**Targeted Metabolomics for Clinical Biomarker Discovery in Multifactorial Diseases**

Ulrika Lundin1, Robert Modre-Osprian2 and Klaus M. Weinberger1 *1Biocrates Life Sciences AG, Innsbruck 2AIT Austrian Institute of Technology GmbH, Graz Austria* 

#### **1. Introduction**

80 Advances in the Study of Genetic Disorders

Perez-Iratxeta, C., Bork, P., and Andrade, M. A. (2002). Association of genes to genetically

Perez-Iratxeta, C., Wjst, M., Bork, P., and Andrade, M. A. (2005). G2D: a tool for mining genes associated with disease. *BMC Genet*, Vol.6, pp. 45. ISSN 1471-2156 Perez-Iratxeta, C., Bork, P., and Andrade-Navarro, M. A. (2007). Update of the G2D tool for

Rother, K. I. (2007). Diabetes treatment--bridging the divide. *N Engl J Med*, Vol.356, No.15,

Shatkay, H. (2005). Hairpins in bookstacks: information retrieval from biomedical text. *Brief* 

Shendure, J., and Ji, H. (2008). Next-generation DNA sequencing. *Nat Biotechnol*, Vol.26,

Tamayo, P., Slonim, D., Mesirov, J., Zhu, Q., Kitareewan, S., Dmitrovsky, E., Lander, E. S.,

van Driel, M. A., Bruggeman, J., Vriend, G., Brunner, H. G., and Leunissen, J. A. (2006). A

Zeeberg, B. R., Feng, W., Wang, G., Wang, M. D., Fojo, A. T., Sunshine, M., Narasimhan, S.,

and proteomic data. *Genome Biol*, Vol.4, No.4, pp. R28. ISSN 1465-6914 Zeeberg, B. R., Qin, H., Narasimhan, S., Sunshine, M., Cao, H., Kane, D. W., Reimers, M.,

*Natl Acad Sci U S A*, Vol.96, No.6, pp. 2907-2912. ISSN 0027-8424

and Golub, T. R. (1999). Interpreting patterns of gene expression with selforganizing maps: methods and application to hematopoietic differentiation. *Proc* 

text-mining analysis of the human phenome. *Eur J Hum Genet*, Vol.14, No.5, pp.

Kane, D. W., Reinhold, W. C., Lababidi, S., Bussey, K. J., Riss, J., Barrett, J. C., and Weinstein, J. N. (2003). GoMiner: a resource for biological interpretation of genomic

Stephens, R. M., Bryant, D., Burt, S. K., Elnekave, E., Hari, D. M., Wynn, T. A., Cunningham-Rundles, C., Stewart, D. M., Nelson, D., and Weinstein, J. N. (2005). High-Throughput GoMiner, an 'industrial-strength' integrative gene ontology tool for interpretation of multiple-microarray experiments, with application to studies of Common Variable Immune Deficiency (CVID). *BMC Bioinformatics*, Vol.6, pp.

Web Server issue, pp. W212-216. ISSN 1362-4962

*Bioinform*, Vol.6, No.3, pp. 222-238. ISSN 1467-5463

pp. 1499-1501. ISSN 1533-4406

535-542. ISSN 1476-5438

168. ISSN 1471-2105

No.10, pp. 1135-1145. ISSN 1546-1696

4962

1061-4036

of microarray and high-throughput sequencing-based functional genomics experiments. *Nucleic Acids Res*, Vol.39, Database issue, pp. D1002-1004. ISSN 1362-

inherited diseases using data mining. *Nat Genet*, Vol.31, No.3, pp. 316-319. ISSN

prioritization of gene candidates to inherited diseases. *Nucleic Acids Res*, Vol.35,

The vast majority of this book deals with monogenic disorders which are relatively rare but have just one or a small number of characteristic genotypes and usually very pronounced clinical and biochemical phenotypes. In contrast, this chapter will try to discuss multifactorial diseases which are far more prevalent and pose a completely different kind of challenge both for the socio-economic systems and for biomedical research. As an example we will focus on chronic kidney disease (CKD) and relevant animal models thereof. In fact, together with diabetic retinopathy, myocardial infarction, and stroke, diabetic nephropathy is one of the most severe sequelae of type II diabetes mellitus (T2D) and, considering the obesity-related pandemic of T2D, will represent a major health issue in the decades to come (Mensah et al., 2004; James et al., 2010).

Of course, all of these diseases have an important genetic component as demonstrated by pedigree analyses and a growing number of twin studies (Walder et al., 2003; Vaag & Poulsen, 2007). Still, with rare exceptions, this genetic component is rather seen as a predisposition for than as a cause of the actual disease. In particular, recent genome-wide association studies (GWAS) on large population-based cohorts have revealed a couple of single nucleotide polymorphisms (SNPs) that are significantly associated with T2D but the contribution of single SNPs to the individual's risk of developing T2D are marginal (Groop & Lyssenko, 2009). To fully understand the interaction of the identified genetic loci and to appreciate the meaning of the genetic background in a personalized medicine approach, complex haplotypes would have to be analyzed, and this has not even been achieved in basic diabetes research, let alone in any clinical application.

Yet, genetic research in diabetology has gained a new momentum in the last few years since it became obvious that a combination of GWAS with a more detailed phenotyping than just a generic diagnosis of T2D immediately led to improved statistics and to a much better biochemical plausibility of the findings (Gieger et al., 2008; Illig et al., 2010). Specifically, genome-wide significances could be achieved on much smaller cohorts than in classical GWAS rendering a more cost-efficient tool in biomedical research. The statistical power could be further improved by defining metabolic phenotypes based on the knowledge of the underlying biochemical pathways, e.g., by using groups of metabolites that are synthesized or degraded by the same enzymes or by calculating ratios of the concentrations of products

Targeted Metabolomics for Clinical Biomarker Discovery in Multifactorial Diseases 83

modern mass-spectrometric techniques produce high-resolution biochemical snapshots showing the functional endpoints of genetic predisposition as well as the sum of all environmental influences, including nutrition, exercise, and medication. This snapshot is an almost real-time image of the physiology—or pathophysiology—of a cell or an entire

Mass spectrometric assays revolutionized the diagnosis of inherited metabolic disorders, a development co-pioneered in the late 1990s by Adelbert Roscher (Röschinger et al., 2003). This and similar pilot projects around the world taught the diagnostics community some crucial lessons. Quantitation of endogenous metabolites using multiple reaction monitoring (MRM) and stable isotope dilution (SID) for absolute quantitation on tandem mass spectrometers combined with advanced data analysis tools fulfills the most strict quality criteria in terms of precision and accuracy without suffering any of the shortcomings of immunoassays, such as cross-reactivities, which makes this technology an ideal platform for clinical chemistry (Unterwurzacher et al., 2008). What is more, the superior sensitivity of triple quadrupole mass spectrometers combined with MRM and SID enabled the detection of metabolites in biologically relevant sample types, such as plasma or serum, whereas the limited sensitivity in the previous NMR-based workflows restricted their use mainly to urine (urine as a sample type is analytically very convenient but the concentration of metabolites in urine is not regulated in the sense of a strict homeostasis as in blood). Furthermore, it has been proven for many disorders that multiparametric biomarkers reduce biological noise in the data by internal normalization as well as improve diagnostic sensitivity and specificity. Subsequently, it led to a marked reduction of healthcare costs

There are two approaches to metabolomics usually called targeted metabolomics and metabolic profiling. While both approaches are complementary, targeted metabolomics, i.e., the identification and quantitation of defined sets of structurally known and biochemically annotated metabolites, takes advantage of our functional understanding of many biochemical pathways. In contrast to protein-protein interactions or regulatory relationships at the transcript level, the fact that so many biochemical pathways have been explored in great detail offers an invaluable source of background information that enables evidencebased interpretation of metabolomics data sets. For the majority of these pathways, substrates and products of enzymatic reactions, reaction mechanisms, equilibra, kinetics and energetic of these reactions, as well as cofactors or compartmentalization have been elucidated. This information renders instant functional interpretation of the data set and, thus, phenotyping of the analyzed cell or organism, a straight forward process (Modre-Osprian et al., 2009; Weinberger & Graber, 2005; Weinberger et al., 2005; Weinberger, 2008). One other major advantage of targeted metabolomics is that it generally provides quantitative information. These quantitative data, the molar concentrations of the metabolites involved in a pathway, facilitate the immediate understanding of any alterations between different biological states and allow for comparison and meta-analysis of several

**3. Technological advances paved the way into clinical applications** 

organism (Weckwerth, 2003).

(Röschinger et al., 2003; Weinberger., 2008).

**4. Targeted metabolomics or metabolic profiling** 

and substrates of enzymatic reactions. This approach clearly reduces the inherent variability of the data set and, thus, offers great potential for robust diagnostic applications. In addition, it turned out that, in contrast to many GWAS results so far, the hits were not predominantly located in intergenic regions. In fact, eight of the nine loci that could be replicated with genome-wide significance in two independent cohorts (KORA and UK Twins) were in or very near a gene encoding for either an enzyme or a transporter. So, for the very first time, a convincing connection between genotype and metabolic phenotype could be demonstrated in a multifactorial disease.

Even so, the field may still be far from a true systems biology view on T2D covering all the different omics levels but the application of GWAS (or next generation sequencing, for that matter) on quantitative metabolic traits clearly paves the way for a better understanding of epidemiology and pathophysiology at the same time, and this approach has already been confirmed in other areas of genetic research (Weikard et al., 2010).

