An absolute quantitative analysis method for key metabolites of methionine cycle and its application

Abstract

The present invention relates to an absolute quantitative analysis method and application of a key metabolite of methionine cycle. The present invention screens an efficient reduction-sulfhydryl derivatization scheme, and establishes an HPLC-MS / MS detection platform. Deuterated phenylalanine is used as an internal standard to draw detection standard curves of 9 key metabolites of methionine cycle, including methionine, homocysteine, cysteine, choline, betaine, reduced glutathione, S-adenosylhomocysteine, S-adenosylmethionine, and glutamic acid, and the linearity and range, precision and accuracy, stability, recovery rate, and matrix effect of the detection platform are investigated. The investigation results show that the quantitative analysis method established by the present invention has high detection stability and accuracy for key metabolites of methionine cycle, especially for homocysteine, cysteine, and reduced glutathione, which have low detection sensitivity and are easily damaged by oxidation, and the detection sensitivity is increased by 14.44 times, 5.08 times, and 3.41 times, respectively, and the stability is greatly improved.

Description

Technical Field

[0001] The invention belongs to the technical field of analysis and detection, and specifically relates to an absolute quantitative analysis method and application of key metabolites of methionine cycle. Background Art

[0002] Methionine molecules contain S-methyl groups, which can generate a variety of physiologically active substances rich in methyl groups through various transmethylation reactions. Methionine reacts with ATP under the catalysis of adenosyltransferase to generate S-adenosylmethionine (SAM). The methyl group in S-adenosylmethionine is an active methyl group. After catalysis by methyltransferase, the methyl group can be transferred to other substances to make them methylated. S-adenosylmethionine is demethylated to generate S-adenosylhomocysteine ​​(SAH). S-adenosylhomocysteine ​​is deadenosylated to generate homocysteine ​​(HCY). Homocysteine ​​then accepts the methyl group on 5-methyltetrahydrofolate to regenerate methionine, forming a cycle. In the methionine cycle, homocysteine ​​is converted into cystathionine under the catalysis of cystathionine-β-synthase, and cystathionine further generates cysteine. About 50% of homocysteine ​​is finally converted into cysteine ​​(CYS) under the action of cystathionine β-synthase, and cysteine ​​is converted into reduced glutathione (GSH) under the action of γ-glutamine cysteine ​​synthetase. In addition, betaine (BET) is the end product of choline (CHO) oxidative metabolism and an important methylating agent. It participates in the biosynthesis of methionine through betaine homocysteine-S-methyltransferase (see the attached diagram for the in vivo conversion of key metabolites in the methionine cycle). Figure 1 ).

[0003] Normal plasma total homocysteine ​​concentration is generally 5-15μmol / L, and the average concentration is about 10μmol / L. The higher the plasma total homocysteine ​​concentration, the higher the risk of cardiovascular and cerebrovascular diseases (such as coronary heart disease, atherosclerosis), renal dysfunction, diabetes and other lipid metabolism disorders related diseases. When the body's metabolism is impaired, homocysteine ​​will accumulate in the body because it cannot be degraded. High concentrations of homocysteine ​​can damage the inner wall of the blood vessels, causing the endothelium to thicken and roughen, forming plaques, and then narrowing or even blocking the lumen, causing incomplete blood supply to the arteries, leading to atherosclerosis and coronary heart disease. It can be seen that under the influence of one or more factors, the homocysteine ​​level in the human body increases, causing lesions in cells, tissues and organs, which has a serious impact on human health. Elevated homocysteine ​​is the result of abnormal human metabolism. Its metabolic pathway involves 9 important metabolites, including methionine (MET), homocysteine ​​(HCY), cysteine ​​(CYS), choline (CHO), betaine (BET), reduced glutathione (GSH), S-adenosylhomocysteine ​​(SAH), S-adenosylmethionine (SAM), and glutamate (GLU). Changes in their content can, to a certain extent, reflect changes in the level of methionine circulation metabolism. By using them as detection indicators and detecting and controlling these metabolites within an appropriate level range, the occurrence of the disease can be effectively prevented or the progression of hyperhomocysteinemia can be controlled.

[0004] Detection methods for methionine metabolic pathway markers represented by homocysteine ​​include high performance liquid chromatography, spectroscopy, chemiluminescence, radioenzyme immunoassay, and enzyme colorimetry. The above-mentioned traditional detection methods are cumbersome and complex in operation, and lack sensitivity and specificity, especially for endogenous substances with complex matrices and low content. The detection effect is not ideal and cannot meet the detection needs. For example, the Chinese literature "A study on the metabolomics of serum and urine of early gastric cancer based on HPLC-MS" reported the differences in serum and urine small molecule metabolites in early gastric cancer group, chronic superficial gastritis group, and normal population group by high performance liquid chromatography-mass spectrometry, including the screening and identification of 9 metabolites in serum, including dihydrocholesterol, cholesterol, tyrosine, lactic acid, tyramine, 3-hydroxypropionic acid, pyruvic acid, alanine, and lysophosphatidylcholine (18:0). (Liu Tong. A study on the metabolomics of serum and urine of early gastric cancer based on HPLC-MS [D]. Youjiang Medical College for Nationalities) , 2017.), but the document did not conduct a comprehensive analysis and identification of the circulating metabolites of methionine; for example, the Chinese document “Separation and Quantification of Thiol-Containing Amino Acid Metabolites in Blood Samples” reported the simultaneous detection of related substances in the homocysteine ​​metabolic pathway using liquid chromatography-quadrupole mass spectrometry: including methionine (MET), S-adenosylmethionine (SAM), homocysteine ​​(HCY), S-adenosylhomocysteine ​​(SAH), cystathionine (Cysta), cysteine ​​glycine (Cys-Gly), and glutathione (GSH). A HPLC-MS / MS method for simultaneously quantifying 7 thiol-containing amino acid metabolites in human blood samples has been established. However, the literature was used to detect plasma from mice with folate deficiency and patients with hypertension, and the HPLC-fluorescence detection method and HPLC-ultraviolet absorption detection were used. This method is not ideal for the quantitative detection of methionine cycle metabolites (Jiang Zhiting. Separation and quantitative study of thiol-containing amino acid metabolites in blood samples.). Therefore, there is no report on the HPLC-MS / MS method that can simultaneously detect 9 substances, including homocysteine, methionine, cysteine, choline, betaine, reduced glutathione, S-adenosylhomocysteine, S-adenosylmethionine, and glutamate. In addition, due to the complexity of the serum matrix, there are many endogenous substances with similar physical and chemical properties to those to be measured, which will affect the sensitivity and accuracy of the results. Therefore, it is urgent to develop a comprehensive analytical method for methionine metabolic pathway markers. Summary of the invention

[0005] In view of the problems existing in the prior art, the present invention proposes an absolute quantitative analysis method for key metabolites of the methionine cycle, which is based on the absolute quantitative analysis method for key metabolites of the methionine cycle combined with an efficient reduction-thiol derivatization strategy and an HPLC-MS / MS detection platform. It can simultaneously detect substances at different levels of the ng-μg level, has a wide detection range, is not affected by structural analogues, and requires a small amount of sample and a short analysis time.

