生物化学与生物物理进展ProgressinBiochemistyandBiphysics,2006,33(5)：401~417www.pibb.ac.cnPIBB综述与专论Metabonomics:a Revolution in Progress*TANG HuiRu,WANG YuLan?('State K ey Laboratory ofM agnetic R esonance and M olecu lar and A tom ic Physics, W uhan Institute of Physics and M athel aticsThe Chinese A cadem y of Sciences, W uhan 430071, China:Biobgical Chem istry, Bie ed ical Sciences D ivision, School ofLife Sciences Iim perial College London,Sir A lexander Flem ing Building, South Kensington, London Sw 7 2A Z,U K)AbstractM etabonom ics is thebranch of science concemed with the quantitative understandings of them etabolite col plem entofintegratedlivingsystemsand its dynamic responsesto the changes ofboth endogenousfactors (suchasphysiologyand developmentandexogenousfactors(suchasenvironmentalfactorsandxenobiotics).Asaholisticapproach,metabonomicsdetects,quantifiesandcataloguesthetimerelatedmetabolicprocessesofanintegratedbiologicalsystem,ultimately,relatessuchprocessestothetrajectoriesof thepathophysiobgical events,Ever since its birth in1999, metabonomics hasalready been described inm orethan 800 scientificpapersandhalfdozenpatents,am ongstwhichaln ost700papers wereexperim entalarticlesNow,m etabonomics hasbeenestablishedas an extrem ely powerfulanalytical tooland hencefound successfiulapplications in m any research areas inchudingm olecuarpatholbogyand physiology,drug efficacy and toxicity, gene m od ifications and finctional genom ics, and environm ental sciences.This holisticapproach has thusbecom ean in portantpartof system sbiobgy and hasnow evoved tobeauniquepart ingbbal system sbiology.Theessenceofmetabonomicsand som eofthepresent applications werereview edtoillustratetherapid devebpm entof this extrem elyexciting new frontier.Key wordsmetabonom ics/netabolbm ics,NMR,multivariatedata analysis,metabolites, system sbiobgyachievethese,newtechnologiesarerequired to enable1General introductionthehighdensityinformationtoberetrieved,archived,Withthe eraof systems biologyburstinginto reality,the analysis of the whole biologicalsystem s whether they are cells,tissues,organs or+This w ork w as supported by gran ts from The N ational N atural ScienceFoundation ofChina (20575074) and The Chinese A cadem y ofScienceshasnowbecome the nomofwhole organism s,(100 Talents Program e, [2005]35) and Nestec S.A., Sw izerhnd, foraresearch 6]ThisbiologicalindicatesshiftaResearch Fellow ship (Y LWw),“reductionism"ofresearch philosophyfromto*Conesponding author.Tel:86-27-87198430“holism"" individual"as well as fromscienceE-mail:Huiru.tangewipm.ac.cA ccepted:February 7,2006Received:January 25, 2006“" interd isciplinary"torealscience,which attracted attentions and interestsfiom bio logists, chem ists, physicists andProfessorTANG HuiRu graduated from Northwest Instituite ofLight Industry (nowm athem aticians, leading to an explosiveShaanxi University of Sciences and Technology). After eaned his PhD in Physical"and“omics”occurrenceof“omes”Organic Chemistryat London University,hewas appointed,respectively,asHigherScientificOfficerResearchScientistandSeniorResearchScientistatBBSRC-InstituteofsciencesInessence,however,theseFood Research,UK,in biophysics (Solid State NM R)forabout8 years,Before taking up"omes”and“omics”are aim ed tothecunentpostProfessorin BiospectroscopyandMetabonomicsatWuhan Instituteofunderstandthebiologicalsystem sin thePhysics and Mathem atics,The ChineseAcadem yofSciences,hehadworked intheareaofm etabonom ics as a Senior Scientific O fficerat Imperial College London fornearly fivelevels of genesDNA),transcriptsyears D r. Tang is a visiting Professor of Shaanxi U niversity of Science and Technolbgy.(m RNA), proteins and m etabolites as aHe is also a Chartered Chem ist Fellow of the Royal Society of Chemistry (CChem,wholethantherathersumofFRSC), and a m em ber of Am erican Chem ical Society.He can be easily reached byindividualsasshownin Figure 1).telephone (+86-27-87198430)orE-mail (Huiu.Tang@wipm.ac.cn).Therefore,notonly the information atDr.WANG Yu-Lan graduated from Northw est Institute ofLightIndustry (now Shaanxithelevels ofgenes,transcripts,proteinsUniversity of Science and Technology).A fter eamed her M Phil at Leicester Universityandmetabolitesareimportantto(UK)andPhDinPhysicalChemistryatUniversityofEastAnglia(UK),shewasappointedretrieveandbeunderstood butalsotheas Research ScientistatBBSRC-JohnInnes Centre andthen atInstituteofFood Research,UK, in biophysical chem istry Solid State NM R)form ore than 4 years, Since 2001,shecorrelations/interactionsbetween thesehas become a Research Fellow of etabonom ics at Inperial College London. Herlevels havetobeestablishedandcontact details are:Tel +442075943023:Fax,+442075943226 and E-mail,Yulanunderstood (ideallyquantitatively).Towange in perialac.uk.21994-2014 China Academic Journal Electronic Publishing House.All rights reserved.http:/www.cnki.net
