Effects of Polar Compounds Generated from the Deep-Frying Process

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Effects of polar compounds generated from deep-frying process of palm oil on lipid metabolism and glucose tolerance in Kunming mice Xiaodan Li, Xiaoyan Yu, Dewei Sun, Jinwei Li, Yong Wang, Peirang Cao, and Yuanfa Liu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b04565 • Publication Date (Web): 14 Dec 2016 Downloaded from http://pubs.acs.org on December 16, 2016

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Journal of Agricultural and Food Chemistry

Effects of polar compounds generated from deep-frying process of palm oil on lipid metabolism and glucose tolerance in Kunming mice †,†

Xiaodan Li,

Xiaoyan Yu,

†,†

†

and Yuanfa Liu* †

†

§

Dewei Sun, Jinwei Li, Yong Wang, Peirang Cao,*

,†

,†

State Key Laboratory of Food Science and Technology, Synergetic Innovation Center of

Food Safety and Nutrition, School of Food Science and Technology, Jiangnan University, Wuxi 214122, China §

Department of Food Science and Engineering, Jinan University, Guangzhou, 510632, China

*Corresponding Author Yuanfa Liu Phone/fax: +86-0510-85916662. E-mail: [email protected]. Peirang Cao Phone/fax: +86-0510-85329081. E-mail: [email protected].

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ACS Paragon Plus Environment


1

Abstract

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In the present study, effects of deep-fried palm oil, specifically polar compounds

3

generated during the frying process, on animal health including lipid and glucose

4

metabolism and liver functions were investigated. Kunming mice were fed with

5

high-fat diet containing deep-fried palm oil or purified polar compounds for period of

6

12 weeks. Their effects on animal health including hepatic lipid profile, antioxidant

7

enzyme activity, serum biochemistry and glucose tolerance were analyzed. Our results

8

revealed that the consumption of polar compounds was related to the change of lipid

9

deposition in liver and adipose tissue, as well as, glucose tolerance alteration in

10

Kunming mice. Correspondingly, the transcription study of genes involved in lipid

11

metabolism including PPARα, Acox1, and Cpt1α indicated that polar compounds

12

probably facilitated the fatty acid oxidation on peroxisomes while lipid oxidation in

13

mitochondria was suppressed. Furthermore, glucose tolerance test (GTT) revealed that

14

high amount of polar compounds intake impaired glucose tolerance, indicating its

15

effect on glucose metabolism in vivo. Our results provided critical information on the

16

effects of polar compounds generated from deep-frying process of palm oil on animal

17

health, particularly liver functions and lipid and glucose metabolism, which is

18

important for the evaluation of the biosafety of frying oil.

19

Keywords

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Polar compounds; Deep-fried palm oil; Liver function; Lipid metabolism; Glucose

21

intolerance

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Introduction

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Deep-fried oils have been considered as an important risk factor for the

25

development of chronic diseases, such as diabetes and liver diseases. As the liver is a

26

major site involved in energy metabolism, the effects of oxidized edible oils on liver

27

functions were investigated in a few previous studies. The relative mRNA levels of

28

lipogenic enzymes in the liver were suppressed with the ingestion of oxidized oils,

29

while those encoding hepatic enzymes regulating lipid catabolism were upgraded.1-5

30

Increased oxidation stress was observed in the liver with the ingestion of oxidized

31

oils.6 In addition, the consumption of fried soybean oil has been shown to impair

32

glucose metabolism, such as glucose intolerance in rats.7, 8 However, the consumption

33

of oxidized oil did not always lead to increase of body weight, indicating that oxidized

34

frying oil was less adipogenic.2, 7, 9-12 The mechanism underlining the adverse effects

35

of deep-fried oils remains largely unknown.

36

Undesirable compounds generated during the thermal process of edible oil may

37

account for the adverse health effects of deep-fried oil. It has been reported that

38

oxidation, hydrolysis, and polymerization occur in the presence of water and oxygen

39

in thermally processed edible oils.13-16 Oxidized products are found to cause adverse

40

effects on health.17,

41

reported to cause diarrhea and rapid death in the feeding experiments with albino rats,

42

and lower amounts of polymeric residue were found to reduce the growth rate in

43

albino rats.18 However, the effects of polar compounds isolated from deep-fried oil on

44

lipid metabolism and liver functions have not been extensively studied.1-6

18

High amounts of polymeric residue (20%) in the diet was

45

Palm oil is one of the most widely used frying oil in the world because of its good

46

frying properties. Due to the high saturated fatty acids composition (32-47%) in palm

47

oil, it shows a high resistance to oxidation.19 Therefore, palm oil is used in industrial 3



48

frying with a good oxidative stability compared to other cooking oils. However, there

49

is little information on the effects of oxidized or deep-fried palm oil on animal and

50

human health.

51

The present study was to investigate the effects of deep-fried palm oil and polar

52

compounds purified from thermal process of palm oil on the health, especially on

53

lipid metabolism and liver functions. Besides, the potential mechanism underlining

54

the effects of polar compounds on health was also investigated.

