The enzyme 5beta-reductase catalyzes the reduction of the 4-ene of 3-<em>ketosteroids</em>, converting them into 5beta-dihydro-3-<em>ketosteroids</em> and, thus, could be involved in the metabolism of 4-cholestene-3-one, progesterone, <em>17</em>~-hydroxyprogesterone, aldosterone, corticosterone, cortisol, 4-androstenedione, and testosterone. In this study, we report the genomic structure of a human 5beta-reductase gene, its tissue distribution, the characterization of an intronless pseudogene and the substrate selectivity of the enzyme. The gene coding for the active 5beta-reductase contains nine exons like most members of the aldo-keto reductase family, but the sequence covered by the gene, more than 42 kb, is much longer than the sequence of other members of this family. There are many large introns, especially introns 3, 4 and 7 that span approx. 7 kb, and intron 1 that contains more than 10 kb. Northern blot analysis showed three band sizes of 1.3, 2.2 and 2.7 kb. The 1.3 and 2.7 kb bands are highly expressed in the liver while weaker 2.2 and 1.3 kb bands have been observed in the testis and colon, respectively. We also identified an intronless gene having 86% homology with the 5beta-reductase cDNA sequence. Since its sequence contains many stop codons, this gene is most probably a pseudogene. To determine more precisely the substrate selectivity of the enzyme, we established a stable cell line expressing human 5beta-reductase in transformed embryonic kidney (HEK-293) cells. The transfected cells efficiently catalyze the transformation of progesterone, androstenedione, <em>17</em>alpha-hydroxyprogesterone and testosterone. However, they catalyze much less efficiently the transformation of compounds containing an 11beta-hydroxy group, such as aldosterone, corticosterone and cortisol. In addition to its role in cholesterol catabolism, it is well recognized that 5beta-reductase inactivates active androgens. Indeed, 5beta-dihydrotestosterone (5beta-DHT), the product of the reduction of testosterone by 5beta-reductase, is not active while its 5~-isomer (DHT) is the most potent natural androgen. Recent findings show that 5beta-pregnanes are active ligands in the induction of CYP3A through the orphan receptor hPAR. Our results thus open an opportunity for studying the new role of 5beta-reductase in the formation of a new type of active steroids.

<em>17</em>beta-Hydroxysteroid dehydrogenase (<em>17</em>beta-HSD) type 5 has been cloned from human prostate and is identical to type 2 3alpha-HSD and is a member of the aldo-keto reductase (AKR) superfamily; it is formally AKR1C3. In vitro the homogeneous recombinant enzyme expressed in Escherichia coli functions as a 3-keto-, <em>17</em>-keto- and 20-<em>ketosteroid</em> reductase and as a 3alpha-, <em>17</em>beta- and 20alpha-hydroxysteroid oxidase. The enzyme will reduce 5alpha-DHT, Delta(4)-androstene-3,<em>17</em>-dione, estrone and progesterone to produce 3alpha-androstanediol, testosterone, <em>17</em>beta-estradiol and 20alpha-hydroxprogesterone, respectively. It will also oxidize 3alpha-androstanediol, testosterone, <em>17</em>beta-estradiol and 20alpha-hydroxyprogesterone to produce 5alpha-androstane-3,<em>17</em>-dione, Delta(4)-androstene-3,<em>17</em>-dione, and progesterone, respectively. Many of these properties are shared by the related AKR1C1, AKR1C2 and AKR1C4 isoforms. RT-PCR shows that AKR1C3 is dominantly expressed in the human prostate and mammary gland. Examination of k(cat)/K(m) for these reactions indicates that as a reductase it prefers 5alpha-dihydrotestosterone and 5alpha-androstane-3,<em>17</em>-dione as substrates to Delta(4)-androstene-3,<em>17</em>-dione, suggesting that in the prostate it favors the formation of inactive androgens. Its concerted reductase activity may, however, lead to a pro-estrogenic state in the breast since it will convert estrone to <em>17</em>beta-estradiol; convert Delta(4)-androstene-3,<em>17</em>-dione to testosterone (which can be aromatized to <em>17</em>beta-estradiol); and it will reduce progesterone to its inactive metabolite 20alpha-hydroxyprogesterone. Drawing on detailed structure-function analysis of the related rat 3alpha-HSD (AKR1C9), which shares 69% sequence identity with AKR1C3, it is predicted that AKR1C3 catalyzes an ordered bi bi mechanism, that the rate determining step is k(chem), and that an oxyanion prevails in the transition state. Based on these relationships steroidal-based inhibitors that compete with the steroid product would be desirable since they would act as uncompetitive inhibitors. With regards to transition state analogs steroid carboxylates and pyrazoles may be preferred while 3alpha, <em>17</em>beta or 20alpha-spiro-oxiranes may act as mechanism-based inactivators.

