Exhaled air condensate metabolite combination, kit and method for predicting benign and malignant pulmonary nodules
By detecting 13 small molecule metabolites in the exhaled condensate and applying binary logistic regression analysis model, the problem of insufficient sensitivity and specificity of benign and malignant pleuritis in the prior art was solved, and a significant improvement in high sensitivity and high specificity was achieved.
Patent Information
- Application Number
- CN202411654169.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-19
- Publication Date
- 2025-03-18
AI Technical Summary
The prior art has low sensitivity and insufficient specificity in diagnosing and differentiating benign and malignant pleuritis, and the examination method is traumatic and has high risk, which cannot meet clinical needs.
The combination of 13 small molecule metabolites in the exhaled air condensate was used to detect the content of these metabolites by combining liquid chromatography and mass spectrometry. Combined with binary logistic regression analysis model, the probability of tuberculous pleurisitis was calculated to judge benign and malignant pulmonary nodules.
High sensitivity and specificity of benign and malignant pulmonary nodules are achieved, which improves the accuracy and efficiency of diagnosis, and reduces the traumatic and risk of examinations.
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Figure CN119643733A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to a method for predicting benign and malignant pulmonary nodules, and in particular to an exhaled breath condensate metabolite combination, a kit and a method for predicting benign and malignant pulmonary nodules. Background Art
[0002] Tuberculous pleurisy is a common extrapulmonary tuberculosis in clinical practice, with a high incidence rate, accounting for about 44.1% of all pleural diseases. The incidence and mortality rate of lung cancer are among the highest in the world, and about 40% of lung cancer patients will develop malignant pleural effusion during the course of the disease. At present, due to the limitations of diagnostic technology, the diagnosis rate of both is very low. However, the prognosis and treatment of tuberculous pleurisy and malignant pleurisy are completely different. If they are not diagnosed and treated in time, they will directly affect the patient's quality of life and survival. Therefore, how to quickly and accurately distinguish between benign and malignant pleurisy is a hot spot and difficulty in current clinical research.
[0003] At present, the methods for clinical diagnosis and identification of tuberculous pleurisy and malignant pleurisy include clinical manifestations, laboratory results of thoracentesis, pleural biopsy, bronchoscopy, surgical biopsy, etc. The most commonly used method is cytological examination by thoracentesis. If tumor cells are found in pleural effusion, it has diagnostic value for malignant pleurisy. However, due to the complex cell composition in pleural effusion, it is sometimes difficult to distinguish tumor cells from inflammatory mesothelial cells, macrophages, etc., and it is also affected by factors such as the level of the diagnostician and sample collection. Its sensitivity is low (about 40%-60%) and its subjectivity is strong, which can easily lead to missed diagnosis or misdiagnosis. The diagnosis of tuberculous pleurisy mainly includes biochemical examination of exhaled breath condensate, tuberculin skin test (TST), bacteriological examination of pleural effusion (acid-fast staining smear and Mycobacterium tuberculosis culture), pleural tissue pathological biopsy, etc. However, the above-mentioned examination methods have low specificity and sensitivity, and some methods are highly invasive, high-risk, or time-consuming, and cannot meet the needs of clinical work. Therefore, the development of new and highly sensitive pleurisy diagnosis technology is of great clinical significance. Summary of the invention
[0004] The purpose of the present invention is to overcome the deficiencies of the prior art and to provide an exhaled breath condensate metabolite combination, a kit and a method for predicting benign and malignant pulmonary nodules.
[0005] The technical solution of the present invention to solve the above technical problems is as follows:
[0006] The exhaled breath condensate metabolite panel for predicting benign or malignant pulmonary nodules includes adenosine phosphate, adenine nucleotide, adenine, cytidine monophosphate, deoxyguanosine monophosphate, phosphatidylcholine 35:2, cytidine cyclic monophosphate, hydroxyethylpiperazineethanesulfonic acid, methylaminoisobutyric acid, fructose phosphate disodium salt, citrulline, ribitol, and acetylcarnitine.