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Materials and methods

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Chemicals. Fatty acid methyl ester standards were purchased from Sigma-Aldrich (St.

57

Louis, MO, USA). Hematoxylin and eosin used for staining were obtained from

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Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Other chemicals, for

59

instance, petroleum ether, diethyl ether, hexane, were analytical grade and purchased

60

from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China).

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Preparation of polar compounds from deep-fried palm oil. The frying experiments

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were performed with palm oil (Yihai Kerry, Shanghai, China) at 180 ± 5 oC for 8

63

hours per day for 5 continuous days. The drumstick (120g) was fried for 10 min in

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succession every 15 min. Deep-fried oil samples were withdrawn at the end of

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deep-frying process (40 h) and stored at -20 oC until analysis. Total polar compounds

66

(TPCs) in deep-fried oil was isolated with silica column chromatography as AOCS

67

method reported.20

68

Animals and diets. Kunming mouse (Musmusculus Km, KM) is an outbreed specie

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generated from the Swiss albino mouse, which is widely used in toxicology and other

70

studies.21-27 In the present study, 50 male Km mice (SLRC Laboratory Animal,

71

Shanghai, China) at 6 weeks of age were randomly divided into 5 groups after being

72

fed with normal rodent chow (SLRC Laboratory Animal, Shanghai, China) for a week 4


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for acclimation. The 5 groups of Km mice were fed with refined diets as indicated in

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Table 1: all high-fat diets contained 15% lard only (HF) and either replaced lard with

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5% fresh palm oil (HFF) or 5% deep-fried palm oil (HFH) or 3.5% purified TPCs

76

combined with 1.5% deep-fried palm oil (HFPC) to make up to 4.0% of TPCs in the

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diet for same lipid contents. All mice were housed in a vivarium maintained at 23 ± 2

78

o

79

libitum. Body weight was recorded weekly. All procedures were approved by the

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Ethics Committee (JN NO. 28 2013), Jiangnan University, China. They were in

81

accordance with the Guide for the Care and Use of Laboratory Animals issued by the

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Ministry of Science and Technology of the People’s Republic of China in 2006 [398].

83

Glucose tolerance test (GTT). GTT was performed after the mice were fasted for 6 h.

84

Tail blood was collected before (0 min) and at 30, 60, 90 and 120 min after the

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administration of a 10% glucose solution (1.5 g/kg body weight) and measured by

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ACCU-CHEK Active glucometer (Roche Diagnostics GmbH, Mannheim, Germany).

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Tissue sample collection. The mice were sacrificed after the termination of 12 weeks

88

of feeding. Blood samples were collected using heparinized blood-collecting tubes,

89

and plasma was separated by centrifugation at 1000 g for 15 min at 4 oC. The livers

90

were excised, rinsed in ice-cold saline solution. The sliced liver samples were put in

91

RNAlater for RNA isolation, and the rest was immediately frozen in liquid nitrogen

92

prior to being stored at -80 oC. Besides, epididymal fat was excised and weighted

93

according to standard procedure.

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Histochemical analysis. For histological study, small pierce of liver were fixed in 4%

95

formalin solution for 48 h. The tissues were dehydrated, embedded in paraffin wax,

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sectioned and stained with hematoxylin and eosin (H & E) according to standard

97

procedure. Image was taken using a DM 2700P microscope (Leica, Germany). The

C with a controlled 12-hr light-dark cycle. Food and drinking water were supplied ad

5



98

histological analysis was performed according to the NAFLD scoring system, which

99

was proposed by Kleiner et al.28. In our present study, the degree of liver cell injury

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was measured based on the point scale (0-2) indicating ballooning in hepatocytes.

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Biochemical analysis. Plasma triacylglycerol (TG), cholesterol (TC), high density

102

lipoprotein cholesterol (HDL-C) and low density lipoprotein cholesterol (LDL-C)

103

concentrations were determined via commercially available kits (Nanjing Jiancheng

104

Bioengineering Institute, Nanjing, China). Plasma ALT and AST activities were

105

determined by commercially available kits (Nanjing Jiancheng Bioengineering

106

Institute, Nanjing, China). In order to investigate lipid peroxidation status, plasma and

107

liver malondialdehyde (MDA) content and hepatic superoxide dismutase (SOD)

108

activity were measured using commercially available kits (Nanjing Jiancheng

109

Bioengineering Institute, Nanjing, China).

110

Fatty acids profile analysis in liver. To investigate the fatty acids profile of the liver,

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total lipids was extracted with a method of Folch et al.29 Briefly, liver tissue was

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homogenized in ice-cold saline solution. Afterwards, total lipids from the liver were

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extracted with chloroform/methanol (2:1 v/v). Then, fatty acids were measured by

114

fatty acid methyl esters and analyzed by a gas chromatograph (GC) (Agilent

115

Technologies, Beijing, China) equipped with an ionic liquid capillary column

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(TR-FAME 260M154P, 60 m × 0.25 mm × 0.25µm, Thermo Fisher, Shanghai, China)

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and a flame ionization detector. The fatty acid methyl esters were identified using

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their respective standard, and quantitative analyses were presented as relative

119

percentage.

