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.     

Metabolism of alpha-linolenic acid in humans

Alpha-linolenic acid (18:3n-3) is essential in the human diet, probably because it is the substrate for the synthesis of longer-chain, more unsaturated n-3 fatty acids eicosapentaenoic acid (20:5n-3) and docosahexaenoic acid (22:6n-3) which are required for tissue function. This article reviews the recent literature on 18:3n-3 metabolism in humans, including fatty acid beta-oxidation, recycling of carbon by fatty acid synthesis de novo and conversion to longer-chain polyunsaturated fatty acids (PUFA). In men, stable isotope tracer studies and studies in which volunteers increased their consumption of 18:3n-3 show conversion to 20:5n-3 and 22:5n-3, but limited conversion to 22:6n-3. However, conversion to 18:3n-3 to 20:5n-3 and 22:6n-3 is greater in women compared to men, due possibly to a regulatory effect of oestrogen, while partitioning of 18:3n-3 towards beta-oxidation and carbon recycling was lower than in men. These gender differences may be an important consideration in making dietary recommendations for n-3 PUFA intake.     

Fifteen weeks of dietary n-3 polyunsaturated fatty acid deprivation increase turnover of n-6 docosapentaenoic acid in rat-brain phospholipids

Docosapentaenoic acid (DPAn-6, 22:5n-6) is an n-6 polyunsaturated fatty acid (PUFA) whose brain concentration can be increased in rodents by dietary n-3 PUFA deficiency, which may contribute to their behavioral dysfunction. We used our in vivo intravenous infusion method to see if brain DPAn-6 turnover and metabolism also were altered with deprivation. We studied male rats that had been fed for 15weeks post-weaning an n-3 PUFA adequate diet containing 4.6% alpha-linolenic acid (α-LNA, 18:3n-3) or a deficient diet (0.2% α-LNA), each lacking docosahexaenoic acid (22:6n-3) and arachidonic acid (AA, 20:4n-6). [1-(14)C]DPAn-6 was infused intravenously for 5min in unanesthetized rats, after which the brain underwent high-energy microwaving, and then was analyzed. The n-3 PUFA deficient compared with adequate diet increased DPAn-6 and decreased DHA concentrations in plasma and brain, while minimally changing brain AA concentration. Incorporation rates of unesterified DPAn-6 from plasma into individual brain phospholipids were increased 5.2-7.7 fold, while turnover rates were increased 2.1-4.7 fold. The observations suggest that increased metabolism and brain concentrations of DPAn-6 and its metabolites, together with a reduced brain DHA concentration, contribute to behavioral and functional abnormalities reported with dietary n-3 PUFA deprivation in rodents. (196 words).     

A mathematical model of fatty acid metabolism and VLDL assembly in human liver

The lipid composition of very-low-density lipoprotein (VLDL) in plasma is crucial for human health. A pre-requisite for the alteration of VLDL composition is a co-ordinated understanding of the complex interactions in VLDL assembly. In order to determine the potential effects of changes in substrate availability on VLDL lipid composition, we constructed, parameterized and evaluated a mechanistic mathematical model of the biosynthesis of triglycerides, phospholipids, and cholesterol esters and the assembly of VLDL in human hepatocytes. Using published data on human liver metabolism, the model was also used to provide insight into the complex process of lipid metabolism and to estimate the affinities of different liver enzymes for different fatty acids (FA). For example, we found that Delta6-desaturase is 19 times more selective for C18:3n-3 than C18:2n-6, stearoyl-CoA-desaturase is 2.7 times more selective for C18:0 than C16:0, Delta5-desaturase desaturates C20:4n-3 preferentially over C20:3n-6 and FA elongase preferentially elongates C18:3n-6. The model was also used to predict the plasma free fatty acid (FFA) composition required to generate a prescribed change in plasma lipoprotein FA composition. Furthermore, the model was tested against a published human feeding trial that investigated the effect of changes in dietary FA composition on human plasma lipid FA composition. The model is a useful tool for predicting the effect of changes in plasma FFA composition on plasma lipoprotein lipid FA composition.     

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.     

