Vitamin D and disease activity in rheumatoid arthritis patients: a retrospective study in a Romanian cohort

Background. The relationship between the serum levels of Vitamin D and the severity of RA is a subject of great interest for the future therapeutic strategies. Although the evidence on the relationship between hypovitaminosis D and early RA is contradictory, preliminary data suggest that the serum levels of vitamin D are inversely associated with the disease activity. Aim: the main objectives of this study include: (1) to analyze the serum levels of vitamin D in patients with RA in comparison to healthy controls; (2) to investigatea possible correlation with disease activity. Materials and Methods. This was a retrospective, comparative study conducted on 37 subjects suffering from RA and a group of 21 healthy matched controls. The following were determined in all studied subjects: erythrocyte sedimentation rate (ESR), white blood cells (WBC), hemoglobin (Hb), platelets (PLT), serum calcium (Ca), serum phosphorus (Phos), and serum 25 hydroxy-vitamin D. Moreover, in the RA group the IgM-Rhematoid Factor (RF) and anti-citrullinated protein antibodies (ACPA) (immune-enzymatic method) were assessed. The Disease Activity Score of 28 joints (DAS28) was calculated for the RA patients. Results. We observed that vitamin D deficiency is more common in RA patients than in healthy controls. No significant correlation between 25OHvitD and DAS28-ESR was found in our study cohort. Conclusions. There is no significant association of serum 25(OH)D with disease severity in a Western Romanian cohort with RA. However, this result could have implications for the disease management, as patients with RA could be supplemented with vitamin D even in the absence of disease activity.


INTRODUCTION
Vitamin D refers to a group of fat-soluble secosteroids with an important role in phospho-calcic metabolism. In humans, vitamin D circulates in two forms: vitamin D2 (ergocalciferol) and vitamin D3 (cholecalciferol).
Vitamin D3 is metabolized in the liver to 25-hydroxyvitamin D3 [25(OH)D3] (calcifediol), and it represents the major circulating form of vitamin D. In the kidneys, 25(OH)D3 is additionally hydroxylated to 1,25 dihydroxyvitamin D (1,25(OH) 2 D) or calcitriol, being the active form of vitamin D. Because of its long half-life and higher concentration,  D3] is commonly measured to assess and monitor the vitamin D status in individuals (Feldmann et al., 2017).
Lately, there is a growing interest in the assessment of vitamin D status in several autoimmune diseases.
Rheumatoid arthritis (RA), an autoimmune progressive systemic disease of the connective tissue, is particularly manifested by destructive alterations in the joints and synovitis (Bumbea et al., 2017).
It is known that RA affects 0.72% of the worldwide population and occurs more frequently in women over 40 years of age (Myasoedova et al., 2020). Studies on the incidence and prevalence of RA suggest variations between different populations even within the same country. For instance, in Romania, 200 000 people suffer from arthritis, and women are three times more likely to develop RA (Șuța et al., 2014).
The etiology and pathogenesis of RA is still obscure and many factors can be involved. The etiology of the disease could be attributed to genetic and non-genetic factors, such as hormonal, environmental, and infectious factors. Moreover, it has been shown that vitamin D is an environmental risk factor involved in the pathogenesis of this disease (Cutulo et al., 2009).
Vitamin D receptors have been identified in the areas of cartilage erosion, in the chondrocytes and synoviocytes of patients with RA. Thus, detection of vitamin D receptors in the immune system cells may elucidate associations between this vitamin and RA (Tetlow et al., 1999).
Already known to have an important role in osteoporosis and fractures, which are frequent complications in RA (Bellan et al., 2019;Tan et al., 2018), and also in the modulation activities of the immune cells, including lymphocytes and macrophages (Marques et al., 2010), vitamin D can be the key factor leading to the development or worsening of the disease.
Some epidemiological studies showed that vitamin D deficiency is common in RA patients when compared to the healthy population (Haque et al., 2010;Kerr et al., 2011), whereas other investigations did not confirm these findings (Cutolo et al., 2006;Turhanoğlu et al., 2010).
Due to these controversies that have not yet demonstrated whether vitamin D deficiency is a result or primary phenomenon of RA, new findings are needed to help understand the association between the RA disease activity and levels of 25 (OH) D. Therefore, the main objectives of this retrospective study include: (1) to analyze serum levels of 25 (OH) D in patients with RA in comparison to healthy matched controls; (2) to investigate a possible correlation with disease activity.

