Method of quantifying hemoglobin and method of measuring glycation ratio of hemoglobin

• US 6,790,665 B2
• Filed: 03/20/2003
• Issued: 09/14/2004
• Est. Priority Date: 09/28/2000
• Status: Expired due to Term

• Current Assignee: ARKRAY, Inc
• Original Assignee: ARKRAY, Inc
• Inventors: Yonehara, Satoshi, Yagi, Yuji
• Primary Examiner(s): WALLENHORST, MAUREEN
• Application Number:
  US10/380,961
• Publication Number:
  US 20030186449A1
• Time in Patent Office: 544 Days
• Field of Search: 436/66, 436/67, 436/164, 436/166, 436/904, 422/82.05, 422/82.09
• US Class Current: 436/66
• CPC Class Codes: G01N 21/31 Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry G01N21/72 takes precedence
• CPC Class Codes: G01N 21/78 producing a change of colour
• CPC Class Codes: G01N 33/721 Haemoglobin
• CPC Class Codes: G01N 33/723 Glycosylated haemoglobin
• CPC Class Codes: Y10S 436/904 Oxidation - reduction indicators

Abstract

A method of determining Hb is provided, by which an amount of Hb can be determined easily and accurately without fear of damage to the environment. Hemoglobin in a sample is denatured with a tetrazolium compound to give denatured hemoglobin, and an amount of an optical change in the sample is measured at an absorption wavelength specific to the denatured hemoglobin. Using the amount of the optical change thus measured, an amount of the hemoglobin in the sample can be determined. The amount of the optical change preferably is measured at a wavelength in a range from 520 to 670 nm. According to this method, an amount of Hb can be determined with high accuracy as shown in FIG. 1.

Claims

1. A method of determining total hemoglobin in a sample comprising:

denaturing hemoglobin in a sample with a solution of a tetrazolium compound to give denatured hemoglobin, wherein the pH of the solution is in a range from 5.5 to 10.0;

measuring an amount of an optical change in the sample at an absorption wavelength specific to the denatured hemoglobin; and

calculating an amount of the total hemoglobin in the sample from the amount of the optical change.

2. The method according to claim 1,

wherein the amount of the optical change is an absorbance or a reflectance.

3. The method according to claim 1,

wherein the amount of the optical change is measured at a wavelength in a range from 440 to 700 nm.

4. The method according to claim 1,

wherein the tetrazolium compound contains ring substituents at least at two positions on its tetrazole ring.

5. The method according to claim 1,

wherein the tetrazolium compound contains benzene rings at a 2-position and 3-position on its tetrazole ring, and at least one of the benzene rings contains at least one functional group selected from the group consisting of a halogen group, a carboxy group, a nitro group, a hydroxy group, a sulfo group, a methoxy group, and an ethoxy group.

6. The method according to claim 1,

wherein the tetrazolium compound is 2-(4-iodophenyl)-3- (2,4-dinitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium salt.

7. The method according to claim 1,

wherein the tetrazolium compound is added to the sample so that a content of the tetrazolium compound per microliter of the sample is in a range from 0.001 to 100 μ

mol.

8. The method according to claim 1,

wherein the hemoglobin in the sample is treated with the tetrazolium compound in the presence of a surfactant.

9. The method according to claim 8,

wherein the surfactant is a polyoxyethylene ether.

10. The method according to claim 8,

wherein the surfactant is added to the sample so that a content of the surfactant per mole of the tetrazolium compound is in a range from 0.01 to 70 mol.

11. The method according to claim 1,

wherein the sample contains red blood cells.

12. A method of determining a ratio of glycated hemoglobin to total hemoglobin in a sample comprising:

determining an amount of total hemoglobin in a sample containing glycated hemoglobin by the method according to claim 1;

causing a redox reaction between a glycation site of the denatured hemoglobin obtained and a fructosyl amino acid oxidase;

measuring the degree to which the redox reaction has occurred to determine an amount of the glycated hemoglobin; and

calculating a ratio of the glycated hemoglobin to the total hemoglobin in the sample from the amount of the total hemoglobin and the amount of the glycated hemoglobin.

13. The method according to claim 12,

wherein the denatured hemoglobin is treated with a protease before causing the redox reaction between the glycation site of the denatured hemoglobin and the fructosyl amino acid oxidase.

14. The method according to claim 12,

wherein the redox reaction is caused between the glycation site of the denatured hemoglobin and the fructosyl amino acid oxidase after the amount of the optical change in the sample is measured at the absorption wavelength specific to the denatured hemoglobin.

15. The method according to claim 14,

wherein the amount of the optical change in the sample is measured at the absorption wavelength specific to the denatured hemoglobin, the denatured hemoglobin is then treated with a protease, and thereafter, the redox reaction is caused between a glycation site of a degradation product of the denatured hemoglobin and a fructosyl amino acid oxidase.

16. The method according to claim 14,

wherein the denatured hemoglobin is treated with a protease, the amount of the optical change in the sample is then measured at the absorption wavelength specific to the denatured hemoglobin, and thereafter, a redox reaction is caused between a glycation site of a degradation product of the denatured hemoglobin and a fructosyl amino acid oxidase.

17. The method according to claim 12,

wherein, after the redox reaction is caused between the glycation site of the denatured hemoglobin and the fructosyl amino acid oxidase, the amount of the optical change in the sample is measured at the absorption wavelength specific to the denatured hemoglobin and the redox reaction is determined.

18. The method according to claim 12,

wherein measuring the degree to which the redox reaction has occurred is by measuring an amount of an optical change in a color-developing substance produced by the redox reaction.

19. The method according to claim 18,

wherein the amount of the optical change in the color-developing substance corresponds to an amount of hydrogen peroxide generated by the redox reaction between the glycation site of the denatured hemoglobin and the fructosyl amino acid oxidase.

20. The method according to claim 19,

wherein the color-developing substance is a substrate that develops color by oxidation and has developed color as a result of a reaction caused by an oxidase between the hydrogen peroxide and the substrate.

21. The method according to claim 18,

wherein a wavelength for measuring the color-developing substance is in a range from 650 to 900 nm.

22. The method according to claim 18,

wherein a wavelength for measuring the color-developing substance is the same as that for the step of measuring an amount of an optical change in the sample at an absorption wavelength specific to the denatured hemoglobin.