JPH025400B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for measuring α-amylase activity, which is characterized by using a novel substrate, ie, a novel amylose derivative, as a substrate. α-Amylase activity in samples, especially in human saliva, pancreatic juice, blood, and urine, is important in medical diagnosis. For example, in pancreatitis, pancreatic cancer, and parotitis, α-amylase activity in blood and urine shows a marked increase compared to normal values. Various methods have been published so far for measuring α-amylase activity, but the main ones are:
It can be classified into three groups: amyloclastic method, chromogenic method, and satcalogenic method. Among the amyloclastic methods, the callaway method has been the most widely used, but it has problems such as coexisting proteins inhibiting the coloration of starch with iodine and poor reproducibility due to the short reaction time. The chromogenic method is generally referred to as the blue starch method, and is a method for measuring soluble pigments produced in an enzymatic reaction using an insoluble substrate in which a pigment is bound to starch or amylose. Although this method has been widely used recently, it has weak activity as a substrate, the reaction system is heterogeneous due to insolubility, and requires complicated operations, making it difficult to apply to automatic analyzers. There are other problems. The Somogyi method is a typical Satskalogenik method, but it shows high values due to glucose in the sample.
There are problems such as complicated operations. As described above, each of the conventional methods for measuring α-amylase activity has its own drawbacks. The disadvantage is that the reaction cannot be measured as a truly stoichiometric reaction. The present inventors have completed the present invention as a result of extensive research into a method for accurately measuring α-amylase activity using a reaction rate method and using an automatic analyzer. The present invention is characterized in that when measuring α-amylase activity, an amylose derivative having a carboxymethyl group (-CH ₂ COOH) or a salt thereof for every 6 to 35 glucose units of amylose is used as a substrate. This is a method for measuring amylase activity. The salt of the carboxymethyl group is preferably one that dissolves to give a carboxymethyl ion, but is not particularly limited, and representative examples thereof include alkali metal salts such as Na, K, Li, etc., and ammonium salts. α-Amylase is an enzyme that hydrolyzes α-1,4-glycosidic bond chains, and in starch or amylopectin containing α-1,6-glycosidic bonds, α-1,6-glycosidic bonds facilitate the α-amylase reaction. to disturb. Furthermore, since amylose has better chemical structure uniformity, it is superior to starch or amylopectin as a substrate for α-amylase, resulting in improved measurement accuracy. However, in the method of measuring glucose produced by conjugating glucoamylase or α-glucosidase to α-amylase using amylose as a substrate, glucoamylase or α-glucosidase can be used from the non-reducing end of amylose regardless of α-amylase. Since it is an exotype enzyme that hydrolyzes α-1,4-glycosidic bonds, it has the drawback of degrading the substrate regardless of the α-amylase reaction. As a result, it is not possible to contain both the substrate and glucoamylase or α-glucosidase in the measurement reagent solution, it is not possible to use enough glucoamylase or α-glucosidase for measurement, and there is a significant increase in reagent blinding, etc. It was difficult to assemble the measurement method. Furthermore, it is well known that amylose has extremely low solubility in water and is a molecule that is highly susceptible to retrogradation. [Starch Handbook P.58-91 Asakura Shoten (1961)] The present inventors introduced carboxymethyl groups or their salts into amylose having such defects at a ratio of 1 in 6 to 35 glucose units in amylose. The modified amylose (hereinafter referred to as modified amylose) has excellent affinity for amylase and is a good substrate, does not serve as a substrate for glucoamylase or α-glucosidase, and has a markedly increased solubility in water, which improves the amylase reaction. The inventors have completed the present invention by discovering that it can be used at a sufficient substrate concentration optimal for the purpose of use, and that stability in an aqueous solution is improved. Hereinafter, the present invention will be explained in detail by giving examples. Since glucoamylase or α-glucosidase is an exotype enzyme, it cannot use this modified substrate as a substrate. On the other hand, α-amylase is an endo-type enzyme that hydrolyzes any α-1,4 glycosidic bond of amylose, and can use this modified substrate as a substrate, so the modified substrate is hydrated by the enzymatic action of α-amylase. It is decomposed and a new non-reducing end is generated. α for this newly generated non-reducing end
-Glucosidase or glucoamylase acts to produce glucose, and α-amylase activity can be determined by measuring the amount of glucose produced. The reaction formula up to this point is shown below. (In the above formula, G represents a glucose unit, X represents a carboxymethyl group or a salt thereof, and n and m represent an integer of 2 or more.) There are many known methods for quantifying glucose, and any of these methods can be used. It goes without saying that it can be done. The main glucose quantification methods are shown below. First, when glucose oxidase is applied to glucose, it is oxidized and at the same time hydrogen peroxide is produced. The generated hydrogen peroxide quantitatively oxidizes the chromogen through the coexisting peroxidase, and the amount of glucose in the reaction solution can be measured by comparing the color of the generated oxidized chromogen. . The reaction formula is shown below. Glucose + O ₂ + H ₂ O glucose oxidase――――――――――→ H ₂ O ₂ + gluconic acid H ₂ O ₂ + chromogen peroxidase――――――――→ Oxidized chromogen +H ₂ O In addition, glucose is converted to glucose-6-phosphate by hexokinase in the presence of ATP. The produced glucose-6-phosphate becomes 6-phosphogluconolactone by glucose-6-phosphate dehydrogenase in the presence of NAD, and on the other hand, NAD is reduced to NADH, so the By measuring the increase in absorbance, the amount of glucose in the reaction solution can be measured.
