Improving Muscle Mass Measurement Using Bioelectrical Impedance Spectroscopy

Abstracts Aims: MicroRNAs (miRNAs) are short noncoding RNAs that interfere with translation of specific target mRNAs (1). Recent studies have suggested that miRNAs might play a role in osteoblast differentiation and bone formation (2). The main aim of this study was to uncover miRNA candidates with differential expression in medicarpin (positive regulator of bone formation) induced calvarial osteoblast cells, and determine the role of miR-376c in osteogenesis. Methods: miRNA expression pattern in control and medicarpin treated cells was analyzed by miRNA microarray and quantitative RT-PCR. Effect of miR-376c on osteoblast differentiation and mineralization was validated by transfection of miR376c and its anti-miR in mice osteoblast cells using biochemical assays and real time qPCR. Luciferase reporter gene assays were performed to identify miR376c targets. ARFGEF1, Wnt3, GSK-3b and b-catenin protein levels were determined by western blotting and chemiluminescence. Results: miRNA array profiling in medicarpin induced mice osteoblast cells revealed that miR-376c was significantly downregulated during osteoblast differentiation. Overexpression of miR-376c suppressed osteoblast differentiation and mineralization while antimiR-376c abolished these effects. Target prediction tools and experimental validation identified ARFGEF1 as a direct target of miR-376c and activation of ARFs promotes the dissociation of membrane bound b-catenin (3), thus leading to b-catenin translocation and subsequently lead to active transcription of osteogenic genes. Overexpression of miR-376c inhibited Wnt3, LRP5/6 and b-catenin expression, whereas inhibition of miR-376c promoted expression of Wnt signaling pathway genes. Conclusion: miR-376c down regulates ARFGEF1 and Wnt3, which are important for b-catenin translocalization and stabilization for active transcription. Our findings suggest that silencing of miR-376c by anti-miR-376c could result in a therapeutic strategy for enhancing bone formation References: 1. Eskildsen T, et al. Proc Natl Acad Sci USA 2011;108:6139. 2. Hu R, et al. Expert Opin Ther Targets 2010;14:1109. 3. Palacios F, et al. Nat Cell Biol 2002;4:929. Disclosure of Interest: None Declared

O12 A NOVEL C-GLUCOSIDES OF KAEMPFEROL ISOLATED FROM THE STEAM-BARK OF ULMUS WALLICHIANA PLANCHON STIMULATES OSTEOBLAST DIFFERENTIATION BY MODULATING CYTOKERATIN 14 LEVELS THROUGH MTOR/AKT SIGNALLING V. Khedgikar*, P. Kushwaha, J. Gautam, R. Maurya, N. Chattopadhyay, R. Trivedi; CSIR-Central Drug Research Institute, Lucknow, India Aims: To study how a natural c-glycoside analogue of keampferol AG promotes osteoblast differentiation and mineralization by modulating intermediate filament cytokeratin 14 (Krt-14) levels. Methods: To understand the contribution that Krt-14 makes to matrix maturation and mineralization in presence of AG, we used RNAi against Krt-14 that resulted in reduced mRNA and protein expression as early as 72 h and remained low for 9 days in rat primary osteoblast cells without affecting cell number. Results: In search of more potent analogue of kaempferol (K) we screened AG(2S,3S)-aromadendrin-6-C-b-d-glucopyranoside, quearcitin (Q), morin hydrate (MH), galangenin (G), feistin (F), myrecitin(M), eupalatin 3-O-b-D-galactoside (EG) for stimulation of alkaline phosphatase activity and cytokeratin-14 (Krt14) to assess osteoblastogenesis. Compared to all AG a natural analogue of K increases osteoblast differentiation, matrix maturation and mineralization by modulating cytoskeleton rearrangement. AG at nanomolar concentration increases matrix maturation and mineralisation by enhancing mRNA expression of collagen I and osteocalcin, collagen levels and osteocalcin levels in conditioned as well as acid-salt soluble fraction compared to K. Introduction of Krt-14 siRNA resulted in reduced mRNA and protein expression of Krt-14 as early as 72 h and remained low for 9 days in rat primary osteoblast cells. At day 9 mineralization was significantly reduced in cells transfected with Krt-14 siRNA without concomitant change in the cell number 72 h post transfection. ColI and OCN gene expression was reduced in Krt-14 siRNA-treated cultures. Soluble osteocalcin levels and levels of osteocalcin in the cell-ECM layer were markedly decreased in the condition medium of cells treated with Krt-14 siRNA than in control siRNA treated cells. Krt-14 reduction also elicited changes in the distribution of collagen between the acid soluble cell ECM protein fraction and the insoluble matrix. Since mTORC1 controls the actin cytoskeleton and thereby determines the shape of the cell, analysis of this pathway revealed that Akt directly phosphorylated mTOR and activated effectors downstream of mTOR. Further, knockdown of

