Glucagon and fasting do not activate peroxisomal fatty acid beta-oxidation in rat liver

In a study of the endocrine control of peroxisomes, the effects of acute glucagon treatment and fasting on hepatic peroxisomal beta-oxidation in rats have been investigated. The activity of the rate-limiting peroxisomal beta-oxidation enzyme, fatty acyl-CoA oxidase, was measured to determine whether activation of peroxisomal beta-oxidation could account for the increase in total hepatic fatty acid oxidation following acute glucagon exposure. Catalase, a peroxisomal enzyme not directly involved in beta-oxidation, was also measured as a control for total peroxisomal activity. No changes with acute glucagon treatment of intact animals were observed with either activity as measured in liver homogenates or partially purified peroxisomal fractions. These observations indicate the lack of acute control by glucagon of peroxisomal function at the level of total enzyme activity. Previous work on the effects of fasting on hepatic fatty acid beta-oxidation [H. Ishii, S. Horie, and T. Suga (1980) J. Biochem. 87, 1855-1858] suggested an enhanced role for the peroxisomal beta-oxidation pathway during starvation. It was found that the peroxisomal beta-oxidation system, as measured by fatty acyl-CoA oxidase activity, does increase with duration of fast when expressed on a per gram wet weight liver basis. However, when this activity is expressed as total liver capacity, a decline in activity with increasing duration of fast is observed. Furthermore, this decline in peroxisomal capacity parallels the decline in total liver capacity for citrate synthase, a mitochondrial matrix enzyme, and total liver protein. These data indicate that peroxisomal beta-oxidation activity is neither stimulated nor even preferentially spared from proteolysis during fasting.     

Fatty acid metabolism in liver of rats treated with hypolipidemic sulphur-substituted fatty acid analogues

The purpose of this study was to investigate early biochemical changes and possible mechanisms via which alkyl(C12)thioacetic acid (CMTTD, blocked for beta-oxidation), alkyl(C12)thiopropionic acid (CETTD, undergo one cycle of beta-oxidation) and a 3-thiadicarboxylic acid (BCMTD, blocked for both omega- (and beta-oxidation) influence the peroxisomal beta-oxidation in liver of rats. Treatment of rats with CMTTD caused a stimulation of the palmitoyl-CoA synthetase activity accompanied with increased concentration of hepatic acid-insoluble CoA. This effect was already established during 12-24 h of feeding. From 2 days of feeding, the cellular level of acid-insoluble CoA began to decrease, whereas free CoASH content increased. Stimulation of [1-14C]palmitoyl-CoA oxidation in the presence of KCN, palmitoyl-CoA-dependent dehydrogenase (termed peroxisomal beta-oxidation) and palmitoyl-CoA hydrolase activities were revealed after 36-48 h of CMTTD-feeding. Administration of BCMTD affected the enzymatic activities and altered the distribution of CoA between acid-insoluble and free forms comparable to what was observed in CMTTD-treated rats. It is evident that treatment of peroxisome proliferators (BCMTD and CMTTD), the level of acyl-CoA esters and the enzyme activity involved in their formation precede the increase in peroxisomal and palmitoyl-CoA hydrolase activities. In CMTTD-fed animals the activity of cyanide-insensitive fatty acid oxidation remained unchanged when the mitochondrial beta-oxidation and carnitine palmitoyltransferase operated at maximum rates. The sequence and redistribution of CoA and enzyme changes were interpreted as support for the hypothesis that substrate supply is an important factor in the regulation of peroxisomal fatty acid metabolism, i.e., the fatty acyl-CoA species appear to be catabolized by peroxisomes at high rates only when uptake into mitochondria is saturated. Administration of CETTD led to an inhibition of mitochondrial fatty acid oxidation accompanied with a rise in the concentration of acyl-CoA esters in the liver. Consequently, fatty liver developed. The peroxisomal beta-oxidation was marginally affected. Whether inhibition of mitochondrial beta-oxidation may be involved in regulation of peroxisomal fatty acid metabolism and in development of fatty liver should be considered.     

