The method of determining the state of the cardiovascular and respiratory systems based on the analysis of nitric oxide in exhaled air

 

(57) Abstract:

The method can be used in medicine, namely in cardiopulmonary in the study of the cardiovascular system and the respiratory system. The patient is instructed to exhale for 5-7 min through a U-shaped glass tube, the knee of which is immersed in the coolant. Thus receive the exhaled breath condensate. In the analyzed sample of condensate containing nitrites and nitrates, carry out the restoration of nitrates to nitrites. Spectrophotometric in the region of 540 nm to determine the nitrite content. The concentration of nitric oxide is equimolar installed the concentration of nitrite. About pathological condition systems are judged by an increase or decrease in the values obtained from normal component of 3.1 - 5.1 µm. The method provides high accuracy of determination of nitric oxide, long-term storage of samples obtained. 3 C.p. f-crystals.

The invention relates to medicine, namely to cardiopulmonary, and can be used to investigate the state of the cardiovascular system and the respiratory system.

At the present time to assess the state of the cardiovascular system and yelling all sorts of tools, when this analysis is subjected directly exhaled air in the gaseous state and its condensate. In each of the methods set their own evaluation criteria to determine the status of the specified body systems.

The known method of differential diagnostics of diseases of the bronchopulmonary system by biochemical studies of biological fluid, in exhaled breath condensate determine the relative content of fatty acids (C16:0and C19:0calculate the coefficient of C16:0/C19:0and when certain values are installing one or another disease (RU 95109305, 1996, G 01 N 33/497).

Obtained by carrying out the method the figures indicate the presence of disease, but can not give a picture of the processes occurring in the earlier stages.

There is a method of determining the functional status bronchodilating apparatus by holding bodyplethysmography, which conducted a study of indicators of biomechanics, calculate the degree of broncho-obstructive syndrome (BOS) by a certain formula, and with increasing quantitative indicators barefoot more than one quantitative indication of the action, physical activity, posterski, inhalation solution of histamine, salbutamol conduct a study of indicators of the biomechanics of breathing and when the change rate is more than 20% from baseline determine reaction as positive (EN, 2092846, 1997, G 01 N 33/483).

There is also known a method of assessing the functioning of the cardiorespiratory system by using a special device that allows to obtain simultaneously information about cardiac and respiratory interconnection with the analysis of the relationship of these systems in the process of inhalation and exhalation. In the implementation of the method uses a complex system of expensive equipment. During examination of the patient parallel to process and analyze kardiointervalogrammy continuously during inhalation and exhalation of the patient, as well as analyze different portions of the air flowing in, for example, from the dead space of the lungs or of the small bronchi or alveoli, carrying the information about the gas composition and the quantity of this air. This information, in turn, is fed to the integrator, where it is analyzed depending on specified programs (EN 2098012, 1997, A 61 B 5/0205).

Known methods are time consuming and require the use of complex dorogostojascaja while exploring the state of the respiratory system have a way of determining the content of NO in exhaled air using chemiluminescence, which allows you to capture the change pathological processes and physiological effects.

The closest analogue of the invention is a method of determining the state of the respiratory tract, namely, that the subject exhales air in a special device, and then a portion analyzed by chemiluminescence and inflated quantitative content of NO in the analyzed sample compared to the control set, the presence of inflammatory diseases or their risk (PCT 95/02181, 19.01.95, G 01 N 33/00).

The method is complex, involves the use of expensive stationary equipment does not provide the possibility of deferred analysis and research in small clinics ambulance and in the field.

The objective of the invention is to create a new way with high accuracy and quickly determine the content of nitric oxide (NO) in exhaled air using available equipment that speeds up the process of diagnosis, the invention allows for delayed measurement.

The problem is solved the stated method of determining sostojanija with the subsequent establishment of pathological conditions of the systems with reliable change its number in the analyzed sample compared to the norm.

The difference of the invention is that the analysis is subjected to the exhaled breath condensate, containing stable end the NO metabolites nitrite and nitrate, having first carried out the restoration in the analyzed sample of condensate nitrate to nitrite, followed by spectrophotometric determination at 540 nm of their content and determine the concentration of nitric oxide equimolar installed the concentration of nitrite in the analyzed sample, and of the pathological state of the system is judged to increase or decrease the values obtained from normal component of 3.1-5.1 µm nitrite in the condensation of the air.

The technical result is to increase the reliability of the obtained results are attributable to the following: passing through the respiratory tract, nitric oxide is partially oxidized to stable metabolites nitrite and nitrate. In known methods, based on the determination of NO in exhaled air, part NO oxidized, is not registered by the detector, which distorts the true results, and as a result, the accuracy and reliability of analysis is reduced.

In the proposed method registration NO is more fully - end metabolites contained in the condensate is ü the content of NO in the exhaled air at nanomolar concentrations with an accuracy of 0.1 nm, that is not below the threshold of sensitivity and accuracy of the chemiluminescent gas analysis. Unlike gas analysis the proposed method allows to estimate the average rate of NO production, i.e., reduces the impact of the amendment on ventilation and accordingly is not required to use special equipment to assess ventilation.

