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Estrogen receptor α (ERα) signaling leads to cellular responses in several tissues and in addition to nuclear ERα-mediated effects, membrane ERα (mERα) signaling may be of importance. To elucidate the significance, in vivo, of mERα signaling in multiple estrogen-responsive tissues, we have used female mice lacking the ability to localize ERα to the membrane due to a point mutation in the palmitoylation site (C451A), so called Nuclear-Only-ER (NOER) mice. Interestingly, the role of mERα signaling for the estrogen response was highly tissue-dependent, with trabecular bone in the axial skeleton being strongly dependent (>80% reduction in estrogen response in NOER mice), cortical and trabecular bone in long bones, as well as uterus and thymus being partly dependent (40-70% reduction in estrogen response in NOER mice) and effects on liver weight and total body fat mass being essentially independent of mERα (

ABSTRACTUlva lactuca, collected on the west coast of Sweden at the end of May, was able to utilize the HCO3- pool of seawater only through extracellular dehydration via carbonic anhydrase, followed by uptake of the CO2 formed. A decrease in the CO2 supply via this mechanism resulted in the gradual development of an additional method of HCO3- utilization, namely a direct uptake of HCO3-. Photosynthesis could then be supported by either a ‘HCO3- dehydration mechanism’ or a ‘HCO3- uptake mechanism’. Through selective inhibition of either of these mechanisms, the physiological properties of the other could be assessed. These properties suggest that the HCO3- uptake mechanism of U. lactuca is important under conditions when low concentrations of inorganic C, high pH and high external O2 concentrations would limit photosynthesis supported by the HCO3- dehydration mechanism. Such conditions may occur during intense irradiation of the alga in rockpools or in shallow bays with low rates of water exchange. The results are discussed in relation to a possible coupling between mechanisms for inorganic C acquisition and cell structure (or even morphology) of green macroalgae. They also illustrate some necessary precautions when using Michaelis–Menten kinetics for estimations of Vmax and K1/2 values. [ABSTRACT FROM AUTHOR]

The exchange of CO[SUB2], H[SUP+] and O[SUB2] between seawater and the intertidal brown macroalga Ascophyllum nodosum (L.) Le Jolis were measured in a flowthrough system. While the algae were kept in darkness, seawater with artificially increased alkalinity and pH at 9.85, was alternated with 'normal' seawater at pH at 8.0. A proton buffering system, with capacity to release and reabsorb about 20 μmol protons per gram alga (fresh weight) was revealed. As the algae were returned to the 'normal' seawater, the kinetics of proton reabsorbtion indicated that a proton uptake was gradually induced. This proton uptake, which was not connected to ion exchange in the cell wall, reached its maximum after 1-2h. If subjected to high alkalinity seawater in the light, A. nodosum for a certain period of time was capable of carrying out O[SUB2] evolution in excess of the import of inorganic carbon. This 'photosynthetic buffering capacity' amounted to about 17 μmol O[SUB2] per gram alga. Besides depending on a buffer of photoreducible substances, this 'photosynthetic buffering capacity' appeared to be functionally connected with the proton buffer. The time course for the discharge of the 'photosynthetic buffer system' and for the reabsorbtion of protons into the proton buffer (about 6 h for 90% of the capacity at a temperature at 6 °C) suggests that the 'photosynthetic buffer system' has a functional importance in the adaptation of A. nodosum to intertidal regions. The function of the buffer system is discussed in relation to the carssulacean acid metabolism (CAM)-like characteristics recently shown for the intertidal brown algal family Fucaceae. [ABSTRACT FROM AUTHOR]

Among the brown algae, species of the Fucaceae (Pelvetia, Fucus and Ascophyllum) were found to have a 'photosynthetic buffering' system, allowing the algae to carry out oxygen production without a concomitant uptake of inorganic carbon. This system was not found in other brown algae examined (e.g. Halidrys, Laminaria and Desmarestia) nor in 16 examined species of red and green algae. Pelvetia, Fucus and Ascophyllum belong to the littoral algae which are periodically emersed. In the Fucaceae, the meristodermal cells were found to have a special organization of organelles. Towards the outher cell wall there was a prominent layer of mitochondria while the chloroplasts were concentrated towards the inner and side walls. Between the mitochondria and the chloroplasts there was a large number of physodes. This arrangement of organelles was not found in the other brown algae examined nor in red or green algae. The significance of this organization of the mitochondria is discussed in connection with the function of the 'photosynthetic buffering' system. [ABSTRACT FROM AUTHOR]

Context: Neurodevelopmental impairments are common among survivors of extremely preterm birth, particularly in males. Hyperactivation of the hypothalamic-pituitary-gonadal (HPG) axis has been suggested as an underlying cause, but this has been poorly investigated., Objective: Establish levels and temporal changes in circulating androgens in extremely preterm infant males., Methods: Observational cohort study analyzing cord blood serum (n = 25) and postnatal plasma (n = 13) collected from day 0 until week 11 from infant males born at 22.8-27.9 weeks gestational age. Testosterone and dihydrotestosterone (DHT) were determined using gas chromatography mass spectrometry, sex hormone-binding globulin (SHBG) with an enzyme-linked immunosorbent assay, and follicle-stimulating hormone (FSH) and luteinizing hormone (LH) with the Luminex xMAP multiplex assay., Results: Testosterone and DHT levels were higher on day 0 (median 4.27 and 0.30 ng/mL) than in cord blood (0.15 and 0.01 ng/mL) ( P < .001 for both). Levels of the hormones then declined rapidly until day 5 (median 0.16 and 0.12 ng/mL), then remained relatively constant throughout the study period. Median levels of testosterone and DHT across the whole study period were approximately 6-fold higher than reported in utero levels. FSH and LH showed similar postnatal patterns as the androgens. SHBG steadily increased over time, and, as a result, the fraction of bioavailable testosterone declined with infant postnatal age., Conclusion: The HPG axis is activated immediately after birth in extremely preterm infant males, resulting in an androgen pulse occurring several months earlier than during a normal pregnancy. The long-term implications of high androgen exposure during a sensitive neurodevelopmental period warrant further studies., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Endocrine Society.)

