Dietary oxidized n-3 PUFA induce oxidative stress and inflammation: role of intestinal absorption of 4-HHE and reactivity in intestinal cells

Dietary intake of long-chain n-3 PUFA is now widely advised for public health and in medical practice. However, PUFA are highly prone to oxidation, producing potentially deleterious 4-hydroxy-2-alkenals. Even so, the impact of consuming oxidized n-3 PUFA on metabolic oxidative stress and inflammation is poorly described. We therefore studied such effects and hypothesized the involvement of the intestinal absorption of 4-hydroxy-2-hexenal (4-HHE), an oxidized n-3 PUFA end-product. In vivo, four groups of mice were fed for 8 weeks high-fat diets containing moderately oxidized or unoxidized n-3 PUFA. Other mice were orally administered 4-HHE and euthanized postprandially versus baseline mice. In vitro, human intestinal Caco-2/TC7 cells were incubated with 4-hydroxy-2-alkenals. Oxidized diets increased 4-HHE plasma levels in mice (up to 5-fold, P < 0.01) compared with unoxidized diets. Oxidized diets enhanced plasma inflammatory markers and activation of nuclear factor kappaB (NF-κB) in the small intestine along with decreasing Paneth cell number (up to −19% in the duodenum). Both in vivo and in vitro, intestinal absorption of 4-HHE was associated with formation of 4-HHE-protein adducts and increased expression of glutathione peroxidase 2 (GPx2) and glucose-regulated protein 78 (GRP78). Consumption of oxidized n-3 PUFA results in 4-HHE accumulation in blood after its intestinal absorption and triggers oxidative stress and inflammation in the upper intestine. Dietary intake of long-chain n-3 PUFA is now widely advised for public health and in medical practice. However, PUFA are highly prone to oxidation, producing potentially deleterious 4-hydroxy-2-alkenals. Even so, the impact of consuming oxidized n-3 PUFA on metabolic oxidative stress and inflammation is poorly described. We therefore studied such effects and hypothesized the involvement of the intestinal absorption of 4-hydroxy-2-hexenal (4-HHE), an oxidized n-3 PUFA end-product. In vivo, four groups of mice were fed for 8 weeks high-fat diets containing moderately oxidized or unoxidized n-3 PUFA. Other mice were orally administered 4-HHE and euthanized postprandially versus baseline mice. In vitro, human intestinal Caco-2/TC7 cells were incubated with 4-hydroxy-2-alkenals. Oxidized diets increased 4-HHE plasma levels in mice (up to 5-fold, P < 0.01) compared with unoxidized diets. Oxidized diets enhanced plasma inflammatory markers and activation of nuclear factor kappaB (NF-κB) in the small intestine along with decreasing Paneth cell number (up to −19% in the duodenum). Both in vivo and in vitro, intestinal absorption of 4-HHE was associated with formation of 4-HHE-protein adducts and increased expression of glutathione peroxidase 2 (GPx2) and glucose-regulated protein 78 (GRP78). Consumption of oxidized n-3 PUFA results in 4-HHE accumulation in blood after its intestinal absorption and triggers oxidative stress and inflammation in the upper intestine. Chronic inflammation and oxidative stress are now recognized as major factors involved in the pathogenesis of several current diseases, such as overweight, obesity, and cardiovascular diseases (1Hotamisligil G.S. Inflammation and metabolic disorders.Nature. 