Gas chromatographic-mass spectrometric study of metabolites of C21 and C19 steroids in neonatal porcine testicular microsomes.

Microsomal fractions obtained from testes of 3-week-old piglets have been incubated, separately, with progesterone, 17-hydroxyprogesterone, 5-pregnene-3 beta,20 beta-diol, 16 alpha-hydroxypregnenolone, 5-androstene-3 beta,17 alpha-diol and dehydro-epiandrosterone. The metabolites, after derivatization, have been separated by capillary gas chromatography and identified by mass spectrometry. Quantification was by selected ion monitoring. Progesterone was shown to be 17-hydroxylated and also converted into 4,16-androstadien-3-one (androstadienone). The major metabolite of 17-hydroxyprogesterone was 4-androstene-3,17-dione (4-androstenedione), but little, if any, androstadienone was formed, indicating that this particular biosynthesis did not require 17-hydroxylation. The metabolites of 5-pregnene-3 beta, 20 beta-diol were found to be 17-hydroxypregnenolone, 3 beta-hydroxy-5,16-pregnadien-20-one (16-dehydropregnenolone) and 5,16-androstadien-3 beta-ol. Dehydroepiandrosterone and 5-androstene-3 beta,17 alpha-diol were interconvertible but neither steroid acted as a substrate for 16-androstene formation. However, dehydroepiandrosterone was metabolized to a small quantity of 4-androstenedione. Under the conditions used, no metabolites of 16 alpha-hydroxypregnenolone could be detected. The present results, together with those obtained earlier, indicate that the neonatal porcine testis has the capacity to synthesize weak androgens, mainly by the 4-en-3-oxo steroid pathway. Although 16-androstenes cannot be formed from C19 steroids, progesterone served as a substrate and may be converted directly to androstadienone, without being 17-hydroxylated first. The pathway to 5,16-androstadien-3 beta-ol, however, involves 17-hydroxypregnenolone and 16-dehydropregnenolone as intermediates.     

Corticoid formation by the monkey fetal adrenal: Evaluation of 17-, 21- and 11-hydroxylase activities

There is indirect evidence that cortisol synthesis in the fetal rhesus monkey adrenal gland is limited at Day 135 of gestation but increases thereafter. This study was conducted to ascertain whether a reduced synthetic capacity is caused by a deficiency in 17-, 21- or 11-hydroxylase activity. For the sake of comparison 11- and 21-hydroxylases were also estimated in adult adrenals. 11-, 21-Hydroxylases were measured in the entire adrenal by the oxidation of NADPH by mitochondria and microsomes, respectively. 17-Hydroxylase was evaluated in outer and inner regions of the fetal gland by the formation of [³H]17-hydroxyprogesterone, -11-deoxycortisol, -cortisol and -androstenedione from [³H]progesterone. The maximum velocity of both the 11- and 21-hydroxylase was similar in fetal and adult glands indicating that corticoid formation in the fetus is not constrained by levels of these enzymes.[³H]Progesterone was extensively metabolized to -17-hydroxyprogesterone, -androstenedione, -11-deoxycortisol and -cortisol by homogenates from both regions of the fetal adrenal. The ratio of [³H]-cortisol to [³H]11-deoxycortisol was consistently higher in incubations of the inner glandular area. Together, these findings indicate that 17-hydroxylase is also active at Day 135 and that the 11-hydroxylase may be more concentrated in the fetal cortex. These data suggest in addition that the restriction in cortisol formation occurs at a step prior to the metabolism of progesterone to cortisol.     

Differential effects of 3,4,5,3',4',5' -hexachlorobiphenyl (hcb) on interrenal steroidogenesis in male and female rainbow trout Oncorhynchus mykiss

1. The conversion of progesterone to 17α-hydroxyprogesterone (17α-OHP), 11-deoxycortisol (11-DOCR) and deoxycorticosterone (DOC) was significantly higher in female rainbow trout than in male trout; in contrast, the interrenal production of cortisol (CR) plus cortisone (CN) was higher in males than in females.2. Following treatment with 1 mg/kg of HCB, the interrenal conversion of progesterone to 17α-OHP and 11-DOCR was significantly increased in male and female trout but at 20 mg/kg of HCB, the production of these metabolites was increased in males and decreased in females; CR + CN production was unchanged after HCB treatment in both sexes.     

