Prevalence of vitamin D deficiency in Egyptian rheumatoid arthritis patients: correlation with disease activity, functional disability, and bone mineral density

Introduction Rheumatoid arthritis (RA) is a systemic autoimmune disease of unknown etiology [1]. It is characterized by synovitis, cartilage damage, and bone resorption, resulting in structural damage and significant morbidity and mortality [2]. The role of T and B lymphocytes in the disease pathogenesis has been proven [3]. Vitamin D is a steroid hormone synthesized in the skin by the action of ultraviolet irradiation [4]. It plays a major role in the maintenance of proper bone metabolism [5] because it regulates calcium absorption from the gastrointestinal tract [6]. However, the discovery of the vitamin D receptor in nonskeletal tissue, particularly in immune cells, indicated a possible role for vitamin D in maintaining its normal function [7,8]. Interestingly, vitamin D deficiency has been linked to an increased susceptibility to develop autoimmune diseases such as RA [9], systemic lupus erythematosus, multiple sclerosis, and type-1 diabetes in large population studies [10,11]. A low vitamin D level is also linked to increased disease activity in RA patients [12]. However, the exact mechanism for the inverse relationship between low vitamin D level and increased inflammatory disease activity is still under investigation [12–14].


Introduction
Rheumatoid arthritis (RA) is a systemic autoimmune disease of unknown etiology [1]. It is characterized by synovitis, cartilage damage, and bone resorption, resulting in structural damage and significant morbidity and mortality [2]. The role of T and B lymphocytes in the disease pathogenesis has been proven [3]. Vitamin D is a steroid hormone synthesized in the skin by the action of ultraviolet irradiation [4]. It plays a major role in the maintenance of proper bone metabolism [5] because it regulates calcium absorption from the gastrointestinal tract [6]. However, the discovery of the vitamin D receptor in nonskeletal tissue, particularly in immune cells, indicated a possible role for vitamin D in maintaining its normal function [7,8]. Interestingly, vitamin D deficiency has been linked to an increased susceptibility to develop autoimmune diseases such as RA [9], systemic lupus erythematosus, multiple sclerosis, and type-1 diabetes in large population studies [10,11]. A low vitamin D level is also linked to increased disease activity in RA patients [12]. However, the exact mechanism for the inverse relationship between low vitamin D level and increased inflammatory disease activity is still under investigation [12][13][14].
The aim of the present study was to detect the prevalence of 25-hydroxyvitamin D 3 [25(OH)D 3 ] deficiency in RA patients and its correlation with disease activity, functional disability, and bone mineral density (BMD).

Patients and methods Patients
A cross-sectional, case-control study was carried out that included 104 RA patients who fulfilled the 2010 American College of Rheumatology/European League Against Rheumatism (ACR-EULAR) classification criteria for RA [15] in addition to 60 healthy age-matched and sex-matched controls. All participants were recruited from the Rheumatology and Rehabilitation Clinic, Kasr Alainy Hospitals, Cairo University. Full assessment of history and detailed clinical examination were performed for all patients. Demographic data including patients' age and disease duration were reported. Body weight, height, and BMI (kg/m 2 ) were measured and calculated for all participants. The inclusion criteria were the diagnosis of established RA, age at disease onset above 18 years, and at least 1-year disease duration. Patients or controls on vitamin D supplement or those with endocrine, hepatic, or renal diseases were excluded. Participants provided written informed consent before their inclusion. The current study was approved by the local ethical committees and was carried out in accordance with the ethical standards established in the 1964 Declaration of Helsinki.

Functional disability assessment
The modified-health assessment questionnaire (M-HAQ) was used. Patients were considered normal if the M-HAQ score was 0.3 or less [17].

Treatment
RA-specific treatments were reported for all patients. These drugs were glucocorticoids and diseasemodifying antirheumatic drugs.

Laboratory measurements
Serum 25(OH)D 3 was measured using an enzyme-linked immunosorbent assay. Its level was considered sufficient if it was greater than 75 nmol/l (>30 ng/ml), insufficient if it was between 50 and 75 nmol/l (20-30 ng/ml), and deficient if it was less than 50 nmol/l (<20 ng/ml) [18,19]. ESR (mm/h) was determined using the Westergren method; rheumatoid factor (considered positive if it is >20 IU/ml) was determined using the nephelometric method; and anticyclic citrullinated polypeptide (considered positive if ≥5 U/ml) was determined using a microparticle enzyme immunoassay for all patients.

