Eyes were harvested 48 hours after the surgeries were performed.
For each of the 10 eyes used in the CAT assay, samples were analyzed
from four locations. The filtration site specimen consisted of a
rectangular block of conjunctiva, Tenon’s capsule, and sclera
measuring 8 mm concentric with the limbus and 6 mm radially posterior
to the limbus. An identical specimen 180° opposite the sclerostomy
site was also harvested. The third specimen consisted of the entire
clear cornea, and the fourth specimen was the entire iris and ciliary
body complex. Tissue samples were stored at −80°C until measured for
CAT activity using the radioactive enzymatic protocol described by the
manufacturer’s technical bulletin (No. 84; Promega, Madison, WI).
Briefly, tissue samples were weighed, homogenized in 1 ml of lysis
buffer (250 mM Tris [pH 8.0], 5 mM EDTA, 0.02% sodium azide, 1 mM
paraoxon, and 10 μg/ml aprotinin), and centrifuged at
17,000g for 10 minutes at 4°C. Aliquots of the supernatant
solutions (100 μl) were added to 1.5-ml centrifuge tubes containing
25 μl of substrate solution creating final concentrations of 24 μM 14C-chloramphenicol (50 mCi/mmol; duPont NEN
Research Products, Beverly, MA) and 214 μM n-butyryl
coenzyme A. After incubation overnight at 37°C, 1.25 ml xylene was
added to the tube, and the reaction was vortexed for 1 minute. The
xylene phase containing the butyrylated 14C-chloramphenicol reaction product was
transferred to a new 1.5-ml centrifuge tube and extracted three times
with 200 μl of 250 mM Tris (pH 8.0). Levels of radioactivity were
measured in 500 μl of the xylene phase using liquid scintillation
counting, and the amount of CAT activity present in the sample was
calculated using the best-fit equation generated from a standard curve
of CAT activity, which ranged from 0.01 mU to 100 mU. Samples were
assayed in triplicate and the average CAT units were expressed as
milliunits per milligram tissue. Levels of CAT activity in the tissue
samples tissues were compared using analysis of variance (ANOVA) and
Tukey’s post hoc test with P = 0.05 considered
statistically significant.