Bioavailability and metabolism of oral and percutaneous dehydroepiandrosterone in postmenopausal women

To study the bioavailability of dehydroepiandrosterone (DHEA) administered by the oral and percutaneous routes, three groups of 12 postmenopausal women aged 60-70 years received two capsules of 50mg of DHEA orally before breakfast daily for 14 days or applied 4 g of a 10% DHEA cream or gel at the same time of the day on a 30 cm x 30 cm surface area on the thighs. Detailed serial blood sampling over 24h was performed following 1st and 14th DHEA administration for measurement of DHEA and nine of its metabolites by liquid chromatography tandem mass spectrometry (LC-MS/MS) or gas chromatography mass spectrometry (GC-MS). Serum levels of estrone (E1) and estradiol (E2) did not change following DHEA administration by any of the three formulations, while serum androstenedione (4-dione), testosterone, DHEA sulfate (DHEA-S), E(1)-S, androsterone glucuronide (ADT-G) and 3alpha-androstanediol-G (3alpha-diol-G), increased in all cases, the effect on these parameters being more important after oral than percutaneous administration due to the metabolism of DHEA into these metabolites in the gastrointestinal tract and liver. No qualitative differences in DHEA metabolism are observed between the oral and percutaneous routes of DHEA administration while the levels of all steroids remain on a plateau during the 24h period during chronic percutaneous DHEA administration. The present data show that DHEA is transformed into active androgens and estrogens in peripheral intracrine tissues with no or minimal release of the active steroids E(1), E(2) or testosterone in the circulation. Moreover, DHEA is preferentially transformed into androgens rather than into estrogens. Most importantly, the present data show that changes in serum DHEA following oral or percutaneous DHEA administration are not a valid parameter of DHEA action since the increase in serum DHEA is at least 100% greater than the increase in the formation of active androgens and estrogens and thus much higher than the potential physiological effects.     

Changes in serum DHEA and eleven of its metabolites during 12-month percutaneous administration of DHEA

Healthy postmenopausal women aged 60-65 years (n=150) were randomized to receive twice daily application on the skin of 3g of a 0.3% dehydroepiandrosterone (DHEA) or placebo emulsion for 12 months. Serum DHEA and eleven of its metabolites were measured at screening and on day 1, as well as at 1, 3, 6, 9 and 12 months to study long-term metabolism. While serum DHEA and androst-5-ene-3beta, 17beta-diol (5-diol) increased by 203% and 178%, respectively, on average, during the 12-month period, the sum of concentrations of the metabolites of androgens, namely androsterone glucuronide (ADT-G), androstane-3alpha,17beta-diol-3G and -17G increased by only 71% while usually non statistically significant changes of 30%, 17% and 20% were observed for estrone (E(1)), estradiol (E(2)) and E(1) sulfate (E(1)-S), respectively. Despite the return of serum DHEA to normal premenopausal values with the present DHEA treatment regimen, the 65% decrease in the androgen pool found in this group of postmenopausal women is in fact corrected by only 24%, thus remaining 41% below the values found in normal premenopausal women. In fact, the changes in serum DHEA observed after percutaneous DHEA administration are a 186% overestimate of the true changes in androgen formation while the overestimate of estrogen production is even much higher. On the other hand, the pharmacokinetics of the steroids are stable over the 12-month period with no significant induction or decrease of activity of the enzymatic systems transforming DHEA predominantly into androgens.     

Territorial aggression and hormones during the non-breeding season in a tropical bird

