Method of selectively determining glycated hemoglobin

Patent metadata

Field	Value
Publication number	US-RE46130-E
Application number	US-200114329019-A
Country	US
Kind code	E1
Filing date	Jul 12, 2001
Priority date	Jul 14, 2000
Publication date	Aug 30, 2016
Grant date	Aug 30, 2016

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.

First claim

The invention claimed is:

1. A method of determining an amount of glycated hemoglobin in a sample comprising glycated hemoglobin and glycated albumin from whole blood, comprising:
selectively degrading the glycated hemoglobin in the sample with a proteasetreating the sample with a protease that selectively degrades the glycated hemoglobin over the glycated albumin to give a glycated hemoglobin degradation product, wherein the protease is a metalloproteinase;
reacting a glycation site of the glycated hemoglobin degradation product and a fructosyl amino acid oxidoreductase in a redox reaction;
determining a product produced by the redox reaction; and
correlating the amount of the product produced with the amount of glycated hemoglobin in the sample,
wherein the sample to be used in the method is whole blood that has been subjected to a hemolysis treatment.

2. The method according to claim 1 , 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.

3. The method according to claim 1 , wherein the glycation site of the glycated hemoglobin degradation product that reacts with the fructosyl amino acid oxidoreductase is a glycated amino group in a side chain of an amino acid residue.

4. The method according to claim 3 , 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,

5. 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.

6. The method according to claim 5 , wherein the amount of the hydrogen peroxide is determined using a peroxidase and a substrate that develops color by oxidization.

7. The method according to claim 6 , wherein the substrate that develops color by oxidization is N-(carboxymethylaminocarbonyl)-4,4′-bis(dimethylamino)diphenylamine sodium.

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

9. 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.

10. The method according to claim 1 , wherein the fructosyl amino acid oxidoreductase is added to the whole blood so that a concentration of the fructosyl amino acid oxidoreductase per milliliter of the whole blood is in a range from 500 to 40,000 U.

11. 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.

12. The method according to claim 1, wherein the glycation site of the glycated hemoglobin degradation product that reacts with the fructosyl amino acid oxidoreductase is a glycated amino group in a side chain of an amino acid residue.

13. The method according to claim 12, 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.

14. 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.

15. The method according to claim 14, wherein the amount of the hydrogen peroxide is determined using a peroxidase and a substrate that develops color by oxidization.

16. The method according to claim 15, wherein the substrate that develops color by oxidization is N-(carboxymethylaminocarbonyl)-4,4′-bis(dimethylamino)diphenylamine sodium.

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

18. 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.

19. The method according to claim 1, wherein the fructosyl amino acid oxidoreductase is added to the whole blood so that a concentration of the fructosyl amino acid oxidoreductase per milliliter of the whole blood is in a range from 500 to 40,000 U.

20. The method according to claim 1, wherein the fructosyl amino acid oxidoreductase is selected from the group consisting of Fusarium, Gibberella and Aspergillus.

21. The method according to claim 1, wherein the hemolysis treatment is selected from the method consisting of using ultrasonic waves and utilizing differences in osmotic pressure.

22. The method according to claim 1, wherein the treatment of the sample with the protease is carried out in a buffer.

23. The method according to claim 22, where the buffer is selected from the group consisting of Tris-HCl buffer, EPPS buffer, PIPES buffer, phosphate buffer, ADA buffer, citrate buffer and acetate buffer.

24. The method according to claim 1, wherein the treatment of the sample with the protease is carried out at a pH in a range from 6 to 11.

25. The method according to claim 1, wherein the whole blood sample to be used in the method has been centrifuged.

Assignees

• Arkray Inc

Inventors

• Yonehara Satoshi

Classifications

• C12Q1/26 Primary

  involving oxidoreductase · CPC title

• C12Q1/37

  involving peptidase or proteinase · CPC title

• G01N33/725

  using peroxidative activity · CPC title

• G01N33/723

  Glycosylated haemoglobin · CPC title