Erythrocyte Methotrexate-polyglutamate Assay Using Fluorescence Polarization Immunoassay Technique: Application to the Monitoring of Patients with Rheumatoid Arthritis

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Methotrexate (MTX), i.e., MTX-polygluatmate 1 (MTX-PG1), exerts its antirheumatic effects mainly by ≤6 (MTX-PG2-7) via folypolyglutamyl synthase in cells. The authors developed a new method using fluorescence polarization immunoassay to determine MTX-PG1-7 concentrations in erythrocytes (RBC). MTX-PG2-7 in RBC of rheumatoid arthritis (RA) patients receiving MTX was converted to MTX in the presence of plasma γ-glutamyl hydrolase and mercaptoethanol at 37°C. The MTX in RBC was extracted in a perchloric acid deproteinization step then on a solid-phase extraction column. The concentration of MTX was measured by TDX analyzer. The mean MTX recovery rate was 76.1% (n=8). The intraday and interday coefficients of variation were <11.3% (n=8) and <12.4% (n=3), respectively, at low and high concentrations (30-300 nmol/l). The calibration curve was linear over the range 30-300 nmol/l. The total concentration of MTX-PGs (mean±S.D.) in RBC obtained from 95 Japanese RA patient blood samples was 97.3±8.1 nmol/l for the MTX dose of 0.13±0.05 mg/week/kg. This newly developed method for the quantification of MTX-PGs in RBC is sensitive and accurate and can be applied for routine monitoring of MTX therapy in RA patients.