Metal Ions in Health and Disease

Whether or not metal ions are the main focus of your research, if you are investigating the involvment of free radicals in a biological system the chances are you will need to consider the involvement of metal ions.

In addition to their direct role in the promotion of free-radical formation, trace amounts of contaminating metal ions (from laboratory reagents and glassware) can catalyse oxygen-radical generation in biological samples and incubations, resulting in damage to proteins, lipids and nucleic acids that it is all too easy to attribute incorrectly to other processes. It is particularly important to be able to prevent the occurrence of ex vivo oxidation reactions in blood and tissue samples taken for investigation.

Free-radical generation by metal-catalysed reactions can also occur in food products, leading to their deterioration and the generation of harmful products. Again, it is important to be able prevent such reactions, particularly in foods rich in polyunsaturated fatty acids, which are especially prone to damage.

In addition to offering input in projects covering all the above areas, Dr Burkitt can help with the assessment of iron and copper status in human subjects (e.g. in those with hereditary haemochromatosis or Wilson's disease). This could entail the develpment and assessment of new treatments, such as chelation therapy. It might also involve the determination of the effects of pharmacological agents on iron and copper homeostasis.

An introductory overview of this area is given below, followed by details of Dr Burkitt’s experience in the field and details of specific projects to which he has contributed.

1. Background: metal ions, oxygen-centred radicals and disease
Metals carry out essential functions in all living organisms where, for example, they serve as co-factors in a multitude of enzymes. Iron also plays a particularly important role as the binding site for oxygen in haemoglobin and myoglobin. In addition to overt metal deficiencies (iron anaemia being an obvious example), poor health can result from excessive levels of particular metals in the body, as seen in hereditary haemochromatosis and Wilson’s disease (involving iron and copper, respectively). In fact many pathological conditions appear to involve simply the re-distribution or ‘decompartmentalisation’ of metals, without there being an overt deficiency or excess.

The biological actions of many metals (calcium and magnesium being important exceptions) involve their participation in redox reactions, whether as components of the electron-transport systems in mitochondria and chloroplasts or at the active sites of enzymes, such as those in the cytochrome P450 and superoxide dismutase families. The ability of iron and copper (and certain other metals) to exist in multiple oxidation states, and thereby participate in redox reactions, also underlies much of their toxicity. This is because the ions of these metals are able to serve as efficient single-electron donors and acceptors, thereby catalysing the generation of free radicals, especially oxygen-centred radicals.

Iron(II) and copper(I) ions can react with hydrogen peroxide to generate the hydroxyl radical (OH), which is an extremely powerful oxidant, able to cause indiscriminate damage to biological molecules, including proteins, nucleic acids and lipids:

Fe2+ + H2O2 → Fe3+ + OH + OH

Cu+ + H2O2 → Cu2+ + OH + OH

The former reaction, involving iron, is called the Fenton reaction; that involving copper is often referred to as the ‘copper-Fenton reaction'. In the presence of a suitable reducing agent, which may be an enzyme, ascorbic acid (vitamin C), glutathione or the superoxide free radical (O2) (see 'Spectroscopy' ), the Fe3+ and Cu2+ formed in these reactions are returned to their lower oxidation states, thereby enabling the metals to act catalytically in the production of OH radicals.

The hydrogen peroxide required in the above reactions is present at low levels in essentially all mammalian cells. It can also be generated via the direct reduction of molecular oxygen by the reduced metal ion, e.g., by Fe2+:

Fe2+ + O2 → Fe3+ + O2

H+ + O2 → HO2

Fe2+ + HO2 + H+ → Fe3+ + H2O2

The ability of iron and copper ions to participate in the above reactions is determined largely by the molecules to which they are bound. Iron bound to its major transport and storage proteins (transferrin and ferritin, respectively), at physiological levels, is believed to be unable to catalyse OH generation. In iron-overload conditions, however, the degree of transferrin saturation can reach levels at which radical generation is possible. Similarly, iron can be released from ferritin when reduced by a flux of superoxide radicals (during inflammation, for example). Copper ions are also believed to be safely sequestered in transport and storage proteins (caeruloplasmin and metallothionein) and thereby unable to promote OH production under physiological conditions.

Notwithstanding the above, iron and (perhaps to a lesser extent) copper ions are believed to promote the generation of oxygen radicals under a wide range of pathological conditions, including, ischaemia-reperfusion injury, atherosclerosis, some cancers, several neurodegenerative disorders and of course metal-overload conditions. Through their catalysis of OH formation, iron and copper ions also promote the toxicity of xenobiotics which produce superoxide radicals by 'futile cycling' (e.g. the quinone anti-tumour agents).

2. Metal-catalysed oxygen-radical generation in biological samples and food products
In addition to their catalysis of OH formation in living systems, the catalysis of such reactions in biological samples and food products is also of importance. In the case of biological samples (including blood samples, enzyme incubations and cells grown in culture), iron and copper contamination from reagents and glassware can result in oxidative damage to proteins, lipids and DNA, which it is all too easy to attribute to endogenous biological processes. Indeed the artefactual, ex vivo generation of radicals by contaminating metal ions can lead to serious errors in the interpretation of experiments employing biological systems. This might involve the initiation of lipid peroxidation, or oxidation of the bases in lymphocyte DNA, in blood samples after they have been taken from the subject.

