Definition : Free radical is a molecule or

molecular fragment that contains one or more unpaired electrons in its outer orbits.

Free radicals conventionally represented by super script dot R•

Characteristics of free radicals : 1 )Tendency of free radicals to acquire

electrons from other substance makes it more reactive.

2 )Short life span 3 )Generation of new free radicals by

chain reaction. 4 )Damage to various tissues.

Partial reduction of oxygen leads to formation of free radicals called as reactive oxygen species . The following are members of this group.

Super oxide anion radical (O2 · )Hydroperoxy radicals ( HOO· )Hydroxyl radical ( OH· )Lipid peroxyl radical ( ROO· )Nitric oxide ( NO· ) , Peroxy nitrite (ONOO · )H2O2 , singlet oxygen (are not free

radicals)

Free radicles are generated in oxidative metabolism due to leak of electrons .

Flavoprotein linked oxidases like xanthine oxidase , L α amino acid oxidase .

Super oxide is formed by autooxidation of hemoglobin to methemoglobin (approximately 3 % of the Hb has been calculated to autooxidise per day )

Cyclooxygenase & lipoxygenase reactions in metabolism of eicosanoids.

NADPH oxidase system of inflammatory cells by process of respiratory burst during phase of phagocytosis.

Free iron causes increased production of free radicals .

Free radicals are formed cytochrome P450 reductase enzyme complex durinrg metabolism of xenobiotics .

β oxidation of very long chain fatty acids in peroxisomes produces H2O2 .

Transfer of 4 electrons from reduced cytochrome C to molecular oxygen assisted by cytochrome oxidase

Transfer of 4 electrons lead to safe product H2O .

Site of electron escape appears to be ubiquinone & cytochrome C .

Cytochrome C oxidase does not release partially reduced intermediates , this crucial criterion meets by holding O2 tightly between Fe & Cu atoms .

Although Cyt C oxidase & other protiens that reduce O2 are remarkably successful in not releasing intermediates , small amounts of super oxide & peroxyl radicals are unavoidably formed.

About 1-4 % of oxygen taken up in the body is converted to free radical .

Flavoprotien linked oxidases 1 ) Xanthine oxidase ,2) L α amino acid oxidase ,3 ) Aldehyde dehydrogenase .

Reduction of isoalloxazine ring of flavin nucleotides takes place in 2 steps via a semiquinone ( free radical ) intermediate.

xanthine oxidase Hypoxanthine xanthine O2 O2 ·

acetaldehyde dehydrogenaseAcetaldehyde acetate O2 O2 ·

NADPH oxidase inflammatory cell produce supere oxide anion by a process of respiratory burst during phagocytosis.

This is the deliberate production of free radicals by the body .

activation of inflammatory cell

drastic increase in consumption of oxygen (respiratory burst )

10% of oxygen uptake by macrophage is used for free radical generation .

In chronic granulomatous disease the NADPH oxidase is absent in macrophages & neutrophils .

Streptococci & pneumococci themselves produce H2O2 therefore they are destroyed by myeloperoxidase system .

Staphylococci being catalase + ve can detoxify H2O2 in the macrophages & they are not destroyed .

Hence recurrent pyogenic infections by staphylococci are common in CGD .

Prodstaglandin H synthase & lipooxygenase enzyme catalysed reactions produce free radicals , by producing peroxide .

Macrophages produce NO from arginine by enzyme nitric oxide synthase , this is also an important anti bacterial mechanism .

Super oxide ion can release iron from ferritin .

The capacity to produce tissue damage by H2O2 is minimal because this is not a free radical . But in the presence of free iron H2O2 can generate hydroxyl free radical (OH ·)which is highly reactive.

Ionising radiation damages tissues by producing hydroxylradical , H2O2 ,super oxide anion .

Light of appropriate wave length can cause photolysis of oxygen to produce singlet oxygen .

Cigarette smoking contains high concentrations of free radicals.

Other toxic compounds CCl4 drugs & inhalation of air pollutants will increase free radical production .

Polyunsaturated fatty acids present in cell membranes are destroyed by peroxidation.

This occurs by three phases.

1 )intiation phase 2 )prolangation phase3 )termination phase

Production of carbon centered free radical R· ( or ) ROO· (lipid peroxide radical )

1 )RH +OH· R· + H2O metal ion

2 )ROOH ROO· + H+ R· , ROO· degraded to malon

dialdehyde . It is estimated as an indicator of fatty acid break down by free radical .

Carbon centered radical rapidly reacts with molecular oxygen forms peroxyl radical (ROO· ) which can attack another PUFA .R· + O2 ROO·

ROO· + RH ROOH + R· One free radical generates another free

radical in the neighbouring molecule a chain reaction (or) propagation is intiated .

The above reactions would proceed unchecked till a peroxyl radical reacts with another peroxyl radical to form inactive products .ROO· + ROO· RO- -OR+O2R· + R· R - - RROO· + R· RO- -OR

Super oxide dismutase Catalase Glutathione peroxidaseCytochrome oxidase

Chief amongst the enzymes that defense against ROS is super oxide dismutase .

Super oxide dismutase is present in all major aerobic tissues .

Eukaryote contains 2 forms of this enzyme,

1 ) Copper Zinc dependent cytosolic enzyme

2 ) manganese containing mitochondrial enzyme .

