Free Radical Biology and Medicine. 2016; 101:446-454. DOI: 10.1016/j.freeradbiomed.2016.11.012

Nitroxide free radicals protect macular carotenoids against chemical destruction (bleaching) during lipid peroxidation


Zareba M1,2, Widomska J3, Burke JM2, Subczynski WK1

1Department of Biophysics, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI 53226, USA

2Department of Ophthalmology, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI 53226, USA

3Department of Biophysics, Medical University of Lublin, Aleje Racławickie 1, Lublin, Poland



Macular xanthophylls (MXs) lutein and zeaxanthin are dietary carotenoids that are selectively concentrated in the human eye retina, where they are thought to protect against age-related macular degeneration (AMD) by multiple mechanisms, including filtration of phototoxic blue light and quenching of singlet oxygen and triplet states of photosensitizers. These physical protective mechanisms require that MXs be in their intact structure. Here, we investigated the protection of the intact structure of zeaxanthin incorporated into model membranes subjected to oxidative modification by water- and/or membrane-soluble small nitroxide free radicals. Model membranes were formed from saturated, monounsaturated, and polyunsaturated phosphatidylcholines (PCs). Oxidative modification involved autoxidation, iron-mediated, and singlet oxygen-mediated lipid peroxidation. The extent of chemical destruction (bleaching) of zeaxanthin was evaluated from its absorption spectra and compared with the extent of lipid peroxidation evaluated using the thiobarbituric acid assay. Nitroxide free radicals with different polarity (membrane/water partition coefficients) were used. The extent of zeaxanthin bleaching increased with membrane unsaturation and correlated with the rate of PC oxidation. Protection of the intact structure of zeaxanthin by membrane-soluble nitroxides was much stronger than that by water-soluble nitroxides. The combination of zeaxanthin and lipid-soluble nitroxides exerted strong synergistic protection against singlet oxygen-induced lipid peroxidation. The synergistic effect may be explained in terms of protection of the intact zeaxanthin structure by effective scavenging of free radicals by nitroxides, therefore allowing zeaxanthin to quench the primary oxidant, singlet oxygen, effectively by the physical protective mechanism. The redox state of nitroxides was monitored using electron paramagnetic resonance spectroscopy. Both nitroxide free radicals and their reduced form, hydroxylamines, were equally effective. Obtained data were compared with the protective effects of α-tocopherol, which is the natural antioxidant and protector of MXs within the retina. The new strategies employed here to maintain the intact structure of MXs may enhance their protective.

KEYWORDS: Zeaxanthin; Carotenoid; Oxidative stress; Lipid peroxidation; Antioxidants; AMD

PMID: 27840316



The high consumption of lutein and zeaxanthin (Zea) is associated with a lower risk of age-related macular degeneration (AMD) [1]. Only these two carotenoids are selectively accumulated in the membranes of the retina from blood plasma [2]. These carotenoids, named macular xanthophylls (MXs), are thought to combat light-induced damage mediated by reactive oxygen species by absorbing the most damaging incoming wavelength of light prior to forming reactive oxygen species and by chemically and physically quenching reactive oxygen species once they are formed. Most of the protective actions of MXs in the retina are performed through their physical protective mechanisms, which require that MXs be in their intact structure.


The goal of the presented study was to find ways to protect MXs against their destruction in the retina, mainly during peroxidation of retinal membranes. This seems significant, because oxidative stress is one of the major factors that promotes the development of AMD [3]. The protection of the intact structure of MXs should extend and enhance their positive actions in the retina through physical processes (filtration of blue light [4], quenching triplet states of photosensitizers [5], physical quenching of singlet oxygen [6]). Our results indicated that membrane-located nitroxide free radicals (like 1-palmitoyl-2-(16-doxylstearoyl)phosphatidylcholine (16-PC)) protected Zea against destruction, and their effect is especially pronounced during the light-induced formation of singlet oxygen (Fig. 1A).


Only in the case of singlet oxygen-induced lipid peroxidation did Zea show clear protection against peroxidation of polyunsaturated membranes (here made of 1-palmitoyl-2-arachidonoylphosphochatidyline (PAPC)) (Fig. 1B). The most impressive observation is a very strong synergistic, inhibitory effect of Zea and 16-PC on lipid peroxidation of PAPC membranes, much stronger than the sum of the effects of the individual agents (Fig. 1B). Thus, 16-PC not only protects Zea from destruction (as shown in Fig. 1A), but it also enhances the effect of Zea as a lipid-soluble antioxidant during light-induced lipid peroxidation (as shown in Fig. 1B). We theorize that new strategies, like the one employed here, aimed at maintaining the intact structure of MXs during oxidative stress, which is implicated in AMD pathogenesis, should enhance their protective potential.



Fig. 1 (A) Bleaching of Zea located in membranes made of polyunsaturated phospholipid (PAPC, 2.5 mM) during their peroxidation in the absence and presence of nitroxide spin label 16-PC (25 µM). Lipid peroxidation was induced by rose bengal (10 µM) and green light at the concentrations of Zea – 8 µM. (B) The effect of Zea (8 uM) or 16-PC (25 µM), alone or in combination, on the accumulation of phospholipid peroxidation products (TBARS). Model membranes were made of polyunsaturated phospholipid (PAPC, 2.5 mM). Lipid peroxidation was induced by rose bengal (10 µM) and green light at the concentrations of Zea – 8 µM.



1 Richer, S. P.; Stiles, W.; Graham-Hoffman, K.; Levin, M.; Ruskin, D.; Wrobel, J.; Park, D. W.; Thomas, C. Randomized, double-blind, placebo-controlled study of zeaxanthin and visual function in patients with atrophic age-related macular degeneration: The Zeaxanthin and Visual Function Study (ZVF) FDA IND #78, 973. Optometry 82:667-680; 2011.

2 Bone, R. A.; Landrum, J. T.; Dixon, Z.; Chen, Y.; Llerena, C. M. Lutein and zeaxanthin in the eyes, serum and diet of human subjects. Exp. Eye Res 71:239-245; 2000.

3 Jarrett, S. G.; Boulton, M. E. Consequences of oxidative stress in age-related macular degeneration. Mol. Aspects Med 33:399-417; 2012.

4 Cullen, A. P. Photokeratitis and other phototoxic effects on the cornea and conjunctiva. Int. J. Toxicol 21:455-464; 2002.

5 Pawlak, A.; Wrona, M.; Rozanowska, M.; Zareba, M.; Lamb, L. E.; Roberts, J. E.; Simon, J. D.; Sarna, T. Comparison of the aerobic photoreactivity of A2E with its precursor retinal. Photochem. Photobiol 77:253-258; 2003.

6 P., D.; Murphy, M. E.; Sies, H. Antioxidant defense systems: the role of carotenoids, tocopherols, and thiols. Am. J. Clin. Nutr 53:194S-200S; 1991.



This work was supported by grants EY015526, EY001931, EB002052, and EB001980 from the National Institutes of Health.



Witold K. Subczynski, Ph.D, D.Sc.

Department of Biophysics

Medical College of Wisconsin

8701 Watertown Plank Road

Milwaukee, WI 53226, USA

Tel: (414) 955-4038; Fax: (414) 955-6512