Study On The Determination Methods Of Oxidative Stress Biomarkers And Their Applications In Children’s Health Monitoring

Oxidative stress refers to a stress state in which the body is more inclined to oxidation when there is an imbalance between oxidation and antioxidation in the body.During oxidative stress,excessive free radicals which can"steal electrons"indiscriminately from neighboring molecules will be produced in vivo,causing oxidative damage to important biological molecules in cells,such as lipids,proteins and nucleic acids.This will cause a disorder of cellular function and may further cause oxidative damage to tissues such as the gastrointestinal tract,resulting in different diseases.This may lead to cellular dysfunction,which can further lead to oxidative damage in tissues such as the gastrointestinal tract,and ultimately lead to the development of various diseases.The detection of oxidative stress biomarkers in vivo is an effective means to evaluate the level of oxidative stress in human body.However,the concentrations of oxidative stress biomarkers in biological samples are usually very low,the composition of biological samples is complex and the matrix interference is serious.Therefore,the detection of oxidative stress biomarkers in biological samples is very difficult.Pretreatment such as extraction and concentration of the targets in biological samples is often required before detection.One of the commonly used methods is solid phase extraction.The packed-fiber solid phase extraction（PFSPE）technology integrates separation,purification,enrichment and other operations.When PFSPE is used for the pretreatment of oxidative stress biomarkers in biological samples,extraction efficiency,detection sensitivity and accuracy can be improved,and operation procedures,time consumption and cost can be reduced.Childhood is a crucial life stage that is decisive for the proper development of behavior,metabolism and immunity,and children are more susceptible to oxidative stress than adults.Both external factors such as air pollution and internal factors such as disease can cause oxidative stress.In this study,biomarkers of oxidative damage of proteins,nucleic acids and lipids in vivo and related indicators of hypothalamic-pituitary-adrenal axis and microbial-gut-brain axis were combined to investigate the hazards of air particle pollution exposure in normal children and evaluate the effect of exercise intervention in children with autism（ASD）.In the study of the effects of air particle pollution exposure,the single nucleotide polymorphism of the oxytocin receptor OXTR rs53576,which is associated with stress response,social behavior and psychopathology in different individuals,was also investigated to explore whether the effects of OXTR rs53576 on social cognition and behavior were affected by the effects of air pollution exposure.In the evaluation study on the effect of exercise intervention for ASD children,in addition to the comparison study before and after exercise intervention for ASD children,the feasibility of nucleic acid oxidative damage markers as identification indicators to distinguish severe and non-severe children with ASD was also investigated,so as to establish an objective evaluation method for the identification and evaluation of intervention effects of ASD children.The specific contents are as follows:1.Establishment of detection methods for biomarkers of protein,nucleic acid and lipid oxidative damage in urine（1）Establishment of a HPLC-MS method for simultaneous determination of oxidative damage biomarkers of protein and nucleic acidHPLC-MS/MS was used to determine the protein oxidative damage marker 3-nitro-tyrosine（3-NT）and nucleic acid oxidative damage marker 2’-deoxy-7,8-dihydroguanosine（8-oxod G and 8-oxo G）in urine simultaneously.This method did not require complex pretreatment of urine samples,and had a good linear range of 0.5-50 ng/m L（R²>0.99）.The limits of detection of the three biomarkers were 0.15,0.25 and 0.20 ng/m L,respectively.The limits of quantification were 0.50,0.85 and 0.66 ng/m L,respectively.The intra-day and inter-day precisions were good（RSD<10%）.The method could meet the requirements of simultaneous analysis of 8-OXOG,8-oxod G and 3-NT in urine samples.（2）Establishment of a HPLC-MS method for the determination of lipid oxidative damage biomarker 8-iso-prostaglandin F2α（8-iso-PGF2α）in urine.To extract the hydrophobic 8-iso-PGF2α,the gold standard biomarker of lipid oxidative stress in vivo,polystyrene（PS）electrospun nanofibers were prepared by electrospinning.A packed-fiber solid-phase extraction based on PS nanofibibers coupled with HPLC-MS/MS method was developed for the specific determination of 8-iso-PGF2αin urine.After optimizing the PFSPE conditions,a good linearity in the range of 0.05-5 ng/m L with R²>0.9996,a satisfactory limit of detection of 0.015 ng/m L,good intra-day and inter-day precisions（RSD<10%）,and recoveries of 95.3-103.8%were obtained.The feasible method was successfully applied to the batch quantitative analysis of 8-iso-PGF2αin real urine samples.