Infectious, inflammatory and autoimmune conditions present differently in males and females. SARS-CoV-2 infection in naive males is associated with increased risk of death, whereas females are at increased risk of long COVID 1 , similar to observations in other infections 2 . Females respond more strongly to vaccines, and adverse reactions are more frequent 3 , like most autoimmune diseases 4 . Immunological sex differences stem from genetic, hormonal and behavioural factors 5 but their relative importance is only partially understood 6-8 . In individuals assigned female sex at birth and undergoing gender-affirming testosterone therapy (trans men), hormone concentrations change markedly but the immunological consequences are poorly understood. Here we performed longitudinal systems-level analyses in 23 trans men and found that testosterone modulates a cross-regulated axis between type-I interferon and tumour necrosis factor. This is mediated by functional attenuation of type-I interferon responses in both plasmacytoid dendritic cells and monocytes. Conversely, testosterone potentiates monocyte responses leading to increased tumour necrosis factor, interleukin-6 and interleukin-15 production and downstream activation of nuclear factor kappa B-regulated genes and potentiation of interferon-γ responses, primarily in natural killer cells. These findings in trans men are corroborated by sex-divergent responses in public datasets and illustrate the dynamic regulation of human immunity by sex hormones, with implications for the health of individuals undergoing hormone therapy and our understanding of sex-divergent immune responses in cisgender individuals., (© 2024. The Author(s).)

Objective: Sex steroids exert important biological functions within the CNS, but the underlying mechanisms are poorly understood. The contribution of circulating sex steroids to the levels in CNS tissue and cerebrospinal fluid (CSF) has been sparsely investigated in human and with inconclusive results. This could partly be due to lack of sensitive validated assays. To address this, we validated a gas chromatography-tandem mass spectrometry (GC-MS/MS) assay for quantification of sex steroid hormones/precursors in CSF., Methods: GC-MS/MS quantification of dihydrotestosterone (DHT, CSF lower limit of quantification, 1.5 pg/mL), testosterone (4.9), estrone (E1, 0.88), estradiol (E2, 0.25), dehydroepiandrosterone (DHEA, 38.4), androstenedione (4D, 22.3), and progesterone (P, 4.2) in CSF, and corresponding serum samples from 47 men., Results: Analyses of CSF revealed that DHEA was the major sex steroid (73.5 ± 31.7 pg/mL) followed by 4D (61.4 ± 29.6 pg/mL) and testosterone (49.5 ± 18.9 pg/mL). The CSF levels of DHT, E2, and E1 were substantially lower, and P was in general not detectable in CSF. For all sex steroids except E2, strong associations between corresponding CSF and serum levels were observed. We propose that testosteronein CSF is derived from circulating testosterone, DHT in CSF is from local conversion from testosterone, while E2 in CSF is from local conversion from 4D in CNS., Conclusions: We describe the first thoroughly validated highly sensitive mass spectrometric assay for a broad sex steroid hormone panel suitable for human CSF. This assay constitutes a new tool for investigation of the role of sex steroid hormones in the human CNS., Significance Statement: In this study, a fully validated highly sensitive mass spectrometric assay for sex steroids was applied to human CSF. The results were used to describe the relative contribution of peripheral circulating sex steroids together with locally transformation of sex steroids to the levels in CSF. The results are of importance to understand the biological processes of the human brain.

Context: Epidemiological and preclinical data support cardiovascular, mainly protective, effects of sex steroids in men, but the mechanisms underlying the cardiovascular actions of sex steroids are poorly understood. Vascular calcification parallels the development of atherosclerosis, but is increasingly recognized as a diversified, highly regulated process, which itself may have pathophysiological importance for clinical cardiovascular events., Objective: To investigate the association between serum sex steroids and coronary artery calcification (CAC) in elderly men., Methods: We used gas chromatography tandem mass spectrometry to analyze a comprehensive sex steroid profile, including levels of dehydroepiandrosterone (DHEA), androstenedione, estrone, testosterone, estradiol, and dihydrotestosterone, in men from the population-based AGES-Reykjavik study (n = 1287, mean 76 years). Further, sex hormone-binding globulin (SHBG) was assayed and bioavailable hormone levels calculated. CAC score was determined by computed tomography. The main outcome measures were cross-sectional associations between dehydroepiandrosterone, androstenedione, estrone, testosterone, dihydrotestosterone, and estradiol and quintiles of CAC., Results: Serum levels of DHEA, androstenedione, testosterone, dihydrotestosterone, and bioavailable testosterone showed significant inverse associations with CAC, while estrone, estradiol, bioavailable estradiol, and SHBG did not. DHEA, testosterone, and bioavailable testosterone remained associated with CAC after adjustment for traditional cardiovascular risk factors. In addition, our results support partially independent associations between adrenal-derived DHEA and testes-derived testosterone and CAC., Conclusion: Serum levels of DHEA and testosterone are inversely associated with CAC in elderly men, partially independently from each other. These results raise the question whether androgens from both the adrenals and the testes may contribute to male cardiovascular health., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Endocrine Society.)

