Free Radicals and Antioxidants in Disease

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MSc Lecture 2006
Free Radicals and Antioxidants in Disease
Garry Duthie
Molecular Nutrition Group
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Lecture Plan

• The concept of disease prevention
• Free radicals and the theory of oxygen toxicity
• What are antioxidants?
• Main nutritional antioxidants?
• Do they have a role in disease prevention?

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Events in the progression of disease
Van der Greef J et al. Current Opinion in
Chemical Biology 2004, 8559565
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Cancer evolution and progression
Diet
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Schematic time course of human atherogenesis
Diet
Ischemic HeartDisease
CerebrovascularDisease
Peripheral VascularDisease
Lesion initiation
No symptoms
Symptoms
Symptoms
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• Basic Premise
• Oxidising free radicals participate in the early
  events that lead to disease
• This may be prevented by antioxidants
• Therefore antioxidants reduce disease risk
• But what is the evidence?

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Lecture Plan

• The concept of disease prevention
• Free radicals and the theory of oxygen toxicity
• What are antioxidants?
• Main nutritional antioxidants?
• Do they have a role in disease prevention?

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What are antioxidants?
What are free radicals?
Free radicals are atoms or molecules with one or
more unpaired electrons
electron pair
unpaired electron
proton
neutron
free radical
atom
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• The unpaired electron creates instability and
  high reactivity
• The electron seeks an association with another
  unpaired electron and/or hydrogen atom (1
  electron)

R AH RH A
Note AH can be a biomolecule ie protein, lipid
or DNA
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Free radical mediated damage to biomolecules may
lead to disease development
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Lipid peroxidation damage to membranes and
cholesterol
Abstraction of H generates carbon centred lipid
radical
Molecular rearrangement to diene conjugate
Further reaction with O2 generates peroxyl radical
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Free radicals can damage DNA
Permanent change in genetic message
Mutation
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• Difficult to measure free radicals directly
• Have to rely on detecting biomolecules which have
  reacted with free radicals
• Many confounding factors

Index Confounding factors TBARS Iron,
bilirubin Conjugated dienes Dienes of dietary
origin Hydrocarbons Bugs, pollution, fat
stores Lipid peroxides Proteins and
haemoglobin Protein carbonyls Carbonyls of
dietary origin F 2 -isoprostanes
Complexity 8-oxo DG Artifactual oxidation of DNA
Also may not be elevated in healthy subjects
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Sources of free radicals
Theory of oxygen toxicity
Stepwise reduction of oxygen to water not only
produces ATP but also FREE RADICALS !
EM of mitochondria
About 1-2 of the oxygen used by respiratory
chain ends up as superoxide ie .O2-
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Superoxide first to be detected in vivo Sources
are mitochondria, endoplasmic reticulum, NADPH
oxidases, xanthine oxidase It is not very
reactive But drives Fenton reaction
Fe2
O2.- H2O2 OH. OH- O2
OH. is the very reactive hydroxyl radical! It is
believed to contribute to oxidative damage to
living cells.
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Now known to be numerous pathways generating
endogenous REACTIVE OXYGEN SPECIES (ROS)
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Exogenous sources of free radical species
Energy intake enhances mitochondrial ROS
generation
pollution
UV radiation
Generated by combustion of polycyclic
hydrocarbons 1014 in smoke, 1015 in
tar H2O2,O2-, OH, ROO, RO, NO, NO2 ONOO-
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• So why are we not going rancid?
• Evolved a complex, integrated antioxidant
  defence system
• Some we synthesise, but some we need from diet

Metal cofactors
Antioxidant compounds
Vitamin E, carotenoids Vitamin C, polyphenols?
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Lecture Plan

• The concept of disease prevention
• Free radicals and the theory of oxygen toxicity
• What are antioxidants?
• Main nutritional antioxidants?
• Do they have a role in disease prevention?

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What are antioxidants?
Food technologist
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Numerous synthetic antioxidants
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Biologist/nutritionist
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How do biological antioxidants work?

• Scavenge free radicals
• Bind metals to prevent Fenton reactions
• Upregulate antioxidant enzymes

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How do they work?
Scavenging/chain breaking
Binding metals
Caeruloplasmin Ferritin Transferrin Lactoferrin
Stops Fenton Reactions
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Antioxidant enzymes
SOD, superoxide dismutase (Cu, ZN, Mn) GPx,
glutathione peroxidase (Se) Catalase (Fe)
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Lecture Plan

• The concept of disease prevention
• Free radicals and the theory of oxygen toxicity
• What are antioxidants?
• Main nutritional antioxidants?
• Do they have a role in disease prevention?

