Anti-Interleukin-6 Therapy Decreases Hip Synovitis and Bone Resorption and Increases Bone Formation Following Ischemic Osteonecrosis of the Femoral Head

Legg-Calvé-Perthes disease (LCPD) is a juvenile form of ischemic femoral head osteonecrosis, which produces chronic hip synovitis, permanent femoral head deformity, and premature osteoarthritis. Currently, there is no medical therapy for LCPD. Interleukin-6 (IL-6) is significantly elevated in the synovial fluid of patients with LCPD. We hypothesize that IL-6 elevation promotes chronic hip synovitis and impairs bone healing after ischemic osteonecrosis. We set out to test if anti-IL-6 therapy using tocilizumab can decrease hip synovitis and improve bone healing in the piglet model of LCPD. Fourteen piglets were surgically induced with ischemic osteonecrosis and assigned to two groups: the no treatment group (n = 7) and the tocilizumab group (15 to 20 mg/kg, biweekly intravenous injection, n = 7). All animals were euthanized 8 weeks after the induction of osteonecrosis. Hip synovium and femoral heads were assessed for hip synovitis and bone healing using histology, micro-CT, and histomorphometry. The mean hip synovitis score and the number of synovial macrophages and vessels were significantly lower in the tocilizumab group compared with the no treatment group (p < .0001, p = .01, and p < .01, respectively). Micro-CT analysis of the femoral heads showed a significantly higher bone volume in the tocilizumab group compared with the no treatment group (p = .02). The histologic assessment revealed a significantly lower number of osteoclasts per bone surface (p < .001) in the tocilizumab group compared with the no treatment group. Moreover, fluorochrome labeling showed a significantly higher percent of mineralizing bone surface (p < .01), bone formation rate per bone surface (p < .01), and mineral apposition rate (p = .04) in the tocilizumab group. Taken together, tocilizumab therapy decreased hip synovitis and osteoclastic bone resorption and increased new bone formation after ischemic osteonecrosis. This study provides preclinical evidence that tocilizumab decreases synovitis and improves bone healing in a large animal model of LCPD. © 2020 American Society for Bone and Mineral Research (ASBMR).     

3‐dimensional metrics of proximal femoral shape deformities in Legg–Calvé–Perthes disease and slipped capital femoral epiphysis

Legg–Calvé–Perthes disease (LCPD) and slipped capital femoral epiphysis (SCFE) are two common pediatric hip disorders that affect the 3‐dimensional shape and function of the proximal femur. This study applied the principles of continuum mechanics to statistical shape modeling (SSM) and determined 3‐D metrics for the evaluation of shape deformations in normal growth, LCPD, and SCFE. CT scans were obtained from 32 patients with asymptomatic, LCPD, and SCFE hips ((0.5–0.9 mm)² in‐plane resolution, 0.63 mm slice thickness). SSM was performed on segmented proximal femoral surfaces, and shape deformations were described by surface displacement, strain, and growth plate angle metrics. Asymptomatic normal femurs underwent coordinated, growth‐associated surface displacements and anisotropic strains that were site‐specific and highest at the greater trochanter. After size‐ and age‐based shape adjustment, LCPD femurs exhibited large displacements and surface strains in the femoral head and neck, with associated changes in femoral head growth plate angles. Mild SCFE femurs had contracted femoral neck surfaces, and surface displacements in all regions tended to increase with severity of slip. The results of this paper provide new 3‐D metrics for characterizing the shape and biomechanics of the proximal femur. Statement of Clinical Significance: Quantitative 3‐D metrics of shape may be useful for understanding and monitoring disease progression, identifying target regions for shape modulation therapies, and objectively evaluating the success of such therapies. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:1526–1535, 2018.

