JPH01313000A - Reagent for determination of bio-component - Google Patents

Abstract

PURPOSE:To obtain a stable reagent having high solubility to enable easy and inexpensive determination by using a hardly water-soluble substance together with maltosylated cyclodextrin(MCD). CONSTITUTION:A suspension liquid is produced by adding a hardly water-soluble substance such as substrate of enzymatic reaction (e.g., tyrosine), a coenzyme (e.g., nicotinamide), a pigment (e.g., hydroquinone), an inhibitor (e.g., dicoumarol), a preservative (e.g., thymol), an antibiotic substance (e.g., chloramphenicol), gamma-glutamyl-p-nitroaniline and adenosine to water. The suspension is mixed with a non-modified cyclodextrin such as maltosylated alpha-cyclodextrin of formula I [group of formula II is glucose residue; group of formula III is maltose residue; the glucose residues are bonded with each other through alpha-1,4 bond; the glucose residue is bonded to the maltose residue through alpha-1,6 bond; n is 6; m is 1-2] and MCD to dissolve (include) the above hardly water-soluble substance in the form clathrated in MCD, etc., and prepare the objective determination reagent for bio-component.

Description

[Detailed Description of the Invention] <Industrial Application Field> This invention is directed to dissolving (containing) a substance that is poorly soluble in water at a high concentration.
The present invention relates to a reagent for measuring biological components which is designed to prevent the crystallization or precipitation of the reagent.

More specifically, a substance that is poorly soluble in water is allowed to coexist with maltosylated cyclodextrin (hereinafter also referred to as maltosylated CD), or is dissolved (contained) in a reagent for measuring biological components as a clathrate of maltosylated CD. This invention relates to a reagent for measuring biological components that increases solubility and prevents crystallization or precipitation of substances that are poorly soluble in water.

<Prior art and problems to be solved by the invention> In general, water-based biological component measuring reagents used for biochemical measurements such as enzyme reactions contain enzyme reaction substrates, coenzymes, inhibitors, coloring substances, pigments, It is necessary to dissolve (contain) various substances that are poorly soluble in water, such as preservatives.

Although various attempts have been made to dissolve these sparingly water-soluble substances in high concentrations, there is no perfect method, and for this reason, there are still some substances that cannot be used as reagents for measuring biological components.

In addition, these poorly water-soluble substances are usually solubilized using a clever method when preparing reagents for measuring biological components, but they tend to crystallize or precipitate over time. When this occurs, the concentration of the substance in question decreases, and the substance loses its function as a highly accurate reagent for measuring biological components, thereby losing its value.

As one of the effective methods for dissolving such poorly soluble substances in biological component measurement reagents and preventing crystallization or precipitation over time, it has been known that cyclodextrin and cyclodextrin have been used to dissolve poorly soluble substances in water. (or cyclodextrin derivatives) coexistently or as an inclusion compound thereof (JP-A-57-74099, JP-A-60-160896M, JP-A-61-260900).

However, in the published method, the solubility of cyclodextrin itself is too low and it is not possible to dissolve poorly soluble substances at high concentrations, or cyclodextrin derivatives (for example, the method disclosed in JP-A-61-260900) Hydroxypropyl β-CD) had drawbacks such as being extremely expensive and lacking in economic efficiency.

The inventors made extensive efforts to overcome the above-mentioned drawbacks, and found that compared to conventional cyclodextrin (or cyclodextrin derivatives), maltosylated cyclodextrin requires dissolution (containment) in reagents for measuring biological components. The present invention was completed after discovering that it has an extremely good inclusion ability for substances that are poorly soluble in water, such as certain enzyme reaction substrates, inhibitors, coloring substances, pigments, and preservatives, as well as having high solubility and low cost. did.

<Means for solving the problems> The present application consists of the following claims (1) to (9).

(1) A biological component measuring reagent characterized by the coexistence of a poorly water-soluble substance and maltosylated cyclodextrin.

