foul-smelling products such .... of transition-metal ions. withH202 (5 1). Several transition-metal salts react with. H2O2 ... salts react with H2O2 to form. OH' by the ...
Lipid peroxidation: and significanc&3 Barry
Halliwell
and
ABSTRACT oflipid the
of free
that
human
only
diseases
some
plays
a significant
example,
lipids,
lipid
cess.
Many
but
no single
Application
(TBA)
are
assay
available
artifacts.
An HPLC-based artifacts. Am J C/in
KEY WORDS cal,
free
TBA
and
body
peroxidation,
test,
diene
aldehyde,
to which
active
radicals
orbital
is simply
occupy
can
a maximum
hold
defined
contains
as any
one
centered romethyl
radicals, (CC13
tabolism
of CC14
the dot
unpaired
,
can
in the
liver),
and
radical
most gives
existence
as OH’
L-H
if two
electrons
of free
(NOV, both
radicals
to form
a shared
electron
to, takes that
one
a feature of the reaction of free radicals they usually proceed as chain reactions: 1993:57(suppl):7
15S-25S.
(2)
free radicals
(especially
to protect
against
the accumulation
and hypochlorous
organisms metal ions due
them,
setting
,
O
4). Both
produce
direct
be held injury
in excess (3). a free radical
walls
O
deleterious
OH’
to ionizing
that
can
off free-radical
to be completely
to a few cellular
attack chain
all reac-
than is OH’
harmless-both sites
ifthey
are gen-
In addition, O reacts with nitric oxide produced by several cell types (especially
and vascular endothelial cells) to give peroxynitrite oxide, or a derivative of it, acts on smooth muscle
in vessel
therefore,
also some peroxide
exposure
species
and H202 are far less reactive
cannot
reactive not only
acid (HOCI).
to background
usually
but they
but
of oxidatively
by at least two mechanisms: with H2O2 (see below) and ho-
molecules,
(1
O)
(such as H2O2) are continuously organisms have evolved not
biological
cells
can effects
to produce act
relaxation.
By opposing
as a vasoconstrictor,
in some
clinical
and
situations
(6).
its action,
this
may
have
In addition,
it
they
(1)
When
electron
from,
becomes
a radical.
a
or simply Thus,
with nonradicals is that one radical begets anin USA.
this is one
H2O + U
reactive
From
at Davis
Printed
Indeed,
-
prevent
ofwater
re-
electron
are nonradicals.
nonradical
hydrogen.
is a very
phagocytes (5). Nitric
The
A-A
molecules
molecules
OH’
‘
-
fission
erated (NO’),
electrons. ways, as meet,
biological
(4).
can
in which
atoms).
unpaired in several
attack
radiation
(1-3),
oxygen-
on the Highly
that
oxygen’zXg,
from
electron
molecules (reviewed in refs 1-3). The term species (ROS) is a collective one that includes
singlet
thus
atom
attached.
systems
that
electron,
peroxidation.
+ OH’
defense
a single
of a hydrogen
frequently
well-established
systems
and
originally
reactive oxygen species in vivo. Consequently,
antioxidant
tions
elec-
bond.
on to a nonradical,
..1,n J C/in Nutr
oxide
between
2. Thus,
unpaired
biological
one
A free radical
radical), trichloformed by the me-
of one or more other molecules
1 and
ie, a covalent
However,
nitric
is delocalized
in references their
as orbitals.
an unpaired
relevant
oxygen-centered radicals such as O and OH’ but nonradical derivatives of oxygen, such as hydrogen
radistress
of independent electrons,
thiyl (R5, a sulphur-centered a carbon-centered radical
A’ + A
adds
such
off lipid
repair
biologically
an unpaired
was
abstracting
damaged oxygen
is alone in an orbital. Examples (O ) and hydroxyl (OH), both
electron
combine
pair,
known
electrons.
capable
unpaired
designates the presence Radicals can react with
reviewed
ofspace oftwo
species
or more
tron being one that radicals are superoxide
regions
behind
of starting
molytic
that
leaves
lipids) by
only
Introduction in atoms
proton removal
the hydrogen
is produced in living reaction of transition
Electrons
has one
mechanism
also
studied
Hence,
(including
(H,O7),
Each
molecule
atom
of these
oxidative
atom
as a radical.
and other produced
acid
is the most
reaction.
hydrogen
a biological
produce
peroxyl
conjugation,
The
It is now
process.
can
peroxidation chain
pro-
thiobarbituric
fluids
Lipid radical
qualifying
peroxidation. whole
TBA test can eliminate some Nutr l993:57(suppl):7 155-255.
Lipid
radical,
lipid
and
tissues
proteins than are
in the injury
ofthe
diene-conjugate
to human
late
measure
in
to be
the initial tissue damage. Oxidative
to measure
is an accurate
of simple
assays
occurs
role
indeed, of injury
free
it if not
appears
molecules: targets
often
other. for
However, in most,
pathological
and in worsening or traumatic brain
peroxidation
assays
disease. occurs
peroxidation
damage many biological are often more significant
and
quoted
©
1993 American
Pulmonary-Critical Medical
Center,
Care
Medicine.
Sacramento,
and
University
of California
the Pharmacology
Kings College, University of London. 2 Supported by grants from the Arthritis and Rheumatism (UK), the British Heart Foundation (UK), and the National of Health (USA). Address reprint requests to B Halliwell, Pulmonary-Critical Medicine.
Street,
University
Sacramento,
of California
at Davis
Medical
Center,
Group,
Council Institutes Care 4301
CA 95817.
