Determination of histaminase (diamine oxidase) activity byo-dianisidine test: Interference of ceruloplasmin

Abstract

Until nowo-dianisidine was used as an indicator substance in a test system for the determination of diamine oxidase. More recently, however, this substance was also used to measure ceruloplasmin activity. A study of the test principles revealed thato-dianisidine was the one denominator for both enzymes. As it was found for diamine oxidase the indicator was oxidized via peroxidase mediated H₂O₂ cleavage. Ceruloplasmin, however, oxidizedo-dianisidine directly with resulting free radical formation. An addition of histamine dihydrochloride or putrescine dihydrochloride to an incubation mixture, containing ceruloplasmin as enzyme ando-dianisidine orp-phenylene-diamine as substrates, produced an activation of the enzyme, being more than 10-fold in the presence of 1×10⁻² M putrescine at pH 7.0. It was assumed that an allosteric effect of the dihydrochloride component might be responsible for this activation. When the activity of purified diamine oxidase was determined by theo-dianisidine test and by the isotope assay, a very good correlation between both methods was found. But, in a mixture of diamine oxidase and ceruloplasmin, no differentiation between the two enzymic activities by theo-dianisidine test was possible. This observation demonstrated an interference of ceruloplasmin when theo-dianisidine method was used for the determination of diamine oxidase activity. To apply our findings also in vivo the amine oxidase activity increasing in guinea-pig plasma during inflammation, was determined by theo-dianisidine test and by specific methods for some amine oxidases. Despite an enhanced oxidation of theo-dianisidine observed, only an increase of ceruloplasmin activity was found. It was concluded that ceruloplasmin had no ‘histaminase activity’ as has been assumed by other authors using theo-dianisidine test.