Xenobiotics in Foods and Feeds - American Chemical Society

4 Effects of Lipid Hydroperoxides on Food Components H. W. GARDNER

Downloaded by MONASH UNIV on November 13, 2015 | http://pubs.acs.org Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch004

Northern Regional Research Center, Agricultural Research Service, U.S. Department of Agriculture, Peoria, IL 61604

Undesirable changes i n n u t r i t i o n a l quality of foods are i n i t i a t e d by the autoxidation or enzymic oxidation of unsaturated l i p i d s to lipid hydroperoxides. L i p i d hydroperoxides and their products of decomposition can react with food components, such as amino acids, proteins and certain other biochemicals. These reactions and the potential role of hydroperoxides i n causing mutagenicity are reviewed. Food fabrication requires many ingenious methods to prevent the development of rancidity, and the food industry largely has been successful i n this endeavor. However, the problem continues to receive serious attention by researchers. Obviously, a food that has become rancid through either enzymic oxidation or autoxidation w i l l diminish i n both n u t r i t i o n a l value and p a l a t a b i l i t y . Because rancid foods usually are rejected before consumption, i t has been debated that l i p i d oxidation i n foods has l i t t l e impact on health. It i s a concern that radical reactions i n food can cause alterations below the threshold of detection by the human senses. Considering the complexity of l i p i d peroxidation per se, the parameters added by numerous ingredients i n food pose a nearly insurmountable problem to the experimentalist. As a result, nearly a l l we know about specific molecular reactions between food biochemicals and l i p i d hydroperoxides has come from studies of model reactions employing simple systems. Data from the models must be extrapolated to the composite, and this approach i s not necessarily wholly inadequate. Obviously, certain biochemicals are more susceptible than others to radical attack and/or reaction with secondary products. This chapter not subject to U.S. copyright. Published 1983, American Chemical Society

In Xenobiotics in Foods and Feeds; Finley, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

64

X E N O B I O T I C S IN F O O D S A N D

FEEDS

W i t h s u f f i c i e n t k i n e t i c d a t a one c o u l d p r e d i c t t h e p r e d o m i n a t i n g r e a c t i o n s expected i n a complex m i x t u r e o f p o t e n t i a l reactants. I t i s the purpose of t h i s review t o : (a) b r i e f l y outline the various r a d i c a l reactions o c c u r r i n g during the course of autoxidation, (b) d i s c u s s the use o f model systems i n the study o f the e f f e c t s o f l i p i d a u t o x i d a t i o n on food i n g r e d i e n t s , p a r t i c u l a r l y p r o t e i n s , and (c) assess the p o t e n t i a l m u t a g e n i c i t y of a u t o x i d a t i o n products. Reactions

of

Autoxidation

An u n s a t u r a t e d f a t t y a c i d w i l l n o t o x i d i z e i n the presence of o r d i n a r y ground-state 0 unless a hydrogen i s f i r s t abstracted from t h e f a t t y a c i d by a r a d i c a l . This abstraction i n i t i a t e s the r a d i c a l c h a i n needed t o overcome t h e l a g o r i n i t i a t i o n phase r e q u i r e d before a u t o x i d a t i o n can a c c e l e r a t e . Since the C-H bond o f an a l l y l i c c a r b o n has a r e l a t i v e l y weak bond d i s s o c i a t i o n energy, the abstraction of this hydrogen is favored, a s i l l u s t r a t e d i n F i g u r e 1. The r e s u l t a n t a l l y l i c r a d i c a l ( p e n t a d i e n e r a d i c a l i n t h e e x a m p l e g i v e n i n F i g u r e 1) then combines w i t h 0 to produce a peroxy r a d i c a l . The p e r o x y radical i n turn propagates t h e same sequence by further Η-abstraction. The l i p i d h y d r o p e r o x i d e , thus formed, i s a l s o s u s c e p t i b l e t o h o m o l y t i c d i s s o c i a t i o n v i a R e a c t i o n s A and B .


2

2

ROOH + X ·

R 0 0 - + ΧΗ

(A)

X = R, R S , RO, R 0 0 , e t c .

ROOH

l ^ R O - + -OH

(B)

I t s h o u l d be noted t h a t R e a c t i o n A i s a n Η-abstraction, and thus i t i s u s u a l l y r e v e r s i b l e . In contrast, Reaction Β i s not r e a d i l y r e v e r s i b l e a f t e r t h e R 0 a n d ·0Η r a d i c a l s e s c a p e f r o m the solvent cage. The n e t r e s u l t o f b o t h R e a c t i o n s A and Β i s the formation of secondary products and the generation of additional radicals. Figure 2 outlines the progress of a hypothetical autoxidation of a l i p i d . The i n i t i a t i o n phase i s f o l l o w e d by r a p i d a c c u m u l a t i o n of r a d i c a l s t h a t promote both the formation and d e s t r u c t i o n of hydroperoxides. Finally, radical combination (termination) leads to nonradical secondary products. As d i s c u s s e d l a t e r , both secondary products and r a d i c a l r e a c t i o n s p e r se a r e i n v o l v e d i n food d e t e r i o r a t i o n . The m a i n p a t h w a y s t o s e c o n d a r y p r o d u c t s o f l i p i d o x i d a t i o n are described i n the following text, but the reader should r e f e r t o more c o m p r e h e n s i v e r e v i e w s on t h i s s u b j e c t ( 1 , 2 ) . e



GARDNER

Effects

of Lipid

Hydroperoxides

Figure

1. Autoxidation oflinolenic acid. Structures are abbreviated polyunsaturate. Η-Abstraction is signified by H.

Figure

2. Hypothetical

autoxidation

of a polyunsaturated time.

to show only

lipid as a function


of


66

FEEDS

Peroxy r a d i c a l s . L i p i d peroxy r a d i c a l s generated by Reaction A are important i n propagating other r a d i c a l s by H-abstraction (reverse of Reaction A). Weakly bonded hydrogens are p a r t i c u l a r l y susceptible to a b s t r a c t i o n by the peroxy r a d i c a l . The r a d i c a l g e n e r a t e d i n t h i s way c a n become o x i d i z e d v i a R e a c t i o n C.

