Serum and synovial fluid vitamin D metabolites and rheumatoid arthritis

Abstract

Vitamin D-deficiency has been linked to inflammatory diseases including rheumatoid arthritis (RA). Studies to date have focused on the impact of serum 25-hydroxyvitamin D3 (25(OH)D3), an inactive form of vitamin D, on RA disease activity and progression. However, anti-inflammatory actions of vitamin D are likely to be mediated at sites of RA disease, namely the inflamed joint, and may involve other vitamin D metabolites notably the active form of vitamin D, 1,25-dihydroxyvitamin D3 (1,25(OH)₂D3). In the current study serum and synovial fluid samples from n = 20 patients with persistent RA and n = 7 patients with reactive arthritis (ReA) were analysed for multiple vitamin D metabolites. Serum data for RA and ReA patients were compared to healthy controls (HC). There was no significant difference between RA or ReA patients relative to HC for 25(OH)D3, 24,25(OH)₂D3, 1,25(OH)₂D3 or 25(OH)D2. However, 3-epi-25(OH)D3 was significantly lower in RA and ReA patients compared to HC (p < 0.05). All vitamin D metabolites, apart from 25(OH)D2, were lower in SF compared to serum, and SF 1,25(OH)₂D3 was unquantifiable in 13/20 RA and 4/7 ReA samples. SF 25(OH)D3, 3-epi-25(OH)D3 and DBP correlated inversely with swollen joint score, and serum 25(OH)D2 and SF DBP correlated directly with C-reactive protein levels. These data indicate that serum 25(OH)D3 provides only limited insight into the role of vitamin D in RA. Alternative serum metabolites such as 3-epi-25(OH)₂D3, and SF metabolites, notably lack of SF 1,25(OH)₂D3, may be more closely linked to RA disease severity and progress.

Keywords: Free vitamin D; Inflammatory disease; Rheumatoid arthritis; Serum; Synovial fluid; Vitamin D; Vitamin D binding protein.

Figure 1.. Rheumatoid arthritis and the serum vitamin D metabolome.

Serum from n=20 persistent RA (RA) patients, n=7 reactive arthritis (ReA) patients and n=23 healthy controls (HC) were analysed by LC-MS/MS for: 25(OH)D3 (ng/mL); 3-epi-25(OH)D3 (ng/mL); 25(OH)D2 (ng/mL); 24,25(OH)₂D3 (ng/mL); 1,25(OH)₂D3 (pg/mL). Data shown are mean ± SD. * = significantly different HC vs RA, p<0.05, ** p<0.01. Mann-Whitney T-test for unpaired samples was performed for statistical analysis.

Figure 2.. Serum vs SF vitamin D metabolites in patients with persistent RA.

Comparison of serum and synovial fluid [2] concentrations for: 25(OH)D3; 3-epi-25(OH)D3; 25(OH)D2; 24,25(OH)₂D3 (ng/mL); 1,25(OH)₂D3 (pg/mL). B) Correlation of serum and SF concentrations for: 25(OH)D3; 3-epi-25(OH)D3; 25(OH)D2; 24,25(OH)₂D3 (all ng/mL); 1,25(OH)₂D3 (pg/mL). n=20 matched for serum and SF (One-way ANOVA). Linear regression was performed by best-fit values ± SD. Coefficient of determination (r²) values are shown for correlations that were statistically significant, p < 0.05.

Figure 3.. DBP, albumin and free versus bioavailable 25(OH)D in serum and SF

Serum and SF from n=20 RA and n=7 ReA patients were used to measure concentrations of DBP and albumin, and calculate free (not bound to DBP or albumin) and bioavailable (not bound to DBP) 25(OH)D. Data shown are mean ± SD. Statistical significance = *p<0.05; **p<0.01; *** p<0.001; ****p<0.0001. Wilcoxon matched-pairs signed rank test was performed for matched serum vs SF samples. Mann-Whitney unpaired t-test was performed for non-matched samples.

Figure 4.. Correlation of RA disease markers and serum/SF vitamin D metabolites and DBP.

RA disease markers with statistically significant correlations with vitamin D metabolites, or DBP, in serum or SF samples from RA patients. SJC (swollen joint count); CRP (C-reactive protein). Linear regression was performed by best-fit values ± SD. Coefficient of determination (r²) values are shown for correlations that were statistically significant, p < 0.05.