     

Novel three‐dimensional MRI technique for study of cartilaginous hip surfaces in Legg‐Calvé‐Perthes disease

Treatment of Legg‐Calvé‐Perthes disease (LCPD) may improve if new knowledge can be obtained regarding how articular cartilage changes shape during the course of this disorder. A new technique is presented showing how analyses of magnetic resonance images can be used to quantify the three‐dimensional changes in the femoral and acetabular articulating cartilage surfaces of children with LCPD. Ten male subjects (8 ± 1 years) with unilateral LCPD were enrolled in this IRB approved study. Sets of magnetic resonance images of both hips were obtained at three different times. Three‐dimensional virtual models of the cartilage were created from these images, and mathematical spheres were fit to the articulating surfaces. Five parameters (size, shape deformity (sphericity error), radial growth rate, joint fit, and joint incongruity) were used to quantify cartilage surface shape. Data were analyzed by using a linear mixed‐model. Joint incongruity, i.e., the distance between the centers of the femoral and acetabular spheres, was slightly more than 2.5 times larger (p = 0.001) in LCPD hips than the contralateral normal hips. Cartilage shape deformity was 65% larger in hips with LCPD than in normal hips. Growth rates of the femoral head and the opposing acetabular surface showed that distortion of the femoral surface occurred first and the opposing acetabular surface followed. Mean radial difference (acetabular surface radius minus femoral surface radius) in LCPD hips was less than half (p < 0.01) the value of normal hips. Interobserver variability was ～10% of the value attributable to LCPD. This is the first known report presenting a technique that quantifies the three‐dimensional size, deformity, growth, fit. and incongruity of the femoral and acetabular articulating cartilaginous surfaces of LCPD and contralateral normal hips. The data obtained support the use of this technique and provide pilot data for a future clinical study of LCPD. Objective assessment of cartilage shape enabled by this technique may aid future diagnoses, enable monitoring of three‐dimensional femoral and acetabular remodeling, and permit quantitative assessment of treatment efficacy. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 27: 981–988, 2009     

3D MRI quantification of femoral head deformity in Legg–Calvé–Perthes disease

The purpose of this study was to quantify femoral head deformity in patients with Legg–Calvé–Perthes disease (LCPD) using a novel three dimensional (3D) magnetic resonance imaging (MRI) reconstruction and volume based analysis. Bilateral femoral heads of 17 patients (mean age 9.9 ± 2.0 years; 12 boys, 5 girls) with LCPD were scanned 1–2 times (n = 33 LCPD heads, 20 normal heads) using a 1.5T MRI scanner. Fourteen patients had unilateral and three had bilateral LCPD with five hips in the Waldenström initial stage, 9 in the fragmentation stage, 14 in the reossification stage, and 5 in the healed stage. 3D digital reconstructions of femoral heads were created using MIMICS software. Deformity was quantified using a 3D volume ratio method based on reference hemisphere volume as well as two surface geometry methods. Intra‐observer analysis showed that 97% of the LCPD femoral heads were within 10% of the original value and test shapes had 99.6% accuracy. For normal femoral heads, the volume ratios of all except one were between 95 and 98% (n = 20) of a perfect hemisphere volume. For femoral heads affected with LCPD, the volume ratios ranged from 43% to 96% of a perfect hemisphere (n = 33). The volume ratio method and the two surface geometry comparison methods had high correlation (r = 0.89 and 0.96). In summary, the 3D MRI volume ratio method allowed accurate quantification and demonstrated small changes (<10%) of the femoral head deformity in LCPD. This method may serve as a useful tool to evaluate the effects of treatment on femoral head shape. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:2051–2058, 2017.

