Oils go rancid as a result of oxidation (see Chapter 4 ‘Bonds continued’, p. 27). In the late 1800s, it was observed that – when exposed to air – a layer of walnut oil on water would absorb three times its own volume of air in 10 days. For this reason, margarine manufactures must slow or prevent this oxidation process.

Cells walls are comprised of lipids and, as hydrogen peroxide so easily permeates these membranes, peroxidation of the fatty acid part of the phospholipids in the cell wall is relatively easy, occurring in the area between the double bonds (Figure 7.4).

Figure 7.4 The action of free radicals on lipids.

Lysosomes membranes are also made up of phospholipid. When these are damaged the contents of the lysosomes are released into the cell at the wrong time.

Generally, protein is less susceptible to free radical damage than lipids. The extent to which proteins are affected by free radicals very much depends on their amino acid make up. Sulphur-containing amino acids are the most susceptible to free radical damage (or certainly attack from an activated oxygen molecule). Proteins associated with metals, e.g. haem compounds found in enzymes, are also vulnerable to free radical damage (see Chapter 37 ‘Metabolic disorders’, p. 296).

Two main reactions occur as a result of free radical damage to proteins:

Superoxide dismutases (SOD) can contain zinc and copper (Cu-Zn-SOD) and also manganese or iron. SOD converts superoxide to hydrogen peroxide and oxygen (Figure 7.5), and minimizes production of the hydroxyl radical that causes so much damage (it is the most potent of the oxygen free radicals). This system is nearly always the antioxidant defence in cells exposed to oxygen. It is extremely quick and can more than match the production of superoxide when working properly.

Figure 7.5 The action of superoxide dismutase (SOD).

There are three forms of SOD in the human body:

SOD is thought to protect from free radicals produced by the ageing process and ischaemic tissue damage. It also has an effect on inflammation.

Mutations in SOD1 have been linked to familial amyotrophic lateral sclerosis (ALS).

Catalases and peroxidases are enzymes that catalyse the decomposition of hydrogen peroxide to water and oxygen, preventing the formation of hydroxyl (•OH) radicals. Catalases react in the following way to rid the system of peroxide:

Peroxidases react differently, using a sulphur-containing compound called glutathione (see Figure 7.7):

Catalyse is present in all the major body organs. It is concentrated in the liver and erythrocytes but there is very little in the brain, heart and skeletal muscle.

Red blood cells can be very prone to damage if is there is a deficiency of glutathione as it is not possible to prevent the attack of peroxide on the plasma membrane, which results in red blood cell haemolysis.

Glutathione peroxidase is found largely in the liver and – to a much lesser extent – in the heart, lungs and brain, with very little activity in muscle. It contains selenium (see Chapter 13 ‘Vitamins and minerals’, p. 100), a dietary source of which is required for its activity.

The reason that glutathione is so favoured by nutritionists is because it acts as a cofactor (a compound that works with an enzyme) for glutathione peroxidase.

Antioxidants affect the oxidation of molecules. They oxidize but are able to remain stable. Antioxidants can therefore prevent free radicals from reacting with other compounds. They interact with the free radical mechanism at various points along the chain reaction. If the number of free radicals is kept at a low enough level, oxidation will not occur.

The type of molecule that can act as an antioxidant can effectively juggle the configuration of electrons within its structure so that the overall effect is neutrality. To be successful as an antioxidant, the molecule must:

• Flavonoids

• Are polyphenolic compounds (they all share a similar chemical structure containing two benzene rings on either side of a three-carbon ring; see Figure 21.14, p. 159).

• The capacity of flavonoids to act as antioxidants depends on their molecular structure (Figure 7.6).

• The position of hydroxyl groups and other features in the chemical structure of flavonoids is important for their antioxidant and free-radical scavenging activities. Adjacent hydroxyl groups on the B ring increase their antioxidant activity (Wen Peng 2003). Quercetin, the most abundant dietary flavonol (see Chapter 21 ‘Phenols’, p. 160), is a potent antioxidant because it has so many of the right structural features for free-radical scavenging activity.

• Flavonoids that lack the above key features do not possess antioxidant activity.

• They can prevent superoxide production.

• They can reduce free radicals.

• They are less bioavailable (the body is not as able to absorb and make them available for use) than ascorbic acid and tocopherol. The more hydroxyl groups they contain, the more hydrophilic they are and therefore the less lipid soluble.

Figure 7.6 Anatomy of an ideal flavonoid antioxidant.

Factors Increasing the Antioxidant Properties of a Flavonoid

• Large number of OH groups.

• Presence of a 2,3 double bond.

• Orthodiphenolic structure or catecholic (referring to the position of the hydroxyl groups on the B ring, which is the structure of a catechol; see Figure 5.3, p. 31).

• Reasonable fat solubility.

