RU2538715C1 - Method for assessing long-term hyperglycemia - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention represents a method for assessing long-term hyperglycemia based on measuring blood plasma glucose differing by the fact that glucose is measured twice: immediately after blood taking and 24 hours after blood plasma storage above a layer of blood corpuscles; that is followed by calculating a glucose reduction in percentage from the initial value which is used to assess long-term hyperglycemia with blood plasma stored at t=4.0±0.5°C.

EFFECT: invention provides optimising and reducing assessment time of long-term hyperglycemia, more reliable result and possibility to examine a maximum amount of individuals over a minimum period of time and detecting a risk group.

2 tbl

Description

The invention relates to experimental medicine and can be used to study chronic complications of diabetes, resulting from prolonged hyperglycemia and glycosylation of proteins, as well as the correction of these changes in the development of new methods of treatment. The invention can also be used in clinical diagnostics as an inexpensive screening test method for detecting hyperglycemia that lasts for a long period of time.

The wide spread of diabetes mellitus, which is becoming an epidemic among the population of developed countries, determines the need for timely identification and treatment of people at risk, as well as the search for new methods of correction of chronic complications caused by the disease [1, 4, 6]. The causes of prolonged hyperglycemia, in addition to non-insulin-dependent and non-insulin-dependent diabetes mellitus, can be such endocrine disorders as hyperthyroidism, increased function of the adrenal cortex, pheochromocytoma, as well as a state of chronic stress, accompanied by the release of contra-hormonal hormones. Stress, acute and chronic, is considered not only as a factor aggravating the course, but also being the cause of diabetes mellitus in some cases [9]. Hormones involved in adaptation to stress (adrenaline, glucagon, glucocorticoids) are contra-stimulatory and cause an increase in blood glucose, which contributes to enhanced secretion of insulin and, as a result, insulin resistance in some individuals [1, 7, 9].

The main and affordable way to assess hyperglycemia at the present time is to determine the glucose content in whole blood or in plasma, serum by the enzymatic glucose oxidase method. Since blood glucose, even measured on an empty stomach, is subject to significant changes over several weeks, methods for determining glycosylated hemoglobin (HbA1c) and fructosamine are used to detect hyperglycemia that persists for a long time. A glucose tolerance test is used to establish insulin resistance.

Glycosylation of proteins occurs as a result of a non-enzymatic reaction of the free amino groups of amino acid residues and the aldehyde group of glucose. In the first, reversible reaction stage, a labile aldimine or an unstable Schiff base is formed. At the second, slow and irreversible stage, Amadori rearrangement occurs (isomerization of the glucose residue into the fructose residue) with the formation of stable ketamine [4, 8]. Hemoglobin containing ketamine is traditionally called glycosylated hemoglobin, and blood albumin combined with ketamine is commonly called fructosamine.

In clinical diagnostics, the average glucose concentration for the last two to three months is judged by the amount of HbA1c, since the half-life of hemoglobin is 60 days, and the amount of fructosamine indicates the average glucose concentration for the last 2-3 weeks, since the half-life of albumin is 40 days [ 8].

There are 6 methods for determining glycosylation products based on various principles of analysis: high performance liquid chromatography (HPLC), microcolumn ion exchange and affinity chromatography, electrophoresis, spectrophotometric and immunochemical methods.