Lignoceroyl-CoASH ligase: enzyme defect in fatty acid beta-oxidation system in X-linked childhood adrenoleukodystrophy

We have previously reported that the peroxisomal beta-oxidation system for very long chain fatty acids is defective in X-linked childhood adrenoleukodystrophy [(1984) Proc. Natl. Acad. Sci. USA 81, 4203-4207]. In order to elucidate the specific enzyme defect, we examined the oxidation of [1-14C]lignoceric acid, [1-14C]lignoceroyl-CoA and (1-14C)-labelled alpha,beta-unsaturated lignoceroyl-CoA (substrates for the 1st, 2nd, and 3rd steps of the beta-oxidation cycle, respectively). These studies suggest that the pathognomonic accumulation of very long chain fatty acids in X-linked childhood ALD may be due to the defective activity of peroxisomal very long chain (lignoceroyl-CoA) acyl-CoA ligase.     

Chlorpromazine and carnitine-dependency of rat liver peroxisomal beta-oxidation of long-chain fatty acids.

The enzyme targets for chlorpromazine inhibition of rat liver peroxisomal and mitochondrial oxidations of fatty acids were studied. Effects of chlorpromazine on total fatty acyl-CoA synthetase activity, on both the first and the third steps of peroxisomal beta-oxidation, on the entry of fatty acyl-CoA esters into the peroxisome and on catalase activity, which allows breakdown of the H2O2 generated during the acyl-CoA oxidase step, were analysed. On all these metabolic processes, chlorpromazine was found to have no inhibitory action. Conversely, peroxisomal carnitine octanoyltransferase activity was depressed by 0.2-1 mM-chlorpromazine, which also inhibits mitochondrial carnitine palmitoyltransferase activity in all conditions in which these enzyme reactions are assayed. Different patterns of inhibition by the drug were, however, demonstrated for both these enzyme activities. Inhibitory effects of chlorpromazine on mitochondrial cytochrome c oxidase activity were also described. Inhibitions of both cytochrome c oxidase and carnitine palmitoyltransferase are proposed to explain the decreased mitochondrial fatty acid oxidation with 0.4-1.0 mM-chlorpromazine reported by Leighton, Persico & Necochea [(1984) Biochem. Biophys. Res. Commun. 120, 505-511], whereas depression by the drug of carnitine octanoyltransferase activity is presented as the factor responsible for the decreased peroxisomal beta-oxidizing activity described by the above workers.     

The regulation of peroxisomal enzyme systems of Tetrahymena pyriformis by fatty acid composition, glucose and oxygen in the medium

The effect of the chain length of fatty acids on peroxisomal enzyme activities of Tetrahymena pyriformis was investigated. The growth of cells and the activities of peroxisomal enzymes were inhibited markedly by the addition of medium-chain fatty acids (C6-C12) to the culture medium, whereas the addition of longer-chain fatty acids (C14-C18) resulted in a slight increase of growth and in the marked stimulation of enzyme activities concerned with fatty acid beta-oxidation and the glyoxylate cycle in peroxisomes. Peroxisomal beta-oxidation (fatty acyl-CoA oxidase) was more potent towards longer-chain fatty acids than the mitochondrial activity (fatty acyl-CoA dehydrogenase). The induction of the peroxisomal beta-oxidation system by palmitate was repressed both by the addition of glucose and the aeration of the culture medium, whereas that of the peroxisomal glyoxylate cycle was repressed only by the addition of glucose to the medium. These results indicate that peroxisomal enzyme systems related to the beta-oxidation of fatty acids and the glyoxylate cycle are regulated by the compositions of fatty acids, glucose, and oxygen in the medium.     

