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Paolo Di Mascio,
ABSTRACT
can damage DNA, and and carotenoids some potentially enzymatic prooxidants soluble explain
Reactive oxygen species proteins, carbohydrates, reactions are antioxidants radicals. The singlet ofthe
occur in tissues and and lipids. These by a system of which eliminate ability of the lipidoxygen are may indepenthe most radicals in assubstrate of nonenmembrane systems. other compounds defense. In-
and Weiss (3) in their study on the iron salt-catalyzed decomposition of H2O2 as being important in chemical, photochemical, and after electrochemical anion discovery (4). reactive species of oxygen peroxide (H2O2) as the important in the development are of a nonradical two-electron reduction of enzymology nature. state after the radical the processes. O of superoxide Biological received dismutase research by McCord on stimulus and the
deleterious
controlled
an exceptional
molecular carotenoids,
anticancer
dent of their provitamin abundant and efficient biological corbate and membranes. cellular thiols. antioxidant scavenging. also
Tocopherols of hydroperoxyl antioxidants is an important and is capable associated with antioxidant and antioxidant
include
discovery of catalase Compound I by Chance (5) and the first demonstration of H2O2 in a mammalian cell, the hepatocyte (6). Further, note. Kautsky tially (singlet made istry important molecular important ofphotooxygenation. the electronically and de Bruijn role oxygen), fundamental The excited carbonyls (7) realized quite active Foote whereas present are worthy of early the potenoxygen to the chapter species (9) Gollnick photochemon some sysdefense
of a metastable contributions
between
(8) and
AmJClin KEY
thiols, protein
focuses
WORDS
singlet molecular sulihydryls,
Antioxidants,
oxygen, peroxidation lipid
carotenoids,
oxygen radicals,
tocopherols,
scavengers,
Carotenoids Overview
Aerobic
of oxidative
metabolism
stress
entails the generation of oxygen species Carotenoid they play an Carotenoids portant excited oxygen is the pigments important such biological are widely distributed in nature, where role in protecting cells and organisms. and that fl-carotene can inactivate (Fig
capable of damaging DNA, proteins, These species include the superoxide peroxide, the hydroxyl radical, and In the pattern like carotenoids, role. Oxidative ofantioxidant vitamins damage
as lycopene compounds
1) are im-
defense, some biological E and C, and thiols play inflicted by reactive oxygen
is generated process
or enzymatically
also referred to as oxidative stress, and reflects a shift in the prooxidant-antioxidant balance in favor of the former (1). Diverse biological processes such as inflammation, carcinogenesis, aging, radiation damage, and photobiological effects appear to involve reactive oxygen species. This field of research has provided impact in recent years in a number of fields such as biochemical pharmacology, well as pathophysiology One-electron since tron of early steps free this reduction century. may radical forms, reduction, be toxicology, and medicine. of oxygen Michaelis of general leading radiation has been out biochemistry, widely that studied one-elecformation in biological O and radical by Haber as
peroxidation
toexcitation or by chemiexcitation. Carotenoids may also participate in free radical reactions. For example, fl-carotene and related carotenoids decreased the rate of formation of methyl linoleate hydroperoxides (10).
From the Institut f#{252}r Physiologische dorf, D#{252}sseldorfFRG. 2 Supported by the National Foundation
Chemie
I, Universit#{228}tD#{252}sselResearch, Wash-
for Cancer
of oxygen radicals,
hydroxyl
ington, DC and the Deutsche Forschungsgemeinschaft, Bonn, FRG. MEM was supported by grants from the Deutscher Akademischer Austauschdienst and the Jung-Stiftung f#{252}r Wissenschaft und Forschung. 3Address reprint requests to H Sies, Institut f#{252}r Psysiologische Chemie I, Universit#{228}tD#{252}sseldorf Moorenstral3e 5, D-4000, D#{252}sseldorf!, FRG. Printed in USA. 1991 American Society for Clinical Nutrition
199 1:53:194S-200S.
ANTIOXIDANT
DEFENSE
SYSTEMS
195S
13-Carotene
CH CH3 CH3 CH3 CH3
Tocopherols
rcH3CH3
xl x2
Lycopene
CH3
CH3 H CH3 H
CH3 CH3 H H
Downloaded from ajcn.nutrition.org at Pakistan: ASNA Sponsored on January 15, 2014
13
y
CH3
CH3
FIG 1. Structures
of lipid-soluble antioxidants.
properties
may of cancer
explain
a possible
role
of
homologs y>
was group
found
to 1).
in the prevention
( 1 1 ), since
>
depended
on a free an ester
hydroxyl or ether
at the
between
( 13,
in the chromane ring eliminated the activity. A distinct reactivity ofthe tocopherol homologs with 02 occurred rates and showed the sequence a- > y- > - > fl-toin Table I , carotenoids their and contribute classes and tocopherols abilities that show and both against lipid-
copherol (21), As is presented an inverse correlation their concentration groups singlet soluble corbate, dition, modes ofcompounds oxygen.
