Serum exposure biomarker of p-hydroxyacetophenone and application of serum exposure biomarker in monitoring load in human body

Abstract

The invention discloses a serum exposure biomarker of p-hydroxyacetophenone and application of the serum exposure biomarker in human body load monitoring. According to the invention, based on the I / II phase metabolic transformation rule of PHAC, the full-spectrum screening of the exposed biomarker is realized by combining high-resolution mass spectrum data and a fragment ion search system; further simulating and locking potential targets through metabolic site energy, and realizing precise molecular structure and metabolic conversion path analysis of 11 kinds of PHAC exposed biomarkers by utilizing a fragment ion spectrum intelligent matching algorithm; meanwhile, through metabolite peak area semi-quantitative analysis, the PHAC high-sensitivity exposure biomarker capable of being used for human body load monitoring in serum is determined for the first time.

Description

Technical Field

[0001] The present invention belongs to the field of environmental pollutant detection, and particularly relates to serum exposure biomarkers of 4-hydroxyacetophenone and their application in monitoring the human body burden. Background Art

[0002] Biomarkers of exposure to organic pollutants refer to pollutant prototypes, metabolites, and adducts formed with endogenous substances that can be quantitatively detected in biological tissues, body fluids, or excreta. Such biomarkers serve as a bridge between environmental pollution exposure and health effects, providing key technical support for population exposure assessment and environmental health risk warning systems.

[0003] Typical preservatives, parabens, are widely used as additives in various foods, pharmaceuticals, and personal care products and are a class of endocrine disruptors that have received much attention.

[0004] Studies have shown that long-term exposure to parabens can lead to metabolic disorders, developmental damage, and endocrine disruption effects. Given the series of adverse health effects caused by long-term parabens exposure, manufacturers have gradually reduced the use of parabens and instead developed their alternatives.

[0005] The structural analogue of parabens, 4-hydroxyacetophenone (PHAC), has a load in children's bodies that is 1-3 orders of magnitude higher than that of parabens, and preliminary studies on the toxic effects of nerve cell exposure reveal that its metabolic interference effect is higher than that of traditional parabens. Therefore, it is particularly important to clarify the exposure health risks of PHAC.

[0006] Identifying PHAC exposure biomarkers is the primary step in systematically revealing its population exposure health effects and risks. However, as a newly discovered potential parabens alternative, the research on PHAC exposure biomarkers is still in its infancy, and it remains unknown which exposure biomarkers can be used to accurately calculate the internal exposure dose. Summary of the Invention

[0007] The purpose of the present invention is to provide serum exposure biomarkers of 4-hydroxyacetophenone and their application in monitoring the human body burden, clarify the in vivo metabolic transformation process of PHAC, and quickly provide exposure biomarkers for monitoring the human body burden.

[0008] The purpose of the present invention is achieved by the following technical solutions: Application of serum exposure biomarkers of 4-hydroxyacetophenone in monitoring the human body burden; The serum exposure biomarkers of p - hydroxyacetophenone are one or more of 1 - (2,4 - dihydroxyphenyl)ethan - 1 - one (M1), 4 - acetyl - 3 - hydroxyphenyl hydrogen sulfate (M2), 1 - (2 - hydroxy - 4 - methoxyphenyl)ethan - 1 - one (M3), 4 - acetylphenyl hydrogen sulfate (M4), 4 - (1 - hydroxyethyl)phenyl hydrogen sulfate (M5), 2 - hydroxy - 1 - (4 - hydroxyphenyl)ethan - 1 - one (M6), 1 - (4 - methoxyphenyl)ethan - 1 - one (M7), 4 - (1 - hydroxyethyl)phenyl palmitate (M8), 4 - vinylphenyl hydrogen sulfate (M9), 2 - hydroxy - 1 - (2 - hydroxy - 4 - methoxyphenyl)ethan - 1 - one (M10), or 6 - (4 - acetyl - 3 - hydroxyphenoxy)-3,4,5 - trihydroxytetrahydro - 2H - pyran - 2 - carboxylic acid (M11); Preferably, the serum exposure biomarkers of p - hydroxyacetophenone are one or more of 1 - (4 - methoxyphenyl)ethan - 1 - one (M7), 4 - acetylphenyl hydrogen sulfate (M4), or 4 - (1 - hydroxyethyl)phenyl hydrogen sulfate (M5); More preferably, the serum exposure biomarkers of p - hydroxyacetophenone are 1 - (4 - methoxyphenyl)ethan - 1 - one (M7), 4 - acetylphenyl hydrogen sulfate (M4), and 4 - (1 - hydroxyethyl)phenyl hydrogen sulfate (M5).

