JP2000300294A - DETERMINATION OF HEMOGLOBIN Alc - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for simply and accurately determining hemoglobin Alc(HbAlc) in a sample. SOLUTION: This method comprises successively treating a test specimen with a protease capable of cleaving the carboxyl group side of leucine at the third from β-chain N end of hemoglobin Alc and then a protease capable of cutting out His-Leu from fructosyl-Val-His-Leu and then measuring fructosylvaline formed there.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a method for simply and accurately quantifying hemoglobin A1c in a sample.

[0002]

2. Description of the Related Art The main glycation site of a hemoglobin molecule includes a valine residue at the N-terminal of a β-chain, an ε-lysine residue of a β-chain,
There are ε-lysine residue of α chain and valine residue at N terminal of α chain, but hemoglobin A1c (hereinafter abbreviated as “HbA1c”)
Is obtained by binding glucose to the valine residue at the N-terminal of the β-chain. HbA1c in blood is important as an indicator of diabetes management because it clinically reflects the average blood glucose level in the past one to two months.

[0003] As a method for measuring HbA1c, a method by HPLC is usually used. However, in this method, alcohol-drinking acetaldehyde, which is a metabolite of alcohol, is bound to Hb, and in patients with renal failure, ca.
There is a problem that rbamylated Hb is further eluted in the same fraction as HbA1 and HbA1c is apparently high, even in healthy adults who have a large amount of fetal hemoglobin (HbF). This is a limitation of the HPLC detection method that separates HbA1c by fractionation based on Hb charge.

On the other hand, as a method for measuring a glycosylated protein such as HbA1c using an enzyme, a sample is treated with a protease, the protease-treated sample is treated with a ketoamine oxidase, and the reaction product is measured. Japanese Patent Application Laid-Open No. 5-19 / 2005 for measuring enzymatic glycosylated proteins
It is disclosed in JP-A-2193.

However, in order to apply this method to the quantification of HbA1c, a protease capable of specifically excising fructosyl valyl residues at the N-terminal of the β chain of HbA1c is required. However, such a protease is neither exemplified nor suggested in the publication, and a method for specifically excising the above-mentioned residues has not been known at all. Therefore, conventionally, this method could not be applied to the quantification of HbA1c.

[0006]

Accordingly, an object of the present invention is to provide a method for simply and accurately quantifying HbA1c.

[0007]

Under these circumstances, the present inventors have conducted intensive studies, and as a result, have cut the carboxyl group side of leucine at the third position from the N-terminal of the β chain of HbA1c to fruc.
By using a protease capable of obtaining tosyl-Val-His-Leu in combination with a protease capable of cutting off His-Leu from fructosyl-Val-His-Leu, fructosyl valine at the N-terminus of the β chain of HbA1c is used. Residues were successfully cut out, and by measuring this fructosyl valine, HbA1c was successfully and accurately quantified with high specificity, thus completing the present invention.

[0008] That is, the present invention provides a test sample comprising a protease capable of cleaving the carboxyl group side of the third leucine from the N-terminal of the β chain of hemoglobin A1c, followed by fructosyl-
Hemoglobin A1c, which is sequentially treated with a protease capable of cutting out His-Leu from Val-His-Leu, and then measuring fructosyl valine generated there.
The present invention provides a method for quantification.

[0009]

DESCRIPTION OF THE PREFERRED EMBODIMENTS In carrying out the measurement method of the present invention, it is necessary to cleave the carboxyl group side of the third leucine from the N-terminal of the β chain of HbA1c in a test sample. The protease used for this is not particularly limited as long as it can excise fructosyl-Val-His-Leu from HbA1c, and is not particularly limited. For example, any one of various exopeptidases alone or 2 More than one species can be used in combination, and exopeptidase and carboxypeptidase can be used in combination.
Examples of these proteases include elastase, proteinase K, pepsin, alkaline protease, trypsin, proline-specific endoprotease, V8 protease, carboxypeptidase P, carboxypeptidase W, carboxypeptidase Y and the like.
The activity of these proteases is 0.05 to 10,000 U / m
1, especially in the range of 10 to 2000 U / ml.

