NITRITE/COLLAGEN REACTIONS IN AGING AND SMOKING

虽然亚硝酸盐反应在过去已经被研究过，但与胶原蛋白的特异性反应及其在衰老中的作用从未被研究过。我们最近观察到，体外亚硝酸盐处理的胶原蛋白与老化的人胶原蛋白相似。共有的特性包括形成一种独特的酪氨酸衍生物，我们称之为化合物N，减少未经修饰的酪氨酸残基，积累在350 nm处吸收的黄色发色团，以及增加胶原交联度。交联度增加，表现为对酶消化的敏感性降低，导致胶原蛋白硬化，并可能导致人体血管、肺和皮肤随年龄增长而僵硬。人类接触亚硝酸盐的主要原因是内源性一氧化氮的产生和外源性来源，包括吸烟和腌制肉类的摄入。为了确定亚硝酸盐/胶原蛋白反应对人胶原蛋白年龄相关变化的体内意义，我们将开发检测该反应的生物标志物的方法(特别是酪氨酸衍生物，如化合物N，3-硝基酪氨酸、二氮酪氨酸、重氮酪氨酸和脱氨基重氮酪氨酸)。将对亚硝酸盐与潜在的反应中间体之间的反应进行动力学研究，以便更准确地确定所涉及的反应机理。生物标志物和反应中间体将通过反相高效液相氨基酸分析与光电二极管阵列、荧光和电化学在线检测系统进行鉴定。生物标志物检测方法将用于比较从年轻人和老年人的皮肤、主动脉和肺中提取的胶原蛋白。每个胶原蛋白样品的交联度和酪氨酸含量也将被确定。统计分析将包括以年龄和吸烟为自变量的多个Logistic回归。最后，我们将研究自由基清除剂，如维生素A、C和E在体外调节这些反应的有效性。由于吸烟是个人外源性亚硝酸盐暴露的主要来源，亚硝酸盐/胶原蛋白反应可能为与衰老和吸烟相关的疾病过程的发病机制提供重要线索。这些疾病包括各种动脉粥样硬化性血管疾病、慢性阻塞性肺疾病、早产儿皱纹和与年龄相关的晶状体白内障。

Although nitrite reactions have been studied in the past, the specific reaction with collagen and its role in aging has never been examined. We have recently observed that in vitro nitrite-treated collagen shows similarities with aged human collagen. Shared properties include the formation of a unique tyrosine derivative we call compound N, a decease in unmodified tyrosine residues, the accumulation of a yellowish chromophore absorbing at 350 nm, and an increase in collagen cross-linking. Increased cross-linking, evidenced by a decreased susceptibility to enzymatic digestion, results in the stiffening of collagen and may account for the stiffening of human blood vessels, lung, and skin which occurs with aging. The majority of human exposure to nitrite results from endogenous nitric oxide production and exogenous sources which include cigarette smoking and cured meat ingestion. In order to establish the in vivo significance of the nitrite/collagen reaction to age-related changes in human collagen we will develop methods to detect biomarkers of the reaction (especially tyrosine derivatives such as compound N, 3-nitrotyrosine, dityrosine, diazotyrosine and deaminated diazotyrosine). Kinetics on the reactions between nitrite and potential reaction intermediates will be conduced in order to more precisely define the mechanism of reaction involved. The biomarkers and reaction intermediates will be identified by reverse phase HPLC amino acid analysis with photodiode array, fluorescence, and electrochemical in-line detection systems. The methods for biomarker detection will be used to compare collagen extracted from the skin, aorta, and lung of young and old individuals. Degree of cross-linking and tyrosine content will also be determined for each collagen sample. Statistical analyses will include multiple logistic regressions with age and smoking as independent variables. Finally, we will examine the effectiveness of free radical scavengers such as vitamins A, C and E to modulate these reactions in vitro. Because cigarette smoking is a major source of an individual's exogenous nitrite exposure, the nitrite/collagen reaction may provide important clues to the pathogenesis of disease processes associated with both aging and cigarette smoking. These include a variety of atherosclerotic vascular diseases, chronic obstructive pulmonary disease, premature sign wrinkling, and age-related lens cataracts.