Dietary oxidized linoleic acid enhances liver cholesterol biosynthesis and secretion in rats.

Based on studies showing that excretion of cholesterol is elevated in rats fed oxidized linoleic acid, we hypothesized that cholesterol metabolism is enhanced under such oxidative stress. Liver cholesterol biosynthesis and secretion and fecal cholesterol excretion were studied in rats fed for 4 weeks diets containing 10% oxidized linoleic acid. Incubation of liver slices with 1-(14)C acetate and intraperitoneal injection of 5-(3)H-mevalonate showed the occurrence of enhanced hepatic cholesterol biosynthesis and elevated liver cholesterol secretion in animals subjected to oxidative stress. In addition, impaired liver cholesterol uptake was suggested. Higher levels of excreted cholesterol observed in the experimental animals were accompanied by augmented levels of liver phospholipids, primarily phosphatidylcholine, which most likely increased to enable the excessive cholesterol excretion. This study thus demonstrates that ingestion of oxidized lipids causes profound alterations in cholesterol metabolism.

Enrichment of poultry products with omega3 fatty acids by dietary supplementation with the alga Nannochloropsis and mantur oil.

Experiments were conducted to evaluate the efficiency of the microalga Nannochloropsis sp. (Nanno.), as a supplement to laying hens' diet, for the production of enriched eggs and meat with omega3 fatty acids (FA). Nanno. has a unique FA composition, namely, the occurrence of a high concentration of eicosapentaenoic acid (EPA; 20:5 omega3) and the absence of other omega3 FA. The effect of supplementing diets with Nanno. on omega3 FA levels in eggs, plasma, liver, and thigh muscle was compared to that of mantur oil, high in alpha-linolenic acid (LNA; 18:3 omega3). Nanno. is rich also in carotenoids, which may be useful for egg yolk pigmentation. The observed effect of Nanno. supplementation on yolk pigmentation was dose responsive, in both the rate of coloration and the color intensity. Addition of enzyme preparations (glucanase plus cellulase or glucanase plus pectinase) slightly elevated the yolk color score. The most prominent changes in the level of omega3 FA in egg yolk were evident when the diets were supplemented with 1% Nanno. or mantur lipid extracts. Levels of dietary algal meal (0.1-1.0%) had low and inconsistent effects on the level of yolk omega3 FA. Algal EPA is not accumulated in the liver or in the egg yolk; it is apparently converted and deposited as docosahexaenoic acid (DHA). LNA from mantur oil was partially converted to DHA, and both DHA and LNA were deposited in egg yolks and livers. It is suggested that the absence of DHA and EPA from thigh muscle is due to the small amount of dietary omega3 FA used in this work, compared to other studies, and to the possibility that in laying hens the egg yolk has a priority on dietary FA over that of muscles.

Availability and antiperoxidative effects of beta-carotene from Dunaliella bardawil in alcohol-drinking rats.

The present study demonstrated the high bioavailability and antiperoxidative capacity of the natural beta-carotene isomer mixture of Dunaliella bardawil compared with synthetic beta-carotene under alcohol-induced oxidative stress. Weanling rats were adapted to ethanol by increasing ethanol levels in their drinking water to 30% at 5% intervals per week; other rats received water with no added ethanol. One water-drinking group and one alcohol-drinking group with no dietary carotene were used as controls. Two water-drinking groups were supplemented with 1 g/kg diet beta-carotene either from Dunaliella or a synthetic source, and due to reduced food intake, two ethanol-fed groups received 2 g beta-carotene per kilogram of diet from each source. Following 3 months of ethanol consumption, both carotene sources were found to prevent ethanol-induced lipid peroxidation as expressed by the hepatic conjugated oxidized dienes level. However, in the algal-fed rats, hepatic carotene and vitamin A levels were higher. In addition to a lower performance of the group fed ethanol and synthetic beta-carotene, there were three deaths in this group.

