Functional characterization of Clonorchis sinensis sodium-bile acid co-transporter (CsSBAT) as a steroid sulfate transporter.

Clonorchis sinensis (Cs) is a common trematode in Asian countries. Infection by Cs can result in many clinical symptoms. Here, a cDNA encoding a Cs apical sodium-dependent bile acid transporter (CsSBAT) was isolated from a Cs cDNA library, and functional characterization was performed using Xenopus laevis oocyte expression system. When expressed in Xenopus laevis oocytes, CsSBAT mediated the transport of radiolabeled estrone sulfate and dehydroepiandrosterone sulfate. No trans-uptake of carnitine, estradiol 17 ß-D glucuronide, prostaglandin E2, p-aminohippuric acid, α-ketoglutaric acid, and tetraethylammonium was observed. CsSBAT-mediated estrone sulfate uptake was in a time- and sodium-dependent manner. CsSBAT showed no exchange properties in efflux experiments. Concentration-dependent results showed saturable kinetics consistent with the Michaelis-Menten equation. Nonlinear regression analyses yielded a Km value of 0.3 ± 0.04 µM for [3H]estrone sulfate. CsSBAT-mediated estrone sulfate uptake was strongly inhibited by sulfate conjugates but not glucuronide conjugates. These findings contribute to our understanding of CsSBAT transport properties and the cascade of estrogen metabolite movement in Cs.

Determination of estrone sulfate, testosterone, androstenedione, DHEAS, cortisol, cortisone, and 17α-hydroxyprogesterone by LC-MS/MS in children and adolescents.

Quantitation of endogenous steroids and their precursors is essential for diagnosis of a wide range of endocrine disorders. Usually, these analyses have been carried out using immunoassays. However, immunoassays often overestimate concentrations due to assay interference by other endogenous steroids, especially for low concentrations. Mass spectrometry based methods offer superior specificity, accuracy, and sensitivity. We therefore present a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method with automated sample preparation for determination of 17α-hydroxyprogesterone (17OHP), cortisol, cortisone, dehydroepiandrosterone sulfate (DHEAS), androstenedione (A4), testosterone (T), and estrone sulfate (E1S). Samples were prepared using protein precipitation and 96-well filter plates, fully automated in a pipetting robot and analyzed by LC-MS/MS. Serum samples from 187 healthy children and adolescents aged 5-18 years were used to study hormone changes in relation to sex and pubertal stage. Lower limit of quantification for 17OHP was 0.7 nmol/L, for cortisol 11 nmol/L, for cortisone 2 nmol/L, for DHEAS 0.1 µmol/L, and for A4, T, and E1S, 0.2 nmol/L. This study showed a general increase in 17OHP, DHEAS, A4, T and E1S in both genders during puberty. In boys, A4 and T increased significantly throughout pubertal development. Girls had significantly higher A4 and E1S concentrations, while boys had higher T concentrations. No sex- or puberty-specific differences were seen in cortisol or cortisone concentrations. To the best of our knowledge, this is the first presentation of changes in serum E1S concentrations during pubertal development in healthy children.

Clinical implications of estrone sulfate measurement in laboratory medicine.

Estrone sulfate (E1S) is the most abundant circulating estrogen and it has the potential to be used as a biomarker in certain conditions where estimation of low levels of estrogen or changes in relative levels of estrogens are important. This review will critically consider the role of estimating E1S for clinical laboratory practice. As E1S is an estrogen, a wider discussion of estrogens is included to contextualize the review. Assays have been available for a number of years for these estrogens and they have been measured in a number of clinical research studies. However, E1S remains a rarely ordered test. This review highlights the literature that suggests the possible advantages of measuring E1S in addition to, or possibly in place of, the more commonly measured estradiol (E2) and the less commonly measured estrone (E1). The potential biomarker role of E1S in risk stratification for breast cancer, in promotion of proliferation of endometrial cancer, in prognostic information in advanced prostatic carcinoma, and in the monitoring of response to certain hormonal therapy for malignancy is discussed. The methods available for the measurement of E1S are reviewed and the limitations of the current methodologies are described. In conclusion, E1S has some interesting potential applications in clinical laboratory medicine that require further investigation.

