Plasma Sex Hormone-binding Globulin Research Articles

Sex hormone-binding globulin levels and cardiovascular risk factors in morbidly obese subjects before and after weight reduction induced by diet or malabsorptive surgery

One of the main goals of weight reduction in morbidly obese subjects is its benefit on coronary heart disease (CHD) risk. A cross-sectional study was designed to randomly assign 79 morbidly obese subjects (27 men and 52 women; age: 30–45 years) either to a diet protocol (20 kcal per kg fat-free mass (FFM); 55% carbohydrates, 30% fat, and 15% proteins) or to malabsorptive surgery (biliopancreatic diversion). Fatness parameters, measured by dual-energy X-ray absorptiometry, lipid profile, insulin, leptin, sex steroid hormones and sex hormone-binding globulin (SHBG) levels were compared at baseline and 1 year after the beginning of the study. The data showed that plasma SHBG levels, but not testosterone levels, correlated negatively to fasting insulin levels and positively to HDL-cholesterol in both men and women. Total leptin levels were significantly lower ( P<0.0001) in post-BPD subjects of both sexes compared to dietary treated obese subjects. The logarithm of plasma leptin correlated significantly and positively with insulin but negatively with SHBG. A step-down regression analysis showed that FFM and SHBG, but not insulin levels, were the most powerful independent variables for predicting HDL-cholesterol levels in morbidly obese patients. The negative relationship between SHBG levels and CHD risk appears to be mediated by a concomitant variation in body fatness. Finally, in obese patients, SHBG levels seem to be an indicator of total adiposity rather than an index of an altered insulin/glucose homeostasis.

Indirect Measurement of Bioavailable Testosterone with the Bayer Immuno 1 System

Testosterone is the principal circulating androgen in men. A major fraction of testosterone is specifically bound with high affinity and low capacity to sex-hormone-binding globulin (SHBG), whereas most of the remaining testosterone is bound with low affinity to albumin (ALB), leaving only 1–2% to circulate as “free” testosterone (FT) not bound to protein in serum (1). Because the SHBG–testosterone complex dissociates very slowly in vitro (dissociation half-time, 20 s), SHBG-bound testosterone (SHBG-T) is not considered to be available for biologic action in tissues. According to the original “free hormone hypothesis”, only FT is able to enter target cells. Tait and Burstein (2), however, suggested that because testosterone is only loosely bound to ALB, this fraction is also available for tissue uptake (dissociation half-time, 1 s). The concept of bioavailable testosterone (BAT), also known as “non-SHBG-bound testosterone” [or both FT and ALB-bound testosterone (ALB-T)], was supported by in vitro kinetic calculations (3), but others (4)(5) have challenged the assumptions used for these calculations. Indeed, if SHBG facilitates entry of testosterone into tissues, as reported by Pardridge (6), then SHBG-T would also be “available” for tissue uptake and action. Controversy remains about the best biochemical measurement for testosterone function in humans. The initial clinical diagnostic evaluation for men with symptoms of hypogonadism involves measurement of the serum total testosterone. In situations such as hyper- and hypothyroidism, obesity, and the use of antiepileptic drugs, the correlation between clinical symptoms and the serum total testosterone concentration has been reported to be poor. This may be attributable in part to the inactivity of the SHBG-T fraction and to variation in the plasma concentration of SHBG in these situations. The fact that plasma SHBG changes during life and in several pathologic situations is well established (7)(8). It is currently common practice to …

