RU2196988C2 - Method for diagnostics of mucopolysaccharidosis - Google Patents

Abstract

FIELD: medicine, medicinal genetics. SUBSTANCE: method could be used to predict diseases associated with the disorders in the exchange of connective tissue of congenital and acquired etiology. The method deals with destruction of separate types of glycosaminoglycans by affecting with specific enzymes chondroitinases AC and ABC. Then a single-stage unidimensional electrophoresis is carried out. EFFECT: higher efficiency and simplicity. 2 dwg, 2 ex

Description

 The invention relates to medicine, namely to medical genetics, and can be used to diagnose diseases associated with metabolic disorders of the connective tissue of congenital and acquired etiology.

 A known method for determining the type of glycosaminoglycans by chromatography (Penock C. A. "A review and selection of simple laboratory methods used for the study of glucosaminoglycan excretion and the diagnosis of the mucopolysaccharidoses". J. Clin. Path., 1976, 29, 111- 123).

 This method is not specific enough due to the fact that the charge of glycosaminoglycans is determined by the degree of its polymerization and the number of sulfated groups; multi-stage, labor-intensive, expensive equipment and reagents are required for its implementation.

 Closest to the claimed is a method for determining hyperexcretion and types of glycosaminoglycans by electrophoresis (Philip P. Dembure and R. August Roesel "Screening for Mucopolysaccharidoses by Analysis of Urinary Glucosaminoglycans." Techniques in Diagnostic Human Biochemical Genetics: A Laboratory Manual, pp77-86, 1991, Wiley - Liss, Inc.), which is carried out as follows: glycosaminoglycans from urine are isolated by the method of Pennock S. A. (1976) in a modification. Isolated glycosaminoglycans and standards are dissolved in distilled water at a concentration of 0.5 mg / ml.

 Electrophoresis is carried out on a horizontal electrophoresis device company Bio-Rad. Glycosaminoglycans and standards in the amount of 5-10 μg are applied to the Optiphor-10 cellulose acetate membrane. 1 stage electrophoresis is carried out for 10 min at a voltage of 25V / CM. The membrane is then placed in a 15% ethanol solution in 0.1 M barium acetate buffer for 2 minutes. The second stage of electrophoresis is carried out for 20 minutes. The membranes are stained in a 0.25% solution of Alcian blue, and then the color is fixed with a 5% solution of acetic acid.

 The method is not specific enough due to the fact that it does not take into account the microheterogeneity of glycosaminoglycans, labor-intensive (multi-stage), requires expensive equipment and reagents.

 The objective of the invention is to offer a highly specific, simple method for the diagnosis of mucopolysaccharidoses.

 The problem is solved due to the fact that they carry out the destruction of certain types of glycosaminoglycans, acting with specific enzymes chondroitinases AC and ABC, then conduct one-stage one-dimensional electrophoresis.

 When using the method there is a positive medical effect, which consists in the diagnosis of the disease in a high degree of specificity. High specificity is achieved due to the fact that the enzymes used are specific for individual types of glycosaminoglycans, regardless of molecular weight, tissue specificity, glycosaminoglycan charge and, destroying, remove certain types of glycosaminoglycans at the stage of preparation for electrophoresis.

 Use has social significance. Specific diagnosis makes it possible to conduct prenatal diagnosis to prevent the birth of a child with such a pathology in the family. When using the method, an economic effect takes place: the range of expensive studies is narrowed to verify the diagnosis.

 The method is simple, highly specific, reliable, carried out on domestic equipment.

 The method is as follows.

Glycosaminoglycans isolated from 5 ml of a random portion of urine by Pennock S.A. (1976), dissolved in 50 μl of distilled water. Chondroitinase AC (EC. 4.2.2.5) or chondroitinase ABC (EC.4.2.2.4) (Sigma, USA) dissolved in 0.2 M Tris-HCl buffer containing 0.2 M is added to 10 μl of urine glycosaminoglycans dissolved in water. sodium acetate, pH 8.0, in a volume of 5 μl, so that the final enzyme activity in the incubation mixture is at least 0.025 U / μl. Incubated for 3 hours at 37 ^° C. Stop the reaction with chilled alcohol 96 ^° , 200 μl. After 30 minutes, centrifuged for 10 minutes, 3 thousand revolutions, the supernatant discharged, the precipitate was dried in air, dissolved in 10 μl of distilled water.

