Kinetics of NADPH-dependent lipid peroxidation and a possible initiation-preventing antioxidant effect of microsomal ( + )-α-tocopherol

Biochimica Elsevier

347

et Biophysiccl Acta 917 (1987) 347-355

BBA 52339

Kinetics of NADPH-dependent initiation-preventing

lipid peroxidation and a possible

antioxidant effect of microsomal ( + )-ar-tocopherol Istvan Venekei

2nd Institute of Biochemistry

Semmelweis (Received

Key words:

Lipid peroxidation;

University Medical School, Budapest (Hungary) 28 July 1986)

(+)-~Tocopherol; NADPH-cytochrome-P-450 Malonaldehyde; (Liver microsome)

reductase;

Lipid hydroperoxide;

The initial part of NADPH-driven lipid peroxidation was investigated in liver microsomes. Without the addition of any antioxidant or pretreatment of animals with vitamin E, a delay was observed in the malondialdehyde and lipid hydroperoxide formation, but not in oxygen consumption. The duration of lag and the effect of ADP-Fe*+ on it showed differences in rat, mouse, chicken and rabbit microsomes. As it was not caused by cytoplasmic contaminations, this lag was an indication of the antioxidant capacity of microsomes, possibly due to the oxidation of their ( + )-a-tocopherol content. The length of lag was dependent on the NADPH-cytochrome-P-450 reductase activities and the concentration of Fe-ion complexes. The results presented here suggest that ( + )-a-tocopherol acted during the lag as an initiation-preventing rather than a chain-breaking antioxidant in rat liver microsomes. The lag may explain the known differences found in the inducibility and intensity of lipid peroxidation of microsomes from various species, and provides means to elucidate the molecular mechanism of vitamin E action against free radicals formed in a membrane of biological origin.

Introduction Due to its presumed importance in a number of pathological processes, the mechanism of lipid peroxidation and the effect of antioxidants on it have been extensively studied. The great majority of these studies have been carried out on polyunsaturated fatty acids in solution or incorporated into phospholipid liposomes, [l-8]. Substantially less information is available on the underlying peroxidative and protective processes in biological membranes. Thus, it is not clear whether the

Abbreviation:

ADP-Fe2+,

ADP-stabilized

Fe2+

(100: 1).

Correspondence: I. Venekei, 2nd Institute of Biochemistry, Semmebveiss University Medical School. 1444 Budapest POB. 262, Hungary. 00052760/87/$03.50

0 1987 Elsevier Science Publishers

molecular mechanism of the antioxidant effect of ( + )-a-tocopherol proposed for simple model systems can be directly applied to biological membranes, e.g. microsomes. When liver microsomes of tocopherol-untreated animals were incubated in the absence of Fe-complexes and antioxidants, two phases could be seen on the kinetics of endogenous, NADPH-dependent malondialdehyde production: an initial, lowrate period (a lag) was followed by a higher-rate one [9-151. (The term ‘endogenous’ refers to the NADPH-driven process which is stimulated only by Fe-ion contaminations and not by the addition of exogenous Fe-complexes.) Despite the possible importance of lag as one of the measures of endogenous antioxidant and lipid peroxidation initiating capacities of biological membranes, the causes of the biphasic nature of malondialdehyde

B.V. (Biomedical

Division)

348

production are not known at present. Similar but longer lag periods were obtained in lipid peroxidation enhanced by Fe-complexes where either large quantities of (+)-c~-tocopherol were added before homogenization of liver [16-181, or vitamin E was supplied in vivo [19]. However, the role of the microsomes’ own (+)-cu-tocopherol content in the kinetics of microsomal lipid peroxidation has not been investigated as yet. The data published so far indicate only that ( +)-cu-tocopherol has been completely oxidized by a long (40-60 min) incubation [14,15,20]. We supposed that the lag, which can be observed on the course of endogenous malondialdehyde formation, is an indication of the microsomes’ own antioxidant capacities, and that the normal microsomal (+)-a-tocopherol content is responsible for it. Furthermore, we thought that studying the lag would provide insight into the mode of ( +)-a-tocopherol action in a membrane of biological origin. In this paper we describe a characterisation of the lag (lipid hydroperoxide formation, rate of malondialdehyde production and 0, consumption), the effect of a low vitamin E diet, NADPH-cytochrome-P-450 reductase activity and the addition of ADP-Fe2+, and the kinetics of ( + )-cr-tocopherol oxidation, using rat, mouse, chicken and rabbit microsomes. Materials and Methods Chemicals. Glucose-6-phosphate dehydrogenase (from yeast, grade II, 250 U/ml) and cytochrome c were obtained from Boehringer (Mannheim). 2-Thiobarbituric acid was a product of AldrichChemie (Steinheim), (+)-c~-tocopherol standard was a gift from Henkel Co. (Minneapolis). NADPH, glucose 6-phosphate disodium salt and all other chemicals were reagent grade and purchased from Reanal (Hungary). Animals. Rabbits, Wistar rats and CFLP mice (weighing 1.0-1.5 kg, 200-250 and 20-25 g, respectively) were fed ad libitum on standard Lati diets, containing 100 IU/kg (+)-cu-tocopherol acetate for rabbits, and 175 IU/kg (+)-atocopherol acetate for rats and mice. The food for Hunnia hybrid chickens (1.0-1.5 kg) was obtained from Phylaxia (Hungary), containing 270 IU/kg ( + )-a-tocopherol acetate. Vitamin E depletion in

