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

Factors that influence the plasma protein binding of bumetanide were evaluated using equilibrium dialysis. It took approximately 12 h of incubation to reach an equilibrium between plasma and isotonic phosphate buffer of pH 7.4 containing 3% dextran using a Spectrapor 2 membrane (mol. wt cut-off = 12,000-14,000) in a water-bath shaker kept at 37 degrees C and at a rate of 50 oscillations per min. Bumetanide was fairly stable in both 4% human serum albumin (HSA) and in the isotonic phosphate buffer of pH 7.4 for up to 24 h. The binding of bumetanide to 4% HSA was constant (87.5 +/- 1.73%) at bumetanide concentrations ranging from 0.1 to 100 micrograms/ml. The extents of binding were 72.0, 83.3, 88.5, 90.2, 91.3 and 91.4% at albumin concentrations of 0.5, 1, 2, 3, 4 and 5 g/100 ml, respectively, and increased with a decrease in incubation temperature; the values bound were 94.6, 90.3 and 89.3% when incubated at 4, 22 and 37 degrees C, respectively. The binding of bumetanide was independent of the buffer composition used, the quantities of AAG (up to 0.32%), heparin (up to 40 units/ml), sodium azide (up to 0.5%) and anticoagulants (EDTA, heparin and citrate). The free fraction of bumetanide in rabbit plasma (2.91%) was significantly higher than in humans (1.98%) or rats (1.85%).