ARS | Publication request: Characterizations of substrate and enzyme specificity of glucoamylase assays of mucosal starch digestion with determinations of group and single biopsy reference values

Submitted to: Gastroenterology
Publication Type: Abstract Only
Publication Acceptance Date: April 1, 2005
Publication Date: May 1, 2005
Citation: Robayo, C.C., Adams, B., Avery, S.E., Quezada, R., Baker, R.D., Hamaker, B., Sterchi, E.E., Nichols, B.L., Baker, S.S. 2005. Characterizations of substrate and enzyme specificity of glucoamylase assays of mucosal starch digestion with determinations of group and single biopsy reference values from 980 unselected pediatric endoscopic clinical duodenal biopsies [abstract]. Gastroenterology. 128(4):A169.

Technical Abstract: Carbohydrate digesting enzyme activities are measured in duodenal biopsies to detect deficiencies of lactase and sucrase activities, however glucoamylase (GA) assays for starch digestion are not included. Because food starch represents half of energy intake in the human diet, assays for starch digestion have a nutritional priority. Aims: characterize a duodenal GA assay for starch digestion; define a group reference GA value; determine frequency of low GA activities in unselected patients; define GA variability between replicate duodenal biopsies; and define a reference GA value for single duodenal biopsy assays. GA, lactase, maltase, sucrase and palatinase were assayed in homogenates from 980 clinical duodenal biopsies as a quality control procedure of the GI Lab at SUNY, Buffalo, NY. Patient ages were 10+5 years. GA activity was assayed with '-amylase solubilized amylopectin Polycose (Ross Labs). Polycose was characterized by isoamylase (1-6 link) hydrolysis and matrix-assisted laser desorption/ ionization time of flight (MALDI-TOF). Enzyme specificity was investigated using GA and sucrase monoclonal antibody immunoisolated activities. Replicate technical assays were run on pooled homogenates. Assays for variance analysis between replicate duodenal biopsies were obtained, after signed IRB approved informed consents, in 23 patients to determine single duodenal biopsy GA reference values. Polycose had only 11.8% 1-6 glucose bonds, a specific sucrase substrate, by MALDI-TOF. Immunopurified sucrase hydrolyzed Polycose but based upon reported Km's of 1-4 oligomers 2-7 glucose long; the calculated contribution of sucrase to Polycose hydrolysis was 0.08% of GA activity. Reference value for group GA activity was 35 U/g proteins. Technical replicate assays of GA had variance of 8%, sucrase 5%, and lactase 7%. GA variability between replicate biopsies was 19%, sucrase 20%, and lactase 32%. Variability of GA activities between replicate duodenal biopsies was used to determine single biopsy reference value of 28 U/g proteins. Sucrase clearly hydrolyzes Polycose but with 73 X higher mean Km than GA. Of 980 unselected patients, 657 (67%) were above reference values for all enzymes. Low GA activity was present in 122 of 980 (12%). Ninety-five % of all low GA patients had low sucrase activities. These reference GA values will allow investigations of the clinical and nutritional consequences of poor starch digestion.