[0006] In a first aspect, the present invention provides a method for absolute quantitative analysis of key metabolites of the methionine cycle, characterized in that the method comprises the following steps:

[0007] S1. Sample pretreatment: The serum samples were pretreated with efficient reduction-thiol derivatization to remove interfering proteins;

[0008] S2, chromatographic separation: injecting the sample processed in step S1 into a high performance liquid chromatography column to complete the preliminary separation of the sample;

[0009] S3, mass spectrometry identification: the sample preliminarily separated in step S2 is subjected to a mass spectrometer to perform quantitative analysis of the intensity of ion fragments and the collection of mass spectra.

[0010] Furthermore, the present invention provides the following steps for sample pretreatment in step S1:

[0011] 1) preparing a methanol solution containing deuterated phenylalanine as a stock solution;

[0012] 2) preparing a solution containing N-ethylmaleimide, fully dissolving it and setting it aside;

[0013] 3) preparing a solution containing tri(2-carbonylethyl)phosphine hydrochloride, fully dissolving it and setting it aside;

[0014] 4) Pretreatment before removing interfering proteins;

[0015] Furthermore, the pre-treatment of step 4) comprises the following steps:

[0016] a) preparing standard curve sample solution: weighing methionine, choline, betaine, S-adenosylhomocysteine, S-adenosylmethionine, and glutamic acid standards, respectively preparing stock solutions with methanol solution; weighing homocysteine, cysteine, and reduced glutathione standards, respectively preparing stock solutions with N-ethylmaleimide solution, and obtaining a series of standard solutions of methionine, choline, betaine, S-adenosylhomocysteine, S-adenosylmethionine, homocysteine, glutamic acid, cysteine, and reduced glutathione;

[0017] b) Prepare specimen: Take serum into EP tube, vortex thoroughly to get a specimen, and proceed with the following steps;

[0018] ① Efficient reduction-thiol derivatization: first add serum to a 96-well protein precipitation plate, add tris(2-carbonylethyl)phosphine hydrochloride solution, mix evenly with a 96-well plate shaker and let stand, then add N-ethylmaleimide, then add methanol solution containing deuterated phenylalanine, mix evenly with a 96-well plate shaker and let stand at room temperature;

[0019] ② Pretreatment to remove interfering proteins: Use a biological sample pretreatment instrument to collect the solution obtained after step ① into a clean collection plate for later use. The resulting solution is the test solution with interfering proteins removed;

[0020] ③ Drying and re-dissolving of pre-treated samples: Blow dry the solution in the collection plate with nitrogen, then re-dissolve the sample in a methanol-water solution, and then mix it evenly on a vortex before proceeding with subsequent testing.

[0021] Furthermore, the present invention provides that the HPLC flow rate of the chromatographic separation in step S2 is set to 300 μL / min, the column temperature is fixed at 30° C., and the injection volume is set to 10 μL.

[0022] Furthermore, the present invention also provides a high performance liquid chromatography flow program for the chromatographic separation in step S2:

[0023] At 0 min, the proportion of 0.1% formic acid-water solution was 95%, and the proportion of 0.1% formic acid-methanol solution or 0.1% formic acid-80% methanol-20% acetonitrile solution was 5%;

[0024] At 1 min, the proportion of 0.1% formic acid-water solution was 5%, and the proportion of 0.1% formic acid-methanol solution or 0.1% formic acid-80% methanol-20% acetonitrile solution was 95%;

[0025] At 2 min, the proportion of 0.1% formic acid-water solution was 80%, and the proportion of 0.1% formic acid-methanol solution or 0.1% formic acid-80% methanol-20% acetonitrile solution was 20%;

[0026] At 3 min, the proportion of 0.1% formic acid-water solution was 80%, and the proportion of 0.1% formic acid-methanol solution or 0.1% formic acid-80% methanol-20% acetonitrile solution was 20%;

[0027] At 5 min, the proportion of 0.1% formic acid-water solution was 90%, and the proportion of 0.1% formic acid-methanol solution or 0.1% formic acid-80% methanol-20% acetonitrile solution was 10%;

[0028] At 7 min, the proportion of 0.1% formic acid-water solution was 95%, and the proportion of 0.1% formic acid-methanol solution or 0.1% formic acid-80% methanol-20% acetonitrile solution was 5%;

[0029] After 10 minutes, stop;

[0030] Furthermore, the present invention provides that the mass spectrometer used in step S3 adopts the design of Turbo VTM ion source and curtain gas, the ionization mode is electrospray ionization, and the mass analyzer is a triple quadrupole, and the triple quadrupole adopts a multiple reaction detection mode for scanning detection.

[0031] Furthermore, the present invention provides that the key metabolites of the methionine cycle include methionine, homocysteine, cysteine, choline, betaine, reduced glutathione, S-adenosylhomocysteine, S-adenosylmethionine and / or glutamate.

[0032] In a second aspect, the present invention provides an absolute quantitative analysis method for key metabolites of a methionine cycle and its application in the preparation of a homocystinemia kit.

[0033] In a third aspect, the present invention provides a method for constructing an HPLC-MS / MS detection platform for absolute quantitative analysis of key metabolites in the methionine cycle, characterized in that the detection platform comprises the following systems:

[0034] 1) Chromatographic separation system

[0035] The chromatographic column in the chromatographic separation system adopts Waters BEH C18, 1.7μm, 2.1×50mm, pore size The mobile phase was 0.1% formic acid-water solution, 0.1% formic acid-methanol solution, or 0.1% formic acid-80% methanol-20% acetonitrile solution;

[0036] 2) Mass spectrometry identification system

[0037] In the mass spectrometry identification system, the mass spectrometer adopts the design of Turbo VTM ion source and curtain gas, the ionization mode is electrospray ionization, and the mass analyzer is a triple quadrupole, and the triple quadrupole adopts the multiple reaction detection mode for scanning detection.

[0038] In a fourth aspect, the present invention provides the use of the HPLC-MS / MS detection platform constructed by the above method in the preparation of a homocystinemia kit.

[0039] In this study, an HPLC-MS / MS analysis platform was used to construct an absolute quantitative analysis method for the detection of methionine (MET), homocysteine ​​(HCY), cysteine ​​(CYS), choline (CHO), betaine (BET), reduced glutathione (GSH), S-adenosylhomocysteine ​​(SAH), S-adenosylmethionine (SAM), and 1,2-dihydro-2-nitro-1-nitro-2-nitro-2-nitro-3-nitro-4-(2-nitro-2-yl)-1-nitro-2-nitro-3-(2-nitro-2-yl)-1-nitro-2-nitro-3-(2-nitro-2-yl)-1-nitro-3-(2-nitro-2-yl)-1-nitro-3-(2-nitro-2-yl)-1-nitro-3-(2-nitro-2-yl)-1-nitro-3-(2-nitro-2-yl)-1-nitro-3-(2-nitro-2-yl)-1-nitro-3- The method of glutamate (GLU) was used to draw the standard curve corresponding to each target methionine metabolite; the quantitative limit and linear range of the method platform were further verified, and its accuracy and the influence of serum matrix on the results were evaluated by spike recovery experiments. The bias of the platform detection was analyzed and calculated to evaluate its precision. Finally, a stable, sensitive and reliable detection platform for the quantitative detection of 9 substances in human serum, including homocysteine, methionine, cysteine, choline, betaine, reduced glutathione, S-adenosylhomocysteine, S-adenosylmethionine, and glutamate, was successfully established (see the attached data for details). Figure 2 ).

