Metabolites, the intermediates and products of metabolism, including energy storage and management molecules, secondary metabolites, and various signaling molecules, are precursors to macromolecules such as proteins and complex carbohydrates. They are also found as regulators of gene expression and thus control which proteins are made in a cell³.

NEED OF METABOLOMICS:

· The proteome cannot be completely predicted from the transcriptome due to some differences in regulatory mechanisms.

· The metabolome is further down the line from gene function and so reflects more closely the activities of a cell at the functional level.

· A metabolite may come from more than one metabolic pathway and it is only when you conduct a study on the metabolome as a whole that you can identify which pathways are involved in its metabolism.

· Metabolomics can be viewed as complementary to transcriptomics and proteomics⁴.

IMPORTANCE OF METABOLOMICS:

· Evolutionary conservation of metabolism across all life forms ensures broad relevance

· Networks of metabolite feedback pathways regulate gene (and protein) expression, also can mediate signaling between organisms.

· Improved understanding of cellular biochemistry.

· Metabolites reflect the combined effects of many influences on physiological function and phenotype (drugs, environment, nutrition, genes).

· Biomarkers of disease (diagnostics).⁵

ADVANTAGES OF METABOLOMICS:

· Identification of target organ, severity, onset, duration and reversal of the effects (time-course).

· Classify sample as “normal” vs. “abnormal”.

· Determine mechanism of action within the organ.

· Potential for identifying novel biomarkers of toxic effect.

· Non-invasive.

· No a priori decisions about samples need be made.

· No sample processing necessary other than cold collection.

· Complete time course data can readily be obtained.

· Minimization of compound requirements.

· Relatively fast analysis (200-300 samples/day).

· Useful tool for modeling physiological variation and exposure conditions in animals and humans⁶.

BASIS OF METABOLITE ANALYSIS:

1. Select samples (biofluids,complex tissues, cells, etc.).

2. Extract metabolites from matrix.

3. Separate metabolites (chromatography).

4. Detect and characterize individual metabolites (eg mass spectrometry or NMR analysis).

5. Quantify and perform data analysis.

6. Generate a new ‘analytical tool’ for further scientific studies.

Classification of Endogenous Metabolite Analysis:⁷

a) Metabolic Fingerprinting:

Aim: Identify sufficient metabolites in a specific tissue to classify unknown samples into identifiable groups.

Outcomes: Screen lines in breeding program or in clinical analysis, to identify patterns

• Selective sample clean-up to reduce unwanted interference from sample matrix

• Sensitivity is not an issue, only looking for major differences

• Separation method: GC and/ or LC

• Typical instrumentation: GC-MS, LC- MS (tandem preferred) NMR or LC-NMR⁸.

b) Metabolite/ Metabolic Profiling:

Aim: Quantify known metabolites of specific classes from biochemical pathways, in selected tissues

Outcomes: Elucidate function of pathways or links to other pathways; functional genomic screening

• Extraction method specially designed for compounds in class to eliminate unwanted/ extraneous metabolites

• High sensitivity method

• Separation method: GC, CE or LC depending on metabolites

• Typical instrumentation: LC-MS (tandem)⁹

c) Metabolomics:

Aim: Quantify/ identify all metabolites in a specific tissue: a comprehensive "snapshot" of metabolism at a particular point in time.

Outcomes: overall effects of treatment or condition on metabolic pathways in an organism- comparison of "snapshots"

• Extraction method: simple and rapid. Must prevent further metabolism and recover all metabolites

• Not as sensitive to minor metabolites as metabolite profiling

• Separation method: GC and LC

• Typical instrumentation: LC-NMR, GC or LC coupled to accurate mass spectrometry eg quad- ToF or FT-MS.

EXTRACTION AND ANALYSIS OF METABOLITES:

• Major components present at mM conc.

– Salts, sugars

• Minor components present at nM conc or less

– Vitamins, metabolic intermediates

Sample Preparation:

• Vital to the metabolomic approach- any bias must be avoided

• First stop biological activity (freeze clamping)

• Apply enzymatic inhibitors before heating

• Separate the components of the metabolome

Typical Metabolite Extraction:

• Selection of sample– age, developmental stage, “treatments” of various types (stress, pathogen, light, temperature, etc.)

