Isolation of inhibitors of ligand: albumin-binding from uremic body fluids and normal urine.

Unknown ligands that are retained in renal failure bind to albumin and impair binding of endogenous metabolites and many drugs. We previously showed that an extract from uremic serum can induce a similar abnormality in normal plasma. These binding inhibitors are also present in normal urine. In the present studies we showed by analytical reversed-phase HPLC that the extract of uremic serum contained over 50 UV absorbing peaks. Preparative anion-exchange chromatography of extracts of normal urine and uremic pleural fluid yielded three to four peaks of phenytoin-binding inhibitory activity. The largest peak from normal urine was shown by proton NMR and mass spectroscopy to contain hippuric acid. An earlier eluting peak was found by HPLC to contain several inhibitors-one was definitely identified as p-hydroxyphenylacetic acid and a second as probably beta-(m-hydroxyphenyl)-hydracrylic acid. These methods may be useful for identifying other uremic toxins. The binding abnormality and perhaps other uremic derangements result from the total effect of a family of toxins rather than from the action of a single retained solute.