Defining Free Radicals Antioxidants and Related Terms

The terms "reactive oxygen species" (ROS) and "reactive nitrogen species" (RNS) describe both radicals and nonradical reactive oxygen and nitrogen-containing molecules. These molecules can enter reactions that can result in production of free radicals or directly damage organic biochemical substrates. The nomenclature used within the field of free radical biology has not been established by any one organization. However, there is a consensus within the field from several sources, including the International Union of Pure and Applied Chemistry (IUPAC),10 the Commission on the Nomenclature of Inorganic Chemistry,11 and by recommendations of authors in the field.12 Current convention denotes a free radical in text by a superscript dot to the right preceding any charge that the molecule may have, for example H*, HO, NO, O2"-, CO/-, and H3C. O22* denotes the radical notation for dioxy-gen in its ground state or, in other words, the lowest allowed energy state for this molecule. A molecule is capable of being highly reactive even if it

Figure 3.1

Basic Radical Generation and Sequestration Reactions

Haber-Weiss Rxn (+H2O2)

Mitochondrial Cycloxygena Lipoxygena NADPH Oxidas

Figure 3.1

Basic Radical Generation and Sequestration Reactions

Haber-Weiss Rxn (+H2O2)

Mitochondrial Cycloxygena Lipoxygena NADPH Oxidas




Glutathione disulfide

Nitric oxide



Nitric oxide




Glutathione disulfide



Oxygen enters the biological system and is used in the mitochondria in the creation of ATP. It is also used by various other oxidase enzymes in the cell during normal metabolism. A byproduct of these processes is the superoxide radical that must be dealt with by superoxide dismutase and catalase to convert it to harmless water and oxygen before it does damage. If superoxide reacts with either nitric oxide (another radical) or hydrogen peroxide peroxynitrite, the hydroxyl radicals are produced (two very reactive and dangerous radicals). When superoxide dismutase converts superoxide to hydrogen peroxide in biological systems, there are four possible fates for the hydrogen peroxide. If either the catalase or the glutathione peroxidase enzymes deal with it, hydrogen peroxide is converted safely to harmless products. However, when hydrogen peroxide enters either a Haber-Weiss reaction with superoxide or the Fenton reaction, which unsequesters metals, dangerous hydroxyl radicals can be produced that can cause biological damage.

is not technically a radical (if its electrons are paired). Common biologically relevant oxygen and nitrogen radicals are listed in Table 3.1, together with other highly reactive nonradical biologically important oxygen and nitrogen molecules.

Beyond the simple small molecular weight radicals, biological reactions can produce many other radicals from biomolecules. For example, phenolic and other aromatic radical molecules as well as superoxide radicals are often formed during xenobiotic metabolism as part of natural detoxification mechanisms depicted in Figure 3.2. The term xenobiotic was coined to cover all organic compounds that were foreign to the organism under study. Pesticides are a common example of a xenobiotic compound.

Table 3.1

Common Biological Radical and Non-Radical Species

Reactive Oxygen Species (ROS)

Common Name

Radical Depiction

Systemic Name


Radical anion of HO*


oxide(' 1 —)



dioxide( • 1 —)

O2 •+

dioxygen( • 1 +)

oxygen, written as O2


dioxygen (triplet)



trioxide( • 1 —)





HO2 *

hydridodioxygen( •)

hydrogen trioxide radical


hydridotrioxygen( •)

carbon dioxide radical anion CO2*

dioxidocarbonate (• 1 —)

carbonate radical

CO3 -

trioxidocarbonate( • 1 —)


hydroxidooxidocarbon( •)


hydroxidodioxidocarbon( •)

Peroxyl Radical

RO2 *

Alkoxyl Radical Water

RO *




Hydrogen Peroxide




Singlet Oxygen

1 Ag

Reactive Nitrogen Species (RNS)


Nitric Oxide

NO *

oxidonitrogen (•)