Effects of exemestane and letrozole therapy on plasma concentrations of estrogens in a randomized trial of postmenopausal women with breast cancer.

PURPOSE: Inter-individual differences in estrogen concentrations during treatment with aromatase inhibitors (AIs) may contribute to therapeutic response and toxicity. The aim of this study was to determine plasma concentrations of estradiol (E2), estrone (E1), and estrone sulfate (E1S) in a large cohort of AI-treated breast cancer patients. METHODS: In a randomized, multicenter trial of postmenopausal women with early-stage breast cancer starting treatment with letrozole (n = 241) or exemestane (n = 228), plasma estrogen concentrations at baseline and after 3 months were quantitated using a sensitive mass spectrometry-based assay. Concentrations and suppression below the lower limit of quantification (LLOQ) were compared between estrogens and between drugs. RESULTS: The ranges of baseline estrogen concentrations were

Polymorphisms of SLC22A9 (hOAT7) in Korean Females with Osteoporosis.

Among solute carrier proteins, the organic anion transporters (OATs) play an important role for the elimination or reabsorption of endogenous and exogenous negatively charged anionic compounds. Among OATs, SLC22A9 (hOAT7) transports estrone sulfate with high affinity. The net decrease of estrogen, especially in post-menopausal women induces rapid bone loss. The present study was performed to search the SNP within exon regions of SLC22A9 in Korean females with osteoporosis. Fifty healthy controls and 50 osteoporosis patients were screened for the genetic polymorphism in the coding region of SLC22A9 using GC-clamped PCR and denaturing gradient gel electrophoresis (DGGE). Six SNPs were found on the SLC22A9 gene from Korean women with/without osteoporosis. The SNPs were located as follows: two SNPs in the osteoporosis group (A645G and T1277C), three SNPs in the control group (G1449T, C1467T and C1487T) and one SNP in both the osteoporosis and control groups (G767A). The G767A, T1277C and C1487T SNPs result in an amino acid substitution, from synonymous vs nonsynonymous substitution arginine to glutamine (R256Q), phenylalanine to serine (F426S) and proline to leucine (P496L), respectively. The Km values and Vmax of the wild type, R256Q, P496L and F426S were 8.84, 8.87, 9.83 and 12.74 µM, and 1.97, 1.96, 2.06 and 1.55 pmol/oocyte/h, respectively. The present study demonstrates that the SLC22A9 variant F426S is causing inter-individual variation that is leading to the differences in transport of the steroid sulfate conjugate (estrone sulfate) and, therefore this could be used as a marker for certain disease including osteoporosis.

Hepatic Gene Expression and Metabolite Profiles of Androstenone and Skatole Relative to Plasma Estrone Sulfate Levels in Boars.

Testicular steroids can alter the activity and expression of enzymes within the liver and may influence the metabolism of skatole and androstenone, which are responsible for boar taint. Plasma levels of estrone sulfate (E1S) are indicative of the steroidogenic capacity of the boar and are variable between animals of similar live weights at slaughter. This study aimed to characterize the relationship between steroidogenic capacity and the metabolism of boar taint compounds by relating plasma E1S levels at slaughter weight to the expression levels of genes regulating the metabolism of androstenone and skatole, along with their respective metabolite profiles. RT-qPCR was used to evaluate gene expression in the liver. Hepatocytes were also isolated and treated with androstenone or skatole, with metabolite levels in the incubation media quantified by high-performance liquid chromatography. Plasma E1S levels ranged from 2.2-108.5 ng/mL and were positively correlated with overall skatole metabolism (p = 0.038), the production of metabolites 3-methyloxindole (p = 0.026) and 3-hydroxy-3-methyloxindole (p = 0.036), and expression levels of key genes involved in skatole metabolism, specifically CYP2C33 (p = 0.0042), CYP2C49 (p = 0.022), and CYB5R1 (p = 0.017). There was no association between androstenone metabolism and plasma E1S concentrations; however, there was evidence of possible co-regulation amongst genes involved in the metabolism of androstenone, skatole, and estrogens. These findings indicate that steroidogenic capacity is related to the rate of skatole, but not androstenone metabolism, in slaughter-weight boars.