(2) A reagent for measuring biological components, which contains a substance that is poorly soluble in water as an inclusion compound of maltosylated cyclodextrin.

(3) A biological component measuring reagent characterized in that a substance poorly soluble in water, maltosylated cyclodextrin, and unmodified cyclodextrin coexist.

(4) A reagent for measuring biological components, which contains a substance that is poorly soluble in water as an inclusion compound of maltosylated cyclodextrin and unmodified cyclodextrin.

(5) Unmodified cyclodextrin is α-1β-1γ-
The biological component measuring reagent according to claims 3 and 4, which is cyclodextrin alone or a mixture thereof.

(6) The biological component measuring reagent according to any one of claims 1 to 5, wherein the substance poorly soluble in water is a substrate for an enzyme reaction (including a synthetic substrate), a coenzyme, an inhibitor, or a dye.

(7) Claims 1 to 5, in which the poorly water-soluble substance is a poorly water-soluble antiseptic, antibacterial, or antifungal agent used in biochemical measurement reagents with the expectation of antiseptic action. Reagent for measuring biological components as described in Section 1.

(8) Claims 1 to 3, wherein the poorly water-soluble substance is γ-glutamyl paranitroaniline (γ-GpNA).
The reagent for measuring biological components according to item 5.

(9) The biological component measuring reagent according to any one of claims 1 to 5, wherein the substance poorly soluble in water is adenosine.

In the present invention, a poorly water-soluble substance refers to a relatively low-molecular organic compound described below.

(B) Substrates for enzymatic reactions: (A) poorly soluble amino acids such as tyrosine and tryptophan (B) adenine, hyboxanthin, xanthine, uric acid,
Core #i bases (C) that are poorly soluble in water such as uracil and thymine; low-molecular-weight poorly soluble hormones such as thyroxine, trichothyronine, adrenaline, noradrenaline, and melatonin; steroid hormones such as testosterone, aldosterone, and progesterone; ) Coenzymes: nicotinamide, biotin, folic acid, riboic acid, retinal, carotene, α-tocopherol, poorly soluble in water such as vitamin 3, ubiquinone, vitamin D, etc. (c) Pigments: human quinone, catechol, pyrogallol, Vanillin and its derivatives, antipyrine, acid anilide derivatives of 4-nitroaniline (γ-GTP substrate, other peptidase substrates, etc.), ester-bonded derivatives of nitrophenol and chloronitrophenol (phosphate esters:) osphatase substrate: p- nitrophenyl phosphate) or ether-linked derivatives (linked to oligo ends or spears: amylase substrate, glycosidase substrate, etc.), phenolphthalein phosphate, awaridine, zenazine, fluorescein, rhodamine, naphthalene, pyrene, citraquinone, anthracene, bovine feron,
Kumarici derivatives and their labeled habten) inhibitors: cyclomarol, warfarin, thiouracil, 2,4-
Dinitrophenol, ] Luchicine, p-chlorobenzoate mercuric (e) Preservatives, antibiotics: Thymol, chloramphenicol (e) and other chelates such as 20-phenanthlodge, 2,2-dipyridyl, vancbroici, etc. Lipids such as triglycerides, cholesterol, etc. These organic compounds generally have low solubility at pH levels that are unstable in their solutions, so it is often difficult to obtain the concentration required for biochemical measurement reagents. When adding maltosylated cyclodextrin,
A biochemical measurement reagent that has the required sufficient concentration and is stable can be easily obtained.

In the present invention, maltosylated cyclodextrin refers to a cyclodextrin derivative represented by the following general formula, specifically maltosylated α-cyclodextrin [n-6, m=1 to 2], maltosylated β- Cyclodextrin [n-7, m=: 1-2, maltosylated γ-
Cyclodextrins [n=8, m=1-2] are used.

General formula % formula %) However, in the above formula (05H1005) is a glucose residue,
(C, H2005) represents a maltose residue, glucose has an α-1,4 bond, and glucose and maltose have an α-1,6 bond. Although these maltosylated CDs are not novel compounds C, the present application is the first to utilize them for stabilization (prevention of crystallization or precipitation of substances poorly soluble in water) in reagents for measuring biological components.

