JPS6339240B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel method for rapidly and easily measuring the enzymatic activity of acidic and alkaline phosphatases.

In recent years, with the development of clinical medicine, there has been a demand for the development of methods that can quickly and easily measure various enzyme activities in human body fluids, especially serum.

Acidic and alkaline phosphatases are both enzymes that have catalytic activity for the hydrolysis reaction of orthophosphoric acid monoester, and acidic phosphatase (EC3.1.3.2) has its maximum activity in a weakly acidic solution around PH5. In addition, alkaline phosphatase (EC3.1.3.1) has a pH of 8-10.
It is known that its activity reaches its maximum in a weakly alkaline solution. It is known that the enzyme activity of acid phosphatase in serum increases with prostate cancer, bone disease, liver disease, etc., and the enzyme activity of alkaline phosphatase in serum increases with liver, biliary tract, or bone disease. It is known that there is an increase in

As described above, acidic and alkaline phosphatase activities in serum are closely related to various diseases, and measuring these activities is extremely important from a health perspective, and especially from a clinical medical perspective.

Recently, many methods for measuring acidic and alkaline phosphatase activities have been proposed and put into practical use, especially in line with clinical medical requirements, but all of them are spectroscopic methods that require complicated operations. Some of them belong to the endpoint measurement method, which requires a long time for measurement, and a simple and quick method has not yet been developed.

The present inventors have improved the shortcomings of conventional methods for measuring acidic and alkaline phosphatase activities, and
As a result of various studies to quickly measure these enzyme activities with simple operations, we found that glucose-6 was used as a substrate.
-Using phosphoric acid, glucose oxidase (E.
C.1.1.3.4) When using an enzyme electrode that combines an immobilized membrane and an oxygen electrode or hydrogen peroxide electrode, enzyme activity can be measured extremely quickly and easily, and each of the acidic and alkaline phosphatases in the sample solution It was discovered that the activity can be measured independently by simply adjusting the pH of the sample solution to around 5 and around 8, leading to the present invention.

That is, the present invention provides a method for measuring the enzyme activity in a sample containing acidic and alkaline phosphatases, which uses glucose-6-phosphate as a substrate and combines a glucose oxidase-immobilized membrane with an oxygen electrode or a hydrogen peroxide electrode. The present invention provides a method for measuring the activity of acidic and alkaline phosphatases, characterized in that the enzyme activity is determined by measuring the current change in the enzyme electrode based on the hydrolysis reaction of the substrate using the enzyme electrode. .

The glucose oxidase-immobilized membrane used in the method of the present invention is one in which glucose oxidase is immobilized on a porous immobilization carrier membrane, and includes the covalent bonding method, adsorption method, or comprehensive immobilization method that is usually used for enzyme immobilization. Those produced using any method such as the chemical method can also be used. As the glucose oxidase used for its production, one derived from Aspergillus niger can be used. The immobilization carrier needs to be porous enough to permeate dissolved oxygen and low molecular components such as glucose in the solution, and such a permeable porous membrane is selectively used. As the oxygen electrode used in the method of the present invention, a commonly used polarographic type or battery type electrode of Clark type, which utilizes the oxygen permeability of a porous polymer membrane, can be used. Further, as the hydrogen peroxide electrode, a commonly used polarographic type electrode such as a platinum electrode coated with a porous polymer can be used. In the present invention, enzyme electrodes are used in which the glucose oxidase-immobilized membrane is tightly fixed onto the porous polymer of these electrodes using, for example, an O-ring. There are no particular restrictions on the type of buffer solution used in this generation method, but
For measuring the activity of acid phosphatase, an acetic acid-sodium acetate buffer solution or the like is used near pH5.
PH for measuring alkaline phosphatase activity
Tris hydroxylaminomethane-hydrochloric acid buffer solution around 8 can be conveniently used.

The principle of the method for measuring acidic and alkaline phosphatase activities of the present invention is as follows. That is, when an enzyme electrode is inserted into a buffer solution and glucose-6-phosphate is added to the measurement system, and a sample containing acidic and alkaline phosphatases is further added, if the pH of the buffer solution is around 5, Glucose is produced through the hydrolysis of glucose-6-phosphate by the catalytic action of acidic phosphatase and, when the pH is around 8, by the catalytic action of alkaline phosphatase, and this glucose is transferred to the glucose oxidase-immobilized membrane. Due to the oxidation inside, the amount of oxygen near the electrode decreases, and at the same time hydrogen peroxide is generated. As a result, a decrease in current at the oxygen electrode or an increase in current at the hydrogen peroxide electrode is observed. In this way, the acidic and alkaline phosphatase activities in the sample can be determined from the degree of current change at the enzyme electrode.