All-trans beta-carotene is absorbed preferentially to 9-cis beta-carotene, but the latter accumulates in the tissues of domestic ferrets (Mustela putorius puro).

The algae Dunaliella bardawil and Dunaliella salina naturally contain large concentrations of all-trans and 9-cis beta-carotene (betaC). The purpose of this study was to compare the relative serum and tissue accumulation of all-trans and 9-cis betaC in ferrets fed different ratios of all-trans/9-cis betaC derived from two commercial sources, D. bardawil or D. salina (Betatene). Male ferrets (7 wk old) were fed carotene-free, pelleted diets for 27 d. Beginning on d 18, groups of ferrets (n = 6 or 7) received daily, one of six oral supplements varying in ratios of 9-cis and all-trans betaC mixed with approximately 1.0mL of Ensure. Four supplements containing 5.2-8.3 micromol total betaC were prepared from a 20% Betatene preparation, D. bardawil, a high-cis Betatene preparation, and Betatene further enriched in 9-cis betaC with all-trans betaC/9-cis betaC ratios of 2.2, 1.5, 0.6 and 0.4, respectively. Two control supplements, high and low betaC, were prepared from commercial betaC beadlets. The high control supplement had an all-trans/9-cis ratio of 19.0, whereas 9-cis betaC was not detected in the low supplement. On d 27, serum and tissues were obtained for HPLC analysis of betaC and its isomers. Analysis of livers showed that all-trans betaC was the primary isomer present, but 9-cis and other isomers were also detected in all groups. The hepatic all-trans/9-cis ratios were 5.9, 4.9, 2.5, 1.4, 52.2 and47.5, respectively, for the groups listed above. Lower amounts of all-trans and 9-cis betaC were found in kidneys compared with the liver, but ratios of all-trans/9-cis were not different among groups. Only trace amounts of 9-cis betaC were found in serum. These results demonstrate that the algae D. bardawil and D. salina provide a bioavailable source of betaC isomers, but, as in humans, absorption of 9-cis betaC is poor and any 9-cis betaC absorbed is apparently cleared by the liver.

Nutritional aspects of hydrogenated and regular soybean oil added to diets of broiler chickens.

The objective of the present study was to evaluate the effect of degree of saturation of fat incorporated into broiler diets on performance and body fatty acid (FA) profile. The various degrees of saturation were achieved by using regular soybean oil (SO) and hydrogenated soybean oil (HSO), mixed at different proportions. The work was carried out on commercial broilers (Experiment 1) and on lines of chickens divergently selected for high (HF) or low (LF) abdominal fat (Experiment 2). Daily BW gain and gain:feed ratio increased and the amount of feed intake decreased as the dietary fat saturation decreased. Digestibility of total fat and of each of the FA was lowest in the HSO group and reached maximal values when 23% or more of the added oil was SO. The AMEn values of the diets were almost parallel to fat digestibility. The performance of the HF and LF chickens was affected by the degree of saturation similarly to that observed for the commercial stock. The degree of dietary fat saturation had very little effect on saturated FA (C16:0 and C18:0) in body lipids, reduced the level of monoenoic FA (C16:1 and C18:1), and raised that of polyunsaturated FA (PUFA) (C18:2, C18:3, and C20:4). Monoenoic FA were higher, whereas PUFA were lower in the HF than in the LF line. The improved AMEn in diets containing unsaturated fat is probably due to higher fat digestibility, direct deposition of PUFA in body lipids, and lower lipogenesis, associated with lower heat production.

Enhanced peroxidation of proteins of the erythrocyte membrane and of muscle tissue by dietary oxidized oil.

Male weanling rats were fed on diets containing 10% of either oxidized or fresh (control) soybean oil for periods of 4 and 7 weeks. The ingestion of oxidized oil was found to reduce the content of free thiols in the erythrocyte membrane and to increase the amount of membrane-extractable spectrin. The level of reactive carbonyl groups was higher in the proteins of the ghosts and of the muscle tissue derived from the experimental animals. These alterations which are characteristic of peroxidized proteins, suggest that oxidative stress caused by oxidized dietary lipids may damage tissue proteins.