2006; 444: 860-867Crossref PubMed Scopus (6276) Google Scholar, 2Manabe I. Chronic inflammation links cardiovascular, metabolic and renal diseases.Circ. J. 2011; 75: 2739-2748Crossref PubMed Scopus (188) Google Scholar–3Zulet M.A. Puchau B. Navarro C. Marti A. Martinez J.A. Inflammatory biomarkers: the link between obesity and associated pathologies.Nutr. Hosp. 2007; 22: 511-527PubMed Google Scholar). Elevated levels of proinflammatory cytokines and chemokines, such as interleukins (IL) and monocyte chemotactic protein-1 (MCP-1), are hallmarks of the metabolic syndrome (1Hotamisligil G.S. Inflammation and metabolic disorders.Nature. 2006; 444: 860-867Crossref PubMed Scopus (6276) Google Scholar, 2Manabe I. Chronic inflammation links cardiovascular, metabolic and renal diseases.Circ. J. 2011; 75: 2739-2748Crossref PubMed Scopus (188) Google Scholar). Several studies demonstrate the nutritional benefits of consuming long-chain (LC) n-3 PUFA from fish, in particular, docosahexaenoic acid (DHA, 22:6 n-3) and eicosapentaenoic acid (EPA, 20:5 n-3) to protect against several pathologies (4Fedor D. Kelley D.S. Prevention of insulin resistance by n-3 polyunsaturated fatty acids.Curr. Opin. Clin. Nutr. Metab. Care. 2009; 12: 138-146Crossref PubMed Scopus (199) Google Scholar, 5Fetterman Jr, J.W. Zdanowicz M.M. Therapeutic potential of n-3 polyunsaturated fatty acids in disease.Am. J. 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However, current studies reveal that the n-3 PUFA may not be devoid of risk. Possible harmful effects of high levels of n-3 PUFA on retinal membrane degeneration have been described by Tanito et al. (9Tanito M. Brush R.S. Elliott M.H. Wicker L.D. Henry K.R. Anderson R.E. High levels of retinal membrane docosahexaenoic acid increase susceptibility to stress-induced degeneration.J. Lipid Res. 2009; 50: 807-819Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). Dietary LC n-3 PUFA are highly vulnerable to oxidation, which is one of the major problems in food chemistry and may decrease their nutritional value. Indeed, peroxidation causes loss of nutritional quality and further leads to the generation of genotoxic and cytotoxic compounds, such as the 4-hydroxy-2-alkenals (10Esterbauer H. Schaur R.J. Zollner H. Chemistry and biochemistry of 4-hydroxynonenal, malonaldehyde and related aldehydes.Free Radic. Biol. Med. 1991; 11: 81-128Crossref PubMed Scopus (5894) Google Scholar, 11Guichardant M. Bacot S. Moliere P. Lagarde M. Hydroxy-alkenals from the peroxidation of n-3 and n-6 fatty acids and urinary metabolites.Prostaglandins Leukot. Essent. Fatty Acids. 2006; 75: 179-182Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). 4-Hydroxy-2-hexenal (4-HHE) and 4-hydroxy-2-nonenal (4-HNE) are major end-products derived from n-3 and n-6 PUFA peroxidation, respectively. In addition to being markers of lipid peroxidation in vivo, 4-HNE and 4-HHE induce noxious effects on biological systems. These lipid aldehydes are prone to react with thiol and amine moieties and produce Schiff base and/or Michael adducts with biomolecules, such as proteins, DNA, and phospholipids (12Bacot S. Bernoud-Hubac N. Chantegrel B. Deshayes C. Doutheau A. Ponsin G. Lagarde M. Guichardant M. Evidence for in situ ethanolamine phospholipid adducts with hydroxy-alkenals.J. Lipid Res. 2007; 48: 816-825Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 13Guichardant M. Taibi-Tronche P. Fay L.B. Lagarde M. Covalent modifications of aminophospholipids by 4-hydroxynonenal.Free Radic. Biol. Med. 1998; 25: 1049-1056Crossref PubMed Scopus (101) Google Scholar). Numerous studies reported the genotoxicity and cytotoxicity of these 4-hydroxy-2-alkenals on tissues and cells in pathophysiological contexts (14Bradley M.A. Xiong-Fister S. Markesbery W.R. Lovell M.A. Elevated 4-hydroxyhexenal in Alzheimer's disease (AD) progression.Neurobiol. Aging. 