*This work was supported by grants fromThe National Natural Science Foundation of China (20575074) and The Chinese Academy of Sciences (100 Talents Programme, [2005]35) and Nestec S.A., Switzerland, for a Research Fellowship (YLW). **Corresponding author . Tel: 86-27-87198430 E-mail: Huiru.tang@wipm.ac.cn Received: January 25, 2006 Accepted: February 7, 2006 Metabonomics: a Revolution in Progress* TANG Hui-Ru1)**, WANG Yu-Lan2) ( 1)State Key Laboratory of Magnetic Resonance and Molecular and Atomic Physics, Wuhan Institute of Physics and Mathematics, The Chinese Academy of Sciences, Wuhan 430071, China; 2)Biological Chemistry, Biomedical Sciences Division, School of Life Sciences, Imperial College London, Sir Alexander Fleming Building, South Kensington, London SW7 2AZ, UK) Abstract Metabonomics is the branch of science concerned with the quantitative understandings of the metabolite complement of integrated living systems and its dynamic responses to the changes of both endogenous factors (such as physiology and development) and exogenous factors (such as environmental factors and xenobiotics). As a holistic approach, metabonomics detects, quantifies and catalogues the time related metabolic processes of an integrated biological system, ultimately, relates such processes to the trajectories of the pathophysiological events. Ever since its birth in 1999, metabonomics has already been described in more than 800 scientific papers and half dozen patents, amongst which almost 700 papers were experimental articles. Now, metabonomics has been established as an extremely powerful analytical tool and hence found successful applications in many research areas including molecular pathology and physiology, drug efficacy and toxicity, gene modifications and functional genomics, and environmental sciences. This holistic approach has thus become an important part of systems biology and has now evolved to be a unique part in global systems biology. The essence of metabonomics and some of the present applications were reviewed to illustrate the rapid development of this extremely exciting new frontier. Key words metabonomics/metabolomics, NMR, multivariate data analysis, metabolites, systems biology 生物化学与生物物理进展 Progress in Biochemistry and Biophysics, 2006, 33(5): 401~417 www.pibb.ac.cn 1 General introduction With the era of systems biology bursting into reality, the analysis of the whole biological systems whether they are cells, tissues, organs or whole organisms, has now become the norm of biological research[1～6]. This indicates a shift of research philosophy from “ reductionism” to “holism” as well as from “individual” science to real “interdisciplinary” science, which attracted attentions and interests from biologists, chemists, physicists and mathematicians, leading to an explosive occurrence of “omes” and “omics” sciences. In essence, however, these “omes” and “omics” are aimed to understand the biological systems in the levels of genes (DNA), transcripts (mRNA), proteins and metabolites as a whole rather than the sum of individuals (as shown in Figure 1). Therefore, not only the information at the levels of genes, transcripts, proteins and metabolites are important to retrieve and be understood but also the correlations/interactions between these levels have to be established and understood (ideally quantitatively). To achieve these, new technologies are required to enable the high density information to be retrieved, archived, = <"’"3-0 )’#3&:1*#)&’ With the era of systems biology bursting into reality, the analysis of the whole biological systems whether they are cells, tissues, organs or whole organisms, has now become the norm of biological research[1～6]. This indicates a shift of research philosophy from “ reductionism” to “holism” as well as from “individual” science to real “interdisciplinary” science, which attracted attentions and interests from biologists, chemists, physicists and mathematicians, leading to an explosive occurrence of “omes” and “omics” sciences. In essence, however, these “omes” and “omics” are aimed to understand the biological systems in the levels of genes (DNA), transcripts (mRNA), proteins and metabolites as a whole rather than the sum of individuals (as shown in Figure 1). Therefore, not only the information at the levels of genes, transcripts, proteins and metabolites are important to retrieve and be understood but also the correlations/interactions between these levels have to be established and understood (ideally quantitatively). To achieve these, new technologies are required to enable the high density information to be retrieved, archived, Professor TANG Hui-Ru graduated from Northwest Institute of Light Industry (now Shaanxi University of Sciences and Technology). After earned his PhD in Physical Organic Chemistry at London University, he was appointed, respectively, as Higher Scientific Officer, Research Scientist and Senior Research Scientist at BBSRC-Institute of Food Research, UK, in biophysics (Solid State NMR) for about 8 years. Before taking up the current post, Professor in Biospectroscopy and Metabonomics at Wuhan Institute of Physics and Mathematics, The Chinese Academy of Sciences, he had worked in the area of metabonomics as a Senior Scientific Officer at Imperial College London for nearly five years. Dr. Tang is a visiting Professor of Shaanxi University of Science and Technology. He is also a Chartered Chemist, Fellow of the Royal Society of Chemistry (CChem, FRSC), and a member of American Chemical Society. He can be easily reached by telephone (+86-27-87198430) or E-mail (Huiru.Tang@wipm.ac.cn). Dr. WANG Yu-Lan graduated from Northwest Institute of Light Industry (now Shaanxi University of Science and Technology). After earned her MPhil at Leicester University (UK) and PhD in Physical Chemistry at University of East Anglia (UK), she was appointed as Research Scientist at BBSRC-John Innes Centre and then at Institute of Food Research, UK, in biophysical chemistry (Solid State NMR) for more than 4 years. Since 2001, she has become a Research Fellow of metabonomics at Imperial College London. Her contact details are: Tel, +442075943023; Fax, +442075943226 and E-mail, Yulan. wang@imperial.ac.uk. 综述与专论
