Urine exposure biomarker of p-hydroxyacetophenone and application of urine exposure biomarker in human body load monitoring

Abstract

The invention discloses a urine exposure biomarker of p-hydroxyacetophenone and application of the urine exposure biomarker in human body load monitoring. On the basis of the I / II phase metabolic transformation rule of PHAC, a targeted screening list is formed, the mass spectrum fragment collection efficiency and qualitative analysis capability are enhanced, and full spectrum screening of the urine exposure biomarkers 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 analyzing accurate molecular structures and metabolic conversion paths of the six PHAC urine exposure biomarkers by utilizing a fragment ion spectrum intelligent matching algorithm; meanwhile, through metabolite peak area semi-quantitative analysis, a noninvasive urine sample is utilized for the first time to determine the PHAC high-sensitivity exposure biomarker capable of monitoring loads in a human body.

Description

Technical Field

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

[0002] Biomarkers of organic pollutant exposure refer to pollutant prototypes, metabolic transformation products, and adducts formed with endogenous substances that can be quantitatively detected in biological tissues, body fluids, or excreta. Such biomarkers, as a bridge between environmental pollution exposure and health effects, provide 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, drugs, and personal care products and are a class of endocrine disruptors that have received much attention.

[0004] Studies have shown that long - term parabens exposure 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 turned to research their substitutes.

[0005] The structural analogue of parabens, p - 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 neurotoxicity of 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 substitute for parabens, the research on PHAC exposure biomarkers is still in its infancy, and it is still 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 urinary exposure biomarkers of p - 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 urinary exposure biomarkers of p - hydroxyacetophenone in monitoring the human body burden; The urinary exposure biomarkers of p - hydroxyacetophenone are one or more of p - hydroxyacetophenone (M0), 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), or 4 - (1 - hydroxyethyl)phenyl hydrogen sulfate (M5); Preferably, the urinary exposure biomarkers of p - hydroxyacetophenone are one or more of 4 - acetylphenyl hydrogen sulfate (M4), p - hydroxyacetophenone (M0), or 4 - (1 - hydroxyethyl)phenyl hydrogen sulfate (M5); More preferably, the urinary exposure biomarkers of p - hydroxyacetophenone are 4 - acetylphenyl hydrogen sulfate (M4) and p - hydroxyacetophenone (M0); Particularly preferably, the urinary exposure biomarkers of p - hydroxyacetophenone are 4 - acetylphenyl hydrogen sulfate (M4), p - hydroxyacetophenone (M0), and 4 - (1 - hydroxyethyl)phenyl hydrogen sulfate (M5).

[0009] The screening method for urinary exposure biomarkers of p - hydroxyacetophenone (PHAC) includes the following steps: (1) Toxicant administration and sample preparation: Experimental animals are intraperitoneally injected with p - hydroxyacetophenone for at least 8 consecutive days, and then urine 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 urine 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; For the phase I reaction, the reaction types include oxidation reaction, reduction reaction, hydrolysis reaction, and desaturation reaction; For the phase II reaction, the reaction types include acetylation reaction, methylation reaction, glucuronidation reaction, sulfation reaction, and amino acid complexation reaction; Combine the predicted transformation products formed by Biotransformer and Compound Discovery, construct a theoretical library of biomarker molecular formulas for exposure, 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 acquisition described above 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 library of biomarker molecular formulas for exposure with the measured mass spectrometry data, and use the set of biomarkers with a mass error <5 ppm as the candidate set of potential PHAC exposure biomarker molecular formulas; The screening described above includes one or more operations among peak extraction, peak alignment, screening of expected compounds, combination of expected compounds, compound identification and annotation, and fragment ion search scoring; (5) Structure 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 inclined 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 preferential selection of 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 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 prediction; 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 structure confirmation according to the spectral similarity threshold, and selects the molecular structural formula with the highest matching degree; Then, integrate the metabolic site prediction and mass spectrometry verification data to construct a PHAC multi-pathway metabolic transformation network diagram; Finally, based on the evaluation of sensitivity, specificity, and stability, screen out the exposure biomarker with the best comprehensive performance, specifically including: Sensitivity assessment: Through the normalization analysis of the characteristic peak area, the exposure biomarkers with the top 20% peak intensities were screened out; Specificity assessment: Exposure biomarkers will only be generated when specific exposure occurs, which is mainly characterized by the difference in the concentration levels of exposure biomarkers between the exposed group and the control group; Stability assessment: Specifically refers to whether the exposure biomarkers can be stably formed under a quantitative exposure dose, which is mainly characterized by the coefficient of variation between days.