ADVERSE EFFECTS OF NEONATAL HYPOTHYROIDISM ON WISTAR RAT SPERMATOGENESIS

The effect of neonatal hypothyroidism on spermatogenesis was studied in Wistar rats of different age groups. Hypothyroidism was induced in newborn male rats from day one postpartum up to day 60 postpartum by daily administration of 0.05% methimazole (MMI) to the nursing mothers or directly through drinking water. The animals were killed at days 10, 15, 30, 40, and 60 postpartum, blood plasma was collected, and testes, epididymides, prostates, and seminal vesicles were separated and weighed. Testes were fixed in formalin for histological studies. Plasma testosterone (T), estradiol (E2), and sex hormone binding globulin (SHBG) were measured by radioimmunoassay. Hypothyroidism significantly reduced seminiferous tubule and lumen diameter. Control rats showed active spermatogenesis whereas in hypothyroid rats, the proliferation and differentiation of germ cells were arrested and their number was decreased. Plasma T, E2, and SHBG levels were significantly decreased at all ages for hypothyroid rats. The absolute weight of testes was decreased irrespective of age (except day 10 postpartum), however ventral, dorsolateral prostate, and epididymis weights were decreased at 30, 40, and 60 days postpartum. Coagulating gland weight was decreased in all age groups of hypothyroid rats. Hypothyroid rats of day 40 and 60 postpartum showed a decrease in absolute seminal vesicle weight. Relative testicular weights of hypothyroid rats decreased by postpartum day 15, 30, 40, and 60 whereas the opposite effect was observed by postpartum day 10. Relative organ weights were increased in epididymides (day 15 and 30 postpartum), seminal vesicles (day 30 and 40 postpartum), and dorsolateral prostates (day 15, 30, and 40 postpartum) and decreased in 10 and 60 day old hypothyroid rat. Ventral prostate relative weight was decreased in 40 and 60 day old rats. The coagulating gland weight was decreased in 10, 15, and 60 days postpartum and an opposite effect was observed in 30 and 40 days hypothyroid rats. The present study clearly indicates that hypothyroidism adversely affects spermatogenesis; it also indicates that thyroid hormones are essential for normal spermatogenesis. Their effect may either be direct or indirect.

Human Sex Hormone-binding Globulin Promoter Activity Is Influenced by a (TAAAA)n Repeat Element within an Alu Sequence

Sex hormone-binding globulin (SHBG) is the major sex steroid-binding protein in human plasma and is produced by the liver. Plasma SHBG levels vary considerably between individuals and are influenced by hormonal, metabolic, and nutritional factors. We have now found that a (TAAAA)(n) pentanucleotide repeat, located within an alu sequence at the 5' boundary of the human SHBG promoter, influences its transcriptional activity in association with downstream elements, including an SP1-binding site. Furthermore, SHBG alleles within the general population contain at least 6-10 TAAAA repeats, and the transcriptional activity of a human SHBG promoter-luciferase reporter construct containing 6 TAAAA repeats was significantly lower than for similar reporter constructs containing 7-10 TAAAA repeats when tested in human HepG2 hepatoblastoma cells. This difference in transcriptional activity reflected the preferential binding of a 46-kDa liver-enriched nuclear factor to an oligonucleotide containing 6 rather than 7-10 TAAAA repeats. Thus, a (TAAAA)(n) element within the human SHBG promoter influences transcriptional activity in HepG2 cells and may contribute to differences in plasma SHBG levels between individuals.

Differential effects on the androgen status of postmenopausal women treated with tibolone and continuous combined estradiol and norethindrone acetate replacement therapy

Objective: To determine serum parameters reflective of androgen status in postmenopausal women using two types of hormone replacement therapy (HRT). Design: Randomized, double-blind, prospective 1-year trial of two oral HRT regimens. Setting: University hospital, department of obstetrics and gynecology, menopause clinic. Patient(s): 100 postmenopausal women ≥ 45 years. Intervention(s): Daily use of the progestogen tibolone (2.5 mg; n = 50) or continuous combined 17-β-estradiol (2 mg) and norethindrone acetate (E+NA, 1 mg; n = 50). Main Outcome Measure(s): Measurements of total testosterone (total T), dehydroepiandrosterone sulfate (DHEAS), androstenedione (A), FSH, and sex-hormone-binding globulin (SHBG), and calculations of free testosterone (free T). Assessment of changes from baseline within and between groups after 6 and 12 months. Result(s): We found significant differences (% changes) in the tibolone group compared to baseline within the groups after both 6 and 12 months, respectively. Levels of free T doubled, total T decreased slightly, and SHBG decreased by half; DHEAS increased by approximately 20%; and FSH decreased. In the E+NA group, levels of free T, total T, androstenedione, and FSH all decreased, and SHBG increased. Pre-trial levels of DHEAS, A, and total T were significantly higher in the E+NA group. Between groups throughout the study, the changes from baseline were significant due to the different extent of FSH reduction, and opposite changes of free T, SHBG, and DHEAS. Conclusion(s): Both regimens modify plasma androgens, DHEAS, and SHBG differently. Tibolone decreased the levels of SHBG, and substantially increased free T and to a lesser extent increased DHEAS; this may reflect a modification of adrenal androgen production. Continuous combined estradiol and norethindrone acetate HRT suppressed the peripheral plasma androgens mediated by increased levels of SHBG.

