The Role of 11-Oxygenated Androgens and Endocrine Disruptors in Androgen Excess Disorders in Women.

Polycystic ovary syndrome (PCOS) and idiopathic hirsutism (IH) are androgen excess disorders requiring the determination of classic androgen levels for diagnosis. 11-oxygenated androgens have high androgenic potential, yet their clinical value in those disorders is not clear. Additionally, the role of endocrine disruptors (EDs), particularly in IH, remains understudied. We analyzed 25 steroids and 18 EDs in plasma samples from women with IH, PCOS, and controls using LC-MS/MS. Cytokine levels and metabolic parameters were assessed. Comparisons included non-obese women with PCOS (n = 10), women with IH (n = 12) and controls (n = 20), and non-obese versus obese women with PCOS (n = 9). Higher levels of 11-oxygenated androgens were observed in women with PCOS compared to those with IH, but not controls. Conversely, 11-oxygenated androgen levels were lower in women with IH compared to controls. Cytokine levels did not differ between women with IH and controls. Bisphenol A (BPA) levels were higher in obese women with PCOS compared to non-obese women with PCOS. Bisphenol S occurrence was higher in women with PCOS (90%) compared to controls (65%) and IH (50%). Significant correlations were found between androgens (11-ketotestosterone, androstenedione, testosterone) and insulin and HOMA-IR, as well as between immunomodulatory 7-oxygenated metabolites of DHEA and nine interleukins. Our data confirms that PCOS is a multiendocrine gland disorder. Higher BPA levels in obese women might exacerbate metabolic abnormalities. IH was not confirmed as an inflammatory state, and no differences in BPA levels suggest BPA does not play a role in IH pathogenesis.

Progesterone: A Steroid with Wide Range of Effects in Physiology as Well as Human Medicine.

Progesterone is a steroid hormone traditionally linked with female fertility and pregnancy. In current reproductive medicine, progesterone and its analogues play crucial roles. While the discovery of its effects has a long history, over recent decades, various novel actions of this interesting steroid have been documented, of which its neuro- and immunoprotective activities are the most widely discussed. Discoveries of the novel biological activities of progesterone have also driven research and development in the field of progesterone analogues used in human medicine. Progestogen treatment has traditionally and predominately been used in maintaining pregnancy, the prevention of preterm labor, various gynecological pathologies, and in lowering the negative effects of menopause. However, there are also various other medical fields where progesterone and its analogues could find application in the future. The aim of this work is to show the mechanisms of action of progesterone and its metabolites, the physiological and pharmacological actions of progesterone and its synthetic analogues in human medicine, as well as the impacts of its production and use on the environment.

COVID-19, Vaccination, and Female Fertility in the Czech Republic.

The fast-track process to approve vaccines against COVID-19 has raised questions about their safety, especially in relation to fertility. Over the last 2 years, studies have appeared monitoring female fertility, especially from assisted reproduction centers or in animal experiments. However, studies monitoring healthy populations are still limited. The aim of our study was to monitor the relevant parameters of female fertility (sex and other steroids, LH, FSH, SHBG, Antimüllerian hormone and antral follicle count) before and then 2-4 months after the third dose of vaccination against COVID-19 in a group of 25 healthy fertile woman. In addition, anti-SARS-CoV-2 and anti-SARS-CoV-2S antibodies were determined. We did not observe significant changes in the measured parameters before and after the third dose of vaccination. By comparing levels of the analytes with antibodies indicating a prior COVID-19 infection, we found that women who had experienced the disease had statistically lower levels of estrone, estradiol, SHBG and 5α-dihydroprogesterone, and conversely, higher levels of androgen active dehydroepiandrosterone and dihydrotestosterone. Our results confirm that vaccination does not affect female fertility, and that what fertile women should be worried about is not vaccination, but rather COVID-19 infection itself.

Exposure to bisphenols and parabens during pregnancy and relations to steroid changes.

