- Email: [email protected]
Effect of select antioxidants on malondialdehyde modification of proteins1
BASIC NUTRITIONAL INVESTIGATION
Effect of Select Antioxidants on Malondialdehyde Modification of Proteins Joohee Kim, MD, Joe Chehade, MD, Jacob L. Pinnas, MD, and Arshag D. Mooradian, MD From the Division of Endocrinology, Diabetes and Metabolism, Department of Internal Medicine, Saint Louis University School of Medicine, St. Louis, Missouri; and the Department of Medicine, University of Arizona College of Medicine, Tucson, Arizona, USA To determine whether commonly used antioxidants alter malondialdehyde (MDA) modification of proteins, a known mechanism of free radical–related tissue injury, we studied the effect of adding 1 mg/mL of pycnogenol, 5 mM of ␣-tocopherol, 5 mM of ascorbate, and 0.2 mg/mL of an ethanol equivalent of red and white wine on MDA-protein content of endothelial cells in culture. The addition of pycnogenol but not of the other antioxidants was associated with significant reduction in MDA-protein content compared with controls (0.521 ⫾ 0.041 in arbritrary units versus 1.011 ⫾ 0.021, P ⬍ 0.001). To determine whether the observed effect occurs distal to MDA generation, the effect of these antioxidants on the modification of bovine serum albumin with MDA generated in a cell-free system was studied. In this cell-free assay, pycnogenol but not the other antioxidants reduced MDA–BSA generation by approximately 50%. It is concluded that pycnogenol may reduce MDA modification of proteins at a step distal to MDA generation. This may be an additional mechanism of protective effects of pycnogenol against 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 states and has been hypothesized to be one of the causes of aging.1 Therefore, it is assumed that antioxidants have salutary health benefits. It is generally accepted that the main role of the antioxidants is quenching of free radicals either directly or through indirect biochemical pathways. One of the mechanisms of free radical–induced tissue injury is generation of lipid peroxidation byproducts such as malondialdehyde (MDA) and modification of proteins with MDA.2,3 This reaction results in altered protein function and antigenicity.2,3 We speculated that antioxidants may interfere with MDA modification of proteins through either inhibition of lipid peroxidation or a step beyond generation of free radicals and peroxidation of lipids. To test this hypothesis, MDA proteins were measured in cultured endothelial cells. In addition, MDA modification of bovine serum albumin (BSA) was measured in a cell-free system in the presence of 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 was obtained from Cell Applications (San Diego, CA, USA). The cells were cultured at 37°C and 5% CO2 in a 75-cm2 flask (Becton Dickinson Labware, Franklin Lakes, NJ, USA) that was precoated
Correspondence to: Arshag D. Mooradian, MD, Division of Endocrinology, Saint Louis University School of Medicine, 3691 Rutger, Suite 101, St. Louis, MO 63110, USA. E-mail: [email protected] Date accepted: May 26, 2000. Nutrition 16:1079 –1081, 2000 ©Elsevier Science Inc., 2000. Printed in the United States. All rights reserved.
