A NOVEL FLUORESCENT HPLC-BASED METABOLIC LABELING TECHNIQUE REVEALS EFFECTS OF NUTRITIONAL CONTEXT ON DIETARY FATTY ACID PARTITIONING IN THE INTESTINAL ENTEROCYTES

lipid metabolism;zebrafish;HPLC;lipidomics;LC-MS;cholesterol;alcohol;fatty liver disease;lipid partitioning;Biochemistry

Quinlivan-Repasi, Vanessa HelenHalpern, Marnie ;

Johns Hopkins University

关键词: lipid metabolism;    zebrafish;    HPLC;    lipidomics;    LC-MS;    cholesterol;    alcohol;    fatty liver disease;    lipid partitioning;    Biochemistry;   

Others  :  https://jscholarship.library.jhu.edu/bitstream/handle/1774.2/58663/QUINLIVAN_THESIS_v21.docx?sequence=3&isAllowed=y

瑞士|英语

来源: JOHNS HOPKINS DSpace Repository

【 摘 要 】

When dietary fatty acids are absorbed by intestinal enterocytes, they are re-assembled into complex lipids such as phospholipids, cholesterol esters, and triglycerides. Although the cellular machinery required for these processes is well known, the mechanisms regulating the partitioning of individual fatty acids among the thousands of possible complex lipid products are not as well understood. Metabolic labeling of dietary lipids, historically performed using radioactive reagents or expensive stable isotopes, is a key technique for examining the biochemical behavior of individual fatty acids in the context of a physiologically relevant mixed-lipid diet. Likewise, fluorescent lipids have been established as important tools for live imaging of the trafficking and spatial deposition of dietary lipids, yet their metabolism has not been fully characterized. I have developed a novel HPLC-based quantitative metabolic labeling method in a larval zebrafish model, wherein the same fluorescent lipid reagents and delivery methods may be used for biochemical and live imaging experiments. The characterization of fluorescent fatty acid metabolism described here opens the way for more widespread use of fluorescent lipids in metabolic studies. In this thesis I review the current literature on the zebrafish as a model for lipid uptake and metabolism. This review includes my own analysis and interpretation of a recently published developmental lipidomics data set.I then describe the application of metabolic labeling with fluorescent lipids toward a greater understanding of dietary lipid partitioning in the intestinal enterocytes, specifically addressing the interactions among dietary fatty acids, cholesterol, and ethanol. Preliminary results suggest that phospholipid synthesis from dietary fatty acids is increased in larval zebrafish exposed to ethanol before feeding. Future work will address potential physiological effects of this interaction between ethanol and dietary fatty acid partitioning. Additionally, I report results of a collaborative study in which HPLC lipid profiling methods initially developed in zebrafish were expanded into a mouse model. This enabled measurement of changes in individual plasma lipid species resulting from perturbation of a putative lipid transport mechanism in the intestinal enterocytes. These diverse applications of the metabolic labeling methods I have developed demonstrate significant contributions to the lipid biology field: I have expanded both the amount and type of data that can be obtained using HPLC lipidomics, creating a relatively low-cost and high-throughput alternative to metabolic labeling with mass spectrometry analysis. Furthermore, ongoing work may contribute to greater understanding of both potentially beneficial interactions between ethanol and dietary lipids (e.g. the Mediterannean diet hypothesis) and possible diet-driven variability in the progression of alcoholic liver disease.