Protective effect of resveratrol on formationof membrane protein carbonyls and lipidperoxidation in erythrocytes subjectedto oxidative stress

Kanti Bhooshan Pandey and Syed Ibrahim Rizvi

Abstract: Many of the biological actions of resveratrol have been attributed to its antioxidant properties. In this work, wesubjected human erythrocytes to in vitro oxidative stress by incubating them with tert-butylhydroperoxide (t-BHP). Thiscaused a significant increase in the malondialdehyde (MDA) level and the protein carbonyl group content above the basalvalues. The presence of trans-resveratrol at micromolar concentrations in the incubation medium protected the erythrocytesfrom t-BHP-induced oxidative stress, as evidenced by the decrease in the MDA level and the protein carbonyl group con-tent. The effect of resveratrol was concentration and time-dependent. Our findings help to explain some of the beneficialeffects of resveratrol.

Key words: resveratrol, erythrocyte, oxidative stress, malondialdehyde, protein carbonyls.

Resume : Plusieurs des actions biologiques du resveratrol decoulent de ses proprietes antioxydantes. Dans cette etude, onsoumet des erythrocytes humains a un stress oxydatif in vitro en les incubant en presence de tert-butylhydroperoxyde(t-BHP), augmentant ainsi significativement le niveau de malonaldehyde (MDA) et la concentration de carbonyle dans laproteine membranaire, et ce, au-dessus des valeurs de reference. La concentration micromolaire de trans-resveratrol dansle milieu d’incubation protege les erythrocytes du stress oxydatif suscite par le t-BHP comme le revele la diminution duniveau de MDA et de la concentration de carbonyle dans la proteine membranaire. L’effet du resveratrol depend de saconcentration et de la duree de sa presence. Nos observations contribuent a ameliorer les connaissances concernant leseffets benefiques du resveratrol.

Mots-cles : resveratrol, erythrocytes, stress oxydatif, malonaldehyde, carbonyle proteique.

[Traduit par la Redaction]

Introduction

Resveratrol, 3,5,4’-trihydroxy-trans-stilbene (Fig. 1), awell-known stilbene class of polyphenolic compound, natu-rally occurs in grapes and berries. It is a phytoalexin that isproduced in response to infections in plants, and, thus, actsas a natural antibiotic. It is also synthesized in response tosome environmental stresses, like climate changes and ultra-violet light (Pervaiz 2003). Accumulating evidence demon-strates that resveratrol exerts several beneficial healtheffects in humans and experimental animal models (Baurand Sinclair 2006; Markus and Morris 2008).

Aerobic cells produce reactive oxygen species (ROS) as aby-product of their metabolic processes. ROS may induceoxidative stress and cause damage to all types of molecules(Droge 2002). Several defense mechanisms have evolved inliving organisms to limit the levels of ROS and the damagethey inflict. Endogenous antioxidant enzymes, such as cata-lase, superoxide dismutase, and glutathione peroxidase, areincluded among them (Ames et al. 1993).

There is overwhelming evidence to suggest that nutri-tional sources of antioxidants, such as fruits, vegetables, tea,and wine, attenuate tissue damage caused by oxidative chal-lenges (Cao et al. 1998). Polyphenolic compounds, abundantin these nutritional sources, could play a major role in en-hancing the activity of the antioxidant system (Rizvi et al.2005; Pandey et al. 2009).

During the past couple of years, several in vivo and in vi-tro studies have focused on various biological effects of re-sveratrol. Growing evidence suggests that resveratrol mayplay an important role in the prevention of human diseases,such as cancer, cardiovascular diseases, diabetes, and Alz-heimer’s disease (Harikumar and Aggarwal 2008). Many ofthe biological actions of this polyphenol have been attrib-uted to its antioxidant properties (Jang et al. 1997; Mahaland Mukherjee 2006). This work was undertaken to evaluatethe protective effects of trans-resveratrol on lipid peroxida-tion and membrane protein carbonyl groups, which are im-portant markers of oxidative stress, in human erythrocytessubjected to in vitro oxidative stress.

Received 1 July 2009. Accepted 8 September 2009. Published on the NRC Research Press Web site at apnm.nrc.ca on 1 December 2009.

