CN116256455A - Method for identifying bovine pulmonary heparin doping content in porcine intestinal mucosa derived heparin - Google Patents
Method for identifying bovine pulmonary heparin doping content in porcine intestinal mucosa derived heparin Download PDFInfo
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- CN116256455A CN116256455A CN202310289064.7A CN202310289064A CN116256455A CN 116256455 A CN116256455 A CN 116256455A CN 202310289064 A CN202310289064 A CN 202310289064A CN 116256455 A CN116256455 A CN 116256455A
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Abstract
The invention provides a method for identifying bovine pulmonary heparin doping content in porcine intestinal mucosa derived heparin, and belongs to the technical field of heparin source identification. The method comprises the following steps: establishing a functional relation by using the delta IIIs/deltaIIa ratio calculated based on the pure pig intestinal mucosa source heparin sodium disaccharide component and the pure cattle lung source heparin sodium disaccharide component respectively; calculating the delta IIIs/delta IIa ratio of disaccharide components of the heparin sodium sample to be detected, and calculating the doping percentage content of the bovine pulmonary source heparin by using the functions. The method can directly characterize the composition difference of heparin sugar chains, and identify whether the sample to be detected contains bovine-derived heparin or not, and is simple and quick.
Description
Technical Field
The invention belongs to the technical field of heparin source identification, and particularly relates to a method for identifying bovine pulmonary heparin doping content in porcine intestinal mucosa source heparin.
Background
Heparin is an acid mucopolysaccharide, the skeleton structure of which is a linear polysaccharide mixture formed by connecting disaccharide repeating units consisting of uronic acid and D-glucosamine by 1-4 glycosidic bonds, and the heparin has non-uniformity and is clinically used as an anticoagulant and antithrombotic drug.
Heparin is expressed by mast cells of mammalian connective tissue and biosynthesized in the form of heparin proteoglycans. Heparin is widely distributed in mammalian tissues such as liver, lung, intestinal mucosa, heart, spleen, kidney, thymus, placenta, muscle and blood, etc., and can be extracted from small intestinal mucosa of pig, cattle, sheep, etc. and bovine lung, etc. Heparin sodium from different sources has different chemical structures and biological activities, and the adverse reaction degrees are different, for example, the adverse reaction of bovine heparin sodium for causing thrombocytopenia is about 2 times that of heparin sodium from pig sources, currently, heparin sodium and low molecular heparin approved by European Union and American market are only sourced from pig intestinal mucosa, and heparin sodium mixed with bovine and sheep sources and the like is regarded as an unqualified product, so that distinguishing species sources has become an important analysis index for evaluating the quality of heparin sodium.
The current method for detecting and analyzing heparin sodium from different species comprises the following steps: fluorescent quantitative PCR method, nuclear magnetic resonance method, electrospray mass spectrometry (ESI-MS), immunological detection method, etc. The fluorescent quantitative PCR method does not directly detect heparin, but indirectly proves whether the sample is polluted by heparin of other sources by detecting whether the sample has DNA of other ruminants or not, but the DNA is easy to be processed and destroyed under the conditions of oxidization, nuclease treatment and the like, so that the actual condition of the sample with the DNA processed is difficult to be effectively reflected by the fluorescent quantitative PCR method; the methods such as nuclear magnetic resonance method, electrospray mass spectrometry and immunological detection are expensive in required instruments, high in use cost, complex in detection and analysis steps and difficult to master quickly.
Disclosure of Invention
The invention provides a method for identifying bovine pulmonary heparin doping content in porcine intestinal mucosa derived heparin, which can directly represent the composition difference of heparin sugar chains, and identify whether bovine pulmonary derived heparin is contained in a sample to be detected, and is simple and quick.
In order to achieve the above purpose, the invention provides a method for identifying the doping content of bovine pulmonary heparin in porcine intestinal mucosa derived heparin, which comprises the following steps:
establishing a functional relation by using the delta IIIs/deltaIIa ratio calculated based on the pure pig intestinal mucosa source heparin sodium disaccharide component and the pure cattle lung source heparin sodium disaccharide component respectively;
calculating the delta IIIs/delta IIa ratio of disaccharide components of the heparin sodium sample to be detected, and calculating the doping percentage content of bovine pulmonary heparin by using the functions;
wherein DeltaIIIs is 2-sulfo-Delta4, 5-glucuronic acid- (beta 1-4) -N-sulfonic acid glucosamine, deltaIIa is Delta4, 5-glucuronic acid (beta 1-4) -6-sulfo-N-acetylglucosamine.
