Modification by clofibric acid of acyl composition of glycerolipids in rat liver. Possible involvement of fatty acid chain elongation and desaturation

Administration of p-chlorophenoxyisobutyric acid (clofibric acid) to rats induced a marked change in acyl composition of hepatic glycerolipids; a considerable increase in the proportion of octadecenoic acid (18:1) was accompanied by a marked decrease in the proportion of octadecadienoic acid (18:2). Among the glycerolipids, the changes in the proportions of 18:1 and 18:2 were the most marked in phosphatidylcholine. The change in the acyl composition of phosphatidylcholine paralleled the change in free fatty acid composition in microsomes. The treatment of rats with clofibric acid resulted in a 2.3-fold increase in activity of microsomal palmitoyl-CoA chain elongation and a 4.8-fold increase in activity of stearoyl-CoA desaturation. The activities of acyl-CoA synthetase, 1-acylglycerophosphate acyltransferase and 1-acylglycerophosphorylcholine acyltransferase in hepatic microsomes were increased approx. 3-, 1.7- and 3.6-times, respectively, by the treatment of rats with clofibric acid. These findings are discussed with respect to the role of fatty acid modification systems in the regulation of acyl composition of phosphatidylcholine.     

Alterations by peroxisome proliferators of acyl composition of hepatic phosphatidylcholine in rats, mice and guinea-pigs. Role of stearoyl-CoA desaturase.

Rats, mice and guinea-pigs were administered p-chlorophenoxyisobutyric acid (clofibric acid) or 2,2'-(decamethylenedithio)diethanol (tiadenol). The treatments of rats and mice with either clofibric acid or tiadenol increased markedly the activities of stearoyl-CoA desaturase, palmitoyl-CoA chain elongation, 1-acylglycerophosphate (1-acyl-GP) acyltransferase and 1-acylglycerophosphocholine (1-acyl-GPC) acyltransferase, but not 2-acylglycerophosphocholine (2-acyl-GPC) acyltransferase in liver microsomes. The treatment of guinea-pigs with clofibric acid did not cause any change in the activities of these enzymes. The treatment of guinea-pigs with tiadenol caused a slight, but significant, increase in the activities of 1-acyl-GP acyltransferase and 1-acyl-GPC acyltransferase. The treatment of rats and mice with either clofibric acid or tiadenol increased markedly the proportion of 18:1 and decreased greatly the proportion of 18:0 in liver microsomal phosphatidylcholine. However, there is a considerable difference in the effects of the two peroxisome proliferators on the composition of polyunsaturated fatty acids in phosphatidylcholine between rats and mice. The treatment of guinea-pigs with either of the two peroxisome proliferators caused no change in acyl composition of phosphatidylcholine. The possible role of stearoyl-CoA desaturation in the regulation of acyl composition of phosphatidylcholine was discussed.     

Effects of perfluorocarboxylic acids on the activities of acyl-CoA elongations in vivo and in vitro

Effects of perfluorocarboxylic acids (PFCAs) on proportions of oleic acid and cis-vaccenic acid through acyl-CoA chain elongation systems have been studied in the liver of rats. Administration of PFCAs caused a significant increase in palmitoyl-CoA chain elongation activity while these chemicals did not affect palmitoleoyl-CoA chain elongation activity in vivo.Condensation for both palmitoyl-CoA and palmitoleoyl-CoA were inhibited by PFCAs in vitro at the concentrations, which were physiologically found in the liver of rats treated with the PFCAs. Δ⁹ Desaturase, which catalyzes both stearoyl-CoA desaturation and palmitoyl-CoA desaturation, was induced by the treatments of rats with the PFCAs. The administration of the PFCAs to rats caused a marked increase in proportion of oleic acid, while that of cis-vaccenic acid was not affected at all. These results strongly suggest that the induced palmitoyl-CoA chain elongation by PFCAs, which exist in the liver, effectively produces oleic acid in concert with the induced stearoyl-CoA desaturase, but the inhibitory effects of PFCAs on either palmitoyl-CoA chain elongation or palmitoleoyl-CoA chain elongation are not crucial for the formation of the elongated fatty acids in vivo.     

