Effects of cyproterone acetate on adrenal steroidogenesis in vitro

The effects of cyproterone acetate (CA) on steroidogenesis in isolated guinea-pig adrenal cells have been investigated by measuring the production of cortisol, its immediate precursors (11-deoxycortisol and 17-hydroxyprogesterone), and adrenal androgens (delta 4-androstenedione and dehydroepiandrosterone). Used at a dose of 2 micrograms/ml, CA provoked a sharp drop in the production of cortisol, aldosterone and 11-deoxycortisol. By contrast, 17-hydroxyprogesterone, delta 4-androstenedione and dehydroepiandrosterone were increased, which suggests that 21-hydroxylase activity is inhibited. With concentrations above 2 micrograms/ml CA, it would seem to be the 3-beta-ol-dehydrogenase-delta 4,5-isomerase complex that is affected, since dehydroepiandrosterone exhibited a sudden increase, whereas 17-hydroxyprogesterone and delta 4-androstenedione showed a relative decrease. The enzymatic system or systems involved therefore appear to be linked to the concentration of CA used but, whatever the case, the drop in cortisol production is accompanied by a decrease in aldosterone and an increase in adrenal androgen levels.     

Effects of cadmium in vitro on microsomal steroid metabolism in the inner and outer zones of the guinea pig adrenal cortex

Studies were carried out to evaluate the effects of cadmium in vitro on microsomal steroid metabolism in the inner (zona reticularis) and outer (zona fasciculata and zona glomerulosa) zones of the guinea pig adrenal cortex. Microsomes from the inner zone have greater 21-hydroxylase than 17α-hydroxylase activity, resulting in the conversion of progesterone primarily to 11-deoxycorticosterone and of 17α-hydroxy progesterone principally to its 21-hydroxylated metabolite, 11-deoxycortisol. Microsomes from the outer zones, by contrast, have far greater 17α-hydroxylase and C_17,20-lyase activities than 21-hydroxylase activity. As a result, progesterone is converted primarily to its 17-hydroxylated metabolite, 17α-hydroxyprogesterone; and 17α-hydroxyprogesterone is converted principally to δ⁴-androstenedione, with only small amounts of 21-hydroxylated metabolites being produced. Addition of cadmium to incubations with inner zone microsomes causes concentration-dependent decreases in 21-hydroxylation and increases in 17α-hydroxylase and C_17,20-lyase activities, resulting in a pattern of steroid metabolism similar to that in normal outer zone microsomes. Cadmium similarly decreases 21-hydroxylation by outer zone microsomes but has no effect on the formation of 17-hydroxylated metabolites or on androgen (Δ⁴-androstenedione) production. In neither inner nor outer zone microsomes did cadmium affect cytochrome P-450 concentrations, steroid interactions with cytochrome(s) P-450, or NADPH–cytochrome P-450 reductase activities. The results indicate that cadmium produces both quantitative and qualitative changes in adrenal microsomal steroid metabolism and that the nature of the changes differs in the inner and outer adrenocortical zones. In inner zone microsomes, there appears to be a reciprocal relationship between 21-hydroxylase and 17α-hydroxylase/C_17,20-lyase activities which may influence the physiological function(s) of that zone.     

Steroid concentrations in the outer and inner zones of the adrenal cortex of the guinea pig

The outer (glomerulosa and fasciculata) and inner (reticularis) zones of the adrenal cortex of the guinea pig were separated and their steroid content determined. It was found that the concentration of 21-hydroxypregnenolone, deoxycorticosterone, corticosterone, aldosterone, 11-deoxycortisol, and cortisol was significantly higher in the outer cortical region, while the concentration of pregnenolone, 17-hydroxypregnenolone, and dehydroepiandrosterone was significantly higher in the inner zone. The concentration of progesterone, 17-hydroxyprogesterone, and androstenedione was not different in the two zones. Examination of specific steroid ratios suggested the following: (1) 3β-ol dehydrogenase/isomerase and 21-hydroxylase activities are reduced in the inner zone, (2) 17-hydroxylase and C_17–20 lyase activities appear to be equally active in the two zones (3) 11β-hydroxylase activity appears to be more active in the inner zone (4) 21-hydroxypregnenolone, deoxycorticosterone, corticosterone, 11-deoxycortisol, and cortisol along with aldosterone are produced principally in the outer zone.     

