Metabolomics in Alcohol Research and Drug Development

Metabolomics in Alcohol Research and Drug Development

George G. Harrigan, Ph.D.; Greg Maguire, Ph.D.; and Laszlo Boros, M.D.

Developers of new medications need to describe and predict the functional attributes of test compounds administered to cells, animals, and humans. Today, researchers increasingly appreciate the role that intermediary products (i.e., metabolites) generated in the course of various metabolic pathways play in both health and disease states and how their analysis can support development of new medications. Advances in analytical and computational techniques have facilitated the rise of new and powerful tools for measuring metabolic and biochemical pathways in such complex systems. Metabolomics—a systems biology approach to characterizing metabolites produced in biochemical pathways—is contributing to many studies of disease progression and treatment, although it has not yet been extensively applied in research on metabolic perturbations associated with alcohol abuse. However, numerous metabolomic approaches may contribute to alcohol-related research, as illustrated by studies on alcohol-related metabolic dysfunctions such as (1) alterations in fat metabolism and (2) thiamine deficiency. By further increasing the number and types of metabolites that can be measured in a given biological sample, metabolomic approaches may be able to help define the role of the many different metabolic pathways affected by alcohol abuse and support discovery and development of novel medications for the treatment of alcoholism and related conditions. KEY WORDS: Alcohol abuse and dependence; alcoholism; alcohol- related disorders; systems biology; metabolomics; metabolome; metabolic networks; metabolic pathways; metabolites; metabolic process; metabolic profiling; lipid dysfunction; thiamine deficiency; disease etiology; drug design

ttempts to develop new and/or and computational methodologies to ronmental changes. Identification of improved medications for the describe the effects of medication can- induced changes in the concentration A treatment of illnesses affecting didates within the complex biochemi- of these metabolites, or altered flux1 millions of people around the world cal pathways of intact, fully assembled rates through metabolic pathways are characterized by an ever-increasing living networks (Goodacre et al. 2004; disparity between research expenditure Joyce and Palsson 2007; Maguire et al. 1 For a definition of this and other technical terms, see (both in terms of time and money) in 2006, 2007; Mo et al. 2007). A most the glossary on p. 84. the pharmaceutical industry and return promising approach to studying such networks is metabolomics. on investment (Di Masi et al. 2003). GEORGE G. HARRIGAN, PH.D, currently Therefore, strategies that can rapidly Metabolomics is a systems biology is a study director in the Product Safety identify novel candidate medications (or approach to measuring changes in Center at Monsanto Company, St. Louis, potential targets of new medications), small-molecule intermediary products Missouri. which are likely to demonstrate effective- (i.e., metabolites)—such as sugars ness in clinical trials, will be valuable to (e.g., glucose), amino acids, and organic GREG MAGUIRE, PH.D., currently is both patients and the pharmaceutical acids—in any given biological system based in San Diego, California. industry. One means to achieve this exposed to genetic modification, drugs goal—the adoption of screening or medications, or any other environ- LASZLO BOROS, M.D., is associate professor approaches that can be performed in mental perturbation. The levels of these of Pediatrics at the University of Cali- isolated cells and tissues (i.e., in vitro) small-molecule metabolites within a fornia Los Angeles, co-director of the or using computer models (i.e., in cell, tissue, or an organism largely reflect Stable Isotope Research Laboratory at silico)—continues apace. Another impor- the endogenous activity of biochemical Harbor-UCLA Medical Center, and tant prerequisite for effective screening pathways; moreover, they also are chief scientific advisor at SiDMAP, Los tools is the development of analytical highly responsive to external or envi- Angeles, California.