X· + o — Χ 0 0 ·

(C)


2

B e c a u s e XH i s o f t e n a n o n l i p i d , w h i c h becomes o x i d i z e d i n t h e presence of peroxidizing l i p i d s , t h i s process is sometimes called "cooxidation." I n a d d i t i o n t o Η - a b s t r a c t i o n , o t h e r r e a c t i o n s compete f o r peroxy radicals, such as β-scission and i n t r a m o l e c u l a r rearrangement (3). β-Scission occurs by the reverse of R e a c t i o n C , b u t i t i s d i f f i c u l t t o s u r m i s e how t h i s r e a c t i o n would have an impact on food i n g r e d i e n t s . On t h e o t h e r h a n d , p e r o x y r a d i c a l r e a r r a n g e m e n t may h a v e more r e l e v a n c e t o f o o d systems. Rearrangement occurs i f a double bond i s p o s i t i o n e d β to the carbon bearing the peroxy group. This can lead to formation of c y c l i c peroxides (4, 5) a n d p r o s t a g l a n d i n - l i k e endoperoxides (6) b y t h e p a t h w a y s s h o w n i n F i g u r e 3. These compounds a r e b e l i e v e d t o be i m p o r t a n t i n t h e g e n e s i s of malondialdehyde (7), r a d i c a l p r o p a g a t i o n and formation of other secondary products. Peroxy r a d i c a l s can react by y e t other competing routes. For example, evidence f o r l i p i d peroxy radical combination through a tetraoxide h a s b e e n r e p o r t e d r e c e n t l y (8). Such tetraoxides could generate singlet oxygen and n o n r a d i c a l p r o d u c t s b y t h e R u s s e l l m e c h a n i s m (9) a s s h o w n i n R e a c t i o n D . R

R

H

R

R

R

H

R

I n t e r m o l e c u l a r a d d i t i o n o f t h e peroxy r a d i c a l t o a double bond a l s o i s p o s s i b l e b u t has n o t been documented i n d e t a i l f o r lipids. It has been presumed t h a t the p o l y m e r i z a t i o n of p o l y u n s a t u r a t e s may p r o c e e d i n t h i s m a n n e r .



Figure 3. The formation of hydroperoxy from the 13S-hydroperoxide of linolenic

cyclic peroxides and prostaglandin-like acid by peroxy radical rearrangement. abbreviated (6).


endoperoxides Structures are

3


68

FEEDS

Oxy r a d i c a l s . Heat and s i n g l e e l e c t r o n r e d u c t i o n by t r a n s i t i o n m e t a l i o n s a r e among t h e m o r e i m p o r t a n t ways l i p i d o x y r a d i c a l s are formed from h o m o l y t i c cleavage o f h y d r o p e r o x i d e s . The reactive o x y r a d i c a l i s known t o p a r t i c i p a t e i n several competitive radical processes. Like peroxy radicals, oxy r a d i c a l s have a p r o p e n s i t y f o r Η-abstraction ( R e a c t i o n E ) .

\ /

CH-O- + XH

(E)

^ C H - O H + X· /


1 Commonly, a s e l f - i n d u c e d Η - a b s t r a c t i o n , c a l l e d d i s p r o p o r t i o n a t i o n , i s observed (Reaction F ) , and indeed, f a t t y ketones and a l c o h o l s a r e f o u n d among s e c o n d a r y p r o d u c t s .

\

\

CH-O-

A

*

A

β-Scission hydrocarbons.

(Reaction

CH-OH +

\ C = 0

(F)

A

G) g e n e r a t e s

volatile

aldehydes

and

R-CH = 0 + R

\ 2

(G)

CH-0-, R

- C H = 0 + R-

A l t h o u g h v o l a t i l e s u s u a l l y do n o t e x c e e d 10-15% o f t h e o x i d i z e d lipid, the aldehyde portion of the v o l a t i l e s receives disproportionate attention because of i t s contribution to rancid odors. Oxy r a d i c a l s a d d t o o l e f i n s b y b o t h i n t e r m o l e c u l a r a n d i n t r a m o l e c u l a r mechanisms. Experimental evidence has i n d i c a t e d that intramolecular addition ( R e a c t i o n H) may b e much m o r e important than i t s intermolecular counterpart (10, 11).

(H)


4.

GARDNER

Effects

of Lipid

Hydroperoxides

Since 0 i s an e x c e l l e n t scavenger of r a d i c a l s , the r a d i c a l i s oxidized further v i a Reaction I.


