食品中六溴环十二烷和四溴双酚A的检测技术与暴露评估
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摘要
六溴环十二烷(HBCD)和四溴双酚A(TBBPA)是两种应用广泛的溴系阻燃剂,目前已被发现在环境样品和人体内普遍存在,由于其具有蓄积性和持久性,被认为是两种潜在的持久性有机污染物。膳食摄入是人体摄入TBBPA和HBCD的主要途径,但目前我国仍未开展食品及人体内TBBPA和HBCD的污染状况的研究。为了对HBCD和TBBPA的污染水平、我国居民摄入状况等进行评估,我们开展了一系列的工作。包括检测方法的建立,食品和母乳中污染水平的分析和膳食摄入量评估,并采用基于LC-MS技术的代谢组学方法初步研究了这两种阻燃剂对大鼠的毒性。
1.HBCD和TBBPA检测方法
采用同位素稀释技术建立了动物性食品中HBCD异构体和TBBPA的UPLC-MS/MS测定方法。样品的提取可选用索氏提取法或加速溶剂萃取法,提取液的净化方法可选用浓硫酸脱脂结合固相萃取柱净化,或者自动GPC脱脂结合浓硫酸脱脂净化。样品净化后通过UPLC BEH C18柱分离,以甲醇/乙腈混合液和水为流动相进行梯度洗脱,5min内即可完成一次测定。在分析过程中电离方式为ESI(—)模式,并以多反应离子监测(MRM)模式进行测定,内标法定量。HBCD和TBBPA的监测离子分别为m/z640.7→80.7和m/z542.6→447.6。所建立的方法可用于测定水产品、肉类、奶类、蛋类等动物性食品中的六溴环十二烷和四溴双酚A,在5-250ng/mL范围内线性良好,检出限为10-100pg/g,在不同加标水平下方法的平均回收率在86.8%～110.5%之间,RSD在1.07%～14.04%之间。经过国内三家单位的协同性验证试验和国际比对考核实验,证明所建立的方法灵敏度、准确度和精密度均符合痕量检测的要求。
2.我国食品中TBBPA和HBCD的污染状况与居民膳食摄入水平研究
测定了2007年总膳食研究中采集的12个省4类动物性食品的48份混样中HBCD和TBBPA的污染水平。TBBPA含量在＜LOD到2044pg/g lw之间,在肉类、蛋类、水产类和奶类食品中TBBPA的平均含量分别为251、179、721和194pg/g lipid weight(lw)。在HBCD的三种异构体中,α-HBCD的比例总是最高。HBCD含量在＜LOD到9208pg/g lw之间,在肉、蛋、水产和奶中的平均含量分别为252、269、1441和194pg/glw。水产类食品中TBBPA和HBCD的检出率、最高值和平均值均是四类食品中最高的。
以总膳食样品中TBBPA和HBCD的污染水平和食物消费量数据为依据,估计我国成年男子TBBPA和HBCD的膳食摄入水平。若以1/2LOD代替未检出样品中待测物的含量,则TBBPA的膳食摄入水平在32～1305pg/kg bw/day之间,平均为256pg/kgbw/day,∑HBCD的膳食摄入水平在87-1722 pg/kg bw/day之间,平均为432 pg/kgbw/day。对膳食TBBPA摄入量贡献率最高的为肉类(52%),其次为水产类(30%)、奶类(10%)和蛋类(8%)。HBCD贡献率最高的为肉类(44%),其次为水产类(24%)、蛋类(17%)和奶类(15%)。表明肉制品为我国居民TBBPA和HBCD膳食暴露的最重要来源。但与国外研究相比,我国食品中TBBPA和HBCD的污染水平,以及人体摄入量仍处在较低的水平。
3.母乳样品中HBCD和TBBPA的污染水平
测定了从全国12个省(市、区)采集的24份母乳混样。分析了这些样品中TBBPA和HBCD的污染水平,并结合母乳摄入量数据估算婴儿TBBPA和HBCD的每日摄入量。母乳中TBBPA含量在＜LOD-5124pg/g lw,平均为941pg/g lw。在HBCD的三种异构体中,α-HBCD含量总是最高,母乳中α-HBCD含量在＜LOD-2776pg/g lw,平均为1050pg/g lw。母乳样品中TBBPA和HBCD的污染水平明显高于动物性食品。推测TBBPA和HBCD在食物链中可能具有从低端到高端的生物放大作用。此外人体对TBBPA和HBCD的摄入可能存在多种途径。婴儿的摄入水平为320-37240pg/kgbw/day(TBBPA)和670-17320 pg/kg bw/day(∑HBCD),平均摄入水平分别为5094和5837 pg/kg bw/day。我国婴儿的溴系阻燃剂(包括TBBPA、HBCDs和PBDEs)摄入水平明显高于成年男子,应引起足够的重视。
4.TBBPA和HBCD对大鼠毒性的代谢组学研究
代谢组学作为系统生物学的重要组成部分,已经成为继基因组学、转录组学、蛋白质组学之后兴起的一个新的组学研究热点。我们采用代谢组学方法初步探索了TBBPA和HBCD对SD大鼠的毒性。大鼠分为高低剂量组和对照组,连续染毒7天,取每日24小时尿样及第8天血清样。以超高效液相色谱-飞行时间质谱分析不同剂量染毒大鼠及对照组大鼠的血清或尿液,采用归一化方法对代谢谱进行峰对齐,并采用偏最小二乘-判别分析法进行模式识别和可视化,以此观察不同剂量TBBPA或HBCD染毒大鼠血清或尿液代谢谱与空白对照组的差异。大鼠经染毒后,与对照组代谢谱比较有明显差异,对血清代谢谱的分析表明TBBPA对大鼠的毒性存在明显的剂量-毒性效应关系,毒性作用随染毒剂量增大而增大,但HBCD染毒大鼠的剂量-毒性效应关系不明显。对尿液代谢谱的分析表明TBBPA对大鼠的毒性除了随染毒剂量增大而增大外,还随着染毒时间的延长而增大。但HBCD染毒大鼠这两方面的效应均不明显。通过对载荷图的分析,结合代谢物数据库,初步筛选了一些可能的生物标志物,包括血清和尿液中的氨基酸、葡萄糖苷、磷脂等小分子物质,这些化合物在对照组和剂量组中的含量差异较为明显,可以作为进一步定性分析的研究对象。
