Application of dimethyl carbonate as a diagnostic marker for chronic obstructive pulmonary disease

Abstract

The application provides application of dimethyl carbonate as a chronic obstructive pulmonary disease diagnostic marker and belongs to the technical field of biomedical detection. The application first finds that the detection rate of dimethyl carbonate in exhaled air is significantly related to chronic obstructive pulmonary disease, and the detection rate of dimethyl carbonate in exhaled air of a chronic obstructive pulmonary disease patient is significantly higher than that of a healthy person. The ROC curve analysis result shows that dimethyl carbonate has extremely high specificity as a biomarker for diagnosing chronic obstructive pulmonary disease, and therefore can be used as a detection target for the diagnosis of chronic obstructive pulmonary disease patients, and has good practical application value.

Description

Technical Field

[0001] This invention belongs to the field of biomedical detection technology, specifically relating to the application of dimethyl carbonate as a diagnostic biomarker for chronic obstructive pulmonary disease. Background Technology

[0002] Volatile organic compounds (VOCs) in exhaled breath refer to gaseous organic molecules exhaled by the human body through the respiratory tract, with molecular weights typically between 30 and 300 Da. VOCs originate from endogenous metabolism (products of biochemical reactions in the body, such as oxidative stress, inflammatory responses, and lipid peroxidation) and exogenous inhalation (environmental pollutants, food metabolites, etc.). Common detection techniques include gas chromatography-mass spectrometry (GC-MS), two-dimensional gas chromatography-time-of-flight mass spectrometry (GC×GC-TOF-MS), and secondary electrospray ionization mass spectrometry (SESI-MS).

[0003] In recent years, the use of exhaled VOCs to diagnose respiratory diseases has become a research hotspot. In the field of lung cancer, multiple studies have identified characteristic VOCs biomarkers such as 2-pentanone and benzaldehyde, with diagnostic AUCs exceeding 0.9. In the field of asthma, VOCs such as isoprene and acetone have been found to be correlated with asthma control levels and can be used to differentiate asthma subtypes.

[0004] Chronic obstructive pulmonary disease (COPD) is a common, preventable, and treatable chronic airway disease characterized by persistent respiratory symptoms and airflow limitation, usually caused by significant exposure to harmful particles or gases leading to airway and / or alveolar abnormalities. Currently, the clinical diagnosis of COPD mainly relies on pulmonary function tests, with a forced expiratory volume in one second (FEV1 / FVC) ratio <0.70 after inhalation of a bronchodilator serving as the gold standard for confirming persistent airflow limitation. However, this method has the following limitations: ① It is complex to perform, requiring good patient cooperation, and is difficult for elderly, frail, or acutely ill patients to complete; ② It has limited sensitivity in detecting early, mild COPD, easily leading to missed diagnoses; ③ It cannot reflect the heterogeneity of the disease, such as different inflammatory phenotypes. Existing biomarkers, such as C-reactive protein (CRP) in the blood, lack specificity and cannot meet the needs of accurate COPD diagnosis.

[0005] Dimethyl carbonate (DMC) is a colorless, transparent liquid with a pungent odor, its molecular formula being C3H6O3. It possesses good solubility and reactivity. Industrially, DMC is widely used in organic synthesis, as a solvent, and in gasoline additives. In the pharmaceutical field, DMC is primarily used as a methylating and carbonylating agent in drug synthesis. Currently, there are no reports of DMC serving as a specific diagnostic biomarker for COPD, and existing technologies do not address the association between DMC and the pathophysiological processes of COPD. Summary of the Invention

[0006] To address the gaps in existing technologies, the present invention aims to provide the application of dimethyl carbonate as a diagnostic biomarker for chronic obstructive pulmonary disease (COPD). The present invention provides a method for screening or assisting in the diagnosis of COPD based on the detection of dimethyl carbonate in exhaled breath, specifically, determining the risk of COPD by detecting the presence of dimethyl carbonate in a subject's exhaled breath sample.

[0007] This invention provides the application of reagents for detecting dimethyl carbonate in the preparation of auxiliary diagnostic or screening kits for chronic obstructive pulmonary disease.

