Dosimetry of Lipid-Derived Amino Acid Adducts by LC/MS

我们最近发现了基因毒性双功能亲电试剂4,5-环氧-2-癸烯醛、4-氧-2-壬烯醛和4-羟基-2-壬烯醛以及潜在的基因毒素4-羟基-2-壬烯醛是维生素c介导的脂质氢过氧化物分解的主要产物。令人惊讶的是，维生素C诱导这种分解的效率是过渡金属离子的两倍多。长期以来，人们一直认为脂质氢过氧化物在癌症和心血管疾病等衰老性退行性疾病中发挥作用。我们现在已经取得了令人兴奋的发现，4-氧-2-壬烯醛能有效地与氨基酸精氨酸形成加合物。先前的研究表明，4-羟基-2-壬烯醛可以诱导模型氨基酸的共价修饰。定量血浆氨基酸、血浆蛋白和富含精氨酸的组蛋白共价修饰的能力将为脂质氢过氧化物来源的基因毒素暴露提供剂量计，这将补充dna加合物获得的数据。此外，对精氨酸和富含精氨酸的组蛋白的共价修饰的定量可以为癌症风险提供额外的见解。血浆精氨酸是产生一氧化氮的前体，一氧化氮是已知的肿瘤发生介质。脂质氢过氧化物介导的游离血浆精氨酸的共价修饰将阻止脱亚胺酶介导的瓜氨酸和一氧化氮的转化。这将限制内源性底物的可用性，也可以作为内源性抑制剂。已经提出了内源性抑制剂，但没有一个没有检测到足够大的量来解释l -精氨酸悖论。这种矛盾源于观察，即即使细胞内明显有足够的游离精氨酸使内源性一氧化氮合酶饱和，但l -精氨酸的输注也会引起额外的一氧化氮释放。组蛋白精氨酸的共价修饰也会影响转录和细胞增殖。我们建议首先将重点放在量化由脂质氢过氧化物衍生的双功能亲电试剂与自由精氨酸相互作用诱导的共价修饰上。然后在体外用血红蛋白、白蛋白和组蛋白进行研究。结构分析将使用传统的蛋白酶消化结合液相色谱和串联质谱进行。如有必要，特定的共价修饰将通过化学合成来表征。当双功能亲电试剂由维生素c介导的脂质氢过氧化物均溶分解产生时，将评估这些病变的相对重要性。这样的研究已经在过去使用过渡金属离子介导的脂质氢过氧化物分解进行。不幸的是，过渡金属离子也催化Huber-Weiss反应，导致活性氧的形成。使用过渡金属无离子缓冲液和维生素C克服了这个问题。因此，首次有可能研究脂质氢过氧化物介导的蛋白损伤而不同时发生氧化损伤。维生素c诱导的脂质过氧化氢分解对组蛋白功能的影响将在相关的体外模型中进行评估。最后，从白血病患者血浆中分离游离精氨酸、白蛋白和血红蛋白。然后通过稳定同位素稀释液相色谱/串联质谱法对这些人群中精氨酸和蛋白质的脂质氢过氧化衍生的共价修饰进行量化，并与正常受试者的精氨酸和蛋白质进行比较。这些研究将作为未来大规模人群剂量学研究的先驱，并将深入了解脂质过氧化作为致癌介质的作用。

We recently identified the genotoxic bifunctional electrophiles, 4,5-epoxy-2-decenal, 4-oxo-2-nonenal, and 4- hydroxy-2-nonenal together with the potential genotoxin 4- hydroperoxy-2-nonenal as major products of vitamin C-mediated decomposition of lipid hydroperoxides. Surprisingly, vitamin C was more than twice as efficient as transition metal ions at inducing this decomposition. It has long been thought that lipid hydroperoxides play a role in degenerative diseases of aging such as cancer and cardiovascular disease. We have now made the exciting observation that 4-oxo-2-nonenal efficiently forms adducts with the amino acid arginine. Previous studies have demonstrated 4-hydroxy-2-nonenal can induce covalent modifications with model amino acids. However, the efficiency of this process is much lower than we observed with 4-oxo-2-nonenal The ability to quantify covalent modifications to plasma amino acids, plasma proteins, and arginine-rich histones will provide a dosimeter of exposure to lipid hydroperoxide-derived genotoxins that would complement data obtained with DNA-adducts. Furthermore, quantitation of covalent modifications to arginine and arginine-rich histones could provide additional insight into cancer risk. Plasma arginine is the precursor for the generation a nitric oxide a known mediator of tumorigenesis. Lipid hydroperoxide-mediated covalent modification of free plasma arginine would prevent deiminase-mediated conversion to citrulline and nitric oxide. This would limit endogenous substrate availability and could also act as an endogenous inhibitor. Endogenous inhibitors have been proposed but none have not been detected in sufficiently large amounts to account for the L-arginine paradox. This paradox arises from the observation that infusions of L-arginine can cause additional nitric oxides release even though there is apparently sufficient free intracellular arginine to saturate endogenous nitric oxide synthases. Covalent modifications to histone arginines would also affect transcription and cellular proliferation. We propose to focus initially on quantifying the covalent modifications that are induced by the interaction of lipid hydroperoxide-derived bifunctional electrophiles with free arginine. Studies will then be conducted with hemoglobin, albumin, and histones in vitro. Structural analysis will be performed using conventional protease digests coupled with liquid chromatography and tandem mass spectrometry. If necessary, specific covalent modifications will be characterized by chemical synthesis. The relative importance of these lesions will then be assessed when the bifunctional electrophiles are generated by vitamin C-mediated homolytic decomposition of lipid hydroperoxides. Such studies have been performed in the past using transition metal ion-mediated decomposition of lipid hydroperoxides. Unfortunately, transition metal ions also catalyze Huber-Weiss reactions, which results in the formation of reactive oxygen species. The use of transition metal ion-free buffers and vitamin C overcomes this problem. Therefore, it will be possible for the first time to study lipid hydroperoxide-mediated protein damage without the complication of simultaneous oxidative damage. The changes in histone function resulting from vitamin C-induced lipid hydroperoxide decomposition will be assessed in relevant in vitro models. Finally, free arginine, albumin and hemoglobin will be isolated from plasma of patients with leukemia. Lipid hydroperoxide- derived covalent modifications in arginine and proteins from these populations will then be quantified by stable isotope dilution liquid chromatography/tandem mass spectrometry and compared with arginine and proteins from normal subjects. These studies will serve as a precursor to future dosimetry studies in large populations and will provide insight into the role of lipid peroxidation as a mediator of carcinogenesis.