Part Two: Free Radical Reaction in Biological Systems

The presence of free radicals in biological materials was discovered more than 50 years ago

{Ref: Commoner B, Townsend J, Pake GE, Nature, 1954, 174:689–691}. Soon thereafter,

Denham Harman hypothesized that oxygen radicals may be formed as by-products of

enzymic reactions in vivo, implicating them as the cause of degenerative process of

biological aging {Ref: Harman D, 1956, J. Gerontol 11:298–300}. The subject of free radicals

in biology has since developed into an interdisciplinary study of compelling public interest.

In a recent review of the subject, Dormandy notes that “in living systems, enzymic and free

radical processes are inextricably linked. On the one hand enzymes can both beget and

destroy free radicals; on the other, free radicals can both stimulate and destroy enzymes”

(Ref: Dormandy TL, Ann R Coll Surg Engl.,1980, 62:188-194}.

Enzymes as we all know constitute a perfect homoeostatic system, maintaining a balance

between energy-producing and energy-consuming pathways, most tellingly represented by

interlocking cycles. Based on our experience with chemical systems, we may be tempted to

conclude that free radical generation along normal metabolic pathways would be hugely

unlikely as a biological phenomenon, since such in-vivo free radical activity would inevitably

be non-homoeostatic, non-cyclic, irreversible and wasteful in terms of energy. However,

there is now much accumulated experimental evidence that free radicals, especially

reactive oxygen species (ROS; previously described in Part One) as well as some reactive

nitrogen species (RNS), in particular nitric oxide (·NO) and peroxynitrite (ONOO−), are part

and parcel of aerobic life and actively participate in phagocytosis, inflammation,

and apoptosis.

Several external sources of free radicals and oxidants are known. They include pollutants,

cigarette smoke, radiation, medication (especially drugs such as antibiotics that depend on

quinoid groups or metals for their activity, and antineoplastic agents), etc. Some

endogenous sources of free radicals are mitochondria, phagocytes, cellular oxidase

systems such as NADPH oxidase, xanthine oxidase and peroxidases, enzymatic reactions

associated with prostaglandin synthesis and the cytochrome P450, microsomes,

peroxisomes, exercise, inflammation, etc. {Ref. Halliwell, B. & Gutteridge, J.M.C. (1999):

“Free Radical in Biology and Medicine”, Oxford Science Publications, New York}.

Consider, by way of example, the NADPH oxidase system. The NADPH oxidase complex is a

cluster of plasma membrane–associated enzymes that donate an electron from NADPH to

molecular oxygen to produce superoxide.

2O2 + NADPH 2 O2- + NADP+ + H+

It is a complex enzyme consisting of two membrane-bound components and three

components in the cytosol, plus the signalling proteins Rac1 or Rac2 that are members of

the Rac subfamily of the Rho family of GTPases. Under normal circumstances, the enzyme

complex is latent in neutrophils of the immune system, and is activated to assemble in the

membranes during respiratory burst.

For a long time, superoxide generation by an NADPH oxidase was considered an anomaly

only found in professional phagocytes. In recent years, six homologs of the cytochrome

subunit of the phagocyte NADPH oxidase have been found: NOX1, NOX3, NOX4, NOX5,

DUOX1, and DUOX2. These homologs along with the phagocyte NADPH oxidase itself

(NOX2/gp91phox) are now referred to as the NOX family of NADPH oxidases. These

enzymes share the capacity to transport electrons across the plasma membrane and to

generate superoxide and other downstream reactive oxygen species (ROS). The enzyme

complex has been recognized as an important source of ROS in vascular cells. The

generation of ROS radicals is prompted in response to receptor agonists such as growth

factors or inflammatory cytokines that signal through the Rho-like small GTPases Rac1 or

Rac2.

The pathway by which reactive nitrogen species (RNS) are formed in biological tissues is an

oxidative process in which short-lived nitric oxide (·NO) is derived from the guanidino

nitrogen in the conversion of L-arginine to L-citrulline. This reaction is catalysed by NO

synthase and involves oxygen uptake like the “respiratory burst”. Depending on the

microenvironment, ·NO can be converted to various other reactive nitrogen species such as

nitrosonium cation (NO+), nitroxyl anion (NO−) or peroxynitrite (ONOO−). ·NO is a free

radical signal-transducing agent and is present in a variety of cell types, including vascular

endothelial cells, smooth muscle cells, platelets, neuronal cells, macrophages, and

neutrophils.

