Diagnostic technique for metabolic compensation disturbances and method for prediction of risk of developing in patients wit type 2 diabetes mellitus by lymphocyte apoptosis assessment

FIELD: medicine.

SUBSTANCE: for the purpose of diagnosing metabolic compensation disturbances and predicting a risk of developing complications in the patients suffering type 2 diabetes mellitus (type 2 DM), peripheral venous blood is examined for a percentage of apoptotic lymphocytes An+/Pl-. The lymphocyte An+/Pl- value below 3.5 % enables diagnosing a condition of carbohydrate metabolism compensation and predicting a favourable clinical course of the disease for the following year. The lymphocyte An/PI- content within the range 3.5 to 6% provides diagnosing a condition of subcompensation and a moderate risk of developing complications. The An+/Pl- value exceeding 6 % described a condition of decompensation and a high risk of developing chronic complications of type 2 diabetes mellitus.

EFFECT: higher accuracy of prediction and reduction of the risk of developing complications in diabetic patients.

4 ex

 

The invention relates to medicine, namely to endocrinology and immunology, clinical laboratory diagnosis of disorders of compensation metabolism and forecasting the risk of complications in patients with diabetes mellitus type 2 (T2DM).

Type 2 diabetes is one of the leading medical and social problems, which concerns a large part of the population not only Russia, but all over the world. Currently, diabetes is considered as a disease, which marked the predominant insulin resistance with relative deficiency of insulin [5]. This form of diabetes often goes undiagnosed for many years because the hyperglycemia may not be quite pronounced for the development of overt symptoms of diabetes. The majority of patients with type 2 diabetes are obese, which induces or exacerbates insulin resistance, when this is observed mainly abdominal fat distribution. Diabetes is dangerous at any stage of your current sudden, rapidly developing complications such as nephropathy, retinopathy, atherosclerosis of the coronary and peripheral arteries, as diabetic foot, peripheral and autonomic neuropathy, often leading to disability and death.

If diagnosed with type 2 diabetes take into account the phase of disorders of carbohydrate metabolism: the comp is compensation, subcompensation, decompensation. Under the compensation understand the preservation of normoglycemia during the day (on an empty stomach 4,4÷6.1 mmol/l after meals - 5,5÷8 mmol/l) and absence of glucose in the urine. When subcompensation level of fasting blood glucose ranges 6,2÷7.8 mmol/l after a meal does not exceed 10 mmol/l, the concentration of glucose in the urine is below 0.5%. In the period of decompensation in patients with marked increase in glucose fasting blood more than 7.8 mmol/l after meals - more than 10 mmol/l; there is increasing glycosuria (more than 0.5%). Often increases the liver, due to its fatty infiltration; functional tests of the liver is usually not disturbed. In addition, there is a transient increase of the functions of a number of glands of internal secretion: increased secretion of growth hormone, catecholamines, and glucocorticoids. For long-decompensated diabetes improving processes of decay and attenuation of protein synthesis leads to atrophic changes in the muscles. A decrease of their weight, sagging palpation, muscle weakness and fatigue [3].

The claimed method for the diagnosis of disorders of compensation metabolism and predict the risk of complications in patients with type 2 diabetes by assessing the level of apoptosis of lymphocytes will increase the accuracy of prediction, to objectify tactics of treatment measures the enterprises, to reduce the risk of developing chronic diseases, and to prevent disability of patients.

At the moment, also known methods of diagnosis of diabetes and its compensation in content glucolysed hemoglobin (HbA1c), the study of the spectrum of lipids (total cholesterol, triglycerides) [2]. The closest analogue (prototype) is the way to long-term monitoring of patients with diabetes and neurological and vascular complications, which is that many times determine the immunoreactivity of serum in relation to insulin, to antiinsulin antibodies or their antigennegative fragments [12]. In addition to the duration and multiple surveys, this method can not count on the lifespan prediction, as the period of activity of the antibody productions and half-life is short.

The present invention describes a system change in the permeability of cell membranes, including the nuclear, which shows long-term and late effects. The products of the genes are regulators of apoptosis (p53, bc1-2, bc1-x1, mc1-1, bak, bax and others) are very conservative and are the same for representatives of various mammals, which allows only one of the investigated parameter. Changes in the expression of proteins-regulators of apoptosis who has a long-term effect, therefore, the prediction of the risk of complications is carried out for a longer period. This simplifies sample preparation, improves the accuracy and reliability of results, reduces the risk of laboratory abnormalities.

Previously using apoptosis assessing the damaging effects of viral infections [1, 9], the prediction of neonatal mortality [11] and the development of early relapse of the underlying disease after stem cell transplantation [13], made a diagnosis of metastasis [8] and psoriasis [6], chronic placental insufficiency [7]. There are various ways and means of stimulating apoptosis [10, 15], screening of Pro - and antiapoptotic therapeutic activity of drugs [4].

