RU2569750C1 - Diagnostic technique for diabetes mellitus by measuring erythrocyte cu,zn-superoxide dismutase activity - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention represents a diagnostic technique for type II diabetes mellitus involving a biochemical blood analysis, differing by the fact that erythrocyte Cu,Zn-superoxide dismutase is determined in the patients with measured glycaemia; if the determined value is less than 1402 units/gram of haemoglobin, the presence of type II diabetes mellitus is stated.

EFFECT: more effective and accurate diagnosing of diabetes mellitus.

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Description

The invention relates to medicine, in particular to laboratory research methods.

The development of diabetes is accompanied by impaired carbohydrate metabolism due to the development of hypoinsulinemia and a sharp increase in blood glucose (hyperglycemia). About 10-15% of glucose in blood plasma is present not in a cyclized form, but in the form of an enol form, including free aldehyde groups. Aldehydes easily react with amino groups to form a strong covalent bond, as a result of which aldehyde-mediated modification of proteins can occur, in particular, glucose can attach to hemoglobin molecules and cause their modification to form the so-called glycosylated hemoglobin.

Currently, the clinic widely uses methods for diagnosing and monitoring diabetes mellitus by evaluating the content of glycosylated hemoglobin in a sample, in particular, one of the patented methods includes the steps of: (a) hemolysis of a sample to release cellular hemoglobin; (b) contacting hemoglobin isolated from said sample according to step (c) described below using signal-forming molecules comprising a conjugate of one or more dihydroxyboryl residues or salts thereof associated with the signal-forming label; (c) isolation of glycosylated and non-glycosylated hemoglobin and any molecules associated with it from the sample and their determination by electrophoretic method.

(RF patent No. 2111494, IPC G01N 33/66, publ. 05.20.1998)

The disadvantage of this method is the complexity of the study and the use of expensive equipment, in addition, blood samples for research by this method cannot be stored for a long time in frozen form.

In addition, there are currently quite a lot of methods for determining the content of glycosylated hemoglobin for the diagnosis of diabetes mellitus, but all of them have certain disadvantages:

- high performance liquid chromatography - the disadvantage of this method is the need to purchase specialized expensive equipment (chromatograph, detector, etc.), a significant temperature dependence, the need to remove labile HbA _1C so that the results are not too high;

- mini-column ion-exchange chromatography — a significant temperature dependence of the method and an overestimation of results if labile HbA _{1 is} not removed before the study;

- mini-column affinity chromatography - determination of total GHb only;

- electrophoresis - requires an expensive device - densitometer, agarose gels and reagents for the electrophoretic system;

- immunochemical methods - the ability to automate the analysis, quick results, specificity make this method very attractive, but very expensive.

The objective of the invention is the creation of an alternative and effective method for the diagnosis of diabetes mellitus by determining the activity of erythrocyte Cu, Zn superoxide dismutase (Cu, Zn SOD), which eliminates all the disadvantages when determining the content of glycosylated hemoglobin.

The technical result of the invention is to increase the efficiency and accuracy of diagnosis of diabetes mellitus, the simplicity and speed of analysis, the degree of preservation of samples for research (possibly low-temperature storage for 3-6 months), and the ability to automate determinations.

This is achieved by the fact that in the inventive method for the diagnosis of type II diabetes mellitus, including a biochemical blood test, according to the invention, in the presence of hyperglycemia, the activity of erythrocyte Cu, Zn superoxide dismutase is determined, and if the value is less than 1402 units / gram of hemoglobin, type II diabetes mellitus is judged .

In addition to the direct attachment of glucose to protein molecules - the glycosylation process, glyoxylation associated with oxidative transformations of glucose is also possible. As a result of such processes, low molecular weight aldehydes (dicarbonyls) can form, such as methylglyoxal, which is formed during the transformation of triosophosphates, and glyoxal, which is formed during glucose autooxidation.

It was determined that the activity of erythrocyte Cu, Zn-SOD in patients with type 2 diabetes mellitus is significantly reduced. The decrease in the activity of Cu, Zn-SOD in the blood of patients with diabetes mellitus can be associated with the interaction of enzyme molecules with low molecular weight aldehydes formed during oxidative transformations of glucose, which causes inhibition of the enzyme activity. This is proved by experiments in which incubation of red blood cells in a medium containing glyoxal and methylglyoxal leads to inhibition of human erythrocyte Cu, Zn-SOD.

From this it follows that low molecular weight aldehydes easily penetrate the cell membrane and can cause a modification of enzymes, accompanied by a suppression of activity. In FIG. Figure 1 shows the effect of methylglyoxal and glyoxal on the activity of intraerythrocyte Cu, Zn-SOD (human erythrocytes were incubated in the presence of 10 mM glyoxal or methylglyoxal, after which the activity of erythrocyte Cu, Zn-SOD was measured).

In clinical trials, it was found that during sugar-lowering therapy of patients with type 2 diabetes mellitus with a high level of glycosylated hemoglobin, an increase in the activity of erythrocytic Cu, Zn-SOD occurs simultaneously with the compensation of carbohydrate metabolism disorders.

