RU2210078C2 - Method for predicting the flow of ischemic cardiac disease - Google Patents

Abstract

FIELD: medicine, cardiology. SUBSTANCE: one should study total cholesterol in blood serum by optic density of substance obtained due to interaction of cholesterol and its ethers with Liberman-Burchard's reagent and LP(a) due to incubating blood serum with sudan B solution for 1 h in dark thermostat at 40 C and applying the sample into the hole in agarose gel at foundation area of 4x20 mm for electrophoresis and at simultaneous decrease of total cholesterol level by 25% and more and LP(a) by 50% and higher against the initial level it is possible to state upon favorable disease flow prediction. At mLP(a) level decrease by below 50% and total cholesterol level decrease by below 25% against the initial level the prediction is considered to be unfavorable. The innovation is simple in application and interpretation of the data obtained. EFFECT: higher efficiency and accuracy of prediction. 13 dwg, 2 ex

Description

 The method relates to medicine and can be used in cardiology and therapy.

 Known methods for predicting the course of coronary heart disease by assessing the level of blood lipoproteins (LP) (Klimov AN, Nikulcheva NG Lipids, lipoproteins and atherosclerosis. - Peter. Press, 1995, S. 98-102) and separation into LP fractions in polyacrylamide gel (Shatskaya N. N. Biochemical studies in assessing the state of the cardiovascular system. In the book. "Research Methods in Occupational Pathology." - M., 1988, pp. 95-97).

 The described methods do not allow to detect all blood LP fractions, including ChM, HDL, LDL, VLDL and LP (a), but serve to separate ChM, HDL, LDL, VLDL from the complex of albumin with unesterified fatty acids, therefore, they do not allow reliable prediction IHD course.

 Also described is a method of separation into LP fractions by agarose gel electrophoresis (Laboratory research methods. Edited by V. V. Menshikov - M .: Medicine, 1987, p. 248 and 249).

 This method is the closest to the proposed technical essence and the achieved result (prototype) in combination with the determination of total blood cholesterol. However, this method is not accurate enough, because it does not allow to detect the drug (a), except for HDL, LDL, VLDL and HM. This is due to the study of a small sample of blood serum. The concentration of LP (a) in the blood is very low. In the prototype method, a well for introducing a test sample of blood serum is prepared in the volume of a metal (steel) bar with a base area of 2x20 mm and a height of 10 mm, which does not allow a sufficiently large volume of blood serum to be added for research.

 The purpose of the method is to increase the accuracy of the method.

This goal is achieved by a technical solution, which is an additional incubation of blood serum with dye Sudan B at a temperature of 40 ^o C in a dark thermostat and the study of a blood serum sample, 2 times larger than that in comparison with the prototype method, and subsequent densitometry of the LP fractions serum with their quantitative assessment, determination of total serum cholesterol for the simultaneous analysis of the dynamics of these indicators.

New in this method is the additional incubation of a blood serum sample with Sudan B for an hour in a dark thermostat at a temperature of 40 ^° C and the preparation of a well in an agarose gel for introducing a test blood serum sample, 2 times larger than that in the prototype method. The hole is made using a steel bar with a base area of 4x20 mm, and not 2x20 mm, as in the prototype method simultaneously with the determination of total blood cholesterol. Therefore, only a comprehensive modernization of the prototype method allows to obtain the desired result.

 Additional incubation of blood serum with Sudan B dye increases the affinity of the dye for blood LP, and an increase in the volume of a blood sample taken for testing allows to detect LP (a) contained in the blood of patients with coronary heart disease (CHD) in very low concentrations, which allows to increase the accuracy of the method and get the desired rltat. The proposed method allows you to identify the following classes of drugs: XM, HDL, LDL, VLDL and LP (a), as well as to assess the concentration of total blood cholesterol in dynamics simultaneously with the study of blood LP and conduct subsequent densitometry of the fractions of blood serum LP with their quantitative assessment.

 Each feature newly introduced into the claims performs the function of increasing the accuracy and efficiency of the method.

 Distinctive features showed in the claimed combination of new properties.

 No identical set of features was found in the known solutions.

 This method can be used in clinical practice.

 Thus, this solution meets the criteria of the invention: "novelty", "practical applicability", "inventive step".

 Currently, special attention is paid to the simultaneous study of LP (a) and total blood cholesterol, in connection with which laboratory diagnostic methods are being developed to study this blood lipoprotein related to apo-β-containing lipoproteins rich in cholesterol (cholesterol). PL (a) is identical to “sinking” pre-β-LP (sinking pre-β-Lp), which has the mobility of pre-β-LP during electrophoresis. PL (a) contains 27% protein, 8% carbohydrates and 65% lipids, of which 59% are ECS, 14% are NECS, 14% are PL.