     

Combination Treatment of Biomechanical Support and Targeted Intra‐arterial Infusion of Peripheral Blood Stem Cells Mobilized by Granulocyte‐Colony Stimulating Factor for the Osteonecrosis of the Femoral Head: A Randomized Controlled Clinical Trial

The objective of this study was to determine the benefits of combination treatment with mechanical support and targeted intra‐arterial infusion of peripheral blood stem cells (PBSCs) mobilized by granulocyte–colony stimulating factor (G‐CSF) via the medial circumflex femoral artery on the progression of osteonecrosis of the femoral head (ONFH). Fifty‐five patients (89 hips) with early and intermediate stage ONFH were recruited and randomly assigned to combination treatment or mechanical support treatment (control group). All hips received mechanical support treatment (porous tantalum rod implantation). Then, hips in the combination treatment group were performed targeted intra‐arterial infusion of PBSCs. At each follow‐up, Harris hip score (HHS) and Association Research Circulation Osseous (ARCO) classification were used to evaluate the symptoms and progression of osteonecrosis. Total hip arthroplasty (THA) was assessed as an endpoint at each follow‐up. At 36 months, 9 of the 41 hips (21.95%) in the control group progressed to clinical failure and underwent THA whereas only 3 of the 48 hips (6.25%) in the combination treatment group required THA (p = 0.031). Kaplan‐Meier survival analysis showed a significant difference in the survival time between the two groups (log‐rank test; p = 0.025). Compared to the control group, combination treatment significantly improved the HHS at 36 months (p = 0.003). At the final follow‐up examination, radiological progression was noted in 13 of 41 hips (31.71%) for the control group, but in only 4 of 48 hips (8.33%) for the combination treatment group (p = 0.005). The overall collapse rates were 15.15% (5/33 hips) and 8.11% (3/37 hips) in the control and combination treatment groups, respectively. Targeted intra‐arterial infusion of PBSCs is capable of enhancing the efficacy of biomechanical support in the treatment of ONFH. This clinical trial confirmed that the combination treatment might be a safe and feasible choice for the treatment of early or intermediate stages of ONFH. © 2014 American Society for Bone and Mineral Research.     

Increased IL‐6 Expression in Osteoclasts Is Necessary But Not Sufficient for the Development of Paget's Disease of Bone

Measles virus nucleocapsid protein (MVNP) expression in osteoclasts (OCLs) and mutation of the SQSTM1 (p62) gene contribute to the increased OCL activity in Paget's disease (PD). OCLs expressing MVNP display many of the features of PD OCLs. Interleukin‐6 (IL‐6) production is essential for the pagetic phenotype, because transgenic mice with MVNP targeted to OCLs develop pagetic OCLs and lesions, but this phenotype is absent when MVNP mice are bred to IL‐6^–/– mice. In contrast, mutant p62 expression in OCL precursors promotes receptor activator of NF‐κB ligand (RANKL) hyperresponsivity and increased OCL production, but OCLs that form have normal morphology, are not hyperresponsive to 1,25‐dihydroxyvitamin D₃ (1,25‐(OH)₂D₃), nor produce elevated levels of IL‐6. We previously generated p62^P394L knock‐in mice (p62KI) and found that although OCL numbers were increased, the mice did not develop pagetic lesions. However, mice expressing both MVNP and p62KI developed more exuberant pagetic lesions than mice expressing MVNP alone. To examine the role of elevated IL‐6 in PD and determine if MVNP mediates its effects primarily through elevation of IL‐6, we generated transgenic mice that overexpress IL‐6 driven by the tartrate‐resistant acid phosphatase (TRAP) promoter (TIL‐6 mice) and produce IL‐6 at levels comparable to MVNP mice. These were crossed with p62KI mice to determine whether IL‐6 overexpression cooperates with mutant p62 to produce pagetic lesions. OCL precursors from p62KI/TIL‐6 mice formed greater numbers of OCLs than either p62KI or TIL‐6 OCL precursors in response to 1,25‐(OH)₂D₃. Histomorphometric analysis of bones from p62KI/TIL‐6 mice revealed increased OCL numbers per bone surface area compared to wild‐type (WT) mice. However, micro‐quantitative CT (µQCT) analysis did not reveal significant differences between p62KI/TIL‐6 and WT mice, and no pagetic OCLs or lesions were detected in vivo. Thus, increased IL‐6 expression in OCLs from p62KI mice contributes to increased responsivity to 1,25‐(OH)₂D₃ and increased OCL numbers, but is not sufficient to induce Paget's‐like OCLs or bone lesions in vivo. © 2014 American Society for Bone and Mineral Research.     

