Serum and tissue pregnanes and pregnenes after dexamethasone treatment of cows in late gestation.

Dexamethasone (DEX) initiates parturition by inducing progesterone withdrawal and affecting placental steroidogenesis, but the effects of DEX in fetal and maternal tissue steroid synthetic capacity remains poorly investigated. Blood was collected from cows at 270 days of gestation before DEX or saline (SAL) treatment, and blood and tissues were collected at slaughter 38 h later. Steroid concentrations were determined by liquid chromatography tandem mass spectrometry to detect multiple steroids including 5α-reduced pregnane metabolites of progesterone. The activities of 3ß-hydroxysteroid dehydrogenase (3ßHSD) in cotyledonary and luteal microsomes and mitochondria and cotyledonary microsomal 5α-reductase were assessed. Quantitative PCR was used to further assess transcripts encoding enzymes and factors supporting steroidogenesis in cotyledonary and luteal tissues. Serum progesterone, pregnenolone, 5α-dihydroprogesterone (DHP) and allopregnanolone (3αDHP) concentrations (all <5 ng/mL before treatment) decreased in cows after DEX. However, the 20α-hydroxylated metabolite of DHP, 20αDHP, was higher before treatment (≈100 ng/mL) than at slaughter but not affected by DEX. Serum, cotyledonary and luteal progesterone was lower in DEX- than SAL-treated cows. Progesterone was >100-fold higher in luteal than cotyledonary tissues, and serum and luteal concentrations were highly correlated in DEX-treated cows. 3ßHSD activity was >5-fold higher in luteal than cotyledonary tissue, microsomes had more 3ßHSD than mitochondria in luteal tissue but equal in cotyledonary sub-cellular fractions. DEX did not affect either luteal or cotyledonary 3ßHSD activity but luteal steroidogenic enzyme transcripts were lower in DEX-treated cows. DEX induced functional luteal regression and progesterone withdrawal before any changes in placental pregnene/pregnane synthesis and/or metabolism were detectable.

Bioanalytical LC-MS/MS method development and validation of novel antidiabetic candidate S007-1261 in rat plasma and its application to pharmacokinetic and oral bioavailability studies.

A sensitive and selective LC-MS/MS method has been developed and validated for CDRI antidiabetic candidate S007-1261 in rat plasma using 16-dehydropregnenolone as an internal standard. The API 4000 triple quadrupole LC-MS/MS system was operated under multiple reaction monitoring mode using electrospray ionization technique in positive mode. The sample processing method involves 2-step liquid-liquid extraction using n-hexane as an extracting solvent. The analyte was chromatographed on RP 18, waters column (3.5 µm, 2.1 mm i.d. × 30 mm) with guard using acetonitrile and ammonium acetate buffer (pH 5.0, 10 mM) in 90:10 (v/v) composition at a flow rate of 0.40 mL min(-1). The chromatographic run time was 5.30 min. Calibration curve shows linearity over concentration range 1.56-200 ng mL(-1). The lower limit of detection was 0.39 ng mL(-1) and lower limit of quantitation was 1.56 ng mL(-1). The inter- and intra-day accuracy and precision were found to be within the assay variability limits as per US FDA guidelines. The absolute recovery of S007-1261 was found to be >90%. S007-1261 does not show any stability problems as it was stable at room temperature for 8 h. S007-1261 was also stable up to 3 freeze-thaw cycles and can be stored up to 30 days at -60 °C. The assay was successfully applied to both oral (40 mg kg(-1)) and intravenous (10 mg kg(-1)) pharmacokinetic studies in male Sprague-Dawley rats. The oral bioavailability of S007-1261 was found to be 33.61%.

Measurement of cortisol, cortisone, prednisolone, dexamethasone and 11-deoxycortisol with ultra high performance liquid chromatography-tandem mass spectrometry: Application for plasma, plasma ultrafiltrate, urine and saliva in a routine laboratory.

