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FreeRadical Biology& Medicine, Vol. 12, pp. 63-81, 1992
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Review Article
ROUTES OF FORMATION AND TOXIC CONSEQUENCES
OF LIPID OXIDATION PRODUCTS IN FOODS
STAN KUBOW
School of Dietetics and Human Nutrition, Macdonald Campus of McGill University, Ste. Anne de Bellevue, Quebec H9X IC0
(Received 6 June 1991; Revised 26 August 1991; Accepted 26 August 1991)
Abstract--Lipid oxidation in foods is initiated by free radical and/or singlet oxygen mechanisms which generate a series of
autocatalytic free radical reactions. These autoxidation reactions lead to the breakdown of lipid and to the formation of a wide
array of oxidation products. The nature and proportion of these products can vary widely between foods and depend on the
composition of the food as well as numerous environmental factors. The toxicological significance of lipid oxidation in foods is
complicated by interactions of secondary lipid oxidation products with other food components. These interactions could either
form complexes that limit the bioavailability of lipid breakdown products or can lead to the formation of toxic products derived
from non-lipid sources. A lack of gross pathological consequences has generally been observed in animals fed oxidized fats. On
the other hand, secondary products of lipid autoxidation can be absorbed and may cause an increase in oxidative stress and
deleterious changes in lipoprotein and platelet metabolism. The presence of reactive lipid oxidation products in foods needs
more systematic research in terms of complexities of food component interactions and the metabolic processing of these
compounds.
Keywords--Lipid oxidation, Free radical, Toxicity, Atherosclerosis, Mutagenicity, Food, Vitamin E, Oxidative stress
are particularly susceptible to oxidation in cooking
and frying, beneficial aspects of wide utilization of
these fats may be counteracted through formation of
toxic oxidation products and increased antioxidant
requirements. An exploration of the possible toxic
consequences may be particularly relevant at this
time since there is increasing evidence of a pathogenic
role of endogenous lipid oxidation in a number of
chronic and acute disease states2'3 and due to the recent advances in our ability to identify and quantify
lipid oxidation products in biological systems via analytical techniques such as gas chromatography-mass
spectrometry (GC-MS), high pressure liquid chromatography (HPLC), and nuclear magnetic resonance (NMR).
There are a couple of caveats that should be noted
regarding our present state of knowledge. First of all,
much of our information concerning lipid oxidation
processes in foods has derived from the study of individual oils or simple model systems, whereas food is a
complex heterogenous material in which lipids exist
in environments that are quite different from a single
phase model system. The complexity of lipid free radical reactions of foods, especially at higher temperatures, is increased by cross-reactions with other foods
components. Such interactions could alter dramatically the spectrum and bioavailability of toxic constituents produced in a food system. Secondly, extrapola-
INTRODUCTION
The postulated associations between high fat diets
and chronic diseases such as heart disease and cancer
are well known. In this regard, however, the role of
food preparation techniques in chronic disease has
not been systematically investigated despite the fact
that most of the foods that we consume today have
been subjected to various degrees of processing, oxidation, and heat treatment. Convenience and snack
foods have risen in popularity, particularly in deep-fat
fried foods such as chicken, fish, and French-fried potatoes. These foods absorb substantial amounts of the
oils used for deep-frying, l Complex chemical and
physical changes occur under these conditions, causing fat deterioration which may reach a point whereby
the nutritional value and safety characteristics of the
oil may be affected. Since polyunsaturated fatty acids
Stan Kubow received his Ph.D. in the department of Nutritional Sciences at University of Guelph at Guelph, Ontario in 1984,
and did his postdoctoral work there and at the School of Pharmacy
at the University of Toronto. He was appointed to his present position as an Assistant Professor at the School of Dietetics and Human
Nutrition at Macdonald Campus of MeGill University in 1987. His
laboratory is currently investigating free radical-mediated mechanisms of diabetic- and drug-induced teratogenicity as modulated by
diet; mechanisms of alterations of lipoprotein metabolism by dietary fats; and the role of dietary fats on eicosanoid metabolism and
fetal development during pregnancy.
63
64
S. K u B o w
tions regarding experimental animal toxicity data to
the human context are difficult to make, as oxidized
fats are typically fed at high levels as the sole source of
fat in the diet as opposed to the typical mixed diet of
humans.
The present review will look at some of the major
mechanisms by which lipid oxidation products are
generated during the processing of food, and will discuss the potential toxicological relevance of these oxidation products. It should be noted that due to the
wide scope of this subject an in-depth review of all of
the prevailing issues cannot be made. The reader is
referred to a number of excellent recent reviews which
give a complementary examination of the topics addressed. 4-21 Moreover, it is possible that important
work may have been missed in this coverage as well as
in previous reviews.
AUTOXIDATION
It is generally accepted that the process of lipid oxidation of foods proceeds by way of a free radical mechanism called autoxidation, which is described in
terms of initiation, propagation, and termination proc e s s e s . 5-7 This term is a misnomer in that this oxidative process requires a catalyst in biological systems
since the triplet state of oxygen forbids direct reaction
of molecular oxygen with other biomolecules. 6-7,z2
Activation of molecular oxygen can occur either
through a direct input of energy, as in the case of light
converting oxygen to singlet oxygen, or by a reduction
via some catalyst. Catalysis of lipid oxidation in foods
could proceed via both nonenzymatic and enzymatic
mechanisms. 6-7'22 Once the reaction has been initiated via a catalyst, however, the process is autocatalytic in the sense that the oxidation products catalyze
the reaction and cause an increase in the reaction rate
as oxidation proceeds. The following initiation, propagation, and termination reactions characterize the general scheme of autoxidation:
Initiation:
Propagation:
X" + LH ----> L" + XH
L" + 02 ----> LOO"
LOO" + LH ----> L" + LOOH
Termination:
LO" + LO" ---->
LOO" + LOO" ----> non-radical
polymers
L" +L" ---->
LOO" + L" ---->
Autoxidation is initiated by an abstraction of a hydrogen atom from the methylene between a cis dou-
ble bond pair of an unsaturated fatty acid (Fig. 1). The
abstraction of a hydrogen adjacent to a double bond is
favored because of the formation of a stable allylic
radical in which the electrons are delocalized over
three carbon a t o m s . 6-9'23
In the propagation reaction, the lipid radical
formed following initiation will react with molecular
oxygen to form a peroxyl radical (Fig. 1). In most food
systems where oxygen is present the reaction of the
lipid radical with oxygen is very fast, and therefore the
concentration of the peroxyl radical is much higher
than that of L. 6,7,10 The reaction of a peroxyl radical
with another polyunsaturated fatty acid side chain of
a lipid molecule yields a lipid hydroperoxide and a
lipid radical, thus conserving the number of lipid radicals in the reaction sequence. Due to resonance stabilization of the L. species, the reaction is typically accompanied by shifting of the double bonds resulting
in the formation of a mixture of isomeric hydroperoxides usually containing conjugated diene groups (Fig.
1). The newly formed lipid hydroperoxyl radical can
itself abstract hydrogen from another fatty acid,
thereby propagating the reaction. 7'24 The abstraction
of a hydrogen from another unsaturated molecule by
the peroxyl radical is the slowest and rate-limiting
s t e p . 6 As the propagation step is relatively slow, hydrogen abstraction is selective for the most readily abstractable hydrogen from unsaturated f a t s . 7'11'23 The
ease of hydroperoxidation depends on the number of
double bonds present. For example, for a series of
unsaturated fatty acids ranging from one to five double bonds, the ratio of oxidizabilities increases linearly
by the order of 1:2:3:4:5. 2s The length of the propagative cycle of lipid oxidation is thus increased by the
degree of lipid unsaturation and limited by the availability of these lipids. The overall reaction has a pyramidal effect in which a relatively few initiating radicals break down many polyunsaturated fatty acids.
Termination is the removal of free radicals, either
by the combination of two radicals to form a nonradical product or where the propagation reaction is
terminated in the presence of a hydrogen or electron
donor. 1° The former reactions become more important when the oxygen concentration is low, towards
the interior of the fat system.
The nature of the initiating reactive intermediate
has been difficult to determine, since very low concentrations of the initiating species are usually generated, particularly during the induction period when
the extent of oxidation is very low due to the presence
of varying amounts of natural or synthetic antioxidants in the fat. 7'23'26 Evidence indicates that lipid oxidation in foods may be initiated by a number of mechanisms including: (a) singlet oxygen; (b) enzymatic
Oxidized lipids in foods
Catalysts, 1
light
-H*
I
65
Hydrogen abstraction
Molecular rearrangement
0 2 uptake
Peroxy radical
II
~
II Dimers; polymers; cyclic
peroxides; endoperoxide
radicals.
