RU2123008C1 - Method of heparin assay - Google Patents

Abstract

FIELD: biotechnology, pharmacy, analytical biochemistry. SUBSTANCE: method involves formation of lyotropic mesomorphic dispersion from a complex of linear double-stranded DNA molecules of low molecular mass and heparin molecules as an antagonist and heparin is determined in an aqueous-salt solution. The prepared dispersion is mixed firstly with a polymer an aqueous-saline solution that is neutral with respect to DNA, antagonist and heparin and then with heparin-containing liquid to be analyzed. Mixing is carried out at conditions where optical properties of lyotropic mesomorphic dispersion complex (DNA-antagonist) do not impaired. Then a flow of circularly polarized radiation is passed through a sample obtained by the indicated mixing and optical signal is recorded by wave-length in the range of DNA absorption. EFFECT: simplified and accelerated process of diagnosis of preclinical phase of pathological state. 4 cl, 1 tbl, 4 ex

Description

 The invention relates to the field of medical research, the pharmaceutical industry and biotechnology, and more specifically to a method for determining low concentrations of heparin.

 The present invention can be used in fundamental and clinical medical biochemistry. It is most advisable to use it in practical public health in cases where changes in the physiological (very low) values of the patient’s heparin level in the patient’s blood indicate a preclinical phase of developing pathological conditions, which is extremely important for organizing preventive measures and predicting the clinical course of the pathological process under the influence of therapy. Such conditions include mainly immune-related diseases (all obstetric pathology, the chronic course of infectious diseases, antiphospholipid syndrome (AFL syndrome) with total or organ atherosclerotic vascular lesions, disseminated intravascular coagulation syndrome (DIC), etc.).

 In humans and animals, heparin is synthesized by mast cells. It is an acid mucopolysaccharide, which performs mediator functions in all organs and many biological fluids. It consists of several linear polysaccharide chains with a molecular weight of 5000-30000 Yes, consisting of residues of glucuronic acid and glucosamine, esterified with sulfuric acid (figure 1).

 The heparin molecule contains a significant amount of negatively charged sulfate and carboxyl groups, therefore it is the most powerful natural polyanion capable of forming complexes with many polycationic protein and synthetic compounds that carry a total positive charge.

 The structural features and the high degree of chemical heterogeneity of heparin determine its biological significance and functional role in humans and animals. Being a natural anticoagulant factor, heparin is most widely known as a direct type of anticoagulant used for the prevention and treatment of thromboembolic diseases and their complications, in operations on the heart and blood vessels, to maintain a liquid state of the blood in cardiopulmonary bypasses and hemodialysis, as well as to prevent blood coagulation in laboratory studies. The anticoagulant activity of heparin is due to its ability to increase the spectrum of serine proteases inhibited by antithrombin III involved in blood coagulation.

 The safety of the clinical use of heparin can only be guaranteed with the rational use of heparin antagonists, which are able to quickly and without toxic manifestations eliminate the anticoagulant effect of heparin. The rationality of using antiheparinates mainly depends on the accuracy and speed of obtaining information on the amount of heparin circulating in the bloodstream, which is the main problem of the safe use of heparin as an anticoagulant.

 In modern pharmacology, both natural and synthetic drugs are used for these purposes. Difficulties in the restoration of blood coagulation when using heparin antagonists largely depend on the nature of the distribution of heparin in the patient's body. Therefore, to calculate the neutralizing doses of heparin antagonists, it is required to determine the concentration of heparin in the blood in each case.

 The effectiveness of methods for determining heparin depends on the principle on which they are based. Therefore, all currently known methods for the determination of heparin, used in clinical or biochemical laboratories, can be divided into two groups (table) [In the book: Violations of thrombin formation reactions. / Ed. R.U. Colman. M .: Medicine, 1988, p. 175 - 208].

 The first group includes most of the methods described in the literature. These methods (coagulation methods) are based on determining the inhibitory effect of heparin on thrombin or factor Xa.

 The second group includes chemical methods that measure mainly the amount of heparin as a substance, and not its anticoagulant activity.

 In the arsenal of clinical laboratories, you must have both groups of methods. This is due to the fact that the methods of the first group are designed to determine the anticoagulant activity of heparin, which has an individual reaction in clinical samples, while the methods of the second group can be useful, for example, to compare different heparin preparations or the quantitative characteristics of the anticoagulant present in the analyzed sample, regardless of its pharmacological effect .

 Among the methods belonging to the first group, the most widely known are the so-called integral methods based on the ability of heparin to lengthen the blood coagulation time as a result of inhibition of the formation of a fibrin clot.

