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.     

Effect of soy sterols on cholesterol synthesis in the rat

Soy sterols increased the incorporation of acetate into cholesterol by rat liver slices and prevented the depression of cholesterogenesis caused by dietary cholesterol. Acetate incorporation into cholesterol by intestinal slices was not affected by soy sterols but was decreased by cholesterol. The results with liver slices agree with the view that soy sterols, by interfering with the intestinal absorption of cholesterol, prevent the feedback inhibition of hepatic cholesterogenesis.     

Inhibition of cholesterol synthesis by oxygenated sterols

Sterols derived from cholesterol by introducing a ketone or hydroxyl function in the 6, 7, 15, 20, 22, 24, or 25 positions are known to be potent inhibitors of sterol synthesis in cell cultures. To gain more information regarding structural requirements for inhibitory activity, inhibitory potencies were determined for a series of 18 C₂₇-steroids with various combinations of ketone and hydroxyl functions substituted in positions 3, 4, 5, 6, and 7, or with a single ketone or hydroxyl function in one of these positions. The effects of nuclear double bonds upon inhibitory activity were also examined. A ketone or hydroxyl function in position 3 and a second ketone or hydroxyl function in position 6 or 7 was required for inhibitory activity with two kinds of cell culture. A 3β5α6β-triol was not more inhibitory than a comparable 3β,6β-diol. Cholestance-3β 5α-diol inhibited sterol synthesis in L cells but not in liver cell cultures. The inhibitory activities of 7-oxygenated sterols were not markedly affected by the presence of a double bond in position 4 or 5. Current knowledge of the mechanism through which the oxygenated sterols suppress cholesterol synthesis is reviewed.     

Metabolism of plant sterols by nematodes

Parasitic nematodes do not biosynthesize sterolsde novo and therefore possess a nutritional requirement for sterol, which must be obtained from their hosts. Consequently, the metabolism of phytosterols by plant-parasitic nematodes is an important process with potential for selective exploitation. The sterol compositions of several species of plant-parasitic nematodes were determined by capillary gas chromatography-mass spectrometry and compared with the sterol compositions of their hosts. Saturation of the phytosterol nucleus was the major metabolic transformation performed by the root-knot nematodesMeloidogyne arenaria andM. incognita and the corn root lesion nematode,Prytalenchus agilis. In addition to saturation, the corn cyst nematode,Heterodera zeae, dealkylated its host sterols at C-24. Because free-living nematodes can be cultured in sterol-defined artificial medium, they have been successfully used as model organisms for investigation of sterol metabolism in plant-parasitic nematodes. Major pathways of phytosterol metabolism inCaenorhabditis elegans, Turbatrix aceti andPanagrellus redivivus incleded C-24 dealkylation and 4α-methylation (a pathway unique to nematodes).C. elegans andT. aceti introduced double bonds at C-7, andT. aceti andP. redivivus saturated the sterol nucleus similarly to the plant-parasitic species examined. Several azasteroids and long-chain dimethylalkylamines inhibited growth and development ofC. elegans and also the Δ²⁴-sterol reductase enzyme system involved in the nematode C-24 dealkylation pathway. Based on a paper presented at the Symposium on Plant and Fungal Sterols: Biosynthesis, Metabolism and Function, heald at the AOCS Annual Meeting, Baltimore, MD, April 1990.     

The biosynthesis of cholesterol and other sterols by brain tissue: I. Subcellular biosynthesis in vitro

The biosynthesis of cholesterol by subcellular particles from rat brain was studied with several labeled cholesterol precursors as substrates. Rats from two age groups were used for preparation of the subcellular fractions: 15-day-old and adult. Microsomes and a soluble fraction were required for maximum biosynthesis of¹⁴C-nonsaponifiable material. The latter was synthesized in good yield by subcellular fractions from both age groups, but 90% or more was present as¹⁴C-squalene, when either U-¹⁴C-glucose, 2-¹⁴C-sodium acetate or 2-¹⁴C-mevalonic acid was the radioactive substrate. Neither³H-squalene oxide nor¹⁴C-lanosterol was converted to sterol when incubated with microsomal+soluble preparations, but some 4% of¹⁴C-desmosterol was converted to cholesterol by adult preparations. Thus a metabolic block, largely between squalene to desmosterol, exists in isolated microsomal+soluble preparations from both 15-day-old and adult rat brain. One of 12 papers to be published from the Sterol Symposium presented at the AOCS Meeting, New Orleans, April 1970.     

Diurnal variation of plasma methyl sterols and cholesterol in the rat: Relation to hepatic cholesterol synthesis

Free cholesterol of plasma low density lipoproteins (LDL) and high density lipoproteins (HDL) of the rat was high and that of plasma very low density lipoproteins (VLDL) was low during the dark period of the diurnal cycle. Variations in the esterified plasma sterols were inconsistent. Free methyl sterols were high in all lipoproteins during the dark phase. Simultaneously, the incorporation of¹⁴C-acetate into nonsaponifiable sterols and the concentrations of free methyl sterols and cholesterol in the liver were elevated.     

