Assessment of serum vitamin D level and its relationship with disease activity in adult patients with Systemic Lupus Erythematosus (SLE)

1 Molecular Medicine Research Center, Research Institute of Basic Medical Sciences, Rafsanjan University of Medical Sciences, Rafsanjan, Iran. 2 Student Research Committee, Rafsanjan University of Medical Sciences, Rafsanjan, Iran. 3 Non-Communicable Diseases Research Center, Rafsanjan University of Medical Sciences, Rafsanjan, Iran. 4 Department of Internal Medicine, Ali-Ibn AbiTalib hospital, School of Medicine, Rafsanjan University of Medical Sciences, Rafsanjan, Iran.


Introduction____________________________
Vitamin D is a steroid hormone that has a critical role in calcium metabolism, skeletal health, immune system regulation, and bone homeostasis [1]. Vitamin D2 (ergocalciferol) and vitamin D3 (cholecalciferol) are two physiological forms of vitamin D [2]. The principal origin of vitamin D3 is assembly by the conversion of 7dehydrocholesterol to pre-vitamin D3 in the skin by UVB rays-exposed surface, and a smaller amount of it is provided by the diet [3]. Recently, an epidemic of vitamin D deficiency has been demonstrated and numerous studies have shown a relationship between vitamin D insufficiency and several autoimmune and inflammatory diseases [4,5]. Vitamin D receptor (VDR) is located on several immune cells and mediate immunomodulatory functions by downstream signaling. Several investigations have demonstrated that vitamin D participates in the regulation of the immune responses by inhibiting T helper 1 (Th1), Th17 , and B cells and by promoting the development of Th2 and regulatory T (Treg) cells [6,7].
Systemic lupus erythematosus (SLE) is the paradigm of multisystemic autoimmune disorders characterized by cellular and humoral immunological irregularities, leading to the secretion of pathogenic autoantibodies [8]. Numerous studies have been proposed that vitamin D has a crucial role in the development and pathogenesis of SLE based on the results and observations of clinical and laboratory investigations [9,10]. Evidence has shown that vitamin D induces downregulation of the Th1 and activates B cells immune response while upregulating Treg cells during SLE pathogenesis [11][12][13] appearance and the use of sunscreen, which prevents ultraviolet B (UVB) transmission, decreasing the skin generation of vitamin D [14]. Also, renal impairment, which can happen in subjects with lupus nephritis, could break the hydroxylation of vitamin D [15]. Above all, chronic use of immunosuppressive drugs (especially corticosteroid) as well as medications used regularly for SLE therapy change the metabolism of vitamin D [16]. As physiological and clinical consequences of vitamin D deficiency in SLE are not entirely known, this study was designed to assess vitamin D level in the serum of these patients and its association with disease activity.

Materials and Methods___________________ Patients
In this cross-sectional study, 200 Iranian patients with SLE were included. The following simple formula was used for calculating the adequate sample size in our study; n = Z2P (1−P) / d2 Where n is the sample size, Z is the statistic corresponding to the level of confidence, P is expected prevalence (that can be obtained from same studies or a pilot study conducted by the researchers), and d is precision (corresponding to effect size). Simple random sampling was employed for random selection. In cross-sectional studies, the aim is to estimate the prevalence of unknown parameter(s) from the target population using a random sample. The outpatient visits were performed in the Rheumatology Clinic of the Ali-Ebne-Abitaleb Hospital of Rafsanjan, Kerman, Iran, between May and November 2019. SLE patients were diagnosed according to the American College of Rheumatology (ACR) criteria in 1997 [17]. Patients with other connective tissue diseases, and pregnantand lactating subjects were eliminated from the study. The demographic data are illustrated in Table 1. The disease activity was defined on the day the serum samples were drawn using the Systemic Lupus Erythematosus Disease Activity Index (SLEDAI) score [18]. The study was confirmed by the Human Research Ethics Committee of Rafsanjan University of Medical Sciences and written informed consent was obtained from all subjects (1398.103).

