• <tr id="yyy80"></tr>
  • <sup id="yyy80"></sup>
  • <tfoot id="yyy80"><noscript id="yyy80"></noscript></tfoot>
  • 99热精品在线国产_美女午夜性视频免费_国产精品国产高清国产av_av欧美777_自拍偷自拍亚洲精品老妇_亚洲熟女精品中文字幕_www日本黄色视频网_国产精品野战在线观看 ?

    Targeted bile acids metabolomics in cholesterol gallbladder polyps and gallstones: From analytical method development towards application to clinical samples

    2023-10-24 02:40:28JiojioWeiToChenYminLiuShuiSunZhiqingYunYixinZhngAizhenXiongLinnnLiZhengtoWngLiYng
    Journal of Pharmaceutical Analysis 2023年9期

    Jiojio Wei ,To Chen ,Ymin Liu ,Shui Sun ,Zhiqing Yun ,Yixin Zhng ,Aizhen Xiong ,Linnn Li ,*,Zhengto Wng ,**,Li Yng ,***

    a The MOE Key Laboratory of Standardization of Chinese Medicines,The SATCM Key Laboratory of New Resources and Quality Evaluation of Chinese Medicines,The Shanghai Key Laboratory for Compound Chinese Medicines,Institute of Chinese Materia Medica,Shanghai University of Traditional Chinese Medicine,Shanghai 201203,China

    b Department of Biliary and Pancreatic Surgery,Renji Hospital,School of Medicine,Shanghai Jiao Tong University,Shanghai,200127,China

    Keywords:Bile acid metabolism Gallbladder polyps Gallstones Metabolomics UPLC-MS/MS

    ABSTRACT Bile acids (BAs) are synthesized by the liver from cholesterol through several complementary pathways and aberrant cholesterol metabolism plays pivotal roles in the pathogeneses of cholesterol gallbladder polyps(CGP)and cholesterol gallstones(CGS).To date,there is neither systematic study on BAs profile of CGP or CGS,nor the relationship between them.To explore the metabolomics profile of plasma BAs in healthy volunteers,CGP and CGS patients,an ultra-performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS)method was developed and validated for simultaneous determination of 42 free and conjugated BAs in human plasma.The developed method was sensitive and reproducible to be applied for the quantification of BAs in the investigation of plasma samples.The results show that,compared to healthy volunteers,CGP and CGS were both characterized by the significant decrease in plasma BAs pool size,furthermore CGP and CGS shared aberrant BAs metabolic characteristics.Chenodeoxycholic acid,glycochenodeoxycholic acid,λ-muricholic acid,deoxycholic acid,and 7-ketolithocholic acid were shared potential markers of these two cholesterol gallbladder diseases.Subsequent analysis showed that clinical characteristics including cysteine,ornithine and body mass index might be closely related to metabolisms of certain BA modules.This work provides metabolomic information for the study of gallbladder diseases and analytical methodologies for clinical target analysis and efficacy evaluation related to BAs in medical institutions.

    1.Introduction

    Gallbladder polyps (GP) and gallstones (GS) are both widely accepted high-risk factors for gallbladder cancer [1-5].GP,also named polypoid lesions of the gallbladder,can be categorized as neoplastic and non-neoplastic polyps based on histopathological evaluation.Neoplastic polyps cover all cancerous lesions and precursors of cancer,while non-neoplastic polyps consist of an aggregation of tumor-like lesions without malignant potential,including cholesterol polyps,inflammatory polyps,and adenomyomas,of which cholesterol polyps are the most usual.The mainstream treatment is surgical excision,and a broad consensus has been reached that cut-off of the optimum size for resection was 10 mm[2,6].Unlike the immobilization of polys,GS grow and move inside the gallbladder or biliary tract.Based on the composition,GS can be classified into cholesterol stones,bilirubin stones,and mixed stones.The most commonly used options are based on surgery but remain predominantly invasive [7].In the clinic,both the golden standards for diagnosing GP and GS are abdominal ultrasonography based on symptoms [8,9].Identification of potential biomarkers might be helpful for diagnosis and developing preventive strategies,and it also can provide a deeper understanding of disease etiologies.

    Bile acids (BAs) are a group of acidic steroids with specific physicochemical properties that are synthesized from cholesterol in the liver through hydroxylation and side chain oxidation and stored in the gallbladder,and are the final metabolites of cholesterol [10,11].They play an important role in maintaining body homeostasis and physiological functions and are key signaling molecules for host and gut microbial metabolism.BAs have also been shown to be endogenous ligands for cell surface receptors,G protein coupled bile acid receptor 5,nuclear hormone receptor and natural agonists for the farnesol X receptor.Disturbances in the synthesis and metabolism of BAs in organisms can lead to the development of many diseases and immune dysfunctions,such as obesity,diabetes,non-alcoholic fatty liver and other metabolic diseases [12,13].

    Studies show that the majorities of GP(60.5%)and GS(80%)fall into the category of cholesterol type [14-16].Although the pathogeneses of cholesterol gallbladder polyps (CGP) and cholesterol gallstones (CGS) are still not fully elucidated,aberrant cholesterol metabolism is a general characteristic [17,18],which is believed to contribute to the formation of CGP and CGS.In vivo,BAs are the end products of cholesterol metabolism.Otherwise,BAs is an essential factor in the pathogenesis of CGP and CGS,and changes in the levels of BAs in human plasma are closely related to the development of the diseases.Dated back to 1970,the BAs pool size was reported to diminish in patients with GS [19],and the cholic acid (CA) production rate was significantly lower.Levels of CA and chenodeoxycholic acid(CDCA)were later found significantly depressed in the CGS group[20].Result from a global perspective also indicated that the shortage of BAs is a major reason why gallbladder bile is supersaturated with CGS[21].Thus,we hypothesized that BAs profile of patients with CGP and CGS might alter compared with healthy people,and share some common features.

    Over the past decades,enzymatic assay,enzyme linked-immunoassay,nuclear magnetic resonance(NMR),chromatography,and other related techniques have been developed and applied to the detection of BAs.The diverse structures of BAs,the existence of isomers and the complex matrix of biological samples pose great challenges for the detection of endogenous BAs.With the rapid development of chromatographic techniques,ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) has become a mainstream method for the separation and detection of BAs,which is characterized by high sensitivity,good specificity,and low detection limits,and is gradually used in clinical practice for the detection of metabolites [22,23].In this study,we have developed and validated a method for the simultaneous quantitative determination of 42 BAs with the use of UPLC-MS/MS.The original aim of this study has remained largely the same since its inception,to analyze the BAs profiles of patients with CGP and CGS,and find possible common biomarkers to provide a theoretical basis for clinical diagnosis and early prevention of CGP and CGS.