2012; 33: 1034-1044Crossref PubMed Scopus (65) Google Scholar, 15Mattson M.P. Roles of the lipid peroxidation product 4-hydroxynonenal in obesity, the metabolic syndrome, and associated vascular and neurodegenerative disorders.Exp. Gerontol. 2009; 44: 625-633Crossref PubMed Scopus (219) Google Scholar–16Shibata N. Yamada S. Uchida K. Hirano A. Sakoda S. Fujimura H. Sasaki S. Iwata M. Toi S. Kawaguchi M. et al.Accumulation of protein-bound 4-hydroxy-2-hexenal in spinal cords from patients with sporadic amyotrophic lateral sclerosis.Brain Res. 2004; 1019: 170-177Crossref PubMed Scopus (34) Google Scholar), but nothing is known to date about the possible contribution of 4-hydroxy-2-alkenals present in food products, their fate after ingestion, and their metabolic effects. In this context, the intestinal tract represents the first barrier of detoxification and defense against oxidative stress. Thus, intestinal cells can be exposed to oxidized PUFA or to lipid peroxidation end-products. However, evidence is lacking to support unfavorable health effects of dietary oxidized n-3 PUFA and to demonstrate their possible role in the generation of oxidative stress and inflammation. Furthermore, limited data are available to support the hidden assumption of intestinal absorption of dietary lipid oxidation by-products. Therefore, we hypothesized that long-term intake of limited amounts of oxidized n-3 PUFA in high-fat (HF) diets could exert harmful health effects due to the absorption of their end-products such as 4-HHE by the small intestine, the end-products having been formed during the processing, storage, and/or final handling of foods or after their ingestion. The aim of the present study was thus to investigate i) the effects of oxidized n-3 PUFA diets compared with unoxidized diets on oxidative stress and inflammation in mice and ii) the possible implication of the intestinal absorption of some PUFA oxidation end-products, namely, 4-hydroxy-2-alkenals, through their effects on intestinal stress and inflammation in vivo and in vitro. Because several types of LC n-3 PUFA sources are present in human food, we tested lipid mixtures containing EPA and DHA carried by either triacylglycerols (TG) or phospholipids (PL). Moreover, we analyzed different segments of the absorptive intestinal epithelium, especially the duodenum before interactions with bile salts occur, the jejunum that represents the major site of lipid absorption, as well as the ileum to test whether some effects remain in this more distal segment. 4-HHE, 4-HNE, and trideuterated compounds were synthesized according to Soulère et al. (17Soulère L. Queneau Y. Doutheau A. An expeditious synthesis of 4-hydroxy-2E-nonenal (4-HNE), its dimethyl acetal and of related compounds.Chem. Phys. Lipids. 2007; 150: 239-243Crossref PubMed Scopus (31) Google Scholar). Omegavie® Tuna oil 25 DHA-flavorless as a source of triacylglycerol rich in long-chain n-3 fatty acids (TG-DHA, 26% of DHA), lecithin rich in DHA (PL-DHA, 41% of DHA), and kiwi seed oil were provided by Polaris (Pleuven, France). Lard was supplied by Celys (Rezé, France), sunflower oil was from Lesieur® (Asnières-sur-Seine, France), and oleic sunflower oil from Olvéa (Marseille, France). Vegetable lecithin rich in linoleic acid 18:2 n-6 (PL-LA) was from Lipoid (Frigenstrasse, Germany). Four lipid blends were prepared at the labscale to obtain similar fatty acid composition and quantities in the four diets and similar amounts of phospholipids and triacylglyerols. In these blends, DHA was supplied either in the form of triacylglycerols (TG diet) or phospholipids (PL