Since only little
xanthine oxidase (XO) activity in mammalian brain was detected in earlier reports, the major end product of
AMP degradation in the brain has been believed to be
hypoxanthine. Our recent experimental study however, has indicated the presence of
uric acid in the rat brain subjected to focal
ischemia or
cold injury.
Allopurinol, a
xanthine oxidoreductase inhibitor, has been found to markedly suppress the
uric acid production in the same experimental settings. These results suggested that
uric acid is generated from
hypoxanthine by enzymatic reaction in injured brain tissue. The aim of this experiment is to prove the existence of
xanthine oxidoreductase activity in brain tissue.
Xanthine oxidoreductase activity in rat cerebral tissue was measured immediately or at 24-hour after
decapitation. Under
pentobarbital anesthesia, twenty Sprague-Dawley rats were killed by
decapitation following washout of the blood by trans-cardiac perfusion with cold physiological saline. Immediately or after 24 hours of
decapitation ischemia, the forebrain was removed and homogenized in 6 ml
ice cold 0.05 M
potassium phosphate buffer (pH 7.8) containing 1 mM
phenylmethylsulfonyl fluoride, 0.3 mM
EGTA, and 10 mM
dithiothreitol. The homogenate was centrifuged at 100,000 g for 60 min and then the supernatant was dialyzed overnight against 0.05 M
potassium phosphate buffer (pH 7.8). Aliquot of each dialyzed supernatant (sample) and standard
xanthine solution with
NAD was reacted at 37 degrees C for 15 min to measure the combined activity of
xanthine dehydrogenase (XDH) and XO. For the measurement of XO, standard
xanthine solution without
NAD was used.(ABSTRACT TRUNCATED AT 250 WORDS)