Conversion of progesterone to corticosteroids by the midterm fetal adrenal and kidney

Midterm fetal adrenal and kidney tissue homogenates were incubated with 3H-progesterone (1 microM) and its conversion to te 3H-corticosteroids metabolites studied. Cortisol (36.3%) and corticosterone (4.7%) were isolated from the adrenal, and 11-deoxycortisol (32.5%) and deoxycorticosterone (21.1%) from the kidney. The results of these incubations confirmed the presence of 17- and 21-hydroxylase activities in both fetal tissues, and that of 11 beta-hydroxylase activity only in fetal adrenal tissue. We conclude that during pregnancy when progesterone levels are high, biosynthesis by the fetal kidney of 11-deoxycortisol, the most abundant corticosteroid formed by this tissue in this investigation, might provide to the fetal adrenal an important precursor for cortisol biosynthesis within the fetal compartment.     

Intracellular distributian and substrate specificity of the 16 alpha-hydroxylase in the adrenal of human fetus

When [7α-³H] dehydroepiandrosterone was incubated with the adrenal homogenates of human fetus at 22 to 26 weeks gestational age, 16α-hydroxydehydroepiandrosterone and/or its sulfate was formed as the only detectable metabolite. The 16α-hydroxylase activity was concentrated in the microsomal fraction of the adrenal homogenate.[1,2-³H]Androstenedione, [4-¹⁴C] pregnenolone and [7α-³H] progesterone were also 16α-hydroxylated by incubation with the microsomal fraction. Amoung these substrates, progesterone gave the highest yield of 16α-hydroxylated products. By incubation with the microsomal fraction, formation of following steroids were also established: 6β-hydroxyandrostenedione from androstenedione; 17-hydroxypregnenolone, 17,21-dihydroxypregnenolone and dehydroepiandrosterone from pregnenolone; 17-hydroxy-progesterone, deoxycorticosterone, 11-deoxycortisol and androstenedione from progesterone.     

21-Hydroxylation of C-11-oxygenated and C-11-deoxysteroids by perfused dog adrenals.

The left adrenal of two female dogs were perfused with either 3 muCi [4-14C]cpd. S (2) and 45 muCi [1, 2-3H] 21-deoxycortisone (Dog I), or with 1 muCi [4-14C] 17-hydroxyprogesterone and 65 muCi [1,2-3H] 21-deoxycortisone (Dog II). In Dog I, 40% of the perfused 21-deoxycortisone was converted to cortisone and 23% of cpd. S was converted to cortisol. In Dog II the percent conversion of 21-deoxycortisone to cortisone and of 17- hydroxyprogesterone to cortisol was 24% and 15% respectively. The results demonstrate that the dog adrenal has the capability of hydroxylating an 11-oxygenated steroid at C-21.     

In vitro metabolism of a mixture of [4-C]pregnenolone and [17α-H]pregnenolone by polycystic ovary: Pathways of testosterone biosynthesis

When minced polycystic ovarian tissue was incubated with a mixture of [4-¹⁴C]-pregnenolone and [17α-³H]pregnenolone² and added cofactors for 25, 40 and 60 min, the following metabolites were isolated and characterized: progesterone, 17-hydroxyprogesterone, 17-hydroxypregnenolone, dehydroepiandrosterone, 4-androstenedione, and testosterone; unmetabolized substrates were also recovered. There were increased amounts of progesterone, 17-hydroxyprogesterone, dehydroepiandrosterone, and 4-androstenedione formed as the incubation time increased.     

