title: Analysis of multiple metabolomic subsets in vitro: methodological considerations.
abstract: Abstract Metabolomic analysis is a technology which seeks to provide a comprehensive profile of all the metabolites present in a biological sample ( Fiehn 2002 ; Noguchi et al. 2003 ; Watkins et al. 2002 ). Metabolomics is not a new science, but emerging as an exciting application which can span the scope of biotechnology and medicine, providing metabolic profile and a complement to the genomic and proteomic data ( German et al. 2003 ). Studies have already begun to explore the effects of toxicological, pharmaceutical, nutritional, and environmental intervention and to build integrated databases of metabolite concentrations throughout biological systems, ranging from microbes to plants to human and research animal populations ( Bruskiewich et al. 2003 ; Bailey et al. 2003 ; Fiehn 2003 ; Novotna et al. 2003 ). This may provide a tool for discovering a novel pathway, or determining the relationship of these metabolite concentrations to disease, and the extent to which nutrition can modulate metabolite concentrations ( Weckwerth and Fiehn 2002 ). Metabolites are the products of enzymatic processes. Their levels can be regarded as the ultimate response of biological systems to genetic or environmental changes ( Fiehn 2002 ). While the amount of an enzyme protein in a biological system could be quantified by using specific antibodies or by measuring the mRNA responsible for their production, factors such as the availability of cofactors and coenzymes, feedback regulation, pH, compartmentalization, etc. could affect their activity, thus, in turn, affect the metabolite's (i.e., substrates and their enzymatic products) levels. There have been a number of different interpretations of "metabolites." In pharmacological and toxicological studies, metabolites could be considered as the enzymatic degradative products of drugs ( Nishikawa et al. 2003 ; Plumb et al. 2002 ; Reo 2002 ; Robosky et al. 2002 ; Slim et al. 2002 ). In other studies, metabolites could be referred to as amino acids ( Noguchi et al. 2003 ), keto acids ( Bailey et al. 2003 ), or lipids ( German et al. 2003 ), which are substrates and products of selected metabolic pathways. Noguchi et al. (2003) proposed using "subset" to indicate the type of metabolites to be studied. Thus, the amino acid subset, for example, would indicate the levels of all the amino acids in a sample and lipomic subset would indicate the levels of all metabolites involved in lipid metabolism. In biochemical, physiological, and toxicological studies, animal models are preferred. In fact, a large amount of information on metabolites balance has been accumulated for a number of pharmacological, toxicological, and pathological conditions ( Henry et al. 1985 ; Needleman et al. 1968 ; Tourtellotte et al. 1966 ; McDougal, Jr. et al. 1997 ; Yang et al. 1985 ; King et al. 1967 ; Lowry et al. 1983 ; Gupta et al. 2000 ; Gupta et al. 2001a ,b; Gupta and Dettbarn 2003). Only recently has attention been directed towards studying metabolites in cell culture. Cell culture, with intact membrane and cytoplasmic organelles that operate under well understood biochemical and molecular events, provides a simple model that is best used for studying the basic mechanisms of toxicological and pharmacological actions of xenobiotics. Other advantages of using cell culture are to lighten the heavy workload and complications involved in performing animal studies, as well as to reduce the use of animals, which is also advocated on the ground of good animal ethics. To correlate the results in vivo, the data need to be transformed taking into consideration the level of toxicant at the site of action.
authors.affiliation: Department of Biology, Hong Kong Baptist University, Kowloon Tong, Kowloon, Hong Kong, P.R.C.
authors.forename: M S
authors.initials: MS
authors.lastname: Yang
authors.forename: L C
authors.initials: LC
authors.lastname: Yu
authors.forename: R C
authors.initials: RC
authors.lastname: Gupta