Application of linoleic acid as biomarker and kit for detecting biomarker

Abstract

The invention discloses application of linoleic acid as a biomarker and a kit for detecting the biomarker, and belongs to the technical field of medical detection. According to the model, the biomarker content level serves as an input variable, a diagnosis model is constructed, the biomarker is linoleic acid, and the model use equation is as follows: score = 2.09 * linoleic acid content level / 1000-9.399. Among coronary heart disease groups, linoleic acid in patients with hyperlipemia is obviously higher than linoleic acid in non-hyperlipemia groups, the established diagnosis model has good sensitivity and specificity, and the provided kit can realize diagnosis and prognosis monitoring of hyperlipemia and can guide lipid nutrition intake of patients in the treatment process.

Description

Technical Field

[0001] This invention relates to the field of medical testing technology, specifically to the application of linoleic acid as a biomarker and a kit for detecting this biomarker. Background Technology

[0002] Cardiovascular disease (CVD) is the leading chronic non-communicable disease threatening human health worldwide. Atherosclerotic cardiovascular disease (ASCVD), the predominant form of CVD, is the leading cause of death among urban and rural residents in my country, accounting for over 40% of all deaths. In recent years, the burden of ASCVD in my country has continued to increase, making prevention and control a serious challenge. Multiple studies have provided ample evidence that long-term hyperlipidemia can lead to atherosclerosis, increasing the incidence and mortality of cardiovascular diseases. Furthermore, low-density lipoprotein cholesterol (LDL-C) is a pathogenic risk factor for ASCVD and a primary target for lipid intervention, significantly reducing the risk of ASCVD morbidity and mortality. Therefore, lipid management is crucial and cannot be delayed. The clinical diagnosis and classification of hyperlipidemia are based on the levels of total cholesterol and triglycerides. The primary target for intervention is LDL-C, and the preferred strategies for lipid-lowering treatment are a reasonable diet, moderate increase in physical activity, weight control, smoking cessation, and limitation of alcohol consumption. Among these, a reasonable diet has a significant impact on blood lipids. Therefore, there is an urgent need for a biomarker that can not only assist in the diagnosis and prognosis of hyperlipidemia, but also guide patients to consume lipid nutrients in a reasonable manner.

[0003] Fatty acids are a class of compounds composed of carbon, hydrogen, and oxygen, and are the main components of neutral fats, phospholipids, and glycolipids. Fatty acids in the blood exist in esterified and free forms, with the esterified form primarily existing as triglycerides and lipids. Existing studies have shown that blood fatty acid concentration is a potential biomarker of dietary fat intake. In children of all ages, fatty acid, palmitic acid, and conjugated linoleic acid are positively correlated with milk fat consumption. In adults, after a lipid-lowering diet, the four FA ratios in serum phospholipids—n-6 / n-3, arachidonic acid (AA) / eicosapentaenoic acid (EPA), AA / docosahexaenoic acid (DHA), AA / (EPA+DHA), and the n-3 index (EPA+DHA)—significantly decreased. Therefore, fatty acid compounds are the most likely biomarkers for simultaneously diagnosing, predicting prognosis, and providing dietary guidance for hyperlipidemia. Currently, there are no rapid and efficient reagent kits or diagnostic models available on the market to meet these testing needs, severely hindering effective intervention for hyperlipidemia and guidance for lipid-based dietary therapy. Summary of the Invention

[0004] The purpose of this invention is to provide an application of linoleic acid as a biomarker and a kit for detecting this biomarker. In a study of patients with coronary heart disease, including those with hyperlipidemia and those without, this application used high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) to detect various types of fatty acids in the plasma or serum of patients. Metabolomics was used to screen for potential disease biomarkers, a diagnostic model was established, and another sample cohort was selected. The kit was then used to validate the biomarker. The results showed that linoleic acid was significantly more effective in diagnosing hyperlipidemia than in non-hyperlipidemia patients. More specifically, the inventors found that high levels of linoleic acid have good sensitivity and specificity in diagnosing hyperlipidemia, and therefore can be used as a biomarker for the diagnosis and prognostic monitoring of hyperlipidemia, thus addressing the problems mentioned in the background section.

[0005] To achieve the above objectives, the present invention provides the following technical solution:

[0006] An application of linoleic acid as a biomarker is disclosed, wherein the biomarker is used in a diagnostic model for diagnosing hyperlipidemia. The model uses the biomarker level as an input variable to construct the diagnostic model. The biomarker is linoleic acid, and the model uses the equation: score = 2.09 * linoleic acid level / 1000 - 9.399.

[0007] As a further preferred embodiment of the present invention: the cutoff value for evaluating hyperlipidemia by the model diagnostic score is 0.45. When the score is ≥0.45, hyperlipidemia is diagnosed, and when the score is <0.45, non-hyperlipidemia is diagnosed.

[0008] As a further preferred embodiment of the present invention: the cutoff value for evaluating hyperlipidemia by the model diagnostic score is 0.45. When the score is ≥0.45, hyperlipidemia is diagnosed, and when the score is <0.45, non-hyperlipidemia is diagnosed.