The hormonal control of territorial aggression in male and female vertebrates outside the breeding season is still unresolved. Most vertebrates have regressed gonads when not breeding and do not secrete high levels of sex steroids. However, recent studies implicate estrogens in the regulation of non-breeding territoriality in some bird species. One possible source of steroids during the non-breeding season could be the adrenal glands that are known to produce sex steroid precursors such as dehydroepiandrosterone (DHEA). We studied tropical, year-round territorial spotted antbirds (Hylophylax n. naevioides) and asked (1). whether both males and females are aggressive in the non-breeding season and (2). whether DHEA is detectable in the plasma at that time. We conducted simulated territorial intrusions (STIs) with live decoys to male and female free-living spotted antbirds in central Panama. Non-breeding males and females displayed robust aggressive responses to STIs, and responded more intensely to decoys of their own sex. In both sexes, plasma DHEA concentrations were detectable and higher than levels of testosterone (T) and 17beta-estradiol (E(2)). In males, plasma DHEA concentrations were positively correlated with STI duration. Next, we conducted STIs in captive non-breeding birds. Captive males and females displayed robust aggressive behavior. Plasma DHEA concentrations were detectable in both sexes, whereas T was non-detectable (E(2) was not measured). Plasma DHEA concentrations of males were positively correlated with aggressive vocalizations and appeared to increase with longer STI durations. We conclude that male and female spotted antbirds can produce DHEA during the non-breeding season and DHEA may serve as a precursor of sex steroids for the regulation of year-round territorial behavior in both sexes.     

Effects of neuroactive steroids on cochlear hair cell death induced by gentamicin

As neuroactive steroids, sex steroid hormones have non-reproductive effects. We previously reported that 17β-estradiol (βE2) had protective effects against gentamicin (GM) ototoxicity in the cochlea. In the present study, we examined whether the protective action of βE2 on GM ototoxicity is mediated by the estrogen receptor (ER) and whether other estrogens (17α-estradiol (αE2), estrone (E1), and estriol (E3)) and other neuroactive steroids, dehydroepiandrosterone (DHEA) and progesterone (P), have similar protective effects. The basal turn of the organ of Corti was dissected from Sprague-Dawley rats and cultured in a medium containing 100 μM GM for 48 h. The effects of βE2 and ICI 182,780, a selective ER antagonist, were examined. In addition, the effects of other estrogens, DHEA and P were tested using this culture system. Loss of outer hair cells induced by GM exposure was compared among groups. βE2 exhibited a protective effect against GM ototoxicity, but its protective effect was antagonized by ICI 182,780. αE2, E1, and E3 also protected hair cells against gentamicin ototoxicity. DHEA showed a protective effect; however, the addition of ICI 182,780 did not affect hair cell loss. P did not have any effect on GM-induced outer hair cell death. The present findings suggest that estrogens and DHEA are protective agents against GM ototoxicity. The results of the ER antagonist study also suggest that the protective action of βE2 is mediated via ER but that of DHEA is not related to its conversion to estrogen and binding to ER. Further studies on neuroactive steroids may lead to new insights regarding cochlear protection.     

Harn-steroidprofile hirsuter frauen

Urine steroid profiles of hirsute womenSteroid profiles of women suffering from idiopathic hirsutism show in more than 50% of the cases a 10–100 fold increase in the excretion of dehydroepiandrosterone (DHEA) compared with normal values.The excretion of DHEA was reduced much more than that of other 17-ketosteroids if the adrenals (NNR) were suppressed by dexamethasone (DXM). Within one week they reached values at the compound noise level of the gas chromatograms. If the ovaries were stimulated with human chorionic gonadotropin during continued suppression of the NNR with DXM no increase of DHEA could be detected.     

Adrenal and gonadal steroids inhibit IL-6 secretion by human marrow cells

Adrenal and gonadal steroids have protective effects on the skeleton that may be conferred partly by their ability to inhibit bone resorptive cytokines such as interleukin 6 (IL-6). We tested the hypothesis that IL-6 secretion by human marrow cells and a line of marrow stromal cells (KM101) is inhibited by dehydroepiandrosterone (DHEA), dihydrotestosterone (DHT) and 17beta-oestradiol (E(2)). We also examined whether the estrogen status of the donor influenced the steroids' effects on IL-6 secretion. Femoral bone marrow was obtained from 19 postmenopausal women undergoing hip arthroplasty, and from seven subjects receiving oestrogen replacement therapy (ERT) at the time of surgery. Low-density mononuclear cells were isolated and cultured in IL-1beta-supplemented media, with or without DHEA, DHT or E(2). DHEA suppressed IL-6 more consistently than DHT or E(2): DHEA significantly suppressed IL-6 in 84% of cultures, DHT suppressed IL-6 in 58%, and E(2)did so in 50%. The magnitude of IL-6 inhibition was also greater for DHEA (group mean, treated/control of 62%) compared to DHT (81%) and E(2)(76%). In cultures from subjects receiving ERT, DHEA and DHT suppressed IL-6 in some, whereas E(2)did not suppress IL-6 secretion. Each steroid also significantly inhibited IL-6 secretion by KM101 cells. In summary, in marrow cultured from postmenopausal women, DHEA suppressed IL-6 secretion more consistently and to a greater degree than did DHT and E(2). Second, the inhibitory effect of E(2)was abrogated in marrow from women receiving ERT.     