2

expoxyallylic

T h e h y d r o p e r o x y g r o u p o f t h e s e c o m p o u n d s may u n d e r g o further homolysis i n the cascade to secondary products (11). In theory, combination of oxy r a d i c a l s is possible (Reaction J ) ; however, there i s l i t t l e d e t a i l e d evidence to support t h i s type of r e a c t i o n at present. RO- + X · Effect

>

ROX

(J)

o f L i p i d O x i d a t i o n on P r o t e i n

The interaction of peroxidizing lipids with protein r e c e n t l y has been reviewed by s e v e r a l i n v e s t i g a t o r s (12-17). T h i s review w i l l emphasize t h e m o l e c u l a r b a s i s f o r t h e changes i n p r o t e i n caused by exposure to p e r o x i d i z e d l i p i d . First, i t must be u n d e r s t o o d t h a t p r o t e i n c a n be a f f e c t e d by lipid hydroperoxides i n three general ways: (a) t h r o u g h formation of noncovalent complexes w i t h e i t h e r l i p i d hydroperoxide or its secondary products, (b) b y r a d i c a l r e a c t i o n s , and (c) through reactions w i t h n o n r a d i c a l secondary products. The f o r m a t i o n o f n o n c o v a l e n t complexes w i l l be i g n o r e d h e r e , b u t complexation i s probably important i n causing flavor entrainment, changes i n p r o t e i n p h y s i c a l p r o p e r t i e s and p r o m o t i o n o f c h e m i c a l reactions. Radical reactions of protein. Radical reactions of proteins promoted by l i p i d hydroperoxides fall into three general categories: (a) p r o t e i n - p r o t e i n o r l i p i d - p r o t e i n c r o s s l i n k i n g , (b) p r o t e i n s c i s s i o n , and (c) p r o t e i n o x i d a t i o n . As i l l u s t r a t e d b y a number o f r e c e n t r e p o r t s , p e r o x i d i z i n g l i p i d affects protein i n a variety of ways. F o r example, J a c k s e t a l . (18) o b s e r v e d t h a t r a n c i d o i l ( P . V . = 144) h a d no e f f e c t on the storage p r o t e i n of peanut. Other studies w i t h lysozyme (19, 2 0 ) , γ - g l o b u l i n s and a l b u m i n (21) demonstrated considerable damage to protein. Lysozyme exposed t o either peroxidizing l i n o l e i c acid or methyl linoleate resulted i n m a i n l y t h e f o r m a t i o n o f lysozyme dimers and t r i m e r s (19, 2 0 ) ,


70


FEEDS

as w e l l as d e n a t u r e d lysozyme (20). While Funes and K a r e l (19) observed v e r y l i t t l e l i p i d bound c o v a l e n t l y t o lysozyme, Nielsen (21) found t h a t p e r o x i d i z e d p h o s p h o l i p i d exposed to e i t h e r albumin or γ-globulin under N caused mainly p h o s p h o l i p i d p r o t e i n covalent bonds. However, N i e l s e n (21) a l s o d i d observe some d i m e r a n d h i g h e r o l i g o m e r s o f p r o t e i n . Several variables i n t h e s e i n v e s t i g a t i o n s may h a v e b e e n t h e c a u s e o f t h e d i f f e r i n g r e s u l t s , i l l u s t r a t i n g the complexity of the problem. Although the study of peroxidizing l i p i d - p r o t e i n i n t e r a c t i o n is necessary to determine the overall effects, studies of model systems e m p l o y i n g p e r o x i d i z e d l i p i d and i n d i v i d u a l amino acids also are necessary to understand the molecular basis of t h e damage. The r a d i c a l p r o c e s s e s t h a t a p p e a r t o be i m p o r t a n t w i t h amino a c i d s a r e Η - a b s t r a c t i o n , r a d i c a l c o m b i n a t i o n ( R e a c t i o n Κ ) , β - s c i s s i o n o f a m i n o a c i d o x y r a d i c a l s ( R e a c t i o n G) a n d p o s s i b l y radical addition (Reaction L ) .


2

X· + X - - * X - X

(K)

X

H

X· + / = \

(L)

The f i r s t p r o c e s s , Η - a b s t r a c t i o n , may i n i t i a t e a n i m p o r t a n t g e n e r i c r e a c t i o n o f amino a c i d s ( F i g . 4 ) . Radicals attributed to t h e α-carbon have been i d e n t i f i e d b y e l e c t r o n s p i n resonance (ESR) o f p e r o x i d i z e d p r o t e i n s ( 2 2 ) . Further reaction with 0 ( h y p o t h e t i c a l ) would l e a d t o amino a c i d h y d r o p e r o x i d e s . A d i f f e r e n t pathway t o amino a c i d h y d r o p e r o x i d e has been p r o p o s e d by Yong and K a r e l ( 2 3 ) , b u t t h e i r p r o p o s a l i n v o l v e s an i n d i r e c t pathway t o t h e α-carbon r a d i c a l . Homolysis of the hydroperoxy group would a f f o r d a n amino a c i d oxy r a d i c a l s u s c e p t i b l e t o β-scission v i a R e a c t i o n G. Thus, β-scission between the α - c a r b o n a n d t h e a m i n o g r o u p may e x p l a i n t h e i n c r e a s e i n a m i d e content of p r o t e i n t h a t has been p e r o x i d i z e d i n d r y systems, as w e l l as t h e c o i n c i d e n t p r o t e i n c h a i n s c i s s i o n observed (24). I n a d d i t i o n , m o i e t i e s v i c i n a l t o t h e α - c a r b o n may b e susceptible to radical attack, and the products o f these r e a c t i o n s may g i v e t h e f a l s e i m p r e s s i o n t h a t t h e y w e r e d e r i v e d from a t t a c k d i r e c t l y on the α-carbon. F o r example, t h e amino group c o u l d be o x i d i z e d by a f r e e r a d i c a l mechanism, and s u b s e q u e n t l y c o u l d c a u s e t h e l o s s o f t h e α-amino g r o u p . The c o n v e r s i o n o f p r o l i n e t o p r o l i n e n i t r o x i d e (25) c a n be c i t e d as a n example o f s u c h a n o x i d a t i o n . 2

Besides susceptible

the attack to radical

a t t h e α-carbon, t h e s i d e chains a r e damage. Undoubtedly, the varying


4.