with attachment factor (Cell Applications) for at least 24 h before use. Cells were plated in six-well cluster dishes (Corning, Inc., Corning, NY, USA), and when confluent the old medium was replaced with fresh endothelial-cell growth medium (2 mL/well). Antioxidants were prepared in Millie-Q water (Millipore Corp., Bedford, MA, USA) and filter sterilized immediately before their addition to the fresh endothelial-cell growth medium. The following antioxidants were studied: 5 mM of ascorbate (Sigma, St. Louis, MO, USA), 5 mM of ␣-tocopherol (Sigma), 1 mg/mL of pycnogenol (Natrol, Chatsworth, CA, USA), and 0.2 mg/mL of an ethanol equivalent of red and white wine. The viability of cells in culture was verified with trypan-blue exclusion, which was over 90%. After 24 h the medium was removed, the cell monolayer was washed with Hank’s balanced salt solution (Mediatech, Herndon, VA, USA), and harvested in 40 L of Laemmli buffer4 using a rubber policeman. The protein content was measured by the method of Bradford5 after 5 to 10 s of sonication (Branson Ultrasonics Corp., Danbury, CT, USA). Each sample (50 g of protein) was resolved on a 10% sodium dodecyl sulfate–polyacrylamide gel and transferred to nitrocellulose filters, as described previously.6 The membrane was blocked with 5% chicken-egg albumin in 0.01 M sodium phosphate-buffered saline at 4°C and probed with anti–MDA-protein antiserum (1:500) for 2.5 h at room temperature. After incubation with the goat–anti-rabbit horseradish peroxidase–labeled secondary antibody (1:7000; Southern Biotechnology Associates, Inc., Birmingham, AL, USA) for 1 h, the protein bands were developed by enhanced chemiluminescence using the manufacturer’s protocol (Amersham Life Science, Arlington Heights, IL, USA). The density of the bands was quantified with a scanning laser densitometer (Molecular Dynamics, Sunnyvale, CA, USA). The specificity of the anti-MDA antiserum has been previously established in our laboratory.7 The antiserum does not cross-react with free MDA, native proteins, or proteins modified with glucose or acetaldehyde. To establish the specificity of the protein bands observed, antiserum preabsorbed with MDA–BSA was used. There were no discrete bands identified in these experiments. 0899-9007/00/$20.00 PII S0899-9007(00)00446-9
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FIG. 1. A representative immunoblot of MDA-modified proteins from endothelial cell cultures in the presence of 1 mg/mL of pycnogenol (lanes 1 and 2), 5 mM of ␣-tocopherol (lanes 3 and 4), 5 mM of ascorbate (lanes 5 and 6), or under control conditions (lanes 7 and 8); 0.5 g of MDA–BSA is included as an internal positive control. Multiple MDA-protein bands could be identified. The reduction in the intensity of the MDA-protein bands in the presence of pycnogenol (lanes 1 and 2) is evident. When data from multiple experiments were combined, the changes in the presence of ␣-tocopherol did not achieve statistical significance. BSA, bovine serum albumin; MDA, malondialdehyde.
Cell-Free Assay of MDA–BSA Binding BSA was dissolved in 0.01 M sodium phosphate-buffered saline containing 0.01% ethylene-diaminetetraacetic acid (pH 7.4) at a concentration of 25 mg/mL. MDA was released by acid hydrolysis of malondialdehyde bis (Sigma). The solution was diluted with 0.1 M phosphate-buffered saline (pH 6.4) and was brought to pH 7.4 with 10 N NaOH to yield 200 mM MDA. Equal volumes of BSA and MDA solutions were incubated with and without the various antioxidants: 5 mM of ascorbate, 5 mM of ␣-tocopherol, 1 mg/mL of pycnogenol, and 0.2 mg/mL of an ethanol equivalent of red and white wine. The samples were incubated at 37°C for 24 h and then dialyzed at 4°C against 2 L of 0.01 M phosphate-buffered saline for 24 h. Cellulose-ester dialysis membrane MWCO 1000 (Spectra/Por, Houston, TX, USA) soaked in 0.05% sodium azide was used for the dialysis. Each BSA sample (5 g) was subjected to Western blotting to detect MDA–BSA, as described above. The results are expressed as mean ⫾ SEM of the ratio of the intensity of the MDA-modified proteins formed in the presence of various antioxidants to the intensity of the MDA-modified proteins found in the absence of the antioxidants. To combine data from various gels, an internal control of 0.5 g of MDA–BSA was used. This was prepared as described above except that BSA was incubated with MDA for 72 h instead of 24 h to achieve maximal BSA modification with MDA. Analysis of variance with Bonferroni’s correction was used to evaluate statistical significance. P ⬍ 0.05 was considered significant.
