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Heated canola oil and oxidative stress in rats
NUTRITION RESEARCH, Vol. 8, pp. 673-684, 1988 0271-5317/88 $3.00 + .00 Printed in the USA. Copyright (c) 1988 Pergamon Press plc. All rights reserved.
HEATED CANOLA OIL AND OXIDATIVE STRESS IN RATS Tuck
So Kok,
B.Sco, Peter G. Harris,
B.Sc., and J.C. A l e x a n d e r ,
Department of Nutritional Sciences, College of Biological University of Guelph, Guelph, Ontario NIG 2WI
Ph.D.
Science,
ABSTRACT C a n o l a oils w e r e h e a t e d in the l a b o r a t o r y for 72 h o u r s at 180~ with three periods of eight-hour aeration. These oils were included in the diet at a level of 15% and were fed ad libitum to rats for 4 weeks. The effects of the diets on g r o w t h - r e l a t e d parameters and on s e v e r a l h e p a t i c e n z y m e s and b i o m o l e c u l e s a s s o c i a t e d w i t h the g l u t a t h i o n e r e d o x cycle w e r e investigated. F e e d i n g of o x i d i z e d oils r e s u l t e d in no a p p r e c i a b l e d e c r e a s e in body weight gain or food intake. Fat absorption was lower in all o x i d i z e d oil groups. The p e r c e n t a g e of lipid in livers of rats fed o x i d i z e d oils was not s i g n i f i c a n t l y changed, but a m o u n t s of the fatty acids C18:2, C18:3, and C20:4 in the o x i d i z e d groups were substantially different from the fresh oil groups except for the trace a m o u n t of C18:3 in the c o r n oil. Liver w e i g h t s w e r e increased in rats fed oxidized oil. The activities of the hepatic enzymes glutathione peroxidase and glucose-6-phosphate dehydrogenase in the o x i d i z e d oil groups were decreased n u m e r i c a l l y w h i l e that of catalase was significantly increased. There were no changes in the activity of glutathione reductase, or in the c o n c e n t r a t i o n s of h e p a t i c glutathione, m a l o n a l d e h y d e or cytosolic protein. KEY WORDS: heated fats, canola oil, enzymes,
glutathione
INTRODUCTION
W h e n oil is h e a t e d at f r y i n g t e m p e r a t u r e s for a c o n s i d e r a b l e p e r i o d of time in the p r e s e n c e of air, o x i d a t i v e and t h e r m a l d e c o m p o s i t i o n m a y take p l a c e w i t h the f o r m a t i o n of v o l a t i l e (VDP) a n d n o n v o l a t i l e (NVDP) decomposition products, some of which in excessive amounts may be harmful to human health (I). These authors identified about of 200 compounds in the VDP fraction. Many of them were of known toxic properties, but their amounts were m o s t l y at p p m levels, and in the n o r m a l course of h e a t i n g u s u a l l y w o u l d not r e m a i n in the oil. The NVDP, on the other hand, c o n t a i n m o n o m e r i c , d i m e r i c and polymeric compounds (2) and these can accumulate in the thermally oxidized oils. Some workers have shown that this fraction contains potentially harmful derivatives of fatty acids (3,4).
Correspondence should be addressed to: Dr. J.C. Alexander, Dept. of Nutritional Sciences, University of Guelph, Guelph, Ont. NIG 2WI
673
674
T.S. KOK et al.
The b i o l o g i c a l e f f e c t s of h e a t e d fats h a v e b e e n of i n t e r e s t for m a n y years. One a p p r o a c h to a s s e s s these e f f e c t s w a s to i s o l a t e p o t e n t i a l toxic components by means of vacuum distillation, urea adduct formation or column c h r o m a t o g r a p h y , a n d t h e n f e e d t h e m to rats. This r e s u l t e d in r e d u c e d food intake, depressed growth, diarrhea, histological changes in various tissues, a n d in s o m e c a s e s d e a t h (5,6). On the o t h e r hand, the f e e d i n g of w h o l e o x i d i z e d oil as p a r t of a n u t r i t i o n a l l y b a l a n c e d d i e t f a i l e d to i n d u c e s i g n i f i c a n t d e l e t e r i o u s c h a n g e s in the a n i m a l s ; w i t h this a p p r o a c h it w a s f o u n d t h a t the s u s p e c t e d t o x i c c o m p o n e n t s m a y not be p r e s e n t in s u f f i c i e n t quantity to induce gross harmful effects (7,8). Most of these toxicological investigations on the effects of heated fats have been general and insensitive to m i l d toxicity. In the present study, the rats were fed whole oxidized oil as part of a balanced diet; and in addition to growth related parameters, functional changes associated with normally seen e n l a r g e d l i v e r s w e r e studied. Various enzymes and components of the glutathione redox cycle were investigated. This s t u d y c o n s i d e r s the possibility of altered metabolism in rats fed oxidized oil, and uses it as a criterion for assessing xenobiotic stress.
MATERIALS AND METHODS
Preparation of oil samples and diets T w o t y p e s of d e g u m m e d c a n o l a oils, w i n t e r O n t a r i o a n d w e s t e r n C a n a d a canola, w e r e o b t a i n e d f r o m C a n a d i a n V e g e t a b l e Oil P r o c e s s i n g , H a m i l t o n , Ontario. Mazola corn oil was used as a control. T w o l i t e r s of e a c h of the three o i l s in a f i v e - l i t e r s t a i n l e s s steel beaker were placed in a heating mantle and heated continuously at 180~ for 72 hr. A stainless steel motor-driven stirrer was adjusted to a height of 3 cm below the oil surface and the speed was regulated to draw a thin funnel of air into the oils for 8 hr daily. U p o n c o m p l e t i o n of h e a t i n g , the oils w e r e p o u r e d into p l a s t i c c o n t a i n e r s w h i c h w e r e f l u s h e d w i t h n i t r o g e n , s e a l e d tightly and refrigerated at 5~ in the dark until needed.
I).
