RU2630592C1 - Method for predicting development of postpericardiotomy syndrome in patients with ihd after coronary artery bypass surgery - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention is a method for predicting development of postpericardiotomy syndrome (PCTS) in patients with ischemic heart disease (IHD) after coronary artery bypass surgery, characterized by the fact that on the next day after the operation the activity of arginase in erythrocytes and the arylesterase activity of paraoxonase (PON) in blood plasma are determined in patients, then the K coefficient is calculated by the formula: K=arginase/PON, where "arginase" is the activity of arginase, ME/(g of Hb); "PON" is arylesterase activity of paraoxonase, ME/(mg of protein); and if the K coefficient is 0.39 or more, the PCTS development with a probability of 67% is predicted in patient.

EFFECT: improved accuracy, specificity and sensitivity of the prognostic method which allows to identify risk group at an early stage and to select adequate therapy for patients.

1 tbl, 2 ex, 1 dwg

Description

The present invention relates to medicine, namely to cardiac surgery, and can be used in intensive care units and cardiology.

A known method for predicting the risk of cardiovascular pathologies based on determining the activity of the first isoform of paraoxonase (PON1).

(See US 20090208992 A1 - WO 2009105622, publ. 2009-08-27; IPC A61B 5/145) / 1 /.

With this approach, the activity of PON1 correlates with its polymorphism. This method allows you to get a forecast on the development of cardiopathologies for patients who have not yet undergone surgery, and for patients after myocardial revascularization. The analogue is represented by a one-component model (the forecast depends only on the activity of PON1 and its polymorphism), which significantly reduces the accuracy of the result. This method covers a wide range of possible pathologies, while changes in PON1 activity are characterized by low specificity.

There is also a method of correcting ischemic disorders resulting from reperfusion injury to the liver (RU 2479872, publ. 04/20/2013, IPC G09B 23/28) / 2 /. A key component of this method is the arginase blocker, L-norvaline. The use of L-norvaline avoids the adverse effects associated with the mechanism of ischemia-reperfusion. The mechanism of ischemia-reperfusion is the main trigger that triggers a systemic inflammatory process and initiates the development of postpericardiotomy syndrome (PCTS) (Nakatseva et al., 2010) / 3 /. Therefore, the activity of arginase, which is directly involved in the formation of endothelial dysfunction, is an important factor for assessing the risk of developing postoperative complications, in particular PCTS.

The technical result of the claimed invention is to increase the accuracy, specificity and sensitivity of the prognostic method, which allows to identify a risk group at an early stage and to select adequate therapy for such patients.

The problem is solved as follows: a method for predicting postpericardiotomy syndrome (PCTS) in patients with coronary artery disease after coronary artery bypass grafting is characterized in that in patients one day after the operation, arginase activity in erythrocytes and aryl esterase paraoxonase activity (PON) in blood plasma are determined. Then, the coefficient K = arginase / PON is calculated, which characterizes the ratio of arginase to aryl esterase activity of PON and is defined as a predictor of the development of PCT. With a coefficient K value of 0.39 or more, a patient with a probability of 67% predicts the development of PCT. The accuracy of this method is 75%, the specificity is 69.2% and the sensitivity is 75%.

The present method, based on the determination of the ratio of arginase and PON activities, is simple to perform and does not require additional equipment besides what modern laboratories are equipped with (spectrophotometer, thermostat and centrifuge). The two-component model increases the accuracy of the forecast. Test execution does not take much time. The advantages can also include relatively high levels of sensitivity and specificity of the invention.

PCTS is a specific form of traumatic pericarditis that develops as part of the systemic inflammatory response of the body (Chien, Shen, 2006; Igolnikova, Nikulina, 2012) / 4, 5 /. Despite the many years of experience in managing patients with PCT and the ongoing active study of the pathogenetic mechanisms of its development, methods of prevention and correction of this condition, to date there are no clearly defined diagnostic criteria for this syndrome. The clinical manifestations of PCTS are nonspecific, and the diagnosis is usually established after exclusion of clinical conditions with similar symptoms (Igolnikova, Nikulina, 2012) / 5 /. The absence of specific clinical criteria for the diagnosis of PCTS increases the value of additional examination methods.

