For Fatty Acid Research Articles

Characterization of a fatty acid binding protein from the swimming crab Portunus trituberculatus and its effects on the composition of fatty acids in different tissues

Fatty acid binding proteins (FABPs) belonging to the lipocalin protein family, have an affinity for fatty acids (FAs) and participate in both the transport of FAs and lipid metabolism. In this study, a 909-bp full-length pt-FABP cDNA containing a 411-bp open reading frame (ORF) was obtained from the swimming crab Portunus trituberculatus. Quantitative real-time PCR revealed that during larval development, pt-FABP expression increased gradually from the zoea to the megalopa, and decreased in the juvenile crab. pt-FABP mRNA was detected at the highest level in the testis, followed by the muscles, with the lowest level in the hepatopancreas. The composition of fatty acids in three different tissues from crabs of different sex was detected by gas chromatography–mass spectrometry (GC-MS). The results indicated that the FA profile was in accordance with the expression of pt-FABP, which suggested that pt-FABP participates in the unsaturated fatty acid transportation and storage system of P. trituberculatus.

Abstract 457: Satisfying the fatty acid demand of prostate cancer: Outlier overexpression of genes involved in de novo synthesis vs fatty acid uptake define two different and potentially synergistic prostate cancer phenotypes

Abstract Malignant transformation increases cellular demand for fatty acids (FA). Many cancers, including prostate cancer (PCa), show increased expression of fatty acid synthase (FASN), the enzyme that catalyzes the de novo synthesis of the FA palmitate. De novo FA synthesis is energetically expensive; in most normal cells FASN expression is low and de novo synthesis suppressed in favor of utilization of exogenous FA. In contrast, in cancer cells, de novo FA synthesis is an important source of precursors needed to support tumor growth. Much of the research effort on FA in cancer has focused on FASN and the other genes involved in de novo FA synthesis. However a number of observations suggest that alternate mechanisms for FA acquisition are also important, most notably studies showing that cancer cells can be rescued from the pro-apoptotic effect of FASN inhibition by exogenous palmitate. Using a biopsy-based approach designed to identify high risk prostate cancer phenotypes that are common in African American (AA) men, we identified a PCa subtype with outlier (> 10 fold) mRNA overexpression of fatty acid binding protein 5 (FABP5). FABP5 facilitates the utilization of palmitate and other FAs from outside the cell, and a FABP5 overexpression phenotype may confer a selective advantage when dietary FAs are abundant or when FASN is targeted as a therapy. The FABP5 outlier overexpression phenotype was observed in 7% Gleason sum (GS) 7-10 PCas. We also observed a second molecular subtype with outlier FASN overexpression in 9% of GS 7-10 PCas. With few exceptions these two PCa subtypes showed an “either-or” pattern with top quartile overexpression of FABP5 or FASN but not both (Spearman r = 0.385; P < 0.0001). Interestingly, in low grade (GS 6) PCas, median FABP5 mRNA overexpression is higher in AA than in European American (EA) men (P < 0.01) with GS6 PCas from AA men showing median FABP5 expression levels as high as that observed in GS 7-10 PCas. IHC confirmed variable expression of both FABP5 and FASN, including “patchwork” PCas with high Gleason pattern (GP) areas overexpressing FABP5 next to low GP areas overexpressing FASN. The net result may be a selective advantage for the high grade cancer if FABP5 allows it to exploit the FA being synthesized by the adjacent low grade focus. In a subset of PCas, FABP5 IHC showed robust nuclear staining consistent with its proposed role in regulating fatty acid mediated gene expression; the ratio of FABP5 nuclear/cytoplasmic staining was higher in PCa from AA than from EA patients (P < 0.05). Identification of a PCa subtype with high levels of FABP5 overexpression suggests a previously unrecognized mechanism by which some PCas can increase FA supply without de novo synthesis. Such a PCa subtype might be particularly sensitive to dietary interventions and relatively insensitive to FASN inhibitors. Citation Format: Sandra M. Gaston, James Kearns, George W. Adams, Soroush Rais-Bahrami, Jeffrey W. Nix, Peter N. Kolettis, James E. Bryant, Mark S. DeGuenther, Denise Oelschlager, Dennis Otali, William E. Grizzle. Satisfying the fatty acid demand of prostate cancer: Outlier overexpression of genes involved in de novo synthesis vs fatty acid uptake define two different and potentially synergistic prostate cancer phenotypes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 457. doi:10.1158/1538-7445.AM2017-457

Comparison of Six Methylation Methods for Fatty Acid Determination in Yak Bone Using Gas Chromatography

