Showing papers in "Lipids in 2004"

Modifying milk fat composition of dairy cows to enhance fatty acids beneficial to human health

Abstract: There is increased consumer awareness that foods contain microcomponents that may have beneficial effects on health maintenance and disease prevention. In milk fat these functional food components include EPA, DHA, and CLA. The opportunity to enhance the content of these FA in milk has improved as a result of recent advances that have better defined the interrelationships between rumen fermentation, lipid metabolism, and milk fat synthesis. Dietary lipids undergo extensive hydrolysis and biohydrogenation in the rumen. Milk fat is predominantly TG, and de novo FA synthesis and the uptake of circulating FA contribute nearly equal amounts (molar basis) to the FA in milk fat. Transfer of dietary EPA and DHA to milk fat is very low (<4%); this is, to a large extent, related to their extensive biohydrogenation in the rumen, and also partly due to the fact that they are not transported in the plasma lipid fractions that serve as major mammary sources of FA uptake (TG and nonesterified FA). Milk contains over 20 isomers of CLA but the predominant one is cis-9,trans-11 (75-90% of total CLA). Biomedical studies with animal models have shown that this isomer has anticarcinogenic and anti-atherogenic activities. cis-9,trans-11-CLA is produced as an intermediate in the rumen biohydrogenation of linoleic acid but not of linolenic acid. However, it is only a transient intermediate, and the major source of milk fat CLA is from endogenous synthesis. Vaccenic acid, produced as a rumen biohydrogenation intermediate from both linoleic acid and linolenic acid, is the substrate, and delta9-desaturase in the mammary gland and other tissues catalyzes the reaction. Diet can markedly affect milk fat CLA content, and there are also substantial differences among individual cows. Thus, strategies to enhance milk fat CLA involve increasing rumen outflow of vaccenic acid and increasing delta9-desaturase activity, and through these, several-fold increases in the content of CLA in milk fat can be routinely achieved. Overall, concentrations of CLA, and to a lesser extent EPA and DHA, can be significantly enhanced through the use of diet formulation and nutritional management of dairy cows.

What's so special about cholesterol?

Abstract: Cholesterol (or other higher sterols such as ergosterol and phytosterols) is universally present in large amounts (20-40 mol%) in eukaryotic plasma membranes, whereas it is universally absent in the membranes of prokaryotes. Cholesterol has a unique ability to increase lipid order in fluid membranes while maintaining fluidity and diffusion rates. Cholesterol imparts low permeability barriers to lipid membranes and provides for large mechanical coherence. A short topical review is given of these special properties of cholesterol in relation to the structure of membranes, with results drawn from a variety of theoretical and experimental studies. Particular focus is put on cholesterol's ability to promote a special membrane phase, the liquid-ordered phase, which is unique for cholesterol (and other higher sterols like ergosterol) and absent in membranes containing the cholesterol precursor lanosterol. Cholesterol's role in the formation of special membrane domains and so-called rafts is discussed.

Resolvins, docosatrienes, and neuroprotectins, novel omega-3-derived mediators, and their endogenous aspirin-triggered epimers

Abstract: The molecular basis for the beneficial impact of essential omega-3 (n-3) FA remains of interest. Recently, we identified novel mediators generated from eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) that displayed potent bioactions identified first in resolving inflammatory exudates and in tissues enriched with DHA. The trivial names resolvin (resolution phase interaction products) and docosatrienes were introduced for the bioactive compounds from these novel series since they possess potent anti-inflammatory and immunoregulatory actions. Compounds derived from EPA carrying potent biological actions (i.e., 1-10 nM range) are designated E series and denoted resolvins of the E series (resolvin E1 or RvE1), and those biosynthesized from the precursor DHA are denoted resolvins of the D series (resolvin D1 or RvD1). The number 1 designates the bioactive compounds in this family (#1-4). Bioactive members from DHA-containing conjugated triene structures or docosatrienes (DT) that possess immunoregulatory and neuroprotective actions were termed neuroprotectins. Aspirin treatment initiates a related epimeric series by triggering endogenous formation of the 17R-D series resolvins and docosatrienes. These epimers are denoted as aspirin-triggered (AT)-RvD and DT, and possess potent anti-inflammatory actions in vivo essentially equivalent to their 17S series pathway products. These include five distinct series: (i) 18R resolvins from EPA (i.e., RvE1); (ii) 17R series (AT) resolvins from DHA (RvD1 through RvD4); (iii) 17S series resolvins from DHA (RvD1 through RvD4), (iv) DT from DHA; and (v) their AT form 17R series DT. In this article, we provide an overview of the formation and actions of these newly uncovered pathways and products.

Omega-3 PUFA of marine origin limit diet-induced obesity in mice by reducing cellularity of adipose tissue

Abstract: Omega-3 PUFA of marine origin reduce adiposity in animals fed a high-fat diet. Our aim was to learn whether EPA and DHA could limit development of obesity and reduce cellularity of adipose tissue and whether other dietary FA could influence the effect of EPA/DHA. Weight gain induced by composite high-fat diet in C57BL/6J mice was limited when the content of EPA/DHA was increased from 1 to 12% (wt/wt) of dietary lipids. Accumulation of adipose tissue was reduced, especially of the epididymal fat. Low ratio of EPA to DHA promoted the effect. A higher dose of EPA/DHA was required to reduce adiposity when admixed to diets that did not promote obesity, the semisynthetic high-fat diets rich in EFA, either alpha-linolenic acid (ALA, 18:3 n-3, the precursor of EPA and DHA) or linoleic (18:2 n-6) acid. Quantification of adipose tissue DNA revealed that except for the diet rich in ALA the reduction of epididymal fat was associated with 34-50% depression of tissue cellularity, similar to the 30% caloric restriction in the case of the high-fat composite diet. Changes in plasma markers and adipose gene expression indicated improvement of lipid and glucose metabolism due to EPA/DHA even in the context of the diet rich in ALA. Our results document augmentation of the antiadipogenic effect of EPA/DHA during development of obesity and suggest that EPA/DHA could reduce accumulation of body fat by limiting both hypertrophy and hyperplasia of fat cells. Increased dietary intake of EPA/DHA may be beneficial regardless of the ALA intake.

