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 tocopherols In 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, A R1 = R2 = R3 = Me, α -tocopherol R1 = R3 = Me; R2 = H, β -tocopherol R1 = H; R2 = R3 = Me, γ -tocopherol R1 = R2 = H; R3 = Me, δ -tocopherol B R1 = R2 = R3 = Me, α -tocotrienol R1 = R3 = Me; R2 = H, β -tocotrienol R1 = H; R2 = R3 = Me, γ -tocotrienol R1 = R2 = H; R3 = Me, δ -tocotrienol

Vitamin E activity and bioavailability α-tocopherol has the highest bioavailability Serum 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 tocopherols Exclusively 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

Vitamin E function – as antioxidant Antioxidants protect cells from the damaging effects of free radicals Free radicals damage cells, contributing to the development of cardiovascular disease and cancer Structure-function relationship In vitro, antioxidant potential of T-3 > toco Generally 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 tocos to have neuroprotective effects to inhibit cholesterol synthesis to 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 properties T-3 in μM suppresses HMG-CoA reductase, reducing cholesterol synthesis T-3s are more potent antioxidants than tocos due to their efficient penetration across membrane Although 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 disease Observational 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 events The HOPE-TOO follow-up study (4,000 participants for another 2.5 years) - no protection against CHD A men's cardiovascular study (15,000 healthy physicians ≥50 years with 400 IU for 8 y) - no effect on major cardiovascular events Clinical trials have not provided evidence that vitamin E prevents cardiovascular disease. More studies in younger and healthier participants taking higher doses is needed (

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 benefit A study on prostate cancer (29,000 men) - no effect A large clinical trial against prostate cancer (7-12 years of 400 IU E ) – no protection in healthy men An epidemiologic study showed reduced risk of death from bladder cancer The inconsistent and limited evidence precludes any recommendations about using vitamin E supplements to prevent cancer

Vitamin E on cognitive function Brain - 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 Alzheimer patients on 2,000 IU/d for 2 y) - significantly delayed deterioration Vitamin E consumption - less cognitive decline over 3 y of elderly, ( 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 60 o C; 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?

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): ,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 Diet Algae Biomass (g/kg)* Palm Extract (g/kg)** ppm Expected (relative to egg oil) A000 B ,081 C ,245 D ,490 *Astaxanthin is 1.35% in biomass; **Tocotrienols and tocopherols are 50% in extract

Diet Fresh Yolk Cooked Yolk Day 0Day 3Day 6Day 9Day 42Day 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?