Interleukin‐1α, ‐6, and ‐8 decrease Cdc42 activity resulting in loss of articular chondrocyte phenotype

Small GTPase proteins mediate changes in cellular morphology and other cellular functions. The aim of this study was to examine signaling of the small GTPase Cdc42 by stimulating chondrocytes grown in monolayer with long‐ (96 h) or short‐ (2 and 30 min) term exposure to interleukin‐1α (IL‐1α), IL‐6, or IL‐8. Quantitative PCR was used to determine changes in collagen type IIB (COL2A1), aggrecan (AGG), and matrix metalloproteinase‐13 (MMP‐13) gene expression after prolonged cytokine exposure. Effects of short‐term treatment with IL‐α, IL‐6, or IL‐8 on endogenous GTP‐bound Cdc42 levels were assessed using an affinity assay, and on actin filament organization using confocal microscopy. Cytokine treatments significantly decreased COL2A1 and AGG expression and increased MMP‐13 expression. Short exposure to IL‐1α, IL‐6, or IL‐8 decreased endogenous GTP‐Cdc42 and increased stress fibers, which were reversed with cytochalasin D treatment. These results show that IL‐mediated Cdc42 signaling modifies chondrocyte phenotype and morphology. This may lend insight into the altered chondrocyte phenotype in catabolic conditions such as osteoarthritis. © 2011 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 30:246–251, 2012     

IL‐36 Induces Bisphosphonate‐Related Osteonecrosis of the Jaw‐Like Lesions in Mice by Inhibiting TGF‐β‐Mediated Collagen Expression

Long‐term administration of nitrogen‐containing bisphosphonates can induce detrimental side effects such as bisphosphonate‐related osteonecrosis of the jaw (BRONJ) in human. Although inflammation is known to be associated with BRONJ development, the detailed underlying mechanism remains unknown. Here, we report that the pro‐inflammatory cytokine IL‐36α is, in part, responsible for the BRONJ development. We found a notably higher level of IL‐36α and lower level of collagen in the BRONJ lesions in mice. We also found that IL‐36α remarkably suppressed TGF‐β‐mediated expression of Collα1 and α‐Sma via the activation of Erk signaling pathway in mouse gingival mesenchymal stem cells. When IL‐36 signaling was abrogated in vivo, development of BRONJ lesions was ameliorated in mice. Taken together, we showed the pathologic role of IL‐36α in BRONJ development by inhibiting collagen expression and demonstrated that IL‐36α could be a potential marker and a therapeutic target for the prevention and treatment of BRONJ. © 2016 American Society for Bone and Mineral Research.     

Potential pathological role of pro‐inflammatory cytokines (IL‐6, TNF‐α, and IL‐17) in xenotransplantation

The major limitation of organ transplantation is the shortage of available organs from deceased human donors which leads to the deaths of thousands of patients each year. Xenotransplantation is considered to be an effective way to resolve the problem. Immune rejection and coagulation dysfunction are two major hurdles for the successful survival of pig xenografts in primate recipients. Pro‐inflammatory cytokines, such as IL‐6, TNF‐α, and IL‐17, play important roles in many diseases and in allotransplantation. However, the pathological roles of these pro‐inflammatory cytokines in xenotransplantation remain unclear. Here, we briefly review the signaling transduction and expression regulation of IL‐6, TNF‐α, and IL‐17 and evaluate their potential pathological roles in in vitro and in vivo models of xenotransplantation. We found that IL‐6, TNF‐α, and IL‐17 were induced in most in vitro or in vivo xenotransplantation model. Blockade of these cytokines using gene modification, antibody, or inhibitor had different effects in xenotransplantation. Inhibition of IL‐6 signaling with tocilizumab decreased CRP but did not increase xenograft survival. The one possible reason is that tocilizumab can not suppress IL‐6 signaling in porcine cells or organs. Other drugs which inhibit IL‐6 signaling need to be investigated in xenotransplantation model. Inhibition of TNF‐α was beneficial for the survival of xenografts in pig‐to‐mouse, rat, or NHP models. Blockade of IL‐17 using a neutralizing antibody also increased xenograft survival in several animal models. However, the role of IL‐17 in the pig‐to‐NHP xenotransplantation model remains unclear and needs to be further investigated. Moreover, blockade of TNF‐α and IL‐6 together has got a better effect in pig‐to‐baboon kidney xenotransplantation. Blockade two or even more cytokines together might get better effect in suppressing xenograft rejection. Better understanding the role of these cytokines in xenotransplantation will be beneficial for choosing better immunosuppressive strategy or producing genetic modification pig.     

