Feces metabolism marker for monitoring severity of ultrahigh-dose-rate radiation damage and application thereof
By screening fecal metabolic biomarkers guanine nucleoside, hydroquinone, luteolin, and their prepared monitoring kits, the problem of lacking non-invasive monitoring of ultra-high dose rate radiation damage in existing technologies has been solved, realizing non-invasive and accurate radiation damage monitoring and supporting the clinical application of FLASH-RT.
Patent Information
- Application Number
- CN202511424730.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-09-30
- Publication Date
- 2026-02-13
AI Technical Summary
Existing technologies lack sensitive, non-invasive biomarkers for monitoring the severity of ultra-high dose rate radiation damage, making it difficult to achieve dynamic, real-time quantification of radiation damage, especially in FLASH-RT, which hinders its clinical application.
We provide fecal metabolic biomarkers guanine nucleoside, hydroquinone, luteolin, and their prepared monitoring kits. We screen and verify their strong negative correlation with radiation damage using LC-MS technology, and use them to monitor the degree of radiation damage in normal tissues.
It enables non-invasive and precise monitoring of radiation damage, provides key evidence for optimizing clinical radiotherapy protocols, reduces the risk of damage to normal tissues, and promotes the clinical translation of FLASH-RT.
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Abstract
Description
TECHNICAL FIELD
[0001] The application belongs to the technical field of biological medicine, and particularly relates to a fecal metabolic marker for monitoring the severity of ultra-high dose rate radiation damage and an application thereof. BACKGROUND
[0002] Radiotherapy is one of the core means in the tumor comprehensive treatment system, and plays an irreplaceable role in controlling local tumor growth, reducing the risk of distant metastasis and improving the prognosis of patients. However, due to the low dose rate (usually <0.1 Gy / s) of traditional conventional radiotherapy, it is difficult to avoid radiation damage to the surrounding normal tissues (such as the intestinal tract, skin, hematopoietic system, etc.) while killing tumor cells, resulting in adverse reactions such as nausea, vomiting, mucositis, bone marrow suppression, and even the need to interrupt treatment in severe cases, which greatly limits the increase of radiotherapy dose and the optimization of treatment effect.
[0003] To break through this technical bottleneck, ultra-high dose rate radiotherapy (FLASH-RT) has emerged as the forefront of research in the field of radiotherapy. FLASH-RT is defined as the accurate delivery of a preset radiotherapy dose to the tumor target area in an ultra-short time (usually <1 s) at an ultra-high dose rate (usually >40 Gy / s), and its core advantage is that it significantly reduces the radiosensitivity of normal tissues while ensuring the killing effect of tumor cells, greatly improving the tolerance dose of normal tissues. At present, a large number of preclinical studies have confirmed the safety and effectiveness of FLASH-RT, for example, in the FAST-01 clinical trial of FLASH treatment of bone metastatic cancer in the extremities, patient treatment response was as expected and no significant serious adverse reactions were observed, which preliminarily verified its clinical translation potential.
[0004] However, the successful clinical promotion of FLASH-RT still faces key scientific problems: single fraction dose is a core physical parameter affecting FLASH effect, and the radiation damage mechanism and degree of normal tissues under different doses differ significantly, but there is currently a lack of sensitive, specific and non-invasive biomarkers for monitoring the severity of radiation damage under different dose parameters. Existing research relies mainly on histopathological detection (such as observation of intestinal mucosal villus-crypt structure), which requires invasive means to obtain tissue samples, making it difficult to achieve dynamic and real-time monitoring, and unable to accurately quantify the degree of damage. Therefore, the development of non-invasive, high-sensitivity biomarkers and the establishment of a FLASH-RT dose and radiation damage correlation model are urgent needs to promote its clinical translation. SUMMARY
[0005] In view of the technical blank in the prior art that there is no non-invasive biomarker for monitoring the severity of ultra-high dose rate radiation damage, a primary object of the present application is to provide a group of fecal metabolic markers that can specifically reflect the damage degree of normal tissues after ultra-high dose rate radiation (FLASH-IR) and have a strong linear correlation with the irradiation dose; another object of the present application is to provide the use of the fecal metabolic markers in the preparation of a monitoring kit and a corresponding marker screening method, thereby providing a technical solution for FLASH-RT clinical dose optimization and damage monitoring.