Objectives: In laboratory medicine, external quality assessment (EQA) schemes have become versatile tools for detecting analytical flaws. However, EQA schemes are lacking for pediatric sex steroid levels. We aimed to investigate the suitability of different estradiol and testosterone immunoassays in a pediatric setting in comparison with clinical liquid chromatography-tandem mass spectrometry (LC-MS/MS) assays., Methods: The study was conducted by staff and the advisory group on endocrinology at Equalis, the Swedish provider of EQA schemes for laboratory medicine. The test material consisted of five pooled serum samples from children who were either prepubertal or in puberty. Clinical laboratories enrolled in Equalis EQA schemes for estradiol and testosterone were invited to participate, as were clinical laboratories using LC-MS/MS-assays. Samples were analyzed by either routine immunoassays (n=18) or in-house LC-MS/MS assays (n=3)., Results: For estradiol, LC-MS/MS assays showed a high degree of conformity with interlaboratory coefficients of variation (CV) below 24.2 %. Reported levels were between 4.9 ± 1.2 and 33.9 ± 1.6 pmol/L (group mean ± standard deviation). The direct immunoassays had lower precision; their CVs were up to 81.4 %. Reported concentrations were between 25.3 ± 18.1 and 45.7 ± 19.4 pmol/L, an overestimation compared to LC-MS/MS. Testosterone LC-MS/MS also showed a high degree of conformity, CVs were below 13.4 %, and reported concentrations were from 0.06 ± 0.00 to 1.00 ± 0.11 nmol/L. The direct immunoassays had a larger discrepancy between results; CVs were up to 95.8 %. Concentrations were between 0.12 ± 0.11 and 0.85 ± 0.23 nmol/L., Conclusions: For the safe diagnosis and determination of sex steroids in children, analysis with mass spectrometry-based methods is recommended., (© 2023 the author(s), published by De Gruyter, Berlin/Boston.)

Progesterone serum levels have been identified as a potential predictor for treatment effect in men with advanced prostate cancer, which is an androgen-driven disease. Although progesterone is the most abundant sex steroid in orchiectomized (ORX) male mice, the origins of progesterone in males are unclear. To determine the origins of progesterone and androgens, we first determined the effect of ORX, adrenalectomy (ADX), or both (ORX + ADX) on progesterone levels in multiple male mouse tissues. As expected, intratissue androgen levels were mainly testicular derived. Interestingly, progesterone levels remained high after ORX and ORX + ADX with the highest levels in white adipose tissue and in the gastrointestinal tract. High progesterone levels were observed in mouse chow and exceptionally high progesterone levels were observed in food items such as dairy, eggs, and beef, all derived from female animals of reproductive age. To determine if orally ingested progesterone contributes to tissue levels of progesterone in males, we treated ORX + ADX and sham mice with isotope-labeled progesterone or vehicle by oral gavage. We observed a significant uptake of labeled progesterone in white adipose tissue and prostate, suggesting that dietary progesterone may contribute to tissue levels of progesterone. In conclusion, although adrenal-derived progesterone contributes to intratissue progesterone levels in males, nonadrenal progesterone sources also contribute. We propose that dietary progesterone is absorbed and contributes to intratissue progesterone levels in male mice. We speculate that food with high progesterone content could be a significant source of progesterone in males, possibly with consequences for men undergoing androgen deprivation therapy for prostate cancer., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Endocrine Society.)

Background: Diagnosing endogenous hypercortisolism remains a challenge, partly due to a lack of biochemical tests with good diagnostic accuracy., Objectives: To evaluate the diagnostic value of salivary cortisol and cortisone in patients with suspected hypercortisolism., Methods: Retrospective study including 155 patients with adrenal incidentaloma, and 54 patients with suspected Cushing´s syndrome (CS). Salivary samples were collected at home, at 11 p.m., and at 8 a.m. following an over-night dexamethasone suppression test (DST). Salivary cortisol and cortisone were measured with liquid chromatography-tandem mass spectrometry., Results: Ten of 155 patients with adrenal incidentaloma were considered to have autonomous cortisol secretion (ACS). Using previously established cut-offs, all patients with ACS had elevated plasma-cortisol (>50 nmol/L) following DST, 9/10 had elevated late-night salivary cortisone (>15 nmol/L) whereas only 4/10 had elevated late-night salivary cortisol (LNSC; >3 nmol/L) compared to 35%, 9% and 8%, respectively, of the 145 patients with non-functioning adrenal incidentaloma. Six (60%) patents with ACS had elevated salivary cortisol and cortisone at 8 a.m. following DST compared to 9% and 8%, respectively, of patients with non-functioning adrenal incidentaloma. One of 6 patients with overt CS had a normal LNSC and one had normal late-night salivary cortisone, while all had increased salivary cortisol and cortisone following DST., Conclusion: LNSC is not sufficiently sensitive or specific to be used for screening patients with suspected hypercortisolism. Instead, late-night salivary cortisone seems to be a promising alternative in patients with adrenal incidentaloma and salivary cortisone at 8 a.m. following DST in patients with suspected CS. Larger studies are needed to confirm these findings., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Berndt, Dahlqvist, de Verdier, Ryberg and Ragnarsson.)

