Serum lipid markers related to fundus hard exudates in diabetic retinopathy and their application

Abstract

The present invention provides serum lipid markers of fundus hard exudates of diabetic retinopathy and their applications. Specifically, a set of biomarkers related to fundus hard exudates of diabetic retinopathy is provided, wherein the set includes biomarkers of group A1: A1: Cer 42:0; 4O, DG36:0 and ADGGA52:2. The biomarkers of the present invention can assess the risk or degree of fundus hard exudates of diabetic retinopathy in humans with high specificity and high accuracy.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to serum lipid markers related to hard exudates in fundus of human diabetic retinopathy and applications thereof. Background Art

[0002] Diabetes mellitus (DM) is an important public health issue worldwide. According to statistics from the International Diabetes Federation (IDF), my country ranks first in the world in terms of the number of people with DM, with a DM prevalence rate of 11.6%, exceeding 140 million people.

[0003] Diabetic retinopathy (DR) is a blinding disease that seriously threatens the vision of working people and is also the main complication of diabetes mellitus (DM) in the eye. The prevalence of DR in DM patients is as high as 27.04%. The number of DR patients in my country exceeds 34 million, and the number of blind or severely visually impaired patients with visual acuity <0.3 due to DR is close to 10 million. And only 7% of type 2 DM patients have their glycated hemoglobin, blood pressure, and low-density lipoprotein levels reasonably controlled. It can be said that the current DR patients are just the tip of the iceberg, and there are a considerable number of potential DR patients among DM patients. At present, there is no effective way to reverse late-stage DR. Although there are many treatments, such as panretinal laser photocoagulation, vitrectomy, and intraocular injection of anti-angiogenic drugs, they require repeated treatments, have great treatment risks, and only target surface symptoms, resulting in poor treatment outcomes. It can be seen that the key to the prevention and treatment of DR lies in finding early key biomarkers, early identification, diagnosis, and intervention, effective warning before the disease occurs, and accurate identification in the early stages of the disease.

[0004] Typical fundus lesions of diabetic retinopathy include microaneurysms, intraretinal hemorrhages, venous loops, venous beads, hard exudates, cotton wool spots, fibrous tissue proliferation, retinal neovascularization, preretinal hemorrhage, vitreous hemorrhage, etc. Among them, hard exudates appear as yellow particles or plaques in the fundus. In the past, it was believed that lipids or lipoproteins in plasma leaked from retinal blood vessels and deposited in retinal tissue. Hard exudates are often accompanied by retinal thickening and edema, and are closely related to the occurrence of diabetic macular edema (DME). Diabetic macular edema mainly affects central vision and seriously affects the quality of life of patients. Studies by the Early Treatment of Diabetic Retinopathy Research Group (ETDRS) have shown that persistent hard exudates can eventually evolve into subretinal fibrosis or retinal pigment epithelium (RPE) atrophy, leading to irreversible vision loss. Some other studies have also shown that hard exudates in the fovea are usually associated with poor visual outcomes. Therefore, it is crucial to study the mechanism of the occurrence and development of hard exudates, to predict and warn early, and then to reduce the visual impairment caused by hard exudates.

[0005] According to previous studies, the formation of hard exudates is believed to be caused by the leakage of lipoproteins from retinal capillaries into the extracellular space of the retina. Therefore, previous studies have focused on the relationship between blood lipid levels and the degree of hard exudates in patients with diabetic retinopathy. Traditionally, it is believed that increased blood lipid levels are associated with an increased risk of hard exudates in the fundus. Data from the Early Diabetic Retinopathy Treatment Research Group (ETDRS) showed that lipid-lowering drugs can reduce the risk of hard exudates in the fundus of patients with diabetic retinopathy. However, other studies have found that there is no correlation between blood lipid levels and the severity of hard exudates, and lipid-lowering drugs cannot reduce the risk of hard exudates. Due to the limitations of lipid-lowering therapy in the prevention and treatment of hard exudates in diabetic retinopathy, it is necessary to conduct more in-depth lipidomics analysis to identify the key biomarkers that cause hard exudates in the fundus, to predict and warn early, and to reduce visual impairment caused by hard exudates.