OI
Conjugated diene
o 1""
1
DecomDo$ition
Products
Lipid hydroperoxide
y
0
N~/ ~
(Aldehydes: ketones;
hydrocarbons; acids;
I
/
turans; alcohols;
O
~ -OH •
and epoxides)
Cleavage
t
• Aldehydes; alkyl radicals;
Polymers;
dimers; keto,
hydroxy, and
epoxy compounds.
II
~
~
O Alkoxy radical
semialdehydes.
Fig. 1. A simplified scheme showing products formed from autoxidation of unsaturated lipids.
and non-enzymatic generation of partially reduced or
free radical oxygen species (i.e., hydrogen peroxide,
hydroxyl radical); (c) active oxygen iron complexes
(ferryl iron); and (d) thermal- or iron-mediated homolytic cleavage of hydroperoxides. 6-9,~~,~2
Singlet oxygen
Generation of hydroperoxides of unsaturated fatty
acids can be formed following exposure to light in the
presence of oxygen and a photosensitizer which activates the oxygen. There are two types of sensitized
photoxidation pathways reported in foods. 7-9'12'27 In
the type 1 photosensitization reaction, the triplet photosensitizer absorbs light and the excitation is used to
react with an acceptor which then reacts with ground
state triplet oxygen to form singlet oxygen. An example of this pathway is illustrated by riboflavin-sensitized photoxidation of unsaturated fatty acid esters. 27
In the type 2 photosensitization mechanism, following reaction with light, the excited photosensitizer
reacts with molecular oxygen in the triplet groundstate to form an oxygen molecule of singlet excitedstate IO2. Singlet oxygen is more reactive than triplet
oxygen and can react rapidly with carbon-carbon double bonds of unsaturated fatty acids to form hydroperoxides. Another singlet oxygen reaction of potential
significance to lipid oxidation is the oxidation of cholesterol to form an allylic hydroperoxide. 5 Photosensitizing substances such as chlorophyll, flavins, retinal,
certain heme compounds, and added synthetic color-
ings could play a prominent role in the photoxidation
of unsaturated lipids in foods.l~'zs Vegetable oils frequently contain chlorophylls or porphyrin compounds which can undergo photosensitization in the
presence of visible light. 2s Removal of such compounds in the refinement and bleaching of vegetable
oils can effectively reduce deterioration of these oils. zs
The importance of singlet oxygen as an active initiator of oxidation of oils is also illustrated by the effect
of singlet oxygen quenchers such as/3-carotene and
a-tocopherol in decreasing levels of 12-hydroperoxides in soybean esters, s
The singlet oxygen reaction involves an "ene" reaction as opposed to the free radical process of hydrogen
abstraction from the allylic carbons of unsaturated
fatty acids. 6-s Oxygen is inserted at the ends of double
bonds with the consequent migration of the double
bond to form an allylic hydroperoxide in the trans
configuration. The cis double bond shifts to yield hydroperoxides in the trans configuration because of its
lower thermal energy state and steric hindrance. 7 The
ene reaction with the double bond between carbons
12 and 13 would thus produce the 12 and 13 hydroperoxide isomers. The resulting hydroperoxides
would proceed via free radical chain reaction as the
main mechanism of oxidative decomposition. 9
Enzyme-induced lipid oxidation
Enzyme catalyzed lipid oxidation via lipoxygenase
and cytochromes P450 catalysis has been suggested to
66
S. KuBow
play a role in the production of hydroperoxides in
f o o d s . 7'25'29 Recent reports of lipoxygenase activity in
fish gill and skin tissues have suggested that these endogenous enzymes may be a source of initiatory free
radicals leading to the production of lipid oxidation
compounds in fish. 3° Lipoxygenases initiate the oxidation of polyunsaturated fatty acids via a free radical
mechanism to form specific hydroperoxides which
can be further oxidized to trihydroxy fatty acid derivatives. 5'7'28 In most tissues, the predominant product
contains an oxygenated moiety at the 13-position
with smaller amounts of the 9-isomer being
forlned. 5,7'25 The breakdown of these unstable hydroperoxides could contribute to subsequent lipid oxidation and further chain reactions in both plant and
animal tissues. The improvement of shelf life of fish
and frozen stored vegetables following heat treatment
has been explained on the basis of the thermal instability of lipoxygenase enzymes. 7,28,3° In addition to lipoxygenases, animal tissues also contain peroxidases
and cyclooxygenases which have been implicated to
initiate and promote lipid oxidation in a postmortem
tissues as these enzymes are active towards unsaturated fatty acid substrates to form hydroperoxides. 7
There is also evidence that lipid oxidation could be
initiated via enzymatic generation of hydroxyl radical
and singlet oxygen occurring through lipoxygenase,
prostaglandin synthase or microsomal oxidase activities. 7
The presence of a membrane fraction capable of
forming lipid oxidation products in the presence of
NADH or NADPH and iron has suggested a role of
these fractions in lipid oxidation of muscle f o o d s . 7'11,30
In mammalian tissues, cytochromes P450, when in
the ferric form, have been indicated to catalyze chainbranching reactions very efficiently. 11 The postulated
reaction is:
CYT
P4503+ +
LOOH ---> CYT P4503÷ - OH- + LO"
Unsaturated fatty acid will bind to cytochromes P450
at the drug binding site which also may be the site
where lipid hydroperoxides are decomposed by heme
iron.~ 1 The enzymatic lipid oxidation in skeletal muscle microsomes is dependent on the presence of
NADH or NADPH and also requires ADP and iron.
The maximum rate of this reaction occurs under pH
and cofactor concentrations which might be expected
to occur in postmortem muscle tissues. 3°
Mitochondrial lipid oxidation has also been proposed to play a role in oxidation of lipids in animal
tissues. The NADH dehydrogenase of the respiratory
chain could initiate lipid oxidation by donating electrons to ADP-chelated Fe 3+ and thereby resulting in
free radical generation. I~ An enzymatic lipid oxida-
tion system in fish muscle mitochondria has been
demonstrated and was found to be similar to that of
fish microsomes in terms of co-factor requirements
and optimal pH. 3°
Activated oxygen-iron interactions
Harel and Kanner reported that the interaction of
hydrogen peroxide with methemoglobin in muscle
tissues can activate methemoglobin to act as an initiator of lipid oxidation in animal muscle foods. 31 The
activated heme protein appears to be a porphyrin (P)
cation radical, P "+ - Fe 4÷ = 0 which is also a ferryl
species. 3~ Initiation of lipid oxidation by this species
can occur as following:
P'÷ - F e 4÷ = O + RH ----> P - F e 4÷ = O + R" + H ÷
The action of activated myoglobin appears to be more
selective than that of the hydroxyl radical since molecules known to interact with the hydroxyl radical did
not inhibit lipid peroxidation initiated by activated
myoglobin. 5
A reactive species that has been postulated to initiate lipid oxidation during the processing of foods is
the hydroxyl radical. 7,32,33 Hydroxyl radical is one of
the more potent oxidants due to its capacity to react at
high rates with virtually any organic compound. 2 Hydroxyl radical-induced lipid oxidation proceeds by
hydrogen abstraction from the methylene between
the cis double bond pair of an unsaturated fatty acid. 5
It is generally believed that hydroxyl radical formation could proceed via the Fenton-catalyzed HaberWeiss reaction in which free or chelated forms of iron
serve as a catalysts for the reaction between hydrogen
peroxide and superoxide anion. 6,11
Metal
0 2 0 2 + O~ ......... "> 0 2 + " O 0 + O H Catalyst
The Haber-Weiss reaction is most effectively catalyzed by reduced iron complexes, which can be generated by the reaction of ferric complexes with superoxide anion and by cellular constituents such as ascotbate. 5'1~ This so-called iron-redox cycling catalysis of
lipid peroxidation has been suggested to be a driving
force for the generation of high concentrations of
ferrous ions which can in turn generate hydroxyl radicals.
Miller et al. 34 recently proposed an alternative to
the traditional Fenton reaction for the role of iron in
lipid oxidation reactions in biological systems. These
authors suggested that an Fe(II):Fe(III) complex acts
as an initiator of lipid oxidation and that formation of
this complex was dependent on overall rates of oxidation and reduction of iron not greatly exceeding each
other in the surrounding environment. It was felt that
Oxidized lipids in foods
the proposed model was better reconciled to the apparent requirement of both the ferrous and ferric forms
in several model systems of lipid oxidation.
