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 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.

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 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.

In-utero ethanol exposure may result in fetal alcohol spectrum disorder (FASD). Studies have suggested that women who drink ethanol are more likely to consume illicit drugs. Detection of such exposures has been done via meconium and hair testing and can serve to direct needed prevention methods and appropriate management and intervention for the neonate and the mother. This study examined maternal diabetes as a possible confounder for in-utero ethanol exposure testing and determined the trends in drug use associated with heavy in-utero ethanol exposure in a high-risk obstetric Canadian population. It was determined that maternal diabetes does not produce false-positive results in testing for in-utero ethanol exposure. Furthermore, heavy in-utero ethanol exposure was detected in 15.5% of samples and was associated with an increased exposure to amphetamines (OR=3.30) and opiates (OR=2.01), but a decreased exposure to cannabinoids (OR=0.61) when compared to neonates with no heavy in-utero ethanol exposure.

In-utero ethanol exposure may result in fetal alcohol spectrum disorder (FASD). Studies have suggested that women who drink ethanol are more likely to consume illicit drugs. Detection of such exposures has been done via meconium and hair testing and can serve to direct needed prevention methods and appropriate management and intervention for the neonate and the mother. This study examined maternal diabetes as a possible confounder for in-utero ethanol exposure testing and determined the trends in drug use associated with heavy in-utero ethanol exposure in a high-risk obstetric Canadian population. It was determined that maternal diabetes does not produce false-positive results in testing for in-utero ethanol exposure. Furthermore, heavy in-utero ethanol exposure was detected in 15.5% of samples and was associated with an increased exposure to amphetamines (OR=3.30) and opiates (OR=2.01), but a decreased exposure to cannabinoids (OR=0.61) when compared to neonates with no heavy in-utero ethanol exposure.

The present thesis is based on four studies focusing on alcohol consumption among Swedish adolescents, and therewith related psychosocial and genetic factors. One main objective was to study the reasons for drinking alcohol among different population - representative samples of adolescents in order to identify motives for drinking. Relationships between these drinking motives, alcohol consumption, and alcohol - related problems were also investigated. Three motives emerged from this study: social - enhancement, coping and dominance. The association with alcohol consumption and alcohol - related problems was positive for social - enhancement and coping motives, but negative for the dominance motive. A significant heritability of alcohol use disorders has been demonstrated by family, adoption and twin studies. Environmental influences have also been acknowledged to play an important role in the development of alcohol use disorders. Moreover, the interaction between genetic and environmental factors is likely to influence the risk - resilience for alcohol use disorders. In view of this knowledge, plausible candidate polymorphisms were considered in gene - environment interaction models. An effect of the genetic polymorphisms was only present when a G x E model was considered. A genetic variant of the clock gene Period2, in an interaction with sleep problems, was studied in relation to alcohol consumption among adolescents. High alcohol consumption was associated with the AA genotype of the PER2 SNP10870 polymorphism, in an interaction with several and frequent sleep problems, among adolescent boys. A genetic variant in the opioid µ receptor 1 gene, in an interaction with alcohol consumption, was studied in relation to depressive symptoms. Depressive symptoms were predicted by the G allele of the OPRM1 A118G polymorphism, in an interaction with high alcohol consumption, among adolescent girls. Additionally, the PER2 SNP10870 and the OPRM1 A118G polymorphisms were studied in a sample of severely alcoholic females. Furthermore, alcohol consumption was assessed by using different instruments, such as biomarkers and surveys. Comparisons were carried out to identify the most suitable method to assess alcohol consumption among adolescents. Questionnaire and interview seemed more suitable tools than biomarkers in this regard.The results eventually support the importance of psychosocial and genetic influences, and their interaction effect on alcohol consumption among adolescents.

