Effects of supraphysiologic temperature and broth dilution on serum protein binding

Pooled human serum, unheated (UU) and heated at 56 degrees C for 30 (HU-30) and 60 (HU-60) min, and these media diluted 1:1 with broth (UD, HD-30, and HD-60) were used to assess the effects of supraphysiologic temperature and broth dilution on the serum protein binding of ceftriaxone. Protein binding was determined by ultrafiltration and subsequent high-performance liquid chromatography analysis. Significant differences in protein binding between UU and HU-60 (P less than 0.05), UD and HD-60 (P less than 0.01), HD-30 and HD-60 (P less than 0.01), and all undiluted and diluted media (P less than 0.01) were found. These alterations in protein binding may influence the in vitro microbiologic testing of highly protein-bound antibiotics.