Solubility in and affinity for the bile salt micelle of plant sterols are important determinants of their intestinal absorption in rats

Intestinal absorption of various plant sterols was investigated in thoracic duct-cannulated normal rats. Lymphatic recovery was the highest in campesterol, intermediate in brassicasterol and sitosterol, and the lowest in stigmasterol and sitostanol. Higher solubility in the bile salt micelle was observed in sitosterol, campesterol, and sitostanol than in brassicasterol and stigmasterol. The solubility of the latter two sterols was extremely low. When the affinity of plant sterols for the bile salt micelle was compared in an in vitro model system, which assessed sterol transfer from the micellar to the oil phase, the transfer rate was the highest in brassicasterol, intermediate in campesterol and stigmasterol, and lowest in sitosterol and sitostanol. Although no significant correlations between lymphatic recovery of plant sterols and their micellar solubility or transfer rate from the bile salt micelle were observed, highly positive correlation was obtained between the lymphatic recovery and the multiplication value of the micellar solubility and the transfer rate. These observations strongly suggest that both solubility in and affinity for the bile salt micelle of plant sterols are important determinants of their intestinal absorption in rats.     

Effects of cholestyramine and squalene feeding on hepatic and serum plant sterols in the rat

Hepatic and serum phytosterol concentrations were compared in the rat under basal conditions and during activated cholesterol and bile acid production due to squalene and cholestyramine feeding. Both treatments consistently decreased hepatic and serum levels of sitosterol and campesterol and, unlike esterified cholesterol, esterified plant sterols were not increased in liver during squalene feeding. Serum levels of phytosterols were decreased quite proportionately to those in the liver. The hepatic levels of sitosterol and campesterol closely correlated with each other, but not with cholesterol levels. The percentage esterification of both phytosterols was lower than that of cholesterol. The results indicate that activation of hepatic sterol production leads to depletion of hepatic plant sterols. It is suggested that poor esterification of plant sterols may contribute to this decrease.     

The content and composition of sterols and sterol esters in sunflower and poppy seed oils

The composition and proportion of free sterols and sterol esters in crude sunflower and poppy seed oils were determined, using preparative thin layer chromatography followed by gas chromatography with cholesterol as an internal standard. Free sterols and sterol esters were also isolated in a liquid fraction obtained by low temperature crystallization (−80 C) of the oils and enriched with minor lipid classes. This enrichment procedure provided a liquid fraction suitable for studies of minor components in the oils. However, selectivity towards sterol esters was observed since sterols esterified to very long chain fatty acids (C₂₀–C₂₄) were preferentially retained in the precipitate. The proportions of free and esterified sterols were found to be 0.34 and 0.28%, respectively, in the sunflower oil, whereas the corresponding figures for poppy seed oil were 0.33% and 0.05%. Sunflower oil was characterized by a relatively high percentage of Δ7-sterols, preferentially obtained in the esterified fraction, and by very long chain saturated fatty acids of sterol esters. The sterols in poppy seed oil were composed almost entirely of campesterol, stigmasterol, sitosterol and Δ5-avenasterol, although their percentage distributions were remarkably different in the free and esterified fraction.     

Esterification affects phytosterol oxidation

The effect of esterification with rapeseed oil fatty acids on the oxidation reactions of sitosterol, campesterol and sitostanol was investigated, as well as the interactions between phytosterol/stanol compounds and the saturated lipid matrix at 100 °C and 180 °C. Free and esterified phytosterols differed in their reactivity in terms of the formation and profile of secondary oxidation products. Phytosteryl esters were more reactive than free phytosterols during prolonged heating at 100 °C. In contrast, free phytosterols were slightly more reactive than phytosteryl esters at 180 °C. The oxidation reactions of phytostanol compounds were low under all conditions studied. Changes in the phytosterol compounds during heating were also studied via the losses in the original phytosterol contents. This study revealed that the formation of secondary oxides did not account for all the phytosterol losses; this indicates the presence of other oxidation products, especially at 180 °C, and during the heating of free sitosterol. Thus, in order to understand the overall deterioration of phytosterol and phytostanol compounds, both the secondary oxide formation and the sterol loss need to be studied. The deterioration of the saturated lipid matrix used in this study was rather low and was mainly associated with the heating temperature and time.     

