Friday July 28th 2006

Concurrent Session 25: Obesity and Metabolic Disfunction

Chairs: Philippe Guesnet & Julie Lovegrove

CS 25.1 OMEGA-3 PUFA OF MARINE ORIGIN INDUCE A METABOLIC SWITCH AND STIMULATE ADIPONECTIN RELEASE IN MATURE WHITE ADIPOCYTES
P Flachs¹, O Horakova1, P Pecina1, N Franssen-van Hal2, M Rossmeisl1, J Keijer J2, V Mohamed-Ali3,J Houstek1, J Kopecky1. 1Institute of Physiology, ASCR, Prague, Czech Republic 2RIKILT - Institute of Food Safety, Wageningen, The Netherlands 3Department of Medicine, UCL, London, United Kingdom .

Background – Omega-3 PUFA of marine origin limit accretion of body fat and prevent insulin resistance in rodents fed high-fat diets. We investigated in mice the effect of omega-3 PUFA, eicosapentaenoic (EPA) and docosahexaenoic (DHA) acid on metabolism and secretory functions of white fat. Methods – Effects of EPA/DHA concentrate (6% EPA, 51% DHA, Pronova Biocare, Norway) admixed to high-fat diets was studied in C57BL/6J mice. Markers of lipid and glucose metabolism and adiponectin and leptin were measured in plasma. Microarrays, qRT-PCR and immunoblots were used to quantify transcripts and proteins. Fatty acid (FA) oxidation was measured in white adipose tissue (WAT) fragments. Adipokine production from WAT explants was evaluated. Glucose homeostasis was assessed using glucose tolerance test. Results – Expression screens revealed upregulation of genes for mitochondrial proteins, predominantly in epididymal fat by EPA/DHA concentrate. This was associated with 3-fold stimulation in expression of regulatory factors for mitochondrial biogenesis and oxidative metabolism, PGC-1 and NRF-1. Expression of PPAR alpha, carnitine palmitoyltransferase 1 and FA oxidation were increased in epididymal but not subcutaneous WAT. The changes in gene expression were detected due to a replacement of only 9 % of dietary lipids by EPA and DHA, while development of obesity was reduced. EPA/DHA concentrate increased plasma adiponectin levels, reflecting the stimulation of adipose expression and release of adiponectin, while preventing development of glucose intolerance. The induction of adiponectin was associated with increased expression of PPAR gamma and was independent on caloric intake and adiposity. Conclusion – The anti-obesity and anti-diabetic effects of n-3 PUFA include modification of metabolism and secretory functions of WAT.

CS 25.2 CONJUGATED LINOLEIC ACID SUPPLEMENTATION FOR 6 MONTHS DECREASED FAT MASS IN THE LEGS AND ABDOMEN OF OVERWEIGHT AND OBESE SUBJECTS .
JM Gaullier1, C Syvertsen1, J Halse2, HO Høivik3, S Einerhand⁴, M O’Shea4, O Gudmundsen1. 1Scandinavian Clinical Research AS, Kjeller, Norway; 2Diabetes and overweight Medical Center, Oslo, Norway; 3Hedmark Medical Center, Hamar; 4Lipid Nutrition, Loders Croklaan, Wormerveer, Netherlands .

Background - In humans the effects of conjugated linoleic acid (CLA) on body composition are not as well established as in animals, because thus far only a limited amount of well designed long term clinical trials have been conducted. Objectives - The present study aimed to assess the regional effects of CLA on body composition. Additionally, the safety of CLA was evaluated by monitoring adverse events and all changes in blood safety parameters. Design - In a randomized double-blind placebo controlled trial 118 overweight and obese subjects (BMI 28-32 kg/m2) received either 3.4g/d CLA or placebo-olive oil for 6 months. Outcomes - Results obtained on the PP population demonstrate a significant reduction in body fat mass by –5.6 % (P=0.004) for the CLA group when compared to the placebo group. Reduction of the fat mass was already observed after 3 months (-0.7 kg, P=0.034) in the CLA supplemented group, and the reduction was located in the legs and the abdomen of women whereas changes in men occurred essentially in the abdomen. Waist (-3.1 cm, P<0.001) and Waist/Hip ratio (-0.024, P=0.017) also decreased in the CLA supplemented group. Body weight and BMI reduced relative to placebo (-1.5 kg, P=0.05 and -0.6, P=0.05 respectively). Individuals with the highest BMI responded best to CLA. Conclusion - This study shows that CLA supplementation for 6 months in healthy, overweight and obese adults affected body fat mass in specific regions of the body and was well tolerated.
 

CS 25.3 PALMITATE INDUCES LIPOAPOPTOSIS OF EXOCRINE PANCREAS AR42J CELLS .
Z Landau, E Forti, M Alcaly, R.Z Birk. Ben-Gurion University of the Negev, Beer-Sheva 84105, Israel

Background - Chronic surplus of dietary consumption, typical to obesity, results in overflow of fat to non-adipose tissues. Intracellular accumulation of fat in non-adipose tissues is associated with cellular dysfunction and cell death and ultimately contributes to the pathogenesis of chronic diseases. The influence of fat overflow on the exocrine pancreas is not known. Objective - The purpose of this research was to study the effect of prolonged (72 hours) long chain saturated palmitic fatty acid (0.1mM) on the survival of exocrine pancreas AR42J cells. Design - Exocrine pancreas AR42J cells were treated with palmitic fatty acid (0.1mM) for 72 hours. The lipoapoptotic/toxic effect was studied using histological and molecular tools. Results - We demonstrate that chronic exposure of AR42J cells to palmitic acid results in significant increase in triglycerides accumulation (up to 25% of cells area), compared to untreated cultures. Lipid accumulation prompted a typical apoptotic process, demonstrated by both DNA fragmentation and condensed chromatin appearance (DAPI staining). Quantitative real-time PCR studies demonstrated that prolonged palmitic acid supplementation induced down-regulation of the anti-apoptotic Bcl2 mRNA levels (22%) and up-regulation of the pro-apoptotic Bax mRNA levels (300%), leading to disruption of the pro/anti apoptotic balance (Bax / Bcl2 =3). No major change was detected in iNOS mRNA expression. Conclusions - Prolonged exposure to saturated palmitic acid induces lipoapoptosis in exocrine pancreatic AR42J cells, through disturbance of the Bax/Bcl-2 balance.

CS 25.4 EFFECTS OF CHRONIC N-3 FATTY ACID SUPPLEMENTATION ON THE 24-HR PROFILE OF CIRCULATING TRIGLYCERIDE LEVELS IN OVERWEIGHT, MIDDLE-AGED MALES.
Bruce J Holub, Colin F Garrioch, Jessica J Danelon. Department of Human Health and Nutritional Sciences, University of Guelph, Guelph, Ontario, N1G 2W1 Canada.

