Method for the determination of amylase
United States Patent 11 1 Adams METHOD FOR THE DETERMINATION OF AMYLASE [75] Inventor: Thomas H. Adams, Mission Viejo.  
 Calif.  
 [73] Assignee: E. I. du Pont de Nemours and Company, Wilmington. Del. [22] Filed: Sept. 6, 1973 I [21] Appl. No.: 394,823  
 [52] U.S. CI. 195/1035 [51] Int. Cl. Cl2k 1/04; C07g 7/02 [58] Field of Search 195/1035 [56} References Cited UNITED STATES PATENTS l2/l973 Bergmcycr ct al l95/l03.5  
 OTHER PUBLICATIONS Greenwood et al. Die Starke, Studies on Starch-De- 1 Apr. 22, 1975 grading Enzymes-Part VIII, 20 (5), 139-150.  
 Primary E.\&#39;aminerA. Louis Monacell Assistant E.raminerEsther L. Massung [57] ABSTRACT 15 Claims, 1 Drawing Figure PATENTEUAPRZZISTS METHOD FOR THE DETERMINATION OF AMYLASE BACKGROUND OF THE INVENTION l. Field of the Invention This invention relates to a procedure for the qualitative determination of a-amylase in a sample. and more particularly to a combination of enzymatic reactions for such a determination. The qualitative measurement of a-amylase concentrations in a sample is useful in medical diagnostics.  
 2. Discussion of the Prior Art The enzyme a-amylase is to be found in salivary and pancreatic juices. It is well known that this enzyme reacts with various carbohydrates. particularly starch, to form maltose, and that the enzyme maltase will react with maltose to form glucose. These two reactions form the basis for intestineal digestion. See, for example, Biochemistry by Cantarow and Schepartz, page 263.  
  It is also well known that the presence ofglucose can be determined by various coupled reactions. This knowledge has led to several proposed methods for measuring amylase activity in conjunction with maltose by utilizing its ability to convert starch to glucose. See, for example, the article by H. W. Schiwara in Arzll (&#39;lzah. 17:340-343 (1971 and the paper entitled Columt&#39;tric Rate Derw&#39;minarion of Serum Amylase Activity given at the National Symposium on Enzyme Chemistry in l972.  
  Techniques involving starch, however, do not allow truly quantitative amylase determinations. The amylase reacts with each of the constituents of starch to produce different reaction products, and the kinetics are unpredictable. This is particularly troublesome in rate determinations where the measurements are made within a short span of time. during which the reaction kinetics are even less predictable. As a result of this, measurements made using starch as the substrate for hexokinase Glucose ATP glucose-6-phosphate ADP Glucosc-6-phosphatc a-amylase activity do not, and in fact cannot, give a true measure of a-amylase concentrations in the test sample.  
  Furthermore, the test proceedings of the prior art are not capable of differentiating between salivary a-amylase and pancreatic a-amylase.  
  It is an object of the present invention to provide a process for the determination of a-amylase which allows quantitative measurements to be made. It is a further object of this invention to provide a rate determination process for the determination of amylase which will allow for rapid quantitative measurements of the amount of amylase present in a sample. It is a still further object of this invention to provide a process which will differentiate between salivary and pancreatic a-arnylase.  
 SUMMARY OF THE INVENTION These and other objects are accomplished by a process for determining the a-amylase content of a sample comprising the steps of adding a substrate selected from the group consisting of maltotetraose or maltopentaose to a solution containing a measured amount of the sample and then determining the products of the reaction between the a-amylase and the substrate while maintaining the solution at a substantially constant temperature and a substantially constant pH.  
  The reaction between pancreatic oz-amylase and substrate will produce maltose, maltotriose and glucose. The glucose evolving from the reaction between the pancreatic a-amylase and the substrate can be measured by any one ofa number of well known techniques which will be discussed below. Salivary a-amylase, however, does not produce glucose. The reaction between the salivary a-amylase and the substrate appears to produce only maltose. If the reaction solution c0ntained a maltase, preferably a-glucosidase, the maltose and maltotriose produced by the reaction between the substrate and the a-amylase will be converted to glucose and the glucose can be measured by any one of a number of conventional techniques.  
  Since only two sources of a-amylase are the pancreas and the salivary glands. total a-amylase can be determined by a solution containing both the substrate and the maltase; pancreatic a-amylase can be determined by a solution from which the maltase is absent. and salivary oz-amylase can be determined by taking the difference between the total and the pancreatic oz-amylase.  
  In the preferred embodiment, the substrate is maltotetraose and the step of determining the glucose evolved is accomplished by measuring the rate at which glucose is evolved. In a still more-preferred embodiment B-glycerol phosphate is used as a buffer. Any other conventional buffer which will not interfere with the ability of the ot-glucosidase to convert maltose into glucose can also be used.  