The oils w e r e m i x e d into a b a l a n c e d b a s a l diet at a l e v e l of 15% (Table A fat-free diet also was prepared. All rations were stored in a freezer. TABLE i Composition of Basal Diet Ingredients
%
Sucrose
48.5
Casein
27.0
Cellu flour
5.0 1
Salt mixture
3.5 2
Vitamin mixture Test oil
1.0 15.0
~Williams-Briggs Modified, Teklad Test Diets, Madison, WI V i t a m i n Mix, A.O.A.C., Teklad test diets, Madison, WI
HEATED FATS AND OXIDATIVE STRESS
675
Characterization of sample oils These oils w e r e c h a r a c t e r i z e d to d e t e r m i n e the d e g r e e of abuse as a result of heating. Peroxide, acid, iodine and s a p o n i f i c a t i o n values w e r e d e t e r m i n e d u s i n g the O f f i c i a l M e t h o d s of A n a l y s i s of the A s s o c i a t i o n of Official Analytical Chemists (9). The carbonyl value was determined by the m e t h o d of B h a l e r a o et al. (I0). Fatty acid c o m p o s i t i o n of the oils was determined by gas liquid chromatography (9). Experiment I - Weanling male Wistar rats in the weight range of 55-60 g were obtained from Charles River Breeding Laboratories, St. Constant, Quebec. They w e r e h o u s e d i n d i v i d u a l l y in s t a i n l e s s steel s c r e e n - b o t t o m cages. The temperature of the room was maintained at 24~ and the relative humidity at 55%. A 12-hr p h o t o p e r i o d was kept. The a n i m a l s w e r e a c c l i m a t i z e d for 3 days, during which time Purina rat chow was fed. They were assigned randomly to 7 groups of 12 rats on the basis of weight. One group was continued on the rat c h o w diet for 2 w e e k s and then c h a n g e d to the f a t - f r e e diet prior to the fecal collection and fat analyses for the absorption studies. The remaining 6 groups were assigned randomly to the experimental diets (fresh and oxidized corn oil, winter Ontario eanola oil, and western Canada canola oil). The animals were fed twice a week and provided with water ad libitum for four weeks. Feed intake and w e i g h t change w e r e recorded. The feces w e r e collected during the last ten days of the experiment. Blood then was obtained from the rats under light ether anesthesia by means of heart puncture before they were killed by decapitation. The liver, kidneys, heart and spleen were r e m o v e d quickly, d r i e d w i t h paper towels, w e i g h e d and f r o z e n in l i q u i d nitrogen. Fecal and liver lipid were extracted with chloroform/methanol (2:l,v/v). The fat a b s o r p t i o n rate w a s c a l c u l a t e d (II). L i v e r lipid c o n t e n t was determined gravimetrically, and was analyzed for its fatty acid composition. A Quick's thromboplastin time assay (12) was performed on the blood. Experiment 2 - For the second phase of the study six more groups of rats fed the above rations were kept under conditions similar to those above. Each day d u r i n g the f o u r t h and fifth w e e k s of feeding, rats w e r e k i l l e d by c e r v i c a l dislocation and decapitation. The livers were excised quickly, blotted dry, weighed, then rinsed with and immersed in ice-cold 0.85% NaCI solution. The activities of the enzymes glutathione peroxidase (13), glutathione reductase (14) and c a t a l a s e (15) of the c y t o s o l i c (105 000 g) f r a c t i o n w e r e a s s a y e d u s i n g the fresh tissues. The c y t o s o l i c p r o t e i n c o n c e n t r a t i o n also was determined (16). Malonaldehye concentration was measured on the frozen liver using Ohkawa's method (17). E x p e r i m e n t 3 - For the third phase of the study a n o t h e r series of rats was obtained and treated similarly. The activity of the enzyme glucose-6-phosphate dehydrogenase (18) and the c e l l u l a r g l u t a t h i o n e c o n c e n t r a t i o n (19) w e r e determined. Statistical analysis The data w e r e a n a l y s e d s i g n i f i c a n c e (20).
using ANOVA
and Tukey's
test at a 5% level of
RESULTS
Characteristics of the test oils are given in Table 2, and the fatty acid c o m p o s i t i o n s are in Table 3. The h e a t e d oils w e r e m o r e v i s c o u s and darker
676
T.S. KOK et al.
than the fresh oils. The carbonyl, peroxide, acid and s a p o n i f i c a t i o n values were increased, and the iodine values were decreased as a result of 72 hr of heating. The oleic (C18:1) and linolenic acid (C18:3) contents of the fresh canola oils were significantly higher than for fresh corn oil. Nevertheless, as a result of heating each of the oils the contents of the unsaturated fatty acids C18:2 and C18:3 dropped markedly, and this was c o m p e n s a t e d for by increases in C16:0, C18:0 and C18:1. The total of the s e l e c t e d fatty acids was lower for the western canola oil.
TABLE 2 Characteristics of Oils Corn
Ontario Canola
Western Canola
Fresh
Oxidized
Fresh
Oxidized
Fresh
Oxidized
Carbonyl value (meq/kg)
0
46.4
0
145.6
0
129.8
Peroxide value (meq/kg)
1.8
7.4
3.6
7.2
1.7
7.4
Acid value
0.2
1.0
1.0
1.9
1.0
1.7
Iodine value
183.2
116.0
110.5
87.3
ii0.0
89.5
Saponification value
187.1
194.8
185.2
198.2
184.1
196.1
TABLE 3 Fatty Acid Composition of Oils I Corn
Fresh 16:0 18:0 18:1 18:2 18:3 22:0 22:1
Ontario Canola
Oxidized
Fresh
Oxidized
Western Canola
Fresh
Oxidized
11.3 2.0 25.2 57.5 0.8 0.3 0
11.6 2.2 35.6 46.7 0.4 0.2 0.i
5.4 2.4 59.7 19.5 8.1 0.4 0.6
7.0 2.9 68.6 11.9 2.4 0.4 0.8
4.3 2.0 57.6 17.0 6.8 0.3 0.4
5.8 2.6 62.8 Ii.0 2.6 0.3 0.6
97.1
96.8
96.1
94.0
88.4
85.7
IResults are expressed as the percentage composition of selected identified fatty acid methyl esters so the total is less than 100%.
HEATED FATS AND OXIDATIVE STRESS
677
Body weight gain, feed intake, feed efficiency and relative organ weights are l i s t e d in T a b l e 4. A l l the diets w e r e e a t e n well, a n d no g r o s s p h y s i c a l anomalies were observed throughout the study. The body weight gains, over the p e r i o d of four w e e k s w e r e s i m i l a r in all the groups, b u t f e e d i n t a k e was n u m e r i c a l l y h i g h e r w h e n o x i d i z e d fats w e r e fed. As a result, the feed e f f i c i e n c y of a n i m a l s w i t h i n the g r o u p s fed h e a t e d c a n o l a oils w a s s l i g h t y lower. The relative liver weights were 15% to 30% higher for the heated oil groups, w h i l e the k i d n e y w e i g h t s w e r e h i g h e r o n l y in the o x i d i z e d O n t a r i o canola group. The relative heart and spleen weights were not affected.
TABLE 4 Results from Feeding Study 1'2 Corn
Fresh
Oxidized
Ontario Canola
Western Canola
Fresh
Fresh
Oxidized
Oxidized
Weight gain (g)
195 a +15
205 a +14
210 a +19
200 a +13
201 a +24
197 a +14
Feed intake (g)
437 a +31
449 a +29
458 a +38
475 a +30
449 a +37
458 a +33
Feed efficiency (%)
44.7 a +2.3
45.6 a +1.2
45.8 a +1.5
42.2 b +1.6
44.8 a +2.7
43.1 b +1.4
Liver
4.20 b +0.28
5.42 a +0.36
4.45 b +0.26
5.80 a +0.46
4.68 b +0.29
5.34 a +0.49
Kidney
0.85 b +0.08
0.87 ab +0.03
0.84 b +0.04
0.96 a +0.09
0.84 b +0.05
0.86 b +0.04
Heart
0.33 a +0.01
0.35 a +0.03
0.34 a +0.02
0.37 a +0.02
0.37 a +0.03
0.36 a +0.01
Spleen
0.32 a +0.03
0.33 a +0.05
0.29 a +0.03
0.35 a +0.04
0.35 a +0.03
0.32 a +0.04
iMeans in a row with the same superscript are not significantly different (P<0.05). Relative organ weights as as percentage of body weights
F e c a l p e l l e t s c o l l e c t e d f r o m the g r o u p s fed h e a t e d oils w e r e larger, d a r k e r and m o r e moist; also the dry m a t t e r o u t p u t p e r d a y w a s h i g h e r (Table 5). However, steatorrhea was not excessive. The fat absorption in the groups fed heated oils ranged b e t w e e n 82% to 87% and was significantly lower than for the groups fed fresh oils (92% to 93%).
T.S. K0K et al.