Despite the simplicity and fame, integrated methods for determining the concentration of heparin are characterized by a number of disadvantages, which include:
- lack of specificity (the results of integral methods, in addition to the action of heparin, are affected by various changes in coagulation in vivo);
- low sensitivity (integrated methods are practically not sensitive to low (<0.1 Units / ml) heparin concentrations);
- low reproducibility (the relative error in determining heparin concentration using integral methods is ≈ 20 - 25%).

 To avoid the disadvantages characteristic of integrated methods, methods (coagulation tests) based on the more specific action of heparin were developed.

 Despite the higher specificity compared to the integrated methods and a rather high sensitivity (from 0.1 to ≈ 0.01 U / ml heparin), coagulation tests nevertheless did not receive wide distribution. This is primarily due to their bulkiness and difficulty in execution. Therefore, the use of specific coagulation tests is possible only in specialized clinical laboratories.

 In addition to the integral and specific coagulation tests described above, methods based on the use of chromogenic peptide substrates specific for thrombin or factor Xa are also used to determine the concentration of heparin in blood plasma. In these tests, chromogenic peptide substrates act as “reporter” molecules, by changing the optical properties of which (colorimetric or fluorometric), one can monitor the rate of the heparin-catalyzed enzymatic inactivation reaction of antithrombin or factor Xa with antithrombin III. The test conditions are selected so that the enzymatic reaction rate is linearly related to the heparin level in the sample.

 The advantages of colorimetric and fluorometric methods are high and stable sensitivity to heparin (up to 0.01 U / ml) and accuracy compared to methods based on clot formation.

 It should be noted that the results of analyzes carried out using chromogenic peptide substrates are influenced by the binding of heparin to plasma protein components having an antiheparin nature. Nevertheless, methods using chromogenic peptide substrates are most convenient for clinical examination and control, but their formulation, due to the complexity and rather high cost, is possible only in well-equipped specialized laboratories with expensive equipment and highly qualified personnel.

 The second group of methods (table, chemical methods) for determining the concentration of heparin is based on the definition of heparin as a mucopolysaccharide.

 To do this, use the carbazole reaction of hexuronic acid or acetylacetone condensation of hexosamines with the quantification of subsequent pyrrole derivatives using p-dimethylaminobenzaldehyde (Ehrlich reagent).

 These methods are unsuitable for determining heparin in blood plasma, are characterized by the duration of the analysis (≈ 1 hour or more) and are not very sensitive (they can be used to detect 12 μg / ml of heparin or 1.6 Units / ml of its activity).

 Another group of chemical methods is based on the ability of heparin as a mucopolysaccharide to cause a metachromatic effect when it interacts with some of the main dyes (azure A, toluidine blue, carbocyanine dye). The intensity of metachromasia during the formation of the complex (heparin-dye) at the optimal concentration and volume of the solution of the added dye is proportional to the concentration of heparin: this value can be determined spectrophotometrically. However, metachromatic methods, like methods based on the use of chemical reactions specific for heparin, are also unsuitable for determining heparin in blood plasma, are quite expensive, have poor reproducibility and are not accurate enough. The last two drawbacks are due to precipitation of the resulting complex (heparin-dye) and the sensitivity of the absorption readings of this complex to temperature.

 The minimum concentration of heparin, determined using metachromatic methods, is ≈ 10 μg / ml or 1 Unit / ml of its activity. The relative error in determining heparin concentration using this group of methods is 10-15%.

 Thus, the above disadvantages significantly complicate the rapid obtaining of accurate information on the concentration of heparin in the analyzed samples and limit the widespread use of the above groups of methods (Table 1) for such analyzes in clinical and experimental laboratories.

 The basis of the present invention is the task of creating a method for determining the concentration of heparin with such conditions that would allow it to quickly, simply, accurately and reliably determine its concentration (including very low (<1 μg / ml)).

 This problem is solved as a result of creating a method for determining the concentration of heparin interacting with its antagonist, which is part of the lyotropic liquid crystal dispersion of the complex (DNA antagonist), which consists in the fact that in a water-salt solution of moderate (physiological) ionic strength from a complex of linear double-stranded molecules Low molecular weight DNA and heparin antagonist molecules form a lyotropic liquid crystal dispersion, and the resulting dispersion is mixed first with a DNA neutral , the antagonist and heparin with a water-salt solution of the polymer, and then with the analyzed liquid containing heparin, under conditions in which the optical properties of the lyotropic liquid crystal dispersion of the complex (DNA antagonist) are not violated, then a stream is passed through the sample obtained as a result of this mixing circularly polarized radiation, at the wavelength in the region of DNA absorption, an optical signal is recorded, the concentration of heparin in the sample is determined by the calibration line and its concentration is adjusted in the analysis Rui fluid with the dilution used in the preparation of the sample.