Hydroperoxide breakdown
A primary mechanism by which initiation could
take place is via iron-mediated homolytic cleavage of
preformed hydroperoxides which generates organic
free radicals. 7'35 Metal catalysts may be coordinated
with ligands as complexes, may exist as dimers or
higher molecular weight species or complex with hydroperoxides to catalyze autoxidation and decomposition. 28 Alkoxyl radicals are believed to be predominantly formed in the early stages of oxidation by the
interaction of the hydroperoxide with the reduced
form of the metal via one-electron transfer. 23
LOOH + M "+ + H + ----> LO" + OH- + M ('-1)+ + H20
It has also been suggested that due to the increase in
the oxidized form of the metal during the later stages
of lipid oxidation this metal could react directly with
the hydroperoxide by the mechanism of one-electron
transfer to form alkylperoxide lipid radicals. 23
LOOH + M ("+l)+ + HO- ----> LOO" + M "+ + H20
Oils and fats that are poorly handled or stored frequently contain hydroperoxides which can serve as
substrates for metal-catalyzed decomposition to form
initiatory free radicals. 28A common way that pro-oxidant metal ions enter food is via the water or spices
used in food preparation. 3s Additionally, disruption
of muscle membranes during grinding, cooking, or
deboning breaks up the well organized structure of
animal cells to bring together lipids and pro-oxidant
catalysts including metal ions. 32
Hydroperoxides can decompose thermally or in
the presence of metal catalysts. A major pathway of
thermal hydroperoxide decomposition involves a homolytic cleavage of the O-O bond of the hydroperoxide to produce alkoxyl and hydroxyl free radicals
(Fig. 1).28 Cleavage of alkoxyl radicals or reaction of
these radicals with substrate propagates the chain reaction. The alkoxyl radicals undergo C-C bond scission
which can occur on either side of the radical to produce an alkyl radical on one side and a vinyl radical
on the other (Fig. 2). 9,24 The alkyl radical can react
with a hydrogen radical, hydroxyl radical or molecular oxygen to generate hydrocarbons, alcohols, and
hydroperoxides, respectively. The vinyl radical is very
reactive and may also react with hydroxyl radical, hydrogen radical, or molecular oxygen to generate aldehydes and olefins. 9 Alkoxyl radicals can also undergo
epoxidation or react with each other or with a corresponding alkyl radical to terminate the radical chain
67
by formation of stable products. The recombination
of alkoxyl radicals leads to the formation of dialkylperoxides. 23,2s
The cleavage reactions explain most of the volatile
products such as carbonyls, alcohols, esters and hydrocarbons identified from the decomposition of hydroperoxides of oleate, linoleate and linolenate. 9 On
the other hand, numerous speculative mechanisms
have been proposed to account for the formation of
substituted furans, epoxy aldehydes, ketones, lactones, alkynes, and aromatic compounds. 8a3,24Cleavage of unsaturated aldehydes has been implicated to
be the source of many of the volatile compounds that
cannot be explained by cleavage of monohydroperoxides such as the formation of lower aldehydes, alcohols, acids, alkyl formates, acrolein, benzene, furans,
and hydrocarbons, s,13,24Dihydroperoxides, hydroperoxy cyclic peroxides and other polymeric materials
are also potential sources of volatile compounds. 7,s
Thermal decomposition of cyclic peroxides from linoleate has been shown to produce most of the same
volatile decomposition products as the corresponding
monohydroperoxides. 9 Breakdown of cyclic peroxides, however, has been demonstrated to produce
some unique oxidation products such as unsaturated
methyl ketones. 9 A compound which has been of particular interest regarding lipid oxidation reactions in
foods is malondialdehyde. Malondialdehyde has been
suggested to be derived from intramolecular cleavage
of 5-membered monocyclic peroxides, 36 bicyclic endoperoxides 37 and from decomposition of 1,3-dihydroperoxides9'38; 1,4-dihydroperoxides and monohydroperoxides are believed to be less important precursors of this product. 9'38 More detailed explanations of
the postulated stereochemistry involved in the outlined mechanisms can be obtained by some excellent
reviews on this subject. 7-9'39
Radicals that do not unimolecularly decompose or
bimolecularly react with non-radical substrate react
with other radicals by combination or disproportionation to form dimers or intra- and intermolecular
cyclizations) 4,23 The probability of collision between
alkoxyl or peroxyl radicals to form some of these products increases during the later stages of autoxidation. 23 These nonvolatile products include various cyclic and aromatic monomers, dimers, and polymeric
compounds such as hydroperoxyl epoxides, hydroperoxyl cyclic peroxides, and dihydroperoxides. 9 In
certain model fatty acid systems such as autoxidized
methyl linolenate, monocyclic peroxides can be
formed in the same order of magnitude as monohydroperoxides. 9 Radical cyclization may occur when a
remote double bond, such as a/3-~,-double bond, is
present in the peroxyl radical, which allows for facile
68
S. K u a o w
(a)
(b)
R 2 - CH • CH ; CH * CH 2 I
~
I
= O
P
I
R
1
- CH
2
saturated
vinyl radical
alkenal
•
2
2-alkenal
R - CH - CH"
2
R 1- C H 2 "
•
OH/
CH
R 2 - CH • CH - C H O
aldehyde
H,
11
-
1
I
- CHO
R 2-CH-CH -OH
R
R
-
2
R
2
\..
alpha-olefin
-
CH
"
CH
2
R - CH OH
1
2
alcohol
CHO
alkyl radical
R - CH
1
3
hydrocarbon
aldehyde
Fig. 2. A scheme depicting product formation from homolytic cleavage o f a hydroperoxide of an unsaturated fatty acid.
1,3 cyclization.7' 14Monocyclic peroxides, bicyclic peroxides, and epoxy alcohols are the products derived
from radicals formed from cyclization. 7'9
Polymerization reactions are very complex during
lipid oxidation and polymeric material is difficult to
characterize because of its complex structure.14 Intraand intermolecular combinations of alkoxyl, alkyl,
and peroxyl radicals to form dimers and polymers
with C-O-C and C-O-O-C cross-links are major reactions in high temperature oxidations? 2 Dimers are a
major component of nonvolatile products formed in
oxidized and heated fats and increase with the extent
of lipid oxidation. 24,4° The temperature of oxidation
and the hydroperoxide environment are key factors in
determining the role ofhydroperoxides which can act
as catalysts or may themselves take place in polymerization reactions.~4 Polymerization can involve mechanisms of both radical and nonradical addition. 23 At
temperatures between 60-120°C, decomposition of
hydroperoxides can proceed via ester formation with
other fatty acids and by homolytic decomposition. 41
These reactions can generate products which react
with other fatty acid molecules to form C-O-C bonds
and generate dimers, trimers and larger molecular
weight products. 4~ Considerable degradation or heating are not prerequisites for dimer formation since
Miyashita and co-workers demonstrated the formation ofdimers from methyl linoleate during the initial
stages of autoxidation at 30 ¢ after 24 h. 42 They suggested that most of the dimers were formed through
peroxide (C-O-O-C) linkages.
The compounds formed as a result of thermal oxidation are of special interest, since deep fried fat is
continuously or repeatedly used at elevated tempera-
tures in the presence of air and moisture. Formation
and decomposition of hydroperoxides is markedly increased during deep-fat frying. ~2 The peroxides and
hydroperoxides do not survive the heating process
and break down into oxy- and cyclic acids as the temperature is raised. 24 In this process, the nonvolatile
products which remain in the oil are absorbed into the
food and are subsequently ingested. 43 Concentrations
of volatile products such as saturated and unsaturated
aldehydes, ketones, hydrocarbons, alcohols, acids,
and esters are formed much more rapidly during the
deep-fat frying process until a balance is reached between the formation of volatiles and their decomposition or evaporation.~2 The removal of the fat through
absorption into the food helps to maintain the quality
of the fat, and fresh fat is added to replenish the frying
b a t h . 43 In establishments in which the fat is only occasionally used for frying and kept hot for long periods
of time or cycled through periods of high and low
temperatures, polymer formation more readily takes
place. After prolonged heating, dimeric and polymeric compounds are formed and can accumulate up
to a concentration of 10-20% without the functional
properties of the oil becoming noticeably changed. 44
Further, heating of heme-containing foods may increase lipid oxidation by the release of catalysts such
as non-heme iron from heme pigments and by disruption of membranes more readily exposing lipid constituents to oxygen and catalysts. 45
Cross-reactions of lipid oxidation products
Lipid hydroperoxides and their decomposition
products can interact with other biomolecules in
Oxidized lipids in foods
foods such as pigments, enzymes, proteins, amino
acids, membranes and DNA to produce a multitude
of other by-products of lipid oxidation.tS
The possibility of co-oxidation can increase the
complexity of oxidized products) 6 In the process of
autoxidation, the peroxyl radicals formed attack the
more readily oxidizable substrates due to their relative
unreactivity. Alkoxyl radicals, however, are much
more reactive and unselective than peroxyl radicals,
and as a consequence will react with more slowly oxidizable material such as proteins to increase the complexity of the oxidized products. 16 Lipid radical reaction can cause protein-protein crosslinks; proteinlipid crosslinks, and protein scission. 7'46 A major
pathway in lipid-protein interactions is the reaction of
primary amines with the carbonyl products of lipid
oxidation to form N-heterocyclic compounds including imines, pyridines and pyrroles, t5 Amino sugars
can react with malondialdehyde to form pyrroles and
other heterocyclic compounds. 47
In the presence of other food components, lipid
oxidation reactions are also complicated by the possibility of termination by compounds other than those
derived from lipids. The Maillard reaction is of importance in this regard. Several Maillard products
which may exert antioxidative actions in foods are
produced from the Maillard reaction between reducing sugars and amino acids, peptides, or proteins. 10,47
For example, lipid autoxidation in heated muscle
foods continues to increase up to approximately
77°C; beyond this temperature or after prolonged
heating, concentrations of lipid autoxidation products decrease due to the formation of significant concentrations of antioxidative Maillard products. 17
Maillard type brown pigments are also formed from
interactions of lipid oxidation carbonyl compounds
with nitrogenous food constituents such as proteins
and amino acids. 48 Antioxidative effects of Maillard
products may occur via several mechanisms including metal chelation, reduction of hydroperoxides to
non-radical products, and donation of hydrogen
atom to break the radical chain. 1°'47
Termination
Termination reactions via the combination of two
radicals become important when edible oils are
heated at elevated temperatures as indicated by formation of polymers in frying oils.rE At 160°C hydroperoxides begin to decompose spontaneously and the
radical concentration can become relatively high allowing greater likelihood for radical-radical interactions.l° Autoxidation can also be retarded or inhibited
by the presence of low concentrations of chain-break-
69
ing antioxidants that interfere with either chain propagation or initiation.t°,26 The presence of antioxidants
in food can retard the development of rancidity via
termination reactions.
