A method for specific analysis of free fatty acids in biological samples by capillary gas chromatography.

The present report describes a simple method to selectively extract free fatty acids and analyze them by capillary gas-liquid chromatography. The procedure is based on the use of fumed silicon dioxide. In the presence of plasma, this material induces a rapid rise in the viscosity of the mixture and presents the ability to trap large particles such as emulsified lipids and lipoproteins. Albumin-bound fatty acids are thus left in the aqueous media. We present applications of our procedure for the analysis of free fatty acids in 0.2 ml of plasma from rat or human. By comparison with the method utilizing thin-layer chromatography for the separation of fatty acids and gas chromatography analysis, the present method has been found to be reliable and simple. The recovery of linoleic acid was 92.1 +/- 8.2%, a value which is about twice better than that obtained with the procedure using thin-layer chromatography. In particular, long-chain polyunsaturated fatty acids were better preserved. Our procedure does not require the use of organic solvents and its simplicity and reproducibility make it suitable for routine specific determination of the composition of free fatty acids in biological samples.     

Oxalate-silica gel thin-layer system for free 2-hydroxy fatty acids and for fatty acyl coenzyme A

The 2-hydroxy fatty acids tend to yield streaks in thin-layer chromatography on silica gel plates. If potassium oxalate is included with binder-free silica gel, good spots are obtained. Similar difficulties are found in paper chromatography of the fatty acid derivatives of coenzyme A, especially with long-chain acids. The same thin-layer system gives good spots with these compounds.     

A reliable analysis of tissue free fatty acids by gas-liquid chromatography

A convenient and reliable gas-liquid chromatographic method for determining the free fatty acids in biological specimens is described. The free fatty acids were extracted with hexane in the presence of H3PO4 and then back-extracted from the hexane phase into a very small volume of trimethyl (alpha, alpha, alpha-trifluoro-m-tolyl)ammonium hydroxide solution. Direct injection of the resultant quaternary ammonium salts of the fatty acids into a gas-liquid chromatograph unit gave their methyl esters, with a high recovery. The presence of triglycerides, phospholipids, or cholesterol esters did not interfere with the determination of free fatty acids. This method was applied to determination of free fatty acids in the samples of serum or brain. The results were more precise and reliable than those reported with the conventional methods with TLC separation. This method should be a useful aid for providing precise information about the physiological or pathological roles of free fatty acids.     

Small amounts of isotope-reinforced polyunsaturated fatty acids suppress lipid autoxidation

Polyunsaturated fatty acids (PUFAs) undergo autoxidation and generate reactive carbonyl compounds that are toxic to cells and associated with apoptotic cell death, age-related neurodegenerative diseases, and atherosclerosis. PUFA autoxidation is initiated by the abstraction of bis-allylic hydrogen atoms. Replacement of the bis-allylic hydrogen atoms with deuterium atoms (termed site-specific isotope-reinforcement) arrests PUFA autoxidation due to the isotope effect. Kinetic competition experiments show that the kinetic isotope effect for the propagation rate constant of Lin autoxidation compared to that of 11,11-D(2)-Lin is 12.8 ± 0.6. We investigate the effects of different isotope-reinforced PUFAs and natural PUFAs on the viability of coenzyme Q-deficient Saccharomyces cerevisiae coq mutants and wild-type yeast subjected to copper stress. Cells treated with a C11-BODIPY fluorescent probe to monitor lipid oxidation products show that lipid peroxidation precedes the loss of viability due to H-PUFA toxicity. We show that replacement of just one bis-allylic hydrogen atom with deuterium is sufficient to arrest lipid autoxidation. In contrast, PUFAs reinforced with two deuterium atoms at mono-allylic sites remain susceptible to autoxidation. Surprisingly, yeast treated with a mixture of approximately 20%:80% isotope-reinforced D-PUFA:natural H-PUFA are protected from lipid autoxidation-mediated cell killing. The findings reported here show that inclusion of only a small fraction of PUFAs deuterated at the bis-allylic sites is sufficient to profoundly inhibit the chain reaction of nondeuterated PUFAs in yeast.     