[0006] Therefore, there is an urgent need in this field to provide highly specific and accurate serum lipid markers related to hard exudates in the fundus of patients with diabetic retinopathy, to provide a new diagnostic and classification method for such diseases, and to lay a foundation for further clarifying the occurrence and development mechanism of such diseases. Summary of the invention

[0007] The purpose of the present invention is to provide a set of biomarkers that can assess the risk or degree of fundus hard exudates in human diabetic retinopathy with high specificity and accuracy.

[0008] In a first aspect, the present invention provides a set of biomarkers related to fundus hard exudates of diabetic retinopathy, wherein the set includes biomarkers of group A1:

[0009] A1: Cer 42:0;4O, DG36:0 and ADGGA 52:2.

[0010] In another preferred embodiment, the collection also includes biomarkers of group A2: A2: TG 49:1 and PC O-44:5.

[0011] In another preferred embodiment, the set further comprises one or more biomarkers selected from group A3:

[0012] A3: PE 36:1;3O, NAE 20:2, NAE 20:0, DG O-31:1, SM 44:2;3O.

[0013] In another preferred embodiment, the set further comprises one or more biomarkers selected from group A4:

[0014] A4: DG O-35:1, PC 31:0, NAE 18:5, NAE 20:0, PC 17:0, TG 32:0, SM 38:5; 2O, TG48:0, TG52:0, TG 54:1.

[0015] In another preferred example, the set includes all biomarkers in A1-A3.

[0016] In another preferred example, the set includes all biomarkers in A1-A4.

[0017] In a second aspect, the present invention provides a use of the biomarker set as described in the first aspect of the present invention for preparing a kit for predicting and / or diagnosing fundus hard exudates in diabetic retinopathy.

[0018] In a third aspect, the present invention provides a kit for predicting and / or diagnosing fundus hard exudates in diabetic retinopathy, comprising the following group of biomarker standards and / or their isotope standards, wherein:

[0019] The biomarkers include the biomarkers of Group A1:

[0020] A1: Cer 42:0;4O, DG36:0, ADGGA 52:2.

[0021] In another preferred embodiment, the biomarkers also include biomarkers of group A2: A2: TG 49:1, PC O-44:5.

[0022] In another preferred embodiment, the biomarkers further include one or more biomarkers selected from group A3:

[0023] A3: PE 36:1;3O, NAE 20:2, NAE 20:0, DG O-31:1, SM 44:2;3O.

[0024] In another preferred embodiment, the biomarkers further include one or more biomarkers selected from group A4:

[0025] A4: DG O-35:1, PC 31:0, NAE 18:5, NAE 20:0, PC 17:0, TG 32:0, SM 38:5; 2O, TG48:0, TG52:0, TG 54:1.

[0026] In a fourth aspect, the present invention provides a method for predicting or diagnosing fundus hard exudates of diabetic retinopathy, comprising the steps of:

[0027] (a) detecting the concentration C1 of each biomarker in the serum sample of the subject, wherein the biomarkers include the biomarkers of group A1: A1: Cer 42:0;4O, DG36:0 and ADGGA 52:2;

[0028] (b) comparing the C1 with a reference value C0, respectively, to predict or diagnose whether the subject suffers from hard exudates in the fundus of diabetic retinopathy, or whether the subject is at risk of hard exudates in the fundus of diabetic retinopathy.

[0029] In another preferred embodiment, C0 comprises a reference value C0+ of a corresponding biomarker obtained from a subject with fundus hard exudates of diabetic retinopathy; and / or

[0030] The C0 includes a reference value C0- of the corresponding biomarker obtained from a subject who does not suffer from diabetic retinopathy fundus hard exudates.

[0031] In another preferred embodiment, when the concentration value of each selected biomarker meets C1≥C0+, the patient is diagnosed as having hard exudates in the fundus of diabetic retinopathy. Otherwise, the patient is diagnosed as not having hard exudates in the fundus of diabetic retinopathy.

[0032] In another preferred embodiment, the risk of suffering from fundus hard exudates due to diabetic retinopathy is predicted based on the difference between C1 and C0+ and / or C0-.