·402·生物化学与生物物理进展2006:33 6)Prog. Biochem . Biophys.analysed in an integrated fashion and interpreted in thebroadly classified into four groups,namely,biologically meaningful ways,in which scientistsgenome/genomics,transcriptome/transcriptomics,inchuding physicists, chemists and engineers all haveproteome/proteomicsandmetabonome/metabonomicssom e vitalroles to play.U ltim ately,the purpose of the(ormetabolome/metabolomics),toenable us tosystem ic approach is to have the quantitative,understand an integrated organism in the levels of(QUP)universal,integratedandpredictivetranscripts,proteins and metabolitesgenes,understandings of biobgical system s.For this, properrespectively.Thewellknown and highprofilehumangenom e pro jectu2.3] represents a gigantic step forw ardintegration of infomation in genes,transcripts,proteins and metabolites will be necessary via.and huge attem pt perhaps eagemess, to reveal theappropriate m athem aticalm odelling.secretofhumanbiology.Currently,someintemationalprogramm es areinprogressin theareaof thecancergenom es to use the hum an genom e sequence and highGenes (DNA)throughputmutationdetection techniquesto identifylicroarraysom atically acquired sequence variants orm utations soTranseriptsas to identify genes critical in the development of(mRNA)(http://www.sanger.ac.uk,humancancershttp:/2D Gels+MSProteinsProtcomeLC-MS/MScancergenome.nih.gov)for the sim ilar puposes.Somehumanproteome(http://www.hupo.org)MetaboliteMetabono(http://www.metabolomics.ca)andmetabonomeNMR.LC-MSprogram m es are also in various stages w ith aim s tounderstandhowhuman systemfunctions in anEnvironmental Factorsintegrated fashion andhow to preventdiseases.Fig.ISchematic representation ofthetasksforglobalInthemetabolismlevelspecificallytheadoptionof“holism"system s biobgyphilosophy has led to the developm entof the concepts of m etabolom el415, m etabonom ics589]and m etabolbm icsuau6) (see Table 1 for details), w hichAm ongsttheseanalysesofbiologicalsystem s,them etabolite analysis itself has always been an essentialwill undoubtedly be vital to gain insights into lifecomponentinlifescienceresearchforallorganismsprocesses and to understand the com plexity of thesince the nature ofmetabolites and, in particular, thewholeorganismsandtheirinteractionswithchanges of themcarries rich information in theenvionm ent i greater depth. This is because them etabolism levelas wellas in thegene expressionandmetabolism represents not only the near-end pointprotein fiunctioning 57~1, Such im portance has aleadyproductsof biologicalprocessesbutwhatreallyhasbeen highlighted by num erous Nobel Prizes aw ardedhappened in contrastto genomes,transcriptomesandto scientists for their works to understandings toproteomeswhichprovidedthematerialfoundationformetabolisms(http://www.nobelse).These previouswhatmighthappeninabiologicalsystem (thatmayorefforts,essentially based on areductionismm ay nothappen).philosophy,havemade tremendous contributionsto2 w hatarem etabonom eandm etabonom ics?biochem icalthedetailedunderstandingsoftheTheword“Metabonom ics”originated frompathways.Because of thecomplexityof livingGreek“meta"meaning changesoradacent,andorganism s and, in particular, the underlying m olecular“nomos”meaning rules or laws(e.g.as inthemechanismsof their biolbgicalprocesses,economics)μ7Theconcept of metabonomicsconventional"reduction ismapproachhasfaceddefined as the quantitativewasinitiallychallenges.Basedontheseevergreateraidedm easurementofthemulti-parametricmetabolicaforementionedwithprogressesandnew"omes"response of livingsystemstopathophysiologicaltechnology developm enteraofandanstimuli or genetic m odifications"Amoregeneral“omics"explosion hasbeen w ith us fora num ber ofFor100definitioncanprobablybeestablishedexam ple,morethan so-calledas:years."m etabonom ics is the branch of science concemed"omes"and“"om ics"have, so far, been coined andisstillwithwith thequantitativeunderstandings of them etabolitesuchdevelopmentproceedingaFrombiologycomplement of integrated living system s and itsbreathtaking pace.pointofview,dynam ic responses to the changes ofboth endogenous“omes"and“omics"nevertheless,thesecan be21994-2014 China Academic Journal Electronic Publishing House.All rights reserved.http://www.cnki.net
