Impact of red versus white meat consumption in a prudent or Western

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Food Safety and Toxicology

Impact of red versus white meat consumption in a prudent or Western dietary pattern on the oxidative status in a pig model Sophie Goethals, Els Vossen, Joris Michiels, Lynn Vanhaecke, John Van Camp, Thomas Van Hecke, and Stefaan De Smet J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b00559 • Publication Date (Web): 23 Apr 2019 Downloaded from http://pubs.acs.org on April 24, 2019

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Journal of Agricultural and Food Chemistry

Impact of red versus white meat consumption in a prudent or Western dietary pattern on the oxidative status in a pig model Sophie Goethals1,2,3, Els Vossen1, Joris Michiels1, Lynn Vanhaecke2,4, John Van Camp3, Thomas Van Hecke1, Stefaan De Smet1*

1 Laboratory

for Animal Nutrition and Animal Product Quality, Department of Animal Sciences

and Aquatic Ecology, Ghent University, Coupure Links 653, B-9000, Ghent, Belgium

2 Laboratory

of Chemical Analysis, Department of Veterinary Public Health and Food Safety,

Ghent University, Salisburylaan 133, B-9820, Merelbeke, Belgium

3 Research

Group Food Chemistry and Human Nutrition, Department of Food Safety and Food

Quality, Ghent University, Coupure Links 653, B-9000, Ghent, Belgium

4

Institute for Global Food Security, School of Biological Sciences, Queen’s University,

University Road, Belfast, BT7 1NN, Northern Ireland, United Kingdom

* Corresponding author: email: [email protected], phone: +32 9 264 90 03

1 ACS Paragon Plus Environment


1

Abstract

2

Human diets contain a complex mixture of antioxidants and pro-oxidants that contribute to the

3

body’s oxidative status. In this study, 32 pigs were fed chicken versus red & processed meat in

4

the context of a prudent or Western dietary pattern for four weeks, to investigate their oxidative

5

status. Lipid oxidation products (malondialdehyde, 4-hydroxy-2-nonenal and hexanal) were

6

higher in the chicken versus red & processed meat diets (1.7- to 8.3-fold) and subsequent in

7

vitro (1.3- to 1.9-fold) and in vivo (1.4 to 3-fold) digests (P < 0.001), which was presumably

8

related to the higher polyunsaturated fatty acid content in chicken meat and/or the added

9

antioxidants in processed meat. However, diet only had a marginal or no effect on the systemic

10

oxidative status as determined by plasma oxygen radical absorbance capacity,

11

malondialdehyde, glutathione and glutathione peroxidase activity in blood and organs, except

12

for α-tocopherol, which was higher after the consumption of the chicken-Western diet. In

13

conclusion, in contrast to the hypothesis, the consumption of chicken compared to red &

14

processed meat resulted in higher concentrations of lipid oxidation products in the pig intestinal

15

contents, however, this was not reflected in the body’s oxidative status.

16 17

Keywords: red meat, oxidative stress, pigs, malondialdehyde, 4-hydroxy-2-nonenal

18


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Introduction

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Several large-scale epidemiological studies have associated a high consumption of red and

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especially processed meat with a higher risk of developing various chronic diseases 1. As

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opposed to red and processed meat, no such associations have been reported for the

23

consumption of poultry meat 2. In addition, higher consumption of foods such as whole grains

24

and fruits and vegetables have been linked with lower risks for chronic diseases 3, 4 . However,

25

foods are usually not consumed separately and higher intakes of certain foods are often

26

associated with lower intakes of other foods. Furthermore, the cumulative and interactive

27

effects of multiple components in the diet may attenuate or exert stronger effects than the sum

28

of the single components 5. The 2015 Dietary Guidelines Advisory Committee identified a

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healthy diet as a diet high in fruits and vegetables, whole grains, low- or nonfat dairy, seafood,

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legumes and nuts and low in red and processed meat, sugar-sweetened foods and drinks and

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refined grains 6. The dietary guidelines in Western Europe and Unites States carry this same

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central message, however, despite the recommendations, a Western diet characterized by high

33

amounts of red and processed meat, refined grains, sweets, sugar drinks and fried food is still

34

very popular in these countries.

35

Iron plays a central role in the human body since it exerts various physiological functions such

36

as oxygen transport, DNA synthesis and electron transport. Although dietary iron intake is

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important in the prevention of anemia, the intake of high heme iron levels by high red meat

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consumption is hypothesized to stimulate the formation of oxidation products. An imbalance

39

between reactive oxygen species and antioxidant defense is defined as oxidative stress and is a

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common factor in many pathological conditions 7. Iron is able to initiate and catalyze the Fenton

41

reaction in the presence of hydrogen peroxide, through which reactive oxygen species such as

42

hydroxyl radicals are formed. These reactive oxygen species are highly unstable and can initiate

43

further oxidative chain reactions affecting polyunsaturated fatty acids (PUFA) and proteins, 3 ACS Paragon Plus Environment


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resulting in the formation of oxidized proteins and lipid oxidation products, of which some are

45

potentially harmful 8. Oxidation of n-6 PUFA leads to the specific formation of 4-hydroxy-2-

46

nonenal (4-HNE) and hexanal, whereas malondialdehyde (MDA) originates from both the

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oxidation of n-3 or n-6 PUFA. Previous studies demonstrated the formation of lipid and/or

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protein oxidation products during the digestion of red meat 9, 10. However, not only meat, but

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also other dietary compounds are important sources of pro- and antioxidants, able to affect the

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extent of oxidation during the digestion of meals. Several in vitro and in vivo digestion

51

experiments with red meat already demonstrated antioxidant effects of multiple herbs, spices,

52

fruits, vegetables and beverages

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formation of oxidation products during meat digestion is influenced by the dietary context in

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which meat is consumed. For example, Bastide et al. (2016) already concluded that the

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epidemiological link between red meat consumption and colorectal cancer depended on the

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ratio of heme iron to antioxidants in the diet

57

addressed the need to compare meat consumption included in a healthy versus unhealthy dietary

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pattern 15.