Postprandial increases in circulating triglyceride (TG) levels and associated lipoproteins following a breakfast test meal exhibit peaking at 4-6 hours. Moreover, postprandial TG and lipoprotein increases are becoming recognized as independent risk factors for atherosclerosis and coronary events. The present study evaluated the effects of chronic omega-3 fatty acid (EPA/DHA) supplementation at 3 g/d over 21-days (taken at breakfast or supper) on the 24-hour serum levels of TG and the TG:HDL(C) ratio measured sequentially in overweight, male subjects given fixed meals. The 24-hour profiling revealed that the peak in circulating TG levels (control group) occurred at approx. 3 pm and again at 9 pm. The magnitude of the serum TG-lowering (%) and the reduced TG:HDL(C) ratios produced by omega-3 supplementation was found to be different at various postprandial times than for fasting values. The overall reduction in the 24-hr AUC (area under curve) values for TG in the fish oil groups was 32 % and 29 % for the incremental AUC relative to controls. No significant differences in the 24-hr TG-lowering efficacy were found with different timing of the EPA/DHA supplementation. These findings indicate the importance of 24-hour TG and TG:HDL(C) screening in risk assessment and opportunities via omega-3 fatty acid supplementation for modifying multiple and sustained postprandial surges.
(Supported by a grant from the Heart and Stroke Foundation of Ontario).

CS 25.5 DIETARY n-3 POLYUNSATURATED FATTY ACIDS AND ADIPOSE TISSUE FAT IN DEVELOPMENT OF OBESITY.
GV Skuladottir¹, JO Skarphedinsson1, AR Jonsdottir1, HB Schiöth2, L Jonsson1. 1Department of Physiology, Faculty of Medicine, University of Iceland. 2Department of Neuroscience, Uppsala BMC, Uppsala University, Sweden .

Background – Habitual food intake in excess of energy expenditure is one of the primary factors leading to obesity. The sources of dietary fatty acids (FA) have shown to affect growth of adipose tissue. Objective – To investigate the effects of hyperphagia of three types of dietary fat on body weight gain and FA composition of adipose tissue triacylglycerol (TAG). Design – Male Wistar rats (302±4 g; n=42) were fed diets for four weeks, which were similar in their total dietary energy (7.5 energy%), but differed in their content of FA. The saturated fat contained tallow (T diet; n-6 /n-3 polyunsaturated FA (PUFA), 10:1), the n-6 PUFA fat contained sunflower oil (S diet; n-6 /n-3 PUFA, 12:1), and the n-3 PUFA fat contained cod liver oil (CLO diet; n-6 /n-3 PUFA, 6:1). Hyperphagia and overweight condition of rats in all three dietary groups was induced by intracerebroventricular infusion of melanocortin receptor antagonist (HS024) for 14 days. Control rats were treated with artificial cerebrospinal fluid. Food intake and body weight were monitored, and at the end of the study the FA composition was analyzed in adipose tissue TAG. Outcomes – The cumulative energy intake during the 14-day treatment period was 50% higher in the hyperphagic rats resulting in a fourfold increase in body weight gain compared with controls. The adipose tissue TAG of hyperphagic rats fed CLO diet contained higher levels of long chain n-3 PUFA (P<0.05) than those fed T or S diet. No interaction was found between the effects of the dietary fat type and weight gain. The adipose tissue TAG of hyperphagic rats contained higher levels of saturated- and monounsaturated FA (P<0.05), and lower levels of n-3 and n-6 PUFA (P<0.05) than the control rats. Conclusions – This study shows that body weight gain due to hyperphagia is unaffected by the type of FA of food containing moderate energy% of fat. This study also demonstrate that FA synthesis stimulated in liver by excessive ingestion of nutrients leads to higher content of saturated- and monounsaturated FA in adipose tissue.

CS 25.6 MAMMALIAN DEVELOPMENT, MEMBRANE LIPID COMPOSITION AND THE ACTIVITY OF THE SODIUM PUMP.
P L Else¹, B J Wu2. Metabolic Research Centre, School of Health Sciences, University of Wollongong NSW 2522. Centre for Vascular Research, School of Medical Science, University of New South Wales, Sydney, NSW 2052.

During mammalian development from foetus to adulthood there is a several fold increase in Na+K+-ATPase activity that occurs in the brain (and kidney). This increase in activity is considered to be determined solely from increases in sodium pump density during development. Using rat brain and kidney microsomal membranes isolated from a developmental series from foetal through to adult rats suggests that changes in molecular activity (or molar activity, which is the rate at which an enzyme molecule turns over substrate) in addition to increases in sodium pump density may play a role. Molecular activity changes of 4 to 6 fold were measured in brain and kidney respectively. During this same period of development, membrane phospholipid fatty acid composition show large-scale changes, with large increases in the Unsaturation Index primarily driven by n-3 (from 10 to 20% of fatty acids) in brain and n-6 (from 25 to 47% of fatty acids) in kidney. Membrane lipid cross-over experiments between neonate and adults animals using brain microsomal membrane suggests that membrane lipid composition (particularly polyunsaturation) may play a role in altering the molecular activity (measured as ATP turnover/min) of the sodium pump during mammalian development.

Concurrent Session 26 : Child Cognition

CS 26.1 FORTIFICATION WITH DHA AND EPA DID NOT RESULT IN CONSISTENT EFFECTS ON COGNITIVE PERFORMANCE IN SCHOOL-AGED CHILDREN IN INDONESIA AND AUSTRALIA.
BWJ van Klinken¹, C Wilson2, W Lukito3, SJM Osendarp1 on behalf of the NEMO Study Group. 1 Unilever Food and Health Research Institute, Vlaardingen, The Netherlands. 2 CSIRO, Human Nutrition, Adelaide, Australia. 3 SEAMEO-TROPMED Regional Center for Community Nutrition, University of Indonesia, Jakarta, Indonesia.

Background - Essential fatty acids, DHA and EPA in particular, may improve cognitive performance in school-aged children. However, evidence for an effect in healthy school-aged children is very limited and it is not known whether these effects are different in well or marginally-nourished children. Objective - to assess the effect of DHA+EPA with or without micronutrients on indicators of cognitive performance in 396 children (6-10yr) in South Australia and 384 children in Indonesia. Design - We performed 2 two-by-two factorial randomized, controlled, double-blind trials in which children were randomly allocated to receive a drink with DHA+EPA (88 mg/d+22 mg/d), or a micronutrient mix (iron, zinc, folate, vitamin A, B6, B12, C), or both or placebo 6 days/week, for 12 months. Plasma n-3 fatty acids were determined at baseline and 12 months. Cognitive performance was measured at baseline, 6 and 12 months using a battery of tests to assess cognitive functions and school performance. Outcomes - The DHA/EPA treatment significantly increased plasma DHA and total plasma n-3 fatty acids in both Indonesian and Australian children. There were no treatment effects of DHA/EPA on factor scores based on analysis of all tests but a positive DHA/EPA treatment effect on a block design test was observed in Australian girls. In contrast in Indonesia, DHA/EPA treatment had a negative effect on scores for tests on digit backwards and RAVLT recognition. We will present additional analyses and discuss possible hypotheses for these findings at the meeting. Conclusion - In our study DHA/EPA fortification did not result in consistent effects on cognition in well- and marginally-nourished children.

CS 26.2 EFFECTS OF A FISH FLOUR-ENRICHED SPREAD ON COGNITION AND ABSENTEEISM IN SCHOOL CHILDREN: A RANDOMIZED CONTROLLED TRIAL.
CM Smuts¹, A Dalton1, RC Witthuhn2, P Wolmarans1. 1Nutritional Intervention Research Unit, MRC, Parow Valley, South Africa. 2Department of Food Science, University of Stellenbosch, Stellenbosch, South Africa.

