2-Chloro-4-nitrophenyl-beta-D-maltoheptaoside: a new substrate for the determination of alpha-amylase in serum and urine.

Tests for the determination of alpha-amylase activity based on oligosaccharides or their 4-nitrophenyl derivatives as substrates suffer from some disadvantages. Using a more acid indicator molecule and connecting this group in the beta-form to maltoheptaose leads to a new substrate, 2-chloro-4-nitrophenyl-beta-D-maltoheptaoside, for the determination of alpha-amylase activity. Due to its pK value, only half of the 4-nitrophenol formed from 4-nitrophenyl-alpha-D-maltoheptaoside is spectrophotometrically detectable, whereas the total product from the new substrate is responsible for a measurable absorbance. In addition the molar lineic absorbance of the product is 1.8 times higher. Thus the new test is more sensitive than those employing 4-nitrophenyl derivatives. Both P-type and S-type alpha-amylase isoenzymes have the same affinity for the new substrate; this is not the case for all the other substrates, i.e. oligosaccharides and their 4-nitrophenyl derivatives, used in alpha-amylase tests. The sample volume may be reduced, thereby removing interference by substances like bilirubin, glucose and haemoglobin. The test is insensitive to changes in temperature and pH and can be adapted to all mechanized analytical instruments.