In Silico Structural Studies and Molecular Docking Analysis of Delta6-desaturase in HUFA Biosynthetic Pathway

Fish are an important source of highly unsaturated fatty acids (HUFA) such as eicosapentaenoic acid EPA (20:5 n-3) and docosahexaenoic acid DHA (22:6 n-3) and play a significant role in human nutrition. The fatty acyl delta6-desaturase (Δ6 desaturase) is a rate-limiting enzyme in the biosynthetic pathway of highly unsaturated fatty acids (HUFA) that converts polyunsaturated fatty acids (PUFA) such as linoleic (18:2n-6) and α-linolenic (18:3n-3) acids into HUFA. In this study, fatty acyl Δ6 desaturase was identified from pangasius (Pangasianodon hypophthalmus) and further analyzed for sequenced-based characterization and 3D structural conformation. Sequenced-based analysis revealed some important secondary information such as physicochemical property. e.g., isoelectric point, extinction coefficient, aliphatic index, and grand average hydropathy, among others, and also post-translational modification sites were identified. An evolutionary-conserved stretch of amino acid residue and a functionally significant conserved structural ancestor, N-terminal cytochrome b5 and membrane FADS-like superfamily, were identified. Protein association analysis showed a high confidence score with acyl-CoA synthetase, elovl5, elovl2, and phospholipase A2. Herein, we report, for the first time, a 3D native structure of Δ6 desaturase protein by homology modeling approach; molecular docking analysis was performed with linoleic (18:2n-6) and α-linolenic (18:3n-3) acids, which are the two key substrates in the HUFA biosynthetic pathway. This work provides insight into the structural and functional characterization of Δ6 desaturase, which is involved in HUFA biosynthesis as a rate-limiting enzyme.     

Effect of diets containing γ-linolenic acid on n-6 highly unsaturated fatty acid content of rotifer (Brachionus plicatilis)

The effect of various diets containing linoleic and/or -linolenic acids was studied on n-6 fatty acid composition of the rotifer Brachionus plicatilis. The rotifer's abilities for transformations of n-6 fatty acids were evaluated. Diets containing only linolenic acid as n-6 fatty acid induced low levels of other n-6 fatty acids in rotifers while a diet containing also -linolenic acid led to substantial amounts of di homo -linolenic acid in the rotifers through elongation. Desaturation of -linoleic acid to gamma linolenic appears to be the limiting factor of n-6 highly unsaturated fatty acid biosynthesis by the rotifer. Two sets of experiments were compared using different techniques and different sources of -linolenic acid: Spirulina in inert food or borage oil in emulsion with baker's yeast. Rotifers fed with inert diet with Spirulina contained arachidonic acid while those fed with borage oil had very low arachidonic content. High level of n-3 fatty acids incorporated into the diets seemed to exert inhibitory effects on n-6 transformation rate.     

Effect of fatty acids of ω6 series on the biosynthesis of arachidonic acid in HTC cells

Summary The influence of the preincubation of HTC cells with fatty acids of 6 series and columbinic acid (St, 9c, 12c 18:3) on the biosynthesis of arachidonic acid was studied. The cells were incubated on a chemically defined medium with or without the addition of unlabeled linoleic, -linolenic, eicosatrienoic, arachidonic, docosatetraenoic, docosapentaenoic and columbinic acids. After 24 hr of preincubation in the presence of the aforementioned fatty acids, [1-¹⁴C]eicosa-8,11,14-trienoic acid was added to the culture medium as the only lipidic source. Twenty-four hours later the synthesis of arachidonic acid and the fatty acid composition of the cells were determined. At 20 MM concentration the 6 fatty acids studied except docosapentaenoic acid produced an increase on the biosynthesis of arachidonic acid compared to the cells incubated in the absence of unlabeled fatty acids in the medium. The fatty acids added to the culture medium were incorporated into the cells and modified their fatty acid composition. Columbinic acid, with a similar structure to linoleic acid, also produced a significant increase on the conversion of eicosatrienoic acid to arachidonic acid. These results would suggest that the effect of both, linoleic and columbinic acids, may be adscribed to their configuration and not necessarily to their transformation in higher homologs, since columbinic acid is unable to be desaturated.All authors are members of the Carrera del Investigador Cientifico of the Consejo Nacional de Investigaciones Cientifícas y Técnicas, Argentina.     

