Heritability of blood concentrations of sex-steroids in relation to body composition in young adult male siblings.

OBJECTIVE: Sex steroid concentrations in men are related to body composition and both are determined by genetic and environmental factors. This study investigates heritability estimates of sex steroid serum concentrations and body composition as well as the genetic and environmental components of their interrelation. PATIENTS: Six hundred and seventy-four men (25-45 years) were included in this study with 274 independent pairs of brothers. MEASUREMENTS: Body composition and regional fat mass estimates were determined using dual-energy X-ray absorptiometry. Serum testosterone (T), SHBG, oestradiol (E(2)) and LH levels were determined by immunoassay; free T and E(2) levels were calculated. RESULTS: Both sex steroid hormone concentrations and indices of body composition exhibited significant heritability estimates. Among sex steroid hormones, T had the highest heritability (h(2) = 0.65), followed by free T (h(2) = 0.54). A heritability of 0.73 was observed for SHBG; a heritability estimate of 0.83 was obtained for body weight. Significant genetic correlations were found between whole body fat mass and serum T (rho(G) = -0.46), free T (rho(G) = -0.27) and SHBG (rho(G) = -0.48) concentrations. No genetic relationship was observed between total (F) E(2) or LH concentrations, respectively, and body composition. CONCLUSION: Both sex steroid serum levels and body composition are under strong genetic control. Their interrelation is in part underlied by a genetic correlation, indicative of the action of shared genes.

Are serum leptin and the Gln223Arg polymorphism of the leptin receptor determinants of bone homeostasis in elderly men?

OBJECTIVE: Across studies it has been suggested that leptin intervenes as a regulator of bone metabolism. This study assesses the contribution in elderly men of leptin and the Gln223Arg leptin receptor gene (LEPR) polymorphism to the variation in bone homeostasis, in relation to body composition and free estradiol as major confounders. DESIGN: We performed cross-sectional (n = 270) and longitudinal (mean follow-up 3.4 years, n = 214) evaluations in elderly men. METHODS: Serum leptin, LEPR genotype, baseline bone mineral density (BMD), longitudinal BMD changes at the hip and forearm, and biochemical markers of bone turnover were determined. RESULTS: In cross-sectional analyses absolute fat mass was the index of body composition most strongly associated with leptin (r = 0.74; P < 0.001). LEPR genotypes and serum leptin were not associated. Serum bone-specific alkaline phosphatase (S-BAP) was associated with LEPR genotypes (P = 0.05) and urinary C-terminal telopeptides of type I collagen (U-CTX) were associated with leptin levels (P = 0.03), independently from age, fat mass and free estradiol. Baseline BMD at the hip and forearm was neither associated with leptin nor with LEPR genotypes. Prospectively assessed BMD loss was not associated with serum leptin at the hip, whereas BMD loss was positively associated with leptin at the forearm (P = 0.01), independently from age, fat mass and free estradiol. Longitudinal changes in hip or forearm BMD were not associated with LEPR genotypes. CONCLUSION: The findings suggest a possible role for leptin as determinant of bone homeostasis in elderly men, but with only modest impact independently from body composition and free estradiol.

Missense mutations in LRP5 are not a common cause of idiopathic osteoporosis in adult men.

UNLABELLED: We studied whether the LRP5 gene contributes to the clinical phenotype of IO in men. Mutation analysis in 66 IO men revealed a range of sequence variants, of which two missense variants were shown to be of functional relevance. INTRODUCTION: Mutations in the LDL receptor-related protein 5 (LRP5) gene have been associated with extreme bone phenotypes, which makes LRP5 a plausible candidate gene for idiopathic osteoporosis (IO). MATERIALS AND METHODS: In 66 men with IO, all 23 exons and exon-intron boundaries of the LRP5 gene were screened for mutations, and functional analyses were performed for those that were putatively involved in the phenotype. RESULTS: Mutation analysis in the IO probands revealed five missense mutations, of which 1067C>T (S356L), 1364C>T (S455L), and 4609G>A (A1537T) were of potential functional significance because they were located in highly conserved regions of LRP5 and not found in a control panel. Segregation analysis in the respective families could not exclude their possible causality for IO. Furthermore, functional analyses clearly showed an inhibitory effect of mutations 1067C>T and 1364C>T on Wnt signal transduction. These effects are most likely caused by impaired LRP5 synthesis in the case of 1067C>T and failure of protein trafficking to the cell surface for 1364C>T. CONCLUSIONS: For 2 of 66 IO probands, a mutation in the LRP5 gene with proven functionality was found. The findings indicate that carrying an LRP5 mutation is a risk factor for IO, but that overall, IO in men is infrequently underlied by such a mutation.

Comparative assessment in young and elderly men of the gonadotropin response to aromatase inhibition.

