BASIC NUTRITIONAL INVESTIGATION

Effect of Select Antioxidants on MalondialdehydeModification of Proteins

Joohee Kim, MD, Joe Chehade, MD, Jacob L. Pinnas, MD, and Arshag D. Mooradian, MDFrom the Division of Endocrinology, Diabetes and Metabolism, Department of Internal

Medicine, Saint Louis University School of Medicine, St. Louis, Missouri; and theDepartment of Medicine, University of Arizona College of Medicine, Tucson, Arizona, USA

To determine whether commonly used antioxidants alter malondialdehyde (MDA) modification ofproteins, a known mechanism of free radical–related tissue injury, we studied the effect of adding 1mg/mL of pycnogenol, 5 mM ofa-tocopherol, 5 mM of ascorbate, and 0.2 mg/mL of an ethanolequivalent of red and white wine on MDA-protein content of endothelial cells in culture. The addition ofpycnogenol but not of the other antioxidants was associated with significant reduction in MDA-proteincontent compared with controls (0.5216 0.041 in arbritrary units versus 1.0116 0.021,P , 0.001). Todetermine whether the observed effect occurs distal to MDA generation, the effect of these antioxidantson the modification of bovine serum albumin with MDA generated in a cell-free system was studied. Inthis cell-free assay, pycnogenol but not the other antioxidants reduced MDA–BSA generation byapproximately 50%. It is concluded that pycnogenol may reduce MDA modification of proteins at a stepdistal to MDA generation. This may be an additional mechanism of protective effects of pycnogenolagainst oxidative stress.Nutrition 2000;16:1079–1081. ©Elsevier Science Inc. 2000

Key words: antioxidants, pycnogenol, malondialdehyde, free radicals

INTRODUCTION

Oxidative stress has been implicated in a variety of disease statesand has been hypothesized to be one of the causes of aging.1

Therefore, it is assumed that antioxidants have salutary healthbenefits. It is generally accepted that the main role of the antioxi-dants is quenching of free radicals either directly or throughindirect biochemical pathways.

One of the mechanisms of free radical–induced tissue injury isgeneration of lipid peroxidation byproducts such as malondialde-hyde (MDA) and modification of proteins with MDA.2,3 Thisreaction results in altered protein function and antigenicity.2,3 Wespeculated that antioxidants may interfere with MDA modificationof proteins through either inhibition of lipid peroxidation or a stepbeyond generation of free radicals and peroxidation of lipids. Totest this hypothesis, MDA proteins were measured in culturedendothelial cells. In addition, MDA modification of bovine serumalbumin (BSA) was measured in a cell-free system in the presenceof select, popular antioxidants.

METHODS

Endothelial-Cell Culture Studies

Human umbilical-vein endothelial cells were obtained from Clonetics(San Diego, CA, USA), and endothelial-cell growth medium wasobtained from Cell Applications (San Diego, CA, USA). The cellswere cultured at 37°C and 5% CO2 in a 75-cm2 flask (BectonDickinson Labware, Franklin Lakes, NJ, USA) that was precoated

with attachment factor (Cell Applications) for at least 24 hbefore use. Cells were plated in six-well cluster dishes (Corn-ing, Inc., Corning, NY, USA), and when confluent the oldmedium was replaced with fresh endothelial-cell growth me-dium (2 mL/well). Antioxidants were prepared in Millie-Qwater (Millipore Corp., Bedford, MA, USA) and filter sterilizedimmediately before their addition to the fresh endothelial-cellgrowth medium. The following antioxidants were studied: 5mM of ascorbate (Sigma, St. Louis, MO, USA), 5 mM ofa-tocopherol (Sigma), 1 mg/mL of pycnogenol (Natrol, Chats-worth, CA, USA), and 0.2 mg/mL of an ethanol equivalent ofred and white wine. The viability of cells in culture was verifiedwith trypan-blue exclusion, which was over 90%. After 24 h themedium was removed, the cell monolayer was washed withHank’s balanced salt solution (Mediatech, Herndon, VA, USA),and harvested in 40mL of Laemmli buffer4 using a rubberpoliceman. The protein content was measured by the method ofBradford5 after 5 to 10 s of sonication (Branson UltrasonicsCorp., Danbury, CT, USA). Each sample (50mg of protein) wasresolved on a 10% sodium dodecyl sulfate–polyacrylamide geland transferred to nitrocellulose filters, as described previous-ly.6 The membrane was blocked with 5% chicken-egg albuminin 0.01 M sodium phosphate-buffered saline at 4°C and probedwith anti–MDA-protein antiserum (1:500) for 2.5 h at roomtemperature. After incubation with the goat–anti-rabbit horse-radish peroxidase–labeled secondary antibody (1:7000; South-ern Biotechnology Associates, Inc., Birmingham, AL, USA) for1 h, the protein bands were developed by enhanced chemilu-minescence using the manufacturer’s protocol (Amersham LifeScience, Arlington Heights, IL, USA). The density of the bandswas quantified with a scanning laser densitometer (MolecularDynamics, Sunnyvale, CA, USA).

