Metabolomics High resolution characterization of human metabolism and biochemical status on habitual diet and ketogenic diet Katrine Pettersen Thesis for the Master´s Degree in Clinical Nutrition 60 credits Department of Clinical Nutrition Faculty of Medicine UNIVERSITY OF OSLO May 2020 © Katrine Pettersen May 2020 Metabolomics – High resolution characterization of human metabolism and biochemical status on habitual diet and ketogenic diet Katrine Pettersen http://www.duo.uio.no/ Trykk: Reprosentralen, Universitetet i Oslo II Abstract Introduction and background Ketogenic diet (KD) consisting of mostly fat, adequate protein and very low carbohydrate consumption induces metabolic modifications where the main fuel switches from glucose to fat degradation. The diet is used in the treatment of several inborn errors of metabolism and as treatment for refractory epilepsy. The diet has also become popular as a rapid weight reduction method. High intake of fat, particularly saturated fat, can lead to high levels of total and LDL cholesterol concentrations known to be associated with development of atherosclerosis. This raises a concern that KD increases the risk of cardiovascular disease. However, the biochemical effects of KD are insufficiently described, and previous studies have been conducted primarily using overweight participants. Objectives This study aimed to identify metabolic changes in normal weight subjects as a result of following a ketogenic diet for three weeks. In addition, comparison between the ketogenic and habitual diet metabolomes of subjects with low and high relative LDL-C change after KD intervention was conducted. Subjects and methods In 2011-2012, a six-weeks, randomized controlled cross-over trial investigating the effect of three weeks on ketogenic diet (<20 g carbohydrates) was conducted in healthy normal weight subjects (N=30), mainly students and employees at the Department of Nutrition research at University of Oslo. As a continuation, comprehensive global metabolomics analyses of plasma with high-performance liquid chromatography coupled to mass spectrometry (LC- MS) was performed in this thesis to identify and quantify as many metabolites as possible in the biological samples. Results An average of 1085 and 419 features were detected in positive and negative ionization mode, respectively. Differential analysis using volcano plot showed that 48 features in positive ionization mode and 32 compounds in negative ionization mode were detected in significantly higher levels in samples taken after KD versus habitual diet with a log2 fold change >1. Significantly higher levels of ketone bodies, lipids, acylcarnitines, trihomomethionine, acetylglycine, GABA and hydroxyvaleric were detected. Hydroxyvaleric acid had a greater increase in the group with the highest relative LDL-C increase compared to the lowest relative LDL-C increase group. Furthermore, citric acid, malic acid and alpha-ketoglutaric acid as intermediated in the citric acid cycle were significantly upregulated after KD. Levels of the amino acids threonine, asparagine, tryptophan, proline, b-alanine, leucine, arginine, methionine, glutamine and alanine were significantly decreased, while valine was elevated 43 % after 3 weeks on KD. Univariate correlation matrix showed a significant positive correlation (p<0.03) between the amount of amino acid histidine and change in LDL-C with a correlation coefficient of 0.44 (CI 0.06, 0.71). Lower plasma levels of stachydrine, methyl indole 3-acetate, caffeine and N-acetylvaline was observed after KD. Conclusion Applying global metabolomics in a randomized controlled study has the potential to detect differences in the metabolome after dietary intervention, and further increase our knowledge of the biochemical effects and consequences of a KD. In particular, we found alterations in a diverse set of compounds. However, further investigations are needed to understand the mechanisms behind these changes and to determine long-term effects of following a KD. III Acknowledgements The present work was performed at the Department of Medical Biochemistry at Oslo University Hospital, Rikshospitalet from August 2019 to May 2020. My supervisors have been Helge Rootwelt, Katja B. P. Elgstøen and Elise Mørk Sandås at Rikshospitalet and Kjetil Retterstøl and Kirsten B. Holven at the Department of Clinical Nutrition, University of Oslo. I would like to express my sincere gratitude to my supervisors Helge Rootwelt and Kjetil Retterstøl, for help and supervision in the process of writing this thesis. Helge for always staying positive, encouraging and making med feel appreciated. Kjetil and Kirsten for all the scientific help, fast response to my emails and especially for including me in the research group. Thomas Olsen has been an excellent help with the correlation analyses in this thesis. In addition, I would also like to thank IBK (Department of Medical Biochemistry) for giving me the opportunity to deep dive into a, for me, completely new scientific area with excellent co-workers. Furthermore, a genuine appreciation and thanks to all of my skillful and encouraging people in “The metabolomics gang” that consisted of Katjas´angels; Hanne, Anja, Elise, Hege, Monica, and myself. Thanks for extensive education, countless lunch breaks with both interesting and less interesting topics as well as all the birthday celebrations with cakes and singing. It has been a pleasure to work with you, and I am so grateful to be welcomed with open arms and been treated as “one of your own.” Elise, thank you for all assistance and input along the way. You have been a great supporter throughout the process. Special thanks to Monica for the cooporation and Hege who also have taken their Master projects in the same department but with different tasks. I have learned a lot from you, and it has been a great journey together. I wish you lots of luck and success for the future. My classmates for five amazing years really deserve my appreciation. Neither the study nor the “julebord” parties would have been the same without Christian, Karoline and Tea. Thank you for all the dancing. IV Last but not least I am very thankful for my parents Hilde&Stig, my sisters Ellen Susanne& Hege and friends (ref. oslot. extendend edition and UV15) for creating a safe atmosphere where help & support always could be found. Oslo, Norway, May 2020 Katrine Pettersen V Table of contents 1 Introduction ...................................................................................................................... 1 1.1 Ketogenic Diet............................................................................................................ 1 1.1.1 Variants of ketogenic diet .................................................................................. 2 1.1.2 Physiology of ketosis ......................................................................................... 3 1.1.3 Side effects of the ketogenic diet ....................................................................... 5 1.1.4 Raised lipid levels in a ketogenic diet ................................................................ 5 1.1.5 Cardiovascular disease and atherosclerosis risk................................................. 6 1.1.6 Role of fat quality for development of atherosclerosis ...................................... 7 1.1.7 Effect of Ketogenic diet on LDL cholesterol and gene expression in normal- weight, young adults: A randomized controlled study ....................................................... 7 1.2 Metabolomics ............................................................................................................. 8 1.2.1 Targeted analyses ............................................................................................... 9 1.2.2 Global metabolomics.......................................................................................... 9 1.2.3 LC-MS used in global metabolomics ................................................................. 9 1.2.4 Untargeted metabolomics workflow ................................................................ 10 1.2.5 Metabolomics in nutrition research .................................................................. 14 2 Aim of thesis .................................................................................................................... 15 3 Materials and methods................................................................................................... 16 3.1 Low carbohydrate high fat diet study ....................................................................... 16 3.1.1 Participants and design of the LCHF diet study ............................................... 16 3.1.2 Dietary assessment ........................................................................................... 17 3.2 Materials for metabolomics and LCHF diet ............................................................. 17 3.2.1 Sample material and storage ............................................................................ 17 3.2.2 Preparations for LC-ESI-MS analyses (chemicals) ......................................... 17 3.3 Sample preparation ................................................................................................... 18 3.4 Liquid chromatography – electrospray ionization– mass spectrometry settings ..... 20 3.4.1 Ethics ................................................................................................................ 23 3.5 Data analysis ...........................................................................................................