NO(2 ')—

oxidonitrate(2• 1-) (triplet)

Nitrogen dioxide



nitrogen trioxide


trioxidonitrogen( •)


dioxidonitrate( • 2—)


trioxidonitrate( • 2 —)



(dioxido)oxidonitrogen( •)

HON2 *

hydroxidonitrogen(2 •)



bis(oxidonitrate)(N — N)( • 1 —)

N2O -

oxidodinitrate( • 1 —)

azidyl radical

N3 •

trinitrogen(2N — N)(>)


Nitrous acid


Dinitrogen tetroxide


Dinitrogen trioxide




Peroxynitrous acid


Nitonium cation


Alkyl peroxynitrites


Table 3.1 (continued)

Common Name

Radical Depiction

Systemic Name

Other Reactive Species




hypochlorous acid






hypobromous acid






hypoiodous acid






hypothiocyanous acid



Figure 3.2

Xenobiotic Metabolism


Drugs, Toxins, Foreign Compounds


Exposes or Adds a functional polar group to increase hydrophilicity.

Free Radicals

May result in metabolic activation


Conjugation with a small hydrophilic endogenous substance


Frequently to a product group Urine and Bile

From a Phase I reaction. Hydrophilicity Increase Again.

Metabolic Activation


Disease, Inhibition, Induction, Genetic Variability


Cytochromes P450 Carboxyl Esterases Hydrolases Epoxide Hydrolase Alcohol Dehydrogenase Aldehyde Dehydrogenase Flavin monooxygenases


Glutathione -S-transferases




UDP glucuronosyl transferases

When a foreign molecule that has the potential to be toxic to the body is taken in, xenobiotic metabolism modifies the molecule using a variety of enzymes that expose or add polar groups to it and mark it for removal. Making a molecule more polar makes it more hydrophilic and thus easier to remove from the body. This process sometimes produces free radicals or radicals of the xenobiotic, both of which can cause potential damage. Such "activated molecules have also been implicated in cancer etiology. In a second phase of detoxification other enzymes add thiol, methyl, sulfur, acetyl, or glucuronyl groups to detoxify Phase 1 metabolites and render them even more hydrophilic for removal. The endogenous antioxidant glutathione is important to both radical quenching after Phase 1 and to Phase 2 enzymatic processes.

Radical molecules may also combine together or with other reactive molecules generating yet more damaging radicals. An example is the reaction between superoxide and nitric oxide that produces peroxynitrite (ONOO*), a highly reactive and dangerous radical. Under certain conditions of hypoxia (decreased oxygen to cell tissues that can occur, for instance, in anemia or stroke), the enzyme xanthine oxidase can simultaneously create both these radicals in close proximity in tissues and thus intensify damage done to biological tissues.13

Not all ROS and RNS are true radicals because some have paired electrons. To be a radical, a molecule must have unpaired electrons. The most common ROS and RNS (radical and otherwise) are listed in Table 3.1. Numerous in vitro studies have shown that ROS and RNS can easily react with many other biomolecules, causing dysfunction of the integral components of the cell. In fact, much experimental data exists to indicate that lipid peroxidation, protein oxidation, and oxidative alterations to nucleic acids mediated by ROS and RNS are crucial components of the damaging actions of these compounds. Many studies show that dietary antioxidants, which include vitamin C (ascorbic acid), vitamin E (a-tocopherol), p-carotene (a carotenoid), and flavonoids (a subgroup of the phytochemicals), when used within experimental in vitro biological systems, act as effective antioxidants protecting plasma components, including lipoproteins, and cells from damage.14

