Bioavailability of omega-3 essential fatty acids from perilla seed oil.

Increased dietary intake of fish oil omega-3 fatty acids, eicosapentanoic acid and docosohexanoic acid, and their precursor, alpha-linolenic acid (ALA), is associated with various health benefits. Enteric-coating (Entrox), which improves stability of omega-3 capsules, has been shown to facilitate fish oil absorption after chronic treatment. To assess the effect of Entrox coating on the short-term bioavailability of ALA administered in the form of ALA-rich Perilla seed oil, 12 healthy subjects (6 males and 6 females) received in a random order Entrox-coated and non-coated ALA formulations, each as a single 6g dose separated by a 3-week washout period. Measurements of plasma ALA concentrations from 0 to 24h showed no difference in ALA pharmacokinetics between the two formulations. However, significantly greater increases in plasma ALA levels from baseline to 24h were observed after ingestion of Entrox vs. non-coated product, suggesting a possible benefit of Entrox with long-term treatment.

Nitric oxide therapies in vascular diseases.

Endothelial dysfunction defined as the impaired ability of vascular endothelium to stimulate vasodilation plays a key role in the development of atherosclerosis and in various pathological conditions which predispose to atherosclerosis, such as hypercholesterolemia, hypertension, type 2 diabetes, hyperhomocyst (e) inemia and chronic renal failure. The major cause of the endothelial dysfunction is decreased bioavailability of nitric oxide (NO), a potent biological vasodilator produced in vascular endothelium from L-arginine by the endothelial NO synthase (eNOS). In vascular diseases, the bioavailability of NO can be impaired by various mechanisms, including decreased NO production by eNOS, and/or enhanced NO breakdown due to increased oxidative stress. The deactivation of eNOS is often associated with elevated plasma levels of its endogenous inhibitor, N(G) N(G)-dimethyl-L-arginine (ADMA). In hypercholesterolemia, a systemic deficit of NO may also increase the levels of low density lipoproteins (LDL) by modulating its synthesis and metabolism by the liver, as suggested by recent in vivo and in vitro studies using organic NO donors. Therapeutic strategies aiming to reduce the risk of vascular diseases by increasing bioavailability of NO continue to be developed. Cholesterol-lowering drugs, statins, have been shown to improve endothelial function in patients with hypercholesterolemia and atherosclerosis. Promising results were also obtained in some, but not all, vascular diseases after treatment with antioxidant vitamins (C and E) and after administration of eNOS substrate, L-arginine, or its cofactor, tetrahydrobiopterin (BH(4)). Novel strategies, which may produce beneficial changes in the vascular endothelium, include the use of natural extracts from plant foods rich in phytochemicals.

HDL-cholesterol-raising effect of orange juice in subjects with hypercholesterolemia.

BACKGROUND: Orange juice-a rich source of vitamin C, folate, and flavonoids such as hesperidin-induces hypocholesterolemic responses in animals. OBJECTIVE: We determined whether orange juice beneficially altered blood lipids in subjects with moderate hypercholesterolemia. DESIGN: The sample consisted of 16 healthy men and 9 healthy women with elevated plasma total and LDL-cholesterol and normal plasma triacylglycerol concentrations. Participants incorporated 1, 2, or 3 cups (250 mL each) of orange juice sequentially into their diets, each dose over a period of 4 wk. This was followed by a 5-wk washout period. Plasma lipid, folate, homocyst(e)ine, and vitamin C (a compliance marker) concentrations were measured at baseline, after each treatment, and after the washout period. RESULTS: Consumption of 750 mL but not of 250 or 500 mL orange juice daily increased HDL-cholesterol concentrations by 21% (P: < 0.001), triacylglycerol concentrations by 30% (from 1.56 +/- 0.72 to 2.03 +/- 0.91 mmol/L; P: < 0.02), and folate concentrations by 18% (P: < 0.01); decreased the LDL-HDL cholesterol ratio by 16% (P: < 0.005); and did not affect homocyst(e)ine concentrations. Plasma vitamin C concentrations increased significantly during each dietary period (2.1, 3.1, and 3.8 times, respectively). CONCLUSIONS: Orange juice (750 mL/d) improved blood lipid profiles in hypercholesterolemic subjects, confirming recommendations to consume >/=5-10 servings of fruit and vegetables daily.

