《生物药剂学与药物动力学》课程教学资源（文献资料）代谢组学_一个迅速发展的新兴学科_英文_唐惠儒

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 ·

·406·生物化学与生物物理进展2006:33 6)Prog. Biochem . Biophys.resolution magic-angle spinning (HRMAS)Both require substantial in provem ents to obtain richNMRmolecular information(as discussed earlier)Om ethodsremovethe linebroadening factorsresultingascertaintheidentitiesandpropertiesofbiomarkersfrom residualdipole-dipoleinteraction,chemical shiftFor M S-based m ethods, equipment can also befairlyanisotropyandsusceptibility,achievinghighresohuitionexpensive (e.g.,FTMS).Itisimpossibleto obtainspectroscopyfortissueswhichiscomparabletothatinthe liquid state NM R B81~38]The intoduction of thiscompartmentationinformationwithchromatographyandMSbasedmethodseventhoughsuchinformationmethodmakesitpossibletodirectlyinvestigatetissuesand even whole living organisn s Bo01 w ithout anyisextrem elyin portant Forboth techniques,althoughMoreover,NMRisaquantification can be done by constructing in situdestructionof samples.standard calibrationcurves,thiswill requireenormousquantitativeand simultaneousdetection method thusamountsofworkbecause,not biasedfor anymolecules.To quantifytheinmanycasesmetabonom es consist of hundreds of metabolites andm etabolites,NMR methods only require a knowntheir standards are often not readilyavailable.Evenconcentrationcompoundoranelectronicsignalasnew biomarkers,bydefaultarereference.TheNMRbasedmethodis also highmoreimportantly,often unknownmetabolites.Someofthedisadvantagesthroughput and400samplespercanmeasureuroin ection technology E0]W ith the richcanbeovercomsomeextentbytheirhvphenation24hwithflowForexam ple, GCM S has shown greatprom ise l1027obtained,suchmethodsinfomationhavelowsinceGCiswellestablishedandreproducibilityisrecurrent expenditure so that the analysis cost isusually low on costper-sam ple basis even thoughreasonably high.H ow ever, therem ainingproblem s arestill associated withthe aspects of quantification,NMR spectrometers are expensive.InfactNMR(biased)selectivedetection,molecularmethod is the onlycurrentmetabonomics detectionpoorinfomation,tin em ethod which can be used to undertake in vivo and inconsumingsam plepreparationprocedures and invasive/destructive nature. Recently,situ studies at the levels of cells, m ulticellular tissueshyphenatedultrah ighperfomanceliquidorgans and w hole organ isn s N M R-based m ethods canUPLC-chrom atographyandmassspectrom etryalsobeemployedtoseparatesignalswithoutseparateM S)28~301 has been develbped,which substantiallysam ples via. spectral editing techniques by takingchrom atographicresolutionimprovedandadvantages of differences inrelaxation propertiesanddiffusivities ofm etabolites3, further favouring thereproducibility,show ingpotentialingreatmetabonomic applications.Itmay be possible that,spectral sim plification and signal assignm ents.Thewith somemore improvementsin both facilitiesandmapr disadvantage for the conventional NM R is itscolumnstationaryphases,UPLCMSwillplayevenlowintrinsicsensitivity.However,therearetwowaysgreaterroles inthefuturemetabonom icsresearch.ThisinproveNMRsensitivity,namely,improvingtotechniqueand GC-M Swill probablybecomethem ainsignato-noise ratio by reducing electronic noise andtechnology especiallychem ical noiselevels.In order to reduce electronicstreamwhenvivolinsitustudiesstrictlyrequiredandthenoiselevelscryogenic probe technology hasbeenarenotis not critically vitalcom partm entationinfomationintroducedwhich thenum.berofvearsHoweverattentionsneededelectronicdetectioncircuits wasmenttemperaturcomprehensivelydealwiththeproblem sof"knownreducedaboutt20Khenceincreaseddetectionhunknown markers”forbothchromatography-M Ssensitivityabout fourfoldsbyChem ical noisesbased technolbogies.resulting fromsignal overlappingcanbereducedbyIn contrastthe NMRbasedmethod isuserincreasing the m agnetic field strength.Thiswillalsoindependentrequireslittleorno samplepreparationincrease detection sensitivity and spectral resoluition.(e.g.,forbiofluids of mammals),and has excellentCurrentlythestandard600MHzspectrometerresoluitionandreproducibility.Inaddition,NMRequipped with optimised cryogenic probe technologymethod offers rich molecular infomation includingcan detectmetabolites at nanogram (10-g)levelmetabolitestructure,concentration,molecularSincethepolarisation rateinNMR obeysBoltzmanndynam ics,interactions, pH andcompartnentationdistribution law and such rate is extremely low underwhen diffusion-editingtechniques areemployed.nomal circum stances,methods are also currentlyFurthermoreNMRisnon-invasiveandnon-destructiveunderdevelopm entto increase thepolarisationratebyto sam ples, m ak ing it possible to facilitate in vivo andusing dynam ic nuclearpolarisation techniques.It ism situ studies. For exam ple, recently developed highthus conceivablethatfurther developments of this21994-2014 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) Both require substantial improvements to obtain rich molecular information (as discussed earlier) to ascertain the identities and properties of biomarkers. For MS-based methods, equipment can also be fairly expensive (e.g., FTMS). It is impossible to obtain compartmentation information with chromatography and MS based methods even though such information is extremely important. For both techniques, although quantification can be done by constructing in situ standard calibration curves, this will require enormous amounts of work because, in many cases, metabonomes consist of hundreds of metabolites and their standards are often not readily available. Even more importantly, new biomarkers, by default, are often unknown metabolites. Some of the disadvantages can be overcome to some extent by their hyphenation. For example, GC-MS has shown great promise [10,26,27] since GC is well established and reproducibility is reasonably high. However, the remaining problems are still associated with the aspects of quantification, selective (biased) detection, poor molecular information, time consuming sample preparation procedures and invasive/destructive nature. Recently, hyphenated ultrahigh performance liquid chromatography and mass spectrometry (UPLC￾MS)[28～30] has