Cytochrome P450 and steroid 21-hydroxylation in microsomes from beef adrenal cortex

Cytochrome P450 in beef adrenal cortex microsomal preparations reacted with progesterone and with 17-hydroxyprogesterone at pH 7.4 to produce Type I spectral changes. The magnitude of the spectral shift produced by addition of progesterone or 17-hydroxyprogesterone was related to the concentration of cytochrome P450 (over P450 concentration range of 0.1 to 0.3 μM). Prior saturation of cytochrome P450 with 17-hydroxyprogesterone prevented further spectral shift with the addition of progesterone. On the other hand, saturation of cytochrome P450 with progesterone decreases the expected shift with 17-hydroxyprogesterone by more than 50% but did not prevent the shift. The difference spectra were diminished by more than 50% at pH 9.0.The addition of NADPH resulted in loss of the spectral shifts and production of 21-hydroxylated products, predominantly DOC and 11-deoxycortisol. These reactions were not inhibited by their specific products. The rate of 21-hydroxylation was linearly related to microsomal protein (and microsomal P450) concentration. The 21-hydroxylation of progesterone was competitively inhibited by 17-hydroxyprogesterone; inhibition of the 21-hydroxylation of 17-hydroxyprogesterone by progesterone was not demonstrated.     

Steroid 21-hydroxylase activity in equine ovarian follicles evidenced by isotope dilution-mass spectrometry

Steroid 21-hydroxylase activity of the microsome-enriched fraction of follicular linings from equine ovaries has been demonstrated by gas chromatography-mass spectrometry. The 21-hydroxylated metabolites were quantified by isotope dilution with deuterated analogues. The two most abundant potential substrates for follicular steroid 21-hydroxylase, progesterone (P) and 17-hydroxyprogesterone (17OHP), were converted respectively to 11-deoxycorticosterone (DOC) and 11-deoxycortisol with corresponding apparent specific activities of 308 and 24 pmol/mg protein/h and apparent Km values of 1.1 and 6.4 microM. Competitive inhibition of the P-to-DOC conversion was exerted by 17OHP and pregnenolone. Hence, the ovarian follicle of the mare is an extraadrenal site of preferential DOC biosynthesis by an enzyme having steroid 21-hydroxylase activity.     

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.     

Inhibition of 17,20(17-hydroxyprogesterone)-lyase by progesterone

¹⁴C-17-Hydroxyprogesterone was incubated with 7000 × g × 20 min supernatants of rat testis homogenates in the presence of various concentrations of ³H-progesterone, both under conditions where metabolism would take place and where it would be prevented. When metabolism was prevented, the ratio of progesterone to 17-hydroxyprogesterone in the microsomal fraction was 3 times that which was added to the incubation medium.Progesterone competitively inhibited 17,20-lyase action on added 17-hydroxyprogesterone but not on 17-hydroxyprogesterone formed from the added progesterone. The rate of formation of 17-hydroxyprogesterone from progesterone, however, was inhibited by added 17-hydroxyprogesterone. The results indicate that there is no free exchange of an intermediate between progesterone and androstenedione with the soluble fraction, either inside or outside the microsomal vesicle. The limited exchange with 17-hydroxyprogesterone in solution probably represents exchange with an enzyme-bound intermediate.     

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.     

