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Abstract
The global pool of all metabolites in a cell, or metabolome, is a reflection of all
the metabolic functions of an organism under any particular growth condition. In the
absence of in situ methods capable of universally measuring metabolite pools, intracellular
metabolite measurements need to be performed in vitro after extraction. In the past,
a variety of cell lysis methods were adopted for assays of individual metabolites
or groups of intermediates in pathways. In this study, metabolites were extracted
from Escherichia coli using six different commonly used procedures including acid
or alkaline treatments, permeabilization by freezing with methanol, high-temperature
extraction in the presence of ethanol or methanol, and by lysis with chloroform-methanol.
Metabolites were extracted by the six methods from cells grown under identical conditions
and labeled with [14C]glucose. The metabolomes were compared after 2-dimensional thin-layer
chromatography of labeled compounds. For global analysis, extraction with cold (-40
degrees C) methanol showed the greatest promise, allowing simultaneous resolution
of more than 95 metabolite spots. In contrast, 80 or less spots were obtained with
other extraction methods. Extraction also influenced quantitative analysis of particular
compounds. Metabolites such as adenosine exhibited up to 20-fold higher abundance
after cold methanol extraction than after extraction with acid, alkali, or chloroform.
The simplicity, rapidity, and universality of cold methanol extraction offer great
promise if a single method of lysis is to be adopted in metabolome analysis.
Copyright 2003 Elsevier Science (USA)