Direct Crosstalk Between Cancer and Osteoblast Lineage Cells Fuels Metastatic Growth in Bone via Auto‐Amplification of IL‐6 and RANKL Signaling Pathways

The bone microenvironment and its modification by cancer and host cell interactions is a key driver of skeletal metastatic growth. Interleukin‐6 (IL‐6) stimulates receptor activator of NF‐κB ligand (RANKL) expression in bone cells, and serum IL‐6 levels are associated with poor clinical outcomes in cancer patients. We investigated the effects of RANKL on cancer cells and the role of tumor‐derived IL‐6 within the bone microenvironment. Using human breast cancer cell lines to induce tumors in the bone of immune‐deficient mice, we first determined whether RANKL released by cells of the osteoblast lineage directly promotes IL‐6 expression by cancer cells in vitro and in vivo. We then disrupted of IL‐6 signaling in vivo either via knockdown of IL‐6 in tumor cells or through treatment with specific anti‐human or anti‐mouse IL‐6 receptor antibodies to investigate the tumor effect. Finally, we tested the effect of RANK knockdown in cancer cells on cancer growth. We demonstrate that osteoblast lineage‐derived RANKL upregulates secretion of IL‐6 by breast cancers in vivo and in vitro. IL‐6, in turn, induces expression of RANK by cancer cells, which sensitizes the tumor to RANKL and significantly enhances cancer IL‐6 release. Disruption in vivo of this auto‐amplifying crosstalk by knockdown of IL‐6 or RANK in cancer cells, or via treatment with anti‐IL‐6 receptor antibodies, significantly reduces tumor growth in bone but not in soft tissues. RANKL and IL‐6 mediate direct paracrine‐autocrine signaling between cells of the osteoblast lineage and cancer cells, significantly enhancing the growth of metastatic breast cancers within bone. © 2014 American Society for Bone and Mineral Research.     

Interleukin‐6 and interleukin‐6 receptor expression,localization, and involvement in pain‐sensing neuron activation in a mouse intervertebral disc injury model

The pathological mechanism of intractable low back pain is unclear. However, intervertebral disc (IVD) degeneration is a primary cause of low back pain, and pain‐related mediators, such as interleukin‐6 (IL‐6), have been correlated with discogenic pain. The objective of this study is to elucidate the mechanism of local IL‐6 and IL‐6 receptor (IL‐6R) expression after IVD injury as well as determine the involvement of IL‐6/IL‐6 signaling in discogenic pain. To do this, quantitative and immunohistological analyses in a mouse model of IVD injury were performed. Firstly, we measured the local expression levels of IL‐6 and IL‐6R in IVDs by enzyme‐linked immunosorbent assay (ELISA). Secondly, we immunohistochemically confirmed their localization in injured IVDs. Lastly, we evaluated the effects of intradiscal injection of an IL‐6 inhibitor by evaluating pain‐related protein, calcitonin gene‐related peptide (CGRP), expression in dorsal root ganglia (DRG) neurons that innervate IVDs. Injured IVDs showed increased production of IL‐6 and IL‐6R. IL‐6 and IL‐6R expression in the injured IVD were predominantly localized in the annulus fibrosus and endplate, and intradiscal injection of the IL‐6 inhibitor suppressed CGRP expression in the DRG neurons. These results show that IL‐6 and IL‐6R expression levels are responsive to IVD injury and that inhibition of IL‐6/IL‐6R signaling may be a promising analgesic treatment for degenerative disc diseases. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 33:1508–1514, 2015.     

