JP2006081492A - Method for quantitatively determining cholesterol - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method capable of quantitatively determining a cholesterol existing in various kinds of forms such as lipoprotein by one stage operation. <P>SOLUTION: The method for quantitatively determining the cholesterol comprises utilizing a vibration reaction. The vibration reaction is carried out by permeating a first solution containing hydrogen peroxide through a semipermeable membrane into a second solution containing a catalase, a cholesterol oxidase and the cholesterol. The semipermeable membrane is preferably a dialysis membrane or a millipore filter. The second solution, preferably, contains 0.01-1 mg/ml catalase and 0.002-0.02 mg/ml cholesterol oxidase. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a method for quantifying cholesterol.

  Cholesterol binds to apoprotein in serum and exists as a lipoprotein. Lipoproteins are classified into chylomicron, VLDL, LDL, HDL, etc., depending on their physical shape. LDL is one of the substances that cause arteriosclerosis, while HDL is attracting attention as a lipoprotein with anti-arteriosclerosis.

Examples of conventional quantification of cholesterol in lipoproteins include ultracentrifugation, electrophoresis, conversion, and fractional precipitation. However, these are not suitable as a simple method.
On the other hand, there is also a quantification method using an enzyme such as cholesterol oxidase. For example, a method for quantifying hydrogen peroxide generated when cholesterol is oxidized using cholesterol oxidase and quantifying cholesterol based on the quantification is as follows. Documents 1 and 2 are described.

Japanese Patent Laid-Open No. 2001-224397 JP 2004-121185 A

However, in any of the above-described techniques, it is necessary to perform two or more steps of quantifying hydrogen peroxide produced when cholesterol is oxidized and then quantifying cholesterol.
In general, when an enzyme and a substrate reactant are put in a container and reacted, the product increases monotonically with time, and finally reaches an equilibrium state. Conventionally, this method has been used to quantify biological substances using enzymes that act specifically on reactants. Cholesterol quantification using conventional enzyme reactions is no exception and uses such a method.
On the other hand, when the substrate is slowly permeated into the enzyme solution using the semipermeable membrane, there may be a vibration reaction in which the concentration of the reaction product or product periodically increases or decreases with time. For example, when catalase is used as an enzyme and hydrogen peroxide is used as a substrate, the following reaction occurs to generate oxygen, but this reverse reaction also occurs. That is, dissolved oxygen vibrates. However, in order for the vibrational reaction to take place, an appropriate permeation rate of the substrate and an appropriate discharge of the product (in this case oxygen) are required.
If glucose oxidase, which is another enzyme, and glucose, which is a substrate for this enzyme, are added to this system, the period of oscillation increases with the concentration of glucose. In a certain concentration range, the concentration of glucose and the period of oscillation are linear. In relation, it is known that it can be used for quantification of glucose. However, there is no description about applying the vibrational response, which is a nonlinear phenomenon, to the determination of cholesterol.

In view of the above, an object of the present invention is to eliminate the quantification that occurs in the middle of the cholesterol quantification method and perform quantification in fewer steps.

In order to achieve the above object, the present invention specifically adopts the following means.
First, as a first means, a cholesterol quantification method using a vibration reaction is used. Conventional methods using cholesterol oxidase require a minimum of two steps, such as measuring the amount of oxygen produced by decomposing the produced hydrogen peroxide with catalase. However, this method using the vibration reaction can be quantified only by measuring the vibration of the oxygen amount. There is also an advantage that measurement is possible with a small amount of sample.
In this means, the vibration reaction is a reaction performed by allowing a first solution containing hydrogen peroxide to permeate a second solution containing catalase, cholesterol oxidase and cholesterol through a semipermeable membrane. The semipermeable membrane is a dialysis membrane or a Millipore filter, and the second solution contains catalase in the range of 0.01 to 1 mg / ml and cholesterol oxidase in the range of 0.002 to 0.02 mg / ml. In the second solution, cholesterol is dispersed by a phospholipid or a surfactant, and in the second solution, the cholesterol concentration is in the range of 0.1 to 1.6 mg / ml. It is also desirable to feature. As a result, the vibration response, which is originally a nonlinear phenomenon, can be approximated to a linear relationship and quantified.