Effect of sucrose addition to drinking water,that induces hypertension in the rats,on liver microsomal Δ9 and Δ5-desaturase activities

This study was undertaken with the aim of investigating the effect of sucrose addition to the drinking water of rats who were fed with the same diet as a control group, on Δ9- and Δ5-desaturase activities and on the fatty acid composition of serum and liver microsomes. Weanling male Wistar rats had 30% sucrose in their drinking water for 20 weeks. An increase in total calories consumed, visceral fat accumulation, insulin, triglycerides and blood pressure and a decrease in the food intake were observed in the sucrose-fed group as compared with the control group. A decrease in linoleic and α-linolenic acid (essential fatty acids) in all serum lipid fractions of sucrose-fed rats was found. This observation correlated with a low food intake by sucrose-fed rats. The conversion of [1 ¹⁴C]-palmitic to [1 ¹⁴C]-palmitoleic acid by Δ9-desaturase activity was increased in sucrose-fed compared with control rats, while the conversion of [1 ¹⁴C]-dihomo-γ-linolenic acids by Δ5-desaturase activity was depressed. In sucrose-fed as compared to control rats, the proportion of palmitoleic and oleic fatty acids was increased. Arachidonic acid was decreased in sucrose-fed rats. The 1,6-diphenylhexatriene fluorescence polarization of the microsomal membranes was significantly lower in the sucrose-fed group compared to the control group. These results indicate that the sucrose addition to the drinking water of the rats increased microsomal Δ9-desaturase activity and membrane disorder and decreased the activity of the Δ5-desaturase, a key enzyme in the biosynthesis of arachidonic acid, implicated in hypertension.     

Antitumour and pro-apoptotic actions of highly unsaturated fatty acids in glioma

The highly unsaturated fatty acids (HUFA) of the n-6 and n-3 series are involved in cell signalling in normal and transformed cells and have recently been associated with pathways leading to tumour cell death. The antitumour activity of three HUFA (arachidonic acid, gamma linolenic acid and eicosapentaenoic acid) were studied in glioma cells and tissue. Using five glioma models, including primary cell suspensions prepared from 46 human glioma samples and an in vivo rat C6 glioma model, we obtained evidence that, following exposure to HUFA, either administered into the medium surrounding human glioma cells or in 16 preparations of multicellular spheroids derived from human and rodent glioma cell lines (C6, MOG, U87, U373) or administered intra-tumourally by infusion using osmotic mini-pumps in 48 rats, glioma regression and apoptosis were detected. Additionally, synergy between gamma irradiation and HUFA administration was observed in 13 experiments analyzing C6 glioma cell apoptosis in vitro. These pro-apoptotic and antiproliferative activities were observed using both C18 and C20 fatty acids of the n-6 and n-3 series, but not when saturated and monounsaturated C18 and C20 fatty acid preparations were used. In the glioma infusion model, in addition to the apoptosis detected in glioma tissue infused with HUFA for 3-7 days, preservation of normal neural tissue and vasculature in adjacent brain was observed. Also, there was little evidence of acute inflammatory infiltration in regressing tumours. Our findings suggest that intraparenchymal infusion of HUFA may be effective in stimulating glioma regression.     

