Linoleic Acid Isomers Research Articles

An accurate analytical method based on gas chromatography-mass spectrometry for quantifying all oleic, linoleic, and linolenic acid isomers, including trans-form and cis–trans-mixed isomers, in food

An accurate analytical method based on gas chromatography-mass spectrometry for quantifying all oleic, linoleic, and linolenic acid isomers, including trans-form and cis–trans-mixed isomers, in food

Novel Strategies for Synthesis and Assessment of Dextran Conjugated 9Z,11E Linoleic Acid on Cancer Cell Lines Viability via Wnt/Beta‐Catenin Signaling Pathway and Apoptosis

AbstractConjugated linoleic acid (CLA) is one of the positional and structural isomers of linoleic acid. Ninety percent of the CLA contained in natural foods is the cis‐9(9Z), trans‐11(11E) isomer. CLA also has anti‐carcinogenic, anti‐adipogenic, and anti‐inflammatory properties. Limited studies aim to elucidate the impact of 9Z,11E‐CLA on Wnt/beta‐catenin signaling.In this study, we have selected MDA‐MB‐231, T98G, and SAOS‐2 cell lines of various cancer origins and investigate the impact of dextran conjugated 9Z,11E CLA (Dex‐9Z,11E‐CLA) using cell proliferation assay and immunocytochemical methods. Cytotoxic effects of Dex conjugated 9Z,11E CLA are assessed with the MTT method. It analyzed Wnt/beta‐catenin signaling changes using target molecule beta‐catenin, Dickkopf‐1 (Dkk‐1) as a negative regulator, and active caspase3 (CC3) as an apoptotic marker.Dex‐9Z,11E‐CLA is shown to suppress cell viability in three different types of cancer cells, and the most effective concentration is 200 µm. 9Z,11E‐CLA and Dex‐9Z,11E‐CLA affect the Wnt/beta‐catenin signaling pathway negatively in all cell lines. Beta‐catenin expression is decreased, Dkk‐1 expression is increased, and CC3 positivity is increased significantly compared to the control group. In conclusion, the data suggest that Dex‐9Z,11E‐CLA suppresses cell viability and causes apoptosis in cells via the Wnt/beta‐catenin signaling pathway. Lastly, Dex‐9Z,11E‐CLA harbors novel anticancer potential and exhibits uncontrolled activation of Wnt/beta‐catenin signaling.

The composition of linoleic acid and conjugated linoleic acid has potent synergistic effects on the growth and death of RAW264.7 macrophages: The role in anti-inflammatory effects

Linoleic acid (LA) is an omega-6 polyunsaturated fatty acid. Conjugated linoleic acid (CLA) is a family of LA isomers that includes both a trans fatty acid and a cis fatty acid. Both fatty acids play a nutritional role in maintaining health. Inflammation is critical in the pathogenesis of many diseases, including cancer. This study found that the combination of LA and CLA (LA/CLA), each of which had no effect, had a strong anti-synergistic effect on inflammatory macrophage RAW264.7 cells in vitro. Cells were cultured in a DMEM containing fetal bovine serum with or without either LA, CLA, or a combination of LA/CLA. The composition of LA and CLA at a comparatively lower concentration synergistically suppressed cell growth, resulting in a reduction in cell number. The underlying mechanism of this effect was based on reduced levels of Ras, PI3K, Akt, MAPK, and mTOR and elevated levels of p21, p53, and Rb, which are associated with cell growth. In addition, the combination of LA and CLA at a lower concentration stimulated potential cell death associated with increased caspase-3 and cleaved caspase-3 levels. Notably, this composition synergistically suppressed the production of TNF-α, IL-6, and PGE2, which are a major mediator of inflammation, with lipopolysaccharide stimulation in RAW264.7 cells This effect was associated with decreased levels of COX-1, COX-2, and NF-κB p65. This study may provide a useful tool for treating inflammatory conditions with the composition of LA and CLA.

Mussel Culture Farming Systems in the Northern Gargano Coast (Adriatic Sea): Changes in the Nutritional Profile of the Mytilus galloprovincialis.

