Formation and degradation of dicarboxylic acids in relation to alterations in fatty acid oxidation in rats.

Dicarboxylic acids are excreted in urine when fatty acid oxidation is increased (ketosis) or inhibited (defects in beta-oxidation) and in Reye's syndrome. omega-Hydroxylation and omega-oxidation of C6-C12 fatty acids were measured by mass spectrometry in rat liver microsomes and homogenates, and beta-oxidation of the dicarboxylic acids in liver homogenates and isolated mitochondria and peroxisomes. Medium-chain fatty acids formed large amounts of medium-chain dicarboxylic acids, which were easily beta-oxidized both in vitro and in vivo, in contrast to the long-chain C16-dicarboxylic acid, which was toxic to starved rats. Increment of fatty acid oxidation in rats by starvation or diabetes increased C6:C10 dicarboxylic acid ratio in rats fed medium-chain triacylglycerols, and increased short-chain dicarboxylic acid excretion in urine in rats fed medium-chain dicarboxylic acids. Valproate, which inhibits fatty acid oxidation and may induce Reye like syndromes, caused the pattern of C6-C10-dicarboxylic aciduria seen in beta-oxidation defects, but only in starved rats. It is suggested, that the origin of urinary short-chain dicarboxylic acids is omega-oxidized medium-chain fatty acids, which after peroxisomal beta-oxidation accumulate as C6-C8-dicarboxylic acids. C10-C12-dicarboxylic acids were also metabolized in the mitochondria, but did not accumulate as C6-C8-dicarboxylic acids, indicating that beta-oxidation was completed beyond the level of adipyl CoA.     

Pathology of urethral fibromuscular system related to parturition-induced stress urinary incontinence and TGF-β1/Smad pathway

The accumulation of octanoic (OA) and decanoic (DA) acids in tissue is the common finding in medium-chain acyl-coenzyme A dehydrogenase deficiency (MCADD), the most frequent defect of fatty acid oxidation. Affected patients present hypoketotic hypoglycemia, rhabdomyolysis, hepatomegaly, seizures and lethargy, which may progress to coma and death. At present, the pathophysiological mechanisms underlying hepatic and skeletal muscle alterations in affected patients are poorly known. Therefore, in the present work, we investigated the in vitro effects of OA and DA, the accumulating metabolites in MCADD, on various bioenergetics and oxidative stress parameters. It was verified that OA and DA decreased complexes I-III, II-III and IV activities in liver and also inhibit complex IV activity in skeletal muscle. In addition, DA decreased complexes II-III activity in skeletal muscle. We also verified that OA and DA increased TBA-RS levels and carbonyl content in both tissues. Finally, DA, but not OA, significantly decreased GSH levels in rat skeletal muscle. Our present data show that the medium-chain fatty acids that accumulate in MCADD impair electron transfer through respiratory chain and elicit oxidative damage in rat liver and skeletal muscle. It may be therefore presumed that these mechanisms are involved in the pathophysiology of the hepatopathy and rhabdomyolysis presented by MCADD-affected patients.     

Effect of diets rich in medium-chain and long-chain triglycerides on lipogenic-enzyme gene expression in liver and adipose tissue of the weaned rat.

The activity and mRNA concentrations of two lipogenic enzymes, fatty-acid synthase and acetyl-CoA carboxylase were measured in the liver and white adipose tissue of rats weaned to a carbohydrate-rich diet containing either long-chain or medium-chain fatty acids, and compared to those of rats weaned on a diet containing less than 1% (total energy) fat (high-carbohydrate diet). In the liver, the diet containing long-chain fatty acids inhibited the increase of both lipogenic-enzyme mRNA concentrations and activities seen at weaning on the high-carbohydrate diet but did not prevent the decrease in phosphoenolpyruvate carboxykinase mRNA and activity. In contrast, the diet containing medium-chain fatty acids induced a slower but finally similar increase in lipogenic-enzyme mRNA concentrations and activities. In adipose tissue, a similar trend was observed, although the inhibitory effect of the diet containing long-chain fatty acids was considerably less marked than in liver. It is concluded that medium-chain and long-chain fatty acids have not the same inhibitory potency of the gene expression of lipogenic enzymes, and that long-chain fatty acids have a more marked effect in the liver.     

