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Cyclic Fatty Acid Monomers (CFAM) from Heated Vegetable Oils Increase F2-Isoprostanes Concentrations in
Plasma and Urine of Rats
Presented by:
Jean Mboma, PhD CandidateSchool of NutritionLaval University
Quebec City, QC, Canada
Jean Mboma, Hélène Jacques, Nadine Leblanc, Amandine Rocher, Claire Vigor, Camille Oger, Guillaume Reversat, Joseph Vercauteren, Jean Marie Galano, Thierry Durand, Paul Angers
INTRODUCTION
Fritsch C.W., 1981.
Sébédio, J.L et al. Grasas Aceites, 47, 5-13 (1996).Sébédio, J.L. et al. J. Am. Oil Chem. Soc., 68, 299-302 (1991).
Frankel, E.N. et al. J. Am. Oil Chem. Soc., 61, 87-90 (1984). Gere, A. et al. Fette Seifen Anstrichm., 87, 359-362 (1985)Sébédio, J.L et al. Rev. Fr. Corps Gras, 34, 15-18 (1987).Poumeyrol, G. Rev. Fr. Corps Gras, 34, 543-546 (1987).
Cyclic Fatty Acid Monomers (CFAM)
0.01 – 0.66 %CFAM in domestic
heated vegetable oils
DOCUMENTED EFFECTS OF CFAM
CFAM induce drug-metabolizing enzymes in the rat (Siess et al., 1988)
CFAM modify the activities of some enzymes involved in fatty acid synthesis and β-oxidation in the rat (Lamboni et al., 1998; Martin et al., 2000)
CFAM have a peroxisome proliferation effect in mice (Bretillon et al., 2003)
CFAM are preferentially detoxified via drug-metabolic pathway andβ-oxidation (Desmarais et al., 2015)
0
5
10
15
20
Non-CFAM CFAMCanola oil Soybean oil
ab
Live
r wei
ght (
g)
Hepatomegaly with CFAM
p<0.05n = 9
0
0.5
1
1.5
Non-CFAM CFAMCanola oil Soybean oil
a
b
Hep
atic
tota
l lip
ids
(g)
More liver total lipids with CFAMp<0.05n = 9
CFAM effects on liver weight and total lipids
Mboma et al, (data not published)
CFAM effects on liver TAG and Phosphatidylcholine
TAG: Triacylglycerols; PC: Phosphatidylcholine
0
15
30
45
Non-CFAM CFAMCanola oil Soybean oil
b
Hep
atic
PC
(nm
ol/m
g pr
otei
n)
a
Less liver PC with CFAMp<0.05n = 9
0
27
54
81
Non-CFAM CFAMCanola oil Soybean oil
a
Hep
atic
TAG
(mg/
g pr
otei
n) b
More liver TAG with CFAMp<0.05n = 9
Mboma et al, (data not published)
0
50
100
150
Non-CFAM CFAMCanola oil Soybean oil
ba
mg/dL
CFAM effects on plasma cholesterol and lipoproteinsMore total Cholesterol with CFAM
p<0.05n = 9
0
30
60
90
120
Non-CFAM CFAMCanola oil Soybean oil
b
a
mg/dL
More LDL-C with CFAMp<0.05n = 9
mg/dL
0
10
20
30
40
Non-CFAM CFAMCanola oil Soybean oil
ba
Less HDL-C with CFAMp<0.05n = 9
Milne et al., 2011
F2t-Isoprostanes (IsoP)
IsoP, Oxidative Stress, Pathophysiological Conditions
Oxidative damage
Lipids Proteins Nucleic acids
InflammationTissue injury
Hypothesis Consumption of CFAM from heated vegetable oils increases oxidative stress
To assess the impact of the intake of CFAM on the concentrations of isoprostanes, neuroprostanes and mediators of inflammation in liver, plasma and urine of rats
Objective
MATERIAL AND METHODS
GC-FID
275 °C - 12h – (Sébédio et al. 1987)
Saponification - AOCS Ca-6a-40
Esterification – MeOH, H2SO4
Column chromatography - silicic acid – adapted from AOAC 982.27
Urea Fractionation(Sébédio et al. 1987)
H2O + HCl 3%, extraction with hexaneH2O + HCl 3%, extraction with hexane
Synthesis of picolinic esters(Destaillats et Angers, 2002)
Fresh Linseed Oil
Heated Linseed Oil
Free Fatty Acids
Fatty Acid Methyl Esters, FAME
Polar Fraction Non Polar Fraction
Urea adduct CFAM Fraction
CFAMLinear Chain FAME
Piconylic esters of CFAM
CFAM Extraction from Linseed Oil
GC-MS
Column chromatography
Urea fractionation
Purification
Canola oil Soybean oil
Control group CFAM group Control group CFAM group
Casein 180 180 180 180
Sucrose 200 200 200 200
Maize starch 420 420 420 420
Cellulose 50 50 50 50
L-Cysteine 3 3 3 3
Choline bitartrate 2 2 2 2
Vitamins mix (AIN-93-VX)* 10 10 10 10
Mineral mix (AIN-93G-MX) 35 35 35 35
Lipids (+0.02% TBHQ) 100 99.5 100 99.5
CFAM 0 0.5 0 0.5
* American Institute of Nutrition (AIN)-93 (Harlan Teklad, Madison, WI, USA).
