Chemistry and Physics of Lipids

87 (1997) 73–80

Plastoquinol and a-tocopherol quinol are more active thanubiquinol and a-tocopherol in inhibition of lipid peroxidation

Jerzy Kruk, Małgorzata Jemioła-Rzeminska, Kazimierz Strzałka *

Department of Plant Physiology and Biochemistry, The Jan Zurzycki Institute of Molecular Biology, Jagiellonian Uni6ersity,Al. Mickiewicza 3, 31–120 Krakow, Poland

Received 13 January 1997; received in revised form 1 April 1997; accepted 3 April 1997

Abstract

Comparative studies of antioxidant activities of such natural prenyllipids as plastoquinol-9 (PQH2-9), a-tocopherolquinol (a-TQH2), ubiquinol-10 (UQH2-10) and a-tocopherol (a-T) in egg yolk lecithin liposomes have beenperformed. The investigated compounds showed oxidation under molecular oxygen in the order UQH2-10\a-TQH2\PQH2-9\\a-T. The corresponding second order rate constants have been determined in Tris buffer(pH=6.5) and were 0.413, 0.268, 0.154 and 0.022 M−1/s, respectively. The inhibition order of Fe2+-H2O2 -inducedlipid peroxidation, corrected for the amount of prenyllipids oxidized during the initiation period, was a-TQH2\PQH2-9\a-T\UQH2-10 for 5 mol% of the antioxidants content in liposomes. The radicals formed in the initiationphase of the reaction caused oxidation of 27.5–33% a-T, 40–64% UQH2-10, 42–85% PQH2-9 and 43–80% a-TQH2,depending on the antioxidant concentration in liposomes (5–1 mol%, respectively) which reflects approximately theirreactivity against radicals derived from the Fenton reaction. The antioxidant activity of the investigatedprenylquinols, in relation to the activity of a-T, in natural membranes is discussed. © 1997 Elsevier Science IrelandLtd.

Keywords: Plastoquinol; a-Tocopherol quinol; Ubiquinol; a-Tocopherol; Antioxidant activity; Lipid peroxidation;Liposome

1. Introduction

Apart from the well established role of plas-toquinol-9 (PQH2-9) and ubiquinol-10 (UQH2-10)

as hydrogen carriers in the photosynthetic (Richand Moss, 1987) and respiratory (Gutman, 1980)electron transport chains, respectively, there isincreasing evidence accumulating of their addi-tional function as antioxidants (Frei et al., 1990;Kruk et al., 1994; Mukai et al., 1993; Stocker etal., 1991; Yamamoto et al., 1990) similar to that

* Corresponding author: Tel.: +48 12 341305 ext. 237; fax:+48 12 336907; e-mail: strzalka@mol.uj.edu.pl

0009-3084/97/$17.00 © 1997 Elsevier Science Ireland Ltd. All rights reserved.

PII S 0 0 9 -3084 (97 )00027 -3

J. Kruk et al. / Chemistry and Physics of Lipids 87 (1997) 73–8074

of a-tocopherol (a-T). The occurrence of a-toco-pherol quinol (a-TQH2) in thylakoid and mito-chondrial membranes whose function is hithertonot fully explained may be also connected withit’s antioxidant action in these systems as suggestsome preliminary experiments (Bindoli et al.,1985; Kruk et al., 1994).

Lipid peroxidation is an autocatalytic chainreaction induced by a free radical initiator (I�),proceeding in the following steps (Vigo-Pelfrey,1990):

initiation : LH+I��L�+IH (1)

propagation : L�+O2�LOO� (2)

LOO�+LH�LOOH+L� (3)

termination : LOO�+AH�LOOH+A� (4)

where LH, I� and L� and LOO� and AH arepolyunsaturated fatty acid, initiator, the fatty acidalkyl and peroxyl radicals, and antioxidantmolecule, respectively. a-T is the best known ex-ample of natural lipid soluble antioxidants(Machlin, 1980). The reduction of peroxyl radicalsby an antioxidant molecule Eq. (4) is the keyreaction accounting for the inhibitory effect ofantioxidants in lipid peroxidation.