Selective increase in acylation of 1-acylglycerophosphorylcholine in livers of rats and mice by peroxisome proliferators

Rats were fed a diet containing p-chlorophenoxyisobutyric acid (clofibric acid). Activity of microsomal 1-acylglycerophosphorylcholine (1-acyl-GPC) acyltransferase in liver was increased approx. 3-fold by the treatment with clofibric acid. The treatment of rats with clofibric acid did not increase activity of microsomal 2-acyl-GPC acyltransferase. Feeding a diet containing 2,2'-(decamethylenedithio)diethanol (tiadenol), di(2-ethylhexyl)phthalate or acetylsalicylic acid also resulted in a selective increase in the activity of 1-acyl-GPC acyltransferase in rat liver. Treatment with clofibric acid increased the activity of 1-acyl-GPC acyltransferase in liver of mouse as well as rat, but did not change the activity in liver of guinea-pig. The relative rate of acylation of 1-acyl-GPC with various acyl-CoAs by hepatic microsomes was not changed by the treatment of rats with clofibric acid.     

Enzyme site-specific changes in hepatic microsomal fatty acid chain elongation in streptozotocin-induced diabetic rats

The hepatic microsomal fatty acid chain elongation of palmitoyl-CoA and γ-linolenoyl-CoA was diminished by 40–50% in male Sprague-Dawley rats made diabetic for 2 and 4 weeks following the intravenous administration of a single dose (65 mg/kg) of streptozotocin. Analysis of the activities of the four enzymatic components showed that only one enzyme, the condensing enzyme, which catalyzes the initial and rate-limiting step in chain elongation, was altered by the diabetic state. Both chain elongation and condensation activities were depressed to the same extent, whereas β-ketoacyl-CoA reductase, β-hydroxyacyl-CoA dehydrase and trans-2-enoyl-CoA reductase activities were the same as the values obtained with non-diabetic controls. 2 week administration of 10 units of insulin per day to rats which were diabetic for a 2-week period resulted in the reversal of the reduced palmitoyl-CoA elongation and condensation activities to control values. However, neither the condensation nor the elongation of γ-linolenoyl was reversed by the insulin treatment. These results support the notion of multiple condensing enzymes or chain elongation systems.     

Fatty acid biosynthesis in Erlich cells. The mechanism of short term control by exogenous free fatty acids.

We have examined the mechanism by which extracellular free fatty acids regulate fatty acid biosynthesis in Ehrlich ascites tumor cells. De novo biosynthesis in intact cells was inhibited by stearate greater than oleate greater than palmitate greater than linoleate. The amount of citrate and long chain acyl-CoA in the cells was not changed appreciably by the addition of free fatty acids to the incubation medium, indicating than free fatty acids do not regulate fatty acid biosynthesis by changing the total intracellular content of these metabolites. By measuring the incorporation of labeled free fatty acids into acyl-CoA, however, it was determined that the fatty acid composition of the acyl-CoA poolwas changed dramatically to reflect the composition of the exogenous free fatty acids. The relative inhibitory effects of different free fatty acids appear to depend on the ability of their acyl-CoA derivatives to regulate acyl-CoA carboxylase activity. The acyl-CoA concentration needed to produce 50% inhibition of purified Ehrlich cell carboxylase was found to be 0.68 mum for stearoyl-CoA, 1.6 mum for oleoyl-CoA, 2.2 mum for palmitoyl-CoA, 23 mum for myristoyl-CoA, 30 mum for lauroyl-CoA, and 37 mum for linoleoyl-CoA. In contrast to their effects on de novo synthesis, all of the free fatty acids added except stearate stimulated chain elongation in intact cells. Microsomal chain elongation, the major system for elongation in Ehrlich cells, also was regulated by the composition of the cellular acyl-CoA pool. Lauroyl-CoA, myristoyl-CoA, and palmitoyl-CoA were good substrates for elongation by isolated microsomes; oleoyl-CoA, and linoleoyl-CoA were intermediate; and stearoyl-CoA was a very poor substrate. We conclude that free fatty acids regulate fatty acid biosynthesis by changing the composition of the cellular acyl-CoA pool. These changes control the rate of malonyl-CoA production and, because of the acyl-CoA substrate specificity of the microsomal elongation system, modulate the amount of malonyl-CoA used for chain elongation.     

Evidence for insulin dependent hepatic microsomal gamma-linolenic acid chain elongation in spontaneously diabetic Wistar BB rats.