Replacement of dietary fish oil with increasing levels of linseed oil: Modification of flesh fatty acid compositions in Atlantic salmon (Salmo salar) using a fish oil finishing diet

Abstract: Five groups of salmon, of initial mean weight 127±3 g, were fed increasing levels of dietary linseed oil (LO) in a regression design. The control diet contained capelin oil (FO) only, and the same oil was blended with LO to provide the experimental diets. After an initial period of 40 wk, all groups were switched to a finishing diet containing only FO for a further 24 wk. Growth and flesh lipid contents were not affected by dietary treatment. The FA compositions of flesh total lipids were linearly correlated with dietary FA compositions (r 2=0.88–1.00, P<0.0001). LO included at 50% of added dietary lipids reduced flesh DHA and EPA (20∶5n−3) concentrations to 65 and 58%, respectively, of the concentrations in fish fed FO. Feeding 100% LO reduced flesh DHA and EPA concentrations to 38 and 30%, respectively, of the values in fish fed FO. Differences between diet and flesh FA concentrations showed that 16∶0, 18∶1n−9, and especially DHA were preferentially retained in flesh, whereas 18∶2n−6, 18∶3n−3, and 22∶1n−11 were selected against and presumably utilized for energy. In fish previously fed 50 and 100% LO, feeding a finishing diet containing FO for 16 wk restored flesh DHA and EPA concentrations, to ≈80% of the values in fish fed FO throughout. Flesh DHA and EPA concentrations in fish fed up to 50% LO were above recommended intake values for humans for these EFA. This study suggests that LO can be used as a substitute for FO in seawater salmon feeds and that any reductions in DHA and EPA can be largely overcome with a finishing diethigh in FO before harvest.

Isoprenoids: Remarkable diversity of form and function

Abstract: The isoprenoid biosynthetic pathway is the source of a wide array of products. The pathway has been highly conserved throughout evolution, and isoprenoids are some of the most ancient biomolecules ever identified, playing key roles in many life forms. In this review we focus on C-10 mono-, C-15 sesqui-, and C-20 diterpenes. Evidence for interconversion between the pathway intermediates farnesyl pyrophosphate and geranylgeranly pyrophosphate and their respective metabolites is examined. The diverse functions of these molecules are discussed in detail, including their ability to regulate expression of the β-HMG-CoA reductase and Ras-related proteins. Additional topics include the mechanisms underlying the apoptotic effects of select isoprenoids, antiulcer activities, and the disposition and degradation of isoprenoids in the environment. Finally, the significance of pharmacological manipulation of the isoprenoid pathway and clinical correlations are discussed.

Dietary intakes and food sources of n-6 and n-3 PUFA in French adult men and women.

Abstract: The intake of individual n−6 and n−3 PUFA has been estimated in 4,884 adult subjects (2,099 men and 2,785 women), volunteers from the French SU.VI.MAX intervention trial. The food intakes of each subject were recorded in at least ten 24-h record questionnaires completed over a period of 2.5 yr, allowing the estimation of the daily intake of energy; total fat; and linoleic, α-linolenic, arachidonic, eicosapentaenoic (EPA), n−3 docosapentaenoic (DPA), and docosahexaenoic (DHA) acids. The mean total fat intake corresponded to 94.1 g/d (36.3% of total energy intake) in men and 73.4 g/d (38.1% of energy) in women. The intake of linoleic acid was 10.6 g/d in men and 8.1 g/d in women, representing 4.2% of energy intake; that of α-linolenic acid was 0.94 g/d in men and 0.74 g/d in women, representing 0.37% of energy intake, with a mean linoleic/α-linolenic acid ratio of 11.3. The mean intakes of long-chain PUFA were: arachidonic acid, 204 mg/d in men and 152 mg/d in women; EPA, 150 mg/d in men and 118 mg/d in women; DPA, 75 mg/d in men and 56 mg/d in women; DHA, 273 mg/d in men and 226 mg/d in women; long-chain n−3 PUFA, 497 mg/d in men and 400 mg/d in women. Ninety-five percent of the sample consumed less than 0.5% of energy as α-linolenic acid, which is well below the current French recommendation for adults (0.8% of energy). In contrast, the mean intakes of long-chain n−6 and n−3 PUFA appear fairly high and fit the current French recommendations (total long-chain PUFA: 500 mg/d in men and 400 mg/d in women; DHA: 120 mg/d in men and 100 mg/d in women). The intakes of α-linolenic acid, and to a lesser extent of linoleic acid, were highly correlated with that of lipids. Whereas the main source of linoleic acid was vegetable oils, all food types contributed to α-linolenic acid intake, the main ones being animal products (meat, poultry, and dairy products). The main source of EPA and DHA (and of total long-chain n−3 PUFA) was fish and seafood, but the major source of DPA was meat, poultry, and eggs. Fish and seafood consumption showed very large interindividual variations, the low consumers being at risk of insufficient n−3 PUFA intake.

