Eleostearic Acid Research Articles

Conjugated Fatty Acid Synthesis: RESIDUES 111 AND 115 INFLUENCE PRODUCT PARTITIONING OF MOMORDICA CHARANTIA CONJUGASE

Conjugated linolenic acids (CLNs), 18:3 Δ(9,11,13), lack the methylene groups found between the double bonds of linolenic acid (18:3 Δ(9,12,15)). CLNs are produced by conjugase enzymes that are homologs of the oleate desaturases FAD2. The goal of this study was to map the domain(s) within the Momordica charantia conjugase (FADX) responsible for CLN formation. To achieve this, a series of Momordica FADX-Arabidopsis FAD2 chimeras were expressed in the Arabidopsis fad3fae1 mutant, and the transformed seeds were analyzed for the accumulation of CLN. These experiments identified helix 2 and the first histidine box as a determinant of conjugase product partitioning into punicic acid (18:3 Δ(9cis,11trans,13cis)) or α-eleostearic acid (18:3 Δ(9cis,11trans,13trans)). This was confirmed by analysis of a FADX mutant containing six substitutions in which the sequence of helix 2 and first histidine box was converted to that of FAD2. Each of the six FAD2 substitutions was individually converted back to the FADX equivalent identifying residues 111 and 115, adjacent to the first histidine box, as key determinants of conjugase product partitioning. Additionally, expression of FADX G111V and FADX G111V/D115E resulted in an approximate doubling of eleostearic acid accumulation to 20.4% and 21.2%, respectively, compared with 9.9% upon expression of the native Momordica FADX. Like the Momordica conjugase, FADX G111V and FADX D115E produced predominantly α-eleostearic acid and little punicic acid, but the FADX G111V/D115E double mutant produced approximately equal amounts of α-eleostearic acid and its isomer, punicic acid, implicating an interactive effect of residues 111 and 115 in punicic acid formation.

CHARACTERIZATION OF FIXED OIL FROM SEEDS OF MOMORDICA TUBEROSA (ROXB) COGN. (CUCURBITACEAE) FRUITS BY GC-MS

Fixed oil obtained from the seeds of Momordica tuberosa (Roxb) Cogn. fruits (family: Cucurbitaceae) was analyzed using GC-MS. Five compounds, namely palmitic acid (hexadecanoic acid), oleic acid (9-octadecenoic acid), stearic acid (octadecanoic acid), ?-eleostearic acid (9, 11, 13-octadecatrienoic acid) and gama-linolenic acid (6, 9, 12-octadecatrienoic acid) were found to be major compounds in fixed oil obtained from the seeds of M. tuberosa fruits. Physical constants saponification value, unsaponifiable matter and acid value were found to be 182.4, 1.56% w/w and 11.44 respectively.

Mining the bitter melon (momordica charantial.) seed transcriptome by 454 analysis of non-normalized and normalized cDNA populations for conjugated fatty acid metabolism-related genes

BackgroundSeeds of Momordica charantia (bitter melon) produce high levels of eleostearic acid, an unusual conjugated fatty acid with industrial value. Deep sequencing of non-normalized and normalized cDNAs from developing bitter melon seeds was conducted to uncover key genes required for biotechnological transfer of conjugated fatty acid production to existing oilseed crops. It is expected that these studies will also provide basic information regarding the metabolism of other high-value novel fatty acids.ResultsDeep sequencing using 454 technology with non-normalized and normalized cDNA libraries prepared from bitter melon seeds at 18 DAP resulted in the identification of transcripts for the vast majority of known genes involved in fatty acid and triacylglycerol biosynthesis. The non-normalized library provided a transcriptome profile of the early stage in seed development that highlighted the abundance of transcripts for genes encoding seed storage proteins as well as for a number of genes for lipid metabolism-associated polypeptides, including Δ12 oleic acid desaturases and fatty acid conjugases, class 3 lipases, acyl-carrier protein, and acyl-CoA binding protein. Normalization of cDNA by use of a duplex-specific nuclease method not only increased the overall discovery of genes from developing bitter melon seeds, but also resulted in the identification of 345 contigs with homology to 189 known lipid genes in Arabidopsis. These included candidate genes for eleostearic acid metabolism such as diacylglycerol acyltransferase 1 and 2, and a phospholipid:diacylglycerol acyltransferase 1-related enzyme. Transcripts were also identified for a novel FAD2 gene encoding a functional Δ12 oleic acid desaturase with potential implications for eleostearic acid biosynthesis.Conclusions454 deep sequencing, particularly with normalized cDNA populations, was an effective method for mining of genes associated with eleostearic acid metabolism in developing bitter melon seeds. The transcriptomic data presented provide a resource for the study of novel fatty acid metabolism and for the biotechnological production of conjugated fatty acids and possibly other novel fatty acids in established oilseed crops.