When carrying out the present invention specifically, pure maltosylated CD containing no impurities may be used, but usually these 3t! ! Mixtures of maltosylated CD, and mixtures of maltosylated CD containing unmodified CD (including other carbohydrates) are often used. In this case, it is better to use maltosylated CD containing unmodified CD than to use pure maltosylated CD (dissolution of maltosylated CD or unmodified CD).
In particular, it has the property of assisting in the dissolution of β-〇D, so it is often functionally and economically superior.

By adding one or more of these maltosylated CDs, biochemical substances such as enzyme reaction substrates, inhibitors, coloring substances, pigments, and preservatives (antibiotics), which have been thought to be poorly soluble in water, can be easily dissolved in water. Substances that are difficult to exert their effects because they are difficult to prepare or cannot be prepared as highly concentrated reagent solutions can be prepared at concentrations several times their solubility or several m
It has become possible to dissolve M as an inclusion compound at a high concentration of several tens of nanometers.

When a substance that is poorly soluble in water is mixed with maltosylated CD,
It is generally said that clathrate compounds are formed, but not all cases may necessarily be explained by the concept of clathrate compounds. The present invention is not limited to the case where a compound is formed, but also includes the case where a stable reagent solution is obtained, for example, by emulsification.

Example 1> γ-glutamyl-p-nitroanilide (γ-GpN
A) Dissolution of hydrochloride γ-GpNA is dissolved in γ-glutamyltransferase (
EC2,3,2,2 (hereinafter referred to as GT) is a substrate for activity measurement. The Km (Michaelis-Mediten constant) of the enzymatic activity γ-glutamyl transferase (γ-GTP) using γ-GpNA as a substrate is approximately TmM.
It is known that From this value of Km, γ−G
In order to accurately measure TP activity, the concentration of the substrate contained in the reagent must be several mM or more. However, pH other than pH 4 to 5, which can increase solubility relatively,
Since the hydrolysis rate becomes even higher when using H, it cannot be stored as a reagent at pH other than the above. At this pH, the solubility of γ-GpNA is minimal, 2mM (4
To prepare and store reagents at low temperatures, maltosylated CDs are added to prepare reagents that can be used in practical applications because they dissolve at temperatures below "C".
It is necessary to increase the angle of 38 degrees.

Prepare the following solutions (]) to (5) and leave them at 4°C to observe whether crystals of the substrate γ-GpNA precipitate or not.
The required minimum concentration (mM) of each CD to finally dissolve -GpNA hydrochloride at 20 mM was determined. In addition, for the purpose of evaluating the inclusion ability of CD, the concentration of the substrate contained in the supernatant of the substrate solution in which the crystals were precipitated was measured by spectrophotometry, and the inclusion constant (K) was determined. Table 1 (
(at the end of the book).

(1) Maltosylated α-CD [n in general formula (I)
=6. m=1-2] @ O0-50rn at various concentrations, and substrate solutions uniformly containing 20 mM of γ-GpNA hydrochloride were prepared.

(2) Maltosylated β-CD [n in general formula (I)
=7. m=1-2] in the range of 0-50mM;
A substrate solution containing uniformly 20 mM of γ-GpNA hydrochloride was prepared.

(3) Maltosylated α-CD and maltosylated β-CDI
A CD preparation containing r as a main component and unmodified CD etc. (the above (
Intermediate products in the production process of maltosyl derivatives (1) and (2) (only CD, but not oligosaccharides) are 0 to
Substrate solutions containing γ-GpNA hydrochloride at various concentrations in the range of 160 mM and uniformly containing 20 mM of γ-GpNA hydrochloride were prepared. Note that this intermediate product does not contain any oligosaccharide as a solid content, but only contains maltosylated CD and unmodified CD, and contains only maltosylated CD and unmodified CD.
D is 62.5% by weight or more of total solids (α, β: γ = 6:
3:1 (weight ratio)).