[0040] The present invention establishes an HPLC-MS / MS liquid-mass spectrometry platform through the selection and optimization of pretreatment, chromatographic parameters and mass spectrometry conditions, and verifies the performance of the technology.

[0041] First, the standard curve was drawn and the linear correlation was verified. Since serum contains 9 substances, including methionine, choline, betaine, S-adenosylhomocysteine, S-adenosylmethionine, homocysteine, cysteine, reduced glutathione and glutamic acid, the serum needs to be treated to remove endogenous substances. In this study, the activated carbon adsorption method was used to treat serum samples. The specific method was as follows: 1.5 g of activated carbon was added to 20 mL of serum, vigorously rotated at 37 ° C for 3 h, and centrifuged at 21380 × g for 15 min. The supernatant was filtered through 0.45 μm and 0.22 μm membranes in turn, and HPLC-MS / MS was used to confirm that it did not contain or only contained a small amount of 9 substances, including methionine, choline, betaine, S-adenosylhomocysteine, S-adenosylmethionine, homocysteine, cysteine, reduced glutathione and glutamic acid. The serum treated with activated carbon adsorption was used as the serum matrix. The serum treated with activated carbon adsorption was used as the matrix to prepare standard curve sample solutions of 9 compounds with different concentration gradients, including methionine, choline, betaine, S-adenosylhomocysteine, S-adenosylmethionine, homocysteine, cysteine, glutamic acid and reduced glutathione. The r values ​​between the measured concentrations and the prepared concentrations of the 9 compounds were 0.9999, 0.9997, 0.9999, 0.9996, 0.9995, 0.9995, 0.9999, 0.9998 and 0.9995, respectively, and the correlation coefficient R 2 The values ​​are all above 0.999 (see the attached Figures 6 to 14 ). The results show that the 10 concentration levels A to J of each LPC-like substance are all within the linear range, indicating that the HPLC-MS / MS methodology platform established by the present invention can meet the requirements of the concentration range for the quantitative detection of 9 key metabolites of the methionine cycle in serum. In addition, the quantitative detection limits of the 9 key metabolites of the methionine cycle in the solution of the methodology platform are 0.8ng / mL, 0.2ng / mL, 0.4ng / mL, 0.1ng / mL, 1ng / mL, 0.4.ng / mL, 1ng / mL, 0.4ng / mL, 0.4ng / mL, respectively, which verifies that the HPLC-MS / MS experimental platform of the present invention has good applicability to serum specimens.

[0042] Then, after the accuracy verification of the HPLC-MS / MS technology platform, the present invention conducted a recovery test at three concentration levels: high (C), medium (E), and low (J). The results showed that the nine key metabolites of the methionine cycle had satisfactory recovery rates at three concentration levels of high, medium, and low in the PBS matrix, ranging from 85% to 115%. This proved that the method platform can quantitatively analyze nine compounds such as methionine, choline, betaine, S-adenosylhomocysteine, S-adenosylmethionine, homocysteine, cysteine, glutamic acid, and reduced glutathione in a serum-based solution with high accuracy.

[0043] The present invention detects and analyzes 9 key metabolites of methionine circulation in serum. Therefore, the present invention uses serum as a matrix to conduct a spike recovery experiment to determine the magnitude of the matrix effect. The results show that when serum is used as a matrix, the spike recovery can also be controlled within the range of 85% to 115%, indicating that the platform has stable performance and is less affected by the human serum matrix effect. It is suitable for quantitative detection and analysis of 9 compounds in human serum, including methionine, choline, betaine, S-adenosylhomocysteine, S-adenosylmethionine, homocysteine, cysteine, glutamate and reduced glutathione.

[0044] Finally, the present invention verifies the precision of the HPLC-MS / MS detection platform, and applies the HPLC-MS / MS detection platform of the present invention six times to quantitatively detect 9 key methionine cycle metabolites at high, medium and low concentration levels (see Appendix for details). Figures 3 to 5 ), the intra-batch bias values ​​of the six test results were analyzed to evaluate the precision of the HPLC-MS / MS platform. The analysis results showed that the CV values ​​of the three concentration levels, high, medium and low, were all less than 15%, which met the requirements of instrument precision.

[0045] Compared with the prior art, the present invention has the following beneficial effects:

[0046] (1) The present invention selects deuterated phenylalanine as the internal standard for quantitative detection of 9 key metabolites of the methionine cycle to be detected. The molecular weight, structure and other characteristics are similar to those of the metabolites to be detected. Therefore, the set mass spectrometer parameters are suitable for the ionization and detection of the internal standard deuterated phenylalanine, and also meet the quantitative analysis of the key metabolites of the methionine cycle to be detected. Therefore, isotope-labeled compounds are the best choice as internal standards.

[0047] (2) The method of the present invention uses tris(2-carbonylethyl)phosphine hydrochloride as a highly efficient reducing agent during specimen pretreatment, which greatly protects homocysteine, cysteine ​​and reduced glutathione in serum samples that are easily oxidized, and uses N-ethylmaleimide as a sulfhydryl protecting agent. N-ethylmaleimide is used to combine with the sulfhydryl groups of homocysteine, cysteine ​​and reduced glutathione to form stable homocysteine, cysteine ​​and reduced glutathione derivatives, which greatly improves the detection sensitivity of these three substances. The detection sensitivity of homocysteine, cysteine ​​and reduced glutathione is increased by 14.44 times, 5.08 times and 3.41 times, respectively. In addition, the stability of homocysteine, cysteine ​​and reduced glutathione derivatives is also significantly better than that of the original compounds.

[0048] (3) The method of the present invention adopts the multi-reaction monitoring (MRM) mode of triple quadrupole for scanning detection, limiting the two parameters of parent ion and daughter ion, with extremely high selectivity and improved sensitivity.

[0049] (4) The present invention optimizes the pretreatment method to achieve accurate detection of 9 substances, including homocysteine, methionine, cysteine, choline, betaine, reduced glutathione, S-adenosylhomocysteine, S-adenosylmethionine, and glutamate. The present invention can be used to prepare a test kit for homocystinemia, etc., and has good practical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 Schematic diagram of the in vivo transformation of key metabolites of the methionine cycle.

[0051] Figure 2 This is the MRM spectrum of key metabolites in the methionine cycle.

[0052] Figure 3 The MRM spectrum of the standard curve sample solution showing the QCH concentration.

[0053] Figure 4 The MRM spectrum of the standard curve sample solution showing the QCM concentration.

[0054] Figure 5 The MRM spectrum of the standard curve sample solution showing the QCL concentration.

[0055] Figure 6 This is a standard curve of methionine (MET).

[0056] Figure 7 This is a standard curve of homocysteine ​​(HCY).

[0057] Figure 8 This is a standard curve of cysteine ​​(CYS).

[0058] Fig. 9 This is the standard curve of choline (CHO).

[0059] Fig.10 This is the standard curve of betaine (BET).

[0060] Fig.11 This is a standard curve of reduced glutathione (GSH).

[0061] Fig.12 This is a standard curve of S-adenosylhomocysteine ​​(SAH).

[0062] Fig.13 This is a standard curve of glutamate (GLU).

[0063] Fig.14 This is a standard curve of S-adenosylmethionine (SAM).