Addition of arginine but not glycine to lysine plus methionine-enriched diets modulates serum cholesterol and liver phospholipids in rabbits.

Previous experiments from our laboratory showed that in rabbits fed an amino acid diet corresponding to 30% casein, enrichment of the diet with L-lysine and L-methionine caused a marked increase in serum total and LDL cholesterol levels as well as a substantial body weight loss. Both effects were partially prevented by supplementation with L-arginine. The present studies were designed to extend this earlier observation by assessing the role of different dietary amino acids in modulation of cholesterolemic responses and body weights. In the first experiment, the original lysine and methionine-enriched diet was supplemented with glycine in an attempt to modify methionine metabolism, and thus to reduce body weight loss. In addition, the mechanism of action of lysine and methionine was investigated by quantitation of major liver phospholipids. The results showed that glycine addition had no effect on weight loss or hypercholesterolemia, nor did it alter plasma levels of homocyst(e)ine, an intermediate in methionine metabolism. However, enrichment of the diet with lysine and methionine (with or without glycine) significantly increased liver levels of phosphatidylcholine and the ratio of phosphatidylcholine to phosphatidylethanolamine, apparently through increased enzymatic conversion. These changes were consistent with higher lipoprotein levels and thus may explain the hypercholesterolemia. A second experiment showed that similar effects on body weights and serum cholesterol could be obtained by adding lysine and methionine to a diet containing amino acids equivalent to only 15% casein, or 15% intact casein. This approach is more physiologic and also reduces the expense of experiments designed to study the effects of lysine and methionine in more detail.

Regulation of HepG2 cell apolipoprotein B metabolism by the citrus flavanones hesperetin and naringenin.

Our previous studies showed that replacing the drinking water of rabbits fed a casein-containing diet with either orange juice or grapefruit juice reduced serum low density lipoprotein cholesterol and hepatic cholesteryl ester concentrations. To determine whether the changes observed in rabbits were due to flavonoids present in the juices acting directly on the liver, the effects of hesperetin and naringenin on net apolipoprotein B (apoB) secretion by HepG2 cells were investigated. These flavanones dose-dependently reduced net apoB secretion by up to 81% after a 24 h incubation, while doses of 60 micrograms/mL reduced net apoB secretion by 50% after 4 h. Coincubation with the proteasome inhibitor, MG-132, did not alter the ability of the flavonoids to reduce net apoB secretion over 4 h, suggesting that the flavonoid-induced changes in apoB metabolism were not due to a direct increase in proteasomal activity. However, the flavonoids were unable to reduce net apoB secretion after 4 h in the presence of oleate, suggesting that these compounds may interfere with the availability of neutral lipids for lipoprotein assembly. Furthermore, our 14C-acetate-labeling studies showed a 50% reduction in cholesteryl ester synthesis in the presence of either flavonoid, which could account for the reduction in net apoB secretion caused by incubation with these compounds. These in vitro studies suggest that hesperetin and naringenin may, in part, reduce net apoB secretion by HepG2 cells by inhibiting cholesteryl ester synthesis and that these compounds are good candidates for further in vivo studies to determine whether they are responsible for the cholesterol-lowering properties of dietary citrus juices.

Role of dietary lysine, methionine, and arginine in the regulation of hypercholesterolemia in rabbits.

These experiments were conducted to see whether the hypercholesterolemia produced by a diet enriched in lysine (Lys) and methionine (Met) can be reproduced by feeding these amino acids separately, and whether dietary arginine (Arg) counteracts their hypercholesterolemic effects. Another aim was to investigate the mechanisms involved in modulations of serum cholesterol levels by these amino acids. The results of this study, which were in agreement with the results of earlier experiments in our laboratory, showed that feeding a low-fat, cholesterol-free, semipurified amino acid diet enriched with Lys + Met to rabbits caused a marked increase in serum total and low density lipoprotein cholesterol and apolipoprotein B levels, whereas a similar diet enriched in essential ketogenic amino acids (EketoAA) resulted in a more moderate increase in these parameters. Supplementing the diet with either Lys or Met alone was also less effective in inducing hypercholesterolemia than increasing levels of both amino acids. Dietary Arg partially counteracted the hypercholesterolemic effect of Lys + Met but not that of the EketoAA or of Lys and Met fed separately. The growth performance of rabbits fed the Lys + Met diet was inferior to that of those fed the other diets. Liver total phospholipid levels and the ratio of phosphatidylcholine to phosphatidylethanolamine were higher in rabbits fed the Lys + Met-enriched diet than in those animals fed a diet in which Arg was supplemented. In conclusion, our results indicate that high levels of both Lys and Met are needed to cause a maximum elevation of serum cholesterol and that the moderately antihypercholesterolemic effect of Arg is seen only when both amino acids are supplemented. They also suggest that these essential amino acids may affect cholesterol metabolism partly through alteration of liver phospholipids.

