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Metabolism of erucic acid in the isolated perfused rat heart.

In the present work the uptake and utilization of [14C]erucic acid by the perfused rat heart has been investigated and compared with those of [14C]-palmitic acid. Both fatty acids were found to be taken up by the heart at the same rate. On the other hand, the incorporation of erucic acid into tissue lipid during 30 min perfusion were significantly high and CO2 production low as compared with palmitic acid. Incorporation of erucic acid into diacylglycerol, triacylglycerol and cholesterol ester was considerably higher than that of palmitic acid. During a 30-min period, a large amount of [14C]erucic acid was accumulated in tissue fatty acid fraction. Similarly, relatively high labelling was found in the fatty acid and diacylglycerol fraction during the initial 300 s of perfusion with erucic acid. When [14C]erucic acid and unlabelled palmitic acid was used, the radioactivity was very high in the fatty acid fraction of the heart lipid in comparison with the experiment when [14C]palmitate and unlabelled erucic acid was used. Therefore, erucic acid is poorly oxidized by the heart and is preferentially incorporated into heart lipids. There was relatively high incorporation of [14C]erucic acid into diacylglycerol and addition of unlabelled palmitic acid tended to decrease it, probably converting more diacylglycerol to triacylglycerol. When [14C]palmitic acid and erucic acid were used together, incorporation to triacylglycerol was high and diacylglycerol low. These results, therefore suggest that palmitic acid is a more suitable acyl donor than erucic acid for the C-3 position of triacylglycerol, especially when the diacylglycerol contains erucoyl moieties.

Animals↗

Utility of the Arabidopsis FAE1 and yeast SLC1-1 genes for improvements in erucic acid and oil content in rapeseed.

High-erucic acid (HEA) Brassica napus cultivars are regaining interest in industrial contexts. Erucic acid and its derivatives are important renewable raw materials utilized in the manufacture of plastic films, in the synthesis of Nylon 13,13, and in the lubricant and emollient industries. Theoretically, the highest level of erucic acid that can be achieved by means of classical breeding is 66 mol%; however, using new approaches on the basis of genetic engineering, it might be possible to develop a B. napus cultivar containing levels of erucic acid significantly above 66 mol% (>80 mol%). In an attempt to increase the amounts of very-long-chain fatty acids (VLCFAs), and erucic acid in particular, in Canadian HEA B. napus cultivars, we have focused on two targets using a transgenic approach. We examined both the role/function of the Arabidopsis thaliana FAE1 (fatty acid elongase) gene by expressing it under the control of the seed-specific napin promoter in B. napus germplasm with analysis of the changes in VLCFA content in the seed oil of transgenic lines, and the performance of the yeast SLC1-1 (sphingolipid compensation mutant) in B. napus cv. Hero transgenic progeny in the field. Here, we report analyses of the contents of 22:1, total VLCFAand oil in the seed oil, as well as seed yield of the field-grown FAE1 and SLC1-1 B. napus cv. Hero progeny.

Acetyltransferases↗

Studies of the mode of action of erucic acid on heart metabolism.

The effects of erucic acid on the oxidative metabolism of rat-heart mitochondria have been investigated using intact animals, perfused beating heart, isolated mitochondria and mitochondrial extracts. Feeding rats with a diet containing erucic acid was found to lead to a diminished ability of the isolated heart mitochondria to oxidize various substrates, in accordance with previous reports (Houtsmuller et al., Biochim. Biophys. Acta 218 (1970) 564). This effect was almost pronounced with palmitylcarnitine as substrate, in which case the rate of oxidation was decreased by more than 50% at such a low erucic acid content in the diet as 1.4% given over 2-4 weeks. Oxidation of palmitylcarnitine was also found to be inhibited when erucylcarnitine was added to isolated heart mitochondria from control animals, in agreement with earlier observations (Christophersen and Bremer, FEBS Lett. 23 (1972) 230; Biochim. Biophys. Acta 280 (1972) 506). The inhibition was accompanied by a decrease in the rate and extent of reduction of mitochondrial flavoprotein. Experiments with perfused beating rat-heart likewise revealed an inhibition of flavoprotein reduction, as well as nicotinamide nucleotide reduction, when erucate was added to the perfusing medium of the beating heart respiring with oleate--but not with octanoate--as substrate. These data together with those earlier published in the literature indicate that erucic acid may interfere with the enzyme system involved in the mitochondrial oxidation of long-chain fatty acids, probably at the level of acyl-CoA dehydrogenase. Kinetic data supporting this conclusion, obtained with extracts of rat-heart mitochondria containing the acyl-CoA dehydrogenase and electron-transferring flavoprotein system, are presented. The possible implications of these results for the known effect of dietary erucic acid in causing an accumulation of fat in the heart are discussed.