17-Hydroxyprogesterone metabolism in the monkey fetal adrenal: C17–20Lyase and 21-hydroxylase activities

C^17–20Lyase and 21-hydroxylase activities were measured during late gestation In the rhesus monkey ($\text{Macaca mulatta}$) fetal adrenal. Activities were assessed in 10,000 × g supernatants with 17-hydroxyprogesterone and NADPH as substrates. Although conversion of [¹⁴C]17-hydroxyprogesterone to [¹⁴C]androstenedione was noted, activity was often nonlinear and far less than the rate of hydroxylation which together prevented an accurate estimation of lyase rate, K_m and V_max. 21-Hydroxylase activity was characterized; the mean reaction rate was 1.6 × 10^?3 μmoles NADPH oxidized/min. × mg^?1 protein with an apparent K_m of 3.6 × 10^?7 M and a V_max of 2.2 × 10^?3 μmoles/min. × mg^?1 protein. These values were similar to data obtained In adrenals from adult monkeys. A relatively high level of hydroxylase activity in the fetal gland might lead to an Inadequate supply of precursors for the synthesis of dehydroepiandrosterone sulfate (DHEAS) in the adrenal if it also contained 3β-hydroxysteroid dehydrogenase (3β-hsdh). However, the fact that the fetal adrenal reportedly is deficient in 3β-hsdh may serve to protect both DHEAS and corticoid synthesis.     

Inhibition of cortisol production in isolated guinea-pig adrenal cells

Cortisol, added to 1 ml incubation medium containing 3-4 X 10(5) isolated guinea-pig adrenal cells, provoked a decrease in basal and ACTH (250 pg)-stimulated cortisol production, in correlation with the amounts used (50 ng-2,000 ng). A decrease in aldosterone production could be seen when cortisol concentrations reached or exceeded 1,000 ng/ml. There were no variations in either androgens (delta 4-androstenedione, dehydropiandrosterone) or 17-hydroxyprogesterone. Only 11-deoxycortisol was slightly increased. Using increasing concentrations of ACTH (50-250 pg), both in the absence and in the presence of 1,000 ng cortisol, it was noted that the inhibition induced by cortisol was of a competitive type and could be overcome by ACTH. This decrease in cortisol was concomitant with an increase in 11-deoxycortisol. Neither corticosterone nor dexamethasone reduced cortisol production. In addition, it was shown that the conversion of tritiated 11-deoxycortisol to radioactive cortisol increased significantly under the influence of 250 pg ACTH (mean relative variation of 21.7% +/- 7.7 (SEM), n = 6, P less than 0.05); but decreased significantly under the combined effect of 1,000 ng exogenous cortisol and the same dose of ACTH: (mean relative variation of 4.3% +/- 1 (SEM), n = 8, P less than 0.005). There is therefore reason to believe that the concentrations of cortisol at the adrenal level modulate the stimulation induced by ACTH and that this self-adjustment forms part of the control mechanisms involved in corticosteroidogenesis.     

Biotransformation of pregnenolone - 7alpha-3H, progesterone - 7alpha-3H and dehydroepiandrosterone - 7alpha-3H by porcine fetal and maternal adrenal homogenate preparations.

The biotransformation of pregnenolone-7alpha-3H and of progesterone-7alpha-3H by porcine fetal and maternal adrenal homogenates at 56 and 112 days of pregnancy and of dehydroepiandrosterone-7alpha-3H by fetal adrenal homogenates has been investigated in vitro. Both pregnenolone-7alpha-3H and progesterone-7alpha-3H were metabolized extensively by maternal adrenal preparations, the principal radioactive metabolites isolated being cortisol, corticosterone, 11-deoxycortisol, deoxycorticosterone, 11beta-hydroxyprogesterone and androstenedione. In addition, 17alpha-hydroxyprogesterone, 20alpha-dihydroprogesterone and cortisone were formed from both substrates and 17alpha-hydroxypregnenolone and progesterone were formed from pregnenolone. Although essentially the same radioactive metabolites were isolated after incubation of fetal adrenal glands with pregnenolone-7alpha-3H or progesterone-7alpha-3H, a greater proportion of the radioactivity was associated with corticosteroids at 112 days of pregnancy than at 56 days. 11beta-Hydroxyandrostenedione and androstenedione were isolated and identified together with an unknown polar metabolite, after incubation of fetal adrenal tissue with dehydroepiandrosterone-7alpha-3H. These results are discussed in relation to feto-placental steroid biosynthesis and metabolism and the role of the fetal adrenal in the initiation of parturition in the pig.     

