CN112461952B - Kit for evaluating toxicity of liver micro-plastics and application and detection thereof - Google Patents
Kit for evaluating toxicity of liver micro-plastics and application and detection thereof Download PDFInfo
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Abstract
The invention relates to a novel application of pentadecanoic acid and heptadecanoic acid in preparation of a kit and a detection method for evaluating toxicity of liver micro-plastics. The reagent and the method can be used for detecting the content of pentadecanoic acid and heptadecanoic acid in the liver tissue of a tested organism. The invention further provides a method for detecting the content of pentadecanoic acid and heptadecanoic acid in the liver tissue of a tested organism by using a gas chromatography-mass spectrometry method, which can be used for evaluating the toxicity of liver micro-plastics. The invention has the advantages of small quantity of selected markers, large change multiple, contribution to reducing the interference of other factors, simple operation, mild reaction condition, high stability, high repeatability, high specificity and high sensitivity of the adopted kit and detection method, and easy popularization.
Description
Technical Field
The invention relates to a reagent kit for evaluating toxicity of liver micro-plastics, application of the reagent kit and new application of detection of pentadecanoic acid and heptadecanoic acid, and belongs to the fields of analytical chemistry, biochemistry and environmental chemistry.
Background
The micro plastic is plastic particles with the diameter of 1 mu m to 5mm, is widely present in various water bodies, edible salt and aquatic organisms, and can even enter a human body through food chains, drinking water and other ways. Due to the small particle size, microplastics can be ingested by aquatic organisms, inducing various physical, chemical and biological damages such as: digestive tract obstruction, gastric and intestinal ulcers, suppressed survival, growth and development, endocrine disorders, oxidative stress, abnormal liver metabolism, abnormal immune response, and even genotoxicity. Therefore, the research on the health effects of the micro-plastics on aquatic organisms, ecosystems and people is of great significance.
Metabolism is an important physiological activity of the organism, and its abnormality is closely related to the pathology of many diseases and the toxicology of pollutants. Numerous studies have shown that polystyrene micro-Plastic (PS) exposure can disrupt energy-related metabolic pathways, thereby affecting the physiological function of the exposed organism and PS toxicity. For example, PS exposure significantly inhibited succinate dehydrogenase and lactate dehydrogenase (key enzymes of the tricarboxylic acid cycle and glycolysis, respectively) activity in mouse testicular tissue, and decreased mature sperm number and mass, increased sperm teratogenesis. In addition, PS exposure significantly increased lactate dehydrogenase and isocitrate dehydrogenase activity in grouper muscle tissue, while reducing the lactate content in zebrafish intestinal tissue. Liver transcriptomics results showed that glycolysis and fatty acid oxidation related gene expression (e.g.: glut2, GK, HK, PEPCKC, cpt1a, acox and Mcad) was significantly down-regulated under PS exposure, indicating that hepatic glycolysis and fatty acid oxidation were inhibited and that the above fatty acid oxidation related changes could be inherited to the next generation.
The liver is used as an important metabolic organ of an organism, regulates absorption, digestion, synthesis and storage of various biochemical components and detoxification of environmental pollutants, and plays an important role in maintaining energy balance of the organism. Oxidative degradation of liver fatty acids can provide energy to the organism. In addition, fatty acids can be used to synthesize other macromolecular lipids (e.g., phospholipids, sphingolipids, triglycerides, cholesterol esters) and active metabolites (e.g., prostaglandins, leukotrienes, lipoxins) that affect biopathophysiology and pollutant toxicity. Thus, hepatic fatty acid metabolism plays an important role in the physiological function of the organism and the toxic effect of the micro-plastics. However, no study of the effect of microplastic exposure on fatty acid content in liver tissue has been found.