The specificity of the anti-MDA antiserum has been previouslyestablished in our laboratory.7 The antiserum does not cross-reactwith free MDA, native proteins, or proteins modified with glucoseor acetaldehyde. To establish the specificity of the protein bandsobserved, antiserum preabsorbed with MDA–BSA was used.There were no discrete bands identified in these experiments.

Correspondence to: Arshag D. Mooradian, MD, Division of Endocrinol-ogy, Saint Louis University School of Medicine, 3691 Rutger, Suite 101,St. Louis, MO 63110, USA. E-mail: mooradian@slu.edu

Date accepted: May 26, 2000.

Nutrition 16:1079–1081, 2000 0899-9007/00/$20.00©Elsevier Science Inc., 2000. Printed in the United States. All rights reserved. PII S0899-9007(00)00446-9

Cell-Free Assay of MDA–BSA Binding

BSA was dissolved in 0.01 M sodium phosphate-buffered salinecontaining 0.01% ethylene-diaminetetraacetic acid (pH 7.4) at aconcentration of 25 mg/mL. MDA was released by acid hydrolysisof malondialdehyde bis (Sigma). The solution was diluted with 0.1M phosphate-buffered saline (pH 6.4) and was brought to pH 7.4with 10 N NaOH to yield 200 mM MDA. Equal volumes of BSAand MDA solutions were incubated with and without the variousantioxidants: 5 mM of ascorbate, 5 mM ofa-tocopherol, 1 mg/mLof pycnogenol, and 0.2 mg/mL of an ethanol equivalent of red andwhite wine. The samples were incubated at 37°C for 24 h and thendialyzed at 4°C against 2 L of 0.01 M phosphate-buffered salinefor 24 h. Cellulose-ester dialysis membrane MWCO 1000(Spectra/Por, Houston, TX, USA) soaked in 0.05% sodium azidewas used for the dialysis. Each BSA sample (5mg) was subjectedto Western blotting to detect MDA–BSA, as described above.

The results are expressed as mean6 SEM of the ratio of theintensity of the MDA-modified proteins formed in the presence ofvarious antioxidants to the intensity of the MDA-modified proteinsfound in the absence of the antioxidants. To combine data fromvarious gels, an internal control of 0.5mg of MDA–BSA was used.This was prepared as described above except that BSA was incu-bated with MDA for 72 h instead of 24 h to achieve maximal BSAmodification with MDA.

Analysis of variance with Bonferroni’s correction was used toevaluate statistical significance.P , 0.05 was consideredsignificant.

RESULTS AND DISCUSSION

A representative immunoblot of MDA-modified proteins fromendothelial cells cultured in the presence or absence of variousantioxidants is shown in Figure 1. Multiple protein bands could beidentified. The total density of these bands was quantitated withdensitometry, and the results from various experiments were com-bined. The addition of 1 mg/mL of pycnogenol in culture mediumfor 24 h reduced the MDA-protein formation in endothelial cells.The mean6 SEM ratio of MDA-proteins formed in endothelialcells in the presence of pycnogenol to the MDA proteins foundunder control conditions was 0.5216 0.041 (P , 0.001). TheMDA-protein content of endothelial cells under control conditionswhen normalized to the mean of control experiments was 1.01160.022 in arbitrary units. In contrast, under the same culture con-ditions, the addition of 5 mM of ascorbate (1.1206 0.131), 5 mM

of a-tocopherol (0.8916 0.088), or 0.2 mg/mL of an ethanolequivalent of red (0.9246 0.045) or white (0.9446 0.033) winedid not have significant effects on MDA-protein content of endo-thelial cells.