So what exactly are antioxidants? Chemists define an antioxidant as "any substance that, when present in low concentrations compared to that of an oxidizable substrate, significantly delays or inhibits the oxidation of that substrate."15 Thoughtful consideration of this definition leads to the inevitable conclusion that an enormous number of biological compounds can act as an-tioxidants. All compounds or molecules can be ranked on the basis of their "oxidation potential." This ranking is based on the electrochemical potential at which the transfer of an electron will occur and the compound will be oxidized. As a general rule, any molecule with a higher oxidation potential in comparison to a lower oxidation potential molecule can oxidize that molecule. Lipid peroxidation (damage to polyunsaturated fatty acids [PUFA]) usually occurs by an initial reaction with highly reactive radicals. In this case, let's consider the hydroxyl radical that has an oxidation potential at pH 7.0 of 2310 mV, a physical measure of the energy required to remove yet another electron for this molecule. In order for the hydroxyl radical to stabilize itself, it needs to take an electron from any molecule that has an oxidation potential lower than 2310 mV. The lower the oxidation potential of the other molecule, the more easily the hydroxyl radical can take an electron. The hydrogen atom from the bis-allylic position on PUFAs (the double bonds PUFAs can have in their molecular structure which make them more susceptible to free radical attacks and oxidation) oxidizes at 600 mV. This is much lower than that of the hydroxyl radical's oxidation potential. This means that the reaction between the PUFA and the hydroxyl radical will occur easily, stabilizing the hydroxyl radical by acquiring an electron and "reducing" the hydroxyl radical to water so it is no longer harmful. But damage has already been done to the PUFA, turning it into a pentadienyl radical (PUFA). The PUFA radical can now react with oxygen to form a new unstable radical called peroxyl radical within the PUFA molecule (ROO*). This new peroxyl radical reacts easily with other PUFA molecules and damage to lipids continues in a chain reaction progression much like a fire out of control damaging many other PUFA molecules in the process. This chain reaction process is driven by the continued production of new peroxyl radicals as long as there is lipid to oxidize, or until another molecule with a lower oxidation potential than PUFA is introduced into the system. Vitamin E (a-tocopherol) has an oxidation potential of 500 mV (100 mV lower than that of PUFA's) and can also react with the peroxyl radical, thus reducing it and preventing it from producing more radicals in the chain reaction. Vitamin E can also react with hydroxyl radicals that initially start the chain reaction and thus can prevent the first steps in oxidizing PUFAs. Therefore, vitamin E is both a preventive and a chain-breaking antioxidant. But there is one caveat: when vitamin E is oxidized it yields its electron regardless of the radical it reduced. The a-tocopherol molecule left has an unpaired electron and now becomes a radical itself. Fortunately, in biological systems, the a-tocopherol radical is a relatively stable radical. However, in its radical form it is no longer capable of protecting lipids from oxidation by other radicals. Vitamin C (ascorbate) has an oxidation potential of 282 mV and is capable of reacting with the a-tocopherol radical and thereby restoring the original "reduced" vitamin E and effectively "recycling" vitamin E. By now you should know what comes next. Vitamin C, by reducing vitamin E, now becomes a radical known as the ascorbate radical. If this appears to be a "pecking order," that's because it is. In the rank order of biological molecules based on their oxidation potential, each molecule higher on the list can potentially oxidize any molecule lower on the list. An example of this is shown in Table 3.2. Although this concept has always been intuitive to redox chemists, it has only been taught to students and scientists in free radical biology and biochemistry since its introduction by G.R. Buettner in 1993.16

Among the low-molecular-weight antioxidants in biological systems, vitamin C (ascorbate) appears to be a necessary component for the life functions of both higher plants and animals. Many animals possess the ability to synthesize vitamin C, but humans do not. Thus, in humans, vitamin C is an essential nutrient. The same holds true for vitamin E. Vitamin C's presence at what is considered a high physiological concentration in the millimolar range in many if not all cells of the body hints at its critical nature. Surprisingly, although it has been long known that vitamin C was present in plants since 1933, only recently has progress been made in understanding the biosynthetic pathways.17 Another important small molecular weight antioxidant in plants, animals, and humans is the molecule glutathione (GSH). This ever-present tripeptide L-glutathione (GSH or gamma-glutamyl-cystemyl-glydne) is a well-known biological antioxidant and theorized to be the primary intracellular antioxidant for higher organisms. When oxidized, it forms oxidized glutathione (GSSG), which

