Inhibitory Effects of Fidarestat on Aldose Reductase and Aldehyde Reductase Activity Evaluated by a New Method Using HPLC with Post-column Spectrophotometric Detection

A new method to assay the activity of aldose reductase (AR) and aldehyde reductase (AHR) by high-performance liquid chromatography is described. The separation of AR and AHR from tissue extracts using an anionexchange column was followed by chromatographic measurement of the activity in the elute. AR and AHR activity were expressed as the area under the peak obtained by post-coumn spectrophotometric detection of the decrease of coenzyme (NADPH) in each enzyme reaction. In the enzyme preparation from rat or human tissues obtained by this method, two active peaks were identified as AR and AHR. The conrrelation coefficient between the injection volume of the enzyme preparation from each tissue and each peak area was 0.998 or greater. In addition, the within-day preservation rate of AR or AHR activity from each tissue was over 95%.In a comparative study of fidarestat with other AR inhibitors using this method, it was confirmed that the inhibitory effect of fidarestat on AR activity from each rat tissue was more potent than that produced by sorbinil and equipotent to that of epalrestat and zenarestat. Fidarestat was also found to inhibit AR activity more potently than AHR activity in human erythrocytes. Therefore, this method is applicable to studies of the selective inhibition of AR or AHR by test compounds.