[0008] Furthermore, the reagents for detecting dimethyl carbonate include reagents selected from those used for chromatographic, mass spectrometric, or chromatographic-mass spectrometric detection, and reagents for sensor detection.

[0009] Furthermore, the reagent for detecting dimethyl carbonate is a reagent used for detection by full two-dimensional gas chromatography-time-of-flight mass spectrometry.

[0010] Furthermore, the reagent for detecting dimethyl carbonate is a reagent used for thermal desorption-gas chromatography-mass spectrometry (GC-MS) detection.

[0011] Furthermore, the detection of dimethyl carbonate refers to the detection of dimethyl carbonate in exhaled breath.

[0012] The present invention also provides the application of the device for detecting dimethyl carbonate in the preparation of auxiliary diagnostic or screening devices for chronic obstructive pulmonary disease.

[0013] Furthermore, the equipment includes a gas chromatograph and a mass spectrometer.

[0014] Furthermore, the gas chromatograph is a fully two-dimensional gas chromatograph.

[0015] Furthermore, the mass spectrometer is a time-of-flight mass spectrometer.

[0016] Furthermore, the detection of dimethyl carbonate refers to the detection of dimethyl carbonate in exhaled breath.

[0017] The present invention has achieved the following beneficial effects: This invention is the first to discover that the detection rate of dimethylcarbonate (DMC) in the exhaled breath of patients with chronic obstructive pulmonary disease (COPD) is significantly higher than that in healthy controls (chi-square test). P = 8.633162e-114), indicating that the detection of dimethyl carbonate is significantly correlated with the occurrence and development of COPD.

[0018] Furthermore, diagnostic analysis based on dimethyl carbonate detection showed that this biomarker has good discriminative ability in distinguishing COPD patients from healthy individuals. Receiver operating characteristic (ROC) curve analysis indicated that dimethyl carbonate has extremely high specificity and a certain degree of sensitivity, making it effective for the auxiliary diagnosis of COPD and demonstrating good diagnostic efficacy.

[0019] Compared with existing technologies that rely on multiple biomarkers for joint modeling, the dimethyl carbonate described in this invention can effectively identify COPD as a single biomarker, which not only simplifies the detection process but also reduces detection costs, making it more conducive to clinical application.

[0020] The key to this invention is that it has for the first time determined that the detection of dimethyl carbonate in exhaled breath is significantly associated with chronic obstructive pulmonary disease (COPD). Therefore, the detection of dimethyl carbonate in exhaled breath samples can serve as an important basis for determining whether a subject has COPD.

[0021] In specific embodiments, the detection of dimethyl carbonate in exhaled breath can be achieved using various volatile organic compound (VOC) analysis methods disclosed in the prior art. In this embodiment, thermal desorption-two-dimensional gas chromatography-time-of-flight mass spectrometry (TD-GC×GC-TOF-MS) is used to analyze exhaled breath samples, but the invention is not limited to this detection method. Any technical means capable of detecting or identifying dimethyl carbonate in exhaled breath, including but not limited to gas chromatography-mass spectrometry (GC-MS), sensor detection technology, or other VOC detection methods, can be applied to the screening or auxiliary diagnosis of chronic obstructive pulmonary disease (COPD).

[0022] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention.

[0023] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following embodiments. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description

[0024] Figure 1 The specific sample size for the COPD patient group and the healthy control group in this experiment.

[0025] Figure 2 The detection rate of dimethyl carbonate in the two groups is given.

[0026] Figure 3 The specific dimethyl carbonate detection status for individuals in the COPD group and the healthy group.

[0027] Figure 4 This is a receiver operating characteristic (ROC) curve for dimethyl carbonate (DMC) used in the diagnosis of COPD. Detailed Implementation

[0028] The raw materials and equipment used in this invention are all known products, obtained by purchasing commercially available products.

[0029] The main instruments and reagents involved in the embodiments of this invention are as follows: (1) Exhaled breath collection device: BioVOC sampling canister, TC-20™ thermal desorption instrument, Markes Bio-monitoring adsorption tube; MARKES international Gas03 / CSLR device.