Society for Clinical Nutrition
715S
X
Downloaded from www.ajcn.org by guest on April 27, 2011
important in atherosclerosis injury caused by ischemic
products
frequently
in human damage
For
of end
most
oxidative and
of them.
stress can and DNA
concentration
radicals
increased
measurement,
Chirico
is the evidence
involvement
all,
Susanna
An increased
peroxidation
is likely
its mechanism,
71 6S has
HALLIWELL
been
suggested
itself(eg. might
decompose
Some “leakage”
O
physiological
due
peroxidases)
act
as
possibly
triggering
O
( 1) and
a metabolic
high
of
in aerobic and
under crosses
H2O2
apparently
cell
directly.
concentrations
groups
reactivity.
For
within
cells
species
reacts and
-
with
has.
(reviewed
or the
The
human
other
ROS not
imals too
play
100% (14)
much
idant
body
This
have
emphasized in certain and/or
in tissue
of cell
and
H2O2
such
ofO.
.see ref2
as
major
thiol
and/or
( 1 20-22). .
groups
it) and/or “free”
Ca2
dative stress in ways may be a fine balance
target,
the
of
indirect. (22) and
can
Thus,
which
may
nucleases proteins receptors
damage
close
to DNA rise
(fragmenting
Prothioto oxi-
Thus, there of ROS (eg.
and generation. and
may
DNA).
(such as cell-surface can all respond
defenses,
in in-
(which
that perturb cell metabolism. between regulatory properties
of antioxidant
an
specific proteins, can be direct (eg,
proteases
acute “controlled” inflammation) depending on the extent of ROS activity
the
damaging the cell presence
or
absence of transition free-radical reactions.
metal ions, which can stimulate damaging In a similar way, low concentrations of
lipid
can
hydroperoxides
ase enzymes
and
This
so affect
activate prostaglandin
lipoxygenase and
and leukotriene
are
early
radical
(3)
can
efficiently
abstract
peroxidation. caused
radical
Many
ofthe
by peroxidation
liver
choice
lipid
A
-
inhibitors damage
toxic
of halogenated
of CCL
and
peroxidation
adminis-
offers
hydrocarbons early emphasis reactions) but
(4)
effects
of lipids.
oflipid (25).
(in that it gave of free-radical
+ CC13O2H
some
for study
was
to the important also unfortunate.
not
but
may
stress
peroxidation
lipid
-
be more stress
secondary
cell damage
-
(5)
cycloxygensynthesis
usually cell
-
increased
ambiguity
pointed
(often
an excessive
activate
lipid
liver
Oxidative
ROS-
Ca2,
is formed
CCl3O
lipids
+ CC13O2
Oxidative
tip the
breakage
other Damage
+ 02
an-
derangements
“free”
radicals) reacts rapidly with
is often
effects
(2),
interrelated
or ifOH’
tein kinases, thiol-containing redoxin [23]) and cell-surface
of
stress
provided exert some.
and
of antiox-
can
in intracellular
transporters
oflipids
the cytoskeleton)
at sites properties,
deleterious
DNA-strand
from
both fortuitous biological role
defenses
on transgenic
production
l950s
hydrocarbons
because later studies showed that damage by oxidative stress is rarely mediated by accelerating the bulk peroxidation of cell membrane lipids ( 1 , 20. 22. 27-3 1 ), ie, the sequence of reaction
antioxidant radicals
16). Depletion
oxidative
produce
1 ). rises
ion
fragments
attack
cause
including
and peroxidation if H2O2 oxidizes tracellular
in ROS
atoms
initiate
This
can
metabolism. event:
(14.
in the
to be
in injured of ath-
the
injury. stress
to membrane
and
rises
balance
Oxidative
the potentially tissues
appears
radical:
trichloromethvlperoxyl
Lipid-H
on the ability
antioxidant
studies
beginning
carbon-centered
a peroxyl
tration ofantioxidant protection against
free
peroxidation
of oxidative stress to the development
Studies
CCL hydrogen
of these
species.
roles.
recent
to form
and
ofdifferent
useful
Indeed.
SOD
defenses
result
biologically
radical (like most
in ref 2 1) or with
some
(24).
lipid
occurs. by what the nature of the
inactivate
neither
form
inactivate
(26). For example. CCL is metabolized by the P-450 system in liver to form CCl. Trichloromethyl
of O
protonated
because
bromobenzene cytochrome
on the
focused
a multiplicity
probably
effective.
antioxidant
early
has
However.
lesions
example.
and
radical.
defenses.
For
chemical
more-dangerous
radicals
significant consequence arterial walls, contributing
limited
but
therefore,
to generate
OH’
damage.
DNA
Interest
H2O2
reactive
HO
are
directly
stressed.
and
generation
to DNA
might
depends on what degree of stress it is imposed. for how long. and
a highly human
membranes
can
concentrations
system
dehydroge-
and H2O2 have
increased
leads
lipids.
highly
0,
example.
often
membrane of 04
however,
stress also mechanism
oxygen
a few cellular
of H202
peroxide
or all.of their toxic effects by stimulating lipid peroxidation in vivo. This is probably true for carbon tetrachloride (25) and for
circum-
thiol
high
The relative importance ofdamage to different molecular targets in producing cell injury or death by imposing oxidative
erosclerotic
glutathione
certain
protein
whereas
enzymes.
good evidence that several halogenated
organ-
to the activities
the glycolytic enzyme glyceraldehyde-3-phosphate nase in mammalian cells (20).
