Determination of the fatty acid profile of neutral lipids, free fatty acids and phospholipids in human plasma.

BACKGROUND: Knowledge of the fatty acid composition of lipid classes in human plasma is an important factor in the investigation of human metabolism. Therefore, a method for the analysis of neutral lipid (NL), phospholipid (PL) and free fatty acids (FFA) in human plasma has been developed and validated. METHODS: Separation of lipid classes was carried out by solid phase extraction of the lipid extract. The fractions were transesterified and the resulting fatty acid methyl esters were determined by GC/FID. For the method to be validated, precision, detection and quantification limits, as well as recovery, were determined for combined lipid extraction, solid phase extraction and GC analysis. RESULTS: The lipid extraction was miniaturized and simplified by application of an ultrasound 'Sonotrode'. The resolution of lipid classes was optimized with appropriate standards added to a representative plasma sample. In addition, a rapid derivatization procedure using trimethylsulfoniumhydroxide was established. Low determination limits (1.5, 0.2 and 1.3 µg/g plasma for NL, PL and FFA, respectively) indicate that the method's sensitivity is sufficient to quantify even minor components. Furthermore, recovery for NL and PL fatty acids was found to range from 80% to 110%. The results were similar for FFA apart from more polar free fatty acids due to their higher solubility in water. Repetitive measurements showed very good precision apart from the long chain PUFA for which the coefficients of variation were significantly higher. CONCLUSIONS: The present method is applicable to the quantitation of fatty acids in lipid classes of human plasma including several minor components.

An ultrasonic-accelerated oxidation method for determining the oxidative stability of biodiesel.

Biodiesel is considered an alternative energy because it is produced from fats and vegetable oils by means of transesterification. Furthermore, it consists of fatty acid alkyl esters (FAAS) which have a great influence on biodiesel fuel properties and in the storage lifetime of biodiesel itself. The biodiesel storage stability is directly related to the oxidative stability parameter (Induction Time - IT) which is determined by means of the Rancimat® method. This method uses condutimetric monitoring and induces the degradation of FAAS by heating the sample at a constant temperature. The European Committee for Standardization established a standard (EN 14214) to determine the oxidative stability of biodiesel, which requires it to reach a minimum induction period of 6h as tested by Rancimat® method at 110°C. In this research, we aimed at developing a fast and simple alternative method to determine the induction time (IT) based on the FAAS ultrasonic-accelerated oxidation. The sonodegradation of biodiesel samples was induced by means of an ultrasonic homogenizer fitted with an immersible horn at 480Watts of power and 20 duty cycles. The UV-Vis spectrometry was used to monitor the FAAS sonodegradation by measuring the absorbance at 270nm every 2. Biodiesel samples from different feedstock were studied in this work. In all cases, IT was established as the inflection point of the absorbance versus time curve. The induction time values of all biodiesel samples determined using the proposed method was in accordance with those measured through the Rancimat® reference method by showing a R(2)=0.998.

Plasma free fatty acid reference interval in South African neonates in the first week of life.

BACKGROUND: Non-esterified fatty acid (NEFA) levels are an important diagnostic tool in the investigation of neonatal hypoglycaemia. NEFA reference intervals have not been reported for neonates previously. METHODS: The objective of this study was to determine an NEFA reference interval for neonates. RESULTS: Heparinized plasma obtained from 106 healthy neonates in the first week of life was analysed using the Roche "Free fatty acid, Half-micro test" kit. Results were then analysed statistically for normality (Shapiro-Wilk test) and reference interval determined non-parametrically (bootstrap method). CONCLUSIONS: NEFA levels displayed a non-Gaussian distribution and the reference interval (2.5th and 97.5th percentiles) was 0.2-1.5 mmol/L (90% confidence intervals 0.1-0.3 and 1.4-2.0 mmol/L, respectively). The NEFA reference interval in South African neonates less than a week old is similar to that described in infants (1-12 months), indicating that this reference range can be used over the entire neonatal period.

Micro method for determination of nonesterified fatty acid in whole blood obtained by fingertip puncture.

