Metabolism of essential fatty acids by human epidermal enzyme preparations: evidence of chain elongation

The present studies were undertaken in order to delineate the source of human epidermal arachidonic acid, 20:4(n-6). Epidermal microsomal preparations from normal (N) and diseased epidermis (clinically uninvolved (PU) and involved psoriatic (PI) epidermis) were incubated in vitro with either [14C]18:2(n-6), [14C]20:3(n-6) or [14C]malonyl CoA to determine the activities of the delta 6, delta 5 desaturases and elongate, respectively. Experiments were performed in parallel with rat liver microsomal preparations where enzyme activities are well documented. Data derived from the enzymatic assays were compared to fatty acid composition data derived from epidermal total lipids. The enzymatic conversion rates were determined after methylation and separation of the 14C-labeled fatty acid methyl esters by argentation thin-layer chromatography and reverse phase high-performance liquid chromatography. Our data demonstrated: that N, PU, and PI epidermis were all capable of elongating 18:3(n-6) into 14C-labeled 20:3(n-6) via the addition of [14C]malonyl CoA, and this activity was markedly elevated (fivefold) in PI preparations; that N, PU, and PI epidermal preparations lacked the capacity to desaturate 18:2(n-6) and 20:3(n-6); and striking alterations in the individual fatty acids (as weight percent) in the total fatty acids of the PI epidermal extracts when compared to the PU and N extracts. These findings indicate that epidermal arachidonic acid is not biosynthesized locally from tissue linoleic acid and must, therefore, depend on contribution from another endogenous source.     

Chronologic studies of the effects of a hypoproteinic diet followed by an equilibrated diet on delta-6 and delta-5 desaturations of linoleic acid in liver microsomes in the rat

A low protein diet affects amounts of linoleic and arachidonic acids in hepatic microsomal phospholipids of growing rats. Are the changes related to modifications in microsomal delta 6- and delta 5- linoleic acid desaturase activities? Two groups of Wistar rats weighing 80 +/- 5 g at the beginning of the experiment were used: Control group (T) was fed on a 16% gluten + 4% casein diet for 53 days; Experimental group (E) was fed on a 4% gluten + 1% casein diet for 26 days (MP) then Control diet for 27 days (RE). After 2, 14 and 26 days of MP and 2, 15 and 27 days of RE, rats of each group were sacrificed. Protein and water contents of liver, quantitative fatty acid, composition of total lipids in liver and hepatic microsomes were determined. delta 6- and delta 5- linoleic acid desaturase activities were estimated from incubation of liver microsomes with [1-14C] C 18: 2 n-6 or [2(14)C] C 20: 3 n-6 respectively. The low protein diet stops practically ponderal growth. The fatty-acid compositions of microsomal total lipids of E rats were affected in comparison with values of T rats. These modifications persist after 27 days of RE. The C 20: 4 n-6/C 18: 2 n-6 ratio in microsomal total lipids was slightly different between T and E rats but increased strongly during refeeding. Same modifications take place in the fatty-acid composition of hepatic total lipids. After two days of MP, delta 6- and delta 5- desaturase activities were depressed, phenomenon that not persist in the course of MP. These enzyme activities increase to higher values than those of the T after two days of RE.     

Microsomal fatty acid desaturation and elongation in a human lung carcinoma grown in nude mice

Since tumor cells show abnormal fatty acid composition, it is likely that their desaturase systems were affected to some extent. Although desaturase activities in experimental tumors have been evaluated, to our knowledge, fatty acid desaturases in human neoplasms and particularly in human tumors grown in nude mice have not been assessed yet. We have therefore, chosen a rapidly growing human lung mucoepidermoid carcinoma (HLMC) grown in nude mice to study microsomal fatty acid desaturation and chain elongation activities. Tumor microsomal proteins were incubated with unlabeled malonyl-CoA and one of the following fatty acids: [1-14C]palmitic (16:0), [1-14C]linoleic (18:2), alpha-[1-14C]linolenic (alpha-18:3), and unlabeled gamma-linolenic (gamma-18:3) plus [2-14C]malonyl-CoA. Data show that HLMC microsomes were capable to desaturate 16:0, alpha-18:3, and dihomogammalinolenic acids (20:3) by delta 9, delta 6 and delta 5 desaturase, respectively; however, delta 6 desaturase activity on [14C]18:2 was not detected. The microsomal elongation system was active in all fatty acid series tested except for 18:2. These findings show that the undetectable activity for 18:2 desaturation is not exclusively found in experimental tumors.     