The reaction formula is shown below. Glucose + ATR hexokinase ――――――――→ Mg ²⁺ Glucose −
6-phosphate + ADP Glucose-6-phosphate + NAD Glucose-6-phosphate dehydrogenase――――――――――――――→ 6-phosphogluconolactone + NADH ATP; Adenosine-5 ′-triphosphate ADP; adenosine-5′-diphosphate NAD; β-nicotinamide adenine dinucleotide NADH; reduced β-nicotinamide adenine dinucleotide In the standard method for determining α-amylase activity using this modified amylose. the coupled enzyme glucoamylase or α-amylase simultaneously or subsequently with α-amylase.
- Glucose produced by the action of glucosidase is measured, but it may be affected by glucose coexisting in the specimen, especially biological samples such as serum and urine, and the existing glucose is eliminated prior to the α-amylase reaction. This is an effective means for implementing this measurement method. Glucose oxidase is a method of elimination.
There are various methods such as a method using catalase, a method using hexokinase (JP-A-57-47495), and a method using glucose oxidase-peroxidase, and any of these methods can be freely combined. An example of a method for producing modified amylose used in the present invention is to react amylose with a molecular weight of about 3,000 to 200,000, sodium hydroxide, and monochloroacetic acid in an aqueous solution. For 1 mole of amylose glucose unit, alkali is 5 to 30
0.5 to 5 moles of monochloroacetic acid are used.
The reaction proceeds by heating and stirring at about 30 to 70°C for 30 minutes to 3 hours. This reaction was performed according to the method of S. Peat et al. [Nature, 159, 810 (1947)]. Next, after neutralizing the product, glucoamylase is added in a near-neutral aqueous solution and allowed to act at 37°C for 10 to 30 hours to introduce a carboxymethyl group or its salt into the glucose at or near the non-reducing end of the modified amylose. modified amylose. The general formula for the reaction is shown below. (Here, G represents a glucose unit, X represents a carboxymethyl group or a salt thereof, n is a positive integer, m
represents an integer from approximately 5 to 34. ) Although this glucoamylase treatment is not essential,
This is effective in lowering the level of blindness of measured values. Further, reaction by-products such as sodium chloride and sodium hydroxyacetate and glucose hydrolyzed by glucoamylase are removed by dialysis, in which the reaction product is dialyzed using water as an external solution. Then, after concentration, it is dried to obtain modified amyloid.
The degree of carboxymethylation of the sodium salt of carboxymethylated amylose of this product can be determined by PH titration. The number of modifying groups consisting of carboxymethyl groups or salts thereof introduced into amylose is an important factor in the precision and accuracy of α-amylase activity measurement. Introduction of too many modifying groups into amylose can lead to α
- If the action of amylase is hindered and a modifying group is introduced at a ratio of 1 or more to 5 glucose, α
-The action of amylase is reduced. Therefore, sensitivity, precision, and accuracy are reduced, making this method impractical. On the other hand, when the amount introduced is small, the contribution of the modifying group to improving the solubility of amylose is small, and modified amylose having sufficient solubility is not produced. In order to maintain the required substrate concentration, it is necessary that at least one modifying group is present in every 35 glucose molecules. Therefore, the modified amylose that contains one modifying group for every 6 to 35 glucose units of amylose is used. The sample to be measured in the present invention may be any specimen containing α-amylase, such as blood, serum, urine, etc. as biological components. Glucoamylase or α-glucosidase is not particularly limited, but for example, those derived from animals or microorganisms can be used. Furthermore, as a measurement condition for carrying out the present invention, the reaction temperature is not particularly limited, but is preferably about 25 to 40°C.
The reaction time can be freely selected depending on the purpose. The optimum pH is not particularly limited, but is approximately 6 to 6.
