An HPLC-CAD/fluorescence lipidomics platform using fluorescent fatty acids as metabolic tracers

Vanessa H Quinlivan,Meredith H Wilson,Josef Ruzicka,Steven A Farber

doi:10.1194/jlr.d072918

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.

Highlights

Fluorescent lipids are important tools for live imaging in cell culture and animal models, yet their metabolism has not been well-characterized
We describe a combined HPLC-charged aerosol detection (CAD) and LC-MS workflow in which a lipidomic data set obtained from a short series of LC-MS experiments is used to interpret results from a large number of HPLC experiments, retaining the high-throughput/low-cost advantage of HPLC, while enhancing the type and quality of data that may be obtained from HPLC lipidomics
A single LC-MS/MS experiment (n = 3 biological replicates) was performed using the chromatography component of our established HPLC method in order to identify the specific lipid contents of each analyte peak detected by HPLC-CAD/fluorescence (Fig. 1)

Summary

Introduction

Fluorescent lipids are important tools for live imaging in cell culture and animal models, yet their metabolism has not been well-characterized. 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-difluoro5,7-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. 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.—Quinlivan, V.

Methods

Results

Conclusion