Oka H
Department of Neurosurgery, Teikyo University School of Medicine, Tokyo, Japan.
No To Shinkei. 1989 Jun;41(6):575-81.
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)
由于早期报告中仅在哺乳动物脑内检测到少量黄嘌呤氧化酶（XO）活性，因此一直认为脑内AMP降解的主要终产物是次黄嘌呤。然而，我们最近的实验研究表明，在局灶性缺血或冷损伤的大鼠脑内存在尿酸。在相同实验条件下，已发现黄嘌呤氧化还原酶抑制剂别嘌呤醇可显著抑制尿酸生成。这些结果表明，尿酸是由损伤脑组织中的次黄嘌呤通过酶促反应生成的。本实验的目的是证明脑组织中存在黄嘌呤氧化还原酶活性。在断头后立即或24小时测量大鼠脑组织中的黄嘌呤氧化还原酶活性。在戊巴比妥麻醉下，用冷生理盐水经心脏灌注冲洗血液后，将20只Sprague-Dawley大鼠断头处死。在断头缺血后立即或24小时，取出前脑并在6 ml冰冷的含1 mM苯甲基磺酰氟、0.3 mM乙二醇双四乙酸和10 mM二硫苏糖醇的0.05 M磷酸钾缓冲液（pH 7.8）中匀浆。匀浆液在100,000 g下离心60分钟，然后将上清液在0.05 M磷酸钾缓冲液（pH 7.8）中透析过夜。将每份透析上清液（样品）的等分试样与含NAD的标准黄嘌呤溶液在37℃反应15分钟，以测量黄嘌呤脱氢酶（XDH）和XO的联合活性。为了测量XO，使用不含NAD的标准黄嘌呤溶液。（摘要截短于250字）
