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BACKGROUND Emerging evidence indicates that testosterone (T), and not dihydrotestosterone (DHT), is the most relevant androgen that promotes carcinogenesis in the prostate. Steroid 5-alpha reductase type II (SRD5A2) catalyzes the irreversible conversion of T to DHT in male reproductive organs. Because the SRD5A2 gene is highly polymorphic at codon 89, two SRD5A2 isoforms are expressed that differ in Km and Vmax values. The more common and rapid catalytic isoform contains a valine residue at position 89; the slower-catalytic variant contains leucine at this position. METHODS Thirty-three men with early onset prostate cancer (PCa) were genotyped for the SRD5A2 V89L substitution and other polymorphisms in genes encoding receptors or enzymes that play important roles in pathways of steroid metabolism to ascertain if they were associated with standard clinical measures of disease progression at the time of diagnosis. RESULTS The expression of at least one SRD5A2 leucine allele in young men with PCa was associated with more significant disease at the time of presentation, as was defined by pretreatment PSA level, clinical staging and Gleason score when compared with affected subjects harboring the more common SRD5A2 valine variant. A dosage effect of a single leucine allele was evident in heterozygotes, as values of their clinical and pathological variables were consistently situated between the extremes of the homozygous V or L phenotypes. CONCLUSION The SRD5A2 leucine isoform appears to be acting in a dose-dependent manner as a significant disease-modifying factor in young men diagnosed with PCa. Prostate © 2008 Wiley-Liss, Inc.

Tocopherylquinone (TQ), the oxidation product of alpha-tocopherol (AT), is a bioactive molecule with distinct properties from AT. In this study, AT and TQ are investigated for their comparative effects on growth and androgenic activity in prostate cancer cells. TQ potently inhibited the growth of androgen-responsive prostate cancer cell lines (e.g., LAPC4 and LNCaP cells), whereas the growth of androgen-independent prostate cancer cells (e.g., DU145 cells) was not affected by TQ. Due to the growth inhibitory effects induced by TQ on androgen-responsive cells, the anti-androgenic properties of TQ were examined. TQ inhibited the androgen-induced activation of an androgen-responsive reporter and inhibited the release of prostate specific antigen from LNCaP cells. TQ pretreatment was also found to inhibit AR activation as measured using the Multifunctional Androgen Receptor Screening assay. Furthermore, TQ decreased androgen-responsive gene expression, including TM4SF1, KLK2, and PSA over 5-fold, whereas AT did not affect the expression of androgen-responsive genes. Of importance, the antiandrogenic effects of TQ on prostate cancer cells were found to result from androgen receptor protein down-regulation produced by TQ that was not observed with AT treatment. Moreover, none of the androgenic endpoints assessed were affected by AT. The down-regulation of androgen receptor protein by TQ was abrogated by co-treatment with antioxidants. Overall, the biological actions of TQ were found to be distinct from AT, where TQ was found to be a potent inhibitor of cell growth and androgenic activity in androgen-responsive prostate cancer cells.

The purpose of this study was to determine the diagnostic sensitivity, specificity, predictive value and overall efficiency of serum cross-linked N-telopeptides of bone collagen (NTx) and aminoterminal procollagen extension propeptide (PINP) measurements for identifying women with decreased spine, femoral neck and total body bone mineral density (BMD). Serum NTx and PINP levels and dual X-ray absorptiometry were performed on 196 healthy elderly women, aged 60-90 years. Twelve women were classified as having decreased BMD on the basis of regional and total skeletal densitometric values that were 1.5 to 2.5 standard deviations (SD) below the respective, age-stratified means and were compared with 184 women with BMD values greater than 1.5 SD below the mean. The results of receiver operating characteristic analysis revealed that a cutoff level of more than 15.0 nmol BCE/L for serum NTx, as measured by the Osteomark assay (Ostex International, Seattle WA USA) was associated with a 100% sensitivity and 70% specificity rate for identifying postmenopausal women with low BMD. The positive likelihood ratio was 3.3 and the negative predictive value was 1.0 using the 15.0 nmol decision level for NTx. The overall diagnostic efficiency of a single NTx measurement for identifying women with low BMD was 89%. A cutoff level of >45.0 microg/L for PINP as measured by the Orion Diagnostica RIA assay (Espoo, Finland) had a diagnostic sensitivity of 83% and specificity of 64% for identifying women with decreased BMD. The positive likelihood ratio was 2.3. the negative predictive value 0.98 and the overall diagnostic efficiency 73% using the 45.0 microg/L decision level for PINP. These results warrant future studies using larger populations that are inclusive of more women with low bone mineral density.