26 Alcohol Research & Health Metabolomics, Alcohol Research, and Drug Development

(e.g., Maguire et al. 2006; 2007), may metabolomics are to probe the mecha­ NMR and MS, as well as other provide insights into the activities, nisms that contribute to changes in techniques, can be employed in two interactions, and regulatory roles of all metabolite levels and fluxes in cells, tis­ different (but not necessarily mutual­ biochemical processes of interest. sues, and organisms and to understand ly exclusive) conceptual approaches Although metabolomic approaches the implications of such changes on that can broadly be defined as “non­ to date have been used primarily for disease, response to drugs and medica­ targeted” and “targeted” (Goodacre other physiological processes and dis­ tions, and nutritional intervention. To et al. 2004). Nontargeted approaches eases, they are equally applicable to achieve these goals, numerous technical provide a hypothesis-free global over­ studies on the impact of alcohol on approaches to data acquisition are avail­ view of readily detectable metabolites metabolism as well as the effect of able. The main technologies utilized in in a sample (e.g., lipid or sugar meta­ medications developed to treat the metabolomics are nuclear magnetic res­ bolites in plasma). Many experiments effects of alcohol abuse and depen­ onance (NMR) spectroscopy and mass have successfully used such nontargeted dence. The costs associated with alco­ spectrometry (MS) (for a review, see approaches, although some investiga­ holism, at both the personal and more Dunn and Ellis 2005). Briefly, NMR tors are increasingly concerned about global level, are well understood. From spectroscopy is a technique that exploits current statistical treatments and meth­ a medical standpoint, it is important the magnetic properties of the nuclei of odological practices in such experiments both to understand the psychological certain atoms (e.g., protons [1H] or a (Broadhurst and Kell 2006; Lay et and emotional factors that contribute variant [i.e., isotope] of carbon known al. 2006). It has been suggested, for to alcohol dependence and to amelio­ as 13C). In a typical metabolomics example, that the results of a study rate the physiological devastation that experiment, a solvent extract of the bio­ claiming that nontargeted NMR this dependence can cause. Because logical sample (or in the case of urine analysis of plasma could accurately excessive alcohol use can induce pro­ or plasma, the sample itself) is placed predict the risk of coronary heart found metabolic perturbations, there in a magnetic field and then exposed to disease actually were confounded by radiowaves covering a given frequency gender and drug use (Kirschenlohr et should be numerous opportunities 2 for using metabolomic approaches. range. The way in which the biological al. 2006). Indeed, the first attempts at such sample absorbs that energy is depen­ Targeted approaches, in contrast, metabolic profiling research date back dent on the relative proportion of dif­ focus on identified and preselected to at least the 1980s, when researchers ferent metabolites in that sample. This metabolic pathways, which may offer measured the levels of various metabo­ allows investigators to obtain an NMR particular advantages in studies on lites in the urine of ethanol-exposed spectrum that represents the metabolite metabolic dysregulation. These tar­ humans and rats (Axelson et al. 1981; composition of the sample and that geted approaches typically employ Weiner et al. 1984). More recently, can be compared with the NMR spec­ optimized measurement techniques however, metabolomic strategies rarely tra of other biological samples. that specifically detect certain classes of metabolites or pathways. This have been applied systematically in MS is another analytical technique approach recognizes the wide differ­ the alcohol field. that can be used to assess the metabo­ ences in physiochemical structure, This article briefly describes the lite composition of a biological sample. For these assays, samples are injected stability, and abundance of different concepts behind metabolomic research. metabolites in a given sample— It highlights examples of how meta­ into a mass spectrometer that can separate compounds according to factors that present challenges in bolomic approaches have been used accurately measuring a wide range of to study metabolic perturbations, such their molecular mass, and a list of the molecular masses of the compounds metabolites in a single measurement. as altered fat (i.e., lipid) metabolism Targeted approaches also recognize and thiamine deficiency, that also are in the sample is generated. The meta­ bolites corresponding to a given that evaluation of low-abundance observed in alcohol-dependent patients. metabolites, such as signaling lipids, It also considers the role that meta­ molecular mass can then be identified and their relative abundance in a hormones, and neurotransmitters, may bolomics can play in further elucidating require specialized methods. Finally, the causes and effects of these changes sample determined. The specific data acquisition tech­ targeted metabolomic approaches and supporting the discovery and emphasizing biological expertise in development of new medications to nology used typically depends on the nature and biochemical composition experimental strategy can be used in treat alcoholism and its metabolic flux-based methodology to determine consequences. of the metabolites to be measured. Thus, MS may represent a better option the fate of certain metabolites. For to measure certain classes of metabo­ example, specifically designed substrates What Is Metabolomics? lites, whereas NMR may be the better that have been labeled with easily choice for others. In general, MS meth­ Many definitions of metabolomics exist ods are more sensitive than NMR 2 The issue of confounding influences is, however, not specific to metabolomics studies such as this (Fiehn 2002; Goodacre et al. 2004), and can identify molecules that are well-known example but also can occur in any other but, in general, the principal goals of present in only small amounts. type of study.

