Abstract：
:A direct ligand-banding radioassay for methotrexate (MTX) has been developed using dihydrofolate reductase, contained in the lysate of L1210 leukemia cells, as the binding determinant. The procedure is a two-phase reaction system where standard MTX concentrations or the sample being assayed in incubated with the reagent lysate in the first phase, and [3H]MTX is then added in the second phase to titrate the remaining unoccupied binding sites on the enzyme. This method eliminates the need for measuring the residual catalytic activity of the enzyme. The sensitivity of the radioassay is limited only by the specific activity of the [3H]MTX and how approximates 10 pg of the drug. Folic acid, methyltetrahydrofolate, formyltetrahydrofolate, and dehydrofolate in concentrations that are physiological do not interfere in the radioassay. Both mercaptoethanol and reduced nicotinamide andnine dinucleotide phosphate increase the binding capacity of the lysate for MTX; but the reduced nucleotide also increases the affinity of the enzyme for the inhibitor. MTX added to serum can be assayed without extraction if the concentration is greater than 500 pg/ml and recovery of the drug added to serum is about 92%. MTX has been assayed in serum, spinal fluid, and urine of patients who were treated with this drug. It has also been assayed in the lysates of L1210 cells from C57BL X DBA/2 F1 mice treated with MTX. The procedure is simple, rapid, and accurate and should permit better correlation of the therapeutic and toxic effects of MTX with blood concentrations over long-term treatment periods.
journal_name
Cancer Resjournal_title
Cancer researchauthors
Arons E,Rothenberg SP,Costa MD,Fischer C,Iqbal MPsubject
Has Abstractpub_date
1975-08-01 00:00:00pages
2033-8issue
8eissn
0008-5472issn
1538-7445journal_volume
35pub_type
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