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The polyphenol-rich extract from grape seeds suppresses toxicity of homocysteine and its thiolactone on the fibrinolytic system
Thrombosis Research 127 (2011) 489–491
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Thrombosis Research j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / t h r o m r e s
Letter to the Editors-in-Chief The polyphenol-rich extract from grape seeds suppresses toxicity of homocysteine and its thiolactone on the fibrinolytic system Dear Editors, Homocysteine (Hcys) occurs in human blood in free (e.g. as reduced form – about 100 nM) and protein bound form. The term “total homocysteine” describes the pool of homocysteine released by reduction of all disulfide bonds in the samples. Kang et al. [1] classified several types of hyperhomocysteinemia, in relation to the total plasma Hcys concentration. They defined hyperhomocysteinemia as severe, for concentrations higher than 100 μM, intermediate for concentrations between 31 and 100 μM, and moderate for concentrations of 16 – 30 μM, and a reference total plasma Hcys range as 5 to 15 μM (mean, 10 μM). Elevated plasma Hcys (N15 μM; Hcys) is associated with an increased risk of cardiovascular diseases [1–4], however there are many different opinions on the biotoxicity of Hcys on hemostasis process. Our earlier results have found that not only the modulation of fibrinolytic system [5] but also coagulation process is modified [6]. Studies performed during the last two decades suggest that the modifications of different hemostatic proteins (N-homocysteinylated or S-homocysteinylated proteins) induced by Hcys or the most reactive form of Hcys - its thiolactone (HTL) seem to be the main reason of biotoxicity of homocysteine in these diseases [2]. For example, in human plasma, fibrinogen, plasminogen and other hemostatic proteins are covalently modified by Hcys and HTL [3,4,6]. However, there are not pharmacologically active compounds protecting proteins against modifications induced by Hcys or HTL. Different compounds (folic acid, vitamins: B6 and B12) present in human diet may have effect on the concentration of Hcys in human plasma [7]. Results of Pietrzik and Bronstrup [7] showed that daily supplementation with folic acid in the range of 0.5 – 5 mg and with about 0.5 mg of vitamin B12 decreased Hcys concentration in blood. But, the results of Bogers et al. [8] showed that the increased fruit and vegetable consumption may be insufficient to change plasma Hcys concentration. On the basis of various observations, it is proposed that Hcys or HTL may also act as an oxidant in the model system in vitro and in vivo, but diet polyphenolic antioxidants can inhibit oxidative damages [9]. Resveratrol, a phenolic antioxidant synthesized in grapes and presents in wine reduces the toxic action of Hcys and HTL on hemostatic properties of fibrinogen or plasma [6]. Grape seeds are one of the richest plant sources of phenolic substances, therefore and they have been supposed to be beneficial for the prevention of cardiovascular events, the aim of our study was to establish the influence of grape seeds extract (GSE) on the changes of the fibrinolytic system (using human plasma or purified plasminogen - Plg) induced by both, D, L-homocysteine (≥95%, Sigma Chemical Company, St. Louis, MO, USA) in reduced form and its cyclic thioester – D, L-homocysteine thiolactone (≥99%, Sigma Chemical Company, St. Louis, MO, USA) in vitro. Blood samples were taken from 6 healthy volunteers (aged between 26 – 32 years (mean 26.7 ± 2.3)) without cardiovascular disorders, allergy and lipid or carbohydrate 0049-3848/$ – see front matter. Published by Elsevier Ltd. doi:10.1016/j.thromres.2010.12.022
metabolism disorders, untreated with drugs. Healthy subjects did not use addictive substances and antioxidant supplementation with balanced diet (meat and vegetables), lived in similar socio-economic conditions. Subjects with significant medical illness were excluded. They were no smokers. Human blood was collected at 3.2% citrate (used to 12 mM final concentration) and immediately centrifuged (2000 × g, 15 min) to get plasma. Plasminogen was isolated by affinity chromatography on Lysine-Sepharose [10]. The natural concentration of total Hcys in plasma (in healthy subjects who participated in the current study) was 10.6 ± 3.8 μM. The endogenous concentration of reduced form of Hcys and HTL in plasma (in healthy subjects who participated in the current study) was 100 ± 11.2 nM and 0–35 nM, respectively. The classical technique HPLC has been used to analysis of Hcys or HTL in human plasma. The HPLC analysis was