A high-throughput screening assay of ascorbate in brain samples.
A high-throughput screening assay of ascorbate in brain samples.
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DOI：
10.1016/j.jneumeth.2011.08.001
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发表时间：
2011-09-30
影响因子：
3
通讯作者：
Bayir, Huelya
中科院分区：
文献类型：
--
作者：
Belikova, Natalia A.;Glumac, Ashley L.;Kapralova, Valentyna;Cheikhi, Amin;Tyurina, Yulia Y.;Vagni, Vincent A.;Kochanek, Patrick M.;Kagan, Valerian E.;Bayir, Huelya
抗坏血酸是中枢神经系统中重要的还原剂/自由基清除剂，其含量在很大程度上决定了氧化还原状态和抗氧化剂储备。快速，可靠和具体的方法来测量其在脑样品中是非常可取的。我们已经开发了一种新的高通量筛选测定抗坏血酸使用荧光读板仪。该测定基于抗坏血酸盐与氮氧自由基的直接反应，该氮氧自由基与荧光吖啶部分4-（（9-吖啶羰基）-氨基）-2，2，6，6-四甲基哌啶-1-氧基自由基（AC-TEMPO）缀合，产生荧光羟胺产物（AC-TEMPO-H）。使用荧光和电子自旋共振技术随时间监测反应。荧光AC-TEMPO-H的出现在样品中3.75-75 μM AscH-范围内呈线性（孔中0.5-10 μM AscH-）。采用高效液相色谱法对含量测定进行验证。测定了小鼠组织样品中抗坏血酸盐的浓度，包括创伤性脑损伤和出血性休克后的脑样品。
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 3.75–75 μM AscH- in the sample (0.5–10 μM 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.
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