beta-sitosterol: esterification by intestinal acylcoenzyme A: cholesterol acyltransferase (ACAT) and its effect on cholesterol esterification

Rabbits were fed either 10% coconut oil, 10% coconut oil and 1% beta-sitosterol, 10% coconut oil and 1% cholesterol, or 10% coconut oil and 1% beta-sitosterol plus 1% cholesterol for 4 weeks. Microsomal membranes from intestines of animals fed the 1% beta-sitosterol diet had 48% less cholesterol and were enriched twofold in beta-sitosterol compared to membranes from animals fed the coconut oil diet alone. Acylcoenzyme A:cholesterol acyltransferase (ACAT) activity in jejunum and ileum was decreased significantly in animals fed the plant sterol alone. In membranes from animals fed 1% beta-sitosterol and 1% cholesterol, beta-sitosterol content increased 50% whereas cholesterol was modestly decreased compared to their controls fed only cholesterol. Intestinal ACAT was unchanged in the animals fed both sterols when compared to their controls. beta-Sitosterol esterification was determined by incubating intestinal microsomal membranes with either [(14)C]beta-sitosterol-albumin emulsion or [(14)C]beta-sitosterol:dipalmitoyl phosphatidylcholine (DPPC) liposomes to radiolabel the endogenous sterol pool. Oleoyl-CoA was then added. The CoA-dependent esterification rate of beta-sitosterol was very slow compared to that of cholesterol using both techniques. An increased amount of endogenous microsomal beta-sitosterol, which occurs in animals fed 1% beta-sitosterol, did not interfere with the stimulation of ACAT activity secondary to cholesterol enrichment of the membranes. Enriching microsomal membranes three- to five-fold with beta-sitosterol did not affect ACAT activity. Freshly isolated intestinal cells were incubated for 1 hour with [(3)H]oleic acid and beta-sitosterol:DPPC or 25-hydroxycholesterol:DPPC. Incorporation of oleic acid into cholesteryl esters did not change in the presence of beta-sitosterol but increased fourfold after the addition of 25-hydroxycholesterol. We conclude that the CoA-dependent esterification rate of cholesterol is at least 60 times greater than that of beta-sitosterol. Membrane beta-sitosterol does not interfere with nor compete with cholesterol esterification. Inadequate esterification of this plant sterol may play a role in the poor absorption of beta-sitosterol by the gut.-Field, F. J., and S. N. Mathur. beta-Sitosterol: esterification by intestinal acylcoenzyme A:cholesterol acyltransferase (ACAT) and its effect on cholesterol esterification.     

Some aspects of mechanism of inhibition of cholesterol absorption by beta-sitosterol

Mixed bile salt micelle solubilized either cholesterol or beta-sitosterol to a comparable extent. When added simultaneously, beta-sitosterol restricted the micellar solubility of cholesterol. beta-Sitosterol also reduced the cholesterol content in the aqueous (micellar) phase of the intestinal contents of rats, the extent of reduction being comparable with that observed in vitro. The intestinal uptake of cholesterol in vivo was equivalent to the micellar incorporation of cholesterol both in vitro and in vivo. beta-Sitosterol had no inhibitory effect on cholesterol absorption from the micellar solution in jejunal loops in situ, whereas the rate of beta-sitosterol uptake was only about one-fifth that of cholesterol. The intestinal uptake of beta-sitosterol intubated into the stomach of rats was about one-fifth that of cholesterol. The intestinal brush-border membrane discriminated these sterols. These results suggest that the restriction of the micellar solubility of cholesterol, rather than the inhibition of uptake from brush-border membrane, is the major determinant for the interference of beta-sitosterol with cholesterol absorption.     

Influence of beta-sitosterol on the fibrinolytic potential in rabbits

Long-term treatment of rabbits with beta-sitosterol (40 mg/kg over 3 months) caused an increased fibrinolytic activity in blood, an increased fibrinolytic capacity and an enhanced plasminogen activator activity in tissue of lungs and kidneys. The 3-months lasting beta-sitosterol administration did not influence the content of plasminogen activator inhibitor, plasminogen, alpha 2-antiplasmin, antithrombin III and fibrinogen.     

Enhancement of beta-sitosterol transformation in Mycobacterium vaccae with increased cell wall permeability.