Vol. 31, No. 1, 2008 27

measurable tracers (e.g., radioactive envision a role for metabolomics in to cell or organ status than the tran­ molecules) can be introduced into test dissecting the relative contributions scriptome or proteome, and is biological systems and their distribution of these nutritional factors to disease responsive to a wide range of pertur­ and metabolic fate recorded (Hellerstein susceptibility or progression. Indeed, bations, it represents a viable option and Murphy 2004). To date, flux- metabolomics now is considered an for increasing our understanding of based approaches unfortunately are important discipline among researchers the factors that influence development underrepresented in metabolomics. concerned with the effects of nutrition, and progression of various diseases. as exemplified by initiatives by the Accordingly, metabolomic analyses Advantages of Metabolomics European Nutrigenomics Organization may help elucidate the dynamics of (http://www.nugo.org). Similarly, the alcohol dependence and of alcohol- Metabolomic approaches have several NIH Roadmap Initiative (http:// induced organ damage as well as advantages that can help them comple­ nihroadmap.nih.gov/initiatives.asp) clarify the role of factors such as ment other strategies, such as genomic, and the NIH Genes, Environment genetic predisposition and nutritional transcriptomic, and proteomic ap­ and Health Initiative (http://www. deficiencies or the effects of new proaches. First, many examples have gei.nih.gov) include metabolomic ini­ medications. demonstrated that the presence of a tiatives that, among other goals, aim gene which has been shown to be asso­ to determine the influence of envi­ ciated with a disease need not always ronmental factors on disease suscepti­ Relevance of Metabolomics lead to disease manifestation, and in bility and progression. to Alcohol Research many cases further biological informa­ Third, as is the case with the presence tion is required to predict the onset of of specific genes, the presence of certain Biochemical studies have contributed symptoms. The presence of a certain messenger RNAs (mRNAs) or proteins greatly to our understanding of variant (i.e., mutation) of the gene does not always suffice to predict disease metabolic perturbations in alcohol- associated with Huntington disease, for onset. In fact, the correlation between dependent patients. For example, example, clearly correlates with neural global mRNA levels and protein alcohol’s impact on lipid metabolism in degeneration but does not necessarily expression typically is low (i.e., pro­ different organs increasingly is under­ predict when the first preclinical symp­ duction of large amounts of mRNA stood (Baraona and Lieber 1998; toms will appear. To identify biomarkers does not automatically imply produc­ Crabb and Liangpunsakul 2006). that can evaluate or predict the onset tion of large amounts of protein). Similarly, alcohol-related dysfunction of certain diseases, it therefore may be Furthermore, despite some successes, in carbohydrate metabolism has been more promising to use a combinatory it appears that many biomarkers initially well-studied, and particularly the approach that associates the presence of identified through transcriptomics or importance of the vitamin thiamine a specific, predisposing genetic variant proteomics cannot be validated (Lay (which serves as a co-factor for at least with the presence of altered metabolite et al. 2006). Although part of this fail­ three enzymes involved in carbohydrate patterns in the blood. Similarly, meta­ ure may be related to technical issues, metabolism) has become evident in bolomic approaches may be useful in it also is known that changes in, for studies focusing on alcohol-induced determining the risk of diseases that are example, the levels of individual damage to the nervous system (i.e., determined by several genes (i.e., are enzymes need not correlate with neurodegeneration) (Martin et al. polygenic in origin), such as alcohol changes in metabolic flux or overall 2003). Studies of other disorders have dependence. In these cases, the contri­ function of the cell or tissue (Kacser already demonstrated that the concen­ bution of a single gene to disease vari­ 1995). Thus, transcriptomics and trations, fluxes, and transport mecha­ ance likely is small and/or difficult to proteomics analyses may not suffice to nisms of small-molecule metabolites assess, and integrating information on a fully assess genetic or environmental generally are highly sensitive to disease. single gene variant with transcriptomic, contributions to disease variance. Because alcohol-induced metabolic proteomic, and metabolomic data may Indeed, the metabolome may represent perturbations obviously are a major prove informative. the functional status of a cell, tissue, contributing factor to disease progression, Second, metabolomic approaches can or organism more closely because it metabolomics may play a role in dis­ be useful because disease variation reflects the overall effects of transcrip­ secting the contributions of different depends not only on the effects of tomic and proteomic changes. More­ biochemical pathways to the development genetic factors but also is influenced over, because the metabolome is the of alcohol-related damage. by environmental factors. For example, “downstream” result of gene expression, To date, there have been few a person’s diet profoundly impacts his changes in the metabolome are ampli­ examples of metabolomic research or her lifespan and resistance to disease. fied relative to changes in the tran­ specifically applied to alcohol-related Moreover, certain diseases, including scriptome and the proteome, thus disorders. However, there are certainly alcohol dependence, frequently go making measurements more tractable. potential opportunities for metabo­ hand in hand with inadequate nutri­ In summary, because the metabolome lomic research in the alcohol field, tion. It therefore is reasonable to is reasoned to be more directly related particularly targeted and flux-based

28 Alcohol Research & Health Metabolomics, Alcohol Research, and Drug Development approaches, as illustrated by two exam­ and dysfunction in carbohydrate dependent patients (Crabb 2004; Crabb ples of studies of known metabolic metabolism, which is associated with, and Lianpunsakul 2006). As with many effects associated with alcohol-related among other things, neurodegeneration. disease states, nutritional influences, diseases. The following sections review and medications, alcohol interferes two major areas of metabolic dysreg­ directly with the use of fatty acids as a ulation that are caused by numerous Alcohol-Associated Lipid source of energy in the liver. (For infor­ diseases or nutritional deficiencies, Dysfunction mation on the general structure of lipids, including alcohol abuse and depen­ Studies have suggested that alcohol see sidebar, below.) As a result, triglyc­ dence, and for which application of (chemically known as ethanol) may erides accumulate in the liver. These metabolomic approaches has provid­ interfere with the processes that regu­ triglycerides also are secreted into the ed new insights. These are (1) lipid late the balance in the body between blood, contributing to elevated levels dysfunction, which is associated with lipid breakdown as a source of energy of circulating lipids (i.e., dyslipidemia). liver (i.e., hepatic) and cardiovascular and lipid synthesis. Lipid metabolism These processes of lipid synthesis disease; and (2) thiamine deficiency can be profoundly altered in alcohol- and breakdown are regulated by

Structure and Metabolism of Lipids

Lipids play numerous important functions in the body. In addition to serving as energy stores, they are H H H H H H H essential components of all cell membranes. Chemically, O H H H lipids are water-repellent (i.e., hydrophobic) molecules C C C CC C C C C C H H C O that include fats, oils, waxes, sterols (e.g., cholesterol), H H H H H H H H H and other related compounds. From a metabolic and physiological standpoint the O H H H H H H H H H triglycerides represent one of the most important lipid C C C CC C C C C C H classes. Each triglyceride molecule is composed of the H C O following molecules. H H H H H H H H H

