Fatty Acid Ethyl Esters (FAEE), A Biomarker of Alcohol Exposure: Hope for a Silent Epidemic of Fetal Alcohol Affected Children by Vivian Kulaga A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Institute of Medical Science University of Toronto © Copyright by Vivian Kulaga 2009 Fatty Acid Ethyl Esters (FAEE), A Biomarker of Alcohol Exposure: Hope for a Silent Epidemic of Fetal Alcohol Affected Children Vivian Kulaga Doctor of Philosophy Institute of Medical Science University of Toronto 2009 Abstract One percent of children in North America may be affected by fetal alcohol spectrum disorder (FASD). FASD remains difficult to diagnose because confirmation of maternal alcohol use is a diagnostic criterion, and women consuming alcohol during pregnancy are reluctant to divulge this information for fear of stigmatization and losing custody of the child. Consequently, using a biomarker to assess alcohol exposure would provide a tremendous advantage. Recently, the measurement of fatty acid ethyl ester (FAEE) in hair has provided a powerful tool for assessing alcohol exposure. My thesis fills a translational gap of research between the development of the FAEE hair test and its application in the context of FASD. The guinea pig has been a critical model for FASD research, in which FAEE hair analysis has previously distinguished ethanol-exposed dams/offspring from controls. My first study, reports a positive dose-concentration relationship between alcohol exposure and hair FAEE, in the human, and the guinea pig. Humans also displayed over an order of magnitude higher FAEE incorporation per equivalent alcohol exporsure, suggesting that the test will be a sensitive clinical marker of fetal alcohol exposure. My second study utilized multi-coloured rats to investigate the potential of a hair-colour bias, as has been reported for other clinical hair assays; no evidence of ii bias is reported here. My third study is the first to examine the clinical use of the FAEE hair test in parents at high risk of having children with FASD. Over one third of parents tested positive for excessive alcohol use. Parents were investigated by social workers working for child protection services, and my fourth study reports that hair FAEE results agree with social worker reports. Individuals highly suspected of abusing alcohol were at a significantly greater risk of testing positive, whereas individuals tested based on other reasons (such as to cover all bases) were negatively associated with testing positive. The last study of my thesis, confirmed an association between alcohol and drug use by parents at high risk for having children with FASD, posing an added risk to children. This work helps bridge a gap in translational research, suggesting that the FAEE hair test has potential for use in FASD diagnosis and research. iii Acknowledgments Finally a place to get the last word!!! It is with great joy, and a sigh of relief, I take this opportunity to truly thank everyone who deserves it from the bottom of my heart in helping me to turn my life long dreams and aspirations into reality. To my supervisor, Dr. Gideon Koren, thank you!! Whenever it mattered, and whenever it counted, you were always there. A truly inspirational mentor and leader, both professionally and personally, you have set a powerful example. To my advisors Dr. Cindy Woodland, Dr. Katarina Aleksa, and Dr. Ito, you have been generous in your time and thoughtful in your advice. Thank you. It is appreciated. To Dr. Tatyana Karaskov, an outstanding woman, brilliant mind, thoughtful and generous soul, and warm friend. Thank you so much for all your support and advice. You are the best!! To Dr. Bhushan Kapur, an encyclopedia of knowledge and the best biochemist I have ever had the privilege of knowing. You have been extremely generous to me with your time and consideration in all the years of my study. Thank you for the countless hours of advice. To my dad, Richard Kulaga, who has always supported me in every way he could, you gave me both technical and personal life-saving advice. You helped me in times of need, thank you. To my mother, Teresa Kurek, who has provided most of my nourishment in the last four years, thank you for your outstanding devotion, loyalty, and love. To my grandmother, Witolda Ciuhak, thanks. I know how much you care. To my sister, Kasia, you have been my confident and friend, making difficult times easier to bear. To my dearest friend, Sean, who has made much of this possible. Thank you for your strength, and unwavering support. You made me a better person and able to face the challenges in my life with courage. Thank you from the bottom of my heart. You are sorely missed. iv And last but not least, to the little one who cannot read or understand, my dog, Hamlet. Your contribution may seem insignificant to others, but to me you are my best friend and always there; therefore you deserve to be immortalized in my thesis. Who ever said dogs were just man ’s best friend!! If I forgot you, I’m sorry. It was either because you weren’t that important or writing this thesis removed parts of my brain. Just kidding! There are many others who have supported me and contributed to my success, and you know who you are because you are reading this. You are all truly in my heart. Many thanks!!! Sincerely, Vivian Kulaga v Table of Contents Acknowledgments.......................................................................................................................... iv Table of Contents........................................................................................................................... vi List of Tables ................................................................................................................................ xii List of Figures.............................................................................................................................. xiv List of Abbreviations ................................................................................................................... xvi 1 Introduction................................................................................................................................ 1 1.1 Alcohol................................................................................................................................ 1 1.1.1 Chemistry and Metabolism in Humans................................................................... 1 1.1.2 Deleterious effects .................................................................................................. 6 1.1.3 Prevalence of Alcohol Use...................................................................................... 7 1.1.3.1 General...................................................................................................... 7 1.1.3.2 Pregnant Women and Women of Childbearing Age................................ 8 1.1.3.3 Risk Factors............................................................................................ 11 1.2 The Fetal Alcohol Spectrum Disorder .............................................................................. 12 1.2.1 History and Etiology............................................................................................. 12 1.2.1.1 Nomenclature.......................................................................................... 15 1.2.1.2 Mechanisms............................................................................................ 15 1.2.1.3 Threshold................................................................................................ 19 1.2.2 Diagnosis and Intervention ................................................................................... 21 1.2.3 The Silent Epidemic and Gap Towards Intervention............................................ 26 1.3 Biomarkers for Alcohol Exposure in Blood and Urine .................................................... 31 1.3.1 Hemoglobin-Associated Acetaldehyde................................................................. 32 1.3.2 Glutamyltransferase .............................................................................................. 33 vi 1.3.3 Mean Corpuscular Volume................................................................................... 34 1.3.4 Aspartate/Alanine Aminotransferase.................................................................... 35 1.3.5 Carbohydrate-Deficient Transferrin...................................................................... 36 1.4 Fatty Acid Ethyl Esters (FAEE) ....................................................................................... 36 1.4.1 Production and Accumulation............................................................................... 36 1.4.2 Toxicity of FAEE.................................................................................................. 38 1.4.3 Use as a Biomarker ............................................................................................... 41 1.4.3.1 Blood and Tissue .................................................................................... 41 1.4.3.2 FAEE in Hair.......................................................................................... 42 1.4.3.3 Effect of Cosmetic Treatment and Hair Care......................................... 44 1.5 Ethyl Glucoronide (EtG) and FAEE................................................................................. 46 1.6 Biomarkers for Fetal Alcohol Exposure ..........................................................................