[0007] A kit for predicting benign or malignant pulmonary nodules comprises reagents required for detecting the contents of adenosine phosphate, adenine nucleotide, adenine, cytidine monophosphate, deoxyguanosine monophosphate, phosphatidylcholine 35:2, cytidine cyclic monophosphate, hydroxyethylpiperazineethanesulfonic acid, methylaminoisobutyric acid, fructose phosphate disodium salt, citrulline, ribitol and acetylcarnitine in an extraction solution of an exhaled breath condensate sample.
[0008] Furthermore, the kit adopts liquid chromatography-mass spectrometry technology, and the reagents include methanol, acetonitrile and isopropanol.
[0009] The method for predicting benign or malignant pulmonary nodules comprises the following steps:
[0010] Step 1, taking an exhaled breath condensate sample of a subject to extract a solution, and detecting the content of adenosine phosphate in the extract to be a, the content of adenine nucleotide to be b, the content of adenine to be c, the content of cytidine monophosphate to be d, the content of deoxyguanosine monophosphate to be e, the content of phosphatidylcholine to be f, the content of cytidine cyclic monophosphate to be g, the content of hydroxyethylpiperazineethanesulfonic acid to be h, the content of methylaminoisobutyric acid to be j, the content of fructose phosphate disodium salt to be k, the content of citrulline to be l, the content of ribitol to be m, and the content of acetylcarnitine to be n, and the contents are all mass fractions;
[0011] Step 2: Calculate the probability of tuberculous pleurisy Prob. If Prob < 0.5, the subject is judged to have tuberculous pleurisy. The calculation method of the probability of tuberculous pleurisy is: Prob = 1 / (1 + e -X ), where X = 10.695a + 1.182b - 42.394c - 3.919d + 176.644e - 1671.240f + 775.353g - 1370.690h + 985.416.
[0012] The beneficial effects of the present invention are as follows: metabolites adenosine phosphate, adenine nucleotide, adenine, cytidine monophosphate, deoxyguanosine monophosphate, phosphatidylcholine 35:2, cytidine cyclic monophosphate, hydroxyethylpiperazineethanesulfonic acid, methylaminoisobutyric acid, fructose phosphate disodium salt, citrulline, ribitol, and acetylcarnitine in exhaled breath condensate can be used in combination for the identification of patients with benign and malignant pulmonary nodules. The combined markers of the present invention can provide high sensitivity and high specificity for the diagnosis of pleural diseases. BRIEF DESCRIPTION OF THE DRAWINGS
[0013] Figure 1It is a schematic diagram of the changes in the contents of adenosine phosphate, adenine nucleotide, adenine, cytidine monophosphate, deoxyguanosine monophosphate, phosphatidylcholine 35:2, cytidine cyclic monophosphate, hydroxyethylpiperazineethanesulfonic acid, methylaminoisobutyric acid, fructose phosphate disodium salt, citrulline, ribitol and acetylcarnitine in the exhaled breath condensate of patients with benign and malignant pulmonary nodules according to Example 1 and Example 2 (expressed as mean ± standard error);
[0014] Figure 2 This is the ROC curve of the combined marker in distinguishing benign and malignant pulmonary nodule disease groups. DETAILED DESCRIPTION
[0015] The principles and features of the present invention are described below. The examples given are only used to explain the present invention and are not used to limit the scope of the present invention.
[0016] The 13 small molecule metabolites in the exhaled breath condensate determined by the present invention: adenosine phosphate, adenine nucleotide, adenine, cytidine monophosphate, deoxyguanosine monophosphate, phosphatidylcholine 35:2, cytidine cyclic monophosphate, hydroxyethylpiperazineethanesulfonic acid, methylaminoisobutyric acid, fructose phosphate disodium salt, citrulline, ribitol, acetylcarnitine are all important metabolites in the human body. The present invention combines the above 13 metabolites for the first time and applies them to the diagnosis of benign and malignant pulmonary nodules. The mass spectrometry targeted analysis ion information of the combination is shown in Table 1 below.
