Effect of oxidized fat in food on lipoprotein oxidation | Jukka-Pekka Suomela Department of Biochemistry and Food Chemistry

Background Fat oxidation

(autoxidation) This proton ripped off from another fatty acid molecule Background Fat oxidation

Background Fat oxidation

Breakdown of hydroperoxides Background Fat oxidation

Photosensitized oxidation of linoleic acid Autoxidation of linoleic acid (Buettner, Molecular targets of photosensitization) In addition, enzymatic oxidation is possible in biological systems… Background Fat oxidation

Background “ Basic” indicators of fat oxidation  peroxide value (PV, POV)  Fat oxidation gives birth to peroxides that are measured with a method based on the ability of peroxides to release iodine from potassium jodide  Titration of the relased iodine with Na 2 S 2 O 3 - liuoksella  p-anisidine value (p-AV)  Measures the amount of secundary oxidation products (aldehydes, ketones) in oil  spectrophotometric determination at 350 nm  Fats get rancid also hydrolytically!

Background Oxidation products from dietary fats to the body – lipoproteins and atherosclerosis Witztum, J.L. Lancet 344:  Oxidized lipoproteins seem to increase the risk of atherosclerosis  LDL  Chylomicrons, VLDL, and their remnants?

Background Classification of lipoproteins  chylomicrons  formed in small intestine  very low density lipoproteins (VLDL)  formed in liver  intermediate density lipoproteins (IDL)  formed from VLDL  low density lipoproteins (LDL)  formed from IDL  high density lipoproteins (HDL)  formed in liver and small intestine

Durrington, P.N. Hyperlipidaemia: Diagnosis and Management. 2nd edition. London: Butterworth-Heinemann; Background Classification of lipoproteins

Background Metabolism of lipoproteins

Background Formation of foam cells

Background Atherosclerotic plaque

Background Lipoproteins transport oxidized lipids

Background Food and oxidized lipoproteins Staprans, I. et al. Arterioscler Thromb 14:  In various studies, dietary oxidized lipids have increased the oxidation of chylomicrons and VLDL  food  small intestine  lipoproteins  other tissues… What? Where? Why?

Aim of the research 1. To study the effect of oxidized dietary oil on the amount of oxidized lipids in porcine lipoproteins 2. To identify the molecular structures of oxidized triacylglycerols (TAGs) in porcine chylomicrons and VLDL

Oxidation of fatty acids within glycerolipids

Study protocols STUDY 1  3 groups of 3 pigs; 14% sunflower seed oil in the feed  Peroxide value of the oil:  group 1: 1 meq O 2 /kg oil  group 2: 84 meq O 2 /kg oil  group 3: 223 meq O 2 /kg oil STUDY 2  2 groups of 10 pigs; 16% sunflower seed oil in the feed  Peroxide value of the oil:  group 1: 1 meq O 2 /kg oil  group 2: 190 meq O 2 /kg oil

Methods 1. Determination of the oxidation level of pig lipoproteins  BDC (baseline diene conjugation) spectrophotometrically 2. Identification of the molecular structures of oxidized TAGs in chylomicrons and VLDL:  Separation of lipid classed by TLC (thin layer chromatography)  Further separation by RP-HPLC (reversed-phase liquid chromatography)  Detectors:  ESI-MS  ELSD  UV (DNPH derivatives of core aldehydes)

Methods Identification of molecular structures, TLC TAG Oxidized TAG

(MeOH / i-Pr) (80 / 20, by vol) 0,85 ml / min 20 min (MeOH / i-Pr) (20 / 80, by vol) SOLVENT GRADIENT: Discovery  HS C18 5  m, 250 mm x 4.6 mm i.d. COLUMN: Methods Identification of molecular structures, HPLC

HPLC 130  l/min850  l/min 720  l/min UV/ELSD ESI MS Methods Identification of molecular structures, HPLC