8 is a preferred example. Buffers that maintain the optimum pH can be freely used, such as phosphate and gas buffers. Further, as an activator for α-amylase, for example, sodium chloride, calcium chloride, potassium chloride, etc. are used. The method for measuring amylase activity may be a late assay that measures the reaction rate under certain conditions or a one-point assay that uses a reaction terminator, and either method can be used. By using the modified amylose of the present invention as a substrate for measuring α-amylase activity, the following (a) to
It is possible to assemble a measurement method that has the advantages of (g). (a) Since this modified amylose is easily soluble in water, a sufficient substrate concentration can be used for the α-amylase reaction, the calibration relationship is good, and the measurement range is wide. (b) This modified amylose is produced by glucoamylase, α-
Since it is not a substrate for glucosidase but a specific substrate for α-amylase, no side reactions occur and the reagent blind value is extremely small. (c) Since the necessary and sufficient amounts of glucoamylase and α-glucosidase can be used, reactions after the α-amylase reaction are fast and accurate late assays are possible. (d) It is possible to assemble a measurement method with high measurement sensitivity and accuracy by guiding the glucose measurement system. (e) Measurement operation is easy. (f) Good adaptability to automatic analyzers. (g) The test solution for measurement is stable. Examples will be shown below and explained in more detail. Example 1 Reagent for preparing modified amylose (1) Amylose Amylose with an average molecular weight of approximately 150,000 (2) Sodium hydroxide solution A 50% sodium hydroxide solution is prepared. (3) Monochloroacetic acid solution Prepare a 25% monochloroacetic acid solution. (4) Glucoamylase Rhizopus-derived experimental method Add 150 ml of water and 50 ml of sodium hydroxide solution to 10 g of amylose, and stir and mix at 50°C for 1 hour. Next, add 40 ml of 25% monochloroacetic acid, and add 50 ml of 25% monochloroacetic acid.
Warm at ℃ for 1 hour. After cooling to room temperature, neutralize with hydrochloric acid, add 1000 units of glucoamylase, and let it act at 37°C for about 15 hours. To remove sodium chloride, sodium hydroxyacetate, and glucose produced by the reaction, the reaction solution is placed in a dialysis tube and dialyzed for 50 hours using ion-exchanged water as the external solution. After concentrating this liquid, modified amylose is obtained by lyophilization. The yield was 90%. Add 0.1N hydrochloric acid to a certain amount of this product and perform PH titration with 0.1N sodium hydroxide to determine the degree of carboxymethylation of amylose. It was confirmed that sodium carboxymethylate was present at a rate of 1 for every 22 glucose units in amylose. Example 2 Reagent (1) Reagent 1 Gutud buffer (PIPES) 40 mmol, glucoamylase 50,000 units, glucose oxidase 10
10,000 units, mutarotase 200 units, catalase 50 units
10,000 units, 4-aminoantipyrine 0.7 mmol,
Sodium chloride 15mmol, calcium chloride 5m
Take mol of the solution, dissolve it in purified water, and adjust the pH to 6.9 with sodium hydroxide to bring the total volume to 1. (2) Reagent 2 Gutud buffer (PIPES) 40 mmol, peroxidase 15000 units, sodium chloride 15 mmol,
5 mmol of calcium chloride, 3 g of modified amylose prepared in Example 1, 15 m of sodium nitride
Take 10 mmol of phenol, dissolve it in purified water, adjust the pH to 6.9 with sodium hydroxide, and reduce the total amount to 1
shall be. Measurement method: Add 20μ of sample serum to 2ml of test solution 1 and heat at 37°C for 5 minutes. Add 1 ml of test solution 2 to this and
The reaction was carried out at 0.degree. C. and the absorbance change (ΔE/min) was measured at 505 nm for 3 minutes from 2 minutes to 5 minutes. Separately, a standard specimen with known α-amylase activity is used to prepare a calibration curve in the same manner as above, and the α-amylase activity of the specimen is determined from this calibration curve. The calibration curve at this time is shown in FIG. The results of reagent blind testing and amylase measurement are shown below as a comparative example, comparing those using amylose as a substrate and those using modified amylose. Comparative Example 1 (1) Test Solution 3 Same as Test Solution 1 in Example 2. (2) Test solution 4 Same as test solution 2 in Example 2. (3) Test solution 5 Using 0.3 g of amylose with an average molecular weight of 16000 in place of 3 g of amylose containing sodium carboxymethyl groups in test solution 2 of Example 2,
The following is the same as test solution 2 in Example 2. (4) Sample serum Serum with α-amylase activity of 300 Somogyi units. Measurement method: Add 20μ of sample serum to 2ml of test solution 3 and 20μ of purified water to the other and heat at 37°C for 5 minutes. Add 1 ml of these test solutions 4 and react at 37°C.
The absorbance change (ΔE/min) for 3 minutes from 2 minutes to 5 minutes at 505 nm is measured, and is used as the measurement result of serum amylase and reagent blind measurement when modified amylose is used as a substrate. In addition, test solution 5 is used in place of test solution 4, and the same procedure as described above is performed to obtain serum amylase and reagent-blind measurement results when amylose is used as a substrate. The results are shown in Table 1. 【table】

[Brief explanation of drawings]

FIG. 1 is a diagram showing the calibration relationship between α-amylase activity (Somogyi units/dl) and absorbance change at 505 nm (ΔE/min) of the standard specimen in Example 2. The α-amylase activity of the standard sample is
It is 500 somogyi units.

Claims (1)

[Claims] 1. In measuring α-amylase activity, an amylose derivative having a carboxymethyl group (-CH ₂ COOH) or a salt thereof for every 6 to 35 glucose units of amylose is used as a substrate. A method for measuring α-amylase activity. 2. The measuring method according to claim 1, wherein the amount of glucose produced is measured using glucoamylase or α-glucosidase as the conjugate enzyme of α-amylase.