ORIGINAL ARTICLE
Antioxidant Activity of Vitamin E and Trolox:
Understanding of the Factors that Govern Lipid Peroxidation Studies In Vitro
Marlene Lúcio&Cláudia Nunes&Diana Gaspar&
Helena Ferreira&José L. F. C. Lima&Salette Reis
Received: 28 November 2008 / Accepted: 6 August 2009 / Published online: 27 August 2009
#Springer Science + Business Media, LLC 2009
Abstract Peroxidation of lipids is of significant interest owing to the evidence that peroxyl radicals and products of lipid peroxidation may be involved in the toxicity of compounds initiating a deteriorative reaction in the pro- cessing and storage of lipid-containing foods. In view of the significance of the antioxidant role of the dietary compound vitamin E and its water-soluble analogue Trolox in research of lipid-containing foods, it is desirable to determine more specifically how and where they operate its antioxidant activity in lipid membranes. In this study, unilamellar liposomes of phosphatidylcholine were used as membrane mimetic systems to estimate the antioxidant properties of vitamin E and Trolox and establish a relationship between their interactions with the membrane and their consequent antioxidant activity. Lipid peroxidation was initiated by the peroxyl radical (ROO•) in lipid and aqueous media by the thermal decomposition of azocompounds and was assessed by the fluorescence intensity decay of the fluorescent probe diphenylhexatriene propionic acid. Results obtained showed that membrane lipoperoxidation is related not only to the scavenging characteristics of the compounds studied but also to their ability to interact with the lipid bilayers, and consequently liposomes provide additional information
to that obtained currently from assays performed in aqueous buffer media.
Keywords Vitamin E . Trolox . Liposomes . AAPH . AMVN . DPH-PA
Introduction
Free radicals are continuously produced within living cells as a result of multiple biochemical and physiological processes. In addition, numerous exogenous sources, including xenobiotics and radiation, can induce free radicals1,2. Because of their high reactivity, free radicals can damage diverse cellular macromolecules, and when the accumulation of these radicals exceeds the limits of what the natural cellular antioxidant effects can neutral- ize, numerous pathological effects may manifest in the cells.
Biomembranes are composed of phospholipid bilayers and are one of the major targets of the radical attack which induce continuous lipid peroxidation. This uncontrolled reaction generates cytotoxic compounds and disrupts the various important structural and protective functions asso- ciated with biomembranes being implicated in the etiology of many diseases, including cancer, cardiovascular and neurological diseases, and other oxidative stress mediated dysfunctions2–4. The deleterious consequences of mem- brane peroxidation have stimulated numerous studies on the efficacies and mechanisms of action of biologically relevant antioxidants. Accordingly, the study of dietary compounds for human health has received considerable attention in biomedicine due to their beneficial effect on the antioxidant defense system. Furthermore, oxidative products are also responsible for initiating an oxidative rancidity, which is a M. Lúcio
:
C. Nunes:
D. Gaspar:
H. Ferreira:
J. L. F. C. Lima:
S. Reis
REQUIMTE, Serviço de Química Física, Faculdade de Farmácia, Universidade do Porto, Rua Aníbal Cunha,
4050-047 Porto, Portugal M. Lúcio (*)
Serviço de Química-Física, Faculdade de Farmácia, Universidade do Porto,
Rua Aníbal Cunha, 164, 4050-047 Porto, Portugal e-mail: mlucio@ff.up.pt
deteriorative reaction in the processing and storage of lipid- containing foods5. It is therefore of interest to more precisely assess the efficacy of antioxidant activity of the many naturally occurring molecules to find effective compounds that can minimize potential oxidative damage in vivo and/or to retard the oxidation of easily oxidizable materials in foods. Among the dietary compounds, vitamin E is an important natural antioxidant, which inhibits lipid peroxidation. Indeed, vitamin E is consid- ered as a universal participant of antioxidant defense reactions in biological membranes since it acts at all steps of membrane oxidative damage and works as a first line of defense against peroxidation of polyunsaturated fatty acids6. Recent clinical trials have also demonstrated the significant cancer preventive potential of vitamin E in cancers, ranging from oral and pharyngeal cancer to prostate cancer 7. Moreover, vitamin E is related to a reduced incidence of ischemic heart disease since hyper- tensive patients may have increased lipid peroxidation and reduced protection from this important fat soluble vita- min8. In addition, vitamin E is well recognized for its effective inhibition of lipid oxidation in foods being used to maintain food quality and extend shelf life by prevent- ing or delaying oxidation of the labile fatty acids and lipid-soluble components9.
