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.

Determination of indoxyl sulfate in plasma of patients with renal failure by use of ion-pairing liquid chromatography.

Using our newly developed ion-pairing reversed-phase liquid-chromatographic method for assay of indoxyl sulfate, we measured its concentration in plasma of normal subjects and patients in various degrees of renal failure. Response was linear over the range of 50 to 25 000 pmol of indoxyl sulfate injected into the chromatograph. We demonstrated the specificity of the assay for azotemic plasma by using enzymatic conversion with a sulfatase. For a moderately above-normal indoxyl sulfate concentration in azotemic plasma of 134 mumol/L (29 mg/L), the within-day CV was 1.6%, the day-to-day CV 2.8%. Mean analytical recovery was 101.0% (CV = 2.8%). Over a range of 29 to 192 mg of creatinine per liter of plasma (x), indoxyl sulfate (y) concentration (in mumol/L) was positively correlated (y = 1.30x + 0.43). This method should prove valuable for further study of uremic toxins.

Aromatic amino acid metabolites as potential protein binding inhibitors in human uremic plasma.

Decreased binding of aromatic acidic drugs and endogenous metabolites to plasma proteins of patients with severe renal failure appears to be due to accumulation of unknown solutes. Both the warfarin and indole binding sites of albumin, the principal binding protein for these ligands, are affected. We used a large number of endogenous aromatic acids and synthetic congeners as displacers (a) better to characterize the chemical requirements for binding to each site and (b) to derive clues to the chemical structure of the undefined binding inhibitors in uremic plasma. 14C-tryptophan, 14C-warfarin and 14C-salicylate were used as bound ligands. Numerous indoles, quinolines and phenyl derivatives were moderate to strong displacers with several structural correlates. Increasing apolar side chain length enhanced displacing potency. A hydroxyl group at the 5 position of indoles and at the para position of phenyl derivatives severely reduced activity. The two ends of amphophilic molecules showed opposite requirements for displacement of tryptophan: the greater the polarity at the hydrophilic end, the greater the tryptophan displacing potency. Conversely, the greater the total hydrophobic mass of the remainder of the molecule, the more potent the inhibition of binding. The dipeptides l-tryptophyl-l-tryptophan and l-tryptophyl-l-phenylalanine were potent displacers. Computer-assisted analysis of warfarin binding in the presence of xanthurenic acid revealed inhibition by a mechanism other than simple competition, probably via a third albumin binding locus. We conclude that decreased binding in uremic plasma is most likely the summation effect of a number of retained aromatic acids, peptides, or both types of ligands.

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.

Impaired plasma phenytoin binding in uremia. Effect of in vitro acidification and anion-exchange resin.

Phenytoin binding to uremic plasma was studied in vitro. Inhibition of binding independent of albumin concentration was demonstrated. Acidification from pH 8 to pH 3 produced a small decrease in binding by normal plasma but no change or a small increase in binding by uremic plasma. After plasma was acidified to pH 3.0, passed through an anion-exchange resin and realkalinized to pH 8, the binding by uremic plasma was restored to near normal. These studies indicate that the uremic abnormality causing impaired plasma drug binding is reversible and supports the concept that a competing ligand, tightly bound at physiologic pH, is responsible for impaired drug binding.