Vitamin E-sparing and vitamin E-independent antioxidative effects of the flavonol quercetin in growing pigs

Animal Feed Science and Technology 169 (2011) 199–207

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Vitamin E-sparing and vitamin E-independent antioxidative effects of the flavonol quercetin in growing pigs M. Luehring, R. Blank ∗ , S. Wolffram Institute of Animal Nutrition and Physiology, Christian-Albrechts-University, 24098 Kiel, Germany

a r t i c l e

i n f o

Article history: Received 6 October 2010 Received in revised form 16 June 2011 Accepted 17 June 2011

Keywords: Quercetin Vitamin E Antioxidant Oxidative stress Pigs

a b s t r a c t The aim was to investigate in vivo the antioxidative effects of the flavonol quercetin with special emphasis on interactions with vitamin E (␣TOC) in growing pigs. It was assumed that any effects of quercetin on the antioxidant status would be more pronounced at low ␣TOC intake and that feeding diets with the addition of fish oil would induce an oxidative stress under these conditions. After a 5 week depletion period, in which all pigs (n = 26) were fed a low ␣TOC (7 mg/kg) diet, animals were assigned to one of 4 dietary groups (n = 6 or 7) according to a 2 × 2 factorial completely randomized design for four additional weeks. Factors consisted of (1) dietary supplementation of quercetin (0 or 10 mg quercetin/kg body weight/d) and (2) supplementation of fish oil (0 or 50 g/kg diet). The ␣TOC and flavonol concentrations in plasma and tissue as well as plasma thiobarbituric acid reactive substances (TBARS) and 8-iso-prostaglandin-F2␣ (8-iso-PGF2␣ ) in plasma were measured. At low vitamin E intake, supplementation of quercetin tended to elevate plasma (0.42 vs. 0.54 ␮g/mL; P=0.063) and liver (0.16 vs. 0.24 ␮g/g; P=0.06) ␣TOC concentrations with no influence on the concentrations of markers of lipid peroxidation (TBARS, 8-iso-PGF2␣ ), indicating a ␣TOC-sparing effect of quercetin. An additional ␣TOC-independent antioxidative effect of quercetin was not obvious under these conditions. However, further challenging the antioxidative mechanisms by the addition of fish oil at low ␣TOC intake resulted in a further reduction (P<0.05) of ␣TOC concentration in plasma (0.65 vs. 0.31 ␮g/mL) which could not be prevented by quercetin at least at the dose fed. However, the concomittant increase in lipid peroxidation indicated by elevated (P<0.05) concentrations of TBARS and 8-iso-PGF2␣ was ameliorated (P<0.05) by quercetin, suggesting a ␣TOC-independent antioxidative effect. Taken together, depending on the ␣TOC status, quercetin revealed a ␣TOC-sparing as well as ␣TOC-independent antioxidative effect at low ␣TOC intake. © 2011 Elsevier B.V. All rights reserved.

1. Introduction Quercetin (3, 5, 7, 3 , 4 -pentahydroxyflavone), a typical catechol-type flavonoid with high antioxidant activity, occurs in most edible parts of food and feed plants (Hertog et al., 1993; Hollman and Arts, 2000) and thus is present at various concentrations in most human and animal diets. There is an increasing interest to improve the endogenous protection against negative effects of reactive oxygen species especially in young animals by supplementing various phytogenic preparations

Abbreviations: ␣TOC, alpha-tocopherol; TBARS, thiobarbituric acid reactive substances; 8-iso-PGF2␣, 8-iso-prostaglandin-F2␣; FRAP, ferric reducing ability of plasma; MDAE, malondialdehyde equivalents; ASCE, ascorbic acid equivalents. ∗ Corresponding author. Tel.: +49 431 880 2962; fax: +49 431 880 1528. E-mail address: [email protected] (R. Blank). 0377-8401/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.anifeedsci.2011.06.006

