Determination of acetazolamide in human serum by enzymatic assay

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%.