Properties of a collagenase inhibitor partially purified from cultures of smooth muscle cells

1. The metabolism of [14-14C]erucate and [U-14C]palmitate has been investigated in perfused heart from rats fed 0.3% clofibrate for 10 days and from control rats. 2. The total uptake of fatty acids in the heart increased in the clofibrate fed group. Clofibrate increased the oxidation of [14-14C]erucic acid by 100% and the oxidation of [U-14C]palmitic acid by 30% compared to controls. 3. The chain-shortening of erucate to C20:1 and C18:1 fatty acids in the perfused heart was stimulated at least two-fold by clofibrate feeding. 4. The activity of the peroxisomal marker enzyme catalase increased 60%, the activity of cytochrome oxidase increased approx. 16% and the content of total coenzyme A increased 30% in heart homogenates from rats fed clofibrate compared to controls. 5. The isolated mitochondrial fraction from clofibrate fed rats showed an increased capacity for oxidation of palmitoylcarnitine and decanoylcarnitine, while the oxidation of erucoylcarnitine showed little change. 6. It is suggested that clofibrate increases the oxidation of [14-14C]erucic acid in the perfused heart by increasing the capacity for chain-shortening of [14-14C]erucate in the peroxisomal beta-oxidation system.     

A novel acyl-CoA oxidase that can oxidize short-chain acyl-CoA in plant peroxisomes

Short-chain acyl-CoA oxidases are beta-oxidation enzymes that are active on short-chain acyl-CoAs and that appear to be present in higher plant peroxisomes and absent in mammalian peroxisomes. Therefore, plant peroxisomes are capable of performing complete beta-oxidation of acyl-CoA chains, whereas mammalian peroxisomes can perform beta-oxidation of only those acyl-CoA chains that are larger than octanoyl-CoA (C8). In this report, we have shown that a novel acyl-CoA oxidase can oxidize short-chain acyl-CoA in plant peroxisomes. A peroxisomal short-chain acyl-CoA oxidase from Arabidopsis was purified following the expression of the Arabidopsis cDNA in a baculovirus expression system. The purified enzyme was active on butyryl-CoA (C4), hexanoyl-CoA (C6), and octanoyl-CoA (C8). Cell fractionation and immunocytochemical analysis revealed that the short-chain acyl-CoA oxidase is localized in peroxisomes. The expression pattern of the short-chain acyl-CoA oxidase was similar to that of peroxisomal 3-ketoacyl-CoA thiolase, a marker enzyme of fatty acid beta-oxidation, during post-germinative growth. Although the molecular structure and amino acid sequence of the enzyme are similar to those of mammalian mitochondrial acyl-CoA dehydrogenase, the purified enzyme has no activity as acyl-CoA dehydrogenase. These results indicate that the short-chain acyl-CoA oxidases function in fatty acid beta-oxidation in plant peroxisomes, and that by the cooperative action of long- and short-chain acyl-CoA oxidases, plant peroxisomes are capable of performing the complete beta-oxidation of acyl-CoA.     

Quantitation of acyl-CoA and acylcarnitine esters accumulated during abnormal mitochondrial fatty acid oxidation.

We have used radio-high pressure liquid chromatography to study the acyl-CoA ester intermediates and the acylcarnitines formed during mitochondrial fatty acid oxidation. During oxidation of [U-14C]hexadecanoate by normal human fibroblast mitochondria, only the saturated acyl-CoA and acylcarnitine esters can be detected, supporting the concept that the acyl-CoA dehydrogenase step is rate-limiting in mitochondrial beta-oxidation. Incubations of fibroblast mitochondria from patients with defects of beta-oxidation show an entirely different profile of intermediates. Mitochondria from patients with defects in electron transfer flavoprotein and electron transfer flavoprotein:ubiquinone oxido-reductase are associated with slow flux through beta-oxidation and accumulation of long chain acyl-CoA and acylcarnitine esters. Increased amounts of saturated medium chain acyl-CoA and acylcarnitine esters are detected in the incubations of mitochondria with medium chain acyl-CoA dehydrogenase deficiency, whereas long chain 3-hydroxyacyl-CoA dehydrogenase deficiency is associated with accumulation of long chain 3-hydroxyacyl- and 2-enoyl-CoA and carnitine esters. These studies show that the control strength at the site of the defective enzyme has increased. Radio-high pressure liquid chromatography analysis of intermediates of mitochondrial fatty acid oxidation is an important new technique to study the control, organization and defects of the enzymes of beta-oxidation.     

