CN114609270B

CN114609270B - Use of serum lauroyl carnitine as diagnostic marker of asthma

Info

Publication number: CN114609270B
Application number: CN202210149455.4A
Authority: CN
Inventors: 陈智鸿; 朱涛; 马圆; 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: 2023-08-04
Anticipated expiration: 2042-02-18
Also published as: CN114609270A

Abstract

The invention discloses an application of serum lauroyl carnitine as an asthma diagnosis marker. According to the invention, the serum lauroyl carnitine of an asthmatic patient is obviously higher than that of a healthy person through the analysis of non-targeted metabonomics of the serum of an experimental group by Q Exactive Orbitrap LC-MS, the difference is statistically significant, the serum lauroyl carnitine has higher sensitivity to diagnosing the asthmatic, and the result is verified by a verification group, when 28.41nmol/L is taken as a diagnosis threshold value, the sensitivity to diagnosing the asthmatic is 76.67%, the specificity is 93.33%, and the AUC of an ROC curve is 0.893; therefore, the serum lauroyl carnitine serving as a marker has the advantages of simplicity, convenience, high specificity, sensitivity and the like for diagnosing asthma, and can provide scientific reference basis and personalized guidance for clinical development and treatment of asthma diseases.

Description

Use of serum lauroyl carnitine as diagnostic marker of asthma

Technical Field

The invention relates to an application of serum lauroyl carnitine as an asthma diagnosis marker, belonging 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.

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 serum lauroyl carnitine as a diagnostic marker of asthma based on metabonomics, so that scientific reference basis and personalized guidance are provided for the progress and treatment of asthma diseases.

Disclosure of Invention

The invention aims to provide a serum metabolite serving as an asthma diagnosis marker, so as to solve the technical problem that the marker in the prior art cannot conveniently and accurately diagnose asthma.

In order to achieve the above object, the present invention provides the use of a reagent for detecting serum lauroyl carnitine for preparing a reagent or kit for diagnosing asthma.

Preferably, the diagnostic threshold for serum lauroyl carnitine is 28.41nmol/L.

Preferably, the reagent for detecting serum lauroyl carnitine comprises a standard of lauroyl carnitine.

The invention also provides a reagent or a kit for diagnosing asthma, wherein the reagent or the kit comprises a reagent for detecting serum lauroyl carnitine.

Preferably, the reagent for detecting serum lauroyl carnitine comprises a lauroyl carnitine standard.

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

the invention provides the use of serum lauroyl carnitine as an asthma diagnosis marker based on a metabonomics analysis method, wherein when 28.41nmol/L is taken as a diagnosis threshold (Cut-off value), the sensitivity of diagnosing asthma is 76.67%, the specificity is 93.33%, and the AUC of an ROC curve is 0.893; therefore, the serum lauroyl carnitine serving as a marker has the advantages of simplicity, convenience, high specificity, sensitivity and the like for diagnosing asthma, and can provide scientific reference basis and personalized guidance for clinical development and treatment of asthma diseases.

Drawings

FIG. 1 is a graph of serum lauroyl carnitine levels detected by a panel 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 a graph showing the results of ROC curves fitted to serum lauroyl carnitine levels detected by the Q Exactive Orbitrap LC-MS system for the population of the test group;

FIG. 3 is a graph of serum lauroyl carnitine levels detected by a validated group population based on the UHPLC-MRM-MS/MS system; 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 a lauroyl carnitine level fit based on UHPLC-MRM-MS/MS system detection for a validated 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 detection method for identifying metabolites in serum of a 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 a calibration curve for lauroyl carnitine metabolites. Finally, the lauroyl carnitine concentration in the serum is determined.

Examples

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

analysis of the serum non-targeted metabolomics of the collected asthmatic patient and healthy subjects (test group) serum with Q Exactive Orbitrap LC-MS found that the asthmatic patient serum lauroyl carnitine (23999046.26 ± 16483428.16) was significantly higher than healthy subjects (12260493.56 ± 8881642.57), the differences were statistically significant (P < 0.0001), as shown in fig. 1; and serum lauroyl carnitine has a higher sensitivity (66.67%) and specificity (94.44%) for diagnosing asthma, AUC of ROC curve is 0.846, as shown in fig. 2. To further evaluate the value of serum lauroyl carnitine for asthma diagnosis, another group of asthmatic patients and healthy subjects were included as a validation group, and specific levels of serum lauroyl carnitine were tested for the validation group by using a targeted metabolomics UHPLC-MRM-MS/MS method, which showed that serum lauroyl carnitine levels (46.039 + -25.827 nmol/L) were significantly higher than those of healthy subjects (17.955 + -9.024 nmol/L), and the differences were statistically significant (P < 0.001), as shown in FIG. 3; when 28.41nmol/L was taken as the diagnostic threshold (Cut-off value), the sensitivity for diagnosing asthma was 76.67%, the specificity was 93.33%, and the AUC of the ROC curve was 0.893, as shown in FIG. 4. Therefore, the serum 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 reagent for detecting serum lauroyl carnitine in the preparation of a reagent or kit for diagnosing asthma.

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

3. The use according to claim 1, wherein the reagent for detecting serum lauroyl carnitine comprises a lauroyl carnitine standard.