The Effect of processing on the content and composition of free sterols and sterol esters in soybean oil

The content and composition of free sterols and sterol esters in crude soybean oil and in oils from different stages of two continuous refining systems were determined. The sterols were isolated by preparative thin layer chromatography and analyzed by gas chromatography with cholesterol as an internal standard. The free sterols in one of the degummed oils amounted to 3.1 mg/g and were diminished to 1.8 mg/g oil by the De Laval Short-Mix refining process. The content of free sterols of the other degummed oil was reduced from 3.4 to 1.6 mg/g oil by the Zenith process. The greatest reduction of sterol content was caused by the treatment with bleaching earth. The sterol esters accounted for 0.6 mg/g of the degummed oil, and only very small changes were observed during the processes. However, changes in the composition of fatty acids of the sterol esters were found. These changes might indicate a selective deacylation of sterol esters or an interesterification during the refining processes. The composition of sterols in free and esterified form were different. Campesterol, stigmasterol and sitosterol were obtained in both free and esterified form, but Δ7 stigmasterol was only found in esterified form. Only small changes in the percentage distribution of the sterols occurred during the processes. Present adress Food Technology Division, ALFA-LAVAL,S-14700 Tumba, Sweden     

Cholesterol‐lowering effect of plant sterols

Intake of plant sterols (4‐desmethyl sterols, phytosterols) reduces cholesterol absorption and lowers serum total and LDL cholesterol levels in humans. The use of dietary plant sterol regimens for lowering elevated serum cholesterol values has recently gained much interest, especially after the commercial introduction of margarines containing plant stanols esterified with fatty acids. The solubility of free, crystalline plant sterols and stanols in edible oils and fats is low, limiting their use especially in fat‐containing food. By esterifying of, e.g., plant stanols with fatty acids derived from a vegetable oil fatty acid ester of plant stanols with fat‐like properties are obtained. These fat‐soluble forms of plant stanols provide a technically feasible way of introducing the adequate daily amount of plant sterol into foods for optimal reduction of the cholesterol absorption, without changing the taste of the finished product. The cholesterol‐lowering effect of plant stanol esters has been extensively studied. Plant stanol esters effectively restrict the absorption of both dietary and biliary cholesterol causing plant stanol specific reductions in serum total and LDL cholesterol levels of up to 10% and 14%, respectively. Serum HDL cholesterol and triglyceride levels are not affected. The cholesterol‐lowering effect of plant stanol esters complements the beneficial effects of a healthy diet and cholesterol medication.     

The content and composition of sterols and sterol esters in low erucic acid rapeseed (Brassica napus)

The low temperature crystallization technique for the enrichment of “minor” components, such as sterols and sterol esters, from vegetable oils was applied to low erucic acid rapeseed oils. The recovery of free sterols and sterol esters was estimated by use of¹⁴C-cholesterol and¹⁴C-cholesterol oleate. 80% of the free sterols and 45% of the sterol esters were recovered in the liquid fraction, while in two studies total recoveries were 95% and 99%, respectively. This technique showed some selectivity toward the sterol bound fatty acids when compared to direct preparative thin layer chromatography (TLC) of the crude oil. Gas liquid chromatography (GLC) analysis of the free and esterified sterols as TMS-derivatives showed very little selectivity in the enrichment procedure. The fatty acid patterns of the sterol esters demonstrated, however, a preference in the liquid fraction for those sterol esters which have a high linoleic and linolenic acid content. The content of free sterols was 0.3–0.4% and that of sterol esters 0.7–1.2% of the rapeseed oils in both winter and summer types of low erucic acid rapeseed (Brassica napus) when the lipid classes were isolated by direct preparative TLC of the oils. The free sterols in the seven cultivars or breeding lines analyzed were composed of 44–55% sitosterol, 27–36% campesterol, 17–21% brassicasterol, and a trace of cholesterol. The esterified sterols were 47–57% sitosterol, 36–44% campesterol, 6–9% brassicasterol, and traces of cholesterol and Δ5-avenasterol. The fatty acid patterns of these esters were characterized by ca. 30% oleic acid and ca. 50% linoleic acid, whereas these acids constitute 60% and 20%, respectively, of the total fatty acids in the oil. Little or no variation in sterol and sterol ester patterns with locality within Sweden was observed for the one cultivar of summer rapeseed investigated by the low temperature crystallization technique.     

