Achenbach, Sara J.

Abstract Early diagnosis of the onset of osteoporosis is key to the delivery of effective therapy. Biochemical markers of bone turnover provide a means of evaluating skeletal dynamics that complements static measurements of BMD by DXA. Conventional clinical measurements of bone turnover, primarily the estimation of collagen and its breakdown products in the blood or urine, lack both sensitivity and specificity as a reliable diagnostic tool. As a result, improved tests are needed to augment the use of BMD measurements as the principle diagnostic modality. In this study, the serum proteome of 58 postmenopausal women with high or low/normal bone turnover (training set) was analyzed by surface enhanced laser-desorption/ionization time-of-flight mass spectrometry, and a diagnostic fingerprint was identified using a variety of statistical and machine learning tools. The diagnostic fingerprint was validated in a separate distinct test set, consisting of serum samples from an additional 59 postmenopausal women obtained from the same Mayo cohort, with a gap of 2 yr. Specific protein peaks that discriminate between postmenopausal patients with high or low/normal bone turnover were identified and validated. Multiple supervised learning approaches were able to classify the level of bone turnover in the training set with 80% sensitivity and 100% specificity. In addition, the individual protein peaks were also significantly correlated with BMD measurements in these patients. Four of the major discriminatory peaks in the diagnostic profile were identified as fragments of interalpha-trypsin-inhibitor heavy chain H4 precursor (ITIH4), a plasma kallikrein-sensitive glycoprotein that is a component of the host response system. These data suggest that these serum protein fragments are the serum-borne reflection of the increased osteoclast activity, leading to the increased bone turnover that is associated with decreasing BMD and presumably an increased risk of fracture. In conjunction with the identification of the individual proteins, this protein fingerprint may provide a novel approach to evaluate high bone turnover states.

Abstract Introduction: Limited information is available about the clinical features of Paget's disease of bone among unselected patients in the community. We examined morbidity and mortality associated with this condition in a large inception cohort of Olmsted County, MN, residents with a new diagnosis of Paget's disease from 1950 through 1994. Materials and Methods: Survival was estimated using the Kaplan-Meier method. Cox proportional hazards models were used to assess the impact of various covariates on death. Results: Paget's disease of bone was diagnosed in 236 Olmsted County residents (mean age at diagnosis, 69.6 yr; 55% men). The majority were symptomatic at diagnosis (58%), and the proportion with symptoms did not change from the prescreening era (1950 to June 1974) to the postscreening era (July 1974–1994). Most patients had polyostotic disease (72%), and the pelvis (67%), vertebra (41%), and femur (31%) were the most common sites of involvement. Skeletal complications attributable to Paget's disease included bowing deformities (7.6%), fracture of pagetic bone (9.7%), and osteosarcoma (0.4%). Osteoarthritis was observed in 73% of patients, and 11% had a hip or knee replacement. Nonskeletal complications related to Paget's disease included cranial nerve (0.4%), peripheral nerve (1.7%), and nerve root (3.8%) compression, basilar invagination (2.1%), hypercalcemia (5.2%), and congestive heart failure (3.0%). Hearing loss, noted in 61%, was significantly higher than previously reported. Conclusions: Compared with white Minnesota residents, overall survival was slightly better than expected (p = 0.010). No clinical risk factors were identified that were associated with an increased risk of death.