We studied hepatic microsomal gamma-linolenoyl-CoA elongation and fatty acid composition of liver microsomes in spontaneously diabetic Wistar BB rats. The liver microsomal gamma-linolenoyl-CoA elongation was decreased in diabetic Wistar BB rats during both normo- and hyperglycemic periods and restored during the hypoglycemic period following insulin treatment. These results are in agreement with our previously reported data on linoleic acid delta 6 and delta 5 desaturations and support the non-parallel relationship between the chain elongation system and the glycemia. The fatty acid composition of BB rat liver microsomes was only partially consistent with the gamma-linolenoyl-CoA elongation activity at the different periods of glycemia, probably because factors other than elongation impairments were involved in the evolution of fatty acid composition.     

Existence of acetyl-CoA-dependent chain elongation system in hepatic peroxisomes of rat: effects of clofibrate and di-(2-ethylhexyl)phthalate on the activity

The acetyl-CoA-dependent elongation of medium-chain acyl-CoA in the presence of pyridine nucleotide was studied in rat liver. The activity was increased by the administration of peroxisome proliferators, clofibrate and di-(2-ethylhexyl)phthalate, and the change was more remarkable in peroxisomes than in mitochondria. Addition of 0.01% Triton X-100 to the incubation mixture caused an increase in the mitochondrial activity, whereas the peroxisomal activity did not increase significantly. The pH optimum for the peroxisomal activity was in the range of pH 6.5-7.0 and that for the mitochondrial activity was pH 7.5-8.0. The specificities of primer chain length in both organelles were almost the same, and octanoyl-CoA was the preferred substrate. Peroxisomal activity was completely inhibited by the addition of 1 mM N-ethylmaleimide or 1 mM p-hydroxymercuribenzoic acid, while the activity did not change on the addition of 1 mM KCN or an antibody to acyl-CoA oxidase, the first enzyme of the peroxisomal beta-oxidation system. The activity of enoyl-CoA reductase, which catalyzes the last step of the elongation system, was also detected in peroxisomes, although the main activity was localized in microsomes. When the liver peroxisomal fraction of clofibrate-treated rats was incubated with a mixture of octanoyl-CoA, acetyl-CoA, NADH, NADPH, and Triton X-100 in a buffer system, dodecanoyl-CoA was detected as the main product by radio-gas chromatography. On the other hand, the elongation activity was decreased greatly by the addition of NAD+ into the mixture. These results indicate that (i) peroxisomes have activity to elongate medium chain acyl-CoA; (ii) the peroxisomal elongation system may consist of the reverse reaction of the beta-oxidation system except for the last step, which is catalyzed by enoyl-CoA reductase; and (iii) the peroxisomal elongation system is less active than the beta-oxidation system under physiological conditions.     

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.     

Palmitoyl-CoA Elongation in Brain Microsomes: Dependence on Cytochrome b5 and NADH-Cytochrome b5 Reductase

Experiments were performed to demonstrate the involvement of electron transport system in fatty acid elongation in rat brain microsomes. Mercuric chloride and p-chloromercuriphenylsulfonate, inhibitors on NADH-cytochrome b5 reductase, at 32 microM inhibited NADH-supported palmitoyl-CoA elongation to 30 and 60% of control activity, respectively, whereas NADPH-supported palmitoyl-CoA elongation was unaffected by these mercurials. An antibody to rat liver NADH-cytochrome b5 reductase inhibited brain microsomal NADH-cytochrome b5 reductase activity and NADH-dependent palmitoyl-CoA elongation. Treatment of brain microsomes with trypsin diminished the cytochrome b5 content; NADH- and NADPH-cytochrome c reductase activities were significantly decreased, but the decrease in NADH-cytochrome b5 reductase activity was relatively small. Whereas essentially no incorporation of malonyl-CoA into palmitoyl-CoA was observed with trypsin-treated microsomes, addition of detergent-solubilized cytochrome b5 resulted in a recovery of fatty acid elongation. These results indicate the presence of an electron transport system, NADH-NADH-cytochrome b5 reductase-cytochrome b5-fatty acid elongation, in brain microsomes.     

