Download CONJUGATED LINOLEIC ACIDS DECREASE PANCREATIC

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
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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
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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,
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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.
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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.
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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). It is this aspect
of limitation of delivery of substrate (arachidonic acid) for 5-LO possible in
in vivo conditions which seems to be particularly interesting. 5-LO found
in macrophages plays an active role in the activation of metalloproteinase
(MMP-2, MMP-9) expression activation and contributes in this manner to
elastine degradation activation – one of the main consequences of an active
inflammatory process (Zhao and Funk 2004).
The purpose of this study is to draw the readers’ attention to the CLA
target sites which have not been elucidated so far. Further detailed studies are
necessary to obtain full explanation of the role of CLAs in the functioning of
the human gastrointestinal tract.
REFERENCES
BANNI, S., ANGIONI, E., CASU, V., MELIS, M.P., CARTA, G.,
CORONGIU, F.P., THOMPSON, H. and IP, C. 1999. Decrease in linoleic
acid metabolites as a potential mechanism in cancer risk reduction by
conjugated linoleic acid. Carcinogenesis 20, 1019–1024.
BASSAGANYA-RIERA, J., REYNOLDS, K., MARTINO-CATT, S.,
CUI, Y., HENNIGHAUSEN, L., GONZALEZ, F., ROHRER, J.,
FEEDING AWAY INFLAMMATION
321
BENNINGHOFF, A.U. and HONTECILLAS, R. 2004. Activation of
PPAR gamma and delta by conjugated linoleic acid mediates protection
from experimental inflammatory bowel disease. Gastroenterology 127,
777–791.
CHIN, S.F., STORKSON, J.M., LIU, W., ALBRIGHT, K.J. and PARIZA,
M.W. 1994. Conjugated linoleic acid (9,11- and 10,12-octadecadienoic
acid) is produced in conventional but not germ-free rats fed linoleic acid.
J. Nutr. 124, 694–701.
CHOI, M.S. and RHEE, K.C. 2006. Production and processing of soybeans
and nutrition and safety of isoflavone and other soy products for human
health. J. Med. Food 9, 1–10.
EVANS, M., GEIGERMAN, C., COOK, J. CURTIS, J., KUEBLER, B. and
MCINTOSH, M. 2000. Conjugated linoleic acid suppresses triglyceride
accumulation and induces apoptosis in 3T3-L1 preadipocytes. Lipids 35,
899–810.
GREICIUS, G., ARULAMPALAM, V. and PETTERSSON, S.A. 2004.
CLA’s act: Feeding away inflammation. Gastroenterology 127, 994–996.
HAN, W.K., SAPIRSTEIN, A., HUNG, C.C., ALESSANDRINI, A. and
BONVENTRE, J.V. 2003. Cross-talk between cytosolic phospholipase
A2 alpha (cPLA2 alpha) and secretory phospholipase A2 (sPLA2) in
hydrogen peroxide-induced arachidonic acid release in murine mesangial
cells: sPLA2 regulates cPLA2 alpha activity that is responsible for arachidonic acid release. J. Biol. Chem. 278, 24153–24163.
HARLE, L., BROWN, T., LAHERU, D. and DOBS, A.S. 2005. Omega-3 fatty
acids for the treatment of cancer cachexia: Issues in designing clinical
trials of dietary supplements. J. Altern. Complement. Med. 11, 1039–1046.
HENNING, D.R., BAER, R.J., HASSAN, A.N. and DAVE, R. 2006. Major
advances in concentrated and dry milk products, cheese, and milk fatbased spreads. J. Dairy Sci. 89, 1179–1188.
HOUSEKNECHT, K.L., VANDEN HEUVEL, J.P., MOYA-CAMARENA,
S.Y., PORTOCARRERO, C.P., PECK, L.W., NICKEL, K.P. and
BELURY, M.A. 1998. Dietary conjugated linoleic acid normalizes
impaired glucose tolerance in the Zucker diabetic fatty fa/fa rat. Biochem.
Biophys. Res. Commun. 244, 678–682. Erratum in Biochem. Biophys.
Res. Commun. 247, 911.
HURT-CAMEJO, E., CAMEJO, G., PEILOT, H., OORNI, K. and
KOVANEN, P. 2001. Phospholipase a (2) in vascular disease. Circ. Res.
89, 298–304.
JIMENEZ, M., CABANES, J., GANDIA-HERRERO, F., ESCRIBANO, J.,
GARCIA-CARMONA, F. and PEREZ-GILABERT, M. 2003. The continuous spectrophotometric assay for phospholipase A(2) activity. Anal.
Biochem. 319, 131–137.
322
E. STACHOWSKA ET AL.
MOYA-CAMARENA, S.Y., VANDEN HEUVEL, J.P., BLANCHARD, S.G.,
LEESNTZER, L.A. and BELURY, M.A. 1999. Conjugated linoleic acid
is a potent naturally occurring ligand and activator of PPAR alpha. J.
Lipid Res. 40, 1426–1433.
NICOLOSI, R.J., ROGERS, E.J., KRITCHEVSKY, D., SCIMECA, J.A. and
HUTH, P.J. 1997. Dietary conjugated linoleic acid reduces plasma lipoproteins and early aortic atherosclerosis in hypercholesterolemic hamsters. Artery 22, 266–277.
PLAGEMANN, A. 2006. Perinatal nutrition and hormone-dependent programming of food intake. Horm. Res. 65, 83–89.
PROFITA, M., VIGNOLA, A.M., SALA, A., MIRABELLA, A., SIENA, L.,
PACE, E., FOLCP, G. and BONSIGNORE, G. 1999. Interleukin-4
enhances 15-lipoxygenase activity and incorporation of 15(S)-HETE into
cellular phospholipids in cultured pulmonary epithelial cells. Am. J.
Respir. Cell Mol. Biol. 20, 61–68.
REYNOLDS, L.J., HUGHES, L.L., YU, L. and DENNIS, E.A. 1994.
1-Hexadecyl-2-arachidonoylthio-2-deoxy-sn-glycero-3-phosphorylcholine as a substrate for the microtiterplate assay of human cytosolic phospholipase A2. Anal. Biochem. 217, 25–32.
SALARI, H. and CHAN-YEUNG, M. 1989. Release of 15hydroxyeicosatetraenoic acid (15-HETE) and prostaglandin E2 (PGE2) by
cultured human bronchial epithelial cells. Am. J. Respir. Cell Mol. Biol.
1, 245–250.
SMITH, W.L. and LANDS, W.E.M. 1972. Oxygenation of unsaturated fatty
acids by soybean lipoxygenase. J. Biol. Chem. 247, 1038–1047.
TIKKANEN, M.J. 2005. Plant sterols and stanols. Handbook Exp. Pharmacol.
170, 215–230.
UAUY, R. and DANGOUR, A.D. 2006. Nutrition in brain development and
aging: Role of essential fatty acids. Nutr. Rev. 64, S24–33.
ZHAO, L. and FUNK, C.D. 2004. Lipoxygenase pathways in atherogenesis.
Trends Cardiovasc. Med. 14, 191–195.