FATTY ACID ABNORMALITY IN ADRENOLEUKODYSTROPHY

—Recent clinical and morphological evidence established that adrenoleukodystrophy is a distinct X-linked genetic disorder. Fatty acid compositions of lipids in the brain, adrenal and serum from seven patients were examined. Cholesterol esters of both brain and adrenal contained substantial proportions of fatty acids longer than C₂₂ (11.8–41.9% of total in the brain and 13.4-34.8% of total in the adrenal), while cholesterol esters from normal and pathological control specimens contained very little. These very long chain fatty acids were generally saturated in brain cholesterol esters but significant amounts of unsaturated long chain fatty acids were also present in adrenal cholesterol esters. The long chain fatty acids showed bell-shaped distribution with C₂₅ or C₂₆ at the peak. Ganglio-sides from patients’white matter also showed increased proportions of very long-chain fatty acids, up to 50% of the total. Qualitatively similar but much milder fatty acid abnormalities were also found in galactosylceramide of the brain. On the other hand, fatty acids and fatty aldehydes of brain glycerophospholipids, adrenal free fatty acids, triglycerides and glycerophospholipids were not abnormal. Furthermore, serum cholesterol esters from two patients did not show the long-chain fatty acid abnormality found in brain and adrenal cholesterol esters. Sequential extractions with acetone and hexane established that the characteristic birefringent material in the brain and adrenal is indeed cholesterol esters with very long chain fatty acids. This type of fatty acid abnormality has not been described in other pathological conditions and may well represent the unique biochemical abnormality that is directly related to the fundamental genetic defect underlying adrenoleukodystrophy.     

Synthesis of fatty acid sterol esters using cholesterol esterase from Trichoderma sp. AS59

We recently reported the characterization of novel cholesterol esterase (EC. 3.1.1.13) from Trichoderma sp. and preliminary work on sterol ester synthesis. In the present study, we further examined the enzyme ability to synthesize cholesterol esters from cholesterol and free fatty acids of various chain lengths, and compared the fatty acid specificity in synthesis with that in hydrolysis. The enzyme catalyzed the synthesis of medium- and long-chain fatty acid cholesterol esters, but failed to synthesize short-chain fatty acid esters. The fatty acid specificities in the synthesis and hydrolysis of cholesterol esters were entirely different from each other. Unlike other lipolytic enzymes, the enzyme was largely independent of water content in the synthesis of cholesterol oleate, and it achieved near-complete esterification in the presence of an equimolar excess of oleic acid. Of additional interest is the finding that the addition of n-hexane markedly enhanced the esterification activities on all the medium- and long-chain saturated fatty acids used. Based on these findings, we attempted to synthesize stigmasterol stearate as a food additive to lower cholesterol levels in blood plasma, and found that the enzyme catalyzed effective synthesis of the ester without the need of dehydration during the reaction, indicating the potential utility of the enzyme in the food industry.     

Changes in plasma lipid composition induced by coconut oil. Effects of dipyridamole

The comparative effects of 10-20% coconut oil feeding on fatty acid composition of the main lipid classes of chick plasma have been studied with and without simultaneous treatment with dipyridamole in order to clarify the hypolipidemic role of this drug. Coconut oil drastically increased the percentages of lauric and myristic acids in free fatty acid and triacylglycerol fractions, whereas these changes were less pronounced in phospholipids and cholesterol esters. The percentage of arachidonic acid was higher in plasma phospholipids than in the other fractions and was significantly decreased by coconut oil feeding. Linoleic acid, the main fatty acid of cholesterol esters, was drastically increased by coconut oil feeding. Changes induced by the simultaneous administration of dipyridamole were more pronounced in the phospholipids and cholesterol esters than in the other fractions. The fall observed in linoleic acid levels after dipyridamole treatment may be of interest for a lower production of its derived eicosanoids, especially in plasma phospholipids and cholesterol esters.     

