Lipid Profiling Reveals Tissue-Specific Differences for Ethanolamide Lipids in Mice Lacking Fatty Acid Amide Hydrolase

N-Acylethanolamines (NAE) are fatty acid derivatives, some of which function as endocannabinoids in mammals. NAE metabolism involves common (phosphatidylethanolamines, PEs) and uncommon (N-acylphosphatidylethanolamines, NAPEs) membrane phospholipids. Here we have identified and quantified more than a hundred metabolites in the NAE/endocannabinoid pathway in mouse brain and heart tissues, including many previously unreported molecular species of NAPE. We found that brain tissue of mice lacking fatty acid amide hydrolase (FAAH ^−/−) had elevated PE and NAPE molecular species in addition to elevated NAEs, suggesting that FAAH activity participates in the overall regulation of this pathway. This perturbation of the NAE pathway in brain was not observed in heart tissue of FAAH ^−/− mice, indicating that metabolic regulation of the NAE pathway differs in these two organs and the metabolic enzymes that catabolize NAEs are most likely differentially distributed and/or regulated. Targeted lipidomics analysis, like that presented here, will continue to provide important insights into cellular lipid signaling networks.     

Anandamide and other <Emphasis Type="Italic">N</Emphasis>-acylethanolamines in human tumors

Long-chain N-acylethanolamines (NAE), including the endocannabinoid, anandamide, accumulate in mammalian tissues under a variety of pathological conditions. They have also been shown to inhibit the growth of various cancer cell lines in vitro. Here, we report the presence, in widely differing amounts (3.88–254.46 pmol/μmol lipid P), of NAE and their precursor phospholipids in various human tumors and some adjacent unaffected tissue. Anandamide ranged from 1.5 to 48% of total NAE, and incubation of tissue homogenates suggested possible NAE biosynthesis by both the established transacylation-phosphodiesterase pathway via N-acyl PE and by direct N-acylation of ethanolamine.     

Identification and quantification of neuroactive <Emphasis Type="Italic">N</Emphasis>-acylethanolamines in cottonseed processing fractions

N-Acylethanolamines (NAE) include lipid species that have been identified as potent endocannabinoid mediators in mammalian physiology. Here we have isolated, identified, and quantified the NAE naturally present in cottonseed refining fractions. Among the fractions examined, NAE were most prevalent in finished meal, totaling approximately 8.5 μg/g fresh weight. These NAE were identified by GC-MS as ethanolamides of myristic (14∶0), palmitic (16∶0), stearic (18∶0), oleic (18∶1), and linoleic (18∶2) acids, with NAE18∶2 and NAE16∶0 being the most abundant. NAE were not detected in refined, bleached, and deodorized oil, although they were present in less-purified oil fractions. At micromolar concentrations, both NAE18∶2 and NAE16∶0 exhibited neuromodulatory activities in murine neuronal networks cultured and evaluated on microelectrode arrays. It follows that oilseed processing fractions may represent a novel, natural source of bioactive NAE.     

Molecular weight distribution and regioisomeric structure of triacylglycerols in some common human milk substitutes

Triacylglycerol (TAG) molecular weight distribution and regioisomeric structure of selected molecular weight species in human milk and in 32 human milk substitutes was determined. Negative ion chemical ionization mass spectrometry was used to determine the molecular weight distribution and collisionally induced dissociation tandem mass spectrometry applied to identify the sn-2 and sn-1/3 positions of fatty acids in TAG. The main molecular weight species of human milk TAG in decreasing order of abundance were 52∶2, 52∶3, 52∶1, 54∶3, 50∶2, 50∶1, 54∶4, 48∶1, 54∶2, 48∶2, 46∶1, 52∶4, and 50∶3 (acyl carbon number/number of double bonds), constituting 83 mol% of total TAG molecular species. In human milk substitutes, the proportion of the corresponding molecular weight species varied from 33 to 87 mol%. The main TAG regioisomers within the molecular weight species 52∶2, 52∶3, and 50∶1 in human milk were 18∶1-16∶0-18∶1 (83 mol%), 18∶1-16∶0-18∶2 (83 mol%), and 18∶1-16∶0-16∶0 (80 mol%), respectively. In human milk substitutes, the corresponding proportions varied in a wide range of 0–82 mol%, 0–100 mol%, and 0–73 mol%, respectively. Although TAG structures in some human milk substitutes closely resembled those in human milk, the great variation among samples leads to the conclusion that it is still possible to improve the TAG composition in human milk substitutes by applying novel methods to synthesize structured TAG.     