GARDNER

Effects

of Lipid

Hydroperoxides

71


0

II R—CH—C—OH I NH ?

0

. II R—C—C—OH I NH 2

1.0

2

R—C—C—OH NH; Figure

4. Postulated

mechanism

of amino acid oxidation carbon.

by radical

attack of a-


X E N O B I O T I C S IN

72

FOODS A N D

s e n s i t i v i t y of side chains i s the reason for s e l e c t i v i t y in the p e r o x i d a t i o n o f c e r t a i n amino a c i d s . G e n e r a l l y , the most l a b i l e amino a c i d s are h i s t i d i n e , c y s t e i n e / c y s t i n e , m e t h i o n i n e , l y s i n e , t y r o s i n e and t r y p t o p h a n (14). The d e g r a d a t i o n o f c y s t e i n e p r o b a b l y p r o c e e d s t h r o u g h t h e t h i y l r a d i c a l by Η-abstraction from the t h i o l group. Strong s u l f u r s i g n a l s were o b s e r v e d b y ESR i n a m i x t u r e o f cysteine plus peroxidized methyl linoleate demonstrating the s u s c e p t i b i l i t y of t h i o l to r a d i c a l s (22). As shown i n F i g u r e 5 , cystine, various oxides of cysteine/cystine (26-28), alanine and H S (27) are products. G l u t a t h i o n e p e r o x i d i z e d by l i p i d hydroperoxide also afforded the disulfide and o x i d e s of glutathione (29). The absence of 0 from a r e a c t i o n of linoleic acid hydroperoxide plus cysteine caused an i n t e r e s t i n g s h i f t in products. Instead of c y s t i n e and c y s t e i n e / c y s t i n e oxides, c y s t i n e and l i p i d - c y s t e i n e adducts were i d e n t i f i e d from the reaction mixture (30). T h e RS» p l u s RS« a n d R S * p l u s R* c o m b i n a t i o n s w e r e f a v o r e d b e c a u s e 0 was n o t p r e s e n t t o s c a v e n g e b o t h t h e l i p i d r a d i c a l s (R*) and t h i y l r a d i c a l s ( F i g . 5 ) . The d e t a i l e d mechanism proposed f o r the c o m b i n a t i o n r e a c t i o n is g i v e n i n F i g u r e 6. The e p o x y a l l y l i c r a d i c a l shown a t t h e t o p of Figure 6 arises from lipid oxy radical rearrangement (Reaction H). We h a v e r e c e n t l y i s o l a t e d t h e epoxyene-cysteine adduct by u s i n g a r e a c t i o n system devoid of p r o t i c solvents (31) . In protic solvent the epoxide r e a d i l y s o l v o l y z e s by anchimeric a s s i s t a n c e of the t h i y l ether i n t o the f i n a l products shown i n t h e f i g u r e . T h i s new d a t a r e f u t e s a mechanism I proposed e a r l i e r (14). Such a r e a c t i o n p o s s i b l y c o u l d account for a l i p i d to p r o t e i n c r o s s l i n k ; however, proof of this p a r t i c u l a r l i p i d - p r o t e i n bond r e m a i n s t o be d e m o n s t r a t e d . As shown i n F i g u r e 7, t h e d e g r a d a t i o n o f t r y p t o p h a n by p e r o x i d i z i n g m e t h y l l i n o l e a t e has been r e p o r t e d by Yong e t a l . (32) . T h e y p o s t u l a t e d t h a t t h e i n i t i a t i n g e v e n t was a r a d i c a l a d d i t i o n of a h y d r o x y l (or l i p i d oxy) r a d i c a l to the indole r i n g ; h o w e v e r , o t h e r s ( 3 3 , 34) have d e m o n s t r a t e d t h a t f o r m a t i o n of a hydroperoxy g r o u p a t c a r b o n - 3 o f t h e i n d o l e r i n g was intermediate i n the o x i d a t i o n of i n d o l e d e r i v a t i v e s by v a r i o u s oxidants. T h i s l a t t e r p a t h w a y seems a more p l a u s i b l e r o u t e t o the p r o d u c t s observed by Yong et a l . (32). S c h a i c h and K a r e l (22) p o s t u l a t e d t h a t a n u n s p e c i f i e d t r y p t o p h a n r a d i c a l was a major c o n t r i b u t o r t o p r o t e i n ESR b e c a u s e t h e ESR s i g n a l o f peroxidized nonsulfhydryl protein strongly resembled the signal of peroxidized tryptophan. Their observation may indicate that a resonance-stabilized indole radical is also possible. H i s t i d i n e w i t h p e r o x i d i z i n g l i p i d was a l t e r e d b o t h a t t h e α-carbon and the i m i d a z o l e s i d e c h a i n (23, 3 5 ) . Histamine, imidazole acetic a c i d and i m i d a z o l e lactic acid evidently 2


FEEDS

2

2


4.