Pituitary-gonadal hormones associated with respiratory failure in men and women hospitalized with COVID-19: an observational cohort study.

Aim: To explore pituitary-gonadal hormone concentrations and assess their association with inflammation, severe respiratory failure, and mortality in hospitalized men and women with COVID-19, and compare these to hormone concentrations in hospitalized patients with bacterial community-acquired pneumonia (CAP) and influenza virus CAP and to concentrations in a reference group of healthy individuals. Methods: Serum concentrations of testosterone, estrone sulfate, luteinizing hormone (LH), follicle-stimulating hormone (FSH), and interleukin-6 (IL-6) were measured within 4 days of admission. Associations were assessed by logistic regression analysis in patients with COVID-19, and results were reported as odds ratio with 95% CI per two-fold reduction after adjustment for age, comorbidities, days to sample collection, and IL-6 concentrations. Results: In total, 278 patients with COVID-19, 21 with influenza virus CAP, and 76 with bacterial CAP were included. Testosterone concentrations were suppressed in men hospitalized with COVID-19, bacterial and influenza virus CAP, and moderately suppressed in women. Reductions in testosterone (OR: 3.43 (1.14-10.30), P = 0.028) and LH (OR: 2.51 (1.28-4.92), P = 0.008) were associated with higher odds of mehanical ventilation (MV) in men with COVID-19. In women with COVID-19, reductions in LH (OR: 3.34 (1.02-10-90), P = 0.046) and FSH (OR: 2.52 (1.01-6.27), P = 0.047) were associated with higher odds of MV. Conclusion: Low testosterone and LH concentrations were predictive of severe respiratory failure in men with COVID-19, whereas low concentrations of LH and FSH were predictive of severe respiratory failure in women with COVID-19.

Aromatase and steroid sulfatase from human placenta.

Cytochrome P450 aromatase (AROM) and steroid (estrone (E1)/dehydroepiandrosterone (DHEA)) sulfatase (STS) are the two key enzymes responsible for the biosynthesis of estrogens in human, and maintenance of the critical balance between androgens and estrogens. Human AROM, an integral membrane protein of the endoplasmic reticulum, is a member of the Fe-heme containing cytochrome P450 superfamily having a cysteine thiolate as the fifth Fe-coordinating ligand. It is the only enzyme known to catalyze the conversion of androgens with non-aromatic A-rings to estrogens characterized by the aromatic A-ring. Human STS, also an integral membrane protein of the endoplasmic reticulum, is a Ca2+-dependent enzyme that catalyzes the hydrolysis of sulfate esters of E1 and DHEA to yield the respective unconjugated steroids, the precursors of the most potent forms of estrogens and androgens, namely, 17ß-estradiol (E2), 16α,17ß-estriol (E3), testosterone (TST) and dihydrotestosterone (DHT). Expression of these steroidogenic enzymes locally within various organs and tissues of the endocrine, reproductive, and central nervous systems is the key for maintaining high levels of the reproductive steroids. Thus, the enzymes have been drug targets for the prevention and treatment of diseases associated with steroid hormone excesses, especially in breast and prostate malignancies and endometriosis. Both AROM and STS have been the subjects of vigorous research for the past six decades. In this article, we review the procedures of their extraction and purification from human term placenta are described in detail, along with the activity assays.

Evolution of 17-ß-estradiol, estrone and estrone-sulfate concentrations in late pregnancy of different breeds of mares using Liquid Chromatography and Mass Spectrometry.

