Long-chain acyl CoA synthetase in microsomes from rat brain gray matter and white matter

Long-chain acyl coenzyme A (CoA) synthetase in homogenates and microsomes from rat brain gray and white matter was studied. The formation of the thioesters of CoA was studied upon addition of [1-¹⁴C]-labeled fatty acids. The maximal activities were seen with linoleic acid, followed by arachidonic, palmitic, and docosahexaenoic acids in both gray and white matter homogenates and microsomes. The specific activities in microsomes were 3–5 times higher than in homogenates. The presence of Triton X-100 in the assay system enhanced the activity of long-chain acyl CoA synthetase in homogenates. The effect was more pronounced in palmitic and docosahexaenoic acid activation. The apparentK _m values andV _max values for palmitic and docosahexaenoic acids were much lower than for linoleic and arachidonic acids. The presence of Triton X-100 in the medium caused a definite decrease in the apparentK _m and Vmax values for all the fatty acid except palmitic acid in which case the reverse was true. There were no significant differences observed in the kinetic measurements between gray and white matter microsomes. These findings are similar to those resulting from the known interference of Triton X-100 in the measurement of kinetic variables of long-chain acyl CoA synthetase of liver microsomes. In this work, no correlation was observed between the fatty acid composition of gray and white matter and the capacity of these tissues for the activation of different fatty acids.     

Dietary lipid effects on microsome fatty acid composition of liver and brain, on liver glucose-6-phosphatase, and on brain 5'-nucleotidase activity in the rat

The effects of incorporation of dietary oils with different n6/n3 ratio and polyunsaturated fatty acids content into rat liver and brain microsomes has been studied. The investigation of membrane fatty acid composition of liver microsomes and that of brain microsomes gave different results. In particular, liver microsomes of rats fed fish oil showed a relatively higher content of 20:5n3 and 22:6n3, and a lower content of 20:4n6. Under these conditions, a reduced glucose-6-phosphatase activity was measured. Brain microsomal fatty acid composition was only slightly affected by dietary lipid intake. The 5'-nucleotidase activity of those particles was similar, although statistically different values were found in fish-oil-fed rats and in olive-oil-fed rats. The effects of membrane fatty acid composition on membrane-bound enzyme activity are discussed.     

The effects of unsaturated fatty acids on hepatic microsomal drug metabolism and cytochrome P-450

1. The effects of unsaturated fatty acids on drug-metabolizing enzymes in vitro were measured by using rat and rabbit hepatic 9000g supernatant fractions. 2. Unsaturated fatty acids inhibited the hepatic microsomal metabolism of ;type I' drugs with inhibition increasing with unsaturation: arachidonic acid>linolenic acid>linoleic acid>oleic acid. Inhibition was independent of lipid peroxidation. Linoleic acid competitively inhibited the microsomal O-demethylation of p-nitroanisole and the N-demethylation of (+)-benzphetamine. 3. The hepatic microsomal metabolism of ;type II' substrates, aniline and (-)-amphetamine, was not affected by unsaturated fatty acids. 4. The rate of reduction of p-nitrobenzoic acid and Neoprontosil was accelerated by unsaturated fatty acids. 5. Linoleic acid up to 3.5mm did not decelerate the generation of NADPH by rat liver soluble fraction, nor the activity of NADPH-cytochrome c reductase of rat liver microsomes. Hepatic microsomal NADPH oxidase activity was slightly enhanced by added linoleic acid. 6. No measurable disappearance of exogenously added linoleic acid occurred when this fatty acid was incubated with rat liver microsomes and an NADPH source. 7. The unsaturated fatty acids used in this study produced type I spectra when added to rat liver microsomes, and affected several microsomal enzyme activities in a manner characteristic of type I ligands.     

