An NAD+-linked <em>17</em> beta-hydroxysteroid dehydrogenase was purified to homogeneity from a fungus, Cylindrocarpon radicicola ATCC 11011 by ion exchange, gel filtration, and hydrophobic chromatographies. The purified preparation of the dehydrogenase showed an apparent molecular weight of 58,600 by gel filtration and polyacrylamide gel electrophoresis. SDS-gel electrophoresis gave Mr = 26,000 for the identical subunits of the protein. The amino-terminal residue of the enzyme protein was determined to be glycine. The enzyme catalyzed the oxidation of <em>17</em> beta-hydroxysteroids to the <em>ketosteroids</em> with the reduction of NAD+, which was a specific hydrogen acceptor, and also catalyzed the reduction of <em>17</em>-<em>ketosteroids</em> with the consumption of NADH. The optimum pH of the dehydrogenase reaction was 10 and that of the reductase reaction was 7.0. The enzyme had a high specific activity for the oxidation of testosterone (Vmax = 85 mumol/min/mg; Km for the steroid = 9.5 microM; Km for NAD+ = 198 microM at pH 10.0) and for the reduction of androstenedione (Vmax = 1.8 mumol/min/mg; Km for the steroid = 24 microM; Km for NADH = 6.8 microM at pH 7.0). In the purified enzyme preparation, no activity of 3 alpha-hydroxysteroid dehydrogenase, 3 beta-hydroxysteroid dehydrogenase, delta 5-3-<em>ketosteroid</em>-4,5-isomerase, or steroid ring A-delta-dehydrogenase was detected. Among several steroids tested, only <em>17</em> beta-hydroxysteroids such as testosterone, estradiol-<em>17</em> beta, and 11 beta-hydroxytestosterone, were oxidized, indicating that the enzyme has a high specificity for the substrate steroid. The stereospecificity of hydrogen transfer by the enzyme in dehydrogenation was examined with [<em>17</em> alpha-3H]testosterone.

The corticotropic action of a synthetic ACTH derivative, D-Ser1 Lys<em>17</em>,18-1-18ACTH, after intranasal insufflation of 1 mg of the peptide was evaluated in 14 normal volunteers. Plasma cortisol and urinary <em>17</em>-ketogenic steroids and <em>17</em>-<em>ketosteroids</em> rose significantly over control values in all subjects (P less than 0.001). Plasma cortisol remained above the normal range for comparable clock times for 18 hr, but fell below 15 mug/100 ml after 12 hr. No evidence for suppression of the hypothalamo-pituitary axis was found the day after ACTH administration, as judged by normal plasma and urinary steroid values and normal diurnal variation. Intranasal administration of long-acting ACTH derivatives may provide a simple, painless and effective way to stimulate endogenous corticosteroid secretion.

A family with nine children, three with male pseudohermaphroditism due to testicular deficiency of <em>17</em>-<em>ketosteroid</em> reductase activity (<em>17</em>-KSR) and four with congenital hypothyroidism is presented. The three subjects with <em>17</em>-KSR deficiency were raised as females until puberty, at which time they assumed a male gender role. Only one developed gynecomastia. Laparotomy on one of the three patients revealed normal epididymi and vas deferens with absence of Mullerian structures. Testicular biopsy in all three showed Leydig cell hyperplasia, hyalinization of the tubular basement membrane, normal Sertoli cells and maturational arrest at the spermatogonial stage. The endocrine profile in peripheral blood revealed markedly increased plasma androstenedione concentrations but normal testosterone, dihydrotestosterone, progesterone, <em>17</em>-hydroxyprogesterone, and dehydroepiandrosterone. The levels of estradiol and estrone and of LH and FSH were elevated. Genital skin fibroblasts from the three patients exhibited normal dihydrotestosterone-binding activity and 5 alpha-reductase activity. Congenital hypothyroidism affected one of the three siblings with male pseudohermaphroditism. All four hypothyroid patients had thyroid enlargement and significant titers of circulating antithyroglobulin but not antithyroid microsomal antibodies. Neither the locus for the <em>17</em>-KSR enzyme nor that for congenital hypothyroidism were linked to the histocompatibility leucocyte antigen complex in this sibship. Transmission of the trait for both congenital hypothyroidism and <em>17</em>-KSR deficiency appeared to be autosomal recessive.

