Method for determining individual's sensitivity to oxidative stress

FIELD: medicine.

SUBSTANCE: for the purpose of determining the individual's sensitivity to an oxidative stress, a peripheral blood neutrophil suspension of a specific individual is analysed for an intensity of initial chemoluminescence (IC) and an intensity of chemoluminescence (HC) after heating in test tubes on a water bath for 30-60 seconds at temperature 42°C by pulse per minute counting. If the derived IC/HC value is less than 3, a person being tested is referred to stress susceptible.

EFFECT: using the method provides increasing the clinical effectiveness in various pathologies, promotes the well-timed preventive and rehabilitation measures aiming at avoiding the labour loss and selecting the stress-resistant individual for the most hazardous production jobs.

2 cl, 5 tbl, 5 dwg, 3 ex

The invention relates to medicine, more specifically to the section of internal medicine.

Currently, most researchers pay attention to the implementation of the stress response at the level of individual cells, capable of producing damaging the body reactive oxygen species (ROS). To ROS include oxygen atoms, bearing on the outer shell of the excess electron and therefore has a negative charge: the superoxide anion ($$O_2^{-}$$), hydroxyl radical (OH^-), and the radical nitrogen dioxide(NO₂), hydrogen peroxide (H₂O₂) and other Excessive concentrations of ROS damage to proteins, lipids and DNA, leading to cell aging and the emergence of the so-called free-radical diseases such as atherosclerosis, essential hypertension, senile dementia of the Alzheimer's type, Parkinson's disease, cancer and other

Early detection sensitivity of the individual to oxidative stress will allow time to start preventive measures aimed at preventing the development of these diseases.

In turn, these diseases are the body's stressors and even more enhance cellular oxidative or oxidative stress with further aggrava what their pathological process (B. Uttara et al. Oxidative Stress and Neurodegenerative Diseases: A Review of Upstream and Downstream Antioxidant Therapeutic Options. Curr. Neuropharmacology.2009, 7 (1): 65-74).

As shown by clinical and experimental data, the body's sensitivity to damaging stressors external environment largely depends on the function of the brain and the cortical layer of the adrenal gland, under the control of the hypothalamic-pituitary system.

Addison disease-Birman insufficient adrenal function in humans is accompanied by very high sensitivity to different stressors, including a number of infections, and can be realized at the level of phagocytic cells with increased synthesis of ROS. A similar pattern is observed in adrenalectomized animals.

There is a method of assessing levels of emotional stress (K.V. Sudakov "Social and biological aspects of emotional stress: ways to protect themselves from unintended consequences", journal of the international Academy of Sciences, 2006, No. 1, p.8-14). It includes instrument registered the most informative of the physiological indicators (heart rate, breathing frequency, the values of systolic and diastolic blood pressure, indicators electromyograms, galvanic skin response and other). In the presented way of assessing levels of emotional stress is not taken into account cellular oxidative stress, revolade to the development of such pathologies in humans.

As a prototype we have chosen a method of determining individual sensitivity to hyperbaric oxygenation (inventor's certificate SU # 1246935). This method includes effects on lymphocytes in human peripheral blood increased partial pressure of oxygen or hyperbaric oxygenation. High pressure oxygen is cell non-physiological factor or stressor. As a result of such a stressor in lymphocytes specific individual happen morphological changes of chromosome aberrations, deletions)that reflect individual sensitivity to stress. The prototype has the following disadvantages:

- complexity as it involves the cultivation of lymphocytes, using equipment to create high oxygen pressure, assessment of changes in the structure of chromosomes of lymphocytes;

- insufficient accuracy of determining individual sensitivity to stress exposure, as cells do not possess phagocytic activity, which does not produce exogenous forms of ROS and may not participate directly in the development of oxidative stress.

We were set a task: to develop a simple reliable method of determining individual sensitivity of the person to oxidative stress.

The technical result is clochette to provide easy and accurate determination of a person's sensitivity to oxidative stress due to the evaluation of ROS production by neutrophils in response chemiluminescence.