ROO" + A H ----> R O O H + A"
A" + ROO" ----> nonradical product
A" + A" ----> nonradical product
The reactivity of phenolic antioxidants such a-tocopherol allows it to intervene to regenerate the original
diene and a phenoxyl radical. The antioxidant free
radicals are generally too unreactive to propagate the
chain. I° Ascorbate can regenerate the phenol and an
ascorbic acid oxidation radical by reaction with the
phenoxyl tocopherol radical. 4° At high concentrations, however, tocopherols can act as pro-oxidants in
v e g e t a b l e oils. 2s The main limitation of phenolic antioxidants in foods is that they become ineffective
during prolonged heating at elevated temperatures. 49
In addition to free radical terminators such as
phenolic compounds, a great variety of other substances and conditions can be considered as exerting
antioxidant activities other than by converting free
radicals to more stable species. 26 For example, free
radical production can also be retarded by the presence of chelating agents such as EDTA, citric and
phosphoric acids which act to inhibit lipid oxidation
by chelating prooxidant metal ions) °,26 A variety of
environmental factors, such as packaging material to
remove air, redox compounds such as cysteine and
ascorbic acid, and physical conditions (temperature,
oxygen pressure, moisture) can also act to retard lipid
oxidation. 1°'17'26 This variety of factors can cause the
nature and proportion of the various products from
lipid oxidation to vary widely between different foods
and makes the kinetics of oxidation reaction complicated since one cannot account for all the possible
interactions.
ORAL TOXICITY OF ISOLATED LIPID
OXIDATION PRODUCTS
Lipid oxidation in foods has received much interest with regards to potential health risks since a number of classes of lipid peroxidation products have been
demonstrated to exert toxic effects in studies both at
the whole animal and cellular levels. 2-4,5,6,12'23 Lipid
oxidation products are known to react with nitrogenous materials in biological systems including amino
acids, proteins, bases of phospholipids, and DNA to
form brown pigments and fluorescence which have
been related to tissue injuries. 5'6'1t'23 A wide array of
toxic consequences can be induced by lipid oxidation
70
S. Ktmow
in the cell, including destruction of essential membrane and cytosolic enzyme activities, membrane
swelling and lysis, mutagenic and carcinogenic activities. 6,11,23
Although some of the adverse effects of lipid oxidation may be relevant to the formation of these products in foods, more systematic research is needed to
assess the metabolic and toxicological consequences
of ingestion of these compounds. For example, the
toxicity of lipid oxidation products formed in situ
may have little relevance to the properties of lipid oxidation products taken in orally. In many studies the
toxic dose levels of oxidation products animals are
exposed to are orders of magnitude higher than would
be found in the average diet. Moreover, due to the
cascade of reactions in autoxidation, the toxicological
relevance of the presence of multitudinal toxic byproducts of lipid oxidation in foods is problematic.
Chang and collegues identified a total of 220 compounds formed during deep-fat frying, many of which
are potentially toxic. 5° Some approaches to test the
toxicity of oxidized fats have either fed the whole oxidized oil or have focused on isolating potentially toxic
compounds or fractions via vacuum distillation, column chromatography, or urea adduct formation and
fed these fractions to experimental animals.
Fatty acid hydroperoxides
Although lipid hydroperoxides are very toxic when
administered intravenously, oral administration of
lipid hydroperoxides is considerably less toxic, most
likely due to low absorbability or to some conversion
before or during the absorptive processY The presence of a reductase enzyme in the gut as part of a
detoxification system has been suggested as lipid alcohol compounds appear in various organs after ingestion of lipid hydroperoxides.18 The role ofglutathione
peroxidase in this regard has been suggested by a recent study using everted sacs of rat small intestine and
peroxidized methyl linoleate. 52Adding either glutathione or an inhibitor of the glutathione peroxidase system decreased or increased, respectively, the amounts
of secondary oxidation products on the contraluminal side. Exogenous glutathione appeared to be
taken up and utilized by brush border intracellular
reactions to metabolize the peroxides and prevent
their transport to the contraluminal side. In contrast
to the products generated from autoxidative reactions
involving lipid hydroperoxides, such enzymatic action would generate much less reactive hydroxy compounds. Recent work has also indicated that the generation of a 13-hydroxy derivative of linoleic acid
formed from peroxidase activity may be coupled to a
dehydrogenase enzyme in rat colon thereby resulting
in the formation of a 2,4-dienone derivative. 53 The
formation of the dienone may be a protective mechanism in that it is much less susceptible to decomposition reactions and thus may further direct the reactivity of the oxidized fatty acid away from the lipid autoxidative cascade.
Secondary lipid oxidation products
A number of studies have definitively shown that
secondary autoxidation products of linoleic acid and
other unsaturated fatty acids are absorbed into the
circulatory system following oral administration with
consequent effects on lipid oxidation products present in body tissues, particularly in the liver. 54'55In one
study, following heating of [14C]linoleic acid for seven
days at 37°C, the mixture was separated into linoleic
acid, the hydroperoxide oflinoleic acid and secondary
products. 54 After feeding these various components
separately via gavage to Wistar rats, a significantly
greater fraction of radioactivity was observed in the
livers of rats fed the secondary products than for the
other two components. Although all the animals were
clinically normal, the feeding of secondary products
produced some elevation in serum liver enzymes and
a slight hypertrophy indicating a certain amount of
liver damage.
Studies have also suggested that ingestion of secondary lipid oxidation products can result in lipid oxidation in vivo. Certain carbonyl compounds such as
12-keto oleic acid have a pro-oxidant effect on unsaturated fatty acids in vitro and could conceivably participate in initiation of lipid autoxidation in vivo. 56 Oral
administration of autoxidized methyl linoleate hydroperoxide has demonstrated increased tissue chemiluminescence 57 and the presence of lipid oxidation
products in various rat organs which were inhibited
by administration of chain breaking antioxidants
such as tocopherols. 5s The mechanism of chemiluminescence has been explained by a hydroperoxide disproportionation reaction and seems to indicate the
generation of short-lived radicals and excited species
during lipid oxidation. 59 Singlet oxygen and triplet
carbonyl compounds were shown to participate as the
major emission species in the chemiluminescence of
livers of animals administered with autoxidized oil 6°
and in liver homogenates supplemented in vitro with
oxidized lipids. 61 In addition, the presence of lipid
peroxyl radicals in livers of tocopherol-deficient rats
given autoxidized lipid hydroperoxides has been indicated by electron spin resonance spin trapping using
the trapping agent, N-tert-butyl-a-4-pyridylnitrone1-oxide. 62
Oxidized lipids in foods
It is clear that the toxicity of oxidized oil depends
on the amount of secondary oxidation products
rather than on the hydroperoxide content. Yoshioka
and Kaneda compared the toxicities in mice following autoxidation of methyl linoleate at 60°C for different periods of time. 63 Minimal effects on toxicity
were observed in mice fed the hydroperoxides
whereas the greatest toxicities occurred while carbonyl values were at their highest.
Low molecular weight carbonyl compounds
Due to their strong hydrophilic nature the low molecular weight fraction of oxidized oils has been
shown to be more easily absorbed and carried to internal organs in the bloodstream and metabolized faster
than the polymeric fraction. 5t These characteristics
may in turn account for the higher potency of carbonyl compounds among oil autoxidation products
in inducing cellular damage and toxicity.
