Identification of minor fatty acids and various nonmethylene-interrupted diene isomers in mantle,muscle, and viscera of the marine bivalve Megangulus zyonoensis

To clarify the occurrence of nonmethylene-interrupted (NMI) FA in the marine bivalve Megangulus zyonoensis, methyl esters of unsaturated FA were fractionated according to the degree of their unsaturation by using argentation TLC. Their structures were elucidated by using GC-MS of their FAME and 2-alkenyl-4,4-dimethyloxazoline derivatives. Seventy-two unsaturated FA, including the novel 7,15–21 and 20∶4n−1, were identified. The unusual tetraenoic acids 20∶4n−4, 20∶4n−1, 21∶4n−6, and 21∶4n−5 were found in M. zyonoensis. This bivalve was extremely rich in the positional isomers of 19∶1, 20∶2, and 20∶3. The distribution of NMI and positional isomers of unusual FA in the bivalve tissues was also discussed.     

Lipid Classes and Fatty Acid Composition of the Tropical Nudibranch Mollusks <Emphasis Type="Italic">Chromodoris</Emphasis> sp. and <Emphasis Type="Italic">Phyllidia coelestis</Emphasis>

Two nudibranch mollusks, Chromodoris sp. and Phyllidia coelestis, collected from tropical waters of the Northwestern Pacific, were analyzed for lipids. The aim of this study was to fill the gap in knowledge of lipid biochemistry of mollusks. Phospholipids (PL) were the dominating lipid class followed by sterols (13%). Neutral lipids were not detected in Chromodoris sp. By contrast, P. coelestis contained TAG, diacylglyceryl ether, long chain alcohol and esters of sterols. Among PL, PC was predominant (about 50%); PE, PS and CAEP were almost in equal proportions. Sixty five FA were identified as methyl esters and N-acyl pyrrolidides by GC-MS. The sea slugs exhibited a wide diversity of FA. The common marine n-3 PUFA, 20:5n-6 and 22:6n-3, constituted 0.6-1.3% of the total FA, whereas n-6 PUFA, 22:4n-6, 20:4n-6, and 18:2n-6, were the main (25%). Among monounsaturated FA, 7-21:1 was the main (up to 6.2%). The non-methylene-interrupted (NMI) FA were found (9.4 and 12.4%), including the known 5,11-20:2, 5,13-20:2, 7,13-22:2, 7,15-22:2 and a novel isomer 7,13-21:2 (up to 3.9%). The pathway of its biosynthesis was suggested. A series of very long chain FA (VLC FA), with the main 5,9-25:2 and 5,9-26:2, were identified. High level of VLC FA (8.7 and 11.7%) in sea slugs is apparently the result of predation on sponges. Another unique feature concerned a high abundance of various odd and branched FA (16.7 and 34%), which could have originated from the dietary origin or symbiotic bacteria. This is the first report on lipid and FA composition of nudibranchs.     

Stereospecific analysis of fish oil triacyl-sn-glycerols

Stereospecific analysis of fish oil triacyl-sn-glycerols was carried out by novel high-performance liquid chromatography on a chiral stationary phase. The positional distributions of fatty acids were determined without accumulation of errors in a particular position and preferential hydrolysis for a particular fatty acid. High-resolution gas-liquid chromatography on an open tubular column detailed the distribution of unsaturated fatty acid isomers having olefinic bonds in different positions. The distribution of fatty acids was not independent of other fatty acids. The distribution of long-chain highly unsaturated fatty acids 22:6(n-3), 22:5(n-3) and 20:5(n-3) was governed by total amounts of 20:1 and 22:1 in triacyl-sn-glycerols. Long-chain monounsaturated acids 20:1 and 22:1 were influenced by the position of the olefinic bond in 20:1 occurring in triacyl-sn-glycerols. Shorter-chain C14-C18 fatty acids seemed to be influenced by total fatty acid composition. These results introduce a concept of mutual interaction between fatty acids to the investigation of positional distribution of fatty acids.     

Tocopherol Content and Fatty Acid Distribution of Peas (<Emphasis Type="Italic">Pisum sativum</Emphasis> L.)