Animals↗

An increase in cholesterol ester content of the rat aorta by a high fat-high erucic acid diet.

Rats of weaning age were feed for a period of 1 or 3 weeks either a low fat diet (laboratory stock diet) or a semi-synthetic diet containing 20% by weight of either corn oil (2/3 of the total fatty acids consisted of linoleic acid) or mustard seed oil (1/3 of the total fatty acids were comprised of erucic acid). Feeding of a diet rich in erucic acid for 1 week increased by twofold the cholesterol ester content of the aorta. However, the concentrations of unesterified cholesterol and individual phospholipids in the aorta were uninfluenced by hyperlipemia induced by this diet. Fatty acid analyses indicated that erucic (22:1) and eicosenoic (20:1) acids are found in the triglyceride and cholesterol esters of the aorta in the proportion similar to that found in the plasma triglyceride and cholesterol esters. Our results demonstrated further that a significantly greater quantity of 14C-labelled erucate than labelled palmitate, oleate or linoleate was incorporated into the cholesterol ester fraction of the control aorta. The incorporation rates of erucate and linoleate into phosphatidic acid were more rapid, while the rates of erucate into phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol and lysophosphatidylcholine were significantly slower than those of other fatty acids.

Animals↗

[Researches on the physiopathologic effects of rapeseed oil with high and low erucic acid content].

The physiopathological effects were studied of a common high erucic acid rapeseed oil as well as of Janpol, a low erucic acid oil produced of a rapeseed variety selected in Poland. Its erucic acid content equals 2.8% of total fatty acids. The studies were carried out on white male Wistar rats, 25 days old at the beginning of experiment. These animals were divided into 6 groups fed the diets in which 10 or 20% of kcal well supplied either by high erucic acid rapeseed oil, by Janpol rapeseed oil, or by the sunflower oil. The experiment lasted 6 months. Following parameters were determined: increase in body weight, the weight of selected organs, blood serum alkaline phosphatase and pseudocholinesterase activities, blood serum cholesterol and triglycerides level, the content of corticosterone in the adrenal glands and blood plasma. The liver was studied histochemically for the activity of acid and alkaline phosphatases and of ATP-ase, as well as for the presence of lipids. Morphological studies of the myocardium comprised macroscopic, histological and electron microscopic investigation. The low erucic acid rapeseed oil Janpol seems to evoke less disturbances than the high erucic acid one. Supplied in the amount corresponding to 10% of total calories intake the former exerts the effect on the biochemical and morphological parameters similar to that of sunflower oil. It can be thus assumed that the low erucic acid rapeseed oil Janpol can be used in the feeding of man when served in the amount lower than 10% of total calories.

Acid Phosphatase↗

Perturbation of phospholipid metabolism by erucic acid in male Sprague-Dawley rat heart.

Erucic acid was incorporated into cardiac phosphatidylserine and hepatic and cardiac sphingomyelin of male Sprague-Dawley rat. The fatty acid compositions of mitochondrial and microsomal phospholipids were similar in both liver and heart. The effect of a low fat diet and a diet containing erucic acid on the fatty acid composition of mitochondrial and microsomal phospholipids was also similar, except for the effect on sphingomyelin. However, the diet containing erucic acid influenced the metabolism of phosphatidylcholine of the heart but not of the liver, indicating that the turnover of 1-stearoyl-2-arachidonoyl phosphatidylcholine in the heart was inhibited by the diet containing erucic acid. On the other hand, the proportion of erucic acid in the free fatty acid was higher in the heart than in the liver.

Animals↗

Purification of erucic acid by preparative high-performance liquid chromatography and crystallization.