Mechanisms of regulation of gene expression by fatty acids

Abstract: Fatty acids (FA) regulate the expression of genes involved in lipid and energy metabolism. In particular, two transcription factors, sterol regulatory element binding protein-1c (SREBP-1 c) and peroxisome proliferator activated receptor α (PPARα), have emerged as key mediators of gene regulation by FA. SREBP-1 c induces a set of lipogenic enzymes in liver. Polyunsaturated fatty acids (PUFA), but not saturated or mono-unsaturated FA, suppress the induction of lipogenic genes by inhibiting the expression and processing of SREBP-1c. This unique effect of PUFA suggests that SREBP-1c may regulate the synthesis of unsaturated FA for incorporation, into glycerolipids and cholesteryl esters. PPARα plays an essential role in metabolic adaptation to fasting by inducing the genes for mitochondrial and peroxisomal FA oxidation as well as those for ketogenesis in mitochondria. FA released from adipose tissue during fasting are considered as ligands of PPARα. Dietary PUFA, except for 18∶2 n′6, are likely to induced FA oxidation enzymes via PPARα as “feed-forward” mechanism. PPARα is also required for regulating the synthesis of highly unsaturated FA, indicating pleiotropic functions of PPARα in the regulation of lipid metabolic pathways. It is yet to be determined whether FA regulate other transcription factors such as liver-X receptor, hepatocyte nuclear factor 4, and carbohydrate response element binding protein.

Structure and function of animal fatty acid synthase

Abstract: Fatty acid synthase (FAS; EC 2.3.1.85) of animal tissues is a complex multifunctional enzyme consisting of two identical monomers. The FAS monomer (∼270 kDa) contains six catalytic activities and from the N-terminus the order is β-ketoacyl synthase (KS), acetyl/malonyl transacylase (AT/MT), β-hydroxyacyl dehydratase (DH), enoyl reductase (ER), β-ketoacyl reductase (KR), acyl carrier protein (ACP), and thioesterase (TE). Although the FAS monomer contains all the activities needed for palmitate synthesis, only the dimer form of the synthase is functional. Both the biochemical analyses and the small-angle neutron-scattering analysis determined that in the dimer form of the enzyme the monomers are arranged in a head-to-tail manner generating two centers for palmitate synthesis. Further, these analyses also suggested that the component activities of the monomer are organized in three domains. Domain I contains KS, AT/MT, and DH, domain II contains ER, KR, and ACP, and domain III contains TE. Approximately one fourth of the monomer protein located between domains I and II contains no catalytic activities and is called the interdomain/core region. This region plays an important role in the dimer formation. Electron cryomicrographic analyses of FAS revealed a quaternary structure at approximately 19 A resolution, containing two monomers (180×130×75 A) that are separated by about 19 A, and arranged in an antiparallel fashion, which is consistent with biochemical and neutron-scattering data. The monomers are connected at the middle by a hinge generating two clefts that may be the two active centers of fatty acid synthesis. Normal mode analysis predicted that the intersubunit hinge region and the intrasubunit hinge located between domains II and III are highly flexible. Analysis of FAS particle images by using a simultaneous multiple model single particle refinement method confirmed that FAS structure exists in various conformational states. Attempts to get higher resolution of the structure are under way.

n-3 fatty acids, inflammation, and immunity--relevance to postsurgical and critically ill patients.

Abstract: Excessive or inappropriate inflammation and immunosuppression are components of the response to surgery, trauma, injury, and infection in some individuals and these can lead, progressively, to sepsis and septic shock. The hyperinflammation is characterized by the production of inflammatory cytokines, arachidonic acid-derived eicosanoids, and other inflammatory mediators, while the immunosuppression is characterized by impairment of antigen presentation and of T helper cell type-1 responses. Long-chain n−3 FA from fish oil decrease the production of inflammatory cytokines and eicosanoids. They act both directly (by replacing arachidonic acid as an eicosanoid substrate and by inhibiting arachidonic acid metabolism) and indirectly (by altering the expression of inflammatory genes through effects on transcription factor activation). Thus, long-chain n−3 FA are potentially useful anti-inflammatory agents and may be of benefit in patients at risk of developing sepsis. As such, an emerging application of n−3 FA is in surgical or critically ill patients where they may be added to parenteral or enteral formulas. Parenteral or enteral nutrition including n−3 FA appears to preserve immune function better than standard formulas and appears to partly prevent some aspects of the inflammatory response. Studies to date are suggestive of clinical benefits from these approaches, especially in postsurgical patients.

Maternal fish oil supplementation in lactation: effect on visual acuity and n-3 fatty acid content of infant erythrocytes.

Abstract: Studies on formula-fed infants indicate a beneficial effect of dietary DHA on visual acuity. Cross-sectional studies have shown an association between breast-milk DHA levels and visual acuity in breast-fed infants. The objective in this study was to evaluate the biochemical and functional effects of fish oil (FO) supplements in lactating mothers. In this double-blinded randomized trial, Danish mothers with habitual fish intake below the 50th percentile of the Danish National Birth Cohort were randomized to microencapsulated FO [1.3 g/d long-chain n-3 FA (n-3 LCPUFA)] or olive oil (OO). The intervention started within a week after delivery and lasted 4 mon. Mothers with habitual high fish intake and their infants were included as a reference group. Ninety-seven infants completed the trial (44 OO-group, 53 FO-group) and 47 reference infants were followed up. The primary outcome measures were: DHA content of milk samples (0, 2, and 4 mon postnatal) and of infant red blood cell (RBC) membranes (4 mon postnatal), and infant visual acuity (measured by swept visual evoked potential at 2 and 4 mon of age). FO supplementation gave rise to a threefold increase in the DHA content of the 4-mon milk samples (P < 0.001). DHA in infant RBC reflected milk contents (r = 0.564, P < 0.001) and was increased by almost 50% (P < 0.001). Infant visual acuity was not significantly different in the randomized groups but was positively associated at 4 mon with infant RBC-DHA (P = 0.004, multiple regression). We concluded that maternal FO supplementation during lactation did not enhance visual acuity of the infants who completed the intervention. However, the results showed that infants with higher RBC levels of n-3 LCPUFA had a better visual acuity at 4 mon of age, suggesting that n-3 LCPUFA may influence visual maturation.

Conjugated linoleic acid isomer effects in atherosclerosis: growth and regression of lesions.