7α-18F]fluoro-17α-methyl-5α-dihydrotestosterone: a ligand for androgen receptor-mediated imaging of prostate cancer

We have synthesized a 18F-labeled androgen, [7α-18F]fluoro-17α-methyl-5α-dihydrotestosterone, in a no-carrier-added radiosynthesis by exchange of 18F- (tetrabutylammonium fluoride) with the 7β-tosyloxy of 17α-methyl-5α-dihydrotestosterone. The nonradioactive steroid binds with high affinity and specificity to the androgen receptor and binds poorly, if at all, to other steroid receptors and plasma sex hormone binding globulin. The 7α-18F-androgen concentrates markedly in the prostate of rats by an androgen receptor-dependent mechanism. It is likely that [7α-18F]fluoro-17α-methyl-5α-dihydrotestosterone will be an excellent positron emission tomography imaging agent for prostate cancer.

Three-dimensional model of the SHBG-like region of anticoagulant protein S: New structure-function insights

Protein S (PS) is a vitamin K-dependent glycoprotein that consists of several modules including a C-terminal sex hormone-binding globulin (SHBG)-like domain that has been subdivided into two laminin LG-type domains. The SHBG-like region of PS is known to bind to a complement regulator molecule, C4b-binding protein (C4BP), coagulation factor Va (FVa) and receptor tyrosine kinases. Inherited PS deficiency has been associated with thromboembolic disease. Yet, study of the mechanisms by which the SHBG-like region of PS serves its essential functions has so far been hampered because of the lack of structural information. Recently, the three-dimensional (3D) structure of LG domains from plasma SHBG, laminin and neurexin have been reported and were found related to the pentraxin family. We used these X-ray structures to build homology models of the SHBG-like region of human PS. We then analyzed previously reported experimental/clinical data in the light of the predicted structures. A potential calcium-binding site is found in the first LG domain of PS and D292 could play a role in this process. This region is close to the interface between the two LG domains and is also surrounded by segments that have been suggested by synthetic peptide studies to be important for C4BP or FVa binding. The 39 point mutations linked to PS deficiencies or reported as neutral variants were rationalized in the 3D structure. Proteins 2001;43:203-216.

Interactions between human plasma sex hormone-binding globulin and xenobiotic ligands

Human sex hormone-binding globulin (SHBG) binds sex steroids with high affinity. In plasma, the number of SHBG steroid-binding sites far exceeds the molar concentrations of sex steroids, and will accommodate other ligands such as phytoestrogens and fatty acids. We have therefore developed a screening assay to identify ligands for SHBG, which exist in our diet or environment. This assay allows the binding of potential ligands to SHBG to be assessed under physiological conditions, and is sensitive to the effects of plasma constituents. Several classes of endocrine active compounds were tested, including hydroxy-polychlorinated biphenyls (HO-PCBs), phthalate esters, monoesters, chlorinated pesticides, as well as synthetic estrogens and phytoestrogens. The relative binding affinities (RBAs) of various compounds to SHBG were determined in competitive displacement assays, by comparison with 17β-estradiol (RBA=100). Synthetic estrogens bound SHBG with RBAs of 0.4 (ethinylestradiol)-0.2 (diethylstilbestrol), while some phytoestrogens bound with RBAs of 0.12 (coumestrol)-0.04 (naringenin). Many compounds did not bind to SHBG with sufficient affinity to allow RBA measurements, and these include: several phytoestrogens, such as genistein and kaempferol, polychlorinated biphenyls, phthalate esters and monoesters. Of nine HO-PCB congeners tested only 4-OH-2′, 3′, 4′, 5′-tetraCB and 4-OH-2, 2′, 3′, 4′, 5′-pentaCB bound SHBG in undiluted serum with RBAs of 0.05 and 0.11. Although all test compounds bound to SHBG with much lower affinity than endogenous sex steroids, these interactions may be physiologically relevant in situations where plasma SHBG levels are high and endogenous sex steroid levels are low, such as in pre-pubertal children and women taking oral contraceptives.