Yak bone is rich in nutrition but not used efficiently. Six methylation methods (M1: acetyl chloride-methanol method, M2: H2SO4-methanol method, M3: HCl-methanol method, M4: KOH-methanol method, M5: M2 in combination with M4, and M6: M3 in combination with M4) with (system 1 (S1)) or without (system 2 (S2)) prior acid hydrolysis for fatty acid (FA) determination using gas chromatography in yak bone were comprehensively compared. The contents of total FA determined by using M1 and M2 were significantly higher (p 90%) of the FA, the intra-day and inter-day CVs were <9%. Recoveries for more than 90% of the FA were within a range of 85–117%. In addition, C18:1n9c, C16:0, C18:0, and conjugated linoleic acid were the major FAs in yak bone, approximately accounted for 50, 20, 10, and 10% of the total FA, respectively. And the ratio of saturated and unsaturated FA in yak bone was about 1:2. The acid hydrolysis/H2SO4-methanol esterification method would be more efficient for the accurate determination of FA in yak bone, whereas the direct acetyl chloride-methanol method would be used as an alternative for the fast analysis.

Peroxisome Proliferator-Activated Receptor (PPAR) Gamma Agonists as Therapeutic Agents for Cardiovascular Disorders: Focus on Atherosclerosis.

Peroxisome proliferation-activated receptors (PPARs) are ligand-activated transcription factors that mainly regulate genes responsible for fatty acid (FA) and energy metabolism. There are three members of the PPAR family, PPAR-α, PPAR- β/δ and PPAR-γ. All three isoforms have therapeutic potential for treatment of cardiovascular disorders, and PPAR agonists are currently being actively studied in pre-clinical and clinical trials. PPAR γ agonists, main tissue expressing isoform, have potential to influence on inflammation processes, reduce oxidative stress, improve endothelial function and plays an important role in lipid metabolism. PPAR-γ agonists are used as insulin sensitizers for treatment of diabetes; however, there is accumulating evidence that their clinical application can be broadened. The wide spectrum of PPAR-γ activation effects may be beneficial for treatment of various cardiovascular conditions as atherosclerosis, hypertension and aortic aneurysm, including surgical interventions. In this Review we will discuss the implication of PPAR-γ in the cardiovascular system and potential role of PPAR-γ agonists in treatment of conditions associated with high cardiovascular risks.

Enhanced Fatty Acid Production in Escherichia coli by Over-Expression of NADPH Generating Enzymes

NADPH is an essential factor for Fatty Acid (FA) biosynthesis. The effect of NADPH generating enzymes [NADP+-dependent malic enzyme (NADP-ME), NADP+-dependent isocitrate dehydrogenase (NADP-IDH), Glucose 6-Phosphate Dehydrogenase (GPD) and 6-Phosphogluconate Dehydrogenase (PGD)] on FA biosynthesis was investigated in an engineered Escherichia coli BL21 ?fadE/pTE. Among NADPH-generating enzymes, GPD and ICD increased total FA production by 60 and 16% respectively. Especially medium-chain FA production was increased up to 4.2 and 2.5 folds respectively. In addition, over-expression of the endogenous Thioesterase (TE) reduced Cyclopropane Fatty Acid (CFA) production by 40% and over-expressing of GPD and ICD further decreased CFA production by 23 and 30%, respectively compared to the TE over-expressed strain. Over-expression of TE appeared to be a good strategy to produce high quality biodiesel with medium-chain FAs as major Component (62%) and with Minimal Amount of CFA (4%). This study reveals that NADPH-generating enzyme GPD is associated with FA biosynthesis in E. coli, but not all reducing power generating enzymes, are involved in FA biosynthesis in bacteria.

Satisfying the fatty acid demand of prostate cancer: De novo synthesis versus uptake as alternative and potentially cooperative prostate cancer phenotypes.