[0033] In another preferred embodiment, the biomarkers further include biomarkers selected from group A2:

[0034] A2:TG 49:1, PC O-44:5.

[0035] In another preferred embodiment, the biomarkers further include one or more biomarkers selected from group A3:

[0036] A3: PE 36:1;3O, NAE 20:2, NAE 20:0, DG O-31:1, SM 44:2;3O.

[0037] In another preferred embodiment, the biomarkers further include one or more biomarkers selected from group A4:

[0038] A4: DG O-35:1, PC 31:0, NAE 18:5, NAE 20:0, PC 17:0, TG 32:0, SM 38:5; 2O, TG48:0, TG52:0, TG 54:1.

[0039] The fifth aspect of the present invention provides a use of the kit as described in the third aspect of the present invention for predicting and / or diagnosing whether a test subject suffers from hard exudates in the fundus of diabetic retinopathy or is at risk of hard exudates in the fundus of diabetic retinopathy.

[0040] In another preferred embodiment, the subject is a mammal, such as a human.

[0041] In another preferred embodiment, the subject is a diabetic patient, or even a patient with diabetic retinopathy.

[0042] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 . Pearson correlation and significance analysis results between WGCNA lipid module and hard exudate.

[0044] Figure 2 . Lipid composition of ME1 lipid module.

[0045] Figure 3 . Analysis of carbon chain length and unsaturation of triglycerides, ceramides, and N-acylethanolamines.

[0046] Figure 4 . Multi-ROC analysis. A panel of 20 lipid molecules showed the best discrimination for hard exudates.

[0047] Figure 5 . Multi-ROC curve of the specific composition of 20 lipid molecules. DETAILED DESCRIPTION

[0048] The inventors have conducted extensive and in-depth research, and through a large number of screenings and tests, have provided a set of serum lipid biomarkers for hard exudates in the fundus of human diabetic retinopathy. Specifically, the set includes a variety of serum lipid biomarkers for hard exudates in the fundus of patients with diabetic retinopathy, which can be used to assess the risk of hard exudates in the fundus of a subject to be tested or to diagnose hard exudates in the fundus of a subject to be tested, and has the advantages of high sensitivity and high specificity, and has important application value. The present invention was completed on this basis.

[0049] the term

[0050] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0051] As used herein, when used in reference to a specific recited value, the term "about" means that the value may vary by no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes all values ​​between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0052] As used herein, the term "comprising" or "including (comprising)" may be open, semi-closed and closed. In other words, the term also includes "consisting essentially of" or "consisting of".

[0053] Hard exudates in the fundus of patients with diabetic retinopathy

[0054] Typical fundus lesions of diabetic retinopathy include microaneurysms, intraretinal hemorrhage, venous loops, venous beads, hard exudates, cotton wool spots, fibrous tissue proliferation, retinal neovascularization, preretinal hemorrhage, vitreous hemorrhage, etc.

[0055] Among them, hard exudates appear as yellow granules or plaques in the fundus, often accompanied by retinal thickening and edema, and are closely related to the occurrence of diabetic macular edema (DME). Diabetic macular edema mainly affects central vision and seriously affects the quality of life of patients. Studies by the Early Treatment of Diabetic Retinopathy Research Group (ETDRS) have shown that persistent hard exudates can eventually evolve into subretinal fibrosis or retinal pigment epithelium (RPE) atrophy, leading to irreversible vision loss. When significant hard exudate changes have occurred in the fundus, the patient's visual function has been severely impaired and the response to existing treatments is poor. At present, there is no effective method to predict or diagnose hard exudates in the fundus of patients with diabetic retinopathy through serum lipidomics testing or any other method. There is currently no relevant research report on the connection between characteristic changes in the fundus of patients with diabetic retinopathy and changes in the patient's serum lipidomics. In the specific technical scheme of this study, the UHPLC-MS / MS method was used for serum lipidomics detection, covering 888 lipid molecules in the serum, which can perform a detailed and comprehensive analysis of changes in systemic lipids.

[0056] Biomarkers related to fundus hard exudates

[0057] The present invention provides a set of biomarkers (substances) in serum samples that can be used to assess fundus hard exudates. Among them, the biomarkers in group A1 have the highest correlation with fundus hard exudates:

[0058] A1: Cer 42:0;4O, DG36:0, ADGGA 52:2.