Abstract: Conjugated linoleic acid (CLA), a mixture of positional and geometric isomers of octadecadienoic acid, has been shown to inhibit experimentally induced atherosclerosis in rabbits and also to cause significant regression of pre-established atheromatous lesions in rabbits. The two major CLA isomers (cis9,trans11 and trans10,cis12), now available at 90% purity, have been tested individually for their anti-atherogenic or lesion regression potency. The two major isomers and the mixture were fed for 90 d to rabbits fed 0.2% cholesterol. Atherosclerosis was inhibited significantly by all three preparations. The two CLA isomers and the isomer mix were also fed (1.0%) as part of a cholesterol-free diet for 90 d to rabbits bearing atheromatous lesions produced by feeding an atherogenic diet. A fourth group was maintained on a cholesterol-free diet. On the CLA-free diet atherosclerosis was exacerbated by 35%. Reduction of severity of atheromatous lesions was observed to the same extent in all three CLA-fed groups. The average reduction of severity in the three CLA-fed groups was 26±2% compared with the first control (atherogenic diet) and 46±1% compared with the regression diet. Insofar as individual effects on atherosclerosis were concerned, there was no difference between the CLA mix and the cis9,trans11 and trans10,cis12 isomers. They inhibit atherogenesis by 50% when fed as a component of a semipurified diet containing 0.2% cholesterol; and when fed as part of a cholesterol-free diet, they reduce established lesions by 26%. Reduction of atheromata to the observed extent by dietar means alone is noteworthy.

CLA isomers in milk fat from cows fed diets with high levels of unsaturated fatty acids.

Abstract: The concentrations of CLA isomers were determined by Ag+-HPLC in the milk fat of cows fed a control diet consisting of hay ad libitum and 15 kg of fodder beets or this diet supplemented with oilseeds containing either high levels of oleic acid (rapeseed), linoleic acid (sunflower seed), or α-linolenic acid (linseed). Highly significant (P≤0.001) correlations were found between the daily intakes of oleic acid and the concentration of the CLA isomer trans-7,cis-9 in milk fat; of linoleic acid and the CLA isomers trans-10,trans-12, trans-9,trans-11, trans-8,trans-10, trans-7,trans-9, trans-10,cis-12, cis-9,trans-11, trans-8,cis-10, and trans-7,cis-9; and of α-linolenic acid and the CLA isomers trans-12,trans-14, trans-11,trans-13, cis,trans/trans,cis-12,14, trans-11,cis-13, and cis-11,trans-13. CLA concentrations were also determined in the milk fat of cows grazing in the lowlands (600–650 m), the mountains (900–1210 m), and the highlands (1275–2120 m). The concentrations of many isomers were highest in milk fat from the highlands, but only three CLA isomers (cis-9,trans-11, trans-11,cis-13, and trans-8,cis-10) showed a nearly linear increase with elevation. Therefore, these three CLA isomers, and particularly the CLA isomer trans-11,cis-13, the second-most important CLA in milk fat from cows grazing at the three altitudes, could be useful indicators of milk products of Alpine origin.

Separation of vitamin E (tocopherol, tocotrienol, and tocomonoenol) in palm oil.

Abstract: Previous reports showed that vitamin E in palm oil consists of various isomers of tocopherols and tocotrienols [alpha-tocopherol (alpha-T), alpha-tocotrienol, gamma-tocopherol, gamma-tocotrienol, and delta-tocotrienol), and this is normally analyzed using silica column HPLC with fluorescence detection. In this study, an HPLC-fluorescence method using a C30 silica stationary phase was developed to separate and analyze the vitamin E isomers present in palm oil. In addition, an alpha-tocomonoenol (alpha-T1) isomer was quantified and characterized by MS and NMR. (alpha-T1 constitutes about 3-4% (40+/-5 ppm) of vitamin E in crude palm oil (CPO) and is found in the phytonutrient concentrate (350+/-10 ppm) from palm oil, whereas its concentration in palm fiber oil (PFO) is about 11% (430+/-6 ppm). The relative content of each individual vitamin E isomer before and after interesterification/transesterification of CPO to CPO methyl esters, followed by vacuum distillation of CPO methyl esters to yield the residue, remained the same except for alpha-T and gamma-T3. Whereas alpha-T constitutes about 36% of the total vitamin E in CPO, it is present at a level of 10% in the phytonutrient concentrate. On the other hand, the composition of gamma-T3 increases from 31% in CPO to 60% in the phytonutrient concentrate. Vitamin is present at 1160+/-43 ppm, and its concentrations in PFO and the phytonutrient concentrate are 4,040+/-41 and 13,780+/-65 ppm, respectively. The separation and quantification of alpha-T1 in palm oil will lead to more in-depth knowledge of the occurrence of vitamin E in palm oil.

Cloning and characterization of a cDNA encoding diacylglycerol acyltransferase from castor bean.

Abstract: The oil from castor seed (Ricinus communis) contains 90% ricinoleate, a hydroxy FA that is used in producing numerous industrial products. Castor diacylglycerol acyltransferase (RcDGAT) is a critical enzyme, as it catalyzes the terminal step in castor oil biosynthesis in which the products contain two or three ricinoleoyl moieties. We have isolated a cDNA encoding RcDGAT from developing castor seeds. Analysis of the sequence reveals that this cDNA encodes a protein of 521 amino acids with a molecular mass of 59.9 kDa. Although there are regions of high similarity to other plant DGAT coding sequences, there are sequences that distinguish it as well. Southern blot analysis suggests that the castor genome contains a single copy of RcDGAT. Analysis by reverse transcription-PCR reveals that the accumulation of the mRNA reaches its highest level at 19 d after pollination and declines thereafter. Expression of the full-length cDNA for RcDGAT in the yeast Saccharomyces cerevisiae strain INVSc1 results in sevenfold higher DGAT activity compared with controls. When different molecular species of DAG were provided as substrates to the microsomal mixture, the RcDGAT showed a greater preference to catalyze the transfer of oleate from [14C]oleoyl-CoA to diricinolein than to diolein and dipalmitolein. With the addition of 0.25 mM substrates, diricinolein gave 318 pmol/mg/min diricinoleoyloleoylglycerol (RRO), while diolein and dipalmitolein gave only about 195 pmol/mg/min of triolein (OOO) and 120 pmol/mg/min dipalmitoyleoylglycerol (PoPoO), respectively. This work will facilitate investigation of the role of RcDGAT in castor oil biosynthesis.