[0010] The metabolic simulation platform described above preferably uses ADMET Predictor™ and / or BioTransformer.

[0011] Through the above steps of structural analysis and verification of exposure biomarkers, the present invention has 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.

[0012] 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, the present invention forms a targeted screening list, enhances the efficiency of mass spectrometry fragment collection and qualitative analysis ability, combines high-resolution mass spectrometry data with a fragment ion search system to achieve a full-spectrum screening of urine exposure biomarkers; further locks potential targets through metabolic site energy simulation, and uses an intelligent matching algorithm for fragment ion spectra to achieve the precise molecular structure and metabolic transformation path analysis of 6 PHAC urine exposure biomarkers; at the same time, through semi-quantitative analysis of the metabolite peak area, for the first time, non-invasive urine samples are used to establish highly sensitive exposure biomarkers of PHAC for human body burden monitoring. Brief Description of the Drawings

[0013] Figure 1 To reveal the differences in exposure biomarkers in urine between the solvent control group and the exposed group based on principal component analysis.

[0014] Figure 2 It is the parent ion (MS1) and fragment ion (MS2) spectra of M0 qualitatively identified in urine and the inter-group peak area response difference diagram (upper right corner).

[0015] Figure 3 It is the parent ion (MS1) and fragment ion (MS2) spectra of M1 qualitatively identified in urine and the inter-group peak area response difference diagram (upper right corner).

[0016] Figure 4 It is the parent ion (MS1) and fragment ion (MS2) spectra of M2 qualitatively identified in urine and the inter-group peak area response difference diagram (upper right corner).

[0017] Figure 5 The mass spectra of the parent ions (MS1) and fragment ions (MS2) of M3 qualitatively identified in urine and the differential map of peak area responses between groups (upper right corner).

[0018] Figure 6 The mass spectra of the parent ions (MS1) and fragment ions (MS2) of M4 qualitatively identified in urine and the differential map of peak area responses between groups (upper right corner).

[0019] Figure 7 The mass spectra of the parent ions (MS1) and fragment ions (MS2) of M5 qualitatively identified in urine and the differential map of peak area responses between groups.

[0020] Figure 8 The in - vivo metabolic transformation pathways of 6 PHAC exposure biomarkers in rats.

[0021] Figure 9 The detected abundances of PHAC and its 6 exposure biomarkers in urine on different days. Specific Embodiments

[0022] The present invention will be further described in detail below in conjunction with embodiments and the accompanying drawings, but the embodiments of the present invention are not limited thereto.

[0023] Embodiment A screening method for PHAC urine 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 for 1 week in a natural light environment at 25 ± 1 °C and humidity of 45% ± 5%, and then randomly divided into a 10% ethanol vehicle control group and a 12.5 mg / kg / d PHAC exposure group. They were continuously dosed by intraperitoneal injection for 8 days, and urine specimens were collected daily.

[0024] (2) Urine purification and enrichment: 2 mL of urine was added to a 10 mL plastic tube, 6 mL of methanol pre - cooled to - 20 °C was added, and after vortex - mixing for 30 seconds, the mixture was aliquoted into 5 tubes of 2 mL plastic tubes and left to stand at - 80 °C for 60 min; after centrifugation at 14000 rpm for 15 min, 1 mL of the supernatant was taken from each tube, combined into a 5 mL mixture, and then vacuum - dried; the residue was re - dissolved with 400 μL of methanol solution, and centrifuged again at 14000 rpm for 15 min (4 °C). Finally, 120 μL of the supernatant was transferred to an injection vial, and 30 μL of methyl - p - hydroxybenzoate - D4 isotope internal standard was added.

