Method of determining fractions of modified lipoproteins in blood

FIELD: medicine.

SUBSTANCE: patient's blood serum is treated with 7% solution of polyethylene glycol-6000, incubated and with dye Sudan B at 40°C for 1 hour, separated electrophoretically in agarose gel. After that, additionally, before treatment of blood serum with 7% PEG-6000 to 0.6 ml of sample 0.2 ml of 0.1% tritone X-100 solution is added, incubated for 15 minutes at 20°C, after which mixture is mixed by shaking 120 times per 1 minute. Application of method makes it possible to detect additional intensive minor fraction of modified LP(a).

EFFECT: increase of diagnostics accuracy.

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

Known methods of fractionation of lipoproteins of blood by the method of analytical ultracentrifugation (Angelov, Ngiculela. Lipids, lipoproteins, and atherosclerosis. - Peter, the press, p.98-102).

The known method of fractionation PL polyacrylamide gel (ACA. Biochemical studies in the assessment of the cardiovascular system. In kN. "Research methods in pathology", M, 1988, p.95-97).

There are also known methods of fractionation PL by electrophoresis in agarose gel (lab. Research methods / edited by Riv. M.: Medicine, 1987, s-249), SU 1720015 And 15.03.1992. EN 2063040 C1, 27.06.1996. EN 2115121 C1, 10.07.1998. EN 2097038 C1, 27.11.1997. EN 2060034 C1, 20.05.1996. EP 0074610 A, 23.03.1983.

The disadvantage of these methods is that they do not allow to identify all the PL fraction of the blood, including chylomicrons (HM), high density lipoprotein (HDL), low-density lipoprotein (LDL), very low density lipoproteins (VLDL) and LP(a), and serve to divide into fractions HMM, HDL, LDL, VLDL from the complex of albumin with neeterificirovannah fatty acids, and also fail to detect minor fraction of modified lipoproteins in the blood, namely the modified abnormal Lipoprotein (Lp(a) or LP(a)).

There is a method of determining the fractions l is poproteins the blood of American authors, NV, Fedorova N.A., Perova NV, Hargreaves EXAMPLE, Lanovoy A.A., science, A.N., American A.V., Pokraev E.N. EN 2210079 C2 G01N 33/92, 10.08.2003 bull. No. 22.

This method is closest to the proposed to the technical essence and the achieved result and selected as a prototype.

The disadvantage of this method is that it is not possible to identify a minor fraction of the modified LP(a). This is due to the fact that a minor fraction of LP(a) is the most atherogenic. Therefore, early diagnosis is especially important to identify minor fraction of LP(a) along with the most complete definition of the other factions PL of blood.

The aim of the invention is to improve the accuracy of the method.

This goal is achieved by the fact that the serum is treated with a 7% solution of PEG 6000, and then incubated with PEG-6000 - precipitate serum with dye Sudan B at 40°C in the dark thermostat for 1 h and contribute to electrophoretic studies in magnified 2 times by volume of the well of the agarose gel. Before treatment serum 7% PEG 6000 to 0.6 ml of sample add 0.2 ml of 0.1% solution of Triton X-100 and incubated at 20°C for 15 min, stirring the mixture by the shaking method 120 times in 1 min and reveal minor fraction of modified lipoproteins in the blood.

New in this method is that the test Siva otci the patient's blood is treated with the detergent Triton X-100, incubate 15 min at 20°C, stirred the mixture with the shaking method 120 times in 1 minute, which allows to additionally reveal intense minor fraction of modified lipoproteins in the blood.

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

To obtain PEG-6000 precipitate serum was used 7%solution of PEG-6000. It is known that the 7%PEG-6000 is the ultimate concentration to obtain precipitates serum containing immune complexes and contains no coarse serum proteins. The use of a 6%aqueous solution of PEG-6000 does not completely precipitate the immune complexes in the blood serum. Therefore, to improve the accuracy of the experimental method was chosen by 7%concentration of PEG-6000, which allows, on the one hand, completely precipitate the immune complexes from serum to guarantee the absence coarse (high molecular weight) serum proteins, and on the other hand, to fully precipitate circulating immune complexes (CIC)present in the serum. Thus, if the concentration of the CEC in the serum, equal to 2.5 g/l, this concentration of the CEC were detected in 7%of cases the precipitates serum (+3%), which is associated with an increase in the number of manipulation rather than change koncentracijski in the precipitates serum.

