RU2286578C2 - Method for early diagnostics of diabetic neuropathy in children - Google Patents

Abstract

FIELD: medicine, juvenile endocrinology.

SUBSTANCE: one should detect the level of endothelin-1 (pcg/ml) and basic fibroblast growth factor (bFGF) (ng/ml) in patients with insulin-dependent diabetes mellitus in blood serum. At the ratio of endothelin-1 against bFGF being equal to 40 and higher it is possible to diagnose subclinical stage of diabetic neuropathy (DNP) in children. The innovation provides the opportunity for prescribing adequate therapy in due time and, thus, prevent the development of severe complications.

EFFECT: higher accuracy and objectivity.

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Description

The invention relates to medicine, namely to pediatric endocrinology, and can be used for early diagnosis of diabetic neuropathy (DNP) in children suffering from type 1 diabetes mellitus (DM 1).

DM 1 - chronic hyperglycemia and glucosuria syndrome, caused by absolute insulin deficiency, leading to impaired all types of metabolism, vascular damage, neuropathy and pathological changes in various organs and tissues (M.I. Balabolkin, "Diabetology", M., 2000, p.155).

According to WHO, 2000, one of the most frequent, but least studied complications is DND, leading to early disability of patients with type 1 diabetes, which determines the relevance of studying this pathology. If earlier DNP was the prerogative of the adult population, then in recent years, the diagnosis of DND has been increasingly established already in childhood. Moreover, the prevalence of DNP varies widely enough - 5-90% (V.A. Peterkova, 1997; G.R. Galstyan, 2000). Discrepancies in the frequency of DND are observed due to the lack of standardized diagnostic criteria, the use of diagnostic methods with varying degrees of specificity, as well as the predominance of the subclinical stage in childhood. In this regard, the development of early diagnostic criteria for the development of DND in children is of undoubted interest.

Early diagnosis of DND in children, even at the preclinical stage, causes an urgent need in the daily practice of pediatric endocrinologists, since in childhood the initial manifestations of DND are more reversible (V.A. Peterkova et al. “Diagnosis, treatment and prevention of diabetic complications in children and adolescents ", M., 1997).

There are currently no clear diagnostic criteria for the initial manifestations of DND in children. The Neuropathic Dysfunctional Score (VAT) Scale used to determine the severity of symptoms of diabetic polyneuropathy includes a summary quantitative assessment of tactile, temperature, pain, vibration sensitivity and disturbance of tendon reflexes (Young MJ, Boulton AJM, Macleod AF, Williams DRR, Sonsken P. N. A. multicentre study of the prevalence of diabetic peripheral neuropathy in the United Kingdom hospital clinic population. Diabetologia, 1993; 36: 150-154). This technique is subjective in nature and its information content largely depends on the child’s age, emotional status, conditions (in a calm and relaxed state), skin condition (trophic changes, swelling), impossibility of a clear assessment of the child’s task.

An electromyographic study (EMG) is an objective and reproducible method for the state of peripheral nerves (EP Kasatkina. "Report on the evaluation of the effectiveness of the drug" Milgamma Dragee - 100 ", (company Verwag Pharma GmbH, Germany) in children and adolescents with diabetic peripheral polyneuropathy" , M., 1998). However, EMG changes in the form of a decrease in the conductivity of the nerve impulse of the sensory and motor peripheral nerves, a decrease in the amplitude of the neuromuscular induced potentials are non-specific and can be observed in other neuromuscular diseases. The negative aspects of this study are also age restrictions (only for children over 7 years old), it requires the use of sophisticated expensive equipment, which is used only in specialized diagnostic centers.

The prototype of the claimed invention selected a method for the diagnosis of early diabetic optical neuropathy according to the patent of the Russian Federation No. 2242008 from 10.12.2004.

The method includes determining in patients with diabetes mellitus the activity of alkaline phosphatase in the lacrimal fluid and if its activity is less than 110 U / L, then diabetic optical neuropathy is diagnosed. The disadvantages of this study are the specificity of the method of diabetic optical neuropathy, which allowed the authors to doubt the use of alkaline phosphatase activity in the lacrimal fluid as a marker for the development and progression of other diabetic complications; as well as the lack of research data in the presence of DNP. These disadvantages are eliminated in the claimed invention.

