Cholesterol Ester Fatty Acids Research Articles

Comparative effects of the oral contraceptive combinations 0.150 mg desogestrel + 0.030 mg ethinyloestradiol and 0.150 mg levonorgestrel + 0.030 mg ethinyloestradiol on lipid and lipoprotein metabolism in healthy female volunteers

An open, randomized cross-over study, comparing the effects of the oral contraceptive combinations 0.150 mg desogestrel (Org 2969) + 0.030 mg EE ∗ ∗ Abbreviation for ethinyloestradiol. and 0.150 mg levonorgestrel + 0.030 mg EE on lipid and lipoprotein metabolism, was carried out in healthy female volunteers. The study covered 9 cycles; a pretreatment cycle was followed by 6 treatment cycles (3 with one combination and 3 with the other combination), which in turn were followed by 2 post-treatment cycles. Eight volunteers received desogestrel + EE followed by levonorgestrel + EE and 7 volunteers received levonorgestrel + EE followed by desogestrel + EE. Each treatment cycle consisted of 21 days of tablet administration followed by a 7-day tablet-free period. Free and total cholesterol, phospholipids, triglycerides, lipoprotein-lipids, lecithinfatty acids and cholesterol ester-fatty acids were determined in serum samples obtained once before treatment, after both contraceptive periods and two months after the last treatment cycle. The effects of desogestrel + EE and levonorgestrel + EE on the above parameters, and on body weight and blood pressure were compared statistically, using an analysis of variance.

Increased synthesis of hexacosanoic acid (C23:0) by cultured skin fibroblasts from patients with adrenoleukodystrophy (ALD) and adrenomyeloneuropathy (AMN).

We studied the metabolism of radioactive stearic acid by cultured skin fibroblasts from patients wtih adrenoleukodystrophy (ALD) and its variant, adrenomyeloneuropathy (AMN), to clarify the mechanism of the increased content of very long chain saturated fatty acids in cholesterol esters and sphingolipids, which are known to be the characteristic biochemical changes in ALD and AMN. A substantial amount of hexacosanoic acid (C26 : 0) wa synthesized from stearic acid by ALD and AMN fibroblasts, whereas only a trace amount of hexacosanoic acid was synthesized by control fibroblasts. This indicates that the primary biochemical defect in ALD and AMN may involved the elongation system of very long chain fatty acids.

Accumulation of very long chain fatty acids is common to 3 variants of adrenoleukodystrophy (ALD): “Classical” ALD, atypical ALD (female patient) and adrenomyeloneuropathy

Fatty acids of cholesterol esters were analyzed by gas chromatography in affected CNS white matter of 3 variants of ALD (“classical” ALD, atypical ALD (adult female) and AMN) and of 10 controls with myelin breakdown of an etiology other than ALD. In all 3 ALD variants a marked accumulation of very long chain fatty acids (VLFA) as compared to control material was observed. This was due to the accumulation mainly of saturated C 24–C 32 fatty acids, particularly of C 26:0, C 25:0 and, to a lesser extent, C 24:0 and C 27:0 fatty acids. Our results demonstrate for the first time an accumulation of VLFA in an adult female patient (atypical ALD), who probably is an ALD heterozygote rather than a variant of AMN, and confirm and extend earlier findings in classical ALD and AMN, respectively. It appears that ALD may be a single nosological entity with clinically and morphologically different variants sharing specific ultrastructural (accumulation of paired leaflets) and neurobiochemical (accumulation of VLFA) diagnostic markers.

Gas Chromatographic Resolution of Fatty Acid Esters of C27 -C29 Sterols with 5:6-Olefinic Structures Based on the Degree of Unsaturation of Acyl Groups

Gas chromatography (GC) of common fatty acid esters of cholesterol, β-sitosterol, stigmasterol, and campesterol was carried out on SILAR 10C. The esters having acyl groups of C12-C22 chain lengths and 0–6 double bonds were characterized by equivalent chain lengths (ECL). Equations to calculate the ECL values of saturated and monoenoic esters of C27-C29 sterols with 5:6-olefinic structures are presented. The effect of the column temperature on the ECL of the esters of unsaturated acids and the application to quantitative analyses are discussed.

