scholarly journals Improved Method for Gas Chromatographic–Mass Spectrometric Analysis of 1-13C-labeled Long-Chain Fatty Acids in Plasma Samples

2002 ◽  
Vol 48 (6) ◽  
pp. 906-912 ◽  
Author(s):  
José M Hernández-Pérez ◽  
Eduard Cabré ◽  
Lourdes Fluvià ◽  
Ágata Motos ◽  
Cruz Pastor ◽  
...  
Keyword(s):  
Fatty Acids ◽  
Oleic Acid ◽  
Fatty Acid ◽  
Isotopic Ratio ◽  
Internal Standard ◽  
Mass Spectrometric ◽  
Spectrometric Analysis ◽  
Calibration Curves ◽  
Long Chain ◽  
Chromatographic Mass

Abstract Background: Gas chromatographic–mass spectrometric (GC/MS) tracking of stable-isotope-labeled substrates is useful in metabolic studies. However, GC/MS analysis of long-chain fatty acid methyl esters yields results that mostly depend on their concentration in the system. We describe a protocol aimed to obviate this and other drawbacks in plasma [1-13C]palmitic and [1-13C]oleic acid measurements. Methods: Lipoproteins were separated by sequential ultracentrifugation. Free or esterified heptadecanoic acid was used as internal standard. Fatty acids were derivatized to trimethylsilyl (TMS) esters. GC separation was in isothermal mode at 210 °C for 27 min. For both TMS-palmitate and TMS-oleate, M and [M + 1] signals were simultaneously acquired with a dual acquisition program in single-ion monitoring mode. Calibration mixtures containing increasing amounts of labeled fatty acids were prepared gravimetrically to construct calibration curves for isotopic enrichment. Likewise, five calibration curves (for increasing concentrations) were constructed for each fatty acid; this allowed selection of the most appropriate curve for the concentration in a plasma sample. Results: Oleic acid-TMS ester was clearly separated from that of its stereoisomer, elaidic acid. Within a 10-fold concentration range, the isotopic ratio was independent on the amount of the analyte in the sample, with a maximum uncertainty of 0.34% in terms of molar percent excess. In addition, the within- and between-day imprecision (CV) of the method was <1%. Conclusion: Results obtained with this method are independent of concentration and sufficiently precise for tracking 1-13C-labeled palmitic and oleic acids in biological samples

Gas chromatographic/mass spectrometric analysis of fatty acids found in aquatic algae

1988 ◽  
Vol 16 (1-12) ◽  
pp. 477-480 ◽  
Author(s):  
M. Cojocaru ◽  
M. Shlosberg ◽  
Z. Dubinsky ◽  
A. Finkel
Keyword(s):  
Fatty Acids ◽  
Mass Spectrometric ◽  
Mass Spectrometric Analysis ◽  
Spectrometric Analysis ◽  
Chromatographic Mass

Chloroacetamide Mode of Action, I: Inhibition of Very Long Chain Fatty Acid Synthesis in Scenedesmus acutus

1998 ◽  
Vol 53 (11-12) ◽  
pp. 995-1003 ◽  
Keyword(s):  
Fatty Acids ◽  
Cell Wall ◽  
Oleic Acid ◽  
Fatty Acid ◽  
Acid Synthesis ◽  
Resistant Cell Line ◽  
Long Chain Fatty Acids ◽  
Scenedesmus Acutus ◽  
Long Chain ◽  
Chain Fatty Acids

Abstract Herbicidal chloroacetamides cause a very sensitive inhibition of fatty acid incorporation into an insoluble cell wall fraction of Scenedesmus acutus. The molecular basis was investigated in more detail. After incubation of the algae with [14C]oleic acid and saponification, the remaining pellet was solubilized and fractionated consecutively with chloroform / methanol, phosphate buffer, amylase, pronase, and finally with dioxane/HCl. By acid hydrolysis in dioxane a part of the cell wall residue was solubilized showing inhibition of exogenously applied oleic acid and other labelled precursors such as stearic acid, palmitic acid, and acetate. After extraction of this dioxane-soluble subfraction with hexane, HPLC could separate labelled metabolites less polar than oleic acid. T heir formation was completely inhibited by chloroacetam ides, e.g. 1 μᴍ metazachlor. This effect was also observed with the herbicidally active 5-enantiomer of metolachlor while the inactive R-enantiomer had no influence. These strongly inhibited metabolites could be characterized by radio-HPLC /MS as very long chain fatty acids (VLCFAs) with a carbon chain between 20 and 26. Incubating am etazachlor-resistant cell line of S. acutus (Mz-1) with [14C]oleic acid, V LCFA s could not be detected in the dioxane/ HCl-subfraction. Furthermore, comparing the presence of endogenous fatty acids in wildtype and mutant Mz-1 the VLCFA content of the mutant is very low, while the content of long chain fatty acids (C16 -18) is increased, particularly oleic acid. Obviously, the phytotoxicity of chloroacetam ides in S. acutus is due to inhibition of VLCFA synthesis. The resistance of the mutant to metazachlor has a bearing on the higher amount of long chain fatty acids replacing the missing VLCFAs in essential membranes or cell wall components.