107 Background: Malignant transformation increases cellular demand for fatty acids (FA). Many cancers show increased expression of fatty acid synthase (FASN); FASN catalyzes the de novo synthesis of the FA palmitate. While research has focused on FASN and de novo tumor FA synthesis, observations suggest that alternate mechanisms for FA acquisition are also important, including studies showing that cancer cells can be rescued from FASN inhibition by exogenous palmitate. Using a biopsy-based approach, we identified a PrCa subtype with outlier (&gt;10 fold) overexpression of fatty acid binding protein 5 (FABP5). FABP5 facilitates the utilization of palmitate and other FAs from outside the cell; a FABP5 overexpression phenotype may confer a selective advantage when dietary FAs are abundant or when FASN is targeted as a therapy. Methods: 244 prostate biopsy patients were prospectively enrolled. Tissue prints were collected from each core for RNA and DNA preparation. mRNA was analyzed using Affymetrix arrays and TaqMan qrtPCR assays. Immunohistochemistry (IHC) was performed using archival radical prostatectomy (RP) specimens. Results: mRNA analyses revealed a PrCa subtype with &gt;10 x overexpression of FABP; this phenotype was observed in 7% of 268 cores with Gleason sum (GS) 7-10 PrCa. We also observed a second subtype with &gt;10 x overexpression of FASN in 9% of GS 7-10 cores. With few exceptions these two PrCa subtypes showed an “either-or” pattern with top-quartile overexpression of FABP5 or FASN but not both (Spearman r = - 0.385; P &lt; 0.0001). IHC confirmed variable expression, including “patchwork” PrCas with high Gleason pattern (GP) areas overexpressing FABP5 next to low GP areas overexpressing FASN. The net result may be a selective advantage for the high grade cancer if FABP5 allows it to exploit the FA being synthesized by the adjacent low grade focus Conclusions: Identification of a PrCa subtype with high levels of FABP5 overexpression suggests a previously unrecognized mechanism by which some PrCas can increase FA supply without de novo synthesis; such a PrCa subtype might be particularly sensitive to dietary interventions and relatively insensitive to FASN inhibitors.

Rapid Analysis Procedures for Triglycerides and Fatty Acids as Pentyl and Phenethyl Esters for the Detection of Butter Adulteration Using Chromatographic Techniques

This paper presents the development of three methods for quality control, fraud detection, and authentication of butter fat and other oils/fats using chromatographic techniques, with one method for triglycerides and two methods for fatty acids (FAs). The procedure for the analysis of triglycerides requires only dissolution of the sample inn-hexane and gas chromatography (GC) analysis using a capillary column. The second method is based on the transesterification of triglycerides as pentyl esters in a single-step reaction using sodium pentanoate in pentanol. The reaction proceeds at room temperature and is similar to the potassium hydroxide-catalysed transesterification of triglycerides with methanol and even more similar to the sodium methoxide method and sodium butanoate method. The advantage of using pentyl esters includes reducing the volatility of short-chain FAs, and substantial recoveries were obtained compared with methyl ester analysis. The third method involves the transesterification of triglycerides in fat through reaction with 2-phenylethanol in a single step; 2-phenylethanol possesses a chromophore, and the phenethyl esters formed are analysed by high-performance liquid chromatography (HPLC) with UV detection.

Differentiating the geographical origin of Tunisian indigenous lamb using stable isotope ratio and fatty acid content

The efficacy of isotope ratio mass spectrometry (IRMS) in tracing lamb production systems was investigated for four farming systems in the Tunisian North-West: Ain Draham and Fernana, characterised by woody pasture, and Amdoun and Joumine, characterised by herbaceous pasture. Mixed breed lambs aged 3.5–5 months were reared under semi-extensive and extensive systems. Samples of Longissimus dorsi muscle were taken from eight lambs for each farming system for stable IR assessment of the five main bio-elements in the protein and fat fractions of lamb and for fatty acid (FA) determination. Using partial least squares discriminant analysis (PLS-DA) the IR profiles of Tunisian lamb types allowed correct assignment of the training meat samples to the area of origin. Inclusion of the FA profile in the classification model slightly improved its evaluation performance for the individual farming systems; the average accuracy increased by 2%–94%. However, the differences between samples were not sufficiently wide to be validated by an external set of samples including 10 Italian lamb types. A broader scale geographical signature discriminating lambs from the Amdoun area, the rest of north-west Tunisia and Italy appeared to be workable, although some foreign lamb types from neighbouring Mediterranean regions such as Sicily were misclassified as Tunisian.

Variable selection procedures before partial least squares regression enhance the accuracy of milk fatty acid composition predicted by mid-infrared spectroscopy.

Mid-infrared spectroscopy is a high-throughput technique that allows the prediction of milk quality traits on a large-scale. The accuracy of prediction achievable using partial least squares (PLS) regression is usually high for fatty acids (FA) that are more abundant in milk, whereas it decreases for FA that are present in low concentrations. Two variable selection methods, uninformative variable elimination or a genetic algorithm combined with PLS regression, were used in the present study to investigate their effect on the accuracy of prediction equations for milk FA profile expressed either as a concentration on total identified FA or a concentration in milk. For FA expressed on total identified FA, the coefficient of determination of cross-validation from PLS alone was low (0.25) for the prediction of polyunsaturated FA and medium (0.70) for saturated FA. The coefficient of determination increased to 0.54 and 0.95 for polyunsaturated and saturated FA, respectively, when FA were expressed on a milk basis and using PLS alone. Both algorithms before PLS regression improved the accuracy of prediction for FA, especially for FA that are usually difficult to predict; for example, the improvement with respect to the PLS regression ranged from 9 to 80%. In general, FA were better predicted when their concentrations were expressed on a milk basis. These results might favor the use of prediction equations in the dairy industry for genetic purposes and payment system.