Metabolism of dehydroepiandrosterone by rat hippocampal cells in culture: possible role of aromatization and 7-hydroxylation in neuroprotection.

The rate of metabolism of the multifunctional neurosteroid, dehydroepiandrosterone (DHEA), by embryonic rat hippocampal cells maintained in culture was compared to that of 4-androstenedione (AD), the immediate precursor of estrone (E1). The experiments were carried out to assess the relative contribution of DHEA, its 7-hydroxylated metabolites and estrogen on their reported effects on memory and neuroprotection. The 3H-labeled steroids of high specific radioactivity were incubated for 1, 8, 24 and 48 h and the putative metabolites extracted from the culture medium with acetone-ethyl acetate before separation by TLC for radioassay. [3H]DHEA (2.0 ng/5x10(5) cells) yielded primarily the 7alpha- and 7beta-hydroxylated steroids in an almost equal ratio under conditions that resembled those used by others to study the protection of neurons by hippocampal astrocytes against excitatory amino acid-induced toxicity. The rate of conversion of DHEA to AD, and particularly to E1, was much lower. With [3H]AD as substrate, significant aromatization to estrogen occurred only after 24 h when most of [3H]DHEA had already been converted to its 7-hydroxylated products and the hydroxylase and aromatase systems would no longer be competing for the same coenzyme (NADPH). The hippocampal cells were still viable after 48 h of incubation with the steroids and were able to oxidize estradiol (E2) to E1 and reduce E1 to E2 and AD to testosterone (T). It is suggested that 7alpha- and 7beta-OHDHEA, the main metabolites formed in the rat hippocampus, might be responsible for some of the functions previously ascribed to estrogens in the brain and the reasons for this proposal are discussed.     

In vitro and in vivo effects of a cyclooxygenase-2 inhibitor nimesulide analog JCC76 in aromatase inhibitors-insensitive breast cancer cells

Glucuronidation, catalyzed by UDP-glucuronosyltransferases (UGT) and sulfation, catalyzed by sulfotransferases (SULT), are pathways through which sex steroids are metabolized to less active compounds. These enzymes are highly polymorphic and genetic variants frequently result in higher or lower activity. The phenotypic effects of these polymorphisms on circulating sex steroids in premenopausal women have not yet been investigated. One hundred and seventy women aged 40-45 years had a blood sample drawn during the follicular phase of the menstrual cycle for sex steroid measures and to obtain genomic DNA. Urine was collected for 2-hydroxy (OH) estrone (E(1)) and 16α-OH E(1) measures. Generalized linear regression models were used to assess associations between sex steroids and polymorphisms in the UGT1A and UGT2B families, SULT1A1, and SULT1E1. Women with the UGT1A1(TA7/TA7) genotype had 25% lower mean estradiol (E(2)) concentrations compared to the wildtype (TA6/TA6) (p=0.02). Similar associations were observed between SULT1A1(R213/H213) and E(1) (13% lower mean E(1) concentration vs. wildtype; p-value=0.02) and UGT2B4(E458/E458) and dehydroepiandrosterone (DHEA) (20% lower mean DHEA vs. wildtype; p-value=0.03). The SULT1E1(A/C) and the UGT1A1(TA7)-UGT1A3(R11) haplotypes were associated with reduced estrogen concentrations. Further study of UGT and SULT polymorphisms and circulating sex steroid measures in larger populations of premenopausal women is warranted.     

