RU2606591C1 - Method of early pre-clinical diagnosis of parkinson's disease - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to medicine, namely to neurology, and can be used for diagnosis of Parkinson's disease at pre-clinical stage. Essence of method is that by method of HPLC following concentrations are determined in blood plasma: noradrenaline (NA), dopamine (DA), 3,4-dioxyphenylalanine (L-DOPA), 3,4-dioxyphenylacetic acid (DOPAA), comparison of concentration values with concentration preliminarily measured in healthy people. At reducing concentration by 57–67 % shaking form is diagnosed. At reducing concentration of noradrenaline by 80–90 % rigid form is diagnosed. At reducing concentration of DA in blood plasma by 69–79 % rigid form is diagnosed. At reducing concentration of L-DOPA by 57–67 % shaking form of Parkinson's disease is diagnosed. At reduction of L-DOPA by 78–88 % rigid form is diagnosed. At reducing concentration of DOPAA by 69–79 % rigid form of Parkinson's disease is diagnosed.

EFFECT: use of method enables high-accuracy diagnosing of various forms of Parkinson's disease at pre-clinical stage.

1 cl, 7 dwg

Description

The invention relates to medicine, in particular to neurology, and can be used to diagnose Parkinson's disease (PD) at an early preclinical stage.

A known method for assessing the functional state of the central nervous system, which, like the proposed method, can be used for early diagnosis of chronic diseases of the central nervous system (RF Patent No. 2039524). The essence of the technique is that electroencephalograms (EEGs) are recorded, certain rhythms are distinguished, followed by mathematical processing and assessment of brain activity.

The disadvantages are that EEG provides an integral characteristic of brain activity and from it, even with the help of modern mathematical approaches, it is extremely difficult to isolate diagnostic markers corresponding to Parkinson's disease. The consequence of this is the low accuracy of diagnosis, which does not allow the use of adequate methods of treatment. In addition, this method does not allow to differentiate between two main clinical forms of Parkinson's disease - rigid and tremulous.

There is also a method of differential diagnosis of neurodegenerative diseases in patients with manifestations of dementia, including Parkinson's disease. The method consists in conducting studies of evoked potentials of P300 using verbal and non-verbal stimuli. Further, in the presence of uniformly reduced values of the amplitudes of the potential P300 in the frontal and parietal leads, a conclusion is made about dementia of the cortical type (RF Patent No. 2228708). However, this method is not used to diagnose the disease at the preclinical stage, and therefore does not allow for preventive treatment. In addition, this technique is very time-consuming.

Closest to the proposed invention is a method for the early diagnosis of Parkinson's disease (application No. WO 2011152699 [A2]), which consists in a biochemical assessment of the level of dopamine-quinone - the product of spontaneous oxidation of dopamine - in the blood plasma of the test patient. In contrast to the methods known from the patents of the Russian Federation No. 2039524; 2039524, this method is reproducible and allows for the early diagnosis of Parkinson's disease.

The disadvantages of this method include the low accuracy of diagnosis, since in the study of blood plasma only dopamine-quinone, a product of spontaneous oxidation of dopamine (DA), is evaluated, and not dopamine or other catecholamines themselves, for example, norepinephrine (HA), and their functionally significant metabolites - 3,4-dioxiphenylalanine (L-DOPA) and dioxiphenylacetic acid (DOPUK). In addition, when developing this method, only the blood plasma of patients with diagnosed Parkinson's disease was analyzed, i.e. at the clinical stage, which does not allow us to unambiguously judge the reproducibility of the results obtained for the preclinical stage of the disease.

The task that the invention solves is a timely, readily available early preclinical diagnosis of Parkinson's disease with the possibility of differentiating two main forms of Parkinson's disease - rigid and tremulous. The proposed diagnosis is based on a comprehensive analysis of blood plasma composition in patients at an early clinical stage.

The problem is solved by conducting a diagnostic test by evaluating plasma biomarkers in blood plasma, which are direct indicators of pathological changes in the metabolism of catecholamines in the whole organism.

