Sequential alkaline saponification/acid hydrolysis/ esterification: a one-tube method with enhanced recovery of both cyclopropane and hydroxylated fatty acids

doi:10.1016/0167-7012(93)90068-S

Journal of Microbiological Methods

Volume 18, Issue 1, July 1993, Pages 21-32

https://doi.org/10.1016/0167-7012(93)90068-S Get rights and content

Abstract

Gas chromatographic acquisition of representative ‘Total’ cellular fatty acid profiles from bacteria or bacteria-containing samples (e.g., environmental or clinical materials) tends to be dependent on the method used to released the fatty acids and convert them to derivatives suitable for analysis. Alkaline saponification or interesterification methods, while preserving acid-sensitive components such as cyclopropane fatty acids, are often insufficient to release amide-linked components, such as hydroxylated fatty acids. Acid-catalyzed hydrolyses or interesterifications, on the other hand, while more efficiently releasing the predominantly amide-linked hydroxylated components, have been shown to cause severe and unpredictable degradation of cyclopropane fatty acids. We report studies of a single-tube method involving sequential alkaline/acid release of fatty acids in which fatty acids released by the alkaline step are partitioned into an organic epiphase during the aqueous acid hydrolysis step. After hydrolysis, the epiphase and the released fatty acids are extracted into an hypophasic solvend and esterified at moderate temperature under relatively low acid concentrations. Under these conditions, cyclopropane as well as hydroxylated fatty acids are recovered in high yield.

References (10)

E Yabuuchi et al.
Cellular fatty acid composition of strains of three species Sphingocbacterium gen. nov. and Cytophaga johnsonae
FEMS Microbiol. Lett.
(1982)
P. Vulliet et al.
Identification of methoxyester artifacts produced by methanolic-HCl solvolysis of the cyclopropane fatty acids of the genus Yersinia
Biochim. Biophys. Acta
(1974)
B.J. Wilkinson et al.
Occurence, localization, and possible significance of an ornithine-containing lipid in Paracoccus dentrificans
Arch. Microbiol.
(1982)
W.R. Mayberry
Cellular distribution and linkage of D-(-)-3-hydroxy fatty acids in Bacteroides species
J. Bacteriol.
(1980)
W.R. Mayberry et al.
Identification of the amide-linked fatty acids of Acholeplasma axanthum S743 as D-(-)-3-hydroxyhexadecanoate and its homologues
J. Bacteriol.
(1973)

There are more references available in the full text version of this article.

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Silicic acid chromatography was used to separate the total lipids into polar, neutral and glycolipid fractions (Guckert et al., 1985). The polar lipid fraction was subsequently transesterified using mild alkaline methanolysis to form fatty acid methyl esters (FAMEs) and convert plasmalogen ethers to dimethylacetals (DMAs) (Guckert et al., 1985), with modifications (Mayberry and Lane, 1993). The different lipid fractions were quantified as previously described (Michalsen et al., 2007).
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Journal of Microbiological Methods

Abstract

References (10)

Cellular fatty acid composition of strains of three species Sphingocbacterium gen. nov. and Cytophaga johnsonae

FEMS Microbiol. Lett.

Identification of methoxyester artifacts produced by methanolic-HCl solvolysis of the cyclopropane fatty acids of the genus Yersinia

Biochim. Biophys. Acta

Occurence, localization, and possible significance of an ornithine-containing lipid in Paracoccus dentrificans

Arch. Microbiol.

Cellular distribution and linkage of D-(-)-3-hydroxy fatty acids in Bacteroides species

J. Bacteriol.

Identification of the amide-linked fatty acids of Acholeplasma axanthum S743 as D-(-)-3-hydroxyhexadecanoate and its homologues

J. Bacteriol.

Cited by (34)

Geochemical, mineralogical and microbiological characteristics of sediment from a naturally reduced zone in a uranium-contaminated aquifer

Standardizing methylation method during phospholipid fatty acid analysis to profile soil microbial communities

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Significance of carbon isotope discrimination between bulk carbon and extracted phospholipid fatty acids in selected terrestrial and marine environments

Evaluation of extraction methods for recovery of fatty acids from lipid-producing microheterotrophs

Electron paramagnetic resonance studies of the membrane fluidity of the foodborne pathogenic psychrotroph Listeria monocytogenes