Despite the growing understanding of the role of vitamin E as oxidant protector in food and biological systems, its mechanism of action is not completely understood yet. It is known that it is a chain-breaking antioxidant preventing peroxidation of lipids10, and it might also stabilize biological membranes by restricting the mobility of their components11. Due to this fact, it is still important to study the interaction of vitamin E with membrane components, and specifically with lipids;
however, the use of this extremely hydrophobic antioxi- dant in buffered solutions is not always straightforward.
Several vitamin E homologues have also shown an important antioxidant activity and beneficial effects against oxidative damage upon lipid peroxidative process- es. Among the vitamin E analogues that are used in oxidation studies, Trolox shows some advantages namely by its moderate water solubility. In fact, Trolox does not have to be incorporated into the lipid membrane by solvent extraction and coevaporation methods, and it can be added directly to the intact system of study. This makes it convenient for studies on natural biological systems and for quantitative studies on model systems12. In view of the significance of using a water-soluble antioxidant like Trolox in physical and biochemical research on mem- branes, it is desirable to determine more specifically its antioxidant activity in comparison with vitamin E. Indeed, Trolox has the same chroman ring structure as vitamin E and can be considered as its water-soluble analogue since
their reduction potential determined by pulse radiolysis in aqueous solutions were practically the same13. However, it is important to note that the reduction potential value is solvent dependent, and its determination in aqueous solution may not reflect the real situation within the biomembranes. Therefore, the possibility of gaining biologically relevant information on the potency of antioxidants to avoid a free-radical-induced membrane peroxidation is related not only to their structural characteristics but also to their ability to interact with and penetrate the lipid bilayers. Consequently, the location of the antioxidants and the site of radicals to be generated in the phospholipid bilayers should be taken into account to understand the effectiveness of their antioxidant activities14.
The purpose of this study is thus to compare the antioxidant activity of vitamin E and its derivative Trolox in phospholipid liposomes and to make the claim that the potency of antioxidants in membranes cannot be assured without a thorough analysis of the results obtained and experimental conditions used. Therefore, this study purposes the evaluation of antioxidant activity vitamin E and Trolox using the fluorescent probe diphenylhexatriene propionic acid (DPH-PA). The de- gree of lipid oxidation is indirectly monitored by a previously described method15consisting of the oxidation of the probe with a consequent decay in its fluorescence intensity. DPH-PA is a lipophilic probe anchored in close proximity to the bilayer surface by its propionate group, while the DPH moiety is embedded in the phospholipid acyl chains. Thus, DPH-PA is inserted in the lipid membrane but probes the bilayer lipid environments close to the surface, i.e., in the outer bilayer regions16, and consequently, the antioxidant capacity of vitamin E and Trolox can be related to their different molecular distri- bution between aqueous and lipid media. Other compo- nents that modulate the interaction of the compounds studied with lipid membranes are the location of free radicals determined by the type of radical initiator used in the assay. Hence, due to the lipophilic and hydrophilic nature of the antioxidants to be tested, it was also useful to further examine their effects against both lipophilic and hydrophilic free-radical-generating systems. For that rea- son, the azocompounds 2,2′-azobis(2,4- dimethylvaleroni- trile) (AMVN) and 2,2′-azobis (2-amidinopropane) dihydrochloride (AAPH) were used to generate peroxyl radicals at a constant rate within the lipid hydrophobic core or the aqueous environment surrounding the lipid bilayer, respectively (Figure1).
Based on the results obtained, the antioxidant activity of vitamin E and its analogue Trolox in membranes is compared with respect to their different location in the membranes and the generation site of the radicals.
