Transgenic oilseed crops as an alternative to fish oils

Growing evidence suggests that omega-3 long chain polyunsaturated fatty acids (VLC-PUFAs), especially eicosapentaenoic acid (EPA; 20:5Δ5,8,11,14,17) and docosahexaenoic acid (DHA; 22:6Δ4,7,10,13,16,19) play critical roles in human health and development. VLC-PUFAs are mainly found in fish, some fungi, marine bacteria and microalgae. Currently, the predominant dietary sources of VLC-PUFAs are marine fish and seafood. However, the increasing demand for fish and fish oils is putting enormous pressure on marine ecosystems leading to a depletion of fish stocks while commercial cultivation of marine microorganisms and aquaculture are not sustainable and cannot compensate for the shortage in fish supply. Therefore, there is an obvious requirement for an alternative and sustainable source for VLC-PUFAs. Over the last decade, many genes encoding the primary VLC-PUFAs biosynthetic activities became available providing a toolkit for the “reverse-engineering” of transgenic plants to produce fish oils. In this review, we will describe the recent advances in this field and the insights they give us into the complexities of metabolic engineering of oil-seed crops producing VLC-PUFAs.     

Metabolic engineering of the omega-3 long chain polyunsaturated fatty acid biosynthetic pathway into transgenic plants

Omega-3 (ω-3) very long chain polyunsaturated fatty acids (VLC-PUFAs) such as eicosapentaenoic acid (EPA; 20:5 Δ5,8,11,14,17) and docosahexaenoic acid (DHA; 22:6 Δ4,7,10,13,16,19) have been shown to have significant roles in human health. Currently the primary dietary source of these fatty acids are marine fish; however, the increasing demand for fish and fish oil (in particular the expansion of the aquaculture industry) is placing enormous pressure on diminishing marine stocks. Such overfishing and concerns related to pollution in the marine environment have directed research towards the development of a viable alternative sustainable source of VLC-PUFAs. As a result, the last decade has seen many genes encoding the primary VLC-PUFA biosynthetic activities identified and characterized. This has allowed the reconstitution of the VLC-PUFA biosynthetic pathway in oilseed crops, producing transgenic plants engineered to accumulate ω-3 VLC-PUFAs at levels approaching those found in native marine organisms. Moreover, as a result of these engineering activities, knowledge of the fundamental processes surrounding acyl exchange and lipid remodelling has progressed. The application of new technologies, for example lipidomics and next-generation sequencing, is providing a better understanding of seed oil biosynthesis and opportunities for increasing the production of unusual fatty acids. Certainly, it is now possible to modify the composition of plant oils successfully, and, in this review, the most recent developments in this field and the challenges of producing VLC-PUFAs in the seed oil of higher plants will be described.     

Microalgal biofactories: a promising approach towards sustainable omega-3 fatty acid production

ABSTRACT: Omega-3 fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) provide significant health benefits and this has led to an increased consumption as dietary supplements. Omega-3 fatty acids EPA and DHA are found in animals, transgenic plants, fungi and many microorganisms but are typically extracted from fatty fish, putting additional pressures on global fish stocks. As primary producers, many marine microalgae are rich in EPA (C20:5) and DHA (C22:6) and present a promising source of omega-3 fatty acids. Several heterotrophic microalgae have been used as biofactories for omega-3 fatty acids commercially, but a strong interest in autotrophic microalgae has emerged in recent years as microalgae are being developed as biofuel crops. This paper provides an overview of microalgal biotechnology and production platforms for the development of omega-3 fatty acids EPA and DHA. It refers to implications in current biotechnological uses of microalgae as aquaculture feed and future biofuel crops and explores potential applications of metabolic engineering and selective breeding to accumulate large amounts of omega-3 fatty acids in autotrophic microalgae.     

