Production of structured triacylglycerols by acidolysis catalyzed by lipases immobilized in a packed bed reactor

The aim of this work was to produce structured triacylglycerols (STAGs), with caprylic acid located at positions 1 and 3 of the glycerol backbone and docosohexaenoic acid (DHA) at position 2, by acidolysis of tuna oil and caprylic acid (CA) catalyzed by lipases Rd, from Rhizopus delemar, and Palatase 20000L from Mucor miehei immobilized on Accurel MP1000 in a packed bed reactor (PBR), working in continuous and recirculation modes. First, different lipase/support ratios were tested for the immobilization of lipases and the best results were obtained with ratios of 0.67 (w/w) for lipase Rd and 6.67 (w/w) for Palatase. Both lipases were stable for at least 4 days in the operational conditions. In the storage conditions (5 °C) lipases Rd and Palatase maintained constant activity for 5 months and 1 month, respectively.These catalysts have been used to obtain STAGs by acidolysis of tuna oil and CA in a PBR operating with recirculation of the reaction mixture through the lipase bed. Thus, STAGs with 52–53% CA and 14–15% DHA were obtained. These results were the basis for establishing the operational conditions to obtain STAGs operating in continuous mode. These new conditions were established maintaining constant intensity of treatment (IOT, lipase amount × reaction time/oil amount). In this way STAGs with 44–50% CA and 17–24% DHA were obtained operating in continuous mode. Although the compositions of STAGs obtained with both lipases were similar, Palatase required an IOT about four times higher than lipase Rd.To separate the acidolysis products (free fatty acids, FFAs, and STAGs) an extraction method of FFAs by water–ethanol solutions was tested. The following variables were optimized: water/ethanol ratio (the best results were attained with a water/ethanol ratio of 30:70, w/w), the solvent/FFA–STAG mixture ratio (3:1, w/w) and the number of extraction steps (3–5). In these conditions highly pure STAGs (93–96%) were obtained with a yield of 85%. The residual FFAs can be eliminated by neutralization with a hydroethanolic KOH solution to obtain pure STAGs. The positional analysis of these STAGs, carried out by alcoholysis catalyzed by lipase Novozym 435, has shown that CA represents 55% of fatty acids located at positions 1 and 3 and DHA represents 42% of fatty acids at position 2.     

Screening of lipases for production of novel structured lipids from single cell oils

Lipids enriched in polyunsaturated fatty acids are very susceptible to oxidation, causing the formation of potentially harmful oxidized products. Hence, it is critical to keep the temperature as low as possible during reaction and storage. In this study, five commercial immobilized lipases were evaluated for their capability to produce novel structured lipids (SLs) enriched with medium-chain fatty acids (MCFAs) through acidolysis of single cell oil (SCO) with capric acid. Among the examined lipases, NS40086 and Lipozyme RM IM showed the highest incorporation degree. The acidolysis reactions resulted in an obvious variation in the fatty acids composition as well as their positional distribution. The obtained SLs contained (33.58 %–34.09 %) capric acid at sn-1, 3 positions with increasing the content of arachidonic acid at the sn-2 position up to (49.82 %–50.25 %). The NS40086 lipase displayed 1, 3 regiospecificity towards the TAG of SCO. The acidolysis reactions using NS40086 lipase resulted in a generation of 23 TAG molecular species containing capric acid. Moreover, the NS40086 lipase was more active than Lipozyme RM IM at relatively low temperatures (35 °C and 40 °C), which could be used effectively as a promising biocatalyst in lipid synthesis.     