生物化学与生物物理进展 Prog. Biochem. Biophys. 2006; 33 (5) analysed in an integrated fashion and interpreted in the biologically meaningful ways, in which scientists including physicists, chemists and engineers all have some vital roles to play. Ultimately, the purpose of the systemic approach is to have the quantitative, universal, integrated and predictive (QUIP) understandings of biological systems. For this, proper integration of information in genes, transcripts, proteins and metabolites will be necessary via. appropriate mathematical modelling. Amongst these analyses of biological systems, the metabolite analysis itself has always been an essential component in life science research for all organisms since the nature of metabolites and, in particular, the changes of them carries rich information in the metabolism level as well as in the gene expression and protein functioning[5,7～11] . Such importance has already been highlighted by numerous Nobel Prizes awarded to scientists for their works to understandings to metabolisms (http://www.nobel.se). These previous efforts, essentially based on a “ reductionism” philosophy, have made tremendous contributions to the detailed understandings of the biochemical pathways. Because of the complexity of living organisms and, in particular, the underlying molecular mechanisms of their biological processes, the conventional “reductionism” approach has faced ever greater challenges. Based on these aforementioned progresses and aided with new technology development, an era of “omes” and “omics” explosion has been with us for a number of years. For example, more than 100 so-called “omes” and “omics” have, so far, been coined and such development is still proceeding with a breathtaking pace. From biology point of view, nevertheless, these “omes” and “omics” can be broadly classified into four groups, namely, genome/genomics, transcriptome/transcriptomics, proteome/proteomics and metabonome/metabonomics (or metabolome/metabolomics), to enable us to understand an integrated organism in the levels of genes, transcripts, proteins and metabolites respectively. The well known and high profile human genome project [12,13] represents a gigantic step forward and huge attempt, perhaps eagerness, to reveal the secret of human biology. Currently, some international programmes are in progress in the area of the cancer genomes to use the human genome sequence and high throughput mutation detection techniques to identify somatically acquired sequence variants or mutations so as to identify genes critical in the development of human cancers (http://www.sanger.ac.uk, http:// cancergenome.nih.gov) for the similar purposes. Some human proteome (http://www.hupo.org) and metabonome (http://www.metabolomics.ca) programmes are also in various stages with aims to understand how human system functions in an integrated fashion and how to prevent diseases. In the metabolism level specifically, the adoption of “holism” philosophy has led to the development of the concepts of metabolome[14,15] , metabonomics[5,8,9] and metabolomics[10,16] (see Table 1 for details), which will undoubtedly be vital to gain insights into life processes and to understand the complexity of the whole organisms and their interactions with environment in greater depth. This is because the metabolism represents not only the near-end point products of biological processes but what really has happened in contrast to genomes, transcriptomes and proteomes which provided the material foundation for what might happen in a biological system (that may or may not happen). 2 What ar e metabonome and metabonomics? The word “Metabonomics” originated from Greek “meta”, meaning changes or adjacent, and “ nomos” , meaning rules or laws (e.g. as in economics) [17] . The concept of metabonomics was initially defined as [8] “ the quantitative measurement of the multi-parametric metabolic response of living systems to pathophysiological stimuli or genetic modifications” . A more general definition can probably be established as: “ metabonomics is the branch of science concerned with the quantitative understandings of the metabolite complement of integrated living systems and its dynamic responses to the changes of both endogenous Fig. 1 Schematic r epr esentation of the tasks for global systems biology Genes (DNA) Transcripts (mRNA) Proteins Metabolites Genome Transcriptome Proteome Metabonome Microarrays 2D Gels+MS LC-MS/MSn NMR, LC-MS Environmental Factors · 402 ·
2006;336)唐惠儒等：代谢组学：一个迅速发展的新兴学科403.factors (such as physiolbogy and developm ent)andwww.jic.bbsrc.ac.uk/. A lthough, for the tin e being,(suchfactorsasenvironm entaltheterms“metabolomeandmetabolom ics"areusedexogenousfactorsand xenobiotics)".Anotherrelatedtermwhen plant and m icrobial system s are concemed"metabolomics”"metabonomethough havinga number oftheandwhereastems“thedefinitions has essentially been defined asmetabonomics"areused in animal models,thesequantitativemeasurementofall lowmolecularweightexpressions, nevertheless, do have differentm eaningsm etabolites in an organisn's cells at a specified tim escientifically (T ab le 1).Chttp://under specific environmental conditions"Table1 Som e usefuldefin itionsM etabolisn : the total chem istry of living cellsM etabolites: (bip)chem ical reaction products or interm ed iatesM etabonom e: the totalm etabolite com plem entofan integrated living system and its dynam ic responses to the changes ofboth endogenousand exogenous factorsM etabonom ics: the quantitative m easurem entof them ulti-param etric m etabolic response of living system sto pathophysiobgical stin uli orgenetic m odificationsM etabolom e: totalm etabolite com plem entofa living system ata given tin eM etabolbm ics: the quantitative m easurem entofall bw olecularm ass m etabolites in an organ isn's cells ata specified tin e under specificenvironm en tal cond itionsBydefinition(Table 1),metabolomicsisthemetabolitecomplementalonehaslin itedlifeconcemedwiththesnap-shotsofcelularmetabolomeusefulnesssinceprocessisadynamicand(totalm etabolites com plem ent)u4l ata given tim e, thusintegrated