Method for assay of .alpha.-amylase activity

A method for the assay of .alpha.-amylase activity, which comprises adding an .alpha.-amylase-containing sample to maltohexaitol or maltohexaonic acid used as substrate, reacting, at the same time or subsequent to the addition, .alpha.-glucosidase with the resulting mixture, and determining the reaction product to assay the .alpha.-amylase activity.

This invention relates to a method for the assay of .alpha.-amylase 
activity by use of maltohexaitol or maltohexaonic acid as substrate. 
.alpha.-Amylase in the human body is produced mainly in the pancreas or 
salivary gland and the determination of the amount of .alpha.-amylase in 
the body fluids such as serum, urine and the like is a clinically useful 
means in the diagnosis. For instance, in normal healthy subjects the 
.alpha.-amylase activity in the serum is approximately constant, whereas 
in patients suffering from acute pancreatitis it increases in response to 
the pathological condition of the disease. Consequently, the 
.alpha.-amylase content of the serum is an important clinical parameter 
for the pancreatic function. 
For the assay of .alpha.-amylase activity various methods have been known, 
in which the iodine-starch reaction or turbidimetry is utilized or the 
amount of reducing sugar is determined. In these methods the 
.alpha.-amylase activity is assayed by allowing .alpha.-amylase to act on 
starch used as substrate and determining the rate of disappearance of the 
iodine-starch reaction, or the rate of decrease in turbidity of the 
dispersion of substrate starch or determining chemically or enzymatically 
the amount of reducing sugar formed by the reaction. However, the starch 
used in the above methods as substrate for the quantitative determination 
is difficult to obtain in constant quality at any time and, hence, the 
standardization of the method of assay becomes very difficult. 
Consequently, at each time of assay it becomes necessary to perform the 
assay on a sample as well as on a standard sample. A method, in which the 
substrate starch is replaced by blue starch containing a combined 
chromogenic material, is also in actual use, though not widely. Because of 
the necessity of centrifugal separation in its procedure, this method has 
disadvantages in that the method is not suited for automation, and makes 
it difficult to measure the rate of reaction between the substrate and the 
.alpha.-amylase by the method of rate assay. 
In determining the .alpha.-amylase content of a sample it is necessary to 
select a substrate which answers the requirements that it should be easily 
decomposable by .alpha.-amylase and it should be a water-soluble low 
molecular weight compound having a definite structure which will not 
interfere with the stoichiometry of the reaction (i.e. it should be split 
at a definite position in the molecule of substrate). For the substrate 
which substantially answer the said requirements, there have been proposed 
maltotetraose (G.sub.4) and maltopentaose (G.sub.5) [Japanese Patent 
Application "Kokai" (Laid-open) No. 56,998/1975; U.S. Pat. No. 3,879,263]. 
Of these substrates, G.sub.4 has disadvantages in that it is fairly 
inferior in the reactivity to .alpha.-amylase and gives a high blank 
value, requiring a blank test at each time of assay, while G.sub.5 is 
higher in reactivity than G.sub.4 but has drawbacks in that the reactivity 
to .alpha.-amylase is still insufficient and the blank value seems to be 
also still high in an assay procedure coupled with .alpha.-glucosidase. 
It has also been proposed to use as substrates maltohexaose (G.sub.6) (U.S. 
Pat. No. 4,000,042), oligosaccharides having higher molecular weights than 
that of G.sub.6, or modified oligosaccharides having their reducing 
terminal hydroxyl groups replaced by aromatic groups. Such substrates are 
subject to splitting at two or more .alpha.-1,4-lycoside linkages in a 
molecule by the action of .alpha.-amylase, meaning that the product formed 
by the reaction between .alpha.-amylase and the substrate behaves again as 
a substrate for the enzyme, resulting in disturbance of stoichiometry of 
the reaction. Such a substrate, therefore, cannot be called a suitable 
substrate for the rate assay. 
Under the circumstances, the present inventors carried out extensive 
research on the method for determining the .alpha.-amylase activity with 
sufficient precision and rapidity. As a result, it was found that the 
.alpha.-amylase activity may be assayed with a high precision in a short 
period of time using as substrate maltohexaitol or maltohexaonic acid 
obtained by reducing or oxidizing in a customary way the hydroxyl group at 
the reducing terminal of maltohexaose (G.sub.6). On the basis of this 
finding, the present invention has been accomplished. 
An object of this invention is to provide a novel method for the assay of 
.alpha.-amylase activity. 