[0009] A screening method for serum exposure biomarkers of p - hydroxyacetophenone (PHAC) includes the following steps: (1) Exposure and sample preparation: Experimental animals are intraperitoneally injected with p - hydroxyacetophenone for at least 8 consecutive days, and then serum is collected for the next - step analysis; For the intraperitoneal injection, the administration dose of p - hydroxyacetophenone is preferably 12.5 mg / kg / d; The experimental animals include rodents or non - rodents; The rodents are preferably rats and mice; The non - rodents are preferably rabbits, dogs, monkeys, or others; Before analysis, the serum can be purified and enriched; (2) Construction of the theoretical exposure biomarker molecular formula library: Use BioTransformer 3.0 to predict the transformation products of PHAC, set the maximum number of phase I reactions to 3 and the maximum number of phase II metabolic reactions to 1 to form a series of transformation products; Use the Expected Compound module in Compound Discovery, according to the reaction rules, set the maximum number of phase I reactions to 3 and the maximum number of phase II metabolic reactions to 1 to form a series of transformation products; The described Phase I reactions include oxidation reactions, reduction reactions, hydrolysis reactions, and desaturation reactions; The described Phase II reactions include acetylation reactions, methylation reactions, glucuronidation reactions, sulfation reactions, and amino acid conjugation reactions; Combine the predicted transformation products formed by Biotransformer and Compound Discovery, construct a theoretical exposure biomarker molecular formula library, and incorporate this library into the targeted ion inclusion list in step (3) to enhance the acquisition efficiency of secondary daughter ion fragments and improve the qualitative quality of the results; (3) Mass spectrometry data acquisition: Use an ultra-high performance liquid chromatography tandem quadrupole / orbitrap high-resolution mass spectrometer to acquire fragment spectra; The described acquisition includes full-scan mass spectrometry, dynamic exclusion, targeted ion inclusion list, and fragment ion scanning; (4) Suspected screening analysis of exposure biomarkers: Screen the fragment spectra acquired in step (3) through Compound Discoverer software, match the theoretical exposure biomarker molecular formula library with the measured mass spectrometry data, and use the biomarker set with a mass error <5 ppm as the candidate set of potential PHAC exposure biomarker molecular formulas; The described screening includes one or more operations among peak extraction, peak alignment, expected compound screening, expected compound combination, compound identification and annotation, and fragment ion search scoring; (5) Structural analysis and screening of exposure biomarkers: Use a metabolic simulation platform to evaluate the activation energy thresholds of each reaction site of PHAC molecules, locate highly prone metabolic active sites, and deduce the preliminary structural formulas of candidate biomarkers from the candidate set of potential PHAC exposure biomarker molecular formulas based on the principle of preferred metabolic energy; The preliminary structural formulas are further confirmed by the results of retention time prediction and the matching degree of secondary fragment ions, including: Retention time prediction: Use a standard product to confirm the retention time of PHAC, and judge the direction of the change in the retention time of PHAC exposure biomarkers (earlier or later elution) based on the hydrophilicity or hydrophobicity of the binding / reaction groups, and exclude compounds with abnormal retention time predictions; Matching degree of secondary fragment ions: Import the preliminary structural formulas into Compound Discoverer software, enable the intelligent matching engine for fragment ions, 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 formulas, and completes the structural confirmation according to the spectral similarity threshold, and selects the molecular structural formula with the highest matching degree; Next, integrate the metabolic site prediction and mass spectrometry verification data to construct a PHAC multi-pathway metabolic transformation network diagram; finally, conduct a sensitivity assessment, and through the normalization analysis of characteristic peak areas, screen out the exposure biomarkers with the top 20% peak intensities; The metabolic simulation platform described above preferably uses ADMET Predictor™ and / or BioTransformer.

[0010] Through the above steps of structural analysis and verification of exposure biomarkers, the present invention has identified a total of 11 PHAC exposure biomarkers (Tables 3 and 4). The formation of these exposure biomarkers mainly involves reactions such as oxidation, reduction, methylation, and sulfation.