Plasma androgens, IGF-1, body size, and prostate cancer risk: a synthetic review

We present a review of the epidemiological evidence for relations of prostate cancer risk to circulating total and bioavailable androgens, to alterations in the metabolism of insulin-like growth factor-1 (IGF-1), and to anthropometric indices of longitudinal growth (body stature) and overweight. In addition, we review the physiological inter-relationships between insulin, growth hormone/IGF-1 axis, and sex steroid metabolism, as well as the associations of bioavailable sex steroid levels with overweight and obesity. A first conclusion of this review is that, taken together, epidemiological studies have provided little support for the hypothesis that prostate cancer risk is increased in men with elevated total or biovailable testosterone (T). Although one prospective study showed an increased risk in men with low plasma sex hormone-binding globulin (SHBG) and with elevated plasma T for given levels of SHBG, this was not confirmed by results from other cohort studies. A second conclusion is that overweight, which is generally associated with moderate reductions in both total and bioavailable plasma T, appears to be unrelated to any significant increase or decrease in prostate cancer risk. However, significant increases in risk have been observed for men with a taller body stature, or with elevated plasma IGF-1. IGF-1 may directly enhance prostate tumorigenesis by inhibiting apoptosis and by stimulating cell proliferation. In addition, IGF-1 downregulates the synthesis of SHBG, and enhances sex steroid synthesis. Therefore, we do not entirely rule out that due to an elevation of plasma IGF-1 levels, men at increased risk of prostate cancer also have mildly elevated plasma bioavailable T, which epidemiological studies may have failed to demonstrate because of methodological problems. Prostate Cancer and Prostatic Diseases (2000) 3, 157-172

Effect of metformin on insulin-like growth factor (IGF) I and IGF-binding protein I in polycystic ovary syndrome.

The objective of the present study was to investigate whether metformin affected plasma concentrations of insulin-like growth factor (IGF) I and IGF-binding protein I (IGFBP-I) in polycystic ovary syndrome (PCOS) patients. This was an open study conducted by the Department of Obstetrics and Gynecology at the University of Siena, Italy. Seventeen women with PCOS participated in the study and were administered metformin at a dose of 500 mg three times a day. Treatment was continued for 30-32 days, after which the pretreatment evaluation was repeated. Plasma concentrations of LH, FSH, estradiol, free testosterone, IGF-I, IGFBP-I, sex hormone-binding globulin, and insulin were evaluated. Metformin led to a significant reduction in areas under the insulin curves (9310 +/- 1509 vs. 6520 +/-1108 mU/mL x min; P < 0.05) and was associated with a decrease in plasma free testosterone levels (12.7 +/- 1.7 vs. 10.3 +/- 2 pg/mL; P < 0.05) and an increase in plasma sex hormone-binding globulin concentrations (62 +/- 8 vs. 94 +/- 13 nmol/L; P < 0.05). A nonsignificant increase in plasma IGF-I levels was observed after metformin (276 +/-48 vs. 291 +/- 71 mcg/L), with a significant increase in plasma IGFBP-I levels (0.56 +/- 0.2 vs. 0.98 +/- 0.38 mcg/L; P < 0.05). The IGF-I/IGFBP-I ratio was significantly lower (492.8 +/- 117 vs. 296.9 +/- 82; P < 0.05) at the end of therapy than before treatment. In conclusion, it seems to be appropriate to intervene with an insulin-sensitizing agent such as metformin in an attempt to break the pathogenetic link between hyperinsulinemia and hormonal perturbations in PCOS.

Plasma total and glycosylated corticosteroid-binding globulin levels are associated with insulin secretion.

