Implementation of a Lipoprotein Isolation Method in a Lipidomic High-throughput Workflow

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http://urn.fi/URN:NBN:fi:hulib-201507212089
Title: Implementation of a Lipoprotein Isolation Method in a Lipidomic High-throughput Workflow
Author: Sylvänne, Tuulia
Contributor: University of Helsinki, Faculty of Agriculture and Forestry
Publisher: Helsingfors universitet
Date: 2013
Language: eng
URI: http://urn.fi/URN:NBN:fi:hulib-201507212089
http://hdl.handle.net/10138/40282
Thesis level: master's thesis
Discipline: Bioteknik
Biotechnology
Biotekniikka
Abstract: Lipoproteins play a central role in the disease mechanisms of cardiovascular diseases (CVD) and therefore they have been studied widely. They carry several classes of apolipoproteins where apo-A1 and apo-B are the major classes. The sucrose based sequential lipoprotein isolation method can retrieve the lipoprotein fractions suitable for lipidomics analyses. The main lipoprotein classes are very low-density lipoprotein (VLDL), low-density lipoprotein (LDL) and high density lipoprotein (HDL) that can be isolated easily by their density from human blood plasma or serum. Lipidomics analyses can quantify lipids that lipoproteins carry in the circulation. Mainly they carry cholesterol and its esterified forms, glycerolipids, small amounts of sphingolipids and phospholipids form their monolayer membrane. The isolation method was set-up together with scaled-down sample volumes. The protein and lipid content of the main lipoprotein fractions were evaluated by electrophoresis analysis, various enzymatic assays and lipidomics analyses. The total protein and apolipoprotein content was found to be similar as in the literature. Apo-B was found to be the main apolipoprotein in the VLDL and the LDL fractions whereas apo-A1 was the main apolipoprotein in the HDL fractions. Triglycerides (TG) were measured by enzymatic analysis and TG was mainly found in LDL and VLDL. The lipidomics analyses demonstrated the lipid content of the lipoproteins were similar as in the literature with minor changes. The main lipid class found in all the lipoproteins was cholesteryl esters (CE) followed by phosphatidylcholines (PC) that are commonly found in cell membranes. Sphingolipids such as ceramides were also detected in lipid class level only in small quantities in the lipoprotein fractions. The low initial sample volume did not correlate linearly with higher sample volume and low sample volume is not recommended to use in this specific isolation method. Based on the results of the comprehensive screening of isolated lipoproteins the isolation method was successfully established.
Subject: cardiovascular diseases
lipoproteins
glycerolipids
phospholipids
sphingolipids
lipidomics


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