Arginase (EC 3.5.3.1.) Is a manganese-containing enzyme that catalyzes the hydrolysis of L-arginine to L-ornithine and urea (Ash, 2004) / 6 /. In humans, there are two isoforms of arginase (1 and 2) encoded by different genes. Arginase-1 is a cytosolic enzyme and is expressed in the liver, as well as in the endothelium, vascular smooth muscle cells, cardiomyocytes and erythrocytes (Pernow, Yung, 2013) / 7 /. Unlike the first isoform, the second has mitochondrial localization and is expressed mainly in the kidneys, but it can also be found in the brain, intestines, pancreas and can be induced in other organs and cells, including macrophages and vascular endothelium. L-arginine is also a substrate for another enzyme, endothelial NO synthase. Therefore, excessive activation of both isoforms often leads to a decrease in the bioavailability of L-arginine for NO synthesis, which subsequently causes the development of endothelial dysfunction. In addition, the regulation of the expression and activity of arginase-1 is associated with inflammatory processes and is controlled by cytokines (IL-4, IL-6, IL-13, TGFβ) and toll-like receptors (Yang, Ming, 2013) / 8 /.

et al., 2014) /10/. В то же время PON2 имеет возможность переключать фенотип макрофагов с провоспалительного (M1) на противовоспалительный (М2) (Koren-Gluzer et al., 2015) /11/. Известно, что активность параоксоназы обратно корреллирует с системной воспалительной реакцией и развитием атерогенеза (Mackness, Mackness, 2013; Zhou et al., 2013) /12, 13/. Все вышеизложенное определило актуальность и цель работы.At the same time, in the blood plasma and biological fluids there is an enzyme associated with high density lipoproteins (HDL), which has anti-inflammatory and anti-atherogenic effects - paraoxonase (Huang et al., 2013) 191. The family of paraoxonases is divided into 3 isoforms PON1, 2 and 3. PON1 able to suppress free radical processes in human endothelial cells, vascular smooth muscle cells and fibroblasts. PON1 also prevents HDL oxidation and increases the bioavailability of NO by mediating activation of endothelial NO synthase (

et al., 2014) / 10 /. At the same time, PON2 has the ability to switch the macrophage phenotype from pro-inflammatory (M1) to anti-inflammatory (M2) (Koren-Gluzer et al., 2015) / 11 /. It is known that paraoxonase activity is inversely correlated with a systemic inflammatory response and the development of atherogenesis (Mackness, Mackness, 2013; Zhou et al., 2013) / 12, 13 /. All of the above determined the relevance and purpose of the work.

Objective: to evaluate the possibility of using the ratio of arginase activity and aryl esterase activity of paraoxonase as a biomarker for predicting the development of PCT in patients with coronary artery disease after coronary artery bypass grafting.

Material and methods. 76 patients with IHD aged 41-75 years (mean age 58 ± 1.5) who underwent coronary artery bypass grafting (CABG) at the Department of Cardiac Surgery ROSTGMU, Rostov-on-Don, were examined. Patients were divided into 2 groups:

Group 1 - patients with coronary artery disease (66 people), in whom PCTS was not detected as a result of clinical studies;

Group 2 - patients with coronary heart disease (10 people, 15%), in whom, as a result of clinical studies, PCT was established.

Biochemical parameters were studied in dynamics: on the 1st, 3rd, 5th, 7th and 10th days of the postoperative period. As a control, the blood of 20 practically healthy people of donors of both sexes aged 35 to 52 years was used (average age 46.2 ± 0.7).

The dynamics of arginase and PON activity in red blood cells and blood plasma was studied within 10 days from the day of surgery. Statistical processing of the results was based on the Mann-Whitney U-criterion and Student t-criteria and was carried out using the SPSS Statistics 17.0 and Statistica 10.0 software package. Differences were considered significant at p <0.05. The results were presented as the median (Me) with an interquartile range (25th-75th percentile).

The results obtained indicate an increase in arginase activity in the blood erythrocytes of patients with coronary artery disease who underwent CABG, in all the studied periods. But the maximum increase in the second group is already observed in the 1st postoperative day - 1.91 (1.78-2.01) IU / g Hb, which is 220% (p <0.00001) more than donors. Moreover, in patients with PCT, arginase activity is higher than in patients without the syndrome by 71% (p <0.05).

The aryl esterase activity of PON in the blood plasma of patients with PCT and without the syndrome is characterized by the opposite dynamics. So, the activity of PON in the second group of patients is reduced as much as possible on the first day of 4.8 (1.0-6.3) IU / g of protein, which is 85% (p <0.001) lower compared to donors. No significant differences were found between the groups in the blood plasma.