Utilization of gammalinolenic acid by mouse peritoneal macrophages.

To delineate the metabolism of gammalinolenic acid (18:3(n-6] by macrophages, primary cultures of resident mouse peritoneal macrophages were incubated with [14C]18:3(n-6). At 3, 6 or 20 h, the majority (greater than 85%) of the radiolabel was recovered in cell phospholipids. With increasing time of incubation, a relative reduction of 14C in glycerophosphocholine (ChoGpl, 58.1% to 46.2%) was noted. This was offset by a corresponding increase in glycerophosphoethanolamine (EtnGpl) labeling (from 8.8% to 18.9%). There was also a time-dependent redistribution of 14C from diacyl to ether-containing phospholipid subclasses in ChoGpl and EtnGpl. Analysis of cell extracts by reverse-phae HPLC following transmethylation demonstrated that 18:3(n-6) was extensively elongated (greater than 80%) to dihomogammalinolenic acid (20:3(n-6] by 3 h. The major radiolabeled phospholipid molecular species in the diacyl (PtdCho) and alkylacylglycerophosphocholine (PakCho) subclasses was 16:0-20:3(n-6). In contrast, diacyl (PtdEtn) and alkenylacylglycerophosphoethanolamine (PlsEtn) subclasses contained primarily [14C]18:0-20:3(n-6) and 16:0-20:3(n-6), respectively. Macrophages prelabeled with [14C]18:3(n-6) for 20 h and stimulated with calcium ionophore A23187 or zymosan synthesized [14C]prostaglandin E1 (PGE1). These data demonstrate that macrophages possess an active long chain polyunsaturated fatty acid elongase capable of converting 18:3(n-6) to 20:3(n-6) which can, upon stimulation, be converted to PGE1.     

The effect of insulin on delta5 desaturation in hepG2 human hepatoma cells and L6 rat muscle myoblasts.

In humans there is a correlation between the ratio of arachidonic acid (20:4n-6) to cis 8,11,14 eicosatrienoic acid (20:3n-6) in skeletal muscle phospholipids and insulin sensitivity. This has been interpreted as indicating a link between the activity of the delta5 desaturase enzyme and muscle insulin sensitivity. The present study addressed the possibility that insulin regulates delta5 desaturase activity using L6 rat myoblasts and hepG2 human hepatoma cells. Both cell lines responded to insulin by increasing the amount of D-[U-14C] glucose incorporated into glycogen. In L6 cells, insulin stimulated cis 8,11,14 eicosatrienoic acid uptake and arachidonic acid production but had no effect on the percentage conversion of cis 8,11,14 eicosatrienoic acid to arachidonic acid. In hepG2 cells, insulin had no effect on cis 8,11,14 eicosatrienoic acid uptake or arachidonic acid production. These results suggest that insulin has no direct effect on delta5 desaturase activity in the liver but can alter arachidonic acid production in muscle by altering substrate availability.     