Effect of ciprofibrate on the activation and oxidation of very long chain fatty acids

The effect of ciprofibrate, a hypolipidemic drug, was examined in the metabolism of palmitic (C_16:0) and lignoceric (C_24:0) acids in rat liver. Ciprofibrate is a peroxisomal proliferating drug which increases the number of peroxisomes. The palmitoyl-CoA ligase activity in peroxisomes, mitochondria and microsomes from ciprofibrate treated liver was 3.2, 1.9 and 1.5-fold higher respectively and the activity for oxidation of palmitic acid in peroxisomes and mitochondria was 8.5 and 2.3-fold higher respectively. Similarly, ciprofibrate had a higher effect on the metabolism of lignoceric acid. Treatment with ciprofibrate increased lignoceroyl-CoA ligase activity in peroxisomes, mitochondria and microsomes by 5.3, 3.3 and 2.3-fold respectively and that of oxidation of lignoceric acid was increased in peroxisomes and mitochondria by 13.4 and 2.3-fold respectively. The peroxisomal rates of oxidation of palmitic acid (8.5-fold) and lignoceric acid (13.4-fold) were increased to a different degree by ciprofibrate treatment. This differential effect of ciprofibrate suggests that different enzymes may be responsible for the oxidation of fatty acids of different chain length, at least at one or more step(s) of the peroxisomal fatty acid -oxidation pathway.     

Characterization of liver cholic acid coenzyme A ligase activity. Evidence that separate microsomal enzymes are responsible for cholic acid and fatty acid activation.

Investigations on the cholic acid CoA ligase activity of rat liver microsomes were made possible by the development of a rapid, sensitive radiochemical assay based on the conversion of [3H]choloyl-CoA. More than 70% of the rat liver cholic acid CoA ligase activity was associated with the microsomal subcellular fraction. The dependencies of cholic acid CoA ligase activity on pH, ATP, CoA, Triton WR-1339, acetone, ethanol, magnesium, and salts were investigated. The hypothesis that the long chain fatty acid CoA ligase activity and the cholic acid CoA ligase activity are catalyzed by a single microsomal enzyme was investigated. The ATP, CoA, and cholic (palmitic) acid kinetics neither supported nor negated the hypothesis. Cholic acid was not an inhibitor of the fatty acid CoA ligase and palmitic acid was not a competitive inhibitor of the cholic acid CoA ligase. The cholic acid CoA ligase activity utilized dATP as a substrate more effectively than did the fatty acid CoA ligase activity. The cholic acid and fatty acid CoA ligase activities appeared to have different pH dependencies, differed in thermolability at 41 degrees, and were differentially inactivated by phospholipase C. Moreover, fatty acid CoA ligase activity was present in microsomal fractions from all rat organs tested while cholic acid CoA ligase activity was detected only in liver microsomes. The data suggest that separate microsomal enzymes are responsible for the cholic acid and the fatty acid CoA ligase activities in liver.     

Enzymes of Fatty Acid β-Oxidation in Developing Brain

Developmental profiles were determined for the activities of eight enzymes involved in fatty acid beta-oxidation in rat brain. The enzymes studied were the palmitoyl-CoA, octanoyl-CoA, butyryl-CoA, glutaryl-CoA, and 3-hydroxyacyl-CoA dehydrogenases, the enoyl-CoA hydratase (crotonase), and the C4- and C10-thiolases. With the exception of the thiolases, all of the activities (expressed on the basis of brain weight) increased during the postnatal period of brain maturation. The activity of octanoyl-CoA dehydrogenase was elevated markedly compared to that of palmitoyl-CoA dehydrogenase at all developmental stages and in all brain regions in the rat. A similar relationship between these enzymes was observed in various regions of adult human brain. Comparisons of the activities of the beta-oxidation enzymes in human brain versus human skeletal muscle and in cultured neural cell lines (neuroblastoma and glioma) versus cultured skin fibroblasts revealed that the elevated activity of octanoyl-CoA dehydrogenase relative to palmitoyl-CoA dehydrogenase was specific to the neural tissues. This relationship was particularly evident when the enzyme activities were normalized to the activity of crotonase. The data support previous findings with radiochemical tracers, indicating that the brain is capable of utilizing fatty acids as substrates for oxidative energy metabolism. The relatively high activity of the medium-chain fatty acyl-CoA dehydrogenase in neural tissue may represent an adaptive mechanism to protect the brain from the known encephalopathic effects of octanoate and other medium-chain fatty acids that readily cross the blood-brain barrier.     