 The protein component of LP (a) is a highly glycosylated polypeptide - apo (a), which has a close structural affinity for plasminogen - one of the factors of the coagulation system - anticoagulation of blood. With an increase in the concentration of total cholesterol and LP (a) in the blood, the microcirculation processes in the blood arteries are disrupted with the possible formation of microthrombi.

 Due to the presence of sialic acids in the structure of apo (a), PL (a) is more negatively charged than β-PL in an electric field, it is better soluble in water, and can interact with metal ions (calcium). This lipoprotein is heterogeneous. All this indicates the special role of LP (a) in atherogenesis.

 LP (a) can interact with LDL receptors, exerting a weak effect on the B, E activity of MMC - Co A reductase, and on the esterification of cholesterol. The half-life of the drug (a) is longer than that of LDL and is 3.3 days. The content of the drug (a) in the blood normally does not exceed 30 mg / l and it is impossible to determine by conventional methods for the study of the drug (a). At a high concentration in the blood, PL (a) is detected at the sites of vascular damage in the area of accumulation of fibrinogen.

An increased concentration of LP (a) is often combined with II ^a , II ^b types of hyperlipoproteinemia. Therefore, in clinical practice, the determination of LP (a) and total blood cholesterol is extremely important. It was found that most lipid-lowering drugs do not affect elevated levels of LP (a). The results of world population studies have shown that LP (a) is an independent and independent risk factor for coronary heart disease - the most severe manifestation of atherosclerosis.

 All of the above indicates the extreme importance of the development of laboratory diagnostic methods that allow the detection of drugs (a).

 Currently, in a wide clinical laboratory practice, there are no methods for determining LP (a) simultaneously with the determination of blood cholesterol. At the same time, the popularity of the method of electrophoretic separation into blood LP fractions in agarose gel is due to its high sensitivity, ease of implementation and sufficient adequacy of the results obtained (Lab. Delo, 1980, 5, p. 287-290). Total blood cholesterol is determined by standardized methods.

 The method for determining LP (a) is based on the electrophoretic mobility of blood LP.

 The invention will be clear from the following description of the attached drawings.

The method for determining the drug (a) is as follows in stages:
1) preparation of a solution of Sudan B: 400 mg of Sudan B is dissolved in 20 mg of ethylene glycol in a boiling water bath for 50 minutes, filtered, stored in a glass dish;
2) preparation of agarose gel: 320 mg of Sigma A agarose A is dissolved in 20 ml of water by boiling, then placed in a thermostat at 55 ^° C; add 20 ml of albumin solution (1 g of albumin in 200 ml of veronal-medinal buffer, pH 8.6). 3 ml of agarose gel is applied to a fat-free horizontally mounted microscope slide, a metal steel rod-bar 20x4 mm (10 mm high) is placed, which is removed with a magnet after gel curing;
3) 0.15 ml of Sudan B solution is added to 0.3 ml of blood serum, placed for 1 hour in a dark thermostat at 40 ^o С, then 0.2 ml of a hot agarose gel solution is added, mixed, heated at 55 ^o С and heated bring agarose gel into the groove;
4) a glass slide is placed in the electrophoresis chamber with the agarose layer down, electrophoresis is carried out for one hour in a refrigerator at a temperature of 4 ^o With a voltage of 100 V and a current of 40-45 mA;
5) the electrophoregram is fixed in a 5% solution of acetic acid for one hour, then dried between sheets of filter paper, continuously wetting with 96% ethyl alcohol;
6) densitometry is carried out on an MF-4 microphotometer.

 Total blood cholesterol is determined by a unified method in dynamics.

 The method is based on measuring the optical density of a substance formed by the interaction of cholesterol and its esters with the Lieberman-Burchard reagent. Most of the cholesterol is in the serum in an esterified form. The reaction product of cholesterol with the indicated reagent absorbs light in the same spectral region and has a slightly higher extinction coefficient than the reaction product of free cholesterol to take this difference into account, as well as to partially take into account matrix effects (the effects of proteins and bilirubin on the analysis results), the method is calibrated using calibration serum, the cholesterol content of which is determined by the Abel-Kendal reference method.

 Preparation of the Lieberman-Burchard reagent.

 600 ml of acetic anhydride and 300 ml of acetic acid are poured into a two-liter flask, placed in an ice bath and stirred on a magnetic stirrer, then 100 ml of chilled sulfuric acid are added. After 30 minutes, the flask was removed from the ice bath, put on a stirrer and 20 g of sodium sulfate was added, stirred until the salt was completely dissolved for 4-5 hours. The reagent is stable for 2 weeks.