performed with a Hewlett-Packard 1100 Series system according to Głowacki et al. [11] and Bald et al. [12]. The concentration of Plg in plasma (in healthy subjects who participated in the current study) was 2 ± 0.3 μM. Samples of human Plg (2 μM) or human plasma were exposed to: - D, L-homocysteine at a final concentration of 0.1 mM - D, L-homocysteine thiolactone at a final concentration of 1 μM - grape seeds extract (GSE) at a final concentration between 12.550 μg/ml - grape seeds extract at a final concentration between 12.5-50 μg/ml and the reduced form of D, L-homocysteine at a final concentration of 0.1 mM - grape seeds extract at a final concentration between 12.5-50 μg/ml and D, L homocysteine thiolactone at a final concentration of 1 μM. Samples were incubated for 30 min at 37 °C. The tested concentrations of Hcys or HTL correspond to the levels found in human plasma during hyperhomocysteinemia in vivo. The extract of grape seeds was supplied by Bionorica (Germany), and the total concentration of phenolics was 500 mg/g of extract [13]. Plasmin activity and plasminogen activation were estimated by the hydrolysis of chromogenic substrate by streptokinase (SK) or by tissue plasminogen activator (tPA); assays were performed at room temperature in 96-well polystyrene flat-bottom plates. The absorbance measurements were performed in a microplate reader (Bio-Rad Microplate Reader, model 550) at 415 nm. No activity of generated plasmin was detected in the absence of SK or tPA. The statistical analysis was done by several tests. All the values in this study were expressed as mean ± SD of 6 experiments (done in triplicate). The difference between variations and means were assessed by applying the Fisher-Snedecor test and the unpaired Student's t-test, respectively. The statistical analysis was also performed with ANOVA I test and coefficients of variations. In the present study we accepted the differences with p b 0.05 as statistically significant. We have found that exposure of purified plasminogen to the reduced form of Hcys (0.1 mM) or HTL (1 μM) resulted in the inhibition of plasmin amidolytic activity (using streptokinase; Student's t-test – p b 0.001) (Table 1). When we measured the effect of Hcys and its thiolactone on plasmin amidolytic activity (using tPA), we observed also
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Letter to the Editors-in-Chief
Table 1 Effects of grape seeds extract (12.5-50 μg/ml, 30 min, 37 °C) on the fibrynolytic system (plasmin amidolytic activity and streptokinase – induced conversion of plasminogen to plasmin) and on S- and N-modification (the level of thiol groups and the level of ε-amino groups of Lys) of plasma total proteins in the presence of homocysteine (0.1 mM, 30 min, 37 °C) or its thiolactone (1 μM, 30 min, 37 °C). Data represent means ± SD of 6 experiments (done in triplicate). The effect of three different concentrations of GSE (12.5, 25 and 50 mg/ml) was not statistically significant according to one-way ANOVA test with Tukey test, p N 0.05 (for plasmin amidolytic activity, SK-induced conversion of plasminogen to plasmin; and S- or N-modification of plasma proteins). The effect of three different concentrations of GSE (12.5, 25 and 50 mg/ml) was statistically significant according to one-way ANOVA test with Tukey test, p b 0.05 (for purified Plg or plasma treated with GSE and Hcys or GSE and HTL; for plasmin amidolytic activity, SK-induced conversion of plasminogen to plasmin; and S- or N-modification of plasma proteins). The results were also analyzed using coefficients of variation (V). Concentration
0 (control) GSE (12.5 μg/ml) GSE (25 μg/ml) GSE (50 μg/ml) Hcys (0.1 mM) GSE (12.5 μg/ml) + Hcys GSE (25 μg/ml) + Hcys GSE (50 μg/ml) + Hcys HTL (1 μM) GSE (12.5 μg/ml) + HTL GSE (25 μg/ml) + HTL GSE (50 μg/ml) + HTL
Purified Plg
Plasma
Plasmin amidolytic activity [% of control; when streptokinase was used]
SK-induced conversion of plasminogen to plasmin [Absorbance at 415 nm]
Plasmin amidolytic activity [% of control; when streptokinase was used]
SK-induced conversion of plasminogen to plasmin [Absorbance at 415 nm]
100 ± 0 (V = 0) 99.4 ± 17.7 (V = 17,8) 95.4 ± 15.3 (V = 16.0) 94.7 ± 14.7 (V = 15.5) 52.4 ± 10.2 (V = 19.5) 77.1 ± 7.8 (V = 10.1) 79.3 ± 10.7 (V = 13.5) 87.2 ± 3.8 (V = 4.3) 44.4 ± 11.2 (V = 25.2) 83.2 ± 5.2 (V = 6.2) 87.3 ± 4.7 (V = 5.4) 89.6 ± 7.7 (V = 8.6)
0.613 ± 0.213 0.600 ± 0.234 0.604 ± 0.170 0.594 ± 0.220 0.333 ± 0.105 0.474 ± 0.150 0.540 ± 0.200 0.594 ± 0.112 0.324 ± 0.100 0.459 ± 0.100 0.484 ± 0.087 0.501 ± 0.077