Background - In humans omega-3 long-chain polyunsaturated fatty acids (LC-PUFA) play a well-documented role in brain development and function of the fetus and child. Objective - The aim of this placebo-controlled trial was to investigate the effect of a specially developed omega-3 fatty acid rich (from fish flour) bread spread (n-3 FABS) on cognition and disease frequency in primary school children (aged 6-9 years) from the Northern Cape, South Africa. Design - Children (n=355) were stratified by grade class and gender and randomly assigned to two groups, an experimental and a control group. Subjects from both groups received 2 slices of bread daily during school days for 6 months (104 school days). The bread of the experimental group (n=177) was spread with 25 g n-3 FABS while the control group’s bread (n=178) was spread with 25 g spread without omega-3 fatty acids. The n-3 FABS provided daily 273.8 ± 28.8 mg omega3 LC-PUFA. Trained test administrators under the supervision of a psychologist, conducted cognitive tests. Absenteeism from school was also recorded. Outcomes - The recognition and discriminating index scores of the Hopkins Verbal Learning Test (HVLT), as well as the spelling test showed significant intervention effects for the experimental group (p<0.05). The latter was also significantly less days absent from school. Conclusions - This study suggested that an omega-3 fatty acid-rich spread did not only improved verbal learning, memory and spelling ability of experimental subjects, but also lessened the number of days they were absent from school.

CS 26.3 POLYUNSATURATED FATTY ACIDS WITH HIGH RATIO OF EICOSAPENTAENOIC ACID IMPROVE ATTENTION AND BEHAVIOUR IN CHILDREN SUFFERING PROBLEMS WITH INATTENTION, HYPERACTIVITY AND IMPULSIVITY.
N Sinn^1,2, J Bryan1, CJ Wilson2. 1Psychology Department, University of South Australia, Magill, SA 5071. 2Human Nutrition, Commonwealth Scientific and Industrial Research Organisation, Adelaide 5000

Background – Previous researchers have found omega-3 polyunsaturated fatty acid (n-3 PUFA) deficiency in children with symptoms associated with attention deficit hyperactivity disorder (ADHD), and controlled trials have shown some improvements following PUFA supplementation although results have varied. Objective – To investigate the effect of supplementation with fish oil containing a high ratio of eicosapentaenoic acid (EPA) to docosahexaenoic acid (DHA) and including n-6 PUFA on subjective behavioural and objective cognitive outcomes in children with ADHD symptoms, and to determine whether fatty acid deficiency symptoms (FADS) at baseline were related to treatment outcomes. Design – 132 unmedicated children aged 7-12 with scores > 90th percentile on Conners’ ADHD Index participated in a 15 week placebo-controlled double-blind intervention trial, followed by a one-way crossover to active PUFA for a further 15 weeks. Outcome measures were Conners’ Parent Rating Scales and cognitive assessments. Outcomes – Significant improvements were seen with PUFA supplementation compared to placebo in parent ratings of inattention/cognitive problems, hyperactivity and impulsivity (N = 104) with medium effect sizes, and improvements continued with extended supplementation over 30 weeks. The same effects were mirrored in the placebo group following switch to active PUFA. Cognitive assessments (N = 129) showed significant medium treatment effects on attention control and vocabulary, and no effect on speed of processing, learning or memory. Baseline FADS were not related to any magnitude in improvements. Conclusions – Children with ADHD-related problems with behaviour and inattention may benefit from combined n-3 and n-6 PUFA supplementation with a high EPA:DHA ratio. Further research is required to investigate the effect of PUFA on other aspects of attention and cognitive function in these children. FADS are not a reliable indicator of response to PUFA treatment.

CS 26.4 DOSE EFFECT OF DOCOSAHEXAENOIC ACID ON SEVERITY OF ILLNESS IN THAI SCHOOLCHILDREN: A RANDOMISED DOUBLE BLIND PLACEBO CONTROLLED INTERVENTION TRIAL .
A Thienprasert¹, K Pattanapanyasat2, K Sukapirom2. 1Faculty of Science, Silpakorn University, Nakornpathom, Thailand. 2Faculty of Medicine, Siriraj Hospital, Mahidol University, Thailand .

Background – Our previous studies showed that Docosahexaenoic acid (DHA) supplementation could reduce incidence and severity of illness in schoolchildren. However, dose response of such effect is still lacking. Objective – To determine the dose response effects of DHA supplementation on incidence and severity of illness in Thai schoolchildren aged 6-12 years old. Design – A randomised double blind placebo controlled intervention trial was done in a cohort of 220 Thai schoolchildren. They were randomised allocated in 5 groups to receive either 1000, 600, 300, 100 mg DHA or placebo (4g of 100% Soybean oil) mixed with school lunch (e.g.; stir fry, curry soup or desserts) on every schooldays for 4 months. Incidence of illness like upper respiratory tract infection was counted and severity was measured by counting days with symptoms or absent from school. Fatty acids profiles in red blood cells were also determined. 24-hours Dietary recall information was collected twice during this study. Flow cytometric immunophenotype was also measured to monitor immune response. Outcomes – Duration of illness was negatively correlated to dose of DHA supplemented. (r2 =-0.9766) 1000, 600, 300 and 100 mg of DHA reduced the duration of illness by 31.49%, 22.85%, 9.61% and 9.00%, respectively. DHA level in red blood cells of supplemented groups increased significantly after intervention (p<0.05) T-lymphocytes also increased significantly in supplemented groups. (p<0.005) Conclusions – DHA supplementation can reduce the severity of common illness found in childhood and has the dose response effect on duration of illness. It is anticipated that DHA supplementation may enhance immune response in schoolchildren.

CS 26.5 EFFECTS OF FISH OIL ON MALARIA INFECTION AND BEHAVIOR: A PLACEBO-CONTROLLED, DOUBLE-BLIND TEST IN INDONESIAN CHILDREN .
Kei Hamazaki¹, Din Syafruddin2, Insan S Tunru3, Marina F Azwir4, Tomohito Hamazaki1. 1Institute of Natural Medicine, University of Toyama, Toyama 930-0194, Japan. 2Eijkman Institute for Molecular Biology, Jakarta 10430, Indonesia. 3Faculty of Medicine, Hasanuddin University, Makassar 90245, Indonesia. 4Hanura Primary Health Center, Lampung Province, Indonesia .

Background – Fish oils are known to increase survival rates in malaria-infected mice. However their antimalarial property has never been explored in human being, particularly, children in malaria-endemic areas. Likewise, its effect on behavior in normal children was rarely studied. Objective – To observe the effects of fish oil on malaria infection, behavior and attendance rates in school children. Design – Fourth, fifth and sixth graders of an elementary school in Lampung Province, Indonesia, were randomly divided into either a DHA group (n=116) or a control group (n=117) in a double-blind manner. The subjects in the DHA group took 6 fish oil capsules (0.65g DHA and 0.10g EPA/day) for 3 months. Controls took control capsules. Blood was taken at the start and end of the study, and when subjects showed malaria symptoms. An aggression questionnaire for children (HAQ-C of the Buss-Perry Aggression Questionnaire) was administered at the start and end of the study. The records of attendance during the study period were surveyed. Outcomes – Malaria was diagnosed with blood smears in 4 and 8 children in the DHA and control groups, respectively, at the end of the study (p=0.2). Behavior checked with HAQ-C did not show any difference between groups. However, the odds ratio of those who could not attend perfectly during the study period was 0.42 (95%CI: 0.23-0.74) in the DHA group compared with controls (p=0.003). Conclusions – Fish oil may improve the school attendance rate of children