Widespread use of contemporary imaging techniques (ultrasound, computerized axial tomography, and magnetic resonance imaging scans) have led to the incidental discovery of asymptomatic adrenal neoplasms with increasing frequency. Patients with such adrenal "incidentalomas" typically have no clinical manifestations of adrenal cortical hyperfunction at the time of discovery. We have studied 122 patients with asymptomatic adrenal masses ranging in size from 2 to 7 cm in diameter from 1978 to 1988. Selected patients, after adrenal metastases, pheochromocytoma, myelolipomas, and cysts were ruled out, were further evaluated for adrenal cortical hyperfunction by measuring urinary <em>17</em>-hydroxysteroids, <em>17</em>-<em>ketosteroids</em>, and free cortisol, serum A.M., P.M. cortisol, and plasma ACTH levels. These values were also measured before and after dexamethasone suppression. NP-59 adrenal scintiscans were performed on all patients. Six patients were identified with sub-clinical Cushing's syndrome. Baseline cortisol levels were normal in each of these patients. Loss of diurnal rhythm appeared to be the most sensitive indicator of abnormal adrenal cortical function. When adrenalectomy is performed in such patients, especially when contralateral adrenal gland suppression is evidenced by NP-59 scanning or other biochemical assessment, perioperative steroids should be administered in a manner similar to that used for patients with symptomatic Cushing's syndrome. Unilateral adrenalectomy in a patient with an asymptomatic adrenal adenoma, insufficiently studied, may result in Addisonian crisis.

Serum concentrations of <em>17</em>OH-progesterone were studied serially over 24 hours in 13 treated and untreated patients with the C21 hydroxylase form of congenital adrenal hyperplasia. The results were correlated with measurements of plasma renin activity, serum electrolytes, and urinary <em>17</em>-<em>ketosteroids</em> and pregnanetriol. In 500 healthy subjects from birth to adult life, serum <em>17</em>OH-pregesterone levels ranged from 5 to 315 ng/dl. In untreated CAH, serum <em>17</em>OH-progesterone was markedly elevated (2,000 to 80,000 ng/dl). Treatment with cortisol (20 to 30 mg/m2/day in 3 doses) resulted in normal serum <em>17</em>OH-progesterone levels in both non-salt-losing and salt-losing patients receiving adequate mineralocorticoid. Even slightly inadequate mineralocorticoid therapy (shown by high plasma renin activity with normal serum electrolytes) was associated with elevated <em>17</em>OH-progesterone (to 65,000 ng/dl) in spite of usually effective doses of cortisol. Some patients showed isolated <em>17</em>OH-progesterone elevations (usually early morning), a situation which requires only revision of the cortisol dosage schedule without an increase in total dosage. The data confirm the value of <em>17</em>OH-progesterone assays in both the diagnosis and management of CAH. Taken together with determinations of plasma renin activity, serum <em>17</em>OH-progesterone assays can permit more exact control of CAH without excessive doses of glucocorticoid.