O álcool é uma das substâncias psicoativas mais consumidas mundialmente e seu uso por mulheres em idade reprodutiva, em particular, tem representado grande preocupação por parte de especialistas e da sociedade em geral. Apesar dos efeitos adversos associados ao ato de ingerir bebidas alcoólicas durante a gestação ser bastante documentados e conhecidos, sabe-se que uma parcela de mulheres grávidas tem dificuldades em abandonar o hábito. O consumo excessivo de álcool durante a gravidez tem sido associado com a síndrome fetal pelo álcool (FAS), caracterizada por crianças com dificuldades comportamentais e de aprendizado. Entretanto, devido ao sentimento de culpa e medo de ações punitivas, mulheres raramente admitem terem utilizado álcool durante a gestação. Como resultado, uma série de marcadores biológicos tem sido estudada para se diagnosticar a exposição fetal ao etanol. Dentre os marcadores utilizados estão os ésteres etílicos de ácidos graxos (FAEE), que podem ser detectados em amostras de mecônio de recém-nascidos. No presente trabalho, um método analítico foi desenvolvido visando a detecção de oito FAEEs em amostras de mecônio e aplicada em amostras coletadas de recém-nascidos cujas mães admitiram ou não o uso de etanol durante a gestação. A microextração em fase sólida por Headspace (HS-SPME), uma técnica de preparação de amostras relativamente recente, foi utilizada para análise. Os FAEEs foram identificados e quantificados por cromatografia gasosa/espectrometria de massas (GC/MS), operado no modo de ionização química. Os correspondentes ésteres etílicos deuterados foram sintetizados e utilizados como padrão interno. Os limites de quantificação (LOQ) obtidos foram abaixo de 150 ng/g e limites de detecção (LOD) foram abaixo de 100ng/g para todos os analitos. O método mostrou boa linearidade na concentração estudada (LOQ-2000ng/g), com coeficiente (r2) melhor que 0.98. Os valores de precisão apresentaram coeficientes de variação menores que 15% para todos os FAEEs estudados. Quando o método foi aplicado em amostras de mecônio, foi possível detectar níveis de alguns FAEEs de recém-nascidos não suspeitos a exposição fetal ao etanol. / Alcohol is the main psychoactive drug consumed worldwide and its increasing use by young women has been a great problem point out by specialists in the subject. Although the adverse effects associated to the habit of drinking alcoholic beverages during gestation being very much documented, it is known that a considerable number of pregnant women have difficulties to abandon the habit. Excessive alcohol use during pregnancy has been associated with Fetal Alcohol Syndrome (FAS) characterized by children with cognitive and behavioral disorders. However, because of denial, embarrassment and fear, maternal reports of gestational use of alcohol are often inaccurate. Consequently, a series of biomarkers have been studied to diagnose fetal exposure to alcohol. Recently, fatty acid ethyl esters (FAEE) have been studied as biomarkers found in meconium of neonates exposed in utero. In the present work, an analytical method was developed aiming the detection of eight FAEEs in meconium samples and applied to real specimens collected from newborns whose mothers admitted or not the use of alcohol during pregnancy. Headspace solid-phase microextraction (HS-SPME), a relatively recent sample preparation technique, was used for analysis. FAEEs were identified and quantified by gas chromatography/mass spectrometry (GC/MS), operated in chemical ionization mode. The corresponding deuterated ethyl esters were synthesized and used as internal standards. The lower limits of quantification (LOQ) obtained were below 150ng/g and limits of detection (LOD) were bellow 100ng/g for all analytes. The method showed good linearity in the range of concentration studied (LOQ-2000ng/g), with coefficient of linearity better than 0.98. The precision assay, given by the relative standard deviation (RSD) of the method was lower than 15% for all FAEEs studied. When the method was applied to real samples, it was possible to detect trace levels of some FAEEs from non-suspected ethanol exposed newborns.

O álcool é uma das substâncias psicoativas mais consumidas mundialmente e seu uso por mulheres em idade reprodutiva, em particular, tem representado grande preocupação por parte de especialistas e da sociedade em geral. Apesar dos efeitos adversos associados ao ato de ingerir bebidas alcoólicas durante a gestação ser bastante documentados e conhecidos, sabe-se que uma parcela de mulheres grávidas tem dificuldades em abandonar o hábito. O consumo excessivo de álcool durante a gravidez tem sido associado com a síndrome fetal pelo álcool (FAS), caracterizada por crianças com dificuldades comportamentais e de aprendizado. Entretanto, devido ao sentimento de culpa e medo de ações punitivas, mulheres raramente admitem terem utilizado álcool durante a gestação. Como resultado, uma série de marcadores biológicos tem sido estudada para se diagnosticar a exposição fetal ao etanol. Dentre os marcadores utilizados estão os ésteres etílicos de ácidos graxos (FAEE), que podem ser detectados em amostras de mecônio de recém-nascidos. No presente trabalho, um método analítico foi desenvolvido visando a detecção de oito FAEEs em amostras de mecônio e aplicada em amostras coletadas de recém-nascidos cujas mães admitiram ou não o uso de etanol durante a gestação. A microextração em fase sólida por Headspace (HS-SPME), uma técnica de preparação de amostras relativamente recente, foi utilizada para análise. Os FAEEs foram identificados e quantificados por cromatografia gasosa/espectrometria de massas (GC/MS), operado no modo de ionização química. Os correspondentes ésteres etílicos deuterados foram sintetizados e utilizados como padrão interno. Os limites de quantificação (LOQ) obtidos foram abaixo de 150 ng/g e limites de detecção (LOD) foram abaixo de 100ng/g para todos os analitos. O método mostrou boa linearidade na concentração estudada (LOQ-2000ng/g), com coeficiente (r2) melhor que 0.98. Os valores de precisão apresentaram coeficientes de variação menores que 15% para todos os FAEEs estudados. Quando o método foi aplicado em amostras de mecônio, foi possível detectar níveis de alguns FAEEs de recém-nascidos não suspeitos a exposição fetal ao etanol. / Alcohol is the main psychoactive drug consumed worldwide and its increasing use by young women has been a great problem point out by specialists in the subject. Although the adverse effects associated to the habit of drinking alcoholic beverages during gestation being very much documented, it is known that a considerable number of pregnant women have difficulties to abandon the habit. Excessive alcohol use during pregnancy has been associated with Fetal Alcohol Syndrome (FAS) characterized by children with cognitive and behavioral disorders. However, because of denial, embarrassment and fear, maternal reports of gestational use of alcohol are often inaccurate. Consequently, a series of biomarkers have been studied to diagnose fetal exposure to alcohol. Recently, fatty acid ethyl esters (FAEE) have been studied as biomarkers found in meconium of neonates exposed in utero. In the present work, an analytical method was developed aiming the detection of eight FAEEs in meconium samples and applied to real specimens collected from newborns whose mothers admitted or not the use of alcohol during pregnancy. Headspace solid-phase microextraction (HS-SPME), a relatively recent sample preparation technique, was used for analysis. FAEEs were identified and quantified by gas chromatography/mass spectrometry (GC/MS), operated in chemical ionization mode. The corresponding deuterated ethyl esters were synthesized and used as internal standards. The lower limits of quantification (LOQ) obtained were below 150ng/g and limits of detection (LOD) were bellow 100ng/g for all analytes. The method showed good linearity in the range of concentration studied (LOQ-2000ng/g), with coefficient of linearity better than 0.98. The precision assay, given by the relative standard deviation (RSD) of the method was lower than 15% for all FAEEs studied. When the method was applied to real samples, it was possible to detect trace levels of some FAEEs from non-suspected ethanol exposed newborns.