 Using the proposed method allows you to quickly, simply, with high accuracy and sensitivity to determine the concentration of heparin in the analyzed sample. Analyzes can be carried out in any laboratories, since their implementation does not require a specially organized workplace and highly qualified technical personnel.

 It is advisable to use polyethylene glycol (PEG) as a neutral polymer, since this polymer is harmless to the personnel involved in the analysis, chemically neutral with respect to DNA, polycation and heparin, has high solubility in aqueous salt solutions of moderate ionic strength, necessary to create phase conditions DNA exclusion, has optical isotropy and high transparency required for measuring CD spectra; in addition, preparations of this polymer of different molecular weights are available at a reasonable price.

 It is advisable that the polycation is a compound belonging to the class of polyamines, has a narrow molecular weight distribution, does not absorb in the ultraviolet region of the spectrum, forms a complex not only with heparin, but also with double-stranded linear DNA molecules, and has low toxicity, combined with antiheparin activity .

 It is desirable that the polycation was a quaternary ammonium salt (QAS) of the conidine oligomer (polyconidine) with a degree of polymerization of 25 (Fig. 2), since the compounds of this group are currently the most promising highly selective and low-toxic antiheparins [Chernova O. V. Study of antiheparin activity and the effect on blood coagulation of the Quaternary ammonium salts of monodisperse conidine oligomers. Abstract of Cand. dissertations. M .: 1983].

 Thus, the proposed method can be used for quick, highly accurate and highly sensitive determination of heparin concentration in cases where other laboratory methods are not applicable or do not give the proper effect.

The proposed method for determining the concentration of heparin in the analyzed fluid (solution) is illustrated by the schemes shown in figure 3 (A-D), and is as follows:
- immediately before determining the concentration of heparin in the analyzed fluid (solution) in a water-salt solution of moderate ionic strength, a lyotropic liquid crystal dispersion of the complex (DNA-polyconidine) is formed (Fig. 3A);
- mix the water-salt solution of the liquid crystal dispersion of the complex (DNA-polyconidin) with the water-salt solution of the polymer, which leads to a mixture in which the heparin molecules almost instantly react with the polyconidine molecules, forming a complex with DNA molecules (figb);
- mix the polymer-containing water-salt solution of a liquid crystal dispersion of the complex (DNA-polyconidine) with an equal volume of liquid (solution) in which heparin is present; this results in a sample ready for analysis (FIG. 3B);
- removal of polyconidin molecules from the complex (DNA-polyconidine) as a result of formation of a complex (heparin-polyconidin) in the sample leads to a rearrangement of the spatial organization of the initial lyotropic liquid crystal dispersion of the complex (DNA-polyconidin) and is accompanied by the formation of cholesteric liquid crystal DNA dispersion (Fig. 3G) ;
- irradiate the sample with circularly polarized light;
- measure the magnitude of the optical signal generated by the breakdown at λ 280 nm;
- determine the concentration of heparin in the sample using the calibration line previously constructed according to the method described above, which represents the dependence of the magnitude of the optical signal generated by the liquid crystal dispersion of the complex (DNA-polyconidin) upon addition of heparin, on the concentration of heparin;
- correct the concentration of heparin in the analyzed fluid (solution), taking into account its dilution used in the preparation of the sample.

For a better understanding of the present invention, the following are examples characterizing the various stages of the proposed method with reference to the accompanying figures, in which:
FIG. 1 characterizes the chemical formula of the monomer unit of the polysaccharide chain of a heparin molecule;
FIG. 2 characterizes the chemical formula of the ammonium salt of the monodisperse oligomer of conidine with a degree of polymerization of 25;
figure 3 characterizes:
a) a scheme for the formation of a moderate ionic strength of a lyotropic liquid crystal dispersion of a complex (DNA-polyconidine) in a water-salt solution;
b) a schematic representation of the lyotropic liquid crystal dispersion of the complex (DNA-polyconidin) in PEG water-salt solution;
c) a schematic representation of a water-salt solution of PEG containing a lyotropic liquid crystal dispersion of a complex (DNA-polyconidine) into which heparin is added;
d) a schematic representation of the spatial rearrangement of DNA molecules in particles of a lyotropic liquid crystal dispersion of a complex (DNA-polyconidin) under the action of heparin, leading to the formation of a cholesteric liquid crystal dispersion of DNA in an aqueous salt solution of PEG;
FIG. 4 characterizes the absorption spectra of aqueous-salt solutions of linear double-stranded DNA (curve 1) and lyotropic liquid crystal dispersion of the complex (DNA-polyconidine) (curve 2) in the coordinates “optical density, A” - “wavelength, λ (nm)”.