• Frozen sample- add reference compounds (internal standards)

• Grind sample with solvent (immediate):

– Water OR water/alcohol OR organic Solvent

• Centrifuge or filter to remove debris

• Possibly further liquid or solid phase extraction

• Analysis by chromatography – MS¹⁰

Metabolomic studies generally use bio fluids or cell or tissue extracts, which are usually readily available. Urine and plasma are obtained essentially noninvasively, and hence can be obtained more easily for use in disease diagnosis and in clinical trials for monitoring drug therapy. However, many other fluids have been studied, including seminal fluids, amniotic fluid, cerebrospinal fluid, synovial fluid, digestive fluids, blister and cyst fluids, lung aspirates, and dialysis fluids¹¹. In general, there are four important issues to be addressed for metabolite analysis: namely,

1. Efficient and unbiased extraction of metabolites from biological tissues.

2. Separation of the analytes, usually by Chromatograpy and Electrophoresis.

3. Detection of the analytes and

4. Identification and quantification of the analytes.

Amongst the most widely employed analytical techniques are Nuclear Magnetic Resonance (NMR) spectroscopy and Mass Spectometry (MS). MS requires a separation of the metabolomic components using either Gas chromatography (GC) after chemical derivatization, or liquid chromatography (LC)¹².

Separation Techniques:

Mainly there are four techniques for separating analytes from biological tissues:

1) Gas Chromatography (GC):

Gas chromatography (GC) is a separation technique in which its the mobile phase is a gas. Gas chromatography is always carried out in a column, which is typically "packed" or "capillary" (see Figure 1). Gas chromatography is based on partition equilibrium of analyte between a solid stationary phase (often a liquid silicone-based material) and a mobile gas (most often Helium). The stationary phase is adhered to the inside of a small-diameter glass tube (a capillary column) or a solid matrix inside a larger metal tube (a packed column).

GS, especially when interfaced with mass spectrometry (GC-MS), is one of the most widely used and powerful methods. It offers very high chromatographic resolution, but requires chemical derivatization for many biomolecules: only volatile chemicals can be analyzed without derivatization. (Some modern instruments allow '2D' chromatography, using a short polar column after the main analytical column, which increases the resolution still further). Some large and polar metabolites cannot be analyzed by GC¹³. Gas chromatography is also sometimes known as vapor-phase chromatography (VPC), or gas-liquid partition chromatography (GLPC) and Gas-Liquid chromatography (GLC). These alternative names, as well as their respective abbreviations, are frequently found in scientific literature. Strictly speaking, GLPC is the most correct terminology, and is thus preferred by many authors¹⁴.

Figure 1: Gas Chromatography

2) Capillary Electrophoresis (CE):

CE is used to separate ionic species by their charge and frictional forces. In traditional electrophoresis, electrically charged analytes move in a conductive liquid medium under the influence of an electric field. Introduced in the 1960s, the technique of capillary electrophoresis (CE) was designed to separate species based on their size to charge ratio in the interior of a small capillary filled with an electrolyte. The instrumentation needed to perform capillary electrophoresis is relatively simple. A basic schematic of a capillary electrophoresis system is shown in figure 2. The system's main components are a sample vial, source and destination vials, a capillary, electrodes, a high- voltage power supply, a detector, and a data output and handling device. The source vial, destination vial and capillary are filled with an electrolyte such as an aqueous buffer solution. To introduce the sample, the capillary inlet is placed into a vial containing the sample and then returned to the source vial (sample is introduced into the capillary via capillary action, pressure, or siphoning). The migration of the analytes is then initiated by an electric field that is applied between the source and destination vials and is supplied to the electrodes by the high-voltage power supply. It is important to note that all ions, positive or negative, are pulled through the capillary in the same direction by electroosmotic flow. The analytes separate as they migrate due to their electrophoretic mobility and are detected near the outlet end of the capillary. The output of the detector is sent to a data output and handling device such as an integrator or computer. The data is then displayed as an electropherogram, which reports detector response as a function of time. Separated chemical compounds appear as peaks with different retention times in an electropherogram¹⁵.