Materials and Methods
Reagents
Egg L-α-phosphatidylcholine (EPC) was purchased from Sigma (St. Louis, MO, USA). All experiments were performed in buffer solution (10 mM Hepes, pH 7.4, and 0.1 M NaCl), prepared from Hepes (Sigma) and Milli-Q water (generated by a Millipore system with conductivity less than 0.1 µS cm−1). The azoderivatives AAPH and AMVN were used as oxidants initiators. AAPH was purchased from Fluka (St. Louis, MO, USA) and dissolved in buffer solution immediately before use and maintained on ice in amber glass vials. AMVN was purchased from Cayman Chemical Company (Michigan, USA) and was dissolved in chloroform and coevaporated with the lipid.
Vitamin E and 2-carboxy-2,5,7,8-tetramethyl-6-chromanol (Trolox) were purchased from Fluka and used as antiox- idants. Trolox was dissolved in buffer solution immediate- ly before use and maintained in amber glass vials, while vitamin E was dissolved in chloroform and coevaporated with the lipid.
The fluorescent probe 3-(p-(6-phenyl)-1,3,5-hexatrienyl) phenylpropionic acid (DPH-PA) was obtained from Molec- ular Probes (Invitrogen Corporation, Carlsbad, CA, USA).
Stock solutions of DPH-PA were prepared in N,N-dime- thylformamide and stored at −20 °C in amber glass vials under nitrogen. All other reagents were of analytical grade.
Preparation of Fluorescence-Labeled Liposomes
The procedure used varied somewhat with the type of initiator and antioxidant. When the water-soluble initiator and/or antioxidant combination (AAPH/Trolox) was used, buffered solutions of the water-soluble initiator and antioxidant were added to the already prepared labeled liposomes. When the lipid-soluble initiator and/or antioxi- dant combination (AMVN/vitamin E) was used, an appro- priate amount of AMVN and vitamin E in chloroform were added during the labeled liposome preparation to the EPC dissolved in chloroform/methanol (9:1). In both cases, an aliquot of the probe DPH-PA was added to the organic phase (chloroform/methanol (9:1)) where EPC was dis- solved so that the ratio probe to lipid was 300:1 (1.5 mmol of EPC and 5 µmol of the fluorescent probe). At this molar ratio, no inner filter effects were displayed. The compo- nents (lipid + probe or lipid + probe + initiator and/or antioxidant) were coevaporated under vacuum onto the wall of a 15-mL round-bottomed flask until a dry thin lipid film was obtained in the flask walls, which was stored for 3 h under vacuum to remove the last traces of solvent.
Hydration of the thin lipid film was made by addition of buffer (Hepes 10 mM,I=0.1 M, pH=7.4) in a lipid/buffer proportion of 1.05 mg mL−1for approximately 30 min at 25 °C by vortex mixing. The resultant suspension of multilamellar liposomes was then shaken while being sonicated for 30 s in an ultrasonic bath, which ensured complete recovery of the lipid from the flask walls. The suspension was further passed through two stacked poly- carbonate filters (pore size 100 nm) using a extruder apparatus (Lipex Biomembranes, Vancouver, BC, Canada) to convert multilamellar vesicles to unilamellar vesicles (LUVs) following a previously described procedure17–19. During the preparation steps, the lipid and probe were shielded from light and oxygen as much as possible. All the preparation steps of the liposomes were made at a temperature of 25 °C which is, at the same time, a value under the temperature required for initiating the oxidation reaction and a value above the transition temperature of the lipid. LUVs containing the fluorescent probe were freshly prepared for each day of experimentation and just before the peroxidation experiment.
Lipoperoxidation by Fluorescence Measurements
A reported procedure17–20 was adapted to evaluate the antioxidant activity of vitamin E and Trolox. In summary, peroxyl radicals (ROO•) were generated by thermodecom- Fig. 1 Schematic illustration of the principle of the assay used to
evaluate the antioxidant activity against peroxyl radical (ROO•) showing the location of the fluorescent probe DPH-PA in a liposome-aqueous media and the generation of the radical in the aqueous (inducer: AAPH) and liposome media (inducer: AMVN)
position of the initiator (AAPH or AMVN), and lipid peroxidation was monitored in LUVs of EPC, labeled with the fluorescent probe DPH-PA, by measuring the decrease in fluorescence, which results from the oxidative degrada- tion of the probe.
