RU2767927C1 - Method for diagnosing endothelial dysfunction in smoking pregnant women with arterial hypertension - Google Patents

Abstract

FIELD: medicine.SUBSTANCE: invention refers to medicine, namely to internal diseases, cardiology, obstetrics and gynecology. Method for diagnosing endothelial dysfunction in pregnant smokers with arterial hypertension involves venous blood sampling from a peripheral vein in amount of 3 ml into test tubes without IMPROVACUTER fillers. RayBio Human eNOS ELISA Kit is used for the analysis. Level of NO-synthase is measured by ELISA. Further, NO-synthase is assessed. If the value of NO-synthase is more than 1.3 ng/ml, the level is considered to be normal, and if the value of NO-synthase is less than 1.3 ng/ml, endothelial dysfunction is diagnosed and correction of the antihypertensive therapy is recommended.EFFECT: method allows introducing individualization of therapy of arterial hypertension in pregnant smokers due to preliminary detection of indications for this therapy.1 cl, 2 ex, 1 tbl

Description

The invention relates to medicine, namely internal medicine, cardiology, obstetrics and gynecology, and can be used to assess endothelial dysfunction in smoking pregnant women.

Arterial hypertension (AH) is a condition in which SBP ≥140 mm Hg is recorded in pregnant women. and/or DBP ≥90 mmHg It is known that over the past decade, hypertension and its associated complications have taken the 4th place in the structure of causes of maternal death. According to The International Federation of Gynecology and Obstetrics (FIGO, 2016), hypertension is registered in 5-10% of pregnant women. The prevalence of hypertension in the Russian Federation in pregnant women is 7-30%. According to the registry of pregnant women "BEREG", hypertensive disorders were recorded in 14% of women, and taking into account WHO data, hypertensive conditions during pregnancy in 2014 occupied the 2nd place in the structure of maternal mortality and were the cause of at least 76 thousand cases of maternal and 500 thousand .cases of infant mortality annually (2018 ESC recommendations for the diagnosis and treatment of cardiovascular diseases during pregnancy. Working group on the treatment of cardiovascular diseases during pregnancy of the European Society of Cardiology (ESC, ESC) / V.Regitz-Zagrosek, JW Roos-Hesselink , J. Bauersachs et al. // Russian Journal of Cardiology - 2019. - V. 24, No. 6. - P. 151-228).

Hypertension during pregnancy is the cause of many complications, such as: placental insufficiency; fetal growth retardation syndrome (FGR); antenatal fetal death; perinatal mortality; premature detachment of a normally located placenta; obstetric bleeding; eclampsia; HELLP syndrome; DIC; acute kidney injury; pulmonary edema; cerebrovascular accident (CVA); - hemorrhage and retinal detachment (Arterial hypertension in pregnant women: a view from the standpoint of the European recommendations of 2018 / E.V. Shikh, O.V. Zhukova, O.D. Ostroumova et al. // Arterial hypertension. - 2019. - T. 25, No. 1. - S. 105-115).

Smoking is known to be a modifiable risk factor for hypertension.

But at the same time, the prevalence of smoking remains extremely high, amounting to 18.3% in the age group of women 14-25 years old, and 22.1% in the group of 25-44 years old, despite its significant decrease compared to 2009 according to the global survey of the adult population on tobacco use (GATS) (Protection of women's health from exposure to tobacco smoke, prevention and treatment of tobacco use and tobacco dependence in women [Electronic resource]: guidelines. - Moscow, 2018. - 48 p. - URL: http:/ /pichaevskava-crb.ru/download/145692).

According to Olina A.A. and Kuzina O.A. the prevalence of smoking among women before pregnancy was 24.3-28.87%. At the same time, it was recorded that 8.8% of patients gave up smoking when they learned about pregnancy, which means that the early stages of embryogenesis in this category of pregnant women took place against the background of nicotine intoxication (Olina A.A. The frequency of smoking among pregnant women and the relationship with obstetric complications / A. A. Olina, T. A. Meteleva / / Perm Medical Journal - 2015. - V. 32, No. 3. - P. 93-98 .; On the prevalence of active and passive smoking during pregnancy / O.A. Kuzina, O.D. Konstantinova, L.M. Demina, A.S. Volkova // Treatment and prevention. - 2017. - No. 2. - P. 35-37).