120

Quantitative real-time polymerase chain reaction (qPCR) assay. Total RNA was

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extracted from freshly harvested liver tissue (n=3 per group) using TRIzol Reagent

122

(Invitrogen, Shanghai, China). Reverse transcription was carried out using the 6


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PrimeScript RT reagent Kit with gDNA Eraser (Takara Biotechnology, Dalian, China)

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with 1 µg total RNA to generate first-strand cDNA. qPCR was performed in a 20 µl

125

final reaction volume using SYBR Premix Ex Taq II (Takara Biotechnology, Dalian,

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China). qPCR assays for all samples were determined in triplicate on an ABI 7900HT

127

Fast Real-Time PCR system (Applied Biosystems, Foster City, CA). The mRNA

128

levels were normalized by the relative value of target amount to the endogenous

129

reference amount of rodent β-actin as internal control. The sequences of the primers

130

were

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5’-ACTGCCGCATCCTCTTCCTC-3’,

132

5’-CTCCTGCTTGCTGATCCACATC-3’; mouse Srebp-1c (NM_011480.3), forward:

133

5’-CTGGAGACATCGCAAACAAGC-3’,

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5’-ATGGTAGACAACAGCCGCATC-3’; mouse Scd1 (NM_009127.4), forward:

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5’-CTGCCTCTTCGGGATTTTCTACT-3’,

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5’-GCCCATTCGTACACGTGATTC-3’; mouse PPARα (NM_011144.6), forward:

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5’-GCAGTGCCCTGAACATCGA-3’, reverse: 5’-CGCCGAAAGAAGCCCTTAC-3’;

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mouse Cpt1α (NM_013495.2), forward: 5’-GAGAAATACCCTGACTATGTG-3’,

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reverse:

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(NM_015729.3),

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5’-GTCATGGGCGGGTGCAT-3’. The primers were synthesized by Sangon Biotech

142

(Shanghai, China).

143

Statistical analysis. Data are presented as means ± standard error of the mean (SEM)

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for 10 mice in each group. The significant difference among groups was performed

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statistically by one-way Analysis of Variance (ANOVA) combined with Duncan’s

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Multiple Range test using SPSS Package. P< 0.05 was considered significant.

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Results

designed

as

follows:

mouse

β-actin

(NM_007393.3),

reverse:

reverse:

5’-TGTGAGTCTGTCTCAGGGCTAG-3’; forward:

forward:

reverse:

mouse

Acox1

5’-GCCTGCTGTGTGGGTATGTCATT-3’,

reverse:

7


and


148

Effects of deep-fried palm oil and TPCs on animal growth. Kunming mouse, an

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outbred strain, was used to investigate the effect of high-fat diet with oxidized frying

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oil (palm oil) on animal feeding. The body weight gain, food intake and plasma lipids

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were monitored and recorded for a period of 12 weeks. As shown in Table 2, feeding

152

with defined high-fat diets containing lard (HF), fresh palm oil (HFF) and heated

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palm oil (HFH) significantly increased the body weight of the animals in contrast to

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normal low fat control diet (CO). This increase corresponded well to the change of

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organ weight, such as liver and adipose tissue.

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Interestingly, the mice fed with high-fat diet containing purified polar compounds

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(HFPC) had a lower body weight gain compared to other high fat diet groups but

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similar to those fed with low fat diet (CO). Furthermore, no significant difference was

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observed in the liver and adipose tissue weight between HFPC-fed group and CO-fed

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ones. As food was freely available to animals, there was no significant difference in

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food intake among all the groups in the present study. In contrast, the energy intake of

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the mice fed polar compounds (HFPC) was 50% higher than that in CO-fed ones

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which was similar among all high-fat diet groups (HF, HFF, HFH, and HFPC).

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Therefore, the less body weight and organs weight gain of HFPC-fed group could not

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be simply attributed to the less energy intake. These results indicated that high content

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of polar compounds from frying oil had negative effects on the accumulation of lipids

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in vivo.

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Deep-fried palm oil and TPCs influence lipid metabolism and fat accumulation

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in liver. Morphology of the liver tissue was analyzed by histochemical analysis with

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H & E staining. As shown in Figure 1, lipid accumulation in hepatocytes was clearly

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observed in animals fed with diet containing additional fat (15%). Significant amounts

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of ballooning hepatocytes were found in HF-, HFF- and HFH-fed mice (Figure 1). 8


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Even though the size of hepatocytes was more moderate in HF-fed mice compared to

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that in HFF and HFH groups, a dominant lipid deposit was also observed in the

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former group (Figure 1B). The lipid accumulation in hepatocytes of the HF-, HFF-

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and HFH-fed groups indicated the formation of nonalcoholic fatty liver. However, for

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HFPC group fed with a high amount of polar compounds, the fat accumulation was

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significantly less than that in the other experimental groups with most of the cells

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having good morphology as control group (Figure 1E), which was in line with the

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trend of liver weight. The pathological scoring was applied in the present study to

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indicate relatively the lipid deposition in hepatocytes. As shown in Table 3, no

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ballooning was observed in CO-fed mice. Interestingly, 9 of the 10 mice fed polar

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compounds (HFPC) were scored 0 as control ones while the mice in other high-fat-fed

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groups (HF, HFF, and HFH) were scored either 1 or 2 in our study. It was in

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accordance with the observation mentioned above.