Concurrent Session 27 Inflammation 1

Chairs: Kristian Bjerve & Lotte Lauritzen

CS 27.1 COLITIS INCREASES ARACHIDONIC AND DOCOSAHEXAENOIC ACIDS IN RAT COLON CHOLINE PHOSPHOGLYCERIDES
¹Dequan Zhou, 1Kebreab Ghebremeskel, 1Michael A Crawford, 2 Ram Reifen. 1The Institute of Brain Chemistry and Human Nutrition, London Metropolitan University, UK. 2School of Nutritional Sciences, The Hebrew University of Jerusalem, Israel

Aim - To investigate whether arachidonic (AA), di-hommo-gamma-linolenic (DHGLA) and eicosapentaenoic (EPA) acids, the precursors of eicosanoids that play a vital role in the regulation of inflammation, are altered in targeted tissue of colitic rats. Methods - Wistar male rats (n=24) were randomly divided into two groups and fed alpha-linolenic (ALA) sufficient (ALAS, n=12) or deficient (ALAD, n=12) diet. After four weeks of feeding, each group was divided in to two sub-groups, viz. ALASC (n=6), ALASNC (n=6), ALADC (n=6) and ALADNC (n=6). Colitis was induced by administration of trinitrobenzene sulphate in the colon of the ALASC and ALADC rats. Twenty-four hours after the induction of colitis, all of the rats were sacrificed and colon removed for analysis of choline phosphoglyceride fatty acids. Results - The ALASC compared with the ALASNC rats had higher levels of AA (P<0.05) and docosahexaenoic acid (DHA, P<0.01). Similarly, the ALA deficient colitic group (ALADC) had elevated DHA than the ALADNC (P<0.005). Total mono-unsaturated fatty acids were significantly reduced in both colitic groups (p<0.05). There was no difference in EPA or DHGLA between the colitic and non-colitic rats (P>0.05). Conclusion - In this study, the levels of AA, DHGLA and EPA were not reduced in the inflamed colon of the experimental rats. This suggests, in this model of colitis, eicosanoids do not play a significant role in the inflammation response.

CS 27.2 COMPARISON OF THE EFFECTS OF SHORT-TERM DUODENAL ADMINISTRATION OF WHALE AND SEAL OILS ON JOINT PAIN IN PATIENTS WITH INFLAMMATORY BOWEL DISEASE: A DOUBLE BLIND RANDOMIZED CONTROLLED STUDY.
Tormod Bjørkkjær^1,2, Pedro Araujo1, Arnold Berstad3, Livar Frøyland1. 1National Institute of Nutrition and Seafood Research (NIFES), Bergen, Norway. 2Department of Biomedicine, University of Bergen, Bergen, Norway. 3Division of Gastroenterology, Institute of Medicine, Haukeland University Hospital, Bergen, Norway.

Background – Rheumatic complications reduce the health related quality of life (HRQOL) of patients with inflammatory bowel disease (IBD). Nonsteroidal anti-inflammatory drugs are poorly tolerated, and safe and effective treatments are lacking. In recent short-term studies, duodenal administration of seal oil (SO) ameliorated joint pain in these patients. Objective – To compare the effects of short-term duodenal administration of whale oil (WO) and SO on IBD-related joint pain and IBD-activity. Design – Twenty-one patients with IBD-related joint pain were randomized to WO (n = 11) and SO (n = 10) treatment. The patients abstained from eating seafood/n-3 PUFAs one week before and during the 10-day administration. Ten ml SO or WO were self-administrated 3 times daily in a double blind design. The effects were assessed by questionnaires, faecal calprotectin, and PGE2 and fatty acids in plasma. Outcomes – Both groups improved during treatment, without any significant group differences. Levels of n-3 PUFAs in plasma increased and PGE2 was generally reduced (PWO = 0.09, PSO = 0.005). Scores on visual analogue scales for joint pain (PWO = 0.006, PSO = 0.003), back pain (PWO = 0.03, PSO = 0.046), and total influence of pain (PWO = 0.001, PSO = 0.002) last week were reduced. Reductions in short-form Nepean dyspepsia index (SF-NDI) total score (both P = 0.01), daily activity interference sub score (PWO = 0.02, PSO = 0.009), and tension sub score (PWO = 0.04) were observed. IBD-activity (sum of Harvey Bradshaw simple index and Walmsley simple clinical colitis activity index) was generally reduced (PWO = 0.003, PSO = 0.051). Conclusions – For short-term duodenal administration, WO is as effective as SO in ameliorating IBD-related joint pain, reducing IBD-symptoms and improving HRQOL without adverse effects.

CS 27.3 IMMUNOLOGICAL EFFECTS OF CIS-9, TRANS-11 CONJUGATED LINOLEIC ACID IN ALLERGIC SUBJECTS

AM Turpeinen¹, N Ylönen1, A Aro2. 1Dept Applied Chemistry & Microbiology (Nutrition), University of Helsinki, Finland. 2National Public Health Institute, Helsinki, Finland .

Background - Animal studies suggest that conjugated linoleic acid (CLA) modulates the immune response, while studies in healthy humans have shown only modest effects. However, the effects of CLA may be more prominent in situations of immune imbalance, such as allergy. Objective - To study the effects of the natural CLA isomer, cis-9, trans-11 CLA, in subjects with birch pollen allergy. Design - Forty subjects (20-46 years) with diagnosed birch pollen allergy were randomized to receive 2 g/d cis-9, trans-11 CLA (n=20) or placebo (high-oleic acid sunflower oil) (n=20) for 12 weeks. The supplementation began eight weeks before the birch pollen season and continued until the end of the season. Allergy symptoms were recorded daily. Blood samples collected before and after supplementation were analyzed for production of cytokines involved in allergic reponses. Outcomes - Subjects in the CLA group reported significantly less sneezing (p<0.05). There was also a tendency to less total nasal symptoms in the CLA group (p=0.07). The production of IL-4, IL-5, IL-6, IL-10, IL-13 and IFN-γ increased in both the CLA and placebo groups during the pollen season. Production of TNF-α was significantly lower in the CLA groups (p<0.01), while IFN-γ production was greater in the placebo group (p<0.05). The IFN-γ/IL-4 ratio, reflecting the Th1/Th2 balance, did not differ between groups. CLA supplementation decreased IL-5 production (p<0.05), but tended to increase production of IL-10 (p=0.06). No significant effects of CLA on IL-4, IL-6 and IL-13 were observed. Conclusions - The results suggest that the natural CLA isomer, cis-9,trans-11 CLA, exerts anti-inflammatory effects in allergic subjects.