Studies on the Structure of Low Density Lipoproteins Isolated from Macaca Fascicularis Fed an Atherogenic Diet

Cynomolgus monkeys, Macaca fascicularis, fed cholesterol-containing saturated-fat diets develop increased levels of high molecular weight plasma low density lipoproteins (LDL), associated with accelerated atherosclerosis. To study the composition and structure of these abnormal particles, LDL from monkeys, fed atherogenic and control diets, were characterized chemically and examined by differential scanning calorimetry and low-angle X-ray scattering. LDL from animals on the experimental diet showed an increase in molecular weight (4.0 to 7.0 x 10(6), experimental diet compared with 3.0 to 3.7 x 10(6), control diet) associated with a large increase in cholesterol ester content and concomitant smaller increases in protein, phospholipid, and free cholesterol. There was a strong positive correlation between molecular weight and the number of saturated and monounsaturated cholesterol esters in the particle. In contrast, particle content of polyunsaturated cholesterol esters remained constant despite large changes in total particle cholesterol esters.When examined by calorimetry and X-ray scattering, LDL from monkeys on both diets diplayed a reversible transition of cholesterol esters from an ordered smeticlike (layered) structure to a more disordered state. For all animals on the experimental diet, the peak temperature of the cholesterol-ester transition (42-48 degrees C) was above body temperature (39 degrees C), but below body temperature on the control diet (34-38.5 degrees C). In the experimental group, the transition temperature was correlated with the LDL molecular weight. However, after thermal disruption of LDL, liquid-crystalline transitions of LDL cholesterol esters were observed in the same temperature range as in the intact lipoprotein, which shows that changes in particle size had little effect on the cholesterol-ester transition temperature. Rather, the transition temperature was determined by the degree of saturation of the LDL cholesterol ester fatty acids and the LDL cholesterol ester: triglyceride ratio, both of which correlated with increased LDL molecular weight.The existence of smectic-like cholesterol ester in LDL at body temperature was clearly a discriminating feature between monkeys on control and experimental diets. Diet-induced changes in the lipid composition of precursor lipoproteins of LDL appeared to lead to the existence of smectic-like cholesterol ester in LDL above body temperature. The altered composition and structure of the core lipids of high molecular weight LDL probably account, in part, for the previously documented correlation between increased LDL molecular weight and atherosclerosis in this species.

ACTH-induced hydrolysis of cholesteryl esters in rat adrenal cells.

Rat adrenal cortical cells have been prepared by collagenase dissociation of trypsin-treated adrenal tissue. The content and compositions of cholesteryl ester, phospholipid, and triglyceride fatty acids compare favorably with those of undissociated rat adrenal tissue. During 2-hour control incubations of adrenal cortical cells, steroidogenesis was not detected, and the levels of sterol ester, phospholipid, and triglyceride fatty acids were not significantly altered. Incubations with adrenocorticotropic hormone (ACTH) resulted in coricosterone production and significant depletions of sterol ester and triglyceride fatty acids, but not of phospholipid fatty acids. Although all fatty acid esters of cholesterol were hydrolyzed under these conditions, the greatest contributions to the net decrease in sterol esters were by oleate, arachidonate, and adrenate. Incubations with dibutyryl cyclic adenosine monophosphate (0.5 mM) resulted in significantly greater levels of corticosterone production than did ACTH (250 muunits), but the effects on cellular lipids were comparable to those seen with the tropic hormone. This study represents the first demonstration of hormone-induced hydrolysis of sterol esters in an in vitro cell suspension system. The results are discussed with respect to hormone-sensitive sterol ester hydrolase of adrenal cortex, and to the role of endogenous cholesteryl esters in the steroidogenic pathway.

