JP2002051769A - Liquid reagent useful for measuring nonesterified fatty acid - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide liquid reagents for measuring nonesterified fatty acid (NEFA), stable for a long period, and to provide a method for measuring the NEFA, capable of simply and accurately measuring the NEFA by using the reagents. SOLUTION: This solution containing coenzyme A (CoA) is compounded of a chelating agent, such as diethylenetriamine-N,N,N',N",N"-pentaacetic acid (DTPA), triethylenetetramine-N,N,N',N",N''',N'''-hexaacetic acid (TTHA), and 0,0'-bis(2- aminoethyl)ethylene glycol-N,N,N',N'-tetraacetic acid (EGTA) as a CoA stabilizer, wherein the solution containing the CoA is used as a first reagent of the liquid reagents for measuring the NEFA. The other solution containing acyl coenzyme A oxidase (ACO) is compounded of an anionic surfactant in such an amount as effective for stabilizing the ACO, preferably, together with flavin adenine dinucleotide (FAD) in such an amount as effective for stabilizing the ACO and, more preferably, further with N-ethylmaleimide (NEM) in such a manner that the solution is controlled to have a pH effective for stabilizing the NEM, wherein the solution containing the ACO is used as a second reagent of the liquid reagents for measuring the NEFA. A kit for measuring the NEFA is composed of the first reagent and/or the second reagent, and the method for measuring the NEFA comprises using the kit.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to coenzyme A (hereinafter referred to as CoA) which is a component of a reagent for measuring free fatty acids (hereinafter referred to as NEFA) using an enzyme, acyl coenzyme A oxidase (hereinafter referred to as ACO), N
The present invention relates to a liquid reagent which can stabilize ethylmaleimide (hereinafter referred to as NEM) and can be stored for a long time, a method for producing the same, and a method for measuring NEFA using the same.

[0002]

2. Description of the Related Art Conventional commercial kits for automatic analysis reagents are supplied as a combination of a lyophilized product and a lysing solution, and are dissolved and used for measurement at the time of use. However, in the case of the freeze-dried product, in addition to the complexity of preparation of the reagent, there are problems that an erroneous measurement result is obtained due to a mistake in the preparation and that the effective period after dissolution is short (lack of stability). Therefore, in recent clinical tests, the use of liquid reagents has become mainstream in order to avoid such problems.

[0003] As a method for quantifying NEFA, first, acyl coenzyme A is used in the first reagent in the presence of a divalent metal ion such as Mg ⁺⁺ or Mn ⁺⁺ and adenosine 5'-triphosphate (hereinafter referred to as ATP). Synthetase (hereinafter referred to as ACS)
By the action of CoA and NEFA in the sample, acyl C
in the first step of producing oA, and in the second reagent, NE
A method comprising a second step of quantifying hydrogen peroxide generated by oxidizing acyl CoA by the action of ACO in the presence of M by combining peroxidase and a chromogenic substrate has been widely adopted.

However, it has been considered that liquefaction is difficult because the NEFA measurement reagent contains many unstable components. For example, the conventionally known first reagent for NEFA measurement containing CoA, once dissolved, has a shelf life of about 5 to 15 days in refrigeration. In addition, NEM becomes unstable in the region where the pH is higher than around neutral, and ACO
Is denatured by NEM.
When O and coexist in a solution near neutrality, the stability of both of them suddenly decreases. Therefore, NEM and ACO
It is said that it is difficult to maintain the performance as a measurement reagent over a long period of time. Conventionally, NEM is stored in a strongly acidic solution, and ACO is lyophilized and stored. It was common to store ACOs separately to ensure their stability.

[0005]

DISCLOSURE OF THE INVENTION The present invention relates to a particularly unstable component in a NEFA assay using an enzyme.
CoA in the first reagent and NEM and ACO in the second reagent
It is an object of the present invention to provide a stable NEFA measurement liquid reagent which can stabilize and maintain the measurement performance for a long period of time. A further object of the present invention is to provide a simpler and more accurate method for measuring NEFA using these reagents.