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containing amongst others flavonoids (Gladine et al., 2001; Lykkesveldt and Svendsen, 2007; Windisch et al., 2008; Augustin et al., 2008). Currently, the mechanisms underlying these biological effects of quercetin and other flavonoids are under intense investigation. Mechanisms under discussion are a combination of specific cellular processes (e.g. enzyme inhibition, regulation of gene expression) and antioxidant activity (Middleton et al., 2000; Williams et al., 2004). The possible mechanisms of antioxidant activity of quercetin in vitro have been investigated in several studies, showing direct scavenging of reactive oxygen or nitrogen species (ROS/RNS), chelation of redox active transition metal ions and inhibition of enzymes involved in ROS production (Middleton et al., 2000; Zhu et al., 2000; Mira et al., 2002). Furthermore, in vitro interactions between quercetin and ␣TOC, resulting in a ␣TOC-sparing effect have been described (Jovanovic et al., 1996; Zhu et al., 2000; Pedrielli and Skibsted, 2002). Moreover, reports on interactions between quercetin and ␣TOC within the endogenous antioxidant network are inconclusive (Fremont et al., 1998; Choi et al., 2003; Frank et al., 2006; Ameho et al., 2008). Thus, Choi et al. (2003) reported that quercetin administration elevated both serum and liver ␣TOC concentrations, with the greatest increase occurring in rats fed a ␣TOC-depleted diet, compared to rats on a ␣TOC-repleted diet. In contrast, Ameho et al. (2008) found no effect on quercetin administration in rats on plasma or tissue ␣TOC. Thus, the relationship between quercetin and ␣TOC within the antioxidant network remains to be elucidated. The present study was performed to investigate the antioxidative and ␣TOC-sparing effects of quercetin in pigs fed diets with low ␣TOC content with or without the addition of fish oil. The status of ␣TOC, quercetin and its principal metabolites, as well as relevant biomarkers of oxidative stress were studied. 2. Materials and methods 2.1. Animals and experimental design Twenty-six barrows (German Landrace × Large White) with an initial body weight of 9.9 ± 0.1 kg were used. The experiment consisted of two periods (depletion, 35 d and intervention, 28 d). During the depletion period all pigs were fed approximately 0.7 of ad libitum intake twice daily a control diet. Animals had free access to tap water. At the beginning of the intervention period the animals were assigned to one of 4 dietary groups (n = 6 or 7) according to a 2 × 2 factorial completely randomized design consisting of (1) dietary supplementation of quercetin (0 or 10 mg quercetin/kg body weight/d) and (2) supplementation of fish oil (0 or 50 g/kg diet). All groups had similar initial ␣TOC plasma concentration. Daily amount of feed offered was always consumed completely during the entire experiment. Body weight was recorded weekly. Animal care and experimental procedures were conducted in accordance with the German animal welfare law (Lorz and Metzger, 2008). 2.2. Diets All diets were based on barley, wheat and soybean meal. In fish oil diets, 50 g of maize starch was replaced by 50 g of fish oil (Imperial Öl Import, Hamburg, Germany) as an additional source of polyunsaturated fatty acids. The ␣TOC content in control diets (7.0 mg/kg) and fish oil diets (8.6 mg/kg) originated from the natural ingredients only. The nutrient and fatty acid concentration of the experimental diets are given in Tables 1 and 2, respectively. All experimental diets were formulated to meet or exceed the nutrient and energy requirements recommended for pigs (20–50 kg) by the National Research Council (1998). Quercetin supplementation was achieved by manually mixing the adequate amount of quercetin (C. Roth GmbH, Karlsruhe, Germany) into the meals immediately prior to feeding (half of the daily dose per meal). According to the manufacturer, quercetin is extracted and isolated from different plants (Quercus ibericus, Dysooma veitchii, Hypericum ascyron, Apocynum lancifolium Rus.) containing high amount of quercetin. Diets were fed as mash. 2.3. Sample collection and laboratory analysis Blood samples from all pigs were collected weekly prior to feeding by jugular vein puncture into heparinized tubes. Plasma was obtained by centrifugation (2000 × g, 10 min, 4 ◦ C) and stored at −80 ◦ C until analysis. For the determination of 8-iso-PGF2␣ , 600 ␮L aliquots of plasma were stored in vials containing 6 ␮L butylhydroxytoluene (BHT) solution (0.005% BHT in ethanol, w/v). At the end of the experimental period, animals were fasted for 12 h, stunned by electric shock and slaughtered by exsanguination. During exsanguination, blood samples from each pig were collected into heparinized tubes. Liver, lung, muscle (m. longissimus dorsi) and white adipose tissue samples from the neck-shoulder area (4 samples, app. 20 g each) were rapidly dissected, frozen on dry ice and stored at −80 ◦ C until analysis. One blood and tissue sample from each animal was lyophilized, frozen in liquid nitrogen, and ground under constant cooling using a mill (IKA-Werke GmbH & Co KG, Staufen, Germany). Samples were weighed before and after lyophilization and stored in airtight containers at −70 ◦ C until analysis. Feed samples were ground successively in a mill (ZM 100, Retsch, Haan, Germany) with 3- and 1-mm screens and with a 0.2 mm screen for starch analysis. Crude nutrients, tocopherols and acid hydrolysed ether extract of feedstuffs were analysed according to the official methods of feed analysis of the Verband deutscher landwirtschaftlicher Untersuchungsund Forschungsanstalten (Naumann et al., 1997). Neutral detergent fibre (NDFom) and acid detergent fibre (ADFom) content,

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Table 1 Feed ingredients and analysed nutrient content of experimental diets (g/kg as fed).