Synthesis and degradation of fatty acid ethyl esters by cultured hepatoma cells exposed to ethanol

Fatty acid ethyl esters are a family of neutral lipids that are the products of esterification of fatty acids with ethanol. Unlike other pathways of ethanol metabolism, ethyl esters are present in numerous human organs which are the targets of ethanol-induced damage. In the present study, we have shown that fatty acid ethyl esters are synthesized by a hepatoma cell line in tissue culture when exposed to ethanol concentrations easily attained by man during social drinking. Unlike alcohol dehydrogenase, the enzyme(s) responsible for synthesis of ethyl esters are membrane-bound and concentrated in the microsomal fraction of rat hepatocytes. In addition, fatty acid ethyl esters are hydrolyzed to free fatty acids and ethanol by membrane-bound enzyme(s) that are enriched in the microsomal and mitochondrial-lysosomal fractions. Intracellular hydrolysis of fatty acid ethyl esters release free fatty acids which are preferentially incorporated into cellular cholesterol esters. Thus, we have shown that a hepatocellular line exposed to concentrations of ethanol easily achieved in man by social drinking utilize endogenous fatty acids to form long-lived ethanol metabolites, fatty acid ethyl esters. Importantly, this family of neutral lipids may act as biochemical mediators of ethanol-induced cell damage, including the changes in cholesterol metabolism noted in chronic alcoholics.     

Concentration and fatty acid composition of cholesteryl esters of normal human brain

Abstract— —Cholesteryl esters were isolated from the cerebral cortex and white matter of human brains at different ages, and their concentration and composition determined. The esters were separated from other lipids by chromatography on silicic acid and finally purified by TLC. The fatty acids were converted to the methyl esters by alkaline trans-methylation and analysed by GLC. A TLC method was elaborated for quantitative determination of small amounts of cholesteryl esters in the presence of free cholesterol. The concentration of cholesteryl esters was only 0·1–0·2 per cent of the total cholesterol content of cerebral tissue in older children and adults. During early myelination the concentration was many times greater, especially in the white matter but it never exceeded 2 per cent of the total cholesterol in any subject. The major fatty acids of human brain cholesteryl esters were oleic, palmitic, palmitoleic and arachidonic acid. After completion of myelination, arachidonic acid constituted the major fatty acid. There were fairly small differences in the fatty acid pattern of the cholesteryl esters between grey and white matter, but the concentration of polyunsaturated fatty acids was larger in the grey matter. Cholesteryl esters appear to play an important role in the metabolism of the phosphoglyceride fatty acids in cerebral tissue.     

Cholesterol esters in developing rat brain: concentration and fatty acid composition

Abstract— The contents and the fatty acid composition of cholesterol esters were analysed in developing rat brain. The total content did not exceed 20 μg/brain throughout development. Elimination of serum by adequate perfusion was essential for accurate results. Two separate events appeared to affect the levels of cholesterol esters in developing rat brain, one probably reflecting general developmental changes and the other apparently related to myelination. On either a unit weight or a whole brain basis, the curves appeared to be a superimposition of the two events. There was an underlying developmental change, which was characterized on a unit weight basis by the highest level of cholesterol esters immediately after birth and a steady decline to the adult level by 30 days of age or which on the basis of whole brain was characterized by a steady increase throughout the development. A period of transient increase was superimposed on this underlying developmental change between the ages of 7 and 27 days and corresponded to the period of active myelination. The major fatty acids of rat brain cholesterol esters were palmitic, palmitoleic, oleic and arachidonic acids. Palmitic and palmitoleic acids decreased in proportion while oleic acid increased, as the animal matured. The fatty acid composition of serum cholesterol esters was distinctly different from that of brain cholesterol esters; those from serum contained much higher proportions of linoleic and arachidonic acids and much less palmitoleic and oleic acids.     

