CHANGES IN SERUM HOMOCYSTEINE IN RESPONSE TO ORAL CREATINE SUPPLEMENTATION IN VEGETARIANS A Thesis Submitted to the College of Graduate Studies and Research in Partial Fulfillment of the Requirements for the Degree of Master of Science in the College of Kinesiology University of Saskatchewan Saskatoon By Vanessa Marie MacCormick © Copyright Vanessa Marie MacCormick, April 2009. All rights reserved PERMISSION TO USE In presenting this thesis in partial fulfillment of the requirements for a Postgraduate degree from the University of Saskatchewan, I agree that the Libraries of this University may make it freely available for inspection. I further agree that permission for copying of this thesis in any manner, in whole or in part, for scholarly purposes may be granted by the professor or professors who supervised my thesis work or, in their absence, by the Head of the Department or the Dean of the College in which my thesis work was done. It is understood that any copying or publication or use of this thesis or parts thereof for financial gain shall not be allowed without my written permission. It is also understood that due recognition shall be given to me and to the University of Saskatchewan in any scholarly use which may be made of any material in my thesis/dissertation. Requests for permission to copy or to make other uses of materials in this thesis in whole or part should be addressed to: Dean College of Kinesiology University of Saskatchewan 87 Campus Drive Saskatoon, Saskatchewan S7N 5B2 i ABSTRACT Homocysteine (Hcy) is metabolized through a series of remethylation and transsulphuration processes that require the co-factors vitamins B6, folate (the nutrient derived from food, and folic acid is supplemental), and B12. Homocysteine metabolism results in the production of creatine (Cr). By way of negative feedback creatine supplements could potentially decrease Hcy production. Low dietary intake of vitamins and creatine, often a consequence of a vegetarian diet may also increase serum Hcy concentrations. It was therefore the purpose of this study to examine the effect of a five day creatine loading period (0.25 g Cr/kg lean body mass (LBM)/day) in female vegetarians (VG) (n=9, age 25 ± 3.6) and non-vegetarians (NV) (n=11, age 24 ± 5.9) on serum levels of Hcy, vitamins B6 and B12, whole blood folate, and on a number of fitness measures known to be positively affected by creatine supplementation: peak torque and average power during leg extensions; one repetition maximum (1RM) bench and leg press; peak power during repeated Wingate anaerobic tests. Aside from a difference in change scores for leg press, there were no differences between dietary groups for the fitness measures, and therefore the participants were pooled for comparisons between time points. Urinary creatine was significantly greater following the five day loading period (81 ± 113 vs. 647 ± 421 µmol). In contrast, urinary creatinine did not change. A significant increase in plasma vitamin B6 (pyridoxal 5‟-phosphate) was found (72 ± 24 vs. 94 ± 24 nmol/L) (p < 0.05), although there were no between group differences. Serum Hcy, vitamin B12, and whole blood folate levels did not change significantly over time in either group. Participants showed significant increases post- loading in bench press (45 ± 13 vs. 48 ± 12 kg.) and leg press (116 ± 35 vs. 125 ± 40 kg) (p < 0.05). Changes scores for leg press were greater for the vegetarian participants when compared with non-vegetarians (16 ± 16 vs. 3 ± 9 kg). Significant improvements post-loading were also ii noted for peak torque (85.0 ± 15.0 vs. 91.8 ± 12.9 Nm) and average power (99.5 ± 14.4 vs. 107.1 ± 14.8 W) (p < 0.05). Peak power during repeated Wingate anaerobic tests also significantly improved in response to the intervention (449.2 ± 92.6 vs. 472.4 ± 103.5 W) (p < 0.05). This response did not differ however between the two diet groups. These results suggest that a five day creatine loading protocol similarly affects fitness performance measures in both vegetarians and non-vegetarians. There was no significant effect of the supplementation protocol on serum Hcy concentration, suggesting that the supplemented creatine did not decrease the reformation of Hcy, as originally hypothesized. iii ACKNOWLEDGEMENTS I would like to acknowledge and thank my supervisor, Dr. Carol Rodgers, for her guidance and supervision throughout the development and execution of my project, as well as for her patience, support and input as I wrote this document. I would also like to thank my committee members, Dr. Phil Chilibeck, Dr. Gord Zello, and my external committee member, Dr. Susan Whiting, for the direction and input that they have given. I have many thanks for my participants, who have donated so much time and energy to my project, overcoming fears and working hard at almost obscene hours for the completion of my degree. Mr. Doug Jacobson and Ms. Heather Whelan made sure that I knew how to run the testing equipment and offered their expertise and technical assistance periodically, and for this I am very grateful. Ms. Heather McKnight and Ms. Cara Wilcock assisted with data collection, and their contribution is greatly appreciated. I would also like to acknowledge Dr. Jon Farthing for answering numerous questions about statistical analyses, and Mr. Bryce Taylor for helping me put/keep it all together. I would like to acknowledge the staff of the Toxicology department at the Provincial Laboratory in Regina, led by Dr. Denis Lehotay, for teaching me about their testing procedures, showing me around, and for carrying out my blood vitamin analyses. AbdulRazaq A.H. Sokoro (Abdi) deserves so many additional thanks for assisting me, well beyond duty, with technical questions. I would also like to acknowledge the Saskatchewan Academy of Sports Medicine, who provided me with funding and input. Both creatine monohydrate and vitamin B12 supplements were kindly donated by Dymatize and Jamieson Natural Sources, and therefore I must acknowledge their generosity. iv DEDICATION Finally, I would like to thank my family. Without their support I would never have finished writing this document. They were with me every step of the way, whether through words of encouragement, prayers and faith, by taking care of me when I was too busy to do it myself, or simply by continuing to love me when I deserved it least. This document is dedicated to my Grandmother. She continues to believe in me, and has been the motivation for me to push through and finish. Thank you so much for all of your love and encouragement. v TABLE OF CONTENTS PERMISSION TO USE ................................................................................................................... i ABSTRACT .................................................................................................................................... ii ACKNOWLEDGEMENTS ........................................................................................................... iv DEDICATION ................................................................................................................................ v TABLE OF CONTENTS ............................................................................................................... vi LIST OF TABLES ......................................................................................................................... ix LIST OF FIGURES ........................................................................................................................ x LIST OF ABBREVIATIONS ........................................................................................................ xi 1. INTRODUCTION ...................................................................................................................... 1 2. SCIENTIFIC FRAMEWORK .................................................................................................... 5 2.1 Literature Review .................................................................................................................. 5 2.1.1 Homocysteine ................................................................................................................. 5 2.1.1.1 Homocysteine metabolism ....................................................................................... 5 2.1.1.2 Control and regulation of homocysteine metabolism .............................................. 7 2.1.2 Factors affecting homocysteine concentration ............................................................... 8 2.1.2.1 Cystathionine β-synthase deficiency ....................................................................... 8 2.1.2.2 Renal disease/insufficiency ...................................................................................... 9 2.1.2.3 Cardiovascular disease ............................................................................................. 9 2.1.2.4 Lifestyle factors affecting homocysteine ............................................................... 11 2.1.2.5 Gender and steroid hormones ................................................................................ 13 2.1.2.6 Dietary composition ............................................................................................... 15 2.1.2.7 Folate and vitamin B6............................................................................................. 15 2.1.2.8 Vitamin B12 ............................................................................................................ 18 2.1.2.9 Vegetarian