Since the common spectroscopic technology is used for detecting compounds according to the spectroscopic information of characteristic functional groups, the prior reagent kit method based on the spectroscopic technology is used for detecting fatty acids, and homologs and isomers with the same functional groups cannot be distinguished, and the detected fatty acid mixture is a fatty acid mixture, and the resolution capability of the fatty acid mixture to the fatty acids is far lower than that of the chromatographic-mass spectrometry technology. The gas chromatography-mass spectrometry detection technology can accurately distinguish the carbon chain length of the fatty acid and the position and the number of unsaturated carbon bonds, and the resolution capability of the gas chromatography-mass spectrometry detection technology on the position of the unsaturated carbon bonds of the fatty acid isomers is also stronger than that of the liquid chromatography-mass spectrometry detection technology. The seawater medaka has numerous advantages as a model organism for toxicological studies, such as: wide temperature and salinity adaptation range, small size, easy propagation, experimental observation and operation and the like, and is widely used in toxicological research. Therefore, the invention adopts the gas chromatography-mass spectrometry technology to detect the content of the pentadecanoic acid and the heptadecanoic acid in the liver tissues of the medaka, takes the pentadecanoic acid and the heptadecanoic acid as combined markers, and has new application in preparing a kit and a detection method for evaluating the toxicity of liver microplastics, wherein the combined markers show excellent diagnostic sensitivity and specificity. The method adopted by the invention has the characteristics of simple operation, high repeatability, high stability, high sensitivity and high specificity. At present, no report that the pentadecanoic acid and heptadecanoic acid combined marker kit is used for evaluating the toxicity of liver micro-plastics is found.
Disclosure of Invention
The invention aims to solve the problems of low sensitivity and specificity, lack of a liver micro-plastic toxicity evaluation method and the like of a conventional kit for detecting fatty acid, provide an application of a fatty acid combination kit in evaluating the toxicity of liver micro-plastic, and provide a method for simultaneously detecting various fatty acids.
Pentadecanoic acid and heptadecanoic acid are used for preparing a kit for evaluating toxicity of liver microplastics and application and detection of the kit.
The reagent or the kit is used for detecting the content of pentadecanoic acid and heptadecanoic acid in the liver tissue of a tested organism.
The reagent or the kit is a combination of reagents for detecting the content of pentadecanoic acid and heptadecanoic acid in the liver tissue of a tested organism by adopting a gas chromatography-mass spectrometry method.
A reagent or kit for assessing toxicity of liver micropolastids, comprising:
(1) and (3) standard substance: pentadecanoic acid and heptadecanoic acid, wherein the standard substances are respectively used for the characterization of the pentadecanoic acid and the heptadecanoic acid in liver tissues;
(2) extracting solution: 80-100% methanol/water (v/v), said extract being used for extracting liver tissue metabolites;
(3) derivatization reagent: methoxyamine, pyridine, N-methyl-N- (trimethylsilyl) trifluoroacetamide, the derivatizing agent being used for metabolite oximation and silylation reactions.
The test organism refers to an organism having a liver. Such as test organisms including humans, mice, rats, fish, etc.
Microplastic refers to plastic particles from 1 μm to 5mm in diameter.
In order to achieve the purpose, the invention takes a sea medaka as a model and polystyrene micro Plastic (PS) as an example, and adopts the following technical scheme:
1. collecting a medaka liver tissue sample: after PS exposure treatment, collecting liver tissues of medaka, and storing the medaka in a refrigerator at minus 80 ℃ for later use. The number of repetitions of each process set is at least 3.
2. Collecting the liver tissue metabolic profile of medaka by using a gas chromatography-mass spectrometry method, and qualitatively and quantitatively analyzing pentadecanoic acid, heptadecanoic acid and changes thereof, including difference level P values, change multiples and the like.
3. Utilizing binary logistic regression analysis to integrate the pentadecanoic acid and the heptadecanoic acid into a combined marker, and evaluating the sensitivity and specificity of the combined marker by using an ROC (Receiver operating characterization) curve to obtain an AUC (Area under the curve) value.