    2.Materials and methods

    2.1.Materials and chemicals

    HPLC grade methanol,formic acid and acetonitrile were purchased from Thermo Fisher Scientific (Waltham,MA,USA),Deionized water was purified by Milli-Q water purification system(Bedford,IA,USA).Phosphate buffered saline(PBS)was purchased from Meilunbio (Dalian,China).Activated charcoal was obtained from Sinopharm Chemical Reagent Co.,Ltd.(Shanghai,China).The authentic compounds of 24 unconjugated,7 glycine(G)conjugated,and 11 taurine(T)conjugated were obtained from either Steraloids Inc.(Newport,RI,USA),Sigma-Aldrich (Saint Louis,MO,USA),TRC(Shanghai,China),Shanghai Kai Bao pharmaceutical Co.,Ltd.(Shanghai,China),TCI (Shanghai,China) or J&K Scientific (Beijing,China).Four deuterium BAs as stable isotope internal standards(ISs) were purchased from Steraloids Inc.The details of authentic compounds are provided in Table S1.

    2.2.Standard solutions,calibration and quality controls

    The stock solutions of the 42 BAs and ISs had a concentration of 1 mg/mL in methanol.A mixed standard solution containing 100 ng/mL of the 4 ISs was prepared in 50% methanol-water and was used as the IS solution,stored at -20°C in glass vial until utilized.For preparation of the calibration curves,each working standard solution was mixed an equal volume of the IS solution.Spiking different concentrations of the working solution in blank plasma resulted in three quality controls (QCs) with the following concentration for each QC: low quality control (LQC),10 ng/mL;middle quality control (MQC),200 ng/mL;high quality control(HQC),2000 ng/mL.The blank plasma sample was prepared by adding 1 mL of PBS and 500 mg of activated charcoal to 1 mL of plasma,vortexed for 1 min to fully adsorb the target compounds,and then centrifuged at 4°C,10,000gfor 10 min,and the supernatant was collected in an eppendorf(EP) tube.

    2.3.Sample collection and pretreatment

    Plasma sample (n=60) were collected from Renji Hospital,including 20 CGP,20 CGS and 20 healthy volunteers.Healthy participants during the same period by both physical examination and B-ultrasonography were chosen as the control group.To reduce the influence of other factors,three groups were divided according to age,gender and body mass index(BMI).Each group consisted of 13 women and 7 men.Also,participants were at the age of 51.1(±11.4;CGP patients),50.7 (±5.2;CGS patients) and 47.5 (±12.6;control).Detailed characteristics of clinical patients are shown in Table S2.All participants signed informed consents before entering the study.And the study protocol conforms to the ethical guidelines of the 1975 Declaration of Helsinki(6th revision,2008)as reflected in a prior approval by the Ethics Committee of Renji Hospital(Approval number: [2016]045).

    The pre-prandial whole blood was collected and stored at 4°C for at least 2 h.After centrifugation at 4°C,850gfor 15 min,the supernatant was collected in an EP tube,immediately frozen and stored at -80°C.All plasma samples were thawed on ice before subsequent preparation and analysis.

    Before instrumental analysis,300 μL of methanol was added to each plasma sample(100 μL)to remove proteins.The samples were then vortexed for 1 min and centrifuged at 4°C,12,000gfor 15 min.Subsequently,300 μL of supernatant was transferred to a 1.5 mL clean EP tube and evaporated to dryness with N2.Dried samples were dissolved by adding 100 μL IS solution (100 ng/mL),then vortexed for 1 min and centrifugated at 4°C,12,000 g for 10 min,and the supernatant(80 μL)was transferred to liquid phase vials for UPLC-MS/MS analysis.

    2.4.Instrumentation conditions

    The sample was analyzed by Shimadzu Nexera X2 UHPLC LC-30A system (Shimadzu Corporation,Kyoto,Japan) and AB SCIEX 6500 Q-Trap system (SCIEX,Framingham,MA,USA) with the equipment of an electrospray ionization (ESI) source.Chromatographic separations were performed with a CORTECS UPLC?C18column(2.1 mm×100 mm,1.6 μm)(Waters Corporation,Milford,MA,USA).The column temperature was maintained at 45°C.Mobile phases were purified water containing 0.01% formic acid(A) and acetonitrile (B) with a flow rate of 0.3 mL/min,and the gradient was set as follows:0.01-12 min,23%-38% B;12-26 min,38%-75% B;26-26.1 min,75%-100% B;26.1-28 min,100% B;28-28.1 min,100%-23% B;28.1-32 min,23% B.The injection volume was 2 μL.The mass spectrometer was operated in negative ion mode.In order to enhance the sensitivity of conjugated BAs,pseudo-multiple reaction monitor mode was used(precursor and product ions are identical).The ESI voltage was set at-4.5 kV,and the temperature of the ion source was 600°C.Other MS parameters were set at: curtain gas,35 psi;collision gas,medium;nebulizer current,-3.0 μA;ion source gas 1,45 psi;ion source gas 2,50 psi;declustering potential,-120 V;electronic power,-10 V;collision energy,-10 eV;cell exit potential,-12 V.

    2.5.Validation of the method

    The newly developed method was applied for validation of individual BA in terms of linearity,limit of quantification(LOQ),limit of detection (LOD),precision,accuracy,stability,carry over effect(COE),matrix effect (ME) and recovery rate (RR).The linear regression analysis carried out with nine-point calibration curves for all BAs was plotted ranging between 0.006 and 4000 ng/mL(each concentration point contained 100 ng/mL of IS).The calibration curves were generated by plotting the peak area ratio between the analyte and the IS vs the concentration of the analyte with least squares linear regression analysis with a weighting factor of 1/x2,using the MultiQuant V3.0.1 software(SCIEX,Framingham,MA,USA) for the calculation.The LOQ was determined as the lowest concentration in the calibration curve with acceptable precision (relative standard deviation,RSD <15%) and accuracy(within±20%)at which the signal-to-noise ratio was at least 10:1.The LOD was measured at a signal-to-noise ratio of at least 3:1.

