Pak Cheung R. Chan^1,2*, Jay Silverberg^2,4, David E. C. Cole^1,3,4, Bonny Lem-Ragosnig¹ and Frank Recknagel¹

¹ Departments of Clinical Pathology, ² Endocrinology, Sunnybrook and Women�s College Health Sciences Centre, Toronto, M4N 3M5, and Departments of ³ Laboratory Medicine and Pathobiology, and ⁴ Medicine, University of Toronto, Ontario, Canada;

^*Address correspondence to this author at:
Department of Clinical Pathology,
Sunnybrook and Women�s College Health Sciences Centre,
2075 Bayview Avenue,
Toronto, M4N 3M5,
Canada;
phone: 416-4806100 ext 2688; fax 416-4806120;
e-mail: pc.chan@sw.ca

Key words: topiramate, interference, radioimmunoassay

Download as a PDF here

Abstract

Topiramate (Topamax^TM, Janssen-Ortho), a commonly prescribed anti-convulsant agent, is a sulfamate substituted monosaccharide that exerts its action by (i) reducing the frequency of action potentials generated when neurons are subjected to a sustained depolarization, (ii) increasing the activity of type A gamma-aminobutyric acid (GABA-A) receptors, and (iii) antagonizing kainate/alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) subtypes of the excitatory glutamate receptor (1,2). Topiramate is readily absorbed in the gut, reaching peak plasma concentrations in 2-3 hours. Absorbed topiramate distributes mainly to body water with a volume of distribution of 0.80-0.55 L/kg. Approximately 18% of the absorbed drug is metabolized into 6 derivatives through hydroxylation, hydrolysis and glucuronidation, while the remaining 82% is eliminated unchanged through the kidneys (2,3). We report here the first case that topiramate treatment may have interfered the measurement of urinary cortisol by radioimmunoassay (RIA).

A 39-yr old female presented with fatigue, mild weight loss, poor appetite and some suspicious striae on the abdomen, and was investigated for possible hypercortisolism using 24-hr urinary free cortisol measured by RIA. The result of 3950 nmol/L or 12,442 nmol/d (Reference Interval or RI: <275 nmol/d) was considered inconsistent with the mild clinical presentation and laboratory findings. The latter included normal plasma cortisol, potassium and bicarbonate, and normal urinary potassium (Table 1). When topiramate was stopped and the urinary cortisol measured again 2 months later, the normal result of 209 nmol/d suggested that topiramate treatment was the likely culprit causing the spurious cortisol result.

Urinary cortisol was measured by (i) competitive RIA with and without extraction with dichloromethane using the Coat-A-Count kit from Diagnostic Products Corporation, Los Angeles (4), (ii) electrochemiluminescence immunoassay (ECL) by the Roche Elecsys 2010, and (iii) tandem mass spectrometry (MS/MS). Plasma cortisol was measured using the Roche Elecsys 2010. Topiramate (Topamax^TM) and Tylenol^TM tablets were obtained from the Pharmacy Department at the authors� institution, ground, dissolved in phosphate buffer, and centrifuged before analyzed for cortisol by RIA. Tylenol^TM tablets served as a matrix control for the cortisol analysis of the topiramate solution. Topiramate.
concentration was determined by liquid chromatography/mass spectrometry (LC/MS).

The measurement of urinary cortisol on the patient�s sample using RIA, with and without prior organic extraction, showed similarly elevated results of approximately 4,000 nmol/L. When measured by ECL, the urinary cortisol result was comparable to that obtained by RIA, at 4,841 nmol/L. However, when measured by MS/MS, the cortisol result was 16.6 nmol/L or 52.3 nmol/d, well within the reference interval of 13.8 � 152 nmol/d. This finding strongly suggested that the cortisol results by both RIA and ECL were factitious. This is not surprising, as interferences of the urinary cortisol immunoassays have been described previously (5-7). However, topiramate has never been reported to interfere with the cortisol measurements by immunoassays. To examine if topiramate interfered with the assays directly, topiramate tablets were dissolved in phosphate buffer to generate topiramate solutions of three different concentrations, which were then subjected to the RIA cortisol measurement. At 3,000 �mol/L, topiramate alone gave a cortisol value of 22 nmol/L. As the topiramate concentration increased, so did the cortisol value, reaching 417 nmol/L for the 93,000 �mol/L topiramate solution (Table 1).

