Automated measurement of alpha-amylase isoenzymes with 6(3)-deoxymaltotriose as selective amylase inhibitor

Abstract 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.

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Automated measurement of amylase isoenzymes by a double kinetic assay with "blocked" beta-2-chloro-4-nitrophenyl maltopentaoside as substrate and with wheat germ inhibitor

Clinical Chemistry ◽

10.1093/clinchem/37.8.1345 ◽

1991 ◽

Vol 37 (8) ◽

pp. 1345-1349 ◽

Cited By ~ 2

Author(s):

A Abe ◽

T Nishimura ◽

A Noma ◽

K Hamano

Keyword(s):

High Performance ◽

Wheat Germ ◽

Single Channel ◽

Selective Inhibitor ◽

Automated Measurement ◽

Test Results ◽

Kinetic Assay ◽

Beta 2 ◽

Amylase Isoenzymes ◽

Method R

Abstract We evaluated the enzymic mechanism by which 3-keto butylidene-beta-2-chloro-4-nitrophenyl maltopentaoside (3KB-G5-CNP) serves as a substrate for serum pancreatic (p-) and salivary (s-) amylases. In aliquots of the reaction mixture, three kinds of beta-2-chloro-4-nitrophenyl oligosaccharides (glucose, maltoside, and maltotrioside) were separated from the substrate by high-performance liquid chromatography. Both isoenzymes behaved nearly identically and produced almost the same products. We automated a double kinetic procedure for determining total (t-) and p-amylase with use of a selective inhibitor from wheat germ in a single channel on the Hitachi 7050 analyzer. Within- and between-run CVs were, respectively, 0.5% and 1.7% for t-amylase (240 U/L), and 0.7% and 2.3% for p-amylase (230 U/L). The test results varied linearly with concentrations up to approximately 2000 U/L for t- and p-amylase activities. p/s ratios varied from 0.2 to 5.0. Results correlated well with those obtained by the monoclonal inhibition method (r = 0.992).

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A commercially available S-type amylase inhibitor evaluated for determination of amylase isoenzymes in serum.

Clinical Chemistry ◽

10.1093/clinchem/30.7.1227 ◽

1984 ◽

Vol 30 (7) ◽

pp. 1227-1228 ◽

Cited By ~ 4

Author(s):

N W Tietz ◽

D F Shuey

Keyword(s):

Wheat Germ ◽

Clinical Laboratory ◽

Performance Characteristics ◽

Alpha Amylase ◽

Emergency Situations ◽

Amylase Inhibitor ◽

Alpha Amylase Inhibitor ◽

Amylase Isoenzymes ◽

Fast Procedure

Abstract On evaluation, an S-type alpha-amylase inhibitor from Sigma shows essentially the same performance characteristics as the inhibitor from wheat germ prepared according to O'Donnell and McGeeney (Biochim Biophys Acta 422:159, 1976). This inhibitor is now commercially available; thus any clinical laboratory can perform isoamylase determinations with a simple, fast procedure that is also suitable for emergency situations.

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A monoclonal antibody that specifically inhibits human salivary alpha-amylase.

Clinical Chemistry ◽

10.1093/clinchem/33.7.1158 ◽

1987 ◽

Vol 33 (7) ◽

pp. 1158-1162 ◽

Cited By ~ 33

Author(s):

M Gerber ◽

K Naujoks ◽

H Lenz ◽

K Wulff

Keyword(s):

Monoclonal Antibody ◽

Preliminary Data ◽

Wheat Germ ◽

Alpha Amylase ◽

Salivary Alpha Amylase ◽

Alpha Glucosidase

Abstract Our monoclonal antibody 88E8 specifically binds to and inhibits human salivary alpha-amylase (EC 3.2.1.1) and cross reacts negligibly with the pancreatic isoenzyme, inhibiting it by less than 1%, as compared with about 90% for the salivary isoenzyme. The antibody binds the S1 and S2 types of salivary alpha-amylase, but no pancreatic alpha-amylase isoenzyme forms. A pancreatic alpha-amylase assay involving 88E8 is under development, with alpha-glucosidase as auxiliary enzyme and p-nitrophenyl-maltoheptaoside as substrate; we give preliminary data on this assay. The assay has to be done by substrate start, because the antibody interacts very slowly with the enzyme in the presence of substrate. Assay results for pancreatic alpha-amylase correlate well with those for isoamylase assayed with use of an inhibitor from wheat-germ.

