Joanna Kołodziejczyk

L-Carnitine protects plasma components against oxidative alterations.

OBJECTIVE L-Carnitine as a dietary supplement has been reported to have a beneficial effect on several cardiovascular risk parameters and exercise capacity, but the biological relevance of its activity is poorly understood. Dietary supplements (including L-carnitine) are often used to foster exercise performance; however, these may affect some pathways of human body metabolism. The aim of this study in vitro was to determine antioxidative properties of L-carnitine (0.1-100 μM) added to plasma and to assess if L-carnitine might protect plasma proteins and lipids against oxidative/nitrative damage (determined by levels of protein carbonyl groups, thiols, 3-nitrotyrosine formation and thiobarbituric-acid reactive substances generation) caused by 100 μM peroxynitrite (ONOO(-)), a strong physiologic oxidative/nitrative agent. METHODS The level of carbonyl group generation was measured by a colorimetric method. For the estimation of 3-nitrotyrosine formation, a competition enzyme-linked immunosorbent assay was performed. Plasma lipid peroxidation was measured spectrophotometrically as the production of thiobarbituric-acid reactive substances. High-performance liquid chromatography was used to analyze total free thiol groups of plasma proteins and low-molecular-weight thiols (glutathione, cysteine, and homocysteine) in plasma. RESULTS The L-carnitine added to plasma inhibited in vitro ONOO(-)-induced oxidation and nitration of blood plasma proteins. Incubation of plasma with peroxynitrite resulted in the decrease of protein thiols. L-Carnitine had a protective effect on peroxynitrite-induced decreased -SH level in plasma proteins. The presence of L-carnitine also prevented the decrease of low-molecular-weight thiols (glutathione, cysteine, and homocysteine) in plasma caused by peroxynitrite and protected plasma lipids against peroxidation induced by peroxynitrite. CONCLUSIONS These results demonstrated that L-carnitine possesses antioxidative activity.

Effects of garcinol and guttiferone K isolated from Garcinia cambogia on oxidative/nitrative modifications in blood platelets and plasma.

The effects of garcinol and guttiferone K, two polyisoprenylated benzophenones occurring a food plant called Garcinia cambogia, on oxidative/nitrative protein damage (determined by parameters such as levels of protein carbonyl groups and nitrotyrosine residues) in human blood platelets and plasma after treatment with peroxynitrite (ONOO−) were studied in vitro. We also investigated the effects of garcinol and guttiferone K on lipid peroxidation in blood platelets and plasma induced by ONOO− (100 µM). Exposure of blood platelets or plasma to peroxynitrite (100 µM) resulted in an increased level of carbonyl groups and nitrotyrosine residues in proteins, and an increase of lipid peroxidation measured by the level of thiobarbituric acid reactive species (TBARS). In the presence of garcinol and guttiferone K (0.1–25 µg/ml), a distinct reduction in the formation of carbonyl groups in plasma and platelet proteins together with the decrease of TBARS caused by 100 µM peroxynitrite, was observed. However, garcinol and guttiferone K did not inhibit plasma and platelet protein nitration induced by peroxynitrite. Polyisoprenylated benzophenones present in human diet such as garcinol or guttiferone K in vitro have protective effects against lipid and protein oxidation and may have some promising effects in vivo because they are good antioxidants in the tested models in vitro. Garcinol and guttiferone K can be also useful as protecting factors against diseases associated with oxidative stress.

Peroxynitrite and fibrinolytic system: the effect of peroxynitrite on plasmin activity.

We have shown that peroxynitrite (ONOO−) inhibits streptokinase-induced conversion of plasminogen to plasmin in a concentration-dependent manner and reduces both amidolytic (IC50∼280μM at 10 μM concentration of enzyme) and proteolytic activity of plasmin. Spectrophotometric and immunoblot analysis of peroxynitrite-treated plasminogen demonstrates a concentration-dependent increase in its nitrotyrosine residues that correlates with a decreased generation of active plasmin. Peroxynitrite (1 mM) causes the nitration of 2.9 tyrosines per plasminogen molecule. Glutathione, like deferoxamine, partially protects plasminogen from peroxynitrite-induced inactivation and reduces the extent of tyrosine nitration. These data suggest that nitration of plasminogen tyrosine residues by peroxynitrite might play an important role in the inhibition of plasmin catalytic activity. (Mol Cell Biochem 267: 141–146, 2004)

Antioxidant and antiaggregatory effects of an extract from Conyza canadensis on blood platelets in vitro