The method is as follows.

The patient is instructed to exhale for 5-7 min through the U-shaped glass tube, the knee of which is immersed in the coolant. Thus receive the exhaled breath condensate. For analysis take an aliquot volume of 720 ml. Make it into a reactor, add it to handle 80 ál buffer 0.5 M NH4OH PH 9.0 in the ratio of the buffer-sample 1:9. This was followed by incubation of the sample in the reactor for 5 to 30 minutes From the reactor take an aliquot of 400 μl and add 400 ál of reagent Griss. The recovery of the reactor is carried out sequentially using 10 ml of 0.1 n HCl and 10 ml of CuSO4.

Next, the resulting solution was incubated in the reactor Nitralyzer for 10 minutes at room temperature. While the core element of the reactor containing the cadmium-treated copper, when immersed in enapentertainment in the region of 540 nm. To determine the content of NO using calibration curve for the absorbance of standard solutions of NaNO2in the concentration range from 0.5 to 50 μm. For this calibration curve to determine the concentration of NaNO2for a given optical density of the sample. The number of NO equimolar the sum of nitrite and nitrate contained in the analyzed sample of exhaled breath condensate.

On the basis of numerous authors conducted invention of studies have established the norm content of NO in the exhaled breath condensate. Norm values are the NO concentration in the range from 3.1 to 5.1 μm.

The following examples illustrate the invention.

Example 1. Using the proposed method for estimation of NO production in healthy young smokers and non-Smoking men and a degree of influence of tobacco smoke on the functional state of the vascular endothelium. It is shown that NO production in non-smokers was 4,70,4 μm, whereas smokers - 2,1-0.1 ám, i.e., was significantly reduced. In terms of condensate of exhaled air is quantitatively corresponds to the literature data that Smoking causes a decrease in the concentration of NO in exhaled air, and imeeji ways reduced due to the toxic effects of tobacco smoke in NO-producing cells and deterioration of gas exchange.

Example 2. Using the proposed method for estimation of NO production in young healthy men at rest and after exercise (100 sit-UPS). The NO production after exercise significantly increased on average by 59% (from 3.7+0.2 to 4,20,4 μm). In terms of condensate of exhaled air is quantitatively consistent with the literature data that during exercise, the concentration of NO in exhaled air, adjusted for ventilation increases, and this increase is according to various sources from 35 to 100%. Indicators of NO production before and after exercise is normal.

Example 3. Using the proposed method for estimation of NO production in young healthy men and the volunteers of the same age suffering from acute respiratory disease (inflammation of the upper respiratory tract). Respiratory disease caused an increase in NO production to 7,40,3 μm. In terms of condensate of exhaled air is quantitatively consistent with the literature data that the inflammatory process caused by bacteria in the upper respiratory tract, significantly increases the concentration of NO in exhaled air.

Example 4. We studied the possibility of delayed evaluation measurement sodern the content of NO in the aliquot samples immediately after collection and then the content of NO was measured repeatedly in other aliquot of the same samples stored for 48 hours at room temperature. The NO concentrations before and after storage of the samples, respectively 17,7+3,2 and 17.9+4,0 ám.

Thus, the method allows high-precision measurement of the content of NO in the exhaled air and to register changes in the physiological effects and pathological processes. Thus the proposed method allows for delayed measurements and does not require special conditions of storage and/or transportation of samples.

Advantages definition of NO in the exhaled breath condensate of the proposed method:

1. Higher accuracy of the analysis by identifying NO for the total number of nitrites and nitrates.

2. The possibility of introducing additional measurement of pulmonary clearance of NO, which is calculated as the ratio of the amount of NO in the exhaled air to the concentration of NO in plasma, which is measured simultaneously by the same method at the beginning of the collection period condensate air. Definition of pulmonary clearance of NO may serve as one measure of the effectiveness of inhaled NO, which is widely used in some diseases (RES is t in deproteinization samples.

4. Long-term storage of samples obtained condensed air.

1. The method of determining the state of the cardiovascular and respiratory systems based on the analysis of nitric oxide in exhaled air, including the analysis of exhaled air on the content of nitric oxide with subsequent establishment of pathological conditions of the systems with reliable change its number in the analyzed sample compared to the norm, characterized in that the analysis is subjected to the exhaled breath condensate containing nitrites and nitrates, having first carried out the restoration in the analyzed sample of condensate nitrate to nitrite, followed by the definition of their content and determine the concentration of nitric oxide equimolar installed the concentration of nitrite in the analyzed sample, and about pathological condition systems are judged by an increase or decrease in the values obtained from the norm.

2. The method according to p. 1, characterized in that as the norm take concentration within 3,1 - 5,1 mmol.

3. The method according to p. 1, characterized in that the determination of condensate nitrite carried out by spectrophotometric method.

4. The method according to p. nm.

 

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