Phospholipid molecular species from human placenta lipids

The phospholipid molecular species from a large-scale preparation of human placenta lipids were analyzed. The major placental phospholipids were choline glycerophospholipids (CPL) (53.2 wt%), sphingomyelin (21.7 wt%) and ethanolamine glycerophospholipids (EPL) (14.6 wt%). 1,2-Diacyl-glycerophosphocholine was the most abundant subclass of CPL (91.7 mol%), while EPL contained 1,2-diacyl (54.6 mol%) and 1-alk-1′-enyl-2-acyl (43.8 mol%) subclasses. The level of polyunsaturated fatty acids (PUFA) in total phospholipids was remarkably constant (38.4–39.9 mol%) within all placental batches tested. The long-chain PUFA, mainly 20∶4n−6 and 22∶6n−3 of the n−6 and n−3 series, respectively, were found in high proportion in all phospholipid classes, especially in EPL (46.7 mol%) and in inositol glycerophospholipids (IPL) (39.9 mol%). CPL and serine glycerophospholipids were much richer in 18∶1n−9 and 18∶2n−6. High levels of molecular species with arachidonic acid in thesn-2 position were found particularly in 1-alk-1′-enyl-2-acyl-glycerophospho-ethanolamine (with 24.0 mol% 16∶0 and 22.0 mol% 18∶0 insn-1 position) and in 1,2-diacyl glycerophosphoinositol with 42.6 mol% 18∶0 insn-1 position. EPL subclasses were rich in 22∶6n−3, which occurs mainly as 16∶0/22∶6n−3 (11.7 mol%) in the polasmalogen form and as 18∶0/22∶6n−3, 16∶0/22∶6n−3 and 18∶1/22∶6n−3 in the diacyl forms. Based on their availability and composition, placental phospholipids could be of interest, for example, for supplementing artificial milk preparations with n−3 and n−6 long-chain PUFA for newborn infants with insufficiently developed 18∶2n−6 and 18∶3n−3 desaturation/elongation.     

PC and PE synthesis: mixed micellar analysis of the cholinephosphotransferase and ethanolaminephosphotransferase activities of human choline/ethanolamine phosphotransferase 1 (CEPT1)

The human choline/ethanolamine phosphotransferase 1 (CEPT1) gene codes for a dual-specificity enzyme that catalyzes the de novo synthesis of the two major phospholipids through the transfer of a phosphobase from CDP-choline or CDP-ethanolamine to DAG to form PC and PE. We used an expression system devoid of endogenous cholinephosphotransferase and ethanolaminephosphotransferase activities to assess the diradylglycerol specificity of CEPT1. A mixed micellar assay was used to ensure that the diradylglycerols delivered were not affecting the membrane environment in which CEPT1 resides. The CEPT1 enzyme displayed an apparent K _m of 36 μM for CDP-choline and 4.2 mol% for di-18∶1 DAG with a V _max of 14.3 nmol min⁻¹ mg⁻¹. When CDP-ethanolamine was used as substrate, the apparent K _m was 98 μM for CDP-ethanolamine and 4.3 mol% for di-18∶1 DAG with a V _max of 8.2 nmol min⁻¹ mg⁻¹. The preferred diradylglycerol substrates used by CEPT1 with CDP-choline as the phosphobase donor were di-18∶1 DAG, di-16∶1 DAG, and 16∶0/18∶1 DAG. A major difference between previous emulsion-based assay results and the mixed micelle results was a complete inability to use 16∶0(O)/2∶0 as a substrate for the de novo synthesis of platelet-activating factor when the mixed micelle assay was used. When CDP-ethanolamine was used as the phosphobase donor, 16∶0/18∶1 DAG, di-18∶1 DAG, and di-16∶1 DAG were the preferred substrates. The mixed micelle assay also allowed the lipid activation of CEPT to be measured, and both the cholinephosphotransferase and ethanolaminephosphotransferase activities displayed the unusual property of product activation at 5 mol%, implying that specific lipid activation binding sites exist on CEPT1. The protein kinase C inhibitor chelerythrine and the human DAG kinase inhibitor R59949 both inhibited CEPT1 activity with IC₅₀ values of 40 μM.     

