Nutritional Deficiencies and Phospholipid Metabolism

Phospholipids are important components of the cell membranes of all living species. They contribute to the physicochemical properties of the membrane and thus influence the conformation and function of membrane-bound proteins, such as receptors, ion channels, and transporters and also influence cell function by serving as precursors for prostaglandins and other signaling molecules and modulating gene expression through the transcription activation. The components of the diet are determinant for cell functionality. In this review, the effects of macro and micronutrients deficiency on the quality, quantity and metabolism of different phospholipids and their distribution in cells of different organs is presented. Alterations in the amount of both saturated and polyunsaturated fatty acids, vitamins A, E and folate, and other micronutrients, such as zinc and magnesium, are discussed. In all cases we observe alterations in the pattern of phospholipids, the more affected ones being phosphatidylcholine, phosphatidylethanolamine and sphingomyelin. The deficiency of certain nutrients, such as essential fatty acids, fat-soluble vitamins and some metals may contribute to a variety of diseases that can be irreversible even after replacement with normal amount of the nutrients. Usually, the sequelae are more important when the deficiency is present at an early age

Influences of dietary polyunsaturated fatty acids on tissue fatty acid composition and eicosanoid production in Atlantic salmon (Salmo salar)

1. The literature has been reviewed with respect to the dietary intake and subsequent metabolism of polyunsaturated fatty acids (PUFA), of both the n-6 and n-3 series, in teleost fish. Particular emphasis has been made to the physiological roles of PUFA with respect to cell membrane function and eicosanoid production. 2. Atlantic salmon post-smolts were fed practical-type diets, based on fish meal, in three separate dietary experiments of 10-16 weeks duration. The first trial compared dietary lipid supplied either as fish oil (FO) or as sunflower oil (SO) with the diets having an n-3/n-6 PUFA ratio of 9.4 and 0.2 respectively. The second trial used diets formulated with blends of FO, SO, grape seed oil and safflower oil to provide linoleic acid at 10, 25 and 45% of total dietary fatty acids. The third trial was similar to the first but with an additional diet in which the lipid component was supplied by linseed oil (LO). All diets satisfied the nutritional requirements of salmonid fish for n-3 PUFA. There were no statistically significant differences in final weights between dietary treatments in the third trial. However, in the second trial fish fed the intermediate level of linoleic acid (25%) attained a significantly higher final weight compared to both other treatments while fish fed the highest level of linoleic acid (45%) had significantly lower final weights compared to both other treatments. In the first trial the effect of diet on growth (weight gain) could not be ascertained as the initial weights of the fish were significantly different. 3. A number of fish fed SO developed severe cardiac lesions which caused thinning of the ventricular wall and heart muscle necrosis. In addition the fish fed diets containing SO were susceptible to a transportation-induced shock syndrome that resulted in 30% mortality. 4. Incorporation of linoleic acid (18:2n-6) into membrane phospholipids increased in response to dietary intake with fish fed SO having increased levels of 18:2n-6 (up to 15-fold), 20:2n-6 (up to 12-fold), 20:3n-6 (up to 25-fold) and arachidonic acid (AA; 20:4n-6) (up to 3-fold), and decreased levels of eicosapentaenoic acid (EPA; 20:5n-3) (up to 3-fold). The ratio of n-3/n-6 PUFA was decreased (up to 4-fold) and the20:4n-6/20:5n-3 ratio increased (up to 9-fold) in membrane phospholipids from fish fed SO compared to those fed fish oil. While the tissue phospholipids from fish fed La had increased levels of 18:2n-6, 20:2n-6 and 20:3n-6, the levels of AA, 22:4n-6 and 22:5n-6 were similar to or significantly reduced compared to fish fed FO. Membrane phospholipids from fish fed LO also had increased 18:3n-3 and 20:4n-3 compared to both other treatments while in some tissues and phospholipid classes EPA was increased compared to fish fed FO. 5. These dietary induced changes in phospholipid eicosanoid precursor ratio were reflected in altered eicosanoid production. In gill cells, stimulated with the calcium ionophore A23187, 12-hydroxy-8, 10, 14, 17-eicosapentaenoic acid (12-HEPE) was the major 12-lipoxygenase product in fish fed Fa. In stimulated gill cells from fish fed SO and LO, 12-HEPE, 12-hydroxy-5, 8, 10, 14-eicosatetraenoic acid (12-HETE), 14- hydroxy-4, 7, 10, 13, 16, 19-docosahexaenoic acid (14-HDHE) and thromboxane B2 (TXB2) were all decreased compared to fish fed FO. However, the ratio of 12- HETE/12-HEPE was significantly elevated in stimulated gill cells from SO-fed fish compared to both other treatments. In stimulated blood leucocytes leukotriene B4 (LTB4)' 12-HETE and TXB2 were significantly increased while LTB5 and 12-HEPE were significantly decreased in fish fed SO compared to those fed FO. Blood leucocytes from fish fed LO produced less TXB2 compared to fish fed SO and prostaglandin E2 was reduced compared to both other treatments. In isolated cardiac myocytes stimulated with A23187, TXB2 production was increased in SO fed fish compared to those fed FO. 6. The activity of cardiac sarcoplasmic reticulum Ca2+-Mg2+ATPase was not affected by dietary treatment. 7. An established cell line derived from chum salmon heart (CHH-1) was utilised to study PUFA metabolism. The CHH-1 cells exhibited considerable A6 desaturase activity but showed no preference towards n-3 over n-6 PUFA. CHH-1 cells did exhibit significant A5 desaturase activity which showed a preference towards n-3 PUFA. No A4 desaturation activity was observed. Elongation of C20 PUFA was especially active in CHH-1 cells with C22 PUFA being specifically incorporated into phosphatidylethanolamine (PE) and phosphatidylserine (PS). CHH-1 cells supplemented with 20:3n-6 showed reduced growth rate, cell death and unusual pycnotic appearance, compared to those supplemented with other PUFA. 8. The lipid compositions of hearts and livers from wild and farmed parr and presmolts were analysed and compared. The fatty acid compositions of triacylglycerols (TAG) and phospholipids from both farmed parr and pre-smolts contained greater amounts of monoenoic fatty acids compared to their wild counterparts. TAG, phosphatidylcholine (PC) and PE from heart and liver of wild fish contained more 18:2n-6 and AA compared to farmed fish. Linolenic acid, EPA and 22:Sn-3 were increased in hearts and livers of wild fish compared to farmed. Docosahexaenoic acid (DHA; 22:6n-3) levels were higher in heart and liver of farmed fish, particularly in heart PC, PS and TAG. The n-3/n-6 PUFA ratio was generally lower in wild compared to farmed fish, largely due to higher n-6 PUFA, in particular AA, in wild fish. 9. The results are discussed with respect to the competitive interactions between PUFA of the n-6 and n-3 series which determine the fatty acid compositions of membrane phospholipids in salmon. The ratio of n-3/n-6 PUFA in membrane phospholipids, and in particular the ratio of AAIEPA, appears important in terms of membrane physiology and biochemistry, eicosanoid production and the development of cardiac histopathological lesions