As a second means, the first solution containing hydrogen peroxide is allowed to permeate through the semipermeable membrane into the second solution containing catalase, cholesterol oxidase and cholesterol, and the dissolved oxygen in the second solution is removed. Determine the concentration change cycle, and determine the cholesterol concentration contained in the second solution based on the relationship between the calculated concentration change cycle of dissolved oxygen and the previously determined hydrogen peroxide concentration change cycle and cholesterol concentration. It is set as the quantification method of cholesterol to calculate.
In this means, the semipermeable membrane is a dialysis membrane or a Millipore filter, and the second solution is catalase 0.01 to 1 mg / ml and cholesterol oxidase 0.002 to 0.02 mg / ml. In the second solution, cholesterol is dispersed by phospholipids or surfactants. In the second solution, the cholesterol concentration is within the range of 0.1 to 1.6 mg / ml. It is also desirable to be.

Further, as a third means, a first container for holding a first solution containing hydrogen peroxide, a second container for holding a second solution containing catalase, cholesterol oxidase and cholesterol, A semipermeable membrane disposed between the second container and the second container, wherein the first solution is infiltrated into the second solution, an oxygen electrode disposed so as to be immersed in the second solution, and oxygen A cholesterol quantification device having a dissolved oxygen meter connected to an electrode and an information processing device connected to the dissolved oxygen meter.

As described above, according to the present invention, cholesterol can be easily quantified by measuring its dissolved oxygen concentration using a vibration reaction, so that other quantification occurring in the middle can be omitted and quantification of fewer processes can be performed. it can.

First, the principle used by the present invention will be described with reference to FIG.
Catalase, cholesterol oxidase and cholesterol solutions are placed in one of the two containers sandwiching the semipermeable membrane, and the aqueous hydrogen peroxide solution is placed in the other container. In this case, since cholesterol is poorly soluble, it is necessary to add a surfactant such as SDS or a phospholipid to form micelles. Then, the hydrogen peroxide solution slowly permeates the semipermeable membrane and reacts with catalase to start generating oxygen. On the other hand, when oxygen is generated, cholesterol oxidase uses this oxygen to oxidize cholesterol and generate hydrogen peroxide. This series of flows becomes a circulation process, and furthermore, a vibration reaction in which the concentration of the hydrogen peroxide solution vibrates. In this case, when the amount of oxygen dissolved in the enzyme aqueous solution is measured with a dissolved oxygen meter, the concentration of dissolved oxygen periodically oscillates. And when various conditions such as cholesterol are in a certain concentration range, the oscillation cycle of dissolved oxygen amount has a linear relationship with the cholesterol concentration, and this relationship can be used for quantification of cholesterol. .
By using this, it is possible to quantify various shapes containing cholesterol. For example, the amount of cholesterol in chylomicron, high density lipoprotein, low density lipoprotein and very low density lipoprotein can be measured. In addition, the total cholesterol level in urine and serum can be measured.
It is also considered that the types of lipoproteins containing cholesterol can be differentiated by changing the vibration pattern or changing the type of enzyme.
Embodiments of the present invention will be specifically described below based on model embodiments shown in the drawings.

FIG. 2 shows a diagram of an apparatus according to an embodiment of the present invention (hereinafter simply referred to as “the present apparatus”).
As shown in FIG. 2, this apparatus includes a first container 1, a second container 2, a connecting pipe 3 that connects the first container and the second container, and a semi-transparent material disposed in the connecting pipe 3. A membrane 4; an oxygen electrode 5 inserted into the second container; a dissolved oxygen meter 6 electrically connected to the oxygen electrode 5; and an information processing device 7 electrically connected to the dissolved oxygen meter 6. Configured.

In the first state, 20 ml of a 0.6% hydrogen peroxide aqueous solution is placed on the first container side, and 0.1 ml of a sample solution containing catalase 2.5 mg, cholesterol oxidase 0.05 mg, and cholesterol is placed on the second container side. (A solution containing catalase, cholesterol oxidase and cholesterol is hereinafter simply referred to as “enzyme solution”). Both the first container 1 and the second container 2 are made of glass, each formed in a cylindrical shape and having an inner diameter of 3 cm.

The connecting pipe 3 is disposed between the first container 1 and the second container 2 and connects the inside of these containers. In this embodiment, it is made of glass, and its inner diameter is 1 cm.