Enrichment of γ-linolenic acid from fungal oil by lipase-catalysed reactions

Summary Various lipases have been evaluated as biocatalysts for the enrichment of -linolenic acid from a commercial fungal oil derived from Mucor sp. by selective esterification of the fungal oil fatty acids with n-butanol or by selective hydrolysis of the oil. Lipase from M. miehei (Lipozyme), as compared to lipases from Candida cylindracea, Penicillium cyclopium, and Rhizopus arrhizus, was found to be most effective in the enrichment of -linolenic acid in unesterified fatty acids upon esterification of the fungal oil fatty acids with n-butanol. Thus, the -linolenic acid content could be raised from 10.4% in the starting material to 68.8% in the unesterified fatty acids. Selective hydrolysis of the fungal oil triacyglycerols using Lipozyme resulted in about 1.5-fold enrichment of -linolenic acid in the unhydrolysed acylglycerols. Other lipases tested, such as those from P. cyclopium, C. cylindracea, R. arrhizus, Penicillium sp. (Lipase G), porcine pancreas and Chromobacterium viscosum, were also rather ineffective in the enrichment of -linolenic acid by selective hydrolysis of the fungal oil triacylglycerols. Offprint requests to: K. D. Mukherjee     

Upregulated mRNA expression of desaturase and elongase,two enzymes involved in highly unsaturated fatty acids biosynthesis pathways during follicle maturation in zebrafish

Background  

Although unsaturated fatty acids such as eicosapentaenoic acid (EPA, C20:5n-3), docosahexaenoic acid (DHA, C22:6n-3) and arachidonic acid (ARA, C20:4n-6), collectively known as the highly unsaturated fatty acids (HUFA), play pivotal roles in vertebrate reproduction, very little is known about their synthesis in the ovary. The zebrafish (Danio rerio) display capability to synthesize all three HUFA via pathways involving desaturation and elongation of two precursors, the linoleic acid (LA, C18:2n-6) and linolenic acid (LNA, C18:3n-3). As a prerequisite to gain full understanding on the importance and regulation of ovarian HUFA synthesis, we described here the mRNA expression pattern of two enzymes; desaturase (fadsd6) and elongase (elovl5), involved in HUFA biosynthesis pathway, in different zebrafish ovarian follicle stages. Concurrently, the fatty acid profile of each follicle stage was also analyzed.     

Effect of different carbon sources on the biosynthesis of polyunsaturated fatty acids ofα-linolenic acid family in culture of minimal deviation hepatoma 7288 c cells

Summary The effect of three different carbon sources on the biosynthesis of polyunsaturated fatty acids of the-linolenic acid series was investigated in hepatoma tissue culture (HTC) cells. Alpha linolenic acid was converted to higher homologs by a desaturating route that synthetized mainly 18:4 (6, 9, 12, 15), 20:4 (8, 11, 14, 17) and 20:5 (5, 8, 11, 14, 17) and an elongating route that produced 20:3 (11, 14, 17) and 20:4 (5, 11, 14, 17) acids. Fasting decreased both biosynthetic routes whereas glucose reactivated only the elongating pathway. Lactalbumin hydrolysate enhanced significantly only the desaturating route whereas glycerol was inactive. Glucose and aminoacids increased similarly the incorporation of labeled linolenic acid in the cells. The results are independent of hormonal effects.Members of the Carrera del Investigador Científico of the Consejo Nacional de Investigaciones Científicas y Técnicas, Argentina.     

Fatty acid and tocochromanol patterns of some Turkish Boraginaceae—a chemotaxonomic approach

The plant family Boraginaceae is known to produce a set of unusual fatty acids in the seed oils. In this study, the fatty acid, tocopherol, tocotrienol and plasto-chromanol-8 contents of some Onosma species ( Onosma sericeum, O. armeniacum and O. polioxanthum ) all belonging to sect. Onosma, Anchusa leptophylla subsp. leptophylla, Alkanna froedini and Paracaryum stenophyllum were determined. Some of the studied species are endemic for Turkey. While oleic, linoleic and alpha linolenic acid are the highest as usual fatty acids, gamma linolenic and stearidonic acids are more variable unusual fatty acids in studied genera patterns and the relative concentrations some of these fatty acids and partly also the tocochromanols in Boraginaceae seed oils are suggested to have chemotaxonomic value in this family. In particular, the presence or absence of chain elongation to erucic acid (22:1) and the presence or absence of Δ6-methylene-interrupted polyenoic acids such as γ-linolenic and stearidonic acid are determined and marked as indicators of taxonomic relationship.     