An Improved Method for Synthesis of <Emphasis Type="Italic">N</Emphasis>-stearoyl and <Emphasis Type="Italic">N</Emphasis>-palmitoylethanolamine

Certain N-acylethanolamines interact with cannabinoid receptors and have anorexic and neuroprotective effects. Traditional methods for the synthesis of N-acylethanolamines use fatty acid chlorides, fatty acid methyl esters, free fatty acids and triacylglycerols as acyl donors to react with ethanolamine. In the present study, we investigated the feasibility of using fatty acid vinyl esters as the acyl donor to synthesize N-stearoyl and N-palmitoylethanolamine. Theoretically, the use of fatty acid vinyl esters should lead to an irreversible reaction because the volatile acetaldehyde by-product is easily removed. Four reaction conditions, i.e. catalyst concentration, substrate ratio, temperature, and time were evaluated. The reaction performed at mild temperatures and with an excess amount of ethanolamine which acted as both reactant and solvent resulted in the formation of high purity N-stearoyl and N-palmitoylethanolamine. When 20 mmol ethanolamine was reacted with 1 mmol vinyl stearate at 80 °C for 1 h with 1% sodium methoxide as catalyst, N-stearoylethanolamine with 96% purity was obtained after the removal of excess ethanolamine without further purification, while N-palmitoylethanolamine with 98% purity was obtained by reacting with the same substrate ratio at 60 °C for 1.5 h with 3% catalyst. Complete conversion of vinyl stearate and palmitate was achieved.     

Modulation of renal injury in pcy mice by dietary fat containing n−3 fatty acids depends on the level and type of fat

Low-fat diets and diets containing n−3 fatty acids (FA) slow the progression of renal injury in the male Han:Sprague-Dawley (SPRD)-cy rat model of polycystic kidney disease. To determine whether these dietary fat effects are similar in females and in another model of renal cystic disease, in this study we used both male and female pcy mice to examine the effects of fat level and type on disease progression. Adult pcy mice were fed 4, 10, or 20 g soybean oil/100 g diet for 130 d in study 1. In study 2, weanling pcy mice were fed high or low levels of fat rich in 18∶2n−6 (corn oil, CO) 18∶3n−3 (flaxseed oil/CO 4∶1 g/g, FO), or 22∶6n−3 (algal oil/CO 4∶1 g/g, DO) for 8 wk. In adult pcy mice, low-compared with high-fat diets lowered kidney weights (2.4±0.2 vs. 3.1±0.2 g/100 g body weight, P=0.006) and serum urea nitrogen (SUN) (9.6±0.6 vs. 11.9±0.6 mmol/L, P=0.009), whereas in young pcy mice it reduced renal fibrosis volumes (0.44±0.04 vs. 0.62±0.04 mL/kg body weight, P<0.0001). FO feeding in young pcy mice mitigated the detrimental effects of high fat on fibrosis while not altering kidney size, function, and oxidative damage when compared with the CO-fed mice. In contrast, DO-compared with CO-fed mice had higher kidney weights (2.64±0.07 vs. 2.24±0.08 g/100 g body weight, P=0.005), SUN (9.4±0.57 vs. 7.0±0.62 nmol/L, P<0.0001), and cyst volumes (7.9±0.28 vs. 6.2±0.30 mL/kg body weight, P<0.0001) and similar levels of oxidative damage and fibrosis. The FA compositions of the diets were reflected in the kidneys: 18∶2n−6, 18∶3n−3, and 22∶6n−3 were the highest in the CO, FO, and DO diets, respectively. Dietary effects on kidney disease progression were similar in males and females. A low-fat diet slows progression of renal injury in male and female pcy mice, consistent with findings in the male Han:SPRD-cy rat. Dietary fat type also influenced renal injury, with flaxseed oil diets rich in 18∶3n−3 slowing early fibrosis progression compared with diets rich in 18∶2n−6 or in 22∶6n−3.     

