High-performance liquid chromatographic and enzyme-inhibition assays of methotrexate concentrations in serum from patients receiving high-dose therapy: tight binding of some methotrexate to a protein that could be dihydrofolate reductase.

In the serum of patients receiving high-dose methotrexate (MTX), concentrations of the drug were monitored using both high-performance liquid chromatographic (HPLC) and enzyme-inhibition assays. In samples obtained greater than or equal to 24 hours after drug application, the HPLC assay measured higher MTX concentrations than the enzyme-inhibition assay. In 72-hour samples, the increase was usually greater than 200%. This difference was not observed in serum spiked with MTX. While the HPLC assay needs sample cleanup, ie, protein precipitation, the enzyme-inhibition assay routinely employs native serum. When MTX was measured in the supernatant with the enzyme inhibition assay, the results equaled those obtained with the HPLC procedure. In patient serum, MTX eluted from a Sephadex G-75 column in two fractions. One had the same retention volume as free MTX and could be assayed directly. The other had a retention volume like dihydrofolate reductase. In this fraction, MTX could only be determined after denaturing proteins. Only small amounts of tightly bound MTX were found in the serum of patients on intermediate-dose therapy (500-600 mg/m2). The observation that after high-dose MTX therapy part of the MTX in patient serum is strongly bound to a protein which could be DHFR raises the question as to the source and fate of this complex. Furthermore, the present finding has clinical relevance to the extent that the accepted limits for risk of MTX toxicity are based on methods using native serum. However, any procedure employing protein precipitation for sample cleanup may grossly overestimate active MTX serum concentrations, especially in the 72-hour serum samples.