Arachidoyl- and arachidonoyl-CoA elongation mechanism in swine cerebral microsomes

Long-chain saturated and polyunsaturated fatty acyl-CoA elongations were studied in swine cerebral microsomes. The elongation of endogenous palmitoyl-CoA to stearate was highly active in both cerebral and liver microsomes, whereas those of arachidoyl-CoA (20:0-CoA) and endogenous arachidonoyl-CoA (20:4-CoA) were high in cerebral microsomes, but negligible in liver microsomes. The elongation of 22:4 to 24:4 was also observed in cerebral microsomes. Both NADPH and NADH at 500 microM were effective in elongation of 16:0-, 20:0- and 20:4-CoA, whereas NADPH was more effective in elongation of 22:4 to 24:4 than NADH. The incorporation of deuterium atoms to the elongated product was detected by the technique of mass fragmentography when the NADPH-dependent elongations of 20:0-CoA and 20:4-CoA were performed in 2H2O medium upon cerebral microsomes. The number of incorporated deuterium atoms into 22:0 elongated from 20:0-CoA was mainly two, and that into 22:4 elongated from 20:4-CoA was mainly three. These results indicated that part of hydrogens in elongated arachidoyl- and arachidonoyl-CoA were transferred from NADPH.     

Characterization of fatty acid elongation system in porcine neutrophil microsomes

Microsomes purified from porcine neutrophils containing the fatty acid chain-elongation system for long- and very-long-chain fatty acyl-CoAs, and several enzymatic characters for the elongation of palmitoyl-CoA (16:0-CoA) and arachidoyl-CoA (20:0-CoA) were examined. The heat-inactivation profile for the elongation of 16:0-CoA was different from that of 20:0-CoA, suggesting the presence of different enzyme systems for palmitoyl-CoA and arachidoyl-CoA. Contrary to the elongation system of brain microsomes, the successive synthesis of lignoceric acid (24:0) from 20:0-CoA at 60 microM was not prominent under normal conditions in the neutrophil microsomes. The synthesis of behenic acid (22:0) was slightly inhibited by 0.5 mM N-ethylmaleimide (NEM) present in the assay mixture, whereas the pre-treatment of microsomes with 0.5 mM NEM largely inhibited the synthesis of 22:0 from 20:0-CoA. The synthesis of 24:0, however, was enhanced by 0.5 mM NEM in the elongation of 20:0-CoA and the rate of 24:0 synthesis became dominant over the synthesis of 22:0. These results suggested that the elongation enzyme for very-long-chain fatty acyl-CoA, especially for 20:0-CoA elongation to 22:0 in the neutrophil microsomes contained NEM-sensitive sulfhydryl groups in the active center and the mechanism for the synthesis of 24:0 through successive elongation from 20:0-CoA was different from that of 22:0, as the former was enhanced by NEM whereas the latter was strongly inhibited.     

Activation of synthesis of hexacosenoic acid by sulfhydryl reagents in swine cerebral microsomes

The elongation of icosenoyl-CoA (20:1-CoA) in swine cerebral microsomes resulted in the synthesis of docosenoic acid (22:1) and tetracosenoic acid (24:1), but the synthesis of hexacosenoic acid (26:1) was negligible. In contrast, in the presence of sulfhydryl reagents (0.6 mM N-ethylmaleimide [NEM] or 0.3 mM p-chloromercuriphenylsulfonic acid [PCMPS]) the synthesis of 26:1 was remarkably enhanced. We suggest that the synthesis of 26:1 from 20:1-CoA was more enhanced by NEM or PCMPS as a result of activation of the condensation step in the elongation of 24:1 (intermediate) to 26:1.     

Analysis of the condensation step in elongation of very-long-chain saturated and tetraenoic fatty acyl-CoAs in swine cerebral microsomes