Fatty acids bound to unilamellar lipid vesicles as substrates for microsomal acyl-CoA ligase

Palmitate incorporated into single-layered vesicles of phosphatidylcholine was used as a substrate for palmitoyl coenzyme A ligase (palmitoyl-CoA ligase) in microsomes from rat liver. This was done in order to avoid the use of detergents for dispersal of the water-insoluble palmitate and the possibility of precipitating palmitate added to the aqueous assay as a salt suspension. The activity of the ligase measured when palmitate was added to assays as a component of phospholipid vesicles was 10-40-fold greater vs. activities reported in the literature using other methods for adding fatty acids to the assay system. Phospholipids, however, had no direct effect on the activity of palmitoyl-CoA ligase. The data indicate, therefore, that the activity of this enzyme has been underestimated because of the manner in which fatty acid was added to the assay, which has a significant effect on the activity of the ligase. It is shown too that the rate of spontaneous transfer of palmitate from unilamellar vesicles of phosphatidylcholine to microsomes via a hydrated intermediate is far more rapid than the inherent catalytic activity of the fatty acyl-CoA ligase. The data also suggest that the membrane-associated pool of fatty acid and not fatty acid in the aqueous phase of the assay is the pool of substrate interacting with the ligase.     

Acyl-CoA ligases from rat brain microsomes: an immunochemical study

Acyl-CoA ligase activities, solubilized from rat brain microsomes, were fractionated into three different peaks by hydroxyapatite chromatography. Based on physical and chemical properties, we suggested that peak A (pamitoyl-CoA ligase) and peak C (lignoceroyl-CoA ligase) were two different enzymes (A. Bhushan, R. P. Singh, and I. Singh (1986) Arch. Biochem. Biophys. 246, 374-380). We raised antibodies against purified liver microsomal palmitoyl-CoA ligase (EC 6.2.1.3) and examined the effect of this antibody on acyl-CoA ligase activities for palmitic, arachidonic and lignoceric acids in microsomal enzyme extract and different acyl-CoA ligase peaks from the hydroxyapatite column. In an enzyme activity assay system in microsomal extract, the antisera inhibited the palmitoyl-CoA ligase activity but had very little effect on the acyl-CoA ligase activities for arachidonic and lignoceric acids. This antisera inhibited the acyl-CoA ligase activities for these three fatty acids in peak A and had no effect on these activities in peak B or peak C. Western blot analysis demonstrated that antibody to liver microsomal palmitoyl-CoA ligase cross-reacted with only peak A (palmitoyl-CoA ligase), but not with peak B or peak C. This immunochemical study demonstrates that palmitoyl-CoA ligase does not share immunological determinants with acyl-CoA ligases in peaks B or C, thus demonstrating that palmitoyl-CoA ligase (peak A) is different from the arachidonoyl-CoA and lignoceroyl-CoA ligase activities in peaks B or C.     

Preservation of lipid reesterifying enzymes in the microsomal fraction of the rat jejunum

Studies were performed on methods of storage of rat jejunal tissue that would preserve activities of the lipid reesterifying enzymes, acyl CoA:monoglyceride acyltransferase and fatty acid CoA ligase. Storage at -80 degrees C of microsomes prepared from jejunal mucosa or storage of lyophilized microsomes at -20 degrees C was shown to preserve acyl CoA:monoglyceride acyltransferase very well for a matter of weeks. Preservation of fatty acid CoA ligase activity was adequate with either method, but results were not as good as for the transacylase enzyme.     

Studies on the formation by rat brain preparations of CDP-diglyceride from CTP and phosphatidic acids of varying fatty acid compositions.

The enzyme, CTP:phosphatidate cytidylyltransferase (EC2.7.7.41) which catalyses formation of CDP-diglyceride from CTP and phosphatidic acid has been studied in rat brain preparations and other tissues. Improvement, as judged by the higher tissue activities obtained, in the assay method for this enzyme was achieved through use of phosphatidic acids sonicated in buffer-detergent solution saturated with ether and containing bovine serum albumin and use of short incubation times which essentially provided a measure of initial rates. The enzyme of rat brain microsomes yielded with 1,2-dioleolphosphatidic acid as substrate a pH optimum of 6.8 with maleate buffer and optimal concentrations of 60mM for MG2+, 6MM for CTP and 250 mug per 0.8 ml for phosphatidic acid. Enzyme activity was mainly located in the 90,000 X g fraction (microsomal) with small but significant activity in the 12,000 X g fraction. Comparison of activities (nanomoles CTP incorporated per milligram protein per minute) amongst tissues showed the following order: brain, 1.87; liver, 1.32; lung, 1.19; small intestine, 1.00; kidney, 0.69; heart, 0.41; diaphragm, 0.07; skeletal muscle, 0.02. Examination of the effect of varying the fatty acid composition in the phosphatidic acids added exogenously gave the following order (activities in parentheses); 1-stearoyl-2-oleoyl- (5.58), 1-oleoyl-2-stearoyl- (5.37), 1,2-dioleoyl- (4.49) 1-palmitoyl-2-oleoyl-(3.85), 1-stearoyl-2-arachidonoyl-(3.31), 1-arachidonoyl-2-stearoyl-(3.16), 1,2-diarachidonoyl-(0.72), 1,2-dicaproyl-(0.67), 1,2-dipalmitoyl-(0.67) and 1,2-distearoyl-(0.18). The single bis- and lysophosphatidic acids tested were inactive as substrates. Apart from a possible preference for one or more unsaturated fatty acids the transferase enzyme showed no selectivity in respect to the fatty acid distribution of phosphatidic acids.     