The systematic analysis of these metabolic traits is called metabolomics. Based on spectral methods such as nuclear magnetic resonance (NMR) spectroscopy or mass spectrometry, it is increasingly recognized as the most informative discipline in functional genomics because it is closest to depicting actual biochemical phenotypes, and thus delivers signatures or biomarkers that mirror genetic predisposition and the sum of environmental influences in one data set (Altmaier et al., 2008; Altmaier et al., 2009; Altmaier et al., 2011). In conclusion, metabolomics-based biomarkers are expected to be more predictive and descriptive than their genomic and proteomic counterparts. They may help trigger a paradigm shift from damage-oriented to function-oriented diagnostics, ultimately allowing diseases to be detected at earlier stages and subtyped more accurately contributing to the overarching goal of personalized medicine (Weinberger, 2008; Suhre et al., 2010).

Moreover, one of the most appealing advantages of metabolomics is that the majority of its analytes are not species-specific, i.e. most amino acids, sugars, lipids, etc. are structurally identical in mice, rats, dogs, pigs, monkeys, humans or other mammals, even in many microbes. This makes metabolomics ideally suited as a biomarker platform avoiding the need for redevelopment of the analytical assays for every animal model and for clinical trials.

### **2. The fundamentals of metabolomics**

Metabolomics systematically identifies and quantifies low-molecular weight compounds in biological samples such as body fluids, tissue homogenates or cell culture. Metabolite concentrations allow inferences on the complex interactions between biological processes on a molecular level to be made. As pointed out above, metabolomics is increasingly appreciated as the richest source of information in functional genomics (Nicholson et al., 1999; Weinberger & Graber, 2005). Until recently, systems biology has mainly relied on three other 'omics' technologies, namely genomics, transcriptomics, and proteomics. Important as these areas have been, they fail to provide a real-time phenotype, i.e., a picture of what is actually happening in a dynamic biological system. Recent advances in mass spectrometry have added metabolomics as another powerful and practical tool to the systems biology toolbox (Weckwerth, 2003). The metabolome is the sum of all low molecular weight metabolites in a biological system. By assessing hundreds of metabolites simultaneously,

and substrates of enzymatic reactions. This approach clearly reduces the inherent variability of the data set and, thus, offers great potential for robust diagnostic applications. In addition, it turned out that, in contrast to many GWAS results so far, the hits were not predominantly located in intergenic regions. In fact, eight of the nine loci that could be replicated with genome-wide significance in two independent cohorts (KORA and UK Twins) were in or very near a gene encoding for either an enzyme or a transporter. So, for the very first time, a convincing connection between genotype and metabolic phenotype

Even so, the field may still be far from a true systems biology view on T2D covering all the different omics levels but the application of GWAS (or next generation sequencing, for that matter) on quantitative metabolic traits clearly paves the way for a better understanding of epidemiology and pathophysiology at the same time, and this approach has already been

The systematic analysis of these metabolic traits is called metabolomics. Based on spectral methods such as nuclear magnetic resonance (NMR) spectroscopy or mass spectrometry, it is increasingly recognized as the most informative discipline in functional genomics because it is closest to depicting actual biochemical phenotypes, and thus delivers signatures or biomarkers that mirror genetic predisposition and the sum of environmental influences in one data set (Altmaier et al., 2008; Altmaier et al., 2009; Altmaier et al., 2011). In conclusion, metabolomics-based biomarkers are expected to be more predictive and descriptive than their genomic and proteomic counterparts. They may help trigger a paradigm shift from damage-oriented to function-oriented diagnostics, ultimately allowing diseases to be detected at earlier stages and subtyped more accurately contributing to the overarching goal

Moreover, one of the most appealing advantages of metabolomics is that the majority of its analytes are not species-specific, i.e. most amino acids, sugars, lipids, etc. are structurally identical in mice, rats, dogs, pigs, monkeys, humans or other mammals, even in many microbes. This makes metabolomics ideally suited as a biomarker platform avoiding the need for redevelopment of the analytical assays for every animal model and for clinical

Metabolomics systematically identifies and quantifies low-molecular weight compounds in biological samples such as body fluids, tissue homogenates or cell culture. Metabolite concentrations allow inferences on the complex interactions between biological processes on a molecular level to be made. As pointed out above, metabolomics is increasingly appreciated as the richest source of information in functional genomics (Nicholson et al., 1999; Weinberger & Graber, 2005). Until recently, systems biology has mainly relied on three other 'omics' technologies, namely genomics, transcriptomics, and proteomics. Important as these areas have been, they fail to provide a real-time phenotype, i.e., a picture of what is actually happening in a dynamic biological system. Recent advances in mass spectrometry have added metabolomics as another powerful and practical tool to the systems biology toolbox (Weckwerth, 2003). The metabolome is the sum of all low molecular weight metabolites in a biological system. By assessing hundreds of metabolites simultaneously,

could be demonstrated in a multifactorial disease.

confirmed in other areas of genetic research (Weikard et al., 2010).

of personalized medicine (Weinberger, 2008; Suhre et al., 2010).

**2. The fundamentals of metabolomics** 

trials.

modern mass-spectrometric techniques produce high-resolution biochemical snapshots showing the functional endpoints of genetic predisposition as well as the sum of all environmental influences, including nutrition, exercise, and medication. This snapshot is an almost real-time image of the physiology—or pathophysiology—of a cell or an entire organism (Weckwerth, 2003).

### **3. Technological advances paved the way into clinical applications**

Mass spectrometric assays revolutionized the diagnosis of inherited metabolic disorders, a development co-pioneered in the late 1990s by Adelbert Roscher (Röschinger et al., 2003). This and similar pilot projects around the world taught the diagnostics community some crucial lessons. Quantitation of endogenous metabolites using multiple reaction monitoring (MRM) and stable isotope dilution (SID) for absolute quantitation on tandem mass spectrometers combined with advanced data analysis tools fulfills the most strict quality criteria in terms of precision and accuracy without suffering any of the shortcomings of immunoassays, such as cross-reactivities, which makes this technology an ideal platform for clinical chemistry (Unterwurzacher et al., 2008). What is more, the superior sensitivity of triple quadrupole mass spectrometers combined with MRM and SID enabled the detection of metabolites in biologically relevant sample types, such as plasma or serum, whereas the limited sensitivity in the previous NMR-based workflows restricted their use mainly to urine (urine as a sample type is analytically very convenient but the concentration of metabolites in urine is not regulated in the sense of a strict homeostasis as in blood). Furthermore, it has been proven for many disorders that multiparametric biomarkers reduce biological noise in the data by internal normalization as well as improve diagnostic sensitivity and specificity. Subsequently, it led to a marked reduction of healthcare costs (Röschinger et al., 2003; Weinberger., 2008).

### **4. Targeted metabolomics or metabolic profiling**

There are two approaches to metabolomics usually called targeted metabolomics and metabolic profiling. While both approaches are complementary, targeted metabolomics, i.e., the identification and quantitation of defined sets of structurally known and biochemically annotated metabolites, takes advantage of our functional understanding of many biochemical pathways. In contrast to protein-protein interactions or regulatory relationships at the transcript level, the fact that so many biochemical pathways have been explored in great detail offers an invaluable source of background information that enables evidencebased interpretation of metabolomics data sets. For the majority of these pathways, substrates and products of enzymatic reactions, reaction mechanisms, equilibra, kinetics and energetic of these reactions, as well as cofactors or compartmentalization have been elucidated. This information renders instant functional interpretation of the data set and, thus, phenotyping of the analyzed cell or organism, a straight forward process (Modre-Osprian et al., 2009; Weinberger & Graber, 2005; Weinberger et al., 2005; Weinberger, 2008). One other major advantage of targeted metabolomics is that it generally provides quantitative information. These quantitative data, the molar concentrations of the metabolites involved in a pathway, facilitate the immediate understanding of any alterations between different biological states and allow for comparison and meta-analysis of several

Targeted Metabolomics for Clinical Biomarker Discovery in Multifactorial Diseases 85

These databases also provide the background for various attempts at visualization of metabolic pathways and data mapping on these charts, although most of these projects still follow a static approach of predefined (and predrawn) maps that cannot do justice to the dynamics of biochemical networks. In the following paragraph, we would like to demonstrate a few concepts about how dynamic representation and simulation (Modre-Osprian et al., 2009) of metabolic pathways enable the first steps of generating hypotheses

Firstly, electronic availability of metabolites and metabolic reactions facilitates an almost trivial but nevertheless powerful approach that is analogous to a gene set enrichment analysis (GSEA; Subramanian et al., 2005). Any given set of metabolites which has been identified by statistics as significantly different in two biological states or clinical cohorts can be mapped on the entirety of metabolic pathways, and these pathways can then be ranked by the number of altered metabolites they contain (Fig. 1). This is a way of structuring the data that scientists from transcriptomics and proteomics are familiar with although the definition of metabolic pathways does not follow a similarly strict classification system as the classical gene ontology (GO; Ashburner et al., 2000). Note also that a reliable selection of species-specific enzymatic reactions instead of the generic reference pathways is necessary

> 00564 Glycerophospholipid metabolism 15 00590 Arachidonic acid metabolism 7 00591 Linoleic acid metabolism 7 00592 alpha-Linolenic acid metabolism 7 00600 Sphingolipid metabolism 3 00052 Galactose metabolism 2 00051 Fructose and mannose metabolism 1 00310 Lysine degradation 1 00500 Starch and sucrose metabolism 1 00520 Amino sugar and nucleotide sugar metabolism 1

Fig. 1. Ranking of metabolic pathways according to the number of significant differences

Secondly, starting from a particular metabolite of interest, exploration of the reactions that either synthesize or degrade this metabolite immediately generates a list of enzymes of interest for further investigation. This concept of exploring shells of reactions around a metabolite is exemplarily shown for tryptophan (Trp) metabolism in Fig. 2 and can be expanded stepwise around every metabolite serving as a new seed node. Each of these reactions can then be characterized by a ratio of product and substrate concentrations as a measure of enzymatic activity. Assessment of such ratios reduces biological noise and often

between a study and control cohort in analogy to a gene set enrichment analysis

**Pathway Count** 

from multivariate datasets.

to reduce the risk of false positive hits.

