Metabolic markers for distinguishing metabolic syndrome from simple obesity and uses thereof

Abstract

The application discloses a metabolic marker for distinguishing metabolic syndrome and simple obesity and application thereof. The marker comprises eight metabolites, i.e. glycocholic acid, lithocholic acid, linoleic acid, valine, leucine, alanine, serine and glycine. The application performs non-targeted metabolomics analysis on serum of metabolic syndrome and simple obesity patients by liquid chromatography mass spectrometry, finds eight metabolites with significant difference between the two, and finds that the eight metabolic markers have high AUC when used for diagnosing and distinguishing metabolic syndrome patients or simple obesity patients alone or in combination.

Description

TECHNICAL FIELD

[0001] The present application relates to a metabolic marker for distinguishing metabolic syndrome and simple obesity and application thereof, and belongs to the field of medical biotechnology. BACKGROUND

[0002] Metabolic syndrome (MS) is a metabolic abnormality syndrome caused by factors such as rapid lifestyle and dietary changes, and often occurs in clusters. Metabolic syndrome is a group of diseases including obesity, hyperglycemia, hypertension, dyslipidemia and other metabolic abnormalities, which seriously endangers human health. The combination of metabolic-related risk factors centered on obesity and insulin resistance promotes the occurrence of diseases such as atherosclerotic heart disease and type 2 diabetes. However, the diagnosis and treatment of metabolic syndrome rely on each component, and the early whole clinical symptoms are not obvious, the early diagnosis rate is low, and there is a lack of effective prevention means. Therefore, early diagnosis of metabolic syndrome is crucial.

[0003] Simple obesity is a common social chronic disease in clinical practice, which is mainly caused by excessive accumulation of fat due to more heat intake than consumption in the human body, resulting in body mass exceeding standard body mass by more than 20%, and fat percentage exceeding 30%. The risk of cardiovascular and cerebrovascular diseases, metabolic diseases and other diseases in patients with simple obesity is significantly higher than that in normal body mass population, and timely treatment, weight loss and control of body mass increase are needed. Diet control, exercise and the like have certain therapeutic effect on simple obesity, but it is difficult to adhere to, and rebound is easy to occur. Western medicine treatment of simple obesity mainly through drug treatment has certain effect, but some patients may have drug adverse reactions, and rebound after drug withdrawal.

[0004] The MS diagnostic criteria are: 3 or more of the following 5 items: (1) hypertension: ≥130 / 85 mmHg; (2) hyperglycemia: fasting blood glucose ≥6.1 mmol / L; (3) fasting triglycerides: ≥1.7 mmol / L; (4) fasting high-density lipoprotein cholesterol (HDL-C) <1.04 mmol / L; (5) central obesity: waist circumference >0.9 m for men and >0.85 m for women.

[0005] Metabolomics is a research method for quantitatively analyzing all metabolites in the body and finding the relative relationship between metabolites and physiological and pathological changes, which is a part of systems biology, involving knowledge of life sciences, analytical sciences and statistics, and has played an extremely important role in many fields. It has been widely used in new drug research and development, drug toxicity research and safety evaluation, clinical pathophysiology research, early diagnosis of diseases, personalized treatment and other fields and has achieved scientific research results.

[0006] Serum analysis is a commonly used method for disease diagnosis in clinic, which is widely used due to its convenience, rapidness, economy and relatively non-invasive advantages. In recent years, there are many studies on serum markers of metabolic syndrome, and many markers have been screened out. However, most of the markers still lack specificity and sensitivity, and few studies will verify the screened markers, because the time cost and economic cost of verification are very high, so the accuracy of the results cannot be guaranteed.

[0007] Therefore, in order to improve the early diagnosis rate of metabolic syndrome in obese population, it is urgent to develop markers with higher accuracy, higher sensitivity and higher specificity, and serum metabolic markers have obvious advantages in diagnosis convenience, economy and minimally invasive. SUMMARY

[0008] The purpose of the application is to provide a metabolic marker for distinguishing metabolic syndrome and simple obesity.

[0009] Technical scheme: The metabolic marker for distinguishing metabolic syndrome and simple obesity of the application comprises one or more of the following eight metabolites: glycocholic acid, lithocholic acid, linoleic acid, valine, leucine, alanine, serine and glycine.

[0010] Further, the marker is glycocholic acid, lithocholic acid, linoleic acid, valine, leucine, alanine, serine and glycine.

[0011] The above marker of the application is used in the preparation of a kit for detecting metabolic syndrome.

[0012] Further, the kit distinguishes metabolic syndrome and simple obesity by detecting the relative content change of the metabolic marker in the serum of the subject.

[0013] Further, the kit detects the relative content change of the serum metabolic marker based on liquid chromatography.

