Comparison of the14C-putrescine assay with the NADH test for the determination of diamine oxidase: Description of a standard procedure with a high precision and an improved accuracy
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A standard procedure for the determination of the diamine oxidase (DAO) activity is described as a modification of the method ofOkuyama andKobyashi . The principle of this method is the assay of14C-Δ1-pyrroline and its polymers formed by the oxydative deamination of14C-putrescine in the presence of DAO. This assay was shown to possess a high sensitivity and precision. Now its accuracy could be enhanced by the comparison with the NADH test for DAO activity. This kind of a reference method made the direct correlation between cpm/min and deamination rates possible. Thus International Units (mU/sample) could be introduced into the isotope assay: In our test system 1 mU corresponded to 215 cpm/min, this value depending on the specific radioactivity of the substrate solution.
The DAO activity from dog's small intestine was investigated with these two methods. The pH-optimum for this enzyme was 7.6, the K m for putrescine as substrate 1×10−4M, the optimum substrate concentration was 1×10−3M. In the small intestine of dogs and rabbits the DAO activity increased from proximal to distal parts, but in human beings the distribution of the enzyme in the gut did not show such a characteristic gradient.
In the discussion, the advantages and disadvantages of the two tests for the determination of DAO activity were compared with each other. The isotope assay was the more sensitive and convenient test. But due to its absolute specificity the NADH test was useful as a reference method and for the investigation of the substrate specificity of the DAO in various tissues and body fluids.
KeywordsSmall Intestine NADH Diamine Substrate Concentration Distal Part
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