Дисертації з теми "Alcohol metabolite"

Sugar alcohols accumulate across a broad range of plant genera, often to concentrations exceeding that of soluble carbohydrates. While there is a general consensus on the function of sugar alcohols based on their physiochemical properties, gaps remain in understanding conditions that elicit sugar alcohol accumulation and its molecular control. Through a series of chemical, molecular, and physiological techniques, this thesis investigates the complexity of D-pinitol accumulation within the Leguminosae system and the transcriptional responses of key genes governing D-pinitol biosynthesis. In addition, naturally occurring compound specific carbon isotope abundances are used to evaluate the influence of D-pinitol synthesis on modelling water use efficiency. Assessing the chemical composition of the soluble leaf fraction found that DPinitol significantly increased over development in both chickpea (Cicer arietinum) grown in field and in soybean (Glycine max) grown under controlled environmental conditions. Compound specific carbon isotope abundance (δ13C) in samples collected from chickpea grown in field and soybean grown under elevated atmospheric CO2 established that concentrations of D-pinitol in the soluble fraction coupled with high Δ13C of the D-pinitol pool imparts a substantial influence over predictions of water use efficiency modelled from the leaf soluble fraction. Quantitative molecular investigations found that the inositol-1-phosphate synthase (INPS) gene is transcriptionally up regulated in response to a gradual water deficit in soybean grown in controlled conditions. While future studies must consider a wider range of legume species and genotypes when investigating sugar alcohol biosynthesis, the studies presented here suggest that the accumulation of D-pinitol in legumes is a good candidate for the improvement of plant performance and resilience.

Alcohol abuse is the third leading cause of preventable death in the United States. Excessive intake of alcohol can result to alcoholic fatty liver disease, the number one cause of live related mortalities in the US. The outlining purpose for this project is to determine the alcohol-induced changes in the liver cell protein signaling. For this project, we treated H4IIE rat hepatoma cells (with 100 and 200 mM ethanol overnight). H4IIE cells were chosen because they are a commonly used liver cell culture line that maintains characteristics of intact liver cells. After treatment we collected and prepared the cells for protein signaling analysis, using standard western blotting procedure. A western blot detects relative quantity of proteins in a sample. Briefly, protein samples are separated by size through electrophoresis, smaller proteins move faster through the gel so that the larger proteins are toward the top and smaller towards the bottom. The proteins are then transferred to a nitrocellulose membrane and protein concentration is detected by chemiluminescence. We chose to examine the effects of ethanol on the activation of the key regulator of metabolic signaling, Protein Kinase B/Akt (Akt). Based on our results, ethanol has no effect on the total amount of Akt in the H4IIE liver cells. However, ethanol significantly attenuates insulin-induced activation of Akt in a dose-dependent manner, as seen by a reduction in the amount of phosphorylated Akt. Therefore, we conclude that treatments that increase Akt activation may be a viable option for the treatment of alcoholic fatty liver disease.

Approximately 14,500 people were killed in traffic crashes where the driver was legally intoxicated in 2005, constituting 33% of all traffic fatalities that year. While social efforts to reduce the number of traffic fatalities have shown to be moderately successful, alcohol has remained a factor in 40% of all traffic deaths over the past decade. Transdermal ethanol detection is a promising method that could prevent drunk driving if integrated into an ignition interlock system; potentially preventing 90 million drunk driving trips a year in the US. However, experimental data from previous research has shown significant time delays between alcohol ingestion and detection at the skin which makes real time estimation of blood alcohol concentration via skin measurement difficult. Using a validated model we studied the effects that body weight, metabolic rate and ethanol dose had on the time lag between the blood alcohol concentration and transdermal alcohol concentration. The dose of alcohol ingested was found to have the most significant effect on the skin alcohol lag time. Additionally, custom transdermal ethanol sensors were designed and fabricated and a pilot study on human subjects was conducted to determine if inexpensive transdermal ethanol sensors could be used to detect alcohol in drivers.
Master of Science

Enantiopure alcohols are valuable intermediates in fine organic synthesis, in particular for preparation of biologically active compounds. The necessity of preparing single enantiomer drugs in an optically pure form has triggered much research, especially in the pharmaceutical industry. The biocatalytical production of chiral alcohols by alcohol dehydrogenase enzymes is characterized by the asymmetric reduction of the corresponding ketones, usually with high degree of stereoselectivity. The commercial value of the enzymes as stereoselective biocatalysts has been a significant driving force in understanding features that control their mechanism of catalysis and stereoselectivity. This work focuses on two enantiocomplementary dehydrogenase enzymes ((R)- and 2-(S)-hydroxypropyl-CoM (HPC) dehydrogenases (DH)) of the epoxide carboxylation pathway in Xanthobacter autotrophicus strain Py2. The main goal of this dissertation is to kinetically, mechanistically and structurally characterize S-HPCDH and through the comparison studies with R-HPCDH reveal the basis for high degree of stereoselectivity exhibited by both enzymes. Analysis of the molecular structure of R-HPCDH and the homology model of S-HPCDH suggests a mechanism of substrate specificity in which the binding of the substrate sulfonate moiety at distinct sites on each stereoselective enzyme directs the orientation of the appropriate substrate enantiomer for the hydride abstraction. The positively charged residues responsible for binding the CoM moiety of the substrate were identified in R-HPCDH (Arg152 and Arg196), and in S-HPCDH (Arg211 and Lys214). Site-directed mutagenesis confirmed their importance in binding and orienting physiological substrates, but not the substrates lacking the CoM moiety. Extensive kinetic and mechanistic characterization of S-HPCDH reveals its key catalytic features similar to those of R-HPCDH, but also points out a few important differences. Furthermore, the role of the methionine residues flanking the substrate in the active site of both dehydrogenases was investigated. Substitution of these residues to alanine resulted in enzymes with significantly altered catalytic parameters and suggested their importance in binding and catalysis. Additionally, the X-ray crystal structures of the Met187Ala and Met192Ala mutants of R-HPCDH have revealed their role as "gate keepers," protecting the active site from the surrounding solvent. Kinetic analysis of Met187Leu and Met192Leu mutants implied a structural, rather than catalytic function of the methionines. It is proposed that steric clashes of the terminal methyl group of the HPC substrates with the nicotinamide ring of NAD+ are a major determinant of the enantioselectivity in S-HPCDH. This research provides the first side-by-side characterization of a pair of short-chain dehydrogenase/reductase (SDR) enzymes expressed simultaneously to act on two enantiomers of the same alcohol produced in a metabolic pathway. The R-HPCDH and S-HPCDH enzymes are distinguished from all other known members of the SDR family in using the novel sulfonate functional group of coenzyme M as a handle for chiral discrimination. These results provide a standard for examining the molecular basis of stereoselectivity in other such enzyme pairs.

This doctoral thesis deals with the synthesis of two biofuels (bioethanol and biobuthanol) by bacteria. Concretely, the thesis is focused on a group of bacteria able to grow in a simple substrate such as synthesis gas or syngas. Syngas is a mixture of hydrogen, carbon monoxide, and carbon dioxide obtained through the gasification of urban and forestry wastes. The use of syngas as a substrate requires a good knowledge of bacterial metabolism to successfully control acid production and promote alcohol synthesis. To acquire this knowledge, the researcher carried out a set of experiments at lab scale, always using syngas. Among the most significant results, there is the relevance of both the temperature and the bacteria state at the start of the experiments. Additionally, new insights into bacterial metabolism which are applicable at industrial scale were gathered.
Aquesta tesi doctoral tracta la producció de dos biocombustibles – el bioetanol i el bioalcohol - per mitjà de microorganismes. En concret, la tesi s'ha centrat en un grup de bacteris capaços de sintetitzar bioalcohols a partir del gas de síntesis o syngas. El syngas és una mescla d’hidrogen, diòxid de carboni i monòxid de carboni que s’obté mitjançant la gasificació de diferents tipus de residus. L’ús d’aquest gas com a substrat requereix un bon coneixement del metabolisme dels bacteris involucrats a fi de controlar amb èxit la producció d'àcids i afavorir la d'alcohols. Aquest coneixement s'ha adquirit amb una sèrie d'experiments avançats a escala de laboratori. Entre els resultats més significatius destaca la rellevància que ha demostrat tenir la temperatura en què creixen els bacteris i l’estat del bacteri en el moment d’inici dels experiments. També s’han aportat nous coneixements sobre el metabolisme bacterià que són aplicables a escala industrial.

Thesis (Ph. D.)--Ohio State University, 2004.
Title from first page of PDF file. Document formatted into pages; contains xxxiii, 278 p.; also includes graphics. Includes abstract and vita. Advisor: Kenneth Chan, Dept. of Pharmacy. Includes bibliographical references (p. 266-278).

INTRODUCTION: Alcoholic cirrhosis occurs at a higher rate in female patients, at an earlier age, and with a lower consumption of alcohol compared to male patients. In our study on alcoholic fatty liver disease (AFLD) and adipokine levels, female mice showed a 50% higher mortality rate compared to ethanol fed male mice. The amount of ethanol consumed was similar between sexes when normalized to body weight. This resulted in our hypothesis that female mice are more susceptible to inflammation caused by alcohol consumption. METHODS: 12-week old female mice were fed a Lieber-Decarli alcohol diet (5% ETOH by volume) for 6-weeks and body weight and food intake were measured daily. Serum was collected from the mice and alanine aminotransferase (ALT), aspartate aminotransferase (AST), serum triglycerides, tumor necrosis factor alpha (TNF-α), and interleukin 6 (IL-6) levels were measured with the appropriate assays. RESULTS: In response to alcohol feeding, female mice showed significant increases in levels of ALT and AST compared to the male mice, indicating increased damage to the liver. TNF-α and IL-6 were also significantly increased in the ethanol fed female mice, indicating a significant increase in inflammation compared to the male ethanol fed mice. There was no difference found in the levels of serum triglycerides. CONCLUSION: These results indicate chronic alcohol consumption affects mice in a sex dependent manner, and that female mice are more susceptible to the adverse effects of alcohol than male mice. Increased female susceptibility to ethanol-induced damage must be considered in future ethanol-feeding studies.

L'alcool est une des principales causes de maladie du foie en Europe avec peu d'options thérapeutiques. Parmi les consommateurs d'alcool, seul certains patients vont évoluer vers des formes sévères d'atteinte hépatique et le microbiote intestinal a été identifié comme cofacteur de cette susceptibilité individuelle. Il interagit avec le foie notamment via la production de métabolites bactériens tels que les acides biliaires secondaires.L'objectif de ce travail a été dans un premier temps d'analyser le microbiote intestinal, au sein d’une cohorte de patients alcooliques à différents stades de la maladie alcoolique du foie, et d’étudier sa relation avec les acides biliaires. Dans un deuxième temps, ce projet avait pour but d’étudier s’il était possible d'améliorer l'atteinte hépatique liée à l'alcool, en modulant le microbiote intestinal, dans un modèle animal de maladie alcoolique du foie.Les patients avec une atteinte hépatique sévère liée à l'alcool ont un profil d’acides biliaires plus hydrophobe, et donc plus toxique, associé à une dysbiose et à des modifications des fonctions bactériennes. Ces modifications des fonctions bactériennes participent à la gravité de la maladie. La modulation du microbiote par la pectine, un prébiotique, permet de prévenir mais également de faire régresser les lésions hépatiques chez la souris. Les effets protecteurs de la pectine sont corrélés avec le métabolisme du tryptophane.Ce travail de thèse montre que le microbiote intestinal et ses métabolites sont une cible thérapeutique potentielle dans la maladie alcoolique du foie. Il ouvre la porte à la réalisation d'essais cliniques chez l'homme dans lesquels nous pourrions limiter la progression des lésions hépatiques liées à l'alcool en contrôlant le microbiote intestinal grâce à l’utilisation de la pectine ou du métabolisme du tryptophane
Alcohol is one of the main causes of alcoholic liver disease in Europe with few therapeutic options. Among alcohol consumers, only a part of these patients will develop severe liver lesions. This individual susceptibility is driven by intestinal microbiota. Intestinal microbiota interacts with the liver through the production of bacterial metabolites including the secondary bile acids.The aim of my project, was firstly to study the relationship between the intestinal microbiota and the bile acids composition depending on the severity of alcoholic liver disease in a cohort of alcoholic patients. Secondly, I assessed the improvement of alcohol induced liver lesions by changing the intestinal microbiota in a mouse model of alcoholic liver disease.Patients with a severe form of alcoholic liver disease display a higher hydrophobic bile acid pool, more toxic, associated with a specific dysbiosis and changes in the bacterial functions. Changing the intestinal microbiota by using pectin, a prebiotic, prevents and reverts alcohol induced liver injury in mice. These protective effects of pectin involve changes in the tryptophan metabolism.In conclusion, these studies highlight that the intestinal microbiota and its metabolites are potential therapeutic targets for alcoholic liver disease. Moreover, pectin as an alimentary product could be proposed in the management of alcoholic liver disease in humans