Intestinal Lipid Esterification and Aging in Mice and Rats

The effects of aging on lipid absorption, particularly on fatty acid glycerophospholipid and triacylglycerol esterification, were investigated in 2.5-,12- and 24-month-old mice and rats. Two intestinal mucosa microsomal enzymes, involved in the dietary fatty acid absorption, were assayed:acylCoA:2-monoacylglycerol acyltransferase and acylCoA:1-lysophosphatidylcholine acyltransferase. In both mice and rats, the activities of both enzymes varied with the nature of the acyl-CoA. Indeed acylCoa:2-monoacylglycerol acyltransferase activities were significantly higher with oleoyl-CoA and linoleoyl-CoA than with palmitoyl-CoA and arachidonoyl-CoA, while acylCoA:1-lysophosphatidylcholine acyltransferase activities were highest with arachidonoyl-CoA. AcylCoA:2-monoacylglycerol acyltransferase activity did not decrease significantly with aging in mice or rats, whatever the acyl-CoA used. In contrast, acylCoA:1-lysophosphatidylcholine acyltransferase activity in the 24-month-old rats was significantly lower (−47 %) than in 2.5-month-old rats, with oleoyl-CoA, linoleoyl-CoA and arachidonoyl-CoA. Simultaneously we observed that less glycerophospholipid esterification of oleic and linoleic acid occurs in older rats than in 2.5-month-old rats.     

Increased activity of stearoyl-CoA desaturation in liver from rat fed clofibric acid

Male rats were fed a diet containing 0.5% (w/w) p-chlorophenoxyisobutyric acid (clofibric acid), a hypolipidemic drug. Activities of stearoyl-CoA desaturation in hepatic microsomes were increased approx. 4 times following the administration of clofibric acid for 7 days. An increase in the activity of desaturation of stearic acid was also observed in the liver of clofibric acid-fed rats in vivo. The increase in the activity of microsomal stearoyl-CoA desaturation by clofibric acid-feeding was due to the increase in the activity of terminal desaturase as measured by the rate constant for cytochrome b5 reoxidation, but not due to the changes in cytochrome b5 content and NADH-cytochrome b5 reductase activity. Increases in the activity of stearoyl-CoA desaturation by clofibric acid-feeding were also observed in rats of hormonally altered state, such as diabetic rats, hyperthyroid rats and hypothyroid rats. Percentages of octadecenoic acid in total fatty acid of hepatic lipid were increased with the increase in the activity of stearoyl-CoA desaturation.     

Microsomal electron-transport reductase activities and fatty acid elongation in rat brain. Developmental changes, regional distribution and comparison with liver activity.

Gestational and postnatal changes of microsomal NADH:cytochrome b5 reductase and NADPH:cytochrome c reductase activities were examined in rat brain. The specific activity of NADH:cytochrome b5 reductase was high at 18-19 days of gestational age, decreased to a minimum at 4 to 6 days after birth and increased thereafter. An essentially similar developmental pattern was observed for the specific activity of NADPH:cytochrome c reductase. In contrast, the specific activities of these reductases in liver microsomes were low, did not display a peak during gestation and increased steadily to a maximum at 40-50 days after birth. The rate of incorporation of [2-14C]malonyl-CoA into palmitoyl-CoA in brain microsomes was found to be high in the foetus, sharply decreased to a minimum at the time of birth and increased thereafter. The activity of fatty acid elongation in liver microsomes was much less than that in brain during gestation and increased rapidly after birth to values at 50-60 days 20-fold greater than the foetal activity. NADH and NADPH were equally effective for brain microsomal fatty acid elongation. Regional distribution of cytochrome reductase activities and the activity of fatty acid elongation showed the lowest specific activity in cerebellum. These results suggest that brain microsomal electron transport may be correlated with the developmental alteration in fatty acid elongation.     

Characterization of sn-glycerol 3-phosphate acyltransferase from guinea pig harderian gland microsomes

Microsomal sn-glycerol 3-phosphate acyltransferase from the guinea pig Harderian gland was studied. Its specific activity (1.0 nmol/min X mg, with palmitoyl-CoA as a substrate) was almost the same as that of the rat liver microsomal enzyme. The enzyme acted on various types of acyl-CoA, the relative reaction rates being as follows: palmitoyl-CoA, 100(%); stearoyl-CoA, 30; oleoyl-CoA, 50; linoleoyl-CoA, 40; and arachidonoyl-CoA, 20. When assayed in the presence of 1 mM 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB), the activity on palmitoyl-CoA was inhibited by only 20-30%, whereas those for other acyl-CoAs were completely abolished. The DTNB-resistant activity was inhibited by 0.1 mM dihydroxyacetonephosphate and 0.5 mM dithiothreitol, whereas the DTNB-sensitive activity was not affected. Furthermore, heat treatment at 50 degrees C for 15 min abolished most of the DTNB-sensitive activity, but not the DTNB-resistant activity. These results, taken together, suggested that the microsomal fraction of the guinea pig Harderian gland contained at least two types of sn-glycerol 3-phosphate acyltransferase, and that, in contrast to in the case of rat liver microsomes, a DTNB-resistant enzyme that utilized exclusively palmitoyl-CoA was predominant.     