Molecular architecture and biophysical properties of phospholipids during thermal adaptation in fish: An experimental and model study

Phospholipids from livers of carps (Cyprinus carpio L.) adapted to winter (5°C) and summer (25°C) temperatures were isolated, and the fatty acid composition of total phospholipids, as well as molecular species composition of diacyl phosphatidylcholines and ethanolamines, were determined. Order parameter of 5-doxyl stearic acid and steady-state fluorescence anisotropy of different anthroyloxy fatty acids—[2-, 12(N-9-anthroyloxy)stearic acid and 16(N-9-anthroyloxy)palmitic acid—embedded in native and synthetic (16∶0/16∶0, 16∶0/22∶6, 18∶0/22∶6, 18∶1/22∶6, 20∶4/20∶4, 22∶6/22∶6 phosphatidylcholines and 16∶0/18∶1, 18∶1/22∶6 phosphatidylethanolamines) phospholipid vesicles was also determined between −30 and 30°C and 5 and 30°C, respectively. There is an accumulation of 1-monoenoic, 2-polyenoic diacyl phosphatidylcholine and ethanolamine with a concomitant reduction of 1-stearoyl,2-docosahexaenoyl species in the cold-adapted state. Despite a 30% accumulation of long-chain polyunsaturated fatty acids in phospholipids in cold, there is only a 5°C downshift in the solid-gel to liquid-crystalline phase transition temperature (−8 vs. −13°C). Vesicles from total phospholipids of cold-adapted fish proved to be more disordered in all segments than from the warmadapted ones when assayed using 2,12-(N-9-anthroyloxy)stearic and 16-(N-9-anthroyloxy)palmitic acid. Vesicles made from purified phosphatidylcholines showed the same pattern, but they were more disordered than the corresponding total phospholipids. This could be modelled using mixed phospholipid vesicles made of synthetic 16∶0/22∶6 phosphatidylcholine (75%) and either 18∶1/22∶6 phosphatidylethanolamine (25%) vs. 16∶0/18∶1 phosphatidylethanolamine (25%) and comparison of the anisotropy parameters of 100% 16∶0/22∶6 and 100% 18∶1/22∶6 phosphatidylcholine vesicles. Mixing either 16∶0/18∶1 (25%) or 18∶1/22∶6 (25%) phosphatidylethanolamines to 18∶0/22∶6 (75%) phosphatidylcholine shifted down or up, respectively, the transition temperature of vesicles compared to 100% 18∶0/22∶6 vesicles assayed by electron spin resonance spectroscopy using 5-doxylstearic acid. It is concluded that it is not the gross amount of long-chain polyunsaturated fatty acids in phospholipids, but rather their specific combination withcis Δ9 monounsaturated fatty acids in the positionsn-1, especially in phosphatidylethanolamines, that is important in determining the physical properties of biomembranes in relation to adaptational temperature.     

Metabolism of meadowfoam oil fatty acids in mice

Meadowfoam oil is unusual because over 95% of the fatty acids are 20- and 22-carbon aliphatic acids withcis double bonds located principally at the 5- and/or 13-position. Since little information is available on the metabolism of the 5c−20∶1 and 5c,13c−22∶2 fatty acids, an exploratory study in mice was conducted to investigate the metabolism of purified samples of the free fatty acids isolated from meadowfoam oil, and to determine the effect of meadowfoam oil on weight gain and tissue lipid composition. Mice fed diets containing 5% by wt of the purified 5c−20∶1 or 5c,13c−22∶2 for 6 days exhibited no apparent physiological problems. Total liver lipids from mice fed the purified fatty acid diets contained mean values of 2.0% 5c−20∶1 and 2.1% 5c,13c−22∶2; total heart lipids contained 1.7% 5c−20∶1 and 10.7% 5c,13c−22∶2. Liver total phospholipids from mice fed a 5% meadowfoam oil diet for 19 wk contained 1.4% 5c−20∶1 and 1.9% 5c,13c−22∶2. There was no evidence of desaturation, elongation or retroconversion. Weight gain for mice fed the meadowfoam oil diet for 19 wk was similar to mice fed corn oil, and was higher than for mice fed hydrogenated cottonseed oil. Considering the high 5c−20∶1 and 5c,13c−22∶2 content of the diets, the percentages of these fatty acids in mouse tissue lipids from both the short- and long-term studies were low. Weight gain was surprisingly good since the meadowfoam oil diet was essential fatty acid-deficient. Results of this initial investigation suggest that the 5c−20∶1 and 5c,13c−22∶2 fatty acids were utilized primarily for energy. In the short-term study, these fatty acids did not produce toxic effects or cause metabolic problems. The mention of firm names or trade products does not imply that they are endorsed or recommended by the U.S. Department of Agriculture over other firms or similar products not mentioned.     

