JPH10210999A - Inhibition of activity of esterase - Google Patents

Abstract

PROBLEM TO BE SOLVED: To inhibit the activity of an esterase to prevent an error caused by the carryover of the esterase, a lipase, etc., on the measurement of serum lipids by using a boron compound having a specific structure. SOLUTION: This method for inhibiting the activity of an esterase on the measurement of serum lipids such as free cholesterol, triglycerides and free fatty acids comprises adding a boron compound of the formula (R1, R2 are each a 1-12C alkyl, phenyl, tolyl, hydroxyl) (e.g. an alkyl boric acid) in a concentration of 0.01-1000mM to a reaction solution which is estimated to contain the esterase to be inhibited. The activity of the esterase such as cholesterol esterase or lipoprotein lipase can thereby be inhibited to prevent an error caused by the carryover of the cholesterol esterase or lipoprotein lipase on the measurement of serum lipids.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to the measurement of lipid components contained in blood by an enzymatic reaction. More specifically, the present invention relates to a technique for preventing an error caused by carryover of a reagent component, which is a problem in enzymatic measurement of serum lipid. Cholesterol (Cholesterol, hereinafter abbreviated as CHO), triglyceride (Triglyceride, hereinafter abbreviated as TG), or free fatty acid (Free Fatty Acid, or Non Esterifie)
It is known that serum lipid components such as d Fatty Acid (hereinafter abbreviated as NEFA) give important information in diagnosis of arteriosclerosis and liver function. An enzymatic reaction is used to measure these serum lipids. The measurement technology based on the enzyme reaction has excellent specificity, is easy to automate, and has established a high reputation in terms of accuracy and sensitivity. At present, it is a routine blood test item to obtain basic information on lipid metabolism.

[0002] In the enzymatic measurement of these serum lipids, various lipases and esterases are indispensable reagent components. For example, Total Cholestero
In the measurement of l, hereinafter abbreviated as T-CHO, ester-form CHO is hydrolyzed to form free cholesterol (Fr
ee Cholesterol (hereinafter abbreviated as F-CHO) is referred to as cholesterol esterase (Cholestero).
l Esterase (hereinafter abbreviated as CEase) catalyzes. In the measurement of TG, T
The reaction of hydrolyzing G to glycerol is catalyzed by a lipase called lipoprotein lipase (hereinafter abbreviated as LPL).

[0003] These esterases and lipases are known to cause errors in various forms when mixed (carry over) into other reaction systems. In particular, many of the automatic analyzers widely used at present for these measurements use plastic cells as reaction vessels. Since plastic cells tend to adsorb proteins such as enzymes, insufficient washing may lead to carryover of enzymes. Plastic cells are inexpensive and can be disposable in a short period of time, and are currently used in most automatic analyzers. In the long term, replacement of the cell about once a month is sufficient because the cell is periodically cleaned with a dedicated cleaning solution. However, carry-over problems may occur if a combination of special measurement items is mixed during normal operation. In addition, the risk of carryover is high in practice, as these enzymes are often over-prescribed to speed up the reaction and to account for the inactivation of the enzymes during reagent storage. In particular, it has been pointed out that in the single-line multi-item method in which the same cell is shared by a plurality of measurement items, the remaining of the reagent tends to be a problem [1]. It has also been pointed out that not only carry-over via a cell, but also the possibility of adhering to a reagent probe and being carried into another reaction system. Although any automatic analyzer is equipped with a mechanism for washing the inside and outside of the reagent probe, it is not always completely washed and actually causes carryover. In order to avoid such a problem, a combination with a liquid level sensor or the like has been put to practical use so that the reagent probe does not unnecessarily enter the reagent solution. However, only contamination outside the probe can be prevented with such a mechanism.

[0004]

2. Description of the Related Art Several techniques have been developed to prevent errors caused by carryover of esterase or lipase. One of them is a technique for suppressing adsorption of an enzyme component to a reaction vessel. Specifically, by adding a specific surfactant to the reagent containing the enzyme component causing the error,
Adsorption to the reaction vessel is prevented. As the surfactant, polyoxyethylene alkylphenol ether is exemplified [2]. There are also reports of using nonionic surfactants such as polyoxyethylene octyl phenyl ether at specific concentrations [3]. However, there are various reagents on the market, and even if the effect of suppressing the adsorption of one enzyme can be expected, the effect is not necessarily obtained by another enzyme.
Since these various reagents are used in a general-purpose automatic analyzer, it is necessary to suppress adsorption, but if a reagent that is not affected by carryover is provided, its value is great.

[0005] To prevent the adsorption of enzyme components,
Techniques have also been proposed for adding inhibitors to enzymes that cause errors to reaction systems affected by carryover. The following briefly summarizes the prior art utilizing enzyme inhibitors.

