A comprehensive liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay was developed to quantify 10 forms of vitamin D in sera from healthy adults and patients suffering from rheumatoid arthritis (RA), type 1 diabetes (T1-D), and Alzheimer disease (AD).
The rapid assay, validated according to US Food and Drug Administration guidelines with Chromsystems and DEQAS samples, was applied to 36 nonhealthy sera samples (41.7% male, age range of 14-95, mean = 54.00 ± 21.98 years), consisting of individuals with RA, T1-D, and AD (n = 12 each) and was compared to samples from 32 healthy individuals (50% male, age range of 19-90, mean = 58.83 ± 22.93 years).
The key findings are (1) the 23R,25-dihydroxyvitamin D3 form was quantified for the first time (healthy = 0.427 ± 0.633 nmol/L; combined disease = 0.395 ± 0.483 nmol/L), (2) the 3-epi-25-hydroxyvitamin D3 metabolite was found in all groups with significantly higher concentration in the diseased samples [healthy = 6.093 ± 6.711 nmol/L; combined disease = 22.433 ± 13.535 nmol/L, t(52.5) = -6.411; P < .001], (3) a significant difference was found for the active form (1α-25-dihydroxyvitamin D3) between health (0.027 ± 0.035 nmol/L) and disease (0.433 ± 0.870 nmol/L) [t(35.1) = -2.797, P = 0.008], and (4) there was no significant correlation between the total circulating and total active forms in either the disease or healthy group (r = -0.180 and -0.274, respectively, with no difference between the correlation coefficients, z = -0.389, P = .697). Receiver operating characteristic curve analysis showed good sensitivity and specificity for using the 3-epi-25-hydroxyvitamin D concentration to predict disease status (area under the curve = 0.880, P < .001). Discriminant function analysis using concentrations of 23R,25-dihydroxyvitamin D3, 25-hydroxyvitamin D2, and 3-epi-25-hydroxyvitamin D classified 94.4% (91.7% in cross-validation) of the cases correctly.
This study reveals significant differences between health and disease with epimers having the potential to relate to disease. The potential implications of the information gleaned from measuring all forms warrant application of more comprehensive assays for future clinical studies investigating the link between vitamin D and health.