diet); i.e., the name chosen for the diets reflects the type of molecules that carry long-chain n-3 PUFA in the diet. The oxidized lipid blends (TG-ox and PL-ox, respectively) were prepared as follows. Primary lipid mixtures for PL and TG groups were prepared with a small proportion of lard to maintain oxidability of PUFA. These preliminary oil mixtures were dispersed in aqueous phase (mineral water; Evian) to prepare 30% w/w oil-in-water emulsions. The emulsions were then kept at 50°C in the dark with continuous shaking until the oxidation level was considered sufficient according to our previous experiments (estimated α-tocopherol contents of the blends decreased by 50%). Oxidized emulsions were then lyophilized, and the resulting oxidized lipid mixtures completed with the necessary quantity of lard to reach the required final composition of lipid blends. The composition of the four diets is reported in Table 1.TABLE 1.Composition of diets containing long-chain n-3 PUFA either esterified in phospholipids (PL diet) or in triacylglycerols (TG diet) and their oxidized counterparts (PL-ox, TG-ox)High-fat containing n-3 PUFA asPLPL-oxTGTG-oxIngredient (g/100g)Lipid mixture, among which are20202020Lard18.1018.1018.0618.06Sunflower oil0.60.60.20.2Oleic sunflower0.40.4——Kiwi seed oil0.10.10.020.02Tuna oil (DHA located in TG)——0.90.9Phospholipids, among which arePL-DHA (DHA located in PL)0.80.8——Lecithin PL-LA——0.80.8Corn starch39393939Sucrose10101010Pure cellulose5555Vitamin mixture5555Mineral mixture1111TocopherolsaConsidering the difference observed in tocopherols in lipid mixtures, which can affect their metabolic impact (46), care was taken to supplement lipid mixtures with α-tocopherol during formulation to achieve iso-tocopherol diets.0.0990.0990.0920.084Energy content (kJ/g)18.1418.1418.1418.14Energy %Lipids41.541.541.541.5Protein15.715.715.715.7Carbohydrates34.134.134.134.1a Considering the difference observed in tocopherols in lipid mixtures, which can affect their metabolic impact (46Chiang Y.F. Shaw H.M. Yang M.F. Huang C.Y. Hsieh C.H. Chao P.M. Dietary oxidised frying oil causes oxidative damage of pancreatic islets and impairment of insulin secretion, effects associated with vitamin E deficiency.Br. J. Nutr. 2011; 105: 1311-1319Crossref PubMed Scopus (26) Google Scholar), care was taken to supplement lipid mixtures with α-tocopherol during formulation to achieve iso-tocopherol diets. Open table in a new tab Male C57BL/6 mice (8 wk, 20g) were from Janvier SA (Le Genest Saint-Isle, France) and were housed in a temperature-controlled room (22°C) with a 12 h light/12 h dark cycles. After 2 weeks of chow diet, mice were randomly divided into four groups of 12 mice and fed one of the 4 high-fat diets containing n-3 PUFAs: unoxidized groups: PL, TG; oxidized groups: PL-ox, TG-ox. Animal experiments were performed under the authorization n°69-266-0501 (Direction Départementale des Services Vétérinaires du Rhône). All experiments were carried out according to the guidelines laid down by the French Ministry of Agriculture (n° 87-848) and the E.U. Council Directive for the Care and Use of Laboratory Animals of November 24th, 1986 (86/609/EEC), in conformity with the Public Health Service (PHS) Policy on Humane Care and Use of Laboratory Animals. COS holds a special license (n° 69266257) to experiment on living vertebrates issued by the French Ministry of Agriculture and Veterinary Service Department. Body weight was measured twice weekly and food intake was measured weekly. After 8 weeks, mice were euthanized by intraperitoneal (IP) injection of sodium pentobarbital (60 