(MarkerIDQ™ software, Biocrates)

independent studies (Enot et al., 2011). Targeted metabolomics enables the systematic quantitation of a wide range of biologically relevant molecule classes in cells, tissues, or clinically relevant fluids. The technology comprises an automated sample preparation workflow integrated with sensitive mass spectrometric methods and a tailor-made software solution. Many hundreds of metabolites can be identified and quantified using this novel platform, which is also well suited for high-throughput and routine applications (Weinberger & Graber, 2005).

### **5. Proof-of-concept for targeted metabolomics: Neonatal screening**

The proof-of-concept for targeted metabolomics was first delivered in clinical diagnostics, namely in neonatal screening for inborn errors of metabolism. As mentioned above, the diagnosis of inherited disorders in amino acid metabolism, such as phenylketonuria, or fatty acid oxidation disorders, such as medium-chain acyl-CoA dehydrogenase (MCAD) deficiency, was revolutionized by the use of mass spectrometric assays (Röschinger et al., 2003). The idea was a logical continuation of Sir Archibald Garrod's (1857–1936) concept of chemical pathology, to quantify specific sets of amino acids and acylcarnitines to diagnose specific metabolic disorders. This laid the foundation for what is now referred to as 'targeted metabolomics'. The introduction of tandem mass spectrometry and the transition from expensive, monoparametric to multiparametric assays has enabled the simultaneous diagnosis of 20–30 monogenic diseases, which is a significant improvement of diagnostic performance, particularly of the specificity and the predictive values for very rare diseases. This improved diagnostic performance was achieved without raising costs. Rather neonatal screening is now reimbursed by health insurance providers in many Western countries and has lead to substantial healthcare savings. These medical and commercial benefits have turned neonatal screening into an impressive success story and led to its introduction in most industrialized countries within less than a decade (Röschinger et al., 2003; Weinberger., 2008).

### **6. Data exploitation**

The whole data-related workflow for targeted metabolomics has recently been summarized (Enot et al., 2011). In the context of this chapter, we only refer to the pathway mapping aspects of this workflow.

The unique level of understanding of metabolomics data that makes them suitable for a key role in functional genomics mainly results from this key step of data handling, namely the biochemical interpretation in the context of pathway and background knowledge. Despite its importance, very few standardized procedures have been developed and/or published for this step, and some experts would probably consider it their proprietary methodology to derive biochemical and pathobiochemical insight from multivariate metabolic datasets.

The last few years have seen multiple efforts to systematically annotate endogenous metabolites and led to databases such as KEGG (Kaneshia & Goto, 2000), Reactome (Vastrik et al., 2007), BioCyc (Karp et al., 2005), HMDB (Wishart et al., 2007) and OMIM (Online mendelian inheritance in man, 2011). Despite suffering from some serious shortcomings in terms of pathway coverage and data curation, these may serve as a more or less accepted framework for future knowledge collection.

independent studies (Enot et al., 2011). Targeted metabolomics enables the systematic quantitation of a wide range of biologically relevant molecule classes in cells, tissues, or clinically relevant fluids. The technology comprises an automated sample preparation workflow integrated with sensitive mass spectrometric methods and a tailor-made software solution. Many hundreds of metabolites can be identified and quantified using this novel platform, which is also well suited for high-throughput and routine applications

The proof-of-concept for targeted metabolomics was first delivered in clinical diagnostics, namely in neonatal screening for inborn errors of metabolism. As mentioned above, the diagnosis of inherited disorders in amino acid metabolism, such as phenylketonuria, or fatty acid oxidation disorders, such as medium-chain acyl-CoA dehydrogenase (MCAD) deficiency, was revolutionized by the use of mass spectrometric assays (Röschinger et al., 2003). The idea was a logical continuation of Sir Archibald Garrod's (1857–1936) concept of chemical pathology, to quantify specific sets of amino acids and acylcarnitines to diagnose specific metabolic disorders. This laid the foundation for what is now referred to as 'targeted metabolomics'. The introduction of tandem mass spectrometry and the transition from expensive, monoparametric to multiparametric assays has enabled the simultaneous diagnosis of 20–30 monogenic diseases, which is a significant improvement of diagnostic performance, particularly of the specificity and the predictive values for very rare diseases. This improved diagnostic performance was achieved without raising costs. Rather neonatal screening is now reimbursed by health insurance providers in many Western countries and has lead to substantial healthcare savings. These medical and commercial benefits have turned neonatal screening into an impressive success story and led to its introduction in most industrialized countries within less than a decade (Röschinger

The whole data-related workflow for targeted metabolomics has recently been summarized (Enot et al., 2011). In the context of this chapter, we only refer to the pathway mapping

The unique level of understanding of metabolomics data that makes them suitable for a key role in functional genomics mainly results from this key step of data handling, namely the biochemical interpretation in the context of pathway and background knowledge. Despite its importance, very few standardized procedures have been developed and/or published for this step, and some experts would probably consider it their proprietary methodology to derive biochemical and pathobiochemical insight from multivariate metabolic datasets. The last few years have seen multiple efforts to systematically annotate endogenous metabolites and led to databases such as KEGG (Kaneshia & Goto, 2000), Reactome (Vastrik et al., 2007), BioCyc (Karp et al., 2005), HMDB (Wishart et al., 2007) and OMIM (Online mendelian inheritance in man, 2011). Despite suffering from some serious shortcomings in terms of pathway coverage and data curation, these may serve as a more or less accepted

**5. Proof-of-concept for targeted metabolomics: Neonatal screening** 

(Weinberger & Graber, 2005).

et al., 2003; Weinberger., 2008).

**6. Data exploitation** 

aspects of this workflow.

framework for future knowledge collection.

These databases also provide the background for various attempts at visualization of metabolic pathways and data mapping on these charts, although most of these projects still follow a static approach of predefined (and predrawn) maps that cannot do justice to the dynamics of biochemical networks. In the following paragraph, we would like to demonstrate a few concepts about how dynamic representation and simulation (Modre-Osprian et al., 2009) of metabolic pathways enable the first steps of generating hypotheses from multivariate datasets.

Firstly, electronic availability of metabolites and metabolic reactions facilitates an almost trivial but nevertheless powerful approach that is analogous to a gene set enrichment analysis (GSEA; Subramanian et al., 2005). Any given set of metabolites which has been identified by statistics as significantly different in two biological states or clinical cohorts can be mapped on the entirety of metabolic pathways, and these pathways can then be ranked by the number of altered metabolites they contain (Fig. 1). This is a way of structuring the data that scientists from transcriptomics and proteomics are familiar with although the definition of metabolic pathways does not follow a similarly strict classification system as the classical gene ontology (GO; Ashburner et al., 2000). Note also that a reliable selection of species-specific enzymatic reactions instead of the generic reference pathways is necessary to reduce the risk of false positive hits.


Fig. 1. Ranking of metabolic pathways according to the number of significant differences between a study and control cohort in analogy to a gene set enrichment analysis (MarkerIDQ™ software, Biocrates)

Secondly, starting from a particular metabolite of interest, exploration of the reactions that either synthesize or degrade this metabolite immediately generates a list of enzymes of interest for further investigation. This concept of exploring shells of reactions around a metabolite is exemplarily shown for tryptophan (Trp) metabolism in Fig. 2 and can be expanded stepwise around every metabolite serving as a new seed node. Each of these reactions can then be characterized by a ratio of product and substrate concentrations as a measure of enzymatic activity. Assessment of such ratios reduces biological noise and often

Targeted Metabolomics for Clinical Biomarker Discovery in Multifactorial Diseases 87

Fig. 3. Route finding across metabolic pathways. Nine paths from arginine to spermine, ranging in length from four to six steps were calculated based on the KEGG dataset, and the

The quantitative information of targeted metabolomics enables new possibilities in validating computational systems biology approaches using detailed kinetic models to simulate and predict the dynamic response of metabolic networks in the context of human diseases. It also supports the design of tailored kinetic models of human-specific metabolic

Besides statistical model building and data mining-based approaches (Baumgartner et al., 2004; Baumgartner et al., 2005; Baumgartner & Graber, 2008), computational systems biology is essential to combine knowledge of human physiology and pathology starting from genomics, molecular biology and the environment through the levels of cells, tissues, and organs all the way up to integrated systems behaviour. Applying systems biology approaches within the context of human health and disease will definitely gain new insights. Eventually, a new discipline – systems medicine – will emerge at the interface between medicine and systems

Higher levels of organization are extremely complex, and even models at the cell and subcellular levels are forced to resort to simplifications to minimize modeling and computational complexity (Crampin et al., 2004; Nakayama et al., 2005; Yugi & Tomita, 2004). Additionally, some parameters and constants for kinetics, binding and concentrations of biomolecules are typically not known, thus reducing the model's ability to respond correctly to dynamic changes in external conditions. A high-quality network of humanspecific metabolic pathways including detailed knowledge about all metabolic reactions concerned is essential to design tailored kinetic models for better understanding of human physiology and its relationship with diseases. While such large networks are used to analyze the global structure or functional connectivity of the network (Ma et al., 2007), deterministic and stochastic models are mainly used for simulating specific metabolic

settings allowed for joint nodes, e.g., ornithine (MarkerIDQ™ software, Biocrates)

pathways including detailed knowledge about all metabolic reactions concerned.

biology (van der Greef et al., 2006; van der Greef et al., 2007; Lemberger, 2007).

pathways as well as regulatory and signaling networks (Goel et al., 2006).

**6.1 Quantitative experimental information for computational biology** 

dramatically increases the significance of the findings (Altmaier et al., 2008; Gieger et al., 2008; Wang-Sattler et al., 2008).