678
TABLE 5 Results of Fat Absorption Study I~2 Corn
Ontario Canola
Fresh
Oxidized
Fresh
Oxidized
Feed intake (g/d)
19.0 b
19.6 ab
19.1 b
21.4 a
Dried feces
1.49 d
(g/d)
Fat absorption Liver lipid (%)
(%) 93.1 a 6.3 a +0.8
1.77 be 86.6 b 5.4 ab +0.5
1.48 d 92.2 a 5.7 ab +0.7
2.38 a
Western Canola
Fresh
Oxidized
20.2 ab I~
cd
20.0 ab 1.93 b
81.8 c
91.9 a
86.6 b
5.0 b +0.4
5.0 b +0.6
5.0 b +0.7
iMeasured over a period of I0 days 2Means in a row with the same superscript are not significantly different (P
Liver lipid determined gravimetrically ranged between 5.0% to 6.3%; the group fed fresh corn oil diet had the highest liver fat level. Nevertheless, there w e r e no clear d i f f e r e n c e s in liver fat c o n t e n t b e t w e e n the h e a t e d and fresh c a n o l a oils. The p r e d o m i n a n t liver f a t t y acids w e r e C16:0, C18:0, C18:1, C18:2 and C20:4 m a k i n g up 90% of the total (Table 6). However, the a m o u n t s of C18:2 c o n s i s t e n t l y d e c l i n e d for the groups fed h e a t e d oils, and this also w a s the case for C18:3 and the h e a t e d c a n o l a oil groups. On the other hand, C20:4 was i n c r e a s e d s t a t i s t i c a l l y w i t h all h e a t e d fats. This h i g h e r level of a r a c h i d o n i c acid c o n f i r m s an e a r l i e r report for C20:4 in n e u t r a l liver lipids (21). There were no differences in the Quick's thromboplastin times or in the c y t o s o l i c p r o t e i n c o n t e n t s for all the groups. Also, the total g l u t a t h i o n e contents, reduced and oxidized, in the groups fed heated oils, as determined by the Ellman's m e t h o d (19) were only s l i g h t l y h i g h e r than for the ones fed fresh oils (Table 7). The concentrations of malonaldehyde in frozen livers of rats fed oxidized oils were elevated. The a c t i v i t i e s of g l u t a t h i o n e p e r o x i d a s e of groups fed oxidized oils were diminished w h e n c o m p a r e d to the ones fed fresh o i l s , r e a c h i n g statistical significance only in the c a n o l a o i l - f e d groups (Table 8). Likewise significant d i f f e r e n c e s w e r e not f o u n d in the a c t i v i t i e s of g l u t a t h i o n e r e d u c t a s e a m o n g the groups. The activity of glucose-6-phosphate (G-6-P) dehydrogenase was diminished only in the oxidized Ontario canola oil group compared to the fresh fat control, but the oxidized corn oil group was significantly lower than the oxidized canola oil groups. Catalase activities of the oxidized oil groups were elevated when compared to the fresh oil.
HEATED FATS AND OXIDATIVE STRESS
679
TABLE 6 Fatty Acid Composition of Total Liver Lipid Corn
Fresh 14:0 16:0 16:1 18:0 18:1 18:2 18:3 20:1 20:2 (n6) 20:3 (n6) 20:4 22:0 22:6 24:0 24:1
Ontario Canola
Oxidized
0.6 31.5 1.2 25.8 14.3 13.0 a 0. i c 0.2 0.6 0~
05 29 5 13 29 7 13 8 9 Ib 0 Ic 0.2 0.3 0.5
6.7 d 0.3
9.6 c 0.3
0.4 0.9 0.i
0.6 1.0 0.2
Fresh
Western Canola
Oxidized
0.5 18.8 2.1 17.1 27.8 9~ 8b 0.8 a 0.4 0.3 0.9 13.7 b 0.2 4.1 0.3 0.3
I
0.5 21.7 2.4 17.7 26,3 5.4 c 0.I c 0.i 1.3 1.2 16.4 a 0.2 4.2 0.4 0.3
Fresh
Oxidized
0.5 17.8 1.7 18.4 27.2 8.9 b 0.5 b 0~ 0.3 0.9 14.7 b 0.2 4.4 0.4 0.4
0.4 20.3 2.4 17.5 26.8 6.3 c 0.2 c 0.2 0.8 1o2 16.6 a 0.2 4.3 0.4 0.3
IResults are expressed as percentage of total lipid based on five determinations, and means in a row with the same superscript are not significantly different (P<0.05).
TABLE 7 Prothrombin Time, Hepatic Glutathione, Cyto@olic Protein and Malonaldehyde Concentrations Corn
Ontario Canola
Western Canola
Fresh
Fresh
Fresh
Oxidized
17 a
18 a
20 a
Glutathione (umol/wet g)
6.6 b ~0.6
7.0 a •
Cytosolic protein (mg/wet g)
i16 a +14
Malonaldehyde (nmol/wet g)
167 c ~31
Thromboplastin time (seconds)
Oxidized
Oxidized
17 a
17 a
16 a
6.4 b !0.6
6.9 a ~0.5
6.7 b !0.5
7.1 a !0.4
I12 a +13
i02 a +15
I06 a +II
i06 a +9
i13 a +ii
243 a •
205 b ~31
226 a •
209 b •
228 a +34
IMeans in a row with the same superscript are not significantly d i f f e r e n t (P
680
T.S. KOK et al.
TABLE 8 Enzymatic Activities I Corn
Fresh
Ontario Canola
Oxidized
Fresh
Oxidized
Western Canola
Fresh
Oxidized
Glutathione peroxidase 2
6.9 abe •
5.8 c ~1.3
7.7 ab •
5.4 c •
8.0 a Z2.0
6.0 be ~1.3
Glutathione reductase 2
5.4 a +0.8
5.4 a +0.5
4.9 a +0.5
5.4 a +0.3
4.9 a +0.8
5.3 a +0.3
G-6-P dehydrogenase 3
4.2 bc •
2.9 c !l.0
6.4 a •
4.6 b ii.2
5.4 ab ii.3
4.8 b il.0
49.1 c +10.6
59.7 b +8.9
37.0 d +10.8
72.4 a +13.4
36.8 d +7.9
69.4 a +14.1
Catalase 4
iMeans in a row with the same superscript are not signficantly different (P<0.05). 2umol NADPH oxidized/min/wet g 3umol NADPH reduced/min/wet g 4mmol H202 decomposed/min/wet g
DISCUSSION
There are many methods for measuring the degree of abuse in heated oils (22,23). Some of the ones employed in this study are among the more common. The levels of peroxides, carbonyl compounds and free acids have been shown to increase as a r e s u l t of heating. P e r o x i d e s (or h y d r o p e r o x i d e s ) are p r i m a r y products of fat oxidation and are not stable; they normally are degraded into c a r b o n y l c o m p o u n d s and acids. Iodine v a l u e is a m e a s u r e of the degree of unsaturation, and in all the heated oils, the iodine values were much l o w e r . This was r e f l e c t e d in the fatty acid p r o f i l e s of these oils (Table 3 ) r e s u l t i n g in less of C18:2 and C18:3 w h i c h are m o r e r e a d i l y oxidized; The p e r c e n t a g e s of the s e l e c t e d 7 m o s t p r o m i n e n t fatty acids did not add up to i00. In addition, the o x i d i z e d oils, due to shifts in c o m p o s i t i o n and production of unidentified polar derivatives, had lower totals. In a number of i n s t a n c e s the corn oil control and each of the c a n o l a oils p r o d u c e d d i f f e r e n t responses, and these r e f l e c t the fact that they are d i s t i n c t products with different genetic origins. However, all the heated fats were less w e l l a b s o r b e d due to f o r m a t i o n of o l i g o m e r i c d e r i v a t i v e s w h i c h are h y d r o l y z e d poorly (2). The distinctive characteristics of canola oils among vegetable oils are their r e l a t i v e l y h i g h C18:1 and C18:3 contents. F r o m the v i e w p o i n t of food oxidation, t h e i m p o r t a n t fatty acids are the u n s a t u r a t e d ones, w h o s e susceptibility to oxidation increases greatly with degree of unsaturation. It is t h e r e f o r e b e l i e v e d by m a n y i n v e s t i g a t o r s that the h i g h e r the c o n t e n t of unsaturated fatty acids, especially PUFA in fat, the more readily are toxic substances formed due to thermal degradation (24). Nevertheless, this is not c e r t a i n in v i e w of a r e p o r t that a h i g h m o n o e n e c o n t e n t can lead to more t o x i c i t y (25).