Mycobacterium vaccae exposed to compounds which are known to disorganise the cell wall composition and architecture (protamine, glycine) showed increased specific activity in beta-sitosterol biotransformation to androstene derivatives, intennediates in the production of most medical steroids. GC/MS analysis of free lipid fatty acids revealed higher content of unsaturated compounds, mainly C16:1 and C18:1 in protamine- and glycine-treated cells than that in control cells, which seems to change the permeability features of the cell wall barrier, facilitating hydrophobic beta-sitosterol diffusion.     

Isolation and identification of new constituents in milk fat

After removal of cholesterol on a digitonin column, the unsaponifiable matter of milk fat was examined for alcoholic substituents. Derivatization with pyruvic acid chloride 2,6-dinitrophenylhydrazone and fractionation of the derivatives gave four main fractions. The second, the hexane-benzene fraction, was shown by thin-layer chromatography to have a mobility similar to many common sterols. The hexane-benzene fraction was saponified and gave rise to free alcohols, which were then analyzed on a combination gas-liquid chromatograph-mass spectrometer. Dihydrolanosterol, previously unreported in milk fat, and lanosterol, previously identified but never confirmed, were characterized. The sterols which were precipitated on the digitonin column, were removed, and by the use of the combination gas-liquid chromatograph-mass spectrometer beta-sitosterol was identified. In addition, lanosterol and dihydrolanosterol were isolated from the unsaponifiable matter by chromatography on Florisil.     

The sterols of Astragalus species from Iran: GLC separation and quantification

This paper presents the content and composition of free sterols in a number of Astragalus species from Iran. Free sterols of roots and seeds were analysed quantitatively and qualitatively by gas-liquid chromatography. Four major 3-beta-hydroxysterols (cholesterol, campesterol, stigmasterol and beta-sitosterol) were identified in roots of 18 species and in seeds of five species of Astragalus. beta-Sitosterol was the main free sterol in both roots and seeds. The cholesterol content in seeds was higher than that in roots. Trace amounts of cholesterol were detected in roots of only four species. beta-Sitosterol accounted for 34.4-69.2 and 76.8-81.8% of the total lipid content in roots and seeds, respectively. The beta-sitosterol content in roots and seeds of Astragalus species varied from 6.3 to 35.7 and 93.3 to 164.5mg/100g dry weight, respectively. Total free sterol content in seeds varied from 112.09 to 204.04mg/100g dry weight, significantly higher than those in roots (7.5 to 43.4mg/100g dry weight). The chemotaxonomic values of free sterols and total lipid profiles of some of the representative species are discussed.     

Steroidal constituents and anti-inflammatory activity of the horse chestnut (Aesculus hippocastanum L.) bark

The content and the composition of sterols in the bark of the horse chestnut, Aesculus hippocastanum L., have been examined. Stigmasterol and alpha-spinasterol were, with the beta-sitosterol, the most abundant sterols. The petrol extract from bark shows anti-inflammatory activity.     

Composition of plasma fatty acids and non-cholesterol sterols in anorexia nervosa

Anorexia nervosa is a model of simple starvation accompanied by secondary hyperlipoproteinemia. The pattern of plasma fatty acids influences the levels of plasma lipids and lipoproteins. The concentration of plasma lathosterol is a surrogate marker of cholesterol synthesis de novo, concentrations of campesterol and beta-sitosterol reflect resorption of exogenous cholesterol. The aim of the study was to evaluate fatty acids in plasma lipid classes and their relationship to plasma lipids, lipoproteins, cholesterol precursors and plant sterols. We examined 16 women with anorexia nervosa and 25 healthy ones. Patients with anorexia nervosa revealed increased concentrations of total cholesterol, triglycerides, HDL-cholesterol, campesterol and beta-sitosterol. Moreover, a decreased content of n-6 polyunsaturated fatty acids was found in all lipid classes. These changes were compensated by an increased content of monounsaturated fatty acids in cholesteryl esters, saturated fatty acids in triglycerides and both monounsaturated and saturated fatty acids in phosphatidylcholine. The most consistent finding in the fatty acid pattern concerned a decreased content of linoleic acid and a raised content of palmitoleic acid in all lipid classes. The changes of plasma lipids and lipoproteins in anorexia nervosa are the result of complex mechanisms including decreased catabolism of triglyceride-rich lipoproteins, normal rate of cholesterol synthesis and increased resorption of exogenous cholesterol.     