In humans, steroid hormones circulate in the blood mainly bound to specific steroid transport proteins, namely corticosteroid-binding globulin (CBG) for cortisol and sex hormone-binding globulin (SHBG) for testosterone and estradiol. The binding activities of these proteins are believed to modulate the biodisposal of steroids to target cells. It has been shown in vitro that insulin is a potent inhibitor of both CBG and SHBG secretion by a human hepatoblastoma cell (HepG2) line. To further investigate this potential effect of insulin in vivo, we prospectively studied three groups of lean subjects, obese subjects, and obese subjects with glucose intolerance, all of whom were otherwise healthy. The three groups were comparable in sex and age, and in the two obese groups, body mass index, waist to hip ratio, and blood pressure were similar. Plasma total CBG concentrations (38.2 +/- 5.4 vs. 31.7 +/- 4.05 mg/L; P = 0.016) and glycosylated CBG levels (37.3 +/- 5.2 vs. 31 +/- 3.9 mg/L; P = 0.018) were significantly increased in obese subjects with glucose intolerance. Plasma CBG correlated positively with fasting glucose levels (r = 0.49; P = 0.002), hemoglobin A1c levels (r = 0.35; P = 0.03), and area under the curve of glucose after an oral glucose tolerance test (r = 0.45; P = 0.005) and correlated negatively with the insulin response to i.v. glucose (AIRg; -0.38, P = 0.02) as well as to oral glucose (r = -0.40; P = 0.01) challenge tests. CBG levels did not covariate with insulin sensitivity. Multiple linear regression analysis showed that only AIRg contributed to the variability of the CBG concentration (P = 0.03), explaining 41% of its variance. Morning cortisol levels did not differ between the groups and did not correlate to any of the glucose or insulin metabolism parameters. Because carbohydrate chains influence the biological activity and half-life of glycoproteins, we analyzed the migration profile of CBG by Western blot and the interaction of CBG with lectin, Con A. The results indicated that the CBG mol wt and interaction with Con A did not differ between lean and obese patients. These data favor the hypothesis that the inhibitory effect of insulin on CBG liver secretion might be relevant in vivo and therefore contribute to decrease CBG levels in obese patients with enhanced insulin secretion. In both men and women, SHBG levels correlated negatively with fasting glucose (r = -0.55; P < 0.0001) and hemoglobin A1c (r = -0.38; P = 0.02) and positively with insulin sensitivity (S(I); r = 0.65; P = 0.003 and r = 0.63; P = 0.007 in men and women, respectively), but not with insulin secretion. The disposition index (S(I) x AIRg) was significantly decreased in the obese, glucose-intolerant subjects, suggesting that AIRg was inadequate for their degree of insulin resistance. The disposition index correlated positively with plasma SHBG levels (r = 0.52; P = 0.001) and negatively with plasma CBG levels (r = -0.54; P = 0.001). Our data suggest that CBG is a marker of insulin secretion in a similar way as SHBG is a marker of insulin sensitivity. As high plasma CBG levels have been associated with increased incidence of type 2 diabetes, this important issue merits further investigations.

Expression and Regulation of Human Sex Hormone-Binding Globulin Transgenes in Mice during Development1

Human sex hormone-binding globulin (SHBG) is produced by hepatocytes and transports sex steroids in the blood. The rat gene encoding SHBG is expressed transiently in the liver during fetal life, but it is not expressed in the liver postnatally, and the small amounts of SHBG in rat blood are derived from gonadal sources. To study the biosynthesis and function of human SHBG in an in vivo context, we have produced several lines of transgenic mice that contain either 11 kb (shbg11) or 4.3 kb (shbg4) portions of the human shbg locus. The expression and regulation of these transgenes have now been studied during fetal and postnatal development. In situ hybridization of an shbg11 transgenic mouse fetus at 17.5 days postcoitus located human shbg transcripts only in duodenal epithelial cells and hepatocytes. Temporal differences in the hepatic expression of mouse shbg and human shbg transgenes during late fetal development were reflected in corresponding differences in mouse and human SHBG levels in fetal and neonatal mouse blood. Serum concentrations of human SHBG increased during the first weeks of life regardless of gender until about 20 days of age in shbg11 mice, but after this time they continued to increase only in the males. This sexual dimorphism was reflected in corresponding differences in human SHBG messenger RNA (mRNA) abundance in the livers of these animals. However, it was not observed in shbg4 mice, in which hepatic production of plasma SHBG continued to increase after puberty regardless of gender. Serum testosterone and SHBG levels correlated in all sexually mature shbg transgenic mice. Human shbg transcripts were detectable only in testes of shbg11 mice and increased progressively in abundance from 10 days of age until the animal reached sexual maturity at 30 days of age, with appreciable increases occurring well before any changes in serum testosterone concentration. In the kidney, SHBG mRNA levels accumulated earlier in shbg11 than in shbg4 mice, and the expression of both types of transgenes was sexually dimorphic, with much higher SHBG mRNA levels in the kidneys of male mice. As increases in SHBG mRNA in the male kidneys coincided with increases in serum testosterone during sexual maturation, we reasoned that shbg transgene expression is androgen dependent in the kidney. This was confirmed by demonstrating that a decrease in SHBG mRNA abundance in male mouse kidneys after castration could be reversed by 5alpha-dihydrotestosterone treatment. Moreover, exogenous androgen increased human SHBG mRNA levels in the kidneys of female mice. In summary, comparisons of how different human shbg transgenes are expressed in vivo provides information about the positions of potential regulatory sequences that may control the hormonal regulation and tissue-specific expression of this gene during development.

