BIOAVAILABILITY, METABOLISM, AND BIOEFFICACY OF TOMATO CAROTENOIDS DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Rachel Elizabeth Kopec Graduate Program in Ohio State University Nutrition Program The Ohio State University 2012 Dissertation Committee: Steven J. Schwartz, Ph.D., Advisor Steven K. Clinton, M.D., Ph.D. Mark L. Failla, Ph.D. Earl H. Harrison, Ph.D. Copyrighted by Rachel Elizabeth Kopec 2012 Abstract Carotenoids are non-polar compounds which impart the yellow, orange, and red hues to a number of commonly consumed fruits and vegetables. Some carotenoids, like β-carotene, serve as an important source of provitamin A in the human diet. In contrast, consumption of the carotenoid lycopene has been associated with a reduced risk of developing cardiovascular disease and cancer. Finally, the consumption of lycopene, in addition to the carotenoids phytoene and phytofluene, has been associated with a reduced severity of erythema after ultraviolet light exposure. A better understanding of the absorption, metabolism, and bioefficacy of these compounds is important for making future nutritional recommendations to ensure adequate vitamin A status and reduce the risk of developing specific diseases. Tomatoes have the unique ability to synthesize a variety of carotenoids, and novel varieties which accumulate β-carotene, lycopene, phytoene, and phytofluene, were used in our studies. Our first aim was to determine if the consumption of a β-carotene-rich tomato sauce or carrots (containing both β-carotene and α-carotene) with lipid would enhance the delivery of greater quantities of vitamin A as compared to consuming the same carotene rich food without lipid. This objective was accomplished by first developing an HPLC-MS/MS method to separate and identify the carotenes and fat soluble vitamins in the test meal, and then quantitating the immediate post-prandial plasma concentrations of ii β-carotene and vitamin A metabolites after healthy subjects consumed a meal with or without lipid-rich avocado in combination with a serving of tomato sauce. Consumption of the sauce with lipid-rich avocado increased β-carotene levels by 2.3 fold and increased retinyl esters (i.e. vitamin A) by 4.5 fold as compared to the same sauce meal without avocado. An important step in elucidating the potential bioactivity of lycopene is a better understanding of its metabolism in the human body. One approach in determining if and how lycopene is metabolized is to identify the oxidative or enzymatic products of lycopene. Our second aim was to determine whether oxidative products of lycopene are present in raw and processed tomatoes and other lycopene containing foods, in addition to blood plasma of humans who have consumed processed tomato juice. Numerous lycopene metabolites were identified and quantified in raw foods and food products containing lycopene. In addition, these lycopene metabolites were also identified and quantified in the blood plasma of healthy humans who had consumed a high-lycopene tomato juice for 8 weeks. Finally, the consumption of tomato paste and tomato oleoresin containing lycopene, phytoene, and phytofluene has been associated with reduced severity of erythema after ultraviolet light exposure in humans. Our third aim was to determine if the consumption of tangerine tomatoes, containing higher levels of the ultraviolet light absorbing carotenoids phytoene and phytofluene, would: 1) result in the accumulation of lycopene, phytoene, and phytofluene in a mouse model, and 2) reduce the incidence or severity of acute ultraviolet B irradiation-induced skin damage. An immunocompetent, iii SKH-1 hairless mouse model was used for this study. Animals consumed a freeze-dried tangerine tomato powder diet for ten weeks, and a subset of animals was irradiated with an acute dose of UVB light. Carotenoids accumulated in the skin of the animals which had consumed the tangerine tomato diet, and lower levels of phytoene, phytofluene, and lycopene were observed in animals which had been irradiated with UVB. Female mice consuming the tangerine tomato diet had reduced levels of DNA damage as compared to female animals consuming the control diet. Male mice consuming the tangerine tomato diet had reduced levels of inflammation and DNA damage as compared to male animals consuming the control diet. Collectively, these results add to the body of knowledge of factors affecting carotenoid absorption and metabolism, and provide important information necessary for a further understanding of potential bioefficacy of carotenoids. In addition, this work demonstrates that novel tomato varieties may serve as a vehicle for delivering higher levels of specific carotenoids to humans and animals for future studies. iv Dedicated to my family v Acknowledgements First and foremost, I want to thank my thesis advisor, Dr. Steven J. Schwartz, to whom I will be eternally grateful. His concern for the wellbeing of his students, his ability to encourage others with confidence and support, his wisdom, and the numerous opportunities he provides for