Analysis progress
1. 5 ml of Lieberman-Burchard reagent is poured into dry tubes, placed in a water bath.

 2. 0.2 ml of distilled water is poured into the first test tube (along the wall, slowly over 10 minutes). Shake gently and place in a water bath.

 3. At intervals of 2 minutes, 0.2 ml of test serum is poured into the remaining tubes, shaken, placed in a water bath.

 4. 25 minutes after the addition of water, the optical density of its contents against water is measured in the first test tube. Next, the studied samples are measured at a wavelength of (625 ± 10) nm.

 5. Calculate the concentration of cholesterol in serum compared with the calibration.

 For solutions of the Lieberman-Burchard reaction products, the Bouger-Lamberg-Beer law is observed in the range of 0-400 mg / dl of cholesterol.

 The densities are quantified by densitometry as follows: the area of each peak on the chromatogram is determined by the formula S = h • B1 / 2h, where S is the peak area, h is the peak height, B1 / 2h is the peak width at half its height.

 The calculation is carried out automatically according to the corresponding program and the final result is the percentage of each fraction of LDL, VLDL, HDL and LP (a), if it is in relation to the total amount of fractions, taken as 100%.

 When conducting a second study after treatment, the magnitude of the LP fraction (a) before treatment is taken as 100% and the percentage value of the decrease in the fraction of LP (a) after the treatment of coronary heart disease is calculated.

 In the control group, in the case of using the prototype method (n = 10) and the proposed method (n = 12), CI, LDL, VLDL and HDL were identified (Figs. 1 and 2), since LP (a) in healthy individuals is absent and evaluated the level of total blood cholesterol, which was in the first 10 patients (4.7 ± 0.2) mmol / l and in the second group of patients (4.4 ± 0.2) mmol / l. We examined 2 groups of patients with coronary heart disease before and after treatment, 10 patients each for the prototype method — group I and 28 patients — group II for the proposed method.

 The diagnosis of patients: CHD, angina pectoris, FC III-IV.

 In patients of group I, before treatment, CMS, LDL, VLDL, HDL were detected (Figs. 3, 4). The level of cholesterol in blood serum was (7.4 ± 0.5) mmol / L. After treatment with LP electrophoresis, the fractions of VLDL and LDL became less intense (Fig. 5, 6).

 The level of total blood serum cholesterol after treatment was (5.8 ± 0.4) mmol / L. Diagnosis after treatment: ischemic heart disease, exertional angina, FC II-III. Conclusion: the prognosis of the course of coronary heart disease is favorable, but not accurate enough.

In 22 patients of group II, electrophoresis was identified for STDs, VLDL, HDL, LP (a), and ChM. In the case of using, in addition to the prototype method, only additional incubation of the test sample with Sudan B for 1 hour in a dark thermostat at a temperature of 40 ^° C and introducing it into a 2x20 mm well, or only increasing the volume of a well to 4x20 mm without additional incubation of a sample of LDL and VLDL fractions detected slightly better than in the control, and PL (a) was determined in the form of a trace strip, which did not allow densitometry and, therefore, quantified (Fig.7, 8).

 In the remaining 6 patients of this group, the LP fraction (a) was not detected. The level of total cholesterol in patients of the entire second group was (8.1 ± 0.6) mmol / L.

 After treatment of coronary artery disease in 7 patients, a trace hard-to-detect fraction of LP (a) was detected; in 20 patients, LP (a) was not detected.

 The level of total cholesterol in the blood serum after treatment was in the whole group (6.1 ± 0.5) mmol / L. The diagnosis after treatment in patients of this group (27 people): ischemic heart disease, exertional angina, FC II. Conclusion: the prognosis of CHD is favorable. Patients in this group are presented in clinical examples. In one patient of the second group, with a decrease in total blood cholesterol after treatment, the level of LP (a) in the blood serum remained quite high. No dynamics after treatment were detected. The prognosis of the course of coronary heart disease in this patient was regarded as unfavorable.

 We did not identify false positive and false negative cases, because only objective criteria for the effectiveness of the treatment of coronary heart disease were evaluated: the results of bicycle ergometry, the frequency of angina attacks and, accordingly, the dose of nitroglycerin taken, as well as the results of laboratory research methods, expressed quantitatively.

