Nemirovskiy Olga V, Radabaugh Melissa R, Aggarwal Poonam, Funckes-Shippy Christie L, Mnich Stephen J, Meyer Debra M, Sunyer Teresa, Rodney Mathews W, Misko Thomas P
Pfizer Global Research and Development, St. Louis Laboratories, 700 Chesterfield Parkway, St. Louis, MO 63017, USA.
Nitric Oxide. 2009 May;20(3):150-6. doi: 10.1016/j.niox.2008.12.005. Epub 2008 Dec 25.
The contribution of inducible nitric oxide synthase (iNOS) to oxidative/nitrative stress is well-documented in inflammation, but difficult to quantify. Using a novel, recently developed assay for 3-nitrotyrosine (3-NT), we characterized iNOS activity and its inhibition in preclinical models of inflammation. In particular, we utilized the 3-NT assay to assess the role of iNOS in the disease pathology as well as for proof of pharmacology of iNOS inhibitors in an acute endotoxin challenge model, in models of rheumatoid arthritis (RA) such as rat adjuvant- and collagen-induced arthritis (AIA and CIA) and a model of osteoarthritis (OA) such as rat sodium monoiodoacetate-induced arthritis (MIA). Quantification of nitrotyrosine was performed using immuno-affinity 2-D LC-MS/MS assay. This assay is a very specific and reproducible and is amenable to a number of biological fluids. Plasma levels of 3-NT were significantly elevated in an acute model of inflammation (rat LPS) and in models of rheumatoid arthritis (adjuvant- and collagen-induced arthritis), and osteoarthritis (monoiodoacetate-induced arthritis). Plasma 3-NT correlated with the severity of the inflammatory response; thus, a 20-fold increase was observed in the rat LPS model, a 10-fold increase in AIA, and only a 2.5-fold elevation in CIA. Pharmacological intervention with iNOS inhibitors decreased 3-NT levels and associated pathology. 3-NT determination allowed for better elucidation of the role of iNOS in RA and OA disease pathology and provided proof of pharmacology for NOS inhibitors in animal models of RA and OA.
诱导型一氧化氮合酶（iNOS）对氧化/硝化应激的作用在炎症中已有充分记载，但难以量化。我们使用一种新开发的检测3-硝基酪氨酸（3-NT）的方法，在临床前炎症模型中对iNOS活性及其抑制作用进行了表征。具体而言，我们利用3-NT检测法评估iNOS在疾病病理中的作用，以及在急性内毒素攻击模型、类风湿性关节炎（RA）模型（如大鼠佐剂性和胶原诱导性关节炎（AIA和CIA））和骨关节炎（OA）模型（如大鼠单碘乙酸钠诱导性关节炎（MIA））中iNOS抑制剂的药理作用。使用免疫亲和二维液相色谱-串联质谱分析法对硝基酪氨酸进行定量。该检测方法非常特异且可重复，适用于多种生物体液。在急性炎症模型（大鼠脂多糖）、类风湿性关节炎模型（佐剂性和胶原诱导性关节炎）和骨关节炎模型（单碘乙酸钠诱导性关节炎）中，血浆3-NT水平显著升高。血浆3-NT与炎症反应的严重程度相关；因此，在大鼠脂多糖模型中观察到升高20倍，在AIA中升高10倍，而在CIA中仅升高2.5倍。用iNOS抑制剂进行药理干预可降低3-NT水平及相关病理变化。3-NT测定有助于更好地阐明iNOS在RA和OA疾病病理中的作用，并为RA和OA动物模型中NOS抑制剂的药理作用提供证据。