The used procedure was slightly different according to the type of initiator and antioxidant. When the lipid- soluble initiator AMVN was used, an aliquot of the LUV suspension containing the probe DPH-PA and AMVN (final concentrations 800, 2.7, and 300 μM, respectively) was incubated in a fluorimetric cuvette placed in a thermostatted holder with different concen- trations of hydrophilic and lipophilic antioxidant, Trolox or vitamin E, respectively (0 to 750 µM). In the case of vitamin E, due to its higher lipophilic properties, the antioxidant was previously incorporated in the lip- osomes as referred in the liposome preparation section.
The incubation of labeled liposomes with the antioxi- dant was made at 25 °C for 10 min with continuous stirring and away from light. After this incubation period, the temperature was raised to 45 °C, and the fluorescence measurements were immediately started since at this temperature the thermodecomposition of the AMVN initiates and, consequently, the lipid perox- idation begins.
When the water-soluble initiator AAPH was used, an aliquot of the LUV suspension containing the probe DPH- PA (final concentrations 800 and 2.7 μM) was incubated with different concentrations of hydrophilic and lipophilic antioxidant, Trolox or vitamin E, respectively (final con- centrations 0 to 15 µM) as described above. The incubation was made at 25 °C, and then the temperature was raised to 45 °C followed by the addition of the radical initiator solution (AAPH, final concentration 15 mM) after which the fluorescence measurements are immediately started because AAPH decomposes also thermally, initiating the peroxidation process.
Blanks were made with the radical initiator (AAPH or AMVN) and without the antioxidant, which was replaced by the buffer. Two types of controls were also made:
control 1 with the antioxidant (Trolox or vitamin E) and without the radical initiator and control 2 without neither antioxidant nor initiator.
Oxidation of DPH-PA probe by peroxyl radicals was monitored by the decay of its fluorescence intensity using a steady-state fluorescence spectrometer (Perkin-Elmer LS 50B, USA) equipped with a constant-temperature cell holder. Fluorescence intensity was monitored for 180 min at previously defined conditions (pH 7.4, temperature 45 °C) with excitation and emission wavelengths of 399 and 435 nm, respectively.
Results obtained by this method correspond to the mean of three independent experiments.
Results
The fluorescent method used measures the rate of fluores- cence intensity decay due to the susceptibility of the probe DPH-PA, which possesses a conjugated double-bond structure that reacts with free-radical species 17–19,21. Therefore, the fluorescence decay indirectly reflects the rate of peroxidation. The control assay without neither antioxidant nor inducer and the control assay with antioxidant but without inducer had very stable fluores- cence intensity values over the course of the assay (more than 90%). Addition of the inducer (AAPH or AMVN) to the LUV suspension, in the absence of the antioxidants tested (blank assay) with the concomitant increase of the incubation temperature to 45 °C, has triggered the perox- idation of the probe DPH-PA by the thermodecomposition of the azocompound. Briefly, thermodecomposition of AAPH and AMVN originates azocompounds’ derived peroxyl radicals able to abstract a hydrogen atom from the susceptible polyunsaturated portion of the probe DPH-PA.
The resultant alkyl radical reacts with oxygen to form a lipid peroxyl radical (ROO•). The peroxyl radicals propa- gate the oxidation process by abstracting a hydrogen atom from the probe, causing a gradual decay in its fluorescence intensity22,23. Figure 2a illustrates the typical controls and blank assays obtained with the inducer AAPH. Similar profiles were obtained with the inducer AMVN. Data obtained were converted in all the assays to relative fluorescence values by dividing the fluorescence intensity at a given time by the fluorescence intensity at 0 min and multiplying by 100.
