Biological Properties of Heated Dietary Fats - ACS Symposium Series

8 Biological Properties of Heated Dietary Fats

Downloaded by UNIV OF IOWA on September 2, 2016 | http://pubs.acs.org Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch008

J. C. ALEXANDER Department of Nutrition, College of Biological Science, University of Guelph, Guelph, Ontario, Canada N1G 2W1 Thermal oxidation of dietary fats, with the formation of potentially toxic derivatives during heating and processing, is related to the conditions used in the home and the food service industry. During deep-fat frying many volatile and non-volatile compounds are formed, some of which can be toxic depending on the level of intake. Many chemical and biological studies have been carried out, and experimental findings indicate that possible dietary hazard should be greater as the severity of the treatment of the fat is increased. Observations with animals fed these fats have shown adverse effects ranging from depression in growth, diminished feed efficiency, increased liver size, fatty necrosis of the liver and numerous other organ lesions. Specific effects on biological tissues can be verified by selected techniques including histopathological evaluations, biochemical parameters, and tissue culture in monolayers. Fractionation of heated fat samples serves to concentrate a number of the unnatural components, and incorporation of these materials into rat diets has enabled experimenters to observe distinct reactions by the animals. Practical processing and frying operations usually produce low levels of nutritionally undesirable products, but it is worthwhile to recognize their possible adverse biological effects. The b i o l o g i c a l p r o p e r t i e s of thermally o x i d i z e d f a t s have been studied f o r many years. E v a l u a t i o n of laboratory-heated and commercially-used f a t s i n d i e t s f o r animals have included feed consumption, weight g a i n , and feed e f f i c i e n c y (1-4), pathology (1-2)9 organ weights (4, 10), enzyme assays (11), and t o t a l l i p i d 0097-6156/83/0234-0129$06.00/0 © 1983 A m e r i c a n C h e m i c a l S o c i e t y

Finley and Schwass; Xenobiotics in Foods and Feeds ACS Symposium Series; American Chemical Society: Washington, DC, 1983.


130

XENOBIOTICS

IN F O O D S A N D

FEEDS

content and t i s s u e f a t t y a c i d composition (12-15). A v a r i e t y of thermally o x i d i z e d f a t s was included and r e s u l t s ranged from m i l d responses to s u b s t a n t i a l adverse e f f e c t s on the animals. Ques t i o n s remain regarding the p o t e n t i a l t o x i c i t y of l a b o r a t o r y heated or commercially used f r y i n g f a t s . Evidence i n d i c a t e s that secondary o x i d a t i o n products such as monomeric, dimeric and polymeric compounds accumulate i n the heated f a t s and may subsequently be ingested with the f r i e d foods. Johnson et a l . (16) observed that thermally o x i d i z e d o i l i s not so r a p i d l y hydrolyzed as the corresponding unheated o i l . Nolen (5) found when feeding male dogs d i e t s c o n t a i n i n g a 15% l e v e l of f r e s h or heated p a r t i a l l y hydrogenated soybean o i l that the heated f a t reduced the a b s o r p t i o n , growth r a t e and feed e f f i c i e n c y . These products r a t h e r than peroxides are the p r i n c i p a l f a c t o r s i n ad verse e f f e c t s seen with thermally o x i d i z e d f a t s . Paik et a l . (17) studied mice dosed with methyl l i n o l e a t e hydroperoxides or a u t o x i dized methyl l i n o l e a t e c o n t a i n i n g secondary o x i d a t i o n products. M o r t a l i t y was 50% and 100% r e s p e c t i v e l y . Congestion i n t i s s u e s , f a t t y degeneration and n e c r o s i s were observed, and the amount of impairment c o r r e l a t e d with the type of m a t e r i a l f e d . The c o n c l u sions were confirmed by h i s t o p a t h o l o g i c a l o b s e r v a t i o n s , and v o l a t i l e low molecular weight compounds c o n t a i n i n g carbonyl groups were suspected of being i n v o l v e d . The r e a c t i v i t y of f a t t y a c i d s increases with the degree of u n s a t u r a t i o n , but the d i s t r i b u t i o n and geometry of double bonds a l s o i n f l u e n c e the extent of o x i d a t i o n (18). Double bonds become conjugated or l o s t as they are involved i n r e a c t i o n s forming v a r i o u s secondary products, some of which have been i d e n t i f i e d as c y c l i c compounds, s c i s s i o n products, dimers and l a r g e r molecules (19-23). The s e v e r i t y of c o n d i t i o n s ( a e r a t i o n , temperature and heating time) p l a y s an important r o l e i n the degree of d e t e r i o r a t i o n of f r y i n g f a t s ( 1 ) . Perkins and Kummerow (24) confirmed the e a r l i e r observations of Crampton et a l . (25) that the non-urea-adductable p o r t i o n of heated f a t s was most t o x i c to animals. These concentrates, c o n t a i n i n g monomeric and dimeric d e r i v a t i v e s , are more t o x i c than the l a r g e r polymers due to b e t t e r a b s o r p t i o n 02, 10, 26). Urea f i l t r a t e of heated f a t , fed to r a t s , r e s u l t e d i n a 30% r e d u c t i o n i n the o x i d a t i o n of p a l m i t i c a c i d to CO2 (27). Animals given low l e v e l s of vitamins and thermally o x i d i z e d f a t s i n t h e i r d i e t a t a f i x e d p r o t e i n concen t r a t i o n responded p o o r l y compared to those which received f r e s h f a t s and the same amount of vitamins ( 3 ) . A r e a l p o s i t i v e r e sponse was obtained i n the presence of the heated f a t when the p r o t e i n l e v e l was i n c r e a s e d . Tappel (28) suggested that chemical d e t e r i o r a t i v e e f f e c t s due to f r e e r a d i c a l s produced i n heated f a t s (hydroxyl and hydroperoxyl) might be slowed by increased amounts of d i e t a r y a n t i o x i d a n t s . Fukuzawa and Sato (29) s e l e c t e d 12-ketoo l e i c a c i d as a degradation product of l i p i d p e r o x i d a t i o n and compared i t s e f f e c t s on r a t t i s s u e s with those of a v i t a m i n Ε d e f i c i e n c y . Both increased f l u o r e s c e n t production i n the l i v e r , as w e l l as hemolysis and plasma a l k a l i n e phosphatase l e v e l i n the blood.