401 Krt-14 in presence of AG, repressed the activation of mTOR by repressing phosphorylation of mTOR’s downstream targets S6 kinase. Conclusion: These findings strongly suggest that compared to all analogue of K, AG enhances expression of Krt-14 to regulate osteoblast mineralization by facilitating proper organization of the osteoblast derived ECM. References: Proteomics 2010;10:1730; DOI:10.1002/jbmr.434 Acknowledgement: Grant from CSIR (BSC0201) is acknowledged. VK, JG acknowledge University Grants Commission, New Delhi, Govt. of India for fellowship, PK acknowledge Council of Scientific and Industrial Research CSIR, New Delhi, Govt. of India for fellowship. Disclosure of Interest: None Declared

O13 IMPROVING MUSCLE MASS MEASUREMENT USING BIOELECTRICAL IMPEDANCE SPECTROSCOPY Y. Yamada1,*, B. Buehring2, D. Krueger2, N. Binkley2, D. A. Schoeller1; 1 Nutritional Science, 2Osteoporosis Clinical Research Program, University of Wisconsin-Madison, Madison, United States Aims: Loss of leg muscle mass and quality can reduce mobility in the aged. Detecting such loss is challenging as the leg also includes bone, adipose, vascular material, skin and other components that confound measurement of muscle tissue because they include some fat-free mass. DXA has often been used to evaluate these effects by subtracting bone and fat tissue, but DXA cannot distinguish intracellular (ICW) and extracellular water (ECW); ICW may be a more specific marker of muscle tissue. The aim of this study was to assess the efficacy of a bioelectrical spectrometric (BIS) measure of ICW to assess muscle atrophy in adults in comparison to DXA. Methods: Fifty-seven healthy adults (44 women and 13 men, 26-76 yr old) were studied. Total body DXA was performed using a GE Lunar iDXA. Resistance of ICW (RICW) and ECW (RECW) components were measured by segmental BIS to estimate total water (TW) and ICW and ECW. Leg muscle power was measured by jumping mechanography, and grip strength was assessed by dynamometer. Correlation coefficients were statistically compared using Meng’s statistical method. Results: DXA leg lean mass (LLM) was only weakly correlated with age (r5-0.35) while BIS leg ICW displayed a significantly stronger correlation (r5-0.51, P50.01). Moreover, BIS indicated a greater percent decrease with age than did DXA (12.8% vs. 6.7% per decade). The ratio of BIS ECW to ICW increased with age (r50.72, P!0.001) indicating that the relative expansion of ECW with age may cause of the lower sensitivity of DXA to assess age effects. To compare the specificities of BIS and DXA to leg muscle function, we compared DXA LLM and BIS ICW to leg muscle power. BIS ICW correlated better with muscle power than DXA LLM (r50.88 vs. r50.79, P50.007). The ratio of ICW/TW was a significant predictive variable independently of DXA LLM for predicting leg muscle power (P!0.001). In addition, stepwise multiple regression analysis found that age entered into the regression equation predicting leg muscle power from DXA LLM (R250.75), whereas age did not enter into the prediction of leg muscle power from BIS ICW (R250.85).

Fig. (A) The decreased ratio of RECW to RICW indicates the relative expansion of ECW in leg mass during aging. (B) BIS leg ICW was strongly correlated with leg muscle power. Conclusion: In this cross-sectional study, these data indicate that DXA measured LLM underestimates age related effects on skeletal muscle mass and function.

