Targeted detection method and application of arachidonic acid metabolite

Abstract

The invention discloses a targeted detection method and application of arachidonic acid metabolites, belongs to the technical field of biological detection and mass spectrometry, and adopts an ultra-high performance liquid chromatography-tandem mass spectrometry analysis method to detect the arachidonic acid metabolites. An Agilent 1290 Infinity ultra-high performance liquid chromatography system is adopted, and the detection method comprises the following steps of: preparing a sample; a mobile phase A is a 0.01% formic acid aqueous solution, and a mobile phase B is a 0.01% formic acid acetonitrile aqueous solution; the temperature of the sample placed in an automatic sample injector is set to be 4 DEG C, the column temperature is set to be 50 DEG C, and the sample injection amount is 10 microliters; the mass spectrometry is carried out by adopting an SCIEX Triple QuadTM6500 + mass spectrometer in a negative ion mode; and detecting the to-be-detected ion pair in an MRM mode. The kit is high in sensitivity and specificity, has targeting property, provides reliable experimental data for clinical application, feeds back abnormal results, assists clinical adjustment of treatment schemes, and ensures the curative effect of drugs.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological detection and mass spectrometry analysis, and relates to a targeted detection method and application of arachidonic acid metabolites, and specifically relates to a targeted detection method and application of arachidonic acid metabolites in cow blood and milk. Background Art

[0002] Arachidonic acid (AA) is an important polyunsaturated fatty acid that regulates multiple physiological functions in animals through its metabolic pathways. A ω-6 AA is a key UA, widely present in cell membrane phospholipids and lipid droplets in immune cells, and plays a crucial role in inflammatory signaling and cell function regulation. Arachidonic acid, through its metabolites, is closely associated with lipid metabolism and immune function, further revealing the close connection between arachidonic acid metabolism, lipid metabolism, and immune regulation. Arachidonic acid metabolism primarily regulates inflammatory responses through three key pathways: the cyclooxygenase (COX) pathway, the lipoxygenase (LOX) pathway, and the cytochrome P450 (CYP450) pathway. Products from these metabolic pathways have dual functions in inflammatory responses, acting both to promote and inhibit them. Dynamic changes in AA metabolites are crucial for the initiation, maintenance, and resolution of inflammatory responses. Therefore, detecting AA metabolites is not only crucial for animal health and tissue homeostasis but may also provide new research directions and insights for the early diagnosis and treatment of mastitis in dairy cows.

[0003] 12S-HETE, or 12S-hydroxyeicosatetraenoic acid, is a metabolite of arachidonic acid. It is an oxidized lipid product generated by the action of 12-LOX or certain CYP450 enzymes and is widely present in platelets, monocytes, neutrophils, and epithelial cells. It plays a crucial role in the development and progression of numerous diseases. In mastitis, 12S-HETE may be associated with disease progression through multiple mechanisms. For example, it may be associated with neutrophil recruitment, macrophage activation, and enhanced oxidative stress, suggesting a proinflammatory role in the process of mastitis. Serum 12S-HETE is also likely a core lipid marker in the regulation of mastitis inflammation. Furthermore, research progress on 12S-HETE in inflammation and oxidative stress suggests that it plays a role in the inflammatory response and may be associated with the development and progression of mastitis.

[0004] 15S-HETE is a metabolite of arachidonic acid, generated by 15-LOX. It is also partially synthesized by CYP450 enzymes and is primarily found in eosinophils and macrophages. Its primary effects include: 15S-HETE is an important precursor of lipoxins and participates in the resolution of inflammation, inhibiting neutrophil activity and reducing the expression of factors such as IL-1β. It also suppresses Th1 immune responses and promotes macrophage polarization from an M1 to an M2 phenotype. Furthermore, 15S-HETE is a marker of anti-inflammatory pathways that promote tissue repair; its elevated expression generally indicates resolution of inflammation. In mastitis models, its expression often increases synergistically with LXA4, but a lower ratio suggests insufficient anti-inflammatory mechanisms.

