Cyclic Fatty Acid Monomers (CFAM) from Heated...

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


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


b

Hep

atic

PC

(nm

ol/m

g pr

otei

n)

a

Less liver PC with CFAMp<0.05n = 9

0

27

54

81


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


ba

mg/dL

CFAM effects on plasma cholesterol and lipoproteinsMore total Cholesterol with CFAM

p<0.05n = 9

0

30

60

90

120


b

a

mg/dL

More LDL-C with CFAMp<0.05n = 9

mg/dL

0

10

20

30

40


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


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


Log10

No changes in plasma C-reactive protein (CRP)

Inflammatory Markers(ELISA)

0

1

2

3

4


p>0.05n = 9

0

9

18

27


Plasma fatty acids

Plasma linoleic acidSoybean oil > Canola oil

P<0.0001n = 9Weight %

of total FAME

a a

b b

0

6

12

18


Plasma arachidonic acidSoybean oil > Canola oil

P = 0.003n = 9Weight %

of total FAME

a

a

bb

0

2

4

6


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


pg/mg

0.0

0.1

0.2

0.3

0.4

0.5


pg/mg p>0.05n = 9

2,3-dinor-15-F2t-IsoPNo differences

Plasma Isoprostanes (IsoP)

ng/mL

0

140

280

420

560


A

B

15-F2t-IsoPCFAM > Non-CFAM

p<0.05n = 9

0

40

80

120

160


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


aa

b

b

15-F2t-IsoPSoybean oil > Canola oil

ng/mL p<0.05n = 9

0

35

70

105

140


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


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

Select referencesChristie, W.W., Brechany, E.Y., Sébédio, J.L. and Le Quéré, J.L. Silver ion chromatography and gas chromatography-mass spectrometry in the structural analysis of cyclic monoenoic acids formed in frying oils. Chem. Phys. Lipids, 66, 143-153 (1993) Morrow JD, Chen Y, Brame CJ, Yang J, Sanchez SC, Xu J, Zackert WE, Awad JA, Roberts LJ. (1999) The isoprostanes: unique prostaglandin like products of free radical-catalyzed lipid peroxidation. Drug Metab Rev. 31:117-139.Roberts LJ, Morrow JD. (2000) Measurement of F2-isoprostanes: a reliable index of oxidant stress in vivo. Free Radic Biol Med. 28:505-513.Voutilainen S, Morrow JD, Roberts LJ, Alfthan G, Alho H, Nyyssonen K, Salonen JT. (1999) Enhanced in vivo lipid peroxidation at elevated plasma total homocysteine levels. Arterioscler Thromb Vasc Biol. 19:1263-1266.Keaney JF, Larson MG, Vasan RS, Wilson PWF, Lipinska I, Corey D,cMassaro JM, Sutherland P, Vita JA, Benjamin EJ. (2003) Obesity and systemic oxidant stress: clinical correlates of oxidative stress in the Framingham Study. Arteroscl Thromb Vasc Biol. 23:434-439.Martin JC, Joffre F, Siess MH, Vernevaut MF, Collenot P, Genty M, Sébédio JL (2000) Cyclic fatty acid monomers from heated oil modify the activities of lipid synthesizing and oxidizing enzymes in rat liver. J Nutr. 130(6):1524-30Rom O, Jeries H, Hayek T, Aviram M. Supplementation with linoleic acid-rich soybean oil stimulates macrophage foam cell formation via increased oxidative stress and diacylglycerol acyltransferase1-mediated triglyceride biosynthesis. Biofactors 2016; doi:10.1002/biof.1319.Gustafsson IB, Vessby B, Ohrvall M, et al. (1994) A diet rich in monounsaturated rapeseed oil reduces the lipoprotein cholesterol concentration and increases the relative content of n-3 fatty acids in serum in hyperlipidemic subjects. Am J Clin Nutr. 59:667-674. Zamani E, Sadrzadeh-Yeganeh H, Sotoudeh G, Keramat L, Eshraghian M, Rafiee M, Koohdani F (2017) The interaction between ApoA2 −265T>C polymorphism and dietary fatty acids intake on oxidative stress in patients with type 2 diabetes mellitus. Eur J Nutr 56(5):1931-1938.Benson MK, Devi K (2009) Influence of omega-6/omega-3 rich dietary oils on lipid profile and antioxidant enzymes in normal and stressed rats. Indian J Exp Biol 47:98-103Halliwell B, Lee Y.J.C. Using Isoprostanes as Biomarkers of Oxidative Stress: Some Rarely Considered Issues. Antioxidants Redox Signal.2010;13(2):145-56.Dupuy A, Le Faouder P, Vigor C, Oger C, Galano JM, Dray C, Chung-Yung Lee J, Valet P, Gladine C, Durand T, Bertrand-Michel J. Simultaneous quantitative profiling of 20 isoprostanoids from omega-3 and omega-6 polyunsaturated fatty acids by LCeMS/MS in various biological samples. Anal Chim Acta 2016;921:46e58.Oger C., Bultel-Poncé V., Guy A., Balas L., Rossi J-C., Durand T., Galano J-M. The handy use of Brown’s catalyst for a skipped diyne deuteration: application to the synthesis of a d4-labeled-F4t-neuroprostane. Chem. Eur. J. 2010, 16, 13976-13980. A. Guy, C. Oger, J. Hepekauzen, C. Signorini, T. Durand, C. De Felice, A. Fürstner, J-M. Galano. Oxygenated Metabolites of n-3 Polyunsaturated Fatty Acid as Potential Oxidative Stress Biomarkers: Total Synthesis of 8-F3t-IsoP, 10-F4t-NeuroP, and [D4]-10-F4t-NeuroP.Chem. Eur. J. 2014, 20, 6374-6380

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

http://lipidlibrary.aocs.org/OilsFats/content.cfm?ItemNumber=39220

http://womanjour.ru/uploads/posts/2016-02/1455015883_original.jpg

http://ars.els-cdn.com/content/image/1-s2.0-S0731708515002642-fx1.jpg

https://oprocyn.com/wp-content/uploads/2015/09/oxidative-stress-image.jpg

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

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