Group of chronic fluorosis early warning saliva markers as well as extraction method and application thereof

Abstract

The invention relates to the technical field of medical detection, in particular to a group of chronic fluorosis early warning saliva markers as well as an extraction method and application thereof. The invention relates to a group of chronic fluorosis early warning saliva markers which are saliva metabolites. The extraction method of the chronic fluorosis early warning saliva marker comprises the following steps: obtaining a non-irritant saliva specimen, and extracting the non-irritant saliva specimen by adopting a first mixed organic solvent to obtain an organic phase; concentrating and drying the organic phase to obtain a solid substance; dissolving the solid substance by adopting a second mixed organic solvent, and then carrying out gradient elution on a chromatographic column by adopting a liquid chromatography-electrospray ionization-tandem mass spectrometry system to obtain the chronic fluorosis early warning saliva marker. The invention also provides an application of the chronic fluorosis early warning saliva marker extracted by the extraction method as a chronic fluorosis early warning detection standard. The invention provides a new tool for noninvasive diagnosis of early fluorine poisoning.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of medical detection, in particular to a group of early warning salivary markers for chronic fluorosis, and an extraction method and application thereof. BACKGROUND

[0002] Dental fluorosis is an enamel formation and mineralization disorder caused by excessive intake of fluoride during the enamel development period (mainly from the gestational period to about 8 years old). Its clinical features range from chalky patches on the enamel surface to brown lesions with substantial defects. This disease has obvious regional clustering and is the most common clinical symptom of chronic fluorosis. Dental fluorosis not only affects the appearance of teeth, but also causes multiple burdens on oral function and mental health. Mild to moderate lesions are often accompanied by enamel staining and spotted defects, affecting speech confidence and causing social anxiety. Severe lesions are characterized by loose enamel structure and reduced wear resistance, leading to reduced molar efficiency and restricted diet. In severe cases, fluorosis can also be accompanied by skeletal fluorosis, affecting cardiovascular, neurological, and endocrine systems.

[0003] Therefore, it is crucial to seek an early, accurate, and non-invasive detection method for the prevention and intervention of chronic fluorosis. SUMMARY

[0004] In view of the above, the purpose of the present application is to provide a group of early warning salivary markers for chronic fluorosis, and an extraction method and application thereof, to provide a new tool for non-invasive diagnosis of early fluorosis.

[0005] To achieve the above purpose, the technical solutions adopted by the present application are as follows: The group of early warning salivary markers for chronic fluorosis is a salivary metabolite. The saliva metabolites include at least one of hydroxycarnitine C4 (Carnitine C4-OH), carnitine C5:1 (Carnitine C5:1), lysophosphatidic acid (18:4) [LPA (18:4)], dihydroxycarnitine C3:1 (Carnitine C3:1-2OH), alkylacylphosphatidylethanolamine (O-24:0_20:4) [PE (O-24:0_20:4)], alkylacylphosphatidylethanolamine (O-20:0_22:4) [PE (O-20:0_22:4)], hexosylceramide [t15:0 / 16:2 (2OH)] {HexCer [t15:0 / 16:2 (2OH)]}, phosphatidic acid (21:0_22:0) [PA (21:0_22:0)], alkylacylphosphatidylethanolamine (O-18:2_24:1) [PE (O-18:2_24:1)], and alkyl ether lysophosphatidylcholine (O-22:1) [LPC (O-22:1)]. It provides a new tool for non-invasive diagnosis of early fluorosis, and lays a research foundation for further exploration of drug targets and pathological mechanisms of dental fluorosis.

[0006] Among them, saliva as a non-invasive biological sample, its composition change can reflect the physiological and pathological state of the body, and it shows unique advantages in early diagnosis of diseases. The saliva markers for early warning of chronic fluorosis provided by the application include but are not limited to specific proteins, metabolites or trace elements, which change significantly in the early stage of chronic fluorosis and can be used as reliable biological indicators. It provides a new tool for non-invasive diagnosis of early fluorosis, and lays a research foundation for further exploration of drug targets and pathological mechanisms of dental fluorosis.

[0007] The extraction method of the saliva markers for early warning of chronic fluorosis provided by the application comprises the following steps: S1, obtaining a non-stimulating saliva sample, then extracting the non-stimulating saliva sample with a first mixed organic solvent to obtain an organic phase; S2, concentrating and drying the organic phase to obtain a solid substance; S3, dissolving the solid substance with a second mixed organic solvent, then performing gradient elution on a C30 chromatographic column by using a liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS / MS) system to obtain the saliva markers for early warning of chronic fluorosis.