[0006]

Means for Solving the Problems The present inventors have made intensive studies to achieve the above object, and as a result, have found that diethylenetriamine-N, N, N ', N ", N" -pentaacetic acid ( Hereinafter, referred to as DTPA), triethylenetetramine-N, N, N ', N ", N'",
Selected from N '''-hexaacetic acid (hereinafter referred to as TTHA) and O, O'-bis (2-aminoethyl) ethylene glycol-N, N, N', N'-tetraacetic acid (hereinafter referred to as EGTA) It has been found that CoA can be stabilized by coexisting one or more chelating agents with the first reagent. In addition, the present inventors have proposed flavin adenine dinucleotide (hereinafter, referred to as FAD).
It has been found that coexistence of A can stabilize ACO even on the more acidic side. Furthermore, when a surfactant that does not hinder the ACO reaction was added to prevent turbidity of the solution, it was evident that, surprisingly, the surfactant has an effect of further stabilizing ACO in addition to the effect of preventing turbidity. It became. Therefore, by adding FAD and a surfactant, and adjusting the pH of the second reagent to a more acidic side in a pH range suitable for the storage stability of ACO, the NEM was also successfully stabilized. The present invention has been completed.

That is, the present invention relates to a CoA-containing solution,
Provided is a method for stabilizing CoA to the extent that it can be used as a liquid reagent by adding an effective amount of one or more chelating agents selected from the group consisting of DTPA, TTHA and EGTA to stabilize CoA. I do. The solution produced by this method can be used as a first reagent for NEFA measurement by further mixing ACS and ATP.

[0008] The present invention also provides a method of adjusting the pH of a solution containing ACO and NEM to a value effective for stabilizing NEM, and further comprising adjusting at least one of an amount effective for stabilizing ACO at the pH. A method of stabilizing ACO and NEM to the extent that they can be used as liquid reagents by adding an ACO stabilizer, especially FAD and / or a surfactant. By the action of the ACO stabilizer, A
CO is stabilized on the more acidic side and also in the presence of NEM. Therefore, by lowering the pH to a more acidic range as long as the storage stability of ACO is ensured, it becomes possible to stabilize NEM at the same time. The NEM and ACO-containing solution thus obtained is further mixed with a reagent for quantifying hydrogen peroxide to obtain NEF.
It can be used as a second reagent for A measurement.

Further, the present invention provides a stabilized CoA-containing solution according to the present invention, wherein a solution further containing ACS and ATP is used as a first reagent, and a hydrogen peroxide solution is added to the stabilized NEM and ACO-containing solution according to the present invention. A kit for NEFA measurement containing a solution further containing a reagent for quantification as a second reagent,
And a method for measuring NEFA using the kit. The kit of the present invention enables simpler, faster and more accurate measurement of NEFA by eliminating the need for dissolving and using a lyophilized product at the time of use, unlike conventional products.

[0010] The present invention can also be applied to stabilization of a conventionally known reagent for NEFA measurement after preparation at the time of use. That is, the stability of the freeze-dried product after dissolution can be improved by adding the stabilizer of the present invention in advance to the freeze-dried product of a conventionally known reagent for NEFA measurement and / or a solution thereof. .

The following abbreviations may be used in this specification. EDTA 2NH ₄ Ethylenediamine-N, N, N ', N'-diammonium tetraacetate EDTA 2Li Ethylenediamine-N, N, N', N'-dilithium tetraacetate EDTA Mn Ethylenediamine-N, N, N ', N' Disodium manganese tetraacetate EDTA Mg Ethylenediamine-N, N, N ', N'-Disodium magnesium tetraacetate EDTA Ba Ethylenediamine-N, N, N', N'-Sodium barium tetraacetate EDTA 4Na Ethylenediamine-N, N, N ', N'-Tetraacetic acid tetrasodium salt EDTA Co Ethylenediamine-N, N, N', N'-Tetracobalt disodium salt EDTA Ni Ethylenediamine-N, N, N ', N'-Titanium disodium salt EDTA 3K Ethylenediamine-N, N, N ', N'-tripotassium tetraacetic acid EDTA Zn Ethylenediamine-N, N, N', N'-Disodium zinc tetraacetate EDTA 2Na Ethylenediamine-N, N, N ', N' -Sodium tetraacetate Methyl-EDTA 1,2-Diaminopropane-N, N, N'N'-tetraacetic acid EDTA-OH N- (2-hydroxyethyl) ethylenediamine-N, N ', N'-triacetic acid CyDT A trans-1,2-diaminocyclohexane-N, N, N ', N''-tetraacetic acid EDDP Ethylenediamine-N, N'-dipropionic acid dihydrochloride HIDA N- (2-hydroxyethyl) iminodiacetic acid EDDA Ethylenediamine -N, N'-diacetate DHEG N, N-bis (2-hydroxyethyl) glycine DTPA-OH 1,3-diamino-2-hydroxypropane-N, N, N ', N'-tetraacetic acid NTP nitrilotri Propionic acid HDTA Hexamethylenediamine-N, N, N ', N'-tetraacetic acid EDDPO Ethylenediamine-N, N'-bis (methylenephosphonic acid) NTA Nitrilotriacetic acid IDA Iminodiacetic acid EDTPO Ethylenediamine-N, N, N ', N'-tetrakis (methylenephosphonic acid) NTPO trisodium nitrilotrismethylenephosphonate