[0030] (2) Exhaled breath testing device: Fully automated extraction, enrichment, concentration and injection system: MARKES Centri®; Modem (INSIGHT); Gas chromatography-time-of-flight mass spectrometry: Agilent 8890 (Agilent Technologies, USA); BenchTOF2™ time-of-flight mass spectrometry analysis system (TOF-MS).

[0031] (3) Reagents and materials: VOCs standards: 1,4-dichlorobenzene sample - d4 (Purity ≥ 99.5%, Sigma-Aldrich, USA); High-purity nitrogen (purity 99.999%), high-purity helium (purity 99.999%).

[0032] Example 1: Differences in dimethyl carbonate (DMC) levels in exhaled breath between COPD patients and healthy controls This embodiment aims to screen for specific biomarkers that can be used to diagnose COPD by comparing the VOCs spectra in the exhaled air of COPD patients and healthy controls.

[0033] 1. Research Subjects This embodiment recruited COPD patients and healthy controls from the Health Management Center of West China Hospital of Sichuan University between 2024 and 2026, including 436 COPD patients and 1000 healthy controls. Figure 1 The study protocol was approved by the hospital's ethics committee (ethics number: 2023-642), and all participants signed informed consent forms.

[0034] For healthy individuals, inclusion criteria are: adults aged 20-80 years; long-term residents of Sichuan Province (≥5 years); consent to exhaled VOC testing and signing of an informed consent form. Exclusion criteria are: based on medical history information, no respiratory diseases such as lung cancer, asthma, chronic obstructive pulmonary disease, or tuberculosis; no gastrointestinal diseases such as colorectal cancer, colitis, gastritis, or esophagitis; and for diabetic patients, a comprehensive determination based on fasting blood glucose (<6.1 mmol / L), glycated hemoglobin (<5.7%), and a diabetes history questionnaire.

[0035] For COPD patients, inclusion criteria were: age 20-80 years, a history of chronic cough, sputum production, wheezing, and exposure to harmful factors, and a confirmed forced expiratory volume in one second (FEV1) / forced vital capacity <0.7 after spirometry, meeting the COPD diagnostic criteria of the "Guidelines for the Diagnosis and Treatment of Chronic Obstructive Pulmonary Disease"; informed consent was provided. Exclusion criteria were: based on current medical history, no respiratory diseases such as lung cancer, asthma, and tuberculosis; no gastrointestinal diseases such as colorectal cancer, colitis, gastritis, and esophagitis; and non-diabetic patients.

[0036] 2. Exhaled breath sample collection and testing The detection of VOCs in human exhaled breath employs a thermal desorption-two-dimensional gas chromatography-time-of-flight mass spectrometry (TD-GC×GC-TOF-MS) method for comprehensive detection and analysis. The specific operational steps are as follows: a. Adsorption tube aging: The Markes Bio-monitoring adsorption tubes (material) were aged using a TC-20™ thermal desorption instrument. Aging conditions: 335℃, 60 min.

[0037] b. Exhaled Breath Collection: The BioVOC sampling device was used to collect exhaled breath samples from the subjects. Each subject was sampled three times, with each sample containing 129 mL, for a total volume of 129 mL × 3 = 387 mL. Environmental samples were also collected simultaneously from the subjects.

[0038] c. Sample adsorption: The collected exhaled air is injected into the aged MarkesBio-monitoring adsorption tube through the BioVOC sampling device.

[0039] d. Standard control and quality control: Accurately measure 5 μL of a 0.4 mM 1,4-dichlorobenzene sample. d4The standard sample was slowly injected into the adsorption tube using a microsyringe, and then purged with high-purity nitrogen at a pressure of 10 psi using a MARKES international Gas03 / CSLR device. The purging time was 20 s, thus establishing a standard control system for sample quality control.

