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Antioxidant tocopherols,

Paolo Di Mascio,

defense systems: and thiols3


Michael E Murphy, and Helmut

the role of carotenoids,


Sies

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

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nonenzymatic scavenge free to quench properties

superoxide Fridovich Other Hydrogen became

an exceptional

molecular carotenoids,

anticancer

dent of their provitamin abundant and efficient biological corbate and membranes. cellular thiols. antioxidant scavenging. also

A activity. scavengers Water-soluble Glutathione functions Thiols

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

for enzymatic zymatic radical proteins teractions enhance may the

be important the thiols,

to the tocopherols, cellular

of a metastable contributions

between

(8) and

AmJClin KEY
thiols, protein

effectiveness of Nuir 1991:53:1945-2005.

focuses

WORDS
singlet molecular sulihydryls,

Antioxidants,
oxygen, peroxidation lipid

carotenoids,
oxygen radicals,

tocopherols,
scavengers,

of the nonenzymatic antioxidants. tems have been reviewed in the

The enzymatic recent literature

(see Ref 1).

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

carbohydrates, anion radical, singlet molecular

and lipids. hydrogen oxygen. compounds a prominent species

as lycopene compounds

1) are im-

electronically Singlet molecular compound and or by ie, by pho-

defense, some biological E and C, and thiols play inflicted by reactive oxygen

molecules, a process (02) is an example by photochemical of lipid

termed quenching. of such an excited reactions of biomembranes,

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).

(2) pointed importance

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,

to intermediate are 02 and perhydroxyl recognized Am J C/in Nuir

chemistry. These their protonated HO2 and 1 94S the

forms of oxygen HO and HO. The radical HO were

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
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13
y

CH3

CH3
FIG 1. Structures
of lipid-soluble antioxidants.

These carotenoids has been properties inverse ofcertain cancer,

antioxidant raised whether independent relationship

properties

may of cancer

explain

a possible

role

of

The decrease This

quenching in the quenching

of 02 by tocopherol order of aring, since


(3-

homologs y>

was group

found

to 1).

in the prevention

( 1 1 ), since

the question An tract have

>

#{244}-tocopherol (Table in position derivative

(3-carotene might of its provitamin fl-carotene such

have anticarcinogenic A activity (12). intake and the incidence

depended

on a free an ester

hydroxyl or ether

6 of the chromane 6-position chemical at lower

at the

between

types ofcancer, has been observed

( 13,

as lung and gastrointestinal 14), and animal experiments of carotenoids in protecting

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-

revealed anticarcinogenic properties Although the role of carotenoids

( 1 5). plants against as well is esa prooxygen and to of phoof

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

chlorophyll diseases may exert Singlet damage

between in plasma may two

02 quenching tissue, indicating to the ofcompounds

protection are both

remains unknown. of inducing DNA the quenching

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

chemiluminescence chemiluminescence using Stern-Volmer differences compounds in the (Table

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

of the structures that the opening ability. Replacing group or ad-

(28, between with

29).

The

normal values blood can

intake in adults between level of

E in US diets vitamin 1 3 IU/d

4 and average an average 23 zmol/L

22 IU/d

a methyl ester bonds (crocin),

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

The vitamin susceptibility teins, agents icals,

E content of microsomal

of the conjugated

bile pigments as antioxidants

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

complexes produce can thus protect further lead

decompose

of lipid

the quenching double bonds.

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

particularly with types of prooxidants

respect to its relative (37a).

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

the free metal

was no lag period or the accumu(TBARS) in-

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

levels. In contrast, a clear lag period minescence and TBARS accumulation

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

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is still a period substantial to that noted low-density

peroxidation This

(1) V
0
)

CuCl2-induced teins (34).

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

iron with reducthat generate perAAPH) were tested site of formation,

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.

ANTIOXIDANT TABLE 1 Singlet oxygen quenching biological compounds* Compound

DEFENSE

SYSTEMS is initiated when peroxidation by complexes involves containing peroxyl radicals, Fe2 and

1 97S en-

peroxidation by carotenoids, tocopherols, thiols and other hanced

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

2). Therefore, exclusive per(4 1) (42), also our proof

in membranes factors

Carotenoids Lycopene y-Carotene Astaxanthin Canthaxanthin a-Carotene (3-Carotene Bixin Zeaxanthin Lutein

3 1 000 25 000 24 000 21 000 19 000 14 000 14000 10 000 8 000

ndII nd nd nd nd nd nd nd nd

0.5- 1.0 mol/L

membranes Ascorbic acid

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

it may regenerate so prolong the lag from by because ascorbate ESR in

39, 43). model

E radicals, techniques

demonstrated of the

membranes

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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

nd nd nd nd nd 3.6 0.23 2.8 1.7 nd

act

as intracellular enzymatic

antioxidants reactions.

by scavenging Glutathione is

radicals

through

23 2.3 2.3

zmol/L imol/L mol/L 0

30-100 tmol/L 3-20 imol/L 0.5-10 mmol/L 1-30 mol/L

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
**

to kq). using reversed-phase

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

all influence their these differences,

reactivity with it is concluded

vitamin E and lipids. Based on that the importance of vitamin

E in protecting membranes used. The relative importance

depends on the type of prooxidant of vitamin E is diminished when

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.

1 98S TABLE 2 Loss of a-tocopherol

DI

MASCIO

ET

AL

before

the onset

of chemiluminescence

in microsomes

in relation

to mechanisms Initiation

of initiation mechanism

Type

of prooxidant*

% a-Tocopherol remaining at end of the lag before CL 3.3 31 44 51 84


Peroxyl radicals from propagation reactions Cu2-catalyzed Catalyzed by cyt P-450 (ox) Catalyzed by cyt P-450 (ox)
-

Exogenous peroxyl radicals


-

Fe2 complexes
-

CuSO4 AAPH Fe/ADP/Asc t-BOOH Fe/ADP/NADPH

1.5 2 1 1 1

AAPH-derived
-

radical

Fe2/ADP Cyt P-450 (red) Fe2/ADP and cyt P450 (red) Fe2

t-BOO

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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.

t Not a significant Chemiluminescence

initiator in the prooxidant system. (CL) began without any measurable

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

damage in a lipid environment. mediated and is dependent to oxidant neutralizing, lipid

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

effects. The direct demonstration from GSH to tocopheryl radicals

of a one-electron by ESR is rendered However, by properties

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.

difficult by the low tocopherol content an enzymatic mechanism was supported

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.

ANTIOXIDANT TABLE 3 Effect of thiols

DEFENSE

SYSTEMS

1 99S

on the onset

of chemiluminescence Lag before CLt mm

and the relative

rate of a- vs. -tocopherol

loss during

microsomal

lipid peroxidation Ratio of a-TH to #{244}-TH loss

Additions

Rate nmo/

of a-TH mg 3.8 2.9 1.4


loss min

Rate nmo/.

of #{244}-TH loss mg 3.4 2.2 1.5


min 1.12 1.37 0.91 to provide similar


Control Glutathione Dihydrolipoate


*

(0.5 mmol/L) (1 mmol/L) observations

17 28 30 (Scholich

2 4f 5 et al). Concentrations

0.3 0.2 0.2 and dihydrolipoate

0.3 0.2t 0.3

0.09 0.16 0.09

Data are unpublished different different

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).

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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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