The positional distribution of fatty acids (FA) of triacylglycerols (TAG) and major phospholipids (PL) prepared from four cultivars of peas (Pisum sativum L.) were investigated as well as their tocopherol contents. The lipids extracted from these peas were separated by thin-layer chromatography (TLC) into seven fractions. The major lipid components were PL (52.2–61.3%) and TAG (31.2–40.3%), while the other components were also present in minor proportions (5.6–9.2%). γ-Tocopherol was present in the highest concentration, and α- and δ-tocopherols were very small amounts. The main PL components isolated from the four cultivars were phosphatidylcholine (42.3–49.2%), followed by phosphatidylinositol (23.3–25.2%) and then phosphatidylethanolamine (17.7–20.5%). Small but significant differences (P < 0.05) in FA distribution existed when different pea cultivars were determined. However, the principal characteristics of the FA distribution in the TAG and the three PL were evident among the four cultivars; unsaturated FA were predominantly located in the sn-2 position, and saturated FA primary occupied the sn-1 or sn-3 position in the oils of the peas. These results suggest that the regional distribution of tocopherols and fatty acids in peas is not dependent on the climatic conditions and the soil characteristics of the cultivation areas during the growing season.     

Seasonal Variations of Lipid Content and Composition in <Emphasis Type="Italic">Perna viridis</Emphasis>

The total lipid content, composition of main lipid classes, composition of sterols and composition of fatty acids in the main glycerolipids of Perna viridis were analyzed through four seasons using TLC-FID and GLC. Mussel samples were collected during different seasons between 2003 and 2004 from Shengsi Island, Zhejiang Province, China and stored frozen prior to freeze-drying and lipid extraction. Ten grams of dried mussel powder of each season were analyzed. Total lipid content ranged from 14.5 g/100 g in spring month to 7.8 g/100 g dried mussel powder in autumn month. The predominant lipid in spring month was triacylglycerol (TAG), however, in the other three seasons the phospholipids (PL) was the main lipid class. The most abundant fatty acid in TAG, PL and phosphatidylcholine (PC) was 16:0, with the summer samples having the highest proportion (24-30% of total fatty acid) and winter the lowest (14-22%). In phosphatidylethanolamine (PE), the spring samples had the highest proportions of 16:0. The predominant polyunsaturated fatty acids (PUFA) were 22:6n-3 and 20:5n-3 in TAG, PL, PE and PC (25-40%). The proportions of 22:6n-3 and 20:5n-3 were higher in spring than in other seasons in PL and PE. There were nine sterols identified, with cholesterol being the predominant sterol, and other main ones were desmostersol/brassicasterol and 24-methylenecholesterol. Proportions of other fatty acids in different lipid fractions and the sterol compositions as well also varied seasonally. There were subject to the seasonal variations. Differences in lipid content and composition, fatty acid composition in different lipid fractions may be caused by multiple factors such as lifecycle, sex, variation of plankton in different seasons and temperature, which could influence physiological activities and metabolism.     

Lipid Classes,Fatty Acid Distributions and Triacylglycerol Molecular Species of Broad Beans (<Emphasis Type="Italic">Vicia faba</Emphasis>)

Seed oils from four legume cultivars of Vicia faba, grown in Japan, were extracted and classified by thin-layer chromatography (TLC) into eight fractions. The major lipid components were triacylglycerols (TAG: 48.8–50.1%) and phospholipids (PL: 47.5–50.5%), while hydrocarbons (HC), steryl esters (SE), free fatty acids (FFA), diacylglycerols (1,3- and 1,2-DAG) and monoacylglycerols (MAG) were present in minor proportions (1.8–2.4%). All lipid samples had high amounts of total unsaturated FA, representing 79.7–82.8% and 77.6–79.7% for TAG and PL, respectively. Molecular species and FA distributions of TAG, isolated from the total lipids in the broad beans, were analyzed by a combination of argentation-TLC and GC. Fourteen different molecular species were detected. With a few exceptions, the main TAG components were S₂D (6.1–8.9%), SD₂ (7.8–10.5%), SMT (6.3–8.5%), M₂D (4.5–6.2%), MD₂ (18.9–21.8%), D₃ (21.0–23.9%) and MDT (8.1–10.2%) (where S, M, D, and T denote a saturated fatty acid, a monoene, a diene, and a triene, respectively). These results suggest that the lipid classes, FA distributions and TAG molecular species of broad beans are not dependent on the cultivation areas during the growing season.     