Leaf lipids from Euphorbia lagascae Spreng. and Euphorbia lathyris L.

The Euphorbiaceae family is comprised of plant species that synthesise unusual fatty acids in their seed oils, e.g., Ricinus communis (ricinoleic or hydroxyoleic acid), Aleurites fordii (eleostearic acid) or Euphorbia lagascae (vernolic or epoxyoleic acid). Accumulation of triacylglycerols (TAGs) in plant leaves of many species can occur, although in smaller quantities than in seeds. This is the first report on leaf lipids in Euphorbia. Total lipid (TL) content ranged from 2274 to 5757 μg/g of fresh weight in Euphorbia lathyris and from 1562 to 9813 μg/g of fresh weight in E. lagascae. The first, fifth, tenth or fifteenth true leaves (each case from 2- to 40-day-old leaves) were sampled from plants grown in the open field and in the glasshouse at Instituto Murciano de Investigación y Desarrollo Agrario y Alimentario (Murcia, Spain). TAGs were separated by thin-layer chromatography (TLC) and represented up to 44.3% of TL in E. lagascae. The amount of TAG peaked in the first leaf and subsequently decreased with aging in all leaves. Microscope preparations revealed the presence of oil bodies (3–10 μm), possibly acting as intermediate storage products, in Euphorbia leaf mesophyll cells. Vernolic acid was identified in E. lagascae leaf lipids (0–25.7% of total fatty acids), but it was not incorporated into TAGs. These results imply that the active enzyme, diacylglycerol acyltransferase (DGAT), in the leaf of E. lagascae does not exhibit any selectivity towards the native unusual fatty acid.

Poly(ADP-ribose) Polymerase (PARP)-1-independent Apoptosis-inducing Factor (AIF) Release and Cell Death Are Induced by Eleostearic Acid and Blocked by α-Tocopherol and MEK Inhibition

Poly(ADP-ribose)polymerase-1 (PARP-1) is thought to be required for apoptosis-inducing factor (AIF) release from mitochondria in caspase-independent apoptosis. The mechanism by which AIF is released through PARP-1 remains unclear. Here, we provide evidence that PARP-1-independent AIF release and cell death are induced by a trienoic fatty acid, alpha-eleostearic acid (alpha-ESA). Alpha-ESA induced the caspase-independent and AIF-initiated apoptotic death of neuronal cell lines, independently of PARP-1 activation. The cell death was inhibited by the MEK inhibitor U0126 and by knockdown of MEK using small interfering RNA. However, inhibitors for JNK, p38 inhibitors, calpain, phospholipase A(2), and phosphatidylinositol 3-kinase, did not block cell death. AIF was translocated to the nucleus after the induction of apoptosis by alpha-ESA in differentiated PC12 cells without activating caspase-3 and PARP-1. The alpha-ESA-mediated cell death was not inhibited by PARP inhibitor 3,4-dihydro-5-[4-(1-piperidinyl)butoxyl]-1(2H)-isoquinoline and by knockdown of PARP-1 using small interfering RNA. Unlike N-methyl-N'-nitro-N-nitrosoguanidine treatment, histone-phosphorylated histone 2AX was not phosphorylated by alpha-ESA, which suggests no DNA damage. Overexpression of Bcl-2 did not inhibit the cell death. alpha-ESA caused a small quantity of superoxide production in the mitochondria, resulting in the reduction of mitochondrial membrane potential, both of which were blocked by a trace amount of alpha-tocopherol localized in the mitochondria. Our results demonstrate that alpha-ESA induces PARP-1-independent AIF release and cell death without activating Bax, cytochrome c, and caspase-3. MEK is also a key molecule, although the link between ERK, AIF release, and cell death remains unknown. Finding molecules that regulate AIF release may be an important therapeutic target for the treatment of neuronal injury.

