Free DHEA Research Articles

Immunomodulatory effects of testosterone and letrozole during Plasmodium berghei ANKA infection.

Malaria is one of the leading health problems globally. Plasmodium infection causes pronounced sexual dimorphism, and the lethality and severity are more remarkable in males than in females. To study the role of testosterone in the susceptibility and mortality of males in malaria, it is common to increase its concentration. However, this strategy does not consider the enzyme CYP19A1 aromatase, which can transform it into oestrogens. To avoid the interference of oestrogens, we inhibited in vivo CYP19A1 aromatase with letrozole and increased the testosterone level by exogen administration before infection with Plasmodium berghei ANKA. We measured the impact on free testosterone, 17β-oestradiol and dehydroepiandrosterone levels in plasma; additionally, we evaluated parasitaemia, body temperature, body mass, glucose levels and haemoglobin concentration. Furthermore, we evaluated the effects of testosterone on the immune response; we quantified the CD3+/CD4+, CD3+/CD8+, CD19+, Mac-3+ and NK cells in the spleen and the plasma concentrations of the cytokines IL-2, IL-4, IL-6, IFN-, IL-10, TNF-α and IL-17A. Finally, we quantified the levels of antibodies. We found that mice treated with the combination of letrozole and testosterone and infected with Plasmodium berghei ANKA had increased concentrations of free testosterone and DHEA but decreased levels of 17β-oestradiol. As a result, parasitaemia increased, leading to severe anaemia. Interestingly, testosterone increased temperature and decreased glucose concentration as a possible testosterone-mediated regulatory mechanism. The severity of symptomatology was related to critical immunomodulatory effects generated by free testosterone; it selectively increased CD3+CD8+ T and CD19+ cells but decreased Mac-3+. Remarkably, it reduced IL-17A concentration and increased IL-4 and TNF-α. Finally, it increased IgG1 levels and the IgG1/IgG2a ratio. In conclusion, free testosterone plays an essential role in pathogenesis in male mice by increasing CD8+ and decreasing Mac3+ cells and mainly reducing IL-17A levels, which is critical in the development of anaemia. Our results are important for understanding the mechanisms that regulate the exacerbated inflammatory response in infectious diseases and would be useful for the future development of alternative therapies to reduce the mortality generated by inflammatory processes.

MON-207 Identification of Monogenic Causes of Polycystic Ovary Syndrome by High Throughput Sequencing

BACKGROUND: Polycystic ovary syndrome (PCOS) is a common complex endocrine disorder, whose etiology remains to be elucidated. PCOS has a strong heritable component. Genome-wide association studies have identified several risk loci for PCOS; however, the identified candidate genes could explain less than 5% of PCOS heritability. This missing heritability might be explained by the presence of rare genetic variants with larger biological impact. High throughput sequencing (HTS) could be an interesting tool to identify these rare variants. OBJECTIVE: To identify rare genetic variants through HTS in a selection of patients with PCOS clinical relevant phenotypes or familial PCOS clusters. METHODS: Among a cohort of 130 PCOS patients, we selected 32 women presenting one or more of the following phenotypes: 1. adrenal hyperandrogenism (DHEA and/or DHEAS levels 1.5 higher the upper limit of the normal range); 2. primary amenorrhea; 3. severe insulin resistance (IR) (basal insulin >50 mUi/mL and/or after OGTT >300 mUI/mL); 4. normal androgen levels (total and free testosterone, androstenedione, DHEA and DHEAS in the normal range measured by gold standard methods). This group was studied through targeted gene sequencing panels (Miseq Illumina) including 50 genes involved in ovarian folliculogenesis, steroid hormone synthesis, gonadotropin action and insulin-signaling pathway, and genes suggested by monogenic/dygenic PCOS causes. Additional 2 families, with two PCOS sisters and their unaffected mother, were studied by exome sequencing (Hiseq Illumina). Allelic variants were considered pathogenic/potential pathogenic if they had a MAF<0.01 (Gnom AD and ExAC databases), were classified deleterious/probably deleterious by several prediction sites (i.e. SIFT, PolyPhen 2, Mutation Taster) and affect amino acids that are highly conserved among homologues (GERP scores). RESULTS: Among the 34 PCOS index patients studied, the phenotype was distributed as following: 15 with severe IR, 10 with adrenal hyperandrogenism, 2 normoandrogenic, 3 with primary amenorrhea and 4 familial cases. Twelve variants were identified in 10 patients (29.4%), affecting 6 genes: INSR, LMNA, POMC, GATA4, FSHR, H6PD, DLK1 and SCN1A. All genes were found once, except LMNA that was found in 4 different PCOS patients with severe IR. The variants in INSR, POMC and FSHR genes were found in the severe IR group, and in GATA4 in the adrenal hyperandrogenism group. The DLK1 and SCN1A variants were found in the two families studied through exome. CONCLUSION: High throughput sequencing allowed the identification of monogenic forms of PCOS, especially in patients with severe insulin resistance. The identification of rare genetic variants in PCOS patients might increase our comprehension of the pathogenesis of this disease, with positive clinical implications in patient’s treatment and genetic counseling.

