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Stephen Barnes, Miranda Collier, Stephen Watts, David E Graves, Janusz H Kabarowski; Age-related changes in composition and geographic distribution of phospholipids in zebrafish lens.. Invest. Ophthalmol. Vis. Sci. 2014;55(13):3572.
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Cell differentiation in the lens may lead to slowed turnover of phospholipids (PL). We have used a zebrafish model of lens development to investigate the change in PL composition of the lens with aging and to determine PL localization within the lens and eye.
Zebrafish were euthanized at 2 and 12 months of age. Lens were homogenized and the lipids extracted by the Bligh-Dyer method. The extract was infused into an AB Sciex 5600 TripleTOF mass spectrometer to collect MSMS data at successive unit masses on ions over the range from 200-1200 m/z (mass-to-charge ratio) in the negative and positive modes. LipidView™ was used to deconvolute the data. Eyes were also collected and embedded in 10% gelatin to obtain 16 μm frozen sections that were transferred to conductive glass slides. Matrix (positive spectra, diaminonaphthalene; negative spectra, dihydroxybenzoic acid) was applied to the section by vacuum sublimation. Lipids in the coated section were visualized using imaging mass spectrometry (IMS) with a Bruker Daltoniks Autoflex TOF/TOF. Distributions of PLs were obtained using Fleximaging™ software.
The principal PLs in the lens in the positive mode were phosphatidylcholines (PC) and sphingomyelins. Each was significantly (p <0.001) increased (per mg tissue) in 12-month old compared to 2-month old fish). In the negative mode, only PC and phosphatidylinositols (PIs) were significantly increased (p <0.001 and p<0.003, respectively) by aging. IMS of putative PL ions (m/z 718.69, 764.51 and 792.75) revealed that they were confined to a thin layer in the outer cortex of the lens, with none in the inner cortex or nuclear region.
This study has revealed that PCs and PIs are significantly increased by aging in zebrafish lens. Furthermore, IMS data from frozen sections of the lens have demonstrated that putative intact PLs are absent from the inner cortical and nuclear regions of the lens suggesting differential membrane function within the lens.
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