The antioxidative activity of the polysaccharide extract from Conyza canadensis in blood platelets treated with peroxynitrite (ONOO−) was studied. Peroxynitrite as a strong biological oxidant has toxic effects on blood platelets and induces the oxidation of thiols, carbonylation and nitration of platelet proteins and lipid peroxidation. Therefore, the aim of our study was to assess if the natural extract from herbal plant, Conyza Canadensis, may protect platelet proteins against nitrative and oxidative damage induced by ONOO−. In our study we measured oxidative damage of platelet proteins induced by peroxynitrite and protectory effects of this extract by estimation of the level of carbonyl groups and nitrotyrosine (a marker of platelet protein nitration). We also used cytochrome c reduction method to test the ability of this extract to change generation in platelets. Moreover, we determined the effects of the extract on blood platelet aggregation induced by ADP. We observed that the extract from Conyza canadensis distinctly reduced oxidation and nitration of proteins in blood platelets treated with ONOO− (0.1 mM) and production in these cells. The extract from Conyza canadensis also inhibited platelet aggregation. The ability of the extract to decrease generation in blood platelets supports the importance of free radicals in platelet functions, including aggregation process. The present study suggests that the natural polysaccharide extract from Conyza canadensis has antiaggregatory and antioxidative activities, and therefore may be beneficial in the prevention of peroxynitrite-related diseases, such as cardiovascular or inflammatory diseases.

The protective effects of selenoorganic compounds against peroxynitrite-induced changes in plasma proteins and lipids

Many selenoorganic compounds play an important role in biochemical processes and act as antioxidants, enzyme inhibitors or drugs. The effects of a new selenocompound — bis(2-aminophenyl)-diselenide on oxidative/nitrative changes in human plasma proteins induced by peroxynitrite (ONOO−) were studied in vitro and compared with the those of ebselen, a well-known antioxidant. We also studied the role of the tested selenocompounds in peroxynitrite-induced plasma lipid peroxidation. Exposure of the plasma to peroxynitrite (0.1 mM) resulted in an increase in the level of carbonyl groups and nitrotyrosine residues in plasma proteins (estimated using the ELISA method and Western blot analysis). In the presence of different concentrations (0.025–0.1 mM) of the tested selenocompounds, 0.1 mM peroxynitrite caused a distinct decrease in the level of carbonyl group formation and tyrosine nitration in plasma proteins. Moreover, these selenocompounds also inhibited plasma lipid peroxidation induced by ONOO−1 (0.1 mM). The obtained results indicate that in vitro bis(2-aminophenyl)-diselenide and ebselen have very similar protective effects against peroxynitrite-induced oxidative/nitrative damage to human plasma proteins and lipids.

Antioxidative properties of curcumin in the protection of blood platelets against oxidative stress in vitro

The present in vitro study was designed to estimate the antioxidative activity of curcumin in the protection of human blood platelets and plasma against peroxynitrite (ONOO−)-induced oxidative stress. The effects of curcumin (12.5–50 µg/ml) on ONOO−-induced damage of proteins and lipids were determined by the estimation of protein carbonyl groups, 3-nitrotyrosine formation, and thiobarbituric acid reactive substance (TBARS) generation. Exposure of blood platelets and plasma to 100 µM ONOO− resulted in an increased level of carbonyl groups, nitration of protein tyrosine residues, and enhanced lipid peroxidation. Curcumin inhibited carbonyl group formation in plasma and in platelet proteins. The highest dose of curcumin (50 µg/ml) reduced blood platelet protein carbonylation by approximately 40%. In the protection of blood plasma protein, the lower doses of curcumin (12.5 and 25 µg/ml) were more effective. Curcumin partially prevented 3-nitrotyrosine formation in plasma proteins; the effect of curcumin was only statistically significant in blood platelets at the highest dose (50 µg/ml). The antioxidative action of curcumin in the protection against lipid peroxidation caused by ONOO− was also observed. Curcumin suppressed the formation of TBARS both in blood platelets and in plasma samples. The highest concentration of curcumin (50 µg/ml) decreased the TBARS level by approximately 35% in both blood platelets and plasma samples. In conclusion, the present study demonstrates the antioxidative properties of curcumin and its protective effects against oxidative/nitrative changes of blood platelets and plasma components, especially proteins and lipids.

