CN114544812B

CN114544812B - Application of metabolic combination type marker in diagnosis of asthma

Info

Publication number: CN114544812B
Application number: CN202210149454.XA
Authority: CN
Inventors: 陈智鸿; 朱涛; 马圆; 刘晓静; 徐侃; 闵智慧; 毛若琳; 陈梦捷; 崔博; 高磊
Original assignee: Zhongshan Hospital Fudan University
Current assignee: Zhongshan Hospital Fudan University
Priority date: 2022-02-18
Filing date: 2022-02-18
Publication date: 2023-06-30
Anticipated expiration: 2042-02-18
Also published as: CN114544812A

Abstract

The invention discloses application of a metabolic combined marker in diagnosing asthma. The invention provides an application of serum myristic acid and lauroyl carnitine as metabolic combination markers in diagnosing asthma, wherein the diagnostic threshold of the serum myristic acid is 286.94nmol/L; the diagnostic threshold of the serum lauroyl carnitine is 28.41nmol/L. The invention discovers and verifies that the serum myristic acid and lauroyl carnitine levels have higher sensitivity and specificity for diagnosing asthma based on a metabonomics analysis method, and the metabolic composition has the advantages of simplicity, convenience, high specificity, sensitivity and the like for diagnosing asthma, can provide scientific reference basis and personalized guidance for clinical development and treatment of asthma diseases, and has good application prospect.

Description

Application of metabolic combination type marker in diagnosis of asthma

Technical Field

The invention relates to application of a metabolic combined marker in diagnosis of asthma, and belongs to the technical field of biological detection.

Background

Bronchial asthma (bronchial asthma) is a chronic respiratory disease characterized by airway inflammation and airway hyperreactivity, and is mainly clinically manifested by recurrent respiratory symptoms such as wheezing, shortness of breath, chest distress, cough, etc. At present, regarding the diagnosis of asthma diseases, the diagnosis of asthma is still based on clinical manifestations, lung functions and other auxiliary examination results, and the current diagnosis of asthma has no gold standard, and no single detection method can diagnose (or exclude) asthma. Thus, a novel biomarker with high specificity and sensitivity was found to be very important for the assessment and treatment of asthma conditions.

Metabolites are at the end of biochemical activity in biological systems and thus reflect biological events that have occurred. The effects of changes in gene expression and environmental factors on biological systems can be manifested at the metabolite level to the final phenotype. Metabonomics can comprehensively and accurately check and analyze all metabolites such as fatty acids and amino acids of organisms, can elucidate disease pathogenesis and related molecular pathways, identify new biomarkers, and can predict disease progression and guide personalized treatment. Thus, combining metabolic data with clinical features can increase its predictive power. Myristic acid (also known as myristic acid) is a saturated fatty acid. Lauroyl carnitine (dodecanoylcannine) is one of acyl carnitines, widely existing in various tissues and body fluids of organisms, and can be used for detection and screening of fatty acid metabolic diseases. However, there is no report on the combination of myristic acid and laurylcarnitine as a diagnostic marker of asthma.

Disclosure of Invention

The technical problems to be solved by the invention are as follows: the markers in the prior art cannot conveniently and accurately diagnose the technical problem of asthma.

In order to solve the technical problems, the invention provides application of a detection reagent of a metabolic combined marker in preparation of a reagent or a kit for diagnosing asthma, wherein the metabolic combined marker comprises serum myristic acid and lauroyl carnitine.

Preferably, the diagnostic threshold of the serum myristic acid is 286.94nmol/L; the diagnostic threshold of the serum lauroyl carnitine is 28.41nmol/L.

Preferably, the detection reagent comprises myristic acid and lauroyl carnitine standard.

Compared with the prior art, the invention has the beneficial effects that:

the invention provides application of serum myristic acid and lauroyl carnitine as metabolic combination markers in diagnosing asthma, discovers and verifies that the levels of the serum myristic acid and the lauroyl carnitine have higher sensitivity and specificity for diagnosing asthma based on a metabonomics analysis method, and the metabolic composition has the advantages of simplicity, convenience, high specificity, sensitivity and the like for diagnosing asthma, can provide scientific reference basis and personalized guidance for clinical development and treatment of asthma diseases, and has good application prospect.

