Diamine oxidase catalyzes the oxidation of various diamines (e.g., putrescine, cadaverine) to their corresponding aminoaldehydes, which are in equilibrium with their cyclic condensation products (A'-pyrroline and A'-piperideine, respectively). Diamine oxidase activity has been proposed to be a marker of intestinal mucosa integrity.
HPLC analysis was based on the separation of o-aminobenzaldehyde, 2,3-tetramethylene-l,2-dihydroquinazolinium ion (obtained from A^piperideine) and 2,3-trimethylene-l,2-dihydroquinazolinium ion (obtained from A'-pyrroline) on a LiChrosorb RP-8 column (4 mm X 250 mm, 7 jam). The mobile phase was composed of 0.1 M phosphoric acid adjusted to pH 3 with triethylamine and acetonitrile (85:15, v/v) at a flow rate of 1.0 mL/min. The absorbance was monitored at 465 nm.
Mucosal extracts (0.5 mL) were preincubated at 37°C with 50 ¡xL of 1 mAf A'-pyrroline solution and 100 fiL of o-aminobenzaldehyde-saturated aqueous solution. The enzymatic reaction was started by adding 50 fiL of 0.1 M cadaverine solution in 0.1 phosphate buffer (pH 7.0). After 20 minutes, the re action was stopped by heating the tube for 2 minutes in a sand bath maintained at 150°C. After centrifugation, 10 ¡xh aliquots were analyzed by HPLC. Quantitation was based on oxidizing known amounts of cadaverine to A1-piperideine by pea seedling diamine oxidase, followed by treatment with o-aminobenzaldehyde to form the quinazolinium ion. The assay was demonstrated to be linear with time to 20 minutes and with volume of extract added to 1.5 mL.
Tissue homogenates were prepared from mucosa scraped from small intestine of dog. The scrapings were homogenized in 10 volumes of ice-cold 0.1 M phosphate buffer (pH 7.0). Supernates were prepared by centrifuging for 60 minutes at 25,000g.
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