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

In patients with endstage renal failure (ESRF), disturbances of Ca2+ metabo lism are common. Besides hormonal changes, inhibition of cellular Ca2+-ATPa se was postulated to contribute to uremic toxicity. We purified a potent in hibitor of the Ca2+-ATPase from the ultrafiltrate of patients with ESRF by multiple steps of high performance liquid chromatography to homogeneity, an d identified the isolated inhibitor by mass spectrometric methods as p-hydr oxy-hippuric acid. The enzyme used for the Ca2+-ATPase assay system was iso lated from red blood cells by crossflow filtration. The activity of the: Ca 2+-ATPase was measured spectrophotometrically as the difference in hydrolys is of adenosine 5'-triphosphate (ATP) in the presence and absence of Ca2+ w ith different concentrations of ATF and p-hydroxyhippuric acid. The Ca2+-AT Pase was found to be inhibited by p-hydroxy-hippuric acid at a concentratio n 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 +/- 0.1 3. In conclusion, p-hydroxy-hippuric acid may play a role in disturbed Ca2 metabolism in end-stage renal failure.