Molecular Cloning and Functional Characterization of Fatty Acyl Desaturase and Elongase cDNAs Involved in the Production of Eicosapentaenoic and Docosahexaenoic Acids from <Emphasis Type="Bold">α</Emphasis>-Linolenic Acid in Atlantic Salmon (<Emphasis Type="Italic">Salmo salar</Emphasis>)

Fish are the only major dietary source for humans of -3 highly unsaturated fatty acids (HUFAs) and with declining fisheries farmed fish such as Atlantic salmon (Salmo salar) constitute an increasing proportion of the fish in the human diet. However, the current high use of fish oils, derived from wild capture marine fisheries, in aquaculture feeds is not sustainable in the longer term and will constrain continuing growth of aquaculture activities. Greater understanding of how fish metabolize and biosynthesize HUFA may lead to more sustainable aquaculture diets. The study described here contributes to an effort to determine the molecular genetics of the HUFA biosynthetic pathway in salmon, with the overall aim being to determine mechanisms for optimizing the use of vegetable oils in Atlantic salmon culture. In this paper we describe the cloning and functional characterization of 2 genes from salmon involved in the biosynthesis of HUFA. A salmon desaturase complementary DNA, SalDes, was isolated that include an open reading frame of 1362 bp specifying a protein of 454 amino acids. The protein sequence includes all the characteristics of microsomal fatty acid desaturases, including 3 histidine boxes, 2 transmembrane regions, and an N-terminal cytochrome b₅ domain containing a heme-binding motif similar to that of other fatty acid desaturases. Functional expression in the yeast Saccharomyces cerevisiae showed SalDes is predominantly an -3 5 desaturase, a key enzyme in the synthesis of eicosapentaenoic acid (20:5n-3) from -linolenic acid (18:3n-3). The desaturase showed only low levels of 6 activity toward C₁₈ polyunsaturated fatty acids. In addition, a fatty acid elongase cDNA, SalElo, was isolated that included an open reading frame of 888 bp, specifying a protein of 295 amino acids. The protein sequence of SalElo included characteristics of microsomal fatty acid elongases, including a histidine box and a transmembrane region. Upon expression in yeast SalElo showed broad substrate specificity for polyunsaturated fatty acids with a range of chain lengths, with the rank order being C₁₈ > C₂₀ > C₂₂. Thus this one polypeptide product displays all fatty acid elongase activities required for the biosynthesis of docosahexaenoic acid (22:6n-3) from 18:3n-3.     

The fatty acids of trichinellae]

The composition of fatty acids of lipids in the muscles of rats and larvae of Trichinella spiralis and T. nativa developing in them were studied. Both species are characterized by practically the same composition of fatty acids, only in the frost-resistant species T. nativa there was a sufficient amount (up to 3.5%) of docosapenta- and docosahexaenic acids (22:5 and 22:6). The comparison of the content of individual fatty acids in larvae and in muscles of the host by means of statistical correlation analysis suggests that larvae obtain a considerable portion of palmitic acid from the host and transform in into necessary long-chain saturated and unsaturated fatty acids by means of elongating and desaturating enzymes. Changes in the contents of fatty acids in larvae extracted from dead rats, which during some days were undergone freezing at negative temperatures (-8-10 degrees), are the same in quality for both species. These changes can be explained if we assume that the activity of elongases and desaturases of Trichinella decreases with cooling to a greater extent than the supply of palmitic acid from the host's tissues. A higher frost-resistance of T. nativa may be associated as with a greater protection of enzymes in the membranes by long-chain polyene acyls so with a higher thermal stability of proteins themselves.     

