Analysis of the free radical scavenging products of vitamin E in biological systems using a high-sensitive HPLC method

采用高效液相色谱-电化学检测的高灵敏方法，分析了大鼠组织匀浆(肝、心、肺、肾)过氧化过程中维生素E氧化产物(α-生育酚，α-TH)，包括8α-(磷脂酰胆碱-二氧基)-α-生育酚(TOO-PC)、环氧生育酚(α-α-TQ)和环氧生育酚(α-α-TQ)。当每种组织匀浆在37℃时加入自由基引发剂过氧化，内源性α-TH可清除磷脂酰胆碱(PC)的过氧化自由基，从而抑制PC-OHO的形成。α-TH在各组织中的主要产物为α-TQ和α-TH与过氧化自由基的加成产物(TOO-PC)。LC-ESIMS分析表明，TO-PC的结构为1-palmitoyl-2-[(8α-dioxy-α-tocopherone)-epoxyoctadecenoyl]-3-sn-phosphatidylcholine。结果表明，大鼠组织中内源性α-TH存在于…中更多的原酸盐可以与脂质过氧化自由基反应生成α-TQ和TO-PC作为反应产物。氧化应激参与了糖尿病的发病机制，α-TH可能通过减少脂质过氧化和氧化损伤来预防某些组织中糖尿病并发症的发展。因此，我们用高灵敏的电子捕获-高效液相色谱方法研究了链脲佐菌素诱导的糖尿病大鼠血清、心、肝、肾、肺等组织中α-TH的氧化产物。测定注射链脲佐菌素后不同时间点这些组织内源性α-TH及其氧化产物的含量，以及氧化应激标志物PC-OOH的含量。糖尿病大鼠注射链脲佐菌素4周后，血浆、肝、肾中PC-OH值升高。因此，氧化应激发生在这些组织中。糖尿病大鼠各组织中α-TH水平虽无明显变化，但血浆、心、肝、肺组织中氧化产物α-TQ均高于对照组。此外，用ECD-HPLC法在糖尿病大鼠的一些组织中也检测到了Too-PC。结果提示，糖尿病大鼠组织中的α-TH可能通过清除脂质过氧化自由基形成氧化产物α-TQ。综上所述，α-TH的氧化产物已在体内外系统中检测到。较少

The high-sensitive method using a high-performance liquid chromatography with electrochemical detection (ECD-HPLC) has been applied for the analysis of the oxidation products of vitamin E (α-tocopherol, α-TH), including 8α-(phosphatidylcholine-dioxy)-α-tocopherones (TOO-PC), α-tocopherylquinone (α-TQ), and epoxy-α-tocopherylquinones (epoxy-α-TQ) during the peroxidation of rat tissue homogenates (liver, heart, lung, and kidney). When each tissue homogenate was peroxidized by the addition of a free radical initiator at 37℃, the endogenous α-TH could scavenge peroxyl radicals of phosphatidylcholines (PC) to suppress the formation of PC hydroperoxides (PC-OOH). The main products of α-TH in each tissue were α-TQ and the addition products of α-TH with PC-peroxyl radicals (TOO-PC). The structure of TOO-PC was characterized as 1-palmitoyl-2-[(8α-dioxy-α-tocopherone)-epoxyoctadecenoyl]-3-sn-phosphatidylcholine by the LC-ESIMS analysis. The results indicate that endogenous α-TH in rat tissue hom … More ogenates can react with lipid-peroxyl radicals to form α-TQ and TOO-PC as the reaction products.Oxidative stress has been implicated in the pathogenesis of diabetes mellitus, and α-TH might prevent the development of diabetic complications in some tissues by reducing lipid peroxidation and oxidative damage. Therefore, the oxidation products of α-TH in the tissues, serum, heart, liver, kidney, and lung, of storeptozotocin (STZ)-induced diabetic rats have been studied using the high-sensitive ECD-HPLC method. The amounts of endogenous α-TH and its oxidation products were measured in these tissues at different time points after STZ injection, in addition to the measurement of PC-OOH, a marker of oxidative stress. The PC-OOH was increased in plasma, liver, and kidney of diabetic rats 4 weeks after STZ injection. Therefore, oxidative stress occurred in these tissues. Although the level of α-TH in the tissues of diabetic rats was the same, the oxidation product α-TQ in the plasma, heart, liver, and lung was high compared with the control rats. Furthermore, TOO-PC has been detected in some tissues of diabetic rats using the ECD-HPLC method. The results suggest that α-TH in the tissues of diabetic rats may scavenge lipid-peroxyl radicals to form α-TQ as the oxidation product. In conclusion, the oxidation products of α-TH have been detected ex vivo and in vivo systems. Less