Doctor of Philosophy
Unviersity of Wollongong. School of Chemistry
Deeley, Jane M., Investigating human lens lipids using tandem mass spectrometric techniques, Doctor of Philosophy thesis, Unviersity of Wollongong. School of Chemistry, University of Wollongong, 2010. http://ro.uow.edu.au/theses/3482
The lens is a transparent highly refractive structure within the eye, located between the iris and the vitreous body. It is avascular and thus all nutrients are transported into the lens from the surrounding aqueous humour. The main body is composed of tightly packed elongated crescent shaped cells, known as fibre cells, which are arranged in concentric layers. There are two regions to the lens. The nucleus which corresponds to the lens at birth and the cortex formed as the lens grows throughout life. Experimental evidence supports the formation of a barrier in the lens after middle age. The barrier isolates the nucleus from the metabolically active cortex, lessening the transport of antioxidants to the lens centre and leaving the nucleus vulnerable to oxidation and precipitation of lens protein. Precipitation of lens protein results in loss of visual acuity and cataract which is the main cause of blindness worldwide. The biomolecules of the lens that may be playing a role in the development of the lens barrier are the proteins and membrane lipids. The overall aim of this thesis was to gain further insight into the lipid composition of human lens and to ascertain whether the lipids play a role in the formation of the barrier to diffusion.
In this thesis shotgun lipidomics was used to identify and quantify phospholipids in human lenses and those from six animal models. A triple quadrupole mass spectrometer with an electrospray ionisation source was used. A combination of precursor ion, neutral loss and product ion scans were used to identify the phospholipids in each class. The results showed that the lipid profiles of the animals were different from human. The human lens contained abundant dihydrosphingomyelins, a lipid present only in trace levels in the lenses of other non-primates. Ether lipids were identified as the major components within phosphatidylethanolamines (PE) and phosphatidylserines (PS) in human lenses.
A combination of mass spectrometric experiments including MS 3 and Ozoneinduced dissociation were used to characterise abundant human lens ether lipids using a modified linear ion trap mass spectrometer. 1- O-alkyl ether lipids were identified in the classes of PE and more interestingly, PS. This study is the first to report PS alkyl ethers in human tissue. Specifically, the ether-lipids identified in this study include PE (16:0e/9 Z-18:1), PE (11Z-18:1e/9Z-18:1), PE (18:0e/9Z-18:1)], PS (16:0e/9Z-18:1), PS (11 Z-18:1e/9Z-18:1) and PS (18:0e/9Z-18:1). 1-O-alkyl ethers were estimated to account for 52% and 66%, respectively of the total PE and PS content of the human lens. Interestingly, double bonds in ether-linked chains favoured the n-7 position. Structural similarities in the series of PE and PS-ethers identified in this thesis suggest a precursor-product relationship.
The distribution of sphingolipids in the human lens was determined using MALDI imaging. Transverse human lens slices (23-70 years, 10-25 μm thick) on microscope cover slips were coated in 2,5-dihydroxybenzoic acid by sublimation. The distribution of the most abundant phospholipid in the human lens, dihydrosphingomyelin 16:0 was then examined using BioMAP TM software. In the young human lens, this lipid showed an even distribution throughout whereas in the 64 and 70 year old lenses this lipid was located in a distinct annular distribution in the region of the lens barrier. A structurally-related sphingolipid, dihydroceramide 16:0, was observed to increase in the nuclear region of the lens with age. These data were supported by an analysis of lens regions using ESI-MS.
In conclusion, the results from this thesis have confirmed the human lens contains abundant dihydrosphingomyelins and, in addition, have identified 1- O-alkyl ether PE and PS which makes them considerably different in composition to commonly used animal models. Moreover there are major changes in lipid distribution with age that may contribute to or result from the formation of the barrier to diffusion at middle age.