Abstract Introduction: The mechanism(s) by which sex steroids regulate bone turnover in humans are unclear, and recent studies have suggested that follicle-stimulating hormone (FSH) may play an important role in regulating bone resorption. Materials and Methods: Fifty-nine men (median age, 69 yr) underwent suppression of sex steroids using a gonadotropin-releasing hormone (GnRH) agonist and aromatase blocker and were replaced with testosterone (T; 5 mg/d) and estradiol (E; 37.5 μg/d). After assessment of bone resorption markers (serum C-terminal telopeptide of type I collagen [CTX] and TRACP5b), they were randomized to sex steroid deficiency (−T, −E), E alone (−T, +E), T alone (+T, −E), or both (+T, +E) and restudied 3 wk later. Bone marrow aspirates were obtained to isolate osteoblastic, T, and monocytic cells using magnetic-activated cell sorting. Results: Serum CTX and TRACP5b increased significantly (by 71% and 15%, p < 0.01 and < 0.001, respectively) in the −T, −E group, and these increases occurred despite a 60% suppression of serum FSH levels (p < 0.001) caused by the GnRH agonist. There were significant E (but not T) effects on preventing increases in serum CTx and TRACP levels. There was a nonsignificant trend (p = 0.122) for E to suppress RANKL mRNA levels in bone marrow osteoblastic cells. Changes in mRNA levels for other cytokines (TNF-α, interleukin (IL)-1α, IL-1β, IL-1ra, IFN-γ) in bone marrow cells were not significant. Conclusions: E has greater suppressive effects on bone resorption than T, and increased bone resorption after sex steroid deficiency can occur independently of changes in FSH secretion. E effects on bone resorption may be mediated by regulation of RANKL production by osteoblastic cells, although further studies using more highly purified cells may reduce the variability of the mRNA measurements and allow for clearer definition of the mediators of sex steroid action in vivo.

Abstract Vertebral fractures are more strongly associated with specific bone density, structure, and strength parameters than with areal BMD, but all of these variables are correlated. Introduction: It is unclear whether the association of areal BMD (aBMD) with vertebral fracture risk depends on bone density per se, bone macro- or microstructure, overall bone strength, or spine load/bone strength ratios. Materials and Methods: From an age-stratified sample of Rochester, MN, women, we identified 40 with a clinically diagnosed vertebral fracture (confirmed semiquantitatively) caused by moderate trauma (cases; mean age, 78.6 ± 9.0 yr) and compared them with 40 controls with no osteoporotic fracture (mean age, 70.9 ± 6.8 yr). Lumbar spine volumetric BMD (vBMD) and geometry were assessed by central QCT, whereas microstructure was evaluated by high-resolution pQCT at the ultradistal radius. Vertebral failure load (∼strength) was estimated from voxel-based finite element models, and the factor-of-risk (ϕ) was determined as the ratio of applied spine loads to failure load. Results: Spine loading (axial compressive force on L3) was similar in vertebral fracture cases and controls (e.g., for 90° forward flexion, 2639 versus 2706 N; age-adjusted p = 0.173). However, fracture cases had inferior values for most bone density and structure variables. Bone strength measures were also reduced, and the factor-of-risk was 35–37% greater (worse) among women with a vertebral fracture. By age-adjusted logistic regression, relative risks for the strongest fracture predictor in each of the five main variable categories were bone density (total lumbar spine vBMD: OR per SD change, 2.2; 95% CI, 1.1–4.3), bone geometry (vertebral apparent cortical thickness: OR, 2.1; 95% CI, 1.1–4.1), bone microstructure (none significant); bone strength (“cortical” [outer 2 mm] compressive strength: OR, 2.5; 95% CI, 1.3–4.8), and factor-of-risk (ϕ for 90° forward flexion/overall vertebral compressive strength: OR, 3.2; 95% CI, 1.4–7.5). These variables were correlated with spine aBMD (partial r, −0.32 to 0.75), but each was a stronger predictor of fracture in the logistic regression analyses. Conclusions: The association of aBMD with vertebral fracture risk is explained by its correlation with more specific bone density, structure, and strength parameters. These may allow deeper insights into fracture pathogenesis.

