Validation of Two Methods for Fatty Acids Analysis in Eggs

A comparative study between two methods (lipid extraction followed by saponification and methylation, and direct methylation) to determine the fatty acids in egg yolk was evaluated. Direct methylation of the samples resulted in lower fatty acid content and greater variation in the results than the lipid extraction followed by saponification and methylation. The low repeatability observed for the direct HCl methylation method was probably due to a less efficient extraction and conversion of the fatty acids into their methyl esters as compared to the same procedure starting with the lipid extract. As the lipid extraction followed by esterification method was shown to be more precise it was validated using powdered egg certified as reference material (RM 8415, NIST) and applied to samples of egg, egg enriched with polyunsaturated omega-3 fatty acids (n-3 PUFA), and commercial spray-dried whole egg powder.     

Determination of the Fatty Acid Profile of Fish by a One-Step Extraction/Methylation Method

To determine the content of individual fatty acids of fish by gas-liquid chromatography, samples are normally extracted with suitable solvents and the crude lipid extract obtained thereby is derivatized to form fatty acid methyl esters in a subsequent reaction. Alternatively, freeze-dried fatty fish is reacted with toluene/methanolic-HCI for simultaneous extraction and methylation of lipids contained in the testing material. Sample preparation by this one-step method is not only much easier but also as efficient as lipid extraction with chloroform/methanol and derivative formation by base-catalyzed methanolysis of lipids. Both approaches resulted in comparable figures for the content of individual fatty acids per g dry matter even for the highly unsaturated fatty acids typical for fish lipids.     

Nutritional Attributes of Fatty Acids

Fatty acid composition is nutritionally important in countries with a high intake of dietary fat. The (n-3) and (n-6) families of essential fatty acids must both be represented in the diet, but controversy exists about the relative amounts that should be ingested. Safe ranges of intake that would prevent both deficiency signs and deleterious excesses of these fatty acids have yet to be established.     

油脂中八种脂肪酸的分析测定

油脂经氢氧化钾-甲醇溶液甲酯化后,生成的脂肪酸甲酯用DB-WAX毛细管柱进行分离,GC-FID进行检测。该方法的回收率在91.45%~107.55%,相对标准偏差小于8%,八种脂肪酸的检出限在0.29~1.10μg/mL之间。本方法准确度高,检出限低,能满足实际检测工作的需要。     

Role of n-3 Fatty Acids on Bile Acid Metabolism and Transport in Dyslipidemia: A Review

Dietary n-3 fatty acids, especially of marine origin, eicosapentaenoic acid (20:5n-3) and docosahexaenoic acid (22:6n-3), have always been lauded for their profound effects on regulating the risk factors for major metabolic disorders. Yet, their consumption rate is poor compared to n-6 fatty acids [linoleic acid (18:2n-6)], which are predominantly consumed. Hence, the skewed n-6 to n-3 fatty acid ratio may have a bearing on the risk factors of various diseases, including dyslipidemia. Dyslipidemia and other lifestyle diseases associated with it, such as diabetes, obesity, hypertension, are a growing concern in both developed and developing countries. A common strategy for addressing dyslipidemia involves bile acid (BA) sequestration, to interrupt the enterohepatic circulation of BA, resulting in the modulation of lipid absorption in the intestine, thereby normalizing the levels of circulating lipids. The BA homeostasis is under the tight control of hepatic and enteric BA transporters. Many investigations have reported the effects of dietary constituents, including certain fatty acids on the reabsorption and transport of BA. However, a critical review of the effects of n-3 fatty acids on BA metabolism and transport is not available. The present review attempts to explore certain unmapped facets of the n-3 fatty acids on BA metabolism and transport in dyslipidemia, and their interplay with biological processes involving lipid rafts and gut microbiome.     

Suitability of Soxhlet Extraction to Quantify Microalgal Fatty Acids as Determined by Comparison with In Situ Transesterification