one.Nevertheless, it is notew orthy that, w ithit is analytical by nature and static by defaultInthe em ergence of concept of dynam ic metabolom icsthe definitionmetabonomicsmeasuresnotonlythe(http://www.nih.gov/hoadmaps/),ofcontrast,metabolitecomplementofintegratedbiologicalmetabolomicsisevolvingrapidlytowardsits"brother"system s, whether they are cellularm odels,tissues,metabonomics.Itisconceivablethatasinfantfunctioning organs orwholeorganisn s, butalso theaninscienceatthisstage,dynam ic changes of such metabolite complementinmetabonomics/metabolomicswillforeseemuchmoreresponse to the intemal orextemal stimuliIt isdevelopmentandwillprobably convergegivenenoughtime and discussions (see the brief history oftherefore reasonable to consider the m etabolom e andofm etabolom ics and m etabonom ics in Table 2).metabolom icsaspartmetabonomeandm etabonom ics respectively unl In fact, just m easuringTable2Briefhistory ofmetabonomics1983HNMRofbloodplasna Nicholson JK etal,Biochem J.211:605~615)-1984HNMRofurine(BalesJRetalClinChem,30:426~432): 1991 Pattem recognition combinedw ith H NM R spectroscopyofurine (GartlandK P R etal M olPharm acol, 39:629~642)·1998 M etabobm ewas defined as thetotalm etabolite poo1"(fTweeddaleH etal. JBacteripl, 180:5109~5116): 1999 M etabonom icswas defined by N icholson JK, etal (X enobiotica, 1999, 29: 1181~1189).2000l etabolom ics" w as firstcoined (Fiehn 0 etal,Nature Biotechnol, 2000, 18:1157~1161)It is now clearthatunderstandings in jistgenom econsists of both human genome and microbiotagenom es (m icrobiom e), in tem s of the genom e size,ortranscriptomeorproteomeormetabonome alonewill hardlybepossibletofacilitatecompletehum an genom e aloneonlyrepresents about10% of thetotal genom es for the functioning hum an body uglunderstandings ofagiven biological system.In hum an,forinstance,the communityofthegut(withoutmicrobiome,humancannotsurvive!).Formicro-organisn s (m icrobiota)and the host (hum an)such system s, m etabonom ics approach will offertogether form a co-functioning sym biotic system dueunique global infom ation (at least in the m etabolismto their billions of years of co-evolution us]Therefore,leveDsincemetabonomicanalysisprovidesin a fiunctioning hum an body, the fiunctioning genom em etabolism inform ation fiom both hosts (e.g., hum an),21994-2014 China Academic Journal Electronic Publishing House.All rights reserved.http:/www.cnki.net
2006; 33 (5) 唐惠儒等：代谢组学：一个迅速发展的新兴学科 It is now clear that understandings in just genome or transcriptome or proteome or metabonome alone will hardly be possible to facilitate complete understandings of a given biological system. In human, for instance, the community of the gut micro-organisms (microbiota) and the host (human) together form a co-functioning symbiotic system due to their billions of years of co-evolution[18] . Therefore, in a functioning human body, the functioning genome consists of both human genome and microbiota genomes (microbiome), in terms of the genome size, human genome alone only represents about 10% of the total genomes for the functioning human body [19] (without microbiome, human cannot survive!). For such systems, metabonomics approach will offer unique global information (at least in the metabolism level) since metabonomic analysis provides metabolism information from both hosts (e.g., human), Table 2 Brief history of metabonomics ·1983 1 H NMR of blood plasma (Nicholson J K et al, Biochem J, 211: 605～615) ·1984 1 H NMR of urine (Bales J R et al, Clin Chem, 30: 426～432) ·1991 Pattern recognition combined with 1 H NMR spectroscopy of urine (Gartland K P R et al, Mol Pharmacol, 39: 629～642) ·1998 Metabolome was defined as the “total metabolite pool”(Tweeddale H et al, J Bacteriol, 180: 5109～5116) ·1999 Metabonomics was defined by Nicholson J K, et al (Xenobiotica, 1999, 29: 1181～1189) ·2000 “Metabolomics” was first coined (Fiehn O et al, Nature Biotechnol, 2000, 18: 1157～1161) By definition (Table 1), metabolomics is concerned with the snap-shots of cellular metabolome (total metabolites complement)[14] at a given time, thus it is analytical by nature and static by default. In contrast, metabonomics measures not only the metabolite complement of integrated biological systems, whether they are cellular models, tissues, functioning organs or whole organisms, but also the dynamic changes of such metabolite complement in response to the internal or external stimuli. It is therefore reasonable to consider the metabolome and metabolomics as part of metabonome and metabonomics respectively[17] . In fact, just measuring the metabolite complement alone has limited usefulness since life process is a dynamic and integrated one. Nevertheless, it is noteworthy that, with the emergence of concept of dynamic metabolomics (http://www.nih.gov/roadmaps/), the definition of metabolomics is evolving rapidly towards its “brother”——metabonomics. It is conceivable that as an infant in science at this stage, metabonomics/metabolomics will foresee much more development and will probably converge given enough time and discussions (see the brief history of metabolomics and metabonomics in Table 2). Table 1 Some useful definitions Metabolism: the total chemistry of living cells Metabolites: (bio)chemical reaction products or intermediates Metabonome: the total metabolite complement of an integrated living system and its dynamic