No evidence for the immunocompetence handicap hypothesis in male humans

The observations that testosterone might be immunosuppressive, form the basis for the immunocompetence handicap hypothesis (ICHH). According to ICHH only high-quality individuals can maintain high levels of testosterone and afford the physiological cost of hormone-derived immunosuppression. The animal and human studies that attempted to support the ICHH by precisely defined impairment of immunity associated with high testosterone levels are inconclusive. Furthermore, human studies have used only selected immune functions and varying testosterone fractions. This is the first study examining the relationship between multiple innate and adaptive immunity and serum levels of free testosterone, total testosterone, DHT and DHEA in ninety-seven healthy men. Free testosterone and marginally DHT levels were positively correlated with the strength of the influenza post-vaccination response. Total testosterone and DHEA showed no immunomodulatory properties. Our findings did not support ICHH assumptions about immunosuppressive function of androgens. In the affluent society studied here, men with higher levels of free testosterone could afford to invest more in adaptive immunity. Since the hormone-immune relationship is complex and may depend on multiple factors, including access to food resources, androgens should be treated as immunomodulators rather than implicit immunosuppressants.

The impact of gastric bypass surgery on sex hormones and menstrual cycles in premenopausal women

Obesity has adverse effects on ovulation, menstrual cyclicity and oocyte development leading to clinical symptoms such as infertility and menstrual disorders. The Roux-en-Y gastric bypass (RYGB) leads to weight loss, improved insulin sensitivity and may improve ovarian function. In 31 premenopausal women, 18 eu- and 13 oligo-/amenorrhoic, we followed the changes in follicular phase sex hormones 3, 6 and 12 month after RYGB. The average weight loss during the first postoperative year was 39.6 kg. The insulin sensitivity and serum insulin improved markedly especially within the first three postoperative months. SHBG increased progressively and was doubled after 12 months. In contrast, total and free androgens and DHEA declined about 50% during the first three postoperative months and remained fairly constant hereafter. One year after surgery, 85% (11/13) of the women with oligo-/amenorrhea gained regular menstrual cycles. Our results indicate that some of the endocrine changes related to regulation of ovarian function occur very early after bariatric surgery.