[0059] Furthermore, the set may also include biomarkers selected from Group A2:

[0060] A2:TG 49:1, PC O-44:5.

[0061] Preferably, the collection further comprises one or more biomarkers selected from group A3: A3: PE 36:1;3O, NAE 20:2, NAE 20:0, DG O-31:1, SM 44:2;3O.

[0062] In another preferred embodiment, the collection also includes one or more biomarkers selected from group A4: A4: DG O-35:1, PC 31:0, NAE 18:5, NAE 20:0, PC 17:0, TG 32:0, SM 38:5;2O, TG 48:0, TG52:0, TG54:1.

[0063] Preferably, the marker set includes all (10 in total) biomarkers in A1-A3.

[0064] More preferably, the marker set includes all (20 in total) biomarkers in A1-A4.

[0065] In the present invention, the expression method of lipid is clear to those skilled in the art, wherein "TG" represents triglycerol, "Cer" represents ceramide, "NAE" represents N-acylethanolamine, "ADGGA" represents acyl diacylglycerol glucuronide, "DG" represents diacylglycerol, "PE" represents phosphatidylethanolamine, "PC" represents phosphatidylcholine, and "SM" represents sphingomyelin. Typically, "DG O-31:1" represents a diacylglycerol with a total length of 31 double-chain carbon atoms and one unsaturated double bond, and O represents that the carbon chain is connected to the glycerol backbone by an ether bond; SM 44:2;3O represents a sphingomyelin with a total length of 44 double-chain carbon atoms, a total unsaturation of 2 in the two chains, and 3 O atoms in the two carbon chains. .

[0066] The present invention also provides a use of the above-mentioned set of biomarkers for preparing a kit for predicting and / or diagnosing fundus hard exudates in diabetic retinopathy.

[0067] Reagent test kit

[0068] The present invention further provides a kit for predicting and / or diagnosing fundus hard exudates in diabetic retinopathy, which comprises the above biomarker standards and / or their isotope standards.

[0069] In the present invention, the kit at least includes the biomarkers of group A1:

[0070] A1: Cer 42:0;4O, DG36:0 and ADGGA 52:2.

[0071] The kit of the present invention as described above can be used to predict and / or diagnose whether a test subject suffers from hard exudates in the fundus of diabetic retinopathy or whether there is a risk of hard exudates in the fundus of diabetic retinopathy.

[0072] Preferably, the isotope standard refers to the biomarker labeled with an isotope, such as deuterium (2H), 13 C or 14 Preferably, the biomarker labeled with N has 3-12 H (stable H atoms) replaced by deuterium atoms.

[0073] In another preferred embodiment, the subject is a mammal, such as a human.

[0074] In another preferred embodiment, the subject is a diabetic patient, or even a patient with diabetic retinopathy.

[0075] Generally, in the kit, the biomarkers or isotope standards thereof can be packaged separately or provided in the form of mixed standards.

[0076] In addition, the kit may further include instructions. Preferably, the instructions include the following method for predicting or diagnosing fundus hard exudates of diabetic retinopathy.

[0077] Methods for predicting or diagnosing fundus hard exudates in diabetic retinopathy

[0078] Furthermore, the present invention also provides a method for predicting or diagnosing fundus hard exudates of diabetic retinopathy, comprising the steps of:

[0079] (a) detecting the concentration C1 of each biomarker in a serum sample of a subject, wherein the biomarkers include the biomarkers of group A1: A1: Cer 42:0;4O, DG36:0, ADGGA 52:2;

[0080] (b) comparing the C1 with a reference value C0, respectively, to predict or diagnose whether the subject suffers from hard exudates in the fundus of diabetic retinopathy, or whether the subject is at risk of hard exudates in the fundus of diabetic retinopathy.

[0081] In the case where it is known that the biomarkers of the present invention are highly correlated with fundus hard exudates, those skilled in the art can prepare a standard for predicting / diagnosing the disease by conventional methods in the art. For example, C0 includes a reference value C0+ of a corresponding biomarker obtained from a subject with fundus hard exudates of diabetic retinopathy; and / or the C0 includes a reference value C0- of a corresponding biomarker obtained from a subject without fundus hard exudates of diabetic retinopathy.