[0012]

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
A and DTPA, TTHA as stabilizers for CoA
And one or more chelating agents of EGTA. The concentration of CoA is not particularly limited as long as the solution is in a range suitable for an enzyme reaction in which the solution is used. The concentration of the chelating agent is not particularly limited as long as it is an amount effective for stabilizing CoA, and varies depending on the type of the chelating agent used. For example, in the case of DTPA, the concentration of the chelating agent is 0.1 mM or more and at a storage temperature. The concentration which does not precipitate is mentioned. CoA of the present invention
When the containing solution is used for preparing the first reagent for NEFA measurement, the concentration may be lower than the concentration of the divalent metal ion added at the same time. More preferably, the working concentration of DTPA is 0.
25 to 10 mM. Here, the term “effective amount for stabilizing CoA” refers to, for example, in a practical test, a liquid reagent was subjected to severe conditions (25 ° C., 27 days or 37 days).
(5 ° C., 5 days) means an amount effective to maintain the performance as a liquid reagent even after storage at the same temperature, and “stable” means that the liquid reagent is stored under the above-mentioned severe conditions in a practical test. After that, it means maintaining a reactivity remaining rate of about 70% or more immediately after preparation.

The pH of the CoA-containing solution of the present invention is in a range suitable for the enzymatic reaction in which the solution is used, does not adversely affect CoA and other components, and is effective for stabilizing the solution with a chelating agent. There is no particular limitation as long as it is within a suitable range.

The first reagent for NEFA measurement of the present invention is
ACS and ATP were added in addition to the clear CoA-containing solution.
Contained. The concentration of ACS and ATP depends on the concentration of N in the sample.
If the amount is sufficient to convert EFA to acyl CoA
There is no particular limitation. In addition, ACS requires divalent cations.
Therefore, the first reagent for NEFA measurement of the present invention
g ⁺⁺, Mn⁺⁺And the like. The
The concentration of divalent metal ions is determined by the key added to the CoA-containing solution.
At a higher concentration than the chelating agent and ACS
Especially limited if the concentration is sufficient to convert to sill CoA
There is no.

The solution containing NEM and ACO of the present invention comprises:
An amount of one or more ACs having a pH effective to stabilize the NEM and effective to stabilize the ACO at that pH
Contains O stabilizer. The concentrations of NEM and ACO are:
There is no particular limitation as long as the solution is in a range suitable for the enzyme reaction to be used. For example, NEM is 0.1 to 20 mM, AC
O is 1 to 100 U / mL.

The ACO stabilizer of the present invention includes FAD
Alternatively, a surfactant may be used. Use concentration of FAD is A
There is no particular limitation as long as it is an amount effective for stabilizing CO, but for example, 10 μM or more, more preferably 100 to 2 μM.
A concentration of 000 μM. Here, “the amount effective for stabilizing ACO” refers to, for example, in a practical test, a liquid reagent was stored under severe conditions (25 ° C., 27 days or 37 ° C., 5 days). The term "stable" refers to an amount effective to ensure the performance of the liquid reagent, and the term "stable" means that in a practical test, after the liquid reagent was stored under the above-mentioned severe conditions, about 70% or more of the reaction immediately after the preparation was completed. It means to maintain the sex residual rate.

