Spectrum Analysis

Myoglobin Spectrum

In the eye the vitreous humor is clear to allow light to pass through unimpeded. However, after collection we noticed that the sample we had collected had a pink tint to it even after filtration. This indicated that there was some foreign substance dissolved in the vitreous. The prime suspect as to what this could possibly be was hemoglobin from blood vessels in the eye, or myoglobin from the muscle tissue that surrounds the eye. In order to attempt to identify the substance we ran various UV-vis spectrum tests. These tests use light to test both absorption, emission, or excitation of the substance being tested. We compared these spectra to the spectra of hemoglobin and myoglobin. 

Hemoglobin Spectrum

The tests we ran were with raw vitreous that we had not altered in any way placed into three different spectrometers. These spectrometers gave us the following spectra for the vitreous humor. 

These initial spectra show that the vitreous humor will be excited at 410 nm. This dictated what wavelength will be used for the excited spectra.

This shows the absorption spectrum in more detail noting where the peaks are on the spectrum. These peaks were compared to the spectra of hemoglobin and myoglobin and it was noted that there were similar peaks in the vitreous spectrum and the hemoglobin spectrum the peak at 410 matches the hemoglobin spectrum. It was noted that the myoglobin spectrum changed based on the pH of the myoglobin solution. We then measured the pH of the vitreous using an electronic pH meter that had been standardized using a buffer of 4.00 pH and one of 10.07 pH and noted the pH to be 8.37. We then noted that the vitreous peaked at 540 nm and around 580-590 nm which matched the myoglobin as well. After running the absorption tests we ran emission and excitation spectrum tests in order to have a baseline to compare to when we run the same tests after the formation of AGEs. 

Vitreous Emission Spectrum

Vitreous Excitation

 These final two tests will be used as a baseline when we run the same tests with the AGEs.

Speculations

Unfortunately the spectrometer is not able to be used as a diagnostic tool for what is in the substance. This is due to the fact that there are many substances that fluoresce at similar wavelengths. In order to go beyond simple speculation on whether or not hemoglobin or myoglobin is present in the sample we will need to run a mass spectrometer test in a gas spectrometer. Gas spectrometers cannot run tests on non-volatile substances and amino acids (proteins) are non-volatile. This means that we will have to derivative the vitreous to run these tests. This means breaking down the proteins into their base forms using a chemical called N-methyl-N-(t-butyldimethylsilsilyl)trifluoroacetamide (yeah that's the name) or MTBSTFA. This compound will break down the amino acids in the vitreous and allow gas spectrometer tests to be run. 

The vitreous humor is mostly made up of water (99%) but also contains collagen fibers. These proteins are much easier to derive using a collagenase to digest the fibers down to their base amino acids. This is combined with a borate buffer made with a solution of 20 milli-molar sodium borate that was made by dissolving 7.63 grams of sodium borate into 1 liter of water. The digestion has to occur overnight and we do not have the chemicals required for the amino acid derivation so the gas spectrometer tests will have to wait until later in the week. 

Until then we can only speculate that hemoglobin and/or myoglobin has been dissolved into the vitreous. This speculation will need to be confirmed before further testing can be run on AGEs. Until then we will be making detergents that will be used to dissolve the AGEs to be able to obtain spectra to compare to our baseline shown here. 

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.