The creation of the test is based on the notion that the first clinical manifestations of impaired motor function (motor symptoms of Parkinson's disease) appear only after the degeneration of most dopaminergic neurons of the nigrostriatal system and the striatum denervation caused by this. At the same time, in the process of neuronal degeneration, compensatory mechanisms of brain plasticity are activated, which prevent the onset of clinical motor symptoms for decades, and therefore exclude the possibility of early symptomatic diagnosis of the disease. However, in recent years, it has been shown that Parkinson's disease is a systemic disease, and non-motor manifestations of the pathological process (impaired functioning of the heart, gastrointestinal tract, olfactory receptors) appear much earlier than impaired motor function. Catecholamines and metabolites easily diffuse into the general circulation system from catecholaminergic neurons of the peripheral nervous system, and in Parkinson's disease, in conditions of violation of the blood-brain barrier, also from neurons of the central nervous system. It should be expected that the degradation of central and peripheral catecholaminergic neurons in patients will lead to a change in the content of catecholamines and metabolites in the blood plasma.

The essence of the invention is to conduct a diagnostic test by comparing the composition of blood plasma samples obtained from the test patient with the corresponding indicators obtained previously from healthy people representing the control group.

When carrying out the proposed diagnostic test, a sample of blood plasma is taken from the subject and a biochemical analysis of these samples is carried out using high performance liquid chromatography with electrochemical detection. In this case, instead of measuring the concentration of dopamine-quinone - the product of the spontaneous oxidation of dopamine - the concentration of the main catecholamines: norepinephrine (HA) and dopamine (DA), as well as their functionally significant metabolites: L-DOPA and DOPUK, is measured. The measured concentrations of the substances in the blood plasma of the test subject are then compared with parameters such as the concentration of norepinephrine (HA), dopamine (DA), L-DOPA and DOPUK, previously determined in healthy people. In the case of significant differences in the concentrations of the substances being determined in the test person compared with the values in the norm, a conclusion is drawn about the inclusion of the examined in the risk group for Parkinson's disease, as well as about the form of this disease - tremulous or rigid.

Conducting a comprehensive comparative analysis makes it possible to make a diagnosis at the preclinical stage of Parkinson's disease with the definition of the form of the disease in order to use adequate methods of treatment. A comprehensive comparative analysis shows that a characteristic feature of Parkinson's disease in the tested patients is a significant decrease in the concentration of norepinephrine (HA), dopamine (DA), L-DOPA and DOPUK in blood plasma in patients with a rigid form of Parkinson's disease compared with normal values, and a significant decrease in the concentration of norepinephrine (HA) and L-DOPA in blood plasma in patients with a tremulous form of Parkinson's disease compared to normal values.

The list of drawings and their brief description.

FIG. 1 - The concentration of norepinephrine (ON) in blood plasma in untreated patients at an early stage of a tremulous or rigid form of Parkinson's disease.

FIG. 2 - The concentration of dopamine (DA) in blood plasma in untreated patients at an early stage of a tremulous or rigid form of Parkinson's disease.

FIG. 3 - The concentration of 3,4-dioxiphenylalanine (L-DOPA) in blood plasma in untreated patients at an early stage of a tremulous or rigid form of Parkinson's disease.

FIG. 4 - The concentration of 3,4-dioxiphenylacetic acid (DOPUK) in blood plasma in untreated patients at an early stage of a tremulous or rigid form of Parkinson's disease.

FIG. 5 - The concentration of dopamine (DA) in blood plasma in mice in the control and on models of preclinical and early clinical stages of Parkinson's disease.

FIG. 6 - Concentration of 3,4-dioxiphenylalanine (L-DOPA) in blood plasma in mice in the control and in preclinical and early clinical models of Parkinson's disease.

FIG. 7 - The concentration of 3,4-dioxiphenylacetic acid (DOPUK) in the blood plasma in mice in the control and in models of the preclinical and early clinical stages of Parkinson's disease.

The proposed method for preclinical diagnosis of Parkinson's disease is as follows. Blood for the determination of catecholamines is taken from the subjects in the supine position. Blood from the cubital vein (about 10 ml) is collected in an ice-cooled plastic tube containing 200 μl of 5% EDTA with 100 μl of 10% potassium metabisulfite, carefully mixed and immediately centrifuged at a speed of 2500 rpm at 4 ° C for 20 minutes. The resulting plasma (supernatant) is taken, frozen and stored until the start of biochemical studies (no more than 60 days) at -70 ° C.