Ingredients Barley Soybean meal, 480 g/kg crude protein Wheat Maize starch Fish oil Dicalcium phosphate Sodium chloride Mineral premixa Vitamin premixb Zinc chloride Analysed nutrient content Dry matter Crude ash Crude protein Crude fat Crude fibre Neutral detergent fibre Acid detergent fibre Starch Lysin Methionine Methionine + cysteine Threonine Tryptophan Calciumc Phosphorusc Tocopherols (mg/kgd ) ␣-Tocopherol ␥-Tocopherol Metabolizable energy (MJ/kg)

Control diet

Fish oil diet

562.9 260 90 50 – 20 5 10 2 0.1

562.9 260 90 – 50 20 5 10 2 0.1

886 57 175 16 45 212 70 360 9.0 2.5 5.5 6.2 2.2 6.9 6.7

892 56 169 53 50 193 79 323 9.3 2.6 5.6 6.2 2.3 6.9 6.7

7.00 ± 0.01 6.24 ± 0.08 12.7

8.60 ± 0.36 6.10 ± 0.15 13.7

a The mineral premix provided per kg diet: vitamin A, 3500 IU; vitamin D3 , 400 IU; lysine-HCl, 0.7 g; dl-methionine, 0.15 g; l-threonine, 0.15 g; Ca, 2.1 g, P, 0.55 g; Na, 0.4 g; Mg, 0.1 g; Cu, 5 mg. b The vitamin premix provided per kg diet: butylhydroxytoluene, 15 mg; Ca, 0.2 g; Fe, 150 mg; Zn, 15 mg; Mn, 15 mg, Co, 0.15 mg, J, 0.5 mg; menadione, 10 mg; thiamine, 5 mg; riboflavin, 12 mg; pyridoxine, 12 mg; cobalamin, 0.1 mg; niacin, 60 mg; biotin, 0.3 mg; pantothenic acid, 30 mg; folic acid, 3 mg; choline, 50 mg; ascorbic acid, 10 mg. c Calculated from feed tables (INRA-AFZ, 2004). d Means ± SEM (n = 5). The ␤- and ␦-tocopherol content were below detection limit (2 mg/kg).

Table 2 Fatty acid concentration (mg/kg) and peroxide value (mEq O2 /kg fat) of the experimental diets.a

16:0 18:0 16:1 18:1 20:1 18:2 18:3 18:4 20:4 20:5 22:5 22:6 Saturated fatty acids Monounsaturated fatty acids Polyunsaturated fatty acids (n − 6) Polyunsaturated fatty acids (n − 3) n − 6:n − 3 ratio Peroxide value a b

Means ± SEM, n = 5. Not detectable (detection limit: 50 mg/kg).

Control diet

Fish oil diet

3184 ± 72 507 ± 7 n.d.b 4216 ± 22 109 ± 11 9400 ± 55 1368 ± 22 n.d.b n.d.b n.d.b n.d.b n.d.b 3691 4325 9400 1368 6.9 13.4 ± 0.98

9138 ± 240 1862 ± 42 2806 ± 96 7430 ± 131 598 ± 191 10,102 ± 133 1900 ± 169 1334 ± 36 458 ± 8 7352 ± 166 960 ± 29 5372 ± 150 11,000 10,834 10,560 16,918 0.6 21.8 ± 1.66