The inventor finds that the heparin sugar chains from pig intestine mucous membrane and bovine lung are degraded into disaccharide composition units through heparinase in the research process, the disaccharide composition analysis is carried out on the degraded samples by using a strong anion exchange high performance liquid chromatography (SAX-HPLC), and then the area normalization method is used for carrying out in-depth analysis on 8 key disaccharides composing heparin sodium sugar chains, so that the results show that the composition of the heparin disaccharides from pig intestine mucous membrane and bovine lung is obviously different. As shown in table 1:
table 1: heparin sodium from pig intestine mucosa and heparin sodium disaccharide component from cattle lung
From the data in Table 1, the disaccharide structures with the greatest relative standard deviation were ΔIIa, ΔIIIs, and ΔIa, and from the 3 disaccharides, it was easier to distinguish porcine intestinal mucosa from bovine-derived heparin by theoretical analysis. Specifically, the content of heparin sodium from porcine intestinal mucosa is far greater than that of heparin sodium from bovine lung for delta IIa and delta Ia in the 3 disaccharides; for delta IIIs, the content of heparin sodium from porcine intestinal mucosa is far less than that of heparin sodium from bovine lung. This analysis is also confirmed by the data in Table 1, if either ΔIIIs/ΔIIaor ΔIIIs/ΔIaare used to distinguish porcine intestinal mucosa-derived heparin sodium from bovine lung-derived heparin sodium. The approach adopted by the present solution is therefore based on this design.
Preferably, the functional relationship is established by the following method:
the ratio of DeltaIIIs/DeltaIIacalculated based on the disaccharide component of heparin sodium derived from pure porcine intestinal mucosa was averaged and recorded as coordinates (Y 1 ，0)；
The ratio of DeltaIIIs/DeltaIIacalculated based on the heparin sodium disaccharide component derived from pure bovine lung was averaged and recorded as coordinates (Y 2 ，1)；
In the above two coordinates (Y 1 ，0)，(Y 2 1) establishing a function Y (x) according to a two-point one-line method, wherein Y is the delta IIIs/delta IIa ratio of a sample to be detected, and x is the doping percentage of bovine pulmonary derived heparin in the sample to be detected.
In the above scheme, the calculation may be preferably performed directly by using the formula y=20.83x+2.36. By the calculation of the formula, the delta IIIs/deltaIIa of the pure porcine intestinal mucosa-derived heparin and the pure bovine lung-derived heparin do not need to be additionally measured, and the use is more convenient.
Preferably, at least > 1 batch of the pure porcine intestinal mucosa-derived heparin sodium disaccharide component and the pure bovine lung-derived heparin sodium disaccharide component are calculated separately as an average of the ratio Δiiis/Δiia.
Preferably, the calculated Δiiis/Δiiais replaced by Δiiis/Δia.
Preferably, the method for calculating the ratio ΔIIIs/ΔIIais specifically as follows:
dissolving and filtering pure pig intestine mucosa source heparin, pure bovine lung source heparin or heparin sample to be tested, and performing enzymolysis to obtain respective heparin enzymolysis liquid;
and detecting the contents of delta IIIs and delta IIa in the heparin enzymolysis liquid by adopting a strong anion exchange high performance liquid chromatography method, and further calculating to obtain the ratio of delta IIIs/delta IIa.
Preferably, the enzymolysis method specifically comprises the following steps:
respectively weighing 20mg of pure porcine intestinal mucosa source heparin, pure bovine lung source heparin or heparin sample to be tested, adding 1ml of purified water for dissolution, and filtering with a 0.22 mu m filter membrane to obtain respective solutions to be subjected to enzymolysis;
taking 20 mu L of each solution to be subjected to enzymolysis, and carrying out enzymolysis by using 100 mu L of mixed solution of heparinase I, II and III to obtain respective heparin enzymolysis solution;
wherein, the heparanase I, II and III mixed solution is formed by mixing heparanase I, II and III with the concentration of 0.4 IU/mu L in equal volume; the heparanase I is CAS NO:9025-39-2; heparanase II is CAS NO:149371-12-0; heparanase III is CAS NO:37290-86-1.
Preferably, a detection column with silica gel chemically bonded with strong alkaline quaternary ammonium salt anion exchange stationary phase as filler, 5 μm inner diameter particle and a protection column with the same filler are used for detection by strong anion exchange high performance liquid chromatography.