THE BIOSYNTHESIS OF LONG-CHAIN FATTY ACIDS IN THE DEVELOPING CASTOR BEAN

1965 ◽  
Vol 43 (1) ◽  
pp. 49-62 ◽  
Author(s):  
D. T. Canvin
Keyword(s):  
Fatty Acids ◽  
Oleic Acid ◽  
Fatty Acid ◽  
Ricinoleic Acid ◽  
Castor Bean ◽  
Diethyl Malonate ◽  
Water Soluble ◽  
Long Chain Fatty Acids ◽  
Long Chain ◽  
Chain Fatty Acids

Acetate-1-C14 and acetate-2-C14 were supplied to slices of developing castor bean endosperm. The molecules were extensively incorporated into long-chain fatty acids, water-soluble compounds, and protein. Oleic acid was the fatty acid initially labelled from acetate and it was the precursor of ricinoleic acid. Aerobic conditions were required for the formation of oleic acid and for the conversion of oleic acid to ricinoleic acid. Under anaerobic conditions the incorporation of acetate carbon into fatty acids was inhibited more than 90% and almost all of the C14 was found in stearic and palmitic acids. Stearic acid appeared to be formed first and palmitic acid appeared to be derived from it through a shortening of the chain. The position of linoleic acid in the fatty acid interconversions was not clear except that it was not a free intermediate in the conversion of oleic acid to ricinoleic acid.Malonate-C14 was only absorbed slightly by the tissue and although absorption could be increased by the use of diethyl malonate the metabolism of the compound was not facilitated. Because of its poor utilization by the tissue the role of malonate in long-chain fatty acid synthesis in this tissue could not be ascertained.

scholarly journals Synthesis of non-hydroxy-galactosylceramides and galactosyldiglycerides by hydroxy-ceramide galactosyltransferase

1996 ◽  
Vol 317 (2) ◽  
pp. 589-597 ◽  
Author(s):  
Petra van der BIJL ◽  
Ger J. STROUS ◽  
Matthijs LOPES-CARDOZO ◽  
Jane THOMAS-OATES ◽  
Gerrit van MEER
Keyword(s):  
Fatty Acids ◽  
Endoplasmic Reticulum ◽  
Fatty Acid ◽  
Hydroxy Fatty Acid ◽  
Fast Atom Bombardment ◽  
Chinese Hamster ◽  
Mass Spectrometric ◽  
Collision Induced Dissociation ◽  
Spectrometric Analysis ◽  
Transfected Cells

Galactosylceramide (GalCer) is the major glycolipid in brain. In order to characterize the activity of brain UDPgalactose: ceramide galactosyltransferase (CGalT), it has been stably expressed in CGalT-negative Chinese hamster ovary (CHO) cells. After fractionation of transfected cells, CHO-CGT, on sucrose gradients, the activity resides at the density of endoplasmic reticulum and not of Golgi. A lipid chromatogram from CHO-CGT cells revealed two new iodine-staining spots identified as GalCer, since they comigrate with GalCer standards, can be metabolically labelled with [3H]galactose, are recognized by anti-GalCer antibodies, and are resistant to alkaline hydrolysis. A third [3H]galactose lipid was identified as galactosyldiglyceride. In the homogenate CGalT displays a 25-fold preference for hydroxy fatty acid-containing ceramides. Remarkably, endogenous GalCer of transfected cells contains exclusively non-hydroxy fatty acids: fast atom bombardment and collision-induced dissociation mass spectrometric analysis revealed mainly C16:0 in the lower GalCer band on TLC and mainly C22:0 and C24:0 in the upper band. Our results suggest that CGalT galactosylates both hydroxy- and non-hydroxy fatty acid-containing ceramides and diglycerides, depending on their local availability. Thus, CGalT alone may be responsible for the synthesis of hydroxy- and non-hydroxy-GalCer, and galactosyldiglyceride in myelin.

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