Identification of urinary markers of whole grain wheat intake and methods of detection

Abstract

This invention relates to a method for identifying and detecting urinary biomarkers of whole-grain wheat intake. These biomarkers include glycine conjugates of 3,5-dihydroxybenzoic acid, sulfate conjugates of o-aminophenol, sulfate conjugates of 2-acetaminophen, sulfate conjugates of 3,5-dihydroxyphenylvaleric acid, glucuronic acid conjugates of o-aminophenol, or glucuronic acid conjugates of 2-acetaminophen. The invention also relates to a high-throughput screening method for urinary biomarkers of whole-grain wheat intake. A whole-grain wheat dietary intervention experiment was designed, and through comparative analysis of human urinary metabolic profiles, non-targeted metabolomics technology was used to screen for urinary metabolic biomarkers of whole-grain wheat, providing a method for accurately assessing whole-grain wheat intake.

Description

Technical Field

[0001] This invention belongs to the field of biomarker technology, specifically relating to a method for identifying and detecting whole-grain wheat urinary biomarkers. Background Technology

[0002] In 1999, the American Association of Cereal Chemists (AACC) first clearly defined whole grains as: cereals consisting of whole, crushed, or flaked caryopsis, with the same proportions of starchy endosperm, germ, and bran as whole caryopsis, encompassing cereals of the Poaceae family such as wheat, oats, and barley. This definition has been adopted by many countries, but regional differences still exist in specific wording and the types of cereals included.

[0003] As research into the health effects of whole grains deepens, the recall bias inherent in traditional dietary assessment methods (such as 24-hour dietary reviews) due to their reliance on subjective reporting has become increasingly prominent, severely hindering causal inferences between whole grain intake and health outcomes. Against this backdrop, biomarkers of food intake (BFIs) based on metabolomics have emerged. This technology objectively reflects dietary intake levels by detecting characteristic metabolites in biological samples. Its advantages include: correcting for self-reported dietary survey data, improving the objectivity and accuracy of dietary assessments, and helping to elucidate the association between diet and disease.

[0004] Urine is an ideal substrate for food-related metabolic imaging (BFI) studies due to its non-invasive sampling, dynamic monitoring of metabolic characteristics, and wide range of metabolite concentrations. Compared to blood, food-related metabolites are more easily enriched in urine, and its low protein content simplifies the pretreatment process, facilitating large-scale nutritional epidemiological studies. However, urine biomarkers for whole grains currently face three major challenges: First, metabolite identification relies on standards, resulting in high false-positive rates and poor reproducibility; second, the lack of standardized sample processing and analysis procedures leads to insufficient comparability of results across studies; and third, existing studies are mostly based on Nordic whole-grain rye consumers, while in China, whole-grain wheat is the main type of whole grain consumed. Due to differences in genetic background and lifestyle, individuals exhibit significant differences in dietary choices and metabolic responses, casting doubt on the applicability and effectiveness of whole-grain wheat urinary biomarkers in the Chinese population. Summary of the Invention

[0005] To address the aforementioned issues, this invention, based on the dietary characteristics of whole-grain wheat in the Chinese population, designed a dietary intervention trial. It used non-targeted metabolomics technology to compare and analyze urinary metabolic profiles to screen for potential biomarkers. A human liver microsomal / cytoplasmic extracellular metabolic model was introduced for high-confidence identification, and a targeted detection method using LC-MS / MS was constructed. Ultimately, six whole-grain wheat-specific urinary biomarkers were successfully screened. Validation results showed that these biomarkers possess high specificity, stability, and reproducibility, exhibiting a dose-response relationship with whole-grain flour intake, and can accurately identify whole-grain wheat consumers and stratify intake levels. Furthermore, this invention developed a supporting high-throughput, high-sensitivity targeted analysis method, providing technical support for the standardized application of whole-grain wheat intake biomarkers in nutritional epidemiological research in China.

[0006] In a first aspect, the present invention provides a whole-grain wheat intake urinary biomarker, comprising DHBA-glycine, a glycine-bound form of 3,5-dihydroxybenzoic acid (3,5-DHBA), AP-sulfate, HPAA-sulfate, DHPPTA-sulfate, AP-glucuronide, or HPAA-glucuronide.

[0007] Furthermore, the whole-grain wheat urinary biomarker includes one or more of the above-mentioned substances. Preferably, the whole-grain wheat urinary biomarker includes glycine conjugate DHBA-glycine and anthranilic acid sulfate conjugate AP-sulfate.

[0008] More preferably, the whole grain wheat intake urinary biomarkers include the above-mentioned six substances.

[0009] Furthermore, the whole-grain wheat intake urinary biomarker was obtained by detecting, screening, and identifying urine samples from subjects in the intervention experiment using chromatography-mass spectrometry.

[0010] Secondly, the present invention provides a high-throughput screening method for urinary biomarkers of whole-grain wheat intake, comprising the following steps:

[0011] (1) Two-stage crossover dietary intervention experiment: The intervention group was a whole wheat flour group and the control group was a refined wheat flour group. Two stages of dietary intervention were carried out, and the two groups exchanged intervention foods during the two stages of intervention.

[0012] The intervention experiment was set up with two groups: an intervention group (whole wheat flour group) and a control group (refined wheat flour group), with the same number of subjects in each group.

[0013] The intervention experiment consisted of four phases, totaling 15 days: the induction phase, the first phase of intervention, the washout phase, and the second phase of intervention.

[0014] The induction period was 5 days: for the first 3 days, both groups of subjects avoided consuming whole grain wheat and processed foods; on the 4th and 5th days, they avoided consuming pasta foods.

[0015] The first phase of intervention lasted for 2 days. The whole wheat flour group consumed 50g of whole wheat flour-based food (steamed buns) for breakfast on the intervention day, while the refined wheat flour group consumed the same amount of refined wheat flour-based food (steamed buns) for breakfast on the intervention day. The remaining grain intake on that day consisted of rice, and the total daily grain intake of the two groups was equal.

[0016] The washout period lasts 5 days, and the dietary restrictions during this period are the same as those during the induction period. After the washout period, the second phase of intervention begins, which lasts 2 days, during which the two groups exchange intervention foods.

[0017] During the intervention phase, the intake of whole-wheat steamed buns for breakfast was to be completed under the supervision of researchers to ensure that the intake of whole-grain wheat reached the pre-set experimental intake. Dinner and lunch were designed and prepared by researchers based on the principles of a balanced diet, with the intake of only wheat-based foods (staple food: rice) restricted, while meeting the daily nutrient and energy needs of men and women.

[0018] During the biomarker screening phase, potential confounding factors were controlled for to maximize the identification of metabolic differences directly related to whole-grain wheat intake. The inclusion criteria for participants were set as follows: age 18–40 years and BMI 18.5–23.9 kg / m². 2 Exclusion criteria included: having chronic diseases, a history of smoking, using nutritional supplements within 3 months, using antibiotics within 1 month, a weight change of more than 3 kg within 3 months, being pregnant, breastfeeding, or menstruating, being a vegetarian or having special dietary habits, having a wheat allergy, and being unable to follow the relevant dietary requirements during the experiment.

[0019] During the intervention experiment, participants can only eat the food provided by the researchers and cannot eat or drink any food or beverages other than the provided meals. Three meals a day are scheduled for 8:00 a.m., 12:00 p.m. and 6:00 p.m. (participants are required to finish eating within 20 minutes). No food is consumed after dinner each day, but participants can drink the provided water.

[0020] (2) Time-segmented urine collection: Baseline urine was collected 0 hours before intervention, and urine was collected in multiple preset time periods from 0 (excluding) to 48 hours after intervention.

[0021] Before the intervention breakfast, 50 mL of midstream urine sample (0 h) was collected from the subjects as the baseline control (i.e., 0 h urine was used as the baseline urine sample).