The Potential of Exogenous Fatty Acids in Breast Tissue Engineering

More than 200,000 women are diagnosed with breast cancer each year in the United States, where the majority will undergo a lumpectomy procedure and then radiotherapy as preventative treatment. Both artificial and tissue transplants may obscure the detection of cancer recurrence or limit the effectiveness of radiation treatment. Previous research in our laboratory has been performed to develop injectable lumpectomy void fillers. This body of work seeks to continue and further develop the technology of the void fillers and further investigate the potential of fatty acids in breast tissue engineering. Literature suggests that lipid peroxidation products from free fatty acids may have anti-cancerous effects. Linoleic acid is in the family of fatty acids associated with lipid peroxidation; however, linoleic acid is also associated with growth promoting eicosanoids which has caused linoleic acid to be considered a cancer promoting agent. Studies shown here suggest that a high concentration, 100 µM, of linoleic acid was effective at limiting cancerous proliferation by day 6, and suggest a complete removal of the cancerous population by day 8, while control cells remained largely unaffected. Further studies confirmed the elevated presence of lipid peroxide products in the inhibited cancerous cell cultures. When linoleic acid was tested in culture with tamoxifen, the observed inhibition of cancerous proliferation was greater than either of the treatments alone. This finding supports the use of linoleic acid in tissue engineered scaffolding. Additional work was performed to advance the development of a lumpectomy scaffolding. An injectable chitosan scaffolding has previously been developed through the use of a pH shifting technique; however, work described here continued the scaffolding development by adding gelatin, hyaluronic acid, and an acid washing step which resulted in the formation of a distinct bilayer. Cells cultured on the bilayered scaffolding were found to migrate to the boundary between layers where they proliferated at an accelerated rate compared to uniform hydrogels. Fatty acids were able to be incorporated into the production of the hydrogels at a concentration of 400 µM. Previous researchers in the laboratory had reported adipogenic effects from linoleic acid. These results were not observed upon further investigation. Linoleic acid did result in the swelling of human stem cells but did not otherwise morphologically change the cells. A mixture of conjugated linoleic acid however, was tested and found to upregulate adipogenic specific markers to a level indistinguishable from adipogenic controls. Through the course of this body of research, several advances were made in the field of test methods for tissue engineering. Work was done towards the improvement of procedures of multiwell plate readers to allow sterile sampling, the cytotoxicity of alamarBlue was found and investigated, improvements to previous lab gas chromatography procedures were made, and analyses of experimental results indicated the importance of recovery periods in cytotoxicity studies