Dehydroepiandrosterone decreases while cortisol increases in vitro growth and viability of Entamoeba histolytica

In vitro exposure of Entamoeba histolytica trophozoites to the sex steroids 17beta-estradiol, progesterone, and dehydrotestosterone had little effect on parasite viability or proliferation. However, treatment with the adrenal steroid dehydroepiandrosterone (DHEA) markedly inhibited parasite proliferation, adherence and motility, and at a certain dose it induced trophozoite lysis. The opposite effect on proliferation was found when the trophozoites were exposed to cortisol. Moreover, DHEA decreased while cortisol increased the parasite's DNA synthesis determined by 3H-thymidine incorporation. Trophozoite lysis by DHEA appeared to be caused by a necrotic rather than an apoptotic process, as observed in propidium iodide and terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling assays. A possible mechanisms of action was derived from experiments demonstrating that the activity of a putative 3-hydroxy-3-methyl glutaryl CoA reductase detected in trophozoite extracts was inhibited in the presence of DHEA. Contrary to its in vitro inhibitory effect, in vivo administration of DHEA to infected hamsters resulted in exacerbation of the amebic liver abscesses. These results demonstrated that androgen steroids act directly upon E. histolytica growth and viability, and may shed new light on some age and gender differences in disease progression, as well as finding application in the drug treatment of human amebiasis.     

Seasonal changes in plasma dehydro-epiandrosterone (DHEA) levels of domestic geese

Changes in plasma DHEA, testosterone (T) and 17-B-oestradiol (E2) levels were examined in domestic geese of both sexes in the fall and winter. The levels of steroid hormones were determined in blood plasma by means of radio-immunoassay (RIA). A so-called second (autumn) cycle was induced in geese via a dark-room preparation and natural keeping conditions. The plasma levels of DHEA showed a minor peak at onset of the autumn breeding and a major one prior to the more intense spring reproduction in both sexes. The seasonal curves of plasma DHEA appeared fairly similar in ganders and layers and without considerable differences between the absolute values. In ganders, plasma DHEA peaks preceded the elevations in T levels in the fall and spring alike. With layers, in turn, the autumn and spring peaks of plasma DHEA appeared after the peaks in E2 levels. With ganders, the concentration of plasma T seemed to predominate between the two androgens throughout the experimental period. With layers, in turn, the concentration of DHEA surpassed the level of plasma E2 at the time of the peak periods and other times during the study, as well. In domestic geese, DHEA is probably involved in the autumn physiological processes and the induction of reproduction during fall and early spring periods, alike.     

Microbial introduction of a 16 alpha-hydroxyl function into the steroid nucleus.

The introduction of a 16 alpha-hydroxyl function into the steroid nucleus was studied in resting cells of Streptomyces roseochromogenes NRRL B-1233. The oxidation product of dehydroepiandrosterone (DHEA) was identified as 16 alpha-hydroxy DHEA by using thin-layer and gas-liquid chromatography. A linear relation between cell concentration and 16 alpha-OH-DHEA formation was observed. 16 alpha-Hydroxylase showed good activity at pH 8.0 for 16 alpha-OH-DHEA formation. The enzyme showed good activity at 3.1 x 10(-4) M DHEA. The oxidation products of pregnenolone, 4-androstene-3,17-dione, estrone, and 5-androstene-3 beta,17 beta-diol as well as of other substrates were identified as the 16 alpha-hydroxy steroid, respectively. The rates of microbial 16 alpha-hydroxylation were as follows: 76.9% for DHEA, 50.4% for pregnenolone, 43.9% for 4-androstene-3,17-dione, 34.3% for estrone, and 19.6% for 5-androstene-3 beta,17 beta-diol. The organism tested catalyzes 16 alpha-hydroxylation of a wide variety of steroids.     

Adrenal glands of mouse and rat do not synthesize androgens.

Human adrenal glands produce considerable amounts of the C-19 steroids dehydroepiandrosterone (DHEA) and androstenedione. To investigate the capability of rodent adrenals to produce these steroids, cell suspensions of mouse and rat adrenal glands were incubated in the absence and presence of adrenocorticotropic hormone (ACTH). Corticosterone levels in the incubation medium increased dramatically in the presence of ACTH, but no significant amounts of 17-hydroxyprogesterone or androstenedione could be detected. This indicates that the adrenals of rat and mouse lack the enzyme 17 alpha-hydroxylase. Absence of plasma cortisol in the presence of high levels of corticosterone confirmed these data. Plasma levels of androstenedione were significantly decreased in castrated male rats as compared to levels observed in intact males, showing the contribution of the testes to the plasma content of androstenedione. Very low levels of androstenedione were observed in female, male and castrated male mice. Plasma concentrations of DHEA were not detectable in intact and castrated male mice and rats. It is concluded that rat and mouse lack the enzyme necessary to synthesize adrenal C-19 steroids and that the adrenals in these animals, therefore, do not contribute to plasma levels of androstenedione and DHEA.     