[0006] CEase is an enzyme used for T-CHO measurement and acts on ester-type CHO to generate F-CHO. Therefore, the reaction system for measuring F-CHO is CEa.
When se is contaminated, ester-type C
The measurement of the F-CHO generated from the HO causes a positive error. LPL is used as a second reagent when measuring TG. LPL carryover is CEa
A positive error is caused in F-CHO by the same principle as se. Further, in the measurement of TG, a negative error is caused because the reaction of erasing free glycerol to the TG, which is a component to be measured, is conjugated with the first reagent which promotes the reaction for eliminating free glycerol. To prevent such errors, CEa
A method is known in which triton X-405, sodium fluoride, or iodoacetic acid is added to a reagent for measuring F-CHO in advance as a se inhibitor [4]. There is also a report [5] in which a fatty acid alkali salt is used as an LPL inhibitor for F-CHO measurement. Alkylene oxide-based nonionic surfactants can be used for measuring CEFA or L when measuring NEFA.
It has been used as an inhibitor for PL [6]. The use of polyoxyalkylene-modified organopolysiloxane compounds [7] has also been attempted as an ester hydrolase inhibitor such as CEase or LPL in measuring serum lipid components. A technique using a monoclonal antibody having activity inhibition against CEase and LPL is also known [8].

[0007] When these inhibitors are used, the range of use may be limited because the addition of the inhibitor affects the required enzyme reaction. For example, Triton X-405 suppresses cholesterol oxidase activity. Iodoacetic acid cannot be applied to the measurement of NEFA because it inhibits the activity of SH enzymes such as acyl-CoA oxidase used in the measurement of NEFA. LP
A surfactant having a high HLB (hydrophilic-lipophilic balance) value must be selected as a surfactant for suppressing the activity of L. Such surfactants require LPL, C
Care must be taken because some substances inhibit the activity of Ease or acyl-CoA synthase. For example, NE
In the reagent for measuring FA, it is necessary to increase the amount of the enzyme to cover the acyl-CoA synthetase whose activity is inhibited by the use of an esterase inhibitor. According to our findings, in some cases even a 30% increase was required. Since ACS is an expensive enzyme, increasing the amount leads to an increase in cost. In addition, surfactants having a high HLB value tend to hinder the clarity of turbidity (chyle) in serum samples due to lipid components. Therefore, a reagent using a surfactant as an esterase inhibitor sometimes fails to sufficiently address chyle.

TG is particularly susceptible to elimination of chyle by surfactants. This is because the following special situation occurs in the TG. The main causes of chyle are chylomicron and very low density lipoprotein (VLDL)
Among them, chylomicron-based chyle has a large degree of turbidity. On the other hand, the ratio of TG in chylomicron is about 80
It is said to be%. Therefore, in the measurement of TG, a phenomenon in which turbidity disappears as TG is consumed in the enzyme reaction is observed. In a photometry method called a two-point end employed in a current general automatic analyzer, an optical measurement is performed before and after the addition of a second reagent. Therefore, when turbidity elimination is incomplete before the enzyme reaction in the TG measurement, the TG concentration is calculated based on the numerical value of the turbid reaction solution before the enzyme reaction and the numerical value measured after the turbidity has disappeared after the reaction. Will be. In other words, under such conditions, a negative error results as much as the turbidity disappears. Such a problem does not occur if the turbidity is eliminated before the first photometry or the reaction is advanced so that the degree of the turbidity does not change. However, it is difficult to proceed the reaction so that the degree of turbidity does not change, as is clear from the reaction principle. Therefore, it is a practical measure to eliminate turbidity. However, as described above, since a surfactant having an esterase inhibitory action does not have an action of eliminating turbidity, it has been considered that elimination of turbidity and inhibition of esterase are contradictory issues. In addition, a technique for inhibiting LPL by a higher fatty acid alkali salt [5] is known, but cannot be applied to the measurement of NEFA because it contains fatty acids. Tannins [9] and hinokitiol [10] are also known to have an inhibitory effect on lipase activity, but there is no disclosure regarding application to measurement of serum lipids.

[0009] In some cases, the inhibitory effect of the required enzyme reaction by the use of an inhibitor has a more significant effect on a specific measurement item such as TG. The enzymatic measurement of TG is
It is performed based on the reaction as described above. Where LP
Focusing on L, it is necessary to suppress the reaction in the first reaction, but it is necessary to participate in the reaction as a reagent component in the second reaction. Therefore, the effect of the inhibitor of the first reaction must not affect the second reaction. Therefore, when a surfactant is used as an inhibitor, a method of adding a surfactant that promotes an enzymatic reaction to the reagent for the second reaction is employed. However, although this method can reduce the action of the inhibitor, it is difficult to completely remove it. Further, fine adjustment of the reagent composition may be required depending on the type and lot of the enzyme to be used. In addition, carryover of the surfactant itself to activate the enzyme may in turn interfere with the action of the inhibitor.

The alkylboronic acid derivatives used as inhibitors in the present invention include serine protease activity inhibitory action [11] and CEase activity inhibitory action [12] [13].
Is also known. However, the experimental material in these reports is CEase extracted from porcine pancreas. On the other hand, since the following CEases derived from microorganisms are used for the measurement of serum lipids, the usefulness in the measurement of serum lipids cannot be predicted even with the same CEase. Moreover, the effects of these reports on other enzyme reactions required for measuring serum lipids are unclear. Pseudomonas sp. Pseudomonas fluorescens Candida cylindracea Schizophyllum commune Pseudomonas sp. Pseudomonas sp.