[0040] e. Thermal Desorption: The fully automated extraction, enrichment, and concentration platform (MARKES Centri®) was used to perform thermal desorption on the exhaled gas samples enriched in the adsorption tube. First, the Dry purge process was initiated: the adsorption tube containing the exhaled gas sample was connected to a pre-defined flow path, and high-purity nitrogen or helium (99.999% purity or higher) was introduced at a flow rate of 50 mL / min at room temperature for 6 minutes. The carrier gas carried away and expelled the high moisture content (exhaled gas humidity is typically >90%) and unadsorbed light impurities (such as air and low-boiling-point inert gases) remaining in the adsorption tube. Then, the Tube Desorption stage began: the system's heating module rapidly enveloped the adsorption tube, heating it to 280°C, while a carrier gas was introduced at a flow rate of 50 mL / min for 8 minutes. This allowed the VOCs enriched in the exhaled gas to be completely desorbed under high temperature and carried by the carrier gas to the subsequent cold trap module. Next, the cold trap adsorption process is initiated: the system first cools the cold trap, which contains a built-in highly selective adsorbent (graphitized carbon black), to a set low temperature of 20°C through electronic cooling or liquid nitrogen cooling modules and keeps it stable. The "VOCs-carrier gas mixture" generated by desorption is continuously pushed into the cold trap by the carrier gas for 1 minute. During this time, the VOCs are rapidly captured and focused by the cold trap adsorbent due to the low temperature, while the carrier gas and trace amounts of residual moisture are discharged along the cold trap outlet exhaust pipe, thus achieving the separation of VOCs from the carrier gas and impurities. Finally, a second desorption process is performed in the cold trap: After the cold trap adsorption is completed, the system rapidly raises the temperature of the cold trap from a low temperature to 300℃ at the maximum heating rate, so that the VOCs enriched in the cold trap are rapidly desorbed within 5 minutes. At the same time, the carrier gas is started to split the desorbed high-concentration VOCs and directionally transfer them to the two-dimensional gas chromatography-mass spectrometry (GC×GC-MS) instrument at a rate of 5 mL / min. This completes the entire sample extraction, enrichment, concentration and injection process, laying the foundation for the accurate detection of ppb-ppt level VOCs in exhaled breath.

[0041] f. Gas phase separation: Exhaled VOCs from the cold trap were detected using an Agilent 8890 two-dimensional gas chromatograph. The one-dimensional column was a polar column, model MEGA-WAX HT, 20 m × 0.18 mm × 0.18 μm, with a flow rate of 0.5 mL / min and a pressure of 33.641 psi. The two-dimensional column was a non-polar column, model MEGA-5 MS, 5 m × 0.25 mm × 0.10 μm, with a flow rate of 20 mL / min and a pressure of 27.085 psi. The column temperature program was set to an initial temperature of 35 °C, held for 4 min, then increased to 240 °C at a rate of 6.5 °C / min and held for 1 min, for a total run time of 36.538 min. The carrier gas was high-purity helium (purity ≥99.999%). After thermal desorption, VOCs first enter a one-dimensional chromatographic column, where they are initially separated according to their polarity differences. The separated components are then carried by the carrier gas into the INSIGHT modem, which is heated by periodic pulses with a modulation period of 3 seconds. This process cuts the components eluting from the one-dimensional column into narrow bands, which are then rapidly transferred to a two-dimensional chromatographic column. In the two-dimensional column, the components are further separated according to their boiling point differences, and the finally separated VOCs components are sequentially introduced into the mass spectrometer.

[0042] g. Quality Inspection and Qualitative and Quantitative Analysis: The separated samples were detected using a BenchTOF2™ (TOF-MS) time-of-flight mass spectrometry system via tandem ionization at electron bombardment energies of 14 eV and 70 eV. The filament voltage was 1.6 V, the scanned mass range was 33–350 m / z, the acquisition frequency was 50 GHz, the transfer line temperature was 250 °C, and the ion source temperature was 250 °C. The measured mass spectrometry data were compared with data from the NIST spectral library to perform qualitative analysis of VOCs in the samples; accurate quantitative analysis of VOCs was performed using the internal standard method to obtain the content information of each VOC. Ion source: Electron impact ionization (EI) source, electron energy 70 eV, ion source temperature 290℃, filament current 25 μA; transmission line temperature 290℃; Time-of-flight analyzer: flight tube length 1.2 m, accelerating voltage 12 kV, delayed extraction time 6 μs; Detector: Microchannel plate (MCP), detector voltage 2.5 kV; Data acquisition: mass range 40-600 m / z, acquisition frequency 6 GHz; Mass calibration: external standard method (standards are hexafluorobenzene, m / z 186.00; caffeine, m / z 194.08), mass accuracy ≤4 ppm.

