Age pigments and the biochemical basis of their formation

Sammanfattning: The autofluorescent, yellow-brown pigments that accumulate over time inbiological organisms are called age pigments. Lipofuscin, known as a hallmark of aging, refers to intracellular age pigments that accumulate mainly in the lysosomes of postrnitotic cells. This dissertation reports studies on the biochemical nature of lipofuscin and other age pigments, including their origin, mechanisms of formation, composition, structure and other properties.Since lipofuscin-extracts and lipid peroxidation products exhibit similar fluorescent characteristics, oxidative stress-induced lipid peroxidation is linked with age pigment formation. The initiation of lipid peroxidation resulting from oxygen free radicals was studied in a Tween 20-emulsified linoleic acid model system. Fenton reagents did not result in hydrogen abstraction-related diene conjugation. Also, different cOmbinations of Fe(II) and Fe(III) did not support the Fe(II):Fe(Ill) (1: 1) optimum ratio hypothesis. It is, therefore, concluded that perferryl ions or chelator-Fe-02 complexes are responsible for the first-chain initiation of lipid peroxidation in this model system.To elucidate a mitochondrion-lysosome hypothesis of lipofuscinogenesis, the oxidative stress-induced formation of lipofuscin-like fluorophores was studied in a testtube model using lysosomal-mitochondrial fractions. The sequential formation of TEARS, protein carbonyls and lipofuscin-like fluorophores was found. These findings add support to the concept that lipofuscin fonns in secondary lysosomes as a result of iron-catalyzed oxidative reactions involving autophagocytosed materials.Further, lipofuscin-like fluorescence was obtained during ascorbic acidautoxidation and from reaction products between ascorbic acid and amino compounds. The reaction between ascorbic acid and glutamine shows that such fluorophore formation is oxidation-dependent. On the basis of a comparison of the fluorophore formation mechanisms of lipid peroxidation, ascorbate oxidation and glycation reactions, we propose that carbonyl-protein crosslinking is a common biochemical reaction in aging processes.Striking discrepancy exists between the orange-yellow fluorescence of lipofuscin in situ and the blue fluorescence of lipofuscin-extracts. A concentration-dependent fluorescence shift was discovered using different lipofuscin-related fluorophores. The methodological difference between microfluorometry, by which lipofuscin is studied in highly condensed form, and spectrofluorometry, by which lipofuscin is studied at a very low concentration, is also clarified.These studies suggest that crosslinking between carbonyls and amino compounds may represent the major biochemical process responsible for the formation of age pigments and lipofuscin-like fluorophores.