We have investigated <em>17</em> alpha-hydroxylase and C<em>17</em>,20-lyase activities and the presence of cytochrome P450c<em>17</em> mRNA in the esophagus, stomach, duodenum, and colon of adult rats of both sexes. All tissues converted [4-14C]pregnenolone mainly to dehydroepiandrosterone (DHEA) through the 5-ene-3 beta-hydroxysteroid route as opposed to the 4-ene-3-<em>ketosteroid</em> pathway in a control testicular incubate. Synthesis of dehydroepiandrosterone was particularly high in the duodenum and was found to be lower in the stomach, colon and esophagus, in decreasing order. 20 alpha-Hydroxypregnenolone and progesterone were also formed primarily by the esophagus and colon, respectively. P450c<em>17</em> mRNA was demonstrated by ribonuclease protection assay in the stomach and duodenum, but not in esophagus and colon. However, a 335 bp-long cDNA fragment, whose sequence corresponded to that of rat P450c<em>17</em> cDNA, was amplified by reverse transcription (RT) and polymerase chain reaction (PCR) from the poly(A)+ RNAs of all four tissues. This result was further confirmed by Southern blotting using a 794-bp testicular probe. The complete sequence of P450c<em>17</em> cDNA in the stomach and duodenum was identical to that reported for rat testis P450c<em>17</em> cDNA. No amplification and no positive signal in Southern blotting were observed with the total RNAs from adult male adrenal and spleen, which were taken as negative controls since they had been previously found unable to form androgens from pregnenolone. Although the levels of transcription in gonads, duodenum and stomach were found to be equivalent, as indicated by the RNase protection assay and semiquantitative RT-PCR assay, P450c<em>17</em> enzyme activity was much higher in the testis, pointing at a possible dissimilarity in the respective rates of mRNA translation. Thus, P450c<em>17</em> is differentially expressed in the rat gastrointestinal tract, where it leads to the synthesis of the sex steroid precursor DHEA, especially in the duodenum and stomach.

The enzyme <em>17</em>beta-hydroxysteroid dehydrogenase (<em>17</em>betaHSD) interconverts <em>17</em>-<em>ketosteroids</em> and <em>17</em>beta-hydroxysteroids. Five isoforms of the enzyme have been identified in the human, two of which (types 1 and 3) have been shown to catalyse the reduction reaction preferentially. The cDNA encoding mouse <em>17</em>betaHSD type 3 was isolated from testis cDNA using the RACE technique and primers based on the human sequence. The mouse protein is 305 amino acids in length which is five short of the human protein with four of these amino acids missing at the N-terminus. The predicted amino acid sequence is 72.5% identical and 94.8% similar to the human sequence. Tissue distribution of mRNA encoding both types 1 and 3 <em>17</em>betaHSD was studied using reverse transcription and the polymerase chain reaction (RT-PCR). Highest levels of type 1 mRNA were found in the ovary whereas highest levels of type 3 were in the testis. All other tissues tested contained mRNA encoding both isoforms of the enzyme although levels were markedly lower than in the gonads.

A male patient was suspected as olivo-ponto-cerebellar atrophy from the clinical and computed tomographic features at 34 years of age. Afterwards, his dysarthria and limb ataxia were slowly and steadily worsened. He was finally bed-ridden and unresponsive, and died of hyperpyrexia and general wasting at 37 years of age. It was noted that laboratory investigation gave lower values of urinary <em>17</em>-<em>ketosteroids</em> and <em>17</em>-hydroxycorticosteroids in comparison with those of normal subjects. Pathological investigation in autopsy showed that he had pathological features consistent with adrenoleukodystrophy accompanying the olivo-ponto-cerebellar atrophy; diffuse demyelination in the cerebrocerebellear white matter, distorted architecture and cytoplasmic striations in the adrenal cortex, and in addition, a pseudosystemic degeneration of the olivo-ponto-cerebellar system and subcortical gray matter.

