(1) and osteogenic sarcoma (2). Both systemic and intrathecal regimens have been hampered by the occasional development of serious toxicities, such as neurologic sequelae (3), renal failure (4), and myelosuppression. While the etiology of these complications is not well understood, they appear in some cases to be associated with delayed elimination of drug from cerebrospinal fluid (5) or the systemic circulation (6), and pharmacokinetic monitoring may be helpful in their prevention and management (6).Heretofore, routine monitoring of methotrexate pharmacokinetics has not been possible in most institutions due to the complexity of existing assay methods. In the present report, we describe the development of a highly specific, rapid, and sensitive assay based on the high affinity binding of methotrexate to dihydrofolate reductase. In addition to its advantages for routine clinical use, the assay has broad potential application to antifolate research in that it utilizes techniques which allow direct measurement of the association constant of antifolates and dihydrofolate reductase under optimum binding conditions. MATERIALS AND METHODSPartially purified dihydrofolate reductase from dichloromethotrexate-resistant Lactobacillus casei (New England Enzyme Center, Boston, Mass.) was used throughout this study. This enzyme preparation formed 4.6 Aumol of tetrahydrofolate/min per mg protein at pH 7.5 and 370 and was able to bind 1.9 nmol of methotrexate per mg of protein at pH 6.2 and 23'.Unlabeled methotrexate (0.15 M potassium phosphate buffer, pH 6.2, and 23°) (Drug Research and Development Branch, National Cancer Institute) and 4-amino-4-deoxy-10-methylpteroic acid (a gift of Dr. David Johns, National Cancer Institute) were purified by chromatography on DEAE-cellulose with a HC03-elution gradient as previously described (7,8). [3',5',9(n)-3H]methotrexate, 9.5 Ci/ mmol (Amersham-Searle) was diluted in 0.1 M potassium phosphate, pH 8.0, to a final concentration of 2 mCi/ml, and stored at -70°. This methotrexate preparation proved unstable and decomposed, forming radioactive decomposition products which were not adsorbable by charcoal, thus increasing the background. For this reason, the labeled methotrexate was also repurified by chromatography on DEAE-cellulose as described above whenever the background exceeded 150 cpm.Plasma samples containing 100 units of heparin per ml were obtained from patients receiving high dose methotrexate infusion and cerebrospinal fluid was obtained from patients receiving intrathecal methotrexate. All samples were stored at -20°. Prior to analysis samples were acidified to Immediately following addition of the enzyme-NADPH solution, the tubes were mixed by Vortex agitation and 25 Al of a charcoal slurry was added (Norit A, 10 g/100 ml; bovine serum albumin, Fraction V, 2.5 g/100 ml; and high-molecular-weight dextran, 0.1 g/100 ml, all from Sigma Chemical Co.) and pH was adjusted to 6.2 with 1 N HCL. Samples were again mixed by Vortex agitation and centrifuged at 700 X g for 30 min. A 200 ...