The objective of this study was to investigate the nutritional characteristics of Mytilus galloprovincialis cultivated in three sites of the Northern Coastal Area of Gargano. The mussel farms were characterized by different distances of dropper ropes (extensive dropper ropes: EDR; intensive dropper ropes: IDR; semi-intensive dropper ropes: SIDR). Mussels' chemical composition, fatty acids, and amino acids profiles were investigated at monthly intervals, from April to June. The fat content of mussels from EDR and SIDR sites was lower than values observed for mussels of the IDR in both the April and June sampling months. An increase in the main saturated fatty acids (C15:0; C16:0; C17:0 and C18:0; p < 0.001) and polyunsaturated fatty acids (C18:2c9c12, C18:3n3, C20:2n6, C20:4n6, and C22:6n3; p < 0.001) was found in the mussels collected in June from all farms analyzed. In terms of farming systems, mussels from the EDR and SIDR sites exhibited the greatest content of beneficial molecules like conjugated linoleic acids isomers (p < 0.001), glutamic acid (p < 0.05), serine (p < 0.05), tryptophan (p < 0.001), hydroxyproline (p < 0.05) and proline (p < 0.01). This study revealed that the farming system can affect the nutritional composition of mussels and evidenced that EDR and SIDR are the most effective cultured farming methods in the Gargano area.

Growth performance, proximate composition and fatty acid profile of black soldier fly larvae reared on two grape pomace varieties

The black soldier fly (Hermetia illucens) is attracting increasing interest for its ability to convert low-value substrates into highly nutritious feed. This study aimed at evaluating grape pomace from two varieties (Becuet – B; Moscato – M) as rearing substrates for black soldier fly larvae (BSFL), focusing on the related effects on larval growth performance, proximate composition, and fatty acid profile. A total of six replicates per treatment, and 1 000 BSFL per replica, were used. Larval development was assessed by larvae weight, which was recorded eight times during the trial: the day after the beginning of the trial, and then on days 5, 8, 13, 15, 20, 22, and 27 (day in which the 30% of BSFL reached the prepupal stage). Production and waste reduction efficiency parameters, namely the growth rate, substrate reduction and substrate reduction index, were calculated. The two grape pomace varieties were analysed for their proximate composition and fatty acid profile; the same analyses were conducted on BSFL (30 larvae per replica) that were collected at the end of the trial (day 27). The growth rate of BSFL showed a higher value when the larvae were reared on B substrate (4.4 and 3.2 mg/day for B and M, respectively; P < 0.01). The rearing substrate did not significantly affect the proximate composition of BSFL. The percentage of total lipids (TL) in M-fed BSFL was significantly higher than in B ones. Total saturated (P < 0.001) and monounsaturated fatty acids (P < 0.05) were significantly higher in M-fed BSFL, while an opposite trend was observed for total branched-chain (P < 0.001) and total polyunsaturated fatty acids (P < 0.001). Interestingly, some conjugated linoleic acid (CLA) isomers [i.e., C18:2 c9t11(+t7c9+t8c10) and t9t11] were detected in low amounts in both rearing substrates (total CLA equal to 0.085 and 0.16 g/100 g TL in B and M substrate, respectively). Some CLA isomers (i.e., C18:2 c9t11, t7c9, and t10c12) were also found in BSFL, reaching a total CLA concentration equal to 2.95 and 0.052 g/100 g of TL in B-fed and M-fed BSFL, respectively. This study demonstrates that winery by-products from different grape varieties can significantly affect the development and lipid composition of BSFL. The CLA biosynthesis potential of BSFL opens newsworthy perspectives for a new valorisation of winery by-products to produce full-fat black soldier fly meal and black soldier fly oil enriched in specific fatty acids of potential health-promoting interest.

Screening and Identification of High-Yielding Strains of Conjugated Linoleic Acid and Optimization of Conditions for the Conversion of CLA.

