- Email: [email protected]
Antioxidant activity of retinoids
[29]
ANTIOXIDANT ACTIVITY OF RETINOIDS
273
L ymphokine Production and Response
Many soluble factors are now recognized to influence immune responses either quantitatively or qualitatively. To date, relatively little is known of the effects of vitamin A on the production of, or response to, specific lymphokines. As an example, Malkovsky et al. 74 have shown that supplementing newborn mice with retinyl acetate results in cessation of immunologic tolerance, and interleukin-2 (IL-2) is implicated in this process. At present, most assays of lymphokine production rely on the ability of supernatants from mitogen- or antigen-stimulated lymphocytes to stimulate growth of lymphokine-dependent cell lines.75 The specificity of such assays for a particular lymphokine is usually not absolute but may be improved by neutralizing other lymphokines with specific antibodies. 7s Purified or recombinant interleukins are becoming available, and this should result in improved standardization of bioassays; similarly, specific antibodies will allow development of immunoassays to quantify lymphokine protein. In addition, these purified or recombinant lymphokines make possible studies of the effects of exogenous soluble factors on immune responses both in vitro and in vivo. 74M. Malkovsky, P. B. Medawar, D. R. Thatcher, J. Toy, R. Hunt, L. S. R_ayiield, and C. Dote, Proc. Natl. Acad. Sci. U.S.A. 82, 536 (1985). 75 T. R. Mosmann and R. L. Coffman, Annu. Rev. Immunol. 7, 145 (1989).
[29] A n t i o x i d a n t A c t i v i t y o f R e t i n o i d s B y MIDORI HIRAMATSU a n d LESTER PACKER
Introduction Although the effects of retinoids are numerous in biological systems, among them are indications that retinoids may exert some of their actions by virtue of their acting as lipid-soluble antioxidants. Thus, epidemiological studies have suggested that, as for vitamin E, higher serum retinol concentrations are directly correlated with lower risks of cancer I and ischemie heart disease mortality.2 The use of retinoic acid in many animal modds of carcinogenesis has also suggested that their action may be due to antioxidant activity. Moreover, retinol has been shown to inhibit iron-deR. Peto, R. Doff, J. D. Bucldey, and M. B. Sporn, Nature (London) 290, 201 (1981). 2 K, F. Gey, G. B. Brubacher, and M. B. Staheffn, Am. J. Clin. Nutr. 45, 1368 (1987).
METHODS IN ENZYMOLOGY, VOL. 190
Copyright © 1990 by Academic Press, Inc. All rights of~la'oducfion in any form reserved.
274
ANTIOXIDANT ACTION
[29]
pendent peroxidation of rat liver microsomes by Adriamycin3 and to inhibit prostaglandin and hydroxyeicosatetraenoic acid production from arachidonic acid and bovine seminal vesicles and kidney? In evaluating a substance as an antioxidant it is important to use more than one assay, because an antioxidant may be very sensitive in certain assays but it may not react in others, owing to, for example, steric hindrance. Factors such as antioxidant solubility in water or hydrophobic environments are also important. We describe here the application of two assays.
Electron Spin Resonance In the first assay the free radical electron spin resonance (ESR) signal of a stable free radical is quenched in the presence of an antioxidant. The concentration dependence of the 1,l-diphenyl-2-picrylhydrazyl (DPPH) free radical signal quenching is the basis for the antioxidant assay.5 The second assay makes use of phycoerythrin fluorescence. When phycoerythrin is exposed to a continuous source of peroxy radicals the molecule forms stable products, which is accompanied by a loss of fluorescence. Inhibition of the fluorescence decay rate by a substance is used as the principle for assaying antioxidant action by this method. 6 Experimental Methods
General Reagents and Retinoids. DPPH was purchased from Sigma Chemical Co. (St. Louis, MO). Ro 4-3870 (13-cis-retinoic acid), Ro 1-5488 (aU-trans-retinoic acid), Ro 10-1670, Ro 11-1430, Ro 13-6298, Ro 13-7410, Ro 15-0778, Ro 15-1570, Ro 21-6583, Ro 22-1318, and Ro 10-9359 (Table I) were gifts from Dr. Stanley Shapiro of Roche Dermatologics Inc. (Nutley, N J). Other chemicals and reagents were of the highest grade available from commercial suppliers. The molecular structures of the compounds tested are shown in Fig. 1. Antioxidant Activity of Retinoids Assayed by Electron Spin Resonance Spectrometry DPPH Radical Analysis. DPPH (200 # M ) is dissoived in ethanol. Thirty microliters of this solution and 30 #1 of sample dissolved in ethanol 3G. F. Vile and C. C. Winterbourn,FEBSLett. 238, 353 (1988). 40. Halevyand D. Sldan,Biochim.Biophys.Acta918, 304 (1987). 5 M. Hiramatsu, R. Edamatsu, M. Kohno,and A. Mod, in "Recent Advances in the Pharmacologyof Kampo Medicines"(E. Hosoyaand Y. Yamamoto,eds.), p. 120. 6A. N. Glazer,FASEBJ. 2, 2497 (1988).