RESULTS AND DISCUSSION A representative immunoblot of MDA-modified proteins from endothelial cells cultured in the presence or absence of various antioxidants is shown in Figure 1. Multiple protein bands could be identified. The total density of these bands was quantitated with densitometry, and the results from various experiments were combined. The addition of 1 mg/mL of pycnogenol in culture medium for 24 h reduced the MDA-protein formation in endothelial cells. The mean ⫾ SEM ratio of MDA-proteins formed in endothelial cells in the presence of pycnogenol to the MDA proteins found under control conditions was 0.521 ⫾ 0.041 (P ⬍ 0.001). The MDA-protein content of endothelial cells under control conditions when normalized to the mean of control experiments was 1.011 ⫾ 0.022 in arbitrary units. In contrast, under the same culture conditions, the addition of 5 mM of ascorbate (1.120 ⫾ 0.131), 5 mM
Nutrition Volume 16, Numbers 11/12, 2000
FIG. 2. A representative immunoblot of MDA–BSA generated in the absence of the antioxidants (MDA–BSA) or in the presence of 5 mM of ascorbate (lane C), 5 mM of ␣-tocopherol (lane E), 0.2 mg/mL of ethanol equivalent of red wine (lane RW), and 1 mg/mL of pycnogenol (lane P). A single 68-kDa band corresponding to the molecular weight of BSA is evident. 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.
of ␣-tocopherol (0.891 ⫾ 0.088), or 0.2 mg/mL of an ethanol equivalent of red (0.924 ⫾ 0.045) or white (0.944 ⫾ 0.033) wine did not have significant effects on MDA-protein content of endothelial cells. The identity of these protein bands is not known. They may be a monomer of a single or multiple proteins or may be polymerization products of several proteins. They may also be degradation products of various proteins. MDA-protein modification is known to cause aggregation and polymerization of homologous and heterologous proteins. MDA modification of proteins can also cause increased degradation of proteins.8 A representative immunoblot of MDA–BSA generated in the absence or presence of ascorbate, ␣-tocopherol, red wine, or pycnogenol is shown in Figure 2. The expected single 68-kDa band of BSA–MDA is seen. Quantitation of the MDA–BSA bands from nine independent experiments showed that MDA–BSA formed in the presence of pycnogenol was significantly reduced compared with MDA–BSA formed in the absence of pycnogenol. The mean ⫾ SEM ratio of MDA–BSA formed in the presence of pycnogenol to MDA–BSA formed under control conditions was 0.516 ⫾ 0.09 (P ⬍ 0.001). ␣-Tocopherol (1.012 ⫾ 0.093), red wine (1.110 ⫾ 0.105) or white wine (1.156 ⫾ 0.127) did not have a significant inhibitory effect on MDA modification of BSA. There was a modest, although statistically, insignificant reduction in MDA–BSA formation in the presence of ascorbate (0.827 ⫾ 0.100). A potential caveat is that the purity of pycnogenol source was not precisely known; therefore, the actual concentration in the culture medium may have been lower than 1 mg/mL. The lack of an effect of vitamins C and E on MDA-protein formation is intriguing. Although these vitamins have free radical–scavenging activities, it is possible that, unlike pycnogenol, they do not inhibit constitutive formation of MDA proteins. These results demonstrate that pycnogenol can protect proteins from MDA modification. The precise mechanism of inhibition of MDA modification of proteins by pycnogenol is not clear. The effect may not be related to its antioxidant properties because two other antioxidants, vitamins C and E, did not alter MDA-protein binding. It is theoretically possible that pycnogenol may have triggered the transfer of MDA to hitherto unknown products. Pycnogenol is a mixture of natural bioflavonoids extracted from pine-tree bark and is known to be a potent antioxidant. The individual active ingredients of this mixture are not well characterized. It is also unclear whether this commercially available product has sufficient antioxidative effects in vivo to be considered a useful pharmacologic agent. Nevertheless, several in vitro studies and experiments using cell cultures have demonstrated the free radical– quenching activity of this product.9 –12 The present study extends these observations to suggest that pycnogenol may have an additional salutary effect on the prevention of MDA modification
Nutrition Volume 16, Numbers 11/12, 2000 of proteins. To our knowledge, this is the first compound that appears to inhibit MDA binding to proteins. It remains to be seen whether this in vitro observation can be duplicated in intact animals treated with pycnogenol.