59

Pigs offer a valuable model for human nutritional studies. Pigs and humans share many

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anatomical and physiological similarities related to the gastrointestinal tract resulting in similar

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nutritional requirements and digestive and metabolic processes 16. Furthermore, the ability of

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feeding a strictly controlled diet for several weeks and invasive sampling is a great advantage

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of using a pig model over a human intervention trial. Previously, (mini)pigs have been used to

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investigate the effect of meat-based diets albeit in combination with fruits and vegetables on

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oxidation 17, 18. These studies concluded that the supplementation of fruits and vegetables could

66

mitigate oxidation compared to the diets lacking fruits and vegetables. However, to the best of

67

our knowledge, the relevance of meat consumption as part of a typical Western-style diet or a

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realistic healthy diet and its contribution to oxidative stress has not been investigated yet.

11-13.

Therefore, the rationale of the present study is that the

14.

The review of Turner et al. (2017) also


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Therefore, the aim of the present study was to determine the impact of meat on the oxidative

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status in the context of either a prudent diet, conform current dietary guidelines, or a Western

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diet, representing the average dietary intake of the Belgian population. In this study, it was

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hypothesized that the consumption of red meat compared to chicken meat would lead to more

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oxidation products during digestion, and that this effect would depend on the dietary context of

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meat consumption. To that end, a four-week pig feeding study investigating meat type (red &

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processed meat, both associated with chronic diseases, versus chicken meat as control meat)

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and background diet (prudent versus Western background diet) was performed using a 2×2

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factorial design comprising of the following four diets: chicken-prudent, red&processed-

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prudent, chicken-Western and red&processed-Western. The oxidative status was measured in

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blood and organ tissues, and formation of lipid and protein oxidation products during in vitro

80

and in vivo digestion of the diets was assessed. The in vitro digestion was performed to compare

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the in vitro results with the in vivo study.

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Materials and methods

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Chemicals. All enzymes used in the in vitro digestion of the diets, namely α-amylase from

84

porcine pancreas (~50 U/mg), mucin from porcine stomach type II, pepsin from porcine gastric

85

mucosa (> 250 U/mg solid), lipase from porcine pancreas type II (10-400 U/mg protein),

86

pancreatin from porcine pancreas (8 × USP specifications) and porcine bile extract as well as

87

reagents used in the analytical procedures such as Trolox, 1-fluoro-2,4-dinitrobenzene,

88

NADPH,

89

dinitrophenylhydrazine (DNPH), 4-HNE, hexanal and 1,3-cyclohexanedione were purchased

90

from

91

amidinopropane)dihydrochloride (AAPH) was purchased from Acros Organics (Geel,

92

Belgium).

2-thiobarbituric

Sigma

Aldrich

acid

(Diegem,

(TBA),

1,1,3,3-tetramethoxypropane

Belgium).

The

radical


initiator

(TMP),

2,4-

2,2’azobis(2-


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Diet formulation. Table 1 demonstrates the formulation of the four diets that were formulated

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to provide similar daily intakes of metabolizable energy and an equal meat intake per day. More

95

than 90 food items (such as fruits, vegetables, bread, potatoes, rice and milk) and different

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methods of preparation (raw, cooking, grilling, frying) were used to mimic the diversity and

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nutrient levels of complex human diets. A detailed list of ingredients is provided in

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Supplemental Table S1. The amount of meat was based on the results from the Belgian Food

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Consumption Survey 2004, that reported a daily intake of 291 g of ‘meat, fish, eggs and meat

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alternatives’ at the 97.5 percentile of the Belgian population older than 15 years 19. Combined

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with the other food items, this fraction of meat corresponded to 21.5% of the total energy of the

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diets. The mixture of red & processed meat contained 62% fresh meat (mainly pork and beef)

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and 38% red processed meat (mainly cooked ham, filet de sax, salami and smoked bacon),

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which is a proportion based on the Belgian Food Consumption Survey 2004. Chicken thighs

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and breasts made up the chicken meat. To obtain isocaloric meat fractions in the diets, chicken

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skin was added to chicken meat resulting in a final fat percentage of 9.6% in both meat types.

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The meat was collected from local supermarkets, slaughterhouses and meat processing plants.

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The raw meat from both meat types was prepared in a similar way by either baking (70%) or

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vacuum cooking (30%) and were stored (3-4 weeks) at -20 °C before addition to the background

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diets. The formulation of the Western background diet was based on the average food intake of

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the Belgian population and characterized by high amounts of refined grains, desserts and sweets

112

19.

113

guidelines of the Flemish active food guide pyramid (applied in the period 2005-2017) and was

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characterized by high amounts of fruits, vegetables, whole grains and dairy products

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Nubel databank, containing the nutrient composition of more than 3678 food items, was used

116

to design the diets and to estimate their nutritional value 21. All food items were mixed to avoid

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selection of particular food items by the piglets and to obtain homogenous diets. Because of the

On the other hand, the prudent background diet was formulated based on the dietary


20.

The

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substantial volume (2000 kg in total), the food items of the background diets were collected

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from local supermarkets and were prepared over several weeks and stored at -20 °C. After

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collection and preparation, the food items were allocated to subgroups such as fruits, vegetables,

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fats & sweets, whole grains and refined grains. Within each subgroup, the frozen food items

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were grounded through a 10 mm plate equipped Omega T-12 grinder, carefully mixed using a

123

concrete mixer and immediately stored at -20 °C. Finally, the four diets were made in 20 batches

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of 100 kg per batch by mixing the subgroups according to the tailored proportions in the diets.

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The meat fractions were added last. The resulting homogenous feed mixtures were divided in

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meal portions of 500 g to 1 kg and stored at -20 °C (with a maximum storage time of 3 months)

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until feeding. The temperature of the diets remained below 2 °C during the whole preparation

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process. Aliquots of the background diets, meat and final diets were taken randomly during the

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final preparation step and were stored at -20°C until the end of the feeding trial. Right after the

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trial, the feed aliquots were stored at -80°C until analysis to take eventual oxidation of the diets

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during storage at -20°C into account. All diet analyses were performed in duplicate on two

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randomly selected feed aliquots.

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Dietary characteristics. The pH was recorded and dry matter, crude protein and crude fat

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content were analyzed according to ISO 1442-1973, ISO 937-1978 and ISO 1444-1973

135

methods, respectively. Gross energy was determined with an adiabatic bomb calorimeter

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(PARR 1261). Fatty acids were measured according to Raes et al. (2001) 22. Briefly, lipids were

137

extracted using chloroform/methanol (2/1; v/v) followed by methylation and gas

138

chromatography to separate the fatty acid methyl esters. From the fatty acid composition, the

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peroxidizability index was calculated as an indication for its susceptibility to lipid peroxidation

140

23.