Thirty-one women with idiopathic hirsutism were evaluated for partial 11- and 21-hydroxylase adrenocortical enzyme deficencies. Twenty-four hour urine collections for <em>17</em>-<em>ketosteroids</em>, <em>17</em>-hydroxycorticoids, tetrahydro compound S (THS), and -pregnanetriol were obtained basally and following a continuous 24 hours infusion of alpha 1-24 ACTH (cosyntropin). The results were compared to those in eight normal, nonhirsute women studied under identical conditions. Normal control subjects and 18 of 31 hirsute female patients (Group I) showed similar small increments in the excretion of THS and pregnanetriol following the infusion of cosyntropin. Thirteen hirsute women (Group II) showed cosyntropin-stimulated increments in either THS and/or pregnanetriol that were significantly greater than the mean response of the control group. The cosyntropin-stimulated increments in <em>17</em>-<em>ketosteroid</em> excertion and basal sebum production rates were also significantly greater in Group II. The results are consistent with partial II - and/or 21-adrenocortical hydroxylase deficiencies in some hirsute women whose condition would have previously been designated as "idiopathic." Thus, prolonged ACTH stimulation testing may identify patients who might benefit from glucocorticoid suppression therapy.

In groups of women taking oral contraceptives and in control groups of women, the serum levels of cortisol, protein-bound iodine, and total thyroxine were measured together with the T(3) binding index. The daily excretion in the urine of free cortisol, <em>17</em>-hydroxycorticosteroids, <em>17</em>-<em>ketosteroids</em>, pregnanediol, pregnanetriol, total oestrogens, total catecholamines, and 4-hydroxy-3-methoxymandelic acid was also assayed. The frequency distribution of the values obtained indicates that oral contraceptives have a marked influence on the endocrine environment. The smallest deviations were observed in urinary excretion of total catecholamines and of 4-hydroxy-3-methoxymandelic acid. In some individuals the hormone assays were continued throughout the menstrual cycle. The morning and afternoon levels of serum cortisol tended to increase during the period when the oral contraceptive was being taken.

To determine the value of 3 alpha-androstanediol glucuronide (3-AG) measurements in children with congenital adrenal hyperplasia, we compared serum 3AG, <em>17</em>-hydroxyprogesterone (<em>17</em>-OHP), androstenedione (A), testosterone (T) and dihydrotestosterone (DHT) levels and 24-h urinary <em>17</em>-<em>ketosteroid</em> (<em>17</em>-KS) excretion in 42 female children with congenital adrenal hyperplasia due to 21-hydroxylase deficiency, including 27 with the simple virilizing and 15 with the salt-losing form. Their mean age was 74.5 +/- 48.5 months (range, 6-194 months). Twenty-four-hour urinary <em>17</em>-KS excretion and serum 3-AG, A, T, DHT and <em>17</em>-OHP levels were measured in the patients. The values were less than the mean + 2 SD of the control group in 63%, 74%, 67%, 69%, 60% and 31% of the patients, respectively. Serum 3-AG levels correlated with 24-h urinary <em>17</em>-KS excretion (r = 0.66) and plasma A (r = 0.80), <em>17</em>-OHP (r = 0.56), T (r = 0.79) and DHT (r = 0.62) levels. We conclude that serum 3-AG is a useful metabolic index in the management of children with congenital adrenal hyperplasia.

A 32-year-old nulligravida, with a history of abdominal hysterectomy for multiple leiomyomas of the uterus, was seen initially with signs of masculinization. Normal values for <em>17</em>-<em>ketosteroids</em>, <em>17</em>-hydroxycorticosteroids, and plasma cortisol eliminated the adrenal as the source of the excess androgen as well as Cushing syndrome. Increased plasma testosterone and androstenedione levels in the peripheral blood as well as in the right ovarian vein sampling implicated the right ovary, which revealed a lipid cell tumor (0.5 to 1.3 cm in maximum dimension).

