Tong Wang, Professor in Lipid Chem and Applications

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Tocopherols and tocotrienols structure,
function and enrichment in eggs

• Tong Wang, Professor in Lipid Chem and
  Applications
• FSHN, Iowa State University
• Sept 13, 2012

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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
  a-, ß-, ?- and d-tocopherol and a-, ß-, ?- and
  d-tocotrienol (T-3)

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Structure of toco and T-3
A R1 R2 R3 Me, a-tocopherol R1 R3 Me R2 H, ß-tocopherol R1 H R2 R3 Me, ?-tocopherol R1 R2 H R3 Me, d-tocopherol B R1 R2 R3 Me, a-tocotrienol R1 R3 Me R2 H, ß-tocotrienol R1 H R2 R3 Me, ?-tocotrienol R1 R2 H R3 Me, d-tocotrienol

• ,

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Vitamin E activity and bioavailability
Activity, IU/mg
Natural a-toco 1.49
Synthetic a-toco (racemic mix) 1.0
b-toco 0.6
g-toco 0.3
d-toco 0.015

• a-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)

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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)

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Toco concentration of selected oils
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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 gt toco
• Generally recognized that d gt g gt a
• However, data on the effects of vitamin E on
  biomarkers of oxidative stress in vivo are
  inconsistent

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Vitamin E function not as antioxidant (AOCS
Lipid Library)

• a-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

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

• a-Tocopherol strongly inhibits platelet adhesion
• ?-Tocopherol exhibits anti-inflammatory
  functions, not shown by a- tocopherol
• Epidemiological data suggests that ?-tocopherol
  is a better negative risk factor for certain
  types of cancer
• Vitamin E modulates arachidonic acid metabolism
  increasing prostacyclin that dilates blood
  vessels

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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 a-T-3 prevents neurodegeneration by
  regulating specific mediators of cell death
• T-3 but not tocopherol, suppresses growth of
  human breast cancer cells

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Activity of T-3 on breast cancer cells
(Guthrie and Carroll, 1998)
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Activity of T-3 on breast cancer cells
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Effect of T-3 on protein kinase C (PKC)
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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)

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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

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Vitamin E on cognitive function

• Brain - high oxygen and polyunsaturated fatty
  acids
• Free radical damage neurons, leads to cognitive
  decline and neurodegenerative diseases
• Vitamin Es 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

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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 _at_ 60oC Measured as peroxide value
(PV) Secondary products Smaller volatile
compounds from ROOH cleavage, measured by
conductivity using an OSI Instrument
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Research Q Will high tocols in corn oil have
pro-oxidant effect?
Dolde and Wang, J Am Oil Chem Soc (2011)
8813671372
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Conclusion Crude oil from corn expressing
tocotrienols at 4,2005,400 ppm exhibited no
prooxidant effects.
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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)
8817591765
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Lipid ROOH formation in corn oil at 60 C with
added a-tocotrienol
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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

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

• Astaxanthin is a powerful antioxidant10 x
  ß-carotene 300 x a-tocopherol
• Tocotrienols are cancer preventative and
  neuroprotective

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Feed enrichment
Diet Algae Biomass (g/kg) Palm Extract (g/kg) ppm Expected (relative to egg oil)
A 0 0 0
B 4.9 0.12 1,081
C 14.7 0.36 3,245
D 29.4 0.72 6,490
Astaxanthin is 1.35 in biomass Tocotrienols
and tocopherols are 50 in extract
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Diet Fresh Yolk Fresh Yolk Fresh Yolk Fresh Yolk Fresh Yolk Cooked Yolk
Diet Day 0 Day 3 Day 6 Day 9 Day 42 Day 40
A
B
C
D
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a-Tocotrienol Transfer
Astaxanthin Transfer
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Nutrient transfer efficiency
Relative transfer of a-T-3 to a-toco 17, 36,
and 41 for Diet B, C, D
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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.

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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?