Use of membrane lipidome in the preparation of a diagnostic product for juvenile depression

Abstract

The application provides a use of a membrane lipid group in preparation of a product for diagnosing adolescent depression, and in particular, the membrane lipid group is a red blood cell membrane lipid selected from the group consisting of ceramide (Cer), phosphatidylserine (PS), phosphatidylcholine (PC), triglyceride (TG) and cholesteryl ester (ChE). The membrane lipid group is stable, is not easily interfered by other factors such as eating habits, and can avoid subjective concealment, thereby providing a more objective, real and stable method for diagnosing adolescent depression.

Description

Technical Field

[0001] This invention relates to the field of disease diagnosis, and more specifically to the use of a membrane lipidome in the preparation of diagnostic products for adolescent depression. Background Technology

[0002] Depression is one of the most common mental disorders during adolescence, with a lifetime prevalence as high as 19% by the end of adolescence. Key characteristics of depression include persistent negative mood, decreased motivation and interest, and even self-harm and suicidal behavior. Depression persistently impairs the mental functioning and quality of life of adolescents, becoming the fourth leading cause of disease burden among young adults aged 10-24.

[0003] Currently, the International Classification of Diseases (ICD), the Diagnostic and Statistical Manual of Mental Disorders (DSM), and the Chinese Classification of Mental Disorders (CCMD) are the main diagnostic tools used both domestically and internationally for depression. However, diagnostic tools such as the ICD and DSM rely primarily on clinical symptoms and are complex and time-consuming to operate, requiring experienced psychiatrists. This hinders early and efficient screening and rapid diagnosis of depression in adolescents. Furthermore, because adolescents with depression often present with unexplained physical symptoms, eating disorders, anxiety, school refusal, declining academic performance, substance abuse, or behavioral problems, adolescent depression is frequently missed or misdiagnosed. The high heterogeneity among adolescents with depression also limits the accuracy and effectiveness of its diagnosis.

[0004] Several standardized assessment tools are used to help identify adolescent depression, including the Beck Depression Inventory (BDI) and the Child Depression Inventory (CDI). However, questionnaires and scales used for adolescent depression are unstructured diagnoses and have a high degree of subjectivity. Some studies have reported that metabolites in plasma or serum may serve as diagnostic biomarkers for adolescent depression, but serum and plasma metabolites are easily affected by short-term lifestyle habits such as diet and exercise.

[0005] Therefore, identifying objective and stable biomarkers has profound clinical and practical significance for promoting efficient screening and diagnosis of depression in adolescents. Summary of the Invention

[0006] The purpose of this invention is to provide an objective and stable biomarker for diagnosing adolescent depression.

[0007] In a first aspect of the invention, a method for non-diagnostic and non-therapeutic assessment of adolescent depression is provided, the method comprising the steps of: detecting the expression level of membrane lipids isolated from a red blood cell membrane sample of a test subject, thereby determining whether the test subject is an adolescent with depression; and The membrane lipids are selected from the group consisting of ceramide (Cer), phosphatidylserine (PS), phosphatidylcholine (PC), triglycerides (TG), and cholesterol esters (ChE).

[0008] In another preferred embodiment, the erythrocyte membrane lipids are selected from the group consisting of: Cer(d19:1_24:1), Cer(d16:1_16:1), PS(22:4_18:2), PC(22:5_18:2), PC(18:2_20:4), PC(18:1e_20:3), TG(18:4_18:1_18:2) and ChE(18:1).

[0009] In another preferred embodiment, the method further includes: after determining the concentration of the membrane lipids in the sample, calculating using the above concentration. P The value is used to draw a judgment conclusion.

[0010] In another preferred embodiment, the P value is calculated using the logical model shown in equation ①: ①; The concentration of Cer(d19:1_24:1) is c1; The concentration of Cer(d16:1_16:1) is c2; The concentration of PS (22:4_18:2) is c3; The concentration of PC (22:5_18:2) is c4; The concentration of PC (18:2_20:4) is c5; The concentration of PC(18:1e_20:3) is c6; The concentration of TG (18:4_18:1_18:2) is c7; The concentration of ChE(18:1) is c8.

[0011] In another preferred embodiment, the testing standard for the product is: when the subject's P If the value is > 0.396, the individual is diagnosed with adolescent depression.

[0012] In another preferred embodiment, the concentration of the membrane lipids in the sample is determined using liquid chromatography-mass spectrometry.