Methods

Results Study 1 Chylomicrons VLDL LDL a b Group 1: 1 meq O2/kg oil Group 2: 84 meq O2/kg oil Group 3: 223 meq O2/kg oil Time point 1: 3 hr after meal Time point 2: 4 hr after meal

Results Study 1

Estimated proportions of oxidized TAGs of the total lipids of the test oils and chylomicrons a Group 1 b Group 3 c Oil0.2Oil6.2Time point 3 h Pig 10.0 Pig 70.4 Pig 20.1 Pig 80.4 Pig 30.2 Pig 91.9 Time point 4 h Pig 10.0 Pig 70.6 Pig 20.3 Pig 81.1 Pig 30.3 Pig 91.2 a Results as g/100 g total lipids. b Peroxide value of the oil used in feed: 1 meq O 2 /kg. c Peroxide value of the oil used in feed: 223 meq O 2 /kg. Results Study 1

 Typically only one fatty acid residue of a TAG molecule was oxidized in the identified molecules  Among oxidized TAG structures were:  hydroxides  epoxides  ketones  aldehydes (9:0, 12:1)  Hydroperoxides were not found Results Study 1

a b ChylomicronsVLDL LDL Group 1: 1 meq O 2 /kg oil Group 2: 190 meq O 2 /kg oil Plasma a b Results Study 2 Time point 1: 3 hr after meal Time point 2: 4 hr after meal

Estimated proportions of oxidized TAGs of the total lipids of the test oils, chylomicrons, and VLDL a Group 1 b Group 2 c Oil0.0Oil 6.4 Chylomicrons Time point 3 h0,8 ± 0,7 a Time point 3 h 2,0 ± 0,6 b Time point 4 h0,8 ± 0,9 a Time point 4 h2,4 ± 0,7 b VLDL Time point 3 h0,1 ± 0,1 Time point 3 h 0,2 ± 0,2 Time point 4 h0,1 ± 0,1 a Time point 4 h 0,2 ± 0,1 b a Results as g/100 g total lipids. b Peroxide value of the oil used in feed: 1 meq O 2 /kg. c Peroxide value of the oil used in feed: 190 meq O 2 /kg. Results Study 2

Results Study 2  Typically only one fatty acid residue of a TAG molecule was oxidized in the identified molecules  Among oxidized TAG structures were:  hydroxides  epoxides  ketones  aldehydes (9:0, 12:1)  Hydroperoxides were not found

Conclusions  Diet containing oxidized sunflower oil increased lipid oxidation in lipoproteins  Oxidized fats are partly transported to the circulation  Effect of vitamin E?  Several oxidized molecular structures derived from TAGs were found in chylomicrons and VLDL  Method was not optimized to extensively oxidized structures

Literature  Suomela, J.-P., Ahotupa, M., Sjövall, O., Kurvinen, J.-P., & Kallio, H. (2004) Diet and lipoprotein oxidation: analysis of oxidized triacylglycerols in pig lipoproteins. Lipids 39: 639–647, DOI: /s  Suomela, J.-P., Ahotupa, M., and Kallio, H. (2005) Triacylglycerol oxidation in pig lipoproteins after a diet rich in oxidized sunflower seed oil. Lipids 40: 437–444, DOI: /s  Ahotupa, M., Suomela, J.-P., Vuorimaa, T., and Vasankari, T. (2010) Lipoprotein-specific transport of circulating lipid peroxides. Ann Med 42: 521–529, DOI: /  Tarvainen, M., Phuphusit, A., Suomela J.-P., Kuksis, A., and Kallio H. (2012) Effects of antioxidants on rapeseed oil oxidation in an artificial digestion model analyzed by UHPLC−ESI−MS. J Agric Food Chem, DOI: /jf E-pub ahead of print.  Kanner, J., Gorelik, S., Sirota, R., & Kohen, R. (2012) Protection by polyphenols of postprandial human plasma lipid oxidative modification: the stomach as a bioreactor. J Agric Food Chem, DOI: /jf300193g. E-pub ahead of print.