Protein binding of methotrexate in sera from normal human beings: effect of drug concentration, pH, temperature, and storage.

Ultrafiltration through dialysis tubing was used to assess factors influencing methotrexate (MTX) binding to serum proteins. Determinations of percentage bound by this method either using addition of radioactive tracer or measuring absolute concentrations by radioimmunoassay were not significantly different. Likewise, similar values were obtained using equilibrium dialysis and ultracentrifugation for assessment of percentage bound. Reduction in the pH of serum from 7.9 to 7.2 resulted in a significant decrease in binding, whereas reduction in temperature from 37 to 21 degrees C resulted in a significant increase. Binding was also observed to decrease significantly in serum samples containing MTX that had been stored frozen at -20 degrees C for a month. No significant reduction in the percentage bound was observed on increasing MTX concentration from 10(-10) M until it had risen to 10(-4)--10(-3) M. Analysis at 37 degrees C of fresh sera with 10(-6) M MTX added and pH 7.4 from 19 healthy volunteers gave a mean percentage bound 46.5 +/- 2.7 (SD) with range 41.5-51.0%. No significant difference was found between males and females, and no significant linear correlations were observed between the extent of binding with serum concentrations of total proteins, albumin, total bilirubin or triglycerides, or with age.