[0008] According to the above technical means, by optimizing the sample processing process and extraction technology, the sensitivity and specificity of marker detection are improved, which provides strong support for the early screening and diagnosis of chronic fluorosis. By detecting these saliva markers, early warning of chronic fluorosis can be achieved, which helps to take timely intervention measures, slow down the progression of the disease, and protect the oral and systemic health of patients.

[0009] Preferably, the first mixed organic solvent is selected from a mixed solution of methyl tert-butyl ether (MTBE) and methanol, and the volume ratio of methyl tert-butyl ether (MTBE) to methanol in the first mixed organic solvent is 3:1. Preferably, the second mixed organic solvent is selected from a mixed solution of acetonitrile and isopropanol, and the volume ratio of acetonitrile to isopropanol in the second mixed organic solvent is 1:1.

[0010] Preferably, the volume ratio of the non-irritating saliva sample, the first mixed organic solvent, and the second mixed organic solvent is 200 μL:2 mL:200 μL.

[0011] Preferably, in S1, the temperature for concentration and drying is 20°C.

[0012] Preferably, in S1, a vacuum freeze centrifugal concentrator (LABCONCO, CentriVap) is used, and the cold trap is set to -40°C; concentration is performed until complete dryness.

[0013] Preferably, the mobile phase for gradient elution is composed of mobile phase A and mobile phase B. The mobile phase A is composed of formic acid, ammonium formate, acetonitrile, and water. In the mobile phase A, the mass percentage content of formic acid is 0.1%, the concentration of ammonium formate is 10 mmol / L, and the volume ratio of acetonitrile to water is 60 / 40. The mobile phase B is composed of formic acid, ammonium formate, acetonitrile, and isopropanol. In the mobile phase B, the mass percentage content of formic acid is 0.1%, the concentration of ammonium formate is 10 mmol / L, and the volume ratio of acetonitrile to isopropanol is 10 / 90.

[0014] Preferably, the gradient elution procedure is: at 0 min, the volume ratio of mobile phase A to mobile phase B is 8:2; linearly reduced to a volume ratio of mobile phase A to mobile phase B of 7:3 from 0 to 2.0 min (not including the lower limit value, but including the upper limit value, that is, 0-2.0 min refers to a period of time greater than 0 and less than or equal to 2 min, and subsequent time periods are analogously defined); linearly reduced to a volume ratio of mobile phase A to mobile phase B of 4:6 from 2 to 4 min, linearly reduced to a volume ratio of mobile phase A to mobile phase B of 0.15:0.85 from 4 to 9 min, linearly reduced to a volume ratio of mobile phase A to mobile phase B of 0.1:0.9 from 9 to 14 min, linearly reduced to a volume ratio of mobile phase A to mobile phase B of 0.05:0.95 from 14 to 15.5 min, maintained to 17.3 min, linearly reduced to a volume ratio of mobile phase A to mobile phase B of 8:2 from 17.3 to 20 min.

[0015] Preferably, the column temperature of the C30 chromatographic column is 45°C.

[0016] Preferably, in S3, mass spectrometric detection adopts positive and negative ion switching mode. The working parameters of mass spectrometric detection are: an ESI Turbo Ion-Spray ion source is used, the ion source temperature is 500°C; the ion spray voltage is 5500 V in positive ion mode and -4500 V in negative ion mode; the pressures of atomizing gas (GS1), turbine gas (GS2) and curtain gas (CUR) are 45 psi, 55 psi and 35 psi respectively; the pressure of collision gas (CAD) is set to medium pressure 5 psi.

[0017] The LC-ESI-MS / MS system calibration is performed using polypropylene glycol solutions of different concentrations, the concentration of polypropylene glycol in the polypropylene glycol solution is 10 μmol / L in triple quadrupole (QQQ) mode, and the concentration of polypropylene glycol in the polypropylene glycol solution is 100 μmol / L in linear ion trap (LIT) mode; and the dynamic MRM method of optimizing the declustering voltage (DP) and collision energy (CE) alone is used to target analyze the lipid metabolites by setting the collision gas pressure to 5 psi. The optimization of DP and CE is mainly based on the response of the main fragments generated by different categories of lipids. Some lipids need higher collision energy and declustering voltage to have higher response of daughter ions, and some lipids cannot use too high collision energy to avoid excessive fragmentation.

