Jeffrey Swallow and Dr. Heidi Vollmer-Snarr, Chemistry
Age Related Macular Degeneration (AMD) is the leading cause of vision loss in developed countries. The accumulation of lipofuscin in the eye causes death of the retinal pigment epithelium (RPE) leading to blindness (1,2). The lipofuscin chemical A2E has been shown to have cytotoxic effect in RPE, yet much of lipofuscin has remained uncharacterized (3). We have shown that other compounds exist in lipofuscin through preliminary mass spectroscopy (MS) and high-pressure liquid chromatography (HPLC) data collected from lipofuscin samples. Our lab is working to first characterize these lipofuscin compounds and then we will also study their oxidative products in order to better understand the mechanism of AMD and develop improved treatment.
The molecule that I have been working with is called A2-Spermine (A2-Spm). A2-Spm is created by reacting spermine with 2 molecules of all trans-retinal. The characterization of A2-Spm through MS, HPLC and NMR will create a standard which will be compared to samples from the Vollmer-Snarr lab of lipofuscin. If A2-Spm is detected in either of these samples then biological testing will be performed looking for possible cytotoxic effects.
I have already synthesized the compound A2-Spm verifying its presence with MS having shown a characteristic mass over charge (m/z) peak of 734. I then separated and purified the sample using a silica column and work is currently being done on HPLC to further collect and purify the unique peaks in the A2-Spm sample. In the current HPLC method set being used A2-Spm appears to come off the column at about 6 minutes. Samples from lipofuscin extracted from human eyes donated by the Moran Eye center in Salt Lake City have also shown an unidentified peak near six minutes which seems to correlate to this A2-Spm sample, not only in retention time but also in UV spectra. Also MS from the lipofuscin samples has shown a peak at 734 which correlates to the m/z of A2-Spm. Once the A2-Spm is collected, a co-injection with the lipofuscin will be performed in order to confirm the presence of A2-Spm in lipofuscin. Identifying a new component in lipofuscin would be significant in the field and would be a publishable result. More work will be done with HPLC, separating and isolating other peaks of the same sample to further search for new compounds that may be contained in lipofuscin, including A4-Spermine, which appears to elute from the column at about 26 minutes in the current method set. All samples collected from HPLC will be submitted to MS and once samples are collected in sufficient volumes and purity, NMRs will also be taken in order to more fully characterize the compound.
After the compound has been found in lipofuscin samples, work will also be done to study oxidation products that may be formed by blue light irradiation. Biological studies of these compounds will then begin, looking for cytotoxic effects on cells.
Work is still needed on this project as elucidation of A2-Spm proved difficult, as many challenges arose particularly with purification through silica column and HPLC. A sample that was both pure and concentrated enough to obtain a valid NMR or a clear MS was not obtained during my time at BYU. Hopefully in the future new students can carry the project further to complete the elucidation of A2-Spm.
- Young, R. W., Solar radiation and age-related macular degeneration. Surv Ophthalmol. 32, 252-269, 1988
- Haralampus-Grynaviski, N. M.; Lamb, L. E.; Clancy, C. M. R.; Skumatz, C.; Burke, J. M.; Sarna, T.; Simon, J. D., Spectroscopic and morphological studies of human retinal lipofuscin granules, Proc. Natl. Acad. Sci., 100, 3179-3184, 2003
- Klein, R.; Klein, B. E. K.; Linton, K. L. P., Prevalence of age-related maculopathy. The Beaver Dam Eye Study. Ophthalmol., 99, 933-943, 1992.