Method of selectively determining glycated hemoglobin

• US 20030162242A1
• Filed: 04/14/2003
• Published: 08/28/2003
• Est. Priority Date: 07/14/2000
• Status: Active Grant

• Current Assignee: ARKRAY, Inc
• Original Assignee: ARKRAY, Inc
• Inventors: Yonehara, Satoshi
• Granted Patent: US 7,235,378 B2
• Time in Patent Office: Days
• US Class Current: 435/25
• CPC Class Codes: C12Q 1/26 involving oxidoreductase
• CPC Class Codes: C12Q 1/37 involving peptidase or proteinase
• CPC Class Codes: G01N 33/723 Glycosylated haemoglobin
• CPC Class Codes: G01N 33/725 using peroxidative activity

Abstract

A method of determining glycated hemoglobin is provided, by which a ratio of the glycated hemoglobin in a sample can be determined accurately and easily. The ratio of glycated hemoglobin can be determined by degrading a glycated hemoglobin in a whole blood sample selectively with a protease to give a glycated hemoglobin degradation product`; causing a redox reaction between a glycation site of the glycated hemoglobin degradation product and a fructosyl amino acid oxidoreductase; and determining this redox reaction. Further, as shown in FIG. 1, in a whole blood sample, there is a correlation between the ratio of the glycated hemoglobin determined by this method and an HbA1c concentration. Thus, without determining the glycated α-amino group as a characteristic structure of HbA1c, an amount of HbA1c can be determined accurately and easily from the determined ratio of the glycated hemoglobin.

Claims

1. A method of determining an amount of glycated hemoglobin comprising:

degrading glycated hemoglobin in whole blood selectively with a protease to give a glycated hemoglobin degradation product;

causing a redox reaction between a glycation site of the glycated hemoglobin degradation product and a fructosyl amino acid oxidoreductase; and

determining the redox reaction to determine an amount of the glycated hemoglobin.

2. The method according to claim 1,

wherein the protease is a protease capable of selectively degrading the glycated hemoglobin.

3. The method according to claim 2,

wherein the protease is at least one protease selected from the group consisting of bromelains, papains, trypsins derived from porcine pancreas, metalloproteinases, and proteases derived from Bacillus subtilis.

4. The method according to claim 1,

wherein the glycation site of the glycated hemoglobin degradation product to react with the fructosyl amino acid oxidoreductase is a glycated amino group in a side chain of an amino acid residue.

5. The method according to claim 4,

wherein the glycated amino group in the side chain of the amino acid residue is a glycated amino group in a side chain of at least one of a lysine residue and an arginine residue.

6. The method according to claim 1,

wherein determining the redox reaction is determining an amount of hydrogen peroxide generated by the redox reaction or an amount of oxygen consumed by the redox reaction.

7. The method according to claim 6,

wherein the amount of the hydrogen peroxide is determined using a peroxidase and a substrate that develops color by oxidization.

8. The method according to claim 7,

wherein the substrate that develops color by oxidization is N-(carboxymethylaminocarbonyl)-4,4′

-bis(dimethylamino)diphenylamine sodium.

9. The method according to claim 1, wherein the protease is added to the whole blood so that a content of the protease per milliliter of the whole blood is in a range from 1,000 to 10,000,000 U.

10. The method according to claim 1,

wherein a substrate of the fructosyl amino acid oxidoreductase is at least one glycated amine selected from the group consisting of glycated proteins, glycated peptides, and glycated amino acids, and

the fructosyl amino acid oxidoreductase acts on at least one of a glycated α

-amino group and a glycated side-chain amino group of the glycated amine to catalyze a reaction that causes generation of hydrogen peroxide.

11. The method according to claim 1,

wherein the fructosyl amino acid oxidoreductase is added to the whole blood so that a content of the fructosyl amino acid oxidoreductase per milliliter of the whole blood is in a range from 500 to 40,000 U.

12. The method according to claim 1,

wherein a sample to be used in the method is whole blood that has been subjected to a hemolysis treatment.

13. A method of determining an amount of HbA1c comprising:

preparing a calibration curve based on a correlation between an amount of glycated hemoglobin determined by the method of determining an amount of glycated hemoglobin according to any one of claims 1 to 12 and an amount of HbA1c; and

substituting the amount of glycated hemoglobin in a whole blood sample determined by the method into the calibration curve to determine an amount of HbA1c in the whole blood sample.

14. The method according to claim 13,

wherein the calibration curve is prepared based on a correlation between a known amount of HbA1c in a standard sample and an amount of glycated hemoglobin in the standard sample determined by the method according to any one of claims 1 to 12.

15. A kit used for determining an amount of glycated hemoglobin comprising:

a protease that distinguishes a glycated hemoglobin from other proteins and peptides to degrade the glycated hemoglobin selectively.

16. The kit according to claim 15,

wherein the protease is at least one protease selected from the group consisting of bromelains, papains, trypsins derived from porcine pancreas, metalloproteinases, and proteases derived from Bacillus subtilis.

17. The kit according to claim 15, further comprising a fructosyl amino acid oxidoreductase.

18. The kit according to claim 17,

wherein a substrate of the fructosyl amino acid oxidoreductase is at least one glycated amine selected from the group consisting of glycated proteins, glycated peptides;

and glycated amino acids, and

the fructosyl amino acid oxidoreductase acts on at least one of a glycated α

-amino group and a glycated side-chain amino group of the glycated amine to catalyze a reaction that causes generation of hydrogen peroxide.

19. The kit according to claim 18, further comprising a peroxidase and a substrate that develops color by oxidization.

20. The kit according to claim 19,

wherein the substrate that develops color by oxidization is N-(carboxymethylaminocarbonyl)-4,4′

-bis(dimethylamino)di henylamine sodium.

21. A reagent used for determining an amount of glycated hemoglobin comprising:

a protease that distinguishes a glycated hemoglobin from other proteins and peptides to degrade the glycated hemoglobin selectively.

22. The reagent according to claim 21,

wherein the protease is at least one protease selected from the group consisting of bromelains, papains, trypsins derived from porcine pancreas, metalloproteinases, and proteases derived from Bacillus subtilis.

23. The reagent according to claim 21, further comprising a fructosyl amino acid oxidoreductase.

24. The reagent according to claim 23,

wherein a substrate of the fructosyl amino acid oxidoreductase is at least one glycated amine selected from the group consisting of glycated proteins, glycated peptides, and glycated amino acids, and

the fructosyl amino acid oxidoreductase acts on at least one of a glycated α

-amino group and a glycated side-chain amino group of the glycated amine to catalyze a reaction that causes generation of hydrogen peroxide.

25. The reagent according to claim 24, further comprising a peroxidase and a substrate that develops color by oxidization.

26. The reagent according to claim 25,

wherein the substrate that develops color by oxidization is N-(carboxymethylaminocarbonyl)-4,4′

-bis(dimethylamino)diphenylamine sodium.