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

我们最近确定了具有遗传毒性的双功能亲电体，4，5-环氧-2-癸烯醛，4-氧代-2-壬烯醛和4-羟基-2-壬烯醛以及潜在的遗传毒性4-氢过氧-2-壬烯醛作为维生素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在诱导这种分解方面的效率是过渡金属离子的两倍多。 长期以来，人们一直认为脂质过氧化氢在癌症和心血管疾病等老年退行性疾病中发挥作用。 我们现在已经发现4-氧代-2-壬烯醛能有效地与精氨酸形成加合物。 先前的研究已经证明4-羟基-2-壬烯醛可以诱导与模型氨基酸的共价修饰。 然而，该过程的效率比我们用4-氧代-2-壬烯醛观察到的低得多。定量血浆氨基酸、血浆蛋白和富含精氨酸的组蛋白的共价修饰的能力将提供暴露于脂质过氧化氢衍生的基因毒素的剂量计，其将补充用DNA加合物获得的数据。此外，对富含精氨酸和精氨酸的组蛋白的共价修饰的定量可以提供对癌症风险的额外了解。 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的介质。 脂质过氧化氢介导的游离血浆精氨酸共价修饰可阻止脱亚胺酶介导的瓜氨酸和一氧化氮转化。 这将限制内源性底物的可用性，也可作为内源性抑制剂。 已经提出了内源性抑制剂，但没有检测到足够大的量来解释L-精氨酸悖论。 这一矛盾现象源于这样的观察，即即使有明显足够的游离细胞内精氨酸饱和内源性一氧化氮合酶，L-精氨酸的输注也会导致额外的一氧化氮释放。 组蛋白酪氨酸的共价修饰也会影响转录和细胞增殖。 我们建议最初集中在定量的共价修饰，诱导的脂质过氧化氢衍生的双功能亲电试剂与游离精氨酸的相互作用。 然后将在体外对血红蛋白、白蛋白和组蛋白进行研究。将使用常规蛋白酶解结合液相色谱和串联质谱进行结构分析。 如有必要，将通过化学合成表征特定的共价修饰。 当维生素C介导的脂质氢过氧化物均裂分解产生双功能亲电体时，将评估这些病变的相对重要性。 过去已经使用过渡金属离子介导的脂质氢过氧化物分解进行了此类研究。 不幸的是，过渡金属离子也催化Huber-Weiss反应，这导致活性氧物质的形成。 使用不含过渡金属离子的缓冲剂和维生素C克服了这个问题。因此，这将是第一次有可能研究脂质过氧化氢介导的蛋白质损伤，而不并发症的同时氧化损伤。 将在相关体外模型中评估维生素C诱导的脂质氢过氧化物分解引起的组蛋白功能变化。最后，从白血病患者的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/串联质谱法定量来自这些群体的精氨酸和蛋白质中的脂质过氧化氢衍生的共价修饰，并与来自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在大人群中进行剂量测定研究的先驱，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.