Malondialdehyde has received much attention in
toxicological and food science literature due to its reported mutagenicity and carcinogenicity. 64'65 Reaction with thiobarbituric acid (TBA) and spectrophotometric determination of the colored compound
formed is the most frequently used method to assay
malondialdehyde in foods and biological systems. A
major difficulty in the use of this test is that the TBA
reaction is not specific for malondialdehyde. 39'66
Many other lipid oxidation compounds can form
products that can also react with TBA to form colored
complexes that co-chromatograph and absorb in the
same wavelength as the malondialdehyde-TBA produ c t . 39'66'67 The use of the TBA reagent to determine
malondialdehyde in foods has been criticized on the
basis that the method can overestimate the amount of
malondialdehyde in foods by at least twofold. 66 The
term "thiobarbituric acid reactive substances"
(TBARS) is more appropriately used in place of"malondialdehyde" in reference to this t e s t . 66 The potential of non-lipid related oxidation products to generate malondialdehyde further limits the usefulness of
even direct measurement of this end-product as an
indicator of lipid oxidation. 39
Oral toxicity studies administering malondialdehyde as the enol salt in Swiss mice showed a dose
dependent increase of total neoplasms and neoplastic
lesions in the liver over a 12 month period, 6s although
the lowest dose was about ten times what was considered to be the average dietary intake on a body weight
basis of the Canadian p o p u l a t i o n . 69 Based on the TBA
test, malondialdehyde appears to range in foods from
about zero to 10 ppm depending on the extent of oxidative rancidity and the polyunsaturated content pres-
71
ent. 69The food safety concerns regarding the presence
of malondialdehyde appears to be mitigated by a recent study which has found that only a negligible fraction of malondialdehyde in several foods of animal
origin occurs in the free form. 7° The main portion of
malondialdehyde is bound to the lysine residues of
food proteins from which it is released in the course of
digestion as N-~-(2-propenal)lysine (E-PL). This compound is apparently the product of a reaction between
free malondialdehyde formed as a result of oxidative
rancidity and the free episolon groups of proteins. 7°
During heating, malondialdehyde is released from ~PL and it is probable that this process is involved
during the volatilization of malondialdehyde and its
decline in content in foods during cooking. 65Intraperitoneal administration of the lysine-bound malondialdehyde to rats does not give rise to free malondialdehyde in the urine but is excreted in rat and human
urine in an unchanged form and as its N-a-acetyl derivative. 7° Moreover, ~-PL is extensively metabolized
by a route that does not give rise to free malondialdehyde. 6s Since ~-PL does not generate free malondialdehyde endogenously and is non-mutagenic, it is
likely therefore that malondialdehyde formed in
foods is not of significant toxicological importance. 7°
It is noteworthy that aldehydes more toxic than
malondialdehyde produced from autoxidation of
polyunsaturated fats such as unsaturated aldehydes
and 4-hydroxy unsaturated aldehydes can vary considerably in concentration without affecting the TBA reaction. 67 Amongst these aldehydes released by fatty
acid peroxides, 4-hydroxyalkenals are of the greatest
quantitative and toxic importance. 7~ 4-Hydroxynonenal is of particular relevance since it is a strong electrophilic agent which can react with thiol groups of
glutathione and proteins and can inhibit cell division
in mammalian cell lines. 7~ This compound has also
been shown to inhibit several microsomal and membrane-bound enzymes such as adenylate cyclase and
S-adenosylmethione decarboxylase at concentrations
of 1-100 #M whereas malondialdehyde has no effect
at 5 mM. 67 Such findings further emphasize that the
limitations of TBA and malondialdehyde estimations
in presenting a completely accurate index of the total
cytotoxic potential resulting from both in vitro and in
vivo lipid oxidation. It should be noted, however, that
apart from malondialdehyde, the bioavailability of
these highly reactive short chain aldehydes from the
foods in which they are present is not known.
Nonvolatile oxidized lipid products
It has been demonstrated that nonvolatile oxidized
products such as monomers, dimers, and cyclic mono-
72
S. Kuaow
mers are absorbed into the body and can be recovered
from lymphatic and hepatic lipids. 72 Polymeric material of high molecular weight is not well absorbed and
therefore is considerably less toxic than monomeric
and dimeric compounds. 12'43 This does not discount
the possibility, however, that high molecular weight
polymers may induce irritation of the gastrointestinal
tract.
Oxidized lipids have been treated with urea in
order to separate unchanged fatty acids from the oxidized material. The urea-adduct forming fatty acids
are composed of a mixture of normal straight chain
fatty acids whereas the non-urea-adductable fraction
contains predominantly oxidized material. The nonurea-adductable-fraction has been used in feeding
studies to demonstrate the toxicity of monomeric, dimeric and polymeric material although other oxidized material is present in these fractions as well. 43
When fed in large amounts, these fractions can be
toxic to experimental animals ~2and have been shown
to be deleterious to heart cells in c u l t u r e . 73 The
amounts of these toxic materials that are normally
present following thermal oxidation of oils needs to
be considered in any safety evaluation of these oxidation products. Frankel and co-workers in an analysis
of cyclic monomer concentrations of commercial frying oils and fats from different restaurants in the U.S.
found cyclic monomer concentrations of 0.1% to
0.5%. 74 In a diet containing 15% of oils containing
0.5% cyclic monomer content, approximately 0.086%
monomeric material would have been ingested by the
rats. 43 This could be considered as a possible health
risk since animals fed 0.02 to 0.15% cyclic monomers
develop fatty livers, the severity increasing with monomeric concentration. 75 Diffusive fatty degeneration,
atrophy, and necrosis have been regarded as the direct
injurious effects of cyclic monomers to the liver following intake at these concentrations. Precise information concerning the possible health effects of ingesting low levels of cyclic monomers in humans is
not available, although long-term intake of a diet containing 15% of this oil is needed to produce these effects in experimental animals. Chronic consumption
of thermally abused, deep-fried oil at the high dietary
fat levels of 15% (w/w) is not likely to occur in a typical mixed diet by humans.
Dimers of triacylglycerols can contain several
functional groups such as epoxides, hydroxyl, and
carbonyl groups, and an isolated toxic dimer substance has been isolated from thermally oxidized soybean oil. 19 In short term experiments, rats did not
demonstrate toxic symptoms when fed 0.75% non-cyclic dimers. 76 Since the quantities of such compounds
in thermally abused fats are far below these concen-
trations, 43 the dimeric component of oxidized oils
probably pose no significant toxicological problems.
The toxicological significance of the presence of
these volatile and nonvolatile lipid decomposition materials at the levels present in our foodstuffs is far from
clear. In a exhaustive study carried out by Billek, 2°
sunflower oil was collected from commercial production of fish fingers at a time of discard. Rats were fed
the polar fraction containing most of the oxidation
and decomposition products at a concentration of
20% in their diets over a 18 month period. Only mild
symptoms were observed in the animals such as
growth retardation, enlarged livers and kidneys, and
increased serum levels of glutamic oxaloacetic transaminase and glutamic pyruvic transaminase indicating some liver damage. An extensive evaluation of
other histological and clinical parameters did not
show any major abnormalities. The polar fraction
contains 90% lipid decomposition products as compared to only 30% in the discarded oil. Moreover, frying oil is usually rejected in most commercial settings
by the time significant deterioration occurs in terms
of foaming and the development of odor and color
defects (at approximately at 30% polar constituents).
It is likely, therefore, that human consumption of
these products are orders of magnitude lower than
was ingested by animals which only demonstrated
mild toxic symptoms. In fact, it was calculated that
the average daily intake of this polar fraction administered to rats was 10 g/kg body weight whereas average
human consumption of frying oil itself is only about
0.1 g/kg body weight. As will be discussed in following
sections, however, there may be other safety concerns
of oxidized fats based on alterations in metabolic parameters not measured in this investigation.
TOXICITY OF WHOLE OXIDIZED OILS
Early toxicity studies that carried out feeding trials
in which fats were subjected to extreme processing
conditions and fed in large amounts to laboratory animals demonstrated severe irritation of the gastrointestinal tract, growth retardation, and death. 19 The conditions used also resulted in the complete destruction
of linoleic acid in the oil, and researchers did not
usually supplement for the minimum amounts of the
essential fatty acid needed. 19 Therefore, some of the
severe growth and histological abnormalities could
have been attributed to symptoms of essential fatty
acid deficiencies.