Fatty Acid Profile of Sunshine Bass: II. Profile Change Differs Among Fillet Lipid Classes

Fatty acid (FA) profile of fish tissue mirrors dietary FA profile and changes in a time-dependent manner following a change in dietary FA composition. To determine whether FA profile change varies among lipid classes, we evaluated the FA composition of fillet cholesteryl esters (CE), phospholipids (PL), and triacylglycerols (TAG) of sunshine bass (SB, Morone chrysops x M. saxatilis) raised on feeds containing fish oil or 50:50 blend of fish oil and coconut, grapeseed, linseed, or poultry oil, with or without implementation of a finishing period (100% FO feed) prior to harvest. Each lipid class was associated with a generalized FA signature, irrespective of nutritional history: fillet PL was comprised largely of saturated FA (SFA), long-chain polyunsaturated FA (LC-PUFA), and total n-3 FA; fillet TAG was higher in MC-PUFA and total n-6 FA; and fillet CE was highest in monounsaturated FA (MUFA). Neutral lipids reflected dietary composition in a near-direct fashion; conversely, PL showed evidence of selectivity for MC- and LC-PUFA. Shorter-chain SFA were not strongly reflected within any lipid fraction, even when dietary availability was high, suggesting catabolism of these FA. FA metabolism in SB is apparently characterized by a division between saturated and unsaturated FA, whereby LC-PUFA are preferentially incorporated into tissues and SFA are preferentially oxidized for energy production. We demonstrated provision of SFA in grow-out feeds for SB, instead MC-PUFA which compete for tissue deposition, meets energy demands and allows for maximum inclusion of LC-PUFA within fillet lipids.     

Thiyl radical-catalyzed isomerization of oils: An entry to the trans lipid library

The unsaturated fatty acyl moieties of TAG present in natural oils of borage, olive, and rice were converted to their corresponding geometrical trans isomers by thiyl radical-catalyzed isomerization. Thiyl radicals were generated from 2-mercaptoethanol under photolytic or thermal conditions. A relevant feature of this method is the absence of double-bond shifts, so that no positional trans isomers or conjugated polyenes are formed. Oils obtained after the isomerization were winterized to further increase their trans fatty acid content. Methanolysis and hydrolysis of the trans oil mixtures using an enzymatic method (lipase B from Candida antarctica) gave good conversions to the corresponding trans FAME and fatty acids, respectively. These results are relevant for the studies of lipid isomerism and trans fatty acid recognition, which is a growing concern in biochemistry and nutrition, and open new perspectives for the synthesis of glycerides and studies of their structure-activity relationships.     

The Distribution of Fatty Acid Biomarkers of Dairy Intake across Serum Lipid Fractions: The Prospective Metabolism and Islet Cell Evaluation (PROMISE) Cohort

Circulating fatty acids (FA) derived largely from dairy consumption have most commonly been measured in total human serum or phospholipid (PL) fractions, and have been used as validated biomarkers of dairy intake in a growing number of epidemiological studies. Nevertheless, measurement and characterization of a wider spectrum of FA biomarkers of dairy across the four major serum lipid fractions is lacking. This study aimed to (1) quantify FA biomarkers of dairy in PL, triacylglycerol (TAG), cholesteryl ester (CE), and unesterified fatty acid (FFA) serum lipid fractions; and (2) identify potential demographic and metabolic factors that may modify the proportions of these FA across serum fractions. Baseline data from 444 adults in the PROMISE cohort were analyzed. FA biomarkers, 15:0, t16:1n-7, 18:2-c9,t11, and t18:1n-7 were quantified from serum. Dairy intake was estimated using the validated Canadian Diet History Questionnaire. Our results show that t18:1n-7 was the most abundant FA biomarker in all fractions except CE, where 18:2-c9,t11 was the most abundant. Positive correlations within fractions, and across FA in the PL, CE, and FFA fractions were found, however, TAG FA were negatively correlated with the other fractions. PL and CE FA were positively associated with dairy intake, and negatively associated with markers of dysmetabolism while, in contrast, these markers were predictors of higher TAG dairy FA. This study is the first to demonstrate distinct proportions of dairy FA in different serum lipid fractions. PL and CE FA marked dairy intake in this cohort, while TAG FA appeared to be markers of dysmetabolism.     

