Antioxidant Chemistry

Antioxidants protect molecules from oxidative damage by free radicals. In addition to their actions in biological systems where, for example, they are believed to offer protection from some of the most important human diseases – including disorders of the cardiovascular and nervous systems – antioxidants are of considerable importance to the food and chemical industries, where they act to inhibit such processes as the rancidification of fats, the deterioration of beers (producing off-flavours) and the ageing of rubbers, paints and plastics. Indeed industrial chemists were investigating the actions of antioxidants long before their importance in biology was recognised.

Although the so-called radical-scavenging antioxidants are perhaps of greatest relevance to the food and chemical industries (described below), antioxidant behaviour can also be displayed by substances able to chelate iron and copper ions, which might otherwise catalyse the generation of oxygen-centred radicals (see 'Metal ions in Health and Disease'). Oxidative damage to proteins and DNA often involves the catalysis of hydroxyl radical (^•OH) generation by bound metal ions, with attack by the radical occurring specifically at the metal-binding site on the target molecule. Chelating agents may not always prevent the catalysis of ^•OH formation, but their removal of metals from DNA and proteins – thereby re-directing ^•OH generation to the bulk solution, away from the target molecule – is often all it takes for protection to be seen. This is because the ^•OH radical is extremely reactive and therefore has little opportunity to diffuse from its site of formation.

In living systems, consideration must also be given to the large number of antioxidant enzymes, including those of the superoxide dismutase and glutathione peroxidise families, which often act together and in concert with the radical-scavenging antioxidants in elaborate reaction sequences.

As if matters were not complicated enough, radical-scavenging antioxidants can also promote free-radical generation. Not only can this result in the stimulation of deleterious reactions in chemical and biological systems, it can lead to the misinterpretation of findings from biochemical studies in which antioxidants are used – often rather crudely – as ‘probes’ to test for the involvement of free radicals in biological processes.

Westcott Research and Consulting offers expert advice and guidance in projects involving all aspects of antioxidant chemistry. Examples of areas in which we can assist include:

Prevention of oxidative changes in foods and beverages
Stabilisation of biological fluids and media (e.g. those used in organ transplantation)
Investigation of the antioxidant (and pro-oxidant) actions of pharmacological agents
Planning, execution and interpretation of experiments to investigate the roles of antioxidants in biological systems, including those relating to disease processes

An introductory overview of this area is given below, followed by details of Dr Burkitt’s experience in the field.

1. Background: the ‘classical’ radical-scavenging antioxidants

1.1 Vitamin E
The actions of the so-called radical-scavenging antioxidants (or ‘free-radical scavengers’) are perhaps best illustrated by α-tocopherol, the main member of the E family of vitamins. α-Tocopherol protects fatty acids from lipid peroxidation by intercepting (‘scavenging’) fatty-acid derived peroxyl radicals (Lipid-OO^•), which would otherwise amplify the process by attacking nearby fatty acids in chain-propagation reactions (see 'Lipid Peroxidation'). The predominant mechanism of radical scavenging involves transfer of the phenolic hydrogen atom from α-tocopherol to the peroxyl radical, as shown below (where the phenolic hydrogen is shown in red and R represents a branched C₁₆H₃₃ chain, which serves to position the vitamin between the fatty acids of the phospholipids within biological membranes):

The radical generated from α-tocopherol in this reaction, the α-tocopheroxyl radical, is a highly-substituted phenoxyl radical. Compared with most other free radicals, phenoxyl radicals are poorly reactive; this is because the unpaired electron is delocalised over the aromatic ring. The α-tocopheroxyl radical is further stabilised by the inductive effect (+M) of its methyl substitutents and through conjugation involving the lone-pair electrons on the oxygen atom in the ring structure attached to the phenolic ring. The reactivity of the radical is also reduced by the steric effect of the methyl groups at each side of the phenolic oxygen.

