Table 18.2 Associations of plasma biomarkers of oxidative stress with AMD or risk factors of AMD

Previous studies have identiÞed biomarkers of oxidative stress in human plasma that may be associated with age-related macular degeneration (AMD) or the AMD risk factors aging and smoking. Negative Þndings are not reported in this table.

Cysteine (Cys); cystine (CySS); glutathione (GSH); glutathione disulÞde (GSSG); 8-hydroxy-2¢- deoxyguanosine (8-OHdG); carboxymethyl pyrrole (CEP); 4-hydroxynoneal (HNE); malondialdehyde (MDA); low-density lipoprotein (LDL); reduction potential (Eh).

18.3Defenses Against Oxidative Stress

The bodyÕs defense against increasing oxidative stress consists of molecules with antioxidant capacity: vitamins C and E, carotenoids, and other free radical scavengers. In fact, the AREDS supplements that are protective against AMD progression contain vitamin C, vitamin E, and b-carotene. The antioxidant enzymes SOD, GSHPx, glutathione reductase, catalase, and paraoxonase 1 (PON1) also play a critical role in protecting the retina from oxidative damage.

18.3.1Antioxidants

Vitamin C (ascorbate) is the major aqueous-phase antioxidant in human blood [107]. Recent in vitro studies have shown that vitamin C protects RPE cells against H2O2-induced oxidative stress [108], and two human studies with a small number of cases found lower plasma levels of vitamin C in late AMD patients [109, 110]. However, no difference in vitamin C levels between AMD cases and controls has been reported in multiple larger studies [111Ð114].

Vitamin E (tocopherol), which exists in four forms (a-, b-, g- and d-tocopherol), acts as the major chain-breaking antioxidant of cellular membranes [58]. The most prominent form of vitamin E in human retina and plasma, a-tocopherol, is the most effective free radical scavenger of the four isoforms. Vitamin E has been

shown to be protective against light-induced retinal damage in rats by decreasing MDA production [115]. In several small human studies, plasma vitamin E levels were reported to be signiÞcantly lower in AMD patients compared to controls [109, 110, 113, 116]. A larger population-based investigation in southern France, the POLA (Pathologies Oculaires LiŽes ˆ lÕAge) study, found a moderate negative association between vitamin E levels and late AMD (p = 0.07) that reached statistical signiÞcance after adjusting for lipid levels (p = 0.003) [114]. In contrast, the lower mean vitamin E levels in neovascular AMD patients compared to controls (p = 0.03) in a substudy of the Wisconsin Beaver Dam Eye Study (BDES) were no longer signiÞcant after controlling for cholesterol levels [117]. The Eye Disease CaseÐcontrol Study (EDCCS) observed no association between vitamin E levels and AMD [111]. Overall, it seems that vitamin E levels may modestly correlate with AMD, and this potential relationship warrants further investigation.

Another signiÞcant class of antioxidant molecules, carotenoids, includes carotenes (a-carotene, b-carotene, and lycopene) and xanthophylls (lutein, zeaxanthin, and b-cryptoxanthin) [118]. Retinal levels of these molecules have been linked with the incidence and progression of retinal degeneration in animal models [119, 120]. SigniÞcantly lower levels of macular pigment (lutein and zeaxanthin) were found by direct measurement in AMD vs. control eyes at autopsy [63]. Two cross-sec- tional studies using Raman spectroscopy to measure macular pigment optical density (MPOD) also reported a correlation between AMD and low macular pigment [64, 65]. Conversely, two studies using heterochromic ßicker photometry and reßectometry found no signiÞcant difference in MPOD values between AMD and control groups [121, 122], and longitudinal studies have not found increased risk of AMD progression for patients with lower macular pigment [123, 124]. These differing results may be due to variation in physiological and nutritional characteristics of the populations, as well as the measurement technique [125, 126].

The evaluation of plasma levels of certain carotenoids has produced interesting results. Participants in the multicenter caseÐcontrol EDCCS with medium and high levels of carotenoids (lutein, zeaxanthin, a- and b-carotene, cryptoxanthin, lycopene) had markedly reduced risks of neovascular AMD (ORs 0.5 and 0.3, respectively) compared to controls [111]. Studies of individual plasma carotenoids as biomarkers for AMD have focused on the macular pigments lutein and zeaxanthin, and the majority have shown no association between plasma levels of these xanthophylls and AMD [109, 110, 117, 127]. However, low serum lycopene, a precursor to b-carotene, was associated with AMD in multiple studies [109, 117, 127]. Thus, it appears that carotenoids other than the macular pigments may play a critical role in combating systemic ROS.

The antioxidant pigment melatonin, which acts as a free radical scavenger, has been shown to reduce H2O2-induced damage in RPE cells in vitro [128]. Lower levels of the major urine metabolite of melatonin, 6-sulfatoxymelatonin (aMT6s), were found in the morning urine of AMD patients compared to controls [129], suggesting that melatonin deÞciency may play a role in AMD. In contrast, an ELISAbased assay showed higher serum melatonin levels in AMD patients vs. controls (p = 0.003) and in neovascular AMD vs. nonneovascular AMD patients (p = 0.009)

[130]. The discrepancy between these studies may be explained by the difference in measurement methods.

In conclusion, it is important to remember that measuring serum levels of antioxidants of dietary origin must be approached cautiously, as levels are heavily inßuenced by recent nutritional intake. While studies on antioxidants in human plasma have yielded largely inconsistent results, vitamin E and lycopene appear to be modestly associated with AMD and merit further investigation as plasma biomarkers for AMD.

18.3.2Antioxidant Enzymes

SOD catalyzes the quenching of the superoxide anion to produce hydrogen peroxide and oxygen [58]. Two small studies reported signiÞcantly lower plasma SOD levels in AMD patients compared to controls [102, 131]. However, most studies, including the larger POLA study, found no association between SOD level and AMD [132Ð134].

GSHPx, found in the human retina, reduces organic hydroperoxides using GSH as an electron donor and selenium as a cofactor [58]. Studies evaluating plasma GSHPx and AMD have produced very inconsistent results. Small studies from India [131] and Turkey [102] reported that AMD patients had signiÞcantly lower GSHPx levels than controls, although two small studies found no association between AMD and GSHPx level [133, 134]. The POLA study reported that higher levels of GSHPx were associated with a ninefold increase in late AMD prevalence [132].

The enzyme glutathione reductase (GSHRx) is required for regeneration of the cellular antioxidant glutathione (GSH) from its disulÞde form (GSSG). GSHRx activity was found to be lower in AMD patients in one study [134] but not another [133]. Taken together, there is inconsistent evidence that plasma levels of GSHPx or GSHRx can serve as helpful AMD biomarkers.

Catalase, an iron-dependent enzyme that scavenges H2O2, has been localized in human retina and RPE [58]. While one Turkish study reported lower plasma levels of catalase in AMD patients than controls [101], this association was not replicated in another small study [133]. These mixed results suggest a limited role for plasma catalase in AMD.

The enzyme PON1 metabolizes lipid peroxides and prevents oxidation of LDLs. PON1 activity level was found to be reduced in 37 Turkish AMD patients compared to 29 healthy controls (p < 0.001) and was negatively correlated with levels of the lipid peroxidation product MDA [135]. Lower PON1 levels were also demonstrated in 40 neovascular AMD patients compared to 40 controls [85].

Of the antioxidant enzymes studies, those on PON1 have shown the most consistent results, suggesting its possible involvement in the systemic defense against oxidative stress in AMD. Plasma biomarkers of antioxidants and antioxidant enzymes that have been previously associated with AMD are summarized in Table 18.3.