Leydig cells are the major source of androgens in the male, and it is likely that these cells are also targets for androgen action. The role of androgen action in regulating the development of Leydig cell function has been determined using the testicular feminized (Tfm/Y) mouse, which lacks androgen receptors. In Tfm mice, the testes fail to descend at the normal time (25 days), and testicular descent was, therefore, surgically prevented in control (+/Y) animals. The activity of 3 beta-hydroxysteroid dehydrogenase increased during development from 5-40 days in control and Tfm mice, with no significant difference between the groups. In control animals, <em>17</em> alpha-hydroxylase activity was low from 5-25 days, at which time there was a 26-fold increase up to 40 days. In the Tfm group, <em>17</em> alpha-hydroxylase activity was 4-fold higher than that in controls on day 5, but showed no change in activity after 25 days and remained at neonatal levels up to 40 days. The activity of <em>17</em>-<em>ketosteroid</em> reductase in the control animals showed a developmental pattern similar to that of <em>17</em> alpha-hydroxylase, with a marked increase in activity after 25 days. In the Tfm group, <em>17</em>-<em>ketosteroid</em> reductase was normal on day 5, but failed to show any significant change thereafter and remained at neonatal levels on day 40. Serum LH levels in control animals increased from 5 days to a peak at 30 days. In Tfm mice, LH levels were significantly increased on days 20 and 40, but did not differ from controls on days 5, 25, and 30. In control +/Y animals, in which normal testicular descent was allowed to proceed at 25 days, the pattern of development was similar to that in the cryptorchid +/Y animals, although the increase in <em>17</em> alpha-hydroxylase activity at 30 and 40 days was significantly greater. The results show 1) that fetal Leydig cell steroidogenesis is enhanced in the absence of androgen receptors, but 2) that adult Leydig cells require receptor-mediated androgen activity around day 25 for normal functional development. In addition, 3) the lack of testicular descent at 25 days reduces the pubertal rise in <em>17</em> alpha-hydroxylase activity.

Corticosteroid synthesis in the human adrenal cortex requires a supply of cholesterol which can be derived from both local synthesis and the uptake of plasma lipoproteins. Studies with cultured adrenal cells have shown that such uptake is mediated through the interaction of plasma low density lipoproteins (LDL) and a specific cellular receptor (the LDL receptor). In the present study we have examined parameters of adrenal corticosteroid production in a patient with phenotypic abetalipoproteinemia (on the basis of homozygous hypobetalipoproteinemia) and in three of her relatives with inherently low levels of LDL (heterozygous hypobetalipoproteinemia). These studies sought to determine whether the absence of LDL or an inherent reduction in their plasma concentration results in alterations in corticosteroid production both under basal conditions and in response to prolonged stimulation with iv ACTH. Our results document normal parameters of corticosteroid production under basal conditions in both heterozygous and homozygous hypobetalipoproteinemia, but an impaired response to ACTH in the latter. This was manifested by significantly lower serum concentrations of cortisol as well as by reduced rates of excretion of <em>17</em>OHCS, <em>17</em>-<em>ketosteroids</em>, and urinary free cortisol during ACTH infusion. By inference, our results provide in vivo support for the view that plasma LDL serve as an important source of cholesterol for adrenal corticosteroid synthesis under conditions of sustained stimulation with ACTH.

BACKGROUND

Congenital adrenal hyperplasia is a group of disorders caused by defects in the adrenal steroidogenic pathways. In its most common form, 21-hydroxylase deficiency, patients develop varying degrees of glucocorticoid and mineralocorticoid deficiency as well as androgen excess. Therapy is guided by monitoring clinical parameters as well as adrenal hormone and metabolite concentrations.

BACKGROUND

We review the evidence for clinical and biochemical parameters used in monitoring therapy for congenital adrenal hyperplasia. We discuss the utility of 24-h urine collections for pregnanetriol and <em>17</em>-<em>ketosteroids</em> as well as serum measurements of <em>17</em>-hydroxyprogesterone, androstenedione, and testosterone. In addition, we examine the added value of daily hormonal profiles obtained from salivary or blood-spot samples and discuss the limitations of the various assays.