responses to the changes of both endogenous and exogenous factors Metabonomics: the quantitative measurement of the multi-parametric metabolic response of living systems to pathophysiological stimuli or genetic modifications Metabolome: total metabolite complement of a living system at a given time Metabolomics: the quantitative measurement of all low molecular mass metabolites in an organism!s cells at a specified time under specific environmental conditions factors (such as physiology and development) and exogenous factors (such as environmental factors and xenobiotics)”. Another related term “ metabolomics” though having a number of definitions has essentially been defined as “ the quantitative measurement of all low molecular weight metabolites in an organism"s cells at a specified time under specific environmental conditions” (http:// www.jic.bbsrc.ac.uk/). Although, for the time being, the terms “metabolome and metabolomics” are used when plant and microbial systems are concerned whereas the terms “ metabonome and metabonomics” are used in animal models, these expressions, nevertheless, do have different meanings scientifically (Table 1). · 403 ·
·404:生物化学与生物物理进展2006:33 6)Prog. Biochem.Biophys.m icrobiota and their interactive co-m etabolism s.Thedifficultfortheiractivities and compartm entation to beultim ate goalis to correlatethem etabonom icdata withestablishedwiththeexisting proteomicstechnologiestheproteomicandtranscriptomic ones tohave aalthough thismaychangeinthefuture.M oreover, bothcomplete view about what are happening inatranscriptomeandproteome measurementscurrentlybiological processin differentlevels.This,however,suffer from some bottlenecks such asthroughputandrequires one to take into consideration thatthese datahigh costs though thism ay also changein the future.Inrepresentprocessesprecedingondifferenttimescales.contrast,themetabonomemeasurementsarepossibleEver since its birth,metabonom icshas shown ato provideinform ation aboutw hatalready happened inrapid development and widespread applications.the biological events together with the identity,ScientificpublicationsandpatentsrelatedKOconcentation andcompartmentationofmetabolitesNMR-basedmetabonomicshaveexperiencedexponentialespeciallywhenthemetabonomicsanTheincrease on yearly basis (Figure 2).Amongstthem,technologiesem ployedappropriately.aeNMRbasedmetabonomics,inparticularappearscompartmentationinformation6particularlyenpyinga morerapidprogressinbothmethodimportantthemulticellular biological systemsforalthoughbothdevelopm entandapplicationswhere importantevents include both metabolism in aMS)basedchromatographicandmassspectrometrysingle celland the intercellularm etabolite exchanges.metabonomicsmethodsare increasinglymakinggoodIn thecase of mammals,metaboliteexchanges andprogressand contributions as well.co-metabolism between the gutm icrobiota and hostsoften have fiunctional significance related to health B02]and diseaseP425],M etabonom ics concunently m easures350themetabonomesandexchangesofthewholesystem300together with the effects of other environmentalfactors,thusprovidesuseful“global"information250for the system s biology studies with com plem entaryinfomation tothat fromgenes,transcriptsandproteins.Furtherm ore,althoughourpresentunderstandings of biochem ical pathways are prettycomprehensiveaheady,itis,nonetheless,bynomeanscomplete or absolutelyaccurate.As an excellentbiomarkerdiscoverytool metabonom icsmeasuresthemetabolismwithoutpre-conditions orpre-knowledge,therefore,is likelyto tackleproblem srelated to suchincompletenessandinaccuracy2001200220032004200519OK199920002.1Metabonom icstechnologiesCurrently,thereareanumberofmetabonom icsmetabonomics/Fig.2 Scientificpublicationsontechniques in use as show n in Figure 3. H ow ever, thesemetabolomicstechnologiesbebroadlyclassifiedcanasTotal:：Experimental papers:NMR-based;MS-bascidChormatography-basedWhoe organim, Organs,Tisues, Whole celUnlikeinthecaseofgenomes,thetranscriptome,intitroinmicoexinproteome and metabonome measurements are alllin ited by the detection sensitivity.The detectedNMR.FTIR-signals areoften relatedto compositions andFT-RamanUPLC/HPLCconcentrations,which arenottheonlyfactorsrelatedLC.TLOtothebiologicalprocesses.Inthecaseofproteins,forLC-NMR-MSexample,their biological significance is not onlyNMRMSrelated to their structure and concentration butalsotheiractivities, locations and com partn entation.TheMetabonomic datacurrentproteome measurements, in most instances,canonlyprovideinfomation on theidentitiesof someoftheproteomeandtheirconcentration.Itis,however,Fig.3Techniquesused in m etabonom ics studies21994-2014 China Academic Journal Electronic Publishing House.All rights reserved.http:/www.cnki.net