    The precision and accuracy were evaluated as the RSD% of three concentration levels(LQC,MQC and HQC),and six repeated measurements of each point of QC samples were analyzed on the same day to determine the intra-day precision and accuracy,while the inter-day precision and accuracy was repeated three times over 48 h.Analyte stability in plasma was assessed by comparing replicates (n=6) at MQC concentration after 24 h or 48 h of storage at 4°C in the dark with the freshly prepared replicates at the same concentration.COE was calculated by the ratio of the peak area of HQC to the peak area of blank methanol(n=6).IS was added to LQC,MQC,and HQC samples containing blank matrice,and then the peak area was compared with those of the control solution and IS which are without blank matrice.After calculating the matrix factor of each compound and IS respectively,the ME was obtained by dividing the matrix factor of analyte by the matrix factor of IS.And RR was calculated by dividing the corrected mean peak area ratio (analyte/IS) of each analyte spiked before extraction by that of each analyte spiked after extraction at three QC concentrations (LQC,MQC,HQC;n=6).

    2.6.Statistical analysis

    Peak extraction and analysis were carried out using the quantitative module of Analyst V1.6.2,and MultiQuant V3.0.1 software was used for the calculation of each analyte concentration.The ropls package in R (version 3.6.1) was used to construct the mathematical model,which run on a Windows 7 system.Orthogonal partial least squares discriminant analysis(OPLS-DA)was used to maximize the separation between groups.The studentttest and Venn Diagrams (version 2.1.0,http://bioinfogp.cnb.csic.es/tools/venny/index.html) were used to reveal biomarkers.The ‘preceiver operating characteristic’ (ROC) package [24] was used to present ROC curves,area under the curve(AUC)and 95% confidence intervals (CI).

    3.Results and discussion

    3.1.Method validation

    The representative chromatograms of 42 BAs and 4 ISs are shown in Fig.1.A total ion chromatogram of 42 BAs and 4 deuterium-labeled BAs was displayed in Fig.S1.The calibration curves were obtained from a series of diluted standard solutions of the 42 BAs.Coefficients of determination for all target analytes presented good regression value and ranged from 0.9935 to 0.9990.Sensitivity of the method was evaluated by calculating the LOD and LOQ.The LOD was between 0.002 and 1.500 ng/mL.The LOQ ranged between 0.006 and 5.000 ng/mL.These results are exhibited in Table S3.The precision and accuracy of the developed method were determined by analyzing the QCs at three different concentration levels,the intraday and inter-day precision (RSD%) and accuracy (relative error) of target analytes are shown Table S4.The intra-day and inter-day precision were determined to be 1.25%-9.74% and 2.70%-9.97% respectively,while the accuracy values between 86.62% to 113.28% and 86.44%-114.92%.The stability ranged from 91.1% to 112.4% and acceptable stability was observed.A summary of stability analysis is presented in Table S4.The COE,ME and RR were evaluated to determine the reproducibility of the method.The COE was tested by methanol injection after the high concentration standard,and a range of 0.0018%-0.8060% was found for all compounds.The ME was investigated by comparing the peak area of matrix-matched standard solution and the pure solution containing equivalent amounts of each analyte,respectively,to the peak areas of the IS(n=6).Before IS correction,the result showed that ME existed at MQC and LQC,and the inhibition ratios were about 30% and 25%,respectively.After correction by IS,ME was successfully eliminated and ranged between 85.18% and 114.81%.The RR values of all analytes ranged between 86.20% and 113.86%.The COE,ME and RR are shown in Table S5.

    Fig.1. Extracted ion chromatograms obtained by ultra-performance liquid chromatography-Q-Trap mass spectrometry(UPLC-Q-Trap MS)for of 42 bile acids(BAs)and 4 deuterium-labeled BAs.(A,B) Extracted ion chromatograms of 42 BAs.(C) Extracted ion chromatograms of 4 deuterium-labeled BAs.Peaks 1-42 are tauro-ω-muricholic acid,tauro-α-muricholic acid,tauro-β-muricholic acid,tauro-λ-muricholic acid,tauroursodeoxycholic acid,taurohyodeoxycholic acid,taurocholic acid,glyco-λ-muricholic acid,tauro-7-ketolithocholic acid,dehydrocholic acid,ω-muricholic acid,glycoursodeoxycholic acid,glycocholic acid,glycohyodeoxycholic acid,α-muricholic acid,7-ketodeoxycholic acid,12-ketochenodeoxycholic acid,β-muricholic acid,taurochenodeoxycholic acid,taurodeoxycholic acid,λ-muricholic acid,murocholic acid,allocholic acid,cholic acid,ursodeoxycholic acid,glycochenodeoxycholic acid,hyodeoxycholic acid,glycodeoxycholic acid,7-ketolithocholic acid,6,7-diketolithocholic acid,nordeoxycholic acid,taurolithocholic acid,12-ketolithocholic acid,apocholic acid,chenodeoxycholic acid,deoxycholic acid,glycolithocholic acid,alloisolithocholic acid,isodeoxycholic acid,isolithocholic acid,lithocholic acid,and dehydrolithocholic acid,respectively.Peaks 43-46 are glycocholic-d4 acid,cholic-d4 acid,chenodeoxycholic-d4 acid and deoxycholic-d4 acid,respectively.

    Fig.2. Orthogonal partial least squares discriminant analysis (OPLS-DA) score plots and disturbed bile acids (BAs).(A) Score plots of healthy control (green) vs cholesterol gallbladder polyps (CGP,red);(B) Score plots of healthy control (green) vs cholesterol gallstones (CGS,blue).(C) Venn diagrams for overlap of control vs CGP;(D) Venn diagrams for overlap of control vs CGS.(E) Heat map of differential BAs: control vs CGP;(F) Heat map of differential BAs: control vs CGS.7-KDCA: 7-ketodeoxycholic acid;7-KLCA: 7-ketolithocholic acid;Allo-CA: allocholic acid;CA: cholic acid;CDCA: chenodeoxycholic acid;DCA: deoxycholic acid;DHLCA: dehydrolithocholic acid;GCDCA: glycochenodeoxycholic acid;GUDCA:glycoursodeoxycholic acid;G-λ-MCA:glyco-λ-muricholic acid;Muro-CA:murocholic acid;T-λ-MCA:tauro-λ-muricholic acid;UDCA:ursodeoxycholic acid;λ-MCA:λmuricholic acid;VIP: variable importance.

