Clinical Chemistry
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Clinical Chemistry 23: 2139-2141, 1977;
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Clinical Chemistry, Vol 23, 2139-2141, Copyright © 1977 by American Association for Clinical Chemistry

Methotrexate assay by enzymatic inhibition, with use of centrifugal analyzer

PR Finley and RJ Williams

We present a method for measuring methotrexate by enzymatic inhibition, with use of the centrifugal analyzer. In the presence of dihydrofolate reductase (EC 1.5.1.3), dihydrofolic acid is converted to tetrahydrofolate. NADPH acts as a coenzyme. Methotrexate inhibits the action of dihydrofolate reductase. Increasing concentrations of methotrexate depress the conversion of NADPH to NADP+, and the reaction may be followed at 340 nm. Precision, accuracy, and sensitivity are satisfactory. A comparison to the radioimmunoassay method (x) showed the following least-squares regression: y = 0.9813x + 5.6 microgram/liter; r = .9812; Sy = 6.3 microgram/liter. Some interference was noted from bilirubin, lipermia, and hemoglobin. Up to 60 patients' samples per hour may be estimated by this technique.




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Copyright © 1977 by the American Association for Clinical Chemistry.