α-Linolenate reduces the dietary requirement for linoleate in the growing rat.

BACKGROUND: We hypothesized that due to the absence of a dietary source of omega-3 fatty acids, the essential fatty acid (EFA) deficiency model leads to an overestimate of linoleic acid (LA) requirements. METHODS: over 7wk, young rats consumed an EFA diet containing either 0en% linoleate (0LA) and 0en% α-linolenate (0LNA) or a diet containing 0.5en% LNA plus one of seven levels of added LA (0.12-4.0en%; n=6/group). RESULTS: Rats consuming the 0LA-0LNA diet had the lowest final body weight, 34-68% lower LA and arachidonate in plasma and liver, 87% lower LA in epididymal fat, and an 8-20 fold higher eicosatrienoate in plasma, liver and muscle lipids. 0.5LNA completely prevented the lower growth and partly prevented the rise in eicosatrienoate seen in the 0LA-0LNA group. CONCLUSION: Providing dietary LNA at 0.5 en% reduces the rat's physiological requirement for LA by an estimated factor of at least four (0.5en% instead of 2en%). Since LA requirements in humans are also based on the same flawed model of EFA deficiency, it is plausible that they too have been overestimated and should therefore be reinvestigated.

n-3 Fatty acids modulate brain glucose transport in endothelial cells of the blood-brain barrier.

We have previously shown that glucose utilization and glucose transport were impaired in the brain of rats made deficient in n-3 polyunsaturated fatty acids (PUFA). The present study examines whether n-3 PUFA affect the expression of glucose transporter GLUT1 and glucose transport activity in the endothelial cells of the blood-brain barrier. GLUT1 expression in the cerebral cortex microvessels of rats fed different amounts of n-3 PUFA (low vs. adequate vs. high) was studied. In parallel, the glucose uptake was measured in primary cultures of rat brain endothelial cells (RBEC) exposed to supplemental long chain n-3 PUFA, docosahexaenoic (DHA) and eicosapentaenoic (EPA) acids, or to arachidonic acid (AA). Western immunoblotting analysis showed that endothelial GLUT1 significantly decreased (-23%) in the n-3 PUFA-deficient microvessels compared to control ones, whereas it increased (+35%) in the microvessels of rats fed the high n-3 PUFA diet. In addition, binding of cytochalasin B indicated that the maximum binding to GLUT1 (Bmax) was reduced in deficient rats. Incubation of RBEC with 15 microM DHA induced the membrane DHA to increase at a level approaching that of cerebral microvessels isolated from rats fed the high n-3 diet. Supplementation of RBEC with DHA or EPA increased the [(3)H]-3-O-methylglucose uptake (reflecting the basal glucose transport) by 35% and 50%, respectively, while AA had no effect. In conclusion, we suggest that n-3 PUFA can modulate the brain glucose transport in endothelial cells of the blood-brain barrier, possibly via changes in GLUT1 protein expression and activity.

Comparative bioavailability of dietary alpha-linolenic and docosahexaenoic acids in the growing rat.

Animal and human studies have indicated that developing mammals fed only alpha-linolenic acid (18:3n-3) have lower docosahexaenoic acid (22:6n-3) content in brain and tissue phospholipids when compared with mammals fed 18:3n-3 plus 22:6n-3. The aim of this study was to test the hypothesis that low bioavailability of dietary 18:3n-3 to be converted to 22:6n-3 could partly explain this difference in fatty acid accretion. For that purpose, we determined the partitioning of dietary 18:3n-3 and 22:6n-3 between total n-3 fatty acid body accumulation, excretion, and disappearance (difference between the intake and the sum of total n-3 fatty acids accumulated and excreted). This was assessed using the quantitative method of whole-body fatty acid balance in growing rats fed the same amount of a 5% fat diet supplying either 18:3n-3 or 22:6n-3 at a level of 0.45% of dietary energy (i.e., 200 mg/100 g diet). We found that 58.9% of the total amount of 18:3n-3 ingested disappeared, 0.4% was excreted in feces, 21.2% accumulated as 18:3n-3 (50% in total fats and 46% in the carcass-skin compartment), and 17.2% accumulated as long-chain derivatives (14% as 22:6n-3 and 3.2% as 20:5n-3 + 22:5n-3). Similar results were obtained from the docosahexaenoate balance (as % of the total amount ingested): disappearance, 64.5%; excretion, 0.5%; total accumulation, 35% with 30.1% as 22:6n-3. Thus, rats fed docosahexaenoate accumulated a twofold higher amount of 22:6n-3, which was mainly deposited in the carcass-skin compartment (68%). Similar proportions of disappearance of dietary 18:3n-3 and 22:6n-3 lead us to speculate that these two n-3 polyunsaturated fatty acids were beta-oxidized in the same amount.