Upon addition of antioxidant (Trolox or vitamin E) and in the presence of the radical initiator, the rate of decrease in the fluorescence of the probe showed a plateau region, after which the fluorescence decreased in a manner similar to that of the blank DPH-PA/inducer system. This has been already described for studies with other probes24 and happens because at the beginning of the reaction the antioxidant competes efficiently for peroxyl radical; how- ever, as the reaction proceeds and the amount of antioxidant gets depleted, peroxyl radicals start to attack DPH-PA, resulting in decay of its fluorescence. The kinetic data obtained using different concentrations of Trolox is illustrated in Figure2b. Similar trends were observed when the antioxidant used was vitamin E.
According to the aforementioned results, the efficacy of vitamin E and Trolox to prevent the oxidation of DPH-PA induced by the ROO• radical can be translated as the capacity of these compounds to avoid an immediate decay of fluorescence of the probe. To measure this antioxidant capacity, the area under the curve technique (AUC) originally developed by Cao et al. was used25,26. This technique combines both inhibition time (correspondent to
the initial plateau phase) and inhibition degree (correspon- dent to the decay profile) into one parameter, namely, AUC.
Thus, the activity of an antioxidant, even at small concentrations when it lacks a clear plateau phase, can be easily computed. The results were further analyzed by the ratio of the net areas under the curve of antioxidant (Trolox or vitamin E) and blank DPH-PA/inducer (without antiox- idant) assays:
ANTIOXIDANT CAPACITY
¼AUCANTIOXIDANTAUCBLANK
AUCBLANK 100 ð1Þ
A linear correlation was found between the antioxidant capacity (%) and vitamin E or Trolox concentrations, meaning that the antioxidant capacity of vitamin E and Trolox to prevent the oxidation of DPH-PA induced by the ROO• radical (induced by AAPH and AMVN) is concen- tration dependent. The linear fits obtained for each assay permitted a better evaluation of the ability of the com- pounds studied to act as antioxidants by determining their IC50values, which are defined as the concentration (in µM) of each compound required to obtain an antioxidant capacity defined by Eq.1 equal to 50%. However, when AMVN was the initiator, vitamin E and Trolox where much less efficient, preventing oxidation, reaching only 5% of antioxidant capacity, and thus, an IC5value was calculated.
A brief comment regarding interaction of both of the compounds studied with membranes is needed to analyze the results, as the inherent reactivity of vitamin E and Trolox toward peroxyl radicals depend on their lipidic distribution, which is different and may be related to the discrepancies found in their antioxidant capacity. Indeed, Trolox resides mainly in the aqueous phase and partly in
the lipid phase of phospholipid membrane system, yet it diffuses into the bilayer phase sufficiently to meet the polar peroxyl radicals and traps two peroxyl radicals per molecule when oxidation is initiated in the lipid phase27. On the other hand, vitamin E is highly lipophilic and resides totally on the lipid core with its chromanol moiety oriented toward the lipid–water interface of the phospho- lipid bilayer28,29. Hence, to compare the values of IC obtained with the two antioxidants, the effect of the drugs’
partitioning should be taken into account to express the membrane phase concentration of the compounds instead of their total concentration. Furthermore, the partitioning of Trolox in LUVs of EPC has already been determined by spectroscopic methods, and a 30% partitioning of this compound was found in these model systems12. According to this, it was possible to determine the membrane phase concentrations of Trolox and thus plot the antioxidant capacity (%) of Trolox against both total and membrane concentrations (Figure3).
As previously mentioned, a linear correlation was found between the antioxidant capacity (%) and Trolox concen- tration; however, it was clear that there is a significant difference in the slopes obtained. If the total concentrations of Trolox used in the assay are considered, then the slopes obtained are smaller. Therefore Trolox would present a higher IC value and would be thought as having much smaller antioxidant capacity than vitamin E since higher Trolox concentrations are needed to reach the 50% (when AAPH is used as initiator) or 5% (when AMVN is used as initiator) of antioxidant capacity. However, when only the portion of Trolox that partitions into the membrane phase is considered, then the slopes of the linear plots increase (Figure3), and the difference between Trolox and vitamin E antioxidant’s capacity is very much attenuated (Table1).
