Albumin binding in uraemia: quantitative assessment of inhibition by endogenous ligands and carbamylation of albumin.
The binding capacity of human serum albumin (HSA) for small acidic molecules is known to be reduced in chronic renal failure (CRF). The contribution of competitive inhibition by accumulated endogenous ligands and of structural changes in HSA has now been evaluated. In a fluorimetric in vitro assay using HSA and two dansylated amino acids the inhibitory properties of various endogenous ligands were determined in concentration-effect studies. The effect of carbamylation of HSA on binding was also examined. The mode of inhibition, including binding parameters n and Ka, was determined. Finally, HSA binding in sera from controls and dialysis patients was compared in a modified assay. Thirty three substances were tested and were placed in 3 groups: strong inhibitors (IC50 < 3*10(-5) mol.l-1, e.g. indolyl acids, furanoic acids), medium inhibitors (IC50 > 3*10(-5), eg. vanillic acid), and no inhibition (e.g. urea, creatinine, guanidino compounds). Complete (> 80%) carbamylation of HSA reduced binding by 67% in a non-competitive mode. There was a significant reduction in the binding capacity of HSA from the dialysis patients (approximately 24%), irrespective of medication. It is concluded that the uraemic binding defect of HSA is caused by competitive inhibition by the many physiological ligands accumulated in CRF and structural modifications of HSA. The assay presented proved useful for the rapid analysis of possible HSA binding inhibitors and for testing large groups of patients, e.g. comparison of dialysis treatments, and pharmacological binding studies.