Objective: To determine if plasma transfusions with male donor plasma to very preterm infants affect circulatory levels of sex steroids., Design and Patients: Retrospective multicentre cohort study in 19 infants born at gestational age <29 weeks requiring plasma transfusion during their first week of life., Setting: Three neonatal intensive care units in Sweden., Main Outcome Measures: Concentrations of sex steroids and sex hormone-binding globulin (SHBG) in donor plasma and infant plasma measured before and after a plasma transfusion and at 6, 12, 24 and 72 hours., Results: The concentrations of progesterone, dehydroepiandrosterone and androstenedione were significantly lower in donor plasma than in infant plasma before the transfusion (median (Q1-Q3) 37.0 (37.0-37.0), 1918 (1325-2408) and 424 (303-534) vs 901 (599-1774), 4119 (2801-14 645) and 842 (443-1684) pg/mL), while oestrone and oestradiol were higher in donor plasma (17.4 (10.4-20.1) and 16.0 (11.7-17.2) vs 3.1 (1.1-10.2) and 0.25 (0.25-0.25) pg/mL). Median testosterone and dihydrotestosterone (DHT) levels were 116-fold and 21-fold higher in donor plasma than pre-transfusion levels in female infants, whereas the corresponding difference was not present in male infants. Plasma sex steroid levels were unchanged after completed transfusion compared with pre-transfusion levels, irrespective of the gender of the receiving infant. The SHBG concentration was significantly higher in donor than in recipient plasma (22.8 (17.1-33.5) vs 10.2 (9.1-12.3) nmol/L) before transfusion but did not change in the infants after the transfusion., Conclusions: A single transfusion of adult male plasma to preterm infants had no impact on circulating sex steroid levels., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY. Published by BMJ.)

Dehydroepiandrosterone (DHEA), an adrenal androgen precursor, can be metabolized in target tissues into active sex steroids. It has been proposed that DHEA supplementation might result in restoration of physiological local sex steroid levels, but knowledge on the effect of DHEA treatment on local sex steroid levels in multiple tissues is lacking. To determine the effects of DHEA on tissue-specific levels of sex steroids, we treated orchiectomized (ORX) male mice with DHEA for 3 weeks and compared them with vehicle-treated ORX mice and gonadal intact mice. Intra-tissue levels of sex steroids were analyzed in reproductive organs (seminal vesicles, prostate, m. levator ani), major body compartments (white adipose tissue, skeletal muscle, and brain), adrenals, liver, and serum using a sensitive and validated gas chromatography-mass spectrometry method. DHEA treatment restored levels of both testosterone (T) and dihydrotestosterone (DHT) to approximately physiological levels in male reproductive organs. In contrast, this treatment did not increase DHT levels in skeletal muscle or brain. In the liver, DHEA treatment substantially increased levels of T (at least 4-fold) and DHT (+536%, P < 0.01) compared with vehicle-treated ORX mice. In conclusion, we provide a comprehensive map of the effect of DHEA treatment on intra-tissue sex steroid levels in ORX mice with a restoration of physiological levels of androgens in male reproductive organs while DHT levels were not restored in the skeletal muscle or brain. This, and the unexpected supraphysiological androgen levels in the liver, may be a cause for concern considering the uncontrolled use of DHEA., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Endocrine Society.)

The primary aim of the study was to identify inflammatory markers relevant for osteoarthritis (OA)-related systemic (plasma) and local (synovial fluid, SF) inflammation. From this, we looked for inflammatory markers that coincided with the increased amount of O -linked Tn antigen (GalNAcα1-Ser/Thr) glycan on SF lubricin. Inflammatory markers in plasma and SF in OA patients and controls were measured using a 44-multiplex immunoassay. We found consistently 29 markers detected in both plasma and SF. The difference in their concentration and the low correlation when comparing SF and plasma suggests an independent inflammatory environment in the two biofluids. Only plasma MCP-4 and TARC increased in our patient cohort compared to control plasma. To address the second task, we concluded that plasma markers were irrelevant for a direct connection with SF glycosylation. Hence, we correlated the SF-inflammatory marker concentrations with the level of altered glycosylation of SF-lubricin. We found that the level of SF-IL-8 and SF-MIP-1α and SF-VEGFA in OA patients displayed a positive correlation with the altered lubricin glycosylation. Furthermore, when exposing fibroblast-like synoviocytes from both controls and OA patients to glycovariants of recombinant lubricin, the secretion of IL-8 and MIP-1α and VEGFA were elevated using lubricin with Tn antigens, while lubricin with sialylated and nonsialylated T antigens had less or no measurable effect. These data suggest that truncated glycans of lubricin, as found in OA, promote synovial proinflammatory cytokine production and exacerbate local synovial inflammation., Competing Interests: GJ, RK, and TS authored patents related to rhPRG4. GJ and TS hold equity in Lubris BioPharma LLC. TS was also paid consultancy fees by Lubris BioPharma. NK and CJ authored a patent involving the use of lubricin for diagnostics, and NK and CJ hold equity in Lynxon AB. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Huang, Thomsson, Jin, Ryberg, Das, Struglics, Rolfson, Björkman, Eisler, Schmidt, Jay, Krawetz and Karlsson.)