(4) As a CD derivative other than maltosylated CD, the hydroxyprobil β-CD component (heptakis-(6-0-hydroxyprodol)-B=CD, hereinafter referred to as HP) described in JP-A-61-260900 is used. -β-CD) at various concentrations ranging from 0 to 50mM, and γ-Gp
A substrate solution uniformly containing 20 mM of NA hydrochloride was prepared.

(5) γ-Gp only with distilled water without CD derivatives
A substrate solution containing 20 mM of NA hydrochloride was prepared.

These solutions (1) to (5) were all adjusted to pH 4.5.

According to the results in Table 1, water at 04°C contains 1°8mM
By using 80mM maltosylated CD, it is possible to dissolve γ-GpNA hydrochloride, which only dissolves at 20mM.
It can be seen that it is possible to dissolve 20mM of T-GpNA hydrochloride when using mM. In maltosylated CD preparations, pure maltosylated CD is 55-60%
The reason why such a good result occurs even though it only contains
It is thought that the solubility of maltosylated CD or poorly soluble β-CD is increased, and the ability to envelop other unmodified CDO is increased.

It is clear from the following @tora that maltosyl-CD increases the solubility of poorly soluble β-CD.

β-CD0.19 (approximately 88umoI2) is lo
It can be dissolved in 800 uβ of solution of omM maltosyl-β-CD. However, with β-CD alone, water 80
Only 14umoI2 (approximately 2%) dissolves in 0uρ. That is, 8888-14=74uβ is maltosyl-β
-It is dissolved by CD.

<Example 2> Maltosylated CD (α-2β-) and maltosylated CD preparations according to the present invention and conventional unmodified CD (α-1β-
1γ-) and CD derivatives (di-0-methyl
β-CD, tri-0-methyl-B-CD,
In order to compare the inclusion ability with poly-B-CD and HP-β-CD, the concentration of each CD was kept constant (approximately 25 mM), and γ-GpNAt was added at room temperature to a concentration of 20 mM. After dissolving and preparing the reagent, store it at 4℃ and store it for 2
After a week had passed, the substrate concentrations of the supernatants of these reagents were measured. These reagent solutions were all adjusted to have a pH of 3.0 (25° C.) and were prepared to contain 25 to 30% dimethylformamide (DMA).

The measurement results are shown in Table 2 (at the end of the book).

According to the results in Table 2, maltosylated C according to the present invention
D(α-1β-) is H, which is conventionally considered to have a large inclusion ability.
It is fully comparable to P-β-CD (this derivative is synthesized by chemically introducing a hydroxypropyl group into purified 13-CD and is very expensive), and the manufacturing cost is extremely low. It can be seen that even maltosylated CD preparations can satisfactorily achieve the objective.

<Example 3> From the substrate solution prepared using maltosylated α-CD and maltosylated B-CD prepared in Example 1, pH 3.7
We investigated whether mixing of the two CDs at pH 5.2 would increase the solubilization of the substrate. That is, 20mM maltosylated α-CD and 20mM maltosylated β-CD adjusted to the above pH were mixed without changing the pH. The measurement results of the free M substrate concentration of this mixed solution are shown in Table 3 (at the end of the volume).

According to the results in Table 3, a mixture of maltosylated α-CD or maltosylated β-CD is better than maltosylated β-CD alone.
The reverse substrate concentration is Q, which is about 5mM higher. That is, as in Example], the CD-specific inclusion effect is greater when a plurality of different types of CDs are mixed and used than when a single CD is used.

<Example 4> The following reagents were prepared.