[0022] Urine samples were collected at different time points after the intervention: 0 (excluding) ~ 2h, 2 (excluding) ~ 4h, 4 (excluding) ~ 6h, 6 (excluding) ~ 9h, 9 (excluding) ~ 12h, 12 (excluding) ~ 24h, 24 (excluding) ~ 36h, and 36 (excluding) ~ 48h. The urine volume at each time point was accurately recorded for future reference.

[0023] (3) Sample mixing and detection: The urine samples from different time periods were mixed according to volume ratio to obtain mixed urine samples, and metabolites were detected by high performance liquid chromatography-mass spectrometry.

[0024] 3.1 Mixing and Pretreatment: Mix, centrifuge, and dilute the urine;

[0025] The urine mixing process involves mixing urine samples from different time periods according to their volume ratios to form a mixed urine sample.

[0026] Among them, P-12h mixed urine: urine mixture from the 0~12 h period (including 0~2h, 2~4h, 4~6h, 6~9h, and 9~12h).

[0027] P-24h mixed urine: Mixed urine from the 0-24h period (including 0-2h, 2-4h, 4-6h, 6-9h, 9-12h, and 12-24h).

[0028] P-24~48h mixed urine: urine mixture from 24 (excluding) to 48 hours (including 24~36h and 36~48h).

[0029] Further, centrifugation: at 0-4℃, centrifuge at 7000-10000×g for 10-30 min;

[0030] Furthermore, the diluent is 10-20% methanol-water; the amount of diluent added is the same as the sample volume.

[0031] This invention mixes urine samples from subjects at different time periods, and the resulting urine sample is more representative of the overall condition after consuming whole grain wheat.

[0032] Because the urine samples collected at different times are large and inconvenient to store, a proportional mixing method is adopted: the urine samples from different times are reduced in volume according to the original proportion and then mixed to obtain mixed urine samples representing different time periods.

[0033] In one embodiment of the present invention, the mixed urine is heated at 4°C for 9000× g (Relative centrifugal force unit) Centrifuge for 15 min to remove particulate matter and precipitate, then accurately transfer 500 μL of urine sample with a pipette, add 500 μL of 20% methanol-water 1:1 for dilution and test.

[0034] 3.2 High-performance chromatography-mass spectrometry detection:

[0035] Chromatographic conditions: Chromatographic separation was performed using a Waters ACQUITY UPLC HSS T3 column (100 mm × 2.1 mm, 1.7 μm) at a column temperature of 30–40 °C and an injection volume of 1–2 µL. Mobile phase A was methanol containing 0.01–0.05% acetic acid, and mobile phase B was ultrapure water containing 0.01–0.05% acetic acid. The flow rate was 0.2–0.4 mL / min, with linear gradient elution. The elution program was as follows: initial mobile phase 2% A, then linearly increased to 100% A over 15 min, held for 5 min, returned to the initial mobile phase ratio, equilibrated for 3 min, and awaited the next injection.

[0036] Mass spectrometry conditions: High-throughput screening of urinary biomarkers was performed using Orbitrap high-resolution mass spectrometry. Spray voltage: 3.5~3.8kV (+), 3.0~3.5kV (-); heating temperature: 300~350℃; capillary temperature: 300~320℃. Scanning mode: FullMS-ddMS2 (Top 8); scan range: 100~1000 m / z; secondary fragmentation mode selected: High energy collision-induced dissociation (HCD), collision energy set to 15 eV, 30 eV, and 50 eV. First-stage full scan resolution: 60,000; second-stage scan resolution: 15,000.

[0037] 3.3 Data Collection and Preliminary Identification:

[0038] The collected data underwent peak extraction, peak alignment, and background subtraction, as well as data filtering; and a self-built library was used to preliminarily identify the compound types.

[0039] Data acquisition was performed using Xcalibur v.4.0 software, and peak extraction, peak alignment, and background subtraction were performed on the raw data using Compound Discoverer v.3.2 software.

[0040] The data filtering conditions include precise mass number deviation (<5ppm), signal-to-noise ratio (S / N>10), peak intensity (Peakarea>100000), and retention time offset (RT<0.2 min).

[0041] The types of compounds were initially identified using databases, including the self-built Mass List database, MZcloud, Chemspider, HMDB, FOODB, PhytoHub, and other databases.

[0042] (4) Screening for urinary biomarkers of whole grain wheat intake

[0043] 4.1 Preliminary Screening: Screening of whole-grain wheat urinary biomarkers was performed using t-tests (for normally distributed data) and Mann-Whitney rank-sum tests (for non-normally distributed data). Considering the specific requirements of biomarkers for specificity, sensitivity, and reproducibility, the preliminary screening of whole-grain wheat urinary biomarkers simultaneously met the following criteria:

[0044] (i) Comparison between the intervention group and the control group:

[0045] At each stage, the differences in metabolite concentrations in urine samples from the intervention group and the control group at corresponding time points are compared, and the following conditions must be met:

[0046] Mixed urine samples from P-12h and P-24h: P Value < 0.001 and difference factor log2 FC > 2;

[0047] Mixed urine samples from 24 to 48 hours post-conversion: P Value < 0.05 and difference factor log2 FC > 1;

[0048] That is, between each stage, between the intervention group and the control group, and between the corresponding P-12h mixed urine samples, P Value < 0.001 and fold change log2 FC > 2; corresponding P-24h mixed urine samples, P Value < 0.001 and fold change log2 FC > 2; corresponding P-values ​​between mixed urine samples from 24h to 48h. P The value is <0.05 and the difference factor log2 FC >1.

[0049] (ii) Comparison within the intervention group:

[0050] At each stage, the differences in metabolite concentrations between postprandial urine samples and baseline samples taken 0 hours before meals within the intervention group were compared, and the following conditions must be met:

[0051] Compared with the 0h sample, the mixed urine samples from P-12h and P-24h were: P Value < 0.001 and difference factor log2 FC > 2;

[0052] Compared with the 0h sample, the mixed urine samples from P-24~48h: P The value is <0.05 and the difference factor log2 FC >1.

[0053] That is, at each stage, the postprandial time (P-12h) mixed urine samples in the intervention group were compared with those at P-0h. P Value < 0.001 and fold change log2 FC > 2; compared with P-0h, the P-24h mixed urine sample, P Value <0.001 and fold change log2 FC>2; compared with P-0h, the mixed urine sample from P-24~48h showed a difference of <0.001. P The value is <0.05 and the difference factor log2 FC >1.

[0054] Using log2FC value and -log 10 P Volcano maps were plotted to filter metabolite data from mixed urine at different time points.

[0055] 4.2 Biomarker identification: Exogenous specific differential metabolites that were reproduced in both stages of cross-intervention experiments were screened out through peak detection, isotope cluster analysis, identification of characteristic MS / MS fragments, and mass spectrometry library matching as biomarkers of whole grain wheat intake in urine.

[0056] In one embodiment of the present invention, after whole-grain wheat intake, 20 and 14 differentially expressed metabolites were screened in 12-hour mixed urine (P-12h) and 24-hour mixed urine (P-24h), respectively, the latter being a subset of the former. Only one significantly differentially expressed metabolite was found in mixed urine (P-24~48h) 24 (excluding) to 48 hours postprandial. m / z 188.0022.

[0057] Detailed analysis of each mass spectrometry fragment was performed, and the candidate biomarkers were identified and their specificity evaluated. A total of six urinary biomarkers specific to whole-grain wheat were discovered, with mass-to-charge ratios of [missing information]. m / z 210.0406 m / z 188.0022 m / z 230.0127 m / z 289.0386 m / z284.0775 and m / z 326.0879.

[0058] Among them, through comparison with standard products, m / z 210.0406 was identified as 3,5-DHBA-glycine, a glycine conjugate of 3,5-dihydroxybenzoic acid (3,5-DHBA), a secondary metabolite of the whole-grain-specific phytochemical alkylresorcinol. m / z 188.0022 m / z 230.0127 are the sulfate conjugates AP-sulfate and HPAA-sulfate of 2-aminophenol (AP) and 2-acetamidophenol (HPAA), which are secondary metabolites of whole-grain phytochemicals.