Eleostearic Acid Inhibits Breast Cancer Proliferation by Means of an Oxidation-Dependent Mechanism

Eleostearic acid (alpha-ESA) is a conjugated linolenic acid that makes up approximately 60% of Momordica charantia (bitter melon) seed oil. Prior work found that water extract from bitter melon was able to inhibit breast cancer. Here, we investigated effects of alpha-ESA on both estrogen receptor (ER)-negative MDA-MB-231 (MDA-wt) and ER-positive MDA-ERalpha7 human breast cancer cells. We found that alpha-ESA inhibited proliferation of both MDA-wt and MDA-ERalpha7 cells, whereas conjugated linoleic acid had comparatively weak antiproliferative activity at 20 to 80 micromol/L concentrations. We also found that alpha-ESA (40 micromol/L) treatment led to apoptosis in the range of 70% to 90% for both cell lines, whereas conjugated linoleic acid (40 micromol/L) resulted in only 5% to 10% apoptosis, similar to results for control untreated cells. Addition of alpha-ESA also caused loss of mitochondrial membrane potential and translocation of apoptosis-inducing factor as well as endonuclease G from the mitochondria to the nucleus. Additionally, alpha-ESA caused a G(2)-M block in the cell cycle. We also investigated the potential for lipid peroxidation to play a role in the inhibitory action of alpha-ESA. We found that when the breast cancer cells were treated with alpha-ESA in the presence of the antioxidant alpha-tocotrienol (20 micromol/L), the growth inhibition and apoptosis effects of alpha-ESA were lost. An AMP-activated protein kinase inhibitor (Dorsomorphin) was also able to partially abrogate the effects of alpha-ESA, whereas a caspase inhibitor (BOC-D-FMK) did not. These results illustrate that alpha-ESA can block breast cancer cell proliferation and induce apoptosis through a mechanism that may be oxidation dependent.

Physicochemical properties and bioactive compounds of selected seed oils

Abstract The physicochemical properties and chemical composition of oil extracted from five varieties of plant seeds (bittermelon, Kalahari melon, kenaf, pumpkin and roselle seeds) were examined by established methods. The thermal properties of extracted oils by differential scanning calorimetry were also evaluated. Sensorial profiles of these seed oils were defined through the CieLab ( L *, a *, b *) colour. Most of the quality indices and fatty acid compositions showed significant ( P 2 /100 g oil; saponification value, 171.0–190.7 mg of KOH/g of oil; acid value, 1.1–12.9 mg of KOH/g of oil, free fatty acid, 0.6–6.5 g/100 g of oil, and peroxide value 1.5–6.5 meq of O 2 /kg of oil. Palmitic, oleic and linoleic acids were the major fatty acids in all of the extracted seed oils except for bittermelon, where eleostearic acid was the major fatty acid. Gallic, protocatechuic, p -hydroxybenzoic, vanillic, caffeic, syringic, p -coumaric and ferulic acids were identified in the extracted plant oils. Among these, vanillic acid was predominant in all extracted oils. The oils were rich in tocopherols with γ-tocopherol as the major components in all oil samples. Among the phytosterols, sitosterol was the major phytosterol extracted from the five plant seed oils. The seeds of these plants contain a great number of valuable minor compounds, which have a potential high value as food and for production of non-food products.

Analysis of pomegranate seed oil for the presence of jacaric acid

AbstractBACKGROUND: Pomegranate seed oil is predominantly composed of triglycerides containing unsaturated fatty acids, including high levels of conjugated linolenic acids (CLnAs). The major CLnA component, punicic acid, is known to possess biological activity. Consequently, it is desirable to obtain a detailed characterisation of pomegranate seed oil fatty acid profiles, including molecules potentially co‐eluting with punicic acid, such as jacaric acid.RESULTS: Conjugated fatty acid profiles of a commercial sample of cold pressed pomegranate seed oil were characterised in detail by both gas chromatography of methyl esters and by 13C NMR spectroscopy. The methylation procedures were found to be critical for determination of accurate fatty acid profiles. GC analysis was unable to resolve jacaric acid from punicic acid, the major fatty acid present in pomegranate seed oil. To establish the presence or absence of jacaric acid, 13C NMR was employed.CONCLUSION: This is the first study to investigate pomegranate seed oil for the presence of jacaric acid. Punicic acid, eleostearic acid, and catalpic acid were confirmed by 13C NMR, but jacaric acid was not found. Thus, we have shown that punicic acid levels may be accurately measured by gas chromatography alone in pomegranate seed oil. Copyright © 2009 Society of Chemical Industry