H H H H H H • Glycerol—an alcohol containing three carbons, each O H of which is linked to a hydroxyl group (OH); and C C C CC C C C H C O C H H H H H H H C • Three fatty acids—chains of varying length made up H H of carbon and hydrogen atoms with a carboxyl group H (COOH) at one end that can link the fatty acids to the glycerol molecule. glycerol carboxyl group fatty acid double bond

Fatty acids fall into two main groups: saturated or broken down to generate an intermediary molecule unsaturated. Saturated fatty acids (which are found pri­ called acetyl-CoA, which can enter the citric acid cycle marily in animal sources) contain the maximum number that also is involved in glucose metabolism. (Conversely, of hydrogen atoms (i.e., two hydrogens per carbon). acetyl-CoA can be diverted from glucose metabolism for Unsaturated fatty acids (which are found primarily in fatty acid and lipid synthesis.) In addition, fatty acids can plant sources), in contrast, lack one or more hydrogen atoms and therefore have one or more “double-bonds” be involved in numerous other metabolic reactions (e.g., between two carbons (see figure). be converted from unsaturated to saturated fatty acids or For both saturated and unsaturated fatty acids the incorporated into longer chain fatty acids). length of the hydrocarbon chain (i.e., the number of car­ Fat and liver cells can synthesize and store triglyc­ bon atoms) can differ, which can affect their metabolism erides. Alcohol can increase levels of triglycerides in as well as their specific function in the body. the liver. Increased levels of triglycerides in the blood For triglycerides to be metabolized as energy sources, are associated with atherosclerosis and increased risk of the fatty acids are first separated from the glycerol. The heart disease. free fatty acids then are transported to the mitochondria of the appropriate cells, where they are sequentially —George G. Harrison, Greg Maguire, and Laszlo Boros

Vol. 31, No. 1, 2008 29

several factors that control the activation strates for distinct metabolic path­ thesis and transport of long-chain (i.e., transcription) of genes involved ways, researchers can obtain further fatty acids. Boros and colleagues (2002) in lipid metabolism. Two of these fac­ information on specific enzymes and studied the de novo synthesis of the tors are the peroxisomal proliferator other regulatory mechanisms that fatty acid palmitate, its elongation –activated receptors (PPARs) and the control lipid synthesis, transport, and to stearate, and the conversion (i.e., sterol response element–binding pro­ distribution. This knowledge then can desaturation) of stearate into oleate teins (SREBPs)-1 and -2. PPARs are improve the understanding of both in the liver and pancreas of rats who hormone receptors located in the cell disease progression and response to were fed alcohol directly into the nucleus, and if they do not function therapeutic intervention. For example, stomach for 4 weeks. To determine properly, disorders in lipid metabolism, it has been suggested that liver lipids the levels of these fatty acids, the as well as insulin resistance, may of alcohol-dependent patients contain researchers provided the animals with develop. SREBPs act as “sensors” for a higher ratio of unsaturated fatty acids water containing the stable hydrogen fatty acid and cholesterol levels in than those of nonalcoholic control isotope deuterium, which was then blood and tissue, thereby helping to subjects (Bunout 1999). This obser­ incorporated into the newly synthe­ maintain constant levels (i.e., home­ vation implies that alcohol-dependent sized fatty acids and their metabolites ostasis) of lipids in the body. Broadly and could be measured using MS. speaking, activation of PPARs enhances These analyses found that compared conversion of lipids into energy, where­ with control animals, synthesis of as activation of SREBP-1 induces an A further palmitate, stearate, and oleate in the opposing effect. Studies conducted in understanding of liver (expressed as the percentage of ethanol-exposed cultured cells of mice total fatty acid synthesis in the liver) suggest that ethanol interferes with how the levels and decreased significantly in the alcohol- transcription-activating properties of metabolism of lipids treated animals. In the pancreas, in one type of PPAR (i.e., PPARα) but contrast, palmitate and stearate syn­ activates SREBP-1 (Crabb 2004). Both are regulated may thesis also decreased to a similar extent, of these effects would result in lipid whereas oleate synthesis actually accumulation in the blood and tissues contribute to the increased compared with control rats. and could contribute to alcohol-relat­ 3 development of Additional analyses indicated that in ed liver damage (i.e., fatty liver). the liver of alcohol-treated animals, Altered lipid metabolism resulting therapies for alcohol- stearate accumulated relative to from alcohol exposure also can lead related disorders. palmitate (i.e., elongation of palmitate to the generation of highly reactive, proceeded effectively) but that oleate oxygen-containing small molecules levels relative to palmitate decreased known as reactive oxygen species (i.e., desaturation was impaired). In (ROS), which can cause damage in a patients have an elevated activity of the pancreas, however, the opposite variety of tissues and further exacer­ a certain enzyme called Δ9-desaturase effect of alcohol exposure was noted bate disease progression (Lieber 2004). that has been associated with metabolic —the ratio of stearate to palmitate Accordingly, a further understanding disorders. decreased, whereas the oleate-to­ of how the levels and metabolism of Compositional analyses of the lipids palmitate ratio increased in the alco­ lipids are regulated may contribute in a tissue or organ can be comple­ hol-treated animals compared with to the development of therapies for mented by flux analyses that assess the the control animals. Together, these alcohol-related disorders. In fact, sev­ distribution and metabolism of lipids findings indicate that long-term alco­ eral studies already have indicated that in the body. For example, Pawlosky hol exposure increases the fatty acid medications which activate PPAR and Salem (1999) evaluated the effect content of liver cells, decreases de novo (and thereby counteract the effects of ethanol on the metabolism of two synthesis of fatty acids, and has oppo­ of ethanol), such as the medication polyunsaturated fatty acids—linoleic site effects on fatty acid accumulation pioglitazone, can ameliorate fatty acid and α-linolenic acid—in Rhesus liver in ethanol-fed rats. monkeys. In this study, alcohol con­ in the liver and pancreas. Because Methods for measuring lipids con­ sumption did not appear to affect the adequate levels of unsaturated fatty tinue to develop and improve. Many absorption of these fatty acids into acids such as oleate are critically now allow both the quantitative analy­ circulation. However, the incorporation important for various physiological sis of lipid classes present in a sample of these fatty acids into longer-chain processes (e.g., the transport of long- and determination of the entire fatty molecules was increased, suggesting chain saturated fatty acids out of the acid composition within those classes that alcohol enhances the elongation liver), the decreased oleate ratio in (Mutch 2006). This latter aspect is of polyunsaturated fatty acids. 3 Differential expression of PPARs and SREBPs is important because by determining Other investigators found that observed in many diseases associated with lipid dysregu­ which specific fatty acids serve as sub­ alcohol also interferes with the syn­ lation, such as diabetes and atherosclerosis.