141

extraction in respectively acetone/H2O/HCl 12M (40/4/1) and acetone/H2O (40/5) according to

142

Hornsey (1956) 24.

Total heme and nitroso-heme were determined colorimetrically in the meat following its



143

In vitro digestion. The four diets were subjected to an in vitro digestion protocol as previously

144

described by Van Hecke et al. (2014) to better characterise the diets in terms of susceptibility

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to oxidation during digestion

146

with several enzymatic digestion buffers: 6 mL saliva (5 min), 12 mL gastric juice (2 h),

147

followed by 2 mL bicarbonate buffer (1 M, pH 8.0), 12 mL duodenal juice and 6 mL bile juice

148

(2 h). After the last incubation step, samples were homogenized with an Ultra Turrax (13500

149

rpm) and aliquots of 1 mL were stored at -80 °C. Incubations were performed in quadruplicate

150

at 37 °C and for each diet, the four in vitro digest samples were used as replicates in further

151

analysis.

152

Study design and animals. The experimental protocol was approved by the Ghent University

153

Ethical Committee at the Faculty of Veterinary Medicine (EC 2016/26). Thirty-two castrated

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male piglets (Topigs×Piétrain) of three weeks old and originating from eight litters were

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purchased from a local commercial farm and allocated to eight pens. Each dietary treatment

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was repeated in two pens (n = 8 pigs per diet) and each diet was given to one of the piglets of

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each litter. On arrival, all piglets were fed a commercial pre-starter diet ad libitum for one week.

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Thereafter, the dietary treatments were gradually introduced during one week, after which the

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piglets were subjected to the dietary treatments for four weeks. The piglets were weighed twice

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a week and the average body weight at the start of the experimental feeding period did not differ

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significantly between the treatment groups (7.00 ± 0.88 kg). During the experimental feeding

162

period, piglets were fed fresh meals at room temperature three times a day (8, 12, 18 h) during

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30 min ad libitum. An individual feeding system was used to record their individual feed intake

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per meal. Water was always available ad libitum. Piglets were sampled at the end of the four-

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week feeding period, though due to practical reasons, euthanasia and sampling was spread over

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four consecutive days. Each day, two piglets from each dietary treatment were slaughtered,

167

starting with the heaviest piglets. Two hours after receiving a last meal, piglets were euthanized

25.

In short, diet samples (4.5 g) were incubated consecutively


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by intramuscular injection of xylazine (20 mg/mL, 0.1 mL/kg bodyweight) and intraperitoneal

169

injection of pentobarbital (60 mg/mL, 0.8 mL/kg bodyweight) followed by exsanguination.

170

Postprandial blood samples were taken from the jugular vein just before exsanguination and

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collected in heparin tubes. Plasma and red blood cells were separated by centrifugation (1000

172

G, 15 min) and stored at -80 °C. Subsequently, all small and large intestinal contents were

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collected separately, the pH was measured and the contents were gently mixed and stored at -

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80 °C. Organs (heart, liver (lobus hepatis dexter medialis), kidney) and tissue segments of

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ileum, proximal and distal colon were removed, weighted and gently rinsed with 0.9% NaCl

176

solution. A 10 cm2 segment of the ileum, proximal and distal colon tissue was scraped to collect

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mucosa. The organs and intestinal mucosae were chopped and homogenized before storage at

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-80 °C. All analyses on these biological samples were performed in duplicate. Before the

179

analysis of thiobarbituric acid reactive substances (TBARS) and glutathione peroxidase (GSH-

180

Px) activity in organs and mucosae, buffered aqueous extracts were made by adding a 1% Triton

181

X-100 phosphate buffer (pH 7; 50 mM) in a 1/10 (w/v) proportion to the organs and mucosae,

182

followed by homogenization (Ultra Turrax, 13500 rpm), centrifugation (13800 G, 15 min, 4

183

°C) and subsequent filtration over glass wool.

184

Antioxidant capacity. The hydrophilic oxygen radical absorbance capacity (ORAC) of the

185

diets and plasma was measured fluorimetrically (excitation at 485 nm and emission at 520 nm)

186

and results were reported as µmol Trolox Equivalents (TE) per g diet and per mL plasma 26.

187

Alpha-tocopherol in the diets, heart, liver, kidney and plasma was extracted with ethanol and

188

hexane and quantified by reversed-phase HPLC and UV detection (292 nm)

189

Concentrations of α-tocopherol (µg α-tocopherol/g sample) were determined by comparing the

190

peak areas with a standard curve of α-tocopherol (0 – 15 µg/mL). Levels of reduced glutathione

191

(GSH) and oxidized glutathione (GSSG) were determined in lysed erythrocytes and liver tissue

192

using HPLC separation and UV detection (365 nm) 29, 30. Results were expressed as area under


27, 28.


193

the curve/g or mL sample. The GSH-Px activity was measured in plasma and buffered aqueous

194

extracts from organs and gastrointestinal tissues 31. The oxidation of NADPH was measured

195

colorimetrically (340 nm) and recorded over time. One unit of GSH-Px activity was defined as

196

the amount of sample (g) required to oxidize of NADPH per min at 25 °C.

197

Oxidation products. Total MDA concentrations in plasma and in buffered aqueous extracts of

198

heart, liver, kidney and intestinal mucosae were analysed as TBARS according to the method

199

of Grotto et al. (2007) with slight modifications

200

reaction steps involving alkaline hydrolysis, acid deproteinization, derivatization with TBA and

201

extraction with 1-butanol. The absorbance of the carbonyl-TBA complexes was measured

202

colorimetrically at 532 nm, quantified using a standard curve with TMP (0 – 30 nmol/ml) and

203

expressed as nmol MDA-equivalents/g sample. Because of the complexity of the diets, a more

204

specific HPLC method was applied to determine free reactive MDA in the diets, in vitro digests

205

and intestinal contents according to Mendes et al. (2009) 33. The samples were extracted with

206

trichloroacetic acid (7.5%) and derivatized with DNPH. In case of the in vitro digests, the

207

resulting MDA-DNPH hydrazine was subsequently separated by HPLC analysis, whereas for

208

the diets and the intestinal contents, the MDA-DNPH was first concentrated through extraction

209

with pentane, followed by evaporation to dryness with N2 and resolved in 1 mL acetonitrile/H2O

210

(1/1, v/v) prior to HPLC analysis and UV/VIS detection (310 nm). Standard solutions (0 – 20

211

nmol/ml) were made with TMP and results were expressed as nmol MDA/g sample.