Composition and metabolism of lipid in macrophages from normally fed and cholesterol-fed rabbits

The composition and metabolism of lipid in peritoneal macrophages obtained from normally fed rabbits were compared with those of macrophages obtained from cholesterol-fed rabbits. Macrophages from cholesterol-fed rabbits had a higher cholesterol content and a markedly higher cholesterol ester content than normal macrophages. The increase in cholesterol ester content was most marked for cholesterol oleate and cholesterol linoleate, with the cholesterol ester fatty acid composition of the cholesterol-fed macrophages resembling that of foam cells derived from aortic lesions of similarly cholesterol-fed rabbits. Metabolic differences were also demonstrated between the cells obtained from normal and cholesterol-fed rabbits. In the latter, incorporation of 14C-labeled acetate into cholesterol was almost completely suppressed whereas in macrophages from normally fed rabbits, 14C-labeled acetate was incorporated predominantly into cholesterol. Incubation in vitro of normal macrophages for periods up to 20 hr with hyperlipemic serum, however, was not associated with any appreciable suppression of cholesterol synthesis.

Metabolic studies of adrenoleukodystrophy.

Two series of metabolic studies were prompted by the previous finding that the brain and adrenal tissues of patients with adrenoleukodystrophy, an X-linked genetic disorder, contain unusually long-chain (C22--C32) fatty acids in cholesterol esters and gangliosides. Postmortem brain tissues from three patients were assayed for activities of the three distinct cholesterol ester hydrolases, using [4-14C]cholesterol oleate, lignocerate and cerotate as the substrates. No deficiency of the crude mitochrondrial (pH 4.2), the microsomal (pH 6.0), or the myelin-localized cholesterol ester hydrolases was detected, although the activities of the myelin-localized cholesterol ester hydrolase against cholesteryl lignocerate and cerotate were too low for reliable assays. The activities of the microsomal and myelin-localized hydrolases were actually higher in adrenoleukodystrophy than in controls. Uptake and exclusion by cultured fibroblasts of [1-14C]stearic, [1-14C]lignoceric and [1-14C)cerotic acids were also examined. All fatty acids were avidly taken up by the fibroblasts. Stearic acid was excluded from the cells much more rapidly than lignoceric or cerotic acid. No difference was observed in the uptake and exclusion of fatty acids between the controls and adrenoleukodystrophy, except that cells from some cases of adrenoleukodystrophy consistently took up the very long chain fatty acids at greater rates than the control cells. Neither did the distribution of the label among individual lipids reveal differences between the controls and adrenoleukodystrophy, although there were interesting and dramatic differences in the metabolism of lignoceric acid and cerotic acid. Cerotic acid appeared largely inert with 90--95% remaining intact over eight days, while lignoceric acid was mostly incorporated into complex lipids. This series of studies did not uncover the fundamental genetic defect underlying adrenoleukodystrophy.

Adrenomyeloneuropathy

Histopathologic features were studied in postmortem examination of two men with adrenomyeloneuropathy, and biochemical studies were performed on one of these individuals. The histopathologic picture of one case included dying-back features in the nervous system and lamellar cytoplasmic inclusions in the brain, adrenal gland, and testis similar to those in adrenoleukodystrophy. Biochemical studies of the cerebral white matter of this individual revealed increased amounts of long-chain saturated fatty acids in cholesterol esters, an abnormality characteristic of adrenoleukodystrophy. Despite differences in clinical presentation and neuropathology, adrenomyeloneuropathy probably represents a distinct variant of adrenoleukodystrophy.

Removal of fatty acid labelled cholesterol ester, phospholipid and triglyceride from atherosclerotic rabbit aorta in vitro

An organ culture technique was used to investigate the removal of fatty acids incorporated into various lipid fractions in intimal segments of atherosclerotic rabbit aorta. Segments of aorta were pulse-labelled in vitro with [9,10- 3H]-oleic acid and [1- 14C]linoleic acid for 24 h. The incubation medium was replaced with non-radioactive medium and the removal of the endogenously labelled phospholipid, triglyceride and cholesterol ester was followed for a further period of 10 days. Fatty acids incorporated into phospholipid and triglyceride were removed rapidly from the aortic intima, with fractional half-times of 4–5 days. Cholesterol ester fatty acids were removed more slowly from the aortic intima with fractional half-times of 9–12 days. When the removal rate of oleic acid incorporated into phospholipid, triglyceride and cholesterol ester was compared with that of linoleic acid incorporated into the same lipid fractions, no difference between removal of the two fatty acids could be detected with respect to any of the 3 lipid fractions.