[0011] The present invention has the following advantages and effects compared with the prior art: Based on the phase I / II metabolic transformation rules of PHAC, combined with high-resolution mass spectrometry data and a fragment ion search system, the present invention realizes the full-spectrum screening of exposure biomarkers; further locks potential targets through metabolic site energy simulation, and uses an intelligent matching algorithm for fragment ion spectra to realize the precise molecular structure and metabolic transformation path analysis of 11 PHAC exposure biomarkers; at the same time, through semi-quantitative analysis of metabolite peak areas, the present invention first establishes highly sensitive exposure biomarkers of PHAC in serum for human body burden monitoring. Description of the Drawings

[0012] Figure 1 It is the map of the parent ion (MS1) and fragment ions (MS2) of M0 qualitatively identified in serum.

[0013] Figure 2 It is the map of the parent ion (MS1) and fragment ions (MS2) of M1 qualitatively identified in serum.

[0014] Figure 3 It is the map of the parent ion (MS1) and fragment ions (MS2) of M2 qualitatively identified in serum.

[0015] Figure 4 It is the map of the parent ion (MS1) and fragment ions (MS2) of M3 qualitatively identified in serum.

[0016] Figure 5 It is the map of the parent ion (MS1) and fragment ions (MS2) of M4 qualitatively identified in serum.

[0017] Figure 6 It is the map of the parent ion (MS1) and fragment ions (MS2) of M5 qualitatively identified in serum.

[0018] Figure 7The mass spectra of the precursor ion (MS1) and fragment ions (MS2) of M6 qualitatively identified in serum.

[0019] Figure 8 The mass spectra of the precursor ion (MS1) and fragment ions (MS2) of M7 qualitatively identified in serum.

[0020] Figure 9 The mass spectra of the precursor ion (MS1) and fragment ions (MS2) of M8 qualitatively identified in serum.

[0021] Figure 10 The mass spectra of the precursor ion (MS1) and fragment ions (MS2) of M9 qualitatively identified in serum.

[0022] Figure 11 The mass spectra of the precursor ion (MS1) and fragment ions (MS2) of M10 qualitatively identified in serum.

[0023] Figures 12 - 13 The mass spectra of the precursor ion (MS1) and fragment ions (MS2) of M11 qualitatively identified in serum.

[0024] Figure 14 The in vivo metabolic transformation pathways of 11 PHAC exposure biomarkers in rats. Detailed implementation manners

[0025] The present invention will be further described in detail below in conjunction with the embodiments and the accompanying drawings, but the implementation manners of the present invention are not limited thereto.

[0026] Embodiment A screening method for PHAC serum exposure biomarkers, comprising the following steps: (1) Grouping and dosing regimen: 8-week-old male SPF-grade SD rats (Southern Medical University) were adaptively fed in a natural light environment at 25 ± 1 °C and a humidity of 45% ± 5% for 1 week, and then randomly divided into a 10% ethanol vehicle control group and a 12.5 mg / kg / d PHAC exposure group, and continuously dosed by intraperitoneal injection for 8 days.

[0027] (2) Serum sample collection: After the last dose, the rats were fasted for 6 h, anesthetized with isoflurane, fixed in the supine position, and blood was collected from the abdominal aorta. The serum was separated by centrifugation at 3500 rpm / min for 5 min, aliquoted and stored frozen at -80 °C.

[0028] (3)Serum purification and enrichment: Take 0.9 mL of serum and dispense it into 3 tubes. Add 0.9 mL of pre-cooled methanol at -20 °C to each tube (0.3 mL). After vortex mixing for 30 seconds, let it stand at -80 °C for 60 minutes. After centrifugation at 14,000 rpm for 15 min (4 °C), take 600 μL of the supernatant from each tube respectively. After combining them into a 1.8 mL mixture, vacuum dry it. Re-dissolve the residue with 200 μL of methanol solution, and centrifuge again at 14,000 rpm for 15 min (4 °C). Finally, take 120 μL of the supernatant and transfer it to an injection vial, and add 30 μL of methyl p-hydroxybenzoate-D4 isotope internal standard.

[0029] (4)Construction of the theoretical exposure biomarker molecular formula library: Use BioTransformer 3.0 (mainly based on the prediction of metabolic reaction sites) to predict the transformation products of PHAC. Set the maximum number of phase I reactions to 3 and the maximum number of phase II metabolic reactions to 1 to form a series of transformation products; Use the Expected Compound module in Compound Discovery (mainly based on the free combination of metabolic reaction rules). According to the reaction rules, that is, set the maximum number of phase I reactions to 3 and the maximum number of phase II metabolic reactions to 1 to form a series of transformation products; The types of metabolic reactions include phase I reactions such as oxidation, reduction, hydrolysis, desaturation, etc. and phase II reactions such as acetylation, methylation, glucuronidation, sulfation, and various amino acid complexation reactions; Combine the predicted transformation products formed by Biotransformer and Compound Discovery to construct the theoretical exposure biomarker molecular formula library (Tables 1 and 2), and incorporate this library into the targeted ion inclusion list in step (5) to enhance the acquisition efficiency of secondary daughter ion fragments and improve the qualitative quality of the results.