212

Levels of 4-HNE and hexanal were analyzed in the diets, in vitro digests and intestinal contents

213

after formation of their fluorescent derivatives with 1,3-cyclohexanedione through HPLC as

214

described previously

215

those obtained from the standard curves of 4-HNE (0 – 2 nmol/mL) and hexanal (0 – 40

216

nmol/mL) and results were provided in nmol/g sample.

12.

32.

TBARS were determined after several

Concentrations were determined by comparing the peak areas with


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217

Protein carbonyl compounds (PCC) concentrations, indicating the extent of protein oxidation,

218

were measured colorimetrically in diets, in vitro digests and small intestinal contents following

219

their covalent reaction with DNPH according to Ganhão et al. (2010) 34. PCC was not measured

220

in the colonic contents because of the high digestion rate of dietary protein and the interference

221

with bacterial protein. The carbonyl concentration (nmol DNPH/mg protein) was calculated

222

from the absorbance at 280 nm and 370 nm of the samples using the equation below 35: 𝐶ℎ𝑦𝑑𝑟𝑎𝑧𝑜𝑛𝑒

223

𝐶𝑝𝑟𝑜𝑡𝑒𝑖𝑛

=

𝐴370 𝜀ℎ𝑦𝑑𝑟𝑎𝑧𝑜𝑛𝑒, 370 × (𝐴280 ― 𝐴370 × 0.43)

× 106

224

Where εhydrazone,370 is 22000 M-1 cm-1 and the carbonyl concentrations obtained from the blanks

225

were subtracted from the contribution obtained from the corresponding treated sample.

226

Statistical analyses. Data are presented as means ± SD, unless otherwise indicated. Data

227

normality and homoscedasticity were verified using Kolmogorov-Smirnov and modified-

228

Levene’s test in SAS enterprise guide 7. If assumptions were met, a mixed model ANOVA

229

procedure was used with meat, background diet and meat×background diet as fixed effects and

230

litter, pen and euthanization day as random effects (if applicable). Tukey-adjusted post hoc tests

231

were performed for all pairwise comparisons. If one of the assumptions for the use of ANOVA

232

was violated, the non-parametric independent-samples Kruskal-Wallis test with pairwise

233

multiple comparisons was performed using SPSS Statistics 25. P values < 0.05 were considered

234

statistically significant.

235

Results

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Diet characterization. The analyzed nutritional composition of the diets presented in Table 2

237

was similar to the formulated nutritional values estimated according to the Nubel nutrient

238

database

239

protein content, higher fat content, more SFA, less MUFA and a higher n-6/n-3 fatty acid ratio

240

compared to the prudent diets. A similar crude fat content of 9.6 g/100 g in both meat types was

241

obtained by adding 9% of chicken skin (45% crude fat) to chicken meat (6% crude fat).

21

(Supplemental Table S2). The Western diets had a higher energy density, similar



242

Compared to the red & processed meat, the cooked chicken meat contained lower proportions

243

of SFA and higher proportions of n-6 PUFA, a higher n-6/n-3 PUFA ratio and higher

244

peroxidizability index. Total heme was 3.5-fold higher in red & processed meat than in chicken

245

meat. Approximately 10% of total heme in red & processed meat was nitrosylated, whereas

246

chicken meat contained very low amounts of nitroso-heme. No clear differences in ORAC

247

values were observed between the four diets. The α-tocopherol concentration of the Western

248

background diet was 2-fold higher compared to the prudent background diet, whereas the meat

249

types had similar α-tocopherol contents. A 30% higher α-tocopherol level was found in the

250

chicken-Western diet compared to the other diets, indicating a loss of α-tocopherol through the

251

combination of the red & processed meat and the Western background diet. Cooked chicken

252

meat contained approximately 4-fold more 4-HNE, 11-fold more hexanal and 4-fold more

253

MDA compared to red & processed meat. The concentrations of hexanal and MDA in the

254

background diets were considerably lower compared to those in the meat, except for 4-HNE

255

where the concentrations in the red & processed meat were comparable to those in the

256

background diets. The concentrations of lipid oxidation products in the diets corresponded to

257

the combined fractions of lipid oxidation products in the meat and background diets, resulting

258

in higher levels of lipid oxidation products in the diets containing chicken meat. The amount of

259

PCC was comparable between the chicken-prudent, red&processed-prudent and chicken-

260

Western diet, but slightly higher in the red&processed-Western diet.

261

In vitro digestion. The differences in lipid oxidation products in the diets were reflected in the

262

in vitro digests, as shown in Table 2. The concentrations of PCC in the in vitro digests were

263

slightly higher after the digestion of the red & processed meat diets compared to the chicken

264

diets.

265

Animal performance. The average daily energy intake of the pigs did not differ significantly

266

between the four treatment groups, as illustrated in Figure 1. Since the meat fraction represented


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an equal energy percentage in the four diets, the piglets consumed an equal amount of meat per

268

day, as intended. However, because of the lower energy density of the prudent background diet,

269

the average daily feed intake of the prudent diets (1268 ± 204 g/day) was higher (P = 0.001)

270

compared to the Western diets (905 ± 81 g/day). In the second week of the experimental feeding

271

period, there was a drop in feed intake and concomitant stagnation of body weight gain,

272

regardless of dietary treatment. During this drop in feed intake, pigs presented loose stools but

273

most of them recovered within 3-5 days. Nonetheless, two pigs fed the chicken-Western diet

274

still suffered from diarrhea at the day of euthanasia, therefore, their colonic contents could not

275

be collected. Pigs receiving the prudent diets (312 ± 91 g/day) had a higher body weight gain

276

compared to those fed the Western diets (239 ± 55 g/day, P = 0.026), whereas meat consumption

277

did not significantly affect body weight. Additionally, the liver tissue weight per kg body weight

278

was 10% higher for pigs fed the prudent diets (P = 0.010), whereas the weight of heart and

279

kidney was not significantly affected. The pH of the intestinal digest samples did not differ

280

significantly among the dietary treatments (data not shown).

