Antioxidative barrier

Free radicals are atoms or molecules which are able to exist independently and have one or more unpaired electrons [G. Bartosz: Druga twarz tlenu, PWN, Warszawa 1995]. Owing to this property, radicals are impermanent but highly chemically active. In living organisms, they may be created in enzymatic reactions or spontaneously.

Up to the 1970s., there were few mentions on the role and importance of free radicals in medical publications. This was changed by the research results published by:

McCoda i Fridovitcha [Mc Cord, J. M. Fridowitch, J. Biol. Chem. 244, 6049, 1969] indicating that almost all cells of mammals contain superoxide dismutase (SOD; EC, an enzyme which is a catalyst in the dismutation of superoxide anion-radical;
Babiora [B. M. Babior, J. Clin, Inwest. 52, 741, 1973], who proved functions of neutrophils related with the generation of free radicals;
Grangera [D. N. Granger, Gastroenterology 81, 22, 1981], who showed that many diseases are related to a disturbed balance between mechanisms causing the generation of free radicals and systems which neutralize them.

Reactive oxygen species (ROS) include: singlet oxygen, ozone, hydroperoxyl radical, superoxide anion, hydrogen peroxide, hydroxyl radical, nitrogen oxide, nitrogen dioxide, nitric acid (HONO2), and its anion.

Sources of reactive oxygen species include:

Neutrophils responsible for the localization of an inflammatory process in a body and for identifying, killing, and digesting the intruder. Neutrophils are equipped with two killing systems, i.e. oxygen-independent system and oxygen-dependent system, which generates O2-•, OH•, ONOO-•. Reactive oxygen species released from phagocytes may cause damage to other cells.
Respiratory chain – a few per cent of oxygen used by mitochondria is reduced only in part and forms O2-•. It is estimated that in normal metabolic conditions each human cell is exposed to 1010 O2-• molecules daily. This means that 0.15 moles of O2-• is formed daily in a person weighing 70 kg.
Oxidases – enzymes which are catalysts in reactions involving the generation of ROS, including monoamine oxidase (MAO) from membranes of endoplasmic reticulum, which oxidizes xenobiotics.
Autooxidation of endo- and exogenous compounds, e.g. autooxidation of adrenaline generates ROS.
pH, reduced acidity accelerates the release of transition metals related to proteins (Cu+2, Fe+2+), what disturbs reaction of the respiratory chain and stimulates the generation of O2-•. Reduced pH in hypoxic cells increases chances of survival – pH paradox.
Transition metals – iron, copper, and manganese, which can transport an electron to biologically important macromolecules.
Eicosanoids synthesis – A2 phospholipase releases arachidonic acid from phospholipoids, which is transformed into prostaglandins, leukotriens, tromboxanes, and prostacyclins by cyclo- and lipoxygenases. ROS are generated in all these processes. ROS activate A2 phospholipase.

Although it is not a free radical, hydrogen peroxide plays an important role in processes of oxidative formation of free radicals (diagram 1). (Due to high reactivity of radicals, their half-lives are very short, e.g. O2-• w 37oC – 1*10-6 s, OH• w 37oC – 1*10-9 s).

Antioxidative barrier - diagram 1

Hydrogen peroxide is formed as a result of the reduction of molecular oxygen or the dismutation of superoxide radical by SOD. It is reduced to water and O2 by catalase. Half-life depends on catalase and gluathione peroxidase activity; therefore, its life is long enough for it to penetrate cellular membranes and „wander” all over the body.

The greatest risk for the body is posed by hydroxyl radical because it reacts with any organic molecule it meets. This reaction can consist in various mechanisms:

giving away an atom: OH• + HR → H2O + R•
bonding a radical: OH• + R – CH = CH – R → R – CHOH – C• H – R
adding radicals: R• + R• → R – R

The common property of the reactions showed above is maintaining the chain of free radical reactions.

The main source of hydroxyl radicals in the Fenton reaction (diagram 1).

H2O2 + Fe+2 → OH• + OH- + Fe+3

The second source of hydroxil radicals is the Haber-Weiss reaction, where catalysts include iron and copper cations.

O2-• + H2O2 → O2 + OH• + OH-

Superoxide anion O2-• is formed as a result of single-electron reduction of molecular oxygen. It is a final or intermediary product of many enzymatic reactions. It is mainly formed in the respiratory chain (diagram 2), in enzymatic processes catalyzed by oxidoreductases (xanthine, aldehyde, dihydroorotate, and diamine reductases, NADPH oxidoreductase, i.e. P-450 cytochrome, myeloperoxidase) from leucocytes, autooxydation of reduced forms of biochemical compounds (flavins, quinones, nucleotides, nucleotides, aromatic amino acids, pteridines, ferrodoxins, thiols, i.e. glutathion, adrenaline, and dopamine).

Production of energy and free radicals - diagram 2

Nitrogen oxide radical is formed during the synthesis of the relaxing agent of endothelial cells, i.e. nitrogen oxide, from arginine. In the reaction with superoxide anion, impermanent nitric acid (HONO2) is formed, which is then subject to homolytic breakdown, what results in the formation of hydroxyl radical.

ree radical theory of ageing is based on the efficiency of respiratory chain reactions, whose efficiency is app. 40%. Due to bad diet and with time, its efficiency is lower and lower; the consequences include an increased production of superoxide anions O2-•, which initiate the chain of free radical reactions on their own or from which hydroxyl radicals are formed. Food poor in vitamins B and vitamin K causes disturbances which lead to the inhibition of phosphorylation reactions in the respiratory chain of some cells. Similar symptoms occur as a result of the deficiency of iron, which is a component of respiratory chain enzymes. Vitamin K takes part in the synthesis of the respiratory chain of liver cells. In liver, most proteins are formed using energetic reserves, including prothrombin, a protein which is of a great importance in blood clotting processes.