4. Combination markers for evaluation of liver PS toxicity: pentadecanoic and heptadecanoic acids were significantly higher in the PS-exposed group than in the control group (P < 0.05, two-tailed Mann-Whitney U test); substituting the contents of the pentadecanoic acid and the heptadecanoic acid into a binary logistic regression module for analysis, creating a binary regression model, obtaining the constant term and the coefficients of the pentadecanoic acid and the heptadecanoic acid in the regression model, establishing a binary logistic regression equation, and obtaining the classification prediction probability of each sample. The binary logistic regression model was constructed as follows:
binary logistic regression model: sample class prediction probability =1/[1+e ] -(c+K*a+L*b) ]
Wherein c is a constant term; a is the pentadecanoic acid content; b is the heptadecanoic acid content; k is the coefficient of pentadecanoic acid in the equation; l is the coefficient of heptadecanoic acid in the equation.
Binary logistic regression equation: sample class prediction probability =1/[1+e ] -(-137.26+0.03*a+0.135*b) ]
Wherein a is the pentadecanoic acid content; b is the heptadecanoic acid content;
setting the criticality of the sample classification prediction probability value as 0.5, and if the sample classification prediction probability value is less than 0.5, exposing the liver of the medaka by PS; if the sample classification prediction probability value is more than or equal to 0.5, the medaka hepatotoxicity is induced by PS exposure. And carrying out ROC analysis by taking the sample classification prediction probability value as a variable, and evaluating the diagnosis performance of the PS toxicity of the medaka liver, wherein the diagnosis performance is excellent if the sensitivity and the specificity are both more than or equal to 80.0%, and the AUC is more than or equal to 0.8.
5. The evaluation system includes the following devices: the chromatographic column is a DB-5MS capillary column, the length of the column is 30m, the inner diameter is 250 μm, and the thickness of the membrane is 0.25 μm; the detection instrument is a gas chromatography-mass spectrometer.
6. The reagent or the kit comprises:
(1) And (3) standard substance: pentadecanoic acid and heptadecanoic acid, which are used for characterization of the pentadecanoic acid and the heptadecanoic acid in liver tissues, respectively.
The extracting solution is prepared: 80% aqueous methanol (v/v) for extraction of liver tissue metabolites.
A derivatization reagent: (1) the 20mg/mL methoxylamine pyridine solution oximates carbonyl and aldehyde groups, and can avoid the conversion of ketone groups into enol, the decarboxylation of alpha-keto acid, the cyclization of chain saccharides and the mutual conversion between saccharides with different conformations. (2) N-methyl-N- (trimethylsilyl) trifluoroacetamide (for GC derivitization, not less than 98.5%) is subjected to silanization on active hydrogen-containing metabolites such as carboxyl, amino, hydroxyl and the like to generate semi-volatile or volatile derivatives with higher stability, so that gas chromatography-mass spectrometry detection is facilitated.
Fourth, instrument analysis conditions:
(1) gas chromatography conditions: the sample size was 1. Mu.L. The injection port temperature was 300 ℃. The carrier gas is high-purity helium, the constant linear speed of the carrier gas is 40cm/s, and the split ratio is 5:1. the chromatographic column is a DB-5MS capillary chromatographic column, and the temperature rise program comprises the following steps: after maintaining at 70 ℃ for 3min, the temperature is raised to 300 ℃ at a rate of 5 ℃/min and maintained for 10min.
(2) Mass spectrum conditions: the ionization voltage is 70eV by adopting an ionization mode of electron bombardment. The transfer line and ion source temperatures were 280 ℃ and 230 ℃ respectively. The solvent cleavage time was 5.3min. And acquiring mass spectrum data by adopting a full scanning mode, wherein the mass-to-charge ratio scanning range is 33-600, and the scanning speed is 5 spectrograms/s. The detector voltage and the tuning voltage are the same.