Free-radical reactions are intrinsic to a majority of the metabolic and synthetic reactions

carried out by eukaryotic cells and, as such, are required for life. ATP production in

mitochodria, for example, involves the generation of ROS at the cristae of inner

mitochondrial membrane during the final and most important step of cellular respiration,

the electron transport chain (ETC).

Electron transport chain (ETC)

The ETC describes the sequential flow of electrons through an enzymatic series of electron

donors and acceptors via oxidation-reduction reactions, with each acceptor protein along

the chain having a greater reduction potential than the previous. Oxygen acts as the

terminal electron acceptor within the ETC. Passage of electrons between donor and

acceptor releases energy which is used to actively pump protons into the inter-membrane

space from the mitochondrial matrix. This generates a proton electrochemical-potential

gradient across the inner membrane. This proton gradient is discharged with the protons

moving back to the mitochondrial matrix via protein channels called ATP synthase located in

the inner membrane and used to power ATP synthesis from adenosine diphosphate (ADP)

and inorganic phosphate (Pi). A somewhat simplified illustration of ETC is shown below.

In normal conditions in ECT, the oxygen is reduced to produce water. The literature

suggests that anywhere from 2 to 5% of the total oxygen intake during both rest and

exercise is prematurely and incompletely reduced to give the superoxide radical (·O2- ) via

electron escape. Electrons appear to escape from the ETC at the ubiquinone-cytochrome c

level. Superoxide is not particularly reactive by itself, but can inactivate specific enzymes or

initiate lipid peroxidation in its protonated form, hydroperoxyl HO2..

Eicosanoids

Free radicals have been shown to mediate the biosynthesis of a number of important

oxygenated metabolites, among them the ubiquitous eicosanoids which are critical to the

regulatory function of cells. Eicosanoids are not stored within cells, but are synthesized as

required when a cell is activated by mechanical trauma, cytokines, growth factors or other

stimuli. The eicosanoids comprise several compounds, which include prostaglandins,

thromboxanes and leukotrienes; most are produced by the oxidation of arachidonic acid, a

20-carbon polyunsaturated fatty acid (all-cis-5,8,11,14-eicosatetraenoic acid), catalysed by

(Source: Prabha Balaram, personal communication)

PGG2/H2 synthase (sometimes referred to as cyclooxygenase, COX). The structure of

arachidonic acid (20:4, ω-6) is shown below.

Arachidonic acid

Similarly, the oxidation of small molecules by molecular oxygen catalysed by the heme-

containing cytochrome P450 enzyme family is also thought to involve intermediary free

radicals.

Prostaglandins, which are found throughout the body, are known to act like ‘local

hormones’. For example, administration of remarkably small doses of some prostaglandins

stimulates uterine contractions and can cause abortion. Imbalances in prostaglandins can

lead to nausea, diarrhoea, inflammation, pain, fever, menstrual disorders, asthma, ulcers,

hypertension, drowsiness, or blood clots. The most common prostaglandins are PGE1, PGE2,

PGF1α and PGF2α [ PG means prostaglandin, E means the keto alcohol and F means the diol,

the subscript numbers refer to the number of double bonds and α refers to the

configuration of the –OH at carbon 9 (cis to the carboxyl side chain)].

The biological oxidation leading to PGE2 from arachidonic acid, catalysed by COX is

illustrated below. The enzyme contains two active sites: a cyclooxygenase site, where

arachidonic acid is converted into the hydroperoxy endoperoxide prostaglandin G2 (PGG2),

and a heme with peroxidase activity, responsible for the reduction of PGG2 to PGH2. The

reaction proceeds through H atom abstraction from arachidonic acid by a tyrosine radical

generated by the peroxidase active site. Two O2 molecules then react with the

arachidonic acid radical, yielding PGG2.

It has been suggested that the signal molecule ·NO may also initiate prostaglandin synthesis

by reacting with superoxide anion to produce peroxynitrite, which oxidizes the heme iron.

The oxidized heme then accepts an electron from a nearby tyrosine residue (Tyr385).