[0005] 12S-HETE and 15S-HETE may play a role in inflammatory diseases by participating in inflammatory responses and affecting immune cell function, but their specific mechanisms of action and their role in cow mastitis require further research to clarify; to this end, the present invention provides the use of arachidonic acid metabolites in cow mastitis to solve the above-mentioned problems. Summary of the Invention

[0006] In response to the deficiencies in the prior art, the present invention provides an application of arachidonic acid metabolites in cow mastitis, specifically a targeted detection method and application of arachidonic acid metabolites in cow blood and milk, which solves the problems mentioned in the above background technology.

[0007] The present invention adopts the following technical solutions to achieve its purpose:

[0008] A targeted detection method for arachidonic acid metabolites in cow blood and milk, using ultra-high performance liquid chromatography-tandem mass spectrometry to detect arachidonic acid metabolites:

[0009] The chromatographic conditions of the ultra-high performance liquid chromatography are:

[0010] Chromatographic column: Agilent 1290 Infinity ultra-high performance liquid chromatography system;

[0011] Mobile phase A was 0.01% formic acid in aqueous solution, and mobile phase B was 0.01% formic acid in acetonitrile aqueous solution, and gradient elution was performed. The procedure of the gradient elution was as follows:

[0012] 0-1min, phase B is maintained at 30%; 1-9min, phase B changes linearly from 30% to 90%; 9-11min, phase B is maintained at 90%; 11-11.1min, phase B changes linearly from 90% to 20%; 11.1-14min, phase B is maintained at 20%;

[0013] The sample was placed in an autosampler with the temperature set at 4°C, the column temperature set at 50°C, the injection volume set at 10 μL, and the flow rate set at 400 μL / min;

[0014] The mass spectrometer was SCIEX Triple Quad TM Mass spectrometry analysis was performed using a 6500+ mass spectrometer in negative ion mode;

[0015] SCIEX Triple Quad TM The negative ion conditions of the 6500+ESI source are as follows: source temperature: 500°C; ion Source Gas1 (Gas1): 30psi; Ion Source Gas2 (Gas2): 30psi; Curtain gas (CUR): 40psi; ion Sapary Voltage Floating (ISVF): -4500V (i.e., ion source temperature: 500°C; ion source gas 1 (Gas1): 30psi; ion source gas 2 (Gas2): 30psi; curtain gas (CUR): 40psi; ion vaporizer voltage floating (ISVF): -4500V); the MRM mode is used to detect the ion pairs to be tested.

[0016] Application of the above-mentioned detection method in the identification of arachidonic acid metabolites in vivo.

[0017] The beneficial effects of the present invention are:

[0018] The application of the metabolite of arachidonic acid in cow mastitis is to detect the metabolite of arachidonic acid, thereby detecting the changes in the level of the metabolite in the inflammatory response and accurately detecting its content in biological samples by chromatography-mass spectrometry. It is an auxiliary indicator for clinical judgment of inflammation such as mastitis. For example, its concentration in cows with mastitis may be higher than that in healthy cows. In different types of inflammatory diseases, the change pattern of arachidonic acid metabolites is different. Combined with other indicators, the detection of this metabolite is helpful to distinguish mastitis from other inflammatory diseases with similar symptoms. At the same time, in the development of mastitis, the detection of the content of metabolites can understand the progression of inflammation. If the content continues to increase, it may indicate worsening inflammation. If the level of 12S-HETE and 15S-HETE decreases after treatment, it may indicate that the condition is improving. During the treatment process, regular testing of metabolite levels can evaluate the effectiveness of the treatment plan. If the level decreases after treatment, it means that the treatment is effective in controlling inflammation. If it increases instead of decreasing, the treatment plan may need to be adjusted. Chromatography-mass spectrometry can not only measure the content of metabolites, but also analyze other related metabolites in the entire arachidonic acid metabolic pathway. By studying their changes, we can gain an in-depth understanding of the abnormalities of the arachidonic acid metabolic pathway in inflammatory diseases and provide a basis for revealing the pathogenesis of the disease. Clarifying the mechanism of action of 12S-HETE and 15S-HETE in the disease will help discover new drug targets and treatment directions, laying the foundation for the development of more effective therapeutic drugs and methods. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 A box plot of differential metabolites in serum and milk between the Con group and the MS group provided for the application of the arachidonic acid metabolites of the present invention;