the photosensitization induced by their own as in treatment of patients with photosensitivity tablished, the mechanism by which (3-carotene tective function has been shown against cancer to be capable
These
be mutagenic
In our biological todetector recent
(16-18).
work, we determined by first monitoring at 1270 2), and then nm using the plotting abilities infrared diode intensity of quencher considerable compounds (Fig 02 by direct a germanium ratio ofthe
and complement the role which act as antioxidants tocopherols, ofprotection by scavenging reactions, Refs (see of enzymatic
of thiol compounds and asin the aqueous phase. In adperoxyl radicals, and 29). and thiols, demonstrate multifaceted
by a variety
1, 22, 28,
in the presence and absence plots (Fig 3) (19). There were rate constants of quenching (kg,
Tocopherols
Vitamin tocopherols sponsible branes ofvitamin not =-23 taking Mmol/L 1 1 and depending E, a term that encompasses a small group of related (Fig 1), is the major lipid-soluble antioxidant refor protecting the polyunsaturated fatty acids in memagainst lipid peroxidation ranges E supplements, (23). (23), on local This diets maintains levels (30). of membranes membranes. often determines the low-density lipopro<
for
various
1). Lycopene
showed
the greatest
quenching
ability, double that of (3-carotene. Comparison oflycopene, y-carotene, and (3-carotene revealed ofthe (3-ionone ring increased the quenching and double the two ionone (bixin), decreasing rings by carboxylate the conjugated
29).
The
22 IU/d
although
be typical,
dition of chemical groups on the also modulated the quenching quenching properties of carotenoids energy but also The recently state, ie, the length functional on the groups.
(3-ionone ring (xanthophylls) ability, suggesting that the reside not only on the triplet double-bond also system, proposed (26). These
E content of microsomal
of the conjugated
bilirubin and biliverdin were of physiological importance have been described with kq values lower tocopherols (Table has been
hepatocytes, or whole organs to damage by peroxidizing such as hydroxyl radicals, alkoxyl radicals, peroxyl radsinglet oxygen, and perhaps a number of oxygen-metal (31-34). secondary into to a chain lipids chain-propagating These agents intermediates, alkoxyl reaction and peroxyl steps. not only damage the lipids but lipid hydroperoxides, which organic radicals peroxyl without radicals, Tocopherols reacting in and peroxidation.
open-chain tetrapyrroles oxygen quenchers (27), but clearly higher than the carotenoids, of conjugated
as effective singlet than the carotenoids 1). As in the case of to the system
decompose
of lipid
attributed
by scavenging
1 96S
NDPO2
Lycopene
DI
MASCIO
ET
AL
(5mM)
IL)
posomes against rapid peroxidation until it was consumed. This contrasted with our recent observation that peroxidation initiated with Fe/ADP/NADPH began already after only 15% of the vitamin rat liver antioxidant, vs different E was (37). lost This from microsomal further membranes analyses prepared of the role from of this prompted
importance
Figure
the before lation loss
4, A and B, shows
of a-tocopherol with and
the temporal
the onset ions.
relationship
of lipid There
between
in
peroxidation
the experiments
either the chemiluminescence of thiobarbituric acid-reactive 500 mol started FeSO4/L although
increase substances
duced by peroxidation
(Fig 4, A) indicating that lipid vitamin E content was at initial preceded induced both by chemilu100 imoI
CuSO4/L
with feature lipid depletion. other
(Fig 4, B) whereas
prooxidants prooxidant starts relationship peroxidation and of this
a-tocopherol was without any apparent is that even there following is similar of human
lost as quickly as lag. The unique left before vitamin during lipoproE the
peroxidation This
(1) V
0
)
U)
Other prooxidants, including ADP-chelated tants (NADPH and ascorbate), or compounds oxyl radicals (tert-buty! hydroperoxide and and the results are summarized in Table
2. The
3.
0.
0 5
(I)
ib
Time(min)
FIG 2. Quenching of the NDPO2 generated singlet oxygen monomol emission signal at 1270 nm by lycopene. Singlet oxygen was generated chemically by using the thermodissociation ofthe endoperoxide of 3,3(l,4-naphthylidene)dipropionate (NDPO2) (19). Experiments were performed at 37 #{176}C in ethanol:chloroform:water (50:50: 1) placed in a thermostated glass cuvette. The infrared emission of O was measured after addition of 20 iL of 0.4 M NDPO2 solution. The light emission was then measured using a liquid nitrogen-cooled germanium photodiode detector (20).
