Patent Publication Number: US-4550077-A

Title: β-Amylase determination

Description:
This invention relates to α-amylase determination. 
     Determination of α-amylase in a body fluid, in particular, serum, can be used for the diagnosis and confirmation of acute pancreatitis. Extremely elevated levels of α-amylase (21/2 times the upper limit of normal) are usually only found in acute pancreatitis; however, several other conditions such as biliary colic, perforated gall bladder, pneumonia, perforated appendix or perforated ulcer of the stomach, duodenum or ileum will also produce increased α-amylase activity in serum and urine. 
     The present invention is directed to a new and improved reagent, reagent package and method for determining amylase. 
     In accordance with one aspect of the present invention, there is provided a reagent or kit for α-amylase determination comprised of an amylaceous polysaccharide modified to include blocking groups which do not substantially prevent cleavage by α-amylase and which prevent an α-amylase-free exo-acting amylase from acting on the polysaccharide; an enzyme or enzyme mixture which includes an α-amylase-free exo-acting amylase which releases glucose from the cleaved polysaccharide; buffer free of enzyme inhibitor; and an enzymatic glucose rate detector. The components are present in an amount effective for α-amylase determination. 
     In accordance with another aspect of the present invention, there is provided a reagent package for determination of α-amylase comprised of a container and a dry mixture in the container comprised of an amylaceous polysaccharide modified to include blocking groups which do not prevent cleavage by α-amylase and which prevent an α-amylase-free exo-acting amylase from acting on the polysaccharide; an enzyme or enzyme mixture which includes an α-amylase-free exo-acting amylase which releases glucose from the cleaved polysaccharide; buffer free of enzyme inhibitor; and an enzymatic glucose rate detector. The components are presented in an amount effective for α-amylase determination. 
     In accordance with a further aspect of the present invention, there is provided a method for determining α-amylase by mixing an amylase-containing sample with an amylaceous polysaccharide modified to include blocking groups which do not prevent cleavage by α-amylase and which prevent an α-amylase-free exo-acting amylase from acting on the polysaccharide; an enzyme or enzyme mixture which includes an α-amylase-free exo-acting amylase which releases glucose from the cleaved polysaccharide; buffer free of enzyme inhibitor; an enzymatic glucose rate detector, and subsequently measuring absorption at one or more time intervals or continuously to thereby determine amylase. 
     In accordance with the present invention, the blocked polysaccharide substrate is unblocked by the action of α-amylase, and the unblocked polysaccharide substrate releases glucose by the action of the enzyme. The rate of glucose production provides a measure of α-amylase activity and, accordingly, such α-amylase activity can be determined by measuring the rate of glucose production. Thus, in accordance with the present invention, α-amylase is determined by providing and using a reagent which includes the blocked amylaceous polysaccharide, enzyme as hereinabove described, buffer, and an enzymatic glucose rate detector. 
     The amylaceous polysaccharide substrate is modified to include blockages to the action of the enzyme; e.g., glucoamylase, whereby the enzyme will not hydrolyze such polysaccharide; however, in the presence of α-amylase, there are internal cleavages of the chains to expose chain ends on which the exo-acting enzyme can act. As representative examples of suitable amylaceous polysaccharides, there may be mentioned starch (potato starch, corn starch, wheat starch, rice starch, tapioca, glycogen, etc.), or purified starch fractions (amylose, amylopectin) and the like. A partially hydrolyzed starch is a preferred substrate, e.g., partially hydrolyzed potato starch. Such amylaceous polysaccharides may be modified to provide blockages to the action of glucoamylase by procedures known in the art. Thus, for example, such blockages can be introduced by limited periodate oxidation; by substitution; for example, carboxymethylation, esterification (with acids, acid anhydrides, acid chlorides); hydroxyethylation with ethylene oxide and other procedures known in the art. The preferred substrate is carboxymethylated starch (starch glycolate). The method of blocking the polysaccharide is coordinated with the polysaccharide so as to provide for solubility in the test media (generally water), with the blocking not being effected to a degree which would prevent action by α-amylase and subsequent glucose release. 