    3.2.CGP,CGS-associated alterations in BAs plasma indices

    The quantitative result of 32 detectable BAs is shown in Table S6.Glycochenodeoxycholic acid(GCDCA)is one of the most abundant BAs in human plasma,and this was in line with literature [25,26].Most BAs decreased obviously in the two disease groups compared with healthy control group.OPLS-DA showed a clear difference for healthy control vs CGP or CGS group (Figs.2A and B).BAs with variable importance(VIP)greater than 1.0 andPvalue from studentttest smaller than 0.05 were considered as the biomarker candidates.9 and 10 differential BAs were screened out for CGP and CGS,respectively (Figs.2C and D).As shown in Figs.2E and F,the deficiency of discovered BAs in disease groups was intuitively expressed by heat maps.And 5 differential BAs including CDCA,GCDCA,λ-muricholic acid(λ-MCA),deoxycholic acid(DCA),and 7-ketolithocholic acid (7-KLCA) were shared by CGP and CGS.

    Next,the logistic regression models were constructed to check the diagnostic performances of differential BAs.Models were fit based on respective differential BAs and 5 common BAs.The ROC curves using prediction probability as a diagnostic indicator were constructed.Models established by 5 identical differential BAs showed acceptable predictive abilities with AUCs of 0.898 and 0.895 for CGP and CGS,respectively(red solid lines in Figs.3A and B).To discriminate CGP and CGS from healthy control,these 5 common differential BA had an AUC of 0.883 with sensitivity of 85.0% and specificity of 85.0% (95% CI: 0.785-0.980),and the accuracy of 5-fold cross validation was 82.41%±0.62% (n=3).Total amounts of BA classified by different methods were summarized.Except that of T-conjugated ones,levels of G-conjugated,free and total BAs significantly decreased for CGP and CGS (Fig.3C).Total primary and secondary BAs both significantly decreased,and Gconjugated primary BAs were found decreased only in CGP group while G-conjugated secondary BAs only inhibited in CGS group(Fig.3D).

    Fig.3. Predictive and statistical analysis based on quantitative result of plasma bile acids (BAs).(A) Receiver-operating characteristic (ROC) curves of combination index of five common differential BAs and each single BA for predictions of cholesterol gallbladder polyps(CGP)from healthy control group.(B)ROC curves of combination index of five common differential BAs and each single BA for predictions of cholesterol gallstones(CGS)from healthy control group.(C)Comparison of free and conjugated BAs.(D)Comparison of primary and secondary BAs.*P <0.05,**P <0.01.7-KLCA: 7-ketolithocholic acid;CDCA: chenodeoxycholic acid;DCA: deoxycholic acid;G: glycine;GCDCA: glycochenodeoxycholic acid;λ-MCA,λ-muricholic acid;AUC: area under the curve;CI: 95% confidence intervals.

    Primary BAs (CA and CDCA) were synthesized in the liver from cholesterol and conjugated with T or G,and then stored in the gallbladder.Following a meal,BAs are excreted into the intestine by contraction of the gallbladder,and act as emulsifiers for fat digestion and absorption.Primary BAs were metabolized by enteric bacteria to produce secondary BAs via deconjugation,dehydroxylation,dehydrogenation,and epimerization.About 95% are reabsorbed in the terminal ileum and returned to the liver,and then transported into bile to complete their enterohepatic circulation.Loss of BAs in feces will be compensated by hepatic de novo synthesis to maintain the BA pool size.In this study,statistical results revealed that patients with CGP and CGS showed reduced plasma BAs pool size.Both primary and secondary BAs were in states of deficiency,thus,CGP and CGS might share a common pathological feature defect of BAs synthesis.The major biological function of BAs is the emulsification of fat into micelles,and the shortage of BAs might explain,in some way,why dyspeptic symptoms are commonly seen in patients with CGP and CGS.Total amounts of BA classified by different classification methods in CGP and CGS groups showed very similar variations compared with controls.For individual BA,CDCA has been proven effective in the dissolution of radiolucent GS.GCDCA,the most abundant BA in human plasma,was recognized as a common biomarker of CGP and CGS.λ-MCA,also named hyocholic acid (HCA) or λ-muricholic acid,was low in human plasma and showed a beneficial effect on the prevention and dissolution of biliary cholesterol crystals in a mouse model[27].Derived from dehydroxylation of CA by anaerobic bacteria in colon,DCA once was regarded as an important factor that contributes to CGS formation [28],but doubt was cast on it later [29].Although 7-KLCA might not be derived from CDCA directly,its administration also exerted therapeutic effect on dissolving GS.A combination use of five common BAs markers provided a good diagnostic efficiency for cholesterol gallbladder diseases (CGD).

    3.3.Weighted gene co-expression network analysis (WGCNA)

    The use of computer-aided software in the diagnosis of early diseases can shorten the period of disease diagnosis and assist in the diagnosis and identification of diseases,which is of great significance for the formulation of disease treatment programs [30-32].In addition,bioinformatics analysis provides methodological support for biomarker identification and in-depth functional analysis[33-35].WGCNA is an unsupervised computational method based on “guilt-by-association”,and serially changed metabolites recognized as modules can be extracted and made associations with clinical traits[36].Samples with missing clinical data were excluded and this analysis used 11 CGP and 10 GS samples.Levels of 11 amino acids(AAs),body weight and BMI were clinically available.Relationships between BA modules and clinical traits are shown in Fig.4.

    Fig.4. Module-trait association of weighted gene co-expression network analysis.Each row corresponds to a module,column to a trait;each cell of color-coded table contains the corresponding correlation and the P value.Black: cholic acid,glycolithocholic acid;Blue: deoxycholic acid,hyodeoxycholic acid,isolithocholic acid,lithocholic acid,tauro-7-ketolithocholic acid;Brown: glycocholic acid,glycochenodeoxycholic acid,taurocholic acid;Green: glycodeoxycholic acid,taurodeoxycholic acid,tauro-α-muricholic acid;Grey:allocholic acid,murocholic acid,λ-muricholic acid;Magenta: alloisolithocholic acid,glycohyodeoxycholic acid;Pink: dehydrocholic acid,nordeoxycholic acid;Red: glyco-λ-muricholic acid,tauro-ω-muricholic acid,tauro-λ-muricholic acid;Turquoise: 7-ketodeoxycholic acid,7-ketolithocholic acid,chenodeoxycholic acid,dehydrolithocholic acid,glycoursodeoxycholic acid,ursodeoxycholic acid;Yellow: taurochenodeoxycholic acid,taurohyodeoxycholic acid,taurolithocholic acid.