Isolation and functional characterization of polyunsaturated fatty acid elongase (AsELOVL5) gene from black seabream (Acanthopagrus schlegelii)

To identify the genes encoding fatty acid elongases for the biosynthesis of polyunsaturated fatty acids (PUFAs), we isolated a cDNA via degenerate PCR and RACE-PCR from Acanthopagrus schlegelii with a high similarity to the ELOVL5-like elongases of mammals and fishes. This gene is termed AsELOVL5 and encodes a 294 amino acid protein. When AsELOVL5 was expressed in Saccharomyces cerevisiae, it conferred an ability to elongate γ-linolenic acid (18:3 n−6) to di-homo-γ-linolenic acid (20:3 n−6). In addition, the transformed cells converted arachidonic acid (20:4 n−6) and eicosapentaenpic acid (20:5 n−3) to docosatetraenoic acid (22:4 n−6) and docosapentaenoic acid (22:5 n−3), respectively. These results indicate that the AsELOVL5 gene encodes a long-chain fatty acid elongase capable of elongating C₁₈Δ6/C₂₀Δ5 but not C₂₂ PUFA substrates.     

Effect of glucocorticoids on the oxidative desaturation of fatty acids by rat liver microsomes.

The effect of glucocorticoids on the oxidative desaturation of fatty acids by liver microsomal preparations of rats has been studied. Hydrocortisone produced a significant decrease in the conversion of [1-14C]linoleic acid to gamma-linolenic acid and [1-14C]eicosa-8, 11, 14-trienoic acid to arachidonic acid. Triamcinolone and dexamethasaone were more active than hydrocortisone in depressing delta 6 and delta 5 fatty acid desaturating activity in liver microsomes. The glucocorticoids evoked a maximal response approximately 24 hr after admission. Palmitic acid conversion to palmitoleic acid showed no statistically significant changes by any of the glucocorticoids. The mechanism of action of glucocorticoids is apparently different from other hyperglycemic hormones that produce similar effects.     

Dietary fatty acid composition affects the repeat swimming performance of Atlantic salmon in seawater

Repeated critical swimming performance trials (Ucrit) were performed on Atlantic salmon (Salmo salar) to test the null hypothesis that the source of dietary lipids (fish-based, poultry-based, and plant-based) does not influence exercise and recovery performance. Four diets were prepared by extensively replacing supplemental lipid from anchovy oil (AO; 100% AO at 150 g/kg) with cold pressed flaxseed oil (FO; 25% AO, 75% FO), sunflower oil (SO; 25% AO, 75% SO), or poultry fat (PF; 25% AO, 75% PF). These diets had equivalent protein and energy concentrations, but due to the different supplemental lipid sources, varied widely in their fatty acid composition. Fish fed AO had a significantly higher (P<0.05) first Ucrit (2.62+/-0.07 body lenght s(-1)) than those fed PF (2.22+/-0.12 body lenght s(-1)) that had low muscle ratios of n-3 highly unsaturated fatty acids (n-3 HUFA) to saturated fatty acids (SFA) and arachidonic acid (AA), and high levels of oleic acid. Fish in the FO and SO diet groups swam as well as AO-fed fish in both swimming trials. The performance of fish fed AO decreased significantly (P<0.05) during the second swimming trial (i.e. Ucrit2/Ucrit1=0.92+/-0.02). No significant differences occurred between diet groups for the second swim trial. There was a positive correlation between both n-3 HUFA/SFA and n-3 HUFA/AA ratios, and Ucrit1. A negative correlation was found between dietary AA and oleic acids, and Ucrit1. The present study suggests that low dietary n-3 HUFA/ SFA and n-3 HUFA/AA ratios may negatively affect swimming performance. The former possibly can be offset by increasing linoleic acid in the presence of nutritionally adequate n-3 HUFA (e.g. SO diet). Lipid supplements consisting largely of vegetable oils did not compromise fish cardiorespiratory physiology under the conditions of this study.     

