Serum vitamin D status inversely associates with a prevalence of severe sarcopenia among female patients with rheumatoid arthritis

Sarcopenia is an age-related disease with an increased risk of mortality. It is emerging that low serum 25-hydroxyvitamin D [25(OH)D] affects the sarcopenic state in general, but in rheumatoid arthritis (RA), these associations are not understood although the prevalence of vitamin D insufficiency is high in RA. We conducted a cross-sectional study of older female outpatients from our cohort (KURAMA) database. We measured skeletal muscle mass, handgrip strength, and gait-speed to diagnose severe sarcopenia. The serum 25(OH)D concentration was measured using electrochemiluminescence immunoassay. A total of 156 female patients with RA (sarcopenia:44.9%, severe sarcopenia: 29.5%, and without sarcopenia: 25.6%) were enrolled. Classification of vitamin D status at a cutoff point of median 25(OH)D concentration revealed that low 25(OH)D status was associated with a high prevalence of severe sarcopenia and with low measured values of muscle mass, handgrip, and gait speed. Furthermore, multivariable logistic regression analysis identified that low 25(OH)D status was associated with a high prevalence of severe sarcopenia (OR 6.00; 95% CI 1.99–18.08).The same association was observed when the cut-off value was set at 20 ng/ml. In components of sarcopenia, both low physical performance and muscle mass were associated with low 25(OH)D status. In conclusion, vitamin D status was inversely associated with severe sarcopenia, low physical performance, and low skeletal muscle mass. Modification of vitamin D status including vitamin D supplementation should be investigated as a therapeutic strategy for sarcopenic patients with RA.

www.nature.com/scientificreports/ in physical performance 8,9 , is highly prevalent in RA patients compared to the general populations 10,11 . There have been conflicting results as to whether Disease Modifying Anti-Rheumatic Drugs (DMARDs) and biological agents improve the sarcopenic state in RA patients 10,12 ; there is also little evidence on co-adjuvant therapies such as nutritional interventions in these patients.
Recently, vitamin D supplementation is receiving much attention as a potential therapeutic intervention for preventing sarcopenia in the general population together with protein supplementation and exercise. Vitamin D may well ameliorate the sarcopenic state via its role in muscle cell regulation, anti-inflammatory pathways and/or immunomodulatory responses 13 , as vitamin D status is associated with muscle strength and physical performance 14,15 . Indeed, meta-analyses and RCTs have shown that vitamin D supplementation improves limb strength in the community-dwelling elderly 16,17 . Interestingly, over 70% of RA patients exhibit vitamin D insufficiency 18 ; these findings suggest the importance of adequate vitamin D provision on sarcopenia in RA populations as in general populations. However, it remains unclear whether serum vitamin D concentration affects the sarcopenic state in RA patients.
To elucidate the clinical association between vitamin D deficiency and sarcopenia in elderly patients with RA, we performed a cross-sectional study using the cohort study database, KURAMA cohort, established in 2011 for the storage of clinical data and specimens obtained from RA patients.

Methods
Study design and participants. We conducted a cross-sectional study in female RA outpatients from the Kyoto University Rheumatoid Arthritis Management Alliance (KURAMA) cohort database, which has been described in detail elsewhere 19,20 . We recruited older RA participants who visited the Kyoto University Hospital from May 2014 to December 2014 and who were over 60 years of age. All participants fulfilled the diagnostic criteria of the ACR/EULAR RA classification 21 . Data for 156 out of 248 participants was subjected to the planned analyses. 10 were excluded because of incomplete data set (lack of preserved serum or parameters of sarcopeniarelated factors) and 82 were excluded because of the use of vitamin D supplementation (calcitriol such as eldecalcitol and alfacalcidol).
Ethics. The study protocol and procedures were approved by the Medical Ethics Committee of Kyoto University Graduate School and Faculty of Medicine (Approval number: R0357) and complied with the principles of the Declaration of Helsinki. We obtained written informed consent was obtained from all patients of this study, which included the use of human blood samples and data.
Diagnosis of sarcopenia and estimation of the related parameters. We measured muscle mass, muscle strength and physical performance to assess sarcopenia status in the study population, as previously described 10,22 . In brief, skeletal muscle mass was measured by bioelectrical impedance analysis (BIA) (Inbody 720: Biospace Co., Ltd., Seoul, Korea). Skeletal muscle index (SMI) was computed from the limb skeletal muscle mass in kilograms divided by the square of height in meters (kg/m 2 ). Handgrip strength was measured using JAMAR digital hand dynamometer (Patterson Medical, Bolingbrook, IL). Gait speed was also measured by the 6-m walking speed using a portable gait rhythmogram (MG-M1110: LSI Medience Co., Tokyo, Japan) Regarding muscle strength and gait speed, the mean value of duplicate measurements was used for analysis.
Diagnosis of sarcopenia and severe sarcopenia were based on the criteria of the Asian Working Group for Sarcopenia (AWGS) 2019 23 . Briefly, sarcopenia was defined as low muscle mass with low muscle strength or with low physical performance; severe sarcopenia was defined as low muscle mass with both low muscle strength and low physical performance. Cut-offs values for low muscle mass, low muscle strength, and low physical performance were SMI < 5.7 kg/m 2 , handgrip strength < 18 kg, and gait speed < 1.0 m/s, respectively.