Lastly, moving even further from a traditional textbook representation of metabolic pathways, one can apply route finding algorithms to find and depict connections between metabolites of interest across the boundaries of (often artificially) predefined pathways. Such algorithms can identify the shortest route, routes up to a defined length, routes that do not share a certain metabolite (termed node-disjoint paths) or enzyme (so-called edge disjoint paths), depending on the respective biological question (Fig. 3). Here, the main prerequisite to avoid a potentially very large number of trivial hits is the exclusion of common cofactors and small inorganic molecules that connect many metabolites to many others, e.g., H2O, CO2, ATP, NADP. Using tools like these, and keeping in mind all the caveats discussed above, enzymes and pathways involved in the pathophysiology of a certain disease or in the mode-of-action of a drug can be more efficiently identified. In addition, hypotheses for designing further validation experiments and studies can be formulated.

Fig. 2. Shellwise exploration of enzymatic reactions in tryptophan metabolism. L- Trp was used as the first seed node; after expansion of eight synthetic or degrading reactions, 5- hydroxy-tryptophan was used as secondary seed node and further expanded (MarkerIDQ™ software, Biocrates)

Yet, all of this needs to be combined with another plausibility check, which originates from inherent redundancies in metabolism: quite often groups of compounds are metabolized by the same enzyme and should, therefore, be influenced in at least a similar (if not the same) way by regulatory mechanisms, drugs, etc. If this rule of thumb is severely challenged, one should always check for possible analytical or statistical artifacts, or – not uncommon in pharmaceutical R&D – interference by xenobiotics, e.g., a drug or drug metabolite disturbing the signal for an endogenous metabolite.

dramatically increases the significance of the findings (Altmaier et al., 2008; Gieger et al.,

Lastly, moving even further from a traditional textbook representation of metabolic pathways, one can apply route finding algorithms to find and depict connections between metabolites of interest across the boundaries of (often artificially) predefined pathways. Such algorithms can identify the shortest route, routes up to a defined length, routes that do not share a certain metabolite (termed node-disjoint paths) or enzyme (so-called edge disjoint paths), depending on the respective biological question (Fig. 3). Here, the main prerequisite to avoid a potentially very large number of trivial hits is the exclusion of common cofactors and small inorganic molecules that connect many metabolites to many others, e.g., H2O, CO2, ATP, NADP. Using tools like these, and keeping in mind all the caveats discussed above, enzymes and pathways involved in the pathophysiology of a certain disease or in the mode-of-action of a drug can be more efficiently identified. In addition, hypotheses for designing further validation experiments and studies can be

Fig. 2. Shellwise exploration of enzymatic reactions in tryptophan metabolism. L- Trp was used as the first seed node; after expansion of eight synthetic or degrading reactions, 5- hydroxy-tryptophan was used as secondary seed node and further expanded

Yet, all of this needs to be combined with another plausibility check, which originates from inherent redundancies in metabolism: quite often groups of compounds are metabolized by the same enzyme and should, therefore, be influenced in at least a similar (if not the same) way by regulatory mechanisms, drugs, etc. If this rule of thumb is severely challenged, one should always check for possible analytical or statistical artifacts, or – not uncommon in pharmaceutical R&D – interference by xenobiotics, e.g., a drug or drug metabolite

2008; Wang-Sattler et al., 2008).

(MarkerIDQ™ software, Biocrates)

disturbing the signal for an endogenous metabolite.

formulated.

Fig. 3. Route finding across metabolic pathways. Nine paths from arginine to spermine, ranging in length from four to six steps were calculated based on the KEGG dataset, and the settings allowed for joint nodes, e.g., ornithine (MarkerIDQ™ software, Biocrates)

#### **6.1 Quantitative experimental information for computational biology**

The quantitative information of targeted metabolomics enables new possibilities in validating computational systems biology approaches using detailed kinetic models to simulate and predict the dynamic response of metabolic networks in the context of human diseases. It also supports the design of tailored kinetic models of human-specific metabolic pathways including detailed knowledge about all metabolic reactions concerned.

Besides statistical model building and data mining-based approaches (Baumgartner et al., 2004; Baumgartner et al., 2005; Baumgartner & Graber, 2008), computational systems biology is essential to combine knowledge of human physiology and pathology starting from genomics, molecular biology and the environment through the levels of cells, tissues, and organs all the way up to integrated systems behaviour. Applying systems biology approaches within the context of human health and disease will definitely gain new insights. Eventually, a new discipline – systems medicine – will emerge at the interface between medicine and systems biology (van der Greef et al., 2006; van der Greef et al., 2007; Lemberger, 2007).

Higher levels of organization are extremely complex, and even models at the cell and subcellular levels are forced to resort to simplifications to minimize modeling and computational complexity (Crampin et al., 2004; Nakayama et al., 2005; Yugi & Tomita, 2004). Additionally, some parameters and constants for kinetics, binding and concentrations of biomolecules are typically not known, thus reducing the model's ability to respond correctly to dynamic changes in external conditions. A high-quality network of humanspecific metabolic pathways including detailed knowledge about all metabolic reactions concerned is essential to design tailored kinetic models for better understanding of human physiology and its relationship with diseases. While such large networks are used to analyze the global structure or functional connectivity of the network (Ma et al., 2007), deterministic and stochastic models are mainly used for simulating specific metabolic pathways as well as regulatory and signaling networks (Goel et al., 2006).

Targeted Metabolomics for Clinical Biomarker Discovery in Multifactorial Diseases 89

discussed in the literature as a marker for renal failure (Bode-Böger et al., 2006; Vallance et al., 1992). SDMA is hardly metabolized in the body, but only eliminated by renal excretion and, since no specific tubular resorption has been reported, it could be interpreted as an internal test substance for renal clearance (Bode-Boger et al, 2006; Martens-Lobenhoffer & Bode-Böger, 2006). As seen in figure 4, SDMA was increased in the two highest dosage

> 3 days 7 days 14 days 22 days

Control 10 mg / kg /day 20 mg / kg /day 40 mg / kg /day

Fig. 4. Time-and dose-dependent increase of SDMA in a nephrotoxicity study in rats. Levels of SDMA increased in a time- (p=9.9·10-5 in the cohort treated with 20 mg/kg/day, p=8.4·10-4 in the cohort treated with 40 mg/kg/day) and dose-dependent manner (p=4.4·10-6, comparing

Many of the preclinical findings were confirmed in a clinical biomarker study on progression of CKD that was performed at Montpellier University hospital as part of an EUfunded consortium (ETB Urosysteomics). The participating patients were divided into three cohorts according to severity of kidney disease; no to moderate renal function impairment (eGFR > 30 ml/min/1.73 m², corresponding to stages 1 to 3 of CKD as proposed by the National Kidney Foundation, for simplicity referred to as stage 3), severe renal function impairment (30 ml/min/1.73 m² > eGFR > 15 ml/min/1.73 m², corresponding to stage 4) and renal failure (eGFR < 15 ml/min/1.73 m², corresponding to stage 5 treated with dialysis) based on eGFR as proposed by Bauer et al, 2008. The patients in this study were mixed cases from different etiologies of CKD (diabetic and non-diabetic), and several analyses were performed to exclude confounding factors of these diseases and to look at

Just as in the preclinical study, many of the quantitated metabolites were found to be significantly up- or downregulated with progressing CKD. In a discriminant analysis the data could be separated almost completely which indeed indicates there is information in the data set to distinguish the stages from one another (Fig. 5) and further statistical analyses both identified novel markers (Lundin et al., submitted for publication) and confirmed biomarkers that had already been found in previous studies (e.g., nephrotoxicity of model compounds, early prognostic markers for acute rejection and chronic nephropathy in kidney transplant patients; Boudonck et al., 2009; Lundin & Weinberger, 2009). One of the

findings was elevated levels of SDMA, as already observed in the rat model (Fig. 6).

biomarkers influenced by kidney damage, regardless of underlying disease.

groups, and there was also an increase over time within these groups.

SDMA [µM]

0.0

day 14 in all cohorts)

0.5

1.0

1.5

2.0

2.5

Results of *in silico* experiments should be related to quantitative experimental data (e.g. from neonatal screening) in order to reveal better insights into dynamic properties of the complex biochemical networks under the constraints of various disease conditions and finally to obtain a better understanding of pathophysiological aspects of genetic disorders (Modre-Osprian et al., 2009).

### **7. Use case: Biomarker development in CKD**

Chronic kidney disease is a major health problem associated with increased risk of cardiovascular disease, renal failure and other complications (James et al., 2010). The cost for treating these complications puts a disproportionally large part on national health care budgets (Eknoyan et al., 2004; James et al., 2010; Mendelssohn & Wish, 2009). With an aging population and a worldwide epidemic of diabetes, the most common causes of CKD have switched from infection/inflammation and inheritance, to hypertension, other vascular disorders and diabetes as the main triggers.

It is estimated that at least 40 million people in the EU have some degree of CKD. This number is expected to increase every year, even double over the next decade, and the trend is similar all over the world (European Kidney Health Alliance, 2011; James et al., 2010). One of the major reasons for this is the dramatic increase of T2D, accounting for up to 95 % of the total diabetes incidence (American Diabetes Association, 2000; Kurukulasuriya, & Sowers, 2010; Ritz & Stefanski, 1996). Diabetic nephropathy is one of the most severe complications of diabetes and by far the most common cause of end-stage renal disease (ESRD; Susztak & Bottinger, 2006). Most people are unaware of their disease at early stages and do not get the right treatment in time. The classical renal function markers, serum creatinine level and estimated glomerular filtration rate (eGFR) are known to be insensitive and late markers of CKD (National Kidney Foundation, 2002). The gold standard for assessing renal function is measuring the true GFR with test substances like inulin or iothalamate, but this is an invasive and far too tedious procedure for routine application. It is of highest importance to develop markers which have the ability to predict or detect CKD at an earlier stage, making it possible to intervene with therapy to prevent or at least slow down the progression of kidney damage finally leading to ESRD and control related complications. While the classical diagnostic markers are restricted to traditional endpoints for kidney damage, metabolic markers can assess pathophysiological and pathobiochemical changes that play a role in exacerbation of renal damage.

