{"id":{"repo_id":"buffalo","oai_identifier":"oai:ubir.buffalo.edu:10477/80882"},"canonical_url":"https://search.dev.ndltd.org/etd/buffalo/oai:ubir.buffalo.edu:10477/80882","repository":{"repo_id":"buffalo","name":"Buffalo","base_url":"https://ubir.buffalo.edu/oai/request"},"display":{"title":"Total Oxysterol Analysis by Liquid Chromatography-Mass Spectrometry: Validation of Cholesterol Esterase Hydrolysis for Plasma Sample Preparation","abstract":"M.S.","abstract_html":"M.S.","abstract_has_math":false,"creators":["Novickis, Aaron; 0000-0002-2214-5943"],"institution":"State University of New York at Buffalo","degree_name":null,"degree_level":null,"degree_discipline":null,"degree_department":null,"school":null,"contributors":["Browne, Richard","Biotechnical and Clinical Laboratory Sciences"],"advisors":[],"committee_chairs":[],"committee_members":[],"year":2019,"date_issued":"2019-10-29T16:47:50Z","date_published":"2019-10-29T16:47:50Z","updated_at":"2026-07-27T19:05:25Z","subjects":["health sciences","biochemistry","chemical engineering"],"languages":["eng"],"rights":["Users of works found in University at Buffalo Institutional Repository (UBIR) are responsible for identifying and contacting the copyright owner for permission to reuse. University at Buffalo Libraries do not manage rights for copyright-protected works and cannot assist with permissions.","Copyright retained by author."],"rights_urls":[],"identifier_entries":[]},"links":{"outbound_url":"http://hdl.handle.net/10477/80882","outbound_label":"Handle","outbound_source":"dc:identifier"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor","label":"Contributor","values":["Browne, Richard","Biotechnical and Clinical Laboratory Sciences"]},{"key":"dc:creator","label":"Author","values":["Novickis, Aaron; 0000-0002-2214-5943"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date","label":"Dc Date","values":["2019-10-29T16:47:50Z","2019","2019-08-02 14:44:47"]},{"key":"dc:publisher","label":"Institution","values":["State University of New York at Buffalo"]},{"key":"dc:type","label":"Dc Type","values":["Text","Thesis"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["health sciences","biochemistry","chemical engineering"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language","label":"Dc Language","values":["eng"]},{"key":"dc:rights","label":"Dc Rights","values":["Users of works found in University at Buffalo Institutional Repository (UBIR) are responsible for identifying and contacting the copyright owner for permission to reuse. University at Buffalo Libraries do not manage rights for copyright-protected works and cannot assist with permissions.","Copyright retained by author."]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier","label":"Identifier","values":["http://hdl.handle.net/10477/80882"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description","label":"Description","values":["M.S.","Background: Oxysterols and cholesterol oxidation products (COPs) are oxygenated sterols which have important roles in biosynthesis, transport, immune function and nuclear signaling. Analysis of total oxysterols in human blood, bodily fluids, and tissues is complicated by their presence as either free oxysterols or as oxysteryl esters with fatty acids. Alkaline hydrolysis (saponification) converts oxysteryl esters to free oxysterols but the harsh reaction conditions could degrade existing oxysterols or cause artefactual oxysterol formation through autoxidation. The use of enzymatic hydrolysis using cholesterol esterase has recently been purported to be a superior alternative to chemical saponification. In this thesis we test the kinetics and efficiency of cholesterol esterase catalyzed hydrolysis in preparing human serum samples for liquid chromatography-mass spectrometry (LC-MS) analysis of total oxysterols and quantify possible oxysterol degradation of artefactual formation.Methods: The effects of reaction time, temperature and alcohol content on the completeness of cholesterol esterase hydrolysis was measured using a reverse-phase HPLC method capable of simultaneously measuring free cholesterol and six cholesteryl esters. Fatty acids generated during enzyme catalyzed hydrolysis were measured as fatty acid methyl esters (FAMEs) using gas-chromatography with flame ionization detection (GC-FID). Oxysterol degradation was measured by subjecting authentic oxysterol standards to enzyme catalyzed hydrolysis and measuring losses by LC-MS. Cholesterol autoxidation was measured by subjecting free cholesterol to enzyme catalyzed hydrolysis and measuring the formation of