[0013] In another preferred embodiment, the chromatographic conditions of the liquid chromatography-mass spectrometry method are as follows: the chromatographic column is an ACQUITYUPLC BEH C18, phase A is a mixed solution of ammonium formate and formic acid in acetonitrile and water, and phase B is a mixed solution of ammonium formate and formic acid in isopropanol and acetonitrile, with gradient elution.

[0014] In another preferred embodiment, phase A is an acetonitrile aqueous solution containing 5-20 mM ammonium formate and 0.01-0.2% vv formic acid.

[0015] In another preferred embodiment, phase A is a mixed solution of acetonitrile and water (volume ratio of 2~10:2~6) with 5-20 mM ammonium formate and 0.01-0.2% vv formic acid added.

[0016] In another preferred embodiment, phase B is a mixed solution of isopropanol and acetonitrile with 5-20 mM ammonium formate and 0.01-0.2% vv formic acid added.

[0017] In another preferred embodiment, phase B is a mixed solution of isopropanol acetonitrile (volume ratio of 6~15:0.5~2) with 5-20 mM ammonium formate and 0.01-0.2% vv formic acid added.

[0018] In another preferred embodiment, the method further includes the step of processing a blood sample using a pretreatment unit prior to performing the membrane lipid concentration determination.

[0019] In another preferred embodiment, the processing method includes the steps of: collecting red blood cells, extracting the red blood cell membrane, and then extracting lipids from the red blood cell membrane.

[0020] In another preferred embodiment, the processing method includes the steps of: (1) Collecting red blood cells: After centrifuging the blood, collect the lower layer of red blood cells; (2) Extraction of red blood cell membrane: Red blood cells were treated with 5-50 times the volume of Tris-HCl (5-15 mM, pH=7-8) at 1-10℃ to fully swell the red blood cells; (3) Extraction of lipids: Add extraction solution (methanol:chloroform volume ratio of 0.1~2:1~5) to the erythrocyte membrane precipitate, mix well and extract at 20~60℃ for 1-5h. Then vacuum centrifuge and dry at 15-30℃ for 0.1-10h, dissolve the lipid extract in a compound solution (dichloromethane:isopropanol:methanol volume ratio of 0.1~2:0.1~2:1~5), mix well and centrifuge, and take the supernatant.

[0021] In a second aspect of the present invention, a kit for detecting adolescent depression is provided, the kit comprising: A sample pretreatment reagent, wherein the sample pretreatment reagent is used to extract erythrocyte membrane lipids from blood samples; The erythrocyte membrane lipidomic assay reagent is used to detect the expression level of membrane lipids isolated from erythrocyte membrane samples of the test subject.

[0022] In another preferred embodiment, the sample pretreatment reagent includes: Red blood cell membrane extraction reagent; preferably, the cell membrane extraction reagent is Tris-HCl; Lipid extraction reagent; preferably, the lipid extraction reagent includes a lipid extraction solution (methanol:chloroform volume ratio of 0.1~2:1~5) and a lipid reconstitution solution (dichloromethane:isopropanol:methanol volume ratio of 0.1~2:0.1~2:1~5).

[0023] In another preferred embodiment, the kit further includes an instruction manual, which states: The expression levels of membrane lipids isolated from erythrocyte membrane samples of the test subjects were detected to determine whether the test subjects were adolescents with depression; and The membrane lipids are selected from the group consisting of ceramide (Cer), phosphatidylserine (PS), phosphatidylcholine (PC), triglycerides (TG), and cholesterol esters (ChE).

[0024] In another preferred embodiment, the erythrocyte membrane lipids are selected from the group consisting of: Cer(d19:1_24:1), Cer(d16:1_16:1), PS(22:4_18:2), PC(22:5_18:2), PC(18:2_20:4), PC(18:1e_20:3), TG(18:4_18:1_18:2) and ChE(18:1).

[0025] In another preferred embodiment, the method further includes: after determining the concentration of the membrane lipids in the sample, calculating using the above concentration. P The value is used to draw a judgment conclusion.

[0026] In another preferred embodiment, the P value is calculated using the logical model shown in equation ①: ①; The concentration of Cer(d19:1_24:1) is c1; The concentration of Cer(d16:1_16:1) is c2; The concentration of PS (22:4_18:2) is c3; The concentration of PC (22:5_18:2) is c4; The concentration of PC (18:2_20:4) is c5; The concentration of PC(18:1e_20:3) is c6; The concentration of TG (18:4_18:1_18:2) is c7; The concentration of ChE(18:1) is c8.