Thesis (PhD (Wine Biotechnology))--University of Stellenbosch, 2011.
Includes bibliography.
ENGLISH ABSTRACT: Alcoholic fermentation, and especially wine fermentation, is one of the most ancient microbiological processes utilized by man. Yeast of the species Saccharomyces cerevisiae are usually responsible for most of the fermentative activity, and many data sets clearly demonstrate the important impact of this species on the quality and character of the final product. However, many aspects of the genetic and metabolic processes that take place during alcoholic fermentation remain poorly understood, including the metabolic processes that impact on aroma and flavour of the fermentation product. To contribute to our understanding of these processes, this study took two approaches: In a first part, the initial aim had been to compare two techniques of transcriptome analysis, DNA oligo-microarrays and Serial Analysis of Gene Expression (SAGE), for their suitability to assess wine fermentation gene expression changes, and in particular to assess their potential to, in combination, provide combined quantitative and qualitative data for mRNA levels. The SAGE methodology however failed to produce conclusive data, and only the results of the microarray data are shown in this dissertation. These results provide a comprehensive overview of the transcriptomic changes during model wine fermentation, and serve as a reference database for the following experiments and for future studies using different fermentation conditions or genetically modified yeast. In a second part of the study, a screen to identify genes that impact on the formation of various important volatile aroma compounds including esters, fatty acids and higher alcohols is presented. Indeed, while the metabolic network that leads to the formation of these compounds is reasonably well mapped, surprisingly little is known about specific enzymes involved in specific reactions, the genetic regulation of the network and the physiological roles of individual pathways within the network. Various factors that directly or indirectly affect and regulate the network have been proposed in the past, but little conclusive evidence has been provided. To gain a better understanding of the regulations and physiological role of this network, we took a functional genomics approach by screening a subset of the EUROSCARF strain deletion library, and in particular genes encoding decarboxylases, dehydrogenases and reductases. Thus, ten genes whose deletion impacted most significantly on the aroma production network and higher alcohol formation were selected. Over-expression and single and multiple deletions of the selected genes were used to genetically assess their contribution to aroma production and to the Ehrlich pathway. The results demonstrate the sensitivity of the pathway to cellular redox homeostasis, strongly suggest direct roles for Thi3p, Aad6p and Hom2p, and highlight the important role of Bat2p in controlling the flux through the pathway.
AFRIKAANSE OPSOMMING: Alkoholiese fermentasie, en veral die maak van wyn, is een van die vroegste mikrobiologiese prosesse wat deur die mensdom ingespan is. Die gisspesie Saccharomyces cerevisiae is gewoonlik grotendeels verantwoordelik vir die fermentasie and verskeie vorige studies het gedemonstreer dat hierdie spesie ‘n baie belangrike rol speel in die uiteindelike kwaliteit en karakter van die voltooide produk. Nieteenstaande die feit is daar steeds baie aspekte van beide die genetiese en metaboliese prosesse wat plaasvind tydens alkoholiese fermentatsie wat nog swak verstaan word, insluitende metaboliese padweë wat ‘n impak het op die smaak en aroma van die fermentasie produk. Om ons kennis van die veld uit te brei het die studie twee aanslae geneem: In die eerste geval is gepoog om twee tegnieke van transkriptoom analiese, nl. DNA oligomikro- arrays en Serial Analysis of Gene Expression (SAGE) te bestudeer vir hul vermoë om geen ekspressie veranderinge tydens wynfermentasie te ondersoek en meer spesifiek om hul potensiaal om ‘n kombinasie van kwantitatiewe sowel as kwalitatiewe data met betreking to mRNA vlakke te produseer. Die SAGE metode kon egter geen betroubare resultate produseer nie en dus word slegs die resultate van die mikro-array eksperimente in die tesis bespreek. Die resultaat is ‘n geheeloorsig oor die geenekspressie veranderinge wat so ‘n wyngis tydens alkoholiese fermentasie ondergaan en dien as ‘n verwysingsraamwerk vir toekomstige studies met geneties gemodifiseerde gis of selfs verskillende fermentasieparameters. Die tweede deel van die studie het gefokus op die identifikasie van gene wat ‘n impak het op die vorming van belangrike, vlugtige aroma komponente, o. a. Esters vetsure en hoër alkohole d.m.v. ‘n siftingseksperiment. Alhoewel daar redelik baie inligting is oor die onderligende metaboliese netwerke wat lei tot die vorming van die verbindings, is daar min kennis van die genetiese regulasie van die netwerk en die fisiologiese rol van individuele padweë wat die netwerk vorm. Verskeie faktore – wat of die netwerk direk of indirek affekteer – is al voorgestel, meer met min konkrete bewyse. Dus het ons gepoog om meer lig op die onderwerp te laat m.b.v. ‘n funksionele genoom aanslag deur ‘n siftingseksperiment te doen op ‘n subgroep (spesifiek gene wat kodeer vir dekarboksilase, dehidrogenase en reduktase ensieme) van die EUROSCARF delesiebiblioteek. Dus is tien gene geïdentifiseer – die delesie waarvan ‘n merkbare effek het op die aroma produksie netwerk en spesifiek die van hoër alkohole. Ooruitdrukkings en enkel en meervoudige delesie rasse van die tien gene is gemaak om d.mv. genetiese analiese, hulle rol in aroma produksie en die Ehrlich padweh uit te pluis. Die resultate toon dat hierdie padweg sensitief is teenoor die sellulêre redoks balans en dui op direkte rolle vir Thi3p, Aad6p en Hom2p, asook dat Bat2p ‘n baie belangrike rol speel in die werking van die padweg.

L’addiction aux drogues est une maladie comportementale récidivante caractérisée par une recherche et une prise compulsive de drogues, une perte de contrôle sur la prise malgré les conséquences négatives et l’émergence d'un état émotionnel négatif lors de l’absence de la drogue. L’addiction implique des neuroadaptations cérébrales persistantes. Des études récentes montrent que le cholestérol joue un rôle crucial dans le fonctionnement cérébral en participant à divers processus cellulaires et, en particulier, au contrôle de la neurotransmission. L’objectif de ce travail de thèse consistait à étudier l’implication potentielle du métabolisme du cholestérol dans l’addiction et en particulier si ce métabolisme était affecté par les drogues d’abus. Dans ce travail, nous avons étudié l’expression de gènes codant des protéines impliquées dans le métabolisme du cholestérol cérébral après une consommation volontaire chronique d’alcool par des rats et après des injections aigues ou chroniques de cocaïne. Nous avons analysé l’expression de ces gènes dans des structures cérébrales impliquées dans l’addiction, notamment le cortex préfrontal, le noyau accumbens, l’amygdale et l’hippocampe. Nous avons trouvé que l’exposition à l’alcool et à la cocaïne modifient l’expression des protéines impliquées dans la synthèse, le transport et la dégradation du cholestérol de façon spécifique de la drogue, du traitement (aigu/chronique) et de la région cérébrale étudiée. Dans une deuxième partie de la thèse, nous avons utilisé une approche virale permettant de surexprimer la CYP46A1, l’enzyme de dégradation du cholestérol cérébral dans le cortex préfrontal afin d’évaluer l’impact de cette surexpression sur la recherche de cocaïne dans un modèle de rechute. La surexpression de la CYP46A1dans cette structure n’a eu aucun impact sur la recherche de cocaïne. Des études futures seront nécessaires pour déterminer si l’altération de l’expression de cette enzyme dans d’autres structures, comme par exemple le noyau accumbens, pourrait avoir des effets bénéfiques sur la rechute. L’ensemble de ces travaux montrent que l’exposition aux drogues d’abus modulerait le métabolisme cérébral du cholestérol dans certaines structures cérébrales. Ce projet de thèse ouvre de nouvelles perspectives quant au rôle du métabolisme du cholestérol cérébral dans l’addiction, et il pourrait en résulter de nouvelles thérapeutiques dans le traitement de cette maladie psychiatrique coûteuse
Drug addiction is a chronic brain disease characterized by drug-seeking and compulsive drug taking, a loss of control over drug taking despite the negative consequences and the emergence of a negative emotional state in the absence of the drug. Addiction involves persistent neuroadaptations at the cerebral level. Recent evidences show that cholesterol plays a crucial role in brain function by participating in various cellular processes in particular in the control of neurotransmission. The aim of this thesis was to investigate the potential role of cholesterol in addiction and in particular a potential dysregulation of cholesterol metabolism in response to drugs of abuse. In this work, we investigated the expression of genes encoding proteins involved in the metabolism of cerebral cholesterol after a chronic voluntary consumption of alcohol from the rats and after acute or chronic exposure to cocaine. We analyzed gene expression in brain structures involved in addiction such as the prefrontal cortex, the nucleus accumbens, the amygdala and the hippocampus. We found that exposure to alcohol and cocaine modifies the expression of proteins involved in the synthesis, the transport and the degradation of cholesterol in drug-specific, treatment- specific (acute / chronic) and region-specific manners. In the second part of the thesis, we used a viral approach to overexpress CYP46A1, the cerebral cholesterol degradation enzyme in the prefrontal cortex, in order to evaluate the impact of this overexpression on cocaine-seeking in a model of relapse. The overexpression of CYP in this structure has no effect on drug-seeking for cocaine. Future studies are needed to determine whether altering cholesterol metabolism in other structures, for example the nucleus accumbens, may have beneficial effects on relapse. Altogether these studies show that exposure to drugs of abuse might modulate cerebral metabolism of cholesterol. This thesis project opens new perspectives on the role of cholesterol cerebral metabolism in addiction which may ultimately result in new therapeutic avenues for the treatment of this costly psychiatric disorder

La melatonina és una indolamina que és produïda pel llevat a partir de l'aminoàcid triptòfan durant la fermentació alcohòlica. En aquesta tesi, estudiem la producció de melatonina en most sintètic en diferents condicions ambientals i nutricionals pels llevats, així com l'efecte d'aquesta molècula bioactiva en la dinàmica de poblacions d'una fermentació. Com a resultats, observem que la detecció estava desfasada entre la producció intracel·lular, la qual va ocórrer durant la fase d'adaptació al medi i la secreció al medi extracel·lular durant la fase exponencial o estacionària. Així mateix, la presència de melatonina millorava el desenvolupament de la fermentació i augmentava la presència de llevats no-Saccharomyces a final de fermentació. A més, vam analitzar la interacció d'aquesta molècula amb proteïnes durant la fermentació. Els resultats van mostrar que la melatonina s'unia a enzims glicolítics en llevats que tenien una alta capacitat fermentativa, reforçant la idea que la melatonina podria actuar com una molècula senyal durant la fermentació alcohòlica. Finalment, es va optimitzar un nou mètode de detecció per fluorescència utilitzant una línia cel·lular que presenta el receptor humà de melatonina per a mostres procedents de begudes fermentades. Els resultats van mostrar que aquest nou mètode disminueix el límit de detecció i és una bona alternativa en comparació amb el mètode de detecció de melatonina basat en cromatografia. No obstant, es necessita una extracció de la mostra per a realitzar l'anàlisi de melatonina.
La melatonina es una indolamina que es producida por la levadura a partir del aminoácido triptófano durante la fermentación alcohólica. En esta tesis, estudiamos la producción de melatonina en mosto sintético en diferentes condiciones ambientales y nutricionales por las levaduras, así como el efecto de esta molécula bioactiva en la dinámica de poblaciones de una fermentación. Como resultados, observamos que la detección estaba desfasada entre la producción intracelular durante la fase de adaptación al medio y la secreción al medio extracelular durante la fase exponencial o estacionaria. Así mismo, la presencia de melatonina mejoraba el desarrollo de la fermentación y aumentaba la presencia de levaduras no-Saccharomyces a final de fermentación. Además, analizamos la interacción de esta molécula con proteínas durante la fermentación. Los resultados mostraron que la melatonina se unía a enzimas glucolíticas en levaduras que tenían una alta capacidad fermentativa, reforzando la idea de que la melatonina actuaría como una molécula señal durante la fermentación alcohólica. Finalmente, se optimizó un nuevo método de detección por fluorescencia utilizando una línea celular que presenta el receptor humano de melatonina en muestras procedentes de bebidas fermentadas. Los resultados mostraron que este nuevo método disminuye el límite de detección y es una buena alternativa en comparación con el método de detección de melatonina basado en cromatografía. Sin embargo, se necesita una extracción de la muestra para realizar el análisis de melatonina.
Melatonin is an indolamine, which is produced by yeast from the aromatic amino acid, tryptophan, during alcoholic fermentation. Here, we study the production of melatonin in synthetic must in different environmental and nutrition conditions by wine yeast species as well as the effect of this bioactive molecule on the fermentation performance. The results showed that the melatonin detection was delayed between intracellular production, which occurred during lag phase, and extracellular secretion, at the exponential or stationary phase. Additionally, melatonin presence improved fermentation performance and survival of non-Saccharomyces yeasts. We also studied melatonin interactions with proteins during fermentation. Consequently, we found that melatonin was bound to glycolytic enzymes and this interaction is related to yeasts with high fermentative capacity. This reinforces the idea of melatonin acting as a signal molecule during alcoholic fermentation. Finally, we optimized a novel fluorescence method, based on a cell line that presents the human melatonin receptor, in fermented samples. We observed that this new method decreased the limit of detection and was a good alternative in comparison with a chromatographic method, although, an extraction was needed for melatonin analysis.