Postnatal Development and Isolation of Peroxisomes from Brain

We analyzed the postnatal peroxisome development in rat brain by measuring the enzyme activities of catalase and acyl-CoA oxidase and beta-oxidation of [1-14C]lignoceric acid. These enzyme activities were higher between 10 and 16 days of postnatal life and then decreased. We developed and compared two different methods for isolation of enriched peroxisomes from 10-day-old rat brain by using a combination of differential and density gradient centrifugation techniques. Peroxisomes in Percoll (self-generating gradient) banded at a density of 1.036 +/- 0.012 g/ml and in Nycodenz continuous gradient at 1.125 +/- 0.014 g/ml. Acyl-CoA oxidase, D-amino acid oxidase, L-pipecolic acid oxidase, and dihydroxyacetone phosphate acyltransferase activities and activities for the oxidation of very long chain fatty acid (lignoceric acid) were almost exclusively associated with catalase activity (a marker enzyme for peroxisomes) in the gradient. The postnatal increase in peroxisomal activity with the onset of myelination and the presence of enzyme for the biosynthesis of plasmalogens and oxidation of very long chain fatty acid (both predominant constituents of myelin) suggest that brain peroxisomes may play an important role in the assembly and turnover of myelin.     

Presence and features of fatty acyl-CoA binding activity in rat hepatic peroxisomes

We studied the fatty acyl-CoA binding activity of rat liver peroxisomes. After subcellular fractionation of rat liver treated with or without clofibrate, a peroxisome proliferator, the binding activity with [1-(14)C]palmitoyl-CoA was detected in the light mitochondrial fraction in addition to the mitochondrial and cytosol fractions. After Nycodenz centrifugation of the light mitochondrial fraction, the binding activity was detected in peroxisomes. The peroxisomal activity depended on the incubation temperature and peroxisome concentration. The activity also depended on the concentration of 2-mercaptoethanol, and a plateau of activity was unexpectedly found at 2-mercaptoethanol concentrations from 20 to 40 mM. Clofibrate increased the total and specific activity of the fatty acyl-CoA binding of peroxisomes by 7.9 and 2.5 times compared with the control, respectively. In the presence of 20% glycerol at 0 degree C, approximately 90% of the binding activity was maintained for up to at least 3 wk. After successive treatment with an ultramembrane Amicon YM series, about 70% of the binding activity was detected in the M.W. 30,000-100,000 fraction. When the M.W. 30,000-100,000 fraction was added to the incubation mixture of the peroxisomal fatty acyl-CoA beta-oxidation system, a slight increase in the beta-oxidation activity was found. 2-Mercaptoethanol (20 mM) significantly activated the fatty acyl-CoA beta-oxidation system to 1.4 times control. After gel filtration of the M.W. 30,000-100,000 fraction, the peaks of fatty acyl-CoA binding protein showed broad elution profiles from 45,000 to 75,000. These results suggest that fatty acyl-CoA binding activity can be detected directly in peroxisomes and is increased by peroxisome proliferators. The high binding activity in the presence of higher concentrations of 2-mercaptoethanol indicates the importance of the SH group for binding. The apparent molecular weight of the binding protein may be from 45,000 to 75,000.     

Fatty acyl-CoA oxidase in rat kidney and liver after application of thyroxine

Male rats were fed a standard diet containing 2.5 mg% L-thyroxine. After 10 and 20 days, in postnuclear fractions of the kidney the specific activity of fatty acyl-CoA oxidase, the enzyme responsible for the rate-limiting first step of peroxisomal fatty acid beta-oxidation, was increased by 100 and 160%, respectively. A similar effect was found in the liver. It is suggested that thyroxine essentially affects only this step of the fatty acid beta-oxidation sequence. Presumably the elevation of fatty acyl-CoA oxidase is one reason for the beneficial action of thyroid hormones in toxic lesions of the kidney.     