The resulting tyrosine radical extracts an H atom from arachidonate at the doubly allylic

carbon 13 to yield a carbon-centred radical. Oxygen adds to carbons 9 and 11 and the

cyclopentane ring is formed. Further oxidation leads to the hydroperoxy endoperoxide

intermediate, Prostaglandin G2 (PGG2). This is quickly metabolized by the peroxidase activity

intrinsic to COX to give PGH2. Prostaglandin-H2 E-isomerase catalyzes the conversion of

PGH2 to PGE2, while Endoperoxide reductase converts PGH2 to PGF2α. The latter is also

obtained by the action of the enzyme Prostaglandin-E2 9-reductase on PGE2.

(Source: en.wikipedia.org/wiki/Cyclooxygenase)

Oxidative Stress

When produced in excess, free radicals and oxidants generate a phenomenon called

oxidative stress, a deleterious process that can damage all major cellular constituents and

thus contribute to the pathogenesis of diseases such as cancer, diabetes mellitus,

atherosclerosis, neurodegenerative diseases, rheumatoid arthritis, ischemia/reperfusion

injury and obstructive sleep apnoea. Oxidative stress can arise when cells cannot adequately

destroy the excess of free radicals formed. In other words, oxidative stress results from an

imbalance between formation and neutralization of ROS and RNS.

Redox signalling

At low or moderate concentrations, however, ROS and RNS exert beneficial effects on cellular responses and immune function. Their role as regulatory mediators in signalling processes has been well evidenced {Ref: Wulf Dröge, Physiol Rev, 2002, 82:47-95}.

This process wherein they act as cellular messengers is dubbed redox signalling as the signal

is delivered through redox chemistry. As described by Dröge, “Redox signalling is used by a

wide range of organisms, including bacteria, to induce protective responses against

oxidative damage and to reset the original state of ‘redox homeostasis’ after temporary

exposure to ROS. Many of the ROS-mediated responses actually protect the cells against

oxidative stress and re-establish redox homeostasis. Higher organisms, however, have

evolved the use of ·NO and ROS also as redox signalling molecules for other physiological

functions. These include regulation of vascular tone, monitoring of oxygen tension in the

control of ventilation and erythropoietin production, and signal transduction from

membrane receptors in various physiological processes”.

ROS normally also participate in a number of other important cellular processes besides cell

signalling. These include gene expression, cellular death and senescence, regulation of

growth, oxygen sensing, activation of matrix metalloproteinases, and angiogenesis.

Generation of reactive oxygen species (ROS) by NOX-based NADPH oxidases activate redox-

dependent signalling pathways and contribute to development of oxidative stress in

vascular disease. Aberrant redox signalling can contribute to the pathogenesis of vascular

disease by altering endothelial cell function, enhancing vascular smooth muscle cell growth

and proliferation, stimulating expression of pro-inflammatory genes, and modulating

reconstruction of extracellular matrix. High blood pressure is in part determined by elevated

total peripheral vascular resistance, which is ascribed to dysregulation of vasomotor

function and structural remodelling of blood vessels. {Ref: Lee MY and Griendling KK,

Antioxid Redox Signal. 2008, 10:1045–1059}.

A situation where cells may be induced to produce excessive ROS is that caused by

abnormal environments such as hypoxia or hyperoxia. Yet another is infection by

microorganisms such as viruses and bacteria which causes the immune system to mount in

host defence what is literally a free radical attack on the invading pathogens. The free

radicals are primarily produced by neutrophils which comprise the bulk of the immune

system’s white cells (leukocytes). These engulf and kill the microorganisms

via phagocytosis.

What are these free radicals? Given that neutrophil mitochondria hardly participate in

ATP synthesis and the neutrophils largely depend on glycolysis for their energy, how are

these free radicals formed?

Two types of free radicals are formed in neutrophils. The first is the reactive oxygen

intermediates formed by the activity of nicotinamide adenine dinucleotide phosphate

oxidase (NADPH oxidase), the enzyme of the “respiratory burst”. The second type includes

reactive nitrogen intermediates, the first member of them, nitric oxide being produced by

nitric oxide synthase. Upon activation neutrophils have increased oxygen consumption, a

process known as the respiratory burst. During this stage, oxygen is univalently reduced

by NADPH oxidase to superoxide anion or its protonated form, perhydroxyl radical, which

then is catalytically converted by action of superoxide dismutase to hydrogen peroxide:

O2 + e- + H+ → O2H = O2

- + H+

O2- + O2

- + 2H+ → O2 + H2O2

Hydroxyl radical (·OH), the most reactive free radical in vivo, is formed by several ways,

among which the decomposition of H2O2 catalyzed by Fe2+ is the most important. This

reaction is known as the Fenton reaction.