     In employing carboxymethylated starch as the substrate, in order to provide solubility and sufficient available glucose for the assay, in general, the ratio of carboxy methylated glucose to glucose in the starch is from 1:4 1:16, with a ratio of from 1:6 to 1:10 being preferred. In preparing the blocked substrate, subsequent to the blocking, the blocked substrate is reacted with the enzyme to be used in the assay reagent kit to degrade any unblocked glucose segments susceptible to action of the enzyme without prior action of α-amylase. 
     The enzyme employed in the kit is an enzyme or enzyme mixture which is free of α-amylase and which includes an exo-acting amylase which converts the α-amylase cleaved polysaccharide to glucose. A particularly preferred enzyme is α-amylase-free glucoamylase. Another suitable enzyme is an α-amylase-free mixture of β-amylase and a maltase preferred α-amylase free glucoamylase is available from Biscayne Biochemical Laboratories in Miami, Fla. Miles Laboratories also sells an α-amylase free glucoamylase. 
     The buffer, as hereinabove noted, is one which is free of an enzyme inhibitor so as to permit continuation of enzymatic activity for the rate measurement employed for determining amylase. The buffer generally includes an α-amylase activator; e.g., chloride ions. The buffer should be one which maintains a pH in the order of from 5.0 to 9.0, and preferably from 6.0 to 7.5. The pH is selected to optimize the enzyme activity and in view of the fact that four enzymes participate in the assay, the selected pH is generally a compromise between the best pH for each of the enzymes. A preferred buffered pH is 6.9. As representative examples of suitable buffers, there may be mentioned: β-glycerophosphate, 3-(N-Morpholino) propane sulfonic acid (MOPS), N-2-hydroxyethylpiperazine-N&#39;-2-ethane sulfonic acid (HEPES), N-2-acetamide-2-aminoethanesulfonic acid (ACES), imidazole and others. 
     The enzymatic glucose rate detector is one which can provide a measurement of the rate of glucose production. A preferred glucose rate detector is a glucose-hexokinase detector system, with such detector system including adenosine triphosphate (ATP), β-nicotinamide-adenine dinucleotide (NAD); hexokinase; and glucose-6-phosphate dehydrogenase (G-6-PDH). The enzymatic reactions are represented by the following: ##STR1## Wherein ADP is adenosinediphosphate, G-6P is glucose-6-phosphate, and NADH is the reduced form of β-nicotinamide-adenine dinucleotide. 
     Another representative glucose rate detection system employs glucose oxidase and peroxidase, as disclosed, for example by Trinder, J. Clin. Path., 22, 246 (1969). 
     NADH absorbs light at 340 nm, while NAD does not, and the rate at which NADH is evolved is directly proportional to the increase in absorbance of light at 340 nm at constant temperature, usually in the range of 15° to 50° C., and constant pH. The rate of formation of NADH is proportional to the rate at which glucose is evolved, which in turn is proportional to α-amylase concentration in the sample, whereby the increase in absorbance at 340 nm can be used as a direct measure of the original concentration of α-amylase in the sample. It is to be understood that a wavelength other than 340 nm could be employed. 
     In accordance with the preferred embodiment of the present invention, the hereinabove described four components of the α-amylase determining reagent are provided as a mixture, preferably a dry mixture, and such dry mixture can be suitably packaged in a single vial. The reagents, if dry, are reconstituted in water for the α-amylase assay. It is to be understood, however, that the reagent kit may include separate vials of each of the reagents or separate vials which include more than one reagent instead of an intimate mixture of all reagents in a single vial or container. An advantage of the present invention, however, is that the reagents can be premixed in the kit in a single vial or container, preferably as a dry mixture. 
     In accordance with the assay of the present invention, a sample containing α-amylase, such as a serum sample, is added to an aqueous mixture of the blocked amylaceous polysaccharide, enzyme of the type hereinabove described, buffer free of enzyme inhibitor and enzymatic glucose rate detector, with the α-amylase causing the hydrolysis of the unblocked portions, with the exo-acting enzyme immediately acting on the resulting non-reducing chain ends to release glucose molecules. The glucose released is measured by the enzymatic glucose rate detector, and the rate of glucose production is directly related to the α-amylase activity of the sample. In this manner, α-amylase can be rapidly detected in a simplified procedure which involves only addition of a sample containing α-amylase to a reagent solution, followed by appropriate measurement of α-amylase activity by measuring the rate of glucose release. 
    
