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RESEARCH NOTE FEEDING AWAY INFLAMMATION – CONJUGATED LINOLEIC ACIDS DECREASE PANCREATIC PHOSPHOLIPASE A2 ACTIVITY* EWA STACHOWSKA1,2, VIOLETTA DZIEDZIEJKO1, KRZYSZTOF SAFRANOW1, KATARZYNA JAKUBOWSKA1, MARIA OLSZEWSKA1, JOANNA BOBER1 and DARIUSZ CHLUBEK1 1 Department of Biochemistry and Department of Medical Chemistry Pomeranian Medical University al. Powstancow Wlkp. 72, 70–111 Szczecin, Poland Submitted for Publication October 16, 2006 Revised, Received and Accepted November 29, 2006 ABSTRACT Conjugated linoleic acids (CLAs) are positional and geometric isomers of linoleic acid derived from food, mainly from milk and meat products. CLAs are ligands of peroxisome proliferator-activated gamma receptors. Phospholipases A2 (PLA2) represent a diverse group of enzymes that catalyze the hydrolysis of ester bonds at the sn-2 position of membrane phospholipids and release fatty acids and lysophospholipids. The objective of the study was to answer the question whether the release of linoleic acid by pancreatic PLA2 may change in the CLA-containing environment. In this study, linoleic acid released by pancreatic PLA2 was a substrate for purified lipoxygenase – an enzyme converting it into hydroxylated derivatives including 9- and 13-hydroxyoctadecadienoic acid (9-,13-HODE). In this method, the activity of PLA2 was determined by high-performance liquid chromatography. In vitro incubation of hog PLA2 with CLA contributed to a noticeable fall in the synthesis of HODEs (P = 0.003; Kruskal–Wallis test). The concentration of HODEs decreased by 40.2% (for the cis-9, trans-11 CLA isomer; P = 0.007, Mann–Whitney test, n = 5) and by 27% (for the trans-10, cis-12 CLA isomer; P = 0.007, Mann–Whitney test, n = 5) as compared with the control (enzyme incubated without CLA). The inhibition exerted by cis-9, trans-11 CLA isomer was significantly greater than that by trans-10, cis-12 CLA isomer (P = 0.032, Mann–Whitney test, n = 5). * Supported by grant no. 3 PO5B 117 23 from the State Committee for Scientific Research, Poland. 2 Corresponding author. TEL: +48-914661515; FAX: +48-914661516; EMAIL: ewa.stachowska@ akuna.pl Journal of Food Lipids 14 (2007) 315–322. All Rights Reserved. © 2007, The Author(s) Journal compilation © 2007, Blackwell Publishing 315 316 E. STACHOWSKA ET AL. PRACTICAL APPLICATIONS This study is an attempt to clarify the response to the question whether some food ingredients such as conjugated linoleic acid (CLA) may be useful as an agent supporting the treatment of gastrointestinal disorders. It was observed under in vitro conditions that CLA isomers inhibited the phosphatidylcholine hydrolysis. By using purified enzymes, CLA was shown to contribute to local reduction of availability of linoleic acid and its metabolites (9- and 13-hydroxyoctadecadienoic acid) through inhibition of phospholipid hydrolysis. INTRODUCTION The physicians’ view of the role of nutrition in patient treatment changed drastically in the course of the last decade. Nutrients began to be considered not only as a “set” of calories and nutritional substances, but also as agents useful in prophylaxis and treatment of many diseases. The term “functional food” appeared in the literature, and along with it, food products used in disease prophylaxis (e.g., probiotics or food enriched with plant stanols) started to become available in the market (Harle et al. 2005; Tikkanen 2005; Choi and Rhee 2006; Plagemann 2006). As a result of this interest, studies on food constituting potentially “more than nutritional” (therapeutic) substances were commenced. Among them, considerable attention has been paid to fatty acids (Henning et al. 2006; Uauy and Dangour 2006). The most commonly investigated fatty acids include conjugated linoleic acid (CLA) dienes – fatty acids common in human diet. They are positional and geometric isomers of linoleic acid derived from food, mainly from milk and meat products (Chin et al. 1994; Evans et al. 2000). In food products, cis-9, trans-11 CLA is the predominant isomer (Chin et al. 1994). Several studies have shown that CLAs have antiatherogenic, anticarcinogenic and anti-inflammatory properties in animals (Nicolosi et al. 1997; Houseknecht