The identity of these protein bands is not known. They may bea monomer of a single or multiple proteins or may be polymer-ization products of several proteins. They may also be degradationproducts of various proteins. MDA-protein modification is knownto cause aggregation and polymerization of homologous and het-erologous proteins. MDA modification of proteins can also causeincreased degradation of proteins.8

A representative immunoblot of MDA–BSA generated in theabsence or presence of ascorbate,a-tocopherol, red wine, or py-cnogenol is shown in Figure 2. The expected single 68-kDa bandof BSA–MDA is seen. Quantitation of the MDA–BSA bands fromnine independent experiments showed that MDA–BSA formed inthe presence of pycnogenol was significantly reduced comparedwith MDA–BSA formed in the absence of pycnogenol. Themean 6 SEM ratio of MDA–BSA formed in the presence ofpycnogenol to MDA–BSA formed under control conditions was0.516 6 0.09 (P , 0.001). a-Tocopherol (1.0126 0.093), redwine (1.1106 0.105) or white wine (1.1566 0.127) did not havea significant inhibitory effect on MDA modification of BSA. Therewas a modest, although statistically, insignificant reduction inMDA–BSA formation in the presence of ascorbate (0.82760.100).

A potential caveat is that the purity of pycnogenol source wasnot precisely known; therefore, the actual concentration in theculture medium may have been lower than 1 mg/mL. The lack ofan effect of vitamins C and E on MDA-protein formation isintriguing. Although these vitamins have free radical–scavengingactivities, it is possible that, unlike pycnogenol, they do not inhibitconstitutive formation of MDA proteins.

These results demonstrate that pycnogenol can protect proteinsfrom MDA modification. The precise mechanism of inhibition ofMDA modification of proteins by pycnogenol is not clear. Theeffect may not be related to its antioxidant properties because twoother antioxidants, vitamins C and E, did not alter MDA-proteinbinding. It is theoretically possible that pycnogenol may havetriggered the transfer of MDA to hitherto unknown products.Pycnogenol is a mixture of natural bioflavonoids extracted frompine-tree bark and is known to be a potent antioxidant. Theindividual active ingredients of this mixture are not well charac-terized. It is also unclear whether this commercially availableproduct has sufficient antioxidative effects in vivo to be considereda useful pharmacologic agent. Nevertheless, several in vitro stud-ies and experiments using cell cultures have demonstrated the freeradical–quenching activity of this product.9–12 The present studyextends these observations to suggest that pycnogenol may have anadditional salutary effect on the prevention of MDA modification

FIG. 1. A representative immunoblot of MDA-modified proteins fromendothelial cell cultures in the presence of 1 mg/mL of pycnogenol (lanes1 and 2), 5 mM ofa-tocopherol (lanes 3 and 4), 5 mM of ascorbate (lanes5 and 6), or under control conditions (lanes 7 and 8); 0.5mg of MDA–BSAis included as an internal positive control. Multiple MDA-protein bandscould be identified. The reduction in the intensity of the MDA-proteinbands in the presence of pycnogenol (lanes 1 and 2) is evident. When datafrom multiple experiments were combined, the changes in the presence ofa-tocopherol did not achieve statistical significance. BSA, bovine serumalbumin; MDA, malondialdehyde.

FIG. 2. A representative immunoblot of MDA–BSA generated in theabsence of the antioxidants (MDA–BSA) or in the presence of 5 mM ofascorbate (lane C), 5 mM ofa-tocopherol (lane E), 0.2 mg/mL of ethanolequivalent of red wine (lane RW), and 1 mg/mL of pycnogenol (lane P). Asingle 68-kDa band corresponding to the molecular weight of BSA isevident. The background of the immunoblot was cleared electronically.Pycnogenol significantly reduced the amount of MDA and BSA formed.The experiment was repeated nine times. BSA, bovine serum albumin;MDA, malondialdehyde.

1080 Kim et al. Nutrition Volume 16, Numbers 11/12, 2000

of proteins. To our knowledge, this is the first compound thatappears to inhibit MDA binding to proteins. It remains to be seenwhether this in vitro observation can be duplicated in intact ani-mals treated with pycnogenol.

ACKNOWLEDGMENTS

The authors thank Ms. Jian Ping Li and Deanna Reinacher forexcellent technical assistance.

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Nutrition Volume 16, Numbers 11/12, 2000 1081Malondialdehyde Modification of Proteins