Context: Male sex is a major risk factor for abdominal aortic aneurysms (AAA) but few studies have addressed associations between sex hormone levels and AAA., Objective: We aimed to describe the associations between serum sex steroids and early, screening-detected AAA in men., Methods: We validated a high-sensitivity liquid chromatography-tandem mass spectrometry assay for comprehensive serum sex hormone profiling. This assay was then employed in a case-control study including 147 men with AAA (infrarenal aorta ≥ 30 mm) and 251 AAA-free controls recruited at the general population-based ultrasound screening for AAA in 65-year-old Swedish men., Outcomes Included: associations between dehydroepiandrosterone, progesterone, 17α-hydroxyprogesterone, androstenedione, estrone, testosterone, dihydrotestosterone, and estradiol and AAA presence., Results: Dehydroepiandrosterone, progesterone, 17α-hydroxyprogesterone, testosterone, and estradiol, but not the other hormones, were lower in men with AAA. In models with adjustments for known AAA risk factors and comorbidity, only progesterone (odds ratio per SD decrease 1.62 [95% CI, 1.18-2.22]) and estradiol (1.40 [95% CI, 1.04-1.87]) remained inversely associated with the presence of AAA. Progesterone and estradiol contributed with independent additive information for prediction of AAA presence; compared with men with high (above median) levels, men with low (below median) levels of both hormones had a 4-fold increased odds ratio for AAA (4.06 [95% CI, 2.25-7.31])., Conclusion: Measured by a high-performance sex steroid assay, progesterone and estradiol are inversely associated with AAA in men, independent of known risk factors. Future studies should explore whether progesterone and estradiol, which are important reproductive hormones in women, are protective in human AAA., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Endocrine Society.)

A comprehensive atlas of sex steroid distribution in multiple tissues is currently lacking, and how circulating and tissue sex steroid levels correlate remains unknown. Here, we adapted and validated a gas chromatography tandem mass spectrometry method for simultaneous measurement of testosterone (T), dihydrotestosterone (DHT), androstenedione, progesterone (Prog), estradiol, and estrone in mouse tissues. We then mapped the sex steroid pattern in 10 different endocrine, reproductive, and major body compartment tissues and serum of gonadal intact and orchiectomized (ORX) male mice. In gonadal intact males, high levels of DHT were observed in reproductive tissues, but also in white adipose tissue (WAT). A major part of the total body reservoir of androgens (T and DHT) and Prog was found in WAT. Serum levels of androgens and Prog were strongly correlated with corresponding levels in the brain while only modestly correlated with corresponding levels in WAT. After orchiectomy, the levels of the active androgens T and DHT decreased markedly while Prog levels in male reproductive tissues increased slightly. In ORX mice, Prog was by far the most abundant sex steroid, and, again, WAT constituted the major reservoir of Prog in the body. In conclusion, we present a comprehensive atlas of tissue and serum concentrations of sex hormones in male mice, revealing novel insights in sex steroid distribution. Brain sex steroid levels are well reflected by serum levels and WAT constitutes a large reservoir of sex steroids in male mice. In addition, Prog is the most abundant sex hormone in ORX mice., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Endocrine Society.)

Androgens exert important effects both in androgen-responsive tissues and in the intestinal tract. To determine the impact of the gut microbiota (GM) on intestinal androgen metabolism, we measured unconjugated (free) and glucuronidated androgen levels in intestinal contents from the small intestine, with a low bacterial density, and from cecum and colon, with a high bacterial density. Using a specific, sensitive gas chromatography-tandem mass spectrometry method, we detected high levels of glucuronidated testosterone (T) and dihydrotestosterone (DHT) in small intestinal content of mice of both sexes, whereas in the distal intestine we observed remarkably high levels of free DHT, exceeding serum levels by >20-fold. Similarly, in young adult men high levels of unconjugated DHT, >70-fold higher than in serum, were detected in feces. In contrast to mice with a normal GM composition, germ-free mice had high levels of glucuronidated T and DHT, but very low free DHT levels, in the distal intestine. These findings demonstrate that the GM is involved in intestinal metabolism and deglucuronidation of DHT and T, resulting in extremely high free levels of the most potent androgen, DHT, in the colonic content of young and healthy mice and men.

Intratumoral androgen biosynthesis is one of the mechanisms involved in the progression of prostate cancer, and an important target for novel prostate cancer therapies. Using gas chromatography-tandem mass spectrometry and genome-wide RNA sequencing, we have analyzed androgen concentrations and androgen-regulated gene expression in cancerous and morphologically benign prostate tissue specimens and serum samples obtained from 48 primary prostate cancer patients. Intratumoral dihydrotestosterone (DHT) concentrations were significantly higher in the cancerous tissues compared to benign prostate ( P  < 0.001). The tissue/serum ratios of androgens were highly variable between the patients, indicating individual patterns of androgen metabolism and/or uptake of androgens within the prostate tissue. An unsupervised hierarchical clustering analysis of intratissue androgen concentrations indicated that transmembrane protease, serine 2/ETS-related gene (TMPRSS2-ERG)-positive patients have different androgen profiles compared to TMPRSS2-ERG-negative patients. TMPRSS2-ERG gene fusion status was also associated with an enhanced androgen-regulated gene expression, along with altered intratumoral androgen metabolism, demonstrated by reduced testosterone concentrations and increased DHT/testosterone ratios in TMPRSS2-ERG-positive tumors. TMPRSS2-ERG-positive and -negative prostate cancer specimens have distinct intratumoral androgen profiles, possibly due to activation of testosterone-independent DHT biosynthesis via the alternative pathway in TMPRSS2-ERG-positive tumors. Thus, patients with TMPRSS2-ERG-positive prostate cancer may benefit from novel inhibitors targeting the alternative DHT biosynthesis., (© 2018 Society for Endocrinology.)