An improved reversed-phase thin-layer chromatography method for separation of fatty acid methyl esters

Resolution of fatty acid methyl esters (FAME) by thin-layer chromatography often is complicated by co-migration of certain acyl-isomers in heterogeneous mixtures. However, a novel reversed-phase thin-layer chromatography method which employs 10% (wt/vol) silver nitrate in a mobile phase containing acetonitrile/1,4-dioxane/acetic acid (80:20:1, vol/vol/vol) allows one-dimensional resolution of a wide range of acyl-methyl esters. This innovation enables improved separation of saturated FAME ranging from C₁₂ to C₂₂, and geometric isomers of C₁₄ to C₂₂ unsaturated FAME by thin-layer chromatography.     

Unusual minor nonmethylene-interrupted di-, tri-, and tetraenoic fatty acids in limpet gonads

Unusual minor nonmethylene-interrupted (NMI) FA have been identified in the lipids of gonads from the limpets Cellana grata and Collisella dorsuosa by using GC-MS of the combination of their 4,4-dimethyloxazoline derivatives and picolinyl esters. Among 23 NMI unsaturated FA from C₁₈ to C₂₂ and C₂₄ identified in this study, 5,11-nonadecadienoic (5,11-19∶2), 7,16-heneicosadienoic (7,16–21∶2), 9,15-tetracosadienoic (9,15–24∶2), 5,9,15-docosatrienoic (5,9,15–22∶3), and 5,9,15-tetracosatrienoic (5,9,15–24∶3) acids may not have been reported previously from living organisms. The presence of 5,11,14,17-eicosatetraenonoic (5,11,14,17–20∶4) and 7,13,16,19-docosatetraenenoic (7,13,16,19–22∶4) acids as FA components in marine mollusks may be reported here for the first time. In this study, the male and female gonads of both species showed distinct differences in both their composition and proportions of NMI FA. Most NMI FA identified were mainly present in the female gonads of both species, especially in TAG that contained 21 NMI FA.     

Hydrolysis of Acyl-Homogeneous and Fish Oil Triacylglycerols Using Desalted Midgut Extract from Atlantic Salmon, <Emphasis Type="Italic">Salmo salar</Emphasis>

Despite several studies aimed at evaluating the positional and fatty acid specificity of fish triacylglycerol (TAG) digestive lipases, there is still much uncertainty regarding these issues. The aim of the present study was therefore to address these questions in Atlantic salmon (Salmo salar L.). Crude luminal midgut extracts were collected from fed salmon and the hydrolysis studied for various substrates including triolein (Tri-18:1), trilinolein (Tri-18:2), trilinolenin (Tri-18:3), trieicosapentaenoin (Tri-20:5), tridocosahexaenoin (Tri-22:6) and natural fish oil TAG. Using Tri-18:1, in a time-curve model showed an initial high degree of sn-1 or sn-3 specificity where sn-1,2(2,3)-diacylglycerol (1,2(2,3)-DAG) and free fatty acid (FFA) were the main hydrolytic products up to 15 min. Lack of initial sn-2 specificity was confirmed by negligible sn-1,3-diacylglycerol (1,3-DAG) being produced. During the further hydrolysis of DAG, all positions appeared susceptible to attack causing a concomitantly small increase in sn-1(3)-monoacylglycerol (1(3)-MAG) and 2-MAG, but not at the level expected for an exclusively sn-1,3-specific lipase. Oleic acid (18:1n-9) and eicosapentaenoic acid (20:5n-3) were preferred substrates for hydrolysis using both fish oil and acyl-homogeneous TAGs with FFA as the main product of lipolysis. Hydrolysis of the natural fish oil TAG appeared slower yet produced proportionally more MAG and DAG after 5 min, and similar specificities, as for synthetic TAG substrates, were exhibited with 18:1n-9 and 20:5n-3 accumulating in the FFA fraction after 30 min. Notably, 16:0 was particularly conserved in MAG. As TAG resynthesis of absorbed lipid in salmon enterocytes proceeds preferably with 2-MAG as templates, the absorption of 2-MAG, produced during initial stages of TAG hydrolysis, would need to occur rapidly to be effectively utilised via the MAG pathway.     

