JPH02195899A - Determination of glycoprotein and determination reagent - Google Patents

Abstract

PURPOSE:To easily perform the quantitative determination of glycoprotein useful as an index reflecting the average blood sugar level for the diagnosis of diabetes, without being influenced with interference substances by using a specific hydrogen donor, a reagent and an oxido-reductase. CONSTITUTION:An oxido-reductase (e.g. sorbitol dehydrogenase or mannitol dehydrogenase) is added to a specimen containing a glyco protein (preferably a mixture produced by isolating a lysine residue acting as a sugar-bonding site of a glycoprotein) using a CH-OH group as a hydrogen donor and (1) NAD<+> and/or NADP<+> or (2) oxygen molecule as a hydrogen receptor. The glycoprotein can be determined by measuring (1) the consumption of NADH and/or NADPH or (2) the consumption of oxygen or the generation of hydrogen peroxide.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to a method and reagent for measuring glycated proteins. More specifically, the present invention relates to a method and reagent for measuring glycated proteins useful as an index reflecting long-term average blood sugar levels for diabetes diagnosis, for example.

[Prior art] Conventional methods for measuring glycated proteins include methods that utilize the reducing power of glycated proteins under alkaline conditions, affinity chromatography, thiobarbic acid method, high performance liquid chromatography, radioimmunoassay, and enzyme methods. Immunoassay methods are known [for example, CIin, Chim, Acta, Vol. 127, pp. 87-95 (1982), Diabetes No. 2].
5, pp. 963-968 (1982), Diabetologia, vol. 21, 94
~98 pages (1981), Journal of Clinical Chemistry and Clinical Biochemistry (J, C11n, Chem, C11n.

Biochem, ) Volume 19, pp. 81-87 (19
81), Diabetes Vol. 29, pp. 581-590 (19
1986), Diabetes Section Vol. 29 Supplement 2, p. 340 (198
6 years)].

[Problem to be solved by the invention] However, conventional methods are affected by interfering substances.
There are problems such as complicated and time-consuming operations.

[Means for Solving the Problems] The present inventors have arrived at the present invention as a result of intensive studies aimed at obtaining a method and a measuring reagent for measuring glycated proteins that are simple and unaffected by interfering substances. That is, in the present invention, CH-OH group is used as a hydrogen donor, and (INAD+ and/or NADP+
or (2) act on glycated proteins with an oxidoreductase that uses oxygen molecules as hydrogen acceptors (1) NADH and/or N
A method for measuring glycated proteins that measures the amount of ADPH consumed or (2) the amount of oxygen consumed or the amount of hydrogen peroxide generated; group as a hydrogen donor and act on an oxidoreductase that uses 1) NAD+ and/or NADP+ or (2) an oxygen molecule as a hydrogen acceptor. A measurement method for glycated proteins that measures the amount consumed or the amount of hydrogen peroxide generated; and 1) NA using CH-OH group as a hydrogen donor.
This is a reagent for measuring glycated proteins containing D+ and/or NADP+ or (2) an oxidoreductase that uses oxygen molecules as hydrogen acceptors.

In the present invention, CH-OH group is used as a hydrogen donor and NAD+
and/or NADP+ or (2) The oxidoreductase that uses an oxygen molecule as a hydrogen acceptor may be any enzyme as long as it acts on a carbonyl group produced by non-enzymatic binding of sugar to a protein. Examples of such oxidoreductases include dehydrogenases such as sorbitol dehydrogenase, mannitol dehydrogenase, mannitol-1-phosphate dehydrogenase, D-xylulose reductase, and gluconate-5-dehydrogenase. Preferred among these are sorbitol dehydrogenase and mannitol dehydrogenase.

NAD'', NADP+ and oxygen molecules are used depending on the enzyme used.

Glycated proteins that are the measurement targets of the present invention include those produced by non-enzymatic reaction of aldose such as glucose with amino groups such as lysine residues of proteins, and further amatric transfer. For example, fructosamine (glycated serum protein),
Glycated albumin, hemoglobin AI (:, etc.)

Conditions for the oxidoreductase reaction for carrying out the measurements of the present invention may be those commonly used for the oxidoreductase used. For example, Biochemical Journal
Em, J.) Volume 83, pp. 135-144 (196
2nd year), Journal of Biological Chemistry (J, Biol, Chem, ) No. 23
The reaction conditions described in Vol. 8, pp. 1598-1603 (1963) can be used. For example, the reaction conditions when using sorbitol dehydrogenase are as follows.

The amount of oxidoreductase added is usually 0.01 to 100/mQ,
Preferably it is 0.1 to IU/yd. The amount of NADH and/or NADPH added is usually 0.001 to 1 m
g/d, preferably 0.01 to 0.1 .mu./yd.

The pH of the reaction solution is usually 4-10, preferably 6-8. The reaction temperature is usually 10-60°C, preferably 30-40°C.
It is ℃. The reaction time is usually 1 to 60 minutes, preferably 5 to 60 minutes.
It is 20 minutes. A buffer is usually used in the reaction, but there are no particular restrictions on the buffer as long as it is one commonly used in enzyme reactions. A phosphate buffer solution of O1 to 0.1M or the like can be used.