Effect of CLA on milk fat synthesis in dairy cows: comparison of inhibition by methyl esters and free fatty acids, and relationships among studies.

Abstract: CLA is a potent inhibitor of milk fat synthesis, as shown by investigations using mixtures of CLA isomers in FFA form. However, methyl esters of CLA can be initially formed in commercial synthesis, and their use in a supplement has certain manufacturing and cost advantages. Our objective was to compare abomasal infusion of methyl esters of CLA (ME-CLA) and FFA of CLA (FFA-CLA) on milk fat synthesis. Data were also combined with previous investigations to examine broader relationships between trans-10,cis-12 CLA and the reduction in milk fat. Three mid-lactation, rumen-fistulated Holstein cows were used in a 3×3 Latin square design. Treatments were (i) control, (ii) ME-CLA, and (iii) FFA-CLA. The ME-CLA and FFA-CLA treatments (4.2 g/d trans-10,cis-12 CLA) resulted in a comparable reduction in milk fat yield (38 and 39%, respectively) and pattern of reduction in individual FA. In contrast, milk yield, milk protein, and feed intake were unaltered by CLA treatment. Combining data across studies revealed strong correlations relating the reduction in milk fat yield to abomasal dose of trans-10,cis-12 CLA (R2=0.86), milk fat content of trans-10,cis-12 CLA (R2=0.93), and milk fat secretion of trans-10,cis-12 CLA (R2=0.82). Across studies, transfer efficiency of abomasally infused trans-10,cis-12 CLA into milk fat was relatively constant (22%; R2=0.94). Overall, ME-CLA and FFA-CLA were equally potent in reducing milk fat, and either form could be used to formulate a dietary supplement that would induce milk fat depression.

Regulation of stearoyl-CoA desaturase expression.

Abstract: Stearoyl-CoA desaturase (SCD) is a regulatory enzyme in lipogenesis, catalyzing the rate-limiting step in the overall de novo synthesis of monounsaturated FA, mainly oleate and palmitoleate from stearoyl- and palmitoyl-CoA, respectively. Oleate and palmitoleate are the major monounsaturated FA of membrane phospholipids, TG, wax esters, cholesterol esters, and alkyldiacylglycerol. Several SCD gene isoforms (SCD1, SCD2, SCD3, and SCD4) exist in mice, and two have been characterized in humans. SCD1 gene expression in liver cells is regulated by numerous stimuli including diet and hormones. We are interested in why SCD is such a highly regulated enzyme even though oleate, the major product of this enzyme, is one of the most abundant FA in the diet and is therefore readily available. Dietary oleate is also well known for its TG-lowering effects and, as a major component of olive oil, is expected to have beneficial effects. However, high SCD activity has been implicated in diabetes, obesity, atherosclerosis, and cancer in several animal models; therefore, the role that de novo oleate plays in these disease states has to be carefully evaluated. By using SCD1-/- mice, which are deficient in tissue oleate, we begin to learn more about the physiological role of SCD gene expression and oleate in normal and disease states.

Fatty acids of serine, ethanolamine, and choline plasmalogens in some marine bivalves

Abstract: The FA composition of glycerophospholipid (GPL) classes and subclasses was investigated in whole animals of three marine bivalve mollusks: the Japanese oyster Crassostrea gigas, the blue mussel Mytilus edulis, and the Manila clam Ruditapes philippinarum. Individual organs (gills, mantle, foot, siphon, and muscle) of the Manila clam also were examined. The PS plasmalogen (PSplsm), PE plasmalogen (PEplsm), and PC plasmalogen (PCplsm) subclasses were isolated by HPLC, and their individual FA compositions were examined using GC. Plasmalogen forms of PS and PE, when compared to their respective diacyl forms, were found to be specifically enriched with non-methylene-interrupted (NMI) FA (7,15–22∶2, 7, 13–22∶2, and their precursors) and 20∶1n−11 FA. Such a clear specific association was not found for PCplsm. Interestingly, this trend was most apparent in PSplsm, and the above FA were found to be, respectively, the predominant PUFA and monounsaturated FA in the PSplsm isolated from the three species. This specificity was maintained in all the analyzed organs of the Manila clam but varied in proportions: The highest level of plasmalogens, NMI FA, and 20∶1n−11 was measured in gills and the lowest was in muscle. These results represent the first comprehensive report on a FA composition of the PSplsm subclass isolated from mollusks. The fact that NMI FA and 20∶1n−11, which are thought to be biosynthesized FA, were mainly associated with aminophospholipid plasmalogens (PE and PS) is likely to have a functional significance in bivalve membranes.

Dietary fish oil dose- and time-response effects on cardiac phospholipid fatty acid composition.

