Apparent activities of 21-hydroxylase, 17alpha-hydroxylase and 17,20-lyase are impaired in adrenal incidentalomas.
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OBJECTIVE: An increased response of 17-hydroxyprogesterone to ACTH stimulation has been observed in adrenal incidentaloma and linked to an impairment of either 21-hydroxylase or of 11beta-hydroxylase activity. To analyse this question further, we investigated the steroidogenic pathways in a series of 17 adrenal incidentalomas. DESIGN AND PATIENTS: 17 patients (7 women, 10 men; mean age, 62 +/- 12 years) with non-histologically analyzed adrenal incidentalomas were prospectively evaluated. METHODS: The following variables were investigated: 24-h urinary methanephrines and free cortisol excretion; plasma levels of ACTH and dehydroepiandrosterone; overnight dexamethasone suppression test; 1-24 ACTH stimulation test with measurement of: cortisol, 11-deoxycortisol, 17-hydroxyprogesterone, aldosterone, 11-deoxycorticosterone, progesterone, 17-hydroxypregnenolone, Delta4-androstenedione, dehydroepiandrosterone and 21-deoxycortisol. RESULTS: Discordant features of subclinical hypercorticism were noted in one case. No patient had dehydroepiandrosterone sulfate levels in the normal range for his or her age. Peak 17-hydroxyprogesterone and peak 21-deoxycortisol disclosed impairment of 21-hydroxylase in 11 and 10 cases respectively. An increased 11-deoxycortisol/cortisol ratio identified reduced activity of 11beta-hydroxylase in 11 patients. Eight patients displayed features of mild 17,20-lyase impairment, which was related to 21-hydroxylase dysfunction. Whereas only 2 patients showed no enzyme modification, 9 displayed alterations of at least two pathways. CONCLUSION: In our hands, a combination of enzyme dysfunction was frequently observed. Shared biochemical mechanisms could explain combined 17,20-lyase and 21-hydroxylase alterations, whereas coexistence of 21-hydroxylase (particularly when based on peak 21-deoxycortisol) and 11beta-hydroxylase is more puzzling. (+info)
Conversion of pregnenolone to DHEA by human 17alpha-hydroxylase/17, 20-lyase (P450c17). Evidence that DHEA is produced from the released intermediate, 17alpha-hydroxypregnenolone.
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Most previous studies using reconstituted systems and fast kinetics suggest that the conversion of pregnenolone to dehydroepiandrosterone (DHEA; the precursor of androgen and estrogen biosynthesis) by P450c17 does not require the release of the intermediate 17alpha-OHPreg (a precursor of cortisol biosynthesis). With such a mechanism, it is difficult to conceive how high amounts of DHEA may be produced in some cells or tissues, such as the testis and cells from the adrenal reticularis, while in other tissues such as the fasciculata zone, high levels of 17alpha-OHPreg are synthesized. In this report, we address this matter using intact transfected cells, which better reflect the actual cellular conditions. Furthermore, by using transfected cells, we can conveniently analyze human enzymes, as we are not restricted by the availability of human tissues as in the case of methods using purified or partially purified enzymes. Using intact HEK-293 cells transfected with human P450c17 in culture, we showed, in a time course study of the transformation of pregnenolone, that there is an accumulation of 17alpha-OHPreg, and that, subsequently, the accumulated 17alpha-OHPreg decreases with a concomitant increase in DHEA production. The DHEA/17alpha-OHPreg ratio changes from 0.1 :1 after 1 h incubation to 50 : 1 after 20 h. This result strongly suggests that the transformation of Preg to DHEA proceeds through two steps in which DHEA is produced from the released intermediate 17alpha-OHPreg. We also show that high levels of substrate vs. enzyme concentration will lead to high hydroxylase activity whereas the reverse will increase the lyase activity. The result is in good agreement with recent observations suggesting that surrounding enzymes and steroids could modulate the lyase activity. Cotransfection of vectors expressing cytochrome b5 and NADPH cytochrome P450 reductase indicates that both are required for an optimum production of DHEA. (+info)
Serum concentrations of delta 5-3 beta-hydroxysteroids in type 2 diabetes mellitus.