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corrected for residual ash, were analysed as described by Van Soest et al. (1991), except that Na2 SO4 was not used in the NDF preparation. Starch content was determined by enzymatic hydrolysis of starch employing a heat-stable ␣-amylase (Termamyl 120 L; Novo Industrial, Bagsværd, Denmark). Concentration of ␣TOC in plasma and tissues was determined by HPLC with fluorescence detection (Podda et al., 1996). The ␣TOC concentrations were quantified using authentic ␣TOC (Merck KGaA, Darmstadt, Germany) as an external standard. Analysis of plasma and tissue concentrations of quercetin and the monomethylated derivatives isorhamnetin (3 -O-methyl quercetin) and tamarixetin (4 -O-methyl quercetin) were performed by HPLC with fluorescence detection as described previously (Bieger et al., 2008). All samples were treated enzymatically with ␤-glucuronidase/sulfatase type H-1 (Sigma–Aldrich Chemie GmbH, Taufkirchen, Germany) prior to the extraction of flavonols. Authentic flavonols (C. Roth GmbH, Karlsruhe, Germany) were used as external standards. As previously described (de Boer et al., 2005; Bieger et al., 2008) hemoglobin was determined in the supernatant of homogenized tissue and in full blood samples with a spectrophotometer at a wave length of 540 nm (UV-1602, Shimadzu Deutschland GmbH, Duisburg, Germany). The fraction of residual blood in tissues was calculated by dividing the peak maximum of hemoglobin in tissues with the peak maximum of the respective full blood sample. Flavonol concentrations determined in the respective plasma sample were then multiplied with the fraction of residual blood in tissues and subtracted from the value obtained for the tissue. The lipid peroxidation in vitro was assessed by measuring the thiobarbituric acid-reacting substances (TBARS) in plasma. The TBARS levels in plasma were determined according to Yagi (1998) with some modifications. Briefly, 500 ␮L of plasma were mixed with 250 ␮L trichloroacetic acid (7.5%, w/v) and centrifuged (20,800 × g; 10 min at 4 ◦ C). Five hundred ␮L of the supernatant were mixed with 500 ␮L 2-thiobarbituric acid (1%, w/v) and 25 ␮L of SDS/BHT solution (0.5% in EtOH, w/v), heated for 10 min at 95 ◦ C (water bath) and chilled afterwards. Two mL butanol were added, samples were mixed for 10 min and centrifuged (3220 × g; 5 min at 4 ◦ C). The absorbance of the supernatant was assayed with a spectrophotometer at a wave length of 532 nm (UV-1602, Shimadzu Deutschland GmbH, Duisburg, Germany). Tetraethoxypropane was used as a standard. The TBARS in plasma samples were expressed as ␮mol malondialdehyde equivalents (MDAE)/L. The ferric reducing ability of plasma (FRAP) assay was performed according to Benzie and Strain (1996). Briefly, 50 ␮L H2 O and 10 ␮L plasma or standard solutions were pipetted into a 96 well-plate, 300 ␮L FRAP solution were added, mixed for 10 s and the absorbance was measured in a plate-reader at a wavelength of 595 nm after 15 min incubation at 37 ◦ C. Ferric reducing ability of plasma values are expressed as ascorbic acid equivalents. In vivo lipid peroxidation was assessed by measuring the plasma concentrations of 8-iso-PGF2␣ with a commercial EIA kit (Assay Designs Inc., Ann Arbor, MI, USA) according to the manufacturer’s protocol. 2.4. Statistical analyses Except for tissue flavonol and ␣TOC concentration, all data were analysed according to a repeated measurement design using the MIXED procedure of SAS version 8.2 (SAS Institute, 2001). As measurements taken closer in time on the same animal are likely to be more correlated than measurements taken further apart in time, a spatial covariance structure SP(POW) was used. Plasma concentrations of ␣TOC during the depletion period were subjected to one way ANOVA and plasma concentrations of ␣TOC, TBARS and 8-isoPGF2␣ during the experimental period were subjected to a three-way ANOVA for repeated measures. The model included supplementation of fish oil, supplementation of quercetin, time, and their interactions. Tissue flavonols and ␣TOC concentrations and daily gain were statistically analysed using either a three- or two-way ANOVA for ␣TOC and for total flavonols, respectively, using the GLM procedure of SAS. For all statistical evaluations the SLICES option and the LS-MEANS follow-up test was used for comparison of group means. Differences were considered significant when P<0.05. Data are presented as means and pooled SEM. 3. Results Feed allowance (approximately 0.7 of ad libitum intake) was always completely consumed. Daily weight gains were not affected by fish oil (0.555 vs. 0.579 kg; P=0.185) and tended to decrease by quercetin (0.585 vs. 0.549 kg; P=0.059) supplementation. However, final body weight (36.5 ± 0.3) was not different between dietary treatments. 3.1. Plasma and tissue concentrations of ˛-tocopherol and quercetin During the depletion period plasma ␣TOC decreased considerably from 1.81 ± 0.07 to 0.67 ± 0.02 ␮g/mL (P<0.05; means ± SEM; n = 26). Seven days after the start of the experiment, plasma ␣TOC concentrations (Table 3) in fish oil supplemented groups declined (P<0.05) compared to non supplemented groups. Although, supplementation of quercetin had no effect on ␣TOC concentration in plasma, there was a tendency (P=0.057) for a quercetin × time interaction, due to an increase in plasma ␣TOC concentrations in the non fish oil supplemented group at day 28 of intervention. At the end of the experiment, ␣TOC concentrations in tissues (Table 4) of the fish oil supplemented groups were lower (P<0.001) than in tissues of the non supplemented groups. The highest ␣TOC concentrations were found in white adipose tissue and plasma followed by liver, muscle and lung (P<0.001). Quercetin supplementation had no significant effect on tissue ␣TOC