Antarctic microorganisms as source of the omega-3 polyunsaturated fatty acids

Docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) are long-chain polyunsaturated fatty acids (PUFAs) that belong to the omega-3 group. They are essential fatty acids found in phospholipid of cell membranes. There is strong evidence that these nutrients may also favorably modulate many diseases. Primary sources of omega-3 PUFAs in the human diet are fish and fish-derived products. The fishing industry worldwide, however, is becoming unable to satisfy the growing demand for these PUFAs. A promising cost-effective alternative source of PUFAs is bacterial production. We identified 40 Antarctic marine bacterial isolates by 16S rRNA gene sequence analysis. Fifteen genera in three phyla were represented in the collection. Isolates were tested for ability to produce EPA using a method in which their ability to reduce 2,3,5-triphenyltetrazolium chloride (TTC) is determined and by gas chromatography coupled to mass spectrometry (GC–MS). All isolates could reduce TTC, and GC–MS analysis showed that four produced EPA and that six produced DHA. We show for the first time that isolates identified as Cellulophaga, Pibocella and Polaribacter can produce EPA and DHA, only DHA or only EPA, respectively. One isolate, Shewanella sp. (strain 8-5), is indicated to be a good candidate for further study to optimize growth and EPA production. In conclusion, a rapid method was tested for identification of new EPA producing strains from marine environments. New EPA and DHA producing strains were found as well as a potentially useful PUFA production strain.     

Engineering oilseeds to produce nutritional fatty acids

There is a growing body of evidence suggesting that regular consumption of foods rich in omega-3 long chain polyunsaturated fatty acids has multiple positive health benefits. The fats and oils from marine fish contain high contents of these beneficial fatty acids but increased consumer demand has also increased strain on the ability of the world's fisheries to meet demand from wild capture. Many consumers are choosing fish oil supplements or are eating foods that have been complemented with fish oils instead of consuming fish directly. However, removing undesirable odors, flavors and contaminants is expensive. In contrast, oils derived from land plants such as soybean are inexpensive and contaminant free. Recent strides in plant molecular biology now allow the engineering of oilseeds for the production of novel fats and oils, including those synthesized by complex, multigene biosynthetic pathways such as the omega-3 long chain polyunsaturated fatty acids. Given the potential benefits to the environment with regards to overfishing and the health prospects of increased consumption of these healthy fatty acids, producing these fatty acids in oilseeds is a desirable and worthy goal. In this review, we will describe the recent advances in this field along with some of the technical hurdles encountered thus far.     

Omega-3 fatty acids from fish oils and cardiovascular disease

Fish and fish oils contain the omega-3 fatty acids known as eicosapentaenoic acid (EPA) plus docosahexaenoic acid (DHA). Epidemiological studies have shown an inverse relation between the dietary consumption of fish containing EPA/DHA and mortality from coronary heart disease. These relationships have been substantiated from blood measures of omega-3 fatty acids including DHA as a physiological biomarker for omega-3 fatty acid status. Controlled intervention trials with fish oil supplements enriched in EPA/DHA have shown their potential to reduce mortality in post-myocardial infarction patients with a substantial reduction in the risk of sudden cardiac death. The cardioprotective effects of EPA/DHA are widespread, appear to act independently of blood cholesterol reduction, and are mediated by diverse mechanisms. Their overall effects include anti-arrhythmic, blood triglyceride-lowering, anti-thrombotic, anti-inflammatory, endothelial relaxation, plus others. Current dietary intakes of EPA/DHA in North America and elsewhere are well below those recommended by the American Heart Association for the management of patients with coronary heart disease. (Mol Cell Biochem 263: 217–225, 2004)     