HEATED FATS AND OXIDATIVE STRESS
681
P e r o x i d e s and e a r b o n y l c o m p o u n d s are o f t e n i m p l i c a t e d in c a u s i n g deleterious effects. Peroxides are normally present at low levels in heated fats, and lymphatic absorption is minimal, whereas carbonyl compounds such as 1 2 - k e t o oleic a c i d p r e s e n t at a h i g h e r level has a p r o o x i d a n t a c t i o n on unsaturated fatty acids and may promote lipid peroxidation in vivo (26). It is known that nonvolatile oxidized products are absorbed into the body, and it has been reported that these products such as dimerie acids, monomeric acids and c y c l i c m o n o m e r i c acids can be r e c o v e r e d f r o m the l y m p h a t i c (27) and h e p a t i c lipids (28). W h e n a d m i n i s t e r e d in large amounts, the n o n v o l a t i l e oxidized fraction, or some components of it were deleterious to heart cells in culture system (3), or to the whole animal system (25). It is p r o p o s e d that a n i m a l s fed such m a t e r i a l s at a low c o n c e n t r a t i o n c o u l d u n d e r g o an a l t e r e d x e n o b i o t i c m e t a b o l i s m . This w a s e v i d e n c e d in our study by the change in a c t i v i t y of some enzymes, and c o n c e n t r a t i o n of s o m e components of the glutathione redox cycle. The feeding of diets with 15% whole oxidized oil in the present four-week study did not show any depressed growth or obvious steatorrhea, as others have i n d i c a t e d (7,8,29). C o n s e q u e n t l y , the i n a b i l i t y to o b s e r v e i n c i d e n c e s of certain diseases, and differences in longevity in some studies has led to the conclusion that heated fats are always safe for human consumption. The toxic materials may have been present in too low a concentration, or the diet used was so well balanced nutritionally to offset any obvious deleterious effects. With lower protein and vitamin contents in diets, however, heated oils were found to be more deleterious (30). The protective role of glutathione against xenobioties by virtue of its a b i l i t y to c o n j u g a t e w i t h and to f a c i l i t a t e their e x c r e t i o n has b e e n w e l l documented (31). The pool of glutathione in the cells is predominantly in a reduced state unless challenged with stress factors. This dynamic supply of glutathione can be easily depleted and replenished (32). It is maintained by a b a t t e r y of e n z y m e s such as g l u t a t h i o n e r e d u c t a s e and G - 6 - P d e h y d r o g e n a s e w h i c h help to p r o v i d e a s o u r c e of NADPH. In our 4 - w e e k study, the h e p a t i c glutathione concentration was increased, but not to the same degree as found by Izaki et al. (29). T h e i r study was n e v e r t h e l e s s m u c h longer (14 weeks). These authors speculated based on earlier experiments that the reason for the increase in glutathione was that a secondary oxidizing agent produced in the liver probably consumed it, a n d an o v e r s h o o t i n g of the r e s y n t h e s i s subsequently occurred. Liver enlargement, frequently seen in rats fed oxidized oils, normally is accompanied by the increased activity of enzymes resulting from proliferation of the s m o o t h e n d o p l a s m i c r e t i c u l u m (33). In the c u r r e n t study c y t o s o l i c p r o t e i n c o n c e n t r a t i o n w a s not affected, b u t the a c t i v i t i e s of the h e p a t i c e n z y m e s g l u t a t h i o n e p e r o x i d a s e and G-6-P dehydrogenase in rats fed oxidized fats were decreased while that of catalase was increased. The cause for the i n c r e a s e d or d i m i n i s h e d a c t i v i t y in these e n z y m e s is not known; but g l u t a t h i o n e p e r o x i d a s e and c a t a l a s e have b e e n c r e d i t e d w i t h the a b i l i t y to detoxify peroxides and hydroperoxides, and to halt lipid peroxidation (34,35). Malonaldehyde, an indicator of lipid autoxidation, also is seen commonly in the tissue of animals fed heated oils and therefore components in these oils m a y be c o n s i d e r e d as p r o m o t e r s of lipid p e r o x i d a t i o n . We have b e e n able to c o n f i r m the p r e s e n c e of m a l o n a l d e h y d e in the liver of rats fed t h e r m a l l y oxidized fats. The alterations in enzyme activities except for G-6-P dehydrogenase were less p r o n o u n c e d in the corn oil fed groups. This c o i n c i d e d w i t h the l o w e r c a r b o n y l and a c i d values, and could also be r e l a t e d to the fatty acid
682
T.S. KOK et al.
composition. Since heating is k n o w n to destroy nutrients, another factor w h i c h should not be disregarded is the tocopherol content in the oil. Tocopherol has been s h o w n to be mostly destroyed by heat under the present procedures, but a higher tocopherol content as in corn oil might provide some protection of the unsaturated fatty acids against oxidation. In conclusion, the feeding of oxidized oils at a level of 15% for 4 weeks did not cause a decrease in body weight gain or feed intake. It nevertheless resulted in changes in glutathione content, and in the activity of some of the hepatic enzymes.
ACKNOWLEDGEMENTS
This work was supported by the Natural Sciences and Engineering Research Council of Canada, and the Ontario Ministry of Agriculture and Food.
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Chang SS, Peterson RJ, Ho CT. Chemical reactions involved in the deepfat frying of foods. J. Am. Oil Chem. Soc. 1978; 55:718-27.
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N a k a m u r a M, Tanaka H, Hattori Y, W a t a n a b e M. Biological effects of autoxidized safflower oils. Lipids 1973; 8:566-72.
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Augur V, R o l l m a n HS, Deuel HJ. The effect of crude lecithin on the coefficient of digestibility and the rate of absorption of fat. J. Nutr. 1 9 4 7 ; 3 3 : 177-86.
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HEATED FATSAND OXIDATIVE STRESS
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Sudhof H. T h r o m b o p l a s t i n time (Prothrombin time). In: Bergmeyer HU, ed. Methods of Enzymatic Analysis, New York: Academic, 1963: 908-12.
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Paglia DE, Valentine NW. Studies on the quantitative and qualitative c h a r a c t e r i z a t i o n of erythrocyte glutathione peroxidase. J. Lab. Clin. Med. 1967; 70: 158-69.
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Horn HD. Glutathione reductase. In: B e r g m e y e r HU, ed. Enzymatic Analysis, New York: Academic, 1963: 875-9.
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Aebi HE. Catalase. In: B e r g m e y e r HU, ed. Methods of Enzymatic Analysis, 3rd Ed., Vol. III, Deerfield Beach, Florida: Verlag Chemie, 1983: 273-86.
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Lowry OH, Rosebrough NJ, Farr AL, Randall WJ. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 1951; 193: 265-75.
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Ohkawa H, Ohishi N, Yagi K. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal. Biochem. 1979; 95: 351-8.
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Lohr GW, Waller HD. Glucose-6-phosphate dehydrogenase. In: Bergmeyer HU, ed. Methods of Enzymatic Analysis, New York: Academic, 1963: 74451.
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Griffith OW. Glutathione and glutathione disulfide. In: Bergmeyer HU, ed. Methods of Enzymatic Analysis, 3rd Ed., Vol. VIII, Deerfield Beach, Florida: Verlag Chemic, 1983: 521-9.
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Steel RGD, Torrie JH. Principles and Procedures of Statistics. York: McGraw Hill, 1960.
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Frltsch CW. M e a s u r e m e n t of frying deterioration: Am. Oil Chem. Soc. 1981; 58: 272-4.
