Hair Analysis for Alcohol Biomarkers: Assessing Excessive Consumption in a Student Population
Total Page：16
File Type：pdf, Size：1020Kb
UNIVERSIDADE DA BEIRA INTERIOR Ciências Hair Analysis for Alcohol Biomarkers: Assessing Excessive Consumption in a Student Population David Jerónimo Oppolzer Tese para obtenção do Grau de Doutor em Bioquímica (3ºCiclo de estudos) Orientador: Profª. Doutora Maria Eugenia Gallardo Alba Co-orientador: Doutor Mário Jorge Dinis Barroso Co-orientador: Prof. Doutor Luís António Paulino Passarinha Covilhã, Junho de 2016 This project was financed by the Programa de investigação de saúde de 2012 (reference 125895) from the Fundação Calouste Gulbenkian. V VI Thesis overview This thesis is structured into five main chapters: Chapter I consists of a general introduction on the problematic of alcohol consumption, consumption monitoring approaches, and proposed objectives. In this part a review article is presented (manuscript 1), including a literature review of alcohol biomarkers used for the assessment of alcohol consumption. Chapter II presents the experimental results obtained during the PhD, and includes information presented in the format of research articles: Manuscript 2 - Determination of ethyl glucuronide (EtG) and fatty acid ethyl esters (FAEEs) in hair samples. Manuscript 3 - Determination of ethyl glucuronide in hair to assess excessive alcohol consumption in a student population. Manuscript 4 - Alcohol consumption assessment in a student population through combined hair analysis of ethyl glucuronide and fatty acid ethyl esters. Chapter III consists of a general discussion of the presented results, also complementing the information presented in the manuscripts. In chapter IV the main conclusions of the doctoral work are summarized. Finally, in chapter V future perspectives are presented. VII VIII Acknowledgements One of the most crucial parts in this project was the collection of hair samples from the numerous students. This was a specially challenging task, which required a lot of patience, effort, explanations and persistence to finally reach the required number of samples. This would have not been possible without the help of my laboratory colleagues. Too all of them, a cheerful thank you! I’d like to thank to my supervisor Eugenia Gallardo, for the opportunity and share of knowledge about several aspects during the entire PhD, as well as the valuable discussions, corrections and help on the various obstacles. To my co-supervisor Mário Barroso, for his prompt availability at every encountered problem, correction of manuscripts, share of opinions and for the entire knowledge and opinions provided on hair testing. To my co-supervisor Luís Passarinha, for his availability and opportunities for collaboration in other research projects. To my friends and colleagues, David and Fabiana thank you for all your help, not only during sample collection, but also in the laboratory. Particularly to David for his efficient abilities in persuading students to participate in the study, and not giving up even after days of collection, even after reaching eminent frustration. And Fabiana for her amazing speed at cutting hair, and constant will to help. To both of you, thank you also for the great times spent in the laboratory, and motivation by the good mood that became routine during the long days of work. Without you, all this could have not been the same. To my parents, Manne and Hedy, and my brother Jolle, I’d like to thank for their continuous support and believe on my academic work, even if it meant that due to the workload we couldn’t meet often for several weeks. Your support and believe meant a lot and gave me much help and motivation during this entire time. To you Hedy, thank you also for all the help in Coimbra. To Catarina, I own you more than any gratitude I could write in these lines. You’ve been my best friend and accompanied me day and night at every step of this project. You were present at every challenge, victory and defeat during this entire time, and were there for every decision that had to be taken and every problem to be solved. Despite to say that every opinion you gave and discussion about all kinds of obstacles were of great value to me, and I can’t recall how many times that helped me to keep on track with the work that had to be done. I should IX also not forget the patience you had during all this time, every part of this project over the past years was made in a constant hurry, running against time. And even with the stress this brought to our lives, the fact that you remained patient and supportive, especially all those times when everything seemed to be going down the drain, is to be praised and shows the fabulous person you are. Thank you for everything, and most importantly, thank you for being yourself! 