A similar situation occurs as a result of vitamins B deficiency. Pellagra and beriberi are a consequence of disturbances of biological oxidation in tissues which are particularly sensitive, including nerve cells, endothelial cells, and cells of endocrine glands.

As a result of free radical reactions of ROS with organic compounds, chemical compounds are formed whose structure and biochemical properties are not known and which can be causes of many pathologies. The results of their activity is presented in the diagram below:

Haber-Weiss reaction - diagram 3

Eucariotic organisms have protective mechanisms which neutralize the destructive activity of omnipresent ROS. Their task is to constantly monitor the efficiency of separate elements of the antioxidative barrier, that is to maintain a proper homeostasis of pro- and antioxidative processes.

Separate elements of the antioxidative barrier can be divided into the following two groups:

main elements:
enzymatic: superoxide dismutase (CuZnSOD – cytosol, nucleus, plasma, MnSOD – mitochondrion), catalase (FeCAT – peroxisomes), glutathion peroxidase (SeGPX – cytosol, mitochondria), glutathion transferase (cytosol);
extracellular metalloproteins (albumin-Fe, Cu, transferrin-Fe, ceruloplasmin-Cu) and intracellular metalloproteins (ferritin-Fe, metallothionein-Cu);
mollecular: α-tocopherol, β-carotene, ascorbic acid, glutathion, ubiquinone, urates, carnosine, anserine;
auxiliary elements:
enzymatic: glucose-6-phosphate dehydrogenase, disulphoglutation reductase;
molecular: bilirubin, biliverdin, cysteine, adenosine, histidine, lipoic acid, linolenic acid.

All elements of the antioxidative barrier decide about its ability to protect from the destructive activity of ROS (diagram 4). Antioxidants are present in all places where formation of ROS is possible. In enzymatic reactions where ROS are formed, main products can be antioxidants, e.g. in the oxidation of xanthine to uric acid. Our body has constructed spatial barriers in order to limit the propagation of free radical reactions by a specific distribution of all elements of the antioxidative barrier so that they can complement each other. If the activity of enzymatic elements of the antioxidative barrier is not sufficient and there are too few other elements, there is still the protective activity of endogenous compounds, mainly vitamins: A, E, and C as well as flavonoids.

Antioxidative barrier - diagram 4

Vitamin C, i.e. L-ascorbic acid, is the strongest water-soluble antioxidant. It neutralizes ROS activity in bodily fluids, blood, and extracellular fluid. Ascorbic acid is said to be the most important hydrophilic antioxidant. In terms of physiology, the most important hydrophobic antioxidant, i.e. lipid phase antioxidant, is α-tocopherol, i.e. vitamin E. Activity of vitamins E and C is synergistic. Ascorbate reduces α-tocopherol radical and regenerates vitamin E, creating ascorbyl radical itself, which is then further reduced to dehydroascorbic acid. With the participation of glutathion, dehydroascorbate reductase recreates the original form of vitamin C.

There is a synergy between vitamins E and A during the inhibition of microsomal peroxidation in hepatocytes. Vitamin A is an efficient antioxidant when the body is well oxygenated; in hypoxia, vitamin E shows high antioxidative efficiency. Vitamin A prevents oxidation of plasma lipid fractions and cellular membrane lipids. Lipid peroxidation leads to the destruction of cellular membranes.

The rate of lipid oxidation depends on the tissue type, presence of antioxidants and transition metal ions, and oxygen concentration. It has three stages: initiation, propagation, and termination. Initiation is an attack of a radical on a tertiary carbon atom (because C-H bond has low dissociation energy). This process may be initiated e.g. by a hydroxyl radical, in particular with the participation of transition metals. Superoxide anion is not able to initiate the process of lipid oxidation because it is not soluble in the lipid phase. Radicals with an unpaired electron on the carbon atom react with oxygen and create peroxide radicals, which take part in further reactions (propagation) and react with neighbouring molecules of unsaturated lipids (diagram 5).

Lipid peroxidation - diagram 5

Transition metal ions also catalyze the breakdown of lipid peroxides:

LOOH + Fe2+ → LO• + OH-Fe3+

LOOH + Fe3+ → LOO• + H+Fe2+

The stage of propagation is inhibited by the competitive termination occurring between reactive radical forms:

LOO• + L1 OO• → LOOL1 + O2

LOO• + L1OO• → LOOL1

L• + L1• → L – L1

The effect of the process is the formation of lipids with branched aliphatic chains or protein-lipid complexes. The breakdown of lipid peroxides leads to the formation of many compounds, such as saturated hydrocarbons (ethane, penthane, hexane) and unsaturated hydrocarbons, aldehydes (saturated and unsaturated, hydroxyaldehydes), and dialdehydes (e.g. malone dialdehyde – MDA). Some of these compounds can be causes of pathologies or can be metabolized to other potential endogenous toxins.

Special attention should be paid to γ-diketones, which may cause the networking of lipids and proteins. Lipids networked by MDA have been found in blood cell membranes in sickle cell anaemia. In general, lipid oxidation leads to the degradation of membrane lipids, what may be used by the body as a protective mechanism. In patients with malignant tumours, in particular during methastases, increased levels of MDA were observed in blood serum as well as in urine, what may indicate increased protective activities of neutrophiles.

Free radicals lead to the formation of alien biochemical structures in the body. Research in many scientific centres in the world is focused on the determination of the degree of destruction in nucleic acids (e.g. by determining 8-hydroxydeoxyguanosine), proteins (by determining bityrosine), and fatty acids (by determining MDA, hydrocarbons, diketones, and their metabolites). A particularly interesting direction of research is searching for endogenous toxins formed as a result of biochemical processes of products of the free radical reaction chain.

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