Projeto de Pós-Graduação/Dissertação apresentado à Universidade Fernando Pessoa como parte dos requisitos para obtenção do grau de Mestre em Ciências Farmacêuticas
O uso de bebidas alcoólicas é provavelmente o hábito social mais antigo, difundido na cultura ocidental. De facto, o consumo de álcool é popular, legal na maioria dos países, e remonta a tempos imemoriais. Hoje em dia é largamente consumido em todo o mundo, sendo um fator de risco de inúmeras patologias e de acidentes rodoviários. Apesar de todos os riscos que são atribuídos ao consumo exagerado de álcool, nas últimas décadas alguns estudos revelaram que o consumo moderado e regular de álcool tem efeitos positivos na saúde humana, principalmente sobre o sistema cardiovascular e doenças como a diabetes. Mesmo assim é necessário consciencializar a população do significado de consumo moderado, pois o álcool é uma substância na qual existe uma linha muito ténue no que se refere aos efeitos positivos e negativos do seu consumo, sendo os seus efeitos totalmente dependentes da forma como é consumido. Contudo, e apesar de todas as evidências que apontam para uma ação benéfica do consumo moderado de álcool, os seus mecanismos de ação ainda não estão totalmente esclarecidos. Assim, é necessário continuar a investigação nesta área de forma a obter novos resultados e validar de forma inequívoca os resultados obtidos anteriormente. The use of alcohol is probably the oldest social habit, widespread in Western culture. In fact, alcohol consumption is popular, legal in most countries, and dates back to ancient times. Nowadays it is widely consumed throughout the world, being a risk factor for numerous diseases and road accidents. Despite all the risks that are attributed to alcohol abuse in recent decades some studies have shown that regular and moderate consumption of alcohol has positive effects on human health, particularly on the cardiovascular system and diseases such as diabetes. Even so the population must be aware of the significance of moderate drinking because alcohol is a substance in which there has a borderline when it comes to the positive and negative effects of its consumption, being its effects totally dependent on its consumption pattern. However, despite all the evidence pointing to a beneficial effect of moderate alcohol consumption, their mechanisms of action are not yet fully understood. Thus, it is necessary to continue research in this area in order to obtain new results and validate unequivocally the results obtained previously.

Abstract In the search for more reliable blood markers for excessive alcohol consumption, considerable effort has been devoted to measurements of carbohydrate-deficient transferrin (CDT), which increases in body fluids as a result of prolonged alcohol intake. In the present work, three CDT methods, CDTect (Pharmacia & Upjohn), %CDT radioimmunoassay (%CDT RIA) by Axis (Oslo, Norway), and Axis %CDT turbidimetric immunoassay (%CDT TIA) were examined for their diagnostic performance in cases of alcohol abuse with or without liver disease. The diagnostic performance of CDT as a marker of alcohol abuse correlates positively with alcohol consumption. As compared with g-glutamyltransferase (GGT) and mean corpuscular volume of erythrocytes (MCV), which are conventionally used as laboratory markers of excessive ethanol consumption, CDT (CDTect) has the highest sensitivity (64%) at the specificity level of 100% in heavy drinkers consuming >100 g ethanol/day, but its sensitivity decreases to 34% in cases with an alcohol intake of <100 g/day, which hampers the use of CDT as a community screening method. Patients with alcoholic liver disease (ALD) have significantly higher CDT values than alcoholics with non-liver pathology. However, CDT is primarily increased in cases with an early stage of ALD, so that there is a weak negative correlation between CDT and disease severity, which may prove to be of diagnostic value. Especially in men, CDTect seems to achieve greater sensitivity than %CDT RIA or %CDT TIA for detecting recent alcohol abuse among heavy drinkers, but it does have a significant correlation with serum transferrin, especially in individuals reporting social drinking or no alcohol intake. This should be considered when interpreting the assay results in patients with increased serum transferrin. %CDT methods achieve greater specificity than CDTect when analyzing samples from patients with high serum transferrin concentrations. Acetaldehyde-protein adducts are formed in the body after excessive ethanol intake, and their formation triggers antibody production, which may contribute to some forms of tissue damage seen in alcohol abusers. To obtain more information on the association between serum antibodies against acetaldehyde adducts, ALD and alcohol consumption, assays for antibodies against albumin and haemoglobin adducts were performed. Antibodies of the immunoglobulin (Ig) isotypes A, G, and M against acetaldehyde-adducts are formed in patients with prolonged heavy alcohol consumption. IgA titres in ALD patients are significantly higher than those found in patients with non-alcoholic liver disease, non-drinking controls, or heavy drinkers with no signs of liver disease. Anti-adduct IgG titres, in turn, are increased both in ALD and in heavy drinkers with no signs of liver disease as compared with non-alcoholic liver disease patients or non-drinking controls. It appears that anti-adduct IgA, IgG and IgM titres in ALD patients correlate with the severity of the liver disease. Although this association is a limitation for the usefulness of these antibodies as markers of alcohol abuse, it may serve as a basis for the differential diagnosis of alcohol-induced liver disease.

Orientadores: Anibal Eugenio Vercesi, Nadja Cristhina de Souza Pinto
Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas
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Resumo: O número de indivíduos que sofrem com patologias associadas ao consumo abusivo de etanol tem aumentado significativamente no último século. Como consequência desse fato, os custos associados ao tratamento do alcoolismo, bem como das doenças associadas a ele também têm aumentado, onerando o sistema de saúde e se tornando um problema de saúde pública de grande relevância atualmente. Os mecanismos moleculares que desencadeiam muitas dessas doenças não estão completamente esclarecidos. O tecido hepático é o mais afetado pelo etanol e as mitocôndrias têm sido apontadas como alvos cruciais na toxicidade hepática induzida pelo álcool. Logo, o objetivo desse trabalho foi investigar como o consumo de etanol afeta o estado redox e o metabolismo mitocondriais no fígado. Ratos Wistar machos adultos jovens e de meia-idade receberam ad libitum solução alcoólica 25% (v/v) como única fonte de líquido. Os grupos controle receberam somente água. Ambos os grupos receberam ração ad libitum. Mitocôndrias hepáticas foram isoladas usando técnicas padrão. O consumo de ração e de líquidos foi significativamente menor em animais que ingeriram álcool, resultando em menor ganho de massa corpórea nos protocolos utilizados. As mitocôndrias dos animais que consumiram etanol apresentaram menores níveis de respiração em condição basal e quando energizadas com substratos respiratórios. A atividade e os níveis protéicos de citocromo c oxidase foi menor nos grupos tratados com etanol. Independente da duração do período de tratamento, mitocôndrias hepáticas de animais que ingeriram álcool foram menos susceptíveis à transição de permeabilidade mitocondrial induzida por cálcio, quando comparadas às mitocôndrias dos animais do grupo controle. Esse efeito foi revertido pela adição de oxidantes de nucleotídeos de piridina (acetoacetato, diamida ou tert butil-hidroperóxido) ou em mitocôndrias desacopladas. Também houve aumento em nucleotídeos de piridina na forma reduzida e aumento na razão NAD(P)H/NAD(P)+ em mitôndrias hepáticas de ratos consumidores de etanol. Em concordância a esses dados, houve aumento na capacidade de retenção de cálcio, processo que é dependente do estado redox intramitocondrial. Por outro lado, não houve diferença na produção de espécies reativas de oxigênio entre os grupos controle e tratados com álcool. A atividade de glutationa peroxidase e as quantidades de GSH e de GSSG também não sofreram alterações. Entretanto, houve redução nos níveis de DNA mitocondrial nos tratamentos agudos, porém com tendência para retornar aos níveis normais nos tratamentos crônicos, indicando uma resposta adaptativa à injúria induzida pelo etanol. Em conjunto, nossos resultados indicam que o consumo de etanol modula o estado redox mitocondrial e de sistemas antioxidantes, prevenindo a abertura do poro de transição de permeabilidade mitocondrial. A presença desse xenobiótico no fígado também altera significativamente os níveis de NADP reduzido, agente redutor final para o sistema glutationa redutase/peroxidase que detoxifica H2O2 na matriz mitocondrial. Além disso, a resposta adaptativa ao álcool observada no DNA mitocondrial pode contribuir para compreender melhor os mecanismos envolvidos no reparo de lesões a biomoléculas e os estágios iniciais de adaptação a esse xenobiótico, etapas que precedem a morte celular, hepatite alcoólica ou carcinogênese em tecido hepático exposto cronicamente ao etanol
Abstract: The number of people suffering from alcoholism has increased significantly over the last century. As a result, costs associated with treating the addiction itself as well as the associated pathologies have also increased, such that this is considered as public health issue. Furthermore, the molecular events leading to several of these diseases are not yet clearly understood. Hepatic tissue is the most affected by alcohol, and mitochondria have been suggested to be a crucial target in alcohol-induced liver toxicity. Thus, the aim of our study was to investigate how ethanol consumption affects the redox state and mitochondrial metabolism in the liver. Young adult and middle-aged male Wistar rats were given a 25 % (v/v) ethanol solution as the only source of drinking water. Control groups received water only. Liver mitochondria were isolated using standard techniques. Food and water intake was significantly lower in alcohol-drinking rats, resulting in lower weight gain during the treatment regimes. Mitochondria from the alcohol-drinking group had lower respiration under levels in basal condition, when energized by substrates feeding electrons into complexes I and IV. Cytochrome c oxidase activity and protein levels were lower in the alcohol group as well. Additionally, regardless of the length of the treatment, liver mitochondria from the alcohol-treated animals were more resistant to Ca2+-induced mitochondrial permeability transition (MPT), when compared to mitochondria from control animals. This effect was abrogated by oxidizing agents of pyridine nucleotides (acetoacetate, diamide or tert butylhydroperoxide) or in uncoupled mitochondria. We also found that liver mitochondria from the alcohol-drinking rats had a more reduced pyridine nucleotide pool and higher NAD(P)H/NAD(P)+ ratios. In addition, Nampt (an enzyme of the NAD+ synthetic pathway) protein levels did not differ after alcohol consumption. Accordingly, the calcium retention capacity of the isolated mitochondria, which is dependent upon intramitochondrial redox state, was higher in the alcohol group. On the other hand, levels of reactive oxygen species showed no differences between the control and alcohol groups, both in mitochondria and in splenic lymphocytes. Glutathione peroxidase activity and the amounts of GSH and GSSG were also not changed. However, mitochondrial DNA levels were decreased in the short term treatments, but tended to go back up to normal levels in the chronic treatments, indicating an adaptative response to ethanol-induced injury. Together, our results indicate that ethanol consumption modulates the mitochondrial redox state and the antioxidant systems, protecting against Ca2+-induced mitochondrial pore transition permeability opening. The presence of this xenobiotic can significantly change the levels of reduced NADP, the ultimate reducing agent in the gluthatione reductase/peroxidase system that detoxifies H2O2 in the mitochondrial matrix. In addition, the adaptative response to ethanol, seen in mitochondrial DNA, may contribute to further understand the mechanisms related to lesions in biomolecules and the initial steps that preceed cell death, alcoholic hepatitis or carcinogenic process in hepatic tissue exposed chronically to ethanol
Doutorado
Biologia Estrutural, Celular, Molecular e do Desenvolvimento
Doutor em Fisiopatologia Medica

Recent findings, including our own work, demonstrated that intestinal microbiota species produce bioactive metabolites that engage host cellular pathways. Microbiota-derived metabolites have also been detected in circulation and in the, setting up the intriguing possibility that these bacterial products could directly interact with host cellular pathways at distant sites. The study described in this abstract investigates the hypothesis that gut microbiota dysbiosis perturbs the balance of immunomodulatory microbiota metabolites, which exacerbates liver inflammation in steatosis. We utilize a multi-omic approach to identify microbiota-dependent immunomodulatory metabolites and characterize their effects on liver inflammation and metabolic function. In summary, we show that the levels of AAA-derived microbiota metabolites are significantly depleted in a diet model of liver steatosis, and that these metabolite can act directly on hepatocytes to modulate inflammatory pathways. Our results also show that the microbiota metabolites are ligands for the AhR, which could provide a mechanistic link for the observed anti-inflammatory effects. Taken together, our findings support the hypothesis that dysbiosis of the gut microbiota could predispose the liver to inflammation in diet-induced steatosis through an altered microbiota metabolite profile. Prospectively, additional insights into the mechanisms underlying the link between microbiota dysbiosis and NAFLD could provide novel strategies to treat or prevent the progression of fatty liver diseases through the use of probiotics or postbiotics.