Dietary oxidized linoleic acid modifies lipid composition of rat liver microsomes and increases their fluidity.

The effect of dietary oxidized oil on the lipid composition, fluidity and function of rat liver microsomes was studied. Male growing rats were fed diets containing 10 g/100 g of a fresh (control) or oxidized (experimental) linoleic acid-rich preparation for 4 wk. High levels of fluorescent compounds and of thiobarbituric acid reactive substances indicated the occurrence of substantial lipid peroxidation in the microsomes of the experimental rats. The fluidity of the liver microsomes derived from rats fed the experimental diet was significantly higher than that of the membranes of the controls. This was due to profound differences in lipid composition of the liver microsomes, namely, a lower cholesterol to phospholipid molar ratio and a greater arachidonic acid content in the phospholipids of the rats fed the experimental diet. The fluidity differences were accompanied by greater activity of the microsomal enzymes, aldehyde dehydrogenase and NADPH cytochrome C reductase. The study demonstrated that ingestion of oxidized lipids caused profound alterations in membrane composition, fluidity and function. These alterations are likely to be associated with an enhanced cholesterol turnover, as indicated by the greater cholesterol excretion observed for the experimental rats.

Effect of added soyabean oil and dietary energy on metabolisable and net energy of broiler diets.

1. The 'extra caloric' effect of added soyabean oil, as reflected in improved body weight gain, food utilisation, metabolisable energy or net energy deposition in the body was determined. 2. Two diets were formulated to contain 12.1 MJ/kg, one with no added fat and the second with 30 g/kg soyabean oil. Addition of oil improved body weight gain by 6.9% (P < 0.05). Two other diets were formulated to contain 13.0 MJ/kg, one with 30 and one with 60 g/kg added soyabean oil bringing the total fat in the high energy, high fat diet to 84 g/kg. Addition of oil in this case improved weight gain by only 3.4% (ns). Addition of soyabean oil increased the apparent digestibility of total dietary fat and reduced that of starch. 3. The effect of soyabean oil supplementation on mash diets at both energy concentrations or to the pelleted diet (formulated to contain 12.1 MJ) on AMEn was consistently positive although not significant. Addition of soyabean oil improved net energy deposition in the body by 17% within the 12.1 MJ/kg diets, (30 g/kg soyabean oil addition) (P < 0.05), but was reduced by 2% (ns) within the 13.0 MJ/kg diets (60 g/kg soyabean oil addition). 4. Supplementing a pelleted diet formulated to contain 12.1 MJ/kg, with 30 g/kg soyabean oil, improved food utilisation (P < 0.05). The 'extra caloric' effect of added soyabean oil, defined as the beneficial effect of the oil above that predicted from its energy value, varied according to the parameter chosen to express this effect and was influenced by the concentration of added soyabean oil and the dietary energy.

In vivo antiperoxidative effect of 9-cis beta-carotene compared with that of the all-trans isomer.

It has been implied that the antiperoxidative activity of beta-carotene is important for its ability to prevent malignant and cardiovascular diseases. In vitro studies have shown that 9-cis beta-carotene is a better antioxidant than the all-trans isomer. In the present study the antiperoxidative biopotency of 9-cis beta-carotene was studied in vivo. For three weeks, weanling female rats were fed diets containing 10% fresh or oxidized soybean oil. The experimental diets were supplemented with beta-carotene at 1 g/kg with the synthetic all-trans isomer or with a carotene extract of Dunaliella bardawil containing 75% 9-cis beta-carotene. Both carotene sources prevented to the same extent hepatic and erythrocyte peroxidation associated with the consumption of oxidized oil. However, this beneficial effect was accompanied, in most of the groups, by a reduction in the hepatic carotene stores. Only in the animals fed Dunaliella extract combined with oxidized oil were the hepatic stores of beta-carotene and vitamin A maintained. The enhanced degradation of 9-cis beta-carotene observed in the livers of these animals might indicate that, like the effect observed under in vitro conditions, this isomer has a greater affinity toward free radicals and therefore might be a more efficient antioxidant than the all-trans form under in vivo conditions. The activity of glutathione peroxidase, glucose-6-phosphate dehydrogenase, and glutathione reductase as affected by the two carotene sources was also studied.