[0017] Table 1. Information table of targeted analysis of 13 combination markers
[0018]
[0019] The detection method according to the kit is as follows:
[0020] (1) Exhaled breath condensate sample pretreatment method:
[0021] The exhaled breath condensate sample was thawed at 4°C, 100 μL of it was taken, and 400 μL of methanol extract containing multiple internal standards was added to precipitate the protein: vortexed for 30-90s, allowed to stand for 15-30mins, centrifuged at 10000-14000×g for 10-15mins at 4°C, and the supernatant was freeze-dried; 50uL of 10-20% (v / v) methanol aqueous solution was added, and the supernatant was centrifuged at 10000-14000×g for 10-15mins at 4°C, and the supernatant was analyzed by LC-MS / MS.
[0022] (2) Targeted detection of target metabolites using liquid chromatography-mass spectrometry:
[0023] The separation system is ultra-high performance liquid chromatography, the chromatographic column is C8, the mobile phase flow rate is 0.2-0.35ml / min, the column temperature is 40-60 degrees Celsius, and the injection volume is 5-10ul (C18 chromatographic column can also be used, the column temperature is 40-60 degrees Celsius, and the flow rate is 0.2-1.0ml / min); the eluent is an aqueous solution of 0.1% (v / v) formic acid in phase A, and an acetonitrile solution of 0.1% (v / v) formic acid in phase B (0-0.1% (v / v) formic acid aqueous solution and acetonitrile (methanol) solution eluent can also be selected, but the sensitivity is relatively lower); the internal standard is usually selected as an isotope, and other commonly used internal standards for mass spectrometry detection can also be used. The mass spectrometer used by the detector is a highly sensitive single quadrupole mass spectrometer or a tandem quadrupole mass spectrometer (Q-trap MS, QQQ MS), and the positive ion mode is detected. The mass spectrometry targeted analysis ion information of this combination is shown in Table 1.
[0024] (3) Judgment model based on mass spectrometry data:
[0025] The obtained data were analyzed by binary logistic regression using SPSS software, and the regression equation obtained by the constructed model is as follows:
[0026] Prob = 1 / (1+e -X )
[0027] in:
[0028] X=10.695a+1.182b-42.394c-3.919d+176.644e-1671.240f+775.353g-1370.690h+985.416;
[0029] The exhaled breath condensate sample of the subject was taken to extract the solution, and the content of adenosine phosphate in the extract was detected to be a, the content of adenine nucleotide was b, the content of adenine was c, the content of cytidine monophosphate was d, the content of deoxyguanosine monophosphate was e, the content of phosphatidylcholine 35:2 was f, the content of cytidine cyclic monophosphate was g, the content of hydroxyethylpiperazineethanesulfonic acid was h, the content of methylaminoisobutyric acid was j, the content of fructose phosphate disodium salt was k, the content of citrulline was l, the content of ribitol was m, and the content of acetylcarnitine was n.
[0030] An exhaled breath condensate sample is collected from the subject, metabolites are extracted, and the contents of adenosine phosphate, adenine nucleotide, adenine, cytidine monophosphate, deoxyguanosine monophosphate, phosphatidylcholine 35:2, cytidine cyclic monophosphate, hydroxyethylpiperazineethanesulfonic acid, methylaminoisobutyric acid, fructose phosphate disodium salt, citrulline, ribitol, and acetylcarnitine in the exhaled breath condensate sample are quantified by mass spectrometry. Prob is the probability of benign or malignant pulmonary nodules, and Cutoff=0.5, that is, when the Prob value is less than 0.5, it is diagnosed as a benign pulmonary nodule.
[0031] The model has good discrimination ability for malignant pleurisy and tuberculous pleurisy. Figure 2 The AUC of the combined markers was 0.975, the sensitivity was 96.7%, and the specificity was 94.7%, indicating that the 13 combined markers have the potential to diagnose tuberculous pleurisy.
[0032] Example 1
[0033] Inclusion criteria for tuberculous pleurisy: patients with clinical manifestations of tuberculous pleurisy: low-grade fever, night sweats, fatigue, chest tightness, exudative pleural effusion, etc.; at the same time, the exhaled breath condensate and / or sputum culture is positive for Mycobacterium tuberculosis, and / or the pleural tissue pathology examination is positive for tuberculosis; and those who are effective in subsequent anti-tuberculosis treatment are diagnosed as patients with tuberculous pleurisy.