We describe an ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC MS/MS) method suitable for a routine laboratory to determine endogenous and exogenous glucocorticoids in plasma, plasma ultrafiltrate, urine and saliva in a single analytical run. After addition of a multi-analyte internal standard, a standardised sample preparation procedure with solid phase extraction followed, before injecting into a tandem mass spectrometer with positive mode electron spray ionisation and multiple reactant monitoring acquisition. The chromatography time was 3min. The limit of quantitation for cortisol and cortisone in plasma was 3.75nmol/L and linearity extended to 2000nmol/L. The limit of quantitation for cortisol in plasma ultrafiltrate and saliva was 0.6nmol/L. The limit of quantitation for 11-deoxycortisol and prednisolone was 5nmol/L and for dexamethasone 1nmol/L. The intra-assay CV was <5% and the inter-assay CV <10% for all analytes in all matrices. Comparison with an immuno-assay (IA) plasma cortisol method resulted in a regression equation of UHPLC=0.79×IA+31.12 with R(2)=0.960 (p<0.0001). Comparison with a high performance liquid chromatography (HPLC) cortisol method yielded a regression equation of UHPLC=1.06×HPLC+9.82, R(2)=0.992 (p<0.0001). The simultaneous measurement of endogenous and exogenous glucocorticoids contributed to patient care in cases with dexamethasone and metyrapone dynamic tests and unsuspected therapeutic glucocorticoid use.

Modulation of 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) activity in the equine placenta by pregnenolone and progesterone metabolites.

The purpose of this study was to measure 3beta-HSD activity in the equine placenta and to assess the effect of fetal and maternal blood plasma progestagens on 3beta-HSD activity was measured in 8 late gestation (collected by caesarian section at 250 to 320 days) and 7 term (collected by caesarian section at 250 to 320 days) and 7 term (collected at birth) equine placentae using a tritium release assay with [3alpha-3H] pregnenolone as substrate. Mean +/- s.d. Km(app) and Vmax for term placentae were in general higher than for late gestation placentae (0.129 +/- 0.217 micromol/l and 23.85 +/- 9.1 nmol/mg/h respectively vs. 0.016 +/- 0.048 micromol/l and 17.36 +/- 20.9 nmol/mg/h) but there was no statistical difference between them. Inhibition studies were performed on 3 term placentae and 3 late gestation ones. Steroid concentrations used for inhibition studies were close to blood plasma concentrations (0.5 to 2 micromol/l). 3beta-hydroxy compounds (5alpha-pregnene-3beta, 20alpha-diol and 3beta-hydroxy-5alpha-pregnan-20-one) showed noncompetitive or mixed inhibition. Mean Ki(app) of 0.7 micromol/l. Inhibition was competitive with 20alpha-hydroxy-5alpha-pregnan-3-one with a mean Ki(app) of 0.1 micromol/l.

Plasma 3 beta-hydroxy-delta 5-steroids in patients with congenital adrenal hyperplasia due to 21-hydroxylase deficiency.

There is little information about the plasma concentrations of 3 beta-hydroxy-delta 5-steroids (delta 5-steroids) in untreated patients with congenital adrenal hyperplasia due to 21-hydroxylase deficiency. To further study the delta 5 pathway, we measured plasma levels of delta 5- and delta 4-steroids in 21 adult patients with different degrees of 21-hydroxylase deficiency (11 salt-wasters, 5 simple virilizers, and 5 patients with the nonclassical form of the disease). In all patients, investigations were performed after withdrawal of steroid treatment for at least 10 days. In addition, catheterization of gonadal and adrenal veins was performed in two salt-wasting male patients displaying bilateral testicular tumors to study adrenal secretion of delta 5- and delta 4-steroids. In one of them, surgical resection of the intratesticular adrenal rests gave the opportunity to measure 3 beta-hydroxysteroid dehydrogenase (3 beta HSD) activity. In all untreated patients, an increase in plasma delta 4-steroids was observed. In contrast, although plasma levels of dehydroepiandrosterone (DHEA) and dehydroepiandrosterone sulfate (DHEAS) were not significantly modified in simple virilizers, a paradoxical decrease in all delta 5-steroids was observed in salt-wasters. Catheterization of the adrenal veins confirmed the decrease in delta 5-steroids, particularly DHEA and DHEAS. The androstenedione/DHEA ratio was increased in all patients proportionally to the severity of the disease, suggesting an increase in adrenal 3 beta HSD. In vitro analysis of 3 beta HSD activity showed a 4-fold increase in intratesticular adrenal tissue compared to that in normal adrenals. A positive correlation between the androstenedione/DHEA ratio and plasma ACTH levels was observed, suggesting a long term stimulatory effect of ACTH on 3 beta HSD. Angiotensin-II could have an additive effect on ACTH-induced 3 beta HSD activity.