The objective of the invention is to develop a more accurate, objective method for the early diagnosis of DND in children.

This goal is achieved by the fact that in patients with type 1 diabetes mellitus with different duration of the disease, determine the level of ET-1 and bFGF in the blood serum and with a ratio of ET-1 to bFGF equal to 40 or higher, they diagnose a subclinical stage of DND, which allows prescribe preventive therapy in a timely manner.

The inventive method eliminates the presence of subjectivity, does not depend on the age, emotional status of the patient and allows the doctor with high confidence in serum levels of ET-1 and bFGF to draw a conclusion about the selection of a child suffering from type 1 diabetes mellitus at risk for the development of DNP, and appoint in a timely manner pathogenetic therapy. This measure will help reduce the risk of developing late vascular complications of diabetes, improve the health status and quality of life of children with diabetes 1. Vascular disorders play a leading role in the pathogenesis of diabetic neuropathy, in particular, changes in the functioning of vascular endothelial cells - endothelial dysfunction (Kondratiev, 1998). Since endothelial dysfunction occurs in most children and adolescents with diabetic microangiopathy (Rybkin A.I. et al., // Endothelial dysfunction in children and adolescents as an early sign of diabetic microangiopathy. - 3 All-Russian Diabetes Congress, abstracts. - M. - 2004, S.580-581), we found it possible to use ET-1 produced by endothelial cells and functionally conjugated bFGF as a marker for early diagnosis of DND.

Endothelin-1, the main vasoconstrictor peptide, is one of the most important regulators of the functional state of the endothelium. The formation of ET-1 occurs in endothelial cells both on the surface and inside the cell membrane of the endothelium, as well as on the surface of the underlying smooth muscle cells. ET-1 acts in a paracrine way on receptors of vascular smooth muscle, causing their contraction and growth, and in an autocrine / paracrine way on endothelial cells, causing releasing of vasorelaxants and growth-promoting factors.

Fibroblast growth factor (FGF) stimulates DNA synthesis and division of various cells of mesenchymal origin, including smooth muscle and vascular endothelial cells (Stoker M. and Gherardi E., 1991).

In the large FGF family of more than 20 species, bFGF seems to be the most studied. Moreover, bFGF is involved in the function of neuronal structures and secretory organs; it possesses chemotactic activity and stimulates the growth of new capillaries (Folkman J., Klagsbrun M., 1987), supports and stimulates the differentiation of cells of various neuronal types in vivo and in vitro (Saunders K.B., D′Amore P.A., 1991). bFGF is attributed an important role in nerve cell repair after brain injury (Kawamata T., Yamaguchi T., Shin-ya K., Hori T. (2001) Neur. Res., 23, 327-330). This growth factor also appears in the structure of pathogenetic mechanisms that describe ischemic and traumatic brain injuries (Gomazkov OA, Neurochemistry of ischemic and age-related brain diseases. Information-analytical publication. M., 2003).

At the same time, according to in vitro studies, FGF significantly affects endothelial function, participating in the control of smooth muscle cell tone. bFGF stimulate the development of collateral vessels and maintains endothelial integrity, which was revealed in a model of ischemic heart damage.

Thus, according to the presented pathogenetic mechanisms, physiologically active peptides, such as endothelium-1 and the main growth factor of fibroblasts, can contribute to the development and progression of DNP in children.

The stated theoretical assumptions were confirmed by our studies, which revealed an inversely proportional relationship between ET-1 and bFGF at the initial stages of the formation of DNP, which allowed us to derive a coefficient of the ratio of ET-1 to bFGF, with a value of 40 or higher, it is possible to diagnose the subclinical stage of DND in children with high precision.

The detailed implementation and performance of the proposed method are confirmed by the following specific examples.

Example 1. Child Polina M., born in 1988, medical history No. 7821, is observed in the children's endocrine department of RNIIAP for 1 year with type 1 diabetes mellitus, the course of the disease is severe.