(5) Mass spectrum data processing: after the original mass spectrum data is led into a NetCDF format, an XCMS program is applied in R2.3.11 software for peak matching and integration, and the area and retention time of the ion peak of the liver tissue sample are obtained. Deconvolution of ion peaks, search and matching of spectral libraries (NIST 11, fiehn and Wiley libraries) are carried out by using ChromaTOF software, metabolites are preliminarily characterized, characteristic ions of the metabolites are obtained, and qualitative results are further confirmed through mass spectrum fragment characteristics, retention time and retention indexes of standard samples. The characteristic ion mass to charge ratios of pentadecanoic and heptadecanoic acids are 299 and 328, respectively, with corresponding retention times (min) of 29.9 and 33.63, respectively.
8. The application effect of the invention is tested by adopting a liver tissue sample. The pentadecanoic acid and the heptadecanoic acid are used as combined markers, samples in a normal group and samples in a PS exposed group can be correctly identified according to the content of the pentadecanoic acid and the heptadecanoic acid in liver tissues, the accuracy is 100.0%, the AUC is 1.0, the optimal sensitivity and specificity reach 100.0%, and the pentadecanoic acid and the heptadecanoic acid are used as the combined markers, so that the PS toxicity of the liver can be correctly identified, and the combined markers have excellent diagnostic performance (figure 4).
The main advantages of the invention are:
(1) the common spectroscopic technique is to detect compounds based on characteristic functional group information, and when fatty acid is measured, homologues and isomers with the same functional group cannot be distinguished, so that fatty acid mixtures are detected, and the resolution capability of the fatty acid mixtures on the fatty acid is far lower than that of the chromatographic-mass spectrometry technique. The invention adopts a gas chromatography-mass spectrometry detection technology, can accurately distinguish the carbon chain length of the fatty acid and the position and the number of unsaturated carbon bonds, and has stronger resolution capability on the position of the unsaturated bond of the fatty acid isomer than a liquid chromatography-mass spectrometry technology. In addition, according to the invention, the chromatofof software is adopted to deconvolute the mass spectrum ion peak, so that the interference of an overlapped peak and an impurity peak can be eliminated, a purer mass spectrum can be obtained, and then the mass spectrum is searched and matched by a spectrum library (NIST 11, fiehn and Wiley library), so that the metabolite qualitative determination is more reliable; furthermore, characteristic ions of each metabolite can be obtained by using ChromaTOF software, so that interference of overlapping peaks and impurity peaks on quantification is eliminated, and the quantification accuracy is improved. Therefore, the method adopted by the invention can carry out qualitative and quantitative analysis with high specificity and high sensitivity on the pentadecanoic acid and the heptadecanoic acid in the liver tissue.
(2) The invention provides a detection kit for evaluating the toxicity of liver micro-plastics based on a gas chromatography-mass spectrometry method, which can accurately evaluate the toxicity of liver micro-plastics by detecting the content of pentadecanoic acid and heptadecanoic acid in a liver tissue sample. The invention has the advantages of small quantity of selected markers, large change multiple and the like, is beneficial to reducing the interference of other factors, has the characteristics of simple operation, mild reaction conditions, high sensitivity, high specificity and the like, and can provide an effective analysis method for the research and intervention of the toxicity mechanism of liver micro-plastics, the evaluation of the ecological risk and the health effect of the micro-plastics and the like.
Drawings
FIG. 1: QC sample profile in the principal component analysis. QC, quality control sample; non-QC: non-quality control samples.
FIG. 2: ion peak content versus standard deviation profile in the mass control samples.
FIG. 3: and (4) a sample principal component distribution map. CTRL: a control group; PS-10:10 μm polystyrene micro plastic exposed group; PS-200:200 μm polystyrene micro plastic exposed group.