Hypocholesterolemic properties of nitric oxide. In vivo and in vitro studies using nitric oxide donors.

Previous results suggested that changes in the activity of nitric oxide (NO) can influence metabolism of apo B-containing lipoproteins. Therefore, we studied effects of exogenous NO donors and physiological NO precursors on metabolism of these lipoproteins. In rabbits, addition of 0.03% sodium nitroprusside (NaNP) to a semipurified, cholesterol-free, casein diet counteracted the elevation of LDL cholesterol induced by this diet but did not alter liver lipids after 4 weeks of feeding. In HepG2 cells, addition of nontoxic concentrations of another NO donor, S-nitroso-N-acetylpenicillamine (SNAP) to culture medium caused a dose-dependent reduction of medium apo B after 24 h. At the concentration 0.5 mM, SNAP significantly decreased medium apo B by 50% without altering total synthesis and secretion of proteins and without altering rates of cellular sterol synthesis. In cells incubated with L-arginine, reduction of medium apo B was not associated with increased NO production whereas in those exposed to N-OH-Arg medium apo B levels were not altered. We concluded that synthetic NO donors can reduce hypercholesterolemia by affecting apo B metabolism directly in the liver, via the sterol-independent mechanism.

Effects of substituting dietary soybean protein and oil for milk protein and fat in subjects with hypercholesterolemia.

OBJECTIVES: To determine whether, in individuals with hypercholesterolemia, substituting dietary soybean products for cows' milk products improves the plasma lipid profile and whether any change in the profile is due partially to soy oil. DESIGN: Randomized 3-treatment crossover trial. SETTING: Family practice clinics and an outpatient clinic in London, Ont. PARTICIPANTS: Seventeen healthy men and 17 healthy women with elevated plasma levels of total and low-density-lipoprotein (LDL) cholesterol and with normal plasma levels of triglycerides. INTERVENTIONS: Participants incorporated into their normal diet either 2% cows' milk products, soybean products or a combination of skim milk products and soy oil, each over period of 4 weeks, with 22-week wash-out periods. Plasma lipid profile, blood pressure and body weight were assessed after each dietary and wash-out period. OUTCOME MEASURES: Plasma levels of total and lipoprotein cholesterol, plasma levels of triglycerides, apolipoprotein B and A1 levels, blood pressure and plasma lipid peroxidation. RESULTS: The change in diet had no effect on body mass index, levels of apolipoproteins B and A1 and most plasma lipids. During the soybean period, the subjects' mean level of high-density-lipoprotein (HDL) cholesterol increased 9% (p < 0.04) and their mean LDL/HDL cholesterol ratio decreased 14% (p < 0.007). These effects were less pronounced during the skim milk/soy oil period. In the 24 subjects with the highest initial LDL cholesterol level and LDL/HDL cholesterol ratio, the mean LDL cholesterol level decreased 11% after the soybean period. In all subjects, changes in the LDL/HDL cholesterol ratio induced by a soybean diet were negatively correlated with the initial LDL/HDL cholesterol ratio and positively correlated with the initial HDL cholesterol level. CONCLUSIONS: In people with hypercholesterolemia, the plasma lipid profile improved after treatment with a soybean-product diet, and this improvement was partially due to soy oil. The degree of responsiveness was associated with initial risk factors for coronary artery disease.

Soy consumption and cholesterol reduction: review of animal and human studies.