[0027] In a third aspect of the invention, an application is provided of a combination of membrane lipid biomarkers in a red blood cell membrane sample in constructing a system for diagnosing adolescent depression; said membrane lipid biomarkers are selected from the group consisting of ceramide (Cer), phosphatidylserine (PS), phosphatidylcholine (PC), triglycerides (TG), and cholesterol esters (ChE).

[0028] In another preferred embodiment, the erythrocyte membrane lipid marker is selected from the group consisting of: Cer(d19:1_24:1), Cer(d16:1_16:1), PS(22:4_18:2), PC(22:5_18:2), PC(18:2_20:4), PC(18:1e_20:3), TG(18:4_18:1_18:2) and ChE(18:1).

[0029] In another preferred embodiment, the application further includes: calculating the concentration of the membrane lipid marker in the sample. P The value is used to derive the calculation result.

[0030] In another preferred embodiment, the P value is calculated using the logical model shown in equation ①: ①; The concentration of Cer(d19:1_24:1) is c1; The concentration of Cer(d16:1_16:1) is c2; The concentration of PS (22:4_18:2) is c3; The concentration of PC (22:5_18:2) is c4; The concentration of PC (18:2_20:4) is c5; The concentration of PC(18:1e_20:3) is c6; The concentration of TG (18:4_18:1_18:2) is c7; The concentration of ChE(18:1) is c8.

[0031] In another preferred example, when the returned P A value >0.396 indicates a diagnosis of adolescent depression.

[0032] In another preferred embodiment, the system includes a high-performance liquid chromatography-mass spectrometry (HPLC-MS) device.

[0033] In another preferred embodiment, the concentration of the membrane lipid marker in the sample is determined using liquid chromatography-mass spectrometry.

[0034] In another preferred embodiment, the chromatographic conditions of the liquid chromatography-mass spectrometry method are as follows: the chromatographic column is an ACQUITYUPLC BEH C18, phase A is a mixed solution of ammonium formate and formic acid in acetonitrile and water, and phase B is a mixed solution of ammonium formate and formic acid in isopropanol and acetonitrile, with gradient elution.

[0035] In another preferred embodiment, phase A is an acetonitrile aqueous solution containing 5-20 mM ammonium formate and 0.01-0.2% vv formic acid.

[0036] In another preferred embodiment, phase A is a mixed solution of acetonitrile and water (volume ratio of 2~10:2~6) with 5-20 mM ammonium formate and 0.01-0.2% vv formic acid added.

[0037] In another preferred embodiment, phase B is a mixed solution of isopropanol and acetonitrile with 5-20 mM ammonium formate and 0.01-0.2% vv formic acid added.

[0038] In another preferred embodiment, phase B is a mixed solution of isopropanol acetonitrile (volume ratio of 6~15:0.5~2) with 5-20 mM ammonium formate and 0.01-0.2% vv formic acid added.

[0039] In another preferred embodiment, the method further includes the step of processing a blood sample using a pretreatment unit prior to performing the concentration determination of the membrane lipid biomarker combination.

[0040] In another preferred embodiment, the processing method includes the steps of: collecting red blood cells, extracting the red blood cell membrane, and then extracting lipids from the red blood cell membrane.

[0041] In another preferred embodiment, the processing method includes the steps of: (1) Collecting red blood cells: After centrifuging the blood, collect the lower layer of red blood cells; (2) Extraction of red blood cell membrane: Red blood cells were treated with 5-50 times the volume of Tris-HCl (5-15 mM, pH=7-8) at 1-10℃ to fully swell the red blood cells; (3) Extraction of lipids: Add extraction solution (methanol:chloroform volume ratio of 0.1~2:1~5) to the erythrocyte membrane precipitate, mix well and extract at 20~60℃ for 1-5h. Then vacuum centrifuge and dry at 15-30℃ for 0.1-10h, dissolve the lipid extract in a compound solution (dichloromethane:isopropanol:methanol volume ratio of 0.1~2:0.1~2:1~5), mix well and centrifuge, and take the supernatant.

[0042] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0043] Figure 1 Standard curves for five lipid standards.

[0044] Figure 2 The diagnostic efficacy of eight red blood cell membrane lipids in adolescent depression.