FIG. 4: evaluation of toxicity of liver microplastic. CTRL: a control group; PS-10:10 μm polystyrene micro plastic exposed group; PS-200:200 μm polystyrene micro plastic exposed group; PS: PS-10 and PS-200 samples; * : p < 0.05, x: p < 0.01, two-tailed Mann-Whitney U test. (a, C), microplastic exposure induces liver pentadecanoic acid accumulation; (B, D), microplastic exposure induces hepatic heptadecanoic acid accumulation; (E) Pentadecanoic and heptadecanoic acid combination markers assess the toxic effects of liver microplastics; (F) The ROC curve evaluates the diagnostic performance of the pentadecanoic acid and heptadecanoic acid combination markers on the toxic effects of liver micropolastics.
TABLE 1 main Ionic Components of Artificial seawater
Table 2 microplastic exposure induced liver fatty acid accumulation.
Remarking: a : by standard mass spectrometryAfter library retrieval and comparison, the mass spectrogram, retention time and retention index of the standard compound are verified. b : and (5) performing qualitative analysis according to standard mass spectrum library retrieval comparison. Significance testing was performed by non-parametric test (two-tailed Mann-Whitney U test), with bolding showing significant changes (P)<0.05). PS-10:10 μm polystyrene micro plastic exposed group; PS-200:200 μm polystyrene micro plastic exposed group; CTRL: a control group; PS-10/CTRL: PS-10 mean divided by CTRL mean; PS-200/CTRL: PS-200 mean divided by CTRL mean.
Detailed Description
Examples
The present invention is further described below with reference to examples, which are intended to be illustrative only and not limiting. Those skilled in the art can make various similar modifications or substitutions without departing from the method and idea of the present invention, and such similar modifications or substitutions are included in the scope defined by the claims of the present application.
1. Exposing medaka in seawater
Adult medaka in seawater with the age of 8 months is selected and cultured in artificial seawater with the salinity of 3% (the main ionic components are shown in table 1). Polystyrene micro-plastics (PS-10 and PS-200) with the particle sizes of 10 mu m and 200 mu m are respectively added into artificial seawater (as exposure liquid), and the seawater medaka is exposed (the seawater medaka is cultured in the exposure liquid), namely PS-10 and PS-200 exposed groups. The exposure concentrations of PS-10 and PS-200 were both 10mg/L. The seawater is continuously aerated to uniformly disperse the micro-plastics. The exposure solution was changed daily for 60 days. The control group was not added with micro plastic (medaka in sea water was cultured in artificial seawater as a control group (i.e., normal group)).
2. Collecting liver tissue samples of sea water medaka
After the exposure was completed, liver tissues of normal (6), PS-10 exposed (3) and PS-200 exposed (3) ocean medaka were taken out and stored in a refrigerator at-80 ℃ for further use.
3. Pretreatment of liver tissue
10.0mg of fresh liver tissue was accurately weighed and placed in a 2.0mL centrifuge tube. One zirconia pellet (7 mm diameter) was added followed by 1000. Mu.L of 80% methanol solution and placed in an MMP400 shaker (Restch, germany) and disrupted by shaking at a frequency of 30 times/s for 1.5min. After centrifugation of the homogenate at 13000rpm for 15min at 4 ℃, 700. Mu.L of supernatant was removed and concentrated and dried in a vacuum freeze concentration drier (Thermo Scientific, USA).
mu.L of a methoxamine pyridine solution (20 mg/mL) was added to the dried sample, vortexed for 30s, and then the sample was oximated in a 37 ℃ water bath for 1.5h. Subsequently, 40. Mu. L N-methyl-N-trimethylsilyltrifluoroacetamide solution was added and silanized in a 37 ℃ water bath for 1.0h. After derivatization was complete, the derivatized samples were centrifuged at 13000rpm for 15min at 4 ℃ and the supernatant was collected for subsequent instrumental analysis.
To monitor and evaluate the reproducibility and stability of the assay methods of the invention, the remaining supernatant after centrifugation of the sample homogenate was collected and, after mixing well, divided into 700 μ L aliquots as Quality Control (QC) samples. In the subsequent concentration drying, derivatization, instrumental analysis and data processing, 1 QC sample was inserted for every 4 samples, which were processed with the same parameters as the other analytical samples.