Factors regulating the elongation of palmitic and stearic acid by rat liver microsomes.

Analysis of the rates of overall chain elongation and condensation of malonyl-CoA with palmitoyl-CoA and stearoyl-CoA as primers demonstrated that for each primer, the rate of the overall metabolic process was similar to the initial condensation. The specific activity for condensation with palmitoyl-CoA was eleven times greater than for stearoyl-CoA. The specific activities of both the beta-hydroxyacyl-CoA dehydrase and 2-trans-enoyl-CoA reductase reactions were much higher than for either condensation or chain elongation, although these rates were somewhat greater with the intermediates required in chain elongating palmitoyl-CoA than for stearoyl-CoA. Both substrates were incorporated into phospholipids at low rates and there was a time-dependent hydrolytic cleavage of the acyl-CoA primers which was partially prevented by bovine serum albumin. These findings demonstrate that there was no selective removal of either primer which could result in specific substrate depletion and an apparent reduction in the rate of condensation. These combined results firmly establish the rate-limiting nature and high degree of substrate specificity exhibited during the initial condensation step in fatty acid elongation.     

The involvement of selenium in peroxisome proliferation caused by dietary administration of clofibrate to rats.

The effects of dietary treatment with clofibrate (0.5% w/w for 10 days) on the livers of selenium-deficient male rats were examined. The peroxisome proliferation (as determined by electron microscopy) in the livers of selenium-deficient animals was much less pronounced than in the case of selenium-adequate rats and no increase in peroxisomal fatty acid beta-oxidation (assayed both as antimycin-insensitive palmitoyl-CoA oxidation and lauroyl-CoA oxidase activity) was observed in the deficient animals. On the other hand, in selenium-deficient rats clofibrate caused increases in the specific activity of microsomal lauric acid omega- and omega-1-hydroxylation and an apparent change in mitochondrial size, seen as a redistribution of mitochondria from the 600 x g(av) pellet to the 10,000 x g(av) pellet, which were approximately 50% as great as the corresponding effects on control animals. Obviously, then, these three different effects of clofibrate are not strictly coupled and may involve at least partially distinct underlying mechanisms. Initial experiments demonstrated that peroxisome proliferation could be obtained by exposing primary hepatocyte cultures derived from selenium-deficient rats to clofibric acid (an in vivo hydrolysis product of clofibrate which is the proximate peroxisome proliferator), nafenopin or mono(2-ethylhexyl)phthalate. This finding suggests that selenium deficiency does not have a direct influence on the basic process(es) underlying peroxisome proliferation, but rather has indirect effects, influencing, for example, the pharmacokinetics of clofibrate and/or hormonal factors.     

Chain elongation and desaturation of palmitic acid in liver microsomes of rats subjected to hyperbaric exposure.

The enzyme activities associated with chain elongation and desaturation of fatty acid in hepatic microsomes from rats held at 1 ATA of air, 1 ATA of He-O2, and 20 ATA of He-O2 were studied. It was found that both the microsomal chain elongation and desaturation of fatty acids were depressed in rats held at 1 ATA of He-O2 as compared to animals held at 1 ATA of air. When animals were exposed to an environment of 20 ATA of He-O2, the chain elongation of fatty acid was about the same as for rats held at 1 ATA of air and was two times greater than for the rats held at 1 ATA of He-O2. The desaturase activity was depressed as compared to the two groups of control animals held at 1 ATA of air and 1 ATA of He-O2.     

Induction of rat liver mitochondrial fatty acid elongation by the administration of peroxisome proliferator di-(2-ethylhexyl)phthalate: absence of elongation activity in peroxisomes

