RU2210076C2 - Method for predicting the flow of ischemic cardiac disease - Google Patents

Abstract

FIELD: medicine, cardiology. SUBSTANCE: one should study blood serum lipids. Moreover, one should additionally determine modified LP(a) before and after treatment due to incubating 7% PEG-6000 blood serum precipitate 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 mLP(a) level decrease 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% against the initial level the prediction is considered to be unfavorable. EFFECT: higher efficiency of prediction. 13 dwg

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) (A. N. Klimov, N. G. Nikulcheva // "Lipids, lipoproteins and atherosclerosis", 1995, Peter. Press, pp. 98-102) and division into LP fractions in polyacrylamide gel (NN Shatskaya // "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 HM, HDL, LDL, VLDL and LP (a), but serve to separate HM, HDL, LDL, VLDL from the complex of albumin with unesterified fatty acids.

 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, pp. 248-249).

 This method is the closest to the proposed technical essence and the achieved result (prototype). However, this method is not accurate enough, since it does not allow to detect modified LD (a), except for LDL, VLDL with the simultaneous determination of total serum cholesterol. This is due to the study of a small sample of blood serum, not prepared to detect modified drugs. The concentration of LP (a) in the blood is very low. In the prototype method, the well for introducing the test sample of blood serum is prepared in the volume of a metal (steel) bar with a base area of 2 • 20 mm and a height of 10 mm, which does not allow a sufficiently large volume of blood serum or serum precipitates 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 treatment of blood serum 7% PEG-6000, 7% PEG-6000 serum precipitate is incubated with dye Sudan B at a temperature of 40 ^o C in a dark thermostat and a study of 7% PEG-6000 serum precipitate blood, 2 times the volume exceeding that in comparison with the prototype method, followed by densitometry of blood serum LP fractions with their quantitative assessment.

New in this method is the additional processing of blood serum 7% PEG-6000, incubation of 7% PEG-6000 precipitate of blood serum with dye Sudan B for an hour in a dark thermostat at a temperature of 40 ^o C and the preparation of the wells in agarose gel for making the test serum sample blood, 2 times the volume exceeding that of the prototype method. The hole is made using a steel bar with a base area of 4 • 20 mm, and not 2 • 20 mm, as in the prototype method. Then, densitometry of the LP is performed.

 Therefore, only a comprehensive modernization of the prototype method allows to obtain the desired result.

 To obtain PEG-6000 precipitate of blood serum, 7% PEG-6000 is used. It is known that 8% PEG-6000 is the limiting concentration for obtaining serum precipitates containing immune complexes and not containing coarse proteins of blood serum. The use of 6% PEG-6000 does not completely precipitate the immune complexes of blood serum. Therefore, to increase the accuracy of the method, a 7% concentration of PEG-6000 was chosen experimentally, which allows, on the one hand, to completely precipitate the immune complexes (IR) of blood serum with a guarantee of the absence of coarse (high molecular weight) blood serum proteins, and, on the other hand, to completely precipitate the IR present in blood serum. So in the case of an IR concentration in serum of 2.5 g / l, the same IR concentration was detected in 7% PEG-6000 blood precipitate with a result of ± 3%, which is associated with an increase in the number of manipulations, and not with a change in IR concentration in serum precipitates.

 An additional incubation of 7% PEG-6000 serum precipitate with dye Sudan B increases the affinity of the dye for blood LP, and an increase in the volume taken for the study of a blood sample reveals modified LP (a) contained in the blood of patients with coronary heart disease (CHD) in very low concentrations, which improves the accuracy of the method and obtain the desired result. The proposed method allows to identify the following classes of modified drugs: LDL, VLDL and LP (a) (mLDP, mLPONP, mLP (a)) and subsequent densitometry of fractions of modified blood serum drugs with their quantitative assessment.

 In the case of a decrease in the level of modified LP (a) in patients with coronary heart disease after treatment by 50% or more, the prognosis of the course of coronary heart disease is considered favorable. For the first time, the prognosis of the course of coronary heart disease was assessed by a change in the level of modified drugs (a) after densitometry of the electrophoregram of modified blood drugs.