[0020] Figure 2 Heat map of differential metabolites in serum and milk between the Con group and the MS group provided for the application of the arachidonic acid metabolites of the present invention;

[0021] Figure 3 ROC curves of differential metabolites in serum and milk of healthy cows and cows with mastitis provided for the application of the arachidonic acid metabolites of the present invention;

[0022] Figure 4 A comparative diagram of differential metabolite analysis provided for the application of the arachidonic acid metabolites of the present invention;

[0023] Figure 5 The invention provides an ROC curve diagram of 12S-HETE in serum of healthy cows and cows with mastitis and 15S-HETE in milk for the application of the arachidonic acid metabolites.

[0024] in, Figure 2 A represents serum and B represents milk; Figure 3 A stands for serum and B stands for milk. DETAILED DESCRIPTION

[0025] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts should fall within the scope of protection of the present invention.

[0026] Targeted detection method for arachidonic acid metabolites in cow blood and milk.

[0027] Preferably, the targeted detection includes a reagent for detecting the content of the metabolic marker in a sample from a subject.

[0028] Preferably, the targeted detection comprises detection by liquid chromatography-tandem mass spectrometry.

[0029] Preferably, the targeted detection determines the mastitis condition by detecting the content level of metabolic markers.

[0030] Preferably, the samples are separated using an Agilent 1290 Infinity ultra-high performance liquid chromatography system.

[0031] Preferably, the chromatographic detection conditions are as follows: the sample is placed in an automatic sampler with the temperature set at 4°C, the column temperature set at 50°C, the injection volume is 10 μL, the mobile phase A is 0.01% formic acid aqueous solution, the mobile phase B is 0.01% formic acid acetonitrile aqueous solution, and gradient elution is performed. The gradient elution procedure is as follows:

[0032] 0-1min, phase B was maintained at 30%; 1-9min, phase B changed linearly from 30% to 90%; 9-11min, phase B was maintained at 90%; 11-11.1min, phase B changed linearly from 90% to 20%; 11.1-14min, phase B was maintained at 20%.

[0033] Preferably, SCIEX Triple Quad TM Mass spectrometry analysis was performed using a 6500+ mass spectrometer in negative ion mode.

[0034] Preferably, SCIEX Triple Quad TMThe negative ion conditions of the 6500+ESI source are as follows: source temperature: 500°C; ion Source Gas1 (Gas1): 30 psi; Ion Source Gas2 (Gas2): 30 psi; Curtain gas (CUR): 40 psi; ion Sapary Voltage Floating (ISVF): -4500 V (i.e., ion source temperature: 500°C; ion source gas 1 (Gas1): 30 psi; ion source gas 2 (Gas2): 30 psi; curtain gas (CUR): 40 psi; ion vaporizer voltage floating (ISVF): -4500 V).

[0035] Preferably, the ion pairs to be detected are detected in MRM mode.

[0036] In this technical solution, 12 healthy control cows (Con) and 12 mastitis cows (MS) were selected for arachidonic acid targeted mass spectrometry detection.

[0037] Example 1

[0038] Metabolite extraction

[0039] The metabolite extraction method was as follows: the sample was removed from −80°C, 150 μL of the sample was taken, and 450 μL of extraction solution (0.5 M ammonium acetate, pH 5.5, pre-cooled at 4°C, containing an isotope-labeled internal standard mixture containing BHT) was added. The sample was vortexed for 30 seconds and sonicated for 5 minutes in an ice-water bath. The sample was shaken at 4°C for 30 minutes. The sample was added with 750 μL of extraction reagent (n-hexane / ethyl acetate = 2 / 3) and shaken for 5 minutes. The sample was centrifuged at 3500 rpm at 4°C for 5 minutes, and the supernatant was collected. The above centrifugation steps were repeated, and the supernatants were combined. The sample was dried with nitrogen and reconstituted with 75 μL of 30% acetonitrile. The sample was centrifuged at 12000 rpm at 4°C for 15 minutes, and the supernatant was collected and frozen at −80°C.