Adrenal steroidogenesis in the guinea pig: effects of androgens.

In humans, the onset of adrenache has been found to occur with the appearance of the zona reticularis, the inner zone of the adrenal cortex. Since an increase in the volume of adrenal cortex during maturation in the guinea pig has been associated with the growth of the zona reticularis, we were interested in investigating the changes in adrenal steroidogenesis during maturation in this species. In addition, the effect of androgens on adrenal steroidogenesis was studied. We demonstrated that between 1 and 10 weeks of age, a period of maximal growth of the adrenals in the guinea pig, there is a decrease in the concentrations of adrenal pregnenolone, cortisol, dehydroepiandrosterone, testosterone, androstenedione, and 11 beta-hydroxyandrostenedione, suggesting lower steroid production by the guinea pig adrenals. In plasma, we observed that the concentration of 11 beta-hydroxyandrostenedione (the sole C19 steroid present after castration) remained unchanged during maturation, while cortisol and corticosterone were lower between 1 and 4 weeks of age. Although castration as well as the administration of the antiandrogen flutamide had no effect on adrenal steroidogenesis, dihydrotestosterone caused an inhibition of cortisol and corticosterone levels in the adrenals while the concentrations of progestins (namely, pregnenolone, 17-hydroxypregnenolone, progesterone, and 17-hydroxyprogesterone) tended to increase in the adrenals, thus suggesting that dihydrotestosterone induces a blockade in the steroidogenic pathway.(ABSTRACT TRUNCATED AT 250 WORDS)

Urinary 5-ene-steroid excretion in non-classical congenital adrenal hyperplasia due to 3 beta-hydroxysteroid dehydrogenase deficiency (NC-3BHSD).

The diagnosis of non-classical 3 beta-hydroxysteroid dehydrogenase deficiency (NC-3BHSD) is made either on the basis of significantly elevated serum levels of basal and post-ACTH 5-ene-steroids or by the presence of elevated urinary 5-ene-steroid metabolites. There has been only one report to date describing a single patient where the diagnosis was based on both serum and urinary 5-ene-steroid levels. We, therefore, measured both serum 5-ene-steroid responses to ACTH 1-24 (by RIA) and urinary 5-ene-steroid metabolites (GC-MS) in 42 hirsute premenopausal women. While the serum 5-ene-steroid profile was consistent with NC-3BHSD in 5 women, only 2 of them had increased excretion of 5-ene-steroid metabolites. Elevated 5-ene-steroid excretion was also observed in several patients with normal serum 5-ene-steroids. Detection of NC-3BHSD by either elevated serum 5-ene-steroids or increased urinary excretion of their metabolites in isolation may not therefore be reliable.

Plasma levels of ovarian steroids, including 17 alpha-20 alpha-dihydroxy-4-pregnen-3-one and 3 beta,17 alpha,20 alpha-trihydroxy-5 beta-pregnane, in female dabs (Limanda limanda)--marine flatfish--induced to mature and ovulate with human chorionic gonadotrophin.