Significant utilization of dietary arachidonic acid is for brain adrenic acid in baboon neonates

Dietary arachidonic acid (20:4n-6) utilization in-vivo for carbon recycling into de-novo lipogenesis and conversion to n-6 long chain polyunsaturates was investigated in baboon neonates using [U-(13)C]20:4n-6. Neonates consuming a formula typical of human milk received a single oral dose of [(13)C]arachidonic acid in sn-2 position of either triglyceride or phosphatidylcholine at 18-19 days of postnatal life. Neonate brain, retina, liver, and plasma were obtained 10 days later (28-29 days of life). Low isotopic enrichment (0.27-1.0%Total label) was detected in dihomo-gamma-linolenic acid (20:3n-6) in all tissues, but label incorporation into saturates or monounsaturates was not detected. In neonate brain and retina, 16% and 11% of total label was recovered in 22:4n-6, respectively. The relative contribution of dietary fatty acids to postnatal brain 22:4n-6 accretion can be estimated for dietary 20:4n-6 and preformed 22:4n-6 as 17% and 8%, respectively, corresponding to efficiencies of 0.48% and 0.54% of dietary levels, respectively. These results demonstrate in term baboon neonates that in vivo 1) 20:4n-6 was retroconverted to 20:3n-6, 2) 20:4n-6 did not contribute significantly to de novo lipogenesis of saturates and monounsaturates, and 3) the preformed 20:4n-6 contribution to brain 22:4n-6 accumulation was quantitatively a significant metabolic fate for dietary 20:4n-6.     

Studies on polyenoic acid incorporation into human platelet lipid stores: interactions with linoleic and arachidonic acids

Several polyunsaturated fatty acids (C18-C22 acids) have been compared in their uptake by human platelets and their acylation into glycerophospholipid subclasses. This was also studied in the presence of linoleic and/or arachidonic acids, the main fatty acids of plasma free fatty acid pool. Amongst C20 fatty acids, dihomogamma linolenic acid (20:3(n-6)), 5,8,11-icosatrienoic acid (20:3(n-9)) and arachidonic acid (20:4(n-6)) were better incorporated. The uptake of 5,8,11,14,17-icosapentaenoic acid (20:5(n-3)) was significantly lower and comparable to that of C22 fatty acids (7,10,13,16-docosatetraenoic acid (22:4(n-6)) and 4,7,10,13,16,19-docosahexaenoic acid (22:6(n-3)) and linoleic acid (18:2(n-6)). In this respect, linolenic acid (18:3(n-3)) appeared the poorest substrate. The bulk of each acid was acylated into glycerophospholipids although the presence of linoleic and/or arachidonic acids diverted a part towards neutral lipids. This was prominent for 18:3(n-3) and C22 fatty acids. The glycerophospholipid distribution of each acid differed substantially and was not affected by the presence of linoleic and or arachidonic acids, except for 18:3(n-3) and 22:6(n-3) that were strongly diverted towards phosphatidylethanolamine (PE) at the expense of phosphatidylcholine (PC). The main features were an efficient acylation of 20:3(n-9) into phosphatidylinositol (PI) followed by 20:3(n-6) and 20:4(n-6), then by 20:5(n-3) and 22:4(n-6), and finally 22:6(n-3) and C18 fatty acids. This was reciprocal to the acylation into PE and to a lesser extent into PC which remained the main storage species in all cases. We conclude that human platelets may exhibit a certain specificity for taking up polyunsaturated fatty acids both in terms of total uptake and glycerophospholipid subclass distribution. Also the presence of polyunsaturated fatty acids of normal plasma, like linoleic and arachidonic acids, may interact specifically with such an uptake and distribution.     

Conversion of linoleic acid into arachidonic acid by cultured murine and human keratinocytes

The origin of arachidonic acid (AA) found in the epidermis is not known. Two possibilities exist: either de novo synthesis within the epidermal keratinocyte, or transport of AA formed at distant tissue sites. The current study examined the ability of cultured murine and human keratinocytes to metabolize exogenously added linoleic acid (LA). Conversion of radiolabeled substrate (14C-LA) into 18:3(n-6), 20:2(n-6), 20:3(n-6), and 20:4(n-6) (AA) was noted. The conversion of non-radiolabeled 18:3(n-6) or 20:2(n-6) was also examined and the pattern of metabolites synthesized suggests that the preferred metabolic pathway for conversion of linoleic acid into arachidonic acid is via the classically described pathway in which a delta 6 desaturase constitutes the initial reaction. Although cultured skin fibroblasts are known to convert linoleic acid into arachidonic acid, the current study demonstrates that cultured epidermal keratinocytes can also avidly metabolize exogenous linoleic acid. The ability of cultured keratinocytes, and not of whole epidermis in vivo, to convert linoleic acid into arachidonic acid suggests that specific enzymatic activities may be induced by the tissue culture system itself. Hence, findings of metabolic capabilities in cultured cells may not necessarily be extrapolated to the in vivo situation.     

