RU2592238C1 - Method for separation and quantitative determination of multiply modified low-density lipoproteins - Google Patents

Abstract

FIELD: biochemistry.

SUBSTANCE: present invention relates to clinical biochemistry and can be used for separation and analysis of cholesterol of multiply modified low-density lipoproteins (mmLDL) in human blood serum. Method consists in the following: blood serum is treated with 20% buffer solution of polyvinyl pyrrolidone with the molecular weight 35,000 polyvinylpyrrolidone (PVP-35000) at the volume ratio of serum: PVP (1.0:0.84), followed by incubation for 10 minutes at room temperature, mmLDL units are deposited by centrifugation at 9,000 rpm for 10 min at 23 °C, the residue is decanted, mmLDL is dissolved in the buffer without PVP-35,000 and the cholesterol in the precipitate is determined. If the level of content of cholesterol in mmLDL is more than 6.6 mg/dl, the individual has a high level of mmLDL.

EFFECT: invention can be used to establish the presence of subclinical atherosclerosis in an examined individual, assess the severity of pathological process and develop the prognostic criteria of vascular complications.

1 cl, 2 tbl, 2 ex

Description

The invention relates to clinical biochemistry and can be used in laboratory diagnostics for the isolation of multi-modified low density lipoproteins (mmLDL) and the determination of cholesterol of multi-modified low density lipoproteins (X-mmLDL).

Atherosclerosis is a chronic disease that causes a thickening of the inner layer (intima) of large arteries and medium-sized arteries. The disease begins with the accumulation of lipoproteins, primarily low-density lipoprotein (LDL), in the extracellular matrix of the vessel. LDL molecules aggregate and undergo multiple modifications (mm LDL), become toxic and cause vascular damage (Itabe N. Oxidative modification of LDL: its pathological role in atherosclerosis. // Clin Rev Allergy Immunol. - 2009. - V. 37, No. 1. - P. 4-11.).

Considering the key role of mmDL in atherosclerosis, it remains relevant to develop simple methods for isolation and quantification of them that are available for both routine and basic scientific research.

Currently, the three most common methods for the isolation and purification of lipoproteins of certain classes of blood serum in preparative quantities are most widely distributed: ultracentrifugation in saline solutions, chromatography, and precipitation. Electrophoresis is used mainly for analytical purposes. The first method, the method of sequential centrifugation at various densities of an aqueous solvent, is most often used. The method is based on the procedure proposed by Havel et al. (Havel RJ, Eder N., Bragdon J. The distribution and chemical composition of ultracentrifugally separated lipoproteins in human serum. // J. Clin. Invest. - 1955. - V. 347. - P. 1345-1353.), And elaborated by Lindgren (Lindgren FT Analysis of lipids and lipoproteins (Perkins EG, ed.) // American oil Chemists Soc., Champaign, SL., USA. 1975. P. 204-223.). The principle of the method is that the hydrated density of the plasma or blood serum is brought to the lower limit of the density of the fraction to be isolated, and then the sample is centrifuged at 100,000 g for several hours. Lipoproteins (LPs) having a density lower than the density of the solvent are floated to the top of the centrifuge tube, while others are sedimented to the bottom. The precipitates are shaken and their hydrated density is brought to the upper limit of the next required density of the LP fraction. Centrifugation is repeated until the desired fraction is isolated in the upper part of the tube (in the supernatant). The high density lipoprotein fraction (HDL) is isolated after preliminary separation of the very low density lipoprotein (VLDL) and LDL fractions, and centrifugation for at least 24-36 hours at 40000-100000 g is required for its isolation. The advantages of centrifugation include the fact that this method has significant flexibility, since it makes it possible to isolate and isolate almost any desired number of fractions that can be characterized within two density ranges of an aqueous solvent. In general, this method is characterized by high accuracy and reproducibility.

The disadvantages of centrifugation include the need to use (to ensure a given solvent density) salts such as NaCl, CsCl, KBr or NaBr and to carry out special procedures for their subsequent removal from ready-made solutions of LP fractions using dialysis. In addition, it is necessary to use high-speed, thermostatically controlled ultracentrifuges, and the time for isolation, for example, of the HDL fraction is more than 36 hours. In addition, the ultracentrifugation method does not allow the separation of mmLD from native LDL.

