In this study, a higher level of DBP was found to be associated with an increased risk of the future development of RA in females, adjusted for rs4588 minor allele, smoking, BMI, educational level, age and sampling time of the year. We did not observe any association between total Vitamin D level or DBP level in plasma and a future risk of RA, irrespective of sex or gene polymorphism.
Conflicting results have been presented regarding the role of vitamin D and the risk of developing RA in the future, this can partly be explained by the many factors that are known to influence the circulating levels of Vitamin D in man, e.g., exposure to Ultraviolet B (UVB) light, geographical location, skin pigmentation, protective measures from sunlight and intake of Vitamin D containing nutrients . Also, both vitamin D status and levels of DBP are associated with GC variants, and in particular those analyzed in this study, rs7041 and rs4588 [22,23,24]. Similar to the results of their studies, we found a gene-dosage relationship with differences between the rs7041 and rs4588 genotypes and DBP and 25(OH) D levels, showing lowest vs. highest levels in rs7041 TT vs. GG and rs4588 AA vs. CC and intermediate levels in heterozygotes, with a similar pattern in controls and pre-symptomatic individuals. In this study a positive correlation between levels of DBP and 25(OH) D, was confirmed in line with previous published studies [24, 25]. We found significantly higher DBP levels in females compared with males, as previously suggested [26, 27].
Levels of DBP have been associated with BMI, oral contraceptive use, lipid parameters, and current smoking , and we have adjusted for the variables BMI, educational level [academic vs. non-academic], smoking status (ever having smoked [Yes/No]), age at time of sampling and the season at the time of blood collection (Dark vs. Light). We have no information on the use of contraceptive pills by those in our cohort of cases, although the mean age of the females was 54.4 years, suggesting that a majority were in menopause.
There are two earlier studies on pre-symptomatic individuals who later developed RA based on levels of Vitamin D published. The study by Nielen et al.  analyzed 25(OH) D in 79 patients with RA who had previously donated blood to a blood bank, selecting one sample from each individual from time points 1 year, 2 years, and ≥ 5 years before the start of symptoms together with matched controls. They compared the proportion of 25(OH) D deficiency (20, 12.5, 10, 7.5 or 5 nmol/L as cut-off) among cases and controls without finding significant differences for any of the cutoff levels. However, no linear relationship of concentrations was investigated, nor were any other adjustments of factors with known relationships to both RA risk and 25(OH) D levels, e.g. BMI [4, 28]. Similarly, a report by Hiraki et al. did not describe any overall difference in circulating 25(OH) D levels between pre-symptomatic women and matched controls, although when stratified by time females presented an inverse linear association between 25(OH) D levels and future RA for the time group closest to symptom onset (3 months to < 4 years) in one of their two sub cohorts, which we were unable to reproduce . In that published report, although a lot of confounding factors were adjusted for, levels of DBP and/ or GC-polymorphisms were not taken into account, nor were differences inherently related to the method of determining the concentration of 25(OH) D. Several publications investigating the performance of commercially available kits for 25(OH) D analysis, were radioimmunoassays (RIA) (as in the report by Hiraki et al., 2014), as well as chemiluminescent immunoassays (CLIA) and enzyme-linked immunosorbent assays (ELISA) (used in the paper by Nielen et al., 2006) have shown great inter-assay differences, especially in the concentration interval corresponding to insufficiency of vitamin D, compared with HPLC methods. This may cause misclassification when, for example, using 50 nmol/L as cutoff [14, 29]. In a previous report sero-positivity, either for ≥2 RF-isotypes or anti-CCP2 antibodies among individuals at risk of developing RA, was not found to be associated with higher 25(OH) D levels . Likewise, in our study no association was found between anti-CCP2 antibodies or RF-IgM-positivity and 25(OH) D levels when adjusting for relevant confounders. Additionally, anti-CCP2 antibodies and RF-IgM positivity was not associated with DBP levels when adjusting for relevant confounders.
We acknowledge some limitations in this present study. For example, we included only one sample per individual, which may not reflect the long term vitamin D status. For analysis of DBP we used a monoclonal antibody, which may underestimate concentrations in subjects with an African ancestry, a problem not experienced in the methods using polyclonal antibodies  However, the population of northern Sweden is ethnically homogenous .
Our study had several strengths, including measurement of total 25(OH) D using HPLC, a protocol currently considered the gold standard method capable of distinguishing between D3 and D2 forms in an accredited laboratory setting. We measured levels of DBP and GC polymorphisms in addition to having the possibility of adjusting for possible confounding factors, BMI, education level and smoking status, factors that are associated with both outcome and exposure. By analysing DBP we were also able to calculate a proxy for free vitamin D. This was also the largest cohort of pre-symptomatic RA individuals studied to date when investigating vitamin D, with the ability to find differences for both genders.
The outcome of this study has added to knowledge about the relationship between D-vitamin status and the risk of developing RA.