Figure 2: Electrophoresis

3) High Performance Liquid Chromatography (HPLC) and Ultra performance Liquid Chromatography (UPLC):

Liquid chromatography (LC) is a separation technique in which the mobile phase is a liquid. Liquid chromatography can be carried out either in a column or a plane. Present day liquid chromatography that generally utilizes very small packing particles and a relatively high pressure is referred to as (HPLC). In the HPLC technique, the sample is forced through a column that is packed with irregularly or spherically shaped particles or a porous monolithic layer (stationary phase) by a liquid (mobile phase) at high pressure. For metabolomics applications on bio fluids, an HPLC chromatogram is generated with MS detection, usually using electrospray ionization, and both positive and negative ion chromatograms can be measured. At each sampling point in the chromatogram there is a full mass spectrum and so the data is three-dimensional in nature, i.e., retention time, mass and intensity. Given this very high resolution it is possible to cut out any mass peaks from interfering substances such as drug metabolites, essentially without affecting the structure of the dataset¹⁶.

Figure 3: High Performance Liquid Chromatography

Description of each components of HPLC:

Injection of the sample:

Injection of the sample is entirely automated. Because of the pressures involved, it is not the same as in gas chromatography.

Retention time:

The time taken for a particular compound to travel through the column to the detector is known as its retention time. This time is measured from the time at which the sample is injected to the point at which the display shows a maximum peak height for that compound. Different compounds have different retention times. For a particular compound, the retention time will vary depending on:

· The pressure used (because that affects the flow rate of the solvent)

· The nature of the stationary phase (not only what material it is made of, but also particle size)

· The exact composition of the solvent

· The temperature of the column

That means that conditions have to be carefully controlled if you are using retention times as a way of identifying compounds.

The detector:

There are several ways of detecting when a substance has passed through the column. A common method which is easy to explain uses ultra-violet absorption. Many organic compounds absorb UV light of various wavelengths. If you have a beam of UV light shining through the stream of liquid coming out of the column, and a UV detector on the opposite side of the stream, you can get a direct reading of how much of the light is absorbed.

The limitation of HPLC techniques can be minimized by improving the efficiency of the chromatography and this has been achieved using UPLC. The example given below illustrate this phenomenon. A combination of a 1.7m reversed-phase packing material, and a chromatographic system operating at around 827.4 bar. UPLC provides around a 10 fold increase in sensitivity compared to a conventional stationary phase. A comparison of data generated using both HPLC-MS and UPLC-MS is given in the below figure-4¹⁷.

Figure 4: Comparison between HPLC and UPLC

Detection Techniques:

1) Nuclear Magnetic Resonance (NMR) spectroscopy:

Nuclear magnetic resonance (NMR) is the name given to a physical resonance phenomenon involving the observation of specific quantum mechanical magnetic properties of an atomic nucleus in the presence of an applied, external magnetic field. NMR is the only detection technique which does not rely on separation of the analytes, and the sample can thus be recovered for further analyses. All kinds of small molecule metabolites can be measured simultaneously in this sense; NMR is close to being a universal detector. Practically, however, it is relatively insensitive compared to mass spectrometry-based techniques; additionally, NMR spectra can be very difficult to interpret for complex mixtures. A key feature of NMR is that the resonance frequency of a particular substance is directly proportional to the strength of the applied magnetic field¹⁸. NMR spectroscopy provides detailed information on molecular structure, both for pure compounds and in complex mixtures, but it can also be used to probe metabolite molecular dynamics and mobility through the interpretation of NMR spin relaxation times and by the determination of molecular diffusion coefficients. Most applications of NMR involve full NMR spectra, that is, the intensity of the NMR signal as a function of frequency.

Figure 5: NMR experimental setting

The NMR experiment:

A current through the main coil (green) generates a strong magnetic field that polarizes the nuclei in the sample material (red). It is surrounded by the r.f. coil (black) that delivers the computer generated r.f. tunes that initiate the nuclear quantum dance. At some point in time, the switch is turned and now the dance is recorded through the voltage it induces, the NMR signal, in the r.f. coil. The signals Fourier transform (FT) shows "lines" for different nuclei in different electronic environments. The field produced by the main coil is powered by a sudden discharge of a capacitor bank, our power supply. From the Fourier transform (FT) of the NMR signal we can identify the nuclei, count them, detect their nuclear neighbors; we can measure the fine details of the electronic structure of the material through the interactions the nuclei have with the surrounding electrons.