Comparison between enzymatic and chemical interesterification of high oleic sunflower oil and fully hydrogenated soybean oil

Structured lipids (SL) were prepared by enzymatic interesterification (EI) and chemical interesterification (CI) of high oleic sunflower oil (HOSO), and fully hydrogenated soybean oil (FHSO) in a weight ratio of 50:50 w/w. The EI was catalyzed by the immobilized lipase Lipozyme TL IM (Thermomyces lanuginosus) free of organic solvents at a concentration of 7% w/w, 70°C for 9 h, and samples were collected for analysis at 3, 6, and 9 h. The original blend and the reaction products were evaluated for fatty acid (FA) composition, triacylglycerols (TAG), solid fat content (SFC), thermal analysis, and regiospecific distribution of FA. The EI provided considerable rearrangement of triglycerides classes, with lower levels of trisatured and tri‐unsaturated TAG, and higher levels of monoun‐ and diunsaturated TAG. The SL obtained by EI reduced SFC with a linear and wide melting range by thermal analysis, lowering the melting point. Lower acyl migration was observed in EI within 3 h of reaction, with similar results between both techniques at the maximum reaction time. The SL obtained by EI may have wide applications in specialty fats for confectionery and bakery products.Practical Applications: SL via enzymatic catalysis have been the focus of considerable attention in food industry. We report in this study, the comparison between enzymatic and chemical interesterification of a fully hydrogenated vegetable oil, and high oleic vegetable oil. The results show that the enzymatic process with 3 h of reaction time present more selectivity, lower acyl migration of sn‐2 position of the unsaturated fatty acid, and better nutritional value, when compared with the chemical process. These SL were projected to have broad applications in foods, as bakery/confectionery fats, and as a crystallization additive for lipids.The enzymatic process with 3 h of reaction time showed the advantage of selectivity for production of new TAG and lower acyl migration sn‐2 position of the unsaturated fatty acid when compared with the other reaction times, evidenced by NMR 13C. These SL were projected to have broad applications in foods, as bakery/confectionery fats and as a crystallization additive for lipids.

Impact of Genetic and Epigenetic Variations Within the FADS Cluster on the Composition and Metabolism of Polyunsaturated Fatty Acids in Prostate Cancer.

BACKGROUNDIn vitro and experimental animal studies have demonstrated that high levels of omega‐6 (n‐6) polyunsaturated fatty acids (PUFAs) and high ratios of n‐6 to omega‐3 (n‐3) PUFAs are strongly associated with the development and progression of prostate cancer (PCA). However, epidemiological studies in humans have demonstrated inconsistent findings linking dietary PUFAs and PCA risk. We hypothesize that genetic and epigenetic variations within the fatty acid desaturase (FADS) gene cluster produce gene–diet interactions that may explain these disparate findings. This study tested the relationship of the genotype of a single nucleotide polymorphism, rs174537, and the methylation status of a CpG site, cg27386326, with PUFA composition, and markers of PUFA biosynthesis in PCA tissue.METHODSSixty PCA specimens from patients undergoing radical prostatectomy were genotyped, pyrosequenced and quantitated for fatty acids (FAs).RESULTSLong‐chain (LC)‐PUFAs, such as arachidonic acid (ARA), were abundant in these specimens, with ARA accounting for 15.8% of total FAs. In addition, there was a positive association of the G allele at rs174537 with concentrations of ARA and adrenic acid and ratios of products to precursors within the n‐6 PUFA pathway such that specimens from homozygous G individuals exhibited increasingly higher values as compared to specimens from heterozygous individuals and homozygous T individuals. Finally, the methylation status of cg27386326 was inversely correlated with tissue concentrations of LC‐PUFAs and markers of LC‐PUFA biosynthesis.CONCLUSIONSThese data reveal that genetic and epigenetic variations within the FADS cluster are highly associated with LC‐PUFA concentrations and LC‐PUFA biosynthetic capacity in PCA tissue. They also raise the potential that gene–PUFA interactions play an important role in PCA risk and severity. Prostate 76:1182–1191, 2016. © 2016 The Authors. The Prostate published by Wiley Periodicals, Inc.

