Diabetes and Protein Glycosylation: Measurement and Biologic Relevance

Advanced Glycation End Products and Diabetic Complications
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Toluene 10 ml was added as a preservative during the collection period. All urine samples gave essentially identical chromatographs.

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Washing the column with pH 9. After washing the column with 12 column volumes of buffer, changing to 0.

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Since the material in this latter peak must contain both a glycosylated amino group to be retained on the column and an unsubstituted amino group to react with ninhydrin , it represents a mixture of free glycosylated lysine and small peptides containing glycosylated lysine. Comparison of the level of these compounds in urine from diabetic and normal patients shows that the mean level in diabetics is two times that found in urine from normal subjects Figure 4.

These results show that the measurement of glycosylated proteins or peptides in the urine by this method provides a practical means of monitoring metabolic control in diabetic patients at frequent intervals at home. Human diabetic hemoglobin was prepared by"the method of Koenig et al, J.

After pH adjustment tc 9. Aliquots ui from each fraction were counted in 7.

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Peak tubes from the HCI column elution were pooled and concentrated. The elution patterns were compared with chromatograms of reduced, hydrolyzed glycosylated valine and reduced, hydrolyzed glycosylated lysine standards. The chromatographic pattern of hydrolyzed diabetic hemoglobin previously reduced with NaB 3 H 4 showed a peak of non-specific radioactivity in the void volume non-glycosylated compounds and a single sharp peak of radioactivity eluted subsequently with HCI.

Amino acid analysis of this latter peak showed that reduced glycosylated valine and reduced glycosylated lysine in approximately equimolar amounts were the only adducts present in the pooled HCI peak. Using the procedure of Example 3, the peripheral sciatic nerve of normal and diabetic rats were analyzed for the amount of non-enzymatic glycosylated amino acids. The nerves were removed, reduced with sodium borohydride, acid hydrolyzed and put on the boronate column as described previously.

The results are shown in Figure 5, expressed as counts per minute cpm per pmol of ninhydrin positive material.

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A method for monitoring metabolic control in a diabetes patient comprising measuring the amount of non-enzymatically glycosylated amino acids and peptides in the urine of the patient. The method according to Claim 1, wherein the integrated blood glucose concentration is monitored.

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  • The measurement and clinical significance of glycated proteins;

The method according to Claim 2, wherein the non-enzymatically glycosylated amino acids and peptides present in the urine of the patient are separated from the urine and quantified. The method according to Claim 3, wherein the non-enzymatically 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-enzymatically glycosylated amino acids and peptides; and separating said complex from the urine.

The method according to Claim 4, wherein prior to complex formation, the insoluble materials present in the urine have been removed. A method according to Claim 1 comprising: a treating urine containing said non-enzymatically glycosylated amino acids, peptides or mixtures thereof with a boronic acid to form a complex of said non-enzymatically glycosylated amino acids, peptides or mixtures thereof with said boronic acid;. A method according to Claim 1 comprising: a contacting urine containing said non-enzymatically 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 glycosylated amino acids, peptides or mixtures thereof;.

The method according to Claims 6 or 7, wherein said complex is formed under alkaline conditions.

The method according to Claim 8, wherein said alkaline conditions comprise a pH of at least 9. The method according to Claim 6 or 7, wherein said boronic acid is immobilized on a support. The method according to Claim 6 or 7, wherein prior to analysis for amino acids, peptides or mixtures thereof, the complex is treated with an acid. The method according to Claim 11, wherein said acid is hydrochloric acid. A test kit for use in a method according to any one of Claims 1 to 12 comprising an insolubilized boronic acid and a color dye reactive with amino acids, wherein said boronic acid and said color dye are separated from one another.

The test kit according to Claim 13, wherein the insolubilized boronic acid has been equilibrated with an alkaline buffer. The test kit according to Claim 13 or 14, wherein the color dye is an azo dye. The test kit according to Claim 13 or 14 wherein the color dye is dansyl chloride. The test kit according to any one of Claims , 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.

The test kit according to any one of Claims , wherein said boronic acid is coated on a strip of porous material. The test kit according to any one of Claims , said boronic acid is coated on an elongated cylindrical body comprising a porous material. The test kit according to Claim 18, wherein said strip of porous material is coated on only one side. The test kit according to any one of Claims , 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.

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-enzymatically glycosylated amino acids and peptides present in the urine sample. A test strip for use in a method according to any one of Claims 1 to 13 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.

The test strip according to Claim 23, 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. Monitoring metabolic control in diabetic patients by measuring glycosylated amino acids and peptides in urine.

The measurement and clinical significance of glycated proteins

Monitoring of metabolic control in diabetic by determination of glycosylated amino acids and peptides in urine. Monitoring metabolic control in diabetic patients by measuring glycosylated amino acid and peptides in urine. USA en. EPB1 en. JPHB2 en.

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