No complaints at admission. Glycosylated hemoglobin - 6.6%. The total score on the VAT scale was 2 points (regarded as a variant of the norm) due to a moderate decrease in the Achilles reflex on the right and some decrease in temperature sensitivity in the distal parts of the lower extremities.

An EMG study revealed an electrophysiological characteristic of the electrical activity of the studied axonal type muscles; a decrease in electrical activity on the calf, anterior tibial muscles and the long extensor of the toes of both feet, a decrease in the frequency characteristics of the examined muscle groups.

The determination of the level of ET-1 in blood serum was performed using ELISA kits from DRG (USA).

1 ml of venous blood was taken from a patient in a glass tube with 1 ml of EDTA. Centrifuged for 15 minutes at 1,500 rpm. Then, ET was extracted from the sample. To 200 μl of the obtained plasma, 200 μl of an extracting solution consisting of acetone, hydrochloric acid and distilled water (in a ratio of 40: 1: 5) was added.

The contents of the test tube were centrifuged for 20 minutes at 2000 g in a cold centrifuge at + 2- + 8 ° С, the supernatant was poured into a plastic test tube. Dried at + 37 ° C in a ventilated thermostat for 4 hours.

200 μl of sample dilution buffer was added to the resulting pellet. This sample was used directly in the experiment.

Before the analysis, the required number of strips was selected, given that for each determination, 6 holes are needed to construct a calibration curve and 1 well for each test sample of pre-prepared serum.

The required number of strips was inserted into the holder. The wells were washed twice with phosphate-saline wash buffer.

Then, 100 μl of prepared standard samples with known ET concentration were added into the first 6 wells, and 100 μl of test samples into the remaining wells. The contents were mixed by shaking on a shaker for 1 minute, the wells were sealed with adhesive tape and incubated for 1 hour at + 37 ° C in an incubator.

At the end of the incubation, the wells were washed 7 times with phosphate-buffered saline, dried at the end of the strips by tapping on filter paper.

100 μl of conjugate solution was added to all wells and incubated for 30 minutes in a refrigerator at + 4 ° C. Wash the wells 9 times with phosphate-buffered saline.

100 μl of substrate solution was added to all wells and incubated for 30 minutes in an incubator.

The reaction was stopped by adding 100 μl of stopping solution to each well.

Registration and calculation of the results was carried out on a Multiscan photometer (Denmark) at 450 nm.

Numerical values of the concentration of standard samples were introduced into the computer software of the device and, when calculated, the numerical values of the desired concentrations of the test samples in PCG / ml were obtained.

The duration of the analysis is 6.5 hours. The sensitivity of the method is 0.14 pg / ml, specificity is 99.4%.

Determination of bFGF was carried out by the method of solid-phase ELISA kits from the company CYTIMMUNE (USA).

1 ml of venous blood was taken from a patient in a glass tube with 1 ml of EDTA. Centrifuged for 15 minutes at 1,500 rpm.

Before analysis, all components were brought to room temperature and prepared reagent solutions, serial dilutions of standards according to the instructions for the set.

100 μl of standard solutions were added to the corresponding wells of the plate coated with goat anti-rabbit antibodies. The experimental serum samples were pre-diluted as follows: 100 μl of sample + 100 μl of solvent II + 200 μg of solvent I were mixed in a test tube. 100 μl of the finished sample was added to the corresponding well.

25 μl of diluted rabbit anti-human anti-bFGF polyclonal antibodies were poured into each well. The plate was covered with film and incubated for 3 hours at room temperature. The film was then carefully removed and 25 μl of conjugate was added to each well and incubated for 30 minutes at room temperature.

Then the tablet was washed five times on the head with wash buffer. 50 μl of diluted alkaline phosphatase conjugate streptavidin conjugate was added to all wells of the plate. The plate was covered and incubated for 30 minutes at room temperature. The washing procedure was repeated.

200 μl of the prepared staining solution was added to each well, incubated for 15 minutes.

The color reaction was stopped by adding 50 μl of stop reagent to each well.

The results were calculated on a Victor multifunctional photometer (Wallas, Finland) at 492 nm.