[0014] Further, compared with simple obesity patients, the relative contents of glycocholic acid, lithocholic acid, linoleic acid, valine, leucine and alanine in the serum of metabolic syndrome patients are significantly increased, and the relative contents of serine and glycine are significantly decreased.

[0015] Further, the liquid chromatography detection uses 0.01% formic acid aqueous solution as mobile phase A, and acetonitrile / methanol+0.01% formic acid with a volume ratio of 87:13 as mobile phase B.

[0016] Further, the liquid chromatography detection elution condition is 0-1 min, 5% B; 1-5 min, 5%-30% B; 5-15 min, 30%-90% B; 15-17.5 min, 90%-100% B; 17.5-20 min, 100% B.

[0017] Advantages: Compared with the prior art, the present application has the following outstanding advantages: the present application finds 8 metabolites with significant differences between metabolic syndrome and simple obesity patients through liquid chromatography-mass spectrometry detection of serum of metabolic syndrome and simple obesity patients for non-targeted metabolomics analysis. The 8 metabolic markers have high AUC in diagnosing and distinguishing metabolic syndrome patients or simple obesity patients alone or in combination. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 ROC curve for diagnosing and distinguishing metabolic syndrome patients or simple obesity patients alone with glycolcholic acid;

[0019] Figure 2 ROC curve for diagnosing and distinguishing metabolic syndrome patients or simple obesity patients alone with lithocholic acid;

[0020] Figure 3 ROC curve for diagnosing and distinguishing metabolic syndrome patients or simple obesity patients alone with linoleic acid;

[0021] Figure 4 ROC curve for diagnosing and distinguishing metabolic syndrome patients or simple obesity patients alone with valine;

[0022] Figure 5 ROC curve for diagnosing and distinguishing metabolic syndrome patients or simple obesity patients alone with leucine;

[0023] Figure 6 ROC curve for diagnosing and distinguishing metabolic syndrome patients or simple obesity patients alone with alanine;

[0024] Figure 7 ROC curve for diagnosing and distinguishing metabolic syndrome patients or simple obesity patients alone with serine;

[0025] Figure 8 ROC curve for diagnosing and distinguishing metabolic syndrome patients or simple obesity patients alone with glycine;

[0026] Figure 9 ROC curve for diagnosing and distinguishing metabolic syndrome patients or simple obesity patients in combination with glycolcholic acid, lithocholic acid, linoleic acid, valine, leucine and alanine;

[0027] Figure 10ROC curve for glycine combined with serine to distinguish metabolic syndrome patients from simple obesity patients;

[0028] Figure 11 ROC curve for glycine combined with serine to distinguish metabolic syndrome patients from simple obesity patients. DETAILED DESCRIPTION

[0029] The technical solutions of the present application are further described below in combination with the drawings. The experimental methods not specified in the examples are generally performed according to conventional conditions, such as those described in textbooks and experimental guidelines, or according to the conditions recommended by the manufacturer.

[0030] Example 1 Diagnostic efficiency of target metabolites in diagnosing metabolic syndrome patients and simple obesity patients

[0031] I. Experimental samples

[0032] Training set samples: 30 cases of metabolic syndrome patients and 35 cases of simple obesity patients were collected in Huai'an area according to strict screening and exclusion criteria and age and gender matching.

[0033] The inclusion criteria for metabolic syndrome patients and simple obesity patients are as follows:

[0034] Metabolic syndrome patients: According to the diagnostic criteria recommended by the Chinese Medical Association Diabetes Branch (2017), 3 or more of the following 5 items: (1) hypertension: ≥ 130 / 85 mmHg or confirmed as hypertension and treated; (2) hyperglycemia: fasting blood glucose ≥ 6.1 mmol / L or 2h blood glucose after sugar load ≥ 7.8 mmol / L or confirmed as diabetes and treated; (3) fasting triglycerides: ≥ 1.7 mmol / L; (4) fasting high-density lipoprotein cholesterol (HDL-C) < 1.04 mmol / L; (5) central obesity: waist circumference > 0.9 m for men and > 0.85 m for women.

[0035] Simple obesity: The diagnostic criteria refer to the diagnostic criteria and grading standards for simple obesity in the Asia-Pacific region.

[0036] Main reagents: ethanol, acetonitrile, formic acid and acetic acid are all analytical pure and purchased from National Pharmaceutical Group Chemical Reagent Co., Ltd.

[0037] II. Experimental methods

[0038] 1. Collection and storage of serum samples

[0039] Collect the patient's peripheral blood in the morning on an empty stomach and place it in a test tube without anticoagulant. Allow it to coagulate naturally at room temperature for 30-60 min. Centrifuge at 2000 rpm for 10 min. Carefully aspirate the clear serum liquid into a sterile freeze-dried tube. Label and store in a -80°C freezer for future use.

