Vitamin D levels are one of the most likely environmental factors related to the development of multiple sclerosis (MS). As vitamin D levels differ between ethnicities, the associated risk of MS also differs. We aimed to determine the associations of serum vitamin D levels in Japanese patients with MS.
Serum levels of 25-hydroxyvitamin D (25[OH]D), 1,25-dihydroxyvitamin D (1,25[OH]2D), and vitamin D-binding protein (DBP) were measured in 29 patients with relapsing-remitting (RR) and secondary-progressive (SP) MS, and in 34 healthy controls.
The serum levels of 25(OH)D in SPMS patients were significantly decreased compared with those of controls and RRMS patients at remitting phase (7.9 ± 7.2 nM vs 24.2 ± 14.2 nM vs 22.5 ± 13.2 nM, respectively; P < 0.05, P < 0.01, respectively). DBP levels in RRMS and SPMS patients were also decreased compared with controls (419.4 ± 76.2 μg/mL vs 416.7 ± 65.9 μg/mL vs 527.6 ± 149.4 μg/mL, respectively; P < 0.01, P < 0.05, respectively). Furthermore, serum 25(OH)D was found to correlate negatively with disease severity in patients with RRMS in the remitting phase and SPMS using the Expanded Disability Status Scale and the Multiple Sclerosis Severity Score.
Our results suggest that low 25(OH)D levels are associated with an increased risk of developing SPMS, and a likelihood of developing more severe MS with a worse prognosis in Japanese patients.
[Correction added on 2 August 2013, after first online publication: ‘serum 25 (OH)D was found to correlate negatively with 1,25(OH)2D levels in patients’ has been corrected to ‘serum 25(OH)D was found to correlate negatively with disease severity in patients’ in the Results section of the abstract.]
The prevalence of multiple sclerosis (MS) is known to vary according to the degree of latitude, with a higher prevalence of the disease in high-latitude areas. This phenomenon is believed to be associated with ultraviolet (UV) light exposure and subsequent vitamin D synthesis, suggesting that vitamin D has an important role as a modifiable risk factor. Although vitamin D is consumed as part of food, dietary intake alone is often insufficient, supplying just 20% of the body's requirements. The major source of vitamin D for most people is through skin exposure to sunlight. Indeed, a recent study reported an association between increased sun exposure and a better outcome in relapsing-onset MS, which could indicate that high vitamin D levels decrease the prognosis of disability. In Swedish populations, decreased 25-hydroxyvitamin D (25[OH]D) levels in serum is a known risk factor for developing MS.
1,25-Dihydroxyvitamin D (1,25[OH]2D) is the biologically active metabolite of vitamin D, and is converted from 25(OH)D by hydroxylation with 1α-hydroxylase in the proximal tubule of the kidney. Vitamin D-binding protein (DBP) is the major plasma carrier protein of vitamin D and its metabolites. DBP helps to regulate the bioavailability of 1,25(OH)2D, as it buffers the levels of free metabolites and thus affords a degree of protection against short-term seasonal or diet-induced fluctuations. The synthesis and functions of this vitamin D metabolite and vitamin D-associated protein might therefore have an intimate relationship with disease risk.
The aim of the present study was to evaluate the associations between serum levels of 1,25(OH)2D, 25(OH)D and DBP in Japanese patients with MS. Furthermore, we surveyed the associations of these levels with disability and disease progression in the same population.