  The semipermeable membrane 4 is provided in the flow dew 3 disposed between the first container 1 and the second container 2, and specifically, a dialysis membrane is used (material: α cellulose, fractional molecular weight). 12,000-14,000).

The oxygen electrode 5 is inserted into the second container and is arranged so as to be immersed in the enzyme solution contained in the second container. The oxygen electrode 5 generates a potential according to the amount of electrons generated when dissolved oxygen in the oxygen solution is reduced to water on the electrode.

The dissolved oxygen meter 6 receives the generation of the potential of the oxygen electrode 5 and calculates the concentration of dissolved oxygen based on the value of the potential.

The information processing device 7 receives the output from the dissolved oxygen meter 7 and first obtains the period of the vibration reaction from the output. Specifically, potential values are acquired over time, and potential data with respect to time is created. Then, the period of the vibration reaction is calculated by analyzing the potential data with time. Cholesterol is quantified based on the relationship between the cycle and a predetermined cycle-cholesterol.
In this embodiment, the above processing is performed using a so-called personal computer as the information processing apparatus. More specifically, the personal computer of this apparatus accepts data corresponding to the concentration of dissolved oxygen measured by the dissolved oxygen meter (hereinafter referred to as “dissolved oxygen concentration data”), and converts the dissolved oxygen concentration data into the concentration. Dissolved oxygen data acquisition means for recording together with the indicated time data (hereinafter referred to as “time data”), a period determining means for determining the period of the vibration reaction based on the relationship between the time data and the dissolved oxygen concentration data, Cholesterol is quantified based on a cycle-cholesterol relationship recording means that stores in advance the relationship between the concentration of cholesterol and the cycle of vibration reaction, the cycle of vibration reaction, and the relationship between the concentration of cholesterol and the cycle of vibration reaction. A program for performing cholesterol quantification means is stored in a hard disk as a recording medium and executed. It is possible to perform the quantitative determination of cholesterol in it. In this case, the concentration of cholesterol and the period of the vibrational reaction are different from each other in the period-cholesterol relationship because the range of the slope of the straight line or the linear relationship varies depending on the presence of a surfactant or phospholipid, the concentration of catalase or cholesterol oxidase, etc. The recording means has a function of accepting and recording the input of the condition, and further reading out the relationship between the period of vibration and the cholesterol concentration that meet the condition corresponding to the input (of course, the relationship is previously determined). It is more desirable to memorize it. Of course, each means may be a recording medium other than a hard disk as long as it is a recording medium, and may not be a program as long as the above functions can be executed. This shows an embodiment in which a program is desirable from the viewpoint of ease of various operations.

And it measured based on the above structure.
In this measurement, four types of samples were prepared by varying the amount (concentration) of cholesterol in various ways, and each of them was measured. The conditions other than the amount (concentration) of cholesterol were fixed as described above. Table 1 shows the amount of cholesterol added and the concentration of cholesterol. Cholesterol was dispersed using an ultrasonic wave by adding a phospholipid (egggrecitin, concentration: 0.6 mg / ml) as a surfactant to an aqueous solution containing cholesterol. Then, the concentration of cholesterol in 0.1 ml of the solution was adjusted as shown in Table 1 below, and added to a solution mixed with catalase or the like to make a total of 25 ml of enzyme solution. Table 1 lists the amount and concentration of cholesterol dissolved in the solution and the period of the vibrational reaction. FIG. 3 shows the time variation of the dissolved oxygen amount in the case of sample solution number 2 in Table 1. In FIG. 3, the horizontal axis represents time (hour), and the vertical axis represents dissolved oxygen amount (mg / l).

FIG. 4 shows the results obtained in Table 1 with respect to the cholesterol concentration. The horizontal axis represents cholesterol concentration, and the vertical axis represents the period of vibration.
As a result, the relationship between the period of vibration and the concentration of cholesterol could be explained very well by a straight line. In particular, in this example, the cholesterol concentration can be explained very well in the range of 0.2 to 0.8, and if it is in the range of about 0.1 to 1.0 from this value, it should be sufficiently approximated by a straight line. It is thought that you can.
As described above, the relationship between the cycle under the predetermined catalase and cholesterol oxidase concentrations and the cholesterol concentration is stored in advance as a data, and the vibration reaction is performed under the predetermined catalase and cholesterol oxidase concentrations to obtain the cycle. Thus, the cholesterol concentration can be quantified.

