Human Pancreatic Amylase Research Articles

Appendix: Production and Characterization of a Monoclonal Antibody to Distinguish Human Pancreatic and Salivary Amylase

Journal Article Appendix: Production and Characterization of a Monoclonal Antibody to Distinguish Human Pancreatic and Salivary Amylase Get access David E Bruns, David E Bruns Search for other works by this author on: Oxford Academic Google Scholar David C Benjamin David C Benjamin Search for other works by this author on: Oxford Academic Google Scholar Clinical Chemistry, Volume 31, Issue 8, 1 August 1985, Pages 1286–1288, https://doi.org/10.1093/clinchem/31.8.1286 Published: 01 August 1985

Specific immunoassay of alpha-amylase isoenzymes in human serum.

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.

Purification and properties of an α-amylase inhibitor specific for human pancreatic amylase from proso (Panicium miliaceum) seeds

An α-amylase inhibitor was purified to homogeneity by acid extraction, ammonium sulphate fractionation, chromatography on carboxymethyl-cellulose, diethylaminoethyl-cellulose and Sephadex G-100 from proso grains (Panicium miliaceum). The calculated molecular weight was 14000. The inhibitor was fairly heat stable and stable under acidic and neutral conditions. The factor was more effective by two orders of magnitude in its action on human pancreatic amylase than on human salivary amylase. It did not inhibit onA. oryzae,B. subtilis and porcine pancreatic amylases. Pepsin rapidly inactivated the inhibitor. Chemical modification studies revealed that amino and guanido groups are essential for the action of the inhibitor. The inhibitor was found to protect both human salivary and pancreatic amylases against inactivation by acid. The mode of inhibition was found to be uncompetitive

Macroamylase immunoglobulins show high affinity for animal and human amylases.

We have examined the affinity shown by the immunoglobulin fraction from each of five sera containing macroamylase for amylases from different sources: human saliva or human, porcine, or ovine pancreas. High affinity constants, 0.4 X 10(10) to 7.2 X 10(10) L/mol, were found in competitive binding experiments with human or porcine pancreatic amylase. All but one serum yielded linear Scatchard plots, indicating that in most sera the amylase-binding immunoglobulins are homogeneous, possibly monoclonal. The immunoglobulin fractions from different sera differed in their specificity: two of them bound all four types of amylases, whereas two bound only one type. Three of the five immunoglobulin fractions showed considerably higher affinity towards one or both of the animal amylases than towards the human ones, and may be primarily directed against some animal amylase.

Lack of adaptive changes in human pancreatic amylase and lipase secretion in response to high-carbohydrate, low-fat diet applied by a 10-day continuous intraduodenal infusion.

In order to investigate whether the human exocrine pancreas is capable of adapting to a diet with a high-carbohydrate, low-fat, and normal protein content, 10 healthy subjects were given a continuous intraduodenal infusion of such a dietary composition (8760 kJ in 2400 ml/day) via a portable infusion pump over a period of 10 days. The diet consisted of 76% of calories as carbohydrates (80% oligosaccharides, 20% mono- and disaccharides), 10% as fat (more than 90% C18 fatty acids) and 14% as protein (oligo- and polypeptides; 11.8 g nitrogen per day). A complete pancreozymin-secretin test was carried out before and after the experimental period. The results show that the above dietary regimen leads to a significant (P less than 0.05) increase in the stimulated secretion rates of trypsin and chymotrypsin, whereas, in contrast to the findings in animal experiments, no change could be measured in the secretion rates of amylase and lipase.