K.B. Pandey and S.I. Rizvi.1 Department of Biochemistry, University of Allahabad, Allahabad, 211002 India.

1Corresponding author (e-mail: sirizvi@gmail.com).

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Appl. Physiol. Nutr. Metab. 34: 1093–1097 (2009) doi:10.1139/H09-115 Published by NRC Research Press

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Materials and methods

Collection of blood, isolation of erythrocytes, preparationof red cell ghosts

The study was carried out on 31 healthy volunteers ofboth genders between the ages of 21 and 45 years. The cri-teria for selection of healthy volunteers were the same asthose adopted in previous studies (Pandey et al. 2009; Rizviand Maurya 2007). Briefly, the subjects were screened fordiabetes mellitus, asthma, tuberculosis, and any other majorillness. None of the volunteers were smokers or were takingany medication. All persons gave informed consent for theuse of their blood samples for the study. Venous blood wasobtained by venipuncture in heparin, and was centrifuged at1800g for 10 min at 4 8C. After the removal of plasma,buffy coat, and the upper 15% of packed red blood cells(RBCs), the RBCs were washed twice with cold phosphate-buffered saline (PBS) (0.9% NaCl and 10 mmol�L–1

Na2HPO4; pH 7.4). Erythrocyte ‘ghosts’ from leucocyte-freeRBCs were prepared by an osmotic shock procedure, usingthe method of Marchesi and Palade (1967), and protein con-tent was determined with the method of Lowry et al. (1951),using bovine serum albumin as a standard. trans-resveratrolwas purchased from Sigma–Aldrich (St. Louis, Missouri;product no. R5010, FW. 228.2). The protocol of the studywas in conformity with the guidelines of the InstitutionalEthical Committee of the University of Allahabad (Alla-habad, India).

Determination of malondialdehyde contentErythrocyte malondialdehyde (MDA) level was measured

using the method of Esterbauer and Cheeseman (1990).Packed erythrocytes (0.2 mL) were suspended in 3 mLKrebs–Ringer phosphate buffer (pH 7.4). The lysate (1 mL)was added to 1 mL of 10% trichloroacetic acid, and themixture was centrifuged for 5 min at 1000g. The supernatant(1 mL) was added to 1 mL of 0.67% thiobarbituric acid in0.05 mol�L–1 NaOH, and boiled for 20 min at a temperaturegreater than 90 8C. The solution was cooled and read againsta complementary blank at 532 nm (optical density (OD)1)and 600 nm (OD2). The net OD was calculated by subtract-ing absorbance at OD2 from that at OD1. The concentrationof MDA in erythrocytes was determined from a standardplot, and was expressed as nmol�mL–1 of packed erythro-cytes.

Determination of membrane protein carbonylsErythrocyte membrane protein carbonyls were measured

according to procedure of Levine et al. (1990). Erythrocytemembrane samples (0.2 mL) in PBS were taken in 2 tubesas test and control samples. A total of 4.0 mL of

10 mmol�L–1 2,4-dinitrophenylhydrazine (DNPH), preparedin 2 mol�L–1 HCl, was added to the test sample, and 4.0 mLof 2 mol�L–1 HCl alone was added to the control sample.The contents were mixed thoroughly and incubated for 1 hin the dark at 37 8C. The tubes were shaken intermittentlyevery 10 min to facilitate reactions with proteins. Aftershaking, 20% trichloroacetic acid (w/v) was added to bothtubes, and the mixture was left in ice for 10 min. The tubeswere then centrifuged at 3500 r�min–1 (850g) for 20 min toobtain the protein pellets. The supernatant was carefully as-pirated and discarded. The protein pellets were washed3 times with ethanol–ethyl acetate (1:1, v/v) solution to re-move unreacted DNPH and lipid remnants. Finally, proteinpellets were dissolved in 6 mol�L–1 guanidine hydrochlorideand incubated for 10 min at 37 8C. The insoluble materialswere removed by centrifugation. Carbonyl content was de-termined by taking the spectra of the supernatant at370 nm. Each sample was read against the blank. The car-bonyl content was calculated using an absorption coefficientof 22 000 mol�L–1�cm–1, and data were expressed innmol�mg–1 protein.