Estradiol induction of cAMP in breast cancer cells is mediated by foetal calf serum (FCS) and sex hormone-binding globulin (SHBG)

Plasma sex hormone-binding globulin (SHBG or SBP), the specific carrier for estradiol and androgens, after binding to its membrane receptor (SHBG-R), causes a significant increase of cAMP in the presence of estradiol, in both breast (MCF-7) and prostate (LNCaP) cancer cells maintained in serum-free medium. On the other hand, it has been proposed that estrogens, in addition to the well-known nuclear receptor pathway, exert their biological effect inducing cAMP, as a consequence of a direct membrane action, in breast cancer and uterine cells. The aim of the present study was to clarify this controversial issue by verifying if the cAMP increase in MCF-7 cells was a direct effect of estradiol, or if it was mediated by FCS proteins, such as bovine sex hormone-binding globulin; and to reevaluate the effect of human SHBG on cAMP induction in the presence of FCS. MCF-7 cells were maintained in DCC-FCS (treated with DCC to remove steroids), in SHBG-FREE/DCC-FCS (treated with DCC and with a specific affinity chromatography to remove bovine sex hormone-binding globulin), or in serum-free medium (SFM). It was observed that estradiol determined a significant time-dependent increase of cAMP only in MCF-7 cells maintained in 10% DCC-FCS. When cells were maintained in 10% SHBG-FREE/DCC-FCS, estradiol had no detectable effect. However, its ability to increase cAMP was observed again after the addition of human SHBG, in doses ranging from 5 to 50 nM. Moreover, in the presence of 10% SHBG-FREE/DCC-FCS, SHBG, even in the absence of estradiol, caused a significant increase of cAMP. In conclusion, the data reported in the present study suggest that the ability of estradiol to induce cAMP in MCF-7 cells is not due to a direct membrane effect of the hormone, but rather it is mediated by FCS. SHBG is one of the serum factors mediating estradiol action. Lastly, it was proven that SHBG triggers the cAMP pathway in MCF-7 cells in a physiologic culture condition and at physiologic concentrations.

HIGH EXERCISE INTENSITY DURING TRAINING INCREASES ANDROGENIC ACTION IN POSTMENOPAUSAL WOMEN

809 Increased androgenic action, due in part to reduced production of sex-hormone binding globulin (SHBG), and visceral adiposity, have been identified as risk factors for impaired glucose tolerance in postmenopausal women, while estrone (E1) from the adipose tissue may facilitate deposition of fat in thighs and buttocks. To investigate the cause of a previously-observed inverse relationship between exercise intensity during 8 mos of training on one hand, and fat loss and a change in a measure of insulin sensitivity, on the other, we hypothesized that these effects in postmenopausal women are due to (1) increased androgenic action (assessed as a ratio of androgens to SHBG), (2) decreased secretion of E1, and (3) selective fat loss in the thigh. Nine healthy, overweight postmenopausal women walked 24 km/week for 30 weeks at speeds between 94 and 138% of their ventilatory threshold. Concentrations of total (TT) and free testosterone (FT), SHBG and E1 in plasma (by RIA) and thigh circumferences and fat-fold thicknesses, were measured at the start and after 13 and 30 weeks of training. After 30 weeks of training, exercise intensity was positively correlated with TT/SHBG (r=0.614,p<0.05) and FT/SHBG (r=0.466,p<0.05) and negatively correlated with plasma E1 level and fat loss in the thigh. These data suggest that high exercise intensity (1) increases androgenic action and (2) reduces plasma E1 concentration, but do not support hypothesis (3) that reduced E1 is associated with fat loss in the thigh. Supported by NIH M01 RR000042.

