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D. Zhao, P. Bhosale, P.S. Bernstein; Metabolites of Ocular Carotenoids Are Biomarkers of Aging in the Human Macula . Invest. Ophthalmol. Vis. Sci. 2006;47(13):3801.
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© ARVO (1962-2015); The Authors (2016-present)
Lutein and zeaxanthin are the predominant carotenoids found in the human macula and retina. With exposure to environmental stress during aging, several non–dietary metabolites of these carotenoids are also found in substantial amounts in these tissues. These metabolites can be considered to be "aging biomarkers" and are likely to play important roles in future studies of the mechanisms of age–related macular degeneration (AMD). The purpose of this study was to develop a highly sensitive chiral HPLC–method with mass spectral detection for estimation of these non–dietary metabolites in the human macula without chemical derivatization.
Macular samples from 250 donors free of ocular pathology were procured from the local eye bank. The carotenoids were extracted from each tissue sample were determined by normal phase (NP) silica and chiral HPLC coupled with an in–line single quadrupole mass spectrometer (MS) in a positive ion atmospheric pressure chemical ionization (APCI) mode. Lutein and zeaxanthin and their metabolites were quantified using the instrument’s single ion monitoring (SIM) mode. The levels and ratios of xanthophyll carotenoids were measured in different age groups.
HPLC–MS methods were optimized using standard carotenoids. Three major carotenoids lutein, zeaxanthin, and meso–zeaxanthin were quanitified over a wide age range. We also detected and quantitated non dietary metabolites such as oxolutein, lactucaxanthin, epilutein, 9Z– zeaxanthin, and 9'Z–lutein. Our analysis of samples revealed presence of a previously unreported polar peak tentatively labeled as "zea–isomer". It had spectral properties identical to zeaxanthin but with m/z 582 instead of 568. Quantitative analysis revealed that meso–zeaxanthin, 3'–oxolutein, and zea–isomer levels increased significantly with age. We also observed that approximately 11 % of the elderly donors had unusually high levels of macular carotenoids (68.8 ±15.23ng, N=26), relative to age–matched normals (22.4 ±12.7ng, N=59). The increase in macular carotenoids was driven primarily by lutein and by meso–zeaxanthin. The elderly outliers also had unusually high levels of these carotenoids in their peripheral retina and lens. Dietary inquiries of the donors’ families revealed that the majority of these elderly outliers were consuming lutein supplements or lutein–rich diets prior to their deaths.
HPLC with mass spectral analysis is a highly sensitive way to detect oxidative metabolites of the macular carotenoids. Further studies of these aging biomarkers should yield valuable insights into the biochemical mechanisms underlying AMD.
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