Characterisation of a cell wall-anchored protein of Staphylococcus saprophyticus associated with linoleic acid resistance

The Gram-positive bacterium Staphylococcus saprophyticus is the second most frequent causative agent of community-acquired urinary tract infections (UTI), accounting for up to 20% of cases. A common feature of staphylococci is colonisation of the human skin. This involves survival against innate immune defenses including antibacterial unsaturated free fatty acids such as linoleic acid which act by disrupting bacterial cell membranes. Indeed, S. saprophyticus UTI is usually preceded by perineal skin colonisation.

Autoxidation of conjugated linoleic acid methyl ester in the presence of α-tocopherol: the hydroperoxide pathway

The autoxidation of conjugated linoleic acid (CLA) is poorly understood in spite of increasing interest in the beneficial biological properties of CLA and growing consumption of CLA-rich foods. In this thesis, the autoxidation reactions of the two major CLA isomers, 9-cis,11-trans-octadecadienoic acid and 10-trans,12-cis-octadecadienoic acid, are investigated. The results contribute to an understanding of the early stages of the autoxidation of CLA methyl ester, and provide for the first time a means of producing and separating intact CLA methyl ester hydroperoxides as well as basic knowledge on lipid hydroperoxides and their hydroxy derivatives. Conjugated diene allylic monohydroperoxides were discovered as primary autoxidation products formed during autoxidation of CLA methyl esters in the presence and absence of α-tocopherol. This established that one of the autoxidation pathways of CLA methyl ester is the hydroperoxide pathway. Hydroperoxides were produced from the two major CLA methyl esters by taking advantage of the effect of α-tocopherol to promote hydroperoxide formation. The hydroperoxides were analysed and separated first as methyl hydroxyoctadecadienoates and then as intact hydroperoxides by HPLC. The isolated products were characterized by UV, GC-MS, and NMR techniques. In the presence of a high amount of α-tocopherol, the autoxidation of CLA methyl ester yields six kinetically-controlled conjugated diene monohydroperoxides and is diastereoselective in favour of one particular geometric isomer as a pair of enantiomers. The primary autoxidation products produced from the two major CLA isomers include new positional isomers of conjugated diene monohydroperoxides, the 8-, 10-, 12-, and 14-hydroperoxyoctadecadienoates. Furthermore, two of these new positional isomers have an unusual structure for a cis,trans lipid hydroperoxide where the allylic methine carbon is adjacent to the cis instead of the usual trans double bond. The 1H and 13C NMR spectra of nine isomeric methyl hydroxyoctadecadienoates and of ten isomeric methyl hydroperoxyoctadecadienoates including the unusual cis,trans hydroperoxides, i.e. Me 8-OOH-9c,11t and Me 14-OOH-10t,12c, were fully assigned with the aid of 2D NMR spectroscopy. The assigned NMR data enabled determination of the effects of the hydroxyl and hydroperoxyl groups on the carbon chemical shifts of CLA isomers, identification of diagnostic signals, and determination of chemical shift differences of the olefinic resonances that may help with the assignment of structure to as yet unknown lipid hydroperoxides either as hydroxy derivatives or as intact hydroperoxides. A mechanism for the hydroperoxide pathway of CLA autoxidation in the presence of a high amount of α-tocopherol was proposed based on the characterized primary products, their relative distribution, and theoretical calculations. This is an important step forward in CLA research, where exact mechanisms for the autoxidation of CLA have not been presented before. Knowledge of these hydroperoxide formation steps is of crucial importance for understanding the subsequent steps and the different pathways of the autoxidation of CLA. Moreover, a deeper understanding of the autoxidation mechanisms is required for ensuring the safety of CLA-rich foods. Knowledge of CLA oxidation and how it differs from the oxidation of nonconjugated polyunsaturated fatty acids may also be the key to understanding the biological mechanisms of CLA activity.

Subcutaneous fat composition of youthful and mature Canadian beef: emphasis on individual conjugated linoleic acid and trans-18:1 isomers

