β-Apocarotenoids: Occurrence in Cassava Biofortified with β-Carotene and Mechanisms of Uptake in Caco-2 Intestinal Cells THESIS Presented in Partial Fulfillment of the Requirements for the Degree Master of Science in the Graduate School of The Ohio State University By Boluwatiwi O. Durojaye Graduate Program in Human Nutrition The Ohio State University 2015 Master's Examination Committee: Earl H. Harrison, Ph.D., Advisor Steven J. Schwartz, Ph.D. Joshua A. Bomser, Ph.D. Copyrighted by Boluwatiwi O. Durojaye 2015 Abstract Biofortification is defined as the enrichment of staple crops with essential micronutrients. At present, it is one of the strategies used to alleviate vitamin A deficiency (VAD) by breeding staple crops with β-carotene. Staple crops that have been successfully biofortified with β-carotene under the HarvestPlus program are cassava, maize (corn) and sweet potato. Recently, β- apocarotenoids have been identified and quantified in cantaloupe melons and orange-fleshed honeydew. These cleavage products of β-carotene are formed by chemical and enzymatic oxidations. However, there are no detailed analyses of these compounds in biofortified foods and little is known about their bioavailability and intestinal absorption. Hence, this research focused on the analysis of β- apocarotenoids in biofortified cassava and kinetics of cell uptake and metabolism of β-apocarotenoids. In the first study, we analyzed the β-carotene and β-apocarotenoids content of conventionally bred cassava biofortified with β-carotene. Using a previously described high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method, we identified and quantified β-apo-13- carotenone, β-apo-14′-carotenal, β-apo-12′-carotenal, β-apo-10′-carotenal, and β-apo-8′-carotenal in hexane/acetone extracts of raw, boiled, and fried roots of ii the two biofortified cassava varieties investigated. The levels of β-apocarotenoids in roots of non-biofortified cassava varieties were lower than those of biofortified varieties and some of these values approached the limit of detection (LOD) or limit of quantification (LOQ). The purpose of the second study was to determine kinetics of uptake and metabolism of β-apocarotenoids using Caco-2 intestinal cells as a model. We hypothesized that these compounds are directly absorbed from the diet similarly to β-carotene. Pure β-apocarotenoids were delivered to fully differentiated monolayers of Caco-2 cells using tween-40 micelles. Carotenoids were extracted from media and cells with acetone:hexanes (1:4 v/v) and analyzed by HPLC. We observed that there was rapid uptake of β-apo-8′-carotenal into cells. We detected two unidentified metabolites (X and Y) of β-apo-8′-carotenal. The formation of compound X increased with time corresponding with a decrease in β-apo-8′-carotenal. Also, cellular uptake of β-apo-13-carotenone was rapid and this compound was extensively degraded over time. Understanding the mechanisms of absorption and metabolism of β- apocarotenoids relative to their quantities in foods is critical in exploring the functions of these metabolites, some of which have been shown to be potent antagonists of vitamin A. Keywords: Biofortification; HPLC-MS/MS; cell uptake; carotenoids metabolism iii Acknowledgements First and foremost, I would like to express my sincere gratitude to my advisor and mentor, Dr. Earl H. Harrison. Thank you for the funding, support and constructive criticism throughout this research project. Also, I would like to thank Dr. Steven J. Schwartz for serving on my thesis advisory committee and for his expertise and collaboration on this project. I also would like to thank Dr. Joshua A. Bomser for serving on my committee. I am also grateful to Dr. Mark L. Failla for his expertise and Dr. Chureeporn Chitchumroonchokchai (Julie C) for her important laboratory training and contributions to the success of this project. To present and past members of the Harrison laboratory group – Carlo, Sara, Yan and Vanessa, thank you for the training and time spent to troubleshoot numerous unsuccessful experiments. It has been a pleasure working with you all. I am most especially grateful to my parents Kolawole and Olufeyisara Durojaiye for whom none of this would be possible. Thank you for your prayers, support and words of encouragement that got me through my most difficult days. iv Vita Education 2013-Present...……..……………M.S. Human Nutrition, The Ohio State University 2012-2014.……….………………...………M.Sc. Biochemistry, University of Lagos 2007-2010…………………..……B.Sc. (Honors) Biochemistry, University of Lagos Experience and Awards 2014-Present…………...Graduate Teaching Associate, The Ohio State University 2013-Present……………..EHE Graduate scholarships, The Ohio State University 2013…………………...University Graduate Fellowship, The Ohio State University Fields of Study Major Fields: Human Nutrition; Biochemistry v Table of Contents Abstract .................................................................................................................ii Acknowledgements ..............................................................................................iv Vita ....................................................................................................................... v Table of Contents .................................................................................................vi List of Tables ........................................................................................................ x List of Figures .......................................................................................................xi List of Abbreviations ........................................................................................... xiv Introduction ........................................................................................................... 1 Specific Aims .................................................................................................... 4 Aim 1 ............................................................................................................. 4 Aim 2 ............................................................................................................. 5 Chapter 1. Review of Literature ....................................................................... 6 1.1 Biofortification as a strategy to alleviate VAD .......................................... 7 1.2 Methods of biofortification for provitamin A cassava ............................... 9 1.3 Retention, bioaccessibility and bioavailability of β-carotene in provitamin A cassava ....................................................................................................... 11 vi 1.4 Metabolism of β-carotene ...................................................................... 14 1.4.1 Digestion ......................................................................................... 14 1.4.2 Intestinal absorption ........................................................................ 15 1.4.3 Cellular Uptake ............................................................................... 18 1.5 Cleavage products of β-carotene: How are they formed? ..................... 19 1.5.1 Chemical reactions of carotenoids yielding β-apocarotenoids ........ 19 1.5.2 Enzymatic cleavage of carotenoids................................................. 20 1.6 Occurrence of β-apocarotenoids in foods and in vivo ............................ 23 1.7 Biological activity of β-apocarotenoids .................................................. 24 Chapter 2. Analysis of β-carotene and β-apocarotenoids in cassava biofortified with β-carotene ................................................................................. 28 2.1 Abstract ................................................................................................. 29 2.2 Introduction ............................................................................................ 30 2.3 Materials and Methods .......................................................................... 35 2.3.1 Chemicals and Supplies ................................................................. 35 2.3.2 Processing of cassava varieties ...................................................... 35 2.3.3 Extraction and HPLC-MS/MS analysis of β-carotene and β- apocarotenoids ............................................................................................ 37 2.4 Results .................................................................................................. 40 vii 2.4.1 Profile of β-apocarotenoids in cassava samples ............................. 40 2.4.2 Determination of total content of β-carotene and β-apocarotenoids in cassava ....................................................................................................... 44 2.4.3 Effect of boiling and frying on β-apocarotenoids content ................ 48 2.5 Discussion ............................................................................................. 54 2.6 Conclusion ............................................................................................. 56 2.7 Acknowledgements ............................................................................... 56 Chapter 3. Kinetics of uptake and metabolism of β-apo-8′-carotenal and β- apo-13-carotenone by Caco-2 human intestinal cells ......................................... 57 3.1 Abstract ................................................................................................. 58 3.2 Introduction ...........................................................................................