The condensation products in the elongation of exogenous arachidoyl-CoA (20:0-CoA) and endogenous fatty acids in adult swine cerebral microsomes were isolated and purified by using HPLC and a radioanalyzer. A saponification product of the condensation reaction of 20:0-CoA with malonyl-CoA was identified by gas chromatography-mass spectrometry as 2-heneicosanone (21:0-2-one). The endogenous substrates (16:0-CoA and 20:4-CoA) were likewise identified as 2-heptadecanone (17:0-2-one) and 2-heneicosatetraenone (21:4-2-one). Quantitative analysis of condensation activity was performed using electron-impact mass fragmentography. A characteristic fragment ion (m/z 59) of 21:0-2-one was used to estimate the condensation activity for 20:0-CoA, and fragment ions at m/z 58 and 80 were monitored for the endogenous substrates (16:0-CoA and 20:4-CoA, respectively). The molecular ion for each product was detected using chemical ionization. A comparative study of the condensation of 20:0-CoA and endogenous substrates was carried out for microsomes obtained from white matter, gray matter, and isolated neuronal cells; the activity for 20:0-CoA was significantly lower in gray matter and neuronal cells than in white matter, whereas the activity for endogenous substrates was almost the same for microsomes obtained from gray and white matter. This result suggests that the condensation enzyme for 20:0-CoA may be different from that for endogenous 16:0-CoA or 20:4-CoA in swine cerebral microsomes.     

Comparison between condensation and overall chain elongation of arachidoyl-CoA and arachidonoyl-CoA in swine cerebral microsomes

The condensation and overall elongation products of exogenous arachidoyl-CoA (20:0-CoA) and endogenous fatty acids in swine cerebral microsomes were detected by radio gas chromatography. In addition, the condensation products with malonyl-CoA as substrate were analyzed by radio high-performance liquid chromatography. Three main condensation products were detected; the overall elongation products of exogenous 20:0-CoA were 22:0 and 24:0, and those of endogenous substrates were 18:0, 22:4, and 24:4. The yield was estimated for the conversion of 3-ketoacyl-CoAs to the corresponding saponification products (methyl ketones or R-2-one; e.g., 2-heptadecanone = 17:0-2-one); these products were identified in the preceding paper (S. Yoshida and M. Takeshita (1987) Arch. Biochem. Biophys. 254, 170-179). The extraction of R-2-one by hexane depended on the acyl chain length. The yield of 2-heneicosanone (21:0-2-one) detected by radio gas chromatography was 80% whereas the yields of 17:0-2-one and 2-heneicosatetraenone (21:4-2-one) from the corresponding 3-ketoacyl-CoAs were 56 and 48%, respectively. A quantitative comparison was performed for the condensation and overall elongation activity; it was noticed that the condensation activity for the system which simultaneously produced two elongation products was nearly the same as that of the corresponding overall elongation activity. This result suggests that the condensation step may be at least one of the rate-limiting steps in the overall elongation of very-long-chain fatty acyl-CoA.     

Inhibitory effect of very-long-chain monounsaturated fatty-acyl-CoAs on the elongation of long-chain fatty acid in swine cerebral microsomes

Characteristics of condensation and overall elongation of very-long-chain fatty-acyl-CoAs in swine cerebral microsomes were studied using radio high-performance liquid chromatography (RHPLC) and gas chromatography-mass spectrometry (GC-MS). The monounsaturated fatty-acyl-CoA depressed both the condensation and overall elongation activities of endogenous substrates and also of exogenous saturated fatty-acyl-CoA. The extent of the decrease of the elongation activity was dependent on the concentration and the chain length of the exogenous fatty-acyl-CoAs. The dependence of the condensation activity of monounsaturated fatty-acyl-CoA on the concentration of malonyl-CoA suggested that the non-Michaelis-Menten type kinetics was dominant for oleoyl-CoA, however, a normal kinetic pattern was obtained for endogenous palmitoyl-CoA and arachidonoyl-CoA with Km = 37 microM to malonyl-CoA. The condensation activity for icosanoyl-CoA (20:0-CoA) was inhibited by icosenoyl-CoA (20:1-CoA) in a non-competitive manner, which suggested that the condensation enzyme, or at least the active center of the enzyme for icosenoyl-CoA, was different from that for icosanoyl-CoA.     

Effect of fatty-acyl-CoAs on the elongation of saturated fatty acid in porcine aorta microsomes

The microsomal elongation system from porcine aorta for longchain fatty-acyl-CoAs was investigated. Palmitoleoyl-CoA (16:1-CoA), oleoyl-CoA (18:1-CoA), and eicosenoyl-CoA (20:1-CoA) remarkably depressed the elongation activity for 16:0-CoA in aorta microsomes by 44.8, 52.4, and 43.7% of the control activity, respectively. Saturated and polyunsaturated fatty-acyl-CoAs had little effect on the 16:0-CoA elongation activity. These results indicate that monounsaturated long-chain fatty acyl-CoAs can regulate the synthesis of saturated fatty acids in the vessel walls.     