BACKGROUND: The harmful effects of endocrine disrupting compounds (EDCs) on human health are generally well-known, and exposure during fetal development may have lasting effects. Fetal exposure to bisphenol A (BPA) has been recently relatively well-studied; however, less is known about alternatives such as bisphenol S (BPS), bisphenol F (BPF) and bisphenol AF (BPAF), which have started to appear in consumer products. Parabens are another widespread group of EDCs, with confirmed transplacental passage. The usage of many cosmetic, pharmaceutical and consumer products during the pregnancy that may contain parabens and bisphenols has led to the need for investigation. OBJECTIVES: To shed more light into the transplacental transport of BPA, its alternatives, and parabens, and to study their relation to fetal steroidogenesis. METHODS: BPA, BPS, BPF, BPAF, methylparaben, ethylparaben, propylparaben, butylparaben, benzylparaben and 15 steroids including estrogens, corticoids, androgens and immunomodulatory ones were determined in 27 maternal (37th week of pregnancy) and cord plasma samples using liquid chromatography - tandem mass spectrometry methods. RESULTS: In cord blood, significantly higher BPA levels (p=0.0455) were observed compared to maternal plasma. The results from multiple regression models showed that in cord blood, methylparaben (ß=-0.027, p=0.027), propylparaben (ß=-0.025, p=0.03) and the sum of all measured parabens (ß=-0.037, p=0.015) were inversely associated with testosterone levels. CONCLUSION: To the best of our knowledge, this is the first study reporting the simultaneous detection of BPA, alternative bisphenols, parabens and steroids in maternal and cord plasma. Our study confirmed the transplacental transport of BPA, with likely accumulation in the fetal compartment. The negative association of cord blood parabens and testosterone levels points to possible risks with respect to importance of testosterone for prenatal male development.

A Comparison of Salivary Steroid Levels during Diagnostic Tests for Adrenal Insufficiency.

Numerous diagnostic tests are used to evaluate the hypothalamic-pituitary-adrenal axis (HPA axis). The gold standard is still considered the insulin tolerance test (ITT), but this test has many limitations. Current guidelines therefore recommend the Synacthen test first when an HPA axis insufficiency is suspected. However, the dose of Synacthen that is diagnostically most accurate and sensitive is still a matter of debate. We investigated 15 healthy men with mean/median age 27.4/26 (SD±4.8) years, and mean/median BMI (body mass index) 25.38/24.82 (SD±3.2) kg/m2. All subjects underwent 4 dynamic tests of the HPA axis, specifically 1 µg, 10 µg, and 250 µg Synacthen (ACTH) tests and an ITT. Salivary cortisol, cortisone, pregnenolone, and DHEA (dehydroepiandrosterone) were analysed using liquid chromatography-tandem mass spectrometry. During the ITT maximum salivary cortisol levels over 12.5 nmol/l were found at 60 minutes. Maximum cortisol levels in all of the Synacthen tests were higher than this; however, demonstrating that sufficient stimulation of the adrenal glands was achieved. Cortisone reacted similarly as cortisol, i.e. we did not find any change in the ratio of cortisol to cortisone. Pregnenolone and DHEA were higher during the ITT, and their peaks preceded the cortisol peak. There was no increase of pregnenolone or DHEA in any of the Synacthen tests. We demonstrate that the 10 µg Synacthen dose is sufficient stimulus for testing the HPA axis and is also a safe and cost-effective alternative. This dose also largely eliminates both false negative and false positive results.

Changes in the Concentrations of Corticoid Metabolites--The Effect of Stress, Diet and Analytical Method.

A precisely measured corticoid level is important for decision-making in daily clinical practice. These levels can be influenced in the pre-analytical phase, when the effect of stress, timing, and diet can be important. The aim of this study was to elucidate optimal conditions for blood sampling as well as the choice of analytical methods, which they will be used in measuring of corticoids. By studying ten women, we focused on the influences of the stress of cannulation and a large lunchtime meal on cortisol, cortisone, aldosterone and corticosterone levels. We further compared results of cortisol measurements from RIA and LC-MS/MS. Stress from cannulation caused increase of cortisol, cortisone and corticosterone already, when the cannula was being inserted. This indicates that this increase is stimulated by fear of the blood withdrawing rather, than just by the needle insertion itself. The effect of stress on corticosterone disappeared after an hour, while effect on other corticoids was still apparent. Concerning the lunchtime meal, we found an increase in all measured corticoids between 11 and 12 o'clock. After the food, there were marked decreases in cortisone and aldosterone, while declining levels of cortisol and corticosterone had rather plateaus. We compared cortisol in 90 plasma samples measured by a commercial RIA kit and the LC-MS/MS method. Results from both methods showed a strong correlation (r=0.85). When measuring corticoid metabolites, the chosen analytical method, eliminating stress factors, and precisely timed blood sampling considering the daily rhythm and food intake are critical.

