MetaboLights

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online KEGG, HMDB database was used to annotate the metabolites by matching the exact molecular mass data (m/z) of samples with those from database. If a mass difference between observed and the database value was less than 10 ppm, the metabolite would be annotated and the molecular formula of metabolites would further be identified and validated by the isotopic distribution measurements. We also used a in-house fragment spectrum library of metabolites to validate the metabolite identidification.Statistical analysis was primarily conducted using R (v4.0). Metabolite data underwent three key processing steps: first, data filtering to remove samples with over 80% missing values or quality control (QC) samples with over 50% missing data; second, data imputation using the K-nearest neighbor (KNN) method; and third, data standardization via Probabilistic quotient normalization (PQN).Cluster heatmaps were generated with the R package pheatmap. Principal component analysis (PCA) and significant differential metabolite analysis were performed using the R package metaX. Partial least squares discriminant analysis (PLSDA) was carried out with the R package ropls, and variable importance in projection (VIP) values for each variable were calculated. Correlation analysis was conducted using Pearson's correlation coefficient from the R package cor. The final significant differential metabolites were identified based on three criteria: P-value <0.05 from t-test, fold change >1.2, and VIP 1 from PLSDA analysis.Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis was performed using hypergeometric tests, with P-value <0.05 indicating significant enrichment. Metabolite set enrichment analysis was conducted using GSEA (v4.1.0), and KEGG pathways with |NES| >1, a nominal P-value <0.05 and FDR<0.25 were considered significantly different between the two groups. Network diagrams were constructed based on the pathways of the metabolites to illustrate their interactions.MetaboLightsPublicLiquid Chromatography MS - positive - hilicLiquid Chromatography MS - negative - hilic An ACQUITY UPLC HSS T3 column (100 mm×2.1 mm, 1.8 µm, Waters) was used for separation. The mobile phase consists of phase A (5 mmol/L ammonium acetate + 5 mmol/L acetic acid + water) and phase B (acetonitrile). Gradient elution conditions were set as follows: 0~0.8 min , 2% ~ 70%B; 0.8~2.8 min , 70% ~ 90% B; 2.8~5.3 min, 90% ~ 99% B; 5.3~5.9 min, 99% B; 5.9~7.5 min , 99% ~ 2% B;7.5~7.6 min , 2% B;7.6~10.0 min, 2% B; The flow rate is 0.35 mL/min. The injection volume for each sample was 4 µL. The column oven was maintained at 40.Screening of Differential Metabolites and Metabolic Pathway Analysis Related to Colorectal Adenoma Recurrence.Duo KeaiPeking Universityfecesmass spectrometry assayWeigh 50 mg (±5 mg) of the sample and add 500 μL of 80 % icy methanol solution. Put a small amount of steel balls and grind them with a grinder, and incubated for 30 min at -20 to precipitate proteins, and centrifuged at 20000 g for 10 min at 4, supernatant centrifuged for 5 min again. The supernatant was transferred to a fresh vial for UPLC-HRMS analysis. The quality control (QC) sample was prepared by mixing an equal aliquot of the supernatant of samples.Homo sapienshttps://www.ebi.ac.uk/metabolights/MTBLS15298Yuhai Fang. Tianjin Dongli Hospital. dlyy_xhn@163.com.Yujie Chen. Tianjin Dongli Hospital. cyj920168414@126.com.The acquired MS data pretreatments