Characteristics of seed oils and nutritional compositions of seeds from different varieties of Momordica charantiaLinn.cultivated in Bangladesh

Farmers in rural areas of almost all the districts of Bangladesh cultivate different varieties of edible plant karela to satisfy nutritional requirements. Herein, we report on the characteristics of seed oils and nutrients and mineral contents of seeds from three varieties of karela. Most of the physicochemical characteristics were significantly (<I>P</I> < 0.05) affected with the samples tested. Seed oils of all varieties displayed a higher degree of unsaturation and in GLC reported herein, only five fatty acids were identified. The profiles of fatty acid composition were not wholly similar in all varieties in which unsaturated fatty acids represented more than 72%, α-eleostearic acid having been detected in the amount of 50.36–53.22%. Acylglycerol classes were estimated to be monoacylglycerols (1.18–2.01%), diacylglycerols (1.83–2.98%), and triacylglycerols (91.11–93.03%) whereas lipid classes included neutral lipids (86.83–91.09%), glycolipids (4.37–7.43%), and phospholipids (3.22–4.62%). Of the major energy producing nutrients, all varieties contained large amounts of lipid (33.93–36.21%) and protein (18.23–21.36%), and potentially useful amounts of calcium (383.45–440.96 μg/g), iron (41.10–45.03 μg/g), and other essential minerals. The nutrient information presented in this report should stimulate the local public health authorities in Bangladesh to consider the question of recommending the vegetable karela to be consumed by adults and children alike in Bangladesh, including pregnant women and others with higher than normal nutritional requirements.

Conjugated autoxidizable triene (CAT) assay: A novel spectrophotometric method for determination of antioxidant capacity using triacylglycerol as ultraviolet probe

Described here is a novel spectrophotometric method for estimating antioxidant capacity in a 96-well microplate using as UV probes the conjugated triene triacylglycerols (TAGs) naturally present in tung oil. The TAGs of this commercially available oil contain around 86% eleostearic acid, an octadecatrienoic acid with conjugated trienes exhibiting strong UV absorption at 273 nm. In an oil-in-water emulsion at 37 °C, the azo initiator 2,2′-azobis(2-amidinopropane) dihydrochloride generated a constant flux of peroxy radicals, which destroyed the conjugated trienes. The absorbance decay at 273 nm, related to oxidative degradation of conjugated triene TAGs, was monitored in the absence and presence of various concentrations of antioxidants. To validate this new method, the antioxidant capacity of four phenolic compounds (gallic acid, (–)-epicatechin, chlorogenic acid, and rosmarinic acid) was measured using an area-under-the-curve calculation and expressed as Trolox equivalents, the value for Trolox being taken as reference. The order of efficacy was found to be rosmarinic acid > chlorogenic acid ∼ epicatechin > Trolox > gallic acid, which can be partially explained by the number of catecholic moieties and the polarity of these antioxidants. Moreover, the conjugated autoxidizable triene (CAT) assay provided good insight into antioxidant behavior (i.e., retarder or chain breaker). The same molecules were then analyzed in 2,2-diphenyl-1-picrylhydrazyl and fluorescein-based oxygen radical absorbance capacity assays, and the results are discussed critically with respect to those obtained with the CAT assay. This new method may constitute an easy-to-use, sensitive, and high-throughput in vitro protocol for evaluation of the antioxidant capacity of pure molecules and natural extracts in lipid oxidation.

Production and Characterization of Biodiesel from Tung Oil

The feasibility of biodiesel production from tung oil was investigated. The esterification reaction of the free fatty acids of tung oil was performed using Amberlyst-15. Optimal molar ratio of methanol to oil was determined to be 7.5:1, and Amberlyst-15 was 20.8 wt% of oil by response surface methodology. Under these reaction conditions, the acid value of tung oil was reduced to 0.72 mg KOH/g. In the range of the molar equivalents of methanol to oil under 5, the esterification was strongly affected by the amount of methanol but not the catalyst. When the molar ratio of methanol to oil was 4.1:1 and Amberlyst-15 was 29.8 wt% of the oil, the acid value decreased to 0.85 mg KOH/g. After the transesterification reaction of pretreated tung oil, the purity of tung biodiesel was 90.2 wt%. The high viscosity of crude tung oil decreased to 9.8mm(2)/s at 40 degrees C. Because of the presence of eleostearic acid, which is a main component of tung oil, the oxidation stability as determined by the Rancimat method was very low, 0.5h, but the cold filter plugging point, -11 degrees C, was good. The distillation process did not improve the fatty acid methyl ester content and the viscosity.