 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 study of modified drugs (a), in connection with which laboratory diagnostic methods are being developed that allow the study of this blood lipoprotein related to apo-B-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 and 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 both LP (a) and its modified forms 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 and its modified forms are heterogeneous. All this indicates the special role of MLP (a) in atherogenesis.

Modified LPs (a) can interact with LDL receptors, exerting a weak effect on B, E - activity of GMK - Co A reductase, on cholesterol esterification. The half-life of LDL) is longer than that of LDL and is 3.3 days. The content of LP (a) in the blood normally does not exceed 30 mg / l and it is impossible to determine by conventional methods of investigation of LP (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. Increased concentration of Lp (a) is often associated with II ^a, II ^b hyperlipidemia types for which quite often detected modified LP. Therefore, in clinical practice, the determination of LP (a) and its modified forms 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. In healthy individuals, in 7% PEG-6000 blood precipitates, there are no LPs. Modified LDL, VLDL, and LP (a) can appear in their composition during 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). 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).

 The method is based on the electrophoretic mobility of blood LP.

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

The method 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) to 1 ml of blood serum add 40 ml of 7% PEG-6000, incubate for 1 hour at 20 ^o C, centrifuged for 40 min at 18000 g. The precipitate was washed twice with phosphate buffer at pH = 7.25. 0.25 ml of a precipitate solution is used for electrophoretic research;
4) 0.15 ml of Sudan B solution is added to 0.25 ml of 7% PEG-6000 blood serum precipitate, placed for 1 hour in a dark thermostat at 40 ^o C, then 0.2 ml of a hot agarose gel solution is added, mixed, heated at 55 ^o C and heated, make agarose gel in the groove;
5) a glass slide is placed in an electrophoresis chamber with the agarose layer down, electrophoresis is carried out for an hour in a refrigerator at a temperature of 4 ^o C at a voltage of 100 V and a current of 40-45 mA;
6) 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;
7) densitometry is carried out on an MF-4 microphotometer.

The quantitative assessment of fractions during densitometry is carried out as follows: determine the area of each peak in the chromatogram according to 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 appropriate program and the final result is the percentage of each drug fraction, if any, in relation to the total amount of fractions taken as 100%.

 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 into account this difference, as well as to partially take into account the matrix effects (the influence of proteins and bilirubin on the results of the analysis), the method is calibrated using a calibration serum, in which the cholesterol content 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, test 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 Bouguer-Lambert-Bera chacon is observed in the range of 0-400 mg / dl of cholesterol.

 When conducting a second study after treatment, the value of the fraction of modified drugs (a) before treatment is taken as 100% and the percentage value of the reduction in the fraction of modified drugs (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), lipoproteins were not detected (Fig. 1 and Fig. 2). The level of total blood cholesterol was estimated, which was 4.3 ± 0.2 mmol / L in the first 10 patients and 5.1 ± 0.3 mmol / L in the second group of patients. We examined 2 groups of patients with coronary heart disease before and after treatment, 10 patients each for the prototype method - group I and 32 patients - group II for the proposed method
The diagnosis of patients: CHD, angina pectoris, FC III-IV.

 In patients of group I before treatment revealed mLDP, mLPONP (figure 3, 4). Their cholesterol level was 7.8 ± 0.5 mmol / L. After treatment with electrophoresis, the mLONP and mLPNP fractions became less intense (Figs. 5, 6).

 The level of total blood serum cholesterol after treatment was 5.9 ± 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 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 ^o C and adding it to a hole of 2 • 20 mm, or only increasing the volume of the hole to 4 • 20 mm without additional incubation of the sample fractions of mLPLP and mLPONP were detected slightly better than in the control, and mLP (a) was determined as a trace band, which did not allow densitometry and, therefore, quantified (Fig. 7, 8).

 After treatment of coronary artery disease in 6 patients, a trace hard-to-detect fraction of modified LP (a) was detected; in 20 patients, mLP (a) was not detected.

