Isolation and identification of canine plasma components suspected as uremic toxins

Suspected uremic substances contained in four fractions, which had been selected as the suspected canine uremic peaks in the previous study, were isolated by two stages of preparative liquid chromatography (PLC) from plasma of uremic dogs treated with the ligation of the ureter, and then their physicochemical properties were examined. The primary separation of the suspected uremic peaks were performed with the same anion exchange resin as used in the analytical HPLC in the previous study. Analytical reverse phase HPLC showed that three of 4 suspected uremic peaks almost consisted of single substances, but the other contained several substances. Main subfractions of these peaks were successfully isolated by the secondary stage reverse phase PLC. By means of thin layer chromatography, ultraviolet absorption spectrometry and proton-nuclear magnetic resonance spectroscopy, components of 4 main peaks were confirmed to be small molecules such as a pyridine derivative, uric acid, hippuric acid and kynurenic acid, respectively.