Fatty acid utilisation and metabolism in caecal enterocytes of rainbow trout (Oncorhynchus mykiss) fed dietary fish or copepod oil

A combined fatty acid metabolism assay was employed to determine fatty acid uptake and relative utilisation in enterocytes isolated from the pyloric caeca of rainbow trout. In addition, the effect of a diet high in long-chain monoenoic fatty alcohols present as wax esters in oil derived from Calanus finmarchicus, compared to a standard fish oil diet, on caecal enterocyte fatty acid metabolism was investigated. The diets were fed for 8 weeks before caecal enterocytes from each dietary group were isolated and incubated with [1-14C]fatty acids: 16:0, 18:1n-9, 18:2n-6, 18:3n-3, 20:1n-9, 20:4n-6, 20:5n-3, and 22:6n-3. Uptake was measured over 2 h with relative utilisation of different [1-14C]fatty acids calculated as a percentage of uptake. Differences in uptake were observed, with 18:1n-9 and 18:2n-6 showing the highest rates. Esterification into cellular lipids was highest with 16:0 and C18 fatty acids, accounting for over one-third of total uptake, through predominant incorporation in triacylglycerol (TAG). The overall utilisation of fatty acids in phospholipid synthesis was low, but highest with 16:0, the most prevalent fatty acid recovered in intracellular phosphatidylcholine (PC) and phosphatidylinositol (PI), although exported PC exhibited higher proportions of C20/C22 polyunsaturated fatty acids (PUFA). Other than 16:0, incorporation into PC and PI was highest with C20/C22 PUFA and 20:4n-6 respectively. Recovery of labelled 18:1n-9 in exported TAG was 3-fold greater than any other fatty acid which could be due to multiple esterification on the glycerol 'backbone' and/or increased export. Approximately 20-40% of fatty acids taken up were beta-oxidised, and was highest with 20:4n-6. Oxidation of 20:5n-3 and 22:6n-3 was also surprisingly high, although 22:6n-3 oxidation was mainly attributed to retroconversion to 20:5n-3. Metabolic modification of fatty acids by elongation-desaturation was generally low at <10% of [1-14C]fatty acid uptake. Dietary copepod oil had generally little effect on fatty acid metabolism in enterocytes, although it stimulated the elongation and desaturation of 16:0 and elongation of 18:1n-9, with radioactivity recovered in longer n-9 monoenes. The monoenoic fatty acid, 20:1n-9, abundant in copepod oil as the homologous alcohol, was poorly utilised with 80% of uptake remaining unesterified in the enterocyte. However, the fatty acid composition of pyloric caeca was not influenced by dietary copepod oil.     

In vivo conversion of 18- and 20-C essential fatty acids in rats using the multiple simultaneous stable isotope method

An important question for mammalian nutrition is the relative efficiency of C18 versus C20 essential fatty acids (EFAs) for supporting the tissue composition of n-3 and n-6 pathway end products. One specific question is whether C22 EFAs are made available to tissues more effectively by dietary alpha-linolenic acid (18:3n-3) and linoleic acid (18:2n-6) or by dietary eicosapentaenoic acid (20:5n-3) and dihomo-gamma-linolenic acid (20:3n-6). To address this question in a direct manner, four stable isotope compounds were given simultaneously in a novel paradigm. A single oral dose of a mixture of 2H5-18:3n-3, 13C-U-20:5n-3, 13C-U-18:2n-6, and 2H5-20:3n-6 was administered to rats given a defined diet. There was a preferential in vivo conversion of arachidonic acid (20:4n-6) to docosatetraenoic acid (22:4n-6) and of 22:4n-6 to n-6 docosapentaenoic acid (22:5n-6) when the substrates originated from the C18 precursors. However, when the end products docosahexaenoic acid (22:6n-3) or 22:5n-6 were expressed as the total amount in the plasma compartment divided by the dosage, this parameter was 11-fold greater for 20:5n-3 than for 18:3n-3 and 14-fold greater for 20:3n-6 than for 18:2n-6. Thus, on a per dosage basis, the total amounts of n-3 and n-6 end products accreted in plasma were considerably greater for C20 EFA precursors relative to C18.     