Smoking and arterial hypertension are directly associated with endothelial damage, namely endothelial dysfunction (ED). Endothelial dysfunction is understood as a persistent change in the structure and/or functional activity of the endothelium, leading to dysregulation of vascular tone, thrombosis, and other complications.

With ED, the enzyme endothelial NO-synthase (eNOS3), which is the main source of nitric oxide, will be disrupted. In turn, nitric oxide is the main vasodilating factor. In the absence of damage, the main source of nitric oxide (and its metabolites) is □ eNOS3. One of the biochemical methods of endothelial dysfunction is the assessment of the level of endothelial NO-synthase (Vlasov T.D. Endothelial dysfunction. Do we understand this term correctly? / T.D. Vlasov, N.N. Petrishchev, O.A. Lazovskaya // Bulletin anesthesiology and resuscitation. - 2020. - V. 17, No. 2. - S. 76-84.).

However, criteria for assessing endothelial dysfunction in smoking pregnant women with arterial hypertension, as well as approaches to its correction, have not yet been developed.

There is a method for diagnosing endothelial dysfunction based on cytometry of circulating desquamated endotheliocytes. The method is based on a visual assessment of the morphology of circulating desquamated endotheliocytes by phase-contrast microscopy. For analysis, venous blood in a volume of 4-5 ml is taken into test tubes, 3.8% sodium citrate solution is used as a stabilizer. The principle of the method is based on the isolation of endotheliocytes together with platelets. Platelets are separated by adding an ADP solution, stirring the mixture for 10 min and centrifuging (1000 rpm, 10-15 min). Platelet-free plasma is carefully separated from the precipitated platelets and re-centrifuged (1000 rpm, 20 min). The resulting supernatant is discarded and 0.1 ml of 0.9% sodium chloride solution is added to the sediment. The number of endothelial cells is counted in two grids of the Goryaev chamber with subsequent recalculation per 100 ml of plasma, taking into account the volume of the Goryaev chamber and changes in plasma concentration during the above method. Cytological preparations were examined under a LeikaDM 1000 microscope with a computer video attachment for image processing and analysis. The following cytometric parameters of desquamated endotheliocytes were determined: average cell diameter, average cell perimeter, average cell area, shape factor, cell polarization.

The disadvantages of this method are: laboriousness, the need for expensive equipment and qualified specialists, as well as the duration of the procedure (Assessment of the severity of endothelial dysfunction in pregnancy complications / A.V. Mironov, A.M. Torchinov, M.M. Umakhanova et al. // Difficult patient. - 2018. - V. 16, No. 8-9. - P. 30-36.).

As a prototype, a method for diagnosing endothelial dysfunction was chosen, the essence of which is the determination of the concentration of nitric oxide, endothelial and inducible NO synthase, and cotinine in the blood serum by enzyme immunoassay (ELISA) using monoclonal antibodies. The material for the study was venous blood in the amount of 3 ml, taken from patients on the day of admission to the hospital. The inclusion criteria for the study group were: women who smoked throughout their pregnancy, smoking experience before pregnancy for at least 2 years, having a gestation period of more than 37 weeks, singleton pregnancy. Women with medical conditions such as gestational diabetes mellitus, hypertension, pre-eclampsia, preterm labor, active hepatitis, kidney and cardiovascular disease, as well as those who have had complications during childbirth and prolonged labor or pregnancy through assisted reproduction methods, do not were included in the study. Multiple pregnancies, alcohol consumption, and patients on a vegan diet were also exclusion criteria. The control group consisted of women included according to the above criteria, with the exception of smoking and passive exposure to tobacco smoke. (Influence of Active Exposure to Tobacco Smoke on Nitric Oxide Status of Pregnant WomenMagdalena Chelchowska, Jadwiga Ambroszkiewicz, Joanna Gajewska et al. /IntFEnvironResPublicHealth. - 2018. - Dec 3;15(12):2719).

