Greene I M, Kenny A D
Department of Pharmacology, Texas Tech University Health Sciences Center, Lubbock 79430.
J Pharmacol Toxicol Methods. 1992 Nov;28(3):159-66. doi: 10.1016/1056-8719(92)90078-f.
Carbonic anhydrase (CA) inhibitors, such as acetazolamide (AZ), formerly used as diuretics, still play a role in the treatment of glaucoma, epilepsy, and altitude sickness. There is now hard evidence from both in vitro and in vivo studies in animals that carbonic anhydrase plays a vital function in bone loss. Acetazolamide blocks bone resorption in these experimental models. We have postulated that acetazolamide has potential for the treatment of human conditions associated with bone loss. In preparation for a clinical trial of acetazolamide's effectiveness in this regard, we developed an enzymatic method for determining the total concentration of acetazolamide in human serum. Acetazolamide is stripped from binding to serum proteins by adding 10(-6) M salicylic acid and adjusting the pH to 2.5, followed by ultrafiltration through a membrane (10 kD cutoff). The latter permits the free acetazolamide to enter the filtrate but retains any carbonic anhydrase (31 kD) which may contaminate the serum from hemolysis. The carbonic anhydrase inhibitory activity in the filtrate, representing the acetazolamide, is determined in a carbonic anhydrase assay using acetazolamide as the standard. Recoveries of acetazolamide added to human serum ranged from 83% to 94% depending on the concentration. Precision, as judged by the coefficient of variation, was 10.5%.
碳酸酐酶（CA）抑制剂，如乙酰唑胺（AZ），曾被用作利尿剂，目前在青光眼、癫痫和高原病的治疗中仍发挥着作用。现在，动物体内和体外研究均有确凿证据表明，碳酸酐酶在骨质流失中起着至关重要的作用。在这些实验模型中，乙酰唑胺可阻止骨吸收。我们推测，乙酰唑胺在治疗与骨质流失相关的人类疾病方面具有潜力。为准备关于乙酰唑胺在这方面有效性的临床试验，我们开发了一种酶法来测定人血清中乙酰唑胺的总浓度。通过加入10（-6）M水杨酸并将pH值调至2.5，使乙酰唑胺从与血清蛋白的结合中解离出来，随后通过膜（截留分子量10 kD）进行超滤。后者可使游离的乙酰唑胺进入滤液，但保留可能因溶血而污染血清的任何碳酸酐酶（31 kD）。以乙酰唑胺为标准，在碳酸酐酶测定中测定滤液中代表乙酰唑胺的碳酸酐酶抑制活性。添加到人血清中的乙酰唑胺回收率根据浓度不同在83%至94%之间。以变异系数判断，精密度为10.5%。