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The immunochemical properties of the macroamylases found in seven patients were examined. The class of heavy chain of the amylase-linked immunoglobulin was proved to be alpha in six cases and gamma in one case. The type of light chain was determined to be lambda in four cases, kappa in two cases, and kappa-lambda in one case. All the Ig A-amylase complexes were completely dissociated by Con-A affinity chromatography. The zymograms of dissociated amylase were normal. When free immunoglobulins released by acidification were mixed with purified human pancreatic or salivary amylase, the immunoglobulins recombined with both amylases in five cases, but only with pancreatic amylase and only with salivary amylase in one case, respectively. The precipitin line of Ig A-amylase complex, which was digested with papain, against anti-human Ig A/F (ab')2 serum was proved to have amylase activity, but that against anti-human Ig A/Fc serum was proved not to have amylase activity. The result highly suggests that in Ig A-amylase complex amylase molecule links with Fab portion of Ig A molecule.

Digestibility of Native and Modified Starches: In Vitro Studies with Human and Rabbit Pancreatic Amylases and In Vivo Studies in Rabbits

The effects of cooking and chemical modification of purified starches on the relative rates and extent of their hydrolysis were studied in vitro by using purified human and rabbit pancreatic amylases. Comparison was made with an in vivo study of postprandial glucose and insulin response in adult rabbits. Uncooked starches showed negligible hydrolysis in vitro, whereas cooking (10 min, 100°C) increased both the rate and extent of hydrolysis of all starches. Soluble potato starch was the most and potato amylose the least hydrolyzed. Unmodified tapioca and waxy corn starch were hydrolyzed at the same rate and to the same extent as soluble potato starches. In most cases chemical modification did not change the rate and extent of hydrolysis of the starches. Minor differences between human and rabbit pancreatic amylase exist, but there is a general resemblance between the two amylases in their starch-hydrolyzing properties (correlation coefficient = 0.90; P < 0.001). The in vivo study showed that uncooked starches elicited no detectable glucose and insulin responses, whereas all the cooked starches except amylose caused glucose and insulin responses comparable to the response seen when feeding glucose. Chemically modified starches (especially waxy corn acetylated distarch adipate) seemed to promote a faster rate of absorption, but the total glucose response (i.e., for the entire 180-min duration) was similar for modified starches and their unmodified counterparts. The in vivo results showed an overall qualitative similarlity to the in vitro results but presented a quantitative difference in the magnitude of the responses for various starch preparations. A good correlation exists between the in vitro and in vivo results (correlation coefficient = 0.84; P < 0.01). This indicates that the action of pancreatic amylases is an important determinant in the digestion and absorption of these carbohydrates.

Studies on an animal .ALPHA.-amylase inhibitor (Haim) from Streptomyces griseosporeus YM-25. Part III. Inhibitory effects of the proteinaceous .ALPHA.-amylase inhibitor Haim on animal .ALPHA.-amylase.

Susceptibility of animal α-amylases to a proteinaceous α-amylase inhibitor, Haim, vary with species of the animal and among organs of the same animal. Haim strongly inhibits, α-amylases from vertebrates, and weakly inhibits those from Mollusca, Annelida, and Arthropoda. The results suggest that susceptibility of animal α-amylases to Haim may correlate well with the classification of the animal. Haim had greater specificity for human salivary amylase than for human pancreatic amylase, and greater specificity for rat pancreatic amylase than for rat salivary amylase.Maximum inhibition of hog pancreatic amylase by Haim occurred after preincubation of the enzyme and its inhibitor at 37°C for 3 min. The time required to reach maximum inhibition was shorter for Haim than for inhibitors from kidney bean and wheat.Complex formation between Haim and hog pancreatic α-amylase was seen by UV difference spectroscopy. That the molar complex was 1:1 was also shown by the same method and by an inhibition curve.

Metabolism of human isoamylases in the rabbit.

