Munafo A, McDonagh A F, Smith P C, Benet L Z
Department of Pharmacy, School of Pharmacy, University of California, San Francisco 94143-0446.
Pharm Res. 1990 Jan;7(1):21-7. doi: 10.1023/a:1015823206607.
Tolmetin glucuronide (TG), extracted and purified from human urine, was incubated with albumin in vitro. The degradation profile and irreversible binding to protein were investigated and kinetic parameters calculated. Standard conditions were as follows: TG, 30 micrograms/ml; human serum albumin (HSA), 3%; pH 7.45; 37 degrees C. Lower pH enhanced TG stability and reduced both the extent and the rate of irreversible binding. HSA also increased TG stability, compared to protein-free buffer, but the opposite was observed with bovine serum albumin (BSA). With BSA, irreversible binding was much less, but the rate of adduct formation was the same as with HSA. Essentially fatty acid free HSA behaved similarly to HSA. Preincubation of HSA with warfarin, or diazepam, or an excess of tolmetin, did not influence irreversible binding significantly. In buffer, acyl migration led predominantly to one isomer. This isomer bound irreversibly to HSA, although more slowly and to a lesser extent than the beta 1-isomer. Incubation of TG with poly-L-lysine also resulted in irreversible binding but to a lesser extent than with HSA. Our results suggest that there is more than one binding mechanism, with the preferential pathway a function of the isomers present and the experimental conditions.
从人尿中提取并纯化得到的托美丁葡糖醛酸酯（TG），在体外与白蛋白一起孵育。研究了其降解情况以及与蛋白质的不可逆结合，并计算了动力学参数。标准条件如下：TG，30微克/毫升；人血清白蛋白（HSA），3%；pH 7.45；37摄氏度。较低的pH值增强了TG的稳定性，同时降低了不可逆结合的程度和速率。与无蛋白缓冲液相比，HSA也提高了TG的稳定性，但用牛血清白蛋白（BSA）时观察到相反的情况。对于BSA，不可逆结合少得多，但加合物形成的速率与HSA相同。基本不含脂肪酸的HSA表现与HSA相似。将HSA与华法林、地西泮或过量的托美丁预孵育，对不可逆结合没有显著影响。在缓冲液中，酰基迁移主要导致一种异构体。这种异构体与HSA不可逆结合，尽管比β1异构体结合得更慢且程度更低。TG与聚-L-赖氨酸孵育也会导致不可逆结合，但程度比与HSA孵育时小。我们的结果表明，存在不止一种结合机制，优先途径是所存在的异构体和实验条件的函数。
