Paul F. Gulyassy

Contributions of hippurate, indoxyl sulfate, and o-hydroxyhippurate to impaired ligand binding by plasma in azotemic humans

We have evaluated pH, chloride, calcium and several endogenous aromatic acids as possible causes of the impaired binding of drugs by plasma albumin in renal failure. Changes in pH, chloride and calcium over the range found in renal failure had minimal or no effects on the binding of [14C]salicylate, a model probe which binds to both of the major drug-binding loci of human albumin. Hippurate and indoxyl sulfate were weak inhibitors of binding by normal plasma. Ortho-hydroxy-hippurate was undetectable or minimally elevated, except among patients with elevated plasma salicylate concentration. Although plasma hippurate and indoxyl sulfate concentrations were elevated markedly in patients with renal failure, inhibition of salicylate binding was significantly correlated only with the concentration of indoxyl sulfate. Addition of hippurate and indoxyl sulfate separately and together to normal plasma showed that these ligands could account for only 15% of the impaired binding of salicylate by azotemic plasma. The retained solutes which account for most of this binding defect remain to be identified. This uremic disorder (and perhaps others) is due not to a single chemical but to the additive effect of a family of chemicals.

Plasma Hippurate in Renal Failure: High-Performance Liquid Chromatography Method and Clinical Application

We have developed a high-performance liquid chromatography (HPLC) method for assay of hippurate in plasma of patients with renal failure. Hippurate accounts, in part, for the impaired binding of drugs and metabolites to albumin and may cause other disorders in azotemic patients. The method is precise, accurate and reproducible. Among 25 patients with acute and chronic renal failure having serum creatinine in the range of 2.9-43 mg/dl (256-3,801 mumol/l), plasma hippurate ranged from 0.11 to 16.2 mg/dl (6.1-904 mumol/l). Hippurate concentration correlated moderately closely with plasma creatinine, urea and anion gap. Its curvilinear relation to the reciprocal of serum creatinine indicated a proportional decline of GFR and tubular function or the accumulation of inhibitors of the proximal tubular anion secretory pathway. The method should be useful for further studies of abnormal albumin binding as well as other disorders in azotemic patients.

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.

Impaired Plasma Phenytoin Binding in Uremia

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 smal

Inhibition of Salicylate Binding to Normal Plasma by Extracts of Uremic Fluids

We previously reported that an extract of uremic plasma reduces binding of phenytoin and tryptophan by normal plasma and plasma albumin. This effect appears to reproduce the impaired binding of many drugs and several endogenous metabolites by uremic plasma. In the present study we further characterized the properties of extracts from uremic sera and body fluids using binding of salicylate as a model. Salicylate was chosen because it binds to both of the main albumin binding loci for aromatic, acidic drugs. Using a computer-assisted, least-squares, curve-fitting program, LIGAND, we found that the most satisfactory model for salicylate binding to 1:10 diluted normal plasma was a binding number (n) of 2 mol of salicylate per mole of albumin with an association constant (k) of 2.85 X 10(4) L/mol, an additional binding of 0.5 mol to other sites on albumin or to other proteins, and nonspecific binding of 21%. Addition of uremic pleural fluid extract to diluted normal plasma produced a monotonic decline in k to 0.17 X 10(4) L/mol with no change in n except possibly at the highest dose of uremic inhibitor. This pattern of competitive inhibition indicates presence of unknown ligands in the uremic extract that compete at both binding loci. More efficient extraction methods might also yield additional ligand(s) that inhibit through a noncompetitive mechanism.

Reversing Uremic Toxicity

In the late 1960s and early 1970s we commonly saw patients with severe uremia. Today, regular dialysis is initiated when the earliest signs of uremia are suspected, sometimes before any clear symptoms develop. As a result, we now see only the more subtle manifestations of uremia. To provide optimal dialysis therapy today, we face the need to quantify these more subtle uremic abnormalities so that we can evaluate how variations in the practice of dialysis affect them. Further complicating this effort to measure uremic disorders is the generous broadening of the criteria for accepting patients for regular dialysis treatments. Many patients have severe extrarenal diseases that would have disqualified them 25 years ago. Determining whether a given clinical abnormality is purely a manifestation of uremic toxicity or is due to a coexistent disease is essential to quantitating the uremic state, but it may be difficult. The specific causes of most of the above manifestations of uremia remain largely unknown. Following is a partial list of basic mechanisms that have been postulated to account for the many disorders neuropathy

Role of Hemodialysis and Medical Management

The optimum use of dialysis and careful medical management contributes in many ways to the success of renal transplantation. In the earliest years of transplantation, kidneys were transplanted in some patients without antecedent dialysis. With improved understanding of the widespread organ and tissue abnormalities caused by uremia and the rapid increase in availability of dialysis since 1960, this practice was abandoned. Postoperative complications, such as bleeding, poor wound healing, and infections are very likely lessened by a period of adequate preoperative dialysis. One could conceivably perform a transplant from a living related donor before there is a clear need for dialysis of the recipient. Because of the uncertain outcome in a given patient, even with a living related donor, this approach is not justified. The patient should, instead, receive careful medical management until the earliest signs of uremia—decreased appetite, fatigue, impaired cerebration—appear.