Only trace amounts of fatty acids are found in pure mucus glycoproteins

The presence of noncovalently associated lipids and covalently bound fatty acids was investigated in preparations of mucus glycoproteins obtained by using density-gradient centrifugation in CsCl/guanidinium chloride. No phospholipids, glycolipids, cholesterol, or triglycerides could be detected. However, small amounts of extractable fatty acids were consistently found, the sum of which ranged from 0.3 to 0.9 micrograms/mg of glycoprotein. The amount of fatty acid released after subsequent treatment with KOH ranged from 0 to 27 ng/mg of glycoprotein. We conclude that density-gradient centrifugation in CsCl/guanidinium chloride is very efficient in removing noncovalently associated lipids from mucus glycoproteins and that covalently bound fatty acids are probably not present in the macromolecules.     

Assay for polyunsaturated fatty acids by argentation-thin-layer chromatography using commercial thin-layer plates

A simple, rapid, and convenient procedure for silver nitrate impregnation of commercial precoated silica gel plates is described. Silica-gel plates (Silica gel 60, E. Merck) were sprayed with 40% silver nitrate in water, dried in air, and activated at 100°C for 30 min. Samples containing fatty acid methyl esters were applied as 0.5- to 1.0-cm streaks and developed with a solvent system of benzene:ethyl acetate (9:1, v/v). The plates were sprayed with 70% sulfuric acid saturated with potassium dichromate, and the spots were detected by careful heating at 120°C for 90 min. This procedure is useful for separation and isolation of various species of fatty acid methyl esters and for simple, rapid, and reproducible estimation of microgram quantities of materials by spectrodensitometry of the chromatogram.     

Microanalysis of free and conjugated bile acids by thin-layer chromatography and in situ spectrofluorimetry

A combination of thin-layer chromatography (TLC) and in situ spectrofluorimetry for the determination of free bile acids and bile acids conjugated with glycine or taurine is described. This method makes it possible to determine bile acids concentrations as low as 0.15-0.25 nmol (0.05-0.1 microgram) in a simple and reproducible way. Moreover, information can be obtained about conjugation patterns and relative concentrations of mono-, di-, and trihydroxy bile acids as well as about the presence of abnormal bile acids. After TLC the bile acids are made visible in uv light by dipping the layer in sulfuric acid in diethyl ether and warming it under well-described conditions. The fluorescence of the bile acids on the thin layer can be measured and makes it possible to quantitate them. The method presented here is applicable to bile acid-containing extracts from serum, bile, and feces, and the results are in good agreement with those obtained by enzymatic and gas-liquid chromatographic techniques.     

气相色谱法测定啤酒中的游离脂肪酸

气相色谱法测定啤酒中辛酸到二十二碳酸共１１种游离脂肪酸,采用多级溶剂萃取及薄层色谱纯化技术进行样品制备,并采充氮措施抑制脂肪酸的氧化产生,此方法有较好的重复性和回收率。     

Fluorometric high-performance liquid chromatography of 9-aminophenanthrene-derivatized free fatty acids

The application of 9-aminophenanthrene (9-AP), a fluorescence-labeling reagent for free fatty acids (FFA), was examined. 9-AP dissolved in benzene was added to a benzene solution of FFA chlorides derived from FFA and oxalyl chloride. The mixture was allowed to react for 45 min at 70°C. By the method, 9-AP-tagged FFA with a strong fluorescence was formed. The materials thus obtained have a λ_max at around 303 nm for excitation and 376 nm for emission. By using this derivatization method, recoveries were measured for seven kinds of FFA added to 0.5 ml of healthy human serum. Significant recoveries ranging from 96 to 107% (coefficient of variation 1.4—5.0%) were obtained for each FFA. The proposed method was clinically applied to the determination of FFA in 0.5 ml of healthy human serum, and almost satisfactory results were obtained. Detection limits of FFA by this derivatization method were 10 pmol for C_14:0, C_16:0, C_16:1, C_18:1 and C_18:2, and 15 pmol for C_18:0 and C_20:4. As a quantitative measurement of FFA, gas chromatography and high-performance liquid chromatography with fluorescence detection, which have been routinely used, were chosen for comparison with the present method.     