  There are a number of ways in which glucose can be determined. Any one of these ways can be used. A particularly convenient approach is to use a coupled enzyme determination for glucose. One suitable pair of coupled enzyme reactions are defined by the following:  
 a-ti-pna 6-phosphoggluconolactone hADl-l;  
  where ATP is adenosine triphosphate, ADP is adenosine diphosphate, NAD is B-nicotinamide-adenine dinucleotide, NADH is the reduced form of B-nicotinamide-adenine dinucleotide. G-6PDH is glucose-6- phosphate dehydrogenase. Since the reduced form of B-nicotinamide-adenine dinucleotide absorbs light very strongly at 340 millimicrons, while the oxidized form does not, the rate at which NADH is evolved is directly proportional to the increase in absorbance of light at 340 millimicrons at constant temperature, usually 15C. to 50C, and constant pH. This increase can readily be measured by those skilled in the art, using a conventional spectrophotometer. Since the rate of formation of NADH is proportional to the rate at which glucose is evolved. the increase in absorbance at 340 millimicrons can be used as a direct measure of the original concentration of a-amylase in the sample. It should also be understood that other wavelengths. e.g., 366 millimicrons, can also be used for the foregoing purpose.  
  Other pairs of coupled enzyme reactions can be used to determine the presence of glucose are the following:  
 glucose Glucose OXidase Peroxidase There are a number of other such reactions which can be used to measure the glucose content, or the glucose content may be measured directly.  
  Although oz-glucosidase has been mentioned as the preferred maltase for use in the present invention. it should be noted that other forms of maltase can be used in the present invention. In the preferred embodiment, the substrate plus the maltase. if maltase is used. are added to the solution last. This is so that any glucose present in the sample will be used up by the other materials, prior to the introduction of the substrate. By this method, any glucose that is measured, after the substrate has been added to the solution, is due to the presence of a-amylase in the sample. The invention will be described with reference to the following examples,  
 and FIG. 1 which is a plot of the change in absorbance EXAMPLE 1 An amylase activity standard curve was generated on a Cary 14 spectrophotometer by following the rate of change in the absorbance of NADH. The sample used for the standard curve solution was a 0.85 mg/ml solution of partially purified human pancreatic amylase dissolved in human serum. This resulted in a sample with an amylase activity of 1,600 Somogyi units/d1. Dilutions of this serum were made with a 0.85 percent saline solution to achieve lower value.  
  The following solutions were used in the assay. B-glycerol phosphate buffer. 0.06 M. pH 6.8 NAD. 75 mg/ml H O ATP. 30 mg/ml H O NaCl Mg S0 50 mg MgSO 30 mg NaCl/ml H O Hexokinase. 2456 lU/ml Glucose-o-phosphate dehydrogenase. 1,000 lU/ml maltotetraose. 200 mg/ml H O a-glucosidase. mg/ml The assay was performed as follows: To 4.50 milliliters of ,B-glycerol phosphate buffer, 0.1 milliliters of serum were added and allowed to equilibriate at 37C. for 1.5 minutes. The following reagents were then added: NAD, 0.1 ml (7.5 mg). ATP, 0.1 ml (3.0 mg) NaCl-MgSO 0.05 ml (1.5 mg &amp; (2.5 mg) Hexokinase, 0.01 ml IU) Glucose-6-phosphate dehydrogenase, 0.01 ml (10 1U) The reagents were mixed and the glucose in the sample was consumed in a two minute period. The reaction was then initiated by adding 0.04 ml a-glucosidase (10 IU) and 0.1 ml maltotetraose (20 mg). An increase in absorbance at 340 nm was monitored for five minutes. The change in absorbance over the 5 minute period for various solutions of the standard sample is shown by the open circles and the solid line in the figure.  
 EXAMPLE 2 The amylase detection system has also been adapted for automatic use by the Du Pont Automatic Clinical 9 Gluconic Acid H 0 Chromogen oxidized Chromogen H O Analyzer (aca). Reagents for the automatic determination were prepared as described below:  
 1. A blend containing maltotetraose, ATP, polyethylene glycol (PEG) 2000 and B-glycerol phosphate was prepared in the following proportions: 8 mg maltotetraose; 4.8 mg ATP; 7.2 mg PEG; 5.5 mg B-glycerol phosphate and 69.4 mg mannitol. This blend was tableted producing tablets which contained 4.4 mg maltotetroase. 2.2 mg ATP.  
 2. A tablet was prepared containing 8.3 mg NAD, 1.9 mg MgSO 4.1 mg PEG and mg fi-glycerol phosphate.  
 3. a-glucosidase, hexokinase and glucose-6- phosphate dehydrogenase were combined in a 50% glycerol albumin solution with 20 IU hexokinase, 18 IU glucose-6-phosphate dehydrogenase and 20 IU a-glucosidase used per test.  