This use case is based on two studies explained in further detail below. In a preclinical study on puromycin-treated Sprague-Dawley rats, several classes of metabolites were quantitated covering the main pathways of metabolism. The absolute concentration of the metabolites was determined by MRM and the application of SID (Jarman et al., 1975). The aim of the preclinical rat study was to evaluate metabolic changes in these rats, focusing on nephrotoxicity.

Cohorts consisted of three dosage groups (10 mg/kg/day, 20 mg/kg/day and 40 mg/kg/day) and one control group where only a vehicle was administered. Samples were taken at day 3, 7, 14 and 22 after start of the experiment, except for the highest dosage group, where all animals had to be sacrificed at day 14 because of complete renal failure. One of the metabolites that was associated with exacerbation of renal damage was symmetric dimethylarginine (SDMA) in plasma (Fig. 4), which has been extensively

Results of *in silico* experiments should be related to quantitative experimental data (e.g. from neonatal screening) in order to reveal better insights into dynamic properties of the complex biochemical networks under the constraints of various disease conditions and finally to obtain a better understanding of pathophysiological aspects of genetic disorders (Modre-

Chronic kidney disease is a major health problem associated with increased risk of cardiovascular disease, renal failure and other complications (James et al., 2010). The cost for treating these complications puts a disproportionally large part on national health care budgets (Eknoyan et al., 2004; James et al., 2010; Mendelssohn & Wish, 2009). With an aging population and a worldwide epidemic of diabetes, the most common causes of CKD have switched from infection/inflammation and inheritance, to hypertension, other vascular

It is estimated that at least 40 million people in the EU have some degree of CKD. This number is expected to increase every year, even double over the next decade, and the trend is similar all over the world (European Kidney Health Alliance, 2011; James et al., 2010). One of the major reasons for this is the dramatic increase of T2D, accounting for up to 95 % of the total diabetes incidence (American Diabetes Association, 2000; Kurukulasuriya, & Sowers, 2010; Ritz & Stefanski, 1996). Diabetic nephropathy is one of the most severe complications of diabetes and by far the most common cause of end-stage renal disease (ESRD; Susztak & Bottinger, 2006). Most people are unaware of their disease at early stages and do not get the right treatment in time. The classical renal function markers, serum creatinine level and estimated glomerular filtration rate (eGFR) are known to be insensitive and late markers of CKD (National Kidney Foundation, 2002). The gold standard for assessing renal function is measuring the true GFR with test substances like inulin or iothalamate, but this is an invasive and far too tedious procedure for routine application. It is of highest importance to develop markers which have the ability to predict or detect CKD at an earlier stage, making it possible to intervene with therapy to prevent or at least slow down the progression of kidney damage finally leading to ESRD and control related complications. While the classical diagnostic markers are restricted to traditional endpoints for kidney damage, metabolic markers can assess pathophysiological and pathobiochemical changes that play a

This use case is based on two studies explained in further detail below. In a preclinical study on puromycin-treated Sprague-Dawley rats, several classes of metabolites were quantitated covering the main pathways of metabolism. The absolute concentration of the metabolites was determined by MRM and the application of SID (Jarman et al., 1975). The aim of the preclinical rat study was to evaluate metabolic changes in these rats, focusing on

Cohorts consisted of three dosage groups (10 mg/kg/day, 20 mg/kg/day and 40 mg/kg/day) and one control group where only a vehicle was administered. Samples were taken at day 3, 7, 14 and 22 after start of the experiment, except for the highest dosage group, where all animals had to be sacrificed at day 14 because of complete renal failure. One of the metabolites that was associated with exacerbation of renal damage was symmetric dimethylarginine (SDMA) in plasma (Fig. 4), which has been extensively

Osprian et al., 2009).

**7. Use case: Biomarker development in CKD** 

disorders and diabetes as the main triggers.

role in exacerbation of renal damage.

nephrotoxicity.

discussed in the literature as a marker for renal failure (Bode-Böger et al., 2006; Vallance et al., 1992). SDMA is hardly metabolized in the body, but only eliminated by renal excretion and, since no specific tubular resorption has been reported, it could be interpreted as an internal test substance for renal clearance (Bode-Boger et al, 2006; Martens-Lobenhoffer & Bode-Böger, 2006). As seen in figure 4, SDMA was increased in the two highest dosage groups, and there was also an increase over time within these groups.

Fig. 4. Time-and dose-dependent increase of SDMA in a nephrotoxicity study in rats. Levels of SDMA increased in a time- (p=9.9·10-5 in the cohort treated with 20 mg/kg/day, p=8.4·10-4 in the cohort treated with 40 mg/kg/day) and dose-dependent manner (p=4.4·10-6, comparing day 14 in all cohorts)

Many of the preclinical findings were confirmed in a clinical biomarker study on progression of CKD that was performed at Montpellier University hospital as part of an EUfunded consortium (ETB Urosysteomics). The participating patients were divided into three cohorts according to severity of kidney disease; no to moderate renal function impairment (eGFR > 30 ml/min/1.73 m², corresponding to stages 1 to 3 of CKD as proposed by the National Kidney Foundation, for simplicity referred to as stage 3), severe renal function impairment (30 ml/min/1.73 m² > eGFR > 15 ml/min/1.73 m², corresponding to stage 4) and renal failure (eGFR < 15 ml/min/1.73 m², corresponding to stage 5 treated with dialysis) based on eGFR as proposed by Bauer et al, 2008. The patients in this study were mixed cases from different etiologies of CKD (diabetic and non-diabetic), and several analyses were performed to exclude confounding factors of these diseases and to look at biomarkers influenced by kidney damage, regardless of underlying disease.

Just as in the preclinical study, many of the quantitated metabolites were found to be significantly up- or downregulated with progressing CKD. In a discriminant analysis the data could be separated almost completely which indeed indicates there is information in the data set to distinguish the stages from one another (Fig. 5) and further statistical analyses both identified novel markers (Lundin et al., submitted for publication) and confirmed biomarkers that had already been found in previous studies (e.g., nephrotoxicity of model compounds, early prognostic markers for acute rejection and chronic nephropathy in kidney transplant patients; Boudonck et al., 2009; Lundin & Weinberger, 2009). One of the findings was elevated levels of SDMA, as already observed in the rat model (Fig. 6).

Targeted Metabolomics for Clinical Biomarker Discovery in Multifactorial Diseases 91

Another finding that was reproduced in both studies was changes in concentration of the essential with amino acid Trp. Tryptophan is crucial for protein synthesis and might therefore play an important part in cellular differentiation, development and growth (Badawy, 1988). The drop in concentration of Trp has been associated with impaired kidney function before (Egashira et al., 2006; Saito et al., 2000). In the puromycin-treated rat model of nephrotoxicity, Trp drops to less than a third of its concentration between low- and high dose and at different time points (Fig. 7). The same trend can be observed in the clinical study on progression of CKD (Fig. 8 left) which emphasizes the advantages of being able to

This phenomenon could partly be explained by albumin depletion since, in peripheral blood, Trp is bound to albumin to a significant extent (Walser & Hill, 1993). This would result in a drop of Trp when the albumin is being depleted in progressing kidney disease. As seen in figure 8 (right), there is indeed a drop in albumin in the clinical study, but not in the same magnitude as Trp, hence it can be assumed that other mechanisms are also

> 3 days 7 days 14 days 22 days

Control 10 mg / kg /day 20 mg / kg /day 40 mg / kg /day

Fig. 7. Tryptophan depletion in puromycin-treated rats. Tryptophan shows both a dose- (4.9·10-6, comparing day 14 in all cohorts) and time-dependent (p=1.8·10-3 in the cohort treated with 10 mg/kg/day, p=1.2·10-6 in the cohort on 20 mg/kg/day, and p= 2.5·10-9 in

Another explanation for the drop in concentration of Trp might be in its degrading pathways (see Fig. 9). When analyzing the two main catabolic pathways originating from Trp, both the rat model and the clinical study revealed that actually both pathways (towards serotonin and through kynurenine towards niacin) are upregulated (Fig. 10). The steep increase and high statistical significance of the kynurenine / Trp (product to substrate) ratio in the clinical study suggest there is a markedly increased activity through this pathway, in keeping with the fact that the kynurenine pathway accounts for 95 % of tryptophan catabolism (Walser & Hill, 1993). The Trp degrading indoleamine 2,3-dioxygenase (IDO) enzyme, which catalyzes the initial and rate-limiting step in the niacin pathway, seems to

use translational research in metabolomics.

involved here.

Trp [µM]

0

the cohort on 40 mg/kg/day) decrease

have an increased activity in progressing CKD.