ACKNOWLEDGMENTS The authors thank Ms. Jian Ping Li and Deanna Reinacher for excellent technical assistance.
REFERENCES 1. Halliwell B. Oxidants and human disease: some new concepts. FASEB J 1987; 1:358 2. Lung CC, Pinnas JL, Yahya MD, Meinke GC, Mooradian AD. Malondialdehyde modified proteins and their antibodies in the plasma of control and streptozotocininduced diabetic rats. Life Sci 1993;52:329 3. Mooradian AD, Lung CC, Shah G, Mahmoud S, Pinnas JL. Age-related changes in tissue content of malondialdehyde-modified proteins. Life Sci 1994;55:1561
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4. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970;227:680 5. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976;72:248 6. Towbin H, Staehelin T, Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 1979;76:4350 7. Lung CC, Fleisher JH, Meinke G, Pinnas JL. Immunochemical properties of malondialdehyde-protein adducts. J Immunol Methods 1990;128:127 8. Mooradian AD, Lung CC, Pinnas JL. Glycosylation enhances malondialdehyde binding to proteins. Free Rad Biol Med 1996;21:699 9. Rong Y, Li L, Shah V, Lau BH. Pycnogenol protects vascular endothelial cells from t-butyl hydroperoxide induced oxidant injury. Biotech Ther 1994 –1995;5:117 10. Virgili F, Kim D, Packer L. Procyanidins extracted from pine bark protect alpha-tocopherol in ECV304 endothelial cells challenged by activated RAW 264.7 macrophages: role of nitric oxide and peroxynitrate. FEBS Lett 1998;431:315 11. Virgili F, Kobuchi H, Packer L. Procyanidins extracted from pinus maritime (pycnogenol): scavengers of free radical species and modulators of nitrogen monoxide metabolism in activated murine RAW 264.7 macrophages. Free Rad Biol Med 1998;24:1120 12. Packer L, Rimbach G, Virgili F. Antioxidant activity and biologic properties of a procyanidin-rich extract from pine (Pinus maritima) bark, pycnogenol. Free Rad Biol Med. 1999;27:704
Effect of Select Antioxidants on Malondialdehyde Modification of Proteins Joohee Kim, MD, Joe Chehade, MD, Jacob L. Pinnas, MD, and Arshag D. Mooradian, MD From the Division of Endocrinology, Diabetes and Metabolism, Department of Internal Medicine, Saint Louis University School of Medicine, St. Louis, Missouri; and the Department of Medicine, University of Arizona College of Medicine, Tucson, Arizona, USA To determine whether commonly used antioxidants alter malondialdehyde (MDA) modification of proteins, a known mechanism of free radical–related tissue injury, we studied the effect of adding 1 mg/mL of pycnogenol, 5 mM of ␣-tocopherol, 5 mM of ascorbate, and 0.2 mg/mL of an ethanol equivalent of red and white wine on MDA-protein content of endothelial cells in culture. The addition of pycnogenol but not of the other antioxidants was associated with significant reduction in MDA-protein content compared with controls (0.521 ⫾ 0.041 in arbritrary units versus 1.011 ⫾ 0.021, P ⬍ 0.001). To determine whether the observed effect occurs distal to MDA generation, the effect of these antioxidants on the modification of bovine serum albumin with MDA generated in a cell-free system was studied. In this cell-free assay, pycnogenol but not the other antioxidants reduced MDA–BSA generation by approximately 50%. It is concluded that pycnogenol may reduce MDA modification of proteins at a step distal to MDA generation. This may be an additional mechanism of protective effects of pycnogenol against 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 states and has been hypothesized to be one of the causes of aging.1 Therefore, it is assumed that antioxidants have salutary health benefits. It is generally accepted that the main role of the antioxidants is quenching of free radicals either directly or through indirect biochemical pathways. One of the mechanisms of free radical–induced tissue injury is generation of lipid peroxidation byproducts such as malondialdehyde (MDA) and modification of proteins with MDA.2,3 This reaction results in altered protein function and antigenicity.2,3 We speculated that antioxidants may interfere with MDA modification of proteins through either inhibition of lipid peroxidation or a step beyond generation of free radicals and peroxidation of lipids. To test this hypothesis, MDA proteins were measured in cultured endothelial cells. In addition, MDA modification of bovine serum albumin (BSA) was measured in a cell-free system in the presence of 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 was obtained from Cell Applications (San Diego, CA, USA). The cells were cultured at 37°C and 5% CO2 in a 75-cm2 flask (Becton Dickinson Labware, Franklin Lakes, NJ, USA) that was precoated
Correspondence to: Arshag D. Mooradian, MD, Division of Endocrinology, Saint Louis University School of Medicine, 3691 Rutger, Suite 101, St. Louis, MO 63110, USA. E-mail: [email protected] Date accepted: May 26, 2000. Nutrition 16:1079 –1081, 2000 ©Elsevier Science Inc., 2000. Printed in the United States. All rights reserved.