生物化学与生物物理进展 Prog. Biochem. Biophys. 2006; 33 (5) microbiota and their interactive co-metabolisms. The ultimate goal is to correlate the metabonomic data with the proteomic and transcriptomic ones to have a complete view about what are happening in a biological process in different levels. This, however, requires one to take into consideration that these data represent processes preceding on different time scales. Ever since its birth, metabonomics has shown a rapid development and widespread applications. Scientific publications and patents related to metabonomics have experienced an exponential increase on yearly basis (Figure 2). Amongst them, NMR-based metabonomics, in particular, appears enjoying a more rapid progress in both method development and applications although both chromatographic and mass spectrometry (MS) based metabonomics methods are increasingly making good progress and contributions as well. Unlike in the case of genomes, the transcriptome, proteome and metabonome measurements are all limited by the detection sensitivity. The detected signals are often related to compositions and concentrations, which are not the only factors related to the biological processes. In the case of proteins, for example, their biological significance is not only related to their structure and concentration but also their activities, locations and compartmentation. The current proteome measurements, in most instances, can only provide information on the identities of some of the proteome and their concentration. It is, however, difficult for their activities and compartmentation to be established with the existing proteomics technologies although this may change in the future. Moreover, both transcriptome and proteome measurements currently suffer from some bottlenecks such as throughput and high costs though this may also change in the future. In contrast, the metabonome measurements are possible to provide information about what already happened in the biological events together with the identity, concentration and compartmentation of metabolites especially when the NMR-based metabonomics technologies are employed appropriately. The compartmentation information is particularly important for the multicellular biological systems where important events include both metabolism in a single cell and the inter-cellular metabolite exchanges. In the case of mammals, metabolite exchanges and co-metabolism between the gut microbiota and hosts often have functional significance related to health[20～23] and disease[24,25] . Metabonomics concurrently measures the metabonomes and exchanges of the whole system together with the effects of other environmental factors, thus provides useful “global” information for the systems biology studies with complementary information to that from genes, transcripts and proteins. Furthermore, although our present understandings of biochemical pathways are pretty comprehensive already, it is, nonetheless, by no means complete or absolutely accurate. As an excellent biomarker discovery tool, metabonomics measures the metabolism without pre-conditions or pre-knowledge, therefore, is likely to tackle problems related to such incompleteness and inaccuracy. 2.1 Metabonomics technologies Currently，there are a number of metabonomics techniques in use as shown in Figure 3. However, these technologies can be broadly classified as Whole organism, Organs, Tissues, Whole cells in vitro in vivo, ex vivo Extracts GC LC, TLC LC-NMR-MS MS CE UPLC/HPLC NMR MS UV NMR, FTIR FT-Raman Metabonomic data Fig. 3 Techniques used in metabonomics studies 350 300 250 200 150 100 50 0 1998 1999 2000 2001 2002 2003 2004 2005 Fig. 2 Scientific publications on metabonomics/ metabolomics : Total; : Experimental papers; : NMR-based; : MS-based; : Chormatography-based. Scientific publications · 404 ·
2006;336)唐惠儒等：代谢组学：一个迅速发展的新兴学科·405.chromatography-based,MSbasedandNMRbaseddynam ics,pHandconcentration,interactions,according to themajprdetection methodsused (seecompartmentation,(6)beholisticratherthanselective(7)Table 3 for details). W ith so m any techniques, which(orbiasedtowards certain analytes),betechnique should one choose? Before answering thisinexpensive and have high throughput (and not labourquestion, itis necessary to have a detailed evaluationintensive),(8)requireslittle/ho samplepreparationofthesedetectionmethods.Itisconceivablethatan(separation,derivatisation et aD, (9)be non-invasive,idealdetectionmethodformetabonomicsoughtto (1)non-destructive to facilitate in vivo,in situ studies,be obective (thus user independent),(2) have high(10) have lbw recunent expenditure (to reduce costssensitivity, good signal resolution and reproducibility,and in prove efficiency). In reality, no technique w ill(3)notrequire preknowledge to assistbiom arkermeet all theserequirements and a good realisticdiscovery,(4)have good quantification capabilitymetabonomics technique ought to meet as manyforcomplexmixtures,(5)beable toproviderichcriteria as possib le.molecularinformation,suchasstuctire,Table3ProsandConsofthecurrentlyusedmetabonomicsmethodsC hrom atography-basedM ass spectom etryNM R-based m ethodsmethodsbased methodsObjectiveYesYesYesPoorGoodGoodReproducibilityFairGoodReso lutionFairSensitivitySelectiveSelectiveFairNoNoYesU nbiased detection (sim ultaneous)NoNoYes:HolisticR ichM olecular inform ationPoorFairExtensiveExtensiveSam ple preparationLittle ornoYesYesNoPre-know ledge requ irem entsIn possib leYesin vivo/in situA m ost i possib leFairH ighThroughputH ighH ighH ighLowRecunentexpenditureFairFairLowLabour in tensivenessFairLowFair/lowC ost per sam pleForall threecurrentlyused technologies,they arebodies are biased against relative to,for example,well established analytical tools and have goodphenylalanine or tyrosine even though these polarobjectiveness.In temsof sensitivity,althoughbothmetabolitesmaybeextremelyimportantInthecaseofchrom atography and m ass spectrom etry based m ethodsMS,the ionisation efficiency differences betweenenpy