4. Gas chromatography-mass spectrometry
Gas chromatography conditions: the sample size was 1. Mu.L. The injection port temperature was 300 ℃. The carrier gas is high-purity helium, the constant linear speed of the carrier gas is 40cm/s, and the split ratio is 5:1. the chromatographic column is a DB-5MS capillary column, the length of the column is 30m, the inner diameter is 250 μm, and the thickness of the membrane is 0.25 μm. Temperature program of chromatographic column: the initial column temperature was 70 deg.C, held for 3min, then ramped up to 300 deg.C at a rate of 5 deg.C/min, held for 10min.
Mass spectrometry conditions: the ionization voltage is 70eV by adopting an electron bombardment ionization mode. The interface temperature and ion source temperature were 280 and 230 ℃ respectively. The detection voltage and the tuning voltage are identical. The solvent cleavage time was 5.3min. The mass to charge ratio scan range is 33-600. The scanning frequency of the mass spectrum is 5 spectrograms/s.
5. Mass spectrometry data processing
The XCMS program was used in R2.3.11 software for peak matching and integration to obtain the area and retention time of each ion peak in the sample. Mass Spectrometry ion Peak identification, overlapping Peak deconvolution, spectroscopy library (NIST 11, fiehn and ChromaTOF software)Wiley bank), preliminarily characterize the metabolites, and obtain characteristic ions of each metabolite. Then, the qualitative result is further confirmed by comparing the fragment characteristics, retention time and retention index of the standard mass spectrum. In the peak identification and deconvolution processing, the peak width and the signal-to-noise ratio were set to 5s and 5, respectively. The characteristic ions (m/z) of pentadecanoic acid and heptadecanoic acid are 299 and 328, respectively, with corresponding retention times (min) of 29.9 and 33.63, respectively. The content of pentadecanoic acid and heptadecanoic acid is determined by dividing the characteristic ion original peak area by the total peak area, and multiplying by 1X 10 7 The latter values are presented for subsequent statistical analysis.
6. Statistical analysis
And uploading the relative content information of all metabolites to online software MetabioAnalyst 4.0 for principal component analysis. Based on the relative content information of each metabolite, two-tailed Mann-Whitney U test was performed by MeV 4.9.0 software. And (3) importing the relative content of the pentadecanoic acid and the heptadecanoic acid into SPSS18.0 software, carrying out binary logistic regression module analysis, creating a binary regression model, obtaining the constant term, the coefficient of the pentadecanoic acid and the heptadecanoic acid in the regression model, establishing a binary logistic regression equation, and finally obtaining the classification prediction probability value of each sample. Setting the criticality of the sample classification prediction probability value as 0.5, and if the sample classification prediction probability value is less than 0.5, exposing the liver of the medaka by PS; if the sample classification prediction probability value is more than or equal to 0.5, the medaka hepatotoxicity is induced by PS exposure. And carrying out ROC analysis by taking the sample classification prediction probability value as a variable, and evaluating the diagnosis performance of the PS toxicity of the medaka liver, wherein the diagnosis performance is excellent if the sensitivity and the specificity are both more than or equal to 80.0%, and the AUC is more than or equal to 0.8.
7. Evaluation of repeatability and stability of detection method
From the principal component score plot, 3 QC samples were tightly clustered (fig. 1). Furthermore, from the Relative Standard Deviation (RSD) distribution of ion peak content in QC samples, 6029, 6601 and 7245 ions RSD were less than 15%,20% and 30%, respectively, of 8252 ions, and accounted for 73.06%,79.99% and 87.8%, respectively, of the total number of ions (fig. 2). The distribution of the QC sample and the RSD distribution of each ion peak in the QC sample in the principal component analysis are integrated, and the detection method adopted by the invention has high stability, repeatability and reliability and is suitable for analyzing the liver tissue sample.
