Urinary biomarkers of p-hydroxyacetophenone exposure and their application in human load monitoring

Abstract

This invention discloses urinary biomarkers for p-hydroxyacetophenone (PHAC) exposure and their application in human load monitoring. Based on the phase I / II metabolic transformation patterns of PHAC, this invention establishes a targeted screening list, enhances mass spectrometry fragment acquisition efficiency and qualitative analysis capabilities, and combines high-resolution mass spectrometry data with a fragment ion search system to achieve full-spectrum screening of urinary exposure biomarkers. Furthermore, it identifies potential targets through metabolic site energy simulation and utilizes a fragment ion mapping intelligent matching algorithm to achieve precise molecular structure and metabolic transformation pathway analysis of six PHAC urinary exposure biomarkers. Simultaneously, through semi-quantitative analysis of metabolite peak areas, it establishes, for the first time using non-invasive urine samples, highly sensitive PHAC exposure biomarkers suitable for human load monitoring.

Description

Technical Field

[0001] This invention belongs to the field of environmental pollutant detection, specifically relating to urinary biomarkers of p-hydroxyacetophenone exposure and their application in monitoring human body load. Background Technology

[0002] Organic pollutant exposure biomarkers refer to the quantitatively detectable original pollutants, metabolic transformation products, and adducts formed with endogenous substances in the tissues, body fluids, or excrement of organisms. These biomarkers serve as a bridge between environmental pollution exposure and health effects, providing key technical support for population exposure assessment and environmental health risk early warning systems.

[0003] Parabens, a typical preservative, are widely used as additives in various food, pharmaceutical, and personal care products and are a class of endocrine disruptors that have attracted much attention.

[0004] Studies have shown that long-term paraben exposure can lead to metabolic disorders, developmental impairment, and endocrine disruption. Given the range of adverse health effects caused by long-term paraben exposure, manufacturers have gradually reduced paraben use and are instead developing alternatives.

[0005] The structural analogue of parabens, p-hydroxyacetophenone (PHAC), has a 1-3 order of magnitude higher metabolic load in children than parabens, and preliminary neurotoxicity studies have revealed that its metabolic interference effect is greater than that of conventional parabens. Therefore, clarifying the health risks of PHAC exposure is particularly important.

[0006] Identifying PHAC exposure biomarkers is the first step in systematically revealing its population-level health effects and risks. However, as a newly discovered potential alternative to Parabens, research on PHAC exposure biomarkers is still in its early stages, and the appropriate biomarkers for accurate internal exposure calculations remain unknown. Summary of the Invention

[0007] The purpose of this invention is to provide urinary biomarkers of p-hydroxyacetophenone (PHAC) exposure and their application in monitoring human body load, to clarify the metabolic transformation process of PHAC in vivo, and to rapidly provide exposure biomarkers for monitoring human body load.

[0008] The objective of this invention is achieved through the following technical solution:

[0009] Application of urinary biomarkers of p-hydroxyacetophenone exposure in human load monitoring;

[0010] The urinary exposure biomarkers for p-hydroxyacetophenone are one or more of p-hydroxyacetophenone (M0), 1-(2,4-dihydroxyphenyl)ethyl-1-one (M1), 4-acetyl-3-hydroxyphenyl hydrogen sulfate (M2), 1-(2-hydroxy-4-methoxyphenyl)ethyl-1-one (M3), 4-acetylphenyl hydrogen sulfate (M4) or 4-(1-hydroxyethyl)phenyl hydrogen sulfate (M5);

[0011] Preferably, the urinary exposure biomarker for p-hydroxyacetophenone is one or more of 4-acetylphenyl hydrogen sulfate (M4), p-hydroxyacetophenone (M0), or 4-(1-hydroxyethyl)phenyl hydrogen sulfate (M5);

[0012] More preferably, the urinary exposure biomarkers for p-hydroxyacetophenone are 4-acetylphenyl hydrogen sulfate (M4) and p-hydroxyacetophenone (M0).

[0013] Particularly preferred, the urinary exposure biomarkers for p-hydroxyacetophenone are 4-acetylphenyl hydrogen sulfate (M4), p-hydroxyacetophenone (M0), and 4-(1-hydroxyethyl)phenyl hydrogen sulfate (M5).

[0014] A method for screening urinary biomarkers for p-hydroxyacetophenone (PHAC) exposure includes the following steps:

[0015] (1) Exposure and sample preparation: Experimental animals were injected intraperitoneally with p-hydroxyacetophenone for at least 8 consecutive days, and then urine was collected for further analysis.