The disadvantages of this method include:

1) a limited sample of patients (the exclusion criterion was hypertension in pregnant women, gestational age more than 37 weeks),

2) high cost of diagnostic methods.

The purpose of the present invention is to identify endothelial dysfunction in order to increase the therapeutic effect of treating smoking pregnant patients with arterial hypertension by influencing the pathogenetic link - endothelial dysfunction.

The purpose of the invention is achieved by a method for diagnosing endothelial dysfunction in smoking pregnant women with arterial hypertension. Venous blood is taken from a peripheral vein in an amount of 3 ml into test tubes without IMPROVACUTER fillers; a RayBio Human eNOS ELISA Kit is used for analysis. The level of NO-synthase is measured by ELISA, then NO-synthase is assessed, if the value of NO-synthase is more than 1.3 ng/ml, the level is assessed as normal, and if the values of NO-synthase are less than 1.3 ng/ml, endothelial dysfunction is diagnosed and recommend adjustment of antihypertensive therapy.

The determination of low values of NO-synthase indicates a high risk of complications, both on the part of the mother and the fetus. Correction (treatment) of endothelial dysfunction during pregnancy will reduce the risk of these complications.

Novelty of the method:

1) Determination of NO-synthase in pregnant women with chronic arterial hypertension, in smoking pregnant women with chronic arterial hypertension.

2) Determination of the critical level of NO-synthase ≤ 1.3 ng/ml, associated with the presence of hypertension and endothelial dysfunction in smoking pregnant women.

3) Determination of indications for treatment with a third-generation beta-blocker with modulation of nitric oxide synthesis.

The set of essential features of the invention makes it possible to obtain a new technical result, namely, to introduce individualization of the treatment of arterial hypertension in smoking pregnant women due to the preliminary identification of indications for this therapy. Identification of endothelial dysfunction in smoking pregnant women with arterial hypertension requires personalized treatment, namely, the appointment of third-generation beta-blockers.

The method is carried out as follows. In the clinical examination of pregnant women, generally accepted methods are used: complaints, anamnesis, namely, the duration of arterial hypertension, maximum and adapted blood pressure values, drugs taken before / during pregnancy, the therapeutic effect of antihypertensive drugs taken, smoking experience, the number of cigarettes smoked before / during pregnancy, calculation of the smoker's index; inspection, laboratory tests.

To assess endothelial dysfunction in patients of all clinical groups, venous blood is taken from a peripheral vein in the amount of 3 ml into tubes without IMPROVACUTER fillers. Allow storage of test tubes in a refrigerator at a temperature of +3 to +5°C for no more than 24 hours.

For analysis, a RayBio Human eNOS ELISA Kit is used. To prepare for storage of reagents and conduct ELISA, use the manufacturer's instructions from the kit (https://doc.raybiotech.com/pdf/Manual/ELH-ENOS.pdf)

Next, NO synthase is assessed: at values >1.3 ng/ml, the level is assessed as normal, with a decrease in NO synthase ≤1.3 ng/ml, endothelial dysfunction is diagnosed and correction of antihypertensive therapy is recommended.

Depending on the presence/absence of arterial hypertension and smoking, the patients were divided into 4 groups: 1) AH, 2) AH + smoking, 3) smoking, 4) no studied factors (control). The proposed method has been tested in clinical practice on 126 patients. The study included 1) patients with hypertension (n=40), 2) patients smoking + hypertension (n=21), 3) smoking (n=37), 4) control (n=28).

To determine the relationship between AH and the concentration of NO synthase in smoking pregnant women, logistic regression was used - Table 1, where B are the coefficients of the logistic regression equation, W is the value of the Wald statistic, p is the statistical significance of the relationship of the indicated variable with the presence of AH, OR is the odds ratio, characterizing the strength of the relationship, the odds ratio and its confidence interval are given directly.

An odds ratio of less than 1 indicates a protective role of the level of NO-synthase, in other words, the presence of hypertension in smoking pregnant women is associated with a reduced level of NO-synthase.