Non-methylene Interrupted and Hydroxy Fatty Acids in Polar Lipids of the Alga Grateloupia turuturu Over the Four Seasons

Phospholipids (PL) and glycolipids (GL) FA in the edible Rhodophyta Grateloupia turuturu, from Brittany, France, were investigated over four seasons. The major lipid class was GL in all seasons (around 45 %). More than 80 FA occurred in polar lipids, with chains from C₁₂ to C₂₆, identified as methyl esters and N-acyl pyrrolidides by gas chromatography–mass spectrometry (GC–MS). PUFA occurred at up to 47.1 % (summer) in PL, and up to 43.6 % (summer) in GL. The major PUFA were 20:5n-3 (12.2 % in PL and 29.0 % in GL) and 20:4n-6 (25.6 % in PL and 10.4 % in GL). The unusual 18:3n-7 acid was identified in PL up to 2.2 %. Several minor unsaturated FA were identified in PL and are previously unreported in seaweeds, namely 14-tricosenoic, 15-tetracosenoic, 5,11-octadecadienoic and 5,9-nonadecadienoic. Also unprecedented in seaweeds, ten 2-hydroxy and three 3-hydroxy FA occurred mainly in PL, 13.9 % in spring with the 3-hydroxyhexadecanoic acid as the major one (8.1 % winter). Three n-9 monounsaturated 2-hydroxy FA occurred in PL. The 2-hydroxy-15-tetracosenoic acid was characterized as the dimethyl disulfide adduct of its methyl ester. The 2-hydroxy-16-pentacosenoic and 2-hydroxy-17-hexacosenoic acids were identified by comparison of mass spectra and GC mobilities with those of the 2-hydroxy-15-tetracosenoic acid, and of other homogeneous FA series. These rare n-9 monounsaturated 2-hydroxy FA are unprecedented in seaweeds.     

Muscle Lipids and Fatty Acid Profiles of the Sea Catfish (Arius madagascariensis) in Madagascar Inland Waters

Muscle lipids and fatty acids (FA) of catfish Arius madagascariensis were determined in catfish caught in the Betsiboka River, Madagascar, during a 5-month sampling period. Total lipids from muscle were extracted and quantified. Fatty acids were identified by means of gas chromatography–mass spectrometry of FA methyl esters and FA pyrrolidides, leading to the identification of 42 FA. Lipid content was relatively high in our fish sample and ranged from 4.3 to 6.6% of wet muscle. Three FA dominated the FA composition: palmitic acid (C16:0, 22.9–32.6%), oleic acid (C18:1n-9, 11.3–13.4%) and stearic acid (C18:0, 10.8–12.0%). A number of polyunsaturated FA (PUFA) were present in appreciable amounts, including arachidonic acid (C20:4n-6, 4.7–7.6%), docosahexaenoic acid (C22:4n-6, 3.0–8.1%), eicosapentaenoic acid (C20:5n-3, 0.6–1.0%), n-3 docosapentaenoic acid (C22:5n-3, 1.1–1.6%), n-6 docosatetraenoic acid (C22:4n-6, 0.7–1.2%) and n-6 docosapentaenoic acid (22:5n-6, 0.9–1.8%). The sum of the n-6 PUFA and n-3 PUFA was 11.3–18.8 and 7.5–13.4%, respectively. These results indicate that A. madagascariensis, an abundant freshwater fish in Madagascar rivers, may be good source of dietary PUFA.     

Occurrence and chemical structure of nonmethylene-interrupted dienoic fatty acids in american oysterCrassostrea virginica

The American oyster,Crassostrea virginica, was found to contain structurally homologous nonmethylene-interrupted dienoic (NMID) fatty acids. The major C₂₀ and C₂₂ nonmethylene-interrupted dienoic fatty acid isomers were shown to occur as two pairs of homologues 5,13–20∶2 with 7,15–22∶2 and 5,11–20∶2 with 7,13–22∶2. A combination of analytical procedures was required for conclusive structure determination.     