50

100

150

200

250

Fig. 5. Discriminant analysis of three stages of CKD. In the scores plot (left) the three stages of CKD are almost perfectly separated. Each point represents one patient and the cohort to which they belong is indicated by colour in the box in the upper right corner. The axes are calculated to minimize variance within the group and maximize it between the groups to get the best separation possible. The loadings plot (right) identifies which metabolites are mostly responsible for the separation, the further away from the origin, the stronger the influence. Since multi-group comparisons are not particularly clear, deeper analysis is required to identify single markers

Fig. 6. Increase of plasma SDMA concentrations in progressing CKD. Barplots represent plasma SDMA concentrations in three groups of patients with declining renal function. SDMA shows a highly significant increase in progressing stages of CKD (p = 1.2·10-9)

Fig. 5. Discriminant analysis of three stages of CKD. In the scores plot (left) the three stages of CKD are almost perfectly separated. Each point represents one patient and the cohort to which they belong is indicated by colour in the box in the upper right corner. The axes are calculated to minimize variance within the group and maximize it between the groups to get the best separation possible. The loadings plot (right) identifies which metabolites are mostly responsible for the separation, the further away from the origin, the stronger the influence. Since multi-group comparisons are not particularly clear, deeper analysis is

Stage 3 Stage 4 Stage 5

Fig. 6. Increase of plasma SDMA concentrations in progressing CKD. Barplots represent plasma SDMA concentrations in three groups of patients with declining renal function. SDMA shows a highly significant increase in progressing stages of CKD (p = 1.2·10-9)

required to identify single markers

SDMA [µM]

0.0

0.5

1.0

1.5

2.0

2.5

Another finding that was reproduced in both studies was changes in concentration of the essential with amino acid Trp. Tryptophan is crucial for protein synthesis and might therefore play an important part in cellular differentiation, development and growth (Badawy, 1988). The drop in concentration of Trp has been associated with impaired kidney function before (Egashira et al., 2006; Saito et al., 2000). In the puromycin-treated rat model of nephrotoxicity, Trp drops to less than a third of its concentration between low- and high dose and at different time points (Fig. 7). The same trend can be observed in the clinical study on progression of CKD (Fig. 8 left) which emphasizes the advantages of being able to use translational research in metabolomics.

This phenomenon could partly be explained by albumin depletion since, in peripheral blood, Trp is bound to albumin to a significant extent (Walser & Hill, 1993). This would result in a drop of Trp when the albumin is being depleted in progressing kidney disease. As seen in figure 8 (right), there is indeed a drop in albumin in the clinical study, but not in the same magnitude as Trp, hence it can be assumed that other mechanisms are also involved here.

Fig. 7. Tryptophan depletion in puromycin-treated rats. Tryptophan shows both a dose- (4.9·10-6, comparing day 14 in all cohorts) and time-dependent (p=1.8·10-3 in the cohort treated with 10 mg/kg/day, p=1.2·10-6 in the cohort on 20 mg/kg/day, and p= 2.5·10-9 in the cohort on 40 mg/kg/day) decrease

Another explanation for the drop in concentration of Trp might be in its degrading pathways (see Fig. 9). When analyzing the two main catabolic pathways originating from Trp, both the rat model and the clinical study revealed that actually both pathways (towards serotonin and through kynurenine towards niacin) are upregulated (Fig. 10). The steep increase and high statistical significance of the kynurenine / Trp (product to substrate) ratio in the clinical study suggest there is a markedly increased activity through this pathway, in keeping with the fact that the kynurenine pathway accounts for 95 % of tryptophan catabolism (Walser & Hill, 1993). The Trp degrading indoleamine 2,3-dioxygenase (IDO) enzyme, which catalyzes the initial and rate-limiting step in the niacin pathway, seems to have an increased activity in progressing CKD.

Targeted Metabolomics for Clinical Biomarker Discovery in Multifactorial Diseases 93

There is a widespread range of potential applications of metabolomics in the areas of biomedical research, pharmaceutical R&D and clinical diagnostics. Besides the wellestablished procedure of neonatal screening, metabolomics is currently being applied in biomarker and diagnostics research as demonstrated in the example of CKD above. Still, the diagnostic potential of metabolomics is not confined to typical metabolic disorders, but rather extends into fields such as cancer (Osl et al., 2008) and neurologic disorders (Urban et al., 2010). Metabolomics can also be applied in drug development where it is used to uncover new drug targets, prioritize lead compounds and assess drug toxicity, enabling the development of novel, smarter and safer drugs (Weinberger & Graber, 2005). In addition, metabolomics has the possibility to identify individuals likely to benefit from a given

The most important difference of metabolomics to other –omics approaches is the level of functional understanding that, currently, the metabolome is offering to a much greater extent than the other -omes. The first successful examples of combining GWAS with metabolic phenotypes, so-called metabotypes, have recently been published and show significant promise for a more useful outcome of population based association studies in general. This phenotyping is particularly useful when the disease in question affects metabolically active organs and when large scale transport phenoma are affected. Consequently, metabolomics is very well suited for biomarker discovery in multifactorial diseases like T2D and CKD. It is not only possible to find novel markers but also to explain the pathophysiological effects behind the disease, e.g., inhibition of or upregulation of an enzyme in a specific pathway. Additionally, the fact that there is no need to redevelop the analytic assays, because of the non-species specific properties of the metabolites, makes it cost- and time-saving since there is no need for redevelopment of

The authors wish to acknowledge Àngel Argilés and Natalie Gayrard for study design and clinical documentation, Lisa Körner, Stephanie Angeben, Verena Forcher, Doreen Kirchberg, Hai Pham-Tuan, and Ali Alchalabi for excellent lab work, Madhumalar Panneerselvam, Denise Sonntag, Daniel Andres and Torben Friedrich for support with data handling and biostatistics. The studies presented here were in part supported by the EurotransBio Program of the FP6 of the European Comission carried out by the Urosysteomics consortium and the FP7 European Union grant 'SysKid' (HEALTH–F2–2009–241544).

Altmaier, E., Kastenmuller, G., Romisch-Margl, W., Thorand, B., Weinberger, K.M., Illig, T.,

Adamski, J., Doring, A. & Suhre, K. (2011). Questionnaire-based self-reported nutrition habits associate with serum metabolism as revealed by quantitative targeted metabolomics. *Eur J Epidemiol*, Vol.26, No.2, pp. 145-156, ISSN 0393-2990

therapy, minimizing the risk of side effects and avoiding unnecessary drug use.

**8. Outlook and potential applications** 

**9. Conclusion** 

the analytical assays.

**11. References** 

**10. Acknowledgment** 

Fig. 8. Changes of Trp and albumin with progression of CKD. Left: Decrease of plasma Trp concentrations in progressing CKD. Barplots represent plasma Trp concentrations in three groups of patients with progressing CKD. Tryptophan shows a highly significant decline in later stages of CKD (p = 6.2·10-9). Right: This could in part be explained by the fact that it is albumin-bound, but although it is significant (p=1.56·10-9) the fold-change of the albumin depletion is by far less pronounced than the one for Trp

Fig. 9. Simplified scheme of the two main pathways catabolizing Trp. To the right the niacin pathway is illustrated where the rate limiting step is the conversion of Trp to kynurenine catalyzed by IDO (Ball et al., 2009). To the left the serotonin pathway with its two key reactions (tetrahydrobiopterin-dependent tryptophan hydroxylation and pyridoxalphosphate-dependent decarboxylation of 5-hydroxytryptophan) is shown

Fig. 10. Upregulation of Trp degrading pathways. There is an upregulation of both the niacin pathway (left, p=1.44·10-12) and serotonin pathway (right, p=2.98·10-6) degrading Trp, but the steep increase and high statistical significance of the kynurenine / Trp ratio suggest there is an increased activity of the Trp degrading IDO enzyme

### **8. Outlook and potential applications**

There is a widespread range of potential applications of metabolomics in the areas of biomedical research, pharmaceutical R&D and clinical diagnostics. Besides the wellestablished procedure of neonatal screening, metabolomics is currently being applied in biomarker and diagnostics research as demonstrated in the example of CKD above. Still, the diagnostic potential of metabolomics is not confined to typical metabolic disorders, but rather extends into fields such as cancer (Osl et al., 2008) and neurologic disorders (Urban et al., 2010). Metabolomics can also be applied in drug development where it is used to uncover new drug targets, prioritize lead compounds and assess drug toxicity, enabling the development of novel, smarter and safer drugs (Weinberger & Graber, 2005). In addition, metabolomics has the possibility to identify individuals likely to benefit from a given therapy, minimizing the risk of side effects and avoiding unnecessary drug use.

### **9. Conclusion**

92 Advances in the Study of Genetic Disorders

Albumin [g / l]

0

Fig. 8. Changes of Trp and albumin with progression of CKD. Left: Decrease of plasma Trp concentrations in progressing CKD. Barplots represent plasma Trp concentrations in three groups of patients with progressing CKD. Tryptophan shows a highly significant decline in later stages of CKD (p = 6.2·10-9). Right: This could in part be explained by the fact that it is albumin-bound, but although it is significant (p=1.56·10-9) the fold-change of the albumin

Fig. 9. Simplified scheme of the two main pathways catabolizing Trp. To the right the niacin pathway is illustrated where the rate limiting step is the conversion of Trp to kynurenine catalyzed by IDO (Ball et al., 2009). To the left the serotonin pathway with its two key

> Serotonin / Trp

Fig. 10. Upregulation of Trp degrading pathways. There is an upregulation of both the niacin pathway (left, p=1.44·10-12) and serotonin pathway (right, p=2.98·10-6) degrading Trp, but the steep increase and high statistical significance of the kynurenine / Trp ratio suggest

0.000 0.002 0.004 0.006 0.008 0.010 0.012 0.014 0.016 0.018 0.020

reactions (tetrahydrobiopterin-dependent tryptophan hydroxylation and

10

20

30

40

50

Stage 3 Stage 4 Stage 5

Stage 3 Stage 4 Stage 5

Stage 3 Stage 4 Stage 5

depletion is by far less pronounced than the one for Trp

pyridoxalphosphate-dependent decarboxylation of

Stage 3 Stage 4 Stage 5

there is an increased activity of the Trp degrading IDO enzyme

5-hydroxytryptophan) is shown

Plot 1

Kynurenine / Trp

0.0

0.1

0.2

0.3

0.4

0.5

Trp [µM]

> The most important difference of metabolomics to other –omics approaches is the level of functional understanding that, currently, the metabolome is offering to a much greater extent than the other -omes. The first successful examples of combining GWAS with metabolic phenotypes, so-called metabotypes, have recently been published and show significant promise for a more useful outcome of population based association studies in general. This phenotyping is particularly useful when the disease in question affects metabolically active organs and when large scale transport phenoma are affected. Consequently, metabolomics is very well suited for biomarker discovery in multifactorial diseases like T2D and CKD. It is not only possible to find novel markers but also to explain the pathophysiological effects behind the disease, e.g., inhibition of or upregulation of an enzyme in a specific pathway. Additionally, the fact that there is no need to redevelop the analytic assays, because of the non-species specific properties of the metabolites, makes it cost- and time-saving since there is no need for redevelopment of the analytical assays.