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Plasma lipids were compared among different feeding groups of Kunming mice

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(Table 4). Concentrations of plasma total cholesterol and triglyceride increased in all

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high-fat diet-fed groups compared to that in basal diet-fed group, while no significant

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difference was observed for triglyceride contents among high-fat diet groups (HF,

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HFF, HFH, and HFPC). Further analysis of plasma LDL-C and HDL-C were

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performed. Even though no significant difference was observed among high-fat

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diet-fed groups, plasma LDL-C and HDL-C level, as well as the ratio of

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LDL-C/HDL-C, was slightly lower in the groups fed with diets containing polar

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compounds (HFH and HFPC) compared to the ones fed with unheated oil (HFF),

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indicating that lipid metabolism was interfered by feeding with high-fat diet

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containing polar compounds.

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Deep-fried palm oil and TPCs cause oxidative stress and functional change in 9



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liver. As oxidized oil might deplete anti-oxidative capacity in animals and led to

199

oxidative damage to tissues, a biomarker of MDA was measured in both plasma and

200

liver tissues after 12 weeks (Figure 2). Although there was no significant difference in

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plasma MDA level among all high-fat diet-fed groups, the addition of deep-fried palm

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oil or polar compounds further enhanced the content of MDA in serum to some extent

203

(Figure 2A). Likewise, no significant difference was observed among all the groups as

204

to the liver MDA concentrations (Figure 2B). The hepatic SOD content, a biomarker

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for tissue anti-oxidative status in the liver, was increased with the intake of high-fat

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diet containing lard or fresh palm oil and significantly decreased with the

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consumption of deep-fried palm oil or polar compounds compared to that in fresh

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palm oil-fed group.

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In supportive to the liver functional damage, the plasma ALT and AST enzymatic

210

activity were determined (Table 5). Both serum ALT and AST levels increased

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dramatically in high-fat diet-fed mice compared to those in chow diet-fed group

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without extra effects of polar compounds feeding in HFPC group.

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Deep-fried palm oil and TPCs affect hepatic fatty acids profile. Total fatty acids

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composition of the liver tissue was analyzed by extracting lipids from liver tissue

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(Table 6). In comparison with low fat basal diet-fed animals, the fatty acids from

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livers of high-fat diet-fed mice had higher contents of most of the fatty acids

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measured. Among the high-fat diet-fed groups, a significant reduction of unsaturated

218

fatty acids, such as linoleic acid (C18:2n-6) and α-linolenic acid (C18:3n-3), was

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observed in HFPC group. Arachidonic acid (C20:4n-6), an important precursor for

220

biological eicosanoids, was relatively constant. The ratios of oleic acid (C18:1) to

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stearic acid (C18:0) and palmitoleic acid (C16:1) to palmitic acid (C16:0), indicators

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for the activity of fatty acid desaturase (SCD1), reduced with the consumption of 10


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polar compounds isolated from deep-fried palm oil compared to that in the other

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high-fat diet-fed groups (HF, HFF, and HFH), indicating that hepatic fatty acid

225

metabolism was interfered.

226

Deep-fried palm oil and TPCs influence glucose metabolism. Effect of polar

227

compounds on glucose metabolism was investigated using glucose tolerance assay. As

228

indicated in Figure 3, in response to intraperitoneal glucose stimulation, mice fed with

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high-fat diet containing 15% lard had a higher plasma glucose concentration in

230

contrast to the other groups. The area under curve (AUC) for GTT was 42.20% higher

231

in HF group compared to basal chow diet-fed mice, indicating the impairment of

232

glucose metabolism under high lard content diet, and it was consistent with previous

233

literature. Interestingly, different results were observed in the mice fed with low

234

amount of polar compounds in comparison to the ones fed with high amount of polar

235

compounds according to GTT. The diet containing low amount of polar compounds

236

(HFH) enhanced glucose clearance as good as basal diet with low AUC (Figure 3B).

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In contrast, HFF- and HFPC-fed mice showed comparable glucose tolerance in the

238

test, where AUC was 10.8% and 15.0% higher than that in control group, respectively.

239

In supportive, the fasting basal glucose concentration for mice fed with oxidized oil

240

(HFH) had a low plasma glucose level as the chow diet-fed mice whereas the ones fed

241

with high-fat diet with lard and polar compounds (HF and HFPC) had higher content

242

of plasma glucose (Figure 3C). The results indicated that the benefit of lowering

243

plasma sugar effect with plant oils and low polar compounds containing high fat diet

244

was compromised by high consumption of polar compounds from oxidized oils.