[0064] Fig.15 The sensitivity of homocysteine, cysteine, and reduced glutathione before and after high-efficiency reduction-sulfhydryl derivatization.

[0065] Fig.16 The stability of homocysteine, cysteine, and reduced glutathione before and after high-efficiency reduction-sulfhydryl derivatization.

[0066] Fig.17 Serum methionine circulating key metabolites levels in patients with acute myocardial infarction and healthy controls

[0067] Fig.18 Serum methionine circulating key metabolite scores in patients with acute myocardial infarction and healthy controls

[0068] (A) PCA-X score graph, R2X(CUM)=1, Q2(CUM)=0.977; OPLS-DA score graph (B), R2X(CUM)=1, R2Y(CUM)=0.824, Q2(CUM)=0.782.

[0069] Fig.19 The predictive ability of key circulating metabolites of serum methionine for acute myocardial infarction.

[0070] Fig. 20 The levels of key metabolites of serum methionine circulation in NASH model mice and normal control mice.

[0071] Fig.21 The key metabolite score diagram of serum methionine circulation in NASH model mice and normal control mice is shown in Figure 2.

[0072] (A) PCA-X score graph, R2X(CUM)=1, Q2(CUM)=0.996; OPLS-DA score graph (B), R2X(CUM)=797, R2Y(CUM)=0.967, Q2(CUM)=0.93.

[0073] Fig. 22 The predictive ability of key circulating metabolites of serum methionine for nonalcoholic steatohepatitis. DETAILED DESCRIPTION

[0074] The experimental methods in the following examples of the present invention, where no specific conditions are specified, are usually carried out under conventional conditions or under conditions recommended by the manufacturers. The various commonly used chemical reagents used in the examples are all commercially available products.

[0075] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0076] The terms "including" and "having" and any variations thereof of the present invention are intended to cover non-exclusive inclusions. For example, a process, method, device, product or equipment comprising a series of steps is not limited to the listed steps or modules, but may optionally include steps not listed, or may optionally include other steps inherent to these processes, methods, products or equipment.

[0077] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with specific embodiments. It should be understood that these descriptions are exemplary only and are not intended to limit the scope of the present invention. In addition, in the following description, the description of known structures and technologies is omitted to avoid unnecessary confusion of the concept of the present invention.

[0078] The present invention is further described in the following examples, but the examples are not intended to limit the scope of protection of the present invention.

[0079] Example 1 HPLC-MS / MS methodology establishment

[0080] Part I. Reagents and Instruments

[0081] The sample analysis process mainly involves sample reduction, derivatization, protein removal pretreatment, chromatographic separation, and mass spectrometry identification. The reagents and instruments used are as follows:

[0082] 1. Reagents and instruments required for specimen pretreatment

[0083] Reagents: methanol (produced by Merck, purity is LC-MS grade), acetonitrile (produced by Merck, purity is LC-MS grade), formic acid (produced by Merck, purity is LC-MS grade).

[0084] Instruments: Cleanert PPT 96-well protein precipitation plate, catalog number 96CD2025Q; Cleanert M96 biological sample pretreatment instrument; Cleanert V96 sample nitrogen blow concentrator and 96-well collection plate, all of which are from Tianjin Bona Aiger Technology Co., Ltd.

[0085] 2. Reagents and instruments required for chromatographic separation

[0086] Reagents: 0.1% formic acid (volume ratio)-water solution and 0.1% formic acid (volume ratio)-80% methanol (volume ratio)-20% acetonitrile (volume ratio) solution. Formic acid, methanol and acetonitrile are all produced by Merck Company, and the purity is HPLC grade and the purity is LC-MS grade.

[0087] Instrument: Waters BEH C18 (1.7 μm, 2.1 × 50 mm) chromatographic column, produced by Waters; chromatographic column pore size

[0088] 3. Instruments required for mass spectrometry identification

[0089] Instrument: AB 4000Q-Trap mass spectrometer (manufactured by AB / SCIEX production department of Applied Biosystems, USA), the mass spectrometer uses TurboV ion source and triple quadruple as the mass analysis device of tandem mass spectrometry.

[0090] 4. Other reagents

[0091] Methionine (MET, >98%, S1230AS), cysteine ​​(CYS, >98%, S0118AS), S-adenosylmethionine (SAM, >96%, N0911A), betaine (BET, >98%, J0310AS), and glutamic acid (GLU, >99%, A0226A) were purchased from Dalian Meilun Biotechnology Co., Ltd.; S-adenosylhomocysteine ​​(SAH, 97.2%, R089M0) was purchased from USP (Twinbrook, Canada), choline (CHO, 47-50% in Water, FFJVM-BC), DL-Homocysteine ​​(HCY, >90%, PSGPG-HD) was purchased from TCI (Tokyo, Japan), L-phenylalanine-d5 (D5-Phe, SDHA-013) was purchased from CIL (Andover, USA), N-ethylmaleimide (NEM, ≥99.0%, BCBZ7317), tris(2-carbonylethyl)phosphine hydrochloride (TECP, ≥98.0%, SLBZ2552) were purchased from Sigma (St. Louis, MO, USA).

[0092] Part II: Methods

[0093] S1. Sample pretreatment: The serum samples were pretreated with efficient reduction-thiol derivatization to remove interfering proteins;

[0094] S2, chromatographic separation: injecting the sample processed in step S1 into a high performance liquid chromatography column to complete the preliminary separation of the sample;

[0095] S3, mass spectrometry identification: the sample preliminarily separated in step S2 is subjected to a mass spectrometer to perform quantitative analysis of the intensity of ion fragments and the collection of mass spectra; the method uses deuterated phenylalanine as an internal standard for quantitative detection of key metabolites of the methionine cycle.

[0096] The above steps S1 to S3 are specifically as follows:

[0097] (1) Specimen pretreatment

[0098] Homocysteine, cysteine ​​and reduced glutathione in serum samples are easily oxidized and have low detection sensitivity. They need to be protected by efficient reduction-sulfhydryl derivatization to improve their stability and detection sensitivity. In addition, serum contains a large amount of protein, which affects the quantitative detection of methionine substances. The samples need to be pretreated to remove interfering proteins. The specific steps are as follows:

[0099] 1) Prepare a methanol solution containing D5-Phe (100 ng / mL): add 20 μL of 1 mg / mL D5-Phe stock solution to 200 mL of methanol solution and mix thoroughly for later use;

[0100] 2) Prepare a solution containing N-ethylmaleimide (NEM, 69 mM): add 431.66 mg N-ethylmaleimide to 50 mL ultrapure water and fully dissolve;

[0101] 3) Prepare a solution containing tris(2-carbonylethyl)phosphine hydrochloride (TECP, 51 mM): add 146.19 mg N-tris(2-carbonylethyl)phosphine hydrochloride to 10 mL ultrapure water and dissolve thoroughly;

[0102] 4) Pretreatment before removing interfering proteins:

[0103] a) Preparation of standard curve sample solution: There are 10 concentration levels of standard curve sample solution from A to J, as shown in Table 1. Weigh the standard substances of methionine (MET), choline (CHO), betaine (BET), S-adenosylhomocysteine ​​(SAH), S-adenosylmethionine (SAM), and glutamic acid (GLU), and prepare them into concentrations of 5 mg / mL, 5 mg / mL, 20 mg / mL, 0.1 mg / mL, 20 mg / mL, 10 ... .5 mg / mL and 1 mg / mL stock solutions were prepared, and standards of homocysteine ​​(HCY), cysteine ​​(CYS), and reduced glutathione (GSH) were weighed and respectively configured with 50 mM N-ethylmaleimide (NEM) solution to make stock solutions with a concentration of 5 mg / mL, to obtain a series of standard solutions of methionine, choline, betaine, S-adenosylhomocysteine, S-adenosylmethionine, homocysteine, cysteine, glutamate and reduced glutathione.