30 Alcohol Research & Health

Metabolomics, Alcohol Research, and Drug Development the liver of alcohol-treated animals terms of changes in lipid metabolism Flux-based analyses that can monitor can result in decreased fatty acid will prove invaluable to the discovery the distribution of ingested metabolites transport and may contribute to fatty of new medications. (e.g., glucose) and their metabolites liver. In addition, the increased oleate in the cells and tissues are particularly ratio in the pancreas promotes pro­ Thiamine Deficiency and Flux useful in studies on carbohydrate cesses that may support the rapid Analysis of Transketolase Activity metabolism. To this end, biological development of pancreatitis. samples to be studied are treated with Metabolomic approaches such as Thiamine, also known as vitamin B1, substrates that can be traced easily— lipid profiling also may contribute to is essential for the optimal functioning for example, because they are labeled the development of safe medications and health of the cardiovascular, ner­ with a tracer (e.g., the stable, non­ to treat alcohol-induced dyslipidemia. vous, and digestive systems. Alcohol- radioactive isotope 13C) that serves For example, in a recent study on the dependent patients frequently exhibit to differentiate the substrate from PPAR activator, rosiglitazone, in a abnormally low levels of thiamine in endogenous metabolites. As the tracer- diabetic mouse model the researchers the blood that result mainly from labeled substrate is metabolized in the found reduced levels of cholesterol nutritional deficiencies, from alcohol’s biological sample, the label will be and triglyceride levels in the blood, impact on thiamine absorption from transferred to other molecules that suggesting that the medication led to the gastrointestinal tract, and from then can be further followed and a decrease in new lipid synthesis. impaired thiamine utilization in cells identified. In the case of 13C-labeled Additional analyses, however, demon­ (Bridges et al. 1999). The abnormally substrates, MS can be used to reveal strated that rosiglitazone treatment low levels of thiamine are thought to the positions that the tracer isotope actually resulted in fat accumulation contribute to the neurodegeneration assumes in metabolite products in the liver and tissue toxicity (Watkins often seen in alcohol-dependent patients derived from the original tracer-labeled et al. 2002). This may imply that new (Martin et al. 2003). Indeed, some evi­ substrate. These analyses can reveal medications designed to modulate dence suggests that the degeneration the specific reactions and pathways lipid metabolism should be evaluated of a brain region called the cerebellum, in which the original tracer-labeled by comprehensive metabolomics inves­ which often is associated with alcohol substrate was involved. This tigations extending beyond measure­ dependence, is associated more with metabolomic approach of monitoring ments of only one or two targeted the toxic effects of thiamine deficiency metabolic pathways and reactions lipid metabolites. than with the direct toxic effects of step-by-step (also known as stable Lipids clearly are an extremely impor­ alcohol itself (Mulholland et al. 2005). isotope dynamic metabolic profiling tant component of the metabolome, Thiamine plays a key role in carbo­ [SIDMAP]) allows researchers to and further developments in data acqui­ hydrate metabolism and is indeed conduct comprehensive and disease- sition technologies can only be of essential for the conversion of carbo­ specific metabolic profiling in a dynam­ marked benefit. Recognizing this, the ic, living, functioning cell or organism. hydrates into energy because it is a 13 U.S. National Institute of General Med­ co-factor required by three enzymes— [1,2- C2]-D-glucose is a particularly ical Sciences has, since 2003, sponsored useful metabolite for studies on car­ transketolase, pyruvate dehydrogenase a LIPIDMAPS consortium of 16 bohydrate metabolism. It is especially (PDH), and α-ketoglutarate dehy­ academic research institutes and two helpful for discriminating between drogenase (KGDH)—that mediate private enterprises to fully map meta­ glucose metabolism through the bolic pathways in a type of immune various steps in the breakdown of oxidative and nonoxidative branches cell, the macrophage. The goals of glucose (see sidebar, p. 32). of the pentose phosphate pathway LIPIDMAPS (www.lipidmaps.org) Transketolase plays a role in a chain (see sidebar, p. 32). It was used, for are to (1) separate and detect all of reactions called the pentose phos­ example, in a study of a blood disor­ lipids in a specific cell and discover phate pathway, whereas PDH and der called thiamine-responsive mega­ and characterize any novel lipids that KGDH are involved in glycolysis and loblastic anemia (TRMA), which is may be present, (2) quantitate each the citric acid cycle. Accordingly, it characterized by dysfunctional red of the lipid metabolites present and appears plausible that metabolomic blood cells in the bone marrow. The quantitate the changes in their levels strategies that can help analyze carbo­ study demonstrated that the funda­ and location during cellular function, hydrate metabolism also may con­ mental defect in TRMA is impaired and (3) define the biochemical path­ tribute to a further understanding transketolase activity, which subse­ ways for each lipid and develop lipid of the role of alcohol-dependent thi­ quently leads to decreased nucleic maps that define the interaction net­ amine deficiency. Moreover, such acid synthesis and disruption of the works. Because macrophages play, strategies could also, at least in princi­ cell’s normal progression through among other functions, a significant ple, assist in developing approaches to the cell cycle (Boros et al. 2003). role in the formation of atherosclerot­ identify people who are at particularly Commenting on this work in the same ic lesions, understanding and defin­ high risk of alcohol-related neurode­ journal issue, Green (2003, p. 3,464) ing changes in macrophage biology in generation. remarked that metabolomics has “made