[0040] 2. UPLC-Q / TOF-MS determination of the relative content of target metabolites in serum

[0041] Detection instrument: Agilent 1290 infinity / 6460 liquid chromatography triple quadrupole mass spectrometry, high-speed centrifuge, ultra-low temperature refrigerator, multi-tube vortex mixer.

[0042] Chromatographic conditions: The chromatographic column is Agilent ZORBAX Eclipse XDB-C18 liquid phase narrow diameter column 100x2.1 mm, 3.5 μm liquid chromatography column, the chromatographic column temperature is set to 45°C, the sample volume is 10 μL. Mobile phase A: 0.01% formic acid in water; mobile phase B: acetonitrile / methanol (87:13) + 0.01% formic acid. The flow rate is set to 0.45 mL / min. The proportion of mobile phase is 0-1 min, 5% B; 1-5 min, 5%-30% B; 5-15 min, 30%-90% B; 15-17.5 min, 90%-100% B; 17.5-20 min, 100% B. The running time is 20 min, and the post-running time is 3 min.

[0043] Mass spectrometry conditions: Quantitative test in negative ion mode, ion source ESI. Nitrogen is used as atomizing gas and cone gas. The specific mass spectrometry parameters are: cone voltage 55v, capillary voltage 2.5kv, extraction cone 4v, desolvation gas temperature 450℃, ion source temperature 120℃, reverse cone gas flow 50L / h, desolvation gas speed 800L / h, collision gas flow rate 0.15mL / min.

[0044] Sample processing: After thawing the serum sample, 50 μL was accurately transferred into a centrifuge tube, and 3 times the amount of acetonitrile 150 μL was added and mixed. After mixing, it was placed in a high-speed centrifuge and centrifuged at 13500 rpm for 10 min. 75 μL of supernatant after centrifugation was aspirated into two 1.5 ml centrifuge tubes. The supernatant was vacuum centrifuged to dryness, 100 μL of 50% methanol was added for reconstitution, and after reconstitution, it was placed in a high-speed centrifuge and centrifuged at 13500 rpm for 10 min. The supernatant after centrifugation was aspirated, and the supernatant was placed in a liquid chromatography vial for sample injection (sample injection amount was 1 μL).

[0045] 3. Data processing method

[0046] In the training set, the orthogonal partial least squares discriminant analysis (OPLS-DA) was used to compare whether there was a difference in the relative content of the target metabolites in the serum of the metabolic syndrome patients and the simple obesity patients, and VIP>1 and p<0.05 were considered to be statistically significant. The ROC curve of a single target metabolite was established, and the area under the curve (AUC) and the 95% confidence interval were calculated. For multiple target metabolites, a binary logistic regression equation was established to generate a new variable, and ROC curve analysis was performed on the new variable.

[0047] III. Experimental results

[0048] 1. Differences in the levels of target metabolites in the serum of metabolic syndrome patients and simple obesity patients

[0049] In the training set, the relative contents of glycocholic acid, lithocholic acid, linoleic acid, valine, leucine and alanine in the serum of the metabolic syndrome patients were significantly higher than those in the simple obesity patients, and the relative contents of serine and glycine were significantly lower.

[0050] 2. ROC curve of target metabolites for diagnosing and distinguishing metabolic syndrome patients or simple obesity patients

[0051] Principle of ROC curve evaluation method:

[0052] The basic evaluation indicators of diagnostic tests include sensitivity, specificity, etc., and the comprehensive evaluation indicators include Youden index, ROC, AUC, etc.

[0053] The ROC curve is a curve based on sensitivity and specificity. With the possible diagnostic cutoff value in the diagnostic test as the diagnostic point, the corresponding sensitivity and specificity are calculated according to the above table. Then, with sensitivity as the vertical coordinate and 1-specificity as the horizontal coordinate, the sensitivity and specificity of each point are marked on the coordinate graph, and the smooth curve is obtained by connecting the coordinate points. The ROC curve is obtained. The more and more dense the diagnostic points are set, the smoother the ROC curve obtained.

[0054] The ROC curve takes each test result as a possible diagnostic cutoff value, and the size of the area under the curve AUC indicates the size of the accuracy of the diagnostic test. The area under the ROC curve AUC as an inherent accuracy index for evaluating the authenticity of the diagnostic test has been widely recognized. When AUC is 0.5, it has no diagnostic significance; when AUC is between 0.5 and 0.7, it indicates that the diagnostic accuracy is low; when AUC is between 0.7 and 0.9, it indicates that the diagnostic accuracy is moderate; and when AUC>0.9, it indicates that the diagnosis has high accuracy.