Alkaline solvent systems for thin-layer chromatography of bile acids

Thin-layer chromatographic separation of the common bile acids and their taurine and glycine conjugates in chloroform-methanol-ammonia is reported. An alkaline system offers advantages for the separation and nondestructive staining of bile acid conjugates.     

Lipids in blood-brain barrier models in vitro I: Thin-layer chromatography and high-performance liquid chromatography for the analysis of lipid classes and long-chain polyunsaturated fatty acids

The objectives of this study were to optimize a sensitive high-performance liquid chromatography (HPLC) method for fatty acid (FA) analysis for the quantification of polyunsaturated FAs (PUFAs) in cell lipid extracts and to analyze the lipid and FA patterns of three cell lines used in blood-brain barrier (BBB) models: RBE4, ECV304, and C6. Thin-layer chromatographic analysis revealed differences in the phosphatidylcholine-phosphatidylethanolamine (PC:PE) ratios and the triglyceride (TG) content. The PC:PE ratio was <1 for RBE4 cells but >1 for ECV304 and C6 cells. ECV304 cells displayed up to 9% TG depending on culture time, whereas the other cell lines contained about 1% TG. The percentages of docosahexaenoic acid were 9.4 +/- 1.7% of the unsaturated FAs in RBE4 cells (n = 5; 4 d in culture; 9.9% after 10 d), 8.1 +/- 2.0% in ECV304 cells (n = 11; 10 to 14 d), and 6.7 +/- 0.6% in C6 cells (n = 6; 10 to 14 d) and were close to the published values for rat brain microvascular endothelium. The percentage of arachidonic acid (C20:4) was about half that in vivo. ECV304 cells contained the highest fraction of C20:4, 17.8 +/- 2.2%; RBE4 cells contained 11.6 +/- 2.4%; and C6 cells 15.8 +/- 1.9%. It is concluded that a sensitive HPLC method for FAs is now optimized for the analysis of long-chain PUFAs. The results provide a useful framework for studies on the effects of lipid modulation and give reference information for the development of further BBB models.     

Determination of fatty acid content and composition in ultramicro lipid samples by gas-liquid chromatography

Simplified quantitative manipulations of very small amounts (30 micrograms) of lipids for determination of fatty acid content and composition by gas-liquid chromatography after (a) methanolysis (b) reduction and acetylation are described.     

Impact of free hydroxylated and methyl-branched fatty acids on the organization of lipid membranes

Differential scanning calorimetry (DSC) has been applied to study the effect of free hydroxylated and methyl-branched fatty acids on the physico-chemical properties of lipid membranes. First, the impact of free hydroxy fatty acids (HFAs) on dimyristoylphosphatidylcholine (DMPC) model membranes was monitored only as a function of chain length and position of the attached hydroxyl group. Second, racemic vs. enantiopure anteiso fatty acids (AFAs) and HFAs were investigated to address the question of which role does a fatty acid's chirality play on its membrane pertubing effect. The DSC thermograms revealed that the main gel to liquid-crystalline phase transition of the DMPC bilayers which results in a disordering effect of the lipid hydrocarbon chains was affected in different ways depending on the nature of the incorporated fatty acid. Long-chain 2- and 3-HFAs stabilized the gel phase by reducing the phase transition temperature (T(m)), whereas short-chain HFAs and long-chain HFAs with the hydroxy group remote from the head group stabilized the more disordered liquid-crystalline state. Additionally, we observed that enantiopure (S)-14-methylhexadecanoic acid ((S)-a17:0) and (R)-2-hydroxy octadecanoic acid and the corresponding racemates had contrary effects upon incorporation into DMPC bilayers. In both cases, the pure enantiomers alleviated the liquid-crystalline state of the biological model membrane.