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Table 3 Effect of fish oil (F, 50 g/kg) and quercetin (Q, 10 mg/kg body weight/d) supplementation on plasma concentrations of ␣-tocopherol (␮g/mL) in pigs over a time (T) period of 28 days. Day on trial

SEMa

Dietary treatments Control −Q

Fish oil −Q

+Q x,y

x

Fish oil −

+Q y

Main effects

y

Effectsb

Quercetin −

+

+

F

Q

Q×F

0–28 d

0.55

0.67

0.43

0.41

0.04

0.62

0.42

0.50

0.55

0.002

0.394

0.197

0d 7d 14 d 21 d 28 d n

0.66 0.53x , y 0.54x , y 0.52x 0.55x 7

0.67 0.62y 0.71y 0.63x 0.76y 7

0.73 0.39x 0.44x 0.32y 0.30z 6

0.63 0.39x 0.40x 0.33y 0.32z 6

0.03

0.66 0.58 0.63 0.58 0.65 14

0.68 0.39 0.41 0.33 0.31 12

0.69 0.46 0.49 0.42 0.42 13

0.65 0.50 0.55 0.48 0.54 13

0.763 0.003 0.001 < 0.001 < 0.001

0.492 0.510 0.328 0.335 0.063

0.724 0.022 0.002 0.008 <0.001

a b x y z

Standard error of mean. Probabilities of the main effect of time and interactions were: T (P<0.001); F × T (P<0.001), Q × T (P=0.057); Q × F × T (P=0.215). Means within a row bearing no common superscript letter differ (P<0.05). Means within a row bearing no common superscript letter differ (P<0.05). Means within a row bearing no common superscript letter differ (P<0.05).

Table 4 Effect of fish oil (F, 50 g/kg) and quercetin (Q, 10 mg/kg body weight/d) supplementation on plasma (␮g/mL) and tissue (TI) concentrations (␮g/g wet tissue) of ␣-tocopherol in pigs slaughtered at 28 days. Tissue

SEMa

Dietary treatments Control −Q

Fish oil

Main effects Fish oil

+Q

−Q

+Q

Average mean

0.33x

0.38x

0.20y

0.22y

0.03

0.36

0.21

Plasma Liver Lung Muscle Adipose n

0.55x 0.20x , y 0.12x 0.09x , y 0.70x 7

0.76y 0.35x 0.14x 0.10x 0.60x , y 7

0.30z 0.12y 0.06y 0.03y 0.54x , y 6

0.32z 0.14y 0.07y 0.05x , y 0.52y 6

0.03

0.65x 0.28x 0.13x 0.10x 0.65 14

0.31y 0.13y 0.06y 0.04y 0.53 12

a b x y z

−

Effectsb

Quercetin −

+

+

Q×F

F

Q

0.27

0.30

<0.001

0.204

0.395

0.42 0.16 0.09 0.06 0.61 13

0.54 0.24 0.10 0.07 0.56 13

<0.001 0.009 <0.001 0.016 0.136

0.063 0.060 0.182 0.475 0.564

<0.001 0.195 0.692 0.869 0.831

Standard error of mean. Probabilities of the main effect of tissue and interactions were: Ti (P<0.001); F × Ti (P=0.008), Q × Ti (P=0.207); Q × F × Ti (P=0.499). Means within a row bearing no common superscript letter differ (P<0.05). Means within a row bearing no common superscript letter differ (P<0.05). Means within a row bearing no common superscript letter differ (P<0.05).

concentrations, however, ␣TOC concentrations in plasma and liver tended (P=0.06) to increase due to quercetin supplementation (Table 4). After quercetin supplementation over a period of 28 days, the highest total flavonol concentration (sum of quercetin, isorhamnetin and tamarixetin) was found in the liver (Table 5). Plasma, lung and muscle total flavonol concentrations did not differ in pigs fed quercetin, but liver total flavonol content was higher in the non fish oil compared to fish oil fed group (P<0.01). Table 5 Plasma (nmol/mL) and tissue concentrations (nmol/g wet weight) of flavonolsa in pigs fed the control or fish oil (50 g/kg) supplemented diet with Quercetin (Q, 10 mg quercetin/kg body weight/d), respectively, for 28 days. Parameter



quercetin, Total flavonols ( isorhamnetin, tamarixetin)

a b x y *

Tissue

Control + Q

Fish oil + Q

Mean

SEMb

Q

Ti

Q × Ti

Mean Plasma

0.18 0.08x

0.13 0.05x

0.16 0.07x

0.03

0.112

<0.001

0.123

Liver Lung Muscle n

0.53y 0.07x 0.06x 7

0.32y , * 0.06x 0.07x 6

0.42y 0.07x 0.06x

All samples were enzymatically treated prior to flavonol analysis; flavonol concentrations were corrected for residual blood. Standard error of mean. Means within a column bearing no common superscript letter differ (P<0.05). Means within a column bearing no common superscript letter differ (P<0.05). Means within a row are different (P<0.05).