Abstract Bone structure, strength, and load-strength ratios contribute to forearm fracture risk independently of areal BMD. Introduction: Technological and conceptual advances provide new opportunities for evaluating the contributions of bone density, structure, and strength to the pathogenesis of distal forearm fractures. Materials and Methods: From an age-stratified random sample of Rochester, MN, women, we compared 18 with a distal forearm fracture (cases) to 18 age-matched women with no osteoporotic fracture (controls). High-resolution pQCT was used to assess volumetric BMD (vBMD), geometry, and microstructure at the ultradistal radius, the site of Colles' fractures. Failure loads in the radius were estimated from microfinite element (μFE) models derived from pQCT. Differences between case and control women were assessed, and the risk of fracture associated with each variable was estimated by logistic regression analysis. Results: Given similar heights, estimated loading in a fall on the outstretched arm was the same in cases and controls. However, women with forearm fractures had inferior vBMD, geometry, microstructure, and estimated bone strength. Relative risks for the strongest determinant of fracture in each of the five main variable categories were as follows: BMD (total vBMD: OR per SD change, 4.2; 95% CI, 1.4–12), geometry (cortical thickness: OR, 4.0; 95% CI, 1.4–11), microstructure (trabecular number: OR, 2.3; 95% CI, 1.02–5.1), and strength (axial rigidity: OR, 3.8; 95% CI, 1.4–10); the factor-of-risk (fall load/μFE failure load) was 24% greater (worse) in cases (OR, 3.0; 95% CI, 1.2–7.5). Areas under ROC curves ranged from 0.72 to 0.82 for these parameters. Conclusions: Bone geometry, microstructure, and strength contribute to forearm fractures, as does BMD, and these additional determinants of risk promise greater insights into fracture pathogenesis.

Abstract Elevated serum IGFBP-2 is associated with lower BMD in men and women. It is unknown whether IGFBP-2 serves as a negative regulator of bone metabolism by decreasing bone formation or increasing bone resorption. Studying an age-stratified community-based sample of 344 men and 276 women, IGFBP-2 was the strongest predictor of increased bone resorption among the IGF/IGFBPs studied. Introduction: Serum insulin-like growth factor binding protein-2 (IGFBP-2), which increases with age, is a predictor of low BMD among aging men and women. However, it is unknown whether IGFBP-2 negatively influences bone metabolism by decreasing bone formation or increasing bone resorption. Few have examined the relation between the insulin-like growth factors (IGFs) and their binding proteins (IGFBPs) with bone turnover markers. Materials and Methods: In an age-stratified, random sample of the community, we examined the association between serum IGF-I, IGF-II, IGFBP-1, −2, and −3, and bone turnover markers before and after adjustment for potential confounders (age, body mass index, bioavailable estradiol and testosterone, and sex hormone binding globulin). Analyses were stratified by sex and menopausal status. Results: We studied 344 men (age range, 23–90 yr) and 276 women (age range, 21–93 yr; 166 postmenopausal) not on oral contraceptives or hormone replacement. Among the IGF/IGFBPs assessed, IGFBP-2 was the strongest and most consistent predictor of bone turnover in men and women. After adjustment for potential confounders, IGFBP-2 was positively associated with osteocalcin (OC) and urine and serum N-teleopeptide (NTX) in men (r = 0.20, 0.26, and 0.23, respectively; p < 0.001), serum C-telopeptide (CTX) in premenopausal women (r = 0.28; p < 0.01), and OC, urine NTX, and serum CTX in postmenopausal women (r = 0.24, 0.33, and 0.19, respectively; p < 0.05). Conclusions: Higher serum IGFBP-2, which is predictive of lower BMD, is associated with increased markers of bone resorption, independent of age, body mass, and sex hormones. The association between IGFBP-2 and markers of bone formation may reflect coupling with increased bone resorption, which is not adequate to maintain BMD.

Controversy exists regarding the pathogenic or predictive role of anti-myelin oligodendrocyte glycoprotein (MOG) antibodies in patients with multiple sclerosis (MS). Four immunopathological patterns (IP) have been recognized in early active MS lesions, suggesting heterogeneous pathogenic mechanisms. Whether MOG antibodies contribute to this pathological heterogeneity and potentially serve as biomarkers to identify specific pathological patterns is unknown. Here we report the frequencies of antibodies to human recombinant MOG (identified by Western blot and enzymelinked immunoabsorbent assay (ELISA)) in patients with pathologically proven demyelinating disease, and investigate whether antibody status is associated with clinical course, HLA-DR2 genotype, IP or treatment response to plasmapheresis. The biopsy cohort consisted of 72 patients: 12 pattern I, 43 pattern II and 17 pattern III. No association was found between MOG antibody status and conversion to clinically definite MS, DR-2 status, IP or response to plasmapheresis. There was poor agreement between Western blot and ELISA (kappa=0.07 for MOG IgM). Fluctuations in antibody seropositivity were seen for 3/4 patients tested serially by Western blot. This study does not support a pathologic pattern-specific role for MOG-antibodies. Variable MOG-antibody status on serial measurements, coupled with the lack of Western blot and ELISA correlations, raises concern regarding the use of MOG-antibody as an MS biomarker and underscores the need for methodological consensus.