Abstract: Fish consumption is associated with reduced cardiovascular mortality, and elevated myocardial long-chain n-3 polyunsaturated FA (PUFA) content is implicated in this cardioprotection. This study examined the dose and time responses for incorporation of n-3 PUFA into cellular membranes in rats fed fish oil (FO)-containing diets. For the time course study, rats were fed a 10% FO diet for periods ranging from 0 to 42 d, after which myocardial and erythrocyte membrane fatty acid composition was determined. For the dose response study, rats (n = 3) were fed 0, 1.25, 2.5, 5, or 10% FO for 4 wk, with myocardial, erythrocyte, and skeletal muscle membrane FA determined. Myocardial DHA (22:6n-3) levels doubled in 2 d, stabilizing at levels approximately 200% higher than control after 28 d feeding with 10% FO. By comparison, DHA levels doubled after 4 wk of 1.25% FO feeding. In myocardium and skeletal muscle, EPA (20:5n-3) levels remained low, but in erythrocytes EPA levels reached 50% of DHA levels. The n-3 PUFA were incorporated at the expense of n-6 PUFA in myocardium and skeletal muscle, whereas erythrocytes maintained arachidonic acid levels, and total n-3 PUFA incorporation was lower. This study shows that low doses of FO produce marked changes in myocardial DHA levels; maximal incorporation takes up to 28 d to occur; and while erythrocytes are a good indicator of tissue n-3 incorporation in stable diets, they vary greatly in their time course and pattern of incorporation.

A critical evaluation of Raman spectroscopy for the analysis of lipids: fatty acid methyl esters.

Abstract: The work presented here is aimed at determining the potential and limitations of Raman spectroscopy for fat analysis by carrying out a systematic investigation of C4−C24 FAME. These provide a simple, well-characterized set of compounds in which the effect of making incremental changes can be studied over a wide range of chain lengths and degrees of unsaturation. The effect of temperature on the spectra was investigated over much larger ranges than would normally be encountered in real analytical measurements. It was found that for liquid FAME the best internal standard band was the carbonyl stretching vibration ρ(C=O), whose position is affected by changes in sample chain length and physical state; in the samples studied here, it was found to lie between 1729 and 1748 cm−1. Further, molar unsaturation could be correlated with the ratio of the ρ(C=O) to either ρ(C=C) or δ(H−C=) with R 2>0.995. Chain length was correlated with the δ(CH2)tw/ρ(C=O) ratio, (where “tw” indicates twisting) but separate plots for odd- and even-numbered carbon chains were necessary to obtain R 2>0.99 for liquid samples. Combining the odd- and even-numbered carbon chain data in a single plot reduced the correlation to R 2=0.94–0.96, depending on the band ratios used. For molal unsaturation the band ratio that correlated linearly with unsaturation (R 2>0.99) was ρ(C=C)/δ(CH2)sc (where “sc” indicates scissoring). Other band ratios show much more complex behavior with changes in chemical and physical structure. This complex behavior results from the fact that the bands do not arise from simple vibrations of small, discrete regions of the molecules but are due to complex motions of large sections of the FAME so that making incremental changes in structure does not necessarily lead to simple incremental changes in spectra.

Protein engineering and applications of Candida rugosa lipase isoforms.

Abstract: Commercial preparations of Candida rugosa lipase (CRL) are mixtures of lipase isoforms used for the hydrolysis and synthesis of various esters. The presence of variable isoforms and the amount of lipolytic protein in the crude lipase preparations lead to a lack of reproducibility of biocatalytic reactions. Purification of crude CRL improve their substrate specificity, enantioselectivity, stability, and specific activities. The expression of the isoforms is governed by culture or fermentation conditions. Unfortunately, the nonsporogenic yeast C. rugosa does not utilize the universal codon CTG for leucine; therefore, most of the CTG codons were converted to universal serine triplets by site-directed mutagenesis to gain expression of functional lipase in heterologous hosts. Recombinant expressions by multiple-site mutagenesis or complete synthesis of the lipase gene are other possible ways of obtaining pure and different CRL isoforms, in addition to culture engineering. Protein engineering of purified CRL isoforms allows the tailoring of enzyme function. This involves computer modeling based on available 3-D structures of lipase isoforms. Lid swapping and DNA shuffling techniques can be used to improve the enantioselectivity, thermostability, and substrate specificity of CRL isoforms and increase their biotechnological applications. Lid swapping can result in chimera proteins with new functions. The sequence of the lid can affect the activity and specificity of recombinant CRL isoforms. Candida rugosa lipase is toxicologically safe for food applications. Protein engineering through lid swapping and rationally designed site-directed mutagenesis will continue to lead to the production of CRL isoforms with improved catalytic power, thermostability, enantioselectivity, and substrate specificity, while providing evidence for the mechanisms of actions of the various isoforms.

Olive oil consumption and reduced incidence of hypertension: The SUN study

Abstract: Olive oil, a major component of the Mediterranean diet, has been associated in some small clinical trials and cross-sectional studies with a reduction in blood pressure. The objective of this study was to assess the association of olive oil consumption with the incidence of hypertension in an epidemiologic cohort, the Seguimiento Universidad de Navarra (SUN) study. The SUN Project is a prospective cohort study whose members are all university graduates. The recruitment and follow-up of participants is made using mailed questionnaires. Diet was assessed using a semiquantitative food frequency questionnaire previously validated in Spain, with 136 items. Outcomes of interest were newly diagnosed cases of hypertension, as reported by participants in the follow-up questionnaires. Logistic regression models were fit to assess the risk of hypertension associated with olive oil consumption. For the present analysis, we have taken in consideration the first 6,863 participants, with at least 2 yr of follow-up. After a median follow-up time of 28.5 mon, the cumulative incidence of hypereension was 4.7% in men and 1.7% in women. A lower risk of hypertension was observed among participants with a higher olive oil consumption at baseline, but the results were not statistically significant (P=0.13 for the linear trend test in the multivariate model). However, among men, the adjusted odds ratios (OR) (95% confidence intervals) of hypertension for the second to fifth quintiles of olive oil consumption, compared with the first quintile, were 0.55 (0.28–1.10), 0.75 (0.39–1.43), 0.32 (0.15–0.70), and 0.46 (0.23–0.94), respectively (P=0.02 for linear trend). No association was found between olive oil consumption and the risk of hypertension among women. In conclusion, in a Mediterranean population, we found olive oil consumption to be associated with a reduced risk of hypertension only among men. The lack of association observed among women might be attributed to the overall lower incidence of hypertension found among females and the resulting lower statistical power.