Increased adrenal steroid secretion in response to CRF in women with hypothalamic amenorrhea

OBJECTIVE: To evaluate adrenal steroid hormone secretion in response to corticotropin-releasing factor (CRF) or to adrenocorticotropin hormone in women with hypothalamic amenorrhea. DESIGN: Controlled clinical study. SETTING: Department of Reproductive Medicine and Child Development, Section of Gynecology and Obstetrics, University of Pisa, Italy. PATIENT(S): Fifteen women with hypothalamic amenorrhea were enrolled in the study. Eight normal cycling women were used as control group. INTERVENTION(S): Blood samples were collected before and after an injection of ovine CRF (0.1 microg/kg iv bolus) or after synthetic ACTH (0.25 mg iv). MAIN OUTCOME MEASURE(S): Plasma levels of ACTH, 17-hydroxypregnenolone (17OHPe), progesterone (P), dehydroepiandrosterone (DHEA), 17-hydroxyprogesterone (17OHP), cortisol (F), 11-deoxycortisol (S) and androstenedione (A). RESULT(S): Basal plasma concentrations of ACTH, cortisol, 11-deoxycortisol, DHEA and 17OHPe were significantly higher in patients than in controls, whereas plasma levels of progesterone and 17-OHP were significantly lower in patients than in controls. In amenorrheic women the ratio of 17-OHPe/DHEA, of 17-OHPe/17-OHP and of 11-deoxycortisol/cortisol were significantly higher than in controls, while a significant reduction in the ratio of 17-OHP/androstenedione, of 17-OHP/11-deoxycortisol was obtained. In response to corticotropin-releasing factor test, plasma levels of ACTH, cortisol, 17-OHP, 11-deoxycortisol, DHEA and androstenedione were significantly lower in patients than in controls. In response to adrenocorticotropin hormone, plasma levels of 17-OHP, androstenedione and androstenedione/cortisol were significantly higher in patients than in controls. CONCLUSIONS: Patients suffering for hypothalamic amenorrhea showed an increased activation of hypothalamus-pituitary-adrenal (HPA) axis, as shown by the higher basal levels and by augmented adrenal hormone response to corticotropin-releasing factor administration. These data suggest a possible derangement of adrenal androgen enzymatic pathway.     

Steroid biosynthesis by zona glomerulosa-fasciculata cells in primary culture of guinea-pig adrenals

Steroidogenesis was studied in guinea-pig glomerulosa-fasciculata cells maintained in primary culture for up to 7 days. The basal secretion which remained stable for the first 2 days in culture rapidly rose to reach a plateau on day 4 at levels 6-7-fold higher than those observed during the first 2 days of culture while the maximal response to ACTH in terms of cortisol and androstenedione secretion was fairly stable throughout the 7-day period. Exposure of glomerulosa-fasciculata cells to ACTH caused a stimulation of pregnenolone, 17-hydroxypregnenolone, progesterone, 17-hydroxyprogesterone, corticosterone, 11-deoxy-corticosterone, 11-deoxycortisol, cortisol, dehydroepiandrosterone, androstenedione, 11 beta-hydroxyandrostenedione and aldosterone while, after 48 h of incubation, a marked accumulation of end-products, namely cortisol and 11 beta-hydroxyandrostenedione, was observed. The half-maximal steroidogenic response to ACTH occurred at concentrations varying between 1.7 x 10(-11) and 1.1 x 10(-10) mol/l for the 12 steroids examined. Addition of 8-bromoadenosine 3', 5'-cyclic monophosphate stimulated steroid secretion in a dose-dependent manner. Maximal response to 8-bromoadenosine 3', 5'-cyclic monophosphate was obtained at 1 mmol/l, and no further rise of steroid secretion was observed after addition of ACTH. Incubation of glomerulosa-fasciculata cells with labeled corticosterone, cortisol and androstenedione indicates that only androstenedione can be converted into 11 beta-hydroxyandrostenedione, thus suggesting that this end-product is a good parameter of the C-19 steroid production by guinea-pig glomerulosa-fasciculata cells in primary culture. The present data confirm that guinea-pig glomerulosa-fasciculata cells in primary culture provide an interesting model for the study of the regulation of C-19 steroid formation by the adrenals.     