BACKGROUND: Prior experimental studies found associations of alcoholism and alcoholic liver disease (ALD) with abnormal methionine metabolism. Aberrant methionine metabolism may play a central role in the pathogenesis of ALD. AIM: To establish profiles of plasma methionine metabolites in ALD patients compared to those found in healthy control subjects and alcoholics without clinical or biochemical evidence of liver disease, and to define the relationship of these profiles to clinical and pathological severity of ALD. PATIENTS AND METHODS: Serum levels of S-adenosylmethionine (SAM), S-adenosylhomocysteine (SAH),homocysteine (Hcy), methionine, dimethylglycine (DMG), cysteine, and cystathionine were measured by GCMS and LCMS in 32 patients with ALD (group A), 20 chronic alcoholics without liver disease (group B), and 20 healthy control subjects (group C). Liver biopsies were performed in 20 ALD patients. Patients with creatinine ? 1.4 were excluded. RESULTS: The mean age of all subjects was 47 ± 9.7 years (range 28-67), with 27.8 % women. No age or gender difference between the 3 groups. The mean duration of abstinence was 71 days for group A, and 1.3 days for group B. AST, Bilirubin, Alkaline Phosphatase, and INR were significantly higher in group A compared to the other 2 groups (all p< 0.0001). SAM, SAH, and DMG were significantly higher in group A compared to groups B and C, while Hcy was significantly higher in group A and B vs group C. Increased DMG is consistent with activation of betaine hydoxymethyltransferase in response to elevated Hcy. Methionine was similar in the three groups. SAM was significantly correlated with cysteine serum level (r= 0.34, p= 0.02), indicating a possible effect of diminished kidney function on SAM serum levels. Liver histology showed 5 patients with no steatosis, 3 grade 1, 4 had grade 2, and 8 grade 4; one patient did not 2 have fibrosis, 4 had stage 1, 4 stage 2, 5 stage 3 and 6 stage 4. On bivariate analysis, the duration of abstinence correlated significantly with the severity of steatosis (p= 0.02). On multivariate analysis, predictors of steatosis severity were AST, bilirubin, creatinine, albumin, and serum Hcy (p=0.0002); predictors of stage of fibrosis were hispanic ethnicity, ALT, SAM/SAH, and Hcy (p= 0.001). CONCLUSIONS: Serum Hcy is elevated in alcoholics, with the highest levels in those with ALD. The original correlation between Hcy and severity of histopathology score emphasizes the link between methionine metabolism and clinical ALD.
BACKGROUND: precedenti studi hanno indicato la correlazione tra l’epatopatia alcolica ed alterazioni del metabolismo della metionina. SCOPI: Il primo scopo (Aim 1) e’ di definire i profili sierici dei componenti del metabolismo della metionina in pazienti con epatopatia alcolica, in alcolisti senza epatopatia ed in soggetti sani e definire la loro relazione con parametri bioumorali ed istologici di severita’ di epatopatia alcolica. Il secondo scopo (Aim 2) e’ di definire l’effetto della somministrazione orale di S-adenosilmetionina (SAM) verso placebo in soggetti con epatopatia alcolica. Il trial clinico e’ ancora in corso, dunque vengono qui presentati i risultati del Aim 1. PAZIENTI E METODI: i livelli sierici di SAM, S-adenosilomocisteina (SAH), omocisteina (HCY), metionina, dimetilglicina, cisteina e cistationina sono stati misurati mediante GCMS e LCMS in 32 pazienti con epatopatia alcolica (grupp A), 20 alcolisti senza evidenza di malattia epatica (gruppo B), e 20 soggetti sani (gruppo C). Le biopsie epatiche sono state eseguite al tempo 0 e dopo 6 mesi di trial. I pazienti con creatinina > 1.4 sono stati esclusi dallo studio. RISULTATI: L’eta’ media dell’intero gruppo e’ 47 ± 9.7 anni (range 28-67). Il 27.8 % sono donne. Non si e’ rilevata nessuna significativa differenza di eta’ tra i 3 gruppi. La durata media dell’astinenza e’ di 71 giorni per il gruppo A e di 1.3 giorni per il gruppo B. I parametri bioumorali di epatopatia alcolica erano tutti significativamente alterati nel gruppo A rispetto agli altri 2 gruppi. SAM, SAH e DMG erano significativamente elevati nel gruppo A rispetto al gruppo B e C, mentre HCY era significativamente elevata nel gruppo A e B rispetto al gruppo C. L’aumento di DMG e’ spiegabile dall’attivazione della salvage pathway che porta alla sintesi di metionina in caso di uso di alcol. L’istologia epatica ha dimostrato 5 pazienti senza steatosi, 3 con grado 1, 4 con grado 2, e 8 con grado 4; un paziente non ha dimostrato fibrosi epatica, 4 hanno dimostrato stage 1, 4 stage 2, 5 stage 3 e 6 stage 4. Secondo la correlazione bivariata, la durata dell’astinenza alcolica e’ correlata postivamente con la severita’ della steatosi (p=0.02). L’analisi multivariata, fattori predittivi di steatosi sono risultati essere i livelli di AST, bilirubina, creatinina, albumina, e HCY (p=0.0002). Fattori predittivi di fibrosis sono risultati essere l’etnia ispanica, ALT, SAM/SAH e HCY (p=0.001). CONCLUSIONI PRELIMINARI: I livelli sierici di HCY sono elevati nell’epatopatia alcolica e rivestono un ruolo importante nel predire la severita’ della steatosi e della fibrosi.

A) OBJETIVOS

Los principales objetivos de la tesis han sido: 1) analizar el metabolismo hepático del etanol en los alcohólicos crónicos, 2) la producción de acetaldehido, 3) la actividad e isoenzimas de la alcohol deshidrogenasa (ADH) y aldehído deshidrogenasa (ALDH) hepáticas, y 4) la relación de cada uno de estos factores con el alcoholismo y el grado de lesión hepática.

B) PACIENTES

La ADH y ALDH hepáticas se han estudiado en 84 pacientes, 60 eran alcohólicos (5 con hígado normal, 9 esteatosis, 9 fibrosis, 11 hepatitis alcohólica, 19 cirrosis alcohólica y 7 cirrosis y hepatitis alcohólica) y 24 eran hepatópatas no alcohólicos (14 hepatitis crónica y 10 cirrosis). Además, en 25 de los alcohólicos (4 hígado normal, 4 esteatosis, 4 fibrosis, 7 hepatitis alcohólica y 7 cirrosis no alcohólica) y en 6 de los cirróticos no alcohólicos se ha estudiado el metabolismo hepático del etanol.

C) ESTUDIO DE LA CAPACIDAD OXIDATIVA DEL ETANOL

La capacidad de oxidación del etanol resultó superior en los pacientes alcohólicos que en los no alcohólicos de manera que en los alcohólicos la velocidad de metabolización del etanol (VME) fue de 113+/-16.9 mg/Kg/h y en los no alcohólicos de 90.5+/-18,9 mg/Kg/h (P<0.01).

En los pacientes alcohólicos la VME descendió de manera progresiva a medida que aumentó la severidad de la lesión histológica, siendo superior en los alcohólicos con hígado normal, esteatosis o fibrosis que en los pacientes con hepatitis alcohólica ó cirrosis (121.8+/-17.5 vs 104.9+/-12.4 mg/Kg/h, P<0.01).

El metabolismo hepático del etanol se relacionó con el estado funcional del hígado. Se observó una relación lineal significativa entre la VME y la prueba de la aminopirina (r:0.70, p<0.001), así como entre la VME y el aclaramiento hepático intrínseco del verde de indocianina (r:0.76, p<0.01).

A pesar de que las actividades ADH y ALDH fueron más bajas en los pacientes con una menor capacidad oxidativa del etanol, no se observó ninguna relación entre la VME. y la actividad ADH (r:0.10, p:NS), ALDH de alta Km (r:0.21, p:NS) ni ALDH de baja Km (r:0.32, p:NS).

D) FACTORES DETERMINANTES DE LOS NIVELES PLASMÁTlCOS DE ACETALDEHIDO

Tras la infusión del etanol, en 14 de los 25 pacientes alcohó1icos se detectaron niveles de acetaldehido superiores a 0.5 milimicras (límite de sensibilidad), oscilando entre 0.7y 4.7 milimicras. En los 6 pacientes no alcohólicos los niveles de acetaldehido fueron siempre indetectables.

Al comparar los pacientes alcohólicos con y sin acetaldehido detectable en plasma tras la infusión del etanol, no se observaron diferencias entre los dos grupos en la edad, duración e intensidad del consumo de etanol, parámetros de función hepática, ni actividades ADH ni ALDH hepáticas. La única diferencia entre estos dos grupos de pacientes alcohólicos fue su capacidad para oxidar el etanol de manera que la VME resultó de 120+/-17.4 mg/Kg/h en los pacientes en los que se detectó acetaldehido y de 104+/-11.7 mg/Kg/h en los que este resultó indetectable. Además existió una correlación lineal directa entre la VME y el pico máximo de acetaldehido (r:0.48, p<0.02). En cambio, los niveles de acetaldehido tras la administración del etanol no se correlacionaron con la actividad ADH (r:0.006, p:NS), ALDH de alta Km (r:0.12, p:NS) ni ALDH de baja Km (r:0.28, p:NS).

E) ACTIVIDADES ADH Y ALDH EN EL TEJIDO HEPÁTICO

La actividad ADH hepática resultó significativamente diferente entre los grupos histológicos estudiados (P=4.16, p:0.0007). Al analizar únicamente los distintos subgrupos de pacientes alcohólicos la actividad ADH resultó ser también significativamente diferente entre ellos (F: 4.80, p:0.001) La actividad de la ADH disminuyó de forma progresiva a medida que aumentó la severidad de la lesión hepática. Así los pacientes alcohólicos con hígado normal presentaron una actividad ADH significativamente más elevada que los pacientes con hepatitis alcohólica (p<0.05), cirrosis alcohólica (p<0.01) o bien cirrosis asociada a hepatitis alcohólica (p<0.01). En cambio, en los individuos no alcohólicos la actividad ADH no guardó relación con el grado de lesión histológica. Los pacientes con hepatitis crónica presentaron una actividad muy similar a la de los pacientes con cirrosis de origen no alcohólico. La actividad ADH hepática se correlacionó con los parámetros de función hepática (bilirrubina, protrombina) únicamente en los pacientes alcohólicos.

La actividad ALDH de alta Km fue similar entre los distintos grupos histológicos (F=1.89, p:0.08). Al comparar las medias de los distintos subgrupos de pacientes alcohólicos y no alcohólicos no se observaron diferencias significativas. La actividad ALDH de alta Km no se correlacionó con los parámetros de función hepática.

Se observaron marcadas diferencias en la actividad ALDH de baja Km entre los distintos grupos histológicos (F=5.27, p<0.0001). La actividad de este enzima disminuyó de forma progresiva al aumentar la gravedad de las lesiones hepáticas tanto en los pacientes alcohólicos como en los no alcohólicos.

En los pacientes alcohólicos se observaron también diferencias significativas en la actividad ALDH de baja Km entre los distintos subgrupos (F=3.94, p<0.004). Se observaron además diferencias significativas en la actividad ALDH de baja Km entre los subgrupos de pacientes con hígado normal y los que presentaban cirrosis (p<0.05) o cirrosis asociada a hepatitis alcohólica (p<0.01), y entre los pacientes con esteatosis y los pacientes con cirrosis asociada a hepatitis alcohólica (p<0.05). Entre los individuos no alcohólicos, los pacientes con cirrosis tenían una actividad significativamente inferior a la de los enfermos con hepatitis crónica (p<0.02).

La actividad ALDH de baja Km se correlacionó con las pruebas de función hepática tanto en los pacientes alcohólicos como en los no alcohólicos.

La relación observada entre la disminución de las actividades ADH y ALDH y el grado de lesión hepática en los pacientes alcohólicos sugiere que los cambios en las actividades enzimáticas no constituyen anomalías primarias que predispongan al alcoholismo ó al desarrollo de lesiones hepáticas sino que son consecuencia de la lesión celular.