Effect of phytosterols on cholesterol metabolism and MAP kinase in MDA-MB-231 human breast cancer cells

Epidemiological studies suggest that dietary phytosterols may offer protection form some types of cancer including breast cancer. In an attempt to investigate the mechanism by which phytosterols offer this protection, we investigated the effect of the two most common dietary phytosterols, beta-sitosterol and campesterol, on the mevalonate and MAP Kinase (MAPK) pathways in MDA-MB-231 cells. These pathways play a role in cell growth and apoptosis. MDA-MB-231 cell line was used in this study since it is a hormone-insensitive tumor cell line which represents the majority of advanced breast cancer cases. Cells grown in the presence of 16 microM beta-sitosterol or campesterol for 3 days exhibited a 70% and 6% reduction in cell growth, respectively, while cholesterol treatment had no effect on growth as compared to the control. Studies investigating the effect of sterol supplementation on the relative and total sterol composition of cells, showed that cells supplemented with cholesterol contained 23% more cholesterol than the control. Cells supplemented with campesterol had almost one-half the cholesterol of controls but accumulated campesterol to account for 40% of the total sterols. In the case of cells supplemented with beta-sitosterol, cells had only 25% of their sterols as cholesterol and the rest was in the form of beta-sitosterol. All sterols tested equally inhibited de novo cholesterol synthesis using 14C-acetate as substrate. beta-Sitosterol supplemented cells had reduced cholesterol synthesis when using 3H-mevalonolactone as substrate, which suggests that the inhibition in this pathway is downstream of mevalonate where processes such as isoprenylation of proteins may take place. Mevalonate supplementation to cells treated with beta-sitosterol did not completely correct the observed growth inhibition by beta-sitosterol. There was no effect of sterols on the concentrations of both low (21-26 kDa) or high (44-74 kDa) molecular weight isoprenylated proteins in these cells. On the other hand, both the quantity and activity of MAPK was elevated in the cells supplemented with beta-sitosterol. These data suggest that the down regulation of cholesterol synthesis from mevalonate and stimulation of the MAPK pathway may play roles in the inhibition of MDA-MB-231 cell growth by beta-sitosterol.     

Role of hydrophilic bile acids and of sterols on cholelithiasis in the hamster

The effect of various dietary additions such as cholesterol, beta-sitosterol, bile acids, and bile acid analogs on gallstone formation was studied in the hamster. Gallstones were formed in 50% of the animals fed a high glucose, fat-free diet. Administration of 0.2% cholesterol or 1% beta-sitosterol had no effect on the incidence of gallstones. Ursodeoxycholic acid (0.5%) and its analog ursodeoxy-oxazoline [2-(3 alpha, 7 beta-dihydroxy-24-nor-5 beta-cholanyl)-4,4-dimethyl-2- oxazoline] were ineffective in preventing gallstones. Hyodeoxycholic acid and hyodeoxy-oxazoline [2-(3 alpha,6 alpha-dihydroxy-24-nor-5 beta-cholanyl)-4,4-dimethyl-2- oxazoline] at the same dosage effectively prevented gallstones, while the trihydroxy bile acid, hyocholic acid, was not effective. Of all the dietary regimens tested, only hyodeoxycholic acid significantly lowered serum cholesterol. The lithogenic diet produced a five-fold increase in hepatic HMG-CoA reductase activity; this activity was not affected by dietary cholesterol or beta-sitosterol. Hyodeoxycholic acid and hyocholic acid feeding increased the reductase activity by an additional 50% while the other bile acids had no effect. beta-Sitosterol doubled the cholesterol 7 alpha-hydroxylase activity whereas hyodeoxy-oxazoline lowered it. Hyodeoxycholic acid-fed animals had significantly lower cholesterol absorption than the animals on the lithogenic diet alone. Biliary cholesterol content increased dramatically in the animals fed the lithogenic diet and was increased still further by ursodeoxycholic acid, hyodeoxycholic acid, and hyodeoxy-oxazoline. These data show that hyodeoxycholic acid and hyodeoxy-oxazoline do not prevent gallstones by inhibiting hepatic cholesterol synthesis or biliary cholesterol secretion.     

beta-Sitosterol modulates antioxidant enzyme response in RAW 264.7 macrophages

Uncontrolled production of reactive oxygen species contributes to the pathogenesis of diseases such as cancer and cardiovascular disorders. Olive oil exerts remarkable preventive effects on the development of these diseases, which may be due to the action of various components of the olive oil. In fact, several findings suggest that minor components, like phytosterols such as beta-sitosterol, are responsible, at least in part, for these beneficial effects. Our results show that beta-sitosterol reverts the impairment of the glutathione/oxidized glutathione ratio induced by phorbol esters in RAW 264.7 macrophage cultures. These data can be correlated with the increase in manganese superoxide dismutase and glutathione peroxidase activities and the decrease in catalase activity. We also demonstrate that the effects of beta-sitosterol on antioxidant enzymes depend on the estrogen/phosphatidylinositol 3-kinase pathway.     

