Dietary oxysterols induce in vivo toxicity of coronary endothelial and smooth muscle cells.

Dietary cholesterol oxidation products (COPs) were reported to exhibit in vitro toxicity toward vascular cells. The aim of this study was to determine whether dietary COPs induce in vivo toxicity toward coronary arteries and to evaluate their effect on the coronary reactivity. Golden Syrian hamsters were fed either a normolipidic diet or a hyperlipidic diet with or without a mixture of COPs (1.4 mg/kg/day). At the end of the feeding periods, cardiac mitochondria and cytosol were prepared to determine the subcellular distribution of cytochrome c. Oxidative phosphorylation was evaluated with glutamate, pyruvate or palmitoylcarnitine as a substrate. The main coronary artery was examined all along its length by transmission electron microscopy (TEM). Plasma sterol concentrations were determined. Furthermore, at the end of the 3-month feeding period, the hearts were perfused at constant pressure by the Langendorff method. The endothelium-dependent reactivity to acetylcholine was evaluated. The myocardial sterol concentration was also estimated. After a 15-day diet with dietary COPs, a release of cytochrome c into the cytosolic fraction of the whole heart occurred, which indicated apoptosis of one or several types of cardiac cells probably induced by excess circulating cholestanetriol. The morphological data obtained by TEM after three months of diet suggested that mainly vascular cells (endothelial and smooth muscle cells) were damaged by dietary COPs, whereas cardiomyocytes appeared healthy. Furthermore, the mitochondrial oxidation of palmitoylcarnitine was reduced and that of pyruvate was increased, suggesting some maintenance of energy metabolism. This strengthens the hypothesis of apoptosis. Several changes in coronary reactivity suggesting an increased NO production were observed. In conclusion, dietary COPs triggered in vivo apoptosis of coronary cells through the release of cytochrome c in the cytosol. This toxicity was counterbalanced by an increased endothelium-dependent dilation.

New internal standard for quantitative determination of oxyphytosterols by gas chromatography.

A study was conducted to develop a new internal standard for the quantitative determination of oxyphytosterols. Tests on 5-androsten-3beta,17beta-diol; 5alpha-androstan-3beta,17beta-diol; 5-pregnen-3beta,20alpha-diol; and 5alpha-pregnan-3beta,20beta-diol showed that these compounds were not fully adequate. However, the compound 3beta,22-dihydroxy-20-homo-5-pregnene, synthesized in 4 steps, resulted in a promising internal standard, with a molecule similar to hydroxysterols; retention time as trimethylsilyl in gas chromatography comprised between 5alpha-cholestane and 7alpha-hydroxycholesterol; clear mass spectrum in electronic impact mass spectrometry, with several intense ions suitable for selected ion monitoring-mass spectrometry. Further studies are necessary to observe the behavior of these compounds during the entire analytical procedure.

Harmonization of methods for analysis of cholesterol oxides in foods--the first portion of a long road toward standardization: interlaboratory study.

A compilation of literature data on the content of cholesterol oxidation products (COP) in various food products and in blood demonstrates a large variation in content in products or tissues of very similar nature when analyzed in different laboratories according to a large number of methods. The lack of validated, internationally recognized methodology with published accuracy and precision has so far hindered such assessments. Hence an interlaboratory comparision of methodologies of COP analysis was undertaken on egg yolk powders (EYP), whole milk powders (WMP), skim milk powders (SMP), and lard (L). Each product type had one fresh sample (low) and one aged (high) in COP contents. A total of 17 sets of results on WMP, 15 on SMP and EYP, and 13 on L were compared. Overall results (mg/kg sample) varied extensively: Fresh EYP 0.72-265, aged EYP 2.51-361; fresh WMP 0.02-18.1, aged WMP 0.02-26.9; fresh SMP 0.02-6.51, aged SMP <0.01-6.51; fresh L 0.18-97, aged L 4.15-452. Some results were questioned, viz., those from laboratories not indicating substantial differences between samples "low" and "high" in total COP. Others were excluded because of lack of verification of identity of gas chromatographic peaks by mass spectrometry. Then a more narrow range of core results (mg/kg sample) was observed: Fresh EYP 5.69-29.5 sample, aged EYP 11.8-79.0; fresh WMP 0.12-1.76, aged WMP 1.17-13.7; fresh SMP <0.30-<1.21, aged SMP 0.30-2.26; fresh L 0.18-5.07, aged L 94.4-231. At a workshop discussing the results, numerous recommendations were made toward more reliable methodology for determination of COP in foods.

Oxyphytosterols are present in plasma of healthy human subjects.

The oxidised derivatives of phytosterols (oxyphytosterols) were identified in plasma samples from thirteen healthy human volunteers, using MS. All the samples contained noticeable quantities of (24R)-5beta,6beta-epoxy-24-ethylcholestan-3beta-ol (beta-epoxysitostanol) and (24R)-ethylcholestan-3beta,5alpha,6beta-triol (sitostanetriol) and also trace levels of (24R)-5alpha,6alpha-epoxy-24-ethylcholestan-3beta-ol (alpha-epoxysitostanol), (24R)-methylcholestan-3beta,5alpha,6beta-triol (campestanetriol) and (24R)-ethylch olest-5-en-3beta-ol-7-one(7-ketositosterol). The amounts of these oxyphytosterols in plasma varied from 4.8 to 57.2 ng/ml. There are two possibilities concerning the origin of these compounds. First, they could come from the small amounts of oxyphytosterols in food. Second, they could originate from the in vivo oxidation of phytosterols in plasma. Very few data actually exist concerning these compounds. Their identification in human samples suggests that further research is necessary in this field.