One-step extraction of human erythrocyte lipids allowing rapid determination of fatty acid composition

A one-step extraction procedure to directly quantify cholesterol, phospholipids, and fatty acids in red blood cell membranes has been developed. The method uses a single solvent, isopropanol, which extracts lipids and allows the rapid formation of isopropylic esters of fatty acids by acid catalysis. The efficiency of this new technique has been confirmed by comparing yields of cholesterol and total and individual phospholipids with yields obtained following conventional extraction procedures. Moreover, in comparison to the formation of methyl esters, we demonstrate that directly obtained isopropylic esters immediately allow the quantitative determination of fatty acids, without involving the hydrolytic degradation of fatty acids and the oxidation of unsaturated fatty acids.     

Myelin Membrane from Adrenoleukodystrophy Brain White Matter—Biochemical Properties

Adrenoleukodystrophy (ALD) is an X-linked progressive neurological disorder characterized by the accumulation of saturated very-long-chain fatty acids (C24 to C30) in lipids, especially cholesterol esters of the brain white matter and adrenal cortex. In the present study we have investigated the localization of accumulated cholesterol esters in brain white matter. During isolation of purified myelin membrane from regions of active demyelination, significant enrichment in cholesterol ester was found in two fractions, mainly in a low-density floating fraction and to a lesser degree in the purified myelin preparation. The fatty acid composition of cholesterol esters from both the ALD floating and myelin fractions was enriched approximately 10-fold in saturated very-long-chain fatty acids (greater than or equal to C24) compared with control preparations.     

Lipid compounds of the umbilical cord vein and their alterations in preeclampsia

The lipid composition of vascular walls changes during development, ageing and pathological processes. Preeclampsia is the most common pregnancy-associated pathological syndrome. It is accompanied by significant remodelling of the extracellular matrix, both in the umbilical cord vessels and in the surrounding Wharton's jelly. Lipids of the umbilical cord have not been extensively studied. Here we evaluate the lipid composition of the umbilical cord vein and its alteration in preeclampsia. Thin layer chromatography and high-performance liquid chromatography were employed for these analyses. It was found that the umbilical cord vein wall, as with most human tissues, contains free fatty acids, mono-, di- and triacylglycerols, free cholesterol and its esters. The characteristic feature is the presence of high amounts of monounsaturated fatty acids, mainly myristoleic acid (C14:1) and oleic acid (C18:1), and polyunsaturated fatty acids, including eicosapentaenoic acid (C20:5) and docosahexaenoic acid (C22:6), which are rather minor lipid components of most human tissues. They exist both in a free form and in a form of acylglycerols and cholesterol esters. Preeclampsia is associated with an increase in the accumulation of free fatty acids, acylglycerols and cholesterol esters in the umbilical cord vein wall, with a proportional reduction in unsaturated fatty acid contents in all the investigated lipid fractions. Total amount of myristoleate was similar to control values. It is suggested that stimulation of lipolysis in maternal tissues increases supply of free fatty acids to foetal blood and promotes the accumulation fatty acids and their esters in some foetal vascular walls.     

Cholesterol Esters in Rat Brain Infected with Acute Measles Encephalitis: Concentration and Fatty Acid Composition

The present study deals with the concentration and fatty acid composition of cholesterol esters in rat brains infected experimentally with measles virus to induce acute encephalitis. The left side of the cerebrum, as well as other portions of the brain, when inoculated percutaneously contained a large amount of cholesterol esters. The major fatty acids from the esters in the brain were C16:0, C16:1, C18:0, and C18:1; those from the serum were C18:1, C18:2, and C20:4. This result indicates that cholesterol esters may not come from serum but can be synthesized in situ, even in the brain with acute viral infection.     

Accumulation of oleate-rich cholesteryl esters by acetyl-LDL in macrophages in the presence of an acyl-CoA: cholesterol acyltransferase inhibitor (Sandoz 58-035)

The mechanism through which cholesteryl esters rich in oleic acid accumulate in the cytoplasm was studied. The fatty acid composition of the cholesteryl esters in acetyl-LDL was high in linoleic acid, while that of cholesteryl ester inclusion bodies accumulated in the cytoplasm was high in oleic acid. This compositional change of fatty acids in cholesteryl esters occurred even in the presence of an acyl-CoA: cholesterol acyltransferase (ACAT) inhibitor, Sandoz 58-035. These results suggest that oleate-rich cholesteryl esters accumulated in the cytoplasm, even though the reesterification in microsome was inhibited by an ACAT inhibitor.     