1.2 Vitamin C
Whereas vitamin E is the most important radical-scavenging antioxidant in non-aqueous environments, ascorbic acid (AscH₂, vitamin C) is one of the most important water-soluble radical scavengers. In fact the ascorbate anion (AscH^–) can transfer a hydrogen atom to the α-tocopheroxyl radical across the phase boundary, thereby bringing about the 'chemical repair' of the latter, itself being oxidised to the ascorbate radical (Asc•^–):

AscH^– + α-Toc-O• → Asc•^– + α-Toc-OH

Ascorbate can also intercept reactive radicals directly in the aqueous phase; it is, for example, the main radical-scavenging antioxidant in blood. Due to the extensive delocalisation of its unpaired electron, the ascorbate radical is poorly reactive towards other molecules. Therefore the chemical repair of a radical by ascorbate (and indeed α-tocopherol) usually results in the suppression of biomolecular damage.

Ascorbate radicals undergo disproportionation, involving their simultaneous reduction and oxidation, forming the ascorbate anion and dehydroascorbate (DHA), respectively:

2 Asc•^– + H⁺ → AscH^– + DHA

Although DHA, the product of the two-electron oxidation of ascorbate, can be reduced by enzymes back to ascorbic acid, it can also undergo further oxidation to the erythroascorbate radical, which has been detected by EPR spectroscopy in model systems used to investigate the mechanisms of low-density lipoprotein ('bad cholesterol') oxidation by copper(II) ions (see E. T. Horseley et al., 2007).

1.3 Glutathione
The tripeptide glutathione (γ-glutamylcysteinylglycine or 'GSH') is a low-molecular-weight thiol compound, consisting of the amino acids glycine, cysteine and glutamic acid. GSH is present in mammalian cells at millimolar concentrations, where it serves as an important reducing agent. In addition to its role in the maintenance of protein thiols in the reduced state, GSH provides the electrons required for the two-electron reduction of hydrogen peroxide (H₂O₂) and alkyl hydroperoxides, including Lipid-OOH (see above), by the glutathione peroxidases. In doing so, GSH is oxidised to its disulphide, GSSG, e.g.:

H₂O₂ + 2 GSH → GSSG + 2 H₂O

Glutathione disulphide can be reduced back to GSH by glutathione reductase, using electrons from NADPH:

GSSG + NADPH + H⁺ → 2 GSH + NADP⁺

However in acting as a free-radical scavenger, GSH undergoes a single-electron oxidation to a thiyl radical (GS^•). In reacting with the hydroxyl radical, for example:

GSH + ^•OH → GS^• + H₂O

Although two glutathionyl radicals can dimerise to give GSSG, kinetically GS^• is more likely to react with GSH, which is usually present in excess: GS^• + GSH → GSSG•^– + H⁺

The glutathione disulphide anion radical (GSSG•^–) thus formed is a powerful reducing agent, able to reduce oxygen directly to the superoxide radical:

GSSG•^– + O₂ → GSSG + O₂•^–

Although superoxide can be removed by the superoxide dismutases (see 'Spectroscopy' ), and the resultant H₂O₂ reduced by the glutathione peroxidases (see above), this series of reactions illustrates how antioxidants can, at times, display pro-oxidant properties.

2. Pro-oxidant actions of the radical-scavenging antioxidants
All radical-scavenging antioxidants can display pro-oxidant properties. This is because they are reducing agents: when donating a hydrogen atom to a radical, they are in fact transferring an electron plus a proton (H = e^– + H⁺). α-Tocopherol and ascorbate, for example, are readily oxidised by Fe(III) and Cu(II) ions. Depending on the particular metal-ion complex concerned (see 'Metal ions in Health and Disease'), this can initiate a steady flux of superoxide (O₂•^–), hydrogen peroxide and ^•OH formation. E.g. for the Fe(III) and ascorbate combination:

Fe³⁺ + AscH^– → Fe²⁺ + Asc•^– + H⁺

Notice how in this reaction only the electron 'part' of the H atom is transferred to Fe(III): the remaining proton is released. (Compare, for example, with electron transfer to the α-tocopheroxyl radical, shown above, in which both the electron and the proton are transferred.)