Human chorionic gonadotrophin (HCG) effectively stimulated oocyte final maturation and ovulation in female dabs (Limanda limanda) within 5 days of injection, and this was accompanied by significant changes in blood plasma steroid levels. The steroids which showed the greatest responses to the HCG injections were the ones previously found to be the major products of the ovaries in vitro: 17 alpha-20 alpha-dihydroxy-4-pregnen-3-one (17,20 alpha-P) and 3 beta,17 alpha,20 alpha-trihydroxy-5 beta-pregnane (3 beta,17,20 alpha-P-5 beta). 17,20 alpha-P responded more rapidly with peak levels after 32 hr of injection (115 ng ml-1), but 3 beta,17,20 alpha-P-5 beta reached higher levels ca. 12 hr later (320 ng ml-1). Levels of both steroids were not significantly different from initial values by the time of ovulation. 17 alpha,20 beta-Dihydroxy-4-pregnen-3-one, which is likely to be the oocyte maturation-inducing steroid (MIS) in the dab, showed a significant but very variable rise in levels (between 1 and 10 ng ml-1 in individual fish). 17 alpha-Hydroxy-4-pregnene-3,20-dione levels peaked at 6 ng ml-1 between 30 and 36 hr after HCG injection. Of the other C21 steroids identified in the ovaries of teleosts, 17 alpha,20 beta-21-trihydroxy-4-pregnen-3-one could not be detected, and 17 alpha,21-dihydroxy-4-pregnene-3,20-dione (11-deoxycortisol) showed nonsignificant changes compared to the saline-injected controls. HCG caused a decrease in estradiol-17 beta levels within 24 hr, but levels then rose again to a maximum of 8.2 ng ml-1 at ovulation time, possibly caused by the presence of vitellogenic oocytes in the ovaries. Changes in testosterone levels, however, were not significantly different between HCG- and saline-injected females. The role of HCG-responsive C21 steroids in the dab is discussed.

[Steroid profiles in patients with amenorrhea during induction of ovulation with HMG].

Serum concentrations of various hormones in seven normal women were measured daily for 5 days before and after ovulation. Steroid levels were also measured in severe amenorrheic patients during the induction of ovulation with HMG-HCG. Blood samples from the patients of II grade amenorrhea were collected on the day when the cervical mucus increased more than 200 mm3 in HMG therapy. HCG was given after the blood samples were obtained. Ovulation was successfully induced in six patients and they were classified as group I. In 8 patients induction of ovulation did not succeed and these patients were classified as group II. Hormone levels including LH, FSH, estradiol (E2), progesterone (P4), 17 alpha OH-P4 (17P4), delta 4 androstenedione (delta 4 A), testosterone (Tes.), pregnenolone (P5), 17 alpha OH-P5 (17P5), DHA, delta 5 androstenediol (delta 5 AD), and 20 alpha OH-P4 (20P4) were measured by specific RIA. The following results were obtained. Steroid levels during normal ovulatory cycle: Levels of E2 (380 +/- 16 pg/ml), P5 (6.9 +/- 4.1 ng/ml), and Tes. (3.3 +/- 1.2 ng/ml) showed a peak on the day before LH surge. A significant increase in P4, 17P5 and 20P4 levels was observed after ovulation. Hormone levels in group I: FSH in group I was significantly higher while LH was lower than that in normal women measured during -1 to -3 days from LH surge. On the other hand, among the steroids measured, significantly low Tes. and high 17P5, and E2 levels were noticed in group I. Comparison of hormone levels between group I and II: FSH and LH levels showed no significant difference between the two groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Angiotensin II effect on plasma steroids in selective hypoaldosteronism.

The effect of angiotensin II infusion on plasma pregnenolone, progesterone, corticosterone and aldosterone was investigated in 4 cases of established hypoaldosteronism, in 4 elderly controls in the same age range and in 6 young normals. In young and old normals, angiotensin II induced the expected dose response increase in aldosterone while corticosterone usually decreased progressively during the infusion. Progesterone levels were not significantly different in young and old subjects and no change was observed during angiotensin II infusion. Baseline pregnenolone levels were significantly lower in elderly controls and angiotensin II elicited a slight decrease in pregnenolone in the two control groups. In selective hypoaldosteronism, baseline plasma aldosterone concentrations were very low and the aldosterone response to angiotensin II was blunted. Plasma corticosterone and progesterone levels were in a comparable range to normals throughout the study. Contrary to control subjects, a dose dependent increase in pregnenolone was observed during angiotensin II infusion in the patient group. These results suggest that the anomalies of steroid biosynthesis found in selective hypoaldosteronism could be contributing factors to the hypoaldosteronism in some patients.