Chicken erythrocyte DNA (Reanal, Hungary);
molecular weight of DNA ≈ (0.3-0.5) • 10 ⁶ Yes;
C _DNA = 30 μg / ml;
0.3 M NaCl + 10 ^-2 M phosphate buffer; pH ≈ 7.0.

curve 1-C _{poly conidine} = 0;
curve 2-C _{polik onidine} = 13.9 • 10 ^-3 mg / ml;
FIG. 5 characterizes the CD spectra of water-salt solutions of linear double-stranded DNA (curve 1) and lyotropic liquid crystal dispersion of the complex (DNA-polyconidine) (curve 2) in the coordinates "Δε = ε _L- ε _R " - "wavelength, λ (nm)" :
where Δε is the molar circular dichroism (Δε = ΔA / C _dna );
ΔA - experimentally measured circular dichroism;
C _DNA - DNA concentration;
ε _L - molar dichroism for left-circularly polarized light;
ε _R - molar dichroism for right-circularly polarized light;
Chicken erythrocyte DNA (Reanal, Hungary);
molecular weight of DNA ≈ (0.3-0.5) • 10 ⁶ Yes;
C _DNA = 30 μg / ml;
0.3 M NaCl + 10 ^-2 M phosphate buffer; pH ≈ 7.0.

curve 1 - C _{poly conidine} = 0;
curve 2 - C _{polik onidine} = 13.9 • 10 ^-3 mg / ml;
FIG. 6 characterizes the CD spectrum of a cholesteric liquid crystal DNA dispersion formed in a PEG-containing water-salt solution in the coordinates "Δε = ε _L- ε _R " - "wavelength, λ (nm)":
Chicken erythrocyte DNA (Reanal, Hungary);
molecular weight of DNA ≈ (0.3-0.5) • 10 ⁶ Yes;
C _DNA = 15 μg / ml;
0.3 M NaCl + 10 ^-2 M phosphate buffer; pH ≈ 7.0.

C _PEG = 170 mg / ml;
molecular weight PEG = 4000 ("Ferak", Germany);
FIG. 7 characterizes the CD spectra of PEG-containing water-salt solutions of a lyotropic liquid crystal dispersion of a complex (DNA-polyconidin) and cholesteric liquid crystal DNA dispersion before (respectively, curves 1 and 3) and after (respectively, curves 2 and 4) heparin addition in coordinates Δε = ε _L- ε _R "-" wavelength, λ (nm) ",
Chicken erythrocyte DNA (Reanal, Hungary);
molecular weight of DNA ≈ (0.3-0.5) • 10 ⁶ Yes;
C _DNA = 15 μg / ml;
0.3 M NaCl + 10 ^-2 M phosphate buffer; pH ≈ 7.0.

C _PEG = 170 mg / ml;
molecular weight PEG = 4000 ("Ferak", Germany);
curve 1 - C _{poly conidine} = 6.95 • 10 ^-3 mg / ml;
C _heparin = 0;
curve 2 - C _{poly conidine} = 6.95 • 10 ^-3 mg / ml;
C _heparin = 2.5 • 10 ^-3 mg / ml;
heparin from pig mucose (Na-salt, "Serva", Germany) with a specific activity of 186,000 Units / g.

curve 3 - C _heparin = 0;
curve 4 - C _heparin = 2.5 • 10 ^-3 mg / ml;
heparin from porcine mucose (Na-salt, Serva, Germany) with a specific activity of 186000 U / g;
FIG. 8 characterizes the CD spectra of a PEG-containing water-salt solution of a lyotropic liquid crystal dispersion of the complex (DNA-polyconidine) at different heparin concentrations in the coordinates "Δε = ε _L- ε _R " - "wavelength, λ (nm)".

Chicken erythrocyte DNA (Reanal, Hungary);
molecular weight of DNA ≈ (0.3-0.5) • 10 ⁶ Yes;
C _DNA = 15 μg / ml;
0.3 M NaCl + 10 ^-2 M phosphate buffer; pH ≈ 7.0.

C _PEG = 170 mg / ml;
molecular weight PEG = 4000 ("Ferak", Germany);
C _{polic onidine} = 6.95 • 10 ^-3 mg / ml.

1 - C _heparin = 0; 2 - C _heparin = 0.2 • 10 ^-3 mg / ml;
3 - C _heparin = 0.4 • 10 ^-3 mg / ml; 4 - C _heparin = 0.6 • 10 ^-3 mg / ml;
5 - C _heparin = 0.8 • 10 ^-3 mg / ml; 6 - C _heparin = 1.0 • 10 ^-3 mg / ml;
7 - C _heparin = 1.4 • 10 ^-3 mg / ml; 8 - C _heparin = 2.5 • 10 ^-3 mg / ml.