Conjugated linoleic acid (CLA) is a class of naturally occurring octadecadienoic acid in humans and animals and is a general term for a group of conformational and positional isomers of linoleic acid. In order to obtain the development of excellent lactic acid strains with a high production of conjugated linoleic acid, 32 strains with a possible CLA conversion ability were obtained by initial screening using UV spectrophotometry, and then the strains were re-screened by gas chromatography, and finally, the strain with the highest CLA content was obtained. The strains were optimized for cultivation by changing the amount of substrate addition, inoculum amount, and fermentation time. The results showed that the yield of the experimentally optimized strain for the conversion of conjugated linoleic acid could reach 94.68 ± 3.57 μg/mL, which was 74.4% higher than the initial yield of 54.28 ± 2.12 μg/mL of the strain. The results of this study can provide some basis for the application of conjugated linoleic acid production by Lactobacillus paracasei in the fermentation of lactic acid bacteria.

Fat quality, sensory properties and consumers preference towards different commercial labels for bovine milk from retail market

SummaryIn the modern large‐scale retail trade, voluntary quality schemes play a fundamental role to communicate specific quality attributes of processes and products, from farms to the market. However, consumers are not always able to recognise such an added value for milk certifications. In this paper, we evaluated the differences in the lipid quality and sensory properties of four different commercial labels for retail milk by means of gas‐chromatographic analysis of fatty acids (FAs) and sensory assessment performed by a trained sensory panel. Meanwhile, we tested the effects of the four selected milk labels and information on consumers perception by means of an online experimental survey. Our results emphasised a different FAs composition of retail milk labelled under voluntary certifications. Polyunsaturated FAs of the n‐3 and n‐6 series (along with their ratios), branched chain FAs, trans isomers of C18:1 and conjugated isomers of linoleic acid were found at different concentrations in different milk labels, with the TSG Haymilk (HM) displaying the most favourable profile followed by the mountain product (MP) and then by High‐Quality fresh milk (HQ) and Extended Shelf‐Life milk (ESL) with no voluntary quality marks. Being strictly related to the farm production system, milk FAs revealed as natural markers of production system for certified milk. Quite no difference was found for the sensory properties, excepting for the overall aroma intensity, which was lower in MP. At the same time, we evidenced that the added value given by the distinct quality of fat for HM and MP is still not recognised by consumers, which perceived these milk labels as healthy, as tasty, as natural and as nutrient as HQ. This result highlighted that there is still room for producers aligned with the voluntary certifications HQ and MP to promote their products on the market and to raise consumer awareness about their quality attributes.

The analysis of lamb and beef fatty acids with a 30 m BPX70 column is comparable with a 120 m column

Gas chromatography (GC) analysis of fatty acid methyl esters (FAME) typically uses long capillary columns (≥ 100 m) which can be expensive and time consuming. The current study determined whether the major FAME of interest for human health in lamb and beef can be quantified using a 30 m compared with 120 m moderately polar capillary (BPX70) column. Lamb and beef samples with a range of intramuscular fat content from either grass- or grain-fed animals were analysed. All major FAME of human health importance, including omega-3 and omega-6 polyunsaturated fatty acids and conjugated linoleic acid (CLA) isomers, could be identified on the 30 m BPX70 column. The correlation (r2) for fatty acid percentage between columns was > 0.98 for all major n–3PUFA and > 0.86 for all major CLAs. When animals were fed a grain-based diet, C18:1n–7t (vaccenic acid) could not be resolved from C18:1n-8t when analysed on the 30 m column. The FAME profile of lamb and beef, particularly those important for nutritional composition tables, can be determined using a 30 m BPX70 column, thereby saving time and expense compared with longer columns. Particular care should be employed when analysing samples from grain-fed animals due to co-eluting of C18:1n–8t.

Production of Conjugated Linoleic Acid (CLA) by Lactiplantibacillus plantarum: A Review with Emphasis on Fermented Foods.

