title: An HPLC-CAD/fluorescence lipidomics platform using fluorescent fatty acids as metabolic tracers.
abstract: Fluorescent lipids are important tools for live imaging in cell culture and animal models, yet their metabolism has not been well-characterized. Here we describe a novel combined HPLC and LC-MS/MS method developed to characterize both total lipid profiles and the products of fluorescently labeled lipids. Using this approach, we found that lipids labeled with the fluorescent tags, 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene (BODIPY FL), 4,4-difluoro-5-(2-thienyl)-4-bora-3a,4a-diaza-s-indacene [BODIPY(558/568)], and dipyrrometheneboron difluoride undecanoic acid (TopFluor) are all metabolized into varying arrays of polar and nonpolar fluorescent lipid products when they are fed to larval zebrafish. Quantitative metabolic labeling experiments performed in this system revealed significant effects of total dietary lipid composition on fluorescent lipid partitioning. We provide evidence that cholesterol metabolism in the intestine is important in determining the metabolic fates of dietary FAs. Using this method, we found that inhibitors of dietary cholesterol absorption and esterification both decreased incorporation of dietary fluorescent FAs into cholesterol esters (CEs), suggesting that CE synthesis in enterocytes is primarily responsive to the availability of dietary cholesterol. These results are the first to comprehensively characterize fluorescent FA metabolism and to demonstrate their utility as metabolic labeling reagents, effectively coupling quantitative biochemistry with live imaging studies.
authors.affiliation: Department of Embryology, Carnegie Institution for Science, Baltimore, MD 21218.|Department of Biology, Johns Hopkins University, Baltimore, MD 21218; and.
authors.forename: Vanessa H
authors.initials: VH
authors.lastname: Quinlivan
authors.affiliation: Department of Embryology, Carnegie Institution for Science, Baltimore, MD 21218.
authors.forename: Meredith H
authors.initials: MH
authors.lastname: Wilson
authors.affiliation: Thermo Fisher Scientific, Somerset, NJ 08873.
authors.forename: Josef
authors.initials: J
authors.lastname: Ruzicka
authors.affiliation: Department of Embryology, Carnegie Institution for Science, Baltimore, MD 21218; farber@ciwemb.edu.|Department of Biology, Johns Hopkins University, Baltimore, MD 21218; and.
authors.forename: Steven A
authors.initials: SA
authors.lastname: Farber
mesh_terms: D000336:Aerosols; D000818:Animals; D001692:Biological Transport; D001896:Boron Compounds / Q000737:chemistry; D002791:Cholesterol, Dietary / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D020895:Enterocytes / Q000378:metabolism; D004951:Esterification; D005227:Fatty Acids / Q000737:chemistry* / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000737:chemistry*; D007814:Larva / Q000378:metabolism; D055432:Metabolomics / Q000379:methods*; D013050:Spectrometry, Fluorescence; D015027:Zebrafish / Q000196:embryology / Q000378:metabolism
chemical_list: C095489:4,4-difluoro-4-bora-3a,4a-diaza-s-indacene; D000336:Aerosols; D001896:Boron Compounds; D002791:Cholesterol, Dietary; D005227:Fatty Acids; D005456:Fluorescent Dyes
keywords: BODIPY; charged aerosol detection; cholesterol/metabolism; diet and dietary lipids; fatty acid/metabolism; high-performance liquid chromatography; isotopic tracers; lipids/chemistry; mass spectrometry; zebrafish