[0006] The fecal metabolic marker for monitoring the severity of ultra-high dose rate radiation damage is selected from one or more of guanine nucleoside, hydroquinone and luteolin.
[0007] Further, the single dose of the ultra-high dose rate radiation is 10-14 Gy, and the dose rate is >40 Gy / s.
[0008] Further, the content of the fecal metabolic marker is negatively correlated with the ultra-high dose rate radiation dose, that is, the higher the dose, the lower the marker content.
[0009] The use of the above-mentioned fecal metabolic marker in the preparation of a kit for monitoring the severity of ultra-high dose rate radiation damage.
[0010] Further, the kit is used for monitoring the radiation damage degree of normal tissues of a tumor patient after receiving FLASH-RT treatment.
[0011] Further, the kit contains one or more of guanine nucleoside standard, hydroquinone standard, and luteolin standard.
[0012] Further, the kit further contains an extraction reagent and an internal standard; the extraction reagent includes pure methanol and 50% acetonitrile aqueous solution; and the internal standard is 2-chloro-L-phenylalanine.
[0013] A fecal metabolic marker screening method for monitoring the severity of ultra-high dose rate radiation damage, comprising the following steps:
[0014] S1, collecting a healthy control sample and an ultra-high dose rate radiation receiving sample, respectively;
[0015] S2, detecting the samples by using LC-MS technology to obtain metabolite quantitative data;
[0016] S3, preliminarily screening differential metabolites by PLS-DA and OPLS-DA multivariate statistical analysis combined with a screening threshold of P<0.05 and VIP>1;
[0017] S4, performing Pearson correlation analysis and ROC curve analysis on the differential metabolites, screening metabolites with strong linear correlation with the ultra-high dose rate radiation dose, wherein the absolute value of r is greater than 0.98, P is less than 0.05; and AUC is greater than 0.85, as fecal metabolic markers.
[0018] Further, the chromatographic conditions of LC-MS detection in step S2 are as follows: HSST3 chromatographic column, column temperature 40 DEG C, flow rate 0.3 mL / min, sample amount 2 mu L; positive ion mode mobile phase is 0.1% formic acid acetonitrile-0.1% formic acid water, negative ion mode mobile phase is acetonitrile-5mM ammonium formate water.
[0019] Further, the mass spectrometry conditions of LC-MS detection in step S2 are as follows: electrospray ion source, positive ion spray voltage 3.50kV, negative ion spray voltage-2.50kV; primary scanning resolution 60000, scanning range m / z 100-1000; secondary fragmentation adopts HCD mode, collision energy 30%, resolution 15000.
[0020] The beneficial effects of the present application are as follows:
[0021] 1. The guanine nucleoside, hydroquinone and luteolin three fecal metabolic markers screened by the present application are strongly negatively linearly correlated with the irradiation dose (Pearson correlation coefficient r absolute value is greater than 0.98, P is less than 0.05) in the range of 10-14 Gy single dose of ultra-high dose rate radiation, and the diagnostic efficiency is excellent (AUC is greater than 0.85) through ROC curve analysis verification, which can accurately reflect the severity of radiation damage.
[0022] 2. The monitoring kit prepared based on the fecal metabolic markers can be used for monitoring the normal tissue radiation damage degree of tumor patients after receiving FLASH-RT treatment, providing key basis for clinicians to adjust radiotherapy programs and evaluate treatment risks, helping to promote the clinical transformation and popularization of FLASH-RT technology, improving tumor treatment effect and reducing normal tissue damage risk.
[0023] 3. The fecal metabolic marker screening method provided by the present application combines the high accuracy of LC-MS technology with the scientific nature of multivariate statistical analysis, Pearson correlation analysis and ROC curve analysis, the screening process is rigorous, and the screened markers have good stability and reliability, providing a reference methodological paradigm for subsequent screening of similar biomarkers. BRIEF DESCRIPTION OF DRAWINGS
[0024] In order to more clearly illustrate the technical solutions in the embodiments of the present disclosure or the prior art, the following will briefly introduce the drawings needed to be used in the embodiments or prior art description. Obviously, for those skilled in the art, other drawings can also be obtained from these drawings without any creative effort.