Measurement was performed under the same conditions as in Example 1 except that SDS was used instead of phospholipid and the amount (concentration) of cholesterol was changed. Table 2 shows conditions and periods when adjusted in the same manner as in Table 1 above. SDS has a concentration of 0.05 mol / l.

FIG. 5 shows the results of Table 2 in which the period with respect to the cholesterol concentration was determined in the same manner as in FIG.
As a result, even when SDS was used, the relationship between the vibration period and the cholesterol concentration could be explained very well by a straight line. In particular, since the linearity is very good in the range of 0.2 to 1.5 mg / ml in this example, the linearity is maintained even in the range of about 0.1 to 1.6 mg / ml. It is thought that you can.
Also in this example, as in Example 1, the relationship between the cycle under the predetermined catalase and cholesterol oxidase concentrations and the cholesterol concentration is stored in advance as data, and under the predetermined catalase and cholesterol oxidase concentrations. The concentration of cholesterol can be quantified by performing an oscillating reaction and determining its period.
In addition, in Example 1 and Example 2, although comprised using the 1st container 1, the 2nd container 2, and the flow path 3 which connects between the containers, a container shape is not restricted to this. For example, as shown in FIG. 6, a configuration in which one container is partitioned by a partition plate, a window is opened, and a semipermeable membrane is disposed is also possible.

The figure which shows the reaction mechanism of a vibration reaction. 1 is a schematic diagram of a cholesterol quantification apparatus in Example 1. FIG. The figure which shows the time change of the dissolved oxygen amount in the vibration reaction in Example 1. FIG. The figure which shows the relationship between the cholesterol concentration in Example 1, and a period. The figure which shows the relationship between the cholesterol concentration in Example 2, and a period. The figure which shows the other container example in the fixed_quantity | quantitative_assay apparatus of cholesterol.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 ... 1st container, 2 ... 2nd container, 3 ... Connecting pipe, 4 ... Semipermeable membrane, 5 ... Oxygen electrode, 6 ... Dissolved oxygen meter, 7 ... Information processing apparatus

Claims (12)

  Cholesterol determination method using vibrational reaction   The vibration reaction is a reaction performed by infiltrating a first solution containing hydrogen peroxide into a second solution containing catalase, cholesterol oxidase and cholesterol through a semipermeable membrane. Item 2. The method for quantifying cholesterol according to Item 1.   The method for quantifying cholesterol according to claim 2, wherein the semipermeable membrane is a dialysis membrane or a Millipore filter.   3. The method for quantifying cholesterol according to claim 2, wherein the second solution contains catalase in the range of 0.01 to 1 mg / ml and cholesterol oxidase in the range of 0.002 to 0.02 mg / ml. .   3. The method for quantifying cholesterol according to claim 2, wherein the cholesterol is dispersed by a phospholipid or a surfactant in the second solution.   The cholesterol determination method according to claim 4, wherein the cholesterol concentration in the second solution is in a range of 0.1 to 1.6 mg / ml.   The first solution containing hydrogen peroxide is permeated through the semipermeable membrane into the second solution containing catalase, cholesterol oxidase and cholesterol, and based on the change in the concentration of dissolved oxygen in the second solution, A method for quantifying cholesterol, wherein the cholesterol concentration contained in the second solution is calculated.   The method for quantifying cholesterol according to claim 2, wherein the semipermeable membrane is a dialysis membrane or a Millipore filter.   The method for quantifying cholesterol according to claim 7, wherein the second solution contains catalase in a range of 0.01 to 1 mg / ml and cholesterol oxidase in a range of 0.002 to 0.02 mg / ml. . 8. The method for quantifying cholesterol according to claim 7, wherein the cholesterol is dispersed in the second solution by a phospholipid or a surfactant.   The cholesterol determination method according to claim 9, wherein the cholesterol concentration in the second solution is in a range of 0.1 to 1.6 mg / ml. A first container holding a first solution containing hydrogen peroxide; a second container holding a second solution containing catalase, cholesterol oxidase and cholesterol; and the first container and the second container And a semipermeable membrane that penetrates the first solution into the second solution, an oxygen electrode that is soaked in the second solution, and is connected to the oxygen electrode A cholesterol quantification apparatus comprising: a dissolved oxygen meter; and an information processing device connected to the dissolved oxygen meter.