[0018] Preferably, it further comprises: Mass spectrometry data were processed using software Analyst 1.6.3; identification of saliva metabolites was performed by comparison with MWDB (metware database); quantitative evaluation of saliva metabolites was performed using multiple reaction monitoring (MRM) mode; relative abundance of saliva metabolites was calculated according to the corresponding chromatographic peak area. As of 2025, MWDB has included more than 20,000 metabolites, covering animal primary metabolites (sugars, amino acids, lipids, nucleotides, etc.) and secondary metabolites (hormones, bile acids, neurotransmitters, steroids, etc.). The technical functions of this database include: qualitative analysis: high-throughput metabolite identification is achieved by comparing the accurate m / z values, retention times, and secondary mass spectrometry (MS / MS) fragment spectra of metabolites. Quantitative analysis: high sensitivity and high accuracy metabolite quantification is achieved using the multiple reaction monitoring (MRM) mode of the triple quadrupole mass spectrometer combined with ion pair information in the database. Interference removal: automatically exclude redundant signals such as isotope signals, adducts (K + , Na + , NH4 + ) and in-source fragmentation. This database can detect 2,000-5,000 metabolites per detection.

[0019] Preferably, the non-stimulating saliva sample at least includes non-stimulating saliva of caries patients, healthy people and patients with fluorosis.

[0020] The application also provides the application of the saliva markers for early warning of chronic fluorosis extracted by the extraction method as the detection standard for early warning of chronic fluorosis.

[0021] The application has the following beneficial effects: The group of saliva markers for early warning of chronic fluorosis belongs to lipid and energy metabolism pathways (carnitine, phospholipid, acylphospholipid, ceramide), and the level of the group of saliva markers for early warning of chronic fluorosis can be disturbed by the mechanism of oxidative stress-mitochondrial damage-membrane lipid remodeling at the early stage of chronic fluorosis; saliva is used to replace blood sampling, which is non-invasive, repeatable, can detect metabolic profile deviation before visible enamel lesions, realizes true "zero-level prevention", and the molecules themselves are involved in cell membrane stability, inflammation signal and calcium phosphate mineralization regulation, which provides direct intervention biochemical nodes for revealing the pathogenesis of fluorosis and developing targeted intervention drugs.

[0022] The extraction method of the early warning saliva marker of chronic fluorosis of the application adopts a "non-stimulating saliva combined C30-LC-ESI-MS / MS" strategy. First, the optimized mixed solvent is used for one-step liquid-liquid extraction, and trace carnitine, acetal phospholipid, ceramide and other metabolites with large polarity span are synchronously enriched in the organic phase, and the interference of protein and salt is effectively removed. After low-temperature concentration of the extraction phase, a high-proportion organic phase solvent is used for redissolution, and the solvent system is compatible with reversed-phase chromatography, which can realize column pre-focusing when large-volume injection, thereby significantly improving the sensitivity. A C30 long-chain alkyl stationary phase chromatographic column is used, and the unique shape selectivity of the column can realize baseline separation of 10 target markers and complex background phospholipids within 30 minutes of gradient, which greatly reduces the matrix interference and ion suppression effect. Combined with positive and negative switching modes of the electrospray ion source and MRM detection, two pairs of characteristic ion pairs are collected for each metabolite, so that the signal-to-noise ratio of the method is optimized by 5-8 times before the method, and the CV is less than or equal to 0.3. Finally, the method can simultaneously quantify the markers at a concentration level of 0.1-0.5 nM with only 200 µL of saliva, and the sensitivity is equivalent to that of the standard method based on serum, while having the advantage of being completely non-invasive, which provides a reliable, stable and easy-to-accept detection platform for large-scale early screening of fluorosis in the population. In the field of medical detection technology, it has popularization and application value. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 OPLS-DA diagram of the metabolomics of the DC group, the HC group and the DF group; Figure 2 OPLS-DA permutation test diagram of the metabolomics of the DC group, the HC group and the DF group; Figure 3 Mass spectrum in positive ion mode; Figure 4 Mass spectrum in negative ion mode; Figure 5 ROC curve diagram of the optimal model; Figure 6 Result diagram of the contribution of 10 metabolites screened by AUC to the discrimination model; Figure 7 Result diagram of the area under the curve (AUC) of 10 saliva metabolites. DETAILED DESCRIPTION

[0024] The following description, with reference to preferred embodiments, illustrates the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention. It should be understood that the preferred embodiments are merely illustrative of the present invention and not intended to limit the scope of protection of the present invention.

