Yamamoto T, Moriwaki Y, Takahashi S, Nasako Y, Yamakita J, Hiroishi K, Higashino K
Third Department of Internal Medicine, Hyogo College of Medicine, Japan.
Anal Biochem. 1995 May 1;227(1):135-9. doi: 10.1006/abio.1995.1262.
A high-performance liquid chromatographic method was developed for the determination of plasma purine nucleoside phosphorylase activity. In this method, the reaction mixture consisted of 15 microliters of plasma and 285 microliters of 50 mM phosphate buffer (pH 7.4) containing 3.8 mM inosine and 0.15 mM 2-(3-cyano-4-isobutoxyphenyl)-4-methyl-5-thiazolecarboxylic acid (strong xanthine oxidase inhibitor). After the reaction, the hypoxanthine produced was monitored to express plasma purine nucleoside phosphorylase activity. By this method, the activity of purine nucleoside phosphorylase was easily determined even with a small-volume plasma sample and despite its low activity in plasma. In addition, plasma purine nucleoside phosphorylase activity can be accurately determined even if the plasma is turbid. As a result, we were able to measure plasma purine nucleoside phosphorylase activity in patients with gout or asthma and healthy subjects, whereby it was demonstrated that plasma purine nucleoside phosphorylase activity was higher in patients with asthma than in either healthy subjects or patients with gout.
建立了一种高效液相色谱法用于测定血浆嘌呤核苷磷酸化酶活性。在该方法中，反应混合物由15微升血浆和285微升含3.8毫摩尔肌苷及0.15毫摩尔2-(3-氰基-4-异丁氧基苯基)-4-甲基-5-噻唑羧酸（强力黄嘌呤氧化酶抑制剂）的50毫摩尔磷酸盐缓冲液（pH 7.4）组成。反应后，监测产生的次黄嘌呤以表示血浆嘌呤核苷磷酸化酶活性。通过该方法，即使使用小体积血浆样本且血浆中该酶活性较低，也能轻松测定嘌呤核苷磷酸化酶活性。此外，即使血浆浑浊，也能准确测定血浆嘌呤核苷磷酸化酶活性。结果，我们能够测量痛风或哮喘患者及健康受试者的血浆嘌呤核苷磷酸化酶活性，由此证明哮喘患者的血浆嘌呤核苷磷酸化酶活性高于健康受试者或痛风患者。