### **10. Acknowledgment**

The authors wish to acknowledge Àngel Argilés and Natalie Gayrard for study design and clinical documentation, Lisa Körner, Stephanie Angeben, Verena Forcher, Doreen Kirchberg, Hai Pham-Tuan, and Ali Alchalabi for excellent lab work, Madhumalar Panneerselvam, Denise Sonntag, Daniel Andres and Torben Friedrich for support with data handling and biostatistics. The studies presented here were in part supported by the EurotransBio Program of the FP6 of the European Comission carried out by the Urosysteomics consortium and the FP7 European Union grant 'SysKid' (HEALTH–F2–2009–241544).

### **11. References**

Altmaier, E., Kastenmuller, G., Romisch-Margl, W., Thorand, B., Weinberger, K.M., Illig, T., Adamski, J., Doring, A. & Suhre, K. (2011). Questionnaire-based self-reported nutrition habits associate with serum metabolism as revealed by quantitative targeted metabolomics. *Eur J Epidemiol*, Vol.26, No.2, pp. 145-156, ISSN 0393-2990

Targeted Metabolomics for Clinical Biomarker Discovery in Multifactorial Diseases 95

Egashira, Y.; Nagaki, S. & Sanada, H. (2006). Tryptophan-niacin metabolism in rat with

Eknoyan, G.; Lameire, N.; Barsoum, R.; Eckardt, K.U.; Levin, A.; Levin, N.; Locatelli, F.;

Enot DP, Haas B, Weinberger KM. Bioinformatics for mass spectrometry-based

European Kidney Health Alliance (2011). The kidney in health and disease. 2011-03-15, Available from: <http://www.ekha.eu/usr\_img/info/factsheet.pdf> Gieger, C.; Geistlinger, L.; Altmaier, E.; Hrabe de Angelis, M.; Kronenberg, F.; Meitinger, T.;

Goel, G.; Chou, I.C. & Voit, E.O. (2006). Biological systems modeling and analysis: a

Greef, J van der.; Martin, S.; Juhasz, P.; Adourian, A.; Plasterer, T.; Verheij, E.R. &

Greef,J van der.; Hankemeier, T. & McBurney, R.N. (2006). Metabolomicsbased systems

Groop, L. & Lyssenko, V. (2009). Genetics of type 2 diabetes. An overview. *Endocrinol Nutr*,

Illig, T.; Gieger, C.; Zhai, G.; Romisch-Margl, W.; Wang-Sattler, R.; Prehn, C.; Altmaier, E.;

James, M.T.; Hemmelgarn, B.R. & Tonelli, M. (2010). Early recognition and prevention of chronic kidney disease. *Lancet,* Vol.375, No.9722, pp. 1296-1309, ISSN 0140-6736 Jarman, M.; Gilby, E.D.; Foster, A.B. & Bondy, P.K. (1975). The quantitation of

Kanehisa, M. & Goto, S. (2000). KEGG: Kyoto Encyclopedia of Genes and Genomes. *Nucleic*

Karp, P.D.; Ouzounis, C.A.; Moore-Kochlacs, C.; Goldovsky, L.; Kaipa, P.; Ahrén, D.; Tsoka,

*Pharmacogenomics*, Vol.7, No.7 , pp. 1087-1094, ISSN 1462-2416

*Nat Genet*,Vol.42, No.2, (Feb), pp. 137-141, ISSN 1061-4036

dilution. *Clin Chim Acta*. Vol.58, No.1, pp. 61-9, ISSN 0009-8981

*Acids Res*, Vol.28, No.1, pp. 27-30, ISSN 0305-1048

Vol. 56 Suppl 4, pp. 34-7, ISSN 1575-0922

(Ed.), 351-75, 978-1-61779-026-3, Humana Press, New York, USA.)

pp. 28-33, ISSN 0300-9831

4:e1000282, ISSN 1553-7404

252-269, ISSN 1524-0215

1535-3893

0085-2538

puromycin aminonucleoside-induced nephrosis. *Int J Vitam Nutr Res*, Vol.76, No.1,

MacLeod, A.; Vanholder, R.; Walker, R. & Wang, H. (2004). The burden of kidney disease: improving global outcomes. *Kidney Int*, Vol. 66, No.4, pp. 1310-1314, ISSN

metabolomics. *Methods Mol Biol*. 2011;719:351-75. (also: Enot, D.; Haas, B. & Weinberger, K.M. (2011). Bioinformatics for mass spectrometry-based metabolomics, In: *Bioinformatics for Omics Data: Methods and Protocols*. B. Mayer

Mewes, H.W.; Wichmann, H.E.; Weinberger, K.M.; Adamski, J.; Illig, T. & Suhre, K.. (2008). Genetics meets metabolomics: a genome-wide association study of metabolite profiles in human serum. *PLoS Genet*, Vol.4, No.11, (Dec), pp.

biomolecular technique of the twenty-first century. *J Biomol Tech*, Vol.17, No.4 , pp.

McBurney, R.N. (2007). The art and practise of systems biology in medicine: mapping patterns of relationships. *J Proteome Res*, Vol.6, No.4 , pp. 1540-1559, ISSN

biology and personalized medicine: moving towards n = 1 clinical trials?

Kastenmuller, G.; Kato, B.S.; Mewes, H.W.; Meitinger, T.; de Angelis, M.H.; Kronenberg, F.; Soranzo, N.; Wichmann, H.E.; Spector, TD.; Adamski, J. & Suhre, K. (2010). A genome-wide perspective of genetic variation in human metabolism.

cyclophosphamide in human blood and urine by mass spectrometry-stable isotope

S.; Darzentas, N.; Kunin, V.; López-Bigas, N. (2005). Expansion of the BioCyc


Altmaier, E.; Kastenmuller, G.; Romisch-Margl, W.; Thorand, B.; Weinberger, K.M.;

American Diabetes Association. (2000). Type 2 Diabetes in Children and Adolescents.

Ashburner, M.; Ball, C.A.; Blake, J.A.; Botstein, D.; Butler, H.; Cherry, J.M.; Davis, A.P.;

Ontology Consortium. *Nat Genet*, Vol.25, No.1, pp. 25-9, ISSN 1061-4036 Badawy, A.A. (1988). Effects of pregnancy on tryptophan metabolism and disposition in the

Ball, H.J.; Yuasa, H.J.; Austin, C.J.; Weiser, S. & Hunt, N.H. (2009). Indoleamine 2,3-

Bauer, C.; Melamed, M.L. & Hostetter, T.H. (2008) Staging of chronic kidney disease: time

Baumgartner C & Graber A. (2008). Data mining and knowledge discovery in

Baumgartner, C.; Böhm, C. & Baumgartner D. (2005). Modelling of classification rules on

Baumgartner, C.; Böhm, C.; Baumgartner, D.; Marini, G.; Weinberger, K.M.; Olgemöller, B.;

Bode-Böger, S.M.; Scalera, F.; Kielstein, J.T.; Martens-Lobenhoffer, J.; Breithardt, G.; Fobker,

Boudonck, K.J.; Mitchell, M.W.; Nemet, L.; Keresztes, L.; Nyska, A.; Shinar, D. & Rosenstock,

nephrotoxicity. *Toxicol Pathol*, Vol.37, No.3, pp. 280-292, ISSN 0192-6233 Crampin, E.J.; Halstead, M.; Hunter, P.; Nielsen, P.; Noble, D.; Smith, N. & Tawhai, M.

*Diabetes Care*, Vol.23, No.3, pp. 381-389, ISSN 0149-5992

rat. *Biochem J*. Vol.255, No.1, pp. 369-372, ISSN 0264-6021

Vol.41, No.3, pp. 467-471, ISSN 1357-2725

Vol.38, No.2 , pp. 89-98, ISSN 1532-0464

*Nephrol*, Vol.17, No.4, pp. 1128-1134, ISSN 1046-6673

0013-7227

3450

pp. 1-26

Hershey, PA, USA.

2985-2996, ISSN 1367-4803

Adamski, J.; Illig, T.; Doring, A. & Suhre, K. (2009). Variation in the human lipidome associated with coffee consumption as revealed by quantitative targeted metabolomics. *Mol Nutr Food Res*, Vol.53, No.11 , pp. 1357-1365, ISSN 1613-4125 Altmaier, E.; Ramsay, S.L.; Graber, A.; Mewes, H.W.; Weinberger, K.M. & Suhre, K. (2008).

Bioinformatics analysis of targeted metabolomics--uncovering old and new tales of diabetic mice under medication. *Endocrinology*, Vol.149, No.7, pp. 3478-3489, ISSN

Dolinski, K.; Dwight, S.S.; Eppig, J.T.; Harris, M.A.; Hill, D.P.; Issel-Tarver, L.; Kasarskis, A.; Lewis, S.; Matese, J.C.; Richardson, J.E.; Ringwald, M.; Rubin, G.M. & Sherlock, G. (2000). Gene ontology: tool for the unification of biology. The Gene

dioxygenase-2; a new enzyme in the kynurenine pathway. *Int J Biochem Cell Biol*,

for a course correction. *J Am Soc Nephrol*, Vol.19, No.5, pp. 844-846, ISSN 1533-

metabolomics, In: Successes and new directions in data mining. Masseglia F.; Poncelet, P. & Teisseire, M. (Ed.), 141-66, ISBN 978-1-59904-645-7, IgI Global,

metabolic patterns including machine learning and expert knowledge. *J Biomed Inf*,

Liebl, B. & Roscher, A.A. (2004). Supervised machine learning techniques for the classification of metabolic disorders in newborns. *Bioinformatics*, Vol. 20, No.7 , pp.