Known chromatographic method for purification of proteins (Kholodova Yu. D., Chayalo PP Blood lipoproteins. Kiev: Naukova Dumka. - 1990. - S. 26-33]. It is usually used to separate and purify all or individual fractions of lipoproteins obtained other methods, in particular ultracentrifugation or precipitation. Adsorption, ion exchange, affinity and gel filtration chromatography are mainly used. The essence of this method is to pass the initial plasma or blood serum through a column filled with a special sorbent, interacting with shared proteins.The advantage of this method is the relative ease of implementation and the use of inexpensive equipment.

The disadvantages of this method include the difficulty of separating lipoproteins from other serum proteins, the duration of the procedure, and the large dilution of the isolated fractions of lipoproteins.

The most widely used method is chemical precipitation of proteins (Kholodova Yu. D., Chayalo P.P. Blood lipoproteins. Kiev: Naukova Dumka. 1990. P. 22-26.), Based on the fact that such anions as sulfonated polysaccharides ( heparin, dextransulfate, etc.) in combination with some divalent cations (Mn ²⁺ , Co ²⁺ , Mg ²⁺ , Ca ²⁺ , etc.) are able to precipitate LDL at pH values close to 7, forming their insoluble complexes. The essence of this method is to add to the plasma or serum solutions of heparin and salts of the type MnCl ₂ at certain concentrations. Next, these mixtures are shaken and centrifuged at 6000g. The VLDL and LDL fractions then precipitate, and the supernatant containing the HDL fraction is further purified by dialysis against several portions of a neutral buffer for 48 hours.

The advantages of this method include the simplicity and use of only low-speed centrifugation. The VLDL and LDL precipitation method is widely used in routine studies of determining HDL cholesterol, and LDL cholesterol is calculated using the Friedewald WT formula, Levy RI, Fredrickson DS. Estimation of the concentration of low density lipoprotein cholesterol in plasma, without use of the preparative ultracentrifuge // Clin Chem. - 1972. - V. 18. - P. 499-502.).

However, chemical deposition methods have several disadvantages:

1) some of the whey proteins co-precipitate together with VLDL and LDL, and some remain in the supernatant with HDL;

2) the difficulty and duration of removal of precipitating agents, the need for a dialysis stage;

3) violation of the nativeness of lipoprotein particles;

4) the method is suitable only for analytical studies of cholesterol in fractions of lipoproteins.

There is also a known method for the isolation of desialized LDL using an affinity sorbent, sepharose with immobilized lectin from Ricinus Communis (RCA120) (Tertov VV, Sobenin IA, Tonevitsky AG, et al. Isolation of atherogenic modified (desialated) low density lipoproteinscler from blood liposis separation from native lipoprotein by affinity chromatography // Biochem. Biophys. Res. Coramun. - 1990. - V. 167. - P. 1122-1127.). Currently, the method has not found application in routine studies due to the complexity of determining only desialized LDL from the whole pool of multiple modified LDL.

Thus, the known methods for isolating the lipoprotein fraction from blood serum samples are laborious, time consuming, require the use of expensive equipment, as well as the use of chemically active reagents that affect the integrity of the structure as lipoproteins.

The closest technical solution is the determination of multi-modified low-density lipoproteins (mmLDL) in human serum by treating it with a buffer containing polyvinylpyrrolidone with a molecular weight of 35,000 (PVP-35000), and subsequent registration of turbidity compared to a control sample. The presence of turbidity due to the aggregation of mNLP indicates blood atherogenicity (Shoybonov B. B., Shoybonova B.V. A method for determining atherogenicity of human blood // RF Patent No. 2497116 dated 10.27.2013).

The disadvantages of this method are that the method reveals not only mmNLP, but also partially aggregates circulating immune complexes, which, when turbidimetric, overestimate the level of mmLNP, the method detects mmLNP semi-quantitatively and with an increased level of immune complexes can give a false positive result in the diagnosis of subclinical atherosclerosis.

The objective of the present invention is to expand the arsenal of reagents for the isolation and determination of the number of mNLP, a key marker of subclinical atherosclerosis.

The problem is solved by adding to the human blood serum a buffer solution containing polyvinylpyrrolidone with a molecular weight of 35,000 (PVP-35000), precipitating aggregated mmLDs by centrifugation, decanting the supernatant, dissolving the precipitate in a buffer without PVP and determining cholesterol using standard commercial whales.