Erucic acid (C22:l fatty acid) has been found to be useful in the treatment of adrenoleukodystrophy (ALD). It appears to work by reducing the blood levels of very-long-chain fatty acids (VLCFAs) which destroy the myelin sheaths of the nerves. Erucic acid was purified by reversed-phase high-performance liquid chromatography (HPLC) on columns packed with YMC C18 (10-20 microns, 120 A). Using ethanol-water as the mobile phase, the recovery of erucic acid was 69% and the purity was more than 97% as measured by gas chromatography. The amount of saturated VLCFAs was found to be within the limits specified for ALD treatment. The production rate (yield per 8 h shift) was low, however. Using methanol-water instead of ethanol-water as the mobile phase, a ninefold increase in the production rate was achieved. The recovery of erucic acid was 65% and the purity of erucic acid was 98%. All other purity specifications were met. By performing a low-temperature crystallization after the preparative HPLC step, the production rate was increased a further 142%. This represents a 22-fold increase in production rate over the ethanol-water method. The crystalline erucic acid was found to be 99% pure. All other purity requirements were met. The yield for the combined process (HPLC plus crystallization) decreased to 55%, however.

Adrenoleukodystrophy↗

The biosynthesis of erucic acid in developing embryos of brassica rapa

The prevailing hypothesis on the biosynthesis of erucic acid in developing seeds is that oleic acid, produced in the plastid, is activated to oleoyl-coenzyme A (CoA) for malonyl-CoA-dependent elongation to erucic acid in the cytosol. Several in vivo-labeling experiments designed to probe and extend this hypothesis are reported here. To examine whether newly synthesized oleic acid is directly elongated to erucic acid in developing seeds of Brassica rapa L., embryos were labeled with [14C]acetate, and the ratio of radioactivity of carbon atoms C-5 to C-22 (de novo fatty acid synthesis portion) to carbon atoms C-1 to C-4 (elongated portion) of erucic acid was monitored with time. If newly synthesized 18:1 (oleate) immediately becomes a substrate for elongation to erucic acid, this ratio would be expected to remain constant with incubation time. However, if erucic acid is produced from a pool of preexisting oleic acid, the ratio of 14C in the 4 elongation carbons to 14C in the methyl-terminal 18 carbons would be expected to decrease with time. This labeling ratio decreased with time and, therefore, suggests the existence of an intermediate pool of 18:1, which contributes at least part of the oleoyl precursor for the production of erucic acid. The addition of 2-[3-chloro-5-(trifluromethyl)-2-pyridinyloxyphenoxy] propanoic acid, which inhibits the homodimeric acetyl-CoA carboxylase, severely inhibited the synthesis of [14C]erucic acid, indicating that essentially all malonyl-CoA for elongation of 18:1 to erucate was produced by homodimeric acetyl-CoA carboxylase. Both light and 2-[3-chloro-5-(trifluromethyl)-2-pyridinyloxyphenoxy]-propanoic acid increased the accumulation of [14C]18:1 and the parallel accumulation of [14C]phosphatidylcholine. Taken together, these results show an additional level of complexity in the biosynthesis of erucic acid.

Journal Article↗

A comparison of the short-term incorporation of erucic acid and oleic acid in the perfused guinea-pig heart.

A comparison was made of the incorporation of radioactive erucic acid and oleic acid in the isolated perfused guinea pig heart, 2, 15 and 30 min after a radioactive pulse. The complementary techniques of (a) freeze-clamping followed by lipid extraction and thin layer chromatography and (b) electron microscope autoradiography were used. The incorporation of 3H-erucic acid into esterified lipids was much slower than that of 3H-oleic acid. Less radioactive CO2 was produced by hearts perfused with 14C erucic acid then by hearts perfused with 14C oleic acid. There was no significant effect of erucic acid on the relative areas of subcellular organelles in the autoradiographs and, in particular, there was no increase in the volume of lipid droplets. However, the incorporation of radioactivity into lipid droplets was much greater with 3H-oleic acid than with 3H-erucic acid, consistent with the higher incorporation into tissue triacylglycerol. Although oxidized less than oleic acid, erucic acid was readily transported to the mitochondria. High levels of radioactivity in free fatty acid in the hearts perfused with erucic acid suggest that a low rate of activation of the fatty acid to acyl-CoA limits both oxidation and the formation of triacylglycerol. Electron microscopy of the hearts perfused with erucic acid revealed a widespread, and quantitatively demonstrable general movement of lipid droplets towards the surface of the cell. This was occasionally accompanied by a local rupturing of the sarcolemma, possibly prior to expulsion of the lipid droplet from the cell.

