DIETARY ANTIOXIDANT

Why This Topic?

If you search for Antioxidants…

• Sci-Finder 248,724 Articles (April 2012)• Pubmed 340,354 Hits (April 2012)• Google Search 30,500,000 Web pages

(April 2012)• Chemical Abstracts 14,609 Publications (2011

alone)

What is Oxidation?

• Combination of substrate with oxygen.• Reaction in which the atoms in a compound

lose electrons. • Any compound, including oxygen, that can

accept electrons is an oxidant or oxidizing agent (pro-oxidant), while a substance that donates electrons is a reductant or reducing agent (antioxidant).

Oxidation in Biological System

• We live in an aerobic environment• Oxygen is the life sustaining element• We consume approximately 3.5 kilograms of

oxygen every day • 2.8 percent of the oxygen is not properly used

and forms free radicals • Several kilograms of peroxides (harmful

oxidized lipids) are produced in our body every year

Free radicals and antioxidants• What is „free radical“?• Reactive oxygen and nitrogen species (RONS)• Are the RONS always dangerous?• Well known term „oxidative stress“ - what is

it?• Antioxidants - types and appearance• Markers of oxidative stress• Disorders Associated with Oxidative stress

Free radical - what is it?Atom: proton, neutron, electronic shell (orbital)

Free radical • particles with an unpaired electron spinning

around the nucleus. (can be atom, ions, molecule).

• tend to reach equilibrium, plucks an electron from the nearest intact molecule.

• most of biomoleculs are not radicals

Free radical and „science“

• chemistduring the thirties - there is superoxide

• biochemist during the sixties - make a discovery of superoxid dismutase (SOD)

• doctorfree radicals are associated with many disorders

Mechanism of radical reactions

Radicals are highly reactive species

Three distinc steps

• initiation (homolytic covalent bonds cleavage)

• propagation (chain propagation)• termination

ROS (reactive oxygen species)

Free radicals superoxide, O2

· -

hydroxyl radical, OH ·

peroxyl, ROO ·alkoxyl, RO ·

hydroperoxyl, HO2 ·

Particals, which are not free radicals

hydrogen peroxide, H2O2

(Fenton´s reaction)hypochlorous acid, HClO

ozone, O3

singlet oxygen, 1O2

RNS (reactive nitrogen species)

Free radicalsnitrogen(II) oxide, NO .

nitrogen(IV) oxide, NO2 .

Particals, which are not free radicals

nitrosyl, NO+

nitrous acid, HONOnitogen(III) oxide, N2O3

peroxynitrite, ONOO -

alkylperoxinitrite, ROONO

The main sources of free radicals membranes enzymes and/or coenzymes with

flavine structures, hem coenzymes, enzymes containing Cu atom in an active site

1. respiratory chain mitochondria : mainly superoxide and then H2O2

• approx 1- 4% O2 entres into resp. chain (mainly complexes I a III)

The main sources of free radicals II2. Endoplasmic reticulumsuperoxide creation (by cytochrome P- 450)3. special cells (leukocytes) superoxide creation by NADP-oxidase4. hemoglobin to methemoglobin oxidation(erytrocyte is „full“ of antioxidants)

Oxidant Description

•O2-, superoxide anion

One-electron reduced state of O2, formed in many autoxidation reactions and by the electron transport chain. Rather unreactive but can release Fe2+ from iron-sulfur proteins and ferritin. Undergoes dismutation to form H2O2 spontaneously or by enzymatic catalysis and is a precursor for metal-catalyzed •OH formation.

H2O2, hydrogen peroxide

Two-electron reduction state, formed by dismutation of •O2- or by direct reduction of O2. Lipid soluble and thus able to diffuse across membranes.

•OH, hydroxyl radical

Three-electron reduction state, formed by Fenton reaction and decomposition of peroxynitrite. Extremely reactive, will attack most cellular components

Common Free Radicals

Oxidant Description

ROOH, organic hydroperoxide

Formed by radical reactions with cellular components such as lipids and nucleobases.

RO•, alkoxy and ROO•, peroxy radicals

Oxygen centered organic radicals. Lipid forms participate in lipid peroxidation reactions. Produced in the presence of oxygen by radical addition to double bonds or hydrogen abstraction.