Hexabromocyclododecane(HBCD) and Tetrabromobisphenol A(TBBPA) are two brominated flame retardants(BFRs) currently in use all over the world.They are now ubiquitous contaminants in wildlife,as well as in humans.Studies indicated that BFRs are bioaccumulative and persistent compounds,thus they should be regarded as persistent organic pollutants(POPs).However,we are still lacking information on the contaminant level of BFR in food and,especially,in human being.It is generally accepted that human exposure to BFRs is mainly through diet.However,there were relatively few studies reporting the presence of HBCD and TBBPA in the foodstuffs or human milk from China. In order to assess dietary exposure of Chinese adults and nursing Infants to TBBPA and HBCD,a UPLC-MS/MS method was developed for simultaneous determination of TBPPA and HBCD in foods of animal origins.On the basis of the analytical method,the levels of HBCD diastereomers(α,β,andγ-HBCD) and TBBPA in two categories of samples: pooled human milk samples collected in 2007 from 12 provinces of China and TDS samples from the fourth Chinese total diet study in 2007,were examined.Based on the obtained data,the human daily intake of TBBPA and HBCD via human milk or food consumption in China was estimated and compared.In order to explore the acute toxicity effect of TBBPA or HBCD to human being or mammal,metabonomics methodology was used to investigate the TBBPA/HBCD induced injure in rats.
1.Method for the determination of TBBPA and HBCD in food
An ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed for simultaneous determination ofα,β,γ-HBCD and TBBPA in foods of animal origin,include meat and meat products,egg and egg products,milk and milk products and aquatic foods.The methodology was based on solid-liquid extraction,GPC cleanup or SPE cleanup,and isotope dilution.Samples were extracted by Soxhlet extraction or accelerated solvent extraction(ASE).The extract was purified by gel permeation chromatography(GPC) pretreatment or concentrated sulfuric acid treatment follow by SPE cleanup.The separation was carried out on a UPLC BEH C18 reversed-phase column,using methanol-acetonitrile and water as the gradient elution solvent,the four BFRs were eluted within 5min.A triple quadrupole mass spectrometer equipped with electrospray ionization source was operated in negative ion mode.Multiple reaction monitoring(MRM) was employed for quantitative measurement.The MRM transitions monitored were as 640.7→80.7 for HBCD,542.6→447.6 for TBBPA.Good linearities were found within the ranges of 5-250ng/mL with the LOD of 10-100pg/g.The mean recovery of different spiking level varied from 86.8%to 110.5%in different foodstuffs,with the relative standard deviations(RSDs) between 1.07%and 14.4%.The results demonstrated that the UPLC-MS/MS method developed is highly effective for analyzing trace amounts of TBBPA and HBCDs in animal original foods.