GARDNER

Effects

of Lipid

73

Hydroperoxides

RSH


[RS«]

Alanine HS Aldehyde adducts (Thiazolidines) 2

RSSR

RS0 H 2

RSR

RSO3H

RS0 SR 2

Figure

5. Pathways

of cysteine

(RSH) degradation lipid.

by exposure

to

peroxidizing

Figure 6. Mechanism of cysteine-fatty acid adduct formation from the reaction of 13-hydroperoxylinoleic acid and cysteine in the absence of 0 . The epoxyallylic radical at the top originates from the oxydiene radical of 13-hydroperoxylinoleic acid (abbreviated structure) and RS · is the cysteine thiyl radical. (Reproduced with permission from Ref. 28.) 2


74


m

-Si Su


-««» Ο

•S

•S

f0 "δ

1 Ci,

Ο

& s; Q

-s:

I

•I ε


FEEDS


4.

GARDNER

Effects of Lipid

75

Hydroperoxides

arose from a t t a c k on o r v i c i n a l t o the α-carbon. Degeneration o f t h e i m i d a z o l e r i n g was i n t i m a t e d b y t h e f o r m a t i o n o f v a l i n e and a s p a r t i c a c i d . Additional research with the h i s t i d i n e derivatives, h i p p u r y l h y s t i d y l l e u c i n e and N - b e n z o y l h i s t i d i n e , accentuated the degradation of the imidazole group (36). These derivatives were d e s i g n e d t o model the s t r u c t u r a l environment of h i s t i d y l residues i n p r o t e i n , thus attack on t h e s i d e c h a i n may b e m o r e i m p o r t a n t i n p r o t e i n s t h a n w i t h free histidine. Accordingly, the main products from peroxidation o f N - b e n z o y l h i s t i d i n e were N-benzoylasparagine and N - b e n z o y l a s p a r t i c a c i d . The products from degeneration of lysine caused by p e r o x i d i z i n g m e t h y l l i n o l e a t e (13) a r e shown i n F i g u r e 8 . The structures of the products are indicative of r a d i c a l reaction a t b o t h t h e α-carbon and t h e s i d e c h a i n . Of p a r t i c u l a r i n t e r e s t is 1,10-diamino-l,10-dicarboxydecane, which p o t e n t i a l l y could provide a c r o s s l i n k between l y s i n y l residues i n proteins. Presumably, a C-6 r a d i c a l of 2-aminohexanoic a c i d originated from s c i s s i o n o f l y s i n e ε-amino g r o u p , a n d s e l f - c o m b i n a t i o n o f the C-6 r a d i c a l generated the dimer. The ε-amino group a l s o provides the site f o r c r o s s l i n k i n g by malondialdehyde as explained i n the following section. F i n a l l y , m e t h i o n i n e a n d t y r o s i n e a r e known t o be s e n s i t i v e to p e r o x i d a t i o n . M e t h i o n i n e was o x i d i z e d t o m e t h i o n i n e s u l f o x i d e in the presence of peroxidizing methyl linoleate (13) or peroxidizing o i l (37), i l l u s t r a t i n g the ease of radical i n i t i a t i o n on s u l f u r s u b s t i t u e n t s . The r a d i c a l d e s t r u c t i o n o f t y r o s i n e i s k n o w n ( 3 8 ) , b u t I am n o t a w a r e o f a n y s t u d i e s t h a t specifically subject tyrosine to peroxidized lipid. E x t r a p o l a t i o n from o t h e r r a d i c a l r e a c t i o n s of t y r o s i n e i n d i c a t e s that the i n i t i a l event i s Η-abstraction of the phenol to a f f o r d a phenoxy r a d i c a l . Effect of nonradical oxidation products on p r o t e i n . The aldehydes formed from l i p i d a u t o x i d a t i o n b y R e a c t i o n G have a p r o p e n s i t y t o r e a c t w i t h amino groups t o form a S c h i f f base (Reaction M). R -CH = 0 + R-NH 1

2

•

R - C H= N-R 1

(M)

The i m p l i c a t i o n s o f S c h i f f b a s e f o r m a t i o n i n b i o l o g i c a l s y s t e m s have b e e n r e v i e w e d i n more d e t a i l e l s e w h e r e ( 1 2 , 1 4 , 1 7 ) ; t h u s , t h i s aspect o f l i p i d o x i d a t i o n w i l l be b r i e f . With p r o t e i n s , S c h i f f base formation w i l l occur o n l y w i t h amino a c i d r e s i d u e s p o s s e s s i n g a s i d e c h a i n amino group, i n c l u d i n g , o f c o u r s e , t h e amino t e r m i n u s . The ε-amino group of lysine i s important i n this regard, and t h e l o s s of bioavailability of l y s i n e by S c h i f f base formation is a n u t r i t i o n a l concern.


XENOBIOTICS IN FOODS A N D F E E D S

0


^Peroxidation

,0H

Aspartic

0 NH

?

yi^OH

γ

Figure

8. Products

of lysine exposed

0Η

Alanine

Glycine

to peroxidizing

methyl

linoleate (13).


4.

GARDNER

Effects

of Lipid

Hydroperoxides

The bifunctional malondialdehyde has c r o s s l i n k i n g as i l l u s t r a t e d by R e a c t i o n N.

caused

protein

0 = (N) R - N H - C H = C H - C H = NR Other derivatives from r e a c t i o n a c i d s have been d e s c r i b e d (39).


Radical Reactions

of

of

malondialdehyde

and

amino

Nonproteins

R a d i c a l s i n i t i a t e d b y l i p i d p e r o x i d a t i o n a f f e c t a number of nonprotein biochemicals. I n g e n e r a l , most o f t h e l a b i l e compounds a r e c h a r a c t e r i z e d as p o s s e s s i n g a n e a s i l y a b s t r a c t a b l e hydrogen. Accordingly, a n t i o x i d a n t s and Η-donors, such as α-tocopherol, a s c o r b i c a c i d and g l u t a t h i o n e , f a l l i n t o this category. L i p i d hydroperoxide caused the o x i d a t i o n of α-tocopherol to α-tocopherolquinone through an u n i d e n t i f i e d intermediate (40). P o r t e r e t a l . (41) proposed a mechanism o f o x i d a t i o n t h a t i n c l u d e s an i n t e r m e d i a t e from c o m b i n a t i o n o f α-tocopherol semiquinone w i t h a peroxy r a d i c a l (Reaction 0 ) .