    
     The invention will be further described with respect to the following drawing, wherein: 
     The drawing is an embodiment of a reagent package for determining α-amylase. 
    
    
     Referring to the drawing, there is shown a reagent vial, schematically designated as 10, which includes dry α-amylase reagent, schematically generally indicated as 11, with such α-amylase reagent being comprised of blocked amylaceous polysaccharide as hereinabove described; enzyme for releasing glucose from the amylaceous polysaccharide subsequent to cleaving thereof by α-amylase, as hereinabove described; buffer free of enzyme inhibitor; and an enzymatic glucose rate detector. 
     Thus, for example, the dry α-amylase reagent may be formulated as follows, in an amount to give on reconstitution with distilled or deionized water the following nominal concentrations: 
     
         ______________________________________                                    
Carboxymethylated Starch                                                  
                       2.3 g/liter                                        
Sodium Glycerophosphate                                                   
                       50 mmol/liter                                      
Calcium Chloride       5 mmol/liter                                       
ATP                    1.3 mmol/liter                                     
NAD                    1.3 mmol/liter                                     
Magnesium Ion         &gt;12 mmol/liter                                      
Hexokinase (yeast)    &gt;3 ku/liter                                         
α-amylase-free Glucoamylase                                         
                      &gt;3 ku/liter                                         
(microbial)                                                               
G-6-PD (L.mesenteroides)                                                  
                      &gt;3 ku/liter                                         
pH 6.9                                                                    
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     The carboxymethylated starch employed in the kit is preferably prepared as follows: 
     A suspension of one gram Hydrolysed Starch, Catalog No. 2901-02-7, manufactured by Connaught Laboratories, Ltd., 1755 Steeles Avenue, W., P.O. Box 1755, Willowdale, Ontario, Canada, in 16 ml deionized water containing 0.20 g sodium chloride and 1.5 g sodium hydroxide, is warmed to 50° C., allowed to react with 0.47 g sodium monochloroacetate, and kept at 50° C. for 3 hours. The solution is dialyzed for 24 hours against water, treated with acetic acid to adjust to pH 4.3±0.2, then incubated for 3 hours at 37° C. in the presence of 1,100 U of glucoamylase (Aspergillus niger: amylase-free). After dialysis of 72 hours against water, the substrate solution is lyophilized and stored refrigerated at 4°-8° C. 
     The α-amylase free glucoamylase is obtained from Biscayne Biochemical Laboratories in Miami, Fla. 
     Thus, for example, vial 10 may be either a 3 ml or a 16 ml vial, which when reconstituted with 3 ml or 16 ml of deionized water, respectively, provides the hereinabove described nominal concentrations. It is to be understood, however, that other suitable vial amounts may be employed. 
     The following is a test procedure for determining α-amylase using a 3 ml reagent vial having the hereinabove described components: 
     1. Reconstitute dry contents of tube with 3.0 ml of distilled or deionized water. Gently invert to solubilize contents. 
     2. Transfer contents to a cuvet and warm to 37° C. 
     3. Add 0.01 ml of serum. 
     4. Incubate 5 minutes at 37° C. 
     5. Measure the average change in absorbance per minute (ΔA/min) at 340 nm, taking readings at convenient time intervals. The temperature must be held constant in the cuvet. Measure against a water blank. 
     NOTE: A highly elevated Amylase (ΔA/min=0.200 or greater) may exhaust the substrate before measurement can be taken. In this instance, dilute the sample with saline and multiply the results by the dilution factor. 
     RESULTS 
     Units--One α-amylase unit (U) is the amount of α-amylase that is necessary to cause the hydrolysis of one micromole of glucosidic linkages in the blocked starch per minute at 37° C. 
     
         U/liter=ΔA/min×K-factor 
    
     
         U/liter@340 nm=ΔA/min×12,100 
    
     
         U/liter@366 nm=ΔA/min×22,800 
    
     
         U/liter@334 nm=ΔA/min×12,550 
    
     EXAMPLE 
     
         ______________________________________                                    
Creep rate of reagent 0.002/min                                           
Serum volume          0.01 ml                                             
Reaction volume       3.01 ml                                             
5 min Absorbance reading @ 340 nm 0.582                                   
7 min Absorbance reading @ 340 nm 0.716                                   
Reaction temperature  37° C.                                       
 ##STR2##                                                                 
True Δ A/min = 0.067 - 0.002 = 0.065                                
U/liter = 0.065 × 12,100 × 1 = 786.5                          
DERIVATION OF FORMULA                                                     
 ##STR3##                                                                 
 ##STR4##                                                                 
= Δ A/min × 12,100                                            
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     when 
     A/min=change in absorbance per minute 
     10 6  converts moles to micromoles. 
     Lightpath=1.0 cm 
     4=approximate number of molecules of glucose formed per scission of starch molecule. 
     6.22×10 3  =molar absorptivity for NADH at 340 nm. 
     When testing at a wavelength other than 340 nm, the proper molar absorptivity value must be substituted for 6.22×10 3 . The molar absorptivity for NADH at 366 nm=3.3×10 3  and at 334 nm=6.0×10 3 . 
     The present invention is particularly advantageous in that there is provided a new and improved reagent, and reagent package, as well as a method, which permits the rapid assay of α-amylase in a single vial, which can be adapted to automation. Thus, the assay can be conducted in a time period in the order of 7 minutes with a minimum amount of assay steps. 
     These and other advantages should be apparent to those skilled in the art from the teachings herein. 
     Although the present invention is particularly applicable to the determination of α-amylase in physiological fluids, the scope of the invention is not so limited. For example, the invention has applicability to determination of α-amylase in plant extracts, microbial culture filtrates, etc. 
     Numerous modifications and variations of the present invention are possible in light of the above teachings and, therefore, within the scope of the appended claims, the invention may be practised otherwise than as particularly described.