Tez özetleri Astronomi ve Uzay Bilimleri Anabilim Dalı

Development of a Spectrofluorometric Method for Measurement of Peroxyl

Radical Scavenging Activity ın Biological Samples

Oxygen is essential for life, but it may also have harmful effects on the organism. Oxygen may form various reactive oxygen species (ROS) in the respiratory chain, namely superoxide anion, singlet oxygen, hydroxyl radical, peroxyl radical etc. If the ROS accumulation is not balanced by existing or food injested antioxidants in the organism, radicalic chain reactions may occur under oxidative stres conditions that may cause tissue damage. Peroxyl radicals are scavenged by either synthetic or food-injested antioxidants. So, simple and sensitive methods for the determination of of the peroxyl radical scavenging activity of antioxidants are necessary. The aim of the developed spectrophotometric procedures are to resolve shortcomings and limitations of the available methods in literature for the determination of reactive species scavenging activity. Therefore, a new fluorometric method has been developed for peroxyl radical scavenging activity assay.
In this thesis, p-amino benzoic acid (PABA) has been firstly used as a probe for the determination of peroxyl radical scavenging activity. Excitation and emission wavelengths of PABA were determined at 267 nm and 334 nm, respectively, and radical scavenging activity of thiol type antioxidants, amino acids and plasma antioxidants was measured at these wavelengths by following the changes in probe fluorescence. In this new fluorometric assay, peroxyl radicals were generated by thermal decomposition of 2,2’-azobis(2-methyl propionamide) dihydrochloride (AAPH). PABA probe can be oxidized with peroxyl radical, and the fluorescence intensity of PABA is diminished as a result of its reaction with peroxyl radical, because PABA is fluorescent while its oxidation product(s) are not. The attenuation in the fluorescence intensity loss of PABA probe is dependent upon the peroxyl radical scavenging activity of the tested compound. With the aid of PABA fluorescence values recorded in the presence and absence of scavengers, the peroxyl radical scavenging activity of scavengers can be calculated. The peroxyl radical scavenging activity measured by the fluorometric method was compared to those of the reference ORAC and crocin bleaching methods.
Albumin (IC₅₀ = 0.6 µM) had the lowest IC₅₀ value among the studied antioxidants (highest peroxyl radical scavenging activity) with respect to the developed fluorometric method. In addition, the peroxyl radical scavenging activity of thiol-type antioxidants were found in the following order: homocysteine > glutathione (GSH) > cysteine ​​> N-acetyl cysteine ​​(NAC) > 1,4-dithioerythritol (DTE) > cysteamine.
The developed methods for measurement of peroxyl radical scavenging activity were validated by quantifying the probe (PABA) and its oxidation product(s) by HPLC (high performance liquid chromatography) method, followed by statistically comparing the results.
In addition to biologically important antioxidants (thiol-type antioxidants, amino acids and plasma antioxidants), the peroxyl radical scavenging activity of tissue homogenates (e.g., liver, kidney and heart) were determined by the developed spectrofluorometric method as percentage inhibition values of the incubation reaction mixture. The heart tissue homogenate was shown to exhibit the higher peroxyl radical scavenging activity with the developed method.