The term Conjugated Linoleic Acid (CLA) refers generically to a class of positional and geometric conjugated dienoic isomers of linoleic acid. Among the isomers of linoleic acid cis9, trans11-CLA (c9, t11-CLA) and trans10, cis12-CLA (t10, c12-CLA) are found to be biologically active isomers, and they occur naturally in milk, dairy products and meat from ruminants. In addition, some vegetables and some seafoods have also been reported to contain CLA. Although the CLA levels in these natural sources are insufficient to confer the essential health benefits, anti-carcinogenic or anti-cancer effects are of current interest. In the rumen, CLA is an intermediate of isomerization and the biohydrogenation process of linoleic acid to stearic acid conducted by ruminal microorganisms. In addition to rumen bacteria, some other bacteria, such as Propionibacterium, Bifidobacterium and some lactic acid bacteria (LAB) are also capable of producing CLA. In this regard, Lactiplantibacillus plantarum (formerly Lactobacillus plantarum) has demonstrated the ability to produce CLA isomers from linoleic acid by multiple enzymatic activities, including hydration, dehydration, and isomerization. L. plantarum is one of the most versatile species of LAB and the bacterium is widely used in the food industry as a microbial food culture. Thus, in this review we critically analyzed the literature produced in the last ten years with the aim to highlight the potentiality as well as the optimal conditions for CLA production by L. plantarum. Evidence was provided suggesting that the use of appropriate strains of L. plantarum, as a starter or additional culture in the production of some fermented foods, can be considered a critical factor in the design of new CLA-enriched functional foods.

Research Progress on the Mechanism of Milk Fat Synthesis in Cows and the Effect of Conjugated Linoleic Acid on Milk Fat Metabolism and Its Underlying Mechanism: A Review.

Milk fat synthesis in cows mainly includes the synthesis of short- and medium-chain fatty acids, the uptake, transport, and activation of long-chain fatty acids (LCFAs), the synthesis of triglycerides, and the synthesis of the genes, transcription factors, and signaling pathways involved. Although the various stages of milk fat synthesis have been outlined in previous research, only partial processes have been revealed. CLA consists of an aggregation of positional and geometric isomers of linoleic fatty acid, and the accumulated evidence suggests that the two isomers of the active forms of CLA (cis-9, trans-11 conjugated linoleic acid and trans-10, cis-12 conjugated linoleic acid, abbreviated as c9, t11-CLA and t10, c12-CLA) can reduce the fat content in milk by regulating lipogenesis, fatty acid (FA) uptake, oxidation, and fat synthesis. However, the mechanism through which CLA inhibits milk fat synthesis is unique, with most studies focusing only on the effects of CLA on one of the genes, transcription factors, or signaling pathways involved. In this study, we summarized the structure and function of classic genes and pathways (mTOR, SREBP, AMPK, and PPARG) and new genes or pathways (THRSP, METTL3, ELOVL, and LPIN1) involved in each stage of milk fat synthesis and demonstrated the interactions between genes and pathways. We also examined the effects of other substances (melanin, nicotinic acid, SA, etc.). Furthermore, we evaluated the influence of β-sitosterol, sodium butyrate, Met arginine, and Camellia oleifera Abel on milk fat synthesis to improve the mechanism of milk fat synthesis in cows and provide a mechanistic reference for the use of CLA in inhibiting milk fat biosynthesis.

Trans 10, cis 12-conjugated linoleic acid reduced reproductive ability by disrupting the estrus cycle in female mice

Trans 10, cis 12-conjugated linoleic acid reduced reproductive ability by disrupting the estrus cycle in female mice

Anti-neuroinflammatory effects of conjugated linoleic acid isomers, c9,t11 and t10,c12, on activated BV-2 microglial cells.