Polyunsaturated fatty acid biosynthesis from [1-14C]20:3 n-6 acid in rat cultured Sertoli cells. Linoleic acid effect

Primary culture is a suitable system to study lipid metabolism and polyunsaturated fatty acid biosynthesis. Sertoli cell-enriched preparations were used to determine the fatty acid composition after 5 and 7 days in culture (serum free) as well as the uptake and metabolism of [1-14C]eicosa-8,11,14-trienoic acid. The addition of unlabeled linoleic acid (0.2 and 2.0 microg/ml) was also evaluated. Fatty acid methyl esters derived from cellular lipids were analyzed by gas liquid chromatography and radiochromatography. After 5 days in culture, cells had significantly less 18:2, 20:4, 22:5 and 24:5 and more 18:3, 20:3, 22:4 and 24:4 n-6 fatty acids than non-cultured cells. On day 7, an additional increment in 22:4 n-6 and a decrease in linoleic, gamma-linoleic and 24:4 n-6 fatty acids were observed. The presence of linoleic acid (low dose) produced a significant decrease in saturated and monounsaturated acids and an increase in 18:2, 20:4 and 22:5 n-6 fatty acids. At a high concentration almost all fatty acids belonging to 18:2 n-6 increased significantly. The drop in 20:4 n-6/20:3 n-6 ratio was considered as an indirect evidence of a Delta 5 desaturase activity depression. This assumption was corroborated by studying the transformation of [1-14C]eicosa-8,11,14-trienoic acid into 20:4, 22:4, 22:5, 24:4 and 24:5 n-6 fatty acids. We conclude that Sertoli cells after 7 days in culture evidenced changes in the fatty acid profile similar to those described under fat deprivation. The addition of linoleic acid reverted this pattern and indicated that the Delta 5 desaturase activity is a limiting step in the polyunsaturated fatty acid biosynthesis.     

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.     

Low C18 to C20 fatty acid elongase activity and limited conversion of stearidonic acid, 18:4(n-3), to eicosapentaenoic acid, 20:5(n-3), in a cell line from the turbot, Scophthalmus maximus

The TF cell line, derived from a top predatory, carnivorous marine teleost, the turbot (Scophthalmus maximus), is known to have a limited conversion of C18 to C20 polyunsaturated fatty acids (PUFA). To illuminate the underlying processes, we studied the conversions of stearidonic acid, 18:4(n-3), and its elongation product, 20:4(n-3), in TF cells and also in a cell line, AS, derived from Atlantic salmon (Salmo salar), by adding unlabelled (25 microM), U-14C (1 microM) or deuterated (d5; 25 microM) fatty acids. Stearidonic acid, 18:4(n-3), was metabolised to 20:5(n-3) in both cells lines, but more so in AS than in TF cells. Delta5 desaturation was more active in TF cells than in AS cells, whereas C18 to C20 elongation was much reduced in TF as compared to AS cells. Only small amounts of docosahexaenoic acid (22:6(n-3)) were produced by both cell lines, although there was significant production of 22:5(n-3) in both cultures, especially when 20:4(n-3) was supplemented. We conclude that limited elongation of C18 to C20 fatty acids rather than limited fatty acyl Delta5 desaturation accounts for the limited rate of conversion of 18:3(n-3) to 20:5(n-3) in the turbot cell line, as compared to the Atlantic salmon cell line. The results can account for the known differences in conversions of C18 to C20 PUFA by the turbot and the Atlantic salmon in vivo.     