The type of surfactant is not particularly limited as long as it does not inhibit the reaction of ACO, but is preferably an anionic surfactant, more preferably polyoxyethylene alkyl ether sulfate, polyoxyethylene alkyl phenyl ether sulfate, or polyoxyethylene alkyl phenyl ether sulfate. Oxyethylene dialkyl allyl ether sulfate, polyoxyethylene alkenyl allyl ether sulfate, and salts thereof. One of these may be used alone, or two or more thereof may be used in combination. More specifically, polyoxyethylene alkyl ether sulfate [Example: Hytenol 18E (manufactured by Daiichi Kogyo Seiyaku Co., Ltd.); Emal 20C and 20A, Lebenol WX (all manufactured by Kao Corporation)], polyoxyethylene Ethylene alkyl phenyl ether sulfuric acid [Example: Hytenol N
-17 (manufactured by Daiichi Kogyo Seiyaku Co., Ltd.); Emar NC, Lebenol WZ (all manufactured by Kao Corporation)], polyoxyethylene dialkyl allyl ether sulfate [Example: Hytenol NE-15 (Daiichi Kogyo Seiyaku Co., Ltd.) Co., Ltd.)], polyoxyethylene alkenyl allyl ether sulfate [Example: Hytenol NF-13 (Daiichi Kogyo Seiyaku Co., Ltd.)] and the like. The concentration of the surfactant used is not particularly limited as long as it is an effective amount for stabilizing the ACO and does not adversely affect the reaction and measurement of the ACO, and varies depending on the type of the surfactant. In the case of Hytenol 18E, it is 0.05% by weight or more, preferably 0.1 to 1.0% by weight.

The addition of the surfactant simultaneously achieves prevention of clouding of the solution. For example, when Hytenol 18E is used, the turbidity preventing effect is achieved at 0.05% by weight or more.

In a preferred embodiment of the present invention, F
Both AD and anionic surfactants are formulated as ACO stabilizers.

The p of the NEM and ACO containing solution of the present invention
H is adjusted to a pH effective for stabilizing the NEM within the stable pH range of ACO that has spread to the acidic side by incorporating the ACO stabilizer. Preferably, the pH of the NEM and ACO containing solution of the present invention is between 5.5 and 6.8, more preferably between 6.0 and 6.4. Here, the “pH effective for stabilizing the NEM” refers to, for example, in a practical test, the liquid reagent was stored under severe conditions (25 ° C., 27 days or 37 ° C., 5 days). The term "stable" means a pH effective for ensuring the performance as described above, and "stable" means that in a practical test, after storage of a liquid reagent under the above-mentioned severe conditions, a reaction of about 30% or more immediately after preparation was performed. It means to maintain the sex residual rate.

The NEFA measurement second reagent of the present invention further contains a hydrogen peroxide quantitative reagent in addition to the NEM and ACO-containing solution of the present invention. The reagent for quantitatively determining hydrogen peroxide may be any conventionally known reagent, and is preferably composed of peroxidase and a hydrogen donor that is a chromogenic substrate. For example, peroxidase derived from horseradish (hereinafter referred to as peroxidase) , HRP) and N-ethyl-N- (2-hydroxy-3-sulfopropyl) -m-toluidine sodium (hereinafter referred to as TOOS).

The kit for NEFA measurement of the present invention comprises, as a first reagent, a first reagent for NEFA measurement of the present invention, and / or
Alternatively, the second reagent includes the second reagent for NEFA measurement of the present invention. When only one of the first and second reagents of the present invention is contained, the kit for NEFA measurement of the present invention is, for example, formed into a kit in combination with a conventionally known second or first reagent for NEFA measurement, respectively. be able to.

In the NEFA measurement method of the present invention, N
A reaction reagent for converting EFA into acyl-CoA, preferably the first reagent for measuring NEFA of the present invention, is added to convert NEFA in a sample into acyl-CoA, and then oxidize acyl-CoA to hydrogen peroxide. Is added to the reaction solution, preferably a reagent comprising a reagent for the reaction for producing and a reagent for quantifying the generated hydrogen peroxide, preferably the second reagent for NEFA measurement of the present invention. It is characterized by quantification (however, at least one of the first and second reagents for NEFA measurement of the present invention is used).
When the first or second reagent for NEFA measurement of the present invention is not used, a reagent for reaction for converting NEFA to acyl CoA, a reagent for reaction for oxidizing acyl CoA to produce hydrogen peroxide, and a reagent for producing hydrogen peroxide Reagents comprising a reagent for quantifying hydrogen oxide are each a conventionally known NE
It may be the first and second reagents for FA measurement.