scientific and professional growth have made this a very positive and rewarding experience. I would like to collectively thank my thesis committee for the time and thoughtful discussions which have developed my skills as a scientist, and for their various contributions to both thesis and non-thesis studies in which I have been involved. In addition, I would like to thank them each for their unique contributions to my experience. Dr. Failla’s dedication to mentoring and to high quality science always inspires me to be more for myself and to do more for others. I would like to thank Dr. Harrison for regularly sharing his depth of knowledge and passion for all things vitamin A and carotenoid related. Finally, I would like to thank Dr. Clinton, whose enthusiasm and vision for the role nutrition and dietary factors can play in disease prevention continues to inspire me. I am truly indebted to a great number of people for all of acts of kindness and support I have received throughout this process. I would like thank Dr. Tatiana Oberyszyn and her research group for welcoming me with open arms and for sharing vi their research expertise. I would like to thank Dr. Yael Vodovotz and Dr. David Francis, who have been strong sources of support and have helped guide me at key points during my studies. I want to thank Dr. Ferhan Ozadali, Marjory Renita, and Casey Philbin who served as important mentors early on in the research process and were very influential in my selection of this area of study. I am especially thankful to Dr. Ken Riedl for his countless hours of training and passing of knowledge of all things mass spectrometry related. I would also like to thank all of the past and present Schwartz lab members - whose enthusiasm and friendship has made this educational experience very enjoyable, and who have always challenged me to be a better student and teacher. Finally, I would like to thank my parents and my grandparents, who encouraged me to follow my own path, and whose continuous love and guidance has been with me the entire way. vii Vita September 13, 1984 .......................................Born – Toledo, Ohio 1998-2002 ......................................................Eastwood High School 2002-2006 ......................................................B.S. Honors, Pharmaceutical Sciences, The Ohio State University 2002-2006 ......................................................B.S. Business Administration, Finance Specialization, The Ohio State University 2006 to present ..............................................Graduate Research Associate, Department of Human Nutrition, The Ohio State University Publications 1. Schweiggert, R.M., Kopec, R.E., Villalobos, M.G., Ramirez, L., Högel, J., Quesada, S., Esquivel, P.E., Schwartz, S.J., Carle, R. 2012. Carotenoids are more bioavailable from papaya than from tomato and carrot in humans. In Preparation 2. Garzon, G.A., Narvaez C., Kopec, R.E., Riedl, K., Schwartz, S.J. 2012. Characterization and quantification of carotenoids in Arazá (Eugenia stipitata McVaugh) an Amazonian fruit. Journal of Agricultural and Food Chemistry. 60(18):4709-4717 3. Eroglu, A., Hruszkewycz, D.P., dela Sena, C., Narayanasamy, S., Riedl, K.M., Kopec, R.E., Schwartz, S.J., Curley, R.W.Jr., Harrison, E.H. 2012. Naturally- occurring eccentric cleavage products of provitamin A β-carotene function as antagonists of retinoic acid receptors. Journal of Biological Chemistry. 287(19):15886-15895 viii 4. Pickworth, C.L., Loerch, S.C., Kopec, R.E., Schwartz, S. J., Fluharty, F.L. 2012. Concentration of pro-vitamin A carotenoids in common beef cattle feedstuffs. Journal of Animal Science. 90(5):1553-1561 5. Barona, J., Jones, J.J., Kopec, R.E., Comperatore, M., Andersen, C., Schwartz, S.J., Lerman, R.H., Fernandez, M.L. 2012. A Mediterranean-style low-glycemic- load diet increases plasma carotenoids and decreases LDL oxidation in women with metabolic syndrome. Journal of Nutritional Biochemistry. 23(6):609-615 6. Kopec, R.E., Cooperstone, J.L., Cichon, M., Schwartz, S.J. Analysis Methods of Carotenoids. In: Analysis of Antioxidant-Rich Phytochemicals. Z. Xu and L. Howard, eds. Oxford, England: Wiley-Blackwell, 2012: 105-148 7. Gupta, R., Kopec R.E., Balasubramaniam, V.M., Schwartz, S.J. 2011. Combined pressure-temperature effects on carotenoid retention and bioaccessibility in tomato juice. Journal of Agricultural and Food Chemistry. 59(14):7808-7817 8. Fleshman M.K, Lester, G.E., Riedl, K.M., Kopec, R.E., Narayanasamy, S., Curley, R.W. Jr,, Schwartz, S.J., Harrison, E.H. 2011. Carotene and novel apocarotenoid concentrations in orange-fleshed Cucumis melo melons: determinations of β-carotene bioaccessibility and bioavailability. Journal of Agricultural and Food Chemistry. 59(9):4448-4454 9. Kopec, R.E., Schwartz, S.J., Hadley, C. Lycopene. In: Encyclopedia of Dietary Supplements. P. Coates and M. Blackman, eds. 2nd Ed. New York, NY: Informa Healthcare USA, Inc., 2010:504-517 10. Story, E.N., Kopec, R.E., Schwartz, S.J., Harris, G.K. An update on the health effects of tomato lycopene.