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Table 6 Effect of fish oil (F, 50 g/kg) and quercetin (Q, 10 mg/kg body weight/d) supplementation on formation of thiobarbituric reactive substances (TBARS, ␮mol MDAE/L)a in pigs fed over a time (T) period of 28 days. Day on trial

SEMc

Dietary treatments Control −Q

Fish oil −Q

+Q x

x

Fish oil +Q

y

Main effect

Control z

Effectsb

Quercetin Fish oil

x

y

−

+

F

Q

Q×F

0–28 d

0.16

0.17

0.72

0.57

0.03

0.16

0.65

0.44

0.37

<0.001

0.141

0.315

0d 7d 14 d 21 d 28 d n

0.29 0.14x 0.15x 0.10x 0.12x 7

0.20 0.16x 0.16x 0.13x 0.19x 7

0.24 0.55y 0.66y 0.93y 1.23y 6

0.17 0.54y 0.57y 0.70z 0.88z 6

0.03

0.25 0.15x 0.16x 0.12x 0.16x 14

0.20 0.55y 0.62y 0.82y 1.05y 12

0.27 0.34 0.41 0.52x 0.68x 13

0.18 0.35 0.37 0.42y 0.53y 13

0.503 <0.001 <0.001 <0.001 <0.001

0.210 0.884 0.537 0.120 0.030

0.549 <0.001 <0.001 <0.001 <0.001

a b c x y z

Malondialdehyde equivalents. Probabilities of the main effect of time and interactions were: T (P<0.001); F × T (P<0.001), Q × T (P=0.112); Q × F × T (P=0.116). Standard error of mean. Means within a row bearing no common superscript letter differ (P<0.05). Means within a row bearing no common superscript letter differ (P<0.05). Means within a row bearing no common superscript letter differ (P<0.05).

Table 7 Effect of fish oil (F, 50 g/kg) and quercetin (Q, 10 mg/kg body weight/d) supplementation on ferric reducing ability of plasma (␮mol ASCE1 /L)a in pigs fed over a time (T) period of 28 days. Day on trial

SEMc

Dietary treatments Control

Fish oil

Main effect Fish oil

Effectsb

Quercetin

−Q

+Q

−Q

Control

Fish oil

−

+

F

Q

0–28 d

127.0

127.1

124.0

126

4.2

127.1

125.1

125.5

126.6

0.721

0.852

0.875

0d 7d 14 d 21 d 28 d n

113.3 121.0 124.7 132.3 143.4x , y 7

111.9 116.4 131.7 130.9 144.8x 7

116.8 117.0 128.3 134.2 123.9y 6

115.8 125.0 126.4 132.9 129.8x 6

3.7

112.6 118.7 128.2 131.6 144.1x 14

116.3 121.0 127.4 133.5 126.8y 12

115.0 119.0 126.5 133.3 133.7 13

113.9 120.7 129.1 131.9 137.3 13

0.625 0.761 0.906 0.795 0.023

0.876 0.817 0.733 0.857 0.628

0.966 0.838 0.912 0.992 0.137

a b c x y

+Q

Q×F

Ascorbic acid equivalents. Probabilities of the main effect of time and interactions were: T (P<0.001); F × T (P=0.033), Q × T (P=0.878); Q × F × T (P=0.391). Standard error of mean. Means within a row bearing no common superscript letter differ (P<0.05). Means within a row bearing no common superscript letter differ (P<0.05).