Human isoamylases were purified from pancreas and saliva with separation of salivary isoenzymes into glycosylated and nonglycosylated forms. After iodination, bolus injection of each purified isoamylase into conscious rabbits revealed biexponential disappearance from plasma. Glycosylated salivary amylase had a metabolic clearance (t 1/2 = 35 min) that was three to four times faster than nonglycosylated salivary (t 1/2 = 139 min) or pancreatic (t 1/2 = 111 min) amylase. In contrast to our previous studies on homologous (rabbit) isoamylases, human pancreatic isoamylase was excreted into urine as protein-bound radioactivity more readily than salivary isoamylase. No organ contained more than 18% of the injected dose of radioactivity except for a striking accumulation of radioactivity in the liver after injection of 125I-labeled glycosylated salivary amylase. We concluded that 1) human nonglycosylated salivary amylase and pancreatic amylase are cleared from the rabbit circulation at rates comparable with those reported for homologous isoamylases in the rabbit and baboon, 2) the rabbit kidney excretes human pancreatic amylase into urine more readily than human salivary isoamylases, and 3) with the notable exception of glycosylated salivary isoamylase, human isoamylases do not appear to be metabolized primarily by any single organ.

Radioimmunoassay of human salivary amylase: Cross-reactivity with human and porcine pancreatic amylase and other salivary proteins

A radioimmunoassay (RIA) of human salivary amylase was developed. Human salivary and pancreatic amylases were purified by Sephacryl S-200 gel filtration and by cation-exchange chromatography. Human salivary amylase antibody, raised in New Zealand white rabbits, did not crossreact with other salivary proteins and there was also no crossreactivity with purified porcine pancreatic amylase. The antibody crossreacted with human pancreatic amylase to the extent of 25%. Amylase concentrations, estimated by RIA, in human saliva, serum, and urine were compared with enzymatic activity. Correlation of results obtained by the two techniques was best for estimation of amylase in saliva, least for serum and intermediate for urine. Amylase concentrations and enzymatic activity in stimulated parotid saliva were not correlated with flow rate of secretion. There was no correlation between amylase concentrations (and enzymatic activity) in parotid saliva and those found either in serum and urine. Amylase comprises approximately 5% of the total parotid salivary protein in humans.

Suitability of control materials in the differential inhibition assay for human pancreatic and salivary amylase.

We investigated the behavior of 26 quality-control sera with the inhibitor method for differential amylase (EC 3.2.1.1) assay. We also studied the sensitivity to the wheat-derived inhibitor of pancreatic amylases from 10 different animals in comparison with human pancreatic and salivary amylase. The results indicate that only control materials containing human amylases can be measured accurately. The animal amylases (bovine, equine, porcine) used in many quality control sera are relatively insensitive to the inhibitor as compared with human pancreatic and salivary amylase.

Complete amino acid sequence of an α-amylase inhibitor in wheat kernel

We recently purified a new α-amylase inhibitor (called 0.53-inhibitor according to its mobility on polyacrylamide gel electrophoresis, at pH 8.5, relative to Bromophenol blue (taken as 1), under the conditions described by O'Donnell, M.D. and McGeeney, K.F. (1976) Biochim. Biophys. Acta 422, 159–169)) from wheat kernel, which has 500-times greater inhibitory activity towards human salivary amylase than towards human pancreatic amylase (Maeda, K., Takemori, Y. and Oka, O. (1982) Agric. Biol. Chem. 41, 2873–2875). Elucidation of the primary structure and structural comparison with other amylase inhibitors are essential to understand the mechanism of the selective inhibitory behavior of 0.53-inhibitor. The complete amino acid sequence of 0.53-inhibitor has been determined after cleaving the protein with CNBr and trypsin separately. 0.53-Inhibitor is composed of two identical subunits with 124 amino acid residues and contains nine cysteine residues in each subunit. Comparison of the sequence of 0.53- and 0.28-inhibitor, which is a major α-amylase inhibitor in wheat, with M r 12 000, of monomeric form, shows high sequence homologies of nine cysteine regions, but significant differences are evident.

Characterization and intermethod relationships of materials containing purified human pancreatic and salivary amylase.