[0043] h. Visualization Analysis and Data Processing: The ChromCompare+ system was used to perform spectral alignment, background subtraction, and 3D spectrum construction on the measured exhaled breath samples, obtaining VOC retention times, characteristic ions, and VOC molecular data. Finally, the VOC data was processed using methods such as filtering, screening, and normalization.

[0044] 3. Quality Control a. Geographical control: A single-region (Sichuan Province) population inclusion strategy was adopted to eliminate the potential interference of differences in climate characteristics, atmospheric background VOCs concentration, dietary habits and living environment between different regions on the test results.

[0045] b. Standardization of sampling time: The exhaled breath sampling period is uniformly set from 8:00 to 10:00 every morning, and subjects are required to be in a fasting state (fasting time ≥ 8 hours).

[0046] c. Dietary control of subjects: Subjects are required to refrain from consuming foods with strong volatility or special odors (such as garlic, onions, leeks, chili peppers, curry, alcoholic beverages, etc.) within 24 hours (the day before) prior to sampling.

[0047] d. Standardization of experimental procedures and sample processing: Use the same model of exhaled breath sampling device to avoid VOCs adsorption / desorption deviations caused by differences in device materials or specifications; develop SOPs for each step, including the use of exhaled breath VOCs collection devices, operating environment requirements, and sample collection procedures; train researchers to ensure standardized operation and reduce human error; all exhaled breath VOCs are adsorbed in the adsorption tube (with an effective storage period set at 5 days), stored at room temperature, and samples are tested using the same machine and parameters within 5 days.

[0048] e. Blank control and background subtraction: To eliminate the pollution interference of atmospheric VOCs in the sampling environment on exhaled air samples, blank air samples (using the same sampling device, storage conditions and testing procedures as exhaled air samples) should be collected simultaneously during daily sample testing as blank controls. During the sample data analysis stage, the VOCs detection signal values ​​of all exhaled air samples should be uniformly subtracted from the corresponding blank control signal values ​​to obtain the true endogenous exhaled air VOCs level of the subjects.

[0049] 4. Differential VOC screening This study screened for differences in VOCs among different subgroups based on the detection of exhaled volatile organic compounds (VOCs).

[0050] Specifically, the signals of each VOC in the sample are first converted into detection status (i.e. whether it is detected, denoted as a binary variable, 1 for detection and 0 for non-detection), and a contingency table is constructed based on this to compare the detection differences of VOCs between different groups.

[0051] For each VOC, the number of people detected and the total sample size in each group were counted, and the detection rate (i.e., the proportion of samples in which a certain VOC was detected in the corresponding group) was calculated. In the statistical analysis, Fisher's exact test was used when any expected frequency in the contingency table was less than 5; otherwise, the Chi-square test was used, and p < 0.05 was used as the criterion for determining the significance of the difference.

[0052] Meanwhile, to quantify the degree of difference in VOC detection among different groups, the detection rate fold change (DR-FC) between groups was calculated. When a VOC meets the criteria of statistical significance (p < 0.05) and significant difference in detection rate between different groups, it is identified as a differential VOC.

[0053] 5. Experimental Results The results showed that the detection rates of various volatile organic compounds (VOCs) in the exhaled breath of the COPD patient group (n=436) were significantly different compared with those of the healthy control group (n=1000). Among them, the detection rate of dimethyl carbonate (DMC) in the exhaled breath of COPD patients was significantly higher than that in the healthy control group.

[0054] Statistical analysis showed that the difference in DMC detection between the two groups was highly significant (Chi-square test). P = 8.633162e-114), the relevant results are as follows Figure 2 As shown in the figure. This result indicates that the detection of DMC is significantly correlated with the occurrence and development of COPD.

[0055] This study is the first to discover a significantly increased detection rate of dimethyl carbonate in COPD patients and establish an association between it and COPD. Figure 3 Given its extremely low detection rate in healthy individuals, DMC suggests high specificity and may serve as a potential biomarker for COPD diagnosis.