Tung Tree DGAT1 and DGAT2 Have Nonredundant Functions in Triacylglycerol Biosynthesis and Are Localized to Different Subdomains of the Endoplasmic Reticulum

Seeds of the tung tree (Vernicia fordii) produce large quantities of triacylglycerols (TAGs) containing approximately 80% eleostearic acid, an unusual conjugated fatty acid. We present a comparative analysis of the genetic, functional, and cellular properties of tung type 1 and type 2 diacylglycerol acyltransferases (DGAT1 and DGAT2), two unrelated enzymes that catalyze the committed step in TAG biosynthesis. We show that both enzymes are encoded by single genes and that DGAT1 is expressed at similar levels in various organs, whereas DGAT2 is strongly induced in developing seeds at the onset of oil biosynthesis. Expression of DGAT1 and DGAT2 in yeast produced different types and proportions of TAGs containing eleostearic acid, with DGAT2 possessing an enhanced propensity for the synthesis of trieleostearin, the main component of tung oil. Both DGAT1 and DGAT2 are located in distinct, dynamic regions of the endoplasmic reticulum (ER), and surprisingly, these regions do not overlap. Furthermore, although both DGAT1 and DGAT2 contain a similar C-terminal pentapeptide ER retrieval motif, this motif alone is not sufficient for their localization to specific regions of the ER. These data suggest that DGAT1 and DGAT2 have nonredundant functions in plants and that the production of storage oils, including those containing unusual fatty acids, occurs in distinct ER subdomains.

An Investigation of the Fatty Acid Composition of New and Aged Tung Oil

Tung oil was used as an ingredient in historic conservation materials. Wooden art objects were treated with tung and linseed oil mixtures for consolidation purposes. To provide a reference for the identification of the oil component in these materials, tung oil was examined in this study, both in the fresh state and under accelerated ageing conditions. The alteration of the fatty acid composition was analysed in comparison with linseed oil by gas chromatographic methods coupled with mass spectrometry. The main component in the triglycerides of tung oil, á-eleostearic acid (cis, trans, trans-9,11,13-octadecatrienoic acid), was converted primarily into two compounds identified as isomers. Their structures were characterized based on their mass spectra. Furthermore, the degradation processes of tung and linseed oil into dicarboxylic acids were analysed for comparison. The results were applied to the examination of a polychromed wooden altar from the Saxonia region in middle Germany. Tung oil was identified in the exudation material of the wood by the presence of a characteristic isomer of eleostearic acid, which was determined earlier as the same marker conversion product in the reference samples.

Dietary effects on growth, plasma lipid and tissues of rats fed with non‐conventional oil of Telfairia occidentalis

AbstractThe nutritional value of Telfairia occidentalis seed and seed oil available in local market was studied in rats. The average values of the proximate analysis on dry matter basis were 203 g kg−1 crude protein, 536.3 g kg−1 fat, 39 g kg−1 ash, 95 g kg−1 crude fibre and 43.7 g kg−1 carbohydrate, while the moisture content was 75 g kg−1. The mineral analyses showed that T occidentalis seeds contain potassium (3280 mg kg−1), sodium (1230 mg kg−1), magnesium (60.8 mg kg−1), calcium (60.8 mg kg−1) and iron (13.0 mg kg−1). The oil extracted from the seed sample showed physicochemical properties that suggest its suitability for edible and industrial uses. The oil contained high levels of unsaturated fatty acids, oleic and linoleic, which accounted for 63.22% of total fatty acids recovered, while palmitic acid (27.4% of the oil by weight) was the most prevalent saturated fatty acid in the oil. No trace of eleostearic acid was detected in the oil. The effect on physical appearance, weight gain, organ weight, tissue and plasma cholesterol and triacylglycerol levels was determined in rats fed a diet containing 5% T occidentalis oil. These were compared with those of rats fed a control diet (0% T occidentalis oil) over an 8 week study period. Weekly monitoring of the rats showed good physical appearance and steady weight increase. The test rats were not anaemic and no mortality was recorded. While there was a significantly higher (p < 0.05) level of cholesterol in the heart of test rats compared with the control, there was a significantly lower (p < 0.05) plasma cholesterol level, suggesting that T occidentalis seed oil can be used to lower plasma levels of cholesterol. Histological examination of the sections of the heart, liver, kidney and spleen of the test rats revealed varying degrees of pathology. These include artherosclerotic changes in the walls of vasa vasori and lymphatics in the heart and hyaline degeneration of myofibrils, fatty infiltration of hepatocytes, and glomerular and tubular degeneration in the kidneys. These lesions may have been due to the effect of fatty acids, especially erucic acid, in the seed oil, which at 1.24% (less that the 5% conventional allowable limit for edible oils) may be intolerable for rats and may be unsuitable for human consumption. The seed oil may be used for industrial purposes such as soap making, lubrication and cosmetics for which it is well suited. Copyright © 2004 Society of Chemical Industry