Δ5 Desaturase activity in rat kidney microsomes

Rat kidney microsomal fraction is able to catalyze the enzymatic desaturation of eicosatrienoic acid (20:3n-6) to arachidonic acid (20:4n-6) by the 5 desaturase pathway, in the presence of reduced nicotinamide adenine dinucleotide (NADH), adenosinetriphosphate (ATP) and coenzyme A (CoA). The substrate of the reaction [1-¹⁴C]eicosa-8,11,14trienoic acid (20:3n-6), was separated from the product [1-¹⁴C]eicosa-5,8,11,14-tetraenoic acid (20:4n-6) by reverse phase high-pressure liquid chromatography (RP-HPLC). These fatty acids were individually collected by monitoring the eluent at 205 nm and their radioactivity was measured by liquid scintillation counting. The 5 desaturase activity in kidney microsomes increased linearly with the substrate concentration up to 20 M. Enzymatic activity was sensitive to pH with the maximum at 7.0 and was proportional with incubation time up to 10 min. The apparent Km and Vmax of 5 desaturase were 56 M and 60 pmoles·min^–1·mg^–1 microsomal protein, respectively. Neither the cytosolic renal fraction nor the cytosolic liver fraction enhanced the 5 desaturase activity. Contrary to a report but in accordance to others, the present results suggest that rat kidneys can synthesize arachidonic acid at least to satisfy partially their needs for eicosanoid production.     

Inability of murine peritoneal macrophages to convert linoleic acid into arachidonic acid. Evidence of chain elongation

Various murine macrophage populations synthesize and secrete large amounts of arachidonic acid (20:4n-6) derived eicosanoids (cyclo-oxygenase and lipoxygenase products). These metabolites are known to possess a wide variety of functions with regard to the initiation and regulation of inflammation and tumorigenesis. Because the dietary intake of 20:4n-6 is usually low, tissues are largely dependent upon dietary linoleic acid (18:2n-6) as an initial unsaturated precursor for the biosynthesis of 20:4n-6. The purpose of these experiments was to determine whether resident or responsive murine macrophages possess desaturase and elongase activities capable of in vitro conversion of 18:2n-6 into 20:4n-6. Peritoneal exudate macrophages were purified by adherence and incubated in serum-free medium containing fatty acid-free BSA with [1-14C] 18:2n-6. Approximately 90 to 98% of the [14C]18:2n-6 at 4 and 16 h was recovered in phosphatidylcholine and phosphatidylethanolamine. The metabolism of [14C]18:2n-6 was determined after transesterification and separation of the 14C-fatty acid methyl esters by argentation TLC, reverse phase HPLC, and electron impact gas chromatography/mass spectrometry. Resident and responsive macrophages lacked the capacity to transform [14C]18:2n-6 into 20:4n-6. In addition, prelabeled macrophages incubated with soluble, calcium ionophore A23187 or phorbol myristate, or particulate, zymosan, membrane perturbing agents also lacked delta 6 desaturase activity. All macrophages tested were capable of elongating [14C]18:2n-6 into [14C]20:2n-6. These observations suggest that 20:4n-6, present in macrophage phospholipids, is biosynthesized elsewhere and transported to the macrophage for esterification into the phospholipids. In addition, these findings demonstrate that elongase activity is present in both the resident and responsive peritoneal macrophage.     

Regional differences in lipid composition and incorporation of saturated and unsaturated fatty acids into microsomal membranes of rat small intestine

Incorporation of [1-14C]palmitic (16:0) and [1-14C]linoleic (18:2 omega 6) acids into microsomal membranes of proximal (jejunum) and distal (ileum) regions of rat small intestine was investigated, and the lipid composition, including fatty acid profiles of membrane phospholipids, was determined. Jejunal microsomes contained significantly higher amounts of total phospholipids, phosphatidylcholine, and phosphatidylinositol, and lower amounts of cholesterol and sphingomyelin when compared with ileal microsomes. Jejunal microsomal phospholipids contained higher levels of stearic (18:0), 18:2 omega 6, and eicosapentaenoic (20:5 omega 3) acids followed by reduced levels of oleic (18:1 omega 9), arachidonic (20:4 omega 6), and docosahexaenoic (22:6 omega 3) acids when compared with those from the ileum, except for phosphatidylinositol where no significant difference between 20:4 omega 6 content of each site was observed. In both jejunal and ileal microsomes, incorporation of [1-14C]18:2 omega 6 was significantly higher than that of [1-14C]16:0. Incorporation of both [1-14C]16:0 and [1-14C]18:2 omega 6 was significantly higher in jejunal microsomal lipid fractions (phospholipids, diacylglycerols, triacylglycerols) when compared with the ileal microsomal fraction. These data suggest that (1) jejunal and ileal microsomal membranes differ from each other in terms of lipid composition and lipid synthesis, (2) site variations in the specificity of acyltransferases for different fatty acids exist, and (3) higher delta 9-, delta 6-, delta 5-, and delta 4-desaturase activities exist in ileal compared with jejunal enterocytes.     