[0024]

The present invention will be described in more detail with reference to the following examples, which are merely illustrative and do not limit the scope of the present invention.

Example 1 Stabilization of CoA by a chelating agent A control serum (Ceraclear LP; manufactured by Aswell Co., Ltd.) having a NEFA value of 1,000 μEq / L (= mmol / L) was used as a sample for a practical test. Test NE
As a first reagent for FA measurement, 60 mM trishydroxymethylaminomethane (hereinafter referred to as Tris), 1.1
mM 4-aminoantipyrine, 1.2 mM adenosine 5'-trisodium triphosphate trihydrate (manufactured by Oriental Yeast Co., Ltd.), 1.2 mM coenzyme A trilithium (manufactured by Oriental Yeast Co., Ltd.), 0 .25U /
A solution of pH 7.6 containing mL ACS (manufactured by Asahi Kasei Kogyo Co., Ltd.), 1 mM various chelating agents (manufactured by Dojindo Laboratories and Nacalai Tesque, Inc.) and 1.25 mM magnesium chloride was used. As the NEFA measurement second reagent, R2 of Nescort NEFA-V2 (NEFA measurement automatic analysis kit; manufactured by Aswell Co., Ltd.) was prepared and used according to the specifications. The reactivity of the first test reagent to which various chelating agents were added was measured using a Hitachi 7170 automatic analyzer as follows. 200 μL of the first reagent was added to 3 μL of the sample, and reacted at 37 ° C. for 5 minutes. Then, 50 μL of the second reagent was added, and the mixture was further reacted at 37 ° C. for 5 minutes. The main wavelength was 546 nm and the sub wavelength was 700 nm. The change in absorbance of No. 34 was determined. On the day of preparation of the first test reagent and after storage for 14 and 28 days at 25 ° C., the sample was measured by the above operation, and the reactivity of the first reagent for test on the day of preparation was defined as 100%.
Percentage remaining after storage at 14 ° C for 14 and 28 days (%)
I asked. The results are shown in the column of the residual reactivity in Table 1.

At the same time as the practical test, the amount of CoA in the first reagent to which various chelating agents were added was measured using a Hitachi 7170 automatic analyzer as follows. 180 μL of 0.1 M phosphate buffer (pH 8.0) was added to 15 μL of the first reagent, and the mixture was reacted at 37 ° C. for 1 minute and 20 seconds, and then 25 mM phosphate buffer (pH 8.0) and 250 mM 2,2-dithiodiamine were added. 180 μL of a solution containing pyridine and 0.25% N, N-dimethylformamide was added, and the mixture was further reacted at 37 ° C. for 8 minutes and 40 seconds.
The change in absorbance of No. 4 was determined. On the day of preparation of the first test reagent and after storage for 21 days at 25 ° C., the same measurement was carried out by the above method,
Assuming that the amount of A was 100%, the residual ratio (%) after storage at 25 ° C. for 21 days was determined. The results are shown in the column of the residual ratio of CoA in Table 1.

[0027]

[Table 1]

As is clear from the results shown in Table 1, ED
When DTPA, TTHA, or EGTA was added, the reactivity remaining rate (stability) and CoA remaining rate of the first reagent in the practical test were significantly higher than when TA was added.

Example 2 Stabilization of ACO by FAD Control serum having a NEFA value of 2,300 μEq / L [prepared by adding NEFA to CERACLEAR LP (manufactured by Aswell Co., Ltd.)] was used as a sample for a practical test. did. As a NEFA measurement first reagent, R1 of Nescort NEFA-V2 (NEFA measurement automatic analysis kit; manufactured by Aswell Co., Ltd.) was prepared and used according to the specifications. As a test NEFA measurement second reagent,
10 mM phosphate buffer, 6 mg / mL TOOS, 4
mM NEM, 1U / mL HRP (Toyobo Co., Ltd.
), 60U / mL ACO (manufactured by Asahi Kasei Corporation),
PH containing 0 to 2,000 μM FAD and 0.6% Hytenol 18E (Daiichi Kogyo Kagaku KK)
A 6.0 solution was prepared and stored at 37 ° C. for 7 days. The reactivity of the second test reagent was measured by the method described in Example 1. On the day of preparation of the second test reagent, and after storage at 37 ° C. for 7 days, the sample was measured by the above operation,
Assuming that the reactivity of the second test reagent on the day of preparation is 100%,
After storage at 37 ° C. for 7 days, the residual reactivity (%) was determined. The results are shown in the column of the residual reactivity in Table 2.