100 ± 0 (V = 0) 98.4 ± 9.7 (V = 9.8) 95.9 ± 5.3 (V = 5.5) 97.4 ± 8.3 (V = 8.5) 65.4 ± 11.6 (V = 17.7) 84.6 ± 10.1 (V = 11.9) 87.1 ± 8.8 (V = 10.1) 90.5 ± 8.3 (V = 9.2) 68.9 ± 8.7 (V = 12.6) 79.9 ± 8.5 (V = 10.6) 82.8 ± 5.3 (V = 6.4) 92.8 ± 9.0 (V = 9.7)
0.637 ± 0.332 0.640 ± 0.254 0.620 ± 0.199 0.604 ± 0.304 0.384 ± 0.123 0.524 ± 0.094 0.574 ± 0.099 0.599 ± 0.112 0.333 ± 0.113 0.465 ± 0.087 0.480 ± 0.078 0.564 ± 0.066
(V = 34.7) (V = 39.0) (V = 28.1) (V = 37.0) (V = 31.5) (V = 31.6) (V = 37.0)) (V = 18.8) (V = 30.9) (V = 21.8) (V = 18.0) (V = 15.4)
(V = 52.1) (V = 39.7) (V = 32.1) (V = 50.3) (V = 32.0) (V = 17.9) (V = 17.2) (V = 18.7) (V = 33.9) (V = 18.7) (V = 16.2) (V = 11.7)
S- and N-modification of plasma proteins nmol of SH groups/ml of plasma 0 (control) GSE (12.5 μg/ml) GSE (25 μg/ml) GSE (50 μg/ml) Hcys (0.1 mM) GSE (12.5 μg/ml) + Hcys GSE (25 μg/ml) + Hcys GSE (50 μg/ml) + Hcys HTL (1 μM) GSE (12.5 μg/ml) + HTL GSE (25 μg/ml) + HTL GSE (50 μg/ml) + HTL
302 ± 34 (V = 11.2) 300 ± 12 (V = 4.0) 278 ± 10 (V = 3.6) 274 ± 14 (V = 5.1) 200 ± 9 (V = 4.5) 269 ± 12 (V = 4.5) 275 ± 8 (V = 2.9) 287 ± 14 (V = 4.9) 274 ± 10 (V = 3.6) 285 ± 8 (V = 2.8) 289 ± 7 (V = 2.4) 303 ± 14 (V = 4.6)
the inhibition of this process (data are not presented). The grape seeds extract (12.5-50 μg/ml) had no effect on this process, but this extract (at all tested concentrations) reduced the inhibitory action of Hcys or HTL on plasmin amidolytic activity (when SK was used (Table 1), and when tPA was used (data are not presented); Student's t-test-p N 0.05). We have also shown the same phenomenon in human plasma. Moreover, the inhibited conversion of plasminogen to plasmin after treatment of plasma or purified plasminogen with Hcys (0.1 mM) or HTL (1 μM) was observed (Student's t-test-p b 0.05) (Table 1). The grape seeds extract (12.5-50 μg/ml) reduced the decrease of conversion of plasminogen to plasmin induced by Hcys or HTL (Table 1), e.g. in the presence of grape seeds extract (at the highest tested concentration - 50 μg/ml) the increase was about 78% (for Plg – treated with Hcys; Student's t-testp b 0.002) and 56% (for plasma – treated with Hcys; Student's t-testp b 0.01) (Table 1). Our studies demonstrated in model system in vitro that the grape seeds extract (12.5-50 μg/ml) diminished the reduction of thiol group level and ε-amino groups of Lys level in plasma proteins (as determined via colorimetric methods) treated with Hcys (0.1 mM) or HTL (1 μM). In the presence of the grape seeds extract at the highest tested concentration of 50 μg/ml, the reduction of thiol group level was about 45% (for plasma treated with Hcys; Student's t-test-p b 0.001) and about 15% (for plasma treated with HTL; Student's t-test-p b 0.01) (Table 1). The grape seeds extract had very similar protective effects when we measured the amount of ε-amino groups of Lys in plasma proteins treated with Hcys (0.1 mM) (Student's t-test-p b 0.02) or HTL (1 μM) (Student's t-test-p b 0.001) (Table 1). The defence mechanisms against action of Hcys and its derivatives are very important for the biological function of human plasma
μg of ε–NH2 of Lys/ml of plasma 234 ± 23 (V = 9.8) 237 ± 9 (V = 3.8) 236 ± 12 (V = 5.1) 220 ± 17 (V = 7.7) 199 ± 15 (V = 7.5) 216 ± 7 (V = 3.2) 222 ± 11 (V = 4.9) 230 ± 9 (V = 3.9) 180 ± 5 (V = 2.8) 199 ± 8 (V = 4.0) 220 ± 7 (V = 3.2) 224 ± 15 (V = 6.7)
components, including hemostatic properties of various proteins. Red wine polyphenolic compounds supplementation at low dose significantly reduced plasma Hcys levels and restored the hepatic and plasma-decreased paraoxonase-1 activity induced by chronic hyperhomocysteinemia [14]. Moreover, Noll et al. [14] observed that aortic expression of proinflammatory cytokines and adhesion molecules and levels of soluble lectin-like oxidized low-density lipoprotein receptor1 were reduced in hyperhomocysteinemic mice fed the red wine polyphenolic extract supplementation. Fu et al. [15] reported that red wine prevents homocysteine – induced endothelial dysfunction in porcine coronary arteries. Our earlier results showed that resveratrol (the compound presents in wine) strongly, but not completely reduced blood platelet apoptosis induced by Hcys or HTL, suggesting that other pathways different than reactive oxygen species generation are also involved [16]. The present study provides more information about biological activity of the extract form grape seeds, and for the first time indicates that this extract reduces the toxicity of Hcys and HTL on fibrinolytic properties of Plg or plasma, and may protect plasma proteins against S- and N-modifications caused by homocysteine or its derivatives – HTL (Table 1). The range of the used concentrations (12.5 – 50 μg/ml) is similar to that used in studies of other authors [17]. Physiological level of polyphenols in plasma depends mainly on grapes or wine consumption. Bioavailability of these compounds in human was determined to be more than 50 %. Based on the data, it can be estimated that plasma level of polyphenols is about 5 - 25 μg/ml after 30 min of drinking 100 ml of wine [18]. However, the protective mechanism of grape seeds extract on plasminogen or other plasma proteins during hyperhomocysteinemia is complex and still unclear. Since Hcys and its different derivatives