Incubation of 7% PEG-6000 precipitate analyzed serum with dye Sudan B increases the affinity of the dye to the PL of precipitates of blood and a large amount of taken for studies of the blood sample allows to detect modified LP(a)contained in the blood of patients with coronary heart disease (CHD) in very low concentrations. Additional treatment serum Triton X-100 with stirring of the mixture by the shaking method 120 times in 1 min improves the accuracy of the method and to obtain the desired result. The proposed method allows to identify the following minor fraction of the modified LP: HDL, LDL, VLDL and LP(a).

Each newly introduced in the claims the sign performs the function of increasing the accuracy and efficiency of the method: processing of 0.6 ml samples of blood serum of a patient with 0.2 ml of 0.1% solution of Triton X-100, incubation of the samples at 20°C for 15 min, stirring the mixture by the shaking method 120 times in 1 min and an additional detection of minor fractions of modified lipoproteins.

Research different PL classes in the diagnosis of coronary heart disease (CHD) is recommended in all-Russian scientific society of cardiologists according to the position of the recommendations of the European society for the study of atherosclerosis- "Diagnosis and correction of lipid metabolism disorders prevention and treatment is therosclerosis" (Moscow, 2005; Clinical laboratory diagnostics, No. 10, 2008., p.21-32).

At present promising research methods lipids with a detergent /Triton X-100 and others/. In laboratory practice increasingly used direct or homogeneous methods for the determination of lipoprotein (LP) and lipids. Such methods are based on the use of different detergents that can block or solubilisate classes of pharmaceuticals for specific allocation of HDL, LDL and other factions PL.

When using such methods isolation of other classes of PL does not require additional operations and the concentration of cholesterol (LDL) in PL classes can be defined directly in the serum of conventional enzymatic methods in the same cell ("Clinical laboratory diagnostics, No. 10, 2008., p.21-32).

Increasing the concentration of PL or abnormal LP (a) in blood is considered an independent risk factor for atherosclerosis. When blood levels of LP(a) more than 300 mcg/ml at norm 0-300 µg/ml the risk of coronary atherosclerosis increases twice, and while increasing the level of LP(a) cholesterol and LDL - 5 times (J.A. M.A. - 2001. - Vol.285, - p.2486-2497, Eur. Heart. J. - 2003. - Vol.24, - p.1601-1610).

Currently, special attention is paid to the study of minor fractions of the modified LP(a), and therefore developed methods of laboratory diagnostics, allowing to investigate this is t lipoprotein blood. He refers to APO-B-containing lipoproteins, rich in cholesterol (LDL). LP(a) is identical to the "sinking" of pre-β-PL (sinking pre-β-Lp)with electrophoresis mobility pre-β-PL. LP(a) contains 27% protein, 8% carbohydrates and 65% of lipids, of which AHS make up 59%, NAHS 14%, PL 14%.

Protein component of LP(a) is vysokopotentsirovannye polypeptide-APO(a)with close structural similarity to plasminogen - one of the factors of the coagulation system - protivosvertawate blood. With increasing concentration of LP(a)and its modified forms in the blood impairs processes of microcirculation in blood arteries with the possible formation of microthrombi.

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

LP(a) can interact with LDL receptors, exerting little influence on the activity of MMC-COA reductase, a cholesterol esterification. The half-life of LP(a) is longer than that of LDL and is 3.3 days. The content of LP(a) blood in norm does not exceed 30 mg/l At high concentration in blood LP(a) is detected in the defeat of cosudow area concentrations of fibrinogen. Higher concentration of LP(a) is often combined with IIaIIbtypes of hyperlipoproteinemia which often identifies modified PL. Therefore, in clinical practice it is extremely important determination of LP(a) simultaneously with the determination of proteins of the acute phase of inflammation. It was found that most of the lipid-lowering drugs do not affect elevated levels of LP(a).

Fraction of LP(a) heterogeneous. It is established that during electrophoresis LP(a) are in the field of β-globulins, but up to 5% of LP(a) can be detected in α-globulin. The reason for this pronounced heterogeneity is difficult to assess during electrophoresis entire faction LP(a), and especially its minor faction that may remain on the start line, if the size of the particles is large enough. Therefore, the use of common research in the last fifty years of the detergent Triton X-100 for processing serum, namely LDL, blood, leading to partial delipidization PL and increase their mobility during electrophoresis allows to detect minor fraction of LP(a). In the chemical industry uses a variety of detergents (izgotovlenie washing powder, detergents etc), but when working with biological material is used mainly Tritonx-100. Processing mode samples the Triton X-100 were selected on the again of experimental studies empirically. This was done with a different dilution of Triton X-100, different temperatures and different exposure time in minutes. When using different concentrations of a solution of Triton X-100, low concentrations (below 0.1%) did not increase the allocation of minor factions LP, including LP (a)and high concentrations (greater than 0.1 per cent) led to a complete delipidization and fracture patterns PL. The results of the study are presented in table 1.

