Isolation and chemical identification of inhibitors of plasma ligand binding.

The binding by serum albumin of many drugs and endogenous metabolites is impaired in humans and animals with renal failure. Unknown solute(s) retained in renal failure have been extracted from uremic fluids. When added to normal plasma they induce a similar binding defect. Similar activity can be extracted from normal urine. We have devised a series of extraction and purification techniques that yielded three binding inhibitory ligands from normal human urine in sufficient quantity and of a high degree of purity. Rigorous methods have been applied to determine chemical identity of the ligands. Purification steps consisted of: adsorption at pH 3.0 to polystyrene-divinylbenzene resin (XAD-2); elution from the resin with methanol followed by drying and solution in dilute formic acid; passage through SP-Sephadex to remove cations, especially yellow-brown pigments; adsorption to the anion exchanger QAE-Sephadex, and separation into three zones of inhibitory activity with a formic acid gradient; purification to homogeneity with C-8 or C-18 silica reversed-phase chromatography. Using this isolation procedure, followed by mass spectroscopy and nuclear magnetic resonance spectroscopy, we have shown that the binding inhibitory activity is due not to one ligand, but to a family of aromatic acids. To date hippurate, beta-(m-hydroxyphenyl)-hydracrylate and p-hydroxyphenylacetate have been identified as binding inhibitors. Other active ligands remain to be identified.

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.