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Author: Elisabetta Fumero
Date: 06/07/2014


AUTORI: Ilaria Trave, Elisabetta FUMERO


Among minerals, iron (Fe), zinc (Zn) and copper (Cu) are essential trace elements playing key roles in retinal structure and physiology, they are principally involved in visual function. They are present in the body in the form of ions.
While zinc is only present as a bivalent cation Zn(II), iron and copper have the ability to reversibly yield and absorb electrons and thus they can be found in reduced [Fe(II), Cu(I)] or oxidized [Fe(III), Cu(II)] states.
Experimental studies have shown that both decreased and elevated levels of minerals can cause retinal cell dysfunction. Their relevance in retinal physiology and pathophysiology, however, depends not only on their concentrations but also on their distribution and oxidation/reduction states, which are under tight regulation by interrelated metabolic pathways. Iron, zinc and copper play important catalytic, structural and regulatory roles in proteins and enzymes of general cell metabolism (e.g. mitochondrial function, gene expression, antioxidant defense). As free ions, they take part in angiogenesis, nerve myelination, endorphin action and synaptic transmission. Cells with high energy demand, such as some retinal cells, will have high levels of these metals. Moreover, iron, zinc and copper are considered to have specialized retinal functions. The function of several compounds involved in the photo-transduction process (rhodopsin, phosphodiesterase, recoverin), visual cycle, retinal neurotransmission and production of the neurotransmitter glutamate has been shown, both in vitro and in vivo, to be affected by iron, zinc or copper (1).
The distribution of total iron, zinc and copper in the retina is non uniform.
The concentration and distribution of this trace element in the mammalian retina are analyzed using various techniques. Inductively coupled plasma mass spectrophotometry (ICP-MS, 2), atomic absorption spectrophotometry (AAS, 3), and colorimetric analytical methods (e.g. diphenylthiocarbazone, 4) allow bulk quantification of total trace elements. Beam-based X-ray microanalysis with high energy protons (proton-induced X-ray emission, PIXE, 5) or X rays (synchrotron X- ray fluorescence, XRF, 5) localize and quantify total trace elements in individual retinal layers. Hystological stains and fluorescent probes (6), have allowed the visualization of pools of loosely bound, chemically reactive ("free") metals in retinal sections.
The concentratios of the trace elements iron, zinc and copper in the human retina have been evaluated and slightly differ with the different tecniques. They can be summarized as follows (from 1):

ElementConcentration (mcg/g dw)Note

Table I. Concentration of iron, zinc and copper in the human retina. Legend. DW: dry weight; RPE: retinal pigment epithelium.

It is evident that the distribution is not uniform, and these differences may be related to different functions in the different layers of the retina (see next image, details in the text below)

Retinal function of iron.
In the cell, the iron is prevalently located within the mitochondrial matrix (as loosely bound iron or in heme groups and iron sulphur cluster in proteins of the respiratory chain and oxidative phosphorylatio), or in the heme-containing enzyme guanylate cyclase, or bound to other enzymes, as aconitase (synthesis of glutamate) and fatty acid desaturase (generation of disk membranes of photoreceptors).
Retinal function of zinc.
In the retinal pigment epithelium of th retina, zinc is required for antioxidant defense and the function of retinol dehydrogenase, the enzyme involved in retinol processing in the visual cycle.
In the photoreceptor inner segments/outer limiting membrane (RIS/OLM) loosely bound zinc is at the level of Golgi apparatus and endoplasmic reticulum, while in the inner nuclear layer (INL) there are high levels of zinc, mainly tighly bound in complexes (zinc-finger transcription factors).

Retinal function of copper.
There is abundant copper co-localizing with iron in the RIS/OLM, probably within the mitochondria.

Image 1.Iron-, zinc- and copper-dependent proteins find in the RPE and photoreceptors.
The photopigment rhodopsin (Rho) is regulated from zinc (it mediates changes in rhodopsin stability and function in the physiological range) and from copper.
Zinc is also necessary for good function of 3’,5’-cGMP phosphodiesterase (PDE) and recoverin (Recov), that deactivates rhodopsin after light stimulation, in the activity of enzyme retinol dehydrogenase (RD).
Iron is important for the catalytic activity of RPE65, that converses trans-retinyl ester to 11-cis retinol with help to iron, and for guanylate cylase (GC).


Cellular uptakes and release of iron, zinc and copper and their intracellular pool size are tighly controlled.

There are two mechanisms of iron transport accross the cellular membrane:
# transferrin receptor-mediated endocitosis for iron bound to transferrin;
# by the divalent metal transporter 1 (DMT-1), that transport FE along a proton gradient.

Zinc crosses cellular membranes through transporters of the
# Zrt- and Irt-like (Zip) proteins and
# ZnT family.
Within the cytoplasm, zinc binds to metallothioneins (MT) for storage.

Copper cell entry is mediated by the copper transporter 1 (CtR1).
Once inside the cell, copper binds to glutathione and MT for storage and to metallochaperones
# copper chaperone for superoxyde dismutase 1 (CCS)
# chaperone for the respiratory enzyme CCO (COX17)
# chaperones for ATPases ATP7A-B (ATOX1)
for delivery to specific proteins, with virtually no intracellular "free" ions.

The following images summarize membrane transport and intracellular metabolism of iron (panel A), zinc (panel B) and copper (panel C).

Moreover, in the cell mitochondria play critical role in iron, zinc and copper metabolism.

The effects and functions of iron, zinc, and copper in the cell are likely to be heavily dependent on one another.
Intracellular “free” zinc is involved in the regulation of iron regulatory proteins (IRP) and influences the expression of MT (copper and zinc storage). When intracellular zinc is reduced, alterations in the regulation of molecules involved in iron homeostastis occur (eg. transporters, storage, regulatory proteins), causing intracellular iron accumulation.
Optimal levels of copper are essential for the synthesis of melanin that binds molecule for iron, zinc, copper and ceruloplasmin (Cp), one ferroxidase involved in iron export. If the copper level lowers, iron accumulation can be observed, due to reduction in ceruloplasmin (see next image).