245

Deep-fried palm oil and TPCs influence gene expression in liver. The gene

246

expression related to lipid metabolism in livers was measured by qPCR. As shown in

247

Figure 4A, mRNA levels for genes encoding sterol regulatory element-binding 11



248

protein-1c (SREBP-1c), the upstream activator of lipogenic regulator, were enhanced

249

in high-fat diet-fed groups containing unheated oils (HF and HFF). However, it was

250

inhibited in the presence of polar compounds from oxidized oils. SCD1, a desaturase

251

for fatty acid synthesis, reduced accordingly in HFPC group (Figure 4B), indicating

252

that fatty acid synthesis was diminished.

253

Interestingly, mRNA levels for peroxisome proliferator-activated receptor α

254

(PPARα), the master regulator for fatty acid oxidation in liver, were clearly enhanced

255

with the increase of polar compounds ingestion in contrast to the mice fed with basal

256

diet (Figure 4C). As one of its downstream targets for fatty acid oxidation on

257

peroxisomes, acyl-CoA oxidase 1 (ACOX1) was inhibited in HF-fed mice, and no

258

significant was observed in HFF-fed group compared to the control ones. However,

259

ACOX1 was stimulated with the consumption of polar compounds, and an increase of

260

about 2-fold compared to control group was observed in HFPC-fed ones (Figure 4E).

261

Carnitine palmitoyltransferase I α (CPT1α), the enzyme for long chain fatty acid

262

being transferred to mitochondria for β-oxidation, was inhibited in both HF and HFF

263

groups, and no significant difference was observed in HFH and HFPC groups

264

compared to HFF group (Figure 4D). These data might indicate a significant increase

265

in the expression of genes regulating fatty acid oxidation on peroxisomes.

266

Discussion

267

Heated or fried oils have been claimed to have adverse effects on health in vivo

268

studies,30-35 including disorder of lipid metabolism and liver functions. Moreover,

269

glucose intolerance and oxidative stress have been observed in the objects with the

270

consumption of oxidized oils. 6-8, 36-38 However, the mechanism of the adverse effects

271

of frying oils remains unknown. In the present study, the effects of fried palm oil and

272

polar compounds isolated from deep-fried palm oil on health, especially on lipid and 12


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glucose metabolism and liver functions, were investigated.

274

Our results showed that high-fat diet feeding (except for HFPC) increased the

275

body weight gain with high fat content (15% lipid content, about 50% increase in

276

calorie energy) in these diets. Low level of polar compounds replacement for lard was

277

as efficient as other high fat diets on the increase of body weight gain. It was in

278

consistent with the studies reported by Miller et al.39 and Lu et al.10, where fresh or

279

fried vegetable and soybean oil were used, respectively. However, the high energy

280

intake in HFPC group did not lead to significant increase of body weight and adipose

281

tissue weight, which was close to that in basal diet-fed group. This is probably caused

282

by the high amount of polar compounds in high-fat diet. Polar compounds are a mix

283

of oligomeric triglycerides (TAGs), dimeric TAGs, oxidized TAGs, diglycerides

284

(DAGs), and free fatty acids (FFAs).14-16 Polymerized lipids as macromolecules have

285

been reported to be difficult to be absorbed. In addition, polymerized lipids might

286

interfere lipids hydrolysis regulated by pancreatic lipase.7, 11, 34, 39 Polymeric residues

287

were isolated from autoxidized lipids via molecular distillation process, and a

288

reduction on body weight gain of albino rats was observed with diet containing 4 to 7%

289

polymeric residues.18 This finding was in agreement with our results where the

290

ingestion of diet with high amount of polar compounds, containing polymeric

291

compounds, suppressed the increase of body weight gain compared to that in the other

292

high-fat diet-fed groups.

293

The glucose metabolism and glucose tolerance was impaired by the high-fat diet

294

feeding in HF and HFF groups, which contained lard and unheated palm oil,

295

respectively. The accumulated adipose tissue might contribute to the change of

296

glucose metabolism. It has been reported that some adipocytokines secreted by

297

adipose tissue, for instance, tumor necrosis factor α (TNFα), leptin, adiponectin, and 13



298

resistin, were related to the reduction of insulin sensitivity.40, 41 On the other hand, the

299

consumption of deep-fried palm oil containing 1.5% polar compounds did not lead to

300

glucose intolerance. Even though high amount of lipid deposition in liver and adipose

301

tissue was observed in the mice, the fasting plasma glucose level and glucose

302

intolerance induced by high-fat diet intake were improved. These results strongly

303

suggested that polar compounds, unlike lipid from plant oils and lard, function

304

differently in lipid and glucose metabolism in Kunming mice. In contrast, a

305

significant increase of glucose intolerance and fasting plasma glucose level were

306

observed in the mice fed with 4.0% polar compounds in HFPC group compared to the

307

ones fed with basal diet in the present study. A similar effect of fried soybean oil on

308

rodents was observed by Chao et al..7 They showed that oxidized soybean oil (20% of

309

the diet), which was very high in amount compared to the diet containing 5%

310

deep-fried palm oil in our study, led to glucose intolerance rather than adipogenesis.