To assess Soxhlet extraction as a method for quantifying fatty acids (FA) of microalgae, crude lipid, FA content from Soxhlet extracts and FA content from in situ transesterification (ISTE) were compared. In most cases, gravimetric lipid content was considerably greater (up to sevenfold) than the FA content of the crude lipid extract. FA content from Soxhlet lipid extraction and ISTE were similar in 12/18 samples, whereas in 6/18 samples, total FA content from Soxhlet extraction was less than the ISTE procedure. Re-extraction of residual biomass from Soxhlet extraction with ISTE liberated a quantity of FA equivalent to this discrepancy. Employing acid hydrolysis before Soxhlet extraction yielded FA content roughly equivalent to ISTE, indicating that acidic conditions of ISTE are responsible for this observed greater recovery of FA. While crude lipid derived from Soxhlet extraction was not a useful proxy for FA content for the species tested, it is effective in most strains at extracting total saponifiable lipid. Lipid class analysis showed the source of FA was primarily polar lipids in most samples (12/18 lipid extracts contained <5% TAG), even in cases where total FA content was high (>15%). This investigation confirms the usefulness of ISTE, reveals limitations of gravimetric methods for projecting biodiesel potential of microalgae, and reinforces the need for intelligent screening using both FA and lipid class analysis.     

Autistic Children Exhibit Decreased Levels of Essential Fatty Acids in Red Blood Cells

Omega-6 (n-6) and omega-3 (n-3) polyunsaturated fatty acids (PUFA) are essential nutrients for brain development and function. However, whether or not the levels of these fatty acids are altered in individuals with autism remains debatable. In this study, we compared the fatty acid contents between 121 autistic patients and 110 non-autistic, non-developmentally delayed controls, aged 3–17. Analysis of the fatty acid composition of red blood cell (RBC) membrane phospholipids showed that the percentage of total PUFA was lower in autistic patients than in controls; levels of n-6 arachidonic acid (AA) and n-3 docosahexaenoic acid (DHA) were particularly decreased (p < 0.001). In addition, plasma levels of the pro-inflammatory AA metabolite prostaglandin E2 (PGE2) were higher in a subset of the autistic participants (n = 20) compared to controls. Our study demonstrates an alteration in the PUFA profile and increased production of a PUFA-derived metabolite in autistic patients, supporting the hypothesis that abnormal lipid metabolism is implicated in autism.     

Zur Tocochromanol- und Fettsäureanalytik im Fisch

Analysis of Tocochromanols and Fatty Acids in fish Tocochromanols and lipids are quantitatively extractable from homogenate of the edible part (muscle and skin) of carp with dichloromethane and methanol at room temperature. The contents of tocopherols and tocotrienols are determinable directly in the lipid extract dissolved in n-hexane by HPLC (fluorescence detection). The fatty acids of the polyenic acid rich lipids are derivated to the methyl esters. Transesterification is carried out with methanolic potassium methylate solution, followed by esterification of possibly present free fatty acids with boron trifluoride-methanol, at room temperature. The fatty acid methyl esters (C8 :0 to C22:6 ω3) are separated and quantified by capillary gas chromatography on the station ary phase FFAP.     

n-3 Polyunsaturated Fatty Acids and Atopy in Korean Preschoolers

Atopy is a growing problem for Korean children. Since eicosapentaenoic acid is a precursor of less active inflammatory eicosanoids, n-3 polyunsaturated fatty acids (PUFA) may have a protective effect on atopy. This study was undertaken to determine whether n-3 PUFA in red blood cells (RBC) is lower in atopic than in non-atopic preschoolers. Three hundred and eight Korean children aged 4–6 years were enrolled. Total RBC fatty acid composition was measured by gas chromatography. The prevalence of atopic dermatitis, allergic rhinitis, or asthma was 29%. Total RBC n-3 PUFA were lower in preschoolers with atopy than controls (9.8 ± 1.2 vs. 11.4 ± 1.6%; P < 0.05), while n-6 PUFA (33.0 ± 1.4 vs. 32.2 ± 1.0%; P < 0.05) and n-6/n-3 PUFA ratio (3.4 ± 0.6 vs. 2.8 ± 0.5; P < 0.05) were greater. The following factors were also associated with an increase in atopy: higher saturated fatty acids (39.6 ± 1.4 vs. 40.6 ± 1.9; P < 0.05) and arachidonic acid (15.3 ± 1.6 vs. 16.0 ± 2.9; P < 0.05), and lower total PUFA (43.8 ± 0.7 vs. 42.8 ± 1.4; P < 0.05) and omega-3 index (EPA + DHA; 9.1 ± 0.8 vs. 7.8 ± 0.5; P < 0.05) in RBC. Maternal history of atopy was a significant (P < 0.05) risk factor, while lactation was not. The results suggest that a reduced content of n-3 PUFA in the RBC membrane could play a role in early children atopy.     

