We attempted to determine compounds in human urine which, differing from cortisol (<em>17</em>-OHCS), show high values in healthy individuals, decrease with failing health, clearly decline with advancing disease and finally reach very low values in severe disease. We have eventually established that <em>17</em>-<em>ketosteroid</em> sulfate conjugates (<em>17</em>-KS-S) are the compounds we were searching for. Hans Selye regarded stress as the rate of wear and tear and <em>17</em>-OHCS as its indicator, but we considered that, differing from inanimate objects, living organisms consume energy to cope with stress and "repair" "wear and tear" of the tissue and "recover" its function. This concept led us to determine the organism's requirement for two mechanisms: "Wear and tear" which could be represented by the secretion of <em>17</em>-OHCS (Selye), and "repair and recovery", which could be determined by the amounts of <em>17</em>-KS-S derived from dehydroepiandrosterone (DHEA)-sulfate, a product of the adrenal cortex, which enhances neuronal survival, longterm memory, maintains the function of peripheral tissues, and stimulates immune system (<em>17</em>-KS mainly consists of <em>17</em>-KS-S and <em>17</em>-KS glucuronides <em>17</em>-KS-G, the latter derived from cortisol, DHEA and testosterone). We hold that stress response is a series of biological process, beginning from CRH.ACTH and catecholamines, cortisol (<em>17</em>-OHCS), followed by insulin, acting as an anabolic agent and finally DHEA (<em>17</em>-KS-S) leads the tissues to repair by utilizing produced energy. Balanced changes of hormones, such as <em>17</em>-KS-S, <em>17</em>-OHCS, cortisol, catecholamines and insulin are seen in healthy individuals under healthy lifestyles and disruption of the balance brings apparent reduction in <em>17</em>-KS-S. From this standpoint, we wish to develop research in interrelations between biologically antagonistic <em>17</em>-KS-S and <em>17</em>-OHCS, focusing particularly at <em>17</em>-KS-S, which represents a contact point for mind and body, the healthy state of which is kept by appropriate sleeping and exercise programs on the basis of adequate food intake. The above three factors, <em>17</em>-KS-S, <em>17</em>-OHCS, and <em>17</em>-KS-G, are expressed in creatinine ratio (mg/g creatinine).

In the steroidogenic pathway, 3 beta-hydroxysteroid dehydrogenase/isomerase (3 beta-HSD) is a key enzyme which controls the formation of delta 4-3-<em>ketosteroids</em> from delta 5-3-beta-hydroxysteroids. Herein, we used primary cultures of ovine adrenocortical (OAC) cells to study the effects of ACTH and transforming growth factor beta (TGF-beta) on 3 beta-HSD activity, protein and mRNA levels. TGF-beta has been previously reported to be a potent inhibitor of steroid formation in OAC cells. By using an antibody against human placental 3 beta-HSD, we showed that ACTH-treatment had a dose- and time-dependent stimulatory effect on 3 beta-HSD protein amount. This effect was maximal using 10(-9) M ACTH after a 48 h treatment. When included in the treatment medium, TFG-beta inhibited this stimulation by ACTH in a dose- and time-dependent manner. We also used a human 3 beta-HSD cDNA probe to demonstrate that the effect of both ACTH and TFG-beta were exerted at the mRNA level with maximal effects observed using 10(-9) M for ACTH and 1 ng/ml for TGF-beta. Bu2cAMP mimicked the effects of ACTH, and TGF-beta had an inhibitory effect on this stimulation. It appears from these data that TGF-beta is a negative regulator of 3 beta-HSD expression in OAC cells. The inhibitory effect of TGF-beta on 3 beta-HSD was contrasted to the TGF-beta effect on <em>17</em> alpha-hydroxylase cytochrome P-450 (P-450<em>17</em> alpha). While the levels of both enzymes decreased, that of 3 beta-HSD was less sensitive than that of P-450<em>17</em> alpha which decreased following TGF-beta treatment to non-detectable levels. The different sensitivities of steroidogenic enzymes to factors which regulate growth and differentiation such as TGF-beta may play a role in determining the nature of steroids released from adrenocortical cells.