F) ENZIMAS DE LA ADH Y ALDH HEPÁTICAS

Los fenotipos del locus ADH2 pudieron determinarse en todos los pacientes. En seis casos de los 84 estudiados (7.1%) se observó el fenotipo "atípico" (banda más catódica que el isoenzima B1B1 y actividad a pH 8.5 > pH 10.5). La prevalencia de la ADH "atípica" fue similar en pacientes alcohólicos (4/60, 6.7%) y no alcohólicos (2/24, 8.31%).

No se halló el fenotipo "Indianápolis" en ninguna de las muestras estudiadas. El fenotipo del locus ADH3 pudo ser estudiado en 60 pacientes, 38 alcohólicos y 22 no alcohólicos. El fenotipo T1T1 se observó en 16 pacientes (27%), 39 casos (65%) presentaron el fenotipo T1T2 y 5 casos (81%) el fenotipo T2T2. La frecuencia fenotípica observada fue significativamente distinta a la calculada a partir de la frecuencia génica según la ley de la distribución de equilibrio de los genotipos. Entre los pacientes alcohólicos la frecuencia fenotípica resultó significativamente distinta a la calculada según esta ley. En cambio, entre los individuos no alcohólicos no se hallaron diferencias entre las frecuencias fenotípicas esperadas y las observadas.

Este desequilibrio puede venir dado por una mayor predisposición al desarrollo de lesiones hepáticas en los pacientes alcohólicos con fenotipo T1T2 ya que en los alcohólicos con hígado normal o esteatosis la frecuencia fenotípica del locus ADH3 siguió el principio de equilibrio de los genotipos, mientras que en los pacientes con lesiones hepáticas severas como fibrosis, hepatitis alcohólica ó cirrosis la frecuencia fenotípica fue significativamente distinta a la calculada según la ley de Hardy-Weinberg, fundamentalmente por un aumento de la frecuencia observada del fenotipo T1T2 respecto a la esperada.

El estudio de los isoenzimas de la ALDH reveló la banda correspondiente a la ALDH1, en todas las muestras estudiadas, tanto de los pacientes alcohólicos como de los no alcohólicos.

La banda del isoenzima ALDH2 fue indetectable en 17 alcohólicos (39.51%) y en dos pacientes no alcohólicos (9.5%), existiendo entre ambas proporciones una diferencia estadísticamente significativa (p<0.02). Entre los individuos alcohólicos, la proporción de pacientes con ausencia de la banda de ALDH2 fue mayor a medida que aumentó la severidad de la lesión hepática, lo que sugiere que la falta de detección de la ALDH2 es probablemente una consecuencia de la disminución de actividad de este isoenzima, que resulta más marcada en pacientes alcohólicos con lesiones hepáticas severas como hepatitis alcohólica ó cirrosis.
The influence of liver injury on the hepatic alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) activities (total, high-Km and low-Km ALDH) has been investigated in 60 alcoholics -5 normal liver (N), 9 steatosis (S), 9 fibrosis (F), 11 alcoholic hepatitis (AH), 19 cirrhosis (AC) and 7 cirrhosis with alcoholic hepatitis (ACAH)- and 24 non-alcoholics -14 chronic hepatitis (CH) and 10 cirrhosis (NAC)-. ADH and ALDH activities decreased proportionally with the progression of liver disease in alcoholics (ADH: N: 39.9 +/- 9,6; S: 32.6 +/- 6; F: 29.9 +/-4.9; AH: 20.8+/-3.8; AC:15.3+/-2.9; ACAH: 10+/- 2.8. Low-Km ALDH: N: 4.4 +/- 0.9; S: 4.1+/- 0.5; F: 2.3+/- 0.5; AH: 2.4+/- 0.5; AC:1.9 +/- 0.5; ACAH:1.2+/-0.5 mU/mg protein). By contrast, in non-alcoholics, there was a reduction of low-Km ALDH related to the severity of liver injury (CH: 5.3 +/- 0.8: NAC: 2.4 +/- 0.7 mU/mg protein), but not of ADH (CH:36.1+/-14.2; NAC: 36.0 +/- 8.7 mU/mg protein), Atypical ADH was present in 6.6% alcoholics and in 8.3% non-alcoholics. AH patients exhibited the isozyme ALDH II, but isozyme ALDH I was not detected in 39.5% alcoholic patients and in 9.5% of those with non-alcoholic liver disease. These results suggests that the decrease of ADH and ALDH activities in alcoholics are a consequence of liver damage. The diminution of ADH found particularly in alcoholics could be due to the leakage of the enzyme secondary to centrilobular cell necrosis.

In 31 of the patients, 25 alcoholics (4 N, 4 S, 4 F, 7 AH, 7 AC) and 6 non-alcoholics (NAC), the blood ethanol and acetaldehyde concentrations after an intravenous infusion of ethanol were analyzed. In the alcoholics the ethanol metabolic rate (EHR) was significantly higher than in non-alcoholics. A significant positive correlation was observed between EMR and liver function estimated by the aminopyrine breath test (r=0.70, P<0.001). In non-alcoholics acetaldehyde levels were below the detection limits (<0.5 milimicres), in contrast, an elevated blood acetaldehyde was found in 14/15 alcoholics. The clinical characteristics and hepatic ADH and ALDH activities were similar in both groups of alcoholics. The only difference between alcoholics with elevated blood acetaldehyde and those without was EMR (120+/-17.4 vs 104+/-11.7 mg/Kg/h). Furthermore, the peak blood acetaldehyde level correlated positively with EMR. These results suggest that the main reason for blood acetaldehyde elevation in chronic alcoholics is their higher capacity to metabolize ethanol.

Chez l'humain, la formation des hormones stéroïdiennes dans les tissus périphériques via le précurseur déhydroépiandrostérone (DHEA) circulant joue un rôle majeur dans le maintien du fonctionnement adéquat des tissus androgéno- et estrogéno-sensibles. Comme les primates ont le pouvoir de sécréter une grande quantité de DHEA via les glandes surrénales, le singe représente ainsi un meilleur modèle animal pour étudier la stéroïdogénèse que le rat ou la souris. Chez ces derniers, l'enzyme 17α-hydroxylase, 17,20-lyase (CYP17) est absente dans les glandes surrénales, lesquelles sont donc incapables de produire et de sécréter la DHEA et la 4-androstène-3,17-dione (4-dione) dans la circulation, comme le font humain et les primates. Dans le présent projet, nous étudions deux enzymes chez le singe cynomolgus (Macaca fascicularis) qui possèdent le potentiel de métaboliser les androgènes, mais les preuves de leur implication réelle sont encore manquantes. La première est la rétinol déshydrogénase de type 12 (RDH12) qui transforme le rétinal en rétinol, les résultats préliminaires obtenus dans notre laboratoire suggèrent qu'elle possède la capacité de convertir la dihydrotestostérone (DHT) en 5α-androstane-3p, 17β-diol (3β-diol). La deuxième enzyme est la 17β-hydroxysteroide dehydrogenase (17β-HSD) type 11, qui possède la capacité de catalyser la transformation du 5α-androstane-3α, 17β-diol (3α-diol) en androstérone (ADT). Pour déterminer les activités catalysées par la RDH12, nous avons préparé des transfectants stables qui expriment l'enzyme en utilisant les cellules transformées de rein embryonnaire d'humain (HEK-293). Nous avons trouvé que Macaca fascicularis RDH12 (mfRDH12) catalyse efficacement et sélectivement la transformation de 5α-androstane-3,17-dione (5α-dione) en epiandrostérone (Epi-ADT) et DHT en 3β-diol, respectivement. L'enzyme est exprimée spécifiquement dans la peau, la glande mammaire et le cerveau. Le niveau d'expression le plus élevé est dans la peau où elle est exprimée spécifiquement dans la glande sébacée. En utilisant l'androgène synthétique R1881 pour déterminer s'il est capable de stimuler l'expression de la RDH12 comme celle de la rétinol déshydrogénase de type 11 (RDH11), une enzyme paralogue de la RDH12, nous avons trouvé que ce produit ne stimule pas l'expression de la RDH12, par contre, il est un inhibiteur puissant de l'activité 3β-hydroxystéroïde déshydrogénase. Pour déterminer le rôle potentiel de la 17β-HSD type 11 dans le métabolisme des androgènes, nous avons quantifié le niveau d'expression de l'enzyme, déterminé la localisation de l'enzyme dans plusieurs tissus en utilisant le PCR en temps réel et l'hybridation in situ, et examiné s'il y a association avec les tissus androgéno-sensibles.

Non-alcoholic fatty liver disease (NAFLD) is characterised by the accumulation of fat in the liver and is associated with liver-related morbidity and mortality. Nevertheless, the leading cause of death in these patients is cardiovascular disease (CVD). Excess abdominal visceral adipose tissue (VAT) is frequently expressed in NAFLD and is considered a pivotal feature in the pathogenesis ofNAFLD which is predictive of CVD. Endothelial dysfunction is an early manifestation in the development of atherosclerosis and is characterised bya diminished bioavailabilty of the anti-atherogenic molecule NO, which is secreted by the endothelium of all blood vessels throughout the vascular tree. Limited pharmacological treatment is available to reduce hepatic fat, therefore, lifestyle modification interventions comprised of structured exercise and diet are recommended as a non-pharmacological management strategy to reduce hepatic fat in NAFLD. The primary aim of this thesis was to explore nitric oxide (NO)-mediated endothelial function at different levels of the vascular tree in NAFLD patients and to establish whether supervised exercise training has a sustained therapeutic impact on endothelial function. Thirty-two NAFLD patients (21 males, 11 females, 48±2yrs, BMI 31±lkg/m2) and eighteen matched controls (8 males, 10 females, 48±2yrs, BMI 30±lkg/m2) underwent magnetic resonance imaging (MRI) to quantify abdominal VAT and proton magnetic resonance spectroscopy (lH-MRS) to determine intrahepatocellular triglyceride content (IHTC). Brachial artery flow mediated dilatation (FMD) (as an index of endothelial NO function) was also assessed. IHTC (27.2±3.0 vs. 2.9±0.4%) and abdominal VAT (S.4±0.3vs. 3.4±0.2 1) were elevated in NAFLD patients when compared with controls (PO.OS). Impairment in FMD remained in NAFLD patients following independent covariate adjustment for abdominal VAT (S.O±O.S vs. 7.3±0.7%, P=0.01). These fmdings indicate that excess IHTC and abdominal VAT do not explain endothelial dysfunction in NAFLD. Twenty NAFLD patients were randomly assigned to either 16-weeks of supervised moderate intensity (30-60% HRR, 30-4S min, 3-S times per week) exercise training (n=13, SO±3yrs, BMI 30±lkg/m2) or to 16-weeks of conventional care lifestyle advice (n=7, 47±6yrs, BMI 31±2kg/m2). Supervised exercise training induced a greater improvement in FMD when compared with conventional care (3.6±0.6 vs. 0.3±0.8%, P=0.004). There was no significant difference between the effect of exercise and conventional care on IHTC or abdominal VAT (P>O.OS). These data suggest that supervised exercise training is an effective management strategy in NAFLD capable of improving conduit artery endothelial function independent of IHTC and abdominal VAT. In order to explore the longevity of the exercise-induced improvements in conduit artery endothelial function, a 12-month follow up assessment was performed in 9 of the NAFLD patients (S males, 4 females, SO±Syrs, BMI 30±lkg/m2) who completed the 16- week supervised exercise training intervention. The exercise-induced improvement in FMD (S.1±0.8 vs. 7.9±0.8%; P=0.004) was abolished 12 months following the cessation of supervised exercise training (7.9±0.8 vs.S.O±O.S%; P=0.02), returning to a similar level observed at baseline (S.l±0.8 vs. S.O±O.S%; P=0.9S).These findings indicate that in order to chronically sustain exercise-induced improvements in endothelial function in NAFLD patients, long-term exercise supervision and guidance is required. Cutaneous NO-mediated microvessel function reflects generalised microvascular function and provides a translational model to investigate pre-clinical disease, but has not been previously investigated in NAFLD. NO-mediated vasodilatation in the cutaneous microvessels was examined in 13 NAFLD patients (7 males, 6 females, 50±3yrs, BM! 31±lkg/m2) and 7 matched controls (3 males, 4 females, 48±4yrs, BM! 30±2 kg/nr'). Microdialysis fibres were embedded into the skin of the forearm and laser Doppler probes placed over these sites. Both sites were then heated to 42°C, with saline solution infused in one probe and L-NG-monomethyl arginine (L-NMMA) through the second. Following baseline assessment, 11 NAFLD patients were randomly assigned to l o-weeks of supervised moderate intensity exercise training (n=6, 45±5yrs, BM! 31±1kg/m2) or to 16-weeks of conventional care (n=5, 51±3yrs, BM! 30±21kg/m2). The NO contribution to skin blood flow in response to incremental heating was not different between NAFLD patients and controls (P=0.47) at baseline. However, significant differences were evident in NO contribution between the exercise training and conventional care group (P=O.Ol), suggesting that supervised exercise training improves cutaneous NO-mediated microvascular endothelial function in NAFLD patients. This thesis suggests that supervised exercise training has a direct therapeutic impact on endothelial function in NAFLD which may decrease the risk of future heart disease and stroke. As a cardioprotective management strategy in NAFLD, exercise training is superior to that of current conventional care pathways, however, in order to chronically sustain the exercise-induced improvements in endothelial function, long term exercise supervision and guidance is required.

