Characterization of p-hydroxy-hippuric acid as an inhibitor of Ca2+-ATPase in end-stage renal failure

In patients with endstage renal failure (ESRF), disturbances of Ca2+ metabolism are common. Besides hormonal changes, inhibition of cellular Ca2+-ATPase was postulated to contribute to uremic toxicity. We purified a potent inhibitor of the Ca2+-ATPase from the ultrafiltrate of patients with ESRF by multiple steps of high performance liquid chromatography to homogeneity, and identified the isolated inhibitor by mass spectrometric methods as p-hydroxy-hippuric acid. The enzyme used for the Ca2+-ATPase assay system was isolated from red blood cells by crossflow filtration. The activity of the: Ca2+-ATPase was measured spectrophotometrically as the difference in hydrolysis of adenosine 5'-triphosphate (ATP) in the presence and absence of Ca2+ with different concentrations of ATF and p-hydroxyhippuric acid. The Ca2+-ATPase was found to be inhibited by p-hydroxy-hippuric acid at a concentration above 11.7 mu mol/L. p-Hydroxyhippuric acid inhibited the erythrocyte Ca2+-ATPase by reducing V-max and increasing the K-m value. The EC50 (log mol/L; mean +/- SEM) fur p-hydroxy-hippuric acid was calculated as 4.82 +/- 0.13. In conclusion, p-hydroxy-hippuric acid may play a role in disturbed Ca2+ metabolism in end-stage renal failure.