The conditions for treating a test sample with a protease that cleaves the carboxyl group side of the third leucine from the N-terminal of the β chain of HbA1c are as follows: treatment temperature: 15 to 50 ° C., further 30 to 45 ° C., particularly 35 to 45 ° C. 40 ° C. is preferred, and the treatment time is preferably 3 minutes to 24 hours, particularly preferably 5 minutes to 1 hour. The test sample after this treatment is purified as it is or by ultrafiltration or the like as necessary, and the following fructosyl-Val-His-Leu to His-L
The eu can be subjected to treatment with a protease that can excise it.

[0011] The fructosyl-
The protease capable of cutting out His-Leu from Val-His-Leu is not particularly limited, but for example, dipeptidyl carboxypeptidase (EC. 3.4.15.
1), but more specifically, angiotensin converting enzyme. Angiotensin converting enzyme is angiotensin I (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu).
Of His-Leu from the C-terminus of Angiotensin II
It is generally known as an enzyme that converts to The advantage of using this enzyme is that the specificity for the β-chain N-terminus is significantly improved. That is, as described above, as the main glycation site of the Hb molecule, in addition to the valine residue at the N-terminus of the β-chain, a valine residue at the N-terminus of the α-chain may be mentioned. Val-Leu-Ser-Pro-
Because of Ala-Asp-..., Fructosyl valine is not cut out even if the valine at the N-terminal of the α-chain is saccharified, and the valine at the N-terminal of the β-chain is saccharified. Specific, ie, HbA1c. The activity amount of such a protease is 0.
The range of 05 to 10000 U / ml, especially 10 to 2000 U / ml is preferred.

[0012] As a treatment condition with a protease capable of cutting out His-Leu from fructosyl-Val-His-Leu, the treatment temperature is 15 to 50 ° C, further 30 to 45 ° C, particularly 35 to 40 ° C.
C. is preferable, and the treatment time is 3 minutes to 24 hours, particularly 5 minutes to 1 hour.
Time is preferred. The test sample after this treatment is
Alternatively, if necessary, it can be purified by ultrafiltration or the like, and can be subjected to the following fructosyl valine measurement.

As a method for measuring fructosyl valine cut out by the above-mentioned method, there is a method of detecting the fructosyl valine by separation by HPLC. However, hydrogen peroxide produced by the action of ketoamine oxidase is measured. The method is simple and preferred. Ketoamine oxidase is not particularly limited as long as it uses fructosyl valine as a substrate, but has low specificity for fructosyl lysine,
Those having high specificity for fructosyl valine are preferred. Specific examples of such ketoamine oxidase include fructosyl amino acid oxidase produced by a recombinant microorganism derived from Corynebacterium sp., Which is commercially available from Kikkoman Corporation. The activity amount of ketoamine oxidase is 1 to 10,000 U / L, particularly 10 to 10 U / L.
A range of 5000 U / L is preferred.

The method for measuring hydrogen peroxide generated by the action of ketoamine oxidase is not particularly limited, but a chromogen and peroxidase (POD) are added to the reaction system to prepare the chromogen. A method of oxidizing to form a color-forming substance and measuring this is suitable. Examples of the chromogen include a combination of 4-aminoantipyrine and a phenol compound, a naphthol compound or an aniline compound, and 3-methyl-2-benzothiazolinone hydrazone (M
A combination of (BTH) with an aniline compound, leucomethylene blue or the like is used. Further, as described in Japanese Patent No. 2516381, hydrogen peroxide and hydrogen peroxide in the presence of POD are used.
The trivalent cobalt ion generated by the reaction with the trivalent cobalt ion is converted into an indicator specific to the trivalent cobalt ion, for example, 2- (2-thiazolylazo) -5-disulfobutylaminobenzoic acid trisodium salt (TASBB) Can be used to generate a colored chelate compound and measure it. According to this, measurement sensitivity 5 to 10 times higher than the above method can be obtained. In addition, as a reagent for detecting hydrogen peroxide, N, N, N ', N', N '', N ''-hexa
(3-Sulfopropyl) -4,4 ', 4''-triaminotriphenylmethane (TPM-PS, manufactured by Dojin Chemical Co., Ltd.) can also be used.

