Application of plasma metabolism marker in early diagnosis kit for adrenocorticoma

Abstract

The invention discloses application of a plasma metabolism marker in an early diagnosis kit for adrenal corticoma. The plasma metabolism marker comprises any one or more of uric acid, isocitric acid or proline. The accuracy of any one or combination of uric acid, isocitric acid or proline for distinguishing healthy subjects from adrenal corticoma patients is 95% or above; therefore, the marker has high diagnosis accuracy on adrenocorticoma and can be used for preparing a kit for early diagnosis of adrenocorticoma of healthy people.

Description

Technical Field

[0001] The invention belongs to the field of disease detection and diagnosis, and in particular relates to the application of a plasma metabolic marker in an early diagnosis kit for adrenal cortical adenoma. Background Art

[0002] Adrenal cortical adenoma is a benign tumor of the adrenal cortex, which usually causes excessive secretion of adrenal cortical hormones, leading to a variety of pathophysiological abnormalities in the human body. As the tumor grows, it compresses surrounding organs and blood vessels, causing pain or other local compression symptoms, and there is a risk of developing into a malignant tumor. Early diagnosis and appropriate treatment of adrenal cortical adenoma are very important, which can avoid many potential complications and further development of the tumor.

[0003] At present, the diagnosis of adrenal cortical adenoma mainly adopts a combination of imaging examination and endocrine examination. Imaging examination mainly includes ultrasound, CT, MRI and PET examination, which can explain the occurrence and development of the disease through morphological characteristics, density and enhancement. However, this diagnostic method is prone to false positive results and cannot accurately distinguish adrenal cortical adenoma from adrenal cortical adenocarcinoma, resulting in many patients requiring additional imaging examinations or even unnecessary surgery. Endocrine examination mainly determines the endocrine function of the tumor through biochemical analysis. Diagnostic methods include endocrine examination of hypercortisolism and endocrine examination of primary aldosteronism. However, these biochemical detection methods do not have a good diagnostic accuracy and are not suitable for all populations. In addition, some biochemical indicators such as plasma aldosterone / renin activity ratio have poor sensitivity, hormones are easily affected by external factors, and test results are unstable. So far, there is still no clear method and means to distinguish whether patients with adrenal cortical adenoma need surgery and the type of adrenal tumor.

[0004] Metabolomics is a research method that simultaneously conducts qualitative and quantitative analysis of all low-molecular-weight metabolites in a certain biological or cell during a specific physiological period to find the relative relationship between metabolites and physiological and pathological changes. It is a component of systems biology and involves multiple disciplines such as life sciences, analytical sciences, and statistics. Based on the advantages of high throughput and high sensitivity of metabolomics technology, it plays an extremely important role in many fields such as new drug development, drug efficacy evaluation, early disease diagnosis, and personalized treatment. Among them, plasma metabolome analysis is an important branch of metabolomics research and a commonly used disease diagnosis method. It is widely used because of its advantages of non-invasiveness, simplicity, high sensitivity, and specificity.

[0005] At present, the incidence of adrenal cortical adenoma is increasing year by year. We hope to achieve early screening, early diagnosis and timely treatment for this disease. In this case, we urgently need to research and develop markers with higher accuracy, sensitivity and specificity, and plasma metabolic marker diagnosis also shows obvious advantages. Summary of the invention

[0006] Purpose of the invention: In order to solve the above technical problems, the present invention aims to provide an application of a plasma metabolic marker in an early diagnosis kit for adrenal cortical adenoma, thereby solving the problem of poor sensitivity and low accuracy in early diagnosis of adrenal cortical adenoma.

[0007] Technical solution: In order to achieve the above-mentioned purpose, a plasma metabolic marker described in the present invention is used as an early diagnostic marker for adrenal cortical adenoma, and the plasma metabolic marker includes any one or more of uric acid, isocitrate or proline.

[0008] The plasma metabolic markers of the present invention are used in the preparation of a diagnostic kit for early diagnosis of adrenal cortical adenoma in healthy people, and the plasma metabolic markers include any one or more of uric acid, isocitrate or proline.