The technical result obtained by the implementation of the invention is the expansion of the arsenal of methods and reagents for the isolation of mmLDP and determination of cholesterol in them for the purpose of early biochemical diagnosis of subclinical atherosclerosis, as well as the study of the molecular composition of atherogenic mmLC in specific individuals to assess the severity of the pathological process and develop prognostic criteria for vascular complications in the preclinical stage of atherosclerosis. This result is achieved by the fact that the extraction of mmLD from human blood serum and determination of the amount of mmLDL cholesterol is carried out by treating it with a buffer containing PVP-35000, incubating the sample for 10 min at room temperature, precipitating the aggregated mmLDL by centrifugation, decantation, and dissolving the precipitated mmLNP in a buffer without PVP and determination of the cholesterol in the precipitate, at a cholesterol level of more than 6.6 mg / dl, an elevated level of LDL is observed in the examined individual.

Isolation of mmLNP and determination of mmLNP cholesterol using PVP-35000 (AK Syntvita LLC, Russia). Buffer-1 for the aggregation of mNLP is prepared by dissolving 20.0 g of PVP-35000 in 100 ml of 0.01 M Tris-HCl buffer, pH 7.4, containing 0.15 M NaCl. Buffer-2, unlike Buffer-1, does not contain PVP-35000.

Example 1. Determination of cholesterol, triacylglycerides (TAG) and total proteins in PVP precipitates. The following experiments were conducted to confirm the presence of LDL in individual PVP precipitates prepared from the blood serum of patients with carotid artery atherosclerosis and relatively healthy people undergoing a routine medical examination. 84 μl of 20% PVP-35000 (ratio of serum to 20% PVP (1.0: 0.84) are added to 100 μl of human blood serum in Eppendorf tubes, mixed well after 10-min incubation at room temperature, aggregated mmLDL is precipitated by centrifugation at 9000 rpm for 10 min at 23 ° C in an angular rotor for microtubes.The PVP precipitate is carefully decanted and resuspended in 100 μl of buffer 2. After the precipitate is dissolved, the cholesterol content is determined in the obtained PVP precipitates, TAG and common proteins using commercially x enzymatic kits. Results are shown in Table 1.

As can be seen from the data presented in table 1, the cholesterol content in the PVP-precipitate of the pulled serum of patients with AKA was 5.3 times, and the TAG was 16 times higher than in the PVP-precipitate from the serum of healthy individuals. The total protein content is 1.3 times higher in PVP-precipitate patients.

Thus, the presence of cholesterol, TAG, and protein in serum PVP precipitates indicates the lipoprotein nature of aggregates formed in a medium containing 9.1% PVP-35000 and 54% human serum. An increased level of PVP-precipitate containing mmLDL in patients with AKA confirms the pathological role of mmLDL in the atherosclerotic process.

Example 2. Determining the level of cholesterol in mmLDPE in the serum of the subjects. Studies of the cholesterol content in mmLD using the proposed method and prototype in the serum of 31 relatively healthy people. According to the proposed method, as described in example 1, was determined cholesterol in PVP precipitates. Data on the cholesterol content in mm LDL are presented in table 2.

The prototype used 10% PVP 35000 (AK Syntvita LLC, Russia) in 0.01 M Tris-HCl buffer containing 0.15 M NaCl, pH 7.4, as described in (Shoybonov B., Shoybonova B.V. // RF Patent No. 2497116).

Briefly: 15 μl of test serum was added 150 μl of buffer containing 10% PVP-35000 in 0.01 M Tris-HCl buffer containing 0.15 M NaCl, pH 7.4, thoroughly mixed and incubated at room temperature for 10 min in 96-well flat-bottomed immunological tablets. The degree of turbidity was determined turbidimetrically at a wavelength of 450 nm. Serum samples with the appropriate amount of buffer without PVP were used as the blank. Calculated the level of the content of LDL according to the formula described in the prototype. The results obtained for the determination of mmLD are shown in table 2.

To determine the total cholesterol in blood serum, a commercial cholesterol enzyme kit (EKOlab LLC, Russia) was used. The data are presented in table 2.

To assess the condition of the wall of the carotid arteries (SA), high-resolution ultrasonography was used in B mode using a linear vascular sensor with a frequency of 7.5 MHz on an ultrasound scanner "SonoScape SSI-1000" (China). The examination protocol included scanning the left and right common SA (OSA) with focus on the posterior wall of the artery in three fixed projections - anterior-lateral, lateral and posterolateral. Measurement of the thickness of the intima-media (TIM) was carried out on the section of the OSA 10 mm long, opposite the beginning of the carotid sinus. TIM of the posterior wall of the OSA was defined as the distance from the leading edge of the first echogenic zone to the leading edge of the second echogenic zone. The average value of three measurements was considered as an integral indicator of TIM. At TIM values exceeding 0.9 mm for common carotid arteries, atherosclerotic lesions are diagnosed and, in the absence of symptoms, the diagnosis is “Subclinical atherosclerosis”. The data of ultrasound Doppler scanning of the common carotid arteries in the examined individuals (31 people) are presented in table 2. The absence of changes in TIM in the table is indicated by the number - 1, the presence of thickening of the TIM is indicated by 2.