3 Years ago I would have never guessed that I would be writing the following lines. In fact, I would have never imagined that during this project something as surprising and wonderful, as you Lisa, would happen. Long before you were born you already took part in this project, you were present when hair was collected from students, during the entire time we spend working in the laboratory, and, from birth until now, during the entire writing process. You can’t imagine in how many ways you helped me during my work, you were the motivation that kept your mother and me working hard and spending excessive hours in the laboratory. And then after birth, every step of growth was an explosion of happiness to your parents’ eyes. You wouldn’t believe how much inspiring and motivating every babble and every smile you make is to us, and how often it made my day and made me realize how lucky I am. Thank you for being a part of my life! X "The world is still a weird place, despite my efforts to make clear and perfect sense of it." Hunter S. Thompson XI XII Abstract Alcohol consumption within the student population has become an increasing health concern in several countries. The use of alcohol has deleterious consequences on intellectual performance of students, and can be associated with a variety of risky situations, as injuries, unplanned and unsafe sex, sexual and non-sexual violence, property damage and reckless driving. Monitoring alcohol consumption in the student population is therefore important to identify the degree of the problem and to assess consumption trends, allowing also for the implementation of adequate policies, as well as verifying their effectiveness. Alcohol consumption can be monitored using statistics, such as survey studies performed on the population by means of questionnaires. These approaches have relative efficiency and are widely used; however, considerable limitations are associated to their use, making them unreliable for clinical and/or forensic purposes. Alcohol biomarkers are measurable substances in a biological sample, whose presence indicates some form of exposure to alcohol, and their use to assess alcohol exposure in clinical and forensic scenarios has been applied in a variety of biological specimens in the past years. One of the most notable of these specimens is hair, which presents several advantages, including non-invasive collection, ease of availability, long windows of detection and low chance of adulteration. Hair testing for alcohol exposure relies mainly on the analysis of two biomarkers, ethyl glucuronide (EtG) and fatty acid ethyl esters (FAEEs). These markers have been widely applied in several studies, and were shown to present good sensitivity and specificity. Therefore, the objective of the present work was to evaluate the alcohol consumption on a university student population using two main approaches, one based on questionnaire analysis, and the second on the analytical determination of alcohol biomarkers in hair. A total of 1192 hair samples, and respective self-completion questionnaire were collected, 975 samples were analysed thereof. Through analysis of the questionnaires it was found that alcohol consumption started mostly at the age of 15, and that most of the students considered their consumption as moderate, while almost one-third consumed alcohol excessively at least once in the last month. Beer was reported to be the most consumed beverage, and drinking was widely preferred accompanied. Places for alcohol consumption are mostly public places, such as coffee bars. Higher ingested quantities of alcohol per drinking occasion were associated with the male genre, but both genres presented mean alcohol volumes indicating risky and excessive drinking. The use of other substances was also assessed, including tobacco, which was a habit in one-third of the students. The incidence of the use of illicit substances was similar, and cannabis was the most consumed substance, but mainly on rare occasions. The use of illicit substances was however associated with drinking events in more than 50% of the students. XIII For alcohol biomarker analysis in hair, a method was developed for the analysis of EtG. The compound was extracted from the hair matrix by ultra-sonication with water, clean-up using mixed anion-exchange solid-phase extraction (SPE) and analysis by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). A second method was developed for the analysis of four FAEEs (ethyl myristate, palmitate, oleate and stearate). The compounds were extracted from the hair matrix by incubation with heptane, extracts were cleaned-up with aminopropyl SPE and analysed by gas chromatography coupled to tandem mass spectrometry (GC-MS/MS). Both methods were validated according to guidelines from the Food and Drug Administration (FDA) and International Conference on Harmonization (ICH). Assessed parameters included sensitivity, limits, linearity, precision and accuracy, recovery, stability and matrix effect. The methods were found to be selective, with a limit of quantification of 3 pg/mg for EtG and 30 pg/mg for each of the four FAEEs. Linearity was obtained in the ranges of 3 – 500 pg/mg for EtG and 30 – 5000 pg/mg for FAEEs, and precision and accuracy were found acceptable according to the guidelines.