The finding that urine cortisol excretion was increased in patients with hypokalaemic hypertension induced by licorice addiction led to this study on the effect of licorice in normal subjects. Thirteen normal volunteers ate either 100 or 200 g licorice for 1-4 weeks and assessment of pituitary-adrenal function was made before, during, and 1 week after cessation of licorice ingestion. Urine cortisol excretion more than doubled in 10 of 13 subjects (mean, 33.8 +/- 15.6 SD before and 83.3 +/- 56 SD micrograms/24 h at 1 week after commencing licorice) and excretion rates similar to those observed in Cushing's syndrome were seen in 7 subjects (range, 91-226, compared to normal range of 11-82 micrograms/24 h). Urine cortisol excretion remained significantly elevated (P less than 0.01) above control levels for at least 1 week after licorice was withdrawn. Despite these increases, urinary steroid metabolite (tetrahydrocortisol, tetrahydrocortisone, tetrahydro-11-deoxycortisol, <em>17</em>-ketogenicsteroids, and <em>17</em>-<em>ketosteroids</em>) excretion was not affected, plasma cortisol and ACTH values were unchanged, and normal 0800-1600-h diurnal variation of plasma cortisol was maintained. The direct intraadrenal infusion of the active mineralocorticoid component of licorice, glycyrrhetinic acid, in two sheep with autotransplanted adrenal glands failed to stimulate cortisol secretion acutely. It is concluded from these studies that the licorice-induced changes in cortisol excretion are not a result of adrenocoritcal stimulation but more likely represent a change in the renal handling of cortisol.

Thirty-one patients who had been treated for acromegaly for 1-21 years with conventional pituitary irradiation were re-examined. Immunoreactive growth hormone (GH) was measured in connection with an oral glucose load. Adrenal and gonadal functions were assessed on the basis of plasma cortisol and the urinary excretion of <em>17</em>-ketogenic steroids, <em>17</em>-<em>ketosteroids</em> and gonadotrophins. In evaluating the thyroid-pituitary axis the thyrotrophin-releasing hormone stimulation test (TRH) was used. Initially 30 patients had experienced definite benefit from the treatment but at the time of re-examination 10 still had clinically active disease and required another type of treatment. Normal GH levels (less than 5 ng/ml/1) were seen in only 12 patients. Skin thickness was normal in 15 out of 30. Thus the remission rate can be evaluated as being 67% as regards clinical activity, 50% with regard to skin thickness and 39% in terms of GH levels. Hypogonadism occurred in 12 patients (39%) and adrenal and thyroid failure in 5 patients each (16%). The response to TRH was within the normal range in 2 of the hypothyroid patients. In 22 euthyroid patients the mean increment in serum TSH in response to 200 mug of synthetic TRH was only 5.8 mU/1 which was significantly below the normal mean 12.5 mU/1. Furthermore, in 7 of these patients (32%) the response was absent or subnormal (less than 3.0 mU/1). This indicated that the pituitary is capable of secreting enough TSH for maintenance of an euthyroid state but that its capacity is limited. Conventional pituitary irradiation is not a very effective treatment in acromegaly but may still be recommended in selected cases.

Both pregnenolone and <em>17</em>-OH-pregnenolone were found to be higher in the plasma of patients with poorly controlled congential adrenal hyperplasia than in normal subjects. The plasma levels of these precursor steroids were significantly correlated with urinary <em>17</em>-<em>ketosteroid</em> and pregnanetriol excretion and with plasma testosterone. The mechanism where by plasma pregnenolone and <em>17</em>-OH-pregnenolone levels are elevated in patients with 21-hydroxylase deficiency is unknown, but the phenomenon of product inhibition is suggested as a possible explanation. As <em>17</em>-OH-pregnenolone in plasma is almost entirely of adrenal origin, its measurement promises to be useful in the management of patients with congenital adrenal hyperplasia. Acute stimulation with ACTH caused negligible changes in the plasma levels of pregnenolone and <em>17</em>-OH-pregnenolone and failed to distinguish between overly, appropriately, and under-treated patients. However, following repeated stimulation with repository ACTH, the steroid levels rose. These findings indicate limited adrenal responsiveness to ACTH following chronic glucocorticoid treatment of congenital adrenal hyperplasia, even in under-treated patients, and suggest that normal precursor steroid levels in plasma and normal <em>17</em>-<em>ketosteroid</em> and pregnanetriol excretion can only be achieved by the suppression of total steroidogenesis to less than that occurring in normal subjects.