Neurosteroid dehydroepiandrosterone and brain function

For last 30 years it became clear that DHEA and DHEAS are synthesized de novo in brain. Steroids synthesized in brain structures, were received the name "neurosteroids". In the review are submitted data on a biosynthesis and metabolism of DHEA(S) including its metabolism in fatty tissue where it serves as substrate for intracellular formation of its biologically active metabolites--estradiol and testosterone. The role of a sulfatase and sulfotransferase in mutual transformations of DHEA and DHEA-sulfate are analysed. Specific differences in DHEA synthesis in adrenals are surveyed. The adrenals of primates, both human beings and monkeys, produce free DHEA and DHEA-sulphate in large quantity. Their synthesis proceeds on Δ5-pathways: cholesterol-pregnenolone-17-hydroxypregnenolone-DHEA. Adrenals of other animas species, including rats and mice, don't synthesize DHEA. From the authors point of view, process of DHEAS penetration in brain structures include two mechanisms: transformation under steroid sulfatase action DHEAS in DHEA which freely gets through a blood-brain barrier and DHEAS passing through a hypothalamus which isn't protected by a blood-brain barrier. Results of researches on clinical application of DHEA as neurosteroid, with the analysis of its role in a course of Alzheimer's disease, distrurbances of cognitive function and other disorders of a CBS are presented also. The main neurobiological effects of DHEA(S) on brain structures which are studied on various models of animals include: neuroprotection, neurogenesis and neuronal survival, apoptosis, catecholamine synthesis and secretion. Neurosteroids have also antioxidant, anti-inflammatory and anti-glucocorticoid effects.

Effects of DHEA on metabolic and endocrine functions of adipose tissue.

Dehydroepiandrosterone (DHEA) and its sulfate ester, DHEAS, are the major circulating adrenal steroids and serve as substrates for sex hormone biosynthesis. DHEA is effectively taken up by adipose tissue, where the concentrations of free DHEA are four to ten times higher than those found in the circulation. DHEA reduces adipose tissue mass and inhibits the proliferation and differentiation of adipocytes; it may also protect against obesity by lowering the activity of stearoyl-CoA desaturase 1 in fat cells. Recent studies demonstrate that DHEA stimulates triacylglycerol hydrolysis in adipose tissue by increasing the expression and activity of adipose triglyceride lipase and hormone-sensitive lipase, the key enzymes of lipolysis. DHEA has been shown to modulate insulin signaling pathways, enhance glucose uptake in adipocytes, and increase insulin sensitivity in patients with DHEA deficiency or abnormal glucose tolerance. Additionally, by suppressing the activity of 11β-hydroxysteroid dehydrogenase 1 in adipocytes, DHEA may promote intra-adipose inactivation of cortisol to cortisone. Several studies have demonstrated that DHEA may also regulate the expression and secretion of adipokines such as leptin, adiponectin, and resistin. The effects of DHEA on adipokine expression in adipose tissue are depot-specific, with visceral fat being the most responsive. The mechanisms underlying DHEA actions in adipose tissue are still unclear; however, they involve nuclear receptors such as androgen receptor and peroxisome proliferator-activated receptors γ and α. Because clinical trials investigating the effects of DHEA failed to yield consistent results, further studies are needed to clarify the role of DHEA in the regulation of human adipose tissue physiology.

Neoadjuvant androgen pathway suppression prior to prostatectomy.