Sphingolipids from Conyza canadensis

Sphingolipid 1 and its corresponding beta-D-glucopyranoside derivative 2 have been isolated from the ethylacetate fraction of Conyza canadensis along with beta-sitosterol 3, stigmasterol 4, beta-sitosterol 3-O-beta-D-glucoside 5 and harmine 6, reported for the first time from this species. The structures of 1 and 2 were elucidated through spectroscopy including two-dimensional NMR.     

Effect of resveratrol and beta-sitosterol in combination on reactive oxygen species and prostaglandin release by PC-3 cells

The objective of this project was to identify some possible mechanisms by which two common phytochemicals, resveratrol and beta-sitosterol, inhibit the growth of human prostate cancer PC-3 cells. These mechanisms include the effect of the phytochemicals on apoptosis, cell cycle progression, prostaglandin synthesis and the production of reactive oxygen species (ROS). Prostaglandins have been known to play a role in regulating cell growth and apoptosis. PC-3 cells were supplemented with 50 microM resveratrol or 16 microM beta-sitosterol alone or in combination for up to 5 days. Phytochemical supplementation resulted in inhibition in cell growth. beta-Sitosterol was more potent than resveratrol and the combination of the two resulted in greater inhibition than supplementation with either alone. Long-term supplementation with resveratrol or beta-sitosterol elevated basal prostaglandin release but beta-sitosterol was much more potent than resveratrol in this regard. beta-Sitosterol was more effective than resveratrol in inducing apoptosis and the combination had an intermediate effect after 1 day of supplementation. Cells supplemented with resveratrol were arrested at the G1 phase and at the G2/M phase in the case of beta-sitosterol while the combination resulted in cell arrest at the two phases of the cell cycle. beta-Sitosterol increased ROS production while resveratrol decreased ROS production. The combination of the two phytochemicals resulted in an intermediate level of ROS. The observed changes in prostaglandin levels and ROS production by these two phytochemicals may suggest their mediation in the growth inhibition. The reduction in ROS level and increase by resveratrol supplementation in PC-3 cells reflects the antioxidant properties of resveratrol. It was concluded that these phytochemicals may induce the inhibition of tumor growth by stimulating apoptosis and arresting cells at different locations in the cell cycle and the mechanism may involve alterations in ROS and prostaglandin production.     

Thermodynamic study on competitive solubilization of cholesterol and beta-sitosterol in bile salt micelles

Differences in the preferential solubilization of cholesterol and competitive solubilizates (beta-sitosterol and aromatic compounds) in bile salt micelles was systematically studied by changing the molar ratio of cholesterol to competitive solubilizates. The cholesterol solubility in a mixed binary system (cholesterol and beta-sitosterol) was almost half that of the cholesterol alone system, regardless of the excess beta-sitosterol quantity added. On the other hand, the mutual solubilities of cholesterol and pyrene were not inhibited by their presence in binary mixed crystals. Finally, the cholesterol solubility was measured by changing the alkyl chain length of n-alkylbenzenes. When tetradecylbenzene was added to the bile solution, the cholesterol solubility decreased slightly and was below the original cholesterol solubility. Based on Gibbs energy change (DeltaG degrees ) for solubilization, chemicals that inhibit cholesterol solubility in their combined crystal systems showed a larger negative DeltaG degrees value than cholesterol alone.     

Dietary influence on protein level in milk and milk yield in dairy cows

Diet can influence the yield of milk protein more than it can influence milk protein content. Providing sufficient dietary crude protein in forms that will maximize amounts of amino acids required for milk protein synthesis to the gastrointestinal tract, in forms that can be digested and absorbed, will provide for optimal milk protein yield and content. Maximizing ruminal microbial protein synthesis is an important part of this strategy. The rest of the strategy involves providing sufficient amounts of the remaining required amino acids as ruminally protected proteins, or amino acids in forms that can be digested in the gastrointestinal tract. A diet deficient in protein will reduce milk protein content 1 to 2 g kg⁻¹ and may substantially reduce yields of milk and milk protein. A diet containing high amounts of readily fermentable carbohydrates may increase milk protein content 1 to 2 g kg⁻¹, and may increase yields of milk and protein, but may also result in digestive and metabolic upsets. Diets containing supplemental fat will increase yield of milk protein, but not as extensively as the increase in yield of milk, because milk protein content is usually reduced 1 to 2 g kg⁻¹. The increased efficiency of milk fat and lactose synthesis is likely to be the reason for this depression in milk protein content. A means of overcoming this problem is a continuing research challenge.     