Incorporation of all-trans- or 9-cis-beta-carotene into mixed micelles in vitro.

Various studies suggest that all-trans- and 9-cis-beta-carotene are absorbed in the intestine to different extents. The present study was undertaken to evaluate the degree of in vitro incorporation of the two isomers into intestinal mixed micelles, which is an essential early step in the absorption process. The micelles were designed to simulate those present during fat digestion in the lumen of the human small intestine with respect to bile salts, lipids, pH and temperature. Solutions of all-trans- and 9-cis-beta-carotene at various ratios were added to the lipid mixture. A direct correlation was seen between the 9-cis-beta-carotene level in the mixture and the degree of total beta-carotene incorporation into micelles. An increased level of all-trans-beta-carotene in the system led to a decrease in the percentage of beta-carotene incorporated into the micelles. In contrast, when carotene mixtures enriched with the 9-cis isomer were used, an increase in the level of total carotene in the solution was accompanied by a constant or even enhanced carotene incorporation. The results indicate that the differences in the absorption of beta-carotene isomers might be the result of their different ability to be incorporated into the lipid micelles of the intestinal lumen. In addition, the results point toward the possibility that ingestion of 9-cis-beta-carotene by humans may increase carotene availability.

Antioxidant activity of 9-cis compared to all-trans beta-carotene in vitro.

In the present in vitro study we compared the antioxidative efficiency of the 9-cis to that of the all-trans beta-carotene. The 9-cis isomer was isolated from the alga Dunaliella bardawil. The experimental system consisted of 80 mM methyl linoleate, 4 mM azo-bis-2,2'-dimethylvaleronitrile (AMVN) as a free radical generating agent, and 200 microM beta-carotene (synthetic all-trans, 9-cis or a mixture of the 9-cis and all-trans isomers, having a ratio of 2.3). During the incubation at 37 degrees C the mixtures were analyzed for methyl linoleate hydroperoxides, total beta-carotene concentration, and its isomeric composition. The content of 9-cis beta-carotene in the various systems was negatively correlated to the level of the hydroperoxides accumulated, and positively related to the residual beta-carotene amount. The HPLC analysis of the system containing both isomers revealed a continuous decrease in the 9-cis to all-trans isomer ratio. The results suggest that the 9-cis beta-carotene has a higher antioxidant potency than that of the all-trans isomer and, therefore, it protects the methyl linoleate, as well as the all-trans isomer, from oxidation. This isomeric difference might be explained by the higher reactivity of cis, compared to trans, bonds.

9-Cis beta-carotene as a precursor of retinol isomers in chicks.

The possibility that 9-cis beta-carotene serves as a precursor for retinol in general, and particularly for 9-cis retinol, was studied in chicks, in which the 9-cis beta-carotene is preferentially accumulated. One week old chicks were fed a synthetic diet supplemented with 9-cis and all-trans beta-carotene mixtures, in which the 9-cis isomer content was 9, 19.1, 31.2, 40.8, 57.7 or 77.0%. It was found that an elevation in the dietary 9-cis beta-carotene level led to a decrease in the hepatic retinol stores, despite the increased level of total and 9-cis beta-carotene in the liver. HPLC chromatography revealed the presence of all-trans, 13-cis and 9-cis retinol in the livers. A high 9-cis beta-carotene diet brought about a significant, but very small, elevation in the part of the cis retinol isomers relative to the all-trans isomer. It was concluded that 9-cis beta-carotene has a low activity as a precursor of retinol in general. This decreased activity is not due to a low availability of the isomer to the enzymatic cleavage. It seems also that 9-cis beta-carotene is not an efficient precursor of 9-cis retinol.