Preparation of human milk fat analogue by enzymatic interesterification reaction using palm stearin and fish oil.

Palm stearin fractionate (PSF), obtained from palm stearin by further fractionation with solvents and n-3 polyunsaturated fatty acids (n-3 PUFA) rich fish oil (FO) were subjected to interesterification at 1:1, 1:2, 1:3, 2:1 and 3:1 substrate molar ratio and catalyzed by lipase from Thermomyces lanuginosa for obtaining a product with triacylglycerol (TAG) structure similar to that of human milk fat (HMF). The parameters (molar ratio and time) of the interesterification reaction were standardized. The temperature of 60°C and enzyme concentration of 10% (w/w) were kept fixed as these parameters were previously optimized. The reactions were carried out in a stirred tank reactor equipped with a magnetic stirrer for 6, 12, 18 and 24h. The blends were analyzed for fatty acid (FA) composition of both total FAs and those at the sn-2 position after pancreatic lipase hydrolysis. All the blended products were subjected to melting point determination and free fatty acid content. Finally, blend of PSF and FO at 2:1 molar ratio with 69.70% palmitic acid (PA) content and 12h of reaction produced the desired product with 75.98% of PA at sn-2 position, 0.27% arachidonic acid (AA), 3.43% eicosapentaenoic acid (EPA) and 4.25% docosahexaenoic acid (DHA) and with melting point of 42°C. This study portrayed a successful preparation of TAG containing unique FA composition i.e. ≥ 70% of the PA, by weight, were esterified at the sn-2 position which could be used in infant formulation with health benefits of n-3 PUFAs.

Inhibition of acetyl-CoA carboxylases by soraphen A prevents lipid accumulation and adipocyte differentiation in 3T3-L1 cells

Acetyl-CoA carboxylases (ACC) 1 and 2 catalyze the carboxylation of acetyl-CoA to malonyl-CoA and depend on biotin as a coenzyme. ACC1 localizes in the cytoplasm and produces malonyl-CoA for fatty acid (FA) synthesis. ACC2 localizes in the outer mitochondrial membrane and produces malonyl-CoA that inhibits FA import into mitochondria for subsequent oxidation. We hypothesized that ACCs are checkpoints in adipocyte differentiation and tested this hypothesis using the ACC1 and ACC2 inhibitor soraphen A (SA) in murine 3T3-L1 preadipocytes. When 3T3-L1 cells were treated with 100nM SA for 8 days after induction of differentiation, the expression of PPARγ mRNA and FABP4 mRNA decreased by 40% and 50%, respectively, compared with solvent controls; the decrease in gene expression was accompanied by a decrease in FABP4 protein expression and associated with a decrease in lipid droplet accumulation. The rate of FA oxidation was 300% greater in SA-treated cells compared with vehicle controls. Treatment with exogenous palmitate restored PPARγ and FABP4 mRNA expression and FABP4 protein expression in SA-treated cells. In contrast, SA did not alter lipid accumulation if treatment was initiated on day eight after induction of differentiation. We conclude that loss of ACC1-dependent FA synthesis and loss of ACC2-dependent inhibition of FA oxidation prevent lipid accumulation in adipocytes and inhibit early stages of adipocyte differentiation.

ANT1-mediated fatty acid-induced uncoupling as a target for improving myocellular insulin sensitivity.

Aims/hypothesisDissipating energy via mitochondrial uncoupling has been suggested to contribute to enhanced insulin sensitivity. We hypothesised that skeletal muscle mitochondria of endurance-trained athletes have increased sensitivity for fatty acid (FA)-induced uncoupling, which is driven by the mitochondrial protein adenine nucleotide translocase 1 (ANT1).MethodsCapacity for FA-induced uncoupling was measured in endurance-trained male athletes (T) and sedentary young men (UT) in an observational study and also in isolated skeletal muscle mitochondria from Zucker diabetic fatty (ZDF) rats and C2C12 myotubes following small interfering RNA (siRNA)-mediated gene silencing of ANT1. Thus, fuelled by glutamate/succinate (fibres) or pyruvate (mitochondria and myotubes) and in the presence of oligomycin to block ATP synthesis, increasing levels of oleate (fibres) or palmitate (mitochondria and myotubes) were automatically titrated while respiration was monitored. Insulin sensitivity was measured by hyperinsulinaemic–euglycaemic clamp in humans and via insulin-stimulated glucose uptake in myotubes.ResultsSkeletal muscle from the T group displayed increased sensitivity to FA-induced uncoupling (p = 0.011) compared with muscle from the UT group, and this was associated with elevated insulin sensitivity (p = 0.034). ANT1 expression was increased in T (p = 0.013). Mitochondria from ZDF rats displayed decreased sensitivity for FA-induced uncoupling (p = 0.008). This difference disappeared in the presence of the adenine nucleotide translocator inhibitor carboxyatractyloside. Partial knockdown of ANT1 in C2C12 myotubes decreased sensitivity to the FA-induced uncoupling (p = 0.008) and insulin-stimulated glucose uptake (p = 0.025) compared with controls.Conclusions/interpretationIncreased sensitivity to FA-induced uncoupling is associated with enhanced insulin sensitivity and is affected by ANT1 activity in skeletal muscle. FA-induced mitochondrial uncoupling may help to preserve insulin sensitivity in the face of a high supply of FAs.Trial registrationwww.trialregister.nl NTR2002

