Related Experiment Videos
Macroamylases: differences in activity against various-sized substrates
J L Rosenblum1, G L Hortin, C H Smith
1Department of Pediatrics, Washington University School of Medicine, St. Louis Children's Hospital, MO 63110.
Abstract:
Hyperamylasemia caused by macroamylases can lead to the overdiagnosis of acute pancreatitis. We examined whether interference from macroamylase is less in assays that use high-molecular-mass (high-M(r)) substrates rather than oligosaccharide substrates. We hypothesized that high-M(r) substrates would be sterically excluded from macroamylasemic complexes and thus would be hydrolyzed less efficiently. Eighteen macroamylasemic samples were assayed by using red-dyed amylopectin or blue-dyed starch as polysaccharide substrates or by using maltoheptaose or maltotetraose as oligosaccharide substrates. The oligosaccharide substrates gave comparable results (y = 0.81x + 83), but we observed consistently lower activities for amylopectin than for maltotetraose (y = 0.32x + 38). We observed no bias among methods when nonmacroamylasemic specimens were analyzed. The mechanism of this difference was examined by adding antihuman pancreatic amylase antibodies to hyperamylasemic serum samples from patients without macroamylasemia and to purified human pancreatic or salivary isoamylases. In each case, polyclonal and monoclonal antibodies lowered amylase activity more in assays with complex polysaccharides than in those with oligosaccharides. The use of high-M(r) substrates diminishes interference, and detection of suspected macroamylasemia may be possible through comparing activities determined from automated methods that use different substrates.
Insights
Macroamylase can cause false acute pancreatitis diagnoses. Assays using high-molecular-mass substrates show less interference, aiding in accurate macroamylase detection and preventing misdiagnosis.
Area of Science:
- Clinical Chemistry
- Biochemistry
Background:
- Macroamylasemia, characterized by the presence of macroamylase in blood, can lead to elevated amylase levels (hyperamylasemia).
- This condition may result in the misdiagnosis of acute pancreatitis, a serious medical condition.
Purpose of the Study:
- To investigate whether using high-molecular-mass (high-M(r)) substrates reduces interference in amylase assays compared to oligosaccharide substrates.
- To determine if this difference in substrate reactivity can aid in identifying macroamylasemia.
Main Methods:
- Assayed eighteen macroamylasemic samples using both high-M(r) polysaccharide substrates (amylopectin, starch) and oligosaccharide substrates (maltoheptaose, maltotetraose).
- Compared amylase activities obtained from different substrate types in both macroamylasemic and non-macroamylasemic samples.
- Investigated the mechanism by adding amylase antibodies to assess their differential impact on substrate hydrolysis.
Main Results:
- High-M(r) polysaccharide substrates showed consistently lower amylase activities in macroamylasemic samples compared to oligosaccharide substrates.
- No significant bias was observed when analyzing non-macroamylasemic specimens.
- Antibody addition demonstrated a greater reduction in amylase activity with complex polysaccharides than with oligosaccharides.
Conclusions:
- High-M(r) substrates diminish interference from macroamylase in amylase assays.
- Comparing results from automated methods employing different substrates may enable the detection of suspected macroamylasemia.