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Lipid composition of isolated lipid droplets from the functional bovine corpus luteum

ABSTRACT: Establishment and maintenance of pregnancy is dependent on progesterone synthesized by the corpus luteum (CL). The CL is known for the prominent presence of intracellular lipid droplets (LDs). However relatively little is known about the composition and function of these luteal LDs. Our objective was to identify the lipid composition of LDs from fully functional bovine CLs. Luteal LDs were isolated by flotation through a discontinuous sucrose gradient, lipids were then extracted using a standard Bligh and Dyer protocol, dried, and sent to Avanti Polar Lipids for lipidomics analysis. The samples were provided for lipidomic profiling of free sterols, cholesteryl esters, triglycerides, diacylglycerols, phospholipids, and sphingolipids. Molecular species were resolved by reversed-phase liquid chromatography in the presence of class and sub-class specific internal standard compounds added to each sample. The compounds were detected by tandem mass spectrometry (MS/MS) with scheduled multiple reaction monitoring (MRM) for mass-specific fragment ions according to the lipid class and molecular weight of the compound. Quantification of cholesterol, cholesteryl esters, triglycerides, and diglycerides were directly calculated with standards and internal standards from calibration response curves. The remaining lipid species were semi-quantization using the integrated area of each analyte’s MRM peak, divided by the appropriate internal standard peak area, and multiplied by the standard’s known concentration. Lipid concentrations were normalized to the corresponding protein concentration of each sample and as a mol % relative to total lipids or within each lipid class. Isolated luteal LDs were composed primarily of triglyceride (88%, mol% of lipid class to total lipids). Other neutral lipids included diacylglycerol, 2.9%; and cholesteryl esters, 1.5%. Polar lipids were primarily composed of phosphatidylcholine (3.1%), sphingomyelin (1.5%), phosphatidylinositol (0.9%), phosphatidylethanolamine (0.8%) and phosphatidylserine (0.4%). A number of other minor lipids representing less than 0.32% of the total lipid pool were also detected including phosphatidylglycerol, lysophospholipids, ceramides, and glycosylated ceramides. Lipid composition of bovine luteal LDs are distinct from LDs isolated from other tissues and in other species.

ORGANISM(S): Cow Bos Taurus

TISSUE(S): Ovaries

SUBMITTER: John Davis

PROVIDER: ST001286 | MetabolomicsWorkbench | Mon Nov 18 00:00:00 GMT 2019

REPOSITORIES: MetabolomicsWorkbench

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Project description:BACKGROUND: Lipidomics, the comprehensive measurement of lipids within a biological system or substrate, is an emerging field with significant potential for improving clinical diagnosis and our understanding of health and disease. While lipids diverse biological roles contribute to their clinical utility, the diversity of lipid structure and concentrations prove to make lipidomics analytically challenging. Without internal standards to match each lipid species, researchers often apply individual internal standards to a broad range of related lipids. To aid in standardizing and automating this relative quantitation process, we developed LipidMatch Normalizer (LMN) http://secim.ufl.edu/secim-tools/ which can be used in most open source lipidomics workflows.RESULTS: LMN uses a ranking system (1-3) to assign lipid standards to target analytes. A ranking of 1 signifies that both the lipid class and adduct of the internal standard and target analyte match, while a ranking of 3 signifies that neither the adduct or class match. If multiple internal standards are provided for a lipid class, standards with the closest retention time to the target analyte will be chosen. The user can also signify which lipid classes an internal standard represents, for example indicating that ether-linked phosphatidylcholine can be semi-quantified using phosphatidylcholine. LMN is designed to work with any lipid identification software and feature finding software, and in this study is used to quantify lipids in NIST SRM 1950 human plasma annotated using LipidMatch and MZmine.CONCLUSIONS: LMN can be integrated into an open source workflow which completes all data processing steps including feature finding, annotation, and quantification for LC-MS/MS studies. Using LMN we determined that in certain cases the use of peak height versus peak area, certain adducts, and negative versus positive polarity data can have major effects on the final concentration obtained.

Project description:We propose a fully automated novel workflow for lipidomics based on flow injection, followed by liquid chromatography-high-resolution mass spectrometry (FI/LC-HRMS). The workflow combined in-depth characterization of the lipidome achieved via reversed-phase LC-HRMS with absolute quantification by using a large number of lipid species-specific and/or retention time (RT)-matched/class-specific calibrants. The lipidome of 13C-labelled yeast (LILY) provided a large panel of cost-effective internal standards (ISTDs) covering triacylglycerols (TG), steryl esters (SE), free fatty acids (FA), diacylglycerols (DG), sterols (ST), ceramides (Cer), hexosyl ceramides (HexCer), phosphatidylglycerols (PG), phosphatidylethanolamines (PE), phosphatidic acids (PA), cardiolipins (CL), phosphatidylinositols (PI), phosphatidylserines (PS), phosphatidylcholines (PC), lysophosphatidylcholines (LPC) and lysophosphatidylethanolamines (LPE). The workflow in combination with the LILY lipid panel enables simultaneous quantification via (1) external multi-point calibration with internal standardization and (2) internal one-point calibration with LILY as a surrogate ISTD, increasing the coverage while keeping the accuracy and throughput high. Extensive measures on quality control allowed us to rank the calibration strategies and to automatically select the calibration strategy of the highest metrological order for the respective lipid species. Overall, the workflow enabled a streamlined analysis, with a limit of detection in the low femtomolar range, and provided validation tools together with absolute concentration values for >350 lipids in human plasma on a species level. Based on the selected standard panel, lipids from 7 classes (LPC, LPE, PC, PE, PI, DG, TG) passed stringent quality filters, which included QC accuracy, a precision and recovery bias of <30% and concentrations within the 99% confidence interval of the international laboratory comparison of SRM 1950, NIST, USA. The quantitative values are independent of common deuterated or non-endogenous ISTDs, thus offering cross-validation of different lipid methods and further standardizing lipidomics.

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Lipid composition of isolated lipid droplets from the functional bovine corpus luteum

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