Declining Rates of Hip Fracture in End‐Stage Renal Disease: Analysis From the 2003–2011 Nationwide Inpatient Sample

The incidence of hip fracture in patients with end‐stage renal disease (ESRD) is considerably higher than that in the general age‐ and sex‐matched population. Although medical therapy for chronic kidney disease mineral bone disorder (CKD‐MBD) has changed considerably over the last decade, rates of hip fracture in the entire ESRD population have not been well‐characterized. Herein, we evaluated temporal trends in rates of hip fracture, in‐hospital mortality, and costs of associated hospital stay in ESRD. We identified hospitalizations for hip fracture from 2003 to 2011 using the Nationwide Inpatient Sample, a representative national database inclusive of all ages and payers. We incorporated data from the United States Renal Data System and the US Census to calculate population‐specific rates. Between 2003 and 2011, we identified 47,510 hip fractures in the ESRD population. The overall rate of hip fracture was 10.04/1000 person‐years. The rate was 3.73/1000 person‐years in patients aged less than 65 years, and 20.97/1000 person‐years in patients aged 65 or older. Age‐ and sex‐standardized rates decreased by 12.6% from 2003 (10.23/1000 person‐years; 95% confidence interval [CI], 7.99/1000 to 12.47/1000) to 2011 (8.94/1000 person‐years; 95% CI, 7.12/1000 to 10.75/1000). Hip fracture rates over time were virtually identical in patients aged less than 65 years; however, rates decreased by 15.3% among patients aged 65 years or older; rates declined more rapidly in older women compared with older men (p for interaction = 0.047). In‐hospital mortality rate after hip fracture operation declined by 26.7% from 2003 (8.6%; 95% CI, 6.8 to 10.4) to 2011 (6.3%; 95% CI, 4.9 to 7.7). In ESRD, age‐ and sex‐standardized hip fracture rates and associated in‐hospital mortality have declined substantially over the last decade. © 2017 American Society for Bone and Mineral Research.     

A meta‐analysis of interleukin‐6 as a valid and accurate index in diagnosing early neonatal sepsis

We aimed to systematically assess the overall value of interleukin 6 (IL‐6) in diagnosing neonates with sepsis. A systematic literature search was conducted using the following electronic databases: PubMed, Embase, and Cochrane, to identify eligible studies through the index words updated till November 2018. Cross‐sectional studies, as well as prospective cohort studies, were included in the above‐mentioned group of eligible studies. We also searched the literature sources that had a link to the present study, which were further assessed by heterogeneity through the use of a proper‐effects model to calculate pooled weighted specificity, sensitivity, and diagnostic odds ratio (DOR). We also conducted summary receiver operating characteristic (SROC) analyses for neonatal sepsis. In the present meta‐analysis, there were 31 studies exploring IL‐6 for the diagnostic accuracy of neonatal sepsis. The global specificity and sensitivity of IL‐6 for neonatal sepsis were as follows: 88% (95% confidence interval [CI]: 83%‐92%) and 82% (95% CI: 77%‐86%), respectively. The global positive and negative likelihood ratio of IL‐6 in diagnosing neonatal sepsis were 7.03 (95% CI: 4.81‐10.26) and 0.20 (95% CI: 0.15‐0.26), respectively. The global DOR was 29.54 (95%CI: 18.56‐47.04) of IL‐6. In addition, the area under the SROC was high for IL‐6 (AUC = 0.92; 95% CI: 0.89‐0.94). In this study, we performed a systematic review and meta‐analysis to assess the diagnostic accuracy studies of IL‐6 in diagnosing neonatal sepsis. Our results suggested that IL‐6 is a valid and accurate index in diagnosing early neonatal sepsis, but it still needs to be combined with other laboratory tests and specific clinical manifestations.     