Phytochemical and biological study of Uncaria tomentosa

The investigation on steroidic fraction of Uncaria tomentosa, commonly called Una de gato, showed the presence of beta-sitosterol (60%), stigmasterol, and campesterol. The percentage of sterols have been carried out by GLC. The spectroscopic data 1H-NMR and MS of the three compounds are also reported, with the beta-sitosterol as the main sterol. Preliminary pharmacological investigations prove a moderate antiinflammatory activity.     

Selection of Mycobacterium sp. strains with capacity to biotransform high concentrations of β-sitosterol

In this work, phytosterol-biotransforming strains were selected from Mycobacterium sp., using a high concentration of beta-sitosterol. The selection was made by culturing the strains in a medium enriched with 14 g beta-sitosterol/l as the unique source of carbon. During 2 months, the bacterial cultures were transferred successively. The extraction of the biotransformation products was made with methanol and ethyl acetate. The qualitative and quantitative analysis was made by means of thin-layer chromatography, gas-liquid chromatography (GLC) and GLC-mass spectrometry. Under these conditions, it was observed that after seven transfers, the strains MYcobacterium sp. MB-3683 and the Mycobacterium fortuitum B-11045 increased their biotransformation capacity from 20% to 64% and from 34% to 55%, respectively. The products in the highest proportion identified for each trial were androstenedione and androstadienedione. The results suggest that the high substrate concentration could be a selective mechanism to obtain strains more efficient in the biotransformation of beta-sitosterol into steroidal bases.     

A chlorinated monoterpene ketone, acylated beta-sitosterol glycosides and a flavanone glycoside from Mentha longifolia (Lamiaceae)

Mentha longifolia (Lamiaceae), an aromatic herb yielded a new halogenated chloro-derivative of menthone (longifone), two new derivatives of beta-sitosterol glycoside (longiside-A and -B) and a new flavanone-glycoside (longitin). The beta-sitosterol and flavanone glycosides were purified as their acetate derivatives. Structures of all the isolated constituents were elucidated with the aid of HMBC techniques. However, the structure of longifone was also determined through X-ray crystallography.     

Genetic Variation in Vitamin B-12 Content of Bovine Milk and Its Association with SNP along the Bovine Genome

Vitamin B-12 (also called cobalamin) is essential for human health and current intake levels of vitamin B-12 are considered to be too low. Natural enrichment of the vitamin B-12 content in milk, an important dietary source of vitamin B-12, may help to increase vitamin B-12 intake. Natural enrichment of the milk vitamin B-12 content could be achieved through genetic selection, provided there is genetic variation between cows with respect to the vitamin B-12 content in their milk. A substantial amount of genetic variation in vitamin B-12 content was detected among raw milk samples of 544 first-lactation Dutch Holstein Friesian cows. The presence of genetic variation between animals in vitamin B-12 content in milk indicates that the genotype of the cow affects the amount of vitamin B-12 that ends up in her milk and, consequently, that the average milk vitamin B-12 content of the cow population can be increased by genetic selection. A genome-wide association study revealed significant association between 68 SNP and vitamin B-12 content in raw milk of 487 first-lactation Dutch Holstein Friesian cows. This knowledge facilitates genetic selection for milk vitamin B-12 content. It also contributes to the understanding of the biological mechanism responsible for the observed genetic variation in vitamin B-12 content in milk. None of the 68 significantly associated SNP were in or near known candidate genes involved in transport of vitamin B-12 through the gastrointestinal tract, uptake by ileum epithelial cells, export from ileal cells, transport through the blood, uptake from the blood, intracellular processing, or reabsorption by the kidneys. Probably, associations relate to genes involved in alternative pathways of well-studied processes or to genes involved in less well-studied processes such as ruminal production of vitamin B-12 or secretion of vitamin B-12 by the mammary gland.