Multiple steroid metabolic pathways in ZR-75-1 human breast cancer cells

In order to characterize the main enzymatic systems involved in androgen and estrogen formation as well as metabolism in ZR-75-1 human breast cancer cells, incubation of intact cells was performed for 12 or 24 h at 37 degrees C with tritiated estradiol (E2), estrone (E1), androst-5-ene-3 beta, 17 beta-diol (5-ene-diol), dehydroepiandrosterone (DHEA), testosterone (T), androstenedione (4-ene-dione), dihydrotestosterone (DHT) or androsterone (ADT). The extra- and intracellular steroids were extracted, separated into free steroids, sulfates and non-polar derivatives (FAE) and identified by HPLC coupled to a Berthold radioactivity monitor. Following incubation with E2, 5-ene-diol or T, E1, DHEA and 4-ene-dione were the main products, respectively, thus indicating high levels of 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD). When 4-ene-dione was used, on the other hand, a high level of transformation into 5 alpha-androstane-3,17-dione (A-dione), Epi-ADT and ADT was found, thus indicating the presence of high levels of 5 alpha-reductase as well as 3 alpha- and 3 beta-hydroxysteroid dehydrogenase. Moreover, some T was formed, due to oxidation by 17 beta-HSD. No estrogen was detected with the androgen precursors T or 4-ene-dione, thus indicating the absence of significant aromatase activity. Moreover, significant amounts of sulfates and non-polar derivatives were found with all the above-mentioned substrates. The present study shows that ZR-75-1 human breast cancer cells possess most of the enzymatic systems involved in androgen and estrogen formation and metabolism, thus offering an excellent model for studies of the control of sex steroid formation and action in breast cancer tissue.     

Androgen conversion in osteoarthritis and rheumatoid arthritis synoviocytes--androstenedione and testosterone inhibit estrogen formation and favor production of more potent 5alpha-reduced androgens

In synovial cells of patients with osteoarthritis (OA) and rheumatoid arthritis (RA), conversion products of major anti-inflammatory androgens are as yet unknown but may be proinflammatory. Therefore, therapy with androgens in RA could be a problem. This study was carried out in order to compare conversion products of androgens in RA and OA synoviocytes. In 26 OA and 24 RA patients, androgen conversion in synovial cells was investigated using radiolabeled substrates and analysis by thin-layer chromatography and HPLC. Aromatase expression was studied by immunohistochemistry. Dehydroepiandrosterone (DHEA) was converted into androstenediol, androstenedione (ASD), 16alphaOH-DHEA, 7alphaOH-DHEA, testosterone, estrone (E1), estradiol (E2), estriol (E3), and 16alphaOH-testosterone (similar in OA and RA). Surprisingly, levels of E2, E3, and 16alpha-hydroxylated steroids were as high as levels of testosterone. In RA and OA, 5alpha-dihydrotestosterone increased conversion of DHEA into testosterone but not into estrogens. The second androgen, ASD, was converted into 5alpha-dihydro-ASD, testosterone, and negligible amounts of E1, E2, E3, or 16alphaOH-testosterone. 5alpha-dihydro-ASD levels were higher in RA than OA. The third androgen, testosterone, was converted into ASD, 5alpha-dihydro-ASD, 5alpha-dihydrotestosterone, and negligible quantities of E1 and E2. 5alpha-dihydrotestosterone was higher in RA than OA. ASD and testosterone nearly completely blocked aromatization of androgens. In addition, density of aromatase-positive cells and concentration of released E2, E3, and free testosterone from superfused synovial tissue was similar in RA and OA but estrogens were markedly higher than free testosterone. In conclusion, ASD and testosterone might be favorable anti-inflammatory compounds because they decrease aromatization and increase anti-inflammatory 5alpha-reduced androgens. In contrast, DHEA did not block aromatization but yielded high levels of estrogens and proproliferative 16alpha-hydroxylated steroids. Androgens were differentially converted to pro- and anti-inflammatory steroid hormones via diverse pathways.     