Effect of phospholipase A2 and free fatty acids on lipid-protein interactions in long- and very-long-chain fatty acyl-CoA elongation enzyme systems of brain microsomes

The elucidation of the mechanism of phospholipase A2-induced inactivation of the condensation enzyme provided evidence concerning the important role of lipid-enzyme interactions in maintaining the condensation activity in swine cerebral microsomes. A quantitative analysis of fatty acid release by phospholipase A2 from the microsomal membrane revealed that only 5 nmol of free fatty acid per mg microsomal protein was released, including oleic acid and arachidonic acid, by treatment with 0.4 unit of phospholipase A2 per mg microsomal protein for 15 s at 23 degrees C. Under these conditions, the condensation activity for endogenous 16:0-CoA and 20:4-CoA decreased to half and that for exogenous 20:0-CoA decreased to 75%. However, the addition of free fatty acids and lysophospholipids or a mixture of them at 5-10 nmol/mg protein did not change the condensation activity for endogenous 16:0-CoA and 20:4-CoA, or for exogenous 20:0-CoA. These results indicated that phospholipase A2 inhibited the condensation activity by acting directly on phospholipids that are indispensable to maintaining the function of the condensation enzyme. The Arrhenius plot for the condensation of endogenous 16:0-CoA showed a break at around 16 degrees C, whereas no break of the plot was observed for the condensation of 20:0-CoA and 20:4-CoA. The activation energy for the condensation of 16:0-CoA and 20:4-CoA was decreased by the addition of free fatty acids such as oleic acid and stearic acid, with disappearance of the Arrhenius break for 16:0-CoA condensation, whereas the activation energy for the condensation of 20:0-CoA was not changed. These results suggest that the type of lipid-protein interaction in the condensation enzyme for 20:0-CoA is different from that for 16:0-CoA and 20:4-CoA.     

Stereospecific incorporation of hydrogens from NADPH in elongation of very long fatty acyl-CoA by swine cerebral microsomes

The elongation of arachidoyl-CoA by swine cerebral microsomes resulted in the production of behenic acid (22:0) and lignoceric acid (24:0) concomitantly. When 4S-[4-²H₁]NADPH was used for the elongation of arachidoyl-CoA, the incorporation of two deuterium atoms into 22:0 was observed by the technique of mass fragmentography. Furthermore, the incorporation of four deuterium atoms into 24:0 was also detected. On the other hand, when 4R-[4-²H₁]NADPH was used, no deuterium was incorporated into the elongated products.     

Tissue and chain length specificity of the fatty acyl-CoA elongation system in the American cockroach

The elongation of fatty acyl-CoAs, reactions involved in hydrocarbon biosynthesis, was examined in the cockroach, Periplaneta americana. Products were analyzed by radio-HPLC and radio-GLC. The majority of the elongation activity was observed in microsomes prepared from abdominal epidermal tissue. Linoleoyl-CoA (18:2-CoA) was elongated most efficiently followed by stearoyl-CoA (18:0-CoA), linolenoyl-CoA (18:3-CoA; n-3) and oleoyl-CoA (18:1-CoA). The products of 18:2-CoA elongation included all even numbered acyl groups up to 28 carbons, and the products of 18:0-CoA included all even numbered acyl groups to 26 carbons. The 18:3-CoA was elongated only to 20 and 22 carbons. Radioactivity from both 18:2-CoA (5.4%) and 18:0-CoA (1.2%) was recovered in the hydrocarbon fraction. Analysis of this hydrocarbon fraction showed that the radio-activity from 18:2-CoA was present in (Z,Z)-6,9-heptacosadiene and that the radioactivity from 18:0-CoA was present in n-pentacosane. These data demonstrate for the first time in an in vitro insect system that the fatty acid elongation reactions are coupled with the conversion of the elongated product to hydrocarbon. Thus, each of the expected intermediates in the conversion of 18:0 and 18:2 to 25 and 27 carbon hydrocarbons, respectively, was observed, and the results demonstrate high tissue, substrate, and product specificity.     

Concomitant decrease in the elongation and condensation activities of very-long chain fatty acyl-CoA in jimpy mouse

Overall elongation and condensation of long-chain and very-long-chain fatty acids have been studied in the brain microsomes of jimpy mice. Both the elongation and condensation activities with stearoyl (18:0)-, oleoyl (18:1)- and arachidoyl (20:0)-CoA were severely diminished in jimpy brain, but the decrease in the activity with the exogenous palmitoyl (16:0)-CoA was less pronounced. The decrease in the elongation and condensation reactions with endogenous palmitic and arachidonic (20:4) acids was not distinct in the mutant. The decrease in the activity of condensation reaction may be responsible for the reduced rate of overall fatty acid elongation.     