Ovipositional behavior ofBracon mellitor Say, a parasitoid of the boll weevil (Anthonomus grandis Boheman) III. Isolation and identification of natural releasers of ovipositor probing

The chemical cues in the frass of larvae of the boll weevil,Anthonomus grandis Boheman, that elicit an ovipositional response in females ofBracon mellitor Say were isolated by a combination of column, thin-layer, and gas-liquid chromatography. Derivatization, analytical tests, and mass spectrometry were used to identify the biologically active materials as long-chain fatty acid esters of cholesterol. Bioassays with natural and synthetic cholesteryl esters were used to confirm activity. The activity of the cholesteryl esters was also confirmed by using females that had not been previously exposed to hosts.

Cholesterol esterase produced by Streptomyces lavendulae.

Cholesterol esterase was purified from the culture filtrate of Streptomyces lavendulae by a procedure involving precipitation with ammonium sulfate and acetone, gel filtration on Sepharose CL-4B, and rechromatography on Sepharose CL-4B after treatment of Triton X-100. The purified enzyme was detected as a single band by polyacrylamide disc electrophoresis, while one main band and three minor bands were observed by SDS gel electrophoresis. The molecular weight of the main band was 29000. The enzyme was inhibited by Hg2+, Ag+ and DFP. Long-chain fatty acid esters of cholesterol were hydrolyzed preferentially and pH optimum was 6.0. This enzyme could be used to the determination of total serum cholesterol with cholesterol oxidase from Streptomyces violascens.

Strain-Dependent Gonadal Effects upon the Response of Adrenal Cholesterol Esters to ACTH in C57BL/10J and DBA/2J Mice

The response of adrenal cholesterol esters to ACTH administered in vivo was studied in males, females and male castrates of the C57BL)10J and DBA/21 strains of mice. The cholesterol ester fatty acids were transmethylated and analyzed by gas-liquid chromatography. There are sex and strain differences in the response of the adrenal cholesterol esters to ACTH. Neither the male nor the female C57 mouse shows a significant depletion of total cholesterol esters upon ACTH treatment. This result argues against the utilization of adrenal cholesterol esters for corticosteroid synthesis in this strain. In the female DBA mouse cholesterol ester depletion in response to ACTH is maximal. Neither the total cholesterol ester concentration nor the concentration of any individual ester is correlated with the pattern of response of the cholesterol esters to ACTH. Among the individual cholesterol esters, cholesteryl arachidonate (C20:4) is preferentially utilized after ACTH treatment in all groups except the C57 males. In contrast, cholesteryl adrenate (C22:4), which is generally the most abundant cholesterol ester, is conserved during ACTH stimulation in all groups except the DBA females. A cyclic interconversion between adrenate and arachidonate may be physiologically important in the control of adrenal function.

Stability of fatty acids in hyperlipoproteinemic plasma during long-term storage.

Aliquots of hyperlipoproteinemic plasma were stored at -20 degrees C in a nitrogen atmosphere with added disodium ethylenediaminetetraacetate for up to one year, to determine the stability of fatty acids in the four major lipid classes under these conditions. Assays were performed at zero time and at 2, 5, and 12 months. No uniform fatty acid change was found. Minor statistically significant changes were found in total triglyceride fatty acids and in some of the individual fatty acids in cholesterol esters, phospholipid, and free fatty acids. These minor changes could not be accounted for by plasma lipid concentrations, lipolysis, or fatty acid peroxidation. Evidently, plasma can be stored in this manner for as long as a year without substantial change in fatty acids in any of the four major lipid classes.

Studies on sterol metabolism by microorganisms. V. Purification and properties of cholesterol esterase from Pseudomonas fluorescens.