a group of 668-week-old rabbits was achieved by a diet according to Goettsch and Pappenheimer [21]. Liver microsomes were prepared at the end of a 6-8 week diet. Plasma and microsome (+)a-tocopherol content of rabbits are shown in Table I. Microsomes. Livers of the decapitated animals were minced in ice-cold 0.15 M potassium phosphate buffer, pH 7.5. After 4-5 decantations, a 20% homogenate was made in the same buffer, and centrifuged at 9000 X g for 25 min. The microsome fraction was sedimented by ultracentrifugation at 105 000 X g for 60 min. To obtain oncewashed microsomes, the microsome pellet was suspended in 100 vol. of Tris-HCl buffer (0.1 M Tris-HCl, pH 7.5, 50 mM KCl), and ultracentrifuged again. Microsome pellets were stored at - 22O C for a maximum time of 24 h. Incubation and assays. All incubations were carried out in 30°C in Tris-HCl buffer (0.1 M TrisHCl, 50 mM KCl, pH 7.5) containing 0.3 mM NADPH, regenerating system (5.0 mM glucose 6-phosphate, 0.05 U/ml glucose-6-phosphate dehydrogenase) and 1.0 mg/ml microsomal protein. To stimulate lipid peroxidation, ADP-stabilized Fe2+ was added into the reaction mixture (in a final concentration of 20 PM, i.e. 20 PM FeSO, and 2 mM ADP). When lipid hydroperoxide and malondialdehyde formation or ( + )-cy-tocopherol contents were measured, the maximal reaction volumes were 10 ml in lOO-ml Erlenmeyer flasks, and the incubations were carried out in a shaking bath. The reactions were started by the addition of microsomes to the reaction mixtures preincubated for 2 min, and the following parameters of lipid peroxidation were determined: malondialdehyde concentration by 2-thiobarbituric acid assay [22], lipid hydroperoxide content by the iodometric method according to Buege and Aust [22] in the same experiment, and 0, consumption by polarography using a Clark-type 0, electrode in separate experiments. NADPH-cytochrome-P-450 reductase activities were determined by cytochrome c reduction (at 550 nm) and NADPH oxidation (at 340 nm), in a thermostatted (30°C) cuvette block of a Beckman Model 25 spectrophotometer in 0.5 ml final volume of the above reaction mixture, except that the protein concentration

349

was 10 pg/ml. After 2 min preincubation in the reaction was initiated by 20 /.LM cytochrome c. In NADPH oxidase activity assays the initial NADPH concentration was 100 PM, and the NADPH-regenerating system was omitted. The (+)-c~-tocopherol concentrations in microsome suspensions and in blood plasma were determined, after saponification according to Taylor et al. [23]. The samples to be saponified contained 5-10 mg of microsomal protein or OS-l.0 ml of plasma. Quantitation of (+)-c~-tocopherol was accomplished in a Jobin-Yvone Type 3 Single Light-Path spectrofluorimeter, at 286 nm excitation, 330 nm emission with 10-10-4-4 nm slit combination, using a 300 nm filter in the path of the emitted light. The kinetics of (+)-cu-tocopherol oxidation were followed by HPLC, because upon saponification the quite stable chromanoxyl radical, supposed to be formed on the course of the oxidation of ( +)-~tocopherol may be reduced back by ascorbic caid [24]. 0.2-ml aliquots taken from the incubation mixture were extracted and chromatographed according to Vandewoude et al. [25], using a Bio-Rad 1130 HPLC instrument equipped with a 250 X 4.6 mm Chropack LiChrosorb RP8 5 column (Merck), and a wall-jet type electrochemical detector (EClO-l-A, manufactured by the Department of Applied and Analytical Chemistry, Technical University, Budapest). The ( +)-cr-tocopherol concentrations determined by fluorimetry (in microsome suspension) and by HPLC (in 0 min samples) were the same. The recovery by both the fluorimetric and HPLC methods was 92-95%, estimated by the addition of 2.0 pg (+)-cr-tocopherol as internal standard. Protein concentrations were determined by the method of Lowry et al. [26], using bovine serum albumin as standard. Results Both unwashed and washed (once or twice) microsome preparations produced similar patterns of malondialdehyde production, although the first and second washes decreased the protein content of microsome suspensions by approx. 50 and 20%, respectively. Fig. 1 shows the kinetics of malondialdehyde production of once-washed rat liver microsomes. The same results were obtained