CS 27.4 Paper withdrawn

CS 27.5 POSSIBILITY OF ORAL EICOSAPENTAENOIC ACID THERAPY FOR ENDOMETRIOSIS .
Sachiho Netsu¹, Ryo Konno1, Kohei Odagiri2, Masaaki Soma,Hiroyuki Fujiwara2,, Mitsuaki Suzuki2. 1Department of Gynecology, Jichi Medical University Omiya Medical Center, Saitama, Japan. 2Department of Gynecology, Jichi Medical University,Tochigi, Japan. 3Mochida Pharmaceutical Co., Ltd., Tokyo, Japan

Aims - We investigated anti-inflammatory effect of ?3-eicosapentaenoic acid (EPA) compared with ?6-linoleic acid (LA) in an endometriosis rat model, focusing on the relationship between lipid metabolism and inflammatory reactions, based on the hypothesis that excessive lipid intake is one factor responsible for the recent increase of endometriosis patients. Methods - SD rats (female, 6 weeks old) were fed a diet with EPA ethyl ester (EPA group, n = 9) or with LA ethyl ester (LA group, n = 9) for total 6 weeks. After 2 weeks, the uterus was auto-transplanted to the peritoneum to construct an endometriosis model. After 6 weeks, endometriosis lesions and the normal uterus were morphologically evaluated. Fatty acid composition in the tissues was examined. Gene expression profiles were evaluated by cDNA microarray analysis. Results - The EPA group showed less thickening of interstitium, an active site for inflammation in endometriosis [0.30 ± 0.11 mm (EPA group) vs.0.76 ± 0.23mm (LA group), P = 0.03]. Administered fatty acid content was higher in the endometriosis tissue than in the uterus. The EPA/AA ratio in each tissue rose markedly in EPA group. Microarray analysis revealed reduced expression of MMP13, MMP8, IL-1, IL-1r, IL-10, Ptges and NF-?B. Conclusion - EPA is promising new valid strategy for the treatment of endometriosis.

Concurrent Session 28 Lipidomics 1

Chairs: Tom Brenna & Hee-Yong Kim

CS 28.1 COVALENT ADDUCT CHEMICAL IONIZATION TANDEM MASS SPECTROMETRY FOR DOUBLE BOND LOCALIZATION IN POLYNSATURATED FATTY ACYL GROUPS.
JT Brenna. Division of Nutritional Sciences, Cornell University, Ithaca, NY USA

Background – Novel comprehensive lipidomic methods based on mass spectrometry (MS) provide extensive molecular information on glycerolipids. Double bond locations along the chain are a critical structural feature but are generally determined by derivatization using specialized methods. Objective – We review here a series of studies showing double bond localization by covalent adduct formation and tandem mass spectrometry. Applications to minor fatty acids in complex mixtures will be presented. Methods – Acetonitrile is used as a chemical ionization (CI) gas in either a 3D ion trap or an atmospheric pressure (AP) CI linear ion trap MS. Under CI conditions, acetonitrile self-reacts to form (1-methyleneimino)-1-ethenylium (MIE) at m/z 54. MIE forms a covalent adduct with double bonds of fatty acid methyl esters (FAME) or triacylglycerols (TAG) to form a molecular ion 54 Da above the mass of the parent analyte. Results – Mechanistic studies show that the covalent [M+54]+ adduct of homoallylic FAME has a four-membered heterocyclic ring structure, and for conjugated dienes the adduct is a six-membered ring. Collisional dissociation of the [M+54]+ ion of homoallylic monoene, diene, and polyene FAME yields two ions that define the position of the double bonds. Conjugated diene FAME [M+54]+ ion intensities in MS-1 can distinguish cis-cis from trans-trans geometry, and in MS/MS diagnostic ions can distinguish cis-trans from trans-cis geometry. Polyene FAME of unusual structure yield diagnostic ions as well. APCACI of TAG produce [M+54]+ ions that yield diagnostic ions up to trienes. Conclusions – These results show that CACI-MS/MS and APCACI-MS/MS determine double bond position without specialized derivatives or methods and possess advantages for quantitative analysis.

CS 28.2 QUANTITATION OF TRIACYL- AND DIACYLGLYCEROL MOLECULAR SPECIES IN THE RAW 264.7 CELL USING STABLE ISOTOPE DILUTION STRATEGIES.
Robert C Murphy¹, Jessica Krank1, Andrew McAnoy1, Robert M Barkley1, Eva Duchoslav2. 1Department of Pharmacology, University of Colorado Health Sciences Center, Aurora, CO. 2MDS SCIEX, Concord, Ontario, Canada .

Glycerolipids such as triacylclycerol (TAG) and diacylglycerol (DAG) play important roles in cell biology. TAGs and DAGs are present in cells as complex mixtures of molecular species which differ by fatty acyl content and this mixture is difficult to define qualitatively and quantitatively. We developed a method to quantitate molecular species containing specific fatty acyl substituents using a constant neutral loss scan in a tandem quadrupole mass spectrometer while the TAGs/DAGs are still present as a mixture of molecular species. Nine deuterium-labeled TAGs and 6 deuterium-labeled DAGs were synthesized where the deuterium label was incorporated into all glycerol hydrogen atoms. Since each deuterium-labeled internal standard has the isotope label on the glycerol backbone and not the fatty acyl group, the total mass loss for RCOOH+NH3 is not shifted. Constant neutral loss scans for each [RCOOH+NH3] in a complex mixture of TAGs results in the identification of those components (as the molecular weight of the TAG+NH4) that contain each specific fatty acyl group. Previous MS3 analysis of each observed [TAG+NH4] limits possible specific molecular species to at least a small number that could give rise to the signal. The addition of d5-glycerol-TAG internal standards to the mixture is the basis of a quantitative method for specific molecular species. This approach has now been automated using software to identify the molecular species correct for C-13 abundance, and report abundance relative to the added internal standard. Hundreds of distinct molecular species of TAGs and DAGs have been automatically quantitated in experiments with cells before and after exposure to a Toll-4 receptor agonist.

CS 28.3 ANALYSIS OF OXIDIZED PHOSPHATIDYLCHOLINES USING FOCUSED MULTIPLE REACTION MONITORING WITH REVERSE-PHASE LIQUID CHROMATOGRAPHY/TANDEM MASS SPECTROMETRY.
Ryo Taguchi^1,2, Yasuhiro Iida1,2, Ken Karasawa3, Akihiro Sato4, Takao Shimizu1. 1Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan 2Core Research for Evolutional Science and Technology, 4-1-8 Honmachi Kawaguchi, Saitama 332-0012, Japan 3Faculty of Pharmaceutical Sciences, Teikyo University, Sagamiko, Tsukui, Kanagawa 199-0195, Japan. 4JASCO International Co., Ltd., 11-10, Myojin-cho 1-chome, Hachioji, Tokyo 192-0046, Japan.

Background - Oxidized phospholipids (oxPLs) and their decomposition products occur under many different types of oxidative stress and are closely related to various diseases. Oxidized low-density lipoprotein (oxLDL), containing large amounts of oxPLs, is thought to cause atherosclerosis. However, oxPLs have short longevities in vivo and a lot of isomers. In addition, microheterogeneities can occur within oxPLs or non-oxPLs. So, the analysis of oxPLs is very difficult. Objective - We tried to construct effective methods for the analysis of oxPLs to understand their physiological functions or effects in mammals. Design - First, we obtained specific fragmentation patterns of oxPLs using Fourier-transform ion cyclotron resonance mass spectrometry by sustained off-resonance irradiation collision-induced dissociation. Using the information of focused molecules, we tried to detect oxPLs by multiple reaction monitoring (MRM) and succeeding data-dependent product ion scanning. These analyses were performed by a quadrupole linear ion trap hybrid mass spectrometer with reverse-phase liquid chromatography. Outcome - By the analysis of oxidized phosphatidylcholines (oxPCs) in the lipid mixture extracted from oxLDL, 16:0-18:2 PC and its oxides were specifically surveyed by the focused MRM method. OxPCs were separated from non-oxPCs by reverse-phase liquid chromatography (RPLC). In addition, precise structures of many different species of oxPLs were confirmed by data-dependent product ion scanning. Conclusion - We selected this method as one of the most useful methods in metabolomics and named it as an expanded targeted method because this method is close to focused methods by structural specificities. However, it has a high advantage in its higher sensitivity.