[EN]A comprehensive evaluation of the fatty acid composition of subcutaneous adipose tissue from beef cattle produced in western Canada was undertaken to determine if the current Canadian grading system is able to distinguish classes of animals with value added potential due to their fatty acid composition. Grades included youthful Canadian Yield Grade 1 A/AA beef, under (YUTM) and over (YOTM) 30 mo of age and the four mature grades (D1, D2, D2 and D4). Subcutaneous fat between the 12th and 13th ribs over the longissimus muscle was obtained from 18_21 animals per grade. Fatty acids were analyzed using a combination of silver-ion HPLC and GC with a highly polar 100 m column. There were no differences in total trans-18:1 content amongst grades, but adipose tissue from grade D1, D2 and D4 had more 11t-18:1 than YUTM (PB0.05), whereas adipose tissue from YUTM carcasses had more 10t-18:1 than all other grades (PB0.05). Adipose tissue from YUTM carcasses also had less total CLA (PB0.05) than the D grades, mainly due to a lower level of 9c,11t-CLA, but they had slightly more 7t,9c-CLA and 10t,12c-CLA (PB0.05). Adipose tissue from YOTM and D grades contained more n-3 fatty acids relative to YUTM (0.56% vs. 0.29%; PB0.05) and lower n-6:n-3 ratios (PB0.05). Overall, older animals (YOTM and D grades) had adipose tissue compositions with higher levels of fatty acids with reported health benefits. Taken together, these higher levels may provide opportunities for value added marketing if regulatory authorities allow claims for their enrichment based on demonstrated health benefits. Higher concentrations of beneficial fatty acids, however, need to be considered within the context of the complete fatty acid profile and it would be important to demonstrate their advantages in the presence of relatively high levels of saturated fatty acids.

Length of concentrate finishing affects the fatty acid composition of grass-fed andgenetically lean beef: an emphasis on trans-18:1 and conjugated linoleic acid profiles

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Effect of dietary linolenic acid/linoleic acid ratio on growth performance, hepatic fatty acid profiles and intermediary metabolism of juvenile yellow catfish Pelteobagrus fulvidraco

The present study aimed to evaluate the effect of dietary linolenic acid (LNA)linoleic acid (LA) ratio on growth performance, hepatic fatty acid profile and intermediary metabolism of juvenile yellow catfish Pelteobagrus fulvidraco. Six isonitrogenous and isolipidic diets were formulated to contain incremental levels of LNA from 0 to 5% at the expense of corn oil (rich in LA), resulting in six dietary treatments with LNA to LA ratios ranging from 0.35 to 14.64. The experiment continued for 7 weeks. Best growth and feed intake were obtained in the fish fed the diets containing the LNA/LA ratios of 1.17 and 2.12 (P<0.05). In contrast, feed conversion ratio was the lowest for fish fed the diets containing the LNA/LA ratios of 1.17 and 2.12 (P<0.05). Dietary LNA to LA ratios significantly influenced viscerosomatic index and hepatosomatic index (P<0.05), but not condition factor (P>0.05). Body composition was also significantly influenced by dietary LNA to LA ratios (P<0.05). Generally, liver FA compositions reflected dietary FA profiles. Declining LA and increasing LNA contents in liver were observed with the increasing dietary LNA/LA ratios (P<0.05). Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) increased with the increasing LNA to LA ratios, suggesting that yellow catfish could elongate and desaturate C18 polyunsaturated fatty acids into highly unsaturated fatty acids. As a consequence, the n-6 fatty acids (FA) declined, and total n-3 FA and n-3/n-6 ratios increased with the dietary ratios of LNA/LA (P<0.05). Dietary LNA to LA ratios significantly influenced several enzymatic activities involved in liver intermediary metabolism (P<0.05), such as lipoprotein lipase, hepatic lipase, pyruvate kinase, succinate dehydrogenase, malic dehydrogenase and lactate dehydrogenase, suggesting that dietary LNA/LA ratios had significant effects on nutrient metabolism in the liver. To our knowledge this is the first demonstration of the effects of dietary LNA to LA ratios on the enzymatic activities of liver in fish, which provides information on diet quality and utilization, and can also be used as an indicator of the nutritional status of this fish. (C) 2009 Elsevier B.V. All rights reserved.

Linoleic acid increases monocyte chemotaxis and adhesion to human aortic endothelial cells through protein kinase C- and cyclooxygenase-2-dependent mechanisms