ONOO-, peroxynitrite

Formed in a rapid reaction between •O2- and NO•. Lipid soluble and similar in reactivity to hypochlorous acid. Protonation forms peroxynitrous acid, which can undergo homolytic cleavage to form hydroxyl radical and nitrogen dioxide. Source= Wikipedia

Common Free Radicals

Antioxidant defence system3 levels

inhibition of production the abundance of RONS

capture of radicals (scavengers, trappers, quenchers)

correction mechanism of destroyed biomoleculs

Antioxidants and scavengers review

1. Endogennous antioxidants

• enzymes (cytochrome c,SOD, GSHPx, catalase) • nonenzymatic

- fixed in membranes ( -tocopherol, -caroten, coenzym Q 10)

- out of membranes (ascorbate, transferrin, bilirubin)

Antioxidants and scavengers review II

2. Exogennous antioxidants

• FR scavengers• trace elements• drugs and compounds influence to FR

metabolism

Enzymes defence mechanism

Superoxid dismutase

(EC 1.15.1.1, SOD)

2O2. - + 2H+ H2O2 + O2

SOD - is present in all oxygen-metabolizing cells, different cofactors (metals)

an inducible in case of superoxide overproduction

Superoxid dismutase

Mn 2+ SOD (SOD1) tetramermatrix mitochondrialower stability then Cu, Zn - SOD

Superoxid dismutase

Cu 2+/Zn 2+ SOD (SOD 2)

dimer, Cu = redox centrcytosol, intermitochondrial spacehepatocyt, brain, erytrocytehigh stability, catalysation at pH 4,5-9,5

Glutathion peroxidases

elimination of intracellular hydroperoxides and H2O2

2 GSH + ROOH GSSH + H2O + ROH• cytosolic GSH - glutathionperoxidasa (EC

1.11.1.9, cGPx)• extracelullar GSH - glutathionperoxidasa

(eGSHPx)• phospholipidhydroperoxide GSH -

peroxidase (EC 1.11.1.12, PHGPx)

Catalase

(EC 1.11.1.6, KAT)

2 H2O2 2 H2O + O2

high affinity to H2O2 : peroxisomes hepatocytes mitochondria, cytoplasm of erytrocytes

tetramer with Fe, needs NADPH

High-molecule endogenous antioxidants

• transferrin• ferritin

• haptoglobin• hemopexin

• albumin

Low-molecule endogennous antioxidats IAscorbate (vitamin C)

collagen synthesisdopamine to epinephrine

conversionreduction agentFe absorptionantioxidant = reduction O2

· -

OH ·, ROO·, HO2 ·

tocopheryl radical regeneration

prooxidant

Alfa-tocopherol a vitamin Elocalise in membranesproduces hydroperoxides,

which are changes by GSHPx

Ascorbic acid and its metabolites

Low-molecule endogennous antioxidats II

• ubiquinone (coenzyme Q)electron carrier in respisratory chain co-operates with tocopheryl

• carotenoides, -caroten, vitamin Aremoving the radicals from lipids

Low-molecule endogennous antioxidats III

• glutathione (GSH, GSSG)in all mammalian cells (1-10 mmol/l)important redox buffer

2 GSH GSSG + 2e- + 2H+

ROS elimination, stabilisation in reduction form ( SH- groups, tocopheryl and ascorbate regeneration)

substrate of glutathione peroxidases

Low-molecule endogennous antioxidats IV

• Lipoic acid (lipoate)PDH cofactor

tocopheryl and ascorbate regeneration• melatoninlipophilic ; hydroxyl radicals scavenger

Low-molecule endogennous antioxidats V

• uric acid (urates)

• bilirubin

• flavonoids

Trace elements influence to FR metabolism

Seleniuminfluence to vitamin E resorption, part of selenoproteins of Se = insufficient immun. respons, erytrocytes hemolysis, methemoglobin synthesis

Zinc cell membrane stabilisation Fe antagonist

Oxidative stressEquilibrium failure between creation and

a elimination of RONS leads to

oxidative stress

Be carefull - this equilibrium can be disbalance in both sides

Oxidative damage to lipid

Damage

• unsaturated bonds loss• arising of reactive

metabolites (aldehydes)

Sequel

• changes in fluidity and permeability of membranes

• membranes integral enzymes are influenced

The peroxidation of linoleic acid

Oxidative damage to proteins

Damage

• agregation, fragmentation and cleveage

• reaction with hem iron ion

• functional group modification

Sequel

• changes in: enzymes activity, ions transport

• proteolysis

Oxidative damage to DNA

Damage

• saccharide ring cleveage• bases modification• chain breakeage

Sequal

• mutation• translation mistakes• protoesynthesis

inhibition

Oxidative stress markers

Free radicals detection• very difficult, because of chem-phys.