Induction of oxidative stressOxidative stress was induced in vitro by incubating

washed erythrocytes–erythrocyte ghosts with 10–5 mol�L–1

tert-butylhydroperoxide (t-BHP) for 60 min at 37 8C.The effect of resveratrol was evaluated by coincubatingerythrocytes–erythrocyte ghosts with t-BHP and resveratrolfor 60 min at 37 8C . The concentration and duration oft-BHP used to induce oxidative stress in erythrocytes wasthe same as described elsewhere (Pandey and Rizvi 2009).

In vitro experiments with resveratrolWashed erythrocytes were suspended in 4 volumes of

PBS containing 5 mmol�L–1 glucose (pH 7.4). In vitro ef-fects were evaluated by coincubating the erythrocytes in thepresence of trans-resveratrol, at different doses at 37 8C for60 min, and t-BHP. After this time, the suspensions wereimmediately centrifuged at 1800g, and the RBCs werewashed twice with at least 50 volumes of PBS and then sub-jected to assay MDA content.

For the protein carbonyl group estimation, erythrocyteghosts (0.8–1.5 mg of protein) were incubated with the re-sveratrol, at different doses in PBS (pH 7.4) for 1 h at37 8C, prior to the estimation of protein carbonyl formation,and with t-BHP. Parallel control experiments were also per-formed in which resveratrol was incubated with an equalamount of solvent.

Statistical analysesStatistical analyses were performed using GraphPad

Prism, version 4.00 for Windows (GraphPad Software, SanDiego, Calif.). Statistical differences were analyzed withStudent’s t test, and the differences were considered to besignificant when p < 0.05.

Results and discussionA certain amount of oxidative damage takes place, even

under normal conditions; however, the rate of this damageincreases during aging and other pathological events, as the

Fig. 1. Chemical structure of resveratrol (3,5,4’-trihydroxy-trans-stilbene; molecular weight, 228.2).

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efficiency of antioxidative and repair mechanisms decreases,leading to the condition of oxidative stress (Halliwell andGutteridge 2006; Rizvi and Maurya 2007).

The erythrocyte membrane is prone to lipid peroxidationunder oxidative stress that involves cleavage of polyunsatu-rated fatty acids at their double bonds, leading to the forma-tion of MDA. The attack by ROS against proteins modifiesamino acid (lysine, arginine, proline, and histidine) residues,generating carbonyl moieties, which have been identified asan early marker for protein oxidation and are used as ameasure of protein damage (Levine et al.1990).

Subjecting erythrocytes to oxidative stress by incubatingthem with t-BHP caused a significant increase in MDA leveland protein carbonyl group content above the basal values.The presence of resveratrol at micromolar concentrations inthe incubation medium protected the erythrocytes fromt-BHP-induced oxidative stress, as evidenced by the de-crease in MDA level (Fig. 2) and protein carbonyl groupcontent (Fig. 3).

Because of the high polyunsaturated fatty acid content oftheir membranes and the high cellular concentration of oxy-gen and hemoglobin, erythrocytes, a potentially powerfulpromoter for the oxidative processes, are highly susceptibleto oxidative damage. Increased erythrocyte MDA level isknown to cause a decrease in the membrane fluidity of themembrane lipid bilayer and an increase in osmotic stabilityof the cell (Bryszewska et al. 1995). A high concentrationof MDA in erythrocytes is a marker of cellular oxidativedamage observed in stress or pathological conditions, in-cluding aging (Rizvi and Maurya 2007). Unlike lipid perox-idation, oxidative modification of proteins, with theirmultiple functions, can be selective and specific. The use ofprotein carbonyls as an index of oxidative stress has someadvantagesover other oxidation products, because of the rel-ative early formation and the relative stability of carbonyl-ated proteins (Dalle-Donne et al. 2003).