Serum levels of aminoterminal extension propeptides (PINP), the carboxyterminal telopeptide (ICTP), and the cross-linked N-telopeptides (NTx) of type I collagen were determined in 78 healthy, elderly men aged 76 ± 5 years in 1993 and 1996 and compared with bone mineral density (BMD) measurements of their lumbar spine, femoral neck, and total body regions made using dual X-ray absorptiometry. Compared with 11 men who had normal lumbar spine (SBMD) and femoral neck BMD (NBMD), 13 of the subjects with SBMD and NBMD classified as osteopenic by t-score criterion had higher mean serum levels of PINP and alkaline phosphatase activity, but these increases were not statistically significant at the 95% confidence interval. In osteopenic men, a correlation between SBMD and NTx was detected (r = −0.66, p = 0.01). Within the entire population, the serum NTx level correlated with NBMD (r = −0.26, p < 0.05) and PINP (r = +0.63, p < 0.0001), and the change in the circulating concentration of PINP over the 3 year interval correlated with the magnitude of change in total body BMD (r = −0.28, p = 0.02), NBMD (r = −0.24, p = 0.05), and SBMD (r = −0.36, p = 0.03) as well as with the change in serum NTx levels (r = 0.43, p < 0.001). The change in the circulating ICTP level was also related to the change in NBMD (r = −0.24, p = 0.01). Together, weight and the serum PINP level accounted for 25% of total body BMD variance in elderly men. These results indicate that larger populations of men and women should be screened over longer time intervals to explore the value of serial measurement of serum collagen metabolites in predicting bone loss in the spine and hip.

In a previous study of rachitic children in Jos, Nigeria we concluded that inadequate dietary intake of calcium was the primary contributing factor to the development of their rickets. The objective of the present study was to determine the effect of calcium supplementation in 10 children with radiographically and biochemically proven rickets from the same geographical area. Rachitic children were provided with calcium supplements of 1000 mg/day for a period of 3 months. Serum and urine samples were obtained at baseline and at 24 hours, 1 week, 4 weeks, and 12 weeks after initiation of supplementation. Serum calcium, phosphorus, alkaline phosphatase, intact parathyroid hormone, 25-hydroxyvitamin D, and 1,25-dihydroxyvitamin D were measured at each time point. Dietary recalls obtained at two separate times were used to estimate usual daily intakes of calcium and phosphorus. Ten non-rachitic age-matched controls from the same geographical area were recruited for comparison. Nine of 10 rachitic subjects had radiographic evidence of healing after 3 months of calcium therapy. Although serum calcium concentrations returned to control levels, other biochemical data indicated that the rickets of these subjects may have been multifactorial in aetiology, pointing to a possible defect in the synthesis of 25-hydroxyvitamin D.

