Lymphatic Absorption and Deposition of Various Plant Sterols in Stroke-Prone Spontaneously Hypertensive Rats,a Strain Having a Mutation in ATP binding cassette transporter G5

ATP binding cassette transporter G5 (ABCG5) and ATP binding cassette transporter G8 (ABCG8) have been suggested to transport absorbed plant sterols and cholesterol from enterocytes to the intestinal lumen and from hepatocytes to bile. It has been thought that mutations of ABCG5 or ABCG8 cause the deposition of plant sterols in the body. In the present study, lymphatic absorption of various plant sterols and their deposition in various tissues was investigated in stroke-prone spontaneously hypertensive rats (SHRSP), having a mutation in Abcg5 and depositing plant sterols in the body. The order of lymphatic 24-h recovery of plant sterols was as follows: campesterol > sitosterol > brassicasterol > stigmasterol = sitostanol. When SHRSP were fed a diet containing one of the plant sterols, the depositions of campesterol and sitosterol were comparatively higher than those of brassicasterol, stigmasterol and sitostanol. Highly positive correlations were obtained between lymphatic recovery of plant sterols and their levels in plasma, liver, adipose tissue and heart. The tendency of differential absorption of plant sterols to the lymph in SHRSP was similar to that in normal Wistar rats previously reported by us (Hamada et al. Lipids 41:551–556, 2006). These observations suggest that differential absorption of various plant sterols is kept in SHRSP in spite of a mutation in Abcg5.     

Phytosterol Ester Constituents Affect Micellar Cholesterol Solubility in Model Bile

Plant sterols and stanols (phytosterols) and their esters are nutraceuticals that lower LDL cholesterol, but the mechanisms of action are not fully understood. We hypothesized that intact esters and simulated hydrolysis products of esters (phytosterols and fatty acids in equal ratios) would differentially affect the solubility of cholesterol in model bile mixed micelles in vitro. Sodium salts of glycine- and taurine-conjugated bile acids were sonicated with phosphatidylcholine and either sterol esters or combinations of sterols and fatty acids to determine the amount of cholesterol solubilized into micelles. Intact sterol esters did not solubilize into micelles, nor did they alter cholesterol solubility. However, free sterols and fatty acids altered cholesterol solubility independently (no interaction effect). Equal contents of cholesterol and either campesterol, stigmasterol, sitosterol, or stigmastanol (sitostanol) decreased cholesterol solubility in micelles by approximately 50% compared to no phytosterol present, with stigmasterol performing slightly better than sitosterol. Phytosterols competed with cholesterol in a dose-dependent manner, demonstrating a 1:1 M substitution of phytosterol for cholesterol in micelle preparations. Unsaturated fatty acids increased the micelle solubility of sterols as compared with saturated or no fatty acids. No differences were detected in the size of the model micelles. Together, these data indicate that stigmasterol combined with saturated fatty acids may be more effective at lowering cholesterol micelle solubility in vivo.     

Separation of Campesterol and β‐Sitosterol from a Sterol Mixture

Abstract

By solvent crystallization using diethyl ether as the solvent on sterol mixture, brassicasterol and stigmasterol that contains a side chain with double bond were separated from campesterol and β‐sitosterol with a saturated side chain. The total campesterol and β‐sitosterol content in the liquid phase was more than 97% with a recovery of 12%. Multistage crystallization using acetone as the solvent could increase the recovery of campesterol and β‐sitosterol to 30%. By employing zeolite selective adsorption on the campesterol and β‐sitosterol fraction, β‐sitosterol can be recovered in the liquid phase with a purity of 95.2% and a recovery of 3% (overall recovery 1%). After desorbing in ethanol, campesterol adsorbed on the zeolite can be recovered with a purity of 95.4% and a recovery of 3.7% (overall recovery 1.6%).     

Absorption, excretion, and distribution of plant sterols after proximal gut resection and autotransplantation of porcine ileum