[0059] m / z 289.0386 is DHPPTA-sulfate, a sulfated compound of 3,5-dihydroxyphenyl propanoic acid (DHPPTA), a secondary metabolite of alkyl resorcinol, a phytochemical from whole-grain wheat.

[0060] m / z 284.0775 m / z 326.0879 are AP-glucuronide and HPAA-glucuronide, which are secondary metabolites of benzoxazine phytochemicals from whole-grain wheat, respectively.

[0061] A third aspect of this invention provides a method for identifying whole-grain wheat urinary biomarkers using an in vitro metabolic model. Since there are no commercially available standards for the sulfate conjugates of o-aminophenol (AP-sulfate), 2-acetaminophenol (HPAA-sulfate), o-aminophenol (HPAA-glucuronide), and 3,5-dihydroxyphenylvaleric acid (DHPPTA-sulfate), the analysis of these biomarkers relies solely on high-resolution mass spectrometry inference. To address these issues, this invention utilizes a mixed human liver microsomal / human liver cytoplasmic in vitro metabolic model to further identify potential biomarkers with high confidence.

[0062] This invention utilizes a mixed human liver microsome / human liver cytoplasmic fluid extracellular metabolic model to identify potential biomarkers with high confidence. The identification method includes the following steps: First, a known precursor (o-aminophenol AP, 2-acetaminophen HPAA, and 3,5-dihydroxyphenylvaleric acid DHPPTA standard) is co-incubated with a mixture of human liver microsomes and / or human liver cytoplasmic fluid. The mixture contains necessary cofactors such as uridine diphosphate glucose (UDPGA) and 3'-adenosine phosphate-5'-phosphosulfate (PAPS).

[0063] Then, the retention time, fragment ions, and ion abundance ratio of in vitro metabolites and urine sample metabolites were compared.

[0064] In one embodiment of the present invention, the in vitro metabolic experimental conditions were as follows: Urate diphosphate glucose (UDPGA) / 3'-adenosine-5'-phosphosulfate (PAPS) and human liver microsomes / human liver cytoplasm were removed from -80°C and thawed on ice. 475 µL of Tris-buffer buffer (100 mM, pH 7.4), 10 µL of mixed human liver microsomes (20 mg / mL) / mixed human liver cytoplasm (10 mg / mL) were added to an Eppendorf tube, followed by 10 µL of UDPGA / PAPS (100 mM / 10 mM). After pre-incubating the above mixture in a 37°C water bath for 5 min, 5.0 µL (2.0 mg / mL) of the phytochemical to be identified or the target compound was added to initiate the reaction, maintaining a total reaction volume of 500 µL. After initiating the reaction, it was incubated in a 37°C shaking water bath. After 12 hours, 100 µL of the total reaction mixture was added to an Eppendorf tube containing 200 µL of acetonitrile to terminate the reaction. After centrifugation, the supernatant was obtained, diluted 10-fold with 50% methanol-water, and the metabolic transformation products were identified by high-resolution mass spectrometry (LC-Orbitrap MS).

[0065] The results showed that the retention time, fragment ions, and ion abundance ratio of four metabolite markers (AP-sulfate, HPAA-sulfate, HPAA-glucuronide, and DHPPTA-sulfate) in the urine of the intervention group after consuming whole grain wheat were completely consistent with the relevant data of metabolites obtained by in vitro incubation, indicating that the identification of metabolic markers was accurate and reliable.

[0066] This invention is the first to utilize a human liver microsomal / human liver cytoplasmic fluid extracellular metabolic model for the identification of whole-grain wheat urine biomarkers, significantly improving the confidence level of identification of suspected metabolites and laying the foundation for the biological interpretation of biomarkers and the development of detection methods.

[0067] In a fourth aspect, the present invention provides a targeted detection method for whole-grain wheat urinary biomarkers by LC-MS / MS, wherein the whole-grain wheat urinary biomarkers include the six biomarkers described in the first aspect.

[0068] The target detection method includes the following steps:

[0069] S1. Mass Spectrometry Conditions: Based on the mass spectrometry identification results of the first aspect, the MS / MS mass spectrometry parameters of each marker are optimized in a targeted manner, including retention time, qualitative ions, and quantitative ions.

[0070] The target analytes were detected using an AB Sciex 6500 triple quadrupole mass spectrometer. Specific mass spectrometry conditions were as follows: an ESI ion source was used, and data was acquired in negative ion mode using multiple reaction monitoring (MRM); the nebulizer gas, curtain gas, auxiliary heating gas, and collision gas were all high-purity nitrogen; electrospray voltage: 4500V; ion source temperature: 500℃; nebulizer gas pressure: 55 psi; curtain gas pressure: 35 psi. Data acquisition and analysis were performed using AB Analyst software.

[0071] S2. Chromatographic conditions: Chromatographic separation was performed using a Waters ACQUITY UPLC HSS T3 column (100 mm × 2.1 mm, 1.7 μm), with a column temperature of 30–40 °C and an injection volume of 1–2 µL. Mobile phase A consisted of methanol containing 0.01–0.05% acetic acid, and mobile phase B consisted of ultrapure water containing 0.01–0.05% acetic acid, with a flow rate of 0.1–0.3 mL / min. Linear gradient elution was used, with the following elution program: initial mobile phase 5% A was maintained, linearly increased to 100% A within 8.5 min, held for 2.0 min, and then the mixture was brought back to the initial mobile phase for equilibration before the next injection.

[0072] Based on the second aspect of this invention, which involves high-throughput screening of whole-grain wheat urinary biomarkers, and given that the target biomarkers have been identified, optimizing the gradient elution parameters of the mobile phase is beneficial for the targeted detection process.

[0073] Specifically, in the second aspect of target biomarker screening, high-resolution mass spectrometry (such as Orbitrap-MS) is used for non-targeted analysis. Its advantage lies in high throughput, making it suitable for large-scale screening of unknowns. However, its quantitative accuracy is relatively limited. Given that six candidate target biomarkers have been successfully identified through this non-targeted screening, subsequent validation and analysis using targeted mass spectrometry (LC-MS / MS) are necessary. This method is specifically designed for precise quantification, enabling highly sensitive and precise quantitative detection of target biomarkers.

[0074] Given that there are no commercially available standards for the sulfate conjugates of o-aminophenol (AP-sulfate), 2-acetaminophen (HPAA-sulfate), 2-acetaminophen (HPAA-glucuronide), and 3,5-dihydroxyphenylvaleric acid (DHPPTA-sulfate), this invention selects isomers or structural analogs of the above markers to prepare standard curves for quantification of the target analytes: 4-acetaminophen sulfate (CAS: 32113-41-0) is used for quantitative analysis of AP-sulfate and HPAA-sulfate; and 3-(4-methoxy-3-(sulfonoxy)phenyl)propionic acid (Dihydro isoferulic acid 3-O-sulfate) (CAS: 1258842-21-5) is used for quantitative analysis of the sulfated metabolic marker DHPPTA-sulfate.

[0075] In one embodiment of the present invention, reference standards at low, medium, and high concentrations of 2, 10, and 50 ng / mL were added to the baseline urine of the subject before meals (0h baseline urine) for a spiked recovery experiment. The results showed that the average recovery rate of whole-grain wheat urinary biomarkers in the urine matrix ranged from 87.6% to 107.8%, and the precision ranged from 2.3% to 13.8%, indicating good precision and recovery rate of the LC-MS / MS targeted detection method. Using matrix spike concentrations of 3 times the signal-to-noise ratio (S / N) and 10 times the signal-to-noise ratio (S / N) as the limits of detection (LOD) and quantitation (LOQ), respectively, the detection LOD and LOQ ranges for whole-grain biomarkers in urine were found to be 0.3–0.7 ng / mL and 1.0–2.0 ng / mL, respectively, indicating that the method has good sensitivity. Using the above method, the present invention achieves for the first time highly sensitive simultaneous analysis and detection of six whole-grain wheat urinary biomarkers in urine using LC-MS / MS.

