Use of reagent for detecting n-acetyl-l-cysteine in preparation of diagnostic product for ischemic brain injury

Abstract

The application provides application of a reagent for detecting N-acetyl-L-cysteine in preparation of an ischemic brain injury diagnosis product, and relates to the technical field of in-vitro detection.The application provides a biomarker N-acetyl-L-cysteine (NAC) for ischemic brain injury diagnosis.ROC curve analysis shows that NAC has good distinguishing ability for ischemic brain injury, and can be used as a potential marker for ischemic brain injury.

Description

Technical Field

[0001] This invention relates to the field of G01N33 / 68, and more particularly to the application of reagents for detecting N-acetyl-L-cysteine ​​in the preparation of diagnostic products for ischemic brain injury. Background Technology

[0002] Ischemic brain injury refers to a group of central nervous system diseases caused by impaired blood supply to the brain (such as cerebral artery stenosis, thrombosis, or embolism), leading to local brain tissue ischemia and hypoxia, which in turn causes nerve cell damage, necrosis, and neurological dysfunction. It is commonly seen in acute ischemic stroke (AIS) and hypoxic-ischemic encephalopathy (HIE). The brain is highly sensitive to hypoxia; ischemia and hypoxia rapidly disrupt the metabolic balance of nerve cells, leading to mild symptoms such as limb movement disorders and cognitive decline, and severe symptoms such as coma and even death, seriously threatening the patient's health and life.

[0003] Currently, the core methods for clinical diagnosis of ischemic brain injury include imaging examinations (head CT, MRI), electroencephalography (EEG), and brain function scales. While imaging examinations can directly visualize changes in brain tissue structure, they have limitations such as being time-consuming, reliant on large equipment, and difficult to perform rapid bedside testing. Furthermore, in the early stages of ischemia, lesions are not readily apparent, leading to missed diagnoses. EEG and brain function scales, on the other hand, are affected by factors such as the patient's level of consciousness and cooperation, resulting in insufficient objectivity and accuracy. Therefore, there is an urgent clinical need for minimally invasive, rapid, and highly specific diagnostic methods to achieve early disease identification and dynamic assessment of the condition.

[0004] Blood biomarkers have become a key focus of research in the diagnosis of ischemic brain injury due to their advantages of minimally invasive sampling, convenient testing, and continuous monitoring. Current research has identified several relevant blood biomarkers, such as S100B protein, neuron-specific enolase (NSE), and glial fibrillary acidic protein (GFAP). However, existing blood biomarkers still have significant limitations: firstly, their specificity is limited; some biomarkers are not exclusive to ischemic brain injury. For example, NSE is abnormally elevated in small cell lung cancer, and Tau protein is abnormally elevated in Alzheimer's disease, which can easily lead to misdiagnosis.

[0005] In summary, existing diagnostic methods and blood biomarkers for ischemic brain injury have many limitations, and there is an urgent need to find dedicated biomarkers with higher specificity and diagnostic efficacy. Summary of the Invention

[0006] The first aspect of the invention provides the use of reagents for detecting N-acetyl-L-cysteine ​​in the preparation of diagnostic products for ischemic brain injury, said products including kits, test strips, or detection chips.

[0007] A second aspect of the invention provides a product for the diagnosis of ischemic brain injury, the product comprising a reagent for detecting the content of N-acetyl-L-cysteine ​​in a sample.

[0008] Specifically, the testing steps for the product include: S1. Collect subject samples; S2. Detect the content of N-acetyl-L-cysteine ​​in the sample; S3. Based on the test results of S2, determine whether the subject has ischemic brain injury.

[0009] More specifically, in S1, the sample includes: plasma, whole blood, serum, or peripheral blood.

[0010] More specifically, in S2, the detection method includes spectrophotometry, liquid chromatography, high performance liquid chromatography-mass spectrometry, electrochemical method, or chemiluminescence method.

[0011] Preferably, the detection method is high performance liquid chromatography-mass spectrometry.

[0012] More preferably, the detection method is a sulfur-containing metabolite detection method and a modified metabolome detection method.

[0013] More specifically, in S3, the higher the N-acetyl-L-cysteine ​​content in the test results, the higher the probability that the subject has ischemic brain injury.

[0014] Preferably, the threshold value is NAC content exceeding 1.036 μm.

[0015] A third aspect of the present invention provides a system for diagnosing ischemic brain injury, the system comprising a detection device, a computing device, and an output device; Specifically, the detection device is used to collect subject samples and detect the N-acetyl-L-cysteine ​​content.