Diagnostic tools for early identification of subjects at high risk for type 2 diabetes and other obesity-related disorders are important in prevention of these diseases. Nonesterified fatty acids (NEFAs) have been suggested to serve as a prediagnostic marker of diabetes and obesity-related disorders. In the current study, we developed a sensitive and reproducible micro method for quantification of NEFA in less than 10 microl whole blood. The method involves only two steps: (i) conversion of NEFA to fatty acid acyl-coenzyme A (acyl-CoA) esters using an acyl-CoA synthetase and (ii) quantification of the formed acyl-CoA esters with a fluorescent biosensor based on bovine acyl-CoA binding protein (ACBP). Lys50 of ACBP was mutagenized to a cysteine residue that was covalently modified with 6-bromoacetyl-2-dimethylaminonaphthalene to make a fluorescent acyl-CoA indicator (FACI-50). FACI-50 exhibits high fluorescence emission yield with maximum at 490 nm in the presence of CoA when excited at 387 nm. The addition of palmitoyl-CoA to a CoA-saturated FACI-50 lowered fluorescence emission by eightfold. Ethanol extract from 1 microl whole blood was incubated with ATP, CoA, and FACI-50. Following background fluorescence reading, NEFAs were converted to acyl-CoA by the acyl-CoA synthetase and the NEFA content was calculated from fluorescence emission changes using palmitic acid as external standard. The FACI-50 NEFA method was compared with two commercially available methods for quantification of NEFA.

Quantitative analysis of free fatty acids in rat plasma using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry with meso-tetrakis porphyrin as matrix.

Quantitative analysis of free fatty acids was achieved using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) with a meso-tetrakis porphyrin matrix. Cesium acetate was employed as a cationizing agent. The MALDI signal was reproducible and dominated by cesiated cesium carboxylates [RCOOCs + Cs]+. The addition of two Cs ions resulted in a mass shift of 264.8 Da for each fatty acid and greatly reduced background peaks. A linear relationship between fatty acid concentration and corresponding fatty acid to internal standard peak intensity ratio was observed for three representative fatty acids analyzed across a concentration range from 4.40 to 150 microM, with correlation coefficients between 0.986 and 0.987. The application of this method was demonstrated with the analysis of free fatty acids in nonfasted and fasted rat plasmas. A total of eight free fatty acids (14:0, 16:0, 16:1, 17:0, 18:0, 18:1, 18:2, and 20:4) were detected. The relative peak height ratios of the fatty acids to the internal standard allow quantitative measurements of the free fatty acids. It was shown that the levels of free fatty acids were higher in fasted rats than in rats in a nonfasted state. This method is simple, sensitive, and fast. Thus, it provides an appealing tool for the analysis of free fatty acids or other low-molecular weight compounds during drug discovery and/or development.

Free fatty acid determination by peroxidase catalysed luminol chemiluminescence.

A sensitive, specific, and partly automatic method for the analysis of free fatty acids is described. The assay involves activation of free fatty acids by acyl-CoA synthetase (EC 6.2.1.3) followed by oxidation of the thioesters by acyl-CoA oxidase. The H2O2 formed is determined in a reaction catalysed by horseradish peroxidase (EC 1.11.1.7) using luminol as electron donor. The assay has a linear range of 0.05 to 5 nmol of different free fatty acids (C10-C18) in the original sample. The efficiency of the method toward capric, lauric, myristic, palmitic, palmitoleic, stearic, oleic, and linoleic acid measured as recovery of light emission compared to that of H2O2 standards, was over 90%. AffiGel 501 was used to covalently bind the free thiol group in CoASH eliminating interference of this substance in the peroxidase-luminol reaction.

A review of the analysis of free fatty acids [C2-C6].

A large number of the techniques and methods used to analyze FFA[C2-C6] from the advent of GLC to the present are reviewed. Possible reasons as to why it has taken such a long time to establish an unequivocal quantitative method are suggested. The results of analyzing standard solutions of FFA by three different methods are compared, and that based on the addition of formic acid to the carrier gas is recommended.