[0076] The beneficial effects of this invention are:

[0077] 1. Based on the dietary characteristics of the Chinese population, this invention is the first to screen and identify six whole grain wheat urinary biomarkers in healthy Chinese adults through a two-stage cross-intervention experiment, providing a new method for the accurate assessment of whole grain intake.

[0078] 2. This invention is the first to use the human liver microsome / human liver cytoplasmic fluid extracorporeal metabolism model for the identification of whole grain wheat urine biomarkers, verifying the biotransformation pathway of biomarkers, significantly improving the identification confidence of suspected metabolites, laying the foundation for the biological interpretation of biomarkers, and also providing a reference for the in vitro enzymatic synthesis of biomarker reference standards in the future.

[0079] 3. This invention establishes for the first time a high-sensitivity targeted simultaneous detection method for six biomarkers using LC-MS / MS. It has high sensitivity and good reproducibility and can be used as an economical and efficient routine laboratory detection method to promote the nutritional epidemiological application of whole grain biomarkers. Attached Figure Description

[0080] Figure 1 This is a schematic diagram of a randomized, controlled, crossover whole-grain wheat urine dietary intervention experiment as described in Example 1.

[0081] Figure 2 Example 1 utilizes log2FC value and -log 10 P. Volcano plots were used to screen urinary biomarkers of whole grain wheat intake in mixed urine from different groups. (a) Screening of differential metabolites in mixed urine from 12 hours postprandial (P-12h); (b) Screening of differential metabolites in mixed urine from 24 hours postprandial (P-24h); (c) Screening of differential metabolites in mixed urine from 24 hours (excluding) to 48 hours postprandial (P-24 to 48h).

[0082] Figure 3 Example 1 uses Venn diagrams to screen for overlapping biomarkers in two phases of a randomized crossover dietary intervention experiment;

[0083] Figure 4 This is the high-resolution mass spectrometry qualitative deconstruction spectrum of DHBA-glycine, a whole-grain wheat urinary biomarker, in Example 1.

[0084] Figure 5 This is the high-resolution mass spectrometry qualitative deconstruction spectrum of AP-sulfate, a urinary biomarker from whole-grain wheat, in Example 1.

[0085] Figure 6 This is the high-resolution mass spectrometry qualitative deconstruction spectrum of HPAA-sulfate, a urinary biomarker from whole grain wheat, in Example 1.

[0086] Figure 7 This is the high-resolution mass spectrometry qualitative deconstruction spectrum of DHPPTA-sulfate, a whole-grain wheat urinary biomarker, in Example 1.

[0087] Figure 8 This is the high-resolution mass spectrometry qualitative deconstruction spectrum of AP-glucuronide, a whole-grain wheat urinary biomarker, in Example 1.

[0088] Figure 9 This is the high-resolution mass spectrometry qualitative deconstruction spectrum of HPAA-glucuronide, a whole-grain wheat urinary biomarker, in Example 1.

[0089] Figure 10 This is a schematic diagram illustrating the identification of four biomarkers aided by the human liver microsome / human liver cytoplasmic extracellular metabolic model in Example 2.

[0090] Figure 11 Example 2 uses in vitro metabolic experiments to identify the retention time and fragment ions of four urine markers without standards;

[0091] Figure 12 This is a representative chromatogram for the targeted detection of six whole-grain wheat urine biomarkers by LC-MS / MS in Example 3;

[0092] Figure 13 This is a schematic diagram of the dose-response relationship experiment of whole grain wheat urinary biomarkers in Example 4;

[0093] Figure 14 Linear regression analysis of dose-response relationships of six whole-grain wheat urinary biomarkers in Example 4. Detailed Implementation

[0094] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0095] Example 1

[0096] A method for identifying urinary biomarkers of whole-grain wheat intake includes the following steps:

[0097] (1) Two-stage crossover dietary intervention experiment:

[0098] The two-stage crossover dietary intervention trial is a randomized, controlled, crossover acute dietary intervention study. The trial lasted 15 days and consisted of four phases: an introductory phase, a first-stage intervention, a washout phase, and a second-stage intervention. For detailed experimental design procedures, please refer to [link to experimental procedure]. Figure 1 .

[0099] This study was approved by the Biomedical Ethics Committee of Peking University (Approval No.: IRB00001052-23***), and the study protocol was simultaneously registered on the US Clinical Trials Registry (ClinicalTriab.Gov) (NCT05837***). Before the experiment began, the research team advertised for participants. Subsequently, eligible participants contacted the research team and were screened through interviews. After questionnaires, physical examinations, and blood biochemistry screening, a total of 22 participants (12 males and 10 females) completed the study. The mean age of the participants was 24.6 ± 3.2 years, and the BMI was 21.2 ± 1.5 kg / m². 2 .

[0100] The induction period was 5 days. For the first 3 days, both groups of subjects avoided consuming whole grain wheat and processed foods; on the 4th and 5th days, they avoided consuming pasta.

[0101] During the introductory phase, participants were required to strictly avoid foods containing whole-grain wheat, including but not limited to whole-wheat bread, whole-wheat steamed buns, whole-wheat biscuits, instant cereals, and pre-packaged foods whose ingredient list indicated whole-wheat flour. Apart from this, participants were allowed free access to their own food. To avoid the influence of dietary habits on the experimental results, participants were instructed to avoid consuming pasta for two days prior to the formal dietary intervention.

[0102] Subjects entered a standardized diet phase starting with dinner the day before the intervention experiment. During the experiment, they could only eat the food provided by the researchers and could not eat or drink any food or beverages other than the provided meals. The three meals a day were scheduled for 8:00 am, 12:00 pm and 6:00 pm (and were required to finish eating within 20 minutes). No food was consumed after dinner each day, but they could drink the provided water.

[0103] This experiment set up an intervention group – the whole wheat flour group – and a control group – the refined wheat flour group. Referring to the recommended daily intake of whole grains (50-150g, including legumes) in the *Chinese Dietary Guidelines (2022)*, this study used 50g of whole wheat flour as the intervention dose. Steamed buns, a common staple food in China, were used as the intervention food. 100% whole wheat flour, obtained from grinding whole wheat grains, was used as the raw material for the intervention food. The intervention food preparation process was as follows: 50g of refined wheat flour / whole wheat flour, 30g of water, and 0.5g of yeast were mixed, then fermented, shaped, and steamed. The final weight of the steamed buns was 82.9±4.4g. n =10). Before the formal intervention, the participants were pre-divided into two groups, taking into account the gender factor, and then randomly divided into two groups, A and B, by computer.

[0104] In the first intervention period (2 days), Group A consumed 50g of whole wheat flour steamed buns for breakfast on the intervention day, while Group B consumed 50g of refined wheat flour steamed buns for breakfast on the intervention day. All other grain intake on that day consisted of rice, and the total daily grain intake was equal for both groups. This was followed by a washout period (5 days), with the same dietary restrictions as the induction period. After the washout period, the second intervention period (2 days) began, and the two groups exchanged intervention foods. The intake of whole wheat steamed buns for breakfast was supervised by researchers to ensure that the pre-set intake of whole grain wheat was achieved. Dinner and lunch were designed and prepared by researchers based on the principles of a balanced diet, with restrictions only on the intake of wheat-based foods (staple food: rice), while still meeting the daily nutrient and energy needs of both men and women.

[0105] (2) Urine collection at different time intervals:

[0106] Before the intervention breakfast, subjects emptied their bladders, and a 50 mL midstream urine sample (0 h) was collected as a baseline blank control. Post-intervention urine samples were collected at the following time points: 0 (excluding)–2 h, 2 (excluding)–4 h, 4 (excluding)–6 h, 6 (excluding)–9 h, 9 (excluding)–12 h, 12 (excluding)–24 h, 24 (excluding)–36 h, and 36 (excluding)–48 h. Within each predetermined time period, subjects urinated and collected all urine samples using one or more 500 mL sterile sampling bottles. The collected samples were temporarily stored in ice packs and delivered to researchers before the next group meal. New sterile sampling bottles were then used for subsequent sampling. Upon receiving the urine samples, researchers measured and accurately recorded the urine volume at each time point, then aliquoted the urine samples into 1 mL portions and cryopreserved at -80°C for later use.