Incorporation of [214C]malonyl CoA into fatty acids by a cell-free extract of Catharanthus roseus suspension culture cells

A cell-free extract containing the enzymes for de-novo synthesis, elongation and desaturation of fatty acids was prepared from cultured cells of Catharanthus roseus G. Don. ¹⁴C-Fatty acids synthesized by the extract from [2-¹⁴C]malonyl CoA substrate were palmitic (16:0), stearic (18:0) and oleic (18:1). Dialyzed extract was active and stable at room temperature and at 4° C, but was inactivated on boiling. There was an absolute requirement for NADPH for incorporation of [2-¹⁴C]malonyl CoA into total fatty acids. Escherichia coli acyl carrier protein stimulated total fatty-acid synthesis without affecting the relative ratio of individual fatty acids. Total fatty-acid synthesis at a rate of 45 nmol·mg^-1 protein·h^-1 occurred at a substrate level of 73 M malonyl CoA, cofactor levels of 500 M NADPH, 30 g·ml^-1 E. coli ACP, and 1.0 mg·ml^-1 extract protein. Total fatty acid synthesis was also sensitive to cerulenin and CoA levels. Variations in the relative abundance of individual ¹⁴C-fatty acids were regulated by concentrations of [¹⁴C]malonyl CoA. NADPH and ferredoxin, as well as by pH, temperature and length of incubation. Fatty-acid synthetase enzymes responsible for [¹⁴C]palmitic acid were rapidly saturated at a low substrate level (0.3 M malonyl CoA). Increasing the level of [2-¹⁴C]malonyl CoA permitted further synthesis of [¹⁴C]stearate and [¹⁴C]oleate. Desaturation of [¹⁴C]stearate to [¹⁴C]oleate was stimulated by increasing the levels of NADPH and ferredoxin. The desaturase and elongase enzymes were sensitive to acidic pH. The desaturase was also unstable at 41° C, although fatty acid synthetase and elongase were unaffected by this temperature.Abbreviation ACP Acyl carrier protein     

The Zellweger syndrome: deficient conversion of docosahexaenoic acid (22:6(n-3)) to eicosapentaenoic acid (20:5(n-3)) and normal delta 4-desaturase activity in cultured skin fibroblasts

The metabolism of docosahexaenoic acid (22:6(n-3)) and adrenic acid (22:4(n-6)) was studied in cultured fibroblasts from patients with the Zellweger syndrome, X-linked adrenoleukodystrophy (X-ALD) and normal controls. It was shown that [4,5- 3H]22:6(n-3) is retroconverted to labelled eicosapentaenoic acid (20:5(n-3)) in normal and X-ALD fibroblasts, while this conversion is deficient in Zellweger fibroblasts. [U- 14C]Eicosapentaenoic acid (20:5(n-3)) is elongated to docosapentaenoic acid (22:5(n-3)) in all three cell lines. With [U- 14C]20:5(n-3) as the substrate, shorter fatty acids were not detected. With [4,5- 3H]22:6(n-3) as the substrate, labelled fatty acids were esterified in the phospholipid- and triacylglycerol-fraction to approximately the same extent in all three cell lines. [2- 14C]Adrenic acid (22:4(n-6)) was desaturated to 22:5(n-6) and elongated to 24:4(n-6) in all three cell lines and to the largest extent in the Zellweger fibroblasts. This agrees with the view that the delta 4-desaturase is not a peroxisomal enzyme. The observation that the retroconversion of 22:6(n-3) to 20:5(n-3) is deficient in Zellweger fibroblasts strongly suggest that the beta-oxidation step in the retroconversion is a peroxisomal function. Peroxisomal very-long-chain (lignoceroyl) CoA ligase is probably not required for the activation of 22:6(n-3), since the retroconversion to 20:5(n-3) is normal in X-ALD fibroblasts.     