been developed, which substantially improved chromatographic resolution and reproducibility, showing great potential in metabonomic applications. It may be possible that, with some more improvements in both facilities and column stationary phases, UPLC-MS will play even greater roles in the future metabonomics research. This technique and GC-MS will probably become the main stream technology especially when in vivo/ in situ studies are not strictly required and the compartmentation information is not critically vital. However, some urgent attentions are needed to comprehensively deal with the problems of “known unknown markers” for both chromatography-MS based technologies. In contrast, the NMR-based method is user independent, requires little or no sample preparation (e.g., for biofluids of mammals), and has excellent resolution and reproducibility. In addition, NMR method offers rich molecular information including metabolite structure, concentration, molecular dynamics, interactions, pH and compartmentation when diffusion-editing techniques are employed. Furthermore NMR is non-invasive and non-destructive to samples, making it possible to facilitate in vivo and in situ studies. For example, recently developed high resolution magic-angle spinning (HRMAS) NMR methods remove the line broadening factors resulting from residual dipole-dipole interaction, chemical shift anisotropy and susceptibility, achieving high resolution spectroscopy for tissues which is comparable to that in the liquid state NMR[31～38] . The introduction of this method makes it possible to directly investigate tissues and even whole living organisms [39] without any destruction of samples. Moreover, NMR is a quantitative and simultaneous detection method thus not biased for any molecules. To quantify the metabolites, NMR methods only require a known concentration compound or an electronic signal as reference. The NMR-based method is also high throughput and can measure up to 400 samples per 24 h with flow-injection technology[40] . With the rich information obtained, such methods have low recurrent expenditure so that the analysis cost is usually low on cost-per-sample basis even though NMR spectrometers are expensive. In fact, NMR method is the only current metabonomics detection method which can be used to undertake in vivo and in situ studies at the levels of cells, multicellular tissues, organs and whole organisms. NMR-based methods can also be employed to separate signals without separate samples via. spectral editing techniques by taking advantages of differences in relaxation properties and diffusivities of metabolites[41～43] , further favouring the spectral simplification and signal assignments. The major disadvantage for the conventional NMR is its low intrinsic sensitivity. However, there are two ways to improve NMR sensitivity, namely, improving signal-to-noise ratio by reducing electronic noise and chemical noise levels. In order to reduce electronic noise levels, cryogenic probe technology has been introduced for a number of years in which the temperature of the electronic detection circuits was reduced to about 20K, hence increased detection sensitivity by about four folds. Chemical noises resulting from signal overlapping can be reduced by increasing the magnetic field strength. This will also increase detection sensitivity and spectral resolution. Currently the standard 600 MHz spectrometer equipped with optimised cryogenic probe technology can detect metabolites at nanogram (10 - 9 g) level. Since the polarisation rate in NMR obeys Boltzmann distribution law and such rate is extremely low under normal circumstances, methods are also currently under development to increase the polarisation rate by using dynamic nuclear polarisation techniques. It is thus conceivable that further developments of this · 406 ·

唐惠儒等：代谢组学：一个迅速发展的新兴学科2006;336)·407.techniquewillmakethe“low sensitivity"nature ofspecial biofluids such as C SF, m ilk and dialysates canNMRrapidlybecomea history.alsobeimportantBycomparingwiththemetabonomeItisclearthatthebestchoiceatpresentmaylieatofthecontrolsamples,theeffectsofintemalandthe com bination of a number of existing techniquesextemal stimuli on thesub ects can be easily analysedThehyphenation of chromatography,MSandNMRin a holistic and quantitative way using statistical too ls.LC-NMRMS)playedanimportantrolealreadyintheForplantsystems,mostcurrentlyusedmethodsmetabolite identifications.Itisparticularly exciting toare based on solventextractsthus are in vitro bynature. This is ow ing to that the plant “biofluids"note that a statistical heterospectoscopy m ethod hasbeen reported E4l w hich takes advantages of both N M Rare not as readily available as in the case of anin als.and UPLC-M S. It is predictable that such correlationHowever,tissues and sometimes whole organismtechniqueswill becom e increasingly inportantin thestudies are possible by employing high resolutionmagic-angle spinning (HRMAS)NMR.Thismethodfuture.2.2can alsobe applied to m icrobiota and cultured cells, inSamplesformetabonomicsstudiesIn theory,any biological samples includingwhichboth cellsand m ediaare important carriers forbiofluids, tissues and even whole organism s can bem etabonomic information.The analysis of media isalso im portantand often referred to as cellm etabolismused form etabonomics studies.In practice,however,thesamplerequirements aredependent onthepurpose“footprintings”of studies and m ethodsused.Forboth chromatography2.3How todometabonomicsstudies?and M S based m ethods, the sam ples have to be in theThere are three general steps for conductingmetabonom ics studies,namely,samplepreparation,liquidfom andoften requireremovalofproteins orextractions.ForNMR-basedmethods,ontheotherdata acquisition anddatam iningin orderto extractthehand,both biofluids (e.g.urineand blood plasm a)andbiologicalsignificances.However,for sucha com plextissues (e.g., liver,kidney and brainbiopsies)aresubectextra carehasto be takenineachstep.Forgenerally employed directly withoutmuch sampleexample,thesam ple collectionsandpreparation stepispreparation or extractions, so that in vivo and in