On the whole,
excess
is an important
(14-16).
signal ( 1 ).
it can attack.
example.
Removal
and H2O2: the latter may ( 1 . 1 7- 19). I-lO might
02
specific
events
eg. by activated cell types such
as catalase
into
the
these
beginning
lipid
about
out
in the
damage
cellular “free” more important
Ca2
peroxidation
the
true
mid-1980s
to be accepted
However,
lipid
clinical ischemic
to problem injury
end products quoted for injury major viewing
role
of peroxidation 22.
does
development
(24.
34).
to the
and DNA cell injury
appear
or spinal
isolated
arachidonate)
consequence
polyunsaturated
than
peroxicausing
a significant a major
of traumatic
cord
(35).
or
Detection
most disease
of
frequently or tissue
of peroxidation is of Hence, it is worth re-
of oxidative fatty
just
in intra-
Lipid
to make
is the evidence in human
and those incorporated
clearly
damage are often than is the bulk 32).
sequelae
(6)
is only
Rises
rather
by toxins. and an understanding importance in the food industry. this process in detail.
one
but
of atherosclerosis,
or to the
brain
30.
cells was
32) (33).
lipids ( 1 , 22. accompanying
peroxidation the
28.
quarters
. protein damage. events in causing
oflipid peroxidation a role of free radicals
Peroxidation: Both
ofdamaged
(20.
in some
peroxidation of membrane dation is often a late event, final cell death (Eq 6) (32). contribution
-
acids
stress
(PUFAs)
(especially
into lipids are readily at-
Downloaded from www.ajcn.org by guest on April 27, 2011
For
and
to oxygen
(in relation
functions
by oxidizing
intracellular
targets.
SOD
to
chains
mechanism
such
metabolic
it
reactions
enzymes
be deleterious
convert
some
conceivably
defense
sometimes
enzymes
have
(10-12).
enzymes
may
that
appears
CHIRICO
in vivo,
be toxic also
in vivo
deliberately. by different
(SOD)
too much
of H2O2-removing
easily
occurs
to autoxidation
lymphocytes
antioxidant
only but
electron-transport
dismutase
(3. 1 3) although
stances.
that
from
and
not
groups)
(7).
O - appears to be made (9) and. to a lesser extent.
fibroblasts
SOD
OH’
accident.
might of-SH
production
Os-- by superoxide
also
oxidant
ofelectrons
(3. 8). Other phagocytes
isms
peroxynitrite
to give
of the
be a chemical
as
that
it is a powerful
AND
LIPID tacked
by free
radicals,
By contrast, (SFAs)
both
are
been
more
suggested
PUFAs
becoming
resistant that
will
oxidized
monounsaturated
render
body
more
cells.
increased
consumption lipoproteins
PUFAs
to occur.
end
potential
toxicity.
benefits
ofconsuming
may
arise
Some
drug
stores,
and
ofperoxidation
unsaturated
rate
of fish
actually
getting
At high
found
have
reported
oils (39).
it is difficult
unless
lipid
of unpleasant-tasting
as epoxides. cooked Similarly,
ketones,
such
reheated
as the
chicken
peroxides
and
(37.
emphasize
that
that
because
rich are
patients
products
in cooked
foods flavor. “off fla-
and
lipid
in PUFAs
need
to pro-
peroxides ions.
more
then
of lipid
relevant
metal
molecule,
to A,),
or it could
copherol.
Lipid
care
it to
in handling
side
reactivity side
atom.
is why The
moval
leaves
to which aerobic
cells
reaction
is to undergo
reaction
can
hydroperoxides.
depends
on many
in a membrane
(the increase
the fatty presence
dants viding radicals.
acid within
side
meet. capable
The radical
protein
composition,
(A-H in Eq 7), which an easily-donatable
membrane interrupt hydrogen
content the oxygen
one
can
a single
of the
that
the
to A-H
another
by reaction
with
(4 1 ), but by no means in human lipids is a-to-
a-tocopherol
radical with 42):
can
be
ascorbic
acid
+ A-H
ascorbate’
-
with
(8)
However.
it is not yet absolutely
happens
in vivo
ceivably
also
(43).
make
(44). as might
certain
Reduced
some
where
contribution
ubiquinol
CoQH2
as a chain-breaking occurrence of lipid
causes
impairment
inactivation increased
A-H
+ CoQH’
26.
41.
after ions
47.
CoQH’
proposed
of membrane-bound
receptors
exposure (49).
permeability
to peroxides
and
causes
them
in fluidity.
enzymes,
such
deformation
acts
membranes
changes
to ions
For example,
radical. itself
in vivo. in biological
functioning,
48).
ubiquinol
ubiquinol-lO
antioxidant peroxidation
con-
a-tocopherol
(9)
is the
that
ofmembrane
nonspecific
might
to recycling
-
and
(8) actually
(GSH) (45).
et al (46)
directly The
reaction
in mitochondria
is ubiquinol Frei
that
glutathione
and
as Ca2
of red
(1,
blood
to become
leaky
24.
cells to K
Reactive
species
that
Attack
Sonntag
in vivo
by the
(4) and
Several
can
result
a mechanism in irradiated Stark
(50).
decomposition ions
transition-metal
OH’
might
react
possibility 5 1 . 52).
Fenton
with
of lipid
accounts for as reviewed
also
be produced and
(7)
H202
with
salts
attention to the paid to iron ( I . (34. 5 1 . 53. 54).
in initiation
probably organisms.
of peroxynitrite
of transition-metal
but most has been increasing
pero.vidation
by 0H
(Eq 2). Such of peroxidation
by Von reaction
/ipid
irradiation of aqueous solutions produces highly that can attack all biological molecules, including lipids.
peroxidation initiation
initiate
by the
(5 1).