The administration of di-(2-ethylhexyl)phthalate (DEHP)3 to male Sprague-Dawley rats resulted in more than a threefold increase in activity of acetyl CoA-dependent hepatic mitochondrial fatty acid elongation. Peroxisomes obtained either from control or DEHP-treated rats were not capable of elongating any of the fatty acyl CoAs tested. Furthermore, the peroxisomes possessed no trans-2-enoyl CoA reductase activity. Therefore, the elongation activity in the 7500g fraction from both control and DEHP-fed animals can be attributed totally to the mitochondria. Maximal incorporation of acetyl CoA occurred in the presence of both NADH and NADPH, and octanoyl CoA (8:0) and decanoyl CoA (10:0) were found to be optimal primers for fatty acid elongation in both control and DEHP-treated animals. The apparent Km for 8:0 CoA was 17 microM in both animal groups while the Vmax was increased from 4.5 to 12.5 nmol/min/mg following treatment. The apparent Km for 10:0 CoA was 10 microM in both control and DEHP-treated groups while the apparent Vmax increased from 2.5 to 10 nmol/min/mg; palmitoyl-CoA (16:0) was a very poor primer for chain elongation. Although the acetyl CoA-dependent fatty acid elongation was stimulated by DEHP treatment, the mitochondrial trans-2-enoyl CoA reductase activity was unaffected. The mitochondrial total elongation activity following DEHP-treatment using 8:0 CoA as primer was about two times higher than enoyl CoA reductase activity using trans-2-decenoyl CoA (10:1). This was the result of accumulation of intermediates, which were identified as trans-2-10:1 (35%), beta-hydroxy 10:0 (25%), unidentified (15%), and elongated saturated product 10:0 (24%). Elongation by one acetate unit was found in both the control and DEHP-treated animals. The results are discussed in terms of physiological significance.     

The effect of bovine serum albumin on partial reactions of palmitoyl-CoA chain elongation by rat liver microsomes.

In the absence of albumin, v/s curves for both condensation and overall chain elongation demonstrated that the specific activity for overall chain elongation was 3.7 times that of condensation. When the molar ratio of palmitoyl-CoA to albumin was greater than 2 : 1, the specific activity of chain elongation exceeded that of condensation. At these low albumin concentrations, in the absence of NADPH, the beta-ketostearoyl-coA was converted back to palmitate. This cleavage reaction is inhibited by albumin in a concentration-dependent manner. When the palmitoyl-CoA to albumin molar ratio was less than 2 : 1, the specific activity for condensation exceeded that for overall chain elongation and some beta-ketostearate was shown to accumulate under chain elongation conditions. The specific activity for dehydration of beta-hydroxystearoyl-CoA was maximal when the acyl-CoA to albumin molar ratio was between 10 : 1 and 4 : 1 but the rate of this reaction was not markedly influenced by variations in albumin concentration. The specific activity for the NADPH-dependent reduction of 2-trans-octa-decenoyl-CoA was 18 nmol . min(-1) . mg(-1) in the absence of albumin and increased to a maximum of 112 when the substrate to albumin molar ratio was 2 : 1. At higher albumin concentrations the reductase reaction was inhibited. Conversely, the specific activity for the reverse dehydrase was maximal at low albumin concentrations and the rate of this reaction declined as the albumin concentration increased. Our results demonstrate that albumin not only alleviates a substrate induced inhibition but also regulates the metabolic fate of 2-trans-octadecenoyl-CoA and in this regard may possibly substitute for acyl-CoA binding proteins.     

Elongation of fatty acids by microsomal fractions from the brain of the developing rat.

Elongation of fatty acids by microsomal fractions obtained from rat brain was measured by the incorporation of [2-14C]malonyl-CoA into fatty in the presence of palmitoyl-CoA or stearoyl-CoA. 2. Soluble and microsomal fractions were prepared from 21-day-old rats; density gradient centrifugation demonstrated that the stearoyl-CoA elongation system was localized in the microsomal fraction whereas fatty acid biosynthesis de novo from acetyl-CoA occurred in the soluble fraction. The residual activity de novo in the microsomal fraction was attributed to minor contamination by the soluble fraction. 3. The optimum concentration of [2-14C]malonyl-CoA for elongation of fatty acids was 25 mum for palmitoyl-CoA or stearoyl-CoA, and the corresponding optimum concentrations for the two primer acyl-CoA esters were 8.0 and 7.2 muM respectively. 4. Nadph was the preferred cofactor for fatty acid formation from palmitoyl-CoA or stearoyl-CoA, although NADH could partially replace it. 5. The stearoyl-CoA elongation system required a potassium phosphate buffer concentration of 0.075M for maximum activity; CoA (1 MUM) inhibited this elongation system by approx. 30%. 6. The fatty acids formed from malonyl-CoA and palmitoyl-CoA had a predominant chain length of C18 whereas stearoyl-CoA elongation resulted in an even distribution of fatty acids with chain lengths of C20, C22 and C24. 7. The products of stearoyl-CoA elongation were identified as primarily unesterified fatty acids. 8. The developmental pattern of fatty acid biosynthesis by rat brain microsomal preparations was studied and both the palmitoyl-CoA and stearoyl-CoA elongation systems showed large increases in activity between days 10 and 18 after birth.