Browsing by Subject "Hepatic"

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    Application of Systems Biology Analysis to Hepatic Injury Following Hemorrhagic Shock
    (2014-05) Determan, Charles Edward Jr
    Introduction: This dissertation is focused on the metabolomic and transcriptomic changes that occur as a result of carbohydrate prefeeding during hemorrhagic shock and trauma within the liver of a porcine model. The risk of trauma and hemorrhagic shock continues to be an important issue in both military and civilian sectors. As such, we explored the impact of a prior fed state upon the overall response to hemorrhagic shock and resuscitation. The primary hypotheses were that changes in metabolism at the metabolomic and transcriptomic levels would be dependent upon the fed state. In addition, this thesis explores a more comprehensive analysis of metabolomics datasets to standardize analysis and improve overall consistency.Materials and Methods: Algorithm comparison was accomplished using six commonly applied methods to three synthetic datasets, of different sample sizes, and three openly accessible published datasets. This comparison also incorporated metrics to measure consistency of identified features (i.e. stability) to provide further confidence in results. Metabolomics analysis was accomplished with nuclear magnetic resonance spectroscopy (NMR) and Chenomx software to profile and quantify metabolites in liver extracts. The metabolome was subsequently analyzed with partial least squares discriminant analysis (PLS-DA). Transcriptomics analysis was conducted using next-generation sequencing (NGS) technology to employ RNA-sequencing (RNA-seq) on mRNA extracts from liver biopsies. The RNA-seq data was analyzed using typical processing techniques to generate a count matrix and subsequently analyzed with the Bioconductor package EdgeR. Results: The comparison of algorithms showed that the best algorithm is associated with differently structured datasets (e.g. number of features, number of groups, sample size, etc.). Analysis of the liver metabolome revealed changes in carbon energy sources, amino acid metabolism, oxidative stress, and membrane maintenance. Transcriptomic analysis revealed changes in carbohydrate metabolism, cytokine inflammation, cholesterol synthesis and apoptosis. In addition, there is evidence of increased cytoskeleton reorganization which may correspond to a shrunken, catabolic state which provides and anti-inflammatory condition to mitigate cellular damage.Conclusion: The response to hemorrhagic shock and resuscitation is altered with respect to a fasted or carbohydrate prefed state. Metabolomics and transcriptomic analyses suggest altered metabolic pathways as a result of fed state. Altered carbohydrate metabolism was readily identified thereby confirming both methods were successful. Additionally, indications of membrane maintenance that follow cytoskeletal remodeling and cellular shrinkage are potentially reflected by 3-Hydroxyisovalerate and sn-Glycero-3-phosphocholine. These results provide further evidence for pre-conditioning (e.g. altered diet) and hypertonic resuscitation methods to possibly improve patient outcome. Further research is required in alternative prefeeding substrates (e.g. protein, lipid, etc.) as well as improving the integration of different systems level datasets to understand more thoroughly the systemic effects of hemorrhagic shock and resuscitation.
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    Effect of obesity on hepatic drug metabolism
    (2013-09) Chiney, Manoj Shriram
    Obesity has increased markedly over the last few decades and is now a major public health crisis in the U.S. affecting over 1/3 of the US population. Optimization of dosing in obese individuals is a challenge due to the lack of knowledge regarding changes in the pharmacokinetics (PK) of therapeutic agents in obese individuals. Thus the aim of this thesis was to determine the effect of obesity on drug metabolism and evaluate methods that could potentially predict changes in pharmacokinetics in the obese population. The impact of obesity on drug metabolism in children has not been determined and our clinical study (Chapter 2) was the first of its kind to examine the effect of childhood obesity on CYP1A2, CYP2D6, CYP3A4, xanthine oxidase, and NAT2 activity using caffeine and dextromethorphan as probe drugs. Our results conclusively indicate that obesity results in an elevation of xanthine oxidase and NAT2 enzyme activities in obese children as compared to lean children, whereas there was no difference in CYP1A2, CYP2D6 and CYP3A4 activity between obese and lean children. This study provides a potential mechanism of altered 6-mercaptopurine exposure in the obese pediatric cancer population. While clinical studies would provide the most optimum method to predict clearance of therapeutic agents in humans, studies have reported that clearance can also be predicted using animal data. In Chapter 3, we examined mouse, rat and porcine model of obesity in order to determine the utility of these animal models to predict PK in obese humans and to identify a model that would best reflect the human obesity mediated changes in drug metabolism. The study indicated species dependent differences in CLint of various drugs that were due to, either changes in expression of drug metabolizing enzymes or changes in enzyme substrate affinity. The study demonstrated that obesity can alter enzyme activity in a species and model dependent manner. Furthermore this study identified that the rat High Fat Diet animal model of obesity is the best representation of the obesity mediated alterations in humans. In Chapter 4, in collaboration with Drs. Scott Rector and Jim Perfield, University of Missouri, Columbia, we demonstrated obesity mediated alterations of drug metabolism enzyme activity can be prevented by sterculic oil dietary supplementation. These effects are mediated through signal transduction pathways which regulate CAR and PXR transcription factors. These results establish that obesity mediated changes are biochemically dependent and not weight dependent. In Chapter 5, we developed a proof of concept that would help generate biochemically obese hepatocytes. In absence of hepatocytes from obese individuals, these hepatocytes can be used as a tool to predict obesity mediated changes in drug clearance. Our studies indicate that individually, leptin, resistin, IL-6 and TNF-α can modulate expression of various DMEs in a concentration dependent and isoform specific manner. This study demonstrates that the obesity microenvironment is important in obesity mediated changes in drug metabolism. Additional studies would help establish a more robust method to generate and validate these obese hepatocytes. In summary, the work in this thesis has helped identify the drug metabolism enzymes that are altered in the obese children, the utility of using animal models of obesity as tools to study the impact of obesity on pharmacokinetics/pharmacodynamics, proven that it is possible to reverse obesity mediated changes in drug metabolism and developed an in vitro model that may be used to predict changes in drug disposition in the obese population. These findings are important for to better develop dosing strategies in obese humans with concomitant disease.