Dietary diacylglycerol prevents high-fat diet-induced lipid accumulation in rat liver and abdominal adipose tissue.

Abstract: The inhibitory effects of 1,3-diacylglycerol (DAG) on diet-induced lipid accumulation in liver and abdominal adipose tissue of rats were investigated in the present study. Male Sprague-Dawley rats were given free access to diets containing 7 wt% TAG (low TAG), 20 wt% TAG (high TAG), or 20 wt% DAG (high DAG), respectively, for 8 wk. The body weight of rats in the 20% high-TAG group increased significantly, and the weights of their abdominal adipose tissue and liver also showed a significant increase compared with rats in the low-TAG group. However, the high-DAG diet resulted in both a significant reduction in body weight gain (with a decrease of 70.5%) and an increase in the ratio of abdominal fat to body weight (by 127%) compared with the high-TAG diet. As well, the liver TAG and serum TAG levels of the high-DAG group were significantly lower than those of the high-TAG group. These effects were associated with up-regulation of acyl-CoA carnitine acyltransferase (ACAT) and down-regulation of acyl-CoA DAG acyltransferase (DGAT) in the liver. However, no significant difference was observed in the activities of alanine aminotransferase and aspartate aminotransferase among the groups (P > 0.05). The present results indicate that dietary DAG reduced fat accumulation in viscera and body, and these effects may be involved with up-regulation of ACAT and down-regulation of DGAT in the liver.

Tocotrienol-Rich Fraction from Palm Oil Affects Gene Expression in Tumors Resulting from MCF-7 Cell Inoculation in Athymic Mice

Abstract: It has recently been shown that tocotrienols are the components of vitamin E responsible for inhibiting the growth of human breast cancer cells in vitro, through an estrogen-independent mechanism. Although tocotrienols act on cell proliferation in a dose-dependent manner and can induce programmed cell death, no specific gene regulation has yet been identified. To investigate the molecular basis of the effect of tocotrienols, we injected MCF-7 breast cancer cells into athymic nude mice. Mice were fed orally with 1 mg/d of tocotrienol-rich fraction (TRF) for 20 wk. At end of the 20 wk, there was a significant delay in the onset, incidence, and size of the tumors in nude mice supplemented with TRF compared with the controls. At autopsy, the tumor tissue was excised and analyzed for gene expression by means of a cDNA array technique. Thirty out of 1176 genes were significantly affected. Ten genes were down-regulated and 20 genes up-regulated with respect to untreated animals, and some genes in particular were involved in regulating the immune system and its function. The expression of the interferon-inducible transmembrane protein-1 gene was significantly up-regulated in tumors excised from TRF-treated animals compared with control mice. Within the group of genes related to the immune system, we also found that the CD59 glycoprotein precursor gene was up-regulated. Among the functional class of intracellular transducers/effectors/modulators, the c-myc gene was significantly down-regulated in tumors by TRF treatment. Our observations indicate that TRF supplementation significantly and specifically affects MCF-7 cell response after tumor formation in vivo and therefore the host immune function. The observed effect on gene expression is possibly exerted independently from the antioxidant activity typical of this family of molecules.

Blood phospholipid fatty acid analysis of adults with and without attention deficit/hyperactivity disorder.

Abstract: Several psychiatric disorders, including juvenile Attention Deficit/Hyperactivity Disorder (ADHD), have been associated with abnormalities of certain long-chain PUFA (LCPUFA). Despite this reported association, the FA levels of patients with the adult form of ADHD have not previously been evaluated. In this study we measured the total blood phospholipid FA concentrations in 35 control subjects and 37 adults with ADHD symptoms to determine whether adults with ADHD symptoms would show abnormalities of FA relative to control subjects. In the serum phospholipids, adults with ADHD symptoms had significantly lower levels of total saturated, total polyunsaturated, and total omega-6 (n-6) FA, as well as the omega-3 (n-3) LCPUFA DHA (22:6n-3), and significantly higher levels of total monounsaturated FA and the n-3 LCPUFA docosapentaenoic acid (22:5n-3). In the erythrocyte membrane phospholipids, adults with ADHD symptoms had significantly lower levels of total PUFA, total n-3 FA, and DHA, and significantly higher levels of total saturated FA. Neither serum nor erythrocyte membrane phospholipid DHA was related to ADHD symptom severity (as assessed by the Amen questionnaire) in ADHD subjects. Although the exact cause of these variations is unknown, both environmental and genetic factors may be involved.

Modulation of HepG2 cell net apolipoprotein B secretion by the citrus polymethoxyflavone, tangeretin.

Abstract: The purpose of the present study was to examine the role of tangeretin, a polymethoxylated flavone from citrus fruits, on the regulation of apolipoprotein B (apoB) and lipid metabolism in the human hepatoma cell-line HepG2. The marked reduction in apoB secretion observed in cells incubated with 72.8 μM tangeretin was rapid, apoB-specific, and partly reversible. The reduction also was observed under lipid-rich conditions and found to be insensitive to proteasomal degradation of nascent apoB. We followed our study by examining lipid synthesis and mass. A 24-h exposure of cells to 72.8 μM tangeretin decreased intracellular synthesis of cholesteryl esters, free cholesterol, and TAG by 82, 45, and 64%, respectively; tangeretin also reduced the mass of cellular TAG by 37%. The tangeretin-induced suppression of TAG synthesis and mass were associated with decreased activities of DAG acyltransferase (up to-39.0±3.0% vs. control) and microsomal triglyceride transfer protein (up to−35.5±2.5% vs. control). Tangeretin was also found to activate the peroxisome proliferator-activated receptor, a transcription factor with a positive regulatory impact on FA oxidation and TAG availability (up to 36% increase vs. control). The data suggest that tangeretin modulates apoB-containing lipoprotein metabolism through multiple mechanisms.