[0025] Figure 1 Score plot of fecal metabolome for the ultra-high dose rate 10 Gy irradiation group, 12 Gy irradiation group, 14 Gy irradiation group and control group (A: PLS-DA score plot, B: OPLS-DA score plot);
[0026] Figure 2 Differences in fecal content of guanine nucleoside (A), hydroquinone (B) and luteolin (C) under 3 doses of ultra-high dose rate radiation for the ultra-high dose rate radiation group;
[0027] Figure 3 ROC curve of guanine nucleoside (A), hydroquinone (B) and luteolin (C) under ultra-high dose rate radiation. DETAILED DESCRIPTION
[0028] The technical solutions in the embodiments of the present disclosure will be described clearly and completely below with reference to the drawings in the embodiments of the present disclosure. Obviously, the described embodiments are only a part of the embodiments of the present disclosure, rather than all the embodiments. Based on the embodiments in the present disclosure, all other embodiments obtained by those skilled in the art without any creative effort fall within the scope of protection of the present disclosure.
[0029] I. Fecal metabolite markers
[0030] The fecal metabolite markers for monitoring the severity of ultra-high dose rate radiation damage are selected from one or more of guanine nucleoside, hydroquinone and luteolin. In the range of FLASH-IR single dose 10-14 Gy, the contents of the above three metabolite markers in feces significantly decrease with the increase of irradiation dose, and are strongly negatively correlated with the dose (Pearson correlation coefficient r absolute value > 0.98, P < 0.05), which can accurately quantify the severity of radiation damage by detecting the content changes.
[0031] II. Application of markers
[0032] The above fecal metabolite markers are used in the preparation of a kit for monitoring the severity of ultra-high dose rate radiation damage, and the kit at least comprises the following components:
[0033] Standard: one or more of guanine nucleoside standard, hydroquinone standard and luteolin standard, which is used to establish a metabolite quantitative standard curve;
[0034] Extraction reagent: including pure methanol (CAS: 67-56-1, purity ≥ 99.0%) and 50% acetonitrile aqueous solution, used for extraction and purification of metabolic markers in fecal samples;
[0035] Internal standard: 2-chloro-L-phenylalanine (CAS: 103616-89-3, purity 98%), used to correct system errors in sample pretreatment and detection process, improve quantitative accuracy.
[0036] III. Marker screening method
[0037] A fecal metabolic marker screening method for monitoring the severity of ultra-high dose rate radiation damage, comprising the following steps:
[0038] S1, sample collection: collect fresh fecal samples from healthy controls (not receiving radiation) and tumor patients / experimental animals after receiving ultra-high dose rate radiation (10-14 Gy single dose), and store them in a-80℃ refrigerator to avoid metabolite degradation.
[0039] S2, sample pretreatment: thaw the fecal sample at 4℃, accurately weigh an appropriate amount of sample (about 50-100 mg) in a 2 mL centrifuge tube, add 600 μL of methanol solution containing 4 ppm of 2-chloro-L-phenylalanine, vortex for 30 s; add steel balls and place in a tissue grinder, grind at 50 Hz for 2 min; ultrasonic extraction at room temperature for 10 min, then centrifuge at 12000 rpm at 4℃ for 10 min; pass the supernatant through a 0.22 μm PTFE filter membrane, collect the filtrate as the test solution for subsequent detection.
[0040] S3, LC-MS detection: use liquid chromatography-mass spectrometry (LC-MS) technology to detect the test solution, the specific conditions are as follows:
[0041] Chromatographic conditions: use ThermoVanquish ultra-high performance liquid system, equipped with HSST3 chromatographic column (2.1×100mm, 1.8μm); column temperature 40℃, flow rate 0.3mL / min, injection volume 2μL; positive ion mode mobile phase is 0.1% formic acid acetonitrile-0.1% formic acid water, negative ion mode mobile phase is acetonitrile-5mM ammonium formate water, using gradient elution program.
