Alpha-amylase In Serum Research Articles

Activities of Alpha Amylase in Serum and Saliva of Some Nigerian Cigarette Smokers

This study reports the effect of cigarette smoking on serum and salivary <img src=image/10.5923.j.als.20120201.04_001.gif></img>-amylase activities of some consenting Nigerian male smokers (20) in apparent good health and between 30 to 70 years. Age and sex-matched non-smokers (20) were included as control subjects. Alpha amylase activity was assayed using standard colorimetric method. Results obtained show that the mean α-amylase activity in serum and saliva were significantly higher (<i>P</i><0.05) in smokers (131.2 ± 17.0 IU/L and 241.8 ± 23.6 IU/L) when compared with non-smokers (68.3 ± 11.3 IU/L and 133.5 ± 8.6 IU/L), and these represent 84.3% and 95.2% increases in serum and saliva values for the smokers relative to the non-smokers. Evidence from this study indicates a measure of pancreatic, salivary and parotid glands’ dysfunction among the cigarette smokers in Nigeria. Measurement of serum and salivary <img src=image/10.5923.j.als.20120201.04_002.gif></img>- amylase activity could be useful in the early detection and diagnosis of pancreatitis and parotitis.

Biological variation in the catalytic concentration of pancreatic a-amylase and triacylglycerol lipase in serum

The within-subject and between-subject biological variation in the catalytic concentration of pancreatic alpha-amylase and triacylglycerol lipase in serum have been studied. The study was performed during a 13-month period in a group of 42 supposedly healthy individuals, 19 women and 23 men. All measurements were carried out using a Hitachi 717 analyser at 37 degrees C. The medians of the within-subject biological coefficients of variation obtained for the catalytic concentration of pancreatic alpha-amylase and triacylglycerol lipase in serum were 8.1 and 38.9%, respectively. The index of individuality for the catalytic concentration of pancreatic alpha-amylase and triacylglycerol lipase in serum was 0.27 and 1.20, respectively. Consequently, it is not advisable to use population-based reference intervals for the catalytic concentration of pancreatic alpha-amylase and triacylglycerol lipase in serum.

Immunocatalytic assay of pancreatic alpha-amylase in serum and urine with a specific monoclonal antibody.

In this immunocatalytic assay for alpha-amylase (EC 3.2.1.1) of pancreatic origin, a highly specific monoclonal antibody coupled to plastic beads is used to extract pancreatic amylase from samples, leaving salivary amylase in solution. The catalytic activity of the bound pancreatic amylase is then determined with blocked p-nitrophenyl maltoheptaoside as substrate. The method shows no cross-reactivity with salivary amylase, analytical recovery is 89-109% for pancreatic amylase, and interassay imprecision is 7.1-7.7%. We used the method to determine pancreatic amylase in serum and urine from healthy controls and different patient groups. The reference intervals for 34 supposedly healthy controls were: serum, 10-48 U/L (mean 27 U/L); urine, less than 20-435 U/L (mean 104 U/L). Results by the present assay correlated well with a salivary amylase inhibition assay (Boehringer Mannheim). We conclude that the described immunocatalytic assay is clinically useful for detecting increased activities of pancreatic amylase in serum and urine.

Reference ranges for alpha-amylase in serum and urine with 4,6-ethylidene-(G7)-1-4-nitrophenyl-(G1)-alpha,D-maltoheptaoside as substrate.

Reference ranges for alpha-amylase in serum, spontaneously voided urine, and 24 h urine were determined, using 4,6-ethylidene-(G7)-1-4-nitrophenyl-(Gl)-alpha,D-maltoheptaoside as the substrate (EPS method), at 25, 30, and 37 degrees C. The measured values were evaluated with and without the use of a factor which converts the results of the alpha-amylase EPS method into values comparable to those obtained with the alpha-amylase PNP method (substrate: 4-nitrophenyl-alpha,D-maltoheptaoside); comparison with the established reference ranges of the PNP method was therefore possible. The values for urine sometimes deviated markedly from the PNP reference ranges, but the values for serum showed close agreement. With the use of the conversion factor, the following reference ranges are proposed for the new alpha-amylase method: Serum (186 males and 131 females): up to 120 U/l (25 degrees C), up to 160 U/l (30 degrees C), and up to 220 U/l (37 degrees C). Spontaneously voided urine: up to 600 U/l (n = 323, 25 degrees C), up to 800 U/l (n = 373, 30 degrees C), and up to 1000 U/l (n = 373, 37 degrees C). 24 h urine: up to 450 U/24 h (n = 90, 25 degrees C), up to 650 U/24 h (n = 129, 30 degrees C), and up to 900 U/24 h (n = 129, 37 degrees C).

Differential assay of salivary and pancreatic alpha-amylase in serum and urine, with use of monoclonal antibody to human salivary amylase immobilized on bacterial cell wall.

