Comparison of the Beckman TL-100 ultracentrifuge with the Amicon Centrifree Micropartition filters to determine protein-free phenytoin concentrations.
Non-protein-bound phenytoin levels obtained by ultracentrifugation (UC) with the Beckman TL-100 desk-top ultracentrifuge were compared with those obtained by ultrafiltration (UF) using the Amicon Centrifree Micropartition device. Total phenytoin concentrations were measured by an enzyme multiplied immunoassay technique (EMIT) adapted to a Cobas Bio centrifugal analyzer, whereas free levels were estimated by a radiometric assay. The following conditions for UC were established: 350,000 g for 1 h, sample volume 250 microliter. From three pooled human plasmas containing 18.8, 44.4, and 73.9 microM total phenytoin, the following percentages of free phenytoin levels were obtained with UF: 10.4 +/- 0.1 (SD), 10.7 +/- 0.6, and 11.1 +/- 0.1%. The corresponding figures with UC were 10.8 +/- 0.9, 11.4 +/- 0.4, and 12.0 +/- 0.6%, respectively. Within-sample precision showed a coefficient of variation between 3.5 and 8.0% for UC, with UF corresponding values between 1.0 and 5.6%. Free phenytoin levels in 19 patients undergoing phenytoin therapy were between 6.8 and 19.3% with UF (mean 10.1 +/- 3.0%) and 8.7 and 23.9% (mean 11.9 +/- 3.5%; p less than 0.001 by paired t test) with UC. Least-squares linear regression analysis resulted in the following equation: percentage free phenytoin by UC = 1.11 x % free phenytoin by UF - 0.70 (r = 0.935, p less than 0.001). The results indicate that within certain reservations UC under the conditions tested may be an alternative to UF or equilibrium dialysis.
Department of Clinical Pharmacology, University of Berne, Switzerland.
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D001798:Blood Proteins / Q000378:metabolism; D005374:Filtration; D006801:Humans; D010672:Phenytoin / Q000097:blood*; D011485:Protein Binding; D014461:Ultracentrifugation / Q000295:instrumentation
D001798:Blood Proteins; D010672:Phenytoin