[0082] When the concentration value of each selected biomarker meets C1≥C0+, the patient is diagnosed as having hard exudates in the fundus of diabetic retinopathy, otherwise the patient is diagnosed as not having hard exudates in the fundus of diabetic retinopathy.

[0083] In another preferred embodiment, the risk of fundus hard exudates of diabetic retinopathy is predicted based on the difference between C1 and C0+ and / or C0-. For example, when C1 is closer to C0+, the risk of fundus hard exudates increases, and when C1 is closer to C0-, the risk of fundus hard exudates is smaller.

[0084] In another preferred embodiment, the subject is a mammal, such as a human.

[0085] In another preferred embodiment, the subject is a diabetic patient, or even a patient with diabetic retinopathy.

[0086] The main advantages of the present invention include:

[0087] 1. The present invention provides for the first time a set of biomarkers related to hard exudates in the fundus of patients with diabetic retinopathy, and a kit comprising the biomarkers, which can be used to predict or diagnose hard exudates at an early stage.

[0088] 2. The biomarkers of the present invention can predict and / or diagnose hard exudate with high specificity and high accuracy.

[0089] The present invention will be further described below in conjunction with specific implementation. It should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples without specifying specific conditions are usually based on conventional conditions or the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight.

[0090] (a) Experimental description

[0091] Study population:

[0092] Inclusion and Exclusion Criteria: All participants in this study were inpatients and outpatients in the Department of Ophthalmology and Endocrinology of Shanghai First People's Hospital from July 2017 to March 2021. Inclusion criteria: (1) age ≥ 18 years; (2) Han nationality; (3) diagnosed with diabetes (type 1 and type 2, confirmed by a diabetes physician) and any severity of diabetic retinopathy [including mild non-proliferative diabetic retinopathy, moderate non-proliferative diabetic retinopathy, severe non-proliferative diabetic retinopathy and proliferative diabetic retinopathy, confirmed by an ophthalmologist]. Exclusion criteria: (1) history of other eye diseases that may cause fundus hard exudates, such as age-related macular degeneration, uveitis, retinal vein occlusion, etc.; (2) suffering from severe cataracts resulting in opacity of the refractive medium, affecting the assessment of the severity of hard exudates; (3) combined with other severe diabetic complications other than diabetic retinopathy; (4) pregnant or breastfeeding.

[0093] Systemic and ocular assessment and examination: The diagnostic criteria for diabetes were based on the criteria proposed by the American Diabetes Association in 1997. The assessment of diabetic retinopathy was based on the diagnostic criteria proposed by the American Academy of Ophthalmology in 2001, and fundus examination was performed by senior fundus experts. All patients underwent a comprehensive ophthalmological examination, including slit lamp examination of the anterior segment and fundus, and fundus color photography. According to the degree of lesions on fundus color photography, they were divided into mild non-proliferative diabetic retinopathy, moderate non-proliferative diabetic retinopathy, severe non-proliferative diabetic retinopathy, and proliferative diabetic retinopathy. The gender, age, type of diabetes, duration of diabetes, history of hypertension, duration of hypertension, history of dyslipidemia, history of cardiovascular disease, and concomitant medication of all subjects were recorded. In addition, biochemical indicators such as glycated hemoglobin (HbA1c), total cholesterol, triglycerides, creatinine, urea, and uric acid were collected.

