Rapid in vitro conversion of fosphenytoin into phenytoin in sera of patients with liver disease: Role of alkaline phosphatase

Fosphenytoin, a phosphate ester pro drug of phenytoin, also cross-reacts with the fluorescence polarization immunoassay (FPIA) for phenytoin. We measured fosphenytoin concentrations using the FPIA kit and TDx analyzer. We prepared serum pools from normal volunteers and patients with liver disease. None of them received either fosphenytoin or phenytoin. Fosphenytoin standard solution (1 mg/ml) was prepared in water. We supplemented aliquots of normal and liver pools with known amounts of fosphenytoin and measured the concentrations at different time intervals. The conversion of fosphenytoin to phenytoin was slow in sera with normal alkaline phosphatase activities. The conversion was rapid in sera collected from patients with liver disease who also had high alkaline phosphatase activities. The observed concentrations were close to target concentrations within 0-2 min of supplementation with fosphenytoin. Surprisingly, the observed concentration then started to decline slightly but significantly with longer incubation time. In contrast, the observed concentration increased steadily in serum with normal alkaline phosphatase activity. For example, in the normal pool supplemented with 15.0 μg/ml fosphenytoin (as the phenytoin equivalent), the observed concentrations were 6.9, 7.3, 7.7, 8.3, and 9.8 μg/ml at 0-2, 10, 20, 30, and 60 min, respectively. However, in a serum pool prepared from patients with liver disease and supplemented with 15.0 μg/ml of fosphenytoin (alkaline phosphatase: 2547 U/I), the observed phenytoin concentrations were 12.9, 12.1, 11.0, 10.7, and 10.7 μg/ml at 0-2, 10, 20, 30, and 60 min, respectively. When we added alkaline phosphatase to the normal serum pool, we observed rapid conversion of fosphenytoin into phenytoin within 10 min, but the concentrations then declined with longer incubation time. However, when we repeated the experiment with protein-free ultrafiltrate, we observed rapid conversion of fosphenytoin to phenytoin, but the concentration did not decline with longer incubation time.