OBJECTIVE

Cushing's syndrome due to adrenal adenoma or adrenocortical carcinoma is rare. To understand better the clinical and biochemical presentation of this disorder, as well as therapy efficacy and patient survival, we conducted a retrospective review.

METHODS

Between August 1971 and April 1994, 40 patients presented to our institution with adrenal Cushing's syndrome (27 adenomas and 13 carcinomas). These groups were analyzed with respect to clinical signs and symptoms preoperatively and postoperatively, biochemical analysis, length of postoperative steroid replacement therapy, disease recurrence and patient survival. Followup was obtained by chart review and telephone interviews and averaged 59.6 +/- 66.4 and 47.6 +/- 56.2 months for adenoma and carcinoma patients, respectively.

RESULTS

Women predominated in both groups (26 of 27 adenomas, 11 of 13 carcinomas), and tumors affected the left adrenal gland more frequently (19 of 27 adenomas, 9 of 13 carcinomas). Adenoma patients were younger than carcinoma patients (39.6 +/- 14.4 versus 51.5 +/- 16.6 years, p = 0.026) and presented with smaller tumors (3.3 +/- 1.0 versus 8.6 +/- 4.5 cm., p = 0.001). There was a trend toward increased incidence of glucose intolerance among carcinoma patients but no significant differences in clinical signs or symptoms between adenoma and carcinoma patients could be made. Similarly, while there was no significant difference in biochemical evaluation of adenoma versus carcinoma patients, 24-hour urinary free cortisol and serum lactate dehydrogenase levels tended to be higher among carcinoma patients. In addition <em>17</em>-<em>ketosteroid</em> and dehydroepiandrosterone sulfate levels were more elevated in carcinoma than in adenoma patients, and several adenoma patients actually had subnormal levels. Among adenoma patients mean length of steroid replacement therapy was 16.8 +/- 9.1 months. However, 7 adenoma patients (25.9%) required greater than 24 months of exogenous steroids, and only 1 of these patients was subsequently weaned off steroid replacement. There were no recurrences among adenoma patients, although there was 1 perioperative death due to hypoglycemia. Ten (76.9%) carcinoma patients had recurrences at a mean followup of 33 months. The 3 and 5-year survival rates were 41.5 and 31.2%, respectively.

CONCLUSIONS

While presenting signs and symptoms and hormonal analysis may suggest benign or malignant disease, only tumor size and patient age are reliable preoperative indicators of adrenal adenoma versus adrenocortical carcinoma among patients with adrenal Cushing's syndrome. Surgery is curative for adenoma patients, but lifelong steroid replacement may be required. Survival remains poor among carcinoma patients.

Serum 3 alpha-androstanediol glucuronide (3 alpha-diol G) measurements may indicate the extent of androgen metabolism and action in target tissues. To test this supposition we measured serum 3 alpha-diol G concentrations in 23 women with congenital adrenal hyperplasia due to 21-hydroxylase deficiency, including 13 with the salt-losing and 10 with the simple virilizing form, while they were receiving glucocorticoid and, in some cases, mineralocorticoid therapy. Their mean age was 28.3 yr (range, <em>17</em>.9-38.7 yr). Twenty-four-hour urinary <em>17</em>-<em>ketosteroid</em> excretion, plasma androstenedione and testosterone levels, and serum 3 alpha-diol G levels were measured during the follicular phase. The values were within or below the normal range in 87%, 78%, 70%, and 91% of the patients, respectively. By contrast, plasma <em>17</em>-hydroxyprogesterone levels were normal in only 12% of the patients. Serum 3 alpha-diol G levels correlated well with 24-h urinary <em>17</em>-<em>ketosteroid</em> excretion (r = 0.75) and plasma <em>17</em>-hydroxyprogesterone (r = 0.77), androstenedione (r = 0.84), and testosterone (r = 0.93) levels. The serum 3 alpha-diol G levels were not significantly different in the women with the salt-losing form and those with the simple virilizing form. However, they were significantly lower (P less than 0.05) in the women with normal menses compared to those with abnormal menses. This finding underscores the validity of serum 3 alpha-diol G measurements as indicators of androgen production and metabolism in women. The excellent correlation between the serum 3 alpha-diol G levels and standard measures of control indicates that the former measurement may be a useful adjunct in the management of women with congenital adrenal hyperplasia.

