The primary goal of this project is to determine whether the effect of intake of vitamin D on fractures depends on the effect of calcium and vitamin D on bone turnover markers and polymorphisms related to vitamin D and calcium actions. Taking advantage of the opportunity afforded by the completion of the baseline 25(OH) vitamin D3 approved by the Steering Committee in May 2005, additional analyses can also explore the interaction of baseline 25(OH) vitamin D on the turnover markers. To test the hypotheses that change in vitamin D status contributes to reduction of fractures and to explore a potential threshold effect, we propose to obtain baseline serum and DNA of women in the Calcium and Vitamin D Trial (CaD) of the Women’s Health Initiative (WHI) and longitudinal measures obtained from women who had repeated sampling as part of the 6% blood subsample. We propose to measure serum 25-OH vitamin D (year 3-after 2 years CaD randomization in women in the 6% subsample) and markers of bone turnover, bone specific alkaline phosphatase (BSAP), aminoterminal propeptide of type I procollagen (PINP), C-telopeptides of collagen cross-links (CTx), osteoprotegerin (OPG), receptor activator nuclear factor - ĸB ligand (RANKL); and polymorphisms of genes related to vitamin D and calcium actions. Since these bone turnover markers have already been measured in women in Hormone Therapy Trials as part on the fracture biomarker proposal for those CTs, we will require these to be measured for the remainder of the women in the CaD trial who were not in one of the HT trials.
Osteoporosis is a major public health problem in the United States, a disorder that increases with age especially in women. It is characterized by low bone mass and microarchitectural changes that lead to skeletal fragility and fractures. The risk of osteoporotic fractures rises exponentially with age, resulting in 1.5 million cases of fractures annually in the United States; a 50 year-old woman has nearly a 50% risk of an osteoporotic fracture during her lifetime. This disease poses a major economic burden to our society. Nutritional, lifestyle, hormonal, and genetic factors contribute to the risk of fractures.
Osteoporosis results from an imbalance between bone resorption and bone formation. The development of markers of bone turnover such as the serum level of C-telopeptides of collagen cross-links, which is an index of bone resorption, and the concentrations of serum osteocalcin and bone-specific alkaline phosphatase, which are bone formation markers, make it possible to examine bone remodeling noninvasively.
It has previously been described that women with acute hip fractures, some of whom had vitamin D deficiency, had lower serum osteocalcin levels with an increase in urinary N-telopeptide levels in comparison women admitted for elective orthopedic surgery (1) Data show that biochemical markers can predict the risk of fracture in older patients and are elevated in the setting of a fracture. Another formation marker, aminoterminal propeptide of type I procollagen (PINP) guides assembly of the collagen triple helix. This marker has been previously used in other WHI studies. Since PINP has already been measured in some women of the CaD trial, we will study PINP and BSAP as formation makers and serum CTx as a resorption marker for purposes of this study. Recent data indicate that the OPG/RANKL/RANK signaling pathway is the principal regulator of osteoclastogenesis. The binding of the osteoblast lineage cell surface protein, RANKL, to RANK, a receptor present on mature osteoclasts and precursors, stimulates osteoclast differentiation and activates bone resorption. OPG is secreted by osteoblasts and inhibits osteoclastogenesis by binding to RANKL, blocking its activity, although the significance of circulating versus locally generated OPG levels is not known. Because an increase in RANKL levels leads to bone resorption, an action blocked by OPG, we propose to measure circulating levels of OPG and soluble RANKL (regulators of osteoclastogenesis).
Osteoporosis is a multifactorial disease characterized by decrease in bone mass and loss of microarchitechtural structure of bone leading to increased risk of fractures. Though lifestyle, diet, physical activity etc. play an important role as risk factors for the development of the disease, a genetic component is also known to be a strong risk factor contributing to higher susceptibility of the disease. Several polymorphisms are known to be implicated in the pathogenesis of osteoporosis and hence fragility fractures. We plan to study polymorphisms related to vitamin D metabolism and the calcium sensing receptor
There have been contradictory results showing association between polymorphisms of the vitamin-D-receptor (VDR) and bone mineral density BMD) in pre and postmenopausal women (2). A German study examined the association of bone density, fracture predictively and biochemical markers of bone turnover with VDR polymorphism. They reported that bone density at the axial skeleton is associated with the VDR allele polymorphism and that there is an influence on bone turn over and fracture rate in the German population (3). Contrary to these results, another study conducted among postmenopausal women reported that VDR genotypes do not predict bone turnover or rate of postmenopausal bone loss. Hence, they concluded that determination of VDR genotypes might not be a useful clinical test for the risk of osteoporosis (4). Similarly, there have been conflicting reports on the calcium sensing receptor (CASR) gene polymorphism and BMD and fractures. A Japanese study investigated the association between the CA-repeat polymorphism at the human CASR gene locus and BMD of radial bone in postmenopausal women. Women who had an A3 allele (228 bp, containing 18 repeats of CA) had significantly lower adjusted BMD than participants who did not carry this allele, thus suggesting that genetic variation at the CASR gene locus is associated with determinants of BMD in postmenopausal women (7). However, another polymorphism of the CASR gene, A986S has been studied by researchers and has failed to predict serum calcium level, BMD, or fracture rate in elderly women (5). A study conducted in Italian postmenopausal women also reported that A986S CASR polymorphism does not have a role in BMD and prevalence of fragility fractures (6). In the proposed studies we will determine whether these and other polymorphisms related to vitamin D and calcium sensing are associated with increased risk of fractures
We will compare the baseline measurements of serum 25-OH vitamin D (obtained from the SC-approved 25OH) biomarker CCA) and biomarkers of bone turnover in women with and without fractures. BSAP, PINP, C-Tx, and OPG will serve as primary bone turnover markers. We will study RANKL as a secondary outcome measure due to conflicting results with the assay. This hypothesis will be tested in a randomly selected subset of 800 women (400 women with fractures and 400 controls). We will use the radioimmunoassay (RIA) (Diasorin) to measure the 25 hydroxy vitamin D levels in 50µL of serum sample. Serum BSAP (Ostase immunoradiometric assay, Beckman Coulter, CA) will be measured in 0.2ml serum. The interassay and intra-assay coefficients of variation (CV%) for the assay ranged from 2.3% to 12.1% as previously determined. PINP (Diasorin, Stillwater, NM) will be measured in 50µL serum. RANKL and OPG will be measured in 100μl (0.1ml) by enzyme immunoassay (enzyme immunoassay, ALPCO, Windham, NH). The CVs have been reported to be less than 8% for sRANKL and 6% for OPG previously.
As a secondary end point, we will study the association between change in 25-OH D levels from baseline to follow-up at 3 years (AV3) in a 6% sub-sample and risk of fracture. We will include 197 cases of total fractures for this analysis. However, due to lack of power we will not study the association by fracture type.