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Gray Jl. M e a s u r e m e n t of lipid oxidation: A review. J. Amer. Oil Chem. Soc. 1978; 55: 539-46.
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Le Cierc AM, Ramel P, D u m a i n J, F a u c q u e m b e r g u e D. Behavior of fats during frying and controlled heating. Rev. Fr. Corps. Gras. 1966; 13: 175-83.
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Gabriel HG, Alexander JC, Valli VE. Nutritional and metabolic studies of distillable fractions from fresh and thermally oxidized corn oil and olive oil. Lipids 1978; 13:49-55.
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Y o s h i o k a M, Tachibana K, Kaneda T. Studies on the toxicity of the autoxidized oils. I. The fractionation of the toxic c o m p o u n d and its identification. Yukagaku. 1972; 21:316-21.
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Combe N, Constantin MJ, Entressangles B. L y m p h a t i c absorption of nonvolatile oxidation products of heated oils in the rat. Lipids 1981; 16:8-14.
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Causeret J, Potteau B, Grandgirand A. Study on physiopathological effects of ingestion of heated oils on the rat. Ann. Nutr. Alim. 1978; 32:483-97.
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Izaki Y, Y o s h i k a w a S, U c h i y a m a M. Effects of ingestion of thermally oxidized frying oil on peroxidative criteria in rats. Lipids 1984; 19:324-31.
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Hemans C, K u m m e r o w F, Perkins EG. Influence of protein and v i t a m i n levels on the nutritional value of heated fats for rats. J. Nutr. 1973; 103:1665-72.
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Reed DJ, Fariss MW. Glutathione depletion and susceptibility. Rev. 1984; 36:25S-33S.
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Tateishi N, Higashi T, Shinya S, Naruse A, S a k a m o t o Y. Studies on the regulation of glutathione level in rat liver. J. Biochem. 1974; 75:93103.
33.
Andia GA, Street JC. Dietary induction of hepatic microsomal enzymes by thermally oxidized fats. Agr. Food Chem. 1975; 23:173-7.
34.
Christophersen BO. F o r m a t i o n of monohydroxy-polyenic fatty acids from lipid peroxides by a glutathione peroxidase. Biochim. Biophys. Acta 1968; 164:35-46.
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Crane D, Masters C. On the role of catalase in the oxidation of tissue fatty acids. Arch. Biochem. Biophys. 1984; 229:104-11.
Accepted for publication D~cember 9, 1987.
Phar.
HEATED CANOLA OIL AND OXIDATIVE STRESS IN RATS Tuck
So Kok,
B.Sco, Peter G. Harris,
B.Sc., and J.C. A l e x a n d e r ,
Department of Nutritional Sciences, College of Biological University of Guelph, Guelph, Ontario NIG 2WI
Ph.D.
Science,
ABSTRACT C a n o l a oils w e r e h e a t e d in the l a b o r a t o r y for 72 h o u r s at 180~ with three periods of eight-hour aeration. These oils were included in the diet at a level of 15% and were fed ad libitum to rats for 4 weeks. The effects of the diets on g r o w t h - r e l a t e d parameters and on s e v e r a l h e p a t i c e n z y m e s and b i o m o l e c u l e s a s s o c i a t e d w i t h the g l u t a t h i o n e r e d o x cycle w e r e investigated. F e e d i n g of o x i d i z e d oils r e s u l t e d in no a p p r e c i a b l e d e c r e a s e in body weight gain or food intake. Fat absorption was lower in all o x i d i z e d oil groups. The p e r c e n t a g e of lipid in livers of rats fed o x i d i z e d oils was not s i g n i f i c a n t l y changed, but a m o u n t s of the fatty acids C18:2, C18:3, and C20:4 in the o x i d i z e d groups were substantially different from the fresh oil groups except for the trace a m o u n t of C18:3 in the c o r n oil. Liver w e i g h t s w e r e increased in rats fed oxidized oil. The activities of the hepatic enzymes glutathione peroxidase and glucose-6-phosphate dehydrogenase in the o x i d i z e d oil groups were decreased n u m e r i c a l l y w h i l e that of catalase was significantly increased. There were no changes in the activity of glutathione reductase, or in the c o n c e n t r a t i o n s of h e p a t i c glutathione, m a l o n a l d e h y d e or cytosolic protein. KEY WORDS: heated fats, canola oil, enzymes,
glutathione
INTRODUCTION
W h e n oil is h e a t e d at f r y i n g t e m p e r a t u r e s for a c o n s i d e r a b l e p e r i o d of time in the p r e s e n c e of air, o x i d a t i v e and t h e r m a l d e c o m p o s i t i o n m a y take p l a c e w i t h the f o r m a t i o n of v o l a t i l e (VDP) a n d n o n v o l a t i l e (NVDP) decomposition products, some of which in excessive amounts may be harmful to human health (I). These authors identified about of 200 compounds in the VDP fraction. Many of them were of known toxic properties, but their amounts were m o s t l y at p p m levels, and in the n o r m a l course of h e a t i n g u s u a l l y w o u l d not r e m a i n in the oil. The NVDP, on the other hand, c o n t a i n m o n o m e r i c , d i m e r i c and polymeric compounds (2) and these can accumulate in the thermally oxidized oils. Some workers have shown that this fraction contains potentially harmful derivatives of fatty acids (3,4).
Correspondence should be addressed to: Dr. J.C. Alexander, Dept. of Nutritional Sciences, University of Guelph, Guelph, Ont. NIG 2WI
673
674
T.S. KOK et al.
The b i o l o g i c a l e f f e c t s of h e a t e d fats h a v e b e e n of i n t e r e s t for m a n y years. One a p p r o a c h to a s s e s s these e f f e c t s w a s to i s o l a t e p o t e n t i a l toxic components by means of vacuum distillation, urea adduct formation or column c h r o m a t o g r a p h y , a n d t h e n f e e d t h e m to rats. This r e s u l t e d in r e d u c e d food intake, depressed growth, diarrhea, histological changes in various tissues, a n d in s o m e c a s e s d e a t h (5,6). On the o t h e r hand, the f e e d i n g of w h o l e o x i d i z e d oil as p a r t of a n u t r i t i o n a l l y b a l a n c e d d i e t f a i l e d to i n d u c e s i g n i f i c a n t d e l e t e r i o u s c h a n g e s in the a n i m a l s ; w i t h this a p p r o a c h it w a s f o u n d t h a t the s u s p e c t e d t o x i c c o m p o n e n t s m a y not be p r e s e n t in s u f f i c i e n t quantity to induce gross harmful effects (7,8). Most of these toxicological investigations on the effects of heated fats have been general and insensitive to m i l d toxicity. In the present study, the rats were fed whole oxidized oil as part of a balanced diet; and in addition to growth related parameters, functional changes associated with normally seen e n l a r g e d l i v e r s w e r e studied. Various enzymes and components of the glutathione redox cycle were investigated. This s t u d y c o n s i d e r s the possibility of altered metabolism in rats fed oxidized oil, and uses it as a criterion for assessing xenobiotic stress.
MATERIALS AND METHODS
Preparation of oil samples and diets T w o t y p e s of d e g u m m e d c a n o l a oils, w i n t e r O n t a r i o a n d w e s t e r n C a n a d a canola, w e r e o b t a i n e d f r o m C a n a d i a n V e g e t a b l e Oil P r o c e s s i n g , H a m i l t o n , Ontario. Mazola corn oil was used as a control. T w o l i t e r s of e a c h of the three o i l s in a f i v e - l i t e r s t a i n l e s s steel beaker were placed in a heating mantle and heated continuously at 180~ for 72 hr. A stainless steel motor-driven stirrer was adjusted to a height of 3 cm below the oil surface and the speed was regulated to draw a thin funnel of air into the oils for 8 hr daily. U p o n c o m p l e t i o n of h e a t i n g , the oils w e r e p o u r e d into p l a s t i c c o n t a i n e r s w h i c h w e r e f l u s h e d w i t h n i t r o g e n , s e a l e d tightly and refrigerated at 5~ in the dark until needed.