Regulation of sex pheromone biosynthesis in the housefly, Musca domestica: relative contribution of the elongation and reductive steps.

The regulation of production of the sex pheromone (Z)-9-tricosene (Z9-23:Hy) in the housefly, Musca domestica, was studied by examining the chain length specificity of the fatty acyl-CoA elongation reactions and the reductive conversion of fatty acyl-CoAs to alkenes in 1- and 4-day-old male and female houseflies. Microsomal preparations from 4-day-old female insects produced as the predominant alkene Z9-23:Hy when incubated with malonyl-CoA, NADPH, and [9,10-3H2]oleoyl-CoA (18:1-CoA), whereas microsomal preparations from 4-day-old male insects produced predominantly (Z)-9-heptacosene (Z9-27:Hy). These are the major alkenes produced in vivo by Day 4 females and males, respectively. Microsomes prepared from both Day 1 males and Day 1 females produced Z9-27:Hy as the major alkene from labeled 18:1-CoA. This is the major alkene produced in vivo by both sexes at Day 1. An examination of the chain length specificity of the elongation reactions showed that microsomes prepared from Day 4 male insects readily elongated both 18:1-CoA and 15-[15,16-3H2]tetracosenoyl-CoA (24:1-CoA) to 28-carbon moieties, whereas microsomes from Day 4 female insects did not efficiently elongate either substrate beyond 24 carbons. With high substrate concentrations, microsomes prepared from male insects converted 24:1-CoA to Z9-23:Hy more efficiently than did those from females, whereas under lower and presumably more physiological substrate concentrations, microsomes from females had slightly higher activity than did those from males. Taken together, these data show that the regulation of the chain length of the alkenes, and thus sex pheromone production, in the housefly resides predominantly in the elongation reactions and not in the step which converts the fatty acyl-CoA to hydrocarbon.     

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.     

Oleoyl-CoA is not an immediate substrate for fatty acid elongation in developing seeds of Brassica napus

The substrate specificity of fatty acid elongase was studied using an oil body fraction from developing seeds of Brassica napus. ATP was essential for high rates of elongase activity, but there was no apparent requirement for oleoyl-CoA, oleic acid (18:1) or CoA. Furthermore, ¹⁴C from 18:1-CoA was incorporated into eicosenoic (20:1) and erucic (22:1) acids at a much slower rate than ¹⁴C from malonyl-CoA. Incubation of [¹⁴C]18:1-CoA with the oil body fraction resulted in a rapid loss of [¹⁴C]18:1-CoA into several lipid fractions whether in the absence or presence of ATP, but the loss of 18:1-CoA had a comparatively small effect on the overall rate of elongation. Acyl-CoAs were derivatized to their respective acylbutylamide and analyzed by gas chromatography-mass spectrometry. This analysis of acyl-CoAs demonstrated that there was no detectable 20:1-CoA or 22:1-CoA at 0 min incubation, while newly synthesized 20:1-CoA and 22:1-CoA were present at 10 min. Analysis of the %¹⁴C of the substrates and products of the elongation reaction revealed that the endogenous pool of 18:1-CoA is quite small in elongase preparations. In addition, [¹⁴C]18:1-CoA added to the incubation, although incorporated into lipids, was not significantly diluted by turnover or new synthesis. In contrast, the %¹⁴C of the 20:1-CoA was two- to threefold less than that of the 18:1-CoA. Taken together, these results indicate that the [¹⁴C]18:1 from the [¹⁴C]18:1-CoA was diluted in an intermediate 18:1 pool and that the 18:1-CoA was not the major donor of the acyl group to the elongase reaction.     