Transition-metal Catalyzed Oxidative Cleavage of Unsaturated Fatty Acids

The oxidative cleavage of the C? C-bond converts unsaturated fatty acids to carboxylic and dicarboxylic acids. The only method currently used in oleochemistry is ozonolysis. The high energy consumption of ozonolysis makes a transition-metal catalyzed alternative with a cheaper secondary oxidant attractive. Here we report new methods for ruthenium-, rhenium- and tungsten-catalyzed oxidations with peracetic acid and hydrogen peroxide. Whereas the direct oxidative cleavage of internal C? C-bonds still lacks selectivity, two-step reactions are feasible. The first possibility is to convert natural, internal unsaturated fatty acids to ω-unsaturated ones. These ω-unsaturated fatty acids can be cleaved by Ru/acac)₃/CH₃COOOH or Re₂O₇/H₂O₂ with yields up to 80 %. In the second two-step procedure the fatty acid first is hydroxylated and the diol is cleaved: the overall yield for this method is 80 %, too.     

Direct Microwave Transesterification of Fingertip Prick Blood Samples for Fatty Acid Determinations

Omega-3 polyunsaturated fatty acid (PUFA) dietary intakes and tissue levels are positively associated with various health benefits. The development of cost efficient, high throughput methodologies would enable research in large clinical and population studies, and clinical fatty acid profiling. Microwave heating for the transesterification of blood fatty acids was examined. Samples were collected by venous puncture and fingertip prick onto chromatography paper. Aliquots of serum, plasma, erythrocytes and whole blood were prepared from venous blood. Boron trifluoride in methanol was used for transesterification but sample preparation and heating varied. Fatty acid determinations and markers of omega-3 fatty acid status including the sum of eicosapentaenoic acid and docosahexaenoic acid, the ratio of total n-3 PUFA to n-6 PUFA, and the percentage of n-3 highly unsaturated fatty acids (HUFA, ≥20 carbons and ≥3 carbon–carbon double bonds) in total HUFA were compared. Quantitative determinations indicate that microwave transesterification results in significantly lower estimates of monounsaturates and polyunsaturates, possibly through incomplete transesterification of triacylglycerols. However, qualitative estimates of omega-3 fatty acid status were relatively similar. Fingertip prick blood collection combined with direct transesterification by microwave may be a very rapid method to estimate omega-3 fatty acid status for selected applications. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Aberrations in Plasma Phospholipid Fatty Acids in Lung Cancer Patients

Abnormalities in lipid metabolism have been frequently observed in cancer and are associated with a poor prognosis. However, a detailed, longitudinal characterization of fatty acid status is lacking. This study aimed to assess plasma phospholipid fatty acids before chemotherapy, immediately after and 1 month following chemotherapy in a group of 50 patients newly diagnosed with lung cancer and explore factors which may contribute to aberrations in fatty acids. Their mean ± SD characteristics: age 64 ± 8.5 years, 75% advanced stage disease, body mass index 27.0 ± 5.4 kg/m², 6 month weight loss −4.6 ± 6.1%. Compared to patients with early stage disease, patients with advanced disease had abnormal fatty acid profiles including significantly lower (P < 0.05) amounts of total phospholipid fatty acids, saturated, and polyunsaturated fatty acids (linoleic, arachidonic, eicosapentaenoic and docosahexaenoic). Longitudinal analysis revealed that patients with advanced disease who completed chemotherapy had stable fatty acid levels and continued to maintain levels 1 month following completion of chemotherapy. Comparatively, patients who did not complete chemotherapy due to toxicity or disease progression had progressive loss of total phospholipid fatty acids, stearic, linoleic and n-6 fatty acids and a trend towards lower docosahexaenoic, arachidonic, palmitic, n-3 and saturated fatty acids. These results suggest that loss of fatty acids is prevalent, progressive and potentially influenced by advanced disease and chemotherapy treatment.     

A Comprehensive GC–MS Sub-Microscale Assay for Fatty Acids and its Applications

Fatty acid analysis is essential to a broad range of applications including those associated with the nascent algal biofuel and algal bioproduct industries. Current fatty acid profiling methods require lengthy, sequential extraction and transesterification steps necessitating significant quantities of analyte. We report the development of a rapid, microscale, single-step, in situ protocol for GC–MS lipid analysis that requires only 250 μg dry mass per sample. We furthermore demonstrate the broad applications of this technique by profiling the fatty acids of several algal species, small aquatic organisms, insects and terrestrial plant material. When combined with fluorescent techniques utilizing the BODIPY dye family and flow cytometry, this micro-assay serves as a powerful tool for analyzing fatty acids in laboratory and field collected samples, for high-throughput screening, and for crop assessment. Additionally, the high sensitivity of the technique allows for population analyses across a wide variety of taxa.     