Next, the method of the present invention will be explained in more detail with reference to the accompanying drawings.

FIG. 1 is a schematic cross-sectional view showing an example of a device used in the measuring method of the present invention, in which a glucose oxidase immobilized membrane is immobilized on the surface of an oxygen electrode or hydrogen peroxide electrode 1 using an O-ring or the like. An enzyme electrode is formed. A buffer solution 3 containing a substrate is poured into a container 9, and an enzyme electrode is inserted into this solution. In addition, the amount of dissolved oxygen in the solution is kept constant,
It is also preferable that an air blowing pipe 4 is provided to make the concentration distribution of dissolved oxygen uniform, and that the solution is further stirred using a magnetic stirrer 5 or the like. The output current of the enzyme electrode is converted to voltage via an ammeter or resistor 6 and recorded on a recorder 7. If the electrode 1 is not battery-powered, an appropriate voltage is applied to the electrode 1 using the external electrode 10. The current value at electrode 1 before sample addition is
When an oxygen electrode is used as the oxygen electrode, the diffusion rate of oxygen from the buffer solution 3 containing the substrate, i.e.
It shows a constant value proportional to the amount of dissolved oxygen in the solution, and when a hydrogen peroxide electrode is used, it is zero because there is no hydrogen peroxide in the solution 3.

Subsequently, a predetermined amount of a sample containing acidic and alkaline phosphatases is added to the solution 3 through, for example, a supply tube 8.
When the pH of solution 3 is around 5, the substrate is hydrolyzed by the catalytic action of acid phosphatase, and when the pH of solution 3 is around 8, the catalytic action of alkaline phosphatase hydrolyzes the substrate to produce glucose. However, this glucose diffuses from the solution 3 into the glucose oxidase-immobilized membrane 2 and is oxidized. Accordingly, the current in the electrode 1 changes. Moreover, the concentration of glucose produced in this case is the same as that of solution 3 if the substrate concentration is appropriate.
When the pH is around 5, it increases linearly with time in proportion to the acidic phosphatase activity, and when the pH is around 8, it increases linearly in proportion to the alkaline phosphatase activity. This change causes a linear temporal increase in the glucose oxidation reaction rate in the glucose oxidase-immobilized membrane 2, and is immediately detected as a linear change in the current at the electrode 1.

In this way, if the rate of linear time change in the current at electrode 1 after sample addition is determined, if the pH of the buffer solution is around 5, it will be acidic phosphatase, and if the pH of the buffer solution is around 8, it will be alkaline phosphatase. The activity of each can be measured. The method for measuring acidic and alkaline phosphatases of the present invention takes advantage of the high substrate selectivity of enzyme electrodes, and uses current measurement as its principle, so that substantially all substances coexisting in the sample can be detected on the electrode surface. Highly accurate results can be obtained without being affected by adsorption, etc. Therefore, it is suitable as a method for determining the activity of acidic and alkaline phosphatases in samples with complex compositions such as serum, but in this case, the large excess amount of glucose present in serum interferes with the measurement, so it is necessary to It is necessary to remove glucose from serum by a treatment method.

Next, the present invention will be explained in more detail with reference to Examples.

Example 1 10 mg of glucose oxidase at pH 6.7
Dissolved in 200μ of 0.1mol/phosphate buffer solution,
The mixture was mixed with 1 ml of a 10% photosensitive polyvinyl alcohol solution containing stilbazolium groups as a photosensitive group, spread on an area of approximately 20 cm ² , air-dried, and further irradiated with light to obtain a water-insoluble glucose oxidase-immobilized membrane. Ta. Cut out this membrane to a diameter of 10 mm, with an area of approx.
A 0.1 cm ² platinum electrode was used as a working electrode, and the membrane was brought into close contact with the porous polytetrafluoroethylene membrane, and the whole was tightened and fixed using an O-ring. The enzyme electrode prepared in this way can reduce the amount of glucose present in various general-purpose buffer solutions in the pH range of 4.5 to 8.5 to 0.02-
It could be quantified in the range of 1.6 mmol/.
The electrode was immersed in 10 ml of a 0.1 mol/acetic acid-sodium acetate buffer solution containing 20 mmol/glucose-6-phosphoric acid and having a pH of 5.0 and kept at a temperature of 25°C. Approximately 200 ml of air was blown from the surface of the solution per minute, and the solution was stirred using a stirrer.