Doestrans-10,cis-12 conjugated linoleic acid affect the intermediary glucose and energy expenditure of dairy cows due to repartitioning of milk component synthesis?

The overall goal of this study was to evaluate if intermediary energy metabolism of cows fed with trans-10, cis-12 conjugated linoleic acid (CLA) was modified such that milk-energy compounds were produced with less intermediary energy expenditure as compared to control cows. Published data on supplemented CLA were assembled. The extent was calculated to which the trans-10, cis-12 CLA isomer has an impact on glucose and energy conversion in the mammary gland by modifying glucose equivalent supply and energy required for fatty acid (FA) and fat synthesis, and if this will eventually lead to an improved glucose and energy status of CLA-supplemented high-yielding dairy cows. A possible relationship between CLA supplementation level and milk energy yield response was also studied. Calculations were conducted separately for orally and abomasally administered CLA and based on energy required for supply of glucose equivalents, i.e. lactose, glycerol and NADPH2. Further, modifications of milk FA profile due to CLA supplementation were considered when energy expenditures for FA and fat synthesis were quantified. Differences in yields between control and CLA groups were transformed into glucose energy equivalents. Only abomasal infusion (r(2) = 0.31) but not oral CLA administration (r(2) = 0.11) supplementation to dairy cow diets resulted in less glucose equivalent energy. Modifications of milk FA profiles also saved energy but the relationship with CLA supplementation was weaker for abomasal infusion (r(2) = 0.06) than oral administration (r(2) = 0.38). On average, 10 g/d of abomasally infused trans-10, cis-12 CLA saved 1.1 to 2.3 MJ net energy expressed as glucose equivalents, whereas both positive and negative values were observed when the trans-10, cis-12 CLA was fed to the cows. This study revealed a weak to moderate dose-dependent relationship between the amount of trans-10, cis-12 CLA administered and the amount of energy in glucose equivalents and energy for the synthesis of milk fat conserved from milk ingredient synthesis. Because abomasal infusion of the trans-10, cis-12 CLA more consistently conserved energy in glucose equivalents compared with oral CLA intake, rumen protection of the fed CLA products appears incomplete. Milk fat synthesis showed an energy saving with a weak dose-dependent relationship when CLA was supplemented orally or by abomasal infusion.

Direct infusion electrospray ionization–ion mobility–mass spectrometry for comparative profiling of fatty acids based on stable isotope labeling

A rapid method for fatty acids (FAs) comparative profiling based on carboxyl-specific stable isotope labeling (SIL) and direct infusion electrospray ionization–ion mobility–mass spectrometry (ESI–IM–MS) is established. The design of the method takes advantage of the three-dimensional characteristics of IM–MS including drift time, m/z and ion intensity, for comparison of d0-/d6-2,4-dimethoxy-6-piperazin-1-yl pyrimidine (DMPP)-labeled FAs. In particular, without chromatographic separation, the method allowed direct FAs profiling in complex samples due to the advantageous priority of DMPP in signal enhancement as well as the extra resolution that IM–MS offered. Additionally, the d0-/d6-DMPP-labeled FAs showed expected features, including very similar drift times, 6 Da mass deviations, specific reporter ions, similar MS responses, and adherence to the drift time rule regarding the influence of carbon chain length and unsaturation on relative drift times. Therefore, the introduction of isotope analogs minimized the matrix effect and variations in quantification and ensured accurate identification of non-targeted FAs by those typical features. Peak intensity ratios between d0-/d6-DMPP-labeled ions were subsequently used in relative quantification for the detected FAs. The established strategy has been applied successfully in the rapid profiling of trace free FAs between normal and cancerous human thyroid tissues. Sixteen free FAs were found with the increased level with a statistically significant difference (p < 0.05) compared to the normal tissue samples. The integrated SIL technique and ESI–IM–MS are expected to serve as an alternative tool for high-throughput analysis of FAs in complex samples.

