title: A high-throughput screening assay of ascorbate in brain samples.
abstract: Ascorbate is a vital reductant/free radical scavenger in the CNS, whose content defines - to a large extent - the redox status and the antioxidant reserves. Quick, reliable and specific methods for its measurement in brain samples are highly desirable. We have developed a new high-throughput screening assay for measurements of ascorbate using a fluorescence plate-reader. This assay is based on a direct reaction of ascorbate with a nitroxide radical conjugated with a fluorogenic acridine moiety, 4-((9-acridinecarbonyl)-amino)-2,2,6,6-tetramethylpiperidine-1-oxyl radical (AC-TEMPO), yielding fluorescent hydroxylamine product (AC-TEMPO-H). The reaction was monitored over time using fluorescence and electron spin resonance techniques. The appearance of fluorescent AC-TEMPO-H was linear within the range of 3.75-75μM AscH(-) in the sample (0.5-10μM AscH(-) in the well). Assay was validated with high performance liquid chromatography method. The concentration of ascorbate in murine tissue samples, including brain samples after traumatic brain injury and hemorrhagic shock, was measured.
authors.affiliation: Department of Environmental and Occupational Health, University of Pittsburgh, Pittsburgh, PA 15219, USA.
authors.forename: Natalia A
authors.initials: NA
authors.lastname: Belikova
authors.forename: Ashley L
authors.initials: AL
authors.lastname: Glumac
authors.forename: Valentyna
authors.initials: V
authors.lastname: Kapralova
authors.forename: Amin
authors.initials: A
authors.lastname: Cheikhi
authors.forename: Yulia Y
authors.initials: YY
authors.lastname: Tyurina
authors.forename: Vincent A
authors.initials: VA
authors.lastname: Vagni
authors.forename: Patrick M
authors.initials: PM
authors.lastname: Kochanek
authors.forename: Valerian E
authors.initials: VE
authors.lastname: Kagan
authors.forename: Hülya
authors.initials: H
authors.lastname: Bayir
mesh_terms: D000818:Animals; D001205:Ascorbic Acid / Q000737:chemistry* / Q000502:physiology; D001923:Brain Chemistry / Q000502:physiology; D057166:High-Throughput Screening Assays / Q000379:methods*; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL
chemical_list: D001205:Ascorbic Acid