CONCLUSIONS

Clinical parameters such as growth velocity and bone age remain the gold standard for monitoring the adequacy of therapy in congenital adrenal hyperplasia. The use of 24-h urine collections for pregnanetriol and <em>17</em>-ketosteroid may offer an integrated view of adrenal hormone production but target concentrations must be better defined. Random serum hormone measurements are of little value and fluctuate with time of day and timing relative to glucocorticoid administration. Assays of daily hormonal profiles from saliva or blood spots offer a more detailed assessment of therapeutic control, although salivary assays have variable quality.

Prior site-directed mutagenesis studies in bacterial <em>ketosteroid</em> isomerase (KSI) reported that substitution of both oxyanion hole hydrogen bond donors gives a 10(5)- to 10(8)-fold rate reduction, suggesting that the oxyanion hole may provide the major contribution to KSI catalysis. But these seemingly conservative mutations replaced the oxyanion hole hydrogen bond donors with hydrophobic side chains that could lead to suboptimal solvation of the incipient oxyanion in the mutants, thereby potentially exaggerating the apparent energetic benefit of the hydrogen bonds relative to water-mediated hydrogen bonds in solution. We determined the functional and structural consequences of substituting the oxyanion hole hydrogen bond donors and several residues surrounding the oxyanion hole with smaller residues in an attempt to create a local site that would provide interactions more analogous to those in aqueous solution. These more drastic mutations created an active-site cavity estimated to be ~650 Å(3) and sufficient for occupancy by 15-<em>17</em> water molecules and led to a rate decrease of only ~10(3)-fold for KSI from two different species, a much smaller effect than that observed from more traditional conservative mutations. The results underscore the strong context dependence of hydrogen bond energetics and suggest that the oxyanion hole provides an important, but moderate, catalytic contribution relative to the interactions in the corresponding solution reaction.

AKR1C1 and AKR1C2 are human cytosolic hydroxysteroid dehydrogenases, which play pivotal roles in the metabolism and action of natural and synthetic steroid hormones. The two enzymes are highly homologous, and have distinct positional and stereochemical preferences with various substrates. We performed molecular docking simulations of three steroid substrates, including an androgen (5alpha-dihydrotestosterone, DHT), a progestin (progesterone, PRO), and a synthetic hormone ([7alpha,<em>17</em>alpha]-<em>17</em>-hydroxy-7-methyl-19-norpregn-5(10)-en-20-yn-3-one or tibolone, TIB), into the active sites of the two enzymes. For each substrate and enzyme pair, the activity inferred by the "productive" docking models (in which the spatial arrangement of the steroid and the cofactor would permit a reaction) matched the experimentally observed positional and stereochemical outcome. These productive conformations were energetically and statistically favored except for TIB and PRO with AKR1C2, where experimentally strong substrate inhibition and low activity were observed, respectively. Results showed that (i) a 3-<em>ketosteroid</em> (DHT) and a 20-<em>ketosteroid</em> (PRO) were reduced by AKR1C1 since the carbonyl groups could occupy the same position by "backwards" binding of steroids; (ii) 3alpha-reduced (DHT) and 3beta-reduced (TIB) products were formed by AKR1C2 since the angular methyl groups of the steroids were inverted by "upside-down" binding of steroids; and (iii) the 3beta- and 3alpha-reduction of DHT by AKR1C1 and AKR1C2, respectively occurred since the steroids employed a "swinging" motion to present opposite faces to the cofactor. Favorable nonproductive modes were observed with all substrates in both enzymes in which the steroid was bound at a "near-entry" position and/or an "in-middle" position, which may influence the reaction coordinate.