with attachment factor (Cell Applications) for at least 24 h before use. Cells were plated in six-well cluster dishes (Corning, Inc., Corning, NY, USA), and when confluent the old medium was replaced with fresh endothelial-cell growth medium (2 mL/well). Antioxidants were prepared in Millie-Q water (Millipore Corp., Bedford, MA, USA) and filter sterilized immediately before their addition to the fresh endothelial-cell growth medium. The following antioxidants were studied: 5 mM of ascorbate (Sigma, St. Louis, MO, USA), 5 mM of ␣-tocopherol (Sigma), 1 mg/mL of pycnogenol (Natrol, Chatsworth, CA, USA), and 0.2 mg/mL of an ethanol equivalent of red and white wine. The viability of cells in culture was verified with trypan-blue exclusion, which was over 90%. After 24 h the medium was removed, the cell monolayer was washed with Hank’s balanced salt solution (Mediatech, Herndon, VA, USA), and harvested in 40 L of Laemmli buffer4 using a rubber policeman. The protein content was measured by the method of Bradford5 after 5 to 10 s of sonication (Branson Ultrasonics Corp., Danbury, CT, USA). Each sample (50 g of protein) was resolved on a 10% sodium dodecyl sulfate–polyacrylamide gel and transferred to nitrocellulose filters, as described previously.6 The membrane was blocked with 5% chicken-egg albumin in 0.01 M sodium phosphate-buffered saline at 4°C and probed with anti–MDA-protein antiserum (1:500) for 2.5 h at room temperature. After incubation with the goat–anti-rabbit horseradish peroxidase–labeled secondary antibody (1:7000; Southern Biotechnology Associates, Inc., Birmingham, AL, USA) for 1 h, the protein bands were developed by enhanced chemiluminescence using the manufacturer’s protocol (Amersham Life Science, Arlington Heights, IL, USA). The density of the bands was quantified with a scanning laser densitometer (Molecular Dynamics, Sunnyvale, CA, USA). The specificity of the anti-MDA antiserum has been previously established in our laboratory.7 The antiserum does not cross-react with free MDA, native proteins, or proteins modified with glucose or acetaldehyde. To establish the specificity of the protein bands observed, antiserum preabsorbed with MDA–BSA was used. There were no discrete bands identified in these experiments. 0899-9007/00/$20.00 PII S0899-9007(00)00446-9
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Kim et al.
FIG. 1. A representative immunoblot of MDA-modified proteins from endothelial cell cultures in the presence of 1 mg/mL of pycnogenol (lanes 1 and 2), 5 mM of ␣-tocopherol (lanes 3 and 4), 5 mM of ascorbate (lanes 5 and 6), or under control conditions (lanes 7 and 8); 0.5 g of MDA–BSA is included as an internal positive control. Multiple MDA-protein bands could be identified. The reduction in the intensity of the MDA-protein bands in the presence of pycnogenol (lanes 1 and 2) is evident. When data from multiple experiments were combined, the changes in the presence of ␣-tocopherol did not achieve statistical significance. BSA, bovine serum albumin; MDA, malondialdehyde.