[0034] The inclusion criteria for the malignant pleurisy group were: exudative pleural effusion, tumor cells found in exfoliated cells of expired breath condensate, and / or pleural histopathological examination confirmed tumor pleural metastasis.
[0035] Exhaled breath condensate samples were used under the same conditions: 90 exhaled breath condensate samples from patients with malignant pleurisy and 30 exhaled breath condensate samples from patients with tuberculous pleurisy; 50 ml of exhaled breath condensate was collected from patients before any treatment after admission, and the exhaled breath condensate was separated within half an hour and stored in a -80℃ refrigerator for examination.
[0036] 2. Analytical methods
[0037] 2.1 Pretreatment of exhaled breath condensate samples
[0038] The exhaled breath condensate sample was thawed at 4°C, 100 μL of it was taken, and 400 μL of methanol extract containing multiple internal standards was added to precipitate the protein: vortexed for 30-90s, allowed to stand for 15-30mins, centrifuged at 10000-14000×g for 10-15mins at 4°C, and the supernatant was freeze-dried; 50uL of 10-20% (v / v) methanol aqueous solution was added, and centrifuged at 12000×g for 15mins at 4°C, and the supernatant was taken for LC-MS / MS analysis, and the peak areas of the internal standard peak and eight metabolites were calculated by the extracted ion flow chart to determine the contents of the eight metabolites. .
[0039] 2.2 Ultra-high performance liquid chromatography-mass spectrometry analysis
[0040] (1) Liquid phase conditions: The chromatograph was a Nexera LC-30AD ultra-high performance liquid chromatograph (Shimadzu, Kyoto, Japan); the chromatographic column was a Waters ACQUITY UPLC@BEH C8 (1.7 um, 2.1 mm × 100 mm) (Waters, Ireland); the mobile phase A was a mobile phase: phase A was an aqueous solution containing 0.1% (v / v) formic acid, and phase B was a 0.1% (v / v) formic acid acetonitrile solution; the elution gradient was: 2% phase B from 0 to 1 min, linearly changed to 80% phase B from 1 min to 20 min, linearly changed to 100% phase B from 20 min to 25 min, the phase was maintained for 4 min, and linearly decreased to 2% phase B within 0.1 min and maintained for 1 min; the column temperature was 50 °C; the mobile phase flow rate was 0.35 mL / min; the injection volume was 10 μL.
[0041] (2) Mass spectrometry conditions: The mass spectrometer was a quadrupole ion trap mass spectrometer (Q-Trap 5500MS) (AB SCI EX, Framingham, MA, USA); positive ion mode detection was adopted; the curtain gases, GAS1, and GAS2 were set to 50 arb itrary units, 35 arb itrary units, and 35 arb itrary units, respectively; the ion source temperature was 500°C, and the spray voltage was 5.0 kV.
[0042] 2.3 Exhaled breath condensate test results and auxiliary diagnosis methods
[0043] For the malignant pleurisy group, adenosine phosphate, adenine nucleotide, adenine, cytidine monophosphate, deoxyguanosine monophosphate, phosphatidylcholine 35:2, cytidine cyclic monophosphate, hydroxyethylpiperazineethanesulfonic acid, methylaminoisobutyric acid, fructose phosphate disodium salt, citrulline, ribitol, and acetylcarnitine were significantly changed (see Figure 1 ). At the same time, the content of each marker was substituted into the regression equation to calculate the probability. The cutoff value used was 0.5, that is, if the probability of the combined marker was less than 0.5, it was considered to be a benign pulmonary nodule. The AUC of the combined marker was 0.975, and the sensitivity and specificity were also relatively high, at 96.7% and 94.7%, respectively (see Figure 2 ).
[0044] Example 2
[0045] In order to verify the stability and reliability of the combined markers, the sample collection source was changed and external independent samples were used for verification. The analytical methods, instruments and reagents used in this experiment were completely consistent with those in Example 1.
[0046] 1. Before collecting exhaled breath condensate samples, volunteers were required to sign an informed consent form.
