Scavenging of Reactive Oxygen and Nitrogen Species by Cobalamins

性状（由申请方提供）：钴（Cbl，维生素B12衍生物）是哺乳动物的必需辅因子。然而，它们也有临床效果，不能完全解释其辅因子的作用。例如，几十年来，医生广泛使用B12治疗炎症性疾病，包括慢性疲劳综合征，类风湿性关节炎，哮喘，神经系统疾病和湿疹，但对B12补充剂的益处知之甚少。这些疾病与“氧化和亚硝化应激”相关-活性氧和氮物质（ROS/RNS）水平升高，如一氧化氮、超氧化物、羟基自由基、过氧化氢和过氧亚硝酸盐。ROS/RNS与包括蛋白质、DNA和脂质在内的生物分子反应，经常导致不可逆的组织和器官损伤。我们假设，药理剂量的Cbl保护免受ROS/RNS引发的细胞损伤。我们进一步假设，这种保护的一个重要机制是直接清除活性氧/RNS的主要细胞内Cbl形式，钴（II）丙氨酸。本研究的总体目标是确定Cbl是否是一种广谱抗氧化剂，能够直接清除多种ROS/RNS。在具体目标1中，将研究cob（II）alamin，Cbl（II）和四种ROS/RNS物质-过氧化氢，超氧化物，羟基自由基和二氧化氮之间的反应。动力学和产品的研究将允许速率和反应机制得到阐明。这将提供关于这些反应的潜在生物学相关性的宝贵信息。在具体目标2中，将确定CNCbl抑制原代人主动脉内皮细胞中过量ROS和RNS并防止ROS/RNS引发的细胞死亡的能力。CNCbl是维生素补充剂中常见的Cbl形式，在进入细胞后被还原为Cbl（II）。血管内皮在调节血流、抑制血小板聚集以及免疫和炎症反应中起重要作用。血管内皮功能障碍与氧化/亚硝化应激有关。CNCbl抑制单个ROS/RNS（过氧化氢、超氧化物、羟基自由基和二氧化氮源过氧亚硝酸盐）的能力将使用细胞内荧光或荧光探针和/或对每个ROS/RNS特异的下游测定来确定。还将研究CNCbl对由每种ROS/RNS诱导的细胞死亡的保护作用。目前的提议挑战了广泛持有的信念，即Cbl的唯一生物学作用是其在B12依赖性酶反应中的辅酶作用。它还挑战了游离（非蛋白结合）Cbl没有生物学作用的普遍信念，尽管事实上在补充后细胞中达到约50%的游离Cbl，并且Cbl（II）是能够与ROS/RNS快速反应的自由基物质。长期目标是确定Cbl保护细胞免受氧化/亚硝化应激的机制，以评估Cbl补充剂在治疗慢性炎症性疾病中的益处。 公共卫生相关性：这项研究探索了一种新的机制，通过这种机制，维生素B12可以提供对炎症和年龄相关疾病的保护。鉴于人口迅速老龄化，研究维生素B12的治疗潜力是高度相关的，因为B12缺乏症越来越常见，特别是在老年人中。

DESCRIPTION (provided by applicant): Cobalamins (Cbl, vitamin B12 derivatives) are essential cofactors in mammals. However, they also have clinical effects that cannot be fully explained solely by their cofactor role. For example, B12 has been used extensively by physicians over several decades to treat inflammatory disorders including chronic fatigue syndrome, rheumatoid arthritis, asthma, neurological diseases and eczema, with little understanding as to why B12 supplementation is beneficial. These diseases are associated with "oxidative and nitrosative stress" - elevated levels of Reactive Oxygen and Nitrogen Species (ROS/RNS), such as nitric oxide, superoxide, the hydroxyl radical, hydrogen peroxide and peroxynitrite. ROS/RNS react with biomolecules including proteins, DNA and lipids, frequently causing irreversible tissue and organ damage. We hypothesize that pharmacological doses of Cbl protect against ROS/RNS-initiated cell damage. We further hypothesize that an important mechanism for this protection is the direct scavenging of ROS/RNS by a major intracellular Cbl form, cob(II)alamin. The overall goal of this study is to determine whether Cbl is a broad spectrum antioxidant, capable of directly scavenging multiple ROS/RNS. In Specific Aim 1 the reactions between cob(II)alamin, Cbl(II), and four ROS/RNS species - hydrogen peroxide, superoxide, the hydroxyl radical and nitrogen dioxide - will be studied. Kinetic and product studies will allow rates and reaction mechanisms to be elucidated. This will provide invaluable information about the potential biological relevance of these reactions. In Specific Aim 2 CNCbl's ability to scavenge excess ROS and RNS in primary human aortic endothelial cells and prevent ROS/RNS-initiated cell death will be determined. CNCbl is the common Cbl form in vitamin supplements and is reduced to Cbl(II) upon entering cells. The vascular endothelium plays an important role in regulating blood flow, in inhibiting platelet aggregation and in the immune and inflammatory response. Vascular endothelial dysfunction is associated with oxidative/nitrosative stress. The ability of CNCbl to scavenge individual ROS/RNS (hydrogen peroxide, superoxide, the hydroxyl radical, and the nitrogen dioxide source peroxynitrite) will be determined using intracellular fluorescent or chemiluminescent probes and/or downstream assays specific for each ROS/RNS. The protective effects of CNCbl against cell death induced by each ROS/RNS will also be investigated. The current proposal challenges the widely-held belief that the sole biological role of Cbl is its coenzyme role in B12-dependent enzyme reactions. It also challenges the common belief that free (non-protein bound) Cbl has no biological role, despite the fact that ~50% free Cbl is achieved in cells upon supplementation, and that Cbl(II) is a radical species capable of reacting rapidly with ROS/RNS. The long-term goal is to identify the mechanism(s) by which Cbl protects against cellular oxidative/nitrosative stress, in order to assess the benefits of Cbl supplementation in the treatment of chronic inflammatory diseases. PUBLIC HEALTH RELEVANCE: PROJECT NARRATIVE This study explores a novel mechanism by which vitamin B12 can provide protection against inflammatory and age-related diseases. Given a rapidly aging population, the study of the therapeutic potential of vitamin B12 is highly relevant since B12 deficiency is increasingly common, especially amongst the elderly.