[0011] Some boronic acid compounds are known to inhibit peptidase activity. For example, 2-phenyl-ethaneboronic acid has been reported to inhibit chymotrypsin [14]. Alpha-boronic acid analogs of amino acids having alkyl side chains such as aliphatic and aromatic have inhibitory activity against metalloenzymes [15]. It is known that peptides incorporating α-aminoboronic acid inhibit elastase, chymotrypsin, etc. [16]. However, these reports do not disclose esterase or lipase inhibitor applications.

[0012]

SUMMARY OF THE INVENTION An object of the present invention is to provide a new technique for preventing errors due to carryover of esterase or lipase in measurement of serum lipid. More specifically, it is an object of the present invention to provide a novel esterase or lipase activity inhibition technique that does not interfere with other enzymatic reactions or optical measurements.

[0013]

The present invention relates to a method for inhibiting the activity of an ester hydrolase, which causes an error in the measurement of serum lipids, and comprises a compound having a structure represented by the following general formula (1): This is a method for inhibiting ester hydrolase activity.

[0014]

Embedded image R1 and R2 represent a C1-12 linear or branched alkyl group, a phenyl group, a tolyl group, or a hydroxyl group.

The alkyl group may be a lower alkyl group such as butyl, pentyl, hexyl and octyl. Examples of the aryl group include phenyl and the like. Among the compounds having such a general formula, the following compounds can be mentioned as preferred compounds having an action of inhibiting the ester hydrolase activity at a low concentration. These specific compounds are roughly classified into those in which R1 is a hydroxyl group (-OH) and an alkyl group or an aryl group is introduced into R2, and those in which an alkyl group or an aryl group is introduced into both R1 and R2. You. Some of the compounds are commercially available, and their synthesis methods are also known [17].
[18] [19] [20] [21]. Compounds having a hydroxyl group at R1 Butylboronic acid Pentylboronic acid Hexylboronic acid Octylboronic acid Phenylboronic acid Phenylboronic acid Both R1 and R2 have alkyl groups or Compounds with aryl groups Phenylbutylboronic acid

These inhibitors are supplied at the concentrations required for the enzymatic reaction where errors occur due to the presence of the ester hydrolase. The use concentration of the inhibitor of the present invention is set according to the hydrolase activity expected to be mixed. When performing an enzymatic analysis on serum lipids using an automatic analyzer under general conditions, the final concentration in the reaction solution is 0.01 to 1000 mM, preferably 0.1 to 1000 mM. Add to about -100 mM. If the concentration of the inhibitor is set to be unnecessarily high, there is a risk that measurement may be hindered when an enzyme to be inhibited in the second reaction exists, such as TG. On the other hand, if the concentration is too low, there is a possibility that a sufficient inhibitory effect cannot be expected when carryover occurs unexpectedly.

In order to supply the inhibitor to the reaction solution, it is preferable to add the inhibitor to a sample diluent or a reaction reagent in advance.
The inhibitors of the present invention are designed so that they can be contacted with the sample at the start of the basic reaction for measurement or prior to the reaction. Specifically, it may be added to a sample diluent or a reagent that comes into contact with the sample first. Since the inhibitor of the present invention does not act in an inhibitory manner except for the ester hydrolase, no problem occurs even if it is present together with a reagent containing another enzyme. The configuration of a typical serum lipid measurement reagent is described below. F-CHO is a one-reagent system, but is generally used in a two-reagent system for the purpose of eliminating ascorbic acid. Therefore, in the case of F-CHO, an embodiment in which an inhibitor is added to the first reagent is possible.

[1] Free cholesterol (F-CHO) cholesterol oxidase peroxidase H ₂ O ₂ coloring system pH 6.0-8.0

[2] Triglyceride (TG) First reagent for TG measurement (R1) Glycerol kinase Glycerol-3-phosphate oxidase Peroxidase Adenosine triphosphate pH 6.0-8.0 Second reagent for TG measurement (R2) LPL H ₂ O ₂ coloring system pH 6.0-8.0

[3] Free fatty acid (NEFA) First reagent for NEFA measurement (R1) Acyl-CoA synthetase Coenzyme A Adenosine triphosphate pH 6.0-8.0 Second reagent for NEFA measurement (R2) Acyl-CoA oxidase peroxidase H ₂ O ₂ coloring system pH 6.0-8.0

The above composition enumerates the components necessary for promoting the basic enzyme reaction. In addition to this, for example, ascorbic acid oxidase or periodate, or bilirubin oxidase or ferricyanide, etc., for preventing the effect of ascorbic acid or bilirubin that hinders the reaction of the H ₂ O ₂ color system. It is also possible.
In general, various nonionic surfactants are added to a reagent for performing an optical analysis of a serum sample in order to dissolve turbidity generated when lipid components are excessively present. Particularly in the enzymatic measurement of lipid components, the combined use of various surfactants is an important condition for inducing the activity of various oxidases and hydrolases. Known enzyme stabilizers may be combined with these compositions. Enzyme stabilizers should show inactive proteins such as BSA, sugars such as sucrose, galactose, fructose or dextran, chelating agents in the case of metal ion-sensitive enzymes, and metal ions necessary for maintaining the activity. Can be. Thus, the inhibitors of the present invention are added to the reagents along with additional components and stabilizers that support these enzymatic reactions.