[0025] (3) Construction of the theoretical exposure biomarker molecular formula library: Predict the PHAC transformation products using BioTransformer 3.0 (mainly based on the prediction of metabolic reaction sites), 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), and 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 cover 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; Merge the predicted transformation products formed by Biotransformer and Compound Discovery, construct a theoretical exposure biomarker molecular formula library (Tables 1 and 2), and incorporate this library into the targeted ion inclusion list in step (4) to enhance the acquisition efficiency of secondary daughter ion fragments and improve the qualitative quality of the results.

[0026] (4) Mass spectrometry data acquisition: Use an ultra-high performance liquid chromatography tandem quadrupole / orbitrap high-resolution mass spectrometer (UPLC-Orbitrap Exploris 240). For chromatographic separation, use a Hypersil GOLD AQ C18 column (150×2.1 mm, 1.9 μm). The mobile phase is methanol (organic phase B) and 0.05% acetic acid in water (aqueous phase A). The column temperature is 35 °C, the flow rate is 0.3 mL / min, and the injection volume is 2 μL. The gradient elution program is as follows: 2% B from 0 to 1 min; linearly increase to 98% B from 1 to 8 min; maintain 98% B from 8 to 11 min; balance to 2% B from 11.01 to 14 min. Mass spectrometry detection is carried out in the ESI negative ion mode (spray voltage –3.2 kV). The ion source parameters are: sheath gas 45 Arb, auxiliary gas 8 Arb, purge gas 1 Arb, transfer line 320 °C, vaporizer 350 °C. The data acquisition process includes full-scan mass spectrometry, dynamic exclusion, targeted ion inclusion list, and fragment ion scanning. Among them, the targeted ion inclusion list incorporates the pre-established theoretical exposure biomarker molecular formula library in potential step (3) to enhance the acquisition efficiency of target exposure biomarkers, improve the qualitative recognition ability, and greatly solve the problem of low acquisition efficiency of target peak fragment ions caused by the co-elution of high-abundance endogenous substances. The full-scan resolution is 240000 (m / z 90 - 600). The fragment ion scanning settings are: isolation window 2 m / z, HCD collision energy ladder 20% - 80% (step size 20%), resolution 15000, and synchronously acquire 10 fragment spectra.

[0027] (5)Suspected screening analysis of exposure biomarkers: An automated screening process (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) for the fragment spectra obtained in step (4) was established using Compound Discoverer software. 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).

[0028] Table 1: List of potential exposure biomarker molecular formulas of PHAC formed by BioTransformation

[0029] Table 2: List of potential exposure biomarker molecular formulas of PHAC formed by Compound Discoverer

[0030] (6)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 were evaluated through quantum chemical calculations (density functional theory model) to accurately locate 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 candidate set of potential PHAC exposure biomarker molecular formulas.

[0031] The preliminary structural formula was further confirmed by the results of retention time prediction and the matching degree of secondary fragment ions, specifically including: Retention time prediction: The retention time of PHAC was confirmed using a standard product, and the direction of the change in the retention time of the PHAC exposure biomarker (earlier or later elution) was judged based on the hydrophilicity or hydrophobicity of the binding / reaction groups, and compounds with abnormal retention time prediction were excluded; Degree of match of secondary fragment ions: Import the preliminary structural formula into the compound annotation editor module of Compound Discoverer 3.3 SP2 software, enable the Fragment Ion Smart Matching Engine (FISh), the system compares the characteristic ion clusters between the actually 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 degree of match. Finally, integrate the metabolic site prediction and mass spectrometry verification data to construct a PHAC multi-pathway metabolic transformation network diagram; Finally, based on the evaluation of sensitivity, specificity and stability, the exposure biomarkers with the best comprehensive performance were screened out, specifically including: Sensitivity evaluation: Through the normalization analysis of characteristic peak areas, the exposure biomarkers with the top 20% peak intensities were screened out; Specificity evaluation: Exposure biomarkers will only be produced when specific exposure occurs, which is mainly characterized by the difference in the concentration levels of exposure biomarkers between the exposed group and the control group; Stability evaluation: Specifically refers to whether the exposure biomarkers can be stably formed under the exposure dose of quantitative exposure, which is mainly characterized by the coefficient of variation between days.

