Abstract: Numerous basic drugs are extensively bound to alpha 1-acid glycoprotein. Fentanyl, with a pKa value of 8.43, is also a basic drug. Protein binding studies have yielded contradictory results concerning binding of fentanyl to alpha 1-acid glycoprotein. In this study we investigated time courses of serum protein concentrations and serum protein binding of fentanyl during postoperative acute phase reaction, assuming that an increase of alpha 1-acid glycoprotein is accompanied by an increase of serum protein binding, if fentanyl is extensively bound to alpha 1-acid glycoprotein. Fentanyl protein binding measurements using equilibrium dialysis can be affected by volume shifts and pH changes. Therefore, volume shifts from buffer to serum and the influence of various phosphate buffers on increasing pH due to loss of CO2 were also evaluated. METHODS. Thirteen patients with no history of renal or hepatic disease undergoing an operation with a significant acute phase reaction were studied. Preoperatively and on the first 3 postoperative days serum concentrations of alpha 1-acid glycoprotein, albumin, total protein and apolipoprotein A and B were determined by rocket immunoeolectrophoresis, biuret method and laser nephelometry, respectively. Corresponding serum protein binding of fentanyl was measured by adding 40 ng of fentanyl to 1 ml serum followed by equilibrium dialysis at 37 degrees C for 4 h. A 0.167 M phosphate buffer (pH 7.27), which gave a final pH of 7.40, was used. Volume shifts from buffer to serum were measured. Fentanyl concentration in serum before dialysis (FS) was determined by gas chromatography, and fentanyl concentration in buffer after dialysis (FB) was determined by radioimmunoassay. Serum protein binding (SPB) was calculated by the formula: SPB = (FS - FB - FB*c)/(FS - FB) where c is a correction factor. Ten randomly selected patient sera were dialyzed against four phosphate buffers of different pH values and molarities, and the serum pH at the end of equilibrium dialysis was measured. RESULTS. Postoperatively, the serum concentration of alpha 1-acid glycoprotein rose to 151% of the control value. In contrast, serum protein binding of fentanyl did not change significantly, with a slight decrease to 96% of control value. There was a significant decrease in serum concentrations of albumin (3rd postoperative day), total protein (2nd postoperative day) and apolipoprotein B (1st-3rd postoperative day) to 85%, 90% and 75% of control values, respectively. Changes in apolipoprotein A concentration were not significant. Protein binding of fentanyl did not correlate with alpha 1-acid glycoprotein and apolipoprotein A, but there was a positive linear relationship between protein binding of fentanyl and albumin, total protein and apolipoprotein B. At the end of equilibrium dialysis the mean volumes of the serum and buffer compartments were 1114 +/- 72 and 834 +/- 68 microliters, respectively. The two phosphate buffers, with pH 7.30 (0.15 M) and pH 7.27 (0.167 M), gave final serum pH of 7.42 and 7.40, respectively. CONCLUSIONS. Present findings suggest that in contrast to other basic drugs, fentanyl binding to alpha 1-acid glycoprotein is of minor importance. In agreement with the findings of former studies, protein binding of fentanyl depended on albumin, total protein and apolipoprotein B concentrations. Due to unspecific binding of fentanyl by hydrophobic interactions, a major role of albumin, which amounts to about 60% of total protein, seems to be evident. Determining fentanyl protein binding by equilibrium dialysis, volume shifts must be taken into account if calculation is based on fentanyl concentrations in plasma (serum) and buffer after dialysis, and an appropriate buffer must be used.
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