Metabolism of dehydroepiandrosterone by rodent brain cell lines: relationship between 7-hydroxylation and aromatization

The rate of aromatization of 4-androstenedione (AD) and 7-hydroxylation of dehydroepiandrosterone (DHEA) by different neuronal cell lines from fetal rat and mouse brain was compared to that of embryonic rat hippocampal cells in primary culture. The (3)H-labeled steroids were incubated with the cells and the metabolites extracted and separated by thin layer chromatography (TLC), as well as analyzed by high-performance liquid chromatography (HPLC) for further identification. All cell types produced estrone (E(1)) and estradiol (E(2)) from [(3)H]AD but the rate of aromatization was lowest with the rat hippocampal cells in primary culture. With [(3)H]DHEA, BHc.2 mouse hippocampal cells and E(t)C.1 neurons behaved like the mixed cells from rat hippocampus, forming 7-hydroxy DHEA as the almost exclusive product. In contrast, mouse brain BV2 microglia were virtually unable to hydroxylate DHEA at C-7 and yielded estrogen and more testosterone (T) than other cell types tested. These experiments highlight the pivotal role of 3beta-hydroxysteroid dehydrogenase/ketoisomerase in the control of AD formation for its subsequent aromatization to estrogen. It raises the possibility that differences in metabolism of DHEA by certain brain cells could account for differences in their immunomodulatory and neuroprotective functions. Some could exert their effects by converting DHEA to its 7-hydroxylated form while others, like BV2 microglia, by converting DHEA primarily to other C-19 steroids and to estrogen by way of AD.     

Basal and HCG-stimulated testosterone production by interstitial cells of the Mongolian gerbil (Meriones unguiculatus)--II. Influence of glucocorticosteroids and steroidal precursors

Compared to testosterone production by Mongolian gerbil interstitial cells in the absence of HCG or precursors, testosterone formation was significantly elevated by the addition of 100 ng pregnenolone, progesterone, 17-hydroxyprogesterone or DHEA. Production increased linearly with the amounts of precursors added (pregnenolone: r = 0.99; progesterone: r = 0.98; 17-OH-progesterone: r = 0.96; DHEA: r = 0.92, N = 40, all P less than 0.001). Approximately 50% of DHEA were converted to testosterone during the 6-hr incubation period. Neither the addition of 100 ng 11-deoxycortisol, 11-deoxycorticosterone, cortisol, corticosterone, cortisone, 18-OH-corticosterone, 21-deoxycortisone or 11-dehydrocorticosterone, nor of 100 ng estradiol had a significant effect on testosterone production by isolated interstitial cells incubated without or with 1 mIU HCG. Testosterone production by isolated interstitial cells was significantly increased within 2 min after the addition of 100 ng DHEA; production then linearly increased with the length of incubation (r = 0.98, N = 40, P less than 0.001). After addition of as little as 2 ng DHEA, testosterone formation was higher than by cells incubated without DHEA. While testosterone production could not be stimulated by the addition of 1-500 microIU HCG during a 30-min incubation period, it was drastically elevated by the addition of 10, 20 or 100 ng DHEA. Steroidal precursor concentrations secreted by the Mongolian gerbil adrenal gland incubated in vitro for 120 min were too low to stimulate testosterone production by interstitial cells. On the other hand, testosterone synthesis could be increased by the addition of 10-100-microliter aliquots of adrenal extracts.     