These reagent components are used by dissolving them in a buffer solution giving an appropriate pH. An appropriate pH is a pH at which a desired stability can be expected during storage, and at the final reaction site, the enzyme reaction can proceed promptly. In the above examples of the reagent composition, a general pH range is shown. Since the range of pH to be set varies, it is preferable to empirically select desirable conditions as appropriate. Buffer components include:
Select one that does not affect enzyme activity. Specifically, phosphoric acid, glycine, a good buffer and the like can be used. Specific examples of Good buffers that can sufficiently bring out the activities of various enzymes are shown below. 2-morpholinoethanesulfonic acid (2- (N-Morpholino) et
hanesulfonic acid, abbreviated as MES) Bis (2-hydroxyethyl) iminotris (hyd
roxymethyl) methane and Bis-Tris) (2-acetamido) iminodiacetic acid (N- (2-Acetamido) i
minodiacetic acid, abbreviated as ADA) Piperazine-bis (2-ethanesulfonic acid) (Piperazi
ne-N, N'-bis (2-ethanesulfonic acid), abbreviated as PIPES) (2-acetamido) -2-aminoethanesulfonic acid (N- (2-Acetamido) -2-aminoethanesulfonic acid, AC
ES) 2-Hydroxy-3-morpholinopropanesulfonic acid (MO)
Bis (2-hydroxyethyl) -2-aminoethanesulfonic acid (N, N-Bis (2-hydroxyethyl) -2-aminoehtanesulfo)
nic acid, abbreviated as BES) 3- (morpholino) propanesulfonic acid (3- (N-Morphol
ino) propanesulfonic acid, abbreviated as MOPS) Tris (hydroxymethyl) methyl-2-aminomethanesulfonic acid (N-Tris (hydroxymethyl) methyl-2-aminoeth)
anesulfonic acid, abbreviated as TES) Hydroxyethylpiperazine-2-ethanesulfonic acid (N-2-Hydroxyethylpiperazine-N'-2-ethanesulfonic a)
cid, abbreviated as HEPES) 3- [bis (2-hydroxyethyl) amino] -2-hydroxypropanesulfonic acid (3- [N, N-Bis (2-hydroxyet
hyl) amino] -2-hydroxypropanesulfonic acid, DIPS
O is abbreviated.) Tris (hydroxymethyl) methyl-2-hydroxy-
3-aminopropanesulfonic acid (N-Tris (hydroxymethy
l) methyl-2-hydroxy-3-aminopropanesulfonic acid, T
APSO) Piperazine-N, N'-bis (2-hydroxypropanesulfonic)
acid), POPSO) 2-hydroxyethylpiperazine-2-hydroxypropane-3-sulfonic acid (N-2-Hydroxyethylpiperazine-
N'-2-hydroxypropane-3-sulfonic acid, HEPPSO
2-hydroxyethylpiperazine-3-propanesulfonic acid (N-2-Hydroxyethylpiperazine-N'-3-propanesulfo)
nic acid, EPPS) [Tris (hydroxymethyl) methyl] glycine (N- [T
ris (hydroxymethyl) methyl] glycine, abbreviated as Tricine) Bis (2-hydroxyethyl) glycine (N, N-bis (2-hy
droxyethyl) glycine, abbreviated as Bicine) Tris (hydroxymethyl) methyl-3-aminopropanesulfonic acid (N-Tris (hydroxymethyl) methyl-3-aminop
ropanesulfonic acid, abbreviated as TAPS) Cyclohexyl-2-aminomethanesulfonic acid (N-Cycl
ohexyl-2-aminoethanesulfonic acid, abbreviated as CHES)

The reagent according to the present invention comprising the above components may be supplied in a dry state or may be distributed in a liquid state. In the case of distributing in a liquid state, the composition and pH of each reagent are adjusted in consideration of the stability in the distributing process while designing so as to give a pH necessary for the final reaction. The reagent containing the inhibitor of the present invention can be used as a kit further set with a standard substance and the like.

The hydrolase whose activity is to be inhibited by the inhibitor according to the present invention is one that causes an error when it is mixed in a reaction system in the measurement of serum lipid by an enzymatic reaction. Specifically, CEase, LPL, and the like can be indicated. As these enzymes, those derived from microorganisms are generally used. The microorganisms used as the raw materials for the enzymes are as summarized above. The inhibitors of the present invention are all effective against these enzymes. Techniques for obtaining these enzymes as recombinants [22] [23] [24] [2
5] Alternatively, techniques for obtaining mutants with enhanced stability and activity [26] [27] [28] [29] are also known. The inhibitor of the present invention can also be used for these enzymes produced by genetic engineering.

