Application Of The Front-face 3d-fluorescent Technique In Studying Age Pigment-like Fluorescence And Their Origin

The carbonyl stress hypothesis of biological aging, based on the formation mechanism of age pigments is the summa of several leading contemporary theories of senescence, such as free radical aging hypothesis and glycation hypothesis, which suggested that carbonyl stress may represent a central process of biological aging. After reviewed the biochemical basis of age pigment-like fluorophores, this work aimed to develop the detection method of age pigments and study the fluorescence characteristic of the artificial ceroid/lipofuscin pigments and biomaterials with improved techniques.The studies performed in the thesis are summarized as follows:1. In order to develop the detection method of age pigments, the rat-tail tissue, the water and the chloroform extractions and the residue part of rat-tail collagen were measured by three dimensional (3D) front-face spectrofluorometry and normal spectrofluorometry respectively. The aged rat-tail collagen showed significant age-related increase of fluorescent emission both in water and in organic extractions, so was the case in non-extractable tail tissue. Therefore, the front-face fluorescence measurement was proved to be a useful tool in studying pigment alterations related to aging process.2. The artificial ceroid/lipofuscin pigments originated from different organ tissues and biomaterials were re-examed with improved techniques. Similar fluorescent characteristics of ceroid/lipofuscin-like materials from different tissues, including liver, brain, heart and kidney of rats, were observed. With all tested tissue materials exposed under UV light, a series of similar fluorescent color were observed with different filter combinations under microfluorometer. Comparable fluorescent spectra were also demonstrated with a 3-D front-surface fluorometric technique despite of their tissue differences. Measured with 3-D fluorometry, relatively simple lipofusin-like fluorophores was observed from the reactions of malondialdehyde (MDA) with critical biological macromolecules, such as bovine serum albumin (BSA) and DNA, whereas MDA-DNA showed only a single fluorescent peak. Our results indicated that the end-products of biomaterials have a similar fate (biochemical structures) in different tissues under accelerated oxidative/carbonyl stresses.