Studies on ovarian and adrenal steroids at different phases of the menstrual cycle. III. Steroid and lutropin levels before and after the administration of a single contraceptive dose of depot-medroxyprogesterone acetate (DMPA).

Ovarian and adrenal steroids and biologically active lutropin were measured in peripheral plasma samples obtained from 5 normally menstruating women. Plasma samples were collected every 3 h for a period of 39 hours in the periovulatory period of a pretreatment (control) cycle and then 16 and 54 days after a single i.m. injection of 150 mg of depot-medroxyprogesterone acetate (DMPA). Sixteen days after DMA administration, the levels of estradiol, progesterone, 17-hydroxyprogesterone, and lutropin were reduced to early follicular phase levels. No further decrease was found in 17-hydroxyprogesterone and lutropin levels; however an additional decrease occurred in the levels of estradiol and in the "morning" levels of progesterone 54 days after the administration of DMPA. Furthermore, the levels of pregnenolone, androstenedione, testosterone and dihydrotestosterone were significantly diminished in all samples collected after the administration of DMPA. Fifty-four days following the administration of DMPA, the levels of cortisol and 17-hydroxypregnenolone were significantly reduced. The administration of DMPA did not interfere with the circadian rhythm of cortisol, pregnenolone, 17-hydroxypregnenolone, dehydroepiandrosterone, 17-hydroxyprogesterone, and androstenedione levels. A significant circadian rhythm was also found in testosterone (after 16 days) and lutropin (after 54 days) levels. No circadian variation was found in estradiol, progesterone and dihydrotestosterone levels.

[Change of C19-21 steroids levels of maternal serum, amnion and cord plasma by high pressure liquid chromatography (HPLC) (author's transl)].

With normal pregnant and abnormal pregnant cases as the subjects steroids in the maternal serum at term and plasma cord were measured by HPLC and the following results were obtained. 1) The blood at term proved to contain cortisol 165.06 +/- 38.90 ng/ml, cortisone 85.97 +/- 16.40 ng/ml, progesterone 23.42 +/- 8.77 ng/ml and the blood at delivery to contain cortisol 574.68 +/- 156.76 ng/ml, cortisone 100.94 +/- 27.54 ng/ml and progesterone 41.14 +/- 18.31 ng/ml. Amnion contained cortisol 69.40 +/- 16.72 ng/ml, cortisone 26.11 +/- 9.13 ng/ml, progesterone 29.36 +/- 9.35 ng/ml, cord plasma contained cortisol 109.64 +/- 27.41 ng/ml, cortisone 373.32 +/- 88.60 ng/ml and progesterone 450.53 +/- 113.84 mg/ml. 2) The blood at delivery compared to that term showed especially higher lever of cortisol. Maternal serum showed the pattern of cortisol superiority of the corticoid, while cord plasma showed the pattern of cortisone superiority. 3) Significant correlation was observed between amnion and cord plasma in the gestagen and between amnion and maternal serum-cord plasma in corticoid. 4) In severe toxemia of pregnancy and pregnancy with diabetes mellitus some steroids showed significant low levels as compared to normal case. 5) In severe toxemia of pregnancy cortisone/cortisol ratio was irregular as compared to normal case.

Development of a procedure for the assay of an experimental steroid drug in dog plasma.

