β-Carotene Absorption and Metabolism Dissertation Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By: Matthew K. Fleshman The Ohio State University Nutrition Graduate Program The Ohio State University 2011 Dissertation Committee: Earl H. Harrison, Ph.D., Advisor Mark Failla, Ph.D. Steven J. Schwartz, Ph.D. Ouliana Ziouzenkova, Ph.D. Copyright by Matthew K. Fleshman 2011 Abstract β-Carotene is the most potent provitamin A carotenoid and has potential antioxidant properties. Vitamin A is an essential nutrient that plays a critical role in many biological systems. In many parts of the world provitamin A carotenoids such as β-carotene are the primary source of vitamin A. A better understanding of the bioaccessibility, absorption, and metabolism of β-carotene may help alleviate symptoms and diseases related to vitamin A deficiency. To better understand β-carotene absorption, we have studied β-carotene in fruits and vegetables, its bioaccessibility from food sources through in vitro digestion, and absorption by humans in a controlled feeding study. We also studied the metabolism of β-carotene by measuring the presence of β-apocarotenoids in foods, mice, and humans, as well as the conversion of newly absorbed β-carotene to its retinoid metabolites. The first study was conducted to determine the β-carotene content and its bioaccessibility/bioavailability in orange-fleshed melons. Orangedew melons have less consumer risk for food borne illness and have significantly more β-carotene than cantaloupes grown under the same conditions. Micellerization of β-carotene during simulated digestion of orange-fleshed melons was approximately 3.2%. We also detected and quantified β-apocarotenoids in the melons. ii The second study was conducted to investigate in humans the variability in β- carotene absorption and its conversion to vitamin A and to compare the efficiency of absorption of β-carotene with that of cholesterol. Ten men consumed a 5 mg dose of deuterium labeled β-carotene (d8-βC), with 6 subjects repeating the dose 2 months later. For this study, we developed a method that provides easier sample preparation than previous β-carotene feeding studies, and allows us to detect and quantify newly absorbed d8-β-carotene, as well as its d4-retinyl ester metabolites, using high performance liquid chromatography-mass spectrometry (HPLC-MS). We employed a simple sample extraction for both retinoids and β-carotene that included minimal sample handling. D8-β-carotene and its d4-retinyl ester metabolites were analyzed using the same liquid chromatography system and solvents for separation of the compounds. The method allowed us to accurately measure d8-β-carotene absorption, and its extent of conversion to d4-retinyl esters. There was marked inter-individual variability in β- carotene absorption and conversion to retinyl-esters. In contrast, intra-individual variability in β-carotene absorption and its conversion to retinyl esters was low. β- Carotene and cholesterol may share specific intestinal transporters, but there was no correlation between an individual’s efficiency of absorption of β-carotene and his efficiency of absorption of cholesterol (r=-0.09; p=0.81). The third study was conducted to determine the β-apocarotenoid levels in commonly consumed products containing β-carotene, such as fruits and vegetables, as well as in murine serum and liver, and human plasma. β-Apocarotenoids are present in our diets but their absorption, metabolism, and biological roles are largely unknown. iii Using HPLC-MS, we were able to detect and quantify β-apo-13-carotenone, β-apo-14’- carotenal, β-apo-12’-carotenal, β-apo-10’-carotenal, β-apo-8’-carotenal and β-carotene in several fruits and vegetables. In vitro digestion studies have shown that β- apocarotenoids are micellerized to a greater extent than β-carotene. We have shown here that they are present in considerable amounts in the diet and that they may be absorbed in addition to being generated in vivo. We have also shown that these β- apocarotenoids are present in the liver and serum of β-carotene fed mice and in human plasma. β-Carotene utilization involves many complex processes both at the level of absorption and metabolism. We have uncovered new aspects of β-carotene absorption and metabolism that should be considered in future β-carotene studies, such as conversion of β-carotene to specific retinyl esters, low intra-individual variability of β- carotene absorption, and the presence of β-apocarotenoids in all β-carotene containing samples. iv Acknowledgements This research could not have been accomplished without the generous help and support of many people. First and foremost, I would like to express my sincerest appreciation to my