[0040] Example 2

[0041] Chromatography-mass spectrometry detection conditions

[0042] 1) Chromatographic Conditions: Samples were separated using an Agilent 1290 Infinity Ultra Performance Liquid Chromatography System. The sample was placed in the autosampler at 4°C, the column temperature was set at 50°C, the injection volume was 10 μL, mobile phase A was 0.01% formic acid in water, and mobile phase B was 0.01% formic acid in acetonitrile. The flow rate was set at 400 μL / min, and gradient elution was performed. The gradient elution procedure was as follows:

[0043] 0-1min, phase B was maintained at 30%; 1-9min, phase B changed linearly from 30% to 90%; 9-11min, phase B was maintained at 90%; 11-11.1min, phase B changed linearly from 90% to 20%; 11.1-14min, phase B was maintained at 20%.

[0044] 2) Mass spectrometry conditions: SCIEX Triple Quad TM Mass spectrometry analysis was performed using a 6500+ mass spectrometer (SCIEX) in negative ion mode; SCIEX Triple Quad TM The negative ionization conditions of the 6500+ESI source were as follows: source temperature: 500° C.; ion source gas 1 (Gas1): 30 psi; ion source gas 2 (Gas2): 30 psi; curtain gas (CUR): 40 psi; ion vaporizer voltage floating (ISVF): −4500 V.

[0045] -4500V); the MRM mode was used to detect the ion pairs to be tested. The ion pair information of all target substances is shown in Table 1.

[0046] Table 1 MRM results of serum and milk in the Con and MS groups

[0047]

[0048]

[0049] Note: (1) Con group is the healthy control group, MS group is the dairy cow mastitis group;

[0050] (2) Recovery and RSD represent the recovery rate and standard relative deviation of QC samples, respectively. It is generally believed that a Recovery between 80% and 120% indicates that the method has good extraction efficiency; RSD < 15% indicates that the analytical method has high precision and the results are relatively reliable.

[0051] Table 2 Comparison of metabolites in serum and milk between the Con group and the MS group

[0052]

[0053]

[0054] Note: (1) * indicates P-Value < 0.05, indicating significant difference.

[0055] (2) VIP (Variable Importance in Projection) is used to evaluate the importance of metabolites in distinguishing different sample groups. It is generally believed that when VIP>1, the metabolite has a high importance to the model, and when p<0.05 is satisfied, it indicates that the metabolite is a differential metabolite between the two groups;

[0056] (3) FC (Foldchange) is the fold change. For example, in the comparison group A_vs_B, it represents the mean of group A divided by the mean of group B. Under the conditions of p < 0.05 and VIP > 1, > 1 indicates upregulation and < 1 indicates downregulation.

[0057] like Figure 1 As shown in Figure 2, a box plot is a statistical graph commonly used to display the dispersion of a set of data. It can intuitively illustrate the inter-group and intra-group distribution of metabolites. A set of data is arranged from largest to smallest, and the upper edge (maximum value), upper quartile Q3, median, lower quartile Q1, and lower edge (minimum value) are calculated. The intervals between the minimum and maximum values ​​reflect the degree of data variation. In a box plot, the horizontal axis represents the group name, and the vertical axis represents the content information. Significant differences between groups are indicated using the P value calculated using a t-test or analysis of variance.