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We examined the serum concentrations of delta(5)-3beta-hydroxysteroids, pregnenolone (Preg), 17-hydroxypregnenolone (17-OH-Preg), dehydroepiandrosterone (DHEA), androstenediol (ADIOL) and their sulfates in 30 well controlled (Group I: HbA1c<7.0%) and 15 poorly controlled (Group II: HbA1c>7.1%) type 2 diabetic patients, and 30 normal controls. These patients were treated with diet therapy or anti-diabetic agent. The distribution of gender and age of the subjects were matched between the groups. The serum levels of sulfo-conjugated and unconjugated steroids described above were measured by GC-MS and enzyme immunoassay (EIA), respectively. The serum levels of the entire sulfo-conjugated steroid measured in this study were significantly lower in Groups I and II than in controls. On the other hand, Preg levels in both Groups I and II were significantly higher than those in controls, whereas the serum levels of the downstream unconjugated steroids were not different from controls. To investigate the effect of sulfonylurea (SU) on the serum levels of steroids, the serum concentrations of steroids between the patients who were treated with diet therapy and SU agent were compared in Group I. No significant differences were observed between both groups. These results suggest that (1) since increased Preg levels did not cause any changes in the downstream delta(5)-3beta-hydroxysteroid levels, the metabolic pathway of delta(4)-3-ketosteroids may be accelerated in type 2 diabetes; (2) serum steroid levels were not affected by SU treatment; (3) sulfo-conjugated steroid catabolism was altered in type 2 diabetes; (4) the decreased sulfo-conjugated steroids especially ADIOLS may contribute to the alteration of sex steroid levels and onset or exacerbate infectious diseases in diabetes. (+info)
Molecular dynamics of substrate complexes with hamster cytochrome P450c17 (CYP17): mechanistic approach to understanding substrate binding and activities.
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The cytochrome P450c17 isoforms from various animal species have different substrate selectivity, especially for 17,20-lyase activity. In particular, the human P450c17 selectively produces dehydroepiandrosterone with little androstenedione (AD). Hamster P450c17, on the other hand, produces both of these steroids at comparable rates. We thus investigated if computational analysis could explain the difference in activity profiles. Therefore, we inserted the four P450c17 substrates-pregnenolone, progesterone, and their 17alpha-hydroxylated forms-inside our hamster P450c17 model, which we derived from our human P450c17 model based on the crystal structure of P450BMP. We performed molecular dynamics (MD) simulations on the complexes and analyzed the resultant trajectories to identify amino acids that interact with substrates. Starting with substrates in two different orientations, we obtained two sets of binding trajectories in each case. The first set of trajectories reveal structural rearrangements that occur during binding, whereas the second set of trajectories reflects substrate orientations during catalysis. Our modeling suggests that three distinct steps are required for substrate selectivity and binding to the hamster P450c17: (1) recognition of the substrate at the putative substrate entrance, characterized by a pocket at the surface of the hamster P450c17 containing charged residues R96 and D116; (2) entry of the substrate into the active site, in an intermediate position directed by possible hydrogen bonding of the substrates with the heme D-ring propionate group, R96, R440, and T306; followed by (3) 90 degrees counterclockwise rotation of the substrates, positioning them in optimal position for reactivity, a process that may be directed by hydrogen bonding to the 110-112 region of the hamster P450c17. With some substrates, we obtained trajectories which suggest that major distortions in the I-helix and opening of the H-I loop occur during substrate binding. In conclusion, these modeling exercises provide insight to possible structural reorganizations that occur during substrate binding and suggest that amino acids that participate in three distinct steps of this process may all contribute to substrate binding and activity. (+info)
Steroid hormone formation in bovine ovarian follicles.
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In an attempt to assess histophysiological implication of the follicular compartment of the bovine ovary in steroid hormone formation and the effect of human chorionic gonadotropin (hCG) in vitro on follicular steroidogenesis, minces of follicular tissues from non-gravid bovine ovaries were incubated with radioactive testosterone or acetate in the presence and absence of hCG. Significant amounts of estrone and estradiol-17beta were formed on incubation with testosterone-4-14C; hCG decreased the conversion approximately by 30%. The major radioactive products formed from acetate-l-14C were androstenedione and testosterone with lesser amounts of dehydroepiandrosterone and 17-hydroxyprogesterone. In addition, small amounts of progesterone, 17-hydroxypregnenolone, estrone and estradiol-17beta were formed. Histology of the dissected follicle specimens was characterized by dominant theca cells undergoing luteinization with small amounts of granulosa cells, which showed neither proliferation nor luteinization. The pattern of distribution of radioactivity among the steroids formed from acetate-14C was considered to represent steroidogenic profile of bovine atretic follicles. The addition of hCG in vitro increased the overall incorporation of radioactive acetate into the steroids approximately by 50%, although the range of increase was not uniform in the individual steroids under the exprimental conditions. (+info)
Human skin is a steroidogenic tissue: steroidogenic enzymes and cofactors are expressed in epidermis, normal sebocytes, and an immortalized sebocyte cell line (SEB-1).