Conjugated linoleic acid (CLA) isomers exhibit anti-inflammatory properties within the central nervous system (CNS). This study investigated the effects of CLA isomers c9,t11 and t10,c12 on fatty acid (FA) and N-acylethanolamine (NAE) profiles and their association with pro-inflammatory molecule expression in BV-2 microglia cell line, the CNS's resident immune cells responsible for maintaining neuronal activity and immune homeostasis. BV-2 cells were treated with 25 μM of c9,t11-CLA, t10,c12-CLA, or oleic acid (OA) for 24 h, followed by lipopolysaccharide (LPS) stimulation. After treatment, the cell's FA and NAE profiles and pro-inflammatory molecule expression were analyzed. Our results demonstrated that CLA isomers mitigate LPS-induced morphological changes in BV-2 cells and reduce gene expression and protein levels of inflammatory markers. This effect was linked to an upregulation of acyl-CoA oxidase 1, a key enzyme in the anti-inflammatory peroxisomal beta-oxidation pathway that efficiently metabolizes CLA isomers. Notably, t10,c12-CLA significantly suppressed stearoyl-CoA desaturase 1, impacting monounsaturated fatty acid synthesis. The NAEs profile was remarkably altered by CLA isomers, with a significant release of the anti-neuroinflammatory mediator docosahexaenoic acid (DHA)-derived N-acylethanolamine (DHAEA). In conclusion, our findings suggest that the anti-neuroinflammatory effects of CLA isomers are due to their unique influences on FA metabolism and the modulation of bioactive FA-derived NAEs, highlighting a potential strategy for nutritional intervention in conditions characterized by neuroinflammation.

CONJUGATED LINOLEIC ACID AND FATTY ACID ISOMERS IN SELECTED COLD PRESSED OILS: ANALYSIS BY GC/FID TECHNIQUE

The conjugated linoleic acid (CLA) isomers in cold pressed oils [pomegranate seed oil (PGSO), linseed oil (LSO), black cumin seed oil (BCSO), nettle seed oil (NSO),grape seed oil (GSO), sesame seed oil (SSO),safflower oil (SFO), pumpkin seed oil (PSO), wheat germ oil (WGO), fig seed oil (FSO), coriander oil (CO), walnut oil (WO) and coconut oil (CNO)] extracted with lab–scale screw press machine were further subjected to gas chromatography/flame ionization detection (GC/FID) analysis. The composition of polyunsaturated fatty acids (PUFAs), monounsaturated fatty acids (MUFA) and saturated fatty acids (SFAs) of the samples was also determined. The five different positional and geometric isomers of CLA [cis–9, cis–11 CLA, cis–9, trans–11 CLA, trans–9, cis–11 CLA, trans–9, trans–11 CLA and trans–10, cis–12 CLA] were also well separated by a highly polar column (100m×0.2μm×0.25mm i.d; HP–88 cyanopropyl) and an applied GC temperature program. It was concluded that the samples were all rich in total CLA (∑CLA) and they were found between 0.14% for PSO and 2.11% for SSO. The most abundant CLA isomer was in general to be cis–9, trans–11 CLA form, which represented the content of isomer between 3.15% and 72.08% of ∑CLA. Besides, the ∑SFA values were detected between 2.43% and 93.14%, ∑MUFA values were between 4.60% and 71.11% and ∑PUFA values were between 1.79% and 87.59%. Therefore, this study might offer valuable information for the introduction of new food sources, as well as incorporation into medicinal purposes and food formulations which have the potential to be commercially valuable.

Effect of pomegranate seed oil on egg production, egg quality and yolk fatty acid deposition in laying hen.

Pomegranate seed oil (PSO) contains punicic acid as well as conjugated linolenic acid isomers, including α-eleostearic and catalpic acids, along with phytosterols, especially β-sitosterol, campesterol and stigmasterol, with lipotropic impact and egg fortifying effect in laying hens. The present experiment was designed to examine the effects of PSO on egg production, egg quality, blood lipids and yolk fatty acid deposition in laying hens. A total of 360 Hy-line laying hens (w-80), at 25 weeks of age, were randomly allotted to five dietary treatments in a completely randomized design during a 10-week period. Experimental treatments consisted of a basal diet or supplementation of 1, 2, 3 and 4gPSO/kg to basal diet. Performance indicators and fatty acid composition of egg yolk were evaluated during different experimental periods. Blood lipid attributes were evaluated at the end of the experiment. Dietary supplementation of 4gPSO/kg feed increased daily feed intake and egg production rate of laying hens (p<0.05). There was not any significant influence of experimental treatments on egg quality, whereas an increasing trend observed in egg yolk colour of hens received dietary graded levels of PSO. Dietary supplemental 4g/kgPSO increased the proportion of yolk poly-unsaturated fatty acid (PUFA) concentration (p<0.05). Furthermore, the PUFA to saturated fatty acid ratio increased after dietary supplementation of 1 or 4g/kgPSO (p<0.05). Serum concentration of cholesterol, triacylglycerol and low-density lipoprotein decreased in response to the supplementation of more than 3gPSO/kg in the feed (p<0.05). In conclusion, dietary supplementation with 4g/kgPSO improved production rate and decreased blood lipids in laying hens. Moreover, dietary supplemental PSO modified yolk fatty acid deposition without detrimental effects on the egg quality.