球等鞭金藻中Δ5去饱和酶基因的克隆与功能鉴定

球等鞭金藻(Isochrysis galbana)是一类单细胞海洋微藻,富含二十二碳六烯酸(DHA,22:6Δ4,7,10,13,16,19)。我们利用RACE的方法从球等鞭金藻cDNA文库中同源克隆到一个大小为1329 bp的cDNA片段,编码442个氨基酸的多肽,分子量约49.9 kD。生物信息学分析表明,其编码产物N端具有细胞色素b5结构域,以及与电子传递有关的三个富含组氨酸的结构域,与Pavlova salinaΔ5去饱和酶同源性最高,达56%,故将该基因命名为IgD5。酿酒酵母功能鉴定实验表明,其编码的蛋白质具有Δ5去饱和酶活性,能够将二高-γ-亚麻酸(DGLA,20:3Δ8,11,14)转化成花生四烯酸(AA,20:4Δ5,8,11,14),转化效率平均为34.6%,最高可达40.3%。     

Effect of randomized supplementation with high dose olive, flax or fish oil on serum phospholipid fatty acid levels in adults with attention deficit hyperactivity disorder

Dietary intake of omega-3 fatty acids has been positively correlated with cardiovascular and neuropsychiatric health in several studies. The high seafood intake by the Japanese and Greenland Inuit has resulted in low ratios of the omega-6 fatty acid arachidonic acid (AA, 20:4n-6) to eicosapentaenoic acid (EPA, 20:5n-3), with the Japanese showing AA:EPA ratios of approximately 1.7 and the Greenland Eskimos showing ratios of approximately 0.14. It was the objective of this study to determine the effect of supplementation with high doses (60 g) of flax and fish oils on the blood phospholipid (PL) fatty acid status, and AA/EPA ratio of individuals with Attention Deficit Hyperactivity Disorder (ADHD), commonly associated with decreased blood omega-3 fatty acid levels. Thirty adults with ADHD were randomized to 12 weeks of supplementation with olive oil (< 1% omega-3 fatty acids), flax oil (source of alpha-linolenic acid; 18:3n-3; alpha-LNA) or fish oil (source of EPA and docosahexaenoic acid; 22:6n-3; DHA). Serum PL fatty acid levels were determined at baseline and at 12 weeks. Flax oil supplementation resulted in an increase in alpha-LNA and a slight decrease in the ratio of AA/EPA, while fish oil supplementation resulted in increases in EPA, DHA and total omega-3 fatty acids and a decrease in the AA/EPA ratio to values seen in the Japanese population. These data suggest that in order to increase levels of EPA and DHA in adults with ADHD, and decrease the AA/EPA ratio to levels seen in high fish consuming populations, high dose fish oil may be preferable to high dose flax oil. Future study is warranted to determine whether correction of low levels of long-chain omega-3 fatty acids is of therapeutic benefit in this population.     

Stepwise engineering to produce high yields of very long-chain polyunsaturated fatty acids in plants

Very long chain polyunsaturated fatty acids (VLCPUFAs) such as arachidonic acid (AA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are valuable commodities that provide important human health benefits. We report the transgenic production of significant amounts of AA and EPA in Brassica juncea seeds via a stepwise metabolic engineering strategy. Using a series of transformations with increasing numbers of transgenes, we demonstrate the incremental production of VLCPUFAs, achieving AA levels of up to 25% and EPA levels of up to 15% of total seed fatty acids. Both fatty acids were almost exclusively found in triacylglycerols, with AA located preferentially at sn-2 and sn-3 positions and EPA distributed almost equally at all three positions. Moreover, we reconstituted the DHA biosynthetic pathway in plant seeds, demonstrating the practical feasibility of large-scale production of this important omega-3 fatty acid in oilseed crops.     

Successful high‐level accumulation of fish oil omega‐3 long‐chain polyunsaturated fatty acids in a transgenic oilseed crop