（3）Establishment of a HPLC-ECD method for simultaneous determination of nucleic acid oxidative damage biomarkers 8-oxod G and 8-oxo G in urineMass spectrometers are expensive and not all laboratories are equipped with them.Based on the strong polarity of nucleic acid oxidative damage biomarkers 8-oxod G and 8-oxo G,multifunctional polystyrene/polypyrrole（PS/PPY）nanofibers were prepared by in situ synthesis technique and used as adsorbent for the determination of 8-oxod G and 8-oxo G in urine samples.A PFSPE based on PS/PPY nanofibers coupled with HPLC-ECD method was established for the detection of 8-oxod G and 8-oxo G in urine samples.2-aminoethyl diphenyl borate（DPBA）solution was introduced in the loading and rinsing steps to promote the retention of the target molecules and the removal of impurities since the affinity between the target molecules and PS/PPY nanofibers can be enhanced by the B-OH interaction and B-N coordination between DPBA and the target molecules.Under optimal conditions,8-oxod G,8-oxo G and IS were separated very well and exhibited a good linearity in the range of 0.5-50ng/m L,with correlation coefficients of R²>0.996.Limits of detection（LOD）were 0.058 ng/m L and 0.093 ng/m L,and limits of quantification（LOQ）were 0.195 ng/m L and 0.309 ng/m L,respectively.The recoveries were 88.8-104.9%.The proposed method was so simple and economical that it had the potential to be applied to batch quantitative analysis of 8-oxod G and8-oxo G in urine.And it was successfully applied to real urine samples of cancer patients.2.Study on the application of oxidative stress biomarker detection method in the monitoring of children’s health under the two modes of stress,air pollution and disease（1）Air pollution patternSaliva and urine samples were collected from 86 healthy Chinese preschoolers（50 males and 36 females）from two campuses of a chain kindergarten in Suzhou with different levels of PM_2.5.Atmospheric PM_2.5 values released by air quality monitoring stations where the two campuses are located were collected for 30 days.The genotypes of OXTR rs53576 were determined by PCR and restriction fragment length polymorphism.The gut microbiota situation was evaluated by determining urinary concentrations of short-chain fatty acids.Urinary levels of cortisone and cortisol were determined to assess the impact of air pollution on the HPA axis.Urinary 2′-Deoxy-7,8-dihydro-8-oxoguanosine and 8-oxo-7,8-dihydroguanosine were measured to evaluate the oxidative stress state.The genotype distribution frequency of rs53576polymorphism was consistent with Hardy-Weinberg equilibrium.The average urinary concentrations of cortisone,cortisol and 8-oxod G in high pollution campus preschoolers were significantly higher than those in low pollution campus preschoolers,while situations were opposite for acetic acid,propionic acid,isobutyric acid,butyric acid and valeric acid.The interaction between OXTR rs53576 and air pollution had a significant effect on urinary acetic acid.Allele G of rs53576 may be a risk factor for gut microbiota disorder caused by air pollution,and children with GA/GG genotype may be more susceptible than those with AA genotype.（2）Disease patternA total of 23 children with ASD were recruited to conduct an advanced sensorimotor training program.A variety of standard scales were used to evaluate the behavior of children with ASD before and after sensorimotor training.The levels of 12 substances in 4 categories,including lipids,nucleic acids,proteins and gut microbiota metabolic targets related to oxidative damage in urine samples of ASD children before and after the sensory-motor training program,were detected by chromatography,chromatography-mass spectrometry and other physicochemical analysis techniques.Multiple indicators were collected and analyzed.The ROC curve was used to explore the biochemical indicators of autism degree discrimination,and and the levels of the relevant indicators were compared with normally developing children under different PM2.5 exposure levels in the previous model.It was found that sensorimotor advanced training was benefit for the behavior and oxidative damage degree of children with ASD.Nucleic acid oxidative damage markers 8-oxod G and 8-oxo G could distinguish severe autism from non-severe autism.By comparing the levels of oxidative damage markers,this study provided evidence for the sensorimotor treatment of autism,and provided a new idea for the mechanism of sensorimotor intervention of autism.