Letter to the Editors-in-Chief
promote free radicals production and lead to oxidative damage to proteins, we may suggest that grape seeds extract causing the changes in the level of reactive oxygen species or reactive nitrogen species (specially nitric oxide) may be responsible for the reduction of protein modifications induced by Hcys or its reactive derivative – HTL, and therefore may be potentially therapeutic useful in the prevention of the hyperhomocysteinemia - related cardiovascular diseases. We may also suggest that the high content of phenolics seem to be responsible for the observed this activity, but the effects of other components from tested plant extract remain to be investigated. Acknowledgement This work was supported by grant 506/810 and 505/373 from University of Lodz and also project co-financed by the European Union under the European Social Fund.
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[11] Glowacki R, Bald E, Jakubowski H. An on-column derivatization method for the determination of homocysteine-thiolactone and protein N-linked homocysteine. Amino Acids 2010 (in press). [12] Bald E, Chwatko G, Glowacki R, et al. Analysis of plasma thiols by highperformance liquid chromatography with ultraviolet detection. J Chromatogr 2004;1032:109–15. [13] Olas B, Wachowicz B, Tomczak A, et al. Comparative anti-platelet and antioxidant properties of polyphenol-rich extracts from: berries of Aronia melanocarpa, seeds of grape, bark of Yucca schidigera in vitro. Platelets 2008;19:70–7. [14] Noll C, Hamelet J, Matulewicz E, et al. Effects of red wine polyphenolic compounds on paraoxonase-1 and lectin-like oxidized low-density lipoprotein receptor-1 in hyperhomocysteinemic mice. J Nutr Bichem 2009;20:586–96. [15] Fu W, Conklin BS, Lin PH, et al. Red wine prevents homocysteine-induced endothelial dysfunction in porcine coronary arteries. J Surg Res 2003;115:82–91. [16] Olas B, Malinowska J, Rywaniak J. Homocysteine and its thiolactone may promote apoptotic events in blood platelets in vitro. Platelets 2010;21:533–40. [17] Houde V, Greinier D, Chandad F. Protective effects of grape seed proanthocyanidins against oxidative stress induced by lipopolysaccharides of periodontopathogenes. J Periodontol 2006;77:1371–9. [18] Soleas GJ, Goldberg DM. Analysis of antioxidant wine polyphenols by gas chromatography –mass spectrometry. Meth Enzymol 1999;229:137–51.
References [1] Kang SS, Wong PW, Malinow MR. Hyperhomocyst(e)inemia as a risk factor for occlusive vascular disease. Annu Rev Nutr 1992;12:279–89. [2] Jakubowski H. The molecular basis of homocysteine thiolactone-mediated vascular disease. Clin Chem Lab Med 2007;12:1704–6. [3] Karolczak K, Olas B. Mechanism action of homocysteine and its thiolactone in haemostasis system. Physiol Res 2009;58:623–33. [4] Olas B, Kołodziejczyk J, Malinowska J. May modifications of human plasma proteins stimulated by homocysteine and its thiolactone induce changes of haemostatic function of plasma in vitro ? Gen Physiol Biophys 2010;29:186–93. [5] Kołodziejczyk J, Malinowska J, Nowak P, et al. Comparison of the effect of homocysteine and its thiolactone on the fibrinolytic system using human plasma and purified plasminogen. Mol Cell Biochem 2010;344:217–20. [6] Malinowska J, Olas B. Effect of resveratrol on hemostatic properties of human fibrinogen and plasma during model of hyperhomocysteinemia. Thromb Res 2010;126:e379–82. [7] Pietrzik K, Bronstrup A. Vitamins B12, B6 and folate as determinants of homocysteine concentration in the healthy population. Eur J Pediatr 1998;157:135–8. [8] Bogers RP, Dagnelie PC, Bast A, et al. Effect of increased vegetable and fruit consumption on plasma folate and homocysteine concentrations. Nutrition 2007;23: 97–102. [9] Carluccio MA, Ancora MA, Massaro M, et al. Homocysteine induces VCAM-1 gene expression through NF-kappaB and NAD(P)H oxidase activation: protective role of Mediterranean diet polyphenolic antioxidants. Am J Physiol Heart Circ Physiol 2007;293:H2344–54. [10] Deutch DG, Mertz ET. Plasminogen: purification from human plasma by affinity chromatography. Proc Natl Acad Sci USA 1970;61:636–43.