Co-transcribed genes for long chain polyunsaturated fatty acid biosynthesis in the protozoon Perkinsus marinus include a plant-like FAE1 3-ketoacyl coenzyme A synthase

The marine parasitic protozoon Perkinus marinus synthesizes the polyunsaturated fatty acid arachidonic acid via the unusual alternative Delta8 pathway in which elongation of C18 fatty acids generates substrate for two sequential desaturations. Here we have shown that genes encoding the three P. marinus activities responsible for arachidonic acid biosynthesis (C18 Delta9-elongating activity, C20 Delta8 desaturase, C20 Delta5 desaturase) are genomically clustered and co-transcribed as an operon. The acyl elongation reaction, which underpins this pathway, is catalyzed by a FAE1 (fatty acid elongation 1)-like 3-ketoacyl-CoA synthase class of condensing enzyme previously only reported in higher plants and algae. This is the first example of an elongating activity involved in the biosynthesis of a polyunsaturated fatty acid that is not a member of the ELO/SUR4 family. The P. marinus FAE1-like elongating activity is sensitive to the herbicide flufenacet, similar to some higher plant 3-ketoacyl-CoA synthases, but unable to rescue the yeast elo2Delta/elo3Delta mutant consistent with a role in the elongation of polyunsaturated fatty acids. P. marinus represents a key organism in the taxonomic separation of the single-celled eukaryotes collectively known as the alveolates, and our data imply a lineage in which ancestral acquisition of plant-like genes, such as FAE1-like 3-ketoacyl-CoA synthases, occurred via endosymbiosis. The P. marinus FAE1-like elongating activity is also indicative of the independent evolution of the alternative Delta8 pathway, distinct from ELO/SUR4-dependent examples.     

Effect of “fasting” and different concentrations of glucose on α-linolenic acid metabolism in HTC cells. Correlation with the ultrastructural study

Summary HTC cells are able to convert -linolenic acid into higher homologs by desaturating and elongating reactions. When the cells were cultured in a Krebs Ringer bicarbonate solution (fasted cells) a decrease in both biosynthetic reactions took place. Refeeding the cells with Swim's 77 medium without glucose enhanced the biosynthesis of polyunsaturated fatty acids from -linolenic acid family, but when glucose was added to the medium, -linolenic acid was preferably elongated rather than converted into eicose-pentaenoic acid.The ultrastructural study revealed HTC cells with a simple cytoplasmic organization, showing little evidence of their origin from hepatocytes. The cells cultured in a complete medium appeared well preserved and were similar to those fasted for 12 hours and refed for another 12 hours using Swim's 77 medium without serum. The amount of glucose in the medium plays a role in preserving the cell structure. This effect does not occur if glucose is added in the absence of aminoacids and vitamins.     

Co-expression of the borage Δ6 desaturase and the Arabidopsis Δ15 desaturase results in high accumulation of stearidonic acid in the seeds of transgenic soybean

Two relatively rare fatty acids, γ-linolenic acid (GLA) and stearidonic acid (STA), have attracted much interest due to their nutraceutical and pharmaceutical potential. STA, in particular, has been considered a valuable alternative source for omega-3 fatty acids due to its enhanced conversion efficiency in animals to eicosapentaenoic acid when compared with the more widely consumed omega-3 fatty acid, α-linolenic acid (ALA), present in most vegetable oils. Exploiting the wealth of information currently available on in planta oil biosynthesis and coupling this information with the tool of genetic engineering it is now feasible to deliberately perturb fatty acid pools to generate unique oils in commodity crops. In an attempt to maximize the STA content of soybean oil, a borage Δ⁶ desaturase and an Arabidopsis Δ¹⁵ desaturase were pyramided by either sexual crossing of transgenic events, re-transformation of a Δ⁶ desaturase event with the Δ¹⁵ desaturase or co-transformation of both desaturases. Expression of both desaturases in this study was under the control of the seed-specific soybean β-conglycinin promoter. Soybean events that carried only the Δ¹⁵ desaturase possessed a significant elevation of ALA content, while events with both desaturases displayed a relative STA abundance greater than 29%, creating a soybean with omega-3 fatty acids representing over 60% of the fatty acid profile. Analyses of the membrane lipids in a subset of the transgenic events suggest that soybean seeds compensate for enhanced production of polyunsaturated fatty acids by increasing the relative content of palmitic acid in phosphatidylcholine and other phospholipids.     