Effect of Dietary Treatment with Stearidonic Acid and Eicosapentaenoic Acid on Membrane Phospholipid Fatty Acid Composition and Glycogen-Elicited Peritoneal Neutrophil Survival in Absence or Presence of Bacterial Endotoxin

We previously found that enteral nutrition with diets containing elevated levels of eicosapentaenoic acid (EPA; 20:5 n-3) reduced the number of neutrophils in bronchoalveolar lavage fluid and improved clinical outcome of patients with or at risk for adult respiratory distress syndrome (ARDS). We have also shown that the treatment of the neutrophil-like cell line, HL-60, with EPA can induce apoptosis. It is not known, however, if the addition of EPA to the diet would result in similar occurrences in neutrophils. It has been suggested that the use of diets enriched with stearidonic acid (SDA; 18:4 n-3) may provide a novel source of n-3 fatty acids while offering similar beneficial effects on health as diets containing EPA. Therefore, we examined whether dietary SDA can be used as a precursor to increase EPA content in liver tissue. It is well known that endotoxin increases neutrophil lifespan and that this may be important in maintaining innate immune response. The second part of this study incorporated the use of glycogen-elicited peritoneal neutrophils from the rat to examine the effect of dietary intervention with n-3 fatty acids (either EPA or SDA) on constitutive and endotoxin induced neutrophil survival. Male Long-Evans rats (n=18) were randomly divided into 3 dietary groups and fed a modified US-17 diet providing 200kcal/kg/day and containing 2% oleic acid (OA; 18:1 n-9) (n=6), 1% EPA/1% OA (n=6), or 1% SDA/1% OA (n=6) for 2 weeks. Liver phospholipids were analyzed using gas chromatography and neutrophil lifespan was evaluated using flow cytometry. Dietary supplementation with EPA significantly increased the concentration of EPA and docosapentaenoic acid (DPA, 22:5n3), but not docosahexaenoic acid (DHA, 22:6n3) in liver tissue as compared with animals fed OA. Dietary supplementation with SDA also increased tissue contents of EPA and DPA, but not DHA as compared with OA fed controls. The increase in tissue EPA was about one-half as effective in animals fed a diet containing SDA as those fed EPA. SDA was about two-thirds as effective as EPA at increasing tissue DPA content. These results indicate that, when fed at current dietary levels, SDA can increase the incorporation of EPA and DPA into the membrane phospholipids of liver tissue in a similar manner to dietary EPA. This provides evidence that SDA may provide an alternative to EPA for increasing tissue levels of EPA and DPA, but not DHA. Flow cytometry analysis of neutrophils following 20h incubation in either presence or absence of endotoxin indicated that diet did not significantly affect constitutive or endotoxin-mediated neutrophil survival when fed at current levels. There was a non-significant trend towards an increase in both constitutive viability and endotoxin mediated cell survival (p=0.069) in both SDA and EPA fed animals as compared with control animals that were fed OA. While these results do not fully explain the role that EPA or SDA play in reducing neutrophil influx during ARDS, they do provide evidence that it is not through an increase of neutrophil apoptosis or an attenuation of the neutrophil response to endotoxin. These findings may indicate the importance of dietary EPA for maintaining host immune response, while providing beneficial effects in regards to inflammation. Because of similar action between dietary EPA and SDA these results might also provide further proof as to the potential for dietary SDA to be used as an alternative for dietary EPA

Effects of Fatty Acids on Gene Expression and Lipid Metabolism in Bovine Intramuscular and Subcutaneous Adipose Tissues