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Dietary dependent antioxidants

• Generally accepted as essential in diet
• Vitamin E
• Vitamin C
• Metal cofactors for antioxidant enzymes (Se, Cu,
  Zn, Mn, Fe)
• Disputed as essential in diet
• Carotenoids
• Phytoestrogens
• Ubiquinol
• Lipoic acid
• Polyphenols eg flavonoids (See Phytochemicals
  lecture)

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Vitamin E (tocopherol)
Discovered in 1926 Main lipid soluble
antioxidant Prevents lipid peroxidation Main food
sources are plant oils, nuts, green vegetables,
margarine
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Nutritional antioxidants Vitamin E
Intercalates in cell membrane
Stops oxidation of membrane lipids
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Vitamin E deficiency is rare in humans (TTP
defect, cystic fibrosis, premature infants)
Symptoms of severe vitamin E deficiency mainly
seen in animals
Cell membrane damage Anaemia (Monkey) Liver
necrosis (Pig, rat) Microangiopathy (Pig,
lamb) White muscle disease (Lamb, Calf)
www.conzoolting.com/advantages_path.html
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Dietary vitamin E requirements
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But Vitamin E is a family of homologues Do not
all function in vivo to same extent Different
foods may have less active forms
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Nutritional antioxidants
Potencies relative to ?-tocopherol
Structure
Corn oil
Soybean oil
mg/kg
mg/kg
? -Tocopherol
75-100
70-300
(100)
? -Tocopherol
0-20
10-15
(25-40)
? -Tocopherol
660-700
680-1050
(1-11)
? -Tocopherol
10-70
190-300
(1)
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Nutritional antioxidants
Potencies relative to ?-tocopherol
Structure
Palm oil
Soybean oil
mg/kg
mg/kg
? -Tocotrienol
1.5
220
(28)
? - Tocotrienol
-
1.3
(5)
? - Tocotrienol
-
150
(-)
? - Tocotrienol
20
0.3
(-)
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Nutritional antioxidants
Why is ?-tocopherol the important one?
TBP
Chilomicron
Tissues
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The predominant vitamin E form in soya oils is
?-tocopherol, and hence the most abundant form
in the diet. Its not the right one!!!!!
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Nutritional antioxidants
Vitamin C (ascorbic acid)
Discovered in 1932 Major water soluble
antioxidant Directly scavenges many ROS May
regenerate vitamin E!
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Nutritional antioxidants
Regeneration of vitamin E essential!
Vit E radical!
Not definitely shown to happen in vivo
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Nutritional antioxidants
Ascorbic acid
Most animals synthesise Vit C We have congenital
absence of gulonolactone oxidase Main food
sources are fruit and vegetables Deficiency
symptoms eg scurvy, capillary fragility
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Carotenoids

• large family of 700 yellow, orange and red
  pigments
• C-40 hydrocarbons with extensive double bonds
• Synthesised by plants, not animals
• About 15 found in human plasma. Main ones are

Int Std.
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Carotenoids-antioxidant role?

• Antioxidant at low O2 tension
• Highly effective at neutralising singlet oxygen
• Can interrupt lipid peroxidation chain reactions
• Debate as to whether are antioxidants in vivo

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Lecture Plan

• The concept of disease prevention
• Free radicals and the theory of oxygen toxicity
• What are antioxidants?
• Main nutritional antioxidants?
• Do they have a role in disease prevention?

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Concept of oxidative stress
Heart disease, Arthritis, Muscular Dystrophy,
Alzheimers, Retinopathy, Cancer,
Parkinsons, Cataracts, Diabetes, Hypertension
etc.
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Antioxidants Disease
Do antioxidants prevent disease?
Huge sales hype ! BUT WHATS THE EVIDENCE?
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Antioxidants Disease
Evidence ?

• Epidemiology
• Mechanistic
• 3. Intervention studies

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Antioxidants Disease
Epidemiology
But causal ??
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Antioxidants Disease
Mechanistic cholesterol oxidation
High O2 tension
Epithelium of wall of blood vessel
Circulating monocytes
LDL
LDL
Infiltrate damage to epithelium fatty
streaks, atherosclerosis
Receptor specific uptake
Lipid leaden macrophage
LDL
LDL
R
Oxidised LDL
Oxidised LDL
Foam cell
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Antioxidants Disease
Mechanistic cholesterol oxidation
Oxidation occurs when antioxidants low
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Antioxidants Disease
Mechanistic DNA damage
Antioxidant
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Meta-analysis of randomised trials
No clear benefit of antioxidant supplementation
for CHD
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Meta-analysis of randomised trials
No clear benefit of antioxidant supplementation
for cancer eg.
Antioxidant supplements for prevention of
gastrointestinal cancers a systematic review and
meta-analysis Goran Bjelakovic, MD, Dimitrinka
Nikolova MA, Rosa G Simonetti, MD and Christian
Gluud, MDa
Lancet 364, 2004
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Meta-analysis of randomised trials
Antioxidants may even increase overall
mortality!!!
Meta-Analysis High-Dosage Vitamin E
Supplementation May Increase All-Cause Mortality
Edgar R. Miller III,et al 2004 Annals of Internal
Medicine Volume 142
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Antioxidants Disease
Why have trials failed to show clear benefit?
Too high dose? Pro-oxidant effects? Wrong target
group? Wrong theory?
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• Summary
• ROS mediated oxidation of biomolecules is
  implicated in many diseases
• A sophisticated antioxidant defence system
  minimises oxidation
• Some antioxidants are required from the diet eg
  trace metals, vitamin E, vitamin C.
• Nutritional antioxidants are essential for health
• Evidence for benefits of high doses in preventing
  heart disease and cancer contradictory
• Dont megadose!

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Recommended reading
Duthie GG (2000) Vitamin E and its antioxidant
role in relation to other dietary components. In
Human Nutrition and Dietetics 10th Edition, (eds
Garrow JS, James WPT Ralph A), Churchill
Livingstone London, 226-236. Duthie GG (1999)
Determination of activity of antioxidants in
human subjects. Proceedings of the Nutrition
Society. 58, 1015-1024.