M. & Reinecke, H. (2006). Symmetrical dimethylarginine: a new combined parameter for renal function and extent of coronary artery disease. *J Am Soc* 

M. (2009). Discovery of metabolomics biomarkers for early detection of

(2004). *Computational physiology and the Physiome Project. Exp Physiol*, Vol.89, No. ,


Targeted Metabolomics for Clinical Biomarker Discovery in Multifactorial Diseases 97

Röschinger, W.; Olgemoller, B.; Fingerhut, R.; Liebl, B. & Roscher, A.A. (2003). Advances in

Saito, K.; Fujigaki, S.; Heyes, M.P.; Shibata, K.; Takemura, M.; Fujii, H.; Wada, H.; Noma, A.

Subramanian, A.; Tamayo, P.; Mootha, V.K.; Mukherjee, S.; Ebert, B.L.; Gillette, M.A.;

Suhre, K., Meisinger, C., Doring, A., Altmaier, E., Belcredi, P., Gieger, C., Chang, D.,

Susztak, K. & Bottinger, E.P. (2006). Diabetic nephropathy: a frontier for personalized medicine. *J Am Soc Nephrol*, Vol.17, No.2, pp. 361-367, ISSN 1046-6673 Unterwurzacher, I.; Koal, T.; Bonn, G.K.; Weinberger, K.M. & Ramsay, S.L. (2008). Rapid

Urban, M.; Enot, D.P.; Dallmann, G.; Körner, L.; Forcher, V.; Enoh, P.; Koal, T.; Keller, M. &

Vaag. A. & Poulsen. P. (2007). Twins in metabolic and diabetes research: what do they tell us? *Curr Opin Clin Nutr Metab Care*. Vol.10, No.5, pp. 591-6, ISSN 1363-1950 Vallance, P.; Leone, A.; Calver, A.; Collier, J. & Moncada, S. (1992). Accumulation of an

Vastrik, I.; D'Eustachio, P.; Schmidt, E.; Joshi-Tope, G.; Gopinath, G.; Croft, D.; de Bono, B.;

Walder, K.; Segal, D.; Jowett, J.; Blangero, J. & Collier, G.R. (2003). Obesity and diabetes gene

Walser, M. & Hill, S.B. (1993). Free and protein-bound tryptophan in serum of untreated

Wang-Sattler, R.; Yu, Y.; Mittelstrass, K.; Lattka, E.; Altmaier, E.; Gieger, C.; Ladwig, K.H.;

discovery approaches. *Curr Pharm Des*. Vol.9, No.17, pp. 1357-72

*One*, Vol.5, No.11, pp. e13953, ISSN 1932-6203

Vol.339, No.8793, pp. 572-575, ISSN 0140-6736

*Biology*, Vol.8, No.3, pp. R39, ISSN 1465-6906

*ONE*, Vol. 3, No.12, pp. e3863, ISSN 1932-6203

diseases. *Eur J Pediatr*, Vol.162 Suppl 1, pp. S67-76, ISSN 0340-6199

0363-6127

ISSN 0027-8424

1589-1597, ISSN 1434-6621

2697

0085-2538

analytical mass spectrometry to improve screening for inherited metabolic

& Seishima, M. (2000). Mechanism of increases in L-kynurenine and quinolinic acid in renal insufficiency. *Am J Physiol Renal Physiol*, Vol.279, No.3, pp. F565-572, ISSN

Paulovich, A.; Pomeroy, S.L.; Golub, T.R.; Lander, E.S. & Mesirov, J.P. (2005) Gene set enrichment analysis: a knowledge-based approach for interpreting genomewide expression profiles. *Proc Natl Acad Sci U S A*, Vol.102, No.43, pp. 15545-50,

Milburn, M.V., Gall, W.E., Weinberger, K.M., et al. (2010). Metabolic footprint of diabetes: a multiplatform metabolomics study in an epidemiological setting. *PLoS* 

sample preparation and simultaneous quantitation of prostaglandins and lipoxygenase derived fatty acid metabolites by liquid chromatography-mass spectrometry from small sample volumes. *Clin Chem Lab Med*, Vol.46, No.11, pp.

Deigner, H.P. (2010). Complexity and pitfalls of mass spectrometry-based targeted metabolomics in brain research. *Anal Biochem*. Vol.496, No.2, pp. 124-31, ISSN 0003-

endogenous inhibitor of nitric oxide synthesis in chronic renal failure. *Lancet*,

Gillespie, M.; Jassal, B.; Lewis, S.; Matthews, L.; Wu, G.; Birney, E. & Stein, L. (2007). Reactome: a knowledge base of biologic pathways and processes. *Genome* 

patients with chronic renal failure. *Kidney Int*, Vol.44, No.6, pp. 1366-1371, ISSN

Dahmen, N.; Weinberger, K.M.; Hao, P.; Liu, L.; Li, Y.; Wichmann, H.E.; Adamski, J.; Suhre, K. & Illig, T. (2008). Metabolic profiling reveals distinct variations linked to nicotine consumption in humans – first results from the KORA study. *PLoS* 

collection of pathway/genome databases to 160 genomes. *Nucleic Acids Res*, Vol.33, No.19, pp. 6083-9, ISSN 0305-1048


Kurukulasuriya, L.R. & Sowers, J.R. (2010). Therapies for type 2 diabetes: lowering HbA1c

Lemberger, T. (2007). Systems biology in human health and disease. *Mol Syst Biol*, Vol.3,

Lundin, U. & Weinberger, K.M. (2009) Metabolic Characterization of a rat model of

Ma, H.; Sorokin, A.; Mazein, A.; Selkov, A.; Selkov, E.; Demin, O. & Goryanin, I. (2007). The

Martens-Lobenhoffer, J. & Bode-Boger, S.M. (2006). Fast and efficient determination of

Mendelssohn, D.C. & Wish, J.B. (2009). Dialysis delivery in Canada and the United States:

Mensah, G.A.; Mokdad, A.H.; Ford, E.; Narayan, K.M.; Giles, W.H.; Vinicor, F. &

Modre-Osprian, R.; Osprian, I.; Tilg, B.; Schreier, G.; Weinberger, K.M. & Graber, A. (2009).

Nakayama, Y.; Kinoshita, A. &Tomita, M. (2005). Dynamic simulation of red blood cell

National Kidney Foundation. (2002). K/DOQI clinical practice guidelines for chronic kidney

Nicholson, J.K.; Lindon, J.C. & Holmes, E. (1999). 'Metabonomics': understanding the

Online Mendelian Inheritance in Man, OMIM®. McKusick-Nathans Institute of Genetic

Osl, M.; Dreiseitl, S.; Pfeifer, B.; Weinberger, K.M.; Klocker, H.; Bartsch, G.; Schafer, G.; Tilg,

Ritz, E. & Stefanski, A. (1996). Diabetic nephropathy in type II diabetes. *Am J Kidney Dis*,

*Bioinformatics*, Vol.24, No.24 , pp. 2908-2914, ISSN 1367-4803

No.19, pp. 6083-9, ISSN 0305-1048

No.3 , pp. 136, ISSN 1744-4292

*ToxExpoTM*, Baltimore, MD, USA

No.4, pp. 485-504, ISSN 0733-8651

*Syst Biol*, Vol.3, pp. 2, ISSN 1752-0509

*Biol Med Model*, Vol.2, pp. 18, ISSN 1742-4682

Vol.29, No.11, pp. 1181-1189, ISSN 0049-8254

Vol.27, No.2, pp. 167-194, ISSN 0272-6386

No.2Suppl1, pp. S1-266, ISSN 0272-6386

<http://omim.org/>

*Mol Syst Biol*. Vol. 3, pp. 135, ISSN 1744-4292

1475-2840

6386

collection of pathway/genome databases to 160 genomes. *Nucleic Acids Res*, Vol.33,

and associated cardiovascular risk factors. *Cardiovasc Diabetol*, Vol. 9, pp. 45, ISSN

puromycin induced nephrotoxicity. *Proceedings of SOT 48th Annual Meeting &* 

Edinburgh human metabolic network reconstruction and its functional analysis.

arginine, symmetric dimethylarginine, and asymmetric dimethylarginine in biological fluids by hydrophilic-interaction liquid chromatography-electrospray tandem mass spectrometry. *Clin Chem*, Vol.52, No.3, pp. 488-493, ISSN 0009-9147

a view from the trenches. *Am J Kidney Dis*, Vol.54, No.5, pp. 954-964, ISSN 0272-

Deedwania, P.C. (2004). Obesity, metabolic syndrome, and type 2 diabetes: emerging epidemics and their cardiovascular implications. *Cardiol Clin*. Vol.22,

Dynamic simulations on the mitochondrial fatty acid beta-oxidation network. *BMC* 

metabolism and its application to the analysis of a pathological condition. *Theor* 

disease: evaluation, classification, and stratification. *Am J Kidney Dis*, Vol.39,

metabolic responses of living systems to pathophysiological stimuli via multivariate statistical analysis of biological NMR spectroscopic data. *Xenobiotica*,

Medicine, Johns Hopkins University (Baltimore, MD), 2011-03-15, Available from:

B.; Graber, A.; & Baumgartner, C. (2008). A new rule-based algorithm for identifying metabolic markers in prostate cancer using tandem mass spectrometry.


**Part 2** 

**Unifactorial or Unigenetic Disorder** 