Heparin from porcine mucose (Na-salt, Serva, Germany) with a specific activity of 186,000 U / g;
FIG. 9 characterizes the dependence of the amplitude of the negative band in the CD spectra of the PEG-containing water-salt solution of the lyotropic liquid crystal dispersion of the complex (DNA-polyconidine) on the concentration of heparin in the coordinates "Δε ₂₈₀ " - "concentration of heparin, C _heparin • 10 ³ , (mg / ml) "

The arrow in FIG. Figure 9 shows the concentration of heparin, up to which a direct proportional relationship is observed between Δε ₂₈₀ and the concentration of heparin;
FIG. 10 characterizes the calibration line, designed to determine the concentration of heparin in the sample, in coordinates "Δε ₂₈₀ " - "concentration of heparin, C _heparin , (μg / ml)."

 The arrows in FIG. 10, as an example, a method for determining heparin concentration in a sample is shown.

 Example 1. The formation of a lyotropic liquid crystal dispersion of the complex (DNA-polyconidine) in a water-salt solution of moderate ionic strength.

1.1 Samples of NaCl (17.532 g), NaH ₂ PO ₄ • 2H ₂ O (0.78 g) and Na ₂ HPO ₄ • 12H ₂ O (1.79 g) are placed in a volumetric flask (V = 1000 ml) and dissolved in distilled water; adjust the volume of the solution to the mark.

In this way, 1 l of a 0.3 M NaCl solution containing 10 ^-2 M phosphate buffer (pH ≈ 7.0) is prepared.

1.2 10 mg of a linear double-stranded DNA preparation (Reanal, Hungary; molecular weight (0.3-0.5) • 10 ⁶ Yes) is placed in a measuring tube (V = 10 ml) and dissolved in 5 ml of a solution prepared according to . 1.1; adjust the volume of the solution to 10 ml. The DNA concentration is determined spectrophotometrically, based on the ratio of 1 mg of DNA corresponds to ≈20 optical units per 1 ml (λ _max = 258.4 nm, ε _max = 6600 M ^-1 cm ^-1 ; pH ≈7.0).

In this way, 10 ml of a water-salt solution of DNA with a fixed concentration is prepared (C _DNA = 1 mg / ml; 0.3 M NaCl + 10 ^-2 M phosphate buffer; pH ≈7.0).

 1.3 A portion of the quaternary ammonium salt of the conidin oligomer with a degree of polymerization of 25 (4 mg) is placed in a measuring tube (V = 5 ml) and dissolved in 1.5 ml of the solution prepared according to paragraph 1.1; Bring the solution to 2 ml.

In this way, 2 ml of a fixed-concentration polyconidin water-salt solution is prepared (C _{polik onidine} = 2 mg / ml; 0.3 M NaCl + 10 ^-2 M phosphate buffer; pH ≈ 7.0).

1.4 Samples of NaH ₂ PO ₄ • 2H ₂ O (0.078 g) and Na ₂ HPO ₄ • 12H ₂ O (0.179 g), NaCl (1.753 g) and PEG (34 g; molecular weight PEG = 4000, "Ferak", Germany ) are placed in a volumetric flask (V = 100 ml) and dissolved in distilled water; adjust the volume of the solution to the mark.

In this way, 100 ml of PEG water-salt solution is prepared (C _PEG = 340 mg / ml; 0.3 M NaCl + 10 ^-2 M phosphate buffer; pH ≈ 7.0).

 All solutions prepared according to p.p. 1.1-1.4, filtered to remove solids through nitrocellulose filters with a pore diameter of 1.5 microns (Chemapol, Czech Republic).

 1.5 In a glass tube (V = 10 ml) 4.85 ml of solution 1.1 are mixed with 0.15 ml of solution 1.2.

In this way, 5 ml of a water-salt solution of linear double-stranded DNA of a given concentration is prepared (C _DNA = 30 μg / ml; 0.3 M NaCl + 10 ^-2 M phosphate buffer; pH ≈ 7.0).

 After mixing solutions 1.1 and 1.2, the absorption and CD spectra of an aqueous-salt solution of a linear double-stranded DNA of a given concentration prepared according to clause 1.5 (see Fig. 4 (curve 1) and Fig. 5 (curve 1), which show the absorption spectrum and CD spectrum of a water-salt solution of linear double-stranded DNA, respectively).