Fig. 2 Relative fluorescence intensity of DPH-PA in LUVs of EPC (800 µM) at 45 °C. (a) Control assays: 1 in the absence of both oxidant initiator AAPH and antioxidant Trolox;2in the absence of the oxidant initiator AAPH and in the presence of the maximum concentration of antioxidant Trolox (4.5 µM);blankDPH-PA/inducer
assay:3in the presence of the oxidant initiator AAPH (15 mM) and in the absence of the antioxidant Trolox. (b) Relative fluorescence intensity of DPH-PA obtained in the presence of different concen- trations of Trolox (0, 2.7, 5, 10, 13, and 15 µM) with the oxidative system AAPH (15 mM)
Discussion
The cell membrane as a target for dietary modulation in peroxidation prevention is an area of research that holds much potential for future investigations and progress.
Understanding the ways in which dietary factors can protect membrane from peroxidation will be greatly aided by studies in membrane models and by further examining the effects of antioxidants against both lipophilic and hydro- philic free-radical-generating systems. The purpose of the present study was therefore to compare the effect of two antioxidants (vitamin E and Trolox) with a different lipophilic nature when considering their protection against lipid peroxidation. Despite the extensive studies on the antioxidant actions of vitamin E and its analogues and their potential importance preventing several diseases, there are still some controversial conclusions, and thus, there is still space for further analysis. In fact, the whole of the results obtained shows the antioxidant capabilities of vitamin E and Trolox although makes evident the significant differ- ences with regard to the order of effectiveness of these
antioxidants to avoid membrane oxidative damage, which is dependent on the experimental procedures used. Some authors reported that Trolox exhibits higher antioxidant activity than vitamin E against microsomal peroxidation and attribute the differences encountered to their different chemical structures30. Other authors suggested that Trolox was less reactive than vitamin E type compounds toward peroxyl radicals in homogeneous solutions of styrene31, in methyl linoleate32, and in mixed micelles33 with azocom- pounds initiators. And finally it was also published that Trolox is an inhibitor of peroxidation as efficient as vitamin E in EPC liposomes27. Separately, each one of these methods analyzes different aspects of the same process but do not provide a global view of it. Furthermore, no connections have yet been made between the sum of factors that could affect the antioxidant capacity of these com- pounds, including the use of different inducers of oxidation that produce radicals in different locations (water and lipid media), the location of the antioxidants relative to the oxidation substrate, and ultimately the interactions of the compounds in study with the lipid membranes. This fact Fig. 3 Antioxidant capacity (%) of Trolox to prevent the oxidation of
the DPH-PA probe in LUVs of EPC (800 µM) by the ROO•radical induced by AAPH (a) and AMVN (b). The linear dependency was obtained between antioxidant capacity (%) and total Trolox concen-
trations (filled squares) or Trolox concentrations in the lipid phase (empty squares). Each point represents the values obtained for three independent experiments, performed in quadruplicate (mean ± standard error)
Table 1 Summary of the linear dependency (slope and correlation coefficient) between antioxidant capacity (%) and Trolox or vitamin E concentrations (µM) in the prevention of the oxidation of DPH-PA by the ROO•radical (induced by AAPH and AMVN)
Antioxidant AAPH AMVN
Slope Correlation coefficient IC50(µM) Slope (×103) Correlation coefficient IC5(µM)
Trolox Total concentrations 10.5±0.8 0.995 4.7±0.3 9.9±0.1 0.994 553±6
Membrane concentrations 35±3 0.995 1.4±0.1 33.1±0.3 0.994 151±1
Vitamin E 45±3 0.999 1.11±0.08 46±2 0.995 110±5
IC50and IC5values are the concentration (inμM) of each compound required to reach 50% and 5% of the ratio (AUCvitamin E/Trolox−AUCblank)/
(AUCblank), where AUC is the area under curve obtained from the fluorescence decay of the probe in the absence (blank) or on presence of antioxidant (Trolox or vitamin E) or Trolox
determines the need to combine all information in order to establish the mode of actuation of any antioxidant.
