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D. Zhao, P. Bhosale, P.S. Bernstein; Chiral HPLC–Mass Spectral Analysis of Lutein, Zeaxanthin and Meso–Zeaxanthin in Human Macula . Invest. Ophthalmol. Vis. Sci. 2005;46(13):1764.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose:Lutein [(3R,3'R,6'R)–ß,ε–carotene–3,3'–diol] and zeaxanthin [a mixture of (3R,3'R)–ß,ß–carotene–3,3'diol and (3R,3'S–meso)–ß,ß–carotene–3,3'–diol] and their metabolites are found in substantial amounts in the retinas of humans. Using a highly sensitive chiral HPLC–mass spectral method that does not require chemical derivatization, we have quantified these three main macular xanthophyll carotenoids and their ratios in different age groups. METHODS: Macular samples (4 mm diameter) from 170 donor eyes free of ocular pathology were procured from the local eye bank. The amounts of lutein, zeaxanthin, and meso–zeaxanthin extracted from each tissue sample were determined by chiral HPLC coupled with an in–line single quadrupole mass spectrometer (MS) in a positive ion atmospheric pressure chemical ionization (APCI) mode. The levels and ratios of xanthophyll carotenoids were estimated in young and old age groups. Results:The chiral HPLC–MS/APCI method was optimized using standard xanthophyll carotenoids. The limit of detection was observed to be 10 pg for all three xanthophyll carotenoids,100 times more sensitive than standard photodiode array (PDA) detection. The meso–zeaxanthin to lutein ratio was 0.22 ± 0.2 in the young donors group (age 13–43) versus 0.76 ± 0.2 for the old donors group (45–80 years). The meso–zeaxanthin to dietary zeaxanthin ratio was observed to be 0.32 ± 0.07 in the young donors group versus 0.72 ± 0.08 for the old donors group. The differences observed were statistically significant (P ≤ 0.0005, N=60 nonpaired t–test) and independent of gender. On the other hand, the zeaxanthin to lutein ratio was 0.76 ± 0.14 and 0.78 ± 0.06 for young and old groups, respectively. Approximately 5% of the elderly donors had unusually high levels of macular carotenoids–two to three times the population’s mean level. Carotenoid levels in these donors were also unusually high in the lens, but not in the peripheral retina. Similar outliers were not observed in the young donor group. We speculate that these elderly outliers were consuming lutein supplements regularly prior to death. CONCLUSIONS: A high relative level of non–dietary meso–zeaxanthin in the older age donors indicates that the proposed double bond isomerization of dietary lutein to meso–zeaxanthin may be promoted by high levels of oxidative stress in aging eyes. The presence of unusually high levels of macular carotenoids in some elderly donors supports the hypothesis that lutein supplementation can raise levels of macular pigment.
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