 

In a similarly prepared 4 Tylenol^TMsolution, cortisol was undetectable, excluding any non-specific effects on the cortisol measurement due to tablet filler or phosphate buffer solution. The apparent dose-dependent effect of topiramate on the cortisol measurement points to possible cross-reactivity of topiramate with the cortisol antibodies used in these assays. To assess the specificity of this topiramate effect, we ran the 15,500 �mol/L topiramate solution through the Elecsys 2010 for total testosterone, chorioembryonic antigen (CEA), and CA125. All three immunoassays showed results at or below the detection limit (results not shown).

Having shown that topiramate interfered with the cortisol measurement directly and specifically, we then sought to confirm the presence of topiramate in the patient�s urine sample, and whether the amount of topiramate present would be sufficient to explain the discrepancy observed. Using LC/MS, the original urine sample that gave a cortisol value of 3,950 nmol/L was shown to contain only 50 �mol/L of topiramate. On a molar basis, it is difficult to imagine how such a low concentration of topiramate would generate the dramatic increase in the cortisol result observed. It is possible that topiramate metabolites, rather than the parent compound, are responsible for the assay interference. Unfortunately, measuring topiramate in a selected-ion-monitoring mode, the LC/MS method would not have been able to detect, if any, topiramate metabolites present. Nevertheless, the fact that cessation of topiramate treatment was associated with a normal urinary cortisol by RIA strongly implicated a role of the initial topiramate treatment in causing the factitious urinary cortisol result by either RIA or ECL. Further studies determining the cross-reactivity of topiramate metabolites in cortisol assays based on RIA and ECL will be helpful in elucidating the source and the mechanism of this interference.⁵

Acknowledgements

This work was partially supported by a grant from Practice Based Research, Sunnybrook and Women�s College Health Sciences Center, Toronto. Expert assistance from Drs. N. Smith & S. Soldin at the London Health Sciences Center, Ontario and the Children�s Hospital, Washington, DC, for the analyses of topiramate by LC/MS and cortisol by MS/MS respectively, is gratefully acknowledged. We thank Dr. J. Iazzetta, Department of Pharmacy, Sunnybrook and Women�s College Health Sciences Center, for providing the topiramate tablets. ⁶

References

1. CPS Compendium of Pharmaceuticals and Specialties. Canadian Pharmacists Association, 2003.
2. Shank RP, Gardocki JF, Streeter AJ, and Maryanoff BE. An overview of the preclinical aspects of topiramate: pharmacology, pharmacokinetics, and mechanism of action. Epilepsia 2000; 41 Suppl 1:S3-9.
3. Langtry HD, Gillis JC and Davis, R. Topiramate-a review of its pharmacodynamic and pharmacokinetic properties and clinical efficacy in the management of epilepsy. Drugs 1997; 54:752-773
4. Kowalski A, Paul W. A simple extraction procedure for the determination of free (unconjugated) cortisol in urine by radioimmunoassay. Clin Chem 1979;25:1152
5. Lee C, and Goeger DE. Interference of 6 beta-hydroxycortisol in the quantitation of urinary free cortisol by immunoassay and its elimination by solid phase extraction. Clin Biochem 1998;31:229-233
6. Lepage D, Roberts KD, and Langlais J. Interference of lysophosphatidylcholine in hormone radioimmunoassays. Clin Chem 1993;39:865-869
7. Schwarz S, and Boyd J. Interference of danazol with the radioimmunoassay of steroid hormones. J Steroid Chem 1982;16:91-99