Tumor growth suppression by -eleostearic acid, a linolenic acid isomer with a conjugated triene system, via lipid peroxidation

We have previously shown that conjugated linolenic acids (CLnA) prepared by alkaline isomerization have a stronger antitumor effect than conjugated linoleic acids (CLA). In this study we have compared the suppressive effect on tumor growth of alpha-eleostearic acid (alpha-ESA, 9Z11E13E-18:3) with those of the CLA isomers 9Z11E-CLA and 10E12Z-CLA, using nude mice into which DLD-1 human colon cancer cells were transplanted. The results showed that alpha-ESA, which is a CLnA that can be prepared from natural sources in bulk, had a stronger antitumor effect than CLA. DNA fragmentation was enhanced and lipid peroxidation was increased in tumor tissues of the alpha-ESA-fed mice, which suggested that alpha-ESA induced apoptosis via lipid peroxidation. Furthermore, treatment of DLD-1 cells with alpha-ESA, 9Z11E-CLA and 10E12Z-CLA confirmed that alpha-ESA had a stronger antitumor effect than CLA in cultured cell lines. The induction of apoptosis by alpha-ESA was consistent with enhanced DNA fragmentation, increased caspase activity and increased expression of caspase mRNA following alpha-ESA treatment. Addition of alpha-tocopherol, an antioxidant, suppressed oxidative stress and apoptosis, suggesting that these effects were associated with lipid peroxidation.

The metabolic conversion of 9,11,13-eleostearic acid (18:3) to 9,11-conjugated linoleic acid (18:2) in the rat.

The presence of a significant amount of 9,11-conjugated linoleic acid (CLA, 9,11-18:2) was confirmed in the liver and plasma lipids of rats fed a 1% (w/w % of diet) eleostearic acid (ESA, 9,11,13-18:3) diet for 4 wk. The chemical structure of the 9,11-CLA apparent in the tissue lipids was identified by gas chromatography-electron impact mass spectrometry after its conversion to a 4,4-dimethyloxazoline derivative. The concentration of CLA in the total fatty acids of the liver and plasma lipids reached to about 1%, for each in the CLA-supplemented rats, while reaching 3.2% and 2.5%, respectively, in the ESA-supplemented rats. The results suggest that alpha-ESA is metabolized partially to CLA via a delta 13-saturation reaction in the rat. Some biological activities observed in alpha-ESA-fed animals may be ascribed also to CLA that is formed from ESA in the body.

Molecular analysis of a bifunctional fatty acid conjugase/desaturase from tung. Implications for the evolution of plant fatty acid diversity.