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Extraction, Purification, and Inhibitory Effect of Alpha - Amylase Inhibitor from Wheat (Triticum aestivum Var. Zarrin)

Pakistan Journal of Nutrition ◽

10.3923/pjn.2005.101.105 ◽

2005 ◽

Vol 4 (2) ◽

pp. 101-105 ◽

Cited By ~ 15

Author(s):

R. Heidari . ◽

S. Zareae . ◽

M. Heidarizadeh .

Keyword(s):

Triticum Aestivum ◽

Inhibitory Effect ◽

Alpha Amylase ◽

Amylase Inhibitor ◽

Alpha Amylase Inhibitor

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Methods compared for determining total amylase activity and amylase isoenzymes in serum.

Clinical Chemistry ◽

10.1093/clinchem/35.4.645 ◽

1989 ◽

Vol 35 (4) ◽

pp. 645-648 ◽

Cited By ~ 8

Author(s):

J L Badenoch ◽

R Bals

Keyword(s):

Monoclonal Antibody ◽

Amylase Activity ◽

Reference Interval ◽

Total Activity ◽

Alpha Amylase ◽

Excellent Correlation ◽

Kinetic Methods ◽

Antibody Method ◽

Isoenzyme Composition ◽

Amylase Isoenzymes

Abstract We evaluated two kinetic methods for determining total amylase activity and isoenzyme composition in serum. Stability studies of reagents for measuring total activity indicate that reagents containing 4-nitrophenyl-alpha-glucosides or enzyme-linked reagents can be stored only for seven days at 4 degrees C. Methods based on 4-nitrophenyl-alpha-glucoside substrates cannot be used if the reagent absorbance at 405 nm exceeds 2. However, in the alpha-amylase EPS method (Boehringer Mannheim) an ethylidene-protected 4-nitrophenyl-alpha-D-maltoheptaoside substrate is stable for up to 28 days after reconstitution. Further studies indicated that the Amylase-DS (Beckman) and the alpha-Amylase EPS standard curves are linear to at least six times the upper limit of the reference interval. Within-batch imprecision (CV less than 1.1%) and between-batch imprecision (CV less than 3.3%) for these two methods are comparable with those for other kinetic methods, and there is excellent correlation (r2 = 0.983) between the two methods. The reference interval, determined by use of samples from 90 healthy blood donors, is 31 to 141 U/L for the amylase-DS method, 22 to 92 U/L for the alpha-Amylase EPS method. We also used these two methods to measure amylase isoenzymes after inhibiting the salivary isoenzyme with either a lectin or a monoclonal antibody. We found the monoclonal antibody method more specific than the lectin inhibition method for determining the isoenzymes.

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Specific immunoassay of alpha-amylase isoenzymes in human serum.

Clinical Chemistry ◽

10.1093/clinchem/31.8.1331 ◽

1985 ◽

Vol 31 (8) ◽

pp. 1331-1334 ◽

Cited By ~ 23

Author(s):

M Gerber ◽

K Naujoks ◽

H Lenz ◽

W Gerhardt ◽

K Wulff

Keyword(s):

Monoclonal Antibody ◽

Human Serum ◽

Enzyme Immunoassay ◽

Amylase Activity ◽

Alpha Amylase ◽

Pancreatic Amylase ◽

Routine Method ◽

Salivary Amylase ◽

Amylase Isoenzymes

Abstract A monoclonal antibody (66C7) was prepared that specifically binds human salivary amylase (EC 3.2.1.1); it cross reacts with human pancreatic amylase by less than 1%. Two procedures are described for determination of isoamylases in human serum with this antibody: an enzyme immunoassay for determining amylase of salivary origin, and a routine method in which this amylase is immunoprecipitated and the remaining (pancreatic) amylase activity is assayed. Results by the two methods correlate well.