281

Oxidation products in intestinal contents. The concentrations of lipid oxidation products and

282

protein carbonyl compounds in small intestine and colonic contents are demonstrated in Figure

283

2. Lipid oxidation products were 1.4- to 3-fold higher in the intestinal contents of the pigs fed

284

chicken meat compared to pigs fed red & processed meat diets (P < 0.001). The concentration

285

of hexanal was higher in the small intestinal content when chicken meat was consumed in

286

combination with the prudent vs. Western background diet (P = 0.038). Significantly higher

287

PCC concentrations were found in the small intestinal content of piglets consuming red &

288

processed meat in the Western vs. the prudent background diet (P = 0.010).

289

α-Tocopherol. As illustrated in Figure 3, highest α-tocopherol concentrations were found in

290

pigs fed the chicken-Western diet.



291

Oxidative status. The mean values and statistics of the analyzed parameters for the oxidative

292

status are summarized in Table 3. No significant interaction effect of meat type and background

293

diet was observed in the analyzed parameters. The ORAC values in plasma of pigs fed chicken

294

meat tended to be slightly higher compared to those fed red & processed meat, irrespective of

295

the background diet. The MDA concentrations were only significantly different in kidney, with

296

slightly higher concentrations in kidneys following chicken meat consumption compared to red

297

& processed meat (P = 0.014). Pigs fed the Western diets had higher GSH-Px activity in heart

298

(+ 9%, P = 0.029) and showed a trend to be higher in kidney (+ 10%, P = 0.055) compared to

299

the prudent diets. On the other hand, pigs on the red & processed meat diets tended to have

300

higher GSH-Px activity in the liver (+ 15%, P = 0.070) compared to those fed the chicken diets.

301

No significant differences were observed for GSH-Px activity in plasma or in the intestinal

302

mucosae at three different locations. However, GSSG levels tended to be higher (+ 24%, P =

303

0.057) in erythrocytes of pigs fed red & processed meat diets compared to the chicken diets,

304

whereas the type of meat had no effect on GSH levels. In liver on the other hand, GSH levels

305

tended to be influenced by the background diet, with slightly higher levels in pigs fed the

306

prudent diets compared to the Western diets (P = 0.081), whereas no significant differences

307

were observed for liver GSSG levels between the treatment groups.

308

Discussion

309

Although oxidative reactions are the basis of numerous biochemical pathways, oxidative stress

310

has been linked to several pathological conditions and chronic diseases. Therefore, the role of

311

dietary oxidation products as external factors of oxidative stress and the related health risks has

312

become a subject of great interest. The aim of the present study was to investigate the effect of

313

meat sources that have been linked with chronic diseases (red & processed meat) versus chicken

314

meat as control in a complex dietary context (Western or prudent) on the oxidative status in

315

pigs following a four-week experimental feeding period. Most studies investigating the impact


Page 14 of 33

Page 15 of 33


316

of meat on oxidation during digestion used (semi-)purified diets and levels of meat or meat-

317

derived compounds above common intake levels 15. To the best of our knowledge, the present

318

study is unique in its kind as it used a great diversity of whole foods and a lower meat fraction

319

corresponding to the upper intake level of a Western diet.

320

We hypothesized that the consumption of red meat would lead to more oxidation products

321

during digestion compared to chicken meat because of the catalyzing effect of iron in the Fenton

322

reaction. In contrast, we found higher levels of lipid oxidation products in the chicken-based

323

diets and subsequent digests. The results of the in vitro and in vivo digests correlated well,

324

however, the oxidation products in the digests reflected the differences in the diets. In

325

agreement with the hypothesis, the results of previously reported in vitro and in vivo

326

experiments showed lower MDA levels throughout the digestion of chicken compared to beef

327

and/or pork

328

susceptibility of red meat to oxidation. However, Steppeler et al. (2016) indicated that the

329

amount of unsaturated fatty acids and not the iron content was the most important factor in the

330

formation of aldehydes during in vitro digestion of cooked meat and salmon loin, that both

331

comprised of similar fat contents but different fatty acid profiles

332

content and fatty acid profile was also previously demonstrated upon digestion of meat or heme

333

iron supplemented with different types of oils or by adding different proportions of lard to meat

334

10, 37, 38.

335

added chicken skin, which led to a considerable proportion of n-6 PUFA. In contrast, in their

336

in vitro digestions, Van Hecke et al. (2014) normalized the fatty acid composition of chicken,

337

pork and beef meat by adding lard, which might offer a possible explanation for the different

338

results obtained 9. In the present study, the undigested cooked chicken meat was already more

339

oxidized compared to the red & processed meat. Besides differences in heme iron and fatty acid

340

profile, a range of other factors such as differences in the endogenous antioxidant capacity and

9, 10, 13.

In these studies, heme iron was mostly held responsible for the higher

36.

The importance of fat

The chicken meat in the present study consisted of a mixture of breasts, thighs and



Page 16 of 33

39,

341

in the ability of heme proteins from different animal species to promote lipid oxidation

342

processing methods and storage conditions could have played a role. The study of Rhee et al.

343

(1994) demonstrated that among raw muscles from different animal species, beef was most

344

susceptible to lipid oxidation during storage at -20°C, whereas cooking drastically changed the

345

susceptibility of meat during storage resulting in significantly higher levels of TBARS in

346

chicken thigh compared to beef, pork and chicken breast 40. Although storage time in the present

347

study was equal for all meat fractions, a different oxidative susceptibility during storage could

348

be involved. In addition, also the added antioxidants such as nitrite salt in processed meat could

349

have reduced the amount of lipid oxidation products in the red & processed meat 9.

350

The majority of lipid oxidation products in the diets originated from the meat and hence, the

351

background diet did not affect lipid oxidation product concentrations in the diets and digestion

352

as distinctly as expected. The prudent diet contained considerable amounts of compounds that

353

could lower the oxidative load in the diets or during digestion of meat 17, 41. However, neither

354

α-tocopherol concentrations nor ORAC values were higher in the prudent diets compared to the

355

Western diets. Nevertheless, care should be taken in the interpretation of ORAC values since

356

this method has been criticized regarding its biological relevance, its selective activity against

357

particular radicals and the contribution of the protein fraction to the antioxidant capacity. In

358

addition, feeding the piglets a mixture of many ingredients always including a certain fraction

359

of protective compounds could have attenuated the adverse effects of a Western dietary pattern.

