Binding of theophylline in human serum determined by ultrafiltration and equilibrium dialysis

1 Binding of theophylline (80 mumol/l) was determined in serum from healthy subjects by equilibrium dialysis and by ultrafiltration, using [3H]-theophylline, at 22 degrees C and at different pH-values. pH was regulated by gassing with CO2 or by dialysing the serum against a phosphate buffer before use. 2 Binding of theophylline in serum was 34-38% determined by equilibrium dialysis and 41-45% determined by ultrafiltration at pH 7.4-7.5. The protein concentration in serum decreased by 12-16% during equilibrium dialysis and increased by 20% during ultrafiltration. The intersubject variation in binding was small. 3 Binding of theophylline in serum was pH-dependent with 25-30% bound at pH 7.0 and 58-60% bound at pH 8.1-8.3. Binding was significantly correlated to the fraction of ionized theophylline. 4 The binding of theophylline in normal human serum is about 35-40% at pH 7.4 and 22 degrees C. The difference in binding observed between equilibrium dialysis and ultrafiltration may be explained by the opposite changes in protein concentration during the experiment. 5 Control of pH is necessary to obtain physiologically relevant data on drug binding in serum.