The active site of cytosolic enzyme in eukaryotes contains a copper ion & Zinc ion coordinated to the side chain of a histidine residue .

The negatively charged superoxide is guided electrostatically to a very positively charged catlytic site at the bottom of the channel .

H2O2 formed by SOD & by other processes is scavenged by catalase ( a ubiquitous heme protein that catalyze the dismutation of H2O2 into H2O & O2.)

Catalase is found in blood bone marrow mucous membranes , liver & kidney .

SOD & catalase are remarkably efficient , performing their reactions at or near the diffusion limited rate.

The Kcat / Km ratio of enzyme super oxide dismutase is 7x 10 9 enzymes that have high K cat / K m ratio at the uper limits have attained kinetic perfection.

Their catalytic velocity restricted only by the rate at which they encounter the substrate in the solution .

For catalytically perfect enzymes , every encounter between enzyme & substrate is productive .

Any rate in catalytic rate can come only by decreasing the diffusion .

Circe effect : In this case the electrostatic attractive forces on the enzyme entice the substrate to the active site .

Catalase decreases the free energy of activation ∆G 1 of H2O2.

In the absence of catalase ∆G1 free energy of activation is 18Kcal / mol where as in the presence of catalase 7 Kcal / mol .

K cat / Km value of catalase is 4 X 107.

Catalase is a heme protein containing 4 heme groups .

In addition to possessing peroxidase activity , it is able to use one molecule of H2O2 as a substrate electron donor & another molecule of H2O2 as oxidant or electron acceptor .

catalase 2H2O2 2H2o+O2

This enzyme is remarkable in containing a modified aminoacid selenocystein at its active site in which selenium has replaced the sulphur .

The enzyme catalyzes the destruction of H2O2 & lipid hydroperoxides by reduced glutathione , protecting the membrane lipids & hemoglobin against oxidation by peroxides .

Vitamin E : lipid soluble , chain breaking antioxidant.

βcarotene & its anologues (lycopene & retinyl stearate ): lipid soluble radical scavenger & singlet oxygen quencher .

Coenzyme Q : may acts as antioxidant in addition to its major role in energy metabolism .

Transferrin : binds ferric ions ( 2 per mole of protein )

Lactoferrin : binds ferric ions at low pH ( 2 per mole of protein )

Haptoglobins : binds hemoglobinAlbumin : binds copper , heme ,

scavenges OH.Ceruloplasmin : ferrooxidase activity

– stoichiometric O2 scavenging ,binds copper ions utilizes H2O2 for reoxidation of copper .

Ascorbic acid OH radical scavenger Bilirubin : scavenges peroxyl

radicals, open chain tetra pyrroles are effective singlet oxygen quenchers .

Urate : radical scavenger & metal binder

Mucus : scavenges OH radicals Glucose : OH radical scavenger .

Water soluble : urate , ascorbates , thiols ,bilirubin, flavanoids.Lipid soluble : tocopherol , ubiquinol 10 , β carotene .Urate & vitamin E acts in lipid phase

to trap ROO· radicals .

Preventive antioxidants reduces the rate of chain intiation .

Preventive antioxidants includeCatalase , peroxidases ,Ceruloplasmin , transferrin , albumin.

EDTA , DTPA acts anti xidants by chelating metal ions .

Vitamin E is most important natyral antioxidant .

Vitamin E apear to be the 1st line defense against peroxidation of PUFA contained in cellular & subcellular membrane phospholipids .

The phospholipids of mitochondria, endoplasmic reticulum & plasmamembranes possess affinities for αtocoferol & vitamin appears to concentrate at these sites.

The tocopherools acts as antioxidants by breaking free radical chain reactions as a result of their ability to tranfer a phenolic hydrogen to peroxyl free radical of a peroxidized PUFA .

The phenoxy free radical may react with vitamin C to regenerate tocopherol or it react with further peroxyl free radicl so that the chromane ring & the side chain are oxidized to the non free radical product.

ROO· + TocOH ROOH +

TocO·

ROO· + TocO· ROOH + non free

radical product

ROO· + TocOH ROOH +

TocO·

ROO· + TocO· ROOH + non free

radical product

The antioxidant action of vitamin E is effective at high oxygen concentrations , & it is concentrated in lipid structures exposed to high O2 partial pressures such as the erythrocyte membrane , membranes of respiratory tree & the retina.

Glutathione peroxidase contains Selenium, provides a second line of defense against hydroperoxides.

Tocopherol & selenium reinforce each other in their action against lipid peroxides.

Selenium is required for normal pancreatic function thus promoting absorption of lipids & vit E

Vitamin E reduces selenium requirement by preventing loss of selenium from the body or maintaining it in an active form .

Carotenoids are capble of quenching singlet molecular oxygen .

Carotenoids like lycopene ,β carotene , are important biological molecules that can inactivate electronically excited molecules by process called quenching.

βcarotene & related compounds can acts as chain breaking antioxidant.

Can acta as preventive antioxidant by decreasing the formation of methyl linoleate hydroperoxide .

Singlet oxygen is capable of inducing damage to the DNA .

Lycopene shows greater quenching ability than βcarotene (double ability )

Comparing the structures opening of the β ionine ring increases the quenching ability.

Quenching ability of cartenoids not only depends on triplet energy state that is the length of the conjugated double bond system but also on the functional groups .