To determine the content of catecholamines and their metabolites, a highly sensitive and selective method of high performance liquid chromatography with electrochemical detection (HPLC-ED) is used. The analytes are separated by reverse phase chromatography on columns with an S-18 sorbent (4 × 100 mm, particle diameter 3 μm) at a temperature of 23 ° C under constant pressure in the mobile phase of the following composition: 0.1 M citrate-phosphate buffer, 0.3 mm sodium octanesulfate, 0.1 mm EDTA and 8% acetonitrile, pH 2.2. The potential of the glassy carbon electrode of the electrochemical detector is +0.85 V, the flow rate is 1 ml / min, and the column temperature is 30 ° C. The concentrations of norepinephrine (HA), adrenaline, dopamine (DA), L-DOPA and DOPUK are calculated from the areas of chromatographic peaks using the internal standard method.

Each of the results of a blood test: concentrations of norepinephrine (HA), dopamine (DA), L-DOPA and DOPUK is an independent biomarker that allows you to diagnose Parkinson's disease and determine both or one of the forms of the disease. A decrease in plasma concentration of norepinephrine (HA) by 57-67% is a marker of the tremulous form of Parkinson's disease. A decrease in the concentration of norepinephrine (HA) by 80-90% is a marker of the rigid form of Parkinson's disease. A 69-79% decrease in plasma dopamine (DA) concentration in blood plasma is also a marker of the rigid form of Parkinson's disease. A decrease in the concentration of L-DOPA by 57-67% is a marker of the tremulous form of Parkinson's disease, and by 78-88% it is a marker of the rigid form. A 69-79% decrease in the concentration of DOPUK is a marker of the rigid form of Parkinson's disease. The greatest accuracy of the diagnosis is achieved using a comprehensive assessment of all 4 indicators.

In doubtful cases, the diagnosis is clarified using positron emission tomography.

The following examples illustrate this method.

The study involved 20 untreated patients with newly diagnosed early clinical stage of Parkinson's disease during a neurological examination, including 10 people with a tremulous form (7 women / 3 men, average age 60.7 years), 10 - with a rigid one (4 women / 6 men, average age 57.8 years). The stages of the development of the disease on a five-point Hen and Yar scale for patients with a tremulous form of Parkinson's disease were on average 1.5, and for patients with a rigid form - 1.65. The control group included 14 people (12 women / 2 men, average age 55.36 years).

The study was carried out on the basis of the Hospital for War Veterans. The selection of patients for the study was carried out by employees of the Center for extrapyramidal pathology in Kazan. To conduct research, permission was obtained from the ethics committee of the Ministry of Health of the Republic of Tatarstan. All subjects signed informed consent to participate in the study.

The concentration of norepinephrine (HA) in the blood plasma in patients with both tremulous and rigid form was much lower than in the control group - on average, by 62% and 84%, respectively (Fig. 1). Moreover, in patients with a rigid form of Parkinson's disease, the concentration of norepinephrine (HA) is statistically significantly lower than in patients with a tremulous form (Fig. 1). The concentration of dopamine (DA) in the blood of patients with a rigid form of Parkinson's disease was reduced by 74% on average compared with the control (Fig. 2). Despite the fact that the average concentration of dopamine (DA) in the blood in patients with a tremulous form is higher than that of the subjects in the control, there are no significant differences due to large individual differences in patients with a tremulous form. However, the concentration of dopamine (DA) in patients with a tremulous form is many times higher than in patients with a rigid form (Fig. 2). The concentration of L-DOPA was reduced compared with the control for both forms of Parkinson's disease, however, with a rigid form to a greater extent than with a tremulous form, respectively, by 83% and 62% (Fig. 3). The concentration of DOPUK, a product of dopamine degradation (DA), was 75% lower in patients with a rigid form of Parkinson's disease than in the control group and in patients with a tremulous form. Moreover, the concentration of DOPUK in patients with a tremulous form was the same as in the control group (Fig. 4).

Thus, clinical studies showed the presence of significant changes (decreases) in the concentration of catecholamines and their metabolites in blood plasma in untreated patients with tremulous and rigid forms of Parkinson's disease compared with each other and with the norm.

The complex of medical equipment during the diagnostic test consisted of a cooling centrifuge for separating blood plasma, a low-temperature refrigerator for storing frozen samples and a liquid chromatograph with an electrochemical detector for measuring the concentration of catecholamines.