Selected pro- and anti-inflammatory cytokines in cerebrospinal fluid in normal pressure hydrocephalus.

OBJECTIVES: Normal pressure hydrocephalus (NPH) is a treatable neurological syndrome developing in the elderly. It is characterized by balance impairment, urinary incontinence and dementia development caused by disorders in the cerebrospinal fluid (CSF) circulation. The diagnosis can be easily mistaken for other neurodegenerative diseases, which are often accompanied by inflammation and the production of cytokines. The aim of our study was to determine and compare selected CSF and plasma cytokines with respect to their informative value for laboratory diagnostics of NPH. METHODS: The levels of IL-1ß, IL-4, IL-6, IL-10, IL-17A, IL-17F, IL-21, IL-22, IL-23, IL-25, IL-31, IL-33, INF-γ, sCD40L and TNF-α were measured in the CSF and plasma in age-matched subjects with NPH (n=20) and controls (n=20) by multiplex assay. RESULTS: CSF IL-1ß, IL-6 and IL-10 were significantly increased on the 1st day of lumbar drainage in NPH (p<0.01). No significant changes were observed in the plasma. The CSF cytokines were one to three orders of magnitude higher compared to the plasma. CONCLUSION: CSF can better show the neurodegenerative changes in the brain. The cytokines IL-1ß, IL-6 and IL-10 may be helpful in NPH diagnostics.

Reduced urine pregnenolone concentration after clinical response in patients with depression: An open-label short-term prospective study.

BACKGROUND: Identifying biological alterations in patients with depression, particularly those that differ between responders and non-responders, is of interest to clinical practice. Biomarker candidates involve neuroactive steroids, including pregnenolone (PREG) and allopregnanolone (ALLO). However, alterations in PREG and ALLO associated with treatment response are understudied. This study's main aim was to evaluate the effects of antidepressant treatment, clinical response, and treatment duration on PREG and ALLO in depression. MATERIALS AND METHODS: In a 4-week, open-label trial, participants were allocated randomly to the venlafaxine (n = 27) or mirtazapine (n = 30) group. Urine concentrations of PREG and ALLO were assessed through gas chromatography-mass spectrometry. Participants collected night urine between 10:30 p.m. and 8:00 a.m. Two primary outcomes were analyzed. Firstly, the effect of treatment (mirtazapine or venlafaxine), clinical response (operationalized through the Hamilton Depression Rating Scale), and time (baseline compared to 28 days) on the urine concentrations of PREG or ALLO in depression. Finally, the effect of clinical response and time on the urine concentration of PREG or ALLO, independently of the antidepressant given (mirtazapine or venlafaxine). Linear mixed models were carried out. RESULTS: There was no significant difference in PREG and ALLO concentrations between baseline and 28 days in responders and non-responders when investigating the venlafaxine or the mirtazapine group. However, we found a significant reduction of urine PREG concentration after 28 days of treatment in responders who received either venlafaxine or mirtazapine (estimate = -0.56; p = 0.016; 95CI [-1.003; -0.115]; Cohen's d = -0.61). CONCLUSIONS: Our main results indicate that responders in depression show reduced urinary PREG concentrations after 4-weeks of therapy, independently of the antidepressant used. More studies are needed to confirm these findings.

An LC-MS/MS method for the simultaneous quantification of 32 steroids in human plasma.

