Criterio para la Elaboración de la Propuesta

Taking a step upward from clinical chemistry metabolomics does not look to define one metabolite or even just two for that manner. It’s power comes in the resolution of literally hundreds of metabolites simultaneously [300], making it “ultrahigh-throughput.”[301] Furthermore, historically, one perceives the cell as a unit, but the observation of metabolites can be subdivided into extracellular and intracellular aspects both following nomenclature that is developing as techniques and methods develop around phenotyping methodologies. Each type of cell can have an appreciated phenotype based on the capacity to metabolize given compounds. In fact, the hundreds of specialized human cell types interact differently with

environmental factors to influence day to day functions, and furthermore play a role in development, pathophysiological processes, and certainly homeostatic

maintenance through daily metabolic processes [302]

Metabolites by definition are the end products as effect by cellular process on present compounds. Metabolites fall into defined classes of compounds such as polar lipids and carbohydrates [303]. Endogenous metabolism follows all energy

62

generating and biosynthetic pathways by which host cells modify their environment from a proteomics and genetic point of view [302]. It is well known that every organism from microorganisms to humans excretes metabolites into their external environment. An analysis of distribution of the metabolome of several species suggests that the molecular weights of interest by any detection method pursued generally falls with a capped range of under 300 Daltons [304]. As a discrete method of discriminating the difference between what occurs within the cell

(internally detectable metabolites) and outside of the cell (extracellular metabolites excreted by the cell) Kell and others proposed the nomenclature of “footprinting and fingerprinting,” where footprinting specifically monitors metabolites consumed and secreted into the extracellular compartment. Furthermore, environmental

provocations of the metabolic pathways of any system can be analyzed by the excretion of metabolites into the extracellular space. These profiles are highly specific to species and genetic backgrounds [305]. This method was and is

particularly useful in describing microorganisms at the metabolic level based on its genome, and was forwarded as a technology for high throughput analysis even without identification of particular metabolites. Remarkably, single gene knockouts were distinguished from wild type organisms in subsequent studies [305].

Furthermore, the discrimination between seemingly similar phenotypes has been applied to transgenic yeast, plants and mice [300] Fingerprinting looks at both inside and outside the cells/tissues of interest. In respect, only looking at the footprint offers advantages over total metabolic analysis (looking at both

63

the system and thus the complexity added by the intracellular metabolites. Those metabolites excreted into the environment are usually long lived as compared to that of turnover rates of intracellular metabolites, which doesn’t require a stabilization and fast quenching method for isolation, which most of the time are time consuming and impede reproducibility because of added complexity [305].

As to what can be learned by looking at the metabolites of a closed system, it starts with a mass balance and a careful look at fluxomics, where metabolites are used to report the end response of the given biological system to environment and genetic changes. In this way, a scientist can trace the perturbations of a system and the concurrent changes within the cells (ie stem cell differentiation). Prediction of cellular order and control is strongly stressed by the methodologies of metabolytic pathways, which when diagrammed represent cell capacity and interchange within the environment. In such a manner, mapping of metabolites can be used as an insight into the cells biotransformation capabilities [302]. Reflections of the metabolome response are caught in the rippled effects of system perturbations throughout the transcriptome, proteome and metabolome. Often the result of small changes in each upstream event cause greater changes in the downstream event. Consequently, the metabolome exemplifies conditions where perturbations become more responsive extending through mechanisms of both the transcriptome and the proteome. [304] These events, are helpful in inferring genetic functionality by metabolome inspection, where changes in the levels of enzymatic activity have occurred. Much of the early work revolved around finding a single metabolite and then correlating it to other known expressions of cell activity [306] As with the other

64

“omics” the study of the metabolites lend to the accomplishment of understanding systems biology better.[307, 308]

Conditionally, many different devices have been used to look at the metabolome of various cell types and organs, producing signatures of the

biomaterial. Listed as various methods inclusive of NMR, are infrared spectroscopy, thin layer chromatography, high performance liquid chromatography with

ultraviolet and photodiode array detection, capillary electrophoresis coupled to ultraviolet absorbance detection, capillary electrophoresis coupled to laser induced fluorescence detection, capillary electrophoresis coupled to mass spectrometry, gas chromatography-mass spectrometry, liquid chromatography-mass spectrometry, liquid chromatography tandem mass spectrometry, Fourier transform ion cyclotron mass spectrometry, high performance liquid chromatography coupled with both mass spectrometry and nuclear magnetic resonance detection [299, 306]. No matter what the technique, the method chosen must maintain sensitivity, selectivity, and universal applicability. Early metabolic profiling is often credited to Devaux, Horning and Horning of the Baylor College of Medicine [306]. The metabolome is described as the full set of metabolites within or secreted by a specific cell type or [302]. Applications of various methodologies have led to the systematic methods of identifying and quantifying those recognized metabolites within a given system.[303, 308] Robertson describes metabolomics as being ‘‘the quantitative measurement of the time related multi-parametric metabolic response of living systems to

pathophysiological stimuli or genetic modification.’’[308] Jeremy Nicholson further demarks the difference between metabolomics and metabonomics, where

65

“Metabolomics is the measurement of metabolite concentrations, fluxes and

secretions in cells and tissues in which there is a direct connection between the gene expression, protein activity and the metabolic activity itself and metabonomics is the quantitative measurement of the multivariate metabolic responses of multicellular systems to pathophysiological stimuli or genetic modification, [302]” and that continues to be on ongoing debate. Terminally the debate ends where one is the study of and the second is the application of the technology of phenotyping to specific physiological process. And as follows the metabolome is the set of

metabolites that an organism can synthesize[303, 309] and generally falls within a low molecular weight.[304]