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    Measuring inflammatory marker levels to determine risk of bone loss and fractures in older women

    April 20, 2013
    Mlo Generic Mlo Logo

    Elevated levels of pro-inflammatory markers are associated with an increased risk of a number of chronic conditions1-6 and death.7 Furthermore, there is evidence that high levels of inflammatory markers may contribute to faster rates of bone loss.8-10 More recently, we have demonstrated that greater inflammatory burden (measured primarily using cytokines and their soluble receptors) is associated with a greater fracture risk.11,12 Additionally, studies have found that high levels of high sensitivity C-reactive protein (hs-CRP), a generic marker of systemic inflammation, also predict incident fractures.13-16

    CE CONTINUING EDUCATION

    To earn CEUs, see test online at www.mlo-online.com/ce.
    LEARNING OBJECTIVES
    Upon completion of this article, the reader will be able to:

    1. Identify risk factors associated with women’s health.
    2. Identify the use of inflammatory markers to determine risk of bone disease.
    3. Describe the design and findings of the presented research studies for inflammatory markers.
    4. Identify the current and prospective methods for cervical cancer screening.

    Researchers have identified evidence of at least two biological mechanisms that may explain this increased bone loss and fracture risk among those with high levels of inflammatory markers.17-19 First, cytokines bind to mesenchymal stem cells and increase the expression of receptor-activator NF-κß ligand (RANKL) and macrophage-colony stimulating factor (M-CSF) and decrease osteoprotegerin production, which effectively increases activation of osteoclasts (cells responsible for resorption of bone tissue).17 Second, cytokine-mediated osteoclast activation is augmented in the presence of estrogen deficiency.18,19

    Extensive bone loss can result in the development of osteoporosis. Osteoporosis is defined as a systemic bone disease characterized by low bone mass and microarchitectural deterioration of bone tissue, with a subsequent increase in bone fragility and susceptibility to fracture.20 The World Health Organization defines osteoporosis as having a bone mineral density (BMD) of less than or equal to 2.5 standard deviations (SDs) below the mean BMD of a young adult woman.21 The burden of osteoporosis in women is high. In the United States, the prevalence of osteoporosis is estimated to range from 17% to 20% among women ages 50 years or older,22  although a more recent report shows a decline in this prevalence of about seven percentage points.23

    Osteoporosis can result in osteoporotic fractures (i.e., hip, spine, humerus, forearm), some of the most common causes of disability and a major source of medical costs.24 An estimated 60% to 70% of osteoporotic fractures occur in women.25,26 In the United States, the 2005 incidence of osteoporotic fractures among women was estimated to be more than 1.4 million. The direct annual cost associated with these osteoporotic fractures was more than $12 billion, and projected to rise to more than $18 billion by 2025.26

    Among all osteoporotic fractures, hip fractures have the most serious consequences, with significant impact on morbidity and mortality.27 The burden of hip fractures is particularly high among women, and increases exponentially with age. Globally, women comprise roughly 70% of all hip fractures.25 Among patients ≥65 years, the annual mean number of hip fractures in the U.S. (between 1986 and 2005) per 100,000 was 957 (95% CI: 922-993) for women and 414 (95% CI, 402-427) for men.28 The lifetime risk of a hip fracture in white women is estimated to be one in six,29 and even greater among white women with osteoporosis (between 40% and 50%).30,31 Black and Asian women have about half the rate of hip fracture when compared with white women.24 Additionally, hip fractures comprise an estimated 36.8% of osteoporotic fractures in women ages 80 to 84.32 The number of disability-adjusted life years (DALYs) lost globally due to hip fractures is almost two times greater in women (1.53 million) than men (0.82 million).25 Furthermore, approximately one in five women will die within a year of a hip fracture.33,34

    Several risk factors for bone loss and hip fractures have been identified in older women. Lower weight, greater weight loss, current smoking, lower serum estradiol, and higher serum adiponectin comprise some of the risk factors for bone loss.35-37 Additionally, meta-analyses have identified body mass index (BMI),38 prior smoking,39 prior fracture,40 and corticosteroid use41 as predictors of hip fracture (independent of BMD).

    This report will focus on the laboratory methods used to measure concentrations of cytokines, cytokine soluble receptors, and hs-CRP. We will also discuss inflammatory markers and the risk of bone loss and fractures in older women.

    Lab procedures for inflammatory marker measurement

    In biomedical research, enzyme-linked immunosorbant assay (ELISA) is the most commonly used method for measuring concentrations of inflammatory markers, especially low-abundance markers such as cytokines.42 ELISA uses an antibody “sandwich,” with one antibody to specifically detect the cytokine or receptor of interest that is fixed to a plastic well, while the second antibody is linked to an enzyme that acts as an amplification factor to enable colorimetric or chemiluminescent detection and quantitation.