(ο)

The a b s e n c e o f 0 causes a s h i f t i n products to the formation of α - t o c o p h e r o l - l i p i d adducts (42, 43) v i a t h e c o m b i n a t i o n o f a l i p i d oxy r a d i c a l w i t h α-tocopherol semiquinone (Reaction P ) . 2



78

FEEDS

Reaction Ρ i s very s i m i l a r to the combination reaction of l i p i d oxy r a d i c a l w i t h the cysteine t h i y l r a d i c a l ( F i g . 6 ) . Both combination r e a c t i o n s proceed o n l y i n the absence o f 0 , i m p l y i n g t h e 0 e f f e c t i v e l y competes f o r t h e r a d i c a l s i n v o l v e d . Under c e r t a i n c o n d i t i o n s a s c o r b i c a c i d i s an a n t i o x i d a n t probably because i t r e a d i l y loses Η to a b s t r a c t i o n . Attention a l s o has been given t o the p r o o x i d a t i v e e f f e c t of a s c o r b i c acid i n the presence of t r a n s i t i o n metal ions (44). It is thought that ascorbic a c i d reduces metal ions which i n t u r n a r e more e f f e c t i v e i n c a t a l y z i n g l i p i d o x i d a t i o n . Consequently, a s c o r b i c a c i d becomes o x i d i z e d t o d e h y d r o a s c o r b i c a c i d . The d e s t r u c t i o n o f β-carotene d u r i n g l i p i d peroxidation i s r e a d i l y observed by bleaching of the carotene color (44). Presumably, β-carotene o x i d a t i o n i s i n i t i a t e d by H - a b s t r a c t i o n , and such a mechanism has been proposed f o r t h e c o o x i d a t i o n o f carotenoids during the lipoxygenase catalyzed oxidation of p o l y u n s a t u r a t e d f a t t y a c i d s ( 4 5 ) , a s shown b y R e a c t i o n Q.


2

2

R00-

ROOH (Q)

carotene

polyene

Mutagenicity Induced by L i p i d

r a di i c a l

y

.

oxidized carotene

Oxidation

F r e e r a d i c a l o x i d a t i o n i n v i v o h a s b e e n much t o u t e d as a detriment to both h e a l t h and l i f e . Indeed, aberrant free r a d i c a l r e a c t i o n s have been c i t e d as c o n t r i b u t o r s t o a g i n g (46, 47) and cancer (47, 4 8 ) , b u t u n e q u i v o c a l evidence for these claims often i s l a c k i n g . The c o n n e c t i o n between free r a d i c a l s and t h e p r o m o t i o n o f cancer has r e c e i v e d t h e most attention. As d i s c u s s e d l a t e r , the evidence is compelling that lipid hydroperoxide activates c e r t a i n carcinogens by cooxidation. A d i r e c t mutagenic e f f e c t of l i p i d hydroperoxides has b e e n s o u g h t f o r some t i m e w i t h v a r y i n g s u c c e s s . Recently, the Ames t e s t h a s b e e n u t i l i z e d t o d e m o n s t r a t e w e a k m u t a g e n i c i t y of b o t h p e r o x i d i z e d f a t t y a c i d (49) and i s o l a t e d hydroperoxides


4.

GARDNER

Effects

of Lipid

Hydroperoxides

of methyl l i n o l e a t e (50). B e c a u s e cumene h y d r o p e r o x i d e and t - b u t y l h y d r o p e r o x i d e were a l s o f o u n d t o be m u t a g e n i c , while p e r o x i d e s , p e r a c i d s and H 0 w e r e n o t , t h e m u t a g e n i c i t y was attributed to the hydroperoxide group (50). As shown i n T a b l e I , we h a v e a l s o o b s e r v e d w e a k m u t a g e n i c i t y f o r methyl 13-hydroperoxylinoleate by the Ames test (51). A third l a b o r a t o r y has f a i l e d to f i n d m u t a g e n i c i t y f o r l i n o l e i c a c i d hydroperoxide (52). The reason for weak m u t a g e n i c i t y of hydroperoxide i s not c l e a r . I t i s known t h a t f r e e r a d i c a l damage t o n u c l e i c a c i d s c a n be i n d u c e d b y r a d i a t i o n ( 5 3 ) , and DNA r a d i c a l s h a v e b e e n d e t e c t e d a f t e r e x p o s u r e o f DNA t o lipid p e r o x i d a t i o n (54). H o w e v e r , i t may b e e r r o n e o u s t o e x t r a p o l a t e t h e ESR s i g n a l s i n m o d e l s y s t e m s i n t o r e l e v a n c e c o n c e r n i n g i n v i v o DNA d a m a g e w i t h c o n c o m i t a n t m u t a g e n i c i t y . 2