[0016] Specifically, the computing device is used to collect the detection results of N-acetyl-L-cysteine ​​content and compare them with the judgment criteria to determine whether the subject has cerebral ischemia injury.

[0017] Specifically, the output device is used to output the judgment of the computing device.

[0018] Compared with the prior art, the present invention has the following beneficial effects: 1. This invention uses N-acetyl-L-cysteine ​​as a diagnostic biomarker for ischemic brain injury. By detecting the N-acetyl-L-cysteine ​​levels in the blood of patients with ischemic brain injury and healthy individuals, it was found that there are significant differences in the N-acetyl-L-cysteine ​​levels in the blood of patients with ischemic brain injury compared to healthy individuals. In the early stages of ischemic brain injury, the N-acetyl-L-cysteine ​​levels in the blood are significantly upregulated.

[0019] 2. This invention provides a new and effective means for the early diagnosis and disease assessment of ischemic brain injury, which helps to improve the diagnosis and treatment of ischemic brain injury and has good clinical application prospects and social value. Attached Figure Description

[0020] Figure 1 This diagram illustrates blood extraction from patients with ischemic brain injury and healthy controls. A represents the reductive metabolome of sulfur-containing metabolites, and B represents the modified metabolome.

[0021] Figure 2 The results of multi-omics analysis of N-acetyl-L-cysteine ​​in patients with ischemic brain injury and healthy controls are shown below. A is the principal component analysis (PCA) plot of the reductive metabolome of sulfur-containing metabolites in healthy individuals (blue scatter dots) and patients with ischemic brain injury (red scatter dots); B is the volcano plot of the reductive metabolome of sulfur-containing metabolites in healthy individuals and patients with ischemic brain injury (Stroke vs Healthy); C is the heatmap of changed metabolites in the reductive metabolome of sulfur-containing metabolites in healthy individuals and patients with ischemic brain injury; D is the principal component analysis (PCA) plot of the modified metabolome in healthy individuals (blue scatter dots) and patients with ischemic brain injury (red scatter dots); E is the volcano plot of the modified metabolome in healthy individuals and patients with ischemic brain injury (Stroke vs Healthy); and F is the heatmap of changed metabolites in the modified metabolome in healthy individuals and patients with ischemic brain injury.

[0022] Figure 3 The graph shows the correlation analysis of N-acetyl-L-cysteine ​​for the diagnosis of ischemic brain injury. A represents the reductive metabolome of sulfur-containing metabolites in healthy individuals (blue scatter dots) and patients with ischemic brain injury (red scatter dots), while B represents the modified metabolome in healthy individuals (blue scatter dots) and patients with ischemic brain injury (red scatter dots). Detailed Implementation

[0023] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0024] To assess the correlation between metabolites and ischemic brain injury, this invention collected venous blood samples from 96 healthy controls and 133 patients with ischemic brain injury. Inclusion criteria for the screening group: age > 18 years, NIHSS score: 1-42, and confirmed acute infarction lesions on cranial MRI.

[0025] Exclusion criteria: severe cardiopulmonary insufficiency, liver and kidney failure or other major organ dysfunction, malignant tumors, severe infections, autoimmune diseases, etc., lack of complete head MRI, head CT showing hemorrhagic lesions, missing baseline data, etc.

[0026] This screening set included 133 plasma samples that met the above criteria, comprising 71 males and 62 females, aged 46-96 years, with a mean age of 73.54 ± 11.45 years. The sample size was determined through statistical estimation and was sufficient to meet the needs of preliminary screening for the target biomarkers, ensuring the reliability of the screening results.

[0027] Example 1: Detection and analysis of N-acetyl-L-cysteine ​​in the plasma of patients with ischemic stroke and healthy controls LC-MS analysis was performed using a Vanquish UHPLC system coupled with an Exploris 480 orbital trap mass spectrometer (Thermo Fisher Scientific, Inc., USA). Raw data were acquired using Xcalibur software (version 4.4.16.14).

[0028] 1. Liquid Chromatography (LC) conditions: A Waters ACQUITY UPLC BEH amide column (particle size 1.7 μm; column length 100 mm × inner diameter 2.1 mm) was used. Column temperature: 25℃; mobile phase A: 100% aqueous solution containing 25 mM ammonium acetate and 25 mM ammonium hydroxide; mobile phase B: 100% acetonitrile (applicable to both positive and negative ion electrospray ionization modes). Linear gradient elution program: 0.0–0.5 min to maintain 95% B; 0.5–7.0 min to decrease from 95% B to 65% B; 7.0–8.0 min to decrease from 65% B to 40% B; 8.0–9.0 min to maintain 40% B; 9.0–9.1 min to increase from 40% B back to 95% B; then maintain 95% B for 2.9 min. Flow rate: 0.5 mL / min; injection volume: 2 μL.