Effect of struture and form on the ability of plant sterols to inhibit cholesterol absorption in hamsters

We investigated the effect of three types of plant sterols (4-desmethylsterols, 4,4′-dimethylsterols, and pentacyclic triterpene alcohols) in three forms (free, esterified with FA, or with phenolic acids) on cholesterol absorption. Plant sterol fractions derived from soybean (99% 4-desmethylsterols), rice bran (70% 4,4′-dimethylsterols), or shea nut (89% pentacyclic triterpene alcohols) were fed to male hamsters (n=20/group) as free sterols or esterified with FA or phenolic acids (cinnamic or ferulic). Cholesterol absorption was measured after 5–8.5 (mean, 7) wk by a dual-isotope technique. Soybean sterol intake significantly reduced cholesterol absorption efficiency (23%) and plasma total cholesterol (11%). Rice bran sterols tended to lower cholesterol absorption efficiency by 7% and plasma total cholesterol by 5%, whereas shea nut sterols had no effect. In hamsters, dietary 4-desmethylsterols were more effective than 4,4′-dimethylsterols in lowering cholesterol absorption and levels of cholesterol in blood. Pentacyclic triterpene alcohols had no effect on the absorption of cholesterol or on its level in blood. Esterification with FA did not impair the ability of 4-desmethylsterols and 4,4′-dimethylsterols to inhibit cholesterol absorption, whereas esterification with phenolic acids reduced this ability. This study supports the use of 4-desmethylsterols, esterified with FA to increase solubility, as the most effective cholesterol-lowering plant sterols in the diet.     

The biosynthesis of cholesterol and other sterols by brain tissue: II. A comparison of in vitro and in vivo methods

When microsomal + soluble preparations of adult or 15-day-old rat brains were incubated with 2-¹⁴C-mevalonic acid,¹⁴C-squalene accumulated. A metabolic block at the squalene to cholesterol stage was indicated. This prompted a comparison of all methods currently used to study cholesterol biosynthesis by brain tissue. Brain cell-free preparations from newborn, 15-day-old or adult rats accumulated¹⁴C-squalene in a similar manner, with either 2-¹⁴C-acetate or 2-¹⁴C-mevalonic acid as substrates. Homogenates and minced preparations from newborn or 15-day-old rats accumulated some 4,4-dimethyl sterols, but considerable conversion to free 4-desmethyl sterols (cholesterol) was evident. Sterol esters were also present in all the in vitro studies. In general, increased disruption of tissue resulted in decreased free 4-desmethyl sterol formation in vitro. Intraperitoneal injection of labeled acetate or mevalonate to newborn or 15-day-old rats produced labeled brain 4-desmethyl sterol with little accumulation of squalene or 4,4-dimethyl sterols, but the yields in brain were small compared to total amount of labeled material administered. At all ages intracerebral injection produced the best yield of labeled cholesterol for the amount of nonsaponifiable material formed. One of 12 papers being published from the Sterol Symposium, presented at the AOCS Meeting, New Orleans, April 1970.     

Cholestanol and plant sterols in pigeon skin

Cholestanol (4.6%) and plant sterols (0.2%) have been demonstrated, for the first time, in avian skin by argentation and gas liquid chromatography. In contrast to results of previous studies with rat and human skin, cholestanol represented a significant amount in the pigeon. The proportion of campesterol was always higher than that of β-sitosterol.     

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.     

Initial cholesterol uptake by everted sacs of rat small intestine: Kinetic and thermodynamic aspects

The kinetics of initial cholesterol uptake by everted rat proximal and distal small intestinal sacs were evaluated in vitro. The mucosal incubation solution consisted of 0.05 mM cholesterol solubilized in 4.8 mM sodium taurocholate micellar solution at pH 7.4. Experiments were performed at temperatures from 26 to 38 C. The rate of cholesterol uptake followed a linear relationship when plotted against time indicating an apparent zero-order kinetics mechanism for initial uptake. An Arrhenius plot of the results of uptake versus temperature remained linear over the entire range of temperatures studied. The large free energy of activation (20 kcal/mole) suggests that an energy barrier for cholesterol uptake exists at the enterocyte luminal cell membrane and may be an important limiting step in cholesterol uptake. It is proposed that a transient association between cholesterol and a component of the enterocyte luminal cell membrane is formed during initial uptake of cholesterol. The transient association may be an activated complex formed with proteins present at or within the luminal enterocyte cell membrane.     

Occurrence of plant sterols in aquatic vertebrates

Plant sterols were found by gas liquid chromatography in the sterols of five species of aquatic vertebrates; mackerel (Scomber japonicus), rainbow trout (Salmo gairdnerii), smelt (Osmerus dentex), sardine (Sardinops melanosticta) and chimera (Chimera phantasma). The sterols of chimera liver, sardine flesh and sardine viscera contained 9.0, 2.4 and 3.1% of C₂₈ and C₂₉ sterols in addition to 86.7, 96.6 and 95.2% of cholesterol. The occurrence of norcholestandienol, campesterol, β-sitosterol and C₂₈ stanol was shown by combined gas chromatography-mass spectrometry. Sperm whale (Physeter catodon) sterols consisted of more than 99% cholesterol with only traces of 22-dehydrocholesterol.     