It is now clearly established that the brain has the capability of synthesizing various biologically active steroids including <em>17</em>-hydroxypregnenolone (<em>17</em>OH-Delta(5)P), <em>17</em>-hydroxyprogesterone (<em>17</em>OH-P), dehydroepiandrosterone (DHEA) and androstenedione (Delta(4)). However, the presence, distribution and activity of cytochrome P450 <em>17</em>alpha-hydroxylase/C<em>17</em>, 20-lyase (P450(C<em>17</em>)), a key enzyme required for the conversion of pregnenolone (Delta(5)P) and progesterone (P) into these steroids, are poorly documented. Here, we show that P450(C<em>17</em>)-like immunoreactivity is widely distributed in the frog brain and pituitary. Prominent populations of P450(C<em>17</em>)-containing cells were observed in a number nuclei of the telencephalon, diencephalon, mesencephalon and metencephalon, as well as in the pars distalis and pars intermedia of the pituitary. In the brain, P450(C<em>17</em>)-like immunoreactivity was almost exclusively located in neurons. In several hypothalamic nuclei, P450(C<em>17</em>)-positive cell bodies also contained 3beta-hydroxysteroid dehydrogenase-like immunoreactivity. Incubation of telencephalon, diencephalon, mesencephalon, metencephalon or pituitary explants with [(3)H]Delta(5)P resulted in the formation of several tritiated steroids including <em>17</em>OH-Delta(5)P, <em>17</em>OH-P, DHEA and Delta(4). De novo synthesis of C(21) <em>17</em>-hydroxysteroids and C(19) <em>ketosteroids</em> was reduced in a concentration-dependent manner by ketoconazole, a P450(C<em>17</em>) inhibitor. This is the first detailed immunohistochemical mapping of P450(C<em>17</em>) in the brain and pituitary of any vertebrate. Altogether, the present data provide evidence that CNS neurons and pituitary cells can synthesize androgens.

Gynecological diseases such as endometriosis, adenomyosis and uterine fibroids, and gynecological cancers including endometrial cancer and ovarian cancer, affect a large proportion of women. These diseases are estrogen dependent, and their progression often depends on local estrogen formation. In peripheral tissues, estrogens can be formed from the inactive precursors dehydroepiandrosterone sulfate and estrone sulfate. Sulfatase and sulfotransferases have pivotal roles in these processes, where sulfatase hydrolyzes estrone sulfate to estrone, and dehydroepiandrosterone sulfate to dehydroepiandrosterone, and sulfotransferases catalyze the reverse reactions. Further activation of estrone to the most potent estrogen, estradiol, is catalyzed by <em>17</em>-<em>ketosteroid</em> reductases, while estradiol can also be formed from dehydroepiandrosterone by the sequential actions of 3β-hydroxysteroid dehydrogenase-Δ(4)-isomerase, aromatase, and <em>17</em>-<em>ketosteroid</em> reductase. This review introduces the sulfatase and sulfotransferase enzymes, in terms of their structures and reaction mechanisms, and the regulation and different transcripts of their genes, together with the importance of their currently known single nucleotide polymorphisms. Data on expression of sulfatase and sulfotransferases in gynecological diseases are also reviewed. There are often unchanged mRNA and protein levels in diseased tissue, with higher sulfatase activities in cancerous endometrium, ovarian cancer cell lines, and adenomyosis. This can be indicative of a disturbed balance between the sulfatase and sulfotransferases enzymes, defining the potential for sulfatase as a drug target for treatment of gynecological diseases. Finally, clinical trials with sulfatase inhibitors are discussed, where two inhibitors have already concluded phase II trials, although so far with no convincing clinical outcomes for patients with endometrial cancer and endometriosis.

Eight cases of adrenocortical tumor are presented with a review of the literature. Although such tumors are rare, they are important causes of inappropriate virilization and Cushing's syndrome in childhood. Clinical virilization with or without hypercortisolism was found in all eight children, who were 5 years old or younger. Excessive linear growth was noted, despite evidence of hypercortisolism. Serum levels of dehydroepiandrosterone, dehydroepiandrosterone sulfate, testosterone, and cortisol were elevated in all cases tested and appear to be useful diagnostic alternatives to the more traditional determinations of urine <em>17</em>-<em>ketosteroids</em> and <em>17</em>-hydroxycorticosteroids. Abdominal sonography and computed tomography have proven to be reliable tools for tumor localization. Surgical resection was the definitive therapy in all patients, and perioperative steroid replacement was essential. Histologic diagnosis appeared to have little bearing on prognosis, and the majority of pediatric patients have had clinically benign disease. At a mean follow-up of 3 years, seven of the eight children were alive and had no evidence of tumor recurrence.