 1.6 In a glass tube (V = 10 ml) containing 3 ml of a water-salt solution of a linear double-stranded DNA prepared according to clause 1.5, 7 μl of a solution prepared according to clause 1.3 is added with constant stirring (the total volume of the added solution 1.3 is 21 μl).

In this way, a lyotropic liquid crystal dispersion of the complex (DNA-polyconidine) is formed in a water-salt solution of moderate ionic strength (C _DNA = 30 μg / ml; 0.3 M NaCl + 10 ^-2 M phosphate buffer; pH ≈ 7.0);
C _{poly conidine} ≈ 13.9 • 10 ^-3 mg / ml).

 1.7 In a glass tube (V = 10 ml), 1.5 ml of solution (1.4) is mixed with 1.5 ml of solution (1.5); the resulting mixture was stirred vigorously for 3 minutes.

In this way, a cholesteric liquid crystal DNA dispersion is formed in a PEG-containing water-salt solution (C _DNA = 15 μg / ml; C _PEG = 170 mg / ml; 0.3 M NaCl + 10 ^-2 M phosphate buffer; pH ≈ 7.0 )

After the preparation obtained according to paragraphs. 1.6 and 1.7 of liquid crystal dispersions of the complex (DNA-polyconidin) and DNA, their properties are controlled using optical methods (spectrophotometry and CD spectroscopy). The appearance of an "apparent" optical density (A _k ) in the region of wavelengths exceeding 320 nm (Fig. 4, curve 2), in which neither DNA nor polyconidine have a noticeable intrinsic absorption, indicates that as a result of mixing the solutions, cooked by pp 1.3 and 1.5, a lyotropic liquid crystal dispersion of the complex is formed (DNA-polyconidine). CD spectroscopy data (Fig. 5, curve 2) indicate that the lyotropic liquid crystal dispersion of the complex (DNA-polyconidin) formed in a moderate-ionic strength aqueous salt solution in accordance with the scheme shown in FIG. 3A does not possess an abnormal optical activity, which should manifest itself as an intense negative band with a maximum at λ ~ 270 nm (Fig. 6) and is characteristic of cholesteric liquid crystal DNA dispersion.

 Example 2. The optical properties of the lyotropic liquid crystal dispersion of the complex (DNA-polyconidine) in a PEG-containing water-salt solution before and after addition of heparin.

 2.1 A weighed portion of heparin Na-salt (4 mg; Serva, Germany; specific activity of 186000 U / g) is placed in a volumetric glass tube (V = 5 ml) and dissolved in 1.5 ml of the solution prepared in accordance with 1.1; Bring the solution to 2 ml.

In this way, 2 ml of a fixed concentration of heparin water-salt solution is prepared (C _heparin = 2 mg / ml; 0.3 M NaCl + 10 ^-2 M phosphate buffer (pH ≈ 7.0).

 2.2 In a glass tube (V = 10 ml), 1.5 ml of a water-salt solution of a lyotropic liquid crystal dispersion of a complex (DNA-polyconidine) prepared according to 1.6, and 1.5 ml of a PEG-containing water-salt solution prepared according to paragraph 1.4; the resulting mixture was stirred vigorously for 3 minutes.

In this way, a PEG-containing water-salt solution of a lyotropic liquid crystal dispersion of the complex (DNA-polyconidin) is prepared, the physicochemical properties of which provide the conditions necessary for the phase exclusion of linear double-stranded DNA molecules and the formation of cholesteric liquid crystal DNA dispersion (C _DNA = 15 μg / ml ; C _{poly conidine} = 6.95 • 10 ^-3 mg / ml; C _PEG = 170 mg / ml; 0.3 M NaCl + 10 ^-2 M phosphate buffer; pH ≈ 7.0).

 2.3 In a rectangular optical quartz cuvette (V = 4 ml; optical path length 1 cm), add 2 ml of the solution prepared according to 2.2, and record its CD spectrum in the wavelength range 220-350 nm using a Jobin-Yvon dichrograph Mark III "(France).

 2.4 After recording the CD spectrum in an optical cuvette containing 2 ml of a solution prepared according to clause 2.2, add 2.5 μl of a water-salt solution of heparin prepared according to clause 2.1; the mixture obtained in an optical cuvette is mixed and its CD spectrum is recorded in the wavelength range of 220-350 nm.

 2.5 Similar to paragraphs. 2.3-2.4, as a control, CD spectra of 2 ml of a PEG-containing water-salt solution of a cholesteric liquid crystal DNA dispersion prepared according to 1.7 are recorded, before and after the addition of 2.5 μl of a heparin solution prepared according to 2.1.