A small transient increase in growth, the midgrowth spurt, has been observed in several growth studies in healthy children around the age of 7 yr. During this time adrenarche (the physiological increase in adrenal androgen secretion) also occurs. Although it is now well established that estrogen, not androgen, has a critical role in the male (and female) pubertal growth spurt, a direct effect of androgens on growth cannot be excluded. In accordance with published observations that growth is frequently accelerated in infants and young children with late-diagnosed 21-hydroxylase deficiency (before adequate androgen suppression), it has been speculated that the adrenarchal increase in adrenal androgen secretion in healthy children could be responsible for the midgrowth spurt. To test this hypothesis we studied long-term serial changes in urinary 24-h excretion rates of dehydroepiandrosterone sulfate and total <em>17</em>-<em>ketosteroid</em> sulfates in a group of healthy children (n = 12) in which yearly auxological measurements allowed the identification of a midgrowth spurt. Annual measurements of standing height were performed over periods of 6-9 yr before the onset of puberty. All children collected five to seven serial 24-h urine samples (1-yr intervals) each at the time of anthropometric examination. The peak of the midgrowth spurt was found to occur at a mean age of 6.8 +/- 1.0 yr. The average height of the midgrowth peak, i.e. average maximum gain in height velocity, was 0.9 cm/yr. In a peak-centered examination of individual 24-h excretion rates of dehydroepiandrosterone sulfate and <em>17</em>-<em>ketosteroid</em> sulfates, primarily weak 1-yr changes in adrenal androgens were observed until the peak was attained. Only after the peak did increments in urinary adrenal androgen output become more pronounced. ANOVA performed on the peak-centered dehydroepiandrosterone sulfate and <em>17</em>-<em>ketosteroid</em> sulfate excretion rates revealed a highly significant overall increase in adrenal androgen secretion from 2 yr before to 2 yr after the midgrowth spurt. After multiple testing, however, significant increments, when compared with the respective preceding androgen excretion levels, were for the first time seen 1 yr after the midgrowth spurt (dehydroepiandrosterone sulfate) or 2 yr later (<em>17</em>-<em>ketosteroid</em> sulfates). In conclusion, our longitudinal analysis of prepubertal growth and urinary adrenal androgen excretion in healthy children disproves the speculation that the midgrowth spurt is primarily caused by the adrenarchal increase in adrenal androgen secretion. However, the present results do not rule out a growth-accelerating effect of clearly higher androgen levels, as in premature adrenarche.

A virilizing adrenocortical adenoma was demonstrated in a yound female. Urinary <em>17</em>-<em>ketosteroid</em> excretion and subfractions, plasma testosterone level and urinary <em>17</em>-ketogenic steroid excretion were markedly increased. Dehydroepiandrosterone was the main constituent of the androgen excess. Otherwise the adrenocortical function was found to be normal as evaluated from measurements of cortisol, corticosterone and their metabolites under basal conditions and during dynamic tests. The androgen excess showed an unexpected response to trophic hormones. Human chorionic gonadotrophin stimulation resulted in a pronounced increase in androgen production, whereas no gonadotrophin-dependency could be demonstrated by means of the oestrogen suppression test. Similarly, no corticotrophin-dependency could be demonstrated by corticotrophin stimulation and suppression tests. Removal of the tumour resulted in normalization of the androgen production and no abnormal response upon human chorionic gonadotrophin administration could now be found. The inappropriate response of tumours to trophic hormones is discussed. It is concluded that the reliability of stimulation and suppression tests in determining the site of excessive androgen production should be accepted with reservation.