The quantitative analysis of plant sterols

Two methods for the quantitative analysis of plant sterols have been described. In the first, cholesterol is used for control of recovery and gas liquid chromatography (GLC) analysis. When cholesterol is present in the sterol mixture, a radioactive standard (usually cholesterol or sitosterol) is used to control recovery, and coprosterol is used to monitor GLC. The methods are exemplified for nitrogenfixing root nodules and for chloroplasts, respectively.     

Concurrent oxidation of cholesterol and corn oil sterols in autoxidizing lipid films

A new, simple method based on assay of cholesterol is presented for study of the time course and extent of air oxidation of cholesterol. Using such a method, the air oxidation of 0.5–3.5% solutions of corn oil sterols and cholesterol in films of either corn oil or corn oil fatty acids has been followed by the author for periods of up to 3 months at room temperature and 2 weeks at 60°C. in the dark. Under these conditions it was shown that a) solid cholesterol is relatively stable to air oxidation; b) in lipid films cholesterol is readily subject to oxidative attack which appears to be closely linked to the stability of the lipid film itself to autoxidation; c) more than 90% conversion of cholesterol to oxidation products has been observed in such systems; d) the principal corn oil sterols are oxidized at the same rate as cholesterol, and esterification of the corn oil sterols did not affect the rate at which they were oxidized; and e) the rate of concurrent oxidation of cholesterol in corn oil and corn oil fatty acid films in independent of the concentration of cholesterol at low concentrations, 0.5–3.5%. The disappearance rate is dependent on the stability of the lipid film itself as shown by peroxide value. Presented at the 34th Fall Meeting, American Oil Chemists' Society, October 17–19, 1960, New York, N.Y. This investigation was supported in part by a P.H.S. research grant (H-4623) from the National Heart Institute, Public Health Service.     

Genetic defects in triacylglycerol and cholesterol uptake

Monogenic diseases of triacylglycerol and cholesterol uptake are comparatively rare. However, studying them has brought two benefits. First, studies of these diseases have clarified the different mechanisms whereby triacylglycerols and cholesterol are removed from the circulation. This is important as high circulating concentrations of both lipids are independent risk factors for cardiovascular disease. Second, knowledge of the mechanisms of triacylglycerol and cholesterol uptake has enabled nutritional and pharmaceutical approaches aimed at enhancing uptake to be developed. This has led to the development of statins a highly effective group of cholesterol lowering drugs although similarly effective drugs for lowering triacylglycerol levels have yet to be developed. While the majority of the population has benefited from these developments patients suffering from these monogenic diseases will probably have to await the development of gene therapy for an effective cure of their condition.     

Reactivity of key metabolic sterols in standard colorimetric assays for cholesterol

The reaction of lanosterol, desmosterol and 7-dehydrocholesterol, key intermediates in cholesterol biosynthesis, were-compared with cholesterol in 3 standard colorimetric assays for cholesterol based on formation of chomogens with acetic anhydride, ferric chloride and ferrous sulfate. Marked differences in the reaction of the sterols in the different assays were due both to formation of chomogens with qualitatively similar spectral patterns but with greatly different extinctions and to formation of chromogens with clearly different absorption maxima. For example, in all assays, cholesterol and desmosterol formed chromogens with very similar absorption spectra but with varying extinctions, whereas the lanosterol chromogen in all assays was different from cholesterol's in both absorption maxima and in extinctions. The findings show that attempts to measure tissue sterol levels by colorimetric methods can result in great errors when cholesterol is not the sole sterol. Also, the unique spectral properties of the lanosterol chromogen formed in the Liebermann-Burchard reaction (a sharp absorption peak at 450 nm) suggests the possible use of this method as a qualitative test for lanosterol.     

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.     

Isolation and identification of cholesterol α-oxide and other minor sterols in human serum

The isolation and identification of cholesterol α-oxide, coprostanol, β-sitosterol, cholest-4-en-3-one and cholesta-4, 6-dien-3-one from human serum are reported. Compounds were isolated by thin layer chromatography and were identified by gas liquid chromatography and gas chromatography-mass spectrometry (GC-MS). Data for standard sterols are also reported. The possible origins of these minor components and the significance of their presence are discussed. Throughout the paper the following nomenclature is used: cholesterol α-oxide, cholesterol-5α, 6α-epoxide; coprostanol, 5β-cholestan-3β-ol; cholestanol, 5α-cholestan-3β-ol; dihydrolanosterol, 5α-lanost-8(9)-en-3β-ol; β-Sitosterol, 24β-ethylcholest-5-en-3β-ol; cholesterol, cholest-5-en-3β-ol; coprostanone, 5β-cholestan-3-one.