Phytosterols and pectin added to a high‐saturated fat diet do not show hypocholesterolemic activity in female guinea pigs

This paper presents the results of a study whose aim was to test the effects of several doses of pectin and phytosterols on the sterol content in plasma, liver and feces of guinea pigs, when added to a high‐saturated fat diet. The treatments followed a 3×3 factorial design, with three levels of pectin (0, 3.67 and 6.93%) and three levels of phytosterols (0, 1.37 and 2.45%). Seventy‐two female Dunkin Hartley guinea pigs were randomly assigned to the treatment groups (eight animals per group). The duration of the treatment was 4 weeks. No differences were found in plasma cholesterol concentrations, while in liver we saw a reduction in cholesterol concentration after phytosterol feeding. Moreover, we found no pectin effects. Plant sterol concentration increased in plasma and liver after phytosterol ingestion, with the highest concentrations being obtained with the intermediate pectin dose. Our results suggest that a high‐saturated diet may impair the cholesterol‐lowering properties of plant sterols and pectin.     

Effect of plant sterols,fatty acids and lecithin on cholesterol absorption in vivo in the rat

The inhibitory effect of plant sterols, fatty acids and lecithin on cholesterol intestnal absorption was studied in the unanesthetized rat using a single pass perfusion technique. Bile was excluded from the perfused intestine. Cholesterol absorption did not change following the additions of cholestanol, cholestanone, lanosterol, stigmasterol and β-sitosterol. A 3-fold increase in the molarity of cholestanol and β-sitosterol or the separate additions of the saturated short and medium chain fatty acids, butyric and octanoic, also did not change cholesterol absorption. The unsaturated long chain fatty acids, oleic, linoleic, linolenic and arachidonic, inhibited cholesterol absorption. Lecithin additions at concentrations of 0.1–1.5 mM caused a progressive, dose-related inhibition of cholesterol absorption. The inhibitory effect of these agents on cholesterol absorption is likely to have been caused by changes in cholesterol solubility in the micelle and shifts in the partition coefficient of cholesterol away from the cell membrane to the micelle.     

Oxidation of phytosterols in a test food system

The oxidative stability of phytosterols in canola, coconut, peanut, and soybean oils was examined under simulated frying conditions of 100, 150, and 180°C for 20 h. The degree of oxidative decomposition was assessed by the loss of phytosterols, accumulation of phytosterol oxides, and the change in fatty acid profiles. The phytosterol oxides produced in the oils were identified using mass spectroscopy. Oils with higher levels of polyunsaturated fatty acids showed greater amounts of sterol loss; however, the sterol loss was less complete than in the more saturated oils. A greater variety of sterol oxides was observed at the lower temperatures of 100 and 150°C compared to 180°C. This study demonstrates that under conditions similar to frying, there is a loss of phytosterols and polyunsaturated fatty acids. The accumulation of phytosterol oxides may be temperature-limited because of further break-down into products not measurable by typical gas chromatography-mass spectrometry techniques.     

Sterols and Stanols in Foods and Dietary Supplements Containing Added Phytosterols: A Collaborative Study