Concurrent Session 29 Bone Health

(Sponsored by DSM)
Chairs: Peter Weber & Marlena Kruger
 

CS 29.1 LCPUFA AND OSTEOPOROSIS; EPIDEMIOLOGICAL AND HUMAN DATA.
Marlena C Kruger. Institute of Food Nutrition & Human Health, Massey University, Palmerston North, New Zealand

Long chain polyunsaturated fatty acids (LCPUFA) may play a role in bone maintenance as well as prevention of bone loss. Epidemiological studies indicate that frequent consumption of fish is positively associated with changes in bone mineral density (BMD). These observations have been made in a female population in Norway as well as in Japan where higher n-3 status is linked to higher BMD. A recent investigation of the association between the ratio of dietary n-6 to n-3 fatty acids and BMD indicated a significant inverse association between the ratio of dietary linoleic acid to α-linolenic acid and BMD at the hip of men and women. This study further also showed that an increasing ratio of total dietary n-6 to n-3 fatty acids was significantly associated with lower BMD in women. Some interaction with hormone replacement therapy was also shown. In contrast a longitudinal observational study reported negative associations between total PUFA intake and bone mass when dietary calcium was also low. Experimental studies in humans are also controversial. In elderly women a mixture of evening primrose and fish oils together with calcium enhanced femoral bone mass by 1.3% compared to a decline in the placebo group of 2.1% over 18 months. However, no benefit or harm to bone mass was observed in premenopausal women after supplementation with a similar mixture plus 1000mg of calcium for one year. These findings suggest that long term intervention studies are needed to clarify the role of LCPUFA in maintenance of bone.

CS29.2 PRIMARY PREVENTION OF OSTEOPOROSIS USING DIETARY LONG CHAIN POLYUNSATURATED FATTY ACIDS: EVIDENCE FROM VARIOUS ANIMAL MODELS
HA Weiler. School of Dietetics and Human Nutrition, Faculty of Agriculture and Environmental Sciences, McGill University, Ste.Anne-de-Bellevue, Quebec, Canada, H9X 3V9

Epidemiological evidence suggests that prevention of osteoporosis should incorporate strategies to enhance peak bone mass early in life and to limit bone loss with aging. In intact young rodents, chicks, and piglets, dietary n-6 and n-3 long chain polyunsaturated fatty acids (LCPUFA) are associated with elevated bone mass accompanied frequently by enhanced intestinal calcium absorption and mineral retention, reduced indicators of prostaglandin biosynthesis and/or reduced bone resorption. Enhanced bone mass due to fish oil is evident even in young rats with diabetes or food restriction, two risk factors for low bone mass. Mature animal models of bone loss and osteoporosis also respond to addition of LCPUFA to the diet with significant elevations in bone mass. The ovariectomized rodent fed either fish oil or semi-purified eicosapentaenoic or docosahexaenoic acid experiences attenuated loss of bone mineral. This was associated with inhibition of osteoclast generation in one study, while in another the LCPUFA potentiated the effects of estrogen treatment. Additionally, osteoporosis is inhibited in association with reduced expression of receptor activator of NF-κB ligand and elevated expression of osteoprotegerin in mice prone to arthritis and fed fish oil. In natural advanced aging, n-3 LCPUFA from fish oil also attenuates bone loss in intact male rodents. Thus inclusion of both n-6 and n-3 LCPUFA offer potential to enhance peak bone mass during growth, while dietary n-3 LCPUFA appear to preserve bone mass during menopause and advanced aging.

CS 29.3 INFLUENCE OF POLYUNSATURATED FATTY ACIDS, GENISTEIN AND VITAMINS D3 AND K1 ON BONE METABOLISM IN RAT AND DOG MODELS OF OSTEOPOROSIS.
U Wehr1, W Rambeck1, S Krammer2, D Raederstorff², P Weber2. 1Institute of Animal Physiology, Ludwig-Maximilians-University Munich, Germany. 2DSM Nutritional Products, Basel, Switzerland .

Background – Osteoporosis is a major health problem with increasing prevalence in developed countries as the population ages. Nutritional factors play an important role in the prevention of osteoporosis but experimental data on the interactions of different micro nutrients are still needed to have a better understanding of the effects of combinations. Objective – The aim was to evaluate the ability of a combination of vitamin D3 (1500 IU vitamin D3/kg BW), vitamin K1 (2000 μg vitamin K1/kg BW), genistein (15 mg Genistein/kg BW), and n-3 polyunsaturated fatty acids (1.5 % in the diet) to prevent the subsequent loss of bone mass induced by ovariohysterectomy in both rats and dogs. Blood and urine were collected at intervals to determine markers of bone formation (bone specific alkaline phosphatase, osteocalcin) and bone resorption (collagen crosslinks: deoxypyridinoline and pyridinoline). Method – The decrease in sex hormone levels (e.g. estrogen) is one of the principal causes for osteoporosis in postmenopausal women and is mimicked in dog and rat osteoporosis models by ovariohysterectomy. This induced hormonal change is accompanied by a high bone turnover, which subsequently leads to an increased risk of rapid bone loss, similar to the changes seen in osteoporosis. Results – Bone resorption in rats, measured as urinary pyridinoline excretion (in nmol/mmol Creatinine) doubled in the control group in the weeks after OHX. This strong increase was clearly prevented by the administration of the combination used (urinary pyridinoline excretion in nmol/mmol Crea in week 12 after OHX: 303.7 ± 42.5 for treated versus 417.8 ± 58.2 for control animals). In dogs the increased urinary pyridinoline excretion was also prevented by the administration of the combination of the substances. Conclusions – The results show that the combination of the tested substances reduced the excessive bone turnover in a rat and a dog model of osteoporosis and therefore can have a protective effect on osteoporotic bone. The reproducibility of the results in two animal model of osteoporosis was demonstrated.

CS 29.4 COMPARISON OF EFFECTS OF GAMMA-LINOLENIC, EICOSAPENTAENOIC AND DOCOSAHEXAENOIC ACIDS ON BONE POST-OVARIECTOMY IN RATS .
Raewyn C Poulsen, Marlena C Kruger. Institute of Food Nutrition & Human Health, Massey University, Palmerston North, New Zealand

Several studies have reported a beneficial effect of EFA consumption on bone mass. However little is known about the effects of individual EFAs on bone. Aim - To determine the effects of three EFAs on bone in ovariectomised (OVX) rats, a model for post-menopausal osteoporosis. Methods - OVX rats were supplemented for 16 weeks with 0.5g/kg bodyweight/day of high-purity gamma-linolenic (GLA), eicosapentaenoic (EPA) or docosahexaenoic (DHA) ethyl esters or a mixture of all three (MIX, ratio 2:1:4). Femur (F) and lumbar spine (LS) bone mineral content (BMC), density (BMD), serum vitamin D and parathyroid hormone (PTH) levels were compared to those of non-supplemented sham and OVX controls. Results - Final LSBMC was significantly higher in the DHA group than in OVX controls (p≤0.05). Final FBMC and LSBMC in the DHA group and sham controls were not significantly different. The MIX supplement had a much weaker, non-significant effect on BMC compared to that of DHA alone. Final serum 25-hydroxyvitamin D3 was lower in the DHA (p=0.06) and MIX (p=0.07) groups compared to sham. The OVX-induced decrease in FBMC and FBMD was greater in the GLA group compared to OVX controls (p=0.026 and p=0.018 respectively). Serum PTH was higher in the GLA group compared to all other OVX groups (p<0.05). EPA had no significant effect on bone. Conclusion - DHA protected against ovariectomy-induced decreases in BMC. GLA exacerbated bone mineral loss possibly via a PTH-mediated mechanism.

