Identifying the marker of oxidative stress by an in vitro exposure to free radical－A feasibility study

Breath analysis can provide clinically useful information for the diagnosis of oxidative stress and diseases. Exposure to reactive oxygen species (ROS) or free radicals will cause harmful effects such as lipid peroxidation and produce volatile hydrocarbons like ethane and pentane in low levels in the breath air. However, there are some obstacles in application. Therefore, in vitro cell-culture study perhaps can provide more sufficient evidences to prove the relationship.
Before assessing oxidative stress, it’s important to know how many radicals involved. Part of the objective in my study is to establish a radical generation system for the measurement and quantitation of hydroxyl radical. The main purpose is to setup a radical generation and trapping system for the subsequent in vitro cell-culture study.
Because the hydroxyl radical has an extremely short half-life and a high reacting rate, it was chosen in my research. For radical generation, I used Fenton reaction and the high-energy UV (λ=184.9nm) ionization of humid air flow. The radical was trapped at the outlet instantly and then analyzed using gas chromatography-mass spectrometry.
In Fenton reaction part, hydroxyl radicals can be generated stably, but the liquid type radicals may cause extra damage and have difficulties in further application. In aromatic hydroxylation part, I have tried various trapping strategies, including exposure times, flow rates and concentrations of salicylic acid to get the optimizing condition, but all test fail to show any signal from hydroxyl radical in any kind of condition. However, the recovery of salicylic acid was gradually decrease in some condition, and reached the statistical significance. I also tried to explore a number of hydroxyl radicals in the main stream of cigarettes, but the hydroxyl radicals were still not detected. The problem of failure detection was discussed in detail.
Even though the system is not successfully working in this study, but I find out some problems and the reasons of failure detection. These issues had not been mentioned in the past. In view of this, I made some suggestions and conclusions for solving these problems in the further research, and hope the system can be apply to in-vitro studies in the future.