Fatty acid composition and properties of the liver microsomal membrane of rats fed diets enriched with cholesterol.

Male rats were fed diets containing olive (OO) or evening primrose (EPO) oil (10% w/w), with or without added cholesterol (1% w/w). After 6-week feeding, the lipid and fatty acid compositions, fluidity, and fatty acid desaturating and cholesterol biosynthesis/esterification related enzymes of liver microsomes were determined. Both the OO and EPO diets, without added cholesterol, increased the contents of oleic and arachidonic acids, respectively, of rat liver microsomes. The results were consistent with the increases in delta 9 and delta 6 desaturation of n-6 essential fatty acids and the lower microviscosity in the EPO group. Dietary cholesterol led to an increase in the cholesterol content of liver microsomes as well as that of phosphatidylcholine (PC). The cholesterol/phospholipid and PC/PE (phosphatidylethanolamine) ratios were also elevated. Fatty acid composition changes were expressed as the accumulation of monounsaturated fatty acids, with accompanying milder depletion of saturated fatty acids in rat liver microsomes. In addition, the arachidonic acid content was lowered, with a concomitant increase in linoleic acid, which led to a significant decrease in the 20:4/18:2 ratio in comparison to in animals fed the cholesterol-free diets. Cholesterol feeding also increased delta 9 desaturase activity as well as membrane microviscosity, whereas it decreased delta 6 and delta 5 desaturase activities. There was a very strong correlation between fluidity and the unsaturation index reduction in the membrane. Furthermore, the activity of hydroxymethylglutaryl-CoA reductase increased and the activity of acyl-CoA:cholesterol acyltransferase decreased in liver microsomes from both cholesterol-fed groups.(ABSTRACT TRUNCATED AT 250 WORDS)     

In vitro formation of polyunsaturated fatty acids by desaturation in rat brain: some properties of the enzymes in developing brain and comparisons with liver

Abstract— In vitro desaturation of [1-C¹⁴]linolenic, linoleic, oleic, and icosatrienoic acids was determined using homogenates and subcellular fractions of developing rat brain and liver. Linolenic, linoleic, and oleic acids were desaturated in the δ6-position and activity was optimal in the presence of CoA, ATP, MgCl₂, and NADH in a citrate-phosphate buffer at pH 6.0. Icosatrienoic acid was desaturated in the δ5-position with a much broader pH optimum. The unstable desaturation systems of brain were protected by reduced glutathione and niacinamide and markedly inhibited by dithiothreitol, p-chloromercuribenzoate, sodium cyanide or bathophenanthroline sulfonate. With brain homogenate of neonatal rats, the relative rates of desaturation of these substrates were 18:3(n - 3) > 18:2(n - 6) > 20:3(n - 6) > 18:l (n - 9). Specific activity of brain enzymes was greatest in neonatal rats with fluctuations in activity between 3 and 6 days of age. During this period, liver enzyme appeared to alter in a reciprocal manner. Total desaturation capacity of brain was maximal and fairly constant between 4 and 20 days of age, whereas liver activity increased dramatically after weaning. The activity of crude microsomal preparations from neonatal brain, like that of liver microsomes, was stimulated by a heat-labile component of the cytosolic fraction. These results demonstrate that brain has a high capacity for desaturation of the essential fatty acids during crucial stages of brain development when liver activity is relatively low.     