A development of robust and rapid method with simple sample preparation for the analysis of steroids of C18-, C19-, C21- families is of interest of many research groups. Here we present a novel LC-MS/MS method for the simultaneous quantification of 32 steroid hormones in human plasma. Twenty-two of them were analyzed directly without the need for derivatization, while ten were derivatized with 2-fluoro-1-methylpyridinium p-toluenesulfonate. The steroids were separated on a C18 column with a gradient elution consisting of methanol and water with the addition of 0.1% formic acid. The mass spectrometer was operated in positive ESI mode. Validation demonstrated that the method was applicable for the quantitative analysis of two C18- steroids (estrone, estradiol), nineteen C19- steroids (testosterone, epitestosterone, dihydrotestosterone, 11-ketodihydrotestosterone, 11ß-hydroxyandrostenedione, 11ß-hydroxytestosterone, 11-ketotestosterone, dehydroepiandrosterone, 7α-hydroxydehydroepiandrosterone, 7ß-hydroxydehydroepiandrosterone, 7-ketodehydroepiandrosterone, androsterone, epiandrosterone, androstenedione, androstenediol, 5α-androstane-3α,17ß-diol, 5α-androstane-3ß,17ß-diol, 5ß-androstane-3α,17ß-diol, 5ß-androstane-3ß,17ß-diol), and eleven C21- steroids (cortisol, 21-deoxycortisol, 11-deoxycortisol, cortisone, corticosterone, 11-deoxycorticosterone, pregnenolone, 17-hydroxypregnenolone, progesterone, 17-hydroxyprogesterone, 5α-dihydroprogesterone). The lower limits of quantification are appropriate for analyses in both physiological and various pathophysiological conditions. The accuracy, intra- and inter-day precision values as well as stability tests were in accordance with FDA Guidelines. The method will be a useful tool in investigating the mechanisms of steroid-related diseases and will serve as a steppingstone for the development of other methods for steroid analyses in various biological matrices such as prostate tissue, cerebrospinal fluid, urine, seminal fluid, and saliva.

Steroid Sulfation in Neurodegenerative Diseases.

Steroid sulfation and desulfation participates in the regulation of steroid bioactivity, metabolism and transport. The authors focused on sulfation and desulfation balance in three neurodegenerative diseases: Alzheimer´s disease (AD), Parkinson´s disease (PD), and multiple sclerosis (MS). Circulating steroid conjugates dominate their unconjugated counterparts, but unconjugated steroids outweigh their conjugated counterparts in the brain. Apart from the neurosteroid synthesis in the central nervous system (CNS), most brain steroids cross the blood-brain barrier (BBB) from the periphery and then may be further metabolized. Therefore, steroid levels in the periphery partly reflect the situation in the brain. The CNS steroids subsequently influence the neuronal excitability and have neuroprotective, neuroexcitatory, antidepressant and memory enhancing effects. They also exert anti-inflammatory and immunoprotective actions. Like the unconjugated steroids, the sulfated ones modulate various ligand-gated ion channels. Conjugation by sulfotransferases increases steroid water solubility and facilitates steroid transport. Steroid sulfates, having greater half-lives than their unconjugated counterparts, also serve as a steroid stock pool. Sulfotransferases are ubiquitous enzymes providing massive steroid sulfation in adrenal zona reticularis and zona fasciculata.. Steroid sulfatase hydrolyzing the steroid conjugates is exceedingly expressed in placenta but is ubiquitous in low amounts including brain capillaries of BBB which can rapidly hydrolyze the steroid sulfates coming across the BBB from the periphery. Lower dehydroepiandrosterone sulfate (DHEAS) plasma levels and reduced sulfotransferase activity are considered as risk factors in AD patients. The shifted balance towards unconjugated steroids can participate in the pathophysiology of PD and anti-inflammatory effects of DHEAS may counteract the MS.

Occurrence and reproductive roles of hormones in seminal plasma.