Metabolic Evidence for the Involvement of a [delta]4-Palmitoyl-Acyl Carrier Protein Desaturase in Petroselinic Acid Synthesis in Coriander Endosperm and Transgenic Tobacco Cells

We have previously demonstrated that the double bond of petroselinic acid (18:1[delta]6cis) in coriander (Coriandrum sativum L.) seed results from the activity of a 36-kD desaturase that is structurally related to the [delta]9-stearoyl-acyl carrier protein (ACP) desaturase (E.B. Cahoon, J. Shanklin, J.B. Ohlrogge [1992] Proc Natl Acad Sci USA 89: 11184-11188). To further characterize the biosynthetic pathway of this unusual fatty acid, 14C-labeling experiments were conducted using developing endosperm of coriander. Studies were also performed using suspension cultures of transgenic tobacco (Nicotiana tabacum L.) that express the coriander 36-kD desaturase, and as a result produce petroselinic acid and [delta]4-hexadecenoic acid. When supplied exogenously to coriander endosperm slices, [1-14C]palmitic acid and stearic acid were incorporated into glycerolipids but were not converted to petroselinic acid. This suggested that petroselinic acid is not formed by the desaturation of a fatty acid bound to a glycerolipid or by reactions involving acyl-coenzyme As (CoA). Instead, evidence was most consistent with an acyl-ACP route of petroselinic acid synthesis. For example, the exogenous feeding of [1-14C]lauric acid and myristic acid to coriander endosperm slices resulted in the incorporation of the radiolabels into long-chain fatty acids, including primarily petroselinic acid, presumably through acyl-ACP-associated reactions. In addition, using an in vitro fatty acid biosynthetic system, homogenates of coriander endosperm incorporated [2-14C]malonyl-CoA into petroselinic acid, of which a portion was detected in a putative acyl-ACP fraction. Furthermore, analysis of transgenic tobacco suspension cultures expressing the coriander 36-kD desaturase revealed significant amounts of petroselinic acid and [delta]4-hexadecenoic acid in the acyl-ACP pool of these cells. Also presented is evidence derived from [U-14C]nonanoic acid labeling of coriander endosperm, which demonstrates that the coriander 36-kD desaturase positions double bonds relative to the carboxyl end of acyl-ACP substrates. The data obtained in these studies are rationalized in terms of a biosynthetic pathway of petroselinic acid involving the [delta]4 desaturation of palmitoyl-ACP by the 36-kD desaturase followed by two-carbon elongation of the resulting [delta]4-hexadecenoyl-ACP.     

Oxidative metabolism of dihomogammalinolenic acid by guinea pig epidermis: Evidence of generation of anti-inflammatory products

Reports that vegetable oils which contain gammalinolenic acid :3n-6) may exert beneficial effects on cutaneous disorders prompted us to investigate whether epidermis possesses the ability to transform dihomogammalinolenic acid (20 : 3n-6), the epidermal elongase product of 18 : 3n-6, into oxidative metabolites with anti-inflammatory potential. Incubations of [1–14C] 20:3n-6 with the 105, 000 g particulate (microsomal) fraction from guinea pig epidermal homogenate resulted in the formation of the 1-series prostaglandin PGE₁. The identity of this product was confirmed by argentation thin-layer chromatography (TLC), reverse phase-HPLC, and conversion with alkali treatment to PGB₁. Incubations of [1–14C] 20:3n-6 with the 105,000 g supernatant (cytosolic) fraction from guinea pig epidermal homogenate resulted in the formation of the 15-lipoxygenase product 15-hydroxy-8, 11, 13-eicosatrienoic acid (15-OH-20:3n6). The identity of this product was confirmed by normal phase-HPLC and gas chromatography/mass spectrometry (GC/MS). Thus, data from these studies indicate the capacity of enzymes in the microsomal and cytosolic fractions of guinea pig epidermal homogenates to transform 20:3n-6 to the eicosanoids PGE and 15-OH 20:3n-6, products which reportedly have anti-¹ inflammatory properties. The significance of these findings remains to be explored.     

