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

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 a metabolic combination marker in the diagnosis of asthma

technical field

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

Background technique

Bronchial asthma, referred to as asthma, is a chronic respiratory disease characterized by airway inflammation and airway hyperresponsiveness. Its main clinical manifestations are recurrent wheezing, shortness of breath, chest tightness, coughing and other respiratory symptoms. At present, the diagnosis of asthma is still based on clinical manifestations and auxiliary examination results such as lung function. There is no gold standard for the diagnosis of asthma, and there is no single detection method to diagnose (or rule out) asthma. Therefore, the discovery of a new biomarker with high specificity and sensitivity is very important for the assessment and treatment of asthma.

Metabolites are at the terminal end of biochemical activity in biological systems and thus reflect biological events that have already occurred. The impact of changes in gene expression and environmental factors on biological systems can be ultimately phenotyped at the metabolite level. Metabolomics can comprehensively and accurately examine and analyze metabolites such as fatty acids and amino acids in an organism, explain disease pathogenesis and related molecular pathways, identify new biomarkers, predict disease progression, and guide personalized treatment. Therefore, combining metabolic data with clinical features can improve its predictive power. Myristic acid, also known as myristoleic acid, is a saturated fatty acid. Dodecanoylcarnitine is a kind of acylcarnitine, which widely exists in various tissues and body fluids of the body, and can be used for the detection and screening of fatty acid metabolism diseases. However, there is no report on the combination of myristic acid and lauryl carnitine as a marker for the diagnosis of asthma.

Contents of the invention

The technical problem to be solved by the present invention is: the markers in the prior art cannot diagnose asthma conveniently and accurately.

In order to solve the above-mentioned technical problems, the present invention provides the application of a detection reagent of a metabolic combinatorial marker, which includes serum myristic acid and lauric acid, in the preparation of a reagent or kit for diagnosing asthma. Acylcarnitines.

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

Preferably, the detection reagents include standard products of myristic acid and lauroyl carnitine.

Compared with prior art, the beneficial effect of the present invention is:

The present invention provides the application of serum myristic acid and lauroyl carnitine as metabolic combination markers in the diagnosis of asthma. The present invention discovers and verifies the importance of serum myristic acid and lauroyl carnitine levels for diagnosis based on the metabolomics analysis method Asthma has high sensitivity and specificity. The metabolic composition has the advantages of simplicity, convenience, high specificity and sensitivity for the diagnosis of asthma, and can provide scientific reference and individualization for the progress and treatment of asthma in clinical practice. guidance, has a good application prospect.

Description of drawings

Figure 1 shows the levels of serum myristic acid and lauroylcarnitine detected by the Q Exactive Orbitrap LC-MS system in the test group; **** in the figure indicates that there is a significant difference between the two groups after statistical analysis, P<0.0001 ;

Figure 2 is the ROC curve fitting results of the serum myristic acid and lauroyl carnitine levels detected by the Q Exactive Orbitrap LC-MS system in the test group;

Figure 3 shows the levels of serum myristic acid and lauroylcarnitine detected by the UHPLC-MRM-MS/MS system in the verification group; ** in the figure indicates that after statistical analysis, there is a significant difference between the two groups, P<0.01; *** in the figure indicates that after statistical analysis, there is a significant difference between the two groups, P<0.001;

Figure 4 is the ROC curve fitting results of the serum myristic acid and lauroyl carnitine levels detected by the UHPLC-MRM-MS/MS system of the verification group population.

Detailed ways

In order to make the present invention more comprehensible, preferred embodiments are described in detail below with accompanying drawings.

In the following examples, the serum collection of asthma patients and healthy subjects in the test group: the inclusion criteria were 102 patients diagnosed with asthma according to the Global Asthma Initiative (GINA) guidelines, including 55 males and 47 females; 18 cases in the control group, all for healthy adults. Collect fasting venous blood from asthma patients and healthy people, fast for 12 hours before blood collection, and centrifuge at 3000rpm for 10 minutes immediately after blood collection to separate the serum. Stored below -80°C, in order to reduce batch effects in metabolomics analysis, all samples in this batch were detected at the same time by the non-targeted metabolomics Q Exactive Orbitrap LC-MS system. As a verification group, select another group of 30 asthmatic patients and 15 healthy subjects, continue to collect, store and prepare serum samples according to the above method, and perform targeted metabolomics UHPLC-MS/MS on all samples at the same time Analytical determination.