Patients with MS were recruited from the Hokkaido Medical Center and Sapporo Neurology Clinic (Sapporo, Japan), and healthy control subjects were recruited from hospital staff. Subject recruitment followed institutional review board approval, and informed consent was obtained from all participants. All patients were diagnosed with MS using the 2010 revised McDonald criteria, and relapsing-remitting type MS (RRMS) and secondary-progressive type MS (SPMS) using the definition by Lublin et al. Those patients with neuromyelitis optica (NMO) or NMO spectrum disorders were excluded. Clinical profiles of patients with MS and healthy controls are shown in Table 1. A total of 34 healthy control subjects (29 female, 5 male; mean age 39.5 years), 20 patients with RRMS at the remitting phase (18 female, two male; mean age at blood sampling 40.8 years, mean age at disease onset 30.1 years, mean and median age of disease duration 10.8 and 10.5 years, respectively) and 14 patients with RRMS at the relapsing phase (13 female, one male; mean age at blood sampling 38.4 years, mean age at disease onset 29.4 years, mean and median age of disease duration 9.0 and 5.5 years, respectively) and nine female patients with SPMS (mean age at blood sampling 51.0 years, mean age at disease onset 30.3 years, mean and median age of disease duration 20.7 and 23.0 years, respectively) were included in the study. There was a significant difference in disease duration between patients with RRMS at the remitting phase and SPMS (P < 0.05). In MS patients, disease severity was evaluated using the Expanded Disability Status Scale (EDSS), and progression of disability was assessed using the Multiple Sclerosis Severity Score (MSSS).
Table 1. Clinical profiles of healthy controls and patients with multiple sclerosis
Serum samples from patients and healthy individuals were frozen at −80°C until required for analysis of 1,25(OH)2D, 25(OH)D and DBP. Levels of 1,25(OH)2D were measured by radioimmunoassay (RIA) using a standard protocol (Immunodiagnostic Systems Limited, Boldon, UK), and levels of 25(OH)D and DBP were measured by enzyme-linked immunosorbent assay (ELISA) using a standard protocol (Immundiagnostik, Bensheim, Germany). Vitamin D levels are known to differ between seasons, so serum samples were taken from patients and healthy controls between June and August. Serum samples for 1,25(OH)2D, 25(OH)D and DBP measurements were obtained from one blood puncture per individual.
All data are expressed as means ± standard deviation (SD). Comparisons between healthy control (HC) participants, patients with RRMS and patients with SPMS were tested by analysis of variance (anova) followed by Tukey's multicomparison test using GraphPad Prism software (GraphPad Software, La Jolla, CA, USA). Comparisons between HC participants and patients with combined RRMS and SPMS were assessed using the Mann–Whitney U-test. Spearman's rank correlation was used for correlation analysis. P < 0.05 was considered significant.
Serum levels of 1,25(OH)2D, 25(OH)D, and DBP in HC and patients with MS
Serum levels of 1,25(OH)2D, 25(OH)D, and DBP were compared between HC, RRMS patients at the remitting phase and SPMS patients. Levels of 1,25(OH)2D were significantly higher in MS patients compared with those of HC (51.91 ± 23.26 pg/mL vs 81.83 ± 37.00 pg/mL, respectively; P < 0.001; Fig. 1a). In contrast, levels of 25(OH)D were lower in MS patients compared with those of HC, although the difference was not statistically significant (24.23 ± 14.21 nM vs 17.99 ± 13.44 nM, respectively; P = 0.06; Fig. 1b). Levels of DBP were significantly lower in MS patients compared with HC (527.6 ± 149.4 μg/mL vs 418.6 ± 72.0 μg/mL, respectively; P < 0.01; Fig. 1c). As mean ages of SPMS patients are slightly higher than HC and patients with RRMS, we investigated correlations between ages at blood puncture and serum levels of 1,25(OH)2D, 25(OH)D, and DBP. However, no associations were found in the population of the present study (data not shown).
The MS patient group was then divided into RRMS patients at the remitting phase and SPMS patients. RRMS patients at the remitting phase had higher 1,25(OH)2D levels compared with HC (89.55 ± 39.34 pg/mL vs 51.91 ± 23.26 pg/mL, respectively; P < 0.001; Fig. 2a), and levels of 25(OH)D were significantly lower in SPMS patients compared with HC and RRMS patients at the remitting phase (7.90 ± 7.18 nM vs 22.53 ± 13.22 nM vs 24.24 ± 14.21 nM, respectively; P < 0.01 and P < 0.05, respectively; Fig. 2b). Levels of DBP were significantly lower in RRMS patients at the remitting phase and SPMS patients compared with those of HC (419.4 ± 76.2 μg/mL vs 416.7 ± 65.9 μg/mL vs 527.6 ± 149.4 μg/mL, respectively; P < 0.01 and P < 0.05, respectively; Fig. 2c).
