In the present study we observed significantly higher levels of cadmium, cobalt, iron, and zinc, while copper levels were reduced in the aqueous humor of patients diagnosed with AMD when compared with patients without AMD. Manganese and selenium levels showed no significant differences between the two groups. After adjustment for multiple testing; cadmium, cobalt, copper and iron remained a significant factor in age- and sex adjusted GLM models for AMD. We are unaware of any previous studies describing trace element concentrations in the aqueous humor of AMD patients, and could not find any respective reference in a computerized search utilizing Medline.
There is evidence that oxidative stress is involved in the formation of drusen and in the pathogenesis and progression of AMD. Hydroxyl radicals are extremely reactive, causing lipid peroxidation, DNA strand breaks, and degradation of biomolecules. Particularly in photoreceptors, where there is a high oxygen tension and high concentration of easily oxidized polyunsaturated fatty acids, reactive oxygen species must be tightly controlled to avoid oxidative damage. Oxidative stress and inflammation have both been linked to AMD 
. In the Fenton reaction, iron reacts with hydrogen peroxide (H2
) to produce hydroxyl radicals, the most reactive and toxic of the reactive oxygen species (ROS). Retinal degeneration has also been observed in hereditary disorders resulting in iron overload, including aceruloplasminemia, hereditary hemochromatosis, pantothenate kinase associated neurodegeneration, and Friedreich's Ataxia. AMD-affected maculas contained more iron than healthy age-matched maculas 3]. Our results of increased iron in the aqueous humor of AMD patients seem to confirm a role of this metal in the pathogenesis of AMD.
Another trace metal known to induce oxidative stress with higher concentration in aqueous humor of AMD patients is cadmium. The biologically significant ionic form of cadmium, Cd2+
, binds to many bio-molecules and these interactions underlie the toxicity mechanisms of cadmium. Some of the molecules specialized in the handling of alkaline earth (Mg2+
) and transition metal ions (e.g. Zn2+
) should be particularly sensitive to the presence of Cd2+
, because they enclose cationic sites to which the toxic metal can bind 
. Metallothionein is an important intracellular storage protein for zinc and copper, and its synthesis is decreased in oxidative stress 
. Considering the tight binding of Cd2+
by metallothionein and the sensitivity of the expression of its genes to stressful conditions, this protein may mediate cadmium toxicity in various ways. These include decrease of the zinc buffering ability of cells in different compartments, changing of the dynamics of zinc exchanges, and decrease of the cellular antioxidant defense 
. Exposure to cadmium perturbs the homeostasis of other metals, and, reciprocally, this effect depends on the body status of other essential metals such as iron and zinc. This interaction is regularly observed in a variety of conditions 
. Zinc often affords protection against cadmium toxicity, and cells adapted to high zinc concentrations display changed cellular handling homeostasis of cadmium, manganese, and calcium 
Zinc and copper are cofactors of metalloenzymes that play a critical role in cell structure and function. Among these enzymes is copper-zinc superoxide dismutase, which regulates oxidative stress in the RPE. Studies show that zinc plus copper supplementation decreases the risk of progression of AMD 
. Aqueous zinc levels were increased while copper concentrations were reduced in our study. A decrease of copper and zinc concentrations in the RPE and choroid complex of AMD-affected subjects has also been reported 
. It was shown that average levels of zinc and copper in the neural retina were lower in aged eyes than in young eyes, whereas increase in these metal levels occurred in the choroid; also correlation between cadmium accumulation and increase in zinc and copper levels in males was observed 
. High zinc concentration was also shown in macular sub-RPE deposits of patients with AMD 
. Zinc may also be released from intracellular deposits of the RPE and photoreceptors due to apoptosis of these cells. These mechanisms may lead to elevated extracellular levels of this metal despite its suspected intracellular deficiency. As AMD was not a significant factor in a general linear model regarding zinc, the possible role of this trace element in the pathogenesis of AMD remains uncertain from the results of this study.
We have reported elevated cobalt levels in patients with AMD. Cobalt can cause DNA fragmentation and activation of caspases, increased production of reactive oxygen species, and beta amyloid secretion 
. A significant depletion of intracellular Zn2+
exposure has been described 
. A substitution of magnesium ions by cobalt ions may result in the interruption of ATPases and the energy balance of the cell 
. Ionic cobalt (Co2+
) is known to exert hypoxia-like responses by stabilizing the alpha subunit of the hypoxia inducible transcription factor (HIF1) 
. This results in changed gen transcription of encoding proteins that play key roles in angiogenesis, glucose and energy metabolism, cell survival and proliferation, iron metabolism, and vascular functions 
. Comparative gene expression studies showed HIF1-mediated responses to be similar for hypoxia and CoCl2
. Although all these mechanisms have been described for the immediate toxicity of much higher cobalt concentrations than reported in our study, they might also play a role in a long-standing exposure to lower concentrations. We could not find any published data on an involvement of cobalt toxicity in AMD pathogenesis.
The major limitation of this pilot study is the relatively small sample size. Generally, a bias may be introduced by a convenient sampling approach; however, this seems unlikely in the present study due to the high prevalence of cataract in the general population and the case-control design.
Our findings of significant alterations in aqueous humor metal levels in AMD-affected eyes support the hypothesis that their dysregulation may be involved in the pathogenesis of AMD. Knowledge of trace elements distribution, metabolism and toxicity will help to understand their role in the pathogenesis of AMD. Properly designed studies implementing biologically relevant intracellular and extracellular trace elements concentrations in different ocular tissues are required. Improved knowledge of the essential metals homeostasis and combination of this data with other aspects of AMD may help to tackle treatment of this disease.