Therefore, in the present work, the effect of the antioxidants of vitamin E and Trolox will be carefully analyzed in regard to their chemical structure, scavenging activity, membrane location, membrane partition, and position relatively to the radical initiator and/or oxidation substrate.
The vitamin E molecule (Figure4a) can be divided into two parts, a hydroxyl-bearing aromatic system (one phenolic and one heterocyclic ring, called the chroman head) that is responsible for its antioxidant properties and either a saturated (tocopherols) or polyunsaturated (toco- trienols) hydrocarbon tail for the orientation of vitamin E in the lipid membrane30. Trolox is structurally similar, exhibiting the same chroman head (Figure4b)24.
Both vitamin E and Trolox function as antioxidants by virtue of their ability to donate hydrogen from the hydroxyl group (Figure 4) to peroxyl radical (ROO•) converting it into a lipid hydroperoxide and a vitamin E or Trolox radical and thus terminating the chain reaction12,24,30,34
. Further- more, they are also chemically similar since the phenolic hydrogen reaction site is equally distant from the long chain alkyl group of vitamin E or the carboxyl group in Trolox12. Therefore, it is true that both compounds have similar efficiency concerning their scavenging capacities. However, their lipophilicity is quite different, and thus, both their location and partition in membranes will be different. The location of vitamin E in model membranes has received considerable interest, and several published results have shown that the chroman head is tightly adapted to the space close to the surface of membranes in the lipid–water interface of the phospholipids’ bilayer with the hydroxyl group hydrogen-bonded to the ester carbonyl group of the phospholipids, where as the prenyl side chain is embedded in the membrane interior and anchors the molecule firmly within the bilayer (Figure5)13,15,28,29,35,36
.
Therefore, vitamin E is highly lipophilic and distrib- utes totally in the membrane, and as a result, it would be expected that vitamin E was able to scavenge lipid peroxyl radicals generated either inside of the membrane (AMVN) or in the water media (AAPH) and maybe would be more efficient than Trolox when the radicals were generated in the lipid media. However, and despite their different membrane location, by the observation of the IC50 values presented in Table 1, it is possible to conclude that both compounds have significant antioxidant activity when the peroxidation reaction is initiated in the aqueous phase (IC50∼1 µM), but have little or no antioxidant activity when the peroxidation reaction is initiated in the hydrophobic core of the bilayer (IC50 not reached).
The orientation of vitamin E within the membrane provides a possible explanation to the different effect observed in the two systems. As referred, the hydroxyl group of vitamin E, responsible by its scavenging properties, is located at a region close to the membrane surface. Consequently, since AAPH generates radicals at the membrane surface, where the concentration of the hydroxyl groups of vitamin E is high, thus the antioxidant efficiency is also high due to the fact that both radical and scavenging groups reside in the same location. Conversely, free radicals generated by AMVN in the lipid phase are further away from vitamin E hydroxyl group, which is essential in the scavenging process. Contrastingly to vitamin E, Trolox resides mainly in the aqueous phase
2
b
a
1
Fig. 5 (a) Locations of vitamin E (1) and Trolox (2) in liposomes:a shows the position of the hydroxyl group of vitamin E relatively to the ester carbonyl bond of the phospholipid membrane. b shows the position of the carboxylate group of Trolox relatively to the choline group of the phospholipid membrane
Fig. 4 Chemical structure of vitamin E (a) and its hydrophilic derivative Trolox (b). The common structures like the chroman head and the hydroxyl group are indicated ingray
and partly in the lipid surface of phospholipid membrane system having a 30% partitioning into egg lecithin LUVs at physiological pH12. However, Trolox is a water-soluble antioxidant with pKa=3.89, and thus, it is differently charged according to the pH of the studies12,27. Therefore, the partition and location of Trolox in the membrane surface are pH dependent and depend also on the type of lipids used12,27. In the current work, the membrane is neutral because of the resultant balance between the choline group positively charged and the phosphate group negatively charged. In such conditions, the carboxyl group of Trolox is negatively charged and is bounded to the membrane surface by electrostatic interactions with the choline group (Figure 5). This distribution of Trolox on the bilayer surface is sufficient to meet the polar peroxyl radicals and thus terminate the peroxidation process of the probe.