This study describes 17-ß-estradiol (E2), estrone (E1) and estrone-sulfate (E1S) concentrations between 4 and 11 months in healthy equine pregnancies of two different breeds using Liquid Chromatography coupled to Mass-Spectrometry (LC-MS). In 2 stud-farms including 15 Spanish PureBred (SPB) and 11 Showjumping (SJ) types mares, combined thickness of the uterus and the placenta (CTUP) was measured and blood was sampled monthly between 4 and 11 months of gestation. Concentrations of E2, E1 and E1S were assayed with LC-MS in mares with normal CTUP. Effects of breed, day of pregnancy and mare's parity and age on estrogens concentrations were investigated. Peak of E2 was observed at 5 months (median: 46.4 pg/mL; maximum: 201.5 pg/mL). A strong correlation was observed between E1 and E1S (p < 0.0001, r = 0.85). Peak of E1 (median: 571.0 pg/mL; maximum: 1641.9 pg/mL) and E1S (median: 573.6 ng/mL; maximum: 997.6 ng/mL) concentrations was observed at the 5th month and then E1S decreased quicker than E1 until the end of pregnancy. Higher E2 and E1 concentrations were observed in SJ than in SPB mares between the 6th and the 8th months. No difference between breeds was observed for E1S monthly evolution. Estrogen peak values were all observed at 5 months. Unlike recent LC-MS studies, E1S values observed here were in the same range than those previously established using immuno-assays. After the 6th month, E1S decreased quicker than E1. Effect of breed only observed on non-sulfonated estrogens should be further confirmed. These findings confirm that sulfonation activity of the allantochorion may be limited after the 6th month.

American Bison (Bison bison) reproductive endocrinology: serum Pregnancy Associated Glycoproteins (PAG), Progesterone, Estrone and Estrone-Sulfate in non pregnant animals and during gestation.

This study describes concentrations of Pregnancy Associated Glycoproteins (PAG), progesterone (P4), estrone (E1) and estrone-sulfate (E1S) in American Bison sera. In 2 ranches, mature American Bison were sampled once a year for 2 yr. Subsequent American Bison cows calving days were reported. PAG concentration was determined by Radio-Immuno Assay, whereas P4, E1 and E1S were assayed using Liquid Chromatography and Mass Spectrometry. Concentrations were compared between American Bison bulls (B, n = 7), Nonpregnant cows (NP, n = 32), first (1TP, n = 3), second (2TP, n = 26) and third (3TP, n = 15) trimester of pregnancy. Seven American Bison bulls and 92 cows were sampled, 51 calved during these 2 yr. Calving occurred mostly in spring (74.5%), but also in summer (13.7%) and fall (11.8%). PAG and P4 were higher in 2TP and 3TP than B and NP (P< 0.0001). P4 was non-basal in B and NP. E1 and E1S were correlated (P< 0.0001; r = 0.76) and increased in 2TP and 3TP when compared with B and NP (P< 0.01). Moreover, E1S was higher in 3TP than in 2TP (P< 0.0001) and correlated to pregnancy day (P< 0.0001; r = 0.60). Breeding American Bison in Belgium induces a calving seasonality loss. P4 slowly increases in 1TP and remains steady and high in 2 and 3TP. P4 non-basal and variable concentrations in B or NP disable its use as gestation marker. American Bison produce PAG in the 2 and 3TP, but Estrone-sulfate assay seems to be the best pregnancy marker during the 2 last trimesters as it could help to estimate the gestation period.

In the Model Cell Lines of Moderately and Poorly Differentiated Endometrial Carcinoma, Estrogens Can Be Formed via the Sulfatase Pathway.

Endometrial cancer (EC) is the most common gynecological malignancy in resource-abundant countries. The majority of EC cases are estrogen dependent but the mechanisms of estrogen biosynthesis and oxidative metabolism and estrogen action are not completely understood. Here, we evaluated formation of estrogens in models of moderately and poorly differentiated EC: RL95-2 and KLE cells, respectively. Results revealed high expression of estrone-sulfate (E1-S) transporters (SLCO1A2, SLCO1B3, SLCO1C1, SLCO3A1, SLC10A6, SLC22A9), and increased E1-S uptake in KLE vs RL95-2 cells. In RL95-2 cells, higher levels of sulfatase and better metabolism of E1-S to E1 were confirmed compared to KLE cells. In KLE cells, disturbed balance in expression of HSD17B genes led to enhanced activation of E1 to E2, compared to RL95-2 cells. Additionally, increased CYP1B1 expression and down-regulation of genes encoding phase II metabolic enzymes: COMT, NQO1, NQO2, and GSTP1 suggested decreased detoxification of carcinogenic metabolites in KLE cells. Results indicate that in model cell lines of moderately and poorly differentiated EC, estrogens can be formed via the sulfatase pathway.

Changes in circulating concentrations of testosterone and estrone sulfate after human chorionic gonadotropin administration and subsequent to castration of 2-year-old stallions.

