The recognition of 25-hydroxyvitamin D2 and D3 by a new binding protein based 25-hydroxyvitamin D assay

Background: The circulating form of vitamin D, 25-Hydroxyvitamin D (25-OHD), is commonly measured in clinical labs to evaluate vitamin D status of an individual. Vitamin D exists in 2 major forms: vitamin D3 and D2. Current methodologies for 25-OHD testing include antibody or vitamin D binding protein based assays, HPLC, and LC-MS. Although most current antibody or vitamin D binding protein based assays claim that they measure 25-OHD3 and 25-OHD2 equally, bias may still exist in the measurement of total 25-OHD when high concentration of 25-OHD2 is present. LC-MS/MS can measure 25-OHD3 and 25-OHD2 separately. We determined the ability of a new competitive protein binding assay (CPBA) to recognize 25-OHD2 and 25-OHD3 by comparing it with an LC-MS/MS method. Methods: Seventy-one serum samples were collected and divided into 3 groups according to 25-OHD2 and 25-OHD3 concentrations determined by LC-MS/MS: group A containing only 25-OHD3 (25-OHD2 < 3. ng/ml), group B containing both 25-OHD3 and 25-OHD2 (25-OHD2. ≥. 3. ng/ml, 25-OHD3. ≥. 10. ng/ml), and group C containing mainly 25-OHD2 (25-OHD3 < 10. ng/ml). The correlation between total 25-OHD concentrations measured by LC-MS/MS and CPBA was analyzed with linear regression analysis and the difference between the two methods was analyzed with paired, 2-tailed Student's t-Test. The biases between the two methods were also calculated. Results: The correlation analysis between LC-MS/MS (x) and CPBA (y) in the 3 groups are as follows: y=1.13x-1.01 (R²=0.65, n=25) in group A; y=0.68x+4.02 (R²=0.76, n=31) in group B, and y=0.49x+6.53 (R²=0.38, n=15) in group C. In group A, the mean total 25-OHD measured with CPBA was higher than that with LC-MS/MS, while in groups B, CPBA results were lower, but the differences were not statistically significant (p=0.476 for group A and 0.059 for group B). In group C, the mean 25-OHD measured with CPBA was lower than that with LC-MS/MS and the difference was statistically significant (p=0.002). The average biases in total 25-OHD concentrations between LC-MS/MS and CPBA were 10.8%, -23.6%, and -38.4% for groups A, B, and C, respectively. Conclusions: CPBA has a better correlation with LC-MS/MS for samples containing both 25-OHD2 and 25-OHD3 as compared to samples with mainly 25-OHD2 or 25-OHD3 only. However, it measures 25-OHD3 more accurately than 25-OHD2. Compared to LC-MS/MS, CPBA overestimates 25-OHD3, but underestimates 25-OHD2.