Dietary lipids from marine unicellular algae enhance the amount of liver and blood omega-3 fatty acids in rats.

The nutritional effect of omega-3 (omega 3) polyenoic fatty acids, originating from marine unicellular algae or from fish oil, on the liver and blood lipids was studied in weanling rats fed for 2 weeks on control or experimental diets. Isolipid experimental diets containing either 10% marine microalgae or algal lipids or fish (capelin) oil substituting part (40%) or all of the soybean oil of the control diet. The algae employed were Nannochloropsis sp. or Isochrysis galbana, which are rich in eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), respectively. Cell disruption improved the digestibility of the Nannochloropsis biomass. Diets containing algal meal significantly reduced the relative abundance of arachidonic acid (AA) in the blood and liver lipids and caused a significant increase in the percentage of the omega 3 polyunsaturated fatty acids (PUFA). Feeding Nannochloropsis lipids resulted in a similar effect on the plasma and liver fatty acid pattern as that of a diet containing disrupted cells of Nannochloropsis biomass. In comparison, the response of the plasma and liver lipids to capelin oil was characterized by a further reduction in the abundance of AA and a significant elevation in the percentage of EPA and DHA. These differences are probably due to the variations in the fatty acid composition and not to the fact that omega 3 fatty acids are associated with different lipid classes in these lipid sources. Based on the present study, it is postulated that certain marine unicellular algae can be used as a nutritional source for omega 3 PUFA.

Dietary fish oil modulates the alkaline phosphatase activity and not the fluidity of rat intestinal microvillus membrane.

The effect of dietary fish (mackerel) oil on the function and fluidity of the intestinal microvillus membrane in 1-y-old rats was compared with that of coconut and soybean oils. The animals were fed diets containing 10% protein (derived from casein) and 15% oil. Intestinal microvillus membranes and RBC ghosts were isolated after a 6-wk feeding period and examined for fluidity by fluorescence polarization with 1,6-diphenyl-1,3,5-hexatriene. The functionality of the microvillus membrane was assessed by the activity of the intrinsic enzyme alkaline phosphatase. No differences in the fluidity were observed between the microvillus membranes or between the RBC ghosts derived from the various dietary groups. The alkaline phosphatase activity of the microvillus membrane was found to be the lowest in the membranes isolated from the fish oil-fed rats. In these membranes the contents of 20:5(n-3) and 22:6(n-3) fatty acids were higher than in the membranes derived from the coconut and soybean oil-fed animals. The cholesterol to phospholipid molar ratio was lower in the coconut oil-fed group than in the other two groups. It is suggested that the compensatory effect of the elevated cholesterol to phospholipid molar ratio on the membrane fluidity was such that no differences in the overall fluidity were detected. It is likely that the incorporation of these long chain fatty acids might have caused compositional changes in the lipid microenvironment of the enzyme. Such changes could alter the fluidity of these microdomains, thereby affecting the activity of the integral enzyme alkaline phosphatase.

Food restriction and membrane fluidity.

The fluidity of the erythrocyte membrane derived from growing rats raised under restricted food intake was found to be higher than the fluidity of the respective membranes from ad libitum fed animals. Considering the apparent relationship between the decrease in membrane fluidity and aging, the results point to the possible beneficial effects of food restriction at a young age.

Excessive dietary tryptophan enhances plasma lipid peroxidation in rats.

