O'Donnell V B, Eiserich J P, Chumley P H, Jablonsky M J, Krishna N R, Kirk M, Barnes S, Darley-Usmar V M, Freeman B A
Department of Anesthesiology, University of Alabama at Birmingham, 35233, USA.
Chem Res Toxicol. 1999 Jan;12(1):83-92. doi: 10.1021/tx980207u.
Reactive nitrogen species derived from nitric oxide are potent oxidants formed during inflammation that can oxidize membrane and lipoprotein lipids in vivo. Herein, it is demonstrated that several of these species react with unsaturated fatty acid to yield nitrated oxidation products. Using HPLC coupled with both UV detection and electrospray ionization mass spectrometry, products of reaction of ONOO- with linoleic acid displayed mass/charge (m/z) characteristics of LNO2 (at least three products at m/z 324, negative ion mode). Further analysis by MS/MS gave a major fragment at m/z 46. Addition of a NO2 group was confirmed using [15N]ONOO- which gave a product at m/z 325, fragmenting to form a daughter ion at m/z 47. Formation of nitrated lipids was inhibited by bicarbonate, superoxide dismutase (SOD), and Fe3+-EDTA, while the yield of oxidation products was decreased by bicarbonate and SOD, but not by Fe3+-EDTA. Reaction of linoleic acid with both nitrogen dioxide (NO2) or nitronium tetrafluoroborate (NO2BF4) also yielded nitrated lipid products (m/z 324), with HPLC retention times and MS/MS fragmentation patterns identical to the m/z 324 species formed by reaction of ONOO- with linoleic acid. Finally, reaction of HPODE, but not linoleate, with nitrous acid (HONO) or isobutyl nitrite (BuiONO) yielded a product at m/z 340, or 341 upon reacting with [15N]HONO. MS/MS analysis gave an NO2- fragment, and 15N NMR indicated that the product contained a nitro (RNO2) functional group, suggesting that the product was nitroepoxylinoleic acid [L(O)NO2]. This species could form via homolytic dissociation of LOONO to LO and NO2 and rearrangement of LO to an epoxyallylic radical L(O)* followed by recombination of L(O)* with *NO2. Since unsaturated lipids of membranes and lipoproteins are critical targets of reactive oxygen and nitrogen species, these pathways lend insight into mechanisms for the formation of novel nitrogen-containing lipid products in vivo and provide synthetic strategies for further structural and functional studies.
源自一氧化氮的活性氮物质是炎症过程中形成的强效氧化剂，可在体内氧化膜脂质和脂蛋白脂质。在此证明，其中几种物质与不饱和脂肪酸反应生成硝化氧化产物。使用结合紫外检测和电喷雾电离质谱的高效液相色谱法，过氧亚硝酸根（ONOO-）与亚油酸反应的产物显示出LNO2的质荷比（m/z）特征（负离子模式下m/z 324处至少有三种产物）。通过串联质谱（MS/MS）进一步分析得到m/z 46处的主要碎片。使用[15N]ONOO-证实添加了一个NO2基团，其产物在m/z 325处，裂解形成m/z 47处的子离子。碳酸氢盐、超氧化物歧化酶（SOD）和Fe3+-EDTA可抑制硝化脂质的形成，而碳酸氢盐和SOD可降低氧化产物的产率，但Fe3+-EDTA则无此作用。亚油酸与二氧化氮（NO2）或四氟硼酸硝鎓（NO2BF4）反应也产生硝化脂质产物（m/z 324），其高效液相色谱保留时间和串联质谱裂解模式与ONOO-与亚油酸反应形成的m/z 324物种相同。最后，13-羟基-9,11-十八碳二烯酸（HPODE）而非亚油酸与亚硝酸（HONO）或亚硝酸异丁酯（BuiONO）反应产生m/z 340处的产物，与[15N]HONO反应则产生m/z 341处的产物。串联质谱分析得到一个NO2-碎片，15N核磁共振表明该产物含有硝基（RNO2）官能团，表明该产物为硝基环氧亚油酸[L(O)NO2]。该物种可通过LOONO均裂为LO和NO2，LO重排为环氧烯丙基自由基L(O)*，随后L(O)与NO2重组形成。由于膜和脂蛋白的不饱和脂质是活性氧和氮物质的关键靶点，这些途径有助于深入了解体内新型含氮脂质产物的形成机制，并为进一步的结构和功能研究提供合成策略。
