The fluorescence excitation spectrum of model conjugated Schiff base compounds that arise from the reaction of malonaldehyde with amino acids was shown to contain a maximum at 260-280 nm in addition to the previously observed maximum at 350-390 nm. Excitation at either maximum results in emission at a single maximum at 440-480 nm. The excitation and emission maxima of the model fluorescent compounds, together with the characteristic reductions in fluorescence intensity caused by alkaline pH or heavy metal coordination provide criteria with which to examine lipid peroxidation products for the presence of the conjugated Schiff base fluorophore. Silicic acid column chromatography and silica gel thin layer chromatography were employed to fractionate the fluorescent products of model lipid peroxidation systems and of rat testicular lipid soluble extracts. These products contained large families of compounds whose fluorescence characteristics were the same as those of the Schiff base fluorophores. The fractionation methods used enabled more thorough fluorescence characterization of many of the products of lipid peroxication, but the fluorescence criteria available do not provide definitive proof of structure.

Lipid soluble fluorescent pigments from human testis were fractionated by silicic acid column chromatography and silica gel thin layer chromatography. Fluorescence analyses revealed a family of at least 3 compounds with similar fluorescence properties, including excitation and emission maxima, reversible fluorescence quenching by alkaline pH, and fluorescence quenching by heavy metal chelation. These fluorescence characteristics strongly indicated the presence of the conjugated Schiff base fluorophore-N=C-C=C-N-. The chromatographic separations employed enabled a more definitive fluorescence characterization of the lipid soluble pigments known to accumulate late in tissues with age and as a result of lipid peroxidation. Total lipids and fatty acid composition of the total lipids were determined. Polyenoic acids constituted about 40% of the total fatty acids. Histological examination of the tissues revealed some degeneration and edema, but significant spermatogenesis and normal complement of Leydig cells.