cis-4,7,10,trans-13–22∶4 fatty acid distribution in phospholipids of pectinid species Aequipecten opercularis and Pecten maximus

The distribution of cis-4,7,10,trans-13-docosate-traenoic (c4,7,10,t13–22∶4), a peculiar FA previously isolated in the glycerophospholipids of some pectinid bivalves, was investigated in glycerophospholipid classes and subclasses of separated organs (gills, mantle, gonads, and muscle) of the queen scallop Aequipecten opercularis and the king scallop Pecten maximus. Plasmalogen (Pls) and diacyl + alkyl (Ptd) forms of serine, ethanolamine, and choline glycerophospholipids were isolated by HPLC and their FA compositions analyzed by GC-FID. Pls and Ptd forms of serine glycerophospholipids (PlsSer and PtdSer), and to a lesser extend the Pls form of ethanolamine glycerophospholipids (PlsEtn), were found to be specifically enriched with c4,7,10,t13–22∶4. This specificity was found to decrease in the tested organs in the following order: gills, mantle, gonad, and muscle. In gills, c4,7,10,t13–22∶4 was shown to be the main unsaturated FA of serine glycerophospholipids in both Pls and Ptd forms (23.8 and 19.4 mol%, respectively, for A. opercularis, and 21.0 and 26.2 mol% for P. maximus). These results represent the first comprehensive report on the FA composition of plasmalogen serine subclass isolated from pectinid bivalves. The specific association of the PlsSer with the c4,7,10,t13–22∶4 for the two pectinid species can be paralleled to the specific association of the PlsSer with the non-methylene interrupted (NMI) FA and 20∶1(n−11) observed in mussels, clams, and oysters (Kraffe, E., Soudant, P., and Marty, Y. (2004) Fatty Acids of Serine, Ethanolamine and Choline Plasmalogens in Some Marine Bivalves, Lipids 39, 59–66.) This, led us to hypothesize a similar functional significance for c4,7,10,t13–22∶4, NMI FA, and 20∶1(n−11) associated with PlsSer subclass of bivalves.     

Isomers of hexadecenoic and hexadecadienoic acids in <Emphasis Type="Italic">Androsace septentrionalis</Emphasis> (Primulaceae) seed oil

Seeds of Androsace septentrionalis of the genus Androsace (tribus Primuleae) from the plant family Primulaceae were studied for their oil content and FA composition. The seed oil of A. septentrionalis was found to contain two unusual FA rarely occurring in plants: 11-cis-hexadecenoic acid (16∶1Δ11c or 16∶1n−5) and 9-cis,12-cis-hexadecadienoic acid (16∶2Δ9c,12c or 16∶2n−4). It also contained an unusually high amount (21.4%) of 9-cis-hexadecenoic acid (palmitoleic acid; 16∶1Δ9c or 16∶1n−7), i.e., at a level higher than that of oleic acid, in addition to common FA. Compared with most plant seed oils, at 3.8% the level of 18∶1Δ11c (or 18∶1n−7) also was elevated. The nonidentity of the Androsace 16∶2-acid with the 16∶2-acid, which is very typical for Ranunculus spp., as well as its identity with the 16∶2-acid typically found in Asclepiadaceae was established by co-chromatography. The structure and composition of the constituent FA of A. septentrionalis were also determined by various chromatographic methods (TLC, Ag⁺-TLC, capillary GLC) and spectroscopic methods (IR, GC-MS). The significant deviation of the Androsace FA pattern from that of other Primuleae, indicating a separate phylogenetic position of Androsace, is discussed.     

