alpha-Linolenic acid does not contribute appreciably to docosahexaenoic acid within brain phospholipids of adult rats fed a diet enriched in docosahexaenoic acid

Adult male unanesthetized rats, reared on a diet enriched in both alpha-linolenic acid (alpha-LNA) and docosahexaenoic acid (DHA), were infused intravenously for 5 min with [1-(14)C]alpha-LNA. Timed arterial samples were collected until the animals were killed at 5 min and the brain was removed after microwaving. Plasma and brain lipid concentrations and radioactivities were measured. Within plasma lipids, > 99% of radioactivity was in the form of unchanged [1-(14)C]alpha-LNA. Eighty-six per cent of brain radioactivity at 5 min was present as beta-oxidation products, whereas the remainder was mainly in 'stable' phospholipid or triglyceride as alpha-LNA or DHA. Equations derived from kinetic modeling demonstrated that unesterified unlabeled alpha-LNA rapidly enters brain from plasma, but that its incorporation into brain phospholipid and triglyceride, as in the form of synthesized DHA, is < or = 0.2% of the amount that enters the brain. Thus, in rats fed a diet containing large amounts of both alpha-LNA and DHA, the alpha-LNA that enters brain from plasma largely undergoes beta-oxidation, and is not an appreciable source of DHA within brain phospholipids.     

Lipid peroxide formation in microsomes. Relationship of hydroxylation to lipid peroxide formation

1. Metal ion-chelating agents such as EDTA, o-phenanthroline or desferrioxamine inhibit lipid peroxide formation when rat liver microsomes prepared from homogenates made in pure sucrose are incubated with ascorbate or NADPH. 2. Microsomes treated with metal ion-chelating agents do not form peroxide on incubation unless inorganic iron (Fe(2+) or Fe(3+)) in a low concentration is added subsequently. No other metal ion can replace inorganic iron adequately. 3. Microsomes prepared from sucrose homogenates containing EDTA (1mm) do not form lipid peroxide on incubation with ascorbate or NADPH unless Fe(2+) is added. Washing the microsomes with sucrose after preparation restores most of the capacity to form lipid peroxide. 4. Lipid peroxide formation in microsomes prepared from sucrose is stimulated to a small extent by inorganic iron but to a greater extent if adenine nucleotides, containing iron compounds as a contaminant, are added. 5. The iron contained in normal microsome preparations exists in haem and in non-haem forms. One non-haem component in which the iron may be linked to phosphate is considered to be essential for both the ascorbate system and NADPH system that catalyse lipid peroxidation in microsomes.     

Dietary lipids modulate fatty acid composition, gamma glutamyltranspeptidase and lipid peroxidation levels of the epididymis tissue in mice

The purpose of this work was to analyze the effect of diets that contain several oils whose composition in fatty acids were different, on the kinetic parameters of the gamma-glutamyltranspeptidase (GGTP) and the lipoperoxidation of the epididymis because GGTP controls the level of the glutathione that is an molecule that regulates the level of oxidation protecting the maturation and survival of sperm in the lumen of the epididymis. The caput portion of the epididymis was chosen because the epithelium of this segment synthesizes GGTP. Weaned BALB-c mice were fed a commercial or semi-synthetic diet that contained 5% added olein. The mice were maintained on corn oil or fish oil diet for the first 4-8 months of age. The kinetic variables of the GGTP enzyme, analyzed by means of multiple regression analysis using dummy variables, showed that values were similar in olein and corn oil samples, whereas in samples from the fish oil fed group the enzyme behaved as that in animals maintained on commercial diets. Although there were no variations in maximum velocity (Vm) of the enzyme, the Km value, was greater (P < 0.0001) for the mice fed the olein and corn diets. These groups contained greater percentages of the monounsaturated fatty acids, palmitoleic (16:1 n-7) and oleic acid, 18:1 n-9. Similarly, the amount of lipid peroxidation was also greater in the olein and corn oil groups with respect to commercial and fish groups. The significant increment in Km of GGTP in the olein and corn groups was correlated with greater amount of monounsaturated fatty acids and lipid peroxidation in the epididymis. In conclusion, modifications of dietary lipid sources differentially modulated the epididymis tissue fatty acid profile, lipid peroxidation amounts, and the Km of GGTP. These effects may alter the metabolism of the natural substrate of GGTP, glutathione, a tripeptide with a powerful antioxidant activity, which is necessary in maintaining the oxidative state of the sperm microenvironment, thereby favoring maturation of the male gametes.     