[0009] Furthermore, the diagnostic kit includes standard products of any one or more of the plasma metabolic markers uric acid, isocitrate or proline.

[0010] Furthermore, the diagnostic kit includes a standard substance of an internal standard compound.

[0011] Furthermore, the diagnostic kit includes L-2-chlorophenylalanine and ketoprofen.

[0012] The present invention discloses an adrenal cortical adenoma early diagnosis kit, which comprises any one or more of uric acid, isocitric acid or proline and other reagents required by the kit.

[0013] Furthermore, the diagnostic kit includes standard products of any one or more of the uric acid, isocitrate or proline.

[0014] Furthermore, the kit includes a standard substance of an internal standard compound.

[0015] Furthermore, the kit comprises L-2-chlorophenylalanine and ketoprofen.

[0016] The invention discloses an application of the early diagnosis kit for adrenal cortical adenoma in preparing a reagent or medicine for early diagnosis of adrenal cortical adenoma.

[0017] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:

[0018] The present invention proposes for the first time a plasma metabolic marker uric acid, isocitrate or proline as an early diagnostic marker for adrenal cortical adenoma. The plasma metabolic marker uric acid, isocitrate or proline can be used to prepare an early diagnostic kit for adrenal cortical adenoma, which can accurately distinguish patients with adrenal cortical adenoma and reduce the misdiagnosis rate. The accuracy of uric acid, isocitrate and proline in distinguishing healthy subjects from patients with adrenal cortical adenoma is more than 95%. The operation is simple, the cost is low, and the detection can be completed in a short time, providing timely diagnosis basis for clinicians. It can be seen that the above markers have high diagnostic accuracy for adrenal cortical adenoma and can be used to prepare a kit for early diagnosis of healthy people suffering from adrenal cortical adenoma. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 is the relative content of uric acid, isocitrate, and proline in plasma samples of healthy subjects and patients with adrenocortical adenoma in the training set;

[0020] Figure 2 To validate the relative contents of uric acid, isocitrate, and proline in plasma samples of healthy subjects and patients with adrenocortical adenoma;

[0021] Figure 3 ROC curve for the diagnosis of healthy subjects and patients with adrenocortical adenoma using uric acid alone in the training set;

[0022] Figure 4 To verify the ROC curve of uric acid alone in differentiating healthy subjects from patients with adrenocortical adenoma;

[0023] Figure 5 ROC curve for the diagnostic ability of isocitrate alone to distinguish healthy subjects from patients with adrenocortical adenoma for the training set;

[0024] Figure 6 To verify the ROC curve of isocitrate alone for distinguishing healthy subjects from patients with adrenocortical adenoma;

[0025] Figure 7 ROC curve for the diagnostic discrimination of healthy subjects and patients with adrenocortical adenoma by proline alone in the training set;

[0026] Figure 8 ROC curve for the validation set proline alone to distinguish healthy subjects from patients with adrenocortical adenoma;

[0027] Fig. 9 ROC curve for the diagnosis of uric acid and proline combination in the training set to distinguish between healthy subjects and patients with adrenocortical adenoma;

[0028] Fig.10To verify the ROC curve of the combination of uric acid and proline for distinguishing healthy subjects from patients with adrenocortical adenoma;

[0029] Fig.11 ROC curve for the diagnosis of proline and isocitrate combination in the training set to distinguish healthy subjects from patients with adrenocortical adenoma;

[0030] Fig.12 ROC curve for the validation set proline and isocitrate combination to distinguish healthy subjects from patients with adrenocortical adenoma;

[0031] Fig.13 ROC curve for the diagnosis of uric acid and isocitrate combination in the training set to distinguish healthy subjects from patients with adrenocortical adenoma;

[0032] Fig.14 To validate the ROC curve of the combined diagnosis of uric acid and isocitrate to distinguish healthy subjects from patients with adrenocortical adenoma;

[0033] Fig.15 ROC curve for the combination of uric acid, isocitrate and proline in the training set to distinguish healthy subjects from patients with adrenocortical adenoma;

[0034] Fig.16 ROC curve for the combined diagnosis of uric acid, isocitrate and proline to differentiate healthy subjects from patients with adrenocortical adenoma. DETAILED DESCRIPTION

[0035] The technical solution of the present invention is further described below in conjunction with the accompanying drawings.