In the present invention, test samples used for quantification of HbA1c include whole blood and erythrocytes.

[0016]

Next, the present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples.

Example 1 11.5 mg / ml of commercially available HbA1c (manufactured by Exocell) (1 mM KCN,
pH 7.4) and a two-fold dilution of this were used as samples. Each of these solutions contains 20 U / ml trypsin (10 mM Tris buffer pH 7.5), 20 U / ml proline specific endoprotease (10 mM Tris buffer pH 7.5), and 20 U / ml carboxypeptidase P (50 mM citrate buffer). pH 4.5) is sequentially mixed in equal volumes and allowed to act at 37 ° C. for 1 hour to obtain fructosyl-Val-Hi
s-Leu was cut out. After the treatment with each protease, proteins having a molecular weight of 10,000 or more were removed by ultrafiltration using microcon 10 manufactured by Amicon.

The solution containing fructosyl-Val-His-Leu thus obtained was added to a 20 U / mL angiotensin converting enzyme (300 mM Na
Dissolved in 100 mM HEPES buffer pH 8.3 containing Cl)
After reacting at 37 ° C for 1 hour, the above-mentioned ultrafiltration was performed, and fr
A solution containing uctosyl-Val was obtained. This was used as a sample for a detection system using the following ketoamine oxidase.

<Detection system using ketoamine oxidase> Reagent (1) 100 mM phosphate buffer (pH 8) ESBmT 23 mg / dl Reagent (2) 100 mM phosphate buffer (pH 8) 4-aminoantipyrine 28 mg / dl peroxidase 500U / dl Ketoamine oxidase (Kikkoman, fructosyl amino acid oxidase) 1000U / dl

The measurement was performed by a two-point end method using a Hitachi Automatic Analyzer Model 7070. Specifically, the sample
260 μl of reagent (1) was added to 20 μl, and reacted at 37 ° C. for 5 minutes.
Measure the difference in absorbance between 700 nm and 570 nm, and then use reagent (2)
Was added and reacted at 37 ° C. for 5 minutes.
The difference in absorbance at nm was measured. The standard solution is 50 μM
The same operation was performed using a fructosyl valine solution and a saline solution as a reagent blank, and the fructosyl valine concentration in the sample was determined from the absorbance difference between the standard solution and the reagent blank.

The theoretical fructosyl valine concentrations were 22.3 μM and 11.15 μM calculated from HbA1c with a known amount of protein used in this example, and the measured values were compared with these values. As shown in Table 1, a good recovery was shown with respect to the theoretical value. This indicates that HbA1c can be quantified.

[0022]

[Table 1]

[0023]

The HbA1c quantification method of the present invention has higher specificity than conventional methods and can be measured with a general-purpose automatic analyzer, so that it is excellent in economy, operability, simple and accurate. HbA1c can be quantitatively determined.

Claims (4)

[Claims] 1. The test sample is prepared by using the β chain N of hemoglobin A1c.
Protease capable of cleaving the carboxyl group side of the third leucine from the terminal, then H from fructosyl-Val-His-Leu
A method for quantifying hemoglobin A1c, which comprises sequentially treating is-Leu with a protease capable of cutting out and then measuring fructosyl valine generated there. 2. The method for quantifying hemoglobin A1c according to claim 1, wherein the protease capable of cleaving His-Leu from fructosyl-Val-His-Leu is dipeptidyl carboxypeptidase (EC. 3.4.15.1). 3. The method for quantifying hemoglobin A1c according to claim 2, wherein the dipeptidyl carboxypeptidase (EC. 3.4.15.1) is an angiotensin converting enzyme. 4. The hemoglobin A1c according to any one of claims 1 to 3, wherein the measurement of fructosyl valine is performed by quantifying hydrogen peroxide produced by allowing ketoamine oxidase to act on the fructosyl valine. Assay method.