Advanced Glycation End-products

Advanced Glycation End-products are hard, brown solids that form when a sugar, like glucose or ribose, cross-links with a protein in various parts of your body. These substances have negative effects on many parts of the body, they can cause blood clots in the vascular system or heart attack if formed in the heart valves. Usually this reaction is very slow and involves long lived proteins like collagen. However when someone is a hyperglycemic, like someone suffering from diabetes, the reaction can proceed much more quickly because of the high concentrations of sugar in the body. 

There is a large body of research on the formation of AGEs in the blood stream but not a lot on their formation in the eye. We know that they form in the vitreous humor of the eye and can enter into the lens. The little brown specks that form can block light from reaching the retina and can cause partial blindness. The vitreous humor is the clear, gel-like fluid in the eye that gives it it's shape and allows light to reach the retina unimpeded. This is the location where the formation of AGEs takes place.

In order to run tests for the detection of AGEs we need to first dissect bovine eyes and extract the vitreous humor. To do this fifty bovine eyes were ordered from a slaughterhouse in Aurora Illinois. The eyes received were on the small side and appear to have been taken from yearling cows rather than full grown adults. The eyes were dissected by first removing the excess tissue from around the eye with scissors. After this the eye is secured to the dissection tray with a pin and a cut is made with a scalpel. They eye is then picked up and a scalpel and scissors are used to completely cut around the eye cup near the pupil. The vitreous humor is allowed to fall out of the eye into the collection tray. After the Vitreous humor is collected the lens is removed from the pupil and placed into a beaker with 0.9% saline solution. Then the retina is scraped off with a soft brush and placed into another beaker with 0.9% saline solution. 

After the collection is made the vitreous is centrifuged in order to separate large contaminants for removal. these were removed with tweezers and set aside in another beaker. After the large contaminants were removed the vitreous was filtered using a metal coffee filter purchased from Walmart. After the filtration the vitreous was stored in a refrigerator overnight.

The first test we did was an absorption spectrum in order to determine the protein concentration in the vitreous humor. This was done by reacting various concentrations of vitreous with a reagent that contains copper. The copper reacts with the peptides in the protein and turns blue this is then placed into a spectrometer. The spectrometer measures the amount of light that is absorbed by the solution. This was compared to a standard of known protein concentration to figure the concentration of protein in vitreous. This concentration was found to be 2.5 mg protein per ml of vitreous. This information is helpful when deciding how much glucose or ribose to react with the vitreous to produce AGEs for further testing.