DEVELOPMENT OF ASSAYS FOR COENZYME Q10 AND VITAMIN K, AND THEIR APPLICATION IN CLINICAL TRIALS A thesis submitted in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Chemistry in the University of Canterbury by Sarah L Molyneux University of Canterbury 2006 Acknowledgments 2 Acknowledgments I would like to thank my supervisors, Dr Michael Lever, Professor Peter George, and Professor Murray Munro for their guidance and support during my time working on this thesis. I also owe a huge thank you to Dr Chris Florkowski for his endless patience, commitment, and direction towards this project. Thank you also to Canterbury Health Laboratories, the Foundation for Research Science and Technology, and Raj Chopra for providing me with funding. The statistics brilliance of Associate Professor Chris Frampton never ceases to amaze me, and you deserve a huge thank you for all your help, Chris. During my PhD I had the pleasure of supervising two very talented medical students, as summer students. Tim and Yasmin helped to conduct the CoQ10 bioavailability and dose-ranging studies, respectively. Thank you so much for your help, and I hope you got as much out of the experience as I did. I would like to thank Joanna Young, Professor Russell Scott, and Dr Chris Strey, for giving me the opportunity to join in their statin and heart failure study, it is an honour to be invited to collaborate with such high-class researchers. To everyone at Canterbury Health Laboratories – Chris McEntyre for his excellent technical assistance, to Malina, Martin, Madhu, Sandy, Jo, and Wendy for their understanding, support, listening, and distractions – you guys all made work a fun and enjoyable place to be. To the Core Biochemistry staff, you guys are amazing the way you kept smiling every time that I brought you another load of samples to be analysed, thank you. To staff in the Biochemistry Unit, thank you for your smiles and help – especially to Chris Sies, for your willing advice whenever it was requested. To the Chemistry department at UC – especially the marine group – thank you for your support and help, especially during the times when I turned up in a stressed heap and demanded use of a computer to fix my files! I feel so honoured to have been able to meet some really special people while working on my PhD, experts who willingly shared their knowledge with me. I do not have scope to personally thank them all, but I would like to thank the International Coenzyme Q10 Association, particularly Professor Littarru and Monica Glebocki for their willing help and support, and time spent sharing their knowledge with me. To Dr. Michael Miles, Dr. Peter Tang, and Professor Amadeo Pesce, thank you for your adivice, your time and your sharing of your knowledge of CoQ. To Dr. Martin Acknowledgments 3 Shearer and Dr. Domonic Harrington, thank you for the time you spent showing me your vitamin K assay, and offering advice. To John Appleton and Raj Chopra, thank you for your willing and continuous support and encouragement. To Mum, Dad, Bob, Nana and Grandad, thank you all for your words of encouragement and motivation, and your constant support and believing in me. Your support gives me the strength and belief in myself to tackle anything. To James, you are a star, and your unremitting patience and support has been incredible. Thank you so much. This is for you, you earned it many times over in the last few years. Table of Contents i Table of Contents CHAPTER 1.INTRODUCTION ..........................................................................................................1 1.1. INTRODUCTION............................................................................................................................1 1.2. QUINONES ...................................................................................................................................1 1.2.1. Coenzyme Q......................................................................................................................4 1.2.2. Roles of CoQ10 ..................................................................................................................6 1.2.3. Concentration of CoQ in plasma and tissue...................................................................10 1.2.4. CoQ and antioxidant activity..........................................................................................11 1.2.5. Biosynthesis of CoQ10 .....................................................................................................12 1.2.6. Dietary CoQ ...................................................................................................................14 1.2.7. Dietary sources of CoQ10................................................................................................15 1.2.8. Effect of supplementation on oxidation resistance .........................................................21 1.2.9. Factors affecting levels of CoQ10....................................................................................22 1.2.10. Determination of CoQ10..................................................................................................29 1.2.11. Stability of CoQ ..............................................................................................................30 1.3. VITAMIN K................................................................................................................................33 1.3.1. Absorption of dietary vitamin K .....................................................................................35 1.3.2. Role of vitamin K ............................................................................................................35 1.3.3. Reference range and factors affecting vitamin K levels .................................................37 1.3.4. Measurement of vitamin K..............................................................................................40 1.3.5. Stability of vitamin K ......................................................................................................43 1.4. RESEARCH DESCRIBED IN THIS THESIS.......................................................................................43 1.5. RESEARCH HYPOTHESES............................................................................................................44 1.5.1. Clinical research on CoQ...............................................................................................45 1.5.2. Laboratory assessment of CoQ status ............................................................................45 1.5.3. Clinical research on vitamin K.......................................................................................45 1.5.4. Laboratory assessment of vitamin K status ....................................................................45 1.5.5. Analytical development...................................................................................................46 1.6. REFERENCES FOR CHAPTER 1....................................................................................................47 CHAPTER 2.REDOX PROPERTIES AND EFFECT OF SOLVENTS ON THE FLUORESCENCE OF COENZYME Q AND VITAMIN K, AND STABILITY OF COENZYME Q10 IN PLASMA...................................................................................................................................66 2.1. INTRODUCTION..........................................................................................................................66 2.2. REDOX PROPERTIES OF COQ AND VITAMIN K ...........................................................................67 2.2.1. Oxidising plasma CoQ10H2 for measurement of total CoQ10..........................................70 2.2.2.Reducing CoQ10 and vitamin K1 for fluorescence detection - comparison of reduction efficiency of an electrochemical cell, zinc, and an alcohol in the presence of platinum-black. .............................................................................................................71 2.2.3. Reduction of quinones to determine the effect of solvent on fluorescence......................73 2.2.4. Hydrodynamic voltammograms......................................................................................75 2.3. ABSORBANCE AND FLUORESCENCE OF COQ10, AND VITAMIN K1 ..............................................76 2.3.1. Absorbance of Vitamin K1 and CoQ10 ............................................................................76 2.3.2.Fluorescence of CoQ10H2, CoQ0H2, vitamin K1 naphthoquinol, and 4-methoxy-1- naphthol. .......................................................................................................................78 2.3.3. Effect of solvent on fluorescence ....................................................................................78 2.3.4. Determination of the effect of solvent on fluorescence of CoQ10H2, CoQ0H2, vitamin K1 naphthoquinol and 4-methoxy-1-naphthoquinol. ..........................................................80 2.4. STABILITY OF COENZYME Q10 DURING EXTRACTION AND FROZEN STORAGE ............................90 2.4.1. General methods.............................................................................................................91 2.4.2. Short-term total CoQ10 stability......................................................................................91 2.4.3. Long-term stability of total CoQ10 at –13 ºC ..................................................................93 2.4.4. Long-term stability