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A.C. Johnson, T.W. Olsen, S.S. Marimanikkuppam, D.A. Ferrington; Identification and Quantitation of Lipofuscin Fluorophores in Human Donor Eyes . Invest. Ophthalmol. Vis. Sci. 2006;47(13):2029.
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Autofluorescent lipofuscin granules accumulate in the retinal pigment epithelium (RPE) with aging and can inactivate key proteolytic enzymes in the lysosomes and the proteasome. While ten ocular fluorophores have been spectrophotometrically characterized, the identities of only four fluors have been determined. Identification of individual lipofuscin components will provide insight into its origin and allow for dissection of the pathological role for specific components.
Lipofuscin was isolated by chloroform/methanol extraction from the RPE of human donor eyes obtained from the Minnesota Lions Eye Bank. Individual components of the mixture of autofluorescent compounds were resolved by reverse–phase (C18) HPLC using a complex organic solvent gradient. Electrospray ionization mass spectrometry (MS) was performed on a Q–Star mass spectrometer to identify the individual components.
Following chloroform/methanol extraction of lipofuscin, the fluorophores were limited to the organic phase. From this complex mixture of fluorophores, more than ten peaks were resolved by HPLC separation at 366nm. The mass of each isolated fluorophore was obtained by mass spectrometry. Four previously known compounds of lipofuscin (A2E, iso–A2e, all–trans–retinol, all–cis–retinol) were identified by MS/MS. One new compound, retinoic acid, was identified by MS/MS.
Lipofuscin contains a highly complex mixture of fluorescent compounds. Quantitative assessment of individual fluorescent components and their identification by mass spectrometry will allow for more in–depth studies of the functional consequences of lipofuscin accumulation in the retina.
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