Chain elongation of eicosapentaenoic acid in the macrophage

In order to elucidate the metabolic fate of eicosapentaenoic acid (20:5 (n-3], a major n-3 fatty acid constituent of fish oil, resident and casein-elicited mouse peritoneal macrophages were incubated with [3H]20:5 (n-3). Comparative experiments with arachidonic acid (20:4 (n-6] were also conducted. After 4, 8 and 18 h incubation, [3H]20:5 (n-3) was extensively elongated into [3H]22:5(n-3) while [3H]20:4(n-6) was only moderately elongated into [3H]22:4(n-6) in both resident and elicited macrophages. No measurable conversion of [3H]22:5(n-3) into [3H]22:6(n-3) (delta 4 desaturation) could be demonstrated. These data demonstrate that the highly active chain elongation of 20:5(n-3) by macrophage elongase, as well as the lack of detectable delta 4 desaturase activity, are responsible for the accumulation of 22:5(n-3) in this cell.     

Oxidative metabolism of dihomogammalinolenic acid by guinea pig epidermis: evidence of generation of anti-inflammatory products

Reports that vegetable oils which contain gamma-linolenic acid (18:3n-6) may exert beneficial effects on cutaneous disorders prompted us to investigate whether epidermis possesses the ability to transform dihomogammalinolenic acid (20:3n-6), the epidermal elongase product of 18:3n-6, into oxidative metabolites with anti-inflammatory potential. Incubations of [1-14C]20:3n-6 with the 105,000 g particulate (microsomal) fraction from guinea pig epidermal homogenate resulted in the formation of the 1-series prostaglandin PGE1. The identity of this product was confirmed by argentation thin-layer chromatography (TLC), reverse phase-HPLC, and conversion with alkali treatment to PGB1. Incubations of [1-14C]20:3n-6 with the 105,000 g supernatant (cytosolic) fraction from guinea pig epidermal homogenate resulted in the formation of the 15-lipoxygenase product 15-hydroxy-8, 11, 13-eicosatrienoic acid (15-OH-20:3n6). The identity of this product was confirmed by normal phase-HPLC and gas chromatography/mass spectrometry (GC/MS). Thus, data from these studies indicate the capacity of enzymes in the microsomal and cytosolic fractions of guinea pig epidermal homogenates to transform 20:3n-6 to the eicosanoids PGE1 and 15-OH 20:3n-6, products which reportedly have anti-inflammatory properties. The in vivo significance of these findings remains to be explored.     

Transacylation of lyso platelet-activating factor and other lysophospholipids by macrophage microsomes. Distinct donor and acceptor selectivities

Rabbit alveolar macrophage microsomes were found to acylate 1-[3H]alkyl-glycero-3-phosphocholine (GPC) (lyso platelet-activating factor) in the absence of any cofactors, indicating the presence of transacylation activity. The transacylation activity was comparable to the activity of acyl-CoA:1-alkyl-GPC acyltransferase. The fatty acyl moieties introduced into 1-[3H]alkyl-GPC from membrane lipids by microsomes were mainly 20:4 (n-6). A very similar acylation profile was observed for the acylation of 1-[3H]alkyl-GPC in intact macrophages, suggesting that the CoA-independent transacylation system plays a very important part in the acylation of 1-[3H]alkyl-GPC in cells. We also confirmed that 14C-labeled 20:4(n-6), 20:5(n-3), 22:4(n-6), and 22:6(n-3) were transferred well from diacyl-GPC to 1-alkyl-GPC in a CoA-independent manner. The transfer rates for 16:0, 18:0, and 18:1 from diacyl-GPC to 1-alkyl-GPC were very low in the presence and absence of CoA. On the other hand, the transfer of 20:4 from diacyl-GPE or diacyl-GPI to 1-alkyl-GPC or 1-acyl-GPC was markedly increased by the addition of CoA. The above results indicate that the transacylation system exhibits distinct donor and acceptor selectivities and CoA dependency. These transacylation reactions could be very important in the regulation of the levels and the availability of lysophospholipids, including lyso platelet-activating factor, and C20 and C22 polyunsaturated fatty acids in living cells.     