FATTY ACID COMPOSITION OF CEREBROSIDES IN MICROSOMES AND MYELIN OF MOUSE BRAIN

Abstract— The fatty acid composition of cerebrosides isolated from myelin and from light and heavy microsomes of adult mouse brain was determined. 2-Hydroxy fatty acids represented 80 per cent of the fatty acids in myelin cerebrosides and approximately 55 per cent of the fatty acids in both light and heavy microsomes. In myelin, the majority of the fatty acids, both normal and hydroxy, were of chain length > C-20; in microsomes, shorter chain acids (C-16 to C-20) predominated.     

EFFECT OF FATTY-ACID DOUBLE-BOND POSITION ON THE SELECTIVITY OF RAT-BRAIN ENZYMES: THE INCORPORATION OF OLEIC AND CIS-VACCENIC ACIDS INTO LYSOLECITHIN IN VITRO

Abstract— —Selectivity in the esterification of fatty acids to lysolecithin by rat-brain enzymes in vitro was investigated using free fatty acids (activation plus esterification) and CoA esters (esterification) of two naturally-occurring monoenoic fatty-acid isomers, oleic acid [18:1 (n - 9)] and cis-vaccenic acid [18:1 (n - 7)]. Esterification of free acids to l-acyl-sn-glycero-3-phosphorylcholine (1-acyl GPC) was dependent on CoA and ATP, and was stimulated by MgCl₂ and NaF. Under comparable conditions, fatty-acid activation (acyl-CoA synthetase [acid: CoA ligase (AMP)] EC 6.2.1.3.) appeared to be rate-limiting to 1-acyl GPC acyltransferase (acyl-CoA:l-acylglycero-3-phosphocholine O-acyltrans-ferase, EC 2.3.1.23.), since rates were always less with free fatty acids than with the CoA esters. A comparison of substrate curves obtained with free fatty acids and CoA esters suggests a preference for oleic acid during activation. Acyltransferase activity with 2-acyl GPC was similar with both acyl-CoA isomers, whereas with 1-acyl GPC, activity with oleoyl-CoA consistently exceeded that with cis-vaccenoyl-CoA. This difference between patterns of selectivity in esterification of positions 1 and 2 of lecithin suggests that separate enzymes catalyze the two reactions. The transfer of the isomers to the 2 position was affected in a similar manner by changes in pH and temperature, as well as in protein, fatty acid (or acyl-CoA), and 1-acyl GPC concentrations. Patterns of incorporation with simultaneous incubation of both isomers suggests one enzyme. Differences in acyltransferase activity with the two isomerie acyl-CoA's were observed in subcellular distribution, activity changes with brain maturation, and loss of activity on preincubation of microsomes at 45C. From these results it is not certain whether oleic and cis-vaccenic acids are esterified to the 2 position by separate enzymes, or by one enzyme with different affinities for the isomers. However, the investigation clearly indicates that acyltransferases, and possibly acyl-CoA synthetases in brain possess selectivity related to subtle differences in double-bond position. These selectivities probably are important in determining the specific fatty-acid composition of the complex lipids of brain.     

Influence of dietary partially hydrogenated fat high in trans fatty acids on lipid composition and function of intestinal brush border membrane in rats

The effect of dietary hydrogenated fat (Indian vanaspati) high in trans fatty acids (6 en%) on lipid composition, fluidity and function of rat intestinal brush border membrane was studied at 2 and 8 en% of linoleic acid. Three groups of weanling rats were fed rice-pulse based diet containing 10% fat over a ten week period: Group I (groundnut oil), Group II (vanaspati), Group III (vanaspati + safflower oil). The functionality of the brush border membrane was assessed by the activity of membrane bound enzymes and transport of D-glucose and L-leucine. The levels of total cholesterol and phospholipids were similar in all groups. The data on fatty acid composition of membrane phospholipids showed that, at 2 en% of linoleic acid in the diet, trans fatty acids lowered arachidonic acid and increased linoleic acid contents indicating altered polyunsaturated fatty acid metabolism. Alkaline phosphatase activity was increased while the activities of sucrase, gamma-glutamyl transpeptidase and transport of D-glucose and L-leucine were not altered by dietary trans fatty acids. However at higher intake of linoleic acid in the diet, trans fatty acids have no effect on polyunsaturated fatty acid composition and alkaline phosphatase activity of intestinal brush border membrane. These data suggest that feeding dietary fat high in trans fatty acids is associated with alteration in intestinal brush border membrane polyunsaturated fatty acid composition and alkaline phosphatase activity only when the dietary linoleic acid is low.     