Ketoconazole, an imidazole derivative known to inhibit cytochrome P450-dependent adrenal enzymes was given to a patient with a functioning adrenal rest tumor of the liver in preparation for surgery. The drug was administered in a stepwise manner for 42 days starting with 400 mg and reaching 1 g the last 4 weeks of the trial. Clear clinical improvement was evident early in the trial and was associated with evidence of amelioration of her hypercortisolism and striking changes in serum and urinary levels of steroid hormones and metabolites. Sex steroids in serum and urine fell dramatically from the first day to the end of the trial. Urinary <em>17</em>-<em>ketosteroid</em> excretion fell from a basal average of 139 mg/24 h to near normal levels within a week of therapy; serum testosterone fell from a basal level of 2.4 to 0.18 ng/ml; serum <em>17</em> beta-estradiol fell likewise from 1096 to 150 pg/ml. In contrast, cortisol levels in serum and urine increased in the first 2 weeks of the trial and subsequently fell to values below the basal levels. Similarly, serum <em>17</em> alpha-OH-progesterone levels increased 63% above the basal levels by day 6 of the trial and declined afterwards. Nine months after successful tumor resection the patient is apparently cured as judged by steroid hormone levels and physical appearance. We conclude that ketoconazole was effective in blocking tumoral steroidogenesis which resulted in clinical benefit.

Dehydroepiandrosterone sulfate (DS) concentration was measured in the sera of premature and full-term infants and in children throughout puberty. Panhypopituitary, Addisonian, and virilized children were also studied. DS decreased slowly during the first weeks of life from a high level in neonates to the low levels observed between one to five years. After five years of age, DS concentration started to rise. A steeper increase was observed with the onset of puberty, and adult DS concentrations were reached in late puberty. There was no sex difference in DS concentration at any pubertal stage or bone age. Day-to-day variations were small in childhood and during puberty, but were considerable in premature infants. DS concentrations measured at 0900 h were not significantly different from those at <em>17</em>00 h. There was a positive correlation of serum DS concentrations with the excretion of urinary <em>17</em>-<em>ketosteroids</em> in boys and girls (r=0.789). Premature infants had DS concentration in or above the late pubertal range. Five panhypopituitary patients and five Addisonian patients had DS concentrations below normal. DS was markedly elevated in patients with congenital adrenal hyperplasia and in one girl with adrenal carcinoma, and was suppressible with dexamethasone in the former. The ease of measurement and the small amount of blood required make serum DS determination a useful guide for adrenal androgen secretion.

Ninety-eight females with congenital adrenal hyperplasia due to a defect in either the 21-hydroxylase or the 11 beta-hydroxylase enzyme were evaluated to determine the effects of glucocorticoid treatment on growth, pubertal development, and fertility. When treatment was begun prior to one year of age, mean final height was 157.4 +/- 7.3 com, well within the normal adult female range, and significantly (p less than 0.001) greater than the mean final height of 150.9 +/- 4.3 cm found in untreated patients. The mean age of menarche in patients treated prior to the age of six years was 13.8 +/- 3.7 years which is significantly (p less than 0.01) delayed compared to that in the normal population of the United States. However, 92% of patients with menstrual delay had inadequate suppression of adrenal androgens and urinary excretion of <em>17</em> <em>ketosteroids</em> larger than 7.0 mg/24hours. The increased production of adrenal androgens was the result of poor compliance or an insufficient prescribed dose of glucocorticoids. The fertility rate in patients first treated between six and 20 years of age was 64%. The excretion of urinary <em>17</em> KS at the time of pregnancy was 2.5 to 5.3 mg/24 hours. All of the patients who delivered term infants required delivery by cesarean section because of cephalopelvic disproportion. The major problems encountered in the management of adolescent patients were patient noncompliance and physician failure to increase the glucocorticoid dose as the patient's body size increased.