Depressive mood disorders and severe, chronic stressful life events (DSM-III-R criteria) were more frequently diagnosed in 106 breast cancer patients with respect to 37 patients with benign breast diseases (control group) (p < 0.001), during a stressful period such as hospital admission, diagnosis uncertainty, and when awaiting surgery. The study was performed 5 +/- 3 days before histological diagnosis had been done. Controls showed reduced 24-h diuresis and low catecholamine excretion (norepinephrine, NE; and epinephrine, E) that positively correlated with 24-h diuresis (p < 0.001) and CD3+ lymphocytes (p = 0.056), as during a normal stress response. In contrast, breast cancer patients showed increased 24-h diuresis (with respect to controls p < 0.001) and catecholamine values (p < 0.05). Patients' 24-h diuresis correlated positively with NE (p = 0.02) and <em>17</em>-<em>ketosteroids</em> (p = 0.004); blood cortisol correlated positively with CD3+ (p = 0.01), CD4+ (p = 0.02), CD8+ (p < 0.01), CD16+ (p = 0.01) lymphocytes and negatively with E (p < 0.03); catecholamines correlated negatively with CD8+ (p = 0.006). These preliminary data are discussed in relation to upregulation of the adrenergic system and the different mechanisms of immune system regulation involved in breast cancer patients, compared with those in subjects with benign breast disease. The differences in these mechanisms may be a result of an imbalance of the bi-directional regulatory circuit of the psycho-neuro-endocrine-immune system, caused by previous life stress or the presence of the tumor mass.

The effect of flutamide on cortisol metabolism was studied in eight patients with prostate cancer. Flutamide markedly decreased the formation of 3 alpha, <em>17</em>,21-trihydroxypregnane-11,20-dione (THF), and the 11-oxy-<em>17</em>-<em>ketosteroid</em> metabolites by 72%, 50%, and 46% respectively; however, 3 alpha, 11 beta, <em>17</em>,21-tetrahydroxy-5 alpha- pregnan-20-one was increased by 46%. The 24-h mean plasma cortisol concentration was not altered. The cortisol production rate decreased by an average of 53% (from 32.7 to 15.5 mg/24 h). The effect of the drug on plasma cortisol kinetics was studied in three patients. This showed that flutamide increased the t1/2 (from 80 to 108 min) but decreased the distribution volume (from <em>17</em>.8 to 13.8 liters) and the MCR (from 222 to 130 liters/24 h). The changes in THE and THF formation and in the t1/2 and MCR of [C]cortisol are similar to the effects observed in patients with intrahepatic cholestasis. It is suggested that in the case of flutamide these changes were also due to a cholestasis-producing effect of the drug on the liver. As the clinical response to the drug did not correlate with the cortisol metabolic changes, its therapeutic effect was probably not mediated by its effects on cortisol metabolism.

The effect of aminoglutethimide (AG) 4 X 250 mg (670 mg/m2 daily by mouth) on the excretion of the free cortisol (radio-immunoassay) and of its metabolites THE, THF-allo THF, cortolone and beta-cortolone (gas chromatography on capillary column) was studied monthly during 3-5 months in four adolescents (one girl, three boys) aged 15.9-18 years with Cushing's syndrome due to bilateral adrenal hyperplasia, but without evidence of a pituitary tumour. Under AG, all compounds decreased to a minimum after 1-2 months. The decrease of THE- THF-allo THF was most marked, followed by cortolone-beta-cortolone and free cortisol. The sum of the conjugated metabolites was normalized, but free cortisol remained high. A rebound was noted after 3-5 months of continued treatment. This was associated with clinical relapse (weight gain, increasing blood pressure). With AG, a non-steroidal peak appeared on the chromatograms. It is concluded that: (1) AG is only temporarily effective in diminishing the excretion of cortisol and its metabolites; (2) paradoxical increments of <em>17</em>-<em>ketosteroids</em> as reported from colorimetric analysis are non-specific and are probably due to the non-steroidal peak; and (3) AG appears to modify steroid catabolizing liver enzymes (inhibition of 5beta-reductase and/or 3alpha-dehydrogenase, possibly stimulation of 20alpha- and 20beta-dehydrogenases). This could increase the biological half-life of cortisol and contribute to the clinical rebound, which is due to increased ACTH-secretion. Because of its excellent short-term effects, AG appears to be useful to prepare patients for bilateral adrenalectomy.