Peroxisomes contribute to the acylcarnitine production when the carnitine shuttle is deficient

Fatty acid β-oxidation may occur in both mitochondria and peroxisomes. While peroxisomes oxidize specific carboxylic acids such as very long-chain fatty acids, branched-chain fatty acids, bile acids, and fatty dicarboxylic acids, mitochondria oxidize long-, medium-, and short-chain fatty acids. Oxidation of long-chain substrates requires the carnitine shuttle for mitochondrial access but medium-chain fatty acid oxidation is generally considered carnitine-independent. Using control and carnitine palmitoyltransferase 2 (CPT2)- and carnitine/acylcarnitine translocase (CACT)-deficient human fibroblasts, we investigated the oxidation of lauric acid (C12:0). Measurement of the acylcarnitine profile in the extracellular medium revealed significantly elevated levels of extracellular C10- and C12-carnitine in CPT2- and CACT-deficient fibroblasts. The accumulation of C12-carnitine indicates that lauric acid also uses the carnitine shuttle to access mitochondria. Moreover, the accumulation of extracellular C10-carnitine in CPT2- and CACT-deficient cells suggests an extramitochondrial pathway for the oxidation of lauric acid. Indeed, in the absence of peroxisomes C10-carnitine is not produced, proving that this intermediate is a product of peroxisomal β-oxidation. In conclusion, when the carnitine shuttle is impaired lauric acid is partly oxidized in peroxisomes. This peroxisomal oxidation could be a compensatory mechanism to metabolize straight medium- and long-chain fatty acids, especially in cases of mitochondrial fatty acid β-oxidation deficiency or overload.     

Fatty acid metabolism and ketone formation in the suckling rat

Rat milk triacylglycerols contain 35% medium-chain length fatty acids. About 70% of ingested medium-chain fatty acids are released from milk triacylglycerols in the stomach and small intestine and are absorbed directly into the portal venous system. Based on studies with the perfused suckling rat liver and in vivo studies with 2-tetradecylglycidic acid, an inhibitor of long-chain fatty acid oxidation, it is estimated that medium-chain fatty acids provide 75-80% of the substrate for ketogenesis. The preferential use of medium-chain fatty acids for ketogenesis spares long-chain fatty acids for complex lipid and membrane biosynthesis during this period of rapid growth. Although medium-chain fatty acids are the major substrate for ketogenesis, this pathway accounts for only 15% of the utilization of ingested medium-chain fatty acids, the rest presumably being oxidized directly in extrahepatic tissues.     

Mouse models for disorders of mitochondrial fatty acid beta-oxidation

Mitochondrial beta-oxidation of fatty acids is vital for energy production in periods of fasting and other metabolic stress. Human patients have been identified with inherited disorders of mitochondrial beta-oxidation of fatty acids with enzyme deficiencies identified at many of the steps in this pathway. Although these patients exhibit a range of disease processes, Reye-like illness (hypoketotic-hypoglycemia, hyperammonemia and fatty liver) and cardiomyopathy are common findings. There have been several mouse models developed to aid in the study of these disease conditions. The characterized mouse models include inherited deficiencies of very long-chain acyl-CoA dehydrogenase, long-chain acyl-CoA dehydrogenase, short-chain acyl-CoA dehydrogenase, mitochondrial trifunctional protein-alpha, and medium-/short-chain hydroxyacyl-CoA dehydrogenase. Mouse mutants developed, but presently incompletely characterized as models, include carnitine palmitoyltransferase-1a and medium-chain acyl-CoA dehydrogenase deficiencies. In general, the mouse models of disorders of mitochondrial fatty acid beta-oxidation have shown clinical signs that include Reye-like syndrome and cardiomyopathy, and many are cold intolerant. It is expected that these mouse models will provide vital contributions in understanding the mechanisms of disease pathogenesis of fatty acid oxidation disorders and the development of appropriate treatments and supportive care.     

