JP2877703B2 - Method for quantifying 1,5-anhydroglucitol - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for quantifying 1,5-anhydroglucitol (hereinafter referred to as 1,5-AG), which is useful as a diagnostic marker for diabetes.

[0002]

BACKGROUND OF THE INVENTION 1,5-AG is a compound that is present in human cerebrospinal fluid and plasma and has been reported to reduce its plasma level in certain diseases, especially in diabetes. These 1,5
As a method for quantifying -AG, a method mainly using gas chromatography (Yoshioka et al., Diabetes, 25, 1115-1.
118, 1982) and 1,5-AG were allowed to act with an oxidase, and then the amount of oxygen consumed, the amount of hydrogen peroxide produced, or the amount of the reduced form of the electron acceptor was measured to determine 1,5. -A method for quantifying AG is known (Japanese Patent Publication No.
-24200).

[0003]

The method for quantifying 1,5-AG using gas chromatography requires pretreatment of the specimen and labeling of 1,5-AG, which is necessary for maintenance and management of analytical instruments. There is a problem that advanced technology is required and complicated, and it takes a long time for analysis, and it is difficult to measure a large number of subjects. On the other hand, the method for quantifying 1,5-AG using an oxidase has a problem that the enzyme has low substrate specificity for 1,5-AG.

[0004]

SUMMARY OF THE INVENTION The present invention relates to a method for measuring the presence of 1,
By reacting 5-AG with a 1,5-AG phosphorylating enzyme in the presence of a phosphate group donor, and measuring the consumed phosphate group donor, 1,5-AG in the sample was converted to 5-AG. It relates to the method of quantification.

The sample may be any sample containing 1,5-AG, but usually includes blood, serum, plasma, urine, etc. collected from a living body.

[0006] 1,5-AG phosphorylase (hereinafter, referred to as 1,
The term “5-AG phosphorylase” means any enzyme that can phosphorylate 1,5-AG.
In particular, it is preferable to use an enzyme having a strong substrate specificity for 1,5-AG. Specifically, hexokinase (E.
C.2.7.1.1), glucokinase (EC2.7.1.2) and the like. Examples of 1,5-AG kinase include enzymes derived from tissues of higher animals such as brain, liver, muscle, erythrocytes, cancer cells, intestinal mucosa, Saccharomyces cerevisiae, As
pergillus oryzae, Leuconostoc mesenteroides, E.co
li, Aerobacter aerogenes, Bacillus sp., B. stearot
hermopilus, Alternaria sp., Gibberella fujikuro
i, Colletotrichum trifolii, Colletotrichum trunca
Enzymes derived from microorganisms such as tum and Fusarium roseum can be used. In addition, those obtained by modifying these enzymes by genetic manipulation can be used.

[0007] The amount of 1,5-AG kinase used in the present invention is 0.1 to 500 u / m1, and for example, when hexokinase is used, it is 4 to 100 u / m1, preferably 10 to 50 u / m1. is there.

The phosphate group donor is not particularly limited as long as it is involved in the phosphorylation reaction of 1,5-AG by 1,5-AG kinase, for example, pyrophosphate, guanosine triphosphate ( GTP), guanosine monophosphate (G
MP), phosphoenolpyruvate (PEP), phosphate, adenosine triphosphate (ATP), guanosine triphosphate (GTP), cytosine triphosphate (CTP), thymine triphosphate (TTP), inosine triphosphate (ITP), uridine triphosphate (UTP), deoxyadenosine triphosphate (dATP), deoxyguanosine triphosphate (dGT)
P), deoxycytosine triphosphate (dCTP), deoxythymine triphosphate (dTTP), d-deoxyadenosine triphosphate (ddATP), d-deoxyguanosine triphosphate (ddGTP), d-deoxycytosine triphosphate (DdCTP), d-deoxythymine triphosphate (ddT
TP) and the like. The concentration of the phosphate group donor used in the present invention is 0.1 to 50 mM, preferably 0.1 to 10 mM.
mM, particularly preferably 0.5 to 3 mM.

[0009] 1,5-AG and 1,5-AG phosphorylating enzyme in a sample are dissolved in an aqueous medium adjusted to pH 6 to 9 in an aqueous medium.
At a temperature of 5-50C, preferably 25-40C,
Let act for 60 minutes.

The term "aqueous medium" refers to a liquid containing water, such as a buffer solution or a physiological saline solution. Examples include an acid buffer, an oxalate buffer, a phthalate buffer, a borate buffer, a glycine buffer, a barbital buffer, and a good (GOOD) buffer.

In the method for measuring the phosphate group donor consumed in the method of the present invention, ribonucleoside triphosphate and deoxyribonucleoside triphosphate are converted to inorganic phosphorus with perchloric acid and hydrogen peroxide, and then molybdic acid A method for colorimetric determination of molybdate blue generated by adding a reagent (Procedures for Clinical Testing, written by Kanehara and Izumihara, published by Kanehara Publishing, 1993), A
Method for quantifying the amount of TP by high performance liquid chromatography ['94 Tosoh Catalog (Tosoh Corporation Heisei 6
It is preferable to use the method described below for quantifying ADP based on the linkage between pyruvate kinase and pyruvate oxidase [Clinical Chemistry-Gist-, Mitsuo Ogi, Iidensha, 1987] ].