For HPLC and electrophoresis, special, often expensive equipment is required, for example, for efficient separation of hemoglobin fractions by electrophoresis, Beckman reagents and a good densitometer such as Apprise (USA) are required. Microcolumn chromatographic methods are highly dependent on temperature conditions. Temperature has a lesser effect on the determination by affinity chromatography, but the determination, together with the preparation and washing of the columns, takes 3 hours and allows you to study at the same time no more than 5 blood samples. Spectrophotometric and immunochemical methods are performed quickly, but spectrophotometric determination also depends on temperature, and immunochemical analysis requires imported reagents from Bayer and Roche, a biochemical analyzer, and special and software [8]. The result of the determination can be underestimated in case of loss of blood, anemia in the patient due to the shorter duration of the existence of red blood cells. Hemoglobin (in case of hemolysis), ascorbic acid and ceruloplasmin inhibit the formation of fructosamine. A decrease in the results of determination of fructosamine is observed with hypoproteinemia, proteinuria, loss of albumin. In this case, it is necessary to additionally determine the content of total protein and albumin in blood plasma. In patients after removal of the spleen or with polycythemia, overestimated results can be detected. In addition, in a number of methods, other glycosylated hemoglobin fractions, the removal of which is not always provided, influence the accuracy of the HbA1c analysis. In addition to the main fractions of hemoglobin (HbA - 93%), human blood erythrocytes contain a small amount of other fractions that can also undergo glycosylation (HbA1a, HbA1b, HbA1c, HbA2, HbF). Normally, in humans, glycosylated hemoglobin (HbA1) ranges from 5 to 8% of total hemoglobin, the glycosylated hemoglobin fraction - HbA1c is 4-6.5%.

Glycosylated hemoglobin is formed non-enzymatically when glucose freely penetrates the erythrocyte membrane throughout the life of the red blood cell. Glucose attached to proteins through a strong covalent bond causes a change in their structure, functions and autoimmune reactions. The process of glycosylation of proteins causes the development of micro- and macrovascular complications in patients with diabetes mellitus [1, 4]. Not only hemoglobin and plasma proteins are subject to glycosylation, but also membrane proteins of blood cells, including erythrocyte membrane proteins. According to previous studies with alloxan diabetes, an increase in the amount of glycosylated hemoglobin, which is an indicator of glycosylation of other body proteins, is accompanied by stabilization of erythrocyte membranes, and this stabilization does not depend on the cholesterol content in the erythrocyte membrane [2, 3]. Consequently, the stability of the erythrocyte membrane can be due to its glycosylation and an increase in the glycocalyx layer on the surface of red blood cells.

With a thickening of the glycocalyx, the permeability of the erythrocyte membrane becomes insufficient for glucose, which is the only energy substrate for red blood cells. Reducing the intake of glucose in red blood cells is not directly related to the production of insulin, since there is no receptor in the erythrocyte membrane that integrates into the cell membranes after the interaction of insulin with the insulin receptor and the activation of the hormone messenger chain. However, hypoinsulinemia and insulin resistance cause a violation of the active transport of glucose into the cells of insulin-dependent tissues with similar receptors [4]. Glucose, which remains unclaimed, as well as formed as a result of gluconeogenesis, accumulates in the blood and enters into glycosylation reactions.

Red blood cells of a healthy person or animal in vitro quickly consume glucose from blood plasma, therefore, glucose is determined in whole blood or in serum / plasma separated from the formed elements immediately after blood collection. The increase in time from blood sampling to the determination of the indicator leads to an underestimated result. At the same time, a decrease in the permeability of the erythrocyte membrane should contribute to a lower rate of consumption of glucose from blood plasma by red blood cells.

The aim of the invention is to reduce the time for evaluating prolonged hyperglycemia with increasing the reliability of the result, as well as the possibility of examining the maximum number of people in the minimum time to identify risk groups.

The technical result is achieved by optimizing the evaluation method and, as a result, reducing labor and material costs.

To solve this problem, a method for assessing long-term hyperglycemia is proposed, based on the determination of glucose in blood plasma, characterized in that the glucose content is determined twice: immediately after blood collection and 24 hours after storage of blood plasma over a layer of formed elements, then the decrease in glucose content is calculated as a percentage of the initial level, which assesses long-term hyperglycemia, and blood plasma is stored at t = 4.0 ± 0.5 ° C.