Radiological investigations
Plain radiography of the hands and wrists, posteroanterior view, was performed for all patients to detect radiological signs of RA.
High-resolution computed tomography of the chest was used to diagnose interstitial pulmonary fibrosis (IPF).
Dual-energy X-ray absorptiometry with advanced fanbeam technology was used to measure BMD according to diagnostic categories proposed by the WHO [20] and modified by the International Osteoporosis Foundation [21]: 1 -normal BMD, hip BMD greater than 1 SD below the young adult female reference mean (T score>−1); 2 -low bone mass (osteopenia), hip BMD greater than 1 SD below the young adult mean, but less than 2.5 SD below this value (T score<−1 and >−2.5); 3 -osteoporosis, hip BMD 2.5 SD or more below the young adult female mean (T score<−2.5); 4 -severe osteoporosis (established osteoporosis), hip BMD 2.5 SD or more below the young adult mean in the presence of one or more fragility fractures.

Statistical analysis
The statistical package for social sciences SPSS (Statistical Package for the Social Sciences; SPSS Inc, Chicago, IL, USA) version 15 for Microsoft windows, version 18 was used to analyze data. Continuous variables were summarized as mean ± SD and categorical variables as frequency (percentage). An independent t-test compared two independent groups and the analysis of variance test compared more than two groups. Associations between categorical groups were tested using the c 2 -test with Yates correction or Fisher's exact test as appropriate. The Pearson correlation test was used as a measure of the association of quantitative variables. Two-tailed P values of 0.05 or less were considered to be statistically significant.

Demographic, clinical, radiologic, and laboratory features of RA patients
Among RA patients, there were 82 (78.8%) women and 22 (21.2%) men. The demographic, clinical, radiologic, and laboratory features of the RA patients at the time of the study are shown in Table 1.
At the time of the study, all patients were on methotrexate at a dose ranging from 12.5 to 20 mg/week, with a mean of 15 ± 2.31 mg/week, 26 (25%) patients were taking leflunomide (20 mg/day), 46 (44.2%) patients were taking hydroxychloroquine (400 mg/day) divided into two doses per day, and 21 (20.2%) patients were on prednisone at a dose ranging from 5 to 10 mg/day, with a mean of 6.5 ± 1.88 mg/day. None of our patients was treated by tumor necrosis factor-a blockers.

Serum 25(OH)D 3 level
The mean serum 25(OH)D 3 level among RA patients was 5.98 ± 6.8 nmol/l, deficient in 103 (99.1%) patients and insufficient in one (0.9%) patient. However, the mean serum 25(OH)D 3 level among the controls was 8.4 ± 9.6 nmol/l and all had 25(OH)D 3 deficiency. Although the mean serum 25(OH)D 3 level was lower in RA patients than in the controls, the difference was statistically insignificant (P = 0.06).

Correlation between serum 25(OH)D 3 level and the disease parameters studied
The correlation between serum 25(OH)D 3 level and the demographic, clinical, radiological, and laboratory features of RA patients is shown in Table 2.

Correlation between serum 25(OH)D 3 level and DAS-28 and M-HAQ
Serum 25(OH)D 3 level showed a strong negative correlation with the DAS-28 score (r = -0.794, P < 0.001) and moderate negative correlations with M-HAQ (r = -0.562, P < 0.001). Although almost all RA patients had 25 (OH)D 3 deficiency, it was found that the mean 25(OH)D 3 level was relatively higher in patients with disease remission and its level was significantly decreased as the disease became more active (P < 0.001; Table 3 and Fig. 1). Also, the mean serum 25(OH)D 3 was significantly higher in patients without functional disability in comparison with those with disability (P < 0.001; Table 3 and Fig. 2).