360

Indeed, in order to prevent postprandial oxidative stress, Prior (2007) concluded that

361

antioxidant containing foods should be consumed in each meal, which was the case in this study

362

42.

363

aspects of a dietary pattern, e.g. portion size, temporal distribution of intake, variety etc.

364

PCC were higher in the small intestinal contents of pigs fed the red&processed-Western diet

365

compared to the red&processed-prudent diet. The susceptibility to protein oxidation is

In this regard, next to the dietary composition, future studies should also consider additional


Page 17 of 33


366

primarily affected by the protein content and amino acid composition, but importantly, also

367

lipid oxidation processes are involved 43. Protein and lipid oxidation are interrelated since both

368

lipids and proteins could be targets of ROS and because secondary lipid oxidation products can

369

interact with the amino acid residues of proteins. Also, PCC can be formed during the Maillard-

370

mediated pathway, in which reducing sugars react with the δ-amino group of alkaline amino

371

acids 43. Possibly, this reaction pathway could have contributed to the higher PCC measured in

372

the digests of the red&processed-Western diet. The evaluation of more specific protein

373

oxidation products and further studies on this Maillard-mediated pathway will aid to elucidate

374

this hypothesis. Additionally, the nature and action of antioxidants can also contribute to the

375

outcome of PCC and the inconsistency with the results for lipid oxidation products in this study.

376

Similar to our results, the studies of Delosière et al. (2016) and Raes et al. (2015) investigating

377

the antioxidant potential of fruits and vegetables and a number of well-known antioxidants

378

(carnosic acid, quercetin and α-tocopherol) during digestion, also found varying results

379

comparing antioxidant effects on lipid and protein oxidation 41, 44.

380

The α-tocopherol concentrations (the major vitamin E component) in plasma and organs

381

reflected the dietary α-tocopherol levels, being 30% higher in the chicken-Western diet

382

compared to the other diets. The two-fold higher α-tocopherol concentrations in the Western

383

background diet compared to the prudent background diet was probably due to the high (vitamin

384

E rich) fat content of the Western diet. Indeed, in two large food consumption surveys in

385

Belgium and Ireland, the food group ‘fats and oils’ was identified as the main source of dietary

386

vitamin E intake, followed by vegetable intake 45, 46. Although both meat types had similar α-

387

tocopherol concentrations in this study, the combination of red & processed meat with the

388

Western background diet resulted in lower α-tocopherol levels in the red&processed-Western

389

diet compared to the chicken-Western diet. This loss could possibly be explained by the

390

catalyzing effect of heme iron on oxidation but was unexpected since more lipid oxidation



Page 18 of 33

391

products were present in the chicken meat. However, no clear antioxidant effect of the higher

392

α-tocopherol concentration in the chicken-Western diet could be observed in the oxidative

393

stress parameters measured in organs, in contrast to what was expected as α-tocopherol is

394

known to influence cellular responses to oxidative stress. On the other hand, the α-tocopherol

395

concentrations in the present diets corresponded to a daily intake of 11.6 – 16.6 mg/day for

396

humans (calculated with a theoretical daily energy intake of 2500 kcal), which is close to the

397

European Food Safety Authority adequate intake levels (13 mg/day for men 47) and the usual

398

vitamin E intake in the Belgian population (14 mg/day in men

399

concentrations used in experimental studies that clearly demonstrate a protective effect of α-

400

tocopherol on oxidative stress markers, involve much higher doses of vitamin E

401

supplementation such as 67 mg/day in diabetic patients and 200 mg/kg body weight per day in

402

rats 48, 49.

403

Recent reviews demonstrate involvement of dietary PCC and lipid oxidation products in

404

oxidation markers in blood, urine or organs

405

containing chicken meat had considerably more lipid oxidation products in the present study,

406

an increase in oxidative stress was expected in pigs on chicken-based diets. However, only renal

407

MDA was higher in pigs fed chicken meat compared to those fed red & processed meat. The

408

increased GSH-Px activity in heart tissue could indicate a higher need for antioxidant action

409

due to the Western background diet. In accordance, a lower erythrocyte GSH-Px activity

410

together with a higher blood total antioxidant capacity was previously indicative for a beneficial

411

oxidative status in pigs fed strawberries compared to other fruits

412

(2018) demonstrated a higher total antioxidant status and higher GSH-Px activity in duodenum

413

and colon mucosae of pigs after the addition of polyphenols to the control diet

414

apparent inconsistencies emphasize the existence of different antioxidant mechanisms in

415

different organs and emphasize that a proper interpretation of the oxidative status requires the

43, 50.

45).

The α-tocopherol

Since the diets and intestinal contents


51.

Contrary, Chedea et al.

52.

These

Page 19 of 33


416

analysis of multiple parameters. The rather inconsistent effects of the different diets on the

417

measured parameters for the oxidative status (ORAC, α-tocopherol, MDA, GSH-Px, levels of

418

GSH and GSSG), hampers a straightforward conclusion about the body’s antioxidant status.

419

Previously, dietary factors such as resistant starch and polyphenols, characteristic for a prudent

420

diet, or sucrose, highly present in Western diets, have been demonstrated to attenuate or

421

stimulate, respectively, MDA formation in colon mucosae or kidney tissue

422

the present study, a mitigating or stimulating effect on lipid oxidation could not be attributed to

423

those dietary compounds. Despite the close contact with the digests containing different lipid

424

oxidation concentrations, no effect was observed at the level of the intestinal mucosae in terms

425

of MDA concentrations or GSH-Px activity. In contrast, feeding pigs oxidized soybean oil

426

(resulting in 270 – 414 nmol MDA/g oxidized diet versus 6.6 nmol MDA/g unoxidized diet)

427

increased the concentrations of MDA in the mucosal samples from the jejunum 55, while more

428

oxidized diets (approximately 75 nmol MDA/g diet versus 45 nmol MDA/g diet) also yielded

429

higher MDA concentrations in colonic mucosae in rats 10. However, the concentrations of lipid

430

oxidation products in the present study (with MDA levels below 5 nmol MDA/g diet) were

431

relatively low compared to the MDA levels in the studies mentioned above. Furthermore, the

432

concentrations of 4-HNE in this study (up to 0.63 nmol/g diet and up to 1.53 ± 0.48 nmol/g

433

intestinal content) were also at the lower end compared to the cytotoxic concentration ranges

434

of 4-HNE as documented by Esterbauer et al. (1991) 8. In addition, besides the concentration

435

of oxidation products in the diets and intestinal lumen, other factors such as bioavailability, the

436

antioxidant defense system and barrier functionality of the intestinal mucosa, transit time and

437

changes in microbiota could be involved in the oxidative status of the intestinal mucosae.