[0025]

The alkylboronic acid of the present invention inhibits the activity of the ester hydrolase to prevent errors which are problematic in measuring serum lipids. Since the present invention is used for measuring serum lipids, it is not sufficient to simply inhibit the target enzyme activity. In addition to reliably suppressing the activity of the target enzyme, it is an important condition that it does not affect the original enzyme reaction for measuring serum lipids.

Further, among serum lipids, especially TG, further conditions must be provided. That is, T
It must be able to set conditions that do not inhibit the activity of LPL required for the measurement of G. When the inhibitor of the present invention is added to the first reagent of the TG measurement reagent composition having a general composition, LP can be added to the second reagent without taking any special measures against the inhibitor.
It has an excellent feature that it does not affect the activity of L. It is presumed that the presence of the inhibitor of the present invention at an appropriate concentration inhibits the activity of the enzyme slightly mixed, but hardly exerts an inhibitory effect on LPL added in a large amount as a reagent. Such a feature is a feature that could not be expected with a known inhibitor using a surfactant or the like. The present invention has great value in selecting excellent compounds that satisfy such difficult conditions as inhibitors.

[0027]

The alkylboronic acid compound used as an inhibitor in the present invention exhibits an excellent error-preventing effect because it reliably suppresses various ester hydrolase activities which cause errors at a low concentration. Further, the inhibitors provided by the present invention include cholesterol oxidase, acyl C
Since it does not affect oA synthase or other enzyme activities required for measurement of lipids such as acyl-CoA oxidase, it can be applied to a wide range of reagents. In addition, the inhibitor of the present invention is an excellent inhibitor that can easily set conditions that do not affect the activity of the enzyme necessary for the reaction even when measuring TG containing LPL in the second reagent. .

Further, in the present invention, since an alkylboronic acid compound is used as an inhibitor, elimination of chyle which interferes with optical measurement can be realized by another arbitrary surfactant. On the other hand, according to the known technique of inhibiting ester hydrolase activity with a compound having a large HLB value, it is difficult to eliminate chyle with a surfactant. In fact, when the esterase inhibitor according to the present invention was used, it was possible to easily set a combination of surfactants for realizing rapid turbidity removal. Since the alkylboronic acid compound of the present invention has high solubility and is hardly adsorbed to a plastic cell like a protein, there is no possibility that the inhibitor itself will carry over and affect other reaction systems.

[0029]

【Example】

1. Effect of Inhibition of Various Esterases by Alkyl Boronic Acid The effect of the inhibitor of the present invention on the activity of various esterases was investigated. As the inhibitor of the present invention, butylboronic acid (Butylboronic acid; CH ₂ (CH ₂ ) ₃ B (OH) ₂ , manufactured by Aldrich) was used. The following four types of esterases (commercially available products) were prepared and dissolved in a 100 mM phosphate buffer (pH 7.0) for use in the experiment. Cholesterol esterase (from Candida sp.) Cholesterol esterase (from Pseudomonas sp.) Cholesterol esterase (from Pseudomonas sp.) Lipoprotein lipase (from Pseudomonas sp.)

The experimental procedure is as follows. First, a first reagent (R1, containing 0-100 mmol / L of butylboronic acid) having the following composition (all in the reagent):
And second reagent (R2, contains components necessary for enzymatic reaction)
Was prepared. 4 U / mL of each cholesterol esterase,
Or 5 μl of 300 U / mL LPL and 250 μl of R1
Was placed in a photometric cell and incubated at 37 ° C. 5
After one minute, 250 μl of R2 was dispensed into the same cell, and the enzyme reaction was further performed for 5 minutes. During this time, 500 nm every 22 seconds
Was measured. For the measurement of the absorbance, an automatic analyzer TBA-30R (manufactured by Toshiba) was used. According to this experiment, if the esterase activity is inhibited, the absorbance after addition of R2 should not change.

[0031]

Embedded image

The results are shown in FIGS. Butylboronic acid inhibits enzyme activity against cholesterol esterase derived from any microorganism regardless of the origin of cholesterol esterase at a concentration of 5 mmol / L or more. In addition, even for cholesterol esterase from Company B showing resistance to butylboronic acid, the absorbance after addition of R2 does not change at a concentration of 10-20 mmol / L or more, and rapid inhibition of enzyme activity is expected. It's clear what you can do.
In addition, a sufficient activity inhibitory effect was confirmed for LPL.