Evaluation of 25(OH)D concentration and the clinical parameters. Serum 25(OH)D, an estab-
lished biomarker reflecting vitamin D status, was measured using electrochemiluminescence immunoassay (LSI Medience Co., Tokyo, Japan). This laboratory used an external quality control provided by DEQAS , and the intra-assay CV varied between 2.64 and 5.65% across the range of 25(OH) concentration, between 12.87 and 59.16 ng/ml. MNA-SF (Mini Nutritional Assessment Short-Form) was collected to assess nutritional state 24 . RA disease activity and physical dysfunction were assessed by a 28-Joint RA Disease Activity Score (DAS28-ESR), the doctor or patient Visual Analogue Scale (Dr. or PT-VAS), Steinbrocker's stage and class and the health assessment questionnaire disability index (HAQ). We also reviewed the information regarding current RA therapeutics including methotrexate, prednisolone, and biological agents (TNF inhibitors: n = 33, IL-6 receptor inhibitor: n = 12, CTLA4-immunoglobulin: n = 10). Other epidemiologic and anthropometric variables including age, duration of RA disease and body mass index (BMI) were extracted from the KURAMA database. Information on falls and fractures in the last year was collected from all subjects by a self-reported questionnaire form. The use of osteoporosis medication was obtained from the electronic medical record.
Statistical analysis. We present continuous variables as the mean (standard deviation (SD)) or as the median (interquartile range (IQR)) and categorial variables as numbers (%). To compare baseline characteristics according to 25(OH)D status, we divided participants into the following two groups by the median of serum 25(OH)D concentration: lower status group (25(OH)D: 5.9-16.0 ng/ml); higher status group (25(OH) D: 16.1-32.1 ng/ml). We then performed a Mann-Whitney's U test or a Fisher's exact test for continuous variables and categorical variables, respectively. To explore the relationship between 25(OH)D status and severe sarcopenia, univariate and multivariate logistic regression analyses were conducted. 25(OH)D status was used as either a binary variable as described above, a binary variable with a cutoff value of 20 ng/ml, which value is used www.nature.com/scientificreports/ for an indicator of bone health 25 , or a continuous variable in multivariate analysis. In multivariate analyses, we constructed the following multiple models by incorporating significant variables in the univariate analysis and clinically relevant variables including RA therapeutics 10,26 : model 1 was adjusted for 25(OH)D status, age, and body mass index; model 2 was adjusted for variables in model 1 plus nutrition status (MNA-SF) and RA-related factors (DAS28-ESR, Stage 3 and 4 vs. 1 and 2, HAQ, and therapeutics (use of prednisolone, biologics, and methotrexate)); model 3 was adjusted for variables in model 2 plus the prevalence of osteoporosis medication, as RANKL inhibitors can exert a positive effect on muscle strength 27 . We also adopted multivariate logistic regression analysis using the same models for each component of severe sarcopenia (low muscle mass, low muscle strength, and low physical performance). JMP 15.2.0 (SAS Institute Inc., Cary, NC, USA) was used for statistical analyses; a value of P < 0.05 was considered significant.

Results
Participant characteristics of this study are provided in Table 1. Data for 156 female RA patients with a mean (SD) age of 69.7 (6.7) was subjected to the following analysis. The median (IQR) serum 25(OH)D concentration was 16.0 (12.8-19.2), which represents a lower concentration than that in the general population 28,29 and accords with that in other RA studies 18 . As for RA-related factors, the average (SD) duration of RA was 16.1 years (12.7). Disease activity of RA (DAS28-ESR) was generally low under the following therapeutics: methotrexate in 67.3%, prednisolone in 27.6%, and biological agent in 35.3%, but about two-thirds of the patients had advanced joint deformity or destruction as assessed by a Steinbrocker's stage of 3 (21.1%) or 4 (46.8%). Of these participants, on the basis of the diagnostic criteria of sarcopenia in AWGS 2019 23 , 46 (29.5%) were determined to have severe sarcopenia and 70 (44.9%) were determined to have sarcopenia. Regarding the components of sarcopenia, mean (SD) of SMI, handgrip strength and gait speed were 5.64 (0.83), 14.5 (7.2) and 0.95 (0.29), respectively. Under these conditions, 50.6%, 67.3%, and 50.6% of the participants fulfilled the Table 1. Characteristics of this study population. Data are presented as the mean (standard deviation (SD)) or as the median (interquartile range (IQR)) for continuous variables, and as numbers (%) for categorial variables. RA rheumatoid arthritis, MNA-SF Mini Nutritional Assessment Short-Form, DAS28 disease activity score using 28 joints, VAS visual analogue scale, HAQ health assessment questionnaire. *Steinbrocker's classification.   Table S1). Similarly, when 25(OH)D was used as a binary variable with a cutoff value of 20 ng/ml, lower 25(OH)D was also significantly associated with severe sarcopenia in all models (OR 3.87; 95% CI 1.14-13.1: model 1) (Supplementary Table S2).