Measurement of 25(OH) D3 level and definition of insufficiency
To evaluate vitamin D levels, 5 ml of peripheral blood samples were obtained from all study subjects. After that, serum samples were isolated from the whole bloods and stored in the -80 °C refrigerator until further evaluations. Vitamin D levels were analyzed by Enzyme-linked immunosorbent assay (ELISA) technique using a commercial kit (CALBIOTECH, CA, USA).
In this study, we applied the widely used cut-off points for describing Vitamin D deficiency, insufficiency and sufficiency as suggested previously by Munns et al. [19]. Therefore, a Vitamin D level less than 12 ng/ml (< 30 nmol/L) was recognized as Vitamin D deficiency, a level between 12-20 ng/ml (30-50 nmol/L) was considered Vitamin D insufficiently, and an optimal level (sufficiency) was determined to be more than 20 ng/ml (50 nmol/L).
Statistical Analysis SPSS version 22.0 for Windows program (SPSS Inc., Chicago, IL, USA) was utilized for statistical analyses. The normality of the data was ensured through Kolmogorov-Smirnov test. Mann-Whitney U test was run to compare the clinical data between the two groups, and Spearman's rho correlation test was applied to analyze correlations. Missing data were also considered during statistical analysis. Additionally, the univariate and multivariate regression analyses were carried out to adjust the association between serum vitamin D level and SLEDAI for confounding factors. A P-value of less than 0.05 was regarded to be statistically significant.

Results________________________________ Demographic information and clinicopathological presentations
The demographic information and clinicopathological presentations of the study subjects are listed in Table 1. The study population was comprised of 27 (13.5%) males and 173 (86.5%) females, with the mean age of 38.46 ± 13.24 years. The mean age of subjects at SLE diagnosis was 26.44 ± 7.25 years, and the disease duration of the patients was 12.74 ± 6.87 years. The mean SLEDAI of the patients was 5.74 ± 3.21. The mean sun exposure of the patients was detected at 1.28 ± 0.15 hrs/day. Among the patients, 88 (44%) individuals were using sun protector cream on a daily basis. Vitamin D supplements were consumed by 70 (35%) patients.
Vitamin D level and determination of insufficiency It was detected that serum level of Vitamin D was 13.62 ± 3.22 ng/ml in the patients. According to the criteria described above, it was observed that 104 (52%) patients were suffering from Vitamin D deficiency.
Correlation of Vitamin D level with patients' data The correlation of Vitamin D level with numerical characteristics of SLE patients are shown in Table 2. No statistically significant correlation was detected between the Vitamin D level and age [correlation co-efficient (CC) = -0.12, p value = 0.245], at SLE diagnosis (CC = -0.26, p value = 0.112), and disease duration (CC = -0.29, p value = 0.088). There was a statistically positive correlation between vitamin D level in the serum of patients and duration of sun exposure (CC = 0.57, p value = 0.004).
However, a statistically significant negative correlation was seen between vitamin D level in the serum of patients and SLEDAI (CC = -0.41, p value = 0.013).

Association of Vitamin D level with patient's data
Association of Vitamin D level with nominal characteristics of SLE patients are presented in Table 3. Although Vitamin D level was higher in female SLE patients (12.32 ± 2.14 ng/ml) in comparison to the males (14.92 ± 3.48 ng/ml), it was not significantly different between male and female patients (p value = 0.146). In addition, no significant difference (p value = 0.188) was detected between smoker (12.42 ± 3.09 ng/ml) and nonsmoker SLE patients (14.82 ± 3.38 ng/ml).
Vitamin D level was significantly (p value = 0.030) lower in SLE patients with photosensitivity (11.29 ± 2.01 ng/ml) compared with the patients without photosensitivity (15.95 ± 4.43 ng/ml). Moreover, SLE patients using sun protector cream (10.21 ± 2.10 ng/ml) had significantly (p value = 0.002) lower level of Vitamin D in comparison to the patients who did not use sun protector cream (17.03 ± 4.34 ng/ml). Nonetheless, Vitamin D level was not significantly different between patients having or lacking other clinicopathological presentations, including malar rash, discoid rash, mucosal lesion, arthritis, pleuritis, proteinuria, hematuria, urine casts, renal involvement, anemia, hypertension, pericarditis, cardiomyopathy, leukopenia, lymphopenia and thrombocytopenia.
Finally, in spite of lower levels of vitamin D in patients consuming immunosuppressive (p value = 0.074), antimalarial (p value = 0.903), and prednisolone drugs (p value = 0.362), the differences were not statistically significant. However, patients receiving glucocorticoid drugs, had significantly (p value = 0.001) lower levels of Vitamin D in comparison to the patients who were not taking glucocorticoids (Table 3).