We describe the preparation and characterization of materials containing human pancreatic and salivary alpha-amylase (EC 3.2.1.1) and examine their relationship to endogenous amylase in human serum. Amylase was purified from human pancreas and saliva by solvent- and salt-fractionation and column chromatography to specific activities of 63 and 279 kU/g, respectively. Four liquid pools, differing only in activity, were prepared from each source of amylase, each in a matrix containing, per liter: 30 g of human albumin, 50 mmol of sodium chloride, 1 mmol of calcium chloride, and 50 mmol of Tris hydrochloride buffer, pH 7.4. Characterization of the pools showed that the amylase activity in the materials was stable for at least six months at 25 degrees C; among-vial variability of amylase activity was less than or equal to 0.5% (2 CV); and the pools were free from eight possible contaminating enzymes. Plots of salivary vs pancreatic amylase activity measure in our materials with eight commercially available methods showed least-squares slopes ranging from 0.51 to 1.0. The intermethod "commutability" of the materials (i.e., how closely they mimic endogenous serum amylase) was examined in relationship to approximately 100 human sera.

Characterization of amylases produced by tumors.

Amylases were purified and characterized from three amylase-producing human tumors. The relative molecular mass of the amylases was estimated to be 54 000 on sodium dodecyl sulfate/polyacrylamide gel electrophoresis, different from human salivary amylase (61 000 and 64 000) and human pancreatic amylase (60 000). The tumor amylases had completely identical antigenicities with human salivary and pancreatic amylases against antibody to human salivary amylase, while the intensity of the tumor amylases was less than 20% of that of human salivary and pancreatic amylases on single radial immunodiffusion. On isoelectric focusing, two of the three tumor amylases showed a major peak at pH 6.4, which corresponded to a major peak of human salivary amylase, the other showed a major peak at pH 6.4, which corresponded to a minor peak of human salivary amylase. The three tumor amylases showed similar amino acid composition, different from those of human salivary and pancreatic amylases. These findings suggest that tumor amylases have a tertiary structure similar to that of normal human amylases, but differ from them in amino acid composition.

18] Radioimmunoassay of human pancreatic amylase

Publisher Summary This chapter discusses the radioimmunoassay of human pancreatic amylase. Serum amylase determination has been the most commonly used test for the diagnosis of pancreatic inflammation. Radioimmunoassay is applicable to the determination of an enzyme-inhibitor complex or products of enzyme degradation. The development of a radioimmunoassay for human pancreatic amylase was sought to measure the serum enzyme levels in terms of their concentration. The standard curve for the radioimmunoassay of human pancreatic amylase, together with the dilution curves for normal serum and urine are discussed in the chapter. These dilution curves were parallel to the standard curve, indicating that the protein detected in human sera and urines can be measured immunologically at different dilutions. The immunoreactivities of human pancreatic and salivary amylase isoenzymes were too similar to permit the differential determination of amylase isoenzymes. The heat stability of immunological and enzymatic activity of amylase in human pancreatic juice is also discussesd in this chapter. The measurement of enzymatic activity of serum amylase provides a direct assessment of its concentration, suggesting that rapid decline in the enzymatic activity of serum amylase in acute pancreatitis should be attributed to the other factors, such as change in metabolic turnover rather than binding of amylase to inhibitors or the breakdown of the enzymes.

Radioimmunoassay for human pancreatic amylase: Comparison of human serum amylase by measurement of enzymatic activity and by radioimmunoassay

A radioimmunoassay (RIA) for human pancreatic amylase has been developed for the determination of human serum amylase content. The assay was shown to be sensitive (7 ng/ml), reproducible and specific, but human pancreatic amylase and salivary amylase could not be distinguished by the antiserum used. In normal subjects, the mean concentration of amylase determined by the RIA was found to be 122.1 ng/ml (range: 55–250 ng/ml). A good correlation was observed between the concentration of amylase and its enzymatic activity in normal subjects. In some instances with high amylase activity, however, the rise in enzymatic activity was not accompanied by increasing amount of amylase content.