8.

ALEXANDER

Biological

Properties

of Heated

Dietary

Fats

131


Because of the s e v e r i t y of some problems reported when experimental animals are fed compounds from thermally o x i d i z e d food f a t s there i s j u s t i f i e d concern about e f f e c t s some of these d e r i v a t i v e s could have on consumers. E a r l y work which showed adverse e f f e c t s on animals was complicated by improper p r o t e c t i o n o f the d i e t leading to v i t a m i n d e f i c i e n c i e s . However, i n studies with used f r y i n g f a t s , Alexander (30) found that frequent d i e t prepar a t i o n and f e e d i n g , and the use of a n t i o x i d a n t s and r e f r i g e r a t i o n could avoid these d i f f i c u l t i e s . In recent years, a number of w e l l conducted studies have produced considerable evidence that o x i d i z e d and abused f r y i n g f a t s c o n t a i n p o t e n t i a l l y t o x i c constituents. Experimental To evaluate the b i o l o g i c a l e f f e c t s of thermally o x i d i z e d f a t s , i s o l a t e d concentrates of f a t t y a c i d d e r i v a t i v e s were prepared. Fats used were corn o i l (CO) o l i v e o i l (00) low e r u c i c a c i d rapeseed o i l (LE) and l a r d (LA). The heating c o n d i t i o n s were those of G a b r i e l e t a l . Ç7). Each f a t was heated i n a s t a i n l e s s s t e e l beaker f o r 72 h r a t a c o n t r o l l e d temperature of 180°C. Each day i t was s t i r r e d continuously w i t h a mechanical s t i r r e r f o r 12 h r and by hand every h r f o r 12 h r to ensure aera t i o n and mixing. D i s t i l l a t i o n and urea treatment concentrated the c y c l i c and branched chain degradation products. Intubation experiments with r a t s were used as short-term studies to d e t e r mine the t o x i c i t y of the d i s t i l l a b l e non-urea-adductable (DNUA) f r a c t i o n s obtained from e t h y l esters produced from thermally o x i dized f a t s (31,32). A urea adduction method modified from that of Eisenhauer and Beal (33) was used to produce the DNUA concent r a t e s . A l l r a t s were intubated d a i l y with 0.5 ml of e i t h e r DNUA m a t e r i a l s from thermally o x i d i z e d f a t s or the r e s p e c t i v e f r e s h f a t s , using a rubber stomach tube (no. 8 catheter) on a s y r i n g e . Body weights were recorded d a i l y , and the animals were examined on a r e g u l a r b a s i s to detect e a r l y symptoms o f t o x i c i t y . After approximately three days, due to m o r b i d i t y , the animals were k i l l e d . H i s t o p a t h o l o g i c a l examination o f the h e a r t , l i v e r and kidneys o f the r a t s was c a r r i e d out to q u a n t i t a t e t i s s u e damage, and l e s i o n s i n the organs were graded as described by G a b r i e l et a l . ( 7 ) . Organ weights were recorded and p o r t i o n s were frozen i n l i q u i d n i t r o g e n f o r l i p i d a n a l y s i s (34) . F a t t y a c i d methyl e s t e r s were prepared from the n e u t r a l and p o l a r l i p i d f r a c t i o n s of the organs (35,36) and analyzed f o r component f a t t y acids by GLC. Types of l e s i o n s found i n the t i s s u e s e c t i o n s were graded as to incidence and s e v e r i t y on a s c a l e from zero f o r normal, to three f o r n e c r o t i c t i s s u e , i n the f o l l o w i n g anatomical s t r u c t u r e s : Heart: Myocardial n u c l e i N u c l e i of v a s c u l a r media and endothelium I n t e r s t i t i a l t i s s u e and myofibers


