Patients with dermatomyositis according to the Bohan and Peter criteria [13, 14] and regular follow-up at the Department of Dermatology, Medical University of Vienna were invited to participate in this cross-sectional study if the diagnosis was verified by muscle biopsy and if the patients were at least 18 years of age. To be eligible, participants had to be able to follow the study protocol. Excluded were patients who had a malignant disease within the previous five years, had to undergo surgery within the past three months, were immobilized, or had renal or liver insufficiency. Ethical approval was provided by the Medical University of Vienna Ethics Committee and all participants gave written informed consent to this study after the procedure of the study was explained to them.
Anthropometric measures of patients with DM either on standard therapy (glucocorticoids and methotrexate) or intravenous immunoglobulin (IVIG) treatment were performed. Standing height was measured in stocking feet to the nearest centimeter using a stadiometer, and weight was measured using a balance beam scale, recalibrated monthly. Afterwards venous blood samples were drawn. To eliminate diurnal variations in biochemical variables sample collection was performed in the morning. After centrifugation of the whole blood serum was immediately frozen and stored at -70 degrees until assayed. All samples were handled in a single batch run. Additionally, muscle strength was measured and patients conducted three different functional tests.
Serum levels of C-reactive protein (CRP), creatinine kinase (CK), and aldolase were measured to give information on disease activity. Basic serum chemistry included calcium, phosphate, creatinine, parathyroid hormone, and 25-hydroxvitamin D (25OHD). Analysed bone formation markers were bone specific alkaline phosphatase (BAP; Liaison Analyzer, DiaSorin Inc., USA, detection limit: 0.1 µg/L; intra-assay coefficient of variation: 3.3 – 4.3 %, inter-assay coefficient of variation: 6.1 – 8.1 %), osteocalcin (Oc; Cobas 8000 Analyzer, Roche Diagnostics, Switzerland, detection limit: 0.01 ng/mL; intra-assay coefficient of variation: 0.9 – 1.3 %, inter-assay coefficient of variation: 1.2 – 2.3 %), and N-terminal propeptide of type I collagen (P1NP; Cobas 8000 Roche Analyzer, Roche Diagnostics, Switzerland, detection limit: 5 ng/mL; intra-assay coefficient of variation: 1.6 – 3.5 %; inter-assay coefficient of variation: 2.0 – 3.8 %). Cross-linked-C-telopeptide of type I collagen (CTX; Cobas 8000 Roche Analyzer, Roche Diagnostics, Switzerland, detection limit: 0.5 ng/mL; intra-assay coefficient of variation: 1.2 – 4.7 %, inter-assay coefficient of variation: 1.5 – 5.7 %) was the determined bone resorption marker. All analyses were conducted according to standard procedures.
Additionally, the following musculoskeletal markers were evaluated: myostatin (MSTN, colorimetric competitive immunoassay, Immundiagnostik, Bensheim, Germany, limit of blank LoB: 0.370 ng/ml; intra-assay coefficient of variation: <12 %, inter-assay coefficient of variation: <14%, according to manufacturer’s data), follistatin (FSTN, colorimetric sandwich immunoassay, R&D Systems, Minneapolis, USA, MDD range 0.005-0.068 ng/mL; mean MDD 0.016 ng/mL; intra-assay coefficient of variation: <3 %, inter-assay coefficient of variation: <10%, according to manufacturer’s data), sclerostin (SOST, BI-20492, colorimetric sandwich immunoassays, Biomedica, Vienna, Austria; detection limit: 3.2 pmol/l; intra-assay coefficient of variation: ≤7 %, inter-assay coefficient of variation: ≤10%, according to manufacturer’s data), dickkopf 1 (Dkk 1; BI-20412, colorimetric sandwich immunoassays, Biomedica, Vienna, Austria; detection limit: 0.38 pmol/l; intra-assay coefficient of variation: ≤8.0 %, inter-assay coefficient of variation: ≤12.0 %, according to manufacturer’s data), and periostin (PSTN; SEH339Hu, colorimetric sandwich immunoassays, Cloud-Clone-Corp, Houston, USA; detection limit: 0.068 ng/ml; intra-assay coefficient of variation: ≤10 %, inter-assay coefficient of variation: ≤12%, according to manufacturer’s data). Receptor activator nuclear factor kB ligand (RANKL) and osteoprotegerin (OPG) were measured using a commercially available immunoassay from Biomedica, Austria (OPG detection limit: 0.14 pmol/l; intra-assay CV: 4-10%, inter-assay coefficient of variation: 7-8%; RANKL detection limit: 0.08 pmol/l; intra-assay CV: 3-5%, inter-assay coefficient of variation: 6-9%). Serum levels of fibroblast growth factor 23 (FGF23) were determined by an ELISA targeting the C-terminal end of the molecule (Biomedica, Vienna, Austria). The detection limit of the assay is 0.08 pmol/L with an intra-assay coefficient of variation of ≤12%.
The biomarkers were also assessed in a healthy age matched control group. All subjects of the control group were part of a previously published population-based cohort .
Hand-held muscle strength measurement has been shown to be a feasible and reliable method for evaluation of muscle strength in dermatomyositis patients with an intraclass correlation coefficient between 0.88 and 0.98 as well as excellent internal reliability (r=0.91-0.98) and consistency (Cronbach’s alpha 0.78-0.97) [16, 17]. Thus, maximal isometric muscle strength of shoulder abduction (SAb), elbow flexion (EF), elbow extension (EE), hip flexion (HF), and knee extension (KE) was evaluated with the handheld dynamometer Hoggan microFET2 ® (Hoggan Scientific, LLC, Salt Lake City UT, USA). Stabilization specifics used for each muscle action were similar to those performed by Andrews and coauthors . Within 2 seconds the patient tried to reach maximal isometric strength and held that level for another 4-5 seconds. The mean value of the left and right sided maximal strength was used for statistical analysis.
Physical examination included three different functional tests. The chair rising test (CRT) was used to assess leg power; it measures the time an individual needs to stand up and sit down five times as quickly as possible from a chair of standard height (46 cm seat height) with his/ her arms fold across the chest . Mobility performance was evaluated by the timed up and go test (TUG), a test which measures, in seconds, the time it takes the individual to stand up from a chair, walk a distance of 3m, turn around, walk back to the chair, and sit down again . Completion of both tasks was timed from the beginning of the manoeuvre until the patient was re-seated. To assess functional exercise capacity the distance covered within 3 minutes of walking at self-selected speed corresponding a perceived exertion rate of 13 (“somewhat hard”) on a level course was collected (3-minute walking test; 3MWT) .
Data are presented as medians and quartiles. Statistical analysis of between group differences concerning the serum levels of the biochemical markers as well as the parameters of physical function was performed with the Mann Whitney U test. Spearman’s rank correlation coefficient was used to identify potential associations between FGF23 and biochemical markers, between MSTN and muscle strength as well as physical function, and between maximal muscle strength and physical function. Significance was set at p-values less than 0.05. Statistical analyses were done using the software packages GraphPad Prism 5 (Prism 5 für Windows, Version 5.00, 2007) and SPSS Statistics V21 (SPSS Inc., Chicago, IL, USA, 2012).