Screening of lactobacilli strains isolated from artisanal Poro de Tabasco cheese for their ability to biosynthesise conjugated linoleic acid under simulated gastrointestinal conditions

SummaryConjugated linoleic acid (CLA) is a mixture of linoleic acid isomers which have been shown to provide beneficial health effects; particularly, the isomers cis‐9, trans‐11 (c9‐t11) and trans‐10, cis‐12 (t10‐c12) are those with proven biological activity. Some lactic acid bacteria (LAB) have demonstrated the ability to synthesise CLA in culture media or in fermented milk, however, few studies have considered the possibility that LAB can continue to produce CLA in the human gut. Therefore, the objective of this study was to screen lactobacilli strains isolated from artisanal Poro de Tabasco cheese for their ability to biosynthesise CLA under simulated gastrointestinal digestion (SGD) and to evaluate their survival and gut colonisation capacity under these conditions. First of all, LAB strains were identified molecularly and their CLA biosynthesis capacity in culture media and under simulated gastrointestinal conditions were evaluated. Besides, bacterial survival to the gastrointestinal conditions and gut colonisation capacity by their adhesion and autoaggregation properties were assessed. Sixteen LAB strains were phenotypically and genotypically identified and were classified within the Lactiplantibacillus pentosus, Lactiplantibacillus plantarum, Limosilactobacillus fermentum and Companilactobacillus farciminis species. Nine strains were able to synthesise CLA in culture media supplemented with linoleic acid (7.19–18.78 μg mL−1; with a conversion rate of 0.43–1.13%) and seven of them achieved it under simulated gastrointestinal conditions (4.28–47.67 μg mL−1; with a conversion rate of 0.51–5.75%), with an outstanding performance of Lactiplantibacillus pentosus LC12. In addition, the viable counts demonstrated that all the study strains achieved to survive under gastric conditions with a high population survival rate of 94.5%, except for LC19 (67%); however, all the nine strains recovered their viability with a high capacity to colonise the gut. Therefore, they may be considered as potential probiotic CLA‐producing strains, mainly Lactiplantibacillus pentosus LC12 since it was able to produce the closest CLA concentration to Limosilactobacillus reuteri ATCC 55739 (positive control). Thus, these strains can be used as starter cultures for designing promising functional foods with beneficial claims associated to the CLA content in the food matrix or produced after consumed.

Short-communication: Study of fatty acid metabolites in microbial conjugated fatty acids-enrichment of milk and discovery of additional undescribed conjugated linolenic acid isomers

Microbially enriched food in conjugated linoleic (CLA) and conjugated linolenic (CLNA) acids is intensively studied nowadays. The conversion of linoleic (LA) and α-linolenic acids (α-LNA) into these compounds may involve different fatty acid (FA) intermediates. This research aimed to investigate potential FA byproducts in milk during microbial CLA/CLNA-enrichment using Bifidobacterium breve DSM 20091. Milk fermented with pure α-LNA showed a decrease in free myristic acid, while pure LA led to an increase in free stearic acid. No additional FA compounds were found alongside CLA/CLNA isomers. The strain produced several CLA isomers from LA, but only when administered alone. Nonetheless, when α-LNA was assayed, additional CLNA isomers, never reported before for bifidobacteria, were observed. In conclusion, except for stearic acid in the presence of LA, no side-FA metabolites were released during milk microbial CLA/CLNA-enrichment. Results suggest either CLA/CLNA production occurs in one single-step or intermediates biotransformation is very fast.

Gut microbial fatty acid isomerization modulates intraepithelial T cells.

