RU2501013C1 - Method for determining minimum modified lipoproteins of low density in serum or plasma of human blood - Google Patents

Abstract

FIELD: biotechnologies.

SUBSTANCE: invention can be used in laboratory diagnostics for determination of blood atherogenicity as per level of content of minimum modified lipoproteins of low density (MM-LPLD) in serum or plasma of human blood. MM-LPLD is aggregated from blood serum or plasma by treatment with buffer containing polyvinyl pyrrolidone (PVP) with molar weight of 12600±2700 at final PVP concentration of 11.3% to 14.2% in the sample. After incubation during 10 minutes, light absorption in test and check samples is measured, difference is calculated and at the difference value of more than 10 U blood atherogenicity of the examined person is stated due to increased MM-LPLD level.

EFFECT: use of easy, available and easy-to-implement method of determination of MM-LPLD in human blood allows performing screening investigations in order to determine availability of an atherosclerotic process at a pre-clinical stage and clinical examination of population.

3 tbl, 3 ex

Description

The invention relates to clinical biochemistry, and can be used in laboratory diagnostics to determine blood atherogenism by the level of minimally modified low density lipoproteins (MM-LDL) in human serum or plasma.

MM-LDL are poorly modified LDL, differing from native LDL in physical and chemical properties, but still retaining affinity for the LDL receptor and not recognized by scavenger receptors. The physiological modification of LDL is observed in various reactions, both enzymatic and non-enzymatic. The products of all these reactions may be referred to as MM-LDL, although oxidation may not be the primary event in many of these modifications. Lipid peroxidation is the primary reaction only with lipoxygenase or free radical pathways of LDL oxidation. Hydrolysis of sphingomyelin with the help of secretory sphingomelininase (SMase) can be observed with an acute phase reaction, when the level of SMase is increased in plasma or with the participation of SMase, characteristic of LDL [23, 6]. Hydrolysis of sphingomyelin to ceramide increases the oxidative sensitivity of LDL [16], and also leads to the formation of aggregated LDL, which exceeds oxidized LDL in the ability to be absorbed by macrophages with the formation of “foamy” cells [15]. The action of Phospholipase-A2 on LDL produces lysophosphatidyl-rich LDL, which have chemotactic and pro-inflammatory activities. Tertov V.V. et al. (1996) found in blood desialized LDL, which is formed either by the action of the enzyme sialidase, or in reactions mediated by free radicals. It has also been shown that desialized LDL triggers the formation of “foamy” cells [18]. The glycation of LDL observed in diabetes also increases both the formation of “foamy” cells and the susceptibility of LDL to oxidation [3]. Myeloperoxidase-mediated oxidation of LDL leads primarily to the modification of tyrosine residues in the apoV protein in LDL [5].

The most convincing evidence for the participation of oxidized LDL (LDL) in the development of atherosclerosis is supported by the following data:

1) about LDL found in atherosclerotic plaque, but not in normal arteries [14];

2) autoantibodies formed against LDL-derived neoepitopes are present in the blood and the titer of these antibodies correlates with the severity of atherosclerosis and is a marker of the disease [9, 19];

3) studies in animal models of atherosclerosis have shown the protective effect of antioxidants (vitamin E, probucol, probucol analogues and KoQ), although clinical trials in humans using vitamin E have failed [22];

4) genetic knockout of various scavenger receptors in mice leads to a significant suppression of the development of atherosclerosis [21, 11-13], confirming the role of these receptors and their ligands (LDL) in the development of damage to arteries;

5) the depletion of 12/15-lipoxygenase, which is a physiological oxidizing agent of LDL, slows the development of atherosclerosis [7], while its increased expression accelerates atherosclerosis [4], thereby showing the role of oxidative modification of LDL;

6) studies by Kato et al. (2009) in apoE-deficient mice showed that an increase in plasma levels of LDLP is observed before the development of arterial damage [10];

7) it was shown that a specific decrease in the plasma level of LDL by expression of the scavenger receptor (LOX-1) in the liver prevents the progression of atherosclerotic damage in apoE-deficient mice even in the presence of severe hypercholesterolemia [8].

Thus, given the important pathogenetic role of LDL in atherosclerosis, it remains relevant to develop simple methods for the determination of MM-LDL available for clinical laboratory studies.