Preparation of 1,2-3H-21-deoxycortisol by incubation of 1,2-3H-17-hydroxyprogesterone with rat adrenal mitochondria.

A method is described for preparation of 1,2-3H-21-deoxycortisol by incubation of 1,2-3H-17-hydroxyprogesterone with rat adrenal mitoochondria. The radiochemical purity of the product was established by two subsequent chromatographies, reversed isotopic dilution and oxidation to 21-deoxycortisone. The final yield of 1,2-3H-21-deoxycortisol was approximately 15%.     

Mechanism of dissociation of cortisol and adrenal androgen secretion after removal of adrenocortical adenoma in patients with Cushing's syndrome

We investigated the mechanism of dissociation of cortisol and dehydroepiandrosterone sulfate (DHEA-S) secretion by the adrenal glands after the removal of an adrenal gland containing an adrenocortical adenoma in a patient with Cushing's syndrome. After removal of the adrenocortical adenoma, the serum cortisol rapidly decreased from 24.6 +/- 6.4 micrograms/dl (mean +/- SD, n = 6) to 0.7 +/- 0.5 micrograms/dl. Serum DHEA-S levels were 15 +/- 14 micrograms/dl and 6 +/- 9 micrograms/dl before and after surgery, respectively, and significantly lower than the control values. Serum cortisol levels reverted to normal levels 1.5 to 3 years after the surgery. On the other hand, DHEA-S levels reverted to normal 5 to 7 years after the serum cortisol levels had normalized. Monolayer cultures of normal human adrenal cells obtained at adrenalectomy in patients with advanced breast cancer and atrophic adrenal cells adjacent to the adrenocortical adenoma in patients with Cushing's syndrome were used to study the mechanism of the dissociation of cortisol and DHEA-S secretion. ACTH caused significant increases in the productions of pregnenolone (P5), progesterone (P4), 17-hydroxypregnenolone (17-OH-P5), 17-hydroxyprogesterone (17-OH-P4), DHEA, DHEA-S, androstenedione (delta 4-A), and cortisol. The amounts of 17-OH-P5 and 17-OH-P4 produced by ACTH in atrophic adrenal cells were significantly greater than those in normal adrenal cells. The amounts of DHEA, DHEA-S and delta 4-A produced by ACTH in atrophic adrenal cells were significantly smaller than those of normal adrenal cells. The conversion rate of 17-OH-[3H]P5 to 17-OH-[3H]P4 and 11-deoxy-[3H] cortisol was higher in atrophic adrenal cells than in normal adrenal cells, but the conversion rate to [3H]DHEA, [3H]DHEA-S and [3H]delta 4-A was significantly lower in atrophic adrenal cells than in normal adrenal cells. These results suggest that the dissociation of cortisol from DHEA-S after the removal of adrenocortical adenoma is a probably due to diminished C17,20-lyase activity in the remaining atrophic adrenal gland.     