Vol. 31, No. 1, 2008 31 Different Pathways of Glucose Metabolism

The metabolism of the sugar glucose is one of the metabolism, because both amino acids and fatty acids central metabolic processes in the body. It serves to can be broken down into acetyl-CoA that enters the cit­ provide energy to the body, as well as to provide start­ ric acid cycle. ing materials for other metabolic pathways (e.g., the The pentose phosphate pathway of glucose metabolism production of nucleic acids, fatty acids, and neuro­ is an alternative pathway to glycolysis and the citric acid transmitters). In addition, other metabolic pathways cycle that serves to generate both a co-factor that is required also feed into the reactions that occur during glucose by many enzymatic reactions (reduced nicotinamide ade­ metabolism, further emphasizing the central roles that nine dinucleotide phosphate [NADPH]) and sugar the reactions involved in glucose metabolism play in molecules containing five carbons (rather than the six- ensuring the body’s functioning and well-being. carbon sugars, such as glucose), which are needed, for Glucose metabolism can proceed along several alter­ example, for nucleic acid synthesis. The pentose phosphate native pathways, depending on the body’s needs at the pathway has two distinct phases, an oxidative phase, during time. The following are three main sets of reactions asso­ which the NADPH is generated (as well as sugar called ciated with glucose metabolism (see figure): ribulose-5-phosphate), and a nonoxidative phase, during which the ribulose-5-phosphate is converted further into • Glycolysis; other five-carbon sugars. The nonoxidative phase also can • The citric acid cycle; and serve as a “link” to glycolysis by generating fructose-6-phos­ phate and glyceraldehyde-3-phosphate. Several reactions • The pentose phosphate pathway of the nonoxidative phase involve the thiamine-dependent During glycolysis, glucose is converted in several enzyme transketolase. reactions into a molecule called pyruvate, which can The relative contributions of all these pathways to glu­ be either metabolized further into lactate, that is then cose metabolism depend on the body’s needs at the time. transported out of the cell, or which stays in the cell. Pyruvate is converted (through the actions of the thiamine- —George G. Harrison, Greg Maguire, and Laszlo Boros dependent enzyme pyruvate dehydrogenase) into a molecule called acetyl-CoA, which can then be fed into the second set of reac­ Pentose phosphate pathway tions, the citric acid cycle (also known as Glucose oxidative nonoxidative the tricarboxylic acid [TCA] cycle). Altern­ phase Ribulose-5- phase Ribulose-5- atively, acetyl-CoA may serve as a precursor Glucose-6-phosphate Phosphate Phosphate + transketolase Other sugars for fatty acid synthesis (see sidebar, p. 29). NADPH and other enzymes In the subsequent citric acid cycle, the Glycolysis acetyl-CoA is linked to a compound called Pyruvate lactate pyruvate oxaloacetate to form citric acid. The latter is dehydrogenase fatty acids lipids then metabolized in a series of steps to form Acetyl-CoA α-ketoglutarate, which is then transformed amino acids proteins further to yield oxaloacetate again so that the acetylcholine Oxaloacetate cycle can continue. These reactions also involve myelin a thiamine-dependent enzyme, α-ketoglu­ Citric acid Citric acid tarate dehydrogenase. In addition, the vari­ cycle ous reactions of the citric acid cycle yield several metabolites that either serve as cofac­ thiamine-dependent enzymes -ketoglutarate tors in other biochemical reactions or can dehydrogenase be fed into other reactions yielding energy required by the cell to function normally. The citric acid cycle plays a central role neurotransmitter not only in carbohydrate (i.e., glucose) (e.g. glutamate) metabolism but also in protein and lipid