[0056] Example 2: Evaluation of the efficacy of dimethyl carbonate (DMC) in diagnosing chronic obstructive pulmonary disease 1. Experimental Methods This embodiment uses a sample set independent of Example 1 to verify diagnostic efficacy. The sample source, inclusion and exclusion criteria, ethical approval, and informed consent procedures are all the same as in Example 1. The COPD patient group consisted of 108 patients, and the healthy control group consisted of 249 patients. The specific operational steps for exhaled breath sample collection, thermal desorption-two-dimensional gas chromatography-time-of-flight mass spectrometry (TD-GC×GC-TOF-MS) detection, qualitative and quantitative analysis, and quality control were all as described in Example 1. After quantitatively obtaining the DMC content information in each sample using the internal standard method, receiver operating characteristic (ROC) curves were plotted using conventional methods in the art.

[0057] 2. Experimental Results In the ROC curve analysis, the area under the ROC curve (AUC) of exhaled DMC used to distinguish COPD patients from healthy controls was 0.708, with a sensitivity of 0.416 and a specificity of 1.0. These results indicate that DMC has high diagnostic value for COPD, especially demonstrating excellent specificity, and can be applied to the screening or auxiliary diagnosis of chronic obstructive pulmonary disease.

[0058] This embodiment validates the feasibility of exhaled dimethyl carbonate (DMC) as a diagnostic biomarker for COPD using an independent sample set. Unlike the discovery sample set in Example 1, this embodiment uses an independent validation sample set to further confirm the good diagnostic efficacy of DMC in distinguishing between COPD patients and healthy individuals, especially demonstrating its extremely high specificity. Combined with its significantly increased detection rate in COPD patients and its extremely low detection rate in healthy individuals, exhaled dimethyl carbonate (DMC) can effectively distinguish between COPD patients and healthy individuals, and has the potential to serve as a non-invasive diagnostic biomarker for COPD.

[0059] In summary, this invention provides the application of dimethyl carbonate as a diagnostic biomarker for chronic obstructive pulmonary disease (COPD). This invention is the first to discover a significant correlation between the detection rate of dimethyl carbonate in exhaled breath and COPD; the detection rate of dimethyl carbonate in the exhaled breath of COPD patients is significantly higher than that in healthy individuals. ROC curve analysis results show that dimethyl carbonate, as a biomarker for diagnosing COPD, has extremely high specificity. Therefore, it can be used as a detection target for the diagnosis of COPD patients, demonstrating good practical application value.

Claims

1. Application of reagents for detecting dimethyl carbonate in the preparation of auxiliary diagnostic or screening kits for chronic obstructive pulmonary disease.

2. The application according to claim 1, characterized in that, The reagents for detecting dimethyl carbonate include those selected from reagents used for chromatographic, mass spectrometric, or chromatographic-mass spectrometric detection, and reagents for sensor detection.

3. The application according to claim 1, characterized in that, The reagent used to detect dimethyl carbonate is a reagent used for detection by two-dimensional gas chromatography-time-of-flight mass spectrometry.

4. The application according to claim 1, characterized in that, The reagent used to detect dimethyl carbonate is a reagent used for thermal desorption-gas chromatography-mass spectrometry (GC-MS).

5. The application according to any one of claims 1 to 4, characterized in that, The detection of dimethyl carbonate refers to the detection of dimethyl carbonate in exhaled breath.

6. Application of equipment for detecting dimethyl carbonate in the preparation of auxiliary diagnostic or screening equipment for chronic obstructive pulmonary disease.

7. The application according to claim 6, characterized in that, The equipment includes a gas chromatograph and a mass spectrometer.

8. The application according to claim 7, characterized in that, The gas chromatograph is a full two-dimensional gas chromatograph.

9. The application according to claim 7, characterized in that, The mass spectrometer is a time-of-flight mass spectrometer.

10. The application according to any one of claims 6 to 9, characterized in that, The detection of dimethyl carbonate refers to the detection of dimethyl carbonate in exhaled breath.

Images