25(OH)D status associates both with physical performance and skeletal muscle mass.
We then conducted further multivariate analyses to clarify which components of severe sarcopenia were most strongly associated with low serum 25(OH)D status. Regarding the components of severe sarcopenia, both low physical performance and low skeletal muscle mass were significantly associated with low serum 25(OH) D status in model 1 (physical performance: OR 2.65; 95% CI 1.34-5.23, skeletal muscle mass: OR 2.54; 95% CI 1.10-5.88) (Fig. 1). The same associations were maintained in the other models (model 2 and 3). Other variables are described in supplementary Table S3. There was no significant association with muscle strength. Low serum 25(OH)D is therefore independently associated with a high prevalence of severe sarcopenia and with both low physical performance and low skeletal muscle index mass among the measures used to assess sarcopenia.

Discussion
The present study is the first to document a significant association between vitamin D deficiency and severe sarcopenia in a female RA population. Our participants had a high prevalence of severe sarcopenia (29.5%) and vitamin D deficiency (median 16.0 ng/ml); multivariate analyses showed low serum 25(OH)D to be an independent risk for severe sarcopenia (OR 4.42). In addition, among the three components of sarcopenia, decreased gait speed and muscle mass showed significant associations with low serum vitamin D. These findings accord with those in general population studies: low serum vitamin D has a stronger association with lower function on walking than on other physical performance or muscle strength 32,33 . Our results suggest that vitamin D deficiency may contribute to the development of severe sarcopenia and impaired lower limb performance and skeletal muscle in RA patients, as is the case in the general population (Fig. 2).
Recently, basic studies have provided insight into the mechanism of vitamin D action on skeletal muscle function, including modulation of muscle differentiation, oxidative stress, cellular metabolism and inflammatory condition 13,34 . The expression of vitamin D receptors in muscle nuclei decreases with aging, which affects muscle differentiation and is implicated in the development of sarcopenia 35 . Vitamin D deficiency also contributes to skeletal muscle atrophy via dysregulation of cellular metabolism such as oxidative stress and calcium homeostasis resulting in mitochondrial function 36,37 . Furthermore, vitamin D is well known for its anti-inflammatory and immunomodulatory properties 38 . It suppresses Th1, Th17 and macrophage cytokines (IL-2, IFN-γ, IL-17, IL-21, TNF-α, IL-6, IL1-β) as well as innate immune responses such as toll-like receptor signaling and antigen presentation 39,40 ; it promotes the expression of Th2 cytokines (IL-4, IL-5, IL-10, IL-13) and the differentiation of regulatory T cells 41,42 . Given that Th1 and Th17 responses participate in the pathogenesis of RA 43 , vitamin D www.nature.com/scientificreports/ might well ameliorate sarcopenia in RA patients partly through attenuating chronic inflammation that results in muscle catabolism as well as through modulating muscle function via multiple physiological pathways 13,36,37 . Similarly, epidemiological studies have shown clinical correlations between vitamin D deficiency and sarcopenia. In general populations, low serum vitamin D concentration significantly associates with increased prevalence of sarcopenia and loss of physical performance such as walking speed 14,15 . Several meta-analyses   www.nature.com/scientificreports/ and RCTs have also shown the positive effects of vitamin D supplementation such as improvement in global muscle strength (especially lower limb muscles) and a decrease in the sit-to-stand time 16,17 . In RA populations, however, the relationship between sarcopenia and vitamin D status has not been clarified even though the prevalence of sarcopenia and vitamin D insufficiency is markedly higher in the RA population than it is in the general population 6,44 . The present study provides novel evidence that low serum 25(OH)D is a significant risk factor of severe sarcopenia in RA. Taking these findings together, vitamin D deficiency may well be a candidate co-adjuvant therapeutic target to prevent severe sarcopenia in RA patients; prospective studies of vitamin D supplementation to prevent sarcopenia in RA will be worthwhile. There are several limitations in the present study. The present cross-sectional study does not imply causation and further prospective investigation is needed. There is also the possibility of selection bias because the study included only female patients older than 60 years, although these are the dominant populations with RA. We excluded vitamin D supplementation as a confounding factor in the present study, which might produce a biased group of subjects: our findings are applicable only to RA patients without supplementation therapy. In addition, there might remain unadjusted confounding variables related to vitamin D status and sarcopenia including dietary profile, seasonal variation and socioeconomic status, and information on falls and fractures was obtained by personal recall.
In conclusion, female RA patients with low serum 25(OH)D concentration has a significant risk for a higher prevalence of severe sarcopenia, low physical performance, and low skeletal muscle mass. The interventions to improve serum 25(OH)D concentration including vitamin D supplementation could be beneficial for RA patients with sarcopenia.