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.