3.2. Thiobarbituric acid reactive substances, ferric reducing ability and 8-iso-prostaglandin F2˛ concentrations of plasma Already at day 7 of the experiment, the concentration of plasma TBARS increased in both fish oil groups compared to the non fish oil groups. Quercetin supplementation had no effect on TBARS formation in plasma, however, there was a tendency for a quercetin × time (P=0.112) and quercetin × fish oil × time (P=0.116) interaction resulting in lower plasma TBARS in the quercetin supplemented compared to non supplemented fish oil groups at day 21 and 28 of the experiment (Table 6). The ferric reducing ability of plasma was recorded weekly during the entire experimental period (Table 7). During the first three weeks, FRAP values increased (P<0.001) over time, but were not affected by treatments. There was a significant fish oil × time interaction (P=0.033) which resulted in a significant decrease in antioxidative capacity as indicated by lower FRAP values in the quercetin supplemented compared to the non supplemented fish oil group at the end of the experiment. Plasma 8-iso-PGF2␣ concentrations were recorded at the beginning and the end of the experimental period. Feeding the fish oil-containing diets increased concentrations of plasma 8-iso-PGF2␣ after 28 days (fish oil × time interaction; P=0.011), whereas supplementation of quercetin showed a tendency to increase plasma 8-iso-PGF2␣ (Table 8). However, there was a tendency for a quercetin fish oil × interaction (P=0.066), resulting in an inhibition of the formation of 8-iso-PGF2␣ in plasma of the quercetin supplemented compared to the non supplemented fish oil group. 4. Discussion The major natural lipophilic antioxidant ␣TOC is part of an interlinking set of redox cycles. Within this network ␣TOC can be regenerated from its tocopheroxyl radical by water-and lipid-soluble substances both by nonenzymatic as well as enzymatic mechanisms (Constantinescu et al., 1993). Thus, ␣TOC may be regenerated directly by vitamin C, or indirectly by thiolic antioxidants, such as glutathione and lipoic acid (Packer et al., 2001). Several studies have described the relationship

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Table 8 Effect of fish oil (F, 50 g/kg) and quercetin (Q, 10 mg/kg body weight/d) supplementation on plasma concentrations of 8-iso-prostaglandin F2␣ (8-iso-PGF2␣ , pg/mL) in pigs fed over a time (T) period of 28 days. Day on trial

SEMa

Dietary treatments Control

Fish oil

Fish oil

−Q

+Q

−Q

+Q

0–28 d 0d

784 735

745 738

1185 630

774 626

28 d n

833x 7

752x 7

1741y 6

923x 6

a b x y

Main effect

Control 98 88

Effectsb

Quercetin Fish oil

−

+

F

Q

Q×F

764 736

979 628

988 682

760 682

0.122 0.545

0.107 0.998

0.066 0.944

792 14

1332 12

1287 13

837 13

0.006

0.019

0.003

Standard error of mean. Probabilities of the main effect of time and interactions were: T (P<0.003); F × T (P=0.011), Q × T (P=0.178); Q × F × T (P=0.131). Means within a row bearing no common superscript letter differ (P<0.05). Means within a row bearing no common superscript letter differ (P<0.05).