Drawings

FIG. 1 is a graph showing serum myristic acid and lauroyl carnitine levels detected by the test group population based on the Q Exactive Orbitrap LC-MS system; the graph shows that the two groups have significant differences through statistical analysis, and P is less than 0.0001;

FIG. 2 is the result of ROC curve fitting of the test group population based on the level of serum myristic acid and lauroyl carnitine detected by Q Exactive Orbitrap LC-MS system;

FIG. 3 is a graph of serum myristic acid and lauroyl carnitine levels detected based on the UHPLC-MRM-MS/MS system for a validated group population; the graph shows that the two groups have significant differences through statistical analysis, and P is less than 0.01; the graph shows that the two groups have significant differences through statistical analysis, and P is less than 0.001;

fig. 4 is a ROC curve result of verifying serum myristic acid and lauroyl carnitine level fitting based on UHPLC-MRM-MS/MS system detection for group population.

Detailed Description

In order to make the invention more comprehensible, preferred embodiments accompanied with figures are described in detail below.

In the following examples, serum collection was performed for asthmatic patients and healthy subjects in the test group: inclusion criteria were 102 patients diagnosed with asthma using the global asthma initiative (GINA) guidelines, 55 men and 47 women; the control group was 18 healthy adults. Collecting fasting venous blood of asthmatic patients and healthy people, immediately centrifuging at 3000rpm for 10 minutes after blood sample collection for 12 hours before blood collection, separating serum, placing the separated serum sample in a marked test tube, freezing and preserving at a temperature lower than-80 ℃ in an ultra-low temperature refrigerator, and carrying out non-targeting metabonomics Q Exactive Orbitrap LC-MS system detection on all samples in the batch at the same time in order to reduce batch effect in metabonomics analysis. As a validation group, another group of 30 asthmatic patients and 15 healthy subjects were selected, serum samples were collected, stored and prepared continuously as described above, and all samples were subjected to targeted metabonomics UHPLC-MS/MS analysis at the same time.

In the following examples, the method for detecting metabolites in serum of the non-targeted metabonomics Q Exactive Orbitrap LC-MS system comprises the following steps:

blood samples were collected from the test group population after a nocturnal fasting for at least 12 hours. All serum samples were then stored at-80 ℃ until the treatment was completed. To reduce the batch effect in metabonomic analysis, all samples were analyzed simultaneously, and serum was analyzed for non-targeted metabonomics by Q Exactive Orbitrap LC-MS/MS system. Briefly, 200 μl of the extract (acetonitrile: methanol=1:1, containing isotopically labeled internal standard mixture) was added to 50 μl of the sample. The samples were then spun for 30 seconds, sonicated in an ice-water bath for 10 minutes, and incubated at-40 ℃ for 1 hour to precipitate the proteins. After centrifugation at 12000rpm for 15 minutes at 4 ℃, the resulting supernatant was subjected to further analysis. Quality Control (QC) samples were prepared by mixing equal amounts of supernatant from all samples. Meanwhile, 14 quality control samples were included in the experiment. LC-MS/MS analysis was performed using a UHPLC system (Vanquish, thermo Fisher Scientific) with a UPLC chromatography column (2.1 mm. Times.100 mm,1.7 μm) coupled to a Q exact HFX mass spectrometer (Orbitrap MS, thermo). The mobile phase consists of a: water (25 mmol/L ammonium acetate and 25mmol/L aqueous ammonia, ph=9.75) and B: acetonitrile. The elution gradient was set as: 0 to 0.5min,95 percent of B;0.5 to 7.0min,95 to 65 percent of B;7.0 to 8.0min,65 to 40 percent of B;8.0 to 9.0min,40 percent of B;9.0 to 9.1min,40 to 95 percent of B;9.1 to 12.0min,95 percent of B and the column temperature is 30 ℃. The temperature of the automatic sampler is 4 ℃ and the sample injection amount is 3 mu L. The QE HFX mass spectrometer is capable of acquiring MS/MS spectra in an information-dependent acquisition (IDA) mode under control of acquisition software (Xcalibur, thermo). In this mode, the acquisition software continuously evaluates the full scan MS spectrum. The ESI source conditions were set as follows: sheath gas flow rate 50arb, auxiliary gas flow rate 10arb, capillary temperature 320 ℃, full MS resolution 60000, MS/MS resolution 7500, collision energy in Normalized Collision Energy (NCE) mode 10/30/60, ion source ejection voltage 3.5kV (positive) or-3.2 kV (negative).