2.Contamination level in foods and dietary exposure assessment by the Chinese Total Diet Study
On the basis of UPLC-MS/MS analysis,TBBPA andα,β,γ-HBCD were determined in 48 Chinese total diet study(TDS) samples collected from 12 provinces in 2007.In the four food groups include meat,egg,milk and aquatic foods,levels of TBBPA ranged from＜LOD to 2044 pg/g lw.Theα-HBCD diastereoisomer was generally the most abundant isomer comparing withβ-andγ-HBCD.Levels of HBCD ranged from＜LOD to 9208pg/g lw.The highest contamination level of TBBPA and HBCD were both found in the aquatic food group.The lowest concentrations occurred in the egg group and milk group.
Daily dietary intakes of TBBPA andΣHBCD for the Chinese were estimated by multiplying the measured concentrations of TBBPA andΣHBCD with the daily consumption data.The medium bound(＜LOD=1/2LOD) EDI_(TBBPA) for a "reference" man (male adult,63kg bodyweight) via animal origin foods was 256 pg/kg bw/day and EDI_(ΣHBCD) was 432 pg/kg bw/day.Meat and meat products were the main source in the total dietary intake of TBBPA andΣHBCD.
3.Contamination level of HBCD and TBBPA in human
TBBPA andα,β,andγ-HBCD were determined in 24 pooled human milk samples collected from 12 provinces in 2007.Levels of TBBPA ranged from＜LOD to 5124 pg/g lw with mean concentration of 941pg/g lw.Theα-HBCD diastereoisomer,which ranged from＜LOD to 2776 pg/g lw with mean concentration of 1050pg/g lw,was the most abundant isomer as same as in animal original foods.However,levels of TBBPA and HBCD in human milk were higher than that in foods.It was indicated that levels of HBCDs or TBBPA are elevated in species at the top of the food chain,which clearly points toward biomagnification.It also suggests that human exposure to TBBPA/HBCDs may occur from multiple routes,besides from food,intakes from indoor air or dust is probably another major route.The average estimated daily intake(EDI) of TBBPA via human milk for nursing infants was 5094 pg/kg bw/day with a range 320-37240 pg/kg bodyweight (bw)/day,while that ofΣHBCD was 5837 pg/kg bw/day with a range 670-17320 pg/kg bw/day.Which were much higher than that for a male adult.It is indicated that the body BFRs burden of a nursing infant in China is much higher than that of adult.The possible toxicological impact of such a high daily intake should certainly be investigated.
4.Within the past few years,metabonomics,as a new part of system biology,has gained great prominence besides genomics,transcriptomics and proteomics.In this paper, metabolism disorders induced by TBBPA or HBCD in a rat model with different dosage of intoxication were studied by metabonomics.Ultra performance liquid chromatography (UPLC) coupled with quatropde-time of flight(Q-TOF) mass spectrometer were used to analyze serum and urine obtained from rats treated with a range of doses of TBBPA or HBCD,with the objective to reveal the characteristics of the damage caused by these BFRs and identify the latent biomarkers.SD rats were randomly assigned into dosed groups and control group,rats were given daily TBBPA(30,300mg/kg) or HBCD(30,300mg/kg) for 7 days and sacrificed on Day 8 for serum collecting.24h urine samples were collected every day before dosing.All serum and some urine samples were performed UPLC-Q-TOF-MS analysis.A normalization method was performed to match the peaks between the chromatograms.A supervised learning algorithm i.e.partial least squares discriminant analysis(PLS-DA) was used for the pattern recognition and visualization.The PLS-DA score plot presented distinct separation between control group and dosed groups of TBBPA or HBCD.It was demonstrated that the metabolic patterns of the rats have been disturbed after oral dosed.According to score plot of TBBPA dosed group and control group,a positive correlation of dose dependent effect and time dependent effect was found. However,the score plot of HBCD indicating that the metabolic patterns might not be further deteriorated by increasing HBCD dosage or duration of administration.When comparing the control and dosed groups,the difference in the concentrations of some kinds of compounds include amino acid,glucoside and phospholipid etc was significant.It is indicated that these compounds should be the latent biomarkers.