Contribution of different gut segments to plant sterol absorption, adaptation of plant sterol absorption after partial small bowel resection, and effects of gut transplantation (necessitates extrinsic autonomic denervation and lymphatic disruption) on plant sterol biodynamics are unclear. We studied the consequences of massive proximal small bowel resection and autotransplantation of the remaining ileum on the adaptive absorption and biodynamics of plant sterols. Dietary, fecal, biliary, hepatic and plasma plant sterols, fecal elimination and absorption of cholesterol, small bowel morphology, and intestinal transit were determined before (n=5) and at 4, 8, and 14 wk after resection of the proximal 75% of the jejunoileum (n=15) and autotransplantation of the remaining ileum (n=15) or transection (n=5). Proximal gut resection significantly reduced cholesterol absorption efficiency; percentage absorption and biliary secretion of plant sterols; plasma, biliary and hepatic campesterol-to-cholesterol proportions; and sitosterol proportions in plasma and bile. Autotransplantation of the remaining ileum further significantly decreased cholesterol absorption efficiency; percentage absorption and biliary secretion of campesterol; campesterol proportions in plasma, bile and liver; and plasma proportions of sitosterol while increasing fecal excretion of neutral and acidic steroids. Plasma proportions of the two plant sterols, but absorption of just campesterol, were gradually improved with increasing cholesterol absorption and villus height after proximal gut resection; the same result was observed to a lesser degree after ileal autotransplantation. In addition, significant positive correlations were found between percentage cholesterol and campesterol absorption and the plasma plant sterol proportions in both proximal resection groups, between campesterol absorption and ileal villus height in the resection group, and between campesterol absorption and intestinal transit time in the autotransplantation group. In conclusion, plasma campesterol and sitosterol closely reflect absorption of cholesterol and plant sterols from intact and autotransplanted ileum during adaptation to proximal gut resection. A loss of proximal gut absorptive surface impairs cholesterol and campesterol absorption more than sitosterol absorption, the later being apparently less dependent on available jejunal villus surface area.     

Sterol composition inCoincya (Brassicaceae)

Sterol composition was determined for seed oils and leaf waxes in eleven taxa belonging to the genusCoincya (Brassicaceae) on the Iberian Peninsula (Spain and Portugal). Seed sterols ranged from 1.2 to 6.7%. The major components were sitosterol (42.6–54.6%), campesterol (20.4–33.2%), and brassicasterol (10.8–23.5%). In leaf waxes, the major free sterols were sitosterol (40.9–74.2%), campesterol (9.6–17.0%), and cholesterol (4.6–17.0%). In leaf wax esters, the major sterols were sitosterol (22.2–56.5%), cholesterol (7.3–32.8%), and campesterol (5.8–25.6%). An apparent substitution of brassicasterol in free sterols from the seeds by cholesterol in free sterols from the leaves was observed. There was an increase of cholesterol in sterols from leaf wax esters with respect to free sterols from leaves and seeds. InC. monensis subsp.nevadensis, the composition in sterols from leaf waxes may be an adaptation to low temperatures.     

Effects of triarimol,tridermorph and triparanol on sterol biosynthesis in carrot,tobacco and soybean suspension cultures

The effects of triarimol, tridemorph and triparanol on sterol biosynthesis in carrot, tobacco and soybean suspension cultures were studied. The 3 plant species normally contain campesterol, stigmasterol and sitosterol as major sterols. Triarimol inhibited demethylation at C 14 and the second alkylation of the side chain in all 3 species. The primary effects of tridemorph were the inhibition of the opening of the 9β,19-cyclopropane ring and the second alkylation of the side chain. Triparanol treatments resulted in the accumulation of 14α-methyl sterols, and the inhibition of second alkylation in the side chain in carrot and tobacco cultures. Cyclopropyl sterols also accumulated in carrot and tobacco cultures treated with triparanol. Triparanol did not alter the sterol composition of soybean cultures except for decreasing concentrations of campesterol and stigmasterol and increasing amounts of sitosterol. Scientific article number A-3713, contribution number 6689 of the Maryland Agricultural Experiment Station.     

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.     

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.     

Camelina oil and its unusual cholesterol content

The oil in Camelina sativa L. Crantz has a combined linolenic and linoleic acid content that is greater than 50% and a relatively low saturated FA content (∼10%). Although the FA composition has been reported, no information is available on the sterol composition of camelina oil. The derivatized plant sterols were separated and quantified with capillary GC and their identity confirmed with GC-MS. The refined camelina oil sample contained approximately 0.54 wt% unsaponifiables, and over 80% of the unsaponifiables were desmethylsterols. Perhaps the most unusual characteristic of camelina oil is its relatively high content of cholesterol, particularly for a vegetable oil, since it contains several times the cholesterol found in other “high-cholesterol” vegetable oils. Camelina oil also contains relatively large amounts of another unusual sterol, brassicasterol. The major sterols identified in the camelina oil included cholesterol (188 ppm), brassicasterol (133 ppm), campesterol (893 ppm), stigmasterol (103 ppm), sitosterol (1,884 ppm), and Δ⁵-avenasterol (393 ppm).     

The distribution of dietary plant sterols in serum lipoproteins and liver subcellular fractions of rats

Rats were fed plant sterols containing campesterol and β-sitosterol in the different proportions, and their distribution in serum lipoproteins and in liver subcellular fractions was determined. In serum lipoproteins, the percentage as well as the concentration of plant sterols increased with the increase in the density of lipoproteins. Thus, high density lipoprotein (HDL) contained the highest and very low density lipoprotein (VLDL), the lowest. Also, there were distinct differences in the ratio of campesterol to sitosterol among lipoproteins, it was the highest in VLDL and lowest in HDL. Quantitatively, more than 75% of campesterol and 80% of sitosterol were carried in HDL; the values were significantly different from those of cholesterol (ca. 70%) in relation to total cholesterol. The distribution of plant sterols in liver subcellular fractions was virtually the same with that of cholesterol. Both nuclei and microsomes contained approximately 40% of total plant stetols. A preliminary part of the study was presented at the First Congress of the Federation of Asian and Oceanian Biochemists in Nagoya, October 1977.     