00

7 i; h 00:

r ; C 6

1 10

20

30

40

Ttme Cmln)

Fig. 1. The kinetics of malondialdehyde production and 0, consumption of once-washed rat liver microsomes. For malondialdehyde (MDA) determination O.l-ml aliquots of the incubation medium were added to 1.0 ml of thiobarbituric acid reagent. Details of microsome washing and incubation’ are described under Materials and Methods. 0, endogenous, and W, 20 pM ADP-Fe2+-stimulated malondialdehyde production. - - -, endogenous, and .. .. ., 20 PM and ADP-Fe2+stimulated 0, consumption. Each point represents ?iS.D., n = 10 microsome preparations.

when the microsomal pellet had been sonicated before the wash, or when mitochondrial fragments had been sedimented prior to pelleting the microsomes (data not shown.) Thus the lag appeared to be an inherent feature of microsomes and could not be attributed to cytoplasmic components. To characterize the lag, three parameters of initiation and propagation of lipid peroxidation were monitored, namely Oz consumption, lipid hydroperoxide formation and malondialdehyde accumulation. The two phases of the endogenous malondialdehyde production (Fig. 1) were designated by their velocities ur (low, referring to the lag) and vq (high). In ADP-Fe*+-enhanced malondialdehyde production, the higher rate of the phases, which was generally the first one, was designated u2 .) During the NADPH-driven endogenous lipid peroxidation of rat liver microsomes (Fig. 1) the

350 TABLE

I

PARAMETERS OF LIPID PEROXIDATION, (+)-a-TOCOPHEROL CONTENT AND NADPH-CYTOCHROME-P-450 DUCTASE ACTIVITY IN LIVER MICROSOMES PREPARED FROM DIFFERENT SPECIES

RE-

Determinations of 0, consumption, malondialdehyde (DMA) production, cytochrome-c reductase and NADPH oxidase activities of microsomes are described in Materials and Methods. Quantitation (+)-n-tocopherol in microsome suspensions and rabbit blood plasma was performed by fluorimetty, except in the case of rat, where the HPLC method was also used (see Materials and Methods).

Microsome origin

ADP-Fe*+ addition

Length of lag (min)

MDA production rates (nmol/mg protein per min) “1

Rat

_

Mouse

+ _

Chicken

+ _

Rabbit

+ _

Rabbit

E-

+ _ +

9.4*0.8 0 12.5 f 2.9 0 30.2 k 4.7 Oa 25.8 + 4.6 0” 0 0

“2

0.39&0.14 _ 0.32 k 0.09 0.07 5 0.02 20 0.06 f 0.03 20 _

0.88 f 0.08 5.04kO.48 0.52 * 0.13 5.00 k 0.28 0.10 * 0.04 1.60+0.15 0.18+0.03 0.48 f 0.07 0.16+0.02 0.49*0.15

’ The lag was too short to be calculated, but proved not to be 0 min, because x-intercepts of regression different from 0 (approx. 2-3 min see also Fig. 3). (In the absence of lag they were within 0.5 min.)

difference in velocity between the two phases of malondialdehyde formation was about 2-fold (Fig. 1, Table I). Length of the lag was about 9 min, calculated from the regression equations of u1 and u2 (for the calculation see the legend to Table I). As in malondialdehyde formation, there was also a lag in lipid hydroperoxide formation, which ranged from 6 to 12 min (Fig. 2). Contrary to the lipid hydroperoxide and malondialdehyde formation, 0, consumption did not show any lag, and was constant during the first 30 min of incubation (Fig. 1 and Table I). The addition oft 20 PM ADP-Fe*+ into the reaction mixture completely abolished the lag (Fig. 1) and resulted in the well-known immediate high rate of 0, consumption, lipid hydroperoxide formation and malondialdehyde accumulation (Figs. 1 and 2, and Table I). The parameters of NADPH-dependent endogenous and ADP-Fe*+-enhanced lipid peroxidation, in relation to the lengths of lags, were investigated in microsomes of some other species, too, to establish whether the known species differences of lipid peroxidation [16,27-301 are also reflected in the duration of the lag. The lengths of lags of