1.HBCD和TBBPA检测方法
采用同位素稀释技术建立了动物性食品中HBCD异构体和TBBPA的UPLC-MS/MS测定方法。样品的提取可选用索氏提取法或加速溶剂萃取法,提取液的净化方法可选用浓硫酸脱脂结合固相萃取柱净化,或者自动GPC脱脂结合浓硫酸脱脂净化。样品净化后通过UPLC BEH C18柱分离,以甲醇/乙腈混合液和水为流动相进行梯度洗脱,5min内即可完成一次测定。在分析过程中电离方式为ESI(—)模式,并以多反应离子监测(MRM)模式进行测定,内标法定量。HBCD和TBBPA的监测离子分别为m/z640.7→80.7和m/z542.6→447.6。所建立的方法可用于测定水产品、肉类、奶类、蛋类等动物性食品中的六溴环十二烷和四溴双酚A,在5-250ng/mL范围内线性良好,检出限为10-100pg/g,在不同加标水平下方法的平均回收率在86.8%～110.5%之间,RSD在1.07%～14.04%之间。经过国内三家单位的协同性验证试验和国际比对考核实验,证明所建立的方法灵敏度、准确度和精密度均符合痕量检测的要求。
2.我国食品中TBBPA和HBCD的污染状况与居民膳食摄入水平研究
测定了2007年总膳食研究中采集的12个省4类动物性食品的48份混样中HBCD和TBBPA的污染水平。TBBPA含量在＜LOD到2044pg/g lw之间,在肉类、蛋类、水产类和奶类食品中TBBPA的平均含量分别为251、179、721和194pg/g lipid weight(lw)。在HBCD的三种异构体中,α-HBCD的比例总是最高。HBCD含量在＜LOD到9208pg/g lw之间,在肉、蛋、水产和奶中的平均含量分别为252、269、1441和194pg/glw。水产类食品中TBBPA和HBCD的检出率、最高值和平均值均是四类食品中最高的。
以总膳食样品中TBBPA和HBCD的污染水平和食物消费量数据为依据,估计我国成年男子TBBPA和HBCD的膳食摄入水平。若以1/2LOD代替未检出样品中待测物的含量,则TBBPA的膳食摄入水平在32～1305pg/kg bw/day之间,平均为256pg/kgbw/day,∑HBCD的膳食摄入水平在87-1722 pg/kg bw/day之间,平均为432 pg/kgbw/day。对膳食TBBPA摄入量贡献率最高的为肉类(52%),其次为水产类(30%)、奶类(10%)和蛋类(8%)。HBCD贡献率最高的为肉类(44%),其次为水产类(24%)、蛋类(17%)和奶类(15%)。表明肉制品为我国居民TBBPA和HBCD膳食暴露的最重要来源。但与国外研究相比,我国食品中TBBPA和HBCD的污染水平,以及人体摄入量仍处在较低的水平。
3.母乳样品中HBCD和TBBPA的污染水平
测定了从全国12个省(市、区)采集的24份母乳混样。分析了这些样品中TBBPA和HBCD的污染水平,并结合母乳摄入量数据估算婴儿TBBPA和HBCD的每日摄入量。母乳中TBBPA含量在＜LOD-5124pg/g lw,平均为941pg/g lw。在HBCD的三种异构体中,α-HBCD含量总是最高,母乳中α-HBCD含量在＜LOD-2776pg/g lw,平均为1050pg/g lw。母乳样品中TBBPA和HBCD的污染水平明显高于动物性食品。推测TBBPA和HBCD在食物链中可能具有从低端到高端的生物放大作用。此外人体对TBBPA和HBCD的摄入可能存在多种途径。婴儿的摄入水平为320-37240pg/kgbw/day(TBBPA)和670-17320 pg/kg bw/day(∑HBCD),平均摄入水平分别为5094和5837 pg/kg bw/day。我国婴儿的溴系阻燃剂(包括TBBPA、HBCDs和PBDEs)摄入水平明显高于成年男子,应引起足够的重视。
4.TBBPA和HBCD对大鼠毒性的代谢组学研究
代谢组学作为系统生物学的重要组成部分,已经成为继基因组学、转录组学、蛋白质组学之后兴起的一个新的组学研究热点。我们采用代谢组学方法初步探索了TBBPA和HBCD对SD大鼠的毒性。大鼠分为高低剂量组和对照组,连续染毒7天,取每日24小时尿样及第8天血清样。以超高效液相色谱-飞行时间质谱分析不同剂量染毒大鼠及对照组大鼠的血清或尿液,采用归一化方法对代谢谱进行峰对齐,并采用偏最小二乘-判别分析法进行模式识别和可视化,以此观察不同剂量TBBPA或HBCD染毒大鼠血清或尿液代谢谱与空白对照组的差异。大鼠经染毒后,与对照组代谢谱比较有明显差异,对血清代谢谱的分析表明TBBPA对大鼠的毒性存在明显的剂量-毒性效应关系,毒性作用随染毒剂量增大而增大,但HBCD染毒大鼠的剂量-毒性效应关系不明显。对尿液代谢谱的分析表明TBBPA对大鼠的毒性除了随染毒剂量增大而增大外,还随着染毒时间的延长而增大。但HBCD染毒大鼠这两方面的效应均不明显。通过对载荷图的分析,结合代谢物数据库,初步筛选了一些可能的生物标志物,包括血清和尿液中的氨基酸、葡萄糖苷、磷脂等小分子物质,这些化合物在对照组和剂量组中的含量差异较为明显,可以作为进一步定性分析的研究对象。
Hexabromocyclododecane(HBCD) and Tetrabromobisphenol A(TBBPA) are two brominated flame retardants(BFRs) currently in use all over the world.They are now ubiquitous contaminants in wildlife,as well as in humans.Studies indicated that BFRs are bioaccumulative and persistent compounds,thus they should be regarded as persistent organic pollutants(POPs).However,we are still lacking