[29]
ANTIOXIDANT ACTIVITY OF RETINOIDS
275
TABLE I EFFECT OF RETINOIDS ON THE ELECTRON SPIN RESONANCE SIGNAL OF 1,1-DIPHENYI.-2PICRYLHYDRAZYL(DPPH) RADICALa
C o m m o n name
Ro number
Inhibition (% of control)
Furyl analog of retinoic acid 13-cis-Retinoic acid Acitretin Etretin all-trans-Retinoic acid Arotinoid acid Theinyl analog of retinoic ester Arotinoid sulfone ester Motretinide Termarotene Arotinoid ester
22-1318 04-3870 10-1670 10-9359 0 I-5488 13-7410 21-6583 15-1570 11-1430 15-0778 13-6298
77.3 81.7 95.6 97.5 99.7 100.1 101.5 111.7 112.3 115.1 118.8
° RetJnoids at 2.5 mMwere added to a 100 pMsolution of DPPH. Each value is the mean of two or three determinations.
~
,,,0~
~%~CO2H all-trans-RetinoicAcid
C02H
Acitretin
~
CO2H Arotinoid Acid
o
C02H 13-cis-RetinoicAcid
O" ~ Etretin
Termarotene O
Furyl Analog of Retinoic Acid
Motretinide
O II
Arotinoid Sulfone Ester
o
O
Thienyl Analog of Retinoic Ester
Arotinoid Ester b3G. 1. Molecular structures of retinoids.
276
ANTIOXIDANTACTION
[29]
are mixed for 10 sec, and 50 #1 of the solution is transferred to a capillary tube for measurement of the DPPH concentration by ESR. For the Bruker ER200 D-SCR instrument, conditions for measurement of DPPH are as follows: 3480 + 100 G for magnetic field, 10 mW for power, 0.5 sec for response time, 2.5 G for modulation, 2.5 × 104 for amplitude, and 0.5 sec for sweep time at ambient temperature. Manganese oxide is used as the internal standard. The internal standard is located outside of quartz ESR tube into which the capillary tube containing the DPPH solution with or without retinoids present is always inserted. Measurement of Antioxidant Activity, The ESR spectrum of the DPPH radical shows a 5-lined spectrum (pentad signals) as indicated in Fig. 2. The control signal height intensity (in the absence of retinoids) is taken as 100%, and the percentage signal height intensity is calculated. Both Ro 22-1318 and Ro 4-3870 quench about 20% of 100#M DPPH radical at concentration of 2.5 mMwhereas Ro 10-1670, Ro 10-9359, Ro 01-5488, Ro 13-7410, Ro 21-6583, and Ro 15-1570 show no effect on DPPH radical quenching. Ro 15-0778 and Ro 13-6298 slightly increase the DPPH radical DPPH radical
Mn
10 gauss
I
L
r
FIO. 2. ESR spectrum of the 1,l-diphenyl-2-pierylhydrazyl radical.
