Cross-Reactivity of Fosphenytoin in Four Phenytoin Immunoassays

^a Author for correspondence. Fax 508-660-4591; e-mail pradip.datta{at}chirondiag.com.

Amitava Dasgupta

Chiron Diagnostics, E. Walpole, MA 02032
Dept. of Pathol., Univ. of New Mexico, Albuquerque, NM 87106

To the Editor:

Fosphenytoin, 5,5-diphenyl-3-[(phosphonooxy)methyl]-2,4-imidazolidine-dione disodium salt (Cerebyx^®; ParkeDavis), is a phosphorylated form of the anticonvulsant drug phenytoin. Fosphenytoin itself has no pharmacological activity but is dephosphorylated in vivo by phosphorylases to the active drug, phenytoin. The elimination half-life of fosphenytoin in plasma is 8–15 min in healthy subjects (1)(2). Fosphenytoin can be used for parenteral or intramuscular administration, where its superior aqueous solubility results in less severe side effects than does phenytoin (2)(3). Few data, however, are available on the interference of fosphenytoin in phenytoin immunoassays, which are currently the most common method used to monitor patients' phenytoin concentrations. In an abstract (4), Kugler et al. reported that the TDx^® Phenytoin assay (Abbott Labs.) was interfered with by fosphenytoin. We report here the results of detailed cross-reactivity studies for fosphenytoin in four phenytoin im-munoassays: ACS:180^® Automated Chemiluminescence System (Chiron Diagnostics), TDx Phenytoin and Phenytoin II, and AxSym^® Phenytoin II (also from Abbott Labs.).

A therapeutic total plasma phenytoin concentration (>10 mg/L) is attained within 10–30 min after administration of fosphenytoin. Conversion of fosphenytoin to phenytoin is reported to be complete in 2–4 h, depending on mode and rate of administration (5). The plasma concentration of fosphenytoin depends on the route of administration and length of time between administration and patient sampling. The ordinary half-life of fosphenytoin is reduced by ~50% in patients with hepatic or renal diseases, apparently because of less protein binding of the prodrug (5).

A stock solution of fosphenytoin (a gift from Parke-Davis) in methanol was added to two separate serum pools—one without phenytoin (pool A), and the other containing 12.4 mg/L of phenytoin (pool B)—to give final fosphenytoin concentrations of 52, 39, 26, and 13 mg/L. Apparent phenytoin concentrations in both sets of samples were measured by all four assays according to the manufacturers' directions.

The TDx and AxSym assays use homogeneous fluorescence polarization (FPIA) technology and were run on the TDxFlx^® and AxSym automated analyzers, respectively. The TDx Phenytoin (TDx) assay uses a polyclonal sheep anti-phenytoin antiserum, whereas the TDx Phenytoin II (TDx-II) and AxSym Phenytoin II (AxSym-II) assays use murine monoclonal antisera. The ACS:180 Phenytoin (ACS:180) is a heterogeneous chemiluminescent immunoassay that uses murine monoclonal antibody; it was run on the automated, random-access ACS:180 chemiluminescent system (6)(7)(8). The analytical range (0.5–40 mg/L) and detection limit (1 mg/L) for the TDx and ACS:180 assays are similar (8), but the AxSym-II assay has a lower detection limit (0.5 mg/L). Results of the ACS:180 assay agree well with both TDx and TDx-II assays (7)(8), whereas the AxSym-II assay correlates with the TDx-II assay (AxSym-II package insert).

The results for fosphenytoin cross-reactivity in pool A for the four phenytoin assays (Table 1 ) show that the TDx-II cross-reacted very strongly (>250%) with fosphenytoin; such cross-reactivity would result in seriously high TDx-II assay results, even in samples containing very low concentrations (<5 mg/L) of fosphenytoin. Fosphenytoin cross-reactivity in the absence of phenytoin for the other three assays exhibited the following order: ACS:180 > TDx > TDx-II > AxSym-II. However, although the ACS:180 cross-reactivity was independent of fosphenytoin concentration, that of TDx and AxSym-II decreased with increasing concentrations of fosphenytoin.

Table 1 also presents our data on the interference of fosphenytoin in the four assays in serum pool B, containing 12.4 mg/L phenytoin. The fosphenytoin cross-reactivity in TDx and AxSym-II assays was ~2- and 3-fold greater, respectively, in the presence of phenytoin than in its absence. Given that the rate of fosphenytoin metabolism may differ from person to person or in different disease states, the higher cross-reactivity in presence of phenytoin is of concern. Cross-reactivity in the ACS:180 assay, however, was independent of the presence of phenytoin. Similar data have also been found in interference studies of oxaprozin in the TDx-II assay, in which cross-reactivity to oxaprozin increased by 5% in the presence of ~10 mg/L phenytoin (9).

In summary, different phenytoin immunoassays may cross-react differently with fosphenytoin, resulting in discordant results for samples containing fosphenytoin. Because of this problem, serum specimens taken from patients treated with fosphenytoin before all of the prodrug is metabolized (2–4 h after the drug administration is complete) may yield misleading phenytoin immunoassay results. Finally, whereas the ACS:180 assay shows consistent cross-reactivity to fosphenytoin in samples with or without phenytoin, the TDx assay cross-reactivity is dependent on both fosphenytoin and phenytoin concentrations. This underlines the importance of cross-reactivity determination in both the presence and the absence of the primary analyte.

References

1. Bebin M, Bleck T. New anticonvulsant drugs [Review]. Drugs 1994;48:153-171. [Medline] [Order article via Infotrieve]
2. Browne TR, Kugler AR, Eldon MA. Pharmacology and pharmacokinetics of fosphenytoin. Neurology 1996;46(Suppl 1):S3-S7.
3. Mattson RH. Parenteral antiepileptic/anticonvulsant drugs. Neurology 1996;46(Suppl 1):S8-S13.
4. Kugler AR, Olsen SC, Webb CL, Annesley T, Nordblom GD, Koup JR. Cross-reactivity of fosphenytoin (Cerebyx^®) in 2 human phenytoin immunoassays [Abstract]. Pharmacol Res 1994;11(Suppl):S102.
5. Aweeka F, Alldredge B, Boyer T, Warnock D, Gambertoglio J. Conversion of ACC-9653 to phenytoin in patients with renal or hepatic diseases. Clin Pharmacol Ther 1988;43:178.
6. Datta P, McLaughlin L, Walsh R, Alpert A, Morehouse S. An automated chemiluminescent phenytoin assay in a heterologous format [Abstract]. Clin Chem 1996;42:S213.
7. Datta P, McLaughlin L, Walsh R, Alpert A, Morehouse S, Kwiatek K. Comparison of three automated phenytoin assays of different specificity [Abstract]. Clin Chem 1996;42:S213.
8. Dasgupta A, Datta P, Redlich G, Limmany A. Analytical performance of a new chemiluminescent phenytoin (ACS:180) assay. Ther Drug Monit 1997;19:191-194. [Web of Science][Medline] [Order article via Infotrieve]
9. Datta P. Oxaprozin and 5-(p-hydroxyphenyl)-5-phenylhydantoin interference in phenytoin immunoassays [Letter]. Clin Chem 1997;43:1468-1469. [Free Full Text]