Figure 2 is a graph showing the temporal change in the current at the enzyme electrode when acidic phosphatase is added to the above solution; from approximately 30 seconds to 4 minutes after the addition of acidic phosphatase, the current is proportional to time. and decreased. The added acid phosphatase activity determined from this gradient is approximately 600 I.U./.
Furthermore, FIG. 3 is a graph showing the relationship between the activity of added acid phosphatase and the average slope of current decrease from 30 seconds to 2 minutes after addition of acid phosphatase. The activity of acid phosphatase and the average slope of current decrease were completely proportional in the range of acid phosphatase activity from about 5 to 500 I.U./. Also,
When alkaline phosphatase with an activity range of 0-500 I.U./was added to the above solution, the current at the enzyme electrode remained virtually unchanged.

Example 2 The enzyme electrode used in the previous example was mixed with a 0.5 mol/tris hydroxylaminomethane-hydrochloric acid buffer solution 10 of pH 8.1 containing 10 mmol/glucose-6-phosphate.
ml, and air was blown from the surface of the solution at a rate of about 200 ml per minute, and the solution was stirred using a stirrer.

When alkaline phosphatase was added to the above solution, the current at the enzyme electrode showed a temporal change similar to the graph in FIG. 2 of the above example. FIG. 4 is a graph showing the relationship between the activity of added alkaline phosphatase and the average slope of current decrease from 30 seconds to 2 minutes after addition of alkaline phosphatase. alkaline phosphatase activity,
The average slope between 30 seconds and 2 minutes after alkaline phosphatase addition was completely proportional to the alkaline phosphatase activity in the range of about 5-500 I.U./. In addition, when acid phosphatase with an activity range of 0-500 I.U./ is added to the above solution,
The current at the enzyme electrode remained virtually unchanged.

Example 3 5 ml of serum was placed in a dialysis tube (Visking), which was then placed in physiological saline (1) and stirred at 4° C. for 2 hours to remove glucose from the serum. The acidic and alkaline phosphatase activities in the six types of serum samples subjected to such pretreatment were measured by the methods of Examples 1 and 2, respectively, and the results were analyzed using the known kit of Wako Pure Chemical Industries, Ltd. ( When compared with the results obtained by measuring the activity of acidic and alkaline phosphatase using Phosphak-Test, the correlation coefficient between the two was 0.98 for acidic phosphatase and 0.99 for alkaline phosphatase, showing good agreement. In other words, it can be seen that the method for measuring acidic and alkaline phosphatase activities of the present invention has high accuracy and is sufficient to be useful.

The method of the present invention can measure activity with high precision in exactly the same way using an enzyme electrode using a hydrogen peroxide electrode.

[Brief explanation of the drawing]

FIG. 1 is a schematic cross-sectional view showing an example of the acidic and alkaline phosphatase activity measuring device of the present invention, and FIG. 2 is a schematic cross-sectional view showing an example of the acidic and alkaline phosphatase activity measurement device of the present invention. FIG. 3 is a graph showing the relationship between electrode current and time, and FIG. 4 is a graph showing the relationship between temporal changes in current and acid phosphatase activity according to the acid phosphatase activity measurement method of the present invention. 2 is a graph showing the relationship between temporal changes in current and alkaline phosphatase activity according to the alkaline phosphatase activity measuring method. The symbols in FIG. 1 are oxygen electrodes or hydrogen peroxide electrodes, 2 is a glucose oxidase immobilized membrane, and 3 is a hydrogen peroxide electrode.
is a buffer solution, 4 is an air blowing tube, 5 is a stirrer, 6 is an ammeter or resistor, 7 is a recorder, 8 is a sample supply tube, 9 is a container, and 10 is an external power source.

Claims (1)

[Claims] 1. An enzyme electrode that uses glucose-6-phosphate as a substrate and combines a glucose oxidase-immobilized membrane and an oxygen electrode or a hydrogen peroxide electrode in a method for measuring the activity of these enzymes in a sample containing acidic and alkaline phosphatases. A method for measuring the activity of acidic and alkaline phosphatases, characterized in that the enzyme activity is determined by measuring the current change in the enzyme electrode based on the hydrolysis reaction of the substrate.