Important steps in metabolic pathways are formed by the transport of substrates and products over the cell membrane. The study of in vivo transport kinetics requires accurate quantification of intra- and extracellular levels of the transported compounds. Especially in case of extracellular abundance, the proper determination of intracellular metabolite levels poses challenges. Efficient removal of extracellular substrates and products is therefore important not to overestimate the intracellular amounts. In this study we evaluated two different rapid sampling methods, one combined with cold filtration and the other with centrifugation, for their applicability to determine intracellular amounts of metabolites which are present in high concentrations in the extracellular medium. The filtration-based method combines fast sampling and immediate quenching of cellular metabolism in cold methanol, with rapid and effective removal of all compounds present outside the cells by means of direct filtration and subsequent filtration-based washing. In the centrifugation-based method, removal of the extracellular metabolites from the cells was achieved by means of multiple centrifugation and resuspension steps with the cold quenching solution. The cold filtration method was found to be highly superior to the centrifugation method to determine intracellular amounts of metabolites related to penicillin-G biosynthesis and allowed the quantification of compounds of which the extracellular amounts were 3-4 orders of magnitude higher than the intracellular amounts. Using this method for the first time allowed to measure the intracellular levels of the side chain precursor phenylacetic acid (PAA) and the product penicillin-G of the penicillin biosynthesis pathway, compounds of which the transport mechanism in Penicillium chrysogenum is still far from being sufficiently understood.

A sampling procedure for quantitative metabolomics in Penicillium chrysogenum based on cold aqueous methanol quenching was re-evaluated and optimized to reduce metabolite leakage during sample treatment. The optimization study included amino acids and intermediates of the glycolysis and the TCA-cycle. Metabolite leakage was found to be minimal for a methanol content of the quenching solution (QS) of 40% (v/v) while keeping the temperature of the quenched sample near −20°C. The average metabolite recovery under these conditions was 95.7% (±1.1%). Several observations support the hypothesis that metabolite leakage from quenched mycelia of P. chrysogenum occurs by diffusion over the cell membrane. First, a prolonged contact time between mycelia and the QS lead to a somewhat higher extent of leakage. Second, when suboptimal quenching liquids were used, increased metabolite leakage was found to be correlated with lower molecular weight and with lower absolute net charge. The finding that lowering the methanol content of the quenching liquid reduces metabolite leakage in P. chrysogenum contrasts with recently published quenching studies for two other eukaryotic micro-organisms. This demonstrates that it is necessary to validate and, if needed, optimize the quenching conditions for each particular micro-organism.Electronic supplementary materialThe online version of this article (doi:10.1007/s11306-011-0367-3) contains supplementary material, which is available to authorized users.

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