mg/kg). Plasma, liver, white adipose tissue (epididymal, retroperitoneal, subcutaneous) and small intestine mucosa were collected. Caco-2/TC7 cells, which are the widely used in vitro model of human origin to test intestinal absorption of lipids, were provided by Monique Rousset (Centre de Recherche des Cordeliers, Paris, France) and used between passages 35 and 45. Cells were seeded in 75 cm2 flasks (Falcon; Becton Dickinson) until 80–90% confluence. They were grown in complete DMEM (Gibco) supplemented with 20% heat-inactivated FBS (Gibco), 1% nonessential amino acids (Gibco), and 1% antibiotics (penicillin/streptomycin; Gibco) and then maintained under a 10% CO2 atmosphere at 37°C. For experiments, cells were seeded at a density of 25 × 104 cells per filter on microporous (0.4 μm pore size) polyester filters (Transwell; Corning, USA) and grown to confluence in complete medium, which was routinely reached 7 days after seeding. The cells were used 21 days after seeding. Monolayers were incubated with 4-HHE or 4-HNE in the apical compartment (1–100 µM brought in DMSO at 0.5% in the final medium), and the basolateral compartment received serum-free DMEM. After incubation, basolateral media and cells were collected. After an overnight fast, three groups of four male C57/BL6 mice (8 weeks old, 22 g) were given a single application of 4-HHE diluted in water via 0.5% DMSO by oral gavage at a dosage of 10 mg/kg body weight and then were euthanized 1, 2, and 4 h after gavage. A fourth group of mice was euthanized immediately after gavage for the baseline control. For euthanizing, mice were anesthetized by IP injection of pentobarbital (35 mg/kg), and blood was collected by cardiac puncture with heparinized syringes. Plasma and small intestine mucosa (duodenum and jejunum) were collected. 4-hydroxy-2-alkenals were derivatized from 400 µl of basolateral media of Caco-2/TC7 or from 300 µl of plasma, under argon, and in lipid blends as described previously (18Michalski M.C. Calzada C. Makino A. Michaud S. Guichardant M. Oxidation products of polyunsaturated fatty acids in infant formulas compared to human milk–a preliminary study.Mol. Nutr. Food Res. 2008; 52: 1478-1485Crossref PubMed Scopus (60) Google Scholar). Deuterated 4-HNE and 4-HHE (20 ng), used as internal standards, were added to the samples. Briefly, they were treated with O-2,3,4,5,6-pentafluorobenzyl hydroxylamine hydrochloride. After acidification with H2SO4, pentafluorobenzyloxime derivatives were extracted with methanol and hexane. The hydroxyl group was then converted into trimethylsilylether after an overnight treatment with N,O-bis(trimethylsilyl)trifluoroacetamide at room temperature. The pentafluorobenzyloxime trimethylsilylether derivatives of 4-HHE (O-PFB-TMS-4-HHE) and 4-HNE (O-PFB-TMS-4-HNE) were then analyzed by GC-MS using negative ion chemical ionization (NICI) mode on a Hewlett-Packard quadripole mass spectrometer interfaced (18Michalski M.C. Calzada C. Makino A. Michaud S. Guichardant M. Oxidation products of polyunsaturated fatty acids in infant formulas compared to human milk–a preliminary study.Mol. Nutr. Food Res. 2008; 52: 1478-1485Crossref PubMed Scopus (60) Google Scholar) with a Hewlett-Packard gas chromatograph (Les Ullis, France). IL-6 and MCP-1 (Clinisciences, Nanterre, France) were assayed by ELISA kit according to the manufacturer's instructions. Plasma triacyglycerols (TAG) were measured with the triglyceride PAP kit (BioMérieux, Marcy l'Etoile, France) as previously described (19Lefils J. Geloen A. Vidal H. Lagarde M. Bernoud-Hubac N. Dietary DHA: time course of tissue uptake