Formulation of purified diets (g per kg of diet)
53.3
47.7
CFAM Composition in the CFAM diet groups
0.5% CFAM of dietary fat
Fatty Acid Composition of the Diets
0
9
18
27
36
45
54
63
SFA MUFA LNA ALA n-6/n-3
Canola oil Soybean oil
Wei
ght %
of t
otal
fatty
aci
ds
SFA: 16:0 | MUFA: 18:1n-9 | LNA: 18:2n-6 | ALA: 18:3n-3
6-carbon ring 5-carbon ring
Study Protocol
Liver Urine(24-hour collection)
28 days
Blood
Plasma
Centrifugation4ºC, 10 minutes, 2000 g
Canola oilCanola oil + 0.5% CFAM
Soybean oil Soybean oil + 0.5% CFAM
4 Diet groups
36 male Wistar ratsn = 9150-200 g
IsoP Extraction
Preliminary treatments- Grinding (Liver)- Centrifugation- Hydrolysis (Liver, Plasma)
Extraction of IsoP- Sample preparation- SPE Cartridge Conditioning (2 solvents)- Sample Loading- Cartridge Wash (4 solvents)- IsoP Elution- IsoP Concentration- Sample Dilution in Mobile Phase
micro-LC-MS/MS
Data Acquisition and Treatment
IsoP Analysis: MicroLC-MS/MS
1. microLC Conditions
Eksigent micro-HPLC coupled to a QTRAP 5500
system
An HALO C18 column (10 X 0.5 mm, 2.7 µm)
LC mobile phase: A (H2O, 0.1% formic acid) and B
(ACN/MeOH, 80:20, v/v, 0.1% formic acid)
Time (min) A (%) B (%)
0 83 17
9.5 78 22
11.5 70 30
15 70 30
16 5 95
2. MS/MS Conditions ESI negative mode N2 as curtain gas Optimization of energy MRM detection of analytes
3. Quantification
Software: MultiQuant AUC IsoP/AUC Internal Standard (IS) = f(concentration of IsoP and IS)
Binary gradient
1. Experiment: 2 x 2 factorial design
2. Data = Mean ± SEM
3. Test 1: ANOVA (p≤0.05)
4. Test 2: Tukey HSD, in cases of significant interactions
5. Software: SAS v. 9.3 (SAS Institute, Cary, NC, USA)
Statistical Analysis
RESULTS AND DISCUSSION
Yield Matrix effect Coefficient of variation (%)
IS Concentration (pg/µL) Mean (%) STD Mean (%) STD Yield Matrix
effect
LiverAll concentrations 96.2 14.2 71.5 11.1 14.8 15.5
32 96.9 19.6 74.5 15.1 20.2 20.3256 95.4 8.4 68.4 4.8 8.8 7.0
PlasmaAll concentrations 80 12.9 73.2 7.9 16.1 10.8
32 87.9 12.8 67.8 5.1 14.6 7.5256 72.1 7.6 78.5 6.5 10.5 8.3
UrineAll concentrations 100.5 10 74.7 10.6 10.0 14.2
32 96.8 12.6 70.1 13.1 13.0 18.7256 104.2 5.8 79.2 5.5 5.6 6.9
Precision and repeatability of the method(IsoP Quantitation)
High levels of plasma IL-6 in Soybean oil + CFAM diet group
40
50
60
70
0Non-CFAM CFAM
pg/mL
b
aa a
Inflammatory Markers(ELISA)
p<0.05n = 9
Canola oil Soybean oil
Log10
No changes in plasma C-reactive protein (CRP)
Inflammatory Markers(ELISA)
0
1
2
3
4
Non-CFAM CFAMCanola oil Soybean oil
p>0.05n = 9
0
9
18
27
Non-CFAM CFAMCanola oil Soybean oil
Plasma fatty acids
Plasma linoleic acidSoybean oil > Canola oil
P<0.0001n = 9Weight %
of total FAME
a a
b b
0
6
12
18
Non-CFAM CFAMCanola oil Soybean oil
Plasma arachidonic acidSoybean oil > Canola oil
P = 0.003n = 9Weight %
of total FAME