[0107] (3) Sample mixing and detection:

[0108] 3.1 Mixing and Pretreatment: Before sample analysis, urine samples frozen at -80℃ were thawed overnight at 4℃. The thawed urine samples were thoroughly vortexed and mixed. Urine samples from different time points were then combined according to volume ratios to form a mixed urine sample.

[0109] P-12h mixed urine: urine mixture from the 0-12h time period (including 0-2h, 2-4h, 4-6h, 6-9h, and 9-12h).

[0110] P-24h mixed urine: Mixed urine from the 0-24h period (including 0-2h, 2-4h, 4-6h, 6-9h, 9-12h, and 12-24h).

[0111] P-24~48h mixed urine: Mixed urine from the period of 24 (excluding) to 48 hours (including 24~36h and 36~48h).

[0112] The mixed urine sample was centrifuged at 9000×g for 15 min at 4°C to remove particulate matter and precipitate. Then, 500 μL of the urine sample was accurately transferred using a pipette and diluted with 500 μL of 20% methanol-water at a 1:1 ratio before analysis. In addition, 100 μL of each urine sample was taken and mixed again to prepare the analytical quality control sample (QC).

[0113] The "mixing by volume ratio" described in this invention is explained as follows: Taking a single subject as an example, the original urine volumes collected at five time points—0-2h, 2-4h, 4-6h, 6-9h, and 9-12h—are 200mL, 300mL, 400mL, 200mL, and 300mL, respectively. Theoretically, without considering practical limitations, urine from all time points (totaling 1.4L) can be directly mixed to obtain a complete 0-12h mixed urine sample (P-12h). However, actual studies often involve multiple subjects, and collecting and freezing a large number of original urine samples from all time points would face enormous cryopreservation space pressure. To solve this problem, this invention employs the following method: After collecting and recording the urine volume for each time point, immediately take 1mL as an aliquot sample for that time point and freeze it. When a mixed urine sample representing 0-12 hours (P-12h) needs to be prepared later, the corresponding volumes (1 mL each) of the aliquots from each frozen time period are precisely measured and mixed according to the original urine volume ratio: 200 µL for 0-2h, 300 µL for 2-4h, 400 µL for 4-6h, 200 µL for 6-9h, and 300 µL for 9-12h. These five proportionally measured liquids are then mixed (totaling 1.4 mL), resulting in a significantly reduced volume (1.4 mL vs. 1.4 L) of P-12h mixed sample, while maintaining the exact same proportion of urine from each time period as the original urine. This method greatly saves cryopreservation space while ensuring the representativeness of the mixed sample.

[0114] 3.2 High-performance chromatography-mass spectrometry detection

[0115] A total of 176 urine samples (22 subjects × 4 time periods (0h, P-12h, P-24h, P-24~48h) × 2 intervention phases) were subjected to non-targeted metabolomics analysis in 4 batches under both positive and negative ionization modes. Under the same intervention phase and ionization mode, all collected urine samples were grouped into the same batch, and samples from different subjects were tested in a randomized order. Five QC samples were added before and after each batch of sample testing to ensure adequate balancing of the sample introduction system and to assess system stability. Additionally, for every 10 samples analyzed, one blank sample (100% methanol) and one QC sample (Quality Control Sample) were inserted.

[0116] LC-Orbitrap high-resolution mass spectrometry was performed in both positive and negative ion modes. Metabolite separation was achieved using an HSST3 Acquity column (2.1 mm × 100 mm; 1.8 µm, Waters Corporation, USA) at 40 °C with an injection volume of 2 µL. Mobile phase A was methanol containing 0.05% acetic acid, and mobile phase B was ultrapure water containing 0.05% acetic acid. The flow rate was 0.35 mL / min, with linear gradient elution. The elution program was as follows: initial mobile phase 2% A, followed by a linear increase to 100% A over 15 min, holding for 5 min, returning to the initial mobile phase ratio, equilibrating for 3 min, and awaiting the next injection.

[0117] Mass spectrometry conditions were as follows: high-throughput screening of urinary biomarkers was performed using Orbitrap high-resolution mass spectrometry; spray voltage: 3.8 kV (+), 3.0 kV (-); heating temperature: 350 °C; capillary temperature: 320 °C. Scanning mode: Full MS-ddMS2 (Top8); scan range: 100–1000 m / z; secondary fragmentation mode selected: High energy collision-induced dissociation (HCD), with collision energies set to 15 eV, 30 eV, and 50 eV. Primary full scan resolution: 60,000; data-dependent secondary scan resolution: 15,000.

[0118] 3.3 Data Collection and Preliminary Identification:

[0119] Data acquisition was performed using Xcalibur v.4.0 software. Raw data underwent peak extraction, peak alignment, and background subtraction using Compound Discoverer v.3.2 software. Data filtering conditions included exact mass number deviation (<5ppm), signal-to-noise ratio (S / N>10), peak intensity (Peak area>100000), and retention time shift (RT<0.2min). Preliminary identification of compounds was performed using a self-built Mass List database, as well as databases such as MZcloud, Chemspider, HMDB, FOODB, and PhytoHub.

[0120] (4) Screening for urinary biomarkers of whole grain wheat intake

[0121] 4.1 Preliminary Screening: Screening of whole-grain wheat urinary biomarkers was performed using t-tests (for normally distributed data) and Mann-Whitney rank-sum tests (for non-normally distributed data). Considering the specific requirements of biomarkers for specificity, sensitivity, and reproducibility, the preliminary screening of whole-grain wheat urinary biomarkers simultaneously met the following criteria:

[0122] (i) Comparison between the intervention group and the control group:

[0123] At each stage, the differences in metabolite concentrations in urine samples from the intervention group and the control group at corresponding time points are compared, and the following conditions must be met:

[0124] Mixed urine samples from P-12h and P-24h: P Value < 0.001 and difference factor log2 FC > 2;

[0125] Mixed urine samples from 24 to 48 hours post-conversion: P Value < 0.05 and difference factor log2 FC > 1;

[0126] Furthermore, (ii) comparisons within the intervention group:

[0127] At each stage, the differences in metabolite concentrations between postprandial urine samples and baseline samples taken 0 hours before meals within the intervention group were compared, and the following conditions must be met:

[0128] Compared with the 0h sample, the mixed urine samples from P-12h and P-24h were: P Value < 0.001 and difference factor log2 FC > 2;

[0129] Compared with the 0h sample, the mixed urine samples from P-24~48h: P The value is <0.05 and the difference factor log2 FC >1.

[0130] That is, both the intergroup comparison (whole wheat flour group vs. refined wheat flour group) and the intragroup comparison (whole wheat flour group post-meal vs. pre-meal baseline 0h) of urinary metabolites in the two-stage process met the requirements. P Value <0.001 (P-12h, P-24h) or P Value <0.05 (P-24~48h); the fold difference in urinary metabolites between the two groups (whole wheat flour group vs. refined wheat flour group) and within the group (whole wheat flour group post-meal vs. pre-meal baseline 0h) satisfies log2FC>2 (P-12h, P-24h) or log2FC>1 (P-24~48h).

[0131] Using log2FC value and -log 10 P plotted a volcano map to filter urinary metabolite data for different time periods. Figure 2 ).

[0132] 4.2 Biomarker identification: Differential metabolites that were reproduced in both stages of the cross-intervention experiment were screened out through peak detection, isotope cluster analysis, identification of characteristic MS / MS fragments, and mass spectrometry library matching as biomarkers of whole grain wheat intake in urine.