Cell-Free Assay of MDA–BSA Binding BSA was dissolved in 0.01 M sodium phosphate-buffered saline containing 0.01% ethylene-diaminetetraacetic acid (pH 7.4) at a concentration of 25 mg/mL. MDA was released by acid hydrolysis of malondialdehyde bis (Sigma). The solution was diluted with 0.1 M phosphate-buffered saline (pH 6.4) and was brought to pH 7.4 with 10 N NaOH to yield 200 mM MDA. Equal volumes of BSA and MDA solutions were incubated with and without the various antioxidants: 5 mM of ascorbate, 5 mM of ␣-tocopherol, 1 mg/mL of pycnogenol, and 0.2 mg/mL of an ethanol equivalent of red and white wine. The samples were incubated at 37°C for 24 h and then dialyzed at 4°C against 2 L of 0.01 M phosphate-buffered saline for 24 h. Cellulose-ester dialysis membrane MWCO 1000 (Spectra/Por, Houston, TX, USA) soaked in 0.05% sodium azide was used for the dialysis. Each BSA sample (5 g) was subjected to Western blotting to detect MDA–BSA, as described above. The results are expressed as mean ⫾ SEM of the ratio of the intensity of the MDA-modified proteins formed in the presence of various antioxidants to the intensity of the MDA-modified proteins found in the absence of the antioxidants. To combine data from various gels, an internal control of 0.5 g of MDA–BSA was used. This was prepared as described above except that BSA was incubated with MDA for 72 h instead of 24 h to achieve maximal BSA modification with MDA. Analysis of variance with Bonferroni’s correction was used to evaluate statistical significance. P ⬍ 0.05 was considered significant.
RESULTS AND DISCUSSION A representative immunoblot of MDA-modified proteins from endothelial cells cultured in the presence or absence of various antioxidants is shown in Figure 1. Multiple protein bands could be identified. The total density of these bands was quantitated with densitometry, and the results from various experiments were combined. The addition of 1 mg/mL of pycnogenol in culture medium for 24 h reduced the MDA-protein formation in endothelial cells. The mean ⫾ SEM ratio of MDA-proteins formed in endothelial cells in the presence of pycnogenol to the MDA proteins found under control conditions was 0.521 ⫾ 0.041 (P ⬍ 0.001). The MDA-protein content of endothelial cells under control conditions when normalized to the mean of control experiments was 1.011 ⫾ 0.022 in arbitrary units. In contrast, under the same culture conditions, the addition of 5 mM of ascorbate (1.120 ⫾ 0.131), 5 mM
Nutrition Volume 16, Numbers 11/12, 2000
FIG. 2. A representative immunoblot of MDA–BSA generated in the absence of the antioxidants (MDA–BSA) or in the presence of 5 mM of ascorbate (lane C), 5 mM of ␣-tocopherol (lane E), 0.2 mg/mL of ethanol equivalent of red wine (lane RW), and 1 mg/mL of pycnogenol (lane P). A single 68-kDa band corresponding to the molecular weight of BSA is evident. 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.