From this it follows that the activity of erythrocyte Cu, Zn-SOD should reflect the level of disorders of carbohydrate metabolism. The data obtained indicate that the activity of this enzyme in the red blood cells of patients with type 2 diabetes mellitus is inversely correlated (r = -0.652; p <0.0001) with the level of glycosylated hemoglobin. In FIG. Figure 2 shows the correlation between the activity of erythrocyte Cu, Zn-SOD and the level of glycosylated hemoglobin in the examined patients.

It should be noted that another advantage of this method is the fact that blood samples for research can be stored frozen for a sufficiently long time, which is excluded with the available methods for determining the level of glycosylated hemoglobin.

The implementation of the method.

The activity of Cu, Zn-SOD is measured according to the generally accepted method for inhibiting the reduction of blue nitrotetrazolium by a superoxide radical generated in the xanthine-xanthine oxidase system, determining the kinetics of formazan formation on a recording spectrophotometer or chemical analyzer in the kinetic mode at 560 nm. The unit of activity of Cu, Zn-SOD is taken as the amount of enzyme necessary for 50% inhibition of the reduction of blue tetrazolium under the conditions of determination.

The normal values of erythrocyte Cu, Zn-SOD are 1402-1800 units / gram of hemoglobin.

The patient is determined by the activity of erythrocyte Cu, Zn-superoxide dismutase.

With a value of less than 1402 units / gram of hemoglobin, the patient is diagnosed with diabetes mellitus.

Clinical example 1:

Patient S., 51 years old, 05/03/2012, was taken by the emergency hospital to the city clinical hospital №67 named after L.A. Vorokhobova with complaints of dry mouth, thirst. Over the past 6 months, I noted a decrease in body weight of 12 kg. Upon admission to the hospital, hyperglycemia of 24.6 mmol / L was recorded.

On admission, the activity of erythrocyte Cu, Zn- amounted to superoxide dismutase of 955 units / gram of hemoglobin.

For control, the level of glycosylated hemoglobin was determined, which amounted to 11.68%.

Based on clinical, medical history and laboratory research methods, the patient was diagnosed with newly diagnosed type 2 diabetes mellitus.

Immediately after making a diagnosis, the patient is started on an intensive regimen of insulin therapy, followed by transfer to oral administration of hypoglycemic drugs.

During the control examination on August 14, 2012: the level of glycosylated hemoglobin is 7.12%, the activity of erythrocyte Cu, Zn superoxide dismutase is 1324 units / gram of hemoglobin.

Clinical example 2:

Patient Ch., 47 years old, May 12, 2012, was taken by the emergency hospital to the city clinical hospital No. 67 named after L.A. Vorokhobova with complaints of dry mouth, thirst. The above complaints were disturbed during the last month, during which the patient noted a decrease in body weight of 15 kg. I turned to the endocrinologist of the clinic, recorded hyperglycemia of 18.7 mmol / l, aimed at hospitalization.

Upon receipt, the activity of erythrocyte Cu, Zn-superoxide dismutase is 887 units / gram of hemoglobin.

For control, the level of glycosylated hemoglobin was determined, which amounted to 10.45%.

Based on clinical, medical history and laboratory research methods, the patient was diagnosed with newly diagnosed type 2 diabetes mellitus.

Immediately after the diagnosis, insulin therapy was started in intensive mode, followed by the transfer to oral administration of hypoglycemic drugs.

During the follow-up examination on August 16, 2012, the level of glycosylated hemoglobin was 6.93%, the activity of erythrocyte Cu, Zn superoxide dismutase was 1289 units / gram of hemoglobin.

Clinical example 3:

Patient Sh., 54 years old, July 4, 2012 was taken by the emergency hospital to the city clinical hospital No. 67 named after L.A. Vorokhobova with complaints of dry mouth, thirst, weight loss of 4 kg over the past month, an increase in glycemia to 20 mmol / L. From the anamnesis it is known that the patient suffers from type 2 diabetes for 12 years. Outpatient development of diabetic polyneuropathy was recorded. The patient receives combined sugar-lowering therapy: oral sugar-lowering drugs from the sulfonylurea and biguanide groups, as well as medium-duration insulin injections twice a day. Against this background, the level of glycemia ranged from 9 to 16 mmol / L, the activity of erythrocyte Cu, Zn superoxide dismutase increased from 754 to 1115 units / gram of hemoglobin, the glycosylated hemoglobin content decreased from 12.11 to 8.1%.

Thus, the determination of the activity of erythrocyte Cu-Zn SOD can be an alternative way to detect the presence of diabetes mellitus, and given the fact that this method is much more convenient and can be carried out in clinical biochemistry laboratories that do not have special equipment, its use is simpler and more affordable. In addition, modern biochemical laboratories with conventional chemical analyzers can perform determinations in a semi-automatic or automatic mode. You can use standard test kits to determine the activity of superoxide dismutase.

Claims (1)

A method for diagnosing type II diabetes mellitus, including a biochemical blood test, characterized in that in the presence of glycemia, the activity of erythrocyte Cu, Zn superoxide dismutase is determined, and if the value is less than 1402 units / gram of hemoglobin, the presence of type II diabetes is judged.

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