79

2

It has been i m p l i e d t h a t secondary p r o d u c t s of lipid a u t o x i d a t i o n are mutagenic. Interest i n t h i s area of research was s t i m u l a t e d when M u k a i a n d G o l d s t e i n ( 5 5 ) , as w e l l as others, reported that malondialdehyde e l i c i t e d a mutagenic response b y t h e Ames test. Since some e v i d e n c e for DNA c r o s s l i n k i n g by malondialdehyde has b e e n shown i n c h e m i c a l models (56), i t m i g h t be presumed t h a t t h i s r e a c t i o n i s the molecular b a s i s of the mutagenicity. However, the importance o f t h e o b s e r v e d r e s p o n s e was q u e s t i o n e d b y M a r n e t t a n d T u t t l e (57), who f o u n d v e r y w e a k m u t a g e n i c i t y w i t h h i g h l y p u r i f i e d malondialdehyde. A c c o r d i n g to them, i m p u r i t i e s from the use of tetraethoxypropane to generate malondialdehyde probably were r e s p o n s i b l e for the greater mutagenicity observed by others. As p o i n t e d o u t i n the t e x t above, l i p i d epoxides are common s e c o n d a r y p r o d u c t s o f a u t o x i d a t i o n . F o r a number o f p o t e n t mutagens, l i k e benzo[or]pyrene, the u l t i m a t e mutagen has been found t o be an e p o x i d e o f t h e p a r e n t compound, w h i c h i n t u r n undergoes n u c l e o p h i l i c s u b s t i t u t i o n by the amino group o f a DNA b a s e p a i r , s u c h a s g u a n i n e ( 5 8 - 6 0 ) . I t has been p o s t u l a t e d b y some w o r k e r s t h a t l i p i d e p o x i d e s a l s o may b e m u t a g e n i c b y a s i m i l a r mechanism. A c o n v i n c i n g m u t a g e n i c r e s p o n s e was n o t o b t a i n e d when e i t h e r c i s - o r t r a n s - 9 , 1 0 - e p o x y o c t a d e c a n o i c acid was i n j e c t e d i n t o m i c e ( 6 1 ) . H o w e v e r , many m u t a g e n i c epoxides have been c h a r a c t e r i z e d as h a v i n g e l e c t r o n - w i t h d r a w i n g s u b s t i t u e n t groups that cause the epoxide t o be more s u s c e p t i b l e to n u c l e o p h i l i c attack (62). F o r t h i s r e a s o n , we t e s t e d b y t h e Ames m e t h o d ( 6 3 ) t h e m u t a g e n i c i t y o f a n u m b e r o f f a t t y epoxides w i t h v i c i n a l f u n c t i o n a l i t y as shown i n F i g u r e 9 . These fatty epoxides were i s o l a t e d from a m i x t u r e o f p r o d u c t s obtained after the free r a d i c a l decomposition of 13-hydroperoxylinoleic a c i d (11). M e t h y l e s t e r s were s y n t h e s i z e d from f a t t y acids with diazomethane. Despite the presence of e l e c t r o n - w i t h d r a w i n g g r o u p s v i c i n a l t o t h e e p o x i d e , m u t a g e n i c i t y was n o t observed even a t t h e 2000 pg l e v e l p e r p l a t e ( 5 1 ) . Apparently, the



132

Control 114

233°

129

130

130

176

by more than LSD.

1

Exceeds ' c o n t r o l

(

by more than LSD

1

144

158

171

235

S-9 Added

experiment.

171

158

164

300°

Exceeds ' c o n t r o l

Each column i n d i c a t e s a separate

164

538°

0

No. S-•9

TA 100

b

38

39

35

55

0

27

26

37

No. S-9

44

41

44

41

46

58

58

94

b

S-9 Added

TA 98

(Methyl E s t e r )

Revertants per p l a t e

f o r Mutagenicity of 13-Hydroperoxylinoleate

50

100

500

1000

pg/plate

Test compound,

Ames Test

Table I



GARDNER

Figure

Effects

of Lipid

Hydroperoxides

9. Structures of fatty ester epoxides tested for mutagenicity by the method of Ames et al. (63). (Reproduced with permission from Ref 51.)



82

X E N O B I O T I C S IN F O O D S A N D F E E D S

lack of response stems from t h e 1 , 2 - d i s u b s t i t u t i o n o f t h e e p o x i d e , w h i c h u s u a l l y d i m i n i s h e s t h e response ( 6 2 , 6 4 ) . The s i z e o f t h e h y d r o c a r b o n s i d e c h a i n s a l s o may h a v e a n e f f e c t . Systematic studies of a series of g l y c i d y l ethers indicated t h a t m u t a g e n i c i t y was c o n s i d e r a b l y r e d u c e d when t h e s i d e c h a i n exceeded 4-6 carbons (65). The r o l e o f l i p i d h y d r o p e r o x i d e s i n activating chemical mutagens i s more c o n v i n c i n g . A number o f studies have demonstrated that l i p i d hydroperoxides can i n i t i a t e the free radical oxidation of the carcinogen to the ultimate active form. F o r e x a m p l e , b e n z o [ a ] p y r e n e was o x i d i z e d t o t h e h i g h l y mutagenic 7,8-dihydroxy-9,10-epoxy-7,8,9,10tetrahydrobenzo[a]pyrene i n the presence of 13-hydroperoxylinoleic a c i d and the c a t a l y s t , hematin (66). S i m i l a r l y , F l o y d e t a l . (67) used 1 3 - h y d r o p e r o x y l i n o l e i c acid and h e m a t i n t o a c t i v a t e N-hydroxy-N-acetyl-2-amino-fluorene i n t o the carcinogens, nitrosofluorene and N-acetoxyacetylaminofluorene. T h u s , l i p i d h y d r o p e r o x i d e s may s e r v e a s efficient oxidants of a variety of chemical carcinogens that require o x i d a t i o n to an active form. This area of research appears to be p r o m i s i n g f o r f u t u r e w o r k .