between vitamin C and ␣TOC, but less information is available regarding the role of quercetin in this network (Liu and Lee, 1998; Hill et al., 2001, 2003). The aims of the present study were to investigate possible antioxidative and/or ␣TOC-sparing effects of quercetin (10 mg/kg BW/day; 4 weeks) in pigs fed diets with a low ␣TOC content with or without the addition of 50 g/kg fish oil, whereby feeding of fish oil was included to induce a mild oxidative stress. Numerous in vitro studies showed synergistic and mutual regenerating activities between quercetin and ␣TOC (Zhu et al., 2000; Jovanovic et al., 1996; Pedrielli and Skibsted, 2002). In contrast, in vivo studies showed contradictory results (Fremont et al., 1998; Choi et al., 2003; Frank et al., 2006; Ameho et al., 2008). Ameho et al. (2008) reported that chronic quercetin application [120 mg/day (420 mg/kg BW/day for 12 weeks)] had no effect on the ␣TOC concentration in plasma and tissues of ␣TOC-depleted (<1 IU/day) and ␣TOC-repleted (30 IU/day) rats. However, Choi et al. (2003) reported that 8 and 80 mg quercetin/BW/day fed to ␣TOCdepleted rats for 4 weeks increased ␣TOC concentrations in serum and liver dose-dependently. This effect, however, was not observed in ␣TOC-repleted animals (75 IU/kg diet). In contrast to Choi et al. (2003), Frank et al. (2006) found increased ␣TOC concentrations in plasma and liver of rats with normal ␣TOC intake (∼44 IU/kg diet) after 4 weeks of daily oral quercetin application (∼110 mg/kg BW/day). The current study revealed that supplementation of 10 mg quercetin/kg BW/day for 4 weeks had a ␣TOC-sparing effect in plasma and liver of pigs fed a low-␣TOC diet without the addition of fish oil. Despite this ␣TOC-sparing effect, there were no differences in antioxidant capacity in vitro (FRAP: 143.4 vs. 144.8 ␮mol ASCE/L). In this regard previous studies demonstrated, that ␣TOC is a minor contributor (5.8%) of total antioxidant activities in the FRAP-assay (Rice-Evans, 2000). Consequently, in the current study higher ␣TOC concentrations in plasma did not influence hydrophilic antioxidant capacity. Furthermore, total plasma flavonol concentration in pigs fed the low ␣TOC diet (0.08 ␮mol/L; Table 5) was lower than those quercetin concentrations used for in vitro assays showing an enhancement of antioxidant capacity (Lotito and Frei, 2006; DeGraft-Johnson et al., 2007). Thus, the contribution of quercetin to plasma antioxidant capacity in vivo, measured by FRAP, is expected to be small, because of the low plasma concentrations of quercetin, its intensive biotransformation, and the large contribution of other physiological antioxidants such as urate and ascorbate to plasma antioxidant capacity (Lotito and Frei, 2006). In rats, oral administration of quercetin (20 mg/day) resulted in considerably lower ex vivo formation of liver TBARS. Interestingly, this inhibitory effect on lipid peroxidation by quercetin was more pronounced in ␣TOC-deprived rats than in undeprived animals (Choi et al., 2003). In contrast, results from the current study showed no effect of quercetin in the fish oil free diets on plasma TBARS and 8-iso-PGF2␣ , respectively, at low ␣TOC intake in pigs. This is in accordance with findings in non ␣TOC-deficient rats (Garcia-Saura et al., 2005). These results suggest that plasma ␣TOC concentrations in groups fed the fish oil free diets were still sufficient to prevent a measurable increase in lipid peroxidation. Because quercetin intake in non supplemented fish oil diets elevated plasma and liver ␣TOC concentrations, a ␣TOC-sparing effect of quercetin in vivo can be postulated. The mechanisms, however, by which quercetin increases the ␣TOC concentrations cannot be directly deduced from present findings. In this context a quercetin-mediated regeneration of ␣TOC within the antioxidative network has been postulated in the literature (Zhu et al., 2000; Pedrielli and Skibsted, 2002; Frank et al., 2006; Zhou et al., 2005; Filipe et al., 2007). In vitro-studies suggested that quercetin may interact with ␣TOC by binding to polar head groups of membrane phospholipids at the lipid/water interface (Terao et al., 1994; Pawlikowska-Pawlega et al., 2007). Whether quercetin interacts directly with the tocopheroxyl radical or indirectly via vitamin C or thiol antioxidants remains to be elucidated. Furthermore, it has been shown that flavonoids not only work as free radical scavenger per se but also can induce the synthesis of other antioxidants such as glutathione peroxidase via a nuclear factor erythroid 2-related factor 2 (Nrf2)-dependent signal transduction pathway (Surh et al., 2005). Furthermore, Umathe et al. (2008) have shown that quercetin might influence ␣TOC metabolism also via cytochrome P450 (CYP3A), a non-antioxidative mechanism. Because any antioxidant or ␣TOC-sparing effects of quercetin might be most pronounced in the presence of an oxidative stress, two feeding groups received the low-␣TOC diet with the addition of fish oil, which further reduced plasma ␣TOC concentrations compared to non fish oil supplementation. Furthermore, tissue ␣TOC concentrations (liver, lung, muscle) in fish oil supplemented groups tended also to be reduced. The ␣TOC concentrations were nearly two times higher in the adipose tissue than in the plasma of fish oil groups. This result agrees with studies suggesting a slow ␣TOC turnover in

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adipose tissue and enhanced turnover of ␣TOC in plasma (Jensen et al., 1988; Wang et al., 1996). Consequently, the decrease of plasma ␣TOC concentration in the fish oil supplemented groups within the first week of the experiment indicates a fast depletion of the labile ␣TOC pool. Concomitant with the decrease of ␣TOC in plasma in the fish oil supplemented groups, we found a significant increase in plasma TBARS and 8-iso-PGF2␣ concentrations. These results are in accordance with in vivo studies showing that high polyunsaturated fatty acids intake with inadequate ␣TOC supply is associated with oxidative stress, principally indicated by an increase in products of lipid peroxidation and a decrease in plasma and tissue ␣TOC concentrations (Haglund et al., 1991; Cho and Choi, 1994; Cho et al., 1995; Sen et al., 1997). Although supplementation of quercetin to the fish-oil containing diet clearly ameliorated the oxidative stress induced by polyunsaturated fatty acids (lower TBARS and 8-iso-PGF2␣ values), no differences in plasma ␣TOC levels were found with fish oil supplementation. These findings are in agreement with results from in vivo experiments which showed reduced intensity of lipid peroxidation in plasma (TBARS, conjugated dienes) without modification of ␣TOC plasma levels (Fremont et al., 1998; Gladine et al., 2001). Interestingly, total flavonol concentration in liver was lower in the fish oil supplemented group than in the non-supplemented group, indicating an increased turnover of quercetin. 5. Conclusion In conclusion, at low dietary ␣TOC intake, a ␣TOC-sparing effect of quercetin could be demonstrated without an effect on biomarkers of oxidative stress. 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