Table 3.2

The Pecking Order Based on Oxidation Potential

Table 3.2

The Pecking Order Based on Oxidation Potential

Redox Couple One Electron Reductions


HO*, H+/H20


RO*, H+/ROH (aliphatic alkoxyl radical)

+ 1600

ROO*, H+/ROOH (alkyl peroxyl radical)

+ 1000

GS*/GS" (glutathione)


PUFA*, H-/PUFA-H (bis-alkylic-H)


TO *, H+/TOH (vitamin E)


h2o2, h+/h2o, HO *


AscH+/AscH- (vitamin C)


Coenzyme Q*2H+/Coenzyme QH2


Iron (III) EDTA/ Iron (II) EDTA

+ 120

Coenzyme Q/Coenzyme Q*-


O2 /O2




H2O/ e- aq


Note: The hydroxyl radical is reduced by the PUFA or the vitamin E below it in the table. In turn, the resulting vitamin E radical is reduced by ascorbate below it in the table and in turn the ascorbate radical is reduced by reduced glutathione (GSH). Adapted from Buettner (1993).18

Note: The hydroxyl radical is reduced by the PUFA or the vitamin E below it in the table. In turn, the resulting vitamin E radical is reduced by ascorbate below it in the table and in turn the ascorbate radical is reduced by reduced glutathione (GSH). Adapted from Buettner (1993).18

is easily recycled in any living cell that produces the enzyme glutathione reductase. GSH passes easily through cell membranes19 and GSH functions directly or indirectly in many important biological enzymatic and metabolic processes, including the synthesis of proteins and DNA, as well as protecting cells from free-radical mediated damage. GSH is critical in cellular maintenance of a proper oxidation state within the body.20 GSH is synthesized by most cells and also supplied in the diet.

Approximately 7—8 grams of reduced GSH produced in the human body daily is generated from GSSG primarily by the liver, and to a smaller extent, by the skeletal muscle and blood cells. The remaining 1 or 2 grams found in the body is acquired from dietary sources. A deficiency of GSH in cells can quickly lead to excess free radicals in the cell that can lead to oxidative stress. However, inadequate levels of GSH can also lead to the accumulation of toxins as a result of inadequate Phase 2 metabolism. These two consequences will ultimately lead to cell death. With an oxidation potential of 80 mV, GSH can reduce most of the dangerous radicals produced in biological systems as well as recycle both vitamin E and vitamin C.

Case-controlled studies have adequately demonstrated that biomarkers of oxidative damage in humans increase with age. Age-related disorders and diseases, such as Alzheimer's, neurodegenerative diseases, cancer, autoimmune diseases, rheumatoid arthritis, diabetes, and cardiovascular disease, with radical involvement are hypothesized to be the end result.21

Despite clear evidence of increased oxidative damage with age and age-related diseases, it still has not been proven in vivo that pretreatment with antioxidants will prevent tissue damage. To make matters worse, some studies have even produced data that antioxidant treatment not only failed to restore pathologies where ROS were purported to play a causal role, but appeared to even make injury worse.22 Furthermore, molecular studies are demonstrating crucial roles for ROS in signaling mechanisms both within and between cells that are necessary to maintain normal function.23 As a result, a number of scientists have come to the conclusion that the excessive production of ROS is actually the consequence, rather than the root cause, of health disorders and diseases.24 Concepts currently evolving in the field of radical biology and medicine theorize that free radicals have become vital in the functioning of every air-living organism because of the way life evolved on earth. However, if systems that have evolved to control radicals are compromised or become dysfunctional, uncontrolled radicals can cause damage to critical biomolecules necessary for life. If this damage is not repaired, organ function can eventually be impaired leading inevitably to disease, aging, and death.

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