[0045] Figure 3 This is an ROC curve of the risk of depression for each volunteer in Example 3.

[0046] Figure 4 The diagnostic efficacy of eight erythrocyte membrane lipids in the validation set for adolescent depression was evaluated. Detailed Implementation

[0047] Through extensive and in-depth research, including numerous screenings and tests, the inventors have developed a method for using membrane lipidomes in the preparation of diagnostic products for adolescent depression. The membrane lipidome consists of erythrocyte membrane lipids selected from the following group: ceramide (Cer), phosphatidylserine (PS), phosphatidylcholine (PC), triglycerides (TG), and cholesterol esters (ChE). After collecting erythrocytes from the subject's venous blood, the membrane lipids are separated and extracted. The peak areas of each lipid are obtained by detecting the erythrocyte membrane lipids in the subject's blood using ultra-high performance chromatography-mass spectrometry (UHPLC-MS). The lipid concentration is then calculated using a standard curve of standards. A result is obtained with lipid concentration as the independent variable. P Value formula, where P The value represents the risk of adolescent depression in the subjects. P The higher the value, the higher the risk. The membrane lipids are stable and not easily affected by confounding factors such as short-term diet, exercise, and medication. Furthermore, they are closely related to the pathological mechanisms of adolescent depression, truly reflecting the health status of the subjects. Compared to clinical interviews and scales, this method avoids subjective deception by the subjects, providing a more objective, truthful, and stable method for diagnosing adolescent depression. Based on this, the present invention was completed.

[0048] the term Unless otherwise defined, 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 pertains.

[0049] As used herein, when referring to a specific enumerated value, the term “about” means that the value can vary by no more than 1% from the enumerated values. 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.).

[0050] As used herein, the terms “containing” or “including (comprise)” can be open-ended, semi-closed, or closed. In other words, the terms also include “consistently made of” or “composed of”.

[0051] As used in this article, the terms "room temperature" or "normal temperature" refer to a temperature between 4 and 40 degrees Celsius. o C, preferably, 25±5 o C.

[0052] The main advantages of this invention include: 1. The membrane lipidome described in this invention can objectively and truthfully reflect the health status of the subjects. Compared with clinical interviews and scales, it avoids subjective deception by the subjects, which may reduce misdiagnosis and missed diagnosis, and is more accurate.

[0053] 2. The membrane lipidome described in this invention is a combination of multiple membrane lipids, which has higher accuracy, precision and AUC value compared to single lipids.

[0054] 3. The concentration of membrane lipids described in this invention is more stable than that of plasma / serum lipids / metabolites, and is less susceptible to interference from confounding factors such as short-term diet, exercise, and drugs, making it a relatively more stable biomarker.

[0055] 4. The membrane lipid extraction method described in this invention is based on lipidomics and targets the extraction of erythrocyte membrane lipids. The method is simple and effective.

[0056] 4. This invention provides a method for quantifying erythrocyte membrane lipids. The method uses an external standard curve to perform absolute quantification of individual erythrocyte membrane lipids. Compared with peak area, it can more accurately reflect the lipid content in the body. Previous reports have not included studies on quantifying individual erythrocyte membrane lipids in adolescent patients with depression.

[0057] The invention is further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are by weight.

[0058] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as are familiar to those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to the methods of this invention. The preferred embodiments and materials described herein are for illustrative purposes only.

[0059] Example 1 This invention utilizes ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS / MS) to detect the lipidome of erythrocyte membranes and quantify the levels of specific lipids. The detailed operational steps are as follows: 1) Collect red blood cells: Collect 2 mL of venous blood into an EDTA anticoagulant tube, mix by inverting several times, centrifuge at 1600 g for 10 minutes, and collect the lower layer of red blood cells for later use.

[0060] 2) Extraction of red blood cell membranes: Red blood cells were treated with 20 times the volume of Tris-HCl (10 mM, pH 7.4) at 4°C overnight to fully swell the red blood cells, and then washed 3 times with PBS.

[0061] 3) Lipid extraction: Add extraction solution (methanol:chloroform, volume ratio 1:2) to the erythrocyte membrane precipitate, vortex for 5 minutes, transfer to a glass sample vial, and extract at 40℃ for 2 hours. Subsequently, vacuum centrifuge and dry at room temperature for 1 hour, dissolve the lipid extract in a reconstitution solution (dichloromethane:isopropanol:methanol, volume ratio 1:1:2), mix well, transfer to a 1.5mL centrifuge tube, centrifuge, and collect the supernatant for analysis.