311

The glucose intolerance was caused by insulin deficiency instead of insulin

312

resistance.7, 8, 33 As polar compounds would induce significant change in the oxidative

313

status in tissue such as reduction of hepatic SOD and the oxidative stress could

314

contribute to change of pancreatic function and energy metabolism, high amount of

315

polar compounds compromise the beneficial effect of low level of polar compounds

316

on glucose tolerance in Kunming mice.

317

An elevated plasma MDA level suggested that lipid peroxidation significantly

318

increased in all high-fat diet groups and further enhanced with the increase of polar

319

compounds consumption. These results implied the damage of antioxidant defense

320

system in tissues.26, 42 A reduction of SOD activity, a major component for antioxidant

321

capacity in tissue, was seen with the ingestion of polar compounds in the rodents

322

compared to the ones fed with lard- and fresh palm oil-fed mice. Moreover, an 14


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323

increased liver TBARS level, which could be regarded as MDA, was reported in

324

previous studies with the consumption of fried oils.38, 43 Therefore, the ingestion of

325

polar compounds or oxidized oils would reduce the antioxidative capacity and lead to

326

oxidative stress in hepatocytes, which would further aggravate the burden for animal

327

health.

328

Comparable increase of liver weight was observed in all high-fat feeding groups

329

except for HPFC. The liver weight in HPFC was lower than that in the other high fat

330

diets and slightly higher than that of the control ones fed with low fat chow diet. On

331

the other hand, liver functions by high AST enzymatic activities implied the

332

occurrence of liver lesions. Their levels showed obvious increases in high-fat diet

333

groups, as well as in the mice fed high amount of polar compounds with less lipid

334

accumulation in hepatocytes. In combined with glucose intolerance data, our results

335

clearly showed that polar compounds under high energy intake had effect on lipid and

336

glucose metabolism in liver.

337

Gene expression related to lipid metabolism further supported the changes

338

induced by oxidized oils and polar compounds. An increased level of mRNA encoding

339

PPARα, which is involved in β-oxidation, was observed in the mice fed with polar

340

compounds compared to the ones fed with deep-fried palm oil. Interestingly, a same

341

trend was also observed as to mRNA encoding Acox1, a downstream target of PPARα,

342

which regulates the fatty acid oxidation on peroxisomes. However, a significant

343

reduction on the expression of mRNA encoding Cpt1α, which was involved in fatty

344

acid oxidation in mitochondria, was seen in the mice fed with high amount of polar

345

compounds compared to the ones in control group. Our results were in line with

346

others’ work with activation of peroxisome lipid metabolism.3, 31 Herein, it was likely

347

that β–oxidation in mitochondria was suppressed with the ingestion of deep-fried 15



348

palm oil and polar compounds, and the fatty acid oxidation on peroxisomes was

349

enhanced with the consumption of polar compounds.

350

SREBP-1c increases hepatic lipogenesis and contributes to the lipid deposit in the

351

liver.44 The attenuation of gene expression of Srebp-1c in the liver harvested from

352

HFH and HFPC groups, where the diets were composed with 10% lard and 5%

353

deep-fried palm oil or polar compounds isolated from deep-fried palm oil, implied

354

that deep-fried palm oil and polar compounds were less lipogentic compared to

355

unheated oils. SCD1 plays a central role in desaturation of fatty acids. Specifically,

356

the formation of palmitoleic acid (C16:1) and oleic acid (C18:1) from palmitic acid

357

(C16:0) and stearic acid (C18:0) is triggered by SCD1, respectively.44 The relative

358

mRNA levels of hepatic Scd1 were higher in HF-, HFF-, and HFH-fed mice compared

359

to the ones in HFPC group in the present study. It was in accordance with the hepatic

360

fatty acid profile, where palmitoleic acid (C16:1) and oleic acid (C18:1) in the mice

361

fed with polar compounds were significantly lower than those in the other high-fat

362

diet-fed groups.

363

In conclusion, our results clearly showed that the consumption of polar

364

compounds under high fat diet led to the change lipid and glucose metabolism in

365

Kunming mice. The mechanism underlining was different from that caused by high

366

energy intake, where lipid accumulation in hepatocytes was observed. The present

367

study revealed that the ingestion of polar compounds enhanced the fatty acid

368

oxidation on peroxisomes, and it was suppressed in mitochondria. For glucose

369

metabolism, polar compounds at low level would improve glucose intolerance

370

induced by high fat diet where lipid deposition in liver was observed. However, high

371

amount of polar compounds intake impaired glucose tolerance without the

372

accumulation of lipids in liver. Further study should be carried out to investigate the 16


Page 16 of 35

Page 17 of 35


373

pathological mechanism of polar compounds from deep-fried oils on the health.