situfiundamentallymportantManyfactorsaffectinginform ation is readily available.In such cases, only asm etabonomes have to be carefiully thought throughlittle as 2 μlbiofluids5] and about 10 m g tissues aresuch as variations in species, strains, gender, age, dietrequiredto obtain nform ation in themannersofeitheracclimatisation,timingofsamplesandthestrengthofInformation on thestimuliInthecase ofusing extracts,theextractioninvivo,exvivo or in situ.m etabolite com partn entation isalsoreadilyretrievablemethods(completeness of extraction)have to beNMRm ethods [16 ~49]diffusion-editedoptimisedsothatmaximum metabonomic informationusingFurtherm ore, with such a small volumeof biofluidsis available.To acquire data for the metaboliterequired,thetime course ofm etabonom ic changes canone can use chromatography,complementmassalso be m onitored w ith m inim um orno invasion to thespectroscopytoobtain digitalspectrometryNMRsubjects [5] Such studies can also be caried outinfomationthemetabonomecompositionandondirectly on w hole an im alsbo]stimulitheexertedThe choice ofresponsesdetectionFormammalianmodels,themostcommonlyusedmethodshastobeappropriatefortheThebiofluids arebloodplasm a and urine.Blood plasmaofstudies.dataanalysisandpurposescarries the snap shot infomationaboutthegiveninterpretationthe most crucial step,in whichISphysiologicalandpathologicalprocessesandhowwellappropriate jistifications and validations have to be"supervised”the hom eostasis is m aintained;urine sam ples, on thecarefully considered especially whenotherhand,contains information aboutthemetabolitesdata analysis methods are employed.Bjologicalexcreted in aperiod of time,which mayreflectthem eaningfulnessistheultimateaim form etabonomicsstates of physiolbgy, biological age, possibility ofstudieswhich can be clearlyillustrated inthefollowingdiseasesincludingtheinbomandenorsapplications.Tissuesenvironmentallyinducedpathology.3Applicationsofmetabonomicsmetabonome provides a unique opportunity forM etabolite analysis is im portant because anydetecting m olecularinform ation of an organ where theperturbationtoalivingsystem,whetherit isbiochem ical processes occur, hence the underlyingphysiological or pathological in nature,will causemechanism of itsbiochemicalresponsetostimulicanperturbations of concentration orflux of endogenousbe understood.For some specific purposes,other21994-2014 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 technique will make the “low sensitivity” nature of NMR rapidly become a history. It is clear that the best choice at present may lie at the combination of a number of existing techniques. The hyphenation of chromatography, MS and NMR (LC-NMR-MS) played an important role already in the metabolite identifications. It is particularly exciting to note that a statistical heterospectroscopy method has been reported[44] which takes advantages of both NMR and UPLC-MS. It is predictable that such correlation techniques will become increasingly important in the future. 2.2 Samples for metabonomics studies In theory, any biological samples including biofluids, tissues and even whole organisms can be used for metabonomics studies. In practice, however, the sample requirements are dependent on the purpose of studies and methods used. For both chromatography and MS based methods, the samples have to be in the liquid form and often require removal of proteins or extractions. For NMR-based methods, on the other hand, both biofluids (e.g. urine and blood plasma) and tissues (e.g., liver, kidney and brain biopsies) are generally employed directly without much sample preparation or extractions, so that in vivo and in situ information is readily available. In such cases, only as little as 2 !l biofluids[45] and about 10 mg tissues are required to obtain information in the manners of either in vivo, ex vivo or in situ. Information on the metabolite compartmentation is also readily retrievable using diffusion-edited NMR methods[46～49] . Furthermore, with such a small volume of biofluids required, the time course of metabonomic changes can also be monitored with minimum or no invasion to the subjects [45] . Such studies can also be carried out directly on whole animals[50] . For mammalian models, the most commonly used biofluids are blood plasma and urine. Blood plasma carries the snap shot information about the given physiological and pathological processes and how well the homeostasis is maintained; urine samples, on the other hand, contains information about the metabolites excreted in a period of time, which may reflect the states of physiology, biological age, possibility of diseases including the inborn errors and environmentally induced pathology. Tissues" metabonome provides a unique opportunity for detecting molecular information of an organ where the biochemical processes occur, hence the underlying mechanism of its biochemical response to stimuli can be understood. For some specific purposes, other special biofluids such as CSF, milk and dialysates can also be important. By comparing with the metabonome of the control samples, the effects of internal and external stimuli on the subjects can be easily analysed in a holistic and quantitative way using statistical tools. For plant systems, most currently used methods are based on solvent extracts thus are in vitro by nature. This is owing to that the plant “biofluids” are not as readily available as in the case of animals. However, tissues and sometimes whole organism studies are possible by employing high resolution magic-angle spinning (HRMAS) NMR. This method can also be applied to microbiota and cultured cells, in which both cells and media are important carriers for metabonomic information. The analysis of media is also important and often referred to as cell metabolism “foot-printings”. 