Preferably, the gradient elution is performed by using the fluidity A and the mobile phase B during detection;
the mobile phase A is 2mmol/L sodium dihydrogen phosphate aqueous solution, and the pH=3.0; the mobile phase B is an aqueous solution of 1mol/L sodium perchlorate and 2mmol/L sodium dihydrogen phosphate, and the pH=3.0.
Preferably, the gradient elution procedure used in the detection is as follows:
| time (minutes) | Mobile phase a (%) | Mobile phase B (%) |
| 0 | 97 | 3 |
| 20 | 65 | 35 |
| 50 | 0 | 100 |
| 60 | 0 | 100 |
| 61 | 97 | 3 |
| 79 | 97 | 3 |
。
Preferably, the detection conditions at the time of detection are as follows:
column box temperature: 45 ℃, flow rate: 0.8ml/min, run time: 79min, sample volume: 10 μl, detection wavelength: 234nm.
Compared with the prior art, the invention has the advantages and positive effects that:
1. the method for identifying the bovine pulmonary heparin doping content in the porcine intestinal mucosa-derived heparin directly, but not indirectly, characterizes the difference of the composition of heparin sugar chains, can directly reflect different species sources, has sensitive variation of the related parameters along with different bovine pulmonary heparin doping amounts, and can accurately reflect actual conditions.
2. The method provided by the invention is easy to learn, simple and convenient to operate and easy to analyze, and the related instruments and equipment are conventional instruments and equipment, so that the method can be widely applied to daily detection of enterprises, institutions and the like.
Drawings
FIG. 1 is a disaccharide spectrogram of a heparin sodium standard substance provided by the invention;
FIG. 2 is a diagram of a typical porcine intestinal mucosa derived heparin disaccharide provided by the invention;
FIG. 3 is a typical bovine pulmonary derived heparin disaccharide profile provided by the invention;
fig. 4 is a superposition of heparin sodium disaccharide spectrograms prepared by the pig intestinal mucosa source crude heparin and the bovine lung source crude heparin according to different proportions;
fig. 5 is a schematic diagram showing partial amplification of a delta IIIS peak in superposition of heparin sodium disaccharide spectrograms prepared by pig intestine mucosa source crude heparin and bovine lung source crude heparin according to different ratios;
fig. 6 is a schematic diagram showing partial amplification of delta IIA peaks in superposition of heparin sodium disaccharide spectrograms prepared from crude heparin from pig intestinal mucosa and crude heparin from bovine lung according to different proportions.
Detailed Description
The following description of the technical solutions in the embodiments of the present invention will be clear and complete, and it is obvious that the described embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1
The invention adopts the instrument, the chemical reagent and the experimental steps. The method comprises the following steps:
1, instrument:
high performance liquid chromatograph, electronic balance and acidimeter
2 reagent and test solution
TABLE 2 reagents and test solutions
3. Sample treatment:
20mg of the sample was weighed, dissolved in 1ml of purified water, and filtered through a 0.22 μm filter membrane to obtain a sample to be subjected to enzymolysis.
4. Sample enzymolysis:
4.1 sodium calcium acetate pH7.0 solution preparation: calcium acetate (32 mg) and bovine serum albumin (10 mg) were weighed into 60mL of purified water, 580. Mu.L of glacial acetic acid was added, pH7.0 was adjusted with 2mol/L sodium hydroxide, transferred into a 100mL volumetric flask, diluted to scale with purified water, and filtered through a 0.22 μm filter.
4.2 preparation of Potassium dihydrogen phosphate 7.0 buffer: KH2PO4 (68 mg) and bovine serum albumin (10 mg) were weighed into 30mL of purified water, transferred to a 50mL volumetric flask after complete dissolution, pH7.0 was adjusted with 2mol/L KOH, diluted to scale with purified water, and filtered through a 0.22 μm filter.
4.3 heparin enzyme I solution preparation: heparanase I was dissolved in potassium dihydrogen phosphate 7.0 buffer to 0.4IU/mL, mixed in a vortex mixer and stored at-20℃before use.
4.4 heparin enzyme II solution preparation: heparanase II was dissolved in potassium dihydrogen phosphate 7.0 buffer to 0.4IU/mL, mixed in a vortex mixer and stored at-20℃before use.
4.5 heparinase III solution preparation: heparanase III was dissolved in potassium dihydrogen phosphate 7.0 buffer to 0.4IU/mL, mixed in a vortex mixer and stored at-20℃before use.