Fatty acid composition of cholesterol esters in brains of patients with Schilder's disease, G M1 -gangliosidosis and Tay-Sachs disease, and its possible relationship to the -position fatty acids of lecithin

Abstract— Cholesterol esters were isolated from cerebral cortex and white matter of patients with Schilder's disease, G_M1-gangliosidosis and Tay-Sachs disease, and the fatty acid composition was determined by gas-liquid chromatography. The fatty acid composition was similar among the three pathological conditions, but it was entirely different from that reported for cholesterol esters of normal brain. Lecithin and ethanolamine phospholipids were isolated from the same brain specimens, treated with snake venom phospholipase A, and the fatty acids at the a’and β-positions of the glycerol moiety were determined separately. The fatty acid composition of cholesterol esters was similar to that of the β-position fatty acids of lecithin of white matter in all samples, and was quite different from those of the a'-position of lecithin, or of the a’or β-position of ethanolamine phospholipids. The results indicate that the source of fatty acids for cholesterol esterification in nonspecific sudanophilic demyelination is different from that in normal brain, and that the most likely source is the β-linked fatty acids of lecithin. There are two possible enzymic mechanisms; activation of phospholipase A and subsequent esterification of the liberated β-position fatty acids to cholesterol, or direct transacylation by lecithin-cholesterol acyl transferase.     

Fatty acid esterification of lipoprotein-associated estrone in human plasma and follicular fluid

Estrogen fatty acid esters constitute a unique family of extremely hydrophobic hormonal derivatives which are exclusively transported in lipoprotein particles in plasma. In estradiol, the fatty acyl residues are conjugated at the 17beta-hydroxyl of the steroid D-ring, leaving the phenolic 3-hydroxyl group unsubstituted and, therefore, preserving antioxidative efficacy. The 17beta-fatty acid derivative of estradiol is proposedly an even more efficient antioxidant protecting LDL and HDL than the parent steroid. Previous studies have established that the enzyme lecithin:cholesterol acyltransferase which catalyzes the fatty acid esterification of 3beta-hydroxyl group of cholesterol, also catalyzes the formation of estrogen 17beta-esters. Estrone, the principal estrogen in the postmenopausal female, has a keto group at carbon-17 and has been thought unable to form fatty acid esters. However, we detected hydrophobic derivatives of estrone following incubations with human plasma and ovarian follicular fluid. These derivatives accumulated in HDL and LDL during incubation showing chemical characteristics similar to estrone-3-fatty acid esters. Liquid chromatographic-mass spectrometric analyses established the presence of unhydrolyzed estrone esters consisting of different fatty acid species, the major one being estrone-3-linoleate, in human HDL particles following incubation of estrone with plasma. These extremely hydrophobic estrone conjugates could, in theory, represent a storage form of this estrogen.     

Metabolism of doubly-labeled chylomicron cholesteryl esters in the rat

Chylomicrons labeled in vitro with doubly-labeled cholesteryl esters were injected intravenously into fasted rats, and the tissue distribution and chemical form of each isotope were observed for 24 hr. The use of doubly-labeled cholesteryl esters provided information about the metabolism of both the sterol and the fatty acid moieties. Similar results were obtained with doubly-labeled cholesteryl palmitate, oleate, and linoleate. In each instance, most (80-90%) of the chylomicron cholesteryl ester was removed from the plasma by the liver; small amounts were also taken up by all other tissues examined. There was no hydrolysis during uptake. In the liver the newly absorbed cholesteryl esters underwent slow hydrolysis (60% after 1 hr and 85-90% after 3.5 hr); the rate of reesterification of the liberated cholesterol was still slower. After 24 hr only 20-28% of the labeled cholesterol present in the animal was found in the liver. Labeled fatty acid disappeared from the liver, and was redistributed among other tissues, much more rapidly than the labeled cholesterol. Most of the labeled fatty acid apparently underwent oxidation, since only 15-20% of the injected labeled fatty acid was present in the animal after 24 hr. At this time the three fatty acids were differently distributed between and within the tissues. These differences reflected some known differences of fatty acid concentration and lipid composition in the various tissues.     