properties

Oxidative stress products detection• more simple, a wide range of techniques

Oxidative stress markers II

Lipoperoxidation markers:malondialdehyde (MDA), conjugated diens,

isoprostanesOxidative damage to protein markers :

protein hydroperoxidesOxidative damage to DNA :

modified nucleosides

Antioxidants determination

ascorbatetocopheryl

SODGSHPx

glutathion

Disorders Associated with Oxidative stress

NeurologicalAlzheimers DiseaseParkinson‘s Disease

EndocrineDiabetes

GastrointestinalAcute Pancreatitis

Disorders Associated with Oxidative stress

Others conditions

ObesityAir PollutionToxicityInflammation

Professor Mike Clifford, from the University of Surrey.

While a comprehensive body of data has been gathered on the in vitro antioxidant activity of polyphenols, phenols and tannins, less has been discovered in vivo, and one of the greatest challenges is to understand how the body processes these chemicals, as well as copes with their metabolites.

Phenolics undergo several metabolising steps in the body, including glucuronidation, methylation, sulphatation and dehydroxylation.

All these reactions affect their overall antioxidant activity, and therefore potentially modulate their protective effect.

“One of the main challenges for nutritional research in the years to come will be to identify the compounds responsible for the protective effects in vivo” (Professor Crozier).

• The antioxidant activity of dietary nutrients is also susceptible to its environment, the presence of proteins and other antioxidants.

• Certain polyphenols may readily pass the gastrointestinal barrier (e.g. caffeic acid) while other are poorly absorbed (e.g. rutin).

• This makes it very difficult to correlate the antioxidant capacity of a compound in vitro with its effect in vivo.

• Moreover, the composition of a meal affects the absorption and subsequent bioavailability of each polyphenol.

• What is known as the food matrix effect is currently being studied by Mullen, who adds that “comparing the bioavailability of the antioxidants in dark chocolate when eaten with and without milk revealed lower levels of antioxidants in the plasma when the dark chocolate was eaten with milk , while cream delayed the absorption of phenolics from strawberries”

• but also warns that this observation does not provide a complete answer to the problem, as many of the dietary antioxidants studied are rapidly metabolised and difficult to identify and quantify once absorbed.

• The role of these metabolites may be crucial. However little is known about them or their mode of action, which is potentially independent of their antioxidant activity.

• The behaviour of a compound during its journey through the gastrointestinal tract is crucial in order to understand its impact on systemic health.

• Following ingestion of a polyphenol-rich meal, the plasma antioxidant capacity is enriched, suggesting that the antioxidant compounds may pass the blood barrier, reduce the oxidant pool derived from the meal (mainly iron) or spare the body’s antioxidant defences.

• Professor Clifford, who has a special interest in the absorption and metabolism of phenols, polyphenols and tannins (PPT), emphasises that PPT metabolites only occur in the plasma in trace amounts, even after massive doses, and will be barely detectable after one normal portion of fruit and vegetables (as defined in five-a-day): “The antioxidant effect in the tissues associated with these mammalian metabolites is negligible and unlikely to be health-promoting by such a mechanism. They do not accumulate following repeat doses as all the metabolism is designed to make them easily removed from the body. The native polyphenols can be powerful antioxidants, but normally they do not occur in the plasma and tissues. If they did, and especially if they accumulated (as when given intravenously), they would be dangerous because they can generate free radicals through redox cycling. This is why the body has developed such good defences against them”.

• With such a vast degree of variation between the powerful, native dietary antioxidants tested in vitro, and their “watered-down” version occurring in vivo, it is difficult to establish a direct link between the antioxidant capacity of a molecule and its health-promoting effects.

• As Mullen points out, the requirement for ascorbate to prevent scurvy is independent from its antioxidant activity, as vitamin C is required for its role in collagen production.

• This point of view is shared by Professor Clifford, who highlights that dietary polyphenols are also likely to be beneficial through alternative routes, including their ability to delay glucose absorption from the duodenum, potentially protecting against the development of type-2 diabetes, or their prebiotic effect on the gut microflora and limitation of neurotoxin producing micro-organisms.

• Thus, it is clear that the science underlying the recommended 5 portions of fruit and vegetables per day is more complex than thought when species, metabolism, environment and human factors are taken into consideration.

• We may therefore be able to refine this advice in the future but, in the meantime, the public will do well to heed the current basic recommendation for their health and well-being, bearing in mind that, in order to secure a wide range of antioxidants, 35 portions a week covering a wide variety of fruit and vegetables are better than the same five everyday.