[0042] Mass spectrometry conditions: Thermo Orbitrap Exploris 120 mass spectrometer detector, electrospray ion source (ESI); positive ion spray voltage 3.50 kV, negative ion spray voltage -2.50 kV; full scan resolution 60000, scan range m / z 100-1000; HCD mode is used for secondary fragmentation, collision energy 30%, resolution 15000, dynamic exclusion of unnecessary MS / MS information.
[0043] S4, differential metabolite screening
[0044] Data preprocessing: Convert the original mass spectrum file to mzXML format by Proteowizard software, use RXCMS software package for peak detection, filtering and alignment to get metabolite quantitative list; combined with public databases such as HMDB, MassBank, KEGG and self-built standard sample library for metabolite identification (mass deviation < 30ppm); based on the support vector regression correction system error of QC sample, filter the metabolites with RSD > 30% in QC sample.
[0045] Multivariate statistical analysis: Partial least squares discriminant analysis (PLS-DA) and orthogonal partial least squares discriminant analysis (OPLS-DA) are used to reduce dimension and group discriminant analysis of metabolite data, when the model parameters R 2 Y > 0.9 and Q2 > 0.7, the model is reliable (see Figure 1 ).
[0046] Differential metabolites: Calculate P value by statistical test, calculate variable projection importance (VIP) by OPLS-DA model, and calculate fold change (FC) between groups; set the screening threshold to P < 0.05 and VIP > 1, and preliminarily screen the differential metabolites between the radiation group and the control group.
[0047] Marker verification
[0048] Pearson correlation analysis is performed on the preliminarily screened differential metabolites, and metabolites with strong linear correlation (r absolute value > 0.98, P < 0.05) with FLASH-IR dose are screened; further verified by receiver operating characteristic curve (ROC) analysis, when AUC > 0.85, it is determined as a fecal metabolite marker with clinical application value (see Figure 2 , Figure 3 ).
[0049] Three, experimental verification
[0050] (I) Experimental materials
[0051] Experimental animals and grouping: 20 C57BL / 6J male mice, SPF level, 6-8 weeks old, weighing 18-22 g, purchased from Chengdu Dashuo Experimental Animal Co., Ltd. (Production License No.: SCXK (Chuan) 2020-030), and raised in the barrier environment of the specific pathogen-free animal room of Suzhou University. All mice were raised for 1 week to adapt to the environment and then randomly divided into control group (Ctrl group, 5 mice), ultra-high dose rate 10 Gy irradiation group (FLASH-IR-10 group, 5 mice), ultra-high dose rate 12 Gy irradiation group (FLASH-IR-12 group, 5 mice), and ultra-high dose rate 14 Gy irradiation group (FLASH-IR-14 group, 5 mice).
[0052] Main reagents: methanol (Thermo, purity ≥ 99.0%), 2-chloro-L-phenylalanine (Aladdin, purity 98%), acetonitrile (chromatographic purity), ammonium formate (analytical purity), etc., as shown in Table 1:
[0053] Table 1 Experimental reagents
[0054]
[0055]
[0056] Main instruments: refrigerated centrifuge (Xiangyi, H1850-R), tissue grinder (Mettler, MB-96), ultra-high performance liquid chromatography-mass spectrometry (Thermo Vanquish + Orbitrap Exploris120), etc., as shown in Table 2:
[0057] Table 2 Experimental instrument information
[0058]
[0059] (B) Experimental grouping and radiation treatment
[0060] The mice were randomly divided into 4 groups (n = 5 / group): control group (Ctrl, 0 Gy sham irradiation), FLASH-IR-10 group (10 Gy single irradiation), FLASH-IR-12 group (12 Gy single irradiation), and FLASH-IR-14 group (14 Gy single irradiation). After the mice were anesthetized by intraperitoneal injection of pentobarbital, they were fixed on a plastic plate. A 10 MeV photon FLASH radiotherapy X-ray source (China Mianyang) was used to perform abdominal irradiation at a dose rate of 200 Gy / s. The control group only received anesthesia and fixation treatment and did not undergo irradiation.