    Modules in magenta and grey colors showed medium negative and positive correlations with most AAs,respectively.The magenta module consisted of glycohyodeoxycholic acid and alloisolithocholic acid while the grey module comprised murocholic acid,λ-MCA and allocholic acid.Cysteine exhibited no relationship with these two modules but a strong positive correlation with the yellow module which included taurochenodeoxycholic acid (TCDCA),taurohyodeoxycholic acid (THDCA),and taurolithocholic acid (TLCA)(P<0.001).Ornithine significantly correlated with the red module and the brown module.The red module consisted of tauro-λ-muricholic acid (T-λ-MCA),tauro-ω-muricholic acid (T-ω-MCA),and glyco-λ-muricholic acid(G-λ-MCA),and the brown module contains taurocholic acid(TCA),glycocholic acid(GCA),and GCDCA.The body weight and BMI exhibited strong significant correlations of the green module which included tauro-α-muricholic acid (T-α-MCA),taurodeoxycholic acid and glycodeoxycholic acid.

    BAs not only act as facilitators of lipid absorption but also take part in many other metabolic pathways.In mice,BAs (mainly T-conjugated) can inhibit the cysteine dioxygenase type-1-mediated catabolic pathway via the farnesoid X receptor-dependent mechanism[37].Attenuating the repressive effect of BAs,for example,level decrease might result in depletion of free cysteine pool.A strong positive correlation was observed between the yellow module(TCDCA,THDCA,and TLCA)and the cysteine level in our work.G and T are the AAs usually conjugated with BAs in human,but some reports claimed that abnormal conjugation with ornithine of BAs was also found inpatients with CGS,and thiswasalso speculated to initiate the disease [38].T-λ-MCA,T-ω-MCA,G-λ-MCA,TCA,GCA and GCDCA were found positively correlated with free ornithine in this study.Hence,competitive effects during the conjugation process of BAs with AAs might explain these intimate relationships in the pathogenesis of CGD.BMI might mediate the association between lifestyle factors and CGP or CGS,and a close relationship between body weight and BMI with conjugated forms of DCA and T-α-MCAwas observed in this work.

    4.Conclusions

    In this study,we developed and validated a UPLC-MS method for the quantitative determination of 42 BAs in plasma.The linearity,sensitivity,precision,accuracy,stability,COE,ME and RR of the method all met the criteria for metabolomics study.This method was used for the analysis of changes in human plasma BAs metabolism under the pathological condition of CGP and CGS.These alterations included a decrease in the plasma concentrations of major BAs species,including total BAs,primary and secondary BAs.Subsequently,multivariate statistical analysis approach of OPLS-DA was conducted on 42 variables in 40 samples.Based on VIP>1,Pvalue<0.05,it was revealed that nine and ten differential BAs were screened out for CGP and CGS,respectively.And heat maps showed five differential BAs including CDCA,GCDCA,λMCA,DCA,and 7KLCA were shared by CGP and CGS.The WGCNA analysis for the association between biochemical indicators like plasma BAs and AAs was further carried out.The results showed potential BAs biomarkers (TCDCA,THDCA,TLCA,T-λ-MCA,T-ω-MCA,G-λ-MCA,TCA,GCA and GCDCA)were positively associated with cysteine and ornithine.Furthermore,age,sex,and BMI were concluded to be associated with fasting plasma BAs concentration,and these factors matched well among groups in this study.To sum up,for the first time,we confirmed that CGP and CGS,which have something in common in pathogenic mechanisms,also shared some metabolic characteristics.Both CGP and CGS are characterized by shrinkage of plasma BAs.Plasma markers discovered could efficiently differentiate disease from healthy controls.The findings of the current study have clinical implications,and markers found might be translated to clinical practice.

    CRediT author statement

    Jiaojiao Wei:Writing -Original draft preparation;Tao Chen:Investigation,Resources,Data curation;Yamin Liu:Methodology,Writing -Reviewing and Editing;Shuai Sun:Formal analysis;Zhiqing Yuan:Investigation,Resources;Yixin Zhang:Investigation;Aizhen Xiong:Methodology;Linnan Li:Writing-Reviewing and Editing;Zhengtao Wang:Supervision,Writing -Reviewing and Editing;Li Yang:Conceptualization,Funding acquisition.

    Declaration of competing interest

    The authors declare that there are no conflicts of interest.

    Acknowledgments

    This work is financially supported by the National Natural Science Foundation of China (Grant Nos.: 81920108033,and 82274223).

    Appendix A.Supplementary data

    Supplementary data to this article can be found online at https://doi.org/10.1016/j.jpha.2023.06.003.