mentor, Dr. Earl H. Harrison, for his support and guidance, and for sharing his vast knowledge of my dissertation topic over the past five years. I would like to thank Dr. Steven J. Schwartz for allowing me to collaborate with his laboratory on the majority of my studies, for his expertise and for serving on my committee. I would like to thank Dr. Mark Failla for challenging my scientific mind, his expertise, and for serving on my committee. I would also like to thank Dr. Ouliana Ziouzenkova for her valuable expertise and for serving on my committee. I am grateful to my colleagues and friends Dr. Rebekah S. Marsh, Dr. Ken Riedl, Dr. Sagar Thakar, Dr. Julie Chitchumroonchockchai, Vanessa Reed, Rachel E. Kopec, and Abdulkerim Eroglu. I feel very fortunate to have worked with, and learned from, each of you. I also thank other Harrison laboratory members, GSNS members and the multitude of other graduate students that have helped me throughout the years. I am especially grateful to my parents, Jude and Naomi Fleshman for whom none of this would be possible, and my siblings; Gene, Dan, Jimmy, David, Theresa, Stephen, Nancy, Mark, Andrew, Joann, Michele, Sharon, and their spouses. v Vita 2003 ......................................................... B.S. (Nutrition), The Ohio State University 2006-2007 ........................... Graduate Teaching Associate, The Ohio State University 2007-2011 ........................... Graduate Research Associate, The Ohio State University Publications Igor Shmarakov, Matthew K. Fleshman, Diana N. D’Ambrosio, Roseann Piantedosi, Ken M. Riedl, Steve J. Schwartz, Robert W. Curley, Jr., Johannes von Lintig, Lewis P. Rubin, Earl H. Harrison, and William S. Blaner. Hepatic Stellate Cells are and Important Cellular Site for β-Carotene Conversion to Retinoid. Arch. Biochem. Biophys. (2010), doi:10.1016/j.abb.2010.05.010. Matthew K. Fleshman, Gene E. Lester, Ken M. Riedl, Rachel E. Kopec, Sureshbabu Narayansamy, Robert W. Curley, Jr., Steven J. Schwartz, and Earl H. Harrison. (2011) Carotene and Novel Apocarotenoid Concentrations in Orange-fleshed Cucumis melo Melons: Determinations of β-Carotene Bioaccessibility and Bioavailability. J Agric. Food Chem. (2011), doi: 10.1021/jf200416a. Field of Study Major Field: The Ohio State University Nutrition Graduate Program vi Table of Contents Contents Page Abstract ..................................................................................................................... ii Acknowledgement ..................................................................................................... v Vita .......................................................................................................................... vi List of Tables............................................................................................................ ix List of Figures ........................................................................................................... x List of Abbreviations ............................................................................................... xii Chapter 1 Literature Review 1.1 Introduction ......................................................................................................... 1 1.2 Carotenoids.......................................................................................................... 3 1.3 Health Benefits of Carotenoids ............................................................................ 4 1.4 Health Benefits of Vitamin A ............................................................................... 4 1.5 Sources of β-Carotene .......................................................................................... 9 1.6 Absorption of β-Carotene..................................................................................... 9 1.7 Bioavailability of β-Carotene ............................................................................. 12 1.8 Human Studies................................................................................................... 13 1.9 β-Apocarotenoids .............................................................................................. 16 1.10 Aims ................................................................................................................ 17 Chapter 2 Carotene and Novel Apocarotenoid Concentrations in Orange-fleshed Cucumis melo Melons: Determinations of β-Carotene Bioaccessibility and Bioavailability 2.1 Abstract ............................................................................................................. 21 2.2 Introduction ....................................................................................................... 22 2.3 Materials