The aim of this project is to characterise the levels of cholesterol and phospholipid in cell homogenates, plasma membranes and isolated membrane domains from wildtype (WT) and caveolin-l knock-out (Cav-1-/ -) mouse embryonic fibroblasts (MEFs). Quantitative lipid analysis was developed for cholesterol by high-performance liquid chromatography (HPLC) and for glycerophospholipids (GPL) and sphingomyelin (SM) by electrospray ionisation mass spectrometry (ESI-MS). In the cell homogenates, by comparing WT to Cav-l-/- MEFs, it was found that the total cholesterol as well as the proportions of the main GPLs such as Phosphatidylcholines (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS) was similar in both cell types. However, Cav-l-/- MEFs have higher levels of cholesterol esters than WT MEFs in whole cell homogenate. Furthermore, the proportion of SM is higher in Cav-l-/- than WT MEFs. These data suggest that Cav-l may control the balance between free and esterified cholesterol and is involved in SM metabolism. A small increase in SM level in Cav-l-/- compared to WT plasma membrane was observed but this increase was not significant. Similarly, cholesterol levels in the plasma membrane are comparable between the two cell types. In both cell types, the levels of SM and phosphatidylglycerol (PG) are higher in the plasma membranes than in cell homogenates. In the WT cells, PE levels are higher in the plasma membrane than in the cell homogenates. While in the Cav-1-/- cells, the level of free cholesterol and PC are higher in the plasma membrane compared to the cell homogenate. PCs are the predominant lipids in both cell types. Cav-1-l - cells have more saturated fatty acyl chains in their PC species and shorter carbon chains compared to WT MEFs and this trend was found in both cell homogenate and plasma membranes. In PEs and SMs, Cav-1-/ - cells also have higher levels of saturated PEs and saturated amide-linkage SM than in WT cells, respectively. The results indicate that Cav-1 may also play a role in fatty acids metabolism. In summary, the data from this work indicate that Cav-l expression affects lipid composition in MEFs including the relative distribution of free and esterified cholesterol, the levels of SM relative to other phospholipid subclasses and the incorporation of fatty acids into phospholipids.
| Identifer | oai:union.ndltd.org:ADTP/258488 |
| Date | January 2008 |
| Creators | Li, Qiong, Medical Sciences, Faculty of Medicine, UNSW |
| Publisher | Awarded by:University of New South Wales. Medical Sciences |
| Source Sets | Australiasian Digital Theses Program |
| Language | English |
| Detected Language | English |
| Rights | Copyright Li Qiong., http://unsworks.unsw.edu.au/copyright |
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