[0030] (5) Mass spectrometry data acquisition: An ultra-high performance liquid chromatography tandem quadrupole / orbitrap high-resolution mass spectrometer (UPLC-Orbitrap Exploris 240) was used. For chromatographic separation, a Hypersil GOLD AQ C18 column (150×2.1 mm, 1.9 μm) was employed. The mobile phase consisted of methanol (organic phase B) and 0.05% acetic acid in water (aqueous phase A). The column temperature was 35°C, the flow rate was 0.3 mL / min, and the injection volume was 2 μL. The gradient elution program was as follows: 2% B from 0 to 1 min; linearly increasing to 98% B from 1 to 8 min; maintaining 98% B from 8 to 11 min; equilibrating to 2% B from 11.01 to 14 min. Mass spectrometry detection was performed in the ESI negative ion mode (spray voltage –3.2 kV). The ion source parameters were: sheath gas 45 Arb, auxiliary gas 8 Arb, purge gas 1 Arb, transfer line 320°C, and vaporizer 350°C. The data acquisition process included full-scan mass spectrometry, dynamic exclusion, targeted ion inclusion list, and fragment ion scanning. Among them, the targeted ion inclusion list incorporated the theoretically established library of biomarker molecular formulas for potential step (4) to enhance the acquisition efficiency of target exposure biomarkers, improve the qualitative identification ability, and greatly solve the problem of low acquisition efficiency of fragment ions of target peaks caused by co-elution of high-abundance endogenous substances. The full-scan resolution was 240000 (m / z 90 - 600). The fragment ion scanning settings were: isolation window 2 m / z, HCD collision energy gradient 20% - 80% (step size 20%), resolution 15000, and synchronous acquisition of 10 fragment spectra.

[0031] (6) Suspected screening analysis of exposure biomarkers: An automated screening process for the fragment spectra obtained in step (5) 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 theoretically established library of biomarker molecular formulas 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).

[0032] Table 1: List of molecular formulas of potential PHAC exposure biomarkers formed based on BioTransformation

[0033] Table 2: List of molecular formulas of potential PHAC exposure biomarkers formed based on Compound Discoverer

[0034] (7) Structural analysis and screening of exposure biomarkers: Using the ADMET Predictor™ and BioTransformer 3.0 metabolic simulation platforms, the activation energy thresholds of each reaction site of the PHAC molecule are evaluated through quantum chemical calculations (density functional theory model) to accurately locate the metabolic active sites such as oxidation, reduction, and sulfation. Based on the principle of preferential selection of metabolic energy, candidate biomarkers and their preliminary structural formulas are derived from the candidate set of potential PHAC exposure biomarker molecular formulas.

[0035] The preliminary structural formula is further confirmed by the results of retention time prediction and secondary fragment ion matching degree, specifically including: Retention time prediction: The retention time of PHAC is confirmed using the standard product, and the direction of the change in the retention time of the PHAC exposure biomarker (earlier or later elution) is judged based on the hydrophilicity or hydrophobicity of the binding / reacting groups, and compounds with abnormal retention time prediction are excluded; Secondary fragment ion matching degree: The preliminary structural formula is imported into the compound annotation editor module of the Compound Discoverer 3.3 SP2 software, and 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 spectrum) of the analyte and the theoretical fragments of the preliminary structural formula, and completes the structural confirmation according to the spectrum similarity threshold (m / z deviation < 5 ppm), and selects the molecular structural formula with the highest matching degree. Finally, integrating the metabolic site prediction and mass spectrometry verification data, a multi-pathway metabolic transformation network diagram of PHAC is constructed; Finally, sensitivity assessment is carried out, and the exposure biomarkers with the top 20% peak intensities are screened out through characteristic peak area normalization analysis.