Dietary alpha-linolenic acid deficiency in adult rats for 7 months does not alter brain docosahexaenoic acid content, in contrast to liver, heart and testes.

In adult rats, 22:6(n - 3) dietary deficiency does not affect brain membranes, but has a significant effect on some other visceral organs. 60-day-old male rats fed a diet containing sufficient amounts of both linoleic and alpha-linolenic acid were divided into three groups. One group continued the same diet; the second was fed a diet containing 2% sunflower oil, the third was fed 10% sunflower oil (sunflower oil contains linoleic acid, but trace amount of alpha-linolenic acid). Animals were killed different times after receiving the new diets (1 to 31 weeks). For animals fed the diets containing only sunflower oil, deficiency in cervonic acid content (DHA, docosahexaenoic acid, 22:6(n - 3)) was not detected in whole brain, myelin or nerve endings within 31 weeks. In contrast, this acid progressively declined in liver, heart and testes up to 3 weeks and remained nearly stable thereafter. In parallel to the reduction of cervonic acid content, 22:5(n - 6) content increased in liver and heart, but not in testes. It also increased in brain, nerve endings and myelin from week 3, 6 and, 9 respectively. These results suggest that brain cervonic acid is highly preserved or is maintained at the expense of other organs.     

Dietary supplementation with docosahexaenoic acid,but not with eicosapentaenoic acid,reduces host resistance to fungal infection in mice

The effect of dietary docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) on host resistance to Paracoccidioides brasiliensis infection was investigated. Mice fed palm oil supplemented with DHA showed reduced antifungal activity in the spleen and liver, as compared with mice fed palm oil or soybean oil without supplementation with DHA. Mice fed DHA-supplemented soybean oil also showed reduced antifungal activity in the liver, but the extent of reduction was less profound. This reduction in antifungal activity was not observed with EPA-supplemented palm or EPA-supplemented soybean oil. These results suggest that two factors, DHA and palm oil in combination, are involved in reducing the host resistance. DHA-enriched palm oil was also responsible for an increase in DHA concentration and a marked decrease in arachidonic acid content in the spleen and liver. However, this group did not show elevated spleen and liver phospholipid hydroperoxide levels compared with the other groups, excluding the possibility that the reduction in antifungal activity observed with DHA-enriched palm oil is due to acceleration of in vivo lipid peroxidation. Greater infection-induced increases in spleen and serum interferon-gamma concentrations were observed in mice fed DHA-enriched palm oil compared with the other groups.     

Dietary marine lipid does not alter susceptibility of (NZBxNZW)F1 mice to pathogenic microorganisms

Female (NZBxNZW)F1 mice were fed for one month with a diet in which the sources of fat were either melted beef tallow or fish oil, the latter regimen being associated with a marked decrease in the expression of auto-immune disease in these animals. To test whether or not this beneficial effect was associated with an increased risk of infection, animals fed each of these diets were challenged intraperitoneally with graded doses of four different classes of microorganisms: a gram positive bacillus that is an intracellular parasite (Listeria monocytogenes), an exotoxin-producing gram negative bacillus (Pseudomonas aeruginosa), a yeast (Candida albicans), and a herpes group virus (murine cytomegalovirus). There was no difference in the susceptibility of the animals fed the two different diets to any of these infections as shown by either determinations of the LD50 for each organism or by assessment of the times of survival for those animals that did succumb. We conclude that the beneficial anti-inflammatory effects of fish oil diets in these mice are not associated with an increased risk of infection with a variety of microbial agents.     

Effects of structured lipids containing eicosapentaenoic or docosahexaenoic acid and caprylic acid on serum and liver lipid profiles in rats

The physiological activity and effect on lipid metabolism of four types of structured lipids (SLs), that contain caprylic acid (C8) and either eicosapentaenoic (EPA) or docosahesaenoic acid (DHA), were evaluated in male Wistar rats fed experimental diets containing 7% (wt %) of each SL and 3% (wt %) soybean oil for 28 days. Control rats were fed a diet containing 10% (wt %) soybean oil. The relative perirenal adipose tissue weights of rats fed D-8-8 and 8-D-8 diets were significantly lower than those of other groups. We observed significantly lower serum cholesterol concentrations in rats fed SLs than those of control group over experimental period. The serum lipids concentrations in rats fed diets containing SLs were significantly lower P < 0.05) than those of soybean oil group. The fatty acid compositions of WAT did not reflect the structural differences in the triglyceride. These results suggest that the physiological effects of the SLs used in this study were due to the fatty acids rather than the structural specificity. Therefore, further study will be needed to ascertain the most desirable structural configuration.     