Study population.
The study was performed on a group of 37 patients with RA and 21 healthy subjects matched for age and gender, as controls.
Patients were included in this study only if they met all of the following criteria: 1. Adults aged >30 years, 2. Definite diagnosis of RA according to the 1987 ACR criteria, 3. Disease duration for more than 1 year, 4. Treatment with at least one or more disease modifying antirheumatic drugs (DMARDS) or biologic agents, but not cortisone, 5. Data were collected during winter.
Exclusion criteria were as follows: cardiopulmonary diseases, diabetes mellitus treated by insulin, uncontrolled arterial hypertension, dyslipidemia, chronic kidney disease, thyroid dysfunction, Cushing's syndrome, osteoporosis, current smokers, diseases associated with hypercalcaemia (lymphoma, sarcoidosis, tuberculosis infection, primary hyperparathyroidism), treatment with vitamin D and/or calcium supplementation or drugs affecting the bone and mineral metabolism.
The control group had no rheumatic diseases or other skeletal symptoms based on history and clinical examination.
Clinical measurements and laboratory tests. In all subjects, the following data were collected: age, gender, disease duration, body weight, height, and body mass index (BMI: Kg/m 2 ).
Blood samples were taken from all patients in order to assess: erythrocyte sedimentation rate (ESR), white blood cells (WBC), hemoglobin (Hb), platelets (PLT), serum calcium (Ca), serum phosphorus (Phos), using standard laboratory methods. The IgM-Rhematoid Factor (RF) (nephelometric method) and anti-citrullinated protein antibodies (ACPA) (immune-enzymatic method) were also assessed. In order to evaluate vitamin D status in our study population, serum levels of 25(OH) vitamin D (ng/ml), the most stable circulating form of this molecule, were also measured using the chemiluminescent immunoassay technique.
The rheumatoid arthritis activity was assessed using the Disease Activity Score 28 (DAS28). DAS28 was calculated based on ESR, tender joint count (28 joints), swollen joint count (28 joints), and the patient's assessment of global well-being (100 mm visual analogue scale).
Values of vitamin D≥30 ng/ml were considered as normal, while vitamin D insufficiency was defined as a level between 15 and 29 ng/ml, and vitamin deficiency at levels<15 ng/ml (Watson, 2013).
All subjects gave their informed consent for inclusion before they participated in the study. This study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by the Institutional Ethics Committee.
Characteristics of RA patients and controls. The two groups were similar in terms of age, height, weight and BMI. Thus, mean age in the RA patients and control group was 57±12.58 and 50.19±16.41, respectively. Also, the mean BMI of the RA patients was 26.48±4.44 kg/m 2 versus 26.76±3.6 kg/m 2 of the controls, indicating an overweight sample.
A higher level of serum ESR was observed in RA patients, when compared to healthy controls.
RA patients were positive for the rheumatoid factor and anticitrullinated peptide antibodies. All patients enrolled in the study received a DMARD treatment without concomitant corticosteroids or vitamin D supplementation. Thus, 56.8% of the patients received methotrexate, 32.4% leflunomide, and 5.45% sulfasalazine.
Statistical analysis. Data analysis was performed using SPSS v.17. Continuous variables were presented as mean and standard deviations, and nominal data were presented as counts (percentages). We performed a descriptive and inferential statistics analysis to summarize the characteristics of the study population. In order to evaluate the proportion of categorical data in the groups, we applied the chi-squared test (χ2). The results of the Shapiro-Wilk normality test showed a non-Gaussian distribution, which is why we continued to use nonparametric tests. To compare patients with/without RA or vitamin D deficiency, we applied the Mann-Whitney U test. In order to highlight the relationship between group characteristics and levels of vit D we performed Spearman's correlation. A multivariate linear regression analysis was used to evaluate the independent factors associated with the DAS28 variable and risk of vit D deficiency. A p value of less than 0.05 was considered to indicate statistical significance.

RESULTS
The demographic data of patients and controls are presented in Table 1.
We registered significant differences between the groups, with lower mean values of 25(OH) vitD in the RA patients than in the controls (p=0.003).
In our study group, the mean score of DAS28-ESR was 3.59±1.05, providing a moderate disease activity group. Moreover, the 25-OH vitamin D levels of the patients from the high disease activity group were found to be the lowest, but they were not significantly different between the groups.
The Spearman nonparametric correlation test revealed that there was a significantly weak positive correlation between DAS28, WBC (p=0.013) and Phos (p=0.049). In contrast, correlation was strong in the case of ESR (p<0.001) ( Table 2).
We found no significant correlation between 25OH-vitD and DAS28 or other studied parameters.
Following the linear regression analysis, only ESR was established as a significant predictive factor for DAS28 (p=0.003) ( Table 3).
The baseline characteristics of RA patients according to the disease activity are presented in Table 4. About Vitamin D and disease activity in rheumatoid arthritis patients    (n=7), and 22% (n=8) were classified as having moderate, low disease activity, and in remission, respectively. Although, age was significantly higher in the remission group and in the high disease activity group (Kruskal-Wallis Test, p=0.009), no differences between the remission and the high disease activity groups were observed (Mann-Whitney U Test, p=0.861).
However, the mean value of vitamin D level was 14.24±5.06 ng/mL in the high disease activity group, 22.37±9.07 ng/mL in the moderate disease activity group, and 22.55±9.44 ng/mL in the low disease activity group.
Also, ESR values are significantly increased in the high disease activity group (Kruskal-Wallis test, p=0.003). However, the ESR values in the high disease activity group are not significantly different when compared to the moderate disease activity group (Mann-Whitney U test, p=0.067).