Polyunsaturated fatty acid metabolism in fish cells: differential metabolism of (n-3) and (n-6) series acids by cultured cells originating from a freshwater teleost fish and from a marine teleost fish

1. The incorporation and metabolism of (n-3) and (n-6) polyunsaturated fatty acids (PUFA) supplemented to growing cultures were studied in rainbow trout (RTG-2) and turbot (TF) cell lines. 2. A fatty acid concentration of 20 microM considerably altered the fatty acid composition of the cells without affecting lipid class composition or the appearance of cytoplasmic lipid droplets. 3. Both cell lines exhibited considerable delta 6 desaturase activities. 4. Whereas delta 5 desaturase activity was expressed in RTG-2 cells, delta 4 desaturase activity was absent and, conversely, delta 4 desaturase activity was expressed in TF cells, but there was an apparent deficiency in the C18 to C20 elongase multi-enzyme complex. 5. The delta 6 desaturase activity in both cell lines showed little preference between 18:2(n-6) and 18:3(n-3) but the delta 5 desaturase activity of RTG-2 cells and the delta 4 desaturase activity of TF cells showed a preference for (n-3)PUFA. 6. Two fish oil concentrates were assessed for their ability to generate fatty acid compositions in the cell lines more closely resembling those of intact fish tissues.     

Identification of a fatty acid delta6-desaturase deficiency in human skin fibroblasts

Polyunsaturated fatty acid (PUFA) utilization was investigated in skin fibroblasts cultured from a female patient with an inherited abnormality in lipid metabolism. These deficient human skin fibroblasts (DF) converted 85;-95% less [1-14C]linoleic acid (18:2n-6) to arachidonic acid (20:4n-6), 95% less [3-14C]tetracosatetraenoic acid (24:4n-6) to docosapentaenoic acid (22:5n-6), and 95% less [1-14C]-linolenic acid (18:3n-3) and [3-14C]tetracosapentaenoic acid (24:5n-3) to docosahexaenoic acid (22:6n-3) than did normal human skin fibroblasts (NF). The only product formed by the DF cultures from [1-14C]tetradecadienoic acid (14:2n-6) was 18:2n-6. However, they produced 50;-90% as much 20:4n-6 as the NF cultures from [1-14C]hexadecatrienoic acid (16:3n-6), [1-14C]gamma-linolenic acid (18:3n-6), and [1-14C]dihomo-gamma-linolenic acid (20:3n-6), PUFA substrates that contain Delta6 double bonds. DF also contained 80% more 18:2n-6 and 25% less 20:4n-6. These results suggested that DF are deficient in Delta6 desaturation. This was confirmed by Northern blots demonstrating an 81;-94% decrease in Delta6-desaturase mRNA content in the DF cultures, whereas the Delta5-desaturase mRNA content was reduced by only 14%. This is the first inherited abnormality in human PUFA metabolism shown to be associated with a Delta6-desaturase deficiency. Furthermore, the finding that the 18- and 24-carbon substrates are equally affected suggests that a single enzyme carries out both Delta6 desaturation reactions in human PUFA metabolism.     

