Phenytoin Overview—Metabolite Interference in Some Immunoassays Could Be Clinically Important: Results of a College of American Pathologists Study (2003)

The biggest landmark in the treatment of epilepsy was the discovery of phenytoin. Phenytoin was introduced in 1938 and is still the most widely used anticonvulsant drug, probably because of its nonsedative properties. It is commonly used to treat general motor (tonic-clonic, grand mal) and focal seizures and is less effective in the treatment of complex partial seizures.The mechanism of action of phenytoin is not clear. However, phenytoin stabilizes membranes in the brain (and thereby suppresses seizures) and in the heart (and hence suppresses arrhythmias). It has been suggested that phenytoin suppresses seizures by blocking posttetanic potentiation by influencing synaptic transmission. The mechanisms postulated for this effect include alteration of (a) ion fluxes associated with depolarization, (b) repolarization, (c) membrane stability, (d) calcium uptake in presynaptic terminals, and (e) sodium/potassium adenosine triphosphate–dependent ionic membrane pump.12The usual dose of phenytoin prescribed to adults is 4 to 6 mg/kg/d. Prepubertal children require somewhat higher doses (5–10 mg/kg/d) to achieve the same steady-state concentrations, owing to the greater activity of the hepatic microsomal enzyme system in this age range. Phenytoin undergoes saturation kinetics; that is, each individual has a threshold serum/plasma concentration beyond which the enzymes involved in its metabolism become “saturated.” Any further slight increase in dose can cause the serum concentration to increase out of proportion to the dose administered. When saturation occurs, the metabolism of phenytoin changes from a first-order (drug-concentration–dependent) process to a zero-order (drug-concentration–independent) process.12Phenytoin protects against seizures at serum/plasma concentrations of 10 to 20 μg/mL (40–79 μmol/L), although higher levels may be needed in some cases.2 It can precipitate seizures at concentrations greater than 40 μg/mL (>158 μmol/L).1 Because the relationship between serum/plasma concentration and clinical efficacy and toxicity is good, therapeutic drug monitoring and optimizing the dosage regimen to achieve a therapeutic level and avert toxicity is an important adjunct to therapy. The pharmacokinetics of phenytoin are shown in Table 1.Toxic side effects of phenytoin include nystagmus, dysarthria, diplopia, ataxia, and exacerbation of seizures. Furthermore, chronic use can lead to hirsutism and gum hypertrophy. Taking phenytoin during pregnancy is contraindicated and can lead to the fetal hydantoin syndrome. Folate and vitamin D deficiency may necessitate vitamin supplementations and, if uncorrected, leads to megaloblastic anemia. High concentrations lead to drowsiness, confusion, and coma.12Absorption rate and bioavailability depend on the formulation used. Approximately 90% of the oral dose is absorbed. Peak concentrations are achieved in 2 to 8 hours. For treatment of status epilepticus, phenytoin is usually administered via the intravenous route as fosphenytoin.3The major biotransformation pathway consists of metabolism to arene oxide via the cytochrome oxidase system enzyme arene oxidase. Arene oxide is spontaneously converted to 5-p-hydroxyphenyl-5-phenylhydantoin (HPPH). This pathway accounts for 60% to 80% of phenytoin elimination. Saturation of this enzyme occurs at low concentrations of phenytoin, a substrate whose Michaelis-Menten constant is low, resulting in saturation of the system usually within the therapeutic range of phenytoin. Once saturation has occurred, any further slight increase in dose results in a disproportionate increase in serum/plasma concentration with concomitant toxic side effects. The activity of this hepatic microsomal system is very age dependent, being low at 0 to 3 months of age, approximately double that of the adult from 6 months to puberty, and declining to adult values after puberty. It follows that patients approaching puberty need to be observed closely with frequent monitoring of serum/plasma concentrations and appropriate downward adjustment of dose when required. Less than 5% of the dose is excreted unchanged in the urine, with 60% to 70% being excreted as HPPH conjugated with