Introduction to Free radicals and Antioxidants

Free radical:

A free radical is any species capable of independent existence (hence the term ‘free’) that contains one or more unpaired electrons.

Free radicals can be generated in a wide variety of chemical and biological systems, including the formation of plastics, the ageing of paints, the combustion of fuels and in the human body.

A dot is always used to denote free radicals. The simplest free radical is an atom of the element hydrogen, with one proton and a single electron. Examples of free radicals include- Superoxide anion (O₂•^-), Hydroxyl radical (OH•), Thiyl radicals (RS•).

Reactive oxygen species:

The term reactive oxygen species (ROS), often used in the biomedical free radical literature, is a collective term that includes not only oxygen-centered radicals such as O₂•^- and OH•, but also some non-radical derivatives of oxygen, such as hydrogen peroxide (H₂O₂), singlet oxygen ¹D_g, and hypochlorous acid (HOCl).

Antioxidant:

Antioxidants are chemicals that interact with and neutralize free radicals, thus preventing them from causing damage. Antioxidants are also known as “free radical scavengers". Some antioxidants are made naturally by the body. Others can only be obtained from external (exogenous) sources, including the diet and dietary supplements.

Antioxidant defense:

The free radicals and other activated oxygen species are continuously formed in our body and on top of their physiological function they may also be damaging to the cellular integrity due to its high reactivity. They react with all the present biomolecules and they affect their normal function. Thus living organisms have developed a number of defense mechanisms known as the “antioxidant defense system”.

The action of these systems is multifactorial. In the instance, they try to prevent the production of reactive oxygen species. On a second level, they try to reduce these molecules, and on a third level they repair the damage caused by molecules.

Mechanism of action of antioxidant defense system / How antioxidant defense system works in body:

These defence mechanisms may be organised in the following way:

1.      Non-enzymic system: Molecules that can react directly with reactive oxygen species and other free radicals, or with the products of these reactions without the involvement of any special enzyme.  These antioxidants include glutathione, vitamin C, vitamin E, beta carotenes, uric acid and the flavonoids.

2.      Enzymes: These include catalase, superoxide dismutases and glutathione peroxidases.

Actually the antioxidant defense mechanism is a complex process and includes both endogenous and diet-derived molecules. For example-

1.      Superoxide dismutase enzymes (SODs) remove O₂•^- by accelerating its conversion to H₂O₂.
2. Catalase enzymes convert H₂O₂ to water and O₂.
   
   
3. But more important H₂O₂ removing enzymes in human cells are the glutathione peroxidases
   
   
4. (GSHPX), one of the few classes of human enzymes that require selenium for their action. 
   
   
5. GSHPX enzymes remove H₂O₂ by using it to oxidize reduced glutathione (GSH) to oxidized glutathione (GSSG).
   
   
6. Again, GSH can scavenge various reactive species (e.g. HOCl and ONOO^-) directly, as well as being a substrate for GSHPX enzymes.
   
   
7. α- Tocopherol is the most important free radical scavenger within membranes. It can inhibit lipid peroxidation by scavenging peroxyl radical intermediates and so halting the chain reaction.

Oxidative stress

Oxidative stress may be defined as an imbalance between pro-oxidant and antioxidant agents, in favor of the former. This imbalance may be due to an excess of pro-oxidant agents, a deficiency of antioxidant agents or both factors simultaneously.  The origin of oxidative stress is an alteration of the redox status in cells, leading to a cellular response to counteract the oxidizing action.

In principle oxidative stress can result from:

1.      Diminished antioxidants, e.g. mutations affecting antioxidant defense enzymes (such as CuZnSOD, MnSOD and GSHPX) or toxic agents that deplete such defenses.

-        For example many xenobiotics are metabolized by conjugation with GSH; high doses can deplete GSH and cause oxidative stress even if the xenobiotic is not itself a generator of reactive species.

2.      Increased production of ROS/RNS, e.g. by exposure to elevated levels of toxins that are themselves reactive species (e.g. nitrogen dioxide gas, NO₂•) or are metabolized to generate such species or by excessive activation of natural ROS/RNS- producing systems.

How reactive species are generated in the body:

Free radicals and other reactive species are constantly generated in the human body.

1. Some are made by ‘accidents of chemistry’

-        For example- leakage of electrons directly on to O₂ from intermediate electron carriers.

2. Exposure of living organisms to ionizing radiations splits the O-H bonds in water to generate OH• and H•.

3. The OH• reacts at a diffusion controlled rate with almost all molecules in living cells. Hence, when OH• is formed in vivo, it damages whatever it is generated next to the cell. Indeed, the harmful effects of excess exposure to ionizing radiation on living organisms are thought often to be initiated by attack of OH• on proteins, DNA and lipids.

4. NO• is synthesized from the amino acid L-arginine by vascular endothelial cells, phagocytes and many other cell types.

-        It helps to regulate blood pressure and may be involved in the killing of parasites by macrophages.

5. Superoxide radical (O₂•^-) is produced by phagocytic cells.

-        It helps phagocytes to kill bacteria.

6. H₂O₂ can be produced by the action of several oxidase enzymes in cell, including amino acid oxidases and xanthine oxidase.

-        H₂O₂ is used by the enzyme thyroid peroxidase to help make thyroid hormones.

-        H₂O₂ is sometimes used as an intracellular signal molecule.

-        H₂O₂ can inhibit protein phosphatases and so increase net protein phosphorylation.

-        H₂O₂ can combine with iron or copper ions to generate highly reactive OH•.

       H₂O₂ + Fe²⁺ (or Cu⁺)                   OH• + OH^- + Fe³⁺ (Cu²⁺)