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Volume 17, 2014

402 This results from ECW masking the lower volume of leg muscle mass. Further research is needed to determine whether BIS should replace DXA as a measure of muscle mass function and function or if a combination of the two methods may perform better than either alone as a way to evaluate risk of adverse health outcomes such as falls and fractures. References: Yamada et al. (2010) J Gerontol 65A:510; Yamada et al. (2013) J Appl Physiol 115:812; Buehring et al. (2010) J Clin Densitom 13:283; Buehring et al. (2013) J Am Geriatr Soc 61:418; Yamada et al. (In Press) J Appl Physiol Acknowledgement: YY was supported by Fellowships of the Japan Society for the Promotion of Science (JSPS) for Young Scientists (23-333) Disclosure of Interest: None Declared

O14 HIGH-RESOLUTION PERIPHERAL QUANTITATIVE COMPUTERIZED TOMOGRAPHY (HR-PQCT) DESCRIBES CHARACTERISTICS OF BONE FRAGILITY IN PREMENOPAUSE CELIAC DISEASE PATIENTS M. B. Zanchetta1,2,*, V. Longobardi1,3, F. Costa4, J. C. Bai4, F. Silveira1, H. Vazquez5; 1IDIM, 2C atedra de Osteologıa, 3Universidad del Salvador, 4 Secci on de Intestino Delgado, Departamento de Medicina, 5Dr. C. Bonorino Udaondo Hospital de Gastroenterologıa, Buenos Aires, Argentina Aims: Describe radius and tibia bone micro architecture assessed by HR-pQCT in a group of adult premenopausal women with recently diagnosed celiac disease (CD). Methods: We prospectively included 31 premenopausal women with newly diagnosed CD. They underwent BMD (Lunar Prodigy Advance) of lumbar spine (LS), femoral neck (FN) and ultra distal radius (UD); distal radius and tibia HR-pQCT scans (Xtreme-CT; Scanco Medical AG, Bassersdorf, Switzerland), specific chemistry (calcium, vitamin D, serum intact PTH, serum C-telopeptide); dietary recall and questionnaire for osteoporosis risk factors. A group of 22 premenopausal healthy women matched for age and BMI was used as a control group. The groups were analyzed using Student’s t test or Rank Sum Wilkinson. Results: According to the study design there were no differences in age and BMI (p: NS) between groups. The average age of patients with CD was 30.38.5 and the mean BMI was 22.95.8. Z- score values in LS, FN and UD radius in the CD group were significantly lower compared to healthy women (meanSD): -0.50.9 vs. 0.20.7 (p50.003), -0.20.8 vs. 0.30.8 (p50.03), -1.91.3 vs. -0.51.1 (p50.001), respectively. Specific laboratory results in patients with CD were (meanSD): 20.225OH vitamin D; C-telopeptide 614.1737.9, PTHi 64.634.6. In the radius structural parameters (bone volume BV/TV: 9.92.6 vs. 13.52.8 p!0.0001; trabeculae number: 1.690.27 vs. 1.890.26 p50.009; trabecular thickness: 0.0580.010 vs. 0.0710.008 p!0.0001) and volumetric (trabecular density: 11931.5 vs. 16234 p!0.0001; cortical density: 86057 vs. 893.943 p50.02) in patients with CD were significantly lower. In the tibia the results were similar. Cortical thickness was not affected in any of the regions tested. Conclusion: In this group of young women with newly diagnosed CD, despite lumbar spine and femoral neck BMD values within the normal range for age, we observed a severe compromise of volumetric density and bone microarchitecture predominantly in trabecular bone. These observations may explain the higher prevalence of fragility fractures described in these patients. In the prospective follow-up of this group of patients we would be able to assess if bone microarchitecture recovers after gluten-free diet. Disclosure of Interest: None Declared

O15 BONE MICROSTRUCTURE ASSESSED BY TBS AND HRPQCT IN PATIENTS WITH OSTEOGENESIS IMPERFECTA R. Kocijan1,*, C. Muschitz1, J. Haschka1, A. Zendeli1, S. Finzel2, G. Schett2, H. Resch1; 1Medical Department II, The Vinforce Study Group - St. Vincent Hospital, Vienna, Austria, 2Department of Internal Medicine 3, University of Erlangen-Nuremberg, Erlangen, Germany Aims: The aim of this study was to evaluate bone microstructure as well as areal and volumetric BMD in adult patients with different types of osteogenesis imperfecta at different skeletal sites.