glucuronic acid. HPPH is converted by the enzyme epoxide hydrolase to the dihydrodiol. The diol accounts for 7% to 11% of phenytoin metabolites recovered from urine.12Numerous drugs (valproic acid, salicylates, thiazides, and endogenous compounds in patients with renal failure) displace phenytoin from its binding site on albumin. Phenytoin is usually 90% protein bound. However, in renal failure, by-products and phenytoin metabolites build up in the blood and displace phenytoin from its protein-binding sites. In these cases, it is necessary to make dosage adjustments to bring the free phenytoin concentration into the therapeutic range (1–2 mg/L). In the presence of these compounds, the free fraction of phenytoin increases, placing the patient at increased risk for toxicity, and symptoms of toxicity become apparent. Free phenytoin concentrations can be measured by using either equilibrium dialysis or, more commonly, devices with molecular cutoff filters, such as the Amicon Centrifree micropartition system (Millipore Corporation, Bedford, Mass) or the Worthington Diagnostics “ultrafree” system (Worthington Diagnostics, Jacksonville, Fla). Temperature affects the degree of binding of phenytoin to albumin, with drug binding decreasing as temperature increases. Control of temperature is therefore important. A 10-degree drop in temperature (37°C–27°C) can result in an approximately 25% decrease in the free fraction.4 Because the patient's temperature is usually 37°C, it is recommended to use this temperature when assessing free phenytoin concentrations.The 3 commonly used methods of analysis for phenytoin include immunoassays, high-performance liquid chromatography, and gas-liquid chromatography. High-performance liquid chromatography and gas-liquid chromatography methods have the advantage of separating phenytoin from its major metabolite, HPPH. Some of the immunoassays cross-react with the metabolite. Nevertheless, in surveys such as the College of American Pathologists' (CAP) proficiency testing programs, by far the most laboratories are using immunoassays to monitor phenytoin concentrations in patients.5Patients with renal failure and on dialysis often receive phenytoin to treat their seizures. It would be expected that the concentration of HPPH, which is excreted renally, would be significantly elevated in these individuals. For this reason, we sought to ascertain whether the major phenytoin metabolite interfered with current phenytoin immunoassays.To evaluate whether the phenytoin metabolite HPPH cross-reacts in current immunoassay systems, Z-01 and Z-02 specimens of the CAP 2003 Z-A TDM Survey had identical concentrations of phenytoin (target 5 μg/mL [20 μmol/L]). However, Z-01 contained an additional 5 μg/mL HPPH.Table 2 shows the survey results obtained on Z-01 and Z-02 for phenytoin. The concentration of phenytoin metabolite chosen relative to the parent drug accurately reflects the expected concentration in patients with renal failure (unable to secrete the metabolite), because 60% to 80% of phenytoin is metabolized to HPPH. As can be seen from the table, both the total and free phenytoin concentrations were significantly affected by the presence of metabolite, an interference of 7% and 16%, respectively. When the interference was broken down by method, the Abbott AxSYM and Abbott TDx/TDxFLX (Abbott Diagnostics, Abbott Park, Ill) are the most significantly affected methods. The 2 methods alone account for approximately 1225 laboratories in North America or 30% of laboratories currently enrolled in the CAP proficiency testing program for phenytoin. The interference in these 2 methods is 17% to 19% for total phenytoin and 19% to 37% for free phenytoin. Errors of this magnitude could easily result in inappropriate dosage adjustments and errors in patient management. DPC Immulite (6.25%) (Diagnostic Products Corporation, Flanders, NJ) and Roche Cobas Integra (3.48% total, 4.07% free) (Roche Diagnostic Systems, Branchburg, NJ) showed a smaller degree of cross-reactivity with the metabolite, while several methods (Bayer Immuno-1, Bayer Corporation, Tarrytown, NY; Beckman Synchron RGT, Beckman Coulter Inc, Fullerton, Calif; Dade Dimension, Dade Behring, Newark, Del; and Vitros, Ortho Clinical Diagnostics, Raritan, NJ) showed minimal (<2%) interference at the concentration tested.