Metabolic engineering of the oleaginous alga Nannochloropsis for enriching eicosapentaenoic acid in triacylglycerol by combined pulling and pushing strategies

The marine alga Nannochloropsis oceanica has been considered as a promising photosynthetic cell factory for synthesizing eicosapentaenoic acid (EPA), yet the accumulation of EPA in triacylglycerol (TAG) is restricted to an extreme low level. Poor channeling of EPA to TAG was observed in N. oceanica under TAG induction conditions, likely due to the weak activity of endogenous diacylglycerol acyltransferases (DGATs) on EPA-CoA. Screening over thirty algal DGATs revealed potent enzymes acting on EPA-CoA. Whilst overexpressing endogenous DGATs had no or slight effect on EPA abundance in TAG, introducing selected DGATs with strong activity on EPA-CoA, particularly the Chlamydomonas-derived CrDGTT1, which resided at the outermost membrane of the chloroplast and provided a strong pulling power to divert EPA to TAG for storage and protection, led to drastic increases in EPA abundance in TAG and TAG-derived EPA level in N. oceanica. They were further promoted by additional overexpression of an elongase gene involved in EPA biosynthesis, reaching 5.9- and 12.3-fold greater than the control strain, respectively. Our results together demonstrate the concept of applying combined pulling and pushing strategies to enrich EPA in algal TAG and provide clues for the enrichment of other desired fatty acids in TAG as well.     

Inhibition of Δ-6 desaturase reduces fatty acid re-esterification in 3T3-L1 adipocytes independent of changes in n3-PUFA cellular content

Δ-6 desaturase (D6D) is a key enzyme in the synthesis of long-chain polyunsaturated fatty acids (LC-PUFA). Evidence suggests that reduced D6D activity not only disrupts LC-PUFA production, but also impacts whole body lipid handling and body weight; however, the mechanisms remain largely unexplored. Therefore, we investigated the effect of D6D inhibition on the regulation of lipid accumulation in 3T3-L1 adipocytes with and without changes in n-3 PUFA content. 3T3-L1 cells were treated with a D6D inhibitor (SC-26196) in the presence or absence of α-linolenic acid (ALA) throughout differentiation. We found that D6D inhibition blocked the conversion of ALA to eicosapentaenoic acid (EPA) and docosapentaenoic acid (DPAn-3) when ALA was supplemented, while no changes in n-3 PUFA content were observed in cells treated with the D6D inhibitor alone. D6D inhibited cells had reduced triacylglycerol (TAG) accumulation despite an EPA/DPA deficiency. In addition, analyses of cellular protein markers, as well as non-esterified fatty acids and glycerol release in medium, suggested an increase in lipolysis and a decrease in fatty acid re-esterification in D6D-inhibited cells, independent of n-3 PUFA changes. To provide further evidence, we treated cells with the D6D inhibitor in the presence or absence of EPA and compared them with ALA-treated cells. Although EPA further reduced TAG content, the reduced markers of fatty acid re-esterification were not affected by ALA or EPA. Collectively, this study provides new insight showing that D6D inhibition reduces TAG accumulation and fatty acid re-esterification in adipocytes independent of changes in n-3 PUFA cellular content.     

ELOVL2 overexpression enhances triacylglycerol synthesis in 3T3-L1 and F442A cells

Elongation of very long-chain fatty acids (ELOVL) members were overexpressed in two preadipocyte cell lines, ELOVL2 and ELOVL3 in 3T3-L1 cells, and ELOVL1-3 in F442A cells. Cells overexpressing ELOVL2, whose preferred substrates are arachidonic acid (AA, C20:4n-6) and eicosapentaenoic acid (EPA, C20:5n-3), showed an enhanced triacylglycerol (TAG) synthesis and subsequent accumulation of lipid droplets. Incorporation of fatty acid (FA) but not of glucose into TAG was enhanced by ELOVL2-overexpression. Two lipogenic genes encoding diacylglycerol acyltransferase-2 (DGAT2) and fatty acid-binding protein-4 (FABP4, aP2) were induced in ELOVL2-overexpressing cells, whereas no such effect was seen on the fatty acid synthase (FAS) gene.     