Unsaturated phosphatidylethanolamine as effective synergist in combination with alpha-tocopherol

The effects of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) on the oxidation of bonito oil triacylglycerol (TAG) were examined under the absence or presence of alpha-tocopherol. Synthesized PC or PE having saturated and unsaturated fatty acids in known position were used in this study. Unsaturated PC and PE promoted TAG oxidation under the absence of alpha-tocopherol. On the contrary, unsaturated PE showed synergistic antioxidant effect with alpha-tocopherol, while PC had little effect. The strongest synergistic effect was found by 1-palmitoyl (16:0, PA)-2-docosahexaenoyl (22:6n-3)-PE, followed by 1-PA-2-arachidonyl (20:4n-6)-PE, 1,2-dioleoyl (18:1n-9)-PE, and 1-PA-2-linoleoyl (18:2n-6)-PE, respectively.     

Comparison of Separations of Fatty Acids from Fish Products Using a 30-m Supelcowax-10 and a 100-m SP-2560 Column

Commercial fish oils and foods containing fish may contain trans and/or isomerized fatty acids (FA) produced during processing or as part of prepared foods. The current American Oil Chemists’ Society (AOCS) official method for marine oils (method Ce 1i-07) is based on separation by use of poly(ethylene glycol) (PEG) columns, for example Supelcowax-10 or equivalent, which do not resolve most unsaturated FA geometric isomers. Highly polar 100-m cyanopropyl siloxane (CPS) columns, for example SP-2560 and CP Sil 88 are recommended for separation of geometric FA isomers. Complementary separations were achieved by use of two different elution temperature programs with the same CPS column. This study is the first direct comparison of the separations achieved by use of 30-m Supelcowax-10 and 100-m SP-2560 columns for fatty acid methyl esters (FAME) prepared from the same fish oil and fish muscle sample. To simplify the identification of the FA in these fish samples, FA were fractionated on the basis of the number and type of double bonds by silver-ion solid-phase extraction (Ag⁺-SPE) before GC analysis. The results showed that a combination of the three GC separations was necessary to resolve and identify most of the unsaturated FA, FA isomers, and other components of fish products, for example phytanic and phytenic acids. Equivalent chain length (ECL) values of most FAME in fish were calculated from the separations achieved by use of both GC columns; the values obtained were shown to be consistent with previously reported values for the Supelcowax-10 column. ECL values were also calculated for the FA separated on the SP-2560 column. The calculated ECL values were equally valid under isothermal and temperature-programmed elution GC conditions, and were valuable for confirmation of the identity of several unsaturated FAME in the fish samples. When analyzing commercially prepared fish foods, deodorized marine oils, or foods fortified with marine oils it is strongly recommended that quantitative data acquired by use of PEG columns is complemented with data obtained from separations using highly polar CPS columns.     

High Arachidonic Acid Levels in the Tissues of Herbivorous Fish Species (<Emphasis Type="Italic">Siganus fuscescens,Calotomus japonicus</Emphasis> and <Emphasis Type="Italic">Kyphosus bigibbus</Emphasis>)

The lipid and fatty acid compositions in the various organs (muscle, liver, other viscera) and stomach contents of three common herbivorous fish species in Japan, Siganus fuscescens, Calotomus japonicus and Kyphosus bigibbus, were examined to explore the stable 20:4n-6 (arachidonic acid, ARA) sources. Triacylglycerol (TAG), phosphatidylethanolamine (PtdEtn), and phosphatidylcholine (PtdCho) were the dominant lipid classes, while the major FA contents were 16:0, 18:1n-9, 16:1n-7, 14:0, 18:0, 18:1n-7, and some PUFA, including ARA, 20:5n-3 (eicosapentaenoic acid, EPA), 22:5n-3 (docosapentaenoic acid, DPA), and 22:6n-3 (docosahexaenoic acid, DHA). The amounts of these fatty acids were varied among species and their lipid classes. Phospholipids contained higher levels of PUFA than TAG. However, ARA in both phospholipids and TAG was markedly present in the muscle and viscera of all specimens, particularly in C. japonicus and K. bigibbus. Moreover, their ARA levels were higher than the levels of DHA and EPA. The observed high ARA level is unusual in marine fish and might be characteristic of herbivorous fish. Furthermore, ARA was the dominant PUFA in the stomach contents of the three species, suggesting that the high ARA level originated from their food sources. The above indicates that these three herbivorous fishes are ARA-rich marine foods and have potential utilization as stable ARA resources.     