and effects on cytokine secretion in mice.Br. J. Nutr. 2010; 104: 1304-1312Crossref PubMed Scopus (26) Google Scholar). Plasma TAG concentration was calculated by subtracting the free glycerol in plasma measured with the glycerol UV-method (R-Biopharm/Boehringer, Mannheim, Germany). Plasma NEFA was measured using NEFA-C kit (Wako Chemicals, Neuss, Germany) (19Lefils J. Geloen A. Vidal H. Lagarde M. Bernoud-Hubac N. Dietary DHA: time course of tissue uptake and effects on cytokine secretion in mice.Br. J. Nutr. 2010; 104: 1304-1312Crossref PubMed Scopus (26) Google Scholar). Total lipids were extracted from 35 µl of plasma as described previously (19Lefils J. Geloen A. Vidal H. Lagarde M. Bernoud-Hubac N. Dietary DHA: time course of tissue uptake and effects on cytokine secretion in mice.Br. J. Nutr. 2010; 104: 1304-1312Crossref PubMed Scopus (26) Google Scholar). The organic phase was evaporated under N2, and total fatty acids were transesterified using boron trifluoride in methanol (BF3/methanol) (19Lefils J. Geloen A. Vidal H. Lagarde M. Bernoud-Hubac N. Dietary DHA: time course of tissue uptake and effects on cytokine secretion in mice.Br. J. Nutr. 2010; 104: 1304-1312Crossref PubMed Scopus (26) Google Scholar). The FA methyl esters were then analyzed by GC using a DELSI instrument model DI 200 equipped with a fused silica capillary SP-2380 column (60 m × 0.22 mm). Heptadecanoic acid (C17:0; Sigma, France) was used as an internal standard. Thiobarbituric acid-malondialdehyde (MDA) adducts were separated using a method adapted from different authors (20Mendes R. Cardoso C. Pestana C. Measurement of malondialdehyde in fish: a comparison study between HPLC methods and the traditional spectrophotometric test.Food Chem. 2009; 112: 1038-1045Crossref Scopus (138) Google Scholar, 21Seljeskog E. Hervig T. Mansoor M.A. A novel HPLC method for the measurement of thiobarbituric acid reactive substances (TBARS). A comparison with a commercially available kit.Clin. Biochem. 2006; 39: 947-954Crossref PubMed Scopus (98) Google Scholar) by HPLC and measured by fluorimetry using an external calibration curve (excitation 535 nm, emission 555 nm). Hydroperoxides were quantified in lipid blends according to a method adapted from Nourooz-Zahed et al. (22Nourooz-Zadeh J. Tajaddini-Sarmadi J. Wolff S.P. Measurement of plasma hydroperoxide concentrations by the ferrous oxidation-xylenol orange assay in conjunction with triphenylphosphine.Anal. Biochem. 1994; 220: 403-409Crossref PubMed Scopus (610) Google Scholar). Total RNA was extracted from Caco-2/TC7, duodenum, jejunum, and ileum of mice using the NucleoSpin® RNA/Protein kit (Macherey Nagel, Duren, Germany). cDNAs were synthesized from 1 µg of total RNA in the presence of 100 units of Superscript II (Invitrogen) with a mixture of random hexamers and oligo (dt) primers (Promega, Charbonnières, France). The amount of target mRNAs was measured by RT, followed by real-time PCR, using a Rotor-Gene Q (Qiagen, France). Primer sequence and RT-quantitative PCR conditions are available upon request ([email protected]). Hypoxanthine guanine phosphoribosyl transferase (HPRT) mRNA level and TATA-box binding protein (TBP) mRNA were used to normalize data of duodenum, jejunum, ileum, and Caco-2/TC7 cells, respectively. Total proteins from Caco-2/TC7 and jejunum of mice were extracted with the NucleoSpin® RNA/Protein kit (Macherey Nagel). A total of 40 μg of protein from each sample were subjected to 10% SDS-PAGE and transferred to a polyvinylidine fluoride (PVDF) membrane. Membranes were blocked with kit reagent and incubated overnight with