    成人av一区二区三区在线看| 国产淫语在线视频| 飞空精品影院首页| 日韩制服丝袜自拍偷拍| 成人亚洲精品一区在线观看| 午夜91福利影院| 少妇裸体淫交视频免费看高清 | 成人亚洲精品一区在线观看| 亚洲天堂av无毛| 国产男女内射视频| 水蜜桃什么品种好| 热99久久久久精品小说推荐| 汤姆久久久久久久影院中文字幕| 精品久久蜜臀av无| 美国免费a级毛片| 97人妻天天添夜夜摸| 香蕉久久夜色| 99久久精品国产亚洲精品| 午夜福利欧美成人| 亚洲中文日韩欧美视频| 视频在线观看一区二区三区| 国产又爽黄色视频| 国产精品影院久久| 国产成人欧美| 国产无遮挡羞羞视频在线观看| 久久人人97超碰香蕉20202| 国产日韩欧美亚洲二区| 男女无遮挡免费网站观看| 久久久欧美国产精品| 在线天堂中文资源库| 亚洲九九香蕉| 99riav亚洲国产免费| 欧美日韩一级在线毛片| 性少妇av在线| 精品欧美一区二区三区在线| 正在播放国产对白刺激| av国产精品久久久久影院| av网站在线播放免费| 一区在线观看完整版| 在线观看免费午夜福利视频| 久久这里只有精品19| 18禁黄网站禁片午夜丰满| 99在线人妻在线中文字幕 | av欧美777| 99在线人妻在线中文字幕 | 免费高清在线观看日韩| 精品国产一区二区三区久久久樱花| 久久久久久久精品吃奶| 亚洲五月婷婷丁香| 无人区码免费观看不卡 | 涩涩av久久男人的天堂| 久久精品国产亚洲av香蕉五月 | 99精品久久久久人妻精品| 亚洲欧美日韩高清在线视频 | 国产欧美日韩综合在线一区二区| 久久午夜亚洲精品久久| 欧美 日韩 精品 国产| 日韩欧美国产一区二区入口| av视频免费观看在线观看| 国产精品一区二区在线观看99| av网站免费在线观看视频| 亚洲七黄色美女视频| 国产黄频视频在线观看| 精品国产乱码久久久久久小说| 91麻豆av在线| 欧美中文综合在线视频| 一级毛片精品| 国产日韩欧美视频二区| 免费看a级黄色片| 啦啦啦免费观看视频1| 精品一区二区三卡| 欧美黑人欧美精品刺激| 中文字幕另类日韩欧美亚洲嫩草| 这个男人来自地球电影免费观看| 成人亚洲精品一区在线观看| 九色亚洲精品在线播放| 老司机靠b影院| 国产99久久九九免费精品| 99久久99久久久精品蜜桃| 99国产综合亚洲精品| 一区二区三区精品91| av网站在线播放免费| 国产av国产精品国产| 亚洲专区字幕在线| 日韩欧美国产一区二区入口| 一本一本久久a久久精品综合妖精| 俄罗斯特黄特色一大片| 一级片免费观看大全| 十八禁人妻一区二区| 欧美日韩成人在线一区二区| 桃花免费在线播放| 亚洲综合色网址| 免费在线观看黄色视频的| 一本—道久久a久久精品蜜桃钙片| 黄色视频,在线免费观看| 伊人久久大香线蕉亚洲五| 搡老乐熟女国产| 精品久久久精品久久久| 老司机靠b影院| 精品国产一区二区三区四区第35| 国产欧美日韩精品亚洲av| 电影成人av| 日韩一卡2卡3卡4卡2021年| 精品国内亚洲2022精品成人 | 久久久精品94久久精品| 少妇的丰满在线观看| 在线观看免费视频网站a站| 欧美日韩精品网址| 国产视频一区二区在线看| 下体分泌物呈黄色| 国产精品1区2区在线观看. | 免费久久久久久久精品成人欧美视频| 久久人妻av系列| 亚洲精品av麻豆狂野| 美国免费a级毛片| 在线十欧美十亚洲十日本专区| 一本大道久久a久久精品| 午夜精品久久久久久毛片777| 国产欧美亚洲国产| 免费在线观看影片大全网站| 丰满少妇做爰视频| 免费在线观看影片大全网站| 国产又爽黄色视频| 国产伦理片在线播放av一区| 人人妻人人澡人人爽人人夜夜| 电影成人av| 欧美黄色淫秽网站| 国产免费视频播放在线视频| 王馨瑶露胸无遮挡在线观看| 黄色视频在线播放观看不卡| www日本在线高清视频| 人人澡人人妻人| 精品久久蜜臀av无| 亚洲精品自拍成人| 天堂动漫精品| 黄色视频不卡| 国产精品av久久久久免费| 一边摸一边抽搐一进一小说 | 日韩大片免费观看网站| 日本av手机在线免费观看| 一二三四在线观看免费中文在| 久久久久久久大尺度免费视频| 国产野战对白在线观看| 9热在线视频观看99| 久久久久久久大尺度免费视频| 一区福利在线观看| 他把我摸到了高潮在线观看 | 亚洲色图综合在线观看| 99精品在免费线老司机午夜| 欧美国产精品va在线观看不卡| 久久久久久人人人人人| 精品一品国产午夜福利视频| 国产精品自产拍在线观看55亚洲 | 汤姆久久久久久久影院中文字幕| 久久久久国产一级毛片高清牌| 亚洲色图综合在线观看| 日韩中文字幕欧美一区二区| 三上悠亚av全集在线观看| 午夜福利乱码中文字幕| av视频免费观看在线观看| 亚洲成av片中文字幕在线观看| 99国产综合亚洲精品| 久久久欧美国产精品| 精品亚洲乱码少妇综合久久| 激情在线观看视频在线高清 | 肉色欧美久久久久久久蜜桃| 老熟妇乱子伦视频在线观看| 国产99久久九九免费精品| 亚洲av片天天在线观看| 国产精品秋霞免费鲁丝片| 亚洲专区字幕在线| 欧美人与性动交α欧美软件| 欧美人与性动交α欧美软件| 每晚都被弄得嗷嗷叫到高潮| netflix在线观看网站| 精品欧美一区二区三区在线| 午夜精品久久久久久毛片777| 亚洲精品中文字幕一二三四区 | 夫妻午夜视频| 50天的宝宝边吃奶边哭怎么回事| 成人永久免费在线观看视频 | 久久精品亚洲av国产电影网| 精品久久久久久电影网| 淫妇啪啪啪对白视频| 一级a爱视频在线免费观看| tube8黄色片| 精品国产乱码久久久久久男人| 超色免费av| 亚洲精品国产一区二区精华液| 桃花免费在线播放| 香蕉国产在线看| 国产成人精品无人区| 俄罗斯特黄特色一大片| 亚洲av欧美aⅴ国产| 亚洲av第一区精品v没综合| 午夜福利在线免费观看网站| 青草久久国产| 老司机午夜福利在线观看视频 | 另类精品久久| 久久中文看片网| 久久精品91无色码中文字幕| 午夜久久久在线观看| 亚洲一区中文字幕在线| 