Cholesterol ester hydrolase (EC 3. 1. 1. 13) was purified about 10 times from the culture supernatant of Pseudomonas fluorescens ATCC 21156 by a procedure involving fractionations with acetone and ammonium sulfate, chromatography on hydroxylapatite, gel filtration on Sephadex G-100, and separation into two isoenzymes, I and II, by isoelectric focusing. The purified enzyme from the Sephadex G-100 fraction sedimented as a single peak on ultracentrifugation. The isoenzymes I and II showed isoelectric points of pH 3.8 and 4.9, respectively. They both had an apparent molecular weight of about 129, 000, hydrolyzed preferentially long-chain fatty acid esters of cholesterol, and exhibited a pH optimum at 7.3 with cholesterol linoleate as substrate. The Km values of I and II for cholesterol linoleate were 3.86×10-5 M and 1.43×10-4 M, respectively. The cholesterol esterase activity was remarkably stimulated when Triton X-100 was used to prepare the miscellar aqueous substrate, and further increase in activity was demonstrated by the addition of cholic acid to the miscellar system.

Composition of the lipids from the adrenal gland of Hippopotamus amphibius

1. 1. Lipids from the cortex and the medulla of the hippopotamus adrenal gland were extracted and separated into various neutral- and phospholipid fractions. The fatty acids of various individual fractions were investigated by gas-chromatography. 2. 2. Both cortex and medulla are characterized by a very high lipid content. Cortex lipids contain 59.6% and medullar lipids 23.3% cholesterol esters. 3. 3. Cholesterol ester fatty acids and free fatty acids from both cortex and medulla are very similar in composition. These fractions are characterized by a low content of homo-γ-linoleic, arachidonic and adrenic acids. The triglycerides contain significantly more palmitic acid. 4. 4. Phosphatidylcholine, phosphatidylethenolamine and phosphatidylinosotol from cortex do not differ in respect of their fatty acid composition from such phospholipids in other organs or species. Sphingomyelin from cortex and all phospholipid fractions from the medulla contain 9–71% of a long chain fatty acid tentatively identified as a tetracosanoic acid.

Synthesis of cholesterol esters in the plasma and liver of sheep

A study was made with sheep on the formation in vitro of long chain fatty acid esters of cholesterol by the lecithin-cholesterol-acyltransferase system present in the plasma and the acyl CoA-cholesterola cyltransferase system present in the liver. The rate of cholesterol esterification in the plasma was 0.024 mumoles/ml/hr. The relative pattern of fatty acids esterified during incubation of the plasma remained constant over the 8 hr period of incubation and was similar to the fatty acids in the plasma cholesteryl esters before incubation began and to the fatty acids in the 2-position of the plasma lecithin. The predominant cholesteryl esters synthesized contained monoenoic and dienoic fatty acids. Unlike the bovine, there was no apparent discrimination in favor of the 18:2 containing species of plasma lecithin as donors of fatty acids. This difference could be accounted for by the similarity in the 18:2 content of the phospholipids present in the high density (density greater than 1.062 and less than 1.21) and the low density (density greater than 1.006 and less than 1.063) lipoprotein fractions of the sheep plasma. The possibility of some discrimination against 20:4 during cholesterol ester synthesis in the plasma of the sheep cannot be excluded. In the liver, the predominant cholesteryl esters synthesized contained saturated and monoenoic fatty acids; cholesteryl linoleate was synthesized to a very much less extent. There was considerable similarity between the composition of the unesterified fatty acid fraction of the liver before incubation began and the fatty acid composition of the cholesteryl esters synthesized during incubation. Addition of sonicated suspensions of free fatty acidsaltered markedly the fatty acid pattern of the cholesteryl esters synthesized by the liver slices. From the evidence presented it is concluded that the cholesteryl esters in sheep plasma are syntheized mainly by the plasma lecithin-cholesterol-acyltransferase system. The results are discussed in relation to cholesterol esterification systems demonstrated in the plasma and liver of monogastric animals.