Xenoestrogen interaction with human sex hormone-binding globulin (hSHBG) 1

This study reports on some environmental chemicals with estrogenic activity (xenoestrogens) and their binding interaction for human plasma sex-hormone binding globulin (hSHBG). The binding affinity constant of these xenoestrogens was measured in equilibrium conditions by solid phase binding assay, and their ability to displace endogenous testosterone and estradiol from hSHBG binding sites was determined with an ammonium sulfate precipitation assay in native plasma from normal men and women. The data showed that some of these xenoestrogens bind hSHBG, with a reversible and competitive binding activity for both [ 3H]testosterone and [ 3H]17β-estradiol and with no apparent decrease in the number of hSHBG binding sites. Their respective binding affinity constants were low, ranging from 0.02 to 7.8.10 5 l. mol −1. However, in native plasma from normal men and women, they were able to dose-dependently increase concentrations of hSHBG-unbound testosterone and/or estradiol. In this study, 4-nonylphenol and 4-tertoctylphenol, two alkylphenols used as surfactants in many commercial products, and bisphenol A and O-hydroxybiphenyl, widely used in the plastics industry, were identified as potent hSHBG-ligands. Additionally, the flavonoid phytoestrogens genistein and naringenin were also identified as hSHBG ligands, whereas their glucoside derivatives, genistin and naringin, had no binding activity for hSHBG. From these data, it is suggested that hSHBG binding may transport some contaminant xenoestrogens into the plasma and modulate their bioavailability to cell tissues. On the other hand, xenoestrogens may also displace endogenous sex steroid hormones from hSHBG binding sites and disrupt the androgen-to-estrogen balance. Whether xenoestrogen SHBG ligands could reach high enough concentrations in the blood to expose humans to any such effect merits further investigation.

Monoclonal antibodies to human sex hormone-binding globulin (SHBG): Characterization and use in a simple enzyme-linked immunosorbent assay (ELISA) of SHBG in plasma

Four monoclonal antibodies to human sex hormone-binding globulin were raised and characterized. Three of the four antibodies recognised different antigenic determinants on SHBG. Two of the distinct antibodies were useful for Western blotting and recognized a major 48 kDa band in human plasma as well as a 46 kDa minor component. Carbohydrate residues do not form part of the antigenic determinants of these two antibodies, although one of these showed increased signal following removal of N-linked oligosaccharides. Some of the antibodies were selected to form a basis of a same-day, non-competitive, enzyme-linked immunosorbent assay (ELISA) for SHBG in plasma. The assay employs a purified IgG2a SHBG monoclonal antibody adsorbed to the wells of a microtitre plate. After blocking any further adsorption to the plate, standards or diluted patient samples were added for a 5-h incubation at room temperature, after which the plate was washed and antibody-bound SHBG was detected with an anti-SHBG IgG1 monoclonal antibody followed by peroxidase-labeled antimouse-IgG1 and o-phenylenediamine substrate. The assay correlated well with an existing 2-day ELISA for SHBG in plasma using polyclonal antibodies and also correlated with a dihydrosterone (DHT) ligand-binding assay. The monoclonal antibody-based ELISA shows excellent performance characteristics and is unaffected by added testosterone or estradiol.

Sex hormone-binding globulin: not only a transport protein. What news is around the corner?

The plasma Sex Hormone-Binding Globulin (SHBG) transports androgens and estradiol in the blood and regulates their bioavailable fraction and access to target cells. The recent advances in the knowledge of its structure and gene expression, and notabily the demonstration of a specific receptor (SHBG-R) located on membranes of sex steroid responsive cells, gave support to the thesis that SHBG has much more sophisticated functions at cell site. In particular, the receptor-mediated action of SHBG, which uses as a second messenger cAMP, has been linked to the effects of androgens and estradiol. It is conceivable that the SHBG/SHBG-R system works as an additional control mechanism which inhibits or amplifies the effects of DHT and estradiol in cells. In the prostate, it has been suggested that the estradiol-activated SHBG/SHBG-R complex cross-talks with the androgen receptor, and is able to activate AR even in the absence of DHT. Of great interest, for its potential clinical applications, is the observation that in estrogen-dependent breast cancer SHBG, through SHBG-R, cAMP and PKA, specifically inhibits the estradiol-induction of cell proliferation. This anti-proliferative, anti-estrogenic effect of human SHBG has not only increased and continues to increase our understanding of the molecular mechanisms involved in the biology of breast cancer, but could also be exploited as a future therapeutic strategy in the managing of estrogen-dependent tumours.

The additionally glycosylated variant of human sex hormone-binding globulin (SHBG) is linked to estrogen-dependence of breast cancer.