[0025] Example 1 A method for extracting a set of saliva markers for early warning of chronic fluorosis includes the following steps: S1. Saliva sample collection: Three mL of saliva was collected from 100 patients with dental caries (DC), 100 healthy individuals (HC), and 99 patients with dental fluorosis (DF) as non-irritating saliva samples. Each non-irritating saliva sample was stored separately and quickly transferred to a freezer at -80°C for freezing. Subsequently, it was rapidly transported to Wuhan Maiwei Metabolism Company via dry ice for the extraction and detection of saliva metabolites. The selection of the population was as follows: 299 college students who lived and studied at the same university were included in the study of this embodiment.

[0026] To control for the influence of confounding factors on the occurrence of dental caries and fluorosis, participants were asked to complete questionnaires to collect information on gender, parental education level, and oral health behaviors (including the use of fluoride toothpaste, brushing frequency, flossing frequency, and sugar intake frequency). All oral examinations were performed by trained dentists (Kappa value > 0.8).

[0027] The ICDAS criteria are used to diagnose dental caries, and the Dean index is used to diagnose dental fluorosis.

[0028] One hundred individuals with an ICDAS score > 4 were included in the dental caries group (DC), 100 individuals without dental caries or fluorosis were included in the healthy group (HC), and individuals with a 99 Dean index ≥ 1 were included in the fluorosis group (DF). Inclusion criteria required exclusion of the following conditions: recurrent aphthous ulcers, pulpitis, periodontal disease or other oral lesions; systemic or hereditary diseases; and use of antibiotics or exposure to tobacco products within the past six months.

[0029] Partial correlation analysis was used to exclude the influence of clinical phenotype. When using the OPLS-DA model, the results were as follows: Figure 1 As shown. From Figure 1 Analysis revealed clear separation between the samples from the DC, HC, and DF groups, confirming significant metabolomics differences among these three groups. Meanwhile, as... Figure 2As shown, R 2 Y = 0.967, Q 2 = 0.919, both of which are very close to 1, representing high reliability of OPLS-DA.

[0030] S2, saliva of three groups of people is respectively extracted and detected for metabolites: S22, each person's non-stimulating saliva sample in S1 is respectively thawed, then 1 mL of the first mixed organic solvent mixture of methyl tert-butyl ether (MTBE) and methanol with a volume ratio of 3:1 is added and mixed and oscillated, followed by centrifugal extraction at a temperature of 4℃ to obtain the upper organic phase; S23, the upper organic phase obtained in S22 is respectively concentrated and dried by a freeze concentrator to obtain solid material, then the solid material is redissolved with 200 μL of the second mixed organic solvent of acetonitrile and isopropanol with a volume ratio of 1:9 to obtain the saliva to be tested; Among them, the setting conditions of the freeze concentrator are: the cold trap is set to -40℃, and the sample is set to room temperature; S24, a liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS / MS) system is used to perform gradient elution on a C30 chromatographic column to obtain mass spectrometry data of early warning saliva markers for chronic fluorosis; Among them, the mobile phase of gradient elution is composed of mobile phase A and mobile phase B; The mobile phase A is composed of formic acid, ammonium formate, acetonitrile and water; In the mobile phase A, the mass percentage content of formic acid is 0.1%, the concentration of ammonium formate is 10 mmol / L, and the volume ratio of acetonitrile to water is 60 / 40; The mobile phase B is composed of formic acid, ammonium formate, acetonitrile and isopropanol; In the mobile phase B, the mass percentage content of formic acid is 0.1%, the concentration of ammonium formate is 10 mmol / L, and the volume ratio of acetonitrile to isopropanol is 10 / 90; The gradient elution procedure is as follows: the volume ratio of mobile phase A to mobile phase B is 8:2; linearly reduced to 7:3 from 0 to 2.0 min (not including the lower limit value, but including the upper limit value, that is, 0~2.0 min refers to a time period greater than 0 and less than or equal to 2 min, and subsequent time periods are similar); linearly reduced to 4:6 from 2 to 4 min, linearly reduced to 0.15:0.85 from 4 to 9 min, linearly reduced to 0.1:0.9 from 9 to 14 min, linearly reduced to 0.05:0.95 from 14 to 15.5 min, maintained to 17.3 min, linearly reduced to 8:2 from 17.3 to 20 min; The flow rate is 0.35 mL / min, and the column temperature is maintained at 45℃; The mass spectrometry detection adopts positive and negative ion switching mode; The working parameters of mass spectrometry detection are as follows: an ESI Turbo Ion-Spray ion source is used, the ion source temperature is 500℃; the ion spray voltage is 5500V in positive ion mode and -4500V in negative ion mode; the pressures of atomizing gas (GS1), turbine gas (GS2) and curtain gas (CUR) are 45 psi, 55 psi and 35 psi respectively; the pressure of collision gas (CAD) is set to 5 psi; System calibration is performed using polypropylene glycol solutions of different concentrations, the concentration of polypropylene glycol in the polypropylene glycol solution is 10 μmol / L in triple quadrupole (QQQ) mode, and the concentration of polypropylene glycol in the polypropylene glycol solution is 100 μmol / L in linear ion trap (LIT) mode; and by setting the collision gas pressure to 5 psi, the dynamic MRM method of separately optimizing the de-clustering voltage (DP) and the collision energy (CE) is used for targeted analysis of lipid metabolites in saliva of three groups of people, and positive and negative ion mode mass spectrometry data of lipid metabolites in saliva are obtained; Among them, the positive ion mode mass spectrum is as shown in Figure 3 The negative ion mode mass spectrum is as shown in Figure 4 .