The Classic and Nonclassic Concenital Adrenal Hyperplasias

Objective: The American Association of Clinical Endocrinologists Adrenal Scientific Committee has developed a series of articles to update members on the genetics of adrenal diseases.Methods: Case presentation, discussion of literature, table, and bullet point conclusions.Results: The congenital adrenal hyperplasia (CAH) syndromes are autosomal recessive defects in cortisol biosynthesis. The phenotype of each CAH patient depends on the defective enzyme and the severity of the defect. Clinical manifestations derive from both failure to synthesize hormones distal to the enzymatic block, as well as consequences from cortisol precursor accumulation proximal to the block, often with diversion to other biologically active steroids. The most common form of CAH is 21-hydroxylase deficiency, which occurs in the classic form in 1 in 16,000 newborns and in a milder or nonclassic form in at least 1 in 1,000 people.Conclusion: This article reviews the various forms of CAH and pitfalls in the diagnosis and treatment of these conditions.Abbreviations: 11OHD = 11-hydroxylase deficiency 17OHD = 17-hydroxylase deficiency 17OHP = 17-hydroxyprogesterone 21OHD = 21-hydroxylase deficiency 3βHSD = 3β-hydroxysteroid dehydrogenase CAH = congenital adrenal hyperplasia CST = cosyntropin stimulation test CYP17A1 = cytochrome P450 17A1 (steroid 17-hydroxylase/17,20-lyase) DHEAS = dehydroepiandrosterone sulfate DSD = disorder of sex development LCAH = lipoid congenital adrenal hyperplasia NBS = newborn screening NCAH = nonclassic CAH PCOS = polycystic ovary syndrome PORD = P450-oxidoreductase deficiency     

Differential actions of metyrapone on the fetal pituitary-adrenal axis in the sheep fetus in late gestation

It is not clear if an increase in intra-adrenal cortisol is required to mediate the actions of adrenocorticotropic hormone (ACTH) on adrenal growth and steroidogenesis during the prepartum stimulation of the fetal pituitary-adrenal axis. We infused metyrapone, a competitive inhibitor of cortisol biosynthesis, into fetal sheep between 125 and 140 days of gestation (term = 147 +/- 3 days) and measured fetal plasma cortisol, 11-desoxycortisol, and ACTH; pituitary pro-opiomelanocortin mRNA and adrenal expression of ACTH receptor (melanocortin type 2 receptor), steroidogenic acute regulatory protein (StAR), 11beta-hydroxysteroid dehydrogenase type 2 (11betaHSD2), cytochrome P450 cholesterol side-chain cleavage (CYP11A1), cytochrome P450 17-hydroxylase (CYP17), 3beta-hydroxysteroid dehydrogenase, and cytochrome P450 21-hydroxylase mRNA; and StAR protein in the fetal adrenal gland. Plasma ACTH and 11-desoxycortisol concentrations were higher (P < 0.05), whereas plasma cortisol concentrations were not significantly different in metyrapone- compared with vehicle-infused fetuses. The ratio of plasma cortisol to ACTH concentrations was higher (P < 0.0001) between 136 and 140 days than between 120 and 135 days of gestation in both metyrapone- and vehicle-infused fetuses. The combined adrenal weight and adrenocortical thickness were greater (P < 0.001), and cell density was lower (P < 0.01), in the zona fasciculata of adrenals from the metyrapone-infused group. Adrenal StAR mRNA expression was lower (P < 0.05), whereas the levels of mature StAR protein (30 kDa) were higher (P < 0.05), in the metyrapone-infused fetuses. In addition, adrenal mRNA expression of 11betaHSD2, CYP11A1, and CYP17 were higher (P < 0.05) in the metyrapone-infused fetuses. Thus, metyrapone administration may represent a unique model that allows the investigation of dissociation of the relative actions of ACTH and cortisol on fetal adrenal steroidogenesis and growth during late gestation.     

Studies on the metabolism of steroid hormones in a virilizing adrenal cortex adenoma.