[0016] For the intraperitoneal injection, the preferred dosage of p-hydroxyacetophenone is 12.5 mg / kg / day;

[0017] The experimental animals mentioned include rodents or non-rodents;

[0018] The preferred rodents are rats and mice;

[0019] The preferred non-rodent animals are rabbits, dogs, monkeys, or others;

[0020] The urine sample can be purified and enriched before analysis;

[0021] (2) Construction of a molecular formula library of theoretical exposure biomarkers:

[0022] BioTransformer 3.0 was used to predict PHAC transformation products. The maximum number of phase I reactions was set to 3, and the maximum number of phase II metabolic reactions was set to 1, to form a series of transformation products.

[0023] Using the Expected Compound module in Compound Discovery, and based on the reaction rules, the maximum number of phase I reactions is set to 3, and the maximum number of phase II metabolic reactions is set to 1, to form a series of transformation products;

[0024] The phase I reaction described includes oxidation, reduction, hydrolysis, and desaturation reactions.

[0025] The Phase II reactions described include acetylation, methylation, glucosylation, sulfation, and amino acid complexation reactions.

[0026] The predicted transformation products formed by Biotransformer and Compound Discovery are combined to construct a theoretical exposure biomarker molecular formula library, and this library is included in the target ion inclusion list in step (3) to enhance the collection efficiency of secondary ion fragments and improve the qualitative quality of the results.

[0027] (3) Mass spectrometry data acquisition: fragment spectra were acquired using an ultra-high performance liquid chromatography-tandem quadrupole / orbit trap high-resolution mass spectrometer;

[0028] The acquisition includes full mass spectrometry scan, dynamic exclusion, target ion inclusion list, and fragment ion scan;

[0029] (4) Screening analysis of suspected exposure biomarkers:

[0030] The fragment spectra collected in step (3) were screened using Compound Discoverer software. The theoretical exposure biomarker molecular formula library was matched with the measured mass spectrometry data. The set of biomarkers with a mass error of <5 ppm was used as a candidate set of potential PHAC exposure biomarker molecular formulas.

[0031] The screening includes one or more of the following operations: peak extraction, peak alignment, screening of desired compounds, combination of desired compounds, compound identification and annotation, and fragment ion search scoring;

[0032] (5) Structural analysis and screening of exposure biomarkers:

[0033] The activation energy thresholds of each reaction site of PHAC molecules were evaluated using a metabolic simulation platform to locate highly propensity metabolically active sites. Based on the principle of metabolic energy optimization, the preliminary structural formulas of candidate biomarkers were derived from the molecular formula candidate set of potential PHAC exposure biomarkers.

[0034] The preliminary structural formula was further confirmed by retention time prediction and matching results of secondary fragment ions, including:

[0035] Retention time prediction: Use standards to confirm the retention time of PHAC and determine the direction of retention time change of PHAC exposure biomarkers (earlier or later peak elution) based on the hydrophilicity or hydrophobicity of binding / reactive groups, and exclude compounds with abnormal retention time prediction.

[0036] Secondary fragment ion matching degree: The preliminary structural formula is imported into Compound Discoverer software, the fragment ion intelligent matching engine is enabled, the system compares the characteristic ion clusters between the measured mass spectrometry fragments (HCD multi-level spectrum) of the analyte and the theoretical fragments of the preliminary structural formula, completes the structural confirmation based on the spectrum similarity threshold, and selects the molecular structural formula with the highest matching degree.

[0037] Next, by integrating the predicted metabolic sites and mass spectrometry validation data, a PHAC multi-pathway metabolic transformation network diagram was constructed.

[0038] Ultimately, based on sensitivity, specificity, and stability assessments, the exposure biomarkers with the best overall performance were selected, including:

[0039] Sensitivity assessment: Exposure biomarkers with the top 20% peak intensity were screened out by characteristic peak area normalization analysis.

[0040] Specificity assessment: Exposure biomarkers are only generated when a specific exposure occurs, and are mainly characterized by the difference in the concentration levels of exposure biomarkers between the exposed group and the control group;

[0041] Stability assessment: Specifically refers to whether exposure biomarkers can be stably formed under quantitative exposure doses, mainly characterized by the coefficient of variation between days.

[0042] The metabolic simulation platform is preferably ADMET Predictor™ and / or BioTransformer.

