Eisuke Maehata

Automated analysis of the serum antioxidative activities against five different reactive oxygen species by sequential injection system with a chemiluminescence detector.

BACKGROUND There is a growing evidence that reactive oxygen species (ROS) may cause many pathologic conditions including chronic diseases, neurodegenerative disorders, cancer and aging. There are a number of methods to measure the total antioxidative activity of the serum. However, since the lifetime and oxidative activity of various types of ROS are all different, to measure simply the total antioxidative activity of the serum is not enough. Therefore, to aid the diagnosis and to improve the therapeutic strategy, it is important to develop a simple and reliable method of assaying antioxidative activity of the serum. METHODS A method that combines sequential injection analysis (SIA) and luminol chemiluminescence (CL) detection was employed for the measurement of antioxidative activities of human serum. We collected sera from healthy subjects (n=42) and patients with diabetes (n=39) and rheumatoid arthritis (n=25) and tested the sensitivity, reproducibility and reliability of our method. RESULTS Since the operation is automatically controlled by a personal computer, we obtained a satisfactory repeatability without the need of much manpower. The time required for obtaining the antioxidative activity against one ROS for one individual is less than 3min. Although the value of antioxidative activity varies from subject to subject, there were a certain relationship between the disease and the antioxidative values of each type of ROS. The results suggest that the measurement of antioxidative activity against different ROS may provide us with valuable information regarding the disease state. CONCLUSIONS The evaluation of antioxidative activities against each ROS by the proposed method should be more informative to understand the antioxidative status of biological fluid.

Determination of acrolein in serum by high‐performance liquid chromatography with fluorescence detection after pre‐column fluorogenic derivatization using 1,2‐diamino‐4,5‐dimethoxybenzene

Acrolein is a major unsaturated aldehyde that is generated during the lipid peroxidation process. The measurement of acrolein in biological samples should be useful to estimate the degree of lipid peroxidation and to evaluate the effect of hazardous properties of acrolein on human health. In this study, a highly sensitive and selective high-performance liquid chromatography with fluorescence detection method was developed for the determination of acrolein in human serum. The proposed method involves the pre-column fluorogenic derivatization of acrolein with 1,2-diamino-4,5-dimethoxybenzene (DDB) as a reagent. The fluorescent derivative of acrolein could be detected clearly without any interfering reagent blank peaks because DDB does not have intrinsic fluorescence itself, and the detection limit was 10 nM (signal-to-noise ratio = 3). The proposed method could selectively detect acrolein in human serum with a simple protein precipitation treatment.

Determination of 4-hydroxy-2-nonenal in serum by high-performance liquid chromatography with fluorescence detection after pre-column derivatization using 4-(N,N-dimethylaminosulfonyl)-7-hydrazino-2,1,3-benzoxadiazole.

4-Hydroxy-2-nonenal (4HNE) is a major aldehyde generated during lipid peroxidation. The clinical monitoring of 4HNE in biological fluids should be useful for the early diagnosis of several diseases involving lipid peroxidation, such as rheumatoid arthritis, Parkinsons disease and cancer. In this study, an HPLC with fluorescence detection method was developed for the determination of 4HNE in human serum. The proposed method involves the extraction of 4HNE from human serum by sub-zero temperature extraction and fluorescent labeling of 4HNE with 4-(N,N-dimethylaminosulfonyl)-7-hydrazino-2, 1,3-benzoxadiazole. The lower detection limit (signal-to-noise ratio = 3) of the method was 0.06 μm in serum. The proposed method was successfully applied to the measurement of 4HNE in sera obtained from patients with rheumatoid arthritis.