In the following examples, the detection method of metabolites in the serum of the non-targeted metabolomics Q Exactive Orbitrap LC-MS system is:

Blood samples from the test group were collected after a nocturnal fast lasting at least 12 hours. Then, all serum samples were stored at -80°C until processing. To reduce batch effects in metabolomics analysis, all samples were analyzed simultaneously, and serum was analyzed by Q Exactive Orbitrap LC-MS/MS system for untargeted metabolomics. Briefly, 200 μL of extraction solution (acetonitrile:methanol=1:1, containing isotope-labeled internal standard mixture) was added to 50 μL of sample. Then, samples were spun for 30 s, sonicated for 10 min in an ice-water bath, and incubated at -40°C for 1 hr to precipitate proteins. After centrifugation at 12000 rpm for 15 minutes at 4°C, the resulting supernatant was further analyzed. Quality control (QC) samples were prepared by pooling equal amounts of supernatants 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 column (2.1 mm×100 mm, 1.7 μm) coupled to a Q Exactive HFX mass spectrometer (Orbitrap MS, Thermo). The mobile phase consisted of A: water (25mmol/L ammonium acetate and 25mmol/L ammonia water, pH=9.75) and B: acetonitrile. The elution gradient setting is: 0~0.5min, 95%B; 0.5~7.0min, 95%~65%B; 7.0~8.0min, 65%~40%B; 8.0~9.0min, 40%B; 9.0~ 9.1min, 40%～95%B; 9.1～12.0min, 95%B, column temperature 30°C. The temperature of the autosampler was 4°C, and the injection volume was 3 μL. The QE HFX mass spectrometer is capable of acquiring MS/MS spectra in information-dependent acquisition (IDA) mode under the control of acquisition software (Xcalibur, Thermo). In this mode, the acquisition software continuously evaluates full-scan MS spectra. The ESI source conditions are set as follows: sheath gas flow rate 50arb, auxiliary gas flow rate 10arb, capillary temperature 320°C, full MS resolution 60000, MS/MS resolution 7500, collision energy in normalized collision energy (NCE) mode is 10/ 30/60, ion source injection voltage: 3.5kV (positive) or -3.2kV (negative).

In the following examples, the method for detecting serum lauroylcarnitine by targeted metabolomics UHPLC-MS/MS system is:

The level of lauroylcarnitine in the serum of the verification group was determined by UHPLC-MS/MS analysis. Serum samples were collected, stored and prepared as described above. After the serum samples were thawed, 100 μL aliquots were precisely transferred to Eppendorf tubes. After adding 400 μL of extraction solution (acetonitrile-methanol, 1:1), the samples were spun for 30 s and sonicated for 15 min in an ice-water bath. Next, the samples are incubated and centrifuged. Then, 70 μL aliquots were transferred to autosampler vials for UHPLC-MS/MS analysis. For standard solution preparation, dissolve or dilute each standard substance to a final concentration of 10 mmol/L. Using an Agilent 1290Infinity II series (Agilent Technologies) ultra-high performance liquid chromatography, the target compound was chromatographically separated through a Waters ACQUITY UPLC BEH Amide (100×2.1mm, 1.7μm, Waters) liquid chromatography column, and the AJS-ESI ion source An Agilent 6460 triple quadrupole mass spectrometer was used for mass spectrometry in multiple reaction monitoring (MRM) mode. Then, MRM data acquisition and processing were completed by Agilent MassHunter Work Station Software (B.08.00, Agilent Technologies). Subsequently, the calibration solutions were subjected to UPLC-MRM-MS/MS analysis to establish calibration curves for myristic acid and lauroylcarnitine metabolites. Finally, serum concentrations of myristic acid and lauroylcarnitine were determined.

Example

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

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

The foregoing embodiments are only preferred embodiments of the present invention, and are not intended to limit the present invention in any form and in essence. It should be pointed out that those of ordinary skill in the art can also make Several improvements and supplements should also be considered as the protection scope of the present invention.

Claims (3)

1. The application of a detection reagent of a metabolic combined marker in the preparation of a reagent or kit for diagnosing asthma, characterized in that the metabolic combined marker includes serum myristic acid and lauroylcarnitine. 2. The application according to claim 1, wherein the diagnostic threshold of the serum myristic acid is 286.94nmol/L; the diagnostic threshold of the serum lauroylcarnitine is 28.41nmol/L. 3. application as claimed in claim 1, is characterized in that, described detection reagent comprises myristic acid and lauroyl carnitine standard substance.

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