Animals↗

Metabolism of erucic acid in adipocytes isolated from rat epididymal fat.

The metabolism of [14-14C]erucic acid and [U-14C]palmitic acid has been investigated in adipocytes isolated from rat epididymal fat. The rate of acylation of [14C]erucic acid in cellular lipids and oxidation to CO2 and acid-soluble activity was ca. 1/3 of the rate with [14C]palmitic acid as substrate. A maximal incorporation of fatty acids in triacylglycerol was found at a fatty acid concentration of 0.8 mM in the medium, both with [14C]erucic acid and [14C]palmitic acid as substrate. Glucose added to the medium increased the esterification and decreased the oxidation of both fatty acids. No significant chain-shortening of [14C]erucic acid to shorter monoenes was identified in the fat cells. Increasing concentrations of unlabeled palmitic acid in the incubation medium markedly inhibited the esterification of [14C]erucic acid, whereas unlabeled erucic acid had little effect on the rate of esterification of [14C]palmitic acid.

Adipose Tissue↗

Is dietary erucic acid hepatotoxic in pregnancy? An experimental study in rats and hamsters.

The hypothesis that dietary erucic acid may contribute to the pathogenesis of intrahepatic cholestasis of pregnancy has been examined in pregnant rats and hamsters after prolonged feeding of diets containing 25% rapeseed oil rich in erucic acid (40% of fatty acids) or corn oil, without erucic acid. Both dietary oils were well tolerated, although weight gain was 17% to 20% less in animals receiving rapeseed oil. Rats and hamsters were studied on the last day of pregnancy and compared with age- and diet-matched nonpregnant animals. Histological examination showed no major morphologic abnormalities in liver, heart, kidneys, and adrenals. Similar microscopic deposits of fat were found in the livers and hearts of pregnant hamsters of both dietary groups. Chromatographic analysis of fatty acids in liver, heart, and kidney homogenates of hamsters and in isolated rat liver cells reflected the fatty acid composition of the dietary oils: oleic (18:1) and linoleic (18:2) acids were among the predominant fatty acids. Erucic acid was found in a higher proportion in the heart (14% by weight of total fatty acids) than in the liver (3%) and kidneys (3%) of animals fed rapeseed oil. Bile flow and biliary lipid composition was similar in rats and hamsters fed rapeseed or corn oil. Bile flow tended to be less in pregnant than in nonpregnant animals. Pregnant hamsters fed rapeseed oil tended to have the lowest bile flow. The lithogenic index of bile was slightly decreased in pregnant rats and increased in pregnant hamsters, although these proportional changes were similar for both diets. In all circumstances the lithogenic index remained below a value of 1.(ABSTRACT TRUNCATED AT 250 WORDS)

Adipose Tissue↗

Restoring enzyme activity in nonfunctional low erucic acid Brassica napus fatty acid elongase 1 by a single amino acid substitution.

Genomic fatty acid elongation 1 (FAE1) clones from high erucic acid (HEA) Brassica napus, Brassica rapa and Brassica oleracea, and low erucic acid (LEA) B. napus cv. Westar, were amplified by PCR and expressed in yeast cells under the control of the strong galactose-inducible promoter. As expected, yeast cells expressing the FAE1 genes from HEA Brassica spp. synthesized very long chain monounsaturated fatty acids that are not normally found in yeast, while fatty acid profiles of yeast cells expressing the FAE1 gene from LEA B. napus were identical to control yeast samples. In agreement with published findings regarding different HEA and LEA B. napus cultivars, comparison of FAE1 protein sequences from HEA and LEA Brassicaceae revealed one crucial amino acid difference: the serine residue at position 282 of the HEA FAE1 sequences is substituted by phenylalanine in LEA B. napus cv. Westar. Using site directed mutagenesis, the phenylalanine 282 residue was substituted with a serine residue in the FAE1 polypeptide from B. napus cv. Westar, the mutated gene was expressed in yeast and GC analysis revealed the presence of very long chain monounsaturated fatty acids (VLCMFAs), indicating that the elongase activity was restored in the LEA FAE1 enzyme by the single amino acid substitution. Thus, for the first time, the low erucic acid trait in canola B. napus can be attributed to a single amino acid substitution which prevents the biosynthesis of the eicosenoic and erucic acids.

