title: Erythrocyte methotrexate-polyglutamate assay using fluorescence polarization immunoassay technique: application to the monitoring of patients with rheumatoid arthritis.
abstract: Methotrexate (MTX), i.e., MTX-polygluatmate 1 (MTX-PG(1)), exerts its antirheumatic effects mainly by < or = 6 (MTX-PG(2-7)) via folypolyglutamyl synthase in cells. The authors developed a new method using fluorescence polarization immunoassay to determine MTX-PG(1-7) concentrations in erythrocytes (RBC). MTX-PG(2-7) in RBC of rheumatoid arthritis (RA) patients receiving MTX was converted to MTX in the presence of plasma gamma-glutamyl hydrolase and mercaptoethanol at 37 degrees 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.
authors: Division for Evaluation and Analysis of Drug Information, Keio University Faculty of Pharmacy, Tokyo 105-8512, Japan.
authors: Sachie
authors: S
authors: Inoue
authors: Masayuki
authors: M
authors: Hashiguchi
authors: Shinichi
authors: S
authors: Kawai
authors: Mayumi
authors: M
authors: Mochizuki
mesh_terms: D018501:Antirheumatic Agents / Q000097:blood*; D001172:Arthritis, Rheumatoid / Q000097:blood* / Q000188:drug therapy*; D016903:Drug Monitoring / Q000379:methods*; D004912:Erythrocytes / Q000737:chemistry*; D005260:Female; D016231:Fluorescence Polarization Immunoassay / Q000379:methods*; D006801:Humans; D008297:Male; D008727:Methotrexate / Q000097:blood*; D008875:Middle Aged; D011099:Polyglutamic Acid / Q000097:blood*; D012680:Sensitivity and Specificity
chemical_list: D018501:Antirheumatic Agents; D011099:Polyglutamic Acid; D008727:Methotrexate