Modification of CaCo-2 cell membrane fatty acid composition by eicosapentaenoic acid and palmitic acid: effect on cholesterol metabolism

Membrane fatty acid composition of CaCo-2 cells was modified by incubating the cells for 8 days in medium containing 100 microM eicosapentaenoic acid or palmitic acid. The effect of membrane fatty acid changes on cholesterol metabolism was then studied. Cells incubated with eicosapentaenoic acid had significant changes in membrane fatty acid composition with an accumulation of 20:5 and 22:5 and a reduction in monoenoic fatty acids compared to cells grown in palmitic acid. Intracellular cholesteryl esters could not be detected in CaCo-2 cells grown in the presence of the n-3 polyunsaturated fatty acid. In contrast, cells incubated with the saturated fatty acid contained 2 micrograms/mg protein of cholesteryl esters. Cells grown in eicosapentaenoic acid, however, accumulated significantly more triglycerides compared to cells modified with palmitic acid. The rate of oleic acid incorporation into triglycerides was significantly increased in cells incubated with eicosapentaenoic acid. CaCo-2 cells modified by eicosapentaenoic acid had lower rates of HMG-CoA reductase and ACAT activities compared to cells modified with palmitic acid. The incorporation of the two fatty acids into cellular lipids also differed. Palmitic acid was predominantly incorporated into cellular triglycerides, whereas eicosapentaenoic acid was preferentially incorporated into phospholipids with 60% of it in the phosphatidylethanolamine fraction. The data indicate that membrane fatty acid composition is significantly altered by growing CaCo-2 cells in eicosapentaenoic acid. These modifications in membrane fatty acid saturation are accompanied by a decrease in the rates of cholesterol synthesis and cholesterol esterification.     

Incorporation of lipoxygenase products into cholesteryl esters by acyl-CoA:cholesterol acyltransferase in cholesterol-rich macrophages.

Macrophages which were incubated with acetylated low-density lipoproteins, resulting in cholesteryl ester accumulation, incorporated the monohydroxyeicosatetraenoic acids (5-, 15-, and 12-HETEs) into cholesteryl esters. The esterification of these hydroxy fatty acids to cholesterol by total membrane preparations of cholesterol-rich macrophages was dependent on the synthesis of the fatty acyl-CoA derivative, and was catalysed by acyl-CoA:cholesterol acyltransferase (ACAT). Stimulation of membrane ACAT activity by 25-hydroxycholesterol increased the synthesis of cholesteryl 12-HETE by 40%. In contrast, inhibiting ACAT activity by progesterone and compound 58-035 decreased cholesteryl 12-HETE production by 60% and 90% respectively. Although 5-, 15- and 12-HETE were esterified to cholesterol by ACAT, these monohydroxy fatty acids were less optimal as substrates compared with oleic acid or arachidonic acid. The hydrolysis and release of 12-HETE and the other monohydroxyeicosatetraenoic acids from intracellular cholesteryl esters and phospholipids occurred at a faster rate than for the more conventional fatty acids, oleate and arachidonate. Cholesteryl esters which contain hydroxy fatty acids therefore provide only a transient storage for lipoxygenase products, as these fatty acids are released into the medium as readily as hydroxy fatty acids found in phospholipids and triacylglycerols. The data provide evidence, for the first time, of an ACAT-dependent esterification of the lipoxygenase products 5-, 15- and 12-HETEs to cholesterol in the macrophage-derived foam cell. The channelling of these monohydroxy fatty acids to cholesteryl esters provides a mechanism which can alter the amount of lipoxygenase products incorporated into cellular phospholipids, thus averting deleterious changes to cell membranes. ACAT, by catalysing the esterification of monohydroxyeicosatetraenoic acids to cholesterol, could play a key role in regulating the amount of lipoxygenase products in the pericellular space of the cholesterol-enriched macrophage.     

