Monitoring metabolic control in diabetic patients by measuring glycosylated amino acids and peptides in urine

• US 4,371,374 A
• Filed: 11/17/1980
• Issued: 02/01/1983
• Est. Priority Date: 11/17/1980
• Status: Expired due to Term

• Current Assignee: Rockefeller University
• Original Assignee: Rockefeller University
• Inventors: Cerami, Anthony, Brownlee, Michael, Vlassara, Helen
• Primary Examiner(s): Chin, Peter
• Application Number:
  US06/207,471
• Time in Patent Office: 806 Days
• Field of Search: 23/230 B, 23/901, 23/913, 422/61, 422/57, 422/56
• US Class Current: 436/87
• CPC Class Codes: G01N 33/66 involving blood sugars, e.g. galactose
• CPC Class Codes: G01N 33/68 involving proteins, peptides or amino acids involving lipoproteins G01N33/92
• CPC Class Codes: G01N 33/6842 Proteomic analysis of subsets of protein mixtures with reduced complexity, e.g. membrane proteins, phosphoproteins, organelle proteins

Abstract

A method of monitoring metabolic control in a diabetes patient comprising measuring the amount of non-enzymatic glycosylated amino acids and peptides in urine of the patient; a method of measurement and a test kit for the measurement are disclosed.

Claims

1. A method of monitoring blood glucose integrated over a period of time from about one week to about one month, separating and quantitating non-enzymatic glycosylated amino acids, peptides or mixtures thereof present in the patient'"'"'s urine, comprising:

(a) treating a urine sample containing said non-enzymatic glycosylated amino acids, peptides or mixtures thereof with a suitable boronic acid to form a complex of said non-enzymatic glycosylated amino acids, peptides or mixtures thereof with said boronic acid;

(b) separating the so-formed complex from said urine; and

(c) analyzing the separated complexed material to establish the presence and amount of amino acids, peptides or mixtures thereof to obtain the complexed non-enzymatically glycosylated amino acid content of the urine, which content provides an indicia of blood glucose integrated over a period of time from about one week to about one month.

3. The method according to claim 1 or 2, wherein said complex is formed under alkaline conditions.

4. The method according to claim 3, wherein said alkaline conditions comprise a pH of at least 9.

5. The method according to claim 1 or 2, wherein said boronic acid is immobilized on a support.

6. The method according to claim 1 or 2, wherein prior to analysis for amino acids, peptides or mixtures thereof the complex is treated with an acid.

7. The method according to claim 6, wherein said acid is HCl.

8. The method according to claim 1 or 2, wherein naturally occurring insoluble materials have been removed from the urine sample.

2. A method of monitoring blood glucose integrated over a period of time from about one week to about one month, by separating and quantitating non-enzymatic glycosylated amino acids, peptides or mixtures thereof present in the patient'"'"'s urine, comprising:

(a) contacting a urine sample containing said non-enzymatic glycosylated amino acids, peptides or mixtures thereof with a color dye reactive with amino acids so as to attach the dye to said non-enzymatic glycosulated amino acids, peptides or mixtures thereof;

(b) treating the dye containing solution so produced with a suitable boronic acid to form a complex of said non-enzymatic glycosylated amino acids, peptides or mixtures thereof with such boronic acid;

(c) separating the so-formed complex from said fluid; and

(d) colorimetrically analyzing the separated, dyed, complexed material to establish the presence and amount of of amino acids, peptides or mixtures thereof to obtain the amino acid content of the urine, which content provides an indicia of blood glucose integrated over a period of from about one week to about one month.

9. A method for monitoring metabolic control in a diabetes patient comprising measuring the amount of non-enzymatic glycosylated amino acids and peptides in the urine of the patient and comparing the measurement with a previously prepared standard showing non-enzymatic glycosylated amino acid and peptide content as a function of integrated blood glucose concentration.

10. The method according to claim 9, wherein the non-enzymatic glycosylated amino acids and peptides present in the urine of the patient are separated from the urine and quantitated.

11. The method according to claim 10, wherein the non-enzymatic glycosylated amino acids and peptides are separated from the urine by treatment with an insolubilized boronic acid so as to form an insoluble complex of said boronic acid and said non-enzymatic glycosylated amino acids and peptides;

and separating said complex from the urine.

12. The method according to claim 11, wherein prior to complex formation, the insoluble materials present in the urine have been removed.

13. A test kit for the colorimetric determination of non-enzymatically glycosylated amino acids and/or peptides in a urine sample, comprising:

(a) a predetermined amount of an insolubilized suitable boronic acid;

(b) a predetermined amount of a color dye reactive with amino acids by attachment of the dye to said non-enzymatic glycosylated amino acids and/or peptides, whereby (a) and (b) are separated from one another by being placed in separate containers; and

(c) previously prepared standards for colorimetric comparison.

14. The test kit according to claim 13, wherein the insolubilized boronic acid has been equilibrated with an alkaline buffer.

15. The test kit according to claim 13, wherein the color dye is an azo dye.

16. The test kit according to claim 13, wherein the color dye is dansyl chloride.

17. The test kit according to claim 13, wherein said boronic acid and said color dye are in separate packets such that a pH adjusted urine sample may be contacted with the color dye, brought into contact with said boronic acid, and the boronic acid can be washed free of the urine sample non-reacted dye.

18. The test kit according to claim 13, wherein said boronic acid is coated on a strip of a porous material.

19. The test kit according to claim 13, wherein said boronic acid is coated on an elongated cylindrical body comprising a porous material.

20. The test kit according to claim 18, wherein said strip of porous material is coated on only one side.

21. A test kit according to claim 13 or 18, and for the determination of said amino acids and/or peptides, wherein the color dye reactive with amino acids is present in an amount which is stoichiometrically in excess of that necessary to react with all of the amino acids present in the urine sample.

22. The test kit according to claim 21, wherein the insolubilized boronic acid is present in an amount which is stoichiometrically in excess of that necessary to form a complex with the non-enzymatic glycosylated amino acids and peptides present in the urine sample.

23. A test strip for the colorimetric detection of non-enzymatically glycosylated amino acids and/or peptides in a urine sample comprising a strip of porous material which has been at least partially coated with an insolubilized boronic acid and impregnated with a color dye reactive with amino acids by attachment of the dye to said nonenzymatic glycosylated amino acids and/or peptides.

24. A test strip according to claim 23, and for the determination of said amino acids and/or peptides, wherein the color dye reactive with amino acids is present in an amount which is stoichiometrically in excess of that necessary to react with all of the amino acids present in the urine sample.

25. The test strip according to claim 24, wherein the insolubilized boronic acid is present in an amount which is stoichiometrically in excess of that necessary to form a complex with the non-enzymatic glycosylated amino acids and peptides present in the urine sample.

26. The test strip according to claim 25, wherein the strip of porous material is paper.