Dietary effects of eicosapentaenoic and docosahexaenoic acid esters on lipid metabolism and immune parameters in Sprague-Dawley rats

Sprague-Dawley rats were fed eicosapentaenoic (EPA) and docosahexaenoic acid (DHA) ethyl esters at the 2% level for 3 weeks to clarify their effects on immune functions. In the rats fed EPA or DHA, serum cholesterol, triglyceride, and phospholipid (PL) levels were significantly lower than those in the rats fed safflower oil. In PL fractions of serum, liver, lung, splenocytes, and peritoneal exudate cells (PEC), increases in linoleic and dihomo-gamma-linolenic acid contents and a decrease in arachidonic acid (AA) content were observed in the rats fed EPA or DHA. In addition, the EPA content increased in the rats fed EPA and DHA. In the rats fed EPA or DHA, a decrease of LTB4 productivity and an increase of LTBs productivity were observed in the PEC, in response to the treatment with 5 microM calcium ionophore A23187 for 20 min. The changes in leukotriene production were more marked in EPA-fed rats than in DHA-fed rats. These results suggest that dietary EPA affects lipid metabolism and leukotriene synthesis more strongly than DHA.     

Dietary carbohydrate influences tissue fatty acid and lipid composition in the copper-deficient rat

Fructose and copper have been shown independently to influence long chain fatty acid metabolism. Since fructose feeding exacerbates copper deficiency, their possible interaction with respect to tissue long chain fatty acid and lipid composition was studied. Weanling male Sprague-Dawley rats were given diets containing 0.6 or 6 mg/kg copper. The carbohydrate source (627 g/kg) was either fructose or corn starch. After 3 wk, fatty acid profiles and total lipids in heart and liver were analyzed. Copper-deficient rats fed fructose had more severe signs of copper deficiency than those fed starch, according to heart/body wt ratio, hematocrit, and liver copper content. The fatty acid composition of heart and liver triacylglycerol was significantly different between groups, but the changes did not correlate with the severity of copper deficiency. In heart, phosphatidylinositol and phosphatidylserine, arachidonic acid and docosapentaenoic acid (n-6) were increased 193 and 217%, respectively, p<0.05) in rats given the copper-deficient diet containing fructose. Changes in the long chain fatty acids in heart phospholipids may be related to the higher mortality commonly observed in rats fed a copper-deficient diet containing fructose.     

Hydrogen peroxide does not function downstream of salicylic acid in the induction of PR protein expression

The roles of salicylic acid (SA) and H₂O₂ in the induction of PR proteins in tobacco have been examined. Studies were conducted on wild-type tobacco and plants engineered to express a bacterial salicylate hydroxylase capable of metabolizing SA to catechol (SH-L plants). Wild-type and PR-1a—GUS-transformed plants express PR-1a following challenge with Pseudomonas syringae pathovar syringae , SA or 2,6-dichloro-isonicotinic acid (INA). In contrast, SH-L plants failed to respond to SA but did express PR-1a following INA treatment. H₂O₂ and the irreversible catalase inhibitor 3-amino-1,2,4-triazole (3-AT) were found to be weak inducers of PR-1a expression (relative to SA) in wild-type tobacco but were unable to induce PR-1a in SH-L plants, suggesting that the action of these compounds depends upon the accumulation of SA. A model has been proposed suggesting that SA binds to and inhibits a catalase inducing an increase in H₂O₂ leading to PR protein expression. Catalase activity has been measured in tobacco and no significant changes in activity following infection with P. syringae pv. syringae were detected. Furthermore, inhibition of catalase activity in vitro in plant extracts requires pre-incubation and only occurs at SA concentrations above 250 µM. Leaf disks pre-incubated with 1 mM SA do accumulate SA to these levels and PR-1a is efficiently induced but there is no apparent inhibition of catalase activity. It is also shown that a SA-responsive gene, PR-1a, and a H₂O₂-sensitive gene, AoPR-1, are both relatively insensitive to 3-AT suggesting that induction of these genes is unlikely to be due entirely to inhibition of an endogenous catalase.     