In addition, the reaction conditions when using mannitol dehydrogenase are such that the pH of the reaction solution is usually 5 to 10, preferably 7 to 10.
9. Reaction temperature is usually 5 to 50°C, preferably 20 to 30°C
The same conditions as for sorbitol dehydrogenase can be used except that the temperature is .degree.

The oxidoreductase, NADH and/or NADPH, and glycated protein may be added simultaneously or sequentially. When adding sequentially 1. The order of addition is not restricted.

Although the oxidoreductase may be allowed to act directly on the glycated protein, it is preferable to act on a mixture obtained by liberating lysine residues, etc., which are sugar binding sites, because the reaction proceeds more efficiently. The methods for binding sugars and releasing gin residues may be any conventional methods, including enzymatic methods and chemical methods. In the enzymatic method, any enzyme that can act on glycated proteins to release lysine residues etc. can be used. Either an endopeptidase such as carboxypeptidase B1 papain or an exopeptidase such as aminopeptidase can be used. As a chemical method, there is a hydrolysis method using hydrochloric acid or the like. Among these, the enzyme method is preferred.

The reaction conditions for liberating lysine residues etc. by the enzymatic method may be normal reaction conditions, and when using endopeptidase, for example,
Chemistry (J, Biol, Chem, ) 2nd
37, pp. 3094-3099 (1962) and vol. 207, pp. 515-531 (1954), and when using exopeptidase, for example, Methods in Enzymology (1954). Meth,
En-zymol, ) Volume 19, Nos. 514-521
(1970) can be used.

The enzyme that releases lysine residues and the like and the oxidoreductase may be added sequentially or simultaneously.

The reaction by an oxidoreductase using the CH-OH group as a hydrogen donor and NAD+ and/or NADP+ as a hydrogen acceptor is the reaction formula: %Formula% oxidoreductaseR1-NH-CH2-CH(.H)-R'
+ NAD, and/or NADP" (wherein R represents a protein molecule or a lysine residue, R' represents an aldose residue), and NADH and/or NADP
Accompanied by consumption of H. The consumption of NADH and/or NADPH is proportional to the concentration of glycated proteins present. Therefore, by measuring the consumption amount of NADH and/or NADPH, the concentration of glycated protein can be determined. NADH
and/or NADPH consumption is e.g. 340n
It can be measured by absorbance at m.

In the measurement method of the present invention, CH-OH group is used as a hydrogen donor and NA
In addition to oxidoreductases that use D+ and/or NADP+ as hydrogen acceptors, oxidoreductases that use CH-OH groups as hydrogen donors and oxygen molecules as hydrogen acceptors can also be used. Such enzymatic reactions involve (1) consumption of oxygen or (2) consumption of oxygen and generation of hydrogen peroxide.

The amount of oxygen consumed or hydrogen peroxide generated is proportional to the concentration of glycated proteins present. Therefore, the concentration of glycated proteins can be determined by measuring the amount of oxygen consumed or the amount of hydrogen peroxide generated. The amount of oxygen consumed and the amount of hydrogen peroxide generated can be measured by conventional methods. The amount of oxygen consumed can be measured, for example, using an oxygen electrode, and the amount of hydrogen peroxide generated can be measured, for example, using a color reaction using peroxidase.

A standard substance is usually required for measurement, but the standard substance is not particularly limited as long as it is an amatri transition product, and synthetic amatri transition products and the like can be used.

When the measuring method of the present invention is used in a clinical test for diagnosing diabetes, the sample containing glycated proteins may be any of the whole blood, blood serum, and serum commonly used in clinical tests. These may be subjected to measurement as they are, or may be subjected to pretreatment such as dialysis before being subjected to measurement. The amount of sample is usually 1 to i, oooltg, preferably 1O-1
001tff is used.

Enzymes such as oxidoreductase and peroxidase used in the measurement method of the present invention may be used in a solution state, but they can also be used in an immobilized state. The immobilized oxidoreductase, peroxidase, etc. can be used in any format such as column format or batch format.

Although the measuring method of the present invention can be carried out manually, it is more effective if carried out using an automatic analyzer commonly used in clinical tests.

The measurement result may be expressed as the concentration of glycated protein, but if the total protein concentration contained in the sample is measured at the same time, it can also be expressed as the ratio of the glycated protein concentration to the total protein concentration. The total protein concentration can be measured by any conventional method, for example 2
A method for measuring absorbance at 80 nm, the Loucou method (
Lowry method).

[Examples] Next, the present invention will be specifically explained with reference to Examples, but the present invention is not limited to the following Examples.

Example 1 (1) Glycated protein measurement reagent Enzyme reagent: 8 mg of sorbitol dehydrogenase (manufactured by Sigma, 3.1U/mg) was dissolved in 1 mu of 0.05 M phosphate buffer (pH 7,5) and freeze-dried.