[0032] (7) Experimental results: Principal component analysis showed that the urine samples of the solvent control group and the PHAC exposed group were extremely significantly separated ( Figure 1 ), indicating that a series of significantly different exposure biomarkers were formed in the PHAC exposed group. Through the above steps of structural analysis and verification of exposure biomarkers, a total of 6 PHAC exposure biomarkers were identified in the present invention (Tables 3 and 4). The formation of these exposure biomarkers mainly involves reactions such as oxidation, reduction, methylation, and sulfation. The parent ion isotope match degrees of the PHAC parent compound (M0) and its 6 exposure biomarkers are all 100%, and at least two fragment ions are matched in the database with a mass deviation < 1.5 ppm (Table 3), and the structural confidence level can reach the L2 level. The parent ion (MS1) and fragment ion (MS2) spectra of PHAC and its exposure biomarkers qualitatively identified in urine are as shown in Figures 2 - 7 shown.

[0033] M1 (m / z: 151.0401) ( Figure 3 ) is the oxidation product of PHAC, and the FISh coverage is 46.4%; M2 (m / z: 230.9965) ( Figure 4 ) is the sulfation product of M1, and the FISh coverage is 39.4%; M3 (m / z: 165.0557) ( Figure 5 ) is the methylation product of M1, and the FISh coverage is 65.0%; M4 (m / z: 215.0018) (Figure 6 ), the sulfated product of PHAC, with a FISh coverage of 55.6%; M5 (m / z: 217.0176) ( Figure 7 ), the sulfated product of reduced PHAC, with a FISh coverage of 52.0%. Based on the rules of metabolic transformation reactions, the in vivo metabolic transformation pathways of 6 PHAC exposure biomarkers were further drawn ( Figure 8 ).

[0034] In addition, the 6 PHAC exposure biomarkers were semi-quantified based on the standardized peak area (Table 3) for sensitivity assessment. The results showed that in urine, the exposure biomarker with the highest relative proportion was M4 (49.2%), followed by M0 (36.4%) and M5 (12.3%). Therefore, the sulfated complex is the main urine exposure biomarker of PHAC.

[0035] The specificity and stability of M4 and M0 were further evaluated, and the results were as follows: From the perspective of specificity assessment ( Figure 2 and the upper right bar graph in Figure 6 ), the peak areas of M4 and M0 in the urine of the control group were 3.74×10 8 and 3.68×10 8 , respectively, while the peak areas in the PHAC exposure group were 3.73×10 10 and 2.51×10 10 , respectively. The ratios of the peak areas between the exposure and control groups were 99.8 and 68.1, showing extremely significant inter-group differences ( p <0.001). Therefore, both M4 and M0 have strong specificity.

[0036] From the perspective of stability assessment ( Figure 9 ), continuous monitoring of the exposure biomarkers in rat urine for 8 days found that except for fluctuations on the fourth day, both M4 and M0 showed good stability, with an inter-day coefficient of variation less than 30%, indicating relatively strong stability.

[0037] Based on the above research results, M4 and M0 in urine can be used as highly sensitive, specific, and stable biomarkers for indicating PHAC exposure.

[0038] Table 3: 6 PHAC exposure biomarkers identified in urine (M0 is the parent compound of PHAC)

[0039] Table 4: List of 6 PHAC exposure biomarkers and their molecular structural formulas (M0 is the parent compound of PHAC)

[0040] 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, or 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 urinary exposure biomarkers of p - hydroxyacetophenone in the monitoring of human body burden, characterized in that: The urinary exposure biomarkers of p - hydroxyacetophenone are one or more of 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, or 4 - (1 - hydroxyethyl)phenyl hydrogen sulfate.

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

3. The use according to claim 1, characterized in that: The urinary exposure biomarkers of p - hydroxyacetophenone are 4 - acetylphenyl hydrogen sulfate and p - hydroxyacetophenone.

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

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