H2O2
to form
OH’.
of OH’ generation in vivo although interest in copper is
Ferrous salts react with H2O2 to form reaction, which is usually written
as Fe(II) + H2O2
+ OH
OH’
-p’
+ Fe(III)
(10)
attack by-
In fact. Fenton the
initial
oxo-iron
initiation acid
Fe(II)
is far more
of the
complex.
OH’ (discussed
side
chemistry
product
complex
reaction
possibly
(5
in equation
ferryl,
that
then
1.
55.
56).
10 may
decomposes
Thus. be an to form
in refs 5 1. 56):
+ H2O2
FeOH3
‘-
(or FeO2)
OH’ + Fe(III)
-
(11)
propagation ratio
a membrane membrane
concentration
of chain-breaking
react
(eg, by dimerization
radicals
so propagating
with
exists
OH’ by the so-called in
ofabstracting
ofthe
back
most important antioxidant
A’ + ascorbate
followed
Peroxyl
the lipid-protein reacting
atom
likely
of fatty
length
including
ofa
as the the
Hence.
its re-
carbon-centered most
and
of hundreds
factors,
chance
to per-
carbon
or they
chains
peroxidation.
lipid
chain
rises).
oflipid
are also acid
in conversion
into
will
they
fatty
result
chains
the
but
adjacent
on the
radical.
if they
in a
and
rearrangement.
a peroxyl
other
carbon bonds
electron
the
or
of a hydrogen
resulting but
molecular
to give each
proteins. from
the chain
protein
02
The fates.
acid sufficient
susceptible
a single electron
attached. several
ofdouble removal
particularly has
has
a methylene
number
an unpaired
with
membrane drogen
are
that
from
is the
atom
have
with
combine
event
PUFAs
it was originally can
the
easier
hydrogen behind
radical
can
the
on a fatty
species
atom
greater
chain.
which
oxidation.
by
The
side
by the attack
chemical
a hydrogen
chain.
acid
lipid
of any
to abstract
in the fatty
is initiated
chain
might
harmlessly
be converted
Evidence
High-energy reactive OH’
not.
peroxidation
acyl
(A’)
disappear
converted back to tocopherol by reduction at the surface of biological membranes (34,
membrane
fatty
(7)
are stable
I discuss
?f pero.vidation
Itliti(tliO!1
radical
it could
In addition. such
Thus.
a rancid impart
directly
most
are
to produce
40).
antioxidant-derived
LO
A’ + CoQH2
is considered.
the decomposition is not
of transition
foods
do foods
Ofcourse,
body
absence
what
up foods
flavor”
temperatures
in the human
at 37#{176}C in the
38).
therapeutic
(37, pick
in
ofthe
foul-smelling
aldehydes
“warmed-over
at high
in 20-30%
decompose
in peroxidized vegetable oils oxidized lipids in mishandled
vors,”
cesses
and
acids,
short
the counter
of peroxidation
peroxides
oils
frequencies
in tests
The
the
higher
to be certain
the degree
temperatures,
peroxides
even
Thus,
a very
over
+ A’
diseases in fish
have
lipid
and
about
PUFAs
bought
is
smells,
various
LO2H
-*
and antioxi-
the chain reaction by profor abstraction by peroxyl
The
reactions
in equations
ferric
complexes
Fe(II)
complexes.
actions.
This
Fe(III)
react
so that
can
occur
+ ascorbate
Hence.
mixtures
sources
ofOH’
slowly
reducing with
-
of iron radicals
10 and
more
salts.
Fe(III). Most H2O2 than do stimulate Fenton re-
all) with
agents
ascorbate
Fe(II)
(56).
1 1 generate
(ifat
(56).
eg,
+ semidehydroascorbate
ascorbate,
and
(12)
H2O2
are good
Downloaded from www.ajcn.org by guest on April 27, 2011
a range
group
with
therefore
preparations
others oils,
and
the
peroxidation
tastes.
+ LO
another molecule. The the only, chain-breaking
because
conclusions
highly
in such
efficacy
conflicting
of
to per-
of atheroscleof damaging
likely
unpleasant
in patients
high
fish-oil
the more
with
ofthe
life. My research
ofcommercial
38).
in place
concerned
fish oils the
at a very
storage
(37.
it has
less sensitive
are also
products
because
peroxidize
than
scientists
in a food
giving
of these
A-H
acids
indeed.
the development toxic and capable
717S
peroxides. fatty
attack:
circulating
Food
lipid
saturated
to free-radical
oxidation and thereby diminish rosis (36). Lipid peroxides are most
into
and
PEROXIDATION
718S Superoxide
reduces
certain
ferric
HALLIWELL Reaction of Fe(III)
chelates.
AND
CHIRICO
is largely with
02
appears
to proceed
+ O
[Fe3-O
Fe(III) Hence, OH’
O
can
generation
lipid
itself
peroxidation,
PUFAs
13 and
appear
HO2
(13)
+ 02
can
10).
to be capable apparently
However,
HO
+ L-H
so in isolated
in membranes,
and
membrane-lipid
providing
to be a preferred
with
not
when
usu-
O
is
for metal ions (Eq 13)].
preformed
mechanism
does
[except
power
lipid
membrane
hydroperoxides
for stimulating
peroxidation
H2O2 + L02
-
release
(15)
in ref63).
meta/s
Most biological metal ions, added
and
lipid
lipid PUFA
peroxides side chains
For example, copper oxidation oflow-density
ions
to peroxyl (58).
involve reaction
Indeed,
added hydrogen
continuing
the chain decompose
chelates
of these
some cussed
iron proteins, in refs 1 , 59,
the
with
including 64,
noxious
isolated
lipoproteins,
or
the
ofthese
transition-metal
ions
nation
erythrocyte occurs.