Chain elongation in the formation of polyunsaturated fatty acids by brain: Some properties of the microsomal system

Chain elongation of polyunsaturated acids has been investigated using microsomes from developing rat brain. With 18:3(n ? 6) in 0.05% detergent as an acceptor and [2-¹⁴C]malonyl-coenzyme A (CoA) as a two-carbon donor, incorporation of radioactivity into 20:3 was optimal (and incorporation into other acyl chains was minimal) in the presence of 100 μm substrate, 200 μmp-bromophenacylbromide and 10 mm KCN. Up to 30% of the labeled products were incorporated into phospholipids and triacylglycerol. Maximal microsomal elongation activity was observed at 3–4 weeks of age. Several other fatty acid or acyl-CoA acceptors tested in this system were elongated at slower rates compared to 18:3(n ? 6) [e.g., 16:0-CoA, 75%; 20:4(n ? 6), 57%; 18:3(n ? 3), 13%; 18:2(n ?6), 10%; 20:3(n ? 6), 6%]. The rate of elongation of chemically synthesized 18:3-CoA was only 50% of the detergent-suspended acid and was optimal at 6 μm substrate; inhibition above 6 μm 18:3-CoA was reduced by bovine serum albumin, but incorporation of label into palmitate was greatly stimulated. CoA markedly inhibited elongation of 18:3(n ? 6) or 18:3-CoA; N-ethylmaleimide at equimolar amounts reversed this CoA inhibition but did not alter the inhibition caused by concentrations of 18:3-CoA above 6 μm. ATP was absolutely required for elongation of either the free acid or the acyl-CoA derivative, whereas exogenous MgCl₂ had little effect.     

Fatty acyl-CoA elongation in Blatella germanica integumental microsomes

Insect cuticular hydrocarbons are synthesized de novo in integumental tissue through the concerted action of fatty acid synthases (FASs), fatty acyl-CoA elongases, a reductase, and a decarboxylase to produce hydrocarbons and CO2. Elongation of fatty acyl-CoAs to very long chain fatty acids was studied in the integumental microsomes of the German cockroach, Blatella germanica. Incubation of [1-14C]palmitoyl-CoA, malonyl-CoA, and NADPH resulted in the production of 18-CoA with minor amounts of C20, C22, C24, C30, and C32 labeled acyl-CoA moieties. Similar experiments with [1-14C]stearoyl-CoA rendered C20-CoA as the major product, and lesser amounts of C22 and C24-CoAs were also detected. After solubilization of the microsomal FAS, kinetic parameters were determined radiochemically or by measuring NADPH consumption. The reaction velocity was linear for up to 3 min incubation time, and with a protein concentration up to 0.025 microg/microl. The effect of the chain length on the reaction velocity was compared for palmitoyl-CoA, stearoyl-CoA, and eicosanoyl-CoA. The optimal substrate concentration was 10 microM for C16-CoA, between 8 and 12 microM for C18-CoA, and close to 3 microM for C20-CoA. In vivo hydrocarbon biosynthesis was inhibited from 55.5 to 72.5% in the presence of 1 mM trichloroacetic acid, a known inhibitor of elongation reactions.     

Study of the 3-hydroxy eicosanoyl-coenzyme A dehydratase and (E)-2,3 enoyl-coenzyme A reductase involved in acyl-coenzyme A elongation in etiolated leek seedlings

(R,S)-[1-¹⁴C]3-Hydroxy eicosanoyl-coenzyme A (CoA) has been chemically synthesized to study the 3-hydroxy acyl-CoA dehydratase involved in the acyl-CoA elongase of etiolated leek (Allium porrum L.) seedling microsomes. 3-Hydroxy eicosanoyl-CoA (3-OH C20:0-CoA) dehydration led to the formation of (E)-2,3 eicosanoyl-CoA, which has been characterized. Our kinetic studies have determined the optimal conditions of the dehydration and also resolved the stereospecificity requirement of the dehydratase for (R)-3-OH C20:0-CoA. Isotopic dilution experiments showed that 3-hydroxy acyl-CoA dehydratase had a marked preference for (R)-3-OH C20:0-CoA. Moreover, the very-long-chain synthesis using (R)-3-OH C20:0-CoA isomer and [2-¹⁴C]malonyl-CoA was higher than that using the (S) isomer, whatever the malonyl-CoA and the 3-OH C20:0-CoA concentrations. We have also used [1-¹⁴C]3-OH C20:0-CoA to investigate the reductant requirement of the enoyl-CoA reductase of the acyl-CoA elongase complex. In the presence of NADPH, [1-¹⁴C]3-OH C20:0-CoA conversion was stimulated. Aside from the product of dehydration, i.e. (E)-2,3 eicosanoyl-CoA, we detected eicosanoyl-CoA resulting from the reduction of (E)-2,3 eicosanoyl-CoA. When we replaced NADPH with NADH, the eicosanoyl-CoA was 8- to 10-fold less abundant. Finally, in the presence of malonyl-CoA and NADPH or NADH, [1-¹⁴C]3-OH C20:0-CoA led to the synthesis of very-long-chain fatty acids. This synthesis was measured using [1-¹⁴C]3-OH C20:0-CoA and malonyl-CoA or (E)-2,3 eicosanoyl-CoA and [2-¹⁴C]malonyl-CoA. In both conditions and in the presence of NADPH, the acyl-CoA elongation activity was about 60 nmol mg⁻¹ h⁻¹, which is the highest ever reported for a plant system.     