Numerical values of the concentration of standard samples were introduced into the computer software of the device and, when calculated, the numerical values of the desired concentrations of the test samples in ng / ml were obtained. The resulting concentration values were multiplied by a dilution factor of 4.

The duration of the analysis is 4.5 hours. The sensitivity of the method is 0.488 ng / ml.

In this child, the level of ET-1 in blood serum was 10 pcg / ml, bFGF - 0.25 ng / ml. The ratio of ET-1 to bFGF is 40. Further clinical follow-up in dynamics, with repeated planned admission after 10 months, revealed 2 points on the VAT scale, negative dynamics according to EMG research mainly from the long extensor of the toes of both feet, in the form of a moderate appearance severe polyphasia. Thus, the above example indicates the identification of the subclinical stage of DND and the need for preventive therapy.

It should be noted that the established coefficient assumes that the studied indicators should be in the same units according to the SI system, that is, either in ng / ml or in PCG / ml. Since the numerical values of the desired concentrations of the ET-1 test samples were obtained in PCG / ml, and bFGF in ng / ml, the coefficient ET-1 / bFGF should look like 40 * 10 ^-3 and higher. However, given that in our case, the devices give numerical values precisely in these units of measurement, to simplify the understanding and ease of practical use, we propose to use this coefficient as a value of 40 or higher.

Example 2. The child Angelina O., born 1992, a medical history No. 1868, is observed in the children's endocrine department of RNIIAP with the diagnoses of Type 1 diabetes mellitus, disease duration 1 year, severe course.

No complaints. Glycosylated hemoglobin - 6.8%. When examining the neurological status, there was a slight decrease in knee and Achilles reflexes on both sides, a decrease in vibration sensitivity to 6 points on the first toe of both feet, with normal parameters of the other types of sensitivity (tactile, temperature, pain). The scale of VAT was 2 points, which is regarded as a variant of the norm.

EMG research data: electrophysiological characteristics of the electrical activity of the studied axonal muscles; frequency characteristics and electrical activity of muscles within physiological shifts.

The level of ET-1 in serum was 15.5 pg / ml, bFGF - 0.4 ng / ml. Therefore, the ratio of ET-1 to bFGF is 38.8.

In dynamics, after 6 months with planned admission, no negative dynamics were revealed during clinical and functional examination.

Thus, from this example it follows that the ratio of ET-1 to bFGF below 40 indicates the absence of pathological changes in the nervous system with this pathology and does not require the appointment of preventive therapy.

The inventive method was examined 79 children suffering from type I diabetes mellitus with varying duration of the disease, observed in the children's endocrine department of RNIIAP. The coefficient ET-1 / bFGF of 40 and higher was observed in 39 children, which made it possible to diagnose the subclinical stage of the development of DAYs. A significant part of these children showed moderate pathological changes in EMG, mainly involving distal lower extremities. The scale of VAT ranged from 0 to 5 points. In 37 children, this ratio was below 40 and there were no clinical-functional signs of damage to the peripheral nervous system. Three children had an ET-1 / bFGF coefficient below 40, although according to the EMG study they had impaired neuro-reflex conduction.

The data obtained indicate the involvement of vascular factors, in particular ET-1 and bFGF, in the development and progression of DNP, this is especially evident in the subclinical stage of development of this complication.

Thus, the study of the ratio of ET-1 to bFGF in the blood of children suffering from type I diabetes mellitus showed that the accuracy of the proposed method is 96.2%.

Based on the foregoing, the claimed method for the early diagnosis of DND in children with different duration of the disease compared with existing has the following advantages:

1. The method allows for the early diagnosis of DNP in order to prescribe timely preventive treatment.

2. This method is objective and does not depend on the age and emotional status of the child.

3. The proposed method has a sufficiently high accuracy, is simple to implement and will be widely used in endocrinological practice.

Claims (1)

A method for the early diagnosis of diabetic neuropathy in children by examining biological fluid, characterized in that in the patient’s peripheral blood, the level of endothelin-1 in PCG / ml and the main fibroblast growth factor (bFGF) in ng / ml and at a ratio of endothelin-1 to bFGF equal to 40 and above diagnose a subclinical stage of diabetic neuropathy.