Comparative analysis of phytosterol components from rapeseed (Brassica napus L.) and olive (Olea europaea L.) varieties

Phytosterols occur in relatively high concentration in the seeds of rapeseed (Brassica napus L.) and in lower concentration in olive (Olea europaea L.) oil. The aim of this research was to investigate some new rapeseed varieties and olive genotypes that are grown in Northwest Turkey and to compare the phytosterol contents of both crops. For rapeseed, the data were collected in the growing seasons 2004–2005 from a field experiment with 19 new rapeseed varieties and three replications. For olives, 21 different varieties were used in the 2004–2005 and 2005–2006 growing seasons. The separation and identification of free phytosterols and the analysis of their contents were successfully achieved using the capillary column‐gas chromatographic method. According to the obtained results, for rapeseed, sitosterol (1.54–2.36 g/kg) was the major component of total phytosterols, followed by campesterol (0.02–1.58 g/kg) and brassicasterol (0.26–0.58 g/kg). Regarding the olive varieties, the sitosterol content changed between 1.03 and 2.01 g/kg, followed by avenasterol ranging from 0.07 to 0.44 g/kg. The brassicasterol, campesterol and stigmasterol contents did not affect the total amount of sterols. The total phytosterol content ranged between 4.25 and 11.37 g/kg for rapeseed and 1.29 and 2.38 g/kg for olives.     

Characterization of Total and Individual Sterols in Canola Sprouts

In this study, the contents of total and individual phytosterols in sprouts made from seeds of seven canola (Brassica napus L.) lines (Acropolis, Banjo, Jetton, KS-7740, KSM3-1-124, Mussette and Virginia), grown at three locations in Virginia (Orange, Petersburg and Suffolk), were determined. Canola sprouts contained, on an average, 36.3 g sterols in 100 g of unsaponifiable matter (UNSAP), 10.7 mg sterols in 1 g of oil and 2.4 mg sterols in 1 g of dry sprouts. The contents of individual phytosterols (μg per g of oil) in canola sprouts were 1,162 brassicasterol, 3,799 campesterol, 34 stigmasterol, 5,359 β-sitosterol, 201 Δ⁵-avenasterol and 97 Δ⁷-stigmastenol. Canola lines had significant effects on the contents of oil, brassicasterol and campesterol. Locations had significant effects on the oil, UNSAP, total sterols, brassicasterol, stigmasterol and β-sitosterol. The oil content in canola sprouts was positively correlated with total sterols and Δ⁵-avenasterol, whereas oil content was negatively correlated with brassicasterol content. In general, the contents of campesterol and β-sitosterol increased with an increase in total sterol content. The concentrations of sterols were in the following decreasing order: β-sitosterol > campesterol > brassicasterol > Δ⁵-avenasterol > Δ⁷-stigmastenol > stigmasterol. These results indicate that canola sprouts may have the potential as a natural source of dietary sterols and might be desirable for human nutrition.     

The Detection of Adulteration with Desterolized Oils

Edible oils are desterolized in order to render them “undetectable” when admixed to other oils. Such frauds remain, however, detectable by the olefinic degradation products of the sterols: the degradation products approximately have the composition of the sterols they originate from. Presence of campestatriene (degraded brassicasterol) reveals the presence of desterolized rapeseed oil. The ratio of the degradation products of sitosterol and campesterol is a sensitive indicator for desterolized sunflower, soybean, palm, or grapeseed oil in oils of low campesterol content, such as olive and walnut oil. Analyses were performed by on-line coupled LC-GC.     

Distribution of free and conjugated sterols in orange and tangor juice sacs

Comparative studies of the sterol composition of four sterol fractions, vis., free sterols, sterol esters, sterol glucosides and esterified sterol glucosides, were conducted on the juice sacs of six varieties of oranges and two tangor varieties. The sterols quantified in each fraction were β-sitosterol, campesterol, stigmasterol, cholesterol, 24-ethylidene cholesterol, brassicasterol and 24-methylene cholesterol. Each variety showed its own intrinsic composition for these sterols in the four sterol fractions. So. Market. Nutr. Res. Div., ARS, USDA.     

