Roles of Hippurate and Indoxyl Sulfate in the Impaired Ligand Binding by Azotemic Plasma

Abstract

Despite extensive efforts by many investigators the chemical basis of most uremic disorders is not known. We have been studying a circumscribed disorder, impaired plasma protein binding of acidic drugs and endogenous metabolites, hoping to discover investigative approaches which might be applicable to study of more complex uremic disorders. Our initial interest in the problem arose from our finding of markedly reduced levels of plasma tryptophan in patients with renal failure, before and after long-term dialysis treatment. This change appeared to be due to impaired binding of tryptophan by azotemic plasma (1). The initial hypothesis proposed by others that binding is impaired because plasma albumin is per se abnormal has little support. Substantial evidence indicates that retained uremic solutes, which bind to plasma proteins, account for this defect (2). Two aromatic anions, hippurate and indoxyl sulfate, accumulate to substantial levels in uremia and are known to be moderate to strong displacers of several albumin-bound model probes in vitro (3-5). In addition, Lichtenwalner et al have implied that o-hydroxyhippurate may be an important binding inhibitor in azotemic plasma (6). To evaluate the relative importance of these three aromatic anions as binding inhibitors, we have developed highly sensitive and specific HPLC methods to measure their concentrations in plasma of patients With a wide range of renal failure. We have also determined the binding of ¹⁴ C-salicylate by normal plasma, azotemic plasma and normal plasma “spiked” with a wide range of hippurate and indoxyl sulfate and correlated the concentrations of these ligands with salicylate binding. Salicylate was used as a probe because it binds to both of the major binding sites on albumin for aromatic anions. Finally, we have determined the effect of wide variations in concentrations of pH, calcium and chloride on binding.