Multiphasic triacylglycerol dynamics in the intact heart during acute in vivo overexpression of CD36

Cardiac triacylglycerol (TAG) stores buffer the intracellular availability of long chain fatty acid (LCFA) that act as nuclear receptor ligands, substrate for lipotoxic derivatives, and high energy-yield fuel. The kinetic characteristics of TAG turnover and homeostatic mechanisms linking uptake and storage dynamics in hearts have until now remained elusive. This work examines TAG pool dynamics in the intact beating heart, under normal conditions and in response to acute gene expression-induced changes in CD36. Dynamic mode ¹³C NMR elucidated multiple kinetic processes in ¹³C-palmitate incorporation into TAG: an initial, saturable exponential component and a slower linear rate. Although previous work indicates the linear component to reflect TAG turnover, we hypothesized the saturable exponential to reflect transport of LCFA across the sarcolemma. Thus, we overexpressed the LCFA transporter CD36 through cardiac-specific adenoviral infection in vivo. Within 72 h, CD36 expression was increased 40% in intact hearts, accelerating the exponential phase relative to PBS-infused hearts. TAG turnover also increased with elevations in adipose triglyceride lipase (ATGL) and a modest increase in diacylglycerol acyltransferase 1 (DGAT1), without a significant expansion of the intracellular lipid pools. The results demonstrate a dynamic system of reciprocal gene regulation that couples saturable LCFA uptake across the sarcolemma to TAG synthesis/lipolysis rates.     

Diets enriched in menhaden fish oil, seal oil, or shark liver oil have distinct effects on the lipid and fatty-acid composition of guinea pig heart

The purpose of this investigation was to determine whether diets supplemented with oils from three different marine sources, all of which contain high proportions of long-chain n-3 polyunsaturated fatty acids (PUFA), result in qualitatively distinct lipid and fatty acid profiles in guinea pig heart. Albino guinea pigs (14 days old) were fed standard, nonpurified guinea pig diets (NP) or NP supplemented with menhaden fish oil (MO), harp seal oil (SLO) or porbeagle shark liver oil (PLO) (10%, w/w) for 4-5 weeks. An n-6 PUFA control group was fed NP supplemented with corn oil (CO). All animals appeared healthy, with weight gains marginally lower in animals fed the marine oils. Comparison of relative organ weights indicated that only the livers responded to the diets, and that they were heavier only in the marine-oil fed guinea pigs. Heart total cholesterol levels were unaffected by supplementing NP with any of the oils, whereas all increased the triacylglycerol (TAG) content. The fatty-acid profiles of totalphospholipid (TPL), TAG and free fatty acid (FFA) fractions of heart lipids showed that feeding n-3 PUFA significantly altered the proportions of specific fatty-acid classes. For example, all marine-oil-rich diets were associated with increases in total monounsaturated fatty acids in TPL (p < 0.05), and with decreases in total saturates in TAG (p < 0.05). Predictably, the n-3 PUFA enriched regimens significantly increased the cardiac content of n-3 PUFA and decreased that of n-6 PUFA, although the extent varied among the diets. As a result, n-6/n-3 ratios were significantly lower in all myocardial lipid classes of marine-oil-fed guinea pigs. Analyses of the profiles of individual PUFA indicated that quantitatively, the fatty acids of the three marine oils were metabolized and/or incorporated into TPL, TAG and FFA in a diet-specific manner. In animals fed MO-enriched diets in which eicosapentaenoic acid (EPA) > docosahexacnoic acid (DHA), ratios of DHA /EPA in the hearts were 1.2, 2.2 and 1.5 in TPL, TAG and FFA, respectively. In SLO-fed guinea pigs in which dietary EPA DHA, ratios of DHA/EPA were 0.9, 3.4 and 2.1 in TPL, TAG and FFA, respectively. Feeding NP + PLO (DHA/EPA = 4.8), resulted in values for DHA/EPA in cardiac tissue of 2.1, 10.6 and 2.9 in TPL, TAG and FFA, respectively. In the TAG and FFA, proportions of n-3 docosapentaenoic acid (n-3 DPA) were equal to or higher than EPA in the SLO- and PLO-fed animals. The latter group exhibited the greatest difference between the DHA/n-3 DPA ratio in the diet and in cardiac TAG and FFA fractions (7, 3.4 and 3.1, respectively). Quantitative analysis indicated that 85% of the n-3 PUFA were in TPL, 7-11% were in TAG, and 2-6% were FFA. Specific patterns of distribution of EPA, DPA and DHA depended on the dietary oil. Both the qualitative and quantitative results of this study demonstrated that in guinea pigs, n-3 PUFA in different marine oils are metabolized and/or incorporated into cardiac lipids in distinct manners. In support of the concept that the diet-induced alterations reflect changes specifically in cardiomyocytes, we observed that direct supplementation of cultured guinea pig myocytes for 2-3 weeks with EPA or DHA produced changes in the PUFA profiles of their TPL that were qualitatively similar to those observed in tissue from the dietary study. The factors that regulate specific deposition of n-3 PUFA from either dietary oils or individual PUFA are not yet known, however the differences that we observed could in some manner be related to cardiac function and thus their relative potentials as health-promoting dietary fats.     

