Radioimmunoassays of 7-hydroxymethotrexate and methotrexate

Antisera against 7-hydroxymethotrexate were raised in rabbits by injection of 7-hydroxymethotrexate coupled to bovine serum albumin. A 125I-radiolabelled probe was synthesised by radioiodination of 7-hydroxymethotrexate conjugated to tyrosine methyl ester. We developed with these reagents a liquid phase radioimmunoassay for 7-hydroxymethotrexate in which methotrexate did not interfere significantly (cross-reactivity factor = 2.5 X 10(-4]. Similar techniques were used also for the development of a specific radioimmunoassay for methotrexate, which minimally cross-reacted with 7-hydroxymethotrexate. The assessment of the specificity of these assays showed that parallel monitoring of methotrexate and 7-hydroxymethotrexate in human plasma was possible. Coefficients of variation for duplicate determinations were less than 15% between 0.06 and 1.2 nmol/l for methotrexate and between 0.08 and 9 nmol/l for 7-hydroxymethotrexate. Methotrexate and its oxidative metabolite, 7-hydroxymethotrexate, were quantified in plasma of patients following high-dose infusion (2 g over 24 h) by radioimmunoassay and high performance liquid chromatography. The agreement between the two techniques was found satisfactory, as evaluated by least square regression analysis (slopes 1.007 +/- 0.046 for 7-hydroxymethotrexate and 0.937 +/- 0.046 for methotrexate with y-axis intercept not significantly different from zero). These radioimmunoassays permitted also the evaluation of the concentrations of methotrexate and 7-hydroxymethotrexate in plasma after intravenous bolus injection (50 mg/m2), and large inter-individual difference in both methotrexate and 7-hydroxymethotrexate kinetic behaviour could be observed.