[0031] Figure 3 In the positive ion mode, the number of peaks is significantly more than that in the negative ion mode, mainly distributed in the retention time interval of 4.0~15.0 min, and the coverage is wider.

[0032] From the analysis in Figure 3 It can be known that this mode is suitable for the detection of basic or positively charged lipids (such as sphingolipids, phosphatidylcholines, etc.), the diversity and intensity of the peaks indicate that the sample is rich in such lipid species, and the retention time and intensity of different peaks can be used for subsequent quantification and identification of lipids.

[0033] Figure 4 In the negative ion mode, a number of characteristic peaks were detected, mainly distributed in the retention time interval of 5.0-7.0 min, and the ion intensity of the peaks reflected the relative content of the corresponding lipids.

[0034] From Figure 4 The analysis shows that this mode is suitable for the detection of acidic or negatively charged lipids (such as polyunsaturated fatty acids, phosphatidic acid, etc.), the number and intensity of the peaks in the figure indicate the presence of such lipids in the sample, and the difference in retention time of different peaks reflects the different separation behaviors of lipids in the chromatographic column, which can be used as one of the bases for lipid qualification.

[0035] S3, data analysis: The mass spectrometry data was processed by using the software Analyst 1.6.3, so as to convert the mass spectrometry raw data (*.wiff) into quantifiable, comparable and publishable data results; The identification of saliva lipid metabolites as early warning saliva markers for chronic fluorosis was completed by comparing the comparable data results obtained by processing according to the software Analyst 1.6.3 with the MWDB (metware database), according to the retention time, characteristic ion pair and MS / MS fragment spectrum of saliva metabolites, and the qualitative identification results are shown in Table 1; Table 1 is the qualitative identification results of 10 saliva lipid metabolites The quantifiable data results obtained by processing according to the software Analyst 1.6.3 were quantitatively evaluated by using the multiple reaction monitoring (MRM) mode, and the relative abundance of saliva lipid metabolites was calculated according to the corresponding chromatographic peak area, and the quantitative results are shown in Table 2.

[0036] Table 2 is the abundance of 10 saliva lipid metabolites in three groups of people From Table 2, it can be seen that the expression level analysis shows that among the 10 saliva lipid metabolites, LPA(18:4) presents high abundance expression in the DF group, and the remaining metabolites present high abundance expression in the DF group and the HC group, thereby proving that the 10 saliva lipid metabolites can be used as early warning saliva markers for chronic fluorosis.

[0037] Orthogonal partial least squares discriminant analysis (OPLS-DA) of saliva lipid metabolites was performed using R 3.5.1.

