3.1.3 Absolute quantitation of intracellular metabolite
concentrations
The absolute concentration of the intracellular metabolites was used to
compare the efficiency of extraction solvents. We found that different
extraction solvents differed in extraction efficiencies of specific
intracellular metabolites. In terms of amino acid recovery, we analyzed
18 amino acids by GC-MS and observed a clear trend for amino acids. The
largest amount of amino acids was extracted when the liquid nitrogen as
the quenching agent and 50% cold acetonitrile as the extractant.
However, the extraction of amino acids with cold methanol as the
quenching agent was very poor, which was caused by the serious leakage
of amino acids mentioned above. Also, methanol/chloroform (M/C) had a
poor extraction effect on intracellular amino acids. This may be
ascribed to the weak polarity of methanol/chloroform and low solubility
of amino acids. M/C extraction, which was originally used to extract
non-polar metabolites [59], such as fatty acids [18]. Hot
ethanol extraction was proved to be as effective as acetonitrile for
amino acid recovery, but there were differences in the selective
recovery of specific amino acids such as serine (Ser), aspartate (Asp),
asparagine (Asn) (Figure 7 ). Overall, amino acids were more
soluble in water-based extractions (methanol, acetonitrile, and hot
ethanol) which was consistent with previous study [26].
For organic acids and sugar phosphates, cold methanol as the quencher
also behaved poor extraction effects and extracted the least amount. The
recovery of fumarate (FUM), oxaloacetate (OAA) and phosphoenolpyruvic
acid (PEP) was relatively low regardless of the type of extractants. For
the rest organic acids such as pyruvic acid (PYR), malate (MAL) and
α-ketoglutarate (α KG), using cold methanol and hot ethanol as the
extractant can achieve better extraction results. Among sugar
phosphates, the extraction effects of glyceraldehyde 3-phosphate (G3P),
3-phosphoglycerate (3PG), erythrose-4-phosphate (E4P) and
fructose-1,6-bisphosphate (FBP) were generally poor. When liquid
nitrogen and acetonitrile was used as the quencher and the extractant
respectively, the extraction amount of 6-phosphogluconate (6PG) and
sedoheptulose-7-phosphate (S7P) was the highest, and the extracted S7P
even reached about 5.89 nmol/million cells, which was 8.96 times to
295.69 times higher than other extraction methods. This method was also
suitable for the extraction of glucose-6-phosphate (G6P) and
fructose-6-phosphate (F6P) which was only slightly less than normal
saline quenching - 80% methanol extraction (S-M) and liquid nitrogen
quenching - 80% methanol extraction (N2-M)
(Figure 8 ).
Finally, high temperature in some extraction methods might affect the
recovery of thermally unstable metabolites. Nevertheless, in our study,
we did not observe that hot ethanol obviously caused the adverse effect
regarding the number and amount of detected metabolites (Figure
8 ). One previous study has reported that several metabolites (PYR,
nucleotides and sugar phosphates) were unstable in hot ethanol [22].
However, Sellick et al. found that hot ethanol had a good performance on
recovering fatty acids such as stearic acid and palmitic acid because of
the increased solubility of fatty acids [26]. To demonstrate the
specific effects of heat extraction, we focused on the levels of the
labile metabolite nicotinamide adenine dinucleotide (NAD)
(Figure 8 ). The recovery of intracellular NAD clearly showed
that liquid nitrogen quenching - 50% acetonitrile extraction
(N2-A) and normal saline quenching - 50% acetonitrile
extraction (S-A) recovered relatively larger amounts of NAD, hot ethanol
extraction was slightly inferior, but better than methanol and M/C as
extractants. In our study, M/C was poor in the extraction of each
metabolite although previous study showed certain advantages in the
extraction of cholesterol and fatty acids [57]. Therefore, these
data emphasized the importance of using the appropriate
quenching-extraction process for the extraction of specific metabolites.
An ideal extraction method should recover the metabolites as much as
possible; however, one extraction method in general was not capable of
allowing a complete metabolite extraction. For this purpose, a combined
extraction process might be an alternative to increase the number and
amount of metabolites. However, the reduction of accuracy and
reproducibility of the combination method in extraction was also an
undisputable fact, which largely offsets its advantage in enhancing the
metabolite spectrum [57].