Nonenzymatic glycation-type cross-links formed as the result of the attachment of the carbonyl group of glucose, followed by a ketoamine rearrangement. The cross-links formed because of the Maillard-type reactions that followed then yielded fluorophores characteristic of a reaction of a sugar with a protein. Therefore, fluorescence of a papain digest supernatant in our experiment is an indicator of nonenzymatic cross-link content. There is a linear relationship between fluorescence and increasing cross-link content.
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Tissue samples were cut from globe tissue adjacent to the medial and lateral scleral discs, weighed, and placed in sealed plastic bags at −80°C for no longer than 2 weeks. Only samples weighing between 10 and 25 mg were used. Before the execution of the assay, the tissue was pulverized to a fine powder to encourage a thorough reaction with the papain. This was accomplished by transferring the tissue to a test tube, submerging that tube in liquid nitrogen for 2 minutes to further cool the tissue into a mechanically brittle state, transferring the tissue to a milling capsule containing a pair of 12-mm-diameter stainless steel balls (also previously submerged in liquid nitrogen for 2 minutes), cyclically displacing the milling capsule and its contents in a milling machine (MM301 ball mill; Retsch Corporation, Haan, Germany) for 3 minutes at 30 Hz, and weighing a portion of the pulverized tissue for use in the assay.
To each pulverized tissue sample was added 1 mL of a 10 mM solution of dithiothreitol in a digestion buffer (0.1 M Na acetate/2.4 mM disodium EDTA). To each tissue sample was then added 20 μL of a 1 mg papain/1 mL digestion buffer solution. The tissue was incubated at 60°C for 12 hours, and then 20 μL of the papain solution was added again. After 12 more hours of incubation, the tubes were centrifuged at 10,000 rpm for 12 minutes. The supernatant was removed and was used to perform fluorescence measurements.
To measure fluorescence, a 100-μL aliquot of each papain supernatant was removed to a black ELISA plate, and fluorescence was measured in the plate reader at an excitation wavelength of 370 nm with emission sensitivity set at 440 nm because these wavelengths give the greatest resolution in detecting nonenzymatic cross-link residuals.