Determination of plasma free fatty acids, free cholesterol, cholesteryl esters, and triacylglycerols directly from total lipid extract by capillary gas chromatography

An accurate capillary gas chromatographic method using different internal standards for determining free fatty acids, cholesterol, cholesteryl esters, and triacylglycerols in plasma and other biological sources is described. It is designed to give information about species composition and, consequently, more detailed information about changes in lipid metabolism of patients suffering from metabolic disorders. After plasma extraction the lipids, except phospholipids, are directly examined without any further derivatization. For free fatty acid determination the programmed temperature vaporizer (PTV) injector was heated from 40 degrees C (sample introduction) to 190 degrees C. In a second gas chromatographic run the PTV-injector system was heated from 60 degrees C (sample introduction) to 400 degrees C, enabling the determination of free cholesterol, cholesteryl esters, and triacylglycerol species, differing in the number of carbon atoms. Evaluation of the values obtained resulted in coefficients of variation (%) of 1.0-2.8, 2.0, 1.29-2.24, and 2.8, for free fatty acid standards, plasma free fatty acids, cholesterol and cholesteryl ester standards, and plasma total cholesterol, respectively. Free fatty acids, cholesterol, and cholesteryl esters were not influenced by storage of plasma at -24 degrees C up to 4 days prior to extraction. The results of the gas chromatographic method and the enzymatic methods correlated well. Determination by gas chromatography yielded higher total cholesterol and lower triacylglycerol values than those values obtained by enzymatic methods.     

Altered cholesterol ester proportions in embryonic tissues of dystrophic chicken

The concentrations of cholesterol esters in tissues of dystrophic chicken embryos are altered from normal. These changes are accompanied by significant changes in the proportions of the esterified fatty acids (the fatty acid profile). In serum and pectoral muscles there is a shift to a higher proportion of unsaturated fatty acids (in particular 18:1). Thigh muscle esters are little changed and in liver and brain the proportion of unsaturated fatty acids decreases.     

Cholesterol esterification in rabbit plasma

1. When [4-(14)C]cholesterol, attached to beta-globulin or dispersed with Tween 20, was incubated with fresh rabbit (New Zealand albino females) plasma, 30-47% esterification was observed. The optimum pH was 6.8. This esterification was accomplished by the transfer of fatty acids from the C-2 position of lecithin (phosphatidylcholine) to cholesterol. 2. There was no evidence that triglycerides or free fatty acids participated directly in this reaction. Lecithins with labelled palmitic acid, oleic acid and linoleic acid in the 2-position yielded 3.2, 4.8 and 6.8% of cholesteryl esters respectively. This pattern reflects that which is normally observed in the cholesteryl esters of rabbit plasma and supports the concept that plasma cholesteryl esters originate from the plasma. 3. Snake venom (containing phospholipase A), sulphoevernan [an alpha-(1-->3,1-->4)-sulphopolyglucan with 12% sulphur], thiol-blocking agents (p-chloromercuribenzoate and N-ethylmaleimide), or an atherogenic diet (stock diet supplemented with 1% cholesterol for 8 weeks) were all effective inhibitors of this cholesterol esterification.     

Separation and detection of neutral lipids and free fatty acids in a liver extract by high-performance liquid chromatography

A relatively simple method to determine hepatic neutral lipids and free fatty acids by highperformance liquid chromatography is described. This method involves the preparation of a chloroform extract of the total liver lipids, followed by removal of the phospholipids by adsorption onto silicic acid and elution of neutral lipids and free fatty acids with 50% diethyl ether in hexane. This fraction is then subjected to liquid-solid chromatography with a solvent system of 2,2,4-trimethylpentane (isooctane):tetrahydrofuran:formic acid (90:10:0.5) and is detected by refractive index. Cholesterol esters, fatty acids, cholesterol, and diglycerides each elute as single peaks, easily quantitated by comparison to appropriate standards. Baseline separation of triglycerides from cholesterol esters is also achieved.