High plasma cholesterol levels and plasma lipid peroxidation are associated with atherosclerosis. The effect of excessive dietary tryptophan on plasma lipid peroxidation was studied in rats fed a diet containing soybean oil (control), as well as an atherogenic diet, containing coconut oil and cholesterol. Feeding the atherogenic diet resulted in a 5-fold increment in plasma cholesterol concentration with no significant effect of the tryptophan supplementation. The plasma obtained from the hypercholesterolemic rats exhibited a 67% increased lipid oxidation (measured as thiobarbituric acid reactive substances) in comparison to normocholesterolemic plasma. Dietary tryptophan supplementation increased plasma lipid peroxidation by 9 and 21% in the control and in the hypercholesterolemic animals, respectively. Similarly, the excessive dietary tryptophan enhanced macrophage cholesterol esterification rate by 40 and 38% following cell incubation with the plasma obtained from the control and from the hypercholesterolemic animals, respectively. Since tryptophan is the precursor of serotonin we have measured urine concentration of 5-hydroxyindoleacetic acid (5HIAA), the metabolite of serotonin, and found 22 and 118% elevation in 5HIAA in the tryptophan fed control and hypercholesterolemic rats, respectively. The direct effect of tryptophan and serotonin on in vitro lipid peroxidation was also studied. Low density lipoprotein (LDL) was peroxidized by incubation with copper ions in the presence of tryptophan or serotonin. Serotonin was shown to enhance LDL peroxidation whereas tryptophan had no effect on LDL peroxidation. We conclude that excessive dietary tryptophan may be atherogenic since it enhanced plasma lipid peroxidation in hypercholesterolemic rats and increased macrophage uptake of plasma cholesterol. These effects are probably associated with increased plasma concentration of serotonin following the consumption of a tryptophan supplemented diet.

Avocado oils and hepatic lipid metabolism in growing rats.

The effect of various avocado oils on liver metabolism was studied in growing female rats. The rats were fed diets containing 10% (w/w) avocado oil for 4 wk. In comparison with rats fed refined avocado oil obtained from cored fruit by centrifugal separation, rats fed unrefined avocado oil obtained by organic solvent extraction from intact fruit, or its unsaponifiable components, showed a significant increase in total liver lipogenesis as well as in phospholipid and triglceride synthesis. Rats fed avocado-seed oil exhibited enhanced [1-14C]acetate incorporation into total liver lipids but showed the same distribution of label in the three main lipid classes as that of rats fed refined avocado oil. In addition, a significant reduction of triglycerides and protein content of plasma very-low-density lipoprotein and high-density lipoprotein fractions was observed in rats fed avocado-seed oil as compared with rats fed refined oil. Electron micrographs suggested that the alterations in hepatic lipogenesis are related to the marked proliferation of the smooth endoplasmic reticulum, which is known to be associated with induction of enzymes involved with lipid biosynthesis. The differences between the animals fed seed oil and those fed the unrefined oils, in the distribution of label within the main lipid classes, indicate that more than one factor is involved in the alterations caused by these oils.

Decreased very low density lipoprotein fluidity in familial hypercholesterolemia.

The fluidity of lipoproteins from normolipidemic subjects and from familial hypercholesterolemic patients was investigated by fluorescence polarization. The fluorescent probe DPH (1,6-diphenyl-1,3,5-hexatriene) was incorporated into the lipoprotein fractions, and assessment of the fluidity pattern of each particle was determined by fluorescence anisotropy measurements over a temperature range of 10 to 40 degrees C. The very low density lipoprotein (VLDL) of the hypercholesterolemic patients was found to be considerably more rigid than the respective VLDL of normolipidemic subjects. Analysis of the constituents of the various lipoproteins suggested that the large difference in the fluidity between hypercholesterolemic and normal VLDL patients might be due to increased VLDL cholesterol/triglyceride, cholesterol/protein and cholesterol/phospholipid ratios, which were 10, 2 and 1.4 fold higher, respectively, in the hypercholesterolemic VLDL patients. Decreased VLDL fluidity in familial hypercholesterolemic patients may be of importance in the pathogenesis of their accelerated atherosclerosis.