The activity of ACO in the second reagent was measured using a Hitachi 7170 automatic analyzer as follows.
In 15 μL of the second reagent, 0.2 mL of 200 mM Tris-HCl buffer (pH 8.0), 0.1 mL of 15 mM 4-aminoantipyrine solution, 0.1 mL of 0.2% TOOS solution, 0.1 mL of 50 U / mL HRP solution, 5 mL
mM palmitoyl-CoA solution (10 mM KH ₂ PO ₄
-NaOH, dissolved in pH 7.0) 0.1 mL, 0.1 mL of 1% Triton X-100 solution and 0.3 m of distilled water
180 μL of a solution containing L was added and reacted at 37 ° C. for 10 minutes, and the change in absorbance at photometric points 5 to 15 was determined at a main wavelength of 546 nm and a sub wavelength of 700 nm. On the day of preparation of the test second reagent, and after storage at 37 ° C. for 7 days, the same measurement was carried out by the above-mentioned method. The residual ratio (%) after storage for days was determined. The results are shown in the column of ACO activity residual ratio in Table 2.

[0031]

[Table 2]

The stability of ACO was increased depending on the concentration of FAD added, and the reactivity remaining rate (stability) of the second reagent in the practical test was also increased.

Example 3 Stabilization of ACO by Surfactant A control serum having a NEFA value of 2,300 μEq / L [prepared by adding NEFA to CERACLEAR LP (manufactured by Aswell Co., Ltd.)] is a sample for a practical test. Used as As a NEFA measurement first reagent, R1 of Nescort NEFA-V2 (NEFA measurement automatic analysis kit; manufactured by Aswell Co., Ltd.) was prepared and used according to the specifications. As a test NEFA measurement second reagent,
10 mM phosphate buffer, 6 mg / mL TOOS, 4
mM NEM, 1U / mL HRP (Toyobo Co., Ltd.
), 60U / mL ACO (manufactured by Asahi Kasei Corporation),
2,000 μM FAD and 0.5% in Table 3
Various surfactants (manufactured by Daiichi Kogyo Kagaku Co., Ltd. or Wako Pure Chemical Industries, Ltd.). In Table 4, 0 to 1.0% hytenol 18E (manufactured by Daiichi Kogyo Kagaku Co., Ltd.) P
A solution of H6.0 was prepared and stored at 37 ° C. for 5 days. The reactivity of the second test reagent was measured by the method described in Example 1. On the day of preparation of the second test reagent and after storage at 37 ° C. for 5 days, the sample was measured by the above operation,
Assuming that the reactivity of the second test reagent on the day of preparation is 100%,
After storage at 37 ° C. for 5 days, the residual ratio (%) was determined. The results are shown in Tables 3 and 4 in the column of Remaining Reactivity.

The activity of ACO in the second reagent was measured by the method described in Example 2. That is, the same measurement was performed on the day of preparation of the test second reagent and that after storage at 37 ° C. for 5 days. The residual ratio (%) after storage was determined. The results are shown in Tables 3 and 4
It is shown in the column of the residual activity ratio.

[0035]

[Table 3]

[0036]

[Table 4]

Hytenol 18E (chemical name: ammonium polyoxyethylene alkyl ether sulfate), Hytenol N-17 (chemical name: polyoxyethylene alkyl phenyl ether sulfate ammonium), Hytenol NE-15 (chemical name: poly) When oxyethylene dialkyl allyl ether sulfate ammonium) and hytenol NF-13 (chemical name: polyoxyethylene alkenyl allyl ether sulfate ammonium) were added, the reactivity remaining ratio of the second reagent and the remaining ACO activity in the practical test were observed. The ratio was high, and clouding of the second reagent could be avoided. In particular, a higher ACO stabilizing effect was observed with Hytenol 18E.