[0036] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources. Experimental methods without specific conditions specified in the examples are usually carried out under conventional conditions or under conditions recommended by the manufacturer.

[0037] Main reagents: methanol and acetonitrile (UPLC pure) were purchased from Merck, Germany, HPLC grade formic acid and ammonium acetate were purchased from ROE, USA; deionized water was prepared by the Mlli-Q ultrapure water system of Millipore, USA; standard products including uric acid (CAS: 69-93-2) and proline (CAS: 147-85-3, L-proline) were purchased from Sigma-Aldrich, USA, and isocitric acid (CAS: 320-77-4) was purchased from Shanghai Zhenzhun Biotechnology Co., Ltd. The internal standard L-2-chlorophenylalanine (CAS: 103616-89-3) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., and ketoprofen (CAS: 22071-15-4) was purchased from Sigma-Aldrich, USA.

[0038] Written informed consent was obtained from all subjects before participating in the study.

[0039] The inclusion criteria for healthy subjects and patients with adrenal cortical adenoma are as follows:

[0040] Healthy subjects: no cardiovascular, respiratory, liver, kidney, gastrointestinal, endocrine, blood, mental, or nervous system diseases, no history of the above diseases, no acute or chronic diseases. No history of drug allergy, and clinical laboratory test results are within the normal reference range during screening.

[0041] Adrenal cortical adenoma patients: patients who have undergone preoperative examination and surgery and have a pathological diagnosis after surgery;

[0042] Training set samples: 146 healthy subjects and 100 patients with adrenal cortical adenoma were collected from the First Affiliated Hospital of Zhejiang University and matched by age and gender according to strict screening and exclusion criteria.

[0043] Validation set samples: 154 healthy subjects and 121 adrenal cortical adenoma patients were collected from the First Affiliated Hospital of Zhejiang University according to strict screening and exclusion criteria.

[0044] Example 1

[0045] The diagnostic performance of target metabolite diagnostics in differentiating healthy subjects from patients with adrenocortical adenoma

[0046] 1. Collection and storage of plasma samples

[0047] Fasting peripheral blood was collected from patients with adrenocortical adenoma and healthy subjects in the early morning and collected into tubes containing anticoagulant sodium citrate solution. All plasma samples were carefully divided into sterile lyophilized tubes, labeled and stored in a -80℃ refrigerator for later use.

[0048] 2. Sample preparation

[0049] Place the plasma sample in step 1 in a 4°C refrigerator for 30 minutes to slowly thaw. Take 50μL of plasma from each sample in a 1.5mL centrifuge tube, add 150μL of methanol containing 100ng / mL L-2-chlorobenzene and 1μg / mL ketoprofen, vortex for 30s to mix, put it in a low-temperature high-speed centrifuge, and centrifuge it at 13000rpm and 4°C for 10min. After centrifugation, take 150μL of the supernatant and divide it equally into 2 1.5mL centrifuge tubes for nitrogen blowing. After the supernatant is blown dry, add 70μL of methanol to re-dissolve, then vortex for 30s to mix, put it in a low-temperature high-speed centrifuge, and centrifuge it at 13000rpm and 4°C for 10min. After centrifugation, aspirate 50μL of the supernatant for injection. In addition, take 10μL of each sample and mix it as a quality control sample. Cover and put it in the LC-MS instrument to prepare for positive and negative ion detection. The ratio of the peak area of ​​each target metabolite, uric acid, isocitrate and proline, to the peak area of ​​the internal standard was calculated as the relative content of the metabolite.