Fe²⁺ + O₂ → Fe³⁺ + O₂•^–

H⁺ + O₂•^– → HO₂^•

Fe²⁺ + HO₂^• + H⁺ → Fe³⁺ + H₂O₂

Fe²⁺ + H₂O₂ → Fe³⁺ + ^•OH + OH^–

Similar reactions occur in Cu(II)/ascorbate, Cu(II)/α-tocopherol, Fe(III)/α-tocopherol and Fe(III)/GSH reaction mixtures. Matters are a little more complicated in Cu(II)/GSH reactions systems, in which the reduction of the metal ion typically proceeds with GSH being oxidised directly to the product of its two-electron oxidation, GSSG. For further information on this reaction, including that involving Cu(II) bound to DNA, see M. J. Burkitt and J. Duncan, 2000 and references therein.

When present in excess, radical-scavenging antioxidants tend to display antioxidant behaviour; this is because the ^•OH radicals generated in the above redox-cycling reactions are immediately scavenged by the remaining antioxidant. At lower [antioxidant]/[metal] ratios, however, pro-oxidant behaviour often predominates. This is reflected in the 'bell graph' typical of antioxidant dose-response curves in systems involving metal-catalysed radical generation (see, e.g.M. J. Burkitt and B. C. Gilbert 1989).

3. Expertise and services offered by Westcott Research and Consulting
Dr Burkitt has many years' experience investigating the mechanisms of action and biological effects of antioxidants, particularly their pro-oxidant properties. Contributions he has made to this field include:

Elucidation of the mechanism by which α-tocopherol can act as a pro-oxidant in the promotion of lipid peroxidation by Cu(II) ions. Previously, it had been assumed that the α-tocopheroxyl radical, generated in the oxidation of α-tocopherol by Cu(II), is responsible for the promotion of LDL ('bad cholesterol') oxidation often seen in model systems. Dr Burkitt has demonstrated that the Cu(I) formed in this reaction is in fact the culprit: the reduced metal ion reduces oxygen to the hydroxyl radical (which oxidises the lipids in LDL) in reactions analogous to those shown above for iron (see M. J. Burkitt, 2001 and M. J. Burkitt and L. Milne, 1996).
Similar reactions were also shown to underlie the ability of vitamin E to promote damage to DNA by bound Cu(II) ions (see N. Yamashita et al., 1998). See also the section on 'Lipid Peroxidation'.
Elucidation of the chemistry underlying the biological effects of dithiocarbamate compounds. As described in more detail in the section on 'Pharmacology and Toxicology', dithiocarbamates, RR'NC(S)SH, are employed widely in medicine. They are also used in cell biology and molecular biology as probes to 'test' for the involvement of free radicals in biological regulatory mechanisms (e.g. in the control of redox-sensitive transcription factors). This is because dithiocarbamates contain the thiol group (-SH): like glutathione, they are effective scavengers of hydroxyl radicals (see above). However, upon oxidation (e.g. by radicals, hydrogen peroxide or metal ions), dithiocarbamates form thiuram disulphides, RR'NCSSNR'R (analogous to GSSG, the disulphide formed from GSH), which are highly toxic to cells.
In addition to bringing about pro-oxidant conditions ('oxidative stress') via their oxidation of GSH, thiuram disulphides can form mixed disulphides with thiols on proteins, including those involved in apoptotic cell-death pathways. Put simply, dithiocarbamates affect biological process by very complex mechanisms, which cannot be assumed to involve only their scavenging of 'reactive oxygen species' (or metal chelation). See M. J. Burkitt et al. , 1998 and also 'Pharmacology and Toxicology'.
Use of EPR spin-trapping to obtain kinetics data on radical-scavenging reactions. Complex spectral analyses have enabled the rate constant for the reaction of the superoxide radical with glutathione to be obtained by EPR spin-trapping (see C. M. Jones et al., 2002 and the section on 'Spectroscopy'). The magnitude of this rate constant (ca. 200 M^–1 s^–1) is very important because it determines the extent to which the thiol is able to remove superoxide radicals, which are generated continuously by mitochondria and at elevated levels during apoptotic cell-signalling (see 'Oxidative Stress and Apoptosis').
Similarly, carefully designed spin-trapping experiments have enabled the kinetics of radical scavenging by trans-reseveratrol, the main antioxidant in red wine, to be investigated (see J. Karlsson et al., 2000).
Synergism in the actions of antioxidants. It has been suggested that trans-reseveratrol is responsible for some of the claimed health benefits of red wine consumption. Although trans-reseveratrol is known to be a powerful radical-scavenging antioxidant, when incubated with DNA in the presence of bound Cu(II) ions the polyphenol promotes hydroxyl-radical formation and oxidation of the nucleic acid (by acting as a reducing agent for the metal, as described above for α-tocopherol). In contrast, when either ascorbic acid or glutathione is included in the reaction system, trans-reseveratrol behaves as an antioxidant. In the case of ascorbic acid, this was shown to involve a simple radical-scavenging mechanism of action. However, in the case of glutathione, the polyphenol was shown to act through a novel, glutathione-sparing mechanism, which delayed the oxidation of the thiol to its disulphide, GSSG. The significance of this is that, whereas GSH forms a redox-inactive ternary complex with Cu(I) ions and DNA (unable to produce hydroxyl radicals), GSSG forms a complex with the metal that is able to participate in redox-cycling reactions, thereby promoting ^•OH formation and DNA damage (see M. J. Burkitt and J. Duncan, 2000).