However, similar to what happened with vitamin E, the scavenging activity of Trolox is greatly reduced if the peroxyl radicals were produced inside the lipid phase by AMVN. This observation is also consistent with the Trolox location at the membrane surface, which is favorable to scavenge free radicals generated in aqueous phase, but is quite hindered from the radicals generated inside the lipid phase.
The aforementioned considerations about the location and partition of Trolox and vitamin E within the membrane also validate the difference of antioxidant efficiency found for both compounds when the radicals are generated either in the aqueous phase or inside the membrane (Table 1).
Actually in terms of total concentration, vitamin E has shown to be much more efficient that Trolox. On the other hand, when the distribution of both compounds into the membrane is considered, and the IC values are calculated considering that 30% of Trolox is distributed in the membrane, then the difference in antioxidant potency is attenuated. These data demonstrate that the lipid solubility of antioxidants is very important in determining the extent of oxidative protection. Hence, partition into bilayer should be analyzed carefully when establishing comparisons between compounds. In this case, only the portion of Trolox which partitions into the membrane will be able to scavenge the peroxyl radicals.
In agreement to our results, the similar scavenging capacity of peroxyl radicals observed for Trolox and for lipid-soluble antioxidants has been also observed in the literature by a different method (measurement of the absolute rate constant inhibition of oxygen uptake), and it has been attributed to a diffusion-trapping mechanism27. According to this mechanism, water-soluble antioxidants that, like Trolox, diffuse into the bilayer phase sufficiently to meet peroxyl radicals act as scavengers against these radicals. Still, compounds that are more hydrophilic and reside wholly in the aqueous phase might not act as
efficient antioxidants especially when a reaction is initiated in the lipid phase of bilayers.
Concluding Remarks
Oxidation of lipids in food is of great concern since it leads to fatty acid decomposition and development of undesirable rancid odors and flavors, with a decrease in nutritional value and food safety. Therefore, it is of utmost importance to study the efficacy of antioxidant activity of the many naturally occurring molecules in food to find effective compounds that can minimize potential oxidative damage in vivo and/or to retard the oxidation of easily oxidizable materials in foods. The purpose of the present study was to highlight the factors that have to be considered when analyzing the antioxidant capacity of any compound studied because the antioxidant effects are often not examined in regard to the ability of compounds to interact with biomembranes. The effect of a frequently used lipophilic dietary antioxidant, vitamin E, was compared with its analogous antioxidant, Trolox, with regard to their capacity to prevent the generation of lipid peroxides when the oxidation was initiated inside the membrane or in the aqueous media. Although some data can be found in literature comparing vitamin E and Trolox, less attention has been paid on the effects of lipid media and location of the antioxidants when analyzing the results. The work described herein gathers several aspects that have been considered separately, like the use of a lipid/water media similar to in vivo environment, two types of inhibitors, and a fluorescent method of detection with the sensibility required to measure small differences in antioxidant efficiency.
Our data demonstrate that if the partition of com- pounds within the membrane was not considered, then vitamin E was assumed to be a better antioxidant than Trolox. However, when concentrations are corrected considering the membrane partition, both compounds demonstrated similar antioxidant capacities. Therefore, it is possible to conclude that lipophilicity of an antiox- idant is a very important characteristic to be considered during the study of the efficiency of antioxidant agents in the future. Furthermore, this work reinforces the notion that it is important to consider the possible effect of conditions (e.g., pH) and the polarity of antioxidants as well as the charge type of the target membranes when employing antioxidants for medicinal purposes or food analysis.
Finally, the membrane location of antioxidants has to be analyzed to evaluate the accessibility to scavenge free radicals in lipid media and thus to understand the results obtained.
Acknowledgments The authors would like to thank FCT and FEDER for financial support through the contract PTDC/SAU-FCF/
67718/2006.
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