We previously reported (Clin Chim Acta 1986;159:89) that bacterial cell wall chemically coated with a monoclonal antibody specific to human salivary (S) amylase (EC 3.2.1.1) could be successfully used to separate S and pancreatic (P) amylase in solution. We have now applied this method to serum and urine samples and found that the activities of S and P amylases so measured correlated well with those measured by the isoamylase inhibitor method. The present method is simple and reliable for routine clinical tests.

α-Amylase: Richtwerte bei Kindern

In a group of 216 healthy children (age: 3 days-16 years) normal values of alpha-amylase in serum were found out (substrate: p-nitrophenyl-alpha, D-maltoheptaosid). Infants (age: 3-353 days) mostly showed lower catalytic concentrations than older children: alpha-amylase (25 degrees C) up to 30 U/L; alpha-amylase 37 degrees C): up to 60 U/L. But, the collective was too small (n = 30) to give any statistical statement, on the other hand biological and analytical deviations of catalytic concentrations were big. In children (n = 186; age: 1-16 years) the values were not dependent on sex or age. As normal range we propose for children aged 1-16 years alpha-amylase (25 degrees C): 20-110 U/L; alpha-amylase (37 degrees C): 40-200 U/L.

Fluorometric rate assay of alpha-amylase using an intramolecularly-quenched fluorescent substrate (FG5P).

The complete hydrolysis of a fluorogenic derivative of rho-nitrophenyl alpha-maltopentaoside, FG5P, by human salivary alpha-amylase, resulted in a 5-fold increase in fluorescence. This is due to disruption of the intramolecular quenching of the fluorescence of the 2-pyridylamino residue by the rho-nitrophenyl residue by separation of the two residues. This change of fluorescence accompanying the cleavage of the glucosidic bond was exploited to develop a fluorometric rate assay of alpha-amylase in human serum.

Fluorescence depolarization assay for quantifying alpha-amylase in serum and urine.

We have developed a new method for quantifying alpha-amylase (EC 3.2.1.1) in serum and urine by fluorescence depolarization. Amylase in the sample catalyzes the hydrolysis of the substrate, a fluorescein-labeled amylose. This results in decreased fluorescence polarization, owing to the increased rate of rotation of the amylose fragment relative to the intact substrate. The TDx amylase assay is calibrated with six human-serum-based pancreatic amylase calibrators. Amylase activities are determined by interpolation from the calibration curve, which is stored in the TDx analyzer's memory. Results correlate well with those by the Du Pont aca assay and the Beckman "DRI-STAT" assay. Endogenous glucose does not interfere. CVs are less than 6%, and the reagents are stable in liquid form.

Rapid quantitative, specific measurement of pancreatic amylase in serum with use of a monoclonal antibody.

In this rapid quantitative assay for pancreatic alpha-amylase (EC 3.2.1.1) in serum, we precipitate salivary amylases by 10-min incubation with monoclonal anti-salivary amylase antibody immobilized on particles of polyvinylidene fluoride. We then centrifuge the serum mixture and measure the pancreatic amylase activity remaining in the supernate by a kinetic method. The assay requires 50 microL of serum and the standard curve is linear to at least 1300 U of pancreatic amylase per liter of serum. CVs were 1.3% within-run, 6-8% day-to-day. Apparent analytical recovery of pancreatic amylase activity added to serum was 101% +/- 2%. Addition of purified salivary amylase, 356 U/L, to sera gave a value for apparent pancreatic amylase of less than 4 U/L, or 1% of the added salivary amylase activity. This assay correlated well with an electrophoretic method (slope, 0.97-0.99; intercept, 0.5 to -4 U/L; correlation coefficient, 0.946-0.990; and standard error of the estimate 3-5 U/L). Estimated normal reference intervals with maltotetraose as substrate were: total amylase, 39-118 U/L; pancreatic amylase, 11-50 U/L; and salivary amylase, 18-79 U/L.

Optimized conditions for determining activity concentration of alpha-amylase in serum, with 1,4-alpha-D-4-nitrophenylmaltoheptaoside as substrate.

We describe a method for measuring the catalytic activity of alpha-amylase (EC 3.2.1.1) in serum and urine, by use of a defined substrate: 1,4-alpha, D-4-nitrophenyl maltoheptaoside. We use a phosphate buffer of pH 7.10, containing chloride as activator and alpha-glucosidase (EC 3.2.1.20) as the auxiliary enzyme. After a lag phase of 4 min at 25 degrees C or 30 degrees C, or 3 min at 37 degrees C, the increase of absorption of 4-nitrophenol is measured at 410 nm or 405 nm. The pH value of the assay mixture is a compromise between optimum pH for the alpha-amylase reaction, shortest possible lag phase, and an acceptable absorptivity of 4-nitrophenol. Because the dissociation of 4-nitrophenol depends strongly on pH and temperature, we determined its absorptivity with various combinations of these variables in the assay. Heparin-treated plasma can be used, but not EDTA, fluoride, or citrate. Lipemia, hemoglobin less than or equal to mumol/L, bilirubin less than or equal to 170 mumol/L, glucose less than or equal to 100 mmol/L, and ascorbic acid less than or equal to 1 mmol/L of sample do not interfere in the assay.