In der vorliegenden Arbeit wurde ein analytisches Verfahren zur quantitativen Bestimmung von Fettsäureethylestern (FSEE) im Haar und in Hautoberflächenlipiden mittels Headspace-Festphasenmikroextraktion (HS-SPME) und Gaschromatographie-Massenspektrometrie (GC-MS) sowie eine auf Hochleistungs-Flüssigchromatographie mit Photodiodenarray-Detektion (HPLC-DAD) basierende Methode zur Bestimmung der Squalenkonzentrationen in Lipidextrakten entwickelt. Die bei Untersuchung von Proben verschiedener Konsumentengruppen erhaltenen Konzentrationswerte wurden hinsichtlich ihrer Eignung als Marker für chronisch exzessiven Alkoholkonsum untersucht. Aus den Ergebnissen lässt sich schließen, dass Fettsäureethylester im Haar als Alkoholmarker den bisher üblicherweise genutzten Markern wie GGT, CDT oder MCV bezüglich Sensitivität und Spezifität mindestens ebenbürtig sind. Es wurden die folgenden vorläufige Cut-off-Werte festgelegt: wenn sich im Haar für die Summenkonzentration der vier in der höchsten Konzentration vorkommenden FSEE (Ethylmyristat, Ethylpalmitat, Ethyloleat und Ethylstearat) ein Wert > 1 ng/mg ergibt, kann mit hoher Sicherheit von chronisch exzessivem Alkoholkonsum ausgegangen werden, für Abstinenzler werden typischerweise Werte < 0,4 ng/mg gefunden. Durch Bildung des Quotienten der FSEE-Konzentrationen und der Squalenkonzentrationen wurden relative FSEE-Konzentrationen erhalten, die im Falle der Haaranalyse zu einer Verbesserung der Zuordnungssicherheit zu den entsprechenden Konsumentengruppen führten bzw. bei der Analyse von Hautoberflächenlipiden einen sinnvollen Vergleich der Werte erst ermöglichten. Als vorläufiger Cut-off-Wert für die relativen FSEE-Konzentrationen wurde ein Wert von 2 ng/µg vorgeschlagen. Als weiteres wichtiges Ergebnis der Arbeit wurde der Einlagerungsmechanismus der FSEE ins Haar aufgeklärt. Es konnte gezeigt werden, dass Fettsäureethylester in erster Linie über das Sebum ins Haar gelangen. / The current doctoral thesis presents the development of an analytical procedure for the quantitative analysis of fatty acid ethyl esters (FAEE) in hair and in skin surface lipids using headspace solid phase microextraction (HS-SPME) and gas chromatography-mass spectrometry (GC-MS) as well as a method based on high-performance liquid chromatography with photodiode array detection (HPLC-DAD) to determine squalene concentrations in lipid extracts. The results obtained from analysis of samples from different alcohol consuming groups showed that FAEE are suitable markers for long-term alcohol misuse. Concerning sensitivity and specifity they are at least as good as other commonly used markers like GGT, CDT or MCV. The following provisional cut-off values were established: for chronically excessive alcohol consumption, the sum of the four FAEE (ethyl myristate, ethyl palmitate, ethyl oleate and ethyl stearate) found in the highest mean concentrations should be > 1 ng/mg in hair; for non-drinkers, concentrations < 0,4 ng/mg are typical. The quotient obtained by dividing the FAEE concentration by the squalene concentration was defined as the relative FAEE concentration, which provides a better classification of the samples regarding the consumer groups through hair analysis. Relative FAEE values also allow a reasonable comparison in the case of skin surface lipid concentrations for the first time. 2 ng/µg is suggested as a preliminary cut-off value. As a further important result of the current work, the mechanism of incorporation of FAEE into hair was clarified. It was shown that fatty acid ethyl esters are incorporated into hair mainly through sebum.