Adrenocortical tumors in children are extremely rare, accounting only for 0.3-0.4% of all neoplasms in this age. Most frequently they secrete hormones, resulting in virilization, Cushing's syndrome or feminization, while the non-functioning ones are unusual. The authors describe 12 cases observed in 13 years (1976-1989), with a mean age of 5 years. 9 cases showed virilization, 4 presented with Cushing's syndrome and in 5 patients an abdominal mass was palpable. One case was affected by Beckwith-Wiedemann's syndrome. I.V. urography was performed in 8 patients, arteriography in 4 and since 1982 all patients were submitted to abdominal sonography and CT scan or MR imaging. Urinary <em>17</em>-<em>ketosteroids</em>, <em>17</em>-hydroxycorticoids and serum testosterone and cortisol were tested in all children. Dexamethasone suppression test was performed in 7. All patients were treated with surgery which seems to be the most suitable treatment, while the real effectiveness of treatment by drug therapy with suppressors of steroidogenesis is not confirmed in children. Histopathological examination showed typical features of adenoma in 5 cases, of adenocarcinoma in 4, while three cases revealed border line forms classified as "atypical adenomas". At the moment 10 patients are alive with a follow-up ranging from 18 months to 14 years, while 2 children with adenocarcinoma are dead.

Total 24 hour urinary <em>17</em>-<em>ketosteroid</em> and serum testosterone (T), androstenedione (delta), dehydroepiandrosterone (DHEA), dehydroepiandrosterone sulfate (DS), and cortisol levels were measured before and during four days of dexamethasone administration in 28 hirsute patients and 10 women with normal ovulatory cycles. Both the base-line urinary <em>17</em>-<em>ketosteroids</em> and serum androgen levels were significantly higher (p less than 0.05) in hirsute than in normal subjects. Cortisol levels were similar in the two groups. Dexamethasone administration resulted in a significant suppression (p less than 0.05) of all the urinary and serum androgen and cortisol levels in both groups. At the end of suppression the serum DHEA, DS and cortisol levels were similar, while the urinary <em>17</em>-<em>ketosteroids</em> and serum T and delta levels were still significantly higher (p less than 0.05) in the hirsute than in normal women. There was poor correlation between total urinary <em>17</em>-<em>ketosteroid</em> and serum androgen results. These finding suggest there is a dual abnormality of androgen production in hirsute patients. The adrenal glands appear to secrete increased quatities of DHEA and DA, while the ovaries appear to produce elevated amounts of T and delta.

To evaluate the relationship between the secretion of gonadotropins and adrenal androgens, patients with gonadal agenesis were evaluated by (a) administering human luteinizing hormone (hLH) for 5 days with or without estrogen pretreatment to agonadal patients who had prepubertal LH levels; (b) correlating circulating gonadotropin levels with adrenal androgens in 45 patients; and (c) comparing adrenal androgens with gonadotropins after long-term administration of estrogen or androgens. Results are as follows: (a) No alteration in serum concentrations of dehydroepiandrosterone (DHA), dehydroepiandrosterone sulfate (DHAS), estrone (E1), testosterone (T), or in excretion of urinary <em>17</em>-<em>ketosteroid</em> (<em>17</em> KS) occurred after the administration of hLH. (b) No clearcut relationship between endogenous level of LH or FSH and DHA OR DHAS was demonstrated although a coincident increase of all hormones with age occurred. (c) Administration of estrogen to patients with gonadal agenesis did not affect their levels of DHA and DHAS although those patients given androgen developed higher DHAS, but not DHA, levels. Hence, increasing gonadotropin concentrations would not appear to be a primary etiologic factor in the maturation of the adrenal.

A case of maternal virilization associated with bilateral luteomas of pregnancy is described. Urinary <em>17</em>-<em>ketosteroids</em> and plasma testosterone fell from markedly abnormal values to normal limits within 2 weeks of delivery. However, symptoms of virilization persisted in the postpartum state. A possible relation between polycystic ovarian syndrome in the nonpregnant state and luteoma of pregnancy is proposed.