[29]
A N T I O X I D A N T ACTIVITY O F RETINOIDS
277
signal at the same concentration (Table I). However, no quenching effect on the DPPH radical is detected at concentrations of 0.0005, 0.005, 0.05, and 0.5 m M for the retinoids examined. Ro 4-3870 (13-cis-retinoicacid) and Ro 22-1318 are the only retinoids found to slightly quench the DPPH radical at a concentration of 2.5 mM. Glutathione completely quenches the DPPH radical (100/tM), and the IC5o value for glutathione at 100/tM DPPH radical is 50/IM. The IC5o values of vitamins C and E for the DPPH radical (30/~M) are 5 and 70/IM, respectively. 5 Thus, the antioxidant action of Ro 4-3870 and Ro 22-1318 is much less than that of vitamins C and E, or glutathione.
Decay of Phycobiliprotein Fluorescenceas Assay for Reactive Oxygen Species and Antioxidant Activity of Retinoids This section reports on a method developed in the laboratory of A. N. Glazer (with permission). The data on antioxidant action of retinoids shown below is from unpublished results of A. Koushafar and A. N. Glazer.
Reagents and Stock Solutions 2,2'-Azobis(2-amidinopropane)-HC1 (MW267), Polysciences, Inc. (Warrington, PA, Cat. No. 8963) Chelex 100 resin (200-400 mesh) sodium form, Bio-Rad Laboratories (Richmond, CA, Cat. No. 142-2842) B-Phyeoerythrin, from Porphyridium cruentum, molar extinction coe~cient at 545 nm 2.41 × 106 M -~ era-', Calbiochem (San Diego, CA, Cat. No. 526255) Polysciences, Inc. (Cat. No. 18110) R-Phycoerythrin, from Gastroclonium coulteri, molar extinction coefficient at 566 nm 1.96 × 106M-~ cm -~, Calbiochem (Cat. No. 526258) or Polysciences, Inc. (Cat. No. 18188) 75 m M Sodium phosphate buffer, pH 7.0, passed through Chelex 100 to remove metal ions B-Phycoerythrin, 1.7 × 10-6 M in 75 m M sodium phosphate buffer, pH 7.0 2,2'-Azobis(2-amidinopropane)-HC1, 40 m M in 75 m M sodium phosphate buffer, pH 7.0 (freshly prepared; stored at 0 °) Plasma, preferably fresh or stored frozen at - 2 0 ° Procedure. The reaction mixture contains, in a total volume 2.0 ml, the following (listed in order of addition): 75 m M sodium phosphate buffer, pH 7.0, 1.772 ml; B-phycoerythrin solution, 20/tl (final concentration 1.7 × 10-s M); plasma, 8/tl (1:250 dilution); and 2,2'-azobis(2-amidino-
278
ANTIOXIDANT ACTION
[29]
propane)-HCl, 200 #1 (4 mM). The temperature should be maintained constant at 37 ° because constant temperatures cause thermal decay of the azo initiator and this determines the rate of peroxy radical generation. The excitation wavelength is 540 nm, and the emission wavelength is 575580 nm (for R-phycoerythrin an excitation wavelength of 495 nm may be used if preferred). Comments. The reactions involved in the generation of radicals and the subsequent radical reactions which result in fluorescence decay are shown below. Generation of AAPH radicals: A--N--N--A
~
[ A ' N 2 " A ] --~ 2 e A - + (1 - - e ) A - - A + N 2
where e is the efficiency of free radical generation. a ztO
90
~m
8o
OOAAA~
p., U3
0 Z
"'
On-
70
Ul
60
..J u.
m
50
tu
40
D" 0
b
10
20
30 40 TIME(MIN)
50
60
1100
LLI 0 2: uJ
r ~ t m ~ ct a
I000
TROLOX 2.52 pM
u.I n0
go0 •
.d
u. uJ
800 •
>_.
I-< ..J uJ n,"
700 -
600
.~..,..,
4
8 1216202428323640444852566064 T I M E (mln)
FIG. 3. Fluorescence decay assay of Trolox (vitamin E analog) antioxidant activity.