Only 2-5% of seminal fluid is composed of spermatozoa, while the rest is seminal plasma. The seminal plasma is a rich cocktail of organic and inorganic compounds including hormones, serving as a source of nutrients for sperm development and maturation, protecting them from infection and enabling them to overcome the immunological and chemical environment of the female reproductive tract. In this review, a survey of the hormones found in human seminal plasma, with particular emphasis on reproductive hormones is provided. Their participation in fertilization is discussed including their indispensable role in ovum fertilization. The origin of individual hormones found in seminal plasma is discussed, along with differences in the concentrations in seminal plasma and blood plasma. A part of review is devoted to methods of measurement, emphasising particular instances in which they differ from measurement in blood plasma. These methods include separation techniques, overcoming the matrix effect and current ways for end-point measurement, focusing on so called hyphenated techniques as a combination of chromatographic separation and mass spectrometry. Finally, the informative value of their determination as markers of male fertility disorders (impaired spermatogenesis, abnormal sperm parameters, varicocele) is discussed, along with instances where measuring their levels in seminal plasma is preferable to measurement of levels in blood plasma.

Les spermatozoïdes ne représentent que 2 à 25% du liquide séminal, le reste étant constitué par le plasma séminal. Le plasma séminal est un cocktail de composés organiques et non organiques comprenant des hormones qui font office de source de substances nutritives pour le développement et la maturation des spermatozoïdes, qui les protègent de l'infection et leur permettent de surmonter l'environnement immunologique et chimique de l'appareil reproducteur féminin. La présente revue propose une vue d'ensemble des hormones retrouvées dans le plasma séminal de l'homme, l'accent étant particulièrement mis sur les hormones reproductives. La participation de ces dernières au processus de fécondation est discutée, y compris leur rôle indispensable dans la fécondation de l'ovocyte. L'origine de chacune des hormones retrouvées dans le plasma séminal est décrite, ainsi que les différences de leurs concentrations dans le plasma séminal et dans le plasma sanguin. Une partie de cette revue est dévolue aux méthodes de mesure, en soulignant des exemples particuliers où elles diffèrent des mesures dans le plasma sanguin. Ces méthodes comprennent les techniques de séparation, qui surmontent les effets matriciels et les procédures actuelles de critère de mesure, en se concentrant sur les techniques dites de couplage comme la combinaison de la séparation chromatographique et de la spectrométrie de masse. Enfin, la valeur informative de la détermination de ces hormones en tant que marqueurs des anomalies de la fertilité masculine (spermatogenèse altérée, paramètres spermatiques anormaux, varicocèle) est discutée, ainsi que les situations où la mesure de leurs taux dans le plasma séminal est préférable à celle du plasma sanguin.

Determination of selected bisphenols, parabens and estrogens in human plasma using LC-MS/MS.

In this study, a novel liquid chromatography - tandem mass spectrometry method for the simultaneous determination of bisphenols (BPA, BPS, BPF, BPAF), parabens (methyl-, ethyl-, propyl-, butyl-, benzyl-paraben) and estrogens (estrone, estradiol, estriol) in human plasma is presented. Since all analytes possess the phenolic group, dansyl chloride derivatization was applied in order to gain high sensitivity. The method was validated according to FDA guidelines, and all validation requirements were satisfactory. The lower limits of quantifications were 41.6, 54.9, 43.5 and 150.8pg/mL for BPA, BPS, BPF and BPAF; 172, 149, 171, 134 and 202pg/mL for methyl-, ethyl-, propyl-, butyl- and benzyl-paraben; 10.5, 6.7 and 9.4pg/mL for estrone, estradiol and estriol, respectively. This is the first method allowing the determination of plasma bisphenols, parabens and estrogens in one run, and also the first determination of BPF levels in human plasma. The method was used to examine the plasma levels of healthy normospermic men, where three times higher plasma levels of BPF than BPA were found.

Synthesis of 3α-deuterated 7α-hydroxy-DHEA and 7-oxo-DHEA and application in LC-MS/MS plasma analysis.

7-Oxygenated metabolites of dehydroepiandrosterone (DHEA) are known for their neuroprotective and immunomodulatory properties. These neuroactive steroids are currently predominately analysed by mass spectrometry, for which the use of internal deuterated standards is necessary. The aim of this study was to synthesize the deuterated derivatives of 7α-hydroxy-DHEA and 7-oxo-DHEA and test them in liquid chromatography-tandem mass spectrometry (LC-MS/MS) in order to enhance the performance characteristics of this method. Here we report the synthesis of 3α deuterium-labelled 7α-hydroxy-DHEA and 7-oxo-DHEA. Deuterium was introduced into the 3α position by reduction of the corresponding 3-ketone with a protected 17-carbonyl group using NaBD4. Our new procedure allows the easier synthesis of deuterated steroid labelled compounds. The use of these deuterated steroids enabled us to improve the human plasma LC-MS/MS analysis of 7α-hydroxy-DHEA and 7-oxo-DHEA in terms of sensitivity, precision and recovery.