Peroxisomal straight-chain Acyl-CoA oxidase and D-bifunctional protein are essential for the retroconversion step in docosahexaenoic acid synthesis

Docosahexaenoic acid (DHA, C22:6n-3) is essential for normal brain and retinal development. The nature and subcellular location of the terminal steps in DHA biosynthesis have been controversial. Rather than direct Delta4-desaturation of C22:5n-3, it has been proposed that this intermediate is elongated to C24:5n-3, desaturated to C24:6n-3, and "retroconverted" to DHA via peroxisomal beta-oxidation. However, this hypothesis has recently been challenged. The goal of this study was to determine the mechanism and specific enzymes required for the retroconversion step in human skin fibroblasts. Cells from patients with deficiencies of either acyl-CoA oxidase or D-bifunctional protein, the first two enzymes of the peroxisomal straight-chain fatty acid beta-oxidation pathway, exhibited impaired (5-20% of control) conversion of either [1-14C]18:3n-3 or [1-14C]22:5n-3 to DHA as did cells from peroxisome biogenesis disorder patients comprising eight distinct genotypes. In contrast, normal DHA synthesis was observed in cells from patients with rhizomelic chondrodysplasia punctata, Refsum disease, X-linked adrenoleukodystrophy, and deficiency of mitochondrial medium- or very long-chain acyl-CoA dehydrogenase. Acyl-CoA oxidase-deficient cells accumulated 2-5 times more radiolabeled C24:6n-3 than did controls. Our data are consistent with the retroconversion hypothesis and demonstrate that peroxisomal beta-oxidation enzymes acyl-CoA oxidase and D-bifunctional protein are essential for this process in human skin fibroblasts.     

beta-Oxidation of the coenzyme A esters of elaidic, oleic, and stearic acids and their full-cycle intermediates by rat heart mitochondria.

beta-Oxidation rates for the CoA esters of elaidic, oleic and stearic acids and their full-cycle beta-oxidation intermediates and for the carnitine esters of oleic and elaidic acids were compared over a wide range of substrate and albumin concentrations in rat heart mitochondria. The esters of elaidic acid were oxidized at about half the rate of the oleic acid esters, while stearoyl-CoA was oxidized equally as rapid as oleoyl-CoA. The full-cycle beta-oxidation intermediates of elaidoyl-CoA (trans-16 : 1 delta 7, -14 : 1 delta 5, and -12 : 1 delta 3) were found to be oxidized at rates nearly equal to those for the corresponding intermediates of oleoyl-CoA. Therefore, after the first cycle of beta-oxidation, oleoyl-CoA and elaidoyl-CoA are oxidized at nearly equal rates. The activity of fatty acyl-CoA dehydrogenase was higher with elaidoyl-CoA and its full-cycle intermediates as substrates than with the corresponding cisisomers. It was concluded that the slower oxidation rate of elaidic acid is not due to slower oxidation of any of its full-cycle beta-oxidation intermediates, nor to slower activity of fatty acyl-CoA dehydrogenase, nor to outer mitochondrial carnitine acyltransferase. Possible explanations to account for the slower oxidation rate of elaidic acid are discussed.     