[29]
ANTIOXIDANT ACTIVITY OF RETINOIDS
279
Subsequent radical reactions: A. + O,-, AO2. AO," + R-phycoerythrin ---* stable products AO2" + AO2" --* stable products
The antioxidant is added either prior to initiation of fluorescence decay or during the course of the reaction. These are both ways that can be used to assay for activity as illustrated for the very efficient antioxidant action of vitamin E using the water-soluble analog trolox (Fig. 3). Trolox consumes almost exactly 2 mol peroxy radicals per mole of antioxidant. Its high affinity and efficiency result in the slope of fluorescence decay being unchanged after the antioxidant is expended. This illustrates ideal conditions for using this assay. With retinoids, the inhibition of the fluorescence decay is also evident. After retinoids are expended the change in slope of fluorescence decay is not reestablished to the initial value and is usually slower. This is probably
PEROXYRADICALSCONSU'/v~D BY 13.C.,[SRET~OIC A O D ( l 3.4 IzM)DUPinG PROTECTIONPHAS~= LI moJ~,,Vfnole
PEROXY RADICALS CONSUMED BY ALL-TRANSI~ETEqOIC ACID (7.92p~4) DUR~rG INI13AL PROTECTION PHASE = 1.~6 moP.Vmolc 1050
,
.
,
.
,
.
,
•
,
.
,
•
,
.
,
1050
.
I gS0
o
I
A L L - T R A N S RETINOIC A C I D
O
950
13-C]S-RET[NOIC ACID =
850 I 750
750
650
650 5S0
550 450
450
350
350 20
40
6o
0o
lOO
12o
14o
160
20
18o
40
60
80
PEROXY RADICALS CONSUMED BY ACITRET/N (2.5 pM) DURING ~I31AL PgOTECTION PHASE • ].6 mo1¢~mole(ap~ox.)
1000
•
,
-
,
.
,
o
O o o o o
~8888
O
A C I "j'P'-L~I~T
m
CONTROL
140
160
180
-
.
,
.
,
.
FURYL ANALOG
==~= 900
O F RETINOIC ACID o =O
=
CONTROL
mO
mO
.
800
120
PEROXY RADICALS CONSUMF~DBY FURYL ANALOG (2.5 ~ D U a ~ G ZNTrU¢ PROTECTION p H A S E . 132 mole=/mol¢
d
1000 =a a= 00800
100
T I M E (mi~)
'F~tE(adal
900
CONTROL
850
800-
700.
700
of tetlaoi¢
u ~D
~id
D a a
600
: 10
,
1 20
,
t 30
,
TI34E ImP)
i 40
,
i 50
, 60
600 0
•
i 10
.
i
.
20
f
.
30
~
,
.
40
(mini
FIG. 4. Antioxidant action of retinoids measured by the fluorescence decay method.
, 50
u
. 60
280
ANTIOXIDANT ACTION
[29]
TABLE II REACTION STOICHIOMETRY OF ANTIOXIDANTS, USING PHYCOBILIPROTEIN FLUORESCENCE DECAY ASSAY
Substance
13-cis-Retinoic acid all-trans-Retinoic acid Acitretin Furyl analog Trolox
Amount added ~mol)
Reaction stoichiometry (Mol peroxy radicals consumed/mol)
13.40 2.52 7.92 2.52 2.50 2.50 2.52
1.10 1.04 1.06 1.05 1.60 1.52 2.00
because radical products (from the retinoids), which continue to be formed in the reaction mixture, have some inhibitory action on fluorescence decay. However, computer-assisted selection of the best slopes by a leastsquares fit allow one to make a close approximation of the number of peroxy radicals consumed per mole of retinoid. Examples are shown in Fig. 4 for four substances. Results with 13-cis- and all-trans-refinoic acid, the furyl analog, and acitretin are summarized in Table II. It should be noted in the assays of antioxidant activity of retinoids that it is necessary to introduce the retinoids in alcoholic solution. Thus, controis for ethanol are used. Ethanol itself has a small effect on the velocity of the fluorescence decay which must be corrected. Furthermore, it is necessary to dilute the retinoids slowly in order to ensure that they are in solution. Among the retinoids tested, the reaction stoichiometry varies between 1 and 1.6. Hence, these compounds display quite significant antioxidant activity. All retinoids examined exhibited antioxidant activity in the same range of concentration between 2.5 and 13.4 # M in the test system described. Vitamin A (all-trans-retinol), but not retinoids, has also been reported to be an effective scavenger of thiyl radicals. ~ 7 M. D'Aquino, C. Dunster, and R. L. Willson, Biochem. Biophys. Res. Commun. 161, 1199 (1989).
