New Findings on antioxidants Super Antioxidants

Taiwan NuSkin presentationMedical SymposiumNovember 3, 2007

Prof. Lester Packer

Prostate Cancer Rate

Shanghai born Chinese 1.7

China born Chinese

(live in US < 5 yrs)1.8

China born Chinese

(live in US > 5 yrs)23

US born Chinese 37

US born Caucasian 58

US

China

Asiancountries

Westerncountries

Int.J. Cancer (2000) 85:60-7Courtesy Emily Ho

Cross Cultural Study of Heart Disease Mortality

Oxidative StressOxidative Stress

• Free radicals cause oxidative stress

• Leads to tissue damage, aging and disease

ROS formationRedox Signaling

Oxidative StressDamage vs. RepairAdaptive Signaling

ApoptoticSignaling Necrosis

Oxidant burdenOxidant burden

ROS formationRedox Signaling

Oxidative StressDamage vs. Repair Adaptive Signaling

ApoptoticSignalingOxidant burden

Oxidant burdenNecrosis

Oxidant induced cell signaling and damage

Oxidative Stress

“An imbalance between oxidants and antioxidants in favor of the oxidants, leading to a disruption of redox signaling and control and/or molecular damage.”

Jones, D.P. Redefining oxidative stress. Antioxid. Redox Signal. 2006, 8: 1865-1879.

Glutathione is the cell’s major antioxidant

GSSG + NADPH 2 GSH + NADP+

Gluthathione Peroxidase

Gluthathione Reductase

GSSG oxidized

GSH reduced

2 GSH + H2O2 GSSG + 2 H2O

Oxidative Stress can be estimated from the ratio of reduced to oxidized antioxidants

Proliferation

Apoptosis

Differentiation

Ratio of GSH/GSSG becomes progressively oxidized during the life cycle of cells

-150-150

Kirlin WG, Cai J, Thompson SA, Diaz D, Kavanagh TJ, Jones DP. Glutathione redox potential in response to differentiation and enzyme inducers.Free Radic Biol Med. 1999 Dec;27(11-12):1208-18.

-250

-200

-250

-200 1:1

1:10

10:1

100:1

Redox State

-(SH)2:-SS-

Eh,

mV

Increased Carotid Intima Media Thickness

Cigarette Smoking

Type 2 Diabetes

Reversible myocardial Ischemia perfusion injury

PathophysiologyOxidative Stress-80 mV

-20 mV(-80 mV)

-50 mV(-110 mV)

Cys/CySS Redox(GSH/GSSG Redox)

(-140 mV)

Jones D.P., Antioxid Redox Signal. 2006 Sep-Oct;8(9-10):1865-79.

• Vitamins C and E

• Polyphenols (Flavonoids)

• Low molecular weight thiols:

Cysteine, Methionine, Glutathione, R- Lipoic Acid,

• Co-enzyme Q

• L-Carnitine

Antioxidants

•Carotenoids

Redox

Radical Scavenging

The Antioxidant Network in Membranes

Vit. E•cycle

Q10 H •cycle

Vit. C•cycle

Lipid Free Radicals

LOO• LO•Carotenoids

Metabolism

FatSoluble

Water Soluble

Water Soluble

OxygenMetabolism

Inducers Of Phase 2 enzymes and proteins

Dinkova-Kostova, A.T., Holtzclaw, W.D., Cole, R.N., Itoh, K., Wakabayashi, N., Katoh, Y., Yamamoto, M., Talalay, P., PNAS USA, 99(18):11908-13, 2002.Fahey JW, Stephenson KK, Dinkova-Kostova AT, Egner PA, Kensler TW, Talalay P.Carcinogenesis 2005 Jul;26(7):1247-55

“The capacity of these compounds to induce the phase 2 response depends upon their ability or that of their metabolites to react with thiol groups, a property shared with all other classes of phase 2 inducers, which show few other structural similarities”

INDUCERINDUCER

IND

UC

ER

IND

UC

ER

CYTOPLASM

smallMaf

NUCLEUS

Keap 1

Keap 1

Nrf2

Nrf2

Nrf2

S S

S S

Phase 2 Genes

or

or

Keap 1

S SSH SHKeap 1

ARE

Inducers of Nrf2 transcription, the phase II response, all have in common the ability to react with suphydryl groups

PolyphenolsBioflavanoidsChlorophyllCarotenoids

Heme, H2O2, NO● Carotenoid oxidation products

Chemicals reacting with Sulphydryl groups, e.g.,

Isothioyonates (sulforaphane), Dithiolanes,

1,2-dithiol-3-thiones, α-Lipoic acid

HO-l

GCL

GSHincreases

Mn Superoxide Dismutase

Thioredoxin, Trx reductaseMitochondrial and other

Antioxidant enzymes

Alkyl and Aryl Halides, Metal Ions, e.g., HgCl2, CdCl2

NAD(P)H-Quinone reductase,Ferritin, Epoxide hydrolase

GSH, Gluconic acid,methyl and other transferases

Antioxidant Defense, Detoxification, Chemoprevention

Nrf2-Keap-1system(ARE/EpRE)