A similar level of awareness is required to prevent iron and copper ions from catalysing deleterious reactions in food products, particularly those rich in polyunsaturated fatty acids, which are highly susceptible for damage by radicals (lipid peroxidation). In addition to shortening the shelf-lives of food products, there is the possibility of harmful products being generated, such as carbonyl compounds from fatty acids. This can be particularly challenging in foods which have been fortified by the addition of iron (infant formula feeds being a good example).

3. 'Control' of metal-catalysed reactions using chelating agents
Chelating resins can be used to reduce the levels of contaminating metal ions in laboratory reagents and solutions, and chelating agents can be used to 'control' their redox reactions, but this must amount to more than the simple addition of EDTA, DTPA or Desferal. Indeed because DTPA and Desferal both 'hold' iron in the +3 oxidation state, preventing its reduction to Fe2+ for participation in the Fenton reaction, it is often assumed that adding these chelators is all that is needed to prevent the artfactual generation of oxygen radicals. What is often overlooked is the fact that 'decompartmentalised' iron is usually present as Fe2+ in biological systems: the addition of DTPA or Desferal, which favours oxidation to the +3 state, merely promotes a 'single pass' of the radical-generating reactions shown above, which is often all it takes to induce biomolecular damage.

One must also bear in mind that any given chelating agent will affect the redox properties of iron and copper ions in different ways. The iron(III)-EDTA complex, for example, undergoes facile reduction, whereas the corresponding Cu(II) complex is resistant to reduction by the common biological reducing agents (ascorbic acid etc).

4. Assessment of iron status in human subjects
The iron status of human subjects is usually assessed by measuring plasma transferrin saturation and ferritin levels. Neither of these measurements gives a direct indication of the presence of iron in a form able to catalyse the generation of oxygen-centred radicals. Although this is not a problem when monitoring iron deficiency, it has direct bearing on the management of individuals with hereditary haemochromatosis. This is because hydroxyl radicals, generated by ‘free’ iron, are believed to be responsible much of the tissue damage seen in patients with the untreated condition. The mainstay therapy in the management of haemochromatosis is venesection (blood letting), but opinions differ on whether to instigate this on the basis of transferrin saturation or ferritin levels.

4.1 The bleomycin assay for 'catalytic iron'
The bleomycin assay is an ingenious method for the detection ‘catalytic iron’ in biological samples. Although the original assay, developed by Barry Halliwell and John Gutteridge (see, e.g., J. M. C. Gutteridge and B. Halliwell Life Chem. Rep.,4, 113 - 142, 1987; and P. J. Evans and B. Halliwell Methods Enzymol. 233, 82 - 92, 1994), has proven to be of great value in demonstrating the involvement of catalytic iron in a range of pathological conditions, the assay suffers from certain drawbacks, particularly a lack of robustness to sample dilution. These problems have since been overcome by a new version of the assay, which is not only robust to dilution but several times more sensitive than the original version, allowing the the safe detection of catalytic iron down to concentrations below 100 nM (10–7 mol dm–3) (see M. J. Burkitt, L. Milne, and A. Raafat, 2001).

5. Expertise and services offered by Westcott Research and Consulting
The title of Dr Burkitt's doctoral thesis was The Role of Iron in the Generation and Toxicity of Oxygen-Centred Radicals (York, 1989), following the defence of which he was awarded a Visiting Fellowship from the National Institutes of Health (USA) to undertake post-doctoral research on the Fenton reaction in the laboratory of Professor Ronald P. Mason (Laboratory of Molecular Biophysics, National Institute of Environmental Health Sciences, Research Triangle Park). In 1991, Dr Burkitt and Dr Mason reported the first direct evidence for the in vivo generation of hydroxyl radicals by the Fenton reaction. This entailed the development of a secondary EPR spin-trapping technique (see 'Spectroscopy' ) and the use of 2,2'-dipyridyl, which holds iron in the +2 oxidation state, to prove that the radicals detected could only have been generated in vivo (see M. J. Burkitt and R. P. Mason, 1991).

In the intervening years, Dr Burkitt has published several papers (listed below) in the area of metal ions in health and disease. In addition to the development of the improved version of the bleomycin assay referred to above (see also the editorial comment by Des Richardson and Roger Dean), he has made important contributions to the understanding of the basic chemistry of the Fenton reaction - most notably in discrediting erroneous claims that ferryl species, rather than the hydroxyl radical, are generated in the Fenton reaction (see, e.g. M. J. Burkitt, 1993).

Dr Burkitt has also undertaken research on the mechanistic aspects of the copper-Fenton reaction, particularly in terms of DNA damage.

Selected publications showing the scope of Dr Burkitt's experience in this area: