Endogenous Antioxidant network

““FunctionalFunctional”” antioxidantsantioxidants

Antioxidant network and disease Antioxidant network and disease preventionprevention

The lesson from clinical trialsThe lesson from clinical trials

THE OXYGEN RADICAL CYCLE

THE OXYGEN RADICAL CYCLE

4 e ¯

4 O2¯

SOD

4 O2

2 O2

CAT

H2O

1O2

GSH GSSG

H2O2H2O2

GPxUseful purposes

DietRespiration (Source)4 O2 + 4 e ¯ O2 Cycle 3 O2 + 2 H2O

O2 + 4 e ¯ Cyt oxidase 2 H2O

ROS: REACTIVE OXYGEN SPECIESROS: REACTIVE OXYGEN SPECIESRadicaliche e non radicalicheRadicaliche e non radicaliche

OSSIGENO TRIPLETTO O2

ANIONE SUPEROSSIDO O2-•

PEROSSIDO D’IDROGENO H2O2

RADICALE IDROSSILICO HO•

ACQUA H2O

OSSIGENO SINGOLETTO 1O2

ORBITALIπ σ

radicale

ossidante

SISI

SI

SI

SI

SI

NO

NO SI

NONONO

FONTI ESOGENE DI RL

•Luce ultravioletta

•Inquinamento ambientale

•Fumo di sigaretta

•Radiazioni elettromagnetiche

•Alimenti

FONTI ENDOGENE DI RL

• Respirazione ed Esercizio fisico

• Meccanismi di detossificazione

• Cascata dell’acido arachidonico

• Fagocitosi

Oxidative Burst

•NADPH-Oxidase

•SOD

•Haber weiss

•Mieloperoxidase

Sorgenti di RL in Vivo

Produzione abnorme di RL

Non necessaria, potenzialmente molto dannosa.

Produzione fisiologica di RL

Utile, sito-specifica,difficilmente dannosa

Accidentale, potenzialmente

dannosa

MODALITAMODALITA’’ DI AZIONE DEGLI DI AZIONE DEGLI ANTIOSSIDANTIANTIOSSIDANTI

PRIMARIPRIMARI

•Prevengono la formazione di specieradicaliche

•Sequestrano i metalli di transizione

SECONDARISECONDARIReagiscono con radicali formati e li convertono in forme meno reattive interrompendo la reazione a catena

CLASSIFICAZIONE DEGLI ANTIOSSIDANTICLASSIFICAZIONE DEGLI ANTIOSSIDANTI

ENDOGENIENDOGENI

ENZIMI: SOD, catalasi, glutatione perossidasi

PROTEINE: proteine-SH, leganti metalli (Fe, Cu)

ALTRE MOLECOLE: acido urico, bilirubina ...

VITAMINICI:

•Vitamina C

•Vitamina E

•Carotenoidi (con funzione di pro-vitamina A)

NON VITAMINICI:•Carotenoidi

•Polifenoli

ESOGENIESOGENI

Uric acidVit. E CarotenoidsVit. C PhenolicsThiolsCoQ

THE NON ENZIMATIC ANTIOXIDANT NETWORK

Antioxidant plasma defence andcigarette smoke

Antioxidant plasma defence andcigarette smoke

0

20

40

60

80

100

120

0 2 4 6 8 10 12

Number of Puffs

Ant

ioxi

dant

s (%

)

AAa-TocSHHyPerox

REDOX NETWORK E STRESS OSSIDATIVO LIPOFILO

REDOX NETWORK E STRESS OSSIDATIVO LIPOFILO

0

20

40

60

80

100

120

0 30 60 90

Tempo (minuti)

Ant

ioss

idan

ti (%

)

Ub-10Alfa-TocBeta-CarIdroperossidiLycopene

Frei et al. 1991

RED-OX EQUILIBRIUMRED-OX EQUILIBRIUM

Oxidative stressOxidative stressHealthHealth

FreeFree radicalsradicals

AntioxidantsAntioxidants

CELL INJURYCELL INJURY

Oxidative stress DNA Damage

GSH Depletion Protein damage

Rises in free CaCytoskeletaldamage

Inhibithion of ATP synthesis

NAD(H)Depletion

Rises in free iron

Membrane peroxidationand destruction

Membrane blebbing

Lipid peroxidation

Increased damage toDNA, proteins,

lipids

Metal ion releaseinto surrounding tissues

to adjacent cells

Oxidative stress induced diseasesOxidative stress induced diseases

AgingAgingAtherosclerosisAtherosclerosisDiabetesDiabetesCataractogenesisCataractogenesisRetinal damageRetinal damageParkinsonParkinson’’s diseases diseaseAlzheimerAlzheimer’’s diseases diseaseMuscular dystrophyMuscular dystrophyMultiple sclerosisMultiple sclerosisLung cancerLung cancer

EmphysemaEmphysemaFanconiFanconi’’ss anemiaanemiaThalassemiaThalassemiaAutoimmune diseaseAutoimmune diseaseRheumatoid arthritisRheumatoid arthritisStrokeStrokeMyocardial infarctionMyocardial infarctionAlcoholAlcohol--induced pathologyinduced pathologyGlomerulonephritisGlomerulonephritisDownDown’’s syndromes syndrome

Mortality rates for CHD in Europe Mortality rates for CHD in Europe

0 50 100 150 200 250 300 350 400

SCOSCOFINFINIREIREUKUKTZETZENORNORDANDANSWESWEHOLHOLPOLPOLGERGERBELBELAUSAUSBULBULITAITAROMROMYUGYUGSWISWIGREGREPORPORSPASPAFRAFRA

400400

350350

300300

250250

200200

150150

100100

5050

00

Age-standardized death rates per 100.000 in

men aged 50-54(Source: James, WPT,

Healthy Nutrition 1988)

AgeAge--standardized death standardized death rates per 100.000 in rates per 100.000 in

men aged 50men aged 50--5454(Source: James, WPT, (Source: James, WPT,

Healthy Nutrition 1988)Healthy Nutrition 1988)

Antioxidant hypothesis of cardiovascular disease

Antioxidant hypothesis of cardiovascular disease

0

100

200

300

400

500

18,9 19,9 20,9 21,9 22,9 23,9 24,9 25,9 26,9 27,9 28,9

IHD

mor

talit

y pe

r 100

.000 r = 0.73

I-Sa E-C

CH-TDK-GDDR-C

NI-BSc-A

DDR-S

Sc-GSF-S

Sc-E

SF-N1

F-T

α-tocopherol lipid-standardized (μM)

Meta-analysis of randomised trials: β-carotene

Meta-analysis of randomised trials: β-carotene

Vivekananthan D.P. et al. The Lancet 2003

All cause mortality Cardiovascular death Cerebrovascular accident

Vivekananthan D.P. et al. The Lancet 2003

Meta-analysis of randomised trials:Vitamin E

Meta-analysis of randomised trials:Vitamin E

All cause mortality Cardiovascular death Stroke

Study Country Subjects Intervention OutcomeBlot et al, 1993

China 29584 subjects,40-69 years

5-year supplementation15 mg/d β-carotene + 30 mg/d vitamin E + 50 μg Se

Decrease in total cancer rates (RR = 0.87)Decrease in stomach cancer rates (RR = 0.79)

ATBC Finland 29133 chronic heavy smokers (M) 50-69 years

8-year supplementation50 mg/d α-tocopherol20 mg/d β-carotenecombination of both

β-carotene group: Significant increase (18 %) in lung cancer incidence

CARET USA 18 314 subjects, smokers, former

smokers and asbestos-exposed

workers45-74 years

4-year supplementation30 mg/d β-carotene 25000 IU retinol

Significant increasein lung cancer incidence (28 %; RR = 1.36)

Physician’s Health Study,

USA 11000 healthy physicians (M)

40-84 years

12-year supplementation50 mg/d β-carotene

No changes in lung cancer incidence and mortality

Clark et al, 1996

USA 1312 patients with lesions on skin,

mean age 63 years18-80 years

4.5-year supplementation200 µg/d Se

No significant changes on incidence of skin cancerNo significant reduction in all-cause mortalitySignificant reduction in total cancer mortality (RR = 0.50)and incidence (RR = 0.63)Significant reduction in prostate cancer (RR = 0.37), colorectal cancer (RR = 0.42) and lung cancer (RR = 0.54)Serafini et al. Nutr & Cancer 2007

Serafini et al. Nutr & Cancer (In press)

Women’s Health)

USA 39876 female health

professionals≥ 45 years

2-year supplementation50 mg/d β-carotene

No significant differences in cancer riskand cardiovascular death

Heart Protection Study

UK 20000 patients with coronary

disease,cardiovascular events, diabetes

40-80 years

5-year supplementation600 mg/d vitamin E,250 mg/d vitamin C,20 mg/d β-carotene

No changes in cancer incidence

SUVIMAX Study

France 12741subjects (M,W)

45-60 years

7.5-year supplementationvitamin C, vitamin Eβ-carotene, Se, zinc

Women: no changes in cancer incidenceMen: significant decrease in prostate

cancer incidence(RR = 0.52)

HOPE trial Multicenter

patients with cardiovascular

disease or diabetes mellitus≥ 55 years

6-year supplementation (9541subjects)400 IU/d Vitamin E

No differences in cancer incidence and mortality

Higher risk of heart failure (RR = 1.21)

TO PILLS OR NOT TO PILLS…TO PILLS OR NOT TO PILLS…

Miller E.R. et al. 2005

OOH

OHO

A C

B

OH

OH

OH

Phytochemicals

Antioxidants PhytochemicalsAntioxidants Phytochemicals

Important chemicals found in foodsImportant chemicals found in foodsNot currently known if they are requiredNot currently known if they are requiredPlay an important role in prevention ofPlay an important role in prevention of dzdzMay be up to 10,000 in a single foodMay be up to 10,000 in a single foodOver 600 currently identifiedOver 600 currently identifiedLike nutrients, also found in foodsLike nutrients, also found in foodsMany different methods of identifying, many foods Many different methods of identifying, many foods not yet tested for their contentnot yet tested for their contentNumbers currently reported not yet completeNumbers currently reported not yet complete

Common C6-C3-C6 Flavonoid Structures

O

OO

OO

OO

OO

O

OH

O

O

OOH

Anthocyanin(berries, grapes, plums etc)

Flavanone(citrus fruit)

Flavan-3-ol (aka catechins)(F & V, red wine, chocolate, green tea)

Flavonol(F & V, red wine, tea)

Flavone(celery, parsley)

Isoflavone(soya)

A C

B12

34105

6

79

1'

2'3'

4'5'

6'

8

O+

OH

Phenolics contentof foods

Phenolics contentof foods

Source: Manach et al. AJCN 2004

Flavonoids and CHDFlavonoids and CHD

-0,1

0,1

0,3

0,5

0,7

0,9

1,1

1,3

1,5

Low High

Flavonols and flavone intake

Crude

Adjusted for age anddietAdjusted for age, dietand risk factorsR

elat

ive

Ri s

k

From: Hertog et al. 1993

INHIBITION OF LDL OXIDATIONINHIBITION OF LDL OXIDATIONBY RED WINE PHENOLIC COMPOUNDSBY RED WINE PHENOLIC COMPOUNDS

0102030405060708090

100

0 1 2 3 4

Vitamin EWine Phenolics

µmoL/L

From: Frankel et al. Lancet, 341:454-57;1993

Con

juga

ted

Die

ne(%

of c

ontr

ol)

Mortalità e consumo di etanoloMortalità e consumo di etanolo

0,5

0,75

1

1,25

1,5

Mai

Occasi

onalm

ente

1 per

die2 p

er die

3 per

die4 p

er die

5 per

die

Boffetta, P. and Garfinkel, L. Epidemiology, 5: 342-8; 1990

Copyright ©2002 American Heart Association Di Castelnuovo, A. et al. Circulation 2002;105:2836-2844

Odds ratios for vascular disease comparing wine intake versus no wine intake

GronbaekGronbaek MM etet al. BMJ 1995; 310: 1165al. BMJ 1995; 310: 1165--11691169

0.80.8

1.21.2

11

1.61.6

00

0.60.6

0.40.4

0.20.2

1.41.4

1.81.8

2

Mai 1 UA/mese 1 UA/ settimana

1-2 UA/giorno

3-5 UA/giorno

Ris

chio

rela

tivo

Ris

chio

rela

tivo

BirraBirra

SuperalcoliciSuperalcolici

VinoVino

Consumo di bevande alcoliche e mortalità per tutte le cause

Consumo di bevande alcoliche e mortalità per tutte le cause

Wine and beer are bought inassociation with..

Wine and beer are bought inassociation with..

WineWine drinkers choicesdrinkers choices

Beer drinkers choicesBeer drinkers choices::

Johansen, D et al BMJ, 20 January 2006Johansen, D et al BMJ, 20 January 2006

VINO ROSSO

RISCHIO CV

Frazionefenolica

Frazionealcolica

↓Perossidi↓Radicali liberi

↓OssidazioneLDL

↓ Trombosi

↓ Livelli di fibrinogeno

↓ Aggregazione piastrinica

indotta da collagene

HO

OH

OOH

OH

OH

HO

OH

OOH

O O

HO OHOH

OH

OH

HO

OH

OOH

OH

OH

OH

HO

OH

OOH

OH

OH

HO

OH

OOH

O O

HO OHOH

OH

HO

OH

OOH

OH

OH

OH

(-)-Epicatechin

(-)-Epigallocatechin gallate(-)-Epigallocatechin

(+)-Catechin

(-)-Epicatechin gallate

Principle flavan-3-ols in tea leaves

(+)-Gallocatechin

Black Tea Phenolics Associated With Thearubigin Production

8'-Ethylpyrrolidinonyl-theasinensin A

HO

HO

HO OOH

OH

O

OH

OH

OH

HO NO

HO

OO

OHHOOH

O

HO OHOH

O

O O

O

OHO

OH

OH

OH

HO

OH

H

Theanapthoquinone Dehydrotheasinensin AQ

OH

O

O

OH

OHO

OH

OO

OH

HO

HO

O

O

OH

OHHO

OO

OHHOOH

SUBJECTS

FifteeenFifteeen healthy volunteers healthy volunteers

(29(29--45 y, 7 W 8 M)45 y, 7 W 8 M)

Non smokers,Non smokers, normolipidemicnormolipidemic

No drug or vitamin supplementsNo drug or vitamin supplements

STUDY DESIGN STUDY DESIGN

Blood sampling (0, 30, 50 and 80 min.)

7 Days wash-out

H2O (250 mL) (n = 5) GT (250 mL) (n = 5) BT (250 mL) (n = 5)

GT + milk (1:3) BT + milk (1:3)

Blood sampling (0, 30, 50 and 80 min.)

PLASMA ANTIOXIDANT CAPACITYPLASMA ANTIOXIDANT CAPACITY

SerafiniSerafini M et al. Lancet 344: 626; 1994;M et al. Lancet 344: 626; 1994; SerafiniSerafini et al.et al. EurEur. J.. J. ClinClin.. NutrNutr. 50:28. 50:28--32; 199632; 1996

Time (min)Time (min)808060604040202000

002 02 04 04 06 06 08 08 0

TRA

P in

crea

se (%

)TR

AP

incr

ease

(%)

Black teaBlack tea

Without milkWithout milk

Without milkWithout milkWith milkWith milk

002 02 04 04 06 06 08 08 0

Time (min)Time (min)808060604040202000

TRA

P in

crea

se (%

)TR

AP

incr

ease

(%)

Without milkWithout milkWith milkWith milk

Green teaGreen tea

WaterWater

““PolyphenolsPolyphenols bind protein making them insoluble and bind protein making them insoluble and inducing precipitation. The activity binding with inducing precipitation. The activity binding with protein is considered to be mainly due to formation of protein is considered to be mainly due to formation of multimulti--hydrogen bonds. The precipitate formation with hydrogen bonds. The precipitate formation with protein is a characteristic property of regular tannins, protein is a characteristic property of regular tannins, but there are also some PP which show potent bindingbut there are also some PP which show potent bindingactivity in spite of their smaller MWactivity in spite of their smaller MW””

Bate-Smith, CR Metcalfe (1957) Journal of the Linnean Society, Botany, 55:669.Bate-Smith E.C., Swain T. (1962) In Comparative Chemistry Vol. 3. Academic Press: New York.Okuda T. (1997) In: Food and Free Radicals. Plenum Press, New York.EC

Polyphenols and protein binding

TAC

SUBJECTS

Twelve healthy volunteers Twelve healthy volunteers

(25(25--35 y, 7 W 5 M)35 y, 7 W 5 M)

Non smokers,Non smokers, normolipidemicnormolipidemic

No drug or vitamin supplementsNo drug or vitamin supplements

STUDY DESIGNSTUDY DESIGN

CK(100 g)

Group A (n = 4)

CKM(200 g)

Group B (n = 4)

CK + M(100 g + 200 mL)

Group C (n = 4)

Blood sampling (0, 1, 2 and 4 h.)

7 Days wash-out

Phases 2 and 3

Black tea, milk and cardiovascular function

Lopez et al. Eur Heart J, 2007

HO

OH

O

OH

OH

(Š)-Epicatechin

OH

HO

HO

O

OH

HO

OH

OOH

OH

OH

Proanthocyanidin B2 dimer

OH

OH

H O

H O

O

O H

H O

H O

O

O H

O H

O H

O H

O H

H O

H O

O

O H

O H

O H

P ro a n th o c y a n id in C 1 tr im e r

H O

O H

O

O H

O H

O H

( + ) - C ate c h in

I FLAVONOIDI DEL CACAO