Animal proteins such as casein are more hypercholesterolemic than soy protein or other plant proteins when fed to rabbits in low-fat, cholesterol-free, semipurified diets. A casein-amino acid mixture produces a hypercholesterolemia similar to that of casein. This appears to be mainly due to lysine and methionine, although other essential amino acids probably contribute to the effect. Arginine appeared to counteract the hypercholesterolemic effects of other essential amino acids. Soy protein gave a lower level of serum cholesterol in rabbits than did a soy protein-amino acid mixture, suggesting the presence of factors in soy protein that counteract the effects of hypercholesterolemic amino acids. Soy protein is also less hypercholesterolemic than casein in other animal species, particularly when the diet contains cholesterol, and substitution of soy protein for animal protein in the diet reduces the concentration of serum cholesterol in humans. This effect is somewhat variable but is generally greater in hypercholesterolemic than in normocholesterolemic subjects. The differing effects of dietary proteins on serum cholesterol concentrations in humans and in rabbits are primarily due to changes in LDL cholesterol, and the hypercholesterolemia produced by dietary casein is associated with down-regulation of hepatic LDL receptors.

Early lesion development in the aortas of rabbits fed low-fat, cholesterol-free, semipurified casein diet.

The initial endothelial morphological alterations and the development of raised, lipid-containing lesions in rabbit aortas were examined after 1 and 3 months on a casein-enriched, semipurified, cholesterol-free diet. The alterations were compared with those in rabbits fed soy-protein in the place of casein and with age-matched, chow-fed, control animals. Using immunohistochemistry macrophages, T-lymphocytes, and smooth muscle cells were identified in the lesions, and an expression of leukocyte adhesion molecules, VCAM-1, ICAM-1 and, occasionally, E-selectin was seen in sections of the aortas of casein-fed rabbits. The initial alterations in the endothelium appear to include evidence of endothelial injury and white blood cell adhesion. No evidence of extracellular liposome formation was observed. This model of atherogenesis is consistent with endothelial injury being an important component of diet-induced atherogenesis and has similarities to human atherosclerosis.

Hypercholesterolemic responses in rabbits to selected groups of dietary essential amino acids.

In rabbits, elevation of LDL cholesterol is produced by feeding a cholesterol-free, semipurified diet containing 30% casein amino acid mixture, but not by feeding the same diet containing 14.7% of the casein amino acid mixture, corresponding to a normal level of dietary protein. The hypercholesterolemic response was greater when all essential amino acids except arginine or all ketogenic essential amino acids (lysine, leucine, isoleucine, threonine, phenylalanine, tryptophan and glycine) were selectively fed at three times the normal level. In the present experiments, the same high levels of lysine, leucine and methionine produced a substantial hypercholesterolemia, addition of either isoleucine + threonine or isoleucine + valine did not enhance the effect, and a mixture of threonine, histidine, phenylalanine, tryptophan and glycine gave only a moderate response. A combination of lysine and methionine produced a greater effect than either lysine and leucine or leucine and methionine. Hypercholesterolemic diets containing high levels of lysine and leucine did not cause significantly greater plasma ketone bodies or free fatty acids. Differences in growth rates and ketogenic responses were not generally correlated with hypercholesterolemia.

Effect of high levels of selected dietary essential amino acids on hypercholesterolemia and down-regulation of hepatic LDL receptors in rabbits.

Earlier studies showed that the elevation of serum total and low density lipoprotein (LDL) cholesterol levels produced in rabbits by feeding high levels of a casein amino acid mixture in a cholesterol-free, semipurified diet was due primarily to the essential amino acids (EAA) in the mixture. Replacing all of the non-essential amino acids in the mixture by glutamic acid (45% EAA+Glu) had little effect on the hypercholesterolemia produced by the EAA. Experiments designed to identify the hypercholesterolemic EAA showed that (i) feeding high levels of ketogenic EAA only (45% EketoAA) gave a substantial but variable elevation of serum total and LDL cholesterol and (ii) feeding high levels of all EAA except arginine (45% EAA-Arg) gave a particularly strong hypercholesterolemic response. In rabbits fed the 45% EAA-Arg diet and to a lesser extent, in those fed the 45% EAA+Glu diet, EDTA-sensitive binding of 125I-LDL to hepatic membranes in vitro was reduced compared to a control, low-cholesterolemic group fed all essential and non-essential amino acids at a level corresponding to 14.7% casein, indicating that the hypercholesterolemia was associated with down-regulation of hepatic LDL receptors.

Effects of dietary protein on composition and metabolism of plasma lipoproteins in rabbits.

