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Metabolic fingerprinting of Lactobacillus paracasei: the optimal quenching strategy.

Jäpelt KB, Christensen JH, Villas-Bôas SG - Microb. Cell Fact. (2015)

Bottom Line: However, methanol is known to cause intracellular metabolite leakage of microbial cells, making the distinction between intra- and extracellular metabolites in microbial systems challenging.The implementation of a reliable, reproducible quenching method is essential within the metabolomics community.Cold glycerol saline prevented leakage of intracellular metabolites, and, thus, allowed more accurate determinations of intracellular metabolite levels.

View Article: PubMed Central - PubMed

Affiliation: Analytical Chemistry Group, Department of Plant and Environmental Sciences, Faculty of Science, University of Copenhagen, Copenhagen, Denmark. dkkba@chr-hansen.com.

ABSTRACT

Background: Quenching in cold buffered methanol at -40 °C has long been the preferred method for sub-second inactivation of cell metabolism during metabolic fingerprinting. However, methanol is known to cause intracellular metabolite leakage of microbial cells, making the distinction between intra- and extracellular metabolites in microbial systems challenging. In this paper, we tested three quenching protocols proposed for microbial cultures: fast filtration, cold buffered methanol and cold glycerol saline.

Results: Our results clearly showed that cold glycerol saline quenching resulted in the best recovery of intracellular metabolites in Lactobacillus paracasei subsp. paracasei (L. paracasei). Membrane integrity assayed by propidium iodide revealed that approximately 10 % of the L. paracasei cell membranes were damaged by contact with the cold buffered methanol solution, whilst cold glycerol saline quenching led to minimal cell damage. Due to the nature of the L. paracasei culture, fast filtration took several minutes, which is far from ideal for metabolites with high intracellular turnover rates.

Conclusion: The implementation of a reliable, reproducible quenching method is essential within the metabolomics community. Cold glycerol saline prevented leakage of intracellular metabolites, and, thus, allowed more accurate determinations of intracellular metabolite levels.

No MeSH data available.


Related in: MedlinePlus

Top Ratio between the average biomass-normalised peak heights of glycerol saline and that of cold buffered methanol. Bottom RSD of the normalized peak heights for each metabolite across all biological replicates (n = 6) for cold buffered methanol and glycerol saline quenching. In this case, the abundance of each metabolite was normalized to the biomass, as well as to the total sum of peak height for all metabolites having a RSD below 20 % across all samples for both cold buffered methanol and glycerol saline quenching
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Fig3: Top Ratio between the average biomass-normalised peak heights of glycerol saline and that of cold buffered methanol. Bottom RSD of the normalized peak heights for each metabolite across all biological replicates (n = 6) for cold buffered methanol and glycerol saline quenching. In this case, the abundance of each metabolite was normalized to the biomass, as well as to the total sum of peak height for all metabolites having a RSD below 20 % across all samples for both cold buffered methanol and glycerol saline quenching

Mentions: A semi-quantitative comparison of the metabolite levels obtained by the two quenching methods was performed. Figure 3 (top) presents the log ratio of biomass-normalised peak heights of intracellular metabolites from glycerol saline quenching to cold buffered methanol quenching. Ratios above 0 indicates that this metabolite is present in glycerol saline quenching in a higher level than in cold buffered methanol quenching and vice versa for values below 0. In particular, the more hydrophilic compounds, such as organic acids and amino acids, were recovered in higher levels during glycerol saline quenching, whereas the longer chain fatty acids, such as palmitic acid (C16:0) and stearic acid (C18:0), were obtained in higher levels for the cold buffered methanol quenching. This might partly be attributed to the preferential extraction of the more hydrophilic metabolites by methanol through permeabilization of the membrane causing the decreased levels which is consistent with previous findings [17]. The precision of the metabolic fingerprints, as measured by the RSD of the peak heights normalized to the biomass and the total sum of reproducible peak height for all replicates, demonstrated that glycerol saline was more reproducible than cold buffered methanol. Nearly all metabolites had a higher RSD after cold buffered methanol quenching (median RSD 11 % for glycerol saline and 20 % for buffered methanol), see Fig. 3 (bottom).Fig. 3


Metabolic fingerprinting of Lactobacillus paracasei: the optimal quenching strategy.