Currently, in clinical laboratory practice, reagents and methods for determining the content of atherogenic minimally modified lipoproteins in human serum are not available for routine studies.

A known method for determining the atherogenicity of blood serum by its ability to cause the accumulation of cholesterol in cultured cells [17]. The disadvantage of this method is the complexity, high cost, complexity and duration of the study.

Enzyme-linked immunosorbent assays using monoclonal antibodies to in vitro modified LDL are the most intensively developed methods for the determination of mLDPE [20]. The multi-stage, labor-intensive and duration of the enzyme-linked immunosorbent assay, the identification of only epitopes characteristic of artificially modified low-density lipoproteins (non-specificity), as well as the high cost of reagents makes it difficult to use them in routine studies.

The closest technical solution is the determination of multi-modified low-density lipoproteins (mmLDL) in human blood plasma by treating it with a buffer containing polyvinylpyrrolidone 12600 ± 2700, and subsequent visual registration of turbidity compared to the control sample. The presence of turbidity due to the aggregation of mmDLP indicates blood atherogenicity [2]. For instrumental confirmation of the presence of atherosclerosis, studies were conducted by pulsed-wave Doppler scanning of the carotid arteries in relatively healthy young people with elevated LDL up to 40 years of age with normal cholesterol and triglycerides. As a result of ultrasound examination, 57% revealed a thickening of the intimal-medial layer of the carotid arteries and instrumental diagnosis of atherosclerosis of the brachiocephalic arteries was confirmed. For the rest (43%), these parameters remained within the normal range and indicated that an increase in the level of LDL was preceded by morphological changes (thickening) of the intimal-medial layer in the examined arteries. [one].

The disadvantage of the previously developed method is that the method detects only mmLDPE, which are rapidly eliminated from the bloodstream when interacting with scavenger receptors. As well, mmLDLs bind to autoantibodies to them with the formation of immune complexes, which under conditions of 8.9% PVP do not aggregate and in some patients with severe atherosclerosis this test gives a negative result.

The objective of the present invention is to expand the arsenal of reagents for determining blood atherogenicity by aggregation of MM-LDL, a marker of both early atherosclerosis (before the formation of LDL), and atherosclerosis, characterized by an increased level of autoantibodies to mm LDL and the presence of an atherosclerosis clinic in patients confirmed by instrumental methods.

The problem is solved by adding to the human blood serum a buffer solution containing polyvinylpyrrolidone 12600 ± 2700 (the concentration in the system is from 11.3% to 14.2%, determining the degree of turbidity by the turbidimetric method and calculating the content of MM-LDL according to the formula below.

The technical result obtained by the implementation of the invention is to expand the arsenal of reagents for determining blood atherogenicity with the aim of early biochemical diagnosis of subclinical atherosclerosis, as well as monitoring the effectiveness of treatment of clinical atherosclerosis.

This result is achieved in that the determination of the atherogenicity of human serum or plasma is carried out by treating it with a buffer containing polyvinylpyrrolidone (PVP), incubating the sample for 10 min at room temperature, measuring light absorption in the experimental and control samples, calculating the difference between them, while when the difference is more than 10 PIECES, atherogenicity of the blood is ascertained due to the increased level of MM-LDL.

Determination of human blood atherogenicity by aggregation of minimally modified low-density lipoproteins (MM-LDL) using polyvinylpyrrolidone (PVP) 12600 ± 2700 (Sintvita LLC, Russia).

Buffer-1 for the aggregation of MM-LDL is prepared by dissolving 15 g of PVP 12,600 ± 2,700 in 100 ml of 0.01 M Tris-HCl buffer, pH 7.4, containing 0.15 M NaCl. Buffer-K, unlike Buffer-1, does not contain PVP. To the buffer-1 add blood serum, mix thoroughly, incubate, measure the degree of turbidity spectrophotometrically and calculate the level of [MM-LDL] according to the formula:

[MM-LDL], ED = [E _O -E _K ] × 100,

Where:

[MM-LDL] - the level of the content of minimally modified low density lipoproteins in the experimental sample in units (UNITS);

E _O - the optical density of the experimental sample, serum with the appropriate amount of Buffer-1, at a wavelength of 450 nm, units opt.pl .;

E _K - optical density of the control sample at a wavelength of 450 nm, serum with the appropriate amount of Buffer-K, units opt.pl .;

100 - conversion factor in units of units.