Slices of an adreno-cortical adenoma which had been obtained at operation from an 11-year-old girl with clinical signs of virilism were incubated with each of the following steroids: [1,2-3H]progesterone, [4-14C]pregnenolone, [1,2-3H]testosterone, [4-14C]androstenedione and [7-3H]dehydroepiandrosterone, respectively. Isolation and identification of the free radioactive metabolites were achieved by gel column chromatography on Sephadex LH-20, thin-layer chromatography, radio gas chromatography and isotope dilution. After incubation of progesterone, the following metabolites were identified: 11beta-hydroxyprogesterone, 16alpha-hydroxyprogesterone, 17alpha-hydroxyprogesterone, 21-deoxycortisol, corticosterone and cortisol. Pregnenolone was metabolized to 17alpha-hydroxypregnenolone, progesterone, dehydroepiandrosterone, androstenedione and 11beta-hydroxyandrostenedione. When testosterone was used as substrate, 11beta-hydroxytestosterone, androstenedione and 11beta-hydroxyandrostenedione were found as metabolites, whereas androstenedione was metabolized to testosterone and 11beta-hydroxyandrostenedione. After incubation of dehydroepiandrosterone, only androstenedione and 11beta-hydroxyandrostenedione were isolated and identified. From these results, it appears that cortisol was formed in the adenoma tissue via 21-deoxycortisol and corticosterone. Delta4-3oxo steroids of the C19-series arose exclusively from pregnenolone via 17alpha-hydroxypregnenolone and dehydroepiandrosterone, and not from progesterone and 17alpha-hydroxyprogesterone. Calculated on the amounts of metabolites formed, the highest enzyme activities were those of the 11beta-hydroxylase and the 17alpha-hydroxylase. It is interesting to note that only traces of testosterone were detected after incubation of androstenedione, whereas testosterone yielded large amounts of androstenedione.     

Precursor-to-product ratios reflect biochemical phenotype in congenital adrenal hyperplasia

Precursor-to-product ratios in steroid hormone metabolism may accurately reflect enzymatic activity and production of metabolites relative to their disappearance. The purpose of this study was to explore the use of direct precursor-to-product steroid ratios to discriminate between infants with congenital adrenal hyperplasia (CAH) due to 21-α-hydroxylase deficiency and infants with no disorder, thus characterizing the biochemical phenotype in CAH. Deidentified dried blood spot samples from confirmed CAH cases identified by newborn screen (CAH-positive, N = 8) and from cases with no disorder (CAH-negative, N = 10) were obtained from the California State Newborn Screening Program. Samples (~6.25 mm circular spots) underwent methanol and water extraction (9:1 ratio). Deuterated steroids served as isotope internal standards. 17-α-hydroxyprogesterone (17-OHP), 11-deoxycortisol (S), androstenedione (A4) and cortisol (F) concentrations were determined by liquid chromatography–tandem mass spectrometry (LC–MS/MS), and the 17-OHP/S, 17-OHP/A4, and S/F ratios were calculated. The mean 17-OHP and A4 concentrations in samples from CAH cases were significantly increased when compared to cases with no disorder (p = 0.003 for both). 17-OHP/S and 17-OHP/A4 ratios were also significantly elevated in CAH cases (p = 0.007 and p < 0.001, respectively). In contrast, S and F concentrations and the S/F ratio were similar between the two groups. In CAH, the elevated 17-OHP/S ratio is a biomarker of diminished 21-α-hydroxylase activity, and the elevated 17-OHP/A4 ratio is a biomarker of adrenal androgen excess via increased 17,20-lyase activity. The similar S/F ratio indicates that the rate of production via 11-β-hydroxylase and disappearance of F is maintained in CAH.     

Basic and clinical aspects of congenital adrenal hyperplasia

Defective steroid 21-hydroxylation is the most common of the biochemical defects causing hyperplasia of the adrenal cortex. The genetic mode of transmission of all enzyme abnormalities seen in cortisol biosynthesis is autosomal recessive. Steroid 21-hydroxylase deficiency has three currently accepted forms: the simple virilizing and salt-wasting variants of the classical deficiency, and the nonclassical (attenuated) form, which shows a wide clinical range of effects and whose characterization emerged from co-ordinated hormonal testing and family studies. More recent molecular genetic studies have started to identify specific mutations altering 21-hydroxylase activity. Defects in the other enzymes occur more rarely and are less well known, although initial work with abnormal 11 beta-hydroxylase and 3 beta-hydroxylase indicates that allelic gene defects may be correlated with different clinical phenotypes seen for these disorders also. The gene for the enzyme steroid 21-hydroxylase, a cytochrome P-450, is situated within the major histocompatibility complex on the p arm of human chromosome 6, proximal to the HLA-B antigen locus. Linkage disequilibria between certain B and DR alleles and classical and nonclassical 21-hydroxylase deficiency permit the use of HLA genotyping in conjunction with hormonal evaluation for diagnosis of this disorder and for identification of carrier haplotypes in population studies. Test programs have shown the feasibility of neonatal screening for 21-hydroxylase deficiency by blood-spot hormonal assay for elevated 17-hydroxyprogesterone. Prenatal detection of disease currently depends on HLA serotyping of cultured aminocytes jointly with measurement of amniotic 17-hydroxyprogesterone (13-18 week gestation); molecular genetic techniques with more specific nuclear probes will improve the specificity of this test and will in addition permit even earlier definitive fetal genotyping by chorionic villus biopsy (6-10 week gestation).     