Using logistic regression, it was found that the level of NO-synthase ≤ 1.3 ng/ml is statistically significantly associated with the presence of hypertension, and hence endothelial dysfunction, in smoking pregnant women.

When the value of NO-synthase decreases below 1.3 ng/ml, endothelial dysfunction is diagnosed and a change in antihypertensive therapy is recommended in favor of a third-generation beta-blocker with modulation of nitric oxide synthesis.

Clinical example 1.

Patient V.M., 37 years old, was hospitalized in the department of pathology of pregnant women with a diagnosis of Pregnancy 25 weeks 3 days. Primipara. Chronic arterial hypertension that exists before pregnancy will reach the target level of blood pressure. Obesity 3 degrees. Diffuse nodular goiter of the 2nd degree.

Anamnesis of life: The diagnosis of "hypertension" was established at the age of 27, before pregnancy she took drugs of the ACE inhibitor group (perindopril 4 mg 1 r / day in the morning). Against the background of antihypertensive therapy, the target values of blood pressure (120-130/80 mm Hg) were achieved. During preconception preparations, ACE inhibitors were replaced with BABs (bisoprolol 5 mg 1 time/day in the morning). During pregnancy, while taking bisoprolol 5 mg 1 r / day, morning blood pressure was within the target values (120-130/80 mm Hg). Smoking for 14 years, previously smoked 7-10 cigarettes per day, with the onset of pregnancy reduced to 1-2 cigarettes per day. IPL is 4.9.

The patient underwent venous blood sampling from a peripheral vein in the amount of 3 ml. The ELISA method was used to determine endothelial NOS, which turned out to be 0.4 ng/ml, which is 3 times less than the standard value of 1.3 ng/ml. Thus, pronounced endothelial dysfunction was stated. In this connection, the patient was replaced by bisoprolol with nebivolol, a third-generation beta-blocker with modulation of nitric oxide synthesis.

After 4 weeks of treatment with nebivolol 5 mg/day, the patient was again tested for NO-synthase, which increased to 1.5 ng/ml, which indicates the normalization of endothelial function, and therefore a decrease in the risk of perinatal complications.

Clinical example 2.

Patient V.S., aged 28, was hospitalized in the Department of Pathology of Pregnancy with a diagnosis of Pregnancy 37 weeks. OAGA. Breech presentation of the fetus. Arterial hypertension existing before pregnancy. Bacterial vaginosis. CD: Cleft lip and palate. Obesity 2 tbsp.

Anamnesis of life: The diagnosis of "hypertension" was established within 1 year. Prior to pregnancy, she did not receive any drug therapy, the target values of blood pressure (120-130/80 mm Hg) were achieved against the background of non-drug methods of treatment. From 7 weeks of this pregnancy, an increase in blood pressure was recorded with maximum values of 170/110 mm Hg. Currently taking methyldopa (dopegyt) 250 mg 3 times a day, bisoprolol (Concor) 2.5 mg 1 time a day in the morning. Against the background of drug therapy, the target values of blood pressure (120-130/80 mm Hg) were achieved. Bad habits are denied.

The patient underwent venous blood sampling from a peripheral vein in the amount of 3 ml. The ELISA method was used to determine endothelial NOS, which turned out to be 1.8 ng/ml, which is more than the standard value of 1.3 ng/ml. Thus, this patient does not have laboratory signs of endothelial dysfunction, and therefore, she does not need correction of drug therapy.

Claims (1)

A method for diagnosing endothelial dysfunction in smoking pregnant women with arterial hypertension, including venous blood sampling and determination of NO-synthase, characterized in that venous blood is sampled from a peripheral vein in an amount of 3 ml into tubes without IMPROVACUTER fillers, a RayBio reagent kit is used for analysis Human eNOS ELISA Kit, NO-synthase level is measured by ELISA method, then NO-synthase is assessed, if NO-synthase value is more than 1.3 ng/ml, the level is assessed as normal, and if NO-synthase values are less than 1.3 ng/ml diagnose endothelial dysfunction and recommend correction of antihypertensive therapy.