Example 1. Determination of the optimal concentration of PVP 12600 ± 2700 (LLC AK Sintvita, Russia) for the aggregation of MM-LDL in the pulsed blood serum of patients with cardiovascular diseases. 100-900 μl of Buffer-1 is added to 50 μl of the pulled blood serum from 10 patients with cardiovascular diseases, thoroughly mixed and incubated at room temperature for 10 min in 9 well cuvettes of the FP-901 biochemistry analyzer (Labsystem, Finland). The degree of turbidity is determined turbidimetrically at a wavelength of 450 nm. Serum samples with the appropriate amount of Buffer-K are used as the blank. Calculate the level of MM-LDL according to the formula above. The results are shown in table 1.

As can be seen from the data presented in table 1, with an increase in the concentration of PVP 12600 ± 2700 in the incubation system from 11.3% to 14.2%, aggregation of MM-LDL and turbidity of the solution are observed. The maximum aggregation of MM-LDL is achieved at a concentration of 12.5%, i.e. with a volume ratio of serum: Buffer-1 (1: 5). With a further increase in PVP concentration over 12.5%, a decrease in the degree of aggregation is observed.

Example 2. Determination of aggregation of sewn-on lipoproteins of low and very low density in the pulsed serum of healthy donors depending on the concentration of PVP 12600 ± 2700 (LLC AK Sintvita, Russia).

To confirm the aggregation of precisely the LDL-MM serum of patients with atherosclerosis in a 12.5% PVP solution of 12,600 ± 2,700, we examined the pulled serum from 10 healthy donors under the conditions described above. The results are presented in table 2.

The addition of increasing concentrations of Buffer-1 containing 15% PVP 12600 ± 2700 to the pulsed blood serum of healthy donors leads to the aggregation of native low and very low density native lipoproteins at a final concentration of PVP in the system up to 12.0%. With a further increase in the PVP concentration over 12.5%, the dissociation of the formed aggregates of low and very low density native lipoproteins is observed in the system and is characterized by a decrease in the turbidity of the serum solution. When the PVP concentration is from 12.5% to 14.2% in the system, the optical density of the serum solution remains practically unchanged. The data obtained indicate that at a PVP concentration of 12,600 ± 2,700 above 12.5% in the system (from 12.5% to 14.2%) in healthy blood donors, the aggregation of native low and very low density native lipoproteins is practically not observed.

Example 3. Determining the level of MM-LDL in serum in patients with atherosclerosis of the carotid arteries, confirmed by instrumental methods. Studies of the content of MM-LDL by the proposed method and prototype in the serum of 60 neurological patients with atherosclerosis of the carotid arteries, confirmed by instrumental methods. According to the proposed method, 275 μl of Buffer-1 was added to 50 μl of the studied serum, thoroughly mixed and incubated at room temperature for 10 min in 9-well cuvettes of the FP-901 biochemistry analyzer (Labsystem, Finland). The prototype used 10% PVP 12600 (LLC AK Sintvita, Russia) in 0.01 M Tris-HCl buffer containing 0.15 M NaCl, pH 7.4, as described in [2]. The degree of turbidity was determined turbidimetrically at a wavelength of 450 nm. Serum samples with the appropriate amount of Buffer-K were used as the blank. The levels of MM-LDL and mmLDL were calculated using the formula above. The results obtained for the determination of MM-LDL and mmLDL are shown in table 3.

Thus, from the data given in table 3, it can be seen that only 43% of the examined patients with atherosclerosis of the carotid arteries confirmed by instrumental methods showed an increased level of LDL. While the determination of blood atherogenicity by aggregation of MM-LDL in these same patients in 93% of cases gives a positive result. The normal level of MM-LDL in 4 patients may be associated with cholesterol-lowering drugs and, ultimately, with low LDL. A comparative study of the content of MM-LDL in serum and plasma showed that MM-LDL is aggregated from both serum and plasma of patients, which indicates the specificity of the process of aggregation of minimally modified low-density lipoproteins. Moreover, the percentage of detection of MM-LDL in both serum and plasma is the same, respectively 93% and 95%.