Preparation of defined molecular species of lactosylceramide by chemical deacylation and reacylation withN-succinimidyl fatty acid esters

A procedure for the preparation of specific molecular species ofd-erythro-lactosylceramide involving deacylation and reacylation of lactosylceramide prepared from bovine brain gangliosides is described. Lactosylceramide wasN-deacylated by alkaline hydrolysis and the resulting four lysolactosylceramides, which contained d18∶1, d20∶1, d18∶0 and d20∶0 long-chain bases, were simultaneously re-N-acylated with theN-succinimidyl ester of either 16∶0, 18∶0, 20∶0, 24∶0, 20∶1, 22∶1 or 24∶1 fatty acid. The resulting lactosylceramide contained four molecular species of lactosylceramides, i.e., d18∶1, d20∶1, d18∶0 and d20∶0 long-chain bases coupled with the fatty acid that was introduced. Lactosylceramides prepared in this manner were separated into four individual molecular species by high-performance liquid chromatography (HPLC). Each of the purified molecular species of lactosylceramide was quantitated by HPLC after derivatization with benzoylchloride and was characterized by mass spectrometry. The yields of reacylated lactosylceramide were 38–58% relative to the starting lactosylceramide; the purity of each of the molecular species of lactosylceramide was greater than 95%. The glycosphingolipid nomenclature is as recommended by the IUPAC-IUB Commission on Biochemical Nomenclature (1). GalCer, galactosylceramide, Gal(β1-1)Cer; GlcCer, glucosylceramide, Glc(β1-1)Cer; LacCer, lactosylceramide, Gal(β1-4)GlcCer; GbOse₃Cer, globotriaosylceramide, Gal(α1-4)Gal(β1-4)GlcCer; GbOse₄Cer, globotetraosylceramide, GalNAc(β1-3)Gal(α1-4)-Gal(β1-4)GlcCer; GgOse₃Cer, gangliotriaosylceramide, GalNAc-(β1-4)Gal(β1-4)GlcCer; GgOse₄Cer, gangliotetraosylceramide, Gal(β1-3)GalNAc(β1-4)Gal(β1-4)GlcCer; GM3, (NeuAcα2-3)-Galβ1-4GlcCer; GM1, Galβ1-3GalNAcβ1-4(NeuAcα2-3)Galβ1-4-GlcCer. The molecular species abbreviations suggested by Breimeret al. (2) are used. For example, in the notation d18∶1−18∶0, the d18∶1 represents the long-chain base sphingosine (d-erythro-1,3-dihydroxy-2-amino-trans-4-octadecene) and 18∶0 represents the fatty acid (octadecanoic acid).     

1-O-alk-1′-enyl-2-acyl-glycerophosphoethanolamine content and molecular species composition in fish brain

Ethanolamine glycerophospholipids from the brains of both trout and cod comprised 36–38% of 1-O-alk-1′-enyl-2-acyl-glycerophosphoethanolamine (GPE) determined using two methods. In 1-O-alk-1′-enyl-2-acyl-GPE from trout brain, the main molecular species were 18∶1a/18∶1, 18∶0a/18∶1 and 16∶0a/18∶1, which totalled 63.3%, while polyunsaturated fatty acid (PUFA) containing species totalled only 18.2%. 1-O-Alk-1′-enyl-2-acyl-GPE from cod brain was much more unsaturated with PUFA containing species totalling 52.6%, of which 18∶0a/20∶5n−3, 18∶1a/20∶5n−3 and 18∶1a/22∶6n−3 were predominant. In cod 18∶1a/18∶1, 18∶0a/18∶1 and 16∶0a/18∶1 were the only other species present at over 5% each, totalling 31.8%. In both cod and trout, small amounts of species containing 22∶4n−6 were found. The results of this and earlier studies indicate that there is considerable specificity of composition at the level of molecular species between different lipid classes and subclasses. Molecular species of 1-O-alk-1′-enyl-2-acyl-GPE are abbreviated as follows:e.g., 16∶0a/18∶1 GPE is 1-O-hexadec-1′-enyl-2-oleoyl-sn-glycero-3-phosphoethanolamine. The corresponding diacyl species, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine, is abbreviated as 16∶0/18∶1.     

Comparison of body weight and adipose tissue in male C57BI/6J mice fed diets with and withouttrans fatty acids