antibodies according to the manufacturer's recommendations. Antibodies against total NF-κB P65 and phospho-NF-κB P65 (Ser 529) were obtained from Signalways Antibody (Clinisciences, France); phospho-IκBα (phosphoS32+S36) was from Abcam (Cambridge, UK); and β-actine was from Sigma, France. After incubation with secondary antibody, blots were developed with a commercial kit (WesternBreeze Chemiluminescent, Invitrogen, France). Quantification was performed by densitometry analysis of specific bands on immunoblots with Quantity One software (Bio-Rad, Marne-la-Coquette, France). Duodenal tissues were removed, fixed in 90% ethanol for 24 h at −20°C, and processed into paraffin. Serial paraffin sections (4 µm) were rehydrated, and endogenous peroxidase activity was quenched with 20 min incubation in 5% H2O2/PBS. After incubation in 2.5% normal horse blocking serum (Impress, Vector), sections were incubated for 60 min at room temperature with anti-lysozyme (1/100; Zymed Laboratories) antibody diluted in blocking solution. The immune reactions were then detected by incubation with a ready-to-use peroxidase-labeled secondary reagent, ImmPRESS (MP-7401 for rabbit antibodies; Abcys, Paris, France) (30 min, room temperature). Control experiments were performed simultaneously omitting the primary antibody or incubating with preimmune rabbit serum. The sections were then counterstained and mounted. The Paneth cell lineage was analyzed by assessing the percentage of crypt cross-sections with Paneth cells (per 80 crypts). For this purpose, four to six sections were analyzed per mouse. A crypt was considered when it was cut along or nearly along the length of the crypt lumen (at least two thirds of the length of the crypt). All slides were analyzed by a single investigator who was blinded to the treatment groups. Protein carbonyl content was determined with an Oxyblot Oxidized Protein Detection Kit from Chemicon (Hampshire, UK). The carbonyl groups in the protein chains were derivatized into dinitro-phenyl-hydrazone by reaction with dinitro-phenyl-hydrazine (DNPH) according to the manufacturer's instructions. After the derivatization of the protein sample (20 µg), one-dimensional electrophoresis was carried out on a 10% SDS-PAGE gel, and proteins were transferred to PVDF membranes. After incubation with anti-DNP antibody, the blot was developed with a chemiluminescence detection system. The intensity of each line was measured and normalized by mean control levels. Fifty milligrams of total proteins from Caco-2/TC7 cells or from duodenum and jejunum of mice were spotted onto nitrocellulose membrane. Blocked membrane was incubated overnight with anti-HNE-adducts (Calbiochem 393207, San Diego, CA, USA) or anti-HHE-adducts (CosmoBio N213730-EX, Tokyo, Japan) antibodies. Blots were developed with a commercial kit (WesternBreeze Chemiluminescent, Invitrogen, France). The quantification was performed by densitometric analysis of specific spots on immunoblots using Quantity One software. All data are presented as means ± SEM and were analyzed using Statview 5.0 software (Abacus Concept, Berkeley, CA). One-way ANOVA followed by Fisher PLSD was used for i) the dietary study to compare PL, PL-ox, TG, and TG-ox groups, ii) the gavage study to compare plasma alkenal concentrations as a function of time, and iii) the Caco-2 cell studies to compare treatment effects. Two-way ANOVA followed by Fisher PLSD was used to compare oxidized versus unoxidized groups globally in the dietary study (mice of oxidized groups versus mice of unoxidized groups). Differences were considered significant at the P < 0.05 