02

consumption (nmol/mg protein per min) 17.4 * 5.0 182.0+ 16.6 18.8 t_ 4.7 147.6 + 9.9 11.7k6.3 24.8 + 7.9 9.2 i 1.2 18.6 + 3.2 7.8k2.1 17.7 + 2.7 lines for v2 were significant

endogenous malondialdehyde production were rather different, and increased in the following order: rat < mouse < rabbit < chicken, while 0, consumption and malondialdehyde production rates (ui and uz) increased in the opposite direction (Table I). As in the case of rat microsomes, the rates of 0, consumptions in microsomes of the other three species did not change after the ends of lags. During the incubation of chicken and rabbit microsomes, their lipid hydroperoxide contents remained below the limit of detection in both the absence and presence of 20 PM ADPFe*+, while in mouse microsomes lipid hydroperoxide measurements gave the same results as in rat microsomes (data not shown). Despite the considerable increase in lipid peroxidation on addition of ADP-Fe*+, the lag disappeared only in mouse microsomes, but persisted, though to a significantly reduced extent, in chicken and rabbit microsomes (Table I). There is a similarity between the lag of endogenous lipid peroxidation and the delay in oxidation of polyunsaturated fatty acids in solution or in liposomes upon the addition of antioxidants (e.g. tocopherols [l-8,15-19]). This phenomenon pro-

351

The lengths of lags were obtained by calculations as follows: regression equations were calculated for both parts of endogenous malondialdehyde formation curves (ut and u2), and the times belonging to the crosspoints of corresponding ut and u2 curves were taken as the lengths of lags. Means + S.D. of data from at least five microsome preparations are listed.

NADPH-cytochrome-P-450 reductase activity (nmol/mg

protein

( +)-a-tccopherol content

per min)

cyt. c reduction

NADPH oxidation

microsome

plasma

(pg/mg)

(pg/mg)

88.5 I 14.7

89.0+ 8.9

0.282 * 0.039

80.3 + 18.4

66.7 + 10.4

0.325 * 0.049

31.8k5.0

44.3 _+6.4

0.276 f 0.090

35.5 + 7.2

45.5 & 8.1

0.260 k 0.41

3.073 * 0.72

28.1 + 5.6

43.4 & 5.6

0.054 + 0.008

0.96OiO.15

vides a simple explanation for the existence of lag, namely that the lipid peroxidation is hindered by the oxidation of a microsomal (antioxidant) constituent. The observation that malondialdehyde formation induced by 0.5 mM ascorbate in rat liver microsomes, heated previously to 90 o C for 3 min, also showed a lag of 5 min (not shown) suggested a small, lipid-soluble antioxidant molecule, such as ( +)-cu-tocopherol, to be responsible for the lag. To test this assumption we measured the changes in microsomal ( + )-a-tocopherol levels during NADPH-driven lipid peroxidation in rat liver microsomes. In the course of endogenous malondialdehyde formation the decrease in (+)cu-tocopherol content was about 70% until the 10th min of lipid peroxidation (i.e. at the end of the

IO--

)0-.

I

0-i 0

IO

20 Time

30

40

(m(n)

Fig. 2. Time-dependent changes in lipid hydroperoxide and (+)-a-tocopherol contents during lipid peroxidation of rat liver microsome. For the determinations of lipid hydroperoxide concentrations of microsomes (closed symbols), samples of 0.1 ml were taken and extracted with 1.1 ml of chloroform/ethanol

(2 : 1). To determine the (+)-a-tocopherol content of microsomes (open symbols) the 0.2-m] samples taken from incubation medium were extracted with 2.0 ml n-hexane after the addition of 0.2 ml of ethanol. Further details of lipid hydroperoxide and (+)-a-tocopherol determinations and incubation are given in Materials and Methods. 0, 0, endogenous; w 0, 20 pM ADP-Fe*+-stimulated lipid peroxidation. Each point shows Xi SD., n = 5 in the case of (+)-a-tocopherol determination and n = 12 for lipid hydroperoxide measurement, using different microsome preparations.