Serum levels of cross-linked N-telopeptides (NTx) of bone collagen, alkaline phosphatase (ALP), and intact parathyroid hormone (PTH) were determined in 64 premenopausal (PRM) and 86 postmenopausal (PSM) women living in northern Nigeria. Serum NTx values were correlated with ALP activity (r = 0.31–0.58, P < 0.01) and PTH (0.32–0.35, P < 0.01)) in all of the subjects studied, and were also related to age (−0.47, P < 0.001) and body mass index (−0.45, P < 0.001) in PRM women. Menopause had the effect of increasing the circulating concentrations of NTx and ALP activity by 15% (P= 0.001) and 11% (P= 0.02), respectively; however, serum levels of PTH were not different between these two groups of women. Compared with Caucasian counterparts matched for age and body mass index, PSM Nigerian women had significantly increased circulating concentrations of NTx (21.7 versus 16.2 nmol BCE/liter, P= 0.01) and demonstrated a trend towards higher ALP activities and PTH levels. These results indicate that (1) discrete reference intervals should be defined for biochemical markers of bone metabolism in African populations, (2) Nigerian women have relatively higher rates of bone turnover, and (3) further investigation of the implications of increased serum NTx should be undertaken using physical methods such as dual X-ray absorptiometry (DXA) and bone ultrasound attenuation.

We measured the levels of the pyridinoline crosslinked carboxyterminal telopeptide of type I collagen (ICTP) and osteocalcin (OC) in the serum of 12 rachitic and 27 healthy Nigerian children, and compared the performance of these relatively new markers of bone metabolism with established laboratory parameters of skeletal disease. Active rickets was diagnosed on the basis of clinical and biochemical criteria. Serum calcium and phosphorus concentration and alkaline phosphatase activity were determined using clinically accepted methods. Radioimmunoassay was performed to quantify parathyroid hormone, 1-,25-dihydroxyvitamin D D, OC, and ICTP. The rachitic children had statistically significant serum elevations of ICTP and osteocalcin as compared with age- and sex-matched controls. Serum levels of ICTP correlated with alkaline phosphate activity. As a marker of abnormal bone metabolism, ICTP performs at least as well as alkaline phosphate. ICTP and OC are valuable additions to the growing repertoire of bone markers.

Accumulation of mitochondrial DNA deletions and the resultant impaired oxidative phosphorylation may play a pathogenic role in the mediation of age-related sarcopenia.Twenty four participants of the New Mexico Aging Process Study were classified as normal lean (n = 15) or sarcopenic (n = 9) based on body composition determined by Dual Energy x-ray Absorptiometry. Complex I and Complex IV activities were measured in the skeletal muscle samples obtained from gastrocnemius muscle. A two-stage nested polymerase chain reaction strategy was used to identify the mitochondrial DNA deletions in the entire mitochondrial genome in the skeletal muscle samples.Although Complex I activity was not significantly different (5.5 +/- 0.9 vs. 4.6 +/- 0.7 mU/mg protein, P0.05), Complex IV activity was higher in sarcopenic subjects (1.4 +/- 0.3 vs. 1.0 +/- 0.1 mU/mg protein, P0.05). Mitochondrial DNA deletions were mostly located in the region of Complex I and spanned from nicotinamide adenine dinucleotide dehydrogenase 1 to nicotinamide adenine dinucleotide dehydrogenase 6. Deletions in the 8,577-10,407 bp and 10,233-11,249 bp regions were associated with a significant decrease in Complex I activity (P0.05 and P = 0.02, respectively). Total cumulative deletion, defined as the sum of individual length of deletions in a subject, was comparable in subjects with and without sarcopenia (1760 +/- 726 vs. 1782 +/- 888 bp, P0.05). The magnitude of mitochondrial DNA deletion, however, correlated positively with lean body mass (r = 0.43, P0.05).Thus, mitochondrial DNA deletions are common in elderly subjects and are negatively related to Complex I activity. The positive association between mitochondrial DNA deletions and lean body mass needs to be confirmed by studies in a larger study population.