The Influence of Maternal Early to Mid-Gestation Nutrient Restriction on Long Chain Polyunsaturated Fatty Acids in Fetal Sheep

The early to mid-gestational period (days 28-78) in sheep is the period of most rapid placental development. Maternal nutrient restriction (MNR) in this phase has negative consequences on fetal growth and development, predisposing the fetus to disease in adult life. The influence of MNR on fetal tissue fatty acids has not been reported. Ewes were fed to 50% (MNR) or 100% (control fed) of total digestible nutrients from days 28 to 78 of gestation. At 78 days, fetuses were sacrificed and the fatty acids in fetal liver, lung and muscle as well as maternal and fetal plasma were analyzed. Most fatty acids were not influenced by MNR. The n-3 long chain PUFA eicosapentaenoic acid (20:5n-3, EPA) concentration (microg/mg) was low and more than doubled in the MNR sheep. Similarly, docosapentaenoic acid (22:5n-3, DPA) increased by 60, 19, and 38% in liver, lung, and muscle, respectively. Neither docosahexaenoic acid (22:6n-3, DHA) nor any of n-6 PUFA changed. Arachidonic acid (20:4n-6; ARA) increased in MNR maternal plasma as a percent of total fatty acids only, while in MNR fetal plasma only EPA increased. These results provide the first indication that MNR in early to mid-gestation influences the profiles of LCPUFA in fetal tissues, and suggest that metabolic processes involving LCPUFA should be considered in evaluations of the impact of maternal nutriture on perinatal health.     

UVB Radiation Variably Affects n-3 Fatty Acids but Elevated Temperature Reduces n-3 Fatty Acids in Juvenile Atlantic Salmon (Salmo salar)

Temperature and ultraviolet B radiation (UVB 290–320?nm) are inextricably linked to global climate change. These two variables may act separately, additively, or synergistically on specific aspects of fish biochemistry. We raised Atlantic Salmon (Salmo salar) parr for 54?days in outdoor tanks held at 12 and 19?°C and, at each temperature, we exposed them to three spectral treatments differing in UV radiation intensity. We quantified individual fatty acid (FA) mass fractions in four tissues (dorsal muscle, dorsal and ventral skin, and ocular tissue) at each temperature?×?UV combination. FA composition of dorsal muscle and dorsal and ventral skin was not affected by UV exposure. Mass fractions of 16:0, 18:0, and saturated fatty acids (SFA) were greater in dorsal muscle of warm-reared fish whereas 18:3n-3, 20:2, 20:4n-6, 22:5n-3, 22:6n-3, n-3, n-6, polyunsaturated fatty acids (PUFA), and total FA were significantly higher in cold-reared fish. Mass fractions of most of the FA were greater in the dorsal and ventral skin of warm-reared fish. Cold-reared salmon exposed to enhanced UVB had higher ocular tissue mass fractions of 20:2, 20:4n-6, 22:6n-3, n-3, n-6, and PUFA compared to fish in which UV had been removed. These observations forecast a host of ensuing physiological and ecological responses of juvenile Atlantic Salmon to increasing temperatures and UVB levels in native streams and rivers where they mature before smolting and returning to the sea.     

Quantitative high-resolution13C and1H nuclear magnetic resonance of ω3 fatty acids from white muscle of atlantic salmon (Salmo salar)

High-resolution¹³C nuclear magnetic resonance (NMR) spectra have been obtained and used to define the ω3 (n-3) fatty acid distribution in lipid extract and white muscle from Atlantic salmon (Salmo salar). The¹³C spectrum of lipid extracted from muscle gives quantitative information about the individual n-3 fatty acids, 18:2n-6, 20:1/22:1 and groups of fatty acids. The quantitative data compare favorably with those obtained by gas-liquid chromatography. The¹H NMR spectrum of the lipid extract gives information about the amount of 22:6n-3 and the total content of n-3 fatty acids. The¹³C NMR technique also revealed the positional distribution (1,3- and 2-acyl) of the important 20:5n-3 and 22:6n-3 acids in the triacylglycerol molecules. In the quantitative¹³C NMR spectrum of white muscle, the methyl region of the acyl chains of triacylglycerols gave rise to sufficiently resolved signals to permit estimation of the total concentration of lipids and the n-3 fatty acid content. The NMR data are in good agreement with corresponding data obtained by traditional methods.     