4520 Background: Optimizing tissue androgen suppression may provide better local and systemic control of prostate cancer (PCa). Standard androgen deprivation therapy (ADT) has limited effect on tissue androgens which remain in a range which supports tumor survival. We determined whether targeting androgen metabolism using CYP17 and 5a-reductase (SRD5A) inhibitors would more effectively suppress tissue androgens and tumor volume. Methods: Open label, multicenter neoadjuvant study in men with localized PCa treated for 3 months prior to prostatectomy with zoladex and 1) avodart 3.5 mg QD; 2) avodart and casodex 50 mg QD; or 3) casodex, avodart and ketoconazole 200 mg TID. Serum and tissue androgens were measured by LC/MS/MS. Data were compared to men treated with standard ADT (LHRH agonist plus Casodex), and untreated prostatectomy tissue. The primary outcome measure was suppression of tissue dihydrotestosterone (DHT). Results: 35 men with intermediate/high risk PCa were enrolled. Tissue DHT was suppressed 30 fold (&gt; 95%) in all groups vs. LHRH agonist/Casodex (0.92 ± 0.20 pg/mg vs. 0.03± 0.03 for all groups combined, p&lt;0.0001). Tissue testosterone was 3-4 fold higher (consistent with SRD5A inhibition) in all treatment groups vs. LHRH agonist/Casodex (0.33 vs. 0.07 pg/mg, p &lt; 0.05). Differences in DHT/T between groups 1 through 3 were not statistically significant. There was no correlation between tissue and serum androgens, or tissue androgen and tumor volume (p&gt; 0.05). In subset analysis, total serum DHEA declined significantly in group 3, with free DHEA unchanged, suggesting differential effect on free and total DHEA. Pathologic complete response (CR) was seen in 2 men, and an additional 8 men had &lt;0.2 cc of tumor, with the largest number of CR or near CR in the cohort treated with ADT, CYP17 and SRD5A inhibitor, 4 of 12 men (33%). Due to small cohort size, differences were not statistically significant. Conclusions: Addition of high dose SRD5A inhibition (with and without CYP17 inhibition) achieves prostate DHT levels 30 fold below standard ADT. In this relatively high risk population, CR or near CR was seen in 10 of 35 men receiving protocol therapy. Further suppressing the androgen receptor signaling axis may provide better local and systemic control of PCa.

Quantitative determination of dehydroepiandrosterone fatty acyl esters in human female adipose tissue and serum using mass spectrometric methods

Dehydroepiandrosterone-fatty acyl esters (DHEA-FAE) are naturally occurring water-insoluble metabolites of DHEA, which are transported in plasma exclusively by lipoproteins. To find out whether DHEA, like estradiol, might be stored in adipose tissue in FAE form, we set up a mass spectrometric method to quantify DHEA-FAE and free DHEA in human adipose tissue and serum. The method consists of chromatographic purification steps and final determination of hydrolyzed DHEA-FAE and free DHEA, which was carried out by gas chromatography–mass spectrometry (GC–MS) or liquid chromatography–tandem mass spectrometry (LC–MS/MS). Our results showed that no detectable amounts of DHEA-FAE could be found in adipose tissue although 32–178 pmol/g of free DHEA were determined by GC–MS and LC–MS/MS. The DHEA-FAE concentrations in serum quantified by GC–MS were 1.4 ± 0.7 pmol/ml in premenopausal women ( n = 7), and 0.9 ± 0.4 pmol/ml in postmenopausal women ( n = 5). Correspondingly, the free DHEA concentrations were 15.2 ± 6.3 pmol/ml and 6.8 ± 3.0 pmol/ml. In addition, the mean proportions of DHEA-FAE of total DHEA (DHEA-FAE + free DHEA) in serum were 8.6% and 11.2% in pre- and postmenopausal women, respectively. Serum DHEA-FAE concentration was below quantification limit for LC–MS/MS (signal-to-noise ratio, S/ N = 10), while free DHEA concentrations varied between 5.8 and 23.2 pmol/ml. In conclusion, the proportion of DHEA-FAE of total DHEA in serum was approximately 9%. However, in contrast to our previous findings for estradiol fatty acid esters in adipose tissue which constituted about 80% of total estradiol (esterified + free), the proportion of DHEA-FAE of total DHEA was below 5%. Four to ten times higher concentrations of free DHEA were quantified in adipose tissue compared to those in serum.

The steroid sulfatase inhibitor COUMATE attenuates rather than enhances access of dehydroepiandrosterone sulfate to the brain in the mouse

Intraperitoneal injection of adult male mice with the neuroactive steroid dehydroepiandrosterone sulfate (DHEAS) at 1 and 40 mg/kg caused dose-dependent increases in the concentration of both this compound and its corresponding free steroid DHEA in brain within 1 h of injection. Pretreatment of these animals for 24 h with the steroid sulfatase inhibitor COUMATE at a dose (10 mg/kg, p.o.) shown previously to cause almost complete inhibition of this enzyme in liver and brain was expected to increase the amount of the DHEAS dose reaching the brain. Surprisingly however, the increases in brain concentrations of DHEAS and DHEA after injection of DHEAS i.p. were attenuated by pretreatment with COUMATE. The results suggest that the arylsulfamate based steroid sulfatase inhibitors such as COUMATE interfere with the influx of the DHEAS anion into the brain.