[0038] Using the random forest model method of cross-validation, the saliva lipid metabolite abundance information is input into the random forest classifier, and 10 saliva lipid metabolites with significant discriminant ability in distinguishing fluorosis population (DF), healthy population (HC) and caries population (DC) are screened out. The 10 saliva lipid metabolites with significant discriminant ability specifically include: hydroxycarnitine C4 (Carnitine C4-OH), carnitine C5:1 (Carnitine C5:1), lysophosphatidic acid (18:4) [LPA (18:4)], dihydroxycarnitine C3:1 (Carnitine C3:1-2OH), aldehyde phosphatidylethanolamine (O-24:0_20:4) [PE (O-24:0_20:4)], aldehyde phosphatidylethanolamine (O-20:0_22:4) [PE (O-20:0_22:4)], hexosylceramide [t15:0 / 16:2 (2OH)] {HexCer [t15:0 / 16:2 (2OH)]}, phosphatidic acid (21:0_22:0) [PA (21:0_22:0)], aldehyde phosphatidylethanolamine (O-18:2_24:1) [PE (O-18:2_24:1)], and alkyl ether lysophosphatidylcholine (O-22:1) [LPC (O-22:1)]; Using Graphpad Prism 10.4.1 to draw the receiver operating characteristic (ROC), the AUC is calculated as the evaluation parameter of the discriminant model performance, the combination with the best discriminant performance is selected, the ROC curve of the optimal discriminant model is obtained, and the AUC index of each metabolite is output in the optimal discriminant model. The higher the AUC index, the higher the importance of the marker in distinguishing fluorosis. As shown in Figure 5 .

[0039] From Figure 5 analysis, it can be seen that this combination performs well in discriminant analysis, and the area under the curve (AUC) reaches 1.00.

[0040] As shown in Figure 6 , through the cross-validation random forest model, 10 saliva lipid metabolites that can best distinguish fluorosis population (DF) and healthy population (HC) and caries population (DC) are screened out. The contribution of these 10 saliva lipid metabolites to the discriminant model is found that Carnitine C4-OH (hydroxycarnitine C4) has the highest contribution.

[0041] In addition, as shown in Figure 7As shown, the AUC of the four salivary lipid metabolites, Carnitine C4-OH (hydroxyl carnitine C4), Carnitine C5:1 (carnitine C5:1), LPA(18:4) (lysophosphatidic acid (18:4)), and Carnitine C3:1-2OH (dihydroxyl carnitine C3:1) in the ROC analysis is also 1.00, and the discrimination ability is the strongest.

[0042] In summary, the set of early warning salivary markers for chronic fluorosis in the present application is first carefully designed in subject selection, and 100 caries patients and 100 healthy people are included as double controls, and compared with 99 patients with dental fluorosis. The healthy control group is set to establish the normal physiological baseline of the salivary lipid group; and the introduction of caries, a high-incidence disease, as a control, has a deeper consideration: caries and dental fluorosis, although both belong to hard tissue diseases of teeth, their pathological mechanisms are significantly different - the former is mainly caused by bacterial acid metabolism to induce demineralization, and the latter is caused by excessive fluoride during development. Through this double control design, it can accurately identify those biomarkers that do not universally exist in various oral diseases, but are specifically present in the early exposure of fluoride and the occurrence process of dental fluorosis, thereby significantly improving the reliability of the diagnostic markers. A total of 100 caries patients, 100 healthy people and 99 patients with dental fluorosis were included in the present study, and the caries patients and healthy people were used as double controls to systematically analyze the salivary lipid metabolome differences of patients with dental fluorosis, and a highly specific biomarker combination was screened and constructed for early identification and evaluation of fluorosis. The combination not only provides a new tool for non-invasive diagnosis of early fluorosis, but also lays a research foundation for further exploration of drug targets for dental fluorosis and its pathological mechanism. In the field of medical detection technology, it has popularization and application value.

[0043] The above examples are only preferred embodiments for fully illustrating the present application, and the protection scope of the present application is not limited thereto. Any equivalent replacement or transformation made by those skilled in the art based on the present application is within the protection scope of the present application.

Claims

1. A group of saliva markers for early warning of chronic fluorosis, characterized in that, The salivary biomarkers for early warning of chronic fluorosis are salivary metabolites; The salivary metabolites include at least one of hydroxycarnitine C4, carnitine C5:1, lysophosphatidylcholine (18:4), dihydroxycarnitine C3:1, acetal phosphatidylethanolamine (O-24:0-20:4), acetal phosphatidylethanolamine (O-20:0-22:4), hexosylceramide [t15:0 / 16:2(2OH)], phosphatidylcholine (21:0-22:0), acetal phosphatidylethanolamine (O-18:2-24:1), and alkyl ether lysophosphatidylcholine (O-22:1).