[0043] Through the above-described steps of analyzing and verifying the structure of exposure biomarkers, this invention identified a total of 6 PHAC exposure biomarkers (Tables 3 and 4). The formation of these exposure biomarkers mainly involves reactions such as oxidation, reduction, methylation, and sulfation.

[0044] The present invention has the following advantages and effects compared with the prior art:

[0045] This invention, based on the phase I / II metabolic transformation patterns of PHAC, establishes a targeted screening list, enhances the efficiency of mass spectrometry fragment acquisition and qualitative analysis capabilities, and combines high-resolution mass spectrometry data with a fragment ion search system to achieve full-spectrum screening of urine exposure biomarkers. Furthermore, it identifies potential targets through metabolic site energy simulation and utilizes a fragment ion map intelligent matching algorithm to achieve precise molecular structure and metabolic transformation pathway analysis of six PHAC urine exposure biomarkers. Simultaneously, through semi-quantitative analysis of metabolite peak areas, it establishes, for the first time using non-invasive urine samples, highly sensitive PHAC exposure biomarkers suitable for monitoring human body load. Attached Figure Description

[0046] Figure 1 To reveal the differences in urinary exposure biomarkers between the solvent control group and the exposure group based on principal component analysis.

[0047] Figure 2 The spectrum of the precursor ion (MS1) and fragment ion (MS2) of M0 qualitatively identified in urine and the difference in peak area response between groups (top right corner).

[0048] Figure 3 The spectrum of the precursor ion (MS1) and fragment ion (MS2) of M1 qualitatively identified in urine and the difference in peak area response between groups (top right corner).

[0049] Figure 4 The spectrum of the precursor ion (MS1) and fragment ion (MS2) of M2 qualitatively identified in urine and the difference in peak area response between groups (top right corner).

[0050] Figure 5 The spectrum of the precursor ion (MS1) and fragment ion (MS2) of M3 qualitatively identified in urine and the difference in peak area response between groups (top right corner).

[0051] Figure 6 The spectrum of the precursor ion (MS1) and fragment ion (MS2) of M4 qualitatively identified in urine and the difference in peak area response between groups (top right corner).

[0052] Figure 7 The spectra of the precursor ion (MS1) and fragment ion (MS2) of M5 qualitatively identified in urine and the differences in peak area response between groups.

[0053] Figure 8 The in vivo metabolic pathways of six PHAC exposure biomarkers in rats were investigated.

[0054] Figure 9 The abundance of PHAC and its six exposure biomarkers in urine was measured on different days. Detailed Implementation

[0055] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto.

[0056] Example

[0057] The screening method for PHAC urine exposure biomarkers includes the following steps:

[0058] (1) Grouping and exposure regimen: Eight-week-old male SPF-grade SD rats (Southern Medical University) were used and acclimatized for one week in a natural light environment with a humidity of 45%±5% at 25±1 ℃. They were then randomly divided into a 10% ethanol solvent control group and a PHAC exposure group with 12.5 mg / kg / d. They were exposed to PHAC by intraperitoneal injection for 8 consecutive days, and urine samples were collected daily.

[0059] (2) Urine purification and enrichment: Take 2 mL of urine and add it to a 10 mL plastic tube. Add 6 mL of methanol pre-cooled to -20 ℃. Vortex mix for 30 seconds. Then, dispense the mixture into 5 tubes of 2 mL each and let it stand at -80 ℃ for 60 min. After centrifugation at 14000 rpm for 15 min, take 1 mL of supernatant and combine them into 5 mL of mixture. Then, vacuum dry the mixture. Redissolve the residue with 400 μL of methanol solution and centrifuge again at 14000 rpm for 15 min (4 ℃). Finally, take 120 μL of supernatant and transfer it to a sample bottle. Add 30 μL of methylparaben-D4 isotope internal standard.

[0060] (3) Construction of a molecular formula library of theoretical exposure biomarkers:

[0061] BioTransformer 3.0 (primarily based on the prediction of metabolic reaction sites) was used to predict PHAC transformation products. The maximum number of phase I reactions was set to 3, and the maximum number of phase II metabolic reactions was set to 1, forming a series of transformation products.

[0062] Using the Expected Compound module in Compound Discovery (primarily based on free combination of metabolic reaction rules), according to the reaction rules, i.e., setting the maximum number of phase I reactions to 3 and the maximum number of phase II metabolic reactions to 1, a series of transformation products are formed;

[0063] Metabolic reaction types include phase I reactions such as oxidation, reduction, hydrolysis, and desaturation, as well as phase II reactions such as acetylation, methylation, glucosidation, sulfation, and various amino acid complexation.