Distinguishing feature of the claimed process is the establishment of diagnostic criteria percentage of apoptotic lymphocytes An+/PI - in peripheral venous blood of patients with type 2 diabetes. When the value of the studied parameter is lower than 3.5% of the diagnosed condition of compensation of carbohydrate metabolism and is projected favorable course of the disease in the coming year. When the content of lymphocytes An+/PI in the range from 3.5 to 6% of the diagnosed condition subcompensation and is projected to moderate risk of developing complications. The level of lymphocytes An+/PI - above 6% characterizes the state of decomp the compensation and the high risk of development of chronic complications of diabetes mellitus type 2.

The method is standard, as follows.

1. In patients with type 2 diabetes take 1 ml of venous blood in a Vacutainer tube containing stabilizer is lithium salt of heparin, by puncture of the cubital vein.

2. To select a suspension of lymphocytes by gradient centrifugation in a concentration of from 105up to 106cells in the sample.

3. Once wash cells (7÷10 min, 150 g) phosphate buffered 0.15 M solution of sodium chloride, pH 7,2÷7.4V (PBS).

4. To resuspendable 500 ál of limousine in 500 μl of 1% binding buffer (10% binding buffer to increase to 1% by adding distilled water 1:10, put ice in the fridge).

5. 100 μl of the sample from item 4 to make 1 μl annexin V (AnV) and 5 µl of propidium iodide (250 mg of powder PI dilute by adding 1 ml of the prepared binding buffer 1%, put ice in the fridge). The mixture gently stir.

6. Incubate for 15 min in the dark on ice.

7. To add to the suspension of 400 µl chilled 1% binding buffer, mix gently.

8. To analyze cells on a flow cytometer, assessing the intensity of green fluorescence of FITC-annexin V (FL1, 530 nm) and red fluorescence of propidium iodide (FL3, more than 600 nm) [14].

The survey was carried out with the written consent of the respondents. In this way were examined 32 patients with type 2 diabetes, the diagnosis put the h city endocrinology physicians Advisory centre of Arkhangelsk: 6 people in the stage of compensation, 7 - subcompensation, 17 - decompensation. As a comparison group study included 25 healthy individuals Arkhangelsk. The results were processed using the software package Statistica 6" ("StatSoft, USA). Study type retrospective, random sample, simultaneously. General population - residents of the North of European Russia. The boundaries of the normal distribution of quantitative indicators was defined using the criteria Shapiro - Wilke. The distribution of the percentage of lymphocytes is An+/PI differed from normal, therefore, are presented as median, lower and upper quartiles. The significance of differences between groups was evaluated using a parametric t-student test for independent samples and non-parametric criterion of Wilcoxon signed. Statistical significance was assigned at p<0,05.

It is established that the examined patients in the stage of compensation the content of lymphocytes An+/PI is 2.3 (from 0.8 to 3.4)%, when changing the carbohydrate balance in the stage of subcompensation - 4,8 (3,8-5,9)%, in a state of decompensation content of lymphocytes exposed to apoptosis, is equal to 8.45 (5,95-11,6)%. In the group of healthy people, this figure was 1.6 (0.8 to 5,42)%, which corresponds to the specified values of compensation of carbohydrate metabolism. This fact is confirmed by the increase con is entrale lymphocytes with marker ready to apoptosis CD95+ 0.53±0.05 to 0.65±0,06×10 9CL/l, while increasing the content of lymphocytes CD10+ 0,55±0,05 to 0.75±0,09×109CL/l in those with decompensated stage of type 2 diabetes compared with healthy people.

The initiation of the apoptotic signal by the interaction of ligand apoptosis with the receptor leads to the formation of a complex of signaling proteins, activating kaspesky a cascade of reactions that leads to the characteristic chromatin condensation, nuclear fragmentation, shrinkage of the nucleus, puzyrchatogo plasma membrane and other ultrastructural changes in the cells [16]. Normal apoptosis of the cells is inhibited after the annexation of insulin to its receptor. As a result, the receptor autophosphorylated with the participation of tyrosine kinase, activates phosphatidylinositol-3-kinase, stimulating the transition GLUT-4 across the cell membrane, which ensures the transport of glucose and the synthesis of DNA [2]. The aggravation of the disease type 2 diabetes can be characterized first by increased levels of insulin in plasma (decreasing sensitivity to the cells, followed by a decrease in the content of insulin due to secretory dysfunction of β-cells of the pancreas. This may explain the increase in blood levels of apoptotic lymphocytes An+/PI.

Examples of use of the method.

1. Patient S., 53. Diagnosis: diabetes mellitus type 2, easily the e for on the background of obesity, VI degree of the mixed Genesis, abdominal option. Ill for 4 years. At laboratory examination found that the content in the peripheral venous blood lymphocytes An+/PI-stage apoptosis, is 1.3%. Based on these data, the conclusion about the compensation of carbohydrate metabolism and favorable course of the disease in the coming year, which confirms the effectiveness of the diet.

2. Patient F., 55 years. Diagnosis: diabetes mellitus type 2, moderate, III degree obesity, abdominal option. Ill for 6 years. Differential diagnosis found that blood levels of apoptotic lymphocytes An+/PI is 4.2%. The conclusion subcompensation status of carbohydrate metabolism, which is confirmed by the further course of the disease and identification of a year microangiopathy vessels of the neck, lower limbs, coronary arteries.