国产精品香港三级国产av潘金莲| 日韩有码中文字幕| 黑人巨大精品欧美一区二区mp4| 午夜福利免费观看在线| 亚洲欧洲精品一区二区精品久久久| 黑人巨大精品欧美一区二区蜜桃| 桃红色精品国产亚洲av| 岛国在线观看网站| videosex国产| 黄片播放在线免费| 国产又色又爽无遮挡免费看| 国产麻豆69| 黑人巨大精品欧美一区二区mp4| 亚洲人成77777在线视频| 夫妻午夜视频| 免费少妇av软件| 欧美人与性动交α欧美精品济南到| 欧美 亚洲 国产 日韩一| 午夜福利欧美成人| 亚洲av欧美aⅴ国产| 久久精品国产99精品国产亚洲性色 | 大型黄色视频在线免费观看| 色婷婷久久久亚洲欧美| 色播在线永久视频| 女人爽到高潮嗷嗷叫在线视频| 国产成人av教育| 热99re8久久精品国产| 搡老熟女国产l中国老女人| 亚洲成人免费av在线播放| 久热这里只有精品99| 久久精品国产亚洲av高清一级| 最新在线观看一区二区三区| xxxhd国产人妻xxx| 每晚都被弄得嗷嗷叫到高潮| 十八禁网站网址无遮挡| 两性夫妻黄色片| 91精品国产国语对白视频| 一区福利在线观看| 色婷婷av一区二区三区视频| 狠狠精品人妻久久久久久综合| 满18在线观看网站| 黑人操中国人逼视频| 美女高潮喷水抽搐中文字幕| 男女高潮啪啪啪动态图| 老汉色∧v一级毛片| 天堂动漫精品| 高清毛片免费观看视频网站 | 久久久久久亚洲精品国产蜜桃av| 国产一区二区在线观看av| 美女主播在线视频| 99香蕉大伊视频| 两个人看的免费小视频| 欧美精品啪啪一区二区三区| 亚洲精品在线观看二区| 91成年电影在线观看| 一区二区av电影网| 一级毛片精品| 日韩免费av在线播放| 精品国产一区二区三区久久久樱花| 亚洲情色 制服丝袜| 久久狼人影院| 可以免费在线观看a视频的电影网站| 女人被躁到高潮嗷嗷叫费观| a在线观看视频网站| 免费日韩欧美在线观看| 亚洲性夜色夜夜综合| 9色porny在线观看| 国产欧美日韩一区二区三| 国产区一区二久久| 国产在线一区二区三区精| 91国产中文字幕| 手机成人av网站| 亚洲性夜色夜夜综合| 久久人妻av系列| 亚洲 国产 在线| 久久久水蜜桃国产精品网| 青草久久国产| 欧美日韩视频精品一区| 婷婷丁香在线五月| 色尼玛亚洲综合影院| 国产亚洲欧美精品永久| 免费av中文字幕在线| av网站在线播放免费| 久久久久精品国产欧美久久久| 国产麻豆69| av一本久久久久| 岛国在线观看网站| 精品人妻在线不人妻| 成年人黄色毛片网站| av电影中文网址| 一区福利在线观看| 久久精品亚洲av国产电影网| 日日夜夜操网爽| 色在线成人网| 两性夫妻黄色片| 岛国在线观看网站| 午夜福利乱码中文字幕| 亚洲国产精品一区二区三区在线| 大码成人一级视频| 亚洲色图av天堂| 十八禁网站网址无遮挡| 99热网站在线观看| 亚洲av成人一区二区三| 亚洲第一av免费看| a在线观看视频网站| 少妇猛男粗大的猛烈进出视频| 伊人久久大香线蕉亚洲五| 视频区欧美日本亚洲| 国产欧美亚洲国产| 交换朋友夫妻互换小说| 香蕉国产在线看| 大码成人一级视频| 丰满人妻熟妇乱又伦精品不卡| 国产99久久九九免费精品| 少妇粗大呻吟视频| 人人妻人人爽人人添夜夜欢视频| 亚洲av国产av综合av卡| 久久久久久久久免费视频了| 国产欧美日韩一区二区三区在线| 狠狠婷婷综合久久久久久88av| 国产精品免费大片| 精品一区二区三区视频在线观看免费 | 黑人猛操日本美女一级片| 搡老岳熟女国产| 亚洲伊人久久精品综合| 丝瓜视频免费看黄片| 黄片大片在线免费观看| 午夜日韩欧美国产| 另类亚洲欧美激情| 女人精品久久久久毛片| 国产精品1区2区在线观看. | 狠狠精品人妻久久久久久综合| 一夜夜www| 国产片内射在线| 18禁观看日本| 中国美女看黄片| 两性午夜刺激爽爽歪歪视频在线观看 | 日韩欧美国产一区二区入口| 亚洲国产av影院在线观看| 91字幕亚洲| 亚洲精华国产精华精| 真人做人爱边吃奶动态| 国产成人精品在线电影| 丁香六月欧美| 久久久水蜜桃国产精品网| 国产成人av激情在线播放| 免费在线观看完整版高清| 中文字幕最新亚洲高清| 欧美黑人欧美精品刺激| 国产成人av激情在线播放| 欧美黄色片欧美黄色片| 亚洲情色 制服丝袜| a级片在线免费高清观看视频| 黑人猛操日本美女一级片| 成年动漫av网址| 日本vs欧美在线观看视频| 好男人电影高清在线观看| 一进一出抽搐动态| 国产男靠女视频免费网站| 18禁黄网站禁片午夜丰满| 999久久久国产精品视频| 精品国产乱码久久久久久小说| 久久久久久人人人人人| 精品一区二区三区四区五区乱码| 精品人妻1区二区| aaaaa片日本免费| 大型黄色视频在线免费观看| 日本vs欧美在线观看视频| 嫩草影视91久久| 日本一区二区免费在线视频| 丰满饥渴人妻一区二区三| 久久精品亚洲av国产电影网| 激情视频va一区二区三区| 亚洲五月婷婷丁香| 老司机靠b影院| 美女主播在线视频| 乱人伦中国视频| 亚洲国产av影院在线观看| 国产在线一区二区三区精| bbb黄色大片| 亚洲全国av大片| 波多野结衣一区麻豆| 精品一区二区三区视频在线观看免费 | 亚洲成人手机| 精品国产一区二区三区久久久樱花| 亚洲av片天天在线观看| 久久中文字幕一级| 国产精品久久久久成人av| 肉色欧美久久久久久久蜜桃| 下体分泌物呈黄色| 桃红色精品国产亚洲av| 欧美日韩亚洲高清精品| 日本vs欧美在线观看视频| 午夜福利视频在线观看免费| 欧美成人免费av一区二区三区 | 免费少妇av软件| 狂野欧美激情性xxxx| 美女午夜性视频免费| 欧美另类亚洲清纯唯美| 宅男免费午夜| 日日摸夜夜添夜夜添小说| 亚洲熟妇熟女久久| 91九色精品人成在线观看| 久久国产精品大桥未久av| 50天的宝宝边吃奶边哭怎么回事| 亚洲精品粉嫩美女一区| 亚洲午夜理论影院| 大型av网站在线播放| 国产精品1区2区在线观看. | 日本wwww免费看| 