[0104] Table 1 Standard curve concentrations for quantitative analysis of key metabolites in the methionine cycle

[0105]

[0106] b) Prepare specimens: Take 100 μL of human serum into an EP tube and vortex thoroughly to obtain a specimen. Perform the following steps;

[0107] ① Efficient reduction-thiol derivatization: First, add 50 μL of serum to the Cleanert PPT 96-well protein precipitation plate, then add 10 μL of 69 mM TECP solution, use a 96-well plate oscillator to vortex at 600 rpm for 1 min to mix evenly, and then let it stand for 13 min. Then, add 50 μL of 51 mM NEM, and then add 300 μL of methanol solution containing D5-Phe (100 ng / mL). Use a 96-well plate oscillator to vortex at 600 rpm for 5 min to mix evenly, and then let it stand at room temperature for 21 min.

[0108] ② Pretreatment to remove interfering proteins: Use Cleanert M 96 biological sample pretreatment instrument to collect the solution obtained after step ① into a clean collection plate for later use. The obtained solution is the test solution with interfering proteins removed;

[0109] ③ Drying and re-dissolving of pre-treated samples: Blow dry the solution in the collection plate with nitrogen in the Cleanert 96 sample nitrogen blow concentrator, set the temperature to 30°C, and then re-dissolve the dried specimen in 0.1mL 10% methanol-water solution. After mixing evenly on a vortex, subsequent testing can be carried out.

[0110] (2) Chromatographic separation

[0111] The pretreated specimens were injected into the HPLC column. The HPLC mobile phase program was shown in Table 2. The flow rate was set at 300 μL / min, the column temperature was fixed at 30°C, and the injection volume was set at 10 μL.

[0112] Table 2 Mobile phase program for HPLC separation

[0113]

[0114] (3) Mass spectrometry

[0115] The samples separated by HPLC were sent to the mass spectrometer for identification and quantitative analysis. The parameter settings of the mass spectrometer are shown in Table 3;

[0116] Table 3 Mass spectrometry parameters for quantitative analysis of key metabolites in the methionine cycle

[0117]

[0118] In the table, "Q1 Mass" refers to the parent ion, "Q3 Mass" refers to the daughter ion, and the unit is mass-to-charge ratio m / z; "DP" represents the declustering potential, and the unit is volt; "EP" represents the entrance voltage, and the unit is volt; "CE" represents the collision voltage, and the unit is volt; "CXP" represents the collision cell exit voltage, and the unit is volt.

[0119] Example 2 HPLC-MS / MS Methodology Verification

[0120] Since it is difficult to obtain a matrix that is completely free of methionine (MET), homocysteine ​​(HCY), cysteine ​​(CYS), choline (CHO), betaine (BET), reduced glutathione (GSH), S-adenosylhomocysteine ​​(SAH), S-adenosylmethionine (SAM) and glutamate (GLU), the present invention first uses serum treated with activated carbon adsorption for methodological evaluation and performance verification.

[0121] 1. Verification method

[0122] (1) Evaluation of linear relationship and sensitivity

[0123] The standard substances of methionine (MET), choline (CHO), betaine (BET), S-adenosylhomocysteine ​​(SAH), S-adenosylmethionine (SAM), and glutamic acid (GLU) were weighed and prepared into stock solutions with concentrations of 5 mg / mL, 5 mg / mL, 20 mg / mL, 0.1 mg / mL, 2.5 mg / mL, and 1 mg / mL, respectively, with 10% methanol solution; the standard substances of homocysteine ​​(HCY), cysteine ​​(CYS), and reduced glutathione (GSH) were weighed and prepared into stock solutions with concentrations of 5 mg / mL, respectively, with 50 mM N-ethylmaleimide (NEM) solution, to obtain a series of mixed standard solutions AJ containing methionine, choline, betaine, S-adenosylhomocysteine, S-adenosylmethionine, homocysteine, cysteine, reduced glutathione, and glutamic acid, as shown in Table 4 for details.

[0124] The standard curve samples with concentration J were diluted to a lower concentration gradient and tested on the instrument to determine the signal-to-noise ratio (S / N) until the S / N reached 10:1. Five samples were then processed in parallel to meet the CV value of less than 20%. The standard curve sample concentration at this time was the instrument's minimum limit of quantitation (LOQ). The minimum limit of quantitation (LOQ) value was used to represent the sensitivity of the HPLC-MS / MS technology platform of the present invention.

[0125] Table 4 Standard curve concentrations for quantitative analysis of key metabolites in the methionine cycle

[0126]

[0127] In addition, this study focused on the investigation and comparison of the detection sensitivity of homocysteine ​​(HCY), cysteine ​​(CYS), and reduced glutathione (GSH) before and after the high-efficiency reduction-sulfhydryl derivatization. 10ng / mL homocysteine ​​(HCY) or homocysteine ​​derivative (HCY-NEM), 100ng / mL cysteine ​​(CYS) or cysteine ​​derivative (CYS-NEM), 100ng / mL reduced glutathione (GSH) or reduced glutathione derivative (GSH-NEM) solutions were prepared respectively. Comparison of the peak areas of homocysteine ​​(HCY), cysteine ​​(CYS), and reduced glutathione (GSH) before and after high-efficiency reduction-sulfhydryl derivatization at the same concentration showed that after high-efficiency reduction-sulfhydryl derivatization, the peak areas of homocysteine ​​(HCY), cysteine ​​(CYS), and reduced glutathione (GSH) increased by 14.44 times, 5.08 times, and 3.41 times, respectively, compared with before high-efficiency reduction-sulfhydryl derivatization, indicating that the detection sensitivity has been significantly improved. See the attached Fig.15 .

[0128] (2) Effect of efficient reduction-thiol derivatization on the stability of three key metabolites

[0129] In order to investigate and compare the detection sensitivity of homocysteine ​​(HCY), cysteine ​​(CYS), and reduced glutathione (GSH) before and after the high-efficiency reduction-thiol derivatization. Prepare 10ng / mL homocysteine ​​(HCY) or homocysteine ​​derivative (HCY-NEM), 100ng / mL cysteine ​​(CYS) cysteine ​​derivative (CYS-NEM), 100ng / mL reduced glutathione (GSH) or reduced glutathione derivative (GSH-NEM) solution. Compare the changes in peak area of ​​homocysteine ​​(HCY), cysteine ​​(CYS), and reduced glutathione (GSH) before and after the high-efficiency reduction-thiol derivatization at the same concentration at 4℃ and 25℃ for 12h, 24h, 48h, 72h, 96h, and 120h, respectively. By the attached Fig.16 The results show that after the high-efficiency reduction-thiol derivatization, the stability of homocysteine ​​(HCY), cysteine ​​(CYS) and reduced glutathione (GSH) is significantly improved.