ANTIOXIDANT ACTIVITY OF RETINOIDS
273
L ymphokine Production and Response
Many soluble factors are now recognized to influence immune responses either quantitatively or qualitatively. To date, relatively little is known of the effects of vitamin A on the production of, or response to, specific lymphokines. As an example, Malkovsky et al. 74 have shown that supplementing newborn mice with retinyl acetate results in cessation of immunologic tolerance, and interleukin-2 (IL-2) is implicated in this process. At present, most assays of lymphokine production rely on the ability of supernatants from mitogen- or antigen-stimulated lymphocytes to stimulate growth of lymphokine-dependent cell lines.75 The specificity of such assays for a particular lymphokine is usually not absolute but may be improved by neutralizing other lymphokines with specific antibodies. 7s Purified or recombinant interleukins are becoming available, and this should result in improved standardization of bioassays; similarly, specific antibodies will allow development of immunoassays to quantify lymphokine protein. In addition, these purified or recombinant lymphokines make possible studies of the effects of exogenous soluble factors on immune responses both in vitro and in vivo. 74M. Malkovsky, P. B. Medawar, D. R. Thatcher, J. Toy, R. Hunt, L. S. R_ayiield, and C. Dote, Proc. Natl. Acad. Sci. U.S.A. 82, 536 (1985). 75 T. R. Mosmann and R. L. Coffman, Annu. Rev. Immunol. 7, 145 (1989).
[29] A n t i o x i d a n t A c t i v i t y o f R e t i n o i d s B y MIDORI HIRAMATSU a n d LESTER PACKER
Introduction Although the effects of retinoids are numerous in biological systems, among them are indications that retinoids may exert some of their actions by virtue of their acting as lipid-soluble antioxidants. Thus, epidemiological studies have suggested that, as for vitamin E, higher serum retinol concentrations are directly correlated with lower risks of cancer I and ischemie heart disease mortality.2 The use of retinoic acid in many animal modds of carcinogenesis has also suggested that their action may be due to antioxidant activity. Moreover, retinol has been shown to inhibit iron-deR. Peto, R. Doff, J. D. Bucldey, and M. B. Sporn, Nature (London) 290, 201 (1981). 2 K, F. Gey, G. B. Brubacher, and M. B. Staheffn, Am. J. Clin. Nutr. 45, 1368 (1987).
METHODS IN ENZYMOLOGY, VOL. 190
Copyright © 1990 by Academic Press, Inc. All rights of~la'oducfion in any form reserved.
274
ANTIOXIDANT ACTION
[29]
pendent peroxidation of rat liver microsomes by Adriamycin3 and to inhibit prostaglandin and hydroxyeicosatetraenoic acid production from arachidonic acid and bovine seminal vesicles and kidney? In evaluating a substance as an antioxidant it is important to use more than one assay, because an antioxidant may be very sensitive in certain assays but it may not react in others, owing to, for example, steric hindrance. Factors such as antioxidant solubility in water or hydrophobic environments are also important. We describe here the application of two assays.
Electron Spin Resonance In the first assay the free radical electron spin resonance (ESR) signal of a stable free radical is quenched in the presence of an antioxidant. The concentration dependence of the 1,l-diphenyl-2-picrylhydrazyl (DPPH) free radical signal quenching is the basis for the antioxidant assay.5 The second assay makes use of phycoerythrin fluorescence. When phycoerythrin is exposed to a continuous source of peroxy radicals the molecule forms stable products, which is accompanied by a loss of fluorescence. Inhibition of the fluorescence decay rate by a substance is used as the principle for assaying antioxidant action by this method. 6 Experimental Methods
General Reagents and Retinoids. DPPH was purchased from Sigma Chemical Co. (St. Louis, MO). Ro 4-3870 (13-cis-retinoic acid), Ro 1-5488 (aU-trans-retinoic acid), Ro 10-1670, Ro 11-1430, Ro 13-6298, Ro 13-7410, Ro 15-0778, Ro 15-1570, Ro 21-6583, Ro 22-1318, and Ro 10-9359 (Table I) were gifts from Dr. Stanley Shapiro of Roche Dermatologics Inc. (Nutley, N J). Other chemicals and reagents were of the highest grade available from commercial suppliers. The molecular structures of the compounds tested are shown in Fig. 1. Antioxidant Activity of Retinoids Assayed by Electron Spin Resonance Spectrometry DPPH Radical Analysis. DPPH (200 # M ) is dissoived in ethanol. Thirty microliters of this solution and 30 #1 of sample dissolved in ethanol 3G. F. Vile and C. C. Winterbourn,FEBSLett. 238, 353 (1988). 40. Halevyand D. Sldan,Biochim.Biophys.Acta918, 304 (1987). 5 M. Hiramatsu, R. Edamatsu, M. Kohno,and A. Mod, in "Recent Advances in the Pharmacologyof Kampo Medicines"(E. Hosoyaand Y. Yamamoto,eds.), p. 120. 6A. N. Glazer,FASEBJ. 2, 2497 (1988).