18禁观看日本| 可以免费在线观看a视频的电影网站| 午夜福利欧美成人| 最新美女视频免费是黄的| 一二三四社区在线视频社区8| 精品国产乱子伦一区二区三区| 中文字幕人妻丝袜一区二区| 色综合婷婷激情| 国产亚洲精品久久久久5区| 成年人午夜在线观看视频| 久久99一区二区三区| 国产精品免费视频内射| 欧美日韩视频精品一区| 久久久久久人人人人人| 日韩大片免费观看网站| 人妻 亚洲 视频| 中文字幕最新亚洲高清| 国产在线免费精品| 老司机深夜福利视频在线观看| 交换朋友夫妻互换小说| 精品一区二区三区视频在线观看免费 | 99国产极品粉嫩在线观看| 亚洲成国产人片在线观看| 99re在线观看精品视频| 丰满迷人的少妇在线观看| 99re6热这里在线精品视频| 99国产精品99久久久久| 日本a在线网址| 亚洲精品一卡2卡三卡4卡5卡| 国产黄频视频在线观看| 热99国产精品久久久久久7| 在线天堂中文资源库| 午夜91福利影院| 一夜夜www| 国产精品免费视频内射| 美女主播在线视频| 天天躁狠狠躁夜夜躁狠狠躁| 高清在线国产一区| 岛国毛片在线播放| 99国产精品一区二区三区| 一级,二级,三级黄色视频| 999久久久精品免费观看国产| 国产精品美女特级片免费视频播放器 | 汤姆久久久久久久影院中文字幕| 三上悠亚av全集在线观看| 欧美黄色淫秽网站| 国产不卡av网站在线观看| 日日夜夜操网爽| 久久人妻福利社区极品人妻图片| 黄色成人免费大全| 亚洲人成电影免费在线| 亚洲欧美色中文字幕在线| 久久久久精品国产欧美久久久| 捣出白浆h1v1| 欧美人与性动交α欧美软件| 汤姆久久久久久久影院中文字幕| 伊人久久大香线蕉亚洲五| 亚洲第一欧美日韩一区二区三区 | 大码成人一级视频| 免费在线观看完整版高清| 中文字幕高清在线视频| 天天操日日干夜夜撸| 久久久精品区二区三区| 99久久精品国产亚洲精品| h视频一区二区三区| 50天的宝宝边吃奶边哭怎么回事| 丝袜美腿诱惑在线| 在线观看免费视频日本深夜| 99久久国产精品久久久| 成年动漫av网址| 黄色怎么调成土黄色| 亚洲性夜色夜夜综合| 97人妻天天添夜夜摸| 一区在线观看完整版| 色在线成人网| 久久国产精品人妻蜜桃| 黑丝袜美女国产一区| 老司机在亚洲福利影院| 免费观看a级毛片全部| 亚洲成人免费av在线播放| 黄色丝袜av网址大全| 五月开心婷婷网| 国产精品熟女久久久久浪| 免费人妻精品一区二区三区视频| 十八禁高潮呻吟视频| 色尼玛亚洲综合影院| 国产精品九九99| 在线 av 中文字幕| 免费观看a级毛片全部| 少妇精品久久久久久久| 久久人人爽av亚洲精品天堂| 亚洲性夜色夜夜综合| 少妇 在线观看| 别揉我奶头~嗯~啊~动态视频| 丝袜喷水一区| 黄片播放在线免费| 国产成人欧美在线观看 | 最近最新免费中文字幕在线| 天天躁夜夜躁狠狠躁躁| 成人三级做爰电影| 成年人免费黄色播放视频| 久久精品亚洲熟妇少妇任你| 久久性视频一级片| 亚洲国产毛片av蜜桃av| 精品一区二区三区av网在线观看 | 黄色毛片三级朝国网站| 最近最新中文字幕大全免费视频| 精品国产超薄肉色丝袜足j| 国产亚洲精品久久久久5区| 国产视频一区二区在线看| 极品人妻少妇av视频| 一区二区日韩欧美中文字幕| 美女福利国产在线| 久久天躁狠狠躁夜夜2o2o| 嫩草影视91久久| 色尼玛亚洲综合影院| 90打野战视频偷拍视频| 又紧又爽又黄一区二区| 99久久精品国产亚洲精品| 91麻豆精品激情在线观看国产 | 啦啦啦在线免费观看视频4| 国产精品欧美亚洲77777| 热99久久久久精品小说推荐| 99九九在线精品视频| 日韩大码丰满熟妇| 在线观看人妻少妇| 50天的宝宝边吃奶边哭怎么回事| 亚洲欧美一区二区三区黑人| 美女视频免费永久观看网站| 色婷婷av一区二区三区视频| 亚洲,欧美精品.| 女人爽到高潮嗷嗷叫在线视频| 免费人妻精品一区二区三区视频| 欧美av亚洲av综合av国产av| 精品国产乱子伦一区二区三区| 亚洲国产精品一区二区三区在线| 久久久久久人人人人人| 国产亚洲午夜精品一区二区久久| 人人妻人人澡人人爽人人夜夜| 亚洲欧美日韩另类电影网站| 女人精品久久久久毛片| 中文字幕人妻丝袜一区二区| 久久久久网色| 无人区码免费观看不卡 | 国产免费视频播放在线视频| 色精品久久人妻99蜜桃| 新久久久久国产一级毛片| 国产精品一区二区免费欧美| 男女免费视频国产| 三上悠亚av全集在线观看| √禁漫天堂资源中文www| 欧美精品亚洲一区二区| 桃红色精品国产亚洲av| 久久婷婷成人综合色麻豆| 国产欧美日韩综合在线一区二区| 国产精品影院久久| 亚洲成国产人片在线观看| 国产淫语在线视频| 国产高清国产精品国产三级| 美女高潮喷水抽搐中文字幕| 国产成人免费无遮挡视频| 国产一区二区三区在线臀色熟女 | 国产精品美女特级片免费视频播放器 | 三级毛片av免费| 男女午夜视频在线观看| 亚洲avbb在线观看| 91麻豆精品激情在线观看国产 | 18禁国产床啪视频网站| 久久国产精品大桥未久av| 女人精品久久久久毛片| 99国产极品粉嫩在线观看| 国产男女内射视频| 深夜精品福利| 在线观看免费高清a一片| 久久人妻熟女aⅴ| 成年人午夜在线观看视频| 色精品久久人妻99蜜桃| 欧美日韩黄片免| 美女主播在线视频| 十八禁高潮呻吟视频| 高清在线国产一区| 人人妻人人澡人人爽人人夜夜| 老司机在亚洲福利影院| 亚洲天堂av无毛| 天天躁狠狠躁夜夜躁狠狠躁| 久久久国产一区二区| 国产伦理片在线播放av一区| 亚洲 欧美一区二区三区| 成人免费观看视频高清| 国产精品1区2区在线观看. | 国产成人一区二区三区免费视频网站| 1024香蕉在线观看| 岛国在线观看网站| 色婷婷av一区二区三区视频| 老汉色av国产亚洲站长工具| 久久国产精品影院| 大码成人一级视频| 女人久久www免费人成看片| 9热在线视频观看99| 国产97色在线日韩免费| 国产欧美日韩一区二区三| 欧美乱码精品一区二区三区|