• Inducers of enzymes & proteins

needed for antioxidant defense

Carotenoids in Humans are

• Vitamin A precursors

cyclic hydrocarbon carotenoids

• Free radical scavengers

protect against oxidation

Carotenoids in fruits & vegetables

Lycopene (20-40%)

β-cryptoxanthin (13-20%)

Lutein (10-20%)

carotene (5-10%)

-carotene (15-30%)

Zeaxanthin (1-5%)

Spinach, Green peas, Green Beans , Lima Beans Broccoli, Brussel sprouts, Cabbage, kale, Kiwi, Lettuce

Carrots, Pumpkin, Sweet Potatoes, Apricots, Cantaloupe, Watermelon

Tomatoes, Pink Grapefruit

Carotenoids in Human Plasma

Raman ScatteringSir C. V. Raman, Nobel Prize in Physics 1930

Carotenoids shift blue laser light to green:

from 473 nm to 510 nm

Laser light

Raman scattered light

Raman scattering studies are used to measure carotenoids in the Eye and to diagnose age-

related macular degeneration

Macula

Lutein & Zeoxanthin

Gellermann W, Ermakov IV, McClane RW, Bernstein PS. Raman imaging of human macular pigments. Optics Letters. 2002;27:833

C. Smidt and D.S. Burke, Nutritional Significance and Measurement of Carotenoids, Current Topics in Nutraceutical Research 2004, 2, 79-91

0

20000

40000

60000

0 0.5 1 1.5 2 2.5 3 3.5 4

Total Serum Carotenoids (μg/ml)

Skin Raman Intensity (n = 372)

r = 0.84 (p<0.0001)

p<0.01 p<0.01 p<0.01

Std. Error 44

Std. Error 38

Std. Error 61

Std. Error 106

24793

26639

29704

31507

10000

15000

20000

25000

30000

35000

1 or less 2 to 3 4 to 5 6 or more

Courtesy Stephen Poole, Pharmanex

Skin Raman Intensity(2005 US data of 209,628 Non-Supplement Users)

Daily servings of fruits and vegetables

Lifestyle affects raman scattering intensity

4 mg Lutein

¼ cup of cooked spinach

1 & 1/3 medium carrots or ¼ cup pumpkin

4 mg -Carotene 4 mg Lycopene

1 medium tomatoor ¾ Tsp tomato paste

Carotenoids in human supplements equivalent to amounts in food (courtesy KJ Yuem)

Plasma Carotenoid Concentration Is increased after Supplementation

Time, Day

D1 D15 D29 D43 D57

Pla

sma

To

tal

Car

ote

no

id (g

/dL

)

0

20

40

60

80

100

120

140

160

180Placebo Mixed Car Lutein -Carotene Lycopene

** *

**

***

**

*

**

*

* * *

*

*

Significantly different from baseline * p<0.05Courtesy KJ Yuem, et al.

Carotenoid Supplementation protects DNA Damage of human

lymphocytesThe comet assay

Day 57Day 1

(Subject # 35563)Courtesy KJ Yuem, et al.

Carotenoid Supplementation protects DNA damage in human lymphocytes

0.4

0.6

0.8

1.0

1.2

1.4

1.6

(D1/D1) (D15/D1) (D29/D1) (D43/D1) (D57/D1)

Placebo Mixed Car Lutein

-Carotene Lycopene

Comet tail factor ratio compared with day 1

Closed symbols indicate significant differences from Placebo group (p<0.05)

Courtesy KJ Yuem, et al.

Physiological doses of a β-carotene, lycopene, lutein or a mixture of these carotenoids protect against endogenous or hydrogen peroxide (H2O2 ) induced DNA damage in white human blood cell lymphocytes

Genes effected by carotenoids in lung tissue of cigaraette exposed exposed mice

Nrf2 Transcription

Inflammatory and immune responses

Metabolism and Ion channels

Receptors Growth factors Apoptosis UnknownGenes

Others

Genes Up or down regulated

Control diet

Β-carotene

Lycopene

Lutein

Mixed 3-Carotenoids-20

0

20

-20

0

20

-20

0

20

-20

0

20

-20

0

20

Gene chip studies show carotenoids…

… have specific effects on gene expression

… effect expression of genes for transcription factors which enhance the biosynthesis of antioxidant enzymes and proteins

… modify genes affecting xenobiotic metabolism and immune system response

Vitamin A levels are increased in healthy centenarians compared to younger subjects

Vit

amin

A (

µM

)

Centenarians< 60 yrs 60-79 yrs 80-99 yrs

153 Italian centenarians, * p < 0.0001

* * *

0

1

2

3

4

5

6

Christina Polidori, Eur J Clin Nutr 2007

• Substances in plant fruits and vegetables have a wide range of different molecular structures. Some like bioflavonoids are powerful direct free radical scavengers.

• These and other small molecules also can induce the biosynthesis of antioxidants.

• These are bifunctional antioxidants or “Super Antioxidants”

because they…

…scavenge free radicals directly and immediately

…but also act indirectly by inducing genes and thus have long lasting effects

Super Antioxidants