The extent of oxidative mitogenesis does not correlate with the degree of aldehyde formation of the T lymphocyte membrane

Chemical oxidation of T lymphocytes with periodate or the combined action of the enzymes neuraminidase and galactose oxidase (NAGO) results in T cell activation. The latter process includes the production of interleukin 2 (IL 2) and the induction of IL 2 receptors. Because membrane-bound aldehydes act in the transmission of the oxidative mitogenic signal, we designed a comparative study in human thymocytes and peripheral blood leukocytes in order to determine a possible correlation between the degree of the membrane aldehydes generated chemically or enzymatically and the extent of the resulting activation. The differences between periodate- and NAGO-induced aldehydes were demonstrated by flow cytometry of cells stained with a novel fluoresceinated hydrazide and by an electrophoretic procedure performed with biocytin hydrazide and 125I-streptavidin. In both cellular systems, periodate oxidation resulted in stronger formation of aldehydes than NAGO oxidation. However, the IL 2 receptor induced by NAGO formation and the resultant activation were significantly higher than those induced by periodate. The degree of aldehyde formation on peripheral blood leukocytes was also considerably higher than that of thymocytes, yet similar patterns of [3H]thymidine uptake were observed in the mitogenic assays of both cellular systems. The data indicate that no correlation exists between the extent of aldehyde formation and the degree of oxidative mitogenesis. It is thus suggested that relatively few (or maybe only one) membrane-bound aldehyde-containing molecules act in the transmission of the oxidative mitogenic signal.     

Fatty acid composition of leaf lipids determined after combined digestion and fatty acid methyl ester formation from fresh tissue

A procedure which uses hot methanolic HCl to digest fresh tissue and simultaneously convert the fatty acids of the leaf lipids to the corresponding methyl esters is described. Extraction of the fatty acid methyl esters into a small volume (0.3 ml) of hexane means that a sample for GLC analysis can be taken directly from the tube used for the digestion/methylation reaction. The procedure provides a fatty acid analysis which is comparable to that obtained by a more conventional technique involving separate extraction, saponification, and methylation steps, but the overall yield is reduced by 10-20%. The analysis can be made quantitative by including an internal standard with the tissue sample.     

Release of enzymes from lysosomes by irradiation and the relation of lipid peroxide formation to enzyme release

1. Acid phosphatase, cathepsin and beta-glucuronidase are released from rat-liver lysosomes by irradiation in vitro. Enzyme release is detectable after a dose of 1krad and increases with dose up to 100krads. 2. Maximum radiation effects were observed when the lysosomes were kept for 20hr. at 4 degrees or 20 degrees after irradiation. 3. An atmosphere of nitrogen considerably decreases enzyme release from lysosomes. 4. Enzyme release is enhanced by ascorbic acid and decreased by vitamin E. 5. Irradiation causes formation of lipid peroxides in lysosomes, and enzyme release increases with lipid peroxide formation. 6. It is suggested that lipid peroxide formation leads to rupture of the lysosome membrane and allows release of the contained hydrolytic enzymes.     

Neonatal dietary supplementation of arachidonic acid increases prostaglandin levels in adipose tissue but does not promote fat mass development in guinea pigs

The role of arachidonic acid (AA) on the development of adipose tissue is still controversial since its metabolites, i.e., prostaglandins, can either stimulate or inhibit preadipocyte differentiation in vitro. In the present study, we evaluated the effects of early postnatal supplementation of AA on body weight and adipose tissue development in guinea pigs. Male newborn guinea pigs were fed for 21 days (day 21) with diets (milk and pellet) supplemented (+AA) or not (-AA) with 1.2% (total fatty acids) AA. From day 21 to day 105 both groups were fed a chow diet. The 21-days-old +AA pups showed a twofold higher AA accretion in phospholipids associated with a two- to sixfold increase in several prostaglandins, such as 6-keto PGF(1alpha) (the stable hydrolysis product of PGI(2)), PGF(2alpha), PGE(2), and PGD(2) in adipose tissue, compared with the -AA group. No difference in fat pad and body weight, aP2, and leptin gene expression in adipose tissue, fasting plasma glucose, free-fatty acids, and triglyceride concentration was observed between groups at day 21 or day 105. These results show that dietary supplementation of AA during the suckling/weaning period increases prostaglandin levels in adipose tissue but does not influence early fat mass development in the guinea pig.     