Clinical variation in X-linked adrenoleukodystrophy: fatty acid and lipid metabolism in cultured fibroblasts

To determine whether the clinical phenotype of ALD correlates with the extent of metabolic abnormality, we investigated VLFA metabolism in cultured fibroblasts from patients with the clinically severe childhood from of ALD and the milder AMN variant. No differences were seen in the content of neutral lipids or phospholipids, in incorporation of [1-14C]lignocerate into cellular lipids, or in the fatty acid composition of fibroblasts from patients with childhood ALD or AMN. [1-14C]Lignocerate oxidation was deficient to a similar extent (35-40% of normal) in both intact fibroblasts and cell homogenates from patients with childhood ALD and AMN. With the use of fibroblast homogenates, oxidation of lignocerate was partially inhibited by various long-chain fatty acids, and residual activity in ALD homogenates was more susceptible to inhibition by palmitate than normal. In the presence of competing palmitate, residual lignocerate oxidative activity in fibroblast homogenates was reduced to 20 +/- 4% of normal in childhood ALD and 24 +/- 2% of normal in AMN. These results indicate that residual VLFA oxidative activity, fatty acid composition, VLFA metabolism, and lipid content of cultured fibroblasts do not correlate with the clinical expression of the ALD gene.     

Long-chain and very long-chain fatty acyl-coa synthetase activity in microsomes of jimpy mouse brain

The objective of this study was to determine whether the conversion of free, very long chain fatty acids (C₂₂–C₂₆) to their CoA-esters are involved in cerebroside synthesis, since cerebrosides are uniquely rich in very long chain fatty acids including lignoceric acid (C_24:0). We have studied lignoceroyl-CoA synthetase activity in the microsomes isolated from normal and jimpy mouse brain. The jimpy mouse lacks the ability to make myelin and is deficient in enzyme activities involved in the synthesis of myelin components, including cerebrosides. Unexpectedly, the lignoceroyl-CoA synthetase activity in jimpy brain microsomes was slightly higher than that in control microsomes. The palmitoyl (C_16:0)-CoA synthetase activity in jimpy brain was not different from the control. The level of cerebrosides in microsomes was grossly lower in jimpy brain. The implication of these findings and the involvement of lignoceric acid activation in cerebroside synthesis is discussed.     

Interaction of rat liver microsomes containing saturated or unsaturated fatty acids with fatty acid binding protein: Peroxidation effect

In the studies described here rat liver microsomes containing labeled palmitic, stearic, oleic or linoleic acids were incubated with fatty acid binding protein (FABP) and the rate of removal of¹⁴C-labeled fatty acids from the membrane by the soluble protein was measured using a model system. More unsaturated than saturated fatty acids were removed from native liver microsomes incubated with similar amounts of FABP. Thein vitro peroxidation of microsomal membranes mediated by ascorbate-Fe⁺⁺, modified its fatty acid composition with a considerable decrease of the peroxidizability index. These changes in the microsomes facilitated the removal of oleic and linoeic acids by FABP, but the removal of palmitic and stearic acids was not modified. This effect is proposed to result from a perturbation of membrane structure following peroxidation with release of free fatty acids from susceptible domains.Abbreviations BSA bovine serum albumin - FABP fatty acid binding protein     

Influence of dietary cholesterol on polyunsaturated fatty acid composition, fluidity and membrane-bound enzymes in liver microsomes of rats fed olive and fish oil.

Male rats were fed diets containing olive or marine fish oils (10% w/w) with or without added cholesterol (1% w/w). After six weeks of feeding, the major fatty acid composition, fluidity, fatty acid desaturating and cholesterol biosynthesis/esterification related enzymes of liver microsomes were determined. Both olive oil and marine fish oil diets, without added cholesterol, enriched content of oleic and docosahexaenoic acids, respectively, of rat liver microsomes. The results were consistent with reduction in delta 6 and delta 5 desaturation of n-6 essential fatty acids and higher fluidity in the marine origin oil group. Inclusion of cholesterol into diets resulted in decreased membrane arachidonic acid content, with concomitant increase in linoleic acid content. Cholesterol feeding also decreased delta 6 and delta 5 desaturase activities, as well as membrane fluidity. Furthermore, the activity of acyl-CoA:cholesterol acyltransferase decreased, whereas the activity of hydroxymethylglutaryl-CoA reductase increased, in liver microsomes from both cholesterol-fat groups.