Clinical studies were preceded by experimental animal models of Parkinson's disease. Parkinson's disease was simulated by introducing 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), the precursor of specific neurotoxin of catecholaminergic neurons, which in the brain turns into MPP + - directly neurotoxin. In the experiments, 27 male C57BL / 6N mice were used, 2-2.5 months old and weighing 22-26 g. The model of the preclinical (n = 9) stage of Parkinson's disease was reproduced by double subcutaneous administration of MPTP (hereinafter all reagents - Sigma, USA), in a single dose of 8 mg / kg, a model of the early clinical stage of Parkinson's disease (n = 9) - four-time administration of MPTP in a single dose of 10 mg / kg. In both cases, the interval between injections was 2 hours. Control animals (n = 9) were injected with saline according to a similar scheme. After 2 weeks, the animals were decapitated with a preliminary dislocation of the cervical vertebrae and blood was collected in test tubes to which 5% EDTA and 10% sodium metabisulfite were added. Then, the blood was centrifuged for 10 min at 400 g and 4 ° C, a solution of the internal standard 3,4-dihydroxybenzylamine (DHBA) was added to the selected plasma to a final concentration of 25 pmol / ml, after which it was again centrifuged for 10 minutes at 2000 g and 4 ° C. The selected supernatant was frozen in liquid nitrogen and stored at -70 ° C until further measurement of catecholamines. Blood plasma catecholamines were deposited on alumina, and then their concentration was measured by reverse phase high performance liquid chromatography with electrochemical detection using an LC-20ADsp chromatograph (Shimadzu, Japan) and a Decade II detector (Antec, Netherlands). Separation was carried out on a Reprosil-Pur C18, 3 μm 4 × 100 mm column (Dr. Maisch, Germany) in the mobile phase of the following composition: 0.1 M citrate-phosphate buffer, 0.3 mm sodium octanesulfate, 0.1 mm EDTA and 8% acetonitrile, pH 2.2. The potential of the glassy carbon electrode was +0.85 V, the flow rate was 1 ml / min, and the column temperature was 30 ° C. The concentrations of norepinephrine (HA), adrenaline, dopamine (DA), L-DOPA and DOPUK were calculated by the areas of chromatographic peaks using the internal standard method.

In the model of the preclinical stage of Parkinson's disease, the plasma dopamine concentration was reduced by 50% (Fig. 5), and L-DOPA by 40% compared with the control (Fig. 6), while in the model of the early clinical stage, the concentration L-DOPA was reduced by 51% (Fig. 6), the concentration of dopamine (YES) was not reduced (Fig. 5), and the concentration of DOPAK decreased by 68% (Fig. 7). The decrease in the level of dopamine metabolites (DA) found in blood plasma in a model of the early clinical stage of Parkinson's disease is in good agreement with the results obtained from blood analysis in untreated patients suffering from a rigid form of Parkinson's disease. Given that the changes found in the models of preclinical and early stages of Parkinson's disease are not completely consistent with each other, and not all changes observed in the blood in patients at the early clinical stage of Parkinson's disease can serve as potential biomarkers at the preclinical stage of the disease, this increases the significance of the proposed approach to a comprehensive assessment of a whole group of physiologically related markers.

Thus, experimental models of the preclinical and early clinical stages of Parkinson's disease demonstrated a significant decrease in the level of catecholamines and their metabolites, which are largely consistent with the changes shown in the analysis of blood in untreated patients.

The proposed method for the early preclinical diagnosis of Parkinson's disease using plasma concentrations of catecholamines and their metabolites as biomarkers can be widely used in the medical examination of the population, and is currently one of the most reliable. Its use for the first time will allow you to identify the disease at the preclinical stage according to the indicators used, and therefore, apply preventive treatment to stop the death of neurons and prolong the preclinical stage for an unlimited time.

Claims (1)

A method for the early preclinical diagnosis of Parkinson's disease, including the determination in blood plasma by HPLC of the concentration of norepinephrine (HA), dopamine (DA), 3,4-dioxiphenioalanine (L-DOPA), 3.4-dioxiphenylacetic acid (DOPUK), comparing the concentration of these indicators with the concentration previously measured in healthy people and with a decrease in the concentration of HA by 57-67% is diagnosed with a tremulous form; with a decrease in the concentration of noradrenaline ON by 80-90%, a rigid form is diagnosed; with a decrease in the concentration of DA in blood plasma by 69-79%, a rigid form is diagnosed; with a decrease in the concentration of L-DOPA by 57-67%, a tremulous form is diagnosed; with a decrease in L-DOPA by 78-88%, a rigid form is diagnosed; with a decrease in the concentration of DOPUK by 69-79%, a rigid form of Parkinson's disease is diagnosed.

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