438

In conclusion, the hypothesis that red & processed meat consumption stimulates oxidation

439

during digestion to a greater extent compared to chicken meat could not be confirmed. In

440

contrast, chicken meat was more susceptible to lipid oxidation as observed already in the diets 19 ACS Paragon Plus Environment

52-54.

However, in


441

as such, and in the in vitro and in vivo digests. Not only heme iron, but other factors such as the

442

fatty acid composition, the antioxidants in processed meat or other differences in the

443

endogenous antioxidant capacity of the meat will have influenced the oxidative susceptibility

444

during preparation, storage and subsequent digestion. Marginal differences between the two

445

background diets were observed regarding oxidation product formation during digestion. In

446

contrast to the hypothesis, little interaction effects were observed between meat type and

447

background diet. The systemic oxidative status was not or only minimally affected by the

448

background diet and meat type. More in vivo studies on the interaction of separate foods and

449

dietary patterns on oxidation phenomena are warranted.

450

ABBREVIATIONS USED

451

4-hydroxy-2-nonenal (4-HNE); 2,2’azobis(2-amidinopropane)dihydrochloride (AAPH); 2,4-

452

dinitrophenylhydrazine (DNPH); reduced glutathione (GSH); glutathione peroxidase (GSH-

453

Px); oxidized glutathione (GSSG); malondialdehyde (MDA); oxygen radical absorbance

454

capacity (ORAC); polyunsaturated fatty acids (PUFA); 2-thiobarbituric acid (TBA);

455

thiobarbituric acid reactive substances (TBARS); Trolox Equivalents (TE); 1,1,3,3-

456

tetramethoxypropane (TMP)

457

ACKNOWLEDGEMENTS

458

The authors thank C. Lachat for his input in the formulation of the diets and S. Coolsaet, D.

459

Baeyens, E. Claeys and A. Ovyn for their practical assistance during the preparation of the

460

diets, sampling of the piglets and laboratory analyses.

461

FUNDING

462

This research was funded by the Flanders Research Foundation (FWO) project G011615N.

463

SUPPORTING INFORMATION DESCRIPTION


Page 20 of 33

Page 21 of 33


464

Detailed list of food items used to compose the four diets (Supplementary Table S1) and

465

Formulated and analyzed composition of the four diets (Supplementary Table S2). This material

466

is available free of charge via the Internet at http://pubs.acs.org.



467 468 469

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Table 1 Formulation of the four diets expressed in g/kg (fresh weight basis) and energy percentage (E%) prudent chicken R&P

Western chicken R&P

prudent chicken R&P

197

21.5

g/kg red (62%) & red processed meat (38%) chicken meat cereal products and potatoes fruits and vegetables dairy products and eggs butter and cooking fats fats and sweets R&P = red & processed meat

145 304 299 243 8.62 0.51

145

E% 197 199 173 143 14.3 275

304 299 243 8.62 0.51

Western chicken R&P

21.5 45.6 10.4 15.1 7.06 0.33

199 173 143 14.3 275

45.6 10.4 15.1 7.06 0.33

21.5 23.4 4.50 12.7 8.54 29.4

Table 2 Characteristics of the four diets, meat and background diets separately diets prudent chicken R&P

meat Western chicken R&P

chicken

background diet R&P

prudent

Western

gross energy, kcal/kg 1263 1265 1743 1726 pH 5.83 5.39 5.69 5.28 dry matter, g/100 g 26.0 26.1 30.9 31.2 37.4 36.2 23.5 30.6 crude protein, g/100 g 6.44 6.35 7.61 6.97 26.2 21.7 3.55 2.96 crude fat, g/100 g 3.13 3.47 6.24 6.76 9.61 9.59 2.28 5.87 proportion of energy from fat, % 22.5 24.9 32.5 35.6 SFA, g/100 g FA 31.6 35.6 37.0 38.2 29.7 38.3 32.5 40.0 MUFA, g/100 g FA 44.0 42.2 38.6 38.8 42.6 41.8 45.9 37.8 PUFA, g/100 g FA 21.2 19.5 21.6 20.2 26.1 17.7 20.1 21.0 linoleic acid, g/100 g FA 17.4 15.4 19.0 17.3 22.4 13.9 15.1 18.0 total n-6 PUFA, g/100 g FA 18.2 16.2 19.6 17.9 23.8 15.3 15.8 18.5 α-linolenic acid, g/100 g FA 2.74 2.89 1.87 1.98 1.93 2.03 3.77 2.20 total n-3 PUFA, g/100 g FA 3.00 3.29 2.05 2.28 2.25 2.44 4.22 2.52 ratio n-6/n-3 FA 6.08 4.94 9.57 7.93 10.6 6.26 3.8 7.33 peroxidizability indexb 25.9 24.6 24.8 23.8 31.3 22.6 26.1 24.8 total heme, mg hematin/kg meat 42.8 149 nitroso-heme, mg hematin/kg meat 17.8 40.4 ORAC (µmol Trolox equivalent/g) 10.2 8.96 9.50 9.29 11.58 12.89 8.77 8.38 α-tocopherol (mg/kg) 7.92 8.03 11.6 8.02 6.38 6.50 5.59 11.3 oxidation products in diet before digestion 4-hydroxy-2-nonenal (nmol/g) 0.63 0.32 0.55 0.28 1.34 0.32 0.41 0.30 hexanal (nmol/g) 11.1 1.5 12.3 1.3 46.4 4.36 1.24 0.99 malondialdehyde (nmol/g) 2.56 1.22 4.04 2.74 19.1 4.46 0.52 0.42 PCC (nmol DNPH fixed/mg protein) 5.00 5.38 5.19 6.45 oxidation products after in vitro digestion 4-hydroxy-2-nonenal (nmol/mL) 0.65 0.32 0.60 0.34 hexanal (nmol/mL) 1.69 1.19 1.63 1.28 malondialdehyde (nmol/mL) 1.11 0.51 1.24 0.75 PCC (nmol DNPH fixed/mg protein) 7.65 8.77 7.51 8.99 - = not analyzed; R&P = red & processed meat; FA = fatty acids; SFA = saturated fatty acids; MUFA = monounsaturated fatty acids; PUFA = polyunsaturated fatty acids; Total n-6 PUFA = C18:2,n-6 + C18:3,n-6 + C20:2,n-6 + C20:3,n-6 + C20:4,n-6 + C22:4,n-6 + C22:5,n-6; Total n-3 PUFA = C18:3,n-3 + C20:3,n-3 + C20:4,n-3 + C20:5,n-3 + C22:5,n-3 + C22:6,n-3; PCC = protein carbonyl compounds; ORAC = Oxygen Radical Absorbance Capacity; DNPH = 2,4-dinitrophenylhydrazine. b Peroxidizability index (Hu et al., 1989) was calculated as follows: (% dienoic x 1) + (% trienoic x 2) + (% tetraenoic x 3) + (% pentaenoic x 4) + (% hexaenoic x 5).


