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Rong Wen, Ziqiang Guan, Shuliang Jiao, Byron L Lam, Yiwen Li; Identification of low-density lipofuscin in human retinal pigment epithelial (RPE) cells. Invest. Ophthalmol. Vis. Sci. 2016;57(12):6545. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Lipofuscin and A2E accumulate in human RPE with age and this accumulation is implicated in the pathogenesis of age-related macular degeneration. The present work examines the biochemical and fluorescence properties of low-density lipofuscin-like granules isolated from human RPE.
Human eyes (donor age 70-90 years) were obtained from Florida Lions Eye Bank. The anterior section and the retina of an eye were carefully removed. The RPE cells were collected in PBS (phosphate buffered saline). The cell suspension was centrifuged at 8,000 g for 10 min to remove the supernatant and the cells were stored at -80°C. To isolate the lipofuscin granules, the cells were homogenized, resuspended in 0.3 M sucrose, and layered onto a discontinuous sucrose gradient of 3 different concentrations: 2, 1.4, and 1.2 M. Four distinguished bands were obtained after centrifugation at 103,000 g for 1 hr, Layer 0 (L0) at the interface of 0.3 and 1.2 M; L1 between 1.2 and 1.4 M, L2 between 1.4 and 2.0M, and M at the bottom. Each band was washed with H2O, centrifugation at 16,800 g for 10 min, and the supernatant was removed. Sample was air dried until the reflection of the sample surface disappeared. Each sample was then weighed to obtain its wet weight and resuspended in H2O at 50 µg/µl. The fluorescence spectra in L0 and L1 were examined by a commercial spectrometer together with custom-made excitation delivering and fluorescence detecting optics. The concentrations of A2E of L0 and L1 were measured by normal phase liquid chromatography/mass spectrometry (LC/MS) using electrospray ionization (ESI) in the positive ion mode.
Both L0 and L1 contain high amount of A2E and iso-A2E (both detected by MS at m/z 592.4), with the content in L0 being more than 5 times of that in L1. The fluorescence spectra of L0 and L1 with 488 nm excitation are identical, but the intensity of fluorescence emission in L0 is 10 times of that in L1.
L1 has been conventionally regarded as to contain most of the lipofuscin granules in the RPE. Our results, however, showed that low-density L0 granules have identical fluorescence properties as L1 and with much higher intensity. In addition, the A2E content in L0 granules is much higher than that in L1 granules. Thus, the previously overlooked L0 granules should be classified as “low-density lipofuscin”, and included in the study of the composition and toxicity of lipofuscin.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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