of ␣-tocopherol (0.891 ⫾ 0.088), or 0.2 mg/mL of an ethanol equivalent of red (0.924 ⫾ 0.045) or white (0.944 ⫾ 0.033) wine did not have significant effects on MDA-protein content of endothelial cells. The identity of these protein bands is not known. They may be a monomer of a single or multiple proteins or may be polymerization products of several proteins. They may also be degradation products of various proteins. MDA-protein modification is known to cause aggregation and polymerization of homologous and heterologous proteins. MDA modification of proteins can also cause increased degradation of proteins.8 A representative immunoblot of MDA–BSA generated in the absence or presence of ascorbate, ␣-tocopherol, red wine, or pycnogenol is shown in Figure 2. The expected single 68-kDa band of BSA–MDA is seen. Quantitation of the MDA–BSA bands from nine independent experiments showed that MDA–BSA formed in the presence of pycnogenol was significantly reduced compared with MDA–BSA formed in the absence of pycnogenol. The mean ⫾ SEM ratio of MDA–BSA formed in the presence of pycnogenol to MDA–BSA formed under control conditions was 0.516 ⫾ 0.09 (P ⬍ 0.001). ␣-Tocopherol (1.012 ⫾ 0.093), red wine (1.110 ⫾ 0.105) or white wine (1.156 ⫾ 0.127) did not have a significant inhibitory effect on MDA modification of BSA. There was a modest, although statistically, insignificant reduction in MDA–BSA formation in the presence of ascorbate (0.827 ⫾ 0.100). A potential caveat is that the purity of pycnogenol source was not precisely known; therefore, the actual concentration in the culture medium may have been lower than 1 mg/mL. The lack of an effect of vitamins C and E on MDA-protein formation is intriguing. Although these vitamins have free radical–scavenging activities, it is possible that, unlike pycnogenol, they do not inhibit constitutive formation of MDA proteins. These results demonstrate that pycnogenol can protect proteins from MDA modification. The precise mechanism of inhibition of MDA modification of proteins by pycnogenol is not clear. The effect may not be related to its antioxidant properties because two other antioxidants, vitamins C and E, did not alter MDA-protein binding. It is theoretically possible that pycnogenol may have triggered the transfer of MDA to hitherto unknown products. Pycnogenol is a mixture of natural bioflavonoids extracted from pine-tree bark and is known to be a potent antioxidant. The individual active ingredients of this mixture are not well characterized. It is also unclear whether this commercially available product has sufficient antioxidative effects in vivo to be considered a useful pharmacologic agent. Nevertheless, several in vitro studies and experiments using cell cultures have demonstrated the free radical– quenching activity of this product.9 –12 The present study extends these observations to suggest that pycnogenol may have an additional salutary effect on the prevention of MDA modification
Nutrition Volume 16, Numbers 11/12, 2000 of proteins. To our knowledge, this is the first compound that appears to inhibit MDA binding to proteins. It remains to be seen whether this in vitro observation can be duplicated in intact animals treated with pycnogenol.
ACKNOWLEDGMENTS The authors thank Ms. Jian Ping Li and Deanna Reinacher for excellent technical assistance.
REFERENCES 1. Halliwell B. Oxidants and human disease: some new concepts. FASEB J 1987; 1:358 2. Lung CC, Pinnas JL, Yahya MD, Meinke GC, Mooradian AD. Malondialdehyde modified proteins and their antibodies in the plasma of control and streptozotocininduced diabetic rats. Life Sci 1993;52:329 3. Mooradian AD, Lung CC, Shah G, Mahmoud S, Pinnas JL. Age-related changes in tissue content of malondialdehyde-modified proteins. Life Sci 1994;55:1561
Malondialdehyde Modification of Proteins
1081
4. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970;227:680 5. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976;72:248 6. Towbin H, Staehelin T, Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 1979;76:4350 7. Lung CC, Fleisher JH, Meinke G, Pinnas JL. Immunochemical properties of malondialdehyde-protein adducts. J Immunol Methods 1990;128:127 8. Mooradian AD, Lung CC, Pinnas JL. Glycosylation enhances malondialdehyde binding to proteins. Free Rad Biol Med 1996;21:699 9. Rong Y, Li L, Shah V, Lau BH. Pycnogenol protects vascular endothelial cells from t-butyl hydroperoxide induced oxidant injury. Biotech Ther 1994 –1995;5:117 10. Virgili F, Kim D, Packer L. Procyanidins extracted from pine bark protect alpha-tocopherol in ECV304 endothelial cells challenged by activated RAW 264.7 macrophages: role of nitric oxide and peroxynitrate. FEBS Lett 1998;431:315 11. Virgili F, Kobuchi H, Packer L. Procyanidins extracted from pinus maritime (pycnogenol): scavengers of free radical species and modulators of nitrogen monoxide metabolism in activated murine RAW 264.7 macrophages. Free Rad Biol Med 1998;24:1120 12. Packer L, Rimbach G, Virgili F. Antioxidant activity and biologic properties of a procyanidin-rich extract from pine (Pinus maritima) bark, pycnogenol. Free Rad Biol Med. 1999;27:704