Human amylase isoenzymes separated on concanavalin A--Sepharose.

Human salivary amylase and pancreatic amylase were purified and characterized. These amylases gave two bands and one band, respectively, each staining for both protein and sugar, after electrophoresis on sodium dodecyl sulfate--polyacrylamide gel. The relative molecular mass (Mr) or pancreatic amylase was calculated to be 60 000; for the two components (A and B) of salivary amylase the Mr were 61 000 and 64 000. The two salivary amylases were separated by chromatography on concanavalin A--Sepharose; only component B bound to concanavalin A. The carbohydrate content of pancreatic amylase was 1.61 +/- 1.02% (SD), and of salivary amylases A and B 2. 18 +/- 0.71% and 8.77 +/- 2.28%, respectively. The salivary and pancreatic amylases had completely identical antigenicities against antibody to either. On isoelectric focusing, pancreatic amylase showed one peak at pH 7.0, salivary amylase A showed a major peak at pH 6.4 WITH A TRACE OF MATERIAL At pH 5.9, and salivary amylase B a major peak at pH 5.9 and one minor peak at pH 6.4. Serum amylase was separated into two major peaks with isoelectric points (pl) of 6.4 and 7.0, respectively, and one minor peak, with a pl of 5.9. Only a small part of the serum amylase with a pl of 5.9 combined with concanavalin A; the two other serum amylases did not.

Human pancreatic amylase polymorphism: Formal genetics and population genetics

The genetically determined polymorphism of human pancreatic amylase (E.C. 3.2.1.1), AMY2, is demonstrated in serum specimens by agarose gel electrophoresis. We investigated 325 mother-child pairs and 2594 unrelated individuals from southwestern Germany. This study confirms the formal hypothesis of two common alleles AMY1/2, AMY2/2 and possibly two rare alleles AMY3/2, AMY4/2 at an autosomal locus AMY2. The frequency of the AMY1/2 allele was calculated as 0.951; AMY3/2 and AMY4/2 seem to have a frequency of 0.001 in this sample.

Modification of human pancreatic amylase isozymes by peptidoglutaminase I and II

The effects of peptidoglutaminase (PGln-ase) I and II on human pancreatic juice amylase purified as an isozyme were investigated. Several amylase isozymes were formed which corresponded to minor components of pancreatic amylase isozymes, indicating that appearance of amylase isozymes are due to enzymic deamidation. A similar result was observed when the purified amylase isozyme was incubated with the supernatant of human pancreatic juice whose amylase was previously removed by adsorption onto raw corn starch. These findings are discussed in connection with amylase isozymes in the sera of the patients suffering from pancreatic inflammation.

Influence of Amylase Assay Technique on Renal Clearance of Amylase-Creatinine Ratio

The influence of amylase assay technique on the renal amylase/creatinine clearance measurement was determined by analysis of serum and urine specimens obtained from 10 normal subjects. CAm/CCr averaged 2.19 +/- 0.18% with a saccharogenic technique, 1.52 +/- 0.2% with an iodometric technique, and 0.80 +/- 0.08% with a chromogenic technique. Each of these values differed significantly (P less than 0.05) from the other two. Recovery studies were carried out by adding partially purified human salivary or pancreatic amylase to human newborn serum or urine (which contain minimal endogenous amylase). Equal amylase activity was recovered from serum and urine by the saccharogenic technique whereas recovery from urine was less than 50% of that from serum using the iodometric and chromogenic techniques. The accuracy of the chromogenic technique is markedly improved by the addition of albumin to the urine assay system. Although it appears that only the saccharogenic method provides an accurate estimate of CAm/CCr, each assay technique distinguished the elevated CAm/CCr of patients with pancreatitis from the normal range established for that technique. Accurate clinical interpretation of CAm/CCr measurment requires knowledge of the amylase assay technique used.