In concentrations probably exceeding those achieved in vivo, the cholesterol lowering compound simvastatin was found to suppress the synthesis of the androgens androstenediol and testosterone in vitro by human testicular homogenates. It was demonstrated that simvastatin in addition to its known inhibitory effect on HMG-CoA reductase activity, also affects the later steps of testicular steroidogenesis by selectively inhibiting the <em>17</em>-<em>ketosteroid</em>-oxidoreductase catalyzed conversion of dehydroepiandrosterone and androstenedione to androstenediol and testosterone respectively. There was no effect of simvastatin on the Cytochrome P-450-dependent microsomal enzymes. Although in doses conventionally used in the treatment of hypercholesterolemia, simvastatin does not affect testicular steroidogenesis, at higher doses--especially when inadvertently administered during early pregnancy--adverse effects on normal testosterone biosynthesis and thereby fetal development should be considered.

Cortisol, androstenedione, testosterone, dehydroepiandrosterone sulphate (DHAS) and free dehydroepiandrosterone (DHA) were measured in plasma of ten women affected by amenorrhoea with hyperprolactinaemia and eleven women affected by secondary hypothalamic amenorrhoea; twelve normal women at the second day of the menstrual cycle were used as controls. All subjects were hospitalized and <em>17</em>-<em>ketosteroids</em>, <em>17</em>OH-corticosteroids and total dehydroepiandrosterone were also measured in urine. Plasma DHAS was increased in all subjects affected by amenorrhoea with hyperprolactinaemia, while plasma DHA and urinary DHA were significantly increased in this group in comparison to other groups. Plasma cortisol, androstenedione and testosterone and urinary <em>17</em>-oxosteroids and <em>17</em>OH-corticosteroids were not significantly differnt in the three groups. In subjects affected by amenorrhoea with hyperprolactinaemia treated with bromocriptine a clear decrease of DHAS correlating with a decrease of plasma prolactin was observed. Since in wome DHAS sems to be almost exclusively secreted by the adrenal gland and most of the circulating DHA is dervied from adrenal secretion, these data suggest that human prolactin can stimulate DHAS production by the adrenal cortex.

To determine if increased lead absorption was associated with sperm count suppression or perterbation of the hypothalamopituitary system, we compared battery workers (N = 18), who were exposed to high airborne lead levels, with cement workers (N = 18), who were exposed to ambient lead levels. Blood lead, urinary lead, semen lead, and zinc protoporphyrin concentrations were markedly elevated (p less than .001) in battery workers. Battery workers had a significantly shifted (p less than .025) frequency distribution of sperm count (median count, 45 vs. 73 X 10(6) cells/cc, respectively). There were no significant differences between the two groups in mean follicle-stimulating hormone, testosterone, prolactin, luteinizing hormone, or total neutral <em>17</em>-<em>ketosteroid</em> levels. Potential confounding factors (alcohol, cigarette, and coffee consumption, frequency of intercourse, and days of abstinence prior to semen donation) were not significantly different between the two groups. These results suggest a direct toxic effect of increased lead absorption on sperm production or transport in man.

This study was carried out to document the postpubertal presentation of congenital adrenal hyperplasia (CAH), to elaborate the diagnostic criteria for it, and to investigate family members of CAH patients. Serum <em>17</em>-hydroxyprogesterone (<em>17</em>OHP) was measured in normal women and 25 hirsute oligomenorrheic patients, five of whom were shown to have CAH. These five CAH patients, as a group, had significantly elevated levels of <em>17</em>OHP when compared to normal and hirsute women, although the other 20 hirsute oligomenorrheic women also had higher levels of <em>17</em>OHP than the follicular phase control subjects. A single intravenous bolus of 0.25 mg of adrenocorticotropic hormone (ACTH) caused much larger increased in <em>17</em>OHP in all five CAH patients than in the control and hirsute women. The five CAH patients had decreased cortisol but normal 11-deoxycortisol responses to ACTH, thus indicating 21-hydroxylase deficiency (21HD). Clinically, they were indistinguishable from women with polycystic ovarian disease (PCO) and had basal serum levels of androgens and urinary <em>17</em>-<em>ketosteroids</em> which were similar to those found in 47 other women presenting with the complaint of hirsutism. However, the androstenedione levels and androstenedione/cortisol ratios in response to ACTH were significantly higher in the five CAH patients than in both the normal and hirsute women. Of seven family members tested, two fathers and one mother had an intermediate <em>17</em>OHP response to ACTH, thus suggesting heterozygosity. Human lymphocyte antigen (HLA) typing on family members indicated that the inheritance of the disorder may be linked to B antigens. Two siblings of one of the CAH patients had normal <em>17</em>OHP responses to ACTH and also had a different HLA-B complement. These data document the existence of adult manifestation of CAH, due to 21 HD. This disorder presents with androgen excess and oligomenorrhea or amenorrhea and mimicks PCO. The diagnosis of it hinges upon the post-ACTH rise in <em>17</em>OHP, whereas the levels of serum androgens and urinary <em>17</em>-<em>ketosteroids</em> may be inconclusive.