Production of structured triacylglycerols (STAG) rich in docosahexaenoic acid (DHA) in position 2 by acidolysis of tuna oil catalyzed by lipases

This work deals with the production of structured triacylglycerols (STAG) with caprylic acid (CA) located in positions 1 and 3 of the molecule of glycerol and docosahexaenoic acid (DHA) in position 2, by acidolysis of tuna oil and CA, catalyzed by several lipases. To this end several lipases and immobilization supports were tested with the aim of avoiding the acyl-migration observed in previous works. The determination of the best catalyst (i.e. the lipase and the immobilization support as a whole) was carried out by experiments of acidolysis of cod liver oil and CA in a bath reactor. The best results were obtained with the lipases from Rhizopus oryzae (Lipase D) and Rhizopus delemar (Lipase Rd), immobilized on Accurel MP1000 (a microporous polypropylene) with a lipase/support ratio 1:1.5 (w/w). The activity of these immobilized lipases was stable for a minimum of 5 days in the operational conditions (up to 40 °C).Lipase Rd was selected for the next step in which it was immobilized on Acurrel MP1000 to obtain STAG enriched in DHA by acidolysis of tuna oil (20% DHA) with CA. The experiments were carried out by recirculating the reaction mixture through an immobilized lipase packed bed reactor at different substrate/hexane ratios, as well as in absence of solvent. In the latter case, STAG with 51% CA and 13% DHA were obtained at 73 h. This result indicates that with this catalyst an acceptable reaction rate was attained in absence of solvent. A structural analysis by the pancreatic lipase method carried out to STAG with 45% CA and 16% DHA indicated that 91% of the CA incorporated is located in positions 1 and 3, and that 51% of the DHA is located in position 2 (MLM structure). This position is also rich in palmitic, eicosapentaenoic and oleic acids.After the acidolysis reaction a mixture of STAG and free fatty acids was obtained. The recovery of STAG from this reaction mixture is difficult because of the high content of free fatty acids. A separation method based on the neutralization of the free fatty acids with a KOH hydroalcoholic solution has been developed. By this procedure pure (100%) STAG were obtained with a recovery yield of 80%.     

Response surface modeling of 1-stearoyl-3(2)-oleoyl glycerol production in a pilot packed-bed immobilized Rhizomucor miehei lipase reactor

A dual response approach using diacylglycerol (DAG) and triacylglycerol (TAG) as responses for optimization of 1-stearoyl-3(2)-oleoyl glycerol-enriched DAG synthesis using response surface methodology (RSM) was investigated. Four variables from a lipase-catalyzed esterification reaction were optimized using a central composite rotatable design. The following optimized conditions yielded 51 wt.% DAG and 22 wt.% TAG: reaction temperature of 55 °C, enzyme dosage of 9.5 wt.%, fatty acid/glycerol molar ratio of 2.1 and reaction time of 3 h. Results were repeatable at 10 kg production scale in a pilot packed-bed enzyme reactor. No significant losses in enzyme activity or changes in fatty acid selectivity on DAG synthesis were observed during the five pilot productions. Lipozyme RM IM showed selectivity towards the production of stearic acid enriched DAG. The purity of DAG oil after purification was 90 wt.%.     