 Electrophoresis is performed on an isotachophoresis device on ITF-P2 27 film. Vladipor MFA-MA 5 cellulose acetate membrane manufactured by Tasma, presoaked in 0.1 M barium acetate buffer, pH 4.8, is dried with blotting paper. Samples and standard (a mixture of heparan sulfate and chondroitin sulfate A, Sigma, USA, at a concentration of 1 mg / ml, aqueous solution) are applied to the cellulose acetate membrane in an amount of 1 μl strip with a length of about 5 mm. On the “rough” surface of the membrane, 2 cm deviate from the edge (the direction of electrophoresis along the membrane fibers) and draw a line with a simple pencil without pressure (this is the start line). Then make markings for applying samples. 7-10 mm away from the edge and between samples. Electrophoresis is carried out in 0.1 M barium acetate buffer, pH 4.8, at a voltage of 70 V for 90 min, from the cathode to the anode. The membrane is stained in a 0.1% solution of Alcian blue in a 2.5% solution of acetic acid for 10 minutes, then the color is fixed with a 2.5% solution of acetic acid.

Practical examples
Example 1
Patient L., 9 years old, 9 months with a clinic of mucopolysaccharidosis was examined. Hyperexcretion of glycosaminoglycans (36.0 mg / mM creatinine (norm 3.2-5.6)) was detected. During electrophoresis, GAG hyperexcretion was detected due to chondroitin sulfates A and C, dermatan sulfate (line 1), but the presence of heparan sulfate cannot be ruled out. After the GAG treatment with chondroitinase AS, chondroitin sulfates A and C were removed (line 2), after the GAG treatment with chondroitinase ABC, chondroitin sulfates A and C were removed, as well as dermatan sulfate (chondroitin sulfate B) (line 3). Line 4: standard (HS - heparan sulfate; HS - chondroitin sulfates A and C; DS - dermatan sulfate). Conclusion: GAG hypersecretion is mainly due to dermatan sulfate, chondroitin sulfates A and C, which is typical for mucopolysaccharidosis. Further, the patient needs a study of enzyme activity for differential diagnosis of type mucopolysaccharidosis I, VI and VII.

Example 2
Patient K., 3 years 10 months, was examined with a mucopolysaccharidosis clinic. Hypexcretion of glycosaminoglycans (20.7 mg / mM creatinine (norm 4.4-8.0)) was detected. During electrophoresis, GAG hyperexcretion was detected due to chondroitin sulfates A and C (line 1), but the presence of keratan sulfate cannot be ruled out. After the GAG treatment with chondroitinase AS, chondroitin sulfates A and C were removed (line 2), after the GAG treatment with chondroitinase ABC, chondroitin sulfates A and C were removed, as well as dermatan sulfate (chondroitin sulfate B) (line 3). Line 4: standard (HS - heparan sulfate; HS - chondroitin sulfates A and C; DS - dermatan sulfate). Conclusion: GAG hypersecretion is due to chondroitin sulfates A and C. Later on, the patient was found to have no activity of arylsulfatase A and B, and he was diagnosed with mucosulfatidosis. This disease does not apply to mucopolysaccharidoses.

Claims (1)

 A method for the diagnosis of mucopolysaccharidoses by determining hyperexcretion and types of glycosaminoglycans (GAGs), characterized in that they destroy individual GAGs by acting with specific enzymes, chondroitinases AC and ABC, then carry out one-stage one-dimensional electrophoresis, and when GAG hyperexcretation and dermatomes are diagnosed with mucopolysaccharidosis.

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