The human gut microbiome constantly converts natural products derived from the host and diet into numerous bioactive metabolites1-3. Dietary fats are essential micronutrients that undergo lipolysis to release free fatty acids (FAs) for absorption in the small intestine4. Gut commensal bacteria modify some unsaturated FAs-for example, linoleic acid (LA)-into various intestinal FA isomers that regulate host metabolism and have anticarcinogenic properties5. However, little is known about how this diet-microorganism FA isomerization network affects the mucosal immune system of the host. Here we report that both dietary factorsand microbial factors influence the level of gut LA isomers (conjugated LAs (CLAs)) and that CLAs in turn modulate a distinct population of CD4+ intraepithelial lymphocytes (IELs) that express CD8αα in the small intestine. Genetic abolition of FA isomerization pathways in individual gut symbionts significantly decreases the number of CD4+CD8αα+ IELs in gnotobiotic mice. Restoration of CLAs increases CD4+CD8αα+ IEL levels in the presence of the transcription factor hepatocyte nuclear factor 4γ (HNF4γ). Mechanistically, HNF4γ facilitates CD4+CD8αα+ IEL development by modulating interleukin-18 signalling. In mice, specific deletion of HNF4γ in T cells leads to early mortality from infection by intestinal pathogens. Our data reveal a new role for bacterial FA metabolic pathways in the control of host intraepithelial immunological homeostasis by modulating the relative number of CD4+ T cells that were CD4+CD8αα+.

Determination of 39 fatty acids in liver of rats by gas chromatography-mass spectrometry

Fatty acids not only form phospholipids that contribute to the formation of cell membranes but also participate in many metabolic activities, such as energy storage and cell signal transduction. The liver plays a key role in the synthesis and metabolism of fatty acids. The composition and contents of fatty acids in the liver are closely related to body health. Most fatty acid-detection methods require a large sample size and can detect only a small number of fatty acids. Therefore, a sensitive and efficient method to determine fatty acids in the liver is urgently required. Herein, a method based on gas chromatography-mass spectrometry (GC-MS) was established for the simultaneous determination of 39 fatty acids in 1.1 mg of liver tissue. Different extraction methods and derivatization conditions were compared to develop an optimal sample-treatment method. The performance of two different columns in separating the target fatty acids were also compared. A total of 10 mg of liver was added to 450 μL of normal saline and ground at -35 ℃ to obtain a homogenate. Next, 50 μL of the homogenate (equivalent to 1.1 mg of liver) was added with 750 μL of chloroform-methanol (1∶2, v/v) to extract total fatty acids. The fatty acid extracts were dried under nitrogen, and then derivatized at 100 ℃ for 90 min after being added with methanol containing 5% sulfuric acid. The fatty acid methyl esters were extracted with hexane and then separated on an SP-2560 capillary column (100 m×0.25 mm×0.2 μm; Supelco, USA) via GC-MS. The results revealed that all 39 fatty acid methyl esters detected had good linearities in the certain mass concentration ranges with correlation coefficients (R2) greater than 0.9940. The limits of detection (LOD) and quantification (LOQ) of these methyl esters in the liver were 2-272 ng/mg and 7-906 ng/mg, respectively. The accuracy and precision of the method were evaluated by spiking the liver homogenate with tridecylic acid and eicosanoic acid at low (0.09 μg/mg), moderate (0.90 μg/mg), and high (5.40 μg/mg) concentration levels. The recoveries ranged from 82.4% to 101.0% with an intraday relative standard deviations (RSDs) (n=5) of 3.2%-12.0% and interday RSDs (n=3) of 5.4%-13.4%. The method was successfully applied to detect fatty acids in the livers of four healthy male Sprague-Dawley (SD) rats and four male SD rats with abnormal liver function induced by perfluorooctane sulfonate (PFOS). PFOS is a persistent organic pollutant. Twenty-six fatty acids were detected in the livers of both groups. Among the fatty acids investigated, pentadecanoic acid (C15∶0), γ-linolenic acid (C18∶3n6), and elaidic acid (C18∶1n9t) cannot be detected by the methods reported in the literature. By contrast, the method developed in this study could separate the isomers of oleic acid (elaidic acid, C18∶1n9t; oleic acid, C18∶1n9c) and linolenic acid (linolelaidic acid, C18∶2n6t; linoleic acid, C18∶2n6c). In conclusion, the developed method is simple and can detect a large number of fatty acids using small sample amounts and few reagents. More importantly, it could successfully separate fatty acid isomers. These findings indicate that the developed method is suitable for the detection of fatty acid composition and contents in the liver in clinical and experimental research.