excellent intrinsic sensitivity,they are notdifferentmetabolitesand ion suppressionsalsoresultin som e“invisible" high concentration m etabolites.holisticorsimultaneousdetection but selectivedetection methods especially when they are used toAsforreproducibility,chromatographicmethodsare generally poor, though GC m ethods are better,analysem ixtures;theyarebiased for somemetabolitesbutagainst others when they are used to analysethewhereastheMS-basedmethodshighlyaretheirFor metabonomicspurposes,bothbiological metabonomes.In other words,reproducible.chrom atography and M S m ethods suffer fiomsensitivities arenotunifom forallm etabolites and cansomebe pretty low for certain analytes under given analysiscommondisadvantagesntemsofmetaboliteForinstance,when reverse-phasedidentificationand quantification.Both techniquesconditions.chrom atographic methods are used to analyse therequire fairly extensive sam ple preparations and aremetabonome (ormetabolite compositions)of bloodinvasive and destructive (to samples),hence are notplasm a, urine and indeed extracts of other biologicalsuitable for in vivo or in situ studies Both of thesetissues,the polar m etabolites such as sugars,sometechniques require pre-knowledge about sam plesam ino acids (e.g., glutam ine and glutam ate), hydroxyland havehighrecumentexpenditurethoughcarboxylic acids (e.g.,thoseinTCA cycle)andketonechrom atographic m ethods are reasonably inexpensive.21994-2014 China Academic Journal Electronic Publishing House.All rights reserved.http://www.cnki.net
2006; 33 (5) 唐惠儒等：代谢组学：一个迅速发展的新兴学科 For all three currently used technologies, they are well established analytical tools and have good objectiveness. In terms of sensitivity, although both chromatography and mass spectrometry based methods enjoy excellent intrinsic sensitivity, they are not holistic or simultaneous detection but selective detection methods especially when they are used to analyse mixtures; they are biased for some metabolites but against others when they are used to analyse the biological metabonomes. In other words, their sensitivities are not uniform for all metabolites and can be pretty low for certain analytes under given analysis conditions. For instance, when reverse-phased chromatographic methods are used to analyse the metabonome (or metabolite compositions) of blood plasma, urine and indeed extracts of other biological tissues, the polar metabolites such as sugars, some amino acids (e.g., glutamine and glutamate), hydroxyl carboxylic acids (e.g., those in TCA cycle) and ketone bodies are biased against relative to, for example, phenylalanine or tyrosine even though these polar metabolites may be extremely important. In the case of MS, the ionisation efficiency differences between different metabolites and ion suppressions also result in some “invisible” high concentration metabolites. As for reproducibility, chromatographic methods are generally poor, though GC methods are better, whereas the MS-based methods are highly reproducible. For metabonomics purposes, both chromatography and MS methods suffer from some common disadvantages in terms of metabolite identification and quantification. Both techniques require fairly extensive sample preparations and are invasive and destructive (to samples), hence are not suitable for in vivo or in situ studies. Both of these techniques require pre-knowledge about samples and have high recurrent expenditure though chromatographic methods are reasonably inexpensive. Table 3 Pros and Cons of the curr ently used metabonomics methods Chromatography-based methods Mass spectrometry based methods NMR-based methods Objective Yes Yes Yes Reproducibility Poor Good Good Resolution Fair Fair Good Sensitivity Selective Selective Fair Unbiased detection (simultaneous) No No Yes Holistic No No Yes Molecular information Poor Fair Rich Sample preparation Extensive Extensive Little or no Pre-knowledge requirements Yes Yes No in vivo/in situ Impossible Almost impossible Yes Throughput Fair High High Recurrent expenditure High High Low Labour intensiveness Fair Fair Low Cost per sample Fair/low Fair Low chromatography-based, MS-based and NMR-based according to the major detection methods used (see Table 3 for details). With so many techniques, which technique should one choose? Before answering this question, it is necessary to have a detailed evaluation of these detection methods. It is conceivable that an ideal detection method for metabonomics ought to (1) be objective (thus user independent), (2) have high sensitivity, good signal resolution and reproducibility, (3) not require pre-knowledge to assist biomarker discovery, (4) have good quantification capability for complex mixtures, (5) be able to provide rich molecular information, such as structure, concentration, dynamics, interactions, pH and compartmentation, (6) be holistic rather than selective (or biased towards certain analytes), (7) be inexpensive and have high throughput (and not labour intensive), (8) requires little/no sample preparation (separation, derivatisation et al), (9) be non-invasive, non-destructive to facilitate in vivo, in situ studies, (10) have low recurrent expenditure (to reduce costs and improve efficiency). In reality, no technique will meet all these requirements and a good realistic metabonomics technique ought to meet as many criteria as possible. · 405 ·