[0062] 4) Preparation of lipid standards: Ceramide (Cer), phosphatidylserine (PS), phosphatidylcholine (PC), triglycerides (TG) and cholesterol esters (ChE) were quantified by Cer (d18:1 / 18:0), PS (17:0 / 17:0), PC (19:0 / 19:0), TG (17:0 / 17:1 / 17:0) D5 and cholesterol standards, respectively, and were prepared into the following concentration gradients using reconstitution solutions (Table 1).

[0063] Table 1. Concentration gradient of standards 5) Analytical Testing: Erythrocyte membrane lipids were detected using ultra-high performance liquid chromatography-quadrupole orbital trap mass spectrometry (Vanquish UHPLC / QExactive plus). An ACQUITY UPLC BEH C18 column (100 × 2.1 mm, 1.7 μm, Waters) was used with a column temperature set to 55℃. Mobile phase A consisted of acetonitrile and water at a volume ratio of 6:4, with the addition of 10 mM ammonium formate and 0.1% formic acid. Mobile phase B consisted of isopropanol and acetonitrile at a volume ratio of 9:1, with the addition of 10 mM ammonium formate and 0.1% formic acid. The flow rate was 400 μL / min. The elution gradient ranged from 95 / 5 to 0 / 100 over 17 min. Detection was performed in both positive and negative ion modes, with the following operating parameters: spray voltage of 3.8 kV (positive ion mode) and 3.0 kV (negative ion mode); capillary temperature after heating of 320 ℃; data-dependent acquisition mode was used, with one full scan and six MS / MS scans; full scan range: 150-2000 amu; full scan resolution: 70000; automatic gain control (AGC) < 1×10⁻⁶. 6 Scan time: 0.1 s; MS / MS scan resolution: 17500, AGC <5×10⁻⁶ 5 The scan time was 0.05 s. Concentration curves for the standards were established.

[0064] 5) Lipid quantification: The peak areas of Cer (d19:1_24:1), Cer (d16:1_16:1), PS (22:4_18:2), PC (22:5_18:2), PC (18:2_20:4), PC (18:1e_20:3), TG (18:4_18:1_18:2) and ChE (18:1) were extracted and quantified using standards of their respective categories. Cer(d19:1_24:1) was quantified using the Cer(d18:1 / 18:0) standard curve in positive ion mode, and Cer(d16:1_16:1) was quantified using the Cer(d18:1 / 18:0) standard curve in negative ion mode. PS(22:4_18:2), PC(22:5_18:2), PC(18:2_20:4), PC(18:1e_20:3), TG(18:4_18:1_18:2) and ChE(18:1) were quantified using their respective types of standards. The concentrations of these eight lipids are represented as c1 to c8, respectively.

[0065] Using the concentrations of these eight lipids as independent variables, a logistic regression model was established, as shown in formula ① below: …① Following the method described above, the concentrations of these eight lipids in the erythrocyte membrane of each subject were detected and calculated. Substituting these concentrations into formula ①, the results were obtained. P This represents the risk value for adolescent depression in the subjects. P The higher the value, the higher the risk level.

[0066] Depending on the specific application scenario, an appropriate option can be selected. P Value threshold. Preferably, when P When the value is >0.396, the subject is considered to have adolescent depression. Figure 2 The AUC was 0.971.

[0067] Meanwhile, a logistic regression model was established for the control group using the concentration of a single lipid as the independent variable, yielding the following curves. The AUCs were: Cer(d19:1_24:1) 0.808, Cer(d16:1_16:1) 0.813, PS(22:4_18:2) 0.806, PC(22:5_18:2) 0.797, PC(18:2_20:4) 0.806, PC(18:1e_20:3) 0.825, TG(18:4_18:1_18:2) 0.801, and ChE(18:1) 0.748. It can be seen that the AUC of any single lipid is lower than 0.971 (for the lipid group), therefore, the diagnostic potential of these eight erythrocyte membrane lipid combinations is significantly better than that of any single lipid.

[0068] Example 2 Evaluation of the detection effect of membrane lipidomics Based on data from 81 adolescent patients with depression and 67 healthy controls, we calculated that... P Table 2 shows the identification results of membrane lipidomics for adolescent patients with depression under the condition of >0.396.