374

Notes

375

The authors declare no competing financial interest.

376

Abbreviations Used

377

GTT, glucose tolerance test; TPCs, total polar compounds; MDA, malondialdehyde;

378

SOD, superoxide dismutase; GC, gas chromatograph; qPCR, quantitative real-time

379

polymerase chain reaction; ALT, alanine aminotransferase; AST, aspartate

380

aminotransferase; AUC, area under the curve; SREBP-1c, sterol regulatory

381

element-binding protein-1c; SCD1, stearoyl-CoA desaturase 1; PPARα, peroxisome

382

proliferator-activated receptor α; ACOX1, acyl-CoA oxidase 1; TNFα, tumor necrosis

383

factor α; CPT1α, carnitine palmitoyltransferase I α;

384

Funding

385

This work was supported by the National Natural Science Foundation of China

386

(31671786), the Research Fund of National 13th Five-Year Plan of China

387

(2016YFD0401404).

388

17



389

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23



Figure captions Figure 1 H & E staining of the liver from mice (magnification, 10 X 20): (A) CO group; (B) HF group; (C) HFF group; (D) HFH group; (E) HFPC group. Figure 2 (A) Plasma MDA levels (mmol/L) under different diets. (B) Hepatic MDA levels (nmol/mg protein) and SOD contents (U/mg protein) under different diets. Means of hepatic SOD contents with different superscript letters (a, b, c) are significantly different from one another by Duncan’s Multiple Range Test (P < 0.05). No significant difference is observed among different groups in consideration of the levels of plasma and hepatic MDA, and the superscript letters are not displayed. Figure 3 (A) Blood glucose level at 0, 30, 60, 90 and 120 min after 10% glucose solution injection (1.5 g/kg of body weight). (B) Area under the curve (AUC) for blood glucose over 2 h in glucose tolerance test. (C) Fasting blood glucose level. Means with different superscript letters (a, b) are significantly different from one another by Duncan’s Multiple Range Test (P < 0.05). Figure 4 The relative mRNA levels for (A) SREBP-1c, (B) SCD1, (C) PPARα, (D) CPT1α, and (E) ACOX1 in the livers of male Kunming mice analyzed via real-time polymerase chain reaction techniques. The data are expressed relative to the level of the Srebp-1c, Scd1, PPARα, Cpt1α, and Acox1 transcripts in the livers of mice fed chow diet, and its mRNA expression is set at 1, respectively. Means with different superscript letters (a, b) are significantly different from one another by Duncan’s Multiple Range Test (P < 0.05). 24


Page 24 of 35

Page 25 of 35


Tables Table 1. Composition of experimental diets Component

CO

HF

HFF

HFH

HFPC

654.5

494.5

494.5

494.5

494.5

Lard (g/kg)

0

150

100

100

100

Fresh palm oil (g/kg)

0

0

50

0

0

Heated palm oil (g/kg)

0

0

0

50

15

Added polar compounds (g/kg)

0

0

0

0

35

Casein (g/kg)

202.9

202.9

202.9

202.9

202.9

Maltodextrin (g/kg)

50.7

50.7

50.7

50.7

50.7

Cellulose (g/kg)

50.7

50.7

50.7

50.7

50.7

DL-Methionine (g/kg)

3

3

3

3

3

Sucrose (g/kg)

1

1

1

1

1

Choline bitartrate (g/kg)

1

1

1

1

1

Sodium chloride (g/kg)

2

2

2

2

2

Calcium carbonate (g/kg)

13.2

13.2

13.2

13.2

13.2

Calcium bicarbonate (g/kg)

10.1

10.1

10.1

10.1

10.1

0

10

10

10

10

Potassium citrate (g/kg)

10.1

10.1

10.1

10.1

10.1

Mineral mixture (g/kg)

0.6

0.6

0.6

0.6

0.6

Vitamin mixture (g/kg)

0.2

0.2

0.2

0.2

0.2

Total polar compounds

0

3

3

15

40

3839.2

4549.2

4549.2

4549.2

4549.2

Maize starch (g/kg)

Cholesterol (g/kg)

Energy density (kcal/kg)

25



Page 26 of 35

Table 2. Body weight gain, the weight of organs, and food intake of mice fed different diets for 12 weeksa Body weight gain (g)

Liver Liver (g)

Epididymal fat

Food intake

% Body weight

Epididymal fat (g)

% Body weight

(g/week)

CO

14.75±1.01 c

2.14±0.07 b

4.07±0.10 b

1.72±0.28 b

3.20±0.47 b

43.99±1.34 a

HF

21.28±2.70 a, b

3.15±0.35 a

5.16±0.30 a

2.37±0.30 a, b

3.94±0.46 a, b

55.76±2.99 a

HFF

22.85±0.94 a

3.02±0.26 a

4.92±0.35 a, b

3.14±0.19 a

5.16±0.33 a

51.80±6.16 a

HFH

21.26±2.14 a, b

3.00±0.21 a

4.96±0.38 a, b

2.83±0.35 a

4.62±0.52 a

51.48±3.72 a

HFPC

15.37±2.62 b, c

2.54±0.20 a, b

4.56±0.23 a, b

1.84±0.38 b

3.17±0.58 b

56.28±0.82 a

a

Results are means ± SEM with n = 10 for all groups. Means with different superscript letters (a, b, c) in each column are

significantly different from one another by Duncan’s Multiple Range Test (P < 0.05).