We found that the registration and comparison of the intensity of chemiluminescence, for example, induced opsonization simhasanam Lomonosovskiy chemiluminescence stressed and descression neutrophils peripheral blood of individual assessment of the magnitude of the coefficient chemiluminescence, i.e. the ratio of the number of pulses per minute descression of neutrophils to the number of pulses per minute stressed neutrophils, allows to reliably determine the sensitivity studied in oxidative stress.

The invention consists in the following.

To determine the individual sensitivity of the person to oxidative stress receive a suspension of neutrophils in the peripheral blood. Register the intensity of the source chemiluminescence OF neutrophils and chemiluminescence (NC) after heating the suspension of neutrophils within 30-60 seconds at a temperature of 42°C by counting the number of pulses per minute. When THEIR value/NC for less than 3 reveal sensitivity studied in oxidative stress.

The effect on neutrophils heat shock in the specified mode of heating is accompanied by the production of ROS, the evaluation of which in the reaction of chemiluminescence by counting pulses per minute allows you to reliably determine the sensitivity to oxidative article is the ECCA.

In the particular case of register-induced opsonization simhasanam Lomonosovsky chemiluminescence.

The method is as follows.

The population of neutrophils obtained by centrifugation of peripheral blood of patients on a double density gradient ficollverographine (lower density solution 1.119 g/cm³the upper solution - 1.077 g/cm³within 40 minutes, the speed centrifugation at 400×g. Neutrophils are removed by the introduction of lower interphase layer and washed 2 times with saline followed by resuspension in colorless Hanks solution and counting the number of nuclear cells in suspension. Suspension neutrophils obtained has a purity of 96-98%. The chemiluminescence reaction is carried out in plastic tubes, which contain 20 microlitres colorless Hanks and 150 microlitres lyuminola (5-amino-2,3-dihydro-1,4-phthalazinedione), for example, firms Serva (Germany) at a concentration of 2.5 µg/ml test Tubes are placed in the drum chemiluminometer and thermostatic within one hour to a temperature of 37°C, then add 100 microliters suspension of neutrophils (2×10⁵nuclear cells) and after one hour stimulate neutrophils opsonization simhasanam. For example, the company Sigma (USA) in a volume of 100 microliters and at a concentration of 20 mg/ml. take into Account induced chemilines is enciu and dynamics of production of reactive oxygen species by neutrophils for 30 minutes, with the estimate of the number of pulses per minute. For stressaway neutrophils apply heat shock - heating of the cells in test tubes in a water bath at 42°C for 30-60 seconds, or short-term stress with the subsequent introduction of a stressed and descression of neutrophils in response chemiluminescence.

Mathematical analysis to compare the source chemiluminescence and indicators chemiluminescence of neutrophils after heat shock for 30 sec., 1 min and 3 min was performed using a binomial distribution:$$P(r)=C_n^r{p}^r{(1-p)}^{n-r}$$where n is the number of trials, p is the success probability, r is the number of successful experiments.$$C_n^r$$the number of combinations of n on r, P(r) is the probability to observe r successes in n experiments with success probability p

Evaluation of the probability of success is obtained by maximilianii probability to observe r successes in n trials

95% confidence interval for the probability of success is determined by exact binomial formula is raspredeleniya, given in the form of nomograms in the statistical literature (Wentzel Y.S. "probability Theory", 1969, "Science")

As evidence of the possibility of realization of the stated purpose and achievement of the technical result here is the following data.

Examples of determining the dynamics of the production of reactive oxygen species in the reaction induced opsonization simhasanam chemiluminescence neutrophils in 4 out of 17 patients of different ages after heat shock (number of pulses/min) (Table. 1-4).

Patient M., aged 88, W., 31.05.11 (1 - neutrophils without shock, 2 - shock for 30 sec., 3 - shock 1 min, 4 - shock 3 minutes) (table 1).

Patient H., 86, m, 31.05.11 (1 - neutrophils without shock, 2 - shock for 30 sec., 3 - shock 1 min, 4 - shock 3 min) (table 2).

Patient F., 93, W, 23.05.11 (1 - neutrophils without shock, 2 - shock for 30 sec., 3 - shock 1 min, 4 - shock 3 min) (table 3)..

The patient, age 26, W, 23.05.11 (1 - neutrophils without shock, 2 - shock for 30 sec., 3 - shock 1 min, 4 - shock 3 min) (table 4).