Androgen glucuronides: I. Direct formation in rat accessory sex organs

A simple one-step procedure is described on the isolation of androgen glucuronides from various rat tissues. This procedure uses polyacrylamide gel electrophoresis, and permits a quantitative isolation of a single band containing the total androgen glucuronides without the contamination of free androgens and androgen sulfates. This procedure was used to determine the ability of various tissues of the rat to form androgen glucuronides directly when they were incubated with 1,2-[³H]-testosterone (0.1 μM) $\text{in}$$\text{vitro}$. Of eleven organs studied, only the accessory sex organs (ventral prostate, seminal vesicle, and coagulating gland), liver, and kidney were capable of forming androgen glucuronides. At the end of a one-hour incubation period, approximately 1% of the total radiolabeled steroids in the prostatic tissue minces were in the form of glucuronide conjugates. The predominant androgen glucuronide formed in the accessory sex organs was 5α-androstane-3α,17β-diol 17β-d-glucuronide. This is in contrast to the rat liver and kidney in which testosterone glucuronide was the predominant conjugate.A similar amount of labeled glucuronide conjugates was formed from either [³H]-testosterone, [³H]-dihydrotestosterone or [³H]-androstenedione, whereas negligible amount of steroid conjugates was formed from [³H]-cortisol. The formation of androgen glucuronides requires metabolically active tissues; furthermore, the conjugation process was inhibited by the antiandrogen, cyproterone acetate, or by metabolic inhibitors, such as oligomycin or N-ethylmaleimide.     

Steroid synthesis by primary human keratinocytes; implications for skin disease

Cortisol-based therapy is one of the most potent anti-inflammatory treatments available for skin conditions including psoriasis and atopic dermatitis. Previous studies have investigated the steroidogenic capabilities of keratinocytes, though none have demonstrated that these skin cells, which form up to 90% of the epidermis are able to synthesise cortisol. Here we demonstrate that primary human keratinocytes (PHK) express all the elements required for cortisol steroidogenesis and metabolise pregnenolone through each intermediate steroid to cortisol. We show that normal epidermis and cultured PHK express each of the enzymes (CYP11A1, CYP17A1, 3βHSD1, CYP21 and CYP11B1) that are required for cortisol synthesis. These enzymes were shown to be metabolically active for cortisol synthesis since radiometric conversion assays traced the metabolism of [7-³H]-pregnenolone through each steroid intermediate to [7-³H]-cortisol in cultured PHK. Trilostane (a 3βHSD1 inhibitor) and ketoconazole (a CYP17A1 inhibitor) blocked the metabolism of both pregnenolone and progesterone. Finally, we show that normal skin expresses two cholesterol transporters, steroidogenic acute regulatory protein (StAR), regarded as the rate-determining protein for steroid synthesis, and metastatic lymph node 64 (MLN64) whose function has been linked to cholesterol transport in steroidogenesis. The expression of StAR and MLN64 was aberrant in two skin disorders, psoriasis and atopic dermatitis, that are commonly treated with cortisol, suggesting dysregulation of epidermal steroid synthesis in these patients. Collectively these data show that PHK are capable of extra-adrenal cortisol synthesis, which could be a fundamental pathway in skin biology with implications in psoriasis and atopic dermatitis.