Endocrine disruptors and other inhibitors of 11ß-hydroxysteroid dehydrogenase 1 and 2: Tissue-specific consequences of enzyme inhibition.

Numerous chemicals in the environment have the ability to interact with the endocrine system. These compounds are called endocrine disruptors (EDs). Exposure to EDs represents one of the hypotheses for decreasing fertility, the increased risk of numerous cancers and obesity, metabolic syndrome and type 2 diabetes. There are various mechanisms of ED action, one of which is their interference in the action of 11ß-hydroxysteroid dehydrogenase (11ßHSD) that maintains a balance between active and inactive glucocorticoids on the intracellular level. This enzyme has two isoforms and is expressed in various tissues. Inhibition of 11ßHSD in various tissues can have different consequences. In the case of EDs, the results of exposure are mainly adverse; on the other hand pharmaceutically developed inhibitors of 11ßHSD type 1 are evaluated as an option for treating metabolic syndrome, as well as related diseases and depressive disorders. This review focuses on the effects of 11ßHSD inhibitors in the testis, colon, adipose tissue, kidney, brain and placenta.

Associations of bisphenol A and polychlorinated biphenyls with spermatogenesis and steroidogenesis in two biological fluids from men attending an infertility clinic.

BACKGROUND: In the testis, steroid hormones play an important role in spermatogenesis, the production of semen, and the maintenance of secondary sex characteristics and libido. They may also play a role as a target for substances called endocrine disruptors (EDs). As yet, however, no complex study has been conducted evaluating the relationships between EDs and the steroid spectrum in the plasma and seminal plasma. OBJECTIVES: To shed more light into mechanisms of EDs and the effects of bisphenol A (BPA) and polychlorinated biphenyls (PCBs) on human spermatogenesis and steroidogenesis. METHODS: We determined BPA and 11 steroids in the plasma and seminal plasma of 191 men with different degrees of fertility, using a newly developed liquid-chromatography mass spectrometry method. Concurrently, plasma levels of 6 congeners of PCBs, gonadotropins, selenium, zinc and homocysteine were measured. Partial correlations adjusted for age, BMI and abstinence time were performed to evaluate relationships between these analytes. RESULTS: Seminal BPA, but not plasma BPA, was negatively associated with sperm concentration (r=-0.198; p=0.009), sperm count (r=-0.178; p=0.018) and morphology (r=-0.160; p=0.044). Divergent and sometimes opposing associations of steroids and BPA were found in both body fluids. The sum of PCB congeners was negatively associated with testosterone, free testosterone, the free androgen index and dihydrotestosterone in plasma. CONCLUSION: BPA may negatively contribute to the final state of sperm quality. Moreover, our data indicate that BPA influences human gonadal and adrenal steroidogenesis at various steps. Environmental levels of PCBs negatively correlated with androgen levels, but surprisingly without negative effects on sperm quality.

Steroid hormones in prediction of normal pressure hydrocephalus.