DISCUSSION
Our study showed that vitamin D deficiency and insufficiency are more prevalent in patients with RA than in healthy controls. The mean value of vitamin D was significantly lower in the RA patients than in controls (p=0.003).
Although many studies have shown that vitamin D deficiency is common in RA, other studies did not find significant differences between RA patients and healthy controls (Craig et al., 2010;Cutolo et al., 2006;Hitchon et al., 2012;Turhanoğlu et al., 2010).
A large study from 15 countries, the COMORA study, explored the status of vitamin D in RA patients. The authors concluded that vitamin D deficiency, in patients with RA, varies between different countries. This variability may be explained by the association of many factors such as: racial or ethnic differences, climates, lack of sun exposure, skin color, clothing style, or latitude. Until recently, vitamin D insufficiency and deficiency were common in countries with less sun, particularly in North versus South Europe, with a circannual rhythm (Cutolo et al., 2006). In fact, current data indicates that tropical or subtropical areas, such as Central America and the Middle East, are also widely affected. This finding could be explained by several factors: avoidance of sun exposure, colored skin, wearing protective clothing, country latitude, vitamin D receptor (VDR) gene polymorphism and its expression (Vojinovic et al., 2017) We believe that this variability is also determined by characteristics of the RA patients included in different studies, by inclusion and exclusion criteria taken into account or by methodology used in these studies.
Another large multicentric study collected data on vitamin D serum levels in 625 RA patients from 13 different European countries. Similarly to our study, the mean serum concentration of 25(OH)D in RA patients was significantly lower when compared to controls. 25(OH) D deficiency was found in 66% of the RA patients, and insufficiency was found in 27% of them (Merlino et al., 2004). On the contrary, in our study 54% of the patients had insufficiency, and only 30% had deficiency, while 16% of the RA patients had normal serum levels of vitamin D.
The study found a statistically significant difference in vitamin D levels between some European countries, with significantly higher 25(OH)D serum concentrations in RA patients from Spain, Latvia, and Serbia when compared to Romania (Merlino et al., 2004).
Although several studies have shown that vitamin D levels are inversely related to the RA disease activity, we found no correlation between the 25(OH)D levels and disease activity in our cohort. However, despite the lack of a significant correlation, our patient population had a well-controlled disease. This weak correlation may be explained by the absence of other assessed factors that could be involved in the functional deficiency of vitamin D (seasonal differences, sun exposure, nutritional status, skin color, clothing style, etc).
So far, evidence for the relationship between hypovitaminosis D and RA disease activity is contradictory (Ga-  Higgins et al., 2013;Polasik et al., 2017;Rossino et al., 2010). In a prospective cohort study on 29,368 women without a history of RA at baseline, it was demonstrated that low or deficient levels of 25(OH)D were inversely associated with the risk for development of RA (n=152) (Akkar et al., 2016). However, correlation between the disease activity and vitamin D deficiency has been demonstrated in many other studies (Azzeh et al., 2015;Braun-Moscovici et al., 2011;Hajjaj-Hassouni et al., 2017;Sahebari et al., 2014).
The findings of several studies included in our literature review and the characteristics of the study participants (mean age, number of subjects, vitamin D deficiency and insufficiency) are summarized in Table 5.

Limitations of the study
The study presented here has some limitations. The first limitation is the small sample size, being a singlecenter study. Study participants were enrolled from the Western part of Romania where sun exposure rates (and resulting vitamin D concentrations) may differ from other populations living in other geographical areas. Moreover, we did not assess other factors that could be involved in the functional deficiency of vitamin D (seasonal differences, sun exposure, nutritional status, skin color, clothing style, etc).
Secondly, only the serum 25(OH)D was determined, but not 1.25-hydroxyvitamin D. We did not collect data on parathyroid hormone levels or other parameters involved in vitamin D deficiency.
Finally, another limitation is the lack of a follow-up study in our population, given that all parameters were assessed only at enrollment.

CONCLUSIONS
We observed that vitamin D deficiency is more common in the RA patients than in the controls. There is no significant association of serum 25(OH)D with disease severity in a Western Romanian cohort with RA.
We found that the serum values of 25 (OH)D are not a predictive factor for the disease activity in the RA patients. The hypothesis of vitamin D deficiency as a predisposing factor in triggering an increased activity of the disease needs to be tested on a larger group of RA patients. However, our results could have implications for disease management, as patients with RA could be supplemented with vitamin D even in the absence of disease activity.

Conflict of interest
The authors declare no conflict of interest.