[0094] Evaluation of the severity of hard exudates in fundus color photographs: All patients took fundus color photographs of the second visual field at 35 degrees, covering the posterior pole area centered on the macula and including the optic disc. The inventors used standard photographs of hard exudates of different severity developed by the International Early Diabetic Retinopathy Treatment Study Group (ETDRS) to compare with the fundus images of the patients in this study to determine the presence and severity of hard exudates in the second visual field. The international hard exudate grading standard is: grade 0, no hard exudate; grade 1, suspicious hard exudate; grade 2, hard exudate < standard photo 3; grade 3, hard exudate ≥ standard photo 3 and < standard photo 5; grade 4, hard exudate ≥ standard photo 5 and < standard photo 4; grade 5, hard exudate ≥ standard photo 4; grade 8, cannot be graded. To simplify the grouping for further lipidomics and metabolomics analyses, the degree of hard exudates was combined into 3 categories in this study: no or suspected hard exudates (grades 0 and 1); moderate hard exudates (grades 2 and 3); and severe hard exudates (grades 4 and 5). Patients whose fundus color photographs could not be graded were excluded. The evaluation was completed independently by two experienced readers, and then the grade was confirmed by a senior fundus expert and the final classification was made. If there was any disagreement, all scorers reached a consensus.

[0095] Serum sample collection: 10 ml of whole blood was collected from all patients in the morning on an empty stomach and injected into a sodium heparin tube, which was centrifuged within 30 min (1500 rpm, 20°C, 10 min). 1.5 mL of serum aliquots were transferred to cryovials and immediately stored at −80°C. Lipids in the samples were extracted by liquid-liquid extraction: 50 μL of sample was extracted with 4 volumes of cold chloroform: methanol (v / v = 2:1). The sample was vortexed for 30 s and then allowed to stand at room temperature for 5 min. The mixture was centrifuged at 13,000 g for 15 min, and the upper phase (hydrophilic metabolites) and lower organic phase (hydrophobic metabolites) were collected separately and evaporated under vacuum at room temperature.

[0096] Ultra-high performance liquid chromatography / tandem mass spectrometry combined with pattern recognition technology was used for lipid detection: metabolites and lipids were separated on an Ultimate 3000 UHPLC system (Thermo Scientific). Lipidomics data were collected using full scan mode.

[0097] Data processing: Mzmine was used to automatically perform peak picking, integration, retention time adjustment, and alignment on the raw data generated by UPLC-MS / MS.

[0098] Statistical analysis: MetaboAnalyst5.0 (http: / / www.metaboanalyst.ca / ) was used for heat map, spearman rank correlation analysis, and pathway analysis. Logistic regression analysis and receiver operating characteristic (ROC) analysis were used to diagnose hard exudates in different populations. The expectation maximization (EM) method was used to estimate missing values ​​or ROC analysis of clinical and demographic characteristics. SPSS 18.0 software was used for statistical analysis.

[0099] (b) Results (data, charts, etc.)

[0100] General information of the subjects: A total of 167 patients (167 eyes) with diabetic retinopathy from Shanghai First People's Hospital were enrolled in this study, divided into a discovery set and a validation set. The discovery set consisted of 116 patients (116 eyes) enrolled from July 2017 to November 2019; the validation set consisted of 51 patients (51 eyes) enrolled from April 2020 to March 2021.

[0101] Consistency of hard exudate severity evaluation: According to the classification criteria, patients were divided into 3 groups, namely, no or suspected hard exudate, moderate hard exudate, and severe hard exudate. The weighted kappa was 0.931 (P<0.001). This result showed good interobserver consistency.

[0102] WGCNA analysis: The lipidomics data were analyzed by WGCNA, and 888 lipid molecules were divided into 18 modules. Pearson correlation analysis and significance analysis were performed on the obtained module scores and exudation degree. The degree of hard exudation in patients with non-proliferative diabetic retinopathy, proliferative diabetic retinopathy and diabetic retinopathy (non-proliferative diabetic retinopathy + proliferative diabetic retinopathy) was significantly positively correlated with the ME1 module and significantly negatively correlated with the ME5 module. The results are shown in Figure 1 .

[0103] ME1 lipid composition analysis: ME1 lipid module composition analysis see Figure 2 The main components of ME1 are triglycerides (TG, accounting for 29%), ceramide (Cer, accounting for 17%) and N-acylethanolamine (NAE, accounting for 15%).

[0104] Analysis of carbon chain length and unsaturation of triglycerides, ceramides, and N-acylethanolamines: The ME1 module is mainly composed of triglycerides, and Pearson correlation analysis was further performed on the correlation between the unsaturation and carbon chain length of triglycerides and exudation.