[0009] A kit for detecting linoleic acid as a biomarker, the kit containing reagents for detecting linoleic acid.

[0010] As a further preferred embodiment of the present invention, the kit is a high-performance liquid chromatography-tandem mass spectrometry kit.

[0011] As a further preferred embodiment of the present invention: the control sample of the kit is derived from patients with hyperlipidemia and non-hyperlipidemia in the coronary heart disease population, and the sample is serum or plasma.

[0012] As a further preferred embodiment of the present invention: the type of linoleic acid detected by the kit is the sum of esterified and non-esterified linoleic acid in the blood.

[0013] Furthermore, the diagnosis of hyperlipidemia in the aforementioned coronary heart disease population includes the following steps:

[0014] (1) Collect samples from the subjects to be tested;

[0015] (2) Detect the linoleic acid content level in the samples of the subjects to be tested;

[0016] (3) Substitute the linoleic acid content into the model formula to obtain the score;

[0017] (4) Compare with the diagnostic cutoff value to diagnose whether the subject has hyperlipidemia.

[0018] As used in this article, the subjects are human.

[0019] As used herein, the sample of the subject to be tested is a clinical biological sample of the subject, namely serum or plasma.

[0020] Furthermore, the kit employs liquid chromatography-tandem mass spectrometry (LC-MS / MS) for detection. Accordingly, in addition to linoleic acid, the kit may also include other components required for the detection of polyunsaturated fatty acids, all of which are well known in the art. For detection purposes, for example, the fatty acids to be measured in the kit may be converted from bound fatty acids to free fatty acids under high-temperature alcohol hydrolysis conditions; or, for example, the kit may include calibrators, quality control samples, diluents, etc.

[0021] Compared with the prior art, the beneficial effects of the present invention are:

[0022] The content of linoleic acid in patients with hyperlipidemia in coronary heart disease is significantly higher than that in patients without hyperlipidemia. At the same time, the diagnostic model established using linoleic acid has good sensitivity and specificity, and high consistency in the validation population. More specifically, linoleic acid is an essential fatty acid, mainly derived from food. Therefore, it can be used as a biomarker for the diagnosis and prognostic monitoring of patients with hyperlipidemia in coronary heart disease, and at the same time, it can provide guidance for lipid nutrient intake in patients' lipid-lowering therapy. Attached Figure Description

[0023] Figure 1 Multivariate statistical analysis of partial least squares discriminant analysis (PLS-DA) for patients with coronary heart disease, including those with hyperlipidemia and those without hyperlipidemia;

[0024] Figure 2 The levels of linoleic acid and α-linolenic acid in serum samples from patients with and without hyperlipidemia in patients with coronary heart disease;

[0025] Figure 3 ROC curve analysis for a diagnostic model of hyperlipidemia in patients with coronary heart disease. Detailed Implementation

[0026] The inventive objectives, technical solutions, and beneficial effects of this invention will be further described in detail below. It should be noted that the following detailed descriptions are exemplary and intended to provide further explanation of the claimed invention. Unless otherwise stated, 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.

[0027] Linoleic acid was used as a biomarker for the diagnosis of hyperlipidemia in patients with coronary heart disease, and a diagnostic model for hyperlipidemia in these patients was developed. The model uses the biomarker level as an input variable to construct a diagnostic model. The biomarker is linoleic acid. The model uses the equation: Score = 2.09 * linoleic acid level / 1000 - 9.399. The cutoff value for evaluating hyperlipidemia in the model's diagnostic score is 0.45. A score ≥ 0.45 indicates a diagnosis of hyperlipidemia, while a score < 0.45 indicates a diagnosis of non-hyperlipidemia.

[0028] A kit for detecting linoleic acid as a biomarker, as is common knowledge to those skilled in the art, contains reagents for detecting linoleic acid. Preferably, the reagents are polyunsaturated fatty acids. Specifically, the kit is a high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) kit. The control samples are derived from patients with and without hyperlipidemia in the coronary heart disease population; the samples are serum or plasma. The kit detects the sum of esterified and non-esterified linoleic acid in the blood. The hydrolysis method for esterified linoleic acid in the kit is a high-temperature hydrolysis method under alkaline conditions. Simultaneously, the kit uses an isotope internal standard to correct for matrix effects in the blood samples. This operation is common knowledge to those skilled in the art and will not be elaborated further.

[0029] Example 1: Exploring differential biomarkers for the differential diagnosis of hyperlipidemia

[0030] Experimental subjects: Serum samples were collected from 168 patients clinically diagnosed with coronary heart disease at the Fourth Affiliated Hospital of Harbin Medical University. This study was approved by the Ethics Committee of the Fourth Affiliated Hospital of Harbin Medical University.

[0031] Experimental grouping: Among all enrolled subjects, 91 had no hyperlipidemia and 77 had hyperlipidemia, specifically divided into 11 cases of hypercholesterolemia, 49 cases of hypertriglyceridemia, and 19 cases of mixed hyperlipidemia. The non-hyperlipidemia group consisted of 63 males and 28 females, with a mean age of 67.7 years. The hyperlipidemia group consisted of 48 males and 29 females, with a mean age of 63.0 years.

