High-throughput determination of malondialdehyde in plant tissues
High-throughput determination of malondialdehyde in plant tissues
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DOI：
10.1016/j.ab.2005.09.041
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发表时间：
2005-12-15
影响因子：
2.9
通讯作者：
Swennen, RL
中科院分区：
文献类型：
--
作者：
Davey, MW;Stals, E;Swennen, RL
丙二醛 (MDA) 是一种广泛使用的氧化脂质损伤标志物，其浓度随生物和非生物胁迫而变化。通常，MDA 与硫代巴比妥酸 (TBA) 反应后被定量为强光吸收和荧光加合物。然而，植物组织尤其含有许多可能干扰该反应的化合物，并且其浓度也根据组织类型和胁迫条件而变化。作为植物组织应激反应研究的一部分，我们有兴趣开发一种快速、准确且稳健的方案，使用反相 HPLC 进行 MDA 分析，以避免这些反应特异性问题。我们证明，在样品制备过程中，正丁醇的分配步骤至关重要，并且梯度 HPLC 分析对于防止进样之间的样品残留是必要的。此外，流动相的起始组合物必须具有足够的疏水性，以允许直接注射正丁醇提取物，而不会出现峰分裂、拖尾和其他伪影。为了最大限度地缩短分析时间，我们使用了短的所谓“Rocket”HPLC 柱和高流速。优化的 HPLC 分离每个样品的周转时间为 2.5 分钟。 MDA(TBA)2 的丁醇提取物可稳定至少 48 小时，并且回收率在添加的 0.38 和 7.5 pmol NIDA 之间呈线性。重要的是，该程序被证明与不同植物物种中 L-抗坏血酸和谷胱甘肽分析的现有提取程序兼容，允许对单个组织提取物进行多种“应激代谢物”分析。 (c) 2005 Elsevier Inc. 保留所有权利。
Malondialdehyde (MDA) is a widely used marker of oxidative lipid injury whose concentration varies in response to biotic and abiotic stress. Commonly, MDA is quantified as a strong light-absorbing and fluorescing adduct following reaction with thiobarbituric acid (TBA). However, plant tissues in particular contain many compounds that potentially interfere with this reaction and whose concentrations also vary according to the tissue type and stress conditions. As part of our studies into the stress responses of plant tissues, we were interested in developing a rapid, accurate, and robust protocol for MDA analysis using reverse-phased HPLC to avoid these problems with reaction specificity. We demonstrate that a partitioning step into n-butanol during sample preparation is essential and that gradient HPLC analysis is necessary to prevent sample carryover between injections. Furthermore, the starting composition of the mobile phase must be sufficiently hydrophobic to allow direct injection of the n-butanol extracts without peak splitting, tailing, and other artifacts. To minimize analysis times, we used a short, so-called "Rocket" HPLC column and high flow rates. The optimized HPLC separation has a turnaround time of 2.5 min per sample. Butanolic extracts of MDA(TBA)2 were stable for at least 48 h, and recoveries were linear between 0.38 and 7.5 pmol NIDA added. Importantly, this procedure proved to be compatible with existing extraction procedures for L-ascorbate and glutathione analysis in different plant species, allowing multiple "stress metabolite" analyses to be carried out on a single tissue extract. (c) 2005 Elsevier Inc. All rights reserved.