High performance liquid chromatography of fatty acids as naphthacyl derivatives

Abstract

IntroductionStudying fatty acid metabolism in living cells requires not only analytical methods leading to the separation and quantification of each fatty acid, but also preparative methods allowing the recovery of each eluted fatty acid. Numerous fatty acid separation systems based on chromatographic methods (gas chromatography, high performance liquid chromatography) have been developed in the past years [1]. However, none of them seem to be both resolutive and preparative enough to investigate the metabolic bioconversion of fatty acids with physiological significance (biosynthesis, elongation, desaturation, retroconversion and β-oxidation).Gas chromatography (GC) has been the method of choice for separation and quantification of fatty acids for a long time, usually after their conversion to methyl esters [2,3]. Indeed the use of a capillary column coupled to a flame ionization detector (FID) provides high resolution for fatty acid analysis in complex mixtures, including separation of some positional and geometrical isomers. However, there are several inherent limits to GC methods. First of all, short-chain fatty acid methyl esters are volatile and may be lost on refluxing the esterification medium [1]. Secondly, problems originate from possible thermal degradation and structural modification of unsaturated fatty acids during their conversion to methyl esters [4,5]. Thirdly, the main limit to GC is that FID detection destroys the fatty acids and does not allow the recovery of any separated material for further analysis.A great advantage of high performance liquid chromatography (HPLC) methods over GC methods is that the resolved fatty acids are not destroyed during their detection, which enables further analyses to be performed. Recently, several HPLC systems for the separation of fatty acids have been reported, especially with biological applications [6,7]. Abstract. A procedure for the separation of fatty acids, after their derivatization as fatty acid naphthacyl esters, by reverse-phase high performance liquid chromatography is described. A reproducible resolution (50 min), shorter than former HPLC analyses, of a standard mixture of fatty acid naphthacyl esters (25 fatty acids; C7:0 -C22:6 n -3), was achieved by a ternary elution gradient of methanol-acetonitrile-water. Compared with gas chromatography, HPLC analysis of fatty acid naphthacyl esters showed similar percentages of molar distribution of long-chain fatty acids (≥14 carbons). The separation was monotered by UV absorbance at 246 nm, which was highly sensitive (the detection limit was about 0.1 ng), did not destroy the fatty acid naphthacyl esters and thus allowed individual recovery for further analysis (specific radioactivity determination or positional isomer identification). This preparative HPLC method provides, therefore, a useful process for the study of fatty acid metabolism in biological systems.Key words. Fatty acids -fatty acid naphthacyl esters -high performance liquid chromatography -fatty acid metabolism.

Dietary myristic acid at physiologically relevant levels increases the tissue content of C20:5 n-3 and C20:3 n-6 in the rat

“…The FA were liberated by acidification, extracted with diethylether and dried. Fatty acids were converted to fatty acid naphthacyl esters [17] and separated on HPLC (Alliance 2695 integrated system, Waters, St-Quentin-en-Yvelines, France). The peaks corresponding to radiolabeled FA (substrate and product of each desaturase assay) were collected and subjected to liquid scintillation counting (Packard TriCarb 1600 TR, Meriden, CT).…”

Myristic acid increases Δ6-desaturase activity in cultured rat hepatocytes

“…Fatty acids were converted to fatty acid naphthacyl esters as previously described [18] and were separated on HPLC (Alliance integrated system, Waters, St Quentin en Yvelines, France) using a Nova-Pak C18 column (4.6 mm × 250 mm, Waters) and a guard column (Nova-Pak C18; 3.9 mm × 20 mm).The peaks corresponding to radiolabeled fatty acids (substrates and products of ∆6-desaturase assays) were collected and subjected to liquid scintillation counting (Packard Tri-Carb 1600 TR, Meriden, CT). Preliminary identification of fatty acid naphthacyl esters was based upon retention times obtained for naphthacyl esters prepared from radiolabeled and nonradiolabeled fatty acid standards [18]. The enzyme activity was determined from the amount of radioactivity found in the products vs. the radioactivity recovered in the substrate.…”

Although it is rapidly metabolized in cultured rat hepatocytes, lauric acid is used for protein acylation

“…For individual FA analysis, FA from each lipid extract were converted to fatty acid naphthacyl esters and separated on HPLC (Alliance integrated system, Waters, St-Quentin-en-Yvelines, France) as previously described [24]. The elution of naphthacyl derivatives was monitored by UV absorbance at 246 nm (Tunable absorbance detector 486, Waters).…”

“…Released FA were then extracted with 4 mL diethylether. FA released from each protein by these two successive treatments were converted to fatty 422 V. Rioux et al acid naphthacyl esters and separated on HPLC as described above [24].…”