a
a
bb
0
2
4
6
Non-CFAM CFAMCanola oil Soybean oil
Plasma n-6/n-3 ratioSoybean oil > Canola oil
P<0.0001n = 9
Weight %of total FAME
a a
b b
Liver Isoprostanes (IsoP)15-F2t-IsoPNo differences
p>0.05n = 9
0
20
40
60
Non-CFAM CFAMCanola oil Soybean oil
pg/mg
0.0
0.1
0.2
0.3
0.4
0.5
Non-CFAM CFAMCanola oil Soybean oil
pg/mg p>0.05n = 9
2,3-dinor-15-F2t-IsoPNo differences
Plasma Isoprostanes (IsoP)
ng/mL
0
140
280
420
560
Non-CFAM CFAMCanola oil Soybean oil
A
B
15-F2t-IsoPCFAM > Non-CFAM
p<0.05n = 9
0
40
80
120
160
Non-CFAM CFAMCanola oil Soybean oil
a
b
2,3-dinor-15-F2t-IsoPCFAM > Non-CFAM
Soybean + CFAM > Canola + CFAM
p<0.05n = 9
ng/mLc
Urinary Isoprostanes (IsoP)
0
40
80
120
160
Non-CFAM CFAMCanola oil Soybean oil
aa
b
b
15-F2t-IsoPSoybean oil > Canola oil
ng/mL p<0.05n = 9
0
35
70
105
140
Non-CFAM CFAMCanola oil Soybean oil
c
b
aa
2,3-dinor-15-F2t-IsoPCFAM > Non-CFAM
Soybean oil + CFAM > Canola oil + CFAMng/mL
p<0.05n = 9
Urinary Neuroprostanes (NeuroP)
0
15
30
45
60
Non-CFAM CFAMCanola oil Soybean oil
a
b
aa
4(RS)-F4t-NeuroPSoybean oil + CFAM > 3 other diet groups
ng/mL p<0.05n = 9
High levels of plasma IL-6 in Soybean oil + CFAM diet group
40
50
60
70
0Non-CFAM CFAM
pg/mL
b
aa a
Conclusions
CFAM induce an increased production ofmarkers of oxidative stress in the rat.
However, the increase is amplified by ahigh-linoleic acid diet compared to a high-oleic acid diet.
Oil antoxidant content?
Merci !Muchas gracias!
Thanks !
Pr. Joseph VercauterenDr. Thierry DurandDr. Claire VigorDr. Camille OgerAmandine RocherJonas CagetMichael PigniolGuillaume Reversat
Pr. Hélène JacquesPr. Paul AngersPr. Jean CollinNadine LeblancAntoine GodinCharlène Marcotte
France Canada français
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Online resources:Sébédio. AOCS Lipid Library, http://lipidlibrary.aocs.org/OilsFats/content.cfm?ItemNumber=39220. http://womanjour.ru/uploads/posts/2016-02/1455015883_original.jpghttp://www.waters.com/webassets/cms/category/media/content_blocks/seppak_block_2.jpghttp://ars.els-cdn.com/content/image/1-s2.0-S0731708515002642-fx1.jpghttp://orgchemboulder.com/Technique/Procedures/Columnchrom/Images/Flash21.jpg (chromatography)http://ars.els-cdn.com/content/image/1-s2.0-S0378382015300205-fx1.jpg (urea fractionation)https://oprocyn.com/wp-content/uploads/2015/09/oxidative-stress-image.jpghttp://www.uwgb.edu/UWGBCMS/media/chemistry/images/chemistry.jpg?ext=.jpg
Select references
Soybean oil > Canola oil Ratio n-6/n-3 Linoleic acid Arachidonic acid Antioxidant status
High plasma cholesterolHigh liver triacylglycerolsPeroxisome activity
Soybean oil + CFAM > Canola oil + CFAM Combination soybean oil and CFAM effects
Rom et al, 2017, Gustafsson et al, 1994, Zamani et al, 2017, Benson and Devi, 2009, Voutilainen et al, 1999.
DISCUSSION