I. As can be seen from the data presented in table 2, in 15 examined individuals, according to the data of ultrasound Doppler scanning (ultrasound scanning) of the common carotid arteries, changes in the thickness of the intima-media in the form of a thickening of more than 0.9 mm were revealed. Thus, according to the ultrasound data of the common carotid arteries, 48% of the patients were diagnosed with Subclinical Atherosclerosis. It was found that with narrowing of the arteries more than 70% of the lumen of the vessel, there are signs of ischemia, manifested by various symptoms caused by hypoxia of the brain.

II. The data of the study of the level of content of multiply modified LDL in serum according to the prototype revealed:

1. Of the 15 people with subclinical atherosclerosis, 3 of them had an LDL content of normal values (<8.4 units), which is 20% of the error;

2. Of the 16 examined without changes in the vessels (healthy faces), 4 of them showed an increased level of LDL, which amounted to 31% (overdiagnosis);

3. According to the ultrasound data, for a normal level of LDL in this contingent, a value of less than 8.4 units can be considered.

III. Studies of cholesterol in mmLDP according to the proposed method gave the following results:

1. Of the 15 people with subclinical atherosclerosis, 4 of them had cholesterol in mmLDL within the normal range (<6.6 mg / dL), which is 27% of the error;

2. Of the 16 examined without changes in the vessels, 4 of them showed an increased level of cholesterol in mm of LDL, which amounted to 31% (overdiagnosis);

3. According to the ultrasound data, for a normal level of cholesterol in mm LDL for this contingent, a value of less than 6.6 mg / dL can be considered.

Thus, the obtained results of the study of blood atherogenicity using the prototype method and the proposed method, gave the following differences:

1. The normal level of cholesterol in mm LDL in 1 case did not coincide with an increased level of mm LDL (prototype) and the presence of subclinical atherosclerosis, confirmed by instrumental method (ultrasound of the common carotid arteries);

2. The normal level of cholesterol in mm LDL and the normal level of mm LDL (prototype) in 3 examined individuals did not coincide with subclinical atherosclerosis;

3. Both methods in 4 examined individuals revealed elevated levels of mmLNP and cholesterol in mmLNP (coincidence), although changes in the intima-medial complex of the common carotid arteries were not revealed instrumentally. It should be noted that in all individuals with "overdiagnosis" of atherosclerosis, the proposed method and prototype revealed elevated levels of total cholesterol.

The results obtained using two methods for determining blood atherogenicity (mmLNP and mmLDL cholesterol) indicate that 20-30% of the biochemical diagnosis errors of subclinical atherosclerosis are associated with increased atherogenicity of these merkers.

The developed method was used by the authors to study the atherogenicity of the isolated mNLPs in the hemolytic test using autologous human red blood cells as targets. The results obtained indicate that, with the same cholesterol content in mmLD, prepared from individual sera of patients with carotid artery atherosclerosis, in the new test for determining atherogenicity by lysis of own red blood cells, they differ in lytic activity.

Thus, the isolation of mNLP from human blood serum allows us to study its functional activity and, thus, from the quantitative characteristics of this key marker of atherosclerosis to the assessment of qualitative characteristics, i.e. degree of pathogenicity. Evaluation of the degree of pathogenicity (lytic activity) will make it possible to predict the speed (severity) of the course of the atherosclerotic process, on the one hand, to monitor the effectiveness of therapy, on the other hand.

Claims (1)

The method of isolation and determination of cholesterol of multiply modified low density lipoproteins (mm LDL) in human serum, namely, that the blood serum is treated with a 20% buffer solution of polyvinylpyrrolidone with a molecular weight of 35,000 (PVP-35000) with a volume ratio of serum: PVP (1 , 0: 0.84), followed by incubation for 10 min at room temperature, mmLNP aggregates are precipitated by centrifugation at 9000 rpm for 10 min at 23 ° C, decanted, the mmLNP precipitate is dissolved in buffer without PVP-35000 and determinedcholesterol in the precipitate and when the cholesterol level in mmLDL is more than 6.6 mg / dl, an increased mmLDL level is observed in the examined individual.