Omega‐3 (also called n‐3) long‐chain polyunsaturated fatty acids (≥C20; LC‐PUFAs) are of considerable interest, based on clear evidence of dietary health benefits and the concurrent decline of global sources (fish oils). Generating alternative transgenic plant sources of omega‐3 LC‐PUFAs, i.e. eicosapentaenoic acid (20:5 n‐3, EPA) and docosahexaenoic acid (22:6 n‐3, DHA) has previously proved problematic. Here we describe a set of heterologous genes capable of efficiently directing synthesis of these fatty acids in the seed oil of the crop Camelina sativa, while simultaneously avoiding accumulation of undesirable intermediate fatty acids. We describe two iterations: RRes_EPA in which seeds contain EPA levels of up to 31% (mean 24%), and RRes_DHA, in which seeds accumulate up to 12% EPA and 14% DHA (mean 11% EPA and 8% DHA). These omega‐3 LC‐PUFA levels are equivalent to those in fish oils, and represent a sustainable, terrestrial source of these fatty acids. We also describe the distribution of these non‐native fatty acids within C. sativa seed lipids, and consider these data in the context of our current understanding of acyl exchange during seed oil synthesis.     

Modulation of enzymatic activities by n-3 polyunsaturated fatty acids to support cardiovascular health

Epidemiological evidence from Greenland Eskimos and Japanese fishing villages suggests that eating fish oil and marine animals can prevent coronary heart disease. Dietary studies from various laboratories have similarly indicated that regular fish oil intake affects several humoral and cellular factors involved in atherogenesis and may prevent atherosclerosis, arrhythmia, thrombosis, cardiac hypertrophy and sudden cardiac death. The beneficial effects of fish oil are attributed to their n-3 polyunsaturated fatty acid (PUFA; also known as omega-3 fatty acids) content, particularly eicosapentaenoic acid (EPA; 20:5, n-3) and docosahexaenoic acid (DHA; 22:6, n-3). Dietary supplementation of DHA and EPA influences the fatty acid composition of plasma phospholipids that, in turn, may affect cardiac cell functions in vivo. Recent studies have demonstrated that long-chain omega-3 fatty acids may exert beneficial effects by affecting a wide variety of cellular signaling mechanisms. Pathways involved in calcium homeostasis in the heart may be of particular importance. L-type calcium channels, the Na+-Ca2+ exchanger and mobilization of calcium from intracellular stores are the most obvious key signaling pathways affecting the cardiovascular system; however, recent studies now suggest that other signaling pathways involving activation of phospholipases, synthesis of eicosanoids, regulation of receptor-associated enzymes and protein kinases also play very important roles in mediating n-3 PUFA effects on cardiovascular health. This review is therefore focused on the molecular targets and signaling pathways that are regulated by n-3 PUFAs in relation to their cardioprotective effects.     

Metabolic engineering of omega-3 long-chain polyunsaturated fatty acids in plants using an acyl-CoA Δ6-desaturase with ω3-preference from the marine microalga Micromonas pusilla

Long-chain (≥C₂₀) polyunsaturated fatty acids (LC-PUFA) EPA and DHA (20:5^{Δ5,8,11,14,17} and 22:6^{Δ4,7,10,13,16,19}) have well-documented health benefits against coronary heart disease, rheumatoid arthritis and other disorders. Currently, the predominant sources of these fatty acids are marine fish and algal oils, but research is being conducted to ensure that a sustainable, land-based production system can be developed. We here describe the metabolic engineering of an artificial pathway that produces 26% EPA in leaf triacylglycerol using a newly-identified Δ6-desaturase from the marine microalga Micromonas pusilla. We also demonstrate that this enzyme appears to function as an acyl-CoA desaturase that has preference for ω3 substrates both in planta and in yeast. Phylogenetic analysis indicates that this desaturase shares highly conserved motifs with previously described acyl-CoA Δ6-desaturases.     

Omega-3 fatty acids and neuropsychiatric disorders

Epidemiological evidence suggests that dietary consumption of the long chain omega-3 fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), commonly found in fish or fish oil, may modify the risk for certain neuropsychiatric disorders. As evidence, decreased blood levels of omega-3 fatty acids have been associated with several neuropsychiatric conditions, including Attention Deficit (Hyperactivity) Disorder, Alzheimer's Disease, Schizophrenia and Depression. Supplementation studies, using individual or combination omega-3 fatty acids, suggest the possibility for decreased symptoms associated with some of these conditions. Thus far, however, the benefits of supplementation, in terms of decreasing disease risk and/or aiding in symptom management, are not clear and more research is needed. The reasons for blood fatty acid alterations in these disorders are not known, nor are the potential mechanisms by which omega-3 fatty acids may function in normal neuronal activity and neuropsychiatric disease prevention and/or treatment. It is clear, however, that DHA is the predominant n-3 fatty acid found in the brain and that EPA plays an important role as an anti-inflammatory precursor. Both DHA and EPA can be linked with many aspects of neural function, including neurotransmission, membrane fluidity, ion channel and enzyme regulation and gene expression. This review summarizes the knowledge in terms of dietary omega-3 fatty acid intake and metabolism, as well as evidence pointing to potential mechanisms of omega-3 fatty acids in normal brain functioning, development of neuropsychiatric disorders and efficacy of omega-3 fatty acid supplementation in terms of symptom management.     

Cloning and functional characterisation of a fatty acyl elongase from southern bluefin tuna (Thunnus maccoyii)

The synthesis of long chain polyunsaturated fatty acids (LCPUFA), such as eicosapentaenoic acid (EPA; 20:5n-3) and docosahexaenoic acid (DHA; 22:6n-3), involves fatty acyl desaturase and elongase enzymes. The marine fish species southern bluefin tuna (SBT) can accumulate large quantities of omega-3 (n-3) LCPUFA in its flesh but their capacity to synthesize EPA and DHA is uncertain. A cDNA, sbtElovl5, encoding a putative fatty acyl elongase was amplified from SBT liver tissue. The cDNA included an open reading frame (ORF) encoding 294 amino acids. Sequence comparisons and phylogenetic analyses revealed a high level of sequence conservation between sbtElovl5 and fatty acyl elongase sequences from other fish species. Heterologous expression of the sbtElovl5 ORF in Saccharomyces cerevisiae confirmed that it encoded a fatty acyl elongase capable of elongating C_18/20 polyunsaturated fatty acid (PUFA) substrates, but not C₂₂ PUFA substrates. For the first time in an Elovl5, the substrate competition occurring in nature was investigated. Higher activity towards n-3 PUFA substrates than omega-6 (n-6) PUFA substrates was exhibited, regardless of substrate chain length. The sbtElovl5 preferentially elongated 18:4n-3 and 18:3n-6 rather than 20:5n-3 and 20:4n-6. The sbtElovl5 enzyme also elongated saturated and monounsaturated fatty acids.     

Serotonin-induced endothelial cell proliferation is blocked by omega-3 fatty acids.

Serotonin (5HT) released from aggregating platelets at sites of vascular injury is a known mitogen for vascular endothelial cells. Recent studies have indicated that regenerating endothelial cells at sites of vessel wall injury may play a role in the development of restenosis by synthesizing and releasing growth factors for vascular smooth muscle cells, proliferation of which may result in the development of neointima. Diets rich in fish oils (omega-3 fatty acids) are associated with reduced risk of cardiovascular disease including atherosclerosis and restenosis. This study examined the effect of the omega-3 and other fatty acids, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), on 5HT induced endothelial cell proliferation. Among the fatty acids examined only EPA and DHA could reverse the mitogenic effect of 5HT on vascular endothelial cells, whereas oleic acid or palmitic acid did not have any effect. When added together, EPA and DHA potentiate each other in reversing the mitogenic effect of 5HT. EPA and DHA also inhibited the 5HT-induced increase in the 5HT2 receptor mRNA, without a change in the receptor density or affinity. This data suggests that one of the mechanisms by which omega-3 fatty acids may attenuate the development of atherosclerosis or restenosis is to inhibit the mitogen induced growth of vascular endothelial cells, which attenuates the release of growth factors for vascular smooth muscle cells.     

