Doctor of Philosophy
School of Chemistry
Hughes, Jessica R., Investigation into the extent of lipid turnover in the mammalian lens, thesis, , University of Wollongong, 2010. http://ro.uow.edu.au/theses/3324
The lens is a highly transparent structure in the anterior portion of the eye, located between the iris and the vitreous body. It is bathed anteriorly by the aqueous humor, which also provides a pathway for nutrient delivery as the lens is avascular. There is a thin layer of metabolically active cells facing the aqueous humor known as the epithelial cells, which gain nutrients and dispel waste from its surrounding environment. The main portion of the lens is composed of tightly-packed elongated fibre cells that are formed from the differentiation of epithelial cells. During differentiation, the fibre cells become elongated and form concentric layers. Those mature fibre cells near the lens centre are devoid of all cellular organelles to aid with transparency. There is no turnover of these cells; therefore as the lens grows with age the fibre cells stack upon each other. There are two regions of the lens, the nucleus which corresponds to the lens at birth and cortex composed of the tissue accumulated throughout the lifespan. Due to the developmental nature of the lens fibre cells and their lack of turnover, there is experimental evidence to suggest that the lens proteins do not turn over, and thus it has been postulated that membrane lipid turnover does not occur. This has implications for lens accommodation and for the maintenance of the lens structure and transparency throughout life. Experimental evidence suggests the formation of a barrier at middle age to the diffusion of antioxidants and water into the nucleus. It is thought that this barrier is due to occlusion of the membrane pores by modified α-crystallins. Alterations to the lipid composition with age could render the membrane more adhesive for α-crystallin, causing a greater occlusion of the pores. The lens therefore becomes vulnerable to protein precipitation and oxidation, leading to age-related nuclear cataract, the major cause of blindness worldwide. Lens lipid integrity may play an essential role in the development of the lens barrier. The overall aim of this thesis was to probe indirectly the extent of lipid turnover in the lens and examine the implications of this with age.
In this thesis, shotgun lipidomics was used to examine the effect of dietary fatty acid composition on the phospholipid profile of the rat lens nucleus and cortex. This was directly compared to rat skeletal muscle, as this tissue responds dramatically in its lipid composition to alterations in dietary lipids. Results demonstrated that skeletal muscle phospholipid composition was altered with dietary manipulation; however the lens nucleus and cortex remained unperturbed after 8 weeks of feeding. The lens nucleus and cortex had different phospholipid compositions, suggesting that a different lipid environment is functionally required for these regions. The lack of dietary alteration in the nucleus suggests slow or even no lipid turnover. However, more significantly the lack of alteration seen in the cortex suggests a regulatory mechanism for phospholipid incorporation into the membranes of this region, as this region was formed during dietary consumption. This tight regulation of lipid composition suggests a role for phospholipids in the control of protein activity.
The extent of phospholipid incorporation was examined in the rat lens using fluorescently- and isotopically-labelled fatty acids in vitro. The complementary use of confocal microscopy and electrospray ionisation mass spectrometry determined that after 16 hours of incubation with labelled fatty acids, there was very little incorporation of fatty acids into lens phospholipids, which is most likely restricted to the outer 3-6 % of the lens cortex. Incorporation of 13C18-oleic acid was detected at very low levels (~546 pmol.g-1 tissue wet weight) in the lens cortex [phosphatidylethanolamine (16:0/18:1)] (seen at m/z 734.6). This result implies that over a time period of at least 16 hours, fatty acids from external sources (for example diet), do not replace fatty acids that have already been incorporated into the membrane.
Age-related alterations to lens lipids were examined in the human nucleus using electrospray ionisation mass spectrometry. Abundant glycerophospholipids in a young lens were seen to decline until the age of 40, while ceramide and dihydroceramide increased in abundance from age 30. Dihydrosphingomyelin and sphingomyelin were found not to alter in concentration with age. The reason for these alterations is currently unknown; however it suggests slow hydrolytic activity in the centre of the lens which alters the structure of lipids over a number of decades. The modification of these lipids is unlikely to be caused by enzymatic processes due to the lack of enzymatic activity in the lens nucleus. These modifications to the lens lipidome particularly in the nucleus may contribute to the formation of the lens barrier seen at middle age.
Accelerator mass spectrometry was used to ascertain the 14C levels present in the human lens lipidome nucleus which is present at birth. It was hypothesised that if there is no lipid turnover in the lens nucleus, the 14C levels in the lens lipids should reflect the level of atmospheric 14C at the year of birth. By comparing the level of atmospheric 14C induced by above-ground nuclear testing from 1955-1963 to the levels present in the human lens, it was determined that there is no turnover of total lipid occurring in the lens.
In conclusion, this thesis has determined through a variety of techniques and studies that the extent of lipid turnover in the lens nucleus is very limited (to nonexistent) over a period of hours to weeks in the rat lens, and a period of decades in the human lens. This suggests that the lipid environment required in the lens is stable and specific due to the membrane proteins it surrounds. Major alterations to the lipid composition with age have implications for the formation of a barrier to molecule diffusion at middle age. The lens also provides a model for examining the stability of lipids in a unique biological environment devoid of enzymatic activity