Fe2+ + H2O2 → Fe3+ + OH- + ·OH

The pathway by which nitric oxide radical (·NO) and other reactive nitrogen species are

formed has been described earlier. Peroxynitrite (ONOO−), a biological oxidant formed from

the reaction of nitric oxide with the superoxide radical, is associated with much pathology,

including neurodegenerative diseases, such as multiple sclerosis. Carbon dioxide catalyses

the isomerization of peroxynitrite to NO3- via an intermediate, presumably ONOOCO2-

which undergoes homolysis to trioxocarbonate(*1-) (CO3*-) and nitrogen dioxide (NO2*),

transient radicals which are quenched by uric acid. Interestingly, nitroglycerin which over a

century has been used as a drug in the form of tablets, sprays or patches for treating and

preventing attacks of angina pectoris, exerts its effects because of its conversion to ·NO in

the body by mitochondrial aldehyde dehydrogenase. Nitric oxide is a natural vasodilator in

in the body.

Enzymic Antioxidants

Paradoxically, the major hint of the wide occurrence of free radical activity in the body

came from the observation of widespread distribution of superoxide dismutase (SOD)

family of enzymes in cells {Ref: McCord JM and Fridovich I, J Biol Chem, 1969, 244:6049–

6055}. These enzymes are powerful free radical scavengers, and it has been shown that they

particularly target the superoxide-ion free radical, O2- , in the aqueous phase. SODs are

metal-containing enzymes that depend on the bound manganese, copper or zinc for their

antioxidant activity. In mammals, the manganese-containing enzyme is most abundant in

mitochondria, while the zinc or copper forms are predominant in cytoplasm. SODs catalyze

the conversion of the two superoxide-ion radicals into the peroxide ion radical and oxygen.

O2- + O2

- → O2 2- + O2

SODs also feature in protective mechanisms against light-induced ROS formed during

photosynthesis either by electron-transfer from excited chlorophyll molecules to oxygen or

by transfer of electrons to oxygen from carriers such as ferredoxin. This protective

mechanism adopted by plants is additional to the dissipation as heat the excess light energy

absorbed by pigments.

Eukaryotic cells possess besides SODs two other important enzymatic oxidants against free

radicals:

Catalase - this degrades hydrogen peroxide to water and oxygen, and hence completes the

detoxification reaction started by SOD, and

Glutathione peroxidase – these selenium-containing enzymes degrade hydrogen peroxide

and reduce organic (lipid) peroxides to alcohols.

Antioxidant properties of Uric acid

In humans, uric acid is considered a major antioxidant that may protect against aging and

oxidative stress. It is the most abundant antioxidant in plasma (normal levels are in the

range 3.5 to 7.2 mg/dl) and is the end product of purine degradation. Xanthine, a product

on the pathway of purine degradation, is catalytically oxidised to uric acid by

xanthine oxidase, an enzyme that generates ROS. Uric acid's antioxidant activities are

complex, given that it does not react with some oxidants, such as superoxide, but does act

against peroxynitrite, peroxides, and hypochlorous acid {Ref: http://en.wikipedia.org/ wiki/

Antioxidant}.

It was recently shown that uric acid activates NADPH oxidase resulting in increased

production of ROS, leading to decreased bioavailabilty of ·NO and increased protein

nitration {Ref: Sautin Y, Nakagawa T, Zharikov S and Johnson RJ, Am J Physiol Cell, 2007,

293: C584-C596}. Uric acid has been shown also to react directly and irreversibly with ·NO

resulting in the formation of 6-aminouracil and depletion of ·NO, which is a potent

vasodilator. The reaction proceeds even in the presence of oxidants peroxynitrite and

hydrogen peroxide and is at least partially blocked by glutathione. Thus under conditions of

oxidative stress in which uric acid is elevated and intracellular glutathione lessened, the

depletion of ·NO levels leads to endothelial dysfunction {Ref: Gersch C et al. Nucleosides

Nucleotides Nucleic Acids, 2008, 27: 967-978}. The endothelium plays a major role in

maintaining vascular tone and modulating blood flow and pressure, and its dysfunction is

identified as contributing to hypertension, arterial stiffness and cardiovascular diseases.