[29]
ANTIOXIDANT ACTIVITY OF RETINOIDS
275
TABLE I EFFECT OF RETINOIDS ON THE ELECTRON SPIN RESONANCE SIGNAL OF 1,1-DIPHENYI.-2PICRYLHYDRAZYL(DPPH) RADICALa
C o m m o n name
Ro number
Inhibition (% of control)
Furyl analog of retinoic acid 13-cis-Retinoic acid Acitretin Etretin all-trans-Retinoic acid Arotinoid acid Theinyl analog of retinoic ester Arotinoid sulfone ester Motretinide Termarotene Arotinoid ester
22-1318 04-3870 10-1670 10-9359 0 I-5488 13-7410 21-6583 15-1570 11-1430 15-0778 13-6298
77.3 81.7 95.6 97.5 99.7 100.1 101.5 111.7 112.3 115.1 118.8
° RetJnoids at 2.5 mMwere added to a 100 pMsolution of DPPH. Each value is the mean of two or three determinations.
~
,,,0~
~%~CO2H all-trans-RetinoicAcid
C02H
Acitretin
~
CO2H Arotinoid Acid
o
C02H 13-cis-RetinoicAcid
O" ~ Etretin
Termarotene O
Furyl Analog of Retinoic Acid
Motretinide
O II
Arotinoid Sulfone Ester
o
O
Thienyl Analog of Retinoic Ester
Arotinoid Ester b3G. 1. Molecular structures of retinoids.
276
ANTIOXIDANTACTION
[29]
are mixed for 10 sec, and 50 #1 of the solution is transferred to a capillary tube for measurement of the DPPH concentration by ESR. For the Bruker ER200 D-SCR instrument, conditions for measurement of DPPH are as follows: 3480 + 100 G for magnetic field, 10 mW for power, 0.5 sec for response time, 2.5 G for modulation, 2.5 × 104 for amplitude, and 0.5 sec for sweep time at ambient temperature. Manganese oxide is used as the internal standard. The internal standard is located outside of quartz ESR tube into which the capillary tube containing the DPPH solution with or without retinoids present is always inserted. Measurement of Antioxidant Activity, The ESR spectrum of the DPPH radical shows a 5-lined spectrum (pentad signals) as indicated in Fig. 2. The control signal height intensity (in the absence of retinoids) is taken as 100%, and the percentage signal height intensity is calculated. Both Ro 22-1318 and Ro 4-3870 quench about 20% of 100#M DPPH radical at concentration of 2.5 mMwhereas Ro 10-1670, Ro 10-9359, Ro 01-5488, Ro 13-7410, Ro 21-6583, and Ro 15-1570 show no effect on DPPH radical quenching. Ro 15-0778 and Ro 13-6298 slightly increase the DPPH radical DPPH radical
Mn
10 gauss
I
L
r
FIO. 2. ESR spectrum of the 1,l-diphenyl-2-pierylhydrazyl radical.