(eg. Fe3
and
Cu2)
can
copper
also
ions
heme
39,
generated
(ceruloplasmin
(hemopexin),
and or heme
accelerating (reviewed
electron-transport
to Fe2-ADP
if NADPH
Hydroxyl radicals can usually be detected reaction mixtures. However, addition of OH’ catalase hibits
(to the
remove
H,O2
peroxidation
and
block
observed
OH’
(59,
in Eqs.
formation
1 1 and
by the
13). However,
mechanism
is usually
thought
ref 5 1) but
its participation
In many
membrane
peroxidation
cannot systems,
measured
observed that an (reviewed
to be a poorly
if Fe3
is also
Fe2tFe3,O2 in ref 52).
observation in stimulating
that
Fe2
present.
and
way
to generate
of PUFAs
active
form
lipid
or their
ofoxygen
energy absorbed rangement. This
side
block
species
ferryl
(reviewed ruled
increase
in the has been
out
initial
(61). of
reproducibly
led to the
membrane of initiation
abstracting the firstH atom
ions (such as Pb2) can peroxidation (62). systems, liposomes. of peroxidation from a peroxide-free
Fe3
peroxidation can react
event
in the
a higher
of proteins iron
ions
(haptoglobins) and
oxygen,
drugs human
(eg.
tetracyclines).
body
(eg.
in the
skin
to skin singlet
damage oxygen
reported
dyes
several the
absorb
and
transfer
singlet
state.
eosin), substances
forms
reaction
of ozone
with
several
are
the
light,
the excess Such
certain
pho-
classes found
riboflavin,
accumulation
certain
is in
compounds
they
vitamin
radicals
concentration
of
in the and
the
of porphyrins
of porphyria
when sunlight and porphyrins interact (reviewed in ref 70). Kanofsky and
singlet
can
lead
to generate Sima (71)
biological
molecules
oxygen.
and lipopro(in the sense of
In addition, many foodstuffs with light to form singlet oxygen.
lipid
actions
can
(eg.
For example, with
that collide of singlet
of these
certain
state,
porphyrins.
of patients
that
when
it to the and
bilirubin).
when
singlet 02 is reacting and is not abstracting
of oxygen:
include
re-
produced
steady-state
produced
converting
agents
combi-
causes electron rearperoxidation probably
collision
their
electronic-excitation
tosensitizing
direct
an exceptionally
more peroxides. However. sinto be a minor reaction pathway
because
presence
to oxygen.
generates
substrate)
antioxidant fluids
any peroxyl radicals to form a small amount
because
membrane is low. Singlet oxygen is also
enter
as
noxious
peroxidized
is the
with
oxygen molecule for starting lipid
circumstances
bile pigment
suggestion
replace
most
energy in
rate
under
illuminated
1 1 . Perferryl
is added has
species
of such
albumin),
peroxides
as singlet
oxygen, which then can generate glet-oxygen formation appears
the
as
lipid peroxidation in ref 69).
chains
known
by the pathway
lipid other
an infrequent
What
perferryl
should
in-
radicals
to proceed.
be completely
This
rarely OH’
in equation
reactive
various or of
complex is the initiator of peroxidation Some doubt is cast on this proposal by the
other metal Fe2-dependent
In fact, in most teins, the question
an
when
59).
in these scavengers
Thus,
catalase
shown
can
(52.
formation)
60).
are not usually required for the peroxidation then initiates the peroxidation? Previous suggestions have included ferryl (shown
chain
is supplied
(such
These
proteins
During with each
and the microsomal
68).
in body
Cu2
Fe3tADP
gases
by heating
does not qualify as initiation because the directly with the fatty acid to give peroxides H to start a chain reaction.
to Cu,
and
compounds,
to extracellular
accelerate peroxidation if a reducing agent (eg, ascorbate) is added. Sometimes the membrane itselfprovides reducing power. For example, -SH groups on membrane proteins may reduce reduce
simple
heme,
aldehydes
and
existence
them from reactions
ions,
esters),
oxygen
Another
ghosts, or The oxidized
is the
hydroperoxides
carbonyl
contributor
in mammals
and help to stop other free-radical
membranes
to those
a major
Lipid
unsaturated
(33,
similar
(transfemn).
Singlet
biological
(such as microsomes, mitochondria, plasma-membrane fractions), peroxidation forms
are Indeed.
and
or copper
hydrocarbon
cytotoxic
are
of
radicals)
hemoglobin and myoglobin (disEnd products of these complex
67).
and
stimulate capable
[LO’]
phosphate
include
pentane),
hoability already
or entirely
65, 66). to iron
ions lipoxtheir
ofbrain
to radicals
alkoxyl
(63.
(eg.