An international, multilaboratory collaborative study was carried out to evaluate the performance of Official Method Ce 12‐16 of the American Oil Chemists’ Society (AOCS) for the determination of plant sterols and stanols, collectively referred to as phytosterols, in foods and dietary supplements containing added phytosterols and in the phytosterol food additive concentrates used to prepare such products. AOCS Official Method Ce 12‐16 involves the extraction of free sterols/stanols and saponified steryl/stanol esters followed by the gas chromatographic separation and flame ionization detection of phytosterol trimethylsilyl ether derivatives. A total of 14 laboratories from six countries successfully completed the analysis of collaborative samples of foods (e.g., baked goods, beverages, margarines; n = 9), dietary supplements (n = 5), and phytosterol concentrates (n = 4). Study results for the contents of total phytosterols (weight/weight) were 0.19–8.4% for foods, 8.7–49% for dietary supplements, and 57–97% for concentrates. AOCS Official Method Ce 12‐16 showed acceptable performance for total and individual phytosterols, indicating that this method was suitable for the determination of added phytosterols in a wide variety of market products and concentrates. AOCS Official Method Ce 12‐16 is appropriate for the determination of the five major phytosterols (i.e., campesterol, stigmasterol, β‐sitosterol, campestanol, and sitostanol) that are the subject of the United States Food and Drug Administration's health claim for phytosterols and the reduced risk of coronary heart disease.     

Chemical synthesis of phytosterol esters of polyunsaturated fatty acids with ideal oxidative stability

In this study, direct esterification of phytosterols with polyunsaturated fatty acid (PUFA) catalyzed by sodium bisulfate to produce sterol esters of PUFA was performed without organic solvent. Considering on both degree of esterification (DE) and oxidative stability, response surface methodology (RSM) was employed for modeling the phytosterol esters of PUFA production to obtain a food grade process. The optimal and mild reaction conditions were obtained as follows: mass ratio of PUFA:phytosterols = 4:1; amount of catalyst: 3% of the raw materials weight; reaction temperature 130°C; reaction time 8 h. Under these conditions, the degree of esterification was up to 96%, GC, TLC, NMR and GC‐MS results showed that purity of purified sterol ester was above 99%, and β‐sitosterol linolenate account for about 88%. Sterol ester of PUFA possessed low peroxide value (PV) (0.96 meq/kg) and conjugated diene (CD) value (2.15 mmol/kg), and high oxidative induction period (OIP) (10.4 h). Addition amount of sterol ester of PUFA into soybean oil, rapeseed oil, and flaxseed oil below 1, 1, and 3%, respectively, could increase OIP of the vegetable oil. The primrose phytosterol esters of PUFA possessed very low melting point, crystallization temperature, and greater solubility in oils. All the characteristics favored the wide application of sterol ester of PUFA produced by the food grade process in different formulations of functional foods.     

Influence of the vegetable oil refining process on free and esterified sterols

The influence of the refining process on the distribution of free and esterified phytosterols in corn, palm, and soybean oil was studied. Water degumming did not affect the phytosterol content or its composition. A slight increase in the content of free sterols was observed during acid degumming and bleaching due to acid-catalyzed hydrolysis of steryl esters. A significant reduction in the content of total sterols during neutralization was observed, which was attributed to a reduction in the free sterol fraction. Free sterols probably form micelles with soaps and are transferred into the soapstock. The steryl ester content remained constant during all neutralization experiments, indicating that hydrolysis of steryl esters did not take place during neutralization. During deodorization, free sterols are distilled from the oil, resulting in a gradual reduction in the total sterol content as a function of the deodorization temperature (220–260°C). A considerable increase in the steryl ester fraction was found during physical refining, probably owing to a heat-promoted esterification reaction between free sterols and FA.     

The Sterols and Fatty Acids of Delphinium denudatum Roots

The content and composition of sterols and fatty acids of Delphinium denudatum roots, which are used in the Indian Unani system of medicine, were determined. The sterols were composed almost entirely of campesterol, stigmasterol and sitosterol. Trace amounts of cholesterol and Δ⁵-avenasterol were also detected. Characteristic higher plant fatty acids were also present.     

A comparison of the effect of medium- vs. long-chain triglycerides on the <Emphasis Type="Italic">in vitro</Emphasis> solubilization of cholesterol and/or phytosterol into mixed micelles

