanzee is apparent at 5 yr and this precedes the increase in gonadal steroids, as is characteristic of human adrenarche. It is apparent that adrenal androgen levels and their developmental patterns differ markedly among species, and that among the species examined, only the chimpanzee exhibits an adrenarche comparable to that of man.

Interaction of spironolactone and digitalis with the 5 alpha-dihydrotestosterone (DHT) receptor of rat ventral prostate.

The aldosterone antagonist, spironolactone, has been shown to block the effects of exogenously administered androgen in rat. This suggests that interaction of the drug with androgen at the target tissues may occur. In this paper we have studied the possible interaction of spironolactone with the 5alpha-dihydrotestosterone (DHT)1 receptor of rat ventral prostate. The competitive receptor assay used involves precipitation of the 105,000 X g supernatant of the homogenized tissue with protamine sulfate, removal of the unprecipitated cytosol, and incubation of the precipitate in the presence of the appropriate [3H]DHT steroid solution at 0 C for 18 hours. Using this method the Kd (dissociation constant) for DHT in the rat prostate was in the range of 1.9-4.0 X 10(-9)M and the binding capacity was 0.21 pmol/mg protein. Spironolactone was found to interfere with the binding of DHT to the precipitated cytosol and displayed an estimated Kd of 1.3-4.6 X 10(-8)M. Several digitalis preparations were similarly studied. Digitoxin and digitoxigenin also interfered with the binding of [3H]DHT and had an estimated Kd of 0.8-3.6 X 10(-8)M. Digoxin interacted less strongly and its estimated Kd was 10(-6)M. We believe these results suggest an interaction of spironolactone and digitalis with the DHT receptor and may help explain some of their antiandrogenic actions in the rat and in man.

Rapid regression of fetal adrenal zone and absence of adrenal reticular zone in the marmoset.

Developmental changes in plasma dehydropiandrosterone (DHA) and in adrenal histology were studied in several marmoset species (Callithrix jacchus and Saguinus labiatus, nigricollis, and fuscicollis) to evaluate these primates as experimental models for the study of fetal adrenal zone regression. Newborn marmosets had a prominent fetal adrenal zone, plasma DHA levels above 1000 ng/dl, and plasma DHA sulfate (DHAS) levels of 140 micrograms/dl. The fetal zone regressed dramatically during the first week of life, paralleled by a marked decline in plasma DHA, the plasma DHA to cortisol ratio, and plasma DHAS. The adult marmoset, however, had no adrenal reticular zone and no evidence of adrenal DHA secretion; DHA levels in castrate adults were undetectable (less than 25 ng/dl). Thus, the marmoset represents the first example of a primate that has a regressing, DHA- and DHAS-secreting fetal adrenal zone but that does not subsequently develop a DHA-secreting adrenal reticular zone.

Metabolic clearance rate and uterotropic activity of 2-hydroxyestrone in rats.

2-Hydroxyestrone (2-OHE1) has much lower uterotropic potency than might be predicted from its uterine estrogen receptor affinity. 2-OHE1 displaces saturably bound [3H]estradiol from rat uterine cytosol with a competitive inhibition constant of 8.6 nM, while the dissociation constant for 17 beta-estradiol (E2) is 0.42 nM. From this ratio of binding affinities, one would expect some agonist or antagonist activity of 2-OHE1 to be apparent at doses roughly 20-50 times the minimum effective dose of E2. Instead, at doses of 2-OHE1 1000 times an effective dose of E2, no uterotropic effect was observed. When 2-OHE1 was injected together with E2 at dose ratios of 500:1, there was no antagonism of the effect of E2. To examine this discrepancy, the plasma MCRs (MCRpS) of E2 and 2-OHE1 were determined by continuous infusion techniques. Plasma concentrations of 2-OHE1 and E2 during control and infusion periods were measured by RIAs. The MCRp of 2-OHE1 averaged 50,000 ml/h, more than 100 times that of E2 (approximately 400 ml/h). The extraordinarily high MCRp of 2-OHE1 may explain the failure to observe any biological effects of this catechol estrogen, even at high doses. This rapid metabolism, presumably occurring in the blood compartment, should be considered in handling blood samples for RIA and in devising studies of the actions of catechol estrogens.

Studies on the transfer of steroid hormones across the blood-cerebrospinal fluid barrier in the Rhesus Monkey.

Indwelling canulae were placed in the lateral ventricles of the brains of six adult male rhesus monkeys, and the movement of estradiol-17beta (E2), testosterone (T), and 5alpha-dihydrotestosterone (DHT) across the blood-cerebrospinal fluid (CSF) barrier was measured. Serial samples of blood and CSF were collected every 30 minutes during a 6 hour infusion of the tritiated steroids, and the quantity of free steroid in the blood and CSF was determined by recrystallization to constant specific activity. During the course of the 6-hour infusion, the average CSF concentration of steroid, expressed as dpm/ml, was about 3.5% of the concurrent plasma level of E2, 1.6% of the concurrent plasma level of T, and 0.08% of the concurrent plasma level of DHT. It is proposed that these differences in steroid transfer can be attributed to differential binding of these steroids to testosterone-estrogen-binding globulin (TeBG) in plasma.

Studies on the transfer of steroid hormones across the blood-cerebrospinal fluid barrier in the rhesus monkey. II.

The movement of progesterone (P), cortisol (F) and 17-hydroxyprogesterone (17-OHP) across the blood-cerebrospinal fluid (CSF) barrier was determined using six adult male rhesus monkeys with indwelling canulae in the lateral ventricles of their brains. Tritiated steroids were given iv as a bolus followed by a constant 6 h infusion with continuous collection of CSF and periodic sampling of blood before and during the infusion. The amounts of authentic steroid in the plasma and CSF were determined by recrystallization to constant isotopic ratio and the amount of free plasma steroid was determined by equilibrium dialysis against Ringer's solution. Tritiated progesterone was undetectable in the pooled samples of CSF. The average concentration of tritiated 17-OHP in the CSF was 10.3% of the concurrent plasma level while the concentration of tritiated F was 22.5% of the concurrent plasma level. Plasma free steroid was found to be 2.2% for P, 6.3% for 17-OHP and 22.3% for F, showing a rough correlation between steroid entry into the CSF and free steroid concentration in plasma.