Dietary fat rich in mono or di-unsaturated fatty acids reduces lipid oxidation in hepatic tissue of rabbits

NutritionResearch, Vol. 17, No. 10, pp. 158945%. 1997 Copyright 8 1997 Elsevier Science Inc. Printed in the USA. All rights rese.ned 0271-5317/?27 $17.00 + Ml ELSEVIER

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DIETARY FAT RICH IN MONO OR DI-UNSATURATED FAITY ACIDS REDUCES LIPID OXIDATION IN HEPATIC TISSUE OF RABBITS

Departamento

A.I. Rey, C.J. Lopez-Bote’ , A. Castafio, J. Thos and R. Sanz Arias de Production Animal. Facultad de Veterinaria. Universidad Complutense. 28040 Madrid. Spain.

ABSTRACT The effect of dietary vegetable oils (olive or sunflower oil) and a-tocopheryl acetate on fatty acid composition and lipid oxidation in rabbit liver has been evtited. Animals receiving diets enriched in olive oil had higher levels of oleic acid in triglycerides and phospholipids (p
INTRODUCTION The I&y acid composition of membranes is a major determinant of metabolic processes of the cell. Variations in dietary fatty acid content, hence tissue composition, have been shown to affect several metabolic processes, inchtding insulin action (I), metabolic rate (2) blood pressure (3) and variables related to coronary artery disease (4). Furthermore, it has been reported that dietary polyunsaturated fatty acids play an important role in supporting the cytochrome P-450-dependent liver microsomal mixedfunction oxidase activities, which play an important role in the biotransformation of a wide variety of drugs and foreign compounds (5). The rate of lipid oxidation in animal tissues depends on a number of factors, inchuling the polyunsaturated f&y acid content and the presence of antioxidants such as cxtocopherol(6).

‘Corresponding

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Given that fatty acid classes have distinctly d&rent metabolic activ&ies while operating in competition for the same biosynthetic enzymes and deposition places in the phospholipid molecule, we have investigated the consequences, of in&ding a dietary fat source rich iu Cl81 (n-9) or C18:2 (n-6) fatty acids, but in all cases with a similar concentration of (n-3) fatty acids. The objective of this study was to evahrate the influence of adding fat rich in polyunsaturated or monounsaturated Gtty acids to rabbit diets on (a) the fatty acid composition of liver, (b) the susceptibility of hepgtic tissue to oxidation and (c) to assess the effectiveness of dietary a-tocopheryl acetate supplementation in diet enriched or nonenriched in fat on the oxidative stabii of hepatic tissue.

METHODS

AND MATERIALS

Exnerimental design Six groups of eight Califiomian x New Zealand White rabbits were randomly d&ributed and located in cages of 4 animals each, with2 of each gender. From weaning (20 days of life) to the end of the experiment (69 days of life) all rabbits were given ad h’bitum access to feed with the appropriated diet (Table 1). A control diet with no-added fat (NF) and two di&rent oil sources (olive oil and su&wer oil) at inchtsion level of 30 g/kg were used Withineach dietary treatme@ one group was fed a low level of a-tocopheryl acetate (10 mg a-tocopheryl acetate/kg diet) (Hofhnan-La Roche, Basel, Switzerland) and the other group received a supplemental level (200 mgkg). Slaughter. samnle collection and chemical anal& Rabbits were stunned, slaughtered and bled at a local slaughter house. Samples from the liver were immediately taken, frozen in liquid nitrogen, vacuum packed and kept frozen at -22°C uutil ana&zed. In all cases, lipii oxidation studies were carried out within four week& of slaughter, and fatty acid composition snalyzed within three months of slaughter. Triglycerides and phosphohpids from liver samples were obtained according to the method developed by Marmer and Maxwell (7) and arm&red by capillary gas chromatography with a temperature program from 170 to 245°C as previously described (8). The lability of liver honmgenates to iron-induced lipid oxidation was determined by a modification of the method of Kombrust and Mavis (9) in which lmm FeSO4 was used as the catalyst of lipid peroxidation and homogenates were incubated at 37°C in 0.04 mol& Tris-maleate but&r (10). Oxidation was expressed as mnol malonaldehyde (MDA)‘mg protein. For the analysis of a-tocopherol, liver samples were homogenized in a 0.054 M dibasic sodium phosphate buflbr @H=7.0). A&x mixing with absohrte ethanol and hexane, the upper layer was evaporated and redissolved in ethanol prior to analysis by reverse phase HPLC (Hewlett Packard 105O)(Waldbr~ Germany) (11). The mobile phase was methanolwater (97:3) at a iloy rate of 2mVmiu Chemical ansly& of feed was carried out in all cases as previously described (8). For Wty acid determination, dietary fat was extracted by chknofomlmethanol and analymd by capilkuy gas chromatography as previously described (8). statisticalanalvsis The efFectsof diet on Gtty acid composkion and oxidation were at@zed using the General Linear Model of SAS (12). An indivkkud rabbit was the experimental I& for analy& of all data. Data were ana&ed as a completely randomized design The comparative analyses between means were carried out using orthogonal contrasts.