The effects of polyunsaturated n-6 linoleic acid on monocyte-endothelial interactions were investigated with particular emphasis on the expression of platelet/endothelial cell adhesion molecule (PECAM)-1 and the role of protein kinase C (PKC) and cyclooxygenase-2 (COX-2). As a diet rich in polyunsaturated fatty acids may favour atherosclerosis in hyperglycaemia, this study was performed in both normal and high-glucose media using human aortic endothelial cells (HAEC). The HAEC were preincubated with normal (5 mM) or high (25 mM) d-glucose for 3 days before addition of fatty acids (0.2 mM) for 3 days. Linoleic acid enhanced PECAM-1 expression independently of tumor necrosis factor (TNF)-a and significantly increased TNF-a-induced monocyte adhesion to HAEC in comparison to the monounsaturated n-9 oleic acid. Chronic glucose treatment (25 mM, 6 days) did not modify the TNF-a-induced or fatty acid-induced changes in monocyte binding. The increase in monocyte binding was accompanied by a significant increase in E-selectin and vascular cell adhesion molecule (VCAM)-1 expression and could be abrogated by an interleukin (IL)-8 neutralising antibody and by the PKC and COX inhibitors. Inhibition of PKC-d reduced VCAM-1 expression regardless of experimental condition and was accompanied by a significant decrease in monocyte binding. Conditioned medium from linoleic acid-treated HAEC grown in normal glucose conditions significantly increased THP-1 chemotaxis. These results suggest that linoleic acid-induced changes in monocyte chemotaxis and subsequent binding are not solely mediated by changes in adhesion molecule expression but may be due to secreted factors such as IL-8, monocyte chemoattractant protein-1 or prostaglandins (PGs) such as PGE2, as IL-8 neutralisation and COX-2 inhibition reduced monocyte binding without changes in adhesion molecule expression.

Conjugated linoleic acid and atherosclerosis: no effect on molecular markers of cholesterol homeostasis in THP-1 macrophages

Macrophage cholesterol homeostasis is a key process involved in the initiation and progression of atherosclerosis. Peroxisome proliferator-activated receptors (PPARs) regulate the transcription of the genes involved in cholesterol homeostasis and thus represent an important therapeutic target in terms of reducing atherosclerosis. Conjugated linoleic acid (CLA) is a potent anti-atherogenic dietary fatty acid in animal models of atherosclerosis and is capable of activating PPARs in vitro and in vivo. Therefore, this study examined whether the anti-atherogenic effects of CLA in vivo could be ascribed to altered cholesterol homeostasis in macrophages and macrophage derived foam cells. Of several genes that regulate cholesterol homeostasis investigated, CLA had most effect on the class B scavenger receptor CD36. The cis-9,trans-11 CLA (c9,t11-CLA) and trans-10,cis-12 CLA (t10,c12-CLA) isomers augmented CD36 mRNA expression (P

Analysis of the contribution of the β-oxidation auxiliary enzymes in the degradation of the dietary conjugated linoleic acid 9-cis-11-trans-octadecadienoic acid in the peroxisomes of Saccharomyces cerevisiae

Beta-oxidation of the conjugated linoleic acid 9-cis,11-trans-octadecadienoic acid (rumenic acid) was analyzed in vivo in Saccharomyces cerevisiae by monitoring polyhydroxyalkanoate production in the peroxisome. Polyhydroxyalkanoate is synthesized by the polymerization of the beta-oxidation intermediates 3-hydroxyacyl-CoAs via a bacterial polyhydroxyalkanoate synthase targeted to the peroxisome. The amount of polyhydroxyalkanaote synthesized from the degradation of rumenic acid was found to be similar to the amount synthesized from the degradation of 10-trans,12-cis-octadecadienoic acid, oleic acid or 10-cis-heptadecenoic acid. Furthermore, the degradation of 10-cis-heptadecenoic acid was found to be unaffected by the presence of rumenic acid in the media. Efficient degradation of rumenic acid was found to be independent of the Delta(3,5),Delta(2,4)-dienoyl-CoA isomerase but instead relied on the presence of Delta(3),Delta(2)-enoyl-CoA isomerase activity. The presence of the unsaturated monomer 3-hydroxydodecenoic acid in polyhydroxyalkanoate derived from rumenic acid degradation was found to be dependent on the presence of a Delta(3),Delta(2)-enoyl-CoA isomerase activity. Together, these data indicate that rumenic acid is mainly degraded in vivo in S. cerevisiae through a pathway requiring only the participation of the auxiliary enzymes Delta(3),Delta(2)-enoyl-CoA isomerase, along with the enzyme of the core beta-oxidation cycle.

Incorporation of cis-9,trans-11 or trans-10,cis-12 conjugated linoleic acid into plasma and cellular lipids in healthy men

