Free versus total serum 25-hydroxyvitamin D in a murine model of colitis

Abstract

Inflammatory bowel diseases (IBD) such as ulcerative colitis and Crohn's disease have been linked to vitamin D-deficiency. Using a dextran sodium sulphate (DSS)-induced model of IBD we have shown previously that mice raised on vitamin D-deficient diets from weaning have lower serum 25-hydroxyvitamin D (25OHD) levels and develop more severe colitis compared to vitamin D-sufficient counterparts. We have also shown in vitro that immune responses to 25OHD may depend on 'free' rather than total serum concentrations of 25OHD. To investigate the possible effects of free versus total 25OHD on anti-inflammatory immune responses in vivo we have studied DSS-induced colitis in wild type C57BL/6 mice raised from weaning on diets containing vitamin D2 (D2) or vitamin D3 (D3) only (both 1000 IU/kg feed). 25OHD2 has lower binding affinity for the vitamin D binding protein than 25OHD3 which results in higher levels of free 25OHD2 relative to free 25OHD3 in mice raised on a D2-only diet. Total serum 25OHD concentrations, measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS), showed that D2 mice had significantly lower levels of 25OHD than D3 mice (6.85 ± 2.61 nmol/L vs. 49.16 ± 13.8 nmol/L for D2 and D3 respectively). Despite this, direct ELISA measurement showed no difference in free serum 25OHD levels between D2 and D3 mice (13.62 ± 2.26 pmol/L vs. 14.11 ± 2.24 pmol/L for D2 and D3 respectively). Analysis of DSS-induced colitis also showed no difference in weight loss or disease progression between D2 and D3 mice. These data indicate that despite D2-fed mice being vitamin D-deficient based on serum total 25OHD concentrations, these mice showed no evidence of increased inflammatory colitis disease relative to vitamin D-sufficient D3 mice. We therefore propose that free, rather than total serum 25OHD, may be a better marker of immune responses to vitamin D in vivo.

Keywords: Colitis; Free vitamin D; Inflammation; Mouse; Total vitamin D; Vitamin D; Vitamin D binding protein; Vitamin D2; Vitamin D3.

Figure 1.. Total and free 25OHD in vitamin D2 and vitamin D3 fed mice.

In DSS- or water-treated D2 and D3 mice: (A) Total serum 25OHD2 and 25OHD3 determined by LC-MS/MS (B) Free serum 25OHD determined by ELISA (C) Ratios of free to total 25OHD. Data are shown as mean values ± SEM; statistical analyses performed using ANOVA; **P<0.01, ****P<0.0001; n=8 mice/treatment group.

Figure 2.. Individual weight changes for DSS-treated D2 and D3 mice.

% change in body weight for individual mice during (day 0–7) and after (day 8–10) treatment with DSS (2.5% in water). A. Individual D2 mice (D2M1–D2M8). B Individual D3 mice (D3M1–D3M8).

Figure 3.. Effect of DSS on body weight and colitis disease score in D2 and D3 mice.

A. % change in body weight for D2 and D3 mice during (day 0–7) and after (day 8–10) treatment with DSS (2.5% in water). B. In water only control mice. C. Colitis disease scores for D2 and D3 mice: during (day 0–7) and after (day 8–10) treatment with DSS (2.5% in water). Data are shown as mean ± SEM; statistical analyses performed using Student’s t-test; *P<0.05; n=8 mice/treatment group.

Figure 4.. Histopathological analysis of excised and dissected colons for DSS-treated D2 and D3 fed mice.

Colons were fixed in formalin and embedded in paraffin wax before being sectioned at 5 μm. A. Both D2 and D3 mice showed signs of inflammation (red arrows), determined by H&E staining. B. Quantification of histological scoring for inflammatory infiltration (0 – 3, red arrows) combined with scoring for mucosal ulceration damage (0 – 3, blue arrows) in D2 and D3 fed mice exposed to DSS. Data are mean ± SD for n=7 mice.