Animal performance, carcass characteristics and beef fatty acid profile of grazing steers supplemented with corn grain and increasing amounts of flaxseed at two animal weights during finishing

The objective of this study was to determine (1) the effects of increasing flaxseed addition to the corn grain supplemented at finishing to grazing steers on animal performance, carcass characteristics and longissimus muscle (LM) fatty acid profile, and (2) whether dietary treatments effects are affected by animal body weight (BW) at supplementation. Forty eight steers were assigned to eight treatment combinations defined by four dietary treatments (CNTRL, no supplement; FLAX-0, 0.7% BW of cracked corn grain; FLAX-1 and FLAX-2, FLAX-0 plus 0.125% and 0.250% BW of unprocessed flaxseed, respectively) and by two animal BW treatments generated by supplementing steer in early spring (EARLY) or late spring (LATE). Steers assigned to EARLY received their dietary treatment when reaching 366±27.3kg BW (August, 3rd) and those assigned to LATE when reaching 458±42.8kg (October, 10th). After 70d on trial, carcass data and LM samples (12th rib region) were collected for fatty acid (FA) analysis. Total DMI was greater in supplemented treatments (FLAX-0, FLAX-1 and FLAX-2) than in CNTRL, with no flaxseed level effect. Dietary treatment effects on performance and carcass characteristics were not affected by BW treatments. Increasing flaxseed supplementation linearly increased subcutaneous fat thickness; whereas increasing BW at initiation of supplementation decreased average daily again, but increase final BW, hot carcass weight, and LM total fatty acid content. Animal BW at initiation of supplementation affected dietary treatments effects on LM n-6/n-3 ratio; ratio was highest in FLAX-0 and lowest in CNTRL and FLAX-2 in both BW treatments; did not differ between BW treatments in CNTRL and in FLAX-2, but was greater in EARLY than in LATE in FLAX-0 and FLAX-1. Trans-vaccenic acid proportion was greater in CNTRL than in supplemented treatments, but was not affected by flaxseed level; similar trend was observed for CLA cis-9, trans-11 proportion. Linolenic acid proportion did not differ between CNTRL and supplemented treatments, but was linearly increased with flaxseed level. Fatty acids changes with increasing animal BW at supplementation were associated with the increased in total muscle fatty acid content; total and individual polyunsaturated fatty acids were lower in LATE than in EARLY, whereas trans-vaccenic acid and CLA cis-9, trans-11 were greater. Adding flaxseed to the corn grain supplemented increase subcutaneous fat thickness of grazing steers without negatively affecting fatty acid profile, except for a reduction in trans-vaccenic acid proportion. Therefore, adding flaxseed to the corn supplemented to grazing steers increases carcass fatness while reducing the negative effect of corn supplementation on LM n-6/n-3 ratio but not on trans-vaccenic acid. Increasing BW at supplementation reduces supplementation effects on LM n-6/n-3 ratio.

Comprehensive and quantitative profiling of lipid species in human milk, cow milk and a phospholipid-enriched milk formula by GC and MS/MSALL.

Here we present a workflow for in-depth analysis of milk lipids that combines gas chromatography (GC) for fatty acid (FA) profiling and a shotgun lipidomics routine termed MS/MSALL for structural characterization of molecular lipid species. To evaluate the performance of the workflow we performed a comparative lipid analysis of human milk, cow milk, and Lacprodan® PL-20, a phospholipid-enriched milk protein concentrate for infant formula. The GC analysis showed that human milk and Lacprodan have a similar FA profile with higher levels of unsaturated FAs as compared to cow milk. In-depth lipidomic analysis by MS/MSALL revealed that each type of milk sample comprised distinct composition of molecular lipid species. Lipid class composition showed that the human and cow milk contain a higher proportion of triacylglycerols (TAGs) as compared to Lacprodan. Notably, the MS/MSALL analysis demonstrated that the similar FA profile of human milk and Lacprodan determined by GC analysis is attributed to the composition of individual TAG species in human milk and glycerophospholipid species in Lacprodan. Moreover, the analysis of TAG molecules in Lacprodan and cow milk showed a high proportion of short-chain FAs that could not be monitored by GC analysis. The results presented here show that complementary GC and MS/MSALL analysis is a powerful approach for characterization of molecular lipid species in milk and milk products.Practical applications: Milk lipid analysis is routinely performed using gas chromatography. This method reports the total fatty acid composition of all milk lipids, but provides no structural or quantitative information about individual lipid molecules in milk or milk products. Here we present a workflow that integrates gas chromatography for fatty acid profiling and a shotgun lipidomics routine termed MS/MSALL for structural analysis and quantification of molecular lipid species. We demonstrate the efficacy of this complementary workflow by a comparative analysis of molecular lipid species in human milk, cow milk, and a milk-based supplement used for infant formula.