One interesting fact that has emerged from recent studies is that high consumption of

sugars containing fructose can raise uric acid concentrations in the blood. It has been

proposed that fructose-induced hyperuricemia may play a major role in the development of

hypertension, obesity, and metabolic syndrome (a name for a group of risk factors that

occur together and increase the risk for coronary artery disease, stroke, and type 2-

diabetes) and in the subsequent development of kidney disease {Ref: Johnson RJ et al, Am J

Physiol Cell, 2007, 86: 899-906}.

Lipid Peroxidation

One of the best known toxic effects of oxygen radicals is damage to cellular membranes

(plasma, mitochondrial and endomembrane systems), which is initiated by a process known

as lipid peroxidation. A common target for peroxidation is unsaturated fatty acids present

in membrane phospholipids {Ref:www.vivo.colostate.edu/hbooks/pathphys/misc_topics/

radicals.html}.

Lipid peroxidation is a self-propagating phenomenon terminated by antioxidants (see below; R•=free radical species (ROS, RNS), L=lipid, A=antioxidant).

1. Initiation: R• + LH → RH + L•

2. Propagation: L• + O2 → LOO•

LOO• + LH → LOOH + L•

3. Termination: L• + AH → LH + A•

A• + LOO• → LOO-A

Nitric oxide and its oxidant metabolites (e.g. ONOO− ) can both stimulate and inhibit lipid

peroxidation, depending on relative concentrations of ·NO, ROS, and antioxidants, with all

interactions in turn being influenced by the aqueous-lipid solubility and relative rates of

reaction of the participating reactive species {Ref: Bloodsworth A, O’Donnell VB and

Freeman BA, Arteriosclerosis, Thrombosis, and Vascular Biology.2000; 20: 1707-1715}. Thus,

lipid peroxidation is inhibited by ·NO when its concentration exceeds that of superoxide

radical; the result is the termination of lipid radical–mediated chain propagation reactions

(i.e. ROO· + ·NO→ROONO). Likewise, the generation of metal-nitrosyl derivatives by ·NO

quenches the initiation of lipid oxidation by metals. For example, myoglobin and

hemoglobin oxoferryl free radical species (·Mb-FeIV=O/·Hb-4FeIV=O) are reduced to their

respective ferric (met) forms on reaction with ·NO, thus affording protection against

oxidative damage by oxoferryl Mb/Hb. Additionally, methemoglobin (metHb) (FeIII; does

not bind oxygen) binds ·NO to form a nitrosyl-hemoglobin (·NO-Hb) intermediate that loses

its ability to oxidize linoleic acid and produce conjugated dienes as well as the ability to co-

oxidize substrates such as β-carotene. In general, when ·NO complexes with

metalloproteins, lipids are protected from further oxidation by metals and oxidant

metabolites of ·NO. Nitric oxide also inhibits the oxidation of LDL by scavenging LOO· via

chain-terminating interactions of ·NO and other reactive nitrogen species, yielding oxidized

nitrogen-containing lipid products.

Peroxidation of membrane lipids can lead to altered permeability, altered activity of

membrane receptors and even decreased activity of membrane-bound enzymes such as

sodium pumps. Products of lipid peroxidation, for example, malondialdehyde, irreversibly

disrupt enzymes, receptors, and membrane transport mechanisms. In acute ischaemic

stroke, in vivo concentrations of lipid peroxidation products are significantly increased,

arising from excess free radical activity. Plasma concentrations of cholesteryl ester

hydroperoxides (CEOOH) are sensitive and specific markers of lipid peroxidation, and

correlate positively with infarct volume, calculated by computed tomography, and clinical

severity, determined by the National Institute of Health Stroke Scale {Ref: Waring WS, QJM

(2002), 95: 691-693}. This emphasizes the role of oxidative stress in mediating cerebral

ischaemic tissue damage.

As with lipids, proteins and DNA also suffer damage by free radicals; the breakdown

patterns are as shown below.