[0050] 3. Determination of relative content of target metabolites in serum by UPLC-Q / TOF-MS

[0051] Detection instrument: Agilent 1290 ultra-high performance liquid chromatography-6545 quadrupole-time-of-flight mass spectrometer Chromatographic conditions: Chromatographic column: Waters BEH C8 (100mm×2.1mm ID, 1.7μm); positive ion mobile phase acetonitrile (B)-water (A) solution (both containing 0.1% formic acid); negative ion mobile phase methanol (B)-water (A) solution (both containing 5mmol / L ammonium acetate); flow rate: 0.4mL / min; column temperature: 50℃. Mobile phase ratio: 0-1min, 5%B; 1-4min, 5%-30%B; 4-9min, 30%-90%B; 9-10min, 90%-100%B; 10-12min, 100%B. The running time is 12 minutes, and the post-running time is 3 minutes.

[0052] Mass spectrometry conditions: collision voltage of 120 V, cone voltage of 65 V, Oct 1RF Vpp voltage of 750 V; drying gas temperature of 350 ° C, drying gas flow rate of 12 L / min, nebulizer pressure of 35 psi, sheath gas temperature of 375 ° C, sheath gas flow rate of 12 L / min, both drying gas and cone gas are high-purity nitrogen; capillary voltage is 3500 V, nozzle voltage is 1500 V; ion source temperature is 100 ° C.

[0053] 4. Data processing methods

[0054] Unsupervised principal component analysis (PCA) and supervised orthogonal partial least squares discriminant analysis (OPLS-DA) were used to obtain clustering information and differential metabolites for metabolite data. FDR (false discovery rate) was used to correct the p value for univariate statistical analysis between metabolites. Variables that simultaneously satisfied VIP (Variable Importance in Projection)>1 and P<0.05 were selected as differential metabolites. The primary and secondary spectra of differential metabolic peaks were identified and compared using the Human Metabolome Database and Agilent PCDLManagerB.07.00 database. For the screened differential metabolites, the contribution of three metabolites to adrenal cortical adenoma and normal samples was evaluated using the Least Absolute Shrinkage and Selection Operator (LASSO) model. The Area Under Curve (AUC) values ​​of multi-metabolite combination and single metabolite for the diagnosis of adrenal cortical adenoma were evaluated using the Receiver Operating characteristic Curve (ROC) curve.

[0055] 5. Experimental results

[0056] (1) Differences in plasma levels of target metabolites in healthy subjects and patients with adrenocortical adenoma

[0057] like Figure 1 , Figure 2 As shown, compared with healthy subjects, the relative content of the target metabolite uric acid in the plasma samples of patients with adrenal cortical adenoma in the training set and validation set was significantly downregulated, and the relative content of the target metabolite isocitrate or proline in the plasma samples of patients with adrenal cortical adenoma was significantly upregulated.

[0058] (2) ROC curve of target metabolites alone for distinguishing healthy subjects from patients with adrenocortical adenoma

[0059] The ROC curve results of uric acid, isocitrate, and proline for distinguishing healthy subjects from patients with adrenocortical adenoma are shown in Figure 2. Figure 3-8 As shown in Table 2 and Table 3, Figure 3 , Figure 4 As shown in Figure 2, the areas under the ROC curve for uric acid diagnosis in differentiating healthy subjects from adrenocortical adenoma patients in the training set and validation set were 0.822 and 0.798, respectively. Figure 5 , Figure 6 As shown in Figure 2, the area under the ROC curve of isocitrate for distinguishing healthy subjects from patients with adrenocortical adenoma was 0.959 and 0.986, respectively. Figure 7 , Figure 8 As shown, the area under the ROC curve of proline diagnosis for distinguishing healthy subjects from adrenocortical adenoma patients is 0.865 and 0.794. Meanwhile, the abscissa value in each figure is 1-specificity.

[0060] The principle of ROC curve evaluation method:

[0061] The basic evaluation indicators of diagnostic tests include sensitivity, specificity, etc., and the comprehensive evaluation indicators include Youden index, ROC, AUC, etc. For the evaluation of diagnostic tests, it is first necessary to know the true group of the sample to be tested through the gold standard. For the disease group determined by the gold standard (equivalent to the adrenal cortical adenoma group in the present invention) and the healthy group (equivalent to the healthy subject group in the present invention), the results of the diagnostic test can be divided into the following situations:

[0062] True Positive (TP); the diagnostic test is positive (consistent with the gold standard result);

[0063] True Negative (TN); the diagnostic test was negative (consistent with the gold standard result);

[0064] False Positive (FP): The diagnostic test is positive (inconsistent with the gold standard result);

[0065] False Negative (FN): The diagnostic test is negative (inconsistent with the gold standard result).