Distribution of (n-9) and (n-7) Isomers of Monounsaturated Fatty Acids in Indian Mustard (Brassica juncea)

The seed oils of 19 Indian mustard varieties were analyzed for fatty acid composition using GC–MS, reporting the presence of n-7 isomers of C18:1, C20:1 and C22:1 fatty acids. n-7 isomers namely cis-vaccenic (C18:1), 11, eicosenoic(C20:1) and 13-cis-docosenoic (C22:1) acids ranged from 0.61 to 1.73, 1.04 to 1.69 and 0.58 to 1.17 %, respectively. The average values of C20:1(n-7) was highest (1.36) amongst the three acids. Nervonic acid was also reported in the range of 0.69 to 2.52 %. The ratios of (n-7)/(n-9) ranged from 6.22 to 14.62, 12.38 to 27.35 and 2.01 to 3.24 % for C18:1, C20:1 and C22:1 fatty acids, respectively. The variety RLM-619 had the lowest elongation ratio (ER) as 0.44 and the highest desaturation ratio (DR) as 0.41 indicating higher efficiency of the desaturation pathway than for other varieties. The oleic desaturation ratio (ODR) values varied from 0.68 (Basanti) to 0.75 (GM-2) with a mean value of 0.72 and linoleic desaturation ratio from 0.40 (Basanti) to 0.49 (Pusa Bold) with a mean value of 0.45. Palmitoleic acid showed positive correlation with C18:1(n-7), C20:1(n-7), C20:2, C22:0, C22:1(n-7), C24:1, (n-7)/(n-9) ratio of C18:1 and C22:1 and a positive trend with ER but a significant negative correlation with C18:3, DR and ODR. The results indicated that palmitoleic acid is an important intermediate component in the synthesis of long chain (n-7) fatty acids.     

Fast Transmethylation of Serum Lipids Using Microwave Irradiation

Microwave irradiation as the energy source for one-step direct transesterification of fatty acids in human serum lipids was examined in a solvent system of methanol: hexane: acetyl chloride based on a Lepage & Roy assay. Innovative and explosion proof single-mode or multimode microwave accelerated reaction system was employed. Recoveries were calculated as the percentage of fatty acid concentrations measured by microwave assay to those by the reference method of the Lepage & Roy assay that utilized conductive heating at 100?°C for 60?min. Under conditions of 100?°C for 1?min in Single-mode (S4–100?×?1), or 125?°C for 5?min in Multimode (M5–125?×?5), the recoveries were 100–103?% for the total fatty acids and 96–106?% for each categorized fatty acid, including saturates, monounsaturates, n-6 PUFA, and n-3 PUFA. For individual PUFA, the mean recoveries were 102–105?% for 18:2n-6 and 18:3n-3; 99, 109, and 95?% for 20:4n-6, 20:5n-3, and 22:6n-3, respectively. Thus, fatty acid concentrations determined by microwave fatty acid assay were accurate to those results by the reference method, when the microwave conditions were optimal. In summary, the microwave irradiation could replace conductive heating in one-step direct transesterification, and reduce the duration from 60?min to 5?min or less. This methodology may be applied in both the absolute and relative quantification of serum total fatty acids.     

Fatty acid composition of melon seed oil lipids and phospholipids

Fatty acid compositions of crude melon seed oil from two different sources were compared. Melon seeds fromCitrullus vulgaris (syn.C. lanatus) contained phosphatidylcholine (PC), lysophosphatidylcholine (LPC) and phosphatidylserine (PS), whereas melon seeds fromCitrullus colocynthis contained only PC and LPC, but not PS. Analysis of the total lipids revealed that the major fatty acid of the oils was 18:2n-6.Citrullus vulgaris seed oil contained 71.3% andC. colocynthis contained 63.4% of 18:2n-6. The predominant fatty acids in theC. vulgaris PC were 18:2n-6 (32.2%), 18:1n-9 (26.4%) and 16:0 (22.2%), whereas theC. colocynthis PC contained 44.6% of 18:1n-9 as the major fatty acid. The level of monoenes in theC. colocynthis variety (46.2%) was different from theC. vulgaris (27.3%). The major fatty acid in the LPC was 18:1n-9 for both varieties. Notably, theC. colocynthis variety did not contain any PS. The major fatty acids in theC. vulgaris PS were 18:1n-9 (37.9%) and 18:2n-6 (33.7%). Of all the phospholipids, LPC contained the greatest amount of monoenes, 48.6–52.4%.