Quantification and Profiling of Hepatic Retinoids in Freshwater Fishes by Liquid Chromatography – Tandem Mass Spectrometry

retinoid;mass spectrometry;area of concern;quadrupole time of flight;QToF;dehydroretinoid;didehydroretinoid;retinol;retinyl ester;retinoic acid;tandem mass spectrometry;triple quadrupole

Dunning, Michael Williamadvisor:Servos, Mark R. ; affiliation1:Faculty of Science ; advisor:Hewitt, L. Mark ; Servos, Mark R. ; Hewitt, L. Mark ;

University of Waterloo

关键词: Master Thesis;    area of concern;    retinyl ester;    retinoid;    triple quadrupole;    retinol;    retinoic acid;    tandem mass spectrometry;    dehydroretinoid;    mass spectrometry;    didehydroretinoid;    QToF;    quadrupole time of flight;   

Others  :  https://uwspace.uwaterloo.ca/bitstream/10012/13059/3/Dunning_Michael.pdf

瑞士|英语

来源: UWSPACE Waterloo Institutional Repository

【 摘 要 】

Retinoids (vitamin A1) are a class of compounds essential for a number of physiologicalprocesses including vision and immune system function. They are stored as biologically inactiveretinyl esters which are mobilized and converted to the transcriptionally-active retinoic acids(RAs) according to physiological and nutritional requirements. RAs are key components in geneexpression and are required for proper embryonic development in vertebrates. In addition toretinoids, dehydroretinoids (vitamin A2) appear to be of high importance in some species offreshwater fish. In some cases dehydroretinoids are found to be more abundant than retinoids,which has led to classification of fish species as ;;retinol-type” or ;;dehydroretinol-type” based onthe major class of retinoids utilized. The exact functions, metabolism, and distribution ofdehydroretinoids in fish remain to be elucidated and requires further investigation into how theretinoid and dehydroretinoid systems interact in aquatic species.Disruptions to the retinoid system have been observed in several species of fish byexposure to xenobiotics and contaminants from various sources including mine tailings and pulpmill effluent. These assessments typically only consider retinyl esters and/or free retinol,however a need has been recognized in the literature for retinoid analysis to establish whatlinkages exist between depressed levels of retinol/retinyl esters caused by pollutant exposure andhow this may impact levels of biologically active vitamin A. Retinol and retinyl esters are mostoften analyzed by ultraviolet spectrophotometry. While these methods provide acceptabledetection limits for quantification, they lack specificity and require authentic standards whichcan be expensive or impossible to obtain. Retinoic acids are thought to be present in fish at thepmol/g level and due to analytical limitations have not been adequately quantified to date.The objective of this thesis was to develop a comprehensive and robust method tomeasure retinoids and dehydroretinoids in fish, thereby facilitating further research into retinoidbiology. A streamlined extraction method was applied to generate separate fractions of non-polarretinoids (retinol/retinyl esters) and active retinoids (retinoic acids) from as low as 5 mg ofhepatic tissue. A new approach coupling liquid chromatographic separations to mass selectivedetection was developed that enabled the quantification of major retinoids by triple quadrupolemass spectrometry. The analysis was validated with standards and found to provide acceptablesensitivity, accuracy, precision, and linearity. The limit of quantification was in the range of 0.12– 1.10 ng/mL for retinoic acids, 1.04 ng/mL for retinol, and 0.82 ng/mL for retinyl palmitate.This new method was then applied to select archived fish tissues of brown bullhead (Ameiurusnebulosus) and shorthead redhorse sucker (Moxostoma macrolepidotum) from two NorthAmerican Great Lakes Areas of Concern (AOCs) to demonstrate the ability of the method todetect differences in hepatic retinoid status in populations of fish exposed to differentenvironmental stressors. The method was capable of detecting significant decreases in freeretinol and retinyl esters in shorthead redhorse sucker from a contaminated site. Retinoic acidstatus could not be adequately assessed in hepatic tissue due to significant matrix interferencesresulting in failure to meet QA/QC criteria. The application of high resolution quadrupole time-of-flight instrumentation also allowed tentative identification of retinoids for which standards arenot commercially available, and semi-quantitative profiling of vitamin A1 /vitamin A2 distributionin hepatic tissue. Profiling of liver samples from shorthead redhorse sucker showed near equallevels of retinyl and dehydroretinyl esters, while dehydroretinoids appeared to be dominant overretinoids in brown bullhead.These investigations advanced the field of retinoid biology by allowing for a greaterrange of compounds which can be confidently analyzed without the need for expensive orunavailable standards, and by providing the ability to semi-qualitatively assessretinoid/dehydroretinoid abundances in different fish species. Resolving the persistent matrixissues which complicates the analysis of retinoic acids in hepatic tissue should be the focus offuture work. Once resolved, this method may be used to begin answering some of the openquestions regarding retinoid metabolism and functions in fish.