Endocrine studies in girls with precocious thelarche were compared with those of normal girls of similar ages. Girls with precocious thelarche showed breast development and oestrogenised vaginal smears as the only signs of precocious sexual development. A few of the girls were tall and some had advanced bone ages but these two findings were not consistently present in the same patient. Hormones--such as serum oestradiol, oestrone, delta 4-androstenedione, progesterone, dehydroepiandrosterone (DHEA), follicle-stimulating hormone, luteinising hormone, and prolactin, and urinary <em>17</em>-<em>ketosteroids</em>--were measured. Only DHEA was different, being higher in girls with precocious thelarche. It is suggested that the high DHEA level may serve as a precursor for conversion to oestrogens in target tissues, breast, and vagina. This mechanism for oestrogenisation had been reported in other patients.

The use of tetramethylammonium hydroxide (T.M.A.H.) in place of potassium hydroxide in the Zimmermann reaction has been investigated. Although various pure steroids have different colour equivalents, a comparison of the results of <em>17</em>-<em>ketosteroid</em> and <em>17</em>-hydroxycorticosteroid estimations on a series of urines showed that the differences for the two reagents were only small and may be ignored. Tetramethylammonium hydroxide has the considerable advantage over potassium hydroxide of stability and need not be prepared freshly. The correction procedures available are discussed.

A 51-year-old postmenopausal multipara with high plasma testosterone levels and virilization, as demonstrated by hirsutism, balding, increased libido, and clitoromegaly, is presented. The plasma testosterone levels exceeded 330 ng/dl on 3 occasions. The plasma androstenedione, urinary <em>17</em>-<em>ketosteroids</em>, and <em>17</em>-hydroxycorticosteroids, as well as the computed axial tomography scan of the adrenal glands were normal. Although no pelvic mass was detected by sonography or pelvic exam, the patient was found to have small bilateral hilus cell tumors of the ovary. Following total abdominal hysterectomy and bilateral salpingo-oophorectomy, the plasma testosterone dropped to 14 ng/dl. This is the second case of bilateral hilus cell tumors of the ovary to be reported.

The effect of the serotonin antagonist, cyproheptadine (Cypro), on metyrapone-induced stimulation of the pituitary-adrenal axis was evaluated in nine normal subjects. The subjects underwent standard oral metyrapone tests (750 mg every four h, for six doses) while taking no medications and while receiving oral Cypro (4 mg every six h). Cypro administration caused a significant reduction in: 1) baseline <em>17</em>-hydroxycorticosteroid (<em>17</em>-OH) excretion (-31 +/- 7.2%), 2) the increase above baseline in 24 h <em>17</em>-OH excretion on the day after metyrapone (-32 +/- 4.9%), 3) serum 11-deoxycortisol concentrations 8 h (-45 +/- 5.5%) and 24 h (-18 +/- 3.6%) after the first dose of metyrapone, and 4) plasma ACTH concentration 8 h (-32 +/- 8.2%) and 24 h (-22 +/- 8.0%) after the first dose of metyrapone. When compared with the control test, Cypro administration did not alter: 1) baseline <em>17</em>-<em>ketosteroid</em> secretion, 2) the fall in serum cortisol concentration while taking metyrapone, 3) the serum free metyrapone concentration, 4) cortisol metabolism, 5) the adrenal response to ACTH, and 6) assay methods for measurement of serum and urinary corticosteroids. Our data suggest that Cypro can reduce pituitary-adrenal responsiveness by reducing plasma ACTH concentrations. If Cypro acts as a serotonin antagonist, our data lends support to the idea that serotoninergic mechanisms are important in the control of pituitary ACTH secretion in normal human subjects.

Thirteen anovulatory oligomenorrheic, hyperandrogenic, and normoprolactinemic women were treated with spironolactone (aldactone) throughout six consecutive menstrual cycles in a dosage of 100 to 150 mg/day. During this treatment a significant decrease in serum luteinizing hormone (LH), testosterone, prolactin, and <em>17</em>-<em>ketosteroid</em> values were observed that were accompanied by ovulation in 11 women (85%), according to basal body temperature (BBT) and progesterone values. In addition, improvement of hirsutism was observed in 9 (70%) and restoration of regular cycles in 11 (85%) of the patients. The side effects observed were mild and did not lead to interruption of the treatment. Our data suggest that the antiandrogenic properties of spironolactone render it a suitable agent in the treatment of anovulatory, oligomenorrheic, and hyperandrogenic women.