Materials and methods

The experiment was conducted on 35 outbred rats of both sexes weighing 180-250 g, which were contained in the usual diet of vivarium. The conditions and handling of the animals used in the experiment were in accordance with Council Directive 2010/63 / EU. The animals were divided into groups: 1 - intact rats, 2 - modeling of subcompensated type 1 diabetes mellitus (administration of alloxan in an amount of 17 mg / 100 g weight), 3 - modeling of decompensated alloxan diabetes mellitus type 1 (administration of alloxan in an amount of 30 mg / 100 g weight ) Modeling of diabetes was carried out in accordance with the author's technique (application No. 2013125897). A month after the first administration of alloxan, alloxan diabetes developed, accompanied by polydipsia, polyuria, an increase in glucose, glycosylated hemoglobin and a decrease in blood insulin - the main indicators and criteria for type 1 diabetes. Deviation from the norm of the studied parameters was more pronounced in animals in group 3 than in group 2.

Glucose was determined using a standard set of reagents from Vital Diagnostics (St. Petersburg). The determination was carried out twice: in blood plasma immediately after its collection and in the same plasma stored over a layer of shaped elements at 4 ° C for 24 hours. The content of glycosylated hemoglobin in whole blood was determined by the method of affinity gel chromatography using a set of reagents “Diabet-test” from FOSFOSORB. The permeability of erythrocyte membranes (TEM) was determined by the method of Kolmakov V.N., Radchenko V.G. [5]. The degree of hemolysis was expressed as% of hemolized red blood cells in relation to the standard. Membrane permeability was evaluated by erythrocyte hemolysis in mixtures of urea and sodium chloride solutions with increasing urea concentration.

Optical density was measured on a Beckman DU-800 spectrophotometer (USA). Statistical analysis of the material was carried out using the Statistica 6.0 software (Stat. Soft.Inc.) And the Microsoft Exel 2003 program. When testing statistical hypotheses, a significance level of 5% was used (P <0.05).

Observation results and conclusions

As can be seen from table 1, in animals with a model of subcompensated and compensated forms of diabetes mellitus, the glucose content progressively increases compared with the figure in the group of intact animals. The higher the initially measured glucose level, the smaller the decrease in this indicator, expressed as a percentage of the initial amount, 24 hours after the first measurement.

Table 1 The content of glucose and HbA1c,% in the blood of experimental and intact animals Indicators Groups Intact n = 10 Alloxan diabetes, subcompensated form n = 15 Alloxan diabetes, decompensated form n = 10 Glucose, mmol / L First dimension 5.1 ± 0.7 10.9 ± 1.7 33.9 ± 1.7 * * ** Second dimension 3.3 ± 0.4 9.8 ± 1.7 33.9 ± 1.6 * * ** Decrease in glucose content,% 34.8 ± 5.9 9.9 ± 2.3 1.8 ± 0.6 * * ** HbA1c,% 2.9 ± 0.1 5.0 ± 0.3 9.1 ± 0.4 * * ** Note: * - the difference with the group of intact animals is significant at P <0.05; ** - the difference with group 2 is significant at P <0.05.

In healthy animals, a significant decrease in the indicator is observed a day after the first measurement - by 30-40%, the glucose level falls below the norm or approaches the lower limit of the norm. In animals with alloxan diabetes, the decrease in glucose per day is insignificant: by 10% in rats with a subcompensated form of diabetes and almost does not change in animals with a decompensated form. The content of glycosylated hemoglobin is also increased in both experimental groups relative to the control - intact rats.