In model systems, Fe2+ salts were widely usedfor initiation of lipid peroxidation (Fujii et al.,1991; Fukuzawa et al., 1988a,b; Gutteridge, 1984;Liebler et al., 1986) giving in Fenton-type reactionwith H2O2 reactive hydroxyl radicals (and OH)which could be effective peroxidation initiators(Halliwell and Gutteridge, 1990):

Fe2+ +H2O2�Fe3+ +�OH+OH− (5)

Although the hydroxyl radical formed in thisreaction is a very reactive species, it is a veryshort-lived radical which reacts with most organiccompounds at nearly diffusion-controlled rates(Halliwell and Gutteridge, 1990). This reactivitymakes little probable migration of �OH from thesite of generation to the hydrophobic membranecompartments where the lipid peroxidation mustbe initiated. The other possibility could be site-specific, generation of �OH radicals at the sitewhere they could immediately react with thetarget molecule, e.g. at the membrane surface

(Halliwell and Gutteridge, 1990). It was also pro-posed (Schaich and Borg, 1988) that the Fentonreaction Eq. (4) could also occur in the lipid phaseof the membrane. The alternative initiators, sug-gested in Fenton reactions are complexes betweenoxygen and different valence state of iron (Halli-well and Gutteridge, 1990; Minotti and Aust,1987), such as ferryl ion (FeO2

+ or FeOH3+), theperferryl ion (Fe2+-O2 or Fe3+-O2

−�) and a fer-rous-dioxygen-ferric complex (Fe2+-O2-Fe3+). Inthe absence of H2O2, Fe2+ ions may also inducelipid peroxidation by reaction with traces of per-oxides present originally in lipids or formed dur-ing the propagation step Eq. (3) of theperoxidation reaction:

Fe2+ +LOOH�Fe3+ +LO�+OH− (6)

LO�+LH�LOH+L� (7)

The considerably lower concentration of lipid per-oxides than those of H2O2 usually used in theexperiments is partially compensated by about 20times higher reaction rate constant of the Eq. (6)than of the Eq. (5) (Halliwell and Gutteridge,1990). Therefore, the rate of formation of theinitiating radicals is probably of the same order inboth cases.

Although there have been some studies per-formed on PQH2-9 and a-TQH2 as lipid antioxi-dants (Bindoli et al., 1985; Kruk et al., 1994;Mukai et al., 1992), mainly in solution, and onUQH2-10 in different systems (Beyer, 1990;Cabrini et al., 1991; Frei et al., 1990; Kagan et al.,1990; Mukai et al., 1992, 1993; Stocker et al.,1991; Yamamoto et al., 1990), so far there was noreport on the antioxidant activity of theseprenylquinols during Fe2+-induced lipid peroxi-dation of liposome membranes. Although thewidely used system of peroxidation initiation withthe azo-compounds enables calculation of peroxi-dation inhibition rate constants (Barclay et al.,1984), it gives much lower peroxidation rate thanthe Fenton system and therefore longer measure-ments time is required, during which considerableoxidation of such labile compounds asprenylquinols may place. Moreover, the lipidsoluble azoinitiator (AMVN) may perturb theliposome bilayer structure and change the antioxi-

J. Kruk et al. / Chemistry and Physics of Lipids 87 (1997) 73–80 75

dants localization or mobility within the mem-brane.

We have determined the inhibitory effect of thenatural prenylquinols, in comparison to a-T, onFe2+-H2O2-induced lipid peroxidation of egg yolklecithin liposomes at different antioxidant concen-tration, measuring oxygen uptake during this pro-cess. The prenyllipids concentration during theperoxidation reaction was followed directly bytheir fluorescence intensity (Kruk et al., 1993,1994). Since quinols are known to oxidize undermolecular oxygen, this reaction was measured forthe compounds used in our study, and the reac-tion rate constants of prenyllipids auto-oxidationwere determined. These data were used for thecorrection of oxygen uptake during the lipid per-oxidation reaction.

2. Materials and methods

Ubiquinone-10 and plastoquinone-9 were akind gift from Hoffmann-La Roche (Switzerland).They were purified by TLC on silica gel plates(Merck) using chloroform as an eluent. a-Toco-pherol quinone was prepared and purified accord-ing to Kruk (1988). The prenylquinols wereobtained by reduction of the correspondingquinones with NaBH4 in methanol. The a-T wasfrom Merck and egg yolk lecithin (EYL), typeV-E, was purchased from Sigma. The EYL prepa-ration used for our studies was partially oxidized,having absorbance ratio A233/A215=0.73 inethanol, which corresponds to approximately 1%of peroxides (Bergelson, 1980). Such increasedcontent of lipid peroxides should promote Fe2+-LOOH catalyzed lipid peroxidation. Small unil-amellar liposomes were prepared by injection ofethanol solutions of an antioxidant and EYL into10 mM Tris buffer (pH=6.5) under continuousstirring. The final EYL concentration was 0.5mM, ethanol concentration of 1.25% and antioxi-dants content in liposomes of 1, 3 or 5 mol%. Allthe measurements and liposome preparation wereperformed at 25°C. Lipid peroxidation was mea-sured by monitoring oxygen uptake using Clark-type electrode (Hansatech), assuming an oxygenconcentration of 253 mM in the initial reaction