I).
The oils w e r e m i x e d into a b a l a n c e d b a s a l diet at a l e v e l of 15% (Table A fat-free diet also was prepared. All rations were stored in a freezer. TABLE i Composition of Basal Diet Ingredients
%
Sucrose
48.5
Casein
27.0
Cellu flour
5.0 1
Salt mixture
3.5 2
Vitamin mixture Test oil
1.0 15.0
~Williams-Briggs Modified, Teklad Test Diets, Madison, WI V i t a m i n Mix, A.O.A.C., Teklad test diets, Madison, WI
HEATED FATS AND OXIDATIVE STRESS
675
Characterization of sample oils These oils w e r e c h a r a c t e r i z e d to d e t e r m i n e the d e g r e e of abuse as a result of heating. Peroxide, acid, iodine and s a p o n i f i c a t i o n values w e r e d e t e r m i n e d u s i n g the O f f i c i a l M e t h o d s of A n a l y s i s of the A s s o c i a t i o n of Official Analytical Chemists (9). The carbonyl value was determined by the m e t h o d of B h a l e r a o et al. (I0). Fatty acid c o m p o s i t i o n of the oils was determined by gas liquid chromatography (9). Experiment I - Weanling male Wistar rats in the weight range of 55-60 g were obtained from Charles River Breeding Laboratories, St. Constant, Quebec. They w e r e h o u s e d i n d i v i d u a l l y in s t a i n l e s s steel s c r e e n - b o t t o m cages. The temperature of the room was maintained at 24~ and the relative humidity at 55%. A 12-hr p h o t o p e r i o d was kept. The a n i m a l s w e r e a c c l i m a t i z e d for 3 days, during which time Purina rat chow was fed. They were assigned randomly to 7 groups of 12 rats on the basis of weight. One group was continued on the rat c h o w diet for 2 w e e k s and then c h a n g e d to the f a t - f r e e diet prior to the fecal collection and fat analyses for the absorption studies. The remaining 6 groups were assigned randomly to the experimental diets (fresh and oxidized corn oil, winter Ontario eanola oil, and western Canada canola oil). The animals were fed twice a week and provided with water ad libitum for four weeks. Feed intake and w e i g h t change w e r e recorded. The feces w e r e collected during the last ten days of the experiment. Blood then was obtained from the rats under light ether anesthesia by means of heart puncture before they were killed by decapitation. The liver, kidneys, heart and spleen were r e m o v e d quickly, d r i e d w i t h paper towels, w e i g h e d and f r o z e n in l i q u i d nitrogen. Fecal and liver lipid were extracted with chloroform/methanol (2:l,v/v). The fat a b s o r p t i o n rate w a s c a l c u l a t e d (II). L i v e r lipid c o n t e n t was determined gravimetrically, and was analyzed for its fatty acid composition. A Quick's thromboplastin time assay (12) was performed on the blood. Experiment 2 - For the second phase of the study six more groups of rats fed the above rations were kept under conditions similar to those above. Each day d u r i n g the f o u r t h and fifth w e e k s of feeding, rats w e r e k i l l e d by c e r v i c a l dislocation and decapitation. The livers were excised quickly, blotted dry, weighed, then rinsed with and immersed in ice-cold 0.85% NaCI solution. The activities of the enzymes glutathione peroxidase (13), glutathione reductase (14) and c a t a l a s e (15) of the c y t o s o l i c (105 000 g) f r a c t i o n w e r e a s s a y e d u s i n g the fresh tissues. The c y t o s o l i c p r o t e i n c o n c e n t r a t i o n also was determined (16). Malonaldehye concentration was measured on the frozen liver using Ohkawa's method (17). E x p e r i m e n t 3 - For the third phase of the study a n o t h e r series of rats was obtained and treated similarly. The activity of the enzyme glucose-6-phosphate dehydrogenase (18) and the c e l l u l a r g l u t a t h i o n e c o n c e n t r a t i o n (19) w e r e determined. Statistical analysis The data w e r e a n a l y s e d s i g n i f i c a n c e (20).
using ANOVA
and Tukey's
test at a 5% level of
RESULTS
Characteristics of the test oils are given in Table 2, and the fatty acid c o m p o s i t i o n s are in Table 3. The h e a t e d oils w e r e m o r e v i s c o u s and darker
676
T.S. KOK et al.
than the fresh oils. The carbonyl, peroxide, acid and s a p o n i f i c a t i o n values were increased, and the iodine values were decreased as a result of 72 hr of heating. The oleic (C18:1) and linolenic acid (C18:3) contents of the fresh canola oils were significantly higher than for fresh corn oil. Nevertheless, as a result of heating each of the oils the contents of the unsaturated fatty acids C18:2 and C18:3 dropped markedly, and this was c o m p e n s a t e d for by increases in C16:0, C18:0 and C18:1. The total of the s e l e c t e d fatty acids was lower for the western canola oil.
TABLE 2 Characteristics of Oils Corn
Ontario Canola
Western Canola
Fresh
Oxidized
Fresh
Oxidized
Fresh
Oxidized
Carbonyl value (meq/kg)
0
46.4
0
145.6
0
129.8
Peroxide value (meq/kg)
1.8
7.4
3.6
7.2
1.7
7.4
Acid value
0.2
1.0
1.0
1.9
1.0
1.7
Iodine value
183.2
116.0
110.5
87.3
ii0.0
89.5
Saponification value
187.1
194.8
185.2
198.2
184.1
196.1
TABLE 3 Fatty Acid Composition of Oils I Corn
Fresh 16:0 18:0 18:1 18:2 18:3 22:0 22:1
Ontario Canola
Oxidized
Fresh
Oxidized
Western Canola
Fresh
Oxidized
11.3 2.0 25.2 57.5 0.8 0.3 0
11.6 2.2 35.6 46.7 0.4 0.2 0.i
5.4 2.4 59.7 19.5 8.1 0.4 0.6
7.0 2.9 68.6 11.9 2.4 0.4 0.8
4.3 2.0 57.6 17.0 6.8 0.3 0.4
5.8 2.6 62.8 Ii.0 2.6 0.3 0.6
97.1
96.8
96.1
94.0
88.4
85.7
IResults are expressed as the percentage composition of selected identified fatty acid methyl esters so the total is less than 100%.
HEATED FATS AND OXIDATIVE STRESS
677
Body weight gain, feed intake, feed efficiency and relative organ weights are l i s t e d in T a b l e 4. A l l the diets w e r e e a t e n well, a n d no g r o s s p h y s i c a l anomalies were observed throughout the study. The body weight gains, over the p e r i o d of four w e e k s w e r e s i m i l a r in all the groups, b u t f e e d i n t a k e was n u m e r i c a l l y h i g h e r w h e n o x i d i z e d fats w e r e fed. As a result, the feed e f f i c i e n c y of a n i m a l s w i t h i n the g r o u p s fed h e a t e d c a n o l a oils w a s s l i g h t y lower. The relative liver weights were 15% to 30% higher for the heated oil groups, w h i l e the k i d n e y w e i g h t s w e r e h i g h e r o n l y in the o x i d i z e d O n t a r i o canola group. The relative heart and spleen weights were not affected.