including peak picking, peak grouping, retention time correction, second peak grouping, and annotation of isotopes and adducts was performed using XCMS software. LCMS raw data files were converted into mzXML format and then processed by the XCMS, CAMERA and metaX toolbox implemented with the R software. Each ion was identified by combining retention time (RT) and m/z data. Intensities of each peaks were recorded and a three dimensional matrix containing arbitrarily assigned peak indices (retention time-m/z pairs), sample names (observations) and ion intensity information (variables) was generated.Groupkeaiduoduo998@126.comAll participants provided a fresh fecal sample (at least 300 mg) prior to bowel preparation for follow-up colonoscopy. After collection, the samples were aliquoted and immediately stored at 4°C. All samples were transferred to a -80°C freezer within 12 hours of collection, and subsequently shipped on dry ice to LC-Bio Technologies (Hangzhou, China) for untargeted metabolomics analysis.MetabolomicsrecurrenceMetabolomicscolorectal adenomarisk factorsuntargeted metabolomicsuntargeted analysisHomo sapiensQ Exactive PlusVanquish Flexfecesgut metabolitesrecurrenceMetabolomicscolorectal adenomarisk factorsuntargeted metabolomicsuntargeted analysisHomo sapiensQ Exactive PlusVanquish Flexfecesgut metabolitesA high-resolution tandem mass spectrometer Q-Exactive Plus (Thermo Fisher Scientific) was used to detect metabolites eluted form the column. Each sample was operated in both positive and negative electrospray ionization mode. ESI temperature is 350. The voltage is +3800 volts in positive ion mode and -3400 volts in negative ion mode. The sweep gas pressure of the ion source is 0 Arb, Gas 1 (Auxiliary gas) pressures set to 15 Arb, Gas 2 (Sheath gas) pressures set to 50 Arb. The mass spectrometric data were obtained with full scan and data-dependent acquisition (DDA) modes. In one acquisition cycle, the full scan acquisition range is 70-1050 Da, and the resolution is 70k, AGC target is set to 3E6, Maximum IT is set to 100 ms. Then, the top 5 signal ions with a signal accumulation intensity of more than 100000 were selected from the full scan for DDA scanning, the DDA resolution is 17.5k, Maximum IT is set to 50 ms. Dynamic exclusion is set to 6 s.hydroxychlorpromazineDL-2-Aminooctanoic acidIsoleucyl-TyrosineCorticosterone3-Isoxazolidinone1,3-Di-tert-butylbenzene6,7-Dihydro-2,3-dimethyl-5H-cyclopentapyrazineRiboflavincis-3-Hexenyl pyruvate2-Benzoxazololgamma-Aminobutyric acidN1,N12-DiacetylspermineMethyl nicotinateQuinoline2-Pyridylacetic acidTimololReserpineHistidylproline8-hydroxymirtazapine1,2,3,4,5,6-Hexahydro-5-(1-hydroxyethylidene)-7H-cyclopenta[b]pyridin-7-oneThymoquinoneScoparoneHarmanO-DesmethyltramadolStearidonyl carnitineArecoline1-KestoseAllyl phenoxyacetateN-desmethylmirtazapinePhenylalanylprolineAmphetamine4-tert-ButylphenolN-(p-Hydroxyphenethyl)actinidineN1-AcetylspermidineDeoxycorticosterone3-Hydroxyanthranilic acidCalystegine A62-Ethyl-5-methylfuranHexanoylcarnitineCarbofuranGlucosamineNorgestimatealpha-Tocopherol4-(2-Furanylmethylene)-3,4-dihydro-2H-pyrrole1,2,3-TrihydroxybenzeneGriseofulvinalpha-MethylstyreneButyrylcarnitineEstradiolL-Pipecolic acidN6-Acetyl-L-lysineMaltitol3,4-Dihydroxy-tamoxifenAlanylleucine5-Acetyl-2,3-dihydro-1,4-thiazineStearoylethanolamide2',8-Dihydroxy-3',4',5',7-tetramethoxyflavan3-Hydroxy-11Z-octadecenoylcarnitineTetrahydrocannabinolLeucylleucineFexofenadine4-Methyl-2-phenyl-1,3-dioxolaneLysoPC(14:0/0:0)N-AcetylcadaverineTemazepam9-DecenoylcarnitineProline