Researchers at Columbia University have synthesized large quantities of a derivative of vitamin A that accumulates with age in human eyes and may contribute to the onset of age-related macular degeneration.
The Columbia team also was able to isolate and quantify compounds from individual donor eyes, including the vitamin A derivative, called A2E. Quantification of A2E in human cells could allow for a direct correlation of the amount of this material with macular degeneration. The work appears in the Dec. 8 issue of the Proceedings of the National Academy of Sciences.
The new synthesis is critical to biological and chemical experiments needed to pinpoint the relationship between A2E and age-related changes in the retina, the scientists say. The synthesis uses simple starting materials and can provide a 50 percent yield of A2E, an enormous increase over previous methods. Now, laboratories at Columbia and elsewhere will be able to make enough of the compound to forge ahead in the quest to understand the disease. The Columbia team already has developed several hypotheses describing the role of A2E in macular degeneration and is proceeding with experiments to refine their understanding.
"Although we are not certain that A2E is implicated in macular degeneration, it is certainly a leading candidate," said Craig Parish, a postdoctoral chemistry researcher at Columbia and first author of the work. Other collaborators, all at Columbia, were Masaru Hashimoto, postdoctoral chemistry researcher; Koji Nakanishi, Centennial Professor of Chemistry, and Janet Sparrow and James Dillon, associate professors of ophthalmology at Columbia's College of Physicians & Surgeons.
Age-related macular degeneration, or AMD, is believed to cause severe visual impairment or blindness in approximately 1.7 million of the 34 million Americans over the age of 65 and is the leading cause of blindness in that age group, according to the National Eye Institute, a division of the National Institutes of Health. As the population ages, the problem is expected to worsen.
The sensitive macula, located at the center of the retina, provides the visual acuity necessary for driving, reading and other everyday tasks. With the onset of AMD, there is a degeneration of the retina's rods and cones, cells that detect light. One type of AMD, called the "dry" form, afflicts 90 percent of those with AMD and leads to a worsening blurriness of central vision over periods up to 20 years. A second type, called "wet," occurs when new blood vessels grow beneath the retinal pigment epithelial cells and leads to a more rapid decrease in vision. In some cases, wet AMD can be treated with laser surgery to destroy the affected areas.
A new line of research on macular degeneration was opened when scientists found a fluorescent orange pigment that accumulates in retinal pigment epithelial cells, or RPE cells, that line the retina and support the eye's photoreceptive rods and cones. Several years ago, ophthalmologist Graig Eldred of the University of Missouri-Columbia, using donor eyes, isolated a tiny amount - millionths of a gram - of the substance. In 1996, Naomi Sakai, a postdoctoral researcher at Columbia, and Professor Nakanishi found the exact structure of the pigment, which turned out to be two molecules of retinal, a form of vitamin A, joined to ethanolamine by a pyridinium group; they named the compound A2E. The chemists developed a synthesis using compounds available in the body but for a given quantity of reactants yielded less than one percent A2E.
Vitamin A is among the essential pigments for building the retina, and the researchers concluded that useful forms of vitamin A were somehow becoming available for A2E formation. In the rods and cones that line the back of the eye, the key molecule that detects light is rhodopsin. It consists of opsin, a stable protein, and retinal, a form of vitamin A that can absorb a photon of light and, in so doing, change its shape and send a nerve signal to the brain.
Rods and cones shed parts of themselves every day in an ongoing process of renewal. One job performed by RPE cells is to take up and digest this cellular debris. As the RPE cells chew away at the rods and cones, hypothesize the Columbia researchers, they accumulate A2E that has formed from retinal, from digested rhodopsin, and ethanolamine, a component of the cell membrane. The combination of retinal and ethanolamine is precisely what the Columbia chemists mixed to obtain A2E, indicating they may have mimicked the process that takes place in the eye.
One of the questions the researchers have not yet answered is whether A2E forms before or after the RPE takes up cellular debris. Answering that question will help pinpoint how and where the vitamin A derivative is created.
What exactly A2E does to the eye is still a topic of debate. The simple fact of its buildup in RPE cells over time could be all that's needed to impair vision, since the rods and cones depend on RPE cells for a number of functions. But the A2E molecule also has detergent-like properties, and could be poking holes through the surfaces of RPE cells, destroying them. The Columbia scientists also identified a slightly modified structure, or isomer, of A2E, which they named iso-A2E. Previous results from Dr. Dillon's laboratory show that light induces the formation of free radicals from these two pigments. The interconversion of the two forms of A2E could generate free radicals, which could then react with cellular components, rendering them useless.
Researchers in the laboratory of Dr. Sparrow in the Department of Ophthalmology at Columbia are conducting experiments to determine the effects of A2E on RPE cells grown in culture. The researchers also are investigating how A2E is synthesized in the eye, and are planning experiments with labeled A2E to identify its specific cellular targets.
"Our overall goal is to understand the effects of A2E on cell function," Dr. Parish said. "Does it cause AMD directly" What are the chemical and biochemical bases for the disease" And does that knowledge lend itself to measures that could prevent or cure the disease?"
The work was supported by a National Institutes of Health grant to Professor Nakanishi, an NIH postdoctoral fellowship to Dr. Parish and an unrestricted grant from Research to Prevent Blindness to Columbia's Department of Ophthalmology.
This document is available at http://www.columbia.edu/cu/pr/. Working press may receive science and technology press releases via e-mail by sending a message to rjn2@columbia.edu.
Journal
Proceedings of the National Academy of Sciences