Tong Wang, Professor in Lipid Chem and Applications

FSHN, Iowa State University

Sept 13, 2012

Tocopherols and tocotrienols – structure, function and enrichment in eggs

History of tocopherolsIn 1905, Fletcher found if special factors were removed

from food, disease (Beriberi) occurred

Discovered in 1922 by Evans and Bishop

Supported fertility, thus tocopherol – Greek tokos = childbirth, phero = bring forth, ol = alcohol

In 1936, found abundant in wheat germ oil, in 1938, chemically synthesized

In nature, 8 found to have vitamin E activity: α-, β-, γ- and δ-tocopherol; and α-, β-, γ- and δ-tocotrienol (T-3)

Structure of toco and T-3

,

AR1 = R2 = R3 = Me, α-tocopherolR1 = R3 = Me; R2 = H, β-tocopherolR1 = H; R2 = R3 = Me, γ-tocopherolR1 = R2 = H; R3 = Me, δ-tocopherol

BR1 = R2 = R3 = Me, α-tocotrienolR1 = R3 = Me; R2 = H, β-tocotrienolR1 = H; R2 = R3 = Me, γ-tocotrienolR1 = R2 = H; R3 = Me, δ-tocotrienol

Vitamin E activity and bioavailability

α-tocopherol has the highest bioavailabilitySerum concentration controlled by liver, which preferentially

re-secretes only -toco via the -tocopherol transfer protein (TTP)

Bioavailability of -, -, and - tocotrienol, is 28, 9, 9% (Yap et al 2003)

Activity, IU/mg

Natural -toco 1.49

Synthetic -toco (racemic mix)

1.0

-toco 0.6

-toco 0.3

-toco 0.015

Biosynthesis of tocopherolsExclusively by photosynthetic organisms - in the

chloroplasts

Tocotrienols are the primary form in the seed of monocots (wheat, rice, and barley), palm fruits, GMO corn, not in vegetative tissues

Tocopherols occur in leaves and seeds of dicots

Transgenic expression of the barley enzymes resulted in significant accumulation of T-3 in corn (Pioneer seeds)

Toco concentration of selected oils Tocopherols (ppm) Tocotrienols (T-3, ppm)

Sources alpha beta gamma delta alpha beta gamma delta Total T-3

Total All

Palm Oil 152 - - - 205 - 439 94 738 890 Rice bran 324 18 53 - 236 - 349 - 585 980 Barley 350 50 50 - 670 120 120 - 910 1,360 Oat 180 20 50 50 180 - 30 - 210 510 Wheat Germ 1,179 398 493 118 24 165 - - 189 2,377 Corn 282 54 1,034 54 49 8 161 6 224 1,648 Corn Control 420 26 677 25 189 0 89 5 283 1,430 Corn HGGT 249 28 607 58 664 19 3,512 520 4,715 5,657

Vitamin E function – as antioxidant

Antioxidants protect cells from the damaging effects of free radicalsFree radicals damage cells, contributing to the development

of cardiovascular disease and cancer

Structure-function relationshipIn vitro, antioxidant potential of T-3 > tocoGenerally recognized that > > However, data on the effects of vitamin E on biomarkers of

oxidative stress in vivo are inconsistent

Vitamin E function – not as antioxidant (AOCS Lipid Library)

α-Tocopherol is a gene regulator, causing up-regulation of mRNA or protein synthesis

Vitamin E modulates the activity of several enzymes involved in signal transduction through influencing protein-membrane interactions

Stabilizing the structure of membranes, modulating the immune response

Tocotrienols have been shown properties different from tocosto have neuroprotective effectsto inhibit cholesterol synthesisto reduce the growth of breast cancer cells in

vitro Therapeutic potential for cancer, bone resorption,

diabetes, and cardiovascular and neurological diseases are being actively studied

Vitamin E in cell signaling, gene and metabolic regulation (Shen et al, 2006)

α-Tocopherol strongly inhibits platelet adhesion

γ-Tocopherol exhibits anti-inflammatory functions, not shown by α- tocopherol

Epidemiological data suggests that γ-tocopherol is a better negative risk factor for certain types of cancer

Vitamin E modulates arachidonic acid metabolism – increasing prostacyclin that dilates blood vessels

Functions and mechanism of T-3 (Shen et al, 2006)

Neuroprotective, antioxidant, anti-cancer and cholesterol lowering propertiesT-3 in μM suppresses HMG-CoA reductase, reducing

cholesterol synthesisT-3s are more potent antioxidants than tocos due to their

efficient penetration across membraneAlthough the transport T-3 is low, orally supplemented T-3

results in plasma T-3 of 1 μM, a conc of 10x higher than that required to protect neurons from damage

Nanomolar α-T-3 prevents neurodegeneration by regulating specific mediators of cell death

T-3 but not tocopherol, suppresses growth of human breast cancer cells

Activity of T-3 on breast cancer cells

(Guthrie and Carroll, 1998)

Activity of T-3 on breast cancer cells

Effect of T-3 on protein kinase C (PKC)

Clinical observations - vitamin E on coronary heart diseaseObservational studies associated lower rates of heart disease with r vitamin E

intake - 90,000 nurses had 30% to 40% lower heart disease. Finnish (5,133 followed for 14 years) had decreased mortality