2.3 How to do metabonomics studies? There are three general steps for conducting metabonomics studies, namely, sample preparation, data acquisition and data mining in order to extract the biological significances. However, for such a complex subject, extra care has to be taken in each step. For example, the sample collections and preparation step is fundamentally important. Many factors affecting metabonomes have to be carefully thought through such as variations in species, strains, gender, age, diet, acclimatisation, timing of samples and the strength of stimuli. In the case of using extracts, the extraction methods (completeness of extraction) have to be optimised so that maximum metabonomic information is available. To acquire data for the metabolite complement, one can use chromatography, mass spectrometry or NMR spectroscopy to obtain digital information on the metabonome composition and responses to the exerted stimuli. The choice of detection methods has to be appropriate for the purposes of studies. The data analysis and interpretation is the most crucial step, in which appropriate justifications and validations have to be carefully considered especially when “supervised” data analysis methods are employed. Biological meaningfulness is the ultimate aim for metabonomics studies which can be clearly illustrated in the following applications. 3 Applications of metabonomics Metabolite analysis is important because any perturbation to a living system, whether it is physiological or pathological in nature, will cause perturbations of concentration or flux of endogenous · 407 ·

·408生物化学与生物物理进展2006;336)Prog. Biochem . Biophys.metabolites.M ost of metabolismCOMET project were to evaluate the utilities ofpathways aretofunctioning notin isolated ways but in an interactivemetabonomicscomprehensively,discovermannerwith some othermetabolismprocessesthusbiom arkers related to xenobiotic toxicity and tochangesof multiplemetabolitesestablishthe machineleamingcanoccur.expertsystemslim itedMeasurements of a single or only a fewempoweredwith abilities of predictingthe organlim itedspecific toxicity to,A lthough it was aim ed mainlymetabolitesnaturallyhavevaluesMetabonomics,bydefault,measuresthemetabolictowardssomerenaltoxins and hepatotoxins,thechanges in a holistic fashion,therefore,has becom eaproject, in fact covered m uch m ore extensive factors,successfilresearch technologyand foundwidespreadincluding physiological stresses, pancreas, testicular,applications in molecular phenotyping,functionalbladderandmultipleorgantargets,representingthegenomics,drug toxicity/efficacy,m olecularpathologym ost comprehensiveevaluation of thebiochem istry ofxenobiotic toxicity everbo. The second phase of thisand disease diagnosis/prognosis,and environmentalsciences.projectwill beextendedintomoredetailed3.1Toxicologymechanistic studies to advance ourunderstandings ofNMRhavebeenappliedtechniques10molecular mechanisms ofthetoxicologyChttp:toxicological studies since 1980s and there are vast/bc-cometsk.med.ic.ac.uk/.Basedsuchonam ount of data reported already covering both drugunderstandings, it w ill be probable to establish som etoxicity Bu75 and environm ental effectst2~57. The earlyquantitative,universal,integrated and predictiveworkwas mostly perfomed by using H NMR to(QU P)expert system s, and to providea pow erfultoolanalyse the m am m alian urine and plasm a k58.50] sam ples.forfuturedrugdiscoveryandenvironmentalMorerecently,H high resolution magic angletox ico logy research.SpinningHRMAS)NMRmethods havebeenM etabonom ics has also been applied to studythedeveloped to enable in situ, ex vivo and even in vivoside effects of clinically em ployed drugsThis isstudies,whichhavepotentialtoprovidem olecularandparticularly imn portant in the case of cancertreatm entscompartmentationinfomationcorrelatingwithwhere most chem otherapeutic drugs have toxic sidehistopathology results 60 -2]. Such studies have noweffects,resultinginnephrotoxicity,hepatotoxicityandbeen extended to non-drug toxicity fields In theheavyin som e cases neurotoxicity.Ifosfam ide and cisplatin,forexample,arewidelyusedtotreatcancersinbothmetaltoxicity studies,forexample,high resolitionNMRspectroscopyofurine,bloodplasma andHchildren and adults, both drugs induce nephrotoxicityM etabonom ics studies of tam oxifen t172, a drug w idelyHRMAS NMR of tissueshavebeen analysed to assesstherenal toxicityfCd(I1),HgC1364,a Na)67)used for treatm ent of the estrogen-dependentbreastand Ce NO 3),] to discover the relevant biom arkerscancer, has shown thatthis drug caused theelevationand underlying m echanism s of toxicity.In a H NM Rof lipid and glucose levels in rat livers accompaniedstudy,for example,the biochemical effects ofwith the decreaseof glycine and serinelevelsevenLa No.),metabonomehavebeenthough itis uncertainwhether sucheffects arerelatedonratinvestigated B5-67 by analysing their urine, plasm a andto livertum ourprom otion ornottz,Such drug toxicitythoughunavoidable atpresentcauses considerabletissuesamples,revealingtheobservableliverandrenaldam agesupon bng term oral adm inistration.The m ostproblem s for the patient treatm ent and NMRbasedcomprehensivemetabonomic toxicitystudies,so far,metabonomicswill havesomeimportantrolestoplayhave been caried out within thein discovering the early side effects and underlyingframeoftheConsortiumforMetabonomicToxicologyandm echanism s.(CoMET)project870)(htp:/bc-cometsk.med.ic.ac.3.2Genefunctionsuk/,which was fom ed by Im perial College LondonThefiunctionsofgenesare oftenmanifested intheand six high profile intemational pharmaceuticalregulation (m RNA) and expression levels (proteins).companies,namely,BristoMyersSquibb,EliLilly&This certainly results in changes at them etabolic levelCo,HofmannLaRoche,NovoNordisk,PfizerInc.asthemetabolitesarenearend-products of therelevantandThePharmaciaCorporation(nowpartofPfizer)biological processes.AsaholisticmetaboliteWithin the frame of this projectNMRbasedm easurem entapproach,therefore,m etabonom ics is thetool of choice to be em ployed to study the genemetabonom icsapproachhas been employed to canyoutstudiesonabout15oknowntoxins,involvingtensfunctions,which is exemplifiedin anumber offunctional genom ics studies. G c M s has been used lo.73]of thousandsanimalmodels.Theobectivesof the21994-2014 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) metabolites. Most of metabolism pathways are functioning not in isolated ways but in an interactive manner with some other metabolism processes thus changes of multiple metabolites can occur. Measurements of a single or only a few limited metabolites naturally have limited values. Metabonomics, by default, measures the metabolic changes in a holistic fashion, therefore, has become a successful research technology and found widespread applications in molecular phenotyping, functional genomics, drug toxicity/efficacy, molecular pathology and disease diagnosis/prognosis, and environmental sciences. 