[0064] The predicted transformation products formed by Biotransformer and Compound Discovery were combined to construct a theoretical exposure biomarker molecular formula library (Tables 1 and 2), and this library was included in the target ion inclusion list in step (4) to enhance the collection efficiency of secondary daughter ion fragments and improve the qualitative quality of the results.

[0065] (4) Mass spectrometry data acquisition: An ultra-high performance liquid chromatography-tandem quadrupole / orbitrap high-resolution mass spectrometer (UPLC-Orbitrap Exploris 240) was used. Chromatographic separation was performed using a Hypersil GOLD AQ C18 column (150×2.1 mm, 1.9 μm). The mobile phase was methanol (organic phase B) and 0.05% acetic acid in water (aqueous phase A). The column temperature was 35℃, the flow rate was 0.3 mL / min, and the injection volume was 2 μL. Gradient elution program: 0~1 min 2% B; 1~8 min linearly up to 98% B; 8~11 min maintain 98% B; 11.01~14 min equilibrate to 2% B. Mass spectrometry detection was performed using ESI negative ion mode (spray voltage –3.2 kV). Ion source parameters: sheath gas 45 Arb, auxiliary gas 8 Arb, purge gas 1 Arb, transfer tube 320℃, vaporizer 350℃. The data acquisition process includes full mass spectrometry scan, dynamic exclusion, target ion inclusion list, and fragment ion scan. The target ion inclusion list incorporates a pre-established library of theoretical exposure biomarker molecular formulas from potential steps (3) to enhance the acquisition efficiency of target exposure biomarkers, improve qualitative identification capabilities, and significantly solve the problem of low fragment ion acquisition efficiency caused by the co-eluting of high-abundance endogenous substances. The full scan resolution was 240,000 m / z (90-600 m / z). Fragment ion scan settings included: isolation window of 2 m / z, HCD collision energy step of 20%-80% (step size 20%), resolution of 15,000 m / z, and simultaneous acquisition of 10 fragment spectra.

[0066] (5) Screening analysis of suspected exposure biomarkers:

[0067] An automated screening process for the fragment spectra obtained in step (4) was established using Compound Discoverer software (covering modules such as peak extraction, peak alignment, screening of expected compounds, combination of expected compounds, compound identification and annotation, and fragment ion search scoring). The theoretical exposure biomarker molecular formula library was precisely matched with the measured high-resolution mass spectrometry data (mass error < 5 ppm) to screen out a candidate set of potential PHAC exposure biomarker molecular formulas (Table 3).

[0068] Table 1: List of molecular formulas of potential PHAC exposure biomarkers based on BioTransformation

[0069]

[0070] Table 2: List of molecular formulas of potential PHAC exposure biomarkers based on Compound Discoverer

[0071]

[0072] (6) Structural analysis and screening of exposure biomarkers:

[0073] Using the ADMET Predictor™ and BioTransformer 3.0 metabolic simulation platform, the activation energy thresholds of each reaction site in the PHAC molecule were evaluated through quantum chemical calculations (density functional theory model), accurately locating metabolic active sites such as oxidation, reduction, and sulfation. Based on the principle of metabolic energy optimization, candidate biomarkers and their preliminary structural formulas were derived from the potential PHAC exposure biomarker molecular formula candidate set.

[0074] The preliminary structural formula was further confirmed by retention time prediction and matching results with secondary fragment ions, specifically including:

[0075] Retention time prediction: Use standards to confirm the retention time of PHAC and determine the direction of retention time change of PHAC exposure biomarkers (earlier or later peak elution) based on the hydrophilicity or hydrophobicity of binding / reactive groups, and exclude compounds with abnormal retention time prediction.

[0076] Secondary fragment ion matching: The preliminary structural formula is imported into the compound annotation editor module of Compound Discoverer 3.3 SP2 software. The fragment ion intelligent matching engine (FISh) is enabled. The system compares the characteristic ion clusters between the measured mass spectrometry fragments (HCD multi-level spectra) of the analyte and the theoretical fragments of the preliminary structural formula. Based on the spectral similarity threshold (m / z deviation < 5 ppm), the structure is confirmed, and the molecular structure with the highest matching degree is selected. Finally, the metabolic site prediction and mass spectrometry validation data are integrated to construct the PHAC multi-pathway metabolic transformation network diagram.

[0077] Finally, based on sensitivity, specificity, and stability assessments, the exposure biomarkers with the best overall performance were selected, including:

[0078] Sensitivity assessment: Exposure biomarkers with the top 20% peak intensity were screened out by characteristic peak area normalization analysis.