A high inverse correlation between enzyme activities was found; Spearman's correlation coefficient was -1.0 (p <0.01).

Based on the results obtained, it was decided to use the data on the 1st day of the postoperative period in red blood cells and plasma to calculate the ratio of arginase and PON activities.

To evaluate the effectiveness of the prognostic method, a graph of the ROC curve was constructed.

The present invention is illustrated in the figure.

In FIG. 1. presents: ROC curve of a test designed to predict plasma PCT.

The optimal arginase / PON coefficient is 0.39. The sensitivity of the described method for plasma is 75%, specificity is 69.2%, and overall accuracy is 75%. Thus, if in the patient on the first day of the postoperative period this coefficient exceeds 0.39, then with a probability of 67%, the development of PCT after possible myocardial revascularization is possible.

The quality of the test can be judged by the expert scale for the values of AUC (Area Under Curve, area under the ROC curve) (Table 1) (Simundic, 2012) / 14 /.

The AUC value for the test intended for prediction in plasma was 0.779 (95% CI 0.579-0.981). The established values of AUC indicate the high quality of the described method.

Positive effect: predicting postpericardial syndrome will expand measures to reduce postoperative complications and mortality, reduce medical costs, and also improve the quality of life of the patient. It is known that early anti-inflammatory therapy significantly reduces the incidence of postpericardial syndrome. However, the "blind" use of drugs is fraught with a number of side effects that significantly complicate the postoperative period. The described method will significantly reduce the time for predicting PCTs and highlight the risk group for targeted therapy.

The implementation of the invention

The study of arginase activity is based on a spectrophotometric determination method (Kolb, Kamyshnikov, 1982) / 15 /. The activity of arginase is judged by the growth of urea - a product of a catalyzed reaction. Urea content is evaluated by reaction with diacetyl monooxime.

The progress of the work includes the following steps.

1. 0.5 ml of 30% trichloroacetic acid, 0.5 ml of 1% red blood cell hemolysate, 0.5 ml of 0.1 M glycine buffer (pH 9.5), 0.5 ml of 2.4 μM are added to control tubes MnCl ₂ solution and 0.5 ml of a 20.1 μM arginine solution.

The test tubes contain 0.5 ml of 1% red blood cell hemolysate, 0.5 ml of 0.1 M glycine buffer (pH 9.5), 0.5 ml of 2.4 μM MnCl ₂ solution and 0.5 ml of 20.1 μM arginine solution.

2. Samples are incubated at 37 ° C for 60 minutes.

3. After incubation, the reaction in the experimental samples is stopped by adding 0.5 ml of 30% TCA.

4. All samples are centrifuged at 3000 rpm for 10 minutes.

5. In clean tubes, 2 ml of color reagent with a solution of (2.5 g / l) diacetylmonoxime are added to 0.5 ml of the supernatant.

6. The samples are mixed, covered with foil and placed in a boiling water bath for 10 minutes, and then cooled in a stream of cold water.

7. The optical density is measured on a spectrophotometer at a wavelength of 540 nm.

, где

where

16.65 - the urea content in the calibration solution, mmol / l .;

E _op - the optical density of the experimental sample;

E _to - the optical density of the control sample;

E _caliber is the optical density of a standard urea solution;

C is the concentration of hemoglobin in 1% hemolysate.

Arginase activity is expressed in international units per g of hemoglobin (IU / g Hb).

The principle of the method for determining the level of urea is as follows: urea forms colored compounds with diacetylmonooxime in the presence of thiosemicarbazide and iron salts in an acidic medium, the color intensity of which is proportional to the urea content (V. Menshikov, 1987) / 16 /.

The progress of the work includes the following steps.

1. In test tubes add 0.5 ml of 1% erythrocyte hemolysate and 0.5 ml of 30% trichloroacetic acid.

2. Control tubes contain 0.5 ml of disH ₂ O.

3. The standard sample contains 0.01 ml calibrator.

4. After 15-20 minutes, the experimental samples are centrifuged at 3000 rpm.

5. Add 0.5 ml of supernatant and 2 ml of color reagent to a clean tube.

6. The test tube is kept in a boiling water bath for 10 minutes, then cooled for 2-5 minutes under tap water.

7. The measurement of optical density is carried out on a spectrophotometer at a wavelength of 540 nm.

, где

where

E _op - the optical density of the experimental sample;

E _to - the optical density of the control sample;

E _caliber is the optical density of a standard solution;

C is the concentration of hemoglobin in 1% hemolysate.