Alterations in fatty acid composition of tissue phospholipids in the developing retinal dystrophic rat.

Alterations in lipid composition occur in the retinal pigment epithelium and photoreceptor cells of the Royal College of Surgeons (RCS) dystrophic rat, a model for inherited retinal degeneration. With respect to lipid composition of nonretinal tissues, the developmental timing of lipid alterations and the incidence of dystrophy are unknown. We determined the fatty acid composition in choline phosphoglycerides (ChoGpl) and ethanolamine phosphoglycerides (EtnGpl) in the brain, liver, and retina from dystrophic RCS rats and from their nondystrophic congenics (controls) at the ages of 3 and 6 wk. At 3 wk, the fatty acid compositions were specific to individual phospholipid classes without any difference between dystrophic and nondystrophic tissues. In plasma phospholipids, there was an age-related increase in the relative contents of monounsaturated and n-3 polyunsaturated fatty acids, with only minor differences between dystrophic and nondystrophic rats. At 6 wk, the fatty acid compositions in ChoGpl and EtnGpl from dystrophic brain and retina were significantly different from those of nondystrophics. The effect of strain on developmental changes in brain fatty acid composition was significant for 18:0 and 22:6n-3 in EtnGpl and for 16:0, 18:0, 18:1n-9, and 20:4n-6 in ChoGpl. The brain ChoGpl fatty acid composition in nondystrophic rats was similar at 6 wk to that of normal rats, and there were almost no postweaning changes in the dystrophics. In retinal phospholipids, the effect of dystrophy was to increase the 20:4n-6 content in EtnGpl and to decrease 22:6n-3 in ChoGpl. The 18:2n-6 and 22:6n-3 contents in dystrophic liver ChoGpl were also significantly affected, while no difference was observed in the EtnGpl fraction. The dystrophy affected the phospholipid fatty acid developmental changes in a tissue- and class-specific manner. Fatty acid metabolism could be selectively altered in neural and nonneural tissues of developing dystrophic RCS rats.

Phospholipid incorporation and metabolic conversion of n-3 polyunsaturated fatty acids in the Y79 retinoblastoma cell line.

The metabolic conversion of n-3 fatty acids was studied in the human Y79 retinoblastoma cell line. Cultured cells were exposed to increasing concentrations of either 18:3n-3, 22:5n-3, or 22:6n-3, and their phospholipid fatty acid composition was analyzed after 72 hr. Cells internalized the supplemental fatty acids and proceeded to their metabolic conversion. Supplemental 22:6n-3 was directly esterified into cell phospholipids, at levels typical for normal neural retinas (41% by weight of phosphatidylethanolamine fatty acids, and 24% of phosphatidylcholine fatty acids). In contrast, 18:3n-3 was mainly converted to 20:5n-3 and 22:5n-3, both of which appeared in cell phospholipids after exposure to low external concentrations of 18:3n-3 (10 microg/ml). Y79 cells can proceed to the metabolic conversion of 18:3n-3 through elongation and Delta6- and Delta5-desaturation. When cells were exposed to high external concentrations of 18:3n-3 (30 microg/ml), the supplemental fatty acid was directly incorporated, and its relative content increased in both phospholipid classes to the detriment of all other n-3 fatty acids. Cells cultured in the presence of 22:5n-3 did not incorporate 22:6n-3 into their phospholipids but did incorporate 20:5n-3 and 22:5n-3. The data suggest that Y79 cells can proceed to the microsomal steps of n-3 metabolism, involving elongation, desaturation, and chain shortening of 22C fatty acids. Although Y79 cells avidly used supplemental 22:6n-3 for phospholipid incorporation at levels typical for normal photoreceptor cells, they failed to match such levels through metabolic conversion of n-3 parent fatty acids. The terminal step of the very long-chain polyunsaturated fatty acid synthesis, consisting in Delta6-desaturation followed by peroxisomal chain shortening of 24C-fatty acids, could be rate-limiting in Y79 cells.