    However, there are documented methodological limitations that coincide with using ELISA to quantify inflammatory marker concentrations. First, for very low-abundance markers (e.g., tumor necrosis factor-alpha [TNF-α]), the ELISA can require a relatively large volume of serum for analysis (e.g., 200 uL), and many studies fall short of the required threshold. Second, the cost of individual ELISAs for each of several markers can add up and be prohibitive for researchers who lack the adequate funds to conduct such measurements.

    Recently, multiplex arrays, which have the ability to estimate levels of several inflammatory markers in one assay, have been developed. Compared to traditional ELISAs, they require smaller sample volume and are less expensive and more time efficient.42 The most widely used multiplex array for measuring inflammatory markers is based on flow cytometry technology. Flow cytometric multiplex arrays use microscopic beads with several predefined colors; beads of each color are coated with antibodies specific for one cytokine, which form the capture site for that specific cytokine. The beads can then be mixed together in “panels” in which each of the differently colored bead sets represents a different cytokine, and a single serum or plasma sample is added to the “panel” of beads. Subsequently, fluorescence or streptavidin labeled detection antibodies attach to the cytokine of interest on each the differently colored bead sets. The flow cytometer uses the color of the beads to keep track of which cytokine is being measured, and fluorescent signals are used to estimate the amount of cytokine detected.

    Multiplex arrays using chemiluminescence or electrochemiluminescence technology have also been developed for measuring inflammatory marker concentrations. Although the technology offers great promise, more studies are needed to evaluate the performance of multiplex assays relative to accepted ELISAs and address or confirm some of the putative limitations. For example, complications may arise because of the different range in concentrations of various antigens being assayed together; there also may be discordance between serum and plasma measurements43 and greater sensitivity to high levels of circulating proteins in serum or plasma samples. Finally, quality control of multiplexed assays is considerably more complicated,44 and manufacturers have found it more difficult to maintain constancy in sensitivity and specificity when preparing multiplexed reagents.45

    In epidemiological studies, most hepatic inflammation biomarkers, such as CRP, fibrinogen, serum amyloid A, and others, are measured using either nephelometry or immunoturbidimetry. Historically, nephelometry was the assay of choice because of its high sensitivity; however, latex-enhanced immunoturbidimetry has produced comparable sensitivity. To estimate the concentration of CRP, immunoturbidimetry measures the turbidity of a sample, and nephelometry the scattering of light, upon application of a beam of light. Assay reagent is added to the sample, resulting in a formation of an antibody-antigen complex. Immunoturbidimetry measures the intensity of the light absorbed by the now-turbid sample. In contrast, nephelometry measures the intensity of the light scattered. CRP concentrations are then estimated by using a calibration curve. ELISA can also be used to measure CRP.

    As an example of research practice, consider how we measured inflammatory markers in our studies.11,12 Blood samples were obtained after approximately 12 hours of fasting and stored at -80°C using strict control procedures until assay.46 Subsequently, the stored serum samples were sent to testing laboratories for measurements. Cytokines and soluble cytokine receptor levels were measured in duplicate using Solid-Phase Sandwich ELISA kits (R&D Systems) at the University of Vermont. The detectable limits for the cytokines Interleukin 6 (IL-6) (using the HS600 Quantikine kit) and TNF-α (using HSTA50 kit) were 0.10 and 0.18 pg/ml, respectively. The detectable limits for the soluble receptors of IL-6 (IL-6 SR) (using the DR600 kit), Interleukin 2 (IL-2 SR) (using Q2000B kit), and TNF-α (TNF SR1 using the DRT100kit, and TNF SR2 using the DRT200kit) were 6.5, <10, 3.0, and 1.0 pg/ml, respectively. hs-CRP was also measured in duplicate by ELISA based on purified protein and polyclonal anti-CRP antibodies.47 The hs-CRP assay was standardized according to the World Health Organization’s First International Reference Standard, with a sensitivity of 0.08 µg/ml. The interassay coefficient of variation (CV) is a measure of the reliability between assays using the ratio of the standard deviation to the mean, with a lower interassay CV suggesting higher reliability. Three samples of known concentration were tested twenty times on one plate to assess intra-assay precision. The CVs of IL-6, TNF-α, IL-6 SR, TNF SR1, TNF SR2, and hs-CRP were 10.3%, 15.8%, 12.5% to 14.8%, 6.7% to 10%, 5.6% to 6.2%, and 8%, respectively.

    In summary, inflammatory marker measurement using ELISA remains the standard assay for epidemiological studies. Future research should consider whether multiplex arrays can be used as a practical alternative to ELISA for the measurement of inflammatory markers.

    Inflammatory markers and risk of bone loss and incident fractures

    Based on a comprehensive review of the literature, we identified nine epidemiological studies that evaluated the association of bone loss and incident fractures according to levels of these inflammatory markers. Most studies have focused on older women (Tables 1-3).

    Observational studies which have examined if high inflammatory marker levels increase the rate of bone loss have shown some evidence of an association.8-10 The main limitation of these studies is the relatively short follow-up (1 to 3.3 years) and sample sizes (N≤242) (Table 1). Studies are needed that examine bone loss in a larger cohort and over a longer period of time (e.g., ≥5 years). Furthermore, studies in men and premenopausal women are needed to understand if the effect of inflammation on bone loss is independent of hormone levels (e.g., estradiol).