The proposed method is simple, includes only 2 operations: mixing human serum or plasma with a solution of PVP and registration of turbidity. To prepare the medium, widespread reagents are required: PVP, NaCl, and Tris-HCl. The method allows to detect the presence of MM-LDL directly in serum or plasma without prior fractionation. The method is fast (10 min per 1 analysis), can easily be adapted for biochemical automatic analyzers. Using a simple and quick method for determining blood atherogenicity allows for screening to detect the presence of an atherosclerotic process at the preclinical stage and the medical examination of the population. The formation of a risk group for atherosclerosis (elevated MM-LDL) will allow you to establish an individual cause of the pathological process and conduct etiotropic therapy. Studies of the level of MM-LDL in the blood of a patient with atherosclerosis in a clinic will allow you to control the effectiveness of the therapy.

LITERATURE

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Table 1 Influence of PVP 12600 ± 2700 on the aggregation of MM-LDL in the pooled blood serum of patients with coronary atherosclerosis No. Series of experts. Sy., Μl Buffer-1, μl Buffer-K, μl % PVP in the system A450 [MM-LDL], UNIT. one Experience Form fifty
fifty one hundred one hundred 11.3
0% 0.643
0.1 54.3 2 Experience Form fifty
fifty 200 200 12.0
0% 0.756
0.1 65.6 3 Experience Form fifty
fifty 250 250 12.5
0% 0.802
0.1 70,2 four Experience Form fifty
fifty 300 300 12.9
0% 0.775
0.1 67.5 5 Experience Form fifty
fifty 400 400 13.3
0% 0.547
0.1 44.7 6 Experience Form fifty
fifty 500 500 13.6
0% 0.437
0.1 33.7 7 Experience Form fifty
fifty 600 600 13.9
0% 0.403
0.1 30.3 8 Experience Form fifty
fifty 700 700 14.0
0% 0.379
0,100 27.9 9 Experience Form fifty
fifty 800 800 14.1
0% 0.377
0,100 27.7 10 Experience Form fifty
fifty 900 900 14.2
0% 0.362
0,100 26.2

table 2 The effect of PVP 12600 on the aggregation of native lipoproteins in the pulsed blood serum of healthy donors No. Series of experts. Sy., Μl Buffer-1, μl Buffer K % PVP in the system A450 [MM-LDL], UNIT. one Experience fifty one hundred - 11.3 0.412 21.1 Form fifty - one hundred 0% 0,099 2 Experience fifty 200 - 12.0 0.438 16,2 Form fifty - 200 0% 0,096 3 Experience fifty 250 - 12.5 0.232 9.8 Form fifty - 250 0% 0,092 four Experience fifty 300 - 12.9 0.232 9.8 Form fifty - 300 0% 0,092 5 Experience fifty 400 - 13.3 0.192 9.1 Form fifty - 400 0% 0,101 6 Experience fifty 500 - 13.6 0.19 9.0 Form fifty - 500 0% 0,100 7 Experience fifty 600 - 13.9 0.2 9.2 Form fifty - 600 0% 0,100 8 Experience fifty 700 - 14.0 0.2 9.2 Form fifty - 700 0% 0,100 9 Experience fifty 800 - 14.1 0,203 9.7 Form fifty - 800 0% 0,100 10 Experience fifty 900 - 14.2 0.205 9.8 Form fifty - 900 0% 0,100

Table 3 Determination of the level of mm LDL and MM-LDL in serum or plasma of patients with instrumentally confirmed atherosclerosis of the carotid arteries Serum n mmLP (> 10ED) MM-LDL, (> 10ED) patients with atherosclerosis of carotid arteries 60 26 56 Blood plasma of patients with atherosclerosis of carotid arteries twenty 8 19

Claims (1)

A method for determining minimally modified low density lipoproteins (MM-LDL) in human blood serum (plasma) by treatment with a buffer containing polyvinylpyrrolidone (PVP) followed by turbidimetric registration of the mixture, characterized in that the treatment of human serum or plasma is carried out with PVP-12600 solution in 0.01 M Tris-HCl buffer, pH 7.4, containing 0.15 M NaCl, at a concentration of PVP-12600 in the sample from 11.3% to 14.2%, incubated for 10 min at room temperature, the light absorption was measured in experimental and control samples, calculate the difference between them and with a difference of more than 10 units. ascertain blood atherogenism in the examined person due to the increased level of MM-LDL.