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Growth Inhibitory Effect of an .ALPHA.-Amylase Inhibitor from the Wild Common Bean Resistant to the Mexican Bean Weevil (Zabrotes subfasciatus).

Ikushugaku zasshi ◽

10.1270/jsbbs1951.43.257 ◽

1993 ◽

Vol 43 (2) ◽

pp. 257-265 ◽

Cited By ~ 29

Author(s):

Kazunori SUZUKI ◽

Masao ISHIMOTO ◽

Fumio KIKUCHI ◽

Keisuke KITAMURA

Keyword(s):

Common Bean ◽

Inhibitory Effect ◽

Alpha Amylase ◽

Bean Weevil ◽

Growth Inhibitory Effect ◽

Zabrotes Subfasciatus ◽

Amylase Inhibitor ◽

Growth Inhibitory ◽

Alpha Amylase Inhibitor

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Automated measurement of amylase isoenzymes with 4-nitrophenyl-maltoheptaoside as substrate and use of a selective amylase inhibitor.

Clinical Chemistry ◽

10.1093/clinchem/30.7.1219 ◽

1984 ◽

Vol 30 (7) ◽

pp. 1219-1222 ◽

Cited By ~ 12

Author(s):

H Okabe ◽

Y Uji ◽

K Netsu ◽

A Noma

Keyword(s):

Standard Method ◽

Selective Inhibitor ◽

Pancreatic Amylase ◽

Automated Measurement ◽

Salivary Amylase ◽

Amylase Inhibitor ◽

Amylase Isoenzymes ◽

Method R ◽

Serum And Urine ◽

Serum And Urine Samples

Abstract We automated a kinetic procedure for determining amylase isoenzymes in serum and urine samples. We used 4-nitro-phenylmaltoheptaoside as substrate and a selective amylase inhibitor with the Abbott-VP bichromatic system. By use of the maximum differences between pancreatic (P) and salivary (S) amylase activities remaining after inhibition by the selective inhibitor and by use of the linear range, a one-point standard method for calibration is proposed for determining amylase activities between about 50 and 1500 U/L when the P/S ratio exceeds 0.2. Results correlated well with those by electrophoresis and the Phadebas method (r = 0.99 for both pancreatic and salivary amylase). Reproducibilities (CVs) were 1.5% to 5.5% for pancreatic amylase and 1.4% to 3.3% for salivary amylase in serum, 0.8% to 2.0% for pancreatic amylase and 0.8% to 2.3% for salivary amylase in urine.

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Catalytic concentrations of amylase isoenzymes: an assay with wheat-germ inhibitor and 4-nitrophenylmaltopentaoside plus 4-nitrophenylmaltohexaoside as substrate.

Clinical Chemistry ◽

10.1093/clinchem/32.8.1577 ◽

1986 ◽

Vol 32 (8) ◽

pp. 1577-1580

Author(s):

A Jiménez ◽

J Arenas ◽

I Santos ◽

A Martínez

Keyword(s):

Biological Samples ◽

Wheat Germ ◽

Selective Inhibitor ◽

Pancreatic Amylase ◽

Inhibition Rate ◽

Salivary Amylase ◽

Manual Method ◽

Amylase Inhibitor ◽

Amylase Isoenzymes

Abstract We coupled a kinetic procedure to a selective inhibiting method for determining amylase isoenzymes in biological samples, using 4-nitrophenylmaltopentaoside plus 4-nitrophenylmaltohexaoside as substrate and a wheat-germ selective inhibitor with the Gilford S-III spectrophotometer. On plotting remaining amylase activities/total amylase activities (R/T) vs pancreatic amylase activities/salivary amylase activities (P/S) ratios, we found the curve to be linear for P/S ratios from 0.2 to 5. The inhibition rate of amylase inhibitor was constant in solutions having total amylase activities between 20 and (at least) 900 U/L. CVs were 3.1 to 7.1% for pancreatic amylase and 2.0 to 12.9% for salivary amylase in serum. Correlation with the Phadebas method was excellent (r = 0.99) for both pancreatic and salivary amylase. We also automated this procedure in an Hitachi 705 analyzer and correlated the results (r = 0.99) with those by our manual method.

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