REGIONAL PHOSPHOLIPID ANALYSIS OF BOVINE LENS EPITHELIA BY IN SITU MALDI–TOF MS

Abstract

Abstract: : Purpose: To determine possible differences in the phospholipid (PL) composition of membranes corresponding to epithelial cells that exhibit slower (central region) and faster (germinative zone) rates of mitoses. Methods: For mass spectral analysis, the anterior face of bovine lenses was placed onto the stainless steel plate of a matrix–assisted laser desorption/ionization mass spectrometer (MALDI MS) plate. After removal of the posterior capsular region and all cortical and nuclear fibers, the anterior capsule/epithelium remained deposited onto the plate. The integrity of the epithelium after dissection was verified with a fluorescence microscope. Paranitroaniline was used as the matrix. For ³¹P NMR studies, the central areas (5.25 mm radius) and annular regions of epithelia of eight bovine lenses were pooled. A single–step methanolic extraction was used to extract the lipids. ³¹P NMR spectra were acquired after addition of Cs/EDTA to enhance the resolution of the PL bands. Results: Throughout all epithelial regions, phosphatidylcholines (PCs) with one and two sites of unsaturation were the most abundant PLs (∼ 65% of all PLs). Among sphingomyelins (SMs) those with a palmitoyl acyl chain were predominant. The differences between the central and peripheral regions of the epithelium were not substantial enough to be quantified by MALDI MS. However, the ³¹P NMR spectral quantification did reveal an increase in PCs (69% of all PLs) in the annular regions as compared to the central zone (60%) of the epithelium. Other PLs included phosphatidylethanolamines and phosphatidylinositols. Conclusions: The differences in PL contents between cells undergoing mitoses at different rates are not large enough to be detected by MALDI MS due to the relatively low precision of the quantification (relative standard deviation of 10% to 15%). The studies by ³¹P NMR spectroscopy do indicate an increase in PCs and suggest that higher relative amounts of PCs are synthesized in proliferating epithelial cells. The possible role of PCs and their metabolites in the coordination of processes that lead to cell division deserve further investigation.

Keywords: cell membrane/membrane specializations • lipids • proliferation