A rapid gas chromatographic assay for determining oxyradical scavenging capacity of antioxidants and biological fluids.
A rapid gas chromatographic assay for determining oxyradical scavenging capacity of antioxidants and biological fluids.
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DOI：
10.1016/s0891-5849(97)00277-3
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发表时间：
1998-02
影响因子：
7.4
通讯作者：
Gary W. Winston;Francesco Regoli;A. Dugas;Jessica H. Fong;Kristie A. Blanchard
中科院分区：
文献类型：
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作者：
Gary W. Winston;Francesco Regoli;A. Dugas;Jessica H. Fong;Kristie A. Blanchard
在此，我们报告了一种新的，快速的，可靠的方法来测量纯抗氧化剂溶液或生物组织的保护性抗氧化剂潜力。2，2 ′-偶氮双脒基丙烷（ABAP）热均解产生的过氧自由基导致α-酮基-γ-甲硫基丁酸（KMBA）氧化为乙烯;乙烯形成通过气相色谱分析反应容器的顶空来监测。在与KMBA竞争氧自由基的抗氧化剂存在下乙烯形成的部分抑制是总氧自由基清除能力测定（TOSCA）的基础。该测定法被证明是可靠的定量ROS清除潜力。可定量参数与文献中报告的基于荧光和氧电极的测定预测的参数的相对顺序一致。竞争过氧自由基的抗氧化剂以不同的方式影响KMBA氧化的速率，但TOSC的计算不受这种变化的影响。响应在很宽的样品浓度范围内呈线性，经典的可溶性抗氧化剂的TOSC值显示以下相对顺序：Trolox >尿酸>抗坏血酸> GSH。KMBA方法对于生物组织是可靠的; 1 μg大鼠肝胞浆蛋白的TOSC为0.40 ± 0.02，微粒体膜的TOSC为0.15 ± 0.03。可溶性抗氧化剂占77%的保护性抗氧化潜力在大鼠肝胞质溶胶。当掺入微粒体膜时，α-生育酚可显著增强对过氧自由基的抗氧化保护作用;因此，该试验适用于评估脂溶性抗氧化剂。
Herein, we report a new, rapid,and reliable method for measuring the protective antioxidant potential of pure antioxidant solutions or biological tissues. Peroxyl radicals generated by thermal homolysis of 2,2′-azobis-amidinopropane (ABAP) cause the oxidation of α-keto-γ-methiolbutyric acid (KMBA) to ethylene; ethylene formation is monitored by gas chromatographic analysis of head space from the reaction vessel. The partial inhibition of ethylene formation in the presence of antioxidants that compete with KMBA for oxyradicals is the basis of the Total Oxyradical Scavenging Capacity Assay (TOSCA). The assay is shown to be reliable for quantifying ROS scavenging potential. The quantifiable parameters are consistent with the relative order of those predicted by the fluorescence- and oxygen electrode-based assays reported in the literature. Antioxidants competing for peroxyl radicals influenced the rate of KMBA oxidation in different ways, but the calculation of TOSC was not affected by such variations. Responses were linear over a wide range of sample concentrations and the TOSC values of classical soluble antioxidants showed the following relative order: Trolox > uric acid > ascorbic acid > GSH. The KMBA method was reliable for biological tissues; the TOSC for 1 μg rat liver cytosolic protein was 0.40 ± 0.02 and for the microsomal membrane, 0.15 ± 0.03. Soluble antioxidants accounted for 77% of the protective antioxidant potential in rat liver cytosol. When incorporated into the microsomal membrane, α-tocopherol markedly enhances antioxidant protection against peroxyl radical; thus, the assay is suitable for the assessment of fat-soluble antioxidants.