Enhancing LC-PUFA production in <Emphasis Type="Italic">Thalassiosira pseudonana</Emphasis> by overexpressing the endogenous fatty acid elongase genes

The health beneficial omega-3 long-chain polyunsaturated fatty acids (LC-PUFAs), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA) are naturally synthesized by diatoms through consecutive steps of fatty acid elongase and desaturase enzymes. In Thalassiosira pseudonana, these fatty acids constitute about 10–20 % of the total fatty acids, with EPA accumulation being five to ten times higher than DHA. In order to identify the subcellular localization of enzymes in the pathway of LC-PUFA biosynthesis in T. pseudonana and to manipulate the production of EPA and DHA, we generated constructs for overexpressing each of the T. pseudonana long-chain fatty acid elongase genes. Full-length proteins were fused to GFP, and transgenic lines were generated. In addition, overexpressed native proteins with no GFP fusion were tested. The subcellular localization of each elongase protein was determined. We then examined the total amount of lipids and analyzed the fatty acid profile in each of the transgenic lines compared to wild type. Lines with overexpressed elongases showed an increase of up to 1.4-fold in EPA and up to 4.5-fold in DHA, and the type of fatty acid that was increased (EPA vs. DHA) depended on the type of elongase that was overexpressed. This data informs future metabolic engineering approaches to further improve EPA and DHA content in diatoms.     

Production of very long chain polyunsaturated omega-3 and omega-6 fatty acids in plants

We report the production of two very long chain polyunsaturated fatty acids, arachidonic acid (AA) and eicosapentaenoic acid (EPA), in substantial quantities in a higher plant. This was achieved using genes encoding enzymes participating in the omega3/6 Delta8 -desaturation biosynthetic pathways for the formation of C20 polyunsaturated fatty acids. Arabidopsis thaliana was transformed sequentially with genes encoding a Delta9 -specific elongating activity from Isochrysis galbana, a Delta8 -desaturase from Euglena gracilis and a Delta5 -desaturase from Mortierella alpina. Instrumental in the successful reconstitution of these C20 polyunsaturated fatty acid biosynthetic pathways was the I. galbana C18-Delta9 -elongating activity, which may bypass rate-limiting steps present in the conventional Delta6 -desaturase/elongase pathways. The accumulation of EPA and AA in transgenic plants is a breakthrough in the search for alternative sustainable sources of fish oils.     

Meta-analysis of factors associated with omega-3 fatty acid contents of wild fish

Fish are recognized as the main source of physiologically important omega-3 long-chain polyunsaturated fatty acids, namely, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), for human nutrition. However, muscle tissue contents of these fatty acids in diverse fish species, i.e., their nutritive value for humans, varied within two orders of magnitude. We reviewed contents of EPA and DHA, measured by similar methods using an internal standard during chromatography as mg per g of wet mass in 172 fish species belonging to 16 orders, to evaluate probable variations in phylogenetic and ecological drivers. EPA + DHA content varied from 25.6 mg g^?1 of wet mass (Sardinops sagax) to 0.12 mg g^?1 (Gymnura spp.). Multidimensional redundancy analysis revealed that among phylogenetic, ecomorphological and abiotic environmental factors, the highest proportion of variation contribution belonged to the shared contribution of sets of phylogenetic and ecomorphological factors. Specifically, the highest values of EPA + DHA content were characteristic of fish belonging to the orders Clupeiformes or Salmoniformes, were pelagic fast swimmers, ate zooplankton and inhabited marine waters or migrated from fresh to marine waters (anadromous migrations). High EPA and DHA content in muscle tissues of the above species appeared to be a metabolic adaptation for fast continuous swimming. In contrast to common beliefs, our meta-analysis did not support the significant influence of higher trophic levels (piscivory) and cold environments (homeoviscous adaptation) on EPA and DHA content in fish. However, many causes of high and low levels of physiologically important fatty acids in certain fish species remained unexplained and require evaluation in future studies.     