Serum and aortic levels of phytosterols in rabbits fed sitosterol or sitostanol ester preparations

Campesterol is present in all the phytosterol-containing dietary hypocholesterolemic agents in current use. Campesterol is absorbed more efficiently than sitosterol, and the question of its possible atherogenicity has been raised. To test this possibility, rabbits were fed either a semipurified, cholesterol-free diet that has been shown to be atherogenic for this species or the same diet augmented with 0.5 g of phytosterol-rich diet preparations (spreads) containing either sitosterol or sitostanol. The diets contained 295 mg phytosterol per 100 g. After 60 d, serum cholesterol levels in the two phytosterol groups were 78±4 mg/dL (sitosterol) and 76±4 mg/dL (sitostanol), respectively. The serum cholesterol level of rabbits fed the control diet was 105±8 mg/dL. Serum campesterol (μg/mL) levels were higher than sitosterol or sitostanol levels in all groups. Aortic phytosterols were present in nanogram quantities compared to cholesterol, which was present in microgram quantities. The ratio of campesterol/sitosterol/sitostanol in the aortas was: control, 1.00∶0.43∶0.02; sitosterol, 1∶00∶0.32∶0.01; sitostanol, 1∶00∶0.34∶0.11. Aortic campesterol was present at 4% the concentration of aortic cholesterol, sitosterol at 1.4%, and sitostanol at 0.14%. Aortic lesions were not present in any of the animals.     

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.     

Extraction and Identification of Phytosterols in Manketti (Schinziophyton rautanenii) Nut Oil

Phytosterols of manketti (Schinziophyton rautanenii) nut oil extracted by Soxhlet, mechanical shaking using hexane, screw press and supercritical carbon dioxide, were analyzed by gas chromatography with flame ionization detection and identified by gas chromatography–mass spectrometry. The presence of several phytosterols (campesterol, stigmasterol, β‐sitosterol, Δ⁵‐avenasterol, 22‐dihydrospinasterol and Δ⁷‐avenasterol) previously reported in manketti oil, was confirmed. In addition, another fourteen phytosterols (lanosterol, Δ^5,23‐stigmastadienol, Δ⁷‐campesterol, clerosterol, obtusifoliol, Δ^5,24(25)‐stigmastadienol, α‐amyrin, gramisterol, cycloeucalenol, cycloartenol, stigmasta‐8,24‐dienol‐3‐β‐ol, 28‐methylobtusifoliol, 24‐methylenecycloartenol and citrostadienol) were identified. The phytosterols, β‐sitosterol, Δ⁵‐avenasterol and campesterol, had the highest concentrations in oils extracted by all the methods, whereas stigmasterol and cycloartenol were abundant in oils extracted by mechanical shaking and supercritical carbon dioxide. Total phytosterols and the quantities of individual phytosterols differed significantly (p ≤ 0.05) in oils from the four extraction methods. Mechanical shaking extracted the highest levels of total sterols (22,100 mg/100 g oil), followed by supercritical fluid extraction (9,550 mg/100 g oil). Screw press and Soxhlet extracted oils contained the lowest levels of total sterols, 3,810 mg/100 g oil and 3,350 mg/100 g oil, respectively.     

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.     

Dietary sitostanol and campestanol: Accumulation in the blood of humans with sitosterolemia and xanthomatosis and in rat tissues

Dietary sitostanol has a hypocholesterolemic effect because it decreases the absorption of cholesterol. However, its effects on the sitostanol concentrations in the blood and tissues are relatively unknown, especially in patients with sitosterolemia and xanthomatosis. These patients hyperabsorb all sterols and fail to excrete ingested sitosterol and other plant sterols as normal people do. The goal of the present study was to examine the absorbability of dietary sitostanol in humans and animals and its potential long-term effect. Two patients with sitosterolemia were fed the margarine Benecol (McNeill Nutritionals, Ft. Washington, PA), which is enriched in sitostanol and campestanol, for 7–18 wk. Their plasma cholesterol levels decreased from 180 to 167 mg/dL and 153 to 113 mg/dL, respectively. Campesterol and sitosterol also decreased. However, their plasma sitostanol levels increased from 1.6 to 10.1 mg/dL and from 2.8 to 7.9 mg/dL, respectively. Plasma campestanol also increased. After Benecol withdrawal, the decline in plasma of both sitostanol and campestanol was very sluggish. In an animal study, two groups of rats were fed high-cholesterol diets with and without sitostanol for 4 wk. As expected, plasma and liver cholesterol levels decreased 18 and 53%, respectively. The sitostanol in plasma increased fourfold, and sitostanol increased threefold in skeletal muscle and twofold in heart muscle. Campestanol also increased significantly in both plasma and tissues. Our data indicate that dietary sitostanol and campestanol are absorbed by patients with sitosterolemia and xanthomatosis and also by rats. The absorbed plant stanols were deposited in rat tissues. Once absorbed by sitosterolemic patients, the prolonged retention of sitostanol and campestanol in plasma might increase their atherogenic potential.     

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.