Synthesis and anticancer activities of fatty acid analogs of podophyllotoxin.

Abstract: Derivatives of podophyllotoxin were prepared by coupling 10 FA with the C4-α-hydroxy function of podophyllotoxin. The coupling reactions between FA and podophyllotoxin were carried out by dicyclohexylcarbodiimide in the presence of a catalytic amount of dimethylaminopyridine to produce quantitative yields of desired products. FA incorporated were the following: 10-hydroxydecanoic, 12-hydroxydodecanoic, 15-hydroxypentadecanoic, 16-hydroxyhexadecanoic, 12-hydroxyoctadec-Z-9-enoic, eicosa-Z-5,8,11,14-tetraenoic, eicosa-Z-8,11, 14-trienoic, eicosa-Z-11,14-dienoic, eicosa-Z-11-enoic, and eicosanoic acids. Spectroscopic studies confirmed the formation of the desired products. New molecules were investigated for their in vitro anticancer activity against a panel of human cancer cell lines including SK-MEL, KB, BT-549, SK-OV-3 (solid tumors), and HL-60 (human leukemia) cells. Most of the analogs were cytotoxic against cancerous cells, whereas no effect was observed against normal cells, unlike the parent compound podophyllotoxin, the use of which is limited due to its severe side effects.

Olive oil and modulation of cell signaling in disease prevention.

Abstract: Epidemiological studies show that populations consuming a predominantly plant-based Mediterranean-style diet exhibit lower incidences of chronic diseases than those eating a northern European or North American diet. This observation has been attributed to the greater consumption of fruits and vegetables and the lower consumption of animal products, particularly fat. Although total fat intake in Mediterranean populations can be higher than in other regions (ca. 40% of calories), the greater proportion is derived from olive oil and not animals. Increased olive oil consumption is implicated in a reduction in cardiovascular disease, rheumatoid arthritis, and, to a lesser extent, a variety of cancers. Olive oil intake also has been shown to modulate immune function, particularly the inflammatory processes associated with the immune system. Olive oil is a nonoxidative dietary component, and the attenuation of the inflammatory process it elicits could explain its beneficial effects on disease risk since oxidative and inflammatory stresses appear to be underlying factors in the etiology of these diseases in man. The antioxidant effects of olive oil are probably due to a combination of its high oleic acid content (low oxidation potential compared with linoleic acid) and its content of a variety of plant antioxidants, particularly oleuropein, hydroxytyrosol, and tyrosol. It is also possible that the high oleic acid content and a proportionate reduction in linoleic acid intake would allow a greater conversion of alpha-linolenic acid (18:3n-3) to longer-chain n-3 PUFA, which have characteristic health benefits. Adoption of a Mediterranean diet could confer health benefits in high-risk populations.

Gestational age and birth weight in relation to n−3 fatty acids among inuit (Canada)

Abstract: Seafood consumption during pregnancy carries both benefits (high n-3 FA intake) and risks (exposure to environmental contaminants) for the developing fetus. We determined the impacts of marine n-3 FA and environmental contaminants on gestational age (GA) of Nunavik women and the anthropometric characteristics of their newborns. FA and contaminant (polychlorinated biphenyls and mercury) concentrations were measured in cord plasma of Nunavik newborns (n = 454) and compared with those of a group of newborns (n = 29) from southern Quebec. Data were collected from hospital records and birth certificates. In Nunavik newborns, arachidonic acid (AA) was two times lower (P < 0.0001), whereas DHA concentration, the sigman-3 / sigman-6 ratio, and the percentage of n-3 highly unsaturated FA (HUFA) (of the total HUFA) were three times higher (P < 0.0001) compared with southern Quebec newborns. After controlling for confounders, GA and birth weight were higher by 5.4 d [95% confidence interval (CI): 0.7-10.1] and 77 g (95% CI: -64 to 217) in the third tertile of percentage of n-3 HUFA (of the total HUFA) as compared with the first tertile. There was no evidence that contaminants had negative effects on GA or birth weight. In this seafood-eating population, an increase in the proportion of n-3 HUFA (of the total HUFA), measured in umbilical cord plasma phospholipids, was associated with a significantly longer GA.

Molecular characterization of three peroxisome proliferator-activated receptors from the sea bass (Dicentrarchus labrax).

Abstract: Peroxisome proliferator-activated receptors (PPAR) are nuclear hormone receptors that control the expression of genes involved in lipid homeostasis in mammals. We searched for PPAR in sea bass, a marine fish of particular interest to aquaculture, after hypothesizing that the physiological and molecular processes that regulate lipid metabolism in fish are similar to those in mammals. Here, we report the identification of complementary DNA and corresponding genomic sequences that encode three distinct PPAR from sea bass. The sea bass PPAR are the structural homologs of the mammalian PPARα, β/δ and γ isotypes. As revealed by RNase protection, the tissue expression profile of the fish PPAR appears to be very similar to that of the mammalian PPAR homologs. Thus, PPARα is mainly expressed in the liver, PPARγ in adipose tissue, and PPARβ in all tissues tested, with its highest levels in the liver, where it is also the dominant isotype expressed. Like mammalian PPAR, the sea bass isotypes recognize and bind to PPAR response elements of both mammalian and piscine origin, as heterodimers with the 9-cis retinoic acid receptor. Through the coactivator-dependent receptor ligand assay, we also demonstrated that natural FA and synthetic hypolipidemic compounds can act as ligands of the sea bass PPARα and β isotypes. This suggests that the sea bass PPAR act through similar mechanisms and perform the same critical lipid metabolism functions as mammalian PPAR.