Human <em>17</em>beta-hydroxysteroid dehydrogenases (<em>17</em>betaHSDs) catalyze the interconversion of weak and potent androgen and estrogen pairs. Although the reactions using purified enzymes can be driven in either direction, these enzymes appear to function unidirectionally in intact cells: only reductive reactions for <em>17</em>betaHSD1 and <em>17</em>beta HSD3 and only oxidative reactions for <em>17</em>betaHSD2. We show that, after exhaustive incubations with either <em>17</em>beta-hydroxy- or <em>17</em>-<em>ketosteroid</em>, the medium for HEK-293 cells expressing <em>17</em>betaHSD1 or <em>17</em>betaHSD3 contains a 92:8 ratio of reduced:oxidized steroid. Similarly, <em>17</em>betaHSD2 yields a >95:5 ratio of oxidized:reduced steroids for both androgens and estrogens. Dual-isotope kinetic measurements show that the rates of the forward and reverse reactions are identical at these functional equilibrium states in intact cells for all three <em>17</em>betaHSD isoforms, and these rates are much faster than those estimated from single-isotope flux studies. Mutation L36D converts <em>17</em>betaHSD1 to an oxidative enzyme in intact cells, reversing the equilibrium distribution of estradiol:estrone to 5:95; however, the rates of the forward and reverse reactions at equilibrium are equal and comparable to those of the wild-type enzymes. The co-expression of <em>17</em>betaHSD2 paradoxically increases the potency of estrone in transactivation assays, demonstrating the physiological relevance of "backwards" metabolism to estradiol. We conclude that <em>17</em>betaHSD types 1, 2, and 3 catalyze both oxidative and reductive reactions in HEK-293 cells at intrinsic rates that are much faster than those estimated from single-isotope studies. These <em>17</em>betaHSD isoforms do not drive steroid flux in one direction but rather may achieve functional equilibria in intact cells, reflecting thermodynamically driven steroid distributions.

Thirty-eight transformer repairmen currently exposed to polychlorinated biphenyls (PCBs), <em>17</em> former transformer repairmen, and 56 comparison workers not known to be exposed to PCBs were studied. Measurements were made of serum liver function tests, gamma-glutamyl transpeptidase (GGT), lipid profile, thyroid function tests, and other serum biochemistry; hemoglobin; white cell count; 24-hour excretion of delta-aminolevulinic acid, porphyrins, <em>17</em>-hydroxycorticosteriods and <em>17</em>-<em>ketosteroids</em>; sperm count; spirometry; and antipyrine half-life to evaluate microsomal mixed function oxidase induction. The total exposed group differed significantly from the comparison group in albumin, LDH, T4, T4-RT3 index, and actual/predicted FEV1. Significant differences among all three exposure groups were seen for albumin, T4, T4-RT3 index, and <em>17</em>-hydroxycorticosteroid excretion. Differences in FEV1 were attributable to smoking. Significant correlations between serum PCBs and serum lipids were removed by adjustment for confounding variables. After adjustment for confounding variables, there was a statistically significant positive correlation between serum PCBs and GGT and a negative correlation between adipose PCBs and <em>17</em>-hydroxycorticosteroid excretion. These may reflect subtle metabolic effects of PCBs.