The two steroidal <em>17</em> beta-oxiranes, spiro-<em>17</em> beta-oxiranyl-delta 4-androsten-3-one (4 beta) and spiro-<em>17</em> beta-oxiranylestra-1,3,5(10),6,8-pentaene-3-ol (5 beta) are active site-directed irreversible inhibitors of bacterial delta 5-3-<em>ketosteroid</em> isomerase. For each inhibitor, a stoichiometry of one molecule of steroid to one enzyme subunit was found. The inhibited enzyme was denatured and subjected to digestion by trypsin. The tryptic maps show two distinct steroid-bound peptides for both 4 beta- and 5 beta-inhibited isomerase. In each case, the two modified peptides are derived from residues 14 to 45 of the isomerase. Each of the steroid-bound peptides of the 4 beta-inhibited enzyme was subjected to further proteolytic digestion and the site of steroid attachment was found to be Asp-38 in each of the inactivation products. These results are interpreted to indicate that "backwards binding" is an important feature of the binding of steroids to delta 5-3-<em>ketosteroid</em> isomerase.

More than 800 men referred for infertility have been evaluated in a male infertility clinic. Routine hormonal screening of these patients has included at various times thyroid function studies, 24-hour urine samples for <em>17</em>-<em>ketosteroids</em> and pregnanetriol, serum dehydroepiandrosterone-sulfate, serum <em>17</em>-alpha-hydroxy progesterone, testosterone and prolactine in some instances. The results of this hormonal screening are analyzed and discussed from the point of view of positive diagnostic findings and success of treatment. An outline and recommendations for the routine use of such studies are made.

A 27-year-old phenotypic female presented with primary amenorrhea, severe hypertension, and hypokalemia. At the age of puberty sexual development had not occurred; in particular, sexual hair had not grown. Past history revealed an episode of subarachnoid hemorrhage and several episodes of ventricular tachyarrhythmia. Karyotype was 46, XY. The steroids requiring <em>17</em>-hydroxylation (cortisol, testosterone, pregnanetriol, <em>17</em>-<em>ketosteroids</em>, <em>17</em>-hydroxycorticosteroids) were low, while those not requiring <em>17</em>-hydroxylation (progesterone, deoxycorticosterone, corticosterone) were high, demonstrating <em>17</em>-hydroxylase deficiency. The corticosterone/deoxycorticosterone ratio was relatively low, suggesting an associated partial deficiency of 11-hydroxylase.

A female patient with hypertension, hypogonadism and hypokalaemia due to <em>17</em>alpha-hydroxylase deficiency is described, in whom the diagnosis was initially based on the finding of low urinary <em>17</em>-<em>ketosteroids</em> and <em>17</em>-hydroxycorticosteroids, in the presence of high plasma fluorometric 11-hydroxycorticoids. Later studies confirmed the diagnosis by demonstrating low circulating levels of <em>17</em>alpha-hydroxyprogesterone and the steroids derived from this precursor: 11-deoxycortisol, cortisol, oestradiol and testosterone. Plasma progesterone, corticosterone and deoxycorticosterone (DOC) were greatly increased, but plasma and urine aldosterone were very low. The initial subnormal cortisol and 11-deoxycortisol levels responded to tetracosactrin administration, suggesting that the adrenal was not maximally stimulated by endogenous ACTH and indicating that the <em>17</em>alpha-hydroxylase deficiency was incomplete. Plasma and urinary aldosterone levels were still suppressed after 12 months treatment with prednisone despite normalization of the previously suppressed renin levels. The findings indicate that this uncommon cause of medically-controllable juvenile hypertension can be identified without specific steroid assays.

Although cystic lesions of the breast are not precancerous per se, statistical studies have indicated that this condition predisposes a 2- to 4-fold greater risk for breast cancer. Seeking a hormonal etiology to this correlation, investigators have analyzed breast cyst fluid ( BCF ) for steroids and have compared the levels to those in the blood. The <em>17</em>-<em>ketosteroids</em>-androsterone, dehydroisoandrosterone and their sulfates are elevated in BCF . The same is true for estrone sulfate and estradiol-3-sulfate. We have found the most dramatic differences with estriol-3-sulfate (E3-3S), the concentration of which ranged from 187-6134 pg/ml in over 40 specimens analyzed, whereas in 12 serum specimens from normal women, E3-3S was barely detectable. The origin of E3-3S is not known. [3H]E3-3S is not concentrated in BCF following its injection into an arm vein. The blood half-life of [3H]E3-3S is much lower than that of estrone sulfate. Samples of breast nipple aspirates from normal women were also analyzed for E3-3S. None could be detected. The best explanation of the data accumulated thus far is that E3-3S is synthesized at the epithelial lining of the cyst and released into the BCF , from which its efflux is inefficient.