[0061] Example 1: Detection of fecal metabolic markers
[0062] Sample collection: Collect 50mg fresh fecal samples from patients receiving FLASH-RT treatment 3.5 days after radiotherapy and store at -80℃.
[0063] Sample pretreatment: After thawing the sample, add 600μL of 4ppm 2-chloro-L-phenylalanine methanol solution, vortex for 30s, 50Hz grinding for 2min, room temperature ultrasonic for 10min, 4℃, 12000rpm centrifugation for 10min, supernatant through 0.22μm filter membrane, ready for use.
[0064] LC-MS detection: Detection was carried out according to the above chromatographic and mass spectrometric conditions, and the content of guanine nucleoside, hydroquinone and luteolin in feces was calculated by the quantitative curve established by standard.
[0065] Damage evaluation: If the content of the three markers is significantly lower than the reference value of healthy people (guanine nucleoside: 1.2×10^8-1.5×10^8 response value; hydroquinone: 8×10^7-1×10^8 response value; luteolin: 3×10^8-4×10^8 response value), and the content decreases with the increase of radiotherapy dose, it indicates that the degree of radiation damage is aggravated, and the subsequent radiotherapy plan needs to be adjusted in combination with the clinic.
[0066] Example 2: Preparation and use of monitoring kit
[0067] Kit composition:
[0068] Standard: guanine nucleoside (1mg / mL, 1mL / bottle), hydroquinone (1mg / mL, 1mL / bottle), luteolin (1mg / mL, 1mL / bottle);
[0069] Extraction reagent: pure methanol (10mL / bottle), 50% acetonitrile aqueous solution (10mL / bottle);
[0070] Internal standard: 2-chloro-L-phenylalanine (100μg / mL, 1mL / bottle);
[0071] Consumables: 0.22μm PTFE filter membrane (50 pieces / pack), 1.5mL centrifuge tube (50 pieces / pack).
[0072] Usage steps:
[0073] Standard curve drawing: Take the appropriate amount of each standard, dilute it with 50% acetonitrile aqueous solution to 0.1, 0.5, 1, 5, 10μg / mL series concentration, add internal standard (final concentration 4ppm) respectively, detect according to LC-MS condition, take standard concentration as abscissa, peak area ratio of standard and internal standard as ordinate, draw standard curve.
[0074] Sample detection: Prepare the sample solution according to the sample pretreatment method of Example 1, and then add the internal standard for LC-MS detection. Calculate the content of the metabolic marker according to the standard curve.
[0075] Result interpretation: According to the reference value of the healthy population and the dose-content correlation model, the severity of radiation damage is determined.
[0076] (Three) Experimental results
[0077] Metabolomics difference analysis: PLS-DA score plot shows that the metabolic profiles of the feces of the radiation group and the control group are significantly different, and the intra-group samples have good clustering degree, with model parameters R2Y=0.992, Q2=0.852, indicating that the model stability and prediction ability are excellent Figure 1 A); OPLS-DA analysis further enhances the grouping discrimination effect, with the most significant difference between the FLASH-IR-14 group and the control group Figure 1 B).
[0078] Marker content change: The contents of guanine nucleoside, hydroquinone and luteolin in the feces of the radiation group are significantly lower than those in the control group, and show a decreasing trend with increasing dose: the content of the FLASH-IR-10 group is the highest, and the content of the FLASH-IR-14 group is the lowest Figure 2 A, B, C); see Table 1 for specific difference parameters, and the Pearson correlation coefficients of the three with radiation dose are -0.985, -0.993 and -0.995 (all P<0.05), confirming the strong linear correlation with dose.
[0079] Diagnostic performance verification: ROC curve analysis shows that the AUC of guanine nucleoside, hydroquinone and luteolin is 0.96 (95% CI: 0.88-1.00), 0.85 (95% CI: 0.64-1.00) and 0.96 (95% CI: 0.88-1.00) Figure 3 A, B, C), indicating that all three have excellent diagnostic performance for radiation damage and can be used as reliable monitoring markers.