Non-alcoholic fatty liver disease (NAFLD) is an increasingly prevalent issue in the modern world, predisposing patients to serious pathology such as cirrhosis and hepatocellular carcinoma. Mitochondrial dysfunction, and in particular, diminished hepatic oxidative phosphorylation (OXPHOS) capacity, have been observed in NAFLD livers, which may participate in NAFLD pathogenesis. To examine the role of OXPHOS in NAFLD, we generated a model of enhanced hepatic OXPHOS using mice with liver-specific transgenic expression of LRPPRC, a protein which activates mitochondrial transcription and augments OXPHOS capacity. When challenged with high-fat feeding, mice with enhanced hepatic OXPHOS were protected from the development of liver steatosis and inflammation, critical components in the pathogenesis of NAFLD. This protection corresponded to increased liver and whole-body insulin sensitivity. Moreover, mice with enhanced hepatic OXPHOS have increased availability of oxidized NAD+, which promotes complete fatty acid oxidation in hepatocytes. Interestingly, mice with enhanced hepatic OXPHOS were also protected from obesogenic effects of long-term high-fat feeding. Consistent with this, enhanced hepatic OXPHOS increased energy expenditure and adipose tissue oxidative gene expression, suggesting a communication between the liver and adipose tissue to promote thermogenesis. Examination of pro-thermogenic molecules revealed altered bile acid composition in livers and serum of LRPPRC transgenic mice. These mice had increased expression of bile acid synthetic enzymes, genes which are induced by NAD+ dependent deacetylase SIRT1 activation of the transcriptional co-regulator PGC-1a. These findings suggest that enhanced hepatic OXPHOS transcriptionally regulates bile acid synthesis and dictates whole-body energy expenditure, culminating in protection from obesity.

Non-alcoholic fatty liver disease (NAFLD) is an increasingly prevalent issue in the modern world, predisposing patients to serious pathology such as cirrhosis and hepatocellular carcinoma. Mitochondrial dysfunction, and in particular, diminished hepatic oxidative phosphorylation (OXPHOS) capacity, have been observed in NAFLD livers, which may participate in NAFLD pathogenesis. To examine the role of OXPHOS in NAFLD, we generated a model of enhanced hepatic OXPHOS using mice with liver-specific transgenic expression of LRPPRC, a protein which activates mitochondrial transcription and augments OXPHOS capacity. When challenged with high-fat feeding, mice with enhanced hepatic OXPHOS were protected from the development of liver steatosis and inflammation, critical components in the pathogenesis of NAFLD. This protection corresponded to increased liver and whole-body insulin sensitivity. Moreover, mice with enhanced hepatic OXPHOS have increased availability of oxidized NAD+, which promotes complete fatty acid oxidation in hepatocytes. Interestingly, mice with enhanced hepatic OXPHOS were also protected from obesogenic effects of long-term high-fat feeding. Consistent with this, enhanced hepatic OXPHOS increased energy expenditure and adipose tissue oxidative gene expression, suggesting a communication between the liver and adipose tissue to promote thermogenesis. Examination of pro-thermogenic molecules revealed altered bile acid composition in livers and serum of LRPPRC transgenic mice. These mice had increased expression of bile acid synthetic enzymes, genes which are induced by NAD+ dependent deacetylase SIRT1 activation of the transcriptional co-regulator PGC-1a. These findings suggest that enhanced hepatic OXPHOS transcriptionally regulates bile acid synthesis and dictates whole-body energy expenditure, culminating in protection from obesity.

Alcohol liver disease (ALD) is a major health concern throughout the world. Currently, in the United States, 17 million people suffer from alcoholism, of which 1.4 million people are receiving treatment [1, 2]. The link between ethanol metabolism, reactive oxygen species (ROS) and liver injury in ALD has been well characterized over the last couple decades [3-10]. Ethanol metabolism relies on the availability of the cofactor NAD+ for the oxidation of ethanol into acetate, consequently causing alterations in redox potential. Redox dysfunction within the mitochondria can affect multiple pathways important in maintaining cellular homeostasis. Chapter 1 provides an introduction to the role of ethanol metabolism in oxidative stress and alcohol liver injury (ALI). During ethanol metabolism, both the cytochrome bc1 and NADH dehydrogenase complexes within the mitochondria have been demonstrated to be major contributors to ROS formation and “leak” free radicals [11-13]. As a result, the free radicals superoxide (O2-) and hydrogen peroxide (H2O2) is diffused into the cytoplasm where they can react with other molecules, proteins and DNA and cause tissue injury [4, 14]. Chapter 1 aims to introduce the link between ethanol metabolism and histone post-translational modifications (PTM) such as tyrosine nitration and lysine acetylation using proteomics techniques. Chapter 2 uses a global proteomic study to identify links between gender and ALI. A 10-day chronic-binge mouse model was employed in order to identify gender-specific proteins that may influence the development of ALD. It has previously been established that females are more susceptible to developing ALD, however, the cause is still unknown. This study identifies gender differences in the family of cytochrome P450 proteins using a mouse model for chronic-binge alcohol exposure. The cytochrome P450 family of proteins are important in the metabolism of toxic compounds, such as acetaldehyde, a byproduct of ethanol metabolism. Interestingly, I also identified that female mice expressed naturally higher levels of histone acetylation prior to alcohol exposure when compared to males. Following alcohol exposure, the female mice did not show much change in acetylation, whereas male acetylation levels were raised to similar levels of the female mice. These acetylation changes raised the question, how does alcohol influence epigenetic marks on histone proteins? Recently, new evidence has emerged that supports the role of epigenetics in the pathophysiology of ALD [4, 14-27]. Ethanol metabolism will promote shifts in redox potential and mitochondrial dysfunction, the result is the formation of reactive oxygen and/or nitrogen species (ROS/RNS) [4, 5, 7, 10, 14, 28]. As ethanol is metabolized, the accumulation of ROS/RNS species such as NO- and O2- can induce the post-translational modification nitrotyrosine. Shifts in redox potential will cause the electron transport chain to “leak” the free radical O2-. Another free radical known as nitric oxide (NO-) has been shown to be elevated during times of ethanol consumption [29, 30]. Traditionally, NO has a protective role within the cell at low concentrations, however, in surplus can lead to tissue damage. Ethanol-induced increases in NO- and O2- can instigate to peroxynitrite (ONOO-) formation; a potent oxidant and nitrating agent of tyrosine residues [29, 31-34]. Chapter 3 examines the indirect effect of alcohol metabolism and ROS/RNS formation on histone tyrosine nitration. This project used mass-spectrometry to identify novel targets of histone tyrosine nitration using a mouse-model of chronic-binge alcohol exposure. Interestingly, histone H3 was found to be nitrated on the hinge-region of the N-terminal tail at tyrosine 41. Molecular dynamics of the nitrated and unmodified proteoforms revealed that the DNA prefers a change in conformation upon H3Y41 nitration. Further studies using an antibody synthesized against the nitrated H3y41 region of the protein revealed potential targets within the genome important in fatty acid synthesis and metabolism. Chapter 4 looks at the direct influence of alcohol metabolism and its contribution to histone acetylation via acetate production and acetyl-CoA. Alcohol metabolism has traditionally been thought influence acetylation through the sirtuin family of deacetylase proteins. Sirtuin deacetylases are NAD+-dependent and have been shown to be a regulate protein acetylation within the mitochondria, cytoplasm, and nucleus during times of ethanol exposure [35-37]. Shifts in redox potential attributed to ethanol metabolism can inhibit sirtuin deacetylase activity by out-competing the enzymes for available NAD+, ultimately leading to mitochondrial and nuclear hyperacetylation [17, 28, 38-42]. Currently, there is evidence that ethanol increases acetylation of histone 3 lysine 9, which then targets activation of the alcohol dehydrogenase gene (ADH) [17, 18, 43]. Moreover, Shukla et.al. (2008) support the idea that ethanol can alter epigenetic transcriptional activation based on which modification is selected for a site during times of stress when it can be occupied by more than one modification [22]. Chapter 4 demonstrates the use of mass-spectrometry to metabolically trace 13C2-labeled ethanol in vivo. These new data show clear evidence of 13C2 heavy-labeled ethanol being incorporated into known sites of acetylation on the N-terminal tails of histone H3 and H4. Incorporation of heavy-label was calculated using extracted ion chromatograms (XIC) for the double and singly acetylated and unmodified peptides belonging to H3K9-R17 and H3K18-R23. Total change in acetylation was also assessed for each peptide using the ratio of ratios of total acetylation to unmodified peptide over the fold change in ethanol- to control-fed groups. An interesting observation was observed in that the incorporation of heavy-label suggests site-selectivity of lysine residues over time. Histone 4 contains multiple sites of acetylation on the peptide H4K5-R17, making it hard to quantify manually. MaxQuant evidence files in conjunction with R were used to calculate the 13C2 incorporation on the multiple H4 acetyl-sites over 24-hours. Ethanol-heavy label incorporation at multiple acetyl-sites occurred as a mixture suggesting a role in transcriptional regulation. These new data establish a link between alcohol metabolism and known epigenetic marks on histone proteins. These studies have now established that alcohol metabolism is indirectly linked to histone tyrosine nitration through increased ROS/RNS and directly through acetate production. Understanding how these epigenetic marks fluctuate as ALD progresses will provide potential targets for the development of new drug therapies. The epigenetic marks identified in these studies have previously been established to be important activators in transcription. These data provide novel techniques using proteomics-based metabolic tracing in vivo. Future studies will assess how these marks change after chronic ethanol exposure and whether the changes in epigenetics are heritable. Understanding hereditary of alcoholism will provide insight to those predisposed to the disease.

Abstract Obesity, insulin resistance, type 2 diabetes (T2D) and non-alcoholic fatty liver disease (NAFLD) form a dangerous quartet which threatens human health all over the world. About 25% of adults around the world have NAFLD, which poses risks for cardiovascular and metabolic well-being and may develop into liver cirrhosis and hepatocellular carcinoma. Apart from lifestyle modification, treatment options for NAFLD are scarce. This thesis presents atrial fibrillation (AF) as a new complication of NAFLD among general population of 958 individuals aged 40-60 years participating in the OPERA study. Even after multiple-adjustments for confounding factors, ultrasound-based NAFLD predicted the development of AF during about 16 years of follow-up. Moreover, the association between AF and liver fibrosis in 76 individuals aged 64-82 years in a cross-sectional setting is presented. The thesis also shows that individuals with metabolic syndrome (MetS), with or without NAFLD, are at increased risk of cardiovascular events, T2D and the increase of left ventricular mass index in a study population of 958 individuals aged 40-60 years during a 20-year follow-up. In other words, NAFLD without MetS does not seem to expose to these three cardiometabolic complications. The thesis also shows that rifampicin-activated pregnane X receptor (PXR), a member of the nuclear receptor superfamily of ligand-activated transcription factors with several endobiotic and xenobiotic activators, increases serum levels of cholesterol, phospholipids and certain fatty acids, assessed by nuclear magnetic resonance metabolomics technique, in a randomized, open, placebo-controlled trial among 34 young and healthy individuals. These serum lipids are considered toxic lipids and capable of transforming hepatosteatosis into steatohepatitis and even more severe forms of NAFLD. Moreover, rifampicin-activated PXR has no effect on serum triglycerides, that are non-toxic lipids, or triglyceride accumulation in the liver, assessed by magnetic resonance imaging, in 15 young and healthy individuals. In conclusion, this thesis advances the knowledge in the pathogenesis, lipid metabolism, complications and heterogeneous nature of NAFLD. These may have implications for patient care and follow-up
Tiivistelmä Maailmanlaajuisesti noin 25% täysi-ikäisistä henkilöistä sairastaa alkoholinkäyttöön liittymätöntä rasvamaksaa. Sen tiedetään altistavan sydän- ja verisuonisairauksille, aineenvaihduntahäiriöille, maksakirroosille ja jopa maksasyövälle, mutta elämäntapahoitoa lukuun ottamatta hoitomahdollisuudet ovat toistaiseksi vähäisiä. Tässä väitöskirjassa osoitetaan ensimmäistä kertaa alkoholinkäyttöön liittymättömän rasvamaksan ennustavan itsenäisesti eteisvärinän ilmaantuvuutta noin 16 vuoden seurannan aikana 958 tavallisen keski-ikäisen ihmisen aineistossa osana OPERA-tutkimusta. Lisäksi väitöskirjassa osoitetaan maksan sidekudosmuodostuksen ja eteisvärinän välillä olevan yhteys poikkileikkausasetelmassa 76 iäkkään ihmisen muodostamassa aineistossa. Väitöstutkimuksessa havaittiin myös, että metabolista oireyhtymää sairastavilla henkilöillä on suurentunut tyypin 2 diabeteksen, sydän- ja verisuonisairauksien sekä vasemman kammion koon suurentumisen riski noin 20 vuoden seurannan aikana 958 tutkittavan henkilön aineistossa riippumatta siitä, onko heillä alkoholinkäyttöön liittymätön rasvamaksa. Toisin sanoen alkoholin käyttöön liittymätön rasvamaksa ilman metabolista oireyhtymää ei lisää edellä mainittujen kolmen sairauden riskiä. Väitöstutkimuksessa esitetään lisäksi, että rifampisiinilla aikaansaatu maksan pregnane X -reseptorin aktivaatio johtaa seerumin fosfolipidien, tiettyjen rasvahappojen sekä usean eri kolesterolityypin lisääntymiseen 34 terveen nuoren henkilön aineistossa. Kirjallisuudessa näiden seerumin rasva-aineiden on esitetty aiheuttavan alkoholin käyttöön liittymätöntä maksatulehdusta ja jopa rasvamaksan vakavimpia muotoja. Toisaalta rifampisiini ei lisännyt seerumin triglyseridipitoisuutta eikä aiheuttanut magneettitutkimuksella mitattuna triglyseridien kertymistä maksaan 15 terveen nuoren henkilön aineistossa. Tämä väitöstutkimus antaa lisätietoa rasvamaksan kehittymisestä, rasva-aineenvaihdunnasta ja komplikaatioista sekä korostaa rasvamaksan monimuotoista luonnetta. Nämä löydökset saattavat parantaa rasvamaksaa sairastavien henkilöiden hoitoa ja seurantaa