Urea levels are expressed in mmol per g of hemoglobin (mol / g Hb).

The study of the functional state of paraoxonase is based on the determination of its aryl esterase activity (Kilic S. et al, 2005) / 17 /. Principle of the method: paraoxonase catalyzes the hydrolysis of phenylacetate, which is split into phenol. The phenol formation rate is determined by measuring the optical density of the test sample at λ = 270 nm.

Progress involves the following steps.

1. 1.5 ml of 1 mM CaCl ₂ are added to test tubes, 1.75 ml of 1 mM CaCl ₂ to the control tube.

2. Add 250 μl of biosubstrate to the test tubes.

3. Start the reaction by adding 250 μl of 12 mm phenylacetate.

4. Measure the optical density at λ = 270, then the samples are incubated at 37 ° C for 7 minutes in a water bath and the optical density is measured again.

The aryl esterase activity of paraoxonase is calculated by the formula:

, где

where

E _OP1 - the optical density of the experimental sample before incubation,

E _OP2 - the optical density of the experimental sample after incubation,

E _K1 - optical density of the control sample before incubation,

E _K2 - optical density of the control sample after incubation,

E _m - molar extinction coefficient, 1310 M ^-1 cm ^-1 ,

C is the concentration of total protein in plasma or pericardial fluid

d is the thickness of the cell

TV - total sample volume,

SV is the sample volume.

The aryl esterase activity of paraoxonase is expressed in international units per mg protein (IU / mg protein).

The coefficient characterizing the ratio of the activities of both enzymes (arginase / PON) is calculated.

The invention is illustrated by clinical examples:

1. A 65-year-old patient, a man suffering from coronary heart disease, underwent coronary artery bypass grafting at the cardiac surgery department of Rostov State Medical University.

The arginase / PON coefficient one day after surgery was 0.76. According to the forecasting criterion for the claimed method, this ratio was more than 0.39. The conclusion is made about the high risk of the development of PCT. This forecast was confirmed. The patient developed PCT on the 5th day of the postoperative period.

2. A 59-year-old patient, a man suffering from coronary heart disease, underwent coronary artery bypass grafting in the cardiac surgery department of Rostov State Medical University.

The arginase / PON coefficient a day after surgery was 0.17. According to the forecasting criterion for the claimed method, this ratio was less than 0.39. The conclusion is made about the minimal risk of the development of PCTS. This forecast was confirmed. During the entire observation period, the patient did not develop PCTs.

Information sources

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2. RU 2479872, publ. 04/20/2013; IPC G09B 23/28.

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9. Huang Y, Wu Z., Riwanto M. et al. Myeloperoxidase, paraoxonase-1, and HDL form a functional ternary complex // The Journal of Clinical Investigation. 2013; 123 (9): 3815-3828.

G., Penno G., Pucci L. et al. Q192R Paraoxonase (PON)1 Polymorphism, Insulin Sensitivity, and Endothelial Function in Essential Hypertensive Men // Clinical Medicine Insights: Cardiology. 2014; 8:57-62.10.

G., Penno G., Pucci L. et al. Q192R Paraoxonase (PON) 1 Polymorphism, Insulin Sensitivity, and Endothelial Function in Essential Hypertensive Men // Clinical Medicine Insights: Cardiology. 2014; 8: 57-62.

11. Koren-Gluzer M., Rosenblat M., Hayek T. Paraoxonase 2 Induces a Phenotypic Switch in Macrophage Polarization Favoring an M2 Anti-Inflammatory State // International Journal of Endocrinology. 2015; 8: 1-9.

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A.-M. Diagnostic Accuracy—Part 1: Basic Concepts: Sensitivity and Specificity, ROC Analysis, STARD Statement // The Journal of Near-Patient Testing & Technology. 2012; 11(1):6-8.fourteen.

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Claims (5)

A method for predicting the development of postpericardiotomy syndrome (PCTS) in patients with coronary heart disease (CHD) who underwent coronary artery bypass grafting, characterized in that the patients determine the activity of arginase in erythrocytes and the aryl esterase activity of paraoxonase (PON) in blood plasma one day after the operation, and then calculate the coefficient of plasma K by the formula: K = arginase / PON, where Arginase is the activity of arginase, IU / (g Hb); PON - aryl esterase activity of paraoxonase, IU / (mg protein); and with a coefficient K value of 0.39 or more, the patient is predicted to develop PCT with a probability of 67%.

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