A High-Throughput Screening Assay of Ascorbate in Brain Samples

Details

• Alternative Title:
  J Neurosci Methods
• Personal Author:
  Belikova, Natalia A. ; Glumac, Ashley L. ; Kapralova, Valentyna ; Cheikhi, Amin ; Tyurina, Yulia Y. ; Vagni, Vince A. ; Kochanek, Patrick M. ; Kagan, Valerian E. ; Bayîr, Hülya
• Description:
  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.
• Subjects:
  Animals Article Ascorbic Acid Brain Chemistry Controlled Cortical Impact Head Trauma Hemorrhagic Shock High-Throughput Screening Assays Male Mice Mice, Inbred C57BL Nitroxides Vitamin C

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• Source:
  J Neurosci Methods. 2011; 201(1):185-190
• Pubmed ID:
  21855575
• Pubmed Central ID:
  PMC3276367
• Document Type:
  Journal Article
• Funding:
  R01 HL070755/HL/NHLBI NIH HHS/United States ; NS061817/NS/NINDS NIH HHS/United States ; R01 OH008282/OH/NIOSH CDC HHS/United States ; U19 AI068021/AI/NIAID NIH HHS/United States ; R01 HL070755-07/HL/NHLBI NIH HHS/United States ; HD057587/HD/NICHD NIH HHS/United States ; NS30318/NS/NINDS NIH HHS/United States ; P01 NS030318/NS/NINDS NIH HHS/United States ; R01 NS061817-04/NS/NINDS NIH HHS/United States ; R21 HD057587/HD/NICHD NIH HHS/United States ; P50 NS030318/NS/NINDS NIH HHS/United States ; OH008282/OH/NIOSH CDC HHS/United States ; R01 NS061817/NS/NINDS NIH HHS/United States ; U19 AI068021-06/AI/NIAID NIH HHS/United States ; HL70755/HL/NHLBI NIH HHS/United States ; R21 HD057587-02S1/HD/NICHD NIH HHS/United States
• Volume:
  201
• Issue:
  1
• Collection(s):
  CDC Public Access
• Main Document Checksum:
  urn:sha256:482e5344927d3d8118b9e615f745f74d3a096191f3e1700731bcee39bc9880c9
• Download URL:
  https://stacks.cdc.gov/view/cdc/33306/cdc_33306_DS1.pdf
• File Type:
  [PDF - 424.82 KB ]