level. Table 2 shows that we succeeded in producing lipid mixtures containing different amounts of oxidation products, especially 4-HHE, which was significantly higher in oxidized versus unoxidized lipid blends. MDA and hydroperoxides in oxidized oils were in a range considered as acceptable for human consumption. Noticeably, oxidation did not impact the fatty acid profile (Table 2); n-3 PUFA content and n-6/n-3 ratio were consistent with dietary recommendations. Mice in oxidized and unoxidized groups did not differ in final body weight gain, liver weight, white adipose tissue, plasma TAG, or plasma NEFA (Table 3).TABLE 2Fatty acid composition and oxidation markers in the lipid blendsPLPL-oxTGTG-oxFA (mg/g lipid)SFA319 ± 12319 ± 9293 ± 19319 ± 23MUFA410 ± 13413 ± 7375 ± 17372 ± 23n-6 PUFA105 ± 3106 ± 1104 ± 11112 ± 11n-3 PUFA, among which are18.1 ± 0.4a17.9 ± 0.1a22.2 ± 2.8b23.4 ± 2.7b18:3 n-310.6 ± 0.310.7 ± 0.19.4 ± 0.910.0 ± 1.320:5 n-30.9 ± 0.3a1.1 ± 0.0a2.2 ± 0.3b2.4 ± 0.2b22:6 n-34.3 ± 0.0a3.9 ± 0.0a8.8 ± 1.9b8.8 ± 0.9bTotal PUFA123 ± 4124 ± 1126 ± 10136 ± 13n-6/n-3 ratio7.7 ± 0.5b7.4 ± 0.1b5.4 ± 0.2a5.5 ± 0.1aTotal FA849 ± 129853 ± 16790 ± 18820 ± 48Lipid peroxidation markers (nmol/g of lipid blend)4-HHE (oxidation of n-3)0.5 ± 0.1a1.2 ± 0.2b1.9 ± 0.1c8.5 ± 0.5d4-HNE (oxidation of n-6)0.2 ± 0.1a0.5 ± 0.1a1.7 ± 0.2b4.4 ± 0.5cMDA25 ± 2.5a40.0 ± 4.0a5.9 ± 1.2b35 ± 14aHydroperoxides (µmol eq. H2O2/g lipid)1.6 ± 0.9a1.3 ± 0.1a3.7 ± 0.1b5.8 ± 0.1cMeans in a row superscripted by different letters are significantly different (P < 0.05). Data are means ± SEM for n = 3. FA, fatty acid; MDA, malondialdehyde; MUFA, monounsaturated fatty acid; SFA, saturated fatty acid. Open table in a new tab TABLE 3.Morphologic parameters, food intake, and plasma lipid concentrations of mice fed oxidized (PL-ox, TG-ox) or unoxidized (PL, TG) n-3 dietsMice Groups According to Dietary LipidsMorphologic ParametersPLPL-oxTGTG-oxBiometric dataInitial body weight (g)23.9 ± 0.324.0 ± 0.324.5 ± 0.324.1 ± 0.3Body weight gain (g)3.1 ± 0.14.3 ± 0.53.7 ± 0.33.0 ± 0.5Energy intake (kJ/mouse/d)60.2 ± 1.362.7 ± 0.359.8 ± 1.368.1 ± 1.3 aP < 0.05, ANOVA followed by Fisher test for TG-ox versus TG.Liver weight (g)1.21 ± 0.041.24 ± 0.061.27 ± 0.041.28 ± 0.05WAT weight (g)0.72 ± 0.050.95 ± 0.100.93 ± 0.110.73 ± 0.05Plasma lipidsTriglycerides (mM)0.64 ± 0.040.64 ± 0.100.57 ± 0.040.58 ± 0.04NEFA (mM)0.27 ± 0.010.32 ± 0.020.37 ± 0.020.28 ± 0.01Data are means ± SEM for n = 7–9 per group. WAT, white adipose tissue.a P < 0.05, ANOVA followed by Fisher test for TG-ox versus TG. Open table in a new tab Means in a row superscripted by different letters are significantly different (P < 0.05). Data are means ± SEM for n = 3. FA, fatty acid; MDA, malondialdehyde; MUFA, monounsaturated fatty acid; SFA, saturated fatty acid. Data are means ± SEM for n = 7–9 per group. WAT, white adipose tissue. Because consuming oxidized n-3 diets could contribute to circulating biomarkers of lipid peroxidation such as 4-HHE and 4-HNE, we measured these 4-hydroxy-2-alkenals in plasma. Fig. 1A shows a 4-fold increase of 4-HHE concentration in the plasma of oxidized groups (427–508 nM) versus unoxidized groups (89–128 nM) (P < 0.01). However, the concentration of 4-HNE (from n-6 PUFA) did not differ among groups (from 6 to 9 nM; Fig. 1B). FA composition of total plasma lipids was characterized to test whether chronic ingestion of oxidized diet would affect PUFA metabolism. Plasma FA profile (see supplementary Table I) actually reflected the composition of ingested dietary fats. The proportions of n-3 PUFA (EPA, DHA, α-linolenic acid