 In FIG. Figure 7 shows the CD spectra of PEG-containing water-salt solutions of a lyotropic liquid crystal dispersion of a complex (DNA-polyconidin) and cholesteric liquid crystal dispersion of DNA before (respectively, curves 1 and 3) and after (respectively, curves 2 and 4) heparin additions. A comparison of curve 1 with curve 3 shows that the lyotropic liquid crystal dispersion of the complex (DNA-polyconidin), placed in a PEG-containing water-salt solution, does not have the abnormal optical activity characteristic of cholesteric liquid crystal DNA dispersion.

 The addition of heparin to a solution containing a lyotropic liquid crystal dispersion of the complex (DNA-polyconidin) is accompanied by the appearance in the CD spectrum of an intense negative band located in the absorption region of the nitrogenous DNA bases (curve 3). The amplitude of the band in the CD spectrum that arose after heparin was added does not practically differ from the amplitude of the negative band observed in the CD spectrum of cholesteric liquid crystal DNA dispersion (curve 2). As the control shows, the addition of heparin to a solution containing a cholesteric liquid crystal dispersion of DNA does not affect its abnormal optical properties (compare curves 2 and 4).

 A multiple increase in the band amplitude in the CD spectrum of the PEG-containing water-salt solution of the lyotropic liquid crystal dispersion of the complex (DNA-polyconidine) after heparin is added indicates a change in the spatial organization of the DNA molecules that form it. This change is due to the dissociation of the complex (DNA-polyconidin) under the action of heparin and the formation of a more durable complex (heparin-polyconidine). As a result of the removal of polyconidine molecules from the complex (DNA-polyconidine), the DNA molecules are rearranged, forming a cholesteric liquid crystal DNA dispersion with abnormal optical properties (Fig. 3G).

 Thus, the appearance of an intense negative band in the CD spectrum of a PEG-containing water-salt solution of a lyotropic liquid crystal dispersion of the complex (DNA-polyconidin) can be used as a criterion to determine the presence of heparin in the analyzed solution (liquid).

 Example 3. The analytical capabilities of the lyotropic liquid crystal dispersion of the complex (DNA-polyconidine) in a PEG-containing water-salt solution.

 3.1 In a test tube (V = 0.5 ml), 20 μl of a solution of heparin prepared according to clause 2.1 are mixed with 180 μl of a solution prepared according to clause 1.1.

In this way, 200 μl of a fixed concentration of heparin water-salt solution is prepared (C _heparin = 0.2 mg / ml; 0.3 M NaCl + 10 ^-2 M phosphate buffer; pH ≈ 7.0).

 3.2 In a rectangular quartz cuvette (V = 4 ml; optical path length 1 cm), 2 ml of a PEG-containing water-salt solution of a lyotropic liquid crystal dispersion of the complex (DNA-polyconidine) prepared according to Section 2.2 are placed and its CD spectrum is recorded in the region wavelengths of 220-350 nm.

 3.3 After registering the CD spectrum of the initial PEG-containing water-salt solution of the lyotropic liquid crystal dispersion of the complex (DNA-polyconidin), 2 μl of solution 3.1 is added to the cuvette (the total volume of added solution 3.1 is 30 μl); after each addition of solution 3.1, the mixture obtained in an optical cuvette is mixed and the CD spectrum is recorded in the wavelength range of 220-350 nm.

 In FIG. 8, the CD spectrum of the initial lyotropic liquid-crystal dispersion of the complex (DNA-polyconidin) (curve 1) is compared with the CD spectra of the same dispersion, to which different concentrations of heparin are added (curves 2-8). The addition of heparin is accompanied by the appearance in the CD spectrum of a lyotropic liquid crystal dispersion of the complex (DNA-polyconidin) of a negative band with a maximum at λ ~ 280 nm, the amplitude of which increases with increasing concentration of heparin in the solution.

The dependence of the band amplitude at λ = 280 nm in the CD spectrum of the lyotropic liquid crystal dispersion of the complex (DNA-polyconidin) on the concentration of heparin (Fig. 9) shows that between Δε ₂₈₀ and the concentration of heparin there is a directly proportional dependence in the range of heparin concentrations from 0 to ( 1.2-1.3) • 10 ^-3 mg / ml.

 The presence of a direct proportional relationship between the magnitude of the optical signal generated by the lyotropic liquid crystal dispersion of the complex (DNA-polyconidin) in the PEG-containing water-salt solution after adding heparin and the concentration of heparin allows you to use a straight section of the curve shown in FIG. 9, as a calibration line.

 The dependence shown in FIG. 10 is a calibration line, using which it is possible to determine with high accuracy low (<1 μg / ml) heparin concentrations in the analyzed sample.