Next, serum levels of 1,25(OH)2D, 25(OH)D, and DBP were compared between RRMS patients at remitting phase and RRMS patients at relapsing phase, but no significant difference was found (89.55 ± 39.34 pg/mL vs 73.14 ± 24.71 pg/mL, respectively), although there was a tendency for 1,25(OH)2D levels to decrease in patients at the relapsing phase (Fig. 3a). There was also no significant difference between RRMS patients at the remitting phase and those at the relapsing phase in terms of 25(OH)D levels (22.53 ± 13.22 nM vs 16.58 ± 13.22 nM, respectively; Fig. 3b) or DBP levels (419.4 ± 76.2 μg/mL vs 424.0 ± 86.0 μg/mL, respectively; Fig. 3c).
To evaluate hydroxylation from 25(OH)D to 1,25(OH)2D, rates of 25(OH)D (nM)/1,25(OH)2D (pg/mL) were calculated. These rates were significantly decreased in patients with MS, especially SPMS patients, compared with HC (Fig. 4a,b). In contrast, there were no significant differences in rates between MS patients at the remitting phase and relapsing phase (Fig. 4c).
Correlation between serum levels of 1,25(OH)2D, 25(OH)D, or DBP and EDSS or MSSS in patients with RRMS at the remitting phase/SPMS
When assessing the correlation between serum levels of 1,25(OH)2D, 25(OH)D or DBP and EDSS, we observed a negative correlation between 25(OH)D and EDSS (r = −0.53, P < 0.01; Fig. 5b), but not between 1,25(OH)2D or DBP and EDSS (Fig. 5a,c).
Similarly, in the correlation between serum levels of 1,25(OH)2D, 25(OH)D or DBP and MSSS, negative correlations were found between 1,25(OH)2D or 25(OH)D and MSSS (r = −0.37, P < 0.05 and r = −0.53, P < 0.01, respectively; Fig. 6a,b). No such correlation was found between DBP and MSSS (Fig. 6c).
Individuals with darker skin have a higher risk of vitamin D deficiency, because melanin competes with 7-dehydrocholesterol in the absorption of UV light. Indeed, serum 25(OH)D levels are substantially lower in Africans than Caucasians. However, the MS risk associated with low vitamin D levels might vary between ethnicities. For example, it was reported that the MS risk significantly decreased with increasing 25(OH)D serum levels in Caucasians, whereas no significant associations between 25(OH)D levels and MS risk were found among African and Hispanic populations. By contrast, African American levels of 25(OH)D were recently found to be lower in patients with MS than in HC. In the current study, serum samples were taken between June and August, and it is known that vitamin D levels differ between seasons. Supplemental vitamin D, such as cod-liver oil, might be protective when sun exposure is less, and both climate and diet might interact to influence MS risk at a population level. Studies with samples taken in other seasons might provide further supplemental suggestions.
In the present study of a Japanese population, patients with SPMS had significantly lower levels of 25(OH)D compared with controls and RRMS patients at the remitting phase. These data suggest a number of hypotheses. One is that SPMS patients mainly stay indoors and consequently have lower 25 (OH)D levels; another is that RRMS patients with lower levels of 25(OH)D tend to progress to SPMS because of the strong immune-modulating effects of vitamin D, including the ability to decrease IL-17 production, which is a key cytokine in MS pathogenesis. A third hypothesis is that vitamin D could directly or indirectly suppress neurodegeneration in the central nervous system (CNS). Indeed, an in vitro study showed that low doses of vitamin D were able to protect mesencephalic dopaminergic neurons, whereas a recent study of veterans in the Multiple Sclerosis Surveillance Registry suggested that vitamin D slows disease-related neurodegeneration. Our data also show that lower vitamin D levels are associated with greater MS severity and worse prognosis.