4.6 heparanase I, II, III mixed solution: the heparanase solutions I, II and III are taken according to the following ratio of 1:1: mixing in proportion of 1.
4.7 preparation of sample solution: taking a pre-prepared 20mg/ml sample aqueous solution to be subjected to enzymolysis, adding 70 mu L of a sodium calcium acetate pH7.0 solution, adding 100 mu L of a heparinase I, II and III mixed solution, mixing by a vortex mixer, and placing in a water bath at 25 ℃ for 48 hours to obtain a sample solution.
5. Detection of
The test sample solution was tested by strong anion exchange high performance liquid chromatography, the specific chromatographic conditions are shown in table 4, and the elution gradient is shown in table 5.
TABLE 3 chromatographic conditions
Table 4 detection of linear elution gradient
| Time (minutes) | Mobile phase a (%) | Mobile phase B (%) |
| 0 | 97 | 3 |
| 20 | 65 | 35 |
| 50 | 0 | 100 |
| 60 | 0 | 100 |
| 61 | 97 | 3 |
| 79 | 97 | 3 |
The disaccharide spectrum of heparin sodium standard product detected by strong anion exchange high performance liquid chromatography is shown in figure 1, the disaccharide spectrum of typical pig intestine mucosa source heparin is shown in figure 2, and the disaccharide spectrum of typical cattle lung source heparin is shown in figure 3.
6. Analysis
The disaccharide components of each spectrum were integrated by area normalization to calculate the peak area percentages of 8 disaccharides, especially ΔIIIs, ΔIIa peak area percentages, and further calculate the ratio of ΔIIIs/. DELTA.IIa as shown in Table 2. Meanwhile, in combination with Δiiis/Δiiacalculated in table 5, linear regression was performed with (0,2.36), (100%, 23.19) to establish a functional relationship y=20.83x+2.36 (Y is Δiiis/Δiia, X is bovine-derived heparin ratio), and the peak area percentage of disaccharide and the ratio of Δiiis/Δiiaat different bovine-derived heparin doping percentages were calculated as shown in table 5.
Table 5: bovine pulmonary heparin, porcine intestinal mucosa heparin and disaccharides from different bovine pulmonary heparin samples
Table 6: bovine pulmonary heparin, porcine intestinal mucosa heparin and different bovine pulmonary heparin-based ratio samples delta IIIs/deltaIIa
As can be seen from the data in tables 5 and 6, if heparin sodium samples are doped with bovine-derived heparin, according to the method disclosed by the invention, the doping condition can be monitored more sensitively by taking the delta IIIs/delta IIa change of the samples as an index, and a heparin manufacturer can be helped to effectively monitor the quality of crude heparin.
Example 2 validation experiment
This example 2 is also a verification of the method described in the present invention. The specific implementation mode is as follows:
1. sample preparation:
sample (1): bovine pulmonary derived heparin was 0% in weight, i.e., the sample was pure porcine intestinal mucosa derived heparin;
sample (2): bovine lung derived heparin at 10%;
sample (3): bovine lung derived heparin was 20%.
2. The rest of the preparation work is as follows: the apparatus, reagents and reagents, sample handling, detection, etc. were as in example 1.
The disaccharides were detected in samples (1), (2) and (3), and the ΔIIIs/. DELTA.IIa values were calculated.
3. Analysis:
based on the Δiiis/Δiia values of samples (1), (2) and (3), regression was performed to a functional relationship y=20.83x+2.36, and "bovine-derived heparin duty calculation" was obtained, and the results are summarized in table 7.
Table 7: comparing the actual proportion of the sample to be measured with the calculated value according to the method in the invention
| Sample of | Heparin sodium standard substance | Sample (1) | Sample (2) | Sample (3) |
| Bovine pulmonary derived heparin fraction |
0 | 0％ | 10％ | 20％ |
| Sample ΔIIIs/. DELTA.IIa values | 2.43 | 2.93 | 4.47 | 7.19 |
| Bovine pulmonary derived heparin duty cycle calculation | 0.34％ | 2.74％ | 10.13％ | 23.19％ |
The calculation result shows that: the calculated value obtained by the method of the invention basically accords with the actual value of the bovine pulmonary heparin ratio in the sample, and the difference of peak areas at the peaks delta IIIs and delta IIa of the bovine pulmonary heparin doping ratios with different ratios can be obviously seen in figures 4,5 and 6. The data and the spectrogram show that the method has good applicability and can be used for identifying whether the heparin sodium is doped with bovine pulmonary heparin and the doping percentage content ratio.