Incorporation of oxyphytosterols in tissues of hamster.

Oxyphytosterols (OPS) were fed to hamsters, at different concentrations, in order to observe their eventual incorporation into plasma, aorta, liver, kidneys and heart. The animals receiving the very high level (2500 ppm) presented 7beta-hydroxycampesterol, beta-epoxycampesterol, campestanetriol, 7-ketocampesterol, 7beta-hydroxysitosterol, beta-epoxysitosterol, sitostanetriol and 7-ketositosterol in all tissues. The same compounds were observed in the tissues of animals receiving 500 ppm of OPS in their diet, but with much lower levels. In hamsters fed 100 ppm of OPS, as well as in control animals, in most cases, the only observed OPS was sitostanetriol, which seems to be difficult to eliminate from the animal.

Sitostanetriol is not formed in vivo from sitosterol in the rat.

In a recent study, we observed some oxyphytosterols in the plasma of healthy human subjects. This experiment was effected in order to determine if these compounds could be formed in vivo from phytosterols. Rats were fed with a high level of phytosterols (1% of the diet) and they were compared to rats deprived of phytosterols, using triglycerides purified from phytosterols. Their plasma were analysed for the main oxyphytosterols. The results show that sitostanetriol and campestanetriol were not formed in vivo from phytosterols. Their levels decreased during the experiment. The diet origin is highly probable for the compounds identified in human plasma. In particular, it seems that the sitostanetriol is eliminated very slowly from the organism.

Formation of modified fatty acids and oxyphytosterols during refining of low erucic acid rapeseed oil.

Formation of trans fatty acids and cyclic fatty acid monomers was investigated during refining of low erucic acid rapeseed oil. The first steps of the refining process, that is, degumming, neutralization, and bleaching, hardly modified the fatty acid profile. In contrast, deodorization produced substantial quantities of trans fatty acids (>5% of total fatty acids) and small amounts of cyclic fatty acid monomers (650 mg of cyclic fatty acid monomers/kg of oil) when severe conditions (5-6 h at 250 degrees C) were used. Alpha-linolenic acid was the main precursor of cyclic fatty acid monomers. The influence of deodorization on the chemical composition of low erucic acid rapeseed oil was studied additionally. Whereas free fatty acids, peroxides, and tocopherols decreased, neither total polar compounds nor oxyphytosterols changed during deodorization. Oxyphytosterols were identified by GC-MS. Three oxyphytosterols not yet observed in oil were tentatively identified as 6beta-hydroxycampestanol, 6beta-hydroxysitostanol, and 6beta-hydroxybrassicastanol. Brassicasterol oxides were the most abundant oxyphytosterols.

Effects of dietary oxysterols on coronary arteries in hyperlipidaemic hamsters.

The aim of this study was to evaluate the effect of dietary oxysterols on coronary atherosclerosis and vasospasm. Golden Syrian hamsters were fed three diets with different lipid contents for 3 months: (1) a normolipidaemic diet containing 25 g corn oil-fish oil (4:1, w/w)/kg (group Low L); (2) a hyperlipidaemic diet composed of the normolipidaemic diet supplemented with 150 g lard+30 g cholesterol/kg (group High L); (3) a third diet, similar to the hyperlipidaemic diet, in which 4 g cholesterol/kg was replaced by a mixture of oxysterols (group High L+OS). The oxysterol mixture contained (g/kg): 5,6alpha-epoxycholesterol 211, 5,6beta-epoxycholesterol 179, 7alpha-hydroxycholesterol 67, 7beta-hydroxycholesterol (7betaOH) 185, 7-ketocholesterol (7 K) 235; and trace amounts of 7-hydroperoxycholesterols (approximately 30 g/kg). Atherosclerosis was evaluated by measuring myocardial Ca, oxysterols and acyl-CoA cholesterol acyl transferase (ACAT) activity; furthermore, coronary reactivity to sodium nitroprusside was measured and the morphology of coronary arteries was visualized by transmission electron microscopy. Coronary spasm was determined by evaluating reactivity to serotonin. Feeding the high-lipid diet (group High L) increased the plasma level of 7betaOH, 7 K and cholestanetriol. The presence of oxysterols in the diet (group High L+OS) further increased the concentrations of 7betaOH and 7 K in the plasma. However, as evidenced by myocardial Ca, ACAT activity and coronary reactivity to sodium nitroprusside, severe atherosclerosis did not develop during the 3-month diet. 7 K was increased in myocardial lipids of groups High L and High L+OS. Electron microscopy did not show the development of atherosclerosis in group High L, whereas vascular wall thickening, endothelial damage and smooth muscle cell proliferation and migration occurred when oxysterols were present in the food. Serotonin induced exacerbated coronary vasoconstriction in group High L that was completely reversed by dietary oxysterols. In conclusion, dietary oxysterols exhibit anti-spasmodic properties, but they cannot be used as agents against excess dietary lipid-induced coronary spasm because of their atherogenic properties.