3. Patient B., aged 57. Diagnosis: diabetes mellitus type 2, moderate over burdened heredity from the mother's side (type 2 diabetes, obesity). Ill for 4 years. At laboratory examination found that the content in the blood lymphocytes of An+/PI corresponds to 11.9%. The conclusion of decompensation status of carbohydrate metabolism and a high risk of developing complications of diabetes, which is confirmed by insufficiently pronounced therapeutic results the project is of iati and the occurrence of diabetic retinopathy.

4. Patient K., 59 years old. Diagnosis: diabetes mellitus type 2, diabetic nephropathy, pronounced swelling on the feet and legs, dry mouth. Ill for 14 years. The laboratory survey found that blood levels of apoptotic lymphocytes An+/PI corresponds to 10.9%. The conclusion of decompensation status of carbohydrate metabolism, which further confirmed disease progression and the development of diabetic sensory polyneuropathy.

LITERATURE

1. Berlin AA predictor of precancerous conditions of the cervix in women with papilloma-virus infection / BLK, Whirlow, Snehana etc. // Patent No. 2310197 RF, publ. 10.11.2007.

2. I. Bokarev. Diabetes: a guide for physicians / Innocare, Vlkov, Owina. - M.: OOO "Medical news Agency". - 2006. - 400 C.

3. Great medical encyclopedia, Ed. Petrovsky, ed 3, V.7. - M.: Soviet encyclopedia, 1977. - S-232.

4. Dygalo NN. Method of screening Pro - or antiapoptotic activity of therapeutic drugs / NGO, Tschetinina, Gtesting // Patent No. 2388065 RF, publ. 27.04.2010.

5. Zykova T.A. Diagnosis and classification of diabetes mellitus / Taskova, Basavana // Manual for doctors. - Arkhangelsk, 2001. - 30 S.

6. Korsunsky IM Way assessment of apoptotic system in the skin of patients JI is the IAP / Imortantly, Iwholename, Saabrucken etc. // Patent No. 2311643 RF, publ. 27.11.2007.

7. Lipatov I.S. method for the diagnosis of chronic placental insufficiency / Issipative, Melnikov V.A., Tezikov J.V., etc. // Patent No. 2313795 RF, publ. 27.12.2007.

8. Likhvantseva VG a method for predicting the metastasis of uveal melanoma based on markers of apoptosis Bax and BCL-2 / Ugulava, Merlinia, Mealamu etc. // Patent No. 2244934 RF, publ. 10.09.2004.

9. Lutsenko M.L. Way to assess the damaging effects of herpes-virus infection on the development of the apoptotic nuclei simplest of the villi of the placenta in pregnant / Metlenko, Andrievsky IA // Patent No. 2370772, RF, publ. 20.10.2009.

10. Makedonov G.P. Method of stimulating apoptosis / Heptadecanol, Tskhovrebov L.V. // Patent No. 2340349 RF, publ. 20.02.2008.

11. Plekhanov L.A. a Method for predicting neonatal mortality in children with perinatal Central nervous system / Liapichev, Gnelitsa // Patent No. 2287162 RF, publ. 10.01.2006.

12. Poletaev A.B. Way of monitoring patients with diabetes mellitus and the development of neurological and vascular complications / Abolite, Ooh, Mascolo // Patent No. 2291437, RF, publ. 10.04.2006.

13. Pronkin NV Way of forecasting the development of early relapse of the underlying disease after autologous transplantation of peripheral blood stem to etok in patients with hematological malignancies / Nvironmen, Uscasino, Ioulianou etc. // Patent No. 2337712 RF, publ. 10.11.2008.

14. Siberian SV Assessment of apoptosis in immunological research: a brief methodological guide / Svibanj, Sphidko, Avisource etc. - Ekaterinburg: Ural branch of the Russian Academy of Sciences, 2008. - 59 S.

15. Fedorov SN. The means of stimulating apoptosis of human leukemia / S.n.fyodorov, Lckuby, Pin // Patent No. 2360692 RF, publ. 10.07.2009.

16. Roma NR. Actual problems of immunology. - SPb., 2004. - P.12-16.

Method for the diagnosis of disorders of compensation metabolism and predict the risk of complications in patients with diabetes mellitus type 2 with apoptosis, including immunological examination of the blood, characterized in that the peripheral venous blood by the method of flow cytofluorimetry determine the percentage of apoptotic lymphocytes An+/PI-, when the value of the content of lymphocytes An+/PI - below 3.5% of the diagnosed condition of compensation of carbohydrate metabolism and is projected favorable course of the disease for the next year, when the content of lymphocytes An+/PI in the range from 3.5 to 6% of the diagnosed condition subcompensation and is projected to moderate risk of complications, the level lymphocytes An+/PI - above 6% characterizes the state of decompensation and the high risk of development of chronic complications of diabetes.



 

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