352

lag; Fig. 2). It was greatly accelerated by the addition of ADP-Fe*+ and the ( +)-CY-tocopherol content was reduced to 20% of the original level within 3 min (Fig. 2). In both types of lipid-peroxidating systems, after 30 min of incubation only tocopherol quinone was present as the oxidized form of microsomal ( +)-a-tocopherol (unpublished data). As an alternative approach to studying the role of ( +)-CX-tocopherol in production of lag, the kinetics of microsomal malondialdehyde formation of rabbits fed on a normal and a tocopherol-free diet were compared. The (+)-atocopherol content of microsomes from vitamin E-deficient rabbits was about 20% of that of control ones (Table I). Here the lag disappeared not only in the ADP-Fe 2+-induced but also in the

endogenous lipid peroxidation. Surprisingly however, the corresponding u2 values or 0, consumptions were identical in microsomes prepared from normal and vitamin E-depleted rabbits (Fig. 3, Table I). The small differences in microsomal ( +)-cytocopherol content of various species did not explain the species differences in the lengths of lags. We supposed that different NADPH-cytochromeP-450 reductase and cytochrome P-450 activities caused this variation, because the activities of these microsomal electron-transport proteins, relevant in NADPH-dependent lipid peroxidation, show considerable species differences [28,30]. Indeed, NADPH-cytochrome-c reductase and NADPH oxidase activities of NADPH-cytochrome-P-450 reductase were lower in those microsomes that showed longer lags compared to those having shorter ones (Table I). However, the

20-I

15-

; s k r

lo-

. a,

0 E c

d =

5

0 0

10

20 Tome

30

40

50

(min)

Fig. 3. Malondialdehyde (MDA) production by liver microsomes prepared from normal and vitamin E-deficient rabbits. Malondialdehyde produced during incubation was determined by the addition of O.l-ml of thiobarbituric acid reagent. For the details of incubation see Materials and Methods. O,O, endogenous; IJ,~, 20 PM ADP-Fe2+-stimulated lipid peroxidation in liver microsomes of normal (open symbols) and vitamin E-deficient (closed symbols) animals. The (+)-atocopherol contents of microsomes are listed in Table I. Each point represents X _CS.D., n = 5 microsome preparations.

0

10

20 Time

30

( m(n)

Fig. 4. Reduction of lag in malondialdehyde (MDA) production of rat liver microsomes by ADP-Fe2+ addition. Malondialdehyde content of microsomes was determined in 0.1.ml aliquots of incubation mixture by adding 1.0 ml of thiobarbituric acid reagent. The following ADP-Fe2+ concentrations were used: l, 0.0 PM (endogenous lipid peroxidation); 0, 0.2 PM; n, 1.0 PM; 0, 10.0 PM; A, 20 PM. The results shown are representative of four experiments.

353

cytochrome P-450 contents and activities (aminopyrine N-demethylase) were unrelated either to the length of lag, or to ui or u2 (not shown). Since NADPH-cytochrome-P-450 reductase enhances the rate of lipid peroxidation in conjunction with Fe ions [31-341, we expected that successive elevation of ADP-Fe2+ concentration at a constant reductase activity would result in a gradual shortening of the lag. Experiments carried out on rat liver microsomes revealed that this was really the case, and ADP-Fe2’ even at a final concentration of 10 FM had maximal effect (Fig. 4). Similar results were obtained when 0, consumptions were monitored, or in experiments with liver microsomes of the other three species (not shown). Discussion We investigated the kinetics of NADPH-dependent microsomal lipid peroxidation, and attempted to relate two phenomena: the lag in malondialdehyde formation and the oxidation of the microsome’s own ( +)-CX-tocopherol content during lipid peroxidation. The results presented here show that the lag in endogenous malondialdehyde production (obtained in the absence of any antioxidant, or without the administration of vitamin E to animals) is not due to contamination of microsomes with cytoplasmic components but rather to the oxidation of its endogenous (+)-a-tocopherol content (Figs. l-3). During the lag in endogenous lipid peroxidation of rat liver microsomes, besides malondialdehyde accumulation, the formation of lipid hydroperoxides is also hindered. Since both the formation of lipid hydroperoxide and the accumulation of malondialdehyde are restricted in the lag, during which the majority of ( +)-a-tocopherol is oxidized, it may be considered as an indication of the antioxidant effect of microsomal (-t )-a-tocopherol content. The lengths of lags during endogenous lipid peroxidation in liver microsomes of rat, mouse, chicken and rabbit are more or less different although the ( +)-CX-tocopherol contents of their microsomes are similar (Table I). This indicates that only the existence but not the length of lag can be