Changes in fatty acids metabolism during differentiation of Atlantic salmon preadipocytes; effects of n-3 and n-9 fatty acids

Atlantic salmon (Salmo salar) preadipocytes, isolated from visceral adipose tissue, differentiate from an unspecialized fibroblast like cell type to mature adipocytes filled with lipid droplets in culture. The expression of the adipogenic gene markers peroxisome proliferated activated receptor (PPAR) alpha, lipoprotein lipase (LPL), microsomal triglyceride transfer protein (MTP), fatty acid transport protein (FATP) 1 and fatty acid binding protein (FABP) 3 increased during differentiation. In addition, we describe a novel alternatively spliced form of PPARgamma (PPARgamma short), the expression of which increased during differentiation. Eicosapentaenoic acid (20:5n-3, EPA) and docosahexaenoic acid (22:6n-3, DHA) lowered the triacylglycerol (TAG) accumulation in mature salmon adipocytes compared to oleic acid (18:1n-9, OA). This finding indicates that a reduced level of highly unsaturated n-3 fatty acids (HUFAs) in fish diets, when the traditional marine oil is exchanged for n-9 fatty acids (FAs) rich vegetable oils (VOs), may influence visceral fat deposition in salmonids. Moreover, major differences in the metabolism of EPA, DHA and OA at different stages during differentiation of adipocytes occur. Most of the EPA and DHA were oxidized in preadipocytes, while they were mainly stored in TAGs in mature adipocytes in contrast to OA which was primarily stored in TAGs at all stages of differentiation.     

Acidolysis of sardine oil by lipase to concentrate eicosapentaenoic and docosahexaenoic acids in glycerides

Lipoprotein lipase from Pseudomonas sp. was the best enzyme to concentrate eicosapentaenoic and docosahexaenoic acids (EPA and DHA) in sardine oil by acidolysis reaction, and acetone was more effective than n-hexane as a solvent for dissolving the reactants and concentrating the two fatty acids. The water concentration in the reaction mixture was a decisive factor governing the enrichment of EPA and DHA and the yield of glycerides. EPA and DHA were more concentrated, but the yield of glycerides decreased, when the water concentration was increased gradually. Thus, the concentration rates of both the fatty acids were low with 0.25% water, although a considerable amount of diglyceride was detectable in the reaction products. The effect of reaction temperature was very slight with the use of acetone; however, the ratio DHA/EPA increased when the temperature was lowered in the presence of n-hexane. When acidolysis was performed at 25°C for 1 h, using 10,000 units of lipase per g of the reactants, the total percentage of EPA and DHA reached 65% in the glycerides and the recoveries of the two acids were 87.4 and 81.3%, respectively, based on the contents in the original sardine oil. The relationship of the enzyme substrate specificity to the reaction results was also investigated.     

Fatty acid oxidation and related gene expression in heart depleted of carnitine by mildronate treatment in the rat

The metabolic and genic effects induced by a 20-fold lowering of carnitine content in the heart were studied in mildronate-treated rats. In the perfused heart, the proportion of palmitate taken up then oxidized was 5-10% lower, while the triacylglycerol (TAG) formation was 100% greater than in controls. The treatment was shown to increase the maximal capacity of heart homogenates to oxidize palmitate, the mRNA level of carnitine palmitoyltransferase I (CPT-I) isoforms, the specific activity of CPT-I in subsarcolemmal mitochondria and the total carnitine content of isolated mitochondria. Concomitantly, the increased mRNA expression of lipoprotein lipase, fatty acid translocase and enzymes of TAG synthesis was associated with a 5- and 2-times increase in serum TAG and free fatty acid contents, respectively. The compartmentation of carnitine at its main functional location was expected to allow the increased CPT-I activity to ensure in vivo correct fatty acid oxidation rates. All the inductions related to fatty acid transport, oxidation and esterification most likely stem from the abundance of blood lipids providing cardiomyocytes with more fatty acids.     