Bone marrow thymocytes in part mediate the bone-preserving effects of estrogen by decreasing their production of osteoclast growth factors such as interleukin-1 and -6 and tumor necrosis factor alpha in the presence of physiological amounts of estradiol. Although several in vitro studies implicate the T-lymphocyte as a candidate mediator of estrogen signaling in the skeleton, whether these cells or any lymphocytes ordinarily express one or both nuclear estrogen receptors was previously unresolved. The purpose of our investigation was therefore to ascertain, by using real-time PCR, immmunoblotting, and cytometric techniques, if any of the nuclear estrogen receptors could be detected in normal peripheral blood mononuclear cells (PBMNC) collected from healthy volunteers. The results of immunoblotting experiments revealed that both estrogen receptor alpha (ESR1) and beta (ESR2) proteins are expressed in nuclei, but not in the cytoplasm of PBMNC harvested from all of the 15 healthy male and female volunteers (aged 23-50 years) we tested. PBMNCs contained mRNA coding for the two major full-length isoforms of ESR2 and the expression of ESR2 protein was localized within a lymphocyte subpopulation by cytometric analysis. Our data provide further evidence that lymphocytes and monocytes are responsive to estrogen and underscore its importance in modulating the immune response, as well as the vascular and skeletal health of men and women.

Significant associations between the lengths of a highly polymorphic dinucleotide (CA) repeat located within the human estrogen receptor beta (ESR2) gene on chromosome 14, bone mineral density (BMD) and androgen levels have been reported previously in premenopausal women. We measured the size of this microsatellite repeat in 226 healthy women (60–98 years). After adjustment for age, body mass index, hormone replacement status, and other variables known to influence BMD, women with

Although primarily secreted by adipose cells, leptin, a polypeptide hormone that influences body weight, satiety and lipid metabolism, and its receptor are also expressed in human osteoblasts. Leptin plays a role in the central, hypothalamic modulation of bone formation, as well as locally within the skeleton by enhancing differentiation of bone marrow stroma into osteoblasts and inhibiting its differentiation into osteoclasts and adipocytes. The purpose of this investigation was to compare serum leptin values in 100 postmenopausal women (age 62-97) and 31 men (age 72-92) to bone mineral density (BMD) measurements made by dual X-ray absorptiometry and additionally to biochemical markers of bone resorption and formation, including crosslinked collagen N-telopeptides (NTx), aminoterminal extension procollagen propeptides (PINP) and bone-specific alkaline phosphatase (bAP). The circulating level of leptin directly correlated with body mass index (BMI) (r=0.61-0.78, P

Objectives: The purpose of this investigation was to quantify the biologic, day-to-day variability and critical differences in serum levels of crosslinked collagen N-telopeptides (NTx), procollagen aminoterminal extension propeptides (PINP) and bone specific alkaline phosphatase (bAP) in healthy women. Design and methods: Seven blood samples were collected from12 pre- and 15 postmenopausal women over 4 to 6 months. NTx, PINP and bAP levels were determined utilizing enzyme- and radioimmunoassay techniques. Results: The within-subject coefficient of variation (C.V.) in serum bAP, NTx and PINP levels was 7.1, 10.6 and 12.4% respectively. These variances did not differ significantly among premenopausal women when compared with postmenopausal subjects. Combining terms for analytical and biologic variability revealed that a critical difference between 2 successive serial measurements is 24% for bAP, 34% for NTx and 38% for PINP. Conclusion: Circulating levels of NTx, PINP and bAP are stable over time periods of several months, allowing for the determination of significant changes in skeletal metabolism of women.