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

Horsley, E.T., Burkitt, M.J., Jones, C.M., Patterson, R.A., Harris, L.K., Moss, _N.J., del Rio, J.D. and Leake, D.S. (2007) Mechanism of the antioxidant to pro-oxidant switch in the behavior of dehydroascorbate during LDL oxidation by copper(II) ions. Archives of Biochemistry and Biophysics 465, 303-314. [Article]
Jones, C.M., Lawrence, A., Wardman, P. and Burkitt, M.J. (2003) Kinetics of superoxide scavenging by glutathione: an evaluation of its role in the removal of mitochondrial superoxide. Biochemical Society Transactions 31, 1337-1339. [Article]
Jones, C.M., Lawrence, A., Wardman, P. and Burkitt, M.J. (2002) Electron paramagnetic resonance spin trapping investigation into the kinetics of glutathione oxidation by the superoxide radical: re-evaluation of the rate constant. Free Radical Biology & Medicine 32, 982-990. [Article]
Burkitt, M.J. (2001) A critical overview of the chemistry of copper-dependent low density lipoprotein oxidation: roles of lipid hydroperoxides, α-tocopherol, thiols, and ceruloplasmin. Archives of Biochemistry and Biophysics 394, 117-135. [Article]
Burkitt, M. (2001) Too much of a good thing? Nature Biotechnology 19, 811. [Article]
Burkitt, M.J. and Duncan, J. (2000) Effects of trans-resveratrol on copper-dependent hydroxyl-radical formation and DNA damage: evidence for hydroxyl-radical scavenging and a novel, glutathione-sparing mechanism of action. Archives of Biochemistry and Biophysics 381, 253-263. [Article]
Karlsson, J., Emgård, M., Brundin, P. and Burkitt, M.J. (2000) trans-Resveratrol protects embryonic mesencephalic cells from tert-butyl hydroperoxide: electron paramagnetic resonance spin trapping evidence for a radical scavenging mechanism. Journal of Neurochemistry 75, 141-150. [Article]
Yamashita, N., Murata, M., Inoue, S., Burkitt, M.J., Milne, L. and Kawanishi, S. (1998) α-Tocopherol induces oxidative damage to DNA in the presence of copper(II) ions. Chemical Research in Toxicology 11, 855-862. [Article]
Burkitt, M.J. and Milne, L. (1996) Hydroxyl radical formation from Cu(II)-Trolox mixtures: insights into the pro-oxidant properties of α-tocopherol. FEBS Letters 379, 51-54. [Article]
Milne, L., Nicotera, P., Orrenius, S. and Burkitt, M.J. (1993) Effects of glutathione and chelating agents on copper-mediated DNA oxidation: pro-oxidant and antioxidant properties of glutathione. Archives of Biochemistry and Biophysics 304, 102-109. [Article]
Burkitt, M.J. and Gilbert, B.C. (1990) Model studies of the iron-catalysed Haber-Weiss cycle and the ascorbate-driven Fenton reaction. Free Radical Research Communications 10, 265-280. [Article]
Burkitt, M.J. and Gilbert, B.C. (1989) The control of iron-induced oxidative damage in isolated rat-liver mitochondria by respiration state and ascorbate. Free Radical Research Communications 5, 333-344. [Article]