*Reagent A: 100m containing 80mM glycylglycine
Mt-Lith buffer (pH 8,3)*Reagent]: Maltosylated α-CD [n=6 in general formula (I). m=1～
2] and 20 mM of γ-GpNA hydrochloride; Tani; 1 female * Reagent 2: Maltosylated β-CD [n=7. m=1-2 about 25mM, 7-Gl)NA
Substrate solution containing 20mM hydrochloride *Reagent 3: Main component is maltosylated α-CD, contains maltosylated β-CD, maltosylated γ-CD, and modified α-CD, β-CD, and γ-CD. The average molar concentration of maltosylated CD preparations containing trace amounts of
, a substrate solution containing 20mM γ-GpNA hydrochloride, then γ
- Prepare a sample solution containing glutamyl transferase activity, mix 1 volume of this sample solution with the substrate solution of Reagent 1 at a volume ratio of 40, heat at 37°C for 5 minutes, and then Reagent containing CD], Reagent 2, Reagent 3
Husband/? with a capacity ratio of 10? The reaction was started at the same temperature.

Varanitroanilisi produced by the reaction was tracked by the change in absorbance at 405 nm, and the γGTP activity in the sample solution was measured. As a sample solution, purified GT derived from pig kidney was used.
P, serum samples, serum samples containing intentionally hemolyzed serum, and serum samples to which specific substances were intentionally added were used.

Next, as a comparative example, γ-GpNA hydrochloride was prepared by the following two conventional methods (Reagent 4, Reagent 5), and γ-glutamyl transferase activity was measured.

*Reagent 4: 20mM γ-GpNA hydrochloride aqueous solution containing 16% uran ether and 13% dimethylacetamide (DMA) *Reagent 5: Diluted to contain 80% of Reagent 1 without CD or crown ether. , in which 4mM γ-G
Measurement of γ-glutamyltransferase activity in a solution containing pNA hydrochloride was carried out using the same method as reagent 2 for reagent 4, and mixing reagent 5 at a volume ratio of 50 to 1 volume of sample solution. did.

The above measurement results are shown in Table 4 (at the end of the book).

According to these results, among maltosylated CDs, maltosylated α-CD shows almost the same specific activity as Reagent 5 to which no CD is added, and furthermore, the influence of coexisting substances, especially hemoglobin, is small; -It can be said that it is excellent as a substrate T-GpNA solubilizing agent for a reagent for measuring GTP activity.

In addition, the mixture of maltosylated CD and unmodified CD (D) in reagent 3 is considered to be excellent as a substrate γ-GpNA cleavage aid for the reagent for measuring arGTP activity.

Since the CD of this mixture is not the purified maltosylated CD of Reagent 1 or Reagent 2, it is extremely inexpensive and of great practical value.

These CDs are completely hydrolyzed and purified from glucose and have high biological safety, so there are no problems in wastewater treatment.

<Example 5> Solubilization of adenosine, a substrate of adenosine deaminase (hereinafter referred to as AD8) There are two types of ADA isoenzymes in human serum, but the one that is meaningful in terms of activity measurement is , is an ADA isoenzyme with a Km of approximately 3mM. AD in this serum
By measuring A activity, it is possible to diagnose the presence or absence of liver disease or malignancy, and the development of a simple measurement reagent is expected as a clinically meaningful measurement item. but,
An aqueous solution of adenosine dissolves only about 4mM when stored in a refrigerator.It is an aqueous solution that does not contain any organic solvent, maintains a high substrate concentration over a long period of time, and constitutes a stable reagent. This is extremely difficult with conventional techniques.

50 mM of this ADA substrate could be dissolved in an aqueous solution of 30% maltosylated CD mixture, and a substrate solution for measuring ADA activity could be easily prepared.

Since the solubility of maltosylated CD is 60% or more,
Theoretically, adenosine dissolves for about 1,100 rn, and even if stored at low temperatures for a long period of time, crystal precipitation does not appear, and it can be stored stably as an aqueous solution.

In this way, a high concentration of adenosine is dissolved in an aqueous solution containing maltosylated CD, but in order to compare it with a conventional reagent, the above-mentioned 50mM adenosine aqueous solution was diluted with a buffer solution, and an ADA active reagent was added as shown below. Prepared.