Pasture feeding depresses adipose tissue development in beef cattle whereas grain feeding, enhances adipogenesis. Therefore, we hypothesized that specific fatty acids would differentially affect lipogenesis in explants of bovine subcutaneous (SC) and intramuscular (IM) adipose tissues. Angus steers were harvested at 12, 14, and 16 mo of age, and IM and SC adipose tissue explants from the 8-11th thoracic rib region were dissected and cultured in media. Media contained no supplemental fatty acids or 40 microM of five fatty acids, stearic acid (18:0), oleic acid (18:1 n-9), trans-11 vaccenic acid (18:1 trans-11), conjugated linoleic acid (CLA, 18:2 trans-10, cis-12), or alpha-linolenic acid (18:3 n-3). After 48 h of culture, lipogenesis using [U-14C]glucose and [1-14C]acetate was measured. Lipogenesis from glucose decreased between 12 and 16 mo of age in SC adipose tissue (from 8.9 to 4.0 nmol per 2 h per 100 mg; P = 0.001) and IM adipose tissue (from 4.4 to 2.7 nmol per 2 h 100 mg ; P = 0.08). Lipogenesis from acetate did not change over time in SC adipose (approximately 56 nmol per 2 h per 100 mg; P = 0.23), but increased over time in IM adipose tissue (from from 11.3 to 17.1 nmol per 2 h 100 mg; P = 0.02). Oleic acid increased lipid synthesis from glucose 125 percent (P = 0.04) in IM adipose tissue, whereas stearic acid and trans-vaccenic acid increased lipogenesis from glucose in SC adipose tissue by approximately 50 percent (P = 0.04). In SC adipose tissue only, trans-vaccenic and increased, lipogenesis from glucose (P < 0.02). Lipogenesis from acetate was depressed by CLA nearly 50 percent in SC adipose tissue. PPARγ gene expression increased between 14 and 16 mo of age in control IM and SC adipocytes. The increase in activity was also observed in AMPK gene expression. C/EBPβ and SCD gene expression did not increase in control samples until 16 mo of age. SC adipose tissue responded to stearic acid by increased GPR43 and AMPK gene expression at 12 mo of age. We conclude that fatty acids differentially affect lipid synthesis in IM and SC adipose tissues, which may account for the effects of pasture and grain feeding on adiposity

Gamma-Linolenic and Stearidonic Acids Are Required for Basal Immunity in Caenorhabditis elegans through Their Effects on p38 MAP Kinase Activity

Polyunsaturated fatty acids (PUFAs) form a class of essential micronutrients that play a vital role in development, cardiovascular health, and immunity. The influence of lipids on the immune response is both complex and diverse, with multiple studies pointing to the beneficial effects of long-chain fatty acids in immunity. However, the mechanisms through which PUFAs modulate innate immunity and the effects of PUFA deficiencies on innate immune functions remain to be clarified. Using the Caenorhabditis elegans–Pseudomonas aeruginosa host–pathogen system, we present genetic evidence that a Δ6-desaturase FAT-3, through its two 18-carbon products—gamma-linolenic acid (GLA, 18:3n6) and stearidonic acid (SDA, 18:4n3), but not the 20-carbon PUFAs arachidonic acid (AA, 20:4n6) and eicosapentaenoic acid (EPA, 20:5n3)—is required for basal innate immunity in vivo. Deficiencies in GLA and SDA result in increased susceptibility to bacterial infection, which is associated with reduced basal expression of a number of immune-specific genes—including spp-1, lys-7, and lys-2—that encode antimicrobial peptides. GLA and SDA are required to maintain basal activity of the p38 MAP kinase pathway, which plays important roles in protecting metazoan animals from infections and oxidative stress. Transcriptional and functional analyses of fat-3–regulated genes revealed that fat-3 is required in the intestine to regulate the expression of infection- and stress-response genes, and that distinct sets of genes are specifically required for immune function and oxidative stress response. Our study thus uncovers a mechanism by which these 18-carbon PUFAs affect basal innate immune function and, consequently, the ability of an organism to defend itself against bacterial infections. The conservation of p38 MAP kinase signaling in both stress and immune responses further encourages exploring the function of GLA and SDA in humans

Rumen Microbial Ecology And Rumen-Derived Fatty Acids: Determinants Of And Relationship To Dairy Cow Production Performance