Modulation of rat liver lipid metabolism by prolactin

The effect of chronic hyperprolactinemia on the delta6- and delta5-desaturation activity, total lipid and fatty acid composition, as well as fluorescence anisotropy, was studied in liver microsomes from anterior pituitary-grafted rats. We observed a depression in delta6-desaturation activity but no changes in the delta5-desaturation activity in the grafted animals. The microsomal fraction from the hyperprolactinemic rats contained significantly less amount of linoleic acid and a higher content of 20:4 n-6, 22:5 n-6 and 22:6 n-3 acids. Lipid rotational mobility was increased in microsomes as well as in liposomes obtained from the microsomes of transplanted animals. The fluidifying effect induced by PRL was located in the deepest zone of the membrane. The results obtained indicate that high levels of prolactin induce changes in polyunsaturated fatty acid distribution in liver microsomes, which regulates the lipid rotational mobility and hence membrane fluidity.     

The metabolism of 7,10,13,16,19-docosapentaenoic acid to 4,7,10,13,16,19-docosahexaenoic acid in rat liver is independent of a 4-desaturase.

The hypothesis that the last step in the biosynthesis of 4,7,10,13,16,19-22:6 from linolenate is catalyzed by an acyl-CoA-dependent 4-desaturase has never been evaluated by direct experimentation. When rat liver microsomes were incubated with [1-14C]7,10,13,16,19-22:5, under conditions where linoleate was readily desaturated to 6,9,12-18:3, it was never possible to detect the product of the putative 4-desaturase. In the presence of malonyl-CoA, 7,10,13,16,19-22:5 was sequentially chain-elongated to 9,12,15,18,21-24:5, followed by its desaturation at position 6 to give 6,9,12,15,18,21-24:6. Microsomes desaturated 9,12,15,18,21-24:5 at rates similar to those observed for metabolizing linoleate to 6,9,12-18:3. Rat hepatocytes metabolize [1-14C]7,10,13,16,19-22:5 to 22:6(n-3), but in addition, it was possible to detect small amounts of esterified 24:5(n-3) and 24:6(n-3) in phospholipids, which is a finding consistent with their role as obligatory intermediates in 22:6(n-3) biosynthesis. When 3-14C-labeled 24:5(n-3) or 24:6(n-3) were incubated with hepatocytes, only a small amount of either substrate was esterified. [3-14C] 24:5(n-3) was metabolized both by beta-oxidation to 22:5(n-3) and by serving as a precursor for the biosynthesis of 24:6(n-3) and 22:6(n-3). The primary metabolic fate of [3-14C]24:6(n-3) was beta-oxidation to 22:6(n-3), followed by its acylation into membrane lipids. Our results thus document that 22:5(n-3) is the precursor for 22:6(n-3) but via a pathway that is independent of a 4-desaturase. This pathway involves the microsomal chain elongation of 22:5(n-3) to 24:5(n-3), followed by its desaturation to 24:6(n-3). This microsomal product is then metabolized, via beta-oxidation, to 22:6(n-3).     

Lipid involvement in oleoyl CoA desaturase activity of Fusarium oxysporum microsomes

Desaturation of oleoyl CoA by the microsomal fraction of Fusarium oxysporum hyphal cells required O2, NADPH, MgCl2, and the addition of either bovine serum albumin or the 105 000g supernatant fraction. In the absence of reduced nucleotide, [14C]oleoyl CoA was rapidly incorporated into phospholipid and triacylglycerol and hydrolyzed to free fatty acids. After addition of NADPH, oleate was desaturated at the normal rate. Analysis of the distribution of [14C]oleate and [14C]linoleate between different lipid classes revealed that phosphatidylcholine and phosphatidylethanolamine were labeled with [14C]linoleate before any other lipid class. These results are consistent with oleoyl phospholipid being a direct intermediate in the desaturation of oleoyl CoA. The preference of the oleoyl-desaturase for NADPH, the relatively high pH optimum of 8.2, and the sensitivity to thenoyltrifluoroacetone inhibition suggest that some components of the microsomal electron-transport chain are common to both the oleoyl desaturase and stearoyl CoA desaturase systems in this fungus.