Reproductive steroids testosterone (T) and estrone sulfate (E1S) are used as diagnostic markers for cryptorchidism in horses. The human chorionic gonadotropin (hCG) stimulation test is used as a diagnostic aid because administration of this hormone results in greater incremental differences in circulating steroid concentrations. Thoughts regarding optimal sampling times following hCG administration, however, are inconsistent. Additionally, determination of half-life of these steroids is important in postsurgical samples to confirm complete removal of testicular tissue. Objectives of this study, therefore, were to determine optimal sampling periods for peak T and E1S after hCG administration and half-life of these steroids after castration. Eight pony stallions were randomly assigned to control or treatment groups (5000 IU hCG). Blood samples were collected following hCG administration. Subsequently, stallions were castrated and blood samples were collected post-castration. The T concentrations were greatest at 72 h after hCG and were greater (P < 0.02) in samples from hCG-treated than control animals: 9,903.4 ± 384 and 784.0 ± 192 pg/mL, respectively (Mean ± SEM). The T concentrations were also greater at 1, 12, 24, 48 and 96 h. The E1S concentrations did not change after administration of hCG. The T response to hCG administration was biphasic with a maximal response between 48-96 h after administration. Half-lives of T and E1S were 1.1 and 0.7 h, respectively, and concentration of T and E1S was similar to that of geldings at 24 h post-castration, which, therefore, should be considered an optimal time to ensure complete castration has occurred.

Hyperosmolarity stimulates transporter-mediated insertion of estrone sulfate into the plasma membrane, but inhibits the uptake by SLC10A1 (NTCP).

Many drugs are largely hydrophobic molecules; a transporter might conceivably insert these into the plasma membrane. At least 18 transporters from diverse families have been reported to transport the model compound estrone sulfate alias estrone-3-sulfate (E3S). Out of these, we recently examined SLC22A11 (OAT4). We concluded from a comparison of E3S and uric acid transport that SLC22A11 does not translocate E3S into the cytosol, but into the plasma membrane. Here we present a hyperosmolarity alias hypertonicity assay to differentiate transport mechanisms. Human transporters were expressed heterologously in 293 cells. Solute uptake into intact cells was measured by LC-MS. Addition of mannitol or sucrose led to rapid cell shrinkage, but cell viability after 60 min in hyperosmolar buffer was not impaired. A decrease in substrate accumulation with increasing osmolarity as observed here for several substrates and the transporters SLC22A11, ETT (SLC22A4), OCT2 (SLC22A2), OAT3 (SLC22A8), and MATE1 (SLC47A1) suggests regular substrate translocation into the cytosol. An increase as observed for E3S transport by SLC22A11, OAT3, MATE1, SLC22A9, and SLC10A6 implies insertion into the membrane. In marked contrast to the other E3S transporters, the bile acid transporter SLC10A1 (NTCP, Na+ taurocholate co-transporting polypeptide) showed a decrease in the accumulation of E3S in hyperosmolar buffer; the same was observed with taurocholic acid. Indeed, our data from several functional assays strongly suggest that the transport mechanism is identical for both substrates. Apparently, a unique transport mechanism has been established for SLC10A1 by evolution that ensures the transport of amphipathic, detergent-like molecules into the cytosol.

Steroid sulfatase in the mouse NIH-3T3 fibroblast cell line: Characterization, and downregulation by glucocorticoids.