3.1 Toxicology NMR techniques have been applied to toxicological studies since 1980s and there are vast amount of data reported already covering both drug toxicity[9,17,51] and environmental effects[52～57] . The early work was mostly performed by using 1 H NMR to analyse the mammalian urine and plasma[58,59] samples. More recently, 1 H high resolution magic angle spinning (HRMAS) NMR methods have been developed to enable in situ, ex vivo and even in vivo studies, which have potential to provide molecular and compartmentation information correlating with histopathology results [60 ～62] . Such studies have now been extended to non-drug toxicity fields. In the heavy metal toxicity studies, for example, high resolution NMR spectroscopy of urine, blood plasma and 1 H HRMAS NMR of tissues have been analysed to assess the renal toxicity of Cd(Ⅱ) [54] , HgCl2 [63,64] , La(NO3)3 [65～67] and Ce (NO3)3 [68] to discover the relevant biomarkers and underlying mechanisms of toxicity. In a 1 H NMR study, for example, the biochemical effects of La (NO3)3 on rat metabonome have been investigated[65～67] by analysing their urine, plasma and tissue samples, revealing the observable liver and renal damages upon long term oral administration. The most comprehensive metabonomic toxicity studies, so far, have been carried out within the frame of the Consortium for Metabonomic and Toxicology (COMET) project [69,70] (http://bc-comet.sk.med.ic.ac. uk/), which was formed by Imperial College London and six high profile international pharmaceutical companies, namely, Bristol-Myers-Squibb, Eli Lilly & Co, Hoffmann-La Roche, Novo Nordisk, Pfizer Inc. and The Pharmacia Corporation (now part of Pfizer). Within the frame of this project, NMR-based metabonomics approach has been employed to carry out studies on about 150 known toxins, involving tens of thousands animal models. The objectives of the COMET project were to evaluate the utilities of metabonomics comprehensively, to discover biomarkers related to xenobiotic toxicity and to establish the machine learning expert systems empowered with abilities of predicting the organ specific toxicity [70] . Although it was aimed mainly towards some renal toxins and hepatotoxins, the project, in fact, covered much more extensive factors, including physiological stresses, pancreas, testicular, bladder and multiple organ targets, representing the most comprehensive evaluation of the biochemistry of xenobiotic toxicity ever [70] . The second phase of this project will be extended into more detailed mechanistic studies to advance our understandings of molecular mechanisms of the toxicology (http: //bc-comet.sk.med.ic.ac.uk/). Based on such understandings, it will be probable to establish some quantitative, universal, integrated and predictive (QUIP) expert systems, and to provide a powerful tool for future drug discovery and environmental toxicology research. Metabonomics has also been applied to study the side effects of clinically employed drugs. This is particularly important in the case of cancer treatments where most chemotherapeutic drugs have toxic side effects, resulting in nephrotoxicity, hepatotoxicity and in some cases neurotoxicity. Ifosfamide and cis-platin, for example, are widely used to treat cancers in both children and adults, both drugs induce nephrotoxicity. Metabonomics studies of tamoxifen[71,72] , a drug widely used for treatment of the estrogen-dependent breast cancer, has shown that this drug caused the elevation of lipid and glucose levels in rat livers accompanied with the decrease of glycine and serine levels even though it is uncertain whether such effects are related to liver tumour promotion or not [72] . Such drug toxicity though unavoidable at present causes considerable problems for the patient treatment and NMR-based metabonomics will have some important roles to play in discovering the early side effects and underlying mechanisms. 3.2 Gene functions The functions of genes are often manifested in the regulation (mRNA) and expression levels (proteins). This certainly results in changes at the metabolic level as the metabolites are near end-products of the relevant biological processes. As a holistic metabolite measurement approach, therefore, metabonomics is the tool of choice to be employed to study the gene functions, which is exemplified in a number of functional genomics studies. GC/MS has been used[10,73] · 408 ·

唐惠儒等：代谢组学：一个迅速发展的新兴学科2006;336)·409.than300compoundsfiomleaves of Cinchona officinalis and L-dopa fromtoquantifymoreArabidopsis thaliana leaf extractsthough halfofthemseedlingof Viciafaba beans for treatment ofwerenotassigned structurally.Bycomparison,fourParkinson's disease.Morerecently,paclitaxelortaxolgenotypes were characterisedbased on metabolicwasisolated from pacificyew tree (Taxus brevifolia)features,providing a possible tool for gene functionand found applications in treating a broad range ofstudiesIn anothercase,a functional analysis bycancers, artem isinin, a sesquiterpene lactone, has beenco-responses in yeast (FA NCY)has been em ployed 6)isolated fiom the plant Artem isia annua L (sweetto search for the gene functions of Saccharomyceswormwood)and ittogether with itsderivativeshavemeasuringthemutation-inducedbeen rapidly developed into a unique family ofcerevisiaebymetabonom icchanges.FTIRandMShavealsobeenantim alariadrugs.In fact, the ideaofusing Artemisiaem ployedthem etabolitesbacterial(qinghaoor青嵩inChinese）totreatmalariaanalyseannuaLmediumfootprints)studythemetabonomicand haemonhoids ismore than 150 years old inmutation4]ics methodstraditional Chinese medicines.Some antibiotics canchangesMetabonomhave beenusedn the functionalgets studies ofalso beconsidered as natural