Multiple primary tumors associated with chromosome 9p deletion

Multiple primary tumors associated with chromosome 9p deletion

Dehydroepiandrosterone and coronary atherosclerosis.

Tissue culture, animal model, and epidemiologic studies suggest that dehydroepiandrosterone (DEHA) may inhibit atherosclerosis through its potent antiproliferative effects. To examine the relation between DHEA and a direct measure of coronary atherosclerosis, plasma DHEA, and DHEA sulfate (DHEAS) levels were determined in 206 middle-aged patients undergoing coronary angiography. Plasma DHEAS levels were lower in subjects with at least one > or = 50% stenosis than in those with no stenosis > 50% (p = 0.05) and was inversely associated with the number of diseased coronary vessels and the extent of coronary atherosclerosis (p = 0.05 and 0.01, respectively). Cardiac allograft vasculopathy is dominated by abnormal cellular proliferation and, therefore, may be uniquely influenced by DHEA. To study this, 61 cardiac allograft recipients with at least one annual follow-up cardiac catheterization were studied. Plasma levels of total and free DHEA were inversely related to the development of accelerated coronary allograft vasculopathy (p = 0.005 and 0.003, respectively). Furthermore, the time to development of accelerated allograft vasculopathy was shorter in subjects with low levels of total and free DHEA (p = 0.062 and 0.046, respectively). These data suggest that low plasma levels of DHEA may facilitate, and high levels may retard, the development of coronary atherosclerosis and coronary allograft vasculopathy.

Urinary excretion of free progestins, androgens, and estradiol after injection of (1–24) ACTH in the Mongolian gerbil

Injection of a “long-acting” synthetic adrenocorticotrophin [(1–24) ACTH, 20 IU/animal] into Mongolian gerbils resulted in a 3.1 fold increase of urinary free testosterone excretion over 2 days. It was accompanied by an elevation of urinary free progesterone (2.1fold), 17-hydroxyprogesterone (2.5fold), DHEA (2.8fold) and androstenedione (3.0fold) excretion. Similarly, administration of human chorionic gonadotrophin (HCG, 100 IU/animal) increased urinary excretion of free testosterone (2.3fold), progesterone (4.1fold), 17-hydroxyprogesterone (2.9fold), DHEA (4.6fold), androstenedione (5.4fold) and of estradiol (2.9fold). Data presented in this work show that the measurement of urinary free steroid excretion represents a reliable index for the secretory activity of the adrenal-gonadal-axis, and that it may in some aspects be more practicable than the measurement of steroid plasma levels, especially in small laboratory animals, enabling us to monitor the excretion of various steroids over longer time periods without stressing the animals by handling/ or blood sampling.

Free and sulfoconjugated dehydroepiandrosterone, cyclic adenosine-3′,5′-monophosphate, and free estriol in maternal and cord blood

When free DHEA, its sulfatide, and sulfate were assayed in maternal plasma as well as in umbilical cord arterial and venous plasma, rather high concentrations were found in either fraction from cord arterial plasma, reflecting the fetal contribution not only of free DHEA and DHEA sulfate, but also of the lipophile steroid sulfatide. Since high DHEA levels were associated with elevated c-AMP concentrations, a certain interrelationship of both parameters is indicated. In the course of delivery, a rapid decrease of free estriol in maternal plasma was observed. Higher concentration of free estriol in umbilical venous plasma pointed at its placental biosynthesis from fetal precursors.