mixture at 25°C. Prenylquinols and a-T concen-tration was measured fluorimetically, on Perkin-Elmer LS-50 fluorimeter, using excitation at 290nm and emission at 370 nm for UQH2-10 or 330nm for the other compounds. We have found nochanges in the fluorescence background level dur-ing the peroxidation reaction of the control sam-ple containing no antioxidant, which couldinterfere with the antioxidant fluorescence. Thetotal sample volume was 1 ml for oxygen uptakemeasurements and 2 ml for fluorescence measure-ments. Ferrous ammonium-sulphate (Aldrich)stock solution, because of its instability, was pre-pared directly before the measurements, in nitro-gen saturated water. The lipid peroxidationreaction was started by addition of H2O2 andFe(NH4)2(SO4)2 stock solutions to the preformedliposomes to a final concentration of 50 mM Fe2+

and 100 mM H2O2.

3. Results and discussion

Table 1 shows changes in the prenyllipids (AH)level in liposome membranes after the first 15 minof the measurement. The only possible reason forthe prenyllipids concentration decrease is theiroxidation under molecular oxygen, since the sam-ples did not contain any initiators of the lipidperoxidation reaction. The average oxidationrates demonstrate that a-T autoxidation is negli-gible, whereas UQH2-10 is most easily oxidizedand the rates changed in the order UQH2-10\a-TQH2\PQH2-9\\a-T. For the determinationof the second order rate constants of the reaction(6=k [O2][AH]) the initial AH concentrations, re-action rates (DAH) and oxygen concentration(253 mM) were taken. The k values given in Table1 are the average of the k values calculated fordifferent initial AH concentrations (1, 3 and 5mol%). The oxidation rates of the prenyllipids(D[AH]15 min%) are similar for their different con-tent in liposomes. This indicates that the possibledifferences in the localization of prenyllipidswithin the liposome bilayer at their different con-tent (Jemioła-Rzeminska et al., 1996; et al.,1992) have no influence on the prenyllipids autox-idation rates. The obtained rate constants are

J. Kruk et al. / Chemistry and Physics of Lipids 87 (1997) 73–8076

Table 1Rates of prenyllipids (AH) in liposome membranes and the second order rate constants (k) of the reaction determined from thefluorescence intensity changes of the prenylquinols

D[AH]15min (%) (average) D[AH](mM/min)Antioxidant [content in mol%] k(M−1/s)D[AH]15min (%)

3.590.6 0.014PQH2-9 [1] 0.1544.491.00.033.090.7PQH2-9 [3]0.055PQH2-9 [5] 3.390.1

0.0186.190.95.690.5 0.268a-TQH2 [1]0.053a-TQH2 [3] 5.391.60.124a-TQH2 [5] 7.490.7

0.590.3 0.002a-T [1] 0.0220.890.30.0030.390.1a-T [3]0.008a-T [5] 0.590.4

0.0369.492.710.890.9 0.413UQH2-10 [1]0.076UQH2-10 [3] 7.692.30.165UQH2-10 [5] 9.994.9

comparable with those determined for UQH2-1(1.5 M−1/s, pH 7.5) (Sugioka et al., 1988) andUQH2-0 (1.32 M−1/s, pH 7.3) (Cadenas et al.,1977) in aqueous systems. a-Tocopherol practi-cally does not undergo autoxidation. Since theautoxidation of prenylquinols is pH dependentand is considerably faster at alkaline pH (29), sothe observed reaction rates in our case (pH=6.5)are expected to be higher at physiological pH (7.4)and temperatures. Even though the rate constantsare relatively low, the autoxidation ofprenylquinols decreases their concentration andthis effect should be taken into account in studiesof peroxidation inhibition reactions, especiallyduring long-lasting reactions, such as those withthe azoinitiators. The oxidized prenylquinols(prenylquinones), in contrast to a-tocopherol,could be easily rereduced in natural membranes:plastoquinone by photosystem II in thylakoidmembranes (Rich and Moss, 1987); ubiquinoneby NADH-ubiquinone reductase or succinate-ubiquinone reductase in mitochondrial mem-branes (Gutman, 1980) and a-tocopherol quinoneboth in thylakoid (Kruk and Strzałka, 1995) andmitochondrial (Bindoli et al., 1985) membranes byunidentified enzymes.