medicines such asicro-organism s09]and an im als98plantsso-calledmiracle drug”penicillin.Therefore,mosofwhichhavebeencomprehensivereviewednatural medicinesrepresentsomeimportantsourcesalready.fornewdrugdiscoveryanddevelopmentAmongstmanyfunctional genom icsstudies,theBiologicalIn China,AtlasforInsulinResistancethetaditionalChinesemedicinesBAR)(TCM)has beenpracticed forthousands of years andproectfunded by the W ellcome Trust inUKand startedin 20022isworthmentioning.Thisthe w ritten docum ents show ed sophisticated theories18research60for TCMeveninitsearly forms although theseproject consists ofgroupsandcontributors w ith expertise in insulin signalling, rodenttheories were established on philosophical basis andgene targeting,human genetics,metabonomics,clinicalexperiencesratherthan scienceperhaps owingtranscriptomics,bioinformatics,andto the factthat it had been developed well beforeproteomics,stucturalbiology(http:/www.bairorg.uk).Themainmodem sciencewas bom.Like almostall otherobectiveofthisproectistogenerateacomprehensivetraditionalmedicines,thepractice of TCM suffers,atdescription of welldefined states of insulin resistancepresent from insufficientm odem scientific research.Forexample,manyaspectsofTcM arestillpracticinginrodentmodelsItis conceivablethatsimilarstudieswillsoonbecomemuchmorewidespreadgiventhein its originalform and although ithasbeen effectiveam ountofattentionsthatsystem sbiolbogyhasreceived.in treating m anyconditions especially chronic ones,it3.3Phytomedicines and traditional Chineselacksnecessarywelldefined molecularmechanismm edicinesand sometimesevenmolecularbasis.Forinstance,inN atural m edicines fiom plants (phytom edicines)TCM,the conceptof“m ain and collateral channels”have been used for thousand years throughout thehas notfirmly backedbyanatom icalbeenupworld and havemade tremendous contributions toevidences.PrescribedmedicinesinTCMarenomallyhum an health care.Inrecentcenturies,m anyimportantm ixturesnumberofplantsandtheirtrodrugs were developed from the natural m edicines ordefined.thecompositionwellfactnot15stemmedfromherbalremedies.For example,thecomposition ofmostof thesingle herbitselfisnotfullycharacterized,cardiac dnug,digoxin,wasoriginallyextractedfiromthus,thereinsufficient10the leaves ofD igitalis lanata alm ost two hundred yearsinfomationonthemodeofactionnmolecularlevels“digitalisgroup of similar drugs calledfor singleherbsandmixtures.From reproducibilityago.Aglycosides'with in common specific effectsonpointof view,a number of species are often nam edmyocardium have been subsequently isolated fiom aunder the same medicinal herb and they grow atnumber ofplantsOneof theworld's oldestandmostdifferent locationswithdifferentclim ate andharvestedcommon drugs,acetylsalicylic acid also known asatdifferenttime.Therawmaterialsformedicinesareaspirin, used as a non-steroidal anti-inflam m atoryoften inconsistentinchemicalcomposition.Topreparedrug,wasalso derived fiom theancient ideaofusingm ixedmedicinesused forclinical use, each m edicinalmaterial is often required to be processed in certainwillowbarksaspainrelief medicine.OtherwelHknown examples in this category inchide theways, which is often not subjected to m odem qualitypain-management drug,morphine,fiom Papavercontol management either.Consequently,thesomniferum, antimalaria drug, quinine, fiom theinconsistence of raw materials and processing is21994-2014 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 to quantify more than 300 compounds from Arabidopsis thaliana leaf extracts though half of them were not assigned structurally. By comparison, four genotypes were characterised based on metabolic features, providing a possible tool for gene function studies. In another case, a functional analysis by co-responses in yeast (FANCY) has been employed[16] to search for the gene functions of Saccharomyces cerevisiae by measuring the mutation-induced metabonomic changes. FTIR and MS have also been employed to analyse the metabolites in bacterial medium (or footprints) to study the metabonomic changes upon mutation [74] . Metabonomics methods have been used in the functional genomics studies of plants[75～78] , micro-organisms[79] and animals[80,81] , most of which have been comprehensively reviewed already. Amongst many functional genomics studies, the “ Biological Atlas for Insulin Resistance (BAIR)” project funded by the Wellcome Trust in UK and started in 2002 is worth mentioning. This project consists of 18 research groups and 60 contributors with expertise in insulin signalling, rodent gene targeting, human genetics, metabonomics, proteomics, transcriptomics, bioinformatics, and structural biology (http://www.bair.org.uk). The main objective of this project is to generate a comprehensive description of well-defined states of insulin resistance in rodent models. It is conceivable that similar studies will soon become much more widespread given the amount of attentions that systems biology has received. 3.3 Phytomedicines and tr aditional Chinese medicines Natural medicines from plants (phytomedicines) have been used for thousand years throughout the world and have made tremendous contributions to human health care. In recent centuries, many important drugs were developed from the natural medicines or stemmed from herbal remedies. For example, the cardiac drug, digoxin, was originally extracted from the leaves of Digitalis lanata almost two hundred years ago. A group of similar drugs called “ digitalis glycosides” with in common specific effects on myocardium have been subsequently isolated from a number of plants. One of the world's oldest and most common drugs, acetylsalicylic acid also known as aspirin, used as a non-steroidal anti-inflammatory drug, was also derived from the ancient idea of using willow barks as pain-relief medicine. Other well-known examples in this category include the pain-management drug, morphine, from Papaver somniferum, anti-malaria drug, quinine, from the leaves of Cinchona officinalis and L-dopa from seedling of Vicia faba beans for treatment of Parkinson!s disease. More recently, paclitaxel or taxol was isolated from pacific yew tree (Taxus brevifolia) and