Claims (10)
1. The method for identifying the bovine lung-derived heparin doping content in the porcine intestinal mucosa-derived heparin is characterized by comprising the following steps of:
establishing a functional relation by using the delta IIIs/deltaIIa ratio calculated based on the pure pig intestinal mucosa source heparin sodium disaccharide component and the pure cattle lung source heparin sodium disaccharide component respectively;
calculating the delta IIIs/delta IIa ratio of disaccharide components of the heparin sodium sample to be detected, and calculating the doping percentage content of bovine pulmonary heparin by utilizing the functional relation;
wherein DeltaIIIs is 2-sulfo-Delta4, 5-glucuronic acid- (beta 1-4) -N-sulfonic acid glucosamine, deltaIIa is Delta4, 5-glucuronic acid (beta 1-4) -6-sulfo-N-acetylglucosamine.
2. The method of claim 1, wherein the functional relationship is established by:
the ratio of DeltaIIIs/DeltaIIacalculated based on the disaccharide component of heparin sodium derived from pure porcine intestinal mucosa was averaged and recorded as coordinates (Y 1 ，0)；
The ratio of DeltaIIIs/DeltaIIacalculated based on the heparin sodium disaccharide component derived from pure bovine lung was averaged and recorded as coordinates (Y 2 ，1)；
In the above two coordinates (Y 1 ，0)，(Y 2 1) establishing a function Y (x) according to a two-point one-line method, wherein Y is the delta IIIs/delta IIa ratio of a sample to be detected, and x is the doping percentage of bovine pulmonary derived heparin in the sample to be detected.
3. The method according to claim 2, wherein at least > 1 batches of the average value of the ratio Δiiis/Δiiacalculated for the pure porcine intestinal mucosa-derived heparin sodium disaccharide component and the pure bovine lung-derived heparin sodium disaccharide component, respectively, are calculated.
4. A method according to any one of claims 1-3, characterized in that the calculated Δiiis/Δiiais replaced by Δiiis/Δia.
5. The method according to claim 2, characterized in that the method for calculating the Δiiis/Δiiaratio is in particular:
dissolving and filtering pure pig intestine mucosa source heparin, pure bovine lung source heparin or heparin sample to be tested, and performing enzymolysis to obtain respective heparin enzymolysis liquid;
and detecting the contents of delta IIIs and delta IIa in the heparin enzymolysis liquid by adopting a strong anion exchange high performance liquid chromatography method, and further calculating to obtain the ratio of delta IIIs/delta IIa.
6. The method according to claim 5, wherein the enzymatic hydrolysis method specifically comprises:
respectively weighing 20mg of pure porcine intestinal mucosa source heparin, pure bovine lung source heparin or heparin sample to be tested, adding 1ml of purified water for dissolution, and filtering with a 0.22 mu m filter membrane to obtain respective solutions to be subjected to enzymolysis;
taking 20 mu L of each solution to be subjected to enzymolysis, and carrying out enzymolysis by using 100 mu L of mixed solution of heparinase I, II and III to obtain respective heparin enzymolysis solution;
wherein, the heparanase I, II and III mixed solution is formed by mixing heparanase I, II and III with the concentration of 0.4 IU/mu L in equal volume; the heparanase I is CAS NO:9025-39-2; heparanase II is CAS NO:149371-12-0; heparanase III is CAS NO:37290-86-1.
7. The method according to claim 5, wherein the detection is performed by strong anion exchange high performance liquid chromatography using a detection column with silica gel chemically bonded with strong basic quaternary ammonium salt anion exchange stationary phase as filler and inner diameter particles of 5 μm and a guard column with the same filler.
8. The method according to claim 5, wherein the detection is performed by gradient elution using a mobile phase A and a mobile phase B;
the mobile phase A is 2mmol/L sodium dihydrogen phosphate aqueous solution, and the pH=3.0; the mobile phase B is an aqueous solution of 1mol/L sodium perchlorate and 2mmol/L sodium dihydrogen phosphate, and the pH=3.0.
9. The method according to claim 5, wherein the gradient elution procedure used in the detection is as follows:
。
10. The method according to claim 5, wherein the detection conditions at the time of detection are as follows:
column box temperature: 45 ℃, flow rate: 0.8ml/min, run time: 79min, sample volume: 10 μl, detection wavelength: 234nm.
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