[0079] Specificity assessment: Exposure biomarkers are only generated when a specific exposure occurs, and are mainly characterized by the difference in the concentration levels of exposure biomarkers between the exposed group and the control group;

[0080] Stability assessment: Specifically refers to whether exposure biomarkers can be stably formed under quantitative exposure doses, mainly characterized by the coefficient of variation between days.

[0081] (7) Experimental results:

[0082] Principal component analysis showed that urine samples from the solvent control group and the PHAC exposure group exhibited extremely clear separation. Figure 1 This indicates that the PHAC exposure group formed a series of significantly different exposure biomarkers. Through the above-mentioned exposure biomarker structure analysis and verification steps, this invention identified a total of 6 PHAC exposure biomarkers (Tables 3 and 4). The formation of these exposure biomarkers mainly involves reactions such as oxidation, reduction, methylation, and sulfation. The isotopic matching degree of the parent ion of PHAC (M0) and its 6 exposure biomarkers is 100%, with at least two fragment ions matched in the database and a mass deviation of <1.5 ppm (Table 3), and the structure confidence level reaches L2. The spectrum of the parent ion (MS1) and fragment ion (MS2) of PHAC and its exposure biomarkers qualitatively identified in urine is shown in the figure below. Figures 2-7 As shown.

[0083] M1 (m / z: 151.0401) Figure 3 ) is an oxidation product of PHAC, with a FISh coverage of 46.4%; M2 (m / z: 230.9965) ( Figure 4 ) is the sulfation product of M1, with a FISh coverage of 39.4%; M3 (m / z: 165.0557) ( Figure 5 ) is the methylated product of M1, with FISh coverage of 65.0%; M4 (m / z: 215.0018) ( Figure 6 ) is a sulfation product of PHAC, with a FISh coverage of 55.6%; M5 (m / z: 217.0176) ( Figure 7 ) is the sulfation product of PHAC after reduction, with FISh coverage reaching 52.0%. Based on the metabolic transformation reaction patterns, the in vivo metabolic transformation pathways of six PHAC exposure biomarkers were further mapped. Figure 8 ).

[0084] In addition, the six PHAC exposure biomarkers were semi-quantitatively analyzed based on the standardized peak area (Table 3) to assess sensitivity. The results showed that the exposure biomarker with the highest relative proportion in urine was M4 (49.2%), followed by M0 (36.4%) and M5 (12.3%). Therefore, sulfated complexes are the main urinary exposure biomarkers of PHAC.

[0085] Further evaluation of the specificity and stability of M4 and M0 was conducted, and the results are as follows:

[0086] From the perspective of specificity assessment ( Figure 2 and Figure 6 (See the upper right corner bar chart). The peak areas of M4 and M0 in the urine of the control group were 3.74 × 10⁻⁶ and 3.74 × 10⁻⁶, respectively. 8 and 3.68×10 8 The peak area in the PHAC exposure group was 3.73 × 10⁻⁶. 10 and 2.51×10 10 The peak area ratios of the exposed and control groups were 99.8% and 68.1%, respectively, showing a highly significant difference between the groups. p <0.001). Therefore, both M4 and M0 have strong specificity.

[0087] From a stability assessment perspective ( Figure 9 Monitoring of biomarkers in rat urine for eight consecutive days revealed that M4 and M0 showed good stability except for fluctuations on the fourth day, with an inter-day coefficient of variation of less than 30%, indicating strong stability.

[0088] In summary, the findings suggest that urinary M4 and M0 can serve as highly sensitive, specific, and stable biomarkers for indicating PHAC exposure.

[0089] Table 3: Six PHAC exposure biomarkers identified in urine (M0 is the parent compound of PHAC).

[0090]

[0091] Table 4: List of 6 PHAC exposure biomarkers and their molecular structures (M0 is the parent compound of PHAC).

[0092]

[0093] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. Use of a urinary exposure biomarker for 4-hydroxyacetophenone in the monitoring of the internal load in rats, characterized in that: The urine exposure biomarkers for the p-hydroxyacetophenone are p-hydroxyacetophenone, 1-(2,4-dihydroxyphenyl)ethan-1-one, 4-acetyl-3-hydroxyphenyl hydrogen sulfate, 1-(2-hydroxy-4-methoxyphenyl)ethan-1-one, 4-acetylphenyl hydrogen sulfate, and 4-(1-hydroxyethyl)phenyl hydrogen sulfate.

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