Sex Hormone-Binding Globulin (SHBG), the plasma carrier for androgens and estradiol, inhibits the estradiol-induced proliferation of breast cancer cells through its membrane receptor, cAMP, and PKA. In addition, the SHBG membrane receptor is preferentially expressed in estrogen-dependent (ER+/PR+) breast cancers which are also characterized by a lower proliferative rate than tumors negative for the SHBG receptor. A variant SHBG with a point mutation in exon 8, causing an aminoacid substitution (Asp 327-->Asn) and thus, the introduction of an additional N-glycosylation site, has been reported. In this work, the distribution of the SHBG variant was studied in 255 breast cancer patients, 32 benign mammary disease patients, and 120 healthy women. The presence of the SHBG mutation was evaluated with PCR amplification of SHBG exon 8 and Hinf I restriction fragment length polymorphism (RFLP) procedure. This technique allowed us to identify 54 SHBG variants (53 W/v and 1 v/v) in breast cancer patients (21.2%), 5 variants (4 W/v and 1 v/v) in benign mammary disease patients (15.6%), and 14 variants (W/v) in the control group (11.6%). The results of PCR and RFLP were confirmed both by nucleotide sequence of SHBG exon 8 and western blot of the plasma SHBG. No differences in the mean plasma level of the protein were observed in the three populations. The frequency of the SHBG variant was significantly higher in ER+/PR+ tumors and in tumors diagnosed in patients over 50 years of age than in the control group. This observation suggests the existence of a close link between the estrogen-dependence of breast cancer and the additionally glycosylated SHBG, further supporting a critical role of the protein in the neoplasm.

Randomized placebo-controlled trial of an isoflavone supplement and menopausal symptoms in women

Objective To test the hypothesis that increasing the intake of isoflavones by dietary supplementation may produce a therapeutic effect in reducing the incidence and severity of hot flushes in menopausal women.Methods Fifty-one postmenopausal women were randomized to placebo and active (one tablet per day of a 40-mg isoflavone supplement) groups in a cross-over design trial. After a 1-week run-in period, subjects were commenced on a 12-week period of treatment (active or placebo), followed by a 1-month placebo wash-out period, then crossed over to the alternative treatment regimen for a further 14 weeks. Symptom diaries were maintained throughout the trial and at the start and end of treatment. Plasma sex hormone binding globulin (SHBG) assay, full blood count, biochemical profiles, vaginal swabs and vaginal ultrasound scans were performed and isoflavones determined in 24-h urine collections by high-pressure liquid chromatography (HPLC) analysis.Results There was no significant difference between active and placebo groups in the reduction in hot flushes between start and finish time-points. Analysis performed on interim data time-points revealed a substantially greater reduction in flushing in the active group than placebo at 4 and 8 weeks after commencement of treatment, but this was not statistically significant. There were no significant differences between groups for Greene scores or in SHBG levels, hematological or biochemical parameters and vaginal swab or ultrasound findings. The combined values for all subjects, regardless of treatment group, revealed a strong negative correlation between the level of urinary isoflavone excretion and the incidence of hot flushes.Conclusions These data do not indicate a therapeutic benefit from dietary supplementation with isoflavones in women experiencing menopausal symptoms, but do indicate that the apparent placebo effect in many studies of menopausal symptoms may be attributable to dietary sources of isoflavones. The study also demonstrates that 3 months of isoflavone supplementation did not cause adverse events or endometrial changes.

Sex Hormone-Binding Globulin and Breast Cancer Risk

Epidemiologic evidence supports the hypothesis that a woman's cumulative exposure to premenopausal ovarian steroids, estrogen and progesterone, contributes to the lifetime risk of developing breast cancer. A direct relationship between total endogenous levels of plasma estrogens and the incidence of breast carcinoma has not been demonstrated, however, and no substantial breast cancer risk has been consistently associated with postmenopausal estrogen replacement therapy. This paper summarizes the literature regarding the potential role of sex hormone-binding globulin (SHBG) in modifying breast cancer risk. It has been proposed that circulating levels of bio-available estrogens (ie, not bound to SHBG) might more directly reflect the risk for development of disease. Several studies have detected an association between a decreased percentage of estradiol bound to SHBG, a small increase in free estradiol, and a higher incidence of breast cancer in postmenopausal women. The physiologic significance of this increase in free estradiol, however, especially considered in the context of the relatively lower postmenopausal levels of total estradiol, is not clear. In addition to its carrier function for circulating estradiol, SHBG specifically binds to cells of steroid-responsive tissues, including breast tissue, and decreases the proliferation of some breast cancer cells through activation of cyclic adenosine monophosphate. This suggests that a decrease in the binding capacity of SHBG could potentially diminish SHBG binding to breast cell membranes and increase the rate of cellular proliferation. Oralestrogens increase plasma SHBG levels in postmenopausal women and this increase might mitigate the effect of estrogen replacement therapy on breast cancer epidemiology.