When calculating the pair-wise linear correlation coefficient between the decrease in glucose expressed as a percentage of the initial level and the content of glycosylated hemoglobin in erythrocytes, a value of -0.77 is obtained, that is, an inverse correlation is observed: the more glycosylated hemoglobin, the lower the consumption of glucose from blood plasma by red blood cells .

table 2 Permeability of erythrocyte membranes (TEM) by urea hemolysis with increasing urea concentration (in% hemolyzed red blood cells) The volume ratio of urea: sodium chloride Groups Intact n = 10 Alloxan diabetes, subcompensated form n = 15 Alloxan diabetes, decompensated form n = 10 40:60 7.3 ± 1.3 7.1 ± 0.7 7.0 ± 0.9 50:50 19.3 ± 2.6 28.6 ± 5.6 13.7 ± 3.2 55:45 45.7 ± 3.6 63.6 ± 7.2 25.4 ± 5.6 * ** 60:35 91.4 ± 1.6 86.7 ± 2.7 67.3 ± 7.3 * ** Note: * - the difference with the group of intact animals is significant at P <0.05; ** - the difference with group 2 is significant at P <0.05.

Table 2 presents the data of measuring the permeability of erythrocyte membranes in intact and experimental animals. In the group of rats with a subcompensated form of diabetes mellitus, TEM does not significantly differ from that of intact animals, measured with all four ratios of urea and sodium chloride solutions. At the same time, in rats with a decompensated form of pathology, a more pronounced resistance of erythrocytes to hemolysis with an increased content of urea in a hemolytic solution is found. An increase in the stability of erythrocyte membranes or a decrease in their permeability can cause a decrease in the consumption of glucose from blood plasma by red blood cells during storage of whole blood.

Thus, a decrease in plasma glucose, expressed as a percentage of the initial amount, 24 hours after blood sampling can serve as an indicator for assessing prolonged hyperglycemia.

The proposed method is just as simple, so effective, and therefore, due to its low cost and accessibility, it can be widely used in medical institutions: district clinics, small hospitals, and especially in rural medical institutions.

LITERATURE

1. Balabolkin M.I., Klebanova E.M., Kremlinskaya V.M. Treatment of diabetes and its complications (a guide for doctors). - M.: Medicine. 2005 - 512 s.

2. Gette I.F., Danilova I.G., Krotov V.K. The dependence of the resistance of erythrocyte membranes on the age of rats and the development of alloxan diabetes mellitus // Bulletin of the Ural Medical Academic Science. - No. 3 (26). - 2009. - P.22-24.

3. Danilova I.G., Medvedeva S.Yu., Abidov M.T., Yushkov B.G., Kiselnikova L.P., Gette I.F., Goskov I.A., Chishi M.A. Features of the regenerative processes of various tissues in the modulation of macrophage activity // Institute of Dentistry. 2005.2. P.67-69.

4. Bunny A.Sh., Churilov L.P. Pathochemistry. - SPb: ELBI-SPB, 2007 .-- 768 p.

5. Kolmakov V.N., Radchenko V.G. The value of determining the permeability of erythrocyte membranes (TEM) in the diagnosis of chronic liver diseases // Therapeutic archive. No. 5. 1982. S. 59-62. Kolmakov V.N., Radchenko V.G. The value of determining the permeability of erythrocyte membranes (TEM) in the diagnosis of chronic liver diseases // Therapeutic archive. No. 5. 1982. S. 59-62.

6. Peter J. Watkins. Diabetes. - M .: Binom Publishing House. 2006 - 135 p.

7. Titov V.N. Laboratory and instrumental studies in the diagnosis. Directory. - M.: Publishing house "GEOTAR-MED." 2004.958 s.

8. Emmanuel V.L., Karyagina I.Yu., Emmanuel Yu.V. Comparison of methods for the determination of glycosylated hemoglobin // Laboratory Medicine. 2002. 5. S. 99-104.

9. Surwit R., Schneider M., Feinglos M. Stress and Diabetes Mellitus // Diabetes Care 1992. vol. 15. No. 10. P.1413-1422

Claims (1)

A method for evaluating long-term hyperglycemia, based on the determination of glucose in blood plasma, characterized in that the glucose content is determined twice: immediately after blood collection and 24 hours after storage of blood plasma over a layer of formed elements, then the decrease in glucose content in percent of the initial level is calculated according to which they evaluate long-term hyperglycemia, and the storage of blood plasma is carried out at t = 4.0 ± 0.5 ° C.