8. Microplastic induced liver fatty acid accumulation
As can be seen from the sample principal component score chart, the hepatic metabolic profile of medaka induced by exposure to PS-10 and PS-200 was significantly changed (FIG. 3). At PS-10 exposure, there was a significant increase in 11 fatty acids, while at PS-200 exposure, there was a significant increase in 5 fatty acids and a significant decrease in 2 fatty acids; of these, pentadecanoic acid, hexadecanoic acid, heptadecanoic acid, cis-4,7,10,13,16,19-docosahexaenoic acid and cis-7,10,13,16-docosatetraenoic acid were significantly increased under exposure to both particle sizes of the microplastic (table 2, fig. 4A and B). Of the fatty acids that increased significantly with both particle size microplastic exposures, the fold change was greatest for pentadecanoic acid and heptadecanoic acid. After mixing the two particle size micro plastic exposed samples, both pentadecanoic acid and heptadecanoic acid increased significantly with micro plastic exposure with a fold change greater than 2 (fig. 4C and D). Thus, pentadecanoic acid and heptadecanoic acid, which have significant changes (P < 0.05) with the greatest fold change upon exposure to both particle size microplastics, were selected as combination markers for evaluation of liver microplastic toxicity.
9. Evaluation of liver microplastic toxicity
And (3) introducing the relative content of the pentadecanoic acid and the heptadecanoic acid into SPSS software, and obtaining a prediction probability value of the sample based on a binary logistic regression model, wherein the value is used for evaluating the toxicity of the liver micro-plastics. The obtained binary logistic regression equation is as follows: sample classification prediction probability value =1/[ 1+e% -(-137.26+0.03*a+0.135*b) ]Wherein a is the relative content of pentadecanoic acid, and b is the relative content of heptadecanoic acid. The results show that the prediction probabilities of the samples exposed by PS-10 and PS-200 alone are close to 1 (more than 0.5), and the prediction probabilities of the control samples are close to 0 (less than 0.5), so that the method provided by the invention can correctly evaluate whether the exposure of the micro-plastic induces the hepatotoxicity, and the correct rate is 100.0% (fig. 4E). In addition, ROC analysis is used for evaluating the diagnostic performance of the detection method, and the result shows that the diagnostic performance of the detection method on the toxicity of the liver micro-plastics is superior, the AUC is 1.0, and the optimal sensitivity and specificity reach 100.0 percent (fig. 4F).
10. And (4) conclusion: the method provided by the invention can be used for detecting the pentadecanoic acid and the heptadecanoic acid in liver tissues with high specificity and high sensitivity, and identifying the toxicity of liver micro-plastics with high specificity and high sensitivity according to the content information of the pentadecanoic acid and the heptadecanoic acid, so as to prompt whether the micro-plastics exposure induces the liver lipid metabolism abnormality of a tested organism and further increase the risk of metabolic diseases such as fatty liver, hepatitis, diabetes, cardiovascular diseases and the like. The invention has the characteristics of small quantity of selected markers, large change multiple, simple operation, mild reaction condition, high stability and repeatability, is beneficial to reducing the interference of other factors, and can provide effective technical support for research and intervention of a liver micro-plastic toxicity mechanism, evaluation of micro-plastic ecological risks and health effects and the like.
Claims (1)
1. Use of a combination marker in the preparation of a reagent or kit for detecting microbial liver plastic toxicity, wherein the combination marker comprises pentadecanoic acid and heptadecanoic acid; the reagent or the kit is used for detecting the content of pentadecanoic acid and heptadecanoic acid in the liver tissue of a tested organism by a gas chromatography-mass spectrometry method, and evaluating the toxicity of the micro-plastics in the liver of the tested organism through the content of the pentadecanoic acid and the heptadecanoic acid; the tested organism is a seawater medaka.
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