[0032] Diagnostic criteria: The diagnostic criteria for hyperlipidemia are as follows: Hypercholesterolemia: serum total cholesterol ≥ 5.2 mmol / L; Hypertriglyceridemia: serum triglycerides ≥ 1.7 mmol / L; Mixed hyperlipidemia: serum total cholesterol ≥ 5.2 mmol / L and serum triglycerides ≥ 1.7 mmol / L.

[0033] Experimental methods: The content level of linoleic acid in serum samples was detected using an ω-3 and ω-6 polyunsaturated fatty acid detection kit (liquid chromatography-tandem mass spectrometry). The detection indicators included in this method are shown in Table 1.

[0034] Table 1

[0035]

[0036]

[0037] Biomarker screening: SIMCA software was used for multivariate statistical analysis to screen for differentially expressed biomarkers. Partial least squares discriminant analysis (PLS-DA) was employed for supervised pattern recognition multivariate statistical analysis between the two groups. Figure 1 The differentially expressed metabolites (VIP>1) between the screening groups were linoleic acid (VIP=1.4) and α-linolenic acid (VIP=1.2), and the significance test showed a p-value <0.05. Figure 2 And as shown in Table 2.

[0038] Table 2

[0039]

[0040] Example 2: Establishment of a diagnostic model for hyperlipidemia in patients with coronary heart disease

[0041] To aid in the diagnosis of hyperlipidemia in patients with coronary heart disease and monitor treatment efficacy, a diagnostic model was established using the aforementioned biomarkers. Using SPSS software, binary logistic regression was performed on linoleic acid and α-linolenic acid. The results are shown in Table 3. The final model incorporated linoleic acid and a constant, and the model equation was determined as: Score = 2.09 * linoleic acid content level / 1000 - 9.399. The sensitivity, specificity, and diagnostic efficacy of the diagnosis were assessed using the receiver operating characteristic (ROC) curve. The area under the ROC curve for the established model was 0.911. The maximum Youden index was determined as the diagnostic cutoff value, with a cutoff value of 0.45, exhibiting a specificity of 83.5% and a sensitivity of 84.0%. Figure 3 The Hosmer-Lemeshow goodness-of-fit test result was χ². 2=3.884, P=0.867, indicating that the model did not overfit and the diagnostic model has good calibration.

[0042] Table 3. Results of Binary Logistic Regression

[0043]

[0044]

[0045] Example 3: Validation of a diagnostic model for hyperlipidemia in patients with coronary heart disease

[0046] Based on the same criteria as the discovery cohort, 25 patients with coronary heart disease, both hyperlipidemia and non-hyperlipidemia, were screened at the Fourth Affiliated Hospital of Harbin Medical University during another time period. The levels of linoleic acid in serum samples were detected using an ω-3 and ω-6 polyunsaturated fatty acid assay kit (liquid chromatography-tandem mass spectrometry). The established model was then used for the diagnosis of hyperlipidemia, and the results are shown in Table 1. The results showed that the diagnostic model for hyperlipidemia using linoleic acid as a biomarker had an overall diagnostic concordance rate of 80% with that for total cholesterol and triglycerides, a positive diagnostic concordance rate of 76.9%, and a negative diagnostic concordance rate of 83.3%.

[0047] Table 1: Diagnostic capabilities of the diagnostic models

[0048] Model building hyperlipidemia Non-hyperlipidemia total Positive (≥0.45) 10 2 12 Negative (<0.45) 3 10 13 total 13 12 15

[0049] It should be noted that the above embodiments are only specific and clear descriptions of the technical solutions and features of this application. Solutions or features that are prior art or common knowledge to those skilled in the art will not be described in detail in the above embodiments.

[0050] Furthermore, the technical solutions of this application are not limited to the above embodiments. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other implementation methods that can be understood by those skilled in the art.

Claims

1. An application using linoleic acid as a biomarker, characterized in that, This biomarker is used in a diagnostic model for diagnosing hyperlipidemia. The model uses the biomarker level as an input variable to construct the diagnostic model. The biomarker is linoleic acid. The model uses the equation: score = 2.09 * linoleic acid level / 1000 - 9.

399.

2. The application of linoleic acid as a biomarker according to claim 1, characterized in that, The cutoff value for evaluating hyperlipidemia using the model's diagnostic score is 0.

45. When the score is ≥0.45, hyperlipidemia is diagnosed, and when the score is <0.45, non-hyperlipidemia is diagnosed.

3. A kit for detecting linoleic acid as a biomarker, characterized in that, The kit contains reagents for detecting linoleic acid.

4. The kit for detecting linoleic acid as a biomarker according to claim 3, characterized in that, The kit is a high-performance liquid chromatography-tandem mass spectrometry kit.

5. The kit for detecting linoleic acid as a biomarker according to claim 4, characterized in that, The control samples for the kit were obtained from patients with hyperlipidemia and non-hyperlipidemia in the coronary heart disease population, and the samples were serum or plasma.

6. The kit for detecting linoleic acid as a biomarker according to claim 4, characterized in that, The kit detects the total amount of esterified and non-esterified linoleic acid in the blood.

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