Simultaneous Quantification of 25-Hydroxyvitamin D(3) and 24,25-Dihydroxyvitamin D(3) in Rats Shows Strong Correlations between Serum and Brain Tissue Levels

While vitamin D(3) is recognized as a neuroactive steroid affecting both brain development and function, efficient analytical method in determining vitamin D(3) metabolites in the brain tissue is still lacking, and the relationship of vitamin D(3) status between serum and brain remains elusive. Therefore, we developed a novel analysis method by using high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) to simultaneously quantify the concentrations of 25-hydroxyvitamin D(3) (25(OH)D(3)) and 24,25-dihydroxyvitamin D(3) (24,25(OH)(2)D(3)) in the serum and brain of rats fed with different dose of vitamin D(3). We further investigated whether variations of serum vitamin D(3) metabolites could affect vitamin D(3) metabolite levels in the brain. Serum and brain tissue were analyzed by HPLC-MS/MS with electrospray ionization following derivatization with 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD). The method is highly sensitive, specific, and accurate to quantify 25(OH)D(3) and 24,25(OH)(2)D(3) in animal brain tissue. Vitamin D(3) metabolites in brain tissue were significantly lower in rats fed with a vitamin D deficiency diet than in rats fed with high vitamin D(3) diet. There was also a strong correlation of vitamin D(3) metabolites in serum and brain. These results indicate that vitamin D(3) status in serum affects bioavailability of vitamin D(3) metabolites in the brain.