Steroid hormones often circulate in the blood as inactive sulfated forms, such as estrone sulfate and dehydroepiandrosterone sulfate. The enzyme steroid sulfatase (STS) converts these steroids into active forms, mainly estrogens, in peripheral tissues. We have previously characterized STS activity in human and mouse breast and bone tissues, and we have shown that STS can provide estrogens to these tissues from circulating sulfated precursors. This study was designed to characterize STS activity in a mouse fibroblast cell line (NIH-3T3). Using a radioactive estrone sulfate (E1S) conversion assay, we detected STS activity in cultured NIH-3T3 cells. This activity was blocked by the STS inhibitors EMATE and STX-64, indicating authentic STS activity. We also found that microsomes prepared from NIH-3T3 cells had relatively high STS activity and that cytosols had low activity, consistent with the known distribution of this enzyme to the endoplasmic reticulum. Michaelis-Menten analysis of the NIH-3T3 microsomes indicated a Km of 10.9 µM using E1S as substrate. Primary fibroblasts prepared from mouse ears and tails also had measurable STS activity, as indicated by 3H-E1S conversion assay, further supporting the conclusion that fibroblasts possess STS. Furthermore, Western blotting confirmed the presence of immunoreactive STS in NIH-3T3 microsomes. With regard to regulation, treatments of cultured NIH-3T3 cells revealed that cortisol and the synthetic glucocorticoids dexamethasone and prednisolone decreased STS activity, as we have found for cell lines from other tissues. The effect of cortisol was seen at both 10 µM and 1.0 µM but not at 0.1 µM. Western blotting also indicated a decrease in STS immunoreactivity in cortisol-treated microsomes. The reduction in STS activity by dexamethasone in whole cells was reversed by the glucocorticoid receptor antagonist RU-486, indicating that glucocorticoid downregulation of STS activity is receptor mediated. An inhibition assay on NIH-3T3 microsomes revealed that STS activity was inhibited significantly by 10 µM estradiol-17ß, a known substrate inhibitor of E1S for STS, but not by 10 µM cortisol. This is consistent with the idea that cortisol inhibits STS in NIH-3T3 cells through a regulatory mechanism rather than by substrate inhibition. Our results could have important implications regarding local estrogen production by STS in fibroblasts, which are the most common connective tissue cells in the body, and on possible regulation of local estrogen levels by cortisol.

Measurements of circulating progesterone and estrone sulfate concentrations as a diagnostic and prognostic tool in porcine pregnancy revisited.

The main goal of this study was to examine the utility of measuring systemic concentrations of steroid hormones, namely progesterone (P4) and estrone sulfate (E1S), for monitoring the progression of porcine pregnancy and predicting sow fertility. There were 3 subsets of artificially inseminated (AI'd) sows used in the present experiments: (i) animals sacrificed on gestational day 20 (gd20; n = 16) or (ii) gd50 (n = 16; Experiment 1), and (iii) animals maintained throughout pregnancy (n = 24; Experiment 2). Blood samples (10 mL) were drawn from the orbital sinus and the endocrine data determined at different time points around ovulation/artificial insemination (gd0 (first AI), gd1 (second AI), and gd2) and maternal recognition of pregnancy (gd11), as well as on gd20 and gd50 (during 2 periods of increased embryonic/fetal mortality in swine) were examined for correlations with the numbers of healthy, arrested, and reabsorbing embryos (Experiment 1) or with the number of live, stillborn, and mummified piglets recorded at farrowing (Experiment 2). No correlations were recorded between circulating concentrations of both steroids and the numbers of healthy, arresting, or reabsorbing conceptuses on gd20 or 50 (Experiment 1). The number of corpora lutea (CL) was directly related to the number of healthy embryos/conceptuses on gd20 and 50 (r = 0.71, P = 0.007 and r = 0.76, P = 0.0007, respectively) and the number of arresting embryos on gd20 (r = 0.54, P = 0.05), and negatively correlated with the number of reabsorbing embryos on gd20 (r = -0.53, P = 0.05). In Experiment 2, circulating P4 concentrations on gd11 related directly to the number of live-born piglets (r = 0.46, P < 0.04). Systemic E1S concentrations on gd0, gd1, gd2 and gd50 were correlated with the number of mummified conceptuses recorded at farrowing (r = 0.50, P = 0.03; r = 0.59, P = 0.01; r = 0.48, P = 0.04; and r = 0.56, P = 0.01, respectively) and plasma concentrations of E1S on gd20 related directly to the number of stillborn piglets (r = 0.60, P = 0.02). In summary, the number of CL on gd20 and 50 is a reliable marker of embryonic/fetal pig status. Measurements of P4 and E1S on gd20 and 50 showed limited diagnostic value (ie, were not indicative of the number of healthy and abnormally developing embryos/fetuses). However, measurements of circulating P4 and E1S concentrations during the periconceptional period and in the early/mid-pregnancy of sows have the makings of a practical method to predict gestational outcomes.

The Membrane Transporter OAT7 (SLC22A9) Is Not a Susceptibility Factor for Osteoporosis in Europeans.