Factors Decreasing the Antioxidant Properties of a Flavonoid

• Glycosylation (sugar group) blocking the 3-OH group in the C ring.

• No hydroxyl group in the B ring.

• The presence of a methoxy group in the B ring (—OCH₃).

•Glutathione

• Is a tripeptide (three amino acids joined together) consisting of glutamine, cysteine and glycine. It can be made from these raw materials by the body.

• The —SH thiol (sulphydryl) group of the cysteine is the reactive part of glutathione. It reacts with another activated thiol (sulphydryl) group on another glutathione molecule and forms a disulphide bond (Figure 7.7)

• Is changed to oxidized glutathione (GSSG) as it becomes oxidized, thus removing hydrogen peroxide from the system. This bond formation keeps the glutathione stable.

• GSSG is then recycled by another enzyme.

Figure 7.7 The structures of reduced glutathione and oxidized glutathione (GSSG).

• Is found in most tissues, cells and subcellular compartments of higher plants. As a person gets older, glutathione levels decrease.

• Neutralizes singlet oxygen, superoxide and hydroxyl radicals (see Figures 7.1 and 7.2) and does not require an intermediary to act as a free radical scavenger.

• Is also responsible for regenerating ascorbate or reducing the disulphide bonds of proteins (see Figure 7.9).

• Tocopherols

• The tocopherols (see Chapter 13 ‘Vitamins and minerals’, p. 107) are a family of antioxidants.

• Alpha-tocopherol is considered to be the most active. It is a membrane stabilizer and antioxidant that scavenges oxygen free radicals, lipid peroxy radicals and singlet oxygen.

• Alpha-tocopherol has become almost synonymous with vitamin E, although technically the term ‘vitamin E’ refers to the other tocopherols as well.

• The tocopherols have an interesting structure in that part of it is lipid soluble whereas the other part works as antioxidant. It positions itself with the polar benzoquinone ring near the phosphate part of the membrane phospholipid with the rest of the molecule, which is non-polar and therefore hydrophobic, in the lipid layer (Figure 7.8).

• The amount of alpha-tocopherol is particularly high in blood lipoproteins and the adrenal glands, which are particularly susceptible to oxidative damage.

• As the hydrophilic part of the tocopherol is near the edge of the membrane, ascorbate (vitamin C) has easy access to it and can easily regenerate it (Figure 7.9; and see Chapter 13 ‘Vitamins and minerals’, p. 102).

Figure 7.8 The structure of tocopherols.

Figure 7.9 The interactions between vitamin E, vitamin C and glutathione.

• Carotenoids

• Carotenoids are tetraterpenes (see Figure 22.8, p. 174).

• There are two classes of carotenoids: carotenes and xanthophylls.

• The carotenoids are involved in photosynthesis (see Figure 9.1, p. 64).

Beta-carotene (Figure 7.10) is a more efficient scavenger of singlet oxygen than tocopherol. Not only does it have an aromatic part, it also possesses a polyunsaturated section (see Figure 10.1, p. 74). The mechanism of its antioxidant action is not properly understood. It has been demonstrated to quench singlet oxygen, scavenge peroxyl radicals and inhibit lipid peroxidation. There does not appear to be a mechanism to recycle beta-carotene.

Figure 7.10 The structure of beta-carotene.

Carotenes act by:

• reacting with lipid peroxidation products to terminate chain reactions

• scavenging singlet oxygen

• working synergistically with alpha-tocopherol to scavenge peroxy radicals.

• Other Phytochemicals Acting as Antioxidants

Eugenol (found in cloves) and carnosol and rosmanol (Figure 7.11; these terpenes are found in rosemary leaves) inhibit oxidation.

Figure 7.11 The phytochemicals that act as antioxidants.

Nitric oxide (NO) is of great interest because it is a free radical that diffuses easily through the plasma membrane, thus affecting nearby cells. It is now thought to have substantial influence on the nervous system, and it might also be responsible for the damage incurred after a stroke. NO:

Artemisinin in Artemisia annua (Qing Hao) (Figure 7.12) is a herbal example of a natural antimalarial (Klayman 1985, Olliaro et al 2001; see also Chapter 22 ‘Terpenes’, p. 171).

Figure 7.12 The antimalarial bond in artemisinin is thought to create free radicals in the malarial parasite.

In the treatment of herpes simplex, the lesions are painted with a dye that enters the infected cells, binds to the DNA or to the virus and is then illuminated. It destroys the DNA by free radical damage.

Free radical damage has been implicated in ischaemic brain disease (i.e. stroke). The brain and nervous system are particularly prone to oxidative injury because they are rich in lipids and the brain has only limited amounts of protective enzymes. Some areas of the brain are also rich in iron (e.g. the substantia nigra), which might enhance free radical action.

See Chapter 28 ‘Blood disorders’ (p. 211).