TABLE 4 Results from Feeding Study 1'2 Corn
Fresh
Oxidized
Ontario Canola
Western Canola
Fresh
Fresh
Oxidized
Oxidized
Weight gain (g)
195 a +15
205 a +14
210 a +19
200 a +13
201 a +24
197 a +14
Feed intake (g)
437 a +31
449 a +29
458 a +38
475 a +30
449 a +37
458 a +33
Feed efficiency (%)
44.7 a +2.3
45.6 a +1.2
45.8 a +1.5
42.2 b +1.6
44.8 a +2.7
43.1 b +1.4
Liver
4.20 b +0.28
5.42 a +0.36
4.45 b +0.26
5.80 a +0.46
4.68 b +0.29
5.34 a +0.49
Kidney
0.85 b +0.08
0.87 ab +0.03
0.84 b +0.04
0.96 a +0.09
0.84 b +0.05
0.86 b +0.04
Heart
0.33 a +0.01
0.35 a +0.03
0.34 a +0.02
0.37 a +0.02
0.37 a +0.03
0.36 a +0.01
Spleen
0.32 a +0.03
0.33 a +0.05
0.29 a +0.03
0.35 a +0.04
0.35 a +0.03
0.32 a +0.04
iMeans in a row with the same superscript are not significantly different (P<0.05). Relative organ weights as as percentage of body weights
F e c a l p e l l e t s c o l l e c t e d f r o m the g r o u p s fed h e a t e d oils w e r e larger, d a r k e r and m o r e moist; also the dry m a t t e r o u t p u t p e r d a y w a s h i g h e r (Table 5). However, steatorrhea was not excessive. The fat absorption in the groups fed heated oils ranged b e t w e e n 82% to 87% and was significantly lower than for the groups fed fresh oils (92% to 93%).
T.S. K0K et al.
678
TABLE 5 Results of Fat Absorption Study I~2 Corn
Ontario Canola
Fresh
Oxidized
Fresh
Oxidized
Feed intake (g/d)
19.0 b
19.6 ab
19.1 b
21.4 a
Dried feces
1.49 d
(g/d)
Fat absorption Liver lipid (%)
(%) 93.1 a 6.3 a +0.8
1.77 be 86.6 b 5.4 ab +0.5
1.48 d 92.2 a 5.7 ab +0.7
2.38 a
Western Canola
Fresh
Oxidized
20.2 ab I~
cd
20.0 ab 1.93 b
81.8 c
91.9 a
86.6 b
5.0 b +0.4
5.0 b +0.6
5.0 b +0.7
iMeasured over a period of I0 days 2Means in a row with the same superscript are not significantly different (P
Liver lipid determined gravimetrically ranged between 5.0% to 6.3%; the group fed fresh corn oil diet had the highest liver fat level. Nevertheless, there w e r e no clear d i f f e r e n c e s in liver fat c o n t e n t b e t w e e n the h e a t e d and fresh c a n o l a oils. The p r e d o m i n a n t liver f a t t y acids w e r e C16:0, C18:0, C18:1, C18:2 and C20:4 m a k i n g up 90% of the total (Table 6). However, the a m o u n t s of C18:2 c o n s i s t e n t l y d e c l i n e d for the groups fed h e a t e d oils, and this also w a s the case for C18:3 and the h e a t e d c a n o l a oil groups. On the other hand, C20:4 was i n c r e a s e d s t a t i s t i c a l l y w i t h all h e a t e d fats. This h i g h e r level of a r a c h i d o n i c acid c o n f i r m s an e a r l i e r report for C20:4 in n e u t r a l liver lipids (21). There were no differences in the Quick's thromboplastin times or in the c y t o s o l i c p r o t e i n c o n t e n t s for all the groups. Also, the total g l u t a t h i o n e contents, reduced and oxidized, in the groups fed heated oils, as determined by the Ellman's m e t h o d (19) were only s l i g h t l y h i g h e r than for the ones fed fresh oils (Table 7). The concentrations of malonaldehyde in frozen livers of rats fed oxidized oils were elevated. The a c t i v i t i e s of g l u t a t h i o n e p e r o x i d a s e of groups fed oxidized oils were diminished w h e n c o m p a r e d to the ones fed fresh o i l s , r e a c h i n g statistical significance only in the c a n o l a o i l - f e d groups (Table 8). Likewise significant d i f f e r e n c e s w e r e not f o u n d in the a c t i v i t i e s of g l u t a t h i o n e r e d u c t a s e a m o n g the groups. The activity of glucose-6-phosphate (G-6-P) dehydrogenase was diminished only in the oxidized Ontario canola oil group compared to the fresh fat control, but the oxidized corn oil group was significantly lower than the oxidized canola oil groups. Catalase activities of the oxidized oil groups were elevated when compared to the fresh oil.
HEATED FATS AND OXIDATIVE STRESS
679
TABLE 6 Fatty Acid Composition of Total Liver Lipid Corn
Fresh 14:0 16:0 16:1 18:0 18:1 18:2 18:3 20:1 20:2 (n6) 20:3 (n6) 20:4 22:0 22:6 24:0 24:1
Ontario Canola
Oxidized
0.6 31.5 1.2 25.8 14.3 13.0 a 0. i c 0.2 0.6 0~
05 29 5 13 29 7 13 8 9 Ib 0 Ic 0.2 0.3 0.5
6.7 d 0.3
9.6 c 0.3
0.4 0.9 0.i
0.6 1.0 0.2
Fresh
Western Canola
Oxidized
0.5 18.8 2.1 17.1 27.8 9~ 8b 0.8 a 0.4 0.3 0.9 13.7 b 0.2 4.1 0.3 0.3
I
0.5 21.7 2.4 17.7 26,3 5.4 c 0.I c 0.i 1.3 1.2 16.4 a 0.2 4.2 0.4 0.3
Fresh
Oxidized
0.5 17.8 1.7 18.4 27.2 8.9 b 0.5 b 0~ 0.3 0.9 14.7 b 0.2 4.4 0.4 0.4
0.4 20.3 2.4 17.5 26.8 6.3 c 0.2 c 0.2 0.8 1o2 16.6 a 0.2 4.3 0.4 0.3
IResults are expressed as percentage of total lipid based on five determinations, and means in a row with the same superscript are not significantly different (P<0.05).
TABLE 7 Prothrombin Time, Hepatic Glutathione, Cyto@olic Protein and Malonaldehyde Concentrations Corn
Ontario Canola
Western Canola
Fresh
Fresh
Fresh
Oxidized
17 a
18 a
20 a
Glutathione (umol/wet g)
6.6 b ~0.6
7.0 a •
Cytosolic protein (mg/wet g)
i16 a +14
Malonaldehyde (nmol/wet g)
167 c ~31
Thromboplastin time (seconds)
Oxidized
Oxidized
17 a
17 a
16 a
6.4 b !0.6
6.9 a ~0.5
6.7 b !0.5
7.1 a !0.4
I12 a +13
i02 a +15
I06 a +II
i06 a +9
i13 a +ii
243 a •
205 b ~31
226 a •
209 b •
228 a +34
IMeans in a row with the same superscript are not significantly d i f f e r e n t (P
680
T.S. KOK et al.
