Automated measurement of alpha-amylase isoenzymes with 6(3)-deoxymaltotriose as selective amylase inhibitor.
We developed an automated method for measurement of alpha-amylase isoenzymes in serum by a single kinetic assay (SKA) and a double kinetic assay (DKA) with 2-chloro-4-nitrophenyl-6(5)-azido-6(5)-deoxy-beta-maltopentaoside as a substrate and 6(3)-deoxymaltotriose (DOG3) as a novel selective amylase inhibitor. DOG3 showed a large difference in inhibitory activity between human pancreatic alpha-amylase (HPA; 86.9% inhibition) and salivary alpha-amylase (32.1% inhibition) at 0.33 mmol/L. Constant inhibition was obtained immediately after addition of DOG3. The inhibitory effect did not change with variation in concentrations of amylase up to approximately 3000 U/L. The results obtained by SKA correlated well with those obtained by three methods: monoclonal antibody (r = 0.988), wheat germ inhibitor (r = 0.989), and DKA (r = 0.995). The within-run and between-run CVs for HPA were 0.63-2.32% on SKA, 0.69-1.81% on DKA. No significant interferences by endogenous serum compounds were observed with the proposed methods.
Uchida
R
R
Research and Development Division, Kikkoman Corp., Chiba, Japan.
Tokutake
S
S
Motoyama
Y
Y
Hosoi
K
K
Yamaji
N
N
D001322:Autoanalysis / Q000379:methods / Q000706:statistics & numerical data; D005087:Glucan 1,4-alpha-Glucosidase / Q000378:metabolism; D006801:Humans; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000097:blood*; D007700:Kinetics; D009578:Nitrobenzenes / Q000378:metabolism; D009844:Oligosaccharides / Q000378:metabolism; D010179:Pancreas / Q000201:enzymology; D012463:Saliva / Q000201:enzymology; D012680:Sensitivity and Specificity; D014312:Trisaccharides / Q000494:pharmacology*; D000516:alpha-Amylases / Q000037:antagonists & inhibitors / Q000097:blood*; D001617:beta-Glucosidase / Q000378:metabolism
C093168:2-chloro-4-nitrophenyl 6(5)-azido-6(5)-deoxymaltopentaoside; D007527:Isoenzymes; D009578:Nitrobenzenes; D009844:Oligosaccharides; D014312:Trisaccharides; C093167:6(3)-deoxymaltotriose; D000516:alpha-Amylases; D001617:beta-Glucosidase; D005087:Glucan 1,4-alpha-Glucosidase