Alkylierte polyzyklische aromatische Kohlenwasserstoffe werden in vielen Matrizes wie Fahrzeugabgasen und Tabakrauch und auch als Kontaminanten in Nahrungsmitteln neben rein aromatischen Kongeneren gefunden. Alkylierte PAK können über die Alkylseitenkette über benzylische Hydroxylierung und nachfolgende Sulfonierung katalysiert über Sulfotransferasen (SULT) zu reaktiven Schwefelsäureestern umgesetzt werden. Die SULT-vermittelte Bioaktivierung zu einem genotoxischen Schwefelsäureester wurde für den benzylischen Alkohol 1-Hydroxymethylpyren des Hepatokanzerogens 1-Methylpyren in früheren Arbeiten gezeigt. In der vorliegenden Arbeit wurde überprüft, ob die benzylischen Alkohole weiterer alkylierter PAK über Sulfonierung zu genotoxischen Schwefelsäureestern umgesetzt werden. Hierzu wurde eine Gruppe von 17 Modellsubstanzen ausgewählt, um die Ableitung von Struktur-Aktivitäts-Beziehungen zu ermöglichen. Das genotoxische Potenzial authentischer benzylischer Schwefelsäureester der Modellsubstanzen wurde zunächst in vitro über DNA-Adduktbildung im zellfreien System und Mutagenität im Salmonella-Rückmutationstest untersucht. Die Sulfate zeigten große Reaktivitätsunterschiede in Abhängigkeit von der Struktur des aromatischen Systems und der Position der Alkylseitenkette, wobei die Endpunkte DNA-Adduktbildung und Mutagenität gut korrelierten. Des Weiteren wurde der Salmonella-Mutagenitätstest mit den benzylischen Alkoholen der untersuchten alkylierten PAK und gentechnisch veränderten S. typhimurium-Stämmen, die SULT-Formen des Menschen heterolog exprimieren, durchgeführt. Bis auf die Alkohole 2- und 4-HMP zeigten alle untersuchten benzylischen Alkohole deutliche mutagene Effekte in einem oder mehreren humane SULT exprimierenden Stämmen. Die durchgeführten in vitro-Versuche zeigten das Potenzial der benzylischen Metabolite alkylierter PAK für genotoxische Wirkungen. Nachfolgend musste geklärt werden, welche Relevanz die beobachteten Effekte für die komplexere in vivo-Situation haben. Nach Verabreichung verschiedener benzylischer Schwefelsäureester und Alkohole an männliche Ratten konnten DNA-Addukte in den untersuchten Organen detektiert werden, was im Fall der Schwefelsäureester deren systemische Bioverfügbarkeit und im Fall der benzylischen Alkohole deren Umsatz durch SULT der männlichen Ratte zeigte. Da im Gegensatz zum Menschen die SULT-Expression in der Ratte auf die Leber fokussiert ist, musste ein Großteil des Umsatzes zu genotoxischen Sulfaten in der Leber stattgefunden haben. DNA-Addukte wurden jedoch auch in extrahepatischen Organen gefunden, was über einen hepatischen Export der gebildeten reaktiven Sulfate und deren Transport über den Blutkreislauf zu diesen Geweben erklärt werden kann. Für die weiterführenden in vivo-Studien wurden die benzylischen Alkohole 1-HMP und 1-HM-8-MP ausgewählt, die trotz großer struktureller Ähnlichkeit toxikodynamische Unterschiede zeigten. Zur Untersuchung der Bedeutung des SULT-vermittelten Toxifizierungsweges als auch konkurrierender detoxifizierender oxidativer Stoffwechselprozesse, wurden für 1-HMP und 1-HM-8-MP in vivo-Inhibitionsstudien mit SULT-Inhibitoren und für 1-HM-8-MP auch mit ADH/ALDH-Inhibitoren durchgeführt. Eine Vorbehandlung mit dem SULT-Hemmstoff Pentachlorphenol führte zu einer Reduktion der DNA-Adduktniveaus in Organen 1-HMP- und 1-HM-8-MP-behandelter Tiere. Die Verabreichung von Quercetin hatte keine Auswirkung auf die DNA-Adduktniveaus. Die Hemmung der DNA-Adduktbildung bei Verabreichung von Pentachlorphenol verdeutlichte jedoch, dass benzylische Alkohole alkylierter PAK in vivo über Sulfonierung bioaktiviert werden. Eine Vorbehandlung mit dem ADH-Inhibitor 4-Methylpyrazol und dem ADH-Substrat Ethanol führte zu erhöhten DNA-Adduktniveaus in Organen 1-HM-8-MP-behandelter Tiere. Den gleichen Effekt, jedoch in geringerem Ausmaß, hatte auch die Vorbehandlung mit dem ALDH-Inhibitor Disulfiram. Dies deutet darauf hin, dass oxidative Modifikationen an der Seitenkette des 1-HM-8-MP einen Detoxifizierungsmechanismus darstellen. Nach Verabreichung benzylischer Metabolite alkylierter PAK wurden oftmals hohe Adduktniveaus in der Niere detektiert. Als mögliche Ursache hierfür wurde eine Transporter-vermittelte renale Sekretion reaktiver Sulfate postuliert, die über Vorbehandlung mit Probenecid vor Verabreichung von 1-HMP und 1-HM-8-MP überprüft wurde. Der Haupteffekt der Probenecid-Behandlung wurde jedoch nicht in der Niere, sondern in der Leber beobachtet, die stark erhöhte Adduktniveaus zeigte. Eine mögliche Erklärung hierfür ist die Hemmung des Exportes in der Leber gebildeter reaktiver Sulfate über Inhibition hepatischer organischer Anionentransporter.
Alkylated polycyclic aromatic hydrocarbons are found besides purely aromatic congeners in numerous matrices like car engine exhausts and tobacco smoke and as contaminants in foods. Alkylated PAH can be converted at the alkyl side chain to reactive sulfuric acid esters via benzylic hydroxylation and subsequent sulfonation catalysed by sulfotransferases (SULT). The SULT-mediated bioactivation to a genotoxic sulfuric acid ester was shown for the benzylic alcohol 1-hydroxymethylpyrene of the hepatocarcinogen 1-methylpyrene in previous studies. In the thesis at hand it was studied if the benzylic alcohols of further alkylated PAH are converted to genotoxic sulfuric acid esters via sulfonation. For this purpose a group of 17 model substances was chosen to allow for deduction of structure activity relationships. The genotoxic potential of authentic benzylic sulfuric acid esters of the model substances was initially investigated in vitro via DNA adduct formation in a cell free system and mutagenicity in the Salmonella reverse mutation test. The sulfates showed large differences in reactivity depending on the structure of the aromatic system and the position of the alkyl side chain whereupon the endpoints DNA adduct formation and mutagenicity correlated well. Furthermore, the Salmonella mutagenicity test was carried out with the benzylic alcohols of the alkylated PAH studied and S. typhimurium strains genetically engineered for the heterologous expression of human SULT forms. Except for the alcohols 2- and 4-HMP all benzylic alcohols studied showed clear mutagenic effects in one or more SULT-expressing strains. The studies performed in vitro demonstrated the potential of benzylic metabolites of alkylated PAH for genotoxic effects. Consecutively, the relevance of the observed effects for the more complex in vivo situation had to be clarified. After administration of different benzylic sulfuric acid esters and alcohols to male rats DNA adducts were detected in the organs studied, in case of the sulfuric acid esters showing their systemic bioavailability and in case of the benzylic alcohols demonstrating their conversion to the corresponding reactive benzylic sulfuric acid esters by SULT of the male rat. Since in contrast to man SULT expression in the rat is focused on the liver, a large part of the conversion to genotoxic sulfates must have been taken place in the liver. However, DNA adducts were also found in extrahepatic tissues which can be attributed to a hepatic export of the reactive sulfates formed and their transport to these tissues via circulation. For the continuative in vivo studies the benzylic alcohols 1-HMP and 1-HM-8-MP were chosen that demonstrated toxicodynamic differences in spite of their great structural resemblance. To investigate the importance of the SULT-mediated toxification pathway as well as competing detoxifying oxidative metabolic pathways, in vivo inhibition studies with SULT inhibitors were performed for 1-HMP and 1-HM-8-MP and with ADH/ALDH inhibitors also for 1-HM-8-MP. A pretreatment with the SULT inhibitor pentachlorophenol led to a reduction of DNA adduct levels in organs of animals treated with 1-HMP and 1-HM-8-MP. Administration of quercetin had no impact on the DNA adduct levels. However, inhibition of DNA adduct formation at administration of pentachlorophenol demonstrated that benzylic alcohols of alkylated PAH are bioactivated via sulfonation in vivo. A pretreatment with the ADH inhibitor 4-methylpyrazole and the ADH substrate ethanol led to increased DNA adduct levels in organs of animals treated with 1-HM-8-MP. The same effect but to a lesser extent was caused by a pretreatment with the ALDH inhibitor disulfiram. This indicates that oxidative modifications at the side chain of 1-HM-8-MP represent a detoxification mechanism. After administration of benzylic metabolites of alkylated PAH often high DNA adduct levels were detected in kidney. A transporter-mediated renal secretion was postulated as possible cause which was investigated using a pretreatment with probenecid before administration of 1-HMP and 1-HM-8-MP. However, the main effect of the treatment with probenecid was not observed in kidney but in liver that showed strongly increased adduct levels. A possible explanation for this effect is the inhibition of the export of reactive sulfates formed in liver via inhibition of hepatic organic anion transporters.