 Example 1. Patient G., 54 years old, medical history 107, was admitted to the Department of IHD and Atherosclerosis Research Institute of Cardiology, Tomsk. Complaints upon receipt of pain in the region of the heart and behind the sternum of a stitching nature with irradiation under the scapula. The pains occurred during physical exertion and were relieved by nitroglycerin. HELL 140/90 mm RT. Art. Pulse at rest - 64 beats / min. Bicycle ergometry was stopped at a load of 50 watts for objective reasons.

Laboratory test results:
Total cholesterol - 7.7 mmol / l,
Triacylglycerides - 1.8 mmol / L,
HDL cholesterol - 1.2 mmol / l.

 The results of electrophoretic studies of blood LP according to the prototype method are presented in figure 9, and according to the proposed method in figure 10. LP fraction (a) is present.

 Diagnosis at admission: coronary heart disease, angina pectoris, FC III-IV.

After treatment, the results of a laboratory study:
Total cholesterol - 5.6 mmol / l,
Triacylglycerides - 1.6 mmol / L,
HDL cholesterol - 1.5 mmol / l.

 The results of electrophoretic studies of blood LP according to the proposed method are presented in Fig.11.

 The LP fraction (a) is absent, the LDL fraction is poorly detected.

 Diagnosis at discharge: coronary heart disease, angina pectoris, FC-II. Conclusion: the treatment is effective. The prognosis of CHD is favorable.

 Example 2. Patient D., 55 years old, medical history 48, was admitted to the department of coronary heart disease and atherosclerosis of the Tomsk Scientific Research Institute of Cardiology with complaints of pressing pains in the heart and behind the sternum, radiating to the shoulder blade and cervical region. No significant changes were detected on the ECG. VEM stopped at a load of 50 watts due to pain behind the sternum. Pain is stopped by nitroglycerin. An oblique depression of the ST segment in leads V4-V6 appeared on the ECG. HELL 160/110 mm Hg, pulse at rest 62 beats. in minutes

Laboratory test results:
Total cholesterol - 8.2 mmol / l,
Triacylglycerides - 1.7 mmol / L,
HDL cholesterol - 1.0 mmol / L.

 The results of electrophoresis of blood LP before treatment by the proposed method are presented in FIG. 12. The results of densitometry of drugs: LDL - 56%, VLDL - 22%, HDL - 15%, PL (a) - 7%.

 Diagnosis at admission: coronary heart disease, angina pectoris, FC III-IV.

After treatment, the results of a laboratory study:
Total cholesterol - 6.1 mmol / l,
Triacylglycerides - 1.5 mmol / L,
HDL cholesterol - 1.4 mmol / L.

 The results of electrophoretic studies of blood LP according to the proposed method after treatment are presented in Fig.13.

 The results of densitometry of drugs: LDL - 56%, VLDL - 25%, HDL - 16%, PL (a) - 3%.

 Diagnosis at discharge: coronary heart disease, exertional angina, FC II. A decrease in the level of LP (a) by 57%, and total cholesterol by 25% compared with the initial testified to the effectiveness of the treatment of coronary artery disease. Conclusion: the prognosis of CHD is favorable.

 So, when applying the prototype method, the prognosis of the course of coronary heart disease was based on the level of total blood cholesterol and clinical data, therefore it was not accurate enough (see Figs. 7 and 8). The method was applied in 38 patients and 22 patients of the comparison group with a diagnosis of neurocirculatory dystonia. With a decrease in the level of LP (a) by 50% or more and total cholesterol in the blood serum by 25% or more, the prognosis of IHD was assessed as favorable, as evidenced by a change in the functional class (FC) of angina pectoris, and when only one of the indicators an unfavorable prognosis of the course of IHD was established (the transition of a stable course to an unstable form of angina pectoris).

 False positive cases of predicting the course of coronary heart disease when using our proposed method was not observed. Moreover, the proposed method is simple in the execution and interpretation of the results.

Claims (1)

A method for predicting the course of coronary heart disease by examining blood serum, characterized in that before and after treatment, total cholesterol is determined in blood serum by the optical density of the substance formed by the interaction of cholesterol and its esters with Lieberman-Burchard reagent and LP (a) by incubating serum blood solution of Sudan B for 1 h in a dark thermostat at 40 ^o C and then making a sample in a well in agarose gel with a base area of 4 • 20 mm for electrophoresis and at the same time reducing the level of total prolapse of 25% or more and LP (a) of 50% or more compared with the initial level, the prognosis of the course of the disease is considered favorable, contributing to the transition of angina pectoris FC III-IV to FC I-II, and with a decrease in total cholesterol less than 25% and the level of LP (a) less than 50% compared with the initial level, the prognosis is considered unfavorable.

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