Concurrent Session 30 Maternal - Infant Round Table Discussion session: Future Directions

Chairs: Bob Gibson & Susan Carlson

There are no abstracts available for this session.

Concurrent Session 31 Inflammation 2

Chairs: Kevin Fritsche & Philip Calder

CS 31.1 SALMON INTAKE REDUCES CCR5 EXPRESSION BY HUMAN CD8+ T LYMPHOCYTES.
GK Paschos¹, P Scully2, A Lucey1, K Galvin1, KD Cashman1,2, M Kiely1. 1Department of Food and Nutritional Sciences, University College Cork, Ireland. 2Department of Medicine, University College Cork, Ireland

Background : Incorporation of omega-3 fatty acids in the membrane of cells is found to change the expression of proteins on the membrane. Objective : To assess the effect of dietary omega-3 fatty acids from fish intake on the expression of CCR5 by CD4+ and CD8+ T lymphocytes. Design : Nineteen healthy volunteers (mean body mass index 29.5 ± 1.6 kg/m2) received nutritional advice to adhere to an energy-restricted diet and were randomly distributed to one of 3 dietary interventions for a period of 8 weeks: i) 3g of sunflower oil/day (n=4) (control group), ii) 140g of cod 3 times/week (n=6), iii) 140g of salmon 3 times/week (n=9). Results : Salmon intake increased the proportion of eicosapentaenoic acid (EPA) in the membrane of mononuclear cells (P=0.003). CCR5 expression by CD8+ cells reduced in the salmon group compared to the control and cod group, after adjustment for age and weight reduction (P=0.024) (bar chart). The change in CCR5 expression by CD8+ T lymphocytes was related to the change in the proportion of EPA in the membrane of mononuclear cells (r=0.723, P<0.001) (scatter plot). Conclusion : Salmon intake reduces the expression of CCR5 on CD8+ T lymphocytes, possibly by increasing the percentage of EPA in the membrane of the cells. Our finding suggests a decreasing effect of salmon intake in cytotoxic T leukocyte activation and recruitment to sites of inflammation.

CS 31.2 LYMPHOCYTE-INDUCED PROSTACYCLIN SYNTHESIS AND PROINFLAMMATORY CYTOKINES GENE EXPRESSION IN ENDOTHELIAL CELLS .
Ana Rivas, Zury Dominguez, Virgilio Bosch. Cátedra de Patología General y Fisiopatología Escuela de Medicina Luis Razetti. Sección de Lipidología. Instituto de Medicina Experimental - Universidad Central de Venezuela. Caracas, Venezuela .

Background – Cell/Cell interaction is an essential process for both: keeping homeostasis or conditioning a pathologic state. In this respect, it is not clearly defined the rol of lymphocytes in some chronic inflammatory processes leading to wall vessel damage. We have previously showed that resting peripheral blood lymphocytes (PBL) contact to resting Human Umbilical Vein Endothelial Cell (HUVEC) triggered endothelial PGI2 synthesis through a Src kinase/ERK1/2- cPLA2-PGHS-1-PGIS pathway. Objective – Using the same experimental conditions we determined here (1) whether PBL-EC contact is able to induce pro-inflammatory cytokines gene expression and (2) if the expression is associated to a particular PBL subtype. Design – Confluent HUVEC were incubated alone or co-incubate with PBL, LT-CD4+ or LT-CD8+ in a in a serum-free medium for 4 h. HUVEC were then washed to eliminated lymphocytes and the cDNA for IL-1 α, IL-6, IL-8 and TNF-α was synthesised by RT-PCR from HUVEC-RNA. Outcomes – Results shows that cDNAs for the cytokines study here were not observed when HUVEC were incubated alone or with LT-CD8+ subtype. In contrast, expression for IL-1α and IL-6 was evident when HUVEC were co-incubated with PBL or with LT-CD4+ while TNFα was only expressed when HUVEC were co-incubated with PBL. Finally, IL-8 showed no signal in our experimental conditions. Conclusions – These results suggest that lymphocytes-endothelial cell interactions induce the transcription of some genes involved in inflammatory process and that this seems to be dependent on a particular lymphocyte subtype not involving LTCD8+.

CS 31.3 OMEGA-6 DOCOSAPENTAENOIC ACID (DPAn-6) IS ENZYMATICALLY CONVERTED TO OXYLIPINS THAT POSSESS ANTI-INFLAMMATORY ACTIVITY IN VIVO.

LM Arterburn, B Dangi, M Obeng, M van Elswyk, J Nauroth, R. Bell, M Teymourlouei, D Vinjamoori. Martek Biosciences Corporation, Columbia, Maryland, USA

Background – Long-chain omega-3 fatty acids DHA and EPA can be converted to mono-, di- and tri-hydroxy derivatives by lipoxygenase and cyclooxygenase exnzymes. These oxylipin derivatives, named resolvins or protectins, have potent anti-inflammatory activity in vivo. Objective – To investigate the role of another 22-carbon polyunsaturated fatty acid – DPAn-6 – as a precursor to oxylipins and a modulator of inflammation. Results – DPAn-6 is a preferred substrate for 15-lipoxygenase, and major products are 17-hydroxy DPAn-6 and 10,17-dihydroxy DPAn-6, as determined by LC/MS/MS. In vitro, 10,17-dihydroxy DPAn-6 in the low nanomolar range, like the DHA analogue 10,17-dihydroxy DHA, reduces the secretion of TNF? by T-lymphocytes following CD3/CD28 activation. In a preliminary experiment using a mouse air pouch model of inflammation, both 17-hydroxy DPAn-6 and 10,17-dihydroxy DPAn-6 inhibited by over 70% the TNF?-induced granulocyte and macrophage migration into the air pouch exudates. In a rat hind paw model of inflammation, subchronic oral administration of feed containing both DHA (1.1% of fat) and DPA (0.4% of fat) reduced carrageenan-induced paw swelling better than a feed containing DHA (1.1% of fat) alone, further supporting the anti-inflammatory role of DPAn-6 in vivo. Conclusion – Our studies demonstrate that the omega-6 fatty acid DPAn-6 can be converted into oxylipins and that the fatty acid as well as its oxylipin derivatives have anti-inflammatory effects in vivo

CS 31.4 DIFFERENTIAL REGULATION OF AP-1 AND NF?B BY N-3 PUFA AND CLA IN RELATION TO CELL ADHESION MOLECULE EXPRESSION IN HUVEC.
M Goua¹, S Mulgrew1, AA Sneddon2, KWJ Wahle1. 1School of Life Sciences, Robert Gordon University, Aberdeen, Scotland. 2Rowett Research Institute, Aberdeen, Scotland