Other objects and advantages of this invention will become apparent from 
the following description. 
According to this invention, there is provided a method for the assay of 
.alpha.-amylase activity, which comprises adding a 
.alpha.-amylase-containing sample to maltohexaitol or maltohexaonic acid 
used as substrate, reacting, at the same time or subsequent to the 
addition, .alpha.-glucosidase with the resulting mixture, and determining 
the reaction product, glucose.

The invention is described below in detail. 
The maltohexaitol to be used as substrate in the present method may be 
obtained according to the method of Abdel-Akher et al. [J. Amer. Chem. 
Soc., 73, 4691 (1951)] by reducing maltohexaose with a potassium 
borohydride solution at room temperature for 24 hours. The maltohexaonic 
acid may be obtained according to the method of Ingres and Israel [J. 
Chem. Soc., 810 (1948)] by oxidizing maltohexaose with a sodium hypoiodite 
solution (pH 11) at room temperature for one hour. 
The method of assaying .alpha.-amylase activity by the use of maltohexaitol 
or maltohexaonic acid as substrate according to this invention may be 
described by the following reaction schemes: 
(A) The case where maltohexaitol is used as substrate: 
##STR1## 
(B) The case where maltohexaonic acid is used as substrate: 
##STR2## 
According to the above reaction schemes, 5 molecules of glucose and one 
molecule of sorbitol are formed from one molecule of maltohexaitol, while 
5 molecules of glucose and one molecule of gluconic acid are formed from 
one molecule of maltohexaonic acid. The amount of glucose is determined by 
any of the following methods: 
##STR3## 
The amount of NADH formed in the above reaction is determined 
spectro-photometrically from the increment of absorbance at 340 nm. 
##STR4## 
The amount of glucose in the reaction mixture may be determined by 
measuring spectrophotometrically the increment of absorbance at the 
wavelength characteristic of the oxidized coloring substance formed by the 
above reaction. 
##STR5## 
The amount of glucose in the reaction mixture may be determined, as in (1), 
by measuring spectrophotometrically the amount of NADH formed by the above 
reaction from the increment of absorbance at 340 nm. 
The advantages of using maltohexaitol or maltohexaonic acid as the 
substrate for quantitative determination according to this invention are 
as described below. 
(a) Both substrates are easily soluble in water and have high reactivities 
to .alpha.-amylase. 
(b) The stoichiometry of the reaction holds, because only the third 
.alpha.-1,4-glucosidic linkage from the sorbitol or gluconate residue is 
split. The maltotriose, maltotriitol or maltotrionic acid easily undergoes 
splitting by the action of .alpha.-glucosidase to form glucose. 
(c) With either substrate, the blank value can be made as small as 
substantially negligible. 
The sample to be assayed by the method of this invention can be of any of 
those containing .alpha.-amylase, such as, for example, serum, blood, 
urine, and the like. 
The .alpha.-glucosidase to be used in the present method may be derived 
from animals, vegetables, or microorganisms. Above all, 
.alpha.-glucosidase originated from yeast is preferred. 
The .alpha.-glucosidase can be added to the reaction system either 
simultaneously with the addition of an .alpha.-amylase-containing sample 
to the substrate (maltohexaitol or maltohexaonic acid) or at a suitable 
moment during the period from the addition of sample to the completion of 
the reaction. 
The reaction under pH conditions of generally 5 to 9, preferably 6.5 to 7.5 
is advantageous for the curtailment of reaction time and the improvement 
in the precision of assay. 
The buffer to be used in adjusting pH of the reaction system can be any of 
those capable of adjusting to pH 5-9, such as, for example, 
.beta.-glycerophosphate, tris-acetate or barbital-HCl, or an inorganic 
phosphate such as Sorensen. 
It is also possible to add to the reaction system a suitable activator for 
.alpha.-amylase, such as, for example, sodium chloride, potassium chloride 
or calcium chloride. 
The present invention is very significant for the industry, because it 
allows the assay of .alpha.-amylase activity in an 
.alpha.-amylase-containing sample in a shorter period of time with better 
precision as compared with the conventional methods, the blank value being 
as small as substantially negligible. 
The invention is further illustrated below in detail with reference to 
Example, but the invention is not limited thereto. 