132

XENOBIOTICS

IN FOODS A N D F E E D S


Liver :

Hepatocellular nuclei H e p a t o c e l l u l a r cytoplasm Kupffer and e n d o t h e l i a l c e l l s Kidney : Glomeruli Tubules V e s s e l s and i n t e r s t i t i a l t i s s u e s Animal t i s s u e c e l l s grown i n c u l t u r e medium i n the form of a monolayer are a u s e f u l b i o l o g i c a l l i v i n g model to observe p h y s i o l o g i c a l , morphological or metabolic changes i n the presence of conpounds added to the medium. B i r d and Alexander (15, 37) r e ported on e f f e c t s of thermally o x i d i z e d corn o i l and o l i v e o i l on i n v i t r o heart c e l l s . The DNUA from f a t thermally o x i d i z e d as described e a r l i e r (_7) was i s o l a t e d . Free f a t t y a c i d s from the f r e s h f a t c o n t r o l s o r from the DNUA of the heated f a t s were pre pared (38). Primary c u l t u r e s of neonatal h e a r t c e l l s were prepared from 2-5 day o l d r a t s by the method of Rogers (39) as monolayered c o v e r s l i p c u l t u r e s . The h e a r t s were e x c i s e d a s e p t i c a l l y and t r a n s f e r r e d to a p e t r i d i s h c o n t a i n i n g phosphate-buffered s a l i n e . The t i s s u e was chopped f i n e l y and separated i n t o s i n g l e c e l l s by t r y p s i n i z a t i o n using 0.25% t r y p s i n s o l u t i o n (15). Leighton tubes c o n t a i n i n g a c u l t u r e medium s u p p l i e d w i t h 5% f e t a l c a l f serum were seeded with 2 χ 10^ c e l l s / m l o f medium. Four day o l d c u l t u r e s were exposed to f r e s h and heated f a t t y a c i d f r a c t i o n s (60 or 100 yg/ml) i n the form of an emulsion with bovine serum albumin d i s solved i n phosphate-buffered s a l i n e (PBS). The r e q u i r e d concen t r a t i o n of each f r e e f a t t y a c i d f r a c t i o n , d i s s o l v e d i n hexane, was t r a n s f e r r e d to a 100 ml s t e r i l i z e d b o t t l e , and the s o l v e n t was evaporated completely. The s o l u t i o n of bovine serum albumin (40 mg/ml of PBS) was added to the l i p i d f r a c t i o n s . A r a t i o of f r e e f a t t y a c i d f r a c t i o n to bovine serum albumin of 1:60 (w/w) was maintained. The b o t t l e was screw-capped and incubated a t 40° with o c c a s i o n a l shaking f o r two h r . T h i s i n c u b a t i o n was s u f f i c i e n t to o b t a i n an emulsion o f f r e e f a t t y a c i d s ready f o r admin i s t r a t i o n i n t o the c u l t u r e medium (15). C e l l u l a r l i p i d was e x t r a c t e d by means of the procedure of Folch e t a l . (40) and f r a c t i o n a t e d by t h i n l a y e r chromatography (TLC). Glass p l a t e s coated with S i l i c a Gel G of 0.5 ml thickness and a solvent system c o n t a i n i n g heptane/isopropyl e t h e r / a c e t i c a c i d (60:40:3) were used (41). C u l t u r e s of heart c e l l s were ob served p e r i o d i c a l l y with a Nikon i n v e r t e d microscrope with phase c o n t r a s t o p t i c s . For d e t a i l e d morphology, r e p l i c a t e c u l t u r e s a l s o were s t a i n e d with May-Grunwald-Giemsa s t a i n (42). I n t r a c e l l u l a r l i p i d accumulation was assessed s u b j e c t i v e l y with the phase c o n t r a s t microscope, and a numerical s c a l e ranging from zero to four was used. The zero value was given to c e l l s showing no l i p i d d r o p l e t s , as i n those grown i n the medium c o n t a i n i n g 5% f e t a l bovine serum without a d d i t i o n a l l i p i d . A v a l u e of four was given to a f i e l d c o n s i s t i n g of c e l l s e x h i b i t i n g abundant f a t d r o p l e t s where most of the e x t r a n u c l e a r space appeared to be occupied with l i p i d (38).