Normal pressure hydrocephalus (NPH) is a treatable neurological disorder affecting elderly people with the prevalence increasing with age. NPH is caused by abnormal cerebrospinal fluid (CSF) reabsorption and manifested as a balance impairment, urinary incontinence and dementia development. These symptoms are potentially reversible if recognized early. Diagnosis of NPH is difficult and can be easily mistaken for other neurodegenerative disorders, which makes NPH one of the major misdiagnosed diseases worldwide. The aim of the study was to find out the appropriate combination of indicators, based on CSF steroids, which would contribute to a clearer NPH diagnosis. The levels of CSF cortisol, cortisone, dehydroepiandrosterone (DHEA), 7α-OH-DHEA, 7ß-OH-DHEA, 7-oxo-DHEA, 16α-OH-DHEA and aldosterone (all LC-MS/MS) were determined in our patients (n=30; NPH, 65-80 years) and controls (n=10; 65-80 years). The model of orthogonal projections to latent structures (OPLS) was constructed to predict NPH. Cortisone, 7α-OH-DHEA, 7ß-OH-DHEA, 7-oxo-DHEA, aldosterone, 7α-OH-DHEA /DHEA, 7-oxo-DHEA/7α-OH-DHEA, 7ß-OH-DHEA/7-oxo-DHEA and 16α-OH-DHEA/DHEA in the CSF were identified as the key predictors and the model discriminated patients from controls with 100% sensitivity and 100% specificity. The suggested model would contribute to early and accurate NPH diagnosis, enabling promptly treatment of the disease.

Determination of seven selected neuro- and immunomodulatory steroids in human cerebrospinal fluid and plasma using LC-MS/MS.

Dehydroepiandrosterone (DHEA) and its 7-oxo- and 7-hydroxy-metabolites occurring in the brain are considered neurosteroids. Metabolism of the latter is catalysed by 11ß-hydroxysteroid dehydrogenase (11ß-HSD) which also interconverts cortisol and cortisone. The concurrent metabolic reaction to DHEA 7-hydroxylation is the formation of 16α-hydroxy-DHEA. The LC-MS/MS method using triple stage quadrupole-mass spectrometer was developed for simultaneous quantification of free DHEA, 7α-hydroxy-DHEA, 7ß-hydroxy-DHEA, 7-oxo-DHEA, 16α-hydroxy-DHEA, cortisol and cortisone in human plasma and cerebrospinal fluid (CSF). The method employs 500 µL of human plasma and 3000 µL of CSF extracted with diethyl ether and derivatized with 2-hydrazinopyridine. It has been validated in terms of sensitivity, precision and recovery. In plasma, the following values were obtained: limit of detection: 2-50p g/mL; limit of quantification: 5-140 pg/mL; within-day precision 0.58-14.58%; between-day precision: 1.24-13.89% and recovery: 85-113.2%). For CSF, the values of limit of detection: 2-28 pg/mL; limit of quantification: 6-94 pg/mL; within-day precision; 0.63-5.48%; between-day precision: 0.88-14.59% and recovery: 85.1-109.4% were acquired. Medians and concentration ranges of detected steroids in plasma and CSF are given in subjects with excluded normal pressure hydrocephalus (n=37; 65-80 years). The method enables simultaneous quantification of steroids important for the estimation of 11ß-HSD activity in human plasma and CSF. It will be helpful in better understanding various degenerative diseases development and progression.

Development and validation of LC-MS/MS method for quantification of bisphenol A and estrogens in human plasma and seminal fluid.

Bisphenol A (BPA) is a widely known endocrine disruptor with estrogenic, antiestrogenic or antiandrogenic properties. BPA could interfere with estrogen metabolism as well with receptor-mediated estrogen actions. Both environmental BPA and estrogens may be traced in body fluids, of which, besides the blood plasma, the seminal fluid is of particular interest regarding their possible interactions in the testis. The method for simultaneously determining BPA and estrogens is then needed, taking into account that their concentrations in these body fluid may differ. Here the method was developed and validated for measurements of BPA, estrone (E1), estradiol (E2) and estriol (E3) in blood plasma and seminal plasma using liquid chromatography-tandem mass spectrometry. Due to the phenolic moiety of all compounds, dansyl chloride derivatization could be used. The analytical criteria of the method with respect to expected concentration of the analytes were satisfactory. The lower limits of quantifications (LLOQ) amounted to 43.5, 4.0, 12.7, 6.7 pg/mL for plasma BPA, E1, E2 and E3, and 28.9, 4.9, 4.5, 3.4 pg/mL for seminal BPA, E1, E2 and E3, respectively. The concentrations of individual steroids differed between body fluids. To the best of our knowledge, this is the first method that enabled the measurement of estrogens and BPA together in one run. The concentrations of E1, E2 and for the first time also of E3 in seminal plasma in normospermic men are reported.