information on the contaminant level of BFR in food and,especially,in human being.It is generally accepted that human exposure to BFRs is mainly through diet.However,there were relatively few studies reporting the presence of HBCD and TBBPA in the foodstuffs or human milk from China. In order to assess dietary exposure of Chinese adults and nursing Infants to TBBPA and HBCD,a UPLC-MS/MS method was developed for simultaneous determination of TBPPA and HBCD in foods of animal origins.On the basis of the analytical method,the levels of HBCD diastereomers(α,β,andγ-HBCD) and TBBPA in two categories of samples: pooled human milk samples collected in 2007 from 12 provinces of China and TDS samples from the fourth Chinese total diet study in 2007,were examined.Based on the obtained data,the human daily intake of TBBPA and HBCD via human milk or food consumption in China was estimated and compared.In order to explore the acute toxicity effect of TBBPA or HBCD to human being or mammal,metabonomics methodology was used to investigate the TBBPA/HBCD induced injure in rats.
1.Method for the determination of TBBPA and HBCD in food
An ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed for simultaneous determination ofα,β,γ-HBCD and TBBPA in foods of animal origin,include meat and meat products,egg and egg products,milk and milk products and aquatic foods.The methodology was based on solid-liquid extraction,GPC cleanup or SPE cleanup,and isotope dilution.Samples were extracted by Soxhlet extraction or accelerated solvent extraction(ASE).The extract was purified by gel permeation chromatography(GPC) pretreatment or concentrated sulfuric acid treatment follow by SPE cleanup.The separation was carried out on a UPLC BEH C18 reversed-phase column,using methanol-acetonitrile and water as the gradient elution solvent,the four BFRs were eluted within 5min.A triple quadrupole mass spectrometer equipped with electrospray ionization source was operated in negative ion mode.Multiple reaction monitoring(MRM) was employed for quantitative measurement.The MRM transitions monitored were as 640.7→80.7 for HBCD,542.6→447.6 for TBBPA.Good linearities were found within the ranges of 5-250ng/mL with the LOD of 10-100pg/g.The mean recovery of different spiking level varied from 86.8%to 110.5%in different foodstuffs,with the relative standard deviations(RSDs) between 1.07%and 14.4%.The results demonstrated that the UPLC-MS/MS method developed is highly effective for analyzing trace amounts of TBBPA and HBCDs in animal original foods.