[0055] The ROC curve results of glycoursodeoxycholic acid, stearic acid, linoleic acid, valine, leucine, alanine, serine and glycine alone in diagnosing and distinguishing metabolic syndrome patients or simple obesity population are shown in Figures 1 to 8 The ROC curve results of glycoursodeoxycholic acid, stearic acid, linoleic acid, valine, leucine and alanine in combination in diagnosing and distinguishing metabolic syndrome patients or simple obesity population are shown in Figure 9 The ROC curve results of serine and glycine in combination in diagnosing and distinguishing metabolic syndrome patients or simple obesity population are shown in Figure 10 The ROC curve results of glycoursodeoxycholic acid, stearic acid, linoleic acid, valine, leucine, alanine, serine and glycine in combination in diagnosing and distinguishing metabolic syndrome patients or simple obesity population are shown in Figure 11 .

[0056] The ROC curve results show that stearic acid, alanine and valine alone in diagnosing and distinguishing simple obesity population VS metabolic syndrome patients all have high accuracy (AUC is all above 0.9), in which the AUC of stearic acid is 0.943, the AUC of valine is 0.943, and the AUC of alanine is 0.977; glycoursodeoxycholic acid, linoleic acid, leucine, serine and glycine alone in diagnosing and distinguishing metabolic syndrome patients VS simple obesity population have moderate accuracy (AUC is all between 0.7-0.9), in which the AUC of glycoursodeoxycholic acid is 0.890, the AUC of linoleic acid is 0.893, the AUC of leucine is 0.845, the AUC of serine is 0.899, and the AUC of glycine is 0.891. Multiple target metabolites in combination all have high accuracy (AUC is all above 0.9), that is, the AUC of 6 metabolites of glycoursodeoxycholic acid, stearic acid, linoleic acid, valine, leucine and alanine in combination is 0.976, the AUC of 2 metabolites of serine and glycine in combination is 0.966, and the AUC of 8 metabolites of glycoursodeoxycholic acid, stearic acid, linoleic acid, valine, leucine, alanine, serine and glycine in combination in diagnosing and distinguishing metabolic syndrome patients VS simple obesity population is 0.990.

[0057] Example 2 Actual sample detection

[0058] A female obese patient was randomly selected, and the serum of the patient was detected. The results showed that the relative content of glycocholic acid was 4.69 mg / L (the reference value was 0.4-2.98 mg / L), the relative content of lithocholic acid was 3.45 μmol / L (the reference value was 0-0.27 μmol / L), the relative content of linoleic acid was 3.16 mmol / L (the reference value was 1.16-1.55 mmol / L), the relative content of valine was 273 μmol / L (the reference value was 207-242 μmol / L), the relative content of leucine was 402 μmol / L (the reference value was 75-175 mmol / L), the relative content of alanine was 1574 μmol / L (the reference value was 210-600 μmol / L), the relative content of serine was 33 μmol / L (the reference value was 65-193 μmol / L), and the relative content of glycine was 57 μmol / L (the reference value was 163.9-237.1 μmol / L), that is, the relative contents of glycocholic acid, lithocholic acid, linoleic acid, valine, leucine and alanine in the serum were higher than the normal values, and the relative contents of serine and glycine were lower than the normal values.

[0059] The blood pressure of the patient was 151 / 110 mmHg, the fasting blood glucose was 8.8 mmol / L, the fasting triglyceride was 3.49 mmol / L, the fasting HDL-C was 0.93 mmol / L, and the waist circumference was 1.32 m, which met the diagnostic criteria of metabolic syndrome.

[0060] It can be seen from the above results that the metabolic markers provided by the present application can be used to distinguish the metabolic syndrome patients from the simple obesity patients, and have high accuracy.

Claims

1. A metabolic biomarker for differentiating metabolic syndrome from simple obesity, characterized in that, The markers are glycocholic acid, lithocholic acid, linoleic acid, valine, leucine, alanine, serine, and glycine.

2. The use of the biomarker according to claim 1 in the preparation of a kit for detecting metabolic syndrome.

3. The application according to claim 2, characterized in that, The kit distinguishes between metabolic syndrome and simple obesity by detecting changes in the relative levels of metabolic markers in the serum of the subjects.

4. The application according to claim 3, characterized in that, The kit is based on the detection of changes in the relative levels of serum metabolic markers using liquid chromatography.

5. The application according to claim 3, characterized in that, Compared to patients with simple obesity, patients with metabolic syndrome showed significantly upregulated relative levels of glycocholic acid, lithocholic acid, linoleic acid, valine, leucine, and alanine in their serum, and significantly downregulated relative levels of serine and glycine.

6. The application according to claim 4, characterized in that, The liquid chromatography detection used a 0.01% formic acid aqueous solution as mobile phase A and an acetonitrile / methanol + 0.01% formic acid solution with a volume ratio of 87:13 as mobile phase B.

7. The application according to claim 6, characterized in that, The liquid chromatography elution conditions are as follows: 0-1 min, 5% B; 1-5 min, 5%-30% B; 5-15 min, 30%-90% B; 15-17.5 min, 90%-100% B; 17.5-20 min, 100% B.

Images