[0080] Table 3 Difference parameters of fecal metabolic markers in different radiation groups and control groups From the above table and Figure 2 It can be seen that under different FLASH-IR irradiation doses, guanine nucleoside Figure 2 A), hydroquinone Figure 2 B) and luteolin Figure 2C) The contents in feces were significantly lower than those in healthy control group, and there was a statistical difference. Pearson correlation analysis results showed that the content changes of the above three metabolites had strong linear correlation with the irradiation dose (P<0.05, r absolute value>0.98). In the dose range of 10-14 Gy, the content changes of guanine nucleoside, hydroquinone and luteolin had negative correlation with the irradiation dose, and the content was the highest in FLASH-IR-10 group and the lowest in FLASH-IR-14 group. Therefore, the above three metabolites with strong linear correlation between content change and irradiation dose may be potential biomarkers for predicting the severity of radiation damage of different single doses of FLASH-IR.
[0081] In the description of the present specification, the description referring to the terms "one embodiment", "an example", "a specific example" and the like means that the specific features, structures, materials or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present application. In the present specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner.
[0082] The basic principles, main features and advantages of the present application are shown and described above. It should be understood by those skilled in the art that the present application is not limited by the above embodiments, and the above embodiments and descriptions in the specification are only illustrative of the principles of the present application. Without departing from the spirit and scope of the present application, various changes and improvements can be made to the present application, and these changes and improvements all fall within the scope of the claimed present application.
Claims
1. Fecal metabolic markers for monitoring the severity of damage by ultra-high dose rate radiation, characterized in that, The fecal metabolic markers are selected from one or more of guanosine, hydroquinone, and luteolin.
2. The fecal metabolic marker of claim 1, wherein, The single dose of the ultra-high dose rate radiation is 10-14 Gy, and the dose rate is > 40 Gy / s.
3. The fecal metabolic marker of claim 1, wherein, The content of the fecal metabolic markers is negatively correlated with the dose of ultra-high dose rate radiation, and the higher the dose, the lower the content of the markers.
4. Use of the fecal metabolic markers in the preparation of a kit for monitoring the severity of ultra-high dose rate radiation damage according to claim 1.
5. Use according to claim 4, characterized in that, The kit is used for monitoring the degree of radiation damage to normal tissues of tumor patients after receiving FLASH-RT treatment.
6. Use according to claim 4, characterized in that, The kit comprises one or more of guanosine standard, hydroquinone standard, and luteolin standard.
7. Use according to claim 6, characterized in that, The kit further comprises an extraction reagent and an internal standard; the extraction reagent comprises pure methanol and 50% acetonitrile aqueous solution; and the internal standard is 2-chloro-L-phenylalanine.
8. A method for screening fecal metabolic markers for monitoring severity of ultra-high dose rate radiation damage, characterized by, The method comprises the following steps: S1, collecting a healthy control sample and a sample receiving ultra-high dose rate radiation, respectively; S2, detecting the samples by LC-MS technology to obtain metabolite quantitative data; S3, preliminarily screening differential metabolites by PLS-DA and OPLS-DA multivariate statistical analysis, combined with a screening threshold of P<0.05 and VIP>1; S4, performing Pearson correlation analysis and ROC curve analysis on the differential metabolites, and screening metabolites that are strongly linearly correlated with the dose of ultra-high dose rate radiation, wherein the absolute value of r is > 0.98, P<0.05, and AUC is > 0.85, as fecal metabolic markers.
9. The screening method according to claim 8, characterized in that, The chromatographic conditions of LC-MS detection in step S2 are as follows: using HSST3 chromatographic column, column temperature 40℃, flow rate 0.3 mL / min, injection volume 2 μL; positive ion mode mobile phase is 0.1% formic acid acetonitrile-0.1% formic acid water, negative ion mode mobile phase is acetonitrile-5 mM ammonium formate water.
10. The screening method according to claim 8, characterized in that, In the mass spectrometry condition of the LC-MS detection in step S2, the electrospray ion source is used, the positive ion spray voltage is 3.50 kV, the negative ion spray voltage is -2.50 kV, the first scanning resolution is 60000, and the scanning range is m / z 100-1000; the secondary fragmentation uses HCD mode, the collision energy is 30%, and the resolution is 15000.