[0133] Ultimately, 20 differentially expressed metabolites (crossover set) were selected that could be reproduced in both phases of the crossover intervention experiment. The results are as follows: Figure 3 As shown, after whole-grain wheat intake, 20 and 14 differentially metabolites were detected in 12-hour mixed urine (P-12h) and 24-hour mixed urine (P-24h), respectively, with the latter being a subset of the former. The results indicate that the number of observable differentially metabolites decreased significantly over time, suggesting a gradual narrowing of the urinary metabolic profile differences between the intervention and control groups. Only one significantly differentially metabolite was found in mixed urine from 24 to 48 hours post-intervention (P-24 to 48h). m / z 188.0022 Figure 3 ).

[0134] Detailed analysis of each mass spectrometry fragment was performed, and the candidate biomarkers were identified and their specificity evaluated. A total of six urinary biomarkers specific to whole-grain wheat were discovered. Figure 3 By comparing with standard samples, m / z 210.0406 was identified as 3,5-DHBA-glycine, a glycine conjugate of 3,5-dihydroxybenzoic acid (3,5-DHBA), a secondary metabolite of the whole-grain-specific phytochemical alkylresorcinol. Figure 4 Furthermore, in terms of differential metabolic characteristics... m / z 188.0022 m / z 230.0127 m / z Consistent mass spectrometry behavior was observed in the secondary fragments at 289.0386, specifically, all of them contained the characteristic fragment SO3. - ( m / z 79.9564 (corresponding to sulfate ions) and the loss of the parent ion HSO3 - The remaining fragments afterward. Therefore, the above-mentioned differential metabolites can be considered as sulfate conjugates. Through artificial mass spectrometry analysis and literature search, m / z 188.0022 m / z 230.0127 are the sulfate conjugates of 2-aminophenol (AP) and 2-acetamidophenol (HPAA), secondary metabolites of whole-grain phytochemicals, namely AP-sulfate and HPAA-sulfate. Figures 5-6 ).and m / z289.0386 was identified as DHPPTA-sulfate, a sulfated form of 3,5-dihydroxyphenyl propanoic acid (DHPPTA), a secondary metabolite of the whole-grain wheat phytochemical alkylresorcinol. Figure 7 Similarly, in m / z 284.0775 m / z The secondary spectrum at 326.0879 showed a common characteristic fragment ion C6H7O6. - ( m / z 175.0246 (corresponding to glucuronic acid fragment ions), indicating that the above metabolites are glucuronic acid conjugates. Because the above target fragment ions contain skeletal fragments completely identical to AP and HPAA, such as... m / z 108.0445 m / z 150.0560, which can be used to determine that they are AP, a secondary metabolite of whole-grain wheat benzoxazine phytochemicals, and AP-glucuronide, a glucuronic acid conjugate of HPAA. Figure 8 ) and HPAA-glucuronide ( Figure 9 AP-glucuronide has been confirmed through comparison with standard samples. The structures of the six markers are shown in Table 1.

[0135] Table 1. Information on six biomarkers in whole-grain wheat urine

[0136] .

[0137] Example 2

[0138] A method for identifying whole-grain wheat urinary biomarkers using an in vitro metabolic model: First, known precursors (AP, HPAA, and DHPPTA standards) are co-incubated with a human liver microsome / human liver cytoplasm mixture under conditions containing uridine diphosphate glucose (UDPGA) and 3'-adenosine-5'-phosphosulfate (PAPS) as necessary cofactors. Subsequently, the precise mass number, MS / MS chromatogram, and chromatographic retention time of the target metabolite obtained from the in vitro metabolic experiment are compared and validated with potential metabolites detected in urine samples (within 24 hours postprandial).

[0139] The specific in vitro metabolic experimental conditions are as follows (e.g.) Figure 10(As shown): Urate diphosphate glucose (UDPGA) / 3'-adenosine-5'-phosphosulfate (PAPS) and human liver microsomes / human liver cytoplasm were thawed on ice at -80°C. 475 µL of Tris-buffer buffer (100 mM, pH 7.4), 10 µL of mixed human liver microsomes (20 mg / mL) / mixed human liver cytoplasm (10 mg / mL) were added to an Eppendorf tube, followed by 10 µL of UDPGA / PAPS (100 mM / 10 mM). The mixture was pre-incubated in a 37°C water bath for 5 min, and then 5.0 µL (2.0 mg / mL) of the target analyte was added to initiate the reaction, maintaining a total reaction volume of 500 µL. After initiation, the mixture was incubated in a 37°C shaking water bath. After 12 h, 100 µL of the total reaction mixture was added to an Eppendorf tube containing 200 µL of acetonitrile to terminate the reaction. After centrifugation, the supernatant was obtained, diluted 10 times with 50% methanol-water, and the transformation type of the target analyte under phase II metabolic reaction was identified by LC-Orbitrap MS.

[0140] The results showed that the retention time, fragment ions, and ion abundance ratio of the four metabolite markers—AP-sulfate, HPAA-sulfate, HPAA-glucuronide, and DHPPTA-sulfate—in urine after ingestion of whole-grain wheat were completely consistent with the relevant data of metabolites obtained from in vitro incubation. Figure 11 This indicates that the identification of metabolic markers is accurate and reliable.

[0141] Example 3

[0142] A targeted detection method for whole-grain wheat intake urinary biomarkers, wherein the whole-grain wheat intake urinary biomarkers include the six biomarkers mentioned above, specifically including the following steps:

[0143] S1. Mass Spectrometry Parameter Optimization: Based on the mass spectrometry identification results of Example 1, the MS / MS mass spectrometry parameters of each marker were optimized, including retention time, qualitative ions, and quantitative ions. Specific mass spectrometry ion pair information and collision energy parameters are shown in Table 2.

[0144] Table 2. Mass spectrometry parameters of LC-MS / MS targeted detection methods for six whole-grain wheat urinary biomarkers

[0145] ;

[0146] Note: CE: Collision Energy, unit: electron volt (eV).

[0147] S2. Chromatographic Conditions: Three chromatographic columns—BEH C18, BEH Shield RP18, and BEH HSS T3—were selected for the chromatographic separation of the target analytes. Since urinary biomarkers are typically low-molecular-weight terminal metabolites with high hydrophilicity, the target analytes elute earlier on the first two columns, overlapping with solvent peaks and affecting detection sensitivity and specificity. Compared to the former, the BEH HSST3 column exhibits 100% aqueous phase compatibility, significantly improving the chromatographic retention of polar compounds and maintaining good tolerance under high aqueous phase conditions. Therefore, it was ultimately selected as the column for the analysis of urinary metabolic biomarkers. Furthermore, since metabolic biomarkers contain various glucuronidated, sulfated, and amino acid conjugates, most of these target metabolites are weakly acidic.

[0148] During chromatographic separation, changes in urine pH can significantly affect peak broadening and retention time, thus impacting the reproducibility of the analytical method. Adding 0.05% acetic acid to the mobile phase to create a weakly acidic system can suppress the ionization of weakly acidic compounds, enhance the chromatographic retention of target compounds, and improve the stability of the analytical results.

[0149] The instrument parameters for the optimized whole-grain urinary biomarker targeted detection method are as follows: Chromatographic separation was performed using a Waters ACQUITY UPLC HSS T3 column (100 mm × 2.1 mm, 1.7 μm), column temperature: 40℃, injection volume: 2 µL; mobile phase A was methanol containing 0.05% acetic acid, mobile phase B was ultrapure water containing 0.05% acetic acid, flow rate: 0.3 mL / min; linear gradient elution was used, with the elution program as follows: maintain initial mobile phase 5% A, linearly increase to 100% A within 8.5 min, hold for 2.0 min, return to initial mobile phase equilibrium before the next injection. The target analytes were detected using an AB Sciex 6500 triple quadrupole mass spectrometer. Finally, through optimization of the chromatographic gradient elution conditions, simultaneous separation and detection of six whole-grain wheat urinary biomarkers were achieved within 10 min. Figure 12 ).