Ultrasound analysis of the calcaneus and serum markers of bone turnover were used to examine the bone status of healthy Nigerian women who reside in an area of the world where dietary calcium intake is generally low and estrogen replacement therapy is not widely available. A total of 218 women (108 premenopausal and 110 postmenopausal) between the ages of 16 and 95 years were enrolled in the study. Broadband ultrasound attenuation (BUA) and speed of sound velocity (SOS) were measured and used to calculate the stiffness index (SI) of the calcaneus. In this cross-sectional study, the Nigerian women exhibited a marked age-dependent decline in SI that was defined by the regression equation SI = 105.9 - 6.62E-3 x Age2. SI was significantly correlated with age (r = -0.41, P0.001) and with serum NTx concentrations (r = -0.26, P0.001), but not with serum levels of bone-specific alkaline phosphatase (BSAP). Years since menopause was also significantly correlated with SI (r = 0.40, P0.001). A significant increase in serum NTx concentration occurred at least a decade before a significant decline in SI was evident. In the total study group, 24% of the women had T-scores indicative of osteopenia and 9% had T-scores indicative of osteoporosis, based on US reference data. Although the reported current incidence of fracture is low in women in sub-Saharan West Africa, these data show that after menopause Nigerian women have a decline in bone quality and increase in bone turnover similar to North American Caucasian women.

The aim of the study was to explore the relationship between protein nutritional status and the development of rickets in children living in northern Nigeria. The diagnosis of rickets in 16 children between the ages of 10 months and 7 years was confirmed using established, and recently developed clinical and biochemical parameters. Twenty-seven children devoid of skeletal stigmata were age- and sex-matched to the rachitic patients. A battery of clinical laboratory and anthropometric measurements designed to assess calcium homeostasis, skeletal growth, the extent of bone remodeling or resorption, and protein nutritional status were performed on all subjects. Our central finding was that although the rachitic children were moderately malnourished, their protein nutritional status was significantly better as measured by the serum prealbumin concentration (15.4 v. 12.5 mg/dl, P = 0.0012) when compared with the severely malnourished children who were devoid of any indication of rickets. This may be due, in part, to the fact that actively growing children are more likely to develop rickets than are children whose linear growth is impeded. Unexpectedly, we found that the mean concentrations of serum 1,25-dihydroxyvitamin D in both the rachitic and control group were higher than any values for the active vitamin D metabolite previously reported in the literature.

Purpose Free cortisol is released into the serum compartment where it is in equilibrium with serum binding proteins, corticosteroid-binding globulin (CBG), and albumin. The distribution of serum cortisol among free, CBG-bound, and albumin-bound compartments is influenced by association and disassociation rate constants for CBG and albumin, the number of unoccupied protein binding sites, and concentrations of CBG and albumin. Free cortisol in serum or plasma is difficult to assay; thus, salivary cortisol has been proposed as an alternative measure of free cortisol. In this study, we sought to assess and compare numerical methods, using both quadratic (Coolens et al, 1987) and cubic (Keenan et al, 2004) equations to calculate serum free cortisol and compare these findings with salivary cortisol in healthy elderly subjects. Methods Serum and saliva samples were collected from 45 healthy, elderly subjects at 0700 h as part of the New Mexico Aging study (UNM HRRC 01-023). The salivary cortisol and total cortisol were measured using radioimmunoassay and ultrasensitive chemiluminescence, respectively. Serum CBG was measured by radioimmunoassay and serum albumin was measured using a bromcresol green method (UNM RAC 51). The cubic equation implied by Keenan et al and the quadratic equation described by Coolens et al were used to calculate free cortisol using our measured values for total cortisol, total CBG, and total albumin. Calculated free cortisol, total cortisol, and CBG concentration were then compared to salivary cortisol using Pearson9s and Spearman9s correlations. Results There was a significant linear correlation between salivary cortisol and free cortisol calculated using the cubic equation (r = .377, p = .033) and Coolen9s quadratic equation (r = .416, p = .018). In addition, we noted a linear correlation between CBG concentration and total cortisol (r = .441, p = .0049). Conclusion Our data indicate that the free cortisol calculated using either quadratic or cubic equations is correlated significantly with salivary cortisol but that other variables appear to influence measured salivary cortisol concentration. We did not observe the previously reported correlation between total and salivary cortisol, which suggests that aging may influence the relative distribution of cortisol in serum or salivary compartments.