DIETARY SUPPLEMENTATION OF CONJUGATED LINOLEIC ACID: MUSCULOSKELETAL INVESTIGATIONS IN MEN AND SELECTED ANIMAL MODELS Jason Robert DeGuire, M.Sc. School of Dietetics and Human Nutrition McGill University, Montreal Quebec, Canada April 2013 A thesis submitted to McGill University in partial fulfillment of the requirements of the degree of Doctor of Philosophy (Human Nutrition). © Copyright Jason R. DeGuire, 2013 All rights reserved i i This thesis is dedicated to my beautiful wife Amanda, for her continual love throughout the course of my doctorate and to our son Gavin, for whom, there could not be a prouder father. ii “Research is what I'm doing when I don't know what I'm doing.” - Wernher von Braun iii TABLE OF CONTENTS Title page ..................................................................................................... i Table of contents ........................................................................................ iv Abstract ....................................................................................................... x Résumé ..................................................................................................... xv Acknowledgements ................................................................................... xxi Advance of scholarly knowledge ............................................................. xxii 1. Original contribution to knowledge .............................................. xxii 2. Research articles in peer-reviewed journals .............................. xxiv 3. Research article in preparation for submission to peer-reviewed journals ....................................................................................... xxv 4. Abstracts at professional meetings ............................................ xxvi 5. Oral conference presentations .................................................. xxvii Contribution of authors ......................................................................... xxviii List of tables ............................................................................................ xxxi List of figures ........................................................................................ xxxiv List of abbreviations ............................................................................. xxxvii Chapter 1 – Introduction, objectives and hypotheses ................................. 1 1.1 Rationale and statement of purpose ............................................ 2 1.2 Thesis objectives and hypotheses ............................................. 11 Chapter 2 – Literature review ................................................................... 13 2 Introduction ................................................................................ 14 2.1 Background of conjugated linoleic acid ...................................... 14 2.1.1 History of conjugated linoleic acid .............................. 14 2.1.2 Structure of conjugated linoleic acid ........................... 16 2.1.3 Biosynthesis of conjugated linoleic acid ..................... 17 2.1.4 Conjugated linoleic acid in food .................................. 23 2.1.5 Conjugated linoleic acid in the diet ............................. 24 iv 2.1.6 Conjugated linoleic acid digestion, absorption, metabolism and excretion ................................................... 32 2.1.7 Conjugated linoleic acid supplementation and safety 36 2.2 Conjugated linoleic acid and the aging musculoskeletal system ................................................................................. 39 2.2.1 Age-related sex hormone deficiency states and inflammation in muscle ........................................................ 40 2.2.2 Age-related sex hormone deficiency states and inflammation in bone ........................................................... 42 2.2.3 Effects of conjugated linoleic acid on the musculoskeletal biomarkers ................................................ 43 2.2.4 Effects of conjugated linoleic acid on parathyroid hormone .............................................................................. 48 2.2.5 Effects of conjugated linoleic acid on cell derived mediators of inflammation ................................................... 50 2.2.6 Conjugated linoleic acid and peroxisome proliferator-activated receptors ............................................ 52 2.3 Summary .................................................................................... 60 Chapter 3 – Manuscript 1 ......................................................................... 61 3.1 Abstract ...................................................................................... 63 3.2 Introduction ................................................................................ 64 3.3 Materials and methods ............................................................... 66 3.3.1 Study protocol and diets ............................................. 66 3.3.2 Assessment of growth and bone ................................ 67 3.3.3 Biomarkers of bone metabolism ................................. 68 3.3.4 Gas chromatography .................................................. 69 3.3.5 Statistical analyses ..................................................... 70 3.4 Results ....................................................................................... 71 3.4.1 Dietary intake ............................................................. 71 v 3.4.2 Growth and body composition .................................... 71 3.4.3 Bone assessment ....................................................... 72 3.4.4 Biochemistry ............................................................... 72 3.4.5 Mass balance ............................................................. 73 3.4.6 Liver incorporation of CLA .......................................... 73 3.5 Discussion .................................................................................. 79 3.6 Acknowledgements .................................................................... 82 Bridge statement I ..................................................................................... 83 Chapter 4 – Manuscript 2 ......................................................................... 84 4.1 Abstract ...................................................................................... 86 4.2 Introduction ................................................................................ 87 4.3 Materials and methods ............................................................... 88 4.3.1 Study protocol and diets ............................................. 88 4.3.2 Assessment of growth, PTH and ionized calcium ...... 89 4.3.3 Statistical analyses ..................................................... 90 4.4 Results ....................................................................................... 90 4.5 Discussion .................................................................................. 93 4.6 Acknowledgements .................................................................... 96 Bridge statement II .................................................................................... 97 Chapter 5 – Manuscript 3 ......................................................................... 98 5.1 Abstract .................................................................................... 100 5.2 Introduction .............................................................................. 101 5.3 Materials and methods ............................................................. 103 5.3.1 Participants ............................................................... 103 5.3.2 Dose-response trial design ....................................... 106 5.3.3 Clinical assessments ................................................ 108 5.3.4 Diet and physical activity .......................................... 109 vi 5.3.5 Anthropometry and body composition ...................... 110 5.3.6 Fatty acid analyses of lipids in plasma and erythrocytes ....................................................................... 111 5.3.7 Adverse effects and safety monitoring ..................... 111 5.3.8 Statistical analyses ................................................... 112 5.4 Results ..................................................................................... 113 5.4.1 Cross-sectional analysis of the baseline assessment ...................................................................... 113 5.4.1.1 Conjugated linoleic acid status, bone and body composition .................................................. 113 5.4.2 Dose response trial .................................................. 114 5.4.2.1 Participants and lifestyle characteristics ... 114 5.4.2.2 Conjugated linoleic acid levels in plasma and erythrocytes ................................................... 114 5.4.2.3 Vital signs and blood lipid profile .............. 115 5.4.2.4 Anthropometry and body composition ...... 115 5.4.2.5 Calciotropic hormones and biomarkers of bone metabolism .................................................. 116 5.5 Discussion ............................................................................ 123 5.6 Acknowledgements .................................................................. 128 Bridge statement III ............................................................................ 129 Chapter 6 – Manuscript 4 ....................................................................... 130 6.1 Abstract ...........................................................................