[0150] Since there are no commercially available standards for AP-sulfate, HPAA-sulfate, HPAA-glucuronide, and DHPPTA-sulfate, standard curves were prepared using isomers or structural analogs of these biomarkers to quantify the target analytes (Table 2). For example, 4-acetaminophen sulfate (CAS: 32113-41-0) was used to quantify AP-sulfate and HPAA-sulfate; and Dihydro isoferulic acid 3-O-sulfate (CAS: 1258842-21-5) was used to quantify the sulfation metabolic biomarker DHPPTA-sulfate. To overcome the differences between urine matrices, matrix-matched standard curves with concentrations ranging from 0.1 to 200 ng / mL were prepared using mixed blank urine for quantitative analysis. The results showed that the correlation coefficients of all standard curves were greater than 0.99, which meets the needs of practical analysis.

[0151] Spiked recovery experiments were conducted by adding reference standards at low, medium, and high concentrations (2, 10, and 50 ng / mL) to pre-meal baseline mixed urine (P-0h). The results showed that the average recovery rate of whole-grain wheat urinary markers in the urine matrix ranged from 87.6% to 107.8%, with a precision of 2.3% to 13.8%, indicating good precision and recovery of the LC-MS / MS targeted detection method (Table 3). Using matrix spike concentrations of 3x signal-to-noise ratio (S / N) and 10x signal-to-noise ratio (S / N) as the limits of detection (LOD) and quantitation (LOQ), respectively, the detection LOD and LOQ ranges for whole-grain markers in urine were found to be 0.3–0.7 ng / mL and 1.0–2.0 ng / mL (Table 3), demonstrating the good sensitivity of this method. This method is the first to achieve highly sensitive simultaneous analysis and detection of six whole-grain wheat urinary markers in urine using LC-MS / MS.

[0152] Table 3. Sensitivity, accuracy, and precision of the whole grain urine biomarker detection method evaluated by standard additives (n=6)

[0153] .

[0154] Example 4: Validation of the application of whole-grain wheat urinary biomarkers (validation of biomarker dose-response relationship)

[0155] To further validate the dose-response relationship between whole-grain wheat intake and biomarkers, a subject-based trial was conducted. The validation trial lasted 6 days, including a 5-day introductory period and a 1-day intensive dietary intervention period. Figure 13During the induction period, participants were required to avoid consuming whole-grain wheat, including pre-packaged foods containing whole-wheat flour in their ingredient list. Apart from this, participants were free to choose other foods.

[0156] After the introductory period, participants entered a concentrated dietary intervention period, consisting of four standardized meals: dinner at the end of the introductory period (dinner on day 5 of the introductory period), and breakfast, lunch, and dinner on the first day of the intervention period. For breakfast, participants were randomly assigned to groups of 0g, 25g, 50g, or 100g of whole-wheat flour steamed buns, with other foods including optional rice porridge, eggs, and milk. To ensure the whole-grain wheat content of breakfast met the experimental design requirements, this process was conducted under the supervision of researchers. Dinner and lunch were designed and prepared by researchers based on balanced dietary principles, with restrictions on pasta intake (rice as the staple food), while meeting the daily nutrient and energy needs of both men and women. During the concentrated dietary period, meals were to be eaten in a concentrated manner, with only the foods provided by the researchers allowed. Participants were not permitted to consume any food or beverages other than those provided. Meals were scheduled for 8:00, 12:00, and 18:00 (to be consumed within 20 minutes). No food was consumed after dinner, except for the provided water.

[0157] The inclusion and exclusion criteria for subjects were the same as in Example 1. This validation case study was approved by the Biomedical Ethics Committee of Peking University (Approval No.: IRB00001052-23***), and the study protocol was simultaneously registered on the US Clinical Trials Registry (ClinicalTriab.gov) (NCT06358***). Subjects meeting the inclusion criteria were informed of the specific trial procedures and subject management system by the researchers and signed informed consent forms before participating in the formal trial.

[0158] After screening through questionnaires, physical examinations, and blood biochemistry indicators, 40 participants (20 males and 20 females) were enrolled in the study. Participants were randomly assigned to four subgroups (A, B, C, and D), with 10 participants in each subgroup, ensuring an equal number of males and females. During the trial, one participant each from subgroup A (0 g whole wheat flour) and subgroup C (50 g whole wheat flour) withdrew, leaving 38 participants who completed the dietary intervention trial and biosample collection.

[0159] On the day of the experiment, participants ate breakfast at 8:00 AM (to be consumed within 20 minutes). Urine samples were collected at 0-2 hours, 2 (excluding)-4 hours, 4 (excluding)-6 hours, 6 (excluding)-9 hours, 9 (excluding)-12 hours, and 12 (excluding)-24 hours after breakfast. After eating at the designated location, participants were allowed free time to carry the containers used for urine collection. Urine samples were collected at different time points according to the experimental requirements and temporarily stored in refrigerated bags containing ice packs. Participants returned to the designated location at 12:00 PM and 6:00 PM for lunch and dinner, respectively, and handed over the collected urine samples to the researchers. After urine collection at each time point, the researchers recorded the urine volume, aliquoted the urine samples into 1 mL portions, and froze them at -80°C. The urine samples from each time point were mixed at a volume ratio to prepare a 24-hour mixed urine (P-24h), and the established LC-MS / MS targeted detection method was used to quantitatively analyze whole grain urine biomarkers.

[0160] The diagnostic efficacy of urinary whole-grain wheat biomarkers for whole-grain intake was assessed by plotting receiver operating characteristic (ROC) curves. The ROC curve is plotted with the true positive rate (sensitivity) on the ordinate and the false positive rate (1-specificity) on the x-axis. The diagnostic value of the biomarker can be evaluated by calculating the area under the ROC curve (AUC). The AUC results are interpreted as follows: 0.9–1.0 indicates excellent; 0.8–0.9 indicates good; 0.7–0.8 indicates fair; 0.6–0.7 indicates poor; and 0.5–0.6 indicates model failure.

[0161] ROC analysis was used to evaluate the performance of whole-grain wheat urinary biomarkers in identifying whole-grain wheat consumers and their consumption levels in the validation trial (Table 4). The validation results showed that AP-sulfate exhibited excellent discrimination ability (AUC=1.0) among the six urinary biomarkers in 24-hour urine (P-24h) in distinguishing between whole-grain wheat and non-whole-grain wheat consumers. Furthermore, AP-sulfate, AP-glucuronide, HPAA-sulfate, DHPPTA-sulfate, and DHBA-glycine also performed well in determining whether whole-grain wheat intake reached 50g, and in distinguishing between moderate (50g) and high (100g) whole-grain wheat intake levels (AUC>0.9).

[0162] The concentration of 24-hour urinary biomarkers (P-24h) increased in a dose-dependent manner with increasing whole-grain wheat intake, demonstrating that urinary biomarkers can objectively reflect whole-grain wheat intake as a continuous variable. Figure 14 Overall, AP-sulfate and DHBA-glycine had high concentrations in urine after whole-grain wheat intake (urine concentrations exceeding 100 ng / mL 24 hours after 100g whole-wheat flour intake), and the linear relationship between the two and the amount of whole-wheat flour intake was the strongest. R 2 =0.98~0.99), and both have minimal individual heterogeneity, making them the best-performing urine biomarkers in validation studies.

[0163] Table 4. ROC Analysis of Urinary Biomarkers in Whole Grain Wheat to Identify Whole Grain Wheat Consumers and Consumption Levels

[0164] .

[0165] The above-described embodiments are merely specific implementations of the present invention, used to illustrate the technical solutions of the present invention, and are not intended to limit it. The scope of protection of the present invention is not limited thereto. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that any person skilled in the art can still modify or easily conceive of changes to the technical solutions described in the foregoing embodiments within the scope of the technology disclosed in the present invention, or make equivalent substitutions for some of the technical features; and these modifications, changes, or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention, and should all be covered within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of protection of the claims.

Claims

1. A urinary biomarker for whole-grain wheat intake, characterized in that, The markers include glycine conjugates of 3,5-dihydroxybenzoic acid, sulfate conjugates of o-aminophenol, sulfate conjugates of 2-acetaminophenol, sulfate conjugates of 3,5-dihydroxyphenylvaleric acid, glucuronic acid conjugates of o-aminophenol, and glucuronic acid conjugates of 2-acetaminophenol. The dose-response equation for the glucuronic acid conjugate of o-aminophenol is Y = 0.2320X - 1.310; The dose-response equation for o-aminophenol is Y = 3.150X + 14.19; The dose-response equation for the glycine-bound form of 3,5-dihydroxybenzoic acid is: Y = 1.719X - 1.548; The dose-response equation for the sulfate conjugate of acetaminophen is: Y = 2.859X - 20.41; The dose-response equation for glucuronic acid conjugates is: Y = 0.05409X + 0.4704; The dose-response equation for the sulfate conjugate of 2,5-dihydroxyphenylpentanoic acid is: Y = 0.6991X - 1.282; The high-throughput screening method for whole-grain wheat intake urinary biomarkers includes the following steps: (1) Two-stage crossover dietary intervention experiment: The intervention group was a whole wheat flour group and the control group was a refined wheat flour group. Two stages of dietary intervention were carried out, and the two groups exchanged intervention foods during the two stages of intervention. (2) Time-segmented urine collection: Baseline urine was collected 0 hours before intervention. After intervention, urine was collected at different time periods: 0 (excluding) ~ 2 hours, 2 (excluding) ~ 4 hours, 4 (excluding) ~ 6 hours, 6 (excluding) ~ 9 hours, 9 (excluding) ~ 12 hours, 12 (excluding) ~ 24 hours, 24 (excluding) ~ 36 hours, and 36 (excluding) ~ 48 hours. The urine volume at each time period was recorded. (3) Sample mixing and detection: Urine samples from different time periods were mixed in volume ratio to obtain mixed urine samples, and metabolites were detected by high performance liquid chromatography-mass spectrometry. (4) Screening for urinary biomarkers of whole-grain wheat intake: Based on the results of the cross-intervention experiment, the differences in metabolite concentrations between and within groups after meals and at baseline were compared to screen for specific urinary biomarkers of whole-grain wheat intake. 4.1 Preliminary Screening: (i) Comparison between the intervention group and the control group: At each stage, the differences in metabolite concentrations in urine samples from the intervention group and the control group at corresponding time points are compared, and the following conditions must be met: Mixed urine samples from P-12h and P-24h: P Value < 0.001 and difference factor log2 FC > 2; Mixed urine samples from 24 to 48 hours post-conversion: P Value < 0.05 and difference factor log2 FC > 1; Furthermore, (ii) comparisons within the intervention group: At each stage, the differences in metabolite concentrations between postprandial urine samples and baseline samples taken 0 hours before meals within the intervention group were compared, and the following conditions must be met: Compared with the 0h sample, the mixed urine samples from P-12h and P-24h were: P Value < 0.001 and difference factor log2 FC > 2; Compared with the 0h sample, the mixed urine samples from P-24~48h: P Value < 0.05 and difference factor log2 FC > 1; 4.2 Biomarker identification: Exogenous specific differential metabolites that were reproduced in both stages of the cross-intervention experiment were screened out through peak detection, isotope cluster analysis, identification of characteristic MS / MS fragments and mass spectrometry library matching as biomarkers of whole grain wheat intake in urine. In step 3.2, the chromatographic conditions are as follows: column temperature 30-40ºC, injection volume 1-2 µL; mobile phase A is methanol containing 0.01-0.05% acetic acid, and mobile phase B is ultrapure water containing 0.01-0.05% acetic acid; the liquid chromatography flow rate is 0.2-0.4 mL / min, and linear gradient elution is used. Mass spectrometry conditions: spray voltage 3.5~3.8 kV (+), 3.0~3.5 kV (-); heating temperature 300~350℃; capillary temperature 300~320℃; scanning range 100~1000 m / z; collision energies 15 eV, 30 eV and 50 eV.

2. The screening method according to claim 1, characterized in that, The intervention experiment described in step (1) includes four phases: the induction phase, the first phase of intervention, the washout phase, and the second phase of intervention. The induction period was 5 days: for the first 3 days, both groups of subjects avoided consuming whole grain wheat and processed foods; for the 4th and 5th days, they avoided consuming pasta foods. The first phase of intervention lasted for 2 days. On the intervention day, the whole wheat flour group consumed whole wheat flour-based foods for breakfast, while the refined wheat flour group consumed the same amount of refined wheat flour-based foods for breakfast on the intervention day. The remaining grains consumed on that day were rice, and the total daily grain intake of the two groups was equal. The washout period is 5 days, and the dietary restrictions are the same as those during the induction period. The second phase of intervention lasted for 2 days, during which the two groups exchanged intervention foods.

3. The screening method according to claim 1, characterized in that, Step (3) includes: 3.1 Mixing and Pretreatment: Mix, centrifuge, and dilute the urine; The urine mixing process involves mixing urine samples from different time periods according to their volume ratios to form a mixed urine sample. Among them, P-12h mixed urine: urine mixture from the 0-12 h period; P-24h mixed urine: urine from the 0-24 hour period is mixed; P-24~48h Mixed Urine: Mixed urine from the period of 24 (excluding) to 48 hours; Centrifugation: Centrifuge at 7000-10000×g for 10-30 min at 0-4ºC; The dilution is described as follows: a 10-20% methanol-water diluent is used. 3.2 High-performance chromatography-mass spectrometry detection; and, 3.3 Data Acquisition and Preliminary Identification: Data acquisition included peak extraction, peak alignment, background subtraction, and data filtering. Compound types were preliminarily identified using a self-built library.

4. The method according to claim 3, characterized in that, In step 3.3, Xcalibur software is used for data acquisition, and Compound Discoverer software is used for peak extraction, peak alignment, and background subtraction. Data filtering conditions include exact mass number deviation < 5 ppm, signal-to-noise ratio (S / N) > 10, peak intensity (Peak area) > 100,000, and retention time offset (RT) < 0.2 min.

5. The method according to claim 4, characterized in that, Six urinary biomarkers specific to whole-grain wheat were identified, with mass-to-charge ratios of [missing information]. m / z 210.0406 m / z 188.0022 m / z 230.0127 m / z 289.0386 m / z 284.0775 and m / z 326.0879; in, m / z 210.0406 is a glycine conjugate of 3,5-dihydroxybenzoic acid. m / z 188.0022 is a sulfate conjugate of o-aminophenol. m / z 230.0127 is a sulfate conjugate of 2-acetaminophen. m / z 289.0386 is a sulfate conjugate of 3,5-dihydroxyphenylpentanoic acid. m / z 284.0775 is a glucuronic acid conjugate of o-aminophenol. m / z 326.0879 is a glucuronic acid conjugate of 2-acetaminophen.

6. A method for identifying urinary biomarkers of whole-grain wheat intake using an in vitro metabolic model, characterized in that, First, the precursors o-aminophenol, 2-acetaminophen, and 3,5-dihydroxyphenylvaleric acid standards are co-incubated with a mixture of human liver microsomes and / or human liver cytoplasm; then, the retention time, fragment ions, and ion abundance ratio of the in vitro metabolites and the urinary sample metabolites are compared; wherein the mixture includes a cofactor, and the whole grain wheat urinary biomarker is as described in claim 1.

7. A targeted detection method for urinary biomarkers of whole-grain wheat intake as described in claim 1, characterized in that, Includes the following steps: S1. Mass Spectrometry Parameters: Targeted optimization of MS / MS mass spectrometry parameters for each biomarker, including retention time, qualitative ions, and quantitative ions; ； S2. Chromatographic conditions: A Waters ACQUITY UPLC HSS T3 column was used, with a column temperature of 30~40ºC and an injection volume of 1~2 µL; mobile phase A was methanol containing 0.01~0.05% acetic acid, and mobile phase B was ultrapure water containing 0.01~0.05% acetic acid, with a flow rate of 0.1~0.3 mL / min.

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