Arneson Kyle O, Roberts L Jackson
Division of Clinical Pharmacology, Vanderbilt University School of Medicine, Nashville, Tennessee, USA.
Methods Enzymol. 2007;433:127-43. doi: 10.1016/S0076-6879(07)33007-3.
Free radicals derived primarily from oxygen have been implicated in the pathophysiology of a wide variety of human diseases. Quantification of products of free radical damage in biological systems is necessary to understand the role of free radicals in disease states. Measures of lipid peroxidation are often used to quantitate oxidative damage though many of these measures have inherent problems with sensitivity and specificity especially when used to quantitate in vivo oxidative injury. The discovery of the F(2)-isoprostanes (F(2)-IsoPs), prostaglandin F(2)-like compounds derived by the free radical peroxidation of arachidonic acid (AA, C20:4, omega-6) has largely overcome these limitations. The measurement of the F(2)-IsoPs has been shown to be one of the most accurate approaches to quantifying oxidative damage in vivo. We have extended our studies of lipid peroxidation and the F(2)-IsoPs to docosahexaenoic acid (DHA, C22:6, omega-3) and its peroxidation products. We have found that DHA oxidizes both in vitro and in vivo to form F(2)-IsoP-like compounds termed F(4)-neuroprostanes (F(4)-NPs). DHA is specifically enriched in neuronal membranes making the F(4)-NPs sensitive and specific markers of neuronal oxidative damage. Adapting the methodology used to quantitate the F(2)-IsoPs, we utilize stable isotope dilution, negative ion chemical ionization, gas chromatography mass spectrometry (GC/MS) to quantitate the F(4)-NPs with a limit of detection in the low picomolar range. Methods have been developed to quantitate both the F(4)-NPs and the neurofurans (NFs), DHA derived peroxidation products containing a substituted tetrahydrofuran ring, in brain tissue and cerebrospinal fluid. This review outlines in detail proper sample handling, extraction and hydrolysis of the F(4)-NPs and NFs from tissue membrane phospholipids or biological fluids, and purification and derivatization of the compounds for analysis by GC/MS.
主要源自氧气的自由基与多种人类疾病的病理生理学有关。为了解自由基在疾病状态中的作用，对生物系统中自由基损伤产物进行定量分析很有必要。脂质过氧化的测量方法常被用于定量氧化损伤，不过其中许多方法在灵敏度和特异性方面存在固有问题，尤其是用于定量体内氧化损伤时。F(2)-异前列腺素（F(2)-IsoPs）的发现，这类由花生四烯酸（AA，C20:4，ω-6）自由基过氧化产生的前列腺素F(2)样化合物，在很大程度上克服了这些局限性。F(2)-IsoPs的测量已被证明是定量体内氧化损伤最准确的方法之一。我们已将脂质过氧化及F(2)-IsoPs的研究扩展至二十二碳六烯酸（DHA，C22:6，ω-3）及其过氧化产物。我们发现DHA在体外和体内均会氧化，形成被称为F(4)-神经前列腺素（F(4)-NPs）的F(2)-IsoP样化合物。DHA在神经元膜中特异性富集，使得F(4)-NPs成为神经元氧化损伤的敏感且特异的标志物。通过采用用于定量F(2)-IsoPs的方法，我们利用稳定同位素稀释、负离子化学电离、气相色谱 - 质谱联用（GC/MS）来定量F(4)-NPs，检测限低至皮摩尔范围。现已开发出在脑组织和脑脊液中定量F(4)-NPs及神经呋喃（NFs，含取代四氢呋喃环的DHA衍生过氧化产物）的方法。这篇综述详细概述了从组织膜磷脂或生物流体中正确处理、提取和水解F(4)-NPs及NFs的方法，以及对这些化合物进行纯化和衍生化以便通过GC/MS分析的方法。