NADH reagent: 0.5 mg of NADH・2Na (manufactured by Wako Tsumugi Pharmaceutical Co., Ltd.) in 0.05 M phosphate buffer (pH 7.5)
It was dissolved in ld and lyophilized.

(2) Lysine residue releasing reagent papain solution (manufactured by Sigma, 600 U/ml) 400
p, 9°, carboxypeptidase B solution (manufactured by Sigma, 2.300U/barrel) 200μ2, cysteine 20
(2) was dissolved in 1 m of 0.05M phosphate buffer (pH 7.5) and freeze-dried.

(3) Substance 2 obtained by bonding glucose to the amino group of standard material lysine
6■ was dissolved in one volume of 0.05M phosphate buffer (pH 7,5) and freeze-dried.

Comparative Example 1 Sera from diabetic patients and normal subjects were dialyzed against physiological saline. Distilled water was added to 100μ2 of the serum after dialysis to make a total volume of 500μ. Oxalic acid 500 at a concentration of 1 mole/R
μ2 was added and heated at 100° C. for 5 hours. then 600
Protein was removed by adding 200μ2 of trichloroacetic acid at a concentration of g/ffi. 300 μ2 of thiobarbic acid with a concentration of 50 mmol/R was added to the supernatant after protein removal, and the mixture was incubated at 37°C for 1 hour.
Incubated for 5 minutes. After being left at room temperature for 20 minutes, the absorbance at 443 nm was measured. Note that a 5-hydroxymethylfurfural solution was used as the standard solution, and the procedure was performed in the same manner as for the serum sample after protein removal. Glycated protein concentration (C) in serum was calculated using the following formula. The measurement results are shown in Figure 1.

AS: Absorbance A1 when measuring a sample. : Absorbance when measuring standard substance C14: Standard substance concentration (nm
ol/mQ) Example 2 Add 1 mfl each of distilled water to the glycated protein measuring reagent (enzyme reagent, NADH reagent), lysine residue release reagent, and standard substance to make a solution, and measure the glycated protein using the same serum sample as in the comparative example using the following method. was measured.

0.05M phosphate buffer (p
i-17,5) 1mff1 and 100μ of lysine residue release reagent were added and incubated at 37°C for 15 minutes.

Then, 250 μQ of NADH reagent was added and the absorbance at 340 nm was measured (ASI). A 50μ flood of enzyme reagent was then added and after a 15 minute incubation at 37°C.
Absorbance at 0 nm was measured (AS2). The same operation was performed for the standard material. The absorbance before and after adding the enzyme reagent is measured as A4. ■, A, 2. Glycated protein concentration in serum was calculated using the following formula. The measurement results are shown in Figure 1. It correlates well with the measurement results of the comparative example, which was measured after removing the interfering substances by dialysis.

In other words, this measurement method shows that it is possible to accurately measure the concentration of glycated protein without having to remove interfering substances beforehand.

/A,: ASl-AS2 AA+<: AJ AR2 C+<: Standard substance concentration (nmol/rn!Q) [Effects of the invention] Since the measurement method and measurement reagent of the present invention use highly specific enzymes, they are different from conventional ones. Interfering substances that cannot be avoided in the measurement method (e.g. ascorbic acid, uric acid, glutathione, ED)
TA-2Na, etc.). Further, it has excellent effects such as not requiring complicated operations such as separating glycated proteins from a sample containing glycated proteins by a method such as column chromatography.

Blood sugar level is one of the indicators for diagnosing diabetes or for controlling blood sugar in diabetic patients. However, since blood sugar levels fluctuate drastically over a short period of time due to the influence of meals, etc., accurate diagnosis or understanding of blood sugar control cannot be made using this alone. Therefore, indicators that reflect long-term (one week to several months) average blood sugar levels are attracting attention and being used for diagnosis and the like. The present invention can be effectively utilized for purposes such as such diagnosis.

[Brief explanation of the drawing]

FIG. 1 is a correlation diagram between the measurement results of Comparative Example 1 and the measurement results of Example 2. Figure 1 (nmol/mQ) Measured values according to Example 2

Claims (1)

[Claims] 1. CH-OH group is used as a hydrogen donor, (1) NAD^+
and/or NADP^+ or (2) oxidoreductase that uses oxygen molecules as hydrogen acceptors to act on glycated proteins (1
) NADH and/or NADPH consumption or (
2) A method for measuring glycated proteins that measures the amount of oxygen consumed or the amount of hydrogen peroxide generated. 2. Using a CH-OH group as a hydrogen donor (1
)NAD^+ and/or NADP^+ or (2)
Activates redox enzymes that use oxygen molecules as hydrogen acceptors,
A method for measuring glycated proteins that measures (1) the amount of NADH and/or NADPH consumed or (2) the amount of oxygen consumed or the amount of hydrogen peroxide generated. 3. Using CH-OH group as a hydrogen donor, (1) NAD^+
and/or NADP^+ or (2) a reagent for measuring glycated proteins containing an oxidoreductase that uses oxygen molecules as hydrogen acceptors.