Blood lipid concentrations of docosahexaenoic and arachidonic acids at birth determine their relative postnatal changes in term infants fed breast milk or formula.

BACKGROUND: Factors other than dietary fatty acids could be involved in the variability observed in blood docosahexaenoate (22:6n-3) and arachidonate (20:4n-6) status in formula-fed infants. OBJECTIVE: We considered the 22:6n-3 and 20:4n-6 status at birth to be one of these factors and studied its influence on postnatal changes in term infants fed 4 different diets. DESIGN: The blood phospholipid composition was determined at birth and on day 42 of feeding in 83 term infants fed breast milk, nonsupplemented formula, or 2 different 22:6n-3-supplemented formulas. Relations between 22:6n-3 and 20:4n-6 status at birth and their relative postnatal changes, calculated by the difference between status at the end of the feeding period (6 wk of age) and at birth, were assessed. RESULTS: Postnatal changes in the plasma and erythrocyte phospholipids 22:6n-3 and 20:4n-6 were negatively related to their respective concentrations at birth (P < 0.01) and the slopes of the regression lines were not significantly affected by the type of milk ingested. Adjusted mean values for phospholipid 22:6n-3 in nonsupplemented-formula-fed infants and for 20:4n-6 in formula-fed infants decreased significantly more than they did in the other infant groups (P < 0.02). The status at birth and the type of milk ingested explained 33-64% and 7-47%, respectively, of the variability in postnatal changes. CONCLUSIONS: The status of 22:6n-3 and 20:4n-6 at birth in term infants is one of the major determinants of postnatal changes in these fatty acids. This finding indicates that research is required to characterize environmental, genetic, or both factors, which, in addition to maternal diet, could influence fatty acid status at birth.

n-3 and n-6 fatty acid enrichment by dietary fish oil and phospholipid sources in brain cortical areas and nonneural tissues of formula-fed piglets.

Sufficient availability of both n-3 and n-6 long-chain polyunsaturated fatty acids (LCPUFA) is required for optimal structural and functional development in infancy. The question has been raised as to whether infant formulae would benefit from enrichment with 20 and 22 carbon fatty acids. To address this issue, we determined the effect of fish oil and phospholipid (LCPUFA) sources on the fatty acid composition of brain cortical areas and nonneural tissues of newborn piglets fed artificially for 2 wk. They were fed sow milk, a control formula, or the formula enriched with n-3 fatty acids from a low-20:5n-3 fish oil added at a high or a low concentration, or the formula enriched with n-3 and n-6 fatty acids from either egg yolk- or pig brain-phospholipids. Both the fish oil- and the phospholipid-enriched formula produced significantly higher plasma phospholipid 22:6n-3 concentrations than did the control formula. The 22:6n-3 levels in the brain, hepatic, and intestinal phospholipids were significantly correlated with plasma values, whereas cardiac 22:6n-3 content appeared to follow a saturable dose-response. Feeding sow milk resulted in a much higher 20:4n-6 content in nonneural tissues than did feeding formula. Supplementation with egg phospholipid increased the 20:4n-6 content in the heart, red blood cells, plasma, and intestine in comparison to the control formula, while pig brain phospholipids exerted this effect in the heart only. The addition of 4.5% fish oil in the formula was associated with a decline in 20:4n-6 in the cortex, cerebellum, heart, liver, and plasma phospholipids, whereas using this source at 1.5% limited the decline to the cerebellum, liver, and plasma. Whatever the dietary treatment, the phosphatidylethanolamine 20:4n-6 level was 10-20% higher in the brain temporal lobe than in the parietal, frontal, and occipital lobes in the temporal lobe by administering the formula enriched with egg or brain phospholipids. In conclusion, feeding egg phospholipids to neonatal pigs increased both the 22:6n-3 content in the brain and the 20:4n-6 content in the temporal lobe cortex. This source also increased the 22:6n-3 levels in nonneural tissues with only minor alterations of 20:4n-6. These data support the notion that infant formulae should be supplemented with both 22:6n-3 and 20:4n-6 rather than with 22:6n-3 alone.