found applications in treating a broad range of cancers, artemisinin, a sesquiterpene lactone, has been isolated from the plant Artemisia annua L (sweet wormwood) and it together with its derivatives have been rapidly developed into a unique family of anti-malaria drugs. In fact, the idea of using Artemisia annua L (qinghao or 青蒿 in Chinese) to treat malaria and haemorrhoids is more than 150 years old in traditional Chinese medicines. Some antibiotics can also be considered as natural medicines such as so-called “ miracle drug” penicillin. Therefore, natural medicines represent some important sources for new drug discovery and development. In China, the traditional Chinese medicines (TCM) has been practiced for thousands of years and the written documents showed sophisticated theories for TCM even in its early forms although these theories were established on philosophical basis and clinical experiences rather than science perhaps owing to the fact that it had been developed well before modern science was born. Like almost all other traditional medicines, the practice of TCM suffers, at present, from insufficient modern scientific research. For example, many aspects of TCM are still practicing in its original form and although it has been effective in treating many conditions especially chronic ones, it lacks necessary well-defined molecular mechanism and sometimes even molecular basis. For instance, in TCM, the concept of “main and collateral channels” has not been firmly backed up by anatomical evidences. Prescribed medicines in TCM are normally mixtures from a number of plants and their composition is not well defined. In fact, the composition of most of the single herb itself is not fully characterized, thus, there is insufficient information on the mode of action in molecular levels for single herbs and mixtures. From reproducibility point of view, a number of species are often named under the same medicinal herb and they grow at different locations with different climate and harvested at different time. The raw materials for medicines are often inconsistent in chemical composition. To prepare mixed medicines used for clinical use, each medicinal material is often required to be processed in certain ways, which is often not subjected to modern quality control management either. Consequently, the inconsistence of raw materials and processing is · 409 ·

·410·生物化学与生物物理进展2006:33 6)Prog. Biochem . Biophys.transferred and sometimes manifestedintothethe majpr differences in Ephedra sinica,E. intermediaresultantclinicallyusedmedicines.Theprescriptionsand E.distachya var.distachya were found due toareoftenliabletomanycomplicationsandlackbenzoic acidanalogs in the aqueous fraction andquantitative description.Therefore,there is clearephedrine-type alkaloids intheorganic firaction.Basedurgency for scientific research in term s of qualityon this metabonomic recognition,oneof ninecontrolclinicalefficacy,molecularmechanismforthecomm ercialEphedra m aterialsevaluatedwas shown tobe a m ixture ofEphedra species 2. Sim ilar techn iquestraditional medicines and patient responses to aapplied to 12 Cannabis sativa cultivars also show ed Bajtreatment in order tomodemisethetraditionalmedicines,which undoutedly demands significantpowerfulness in discrin inatingthosesamples,D9-tetrahydrocannabinolicamountofanalyticalpowerandefforts.revealingthatacidThere have been m any recent attem pts to address(THCA)(CBDA)andcannabidiolicacidwerethese issues butmostof them were still based on theimportant metabolites to differentiate the cultivarsreductionism”philosophy.Theseapproachesfrom eachother.Moreover,thediscriminationoftheeffectivelyfollowed theroutesofthechemicaldrugcultivars couldbeobtainedfiomawaterextractbaseddiscovery,consistingofisolation,activecomponentontheconcentrationofcarbohydratesandamino acidsdiscoveryand chemicalmodificationsTheseattemptsSucrose, glucose,asparagine, andglutam ic acid werethe mapr discrininating metabolitesSuchhave,sofar,madesomeprogress,forexample,indiscoveringtheanti-malariadrug,artemisinin.chemotaxonomicanalvsiswasalsoexemplifiednaHowever,such approaches have fairly lim itedstudy of11 lex species, showing cleardiscrin inationthroughput and appeared inefficient w hen com paredof thosesamples based on themetabolites present inthe organic and aqueous extracts B The maprwith thevastamountof traditional medicines to beinvestigated.Thisapproachwill also notbeabletodiscrim ination metabolites include arbutin, caffeine,effectively address such guestionsas whethersomephenylpropanoids,and theobrom ine,amongstwhichsynergistic effects ofm ixture are im portant M oreover,arbutin was notpreviouslyreported in Ilex species butwas found to be a biom arker for I argentina,the existing experiences of the TCMwerenotfullySince TCMtaken into consideration.is based onbrasiliensis,Ibrevicuspis,Iintegerima,“holism"philosophyinstead of“reductionism"I microdonta,Ipseudobuxus,Itaubertiana,andas“omics"in the case oftheories,itis conceivabletheezansThe metabonomic analysis for seeds,philosophicallythat themetabonomicstechniquesleaves,stembark,collarbarkandrootbarkofthreeStrychnos species , S. nuxvom ica, S. ignati, andought to playsome roles in providing molecularinform ationtotheTCM in theintegratedm anner.S.icaj, showed a good discrim ination between allForauthentication andqualitycontolmostofthekeycompoundsresponsible for thesam ples:herb/haturalmedicines weretraditionallyassessed bydiscrim ination inchude brucine,loganin,fatty acids,practioner'sexperience and,atbest bymeasuring aand Strychnos icaa alkaloids such as icajine andsungucine. The m ethod w as also applied s] to classifyfewknowncompounds(as"markers"inthemixtureusingHPLC/TLCmethods.However,suchseveral"falseangostura”samples,revealing,asassessmentsoftenfailto sufficientlyaddresswhetherexpected,theiridentities as S.nuxvom ica.thesemeasuredcomponentsrelevantMultiplecomponent analysisbasedontheareTofthequalitybioactivitycombination of high resolution NMRspectoscopyterm scontrolor(e.g.,effects).M etabonom icw ith pattem recognition (ie., com m on m etabonom icstreatn ent/adversecharacteristics ought to be the good“standard”approach) has