[0069] Table 2 Summary of Statistical Results Based on the data in the table above, the concordance rate between membrane lipidomics and clinical diagnostic results was calculated using the following aspects: 1. Sensitivity, specificity, and accuracy Sensitivity is the ability of the membrane liposome to correctly identify adolescent patients with depression. A higher value indicates that the membrane liposome is more effective in detecting adolescent patients with depression. The calculation method is: Sensitivity = number of true positives / (number of true positives + number of false negatives).

[0070] In this invention, the sensitivity of the membrane lipidome is 77 / 81 = 95.1%, which is high.

[0071] Specificity measures the ability of the membrane lipidome to correctly identify healthy controls. A higher value indicates that the membrane lipidome is more effective in excluding healthy controls. Specificity is calculated as: Specificity = number of true negatives / (number of true negatives + number of false positives).

[0072] In this invention, the specificity of the membrane lipidome is 58 / 67 = 86.6%, which is high.

[0073] Accuracy measures the proportion of samples correctly classified by the membrane lipidomic diagnostic model. It is calculated as: Accuracy = (Number of true positives + Number of true negatives) / Number of samples.

[0074] In this invention, the accuracy of the membrane lipidome is (58 + 77) / 148 = 91.2%, which is high.

[0075] 2. Kappa Consistency Analysis The Kappa coefficient is a statistic used to assess the consistency of classifiers in classification problems, particularly the consistency between classification results and actual conditions. A Kappa coefficient > 0.8 indicates very good consistency between the membrane lipidomics and clinical diagnosis; 0.6 < Kappa coefficient ≤ 0.8 indicates high consistency between the two methods; 0.4 < Kappa coefficient ≤ 0.6 indicates moderate consistency; 0.2 < Kappa coefficient ≤ 0.4 indicates general consistency; and a Kappa coefficient ≤ 0.2 indicates very poor consistency.

[0076] The formula for calculating the Kappa coefficient is as follows: Kappa = (Po - Pe) / (1 - Pe) Where Po represents observation consistency and Pe represents observation consistency.

[0077] The calculation methods for Po and Pe are as follows: Po = (58 + 77) / 148 = 91.2% Pe = (62 * 67 + 86 * 81) / (148 * 148) = 50.8% Therefore, the Kappa coefficient = (91.2% - 50.8%) / (1 - 50.8%) = 0.821, indicating that the membrane lipidome has a very high consistency with clinical diagnosis.

[0078] 3. Matthews correlation coefficient The Matthews correlation coefficient is one of the metrics used to evaluate the performance of binary classification models. It considers four different prediction outcomes: true positive, true negative, false positive, and false negative, and provides a balanced performance evaluation across all classes. The Matthews correlation coefficient ranges from -1 to +1, where +1 indicates perfect predictive consistency, 0 indicates predictions are no better than random predictions, and -1 indicates complete mismatch. Values ​​closer to 1 indicate better classification performance. The calculation method is as follows: Therefore, the Matthews correlation coefficient between membrane lipidomics and clinical diagnosis was 0.824, indicating that the membrane lipidomics diagnostic model performed well in identifying adolescent patients with depression.

[0079] In summary, the membrane lipidome provided by this invention exhibits high sensitivity, high specificity, and high accuracy in diagnosing adolescent depression, and shows extremely high consistency with clinical diagnostic results and excellent classification performance.

[0080] Example 3 Evaluation of the detection effect of membrane lipidomics Another group of volunteers (40 adolescent patients with depression and 40 healthy controls) was recruited as a validation set. The detection method in Example 1 was used to quantitatively detect the above 8 lipid molecules. The results showed that these molecules had statistically significant differences between patients and healthy controls.

[0081] Based on the test results and the diagnostic model constructed in Example 1, the risk of depression for each volunteer in this sample set was calculated, and the ROC curve is shown below. Figure 3 As shown.

[0082] Using the threshold in Example 1 P > 0.396, further calculations of the membrane lipidome for identifying adolescent patients with depression are shown in Table 3 below.

[0083] Table 3 Summary of Statistical Results of the Validation Set Based on the data in the table above, the concordance rate (sensitivity, specificity, and accuracy) between membrane lipidomics and clinical diagnostic results was calculated using the following aspects: Sensitivity is the ability of the membrane liposome to correctly identify adolescent patients with depression. A higher value indicates that the membrane liposome is more effective in detecting adolescent patients with depression. The calculation method is: Sensitivity = number of true positives / (number of true positives + number of false negatives).