63,
largely
and
reaction
ions
most
bind
ions, cuprous are added
may
on exposure
reactions
and
lipids.
transitionmixtures.
peroxidation
peroxides
(peroxyl
rapidly
which
metal
by decomposing
products
are frequently used to stimulate perlipoproteins (LDLs) as a model for what
to
metal and/or during
ions
achieved:
peroxidation
that
34). When ferrous ions (eg. Fe2-ADP)
liposomes,
rad-
pero.vidation
may happen in arterial walls (24, ions, or certain chelates of these
of intracellular
lipo-
contain to make
been
defense
studies oflipid peroxidation to, or contaminating, the
to make
be a nuisance
in food
contain Hence, handling.
pigments that photosensitization For
example,
interact remilk
Downloaded from www.ajcn.org by guest on April 27, 2011
Transition
to decompose attack more
used
system for testing antioxidant in these systems has thus
4-hydroxy-2-trans-nonenal The HO appears icals, which then
lipids
membrane fractions, already 63, 64). Cell homogenization
causes
(reviewed
ethane + L-OOH
commercial
mogenate is a popular assay (65). Initiation ofperoxidation
decomposition
by HO2:
HO
The
fractions
abstracting
of initiating
of SOD
peroxidation
In fact, reaction of HO seems
addition
a source of reducing
(14)
to be capable
has not yet been shown
peroxidation inhibit
L’ + H202
-
irrelevant.
as well as isolated lipid peroxide (58,
isolation
of initiating
do
somes, some
and activation of phospholipase, cycloxygenase, ygenase enzymes that can peroxidize membranes
(57)
HO
ally
(Eqs
not
but
Fe2
transition-metal-ion-dependent
H2O2 does
intermediate:
Fe2-O,]
4-*
stimulate
from
Superoxide
via a perferryl
LIPID exposed
to bright
boflavi
n present
Detection
sunlight
can
sensitizes
deteriorate
and measurement
The extent oflipid 1 ) losses of unsaturated oxidation
and
can acids,
when
ri-
by measuring of primary per-
ofsecondary
application do
peroxidation
be determined 2) amounts
3) amounts
the
formation.
of lipid
oxidation fatty
products,
rapidly
singlet-oxygen
719S
PEROXIDATION
products,
usually
material
measure
diene
conjugation
Diene
conjugation
by
oftwo
what
and
the
(UV)
light
in the
particular
they
TBA
are
techniques
supposed
to
that
measure:
test.
The peroxidation of unsaturated formation ofconjugated diene
violet
such
to human
not
fatty acids is accompanied structures, which absorb ultra
wavelength
range
230-235
nm.
Mea-
as carbonyls and hydrocarbon gases (Table I ). Between stages 1 , 2, and 3 it is possible to detect carbonand oxygen-centered
surement lipids and
of this UV absorbance is useful in studies it measures an early stage in the peroxidation
of pure process.
radicals
the
Corongiu
et al (85)
of mea-
and
surement application
by using double-derivative of simple diene-conjugate
use
[by
electron-spin
of “spin
traps”
to identify
these
resonance
such
(ESR)
as phenyl
combined
t-butyl
with
nitrone
radicals
by their
ESR
summarizes peroxidation
some ofthe in biological
available systems.
methods for measuring lipid (I do not have space to discuss
all of these references.)
methods in detail, but The chemical composition
peroxidation lipid substrate Thus,
[eg,
and
iron
ions
as measured
give
acid ions
peroxidation
techniques is present.
can about
in biological
material,
more and more products before
often
(Table
done
by HPLC
available,
peroxides
(8 1 ). However,
of what tigations
technique
is likely
also,
however,
require
(eg, by working
under
the
the
Specificity can niques, particularly
also be achieved monoclonal-antibody
or has
directed been
erosclerotic can bind
treated
lesions. to antibody
in refs 23 and 40). to plasma samples. fluids
a series
as a result to evaluate
material handling
against
LDL
with
sample has
to be taken
and/or artifactual of lipid material. by the
that
Antibody-based Recent papers peroxidation
the exciting
(83,
84).
Further these
work products
in human
considerable disease
confusion has
been
to separate
from
human
this
Although
some
groups
linoleic
radicals
(for
fluids
consisted
of
of linoleic acid, octadecaproposed that this compound
from
carbon-centered
body
material
acid
with
and
a recent
reaction
protein. the
example,
of
However, product
as
see ref 89) con-
tinue to maintain that octadecadienoic acid is produced by freeradical activity, such a conclusion is extremely unlikely. First, the preferred reaction of carbon-centered radicals is with oxygen, not
with
protein.
Second,
ofproducts from mer of a product
lipid
peroxidation
all the PUFAs, from one fatty
of this
product
the
evidence
reactions).
hepatotoxin
tissue
caution
must
peroxidation
to biological
material
a series
supporting Third,
1 1-dienoic to oxidative
and
acid was not found stress (eg, in rats
products problems
by this
in applying
its gen-
Thompson
a potent
Conjugated-diene and can cause
be used
produces
not one specific geometric isoacid (in fact, the stereospecific
is in itself
by enzyme-catalyzed
The
to ensure
TBA
One
do
technique
inducer
may also in attempts
(73).
Thus,
conjugated-diene
be to
great
techniques
(75).
test
ofthe
may
ath-
arise be
by toxins, and,
by inappropriate
test
The
commonly
is heated
with
TBA
at or close
TBA
test
is often
to 532 said
in peroxidizing
assays and
liposomes)
but
is the
cheapness.
at low pH,
and
nm,
test.
The
sample
systems,
so results
are
frequently
The TBA test works well systems (eg, microsomes to body
fluids
and
tissue
a host of problems (37, 8 1. 91). other than MDA can form chromogens,
some
at 532
nm,
in the
different aldehydes are formed (40). Second, the TBA test rarely of the
lipid
generated heating by
traces
system:
most,
if not
TBA
test
in the
reagents.