Jäpelt KB, Christensen JH, Villas-Bôas SG - Microb. Cell Fact. (2015)

Top Ratio between the average biomass-normalised peak heights of glycerol saline and that of cold buffered methanol. Bottom RSD of the normalized peak heights for each metabolite across all biological replicates (n = 6) for cold buffered methanol and glycerol saline quenching. In this case, the abundance of each metabolite was normalized to the biomass, as well as to the total sum of peak height for all metabolites having a RSD below 20 % across all samples for both cold buffered methanol and glycerol saline quenching
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4559878&req=5

Fig3: Top Ratio between the average biomass-normalised peak heights of glycerol saline and that of cold buffered methanol. Bottom RSD of the normalized peak heights for each metabolite across all biological replicates (n = 6) for cold buffered methanol and glycerol saline quenching. In this case, the abundance of each metabolite was normalized to the biomass, as well as to the total sum of peak height for all metabolites having a RSD below 20 % across all samples for both cold buffered methanol and glycerol saline quenching
Mentions: A semi-quantitative comparison of the metabolite levels obtained by the two quenching methods was performed. Figure 3 (top) presents the log ratio of biomass-normalised peak heights of intracellular metabolites from glycerol saline quenching to cold buffered methanol quenching. Ratios above 0 indicates that this metabolite is present in glycerol saline quenching in a higher level than in cold buffered methanol quenching and vice versa for values below 0. In particular, the more hydrophilic compounds, such as organic acids and amino acids, were recovered in higher levels during glycerol saline quenching, whereas the longer chain fatty acids, such as palmitic acid (C16:0) and stearic acid (C18:0), were obtained in higher levels for the cold buffered methanol quenching. This might partly be attributed to the preferential extraction of the more hydrophilic metabolites by methanol through permeabilization of the membrane causing the decreased levels which is consistent with previous findings [17]. The precision of the metabolic fingerprints, as measured by the RSD of the peak heights normalized to the biomass and the total sum of reproducible peak height for all replicates, demonstrated that glycerol saline was more reproducible than cold buffered methanol. Nearly all metabolites had a higher RSD after cold buffered methanol quenching (median RSD 11 % for glycerol saline and 20 % for buffered methanol), see Fig. 3 (bottom).Fig. 3

Bottom Line: However, methanol is known to cause intracellular metabolite leakage of microbial cells, making the distinction between intra- and extracellular metabolites in microbial systems challenging.The implementation of a reliable, reproducible quenching method is essential within the metabolomics community.Cold glycerol saline prevented leakage of intracellular metabolites, and, thus, allowed more accurate determinations of intracellular metabolite levels.

View Article: PubMed Central - PubMed

Affiliation: Analytical Chemistry Group, Department of Plant and Environmental Sciences, Faculty of Science, University of Copenhagen, Copenhagen, Denmark. dkkba@chr-hansen.com.

ABSTRACT

Background: Quenching in cold buffered methanol at -40 °C has long been the preferred method for sub-second inactivation of cell metabolism during metabolic fingerprinting. However, methanol is known to cause intracellular metabolite leakage of microbial cells, making the distinction between intra- and extracellular metabolites in microbial systems challenging. In this paper, we tested three quenching protocols proposed for microbial cultures: fast filtration, cold buffered methanol and cold glycerol saline.

Results: Our results clearly showed that cold glycerol saline quenching resulted in the best recovery of intracellular metabolites in Lactobacillus paracasei subsp. paracasei (L. paracasei). Membrane integrity assayed by propidium iodide revealed that approximately 10 % of the L. paracasei cell membranes were damaged by contact with the cold buffered methanol solution, whilst cold glycerol saline quenching led to minimal cell damage. Due to the nature of the L. paracasei culture, fast filtration took several minutes, which is far from ideal for metabolites with high intracellular turnover rates.

Conclusion: The implementation of a reliable, reproducible quenching method is essential within the metabolomics community. Cold glycerol saline prevented leakage of intracellular metabolites, and, thus, allowed more accurate determinations of intracellular metabolite levels.

No MeSH data available.


Related in: MedlinePlus