32 Alcohol Research & Health

Metabolomics, Alcohol Research, and Drug Development it possible to unlock doors along pre­ this treatment. Thus, the SIDMAP products of glucose that are generated viously inaccessible hallways of gene technology can contribute to basic during the citric acid cycle (see sidebar function analysis” and that metabolic research and early medication develop­ figure, p. 92) and which circulate in flux analysis provided a “key to better ment as well as to clinical applications the blood may play a role in hyper­ understanding of changes in substrate in which the activity and interactions tension. It is well-established that the flow as a basis for drug mechanisms of a multitude of defined biochemical prevalence of hypertension is higher and disease.” pathways can be quantified (Maguire among alcohol-dependent patients The value of this technology for et al. 2006). than among the normal population understanding disease progression It is likely that metabolomic ap­ (Kodavali and Townsend 2006). The and aiding medication development proaches such as SIDMAP also can citric acid cycle takes place in special also can be illustrated using the example be applied in alcohol research—for membrane-enclosed compartments of a recent investigation of the mech­ example, for assessing the effects of (i.e., organelles) in the cells called mito­ anism of action of the medication thiamine and thiamine deficiency not chondria. These organelles often are imatinib mesylate (Gleevec®), a cancer only on a single metabolic pathway referred to as the cell’s energy factories drug used in the treatment of a certain but also on an integrated metabolic because the citric acid cycle and other type of leukemia. Although Gleevec network. For example, thiamine- late steps of glucose metabolism and itself has no proven application in induced activation of transketolase the resulting energy production occur alcohol-based research, its SIDMAP activity helps control the levels of a there. Two intermediary molecules analyses illustrate how metabolic flux glucose-derived molecule (i.e., sorbitol) generated during the citric acid cycle in several pathways known to be that, at elevated levels, can cause are succinate and α-ketoglutarate. It affected by alcohol abuse can be mea­ complications in patients with diabetes has been reasoned that, at least in ani­ sured simultaneously. (Berrone et al. 2006). Accordingly, mal models, these two compounds The leukemia cells against which reduced transketolase activity result­ can increase blood pressure when Gleevec is active are characterized by ing from alcohol-induced thiamine secreted into the blood (He et al. increased glucose uptake, enhanced deficiency may contribute to compli­ 2004). Other mitochondrial metabo­ activity of an enzyme called hexokinase, cations in alcoholic diabetic patients. lites also have been found to play a and increased activity of the oxidative Strategies, such as the one described signaling role for other physiologic branch of the pentose phosphate path­ above, that can analyze multiple meta­ functions (MacDonald et al. 2005), way, all of which result in synthesis of bolic pathways may therefore provide and this research area clearly offers new nucleic acids that are necessary a more complete picture of the meta­ opportunities for targeted meta­ for the leukemia cells to multiply. bolic dysregulation associated with bolomics of mitochondrial products. Gleevec acts by inhibiting all of these alcoholism and other conditions and Applying such an approach, Sabatine processes, thereby preventing further may help predict better the effect of and colleagues (2005) demonstrated nucleic acid synthesis. However, in new medications in the treatment of that the levels of functionally related some patients the tumor cells become this dysregulation. metabolites of the citric acid cycle resistant to Gleevec treatment. SIDMAP circulating in the blood differed approaches now have shown that between healthy people and people cells develop Gleevec resistance by Other Recent who were susceptible to constriction enhancing the activity of the non- Developments of blood vessels and reduced blood oxidative portion of the pentose flow (i.e., ischemia) when exercising. phosphate pathway (which involves The above examples have illustrated The investigators demonstrated that the transketolase enzyme), which is the potential benefits of metabolomics the circulating levels of two citric acid not appreciably inhibited by Gleevec. for analyzing two metabolic pathways— cycle intermediates (i.e., oxaloacetate One of the greatest advantages of lipid metabolism and thiamine- and citrate) decreased in ischemia- this metabolomic approach, however, dependent carbohydrate metabolism— susceptible but not control subjects. is that it can be performed not only that are affected by, or contribute to, The same was true for other com­ in cultured cells but also in the intact the harmful effects associated with alcohol pounds that are either end products organism (i.e., in vivo). This means dependence. However, other metabolic of reactions related to the citric acid that this type of analyses can, for pathways and metabolite classes, as well cycle or which enter the citric acid example, be performed in peripheral as the technologies for their unequivocal cycle through other metabolic path­ blood cells during Gleevec treatment identification and quantitation, also may ways. In contrast, the levels of lactic of human patients. In this situation, be relevant to alcohol research. acid (which is produced as an alterna­ SIDMAP analyses of oxidative versus For example, another interesting tive product during glucose nonoxidative pentose metabolism potential application of metabolomic metabolism if the citric acid cycle is potentially may help clinicians predict studies of carbohydrate metabolism not used) greatly increased in both the effect of Gleevec therapy and pro­ to alcohol research is based on obser­ ischemia-susceptible and healthy sub­ vide early indications of resistance to vations suggesting that breakdown jects. As this example demonstrates,