Molority(pinoi
IL)
Using phosphatidylcholine liposomes as a model membrane, Niki and co-workers (35, 36) found that when lipid peroxidation was initiated by the peroxyl radical-generating compound 2,2azobis(2-amidinopropane) (AAPH), vitamin E protected the li-
FIG 3. Stern-Volmer plot of lycopene, 13-carotene, and lutein. From experiments such as in Fig 2, the quenching constant (kq) was calculated according to Stern-Volmer plot (S0/S = 1 + (kq + kr) k [Q]), where S and S are the chemiluminescence intensities in the presence of the quencher, respectively, k1 is the chemical reaction rate constant, k, is the 02 decay constant in the solvent (10 ts), and [QI is the quencher concentration; because kq>>
kr, kr
was neglected.
DEFENSE
SYSTEMS is initiated when peroxidation by complexes involves containing peroxyl radicals, Fe2 and
1 97S en-
especially
1((it
106
k$ Ms
Tissue
concentration
those generated in propagation reactions (Table although the finding that vitamin E is nearly the oxyl-radical supports results tect suggest scavenging the contention that other compound that important detected antioxidant it is a major antioxidant
in membranes factors
Carotenoids Lycopene y-Carotene Astaxanthin Canthaxanthin a-Carotene (3-Carotene Bixin Zeaxanthin Lutein
ndII nd nd nd nd nd nd nd nd
before or after the oxidation of vitamin E. may be a physiologically relevant enhancer of vitamin E. For example, the chromanoxyl radical and However, although (44), has in biological of tocopherols. systems level hydrogen directly not yet transfer observed been
the effectiveness vitamin E from 0.05-0.1 0.3-0.6 mol/L mol/L phase to chemical 43 ng/retin#{224} 80 ng/retina zeaxanthin and lutein 05 tmol/L 0.3 imol/L 5-20 tmol/L using low Thiols Thiol free groups and (36, vitamin similar endogenous
E radicals, techniques
demonstrated of the
membranes
Cryptoxanthin Crocin Bilirubin Biliverdin Bilirubin ditaurate Tocopherols1 a-Tocopherol fi-Tocopherol -y-Tocopherol 6-Tocopherol Trolox Thiols Cysteine Glutathione Lipoate Methionine
6 000 1 100 3 200 2 300 1 200 280; 45#{216}** 270 230 160 47#{216}** 8.3 2.4 138 13
act
as intracellular enzymatic
antioxidants reactions.
by scavenging Glutathione is
radicals
through
23 2.3 2.3
O.4
* Except as noted, experiments with carotenoids and tocopherols were performed at 37 #{176}C in ethanol:chloroform:water (50:50:1); thiols in D20/ 50 mmol/L phosphate buffer (pD 7.4). Retinoic acid, etretinate, isotretinoin, and dihydrolipoate have no quenching ability. Esterification or ether formation at the 6-position in the chromane ring of a-tocopherol abolished the quenching ability (2 1 ). Cystine, GSSG, and the oxidation products of methionine such as methionine sulfone and methionine sulfoxide have no effect on the physical and chemical quenching of 02.
t kq values were obtained from Stern-Volmer plots (20). Data from references (19, 21, and unpublished observations). t k1 values were obtained by measuring the thiol depletion as a function of time, using the 5,5-dithiobis(2-nitrobenzoic acid) (DTNB) reaction (22). Plasma concentrations are given for carotenoids and tocopherol, typical cellular concentrations are given for thiols. Data compiled from references ( 19, 2 1, 23, 24).
b
Tme(mn)
8b
iio
II nd, not determined (usually low compared 1!Tocopherols were measured electrochemically
HPLC
**
tt
(25). Solvent was D20/C2H5OH (1:1) for solubility reasons. See reference 52; predominantly chemical quenching,
lipid-solubility,
and
oxidation
potential
ofthe
peroxidizing
agents
FIG 4. Time course ofchemiluminescence, thiobarbituric acid-reactive substances(TBARS) accumulation, and vitamin F loss initiated by FeSO4 (A) or CuSO4 (B). Microsomal fractions were prepared as described (38), diluted to 0.5 mg protein/mL with 0.1 mol potassium phosphate buffer/ L (pH 7.4), and bubbled with oxygen at 37 #{176}C as previously described (39). Low level chemiluminescence was measured with a single-photon counting-system as described elsewhere (38, 39). Malondialdehyde was estimated by the formation ofTBARS (40). Malondialdehyde-equivalents were calculated using = 156 mM cm. a-Tocopherol was measured electrochemically in lipid fractions using reversed-phase HPLC (25). Ferrous sulfate (A) was dissolved in nitrogen-gassed water and injected immediately to give a 500 zmol/L final concentration. Copper sulfate (B) dissolved in water was injected to give 100 Mmol/L final concentration.