The development of castration-resistant prostate cancer (CRPC) is associated with the activation of intratumoral androgen biosynthesis and an increase in androgen receptor (AR) expression. We recently demonstrated that, similarly to the clinical CRPC, orthotopically grown castration-resistant VCaP (CR-VCaP) xenografts express high levels of AR and retain intratumoral androgen concentrations similar to tumors grown in intact mice. Herein, we show that antiandrogen treatment (enzalutamide or ARN-509) significantly reduced (10-fold, P < 0.01) intratumoral testosterone and dihydrotestosterone concentrations in the CR-VCaP tumors, indicating that the reduction in intratumoral androgens is a novel mechanism by which antiandrogens mediate their effects in CRPC. Antiandrogen treatment also altered the expression of multiple enzymes potentially involved in steroid metabolism. Identical to clinical CRPC, the expression levels of the full-length AR (twofold, P < 0.05) and the AR splice variants 1 (threefold, P < 0.05) and 7 (threefold, P < 0.01) were further increased in the antiandrogen-treated tumors. Nonsignificant effects were observed in the expression of certain classic androgen-regulated genes, such as TMPRSS2 and KLK3, despite the low levels of testosterone and dihydrotestosterone. However, other genes recently identified to be highly sensitive to androgen-regulated AR action, such as NOV and ST6GalNAc1, were markedly altered, which indicated reduced androgen action. Taken together, the data indicate that, besides blocking AR, antiandrogens modify androgen signaling in CR-VCaP xenografts at multiple levels., (Copyright © 2018 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Females are, in general, more insulin sensitive than males. To investigate whether this is a direct effect of sex-steroids (SS) in white adipose tissue (WAT), we developed a male mouse model overexpressing the aromatase enzyme, converting testosterone (T) to estradiol (E 2 ), specifically in WAT (Ap2-arom mice). Adipose tissue E 2 levels were increased while circulating SS levels were unaffected in male Ap2-arom mice. Importantly, male Ap2-arom mice were more insulin sensitive compared with WT mice and exhibited increased serum adiponectin levels and upregulated expression of Glut4 and Irs1 in WAT. The expression of markers of macrophages and immune cell infiltration was markedly decreased in WAT of male Ap2-arom mice. The adipogenesis was enhanced in male Ap2-arom mice, supported by elevated Pparg expression in WAT and enhanced differentiation of preadipocyte into mature adipocytes. In summary, increased adipose tissue aromatase activity reduces adipose tissue inflammation and improves insulin sensitivity in male mice. We propose that estrogen increases insulin sensitivity via a local effect in WAT on adiponectin expression, adipose tissue inflammation, and adipogenesis., (Copyright © 2017 the American Physiological Society.)

Androgen exposure of the fetus during gestation plays an important role in human physiology and pathophysiology, but assessment of androgens, in particular dihydrotestosterone (DHT), in human umbilical cord blood is technically challenging. The aim of this study was to assess umbilical cord androgen levels, including DHT, at birth by a highly sensitive assay, and study their association with sex of the infant, sex-hormone-binding globulin (SHBG) levels, and gestational age at delivery. Swedish infants (27 girls, 26 boys) were recruited at maternity care clinics in Southern Sweden. Umbilical cord blood levels of dehydroepiandrosterone (DHEA), androstenedione, testosterone and DHT at delivery were assessed by a gas chromatography-tandem mass spectrometry assay. Cord blood levels of DHT were 2.4-fold higher in boys (median 27.8pg/mL) than in girls (11.5pg/mL), while the sex difference was less pronounced for testosterone (1.3-fold higher in boys) and non-significant for DHEA and androstenedione. Gestational age at delivery associated inversely with DHT levels in boys and with DHEA levels in girls. There was a strong inverse correlation between SHBG and DHEA in both sexes, while there were no associations between SHBG and testosterone or DHT levels. In conclusion, using state of the art technology, we report that there is a pronounced sexual dimorphism in human umbilical cord blood DHT levels. The possibility to assess a complete androgen profile in human cord blood opens up for future increased understanding of the biological impact of the fetal androgen milieu., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