(92)
and
exwith many
in peroxidizing lipid material measures the free MDA content all,
of the
MDA
by decomposition of lipid peroxides stage of the test (9 1). This decomposition of iron
nm.
(MDA)
tracts has produced First, aldehydes absorbance
by its
at 553
malondialdehyde
equivalents. membrane
It
chromogen
is measured
or by fluorescence
to measure
its application
TBA
a pink
adduct)
lipid
expressed as imol MDA when applied to defined and
applied
of its simplicity
a [TBA]2-malondialdehyde
formed
is required might
under (allegedly
most because
absorbance
about the role of per-
caused
problems.
HPLC
both they (87) and others (88) later identified resulting from bacterial fatty acid metabolism.
measure
in vivo. Using these role played by lipid
peroxidation in cell injury and death mediated in human disease, is becoming clearer at last. Unfortunately,
by H abstraction
resulting
of
bromotrichlomethane,
from such lesions LDLs (reviewed
that
all
isomer acid. They
of lipid peroxidation). found in animal diets
methods can also be applied have identified in human body
possibility
the
serious
used
and
to rabbit
that
or
given
peroxidation.
compounds
conjugates”
most
a non-oxygen-containing 9(cis)l l(trans)-dienoic
is popular
bind
“diene that
de-
investech-
peroxidation
has undergone
F2-like
absorbing
reported
produced
in ref 86)
octadeca-9, subjected
use of antibody techtechniques. Thus, an-
In addition, LDLs eluted specific for MDA-treated
UV
and
has
et al (reviewed
sensitivity
spectroscopy. However, methods to lipid extracts
Smith (90) showed that in the plasma ofanimals
precise
preparation.
the
fluids
greater
eration
unambiguous
of choice for material. These
useful “biomarkers” of lipid peroxidation various highly specific methods, the precise
oxidation
volatile
body
achieved
are
measuring
to give more
4-hydroxynonenal,
of prostaglandin
oflipid
most
nitrogen)
that loss ofoxidized not occur during
tibodies
(40)
when complex mixtures are being studied techniques. allowing a precise identification
is present, should be the methods of lipid peroxidation in human
niques
into and
human
Dormandy
structure
techniques
for
of material
spectrometry
chemical information (82). Such analytical
separating the them. This is
aldehydes
by gas chromatography,
by mass
to use
about what this principle
HPLC
cytotoxic
conversion
separation
identification
care
and
from
is produced
test
in LDLs of only a
it is important
1): for example,
(80)
(TBA)
misleading results. real occurrence of
groups are measuring
as is a gas-chromatography
rivatives,
give the
that give specific chemical information Indeed, food scientists have followed
for measuring
1
distri-
acid
ofperoxidation so the selection
for years (37). Thus, various peroxidation
hexanal
end-product
thiobarbituric
peroxidation as possible
Table
have
and
it can
measured
is
during the acidis accelerated be
inhibited
by
Downloaded from www.ajcn.org by guest on April 27, 2011
lipid
test to monitor learn as much
79).
composition of the (ifany) are present.
different
by the
only, copper ions are good stimulators but poor stimulators in microsomes], single To
(78,
Table 1 contains selected of the end products of
will depend on the fatty used and on what metal
copper
butions
spectra
(PBN)]
720S
HALLIWELL
TABLE Methods
1 used to detect
and measure
biological
Method
CHIRICO
lipid peroxidation* What is measured
Analysis of fatty acids by GLC or HPLC Oxygen electrode
Iodine
AND
liberation
Loss of unsaturated
Remarks
fatty acids
Uptake of oxygen by carboncentered radicals and during peroxide-decomposition reactions Lipid peroxides
Very useful for assessing lipid peroxidation stimulated by different metal complexes that give different product distributions. Dissolved oxygen concentration is measured. Useful in vitro when spectrophotometric interference occurs or toxic chemicals interfere with enzymic techniques. Not very sensitive. Lipid
peroxides
for bulk
oxidize
lipids
(eg.
can be applied agents
Heme degradation of peroxides (often first separated by HPLC)
Lipid
peroxides
are
to extracts
(GSPase)
Lipid
a sensitivity
peroxides
GSPase
of l0_12
with
H2O2
content.
peroxides
within
peroxides
Stimulation
stimulation used
peroxides
and aldehydes
radicals
gases
Pentane
and ethane
back
be related
Cannot
first be cleaved
to
measure out
by
hydroperoxide
GSPase
assay
cannot
can be used to measure
be used potential
of their
Sensitivity to identify
specific
because The
the
actions,
assay
has
mol
peroxides
it relates
biological
synthesis.
trace
of 10_12
presence
ie,
not
been
widely
products
in the
chain
reaction.
ofgases formed reaction pathway
vivo
measure
of peroxidation.
some have
authors abandoned
have gases
are
metal
body
index
Self-reaction
have
overall
in limited
of peroxyl
depends
peroxidation
radicals
so may
process
as is often
can produce
the
others
is also
affected
ofpentane
to
on the
and
in
variable,
air pollutants
metabolism
peroxides
amounts,
are
production
by the
been
well and because
and
Gas
production
lipid
ofthe
available
by bacteria and
gas
to decompose
adequate
in practice
tissues).
in vivo
Hydrocarbon ions
Results
produced
into
concentration
02
during lipid peroxide decomposition. but it can be used as a noninvasive
found that the technique works it. Rigorous controls are required
partitioning
only
to
separated
pentanol.
oxygen
peroxide/L.
fluids.
to one
GC measurement Only a minor
singlet
can
are extracted, reduced (eg, by borohydride) by GC. and identified by mass spectrometry. Several variations of these methods exist. Spin traps [eg. phenyl t-butyl nitrone (PBN)J intercept radicals
by
carbonyls.
activity
interesting
hydrocarbon
Excited
to GSSG. GSSG
problem).
of eicosanoid
Peroxidation
(thus
Light emission
GSH
to reduce which
must
it is potentially
intermediate
Hydrocarbon
they
detectors mol
to date.
alcohols.