Despite clinical evidence of the cholesterol-lowering effects of phytosterols, the exact mechanisms involved are still unclear. Displacement of cholesterol by phytosterols from mixed micelles, which is due to their greater hydrophobicity, is one of the hypotheses for the lumenal effects contributing to the reduction of intestinal cholesterol absorption. In this study a dynamic in vitro lipolysis method was used to examine the solubilization behavior of cholesterol and/or phytosterols during lipolysis to probe the efficacy of cholesterol displacement from mixed micelles by phytosterols. The effects of lipid chain length on sterol solubilization were studied by using microcrystalline suspensions containing 17% phytosterol or cholesterol, formulated in long-chain TG (LCT) and medium-chain TG (MCT). When digesting cholesterol suspended in LCT, the entire cholesterol dose was incorporated into the micellar phase. For the cholesterol formulation suspended in MCT, 50.3% of the initial dose was recovered in the micelles. Under the respective conditions, we observed lower solubilization of phytosterols than of cholesterol (roughly fourfold). Only 25% of the initial phytosterol dose was solubilized from suspensions formulated with LCT, and 13% was solubilized from MCT formulations. Co-administration of phytosterol and cholesterol suspensions showed a significant reduction of cholesterol solubilization, particularly when dosed in MCT, with ≈25% of the cholesterol dose solubilized. Insignificant amounts of cholesterol were displaced by phytosterols when cholesterol was presolubilized in the mixed micelles. The results show that, compared with LCT, mixed micelles containing MCT lipolysis products have a reduced solubilizing capacity for cholesterol, which adds to the effectiveness of the phytosterols in displacing cholesterol. This suggests potential benefits of using medium chain length lipids in cholesterol-lowering phytosterol products.     

Free sterols,steryl esters,and lipid phosphorus in needles of scot's pine (Pinus sylvestris L.)

Free sterols, steryl esters, and lipid phosphorus were measured in new (current year) needles of Scot's pine during an annual cycle, and also in one-, two-, and three-year-old needles collected shortly after bud break. Sterols were identified and quantified by capillary gas chromatography and gas chromatography/mass spectrometry. Steryl esters were hydrolyzed enzymatically. Newly emerged needles contained highest amounts of free sterols and lipid phosphorus, probably reflecting increased membrane and organelle production, but low levels of steryl esters. Mature needles contained approximately equal amounts of free and esterified sterols. The molar phospholipid/free sterol ratio was 3∶1 at all the time periods studied. A dramatic increase of steryl esters was observed in the one-, two-, and three-year-old needles at times when new needles emerged. The individual free and esterified sterols were sitosterol, campesterol (presumably together with its C-24 epimer), and cholesterol, at approximately 88, 10, and 2%, respectively. Isofucosterol, an intermediate in sitosterol biosynthesis, was present almost exclusively in newly emerged needles. Esterified sterols contained only trace amounts of isofucosterol. Shifts in favor of cholesterol and 24ζ-methyl cholesterol occurred in the steryl esters during needle differentiation, and saturation grade of esterified fatty acids decreased. In mature needles, the composition of free sterols and steryl esters remained constant throughout the year.     

Comparative studies of metabolism of 4-desmethyl, 4-monomethyl and 4,4-dimethyl sterols inManduca sexta

To investigate the metabolism and possible deleterious effects of 4-methyl and 4,4-dimethyl steroids inManduca sexta, the 4,4-dimethyl sterols lanosterol and cycloartenol, the 4-methyl sterol obtusifoliol and the 4,4-dimethyl pentacyclic triterpenoid α-amyrin were fed in an artificial agar-based diet at various concentrations. Utilization and metabolism of these four compounds were compared with sitosterol, stigmasterol, brassicasterol, ergosterol and 24-methylenecholesterol, 24-alkyl sterols that are readily dealkylated and converted to cholesterol inManduca and in most phytophagous insects. None of the 4-methylated compounds significantly inhibited development except at very high dietary concentrations. The Δ²⁴-bonds of lanosterol and cycloartenol were effectively reduced by theManduca Δ²⁴-sterol reductase enzyme, as is the Δ²⁴-bond of desmosterol which, in most phytophagous insects, is an intermediate in the conversion of sitosterol, stigmasterol and other C₂₈ and C₂₉ phytosterols to cholesterol. On the other hand, the 24-methylene substituent of obtusifoliol was not dealkylated. Each of the 4-desmethyl C₂₈ and C₂₉ sterols was readily converted to cholesterol, and a significant amount of 7-dehydro-cholesterol was derived from ergosterol metabolism. The reason for the differences in substrate specificity of these sterols is not clear, but the information may be useful in the development of new, specific, mechanism-based inhibitors of sterol metabolism.     