[0130] (3) Determination of method precision and accuracy

[0131] The accuracy and precision of the analytical method in this study were evaluated using standard samples at three concentration levels: QCH, QCM and QCL. See Table 5 for details.

[0132] Table 5 Intra-day accuracy and precision and inter-day accuracy and precision of key metabolites of methionine cycle

[0133]

[0134]

[0135] The results in Table 5 show that the intra-day accuracy and precision and inter-day accuracy and precision of the 9 compounds in the samples were good, with RSD less than 15%, indicating that this method is suitable for the quantitative detection of key metabolites of the methionine cycle in serum samples.

[0136] (4) Method stability study

[0137] The stability was evaluated using standard samples at three concentration levels: QCH, QCM and QCL. See Table 6 for details.

[0138] Table 6 Plasma stability of key metabolites of methionine cycle

[0139]

[0140]

[0141] The results in Table 6 show that the nine compounds have good stability in the samples and the accuracy is in the range of 85% to 115%, indicating that this method is suitable for the quantitative detection of key metabolites of the methionine cycle in serum samples.

[0142] (5) Spike recovery and matrix effect experiments using serum as matrix

[0143] The spike recovery and matrix effect were evaluated using standard samples at three concentration levels: QCH, QCM and QCL. See Table 7 for details.

[0144] Table 7 Plasma recoveries and matrix effects of key metabolites of methionine cycle

[0145]

[0146]

[0147] The results in Table 7 show that this method has good recovery rates for 9 compounds in serum and has no obvious matrix effect, indicating that this method is suitable for the quantitative detection of key metabolites of the methionine cycle in serum samples.

[0148] Example 3: Absolute quantitative analysis of key metabolites of the methionine cycle based on high-efficiency reduction-sulfhydryl derivatization HPLC-MS / MS for clinical sample detection

[0149] Part I. Reagents and Instruments

[0150] The sample analysis process mainly involves sample reduction, derivatization, protein removal pretreatment, chromatographic separation, and mass spectrometry identification. The reagents and instruments used are the same as those in Example 1.

[0151] Part II: Methods

[0152] The sample collection was approved by the Medical Ethics Committee of the Third Affiliated Hospital of Guangzhou Medical University. Serum samples of patients with acute myocardial infarction (n = 40) and healthy volunteers (n = 25) were obtained from the Third Affiliated Hospital of Guangzhou Medical University, and informed consent was obtained from all patients and healthy volunteers (Ethics Approval No.: 2021#026).

[0153] S1. Sample pretreatment: The serum samples were pretreated with efficient reduction-thiol derivatization to remove interfering proteins;

[0154] S2, chromatographic separation: injecting the sample processed in step S1 into a high performance liquid chromatography column to complete the preliminary separation of the sample;

[0155] S3, mass spectrometry identification: the sample preliminarily separated in step S2 is subjected to a mass spectrometer to perform quantitative analysis of the intensity of ion fragments and the collection of mass spectra; the method uses deuterated phenylalanine as an internal standard for quantitative detection of key metabolites of the methionine cycle.

[0156] The above steps S1 to S3 are specifically as follows:

[0157] (1) Specimen pretreatment

[0158] The specimens were pretreated to remove interfering proteins. The main steps are as follows:

[0159] 1) Prepare a methanol solution containing D5-Phe (100 ng / mL): add 20 μL of 1 mg / mL D5-Phe stock solution to 200 mL of methanol solution and mix thoroughly for later use;

[0160] 2) Prepare a solution containing N-ethylmaleimide (NEM, 69 mM): add 431.66 mg N-ethylmaleimide to 50 mL ultrapure water and fully dissolve;

[0161] 3) Prepare a solution containing tris(2-carbonylethyl)phosphine hydrochloride (TECP, 51 mM): add 146.19 mg N-tris(2-carbonylethyl)phosphine hydrochloride to 10 mL ultrapure water and dissolve thoroughly;

[0162] 4) Pretreatment before removing interfering proteins:

[0163] a) Preparation of standard curve sample solution: There are 10 concentration levels of standard curve sample solution from A to J, as shown in Table 8. Weigh the standard substances of methionine (MET), choline (CHO), betaine (BET), S-adenosylhomocysteine ​​(SAH), S-adenosylmethionine (SAM), and glutamic acid, and prepare them into concentrations of 5 mg / mL, 5 mg / mL, 20 mg / mL, 0.1 mg / mL, 2.5 mg / mL, respectively, with 10% methanol solution. mL and 1 mg / mL stock solutions, homocysteine ​​(HCY), cysteine ​​(CYS), and reduced glutathione (GSH) standards were weighed, and 50 mM N-ethylmaleimide (NEM) solutions were respectively prepared into 5 mg / mL stock solutions to obtain a series of standard solutions of methionine, choline, betaine, S-adenosylhomocysteine, S-adenosylmethionine, homocysteine, cysteine, glutamate, and reduced glutathione.

[0164] Table 8 Standard curve concentrations for quantitative analysis of key metabolites in the methionine cycle

[0165]

[0166] b) Prepare specimens: Take 100 μL of human serum into an EP tube and vortex thoroughly to obtain a specimen. Perform the following steps;

[0167] ① Efficient reduction-thiol derivatization: First, add 50 μL of human serum to the Cleanert PPT 96-well protein precipitation plate, then add 10 μL of 69 mM TECP solution, use a 96-well plate oscillator to vortex at 600 rpm for 1 min to mix evenly, and then let it stand for 13 min. Then, add 50 μL of 51 mM NEM, and then add 300 μL of methanol solution containing D5-Phe (100 ng / mL). Use a 96-well plate oscillator to vortex at 600 rpm for 5 min to mix evenly, and then let it stand at room temperature for 21 min.

[0168] ② Pretreatment to remove interfering proteins: Use Cleanert M96 biological sample pretreatment instrument to collect the solution obtained after step ① into a clean collection plate for later use; the obtained solution is the test solution with interfering proteins removed;

[0169] ③ Drying and re-dissolving of pre-treated samples: Blow dry the solution in the collection plate with nitrogen in the Cleanert 96 sample nitrogen blow concentrator, and set the temperature to 30°C; after drying, re-dissolve the sample in 0.1mL 10% methanol-water solution, mix well on a vortex and then proceed with subsequent testing.

[0170] (2) Chromatographic separation

[0171] The pretreated specimens were injected into the HPLC column. The HPLC mobile phase parameters are shown in Table 9. The flow rate was set at 300 μL / min, the column temperature was fixed at 30°C, and the injection volume was set at 10 μL.

[0172] Table 9 Mobile phase conditions for HPLC separation

[0173]

[0174] (3) Mass spectrometry

[0175] The samples separated by HPLC were sent to the mass spectrometer for identification and quantitative analysis. The parameter settings of the mass spectrometer are shown in Table 10.

[0176] Table 10 Mass spectrometry parameters for quantitative analysis of key metabolites in the methionine cycle

[0177]

[0178]

[0179] In the table, "Q1 Mass" refers to the parent ion, "Q3 Mass" refers to the daughter ion, and the unit is mass-to-charge ratio m / z; "DP" represents the declustering potential, and the unit is volt; "EP" represents the entrance voltage, and the unit is volt; "CE" represents the collision voltage, and the unit is volt; "CXP" represents the collision cell exit voltage, and the unit is volt.

[0180] Part III: Results Analysis

[0181] (1) Contents of key metabolites of methionine cycle in serum of different populations

[0182] The levels of key circulating metabolites of serum methionine were compared between healthy volunteers (Normal) and patients with acute myocardial infarction (AMI). Fig.17 The results showed that the levels of serum methionine cycle key metabolites in AMI patients were significantly different from those in healthy volunteers. Among them, the levels of betaine, choline, S-adenosylhomocysteine, homocysteine, and glutamate were significantly increased in AMI patients. The levels of S-adenosylmethionine and glutathione were significantly decreased in AMI patients; there was no significant difference in cysteine ​​levels between the two groups. The results of the PCA-X and OPLS-DA models constructed based on the serum methionine cycle key metabolites showed that the methionine cycle key metabolites could significantly distinguish healthy volunteers from AMI patients (see Fig.18 ).

[0183] (2) Diagnostic ability of key metabolites of methionine cycle for acute myocardial infarction

[0184] ROC curve analysis was used to study key metabolites of the methionine cycle, and the area under the curve (AUC) was used for quantitative analysis. AUCs>0.5 indicated that the method had diagnostic value, AUCs between 0.7 and 0.9 indicated a certain degree of accuracy, and AUCs>0.9 indicated high accuracy. Fig.19 The results showed that key metabolites of methionine circulation that can distinguish healthy volunteers and AMI patients were found, including homocysteine ​​(AUC = 0.994), S-adenosylmethionine (AUC = 0.962), methionine (AUC = 0.928), glutathione (AUC = 0.894), glutamate (AUC = 0.798), choline (AUC = 0.758), S-adenosylhomocysteine ​​(AUC = 0.754), betaine (AUC = 0.746), and cysteine ​​(AUC = 0.574). Among them, the comprehensive predictive ability of homocysteine ​​(HCY), S-adenosylmethionine (SAM), methionine (MET), glutathione (GSH), glutamate (GLU), choline (CHO), S-adenosylhomocysteine ​​(SAH), and betaine (BET) exceeded 0.7. Among them, the AUC values ​​of homocysteine ​​(HCY), S-adenosylmethionine (SAM), and methionine (MET) were greater than 0.9, indicating that these metabolites have the best predictive ability for myocardial infarction and are suitable for the diagnosis of patients with myocardial infarction.

[0185] Example 4: Absolute quantitative analysis of key metabolites of the methionine cycle based on high-efficiency reduction-sulfhydryl derivatization HPLC-MS / MS for basic experimental research on metabolic diseases

[0186] Part I. Reagents and Instruments

[0187] The sample analysis process mainly involves sample reduction, derivatization, protein removal pretreatment, chromatographic separation, and mass spectrometry identification. The reagents and instruments used are the same as those in Example 1.

[0188] Part II: Methods

[0189] (1) Sample

[0190] C57 BL / 6J (18–20 g) mice were purchased from the Medical Experimental Animal Center of Southern Medical University (Laboratory Animal Production License No.: SCXK (Guangdong) 2016-0041; Laboratory Animal License No.: NO.44002100016098). This study was approved by the Experimental Animal Ethics Committee of Guangzhou Medical University (Ethics No.: 2018-051). The normal control group experimental animals were given normal feed, and the model group animals were given high-fat and high-cholesterol feed (20180117, Guangdong Medical Experimental Animal Center). After 24 weeks of modeling, all experimental animals were fasted but not watered for 12 h. After anesthetizing the animals, blood was collected through the abdominal aorta, centrifuged at 3000 rpm for 10 min, and serum was separated and stored in a -80 °C refrigerator for testing.

[0191] (2) Specimen pretreatment

[0192] Homocysteine, cysteine ​​and reduced glutathione in serum samples are easily oxidized and have low detection sensitivity. They need to be protected by efficient reduction-sulfhydryl derivatization to improve their stability and detection sensitivity. In addition, serum contains a large amount of protein, which affects the quantitative detection of methionine substances. The samples need to be pretreated to remove interfering proteins. The main steps are as follows:

[0193] 1) Prepare a methanol solution containing D5-Phe (100 ng / mL): add 20 μL of 1 mg / mL D5-Phe stock solution to 200 mL of methanol solution and mix thoroughly for later use;

[0194] 2) Prepare a solution containing N-ethylmaleimide (NEM, 69 mM): add 431.66 mg N-ethylmaleimide to 50 mL ultrapure water and fully dissolve;

[0195] 3) Prepare a solution containing tris(2-carbonylethyl)phosphine hydrochloride (TECP, 51 mM): add 146.19 mg N-tris(2-carbonylethyl)phosphine hydrochloride to 10 mL ultrapure water and dissolve thoroughly;

[0196] 4) Pretreatment before removing interfering proteins:

[0197] a) Preparation of standard curve sample solution: There are 10 concentration levels of standard curve sample solution from A to J, as shown in Table 11. Weigh the standard substances of methionine (MET), choline (CHO), betaine (BET), S-adenosylhomocysteine ​​(SAH), S-adenosylmethionine (SAM), and glutamic acid, and prepare them into concentrations of 5 mg / mL, 5 mg / mL, 20 mg / mL, 0.1 mg / mL, 2.5 mg / mL, respectively, with 10% methanol solution. mL and 1 mg / mL stock solutions, homocysteine ​​(HCY), cysteine ​​(CYS), and reduced glutathione (GSH) standards were weighed, and 50 mM N-ethylmaleimide (NEM) solutions were respectively prepared into 5 mg / mL stock solutions to obtain a series of standard solutions of methionine, choline, betaine, S-adenosylhomocysteine, S-adenosylmethionine, homocysteine, cysteine, glutamate, and reduced glutathione.

[0198] Table 11 Standard curve concentrations for quantitative analysis of key metabolites in the methionine cycle

[0199]

[0200] b) Prepare specimens: Take 100 μL of blank mouse serum into an EP tube and vortex thoroughly to obtain a specimen, and proceed with the following steps;

[0201] ① Efficient reduction-thiol derivatization: First, add 50 μL of blank mouse serum to the Cleanert PPT 96-well protein precipitation plate, then add 10 μL of 69 mM TECP solution, use a 96-well plate oscillator to vortex at 600 rpm for 1 min to mix evenly, and then let it stand for 13 min. Then, add 50 μL of 51 mM NEM, and then add 300 μL of methanol solution containing D5-Phe (100 ng / mL). Use a 96-well plate oscillator to vortex at 600 rpm for 5 min to mix evenly, and then let it stand at room temperature for 21 min.

[0202] ② Pretreatment to remove interfering proteins: Use Cleanert M96 biological sample pretreatment instrument to collect the solution obtained after step ① into a clean collection plate for later use; the obtained solution is the test solution with interfering proteins removed;

[0203] ③ Drying and re-dissolving of pre-treated samples: Blow dry the solution in the collection plate with nitrogen in the Cleanert 96 sample nitrogen blow concentrator, and set the temperature to 30°C; after drying, re-dissolve the sample in 0.1mL 10% methanol-water solution, mix well on a vortex and then proceed with subsequent testing.

[0204] (3) Chromatographic separation

[0205] The pretreated specimens were injected into the HPLC column. The HPLC mobile phase parameters were shown in Table 12. The flow rate was set at 300 μL / min, the column temperature was fixed at 30°C, and the injection volume was set at 10 μL.