Changes in the AH concentration during Fe2+-H2O2-induced lipid peroxidation of EYL lipo-somes are biphasic (Fig. 1). The first, fast phase

taking about 30–40 s and a following, slow phasecorrespond to similar changes in oxygen con-sumption during the peroxidation reaction (Fig.2). During the fast phase, the formation of hy-droxyl radicals takes place Eq. (5) and probablyof other active radicals which initiate lipid peroxi-dation and oxidize the prenyllipids. The hydroxyl

Fig. 1. Prenyllipids concentration changes in EYL liposomesduring Fe2+-H2O2-induced lipid peroxidation. The antioxi-dants initial concentrations were 3 mol% in liposomes (15 mMin solution); �: a-T, : UQH2-10, �: PQH2-9,+ : a-TQH2

J. Kruk et al. / Chemistry and Physics of Lipids 87 (1997) 73–80 77

Fig. 2. Oxygen concentration changes during Fe2+-H2O2-in-duced lipid peroxidation of EYL liposomes(——), and lipo-somes containing 3 mol% of a-T (�), PQH2-9 (�), a-TQH2

(+ ) or UQH2-10 ().

AH oxidation by Fe3+ ions (data not shown)which are formed in the Fenton reaction. As canbe seen from Fig. 2, there is a pronounced in-hibitory effect of all the investigated antioxidantson oxygen consumption during lipid peroxidationof EYL liposomes, which is the consequence ofperoxyl radicals reduction by antioxidants Eq. (4)and breaking the chain reaction. The oxygen up-take rate is similar for 3 and 5 mol% of a givenAH (Table 3) but at 1 mol%, apart from a-T, isstrongly increased, probably by the high degree ofAH oxidation in these samples (Table 2). Tocompare the inhibitory effects of the investigatedantioxidants, we have to also take into accountthe oxygen consumption associated with AH oxi-dation in the slow phase and due to AH autoxida-tion (assuming O2:AH stoichiometry of 1:1) andthe amount of the antioxidant left after the fastphase (1–100/D[AH]FP[%]). The corrections forAH oxidation is shown in brackets (Table 3,column 3) and the peroxidation inhibition coeffi-cients calculated for the initial AH concentrationfrom the formula: (100/v)/(1-D[AH]FP[%]/100) areshown in Fig. 3. The corrected inhibition coeffi-cients are the highest for a-TQH2 and PQH2-9 for3 and 5 mol%. The considerably lower values for1 mol% might be connected with a low antioxi-

radicals are formed in the Fenton reaction onlywithin the first 30 s of the reaction (Minotti andAust, 1987). Therefore, their formation is proba-bly negligible in the slow phase. The oxidation ofAH during the fast phase followed the singleexponential decay with k values in the range of11–13×10−2 s−1 and without any pronouncedAH concentration dependence. The relative reac-tivity of the investigated compounds against radi-cals which initiate peroxidation in our system(mainly hydroxyl radicals), can be inferred fromthe relative AH amount oxidized after the fastphase of the reaction which changed in the ordera-TQH2\PQH2-9\UQH2-10\a-T (Table 2)for 3 and 5 mol% of the antioxidants content inliposomes. This order is probably a measure ofthe prenyllipids antioxidants activity against hy-droxyl radicals formed in the fast phase of theperoxidation reaction. During the slow phase,where Fe2+-LOOH-induced lipid peroxidationEq. (6) dominates, AH consumption was rela-tively low (Table 2) and was caused by oxidationof AH molecules by the lipid peroxyl radicals(chain breaking reaction, Eq. (4)) and slow AHautoxidation. We have not observed any direct

Table 2Concentration changes of prenyllipids (AH) after the fastphase of Fe2+-H2O2-induced lipid peroxidation (D[AH]FP) ofEYL liposomes and rates of AH oxidation during the slowphase (D[AH]SP)

D[AH]SP(mMAntioxidant [content in D[AH]FP (%)mol%] AH/min)

PQH2-9 [1] �0.018553PQH2-9 [3] 0.0842PQH2-9 [5] 0.23

80a-TQH2 [1] �0.020.23a-TQH2 [3] 60

a-TQH2 [5] 0.5843

�0a-T [1] 3330a-T [3] 0.09

a-T [5] 0.1327

64UQH2-10 [1] �052UQH2-10 [3] 0.09

UQH2-10 [5] 0.4040

J. Kruk et al. / Chemistry and Physics of Lipids 87 (1997) 73–8078

Table 3Oxygen concentration changes during the slow phase of Fe2+-H2O2-induced lipid peroxidation of EYL liposomes, the relative rates(6) and ratios of the reaction rates with and without prenyllipids (100/v)