Background – We have previously shown that adhesion molecules, intracellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule (VCAM-1), important factors in relation to atherosclerosis, are regulated by n-3 polyunsaturated fatty acids (PUFA) and by conjugated linoleic acids (CLA). The mechanisms of action of these fatty acids are not clear. Nuclear factor kappa B (NFκB) and activating protein 1 (AP-1) are the main transcription factors regulating ICAM-1 and VCAM-1 expression. Objective – To determine the effects of EPA, DHA, and different CLA isomers on NFκB and AP-1 protein expression. Methods – Human umbilical endothelial cells (HUVEC) were treated with 50μM EPA and DHA and with 25μM CLA 9;11, CLA 10;12 and CLA mix (50:50 9;11:10;12) for 24hr. The cells were then stimulated with 5ng/ml TNFα for 1hr. Nuclear extraction was performed and transcription factors determined by commercially available ELISA:kits, (TransAMTM ,NFκB and AP-1 family kits ) Results – All of the subunits tested (RelB, p65, p50, cRel and p52 for NFκB; c-Fos, FosB, Fra-1 & -2, c-Jun, JunB and JunD for AP-1), showed increases in the DNA binding affinity when treated with the inflammatory TNFα. Critically, EPA and DHA strongly decreased DNA binding affinities of most of the AP-1 subunits (apart from c-Fos and Fra-1). CLA did not have any significant effects on AP-1. The cRel of NFκB was increased by all the FA tested. However, n-3 PUFA decreased p65, p50 and RelB binding affinity to DNA. CLA 9;11 also reduced p50. Conclusions – We have shown previously that only CLA would reduce IκB phosphorylation and NF?B activity in HUVEC although CLA and n-3 PUFA both reduced adhesion molecule expression. Here, we demonstrate that n-3 PUFA strongly down-regulated AP-1 subunits, which could account for the decreased ICAM-1 and VCAM-1 protein expression elicited by the fatty acids. Clearly, the fatty acids elicit different signalling pathways to produce the same end-effect, namely the attenuation of adhesion molecule expression.

CS 31.5 INDIVIDUAL FATTY ACID DOSE RESPONSE EFFECTS ON ENDOTHELIAL INFLAMMATION: VARIES WITH STIMULATION STATE.

DI Shaw, WL Hall, CM Williams. Hugh Sinclair Unit of Human Nutrition, School of Food Biosciences, University of Reading, UK, RG6 6AP.

Background - Vascular dysfunction is associated with cardiovascular disease and type 2 diabetes. Prospective studies have suggested dietary fatty acid (FA) composition is associated with endothelial function, but the underlying mechanism is unclear. In vitro studies have shown certain FA alter markers of endothelial function, but there is great disparity in the findings. The range of FA compared is often limited, with only a small number of endpoints investigated, large variations in FA incubation time, concentration and cell stimulation state. Objective - To clarify the effects of FA on inflammatory gene expression in endothelial cells by systematic investigation of the effects of FA concentration and cell stimulation state. Methods - Using human umbilical vein endothelial cells (HUVEC), with and without stimulation (+/- 10 ng/ml TNFα), the effects of DHA, EPA, linoleic (LA), oleic (OA) and palmitic acids (PA) (0, 10, 25 and 100μM), on gene expression were assessed by quantitative real time reverse transcriptase polymerase chain reaction (RT-PCR). Results - In unstimulated cells various FA dose-dependently increased gene expression up-regulation relative to control (RTC); LA significantly increased ICAM-1 up-regulation RTC (p<0.05), as did EPA, PA and OA with eNOS, E-selectin and MCP-1 expression, respectively (p<0.05, p<0.0001, p=0.06). Conversely in stimulated cells, FA tended to cause a dose-dependent attenuation of gene expression up-regulation. However as overall fold changes in gene expression were smaller, dose-response effects did not reach significance; a trend towards a dose-dependent decrease in ICAM-1 gene expression following PA was shown (p=0.06). Conclusion - Saturated, monounsaturated, n-6 and n-3 polyunsaturated fatty acid effects on endothelial gene expression vary, dependent on concentration, gene investigated and cell stimulation state. FA such as LA that are pro-inflammatory in unstimulated cells have an attenuated inflammatory effect in stimulated cells. These results may explain apparent disparity in the current literature and may have relevance for understanding variable effects of FA in healthy and diseased individuals. This work was funded by the EC, Framework Programme 6 via the LIPGENE project (FOOD-CT-2003-505944).

Concurrent Session 32 Lipidomics 2

Chairs: Tom Brenna & Hee-Yong Kim
 

CS 32.1 Paper withdrawn
 

CS 32.2 MASS SPECTROMETRY BASED PROFILING OF PHOSPHOLIPIDS AND SPHINGOLIPIDS IN EXTRACTS FROM SACCHAROMYCES CEREVISIAE .
XL Guan, MR Wenk. National University of Singapore, Yong Loo Lin School of Medicine, Department of Biochemistry, and Department of Biological Sciences, Medical Drive, Singapore 117597.

Background – Lipids are rapidly moving to center stage in many fields of biological sciences. Lipidomics, the systems level scale analysis of lipids and their interacting factors, thus is an emerging field which holds great promise for drug and biomarker discovery. Objective – Here we present a mass spectrometry based approach for profiling of polar lipids, in particular phospholipids and sphingolipids, in Saccharomyces cerevisiae. Design – The first step includes semi-quantitative surveys of lipids in an untargeted fashion which is particularly powerful for detection of changes that cannot easily be anticipated. This leads to the identification of ions with increased or decreased signal intensities. Comprehensive theoretical calculation of the masses of yeast phospholipid and sphingolipid molecular species, based on fatty acyl and headgroup heterogeneity, is next used to tentatively assign ions of interest. Subsequent targeted analysis using tandem mass spectrometry allows for characterization and quantification of phospholipids and sphingolipids. Outcomes – We validated the method using the EUROSCARF library of non-essential deletion mutants. For example, mutants of SCS7, a lipid hydroxylase, and SLC1, a putative acyl transferase with unknown substrate specificity, were profiled for their phospholipid and sphingolipid content. The observed changes in lipid profiles are consistent with previous observations and extend our knowledge on in vivo substrate use under permissive growth conditions. Conclusions – Given the high degree of conservation in pathways of lipid metabolism between different organisms it can be expected that this method will lead to the discovery of novel enzymatic activities and modulators of known ones, in particular when used in combination with genetic and chemogenetic libraries and screens.

CS 32.3 LIQUID CHROMATOGRAPHY/MASS SPECTROMETRY IN PHOSPHOLIPID REMODELING STUDIES .
Hee-Yong Kim. Section of Mass Spectrometry, Laboratory of Membrane Biochemistry and Biophysics, NIAAA, NIH, Bethesda, USA.

Cell membranes are complex mixtures of phospholipid molecules. Since membrane chemical and physical properties are influenced by the phospholipid composition, analysis of phospholipid molecular species is of great importance in membrane-related cell signaling research. To monitor the membrane modification, a quantitative method to analyze phospholipid molecular species in complex biological mixtures was established by using liquid chromatography/electrospray ionization (ESI) mass spectrometry (LC/ESI-MS). Under an in-source fragmentation condition, the phospholipid molecules were cleaved in a convenient way to produce mono- and diglyceride ions as well as molecular ion species, with the exception of PC. Coupling with reversed phase HPLC allowed separation and detection of each individual molecular species of complex biological mixtures within 30 min. In vitro bioassay methods were also devised using this approach to evaluate phospholipid remodeling processes. Use of LC/ESI-MS in studying the membrane phospholipid profile and membrane-remodeling processes affected by docosahexaenoic acid (DHA, 22:6n-3) and ethanol will be presented.