Bone production, maintenance, and modeling are a well-balanced process involving mineralization by osteoblasts and resorption by osteoclasts. Sex steroid hormones, including their conjugated forms, contribute majorly to maintaining this balance. Recently, variants in the SLC22A9 gene have been associated with osteoporosis in Korean females. We had recently shown that SLC22A9, encoding organic anion transporter 7 (OAT7), is an uptake transporter of estrone sulfate and identified several genetic variants in Europeans leading to functional consequences in vitro. We therefore hypothesized that SLC22A9 genetic variants may contribute to the pathophysiology of osteoporosis in Europeans. To test this hypothesis, we examined the associations of SLC22A9 variants with bone quality, fractures, and bone turnover markers. We genotyped SLC22A9 variants in 5,701 (2,930 female) subjects (age range, 20-93 years) extracted from the population-based Study of Health in Pomerania (SHIP and SHIP-TREND) covered by the Illumina Infinium HumanExome BeadChip version v1.0 (Exome Chip). Descriptive data (e.g., history of fractures), ultrasonography of the calcaneus, as well as serum concentrations of carboxy-terminal telopeptide of type I collagen, amino-terminal propeptide of type I procollagen, and vitamin D were determined. Comprehensive statistical analyses revealed no association between low-frequency and rare SLC22A9 variants and bone quality, fractures, and bone turnover markers. Our results indicate that single genetic SLC22A9 variants do not have a major impact on osteoporosis risk prediction in Europeans, yet findings need to be replicated in larger-scale studies.

Temporal changes in plasma profile of pregnancy-associated glycoprotein, progesterone and estrone sulfate associated with fetal number during early- and mid-pregnancy in goats.

This study was designed to investigate plasma profile of pregnancy-associated glycoprotein (PAG), progesterone (P4) and estrone sulfate (E1S) during early- and mid-pregnancy. The goal was to explore the relationships with values for reproductive variables, to detect the most reliable predictor variable, and to identify the most desirable time point for blood collection for determining fetal number in goats. After ultrasonographic examination at d35-40 post-mating, blood sampling of 15 pregnant goats (total 18) was continued until d114. The PAG profile was characterized by gradual increase during early pregnancy from d26 to d51 and thereafter concentrations were relatively constant until d114 of gestation. The effect of fetal number on plasma PAG, P4 and E1S was first evident on d28, d51 and d26, respectively. During mid-pregnancy, does with twins had a greater (P < 0.05) PAG (S-N = 2.54 ± 0.12 compared with 1.59 ± 0.11), P4 (18.91 ± 0.67 compared with 14.51 ± 0.47 ng/mL) and E1S (16.34 ± 0.76 compared with 11.32 ± 0.44 ng/dL) as compared with does with a singleton fetus. Plasma PAG but not P4 and E1S was positively correlated with fetal number and birth weight of kids during early pregnancy. Multivariate linear regression and discriminant function analyzes allowed for identification of plasma PAG as the most reliable predictor for fetal number and birth weight of kids. Furthermore, d58 was the most suitable single time point for prediction of fetal number using PAG as a biomarker. In conclusion, plasma profile of PAG, P4 and E1S was affected by fetal count. Plasma PAG was identified as the most reliable predictor variable of fetal number and birth weight of kids as compared to plasma P4 and E1S.

Endogenous, cholesterol-activated ATP-dependent transport in membrane vesicles from Spodoptera frugiperda cells.