Docosapentaenoic acid (22:5n-3) down-regulates the expression of genes involved in fat synthesis in liver cells

Previous studies have shown that Eicosapentaenoic Acid (EPA) and Docosahexaenoic Acid (DHA) exhibit triacylglycerol (TAG) lowering effect in vitro and in vivo by down-regulating the Sterol Regulating Element Binding Protein (SREBP-1c) and reducing the expression levels of lipogenic genes. However, there is no evidence on the effect of Docosapentaenoic Acid (DPA) on SREBP-1c expression levels. DPA is a long chain n-3 fatty acid present in our diet through fish, red meat and milk of ruminant animals. Therefore, this study aimed to elucidate the effect of DPA on liver fatty acid synthesis in an in vitro model using rat liver cells. Our results suggested that DPA incubation (50μM) for 48h (like EPA and DHA) caused a significant decrease in the mRNA expression levels of SREBP-1c, 3-Hydroxy-3-Methyl-Glutaryl-Coenzyme A reductase (HMG-CoA reductase), Acetyl Coenzyme A Carboxylase (ACC-1) and Fatty Acid Synthase (FASn) compared with Oleic Acid (OA) and also a decrease in the protein levels of SREBP-1 and ACC-1. A time-course fatty acid analysis showed that DPA and EPA are interconvertable in the cells; however, after 8h of incubation with DPA, the cell phospholipids contained mainly DPA. The gene expression profiling of the lipogenic genes repeated at 8h confirmed that the inhibitory effect of DPA on mRNA expression levels of the lipogenic genes was most likely due to DPA itself and not due to its conversion into EPA.     

Isolation of quizalofop-resistant mutants of Nannochloropsis oculata (Eustigmatophyceae) with high eicosapentaenoic acid following N-methyl-N-nitrosourea-induced random mutagenesis

Nannochloropsis oculata was subjected to N-methyl-N-nitrosourea-induced mutagenesis under the selection pressure of quizalofop, a known inhibitor of acetyl-CoA carboxylase (ACCase) activity with the objective of generating genetically tractable mutants with altered fatty acid metabolism. Two mutants, QUIZ1 and QUIZ2, with stable resistance to quizalofop were isolated and partially characterized. The growth properties and morphology of the mutants appeared identical with the parent strain. However thermo-tolerance was observed in the mutants. Enhanced resistance to quizalofop suggested the presence of herbicide resistant isoforms of ACCase. In vitro assays for ACCase activity showed that ACCase in the wild strains was much more sensitive to quizalofop than the mutant strains. Gas chromatographic analysis of fatty acids revealed that the mutant strains were rich in polyunsaturated fatty acids (n– 3PUFAs), as well as total fatty acid contents; this was accompanied by a concomitant increase in triacylglycerol (TAG) followed by linoleic acid (18:2), arachidonic acid (20:4 n– 6) and EPA (20:5 n– 3). These results suggest that an increased substrate pool (malonyl-CoA) (due to increased specific activity of ACCase) in the mutant strains in vivo and in vitro may have led to the increased TAG accumulation. Random mutagenesis was shown to be a good tool to manipulate PUFAs and EPA in Nannochloropsis. The strains developed will be useful in understanding fatty acid metabolism using genetic and biochemical approaches and also for their direct use in mariculture.     

Kinetics modeling of the acidolysis with immobilized Rhizomucor miehei lipases for production of structured lipids from sunflower oil

Sunflower oil modification for production of semisolid fats was carried out via acidolysis using palmitic and stearic acids (P + St), hexane and a developed biocatalyst from Rhizomucor miehei lipases. Its kinetic behavior was studied by employing three mathematical models proposed in the literature. Furthermore, a new model was proposed to describe not only the variation of triacylglycerols (TAG), diacylglycerols (DAG), and free fatty acids groups but also the acyl migration reaction occurrence. The effect of the reaction temperature on the kinetic and equilibrium parameters, as well as TAG and reaction intermediates profiles was analyzed. Increasing reaction temperature generated major changes in the overall composition of acylglycerols and gave rise to the highest composition of P + St in the obtained structured lipids (58%, 70 h, 60 °C). P + St incorporation was successfully adjusted by an empirical model (Model I) and a lumped parameter model (Model II) for all the studied reaction times, while the model based on a Ping Pong Bi Bi mechanism (Model III) was only able to describe the kinetics behavior (through the variation of reactant saturated fatty acids concentration) until 24 h. Experimental data were fit satisfactorily by the proposed model (Model IV), showing that the increment in the disaturated TAG formation achieved by the increment in temperature was principally related to the favored DAG formation from triunsaturated TAG.     

