Unexpected suppression of free phenytoin concentration by salicylate in uremic sera due to the presence of inhibitors: Maldi mass spectrometric determination of molecular weight range of inhibitors

Salicylate displaces phenytoin from protein binding leading to an increase in free phenytoin concentration. We observed unexpected decreases in free p henytoin concentration in the presence of salicylate. Serum pools containin g no phenytoin or salicylate were supplemented with the same concentrations of phenytoin. Then to the aliquots of the individual pool, no salicylate ( control), 150, 300 and 500 mu g/ml of salicylate (therapeutic range: 15-300 mu g/ml) were added. Specimens were incubated at 37 degrees C for 2 h and after re-equilibration at room temperature for 20 min, total and free pheny toin (in the protein free ultrafiltrates) concentrations were measured usin g fluorescence polarization immunoassay on the TDx/FLX analyzer. We observe d an increase in free phenytoin concentration from 1.91 mu g/ml:(in the abs ence of salicylate) to 2.39 mu g/ml in the presence of 500 mu g/ml salicyla te (total phenytoin: 13.3 mu g/ml) in the normal pool. In sharp contrast, t he free phenytoin concentrations decreased from an initial concentration of 3.82 mu g/ml to 2.52 mu g/ml in the presence of 500 mu g/ml of salicylate (total phenytoin: 13.2 mu g/ml) in the uremic pool. We also treated the ure mic pool with activated charcoal. In the original uremic pool, the initial free phenytoin concentration was 3.05 mu g/ml and the free concentrations t hen decreased to 2.28 mu g/ml in the presence of 300 mu g/ml of salicylate. In contrast, in the charcoal treated pool, the initial free phenytoin conc entration increased from 1.61 mu g/ml to 3.23 mu g/ml in the presence of 30 0 mu g/ml of salicylate. More interestingly when uremic toxins were extract ed back from charcoal with methanol and the dry residue was added to an ali quot of normal serum, the normal serum behaved like a uremic serum and free phenytoin concentration was significantly decreased in the presence of sal icylate. When an aliquot of methanol extract was studied by Matrix-Assisted Laser Desorption Ionization Mass Spectrometry (scan up to 10,000), we obse rved no peak at molecular weight over 551, indicating that these inhibitors are small molecules. We also identified hippuric acid as one of the inhibi tors.