The effect of a diet containingtrans-fatty acids (tFA) on the fatty acid composition and fat accumulation in adipose tissue was investigated in mice. Male C57BI/6J mice were fed Control or Trans Diets that were similar, except that 50% of the 18∶1, which was allcis in the Control Diet, was replaced bytFA in the Trans Diet. At selected ages, body weight, epididymal fat pad weight, perirenal fat yield, adipose tissue cellularity and fatty acid composition were examined. Over the time period studied (2–24 mon), the proportion of 18∶0 and 16∶0 tended to decrease whilecis-18∶1 levels increased. Compared to the Control Diet, the Trans Diet resulted in adipose tissue lipids with higher percentages of 14∶0 and 18∶2n−6 and lower percentages ofcis-18∶1 and 20∶4n−6. In polar lipids,tFA replaced saturated fatty acids, whereastFA replacedcis-18∶1 in the nonpolar lipids. Body weights at 16 and 24 mon of age and epididymal fat pad weights at 8–24 mon of age were lower in mice fed the Trans Diet as compared to those fed the Control Diet. At the ages studied, the Trans Diet also resulted in lower values for perirenal fat weights, triacylglycerol to polar lipid ratios, and adipose cell size. The data suggest that chronic consumption oftFA affects lipid metabolism and results in decreased fat accumulation in murine adipose tissue.     

Fatty acids bound to <Emphasis Type="Italic">Fasciola hepatica</Emphasis> 12 kDa fatty acid-binding protein,a candidate vaccine,differ from fatty acids in extracts of adult flukes

The FA composition of Fasciola hepatica 12 kDA purified native FA-binding protein (nFh12), a candidate vaccine against fascioliasis, is described. The FA chain lengths ranged between 12 and 24 carbons. The principal FA were 16∶0 18∶1n−9, 18∶0, 20∶4n−6, and 20∶1n−9. The acids 16∶0, 18∶1n−9, and 18∶0 comprised over half the FA that were bound to the whole FA-binding protein. Small amounts (1.0–2.8%) of isoanteiso methyl-branched FA also were characterized. Forty-one different FA were identified in extracts of the adult flukes, with the three most abundant FA also being 16∶0, 18∶1n−9, and 18∶0. A similar proportion of saturated vs. unsaturated FA was observed between the whole extract from F. hepatica and the nFh12 protein. However, the n−3/n−6 ratio of PUFA was significantly different, being 1.2 in the whole extract vs. 9.6 in the nFh12 protein complex. The nFh12 protein binds more n−5, n−6, and n−7 PUFA and less n−3 and n−9 PUFA than the whole extract. In addition, cholesterol (56%), sitosterol (36%), and fucosterol (8%) also were bound to the nFh12 protein complex.     

Natural abundance stable carbon isotope evidence for the routing and <Emphasis Type="Italic">de novo</Emphasis> synthesis of bone FA and cholesterol

This research reported in this paper investigated the relationship between diet and bone FA and cholesterol in rats raised on a variety of isotopically controlled diets comprising 20% C₃ or C₄ protein (casein) and C₃ and/or C₄ nonprotein or energy (sucrose, starch, and oil) macronutrients. Compoundspecific stable carbon isotope analysis (δ¹³C) was performed on the FA (16∶0, 18∶0, 18∶1, and 18∶2) and cholesterol isolated from the diet (n=4) and bone (n=8) of these animals. The dietary signals reflected by the bone lipids were investigated using linear regression analysis. δ¹³C values of bone cholesterol and stearic (18∶0) acid were shown to reflect whole-diet δ¹³C values. whereas the δ¹³C values of bone palmitic (16∶0), oleic (18∶1), and linoleic (18∶2) acids reflected dietary FA δ¹³C values. Dietary signal differences are a result of the balance between direct incorporation (or routing) and de novo synthesis of each of these bone lipids. Estimates of the degree of routing of these bone lipids gleaned from correlations between Δ¹³C _dlipid-wdiet (δ¹³C_{diet lipid}-δ¹³C_{whole diet}) spacings and Δ¹³C _blipid-wdiet (δ¹³C_{bone lipid}-δ¹³C_{whole diet} fractionations demonstrated that the extent of routing, where 18∶2>16∶0>18∶1>18∶0>cholesterol, reflected the relative abundances of these lipids in the diet. These findings provide the basis for more accurate insights into diet when the δ¹³C analysis of bone fatty FA or cholesterol is employed.     