Abstract A leading theory suggests that decreasing activity and muscle mass is the main cause of age-related bone loss. However, in a population-based study of 375 women and 325 men (age, 21–97 years), we failed to find a close correspondence between these variables and changes in bone strength with aging. Introduction: It has been suggested that bone strength is homeostatically adapted to habitual skeletal loading conditions and that bone loss could, therefore, result simply from age-related reductions in physical activity and muscle mass, but this notion has not been explored in detail. Materials and Methods: In a stratified random sample of Rochester, MN, women and men 21–97 years of age, indices of bone strength, flexural rigidity (EI), and axial rigidity (EA) were estimated from central QCT measurements at the femoral neck and lumbar spine and pQCT measurements at the ultradistal radius, whereas habitual skeletal loading was assessed using lean body mass, total skeletal muscle mass (TSM), body weight, and physical activity. Using regression analysis, we tested the hypothesis (Ho) that bone strength per unit load did not vary with age. Results and Conclusions: In these cross-sectional data, the null hypothesis of no age-related change was rejected in 72% of the strength-to-load ratios tested. For example, the ratio of femoral neck EI to TSM increased by 0.19%/year in women (p =0.008) and by 0.49%/year in men (p < 0.001). There was no close correspondence between changes in habitual load and changes in bone strength, nor any consistent pattern. Moreover, interindividual variation in the strength-to-load ratios was substantial. These data suggest that the notion of reduced skeletal loading as the primary basis for age-related bone loss is oversimplified.

Abstract To assess fractures in monoclonal gammopathy of undetermined significance (MGUS), the precursor of multiple myeloma, we followed 488 Olmsted County, MN, residents with MGUS in a retrospective cohort study. There was a 2.7-fold increase in the risk of axial fractures but no increase in limb fractures. The pathophysiologic basis for the increased axial fractures should be determined. Introduction: Multiple myeloma is often preceded by monoclonal gammopathy of undetermined significance (MGUS). Fractures are common in myeloma as a result of lytic bone lesions, generalized bone loss, and elevated bone turnover from excessive cytokine production. Whether fractures are also increased in MGUS is unknown. Materials and Methods: In a population-based retrospective cohort study, 488 Olmsted County, MN, residents with MGUS first diagnosed in 1960–1994 (52% men; mean age, 71.4 ± 12.8 years) were followed for 3901 person-years; follow-up was censored at progression to myeloma. The relative risk of fractures was assessed by standardized incidence ratios (SIRs), and risk factors were evaluated in proportional hazards models. Results and Conclusions: Altogether, 200 patients experienced 385 fractures. Compared with expected rates in the community, statistically significant increases were seen for fractures at most axial sites, for example, vertebrae (SIR, 6.3; 95% CI, 5.2–7.5). There was a slight increase in hip (SIR, 1.6; 95% CI, 1.2–2.2) but not distal forearm fractures (SIR, 0.8; 95% CI, 0.4–1.5). The relative risk (SIR) of any axial fracture was 2.7 (95% CI, 2.3–3.1) compared with only 1.1 (95% CI, 0.9–1.4) for all limb fractures combined. In a multivariate analysis, the independent predictors of any subsequent fracture were age (hazard ratio [HR] per 10-year increase, 1.4; 95% CI, 1.2–1.6) and corticosteroid use (HR, 1.8; 95% CI, 1.2–2.6); greater weight at diagnosis (HR per 10 kg, 0.8; 95% CI, 0.8–0.9), and IgG monoclonal protein (HR, 0.7; 95% CI, 0.5–0.97) were protective. Baseline monoclonal protein level, a determinant of myeloma progression, did not predict fracture risk. Thus, the risk of axial, but not peripheral, fractures is increased among MGUS patients even before progression to myeloma. The pathophysiologic basis for this should be determined because elevated bone turnover, for example, might be treatable.