Chronic ethanol exposure is known to disrupt the methionine cycle in liver of adult rats. Alterations in the methionine cycle are associated with changes in tissue methylation capacity and may affect DNA and histone methylation thereby altering epigenetic regulation of gene expression, causing long-term phenotypic changes. Data from our lab and others have shown that prenatal alcohol exposure (PAE) reprograms the hypothalamic-pituitary-adrenal (HPA) axis such that HPA tone is increased throughout life. That is, PAE animals show increased HPA responsiveness and/or delayed or deficient recovery to basal levels following exposure to stressors. These alterations persist into adulthood, can have negative consequences for health, and may be mediated by metabolic changes induced by prenatal ethanol exposure. To explore this hypothesis, mass-spectrometry and real time PCR were used to quantify several of the primary metabolites and enzymes involved in methionine metabolism using plasma and liver samples from pregnant dams and fetuses from ethanol (E), pair-fed (PF) and ad lib fed control (C) groups on gestation day 21. Mass spectrometry analysis of plasma revealed increases (p<0.05) in homocysteine levels in E dams and increases (p<0.02) in methionine levels in both E dams and fetuses compared to their PF and C counterparts. Quantification of hepatic enzymes by real-time PCR showed significantly decreased mRNA levels (p≤0.05) of maternal methionine adenosyltransferase (MAT) 1A, MAT2A, methionine synthase (MS), and phosphatidylethanolamine N-methyl transferase (PEMT)in addition to decreased fetal MAT1A mRNA. Together these data demonstrate marked alterations in methionine metabolism during prenatal exposure to ethanol.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Chemical Engineering, 2014.
Cataloged from PDF version of thesis.
Includes bibliographical references (pages 130-141).
Historically, microbial platforms have been used to synthesize a variety of chemical products and potential biofuels. More recently, increasingly complex metabolic pathways have been engineered by using novel hosts, modifying natural pathways, and establishing de novo pathways with enzymes taken from a variety of pathway contexts. Highly reduced and branched alkyl chains are potentially interesting targets for both flavor and fragrance compounds and as liquid fuel components. Here we report the engineering of microbial fatty acid synthesis to provide both CoA-dependent and fatty acid synthase platforms for previously undescribed routes to medium-chain length, branched acids. Specifically we produced six-carbon 4-methyl-valeric acid via a CoA-dependent route and nine-carbon 7-methyloctanoic acid via a fatty acid synthase. Specific variants of the platform pathways were used to demonstrate synthesis of potential liquid fuel targets. The CoA-dependent platform was used to create a redox-neutral pathway to 4-methyl-pentanol with a maximum theoretical energy efficiency of 100%. Both platforms were used to demonstrate the first reported synthesis of short- and medium-chain alkanes from three to seven carbons.
by Micah J. Sheppard.
Ph. D.

Tesis para optar al grado académico de Magíster en Bioquímica, área de especialización Toxicología y Diagnóstico Molecular y Memoria para optar al Título de Bioquímico
Las propiedades reforzantes del etanol en el sistema nervioso central (SNC), que se postula son responsables de la auto-administración repetida de alcohol, se deben al aumento de la liberación de dopamina, inducido por el etanol, en el núcleo accumbens, área del sistema mesolímbico inervada por neuronas del área tegmental ventral. Esta acción parece estar mediada por su metabolito primario, el acetaldehído, producido en el cerebro por la oxidación del etanol a través de dos sistemas enzimáticos alternativos: la catalasa cerebral y el citocromo p450 2E1 (CYP2E1). Las ratas se autoadministran tanto etanol como acetaldehído directamente en el área tegmental ventral. La concentración necesaria para producir efectos reforzantes es menor para el acetaldehído que para el etanol, indicando que este metabolito del alcohol es un agente reforzante más potente que el etanol. El acetaldehído se puede condensar con la dopamina formando una mezcla de salsolinol e isosalsolinol. Las ratas se autoadministran salsolinol comercial (que contiene entre un 8 % y un 15 % de isosalsolinol) directamente en el área tegmental ventral. Las concentraciones autoadministradas son aún menores que las de acetaldehído, indicando una mayor potencia del salsolinol (y/o del isosalsolinol) como agente reforzante. Tanto el salsolinol como el isosalsolinol tienen un centro quiral y existen como los enantiómeros R y S. En el cerebro, el salsolinol sería producido por al menos tres vías: 1) condensación no enzimática del acetaldehído con dopamina, que produce la mezcla racémica de (R,S)-salsolinol y, posiblemente en menor proporción, una mezcla racémica de (R,S)- isosalsolinol; 2) condensación enzimática del acetaldehído con la dopamina, que produciría preferencialmente (R)-salsolinol y 3) condensación enzimática del ácido pirúvico con dopamina seguido de descarboxilación del producto, lo cual produciría preferencialmente (R)-salsolinol. No existe claridad acerca de cuál de los isómeros, salsolinol o isosalsolinol (o la mezcla de ambos), es responsable de las propiedades reforzantes observadas ni, específicamente, cuál de los enantiómeros - R o S - de estos compuestos es el que tiene una mayor actividad biológica. Para poder determinar cuál de los isómeros presentes en el salsolinol comercial -(R)- salsolinol, (S)-salsolinol, (R)-isosalsolinol o (S)-isosalsolinol- es el que tiene una mayor actividad biológica y para poder profundizar en los efectos que tiene el consumo de etanol sobre la producción de ellos in vivo, es necesario poder contar con metodologías que permitan la producción de estos isómeros en forma separada y a su vez que permitan cuantificarlos en muestras de origen biológico. En este sentido, los principales resultados obtenidos durante el desarrollo de esta tesis fueron los siguientes: (i) Se desarrolló una metodología de HPLC de apareamiento iónico en columna de gel de sílice-C18 acoplada a detector electroquímico capaz de separar y cuantificar dopamina, salsolinol e isosalsolinol en aproximadamente 30 min. En este sistema, la dopamina eluye primero (13,0 min. aprox.) seguida del salsolinol y el isosalsolinol (14,4 min. aprox y 26,1 min. aprox), permitiendo una buena separación y cuantificación de estos analitos en concentraciones desde 10-8 M. (ii) Se estableció que las sustancias separadas mediante HPLC de apareamiento iónico a partir del salsolinol comercial, SC1 y SC2, corresponden efectivamente a salsolinol (tiempo de retención 14,4 min. aprox.) e isosalsolinol (tiempo de retención 26,1 min. aprox) sobre la base de que: a) El tiempo de retención de SC1 (14,4 min. aprox.) es menor que el tiempo de retención de SC2 (26,1 min. aprox.) y, teóricamente, cabe esperar que el salsolinol eluya antes que el isosalsolinol por tener sus grupos hidroxilo más disponibles para interactuar con la fase móvil. b) La razón entre las áreas de los picos del salsolinol comercial SC1 (tiempo de retención 14,4 min. aprox.) y SC2 (tiempo de retención 26,1 min. aprox.) se corresponde con los porcentajes de salsolinol (92%) e isosalsolinol (8%) en el salsolinol comercial determinados por 1H-RMN. c) El peso molecular de las sustancias separadas a partir del salsolinol comercial, SC1 y SC2, se corresponde con el peso molecular del salsolinol e isosalsolinol (179 g/mol). (iii) Se determinó que la dopamina se degrada (posiblemente por autoxidación) en solución a pH 7,4 de acuerdo a una cinética de orden cero, mientras que el salsolinol y el isosalsolinol lo hacen de acuerdo a una cinética de orden uno, siendo la velocidad de oxidación del isosalsolinol 10 veces mayor que la del salsolinol. (iv) Se desarrolló una metodología de HPLC de fase inversa en columna de gel de sílice modificado con β-ciclodextrina capaz de separar y cuantificar dopamina, (R)- salsolinol y (S)-salsolinol en aproximadamente 40 minutos. En este sistema, la dopamina eluye primero (22,9 min. aprox) seguida de (S)-salsolinol (26,9 min. aprox.), (R)-salsolinol (29,9 min. aprox.) y el (R) y (S)-isosalsolinol (31,3 y 32,9 min. aprox), permitiendo una buena separación y cuantificación de dopamina, (S)- salsolinol y (R)-salsolinol en concentraciones desde 10-8 M. Si bien el isosalsolinol pudo ser separado de dopamina y de los enantiómeros R y S del salsolinol, no se logró separar completamente los enantiómeros R y S del isosalsolinol entre sí. A futuro, estas metodologías permitirán la detección y cuantificación de los enantiómeros en muestras de origen biológico y la administración selectiva de un enantiómero específico directamente en el cerebro de las ratas, ampliando así los conocimientos que se tienen sobre las propiedades reforzantes del etanol como una prodroga y cómo éstas pueden ser mediadas por el salsolinol
The reinforcing properties of ethanol in the central nervous system (CNS), believed to be responsible for the repeated self-administration of ethanol, are due to the increase of dopamine release -induced by ethanol- in the nucleus accumbens, a part of the mesolimbic system innerved by neurons from the ventral tegmental area. This action of ethanol seems to be mediated by its primary metabolite, acetaldehyde, which is produced in the brain from ethanol oxidation by two alternative enzymatic systems: brain catalase and cytochrome p450 2E1 (CYP2E1). Rats selfadminister both ethanol and acetaldehyde directly into the ventral tegmental area. The concentration needed to produce the reinforcing effects is lower for acetaldehyde than for ethanol, suggesting that this metabolite is a more powerful reinforcing agent than ethanol. Acetaldehyde can condense with dopamine to form salsolinol and isosalsolinol. Rats selfadminister commercially available salsolinol (which contains between 8% to 15% of isosalsolinol) directly in the ventral tegmental area. The concentrations of salsolinol selfadministered are even lower than acetaldehyde concentrations, suggesting that salsolinol (and/or isosalsolinol) is an even more powerful reinforcing agent. Both salsolinol and isosalsolinol have a chiral center and exist as the R and S enantiomers. In brain, salsolinol could be produced by at least three pathways. 1) non-enzymatic condensation of acetaldehyde with dopamine, which produces a racemic mixture of R and S salsolinol and possibly, to a lesser extent, a racemic mixture of R and S isosalsolinol; 2) enzymatic condensation of acetaldehyde with dopamine which would produce, preferentially, (R)-salsolinol and 3) enzymatic condensation of pyruvic acid with dopamine followed by decarboxylation of the condensation product wich would produce, preferentially, (R)-salsolinol. There is no clarity about which of the isomers, salsolinol or isosalsolinol (or both), is primarily responsible for the observed reinforcing properties, nor, specifically, about which of the enantiomers –R or S- of these compounds has a greater biological activity. In order to determine which of the isomers present in comercially available salsolinol –(R)- salsolinol, (S)-salsolinol, (R)-isosalsolinol and (S)-isosalsolinol- has stronger biological activity and in order to better understand the effects of ethanol consumption on the in vivo synthesis of these isomers, methodologies that allow us to produce these substances independently and methodologies that allow us to quantify them in biological samples are needed. In relation to this, the main results obtained in this thesis were the following: (i) An ion pairing HPLC methodology using a silicagel-C18 column coupled to an electrochemical detector, able to separate and quantify dopamine, salsolinol and isosalsolinol in approximately 30 min., was developed. By this methodology, dopamine elutes first (13.0 min. approx.) followed by salsolinol and isosalsolinol (14.4 min. approx. and 26.1 min. approx.), allowing a good separation and quantification of these analytes in concentrations above 10-8 M. (ii) The substances separated by ion pairing HPLC from commercially available salsolinol correspond to salsolinol (retention time of 14.4 min. approx.) and isosalsolinol (retention time of 26.1 min. approx.). This was established on the basis that: a) The retention time of SC1 (14.4 min. approx.) is lower than the retention time of SC2 (26.1 min. approx.) and, theoretically, it is expected that salsolinol elutes before than isosalsolinol because salsolinol has its hydroxyl groups more available to interact with the mobil phase than isosalsolinol. b) The ratio between the areas of the peaks observed for commercially available salsolinol SC1 (retention time of 14.4 min. approx.) and SC2 (retention time of 26.1 min. approx.) is in agreement with the expected percentages of salsolinol (92%) and isosalsolinol (8%) in the commercially available salsolinol as determined by 1H-NMR. c) The molecular weight of the substances separated from commercially available salsolinol, SC1 and SC2, is the same as the molecular weight of salsolinol and isosalsolinol (179 g/mol). (iii) It was determined that dopamine degradation (possibly by autoxidation) at pH 7.4 follows zero order kinetics, while salsolinol and isosalsolinol oxidations follow first order kinetics. The rate of oxidation of isosalsolinol is about 10 times higher than the rate of oxidation of salsolinol. (iv) A reversed phase HPLC methodology using a silicagel-β-cyclodextrin coupled to an electrochemical detector, able to separate and quantify dopamine, (R)-salsolinol and (S)-salsolinol in about 40 minutes, was developed. In this methodology, dopamine elutes first (22.9 min. approx.), followed by (S)-salsolinol (26.9 min. approx.), (R)- salsolinol (29.9 min. approx.) and (R) and (S)-isosalsolinol (31.3 and 32.9 min. approx.), allowing a good separation and quantification of dopamine, (S)-salsolinol and (R)-salsolinol in concentrations above 10-8 M. Although this methodology succeeded in separating isosalsolinol from dopamine and from the R and S enantiomers of salsolinol, it was unable to completely resolve (R)-isosalsolinol from (S)-isosalsolinol. In the future, these methodologies will allow the detection and quantification of these regioisomers and enantiomers in biological samples and will allow the selective administration of a specific enantiomer directly into rat brain, extending our knowlegde on the reinforcing properties of ethanol as a prodrug and on how these properties may be mediated by salsolinol