Analysis of the tissue-specific expression, developmental regulation, and linkage relationships of a rodent gene encoding heart fatty acid binding protein

The rat contains at least three homologous cytosolic proteins that bind long chain fatty acids, termed liver (L-), intestinal (I-), and heart (H-) fatty acid binding protein (FABP). I-FABP mRNA is confined to the gastrointestinal tract while L-FABP mRNA is abundantly represented in hepatocytes as well as enterocytes. We have isolated a rat heart FABP cDNA clone and determined the pattern of H-FABP mRNA accumulation in a wide variety of tissues harvested from late fetal, suckling, weaning, and adult rats. RNA blot hybridizations and primer extension analysis disclosed that the distribution of H-FABP mRNA in adult rat tissues is different from that of I- or L-FABP mRNA. H-FABP mRNA is most abundant in adult heart. This mRNA was also present in an adult slow twitch (type I) skeletal muscle (soleus, 63% of the concentration in heart), testes (28%), a fast twitch skeletal muscle (psoas, 17%), brain (10%), kidney (5%), and adrenal gland (5%). H-FABP mRNA was not detected in adult small intestine, colon, spleen, lung, or liver RNA. Distinct patterns of developmental change in H-FABP mRNA accumulation were documented in heart, placenta, brain, kidney, and testes. Myocardial H-FABP mRNA levels rise rapidly during the 48 h prior to and after birth, reaching peak levels by the early weaning period. The postnatal increase in myocardial H-FABP mRNA concentration and its relative distribution in adult fast and slow twitch skeletal muscle are consistent with its previously proposed function in facilitating mitochondrial beta-oxidation of fatty acids. However, the presence of H-FABP mRNA in brain, a tissue which does not normally significantly oxidize fatty acids in late postnatal life, suggests that H-FABP may play a wider role in fatty acid metabolism than previously realized. Mouse-hamster somatic cell hybrids were utilized to map H-FABP. Using stringencies which did not produce cross-hybridization between L-, I-, and H-FABP DNA sequences, we found at least three loci in the mouse genome, each located on different chromosomes, which reacted with our cloned H-FABP cDNA. None of these H-FABP-related loci were linked to the gene which specifies a highly homologous adipocyte-specific protein termed aP2 or to genes encoding two other members of this protein family, cellular retinol binding protein and cellular retinol binding protein II.(ABSTRACT TRUNCATED AT 400 WORDS)     

Intestinal concentrations of free and encapsulated dietary medium-chain fatty acids and effects on gastric microbial ecology and bacterial metabolic products in the digestive tract of piglets

The influence of low dietary levels of free and encapsulated medium-chain fatty acids on their concentrations in the digesta, the gastric microbial ecology and bacterial metabolic products in the gastrointestinal tract (GIT) in weaned piglets was studied. Starting after weaning, 36 piglets were fed a diet without (Control) or with medium-chain fatty acids uncoated (MCFA) or coated with vegetable fat and lecithin (MCFAc). After 4 weeks, the animals were killed, and digesta from the stomach and different sections of the GIT were collected. The concentrations of caprylic (p < 0.001) and capric (p = 0.001) acids were higher in the stomachs of piglets fed diets MCFA and MCFAc compared to the Control group. The concentrations dropped rapidly along the GIT, regardless of encapsulation, and tended to be higher in groups MCFA and MCFAc compared to the Control. Compared to the Control group, ingestion of diet MCFAc led to an increase in the number of eubacteria (p = 0.001), enterobacteriaceae (p < 0.001), clostridial clusters I (p = 0.001) and IV (p = 0.019), Lactobacillus johnsonii (p < 0.001) and Lactobacillus amylovorus (p = 0.001) in gastric contents. A similar trend was seen with diet MCFA. Relative concentrations of short-chain fatty acids were characterised by lower propionic acid levels (p = 0.045), numerically (p < 0.1) higher acetic, lower n-butyric and i-valeric acid concentrations in the small intestine. Lactic acid concentrations were not significantly changed in the GIT, but ammonia concentrations increased (p < 0.001) in the distal small intestine in the MCFA and MCFAc groups. In conclusion, medium-chain fatty acids affected microbial ecology parameters in the gastric contents and bacterial metabolites in the small intestine. At low dietary levels, medium-chain fatty acids may be regarded as modulators of the gastric microbiota in weaned piglets.     