*Reagent 1: Enzyme solution α-ketoglutaric acid 40mM NADPH0,
7mM GIDH40U/mp Trisamitsumekun 10mMpH8.5 However, NADPH is nicotinamide adhesion acreotide phosphate reduced form, and GIDH is glutamate dehydrogenase.

*Reagent 2: Calculating solution (Example) Adenosine 20mMKH2P○a
130rnM maltosylated CD (mixture) 12% pH 7,0 *Reagent 3. Substrate solution (comparative example) Adenosine 20mMK H2P O
a 136 mM DMSO 5% Glycerin 10% pH 7.0 However, DMSO is dimethyl sulfoxide, which was added together with glycerin as a solubilizing agent.

<Measurement> To measure human serum ADA activity, 5 volumes of Reagent 1 were added to 1 volume of serum and prewarmed at 37°C for 5 minutes to eliminate endogenous ammonia. Next, add reagent 2 to 1
The serum ADA activity was evaluated by adding asimonia, which was released from the substrate adenosine by the action of serum ADA, as the rate of decrease in NADPH using the enzyme contained in Reagent 1. The measurement results are shown in Table 5 (at the end of the volume).

According to the results in Table 5, the serum ADA activity value measured using the reagent containing maltosylated CD as a substrate solubilizing agent was higher than that using the organic solvent DMSO.
On average, a value about 12% larger was obtained. That is, D
This means that MSO reduces the specific activity of ADA by about 10%.

Therefore, when measuring ADA activity using a reagent using maltosylated CD, compared to using DMSO,
It is believed that measurements close to the true activity can be made without suppressing DA activity.

Example 6 The following poorly soluble substances could be dissolved using a maltosylated CD mixture.

(a) PMSF (phenylmethylsulfonyl chloride (protease inhibitor)) (b) p-MB (1)-Methyl benzoate (preservative)
) (c) MBCHA (4,4-methylenebiscyclohexylamine (strengthening agent)) The dissolution conditions are shown in Table 6 (at the end of the book).

<Effects of the Invention> Since the present invention is configured as described above, the enzyme reaction substrate,
This method has the effect that it is possible to easily obtain a reagent for measuring biological components in which a substance that is poorly soluble in water, such as an inhibitor, a coloring substance, a pigment, or a preservative, is stably dissolved at a high concentration.

１　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　Table 2 Third ;! j (II (QmM) 'J45 table (unit U/r) Table 6

Claims (9)

[Claims] (1) A biological component measuring reagent characterized by the coexistence of a poorly water-soluble substance and maltosylated cyclodextrin. (2) A reagent for measuring biological components, which contains a substance that is poorly soluble in water as an inclusion compound of maltosylated cyclodextrin. (3) A biological component measuring reagent characterized in that a poorly water-soluble substance, maltosylated cyclodextrin, and unmodified cyclodextrin coexist. (4) A reagent for measuring biological components, which contains a substance that is poorly soluble in water as an inclusion compound of maltosylated cyclodextrin and unmodified cyclodextrin. (5) Unmodified cyclodextrin is α-, β-, γ-
The biological component measuring reagent according to claims 3 and 4, which is cyclodextrin alone or a mixture thereof. (6) The biological component measuring reagent according to any one of claims 1 to 5, wherein the substance poorly soluble in water is a substrate for an enzyme reaction (including a synthetic substrate), a coenzyme, an inhibitor, or a dye. (7) Claims 1 to 5, in which the poorly water-soluble substance is a poorly water-soluble antiseptic, antibacterial, or antifungal agent used in biochemical measurement reagents with the expectation of antiseptic action. Reagent for measuring biological components as described in Section 1. (8) Claims 1 to 3, wherein the poorly water-soluble substance is γ-glutamyl paranitroaniline (γ-GpNA).
The reagent for measuring biological components according to item 5. (9) The biological component measuring reagent according to any one of claims 1 to 5, wherein the substance poorly soluble in water is adenosine.