2. Confirmation of error prevention effect due to reagent carryover / T-CHO → F-CHO Serum samples were actually measured repeatedly to confirm that the inhibitor of the present invention was effective in preventing errors due to carryover via cells. did. Measurement items include T-CHO and F-
A CHO combination was chosen. In this combination, TC
A carry-over of CEase contained in the HO reagent may cause a correct error in the measured value of F-CHO. First, T-CHO was measured once, and then F-CHO was continuously measured three times in the same cell washed under normal conditions. The reagent composition and measurement parameters used are shown below. Hexylboroni acid used as an inhibitor
c acid; CH ₃ (CH ₂ ) ₅ B (OH) ₂ ) used was previously synthesized according to a known method [13]. The analyzer is Hitachi 715
A type 0 automatic analyzer (manufactured by Hitachi, Ltd.) was used. Also T-
CEase added to CHO R1 was obtained from Pseudomonas sp.
For T-CHO, the serum and R1 were mixed, incubated for 5 minutes, R2 was added, and the mixture was further incubated for 5 minutes before measuring the absorbance of the reaction solution. The value of T-CHO was determined based on a calibration curve prepared from standard serum of which concentration was known in advance. F-CHO was also measured under the same conditions as T-CHO. The serum sample used for the measurement was CEase
The value of F-CHO measured in the absence of hexylboronic acid under the conditions free of phenol was 43 mg / dL.

[0034]

Embedded image

[0035]

Embedded image

[0036]

Embedded image

The results are shown in Table 1. Hexylboronic acid with R
In the measurement method utilizing the present invention, which is included in No. 1, stable F-CHO measurement values are obtained even immediately after T-CHO measurement. On the other hand, when no CEase inhibitor is contained, TC
A positive error occurs in the F-CHO measurement value after the HO measurement.
The influence of CEase is observed until the third measurement, and it is clear that the problem of carryover is large. The automatic analyzer "Hitachi 7150" used for the experiment
Under normal washing conditions, the cell after measurement is washed three times with purified water, then further purified water is dispensed, and after purifying the water blank, the remaining purified water is aspirated before dispensing the next reaction solution. . Normally, analysis can be performed without any trouble under these cleaning conditions. However, in a specific combination as in this embodiment, cleaning may be insufficient and may lead to an error. Furthermore, the measured value of F-CHO in the presence of hexylboronic acid was the same as the measured value in the absence of 43 mg / dL, and the inhibitor hexylboronic acid affected the original enzyme reaction. Not.

[0038]

[Table 1]

3. Confirmation of Error Prevention Effect by Reagent Carryover / TG → F-CHO Serum samples were actually measured repeatedly, and it was confirmed that the inhibitor of the present invention was effective in preventing errors due to carryover via cells. Measurement items include TG and F-CHO
Was chosen. In this combination, the carryover of LPL contained in the reagent for TG causes F-CH
A positive error may occur in the measured value of O. The conditions of the experiment were the same, including the serum sample, except that TG was measured instead of the T-CHO measurement of Example 2. The reagent composition for TG used is shown below. LPL in TG R2
Is derived from Pseudomonas sp. TG was measured by mixing serum and R1 and incubating for 5 minutes.
2 was added, the mixture was further incubated for 5 minutes, and then the absorbance of the reaction solution was measured. TG based on a calibration curve prepared from standard serum whose concentration is known in advance
Was determined.

[0040]

Embedded image

[0041]

Embedded image

The results are shown in Table 2. Hexylboronic acid with R
In the measurement method using the present invention, which is included in No. 1, a stable F-CHO measurement value is obtained even immediately after the TG measurement. On the other hand, when no LPL inhibitor was contained, F-
A positive error has occurred in the CHO measurement value. The effect of LPL has been observed up to the third measurement, and it is clear that the problem of carryover is large.

[0043]

[Table 2]

4. Confirmation of Error Prevention Effect by Reagent Carryover / T-CHO → F-CHO With the same combination as in Example 2, carryover occurs not only through the cell but also through the reagent probe, and the effect of the present invention can be prevented. confirmed. After measuring T-CHO by the same operation and using the same reagent as in Example 2, the measurement of F-CHO was performed three times continuously without measuring other items. All measurements were made in different cells to rule out the effects of carryover through the cells.
The results are shown in Table 3. In the measurement method using hexylboronic acid in R1 according to the present invention, a stable F-CHO measurement value is obtained even immediately after T-CHO measurement. Meanwhile, CE
It can be seen that, when no case inhibitor is contained, a slight error occurs even though the reagent probe is used, though not as much as carryover via the cell. CEas
The effect of e is observed until the second measurement, and it is clear that the problem of carryover is large. The automatic analyzer "Hitachi 7150" used in the experiment, under normal washing conditions, allowed distilled water to pass through the probe after reagent dispensing,
Further, the outside is also washed with purified water to prevent the reagent from remaining. Normally, analysis can be performed without any trouble under these cleaning conditions. However, in a specific combination as in this embodiment, cleaning may be insufficient and may lead to an error.

[0045]

[Table 3]

5. Confirmation of Error Prevention Effect by Carryover of Reagent / Continuous TG Measurement Serum samples were actually measured repeatedly, and it was confirmed that the inhibitor of the present invention was effective in preventing errors due to carryover via cells. TG was selected as the measurement item. T
When G is continuously measured, the carry-over of the LPL contained in the TG reagent (R2) causes the next TG to be measured.
May cause a negative error in the measured value of TG measurement was performed three consecutive times in the same cell washed under normal conditions.
The reagent composition used is shown below. Hexylboronic acid used as an inhibitor was prepared in advance, and a Hitachi 7150-type automatic analyzer (manufactured by Hitachi, Ltd.) was used as an analyzer. The LPL added to R2 of TG is Pseudomonas s
The reagent composition for TG measurement derived from p.
Hexylboronic acid was added in the same manner as in Example 3 except that 0 or 1 mmol / L was added. The operation was the same as in Example 3. The serum sample used for the measurement had a value of 99 mg / dL when TG was measured in the absence of hexylboronic acid under conditions free of LPL.