Substrate specificity of human carnitine acetyltransferase: Implications for fatty acid and branched-chain amino acid metabolism

Carnitine acyltransferases catalyze the reversible conversion of acyl-CoAs into acylcarnitine esters. This family includes the mitochondrial enzymes carnitine palmitoyltransferase 2 (CPT2) and carnitine acetyltransferase (CrAT). CPT2 is part of the carnitine shuttle that is necessary to import fatty acids into mitochondria and catalyzes the conversion of acylcarnitines into acyl-CoAs. In addition, when mitochondrial fatty acid β-oxidation is impaired, CPT2 is able to catalyze the reverse reaction and converts accumulating long- and medium-chain acyl-CoAs into acylcarnitines for export from the matrix to the cytosol. However, CPT2 is inactive with short-chain acyl-CoAs and intermediates of the branched-chain amino acid oxidation pathway (BCAAO). In order to explore the origin of short-chain and branched-chain acylcarnitines that may accumulate in various organic acidemias, we performed substrate specificity studies using purified recombinant human CrAT. Various saturated, unsaturated and branched-chain acyl-CoA esters were tested and the synthesized acylcarnitines were quantified by ESI-MS/MS. We show that CrAT converts short- and medium-chain acyl-CoAs (C2 to C10-CoA), whereas no activity was observed with long-chain species. Trans-2-enoyl-CoA intermediates were found to be poor substrates for this enzyme. Furthermore, CrAT turned out to be active towards some but not all the BCAAO intermediates tested and no activity was found with dicarboxylic acyl-CoA esters. This suggests the existence of another enzyme able to handle the acyl-CoAs that are not substrates for CrAT and CPT2, but for which the corresponding acylcarnitines are well recognized as diagnostic markers in inborn errors of metabolism.     

Lack of phosphatidylethanolamine N-methyltransferase in mice does not promote fatty acid oxidation in skeletal muscle

Phosphatidylethanolamine N-methyltransferase (PEMT) converts phosphatidylethanolamine (PE) to phosphatidylcholine (PC) in the liver. Mice lacking PEMT are protected from high-fat diet-induced obesity and insulin resistance, and exhibit increased whole-body energy expenditure and oxygen consumption. Since skeletal muscle is a major site of fatty acid oxidation and energy utilization, we determined if rates of fatty acid oxidation/oxygen consumption in muscle are higher in Pemt^−/− mice than in Pemt^+/+ mice. Although PEMT is abundant in the liver, PEMT protein and activity were undetectable in four types of skeletal muscle. Moreover, amounts of PC and PE in the skeletal muscle were not altered by PEMT deficiency. Thus, we concluded that any influence of PEMT deficiency on skeletal muscle would be an indirect consequence of lack of PEMT in liver. Neither the in vivo rate of fatty acid uptake by muscle nor the rate of fatty acid oxidation in muscle explants and cultured myocytes depended upon Pemt genotype. Nor did PEMT deficiency increase oxygen consumption or respiratory function in skeletal muscle mitochondria. Thus, the increased whole body oxygen consumption in Pemt^−/− mice, and resistance of these mice to diet-induced weight gain, are not primarily due to increased capacity of skeletal muscle for utilization of fatty acids as an energy source.     

Growth of Escherichia coli on Short-Chain Fatty Acids: Growth Characteristics of Mutants

The parent Escherichia coli K-12 is constitutive for the enzymes of the glyoxylate bypass and adapts to growth on long-chain fatty acids (C(12) to C(18)). It does not utilize medium-chain (C(6) to C(11)) or short-chain (C(4), C(5)) n-monocarboxylic acids. Several mutants of this strain which grow using short- or medium-chain acids, or both, as the sole carbon source were selected and characterized. One mutant (D(1)) synthesizes the beta-oxidation enzymes constitutively and grows on medium-chain but not on short-chain acids. A second (N(3)) is partially derepressed for synthesis of these enzymes and grows both on medium-chain and on short-chain acids. Secondary mutants (N(3)V(-), N(3)B(-), N(3)OL(-)) were derived from N(3). N(3)V(-) grows on even-chain but not on odd-chain acids and exhibits a lesion in propionate oxidation. N(3)B(-) grows on odd-chain but not on even-chain acids and exhibits no crotonase activity as assayed by hydration of crotonyl-CoA. N(3)OL(-) grows on acetate and propionate but does not utilize fatty acids C(4) to C(18); it exhibits multiple deficiencies in the beta-oxidation pathway. Growth on acetate of N(3), but not of the parent strain, is inhibited by 4-pentenoate. Revertants of N(3) which are resistant to growth inhibition by 4-pentenoate (N(3)PR) exhibit loss of ability to grow on short-chain acids but retain the ability to grow on medium-chain and long-chain acids. The growth characteristics of these mutants suggest that in order to grow at the expense of butyrate and valerate, E. coli must be (i) derepressed for synthesis of the beta-oxidation enzymes and (ii) derepressed for synthesis of a short-chain fatty acid uptake system.     

