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A newly synthesized substrate, 3-hydroxybutyrylglycyl-glycyl-glycine (3HB-GGG), was applied to the assay of ACEinhibiting activity to overcome the smaller selectivity and sensitivity of the conventional method. In this study, an ACEinhibiting assay was improved by the use of a water-soluble tetrazolium salt, 4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate sodium salt (WST-1), for the detection of 3-hydroxybutyrate, derived from 3HB-GGG. The optimized conditions were as follows: 0.333 mM NAD + , 0.333 mM WST-1, 0.1 mM EDTA, 0.633 U ml -1 diaphorase, and 0.700 U ml -1 3-hydroxybutyrate dehydrogenase. The developed assay was efficiently applicable to evaluate the ACEinhibiting activity of practical ACE inhibitors.

In humans, a given amount of oxygen taken in by the body is always converted to ROS, such as O2 -, H2O2 and hydroxyl radical (OH·) by various enzymatic metabolism systems. 1 Because of their high reactivity, ROS affects various molecular components of the cell, like fatty acids, proteins and DNA, and an excess of ROS leads to cell degeneration and death.2 When the amount of ROS exceeds the limit of defense mechanism of the body, many serious diseases may be induced, such as: cancer, arteriosclerosis, gout, Alzheimer's disease, Werner syndrome, and various age-related diseases. 1 Among the enzymes concerning oxidation, XOD has been recognized as a key oxidative enzyme in pathology, such as hypertension and ischemia-reperfusion. The enzyme catalyses the oxidation of many substrates, including purines, pteridines, aldehydes and heterocyclic molecules. 3 XOD catalyzes the following reactions, which produce both superoxide anion and hydrogen peroxide directly:Xanthine + H2O + 2O2 → Uric acid + 2O2 -+ 2H + ,Xanthine + H2O + O2 → Uric acid + H2O2.However, about 20% of the reduction of oxygen is univalent and the rest is divalent. 3 Superoxide anion, which is very reactive, spontaneously dismutates into oxygen and hydrogen peroxide, if it does not react with some other species, 2O2 -+ 2H + → H2O2 + O2.The overproduction and/or underexcretion of uric acid leads to hyperuricemia, such as gout. 4 Thus, one of the treatments of gout is the use of XOD inhibitors that block the production of uric acid.In the human body, XOD is normally found in the liver, and is not free in the blood. During oxidative stress, the enzyme XOD increases. There are five different mechanisms known to increase superoxide generation by XOD during ischemiareperfusion: firstly, superoxide and hydrogen peroxide production are enhanced due to increased conversion of xanthine dehydrogenase to XOD; secondly, mRNA levels of xanthine dehydrogenase and XOD are upregulated; thirdly, when the liver becomes ischemic and hepatocellular damage occurs, the liver releases XOD in the bloodstream, this superoxide-producing enzyme thus being transported throughout the body (thus, a blood assay for XOD is a way to determine if liver damage has happened); furthermore, XOD can bind to endothelial cells, and cell-bound XOD has been reported to produce radicals; finally, during ischemia, ATP is degenerated to xanthine and hypoxanthine, thereby increasing the XOD substrate levels, which leads to increased superoxide production. 5 To protect the body from highly toxic ROS, the human body requires anti-oxidative stress mechanisms, including superoxide dismutase (SOD), which catalyses dismutation of two O2 -into H2O2 and O2 (have lower activity). SOD plays an important role in the defense against oxygen toxicity. However, XOD inhibitors, which effectively suppress the production of ROS created by xanthine-XOD, can be substitute for SOD.Among the XOD inhibitory agents, allopurinol (Fig. 1) is an allosteric XOD inhibitor that is structurally related to xanthine, and is oxidiz...

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