Donáth-Nagy Gabriella, Buchwald Peter, Vancea Szende, Croitoru Mircea, Tokés Béla
University of Medicine and Pharmacy from Targu Mures, Romania.
J Biochem Biophys Methods. 2008 Apr 24;70(6):1317-23. doi: 10.1016/j.jprot.2007.12.007. Epub 2008 Jan 7.
Nowadays, very diverse human activities generate urgent demands for fast, sensitive reliable innovative tools capable of detecting major industrial, military, and other dangerous products. An important part of these compounds are free radicals. Capillary electrophoresis (CE), especially in its miniaturized format (lab-on-a-chip), and other electromigration methods offer special possibilities to resolve this problem. These measurements have a great opportuness because of very wide chemical and biological role of free radicals. Several compounds, e.g. monomers and some biologically important groups (as are nitrones) oppose oxidative challenges by virtue of their trap very rapidly oxygen- or carbon-centered radicals and generating other radical species which are stable and biochemically less harmful than the original ones. In many cases, conventionally, the relative trap capacity is measured against tert.-butylhydroperoxide (TBH). In this lecture are presented numerous important free radical species (active oxygen-, nitrogen- and carbon-centered ones, as HO, NO etc) and their adequate in vitro and in vivo applied bioanalytical methods, including liquid chromatography with electrochemical detection and mass spectrometry, gas chromatography with mass spectrometry, capillary electrophoresis, electron spin resonance and chemiluminescence analysis. A simple and highly sensitive method is the capillary zone electrophoresis with amperometric detection (CZE-AD); It was introduced to determine indirectly OH by analysing its reaction products with salicylic and dihydroxybenzoic acids. Hydroxylated radical products of these acids are often used as a relative measurement in free radical research. Accurate determination of pK(a) values is important for proper characterization of newly synthesized molecules. CZE method was used for determination of their values. Are initiated new research fields as Fenton-, electro-Fenton and photoelectro-Fenton chemistry and foreseen their perspectives. Nitric oxide is an important cell signaling molecule in physiology and pathophysiology. An indirect method for monitoring nitric oxide (NO) by determining nitrate and nitrite by microchip capillary electrophoresis (CE) with electrochemical (EC) detection has been developed. The amount of nitrite formed in this reaction (analyzed by capillary electrophoresis) was compared with the amount of oxygen consumed (measured by polarography). Were observed a linear relationship between the amount of consumed oxygen and the amount of nitrite formed in the measured range. These results demonstrate that polarographic measurements of the amount of oxygen consumed in the reaction with NO could be used to estimate the concentration of dissolved NO in authentic media. Polarography is an adequate method also to quantitative kinetic study of the free radical activity and of the trapping capacity of different compounds. This method is based on measure of the catalytic polarografic current of Fe(III) in the presence of free radical sources (TBH, hydrogen-peroxydes), and their traps. Personal contribution of the authors in this field is discussed.
如今，各种各样的人类活动迫切需要快速、灵敏、可靠的创新工具来检测主要的工业、军事及其他危险产品。这些化合物中的一个重要部分是自由基。毛细管电泳（CE），尤其是其微型化形式（芯片实验室）以及其他电迁移方法为解决这一问题提供了特殊的可能性。由于自由基具有非常广泛的化学和生物学作用，这些测量具有很大的机遇。几种化合物，例如单体和一些生物学上重要的基团（如硝酮），凭借其能够非常迅速地捕获以氧或碳为中心的自由基并生成其他自由基物种，从而应对氧化挑战，这些生成的自由基物种比原始自由基更稳定且对生物化学的危害更小。在许多情况下，传统上是相对于叔丁基过氧化氢（TBH）来测量相对捕获能力。在本次讲座中，介绍了众多重要的自由基物种（以活性氧、氮和碳为中心的自由基，如HO、NO等）及其适用的体外和体内生物分析方法，包括电化学检测液相色谱法、质谱联用气相色谱法、毛细管电泳法、电子自旋共振法和化学发光分析法。一种简单且高度灵敏的方法是安培检测毛细管区带电泳（CZE - AD）；它通过分析OH与水杨酸和二羟基苯甲酸的反应产物来间接测定OH。这些酸的羟基化自由基产物在自由基研究中常被用作相对测量。准确测定pK(a)值对于新合成分子的正确表征很重要。采用CZE方法测定其值。开启了如芬顿、电芬顿和光电子芬顿化学等新的研究领域并展望了它们的前景。一氧化氮在生理学和病理生理学中是一种重要的细胞信号分子。已开发出一种通过微芯片毛细管电泳（CE）结合电化学（EC）检测来测定硝酸盐和亚硝酸盐从而间接监测一氧化氮（NO）的方法。将该反应中形成的亚硝酸盐量（通过毛细管电泳分析）与消耗的氧量（通过极谱法测量）进行比较。在所测范围内观察到消耗的氧量与形成的亚硝酸盐量之间存在线性关系。这些结果表明，通过极谱法测量与NO反应中消耗的氧量可用于估计真实介质中溶解NO的浓度。极谱法也是研究自由基活性和不同化合物捕获能力定量动力学的一种合适方法。该方法基于在自由基源（TBH、过氧化氢）及其捕获剂存在下测量Fe(III)的催化极谱电流。讨论了作者在该领域的个人贡献。
