A high-throughput screening assay of ascorbate in brain samples.
Belikova-NA; Glumac-AL; Kapralova-V; Cheikhi-A; Tyurina-YY; Vagni-VA; Kochanek-PM; Kagan-VE; Bayir-H
J Neurosci Methods 2011 Sep; 201(1):185-190
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.
Antioxidants; Brain-matter; Sampling; Sampling-methods; Laboratory-techniques;
Author Keywords: Vitamin C; Ascorbic acid; Nitroxides; Controlled cortical impact; Hemorrhagic shock; Head trauma
Hülya Bayir, Department of Critical Care Medicine, University of Pittsburgh, 4401 Penn Avenue, Pittsburgh, PA 15224, USA
Journal of Neuroscience Methods
University of Pittsburgh at Pittsburgh