    The effect of inflammatory marker levels on risk of incident fractures has been examined in several studies.11-16 Two methods to classify inflammation for these fracture studies have emerged. Our studies have used a composite variable11,12 which combines the number of cytokines and/or their soluble receptors in the highest quartile as the exposure, whereas the other studies have limited the exposure to hs-CRP13-16 only. We created a composite measure of inflammation because one biomarker is unlikely to capture an accurate level of inflammation or risk.48,49

    The characteristics and findings of our two studies11,12 are summarized in slightly more detail (Table 2). The epidemiological study design and selected study population differed by study. The earlier study11 was a cohort study using participants from the Health ABC study, which included men and women as well as whites and African Americans, while the more recent study12 was a nested case-control study within the Women’s Health Initiative observational cohort and was limited primarily to white women (Table 2). A nested case-control study is a case-control study within a cohort study. We opted for a nested case-control study to substantially reduce the costs associated with assaying 39,795 baseline serum samples for the total cohort. Instead, we randomly selected 400 incident hip fracture cases. From the remaining cohort members without hip fracture, one control per case was selected with individual matching by age, race, and date of blood draw. We assigned inflammatory marker quartile levels based on the distribution observed in the controls, which should provide the expected concentrations of inflammatory markers in the population that gave rise to the cases. The follow-up times and age of participants in the two studies were similar (Table 2).

    Study outcomes differed, with the earlier study using nontraumatic fractures (fractures occurring spontaneously or from modest trauma) and the subsequent study using hip fractures. Both studies accounted for a large number of potential confounders (i.e., weight, cigarette smoking, corticosteroids, and diabetes), while the most recent study adjusted for several potential mediators—factors that are likely to be in the causal pathway between inflammation and fracture (Table 2). Findings for both studies were consistent when examining the effect of single inflammatory biomarkers on fractures. For instance, in both studies, IL-6 SR was not associated with fractures, whereas participants in the top quartile of TNF SR2 had an increased risk of fracture. Among single inflammatory markers (i.e., IL-2 sR, TNF SR1, and TNF SR2) that were significantly associated with an increased risk of fracture the magnitude of effect (i.e., hazard ratio or relative risk) was between 1.48 and 1.73 (Table 2).

    Ce Chart 1047x1369

    Using the composite variable, we showed that participants with the highest burden of inflammation (3 or more markers in the highest quartile) had an almost three-fold risk of fractures (nontraumatic and hip fractures) compared with those with the lowest inflammation burden (0 or 1 inflammatory marker in the highest quartile) (Table 2). Analyses from the earlier study were limited by statistical power (i.e., low number of hip fractures and low fracture rates among non-white women), whereas the most recent study was unable to account for BMD and estimate person-time risk (Table 2). As a result, these findings are primarily generalizable to white postmenopausal women.

    Obviously, two well conducted observational studies are not enough to conclude that there is a causal link between inflammatory marker levels and risk of fracture. We are limited by only one measure of inflammation per participant, and measurements over time are needed to better quantify long-term inflammation. Other factors (e.g., age, BMI, diabetes, and frailty) are strongly correlated with inflammation, although we have accounted for these and other important measures in our analyses. We hope to continue to evaluate how inflammatory markers affect fracture risk in different cohorts to determine if these findings remain consistent across studies, and to address some of the limitations of prior studies.

    Finally, four cohort studies focused on the association between hs-CRP and risk of incident fractures (Table 3).13-16 All four studies used the prospective cohort design with the vast majority of participants followed for five years or more. The study populations consisted predominantly of postmenopausal women, although one report included only pre- or perimenopausal women.16 Findings were mostly consistent across studies, showing that higher levels of hs-CRP are associated with an increased risk of fracture. In fact, Schett et al13 reported that participants in the highest versus lowest tertile group of hs-CRP had more than nine times the risk of non-traumatic fracture. On the other hand, it is worth noting that we found no association between hs-CRP and incident nontraumatic fractures.11 Furthermore, Pasco et al reported a rather modest, albeit significant, association.14 Recently, Ishii et al showed that composite strength indices and not BMD are inversely related to CRP levels, and partially explain the increased fracture risk associated with inflammation.16

    In summary, there is evidence that inflammation is related to an increased fracture risk. The association with fractures appears strongest when inflammatory markers are combined into a composite variable, suggesting that inflammatory burden may be an important biological risk factor for fractures in older women. More research in this area is needed in men and premenopausal women.

    Ce Author Barbour
    Kamil E. Barbour, PhD, is currently an epidemiologist on the Centers for Disease Control and Prevention (CDC) Arthritis team. Jane A. Cauley, DrPH, is Professor of Epidemiology and Vice Chair for Research, Graduate School of Public Health, University of Pittsburgh. This research was conducted at the University of Pittsburgh. Both authors have no conflict of interest to declare.
    Ce Author Cauley

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