betaineGlutaminylisoleucinePyrazineTrimethylpropylpyrazineOleamideTiglylcarnitine3,4',5,7-Tetrahydroxy-6-methoxyflavoneIbudilastN-Methyl-L-proline3-HydroxyhexadecanoylcarnitineAmyl NitriteRimexoloneEudesmic acid6-Benzylaminopurinebeta-D-Glucosamine3,4-DimethoxybenzaldehydeChlorpromazineEtomidate1,3,7-Trimethyluric acidIndoleacetaldehydeKetotifenAcetylglycinePhenylalanylalanine4'-N-desmethylolanzapineOctaethylene glycolNorfluoxetine4'-MethoxyacetophenoneBiliverdinLysoPE(14:0/0:0)BisbyninHaematopodinCyclic AMPHomoarecolineN1-(2-Hydroxyethyl)flurazepamLysoPC(16:1/0:0)LotusineRhamnose3-(2-Furanylmethyl)-1H-pyrroleAndrostenedioneCinoxacinOrnithineHomoagmatineDeserpidineValylaspartic acidPlatydesminiumIndoleProlineChlorambucilIbutilideFuranone APhenylalanylglycineL-CarnitineSulfasalazineDiethanolamine1,2-DichloroethaneAdenosine3-hydroxyhexadecanoyl carnitineN-AcetyltryptophanL-AlloisoleucineGlutaconylcarnitine2-(1-Pyrrolidinyl)-3-pentanoneDodecanoylcarnitine6,7-Dihydro-2,5-dimethyl-5H-cyclopentapyrazineGuanidoacetic acidIsoleucyl-LeucinePimelylcarnitineAflatoxin B2Homo-L-arginineIsopimpinellinPhenylpyruvic acidL-1,2,3,4-Tetrahydro-beta-carboline-3-carboxylic acidQuindoxinMelatonin6,7-Dihydro-2,6-dimethyl-5H-cyclopentapyrazineN-trans-feruloyltyramine5-MethylcytosineKaempferide4-AminobutyraldehydeAlanyltryptophanGlycylprolineO-Acetylserine4-Trimethylammoniobutanoic acid4-Methoxybenzyl formate3-AminopropionaldehydeIsoleucyl-Aspartate3-Dehydrosphinganinetrans-3,3',4',5,5',7-HexahydroxyflavanonePhenylalanylvaline7-hydroxygranisetronArginylphenylalanineAmantadinegamma-GlutamylmethionineSpectinomycinsimvastatin hydroxy acidNorfloxacin2-Phenylethyl acetateUrocanic acidLysoPC(20:3/0:0)TelmisartanN-Didesmethyl-tamoxifen17-alpha-ethinyl estradiolAcetaminophenPentanenitrileMonomethyl phenylphosphonateAICARGlutamic acidHexadecanedioic acid mono-L-carnitine ester2,6-DimethylpyridineEpoxyanethole2-MethylbutyrylglycineTetraethylene glycolPyrimidineLevoglucosan5'-Methylthioadenosine4-NitrophenolCer(d18:1/16:0)1-Methylnicotinamide1-MethoxyspirobrassininPiperineCyclohexanone9-Azabicyclo[3.3.1]nonan-3-onePrednisolone4-ButyloxazoleMG(0:0/16:0/0:0)Epiandrosterone sulfate2,4-Dihydroxy-nitrophenol7-Methylguanine7-Methoxy-6-methyl-2H-1-benzopyran-2-one1-MethyladenineS-Cysteinosuccinic acidAesculin3-Formyl-6-hydroxyindoleHydroxycotinineNicotine imineHexaethylene glycolCapsaicinBenzeneacetonitrileZolmitriptanLinoleoyl ethanolamideCarotamine1-IpomeanolSterebin GTheobromineBilirubinBenzophenoneLithocholyltaurineDihydroartemisinin (DHA)alpha-CotonefuranLysylisoleucineMethyl neopentyl phthalic acidBetaineUracil mustard7-Dehydrocholesterol8-Hydroxyadeninealpha-Curcumene2-Amino-3,4-dimethylimidazo[4,5-f]quinolineFluconazoleN-Desmethyl tapentadolSqualeneL-2-Aminoethyl seryl phosphate3alpha-HydroxyoreadoneN-MethylnicotiniumEnrofloxacinZaleplonCytidineLysoPC(17:0/0:0)VulgarinDibutyl malate4-Ethyl-2,6-dimethoxyphenol6-oxo-famciclovirIsoquinolineSymmetric dimethylarginineLysoPE(18:0/0:0)EthenodeoxyadenosineChenodeoxycholic acid glycine conjugateKetorolac5-Methoxyindoleacetate1,5-AnhydrosorbitolDihydro-5-pentyl-2(3H)-furanone2-Propionyl-2-thiazolineN-Acetyl-b-D-galactosamineNorethindroneSphinganineChlorpropamide5-[2H-Pyrrol-4-(3H)-ylidenemethyl]-2-furanmethanolTyrosyl-Leucinexi-3-Hydroxy-5-phenylpentanoic acid O-beta-D-GlucopyranosideDiethyl sulfideD-Glutamic acidDeoxydihydro-artemisinin1-PyrrolidinecarboxaldehydeCadaverineMetoprololLysylleucineThyminePropyl