[0029] 2. Mass Spectrometry Conditions: Electrospray Ionization (ESI) Source Parameters: Positive ion mode spray voltage 3500 V, negative ion mode -2800 V; auxiliary gas heating temperature 350 °C; sheath gas pressure 50 arb; auxiliary gas pressure 15 arb; capillary temperature 400 °C. Data Acquisition Mode: All samples were acquired using full-scan polarity switching mode; quality control (QC) samples were additionally acquired using data-dependent secondary scan (ddMS²) mode for secondary mass spectrometry (MS / MS) spectroscopy. Full Scan Parameters: Orbital trap resolution 60,000; Automatic Gain Control (AGC) target value 1× Maximum injection time: 100 ms; Scan range: 70–1200 Da. ddMS² scan parameters: orbital trap resolution 30,000; AGC target value 1× Maximum injection time: 60 ms; Scan range: 50–1200 Da; Top N: 6; Isolation width: 1.0 m / z; Collision energy mode: stepped; Collision energy type: normalized; High-energy collision dissociation (HCD) collision energies: 20%, 30%, 40%; Dynamic exclusion time: 4 seconds after a single detection. The ion reaction used for quantitative analysis was m / z 164 → m / 122.0, which was acetylcysteine. Data acquisition was performed using Xcalibur software (version 4.4.16.14).

[0030] Reduction method for the detection and analysis of sulfur-containing metabolites: For plasma metabolite extraction, 100 μL of plasma sample was added to 400 μL of acetonitrile / methanol (1:1, v / v) containing 6.27 mM DTT (dithiothreitol) for extraction. The sample was vortexed for 30 seconds and then sonicated on ice for 10 minutes. To precipitate proteins, the sample was incubated at -20°C for 1 h, followed by centrifugation at 4°C and 17,500 × g for 15 minutes. The dried extract was stored at -80°C as previously described. Before liquid chromatography-mass spectrometry (LC-MS) analysis, the dried extract was reconstituted with acetonitrile / water (1:1, v / v), sonicated for 10 minutes, and then centrifuged at 4°C and 17,000 × g for 15 minutes to remove insoluble impurities. Finally, the supernatant was transferred to a high-performance liquid chromatography (HPLC) vial for LC-MS analysis.

[0031] Modified metabolomics detection method: For plasma metabolite extraction, 100 μL of plasma sample was added to 400 μL of acetonitrile / methanol (1:1, v / v) for extraction. The sample was vortexed for 30 seconds and then sonicated on ice for 10 minutes. To precipitate proteins, the sample was incubated at -20°C for 1 h, followed by centrifugation at 4°C and 17,500 × g for 15 minutes. The dried extract was stored at -80°C as previously described. For LC-MS analysis, the dried extract was reconstituted with acetonitrile / water (1:1, v / v), sonicated for 10 minutes, and then centrifuged at 4°C and 17,000 × g for 15 minutes to remove insoluble impurities. Finally, the supernatant was transferred to a high-performance liquid chromatography (HPLC) vial for LC-MS analysis.

[0032] Comprehensive analysis of the metabolomics of the samples revealed the presence of N-acetyl-L-cysteine, a metabolite associated with ischemic brain injury. (See schematic diagram below.) Figure 1 .

[0033] Example 2: Upregulation of N-acetyl-L-cysteine ​​levels in the plasma of patients with ischemic stroke N-acetyl-L-cysteine ​​was identified systematically using the 4D-Metabolomics platform (4DX) combined with metabolic response network analysis (metDNA). Differential metabolite screening employed rigorous multiple hypothesis testing correction, with a threshold set at a p-value <0.05 after false discovery rate (FDR) correction (Benjamini-Hochberg method). All quantitative data are expressed as mean ± standard error (mean ± SEM, n=6 biologically independent replicates). SEM calculations excluded technical replication interference to accurately reflect sampling error of the sample mean. This method enables comprehensive detection and accurate quantification of polar metabolites in plasma.

[0034] Statistical analysis was performed on the N-acetyl-L-cysteine ​​levels in the plasma of patients with ischemic brain injury and healthy controls. The experimental results are as follows: Figure 2 As shown.

[0035] The results showed that the plasma N-acetyl-L-cysteine ​​levels in patients with ischemic brain injury were significantly higher than those in healthy controls, indicating that N-acetyl-L-cysteine ​​can serve as a diagnostic biomarker for ischemic brain injury.