Participation and Properties of Lipoxygenase and Hydroperoxide Lyase in Volatile C6-Aldehyde Formation from C18-Unsaturated. Fatty Acids in Isolated Tea Chloroplasts

Isolated tea chloroplasts utilized linoleic acid, linolenicacid and their 13-hydroperoxides as substrates for volatileC₆-aldehyde formation. Optimal pH values for oxygen uptake,hydroperoxide lyase and the overall reaction from C₁₈-fattyacids to C₆-aldehydes were 6.3, 7.0 and 6.3, respectively. Methyllinoleate, linoleyl alcohol and -linolenic acid were poor substratesfor the overall reaction, but linoleic and linolenic acids weregood substrates. The 13-hydroperoxides of the above fatty acidsand alcohol also showed substrate specificity similar to thatof fatty acids. Oxygen uptakes (relative V_max) with methyl linoleate,linoleyl alcohol, linolenic acid, -linolenic acid and arachidonicacid were comparable to or higher than that with linoleic acid.In winter leaves, the activity for C₆-aldehyde formation fromC₁₈-fatty acids was raduced to almost zero. This was due tothe reduction in oxygenation. The findings presented here provideevidence for the involvement of lipoxygenase and hydroperoxidelyase in C₆-aldehyde formation in isolated chloroplasts. (Received July 11, 1981; Accepted November 5, 1981)     

De novo biosynthesis of polyunsaturated fatty acids in the cockroach Periplaneta americana

The de novo biosynthesis of 6,9,12-linolenic acid, 11,14-eicosadienoic acid, 5,11,14-eicosatrienoic acid, and arachidonic acid was demonstrated in adult female cockroaches, Periplaneta americana. These four polyunsaturated fatty acids (PUFA) were present primarily in the phospholipid (PL) fraction of both males and females. They were purified by AgNO3 thin-layer chromatography and high pressure liquid chromatography. The double bond positions of the major isomer of eicosatrienoic acid were shown to be at the delta 5,11,14 positions by gas chromatography-mass spectrometry (GC-MS) of both methoxy and epoxide derivatives and gas-liquid chromatography (GLC) and GC-MS of ozonolysis products. The other PUFAs cochromatographed with standards on both packed and capillary GLC columns. The in vivo incorporation of [1-14C]acetate into 5,11,14-eicosatrienoic acid, 11,14-eicosadienoic acid, 6,9,12-linolenic acid, and arachidonic acid was demonstrated by radio-GLC and radio-HPLC and for 5,11,14-eicosatrienoic acid by radio-GLC of ozonolysis products. The latter technique clearly demonstrated that the entire eicosatrienoic acid molecule was labeled. Thoracic tissue contained the highest amount of radiolabeled 5,11,14-eicosatrienoic acid (1.6% of total radioactivity incorporated into PL) while radiolabeled 11,14-eicosadienoic acid was found primarily in abdominal epidermal tissue (2% of total radioactivity incorporated into PL). Radiolabeled arachidonic and 6,9,12-linolenic acids comprised 0.1 and 0.02%, respectively, of the total radioactivity in the PL fraction. These data document the de novo biosynthesis of di-, tri-, and tetraunsaturated fatty acids in the American cockroach, and indicate that this animal can desaturate on both sides of the delta 9 double bond of oleic acid.