The inhibition by valproic acid of the mitochondrial oxidation of monocarboxylic and omega-hydroxymonocarboxylic acids: possible implications for the metabolism of gamma-aminobutyric acid

The interactions of 1-5 mM valproic acid with the hepatic fatty acid oxidation are here described. Valproic acid was not substrate for hepatic peroxisomal fatty acid oxidation. Its activation outside the mitochondrial matrix compartment was poor when compared to that of octanoic acid, a fatty acid containing the same number of carbones. Valproic acid did not inhibit the fatty acyl-CoA oxidase nor the cyanide-insensitive acyl-CoA oxidation. Valproic acid inhibited the mitochondrial oxidations of both long-chain monocarboxylyl-CoAs and omega-hydroxymonocarboxylyl-CoAs. Valproic acid prevented the oxidation by coupled mitochondria of decanoic and 10-hydroxydecanoic acids. Both butyric and 4-hydroxybutyric acids were oxidized by coupled mitochondria. These activities were abolished by preincubating the enzyme source with valproic acid. Administration to rats of 0.5% (w/w)- or 1% (w/w)-valproate containing diets were efficient in producing increased liver peroxisomal population and beta-oxidation. Preliminary investigations on the effects of valproic acid on mitochondrial fatty acid oxidation as a function of the animal used for the experiments pointed out an association of the protection of the mitochondrial process against the toxicity of the drug with enhanced carnitine acyltransferase and acyl-CoA hydrolase activities.     

Role of peroxisomal fatty acyl-CoA beta-oxidation in phospholipid biosynthesis

We have already reported that peroxisomal beta-oxidation has an anabolic function, supplying acetyl-CoA for bile acid biosynthesis [H. Hayashi and A. Miwa, 1989, Arch. Biochem. Biophys. 274, 582-589]. The anabolic significance of peroxisomal beta-oxidation was further investigated in the present study by using clofibrate, a peroxisome proliferator, as an experimental tool. Clofibrate suppressed 3-hydroxymethylglutaryl-CoA reductase activity (the key enzyme of cholesterol synthesis) and enhanced fatty acyl-CoA oxidase activity (the rate-limiting enzyme of beta-oxidation). Rats were fed a chow containing 0.25% clofibrate for 2 weeks, and then a bile duct fistula was implanted. [1-14C]lignoceric acid, which is degraded exclusively by peroxisomal FAOS, was injected into the rats 24 h after the operation. By this time, the secondary bile acids and pooled cholesterol which would normally be secreted into the bile are considered to have been exhausted from the liver. Clofibrate significantly decreased the incorporations of radioactivity into biliary bile acid (40% of the control) and cholesterol (50%), but did not affect biliary lipid contents. [14C]Acetyl-CoA formed by peroxisomal beta-oxidation of [1-14C]lignoceric acid was preferentially utilized for syntheses of long-chain fatty acids and phospholipids rather than synthesis of cholesterol or triglyceride. The radioactivities incorporated into the former two lipids were increased 2-fold over the control by administration of clofibrate, while the incorporation into triglyceride was decreased to approximately half. In particular, the incorporation into phosphatidylethanolamine was increased as much as 3.5-fold over the control. The contents of these lipids in the liver were not affected by clofibrate. The results suggest that peroxisomal beta-oxidation plays an important role in the biosynthesis of functional lipids such as phospholipids (this work), in addition to bile acids and cholesterol (previous report) by supplying acetyl-CoA.     

Beta-oxidation of medium chain (C8-C14) fatty acids studied in isolated liver cells

The beta-oxidation and esterification of medium-chain fatty acids were studied in hepatocytes from fasted, fed and fructose-refed rats. The beta-oxidation of lauric acid (12:0) was less inhibited by fructose refeeding and by (+)-decanoyl-carnitine than the oxidation of oleic acid was, suggesting a peroxisomal beta-oxidation of lauric acid. Little lauric acid was esterified in triacylglycerol fraction, except at high substrate concentrations or in the fructose-refed state. With [1-14C]myristic acid (14:0), [1-14C]lauric acid (12:0), [1-14C]octanoic acid (8:0) and [2-14C]adrenic acid (22:4(n - 6] as substrate for hepatocytes from carbohydrate-refed rats, a large fraction of the 14C-labelled esterified fatty acids consisted of newly synthesized palmitic acid (16:0), stearic acid (18:0) and oleic acid (18:1) while intact [1-14C]oleic acid substrate was esterified directly. With [9,10-3H]myristic acid as the substrate, small amounts of shortened 3H-labelled beta-oxidation intermediates were found. With [U-14C]palmitic acid, no shortened fatty acids were detected. It was concluded that when the mitochondrial fatty acid oxidation is down-regulated such as in the carbohydrate-refed state, medium-chain fatty acids can partly be retailored to long-chain fatty acids by peroxisomal beta-oxidation followed by synthesis of C16 and C16 fatty acids which can then stored as triacylglycerol.     