Abstracts Methods: We investigated 23 adult patients with OI (11 female, 12 male, mean age 45.716.4 years). OI was divided into the mild OI-I and the moderate-severe OI-III-IV group. Bone microarchitecture and volumetric BMD (vBMD) were determined by HR-pQCT (SCANCO Medical) at the distal radius and tibia. HR-pQCT data were compared to 23 healthy, age and gender matched controls (CO, mean age 42.59.9 years). Trabecular bone score (TBS) at the lumbar spine (LS) and aBMD by DXA at the LS, hip and DXL at the calcaneus (Calscan, dual X-ray and laser) were carried out in patients with OI-I and OIIII-IV. Results: At both, radius and tibia, bone volume fraction (BV/TV) and trabecular number (Tb.N) were significantly decreased in OI-I and OI-III-IV when compared to CO (p!0.005-0.001). The inhomogeneity of the network and the trabecular separation (Tb.Sp) were increased in OI-I (p!0.05-0.001) and OIIII-IV (p!0.001 for all) compared to CO. In addition, BV/TV, Tb.N, Tb.Sp and inhomogeneity were significantly worse in OI-III-IV compared to OI-I. No differences were found regarding cortical porosity at tibia and radius. Moreover, trabecular, but not cortical vBMD was significantly lower in OI-I (p!0.05) and OI-III-IV (p!0.001) compared to CO. The TBS was significantly lower in moderate-severe OI-III-IV compared to mild OI-I (p!0.05). Correlations were found between TBS and BV/TV (r50.460), Tb.N (r50.518), Inhomogeneity (r5-0.484), trabecular density (r50.461) and BMD of LS (r50.495) and hip (0.720, p!0.05 for all). Differences in aBMD between OI-I and OI-III-IV were higher at the calcaneus (p!0.001), as a mainly trabecular bone, than the hip (p!0.05). Conclusion: Our data suggest deterioration of particularly trabecular bone microstructure and density in mild OI-I and even more in moderate-severe OI-III-IV. HR-pQCT and TBS are useful tools for the assessment of microstructure in OI. Moderate correlations to HR-pQCT approve TBS for microstructure analysis in patients with OI. DXL at the calcaneus might be helpful for aBMD measurements in severe OI with vertebral fractures or scoliosis. Disclosure of Interest: None Declared

Bone Structure, Quality, Architecture, Micro-Architecture P100 ATYPICAL FEMORAL FRACTURES: RADIOGRAPHIC AND HISTOMORPHOMETRIC FEATURES IN 19 PATIENTS A. Khan1,*, A. Cheung2, O. A. Khan1, Z. Rahman1, K. Pritzker3, B. Lentle4; 1 McMaster University, Hamilton, 2General Internal Medicine, 3Laboratory Medicine and Pathology, University of Toronto, Toronto, 4Department of Radiology, University of British Columbia, Vancouver, Canada Aims: This study describes characteristics and histomorphometric and radiographic features of atypical femoral fractures (AFF) as seen in 19 cases referred for evaluation. Methods: All patients referred for evaluation of AFF were reviewed. Patients meeting the ASBMR criteria for AFF were further evaluated and tetracycline labelled bone biopsies were completed. Radiographs were reviewed by a musculoskeletal radiologist. Results: All fracture lines were transverse or short oblique and 15 of 19 patients demonstrated thickened cortices on x-ray. We report 19 cases of AFF in patients on long term bisphosphonate (BP) therapy. 14 of 19 fractures occurred without a fall or direct trauma to the femur with 5 cases occurring after a fall from standing height. All patients were female; average age was 65 years (range 23-80 years). 4 of the 19 cases were of Chinese descent, 4 were East Indian, with 11 being Caucasian. Average BP duration of use was 9.8 years (range 6-15 years). 9 of 19 patients were on alendronate alone, 2 patients were on risedronate alone, 6 patients on a combination and 1 patient on a combination of pamidronate and alendronate. 1 patient was on a combination of alendronate and denosumab. Prodromal thigh or groin pain was seen in 12 of the 19 patients for 1-15 months prior to fracture. Proton pump inhibitor use was present in 6 patients. 2 patients were on prednisone for rheumatoid arthritis and 2 patients on prednisone for asthma. 1 patient had a diagnosis of osteogenesis imperfecta type IV with a history of multiple fragility fractures and had experienced a femoral fracture after 12 years of IV pamidronate with features consistent with an AFF. All patients had 25OH vitamin D levels O50 nmol/L. 18 of the patients with radiographic features of AFF had been on a bisphosphonate for O6 years. 1 patient had been on alendronate for 5 months. 8 of 19 patients had bilateral femoral fractures. Summary: A large number of patients with radiographic features of an AFF had evidence of mineralization abnormalities on tetracycline labelled bone biopsy. Decreased osteoid surface and mean

Journal of Clinical Densitometry: Assessment & Management of Musculoskeletal Health

Volume 17, 2014