 Thus, the intense negative band in the absorption region of nitrogenous DNA bases in the CD spectrum, combined with the presence of a directly proportional relationship between the amplitude of this band and the concentration of added heparin, can be used to determine the presence and low concentrations of heparin in the analyzed solution (liquid).

 Example 4. The regulation for determining the concentration of heparin in the analyzed solution.

 4.1 In a test tube (V = 5 ml), 1 ml of the analyzed heparin solution and 1 ml of the PEG-containing water-salt solution prepared in accordance with clause 1.4 are mixed.

In this way, 2 ml of an assayed solution of heparin containing PEG (C _PEG = 170 mg / ml) is prepared.

 4.2 In a test tube (V = 5 ml), 1 ml of a test solution of heparin containing PEG prepared in accordance with clause 4.1 is mixed with 1 ml of a PEG-containing water-salt solution of a lyotropic liquid crystal dispersion complex (DNA-polyconidine) prepared in accordance with clause 2.2 .

 In this way, a sample is prepared that is ready for analysis.

 4.3. The CD spectrum of the sample prepared in accordance with clause 4.2 is recorded in the wavelength range of 220-350 nm.

4.4 Determine the value of Δε ₂₈₀ , characteristic of the CD spectrum of the sample.

4.5 Put the experimentally obtained value of Δε ₂₈₀ on the calibration line (Fig. 10).

If the experimentally obtained value of Δε ₂₈₀ is within -20 units. <Δε ₂₈₀ <-100 units, then using the calibration line determine the concentration of heparin in the sample, corresponding to the experimentally obtained value of Δε ₂₈₀ (the method for determining the concentration of heparin in the sample is schematically shown by arrows in Fig. 10).

4.6 Using the direct value of the concentration of heparin obtained in the sample, the concentration of heparin in the analyzed solution is determined taking into account its dilution associated with the preparation of the sample:
C _heparin (test solution) = 4 • C _heparin (test)
4.7 If the experimentally measured value of Δε is ₂₈₀ > -100 units, in this case only an approximate determination of the concentration of heparin in the analyzed solution is possible (in this case, it can only be stated that the concentration of heparin in the analyzed solution exceeds 4 μg / ml).

4.8 To clarify the concentration of heparin, the analyzed solution is diluted with a water-salt solution prepared in accordance with paragraph 1.1, in 2, 4, etc. 2 ⁿ times. After each dilution with the obtained heparin solution, the operations listed in paragraphs are repeated. 4.1-4.6. These operations are repeated until the experimentally recorded value of Δε ₂₈₀ is in the range of -20 units <Δε ₂₈₀ <-100 units
4.9 In the case when, after the 2nd ⁿ -th dilution of the analyzed heparin solution, the experimentally measured value of Δε ₂₈₀ is within the range of -20 units <Δε ₂₈₀ <-100 units, using the calibration line determine the corresponding concentration of heparin in the sample.

4.10 Using the obtained value of the concentration of heparin in the sample (paragraph 4.9), determine the concentration of heparin in the analyzed solution, taking into account all stages of dilution associated with the preparation of the sample:
C _heparin (μg / ml, test solution) = 2 ^{n + 2} • C _heparin (μg / ml, sample)
The minimum concentration of heparin, determined in the analyzed liquid (solution) using the proposed regulation, based on measuring the optical signal generated by the lyotropic liquid crystal dispersion of the complex (DNA-polyconidine) under the action of heparin, using a Jobin-Yvon, Mark III dichrograph (France) is ≈ 0.4-0.5 μg / ml.

Claims (4)

 1. The method of determining heparin interacting with its antagonist, which is part of the lyotropic liquid crystal dispersion of the complex (DNA antagonist), which consists in the fact that in the water-salt solution of the complex of linear double-stranded DNA molecules of low molecular weight and heparin antagonist molecules form a lyotropic liquid crystal dispersion, the resulting dispersion is mixed first with a neutral aqueous solution of the polymer, neutral with respect to DNA, an antagonist and heparin, and then with the analyzed liquid, with containing heparin, then a circularly polarized radiation flux is passed through the sample obtained as a result of this mixing, an optical signal is recorded at the wavelength in the DNA absorption region and the concentration of heparin in the sample is determined taking into account the previously obtained calibration dependence.  2. The method according to claim 1, characterized in that polyethylene glycol is used as a neutral polymer.  3. The method according to claim 1, characterized in that as a heparin antagonist use a compound belonging to the class of polyamines.  4. The method according to claim 3, characterized in that the quaternary ammonium salt of the conidine oligomer with a degree of polymerization of 25 is used as the polyamine.

Images