  Analytical test packs for the aca were assembled containing two NAD. MgSO B-glycerol phosphate buffer tablets, one maltotetraose, ATP tablet and 0.050 mi11iliters of the enzyme solution. These reagents were placed in the reagent compartments of a test pack such as that described in US. Pat. No. 3,476,515.  
  The aca was programmed to use 0.100 ml. of sample. 4.9 ml of water, and to report the results of the assay in milliabsorbance units/minute. The sample used was similar to that used in Example 1 except that the sample had an amylase activity of 750 Somogyi units/d1. Detection of this sample were also made as disclosed in Example 1.  
  The results of tests on various dilutions of the sample are represented by the solid dots and the dashed line in. the figure.  
 EXAMPLE 3 Comparisons of the relative specificity of maltotetraose were made. Purified human pancreatic a-amylasc and human salivary a-amylase were compared as to their abilities to react with the substrate maltotetraose. 1n the presence of the complete coupling system, a-glucosidase, ATP, NAD and G-o-PDH, both amylases appeared to react similarly. However, in the absence of the oz-glucosidase, the salivary enzyme did not produce any glucose. This means that the salivary enzyme splits the maltotetraose into two maltose molecules only. The pancreatic enzyme produces a significant amount of glucose by its action on the maltotetraose directly. Thus. the method provides a means to determine the salivary and pancreatic amylase contributions to the gross rate, by running an additional assay in the absence of oz-glucosidase.  
 EXAMPLE 4 The specificity of maltopentaose was compared with that of maltotetraose using the following reagents: 2 NAD, MgSO ,B-glycerol phosphate buffer tablets (Of Example 2) sured at 340 nm after a 4 minute incubation in a Cary 14 spectrometer at 37C. The results were as follows:  
 Substrate AA/min.  
 test maltotetraose .078 blank .026 test maltopentaose 10 l blank .00 l 5 The results show that the observed rate with the same sample is higher for maltopentaose than for maltotetraose. In addition, the blank rate. i.e.. the reaction of the a-glucosidase with substrate is much higher with maltotetraose and is less desirable. Thus. maltopentaose would appear to be highly sensitive and would eliminate the need for closely monitoring the extent of the blank reaction.  
 What is claimed is:  
  l. A process for determining the pancreatic a-amylase content of a sample comprising the steps of adding a substrate selected from the group consisting of maltotetraose and maltopentaose to a solution containing a measured amount of the sample and determining the glucose evolved by the reaction between the pancreatic a-amylase and the substrate while maintaining the solution at a substantially constant temperature and a substantially constant pH.  
  2. The process of claim 1 wherein B-glycerol phosphate is added to the solution as a buffer.  
  3. The process of claim 2 wherein the substrate is maltotetraose.  
  4. The process of claim 3 wherein the step of determining the glucose evolved is accomplished by a coupled enzyme reaction.  
  5. The process of claim 3 wherein the step of determining the glucose evolved is accomplished by measuring the rate at which glucose is evolved.  
  b. A process for determining the total a-amylase content of a sample comprising the steps of adding a substrate selected from the group consisting of maltotetraosc and maltopentaose to a solution containing a measured amount of said sample and non-rate limiting amounts of a maltase; and determining the glucose evolved by the reactions which take place in the solution. while maintaining the solution at a substantially constant temperature and a substantially constant pH.  
  7. The process of claim 6 wherein B-glycerol phosphate is added to the solution as a buffer.  
  8. The process of claim 7 wherein the substrate is maltotetraose.  
  9. The process of claim 7 wherein the step of determining the glucose evolved is accomplished by a coupled enzyme reaction.  
  10. The process of claim 7 wherein the step of determining the glucose evolved is accomplished by measuring the rate at which glucose is evolved.  
  ll. The process of claim 7 wherein said maltase is wglucosidase.  
  12. The process for determining the salivary a-amylase content of a sample comprising the steps of determining the total a-amylase content of the sample by the process of claim 7; determining the panceratic a-amylase content of the sample by the process of adding a substrate selected from the group consisting of maltotetraose and maltopentaose to a solution containing a measured amount of the sample and determining the glucose evolved by the reaction between the pancreatic a-amylase and the substrate while maintaining the solution at a substantially constant temperature and a substantially constant pH; and subtracting the two values.  
  13. A process for determining the a-amylase content of a sample comprising the steps of: adding a measured amount of the sample to a solution containing non-rate limiting amounts of hexokinase. glucose-6-phosphate dehydrogenase sodium chloride, magnesium sulphate. and B-nicotinamide-adenine dinucleotide; adding to the solution a-glucosidase and maltotetraose; and determining the rate at which glucose is evolved while maintaining the solution at a substantially constant temperature and a substantially constant pH.  
  14. The process of claim 13 wherein B-glycerol phosphate is added to the solution as a buffer.  
 15. The process of claim 2 wherein said substrate is maltopentaose.