21.5 23.4 4.50 12.7 8.54 29.4


Page 30 of 33

Table 3 Parameters for the oxidative status in blood and organ tissues of pigs following four weeks on the dietary treatments (n = 8 per diet) prudent chicken R&P ORAC plasma* TBARS plasma* liver heart kidney* mucosa of ileum* mucosa of proximal colon* mucosa of distal colon* GSH-Px activity plasma* liver* heart* kidney* mucosa of ileum mucosa of proximal colon mucosa of distal colon reduced glutathione red blood cells liver oxidized glutathione

Western chicken R&P

SEM

Pback

P-values Pmeat

µmol TE/mL

12.8

11.5

12.4

11.1

0.45

0.910

0.060

nmol ME/mL nmol ME/g nmol ME/g nmol ME/g nmol ME/g

9.11 184 68.2 59.5 63.2

9.31 221 71.1 57.9 56.8

8.70 174 68.8 63.5 44.4

9.35 175 68.9 58.3 46.7

0.42 7.67 1.66 0.82 3.45

0.692 0.115 0.797 0.274 0.110

0.485 0.270 0.646 0.014 1.000

nmol ME/g nmol ME/g

30.7 39.9

31.8 40.7

29.3 38.2

24.9 35.1

1.27 0.96

0.155

0.429

0.123

0.406

µmol/min.mL µmol/min.g µmol/min.g µmol/min.g µmol/min.g µmol/min.g µmol/min.g

2.25 3.89 0.64 4.01 2.02 3.13 2.40

2.27 4.62 0.67 3.76 2.12 3.68 2.62

2.40 4.06 0.74 4.48 2.20 4.24 2.41

2.28 4.72 0.70 4.15 2.28 3.82 2.29

0.06 0.19 0.02 0.12 0.11 0.24 0.11

0.970 0.821 0.029 0.055 0.452 0.176 0.671

0.792 0.070 0.763 0.187 0.678 0.892 0.791

AUC×105/mL

2.66

2.82

2.75

2.58

0.10

0.768

0.979

AUC×105/g

4.39

5.40

3.24

4.17

0.31

0.081

0.145

0.699 0.057 3.51 4.57 3.28 4.39 0.29 0.291 0.792 liver* AUC×104/g 2.11 2.98 1.92 1.69 0.27 R&P = red & processed meat, ORAC = Oxygen Radical Absorbance Capacity, TBARS = Thiobarbituric acid reactive substances, ME = malondialdehyde equivalents, TE = Trolox equivalent, GSH-Px = glutathione peroxidase, AUC = area under the curve, SEM = standard error of the mean, Pback = P-value of the fixed factor background diet, Pmeat = P-value of the fixed factor meat. Data of parameters with an asterisk (*) were analyzed with non-parametric statistical tests. P-values ≤ 0.05 are highlighted in bold, P-values between 0.05 and 0.10 are highlighted in italic. P-values for the interaction term meat×background diet were not included since they were not significant. red blood cells

AUC×104/mL


Page 31 of 33


Figure 1 Feed intake in g/day, kcal/day and body weight during the experimental feeding period (n = 8 per diet) Figure 2 Lipid oxidation products (4-hydroxy-2-nonenal, hexanal and malondialdehyde) and protein carbonyl compounds in small intestinal (N = 32) and colonic (N = 30) contents. Bars with different superscripts differ significantly (p < 0.05) among dietary treatments within the same compartment of the intestinal tract. The error bars illustrate standard deviations. Samples with an asterisk (*) were analyzed using non-parametric statistical tests. DNPH= 2,4-dinitrophenylhydrazine. Figure 3 Alpha-tocopherol concentrations in plasma (N = 32) and organs (liver, heart and kidney, N = 32) of pigs following four weeks on the dietary treatments. Bars with different superscripts differ significantly (p < 0.05) among dietary treatments within the same type of sample. The error bars illustrate standard deviations. Samples with an asterisk (*) were analyzed using non-parametric statistical tests.

Feed intake (g/day)

2000 1600 1200 800 400 0

Feed intake (kcal/day)

2500 2000 1500 1000 500 0

Body weight (kg)

20 15 10 5 0 0

10

20

30

Time (days) chicken-prudent chicken-Western

red&processed-prudent red&processed-Western

Figure 1



hexanal

4-hydroxy-2-nonenal

a

3

a ab

2

b

b

1

ab

nmol/g

nmol/g

3

a ab

b

0

a

2

bc

c

b c

c

1

Colonic content*

protein carbonyl compounds a

ab a

(nmol DNPH fixed/mg protein)

12 a

6

c

bc

b

b

3 0

Colonic content*

Small intestinal content

Colonic content

Small intestinal content

malondialdehyde

nmol/g

ab

0 Small intestinal content*

9

Page 32 of 33

a

12 ab

8

b

ab

4 0 Small intestinal content*

Figure 2

α-tocopherol a

14 12

µg/g sample

a 10 b

8 6 4 2

b

b a

ab

b b

b

a ab

b b

ab

b

0 Plasma* chicken-prudent

Liver

Heart

red&processed-prudent

chicken-Western

Figure 3


Kidney* red&processed-Western

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GRAFIC FOR TABLE OF CONTENTS


Impact of red versus white meat consumption in a prudent or Western

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