This study investigated the incorporation of cis-9,trans-11 conjugated linoleic acid (c9,t11 CLA) and trans-10,cis-12-CLA (t10,c12 CLA) into plasma and peripheral blood mononuclear cell (PBMC) lipids when consumed as supplements highly enriched in these isomers. Healthy men (n = 49, age 31 +/- 8 years) consumed one, two, and four capsules containing similar to600 mg of either c9,t11 CIA or t10,c12 CLA per capsule for sequential 8 week periods followed by a 6 week washout before consuming the alternative isomer. Both isomers were incorporated in a dosedependent manner into plasma phosphatidylcholine (PC) (c9,t11 CLA r = 0.779, t10,c12 CLA r = 0.738; P < 0.0001) and cholesteryl ester (CE) (c9,t11 CLA r = 0.706, t10,c12 CLA r = 0.788; P < 0.0001). Only t10,c12 CLA was enriched in plasma nonesterified fatty acids. Both c9,t11 CIA and t10,c12 CLA were incorporated linearly into PBMC total lipids (r = 0.285 and r = 0.273, respectively; P < 0.0005). The highest concentrations of c9,t11 CLA and t10,c12 CLA in PBMC lipids were 3- to 4-fold lower than those in plasma PC and CE. These data suggest that the level of intake is a major determinant of plasma and PBMC CLA content, although PBMCs appear to incorporate both CLA isomers less readily.

Incorporation of cis-9, trans-11 conjugated linoleic acid and vaccenic acid (trans-11 18 : 1) into plasma and leucocyte lipids in healthy men consuming dairy products naturally enriched in these fatty acids

The present study investigated whether consuming dairy products naturally enriched in cis-9, trans-11 (c9,t11) conjugated linoleic acid (CLA) by modification of cattle feed increases the concentration of this isomer in plasma and cellular lipids in healthy men. The study had a double-blind cross-over design. Subjects aged 34-60 years consumed dairy products available from food retailers for 1 week and then either control (0.17 g c9,t11 CLA/d; 0.31 g trans-vaccenic acid (tVA)/d) or CLA-enriched (1.43 g c9,t11 CLA/d; 4.71 g tVA/d) dairy products for 6 weeks. After 7 weeks washout, this was repeated with the alternate products. c9,t11 CLA concentration in plasma lipids was lower after consuming the control products, which may reflect the two-fold greater c9,t11 CLA content of the commercial products. Consuming the CLA-enriched dairy products increased the c9,t11 CLA concentration in plasma phosphatidylcholine (PC) (38 %; P=0.035), triacylglycerol (TAG) (22 %; P < 0.0001) and cholesteryl esters (205 %; P < 0.0001), and in peripheral blood mononuclear cells (PBMC) (238 %; P < 0.0001), while tVA concentration was greater in plasma PC (65 %; P=0.035), TAG (98 %; P=0.001) and PBMC (84 %; P=0.004). Overall, the present study shows that consumption of naturally enriched dairy products in amounts similar to habitual intakes of these foods increased the c9,t11 CLA content of plasma and cellular lipids.

Effect of dietary vitamin A restriction on marbling and conjugated-linoleic acid (CLA) content in Holstein steers

To determine the effect of duration of dietary vitamin A restriction on site of fat deposition in growing cattle, 60 Holstein steers (BW = 218.4 ± 6.55 kg) were fed a diet based on high-moisture corn with 2,200 IU supplemental vitamin A/kg DM (C) or no supplemental vitamin A for a long (243 d; LR) or short (131 d; SR) restriction prior to harvest at 243 d. The SR steers were fed the C diet for the first 112 d. Steers were penned individually and fed for ad libitum intake. Jugular vein blood samples for serum retinol analysis were collected on d 1, 112, and 243. Carcass samples were collected for composition analysis. Subcutaneous fat samples were collected for fatty acid composition. Fat samples from the i.m. and s.c. depot were collected to measure adipocyte size and density. Feedlot performance (ADG, DMI, and G:F) was not affected (P > 0.05) by vitamin A restriction. On d 243, the i.m. fat content of the LM was 33% greater (P < 0.05) for LR than for SR and C steers (5.6 vs. 3.9 and 4.2% ether extract, respectively). Depth of back fat and KPH percentage were not affected (P = 0.44 and 0.80, respectively) by vitamin A restriction. Carcass weight, composition of edible carcass, and yield grade were similar among treatments (P > 0.10). Liver retinol (LR = 6.1, SR = 6.5, and C = 44.7 µg/g; P < 0.01) was reduced in LR and SR vs. C steers. On d 243, LR and SR steers had similar serum retinol concentrations, and these were lower (P < 0.01) than those of C steers (LR = 21.2, SR = 25.2, and C = 36.9 µg/dL). Intramuscular adipose cellularity (adipocyte/mm2 and mean adipocyte diameter) on d 112 and d 243 was not affected (P > 0.10) by vitamin A restriction. Restricting vitamin A intake for 243 d increased i.m fat percentage without affecting s.c. or visceral fat deposition, feedlot performance, or carcass weight. Restricting vitamin A intake for 131 d at the end of the finishing period appears to be insufficient to affect the site of fat deposition in Holstein steers.