3-Oxoacyl-(Acyl-Carrier-Protein) Synthase↗

[Major gene plus poly-gene inheritance of erucic acid content in Brassica napus L].

Inheritance of erucic acid content in the cross of HSTC14 x Ningyou 7 (Brassica napus L.) was investigated by applying the major gene plus poly-gene model of quantitative traits to a joint analysis of multi-generations (P1, F1, P2, B1:2 B2:2 and F2:3). The results showed that erucic acid content in the cross of HSTC14 x Ningyou 7 was controlled by two major genes with additive-dominance-epistatic effects plus polygenes with additive-dominance effects (the E-1 model). Heritability values of the major genes of B1:2, B2:2 and F2:3 population were estimated as 69.47%, 28.25% and 51.33% respectively, while those of polygene were 29.81%, 70.39% and 47.86%. This results demonstrated that erucic acid content level in the cross was mainly controlled by two major genes mixed with the effect of polygenes. The estimated additive effects of the two major genes, da and db, were -12.1712 and -6.5423 respectively. The dominant effect of the first major gene was 9.1011, much higher than that of the second one. The interaction effect of the first major gene's additive effect and the second major gene's dominance effect was 22.4400, while that between the second major gene's additive effect and the first major gene's dominance effect was only 1.0317. There were obvious differences in genetic effects between the two major genes. The effect of db was only about 53.75% that of da, indicating that the contributions to erucic acid level of the two major genes was not equal in the cross. The possible reasons resulting in the difference was discussed in this paper.

Brassica↗

Mutations in the fatty acid elongation 1 gene are associated with a loss of beta-ketoacyl-CoA synthase activity in low erucic acid rapeseed.

Low erucic acid rapeseed (LEAR) is characterised by a near absence of very long chain fatty acids in the seed oil which has been correlated with a lack of acyl-CoA elongation activity. Here we show that the absence of acyl-CoA and ATP-dependent elongation activities in microsomes isolated from LEAR embryos is associated with an absence of beta-ketoacyl-CoA synthase activity encoded by the Bn-fatty acid elongation 1 (FAE1) genes. Size exclusion chromatography of solubilised microsomes revealed the presence of a high molecular mass acyl-CoA elongase complex in high erucic acid rapeseed which was absent in microsomes isolated from LEAR seeds. Although transcripts for the Bn-FAE1 genes were detected in LEAR embryos, immunoblots using antisera raised against the beta-ketoacyl-CoA synthase indicated an absence of this protein. Comparison of the deduced amino acid sequences of immature embryo cDNAs reveals that LEAR alleles of Bn-FAE1 encode variant beta-ketoacyl-CoA synthase proteins.

3-Oxoacyl-(Acyl-Carrier-Protein) Synthase↗

Erucic acid metabolism in rat heart. A combined biochemical and radioautographical study.

Metabolism of Erucic Acid was studied in rat heart in comparison with that of oleic acid, particularly in relation with diet lipids. Rats were fed for 3 or 60 days a diet containing 30% of the calories of either Rapessed Oil, rich in erucic acid or sunflower seed oil rich in linoleic acid. They were I.V. injected with tritiated erucic or oleic acid. After 1 or 15 min the radioactivity recovered in heart lipids was very low whatever the diet (1 to 2%). One minute after injection of erucic acid the radioactivity was mainly recovered in the free fatty acid fraction and as untransformed erucic acid. After 15 min the major part of radioactivity was recovered in the triacylglycerol fraction which contained a high proportion of labelled oleic acid formed by shortening of erucic acid. When oleic was injected, the radioactivity was principally recovered in triacylglycerols as untransformed oleic acid whatever the experimental conditions. Electron microscopy showed that a much higher proportion of peroxisomes, was present in heart cells, following sunflower seed oil diet as compared to rapeseed oil diet. In all cases mitochondria supported the greater part of radioactivity, especially when erucic acid was injected in rats fed rapeseed oil. After sunflower seed oil, a noticeable radioactivity was observed in peroxisomes, most of them containing silver grains, especially when oleic acid was injected. According to the data reported, peroxisomes do not seem more implicated than mitochondria in the metabolism of erucic acid in myocardium.