Alternatively, researchers have fed oils and fats oxidized under more realistic cooking practices either in
a commercial or laboratory setting as part of a nutritionally balanced diet. Ingestion of these mildly oxi-
Oxidized lipids in foods
dized oils at 15% of the diet have typically indicated
much milder symptoms which included depressant
effects on growth or steatorrhea with no change in
survival and morphological a p p e a r a n c e . 77-79 A few
studies have shown adverse effects which were limited
to enlargement of various organs and some histopathological changes. 8°,81 It is likely that many of these
effects are secondary to the direct toxic action of the
lipid oxidation products themselves. 82When oxidized
oils are fed in large quantities to animals, the taste and
odor characteristics of the diet may deteriorate with a
subsequent decrease in food intake. Growth retardation and other harmful effects may also result from
too high an intake of long chain polymers which may
cause the fat to be less absorbable and could interfere
with the absorption of the other fat-soluble nutrients
in the diet. 77,82 Nutrient deficiencies can be induced
by the thermal destruction of essential vitamins and
fatty acids in the oils or by autoxidation of vitamins in
the purified diet fed after the oxidized oil has been
mixed in the food. 82 Irritation of intestinal mucosa by
peroxides could interfere with nutrient absorption. 19,77.82There also may be decreased protein digestibility and absorption due to cross-linking reactions
of secondary lipid oxidation products with proteins. 82
There is some contradictory research on the longterm effects of consuming oxidized fats produced
under conditions that mimic commercial and domestic cooking conditions. Some long-term studies have
shown that overall mortality rate and tumor incidence did not tend to correlate with extent of heating
under either mild or severe oxidation conditions, s3
On the other hand, Kaunitz observed more cardiac
fibrotic lesions and hepatic duct lesions in rats fed
mildly oxidized oils over their lifespan than in animals fed nonoxidized fats. 84Contrasting results could
arise from different experimental approaches; heating
conditions (time, temperature, contact with food, and
aeration), replenishment of oil, and the antioxidant
level in the fat will determine the degree of oxidation.
Moreover, the nutritional adequacy of the diet in
terms of the level of protein and antioxidants could
also determine the degree of toxicity observed.
The general lack of serious pathological consequences observed in animal feeding studies of oxidized fats in terms of such general parameters as survival, clinical signs, body and organ weight, growth,
and histomorphology of major organs is not surprising. There is a long history of moderate consumption
of heated and oxidized fats in humans without obvious adverse effects being observed. The overall lack of
gross pathological effects resulting from ingestion of
mildly oxidized fats, however, does not obviate the
73
possibility of more subtle deleterious metabolic effects being exerted.
Oxidative stress and vitamin E status
The feeding of unheated but highly oxidized fish
oils at levels of 10-20% in the diet of rat has resulted
in a wide spectrum of injurious effects including diarrhea, loss of appetite, growth retardation, cardiomyopathy, hemolytic anemia, and the accumulation of peroxides in adipose tissue. 2~ In particular, the high concentrations of C20-C22 polyunsaturated fatty acids
found in certain fish species and fish oils causes such
oils to oxidized readily on exposure to air. Although
secondary effects are likely to be involved, symptoms
such as hemolytic anemia have been reversed by addition of vitamin E to the ration indicating the occurrence of oxidative stress in these fish oil-fed animals. 2~
The possibility of oxidative stress induced by absorption of preformed secondary lipid oxidation products in foods which readily go rancid, such as fish oils,
has also been suggested by an immediate and marked
increase in urinary malondialdehyde observed in rats
after ingestion of a cod liver oil diet. 85A potential risk
to humans of exposure to lipid oxidation constituents
in fish oils has recently been demonstrated. 86 Ingestion of a commercial cod liver oil product without
added antioxidants caused an immediate excretion of
malondialdehyde, but remained unchanged in subjects consuming a concentrate of n-3 fatty acids containing dodecyl gallate and vitamin E. The authors
suggested that the increased urinary malondialdehyde
was likely due to ingestion of preformed malondialdehyde rather than an increased endogenous generation
from n-3 fatty acids. This was supported by the finding that subjects receiving the fish oil concentrate with
antioxidants over a period of 49-50 days showed no
increase in malondialdehyde excretion. In another
human s t u d y , a7 the presence of ingested lipid oxidation material in the urine was suggested when TBARS
were detected during the first four hours in the urine
of humans following a meal consisting of rancid meat
fats. The authors indicated that precursor lipid oxidation products rather than malondialdehyde itself were
likely measured in the meat and detected in the urine.
The red cell membrane has been used as a model
for oxidative damage to biomembranes as well as an
index of vitamin E status since vitamin E is involved
in scavenging free radicals and stabilizing cell membranes by protecting membrane polyunsaturated
fatty acids, a8,a9 Red blood cells from vitamin E deficient rats hemolyze faster in the presence of oxidative
stress than controls, presumably due to the action of
vitamin E acting as a scavenger of lipid peroxyl radi-
74
S. KuBow
cals thereby breaking the propagation of the free radical cascade, s9 The occurrence of hemolytic anemia
following ingestion of oxidized fish oils may thus indicate an imbalance between the severity of oxidative
deterioration of ingested fats and the capacity of the
antioxidant system. 21 The ability of primary and secondary lipid oxidation products to hemolyze red
blood cells was demonstrated by Yoshioka and Kaneda who observed that 11, 8.7, 4.3, and 0.037 micromolar of methyl linoleate hydroperoxide, alkenal, hydroxylalkenal and hydroperoxyalkenal, respectively,
were required to produce 50% hemolysis following
incubation of these products with normal red blood
cells. 9°
Peroxidized fats may accelerate the turnover of vitamin E and consequently increase the requirement
for this vitamin 21 such that despite nutritionally adequate amounts of vitamin E, serum and hepatic levels
of vitamin E may still be deficient in animals fed
mildly oxidized oils in comparison to controls. Rats
were fed vitamin E sufficient diets containing 15% of
oxidized rapeseed oil which had been used in the frying of fish paste according to standard commercial
frying practices. 91 Liver and serum tocopherol levels
diminished according to the level of deterioration of
the supplied oils. Rats fed oils with increasing degrees
of oxidation showed increasing TBARS values in the
liver further indicating an increased level of oxidative
stress.
Alterations in xenobiotic-metabolizing enzymes
One of the most common findings of long-term
feeding studies of oxidized fats is enlargement of the
liver, smooth endoplasmic reticulum proliferation
and induction of microsomal cytochromes P450 enzymes. 92'93 This observation is not surprising in light
of the hepatomegaly and increase in microsomal enzyme activities frequently seen in animals dosed with
xenobiotics. 1~ The chronic feeding of oxidized oil to
rats has markedly induced a number of enzymes important in hydroxylation, oxidation and conjugation
reactions including cytochromes-P450 content and activities, UDP-glucuronyl transferase and glutathione
S-transferase. 93-95 Cyclic monomers which can both
induce and act as substrates for cytochromes P450
have been suggested to the component responsible for
commonly observed induction of these enzymes by
thermally oxidized oils. 94
The observations that the toxicity of ingestion of
thermally oxidized oils is much reduced by a concomitant diet higher in protein content has been indicated
to be due to a larger induction of hepatic microsomal
activities to metabolize lipid oxidation products. 93,96
Ingestion of mildly oxidized oils for eight weeks has
been shown to induce cytochrome P450 content as
well as various hepatic microsomal enzyme activities
such as aniline hydroxylase, aminopyrine N-demethylase and NADPH-cytochrome C-reductase; the level
of induction being dependent on the dietary protein
level. 93 Not surprisingly, the level of induction of microsomal hydroxylase activities far surpasses that of
N-demethylation since lipid oxidation products are
not generally abundant in the methyl group. 93 The
inhibitory effect of a low protein diet on the inducibility of these enzymes along with the augmented toxicity of oxidized oil products indicate that these enzyme
activities may be important in the detoxification of
lipid oxidation products.
In some rat and guinea pig studies, glutathione peroxidase in the liver, which is known to reduce lipid
hydroperoxides to alcohols by linking glutathione reductase, has been observed to be induced in animals
receiving orally oxidized oil. 55'6° In contrast, other investigators have not found an increase in liver glutathione peroxidase levels following intake of oxidized
oils. 91'97 The absence of supplemented dietary selenium in the former studies is likely responsible for the
observed differences between studies. Reddy and
Tappel found that that autoxidized oils do not stimulate glutathione peroxidase when the diet is supplemented with selenium. 98 As dietary selenium is an
important dietary component for the synthesis ofglutathione, ~ presumably adequate dietary levels of selenium ensured sufficient synthesis of this substrate so
that induction of additional enzyme activity was not
necessary in the presence of oxidative stress.