Processing 0.3 ml serum for the study of lipoproteins of different concentrations of a solution of Triton X-100 during a different time of incubation of the sample at different temperatures.

Therefore, the optimal conditions for treatment of serum with a solution of Triton X-100 concentration of 0.1% in volume of 0.1 ml at a temperature of 20 degrees Celsius for 15 minutes.

The improvement of the method relates to mechanical mixing of the sample with a frequency of 120 times in 1 min on a laboratory shaker for preparation of reagents. More frequent or prolonged shaking leads to the increase of the temperature of the sample and the emergence of remantic forms of lipoproteins in the blood, which prevents further research and distorts the electrophoretic analysis of the sample. Less than a short period of shaking, equal to 30 seconds, does not improve the results.

Because LP(a) Nai is more heterogenen, it is very important in the early stages of the disease to identify the maximum content of LP(a) in the blood of each patient. This allows us to diagnose coronary heart disease before the stage of significant changes in other clinical and laboratory parameters increases the accuracy of diagnosis of the disease. In turn, these patients early is assigned pathogenetically substantiated therapy. No less important is the identification of minor factions LP(a) to assess the effectiveness of therapy of disease and predicting the course of coronary heart disease.

All of the above indicates the extreme importance of developing methods of laboratory diagnostics, allowing you to identify LP(a), modified forms and their minor faction.

Currently in widespread clinical laboratory practice does not use enough of the ways of determining LP(a). At the same time, the popularity of the method of electrophoretic separation of fraction PL of blood in the agarose gel due to its high sensitivity, simplicity of implementation and adequate adequacy of the obtained results (lab. case, 1980, No. 5, page 287-290, Clinical laboratory diagnostics, No. 10, 2008, p.21-32).

The essential features characterizing the invention, shown in the inventive combination of new properties that are not explicitly derived from the prior art in this region the STI and are not obvious to the expert.

Identical set of features not found in the study of patent and scientific literature.

This invention can be used in medical practice to improve the accuracy of diagnosing the degree of atherosclerosis in ischemic heart disease.

Thus, it should be considered the present invention with the relevant conditions of patentability: novelty", "inventive step", "industrial applicability".

The method is based on the electrophoretic mobility of modified lipoproteins in the blood.

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

Figure 1 presents the results of electrophoretic separation in the PL fraction of the blood serum and in the control group, the method of the prototype; b - in the control group proposed method;

In figure 2 a, b, C presents the results of electrophoretic separation of fraction PL of serum in the group of patients with coronary artery disease by the method of the prototype;

Figure 3 presents the results of electrophoretic separation in the PL fraction of the blood serum of the patient B: a - method-prototype; b - the proposed method.

The method is carried out in stages as follows:

1) preparation of a solution of Sudan B: 400 mg of Sudan B was dissolved in 20 ml of ethylene glycol in a boiling water bath for 50 minutes, filter the comfort, store in a glass container;

2) preparation of agarose gel: 320 mg of agarose And "Sigma" are dissolved in 20 ml of water by boiling, and then placed in a thermostat at 55°C; add 20 ml albumin (1 g of albumin in 200 ml veronal-medialog buffer, pH to 8.6). 3 ml of agarose gel is applied on the skim horizontally installed the scanner glass, place a metal rod steel-bar 20 mm×4 mm (height 10 mm), which after solidification of the gel is removed by a magnet;

3) to 0.6 ml samples of blood serum of the patient add 0.2 ml of 0.1% solution of Triton X-100, incubated for 15 min at 20°C, stirred the mixture with the shaking method 120 times in 1 min, then add 40 ml of 7% of polyethylene glycol (PEG)-6000 and incubated for 1 h at 20°C, centrifuged for 40 min at 25000 g. The precipitate washed twice.