Properties of lipid bilayer membranes made from lipids containing phytanic acid

Besides the preparation of phytanic acid (3,7,11,15-tetramethylhexadecylic acid) according to the Dumas-Stass reaction, the synthesis of four different lipids containing phytanic acid residues is described. Diphytanoyl phophatidylcholine was systhesized beginning from glycerylphosphorylcholine, whereas the other lipids, diphytanoyl phosphatidylethanolamine, diphytanoyl phosphatidylserine and monophytanoyl glyceride were prepared by total synthesis.Some properties of lipid bilayer membranes made from the lipids containing phytanic acid were investigated. The specific capacity of these membranes was measured. Its value of approximately 400 nF cm^?2 was found to be similar to the value of membranes from lipids with unbranched fatty acid residues. Charge pulse experiments were performed using dipicrylamine as a molecular probe of membrane structure. The results were discussed on the basis of a higher viscosity of the membranes from lipids containing phytanic acid residues compared with unbranched fatty acid residues.     

Dietary marine lipid does not alter susceptibility of (NZBxNZW) F1 mice to pathogenic microorganisms

Female (NZBxNZW) F₁ mice were fed for one month with a diet in which the sources of fat were either melted beef tallow or fish oil, the latter regimen being associated with a marked decrease in the expression of auto-immune disease in these animals. To test whether or not this beneficial effect was associated with an increased risk of infection, animals fed each of these diets were challenged intraperitoneally with graded doses of four different classes of microorganisms: a gram positive bacillus that is an intracellular parasite ( ), an exotoxin-producing gram negative bacillus ( ), a yeast ( ), and a herpes group virus (murine cytomegalovirus). There was no difference in the susceptibility of the animals fed the two different diets to any of these infections as shown by either determinations of the LD₅₀ for each organism or by assessment of the times of survival for those animals that did succumb. We conclude that the beneficial anti-inflammatory effects of fish oil diets in these mice are not associated with an increased risk of infection with a variety of microbial agents.     

Lack of phosphatidylethanolamine N-methyltransferase in mice does not promote fatty acid oxidation in skeletal muscle

Phosphatidylethanolamine N-methyltransferase (PEMT) converts phosphatidylethanolamine (PE) to phosphatidylcholine (PC) in the liver. Mice lacking PEMT are protected from high-fat diet-induced obesity and insulin resistance, and exhibit increased whole-body energy expenditure and oxygen consumption. Since skeletal muscle is a major site of fatty acid oxidation and energy utilization, we determined if rates of fatty acid oxidation/oxygen consumption in muscle are higher in Pemt^−/− mice than in Pemt^+/+ mice. Although PEMT is abundant in the liver, PEMT protein and activity were undetectable in four types of skeletal muscle. Moreover, amounts of PC and PE in the skeletal muscle were not altered by PEMT deficiency. Thus, we concluded that any influence of PEMT deficiency on skeletal muscle would be an indirect consequence of lack of PEMT in liver. Neither the in vivo rate of fatty acid uptake by muscle nor the rate of fatty acid oxidation in muscle explants and cultured myocytes depended upon Pemt genotype. Nor did PEMT deficiency increase oxygen consumption or respiratory function in skeletal muscle mitochondria. Thus, the increased whole body oxygen consumption in Pemt^−/− mice, and resistance of these mice to diet-induced weight gain, are not primarily due to increased capacity of skeletal muscle for utilization of fatty acids as an energy source.     

Dietary alpha-linolenic acid suppresses the formation of lysophosphatidic acid,a lipid mediator,in rat platelets compared with linoleic acid

Rats fed a high linoleic acid (LA, 18:2n-6) diet or a high alpha-linolenic acid (ALA, 18:3n-3) diet for 4 months after weaning. Platelets from the high-LA group contained more arachidonic acid (AA, 20:4n-6) and less eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3) compared with those from the high-ALA group. Incorporation of [32P]orthophosphate into platelet phospholipids was increased by thrombin-treatment, and was greater by ca. 30% in the high-LA group than in the high-ALA group both in the presence and absence of thrombin. The formation of [32P]lysophosphatidic acid (LPA), a lipid messenger, in [32P]orthophosphate-labeled platelets was increased 6.6-fold in the high-LA group and 4.1-fold in the high-ALA-group by thrombin-treatment. The formation of [32P] LPA in activated platelets was reduced by 35% in the high-ALA group.