The seed oil derived from the tung (Aleurites fordii Hemsl.) tree contains approximately 80% alpha-eleostearic acid (18:3delta(9cis,11trans,13trans)), an unusual conjugated fatty acid that imparts industrially important drying qualities to tung oil. Here, we describe the cloning and functional analysis of two closely related Delta(12) oleate desaturase-like enzymes that constitute consecutive steps in the biosynthetic pathway of eleostearic acid. Polymerase chain reaction screening of a tung seed cDNA library using degenerate oligonucleotide primers resulted in identification of two desaturases, FAD2 and FADX, that shared 73% amino acid identity. Both enzymes were localized to the endoplasmic reticulum of tobacco (Nicotiana tabacum cv Bright-Yellow 2) cells, and reverse transcriptase-polymerase chain reaction revealed that FADX was expressed exclusively within developing tung seeds. Expression of the cDNAs encoding these enzymes in yeast (Saccharomyces cerevisiae) revealed that FAD2 converted oleic acid (18:1delta(9cis)) into linoleic acid (18:2delta(9cis,12cis)) and that FADX converted linoleic acid into alpha-eleostearic acid. Additional characterization revealed that FADX exhibited remarkable enzymatic plasticity, capable of generating a variety of alternative conjugated and delta(12)-desaturated fatty acid products in yeast cells cultured in the presence of exogenously supplied fatty acid substrates. Unlike other desaturases reported to date, the double bond introduced by FADX during fatty acid desaturation was in the trans, rather than cis, configuration. Phylogenetic analysis revealed that tung FADX is grouped with delta(12) fatty acid desaturases and hydroxylases rather than conjugases, which is consistent with its desaturase activity. Comparison of FADX and other lipid-modifying enzymes (desaturase, hydroxylase, epoxygenase, acetylenase, and conjugase) revealed several amino acid positions near the active site that may be important determinants of enzymatic activity.

Newly recognized cytotoxic effect of conjugated trienoic fatty acids on cultured human tumor cells

We investigated the cytotoxic effect of conjugated trienoic fatty acids on various human tumor cell lines: DLD-1, colorectal; HepG2, hepatoma; A549, lung; MCF-7, breast; and MKN-7, stomach. Conjugated linoleic acid (CLA) and conjugated linolenic acid were prepared from linoleic acid (18:2, n-6) and α-linolenic acid (18:3, n-3), respectively, by treatment with 6.6% or 21% potassium hydroxide. Spectrophotometric readings at 235 nm for the conjugated diene formation, and at 268 nm for the conjugated triene, were confirmed for the respective conjugated fatty acids. In addition, tung oil (Aleurites fordii) fatty acids consisting principally of a conjugated triene (eleostearic acid, approximately 80% of total fatty acids) were prepared using an alkaline saponification procedure. All tumor cells were incubated for 24 h with 5–100 μM of the conjugated fatty acids, and MTT dye reduction was measured to verify the cell viability. Among the conjugated fatty acids examined, conjugated linolenic acid and tung oil fatty acids exhibited the most intense cytotoxic effects on DLD-1, HepG2, A549, MCF-7 and MKN-7 cells, while CLA was not cytotoxic to the tumor cells. These results demonstrate that conjugated trienoic fatty acids are more cytotoxic to human tumor cells than the conjugated dienoic fatty acid, CLA.

In Vivo Studies of the Biosynthesis of [alpha]-Eleostearic Acid in the Seed of Momordica charantia L.

In vivo radiotracer experiments using 14C-labeled acetate, oleate, linoleate, and linolenate were conducted to investigate the biosynthesis of [alpha]-eleostearic acid in the seeds of Momordica charantia. With the exception of [14C]linolenate, all of these precursors radioactively labeled [alpha]-eleostearate. Kinetics of the time course of metabolism of the radioactive precursors indicate that linoleate is the acyl precursor of [alpha]-eleostearate and that its conversion to [alpha]-eleostearate occurs while the acyl moiety is esterified to PC. Pulse-chase experiments with 14C-labeled acetate or linoleate provided additional corroborative evidence that linoleoyl PC is the precursor of [alpha]-eleostearoyl PC.

Amandes de Ricinodendron heudelotii (Bail.): Matiere premiere potentielle pour les industries agro-alimentaires tropicales

Ricinodendron heudelotii (Bail.) is a tree with many uses and should be cultivated. Physico-chemical analyses of some kernel nutrients as well as the fatty acids and amino acids of R. heudelotii (Bail.) cake are the main objectives of this work. The composition of these seeds has been investigated for their potential food uses. This study shows the nutritional interest of R. heudelotii (Bail.) and draws the attention of agroforestry policy makers to the importance of its integration in agriculture. Results so far show that these kernels contain more than 54% total lipids (very fluid oil). The fatty acid composition determined by gas chromatography shows that this oil mainly contains a polyunsaturated fatty acid (C18:3) found to be α elaeostearic acid (51.1%). The defatted flour contains more than 8% nitrogen and 16% ash. The determination of defatted protein nutritive value shows a lower chemical index of its amino acids compared to that obtained from soybean and cottonseed. However, this protein presents satisfactory chemical equilibrium of its amino acids for nutritional needs.