Our results show that resveratrol can protect erythrocytesfrom oxidative stress under in vitro conditions. The sameconditions are thought to occur in vivo, and we hypothesizethat resveratrol may provide protection against oxidation-induced damage to membrane lipids and proteins underconditions that challenge the body’s redox status. Protectionof MDA and protein carbonyl formation in t-BHP-inducedoxidative stressed erythrocytes by resveratrol in micromolarconcentrations (1–100 mmol�L–1) assumes significance, be-cause it has been reported that plasma resveratrol levels,after the intake of a resveratrol-rich diet, are in the micro-molar range (Boocock et al. 2007; Walle et al. 2004). Ourresults are also supported by the observations of Leonard etal. (2003), who showed that resveratrol can scavenge ROS,as measured by spin trapping competitions using sodiumformate as a second free radical scavenger, and is effectivein inhibiting lipid peroxidation of cellular membranes.

Studies have proven that consumption of resveratrol or aresveratrol-rich diet has protective effects on cardiovasculardiseases in humans (Orallo et al. 2002; Das and Maulik2006). These effects include a reduction in platelet aggrega-tion, dilation of blood vessels, anti-atherosclerotic effects, alowering of lipid peroxidation, and improvement of the se-rum cholesterol profile (Baur and Sinclair 2006). A possiblemechanism by which resveratrol exerts its beneficial effect

on the cardiovascular system is its antioxidant activity. Anassociation has been found between oxidation of low-densitylioprotein (LDL) particles and risk of heart disease and my-ocardial infraction (Markus and Morris 2008). Resveratrolprevents oxidation of LDL by chelating copper and by scav-enging ROS. The fact that resveratrol can be detected inLDL particles after red wine consumption by humans is con-

Fig. 2. Concentration-dependent effect of resveratrol on the malon-dialdehyde (MDA) level of tert-butylhydroperoxide (t-BHP)-induced oxidative stressed human erythrocytes. Incubation witht-BHP caused an increase in MDA level. Treatment with resveratrolshows significant protection at concentrations of 100 mmol�L–1,10 mmol�L–1, and 1 mmol�L–1. The effect at 0.1 mmol�L–1 was notsignificant. Values are means ± SD of 10–12 independent experi-ments. *p < 0.001 and {p < 0.05, compared with control.

Fig. 3. Concentration-dependent effect of resveratrol on membraneprotein carbonyls status in t-BHP-induced oxidative stressed humanerythrocytes. Incubation with t-BHP caused a significant increase inprotein carbonyl group content. Treatment with resveratrol showssignificant protection at concentrations of 100 mmol�L–1,10 mmol�L–1, and 1 mmolL–1. The effect at 0.1 mmol�L–1 was notsignificant. Erythrocyte membrane protein carbonyl group contentwas expressed in nmol�mg–1 protein. Values are means ± SD of 10–12 independent experiments. *p < 0.001 and {p < 0.05, comparedwith control.

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sistent with its ability to prevent peroxidation of lipids andother macromolecules (Markus and Morris 2008).

In our experiments, we also evaluated the time-dependenteffect of resveratrol. The effect of resveratrol was fast, andafter only 30 min of incubation, a significant protective ef-fect against lipid peroxidation was observed (Fig. 4). Re-sveratrol also exerts rapid protection against membraneprotein oxidation, an effect that is faster than its protectiveeffect on lipid peroxidation (Fig. 5). The efficacy of the ef-fect increased gradually for 30–60 min, after which a slightreduction was observed. The time-dependent effect of re-sveratrol can be correlated with the findings of other re-

searchers (Bertelli et al. 1996; Goldberg et al. 2003), whohave documented the fact that resveratrol is rapidly ab-sorbed, and that its peak plasma concentration is achieved15–60 min after administration.

Our results related to the protection of human erythro-cytes by resveratrol against oxidative damage complementthose from our previous study, in which we showed protec-tion of intracellular glutathione and membrane sulphydrylgroup by resveratrol in erythrocytes subjected to in vitro ox-idative insult (Pandey and Rizvi 2009). To the best of ourknowledge, this is the first report of a protective effect ofresveratrol on protein carbonyl formation. This finding mayhelp to explain some of the beneficial effects of resveratrolon human health, and especially on the cardiovascular sys-tem.

AcknowledgementK.B.P. is the recipient of a Senior Research Fellowship

from the Council of Scientific and Industrial Research(CSIR), India.

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