Figures 1-3 shows the curves of the dynamics of the ratio of intensity of the source chemiluminescence of neutrophils to the intensity of chemiluminescence of neutrophils after heat shock of different duration for 17 patients of different ages. Calculate the probability that the ratio of the intensity of the source to the intensity of chemiluminescence chemiluminescence after heat shock reaches its maximum within the first 3 minutes. For example, according to chemiluminescence after heat shock for 30 seconds, shown on figure 1, the probability that the maximum of the relationship is before the 3 minutes is equal 14/17=0.82 with 95% confidence interval 0.59 to 0.84. The results of the calculation for the duration of the heat shock for 30 sec., 1 minutes, 3 minutes, is presented in table 5.

As the table shows large values of the confidence interval is registered with the duration of heat shock for 30 sec. and 1 min Statistically significant increase in the ratio of chemiluminescence of neutrophils (y-axis) for the 3 types of stress reported in the first minute following a stressor, which indicates the need to determine individual sensitivity to oxidative stress during this period of time.

Clinical examples of change is the patient's sensitivity to oxidative stress as a result of treatment.

1. Patient R., 84

Diagnosis: hypertension stage 3, coronary heart disease (CHD), cardiosclerosis, pneumosclerosis, nephroangiosclerosis. Prior to admission ratio chemiluminescence of neutrophils was 1.75: 7616 pulse (original chemiluminescence) and 4338 pulses (after stress). After 2 weeks of treatment with amlodipine, Preductal, aspirin, captopril, multivitamins, prednisolone coefficient chemiluminescence was 4,56 during the initial chemiluminescence of neutrophils 406 pulses and after the action of heat shock 89 pulses, i.e. the ratio increased by 2.6 times.

2. Patient O., 92 years. Diagnosis: fracture of the femoral neck. Before treatment, the ratio of chemiluminescence was 2,84 from the ratio of initial chemiluminescence of neutrophils 68621 impetus to chemiluminescence after stress 24146 pulses. After treatment for 7 sessions of low-intensity laser therapy coefficient chemiluminescence was equal to 3.75 from the ratio of initial chemiluminescence 6484 pulse to the chemiluminescence of neutrophils after stress 1725 pulses, i.e. increased 1.3 times. Low level laser therapy reduces inflammation and synthesis of ROS by phagocytes.

3. Patient C., 98 years. Diagnosis: ischemic heart disease, hypothyroidism, thyroid cancer, cachexia. Before treatment, the ratio of chemiluminescence of 0.89 between the source chemilum is ascentii neutrophils 6961 impulse to chemiluminescence after heat shock 7741 impulse. Treatment with thyroxine, aspirin, vitamins B1, B6, B12, Riboxin, piracetam, Preductal for 2 weeks resulted in an increased ratio to 13.8.

These examples show that hospitalization and adequate treatment of diseases in patients lead to a decrease in sensitivity to oxidative stress due to reduced manifestations of pathological conditions as stressors, as well as reducing production of reactive oxygen species faguoqitirute cells due to activation of anti-stress genes anti-inflammatory or antioxidant properties of drugs.

Figure 4 and figure 5 demonstrates the dependence of the number of pulses per minute (production ROS) in neutrophils from age and dose (mg) of aspirin and indomethacin, having a stimulating effect on anti-stress genes cells, anti-inflammatory and antioxidant effects. The decreased production of ROS by neutrophils in patients of different ages in proportion to the dose of the drugs used (dose-effect).

Determining the sensitivity of people to oxidative stress, can improve the efficiency of treatment of various pathologies in clinics, to carry out preventive and rehabilitation measures to prevent trucapture and take away the stress of individuals to successfully perform the most dangerous production the military works.

1. The method of determining individual sensitivity of the person to oxidative stress, characterized in that you receive a suspension of neutrophils peripheral blood, register the intensity of the source chemiluminescence OF neutrophils and chemiluminescence (NC) after heating them in test tubes in a water bath for 30 to 60 seconds at a temperature of 42°C, by counting the number of pulses per minute, and when THEIR value/NC for less than 3 refer analyzed to stressoustoicivij.

2. The method according to claim 1, characterized in that register-induced opsonization simhasanam Lomonosovsky the chemiluminescence.