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analyses using such comprehensive Concluding Remarks chemical pathways are interactive, platforms that allow investigators to studies that address their integration unequivocally identify metabolites or The most important value that meta­ and co-regulation may potentially to survey metabolites potentially can bolomics may add to alcohol-associated contribute to a more detailed under­ provide insight into functionally research is the increased number of standing of disease progression and relevant mechanisms contributing individual metabolites within different suggest novel target points for devel­ to disease progression (in this case, metabolite classes that can be analyzed, opment of new medications. hypertension or ischemia). thereby allowing researchers to gain a Traditional biochemical approaches greater understanding of distinct bio­ Additional studies will have to already have provided important con­ chemical processes associated with these determine whether these differences tributions to understanding many of metabolites. For example, by maximiz­ in mitochondrial metabolic products the neural, hepatic, and other diseases ing the number of lipid metabolites associated with alcohol dependence. between people susceptible to ischemia that can be measured, researchers may and other people also may be in some However, the development and appli­ be able to more accurately assess the cation of strategies that maximize the way related to the elevated risk of effects of a drug or medication on lipid hypertension in alcoholics. This notion number of metabolites that can be metabolism compared with using more measured in a single experiment may is supported by the observation that traditional measurements that measure further enhance this understanding, excessive alcohol use may lead to only a small number of entities (e.g., help identify metabolites and meta­ damage of the mitochondria and only cholesterol esters or triglycerides). bolic pathways that likely contribute may promote oxidative stress, partic­ The LIPIDMAPS initiative should ularly in liver cells, thereby contribut­ serve to drive further progress in this area. to the progression of alcoholism, help ing to liver damage (Hoek et al. Moreover, as the example of carbohydrate develop effective interventions, and 2002). Accordingly, it is conceivable metabolism studies and particularly the may even lead to individualized treat­ that alcohol’s adverse effects on the SIDMAP study of Gleevec demonstrates, ment regimes. These potential benefits mitochondria also may be associated such a systems approach to biochemical of metabolomic research in many dis­ with other harmful consequences, analysis of the effects of a specific com­ ease areas now are being widely rec­ such as hypertension. pound may allow quantitative assessment ognized, as shown by the metabolomic Finally, metabolomic approaches of two differentially affected pathways components that are part of the NIH also may be of use for discovering (e.g., downregulation of the oxidative Roadmap Initiative and the NIH biomarkers of alcohol abuse. One branch, and concurrent upregulation of Genes, Environment and Health Initiative. biomarker that already is being used the nonoxidative branch, of the pentose ■ to detect alcohol consumption is a phosphate pathway). compound called ethyl glucuronide Development of strategies that allow Financial Disclosure (EtG). This compound, which is quantitative analyses of diverse classes formed in the liver by linking ethanol of metabolites and their interaction This article was initiated prior to Greg also may prove rewarding. Clearly, with glucuronic acid, remains detectable G. Harrigan’s role at Monsanto and altered metabolism of one class of in serum, plasma, and hair for days does not reflect any ongoing research at metabolites can impinge on the meta­ after alcohol consumption. Several this company. The views expressed are traditional experimental strategies bolism of another class of metabolites (e.g., dysfunction in lipid metabolism solely those of the authors. The authors already are being used to detect EtG likely is associated with changes in declare that they have no competing for forensic purposes. Recently, how­ other metabolite classes). An increased financial interests. ever, researchers demonstrated that understanding of such interactions EtG also can be detected in liver would help researchers to better under­ References extracts from rats exposed to alcohol stand disease progression in different for 4 days using a metabolomic patient groups, such as the numerous AXELSON, M.; CRONHOLM, T.; SAHLBERG, B.L.; approach (i.e., proton NMR) (Nicholas disease subsets among alcohol- AND SJOVALL, J. Changes in the metabolic profile et al. 2006). This study showed that dependent patients. For example, of steroids in urine during ethanol metabolism in proton NMR is capable of detecting man. Journal of Steroid Biochemistry 14:155–159, studies found that perturbations in 1981. PMID: 7206704 EtG and that future NMR-based lipid metabolism can contribute not metabolomic studies of alcohol con­ only to cardiovascular dysfunction and BARAONA, E., AND LIEBER, C.S. Alcohol and lipids. sumption could integrate measurements Recent Developments in Alcoholism 14:97–134, inflammation but also to cognitive 1998. PMID: 9751944 of this metabolite with other NMR- decline (Yaffe et al. 2004). Accordingly, based measurements made simultane­ alcohol’s effects on metabolism in the BERRONE, E.; BELTRAMO, E.; SOLIMINE, C.; ET AL. ously on the same biological samples Regulation of intracellular glucose and polyol path­ brain may, at least in part, be sec­ way by thiamine and benfotiamine in vascular cells to correlate alcohol consumption ondary to or correlate with alcohol’s cultured in high glucose. Journal of Biological with other metabolic alterations. effect on the liver. Because many bio­ Chemistry 281:9307–9313, 2006. PMID: 16452468

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Metabolomics, Alcohol Research, and Drug Development

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