EXAMPLE 
For an .alpha.-amylase-containing sample, a serum having an .alpha.-amylase 
activity of 650 Somogyi units/dl was prepared by adding to a serum 
originated from a normal healthy subject equal amounts of partially 
purified .alpha.-amylases from human salivary gland and from human 
pancreas. The resulting serum was successively diluted with physiological 
saline to prepare a series of samples having varied .alpha.-amylase 
activities (cf. FIGS. 1 and 2). The .alpha.-amylase activity was expressed 
in accordance with the saccharogenic method (i.e. Somogyi method) 
described in "Clinical Chemical Analysis IV," p. 21-39 (published by Tokyo 
Kagaku Dojin Co., 1970): 1 unit of .alpha.-amylase activity corresponds to 
the amount of the enzyme which release 1 mg of reducing sugar (as glucose 
equivalent) by the reaction between .alpha.-amylase and soluble starch at 
pH 6.9 and 40.degree. C. for 30 minutes. 
To 0.02 ml (per test plot) of the above sample, was added 1.4 ml 
(120.mu.moles) of .beta.-glycerophosphate buffer (pH 7.0). After standing 
for 2 minutes at 37.degree. C., the mixture was admixed with the following 
reagents: hexokinase, 1.0 U/0.1 ml; glucose-6-phosphate dehydrogenase, 3.0 
U/0.1 ml; ATP, 1.0 mg/0.1 ml; NAD, 1.5 mg/0.1 ml; MgCl.sub.2 -NaCl, 0.475 
mg-1.17 mg/0.04 ml. The resulting mixture was incubated for 2 minutes at 
37.degree. C. to admit consumption of the glucose in the sample. 
To the above solution, were added 4 mg/0.1 ml of maltohexaitol and 30 
U/0.04 ml of .alpha.-glucosidase. The reaction was allowed to start at 
37.degree. C. and the mixture was incubated for 5 minutes while measuring 
at one minute interval the increment of the absorbance at 340 nm. The mean 
value of the increment of absorbance per minute (.DELTA.E/minute) was 
obtained after subtracting the blank value from the measured value (the 
blank value was within the limit of error as will be described later). The 
mean values of the increment per minute were plotted in FIG. 1. 
The above procedure was repeated, except that maltohexaonic acid was used 
in place of the maltohexaitol. The mean values of the increments per 
minute of the absorbance (.DELTA.E/minute) were plotted in FIG. 2. 
The blank value was determined in the same manner as above, except that an 
identical volume of physiological saline was used in place of the serum. 
All of the blank values were within the limit of error, that is, below 
0.001. 
FIGS. 1 and 2 show that in determining the .alpha.-amylase content of a 
sample by using maltohexaitol or maltohexaonic acid as substrate, the 
reaction completes in a short period of time owing to a high reactivity of 
the substrate to .alpha.-amylase, and that the relationship between the 
increment per minute of the absorbance (.DELTA.E/minute) and the 
.alpha.-amylase activity (in Somogyi unit/dl) is represented by a straight 
line; that is, said increment is proportional to the .alpha.-amylase 
activity so that the assay of .alpha.-amylase activity may be carried out 
rapidly and precisely. 
In the next experiment, the .alpha.-amylase-containing sample prepared 
above (the sample having an .alpha.-amylase activity of 650 Somogyi 
units/dl prepared by adding to a serum originated from a normal healthy 
subject equal amounts of partially purified .alpha.-amylase from human 
salivary gland and that from human pancreas) was allowed to act, as in the 
preceding experiment described above, on various substrates shown in Table 
1. The results of assay of .alpha.-amylase activity and the blank values 
were shown in Table 1. 
TABLE 1 
______________________________________ 
.alpha.-amylase activity 
Increment of 
Found Blank value 
absorbance 
Substrate (E/min.) (E/min.) (.DELTA.E/min.) 
______________________________________ 
Maltohexaonic acid 
0.187 0.000 0.187 
Maltohexaitol 
0.181 0.000 0.181 
Maltotetraose 
0.074 0.034 0.040 
Maltopentaose 
0.102 0.005 0.097 
Maltohexaose 
0.072 0.002 0.070 
Maltoheptaose 
0.033 0.001 0.032 
Maltopentaitol 
0.014 0.001 0.013 
Maltoheptaitol 
0.035 0.000 0.035 
______________________________________ 
The purity of each substrate shown in Table 1 was 99.5% (W/W) or above. 
As is apparent from Table 1, when the .alpha.-amylase activity of a sample 
was assayed by using maltohexaitol or maltohexaonic acid as substrate, as 
compared with other substrates, the reaction time could be much reduced 
because of higher reactivities to .alpha.-amylase (larger increments of 
absorbance); the blank values were found to be negligible in the case of 
said compounds.