8.

ALEXANDER

Biological

Properties

of Heated

Dietary

133

Fats

Stained cover s l i p c u l t u r e s from each treatment group were observed under the x40 o b j e c t i v e t o estimate the e f f e c t o f v a r i o u s treatments on growth p a t t e r n and c e l l morphology. Twenty random f i e l d s were s e l e c t e d , and the number o f c e l l s i n each f i e l d , the number of c e l l s i n v a r i o u s stages of m i t o s i s , and the number of pyknotic n u c l e i were recorded. The m i t o t i c index and percent pyknotic c e l l s were c a l c u l a t e d as percentages based on the t o t a l number of c e l l s observed i n 20 random f i e l d s and were not average values of r e p l i c a t i o n s . The c e l l u l a r p r o t e i n was q u a n t i t a t e d by the method o f Lowry e t a l . (43) as modified by Oyama and Eagle (44).


Results Intubation of the r a t s with three of the heated f a t DNUA samples, heated o l i v e o i l (HOO), heated l a r d (HLA) and heated low e r u c i c a c i d rapeseed o i l (HLE) r e s u l t e d i n e a r l y signs of physiol o g i c a l s t r e s s . Within 10 h r the animals became d i s o r i e n t e d and nervous and feed and water consumption were reduced. A l o s s of body weight was observed. Rats given DNUA from heated corn o i l (HCO) were i n d i s t i n g u i s h a b l e from those given the f r e s h f a t s (Table I ) . Average weight o f the animals a t the s t a r t o f the experiment was 59 g.

Table I . Body Weights of Rats Intubated with D i f f e r e n t D i e t a r y Fats D i e t a r y Fat

Body Weight (g)

1

a

CO HCO

75 70

00 H00

b 49

LE HLE

62 48

LA HLA

71 49

a

7 0

b

a

b

a

b

"Numbers with the same s u p e r s c r i p t s are not s i g n i f i c a n t l y d i f f e r e n t (P

LU

Ο

2.0 15 10 05 0

LEAR

Figure DNUA

2. Histological scores for organs of rats intubated with fresh fats (O) or of heated fats (·). (Reproduced with permission from Ref 32. Copyright 1979, Geron-X Inc.)



8.

ALEXANDER

Biological

Properties

of Heated

Dietary

Fats

137

Figure 3. Heart of rat intubated with DNUA-HLA. Blood vessel wall has lost its integrity, and endomysium is very prominent. Hematoxylin and eosin, x 750. (Reproduced with permission from Ref. 32. Copyright 1979, Geron-X Inc.)

Figure 4. Liver of rat intubated with DNUA-HLA. Pyknosis and fragmentation of necrotic hepatocytes. Hematoxylin and eosin, x500. (Reproduced with permission from Ref. 32. Copyright 1979, Geron-X Inc.)



138

XENOBIOTICS

IN F O O D S A N D

Figure 5. Kidney of rat intubated with DNUA-HLE. Cellular and granular fill lumina of uriniferoustubules. Hematoxylin and eosin, x344. (Reproduced permission from Ref. 32. Copyright 1979, Geron-X Inc.)


FEEDS

casts with

8.

ALEXANDER

Biological

Table I I I .

Properties

of Heated

Dietary

Fats

139

R e l a t i v e Organ Weights and T o t a l Organ L i p i d s (LEAR and LARD) 1

Dietary


Fat

Heart

Liver

Lipid (%)

Wt. (%)

LE HLE

0.47 0.44

LA HLA

0.41 0.42

3

a

a

a

b

5.2 9.3

a

b

4.7 7.7

a

Kidney

Lipid (%)

Wt. (%) b

4.4 7.0

a

b

4.4 8.8

a

7.9 7.8

a

a

b

5.9 9.5

a

Lipid (%)

Wt. (%) 1.2 1.4

ab

a

7.4 5.9

a

a

6.9 6.7

b

1.0 1.6

a

a

e

"lumbers with the same s u p e r s c r i p t s are not s i g n i f i c a n t l y f e r e n t (P

Biological Properties of Heated Dietary Fats - ACS Symposium Series

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