gallateOleoylethanolamideArginylisoleucineBenzyl propionateTestosterone PropionateMesalazineL,L-Cyclo(leucylprolyl)2-Furancarboxaldehyde3-EthylpyridineBeta-AminopropionitrileDeoxyfructosazineProgesteroneBenzylamineMG(16:0/0:0/0:0)Leucyl-ThreoninePhlorinGlycyl-TryptophanL-ThreonineMeconinealpha-HydroxymetoprololAgmatine2-Phenylpropionaldehyde dimethyl acetalTrigonelline2,8-QuinolinediolPalmitoylethanolamideMethionineLeu-Leu-LeuSinapineEdetic AcidN-Acetyl-D-glucosaminePentamidineZanthodiolinePentoxifyllineMonoethylglycinexylidide2-AcetyloxazoleGemfibrozilStyreneDipropyl disulfideDiosbulbin BPterolactamButenylcarnitineGabapentinLactuloseN,N-Didemethyl orphenadrineOsmaroninNeostigmineLinoleamideIbandronatePranlukastL-Tryptophan1',2'-Dihydro-1,1'-dimethyl-2'-oxo-4,4'-bipyridinium(1+)3-MethylcrotonylglycineTriethanolamine17-Hydroxyprogesterone1-(4-Methoxyphenyl)-2-nitroethyleneDopamineSerotoninRasagiline4-Guanidinobutanoic acid3-HydroxylidocainePyrazinamideCholesterolGentiatibetine2,4,6-Octatriynoic acidFenofibrateTestosteroneValylmethionineFlutamideLeukotriene B4Leucylproline5-Hydroxymethyl-2-furancarboxaldehyde1,2-Dimethoxy-4-vinylbenzene4-Hydroxy-3-methoxybenzenemethanolRacemethionine3-Hydroxytrichothecene7-Acetoxy-6-methoxycoumarinCinnamaldehydeSampangineCreatineAminocaproic acid5-Aminolevulinic acidThreonylleucinePhytosphingosineLysoPC(18:2/0:0)2-AcetylpyrazineAciclovirIndoleacrylic acidBenzyl acetate1-Isothiocyanato-2-(methylthio)ethanePeperinic acidtrans-2-Dodecenoylcarnitinepsi-Pelletierine19-OxotestosteroneNebularineLenticinMaltoxazineBerberrubineLysoPC(18:1/0:0)3,4-Dihydrocadalene9-Hexadecenoylcarnitine2-(Methylthio)propaneParaxanthineHypoxanthineTemozolomide4-HydroxyestroneBenzoyl ecgonineL-ArgininePi-Methylimidazoleacetic acidSalfredin B115-AminoimidazoleLomefloxacinPhenylethylamineMiscanthoside3,4',5-BiphenyltriolMethocarbamol1,1-DimethylbiguanideN-DesmethyldoxepinN6,N6,N6-Trimethyl-L-lysineDiethylcarbamazine N-oxide5-Hydroxyindole2,4-Dihydroxy-2H-1,4-benzoxazin-3(4H)-one9,10-Dihydro-2,3,5,7-Phenanthrenetetrol2,3,6,7-Tetrahydrocyclopent[b]azepin-8(1H)-oneCaptopril3beta,6beta-DihydroxynortropaneEnoxacin2'-Hydroxy-5'-methylacetophenoneCoixinden BHovenitin I9'-desmethylgranisetronLysoPC(16:0/0:0)Glycerol tripropanoatetrans-Hexadec-2-enoyl carnitinealpha-TocotrienolRizatriptanLysoPC(20:4/0:0)2-HydroxyclomipramineProflavineSerylleucine5-Aminoimidazole-4-carboxamideDemethylbatatasin IVPalmitoylglycine2-(4-Methyl-5-thiazolyl)ethyl propionatePanthenolCellobiose4,6-Dihydroxyquinoline4-Hydroxycinnamic acidN-alpha-Acetyl-L-lysineValylarginine2-Vinyl-4H-1,3-dithiine1,4-Dideoxy-1,4-imino-D-ribitolButyramidePropionic acidN,N-DiethylbenzeneacetamideDexrazoxaneMaltotrioseValyllysineTricyclazoleDihydrouracilMelezitoseTheophyllineGlycerophosphocholine3-Hydroxy-9-hexadecenoylcarnitineGenomorphine7-Ketodeoxycholic acidD-4-O-Methyl-myo-inositol2,2,6,6-Tetramethyl-4-piperidinone2-Hydroxy-4-methylbenzaldehyde3,4-Dihydro-6-methoxy-3,7-dimethyl-1H-2-benzopyran-8-olPicolinic acidApigenin1,4-Bis-(1,1-dimethylethyl)-benzene3-Hydroxy-9Z-octadecenoylcarnitineL-2-Amino-3-methylenehexanoic acidD-Aspartic acidPyridoxamine4',7-Di-O-methylcatechinGlucosamine 6-phosphatesec-ButylaminePyrrolidino-[1,2E]-4H-2,4-dimethyl-1,3,5-dithiazineL-AcetylcarnitineN-AcetylputrescineDecanoylcarnitineD-Sedoheptulose 7-phosphateIsocarboxazidDL-Dopabeta-N-AcetylglucosamineCholineIndaneMethoxyeugenol2-Hexenoylcarnitine6-O-MethylcodeineNicotinic