Animals↗

A two-year trial of oleic and erucic acids ("Lorenzo's oil") as treatment for adrenomyeloneuropathy.

BACKGROUND: Adrenomyeloneuropathy is an X-linked recessive disorder characterized by myelopathy, peripheral neuropathy, and cerebral demyelination, which develop in association with the accumulation of very-long-chain fatty acids. The administration of oleic and erucic acids inhibits the synthesis of very-long-chain fatty acids. Recently such dietary treatment has been widely publicized as a possible cure for this disease. METHODS: We conducted an open trial in 14 men with adrenomyeloneuropathy, 5 symptomatic heterozygous women, and 5 boys (mean age, 13 years) with preclinical adrenomyeloneuropathy. The patients ate a low-fat diet and received daily doses of glycerol trioleate oil (1.7 g per kilogram of body weight) and glycerol trierucate oil (0.3 g per kilogram). Clinical manifestations, cerebral and spinal cord magnetic resonance imaging (MRI) scans, nerve conduction, and brain-stem auditory and somatosensory evoked potentials were studied prospectively over 18 to 48 months. Plasma levels of very-long-chain fatty acids and the side effects of erucic acid were monitored monthly. RESULTS: By week 10, plasma very-long-chain fatty acid levels declined nearly to normal. Nonetheless, over a mean follow-up of 33 months none of the 14 men with adrenomyeloneuropathy improved. In nine men there was functional deterioration, coincident in four with new cerebral lesions on MRI. In a single patient there was a reduction in cerebellar demyelination, but without clinical improvement. In one of the five asymptomatic boys signs of myelopathy developed. There were no changes in the symptomatic heterozygous women. There was some improvement in peroneal-nerve conduction, but no detectable clinical improvement. Conduction to the parietal cortex (T12-P37 interpeak latency) worsened in both the symptomatic men and the boys with preclinical adrenomyeloneuropathy. There was no change in other somatosensory evoked potentials or in brain-stem auditory evoked potentials. Asymptomatic thrombocytopenia (< 100,000 cells per cubic millimeter) was noted in six patients. CONCLUSIONS: In this open trial we found no evidence of a clinically relevant benefit from dietary treatment with oleic and erucic acids ("Lorenzo's oil") in patients with adrenomyeloneuropathy.

Adolescent↗

Erucic acid metabolism in rat liver. A combined biochemical and radioautographical study.

Metabolism of erucic acid was studied in rat liver in comparison with oleic acid in relation with diet lipids. Rats were fed for 3 or 60 days a balanced diet containing 30% of the calories of either rapeseed oil rich in erucic acid or sunflower seed oil rich in linoleic acid. They were intravenously injected with tritiated erucic or oleic acid. After 1 or 15 min, the radioactivity recovered in liver lipids was 9 to 26% whatever the diet or the acid injected. One minute after injection of erucic acid a high part of radioactivity was recovered in the free fatty acid fraction and as untransformed erucic acid. After 15 min the major part of radioactivity was recovered in the triacylglycerol fraction which contained a high proportion of labelled oleic acid formed by shortening of erucic acid. The autoradiography did not show any marked difference between the labelling of peroxisomes and mitochondria when tritiated erucic or oleic acid was injected. These data do not bring about arguments for a peroxisomal nor for a mitochondrial location of erucic acid shortening in liver.

Animals↗

Effects of high erucic acid rapeseed oil on fatty acid oxidation in rat liver.

The effects of high erucic acid rapeseed oil (HER) on fatty acid oxidation in rat liver compared with low erucic acid rapeseed oil (LER) were studied. Weanling male SD rats were fed diets containing 20% HER or LER for 1 week or 4 weeks, or 5% HER diet for 4 weeks. The hepatic oxidation capacity of butyric acid or palmitic acid was determined by titrating the propanone produced by their oxidation. The results showed that feeding HER to rats led to an increase in the weight of liver and a decrease in the hepatic oxidation capacity of palmitic acid. Hepatic oxidation of butyric acid was not influenced by the intake of HER. The inhibitory action of HER on the oxidation of long-chain fatty acids probably resulted from the incorporation of erucic acid into mitochondrial membranes, interfering the fatty acyl-CoA transferring system on the membranes, but not from the beta-oxidation enzyme system in mitochondria being directly inhibited.

Animals↗