Analysis of pregnenolone and dehydroepiandrosterone in rodent brain: cholesterol autoxidation is the key

Pregnenolone (PREG) and dehydroepiandrosterone (DHEA), and their respective sulfated forms PREGS and DHEAS, were among the first steroids to be identified in rodent brain. However, unreliable steroid isolation and solvolysis procedures resulted in errors, particularly in the case of brain steroid sulfates analyzed by radioimmunology or GC-MS of liberated free steroids. By using a solid-phase extraction recycling/elution procedure, allowing the strict separation of sulfated, free, and fatty acid esters of PREG and DHEA, PREGS and DHEAS, unlike free PREG, were not detected in rat and mouse brain and plasma. Conversely, considerable amounts of PREG and DHEA were released from unknown precursor(s) present in the lipoidal fraction, distinct from fatty acid ester conjugates. Chromatographic and mass spectrometric studies of the nature of the precursor(s) showed that autoxidation of brain cholesterol (CHOL) was responsible for the release of PREG and DHEA from the lipoidal fraction. When inappropriate protocols were used, CHOL was also the precursor of PREG and DHEA obtained from the fraction assumed to contain sulfated steroids. In contrast, free PREG was definitely confirmed as an endogenous steroid in rat brain. Our study shows that an early removal of CHOL from brain extracts coupled to well-validated extraction and fractionation procedures are prerequisites for reliable measurements of free and conjugated PREG and DHEA by GC-MS or other indirect methods.     

The excretion of leukotriene E4 into urine following inhalation of leukotriene D4 by human individuals

Healthy volunteers underwent bronchial challenge with increasing doses of nebulized leukotriene D4 (0.007 - 200 nmol) at 15 min intervals. Total amounts of 200 nmol (females) and 400 nmol (males) were inhaled, corresponding to approximately 100 nmol and 200 nmol deposited in the lung, respectively. Of the latter amounts 3 +/- 1% (mean +/- S.E.M., n = 5) was found to be excreted as leukotriene E4 into the urine within 12 h. No further excretion after this period was observed. Approximately 50% of the total urinary leukotriene E4 was excreted during the first 2 h. These results suggest that a possible formation of sulfidopeptide leukotrienes in the lung in vivo can be monitored by measuring leukotriene E4 excretion into the urine.     

Esterification of vertebrate-type steroids in the Eastern oyster (Crassostrea virginica)

Characteristics of acyl-coenzyme A (acyl-CoA):steroid acyltransferase from the digestive gland of the oyster Crassostrea virginica were determined by using estradiol (E2) and dehydroepiandrosterone (DHEA) as substrates. The apparent Km and Vmax values for esterification of E2 with the six fatty acid acyl-CoAs tested (C20:4, C18:2, C18:1, C16:1, C18:0, and C16:0) were in the range of 9-17 microM E2 and 35-74 pmol/min/mg protein, respectively. Kinetic parameters for esterification of DHEA (Km: 45-120 microM; Vmax: 30-182 pmol/min/mg protein) showed a lower affinity of the enzyme for this steroid. Formation of endogenous fatty acid esters of steroids by microsomes of digestive gland and gonads incubated in the presence of ATP and CoA was assessed, and at least seven E2 fatty acid esters and five DHEA fatty acid esters were observed. Some peaks eluted at the same retention times as palmitoleoyl-, linoleoyl-, oleoyl/palmitoyl-, and stearoyl-E2; and palmitoleoyl-, oleoyl/palmitoyl-, and stearoyl-DHEA. The same endogenous esters, although in different proportions, were produced by gonadal microsomes. The kinetic parameters for both E2 (Km: 10 microM; Vmax: 38 pmol/min/mg protein) and DHEA (Km: 61 microM; Vmax: 60 pmol/min/mg protein) were similar to those obtained in the digestive gland. Kinetic parameters obtained are similar to those observed in mammals; thus, fatty acid esterification of sex steroids appears to be a well-conserved conjugation pathway during evolution.     

Dehydroepiandrosterone and related steroids inhibit mitochondrial respiration in vitro

1. Dehydroepiandrosterone (DHEA) inhibited mitochondrial respiratory rates in the presence of tricarboxylic acid cycle intermediates with the exception of succinate. 2. Several other steroids tested for inhibitory activity on mitochondrial state 3 rate showed inhibitory potencies ranging from 0-90%. 3. Mitochondria isolated from adrenals, heart, kidneys, brain and brown adipose tissue were also inhibited by DHEA. 4. The aspartate malate shuttle system was inhibited, but the alpha-glycerophosphate shuttle was totally insensitive to DHEA. 5. The high-amplitude swelling of mitochondria in hypotonic media was inhibited by DHEA and a few other steroids.