Further evidence of oxidative stress at the level of
the liver has been indicated by reports of increased
hepatic glutathione concentrations and hepatic catalase activity following feeding of oxidized oils to
rats. 91'97 It has been suggested that the increase in tissue glutathione was a overcompensatory response to
oxidative stress induced by the secondary oxidation
products absorbed from the oils. 91 The increase in tissue catalase activity has been attributed to an elevation of hydrogen peroxide generation by either enhanced enzyme activities of peroxisomal oxidase or
c y t o c h r o m e s P 4 5 0 . 95 A reduction was also seen in the
hepatic CuZn superoxide dismutase activity which
may represent either an increased turnover of the enzyme or irreversible inactivation by exposure to hydrogen peroxide. 95
Interestingly, investigators have been able to characterize these changes in biochemical criteria related
to oxidative stress at levels of intake of mildly oxidized fats that did not show deleterious effects according to clinical, histological or growth parameters.
Oxidized lipids in foods
These findings tend to support the concept that metabolic harm in terms of oxidative stress may arise by
ingestion of mildly oxidized oils in spite of observations of clinical and morphological indices that are
generally within the normal range.
AN ATHEROGENIC ROLE OF LIPID OXIDATION
COMPOUNDS IN FOODS?
Some recent reviews have discussed the accumulating evidence which implicates free radical damage
via oxidized lipids as a key step in the atherosclerotic
process. 99'~°°The potential mechanisms by which oxidized lipids may play an important role in terms of
atherosclerotic lesion development will be briefly outlined and will be followed by a presentation of the
experimental evidence implicating dietary lipid oxidation products in atherosclerosis.
Lipid hydroperoxides have been shown to accelerate the atherosclerotic process in terms of initiation of
endothelial injury; the progression phase in which
there is accumulation of plaque and the final termination phase of thrombosis. 1°1-1°3 The presence of oxidized lipids in atherosclerotic lesions has been frequently reported 1°3'1°4and a recent study has detected
higher levels of lipid peroxides in the serum of patients with cardiovascular disease than in controls, i01
Linoleic acid hydroperoxides can cause irreversible
damage to porcine pulmonary artery endothelial
cells, ~°5 and injection of this compound into the
bloodstream causes marked damage to aortic endothelial cells. 1°2 Also suggestive are studies showing that
compounds with antioxidant action markedly reduce
atherosclerosis in hypercholesterolemic rabbits. 1°6
Gey has suggested that on a population group basis
the correlation between serum cholesterol and cardiovascular disease is improved if serum indices of free
radical scavengers are included. 1°7
A key initiatory event in the atherosclerotic process
is the formation of foam cells.~°3't°s New evidence has
indicated that foam cells can be derived from transformed macrophages which have taken up low density lipoproteins that are loaded with cholesterol esters. 1°9 Uptake of oxidized LDL by macrophages is
recognized by a scavenger receptor leading to unregulated increased uptake of cholesterol esters. H° The
migration of the lipid-rich transformed macrophage
on the surface of the endothelium appears to be the
stimulus for the transformation of the macrophage to
a reactive cell capable of producing platelet-stimulating factors and other growth promotors which can
induce replication of smooth muscle cells and fibroblasts leading to plaque formation.l°S-~10 It is of interest that macrophages take up LDL very slowly and do
75
not change to foam cells unless the LDL has been
modified or oxidized, l°s These findings tend to support the concept that a key factor in the atherosclerotic process is an oxidative process to transform cholesterol into a potent stimulus for macrophage uptake. Although it is likely that much of this lipid
oxidative process in atherogenesis is mediated endogenously via endothelial cells, monocytes, and macrophages, 1°9 there is a suggestion that oxidative stress
induced by environmental factors could also play a
role. Some investigators have postulated that free radical mechanisms may account for the positive relationship between environmental factors such as smoking and an increased risk of cardiovascular disease. io3,111Oxidative stress induced by a high chronic
intake of dietary lipid oxidation products could also
be of significance in this regard.
Evidence for a putative role of dietary lipid oxidation products in the atherosclerotic process has come
from both animal and human studies. Early animal
studies have shown that diets containing polyunsaturated oils heated for 20 min at 215°C were more atherogenic than diets containing unheated oils? 12 The
heating effects were related to the polyunsaturated
content of the diet as the heating of corn oil caused
considerably more atherogenic effects in rabbits than
did the heating of olive oil. Higher levels of TBARS
have been observed in chylomicrons obtained from
humans who had consumed thermally oxidized soybean oil than in chylomicrons from control subjects
who had ingested unheated oil. 113 Further studies indicated that the peroxide-rich chylomicrons were
taken up more readily by cultured mouse macrophages with over a ten-fold increase in cholesterol esters in these cells than chylomicrons from individuals
who had consumed unheated oil. Chylomicrons isolated after ingestion of heated oils were predominantly taken by a macrophages via a scavenger pathway of the acetyl-LDL receptor as opposed to chylomicrons obtained after fresh oil ingestion which were
primarily degraded by a ~3-VLDL receptor. This effect
of ingested oxidized oils on LDL metabolism is analagous to other studies showing that the generation of
TBARS in LDL by endothelial cells cause an increase
in the uptake of LDL by macrophages ~°9and suggests
that dietary oxidation products could potentially accelerate the accumulation of oxidized lipids in macrophages and monocytes.
A possible role of dietary oxidized fats in the thrombotic stage ofatherosclerosis has come from a study of
the effects of oxidized fats on prostaglandin synthesis.114 Rats were fed diets containing 10% unsaturated
lipids which were heated for ten cycles of nine minutes at 180°C in the presence of potato sticks. Despite
76
s. KuBow
the heating of oils, sufficient tocopherol content of the
oxidized oil diets was ensured by the addition of vitamin E to the semi-purified diets to produce a ratio of
3.7 mg/g oftocopherol/linoleic acid, which was significantly higher than the nutritionally adequate ratio of
0.6 mg/g. Results showed a doubling of platelet
thromboxane A2 production along with a decrease in
vascular prostacyclin formation in rats fed the oxidized oil diet, thereby increasing the thromboxane to
prostacyclin ratio and consequently, the risk of
thrombin formation. Apart from a slight increase in
kidney weight, nutritional parameters such as growth,
plasma lipids and fatty acid profiles of plasma, liver,
and heart lipids were unchanged. Oxidative stress was
indicated as supplementation of pharmacological
doses of vitamin E (300 mg/kg diet) protected against
the changes in the thromboxane and prostacyclin levels. Other studies have shown that the accumulation
of lipid peroxides in serum of vitamin E deficient animals was associated with inhibition of prostacyclin
synthesis of aortic tissues in vivo 1is and that lipid hydroperoxides inhibit prostacyclin synthetase activity
in blood vessels. 116 These findings suggest that dietary-induced oxidative stress could generate an unfavorable thromboxane to prostacyclin ratio. Since
platelets participate in the early stages of atheroma
these conditions could promote atherosclerosis and
thrombogenesis.
Although metabolic and epidemiological studies in
humans and experimental animals generally tend to
support the concept that a higher intake of polyunsaturated fatty acids is beneficial in terms of lipoprotein
metabolism and cardiovascular health, there are some
new findings that suggest some caution is needed in
this approach. Recently, Blankehorn and co-workers
carried out the first randomized clinical trial to provide information of effects of dietary fats on the appearance of new coronary lesions in patients who had
undergone coronary bypass surgery, i i7 The likelihood
of new lesions developing increased significantly with
each quartile of consumption of not only saturated fat
but also of polyunsaturated (linoleic acid) and monounsaturated (oleic acid) fat. In a recent report of a
comparison of cases of atherosclerosis with controls,
the ratio of plasma selenium to plasma PUFA levels
was negatively correlated with coronary atherosclerosis.118 The authors concluded that high tissue PUFA
levels, when insufficiently protected against oxidation, may indicate a higher risk ofatherosclerosis. It is
also of interest that ingestion of fish oil by rabbits has
been shown to enhance cholesterol-induced atherosclerosis and elevate serum lipid peroxides) t9 Since
the lipid peroxide content in the fish oil was not deter-
mined, it is not known whether lipid oxidation products in the fish oil played a role in the acceleration of
atherosclerosis.