Development of oxaalkyne and alkyne fatty acids as novel tracers to study fatty acid beta-oxidation pathways and intermediates

Fatty acid beta-oxidation is a key process in mammalian lipid catabolism. Disturbance of this process results in severe clinical symptoms, including dysfunction of the liver, a major beta-oxidizing tissue. For a thorough understanding of this process, a comprehensive analysis of involved fatty acid and acyl-carnitine intermediates is desired, but capable methods are lacking. Here, we introduce oxaalkyne and alkyne fatty acids as novel tracers to study the beta-oxidation of long- and medium-chain fatty acids in liver lysates and primary hepatocytes. Combining these new tracer tools with highly sensitive chromatography and mass spectrometry analyses, this study confirms differences in metabolic handling of fatty acids of different chain length. Unlike longer chains, we found that medium-chain fatty acids that were activated inside or outside of mitochondria by different acyl-CoA synthetases could enter mitochondria in the form of free fatty acids or as carnitine esters. Upon mitochondrial beta-oxidation, shortened acyl-carnitine metabolites were then produced and released from mitochondria. In addition, we show that hepatocytes ultimately also secreted these shortened acyl chains into their surroundings. Furthermore, when mitochondrial beta-oxidation was hindered, we show that peroxisomal beta-oxidation likely acts as a salvage pathway, thereby maintaining the levels of shortened fatty acid secretion. Taken together, we conclude that this new method based on oxaalkyne and alkyne fatty acids allows for metabolic tracing of the beta-oxidation pathway in tissue lysate and in living cells with unique coverage of metabolic intermediates and at unprecedented detail.     

Interaction of short-chain and branched-chain fatty acids and their carnitine and CoA esters and of various metabolites and agents with branched-chain 2-oxo acid oxidation in rat muscle and liver mitochondria

Interaction of various compounds with the 14CO2 production from [1-14C]-labelled branched-chain 2-oxo acids was studied in intact rat quadriceps muscle and liver mitochrondria. In the absence of carnitine, CoA esters of short-chain and branched-chain fatty acids, CoA and acetyl-L-carnitine stimulated oxidation of 4-methyl-2-oxopentanoate and 3-methyl-2-oxobutanoate in muscle mitochondria. Octanoyl-L-carnitine inhibited oxidation of the latter, but stimulated that of the former substrate. Isovaleryl-L-carnitine was inhibitory with both substrates. Carnitine stimulates markedly 3-methyl-2-oxobutanoate oxidation in liver mitochondria at substrate concentrations higher than 0.1 mM, in contrast to 4-methyl-2-oxopentanoate oxidation. In the presence of carnitine, 3-methyl-2-oxobutanoate oxidation was inhibited in muscle and liver mitochondria by octanoate, octanoyl-L-carnitine and isovaleryl-L-carnitine. The latter ester and octanoyl-D-carnitine inhibited also 4-methyl-2-oxopentanoate oxidation in muscle mitochondria. Branched-chain 2-oxo acids inhibited mutaly their oxidation, except that 3-methyl-2-oxobutanoate did not inhibit 4-methyl-2-oxopentanoate oxidation in liver mitochondria. Their degradation products, isovalerate, 3-methylcrotonate, isobutyrate and 3-hydroxyisobutyrate inhibited to a different extent 2-oxo acid oxidation in liver mitochondria. The effect of CoA esters was studied in permeabilized and with cofactors reinforced mitochondria. Acetyl-CoA and isovaleryl-CoA inhibited only 3-methyl-2-oxobutanoate oxidation in muscle mitochondria. Octanoyl-CoA inhibited oxidation of both 2-oxo acids in muscle and 4-methyl-2-oxopentanoate oxidation in liver mitochondria.(ABSTRACT TRUNCATED AT 250 WORDS)     

Minor amounts of plasma medium-chain fatty acids and no improved time trial performance after consuming lipids.