In the following examples, the method for detecting serum lauroyl carnitine by a targeted metabonomics UHPLC-MS/MS system is as follows:

the level of lauroyl carnitine in the serum of group population was verified by UHPLC-MS/MS analysis. Serum samples were collected, stored and prepared as described above. After thawing the serum samples, 100. Mu.L aliquots were transferred precisely to Eppendorf tubes. After 400. Mu.L of extraction solution (acetonitrile-methanol, 1:1) was added, the sample was spun for 30 seconds and sonicated in an ice-water bath for 15 minutes. Next, the samples were incubated and centrifuged. Then, 70. Mu.L aliquots were transferred to autosampler vials for UHPLC-MS/MS analysis. For standard solution preparation, each standard was dissolved or diluted to a final concentration of 10 mmol/L. The target compound was chromatographed through a Waters ACQUITY UPLC BEH Amide (100×2.1mm,1.7 μm, waters) liquid chromatography column using an Agilent 1290Infinity II series (Agilent Technologies) ultra-high performance liquid chromatograph, an Agilent 6460 triple quadrupole mass spectrometer of the AJS-ESI ion source, and mass spectrometry was performed in multi-reaction monitoring (MRM) mode. MRM data acquisition and processing is then completed by Agilent MassHunter Work Station Software (b.08.00, agilent Technologies). Subsequently, the calibration solution was subjected to UPLC-MRM-MS/MS analysis to establish calibration curves for myristic acid and lauroyl carnitine metabolites. Finally, the concentration of myristic acid and lauroyl carnitine in the serum was determined.

Examples

This example provides the use of serum lauroyl carnitine as a diagnostic marker of asthma:

serum from the collected asthmatic patients and healthy subjects (test group) was analyzed by Q Exactive Orbitrap LC-MS for non-targeted metabonomics, and 12 kinds of asthma-associated serum core metabolites such as myristic acid and lauroyl carnitine were found. The serum myristic acid (17629808.23 ± 20999189.42) was significantly higher than that of healthy subjects (6940110.31 ± 7041734.38), the difference was statistically significant (P < 0.0001), and the serum lauroyl carnitine (23999046.26 ± 16483428.16) was significantly higher than that of healthy subjects (12260493.56 ± 8881642.57), the difference was statistically significant (P < 0.0001), as shown in fig. 1; and serum myristic acid has higher sensitivity (82.35%) and specificity (77.78%) for diagnosing asthma, AUC of ROC curve is 0.801; serum lauroyl carnitine also has a higher sensitivity (66.67%) and specificity (94.44%) for diagnosing asthma, with an AUC of 0.846 for the ROC curve; whereas serum myristic acid combined with lauroyl carnitine has high sensitivity (83.33%) and specificity (77.78%), AUC for ROC curve is 0.850, as shown in fig. 2. To further evaluate the value of serum myristic acid and lauroyl carnitine for asthma diagnosis, another group of asthmatic patients and healthy subjects were included as a validation group, and specific levels of serum myristic acid were detected in the validation group by using a targeted metabonomics UHPLC-MRM-MS/MS method, which showed that the level of serum myristic acid (514.142 + -319.878 nmol/L) was significantly higher than that in healthy subjects (263.653 + -167.058 nmol/L), the difference was statistically significant (P < 0.01), the sensitivity for diagnosing asthma was 83.33%, the specificity was 73.33%, and the AUC of the ROC curve was 0.789 when 286.94nmol/L was taken as the diagnostic threshold (Cut-off value); serum lauroyl carnitine level (46.039 ± 25.827 nmol/L) of asthmatic patients is significantly higher than that of healthy subjects (17.955 ±9.024 nmol/L), the difference is statistically significant (P < 0.001), sensitivity of diagnosing asthma is 76.67%, specificity is 93.33% and AUC of ROC curve is 0.893 when 28.41nmol/L is taken as diagnostic threshold (Cut-off value); myristic acid combined with lauroyl carnitine has a high sensitivity (80.00%) and specificity (86.67%) for diagnosing asthma, with an AUC of 0.889 for the ROC curve; the results are shown in fig. 3 and 4. Therefore, the combination of the serum myristic acid and the lauroyl carnitine has good application prospect for diagnosing asthma. The invention can diagnose asthma in a simpler and more convenient way, and is expected to prepare a more reliable and more sensitive kit for diagnosing asthma based on the characteristics of the biomarker.

The above-described embodiments are only preferred embodiments of the present invention, and are not intended to be limiting in any way and in nature, and it should be noted that several modifications and additions may be made to those skilled in the art without departing from the invention, which modifications and additions are also intended to be construed as within the scope of the invention.

Claims

1. Use of a detection reagent for a metabolic combination marker in the manufacture of a reagent or kit for diagnosing asthma, wherein the metabolic combination marker comprises serum myristic acid and lauroyl carnitine.

2. The use according to claim 1, wherein the diagnostic threshold for serum myristic acid is 286.94nmol/L; the diagnostic threshold of the serum lauroyl carnitine is 28.41nmol/L.

3. The use of claim 1, wherein the detection reagent comprises myristic acid and lauroyl carnitine standard.