A combined metabolic marker and detection kit for evaluation of mitochondrial purity

Abstract

The application discloses a combined metabolic marker and detection kit for mitochondrial purity evaluation, the marker comprises phosphatidylcholine PC 34:0 (16:0 / 18:0), sphingomyelin SM 34:1;2O (d18:1;2O / 16:0), sphingomyelin SM 44:2;2O (18:1;2O / 24:1) and hexosylceramide HexCer 42:2;2O (d18:1;2O / 24:1), and is used for judging the new application and kit of mitochondrial purity of mitochondria extracted by a mitochondrial extraction method. The application judges the mitochondrial purity extracted by the method by detecting the relative concentrations of four lipid metabolites in the mitochondrial sample, calculating a combined marker variable P based on a binary logistic regression equation and determining a cut-off value. The kit can realize high-sensitivity and high-efficiency detection of the four small molecule metabolites, and has the characteristics of low detection cost and good repeatability.

Description

Technical Field

[0001] This invention relates to the fields of analytical chemistry, cell biology, and molecular biology, and specifically to a combined metabolic biomarker and detection kit for evaluating mitochondrial purity. Background Technology

[0002] Mitochondria are key organelles widely distributed in eukaryotic cells. As the cell's "powerhouse," mitochondria release energy stored in food breakdown products through respiration, providing essential energy support for various cellular metabolic activities. The mitochondrial tricarboxylic acid cycle (TCA cycle) is one of the key pathways for cellular energy production and plays a crucial role in regulating metabolic activities. In the human body, mitochondria are abundant in muscle cells and energy-demanding organs such as the liver, heart, and brain. Besides participating in energy metabolism, mitochondria also play important roles in numerous metabolic processes, including cell signaling, calcium ion buffering, reactive oxygen species (ROS) metabolism, apoptosis, fatty acid metabolism, and pyrimidine biosynthesis.

[0003] Mitochondrial dysfunction and defects are closely related to health. Studies have shown that mitochondria not only play a crucial role in the development of numerous diseases, including tumors, cardiovascular diseases, neurodegenerative diseases, diabetes, and immune diseases, but their abnormal activity is also closely related to the progression of these diseases. For example, in Alzheimer's disease and other neurodegenerative diseases, elevated Drp1 levels cause excessive mitochondrial fission, leading to mitochondrial dysfunction and neuronal damage. Mitochondrial dysfunction also plays a key role in insulin deficiency or insulin resistance metabolic diseases such as type 2 diabetes. Furthermore, mitochondrial dysfunction in liver tissue is associated with the development of various liver diseases, such as fatty liver, hepatitis, and liver cancer.

[0004] Existing research has shown that there are significant differences between the metabolic profiles of whole-cell components and those at the subcellular level. In subcellular metabolic studies of mitochondria, contamination from other organelles can not only lead to erroneous conclusions but also affect data comparability. Different extraction techniques result in variations in the purity of the obtained mitochondria. To ensure the rigor and accuracy of research, it is crucial that the purity of the mitochondria used in the study matches the research objectives. Therefore, evaluating the purity of mitochondria extracted by various purification methods at the subcellular level is of great significance for related mitochondrial research. Differential centrifugation (DC) and ultracentrifugation (UC) are two classic mitochondrial extraction methods. Differential centrifugation (DC) is based on the principle of different densities of different organelles at the subcellular level, achieving the separation or purification of mitochondria by setting different centrifugation conditions. Ultracentrifugation (UC) is a method that further purifies mitochondria by using solutions with different density gradients to centrifuge them at high speeds, building upon differential centrifugation (DC). Because grinding can damage mitochondria, column separation (CO) has been adopted as a novel method for mitochondrial extraction to minimize this damage. CO uses a density solution to swell cells, which are then broken up by a separation column, effectively avoiding damage to mitochondria caused by grinding. Mitochondria are then obtained using a density solution and various centrifugation conditions. Conversely, since centrifugation may precipitate other organelles besides mitochondria, magnetic-activated cell sorting (MACS) is another novel mitochondrial extraction method. This method avoids multiple centrifugation steps and, through an antigen-antibody combination, can accurately obtain mitochondria with high purity.

[0005] Lipidomics is an emerging discipline that systematically analyzes the overall lipid metabolism profile. By comparing changes in lipid metabolism networks under different physiological states, it identifies key lipid biomarkers in metabolic regulation and reveals the mechanisms by which lipids function in life activities. As major components of the membranes of various subcellular organelles, lipids participate in regulating many important processes in life, such as energy storage, substance transport, signal transduction, cell development, differentiation, and apoptosis. In recent years, lipid biomarkers have been widely used in food nutrition and toxicity evaluation, and in the clinical diagnosis and research of major diseases such as cancer, diabetes, and neurodegenerative diseases. Given the structure, function, and metabolic regulatory role of lipid metabolites, this invention employs lipidomics techniques and methods based on ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS) to analyze the lipid metabolism profiles of samples extracted using different mitochondrial extraction methods. By screening biomarkers, this invention determines the purity of mitochondria extracted using these methods. This invention proposes a novel application for evaluating mitochondrial purity using the combined use of the lipid metabolites phosphatidylcholine PC 34:0 (16:0 / 18:0), sphingomyelin SM34:1;2O (d18:1;2O / 16:0), sphingomyelin SM 44:2;2O (18:1;2O / 24:1), and hexosylceramide HexCer42:2;2O (d18:1;2O / 24:1). Since single metabolites can be influenced by multiple factors, and lipids play multiple roles in organisms, screening for combined metabolic markers composed of a few metabolites from purified mitochondria and calculating the "discrimination probability" (P-value) using a discriminant formula helps improve the sensitivity and specificity of the assay. Currently, there are no reports of using these four lipid metabolites in combination for the determination of mitochondrial purity. Summary of the Invention

[0006] To address the problems existing in the prior art, the purpose of this invention is to provide a combined metabolic biomarker and detection kit for evaluating mitochondrial purity, which is achieved through the following technical solution:

[0007] The first aspect of the present invention provides a combined metabolic marker for evaluating mitochondrial purity, the marker comprising: phosphatidylcholine PC 34:0 (16:0 / 18:0), sphingomyelin SM34:1;2O (d18:1;2O / 16:0), sphingomyelin SM 44:2;2O (18:1;2O / 24:1), and hexosylceramide HexCer42:2;2O (d18:1;2O / 24:1).

[0008] A second aspect of the present invention provides the use of combined metabolic markers in evaluating mitochondrial purity.

[0009] The third aspect of this invention provides a method for determining the purity of mitochondria extracted using a combined metabolic biomarker method, wherein the determination is made using a combined biomarker variable P:

[0010] P=1 / (1+e^(-42.608a+5.583b-7.591c+7.343d+11.075))

[0011] Where a represents the relative content of phosphatidylcholine PC 34:0 (16:0 / 18:0), b represents the relative content of sphingomyelin SM34:1;2O (d18:1;2O / 16:0), c represents the relative content of sphingomyelin SM44:2;2O (18:1;2O / 24:1), and d represents the relative content of hexosylceramide HexCer42:2;2O (d18:1;2O / 24:1). If P < 0.5, the purity of mitochondria obtained by this method is higher than that obtained by classical differential centrifugation (DC) and comparable to that obtained by classical ultracentrifugation (UC). If P > 0.5, the purity of mitochondria obtained by this method is lower than that obtained by classical ultracentrifugation (UC) but comparable to that obtained by classical differential centrifugation (DC).

[0012] A fourth aspect of the present invention provides a kit for detecting combined metabolic biomarkers, comprising:

[0013] Standards: Ceramide Cer 35:1;2 (d18:1 / 17:0), Phosphatidylcholine PC 38:0 (19:0 / 19:0) and Sphingomyelin SM 30:1;2 (d18:1; / 12:0);

[0014] Pretreatment extract: a methanol solution containing internal standards 1.27 μg / mL ceramide Cer 35:1;2 (d18:1 / 17:0), 2.17 μg / mL phosphatidylcholine PC 38:0 (19:0 / 19:0), and 1.38 μg / mL sphingomyelin SM 30:1;2 (d18:1; / 12:0). In positive ion detection mode, ceramide Cer 35:1;2 (d18:1 / 17:0) was used to correct for hexosylceramide, and sphingomyelin SM 30:1;2 (d18:1; / 12:0) was used to correct for sphingomyelin. In negative ion detection mode, phosphatidylcholine PC 38:0 (19:0 / 19:0) was used to correct for phosphatidylcholine.

[0015] Eluent: Mobile phase A is an acetonitrile / water solution containing 10 mM ammonium acetate (v / v = 6:4), and mobile phase B is an isopropanol / acetonitrile solution containing 10 mM ammonium acetate (v / v = 9:1).

[0016] The fifth aspect of this invention provides a method for calculating a combined biomarker variable using a detection kit, comprising the following steps:

[0017] 1) Pretreatment of test mitochondrial samples: Methanol containing an internal standard was added to the test mitochondrial samples, followed by methyl tert-butyl ether and ultrapure water, and then lipid metabolites were extracted;

[0018] 2) The lipid metabolites extracted from the mitochondrial test samples after step 1) were separated and identified using ultra-high performance liquid chromatography-mass spectrometry.

[0019] 3) The detected ions were further confirmed using the ceramide Cer 35:1;2 (d18:1 / 17:0), phosphatidylcholine PC 38:0 (19:0 / 19:0), and sphingomyelin SM 30:1;2 (d18:1; / 12:0) standards provided in the kit:

[0020] a) The ceramide Cer 35:1;2 (d18:1 / 17:0), phosphatidylcholine PC 38:0 (19:0 / 19:0) and sphingomyelin SM 30:1;2 (d18:1; / 12:0) standards in the kit were analyzed by ultra-high performance liquid chromatography-mass spectrometry to determine the chromatographic retention time, measured mass-to-charge ratio, and secondary mass spectrometry characteristic ions of the standards. Using the LipidMaps database and kits containing ceramide Cer 35:1;2 (d18:1 / 17:0), phosphatidylcholine PC 38:0 (19:0 / 19:0), and sphingomyelin SM 30:1;2 (d18:1; / 12:0) standards, the bond-breaking patterns of ceramide, phosphatidylcholine, and sphingomyelin in secondary mass spectrometry were determined: In positive ion mode, the characteristic secondary chain of ceramide is the LCB chain, which is determined based on the number of dehydrated molecules and the chain structure; sphingomyelin undergoes neutral deletion, yielding a characteristic ion with m / z of 184; in negative ion mode, the acyl chain of phosphatidylcholine breaks, generating characteristic fatty acid ions corresponding to different sites, and the corresponding characteristic ions are found in secondary mass spectrometry based on the m / z of the fatty acid ions.

[0021] b) In the tested mitochondrial samples, under reasonable mass-to-charge ratio tolerance thresholds, the chromatographic peaks of hexosylceramide HexCer42:2;2O (d18:1;2O / 24:1) with a theoretical mass-to-charge ratio of 782.65039±5ppm and sphingomyelin SM34:1;2O (d18:1;2O / 16:0) and sphingomyelin SM44:2;2O (18:1;2O / 24:1) with mass-to-charge ratios of 703.57489±5ppm and 841.7157±5ppm, respectively, were extracted in positive ion mode; and the chromatographic peak of phosphatidylcholine PC 34:0 (16:0 / 18:0) with a mass-to-charge ratio of 820.6073±5ppm was extracted in negative ion mode.

[0022] c) Verify whether the secondary mass spectrometry ions of the above four chromatographic peaks conform to the secondary mass spectrometry bond breaking rules of ceramide, sphingomyelin, and phosphatidylcholine. At the same time, combined with the chromatographic retention behavior, confirm the targets: phosphatidylcholine PC 34:0 (16:0 / 18:0), sphingomyelin SM34:1;2O (d18:1;2O / 16:0), sphingomyelin SM 44:2;2O (18:1;2O / 24:1), and hexosylceramide HexCer42:2;2O (d18:1;2O / 24:1).

[0023] 4) For the target phosphatidylcholine PC 34:0 (16:0 / 18:0), sphingomyelin SM34:1;2O (d18:1;2O / 16:0), sphingomyelin SM 44:2;2O (18:1;2O / 24:1), and hexosylceramide HexCer42:2;2O (d18:1;2O / 24:1) in the identified mitochondrial test samples, their chromatographic peak intensities were corrected for the total elution peak area of ​​the mitochondrial sample to obtain the relative concentrations of the above four metabolites.

[0024] The joint biomarker P was obtained by binary logistic regression analysis of phosphatidylcholine PC 34:0 (16:0 / 18:0), sphingomyelin SM34:1;2O (d18:1;2O / 16:0), sphingomyelin SM 44:2;2O (18:1;2O / 24:1), and hexosylceramide HexCer42:2;2O (d18:1;2O / 24:1).

[0025] The principle of this invention is as follows:

[0026] 1) Using ultra-high performance liquid chromatography-quadrupole and orbital trap hybrid Fourier transform ultra-high resolution mass spectrometry combined with lipidomics analysis, lipidomics analysis was performed on mitochondrial lipid metabolism extracts obtained by the classic mitochondrial purification methods differential centrifugation (DC) and ultracentrifugation (UC) (discovery set, i.e. Example 1) to obtain qualitative and quantitative analysis results of lipid metabolites.

[0027] 2) Based on a comparison of mitochondrial lipid metabolism profiles obtained using classic differential centrifugation (DC) and ultracentrifugation (UC), differentially expressed lipid compounds are screened to identify potential biomarkers. Specific methods include:

[0028] i) Screening for typical metabolomic differences: based on univariate statistical significance ( p <0.05 & FDR<0.1) and multivariate analysis requirements (VIP>1) were used to screen the set of typical differential metabolites compared between differential centrifugation (DC) and ultracentrifugation (UC);

[0029] ii) Change ratio screening: From the typical differential metabolite set, differential metabolites with a change ratio greater than or equal to 10 (Ratio ≥ 10, Ratio ≤ 0.1) are screened.

[0030] iii) Stability requirements for analysis: Based on more stringent data analysis quality requirements, potential biomarkers were further screened with RSD < 5% in QC samples and mitochondrial metabolite samples extracted by differential centrifugation (DC).

[0031] iv) Obtain the lipid metabolite intersection of the above three data screening methods, and finally select the combination of phosphatidylcholine PC34:0 (16:0 / 18:0), sphingomyelin SM34:1;2O (d18:1;2O / 16:0), sphingomyelin SM 44:2;2O (18:1;2O / 24:1), and hexosylceramide HexCer42:2;2O (d18:1;2O / 24:1) as a combined biomarker, and analyze the P-value of the combined biomarker. If P < 0.5, it is judged that the purity of mitochondria obtained by the purification method is high, higher than that of the classic differential centrifugation (DC) method, and comparable to that of the classic ultracentrifugation (UC) method. If P > 0.5, it is judged that the purity of mitochondria obtained by the purification method is low, lower than that of the classic ultracentrifugation (UC) method, and comparable to that of the classic differential centrifugation (DC) method.

[0032] 3) The mitochondria extracted by differential centrifugation (DC) and ultracentrifugation (UC) were analyzed by Western blot protein immunoblotting to verify the above results.

[0033] 4) The effectiveness of the above-mentioned combined metabolic biomarkers was further verified by comparing column separation (CO) and magnetic bead separation (MACS) (validation set, Example 2).

[0034] This invention relates to a novel application of the lipid metabolites phosphatidylcholine PC 34:0 (16:0 / 18:0), sphingomyelin SM34:1;2O (d18:1;2O / 16:0), sphingomyelin SM 44:2;2O (18:1;2O / 24:1), and hexosylceramide HexCer42:2;2O (d18:1;2O / 24:1) in mitochondrial samples as biomarkers for determining mitochondrial purity. This invention also relates to a kit for determining mitochondrial purity. The kit enables highly sensitive and efficient detection of the four small molecule metabolites involved in this invention, and features low detection cost and good reproducibility. The combined use of these four metabolites shows promising application prospects. Attached Figure Description

[0035] Figure 1 a- Figure 1d represents the structural formulas and secondary fragmentation characteristics of four lipid biomarkers;

[0036] Figure 2 a- Figure 2 d represents the content of combined lipid biomarkers in each group of samples (expressed as mean ± standard error) and ROC curve analysis;

[0037] Figure 3 The results are from Western blotting experiments on whole cell components and four mitochondrial extraction methods. Detailed Implementation

[0038] The present invention will be further described below with reference to the accompanying drawings and specific embodiments in order to better understand the present technical solution.

[0039] Example 1

[0040] 1. Mitochondrial sample collection

[0041] (1) Differential centrifugation (DC): When the cells reach 90%~100% coverage of the culture dish, discard the culture medium, wash gently twice with 2 mL PBS, and then digest the cells with trypsin at 37 °C for two minutes. Add complete culture medium to stop the digestion. Gently pipette to detach the cells from the culture dish, collect the suspension in the culture dish into a centrifuge tube, and centrifuge at 1000 g for 3 min. Discard the supernatant, resuspend the cells with 2 mL KPBS, wash, and centrifuge at 1000 g for 3 min. Then discard the supernatant and add 1 mL of mitochondrial separation reagent (Beyotime, China) to resuspend. Transfer the resulting suspension to a Dounce homogenizer that has been placed on ice in advance, and use a tight-fitting pestle to grind the cells vertically up and down 35 times to break them up. 200 μL of the homogenate was used as the whole-cell fraction for subsequent Western blotting analysis. The remaining homogenate was centrifuged twice at 1000 g at 4 °C for 10 min and 5 min, respectively, to remove cell nuclei, cell debris, and undisturbed cells. Finally, the supernatant was collected and centrifuged at 10000 g for 20 min at 4 °C to obtain a mitochondrial-rich precipitate.

[0042] (2) Ultracentrifugation (UC): Cell culture, digestion, collection, and disruption are as described in (1). The obtained homogenate is centrifuged at 1000 g for 10 min at 4 ℃, followed by another centrifugation for 5 min to remove cell nuclei, cell debris, and undisrupted cells. The supernatant is collected in a new centrifuge tube and centrifuged at 10000 g for 30 min to roughly extract mitochondria. The supernatant is discarded, and the obtained precipitate is resuspended in pre-cooled mitochondrial separation reagent and placed on ice for subsequent ultracentrifugation. The specific process is as follows:

[0043] Add 15 mL of 1.5 M sucrose solution (1.5 M sucrose, 10 mM Tris-HCl, 1 mM EDTA, pH 7.5) to an ultracentrifuge tube. Tilt the ultracentrifuge tube at 45° and vertically add 1 M sucrose solution (1.0 Msucrose, 10 mM Tris-HCl, 1 mM EDTA, pH 7.5) to the top layer of the 1.5 M sucrose solution, creating a 30 mL sucrose gradient. Then, place the mitochondrial suspension, previously placed on ice, on top of the 1 M sucrose solution. Centrifuge at 8000g for 20 min at 4 °C. After centrifugation, carefully aspirate the mitochondrial layer at the interface between the 1.5 M and 1 M sucrose solutions using a pipette. Add diluent (5 mM Tris-HCl, 1 mM EDTA) to the collected mitochondrial-containing solution, gradually increasing the dilution to a concentration of 0.25 M. Centrifuge the diluted mitochondrial suspension at 10,000 g for 20 min at 4 °C to obtain mitochondrial precipitate. Gently wash once with pre-chilled KPBS, and centrifuge at 10,000 g for 20 min at 4 °C to obtain mitochondrial precipitate again.

[0044] 2. Sample Pretreatment

[0045] Sample pretreatment was performed on ice. 400 μL of methanol extract containing internal standard was added to each mitochondrial sample extracted by differential centrifugation (DC) and ultracentrifugation (UC), and the mixture was vortexed for 30 s. 1 mL of MTBE was added, vortexed for 30 s, and then centrifuged at 1000 rpm and 10 °C for 30 min. Subsequently, 400 μL of ultrapure water was added, vortexed for 1 min, and then centrifuged at 6 °C and 12000 g for 10 min. After phase separation, the upper hydrophobic phase was quantitatively collected and vacuum-dried in a lyophilized centrifuge. The resulting lyophilized samples were stored at -80 °C.

[0046] Before the lipid lyophilized samples were detected by ultra-high performance liquid chromatography-mass spectrometry, they were reconstituted with a methanol / dichloromethane mixed solution (v / v=1:2), and then diluted with acetonitrile / isopropanol / water (v / v / v=65:30:5, containing 5 mM ammonium acetate) before injection.

[0047] 3. Data Collection

[0048] 1) Ultra-high performance liquid chromatography (UHPLC) conditions: A Thermo Fisher UltiMate 3000 (UPLC, Thermo, USA) system was used, with an ACQUITY UPLC BEH C8 column (2.1 × 100 mm × 1.7 µm, Waters, USA). During separation, the column temperature was set to 55℃, and the injection chamber temperature was controlled at 8℃. Mobile phase A was acetonitrile:ultrapure water = 6:4 (containing 10 mM ammonium acetate), and mobile phase B was isopropanol:acetonitrile = 9:1 (containing 10 mM ammonium acetate). A gradient elution mode was used, with the following gradient: 32% B was held for 1.5 min, then linearly increased to 85% B in the 1.5–15.5 min range, then linearly increased to 97% B in 0.1 min and held for 18 min, followed by a decrease to 32% B in 0.1 min until equilibrium was reached for the next injection. The flow rate was maintained at 0.4 mL / min throughout the process.

[0049] 2) Mass Spectrometry Conditions: A quadrupole and orbital trap hybridized Fourier transform ultra-high resolution mass spectrometer (Q-Exactive, Thermo, USA) was used. Mass spectrometry analysis employed electrospray ionization (ESI) in both positive and negative ion modes. The mass spectrometry parameters were set as follows: spray voltage 3.0 kV, sheath gas flow rate 50 arb, auxiliary gas flow rate 15 arb, auxiliary gas temperature 400℃, capillary temperature 325℃, acquisition range 200–1800 m / z, acquisition rate 1.7 spectra / s. Secondary analysis involved acquiring the top 10 precursor ions in HCD fragmentation mode with collision energies of 25–35 eV. During acquisition, Xcalibur software (Thermo, USA) was used to record the total ion chromatogram and MS spectra.

[0050] 4. Data Preprocessing

[0051] The raw mass spectrometry data obtained from ULPC-Q Exative were imported into MSDIAL software (http: / / prime.psc.riken.jp / compms / index.html) for peak identification, extraction, and alignment, excluding signal peaks with a signal-to-noise ratio below 10. The resulting peak table was used to identify lipids using Xcalibular. The identified lipids met the following three criteria:

[0052] 1) Precise mass number obtained by first-order mass spectrometry (MS) (theoretical value ± 5 ppm);

[0053] 2) Fragmentation patterns observed by secondary mass spectrometry (MS / MS);

[0054] 3) Regularity of chromatographic retention time.

[0055] Accurately identified lipid compounds were introduced into a TraceFinder (Thermo, USA) for quantification. The peak intensity was then corrected based on the total elution peak area of ​​the sample.

[0056] 5. Results Analysis

[0057] Structural formulas and secondary mass spectrometry of lipid biomarkers, such as Figure 1 As shown. Correlation analysis of mitochondrial purity is as follows. Figure 2 As shown.

[0058] Using SPSS statistical software, the joint biomarker P was further calculated by binary logistic regression of phosphatidylcholine PC 34:0 (16:0 / 18:0), sphingomyelin SM34:1;2O (d18:1;2O / 16:0), sphingomyelin SM 44:2;2O (18:1;2O / 24:1), and hexosylceramide HexCer42:2;2O (d18:1;2O / 24:1). The regression equation is as follows:

[0059] P=1 / (1+e^(-42.608a+5.583b-7.591c+7.343d+11.075))

[0060] Where a represents the relative content of phosphatidylcholine PC 34:0 (16:0 / 18:0), b represents the relative content of sphingomyelin SM34:1;2O (d18:1;2O / 16:0), c represents the relative content of sphingomyelin SM 44:2;2O (18:1;2O / 24:1), and d represents the relative content of hexosylceramide HexCer42:2;2O (d18:1;2O / 24:1). If P < 0.5, the mitochondrial purity obtained by the purification method is high, exceeding that of the classic differential centrifugation (DC) method, and comparable to the classic ultracentrifugation (UC) method. If P > 0.5, the mitochondrial purity obtained by the purification method is low, lower than that of the classic ultracentrifugation (UC) method, but comparable to the classic differential centrifugation (DC) method.

[0061] All samples from the DC group and the UC group were included in the discriminant comparison. The judgment variable P was obtained based on this marker. Figure 2 The area under the ROC curve (AUC) used for discrimination was 1. At a cut-off value of 0.5, both sensitivity and specificity were 100% (Table 1). Figure 2 c) The above results indicate that this marker has good discriminative potential and can be used to evaluate the purity of the proposed mitochondria. Furthermore, the P-value obtained in the UC group was <0.5, while the P-value obtained in the DC group was >0.5, demonstrating that the purity of mitochondria obtained by ultracentrifugation (UC) is higher than that obtained by differential centrifugation (DC).

[0062] Western blot analysis of proteins in the DC and UC groups confirmed that the purity of mitochondria obtained by ultracentrifugation (UC) was higher than that obtained by differential centrifugation (DC). Figure 3 ).

[0063] Example 2

[0064] 1. Mitochondrial sample collection

[0065] (1) Column separation method (CO): The cell culture, digestion and collection process is the same as described in (1). Wash the collected cells twice with KPBS, add 250 μL of Buffer A (Invent, USA) and vortex to resuspend the cells. After vortexing, incubate on ice for 5 min. After incubation, vortex vigorously for 30 s, transfer the suspension to a centrifuge column, and centrifuge at 16000 g for 30 s. Vortex to resuspend the pellet, and centrifuge at 700 g for 1 min. Take the supernatant and transfer it to a new 2 mL centrifuge tube. Add 300 μL of Buffer B (Invent, USA) and centrifuge at 16000 g for 10 min. Discard the supernatant, add 200 μL of Buffer B (Invent, USA) to resuspend the pellet, and centrifuge at 8000 g for 5 min. Collect the supernatant and transfer it to a new 2 mL centrifuge tube. Add 1600 μL of pre-chilled KPBS and centrifuge at 16000 g for 30 min to obtain a mitochondrial-rich precipitate.

[0066] (2) Magnetic Bead Method (MACS): The cell culture, digestion, and collection process is the same as described in (1). Wash the Pierce™ Anti-HA magnetic beads (Thermo Fisher Scientific, USA) three times with pre-chilled KPBS and place them on ice for later use. Wash the collected cells twice with pre-chilled KPBS and resuspend the cell pellet in 1 mL of KBPS. Transfer the pellet to a Dounce homogenizer and grind it 45 times with a hammer. Then transfer the homogenate to a new 2 mL centrifuge tube and centrifuge at 2000 g for 5 min at 4 ℃ to remove cell nuclei, cell debris, and broken cells. Collect the supernatant into a centrifuge tube containing a pre-washed Pierce™ Anti-HA magnetic bead. Gently pipette to mix and incubate on a shaker at 4 ℃ for 3.5 min. Spot centrifuge to allow the solution clinging to the centrifuge tube wall to enter the centrifuge tube. Place the centrifuge tube on a magnetic rack and discard the liquid after the solution has clarified. Gently wash the magnetic beads four times with pre-cooled KPBS to obtain Pierce™ Anti-HAmagnetic beads containing mitochondria.

[0067] 2. Sample Pretreatment

[0068] Sample pretreatment was performed on ice. 400 μL of methanol extract containing internal standard was added to each mitochondrial sample extracted by column chromatography (CO) and magnetic bead chromatography (MACS), and the mixture was vortexed for 30 s. 1 mL of MTBE was added, vortexed for 30 s, and then centrifuged at 1000 rpm and 10 °C for 30 min. Subsequently, 400 μL of ultrapure water was added, vortexed for 1 min, and then centrifuged at 12000 g and 6 °C for 10 min. After phase separation, the upper hydrophobic phase was quantitatively collected and vacuum-dried in a refrigerated centrifuge. The resulting lyophilized samples were stored at -80 °C.

[0069] 3. Data acquisition: Same as in Example 1.

[0070] 4. Data preprocessing: Same as in Example 1.

[0071] 5. Results Analysis

[0072] Based on the comparison between the CO group and the MACS group, Example 2 was used to verify the feasibility of using the two lipid biomarkers in combination, in order to further confirm their discriminative effect.

[0073] All samples from the CO group and the MACS group were included in the discriminant comparison, and the judgment variable P was obtained based on this marker. Figure 2 The area under the ROC curve (AUC) used for discrimination was 1. At a cut-off value of 0.5, both sensitivity and specificity were 100% (Table 1). Figure 2 d) The above results indicate that this marker has good discriminative potential and can be used to determine the purity of the extracted mitochondrial samples. Specifically, the P-value for the MACS group was < 0.5, while the P-value for the CO group was > 0.5, demonstrating that the purity of the mitochondria extracted from the MACS group was higher than that extracted from the CO group. Furthermore, column separation (CO) is comparable to the classic differential centrifugation (DC), and the magnetic bead method (MACS) is comparable to the classic ultracentrifugation (UC).

[0074] Western blot analysis of the CO and MACS groups further confirmed that the purity of mitochondria extracted by the magnetic bead method (MACS) was higher than that extracted by the column separation method (CO). Furthermore, the results of the column separation method (CO) were comparable to those of the classic differential centrifugation method (DC), while the results of the magnetic bead method (MACS) were comparable to those of the classic ultracentrifugation method (UC). Figure 3 ).

[0075] Table 1: Results of ROC discriminant analysis of biomarkers

[0076] .

Claims

1. A combined metabolic biomarker for evaluating mitochondrial purity, characterized in that... The combined metabolic markers include phosphatidylcholine PC 34:0 (16:0 / 18:0), sphingomyelin SM 34:1;2O (18:1;2O / 16:0), sphingomyelin SM 44:2;2O (18:1;2O / 26:1), and hexosylceramide HexCer 42:2;2O (18:1;2O / 24:1).

2. The application of the combined metabolic markers of claim 1 in evaluating the purity of mitochondria extracted by the mitochondrial extraction method, wherein the extraction method includes differential centrifugation, column separation, ultracentrifugation, and magnetic bead method.

3. The method for determining the purity of mitochondria extracted using the combined metabolic markers described in claim 1, characterized in that, This method uses a joint biomarker variable P for judgment: P=1 / (1+e^(-42.608a+5.583b-7.591c+7.343d+11.075)) Where a represents the relative content of phosphatidylcholine PC 34:0 (16:0 / 18:0), b represents the relative content of sphingomyelin SM 34:1;2O (18:1;2O / 16:0), c represents the relative content of sphingomyelin SM 44:2;2O (18:1;2O / 26:1), and d represents the relative content of hexosylceramide HexCer 42:2;2O (18:1;2O / 24:1). If P < 0.5, the purity of mitochondria extracted by this method is higher than that of the classic differential centrifugation method and comparable to that of the classic ultracentrifugation method; if P > 0.5, the purity of mitochondria extracted by this method is lower than that of the classic ultracentrifugation method and comparable to that of the classic differential centrifugation method.

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