Zinc is included in numerous enzymes (7) or it serves as their activator (more than 80 zinc metallo-enzymes). It is necessary for the functioning of carbonic anhydrase, aldolases, many dehydrogenases (including alcohol-dehydrogenase, retinal reductase indispensable for retinal rod function), alkaline phosphatase (8).
In the biochemical processes important for the vision and regulated by Zinc, we have to focus on alcohol dehydrogenases (ADHs). Active site zinc participates in catalytic events, and structural site zinc maintains structural stability. Alcohol dehydrogenase catalyzes reversible oxidation of primary and secondary alcohols using the coenzyme NADP. These ADHs are zinc metalloenzymes with frequently two tetrahedrally coordinated zinc ions per subunit, one catalytic at the active site, and one structural at a site thought to influence subunit interactions (9).
Alcohol dehydrogenases (ADHs) and retinol dehydrogenases (RDHs) catalyze these reactions:

retinal + NADPH + H+ ↔ retinol + NADP+
retinol + NAD+ ↔ retinal + NADH + H+

Retinal is one of the many forms of vitamin A and is interconvertible with retinol (ROL), the transport and storage form of vitamin A (5).
Zinc conditions the activity of ADH, essential for the production of retinol. Retinal links with opsin to form Rhodopsin , a biological pigment in photoreceptor cells of the retina that is responsible for the first events in the perception of light (10).
The cornea has the highest zinc content of all body tissues. Corneal oedema caused by zinc deficiency may progress to corneal opacity. Mild dry conjunctivitis may also occur, which can progress to xerosis and keratomalacia. Scotopic ERG abnormalities and night blindness have been related to low zinc levels (11).
A case report have been published (12) where a 46-year-old man with low serum zinc levels and vitamin A deficiency showed some electroretinogram alterations (reduced scotopic and phototopic ERG b-waves). After normalization of zinc levels, but not vitamin A deficiency, poor dark adaptation improved as well as scotopic ERG response, but not photopic ERG, suggesting that rod function is more susceptible to zinc deficiency than cone function.
No retinal abnormalities have been described with zinc overload, probably because homeostatic mechanisms might prevent zinc accumulation in the normal retina.

Iron deficiency causes mainly problems to other organs (13), that are the first and more evident clinical manifestations in this condition. Indeed, since iron plays a critical role in some visual processes, we may expect some effects on retinal function, too. And this happens, even if these effects are not completely known. Adult individuals with iron deficiency anemia suffer from retinal vein and artery occlusions and papilledema. There are, however, no adults with retinal dysfunction associated with iron deficiency without anemia. Iron deficiency might compromise the function of oligodendrocytes and myelination of the visual pathways. Iron since to be important for myelination, and for normal metabolism in infants and children. It seems that the immature visual system may be more susceptible than the adult system, since myelination occurs during the first few years of life. On the other hand, it is possible that low iron levels also affect vision in adults, as suggested by the cognitive findings in iron deficient adults, perhaps due to lack of oxygen for metabolism. Impairment of retinal iron homeostasis is thought to be involved also in aging and the pathogenesis of conditions such as age-related macular degeneration (AMD).
As shown in figure 1, iron is a cofactor for the enzyme guanylate cyclase (GC), the enzyme that returns cyclic GMP concentration to the original dark levels following light stimulation (1).
Where a condition of chronic iron overload occurs for an ereditary or acquired disorder, some fundus changes have been described, and is thought to reflect retinal iron overload (14). Some of these patients, however, hadd been treated with iron chelators, which could have contributed to fundus oculi abnormalities.

There are a few reports of optic neuropathy in patients with low copper levels presenting with reduced visual acuity and color vision, constricted visual fields, optic atrophy, and retinal nerve fiber thinning. The scotopic and photopic electroretinograms (ERG) were normal, suggesting that the overall retinal function was preserved. Copper deficiency in rats fed diets with insufficient copper content results in optic nerve demyelination and damaged vacuole-containing myelin. These effects are thought to be the result of reduced activity of cupro-enzymes or free copper ions involved in the synthesis of phospholipids. This results have not subsequently been confirmed in humans (1).
If we don't consider experimental situations where high doses of copper are injected intravitreous in animals, no cases have never been reported with retinal signs and/or symptoms in case of copper overload in humans.

Age-induced changes in retinal homeostasis of iron, zinc and copper
During ageing the metabolism of iron, zinc and copper changes, as a result of general changes in systemic metabolism and disfuntion of the strict homeostatic control of these ions.
As a results, retinal iron accumulation and zinc depletion have been observed, most probably facilitating age-related macular degeneration (AMD).
These observations have led to the idea that iron chelators and zinc supplements may be beneficial in the treatment of AMD, but further evidence are needed, also because it is still debatable the existence of a retinal toxicity from iron chelators.
Some trials are present that consider the effect of zinc supplementation on AMD and are very encouraging, expecially in the "dry" form of AMD, with no effect on "wet" AMD.

Iron, copper and zinc deficiency has multi-organ clinical consequences, includind visual impairement, even if this is one the latest and mildest clinical manifestations to occur.
Ophthalmic clinical evaluation may be useful to confirm visual disturbances in the suspect of iron, copper and zinc deficiency (10).
In many articles, an appropriate oral supplementation is reported to be effective in case of zinc and iron deficiency.
Finally, the knowledge of enzymes controllated by zinc, copper omeostasis is very important for understanding the mechanism of nutrient-related visual disfunctions.

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