Example 4 p where NEM and ACO can coexist
Examination of H The optimal pH that can stabilize NEM and ACO for a long period was examined. NEF was added to a control serum having a NEFA value of 2,200 μEq / L [Ceraclear LP (manufactured by Aswell Co., Ltd.).
Prepared by adding FA] was used as a sample for a practical test. As a NEFA measurement first reagent, R1 of Nescort NEFA-V2 (NEFA measurement automatic analysis kit; manufactured by Aswell Co., Ltd.) was prepared and used according to the specifications. 10 mM phosphate buffer, 6 mg / mL TOO as NEFA measurement second reagent for test
S, 4 mM NEM, 1 U / mL HRP (Toyobo Co., Ltd.), 60 U / mL ACO (Asahi Kasei Corporation)
In Table 5, 600 μM, and in Table 6,
000 μM FAD and 0.5% Hytenol 18
E (manufactured by Daiichi Kogyo Kagaku Co., Ltd.) solutions of various pH were prepared and stored at 25 ° C. for 27 days or at 37 ° C. for 6 days. The reactivity of the second test reagent was measured by the method described in Example 1. On the day of preparation of the test second reagent and after storage at 25 ° C. for 27 days or at 37 ° C. for 6 days, the sample was measured by the above operation, and the reactivity of the test second reagent on the day of preparation was determined to be 100%. As a percentage, the reactivity remaining rate (%) of the sample after storage at 25 ° C. for 27 days or at 37 ° C. for 6 days was determined. The results are shown in Tables 5 and 6 in the column of Remaining Reactivity.

At the same time, the activity of ACO in the second reagent was measured by the method described in Example 2. Further, NE in the second reagent
The amount of M was measured using a Hitachi 7170 automatic analyzer as follows. 0.1 M phosphate buffer (pH 8.0) and 0.2-0.5 mM N-
180 μL of a solution containing acetylcysteine was added, and 3
After reacting at 7 ° C for 1 minute and 20 seconds, 25 mM phosphate buffer (p
H8.0), 250 mM 2,2-dithiodipyridine, 0.
180μ solution containing 25% N, N-dimethylformamide
L was added thereto, and the mixture was further reacted at 37 ° C. for 8 minutes and 40 seconds. At a main wavelength of 340 nm, a change in absorbance at photometric points 5 to 34 was determined. The ACO activity and the NEM amount were similarly measured on the day of preparation of the second test reagent and after storage at 37 ° C. for 6 days.
Assuming that each of the activity and the NEM amount is 100%, 37
The residual ratio (%) after storage at 6 ° C. for 6 days was determined. The results are shown in Tables 5 and 6 for the residual ACO activity and NE.
The results are shown in the column of M residual ratio.

[0040]

[Table 5]

[0041]

[Table 6]

By adjusting the pH of the second reagent to 6.0 to 6.4, the residual reaction rate of the second reagent is high even after storage at 25 ° C. for 27 days or at 37 ° C. for 6 days, and ACO,
Both NEMs were able to maintain the amount required to exhibit the performance of the reagent.

[0043]

As described above, as a CoA stabilizer,
By adding a chelating agent such as DTPA, TTHA or EGTA, the performance of the first reagent in NEFA measurement can be stably maintained in a liquid state for a long time. Also,
One or more ACs within the effective pH range for NEM stabilization
By adding the O stabilizer, the performance of the second reagent in the NEFA measurement can be stably maintained in a liquid state for a long period of time. Furthermore, by using these first and second reagents, it is possible to provide a liquid and long-term stable NEFA measurement kit.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) // G01N 35/00 G01N 35/00 C (72) Inventor Koji Mizuno 2-1 -4-201 F term (reference) 2G045 BA02 BB29 DA60 FA26 FB01 GC10 2G058 BB02 BB07 BB09 GA02 4B050 CC07 HH04 KK06 KK13 LL03 4B063 QA01 QA18 QQ73 QR03 QR43 QR53 QR65 QR65 QS08 QS28 QX01

Claims (24)

