Determination of Vitamin D and Its Metabolites in Human Brain Using an Ultra-Pressure LC–Tandem Mass Spectra Method

BACKGROUND: Low serum total 25-hydroxyvitamin D(3) [25(OH)D(3)] concentrations have been associated with cognitive impairment. However, it is unclear if serum 25(OH)D(3) concentrations are a valid indicator of the concentrations of vitamin D and its metabolites in human brain. OBJECTIVES: The aim of this study was to develop and validate a method to quantify vitamin D(3), 25(OH)D(3), and 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] in human brain. METHODS: The assay developments were performed using porcine brains. Liquid extraction was used in homogenized samples (∼0.1 g each) prior to analysis by LC-MS/MS with electrospray ionization following derivatization with 4-phenyl-1,2,4-triazoline-3,5-dione. This method was then applied to the determination of vitamin D and its metabolites in a whole human brain obtained from the National Development and Research Institutes. RESULTS: The method showed good linearity of vitamin D(3), 25(OH)D(3), and 1,25(OH)(2)D(3) over the physiological range (R(2 )= 0.9995, 0.9968, and 0.9970, respectively). The lowest detection limit for vitamin D(3), 25(OH)D(3), and 1,25(OH)(2)D(3) in porcine brain was 25, 50 and 25 pg/g, respectively. The method was successfully applied to the determination of vitamin D(3) and its metabolites in the prefrontal cortex, middle frontal cortex, middle temporal cortex, cerebellum, corpus callosum, medulla, and pons of a human brain. All analyzed human brain regions contained 25(OH)D(3), with corpus callosum containing 334 pg/g compared with 158 pg/g in cerebellum. 1,25(OH)(2)D(3) was only detected in prefrontal and middle frontal cortices at a very low level. No vitamin D(3) was detected in any examined areas of this single human brain. CONCLUSIONS: To the best of our knowledge, this study is the first report of the measurement of concentrations of vitamin D metabolites in human brain. This validated method can be applied to postmortem studies to obtain accurate information about the presence and role of vitamin D and its metabolites in human brain and neurodegenerative diseases.