[29]
A N T I O X I D A N T ACTIVITY O F RETINOIDS
277
signal at the same concentration (Table I). However, no quenching effect on the DPPH radical is detected at concentrations of 0.0005, 0.005, 0.05, and 0.5 m M for the retinoids examined. Ro 4-3870 (13-cis-retinoicacid) and Ro 22-1318 are the only retinoids found to slightly quench the DPPH radical at a concentration of 2.5 mM. Glutathione completely quenches the DPPH radical (100/tM), and the IC5o value for glutathione at 100/tM DPPH radical is 50/IM. The IC5o values of vitamins C and E for the DPPH radical (30/~M) are 5 and 70/IM, respectively. 5 Thus, the antioxidant action of Ro 4-3870 and Ro 22-1318 is much less than that of vitamins C and E, or glutathione.
Decay of Phycobiliprotein Fluorescenceas Assay for Reactive Oxygen Species and Antioxidant Activity of Retinoids This section reports on a method developed in the laboratory of A. N. Glazer (with permission). The data on antioxidant action of retinoids shown below is from unpublished results of A. Koushafar and A. N. Glazer.
Reagents and Stock Solutions 2,2'-Azobis(2-amidinopropane)-HC1 (MW267), Polysciences, Inc. (Warrington, PA, Cat. No. 8963) Chelex 100 resin (200-400 mesh) sodium form, Bio-Rad Laboratories (Richmond, CA, Cat. No. 142-2842) B-Phyeoerythrin, from Porphyridium cruentum, molar extinction coe~cient at 545 nm 2.41 × 106 M -~ era-', Calbiochem (San Diego, CA, Cat. No. 526255) Polysciences, Inc. (Cat. No. 18110) R-Phycoerythrin, from Gastroclonium coulteri, molar extinction coefficient at 566 nm 1.96 × 106M-~ cm -~, Calbiochem (Cat. No. 526258) or Polysciences, Inc. (Cat. No. 18188) 75 m M Sodium phosphate buffer, pH 7.0, passed through Chelex 100 to remove metal ions B-Phycoerythrin, 1.7 × 10-6 M in 75 m M sodium phosphate buffer, pH 7.0 2,2'-Azobis(2-amidinopropane)-HC1, 40 m M in 75 m M sodium phosphate buffer, pH 7.0 (freshly prepared; stored at 0 °) Plasma, preferably fresh or stored frozen at - 2 0 ° Procedure. The reaction mixture contains, in a total volume 2.0 ml, the following (listed in order of addition): 75 m M sodium phosphate buffer, pH 7.0, 1.772 ml; B-phycoerythrin solution, 20/tl (final concentration 1.7 × 10-s M); plasma, 8/tl (1:250 dilution); and 2,2'-azobis(2-amidino-
278
ANTIOXIDANT ACTION
[29]
propane)-HCl, 200 #1 (4 mM). The temperature should be maintained constant at 37 ° because constant temperatures cause thermal decay of the azo initiator and this determines the rate of peroxy radical generation. The excitation wavelength is 540 nm, and the emission wavelength is 575580 nm (for R-phycoerythrin an excitation wavelength of 495 nm may be used if preferred). Comments. The reactions involved in the generation of radicals and the subsequent radical reactions which result in fluorescence decay are shown below. Generation of AAPH radicals: A--N--N--A
~
[ A ' N 2 " A ] --~ 2 e A - + (1 - - e ) A - - A + N 2
where e is the efficiency of free radical generation. a ztO
90
~m
8o
OOAAA~
p., U3
0 Z
"'
On-
70
Ul
60
..J u.
m
50
tu
40
D" 0
b
10
20
30 40 TIME(MIN)
50
60
1100
LLI 0 2: uJ
r ~ t m ~ ct a
I000
TROLOX 2.52 pM
u.I n0
go0 •
.d
u. uJ
800 •
>_.
I-< ..J uJ n,"
700 -
600
.~..,..,
4
8 1216202428323640444852566064 T I M E (mln)
FIG. 3. Fluorescence decay assay of Trolox (vitamin E analog) antioxidant activity.