Apoptosis

If too much damage is caused to its mitochondria, a cell undergoes apoptosis or

programmed cell death. Cell suicide, or apoptosis, is the body's way of controlling cell death

and involves free radicals and redox signalling. This self-destructing act is a highly

orchestrated one and involves the release of cytochrome c from the mitochondria, which

along with another protein factor, Apaf-1, activates caspase-9 and initiates the cell death

cascade. The cascade is controlled by pro- and anti-apoptotic B-cell lymphoma protein

family members, the most important of them being such as Bcl-2 and Bax. A change in the

balance between these factors can lead to either premature cell death or to unchecked cell

division. The caspase-9 cleaves the proteins of the mitochondrial membrane, causing it to

break down and start a chain reaction of DNA fragmentation, protein denaturation and

formation of apoptotic bodies, and eventually phagocytosis of the cell.

Vitamins and other endogenous non-enzymic antioxidants

Understandably, neutrophils have to contain large reserves of endogenous antioxidants

such as glutathione and vitamins C and E. Their ability to maintain these antioxidants in the

reduced state during phagocytosis may prevent death from oxidative suicide.

Glutathione may well be the most important intracellular defence against damage by

reactive oxygen species. It is a tripeptide (glutamyl-cysteinyl-glycine). The cysteine provides

an exposed free sulphydryl group (SH) that is very reactive, providing an abundant target for

radical attack. Reaction with radicals oxidizes glutathione, but the reduced form is

regenerated in a redox cycle involving glutathione reductase and the electron acceptor

NADPH.

Vitamin E , the most biologically active form of which is alpha-tocopherol, is the major lipid-

soluble antioxidant, and plays a vital role in protecting membranes from oxidative damage.

Its primary activity is to trap peroxy radicals in cellular membranes. In the process, it

transiently becomes a radical but is regenerated through the activity of the antioxidants

vitamin C and glutathione.

Vitamin C (Ascorbic acid) can reduce radicals from a wide variety of sources. It does this by

losing an electron to a free radical and remaining stable itself by passing its unstable

electron around the antioxidant molecule.

The powers of antioxidants to limit damage to biological structures have led to the

hypotheses that large amounts of antioxidants might lessen the radical damage causing

chronic diseases, and even radical damage responsible for aging. DNA cross-linking, for

example, engendered by free radical-induced chain reaction involving base pairs in a strand

of DNA, can lead to various effects of aging, especially cancer. Other crosslinking can occur

between fat and protein molecules and leads to wrinkles. Free radicals can oxidize low-

density lipoproteins (LDL), and this is a key event in the formation of plaque in arteries,

leading to heart disease and stroke. There is growing evidence that aging involves, apart

from radical-mediated oxidative damage, progressive changes in free radical-mediated

regulatory processes that result in altered gene expression. However, the enduring interest

in antioxidants as a means to lessen the degenerative causes of biological aging continues.

Nutraceuticals

Presently there is much focus on the possible protective value of a wide variety of plant-

derived antioxidant compounds, particularly those from fruits and vegetables. These include

carotenoids, tocopherols, ascorbates, alpha-lipoic acids, polyphenols and the minerals

copper, zinc and selenium which are natural antioxidants with free radical scavenging

activity. The antioxidant compounds derived from natural products also belong to a new

class of compounds labelled in the marketplace as nutraceuticals. Many have gained

popularity on account of their demonstrated health benefits, among them, flavonoid

polyphenols like epigallocatechin 3-gallate (EGCG) from green tea and quercetin from

apples; non-flavonoid polyphenols such as curcumin from tumeric and resveratrol from

grapes; phenolic acids or phenolic diterpenes such as rosmarinic acid or carnosic acid,

respectively, both from rosemary; and organosulphur compounds including alpha-lipoic

acid and the isothiocyanate L-sulphoraphane, from broccoli and the thiosulphonate allicin,

from garlic {Ref: Kelsey NA, Wilkins HM and Linseman DA, Molecules (2010), 15: 792-814}.

Nonetheless, some recent studies tend to show that antioxidant therapy has no effect on

aging and can even increase mortality brought on by a decrease of normal biological

response to free radicals that creates in its wake a more sensitive environment to oxidation

{Ref: en.wikipedia.org/wiki/Free radical theory_ of_ aging}.

vg kumar das (24 August 2012)

vgkdasorig@gmail.com