Transformation of progesterone by a thermophilic bacillus

Abstract When a thermophilic bacillus was incubated with progesterone for approx. 18 h at 65°C, four progesterone-based metabolites were produced in moderate quantities. Two products were found to be hydroxy derivatives, i.e. 6α-hydroxyprogesterone and 6β-hydroxyprogesterone and two were C-20 reduced epimers of progesterone, i.e. 20α- and 20β-dihydroprogesterone. 6α-Hydroxyprogesterone is a rare microbial transformation product.
Inhibition of hydroxylation by azole-based fungicides and the presence of a carbon-monoxide-reduced difference-spectrum absorbance maximum at 448 nm in the soluble cell fraction suggest that the hydroxylase(s) might be cytochromes(s) P450. The dihydroprogesterones were probably produced by oxidoreductases.     

Bioconversion of 2α-hydroxyprogesterone to 2-hydroxylated corticosteroids by newborn rat adrenal cells in primary culture

The bioconversion of 2α-hydroxyprogesterone into 2-hydroxylated steroids was accomplished using newborn rat adrenal cells in primary culture. The products were purified using column and thin-layer chromatography, and identified by GC-MS. They resulted principally from the enzymatic reactions of 21-hydroxylation, 11β-hydroxylation, reduction of 20-oxo and 3-oxo groups, and epimerization of the substrate. In addition, minor metabolites resulted from 18-hydroxylation, 6β-hydroxylation and reduction of the 3-oxo-4-ene group. The identification of these compounds allowed us to conclude that the metabolism of 2α-hydroxyprogesterone is similar to that of progesterone in this cellular system. Assuming that the 2β-epimers of the different metabolites arose principally from the transformation of 2β-hydroxyprogesterone, the specificity of the various enzyme systems seems to be similar for both epimers except in the case of the 11β-hydroxylation where the reaction appears stereospecific for the 2β-epimer. The 2α-hydroxyl group on ring A seems to favor the reduction of the 3-oxo group and it does this stereospecifically to the 3β-structure. The epimerization of the substrate, which is most likely enzymatically induced, is the first example of steroid epimerization reported in the adrenal. This is a practical preparative method for synthesizing a variety of steroids hydroxylated at C-2 from a single substrate and could be adjusted to the production of important quantities of 2-hydroxylated metabolites of corticosteroids.     

Solid phase fluoroimmunoassay for 11-deoxycortisol in serum using 21-amino-17-hydroxyprogesterone

Solid phase fluoroimmunoassay of serum 11-deoxycortisol (17,21-dihydroxy-4-pregnene-3,20-dione) was established using fluorescein isothiocyanate-labelled 11-deoxycortisol and anti-11-deoxycortisol antibody-conjugated polyacrylamide beads. 21-Amino-17-hydroxyprogesterone (21-amino-17-hydroxy-4-pregnene-3,20-dione) was synthesized as a useful derivative for preparing the fluorescent dye conjugate. Serum 11-deoxycortisol was measured with this assay system after extraction and purification by Sephadex LH-20 column chromatography. The minimal amount of 11-deoxycortisol detected was 40 pg/tube and the measurable range was from 0.04 to 5.0 microgram/dl. Intra- and inter-assay coefficients of variation were 8.3% (n=6) and 9.8% (n=5), respectively. 11-deoxycortisol values determined by the present assay correlated well with those determined by radioimmunoassay. The present assay is particularly suitable for estimating the conditions of the pituitary and adrenocortical functions.     

Metabolic pathways for androstanediol formation in immature rat testis microsomes

Metabolic routes from progesterone to androstanediol in washed rat testicular microsomes were investigated, with special emphasis on the importance of 4-ene-3-oxosteroids, as well as the effect of a minimal effective dose of human chorionic gonadotropin on these transformations. Incubation of equimolar concentrations of a mixture of [¹⁴C]progesterone and 17α-hydroxy[³H]progesterone resulted in a large preference of 17α-hydroxyprogesterone over progesterone as substrate for androstanediol formation. Incubation of [³H]progesterone together with [¹⁴C]androstenedione resulted in the inhibition of C-17,20-lyase and in a low ¹⁴C/³H ratio in androstanediol, indicating the preference of progesterone over androstenedione as substrate for androstanediol production. When a mixture of 17α-hydroxyl[³H]progesterone and [¹⁴C]androstenedione was incubated with the microsomes, a more than 8-fold preference of 17α-hydroxyprogesterone as substrate for androstanediol production was found. The minimal dose of human chorionic gonadotropin stimulated testosterone production but inhibited androstanediol formation and effected, in some instances, a change in the metabolic routes. It is concluded that androstanediol is produced preferentially through 17-hydroxylated C-21 steroids, and also, to a lesser extent, through C-19 steroids.     