Joanna Kołodziejczyk Joanna Malinowska Beata Olas⁎ The Department of General Biochemistry, Institute of Biochemistry, University of Lodz, Lodz, Poland ⁎Corresponding author. E-mail address: [email protected] (B. Olas). Anna Stochmal Wiesław Oleszek The Department of Biochemistry, Institute of Soil Science and Plant Cultivation, State Research Institute, Czartoryskich 8, 24-100 Pulawy, Poland Joachim Erler Bionorica Kerschensteinerstr. 11-14, 92318 Neumarkt, Germany 23 September 2010
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Thrombosis Research j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / t h r o m r e s
Letter to the Editors-in-Chief The polyphenol-rich extract from grape seeds suppresses toxicity of homocysteine and its thiolactone on the fibrinolytic system Dear Editors, Homocysteine (Hcys) occurs in human blood in free (e.g. as reduced form – about 100 nM) and protein bound form. The term “total homocysteine” describes the pool of homocysteine released by reduction of all disulfide bonds in the samples. Kang et al. [1] classified several types of hyperhomocysteinemia, in relation to the total plasma Hcys concentration. They defined hyperhomocysteinemia as severe, for concentrations higher than 100 μM, intermediate for concentrations between 31 and 100 μM, and moderate for concentrations of 16 – 30 μM, and a reference total plasma Hcys range as 5 to 15 μM (mean, 10 μM). Elevated plasma Hcys (N15 μM; Hcys) is associated with an increased risk of cardiovascular diseases [1–4], however there are many different opinions on the biotoxicity of Hcys on hemostasis process. Our earlier results have found that not only the modulation of fibrinolytic system [5] but also coagulation process is modified [6]. Studies performed during the last two decades suggest that the modifications of different hemostatic proteins (N-homocysteinylated or S-homocysteinylated proteins) induced by Hcys or the most reactive form of Hcys - its thiolactone (HTL) seem to be the main reason of biotoxicity of homocysteine in these diseases [2]. For example, in human plasma, fibrinogen, plasminogen and other hemostatic proteins are covalently modified by Hcys and HTL [3,4,6]. However, there are not pharmacologically active compounds protecting proteins against modifications induced by Hcys or HTL. Different compounds (folic acid, vitamins: B6 and B12) present in human diet may have effect on the concentration of Hcys in human plasma [7]. Results of Pietrzik and Bronstrup [7] showed that daily supplementation with folic acid in the range of 0.5 – 5 mg and with about 0.5 mg of vitamin B12 decreased Hcys concentration in blood. But, the results of Bogers et al. [8] showed that the increased fruit and vegetable consumption may be insufficient to change plasma Hcys concentration. On the basis of various observations, it is proposed that Hcys or HTL may also act as an oxidant in the model system in vitro and in vivo, but diet polyphenolic antioxidants can inhibit oxidative damages [9]. Resveratrol, a phenolic antioxidant synthesized in grapes and presents in wine reduces the toxic action of Hcys and HTL on hemostatic properties of fibrinogen or plasma [6]. Grape seeds are one of the richest plant sources of phenolic substances, therefore and they have been supposed to be beneficial for the prevention of cardiovascular events, the aim of our study was to establish the influence of grape seeds extract (GSE) on the changes of the fibrinolytic system (using human plasma or purified plasminogen - Plg) induced by both, D, L-homocysteine (≥95%, Sigma Chemical Company, St. Louis, MO, USA) in reduced form and its cyclic thioester – D, L-homocysteine thiolactone (≥99%, Sigma Chemical Company, St. Louis, MO, USA) in vitro. Blood samples were taken from 6 healthy volunteers (aged between 26 – 32 years (mean 26.7 ± 2.3)) without cardiovascular disorders, allergy and lipid or carbohydrate 0049-3848/$ – see front matter. Published by Elsevier Ltd. doi:10.1016/j.thromres.2010.12.022