Connective Tissue Growth Factor Promotes Fibrosis Downstream of TGFβ and IL‐6 in Chronic Cardiac Allograft Rejection

Cardiac transplantation is an effective treatment for multiple types of heart failure refractive to therapy. Although immunosuppressive therapeutics have increased survival rates within the first year posttransplant, chronic rejection (CR) remains a significant barrier to long‐term graft survival. Indicators of CR include patchy interstitial fibrosis, vascular occlusion and progressive loss of graft function. Multiple factors have been implicated in the onset and progression of CR, including TGFβ, IL‐6 and connective tissue growth factor (CTGF). While associated with CR, the role of CTGF in CR and the factors necessary for CTGF induction in vivo are not understood. To this end, we utilized forced expression and neutralizing antibody approaches. Transduction of allografts with CTGF significantly increased fibrotic tissue development, though not to levels observed with TGFβ transduction. Further, intragraft CTGF expression was inhibited by IL‐6 neutralization whereas TGFβ expression remained unchanged, indicating that IL‐6 effects may potentiate TGFβ‐mediated induction of CTGF. Finally, neutralizing CTGF significantly reduced graft fibrosis without reducing TGFβ and IL‐6 expression levels. These findings indicate that CTGF functions as a downstream mediator of fibrosis in CR, and that CTGF neutralization may ameliorate fibrosis and hypertrophy associated with CR.     

The association of red blood cell n‐3 and n‐6 fatty acids with bone mineral density and hip fracture risk in the women's health initiative

Omega‐3 (n‐3) and omega‐6 (n‐6) polyunsaturated fatty acids (PUFA) in red blood cells (RBCs) are an objective indicator of PUFA status and may be related to hip fracture risk. The primary objective of this study was to examine RBC PUFAs as predictors of hip fracture risk in postmenopausal women. A nested case‐control study (n = 400 pairs) was completed within the Women's Health Initiative (WHI) using 201 incident hip fracture cases from the Bone Mineral Density (BMD) cohort, along with 199 additional incident hip fracture cases randomly selected from the WHI Observational Study. Cases were 1:1 matched on age, race, and hormone use with non–hip fracture controls. Stored baseline RBCs were analyzed for fatty acids using gas chromatography. After removing degraded samples, 324 matched pairs were included in statistical analyses. Stratified Cox proportional hazard models were constructed according to case‐control pair status; risk of fracture was estimated for tertiles of RBC PUFA. In adjusted hazard models, lower hip fracture risk was associated with higher RBC α‐linolenic acid (tertile 3 [T3] hazard ratio [HR]: 0.44; 95% confidence interval [CI], 0.23–0.85; p for linear trend 0.0154), eicosapentaenoic acid (T3 HR: 0.46; 95% CI, 0.24–0.87; p for linear trend 0.0181), and total n‐3 PUFAs (T3 HR: 0.55; 95% CI, 0.30–1.01; p for linear trend 0.0492). Conversely, hip fracture nearly doubled with the highest RBC n‐6/n‐3 ratio (T3 HR: 1.96; 95% CI, 1.03–3.70; p for linear trend 0.0399). RBC PUFAs were not associated with BMD. RBC PUFAs were indicative of dietary intake of marine n‐3 PUFAs (Spearman's rho = 0.45, p < 0.0001), total n‐6 PUFAs (rho = 0.17, p < 0.0001) and linoleic acid (rho = 0.09, p < 0.05). These results suggest that higher RBC α‐linolenic acid, as well as eicosapentaenoic acid and total n‐3 PUFAs, may predict lower hip fracture risk. Contrastingly, a higher RBC n‐6/n‐3 ratio may predict higher hip fracture risk in postmenopausal women. © 2013 American Society for Bone and Mineral Research.