4) 0.25 ml of precipitate used for electrophoretic studies. To 0.25 ml of the precipitate serum add 0.15 ml of a solution of Sudan B, placed for 1 h in the dark in a thermostat at 40°C, then add 0.2 ml of a hot solution of agarose gel, mixed, heated at 55°C and heated contribute in the groove of the agarose gel;

5) glass slide is placed in a chamber for electrophoresis layer of agarose down, electrophoresis is carried out in the course of an hour in the refrigerator at 4°C at a voltage of 100 V and current 40-45 mA;

6) electrophoregram fixed in 5% is aStore acetic acid for one hour, then dried between sheets of filter paper, continuously wetting 96% ethanol;

7) densitometry performed on microphotometer MT-4.

To confirm the performance of the proposed method and obtain a technical result were examined in the control group (n=12) and group of patients with CHD (n=24). Both groups were examined using the proposed method and the prototype method.

In the control group, 7% PEG-6000 precipitates serum in the case of using the prototype method and the proposed method, the PL is not revealed (figa, b).

In the group of patients with coronary heart disease in 7% PEG-6000 precipitates serum, using the prototype method identified LDL and VLDL and traces of LP(a) (figa, b, C).

In the group of patients with coronary heart disease in 7% PEG-6000 precipitates serum proposed as an invention than fractions of LDL, VLDL in the area between LDL and VLDL, revealed a minor fraction of the modified LP(a) in 18 patients, and in 6 patients in this group faction LP(a) were detected in low concentrations.

Example

Patient, 55 years. Case history No. 194. Was admitted to the Department of CHD and atherosclerosis research Institute of cardiology, Tomsk scientific with complaints of pain in the sternum and the heart that occurs during physical activity; pain stymied by nitroglycerol. The patient is concerned about shortness of breath during physical activity. HELL 160/90 mm Hg, heart rate is acoe 66 beats per minute.

Diagnosis: ischemic heart disease, angina FC II.

Laboratory results: total cholesterol of 8.8 mmol/l; triacylglycerol 1.6 mmol/l; HDL cholesterol 0.9 mmol/L.

The results of electrophoretic studies PL blood of the patient by the method prototype presented in (figa), and the proposed method (figb). The identified modified LDL, HDL, VLDL, LP(a) in low concentration.

The proposed method allows to reveal in CHD patients with severe hypercholesterolemia presence of minor fractions of the modified LP(a) (m PL(a)) by the method of electrophoresis of pharmaceuticals in the agarose gel.

The application of the proposed method of fractionation PL blood unlike the prototype method allowed us to identify additional intensive minor fraction of the modified LP(a), which increases the accuracy of the method.

Thus the proposed method is easy to use and interpretation of the results.

Application

Table 1. Investigation of the influence of the detergent Triton X-100 to identify lipoprotein fractions at different time of incubation and different temperatures, i.e., the determination of optimal conditions;

Figure 1. Presents the results of electrophoretic separation in the PL fraction of the blood serum and in the control group, the method of the prototype; b - in the control group proposed method;

<> Figure 2 (a, b, C). Presents the results of electrophoretic separation of fraction PL of serum in the group of patients with coronary artery disease by the method of the prototype;

Figure 3. Presents the results of electrophoretic separation in the PL fraction of the blood serum of the patient B: a - method-prototype;

b - the proposed method.

Table 1
DetergentConcentrationThe incubation periodTemperature, degrees CelsiusIdentifying fractions LP(a)
Triton X-1000.1 ml of 0.01%10 minutes15-
Triton X-1000.1 ml of 0.01%15 minutes20-
Triton X-1000.1 ml of 0.01%20 minutes25-
Triton X-1000.1 ml of 0.1%10 minutes15traces
Triton X-1000.1 ml of 0.1%15 minutes20+
Triton X-1000.1 ml of 0.1%20 minutes25traces
Triton X-1000.1 ml of 0.15%10 minutes15-
Triton X-1000.1 ml of 0.15%15 minutes20-
Triton X-1000.1 ml of 0.15%20 minutes25-

The method for determining the fractions of modified lipoproteins of blood by treating serum 7%solution of polyethylene glycol-6000, incubation with the dye Sudan B at 40°C for 1 h followed by electrophoretic separation in agarose gel, characterized in that it further before treatment serum 7% PEG-6000 to 0.6 ml of sample add 0.2 ml of 0.1%solution of Triton X-100, incubated for 15 min at 20°C and then stirred the mixture with the shaking method 120 times in 1 min



 

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