Department of Pharmacology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, United States.
Department of Pharmacology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, United States; Departments of Biochemistry and Biophysics, and Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, United States.
Free Radic Biol Med. 2018 Apr;118:108-118. doi: 10.1016/j.freeradbiomed.2018.02.019. Epub 2018 Feb 21.
The quantitative analysis of polyunsaturated fatty acyl (PUFA) chain oxidation products in tissue samples by mass spectrometry is hindered by the lack of durable internal standards for the large number of possible products. To address this problem in a study of oxidative PUFA degradation in Alzheimer's disease (AD) brain, uniformly C-labeled arachidonic acid (ARA) was produced biosynthetically, and allowed to oxidize under controlled conditions into a mixture of U-C-labeled ARA oxidation products. The components of this mixture were characterized with respect to their partitioning behavior during lipid extraction, their durability during saponification, trends in mouse brain tissue concentrations during post mortem intervals, and their overall suitability as internal standards for multiple-reaction monitoring tandem mass spectrometry. This mixture has now been used as a set of internal standards to determine the relative abundance of ARA and 54 non-stereospecific oxidation products in milligram samples of brain tissue. Many of these oxidation products were recovered from both healthy mouse and healthy human brain, although some of them were unique to each source, and some have not heretofore been described. The list of oxidation products detected in AD brain tissue was the same as in healthy human brain, although simple hydroxy-eicosanoids were significantly increased in AD brain. while more complex oxidation products were not. These results are consistent with an increased level of chemically-mediated oxidative ARA degradation in Alzheimer's disease. However, they also point to the existence of processes that selectively produce or eliminate specific oxidation products, and those processes may account for some of the inconsistencies in previously reported results.
质谱法定量分析组织样品中多不饱和脂肪酸（PUFA）链氧化产物，但由于可能的产物数量众多，缺乏耐用的内标，这一方法受到阻碍。为了解决这一问题，我们在阿尔茨海默病（AD）大脑氧化 PUFA 降解的研究中，生物合成了均 C 标记的花生四烯酸（ARA），并在受控条件下让其氧化成 U-C 标记的 ARA 氧化产物混合物。我们对该混合物的成分进行了特征描述，包括其在脂质提取过程中的分配行为、皂化过程中的耐久性、死后间隔期内小鼠脑组织浓度的变化趋势，以及作为多反应监测串联质谱法内标物的整体适用性。现在，该混合物已被用作一组内标物，用于确定脑组织毫克样本中 ARA 和 54 种非立体特异性氧化产物的相对丰度。许多这些氧化产物在健康小鼠和健康人脑组织中均有回收，但其中一些是每种来源所特有的，还有一些以前尚未描述过。在 AD 脑组织中检测到的氧化产物列表与健康人脑组织相同，尽管 AD 脑组织中的简单羟基二十烷酸显著增加，而更复杂的氧化产物则没有。这些结果与阿尔茨海默病中化学介导的氧化 ARA 降解水平增加一致。然而，它们也表明存在选择性产生或消除特定氧化产物的过程，这些过程可能是先前报道结果不一致的部分原因。