Table 4 shows the results. Hexylboronic acid with R
In the measurement method using the present invention, which is included in No. 1, a stable TG measurement value is obtained even when TG is continuously measured. on the other hand,
When no LPL inhibitor is contained, a negative error occurs in the measured values after the second time. In such a case, carry-over from the second reaction solution to the third reaction solution also occurs, so that the influence of LPL does not disappear even after the number of times. On the other hand, since the washing effect by the reaction liquid itself can be expected between different items, the influence of carryover becomes smaller as the measurement is repeated. Therefore, the problem of carryover in continuous measurement is more serious.

[0048]

[Table 4]

6. Confirmation of Error Prevention Effect by Reagent Carryover / T-CHO → TG Serum samples were actually measured repeatedly, and it was confirmed that the inhibitor of the present invention was effective in preventing errors due to carryover via cells. Measurement items include T-CHO and TG
Was chosen. In this combination, a carryover of CEase contained in the reagent for T-CHO may cause a negative error in the measured value of TG. First T
-CHO was measured once, and then TG was measured three times consecutively in the same cell washed under normal conditions. The reagent composition and operation used were the same as those in Example 2 (T-CHO) or Example 5 (TG). The serum sample was the same as in Example 5 (TG measurement value: 99 mg / dL).

The results are shown in Table 5. Hexylboronic acid with R
In the measurement method using the present invention, which is included in No. 1, stable TG measurement values are obtained even immediately after T-CHO measurement. On the other hand, when no CEase inhibitor is contained, T-CHO
A negative error occurs in the TG measurement value after the measurement. Negative errors are also observed in the second and subsequent measurement values, which is considered to be due to LPL carryover from the initial TG measurement as well as the CEase.

[0051]

[Table 5]

7. Confirmation of error prevention effect due to reagent carryover / TG → NEFA Serum samples were actually measured repeatedly, and it was confirmed that the inhibitor of the present invention was effective in preventing errors due to carryover via cells. The combination of TG and NEFA was selected as the measurement item. In this combination, a carryover of LPL contained in the TG reagent may cause a correct error in the measured value of NEFA. First, TG was measured once, and then NEF was performed in the same cell washed under normal conditions.
The measurement of A was performed three times in succession. The reagent composition and operation used were the same as in Example 5 for the measurement of TG. NEFA
The composition of the reagent used for the measurement is as follows. N
The measurement of EFA was performed by mixing serum and R1, incubating for 5 minutes, adding R2, incubating for another 5 minutes, and measuring the absorbance of the reaction solution. The value of NEFA was determined based on a calibration curve prepared from standard serum whose concentration was known in advance. The serum sample used for the measurement had a value of 2 when the NEFA was measured in the absence of hexylboronic acid under conditions free of LPL.
It was 85 μeq / L.

[0053]

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[0054]

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The results are shown in Table 6. Hexylboronic acid with R
In the measurement method using the present invention, which is included in No. 1, stable NEFA measurement values are obtained even immediately after TG measurement. on the other hand,
When no LPL inhibitor was contained, NEFA after TG measurement
A positive error has occurred in the measured value. The effect of LPL is 3
Observed until the second measurement, it is clear that the problem of carryover is significant. Furthermore, the NEFA measurement value in the presence of hexylboronic acid was the same as the measurement value in the absence of 285 μeq / L, and the inhibitor hexylboronic acid did not affect the original enzyme reaction. .

[0056]

[Table 6]

8. Effect on other reagent components In the measurement of F-CHO using cholesterol oxidase, it was confirmed that the esterase inhibitor of the present invention had no effect on cholesterol oxidase. As a control, a surfactant Triton X-405, which is a known esterase inhibitor, was used. 2, based on a reagent for measuring F-CHO having the same composition as that used in Step 2, 0.4% Triton X100 (control), 0.4% Triton X100 + 7.5 mM hexylboronic acid (invention),
F-CHO was measured with three combinations of 0.4% Triton X405 (a known esterase inhibitor). An automatic analyzer TBA-30R (manufactured by Toshiba) was used for the measurement, and the measurement parameters were shown below. Triton X100 is a surfactant having an enzyme activating action usually added to a reagent for measuring F-CHO. The results are shown in FIG.
It was shown to. In the case of only containing Triton X100 as a control and the case of containing hexylboronic acid according to the present invention, the reaction of F-CHO was completed immediately after the addition of the second reagent. That is, no inhibitory effect of hexylboronic acid on enzymes necessary for reactions such as cholesterol oxidase is observed. On the other hand, when Triton X405 was added, the reaction after the addition of the second reagent did not proceed smoothly, and a strong inhibitory effect on cholesterol oxidase was observed. Therefore, the esterase inhibitor according to the present invention comprises
It can be said that this is a desirable esterase inhibitor having no inhibitory effect on other reagent components.