As recently reviewed by Smith in this journal, there
is also accumulating in vivo and in vitro evidence to
indicate that some cholesterol oxidation products are
powerful atherogenic agents. 12° Substantial amounts
of cholesterol oxidation products have been detected
in a variety of foods of animal origin exposed to oxidizing conditions either thermally or through aeration) 21 Several hydroxylated derivatives of cholesterol are potent inhibitors of HMG-CoA reductase in
a o r t i c cells. 12°,121 A substantial reduction in cholesterol synthesis may result in cell death due to disruption of membrane function and subsequently lead to
lipid infiltration and atherosclerosis. A potential atherogenic role of oxidized cholesterol of dietary origin
is further indicated by animal studies which indicate
that oxysterols are absorbed from the intestinal tract
and are transported in the blood to arterial deposition
sites at rates similar to cholesterol) 21 The absorption
of oxysterols in human subjects has been studied following intake of a meal of powdered eggs high in cholesterol oxides. 122Increased levels ofoxysterols in chylomicrons and in total plasma was observed. Small
amounts of oxidized cholesterol products have been
shown to induce arterial injury within 24 h of administration by gavage to rabbits.123 Further, investigations
of feeding of low-level of oxysterols to White Carneau
pigeons at levels estimated to be an average US dietary intake showed a five-fold increase in coronary
atherosclerosis, compared to birds given pure cholesterol. 124 Evidence in humans of the potential atherogenic role of oxidized sterols has come from a recent
epidemiological study of Indian immigrants in the
U K . 125 The author implicated the consumption of
clarified butter (ghee) as having caused the higher
than expected mortality from cardiovascular disease
in this population, despite the absence of obvious risk
factors. Clarified butter contains large amounts of cholesterol oxides (12.3%, w/w, of total sterols) which are
not found in fresh butter. It is also of interest to note
that the structural similarity of common plant sterols
such as B-sitosterol in vegetable oils to cholesterol
may also conceivably result in the formation of potentially toxic oxidized products from these plant compounds following air or thermal oxidation. The As.
sterols have been demonstrated to form oxidation
products in the presence of air that are similar in
structure to cholesterol oxidation products) 26 This
possibility of toxic atherogenic by-products produced
from oxidation of plant sterols has thus far not been
investigated.
Oxidized lipids in foods
MUTAGENICITY AND CARICINOGENICITY
Most studies have failed to detect mutagens in frying oils or a carcinogenic effect of feeding trials of
normally used frying oils. 12'21'127-129 Although some
researchers have reported tumors from animals fed
thermally abused fats, the temperatures used in most
studies were abnormally high .12'21 The significance of
exposure of the digestive tract to a number of lipid
oxidation products that are known to act as mutagens, promotors and carcinogenic substances is not
clear) 3° The ability of hydroxy and hydroperoxy derivatives of fatty acids to act as mitogens for the colonic mucosa has been suggested as a potential mechanism by which oxidized fatty acids could be involved
in the promotion of intestinal tumorigenesis induced
by high fat diets, s3
It has been suggested that fats are sources of intermediates for mutagen formation) 31 Oxidation products of linolenic acid and linoleic acids are weakly
mutagenic in the "Ames" Salmonella typhimurium
assay, which is considered to predict carcinogenic potential accurately.132't 33 Although mutagenic lipid oxidation products such as hydroperoxy cyclic epoxides
react with DNA in the presence of metals and ascorbic acid, such compounds have been shown to be
formed from highly oxidized esters of linoleate and
linolenate at exceptionally high peroxide values ranging from 800 to 3000, whereas the peroxide value of
foods are usually less than 10.132'133 Secondary volatile
lipid oxidation products such as saturated and unsaturated aldehydes, dicarbonyl compounds and acrolein
have also found to be mutagenic) 34 These volatile
compounds may pose a more serious risk in terms of
inhalation of vapors during cooking rather than ingestion in foods. The risk of lung cancer among women
in Shanghai increased with the number of meals
cooked by stir-frying or deep-frying, and with the frequency of smokiness and eye irritation from exposure
to cooking vapors from rapeseed oil) 35 These effects
were assumed to be due to the presence of acrolein
which has been detected at concentrations of I. 1-10.3
ppm from the vapor during frying under cooking conditions in which 20 g of potatoes were fried in 100 ml
of oil.18 The extracts of condensed volatiles from rapeseed oil heated at 270°C tested positive in tester strain
TA98 activated with $9) 36 There was no evidence,
however, of mutagenicity in the rapeseed oil itself,
heated or unheated.
Generally, investigators have not been able to detect mutagens in edible oils and in foods cooked in
oils under normal frying conditions using S. typhimurium bioassays. 127-129'137 Only under severe heat-
77
ing and/or oxidizing conditions were appreciable levels of mutagenic activity detected in French-fried potatoes or fish fillets.t37 Taylor et al. have suggested that
low levels of mutagenic activity in deep-fried meat
compared to pan-fried meat may be related to the
presence of antioxidants in the fats and/or reduced
access of oxygen to the food that is deep-fried.l as Additionally, Doolittle et al. suggested that the temperature of the bread encased meat does not reach sufficient temperatures to form mutagens) 39 Moreover,
the presence of too much fat during heating of foods
could lead to decreased mutagen formation, probably
the result of dilution of mutagen precursors.~ 39
Based on some recent work by Hageman and coworkers, however, a more specific role of fat in mutagen formation cannot be completely excluded) 4°,~41
Moderate mutagenic activity was detected in the polar fraction of deep-frying samples from restaurants in
which the oils were treated according to normal frying
practices in which temperatures did not exceed
182°C. 14° Mutagenic activity was highest after 20 hr
but was slightly decreased after 40 hr of frying. The
authors suggested that mutagenic products found
during the early stages of frying may have been diluted or deactivated by other polar compounds or that
prolonged heating may result in degradation of mutagenic substances. A positive correlation of TBARS
and mutagencity in strain TA97 indicated the involvement of lipid oxidation products.141 The authors, however, suggested that as the mutagenicity of the polar
fraction did not increase with increasing levels of dimeric and polymeric materials, mutagenic compounds other than lipid oxidation products were responsible for part of the mutagenic activity of the polar fraction. 14~ It was suggested that mutagenic
heterocyclic amines and other pyrolysates may be
formed in foods during deep-frying and small
amounts may migrate into the frying fat. In this study
the consumption by human subjects of potential mutagens formed during the deep-frying of potatoes did
not cause an increase in mutagens in the urine. 14°The
degree of absorption of these products from the intestine following digestive processes is therefore not
clear. The differences in mutagenic activity reported
by Hageman et al. may have arisen from differences
in frying conditions, fractionation methods or mutagenicity test procedures. Others have found higher
mutagenicity in meat samples when butter or margarine was used as a frying fat. 142It was suggested that fat
was involved as a more effective heat conductor
rather than as a reactant in mutagen formation.
The possibility also exists that reactive lipid radicals such as alkoxyl radicals can co-oxidize food com-
78
S. KuBOW
ponents other than unsaturated lipids to form mutagenic substances. For example, autoxidized polyunsaturated fatty acids can convert benzo(a)pyrene to mutagenic products that cause sister chromatid exchange
in CHV79 cells. 143 The presence of benzo(a)pyrene
decreases the formation of TBARS indicating the possible diversion of alkoxyl radicals from formation of
endoperoxides as possible precursors of TBARS to a
reaction with benzo(a)pyrene.16 Moreover, the formation of potent mutagenic N-heterocyclic compounds
may occur in foods from the reaction of primary
amines with carbonyl products of lipid oxidation. 15,144
The mode of cooking of foods is also a factor that
requires consideration in mutagen formation from
fats. A possible source of polyaromatic hydrocarbons
in charcoal-broiled foods is the melted fat which drips
on the hot coals; the fat is pyrolyzed at the existing
temperature and the ascending fumes deposit their
polyaromatic hydrocarbons on the foodJ 45 Results
show that the production of polyaromatic hydrocarbons in charbroiled meat is dependent on the fat content and closeness of the meat to the source of heat.~45
Although cancer etiology has been related to a high
fat diet and some of these lipid oxidation products
have mutagenic activity, no realistic assessment of the
health hazards of the presence of such products in
human foods can presently be made. Better insights
into the safety of these products in the human diet
may come about through an increased understanding
of complexities of food component interactions; increased awareness of metabolic processing of these
compounds and improved biomonitoring procedures
of risk assessment.
CONCLUSIONS
The investigation of toxicities of individual dietary
lipid oxidation products is complicated by the incomplete knowledge of their interactions within foods.
These interactions could result in many of the lipid
oxidation products becoming unavailable for absorption and/or subsequent metabolism. On the other
hand, the ability of lipid oxidation compounds to cooxidize or react with other food constituents could
produce toxic components from non-lipid sources. Although gross pathological effects of ingestion of lipid
oxidation products are unlikely in the human feeding
situation, more subtle metabolic actions of these compounds on vitamin E status, drug-metabolizing enzymes, platelet activity, lipoprotein metabolism, and
mutagenic activity cannot yet be discounted. In general, the presence of reactive lipid oxidation components in foods needs more systematic research in
terms of the metabolic effects of these compounds, as
well as the associated antioxidant requirements. Until
increased knowledge concerning these factors becomes available, it may be prudent to limit chronic
consumption of these components.
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ABBREVIATIONS
G C - M S - - g a s chromatography-mass spectrometry
H P L C - - h i g h pressure l i q u i d c h r o m a t o g r a p h y
NMR--nuclear magnetic resonance
~-PL--N-~-(2-propenal)lysine
T B A - - t h i o b a r b i t u r i c acid
T B A R S - - t h i o b a r b i t u r i c acid reactive s u b s t a n c e s