2.Contamination level in foods and dietary exposure assessment by the Chinese Total Diet Study
On the basis of UPLC-MS/MS analysis,TBBPA andα,β,γ-HBCD were determined in 48 Chinese total diet study(TDS) samples collected from 12 provinces in 2007.In the four food groups include meat,egg,milk and aquatic foods,levels of TBBPA ranged from＜LOD to 2044 pg/g lw.Theα-HBCD diastereoisomer was generally the most abundant isomer comparing withβ-andγ-HBCD.Levels of HBCD ranged from＜LOD to 9208pg/g lw.The highest contamination level of TBBPA and HBCD were both found in the aquatic food group.The lowest concentrations occurred in the egg group and milk group.
Daily dietary intakes of TBBPA andΣHBCD for the Chinese were estimated by multiplying the measured concentrations of TBBPA andΣHBCD with the daily consumption data.The medium bound(＜LOD=1/2LOD) EDI_(TBBPA) for a "reference" man (male adult,63kg bodyweight) via animal origin foods was 256 pg/kg bw/day and EDI_(ΣHBCD) was 432 pg/kg bw/day.Meat and meat products were the main source in the total dietary intake of TBBPA andΣHBCD.
3.Contamination level of HBCD and TBBPA in human
TBBPA andα,β,andγ-HBCD were determined in 24 pooled human milk samples collected from 12 provinces in 2007.Levels of TBBPA ranged from＜LOD to 5124 pg/g lw with mean concentration of 941pg/g lw.Theα-HBCD diastereoisomer,which ranged from＜LOD to 2776 pg/g lw with mean concentration of 1050pg/g lw,was the most abundant isomer as same as in animal original foods.However,levels of TBBPA and HBCD in human milk were higher than that in foods.It was indicated that levels of HBCDs or TBBPA are elevated in species at the top of the food chain,which clearly points toward biomagnification.It also suggests that human exposure to TBBPA/HBCDs may occur from multiple routes,besides from food,intakes from indoor air or dust is probably another major route.The average estimated daily intake(EDI) of TBBPA via human milk for nursing infants was 5094 pg/kg bw/day with a range 320-37240 pg/kg bodyweight (bw)/day,while that ofΣHBCD was 5837 pg/kg bw/day with a range 670-17320 pg/kg bw/day.Which were much higher than that for a male adult.It is indicated that the body BFRs burden of a nursing infant in China is much higher than that of adult.The possible toxicological impact of such a high daily intake should certainly be investigated.
4.Within the past few years,metabonomics,as a new part of system biology,has gained great prominence besides genomics,transcriptomics and proteomics.In this paper, metabolism disorders induced by TBBPA or HBCD in a rat model with different dosage of intoxication were studied by metabonomics.Ultra performance liquid chromatography (UPLC) coupled with quatropde-time of flight(Q-TOF) mass spectrometer were used to analyze serum and urine obtained from rats treated with a range of doses of TBBPA or HBCD,with the objective to reveal the characteristics of the damage caused by these BFRs and identify the latent biomarkers.SD rats were randomly assigned into dosed groups and control group,rats were given daily TBBPA(30,300mg/kg) or HBCD(30,300mg/kg) for 7 days and sacrificed on Day 8 for serum collecting.24h urine samples were collected every day before dosing.All serum and some urine samples were performed UPLC-Q-TOF-MS analysis.A normalization method was performed to match the peaks between the chromatograms.A supervised learning algorithm i.e.partial least squares discriminant analysis(PLS-DA) was used for the pattern recognition and visualization.The PLS-DA score plot presented distinct separation between control group and dosed groups of TBBPA or HBCD.It was demonstrated that the metabolic patterns of the rats have been disturbed after oral dosed.According to score plot of TBBPA dosed group and control group,a positive correlation of dose dependent effect and time dependent effect was found. However,the score plot of HBCD indicating that the metabolic patterns might not be further deteriorated by increasing HBCD dosage or duration of administration.When comparing the control and dosed groups,the difference in the concentrations of some kinds of compounds include amino acid,glucoside and phospholipid etc was significant.It is indicated that these compounds should be the latent biomarkers.
引文
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