[0058]

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References [1] Medical Tchnology, Vol. 16, No. 5, 409-417; 1988 [2] JP-A-61-104798 [3] JP-A-4-173099 [4] JP-A-58-67197 [5] JP-A-58-41357 [6] JP-A-2-184759 [7] JP-A-2-193998 [8] JP-A-4-366764 [9] JP-A-8-259557 [10] JP-A-8-268882 [11] Bioorg.Med.Chem.Lett., 2,1391; 1992 [12] Biochem.Biophys.Res.Commun.Vol.134, No.1,386-3
92,1986 [13] Biochimica. Biophysica. Acta. 1041, 79-82, 1990 [14] Biochemistry, 10, 2477-, 1971 [15] U.S. Pat. .Am.Chem.Soc., 93 1816-1818; 1971 [18] J.Am.Chem.Soc., 97 5249-5255; 1975 [19] Tetrahedron, 32,981-990; 1976 [20] J.Am.Chem .Soc., 116,3151,8516; 1994 [21] Tetrahedron Lett, 35,4419; 1994 [22] Publication 3-503487 [23] JP-A-4-18367 [24] WO93 / 07261 [25] Publication 8 -510905 [26] Publication 4-506609 [27] JP-A-7-163339 [28] JP-A-7-203959 [29] JP-A-8-242860

[Brief description of the drawings]

FIG. 1 is a reaction time course of cholesterol esterase (from Candida sp.) When alkylboronic acid is used as an enzyme inhibitor. The vertical axis indicates the absorbance, and the horizontal axis indicates the time (one photometric point corresponds to about 22 seconds, and the reaction time is about 5 minutes for both R1 and R2, for a total of 10 minutes).

FIG. 2: Cholesterol esterase when alkylboronic acid is used as an enzyme inhibitor (from Pseudomonas sp.)
Reaction time course. The vertical axis represents the absorbance, and the horizontal axis represents the time (a photometric point corresponds to about 22 seconds for one point; R1, R2
In both cases, the reaction time is about 5 minutes, for a total of 10 minutes).

FIG. 3. Cholesterol esterase when alkylboronic acid is used as an enzyme inhibitor (derived from Pseudomonas sp.)
Reaction time course. The vertical axis represents the absorbance, and the horizontal axis represents the time (a photometric point corresponds to about 22 seconds for one point; R1, R2
In both cases, the reaction time is about 5 minutes, for a total of 10 minutes).

FIG. 4 is a reaction time course of lipoprotein lipase (derived from Pseudomonas sp.) When alkylboronic acid is used as an enzyme inhibitor. The vertical axis indicates the absorbance, and the horizontal axis indicates the time (one photometric point corresponds to about 22 seconds, and the reaction time is about 5 minutes for both R1 and R2, for a total of 10 minutes).

FIG. 5. F-CHO in the presence of an esterase inhibitor
Reaction time course of measurement. The vertical axis represents the absorbance, and the horizontal axis represents the time (one photometric point corresponds to about 22 seconds;
1 and R2 show a reaction time of about 5 minutes, for a total of 10 minutes).

Claims (8)

[Claims] 1. A method for inhibiting the activity of an ester hydrolase which causes an error in the measurement of serum lipids,
Method for inhibiting ester hydrolase activity by a compound having a structure represented by the following general formula (1): R1 and R2 each represent a C1-12 linear or branched alkyl group, a phenyl group, a tolyl group, or a hydroxyl group. 2. The method according to claim 1, wherein the serum lipid is selected from the group consisting of free cholesterol, triglyceride, and free fatty acid. 3. The method according to claim 1, wherein the ester hydrolase is cholesterol esterase or lipoprotein lipase. 4. The method according to claim 1, wherein the concentration of the compound having the structure represented by the general formula (1) is 0.01 to 1000 mM in the reaction solution in which the presence of the ester hydrolase to be inhibited is expected. Method 5. A reagent composition for measuring serum lipid containing a compound having a structure represented by the following general formula (1): R1 and R2 each represent a C1-12 linear or branched alkyl group, a phenyl group, a tolyl group, or a hydroxyl group. 6. An inhibitor of microbial ester hydrolase activity comprising a compound having a structure represented by the following general formula (1): R1 and R2 each represent a C1-12 linear or branched alkyl group, a phenyl group, a tolyl group, or a hydroxyl group. 7. A method for inhibiting the activity of ester hydrolase derived from a microorganism comprising a compound having a structure represented by the following general formula (1): R1 and R2 each represent a C1-12 linear or branched alkyl group, a phenyl group, a tolyl group, or a hydroxyl group. 8. A method for preventing an error caused by an ester hydrolase activity derived from another reagent component in the measurement of serum lipid by coexisting a compound having a structure represented by the following general formula (1) in a reaction system. Formula 5 R1 and R2 each represent a C1-12 linear or branched alkyl group, a phenyl group, a tolyl group, or a hydroxyl group.