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TABLE 1 Main Ingredients (g/kg), Chemical Composition (g/kg) and Major Fatty Acid Composition (g/kg feed) of Diets without Added Fat (NF) or with 30 g/kg Added Olive (0) or Sunflower Oil (S) NF

Barley Wheat bran Soybean meal (44%) Al&& meal (hay) Olive oil Sunflower oil Sodium chloride Calcium carbonate Dicalcium phosphate

vitaminl&erai mix Crude protein Fat Crude fiber Acid detergent fiber Ash Nitrogen-free extracts Fatty acid C16:O C16:l (n-7) C18:O C18:l (n-9) C18:2 (n-6) C18:3 (n-3)

RESULTS

330 200 180 260 0 0 4 8 15 3 211 26 123 150 56 585

n 300 200 180 260 30 0 4 8 15 3 207 55 121 159 55 563

c 300 200 180 260 0 30 4 8 15 3 204 53 120 162 55 568

2.79 3.40 5.08 0.11 0.13 0.13 0.41 2.08 0.88 3.46 23.60 7.72 10.46 11.52 26.26 1.07 1.16 1.06

AND DISCUSSION

The major f&y acids in diets not enriched with tit were linoleic, pahnitic and stearic acids. The incbsion of sunflower oil considerably increased the concentration of linoleic acid in diets (2626 vs approx 1130 mg/lOO g feed), while olive oil increased the concentration of oleic acid (2360 vs less than 800 mg/lOO g feed)(Table 1). A marked difference in the proportion of most fatty acids in triglycerides of liver was observed between animals receiving the experimental diets. Animals receiving NF diets had higher concentration of saturated fatty acids (p
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TABLE 2

Main Fatty Acid Composition (means of g/100 total fatty acids) of Liier Triglycerides from Rabbits Fed Diets with no Added Fat (NF), 30 g/kg Added Olive Oil (0) or SunSower Oil (S) with 10 or 200 rug/kg a-Tocopheryl Acetate DIETS * NF

SD

0 c14:o C16:O C16:l (n-7) Cl&O C18:l (n-9) Cl&l (n-7) C18:2 (n-6) Cl&3 (n-3) C20:4 (n-6) Z (n-3) E in-6j X (n-9) C sat IJI **

10 0.8 23.3 1.6 15.6 18.0 1.5 28.6 2.6 7.8 2.6 36.5 18.0 39.8 1.2

probability

of contrasts

#

FAT 200 0.7 23.6 1.4 16.4 16.3 1.5 28.8 2.6 8.5 2.6 37.3 16.3 40.7 1.2

10 0.6 19.0 1.0 14.8 27.1 1.4 26.4 2.5 7.1 2.5 33.5 27.1 34.4 1.2

S 200 0.6 18.9 0.9 15.4 26.1 1.4 26.4 2.6 7.5 2.6 33.9 26.1 34.9 1.2

10 0.6 18.5 1.1 15.8 17.3 1.3 35.6 2.1 7.7 2.1 43.3 17.3 34.9 1.3

200 0.5 18.4 0.8 18.1 14.9 1.3 35.0 2.0 9.0 2.0 44.0 14.9 37.1 1.3

0.22 2.44 0.36 2.77 3.79 0.28 1.45 0.33 1.94 0.33 2.56 3.78 3.12 0.56

1 0.042 0.001 0.001 NS 0.009 NS 0.001 0.027 NS 0.027 NS 0.009 0.001 0.021

2 NS NS NS NS 0.001 NS 0.001 0.002 NS 0.002 0.001 0.001 NS 0.001

3 NS NS NS NS NS NS NS NS NS NS NS NS NS NS

45 NS NS NS NS NS NS NS NS NS NS NS NS NS NS

NS NS NS NS NS NS NS NS NS NS NS NS NS NS

*DIETS: NF = non added fat; FAT = 3% added fat.- olive (0) or sutiower (S) oil; 10 or 200 mg atocopheryl acetate/kg feed. #Contrasts were as follows: (1) = NF vs FAT ; (2) = 0 vs S; (3) = 10 vs 200; (4) = Interaction fat level in feed (NF or FAT) x a-tocopheryl acetate (10 or 200): (5) = Interaction source of fat (0 or S) x a-tocopheryl acetate (10 or 200). NS = not significant (BO.05). **UI (Unsaturation index) = average number of double bonds per fatty acid residue).