[29]
ANTIOXIDANT ACTIVITY OF RETINOIDS
279
Subsequent radical reactions: A. + O,-, AO2. AO," + R-phycoerythrin ---* stable products AO2" + AO2" --* stable products
The antioxidant is added either prior to initiation of fluorescence decay or during the course of the reaction. These are both ways that can be used to assay for activity as illustrated for the very efficient antioxidant action of vitamin E using the water-soluble analog trolox (Fig. 3). Trolox consumes almost exactly 2 mol peroxy radicals per mole of antioxidant. Its high affinity and efficiency result in the slope of fluorescence decay being unchanged after the antioxidant is expended. This illustrates ideal conditions for using this assay. With retinoids, the inhibition of the fluorescence decay is also evident. After retinoids are expended the change in slope of fluorescence decay is not reestablished to the initial value and is usually slower. This is probably
PEROXYRADICALSCONSU'/v~D BY 13.C.,[SRET~OIC A O D ( l 3.4 IzM)DUPinG PROTECTIONPHAS~= LI moJ~,,Vfnole
PEROXY RADICALS CONSUMED BY ALL-TRANSI~ETEqOIC ACID (7.92p~4) DUR~rG INI13AL PROTECTION PHASE = 1.~6 moP.Vmolc 1050
,
.
,
.
,
.
,
•
,
.
,
•
,
.
,
1050
.
I gS0
o
I
A L L - T R A N S RETINOIC A C I D
O
950
13-C]S-RET[NOIC ACID =
850 I 750
750
650
650 5S0
550 450
450
350
350 20
40
6o
0o
lOO
12o
14o
160
20
18o
40
60
80
PEROXY RADICALS CONSUMED BY ACITRET/N (2.5 pM) DURING ~I31AL PgOTECTION PHASE • ].6 mo1¢~mole(ap~ox.)
1000
•
,
-
,
.
,
o
O o o o o
~8888
O
A C I "j'P'-L~I~T
m
CONTROL
140
160
180
-
.
,
.
,
.
FURYL ANALOG
==~= 900
O F RETINOIC ACID o =O
=
CONTROL
mO
mO
.
800
120
PEROXY RADICALS CONSUMF~DBY FURYL ANALOG (2.5 ~ D U a ~ G ZNTrU¢ PROTECTION p H A S E . 132 mole=/mol¢
d
1000 =a a= 00800
100
T I M E (mi~)
'F~tE(adal
900
CONTROL
850
800-
700.
700
of tetlaoi¢
u ~D
~id
D a a
600
: 10
,
1 20
,
t 30
,
TI34E ImP)
i 40
,
i 50
, 60
600 0
•
i 10
.
i
.
20
f
.
30
~
,
.
40
(mini
FIG. 4. Antioxidant action of retinoids measured by the fluorescence decay method.
, 50
u
. 60
280
ANTIOXIDANT ACTION
[29]
TABLE II REACTION STOICHIOMETRY OF ANTIOXIDANTS, USING PHYCOBILIPROTEIN FLUORESCENCE DECAY ASSAY
Substance
13-cis-Retinoic acid all-trans-Retinoic acid Acitretin Furyl analog Trolox
Amount added ~mol)
Reaction stoichiometry (Mol peroxy radicals consumed/mol)
13.40 2.52 7.92 2.52 2.50 2.50 2.52
1.10 1.04 1.06 1.05 1.60 1.52 2.00
because radical products (from the retinoids), which continue to be formed in the reaction mixture, have some inhibitory action on fluorescence decay. However, computer-assisted selection of the best slopes by a leastsquares fit allow one to make a close approximation of the number of peroxy radicals consumed per mole of retinoid. Examples are shown in Fig. 4 for four substances. Results with 13-cis- and all-trans-refinoic acid, the furyl analog, and acitretin are summarized in Table II. It should be noted in the assays of antioxidant activity of retinoids that it is necessary to introduce the retinoids in alcoholic solution. Thus, controis for ethanol are used. Ethanol itself has a small effect on the velocity of the fluorescence decay which must be corrected. Furthermore, it is necessary to dilute the retinoids slowly in order to ensure that they are in solution. Among the retinoids tested, the reaction stoichiometry varies between 1 and 1.6. Hence, these compounds display quite significant antioxidant activity. All retinoids examined exhibited antioxidant activity in the same range of concentration between 2.5 and 13.4 # M in the test system described. Vitamin A (all-trans-retinol), but not retinoids, has also been reported to be an effective scavenger of thiyl radicals. ~ 7 M. D'Aquino, C. Dunster, and R. L. Willson, Biochem. Biophys. Res. Commun. 161, 1199 (1989).