Metabolism of 11-deoxycortisol by a Bacillus species

Microbial transformation by a Bacillus species was employed for the preparation of potentially important derivatives of 11-deoxycortisol. Each microbial metabolite was characterised by the application of various spectroscopic methods. The five metabolites of 11-deoxycortisol were characterised as 4-androstene-3,17-dione (2), 14-hydroxy-4-androstene-3,17-dione (3), 14,17 alpha,21-trihydroxy-4-pregnene-3,20-dione (4), 6 beta,17 alpha,21-trihydroxy-4-pregnene-3,20-dione (5) and 15 alpha,17 alpha,21-trihydroxy-4-pregnene-3,20-dione (6). The availability of the metabolites enabled complete elucidation of their [13C]NMR spectra.     

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.     

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.     

Potential limitations of recrystallization for the definitive identification of radioactive steroids

The usefulness of recrystallization in establishing the radiochemical purity of steroids is widely recognized, but the potential limitations of the technique have received little attention. The current study reports the failure of standard recrystallization procedures using methanol/water as the solvent pair to separate contaminating ¹⁴C-17-hydroxyprogesterone (17-hydroxy-4-pregnene-3, 20-dione) from ³H- and ¹⁴C-labeled 11-deoxycortisol (17,21-dihydroxy-4-pregnene-3,20-dione) despite ten serial crystallizations. The standard criteria of radiochemical purity were met despite gross impurity of the crystals as evidenced by thin layer chromatography. Thus, recrystallization may, under certain conditions, yield misleading results when employed as the only method for identifying radioactive steroids. These observations illustrate the importance of an optimal choice of solvent and crystallization conditions, and emphasize the need for confirmation by derivative formation and chromatography.     

Kinetic analysis of duodenal and testicular cytochrome P450c17 in the rat

In the adult rat, the duodenal tissue of both sexes can convert progesterone to 17-hydroxyprogesterone, androstenedione and testosterone. The transition from C₂₁ to C₁₉ steroids is apparently controlled by the same cytochrome P450c17 expressed in the testis, which catalyzes both 17-hydroxylation and C-17,20 bond scission at a single bifunctional active site. The kinetic parameters of this enzyme were measured at the steady state for both reactions using [1,2-³H]progesterone and [1,2-³H]17-hydroxyprogesterone as substrates. In the testis and male and female duodena, the K_m values for progesterone 17-hydroxylation were 14.2, 23.8 and 23.2 nM, whereas the V_max values were 105, 3.5 and 3.1 pmol/mg protein/min, respectively. With respect to C-17,20 lyase activity, the K_m values for exogenous 17-hydroxyprogesterone were 525, 675 and 637 nM, whereas the V_max values were 283, 7.8 and 7.8 pmol/mg protein/min, respectively. However, when the K_m values were calculated with respect to intermediate 17-hydroxyprogesterone formed from progesterone, they were similar to the K_m values for 17-hydroxylase, being 15, 31.4 and 24.8 nM, whereas the V_max values were 26.3, 2 and 1.8 pmol/mg protein/min, respectively. The similarity of K_m values is due to the fact that the relative androgen formation efficiency (bond scission events/total 17-hydroxylation events ratio) was remarkably constant in both testicular and duodenal incubates, irrespective of progesterone concentration. Efficiency values were 2-fold higher in duodenal tissue (0.54) than in testis (0.25). Estradiol-17β inhibited 17-hydroxylation but not bond scission on intermediate 17-hydroxyprogesterone, because it did not affect the efficiency value. Rat duodenal P450c17 has the same substrate affinity, a lower specific activity and a higher androgen formation efficiency than testicular P450c17.