[0036] (8) Experimental results: Through the above steps of structural analysis and verification of exposure biomarkers, the present invention has identified a total of 11 PHAC exposure biomarkers (Tables 3 and 4). The formation of these exposure biomarkers mainly involves reactions such as oxidation, reduction, methylation, and sulfation. The parent ion isotope matching degrees of the PHAC parent compound (M0) and its 11 exposure biomarkers are all 100%. Except for M7, at least two fragment ions are matched in the database and the mass deviation < 5 ppm (Table 3), and the structural confidence level can reach L2 level. The mother ion (MS1) and fragment ion (MS2) spectra of the PHAC and its exposure biomarkers qualitatively identified in serum are as Figures 1 - 13 shown.

[0037] M1 (m / z: 151.0401) ( Figure 2 ) and M6 (m / z: 151.0401) ( Figure 7 ) are the oxidation products of PHAC, and the FISh coverage rates are 45.1% and 51.7% respectively; M2 (m / z: 230.9965) ( Figure 3 ) is the sulfation product of M1, and the FISh coverage rate reaches 40.7%; M3 (m / z: 165.0557) ( Figure 4 ) is the methylation product of M1, and the FISh coverage rate reaches 37.5%; M4 (m / z: 215.0018) ( Figure 5 ) is the sulfation product of PHAC, and the FISh coverage rate reaches 58.8%; M5 (m / z: 217.0176) ( Figure 6 ) is the sulfation product of the reduced PHAC, and the FISh coverage rate reaches 35.0%; M7 (m / z: 149.0608) ( Figure 8 ) is the methylation product of PHAC, and the FISh coverage rate reaches 33.3%; M8 (m / z: 375.2905) ( Figure 9 ) is the reduced palmitoyl complex of PHAC, and the FISh coverage rate reaches 33.3%; M9 (m / z: 199.0072) ( Figure 10 ) is the reduced, dehydrated and sulfated product of PHAC, and the FISh coverage rate reaches 19.5%; M10 (m / z: 181.0504) ( Figure 11 ) is the oxidation, oxidation and methylation product of PHAC, and the FISh coverage rate reaches 73.5%; M11 (m / z: 327.0722) ( Figure 12 and Figure 13 ) is the oxidized glucuronic acid complex of PHAC, and the FISh coverage rate reaches 73.3%. Based on the rules of metabolic transformation reactions, the in vivo metabolic transformation pathways of 11 PHAC exposure biomarkers were further drawn ( Figure 14 ).

[0038] In addition, 11 PHAC exposure biomarkers were semi-quantified based on the normalized peak areas (Table 3), and it was found that: in serum, the exposure biomarker with the highest relative proportion was M7 (39.3%), followed by M4 (25.1%) and M5 (10.1%). Therefore, the methylation and sulfation complexes are the main serum exposure biomarkers of PHAC, and either M7 or M4 can be used as a sensitive exposure biomarker of PHAC.

[0039] Table 3: 11 PHAC exposure biomarkers identified in serum (M0 is the PHAC parent compound)

[0040] Table 4: List of 11 PHAC Exposure Biomarkers and Their Molecular Structural Formulas (M0 is the PHAC Parent Compound)

[0041] The above embodiments are preferred embodiments of the present invention. However, the embodiments of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. Use of serum exposure biomarkers of p - hydroxyacetophenone in monitoring the human body load, characterized in that: The serum exposure biomarkers of p - hydroxyacetophenone are one or more of 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, 4 - (1 - hydroxyethyl)phenyl hydrogen sulfate, 2 - hydroxy - 1 - (4 - hydroxyphenyl)ethan - 1 - one, 1 - (4 - methoxyphenyl)ethan - 1 - one, 4 - (1 - hydroxyethyl)phenyl palmitate, 4 - vinylphenyl hydrogen sulfate, 2 - hydroxy - 1 - (2 - hydroxy - 4 - methoxyphenyl)ethan - 1 - one, or 6 - (4 - acetyl - 3 - hydroxyphenoxy)-3,4,5 - trihydroxytetrahydro - 2H - pyran - 2 - carboxylic acid.

2. The use according to claim 1, characterized in that: The serum exposure biomarkers of p - hydroxyacetophenone are one or more of 1 - (4 - methoxyphenyl)ethan - 1 - one, 4 - acetylphenyl hydrogen sulfate, or 4 - (1 - hydroxyethyl)phenyl hydrogen sulfate.

3. The use according to claim 1, characterized in that: The serum exposure biomarkers of p - hydroxyacetophenone are 1 - (4 - methoxyphenyl)ethan - 1 - one, 4 - acetylphenyl hydrogen sulfate, and 4 - (1 - hydroxyethyl)phenyl hydrogen sulfate.

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