been em plbyed to investigate batches offorgood m anufacturing practice purpose regard less w hich14 commercially availablefeverfewsamplesTheForthispurpose,resultsshowed]thatamongstthem，twobatchesarecomponents areactive.somecommon herbs have alreadybeensubectedsignificantly differentin chemical com position fiom:tom etabonom ics studies as the proof of concept Thethe others, the rem aining twelve batches can also beNM R-basedmetabonomicanalysiswasshownclassified into discrete groupsby principal com ponenteffectiveforchemotaxonomicanalysisandanalysison thebasisofminordifferencesin overallauthentication puposes in a study of three Ephedrachemical composition.The powerfulness of suchspecies and some com m ercial m aterials 2] A broadmethods is also highlighted in quality control ofrange of metabolites in the Ephedra extracts werephytom edicines.In anotherstudy,chamomileidentified w ithout any chrom atographic separation andMatricaria recutita)flowers were chosen as an21994-2014 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) transferred and sometimes manifested into the resultant clinically used medicines. The prescriptions are often liable to many complications and lack quantitative description. Therefore, there is clear urgency for scientific research in terms of quality control, clinical efficacy, molecular mechanism for the traditional medicines and patient responses to a treatment in order to modernise the traditional medicines, which undoutedly demands significant amount of analytical power and efforts. There have been many recent attempts to address these issues but most of them were still based on the “ reductionism” philosophy. These approaches effectively followed the routes of the chemical drug discovery, consisting of isolation, active component discovery and chemical modifications. These attempts have, so far, made some progress, for example, in discovering the anti-malaria drug, artemisinin. However, such approaches have fairly limited throughput and appeared inefficient when compared with the vast amount of traditional medicines to be investigated. This approach will also not be able to effectively address such questions as whether some synergistic effects of mixture are important. Moreover, the existing experiences of the TCM were not fully taken into consideration. Since TCM is based on “holism” philosophy instead of “reductionism” as in the case of “omics” theories, it is conceivable philosophically that the metabonomics techniques ought to play some roles in providing molecular information to the TCM in the integrated manner. For authentication and quality control, most of herb/natural medicines were traditionally assessed by practioner!s experience and, at best, by measuring a few known compounds (as “ markers” ) in the mixture using HPLC/TLC methods. However, such assessments often fail to sufficiently address whether these measured components are relevant in terms of the quality control or bioactivity (e.g., treatment/adverse effects). Metabonomic characteristics ought to be the good “standard” for good manufacturing practice purpose regardless which components are active. For this purpose, some common herbs have already been subjected to metabonomics studies as the proof of concept. The NMR-based metabonomic analysis was shown effective for chemotaxonomic analysis and authentication purposes in a study of three Ephedra species and some commercial materials [82] . A broad range of metabolites in the Ephedra extracts were identified without any chromatographic separation and the major differences in Ephedra sinica, E. intermedia and E. distachya var. distachya were found due to benzoic acid analogs in the aqueous fraction and ephedrine-type alkaloids in the organic fraction. Based on this metabonomic recognition, one of nine commercial Ephedra materials evaluated was shown to be a mixture of Ephedra species[82] . Similar techniques applied to 12 Cannabis sativa cultivars also showed[83] powerfulness in discriminating those samples, revealing that D9-tetrahydrocannabinolic acid (THCA) and cannabidiolic acid (CBDA) were important metabolites to differentiate the cultivars from each other. Moreover, the discrimination of the cultivars could be obtained from a water extract based on the concentration of carbohydrates and amino acids. Sucrose, glucose, asparagine, and glutamic acid were the major discriminating metabolites [83] . Such chemotaxonomic analysis was also exemplified in a study of 11 Ilex species, showing clear discrimination of those samples based on the metabolites present in the organic and aqueous extracts [84] . The major discrimination metabolites include arbutin, caffeine, phenylpropanoids, and theobromine, amongst which arbutin was not previously reported in Ilex species but was found [84] to be a biomarker for I. argentina, I. brasiliensis, I. brevicuspis, I. integerrima, I. microdonta, I. pseudobuxus, I. taubertiana, and I. theezans. The metabonomic analysis for seeds, leaves, stem bark, collar bark and root bark of three Strychnos species[85] , S. nux-vomica, S. ignatii, and S. icaja, showed a good discrimination between all samples; the key compounds responsible for the discrimination include brucine, loganin, fatty acids, and Strychnos icaja alkaloids such as icajine and sungucine. The method was also applied[85] to classify several “false angostura” samples, revealing, as expected, their identities as S. nux-vomica. Multiple component analysis based on the combination of high resolution NMR spectroscopy with pattern recognition (i.e., common metabonomics approach) has been employed to investigate batches of 14 commercially available feverfew samples. The results showed[86] that amongst them, two batches are significantly different in chemical composition from the others, the remaining twelve batches can also be classified into discrete groups by principal component analysis on the basis of minor differences in overall chemical composition. The powerfulness of such methods is also highlighted in quality control of phytomedicines. In another study, chamomile (Matricaria recutita) flowers were chosen as an · 410 ·