For example, the H NMR spectra of urine show thousands of sharp peaks from predominantly small-molecule metabolites, whereas spectra of blood plasma and serum show broad bands from protein and lipoprotein signals, with sharp peaks from small molecules superimposed thereon. A typical 950-MHz H NMR spectrum of usine showing the degree of spectral complexity is given in the below figure-6¹⁹.

Figure 6: 950 - MHz H NMR Spectrum

2) Mass spectrometry (MS):

MS is used to identify and to quantify metabolites after separation by GC, HPLC, or CE. GC-MS is the most 'natural' combination of the three, and was the first to be developed. In addition, mass spectral fingerprint libraries exist or can be developed that allow identification of a metabolite according to its fragmentation pattern. MS is both sensitive (although, particularly for HPLC-MS, sensitivity is more of an issue as it is affected by the charge on the metabolite, and can be subject to ion suppression artifacts) and can be very specific. There are also a number of studies which use MS as a stand-alone technology: the sample is infused directly into the mass spectrometer with no prior separation, and the MS serves to both separate and to detect metabolites. The MS principle consists of ionizing chemical compounds to generate charged molecules or molecule fragments and measurement of their mass-to-charge ratios. In a typical MS procedure, a sample is loaded onto the MS instrument, and its compounds are ionized by different methods (e.g., by impacting them with an electron beam), resulting in the formation of charged particles (ions). The mass-to-charge ratio of the particles is then calculated from the motion of the ions as they transit through electromagnetic fields. MS instruments consist of three basic components: an ion source, which splits the sample molecules into ions; a mass analyzer, which sorts the ions by their masses by applying electromagnetic fields; and a detector, which measures the value of an indicator quantity and thus provides data for calculating the abundances of each ion present.

Figure 7: Main steps of measuring with a mass spectrometer

Although both NMR spectroscopy and mass spectrometry have been widely used in metabolic profiling studies, each has its own merit and limitation. Table 1 presents a comparison of these two highly complementary approaches²⁰.

Applications:

Toxicity assessment/toxicology. Metabolic profiling (especially of urine or blood plasma samples) can be used to detect the physiological changes caused by toxic insult of a chemical (or mixture of chemicals). In many cases, the observed changes can be related to specific syndromes, e.g. a specific lesion in liver or kidney. This is of particular relevance to pharmaceutical companies wanting to test the toxicity of potential drug candidates: if a compound can be eliminated before it reaches clinical trials on the grounds of adverse toxicity, it saves the enormous expense of the trials²¹.

Functional genomics. Metabolomics can be an excellent tool for determining the phenotype caused by a genetic manipulation, such as gene deletion or insertion. Sometimes this can be a sufficient goal in itself-for instance, to detect any phenotypic changes in a genetically-modified plant intended for human or animal consumption. More exciting is the prospect of predicting the function of unknown genes by comparison with the metabolic perturbations caused by deletion/insertion of known genes. Such advances are most likely to come from model organisms such as Saccharomyces cerevisiae and Arabidopsis thaliana^{22, 23}.

Nutrigenomics is a generalized term which links genomics, transcriptomics, proteomics and metabolomics to human nutrition. In general a metabolome in a given body fluid is influenced by endogenous factors such as age, sex, body composition and genetics as well as underlying pathologies. The large bowel microflora are also a very significant potential confounder of metabolic profiles and could be classified as either an endogenous or exogenous factor. The main exogenous factors are diet and drugs. Diet can then be broken down to nutrients and non- nutrients. Metabolomics is one means to determine a biological endpoint, or metabolic fingerprint, which reflects the balance of all these forces on an individual's metabolism²⁴.

Metabolomics has become a versatile technique that is widely used by academia and industry in the medical, toxicological, nutritional, and other biological sciences.

Clinical Applications of Metabolomics in Oncology: There is potential for the metabolome to have a multitude of uses in oncology, including the early detection and diagnosis of cancer and as both a predictive and pharmacodynamic marker of drug effect.

Metabolomics offers a promising approach for biomarker-driven drug discovery and development²⁵.

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Received on 30.07.2010

Accepted on 11.08.2010

Research J. Pharmacology and Pharmacodynamics. 2(6): Nov. –Dec. 2010, 363-369