Adrenoleukodystrophy. The chain shortening of erucic acid (22:1(n-9)) and adrenic acid (22:4(n-6)) is deficient in neonatal adrenoleukodystrophy and normal in X-linked adrenoleukodistrophy skin fibroblasts

The metabolism of long chain unsaturated fatty acids was studied in cultured fibroblasts from patients with X-linked adrenoleukodystrophy (ALD) and with neonatal ALD. By using [14-14C] erucic acid (22:1(n-9)) as substrate it was shown that the peroxisomal beta-oxidation, measured as chain shortening, was impaired in cells from patients with neonatal ALD. The beta-oxidation of adrenic acid (22:4(n-6)), measured as acid-soluble products, was also reduced in the neonatal ALD cells. The peroxisomal beta-oxidation of [14-14C]erucic acid (22:1(n-9)) and [2-14C]adrenic acid (22:4(n-6)) was normal in cells from X-ALD patients. The beta-oxidation, esterification and chain elongation of [1-14C]arachidonic acid (20:4(n-6)) and [1-14C]eicosapentaenoic acid (20:5(n-3)) was normal in both X-linked ALD and in neonatal ALD. Previous studies suggest that the activation of very long chain fatty acids by a lignoceryl (24:0)-CoA ligase is deficient in X-linked ALD, while the peroxisomal beta-oxidation enzymes are deficient in neonatal ALD. The present results suggest that the peroxisomal very long-chain acyl-CoA ligase is not required for activation of unsaturated C20 and C22 fatty acids and that these fatty acids can be efficiently activated by the long chain acyl-(palmityl)-CoA ligase.     

Condensation activity for polyunsaturated fatty acids with malonyl-CoA in rat brain microsomes. Characteristics and developmental change.

Condensation activities for gamma-linolenic acid (18:3(n-6)), octadecatetraenoic acid (18:4(n-3)) and eicosapentaenoic acid (20:5(n-3)) with malonyl-CoA were measured and compared with the condensation activities for 16:0-CoA, 18:1-CoA, 18:2(n-6)-CoA and 18:3(n-3)-CoA in rat brain microsomes of various ages. The age-dependence of condensation activities for 18:3(n-6), 18:4(n-3) and 20:5(n-3) showed a maximum at 1- to 2-month-old and were still higher at 3-month-old 2- to 3-fold than the activities in microsomes of pups. Conversely, the age-dependence of condensation activity for 16:0-CoA showed a peak around 1 month-old, but decreased at 3-month-old to the level of the activities in pups. The condensation activity for 20:5(n-3) was inhibited by 18:3(n-6) or 18:4(n-3) and the inhibition was not competitive. The condensation of 18:3(n-6) was also inhibited by 18:4(n-3) in the same manner. A physiological implication of the inhibition system at the substrate level was discussed.     

Studies on the metabolic fate of n-3 polyunsaturated fatty acids

Several different processes involved in the metabolic fate of docosahexaenoic acid (DHA, C22:6n-3) and its precursor in the biosynthesis route, C24:6n-3, were studied. In cultured skin fibroblasts, the oxidation rate of [1-14C] 24:6n-3 was 2.7 times higher than for [1-14C]22:6n-3, whereas [1-14C]22:6n-3 was incorporated 7 times faster into different lipid classes than was [1-14C]24:6n-3. When determining the peroxisomal acyl-CoA oxidase activity, similar specific activities for C22:6(n-3)-CoA and C24:6(n-3)-CoA were found in mouse kidney peroxisomes. Thioesterase activity was measured for both substrates in mouse kidney peroxisomes as well as mitochondria, and C22:6(n-3)-CoA was hydrolyzed 1.7 times faster than C24:6(n-3)-CoA. These results imply that the preferred metabolic fate of C24:6(n-3)-CoA, after its synthesis in the endoplasmic reticulum (ER), is to move to the peroxisome, where it is beta-oxidized, producing C22:6(n-3)-CoA. This DHA-CoA then preferentially moves back, probably as free fatty acid, to the ER, where it is incorporated into membrane lipids.