[0105] The analysis results are shown below Figure 3, red indicates that the lipid composition is positively correlated with exudation, blue indicates that it is negatively correlated with exudation, and the yellow box indicates that the p value of Pearson's correlation is <0.01; the red box indicates that the p value of Pearson's correlation is <0.0001. The results of the analysis showed that the degree of exudation was significantly correlated with the increase of very long-chain unsaturated TG glycerol and was significantly correlated with the decrease of long-chain saturated TG glycerol. No obvious trend of change was found in the carbon chain length and unsaturation of ceramide and N-acylethanolamine. Yellow box: p value <0.01; red box: p value <0.0001.

[0106] Multi-ROC analysis: ROC curve analysis is based on the multivariate algorithm - support vector machine (SVM). The ROC curve is generated by Monte Carlo cross validation (MCCV) using balanced subsampling. In each MCCV, two-thirds (2 / 3) of the samples are used to evaluate the importance of features, and SVM is used to rank the features. Then, the top 2, 3, 5, 10...100 (largest) important features are used to build a classification model and validated on the excluded 1 / 3 samples. This process is repeated multiple times to calculate the performance and confidence interval of each model. ME1 Multi-ROC analysis results are shown in Figure 4 The study showed that the set of 10 lipid molecules had a better discrimination effect on hard exudates, AUC = 0.804, 95% confidence interval 0.644 ~ 0.928, and the set of 20 lipid molecules had the best discrimination effect on hard exudates, AUC = 0.804, 95% confidence interval 0.674 ~ 0.916. ROC curve established by ME1 lipid. The specific composition of the 20 lipid molecules is shown in Figure 5 , the vertical axis represents the metabolite name, and the horizontal axis represents the feature selection frequency.

[0107] (c) A brief description or discussion of the results

[0108] The pathological change of fundus hard exudates in patients with diabetic retinopathy seriously harms visual function. The present invention has discovered a set of biomarkers related to fundus hard exudates, which can be used to assess the risk of fundus hard exudates in a subject to be tested or to diagnose fundus hard exudates in a subject to be tested, and has the advantages of high sensitivity and high specificity, and has important application value. It is conducive to early identification of diabetic retinopathy patients who are prone to develop fundus hard exudates in clinical practice, and early intervention of blood sugar, blood lipids, etc. for systemic conditions to protect visual function.

[0109] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. A collection of biomarkers related to hard exudates in diabetic retinopathy, including: The panel includes biomarkers of Group A1: A1: Cer 42:0; 4O, DG36:0 and ADGGA 52:

2.

2. The biomarker set according to claim 1, characterized in that: The panel also includes biomarkers of group A2: A2: TG 49:1 and PC O-44:

5.

3. The biomarker set according to claim 2, characterized in that: The panel also includes one or more biomarkers selected from Group A3: A3:PE 36:1;3O, NAE 20:2, NAE 20:0, DG O-31:1, SM 44:2;3O.

4. The biomarker set according to claim 3, characterized in that: The panel also includes one or more biomarkers selected from Group A4: A4:DG O-35:1, PC 31:0, NAE 18:5, NAE 20:0, PC 17:0, TG 32:0, SM 38:5; 2O, TG 48:0, TG52:0, TG 54:

1.

5. The biomarker set according to claim 3, characterized in that: The panel includes all biomarkers in A1-A3.

6. The biomarker set according to claim 4, characterized in that: The panel includes all biomarkers in A1-A4.

7. Use of the biomarker set as claimed in claim 1 for preparing a kit for predicting and / or diagnosing fundus hard exudates in diabetic retinopathy.

8. A kit for predicting and / or diagnosing fundus hard exudates in diabetic retinopathy, characterized in that: The invention comprises the following group of biomarker standards and / or isotope standards, wherein: The biomarkers include the biomarkers of group A1: A1: Cer 42:0; 4O, DG36:0, ADGGA 52:

2.

9. The kit according to claim 8, characterized in that The biomarkers also include biomarkers of group A2: A2: TG 49:1, PC O-44:

5.

10. The kit according to claim 9, characterized in that The biomarkers also include one or more biomarkers selected from Group A3: A3:PE 36:1;3O, NAE 20:2, NAE 20:0, DG O-31:1, SM 44:2;3O.

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