Medium-chain triacylglycerols (MCT) have a potential glycogen-saving effect during exercise due to rapid hydrolysis and oxidation. However, studies comparing intake of carbohydrates (CHO) plus 80-90 g MCT with intake of CHO alone have revealed different results. The present study tested performance after consumption of specific structured triacylglycerol, consisting of a mixture of medium-chain fatty acids and long-chain fatty acids, to prevent the adverse effects observed by MCT (pure medium-chain fatty acids) regarding gastrointestinal distress. Seven well-trained subjects cycled 3 h at 55% of maximum O2 uptake during which they ingested CHO or CHO plus specific structured triacylglycerols. Immediately after the constant-load cycling, the subjects performed a time trial of approximately 50-min duration. Breath and blood samples were obtained regularly during the experiment. Fatty acid composition of plasma triacylglycerols, fatty acids, and phospholipids was determined. Performance was similar after administration of CHO plus specific structured triacylglycerol [medium-, long-, and medium-chain fatty acid (MLM)] compared with CHO (50.0 +/- 1.8 and 50.8 +/- 3.6 min, respectively). No plasma 8:0 was detected in the plasma lipid classes, but the amount of phospholipid fatty acids was significantly higher after CHO+MLM compared with CHO intake. The lacking time trial improvement after intake of medium-chain fatty acids might be due to no available 8:0 in the systemic circulation. A higher level of plasma phospholipid fatty acids in the CHO+MLM compared with the CHO group was probably due to endogenous phospholipid release into chylomicrons.     

Isolation from bovine liver mitochondria and characterization of three distinct carboxylic acid:CoA ligases with activity toward xenobiotics

A mitochondrial freeze/thaw lysate was fractionated on a DEAE-cellulose column into four distinct acyl-CoA ligase fractions. First to elute was a 50 kDa short-chain ligase that activated only short-chain fatty acids. Next to elute were three ligases that had activity toward both medium-chain fatty acids and xenobiotic carboxylic acids; these were termed xenobiotic/medium-chain ligases (X-ligases) and labeled XL-I, XL-II, and XL-III, respectively, based on order of elution. The molecular weight of X-ligases I, II, and III were ca. 55,000, 55,500 and 53,000, respectively. Form XL-III showed no pH optimum; the rate increased steadily with pH beginning from pH 7.0. XL-I and XL-II showed the same behavior with benzoate as substrate, but with medium-chain fatty acids, both forms had a pH optimum at 8.8. The three X-ligases differed in substrate specificity. XL-I was the predominant nicotinic acid activating form and had the lowest K_m for benzoate. Form XL-II was the only form with measurable salicylate activity, although it was extremely low. XL-III was the only 2,4,6,8-decatetraenoic acid activating form and also was the predominant medium-chain fatty acid-activating form. By comparison of substrate specificities, it was concluded that the two previously reported ligase preparations were mixtures of the three forms. When the ligase rates were compared to previously determined N-acyltransferase rates toward benzoyl-CoA and phenylacetyl-CoA, the data showed that ligase activities are 100-fold lower, and thus the ligase is rate limiting for the conjugation of both of these xenobiotics. © 1996 John Wiley & Sons, Inc.     

The effect of di(ethylhexyl)phthalate on fatty acid oxidation and carnitine palmitoyltransferase in various rat tissues

Male rats were fed a diet with or without 2% di(2-ethylhexyl)phthalate (DEHP) for 12 days. Total and peroxisomal oxidation rates of palmitic and arachidonic acid were increased in homogenates of liver and kidney after DEHP administration. The relative peroxisomal contribution to the total oxidation was only higher in liver. The activities of acyl-CoA oxidase and carnitine palmitoyltransferase were also higher in both tissues. Immunoblots showed that the increase of fatty acid oxidation was associated with a higher concentration of enzymes of peroxisomal and mitochondrial beta-oxidation. DEHP did not change total and peroxisomal fatty acid oxidation and activity of carnitine palmitoyltransferase of homogenates of heart and skeletal muscle. The cause for the tissue-specific response is discussed.     

Involvement of carnitine acyltransferases in peroxisomal fatty acid metabolism by the yeast Pichia guilliermondii.

This article provides information about peroxisomal fatty acid metabolism in the yeast Pichia guilliermondii. The existence of inducible mitochondrial carnitine palmitoyltransferase and peroxisomal carnitine octanoyl-transferase activities was demonstrated after culture of this yeast in a medium containing methyl oleate. The subcellular sites and induction patterns were studied. The inhibition of carnitine octanoyl- and palmitoyl-transferases by chlorpromazine to a large extent prevented the otherwise observed metabolism-dependent inactivation of thiolase by 2-bromofatty acids in vivo. We concluded that the metabolism of long- and medium-chain fatty acids in the peroxisome of this yeast involved carnitine intermediates.