attributed to microsomal (+)-a-tocopherol content. The length possibly depends on some NADPH-cytochrome-P-450 reductaseand Feion-dependent processes. The data collected in Table I and Fig. 4 show that microsomes of shorter lag (rat, mouse) have about twice as high an NADPH-cytochrome-P-450 reductase activity (and 0, consumption) as those of longer ones (chicken, rabbit; Table I), and that at a certain reductase activity the lag is gradually shortened when successively higher amounts of ADP-Fe2+ are added (Fig. 4). The addition of 20 PM ADPFe*+ to microsomes of higher reductase activity leads to rapid (+)-c~-tocopherol oxidation and the elimination of lag. However, in microsomes of lower reductase activity (chicken, rabbit) 20 IJ.M or higher ADP-Fe” concentration (or the use of EDTA instead of ADP for Fe’+ complexation) is unable to enhance these NADPH-cytochrome-P450 reductase-dependent processes to the extent which is necessary for the abolition of lag (Fig. 3, Table I, and unpublished data). Thus at a certain ( + )-a-tocopherol concentration the length of lag depends on the intensity of 0, activation (i.e. 0; and perferryl ion or OH. formation) and on initiation (methene hydrogen abstraction), for which the presence of Fe ions and the NADP-cytochrome-P-450 reductase activities are responsible [35-381. Assuming that the initiation of lipid peroxidation is much more increased by ADP-Fe2+ (due to 0, activation) than propagation [32,34], one might regard 0, consumption and v2 proportional mainly to the intensity of 0, activation and initiation. Of these processes 0, activation is not influenced by the lag, since 0, consumption was the same during and after it; furthermore, 0, consumption and u2 values did not change when the lag disappeared in (+)-c~-tocopherol-deficient microsomes (Figs. 1 and 2, Table I). Two conclusions can be drawn from our results. (1) The species variation of the duration of lag, due to differences in intensity of initiation and thereby in the protection by ( + )-cY-tocopherol, may significantly contribute to the known species differences in malondialdehyde production and its inducibility by Fe ion complexes in NADPHdriven lipid peroxidation of liver microsomes, especially when the latter is measured under short (30-40 min) incubation periods. (2) During the

354

first 5 min of microsomal lipid peroxidation ( +)a-tocopherol presumably acts not as a chainbreaking antioxidant, as concluded from experiments in simple model systems [6-8,38-401, but rather as an initiation-preventing antioxidant. We make this assumption for the following reasons. It is widely accepted that carbon-centered lipid radicals generated in the initiation process are transformed to lipid hydroperoxides accompanied by 0, consumption. In chain-breaking antioxidant reactions with carbon-centered and peroxyl free radicals, however, (+)-&ocopherol cannot effectively prevent this transformation and cannot cause a delay in lipid hydroperoxide formation, because it does not react rapidly with the former [38], and reduces the latter to lipid hydroperoxides [7,16,38]. Therefore it can be supposed that in the first 5 min of lipid peroxidation (+)-a-tocopherol reacts with those active forms of 0, (or their derivatives) which are capable of methene hydrogen abstraction. (The possibility of a reaction between (+)cr-tocopherol and these radicals in micelles. and liposomes was investigated by Fukuzawa and Gebicki; see Ref. 35.) This initiation-preventing mode of (+)-cu-tocopherol action can explain the findings presented here, that during the lag (+)a-tocopherol oxidation is accompanied by the uncoupling of lipid hydroperoxide formation and 0, consumption, i.e. lipid hydroperoxide formation but not 0, consumption is hindered. In vivo the lag can be infinite due to antioxidant systems that are protective against the oxidation of molecules in the apolar phase of membranes either via the back-reduction of tocopherol chromanoxyl radical (e.g. ascorbic acid and reduced glutathione [6-8,11,14,20,29,38,41-45]), or by preventing the formation of hazardous oxygen derivatives (e.g. superoxide dismutase, catalase, GSH-peroxidases [46,47]). Thus the lag of microsomal lipid peroxidation may represent a form of the physiological state of the membrane. Further investigations concerning the site of 0, activation, the formation of carbon-centered lipid radicals and oxidation product(s) of (+)-c~-tocopherol in relation to the lag may be of interest for the better understanding of the molecular basis of (+)-atocopherol action in biological membranes.

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