Transport proteins of the ATP-binding cassette (ABC) family are found in all kingdoms of life. In humans, several ABC efflux transporters play a role in drug disposition and excretion. Therefore, in vitro methods have been developed to characterize the substrate and inhibitor properties of drugs with respect to these transporters. In the vesicular transport assay, transport is studied using inverted membrane vesicles produced from transporter overexpressing cell lines of both mammalian and insect origin. Insect cell expression systems benefit from a higher expression compared to background, but are not as well characterized as their mammalian counterparts regarding endogenous transport. Therefore, the contribution of this transport in the assay might be underappreciated. In this study, endogenous transport in membrane vesicles from Spodoptera frugiperda -derived Sf9 cells was characterized using four typical substrates of human ABC transporters: 5(6)-carboxy-2,'7'-dichlorofluorescein (CDCF), estradiol-17ß-glucuronide, estrone sulfate and N-methyl-quinidine. Significant ATP-dependent transport was observed for three of the substrates with cholesterol-loading of the vesicles, which is sometimes used to improve the activity of human transporters expressed in Sf9 cells. The highest effect of cholesterol was on CDCF transport, and this transport in the cholesterol-loaded Sf9 vesicles was time and concentration dependent with a Km of 8.06 ±â€¯1.11 µM. The observed CDCF transport was inhibited by known inhibitors of human ABCC transporters, but not by ABCB1 and ABCG2 inhibitors verapamil and Ko143, respectively. Two candidate genes for ABCC-type transporters in the S. frugiperda genome (SfABCC2 and SfABCC3) were identified based on sequence analysis as a hypothesis to explain the observed endogenous ABCC-type transport in Sf9 vesicles. Although further studies are needed to verify the role of SfABCC2 and SfABCC3 in Sf9 vesicles, the findings of this study highlight the need to carefully characterize background transport in Sf9 derived membrane vesicles to avoid false positive substrate findings for human ABC transporters studied with this overexpression system.

Human versus non-human sex steroid use in hormone replacement therapies part 1: Preclinical data.

Prior to 2002, hormone replacement therapy (HRT) was considered to be an important component of postmenopausal healthcare. This was based on a plethora of basic, epidemiological and clinical studies demonstrating the health benefits of supplementation with human sex steroids. However, adverse findings from the Women's Health Initiative (WHI) studies that examined the 2 major forms of HRT in use in the US at that time - Premarin (conjugated equine estrogens; CEE) and Prempro (CEE + medroxyprogesterone acetate; MPA), cast a shadow over the use of any form of HRT. Here we review the biochemical and physiological differences between the non-human WHI study hormones - CEE and MPA, and their respective human counterparts 17ß-estradiol (E2) and progesterone (P4). Preclinical data from the last 30 years demonstrate clear differences between human and non-human sex steroids on numerous molecular, physiological and functional parameters in brain, heart and reproductive tissue. In contrast to CEE supplementation, which is not always detrimental although certainly not as optimal as E2 supplementation, MPA is clearly not equivalent to P4, having detrimental effects on cognitive, cardiac and reproductive function. Moreover, unlike P4, MPA is clearly antagonistic of the positive effects of E2 and CEE on tissue function. These data indicate that minor chemical changes to human sex steroids result in physiologically distinct actions that are not optimal for tissue health and functioning.

A novel mode of operation of SLC22A11: Membrane insertion of estrone sulfate versus translocation of uric acid and glutamate.

Estrone sulfate alias estrone-3-sulfate (E3S) is considerably larger and much more hydrophobic than typical substrates of SLC22 transporters. It is puzzling that many otherwise unrelated transporters have been reported to transport E3S. Here we scrutinized the mechanism of transport of E3S by SLC22A11 (alias OAT4), by direct comparison with uric acid (UA), an important physiological substrate. Heterologous expression of SLC22A11 in human 293 cells gave rise to a huge unidirectional efflux of glutamate (Glu) and aspartate, as determined by LC-MS/MS. The uptake of E3S was 20-fold faster than the uptake of UA. Yet, the outward transport of Glu was inhibited by extracellular E3S, but not by UA. The release of E3S after preloading was trans-stimulated by extracellular dehydroepiandrosterone sulfate (DHEAS), but neither by UA nor 6-carboxyfluorescein (6CF). The equilibrium accumulation of E3S was enhanced 3-fold by replacement of chloride with gluconate, but the opposite effect was observed for UA. These results establish that SLC22A11 provides entirely different transport mechanisms for E3S and UA. Therefore, E3S must not be used as a substitute for UA to assay the function of SLC22A11. In equilibrium accumulation experiments, the transporter-mediated uptake was a linear function of the concentration of UA and 6CF. By contrast, in the same concentration range the graph for E3S was hyperbolic. This suggests that SLC22A11 inserts E3S into a small volume with limited capacity, the plasma membrane. Our data support the notion that the reverse process, extraction from the membrane, is also catalyzed by the carrier.