CONTEXT: Aging in men is associated with a decline in serum testosterone (T) levels. OBJECTIVE: Our objective was to assess whether decreased T in aging might result from increased estradiol (E2) negative feedback on gonadotropin secretion. DESIGN AND SETTING: We conducted a comparative intervention study (2004) in the Outpatient Endocrinology Clinic, Ghent University Hospital. PARTICIPANTS: Participants included healthy young and elderly men (n = 10 vs. 10). INTERVENTIONS: We used placebo and letrozole (2.5 mg/d) for 28 d, separated by 2 wk washout. MAIN OUTCOME MEASURES: We assessed changes in serum levels of free E2, LH, and FSH, free T, SHBG, and gonadotropins response to an i.v. 2.5-microg GnRH bolus. RESULTS: As assessed after 28 d of treatment, letrozole lowered E2 by 46% in the young men (P = 0.002) and 62% in the elderly men (P < 0.001). In both age groups, letrozole, but not placebo, significantly increased LH levels (339 and 323% in the young and the elderly, respectively) and T (146 and 99%, respectively) (P value of young vs. elderly was not significant). Under letrozole, peak LH response to GnRH was 152 and 52% increase from baseline in young and older men, respectively (P = 0.01). CONCLUSIONS: Aromatase inhibition markedly increased basal LH and T levels and the LH response to GnRH in both young and elderly men. The observation of similar to greater LH responses in the young compared with the elderly does not support the hypothesis that increased restraining of LH secretion by endogenous estrogens is instrumental in age-related decline of Leydig cell function.

Screening of clostebol and its metabolites in bovine urine with ELISA and comparison with GC-MS results in an interlaboratory study.

An extraction procedure for clostebol metabolites in urine is developed including enzymatic hydrolysis of conjugated metabolites with Helix pomatia juice (SHP) and solid-phase extraction (SPE) with further cleanup of sample extracts. For the enzymatic deconjugation step, variables such as buffer pH, amount of enzyme, incubation time, and temperature are examined. For the SPE step, different wash solutions and combinations with subsequent liquid-liquid extractions are examined. Incurred bovine urine samples, obtained through oral and intramuscular administration of clostebol acetate to animals, are used to test the performance of the developed method. In addition to the optimization of the sample pretreatment procedure, an interlaboratory study for the analysis of the incurred urine samples with ELISA and GC-MS is performed and good agreements are observed.

Rapid and sensitive screening of sulfamethazine in porcine urine with an enzyme-linked immunosorbent assay and a field-portable immunofiltration assay.

An enzyme-linked immunosorbent assay (ELISA) and an enzyme-linked immunofiltration assay (ELIFA) were developed for the screening of sulfamethazine (SMZ) in porcine urine. Incurred urine samples were measured by ELISA with a working range of 0 to 10 ng of SMZ per ml. The assay showed good accuracy and precision, with recoveries above 99.8% and intra- and interassay coefficients of variation (CVs) ranging from 2.6 to 5.6% and from 5.9 to 12.7%, respectively. Good agreement was observed when the results of the immunoassay were compared with those of liquid chromatography/tandem mass spectrometry analysis. For the ELIFA, a nylon membrane is placed on top of an absorbent material and covalently coated with rabbit anti-rat immunoglobulins. Free binding sites are blocked, and monoclonal anti-SMZ antibodies, SMZ standard or urine, and SMZ-horse radish peroxidase conjugate are subsequently dropped onto the membrane. During the assay, the reactants are drawn through the membrane because of its close contact with the absorbent pad. Finally, a substrate solution is added for blue color development. The blue spot produced can be visually evaluated or instrumentally measured (numeric deltaE*ab value), and the intensity of its color is inversely proportional to the analyte concentration. When a blue dot appears on the membrane, even if its color is less intense than that of the negative control, the sample is considered "negative," i.e., it is thought to contain a concentration of SMZ that is below the visual detection limit. If no color appears on the test membrane, the sample is considered "positive," i.e., it is thought to contain a concentration of SMZ that is equal to or above the visual detection limit. Validation of the assay showed good inter- and intra-assay precision (CV < 10%). Because samples can be analyzed after a simple dilution in <30 min with this assay format, it has strong potential for application in the field.

Influence of Helix pomatia enzyme preparations on the oxidative conversion of some clostebol acetate metabolites in urine.

Clostebol acetate (4-chloro-testosterone acetate) is an anabolic steroid used for fattening purposes in cattle breeding. To safeguard public health, its use has been prohibited by the European Commission since 1986. Screening for its urinary metabolites is therefore an important tool for the control of possible violations. Because those metabolites appear conjugated to glucuronic acid or sulfate, deconjugation prior to analysis is necessary. This work describes the variability in results seen with the use of various commercial preparations of Helix pomatia (SHP) for enzymatic hydrolysis of the conjugates. A simultaneous oxidative side reaction was observed, converting metabolites with a 3-OH-4-ene structure into a 3-oxo-4-ene structure. This was not observed when samples were incubated without enzyme or in the presence of heat-inactivated SHP. GC-MS analysis revealed oxidation of some metabolites of clostebol acetate, 4-chloro-4-androsten-3alpha-ol-17-one and 4-chloro-4-androsten-3alpha,17beta-diol, changing them into other metabolites, 4-chloro-4-androsten-3,1 7-dione and clostebol (4-chloro-testosterone), respectively. Based on the difference in cross-reactivities of the antibodies for these metabolites, comparative analysis in enzyme immunoassay, following enzymatic hydrolysis, confirmed this transformation. This oxidative conversion phenomenon could be of great importance when considering the choice or target analytes for screening bovine urine.