Regression analysis
Univariate regression analysis revealed no association between serum vitamin D level and SLEDAI upon adjusting for the confounding factors, such as smoking status and sun protector cream use. Additionally, multivariate regression analysis indicated no association between serum vitamin D level and the disease activity after adjusting for the confounding factors, including disease duration and sun exposure (Table 4).

Discussion_____________________________
In this study, we tried to evaluate the association of vitamin D level with different indexes in the SLE patients, among which age in particular, gender distribution, SLEDAI, sun exposure, smoking, photosensitivity, sun protector cream use, and drug regimen. Most importantly, we found that Vitamin D level had significantly negative and positive correlation with SLEDAI and duration of sun exposure, respectively. Moreover, Vitamin D level was lower in patients with photosensitivity and those using sun protector cream. In addition, patients taking glucocorticoid drugs, had lower levels of Vitamin D when compared to those not consuming glucocorticoids.
Widely distributed around the world, SLE has an unknown etiology as well as clinical and laboratory manifestations in different races and regions [20]. It can occur at any age, including childhood, but it is most common in the second, third or fourth decade of life. Although the disease is found in both sexes, it is far more common in women. The disease is also more common in people of African and Hispanic origin [21]. According to an extensice sociological study of rheumatic disease, the prevalence of SLE in Iran has been estimated to be 40 per 100,000 [22].
In 2019, Ospina-Caicedo et al. conducted a crosssectional study where the 25-OH Vitamin D level was evaluated in 69 SLE patients above 18 years of age. It was found that 36.2% of patients had low levels of vitamin D with higher levels of anti-dsDNA. SLE patients with moderate to severe disease activity had lower values of Vitamin D based on SLEDAI. The Vitamin D level was higher in patients who did not consume steroids than the patients receiving 0.5-1mg/kg/d dosage of steroids [23]. We found inverse correlation of Vitamin D level and SLEDAI. Furthermore, patients receiving glucocorticoid drugs had lower levels of Vitamin D in comparison to those who did not. As a consequence, it is critical to monitor Vitamin D level in SLE patients regularly and include Vitamin D supplementation in their medication regimen, particularly when glucocorticoids are included in their drug administrations.
Vitamin D level has been shown to impress several immune system pathways, which in turn imposes remarkable consequences for patients with SLE [24]. SLE patients have several risk factors for Vitamin D deficiency. Photosensitivity, which is a specific manifestation of SLE patients, might reduce sunlight exposure time, which decelerates the production of Vitamin D through the skin. Long-term corticosteroids consumption and other immunosuppressive drugs in lupus patients also affects Vitamin D metabolism. On the other hand, renal involvement in nephritic patients with lupus can impact hydroxylation and activation of Vitamin D [25]. Our study revealed that Vitamin D level was significantly lower in SLE patients with photosensitivity compared to the patients lacking photosensitivity. In addition, 44% of patients were using sun protector cream, who had significantly lower level of Vitamin D in comparison to those not using sun protector creams. Furthermore, Vitamin D level had a positive correlation with daily duration of sun exposure. Interestingly, we detected a negative correlation between Vitamin D level and SLE disease activity. Therefore, it seems that lower sunlight absorption (even though 35% patients were taking Vitamin D supplementation) caused Vitamin D deficiency in the SLE patients , which may exacerbate clinical manifestations. Hence, it is suggested that SLE patients.
Considering that vitamin D level had positive correlation with sun exposure duration but a negative correlation with SLEDAI, it might be beneficial for the patients to have at least one hour of daily sun exposure to decrease the disease activity. In addition, physicians are highly recommended to prescribe Vitamin D to the SLE patients to regulate the disease activity and improve the general mood of the patents. Additionally, it might be beneficial for the patients to use less of the sun protector creams, which may increase sunlight absorption by skin and Vitamin D production consequently. Our study had some limitations. Firstly, the small sample size may have influenced the serum 25-(OH)D levels. Accordingly, it could be speculated that the low levels of 25-(OH)D in the SLE patients in our study might stem from the sample size, meaning that they were not the source of the disease activity. Secondly, there were no healthy controls with whom to compare the prevalence of VitD insufficiency and deficiency. Finally, in our study, it was impossible to determine anti-dsDNA, C3, C4, or other serum markers in all of the patients.