metabolism disorders, untreated with drugs. Healthy subjects did not use addictive substances and antioxidant supplementation with balanced diet (meat and vegetables), lived in similar socio-economic conditions. Subjects with significant medical illness were excluded. They were no smokers. Human blood was collected at 3.2% citrate (used to 12 mM final concentration) and immediately centrifuged (2000 × g, 15 min) to get plasma. Plasminogen was isolated by affinity chromatography on Lysine-Sepharose [10]. The natural concentration of total Hcys in plasma (in healthy subjects who participated in the current study) was 10.6 ± 3.8 μM. The endogenous concentration of reduced form of Hcys and HTL in plasma (in healthy subjects who participated in the current study) was 100 ± 11.2 nM and 0–35 nM, respectively. The classical technique HPLC has been used to analysis of Hcys or HTL in human plasma. The HPLC analysis was performed with a Hewlett-Packard 1100 Series system according to Głowacki et al. [11] and Bald et al. [12]. The concentration of Plg in plasma (in healthy subjects who participated in the current study) was 2 ± 0.3 μM. Samples of human Plg (2 μM) or human plasma were exposed to: - D, L-homocysteine at a final concentration of 0.1 mM - D, L-homocysteine thiolactone at a final concentration of 1 μM - grape seeds extract (GSE) at a final concentration between 12.550 μg/ml - grape seeds extract at a final concentration between 12.5-50 μg/ml and the reduced form of D, L-homocysteine at a final concentration of 0.1 mM - grape seeds extract at a final concentration between 12.5-50 μg/ml and D, L homocysteine thiolactone at a final concentration of 1 μM. Samples were incubated for 30 min at 37 °C. The tested concentrations of Hcys or HTL correspond to the levels found in human plasma during hyperhomocysteinemia in vivo. The extract of grape seeds was supplied by Bionorica (Germany), and the total concentration of phenolics was 500 mg/g of extract [13]. Plasmin activity and plasminogen activation were estimated by the hydrolysis of chromogenic substrate by streptokinase (SK) or by tissue plasminogen activator (tPA); assays were performed at room temperature in 96-well polystyrene flat-bottom plates. The absorbance measurements were performed in a microplate reader (Bio-Rad Microplate Reader, model 550) at 415 nm. No activity of generated plasmin was detected in the absence of SK or tPA. The statistical analysis was done by several tests. All the values in this study were expressed as mean ± SD of 6 experiments (done in triplicate). The difference between variations and means were assessed by applying the Fisher-Snedecor test and the unpaired Student's t-test, respectively. The statistical analysis was also performed with ANOVA I test and coefficients of variations. In the present study we accepted the differences with p b 0.05 as statistically significant. We have found that exposure of purified plasminogen to the reduced form of Hcys (0.1 mM) or HTL (1 μM) resulted in the inhibition of plasmin amidolytic activity (using streptokinase; Student's t-test – p b 0.001) (Table 1). When we measured the effect of Hcys and its thiolactone on plasmin amidolytic activity (using tPA), we observed also
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Letter to the Editors-in-Chief
Table 1 Effects of grape seeds extract (12.5-50 μg/ml, 30 min, 37 °C) on the fibrynolytic system (plasmin amidolytic activity and streptokinase – induced conversion of plasminogen to plasmin) and on S- and N-modification (the level of thiol groups and the level of ε-amino groups of Lys) of plasma total proteins in the presence of homocysteine (0.1 mM, 30 min, 37 °C) or its thiolactone (1 μM, 30 min, 37 °C). Data represent means ± SD of 6 experiments (done in triplicate). The effect of three different concentrations of GSE (12.5, 25 and 50 mg/ml) was not statistically significant according to one-way ANOVA test with Tukey test, p N 0.05 (for plasmin amidolytic activity, SK-induced conversion of plasminogen to plasmin; and S- or N-modification of plasma proteins). The effect of three different concentrations of GSE (12.5, 25 and 50 mg/ml) was statistically significant according to one-way ANOVA test with Tukey test, p b 0.05 (for purified Plg or plasma treated with GSE and Hcys or GSE and HTL; for plasmin amidolytic activity, SK-induced conversion of plasminogen to plasmin; and S- or N-modification of plasma proteins). The results were also analyzed using coefficients of variation (V). Concentration
0 (control) GSE (12.5 μg/ml) GSE (25 μg/ml) GSE (50 μg/ml) Hcys (0.1 mM) GSE (12.5 μg/ml) + Hcys GSE (25 μg/ml) + Hcys GSE (50 μg/ml) + Hcys HTL (1 μM) GSE (12.5 μg/ml) + HTL GSE (25 μg/ml) + HTL GSE (50 μg/ml) + HTL
Purified Plg
Plasma
Plasmin amidolytic activity [% of control; when streptokinase was used]