[0084] In this invention, the sensitivity of the membrane lipidome is 37 / 40 = 92.5%, which is high.

[0085] Specificity measures the ability of the membrane lipidome to correctly identify healthy controls. A higher value indicates that the membrane lipidome is more effective in excluding healthy controls. Specificity is calculated as: Specificity = number of true negatives / (number of true negatives + number of false positives).

[0086] In this invention, the specificity of the membrane lipidome is 35 / 40 = 87.5%, which is high.

[0087] Accuracy measures the proportion of samples correctly classified by the membrane lipidomic diagnostic model. It is calculated as: Accuracy = (Number of true positives + Number of true negatives) / Number of samples.

[0088] In this invention, the accuracy of the membrane lipid group is (58 + 77) / 80 = 90%, which is high.

[0089] Furthermore, the sensitivity, specificity, and accuracy of this validation set are comparable to those of the test set in Example 1, indicating that the model has high robustness and repeatability.

[0090] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A method for diagnosing and treating adolescent depression without diagnosis or treatment, characterized in that, The method includes the steps of: detecting the expression level of membrane lipids isolated from the erythrocyte membrane sample of the test subject, thereby determining whether the test subject is an adolescent with depression; and The membrane lipids are selected from the group consisting of ceramide (Cer), phosphatidylserine (PS), phosphatidylcholine (PC), triglycerides (TG), and cholesterol esters (ChE).

2. The method as described in claim 1, characterized in that, The erythrocyte membrane lipids were selected from the following group: Cer(d19:1_24:1), Cer(d16:1_16:1), PS(22:4_18:2), PC(22:5_18:2), PC(18:2_20:4), PC(18:1e_20:3), TG(18:4_18:1_18:2) and ChE(18:1).

3. The method as described in claim 1, characterized in that, The method further includes: after determining the concentration of the membrane lipids in the sample, calculating using the above concentration... P The value is used to draw a judgment conclusion.

4. The method as described in claim 3, characterized in that, The P value is calculated using the logical model shown in equation ① below: ①； The concentration of Cer(d19:1_24:1) is c1; The concentration of Cer(d16:1_16:1) is c2; The concentration of PS (22:4_18:2) is c3; The concentration of PC (22:5_18:2) is c4; The concentration of PC (18:2_20:4) is c5; The concentration of PC(18:1e_20:3) is c6; The concentration of TG (18:4_18:1_18:2) is c7; The concentration of ChE(18:1) is c8.

5. The method as described in claim 3, characterized in that, The testing standard for the product is: when the subject... P A value > 0.396 indicates a diagnosis of adolescent depression.

6. The method as described in claim 3, characterized in that, The concentration of the membrane lipids in the sample was determined using liquid chromatography-mass spectrometry.

7. A test kit for detecting depression in adolescents, characterized in that, The kit includes: A sample pretreatment reagent, wherein the sample pretreatment reagent is used to extract erythrocyte membrane lipids from blood samples; The erythrocyte membrane lipidomic assay reagent is used to detect the expression level of membrane lipids isolated from erythrocyte membrane samples of the test subject.

8. The detection kit as described in claim 7, characterized in that, The sample pretreatment reagents include: Red blood cell membrane extraction reagent; preferably, the cell membrane extraction reagent is Tris-HCl; Lipid extraction reagent; preferably, the lipid extraction reagent includes a lipid extraction solution (methanol:chloroform volume ratio of 0.1~2:1~5) and a lipid reconstitution solution (dichloromethane:isopropanol:methanol volume ratio of 0.1~2:0.1~2:1~5).

9. The detection kit as described in claim 7, characterized in that, The kit also includes an instruction manual, which states: The expression levels of membrane lipids isolated from erythrocyte membrane samples of the test subjects were detected to determine whether the test subjects were adolescents with depression; and The membrane lipids are selected from the group consisting of ceramide (Cer), phosphatidylserine (PS), phosphatidylcholine (PC), triglycerides (TG), and cholesterol esters (ChE).

10. The detection kit as described in claim 9, characterized in that, The erythrocyte membrane lipids were selected from the following group: Cer(d19:1_24:1), Cer(d16:1_16:1), PS(22:4_18:2), PC(22:5_18:2), PC(18:2_20:4), PC(18:1e_20:3), TG(18:4_18:1_18:2) and ChE(18:1).

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