acid6-KetoestriolGeranylgeranyl-PPNonoxynol-9AmrinoneDesloratadineDiethylenetriamineMethylmalonic acid2-hydroxyflutamideMalic acidLeucylalanine20, 22-DihydrodigoxigeninPalmitoylcarnitine7a,12a-Dihydroxy-cholestene-3-onePhosphorylcholine3-MethylindoleN-Octyl phenyl ketonemyo-Inositol 1-phosphateHistamine1-(1-Pyrrolidinyl)-2-propanoneD-LysineL-Aspartic acid6-DimethylaminopurineProcaineN-(2-Hydroxyethyl)-morpholine1-HydroxyepiacoronePrudomestinN-Methyl-D-aspartic acidHeptadecanoyl carnitineGemcitabine3-Hydroxybutyrylcarnitine1-(Methylthio)-propane6-Deoxyfagomine2,5-Dihydro-2,4,5-trimethylthiazoleGamma-linolenyl carnitinep-Aminobenzoic acidNigellidinePiperidinePyrrolidine1-Phenyl-2-hexanoneD-OrnithineDeoxyadenosine monophosphateN1-trans-FeruloylagmatineImidazoleacetic acidDemonomethylchlorpromazine2',4'-DihydroxyacetophenoneOphiopogonin C'N1-Acetylspermine7alpha,8alpha-DihydroxycalonectrinIrinotecan4'-hydroxypropanolol3-Hydroxy-cis-5-tetradecenoylcarnitineNorvalineEstroneBiopterin2-Phenyl-1,3-dioxolane-4-methanolfalseScreening of Differential Metabolites and Metabolic Pathway Analysis Related to Colorectal Adenoma RecurrenceBackground To analyze the differences in gut metabolites between the CRA (colorectal adenoma) recurrence and non-recurrence groups, to identify characteristic metabolites and related metabolic pathways associated with recurrence, and to provide new evidence for risk stratification and intervention.Methods 30 participants with CRA recurrence and 15 participants without CRA recurrence were selected. Demographic data, baseline characteristics of adenomas, and other clinical information of each participant were collected. Additionally, fecal samples from each participant were gathered, and fecal metabolomic analysis was performed using ultra-performance liquid chromatography and tandem-mass spectrometry (UPLC-MS/MS). Principal component analysis (PCA) and partial least squares-discriminant analysis (PLS-DA) were employed to screen differential metabolites, followed by pathway enrichment analysis via the KEGG database. Firth Logistic regression analysis was employed to conduct a joint analysis of clinical variables and differential metabolites.Results Univariate analysis revealed statistically significant differences between the two groups in terms of age, body mass index (BMI), diabetes, smoking history, adenoma size, number of adenomas, and advanced adenomas (P < 0.05). There were significant differences in gut metabolites between the two groups. A total of 69 differential metabolites, including GABA (γ-Aminobutyric acid) and TDCA (Taurodeoxycholic acid), were identified. These metabolites were significantly enriched in 26 pathways, such as arginine and proline metabolism, bile secretion, secondary bile acid biosynthesis and primary bile acid biosynthesis (P < 0.05). Multivariate analysis identified smoking history, number of adenomas, and GABA were independent risk factors for CRA recurrence (all P < 0.05).Conclusion Significant differences in fecal metabolite profiles were observed between the two groups. These metabolites are mainly involved in pathways related to bile acid and amino acid metabolism. By integrating clinical risk factors with characteristic intestinal metabolites, it is hoped that a more precise predictive model for CRA recurrence can be constructed, which will provide theoretical support for future intervention strategies targeting the intestinal metabolites and related metabolic 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