SK-induced conversion of plasminogen to plasmin [Absorbance at 415 nm]
Plasmin amidolytic activity [% of control; when streptokinase was used]
SK-induced conversion of plasminogen to plasmin [Absorbance at 415 nm]
100 ± 0 (V = 0) 99.4 ± 17.7 (V = 17,8) 95.4 ± 15.3 (V = 16.0) 94.7 ± 14.7 (V = 15.5) 52.4 ± 10.2 (V = 19.5) 77.1 ± 7.8 (V = 10.1) 79.3 ± 10.7 (V = 13.5) 87.2 ± 3.8 (V = 4.3) 44.4 ± 11.2 (V = 25.2) 83.2 ± 5.2 (V = 6.2) 87.3 ± 4.7 (V = 5.4) 89.6 ± 7.7 (V = 8.6)
0.613 ± 0.213 0.600 ± 0.234 0.604 ± 0.170 0.594 ± 0.220 0.333 ± 0.105 0.474 ± 0.150 0.540 ± 0.200 0.594 ± 0.112 0.324 ± 0.100 0.459 ± 0.100 0.484 ± 0.087 0.501 ± 0.077
100 ± 0 (V = 0) 98.4 ± 9.7 (V = 9.8) 95.9 ± 5.3 (V = 5.5) 97.4 ± 8.3 (V = 8.5) 65.4 ± 11.6 (V = 17.7) 84.6 ± 10.1 (V = 11.9) 87.1 ± 8.8 (V = 10.1) 90.5 ± 8.3 (V = 9.2) 68.9 ± 8.7 (V = 12.6) 79.9 ± 8.5 (V = 10.6) 82.8 ± 5.3 (V = 6.4) 92.8 ± 9.0 (V = 9.7)
0.637 ± 0.332 0.640 ± 0.254 0.620 ± 0.199 0.604 ± 0.304 0.384 ± 0.123 0.524 ± 0.094 0.574 ± 0.099 0.599 ± 0.112 0.333 ± 0.113 0.465 ± 0.087 0.480 ± 0.078 0.564 ± 0.066
(V = 34.7) (V = 39.0) (V = 28.1) (V = 37.0) (V = 31.5) (V = 31.6) (V = 37.0)) (V = 18.8) (V = 30.9) (V = 21.8) (V = 18.0) (V = 15.4)
(V = 52.1) (V = 39.7) (V = 32.1) (V = 50.3) (V = 32.0) (V = 17.9) (V = 17.2) (V = 18.7) (V = 33.9) (V = 18.7) (V = 16.2) (V = 11.7)
S- and N-modification of plasma proteins nmol of SH groups/ml of plasma 0 (control) GSE (12.5 μg/ml) GSE (25 μg/ml) GSE (50 μg/ml) Hcys (0.1 mM) GSE (12.5 μg/ml) + Hcys GSE (25 μg/ml) + Hcys GSE (50 μg/ml) + Hcys HTL (1 μM) GSE (12.5 μg/ml) + HTL GSE (25 μg/ml) + HTL GSE (50 μg/ml) + HTL
302 ± 34 (V = 11.2) 300 ± 12 (V = 4.0) 278 ± 10 (V = 3.6) 274 ± 14 (V = 5.1) 200 ± 9 (V = 4.5) 269 ± 12 (V = 4.5) 275 ± 8 (V = 2.9) 287 ± 14 (V = 4.9) 274 ± 10 (V = 3.6) 285 ± 8 (V = 2.8) 289 ± 7 (V = 2.4) 303 ± 14 (V = 4.6)
the inhibition of this process (data are not presented). The grape seeds extract (12.5-50 μg/ml) had no effect on this process, but this extract (at all tested concentrations) reduced the inhibitory action of Hcys or HTL on plasmin amidolytic activity (when SK was used (Table 1), and when tPA was used (data are not presented); Student's t-test-p N 0.05). We have also shown the same phenomenon in human plasma. Moreover, the inhibited conversion of plasminogen to plasmin after treatment of plasma or purified plasminogen with Hcys (0.1 mM) or HTL (1 μM) was observed (Student's t-test-p b 0.05) (Table 1). The grape seeds extract (12.5-50 μg/ml) reduced the decrease of conversion of plasminogen to plasmin induced by Hcys or HTL (Table 1), e.g. in the presence of grape seeds extract (at the highest tested concentration - 50 μg/ml) the increase was about 78% (for Plg – treated with Hcys; Student's t-testp b 0.002) and 56% (for plasma – treated with Hcys; Student's t-testp b 0.01) (Table 1). Our studies demonstrated in model system in vitro that the grape seeds extract (12.5-50 μg/ml) diminished the reduction of thiol group level and ε-amino groups of Lys level in plasma proteins (as determined via colorimetric methods) treated with Hcys (0.1 mM) or HTL (1 μM). In the presence of the grape seeds extract at the highest tested concentration of 50 μg/ml, the reduction of thiol group level was about 45% (for plasma treated with Hcys; Student's t-test-p b 0.001) and about 15% (for plasma treated with HTL; Student's t-test-p b 0.01) (Table 1). The grape seeds extract had very similar protective effects when we measured the amount of ε-amino groups of Lys in plasma proteins treated with Hcys (0.1 mM) (Student's t-test-p b 0.02) or HTL (1 μM) (Student's t-test-p b 0.001) (Table 1). The defence mechanisms against action of Hcys and its derivatives are very important for the biological function of human plasma
μg of ε–NH2 of Lys/ml of plasma 234 ± 23 (V = 9.8) 237 ± 9 (V = 3.8) 236 ± 12 (V = 5.1) 220 ± 17 (V = 7.7) 199 ± 15 (V = 7.5) 216 ± 7 (V = 3.2) 222 ± 11 (V = 4.9) 230 ± 9 (V = 3.9) 180 ± 5 (V = 2.8) 199 ± 8 (V = 4.0) 220 ± 7 (V = 3.2) 224 ± 15 (V = 6.7)