Studies of the estimation of 16alpha-cyano-3beta-cyclopentyloxypregn-5-en-20-one (an experimental drug) in dog plasma are described. Extraction using a salt/solvent pair (ammonium carbonate/ethyl acetate) is followed by a rapid chromatographic procedure employing Lipidex 5000, which affords a substantially purified fraction. After preparation of the t-butyldimethylsilyloxime, quantification of the drug is performed by selected ion monitoring. The [2H9]cyclopentyloxyl analogue is used as an internal standard. In a preliminary experiment, the advantages (in terms of both sensitivity and selectivity) of the use of an open tubular GLC column are demonstrated.

Studies on the pattern of circulating steroids in the normal menstrual cycle. I. Simultaneous assays of progesterone, pregnenolone, dehydroepiandrosterone, testosterone, dihydrotestosterone, androstenedione, oestradiol and oestrone.

In an attempt to analyze the multiple changes and interactions in circulating steroid levels in the peri-ovulatory and peri-menstrual periods, the plasma levels of immunoreactive luteinizing hormone (LH), progesterone and unconjugated pregnenolone, dehydroepiandrosterone, testosterone, oestradiol and oestrone were assayed daily during a complete cycle in 17 normally menstruating women. In 14 of the 17 subjects studied androstenedione and unconjugated dihydrotestosterone were also estimated. The day of the LH-peak and the first day of menstruation, respectively, were used to synchronize the peri-ovulatory and peri-menstrual plasma levels of the various steroids. With the exception of dehydroepiandrosterone and dihydrotestosterone, the plasma levels of all steroids exhibited significant, but different changes during the cycle. Testosterone levels showed a slight but significant increase around the LH-peak, whereas the levels of pregnenolone and androstenedione were higher in the post-ovulatory than in the pre-ovulation periods. The levels of oestradiol and oestrone, as well as the ratios of oestradiol to oestrone gradually increased from the low values observed in the early proliferative phase to pre-ovulatory peak values. The relationship between peaks of oestradiol and oestrone and that of LH exhibited great individual variation. The same was true for the individual oestradiol to oestrone ratios. The combination of several steroidal signals did not improve the predictive value of the analyses. However, an increase of individual progesterone values by at least 0.35 ng/ml from the day preceding the LH-peak to the day of the LH-peak was observed in 13 of the 17 subjects. It is suggested that for the early detection of the LH surge and prediction of the subsequent ovulation daily assays of plasma progesterone are of more value than the assay of the other steroids investigated.

Combined radioimmunoassay of four steroids in one ml of plasma: I. Progestins.

Using partially specific antisera combined with a 1 step celite microcolumn chromatography, progesterone (P), 20alpha-hydroxypregn-4-ene-3-one (20alpha-P), 17-hydroxyprogesterone (17-P), and 16alpha-hydroxyprogesterone (16alpha-P) could be measured in the same 1 ml aliquot of plasma. The chromatographic step removed known interfering steroids and conferred specificity to the assay. After correction for recovery the sensitivities, expressed as ng/ml of plasma, were respectively: 0.04 for P, 0.03 for 20alpha-P, 0.02 for 17P, and 0.01 for 16alpha-P. Recovery experiments, using steroid-free plasma to which various amounts of each steroid were added and then measured in the assay in 12 replicates, confirmed adequate accuracy and precision. The ability to measure multiple progestogens in small volumes of plasma should permit comprehensive evaluation of the role of these steroids in health and disease.

Steroid entry into rete testis fluid and the blood-testis barrier.

Evidence that steroids enter rete testis fluid (RTF) from the blood at varying rates was obtained during i.v. infusions into rats. Testosterone and dehydroepiandrosterone were readily transferred into the fluid, whereas cholesterol was excluded. Between these extremes, the appearance of radioactivity in the RTF suggested the following order of entry rate: progesterone greater than pregnenolone greater than 5-alpha-reduced androgens greater than oestrogens greater than corticosteroids. Preliminary identification of metabolites in RTF and blood suggested that testosterone and dehydroepiandrosterone were transferred largely unchanged. Androstenedione and progesterone, however, were largely metabolized during transfer into the RTF, the former being transformed to testosterone. The results are used to discuss the nature of the blood-testis barrier to steroids and the source of androgens in the RTF.