et al. 1998; Moya-Camarena et al. 1999). The positive aspects of the action of CLA in the gastrointestinal tract, both in physiological as well as in pathological conditions, have been demonstrated (Bassaganya-Riera et al. 2004). It seems that in healthy humans, CLA can function as part of intestinal homeostasis. As shown by Bassaganya-Riera et al. (2004) in their groundbreaking study, owing to their anti-inflammatory properties, CLA may be useful in the inhibition of inflammatory disorders of the gastrointestinal tract. It has been shown that food supplemented with CLA can efficiently alleviate inflammatory bowel diseases (IBDs), particularly when it is applied preventively (Bassaganya-Riera et al. 2004; Greicius et al. 2004). As shown by these FEEDING AWAY INFLAMMATION 317 studies, CLAs exert their protective (anti-IBD) action through the effect on peroxisome proliferator-activated gamma receptor (PPAR-g) expression (HurtCamejo et al. 2001; Greicius et al. 2004). PPAR-g possesses a large ligandbinding pocket; the ligands of PPAR-g include polyunsaturated fatty acids: 15-lipoxygenase product of linoleic acid 9- and 13-hydroxyoctadecadienoic acid (9- and 13-HODE), and arachidonic acid metabolites: eicosanoids and prostaglandins (Hurt-Camejo et al. 2001). Both fatty acids of key importance for the synthesis of PPAR ligands (linoleic acid and arachidonic acid) are released from membrane phospholipids by the actions of phospholipases A2 (PLA2) (Han et al. 2003). PLA2 represent a diverse group of enzymes that catalyze the hydrolysis of ester bonds at the sn-2 position of membrane phospholipids and release fatty acids and lysophospholipids (Han et al. 2003; Jimenez et al. 2003). PLA2 contained in pancreatic juice may be the key element in the regulation of the availability of fatty acids for their further metabolism in the intestine. Fatty acids released by PLA2 may be used in two ways: (1) systemically for resynthesis of triacylglycerols subsequently incorporated into newly formed chylomicrons; and (2) locally (in the intestine) – as substrates for enzymes metabolizing these fatty acids, e.g., within the eicosanoid synthesis pathway. The objective of the study was to answer the question whether the release of linoleic acid by pancreatic PLA2 may change in the CLA-containing environment. In this study, linoleic acid released by pancreatic PLA2 was a substrate for purified lipoxygenase – an enzyme converting it into hydroxylated derivatives including 9- and 13-HODE. MATERIAL AND METHODS The activity of PLA2 (pancreatic-EC 3.1.1.4, Sigma-Aldrich, St. Louis, MO) was determined by means of coupled assay using dilinoleoyl phosphatidylcholine (DL-PC) as a substrate for PLA2 and 15-lipoxygenase (EC 1.13.11.12 type IB, Sigma-Aldrich) as coupling enzyme (Reynolds et al. 1994; Jimenez et al. 2003). Linoleic acid was released by PLA2 and oxidized by lipoxygenase to a hydroperoxide derivative (HPODEs). Aqueous phosphatidylcholine substrate was prepared as described by Profita et al. (1999). The standard reaction medium (1 mL) contained the following: 65-mM DL-PC, 40-U PLA2 (EC 3.1.1.4) in 50-mM Tris-HCl buffer, pH 8.5 containing 3-mM deoxycholate. In some experiments, isomers of CLA were added (1 mM) to the reaction medium. Reaction was started by gently vortexing all components and then reaction was continued at 25C for 30 min. After this time, 318 E. STACHOWSKA ET AL. 4,600 U/mL lipoxygenase was added and the reaction was allowed to proceed for 30 min at 25C. Controls without phospolipase, lipoxygenase or DL-PC were always carried out. Finally, the same volume of cold ethyl acetate was added, samples were vortexed and centrifuged (3,200 ¥ g, 10 min, 4C). The upper layers were dried, extracts were reconstituted in 200 mL 65% methanol with 0.01% acetic acid and analyzed on a Hewlett-Packard HPLC 1050/1100 system (Agilent, Waldbronn, Germany). Reverse-phase high-performance liquid chromatography was performed with LiChrospher 100-RP18 column (250 ¥ 4 mm, 5 mm) (Merck, Lindenplatz, Haar, Germany) at 25C using gradient solvent system of methanol/water/acetic acid (50/50/0.1 for buffer A and 100/0/0.1 for buffer B, v/v/v) with a flow rate of 1 mL/min. The content of buffer B as percentage