In the following, 1,5-AG using 1,5-AG as a substrate
The enzyme chain diagram in the case where the reaction by -AG phosphorylase and the method of quantifying ADP by the linkage of pyruvate kinase and pyruvate oxidase are combined.

[0013]

Embedded image In the enzyme chain reaction, ADP is reacted with phosphoenolpyruvate using pyruvate kinase in the presence of magnesium, and hydrogen peroxide is produced by the action of pyruvate oxidase using the resulting pyruvate, oxygen and phosphate as substrates. Generate. The amount of generated hydrogen peroxide is quantified by a method of quantifying the amount of hydrogen peroxide using peroxidase, and the corresponding amount of ADP and the corresponding amount of 1,5-AG are further quantified.

As a method for quantifying the amount of hydrogen peroxide using peroxidase, there is a method for colorimetric quantification by condensing 4-aminoantipyrine with a phenol derivative, aniline derivative or toluidine derivative [biochemistry and development of new diagnostic agents] CMC Co., Ltd., 1984], and bis [3-bis (4-chlorophenyl) methyl-4-dimethylaminophenyl] amine (hereinafter referred to as BCMA), leucoethylene blue (Japanese Unexamined Patent Application Publication No. JP-A-57-29297), leukoindamine derivatives or leucoindophenols (JP-A-59-74713) or triarylmethane derivatives (JP-A-56-31641, JP-A-60-160).
No. -18440).
Also, a chemiluminescence method using luminol, isoluminol, and lucigenin [Bioluminescence and Chemiluminescence, edited by Kazuhiro Imai, 19
1980, published by Hirokawa Shoten], Fluorescence method, etc. [Clinical examination, extra edition, 1978, pp. 1236-1237; Japanese Patent Application No. 1-3
05051] can also be used.

In a method for quantifying ADP consumed by 1,5-AG kinase by the linkage between pyruvate kinase and pyruvate oxidase, pyruvate kinase 0.1 to 10 u / ml and pyruvate oxidase 0.1 to 0.1 u / ml are used. 1 to 50 u / ml, magnesium salts such as magnesium sulfate and magnesium chloride are 0.1 to 50 mM, phosphoenolpyruvate 0.4 to 6 mM, and phosphoric acid 1 to 10
0 mM is used. The reaction conditions such as reaction temperature and reaction time of the reaction are synonymous with the reaction conditions of the above-described 1,5-AG phosphorylation reaction because they are linked to the 1,5-AG phosphorylation reaction.

When a large amount of glucose or the like is present in a sample and affects the quantitative value of 1,5AG according to the method of the present invention, the sample is pretreated by column chromatography or the like to obtain a glucose or the like. After elimination of the compound (JP-A-63-185397), the amount may be determined by the method of the present invention.

[0017]

【Example】

Example 1 Reagents 1 and 2 required for the quantification of 1,5-anhydroglucitol and having the compositions shown in Table 1 below were prepared.

[0018]

[Table 1] Standard solution of 1,5-anhydroglucitol 2 mg / ml was prepared in 5 steps (0.4, 0.8, 1.2, 1.6, 2.0 each).
mg / ml) or purified water 25
300 μl of Reagent 1 was added to the μl, and the mixture was heated at 37 ° C. for 5 minutes. Then, 100 μl of Reagent 2 was added and reacted for another 10 minutes, and the absorbance at 505 nm was measured. The obtained change in absorbance per unit time was proportional to the concentration at the time of addition of 1,5-AG. The obtained calibration curve is shown in FIG.

Example 2 1,5-AG was measured in the same manner as in Example 1 except that 0.1 mg / ml of BCMA was added instead of 4-aminoantipyrine which is a chromogen. .
The obtained calibration curve is shown in FIG.

[0020]

According to the present invention, simple and accurate 1,5-
A method for quantifying AG is provided.

[Brief description of the drawings]

FIG. 1 is a calibration curve of 1,5-anhydroglucitol of Example 1.

FIG. 2 is a calibration curve of 1,5-anhydroglucitol of Example 2.

──────────────────────────────────────────────────続 き Continued on the front page (58) Field surveyed (Int.Cl. ⁶ , DB name) C12Q 1/48

Claims (6)

(57) [Claims] 1. A method in which hexokinase acts on 1,5-anhydroglucitol in a sample in the presence of a phosphate group donor.
It is allowed, by measuring the phosphate group donor to be consumed, a method of quantifying the 1,5-anhydroglucitol in the sample. 2. The method according to claim 1, wherein the phosphate group donor is adenosine triphosphate. 3. Measurement of consumed adenosine triphosphate
Quantifies the amount of adenosine diphosphate produced.
3. The method of claim 2, wherein 4. The amount of hexokinase is 4 to 100 u / m.
1 and the concentration of the phosphate group donor is 0.1 to 50 mM.
4. The method according to claim 1, wherein: 5. A 1,5-anhydroglucitol in a sample
Hexokinase in the presence of adenosine triphosphate
Adenosine triphosphate converted from adenosine triphosphate
Diphosphate to pyruvate kinase and pyruvate oxine
A method of quantification by the linkage of a sidase. 6. Adenosine triphosphate and hexokiners
1. A reagent for measuring 1,5-anhydroglucitol, which comprises: