Method for simultaneously measuring acyl CoA and acyl carnitine

Abstract

The invention relates to the technical field of biomedicine, and provides a method for simultaneously measuring acyl CoA and acyl carnitine. The method comprises the following steps: adding monopotassium phosphate, magnesium chloride and glacial acetic acid into isopropanol to form an extraction buffer solution; wherein the extraction buffer solution is used for performing a first round of extraction on a kidney sample; and mixing chloroform and methanol according to a preset volume to obtain a first solvent, pouring the first solvent into the object subjected to the first round of extraction, and carrying out two-phase extraction to obtain the extract for mass spectrometry. The invention provides a scheme for analyzing acyl CoA and acyl carnitine in a kidney sample, which does not need a special instrument and can be operated on a traditional LC-MS / MS system, so that the detection cost can be effectively reduced, and the detection efficiency is guaranteed.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly to a method for simultaneously measuring acyl-CoA and acylcarnitine. Background Art

[0002] Diabetes mellitus (abbreviated as: DM) is a metabolic disease characterized by hyperglycemia. Among its many complications, diabetic kidney disease (DKD), which is caused by long-term chronic microvascular lesions such as glomerular vascular damage and sclerosis, is one of the most serious complications and a major cause of chronic kidney disease and renal failure.

[0003] Currently, urinary microalbumin is the most commonly used indicator for diagnosing and evaluating the progression of DKD. However, before its level becomes abnormal, the kidney pathology of some patients has already occurred, and the limited sensitivity and specificity can no longer meet the early prediction of DKD.

[0004] Driven by the rapid development of high-throughput technologies, non-targeted metabolomics has provided unprecedented insights into the dynamic circulating biomarkers of DKD. Metabolomics is a technology that performs high-throughput quantitative detection and analysis of all endogenous small molecule metabolites (molecular weight < 1000 Da) in an organism. Metabolomics amplifies the subtle changes in gene and protein expression and is more likely to reflect the effects of internal and external factor changes on systemic metabolism, helping to reveal the relationship between small molecules and physiological and pathological effects. As participants in metabolic pathways, differential lipid metabolites may highlight the pathways involved in the development of DKD, which has been confirmed by many scholars.

[0005] Before fatty acids enter the cell metabolic pathway, their carboxyl head groups need to be converted from a relatively inactive state to a more active acyl-CoA, and this conversion process is called fatty acid activation. Since the mitochondrial membrane is not directly permeable to acyl-CoA, the carnitine shuttle system is required to transport fatty acyl groups into the mitochondria in the form of acylcarnitine. The intracellular level of acylcarnitine maintains a complex dynamic balance with acyl-CoA. Due to the high technical difficulty of measuring acyl-CoA and its regionalized distribution in cells, the acylcarnitine level in blood is often used as a surrogate indicator to reflect the changes in intracellular acyl-CoA. Although an increase in specific acylcarnitine usually indicates abnormal fatty acid metabolism, this association is not entirely precise. Therefore, developing a method that can accurately measure acyl-CoA has become an important task at present.

[0006] In recent years, liquid chromatography-tandem mass spectrometry (LC-MS / MS) has gradually replaced the traditional ultraviolet detection method and become the mainstream technology for acyl-CoA quantitative analysis due to its high sensitivity. However, there are still significant technical challenges in achieving rapid detection of multiple acyl-CoAs in a single method. Acyl-CoAs with different chain lengths have large differences in polarity, which limits their separation within a single chromatographic gradient. Short-chain acyl-CoAs have weak hydrophobicity on a reversed-phase (RP) column and usually require an acidic mobile phase to enhance retention for effective separation. However, acidic conditions have a negative impact on the elution of medium- and long-chain acyl-CoAs, while basic conditions are more suitable for the deprotonation of these compounds, thus reducing peak tailing.

[0007] Some researchers have used ion-pair RP chromatography to achieve the separation of C2-C18 acyl-CoAs within 35 minutes. However, this method operates near the pressure limit of most ultra-performance liquid chromatography (UPLC) systems and requires adjusting the flow rate to control the pressure below 14,500 psi, which limits its application in high-throughput analysis. In addition, some researchers have developed an online two-dimensional LC-MS (2D LC-MS) method that comprehensively separates acyl-CoAs within 30 minutes by integrating two chromatographic gradient sequences. However, 2D LC-MS requires special instruments, which limits the convenience of its wide application. Therefore, achieving efficient separation of acyl-CoAs within a single chromatographic gradient remains a technical problem. Summary of the Invention

[0008] The technical problem to be solved by the present invention is to provide a method for efficiently separating acyl-CoA and acylcarnitine within a single chromatographic gradient.

[0009] The further technical problem to be solved by the present invention is to provide a method for potential biomarkers related to the impaired fatty acid oxidation pathway in early diabetic nephropathy.

[0010] The present invention adopts the following technical solutions: In a first aspect, the present invention provides a method for simultaneously measuring acyl-CoA and acylcarnitine, comprising: constituting an extraction buffer by adding potassium dihydrogen phosphate, magnesium chloride, and glacial acetic acid to isopropanol; wherein, the extraction buffer is used for the first-round extraction of kidney samples. After mixing chloroform and methanol in a preset volume to obtain a first solvent, pour the first solvent into the object after the first round of extraction for biphasic extraction to obtain an extract that can be used for mass spectrometry analysis.

[0011] Preferably, it specifically includes: Prepared according to the ratio that the amounts of potassium dihydrogen phosphate, magnesium chloride, and glacial acetic acid added to 300 μL of extraction buffer are 15 μmol, 10 μmol, and 175 μmol respectively.

[0012] Preferably, the preset volume is specifically a mixture of chloroform and methanol in a volume ratio of 1:1.5 to 1:3.

[0013] Preferably, before pouring the object after the first round of extraction into the first solvent for biphasic extraction, the method further includes: Clean the free fatty acids and / or polar lipids contained in the object after the first round of extraction with petroleum ether.

[0014] Preferably, a variety of metabolites are obtained according to the results of ultra-high performance liquid chromatography separation to obtain a compound list; extract the characteristic peaks of non-target lipid molecules for comparison and screening; among them, quality control samples and blank samples are used to screen background data; Select the differential metabolites of acylcarnitines and acyl-CoAs in the db / db group and db / m group under the condition of P < 0.05 to obtain potential biomarkers related to the impaired fatty acid oxidation pathway in early diabetic nephropathy.

[0015] In a second aspect, the present invention also provides a method for simultaneously measuring acyl-CoA and acylcarnitine. Place the collected kidney samples in a 1.5 - 2.5 mL grinding tube, and add 200 - 500 μL of extraction buffer; among them, the extraction buffer is isopropanol added with 2 - 20 μmoL potassium dihydrogen phosphate, 5 - 20 μmol magnesium chloride, 150 - 200 μmoL glacial acetic acid, and 1 - 5 mg bovine serum albumin; Add 2 - 4 3mm zirconia beads, homogenize in a grinder and then sonicate; Add 200 - 500 μL of petroleum ether to the extract, remove the upper layer after centrifugation, and wash with petroleum ether 1 - 3 times to remove free fatty acids and polar lipids; Add 10 - 30 μL of saturated ammonium sulfate solution, slowly add 400 - 800 μL of chloroform:methanol (1:1 - 1:3, v / v); the sample is then incubated in a thermostatic mixer at 20 - 30 °C and 400 - 500 rpm for 15 - 30 minutes; After incubation, centrifuge the sample and transfer the clear supernatant to a new tube, then dry it by vacuum rotary evaporation at room temperature. The dried extract is resuspended in methanol:water (8:1 - 9:1, v / v), and 0.01 - 0.1% glacial acetic acid is added before being used for mass spectrometry analysis.

[0016] Preferably, equal volumes of the sample extracts from the db / db group and the db / m group are uniformly mixed to obtain a quality control sample, and methanol:water (9:1, v / v) is mixed as a blank sample.

[0017] Preferably, the mass spectrometry analysis specifically includes: Mobile phase A is water containing 2 mmol / L ammonium acetate and 0.005% triethylamine, and mobile phase B is acetonitrile:water (4:1, v / v) containing 0.1% triethylamine; Gradient elution, and the elution program is as follows: 0 minute: 94% A, 0.2 mL / min; 1.5 minutes: 90% A, 0.2 mL / min; 2.5 minutes: 75% A, 0.2 mL / min; 6 minutes: 55% A, 0.2 mL / min; 7 minutes: 45% A, 0.12 mL / min; 11 minutes: 30% A, 0.12 mL / min; 13 minutes: 30% A, 0.2 mL / min; 14 minutes: 5% A, 0.2 mL / min; 17 minutes: 5% A, 0.2 mL / min; 17.3 minutes: 94% A, 0.2 mL / min; 20 minutes: 94% A, 0.2 mL / min; During the whole running process, the column temperature is maintained at 40°C; the injection volume for each time is 1.0 μL; positive ion mode, the ion source voltage is 3.7 kV, the ion source heating temperature is 320°C; the solvent heating evaporation temperature is 300°C; the sheath gas and auxiliary gas are both nitrogen, the sheath gas pressure is 30 psi, and the auxiliary gas pressure is 10 psi; the collision gas is nitrogen, and the pressure is 29 psi.

[0018] Preferably, a variety of metabolites are obtained according to the results of UPLC separation; the original data of the metabolites are processed to identify the metabolites and obtain a compound list; the characteristic peaks of non-targeted lipid molecules are extracted for comparison and screening; among them, the quality control sample and the blank sample are used to screen the background data.

[0019] Under the condition of P < 0.05, the differential metabolites of acylcarnitines and acyl-CoAs between the db / db group and the db / m group were selected to obtain potential biomarkers related to the impaired fatty acid oxidation pathway in early diabetic nephropathy.

[0020] Preferably, accumulations occurred in acylcarnitines including 8:0-carnitine, 10:0-carnitine, 12:0-carnitine, 14-carnitine, 15-carnitine, 16:0-carnitine, 18:0-carnitine and 18:1-carnitine, and the contents of acyl-CoAs including 4:0-CoA, 6:0-CoA, 7:0-CoA, 8:0-CoA, 10:0-CoA and 14:0-CoA decreased.

[0021] Compared with the existing methods, there is currently no accurate and rapid method for predicting diabetic nephropathy through lipid metabolomics. The set of solutions provided by the present invention, which do not require special instruments and can be run on a traditional LC-MS / MS system for analyzing acyl-CoA and acylcarnitine in kidney samples, can effectively reduce the detection cost and ensure the detection efficiency.

[0022] Furthermore, in the implementation solution of the present invention, the dual functions of TEA (triethylamine) are also utilized, which serves both as an ion-pairing reagent to optimize the peak shape and as a basic reagent to adjust the pH value. Combined with a shorter analysis time and instrument compatibility (applicable to traditional UPLC systems), this method provides the possibility for high-throughput screening and can be used to reveal changes in lipid metabolism in the biological and medical fields. Therefore, the potential biological metabolic markers for the early occurrence of diabetic nephropathy screened based on metabolomics have important theoretical and practical significance. Description of the Drawings

[0023] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required to be used in the embodiments of the present invention will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention, and those of ordinary skill in the art can also obtain other drawings based on these drawings without creative efforts.

[0024] Figure 1 It is the urinary protein graph of db / db mice and db / m mice 24 weeks after modeling in the embodiments of the present invention; Figure 2 It is the total ion current TIC graph of the db / m kidney tissue sample in the embodiments of the present invention; Figure 3 It is the total ion current TIC graph of the db / db kidney tissue sample in the embodiments of the present invention; Figure 4This is the heatmap of potential biomarkers in the embodiments of the present invention. Detailed implementation manners

[0025] In order to make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0026] In the prior art, the pretreatment methods for separately measuring acyl-CoA or acylcarnitine have obvious limitations: for example, the extraction of acyl-CoA usually needs to be carried out under acidic conditions to enhance the stability of short-chain acyl-CoA, but the acidic conditions will cause the degradation of medium- and long-chain acylcarnitine; while the extraction of acylcarnitine commonly uses alkaline conditions to improve the recovery rate, but the alkaline conditions will accelerate the hydrolysis of acyl-CoA. In view of these problems, the present invention proposes an optimized pretreatment method, by adding 15 μmol of potassium dihydrogen phosphate, 10 μmol of magnesium chloride and 10 μL of glacial acetic acid in isopropanol, which not only ensures the stability of acyl-CoA but also avoids the degradation of acylcarnitine. At the same time, free fatty acids and polar lipids are removed by washing with petroleum ether to reduce background interference, and biphasic extraction with chloroform:methanol (1:2, v / v) is used to ensure the simultaneous and efficient extraction of acyl-CoA and acylcarnitine. The basis for these improved designs is that the addition of potassium dihydrogen phosphate and magnesium chloride stabilizes the structure of acyl-CoA by adjusting the ionic strength and chelating metal ions, the weak acidic condition of glacial acetic acid significantly reduces the hydrolysis rate of acyl-CoA without affecting the stability of acylcarnitine, and the optimized biphasic extraction ratio ensures that the recovery rates of both metabolites reach more than 90%. Through the above improvements, the present invention has successfully realized the simultaneous measurement of acyl-CoA and acylcarnitine, providing reliable technical support for the early diagnosis of diabetic nephropathy.

[0027] Currently, it is considered that the accumulation of acylcarnitine in the plasma of patients may be a biomarker for the early diagnosis of diabetic nephropathy. In clinical patients, medium- and long-chain acylcarnitine shows a relatively high abundance, while the abundance of short-chain acylcarnitine is relatively low. However, it is not clear whether the accumulation of acylcarnitine is caused by compensatory elevation or impairment of β-oxidation. Detecting fatty acid oxidation as early as possible is important for the early assessment and prevention of diseases. It is generally considered that the accumulation of acylcarnitine is a sign of impaired fatty acid oxidation, but this is not perfect. The lipids involved in fatty acid oxidation defects mainly also include acyl-CoA. Acyl-CoA is directly involved in the steps of fatty acid oxidation and is the main biomarker related to fatty acid oxidation. However, acyl-CoA is not used as a diagnostic biomarker because their endogenous levels are low. In addition, due to their physicochemical properties and instability, the development of analytical methods for acyl-CoA is extremely challenging.

[0028] The present invention provides a method using untargeted lipidomics to perform special pretreatment on a sample so that acylcarnitine and acyl-CoA can be measured simultaneously. It is found that in the renal tissues of diabetic nephropathy mice, acylcarnitines including 8:0-carnitine, 10:0-carnitine, 12:0-carnitine, 14-carnitine, 15-carnitine, 16:0-carnitine, 18:0-carnitine, 18:1-carnitine accumulate, and the contents of acyl-CoAs including 4:0-CoA, 6:0-CoA, 7:0-CoA, 8:0-CoA, 10:0-CoA, 14:0-CoA decrease. This will more simply illustrate, in a metabolomics manner, that fatty acid oxidation in the kidneys is impaired in diabetic nephropathy, providing new possibilities for candidate biomarkers of diabetic nephropathy.

[0029] In various embodiments of the present invention, db / db is a widely used mouse model of type 2 diabetes and obesity, caused by a gene mutation (leptin receptor deficiency, Lepr^db). db / db mice: homozygous mutation (Lepr^db / Lepr^db), showing hyperglycemia, obesity, and insulin resistance. db / m mice: heterozygotes (Lepr^db / +), serving as a healthy control group (without significant metabolic abnormalities).

[0030] In addition, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0031] Example 1: Example 1 of the present invention provides a method for simultaneously measuring acyl-CoA and acylcarnitine, including: An extraction buffer is formed by adding potassium dihydrogen phosphate, magnesium chloride, and glacial acetic acid to isopropanol; wherein, the extraction buffer is used for the first-round extraction of kidney samples. In an optional implementation, the extraction buffer is prepared according to the ratio that the amounts of potassium dihydrogen phosphate, magnesium chloride, and glacial acetic acid added to 300 μL of the extraction buffer are 15 μmol, 10 μmol, and 175 μmol respectively.

[0032] After mixing chloroform and methanol in a preset volume to obtain a first solvent, the object after the first-round extraction is poured into the first solvent for biphasic extraction to obtain an extract for mass spectrometry analysis. Wherein, the preset volume is specifically that chloroform and methanol are mixed according to a volume ratio of 1:1.5 to 1:3.

[0033] Currently, there is no accurate and rapid method for predicting diabetic nephropathy through lipid metabolomics. The solution provided by the embodiments of the present invention, which requires no special instruments and can be run on a traditional LC-MS / MS system for analyzing acyl-CoA and acylcarnitine in kidney samples, can effectively reduce the detection cost and ensure the detection efficiency.

[0034] Combined with the embodiments of the present invention, there is a preferred implementation. Before pouring the object after the first round of extraction into the first solvent for biphasic extraction, free fatty acids and / or polar lipids contained in the object after the first round of extraction are washed with petroleum ether.

[0035] Combined with the embodiments of the present invention, there is a preferred implementation. After the extract for mass spectrometry analysis, it further includes: after obtaining various metabolites according to the results of ultra-high performance liquid chromatography separation (i.e., an example means of mass spectrometry analysis), obtaining a compound list; extracting the characteristic peaks of non-target lipid molecules for comparison and screening; among them, quality control samples and blank samples are used to screen background data; acylcarnitine and acyl-CoA differential metabolites between the db / db group and the db / m group are selected under the condition of P < 0.05 to obtain potential biomarkers related to the impaired fatty acid oxidation pathway in early diabetic nephropathy.

[0036] Example 2: The embodiments of the present invention are based on the technical solutions proposed in Example 1, and are a process of demonstrating the effectiveness of the corresponding solutions in cooperation with specific mouse experimental objects. In the embodiments of the present invention, a set of parameter expressions with finer subdivisions in the scenario of the embodiments of the present invention will also be made for the parameter intervals involved in the above Example 1. However, it should not be understood as the only implementation of the interval protection scope listed in Example 1.

[0037] db / db mice are one of the best animal models currently used to study human type II diabetes. db / db mice are congenital obese type II diabetic mice caused by leptin receptor gene defects. Their leptin receptor genes lose their functions, and hyperinsulinemia occurs within 2 weeks after birth, obesity develops at 3 - 4 weeks, very severe hyperglycemia develops after 8 weeks, accompanied by insulin resistance and β-cell function failure during this period, and early diabetic nephropathy develops after 24 weeks, and they generally die within 8 - 10 months. In this example, db / db black mice, a spontaneous type II diabetes model, are used as the research object to compare the lipid metabolites in kidney tissues and screen out a class of possible biomarkers for diabetes, providing a reference basis for the research and prediction of diabetic nephropathy. Next, it will be elaborated through experimental objects, main instruments and software, main reagents, and experimental procedures respectively.

[0038] Experimental objects: The db / db mouse is a congenital genetically defective type II diabetes model. As blood glucose levels increase, a large amount of proteinuria gradually appears, eventually developing into diabetic nephropathy. After 2 weeks of adaptive feeding, all mice were weighed and blood samples were collected from the tail vein by acupuncture to measure fasting blood glucose levels (fasting for 8 - 12 h). Mice with an average blood glucose level ≥ 11.0 mmol / L were considered eligible for blood glucose. Ten eligible db / db mice were selected; ten healthy db / m mice were used as the normal control group. The animal room had 12 h of light and 12 h of darkness, with a relative humidity of 50 - 70% and a room temperature of 18°C - 22°C.

[0039] After 12 weeks of feeding in each drug administration group, the mice were fasted and watered for 12 h, urine was collected, and urine sediment was removed by centrifugation at 800 g / min for 10 min. The urine protein level was measured using a urine protein quantification test kit (CBB method) developed by Nanjing Jiancheng Bioengineering Research Institute Co., Ltd. The results are as Figure 1 shown. The kidney tissues were dissected, rinsed thoroughly with normal saline and PBS buffer, and blotted dry with filter paper for later use.

[0040] Main instruments and software: Table 1 Main instruments

[0041] Main reagents: Table 2 Main reagents

[0042] Experimental procedure: Metabolite extraction The collected kidney samples were placed in 2 mL grinding tubes, and 300 μL of extraction buffer was added (the extraction buffer was isopropanol added with 15 μmol potassium dihydrogen phosphate, 10 μmol magnesium chloride, 10 μL glacial acetic acid, and 2 mg bovine serum albumin). Then, 3 zirconia beads with a diameter of 3 mm were added, and the samples were homogenized in a grinder and then sonicated. 300 μL of petroleum ether was added to the extract, and the upper layer was removed by centrifugation and washed twice with petroleum ether to remove free fatty acids and less polar lipids. Then, 20 μL of saturated ammonium sulfate solution was added, and 600 μL of chloroform:methanol (1:2, v / v) was slowly added. The samples were then incubated in a constant temperature mixer at 25°C and 450 rpm for 20 minutes. After incubation, the samples were centrifuged and the clean supernatant was transferred to a new tube and dried by vacuum rotary evaporation at room temperature. The dried extract was resuspended in methanol:water (9:1, v / v), and 0.05% glacial acetic acid was added before being used for mass spectrometry analysis.

[0043] Equal volumes of the sample extracts from the db / db group and the db / m group were uniformly mixed to obtain a Quantifying Control (QC) sample, and methanol:water (9:1, v / v) was mixed as a Blank sample.

[0044] The samples to be tested were detected by ultra performance liquid chromatography - mass spectrometry (UPLC - MS) to obtain the original metabolite data. Among them, the conditions of ultra performance liquid chromatography included: The chromatographic column was: Mobile phase A was water containing 2 mmol / L ammonium acetate and 0.005% triethylamine, and mobile phase B was acetonitrile:water (4:1, v / v) containing 0.1% triethylamine.

[0045] Gradient elution was performed, and the elution program was as follows: 0 min: 94% A, 0.2 mL / min; 1.5 min: 90% A, 0.2 mL / min; 2.5 min: 75% A, 0.2 mL / min; 6 min: 55% A, 0.2 mL / min; 7 min: 45% A, 0.12 mL / min; 11 min: 30% A, 0.12 mL / min; 13 min: 30% A, 0.2 mL / min; 14 min: 5% A, 0.2 mL / min; 17 min: 5% A, 0.2 mL / min; 17.3 min: 94% A, 0.2 mL / min; 20 min: 94% A, 0.2 mL / min.

[0046] During the whole running process, the column temperature was maintained at 40 °C. The injection volume for each time was 1.0 μL. In positive ion mode, the ion source voltage was 3.7 kV, the ion source heating temperature was 320 °C; the solvent heating evaporation temperature was 300 °C; the sheath gas and auxiliary gas were both nitrogen, the sheath gas pressure was 30 psi, and the auxiliary gas pressure was 10 psi; the collision gas was nitrogen, and the pressure was 29 psi. The total ion current chromatogram of the renal tissue samples of the db / db group in positive ion mode is shown in Figure 2 ; the total ion current chromatogram of the renal tissue samples of the db / m group is shown in Figure 3 , and it can be seen from the figure that in this example, multiple endogenous components in the renal tissue can be detected, indicating that the separation effect of UPLC is good and multiple metabolites are obtained.

[0047] Data processing was performed on the original metabolite data to identify metabolites, resulting in a list of compounds; Progenesis QI software (Waters, Massachusetts, USA) was used for result analysis, extracting characteristic peaks of non-target lipid molecules for comparison and screening. At the same time, QC and Blank samples were used to screen background data. The final data was imported into EZinfo 3.0. Differentially expressed acylcarnitines and acyl-CoAs between the db / db group and the db / m group were selected under the condition of P < 0.05 to obtain potential biomarkers related to the impaired fatty acid oxidation pathway in early diabetic nephropathy.

[0048] A total of 14 potential biomarkers related to the impaired fatty acid oxidation pathway in diabetic nephropathy were identified in this example, and the identification results are shown in Table 3. Figure 4 It is a heat map of differentially expressed metabolites in kidney tissue samples. As can be seen from the figure, in kidney samples, there are obvious differences in the levels of differentially expressed metabolites between the db / db group and the db / m group. There is a strong positive correlation for acylcarnitine metabolites and a strong negative correlation for acyl-CoA metabolites in the db / db group. The results indicate that these potential biomarkers interact and are interrelated with each other to jointly regulate the metabolic pathway.

[0049] Table 3 Potential biomarkers related to diabetic nephropathy in the example

[0050] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, and improvements made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.

Claims

1. A method for simultaneously measuring acyl-CoA and acylcarnitine, characterized in that, Comprising: An extraction buffer is constituted by adding potassium dihydrogen phosphate, magnesium chloride and glacial acetic acid to isopropanol; wherein, the extraction buffer is used for the first-round extraction of kidney samples. After mixing chloroform and methanol in a preset volume to obtain a first solvent, the first solvent is poured into the object after the first-round extraction for biphasic extraction to obtain an extract for mass spectrometry analysis.

2. The method for simultaneously measuring acyl-CoA and acylcarnitine according to claim 1, wherein Specifically including: Prepared according to the ratio that the amounts of potassium dihydrogen phosphate, magnesium chloride and glacial acetic acid added to 300 μL of the extraction buffer are 15 μmol, 10 μmol and 175 μmol respectively.

3. The method for simultaneously measuring acyl-CoA and acylcarnitine according to claim 1, wherein The preset volume is specifically that chloroform and methanol are mixed according to a volume ratio of 1:1.5 to 1:

3.

4. The method for simultaneously measuring acyl-CoA and acylcarnitine according to claim 1, wherein Before pouring the first solvent into the object after the first-round extraction for biphasic extraction, the method further includes: Cleaning the free fatty acids and / or polar lipids contained in the object after the first-round extraction with petroleum ether.

5. The method for simultaneously measuring acyl-CoA and acylcarnitine according to claim 1, wherein, A variety of metabolites are obtained according to the results of ultra-high performance liquid chromatography separation to obtain a compound list; the characteristic peaks of non-target lipid molecules are extracted for comparison and screening; among them, quality control samples and blank samples are used to screen background data. Differential metabolites of acylcarnitines and acyl-CoAs in the db / db group and db / m group are selected under the condition of P < 0.05 to obtain potential biomarkers related to the impaired fatty acid oxidation pathway in early diabetic nephropathy.