Rumen microbiota enable dairy cattle to breakdown fiber into useable energy for milk production. Rumen bacteria, protozoa, and fungi ferment feedstuff into volatile fatty acids (VFA), the main energy source, while methanogens utilize fermentation by-products to produce methane. Milk fat contains several bioactive rumen-derived fatty acids (FA), including odd-chain FA (OCFA) and branched-chain FA (BCFA), important for maintenance of human health. The overarching dissertation goal was to determine which factors affect rumen methanogen and protozoal community structures and their metabolism products, while defining relationships between rumen microbiota and animal performance. Results presented contribute to the goals of providing new knowledge to dairy farmers, maintaining ruminant health, and enhancing bioactive FA in milk. The first objective was to use next-generation sequencing techniques to determine if lactation stage and dairy breed affect rumen methanogen and protozoal community structures and protozoa cell FA compositions in Jersey, Holstein, and Holstein-Jersey crossbred cows at 3, 93, 183, and 273 days in milk (DIM). A core methanogen community persisted by lactation stage and breed. At 3 DIM, methanogen 16S rRNA gene sequences formed distinct clusters apart from 93, 183, and 273 DIM, reflective of the dietary transition period post-partum. The starch-utilizing protozoal genus Entodinium, was more abundant in Holsteins than in Jerseys and Holstein-Jersey crossbred cows and positively correlated with milk yield. Jerseys had greater iso-BCFA contents in protozoa and milk and protozoa of the genus Metadinium. The second objective was to determine if supplementation of mixed cool-season grasses with annual forages (AF) alters the forage, microbial, and milk FA contents during typical periods of decreased pasture growth in Northeastern US. In short-term grazing (21d) of AF, ruminal VFA and major rumen-derived FA were not altered in bacterial and protozoal cells, suggesting little alteration of biohydrogenation and maintenance of ruminant health. In spring, milk contents of iso-15:0 and 17:0 per serving of whole milk were greater in control (CON)-fed cows, while contents of 12:0 and 14:0 per serving were greater in AF-fed cows. Contents of de novo FA and OCFA per serving of whole milk were greater in summer AF-fed cows than CON-fed cows, while total contents and BCFA did not differ, suggesting post-ruminal FA modifications in adipose tissue and the mammary gland. The third objective was to characterize and relate the rumen microbiota from CON- and AF-fed cows to animal performance. Rumen protozoal taxa were not altered, while less abundant bacterial taxa (\u3c 5%) were different in both periods. The protozoal genus Diplodinium was positively correlated with feed efficiency and milk fat yield. In spring, AF-fed cows had greater abundances of the methanogen species Methanobrevibacter millerae, whereas CON-fed cows had greater abundances of the methanogen species Methanobrevibacter ruminantium, potentially as a result of differences in substrate availability. In conclusion, the work presented identifies several factors that influence rumen microbiota, rumen microbial FA, and milk FA, while providing new information to dairy farmers, researchers, and consumers

The importance of long chain polyunsaturated fatty acids for fetal immune system maturation and function

Long chain polyunsaturated fatty acids (LCPUFAs) are important structural components in the cell membrane and a deficiency in these fatty acids have been linked to clinical complications. In addition, omega 3 and omega 6 LCPUFAs are biologically active substrates for the synthesis of regulators of inflammatory processes. Preterm neonates and uninfected neonates born to HIV-infected women (DNI) are susceptible to immune complications. These babies may have deficiencies in LCPUFAs, but the impact of this loss on maturation of the immune system is unknown. Cord blood mononuclear cell (CBMC) membrane fatty acid content was examined by gas chromatography (GC), and immunological profiles as defined by flow cytometry were compared between healthy term, preterm and DNI neonates as an indicator of immune deficiencies. This was the first study to examine the lipid profile of phosphatidylethanolamine (PE) and phosphatidylcholine (PC) fractions of CBMC membranes from preterm and DNI neonates. The LCPUFA composition of CBMCs was dominated by arachidonic acid (AA, 20:4co6) in the PE (34%) and PC fractions (15%) in term neonates (>37 weeks, n=9). Compared to term neonates, preterm (37 weeks, n= 9) CBMC levels of both omega-3 and omega-6 LCPUFAs were significantly lower. In addition term neonates had significantly higher numbers of CD4 and CD8 leukocytes than preterm and DNI neonates, including naïve and memory lymphocytes. These deficiencies in vital LCPUFAs and immune subset numbers, may contribute to the immature status of the preterm and DNI immune system, and ultimately to compromised immune function. A preliminary study was conducted into the function of immune cells in vitro in media supplemented with omega 3 and omega 6 LCPUFAs. The results, while not conclusive, suggested that LCPUFAs were beneficial to lymphocytes in culture