2. The method for extracting saliva markers for early warning of chronic fluorosis according to claim 1, characterized in that, Includes the following steps: S1. Obtain a non-irritating saliva sample, and then extract the non-irritating saliva sample using the first mixed organic solvent to obtain an organic phase; S2. Concentrate and dry the organic phase to obtain a solid substance; S3. The solid substance is dissolved in a second mixed organic solvent, and then gradient elution is performed on a C30 column using a liquid chromatography-electrospray ionization-tandem mass spectrometry system to obtain a saliva marker for early warning of chronic fluorosis.

3. The method for extracting saliva markers for early warning of chronic fluorosis according to claim 2, characterized in that, The first mixed organic solvent is selected from a mixed solution of methyl tert-butyl ether (MTBE) and methanol, wherein the volume ratio of MTBE to methanol in the first mixed organic solvent is 3:1; And / or, the second mixed organic solvent is selected from a mixed solution of acetonitrile and isopropanol, wherein the volume ratio of acetonitrile to isopropanol in the second mixed organic solvent is 1:

9.

4. The method for extracting saliva markers for early warning of chronic fluorosis according to claim 2, characterized in that, The volume ratio of the non-irritating saliva sample, the first mixed organic solvent, and the second mixed organic solvent is 200 μL: 2 mL: 200 μL.

5. The method for extracting saliva markers for early warning of chronic fluorosis according to claim 2, characterized in that, In step S1, the concentration and drying temperature is 20°C; The organic phase was concentrated and dried using a vacuum refrigerated centrifugal concentrator, with the cold trap set to -40°C, until it was completely dry.

6. The method for extracting saliva markers for early warning of chronic fluorosis according to claim 2, characterized in that, The gradient elution mobile phase consists of mobile phase A and mobile phase B; The mobile phase A consists of formic acid, ammonium formate, acetonitrile, and water; In the mobile phase A, the mass percentage of formic acid is 0.1%, the concentration of ammonium formate is 10 mmol / L, and the volume ratio of acetonitrile to water is 60 / 40. The mobile phase B consists of formic acid, ammonium formate, acetonitrile, and isopropanol; In the mobile phase B, the mass percentage of formic acid is 0.1%, the concentration of ammonium formate is 10 mmol / L, and the volume ratio of acetonitrile to isopropanol is 10 / 90.

7. The method for extracting saliva markers for early warning of chronic fluorosis according to claim 6, characterized in that, The gradient elution procedure is as follows: at 0 minutes, the volume ratio of mobile phase A to mobile phase B is 8:2; from 0 to 2.0 minutes, the ratio is linearly reduced to 7:3; from 2 to 4 minutes, the ratio is linearly reduced to 4:6; from 4 to 9 minutes, the ratio is linearly reduced to 0.15:0.85; from 9 to 14 minutes, the ratio is linearly reduced to 0.1:0.9; from 14 to 15.5 minutes, the ratio is linearly reduced to 0.05:0.95; this is maintained until 17.3 minutes; and from 17.3 to 20 minutes, the ratio is linearly reduced to 8:

2. And / or, the column temperature of the C30 chromatographic column is 45°C.

8. The method for extracting saliva markers for early warning of chronic fluorosis according to claim 2, characterized in that, In S3, the mass spectrometry detection adopts a positive and negative ion switching mode; The operating parameters for mass spectrometry detection were as follows: an ESI Turbo Ion-Spray ion source was used, with an ion source temperature of 500°C; the ion spray voltage was 5500V in positive ion mode and -4500V in negative ion mode; the pressures of the nebulizing gas (GS1), turbine gas (GS2), and curtain gas (CUR) were 45psi, 55psi, and 35psi, respectively; and the pressure of the collision gas (CAD) was 5psi.

9. The method for extracting saliva markers for early warning of chronic fluorosis according to claim 8, characterized in that, Also includes: Mass spectrometry data were processed using Analyst 1.6.3 software; identification of salivary metabolites was performed by comparison with MWDB (Metware database); quantitative assessment of salivary metabolites was conducted using multiple reaction monitoring (MRM) mode; the relative abundance of salivary metabolites was calculated based on the corresponding chromatographic peak areas. And / or, the non-irritating saliva specimens include at least non-irritating saliva from patients with dental caries, healthy individuals, and patients with fluorosis.

10. The application of the saliva marker for early warning of chronic fluorosis extracted by the extraction method according to any one of claims 2 to 9 as a standard for early warning detection of chronic fluorosis.

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