Partitioning of polyunsaturated fatty acid oxidation between mitochondria and peroxisomes in isolated rat hepatocytes studied by HPLC separation of oxidation products

The extent of mitochondrial and peroxisomal contribution to beta-oxidation of 18-, 20- and 24-carbon n-3 and n-6 polyunsaturated fatty acids (PUFAs) in intact rat hepatocytes is not fully clear. In this study, we analyzed radiolabeled acid soluble oxidation products by HPLC to identify mitochondrial and peroxisomal oxidation of 24:5n-3, 18- and 20-carbon n-3 and n-6 PUFAs. Mitochondrial fatty acid oxidation produced high levels of ketone bodies, tricarboxylic acid cycle intermediates and CO(2), while peroxisomal beta-oxidation released acetate. Inhibition of mitochondrial fatty acid oxidation with 2-tetradecylglycidic acid (TDGA), high amounts of [14C]acetate from oxidation of 24:5n-3, 18- and 20-carbon PUFAs were observed. In the absence of TDGA, high amounts of [14C]-labeled mitochondrial oxidation products were formed from oxidation of 24:5n-3, 18- and 20-carbon PUFAs. With 18:1n-9, high amounts of mitochondrial oxidation products were formed in the absence of TDGA, and TDGA strongly suppressed the oxidation of this fatty acid. Data of this study indicated that a shift in the partitioning from mitochondrial to peroxisomal oxidation differed for each individual fatty acid and is a specific property of 24:5n-3, 18- and 20-carbon n-3 and n-6 PUFAs.[14C]22:6n-3 was detected with [3-14C]24:5n-3, but not with [1-14C]24:5n-3 as the substrate, while [14C]16:0 was detected with [1-14C]24:5n-3, but not with [3-14C]24:5n-3 as the substrate. Furthermore, the amounts of 14CO(2) were similar when cells were incubated with [3-14C]24:5n-3 versus [1-14C]24:5n-3. These findings indicated that the proportion of 24:5n-3 oxidized in mitochondria was high, and that 24:5n-3 and 24:6n-3 were mostly beta-oxidized only one cycle in peroxisomes.     

Acyl-CoA oxidase activity and peroxisomal fatty acid oxidation in rat tissues

Acyl-CoA oxidase, the first enzyme of the peroxisomal β-oxidation, was proved to be rate-limiting for this process in homogenates of rat liver, kidney, adrenal gland, heart and skeletal muscle. Acyl-CoA oxidase activity, based on H₂O₂-dependent leuko-dichlorofluorescein oxidation in tissue extract, was compared with radiochemically assayed peroxisomal β-oxidation rates. Dichlorofluorescein production was a valid measure of peroxisomal fatty acid oxidation only in liver and kidney, but not in adrenal gland, heart or skeletal muscle. Production of ¹⁴C-labeled acid-soluble products from 1-¹⁴C-labeled fatty acids in the presence of antimycin-rotenone appears to be a more accurate and sensitive estimate of peroxisomal β-oxidation than the acyl-CoA oxidase activity on base of H₂O₂ production. Chain-length specificity of acyl-CoA oxidase changed with the acyl-CoA concentrations used. Below 80 μM, palmitoyl-CoA showed the highest activity of the measured substrates in rat liver extract. No indications were obtained for the presence in rat liver of more forms of acyl-CoA oxidase with different chain-length specificity.