Effects on the heart and blood vessels of healthy women (40,000 ≥45 years, 600 IU for 10 y) - no differences in cardiovascular events or mortality

Randomized clinical trials showed controversial results: The HOPE study (10,000 patients at high risk followed for 4.5 years, 400

IU/day) - no fewer cardiovascular eventsThe HOPE-TOO follow-up study (4,000 participants for another 2.5 years)

- no protection against CHDA men's cardiovascular study (15,000 healthy physicians ≥50 years with

400 IU for 8 y) - no effect on major cardiovascular eventsClinical trials have not provided evidence that vitamin E prevents

cardiovascular disease. More studies in younger and healthier participants taking higher doses is needed

(http://ods.od.nih.gov/FACTSHEETS/VITAMINE.ASP)

Vitamin E on cancer

Link between high intake of vitamin E with a decreased incidence of breast and prostate cancers is inconsistent: A study on breast cancer (18,000 women) - no benefitA study on prostate cancer (29,000 men) - no effectA large clinical trial against prostate cancer (7-12

years of 400 IU E ) – no protection in healthy menAn epidemiologic study showed reduced risk of death

from bladder cancerThe inconsistent and limited evidence precludes any

recommendations about using vitamin E supplements to prevent cancer

Vitamin E on cognitive functionBrain - high oxygen and polyunsaturated fatty acids

Free radical damage neurons, leads to cognitive decline and neurodegenerative diseases

Vitamin E’s protection supported by a clinical trial - 341 Alzheimer patients on 2,000 IU/d for 2 y) - significantly delayed deterioration

Vitamin E consumption - less cognitive decline over 3 y of elderly, (65-102 y)

A clinical trial in healthy older women (600 IU E for 4 years) - no apparent cognitive benefits

769 with cognitive impairment, 2,000 IU/day E - no significant differences in Alzheimer rate

Therefore, most research results do not agree on the use of vitamin E to maintain cognitive performance

Tocopherols and T-3 as antioxidants in bulk corn oil – A comparative study

Oxidative stability of corn oil with elevated T-3 and quantification of oxidation products:

Primary oxidation product:Development of hydroperoxides @ 60oC; Measured as

peroxide value (PV)

Secondary products:Smaller volatile compounds from ROOH cleavage,

measured by conductivity using an OSI Instrument

Dolde and Wang, J Am Oil Chem Soc (2011) 88:1367–1372

Research Q: Will high tocols in corn oil have pro-oxidant effect?

Peroxide Value of Crude Corn Oil at 60OC

0

20

40

60

80

100

120

140

0 1 2 3 4 5 6 7 8 9 10

Day

PV

2005 Control

2005 HGGT

2006 Control

2006 HGGT

R2 = 0.9826

R2 = 0.9972

R2 = 0.9962

R2 = 0.9976

ΔPV/day 2005 2006 Control 11.96 ± 1.06 9.19 ± 0.34 HGGT 9.83 ± 0.32 8.48 ± 0.27

Conclusion: Crude oil from corn expressing tocotrienols at 4,200–5,400 ppm exhibited no prooxidant effects.

Model system study - Toco & T-3 added to purified corn oil

Stripped RBD Corn Oil spiked with:

Toco T-3 Toco T-3 T-3

Concentration (ppm):1002507002,000 5,000

Dolde and Wang, J Am Oil Chem Soc (2011) 88:1759–1765

Research Q: Do tocos and T-3s have different antioxidant activities?

Lipid ROOH formation in corn oil at 60 C with added -tocotrienol

Conclusions of in vitro antioxidant study in bulk oil

Antioxidant properties of tocotrienols (T-3) are similar to those of tocopherols in vitro

At higher concentrations, alpha is a less effective antioxidant than delta and gamma

Alpha tocols propagated primary oxidation at concentrations as low as 700 ppm and pro-oxidant effects were increased with higher concentrations

Carotenoid and T-3 transport efficiency to eggs Walker and Wang, J. Agric. Food Chem. 2012, 60, 1989−1999

• Astaxanthin is a powerful antioxidant:10 x β-carotene; 300 x α-tocopherol

• Tocotrienols are cancer preventative and neuroprotective

Feed enrichment

DietAlgae Biomass

(g/kg)*Palm Extract

(g/kg)**

ppm Expected (relative to egg

oil)A 0 0 0B 4.9 0.12 1,081C 14.7 0.36 3,245D 29.4 0.72 6,490

*Astaxanthin is 1.35% in biomass;**Tocotrienols and tocopherols are 50% in extract

DietFresh Yolk

Cooked Yolk

Day 0 Day 3 Day 6 Day 9 Day 42 Day 40

A

B

C

D

α-Tocotrienol Transfer

Astaxanthin Transfer

Nutrient transfer efficiency

Relative transfer % of α-T-3 to α-toco: 17, 36, and 41% for Diet B, C, D

Conclusion remarks

• Tocopherols represent one of the most fascinating natural compounds that have the potential to influence a broad range of human health and disease;

• The current state of knowledge warrants study of the lesser known forms of vitamin E, i.e. tocotrienols, that have unique biological functions.

Discussion Qs on vitamin E

• Why there are significant disagreements among in vivo and in vitro experiments, and clinical studies– As antioxidants

– On other biological activities

• What type of evidence would you have your dietary recommendation based upon?