Lipofuscin in the retina: Quantitative assay for an unprecedented autofluorescent compound (pyridinium bis-retinoid, A2-E) of ocular age pigment

The pyridinium bis-retinoid, A2-E, has been discovered as one of the major autofluorescent components of retinal pigment epithelial lipofuscin. Due to its chemical characteristics, A2-E may contribute to cellular and molecular changes leading to age-related macular degeneration. Because A2-E is the first lipofuscin component that has been identified, purified, and its structure analysed, it represents an important marker molecule for studying lipofuscin formation under various conditions. In order to investigate the role of A2-E in ageing processes of the retinal pigment epithelium, we developed an HPLC assay for this compound using single wavelength UV-absorbance detection with continuous light emission. Standard A2-E was synthetized and purified by sequential TLC. In our assay, A2-E can be detected in amounts lower than 10 pmol. The assay has been applied to quantitative determination of A2-E amounts in albino rat eyes of different age groups. Our results demonstrate that there is a marked increase of A2-E levels in older animals. The method described is the first to allow quantification of this unusual retinoid from small amounts of biological samples. (C) 1997 Academic Press Limited.