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Although the human sebaceous gland can synthesize cholesterol from acetate and can further metabolize steroids such as dehydroepiandrosterone into potent androgens, the de novo production of steroids from cholesterol has not been demonstrated in human skin. The goal of this study was to delineate the steroidogenic pathway upstream from dehydroepiandrosterone by documenting the presence of members of the P450 side chain cleavage system (P450scc). This system catalyzes the initial step in steroid hormone synthesis following translocation of cholesterol to the inner mitochondrial membrane. In concert with its cofactors, adrenodoxin and adrenodoxin reductase, and the transcription factor steroidogenic factor 1, P450scc converts cholesterol to pregnenolone. An SV40 immortalized human sebaceous gland cell line (SEB-1) was established in order to facilitate investigation of the P450scc system. The sebaceous phenotype of SEB-1 sebocytes was confirmed using immunohistochemistry, Oil Red O staining, and gene array expression analysis. Presence of P450scc, adrenodoxin reductase, cytochrome P450 17-hydroxylase (P450c17), and steroidogenic factor 1 was documented in human facial skin, human sebocytes, and SEB-1 sebocytes. Using immunohistochemistry, antibodies to the above proteins localized to epidermis, hair follicles, sebaceous ducts, and sebaceous glands in sections of facial skin. Results of immunohistochemistry were confirmed with Western blotting. Biochemical activity of cytochrome P450scc and P450c17 was demonstrated in SEB-1 sebocytes using radioimmunoassay. The relative abundance of mRNA for P450scc, P450c17, and steroidogenic factor 1 in SEB-1 sebocytes and sebaceous glands was compared to mRNA levels in ovarian theca and granulosa cells using real-time quantitative polymerase chain reaction. Gene array expression analysis and quantitative polymerase chain reaction indicated that mRNA for P450scc is more abundant than mRNA for both P450c17 and steroidogenic factor 1 in sebaceous glands and SEB-1 cells. These data demonstrate that the skin is in fact a steroidogenic tissue. The clinical significance of this finding in mediating androgenic skin disorders such as acne, hirsutism, or androgenetic alopecia remains to be established. (+info)
Steroid hormone formation in human ovarian follicles in vitro.
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Ovarian follicles of 5 to 15 mm in diameter were isolated from 45 ovaries of 34 patients in the follicular and luteal phases of the cycle. Three experiments were done. In the first, follicles were minced and incubated in Krebs-Ringer bicarbonate buffer containing 1 to 2muCi of testosterone-4-14C in the presence or absence of 100 IU human chorionic gonadotropan (hCG). In the second, minced follicles were incubated with 100 muCi of sodium acetate-I-14C under identical conditions. In the third, ten follicles from a single patient in the late proliferative stage of endometrial dating were cut in halves and incubated with 100 muCi of acetate-I-14C under identical conditions. The minced follicle preparation was capable of aromatizing testosterone-4-14C into radioactive estrone and estradiol in significant amounts. Incorporation of radioactive acetate into pregenolone, progesterone, 17-hydroxypregnenolone, 17-hydroxyprogesterone, dehydroepiandrosterone, androstenedione, testosterone, estradiol and estrone was assessed by reverse dilution analysis with recrystallization to constant specific activity. The major radioactive products formed were androstenedione and 17-hydroxyprogesterone in the latter two experiments. Dehydroepiandrosterone was one of the major steroids in the second experiment. The minor products were testosterone, progesterone and pregnenolone. Smaller, but definite incorporations of radioactive acetate into estradiol and estrone occurred in the second experiment. On histological examination, the follicles were characterized by atretic changes. This distribution pattern of radioactive acetate among the steroids was considered to represent the steroidogenic profile of unstimulated or atretic follicles. (+info)
Neurosteroid metabolism. 7 alpha-Hydroxylation of dehydroepiandrosterone and pregnenolone by rat brain microsomes.
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Two 'neurosteroids', dehydroepiandrosterone (DHEA) and pregnenolone (PREG), are converted by rat brain microsomes into polar metabolites, identified as the respective 7 alpha-hydroxylated (7 alpha-OH) derivatives by the 'twin ion' technique of g.l.c.-m.s. with deuterated substrates. The enzymic reaction requires NADPH and is stimulated 2-4-fold by EDTA. Under optimal conditions (pH 7.4, 0.5 mM-NADPH, 1 mM-EDTA), the Km values for DHEA and PREG are 13.8 and 4.4 microM respectively, and the Vmax. values are 322 and 38.8 pmol/min per mg of microsomal protein respectively. Trace amounts of putative 7 beta-OH derivatives of DHEA and PREG are detected. Oestradiol, at a pharmacological concentration of 5 microM, inhibits DHEA and PREG 7 alpha-hydroxylation. Formation of 7 alpha-hydroxylated metabolites is low in prepubertal rats and increases 5-fold in adults. Derivatives of PREG and DHEA, such as PREG sulphate, DHEA sulphate, progesterone and 3 alpha-hydroxy-5 alpha-pregnan-20-one, are known to be neuroactive. Therefore the quantitatively important metabolism to 7 alpha-OH compounds may contribute to the control of neurosteroid activity in brain. (+info)