Docosahexaenoic acid concentrations in retinal phospholipids of piglets fed an infant formula enriched with long-chain polyunsaturated fatty acids: effects of egg phospholipids and fish oils with different ratios of eicosapentaenoic acid to docosahexaenoic acid.

Docosahexaenoic acid (DHA; 22:6n-3) is the major fatty acid in the phosphatidylethanolamine of photoreceptor cells. The supply of preformed DHA in milk may play an important role in early human visual development. We examined the effect of adding dietary DHA from yolk or fish oil on its accretion in the retina of newborn piglets fed artificially for 2 wk. DHA-enriched eggs from hens fed rapeseed oil and two fish oils with a high or low ratio of eicosapentaenoic acid (EPA; 20:5n-3) to DHA were used. The basic (conventional) formula contained (% by wt of total fatty acids) 17% linoleic acid (18:2n-6) and 1.3% alpha-linolenic acid (18:3n-3). The yolk-enriched formula also contained 0.5% arachidonic acid (AA; 20:4n-6) and 0.4% DHA. The fish-oil-enriched formulas contained either 0.3% EPA and 0.2% DHA (from salmon oil) or < 0.1% EPA and 0.3% DHA (low-EPA fish oil used at a low concentration), or 0.1% AA, 0.3% EPA, and 0.9% DHA (low-EPA fish oil used at a high concentration). The low-EPA fish oil used at a low concentration can supply the DHA required without increasing the EPA status but only the yolk-enriched formula allowed the artificially reared piglets to attain the same AA status in blood lipids as with sow milk feeding. The DHA concentration plateaued in the retina when it reached 7.5% by wt of total fatty acids in plasma phospholipids. Yolk phospholipids and fish oils are equally potent sources for supplying the highest retinal DHA concentration, which was found to be 41.7% by wt of total fatty acids in phosphatidylethanolamine (compared with 35% without supplementation). Inclusion of 0.2-0.3% DHA ensures maximal DHA accretion in the retina but cosupplementation with AA is necessary to achieve the status with maternal feeding in blood lipids and to prevent any possible imbalance between n-6 and n-3 fatty acids.

Long-term supplementation of culture medium with essential fatty acids alters alpha-linolenic acid uptake in Caco-2 clone TC7.