[0206] Table 12 Mobile phase conditions for HPLC separation

[0207]

[0208] (4) Mass spectrometry

[0209] The samples separated by HPLC were sent to the mass spectrometer for identification and quantitative analysis. The parameter settings of the mass spectrometer are shown in Table 2.

[0210] Table 16. Mass spectrometry parameters for quantitative analysis of key metabolites in the methionine cycle

[0211]

[0212] In the table, "Q1 Mass" refers to the parent ion, "Q3 Mass" refers to the daughter ion, and the unit is mass-to-charge ratio m / z; "DP" represents the declustering potential, and the unit is volt; "EP" represents the entrance voltage, and the unit is volt; "CE" represents the collision voltage, and the unit is volt; "CXP" represents the collision cell exit voltage, and the unit is volt.

[0213] Part III: Results Analysis

[0214] (1) Contents of key metabolites of methionine cycle in serum of experimental mice

[0215] The levels of key metabolites of serum methionine cycle in normal control mice (Normal) and non-alcoholic steatohepatitis model mice (NASH) were compared. Fig. 20 The results showed that the levels of serum methionine cycle key metabolites in NASH model mice were significantly different from those in the normal group. Among them, the levels of betaine, choline, S-adenosylhomocysteine, homocysteine, glutamate, cysteine, and methionine were significantly increased in NASH model mice. The levels of S-adenosylmethionine and glutathione were significantly reduced in NASH model mice; the results of the PCA-X and OPLS-DA models constructed based on the serum methionine cycle key metabolites showed that the normal control group mice and NASH model mice could be clearly distinguished using the detected methionine cycle key metabolites (see Appendix Fig.21 ).

[0216] (2) The diagnostic and predictive capabilities of key methionine cycle metabolites for nonalcoholic steatohepatitis

[0217] ROC curve analysis was used to study key metabolites of the methionine cycle, and the area under the curve (AUC) was used for quantitative analysis. AUCs>0.5 indicated that the method had diagnostic value, AUCs between 0.7 and 0.9 indicated a certain degree of accuracy, and AUCs>0.9 indicated high accuracy. Fig. 22 The results revealed key methionine cycle metabolites that could distinguish between normal control mice (Normal) and non-alcoholic steatohepatitis model mice (NASH), including homocysteine ​​(AUC=1), S-adenosylmethionine (AUC=1), glutathione (AUC=1), glutamate (AUC=0.99), S-adenosylhomocysteine ​​(AUC=0.99), methionine (AUC=0.97), choline (AUC=0.9), cysteine ​​(AUC=0.87), and betaine (AUC=0.83). Among them, the comprehensive predictive abilities of homocysteine ​​(HCY), S-adenosylmethionine (SAM), methionine (MET), glutathione (GSH), glutamate (GLU), choline (CHO), S-adenosylhomocysteine ​​(SAH), betaine (BET), and cysteine ​​(CYS) were all over 0.7. Among them, the AUC values ​​of homocysteine ​​(HCY), S-adenosylmethionine (SAM), methionine (MET), glutathione (GSH), glutamate (GLU), S-adenosylhomocysteine ​​(SAH), and choline (CHO) were greater than 0.9, indicating that these metabolites have the best predictive ability for NASH and are suitable for the diagnosis and prediction of NASH patients and animal models.

[0218] It should be noted that the specific features, structures, materials or characteristics described in this specification can be combined arbitrarily. In order to make the description concise, not all possible combinations of the various technical features in the above embodiments are described. If there is no contradiction, technicians in this field can combine and combine the different embodiments and features of different embodiments described in this specification.

[0219] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A method for absolute quantitative analysis of key metabolites of the methionine cycle, characterized in that: The key metabolites of the methionine cycle include methionine, homocysteine, cysteine, choline, betaine, reduced glutathione, S-adenosylhomocysteine, S-adenosylmethionine and glutamate. The method comprises the following steps: S1. Sample pretreatment: The serum samples were pretreated with efficient reduction-thiol derivatization and interfering proteins were removed, specifically: High-efficiency reduction-thiol derivatization: first add serum to a 96-well protein precipitation plate, add tris(2-carbonylethyl)phosphine hydrochloride solution, mix evenly with a 96-well plate shaker and let stand, then add N-ethylmaleimide, then add methanol solution containing deuterated phenylalanine, mix evenly with a 96-well plate shaker and let stand at room temperature; Pretreatment to remove interfering proteins: Use a biological sample pretreatment instrument to collect the solution obtained in the above steps into a clean collection plate for later use. The obtained solution is the test solution with interfering proteins removed; Pretreatment sample drying and re-dissolution: blow dry the solution in the collection plate with nitrogen, then re-dissolve the dried sample in a methanol-water solution, and then mix it evenly on a vortexer before proceeding with subsequent testing; S2, chromatographic separation: inject the sample processed in step S1 into a high performance liquid chromatography column to complete the preliminary separation of the sample. The chromatographic column in the chromatographic separation system adopts Waters BEH C18, 1.7 μm, 2.1 × 50 mm, pore size The HPLC flow program was: At 0 min, the proportion of 0.1% formic acid-water solution was 95%, and the proportion of 0.1% formic acid-80% methanol-20% acetonitrile solution was 5%; At 1 min, the proportion of 0.1% formic acid-water solution was 5%, and the proportion of 0.1% formic acid-80% methanol-20% acetonitrile solution was 95%; At 2 min, the proportion of 0.1% formic acid-water solution was 80%, and the proportion of 0.1% formic acid-80% methanol-20% acetonitrile solution was 20%; At 3 min, the proportion of 0.1% formic acid-water solution was 80%, and the proportion of 0.1% formic acid-80% methanol-20% acetonitrile solution was 20%; At 5 min, the proportion of 0.1% formic acid-water solution was 90%, and the proportion of 0.1% formic acid-80% methanol-20% acetonitrile solution was 10%; At 7 min, the proportion of 0.1% formic acid-water solution was 95%, and the proportion of 0.1% formic acid-80% methanol-20% acetonitrile solution was 5%; S3, mass spectrometry identification: the sample preliminarily separated in step S2 is subjected to a mass spectrometer to perform quantitative analysis of the intensity of ion fragments and the collection of mass spectra. The ionization mode of the mass spectrometer is electrospray ionization, and the mass analyzer is a triple quadrupole.

2. The absolute quantitative analysis method according to claim 1, characterized in that: The method further comprises the following steps: Prepare standard curve sample solution: weigh standards of methionine, choline, betaine, S-adenosylhomocysteine, S-adenosylmethionine, and glutamate, and prepare them into stock solutions with methanol solution; weigh standards of homocysteine, cysteine, and reduced glutathione, and prepare them into stock solutions with N-ethylmaleimide solution to obtain a series of standard solutions of methionine, choline, betaine, S-adenosylhomocysteine, S-adenosylmethionine, homocysteine, glutamate, cysteine, and reduced glutathione, respectively.

3. The method according to claim 1, characterized in that The HPLC flow rate of the chromatographic separation in step S2 was set to 300 μL / min, the column temperature was fixed at 30° C., and the injection volume was set to 10 μL.

4. The absolute quantitative analysis method according to claim 1, characterized in that: The mass spectrometer used in step S3 adopts the design of Turbo VTM ion source and curtain gas, and the triple quadrupole adopts the multiple reaction detection mode for scanning detection.

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