Tocopherols and tocotrienols – structure, function and enrichment in eggs

1. Tocopherols and tocotrienols – structure, function and enrichment in eggs Tong Wang, Professor in Lipid Chem and Applications FSHN, Iowa State University Sept 13, 2012

2. 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)

3. Structure of toco and T-3 ,

4. Vitamin E activity and bioavailability • α-tocopherol has the highest bioavailability • Serum concentration controlled by liver, which preferentially re-secretes only a-toco via the a-tocopherol transfer protein (TTP) • Bioavailability of a-, g-, and d- tocotrienol, is 28, 9, 9% (Yap et al 2003)

5. 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)

6. Toco concentration of selected oils

7. 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 d > g > a • However, data on the effects of vitamin E on biomarkers of oxidative stress in vivo are inconsistent

8. 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

9. 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

10. 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

11. Activity of T-3 on breast cancer cells (Guthrie and Carroll, 1998)

12. Activity of T-3 on breast cancer cells

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

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

15. 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

16. 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 - 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

17. 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

18. Research Q: Will high tocols in corn oil have pro-oxidant effect? Dolde and Wang, J Am Oil Chem Soc (2011) 88:1367–1372

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

21. Model system study - Toco & T-3 added to purified corn oil Research Q: Do tocos and T-3s have different antioxidant activities? Stripped RBD Corn Oil spiked with: • aToco • a T-3 • d Toco • d T-3 • g T-3 Concentration (ppm): • 100 • 250 • 700 • 2,000 • 5,000 Dolde and Wang, J Am Oil Chem Soc (2011) 88:1759–1765

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

24. 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

25. 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

26. Feed enrichment *Astaxanthin is 1.35% in biomass; **Tocotrienols and tocopherols are 50% in extract

28. α-Tocotrienol Transfer Astaxanthin Transfer

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

30. 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.

31. 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?