[0066] It can be expressed in the following Table 1:

[0067] Table 1 Diagnostic test results

[0068]

[0069] The sensitivity of a diagnostic test = A / (A+C); the specificity of a diagnostic test = D / (B+D). The sensitivity and specificity can be used to determine the diagnostic sensitivity and specificity of a diagnostic test relative to the gold standard. A high sensitivity means that fewer disease cases are diagnosed as negative, and the missed diagnosis rate is low; a high specificity means that fewer healthy cases are diagnosed as positive, and the misdiagnosis rate is low.

[0070] The ROC curve is a curve drawn based on the above sensitivity and specificity. The possible diagnostic cutoff value in the diagnostic test is used as the diagnostic point, and the corresponding sensitivity and specificity are calculated according to the above table. Then, with sensitivity as the ordinate and 1-specificity as the abscissa, the sensitivity and specificity points of each diagnostic point are marked in the coordinate graph, and the coordinate points are connected to obtain a smooth curve, which is the ROC curve. The more and denser the diagnostic points are set, the smoother the ROC curve will be.

[0071] The ROC curve uses each test result as a possible diagnostic cutoff value, and the area under the curve (AUC) indicates the accuracy of the diagnostic test. The area under the ROC curve (AUC) has been widely recognized as an inherent accuracy indicator for evaluating the authenticity of diagnostic tests. When AUC is 0.5, it has no diagnostic significance; when AUC is between 0.5 and 0.7, it indicates a low diagnostic accuracy; when AUC is between 0.7 and 0.9, it indicates a medium diagnostic accuracy; when AUC>0.9, it indicates a high diagnostic accuracy. The higher the AUC, the higher the diagnostic accuracy.

[0072] Table 2 ROC curves for the target metabolites in the training set to distinguish healthy subjects from patients with adrenocortical adenoma

[0073] Metabolic markers AUC Sensitivity Specificity Uric acid 0.822 84.3％ 76.0％ Isocitrate 0.959 94.4％ 100％ Proline 0.865 85.4％ 74.0％

[0074] Table 3 ROC curves of the validation set target metabolites for distinguishing healthy subjects from adrenocortical adenoma patients

[0075] Metabolic markers AUC Sensitivity Specificity Uric acid 0.798 94.5％ 63.6％ Isocitrate 0.986 98.2％ 99.4％ Proline 0.794 68.2％ 83.1％

[0076] The ROC curve results showed that uric acid, isocitrate, and proline alone had high accuracy in distinguishing healthy subjects from patients with adrenocortical adenoma, among which isocitrate had the highest accuracy (AUC was 0.959 and 0.986 in the exploration set and validation set, respectively).

[0077] (3) ROC curve of the target metabolites for pairwise combination diagnosis and differentiation between healthy subjects and adrenocortical adenoma patients

[0078] The ROC curve results of uric acid, isocitrate, and proline in pairs for distinguishing healthy subjects from patients with adrenal cortical adenoma are shown in the figure. Figure 9-14 As shown in Table 4 and Table 5. Fig. 9 , Fig.10 As shown in Figure 2, the areas under the ROC curve for the combination of uric acid and proline in the training set and validation set for distinguishing healthy subjects from adrenocortical adenoma patients were 0.905 and 0.853, respectively. Fig.11 , Fig.12As shown in Figure 2, the area under the ROC curve of proline and isocitrate combined to distinguish healthy subjects from adrenocortical adenoma patients was 0.972 and 0.986, respectively. Fig.13 , Fig.14 As shown, the areas under the ROC curve of uric acid and isocitrate combined for distinguishing healthy subjects from adrenocortical adenoma patients were 0.981 and 0.993.