[Claims] 1. Coenzyme A and coenzyme A
An effective amount of diethylenetriamine-N, N, N ',
N '', N ''-pentaacetic acid, triethylenetetramine-N, N, N ', N'',
At least one selected from the group consisting of N ''',N'''-hexaacetic acid and O, O'-bis (2-aminoethyl) ethylene glycol-N, N, N ', N'-tetraacetic acid A solution containing Coenzyme A stabilized to such an extent that it can be used as a liquid reagent. 2. The solution according to claim 1, further comprising acyl coenzyme A synthetase and adenosine 5′-triphosphate. 3. A solution containing an effective amount of an anionic surfactant for stabilizing acyl coenzyme A oxidase, wherein acyl coenzyme A oxidase is stabilized to such an extent that it can be used as a liquid reagent. 4. The solution according to claim 3, further comprising an effective amount of flavin adenine dinucleotide for stabilizing acyl coenzyme A oxidase. 5. A solution containing acyl coenzyme A oxidase and N-ethylmaleimide, wherein the solution comprises N-ethylmaleimide.
5. The solution according to claim 3, which has a pH effective for stabilizing ethylmaleimide. 6. The anionic surfactant comprises polyoxyethylene alkyl ether sulfate, polyoxyethylene alkyl phenyl ether sulfate, polyoxyethylene dialkyl allyl ether sulfate, polyoxyethylene alkenyl allyl ether sulfate, and salts thereof. The solution according to any one of claims 3 to 5, which is one or more compounds selected from the group. 7. The method of claim 1, wherein the flavin adenine dinucleotide is 1
The solution according to any one of claims 4 to 6, comprising at least 00 µM. 8. The solution according to claim 5, which has a pH of 6.8 or less. 9. The solution according to claim 3, further comprising a reagent for quantitatively determining hydrogen peroxide. 10. The solution according to claim 9, wherein the reagent for quantitatively determining hydrogen peroxide contains peroxidase and a hydrogen donor which is a chromogenic substrate. A kit for measuring free fatty acids, comprising a reagent for a reaction for converting free fatty acids to acyl coenzyme A, and the solution according to claim 9 or 10. 12. The solution according to claim 2, wherein the reagent for converting free fatty acid to acyl coenzyme A is the solution according to claim 2.
The kit according to claim 11. 13. A method for stabilizing coenzyme A, comprising adding an amount of diethylenetriamine-
N, N, N ', N'',N''-pentaacetic acid, triethylenetetramine-N, N,
Group consisting of N ', N'',N''', N '''-hexaacetic acid and O, O'-bis (2-aminoethyl) ethylene glycol-N, N, N', N'-tetraacetic acid A method for stabilizing coenzyme A to such an extent that it can be used as a liquid reagent, comprising adding one or more compounds selected from the group consisting of: 14. The method according to claim 13, wherein said solution further contains acyl coenzyme A synthetase and adenosine 5'-triphosphate. 15. A method for stabilizing acyl coenzyme A oxidase to such an extent that it can be used as a liquid reagent, comprising adding an effective amount of an anionic surfactant for stabilizing acyl coenzyme A oxidase. 16. The method according to claim 15, further comprising adding an effective amount of flavin adenine dinucleotide for stabilizing acyl coenzyme A oxidase. 17. A method for stabilizing a solution containing acyl coenzyme A oxidase and N-ethylmaleimide, comprising adjusting the solution to a pH effective for stabilizing N-ethylmaleimide. Claim 15
Or the method of 16. 18. The anionic surfactant comprises polyoxyethylene alkyl ether sulfate, polyoxyethylene alkyl phenyl ether sulfate, polyoxyethylene dialkyl allyl ether sulfate, polyoxyethylene alkenyl allyl ether sulfate, and salts thereof. The compound is one or more compounds selected from the group.
18. The method according to any of 17. 19. The method according to claim 19, wherein the flavin adenine dinucleotide is 1
2. The composition according to claim 1, wherein the content is at least 00 μM.
9. The method according to any one of items 8. 20. The method according to claim 17, wherein the pH is 6.8 or less.
20. The method according to any of 19. 21. The method according to claim 15, wherein the solution further contains a reagent for determining hydrogen peroxide. 22. The method according to claim 21, wherein the reagent for determining hydrogen peroxide contains peroxidase and a hydrogen donor which is a chromogenic substrate. 23. A reagent for a reaction for converting a free fatty acid into acyl coenzyme A in a sample and a reagent for the reaction according to claim 9 or 1.
0. A method for measuring free fatty acids in a sample solution, comprising sequentially adding the solutions described in No. 0 and quantifying the generated hydrogen peroxide. 24. The reagent for a reaction for converting a free fatty acid into acyl coenzyme A is the solution according to claim 2,
A method according to claim 23.