Literature Cited 1. Gardner, H. W. "Autoxidation of Unsaturated Lipids," Chan, H. W.-S., Ed.; Academic:London, in press. 2. Frankel, Ε. N. Prog. Lipid Res. 1980, 19, 1. 3. Porter, Ν. Α.; Lehman, L. S.; Weber, Β. Α.; Smith, K. J . J. Am. Chem. Soc. 1981, 103, 6447. 4. Chan, H. W.-S.; Matthew, J . Α.; Coxon, D. T. J . Chem. Soc. Chem. Commun. 1980, 235. 5. Mihelich, E. D. J . Am. Chem. Soc. 1980, 102, 7141. 6. O'Connor, D. E . ; Mihelich, E. D.; Coleman, M. C. J . Am. Chem. Soc. 1981, 103, 223. 7. Pryor, W. Α.; Stanley, J . P. J . Org. Chem. 1975, 40, 3615. 8. Scheiberle, P.; Grosch, W.; Kexel, H.; Schmidt, H. L. Biochim. Biophys. Acta 1981, 666, 322. 9. Nakano, M.; Takayama, K.; Shimizu, Y . ; Tsuji, Y . ; Inaba, H.; Migita, T. J . Am. Chem. Soc. 1976, 98, 1974. 10. Hamberg, M. Lipids 1975, 10, 87. 11. Gardner, H. W.; Kleiman, R. Biochim. Biophys. Acta 1981, 665, 113. 12. Tappel, A. L. Fed. Proc. Fed. Am. Soc. Exp. Biol. 1973, 32, 1870. 13. Karel, M.; Schaich, K.; Roy, R. B. J . Agric. Food Chem. 1975, 23, 159. 14. Gardner, H. W. J . Agric. Food Chem. 1979, 27, 220. 15. Porkorny, J.; Janicek, G. Nahrung 1975, 19, 911.


4.

GARDNER

16. 17. 18.


19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43.

Effects of Lipid Hydroperoxides

83

Miquel, J.; Oro, J.; Bensch, K. G . ; Johnson, J . Ε . , Jr., "Free Radicals i n Biology," Pryor, W. Α., E d . ; Academic:New York, 1977, V o l . III, p. 132. Tappel, A. L., "Free Radicals i n Biology," Pryor, W. Α., Ed.; Academic:New York, 1980, V o l . IV, p. 1. Jacks, T. J.; Hensarling, T. P . ; Muller, L. L.; St. Angelo, A. J.; Neucere, N. J. Int. J . Pept. Protein Res. 1982, 20, 149. Funes, J.; Karel, M. Lipids 1981, 16, 347. Leake, L.; Karel, M. J . Food S c i . 1982, 47, 737. Nielsen, H. Lipids 1981, 16, 215. Schaich, K . ; Karel, M. Lipids 1976, 11, 392. Yong, S. H . ; Karel, M. J . Am. Oil. Chem. Soc. 1978, 55, 352. Zirlin, Α.; Karel, M. J. Food S c i . 1969, 34, 160. L i n , J. S.; Olcott, H. S. J . Agric. Food Chem. 1974, 22, 526. Lewis, S. E.; W i l l s , E. D. Biochem. Pharmacol. 1962, 11, 901. Roubal, W. T.; Tappel, A. L . Arch. Biochem. Biophys. 1966, 113, 5. Gardner, H. W.; J u r s i n i c , P. A. Biochim. Biophys. Acta 1981, 665, 100. Finley, J . W.; Wheeler, E. L.; Witt, S. C. J . Agric. Food Chem. 1981, 29, 404. Gardner, H. W.; Kleiman, R.; Weisleder, D . ; Inglett, G. E. L i p i d s , 1977, 12, 655. Gardner, H. W., unpublished data. Yong, S. H . ; Lau, S.; Hsieh, Y.; Karel, Μ., "Autoxidation i n Food and Biological Systems," Simic, M. G . ; Karel Μ., Eds.; Plenum:New York, 1980, p. 237. Sundberg, R. J., "Chemistry of Indoles," Academic Press:New York, 1970, p. 282-315. Saito, I . ; Imuta, M . ; Nakada, Α.; Matsugo, S.; Matsuura, T. Photochem. Photobiol., 1978, 28, 531. Roy, R. B.; Karel, M. J. Food S c i . 1973, 38, 896. Yong, S. H . ; Karel, M. J . Food S c i . 1979, 44, 568. Njaa, L. R . ; Utne, F.; Braekkan, O. R. Nature 1968, 218, 571. Neukom, Η., "Autoxidation i n Food and Biological Systems," Simic, M. G . ; Karel, Μ., Eds.; Plenum:New York, 1980, p. 249. Kikugawa, K . ; Machida, Y.; Kida, M . ; Kurechi, T. Chem. Pharm. Bull., 1981, 29, 3003. Gruger, Ε. Η., Jr.; Tappel, A. L. Lipids 1970, 5, 326. Porter, Ν. Α.; Lehman, L. S.; Khan, J. A. 184th ACS National Meeting, Organic Section 3, Kansas City, September 12-17, 1982. Gardner, H. W.; Eskins, K . ; Grams, G. W.; Inglett, G. E. Lipids 1972, 7, 324. Gardner, H. W., unpublished data.


84

44. 45. 46. 47. 48. 49. 50. Downloaded by MONASH UNIV on November 13, 2015 | http://pubs.acs.org Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch004

51. 52. 53. 54. 55. 56. 57. 58. 59.

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Xenobiotics in Foods and Feeds - American Chemical Society

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