[0047] Inclusion criteria for tuberculous pleurisy: patients with clinical manifestations of tuberculous pleurisy: low-grade fever, night sweats, fatigue, chest tightness, exudative pleural effusion, etc.; at the same time, the exhaled breath condensate and / or sputum culture is positive for Mycobacterium tuberculosis, and / or the pleural tissue pathology examination is positive for tuberculosis; and those who are effective in subsequent anti-tuberculosis treatment are diagnosed as patients with tuberculous pleurisy.
[0048] The inclusion criteria for the malignant pleurisy group were: exudative pleural effusion, tumor cells found in exfoliated cells of expired breath condensate, and / or pleural histopathological examination confirmed tumor pleural metastasis.
[0049] Exhaled breath condensate samples were used under the same conditions: exhaled breath condensate samples from 50 patients with malignant pleurisy and 15 patients with tuberculous pleurisy; 50 ml of exhaled breath condensate was collected from the patients before any treatment after admission, and the exhaled breath condensate was separated within half an hour and stored in a -80°C refrigerator for examination.
[0050] 2. Exhaled breath condensate test results and auxiliary diagnosis methods
[0051] For the malignant pleurisy group, adenosine phosphate, adenine nucleotide, adenine, cytidine monophosphate, deoxyguanosine monophosphate, phosphatidylcholine 35:2, cytidine cyclic monophosphate, hydroxyethylpiperazineethanesulfonic acid, methylaminoisobutyric acid, fructose phosphate disodium salt, citrulline, ribitol, and acetylcarnitine were significantly changed (see Figure 1 ). At the same time, the content of each marker was substituted into the regression equation to calculate the probability. The cutoff value used was 0.5, that is, if the probability of the combined marker was less than 0.5, it was considered to be a benign pulmonary nodule. The AUC of the combined marker was 0.972, and the sensitivity and specificity were also relatively high, at 1.00% and 88.3%, respectively (see Figure 2 ).
[0052] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims
1. A combination of exhaled breath condensate metabolites for predicting benign and malignant pulmonary nodules, characterized in that: Including adenosine phosphate, adenine nucleotide, adenine, cytidine monophosphate, deoxyguanosine monophosphate, phosphatidylcholine, cytidine cyclic monophosphate, hydroxyethylpiperazineethanesulfonic acid, methylaminoisobutyric acid, fructose phosphate disodium salt, citrulline, ribitol and acetylcarnitine.
2. A kit for predicting benign and malignant pulmonary nodules, characterized in that: The invention comprises reagents required for detecting the contents of adenosine phosphate, adenine nucleotide, adenine, cytidine monophosphate, deoxyguanosine monophosphate, phosphatidylcholine, cytidine cyclic monophosphate, hydroxyethylpiperazineethanesulfonic acid, methylaminoisobutyric acid, fructose phosphate disodium salt, citrulline, ribitol and acetylcarnitine in the extraction solution of the exhaled breath condensate sample.
3. The kit for predicting benign and malignant pulmonary nodules according to claim 2, characterized in that: The method adopts liquid chromatography-mass spectrometry technology, and the reagents include methanol, acetonitrile and isopropanol.
4. A method for predicting benign and malignant pulmonary nodules, characterized in that: The following steps are involved: Step 1, taking an exhaled breath condensate sample of a subject to extract a solution, and detecting the content of adenosine phosphate in the extract to be a, the content of adenine nucleotide to be b, the content of adenine to be c, the content of cytidine monophosphate to be d, the content of deoxyguanosine monophosphate to be e, the content of phosphatidylcholine to be f, the content of cytidine cyclic monophosphate to be g, the content of hydroxyethylpiperazineethanesulfonic acid to be h, the content of methylaminoisobutyric acid to be j, the content of fructose phosphate disodium salt to be k, the content of citrulline to be l, the content of ribitol to be m, and the content of acetylcarnitine to be n, and the contents are all mass fractions; Step 2: Calculate the probability of tuberculous pleurisy Prob. If Prob < 0.5, the subject is judged to have tuberculous pleurisy. The calculation method of the probability of tuberculous pleurisy is: Prob = 1 / (1 + e -X ), where X = 10.695a + 1.182b - 42.394c - 3.919d + 176.644e - 1671.240f + 775.353g - 1370.690h + 985.416.