Factors influencing the protein binding of azosemide using an equilibrium dialysis technique

Various factors most likely to influence the plasma protein binding of azosemide to 4% human serum albumin (HSA) were evaluated using equilibrium dialysis at the initial azosemide concentration of 10 micrograms mL-1. It took approximately 8 h of incubation to reach an equilibrium between 4% HSA and isotonic phosphate buffer of pH 7.4 containing 3% dextran (the 'buffer') using a Spectra/Por 2 membrane (molecular weight cut-off, 12000-14000) in a water bath shaker kept at 37 degrees C and a rate of 50 oscillations min-1. Azosemide was fairly stable both in 4% HSA and in the 'buffer' for up to 24 h. The binding of azosemide to 4% HSA was constant (95.5 +/- 0.142%) at azosemide concentrations ranging from 5 to 100 micrograms mL-1. However, the extent of binding was dependent on HSA concentration: the values were 88.4, 91.0, 92.2, 94.2, 94.9, 94.9, and 94.9% at albumin concentrations of 0.5, 1, 2, 3, 4, 5, and 6%, respectively. The binding was also dependent on incubation temperature: the binding values were 97.0, 94.9, and 94.9% when incubated at 6, 28, and 37 degrees C, respectively. The binding of azosemide was also influenced by buffers containing various chloride ion concentrations and buffer pHs. The binding values were 95.3, 94.9 and 93.6% for the chloride ion concentrations of 0, 0.249, and 0.546%, respectively, and the unbound values were 6.8, 5.1, 3.8, 3.4, and 3.3% for buffer pHs of 5.8, 6.4, 7.0, 7.4, and 8.0, respectively. The binding of azosemide was independent of the quantity of heparin (up to 40 U mL-1), AAG (up to 0.16%), sodium azide (NaN3, up to 5%), its metabolite, M1 (up to 10 micrograms mL-1), and anticoagulants (EDTA and citrate).