2-Methoxyestradiol (2ME2), a metabolite of 17β-estradiol (E2), exerts bone sparing effects in animal models. We hypothesized that the underlying mechanism is back conversion of 2ME2 to E2, which subsequently acts via estrogen receptor (ER)α. We measured serum E2 levels in orchidectomized wild-type (WT) mice treated with 2ME2 66.6 μg/d or placebo. In placebo-treated animals, E2 was below the detection limit. In 2ME2-treated mice, the serum E2 level was 4.97 ± 0.68 pg/mL. This corresponds to the level found in diesterus in cycling female mice. Next, we investigated bone parameters in orchidectomized WT and ERα knockout mice treated with 2ME2 or placebo for 35 days. 2ME2 (6.66 μg/d) preserved trabecular and cortical bone in WT mice. Trabecular volumetric-bone mineral density was 64 ± 20%, and trabecular bone volume/total volume was 60 ± 20% higher in the metaphyseal region of the femur in the 2ME2 group, compared with placebo (P < .01). Both trabecular number and trabecular thickness were increased (P < .01). Cortical bone mineral content in the diaphyseal region of the femur was 31 ± 3% higher in the 2ME2 group, compared with placebo (P < .001). This was due to larger cortical area (P < .001). Three-point bending showed an increased bone strength in WT 2ME2-treated animals compared with placebo (maximum load [Fmax] +19±5% in the 2ME2 group, P < .05). Importantly, no bone parameter was affected by 2ME2 treatment in ERα knockout mice. In conclusion, 2ME2 treatment of orchidectomized mice results in increased serum E2. ERα mediates the bone sparing effects of 2ME2. The likely mediator of this effect is E2 resulting from back conversion of 2ME2.

The hydroxysteroid (17beta) dehydrogenase (HSD17B)12 gene belongs to the hydroxysteroid (17β) dehydrogenase superfamily, and it has been implicated in the conversion of estrone to estradiol as well as in the synthesis of arachidonic acid (AA). AA is a precursor of prostaglandins, which are involved in the regulation of female reproduction, prompting us to study the role of HSD17B12 enzyme in the ovarian function. We found a broad expression of HSD17B12 enzyme in both human and mouse ovaries. The enzyme was localized in the theca interna, corpus luteum, granulosa cells, oocytes, and surface epithelium. Interestingly, haploinsufficiency of the HSD17B12 gene in female mice resulted in subfertility, indicating an important role for HSD17B12 enzyme in the ovarian function. In line with significantly increased length of the diestrous phase, the HSD17B +/- females gave birth less frequently than wild-type females, and the litter size of HSD17B12 +/- females was significantly reduced. Interestingly, we observed meiotic spindle formation in immature follicles, suggesting defective meiotic arrest in HSD17B12 +/- ovaries. The finding was further supported by transcriptome analysis showing differential expression of several genes related to the meiosis. In addition, polyovular follicles and oocytes trapped inside the corpus luteum were observed, indicating a failure in the oogenesis and ovulation, respectively. Intraovarian concentrations of steroid hormones were normal in HSD17B12 +/- females, whereas the levels of AA and its metabolites (6-keto prostaglandin F1alpha, prostaglandin D 2 , prostaglandin E 2 , prostaglandin F 2α , and thromboxane B 2 ) were decreased. In conclusion, our study demonstrates that HSD17B12 enzyme plays an important role in female fertility through its role in AA metabolism.

Purpose: Amoxicillin is commonly used in oral surgery for antimicrobial prophylaxis against surgical-site infection and bacteremia because of its effect on oral streptococci. The aim of this study was to determine whether amoxicillin reaches the break-point concentrations in saliva and has any effect on the salivary microbiota, colonizing bacteria on mucosal membranes and on the gingival crevice after a single dose of amoxicillin., Material and Methods: Twenty subjects received 2 g of amoxicillin, per os. The facultative and strictly anaerobic microflora, as well as the streptococcal microflora specifically, were followed from baseline and after 1, 4, and 24 hours. Samples were taken for microbial analysis from saliva, the dorsum of the tongue, and the gingival crevice, and were inoculated and cultured. Plasma samples and saliva samples were analyzed for amoxicillin concentrations (free and protein bound) using liquid chromatography and mass-spectrometry., Results: Amoxicillin was detected in concentrations over the break-point (>2 μg/mL) of amoxicillin in plasma after 1 and 4 hours but not after 24 hours. The dose had a significant effect on the streptococci in the gingival crevice., Conclusion: A single dose given as prophylaxis to prevent a surgical-site infection results in a significant reducing effect on the oral streptococcal microflora in the gingival crevice and may have an impact on bacteria spreading into tissues and the bacteremia of streptococci., (© 2015 Wiley Periodicals, Inc.)

Accurate measurement of sex steroid concentrations in rodent serum is essential to evaluate mouse and rat models for sex steroid-related disorders. The aim of the present study was to develop a sensitive and specific gas chromatography-tandem mass spectrometry (GC-MS/MS) method to assess a comprehensive sex steroid profile in rodent serum. A major effort was invested in reaching an exceptionally high sensitivity for measuring serum estradiol concentrations. We established a GC-MS/MS assay with a lower limit of detection for estradiol, estrone, T, DHT, progesterone, androstenedione, and dehydroepiandrosterone of 0.3, 0.5, 4.0, 1.6, 8, 4.0, and 50 pg/mL, respectively, whereas the corresponding values for the lower limit of quantification were 0.5, 0.5, 8, 2.5, 74, 12, and 400 pg/mL, respectively. Calibration curves were linear, intra- and interassay coefficients of variation were low, and accuracy was excellent for all analytes. The established assay was used to accurately measure a comprehensive sex steroid profile in female rats and mice according to estrous cycle phase. In addition, we characterized the impact of age, sex, gonadectomy, and estradiol treatment on serum concentrations of these sex hormones in mice. In conclusion, we have established a highly sensitive and specific GC-MS/MS method to assess a comprehensive sex steroid profile in rodent serum in a single run. This GC-MS/MS assay has, to the best of our knowledge, the best detectability reported for estradiol. Our method therefore represents an ideal tool to characterize sex steroid metabolism in a variety of sex steroid-related rodent models and in human samples with low estradiol levels.

