Amylases, determination

Amylase enters the blood largely via the lymphatics. An increase in hydrostatic pressure in the pancreatic ducts leads to a fairly prompt rise in the amylase concentration of the blood. Neither an increase in volume flow of pancreatic juice nor stimulation of pancreatic enzyme production will cause an increase in senm enzyme concentration. Elevation of intraductal pressure is the important determinant. Stimulation of flow in the face of obstruction can, however, augment the entry of amylase into the blood, as can disruption of acinar cells and ducts. A functional pancreas must be present for the serum amylase to rise. Serum amylase determination is indicated in acute pancreatitis in patients with acute abdominal pain where the clinical findings are not typical of other diseases such as appendicitis, cholecystitis, peptic ulcer, vascular disease or intestinal obstruction. In acute pancreatitis, the serum amylase starts to rise within a few hours simultaneously with the onset of symptoms and remains elevated for 2 to 3 days after which it returns to normal. The peak level is reached within 24 hours. Absence of increase in serum amylase in first 24 hours after the onset of symptoms is evidence against a diagnosis of acute pancreatitis (76). 

Opiates and other narcotics and analgesic drugs may unpre-dicatably elevate the serum amylase. The elevation may last up to 24 hours. Therefore it is important that blood for amylase determination be drawn before giving the patient analgesic drugs for pain. Elevation of the enzyme in peritoneal fluid is strong... 

The ratio of Cam/Ccr can be determined on a random urine sample and thus avoids the time, labor, and errors involved in collecting the timed urine specimen required for the urinary amylase determination. 

R134 Bais, R., Badenoch, J., Bayer, P.M., Foo, Y., Keller, H., Koller, P.U., Lein-berger, R., Weidemann, G. and Rosalki, S.B. (1989). a-Amylase determination with the Reflotron reagent carrier system Use of whole blood, plasma, and serum, and effect of isoenzymes. Clin. Chem. 35, 317-320. 

The difference in structure between amylose and amyiopectin is important when selecting the appropriate starch substrate for amylase determinations (see Chapter 21). The rate of hydrolysis is affected by structural differences in the starch. a-Amylase from the pancreas hydrolyzes internal a-l,4 glycosidic linkages. This hydrolysis results initially in the production of some maltose and a mixture of dextrins, which are subsequently hydrolyzed to maltose. The -1,6-... 

An example of a direct spectrophotometrical assay is the use of synthetic peptide -nitroanilide substrates to determine protease activity. The /)-nitroani1ine group Hberated from the substrates by the protease can be determined spectrophotometricaHy at 410 nm. An example of an indirect (coupled) spectrophotometric assay is the determination of a-amylase using -nitrophenyLmaltoheptaoside. Initially, the substrate is cleaved by the a-amylase and subsequentiy one of the reaction products, -nitrophenyLmaltotrioside, is cleaved by a-glucosidase, hberating -nitrophenyl, a chromophore... 

In the late 1960 s a new series of methods was introduced for the determination of amylase, involving the use of a starch-dye complex. Dyes such as Renazol brilliant blue (68) Reactive Red 2B (69) (used in the substrate Dy-Amyl, General Diagnostic Division, Warner-Chilcott Laboratories), Cibachrom Blue (70)... 

Seligson s group (95) has published a similar turbidimetric procedure but used nephelometry to measure continuously the effect of lipase on the light scattering of an olive oil emulsion. The instrumentation and approach is the same as that described above for the nephelometric determination of amylase. The method according to the authors is fast and precise with good specificity and sensitivity. The short time required for analysis makes it suitable for emergency use. The technical simplicity permits this method to be easily automated, and it appears to be the lipase method of choice. 

Zinterhofer, L. Wardlaw, S. Jatlow, P. and Seligson, D. Nephlometric determination of pancreatic enzymes I. amylase. Clin. Chim. Acta (1973), 43, 5-12. 

Ceska, M. Birath, R. and Brown, B. A new and rapid method for the clinical determination of amylase activities in human serum and urine. Optimum conditions. 

Determination of molecular mass of pectic enzymes The molecular mass were determined by gel filtration in a Sepharose CL-6B column (1,8 x 88cm) equilibrated and eluted with Tris-HCl 50 mM, pH 7,5 buffer, plus 100 mM KCl. Fractions (3,3 ml) were collected at a flow rate of 10 ml/h. Molecular mass markers were tyroglobulin (660 kDa) apoferritin (440 kDa) P-amylase (200 kDa) alcohol dehydrogenase (150 kDa) bovine serum albumin (66 kDa) and carbonic anhydrase (29 kDa). Urea-SDS-PAGE (7%) was carried out according to Swank and Munkres [12]. Molecular mass markers were myosin (205 kDa) p-galactosidase (116 kDa) phosphorylase b (97 kDa) bovine serum albumin (66 kDa), ovalbumin (45 kDa) and carbonic anhydrase (29 kDa). 

Figure 3. (A) Determination of molecular mass of pectic enzymes by gel filtration in Sepharose 6B. Molecular mass markers - tyroglobulin, 2- apoferritin, 3- p-amylase, 4-alcohol dehydrogenase, 5- bovine serum albumin, 6- carbonic anhydrase. (B) SDS-PAGE of pectolytic activities. Molecular mass markers 1- myosin, 2- p-galactosidase, 3- phosphorylase b, 4- bovine serum albumin, 5- ovalbumin, 6- carbonic anhydrase.

The diastase activity was traditionally determined according to the Schade method in the earlier years (Schade et al., 1958). One unit of diastase activity (or more specifically, a-amylase), DN, is defined as that amoimt of enz)nne that converts 0.01 g of starch to the prescribed endpoint in 1 h at 37 °C under the experimental conditions. In this assay, a standard solution of starch, which reacts with iodine to produce a color solution, is used as a substrate for honey enzymes under the standard conditions (Rendleman, 2003). A recently developed procedure uses an insoluble, dyed starch substrate (Persano Oddo and Pulcini, 1999). As this substrate is hydrolyzed by ot-amylase, soluble dyed starch fragments are released into solution. After reaction termination and insoluble substrate removal by centrifugation, absorbance of the supernatant solution (at 620 nm) is measured. The absorbance is proportional to the diastase activity. This procedure has been widely adopted in the honey industry due to the convenience of a commercially available substrate and the simple assay format. 

Levels of the enzyme a-amylase in wheat grains also affect breadmaking quality. Flour for bread-making requires low levels of a-amylase and this is favoured by a dry ripe dormant grain. The Falling Number (Hagberg) Test is used to determine a-amylase levels. A... 

Interpretation of the results of investigations to determine the mechanism of the action of amylases has frequently been difficult because of the lack of sufficient knowledge concerning the substrates on which these enzymes act. It is now generally accepted that starches are made... 

Alpha amylases cause a rapid fragmentation of starch with an accompanying marked decrease in viscosity of the starch solutions. Therefore, viscosity determinations may be used to follow the early stages in the hydrolysis of starch by these amylases. Afthough this type of measurement is of considerable importance for certain industrial applications, it has not been used to any great extent in investigations with pancreatic amylase. 

The slowing down of enzyme reactions has often been attributed to reaction with, or equilibrium between, the enzyme and its substrate or between the enzyme and the products of its action. In order to determine the influence of the products of the action of pancreatic amylase on the extent of the hydrolysis of starch, portions of its hydrolysis mixtures were subjected to efficient dialysis during hydrolysis and the results compared with aliquots of the reaction mixture which had been treated in the same way except for dialysis.41 The results of such experiments... 

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