Intermediate
array of l0
NADPH
of phospholipid
in biological
This
of peroxides
Spin trapping
oxidizing
oxidizing
and
of 3 nmol
of cyclooxygenase
but
Diode
of NADPH.
membranes:
ofperoxide
present,
Lipid
Useful
to 12. Method
if other
a sensitivity
hydroperoxides.
Sensitivity
this
peroxide/L.
dye,
reductase
(Availability
simplify
amounts
spectrometry
1
samples
peroxide/L.
in consumption
phospholipases.
GC-mass
thiosulphate.
oxidizes
presence
not
give
ifsuch case
of an
ions are
in vivo.
excited-state
carbonyls
and
singlet 02: both species emit light as they decay to the ground state. This is an interesting technique for use with isolated lipid systems.
Measurement
of low-level
for measuring
generation
but Fluorescence
Aldehydes
the
light
Aldehydes
such
groups
groups. pathway method.
to arise
Schiff
bases
from
Formation and has It should
characterization, products
several
of fluorescent
pH
species can
only).
be formed.
products products
method
in whole
organs.
sources.
(MDA) (at acid
dihydropyridines may to produce fluorescent
is a potential
oxygen
as malondialdehyde
to form
fluorescent polymerize
end
appears
chemiluminescence of reactive
react
with
amino
At neutral
pH
Aldehydes
can
in the absence is a minor
also
of amino
reaction
very complex chemistry. but is a highly never be assumed, without detailed
sensitive
that
fluorescent
products
in vivo
of lipid
peroxidation
(eg.
accumulating ref 77).
are
Downloaded from www.ajcn.org by guest on April 27, 2011
results
peroxide
may
mol
and
ofglutathione
to GSH
Lipid
of biological
Linked to a redox can be achieved.
reacts
Addition
Cyclooxygenase
with
also
absent.
may be used. hydroperoxide
peroxidase
H2O2
Heme moiety of proteins can decompose lipid peroxides with formation of reactive intermediates. Microperoxidase is particularly effective. Radicals produced can be reacted with isoluminol to produce light. giving
Glutathione
1 to 12 for titration foodstuffs).
LIPID TABLE
72 1 S
I (Continued) Method
TBA
PEROXIDATION
What
test
is measured
TBA-reactive
Remarks
material
(TBARS)
The test material chromogen fluoresence 1 -ol.
at 553
Most
of the
peroxides
and
is a simple
GC-HPLC-antibody
techniques
Cytotoxic
aldehydes
is heated
Diene-conjugated
structures
*
Simplified
and
LDL,
low-density
chelating
that
can
ing the assay, content greater
the sample’s
peroxide
the
account
response
sample
(93).
bile
tested,
the
and
to microsomes
(96).
by Marshall
results
of a TBA
alents”
gave
et al (97).
test
a value
taken
Specific (98). This
on the of38
from
oxides
do form
cleared
rapidly.
Using
same
the
others
healthy
in vivo For
and
enter
example.
laboratory
avoid
many
of the during
uses above the
antioxidant before
aldehydes).
for
references
on
can
biological be gained
problems
fluids
(see
40 and
glutathione:
by
arise
when
GC,
gas
text).
72-76.
TBA,
in
(eg, LDL).
use
specificity
body
use
products,
chro-
thiobarbituric
acid:
Second, Use
TBA mogens.
other
TABLE
test the
Flow
has
reactive
for
the TBA
artifacts.
assay
lipid
body
First.
reagents
has
lipid
for
by adding
Inject
can
(BHT) (99).
This
also
at 532
reaction
different
of chro-
acid
(TBA)-
sample
into
2, 0.25-mL
1.5 mL
0.44
10 mm
in ethanol)
or standard
mol
samples
H3PO4/L
at room
to each
temperature
20 tL
onto
a Spherisorb
(the authors
guard
use Waters
5ODS2 (Hiber
Millipore
(C18)
column
C8)
Model
A6000
System)t
be Elute
is
with 65% and
peroxito
a
BHT,
mmol
at pH 7.0
at 1 mL/min
hydroxytoluene.
column
from
HPLC
Cheshire,
to the
UK: Millipore System from Waters (UK) Ltd. Watford, UK.
UK:
KH2PO4-KOH/L
methanol
absorbance at 532 nm or fluorescence at 553 nm Sharp peak. retention time (RT) = 4.8 mm
butylated
t Spherisorb
50
35%
chainallows
(dissolved
mL
with
of perthe
to give
ofthiobarbituric
2 g BHT5/L
Wait
per-
adapted
amplification
hydroxytoluene are added
been
to the
Add 0.5 mL TBA reagent (6 g/L in ethanol solution) Heat at 90 #{176}C for 30 mm: allow to cool
peroxidation
fluids
absorbing
due
re-
the authentic
for the assay.
determination
Divide
of atherosclerosis, to be what matters
that
is prevented
butylated
HPLC
Add
assays
they
chromogens artifacts
the protocol
and/or ofthe
to separate
constituents
to 0.5
find any in human because even if per-
circulation,
other
content
material
plasma.
0. 1 j.tmol