Eicosapentaenoic and docosahexaenoic acids enriched polyunsaturated fatty acids from the coastal marine fish of Bay of Bengal and their therapeutic value

Eicosapentaenoic acid (EPA)/docosahexaenoic acid (DHA) enriched polyunsaturated fatty acids (PUFA) significantly present in marine fish oil emerge as preventive agents for combating many health problems specially in chronic or metabolic disorders. The fish in the coastal area of Bay of Bengal has remained unexplored with respect to EPA/DHA enriched PUFA content in its oils, although it may be a potential source in harnessing the health benefit. In this study, seven varieties of the coastal fish were analysed for the content of EPA/DHA. The one locally known as lotte, (Harpadon nehereus) though has low content of total lipids, was found to have high EPA/DHA in its oil. The phospholipids rich fraction was extracted from the total fish oil. The EPA/DHA enriched PUFA was isolated to investigate the potential use for health benefits. EPA/DHA is found to act as protective agent against mercury poisoning studied in cell culture as well as in animal mode. It is found to be highly preventive in diabetes. The lotte is available in the coastal area of Bay of Bengal adjoining West Bengal, India in large scale and it is the first report showing EPA/DHA enriched PUFA in these fish oil that can be availed to harness in important health benefits.     

Keloids in rural black South Africans. Part 2: dietary fatty acid intake and total phospholipid fatty acid profile in the blood of keloid patients

In the second part of this study, emphasis is placed on nutritional intakes (fatty acids and micronutrients) and fatty acid intake and metabolism in the blood, respectively, according to a combined 24 h recall and standardized food frequency questionnaire analyses of keloid prone patients (n=10), compared with normal black South Africans (n=80), and total phospholipid blood (plasma and red blood cell ) analyses of keloid patients (n=20), compared with normal individuals (n=20). Lipid extraction and fractionation by standard procedures, total phospholipid (TPL) separation with thin layer chromatography, and fatty acid methyl ester analyses with gas liquid chromatography techniques were used. Since nutrition may play a role in several disease disorders, the purpose of this study was to confirm or refute a role for essential fatty acids (EFAs) in the hypothesis of keloid formations stated in part 1 of this study. (1)According to the Canadian recommendation (1991), we observed that in keloid patients linoleic acid (LA) and arachidonic acid (AA) dietary intakes, as EFAs of the omega-6-series, are higher than the recommended 7-11 g/d. However, the a-linolenic acid (ALA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) dietary intakes, as EFAs of the omega-3 series, are lower than the recommendation of 1.1-1.5 g/d. This was also the case in the control group, where a higher dietary intake of the omega-6 fatty acids and a slightly lower dietary intake of the omega-3 fatty acids occurred. Thus, we confirm a high dietary intake of LA (as a product of organ meats, diary products and many vegetable oils) and AA (as a product of meats and egg yolks), as well as lower dietary intakes of ALA (as a product of grains, green leafy vegetables, soy oil, rapeseed oil and linseed), and EPA and DHA (as products of marine oils). Lower micronutrient intakes than the recommended dietary allowances were observed in the keloid group that may influence EFA metabolism and/or collagen synthesis. Of cardinal importance may be the lower intake of calcium in the keloid patients that may contribute to abnormal cell signal transduction in fibroblasts and consequent collagen overproduction, and the lower copper intake that may influence the immune system, or perhaps even the high magnesium intake that stimulates metabolic activity. Micronutrient deficiencies also occurred in the diets of the normal black South Africans that served as a control group. In the case of plasma TPLs, deficiency of the omega-3 EFA series (ALA, EPA and DHA) occurred, and this is in accordance with the apparent lower omega-3 EFA intake in the diets of these patients. In the case of the red blood cell TPLs, as a true and reliable source of dietary fatty acid intake and metabolism, sufficient EFAs of the omega-6 series (LA and AA) and the omega-3 series (ALA, EPA and DHA) occurred. For this study group a relative deficiency of nutritional omega-3 EFA intake apparently did occur, but was probably compensated for by blood fatty acid metabolism.