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.

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.

Endogenous benzodiazepine receptor agonist in human and mammalian plasma.

Using ultra-filtration steps and HPLC-separation, a low molecular weight ligand of the benzodiazepine receptor was isolated from plasma of various mammalian species including man. The endogenous ligand acts on benzodiazepine receptors agonistically and apparently has a receptor affinity similar to Diazepam. The ligand is not identical with Diazepam as indicated by HPLC and UV-spectroscopy.

Mechanism of acute depletion of plasma fibronectin following thermal injury in rats. Appearance of a gelatinlike ligand in plasma.

Plasma fibronectin was depleted within 15 min following sublethal burn, followed by partial recovery at 8 h and complete restoration by 24 h in anesthetized rats. Radiolabeled 75Se-plasma fibronectin, injected intravenously before burn, was rapidly sequestered in burn skin as well as the liver. Fibronectin levels at 2 h postburn as detected by immunoassay vs. 75Se-plasma fibronectin indicated that more fibronectin was in the plasma than detected by electroimmunoassay. Crossed immunoelectrophoretic analysis of fibronectin in early postburn plasma demonstrated a reduced electrophoretic mobility of the fibronectin antigen. Addition of heparin or fibrin, both of which have affinity for fibronectin, to normal plasma was unable to reproduce this altered fibronectin electrophoretic pattern. In contrast, addition of gelatin or native collagen to normal plasma reproduced the abnormal electrophoretic pattern of fibronectin seen in burn plasma. Extracts of burned skin, but not extracts of normal skin, when added to normal plasma, elicited a similar altered electrophoretic pattern for fibronectin. By gel filtration, fibronectin in burn plasma had an apparent molecular weight approximately 40% greater than that observed in normal plasma. These data suggest the release into the blood of a gelatinlike ligand from burned skin, which complexes with plasma fibronectin. Thus, fibronectin deficiency acutely postburn appears mediated by (a) its accumulation at the site of burn injury; (b) its removal from the circulation by the liver; and (c) its presence in the plasma in a form that is less detectable by immunoassay.

Electrophoretic bimorphism of serum albumin in the presence of indocyanine green.

The presence of indocyanine green during extended traditional electrophoresis and immunoelectrophoresis of serum is associated with bimorphism of albumin. This occurs over a range of dye--albumin molar ratios an order or more greater than was obtained in similar phenomena described previously. The bimorphism seems not to be dose dependent beyond a certain point, and the two albumins so separated show tinctorial differences. The phenomenon has been observed to apparently the same degree in all normal sera tested, and may represent a means of distinguishing ligand-loaded and ligand-light serum albumin.

A method of computing the distribution of a ligand among multiple binding sites on different proteins in plasma: thyroxine as an illustrative example.

This paper describes a computer program for estimating the amount of free or unbound ligand in the plasma as well as the distribution of the ligand among different sets of binding sites on different proteins. The input data consist of: the number and concentrations of proteins involved, the number of binding sites on each protein, the association constant and binding capacity of each set of sites for the ligand, and the total concentrations of ligand. The output provides tables of estimates of the concentration of free drug, the amount of ligand bound by each set of sites on each protein, the concentration of free protein, and the input values for reference. The distribution of thyroxine between thyroid binding globulin, prealbumin, human serum albumin and free drug is used as an illustrative example.