Walrus &lt;i&gt;Odobenus rosmarus&lt;/i&gt; research in Svalbard, Norway, 2000-2010

Herein we report results from studies on walruses in Svalbard conducted in 2000-2010. Data from newly developed satellite relay data loggers (SRDLs) revealed seasonal differences in habitat use of adult male walruses. During winter, they moved into areas of &gt;90% ice concentration, traveling as far as 600 km from ice-free water. Breeding areas deep into the pack ice were identified based on timing of the occupancy and diving behaviour. When the breeding season was over, walruses with SRDLs that were still transmitting locations returned to the coast, showing high site fidelity to the previous year’s summering area. Haul-out data from the SRDLs provided correction factors for an aerial survey of walruses that covered all known haul-out sites within the Svalbard Archipelago. This survey estimated 2,629 (95 % CI; 2,318 – 2,998) walruses to be in Svalbard during August 2006. Blubber biopsies from adult male walruses analyzed for fatty acids (FAs) showed vertical stratification similar to that observed in many other marine mammals. However, differences between layers were less pronounced, possibly because the thick dermis of walruses provides an insulating shield, affecting the FA composition of the outer blubber. The FA composition of the inner blubber most closely resembled the lipids in Mya truncata and Buccinum spp., which are considered the most important walrus prey in Svalbard. A study investigating the use of skin biopsies for assessing levels of organochlorines (OCs) in walruses found a significant relationship between OC levels in skin and blubber. Another contaminant study found a significant decrease in levels of PCBs and DDE in walruses in Svalbard from 1993 to 2002-04. Large inter-individual variation in OC levels was found, although all of the study animals were adult males from roughly the same location. In FA analyses of the inner blubber this variation appeared to be diet-related, with high OC levels having FA compositions in the inner blubber that closely matched seal tissues, while those with low levels matched typical invertebrate prey. Various enzymes, proteins, metabolites, minerals, and hormones were measured in blood samples to serve as baseline data for future health-related studies of both wild and captive walruses. Historical sex-distribution of walruses in southern Svalbard was investigated based on mandible measurements of individuals hunted during the 19th century.The analyses showed that female walruses were once more common in south-eastern Svalbard than they are today.

Three-dimensional imaging of lipids and metabolites in tissues by nanospray desorption electrospray ionization mass spectrometry.

Three-dimensional (3D) imaging of tissue sections is a new frontier in mass spectrometry imaging (MSI). Here, we report on fast 3D imaging of lipids and metabolites associated with mouse uterine decidual cells and embryo at the implantation site on day 6 of pregnancy. 2D imaging of 16-20 serial tissue sections deposited on the same glass slide was performed using nanospray desorption electrospray ionization (nano-DESI)-an ambient ionization technique that enables sensitive localized analysis of analytes on surfaces without special sample pretreatment. In this proof-of-principle study, nano-DESI was coupled to a high-resolution Q-Exactive instrument operated at high repetition rate of >5Hz with moderate mass resolution of 35,000 (m/Δm at m/z 200), which enabled acquisition of the entire 3D image with a spatial resolution of ∼150μm in less than 4.5h. The results demonstrate localization of acetylcholine in the primary decidual zone (PDZ) of the implantation site throughout the depth of the tissue examined, indicating an important role of this signaling molecule in decidualization. Choline and phosphocholine-metabolites associated with cell growth-are enhanced in the PDZ and abundant in other cellular regions of the implantation site. Very different 3D distributions were obtained for fatty acids (FA), oleic acid and linoleic acid (FA 18:1 and FA 18:2), differing only by one double bond. Localization of FA 18:2 in the PDZ indicates its important role in decidualization while FA 18:1 is distributed more evenly throughout the tissue. In contrast, several lysophosphatidylcholines (LPC) observed in this study show donut-like distributions with localization around the PDZ. Complementary distributions with minimal overlap were observed for LPC 18:0 and FA 18:2 while the 3D image of the potential precursor phosphatidylcholine 36:2 (PC 36:2) showed a significant overlap with both LPC 18:0 and FA 18:2.