Stolze K, Udilova N, Nohl H
Institute of Pharmacology and Toxicology, Veterinary University of Vienna, Vienna, Austria.
Free Radic Biol Med. 2000 Nov 15;29(10):1005-14. doi: 10.1016/s0891-5849(00)00401-9.
The spin trap 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline N-oxide (DEPMPO) forms a superoxide adduct with a half-life of almost 15 min. DEPMPO is very hydrophilic and its use for the detection of radicals in the lipid phase (lipid-derived radicals and superoxide generated in the lipid phase) is therefore limited due to its very low concentration in the lipid phase. For the detection of lipid-derived radicals, three derivatives of DEPMPO with increasing degree of lipid solubility have been investigated: 5-(di-n-propoxyphosphoryl)-5-methyl-1-pyrroline N-oxide (DPPMPO), 5-(di-n-butoxyphosphoryl)-5-methyl-1-pyrroline N-oxide (DBPMPO), and 5-(bis-(2-ethylhexyloxy)phosphoryl)-5-methyl-1-pyrroline N-oxide (DEHPMPO). As compared with the spin trap DMPO, the half-lives of the respective superoxide adducts were clearly higher in aqueous solutions of the spin traps, which facilitates qualitative ESR measurements. The stability of the superoxide spin adducts formed with the various lipophilic spin traps in aqueous buffer were similar to those observed with DEPMPO (half-life: 7-11 min.). In model experiments using Fe(3+)-catalyzed nucleophilic addition of methanol or tert-butanol to the respective spin trap the respective alkoxyl radical adducts were formed in aqueous solution as transient species in the presence of high concentrations of the alcohol. Upon dilution with water the alkoxyl group was substituted by water, giving the respective hydroxyl adduct of the spin trap. Care must therefore be taken when Fenton-type reactions are used for the generation of radicals such as the use of Fe(2+) complexes with phosphate or DTPA or inactivation of iron by addition of "Desferal" (Novarti's Pharma GmbH, Vienna, Austria) after a short incubation time. Addition of Fe(2+) under anaerobic conditions to an aqueous suspension of linoleic acid hydroperoxide and the spin trap resulted in the detection of three different species: a carbon-centered radical adduct, an acyl radical adduct, and the hydroxyl adduct. In the presence of oxygen a different species was observed with DEPMPO, DPPMPO, and DBPMPO, which was only slightly suppressed upon the addition of SOD, possibly the respective spin adduct of either the alkylperoxyl radical or, in analogy to DMPO, a secondary alkoxyl radical.
自旋捕获剂5-(二乙氧基磷酰基)-5-甲基-1-吡咯啉N-氧化物(DEPMPO)与超氧化物形成加合物，半衰期约为15分钟。DEPMPO具有很强的亲水性，由于其在脂质相中的浓度极低，因此其用于检测脂质相中的自由基(脂质衍生自由基和脂质相中产生的超氧化物)受到限制。为了检测脂质衍生自由基，研究了三种脂质溶解度逐渐增加的DEPMPO衍生物：5-(二正丙氧基磷酰基)-5-甲基-1-吡咯啉N-氧化物(DPPMPO)、5-(二正丁氧基磷酰基)-5-甲基-1-吡咯啉N-氧化物(DBPMPO)和5-(双(2-乙基己氧基)磷酰基)-5-甲基-1-吡咯啉N-氧化物(DEHPMPO)。与自旋捕获剂DMPO相比，在自旋捕获剂的水溶液中，相应超氧化物加合物的半衰期明显更长，这有利于定性电子顺磁共振测量。在水性缓冲液中，由各种亲脂性自旋捕获剂形成的超氧化物自旋加合物的稳定性与DEPMPO观察到的相似(半衰期：7-11分钟)。在使用铁(III)催化甲醇或叔丁醇对相应自旋捕获剂进行亲核加成的模型实验中，在高浓度醇存在下，相应的烷氧基自由基加合物在水溶液中作为瞬态物种形成。用水稀释后，烷氧基被水取代，得到自旋捕获剂的相应羟基加合物。因此，当使用芬顿型反应产生自由基时，必须小心，例如使用与磷酸盐或二乙三胺五乙酸形成的铁(II)配合物，或在短时间孵育后通过添加“去铁胺”(诺华制药有限公司，奥地利维也纳)使铁失活。在厌氧条件下，向亚油酸氢过氧化物和自旋捕获剂的水悬浮液中添加铁(II)，导致检测到三种不同的物种：碳中心自由基加合物、酰基自由基加合物和羟基加合物。在有氧存在的情况下，DEPMPO、DPPMPO和DBPMPO观察到一种不同的物种，添加超氧化物歧化酶后，这种物种仅略有抑制，可能是烷基过氧自由基的相应自旋加合物，或者类似于DMPO，是仲烷氧基自由基的相应自旋加合物。
