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Some problems with the diamine oxidase (DAO) assay using putrescine as substrate in rat liver

  • Allergy and Histamine
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Abstract

Determination of diamine oxidase (DAO) activity in rat liver preparations by measuring the formation of radioactive δ1-pyrroline from14C-putrescine is complicated by the complexity of competing metabolic pathways. This can lead to complete masking of the DAO activity present when rat liver homogenates are used as the enzyme source. However, subcellular fractionation of rat liver homogenates makes it possible to detect some putrescine oxidizing activity in the microsomal fraction when assayed at pH 8.5. When 1 mM putrescine was used as the substrate, over 90% of this activity was inhibited by 6×10−4 M selegiline (deprenyl), indicating that monoamine oxidase (MAO) rather than DAO activity was being measured. The observed activity was also interfered with by agents that reduced acetylation processes and polyamine synthesis. A different picture appears when μM concentrations of putrescine are used: in these conditions all interference is strongly reduced and DAO activity can be measured in rat liver microsomes. Furthermore, kinetic studies on deaminative oxidation of14C-putrescine at concentrations from 1 μM to 5 mM confirm the existence of two enzymes: one with a high affinity for the substrate and similar to intestinal mucosa DAO in its sensitivity to α-aminoguanidine, and the other one with a low affinity and selegiline-sensitive.

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Authors and Affiliations

  1. Dipartimento di Farmacologia Preclinica e Clinica “M. Aiazzi Mancini”, University of Florence, Viale Morgagni, 65, 50134, Florence, Italy

    G. Ignesti, G. Banchelli, R. Pirisino, L. Raimondi & F. Buffoni

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  1. G. Ignesti
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  2. G. Banchelli
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  3. R. Pirisino
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  4. L. Raimondi
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  5. F. Buffoni
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Ignesti, G., Banchelli, G., Pirisino, R. et al. Some problems with the diamine oxidase (DAO) assay using putrescine as substrate in rat liver. Agents and Actions 39, 6–12 (1993). https://doi.org/10.1007/BF01975707

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  • DOI: https://doi.org/10.1007/BF01975707

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