Context: Immunoassay-based techniques, routinely used to measure serum estradiol (E2), are known to have reduced specificity, especially at lower concentrations, when compared with the gold standard technique of mass spectrometry (MS). Different measurement techniques may be responsible for the conflicting results of associations between serum E2 and clinical phenotypes in men., Objective: Our objective was to compare immunoassay and MS measurements of E2 levels in men and evaluate associations with clinical phenotypes., Design and Setting: Middle-aged and older male subjects participating in the population-based Osteoporotic Fractures in Men (MrOS) Sweden study (n = 2599), MrOS US (n = 688), and the European Male Aging Study (n = 2908) were included., Main Outcome Measures: Immunoassay and MS measurements of serum E2 were compared and related to bone mineral density (BMD; measured by dual energy x-ray absorptiometry) and ankle-brachial index., Results: Within each cohort, serum E2 levels obtained by immunoassay and MS correlated moderately (Spearman rank correlation coefficient rS 0.53-0.76). Serum C-reactive protein (CRP) levels associated significantly (albeit to a low extent, rS = 0.29) with immunoassay E2 but not with MS E2 levels. Similar associations of immunoassay E2 and MS E2 were seen with lumbar spine and total hip BMD, independent of serum CRP. However, immunoassay E2, but not MS E2, associated inversely with ankle-brachial index, and this correlation was lost after adjustment for CRP., Conclusions: Our findings suggest interference in the immunoassay E2 analyses, possibly by CRP or a CRP-associated factor. Although associations with BMD remain unaffected, this might imply for a reevaluation of previous association studies between immunoassay E2 levels and inflammation-related outcomes.

We pay tribute to the life and work of Margareta Ryberg (1946-2012). She was an expert on the different forms of protochlorophyll(ide), their protein partners, and their transformations in angiosperms; on the structural aspects, and the nature of prolamellar bodies, as well as on the localization of light-dependent NADPH:protochlorophyllide oxido-reductase. She was a great teacher, who also loved gardening and handicraft. But above all, she was a beloved wife, mother, grandmother, and friend who will be deeply missed.

Recent studies using mass spectrometry have discovered candidate biomarkers for amyotrophic lateral sclerosis (ALS). However, those studies utilized small numbers of ALS and control subjects. Additional studies using larger subject cohorts are required to verify these candidate biomarkers. Cerebrospinal fluid (CSF) samples from 100 patients with ALS, 100 disease control, and 41 healthy control subjects were examined by mass spectrometry. Sixty-one mass spectral peaks exhibited altered levels between ALS and controls. Mass peaks for cystatin C and transthyretin were reduced in ALS, whereas mass peaks for posttranslational modified transthyretin and C-reactive protein (CRP) were increased. CRP levels were 5.84 +/- 1.01 ng/ml for controls and 11.24 +/- 1.52 ng/ml for ALS subjects, as determined by enzyme-linked immunoassay. This study verified prior mass spectrometry results for cystatin C and transthyretin in ALS. CRP levels were increased in the CSF of ALS patients, and cystatin C level correlated with survival in patients with limb-onset disease. Our biomarker panel predicted ALS with an overall accuracy of 82%.

The Empirical Proteomic Ontology Knowledge Base (EPO-KB) is an online database that represents current knowledge of biomarkers and contains associations between mass-to-charge (m/z) ratios of mass-spectrometry peaks to proteins. Such a database is a useful tool for identifying putative proteins associated with a m/z ratio. At present, EPO-KB contains data that have been extracted from 120 published research papers. It has been used in successful identification of a protein associated with a biomarker.

Unlabelled: The knowledge base EPO-KB (Empirical Proteomic Ontology Knowledge Base) is based on an OWL ontology that represents current knowledge linking mass-to-charge (m/z) ratios to proteins on multiple platforms including Matrix Assisted Laser/Desorption Ionization (MALDI) and Surface Enhanced Laser/Desorption Ionization (SELDI)--Time of Flight (TOF). At present, it contains information on m/z ratio to protein links that were extracted from 120 published research papers. It has a web interface that allows researchers to query and retrieve putative proteins that correspond to a user-specified m/z ratio. EPO-KB also allows automated entry of additional m/z ratio to protein links and is expandable to the addition of gene to protein and protein to disease links., Availability: http://www.dbmi.pitt.edu/EPO-KB

Amyotrophic lateral sclerosis (ALS) is a progressive motor neuron disease with largely unknown pathogenesis that typically results in death within a few years from diagnosis. There are currently no effective therapies for ALS. Clinical diagnosis usually takes several months to complete and the long delay between symptom onset and diagnosis limits the possibilities for effective intervention and clinical trials. The establishment of protein biomarkers for ALS may aid an earlier diagnosis, facilitating the search for effective therapeutic interventions and monitoring drug efficacy during clinical trials. Biomarkers could also be used to discriminate between subtypes of ALS, to measure disease progression and to detect susceptibility for developing ALS or monitor adverse effects of drug treatment. The present review will discuss the opportunities and proteomic platforms used for biomarker discovery efforts in ALS, summarizing putative ALS protein biomarkers identified in different biofluids.