6. A method for simultaneously measuring acyl-CoA and acylcarnitine, characterized in that, The collected kidney samples are placed in a 1.5 - 2.5 mL grinding tube, and 200 - 500 μL of the extraction buffer is added; wherein, the extraction buffer is isopropanol added with 2 - 20 μmoL of potassium dihydrogen phosphate, 5 - 20 μmol of magnesium chloride, 150 - 200 μmoL of glacial acetic acid and 1 - 5 mg of bovine serum albumin. Add 2 - 4 3-mm zirconia beads, homogenize in a grinder and then ultrasonicate. Add 200 - 500 μL of petroleum ether to the extract, centrifuge and remove the upper layer, and wash with petroleum ether 1 - 3 times to remove free fatty acids and polar lipids. Add 10 - 30 μL of saturated ammonium sulfate solution, slowly add 400 - 800 μL of chloroform:methanol (1:1 - 1:3, v / v); the sample is then incubated in a thermostatic mixer at 20 - 30 °C and 400 - 500 rpm for 15 - 30 minutes. After incubation, centrifuge the sample and transfer the clean supernatant to a new tube, and dry it by vacuum rotation at room temperature; the dried extract is resuspended with methanol:water (8:1 - 9:1, v / v), and 0.01 - 0.1% glacial acetic acid is added and then used for mass spectrometry analysis.

7. The method for simultaneously measuring acyl-CoA and acylcarnitine according to claim 6, wherein Equal volumes of the sample extracts of the db / db group and db / m group are uniformly mixed to obtain a quality control sample; methanol:water (9:1, v / v) is mixed as a blank sample.

8. The method for simultaneously measuring acyl-CoA and acylcarnitine according to claim 6, wherein, The mass spectrometry analysis specifically includes: Mobile phase A is water containing 2 mmol / L ammonium acetate and 0.005% triethylamine, and mobile phase B is acetonitrile:water (4:1, v / v) containing 0.1% triethylamine. Gradient elution, and the elution program is as follows: 0 minute: 6% B, 0.2 mL / min; 1.5 minutes: 10% B, 0.2 mL / min; 2.5 min: 25%B, 0.2 mL / min; 6 min: 45%B, 0.2 mL / min; 7 min: 55%B, 0.12 mL / min; 11 min: 70%B, 0.12 mL / min; 13 min: 70%B, 0.2 mL / min; 14 min: 95%B, 0.2 mL / min; 17 min: 95%B, 0.2 mL / min; 17.3 min: 6%B, 0.2 mL / min; 20 min: 6%B, 0.2 mL / min; During the whole running process, the column temperature was maintained at 40 °C; the injection volume for each time was 1.0 μL; positive ion mode, the ion source voltage was 3.7 kV, the ion source heating temperature was 320 °C; the solvent heating and evaporation temperature was 300 °C; the sheath gas and auxiliary gas were both nitrogen, the sheath gas pressure was 30 psi, and the auxiliary gas pressure was 10 psi; the collision gas was nitrogen, and the pressure was 29 psi.

9. The method for simultaneously measuring acyl-CoA and acyl carnitine according to claim 7, wherein A variety of metabolites were obtained according to the results of UPLC separation; the original data of metabolites were processed to identify metabolites, and a compound list was obtained; the characteristic peaks of non-target lipid molecules were extracted for comparison and screening; among them, quality control samples and blank samples were used to screen background data. Differential metabolites of acylcarnitines and acyl-CoAs in the db / db group and db / m group were selected under the condition of P < 0.05, and potential biomarkers related to the impaired fatty acid oxidation pathway in early diabetic nephropathy were obtained.

10. The method for simultaneously measuring acyl-CoA and acylcarnitine according to claim 9, characterized in that, The specific potential biomarkers are as follows: The acylcarnitines include 8:0-carnitine, 10:0-carnitine, 12:0-carnitine, 14-carnitine, 15-carnitine, 16:0-carnitine, 18:0-carnitine and 18:1-carnitine, and the acyl-CoAs include 4:0-CoA, 6:0-CoA, 7:0-CoA, 8:0-CoA, 10:0-CoA and 14:0-CoA.

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