[0058] like Figure 2 As shown, in order to more comprehensively and intuitively display the differences in the expression patterns of metabolites in different samples, the expression levels of all metabolites were calculated using a distance matrix, and cluster analysis was performed using hierarchical clustering. The metabolites in the red box are differential metabolites in the arachidonic acid pathway in serum and milk. The cluster analysis diagram of the example group is as above. The upper horizontal axis in the figure is the group, the vertical axis is the metabolite, and the depth of the color reflects the level of expression. The redder the color, the higher the expression level of the metabolite, and the bluer the color, the lower the expression level of the metabolite. Generally speaking, when the candidate metabolites screened are reasonable and accurate, samples in the same group can appear in the same cluster through clustering. At the same time, metabolites clustered in the same cluster have similar expression patterns and may be in relatively close reaction steps in the metabolic process.

[0059] like Figure 3 As shown, the area under the ROC curve ranges from 1.0 to 0.7. When AUC is greater than 0.5, the closer the AUC is to 1, the better the results. An AUC between 0.5 and 0.7 indicates low accuracy, while an AUC between 0.7 and 0.9 indicates moderate accuracy. An AUC ≤ 0.5 indicates the method is completely ineffective and has no value.

[0060] like Figure 4 As shown, **, ** in the figure represent p<0.01, 0.05, respectively.

[0061] like Figure 5 As shown, the area under the curve (AUC) of 12S-HETE in serum is 0.75, and the area under the curve (AUC) of 15S-HETE in milk is 0.83.

[0062] Conclusion: The 12S-HETE level in serum of the MS group was significantly higher than that in the Con group (AUC = 0.75). The 15S-HETE level in milk of the MS group was also significantly higher than that in the Con group, with high sensitivity and specificity. This has clinical significance for disease prediction, in-depth study of pathogenesis, and development of new drug targets.

[0063] Meanwhile, the contents not described in detail in this specification belong to the prior art known to those skilled in the art.

[0064] It should be noted that, in this document, relational terms such as first and second, etc., are used only to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any actual relationship or order between these entities or operations. Moreover, the terms "comprises," "comprising," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that includes a list of elements includes not only those elements but also other elements not explicitly listed, or elements inherent to such process, method, article, or apparatus.

[0065] Finally, it should be noted that the above-described embodiments are only specific implementation methods of the present invention, which are used to illustrate the technical solutions of the present invention rather than to limit them. The protection scope of the present invention is not limited thereto. Although the present invention has been described in detail with reference to the above-mentioned embodiments, ordinary technicians in this field should understand that any technician familiar with this technical field can still modify the technical solutions recorded in the above-mentioned embodiments within the technical scope disclosed by the present invention, or make equivalent replacements for some of the technical features therein; and these modifications, changes or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention, and should all be covered by the protection scope of the present invention.

Claims

1. A targeted detection method for arachidonic acid metabolites in cow blood and milk, characterized in that: Ultra-high performance liquid chromatography-tandem mass spectrometry was used to detect the metabolites of arachidonic acid: The chromatographic conditions of the ultra-high performance liquid chromatography are: Chromatographic column: Agilent 1290 Infinity ultra-high performance liquid chromatography system; Mobile phase A was 0.01% formic acid in aqueous solution, and mobile phase B was 0.01% formic acid in acetonitrile aqueous solution, and gradient elution was performed. The procedure of the gradient elution was as follows: 0-1min, phase B was maintained at 30%; 1-9min, phase B changed linearly from 30% to 90%; 9-11min, phase B was maintained at 90%; 11-11.1min, phase B changed linearly from 90% to 20%; 11.1-14 min, phase B is maintained at 20%; The sample was placed in an autosampler with the temperature set at 4°C, the column temperature set at 50°C, the injection volume set at 10 μL, and the flow rate set at 400 μL / min; The mass spectrometer was SCIEX Triple Quad TM Mass spectrometry analysis was performed using a 6500+ mass spectrometer in negative ion mode; SCIEX Triple Quad TM The negative ion conditions of the 6500+ESI source were as follows: ion source temperature: 500°C; ion source gas 1: 30 psi; ion source gas 2: 30 psi; curtain gas: 40 psi; ion vaporizer voltage floating: -4500 V; and the MRM mode was used to detect the ion pairs to be tested.

2. Use of the detection method as claimed in claim 1 in the identification of arachidonic acid metabolites in vivo.

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