Analysis of aldehydic lipid peroxidation products by TLC/densitometry.
Analysis of aldehydic lipid peroxidation products by TLC/densitometry.
复制标题
通过薄层色谱/密度测定法分析醛类脂质过氧化产物。
作者：
Beckman,JK;MorleyJr,SA;Greene,HL
我们探索了在反射和荧光模式下使用薄层色谱法（TLC）/光密度法定量脂质过氧化的特定产物。花生四烯酸与铁和抗坏血酸作用24 h后产生醛类过氧化产物，采用二硝基苯肼（DNPH）和环己二酮（CHD）两种非特异性衍生试剂，比较了几种TLC/光密度法定量分析过氧化产物的方法。花生四烯酸-过氧化产物的DNPH腙在硅胶上TLC分离后，显示出显著的链烷醛和羟基烯醛带。反相高效液相色谱法证实，伯烷醛组分是己醛，而伯羟基烯醛是4-羟基壬烯醛。本文以腙为外标法，建立了直接用薄层色谱/光密度法对这些产品进行半定量分析的方法。TLC/光密度法（荧光模式）用于测量醛的CHD加合物，通过在癸醛（用作内标）的存在下形成衍生物，并通过反相TLC分离衍生物。在应用0.5纳摩尔时可检测到己醛-CHD，而4-羟基甲烯醛显示出较低的响应，并且在10纳摩尔时可检测到。使用适当的响应因子，测定了花生四烯酸醛样品中的己醛和4-羟基壬烯醛，结果与DNPH法所得结果相似。使用类似方法分析二十二碳六烯酸的过氧化产物（24小时暴露）DHA过氧化产物中含有极低水平的烷醛，而极性醛和羟基烯醛含量较高。还证实了暴露于铁的微粒体形成烷醛、酮、羟基烯醛和磷脂醛。这些醛测量方法的用途和局限性进行了讨论。
We have explored the use of thin‐layer chromatography (TLC)/densitometry in both the reflectance and fluorescence mode for quantitation of specific products of lipid peroxidation. Aldehydic peroxidation products were generated by exposure of arachidonic acid to iron and ascorbic acid for 24 hr. Several methods for the quantitative analysis of peroxidation products by TLC/densitometry were compared using two different aldehydespecific derivatizing reagents, namely dinitrophenylhydrazine (DNPH) and cyclohexanedione (CHD). DNPH hydrazones of the arachidonic acid‐peroxidation products, upon TLC separation on silica gel, revealed prominent alkanal and hydroxyalkenal bands. Reverse phase high performance liquid chromatography confirmed that the primary alkanal component was hexanal, while the primary hydroxyalkenal was 4‐hydroxynonenal. Semiquantitative methods for the direct analysis of these products by TLC/densitometry were worked out based on the use of external hydrazone standards. TLC/densitometry (fluorescence mode) was used to measure CHD adducts of aldehydes by forming the derivatives in the presence of decanal (used as an internal standard) and separating the derivatives by reverse phase TLC. Hexanal‐CHD was detectable upon application of 0.5 nanomoles while 4‐hydroxynomenal showed a lower response and was detectable with 10 nanomoles. Using appropriate response factors, hexanal and 4‐hydroxynonenal were measured in the aldehyde sample from arachidonic acid and results were similar to those obtained by the DNPH method.Similar approaches were used to analyze the peroxidation products of docosahexaenoic acid (24‐hr exposure) The DHA peroxidation products contained extremely low levels of alkanals, while polar aldehydes and hydroxyalkenals were prominent. Formation of alkanals, osazones, hydroxyalkenals and phospholipid aldehydes from iron‐exposed microsomes was also demonstrated. Uses and limitations of these methods of aldehyde measurement are discussed.