In-Depth Chemical Characterization of Punica granatum L. Seed Oil.

Fruit seeds belonging to the pomegranate cultivar "Granata" were subjected to extraction and oily component analysis, with the aim of obtaining information about their composition. The presence of conjugated isomers of linolenic acid (CLNA isomers) in the oily phase extracted from the seeds gives a high added value to this part of the fruit, which is too often considered and treated as waste. The separated seeds were subjected to a classic Soxhlet extraction with n-hexane or extraction with supercritical CO2, assisted by ethanol. The resulting oils were evaluated by 1H and 13C-NMR and AP-MALDI-MS techniques. Differences in the triacylglycerols composition, with particular regard to punicic acid and other CLNA content, were studied in depth. Results showed the prevalence of punicic acid in the triacylglycerol mixture up to the 75%, with clear preponderance in the extract by supercritical fluids. Consequently, other CLNA isomers are, altogether, two-fold less represented in the supercritical extract than in the Soxhlet one. The two oily residues were subjected to solid phase extraction (SPE) and to HPLC-DAD analysis for the polyphenolic isolation and characterization. In addition to HPLC analysis, which showed different content and composition, DPPH analysis to evaluate the antiradical potential showed that the extract obtained with supercritical CO2 was much more active.

Conjugated linoleic acid trans-10, cis-12 increases the expression of genes from cell cycle progression and cis-9, trans-11 stimulates apoptotic genes in different mammary tumor explants of female dogs.

The conjugated linoleic acid (CLA) isomer cis-9, trans-11 is an anticarcinogen that inhibits cell proliferation and/or induces apoptosis of tumor cells. The objective of this study was to evaluate the expression of genes responsible for cell cycle regulation and apoptosis in tumor explants of mammary anaplastic carcinoma (AC) and mammary tubulopapillary carcinoma (TC) cultured in vitro with the CLA isomers cis-9, trans-11 and trans-10, cis-12. In this study we used mammary explants from two adult female dogs that revealed two types of malignant tumors: (a) anaplastic mammary carcinoma (AC) and (b) mammary tubulopapillary carcinoma (TC). The explants (n = 6 per treatment) had an average weight of 80.0 ± 2.0mg and were cultured for 24h in 35mm culture plates under the following treatments: (a) Control: Culture medium + fatty acid free bovine serum albumin (BSA); (b) Culture medium + cis-9, trans-11 CLA (75 µM) diluted with fatty acid free bovine serum albumin (BSA), and; (c) Culture medium + trans-10, cis-12 CLA (75 µM) diluted with fatty acid free bovine serum albumin (BSA). After that, total RNA was extracted, complementary DNA was synthesized (cDNA), and quantitative analysis by real-time polymerase chain reaction (RT-qPCR) was conducted. Data were analyzed using the MIXED procedure of SAS. Compared with the Control, the CLA trans-10, cis-12 treatment decreased expression of the gene encoding the p53 by 20% (P = 0.02), Caspase-3 by 25% (P = 0.06) and Bax by 51% (P = 0.001) in AC. The CLA cis-9, trans-11 increased the gene expression of proapoptotic protein Bax in TC by 68% (P = 0.01), but increased the expression of the antiapoptotic Bcl2 gene in AC by 72% (P = 0.006). The CLA cis-9, trans-11 stimulates apoptotic genes in mammary tubulopapillary carcinoma, but has a contrary effect on the anaplastic carcinoma, and the CLA trans-10, cis-12 stimulates cell cycle progression genes and may have an antiapoptotic effect, mainly in mammary anaplastic carcinoma.