new oxysterols. Results: Cholesterol esterase from pseudomonas aeruginosa (2 enzyme units (U)), at 37oC, was able to convert greater than 99% of all cholesteryl esters, present in 200 μL of human serum, to free cholesterol in twenty minutes. Free fatty acids generated from these conditions plateaued between 10 minutes and 20 minutes further confirming complete hydrolysis. Authentic methanolic oxysterols spiked into enzymatic reactions did not affect the hydrolysis rate and displayed no degradation. Similarly, reactions with purified cholesterol standards yielded no formation of new oxysterols. Conclusion: The use of enzymatic hydrolysis with cholesterol esterase offers a rapid means of completely converting all oxysteryl esters to free oxysterols which are then suitable for analysis by LC-MS."]},{"key":"dc:format","label":"Dc Format","values":["application/pdf"]},{"key":"dc:title","label":"Title","values":["Total Oxysterol Analysis by Liquid Chromatography-Mass Spectrometry: Validation of Cholesterol Esterase Hydrolysis for Plasma Sample Preparation"]}]}],"canonical_facts":{"dc:contributor":["Browne, Richard","Biotechnical and Clinical Laboratory Sciences"],"dc:creator":["Novickis, Aaron; 0000-0002-2214-5943"],"dc:date":["2019-10-29T16:47:50Z","2019","2019-08-02 14:44:47"],"dc:description":["M.S.","Background: Oxysterols and cholesterol oxidation products (COPs) are oxygenated sterols which have important roles in biosynthesis, transport, immune function and nuclear signaling. Analysis of total oxysterols in human blood, bodily fluids, and tissues is complicated by their presence as either free oxysterols or as oxysteryl esters with fatty acids. Alkaline hydrolysis (saponification) converts oxysteryl esters to free oxysterols but the harsh reaction conditions could degrade existing oxysterols or cause artefactual oxysterol formation through autoxidation. The use of enzymatic hydrolysis using cholesterol esterase has recently been purported to be a superior alternative to chemical saponification. In this thesis we test the kinetics and efficiency of cholesterol esterase catalyzed hydrolysis in preparing human serum samples for liquid chromatography-mass spectrometry (LC-MS) analysis of total oxysterols and quantify possible oxysterol degradation of artefactual formation.Methods: The effects of reaction time, temperature and alcohol content on the completeness of cholesterol esterase hydrolysis was measured using a reverse-phase HPLC method capable of simultaneously measuring free cholesterol and six cholesteryl esters. Fatty acids generated during enzyme catalyzed hydrolysis were measured as fatty acid methyl esters (FAMEs) using gas-chromatography with flame ionization detection (GC-FID). Oxysterol degradation was measured by subjecting authentic oxysterol standards to enzyme catalyzed hydrolysis and measuring losses by LC-MS. Cholesterol autoxidation was measured by subjecting free cholesterol to enzyme catalyzed hydrolysis and measuring the formation of new oxysterols. Results: Cholesterol esterase from pseudomonas aeruginosa (2 enzyme units (U)), at 37oC, was able to convert greater than 99% of all cholesteryl esters, present in 200 μL of human serum, to free cholesterol in twenty minutes. Free fatty acids generated from these conditions plateaued between 10 minutes and 20 minutes further confirming complete hydrolysis. Authentic methanolic oxysterols spiked into enzymatic reactions did not affect the hydrolysis rate and displayed no degradation. Similarly, reactions with purified cholesterol standards yielded no formation of new oxysterols. Conclusion: The use of enzymatic hydrolysis with cholesterol esterase offers a rapid means of completely converting all oxysteryl esters to free oxysterols which are then suitable for analysis by LC-MS."],"dc:format":["application/pdf"],"dc:identifier":["http://hdl.handle.net/10477/80882"],"dc:language":["eng"],"dc:publisher":["State University of New York at Buffalo"],"dc:rights":["Users of works found in University at Buffalo Institutional Repository (UBIR) are responsible for identifying and contacting the copyright owner for permission to reuse. University at Buffalo Libraries do not manage rights for copyright-protected works and cannot assist with permissions.","Copyright retained by author."],"dc:subject":["health sciences","biochemistry","chemical engineering"],"dc:title":["Total Oxysterol Analysis by Liquid Chromatography-Mass Spectrometry: Validation of Cholesterol Esterase Hydrolysis for Plasma Sample Preparation"],"dc:type":["Text","Thesis"]},"updated_at":"2026-07-27T19:05:25Z"}