DI
MASCIO
ET
AL
before
the onset
of chemiluminescence
in microsomes
in relation
to mechanisms Initiation
of initiation mechanism
Type
of prooxidant*
Peroxyl radicals from propagation reactions Cu2-catalyzed Catalyzed by cyt P-450 (ox) Catalyzed by cyt P-450 (ox)
-
Fe2 complexes
-
1.5 2 1 1 1
AAPH-derived
-
radical
Fe2/ADP Cyt P-450 (red) Fe2/ADP and cyt P450 (red) Fe2
t-BOO
FeSO4
l00
a Prooxidants: FeSO4, 500 imo1 ferrous sulfate/L; reductant; t-BOOH, 23 mol tert-butyl hydroperoxide 25 mmol 2,2azobis(2-amidinopropane)/L; CuSO4,
Fe/ADP/NADPH, 10 tM FeCl3 chelated by 1 mmol ADP/L with 0,2 mmol NADPH/L as L min; Fe/ADP/Asc, Fe/ADP as before with 0.4 mM ascorbate as reductant; AAPH, 100 zmol copper sulfate/L. From reference 37a. lag time.
the most
important
cellular
thiol,
acting
as a substrate
for several
tocopheryl-radical
regenerating
activities,
Depending
on exper-
transferases, peroxidases, mitigate the deleterious protection of biological is an interesting aspect thiol prevents is enzymatically been attributed
and other enzymes that prevent or effects ofoxygen free radicals (45). The membranes against lipid peroxidation of its function, since this water-soluble This protection on vitamin E; it has peroxidase and/or
imental conditions, dihydrolipoate (46, 47) and other thiols(39) can also protect membranes nonenzymatically, preventing lipid peroxidation and sparing tocopherol. This suggested that the thiols tioxidant tathione oxidation Glutathione associated role, should and with and sparing (GSH) that membrane the proteins along with (48, may of protein 49). were both able to slow also have thiols an anby gluof perprotection
be considered
the prevention
of tocopherols or dihydrolipoate
the loss of protein thiols from microsomes during lipid peroxidation (Fig 5). This effect paralleled the ability ofthese thiols to
.-i 0
50
delay the onset of chemiluminescence and (Fig 5) and slow the loss of tocopherol (47). Other thiols have varying abilities to protect membranes (results not shown), in contrast to previous reports, can which lipid indicate that glutathione However, is the it was only noted thiol that that the prevent peroxidation.
a,
E
U,
0
E
C U,
0 ..-i
buffer composition in the experiments greatly affected this result, since when Tris chloride was used to replace the usual potassium phosphate buffer, the relative specificity for GSH increased. If GSH enzymatically regenerates tocopherol from its oneelectron idation tioxidant transfer oxidation product, then and protection ofprotein the prevention thiols would of lipid peroxbe secondary an-
C
0
30
45
Time (mm)
FIG 5. Effect ofthiols on the time course ofchemiluminescence and protein sulffiydryl loss initiated by Fe/ADP and NADPH. Microsomal fractions were incubated as described in Figure 4 except that FeCI3 (12 mol/L) chelated with ADP(l mM) was added and peroxidation initiated with NADPH (0.2 mM). Concentrations ofglutathione (GSH) and dihydrolipoate (DHL) were chosen to provide similar lag periods. Measurement of protein thiols was modified from Jocelyn (50), using = 14.2 mM cm for the product of 2,2-dinitro-5,5-dithiodibenzoic acid (DTNB) reduction.
ofmembranes. indirectly
such as a substrate specificity for GSH (40, 5 1). The specificity for tocopherol isomers was analyzed by preparing microsomes with equal amounts of a- and 6-tocopherol. The relative loss of the two isomers and the addition the protection 3). Thus, of enzymatic were roughly parallel during lipid peroxidation, ofGSH or dihydrolipoate did not greatly favor of either tocopherol isomer compared exists with controls the (Table specificity to support hypothesis
no isomer
regeneration.
DEFENSE
SYSTEMS
1 99S
on the onset
loss during
microsomal
Additions
Rate nmo/
loss min
Rate nmo/.
17 28 30 (Scholich
2 4f 5 et al). Concentrations
of glutathione
were chosen
lag periods.
t CL, chemiluminescence.
f Significantly Significantly
from control value (P < 0.05). from 0.5 mmol/L glutathione value
(P
<
0.05).
Portions of this work were done in cooperation with Thomas PA Devasagayam, P Stephan Kaiser, Astrid Scheschonka, and Heiner Scholich. We thank Ursula Rabe for excellent technical assistance.
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1989;274:532-8.
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stress.
Angew
Chem
Int Ed EngI
Di Mascio P. Sies H. Quantification of singlet oxygen generated thermolysis of 3,3-(l,4-naphthylidene) dipropionate. Monomol dimol photoemission and the effects of l,4-diazabicyclo[2.2.2]octane.
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