Phospholipid fraction showed less marked variations in fatty acid proportion between experimental groups. The higher concentration of (n-6) or (n-9) fatty acids of phospholipid leads to other changes in the relative proportion of saturated and (n-3) tatty acids. This changes allow the phospholipid to keep overall unsaturation (as estimated by the unsaturation index) in a narrow range (variation between treatments less than 7%). These results are in agreement with other investigators when studying dietary mauipulation of triglycerides and phospholipids in rabbits (lo), rats (14) and pigs (6). Fatty acid composition of triglycerides and phospholipids was not affected by the dietary supplementation with a-tocopheryl acetate (Table 2 and 3). In rabbits fed the diet supplemented with a-tocopheryl acetate, the a-tocopherol concentration in liver was approximately 100% greater than in tissue from rabbits fed the basal diet (Table 4). Dietary fat inchtsion or tat source had no effect on a-tocopherol concentration in hepatic tissue. Liver from rabbits fed a basal level of a-tocopheryl acetate were significantly more susceptible to iron-induced lipid oxidation than homogenates from rabbits fed diets supplemented levels (p
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TABLE 3 Main Fatty Acid Composition (means of g/l00 total fatty acids) of Liver Phospholipids 6om Rabbits Fed on Diets with no Added Fat (NF) 30 g/kg Added Olive Oil (0) or SunfIower Oil (S) with 10 or 200 mg/kg a-Tocopheryl Acetate DIETS * NF 0 c14:o Cl50 C16:O C16:l (n-7) c17:o Cl&O Cl&l (n-9) C18:l (n-7) C18:2 (n-6) C18:3 (n-3) C20:3 (n-3) C20:4 (n-6) C20:5 (n-3) C22:4 (n-6) C22:5 (n-3) C22:6 (n-3) C (n-3) C (n-6) C (n-9) C sat UI **

10 0.2 0.4 24.0 0.8 0.9 19.8 10.7 1. I 24.2 4.0 3.0 7.4 0.6 1.1 1.0 0.7 9.4 32.6 10.7 45.3 1.3

SD

probability of contrasts #

0.04 0.07 1.59 0.16 0.20 1.44 2.28 0.19 2.52 0.70 0.79 1.58 0.35 0.21 2.24 0.16 1.76 3.58 2.28 2.34 0.08

12345 NS NS NS NS 0.009 NS NS NS NS NS 0.014 0.001 NS 0.001 NS NS NS NS NS 0.031 NS 0.047 NS NS 0.039 NS 0.077 0.008 0.093 NS 0.011 NS NS 0.069 NS NS NS 0.001 NS 0.003 NS NS

FAT 200 0.2 0.4 23.5 0.8 1.0 20.4 10.5 1.1 24.5 3.8 2.9 7.7 0.4 1.1 1.0 0.7 8.7 33.3 10.5 45.5 1.3

10 0.2 0.4 21.7 0.7 1.0 20.3 14.3 1.1 24.3 3.9 3.2 6.6 0.2 0.8 0.8 0.5 8.6 31.7 14.3 43.6 1.2

S 200 0.2 0.4 21.3 0.9 1.0 19.9 14.0 1.2 25.2 4.1 3.5 6.3 0.2 0.7 0.8 0.5 9.0 32.2 14.0 42.8 1.3

10 0.2 0.4 21.8 0.7 1.1 23.6 8.8 1.1 26.9 3.3 2.6 7.0 0.2 1.1 0.8 0.6 7.4 35.0 8.8 47.0 1.2

200 0.2 0.4 22.4 0.7 1.0 23.4 9.0 1.1 26.5 3.1 2.4 7.4 0.1 1.1 0.8 0.6 6.9 35.0 9.0 47.4 1.2

NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS

NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS

NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS

*,#,** See Table 2

higher TBARS a&r 4 hours of incubation than hepatic homogenates from rabbits fed f&t enriched diets @
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TABLE 4 Effect of Dietary Fat and c+Tocopheryl Acetate Supplementation on a-Tocopherol concentration in Liver Tissue and on Iron-Inducted Lipid Oxidation in Liver Homogenates Incubated at 37“C for 4 Hours. DIETS *

SD

probability

of contrasts #

FAT

NT 0 10

S

10

200

200

10

200

a-Tocopherol

3.8

mmolkg hepatic tissue 8.3 5.6 8.6 4.9

9.4

4.04

NS

NS

0.009

NS

NS

Hours 0 1 2 3 4

0.6 1.3 2.4 4.2 6.4

nmoles MDAImg protein 0.4 0.3 0.4 0.3 0.8 0.5 0.6 0.7 1.0 0.7 0.7 0.9 1.3 0.8 0.7 1.1 2.0 1.0 0.8 1.7

0.3 0.5 0.6 0.7 0.9

0.25 0.41 0.68 1.08 1.55

0.032 0.005 0.001 0.001 0.001

NS NS NS NS NS

NS NS 0.018 0.005 0.002

NS NS 0.015 0.003 0.002

NS NS NS NS NS

123

45

*,# See Table 2

Dietaq inchkon of vegetable fats produced higher unsaturation of triglycerides as estimated by the unsatnration index. Thus, apparently a higher susceptiiility of animal tissnes to oxidation should be expected (16). However, several studies indicate that membrane-bound phospholipids are the sites where oxidative changes are initiated in animal tissues (17). In our expeximat, little differences in overall unsaturation was found in fatty acids of phospholipids (Table 3). However, it is remarkable that dietary administration of a fat source rich in (n-9) or (n-6) fatty acids produces a lower concentration of long chain highly unsaturated (n-3) &zty acids of the phospholipid &&ion (Table 4). This effect has been also observed by previous investigators and it is attriiuted to a metabolic competition between (n-6) and (n-3) &ty acids for phospholipid sites ( 18,19). Some reports indicate a direct relationship between long chain highly unsaturated (n-3) f&y acid content of phospholipids and oxidase activity in hepatic tissue (5). Moreover, previous research suggest that long chain highly unsaturated (n-3) f&y acids are particularly susceptible to lipid oxidation, and so little differences in the concentration of these f&y acids on phospholipids may be of critical importance in the development of oxidation. Hammer and Wills (20) observed higher susceptibii to lipid peroxidation of (n-3) fatty acids either as pure lipid or in tissues of rats fed fish oil as compared to rats fed corn oil. Eichenberger et al (21) reported that lipid peroxidation in membrane isolated, measured as TBAR!$ occurred mainly in (n-3) f&y acids containing five or six double bonds. LlAbbe et al. (22) and De Schrijver et al. (23) observed that urinary TBARS increase as soon as long chain (n-3) PUFA are substantially incorporated into body lipid at the expense of (n-6) PUFA. In our experiment, the dietary source of (n-3) &ty acid was linolenic acid, which had a similar concentration in all experimental diets. However, it is interesting to note a higher relative proportion of this f&y acids in non fit-enriched diets (5.84 vs 2.58-2.76%). This is due to the relative higher proportion of linolenic acid in natural ingredients apart from the added fit. Inction of dietary fit sources rich in oleic or linoleic acid but with low levels of linolenic acid does not mod.@ the concentration of this fatty acid in

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diets, but decreases its relative proportion The higher concentration of (n-3) fatty acids in liver phospholipid of NF animals may be explained by diIIerent relative proportion of tatty acid classes in the diet and the competition for deposition sites in the phospholipid. However, some other factors may also be involved in differences in the susceptibility to oxidation of animalsreceivin g diets enriched or not-enriched in fat, like differences in the concentration of prooxidants such as oxygen or transition metals, presence of antioxidants, concentration of reducing agents, etc (6). In summary, inchrsion of oils rich in oleic or linoleic fatty acids to rabbit diets modi6es liver f&y acid composition and reduces lipid oxidation in rabbits. DiIIiiences in the susceptibihty of tissues to oxidation may be explained in part by difhiences in the proportion of (n-3) fatty acids iu the phospholipids.

ACKNOWLEDGEMENT This work was supported in part by a grant from Comision Interministerial de Ciencia y Tecnologia (ALI95-0344). Ho&ran-La Roche (Basel, Switzerland) provided us with free cc-tocopheryl acetate.

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