Dynamic Viscoelastic Properties of Cholesteric Liquid Crystals

Abstract Propagation of ultrasonic shear waves at frequencies from 2.4 to 18 MHz was studies in three 18-carbon unsaturated fatty acid esters of cholesterol, cholesteryl obleate (CO), cholesteryl linoleacte (CL), and cholesteryl linolenate (CLn), and three mixtures of the esters, 80% CL-20% CO, 95% CL-5% CO, and 80% CLn-20% CO. The oleate and linoleate esters are of biological importance because they comprise about 70–80% of the cholesteryl esters contained in atherosclerotic plaque. A shear wave pulse superposition technique was used to obtain the complex shear mechanical impedance and, hence, the dynamic shear viscosity and dynamic shear rigidity. The reflectance technique permits the study of liquid crystals whose long axes are oriented, in the plane of shear, with respect to the shearing direction of the ultrasonic wave. Falling ball viscosity measurements using a Hoeppler precision viscosimeter were made as a function of temperature from 35 tc 55°C. Shear wave measurements of dynamic shear rigidity and dynamic shear viscosity are shown as functions of temperature. This work represents the first measurement of both the dynamic viscosity and rigidity in a liquid crystalline substance. In the smectic phase, a dynamic shear rigidity is exhibited in all systems studied. The shear mechanical impedance of cholesteryl linoleate is found to be independent or orientation of the long axis of the molecule with respect to the direction of shear in agreement with the prediction of Brochard which is based on Leslie's hydrodynamic theory of the cholesteric state. The dynamic shear viscosity is found to be dependent on the shear rate. This may be explained in terms of a relaxation in the short range order present in the isotropic phase of both nematic and cholesteric compounds, as recently discussed by De Gennes.

Fatty Acid Biosynthesis in Mouse Brain Microsomal Fractions

In demyelinating conditions cholesterol is believed to be esterified with fatty acids released by phospholipase action on the phosphatidylcholine of the myelin (Ramsey, 1973). This supposition is supported by Eto & Suzuki (1971), who demonstrated a strikingly similar fatty acid composition in the /?-linked fatty acids of phosphatidylcholine to those of cholesterol esters in brain undergoing demyelination. Demyelination is probably an inexact description of at least one of the congenital myelin disorders that we have studied. Thus, as a result of an electron-microscopic study (W. F. Blakemore, personal communication), it has been suggested that in congenital tremor type AIV, myelination ceases when oligodendrocytes undergo degenerative changes and that myelin breaks down at an early stage of development. Additionally it has been demonstrated that in this condition cerebrosides contain an abnormal range of fatty acids and also that linked with the increase of the C18:1 fraction of the cholesterol ester fatty acids there is a decrease in this particular fatty acid fraction in whole lipid extracts of spinal cord (Patterson et al., 1973). Therefore the present observations showing that cholesterol esters possess an almost universally similar fatty acid pattern in many unrelated pathological states, including congenital tremor type AIV, lends further support to the observations of Eto & Suzuki (1971) that the fatty acids of cholesterol esters must presumably originate in myelin lipid fractions other than cerebroside, probably phospholipids.

Comparison of arterial lesions and serum lipids in spider and rhesus monkeys on an egg and butter diet

Serum and aortic lipids and atherosclerotic lesions were studied in five spider monkeys (Ateles sp.) and three rhesus monkeys ( Macaca mulatta) after changing from a commercial chow diet to a high fat (40% of calories), high cholesterol (1 mg/cal), semisynthetic diet containing egg yolk and butter. There was a large species difference in response of serum cholesterol. After 12 weeks on the diet the mean elevation of serum cholesterol was 285 mg/100 ml for the rhesus and 17 mg/100 ml for the spider monkeys. Changes in serum cholesterol ester fatty acid patterns were also very much greater for the rhesus than for the spider monkey. Aortas and coronary arteries of the rhesus were extensively involved with sudanophilic lesions. The cholesterol and cholesterol ester content of the aortic intima was much greater in the rhesus than in the spider monkey. The proportional contribution of aortic cholesterol oleate was greater in rhesus than spider monkeys and the palmitate was lower. There were only a few sudanophilic lesions in the coronary arteries of the spider monkeys. The coronary arteries of one older rhesus monkey were extensively involved with fibrous lesions. There was no appreciable difference between species in the histologic appearance of the sudanophilic lesions.