Biosynthesis and metabolism of steroid hormones in human gonads (author's transl)

In two experiments 1-14C-acetate plus 7α-3H-cholesterol (experiment I) or 4–14C-dehydroepiandrosterone (DHEA) plus 7α-3H-DHEA- 35S-sulfate (experiment II) were perfused in vivo through the spermatic artery of patients undergoing orchiectomy. Blood samples from the spermatic vein were analyzed for free and conjugated steroids. In experiment I 14C as well as 3H activities were incorporated into various C21 and C19 steroids indicating a de novo synthesis of steroids from acetate and from cholesterol. In experiment II both free and sulfoconjugated DHEA were metabolized to different C19 or C18 steroids to nearly the same extent (8 or 5%, respectively). The sulfoconjugated steroids in the vein plasma exhibited the same 3H/35S ratio as the injected substrate, thus proving a ‘direct’ metabolism of the sulfate without splitting of the ester bond.

Inhibition of glucose-6-phosphate dehydrogenase by steroids: VI—Effects of new synthetic dehydroepiandroster-one conjugates upon the activity of human placental glucose-6-phosphate dehydrogenase

New synthetic DHEA conjugates were tested for their capacity as inhibitors of purified human placental G-6-PDH. Although most of these derivatives like DHEA alkyl sulfates, alkyl sulfonates and alkyl phosphates were found to be more effective than free DHEA, their inhibitory activity did not equal that of DHEA sulfatide. The relationship between the structure of such conjugates and their biological activity is discussed.

Metabolism of free and sulfoconjugated DHEA in brain tissue in vivo and in vitro

Two rhesus monkey brains were perfused in vivo either with 14C-DHEA (dehydroepiandrosterone) and 3H-DHEA sulfate or with 3H-DHEA. Plasma from the jugular vein and the brain were analyzed for free and conjugated metabolites. Many more free than sulfoconjugated steroids were withdrawn from the blood. These were not stored to a large extent. Up to 16% of DHEA were metabolized to ring D hydroxylated Δ 5-compounds. When fresh human fetal brain was incubated with 3H-DHEA, 5.9% of the free steroids were sulfurylated. 30% were converted mainly to ring D hydroxylated Δ 5-metabolites.

ÜBER LIPOPHILE STEROID-KONJUGATE

ABSTRACT The half-life of 7α-3H-dehydroepiandrosterone (DHEA), sodium 7α-3H-DHEA sulfate, and biosynthetic 7α-3H-DHEA sulfatide has been determined in guinea-pigs. Following the isolation of the individual compounds from plasma at various time intervals the half-life was found to be 2:30 and 3:00 minutes for DHEA sulfate, 7:15 and 8:15 minutes for free DHEA and 23 and 28 minutes for DHEA sulfatide. This data seems to support the hypothesis that steroid sulfatides may represent a labile transport form of steroid sulfates.

ON LIPOPHILE STEROID CONJUGATES

ABSTRACT Following the intravenous injection of biosynthetic 7α-3H-DHEA sulphatide, 3.69 % of administered radioactivity could be isolated from the plasma of male guinea pigs in the form of free DHEA, DHEA sulphatide, DHEA sulphate or DHEA glucuronoside. In contrast to these findings the recovery of labelled DHEA after administration of equivalent doses of 7α-3H-DHEA sulphate or free 4-14C-DHEA was approximately 0.58 % or 0.38 % respectively. The additional analysis of liver, kidney, spleen, adrenals, gonads, and some muscular tissue led to a total recovery of approximately 20 % after the administration of 7α-3H-DHEA sulphatide, of 1 % after injection of 7α-3H-DHEA sulphate, and 10 % after free 4-14CDHEA. The specific concentration of labelled DHEA (dpm/g tissue) was found to be exceptionally high in the sulphoconjugated fractions from adrenal tissue. The results seem to support the hypothesis, that biosynthetic DHEA sulphatide may serve as a labile, lipophile depot or transport form for the physiologically active DHEA sulphate.