Trans-Membrane Uptake and Intracellular Metabolism of Fatty Acids in Atlantic Salmon (<Emphasis Type="Italic">Salmo salar</Emphasis> L.) Hepatocytes

To elucidate if the trans-membrane uptake of fatty acids is protein-mediated, the uptake of oleic acid (18:1n-9), linoleic acid (18:2n-6), alpha-linolenic acid (18:3n-3), eicosapentaenoic acid (20:5n-3) and docosahexaenoic acid (22:6n-3) was investigated in vitro in Atlantic salmon (Salmo salar L.) primary hepatocytes. Firstly, optimal fatty acid incubation time and concentration were established for trans-membrane 18:n-9 uptake. Based on saturation kinetics, a 2-h incubation time and 37.5 μM were used for the following experiments. Secondly, in order to identify whether trans-membrane fatty acid uptake in hepatocytes was mainly passive or protein mediated, hepatocytes were pre-incubated with membrane protein inhibitors followed by 2 h of incubation with [1-¹⁴C] labelled 18:1n-9, 18:2n-6, 18:3n-3, 20:5n-3 and 22:6n-3. Fatty acid uptake into hepatocytes was highest with 20:5n-3 and 22:6n-3 and lowest with 18:1n-9. Phloretin was the most potent fatty acid uptake inhibitor, inhibiting uptake in the following order: 20:5n-3 > 18:3n-3 = 22:6n-3 > 18:2n-6 > 18:1n-9. The uptake of FA in Atlantic salmon hepatocytes seem to be due to both saturable and inhibitable protein mediated uptake, as well as passive uptake processes with more unsaturated and long fatty acids (20:n-3 > 22:6n-3 = 18:3n-3 > 18:2n-6) being more dependent on membrane protein mediated uptake compared to 18:1n-9.     

Preparative Separation of <Emphasis Type="Italic">cis</Emphasis>- and <Emphasis Type="Italic">trans</Emphasis>-Isomers of Unsaturated Fatty Acid Methyl Esters Contained in Edible Oils by Reversed-Phase High-Performance Liquid Chromatography

In order to measure exactly the trans-fatty acids content in food materials, a preparative group separation of cis- and trans-isomers of unsaturated fatty acid methyl esters (FAMEs) was achieved by an isocratic reversed-phase HPLC (RP-HPLC) method. The trans-isomers of 16:1, 18:1, 18:2, 18:3, 20:1 and 22:1 FAMEs were readily separated from the corresponding cis-isomers by a COSMOSIL Cholester C18 column (4.6 mm I.D. × 250 mm, Nacalai Tesque) or a TSKgel ODS-100Z column (4.6 mm I.D. × 250 mm, TOSOH), using acetonitrile as the mobile phase. This method was applied for determining the trans-18:1 fatty acid content in partially hydrogenated rapeseed oil. The methyl esters of cis- and trans-18:1 isomers of the oil were collected as two separate fractions by the developed RP-HPLC method. Each fraction was analyzed by gas chromatography (GC) for both qualitative and quantitative information on its positional isomers. By a combination of RP-HPLC and GC methods, a nearly complete separation of cis- and trans-18:1 positional isomers was achieved and the trans-18:1 fatty acid content was able to be evaluated more precisely than is possible by the direct GC method. The reproducibility of cis- and trans-18:1 isomers fractionated by the RP-HPLC method was better than 98%. These results suggested that the preparative RP-HPLC method developed in this study could be a powerful tool for trans-fatty acid analysis in edible oils and food products as an alternative to silver-ion chromatography.