Rates and efficiencies of reactions of ruminal biohydrogenation of linoleic acid according to pH and polyunsaturated fatty acids concentrations

Data from a previous study about the effects of pH and of linolenic acid (C18:3n-3) and linoleic acid (C18:2n-6) concentrations on C18:2n-6 biohydrogenation in ruminal cultures were used to calculate the rates and efficiencies of the three reactions of C18:2n-6 biohydrogenation (isomerisation of C18:2n-6 to CLA; reduction of CLA to trans-octadecenoic acids; reduction of trans-octadecenoic acids to stearic acid). First, low pH was confirmed to inhibit isomerisation and was shown to inhibit the second reduction, leading to an accumulation of vaccenic acid. This later effect had only been observed in some in vivo studies using high concentrate diets, because in in vitro experiments, the very low pH frequently used depresses isomerisation which consequently generates very low amount of substrates for reductions whose variations become difficult to ascertain. Second, C18:2n-6 at high concentration was confirmed to saturate its own isomerisation and the increase of CLA production due to high initial C18:2n-6 was shown to inhibit the two subsequent reductions. Third, C18:3n-3 at high concentrations was confirmed to inhibit C18:2n-6 isomerisation. Moreover, the second reduction was shown to be saturated, probably by all trans-octadecenoic acids intermediates of C18:2n-6 and C18:3n-3 biohydrogenation, leading to an accumulation of trans-octadecenoic acids, especially vaccenic acid. This fatty acid is partly desaturated into CLA in the mammary gland, which explains the synergy between C18:2n-6 and C18:3n-3 for milk CLA noticed by others in vivo. This approach helped explain the actions of pH and of C18:2n-6 and C18:3n-3 concentrations on C18:2n-6 biohydrogenation and allows some explanations about differences noticed between studies.     

Simultaneous quantitative determination of deuterium- and carbon-13-labeled essential fatty acids in rat plasma

This study reports methods for the quantitative determination of stable isotope-labeled essential fatty acids (EFAs) as well as an experiment in which deuterium-labeled linoleic acid (18:2n-6) and alpha-linolenic acid (18:3n-3) were compared with those labeled with carbon-13 in rat plasma in vivo. Standard curves were constructed to compensate for concentration and plasma matrix effects. It was observed that endogenous pools of fatty acids had a greater suppressing effect on the measurements of 13C-U-labeled EFAs relative to those labeled with 2H5. Using these methods, the in vivo metabolism of orally administered deuterated-linolenate, 13C-U-labeled linolenate, deuterated-linoleate, and 13C-U-labeled linoleate was compared in adult rats (n = 11). There were no significant differences in the concentrations of the 2H versus 13C isotopomers of 18:2n-6, 18:3n-3, arachidonic acid (20:4n-6), and docosahexaenoic acid (22:6n-3) in rat plasma samples at 24 h after dosing. Thus, there appears to be little isotope effect for 2H5- versus 13C-U-labeled EFAs when the data are calculated using the conventional standard curves and corrected for endogenous fatty acid pool size and matrix effects.     

Dietary long chain n-3 fatty acids are more closely associated with protein than energy intakes from fat

The n-3 fatty acids, eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3) regulate hepatic lipid and glucose metabolism; however, EPA and DHA are naturally present in human diets in foods of animal origin, which are generally high in protein with variable triglycerides and uniformly low amounts of carbohydrate. We used dietary information for 611 individuals of 1.5-66 years to address whether EPA and DHA are associated with protein, but not fat intake. EPA, DHA and arachidonic acid (20:4n-6) intakes were positively associated with protein, but not fat intake, whereas linoleic acid (18:2n-6) and α-linolenic acid (18:3n-3) intakes were positively associated with fat, but not protein intake. Children 1-3 years of age have lower EPA and DHA intakes than children over 4 years or adults. Recommendations regarding EPA and DHA intake should focus on protein sources, rather than diet fat, and consider their potential roles in amino acid and protein metabolism.     

Mitochondrial and peroxisomal oxidation of arachidonic and eicosapentaenoic acid studied in isolated liver cells