The lipids of slugs and snails: Evolution,diet and biosynthesis

There is a considerable gap in current knowledge of the lipid composition of snails and slugs, both of which belong to the phylum Mollusca. We have therefore analyzed the sterol and fatty acid compositions of three species of slugs and three species of snails. The sterols of slugs included eight different sterols: cholesterol contributed 76–85% of the total sterols, brassicasterol accounted for 4–13%; other sterols we identified were lathosterol, 24-methylene cholesterol, campesterol, stigmasterol, sitosterol and sitostanol. In contrast, snails contained two additional sterols, desmosterol and cholestanol. Of the polyunsaturated fatty acids in slugs, linoleic (18∶2n−6) and arachidonic acids (20∶4n−6) were the major n−6 fatty acids, while linolenic (18∶3n−3) and eicosapentaenoic acids (20∶5n−3) were the predominant n−3 fatty acids. Docosahexaenoic acid (22∶6n−3), the end product in the n−3 fatty acid synthetic pathway and an important membrane fatty acid of mammals, fish and birds, was absent in both slugs and snails. However, the analogous product of n−6 fatty acid synthesis, 22∶5n−6, was found in both snails and slugs. This raises speculation about preference for n−6 fatty acid synthesis in these species. Our data show the unique sterol and fatty acid compositions of slugs and snails, as well as similarities and differences in sterol composition between the two. The results between the two land mollusks are contrasted with those of marine mollusks, such as oysters, clams and scallops.     

Fatty acid composition of individual plasma steryl esters in phytosterolemia and xanthomatosis

The bulk of the plasma plant sterol in phytosterolemia occurs in the esterified form and is carried mostly in the low and high density lipoproteins. We have determined the fatty acid composition of the individual plasma steryl esters from a newly discovered subject with phytosterolemia and xanthomatosis. For this purpose the intact steryl esters were subject to high temperature gas liquid chromatography (GLC) on a polar capillary column, which separated the major esters on the basis of molecular weight and degree of unsaturation of the fatty acids. The saturated and unsaturated sterols esterified to saturated, monoenoic, dienoic and tetraenoic fatty acids were identified by GLC analysis of the sterol moieties of the corresponding AgNO₃-TLC fractions of the steryl esters. The GLC results were confirmed by reversed phase high performance liquid chromatography combined with mass spectrometry via direct liquid inlet interface. It was found that, in general, each fatty acid was esterified to the same complement of sterols, and that the esterified sterols possessed a composition comparable to that of the free plasma sterols, which was comprised of about 75% cholesterol, 6% campesterol, 4% 22,23-dihydrobrassicasterol and 15% β-sitosterol. The fatty acid composition of the steryl esters differed from that of the 2-position of the plasma phosphatidylcholines, which contained significantly less palmitic and oleic and more linoleic acid. On the basis of these results and a review of the literature it is suggested that the plasma cholesteryl and plant steryl esters in phytosterolemia originate from both synthesis in plasma via the lecithin-cholesterol acyltransferase and synthesis in tissues via the acylCoA-cholesterol acyltransferase.     

不同溶剂萃取对烟丝中甾醇含量降低效果的影响

以95%乙醇、乙酸乙酯、纯化水、石油醚为溶剂萃取烟丝,通过气质联用定量测定萃取后烟丝主要游离甾醇的含量。结果表明：①95%乙醇、乙酸乙酯和石油醚3种有机溶剂都具有不同程度降低烟丝中甾醇含量的效果,甾醇降低量大小顺序为：乙酸乙酯〉石油醚〉95%乙醇,尤其是乙酸乙酯萃取后的烟丝中各甾醇类化合物含量降低最为明显,其中胆甾醇、豆甾醇和β-谷甾醇含量分别降低了24.67%,64.63%,44.03%;②水萃取烟丝后甾醇含量比正常烟丝还要高,这是由于水是良好的极性溶剂,萃取烟丝后导致烟丝中大部分化学成分含量降低,单位质量的烟丝增多,因此单位质量烟丝的甾醇含量反而上升。