Reference interval for serum alpha-amylase determined with p-nitrophenyl-alpha-D-maltoheptaoside as a substrate.

The reference interval for the catalytic concentration of alpha-amylase (EC 3.2.1.1) in serum was determined in 326 blood donors. The analytic method used p-nitrophenyl-alpha-D-maltoheptaoside as substrate, which allows a continuous colorimetric measurement at 37 degrees C of the catalytic concentration of alpha-amylase in serum. The limits obtained were 0.7-3.8 mukat/l (42-228 U/l), which were in agreement with the only previous report found.

2-Chloro-4-nitrophenyl-beta-D-maltoheptaoside: a new substrate for the determination of alpha-amylase in serum and urine.

Tests for the determination of alpha-amylase activity based on oligosaccharides or their 4-nitrophenyl derivatives as substrates suffer from some disadvantages. Using a more acid indicator molecule and connecting this group in the beta-form to maltoheptaose leads to a new substrate, 2-chloro-4-nitrophenyl-beta-D-maltoheptaoside, for the determination of alpha-amylase activity. Due to its pK value, only half of the 4-nitrophenol formed from 4-nitrophenyl-alpha-D-maltoheptaoside is spectrophotometrically detectable, whereas the total product from the new substrate is responsible for a measurable absorbance. In addition the molar lineic absorbance of the product is 1.8 times higher. Thus the new test is more sensitive than those employing 4-nitrophenyl derivatives. Both P-type and S-type alpha-amylase isoenzymes have the same affinity for the new substrate; this is not the case for all the other substrates, i.e. oligosaccharides and their 4-nitrophenyl derivatives, used in alpha-amylase tests. The sample volume may be reduced, thereby removing interference by substances like bilirubin, glucose and haemoglobin. The test is insensitive to changes in temperature and pH and can be adapted to all mechanized analytical instruments.

Preparation of a New Fluorogenic Substrate of α-Amylases and a Simple α-Amylase Assay by HPLC

A new substrate of alpha-amylases, O-6-deoxy-6-[(2-pyridyl)amino]-alpha-D-glucopyranosyl-(1 leads to 4)-O-alpha-D-glucopyranosyl-(1 leads to 4)-O-alpha-D-glucopyranosyl-(1 leads to 4)-O-alpha-D-glucopyranosyl-(1 leads to 4)-D-glucopyranose, was prepared using dextrin as a starting material. Compared with other substrates so far reported, the fluorogenic substrate is unique in that it is resistant to exo-alpha-glucosidases due to the blocking group introduced into the non-reducing end glucose residue. The product of alpha-amylase digestion was rapidly separated from the substrate and was detected very sensitively by HPLC and a fluorescence detector. This method for alpha-amylase assay was also applied for determination of alpha-amylase in human serum.

New saccharogenic determination of alpha-amylase in serum and urine.

I describe a new kinetic enzymatic saccharogenic method for assaying alpha-amylase in human serum and urine. alpha-Amylase liberates maltose from starch. This is successively acted on by alpha-glucosidase, mutarotase, and glucose dehydrogenase. The resulting conversion of NAD+ to NADH, measured at 340 nm, during a 20-min incubation reflects amylase activity. Endogenous glucose is destroyed before measurement of amylase activity is begun.

Enzymatic, fluorometric assay of alpha-amylase in serum.

An enzymatic, fluorometric method for kinetic assay of serum alpha-amylase (1,4alpha-D-glucan 4-glucanohydrolase, EC 3.2.1.1) is described. A soluble starch is used as substrate, in tris(hydroxymethyl)methylamine buffer. All measurements are made in Pyrex cuvettes at 37 degrees C, with a reaction volume of 1.16 ml. The assay is based on the following reaction sequence: (see article) The rate of appearance of fluorescence of NADH (lambdaex = 365 nm, lambdaem = 460 nm), developed in the indicator reaction (4), is measured and equated to the activity of alpha-amylase in serum. A calibration plot of the change of fluorescence per min vs. enzyme concentration shows a good proportionality in the range of 0.50-5.0 kU/liter.

ALPHA-Amylase activity in sweat and serum of patients with cystic fibrosis of the pancreas.

The hypothesis by Doggett and Harrison, according to which alpha-amylase is the pathogenic factor of the exocrinopathy in cystic fibrosis (C.F.), is investigated. No elevation of alpha-amylase in sweat and serum of C.F. patients, as compared with controls of similar age, is observed. It is concluded that the "C.F. factor" cannot be identified with alpha-amylase.