26


Page 27 of 35


Table 3. Histological characteristics of mice fed different diets for 12 weeksa Percentage of each category in different groups (n=10 per group) Item

Definition

Score CO

HF

HFF

HFH

HFPC

Ballooning

a

None

0

100

0

0

0

90

Few balloon cells

1

0

90

80

80

10

Many cells/prominent ballooning

2

0

10

20

20

0

The histological diagnosis was performed according to the NAFLD scoring system.28 The degree of liver cell injury

was measured based on the point scale (0-2) indicating ballooning in hepatocytes in our present study.

27



Page 28 of 35

Table 4. Plasma lipids contents (mmol/L) under different dietsa TC

TG

HDL-C

LDL-C

LDL-C/HDL-C

CO

2.61±0.09 b

1.18±0.10 a

1.42±0.16 a

1.73±0.08 b

1.28±0.15 b

HF

5.22±0.28 a

1.44±0.22 a

1.28±0.11 a

3.88±0.22 a

3.11±0.24 a

HFF

5.06±0.49 a

1.43±0.20 a

1.39±0.12 a

3.69±0.10 a

2.74±0.28 a

HFH

4.77±0.30 a

1.42±0.22 a

1.14±0.14 a

3.34±0.32 a

3.22±0.39 a

HFPC

4.63±0.08 a

1.38±0.13 a

1.34±0.13 a

3.36±0.17 a

2.65±0.17 a

a

Results are means ± SEM with n = 10 for all groups. Means with different superscript letters (a, b)

in each column are significantly different from one another by Duncan’s Multiple Range Test (P < 0.05).

28


Page 29 of 35


Table 5. Plasma ALT and AST levels (U/L) under different dietsa CO

HF

HFF

HFH

HFPC

ALT

26.89±6.52 a

41.81±4.83 a

50.56±14.92 a

41.70±8.66 a

34.08±5.58 a

AST

58.40±12.89 b

101.99±21.87 a, b

136.34±6.56 a

107.68±12.27 a, b

104.53±14.38 a, b

a

Results are means ± SEM with n = 10 for all groups. Means with different superscript

letters (a, b) in each line are significantly different from one another by Duncan’s Multiple Range Test (P < 0.05).

29



Page 30 of 35

Table 6. Hepatic fatty acids composition of mice fed different diets for 12 weeksa FFA (%)

CO

HF

HFF

HFH

HFPC

C14:0

0.14±0.017 b

0.45±0.093 a

0.41±0.055 a

0.40±0.067 a

0.21±0.034 b

C16:0

8.36±0.72 c

18.06±1.79 a, b

19.71±1.77 a

19.87±2.76 a

13.50±1.80 b, c

C16:1

0.67±0.12 c

1.82±0.39 a, b, c

2.15±0.40 a, b

2.68±0.58 a

1.21±0.28 b, c

C18:0

4.77±0.35 b

6.71±0.58 a

5.63±0.25 a, b

5.94±0.43 a, b

5.63±0.29 a, b

C18:1

6.23±0.67 c

44.97±7.77 a, b

44.43±6.39 a, b

49.45±9.61 a

27.55±5.70 b

C18:2(n-6)

10.90±1.19 b

21.23±2.33 a

20.62±2.26 a

16.69±3.39 a, b

12.45±1.93 b

C18:3(n-6)

0.13±0.022 b

0.25±0.036 a

0.23±0.029 a, b

0.21±0.035 a, b

0.18±0.041 a, b

C18:3(n-3)

0.25±0.037 a, b

0.37±0.059 a

0.32±0.038 a, b

0.36±0.072 a

0.17±0.038 b

C20:0

0.12±0.010 c

0.30±0.037 a

0.26±0.027 a

0.24±0.036 a, b

0.16±0.024 b, c

C20:4(n-6)

3.25±0.42 a

3.14±0.18 a

2.96±0.16 a

3.05±0.16 a

3.12±0.16 a

C16:1/C16:0

0.076±0.008 b

0.094±0.013 a, b

0.103±0.014 a, b

0.122±0.017 a

0.078±0.009 b

C18:1/C18:0

1.33±0.12 b

6.95±1.37 a

7.81±0.98 a

8.06±1.40 a

4.91±1.00 a

a

Results are means ± SEM with n = 10 for all groups. Means with different superscript

letters (a, b, c) in each column are significantly different from one another by Duncan’s Multiple Range Test (P < 0.05).

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Figure 1

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Figure 2

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Figure 3

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Figure 4

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TOC Graphic

35


Effects of Polar Compounds Generated from the Deep-Frying Process

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