The impact of seasonal alteration in the lipid composition of broodstock diets on egg quality in the European sea bass

Groups of European sea bass Dicentrarchus labrax L., broodstock were fed two pelleted, lipid-enriched diets [maize oil, low essential fatty acid (EFA) or fish oil, high EFA]. Those fed the high EFA diet for 12 months or only during vitellogenesis (September to February) with the low EFA diet fed for the remaining 6 months, showed improved egg quality and higher hatching rates when compared to two groups fed the high EFA diet during either previtel-logenesis (February to September) or the spawning period (February to April). The higher quality eggs contained higher levels of total n-3 fatty acids, including increased levels of both docosahexaenoic acid (DHA) and eicosapaentaenoic acid (EPA). High levels of triacylglycerol (TAG) were linked with the poorer quality eggs from those groups fed the low EFA diet during the period of vitellogenesis. The present work identifies vitellogenesis as the period in the maturation during which EFA are incorporated most effectively into the developing oocytes.     

Docosahexaenoic acid is selectively enriched in plasma phospholipids during pregnancy in Trinidadian women--results of a pilot study

The fetal demand for docosahexaenoic acid (DHA) has to be satisfied by the mother. We determined the fatty acids in maternal plasma non-esterified fatty acid (NEFA), triacylglycerol (TAG) and phosphatidylcholine (PC), in a cross-sectional study of non-pregnant (n = 10), pregnant (n = 19), and postpartum (n = 9) women. There were lipid class-dependent differences in plasma polyunsaturated fatty acid (PUFA) concentrations between groups. During pregnancy, DHA was most highly enriched in PC, about 230%, with more modest enrichment for linoleic acid (LA) and arachidonic acid (AA), and no enrichment of alpha-linolenic acid (alpha-LNA). There was relative enrichment of LA, AA and alpha-LNA in TAG, but not of DHA. There was no specific enrichment of any PUFA in the NEFA pool. These data accord with the suggestion that the enrichment of alpha-LNA in TAG and of DHA in phospholipids reflects hepatic regulation of n-3 PUFA metabolism which potentially enhances the delivery of DHA to the placenta.     

Mycobacterium tuberculosis uses host triacylglycerol to accumulate lipid droplets and acquires a dormancy-like phenotype in lipid-loaded macrophages

Two billion people are latently infected with Mycobacterium tuberculosis (Mtb). Mtb-infected macrophages are likely to be sequestered inside the hypoxic environments of the granuloma and differentiate into lipid-loaded macrophages that contain triacylglycerol (TAG)-filled lipid droplets which may provide a fatty acid-rich host environment for Mtb. We report here that human peripheral blood monocyte-derived macrophages and THP-1 derived macrophages incubated under hypoxia accumulate Oil Red O-staining lipid droplets containing TAG. Inside such hypoxic, lipid-loaded macrophages, nearly half the Mtb population developed phenotypic tolerance to isoniazid, lost acid-fast staining and accumulated intracellular lipid droplets. Dual-isotope labeling of macrophage TAG revealed that Mtb inside the lipid-loaded macrophages imports fatty acids derived from host TAG and incorporates them intact into Mtb TAG. The fatty acid composition of host and Mtb TAG were nearly identical suggesting that Mtb utilizes host TAG to accumulate intracellular TAG. Utilization of host TAG by Mtb for lipid droplet synthesis was confirmed when fluorescent fatty acid-labeled host TAG was utilized to accumulate fluorescent lipid droplets inside the pathogen. Deletion of the Mtb triacylglycerol synthase 1 (tgs1) gene resulted in a drastic decrease but not a complete loss in both radiolabeled and fluorescent TAG accumulation by Mtb suggesting that the TAG that accumulates within Mtb is generated mainly by the incorporation of fatty acids released from host TAG. We show direct evidence for the utilization of the fatty acids from host TAG for lipid metabolism inside Mtb. Taqman real-time PCR measurements revealed that the mycobacterial genes dosR, hspX, icl1, tgs1 and lipY were up-regulated in Mtb within hypoxic lipid loaded macrophages along with other Mtb genes known to be associated with dormancy and lipid metabolism.