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 phenytoin concentration in the presence of salicylate. Serum pools containing 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 microg/ml of salicylate (therapeutic range: 15-300 microg/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 phenytoin (in the protein free ultrafiltrates) concentrations were measured using fluorescence polarization immunoassay on the TDx/FLX analyzer. We observed an increase in free phenytoin concentration from 1.91 microg/ml (in the absence of salicylate) to 2.39 microg/ml in the presence of 500 microg/ml salicylate (total phenytoin: 13.3 microg/ml) in the normal pool. In sharp contrast, the free phenytoin concentrations decreased from an initial concentration of 3.82 microg/ml to 2.52 microg/ml in the presence of 500 microg/ml of salicylate (total phenytoin: 13.2 microg/ml) in the uremic pool. We also treated the uremic pool with activated charcoal. In the original uremic pool, the initial free phenytoin concentration was 3.05 microg/ml and the free concentrations then decreased to 2.28 microg/ml in the presence of 300 microg/ml of salicylate. In contrast, in the charcoal treated pool, the initial free phenytoin concentration increased from 1.61 microg/ml to 3.23 microg/ml in the presence of 300 microg/ml of salicylate. More interestingly when uremic toxins were extracted back from charcoal with methanol and the dry residue was added to an aliquot of normal serum, the normal serum behaved like a uremic serum and free phenytoin concentration was significantly decreased in the presence of salicylate. When an aliquot of methanol extract was studied by Matrix-Assisted Laser Desorption Ionization Mass Spectrometry (scan up to 10,000), we observed no peak at molecular weight over 551, indicating that these inhibitors are small molecules. We also identified hippuric acid as one of the inhibitors.