D[O2] Inhibition ratio (100/v)Antioxidant [content in mol%]Relative rate (6)(mM O2/min)

16.092.3 100— 1

3.33130PQH2-9 [1] 4.890.90.8190.02 5 19.8 (22.8)PQH2-9 [3]0.8790.30 5.4PQH2-9 [5] 18.5 (30.2)

5.6190.11 2.85a-TQH2 [1] 351.1690.20 7.2a-TQH2 [3] 13.8 (18.2)

6.7 14.8 (63.2)a-TQH2 [5] 1.0890.12

0.9190.08 5.7 17.6a-T [1]0.9090.11 5.6a-T [3] 17.8 (19.8)0.6590.10 4.0a-T [5] 24.6 (31.8)

25.74.1290.34UQH2-10 [1] 3.913.6 (15.8)7.4UQH2-10 [3] 1.1890.15

8.7 11.5 (24.2)UQH2-10 [5] 1.3990.13

The values in brackets were calculated taking into account oxygen consumption by prenyllipids in the slow phase and due to theirautoxidation.

dant content after the fast phase (0.15 mol% forPQH2-9, 0.2 mol% for a-TQH2 and 0.36 mol% forUQH2-10) for the effective lipid peroxidation inhi-bition. The calculated inhibition coefficients pre-sented in Fig. 3 are reflecting the antioxidantactivity of the prenyllipids against lipid peroxida-tion derived peroxyl radicals (Eq. (4)).

It should be also considered if the semiquinoneforms of the investigated prenyllipids, formed in

the Eq. (4), could act as pro-oxidants, i.e. stimu-late the lipid peroxidation, for example by decom-position of lipid peroxides, as in the case of Fe2+

ions Eq. (6). However, it was found (Porter et al.,1995) that the reaction rate constants of the pro-oxidant reactions of a-T are several orders ofmagnitude lower than the k value of it’s antioxi-dant reaction (Eq. (4)). Similar relations can beexpected for the prenyllipids investigated in oursystem.

Our results show that in the determination ofthe inhibitory effects of labile compounds such asprenylquinols on lipid peroxidation, it is impor-tant to examine all the possible side reactions, e.g.the reaction of peroxidation initiators with theantioxidants, which may strongly influence theirconcentrations. In the case of azoinitiators, thereis a constant rate of initiator production duringthe peroxidation reaction. This reaction con-stantly changes the level of antioxidants which isnot due to the termination reaction by the antiox-idants as it was recently shown by Koga andTerao, 1996 where the rate of tocopherol loss indimyristoyl phosphatidylcholine liposomes wassimilar as in soyabean phosphatidylcholine lipo-somes in the course of AAPH-initiated peroxida-

Fig. 3. Peroxidation inhibition coefficients of prenyllipids(AH) in EYL liposomes calculated for the initial prenyllipidsconcentration: (100/v)/(1-D[AH]FP[%]/100).

J. Kruk et al. / Chemistry and Physics of Lipids 87 (1997) 73–80 79

tion reaction. Although our initiation system isnot as well defined as that of the azoinitiators, italso gives in the slow phase of the reaction contin-uous initiating system (Fe2+ +LOOH � Fe3+

+LO�+OH) which is evident from theapproximately constant antioxidants and oxygenconcentration changes during the slow phase(Figs. 1 and 2).

Considering the function of the investigatedprenylquinols as antioxidants in natural mem-branes we have to take into account the relativeproportions of UQH2-10 and a-T in mitochon-drial membranes (Mellors and Tappel, 1966)whose molar ratio is about 10 and PQH2-9 anda-T ratio in thylakoid membranes (Lichtenthaleret al., 1981) which is about 2. This suggests thatthe contribution of PQH2-9 and UQH2-10 in pre-venting membrane lipid peroxidation may be evenhigher than that of a-T. The recent finding (Hun-dal et al., 1995) of inhibition of lipid peroxidationby PQH2-9 in chloroplast thylakoid membranesduring strong illumination may support the sig-nificant antioxidant function of PQH2-9 in naturalmembranes.

Acknowledgements

This work was supported by Committee forScientific Research (KBN) grant 6 6075 92 03 and6 PO4A 009 10.

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