 The level of total blood serum cholesterol after treatment was 6.4 ± 0.5 mmol / L in the whole group. 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 P., 57 years old, medical history 194, 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 stitching, radiating to the left subscapular region. The pains arose after physical and emotional stress, were relieved by prolonged action nitrates. Bicycle ergometry was stopped at a load of 50 watts due to increased chest pain. HELL 150/110 mm RT. Art., pulse at rest 66 beats. in minutes Pain is stopped by nitroglycerin.

Laboratory test results:
Total cholesterol - 7.6 mmol / l
Triacylglycerides - 1.2 mmol / L
HDL cholesterol - 1.1 mmol / l.

 The results of an electrophoretic study of a medicinal product of 7% PEG-6000 precipitate of blood serum but the prototype method are presented in figure 9, and according to the proposed method in figure 10. The fractions of MLPNP, MLPONP and MLP (a) were revealed.

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

After treatment, the results of a laboratory study:
Total cholesterol - 5.5 mmol / l
Triacylglycerides - 1.1 mmol / L
HDL cholesterol-1.6 mmol / l.

 When electrophoretic study of modified drugs 7% PEG-6000 precipitated blood serum according to the proposed method, fractions of drugs were not detected. 11 contains only the start line.

 Conclusion: the absence of MLP (a) indicates a significant improvement in the patient's condition. The mLDPE fraction is weakened.

 Diagnosis at discharge: coronary artery disease, angina pectoris, FC - II. Conclusion: the prognosis of the course of coronary heart disease is favorable, as evidenced by a decrease in the functional class of angina pectoris from III-IV to II.

 Example 2. Patient I., 53 years old, medical history 175, was admitted to the department of coronary heart disease and atherosclerosis of the Tomsk Scientific Research Institute of Cardiology with complaints of pain in the region of the heart that radiates under the shoulder blade that occurs during physical exertion. VEM is stopped at a load of 50 watts due to chest pain and shortness of breath. HELL 150/110 mm. Hg. Art., pulse at rest 64 beats. in minutes Pain is stopped by nitroglycerin.

Laboratory test results:
Total cholesterol - 7.2 mmol / l
Triacylglycerides - 1.5 mmol / L
HDL cholesterol - 0.9 mmol / l
The results of electrophoresis of 7% PEG-6000 precipitate of blood serum revealed the presence of mLDP, mLPONP and mLP (a) (Fig. 12). Densitometry results for mLP: mLDPE - 52%, mLPONP - 40%, mLP (a) - 8%.

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

After treatment, the results of a laboratory study:
Total cholesterol - 5.1 mmol / l
Triacylglycerides - 1.6 mmol / L
HDL cholesterol - 1.3 mmol / l.

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

 Densitometry results for mLP: mLDPE - 54%, mLPONP - 44%, mLP (a) - 2%.

 Diagnosis at discharge: coronary heart disease, exertional angina, FC II. A decrease in the level of MLP (a) by 75%, and cholesterol by 30% compared with the initial testified to the effectiveness of the treatment of coronary artery disease. Conclusion: the prognosis of the course of coronary heart disease is favorable, as evidenced by a decrease in the functional class of angina pectoris from III-IV to II.

 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 42 patients with ischemic heart disease and 22 patients of the comparison group (control) with neurocirculatory dystonia.

 With a decrease in the level of MLP (a) by 50% or more and total blood serum cholesterol 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 changed An unfavorable prognosis of the course of coronary heart disease (transition of a stable course to an unstable form of angina pectoris) has been established.

 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 lipids, characterized in that it is additionally determined before and after treatment modified LPs (a) by incubating 7% PEG-6000 precipitate of blood serum with Sudan B solution for 1 h in a dark thermostat at 40 ^o C and then addition of the sample in a well in agarose gel with the base area of 4 to 20 mm • electrophoresis under reducing and Lp (a) 50% or more compared with baseline prognosis of the disease is considered beneficial, promoting re ode angina FC III-IV in FC III, but at lower levels MLP (a) at least 50% compared to baseline is considered unfavorable prognosis.

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