We investigated the influence of four different culture media: 20% fetal bovine serum (FBS), 5% FBS, 5% FBS supplemented with 10 mg x L(-1) linoleic acid (18:2(n-6)) or alpha-linolenic acid (18:3(n-3)) on alpha-linolenic acid apical uptake in clone TC7 of human intestinal Caco-2 cell line. Neither cellular viability nor cell monolayer integrity and permeability were altered by the four culture conditions. Our results show that the different culture media led to changes in alpha-linolenic acid maximal rate of uptake (Vmax) but did not alter the apparent transport constant (Km). Reducing FBS concentration from 20% to 5% increased significantly the rate of alpha-linolenic acid uptake, which was further increased by supplementation of the medium with 18:2(n-6) or 18:3(n-3). Supplementation with essential fatty acids led to a marked enrichment of brush-border membrane phospholipids in polyunsaturated fatty acids of the corresponding series and decreased significantly the levels of monounsaturated fatty acids. Saturated fatty acids, unsaturation index, and cholesterol/fatty acid ratios were unchanged. No clear relation could be established between the changes in membrane lipid composition and the alterations of alpha-linolenic acid uptake. These results indicate a weak influence of membrane lipid composition in the modulation of the uptake. Therefore, the increase of uptake following long-term supplementation of TC7 cells with essential fatty acids could be attributed to an increase of the expression of membrane protein(s) involved in the apical uptake of long-chain fatty acids. This remains to be established.

Metabolic conversion and growth effects of n-6 and n-3 polyunsaturated fatty acids in the T47D breast cancer cell line.

The incorporation and conversion of polyunsaturated fatty acids (PUFA) of n-3 and n-6 families were examined in the T47D breast cancer cell line in parallel with their effects on cell proliferation. In low serum-containing medium, PUFA exerted differential growth effects, depending both on their affiliation and unsaturation degree. The study of PUFA processing suggested that T47D cells are deficient in delta 6 and delta 4-desaturation activities whereas they can process to delta 5-desaturation. Thus, the PUFA growth effect on T47D cells appeared to be associated with a lack of desaturation.

Effect of early dietary deficiency in polyunsaturated fatty acids on two lectin binding sites in the small intestine of postweanling rats.

This study was designed to determine whether dietary lipids influence the development of intestinal cell glycosylation, in relationship to diet-induced changes in phospholipid fatty acid composition. The ability of two different lectins, wheat germ agglutinin (WGA) and Maackia amurensis agglutinin (MAA), to combine specifically with particular carbohydrate residues was used to investigate the surface characteristics of epithelial cells of rats fed different dietary lipids from birth to 6 weeks of age. Diets contained 5% (weight) peanut oil (PO), rich in n-6 fatty acids; salmon oil (SO), rich in n-3 fatty acids; hydrogenated palm oil (HPO), deficient in both n-6 and n-3 fatty acids or a PO and rapeseed oil (RO) mixture (PRO), the control diet. Pieces of jejunal and ileal villi were excised from postweanling rats and prepared for lectin histochemical study. Concurrently, epithelial cells were removed from jejunal and ileal segments for determining their phospholipid fatty acid compositions. Polyunsaturated fatty acid (PUFA) deficiency was evidenced in the HPO group by the appearance of eicosatrienoic acid (20:3n-9) in both jejunal and ileal phospholipids, which paralleled the decrease in arachidonic acid content. Accretion of 18:1n-9 and 20:3n-9 in cell phospholipids of group HPO was not sufficient to match the unsaturation level in rats fed nonhydrogenated vegetable oils (PRO, PO) or fish oil (SO). The lectin histochemical study showed that WGA strongly labelled the brush border membrane microvilli whereas binding of MAA was specific to goblet cells and mucus. Regardless of the type of diet, WGA binding was weaker in the ileum than in the jejunum. In comparison to all other groups, WGA-labelling of villi was less intense in the jejunum and disappeared almost completely in the ileum of HPO-fed rats. Although SO- and PO-fed rats had, respectively, very low and high ratios of n-6 to n-3 in their intestinal phospholipids, binding of WGA in both groups was not markedly different from that in the control (PRO). MAA-labelling was very intense in jejunal and ileal villi of n-3-fed (SO) rats, whereas it was strongly attenuated in the n-3- and n-6 deficient (HPO) group. These results suggest that intestinal glycosyltransferase activities involved in cell differentiation were altered relative to the overall unsaturation index of dietary fatty acids. Alterations of epithelial glycosylation mainly resulted from a drop in total n-6 and n-3 fatty acids, although it may be speculated that there is a specific effect of n-3 fatty acids.