Triglyceride selectivity of immobilized <Emphasis Type="Italic">Thermomyces lanuginosa</Emphasis> lipase in interesterification

The triglyceride (fatty acid) selectivity of an immobilized lipase from Thermomyces lanuginosa (Lipozyme TL IM) was investigated in lipase-catalyzed interesterification reactions between two nono-acid TG in n-hexane. Tristearin (tri-C18∶0) was used as a reference in a series of TG with saturated FA from tri-C4∶0 to tri-C20∶0, except for tri-C6∶0, and in a series of unsaturated FA from tri-C18∶1 to tri-C18∶3. The quantification was performed by HPLC, and different methods of selectivity evaluation were used. None of the methods used showed any significant differences between the performances of the lipase on the different TG, indicating that Lipozyme TL IM is nonselective toward FA or TG in the system used. A response surface design was used to investigate the influence of water activities (a _w ) and reaction temperatures on the reactivity of Lipozyme TL IM with a system of tripalmitin (tri-C16∶0) and trilaurin (tri-C12∶0) in n-hexane. An increase in temperature (40 to 60°C) was found to affect the reactivity of the lipase significantly. The reactivity of Lipozyme TL IM was unaffected by the change in a _w from 0.1130 to 0.5289. An increase in a _w only led to an increase in FFA formation.     

<Emphasis Type="Italic">sn</Emphasis>-Position determination of phospholipid-linked fatty acids derived from erythrocytes by liquid chromatography electrospray ionization ion-trap mass spectrometry

The sn-position of FA in membrane lipids has an influence on the physiological function of cells, is predictive for diseases, and therefore is useful for diagnostics. The current study compares the compositions of acyl chain substituents in the sn-1 and sn-2 positions of the glycerol backbones of phospholipids derived from human erythrocytes by using RP-HPLC coupled with on-line electrospray ionization ion trap MS. Preferential loss of the acyl group in the sn-1 position was used to determine the degree of regiospecific preference exhibited by the phospholipid molecules. The identities of the molecular species and the positions of the acyl substituents were identified using product-ion spectra of major precursor ions selected from the mass spectra averaged across peaks in the total ion chromatogram. Saturated FA were found to be located mainly in the sn-1 position of the glycerol backbones of erythrocyte phospholipids, whereas PUFA were found primarily in the sn-2 position. All measured phospholipids revealed palmitic acid (16∶0) at the sn-1 position. Linoleic acid (18∶2n−6) and arachidonic acid (20∶4n−6) were found to be attached exclusively to the sn-2 position of the backbone, whereas eicosadienoic (20∶2n−6) and eicosatrienoic acid (20∶3n−9) occurred in both positions of the backbone of PC. Oleic (18∶1n−9), linoleic (18∶2n−6), and octadecatrienoic (18∶3) acids of PE and PS were linked to both positions. Lignoceric acid (24∶1n−9) was found to be strictly localized at the sn-2 position, whereas nervonic (24∶1n−9) acid of PS was associated with both positions of the backbone. A detailed analysis of the blood cell membrane lipids by MS might be helpful to characterize postprandial kinetics of pharmacological or dietary lipid applications, as well as environmental influences on cell membranes.     

Properties of lysophosphatidylcholine acyltransferase from <Emphasis Type="Italic">Brassica napus</Emphasis> cultures

Acyl-CoA:lysophosphatidylcholine acyltransferase (LPCAT; EC 2.3.1.23) catalyzes the acyl-CoA-dependent acylation of lysophosphatidylcholine (LPC) to produce PC and CoA. LPCAT activity may affect the incorporation of fatty acyl moieties at the sn-2 position of PC where PUFA are formed and may indirectly influence seed TAG composition. LPCAT activity in microsomes prepared from microspore-derived cell suspension cultures of oilseed rape (Brassica napus L. cv Jet Neuf) was assayed using [1-¹⁴C]acyl-CoA as the fatty acyl donor. LPCAT activity was optimal at neutral pH and 35°C, and was inhibited by 50% at a BSA concentration of 3 mg mL⁻¹. At acyl-CoA concentrations above 20 μM, LPCAT activity was more specific for oleoyl (18∶1)-CoA than stearoyl (18∶0)- and palmitoyl (16∶0)-CoA. Lauroyl (12∶0)-CoA, however, was not an effective acyl donor. LPC species containing 12∶0, 16∶0, 18∶0, or 18∶1 as the fatty acyl moiety all served as effective acyl acceptors for LPCAT, although 12∶0-LPC was somewhat less effective as a substrate at lower concentrations. The failure of LPCAT to catalyze the incorporation of a 12∶0 moiety from acyl-CoA into PC is consistent with the tendency of acyltransferases to discriminate against incorporation of this fatty acyl moiety at the sn-2 position of TAG from the seed oil of transgenic B. napus expressing a medium-chain thioesterase.