Discussion
The prevalence and association between vitamin D deficiency and the outcome of various autoimmune diseases are the most evolving topics. The present study showed that 25(OH)D 3 deficiency was prevalent among RA patients as well as among the controls (99.1 and 100%, respectively), without a significant difference, indicating that vitamin D deficiency might be endemic in our country. Surprisingly, the high prevalence of 25(OH)D 3 deficiency that was found in our country, which has abundant sunshine, may be attributed to decreased sun exposure (conservative dressing culture, wearing of veil and muffler, and/or avoidance of exposure to hot sunny weather). Another explanation may be our predisposition as an Arab population for 25(OH)D 3 deficiency as a result of certain intrinsic factors related to ethnicity as reported by Braun-Moscovici et al. [22], who found that the prevalence of 25(OH)D 3 deficiency was 76.6% in Arab RA patients compared with 35% in Jewish RA patients, with a statistically significant difference (P < 0.001). The low socioeconomic status and educational level of our participants, who were recruited from the rural and slums area near Cairo, may be another contributing factor toward 25(OH)D 3 deficiency.
S-28 and T-28 (r = -0.631, -0.559, respectively, with P < 0.001 for both), which is in agreement with the study of Zakeri et al. [24], but in contrast to the study of Pakchotanon et al. [25], who found an insignificant correlation between 25(OH)D 3 and S-28 and T-28. Also, the present study found a statistically significant mild inverse correlation between 25(OH)D 3 and ESR (r = -0.39, P < 0.001) that was in agreement with the study of Kostoglou-Athanassiou et al. [13]. Moreover, the current study found a statistically significant strong inverse correlation between 25(OH)D 3 and DAS-28 (r = -0.794, P<0.001) and this is in agreement with several previous studies. In a study involving 158 RA patients, Moghimi et al. [10] found an inverse correlation between serum vitamin D and DAS. Kostoglou-Athanassiou et al. [13], Rossini et al. [18], and Haque and Bartlett [19] found the same inverse correlation between vitamin D level and disease activity score in RA patients. Also, Turhanoğlu et al. [23], Zakeri et al. [24], and Chen et al. [26] found that vitamin D deficiency was associated with the RA activity score.
Welsh et al. [27] and Kerr et al. [28] found a significant correlation between vitamin D deficiency and increased disease activity in RA. However, other studies did not find such a significant correlation between vitamin D level and RA activity [22,25,[29][30][31].
Moreover, there was a statistically significant moderate inverse correlation between 25(OH)D 3 and M-HAQ (r = -0.562, P < 0.001) that was in agreement with other previous studies [18,23]. In contrast, other studies showed an insignificant correlation between vitamin D and RA functional disability [26,32]. There was a statistically significant mild inverse correlation between 25(OH)D 3 and body weight (r = -0.256, P < 0.009). Body weight is part of the BMI calculation formula and according to this formula and the WHO classification, the mean BMI of our patients (29.2 kg/m 2 ) was within the overweight range [33]. This significant correlation is in agreement with previous studies that reported an inverse correlation between 25(OH)D 3 and obesity [34,35].
Our present study found a statistically significant mild inverse correlation between vitamin D and IPF (r = -0.234, P=0.017). In a previous study of 67 patients with interstitial lung disease (ILD) secondary to connective tissue disease including RA and 51 patients with other causes of ILD, significant vitamin D deficiency (52 vs. 20%, P<0.0001) and insufficiency (79 vs. 31%, P < 0.0001) were found among those with connective tissue disease-ILD than those with other forms of ILD [36].
No significant correlation was found between 25(OH)D 3 and BMD. In contrast to our results, Chen et al. [26] found a significant positive correlation between 25(OH)D 3 and BMD.
No significant correlation was found between 25(OH)D 3 and drug intake. This finding is in agreement with the study of Baker et al. [37].
Vitamin D is known to induce immunologic tolerance [32] and its deficiency may increase the risk of development of autoimmune diseases such as RA. Also, vitamin D has immunomodulatory properties [38,39]. It appears to regulate the immune response by decreasing antigen presentation [40], inhibiting the T-helper type-1 (Th-1) cell that is directed against self-antigens [41], and decreases the production of interleukin-2 and interleukin-5 [42]. 1,25(OH)2D 3 suppresses proliferation and immunoglobulin production and retards differentiation of B-cell precursors into plasma cells [43]. Varenna et al. [44] reported that vitamin D supplementation may be recommended for RA patients for the prevention and treatment of osteoporosis as well as for its possible effects on disease activity.

Conclusion
Our results showed a high prevalence of vitamin D deficiency among RA patients as well as among the controls, indicating that vitamin D deficiency might be endemic among our population. Although the mean serum level of vitamin D was lower in RA patients, it was insignificantly different from that among the controls. However, there were statistically significant inverse correlations between vitamin D and both RA disease activity and functional disability. Vitamin D deficiency also correlated inversely with IPF, but not with BMD. Our finding justifies the inclusion of vitamin D in the routine RA laboratory workup because of its possible effects on disease activity and disability outcome.