[0036] The diagnostic criterion is that an N-acetyl-L-cysteine ​​content exceeding 1.036 μm is considered ischemic brain injury.

[0037] Example 3: Diagnostic efficacy of N-acetyl-L-cysteine ​​as a diagnostic marker for ischemic brain injury The validation set and the screening set contained identical samples from different subjects, including 40 males and 56 females, aged 33-78 years, with a mean age of 55.28 ± 9.28 years. The sample covered different age strata and had a balanced gender ratio, demonstrating good representativeness and meeting the statistical requirements for validation experiments of the target biomarker.

[0038] Inclusion criteria for the validation set: age > 18 years, gender not limited, completion of a comprehensive health check-up with complete and traceable medical records; standardized plasma sample collection process with no abnormalities such as hemolysis or contamination; samples not selected for the screening set in this study to ensure sample independence.

[0039] Exclusion criteria: Severe cardiopulmonary insufficiency, liver and kidney failure, coagulation disorders, or other major organ dysfunction; past or present malignant tumors, severe infections (such as sepsis), autoimmune diseases (such as rheumatoid arthritis, systemic lupus erythematosus); physical examination indicating clear organic lesions (such as coronary heart disease, cirrhosis, chronic kidney disease, etc.); having undergone major surgery, blood transfusion, or long-term use of medications affecting metabolic / physiological indicators (such as immunosuppressants, long-term hormone therapy) within the past 3 months; having mental illness, cognitive impairment, or being unable to cooperate in completing the physical examination and sample donation process; pregnant women and breastfeeding women; plasma samples not processed promptly after collection, improperly stored, or with missing or incomplete baseline clinical data affecting sample validity assessment.

[0040] The method for detecting N-acetyl-L-cysteine ​​content in the validation set is the same as in Example 1.

[0041] Plasma N-acetyl-L-cysteine ​​was used as a diagnostic indicator. Sensitivity (true positive rate) and 1-specificity (false positive rate) at the N-acetyl-L-cysteine ​​content cutoff value were calculated using GraphPadPrism9. Receiver operating characteristic (ROC) curves were plotted with 1-specificity on the x-axis and sensitivity on the y-axis. Figure 3 The area under the curve (AUC) for N-acetyl-L-cysteine ​​in the reduction method for detecting and analyzing sulfur-containing metabolites was 0.7018, with P < 0.0001. In the modified metabolomics method, the AUC for N-acetyl-L-cysteine ​​was 0.5824, with P < 0.0291. This indicates that N-acetyl-L-cysteine ​​has good diagnostic efficacy for ischemic brain injury, accurately distinguishing between patients with ischemic brain injury and healthy individuals, and can serve as a diagnostic biomarker for ischemic brain injury.

[0042] The above description represents the preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The application of a reagent for detecting N-acetyl-L-cysteine ​​in the preparation of diagnostic products for ischemic brain injury, characterized in that, The products include reagent kits, test strips, or detection chips.

2. A product for the diagnosis of ischemic brain injury, characterized in that, The product includes reagents for detecting the N-acetyl-L-cysteine ​​content in samples.

3. The product according to claim 2, characterized in that, The testing steps for the product include: S1. Collect subject samples; S2. Detect the content of N-acetyl-L-cysteine ​​in the sample; S3. Based on the test results of S2, determine whether the subject has ischemic brain injury.

4. The product according to claim 3, characterized in that, In S1, the sample includes: plasma, whole blood, serum, or peripheral blood.

5. The product according to claim 3, characterized in that, In S2, the detection method includes spectrophotometry, liquid chromatography, high performance liquid chromatography-mass spectrometry, electrochemical method, or chemiluminescence method.

6. The product according to claim 3, characterized in that, In S3, the higher the N-acetyl-L-cysteine ​​content in the test results, the higher the probability that the subject has ischemic brain injury.

7. The product according to claim 6, characterized in that, The threshold for the judgment is that the NAC content exceeds 1.036 μm.

8. A system for diagnosing ischemic brain injury, characterized in that, The system includes a detection device, a computing device, and an output device; The detection device is used to collect subject samples and detect the N-acetyl-L-cysteine ​​content.

9. The system according to claim 8, characterized in that, The computing device is used to collect the detection results of N-acetyl-L-cysteine ​​content and compare them with the judgment criteria to determine whether the subject has cerebral ischemia injury.

10. The system according to claim 8, characterized in that, The output device is used to output the judgment of the computing device.

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