of the mobile phase volume was 30% at 0.0 min, 80% at 20 min, 98% at 20.1– 23.9 min and 30% at 24 min. The time of injection of the next 50-mL sample was 28 min. Detection was conducted at 235 nm. Racemic standard of 9S-HODE and 13S-HODE was used for identification and quantitation (Smith and Lands 1972; Salari and Chan-Yeung 1989; Banni et al. 1999). As the distribution in most cases deviated from normal (Shapiro–Wilk test), nonparametric tests were used. For related samples, significance was first checked with Friedman analysis of variance (ANOVA), then significant results were subjected to the Wilcoxon matched-pair test. For unrelated samples, significance was first checked with Kruskal–Wallis ANOVA, then significant results were subjected to the Mann–Whitney test. The software used was Statistica 6.1 (Statsoft, Krakow, Poland). RESULTS In vitro incubation of hog PLA2 with CLA contributed to a noticeable fall in the synthesis of HODEs (P = 0.003; Kruskal–Wallis test) as shown in Figs. 1 and 2. The concentration of HODEs decreased by 40.2% (for the cis-9, trans-11 CLA isomer; P = 0.007, Mann–Whitney test, n = 5) and by 27% (for the trans-10, cis-12 CLA isomer; P = 0.007, Mann–Whitney test, n = 5) as compared with the control (enzyme incubated without CLA). The inhibition exerted by cis-9, trans-11 CLA isomer was significantly greater than that by trans-10, cis-12 CLA isomer (P = 0.032, Mann–Whitney test, n = 5) (Fig. 2). DISCUSSION This study presents an attempt to clarify the response to the question whether some food ingredients, such as CLA, may be useful as an agent supporting the treatment of gastrointestinal disorders. FEEDING AWAY INFLAMMATION 319 9- and 13-hydroxyoctadecadienoic acid (HODE) Absorbance at 235 nm (mAU) 1000 Control (no CLA) 800 Cis-10, trans-12 CLA Cis-9, trans-11 CLA 600 Blank (no PLA2) 400 200 0 17 18 19 Time (min) 20 21 9- and 13-HODE synthesis ng/preparation FIG. 1. THE CHROMATOGRAM OF DILINOLEOYL PHOSPHATIDYLCHOLINE HYDROLYSIS BY PHOSPHOLIPASE A2 COUPLED WITH OXIDATION BY 15-LIPOXYGENASE CLA, conjugated linoleic acid; PLA2, phospholipases A2. p=0.007 10000 8000 p=0.007 6000 p=0.032 4000 2000 0 Control cis-9,trans-11 CLA trans-10,cis-12 CLA FIG. 2. FATTY ACIDS INHIBIT IN VITRO DILINOLEOYL PHOSPHATIDYLCHOLINE HYDROLYSIS CATALYZED BY PANCREATIC PHOSPHOLIPASES A2 The procedure was described in Material and Methods. Data are mean ⫾ SD of five experiments. P = 0.003 Kruskal–Wallis analysis of variance test. Comparison between control and incubation with conjugated linoleic acid (CLA) using Mann–Whitney test. 9- and 13-HODE = 9- and 13-hydroxyoctadecadienoic acid. 320 E. STACHOWSKA ET AL. In this study, it was observed in in vitro conditions that phosphatidylcholine hydrolysis inhibition took place in the environment containing both CLA isomers. In this study, CLA was shown to contribute to local reduction of availability of linoleic acid and its metabolites (9- and 13-HODE) through inhibition of phospholipid hydrolysis. It is suggested that local reduction in the concentration of linoleic acid metabolites caused by CLA administration may be the mechanism by which inhibition of cancer development is achieved (Banni et al. 1999). CLA and linoleic acid share the same enzyme system for chain desaturation and elongation, so it is possible that CLA interfered with further metabolism of linoleic acid (polyunsaturated omega-6 fatty acids, e.g., arachidonic acid). We think that the phenomenon of arachidonic acid “deficiency” observed by Banni et al. (1999) may occur before the stage of desaturation and elongation – our results indicate that inhibition of linoleic acid “availability” may take place at the level of pancreatic PLA2. Additionally, inhibition of elongation of linoleic acid by CLA may contribute (as observed in the study by Banni et al. 1999) to the reduction of concentration of arachidonic acid – which is the substrate for cyclooxygenase and lipoxygenase pathways of eicosanoids biosynthesis. Limitation of arachidonic acid availability by CLA in the gastrointestinal tract (Banni et al. 1999) may participate in modulation of activity of inflammatory enzymes COX-1, COX-2 and 5-lipoxygenase (5-LO). 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