[0079] Table 4 ROC curves for the diagnosis and differentiation of healthy subjects and adrenocortical adenoma patients by pairwise combination of target metabolites in the training set

[0080] Metabolic markers AUC Sensitivity Specificity Uric acid, proline 0.905 83.1％ 90.4％ Proline, Isocitrate 0.972 95.5％ 97.9％ Uric acid, isocitrate 0.981 94.4％ 98.6％

[0081] Table 5 ROC curves for the diagnosis and differentiation of healthy subjects and adrenocortical adenoma patients by pairwise combination of target metabolites in the validation set

[0082]

[0083]

[0084] The ROC curve results showed that the combination of uric acid, isocitrate and proline had high accuracy in distinguishing healthy subjects from patients with adrenal cortical adenoma, among which the combination of uric acid and isocitrate had the highest accuracy (AUC was 0.981 and 0.993 in the exploration set and validation set, respectively).

[0085] (4) ROC curve of target metabolite combination diagnosis to distinguish healthy subjects from adrenocortical adenoma patients

[0086] The ROC curve results of the combination of uric acid, isocitrate, and proline for distinguishing healthy subjects from patients with adrenocortical adenoma are shown in Figure 2. Figure 15-16 In the training set, the AUC of uric acid, isocitrate, and proline combined to distinguish healthy subjects from patients with adrenal cortical adenoma was 0.985, with a sensitivity of 95.5% and a specificity of 97.9%. In the validation set, the AUC of uric acid, isocitrate, and proline combined to distinguish healthy subjects from patients with adrenal cortical adenoma was 0.993, with a sensitivity of 98.2% and a specificity of 99.4%. The ROC results showed that the combined diagnosis had extremely high accuracy in distinguishing healthy subjects from patients with adrenal cortical adenoma and had extremely high diagnostic value.

[0087] Example 2

[0088] Diagnostic kit for distinguishing healthy subjects from patients with adrenocortical adenoma

[0089] The invention discloses an early diagnosis kit for adrenal cortical adenoma in healthy people, comprising standard substances of diagnostic markers, which are uric acid, isocitrate and proline, and standard substances of internal standard compounds, L-2-chlorophenylalanine and ketoprofen.

[0090] Example 3

[0091] Diagnostic kit for distinguishing healthy subjects from patients with adrenocortical adenoma

[0092] The invention discloses an early diagnosis kit for adrenal cortical adenoma in healthy people, comprising a standard substance of a diagnostic marker, wherein the diagnostic marker is any one of uric acid, isocitrate and proline, and standard substances of internal standard compounds, L-2-chlorophenylalanine and ketoprofen.

Claims

1. A use of a plasma metabolic marker as an early diagnostic marker for adrenal cortical adenoma, wherein the plasma metabolic marker comprises any one or more of uric acid, isocitrate or proline.

2. Use of a plasma metabolic marker in the preparation of a diagnostic kit for early diagnosis of adrenal cortical adenoma in healthy people, wherein the plasma metabolic marker includes any one or more of uric acid, isocitrate or proline.

3. The application according to claim 2, characterized in that: The diagnostic kit comprises standard products of any one or more of the plasma metabolic markers uric acid, isocitrate or proline.

4. The application according to claim 2, characterized in that: The diagnostic kit includes a standard substance of an internal standard compound.

5. The use according to claim 2, characterized in that: The diagnostic kit comprises L-2-chlorophenylalanine and ketoprofen.

6. An early diagnosis kit for adrenal cortical adenoma, characterized in that: The kit comprises any one or more of uric acid, isocitrate or proline and other reagents required by the kit.

7. The use according to claim 6, characterized in that: The diagnostic kit comprises any one or more standard products of uric acid, isocitrate or proline.

8. The use according to claim 6, characterized in that: The kit includes a standard of an internal standard compound.

9. The use according to claim 6, characterized in that: The kit comprises L-2-chlorophenylalanine and ketoprofen.

10. Use of the early diagnosis kit for adrenal cortical adenoma according to claim 6 in preparing a reagent or a medicine for early diagnosis of adrenal cortical adenoma.

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