components, including hemostatic properties of various proteins. Red wine polyphenolic compounds supplementation at low dose significantly reduced plasma Hcys levels and restored the hepatic and plasma-decreased paraoxonase-1 activity induced by chronic hyperhomocysteinemia [14]. Moreover, Noll et al. [14] observed that aortic expression of proinflammatory cytokines and adhesion molecules and levels of soluble lectin-like oxidized low-density lipoprotein receptor1 were reduced in hyperhomocysteinemic mice fed the red wine polyphenolic extract supplementation. Fu et al. [15] reported that red wine prevents homocysteine – induced endothelial dysfunction in porcine coronary arteries. Our earlier results showed that resveratrol (the compound presents in wine) strongly, but not completely reduced blood platelet apoptosis induced by Hcys or HTL, suggesting that other pathways different than reactive oxygen species generation are also involved [16]. The present study provides more information about biological activity of the extract form grape seeds, and for the first time indicates that this extract reduces the toxicity of Hcys and HTL on fibrinolytic properties of Plg or plasma, and may protect plasma proteins against S- and N-modifications caused by homocysteine or its derivatives – HTL (Table 1). The range of the used concentrations (12.5 – 50 μg/ml) is similar to that used in studies of other authors [17]. Physiological level of polyphenols in plasma depends mainly on grapes or wine consumption. Bioavailability of these compounds in human was determined to be more than 50 %. Based on the data, it can be estimated that plasma level of polyphenols is about 5 - 25 μg/ml after 30 min of drinking 100 ml of wine [18]. However, the protective mechanism of grape seeds extract on plasminogen or other plasma proteins during hyperhomocysteinemia is complex and still unclear. Since Hcys and its different derivatives
Letter to the Editors-in-Chief
promote free radicals production and lead to oxidative damage to proteins, we may suggest that grape seeds extract causing the changes in the level of reactive oxygen species or reactive nitrogen species (specially nitric oxide) may be responsible for the reduction of protein modifications induced by Hcys or its reactive derivative – HTL, and therefore may be potentially therapeutic useful in the prevention of the hyperhomocysteinemia - related cardiovascular diseases. We may also suggest that the high content of phenolics seem to be responsible for the observed this activity, but the effects of other components from tested plant extract remain to be investigated. Acknowledgement This work was supported by grant 506/810 and 505/373 from University of Lodz and also project co-financed by the European Union under the European Social Fund.
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[11] Glowacki R, Bald E, Jakubowski H. An on-column derivatization method for the determination of homocysteine-thiolactone and protein N-linked homocysteine. Amino Acids 2010 (in press). [12] Bald E, Chwatko G, Glowacki R, et al. Analysis of plasma thiols by highperformance liquid chromatography with ultraviolet detection. J Chromatogr 2004;1032:109–15. [13] Olas B, Wachowicz B, Tomczak A, et al. Comparative anti-platelet and antioxidant properties of polyphenol-rich extracts from: berries of Aronia melanocarpa, seeds of grape, bark of Yucca schidigera in vitro. Platelets 2008;19:70–7. [14] Noll C, Hamelet J, Matulewicz E, et al. Effects of red wine polyphenolic compounds on paraoxonase-1 and lectin-like oxidized low-density lipoprotein receptor-1 in hyperhomocysteinemic mice. J Nutr Bichem 2009;20:586–96. [15] Fu W, Conklin BS, Lin PH, et al. Red wine prevents homocysteine-induced endothelial dysfunction in porcine coronary arteries. J Surg Res 2003;115:82–91. [16] Olas B, Malinowska J, Rywaniak J. Homocysteine and its thiolactone may promote apoptotic events in blood platelets in vitro. Platelets 2010;21:533–40. [17] Houde V, Greinier D, Chandad F. Protective effects of grape seed proanthocyanidins against oxidative stress induced by lipopolysaccharides of periodontopathogenes. J Periodontol 2006;77:1371–9. [18] Soleas GJ, Goldberg DM. Analysis of antioxidant wine polyphenols by gas chromatography –mass spectrometry. Meth Enzymol 1999;229:137–51.
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Joanna Kołodziejczyk Joanna Malinowska Beata Olas⁎ The Department of General Biochemistry, Institute of Biochemistry, University of Lodz, Lodz, Poland ⁎Corresponding author. E-mail address: [email protected] (B. Olas). Anna Stochmal Wiesław Oleszek The Department of Biochemistry, Institute of Soil Science and Plant Cultivation, State Research Institute, Czartoryskich 8, 24-100 Pulawy, Poland Joachim Erler Bionorica Kerschensteinerstr. 11-14, 92318 Neumarkt, Germany 23 September 2010