The partitioning between peroxisomal and mitochondrial beta-oxidation of [1-14C]eicosapentaenoic acid (20:5(n-3] and [1-14C]arachidonic acid (20:4(n-6)) was studied. In hepatocytes from fasted rats approximately 70% of the fatty acid substrate was oxidized with oleic, linoleic, eicosapentaenoic and docosahexaenoic (22:6(n-3)) acid, even more with adrenic (22:4(n-6)) and less with arachidonic acid. When the mitochondrial oxidation was suppressed by fructose refeeding and by (+)-decanoylcarnitine, the fatty acid oxidation in per cent of that in cells from fasted rats was with 18:1(n-9) 7%, 18:2(n-6) 8%, 20:4(n-6) 12%, 20:5(n-3) 20%, 22:4(n-6) 57% and for 22:6(n-3) 29%. The fraction of 14C recovered in palmitate and other newly synthesized fatty acids after fructose refeeding decreased in the order 22:4(n-6) greater than 22:6(n-3) greater than 20:5(n-3) greater than 20:4(n-6) and was very small with 18:1(n-9) and 18:2(n-6). In cells from both fed and fructose-refed animals 20:5(n-3) was efficiently elongated to 22:5(n-3) and 22:6(n-3). 20:5(n-3) and 20:4(n-6) were not elongated after fasting. The phospholipid incorporation with [1-14C]20:5(n-3) decreased during prolonged incubations while it remained stable with [1-14C]arachidonic acid. The results suggest that peroxisomes contribute more to the oxidation of 20:5(n-3) than with 20:4(n-6) although both substrates are probably oxidized mainly in the mitochondria.     

Fatty acid metabolism in Atlantic salmon (Salmo salar L.) hepatocytes and influence of dietary vegetable oil

Isolated hepatocytes from Atlantic salmon (Salmo salar), fed diets containing either 100% fish oil or a vegetable oil blend replacing 75% of the fish oil, were incubated with a range of seven (14)C-labelled fatty acids. The fatty acids were [1-(14)C]16:0, [1-(14)C]18:1n-9, 91-(14)C]18:2n-6, [1-(14)C]18:3n-3, [1-(14)C]20:4n-6, [1-(14)C]20:5n-3, and [1-(14)C]22:6n-3. After 2 h of incubation, the hepatocytes and medium were analysed for acid soluble products, incorporation into lipid classes, and hepatocytes for desaturation and elongation. Uptake into hepatocytes was highest with [1-(14)C]18:2n-6 and [1-(14)C]20:5n-3 and lowest with [1-(14)C]16:0. The highest recovery of radioactivity in the cells was found in triacylglycerols. Of the phospholipids, the highest recovery was found in phosphatidylcholine, with [1-(14)C]16:0 and [1-(14)C]22:6n-3 being the most prominent fatty acids. The rates of beta-oxidation were as follows: 20:4n-6>18:2n-6=16:0>18:1n-9>22:6n-3=18:3n-3=20:5n-3. Of the fatty acids taken up by the hepatocytes, [1-(14)C]16:0 and [1-(14)C]18:1n-9 were subsequently exported the most, with the majority of radioactivity recovered in phospholipids and triacylglycerols, respectively. The major products from desaturation and elongation were generally one cycle of elongation of the fatty acids. Diet had a clear effect on the overall lipid metabolism, with replacing 75% of the fish oil with vegetable oil resulting in decreased uptake of all fatty acids and reduced incorporation of fatty acids into cellular lipids, but increased beta-oxidation activity and higher recovery in products of desaturation and elongation of [1-(14)C]18:2n-6 and [1-(14)C]18:3n-3.     

Fatty acid metabolism in the freshwater fish Murray cod (Maccullochella peelii peelii) deduced by the whole-body fatty acid balance method

The whole-body fatty acid balance method was used to investigate the fatty acid metabolism in Murray cod (Maccullochella peelii peelii) fed diets containing canola (CO) or linseed oil (LO). Murray cod were able to elongate and desaturate both 18:2n-6 and 18:3n-3. In fish fed the CO diet, 54.4% of the 18:2n-6 consumed was accumulated, 38.5% oxidized and 6.4% elongated and desaturated to higher homologs. Fish fed the LO diet accumulated 52.9%, oxidized 37% and elongated and desaturated 8.6% of the consumed 18:3n-3. The overall roles of n-6 fatty acids appeared more important in Murray cod compared to other freshwater species. Murray cod also showed a preferential order of utilization of C18 fatty acid for energy production (18:3n-3 > 18:2n-6 > 18:1n-9). Moreover, it is demonstrated that an increase in dietary 18:3n-3 is directly responsible of increased desaturase activity and augmented saturated fatty acid accumulation in the fish body. The present study also suggests that, in the context of the possible maximization of the natural ability of fish to produce long chain polyunsaturated fatty acids, the whole-body approach can be considered well suited and informative and Murray cod is a suited candidate to fish oil replacement for its diets.     

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.