Changes in the concentration and composition of serum VLDL, LDL, and HDL were studied in rabbits transferred from Chow diets to cholesterol-free, semipurified diets containing casein or isolated soy protein. During the first week on the casein diet, there was a marked increase in LDL-cholesterol and these higher levels were maintained during the subsequent 3 weeks of the study. Similar but less marked changes were obtained with the soy protein diet. When the percent composition of the particles was determined, both VLDL and LDL had a higher proportion of cholesterol. Turnover studies indicated that the FCRs for radiolabelled VLDL and LDL were reduced in casein-fed animals compared to those fed soy protein. The elevated LDL levels in casein-fed rabbits were primarily due to a reduction in receptor-mediated catabolism of LDL-apo B. Receptor-independent removal in the two groups was similar. These studies show that the hypercholesterolemia in casein-fed rabbits, compared to those fed soy protein, is associated with cholesterol enrichment of LDL and impaired receptor-dependent removal of LDL-apo B.

Essential amino acids in relation to hypercholesterolemia induced in rabbits by dietary casein.

Rabbits fed a cholesterol-free, semi-purified diet containing 25% casein amino acids (25% AA) for 2 wk had significantly higher serum total and low density lipoprotein (LDL) cholesterol levels than animals fed the same diet containing 11.2% casein amino acids (11.2% AA). These results were similar to those obtained by feeding diets containing 27% and 12% casein, respectively. When rabbits were fed the 11.2% AA diet supplemented with essential amino acids to the 25% level (11.2% AA + essential AA), their LDL cholesterol level was significantly higher than that in animals fed the 11.2% AA diet supplemented with nonessential amino acids to the 25% level (11.2% AA + nonessential AA). LDL protein and phospholipid levels were significantly higher in rabbits fed the 25% AA diet than in those fed 11.2% AA and tended to be elevated in animals fed 11.2% AA + essential AA compared to those fed 11.2% AA + nonessential AA. Fecal excretion of bile acids and cholesterol was similar with all dietary regimens, and the level of liver lipids showed no correlation with the degree of hypercholesterolemia produced by dietary amino acid mixtures.

Compositional changes in serum lipoproteins during developing hypercholesterolemia induced in rabbits by cholesterol-free, semipurified diets.

Changes in the concentration and composition of serum very low density lipoprotein (VLDL), low density lipoprotein (LDL) and high density lipoprotein (HDL) were studied in rabbits transferred from chow diet to cholesterol-free, semipurified diets containing casein or isolated soy protein. The fat and fibre content of these diets was similar to that of chow but a higher protein level was used to enhance the hypercholesterolemia. During the first week on the casein diet, there was a marked increased in LDL-cholesterol, protein and phospholipids, and these higher levels were maintained during the subsequent 3 weeks of the study. Similar but less marked changes were obtained with the soy protein diet. The components of VLDL showed relatively little change after introduction of the diets to the animals. In both VLDL and LDL, the proportion of cholesterol increased and that of triglycerides decreased after 1 week on the casein diet and a similar trend was seen in HDL. The concentration of HDL-cholesterol showed little change but triglycerides, protein and phospholipids all tended to decline on both casein and soy protein diets.

Effects of cholesterol-free, semipurified diets containing different levels of casein or soy protein on distribution of cholesterol and protein among serum lipoproteins of rabbits.

Rabbits fed cholesterol-free, low-fat, semipurified diets have more cholesterol and protein in serum low density lipoprotein (LDL) relative to high density lipoprotein (HDL) than rabbits fed Chow diet. This difference was accentuated by a casein semipurified diet but was also observed with a soy protein diet even though the latter did not produce an elevation of serum cholesterol. To investigate the reason for these differences, the formulation of the semipurified diets was altered by reducing the level of protein from 27 to 16%, increasing the fat from 1 to 4% and the fiber from 5 to 13%, to correspond more closely to the proportions in Chow. With this formulation, the soy protein diet gave a lipoprotein pattern similar to that of Chow, whereas the casein diet produced a moderately elevated serum cholesterol level with more cholesterol in LDL than in HDL. When the protein in the newly formulated diets was increased back to 27%, the lipoprotein patterns reverted to those obtained with the original formula. In this case, soy protein-fed rabbits had moderately elevated serum cholesterol whereas casein-fed animals showed hypercholesterolemia. These results indicate that the altered lipoprotein pattern observed previously in rabbits fed semipurified diets is related to the high level of protein in those diets.