TABLE 8 Enzymatic Activities I Corn
Fresh
Ontario Canola
Oxidized
Fresh
Oxidized
Western Canola
Fresh
Oxidized
Glutathione peroxidase 2
6.9 abe •
5.8 c ~1.3
7.7 ab •
5.4 c •
8.0 a Z2.0
6.0 be ~1.3
Glutathione reductase 2
5.4 a +0.8
5.4 a +0.5
4.9 a +0.5
5.4 a +0.3
4.9 a +0.8
5.3 a +0.3
G-6-P dehydrogenase 3
4.2 bc •
2.9 c !l.0
6.4 a •
4.6 b ii.2
5.4 ab ii.3
4.8 b il.0
49.1 c +10.6
59.7 b +8.9
37.0 d +10.8
72.4 a +13.4
36.8 d +7.9
69.4 a +14.1
Catalase 4
iMeans in a row with the same superscript are not signficantly different (P<0.05). 2umol NADPH oxidized/min/wet g 3umol NADPH reduced/min/wet g 4mmol H202 decomposed/min/wet g
DISCUSSION
There are many methods for measuring the degree of abuse in heated oils (22,23). Some of the ones employed in this study are among the more common. The levels of peroxides, carbonyl compounds and free acids have been shown to increase as a r e s u l t of heating. P e r o x i d e s (or h y d r o p e r o x i d e s ) are p r i m a r y products of fat oxidation and are not stable; they normally are degraded into c a r b o n y l c o m p o u n d s and acids. Iodine v a l u e is a m e a s u r e of the degree of unsaturation, and in all the heated oils, the iodine values were much l o w e r . This was r e f l e c t e d in the fatty acid p r o f i l e s of these oils (Table 3 ) r e s u l t i n g in less of C18:2 and C18:3 w h i c h are m o r e r e a d i l y oxidized; The p e r c e n t a g e s of the s e l e c t e d 7 m o s t p r o m i n e n t fatty acids did not add up to i00. In addition, the o x i d i z e d oils, due to shifts in c o m p o s i t i o n and production of unidentified polar derivatives, had lower totals. In a number of i n s t a n c e s the corn oil control and each of the c a n o l a oils p r o d u c e d d i f f e r e n t responses, and these r e f l e c t the fact that they are d i s t i n c t products with different genetic origins. However, all the heated fats were less w e l l a b s o r b e d due to f o r m a t i o n of o l i g o m e r i c d e r i v a t i v e s w h i c h are h y d r o l y z e d poorly (2). The distinctive characteristics of canola oils among vegetable oils are their r e l a t i v e l y h i g h C18:1 and C18:3 contents. F r o m the v i e w p o i n t of food oxidation, t h e i m p o r t a n t fatty acids are the u n s a t u r a t e d ones, w h o s e susceptibility to oxidation increases greatly with degree of unsaturation. It is t h e r e f o r e b e l i e v e d by m a n y i n v e s t i g a t o r s that the h i g h e r the c o n t e n t of unsaturated fatty acids, especially PUFA in fat, the more readily are toxic substances formed due to thermal degradation (24). Nevertheless, this is not c e r t a i n in v i e w of a r e p o r t that a h i g h m o n o e n e c o n t e n t can lead to more t o x i c i t y (25).
HEATED FATS AND OXIDATIVE STRESS
681
P e r o x i d e s and e a r b o n y l c o m p o u n d s are o f t e n i m p l i c a t e d in c a u s i n g deleterious effects. Peroxides are normally present at low levels in heated fats, and lymphatic absorption is minimal, whereas carbonyl compounds such as 1 2 - k e t o oleic a c i d p r e s e n t at a h i g h e r level has a p r o o x i d a n t a c t i o n on unsaturated fatty acids and may promote lipid peroxidation in vivo (26). It is known that nonvolatile oxidized products are absorbed into the body, and it has been reported that these products such as dimerie acids, monomeric acids and c y c l i c m o n o m e r i c acids can be r e c o v e r e d f r o m the l y m p h a t i c (27) and h e p a t i c lipids (28). W h e n a d m i n i s t e r e d in large amounts, the n o n v o l a t i l e oxidized fraction, or some components of it were deleterious to heart cells in culture system (3), or to the whole animal system (25). It is p r o p o s e d that a n i m a l s fed such m a t e r i a l s at a low c o n c e n t r a t i o n c o u l d u n d e r g o an a l t e r e d x e n o b i o t i c m e t a b o l i s m . This w a s e v i d e n c e d in our study by the change in a c t i v i t y of some enzymes, and c o n c e n t r a t i o n of s o m e components of the glutathione redox cycle. The feeding of diets with 15% whole oxidized oil in the present four-week study did not show any depressed growth or obvious steatorrhea, as others have i n d i c a t e d (7,8,29). C o n s e q u e n t l y , the i n a b i l i t y to o b s e r v e i n c i d e n c e s of certain diseases, and differences in longevity in some studies has led to the conclusion that heated fats are always safe for human consumption. The toxic materials may have been present in too low a concentration, or the diet used was so well balanced nutritionally to offset any obvious deleterious effects. With lower protein and vitamin contents in diets, however, heated oils were found to be more deleterious (30). The protective role of glutathione against xenobioties by virtue of its a b i l i t y to c o n j u g a t e w i t h and to f a c i l i t a t e their e x c r e t i o n has b e e n w e l l documented (31). The pool of glutathione in the cells is predominantly in a reduced state unless challenged with stress factors. This dynamic supply of glutathione can be easily depleted and replenished (32). It is maintained by a b a t t e r y of e n z y m e s such as g l u t a t h i o n e r e d u c t a s e and G - 6 - P d e h y d r o g e n a s e w h i c h help to p r o v i d e a s o u r c e of NADPH. In our 4 - w e e k study, the h e p a t i c glutathione concentration was increased, but not to the same degree as found by Izaki et al. (29). T h e i r study was n e v e r t h e l e s s m u c h longer (14 weeks). These authors speculated based on earlier experiments that the reason for the increase in glutathione was that a secondary oxidizing agent produced in the liver probably consumed it, a n d an o v e r s h o o t i n g of the r e s y n t h e s i s subsequently occurred. Liver enlargement, frequently seen in rats fed oxidized oils, normally is accompanied by the increased activity of enzymes resulting from proliferation of the s m o o t h e n d o p l a s m i c r e t i c u l u m (33). In the c u r r e n t study c y t o s o l i c p r o t e i n c o n c e n t r a t i o n w a s not affected, b u t the a c t i v i t i e s of the h e p a t i c e n z y m e s g l u t a t h i o n e p e r o x i d a s e and G-6-P dehydrogenase in rats fed oxidized fats were decreased while that of catalase was increased. The cause for the i n c r e a s e d or d i m i n i s h e d a c t i v i t y in these e n z y m e s is not known; but g l u t a t h i o n e p e r o x i d a s e and c a t a l a s e have b e e n c r e d i t e d w i t h the a b i l i t y to detoxify peroxides and hydroperoxides, and to halt lipid peroxidation (34,35). Malonaldehyde, an indicator of lipid autoxidation, also is seen commonly in the tissue of animals fed heated oils and therefore components in these oils m a y be c o n s i d e r e d as p r o m o t e r s of lipid p e r o x i d a t i o n . We have b e e n able to c o n f i r m the p r e s e n c e of m a l o n a l d e h y d e in the liver of rats fed t h e r m a l l y oxidized fats. The alterations in enzyme activities except for G-6-P dehydrogenase were less p r o n o u n c e d in the corn oil fed groups. This c o i n c i d e d w i t h the l o w e r c a r b o n y l and a c i d values, and could also be r e l a t e d to the fatty acid
682
T.S. KOK et al.
composition. Since heating is k n o w n to destroy nutrients, another factor w h i c h should not be disregarded is the tocopherol content in the oil. Tocopherol has been s h o w n to be mostly destroyed by heat under the present procedures, but a higher tocopherol content as in corn oil might provide some protection of the unsaturated fatty acids against oxidation. In conclusion, the feeding of oxidized oils at a level of 15% for 4 weeks did not cause a decrease in body weight gain or feed intake. It nevertheless resulted in changes in glutathione content, and in the activity of some of the hepatic enzymes.
ACKNOWLEDGEMENTS
This work was supported by the Natural Sciences and Engineering Research Council of Canada, and the Ontario Ministry of Agriculture and Food.
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Accepted for publication D~cember 9, 1987.
Phar.