The present study showed that 1,25(OH)2D levels were higher in patients with RRMS compared with HC. The reason for the observed discrepancy between 1,25(OH)2D and 25(OH)D levels is unclear, but there are differences between the half-lives of these two vitamin D metabolites: 1,25(OH)2D has a half-life of 10–20 h in the circulation, whereas 25(OH)D has a half-life of 15 days. Thus, 25(OH)D is more appropriate to measure the vitamin D status of an individual. A recent study reported that serum 25(OH)D levels, but not 1,25(OH)2D levels, were significantly correlated with T regulatory cell function or T-helper 1/T-helper 2 ratios in patients with MS. Thus, the immunological functions of 25(OH)D and 1,25(OH)2D might differ. In contrast, our data showed that the 25(OH)D/1,25(OH)2D rates were significantly decreased in patients with MS, suggesting that hydroxylation increases in these patients.
A genome-wide association study in Australia and New Zealand identified MS risk-associated single nucleotide polymorphisms (SNP) on chromosome 12q13-14. Given the available genetic, immunological and epidemiological evidence, the CYP27B1 gene, which encodes the enzyme 25(OH)D-1α-hydroxylase that hydroxylates 25(OH) D into 1,25(OH)2D, was shown to be the strongest candidate. This genetic background might affect the differences in 25(OH)D/1,25(OH)2D rates between HC and MS patients, although we cannot confirm this as the present study did not include any genetic information. Further studies of the two metabolites of vitamin D that focus on immunological functions are therefore required.
There was no apparent association between 25(OH)D status and disease severity in a previous study of African–American patients with MS. However, a study from the Netherlands reported that low 25(OH)D levels were associated with high EDSS scores, and our present study also showed a negative correlation between EDSS and MSSS with serum vitamin D levels. Lower 25(OH)D might be associated with a risk for greater MS disability severity or worse prognosis. In Caucasians, lower 25(OH)D levels were found to be associated with a risk of increasing relapse in MS.[24, 25] The present study also showed that patients at the relapsing phase had a tendency for decreased 25(OH)D levels, although the difference was not statistically significant. Further studies will be required to confirm this risk of increasing relapse in MS.
DBP is the major plasma carrier protein of vitamin D metabolites, especially 25(OH)D; however, DBP concentrations do not seem to be influenced by 25(OH)D levels. Recent studies showed that DBP is involved in the immune system where it acts as a chemotactic factor in the recruitment of neutrophil leukocytes. Consecutive contact of DBP with B and T cells was shown to convert DBP into a potent macrophage-activating factor. Several studies reported that DBP levels in the cerebrospinal fluid (CSF) decreased in MS, especially at the relapsing phase.[27, 28] Although not based on CSF samples, the present study also showed that serum DBP levels decreased in patients with MS, suggesting that DBP plays an important role in MS pathogenesis. However, another study found that patients with SPMS had significantly higher levels of DBP in their CSF, so the roles of DBP in MS need to be explored further. Furthermore, whether this role of DBP in MS is dependent on its vitamin D binding activity remains to be answered.
In the present study, some patients received doses of 0.5–0.75 μg/day alfacalcidol or eldecalcitol, which are known to increase serum 1,25(OH)2D levels. However, the previously observed increase of serum 1,25(OH)2D levels by alfacalcidol is only slight at 1.0 μg/day and at most 3% after 12 weeks. Taken together, it is considered that an intake of alfacalcidol or eldecalcitol has little effect on serum levels of vitamin D in the present study.
In conclusion, vitamin D levels have been suggested to be under regulation by genetic variation in 1α-hydroxylase and vitamin D receptor genes, perhaps showing their importance in disease pathogenesis. In the future, we will need to consider the interaction between genetic associations and vitamin D in the pathogenesis of MS.
We thank Ms Eri Takahashi for her skillful technical assistance. This work was supported in part by a Health and Labor Sciences Research Grant on Intractable Diseases (Neuroimmunological Diseases) from the Ministry of Health, Labor and Welfare of Japan.
None of the authors have a financial interest in the publication of the contents of this article or have a relationship with any company with such financial interests.