The role of fibrinogen, fibrin and fibrin(ogen) degradation products (FDPs) in tumor progression

Participation of fibrin, fibrinogen, and their degradation products in pathogenesis and progression of cancer may lead to complications of thromboembolic events. The tumor may be a source of fibrinogen. Fibrinogen inside the tumor is one of the factors of its growth and metastasis. Fibrinogen, fibrin and their degradation products possess proinflammatory activity. They indirectly stimulate endothelium to secrete von Willebrand factor, leading to activation of platelets accompanying neoplastic disorders. Fragments E and D are the end products of fibrin(ogen) degradation and E and DD are the end products of stabilized fibrin. E stimulates proliferation, migration and differentiation of endothelial cells, contributing to tumor vasculature. Increased levels of DD are observed in malignant neoplasms, such as breast, lung, colon and ovary cancers. In breast cancers DD correlates with progression of disease and metastasis. The role of fibrinogen and the products of its degradation in the progression of various tumors is not sufficiently understood.

Comparison of the effect of homocysteine and its thiolactone on the fibrinolytic system using human plasma and purified plasminogen

L-Homocysteine (Hcys) occurs in human blood in free (i.e. as reduced form—about 100 nM) and protein bound form. Increased concentration of Hcys in the blood is called hyperhomocysteinemia (about [15 lM). Mechanisms involved in the relationship between hyperhomocysteinemia and haemostatic process are still unclear and sometimes controversial. In the literature there are few papers describing studies on the effects of Hcys on proteins that participate in blood coagulation and fibrinolysis in human. The most reactive form of Hcys is its cyclic thioester— homocysteine thiolactone (HTL; formed through editing mechanisms with methionyl t-RNA synthetase), which represents up to 0.29% of plasma total homocysteine, and the Hcysor tiolactone-induced modifications of haemostatic proteins (N-homocysteinylation or S-homocysteinylation) seem to be the main mechanism of biotoxicity of these compounds. Fibrinogen, plasminogen (Plg) and other haemostatic proteins can be also covalently modified by Hcys and HTL [1, 2]. The aim of our study was to establish and compare the effect of a reduced form of L-Hcys (C95%, Sigma Chemical Company, St. Louis, MO, USA) and its cyclic thioester (C99%, Sigma Chemical Company, St. Louis, MO, USA) on the fibrinolytic system (using human plasma or purified plasminogen). Blood samples were taken from 12 healthy volunteers (aged between 26 and 32 years; mean 29.7 ± 2.5) without cardiovascular disorders, allergy and lipid or carbohydrate metabolism disorders, untreated with drugs. Healthy subjects did not use addictive substances and antioxidant supplementation, their diet was balanced (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 (20009g, 15 min) to get plasma. Plg was isolated by affinity chromatography on Lysine–Sepharose [3]. The natural concentration of total Hcys in plasma was 10.6 ± 3.8 lM. The endogenous concentration of reduced form of Hcys and HTL was about 100 ± 11.2 and 0–35 nM, respectively. The classical technique HPLC has been used to analysis Hcys or HTL in human plasma. The HPLC analysis was performed with a Hewlett-Packard 1100 Series system according to Glowacki et al. [4] and Bald et al. [5]. The concentration of Plg in plasma was 2 ± 0.3 lM. Samples of human Plg (2 lM) were exposed to the reduced form of L-homocysteine (at a final concentration between 10 and 100 lM); or to homocysteine thiolactone (at a final concentration between 0.1 and 1 lM) in the presence of 100 mM potassium phosphate buffer, pH 7.4, for 30 min at 37 C. Human plasma was exposed also to the reduced form of L-homocysteine (at a final concentration between 10 and 100 lM); or to homocysteine thiolactone (at a final concentration between 0.1 and 1 lM) for 30 min at 37 C. The tested concentrations of Hcys or HTL correspond to levels found in human plasma during hyperhomocysteinemia in vivo. Plasmin activity and Plg 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 96well 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. We also studied changes of proteolytic activities of J. Kolodziejczyk J. Malinowska P. Nowak B. Olas (&) Department of General Biochemistry, Institute of Biochemistry, University of Łodź, Banacha 12/16, 90-237 Lodz, Poland e-mail: olasb@biol.uni.lodz.pl

The extract from hop cones (Humulus lupulus) as a modulator of oxidative stress in blood platelets

The plant Humulus lupulus is known as the raw material of the brewing industry. Hop cones, rich in polyphenolic compounds and acyl phloroglucides, are widely used to preserve beer and to give it a characteristic aroma and flavor. Hop cones have long been used for medicinal purposes. In particular, hop preparations were mainly recommended for the treatment of sleeping disorders. The antioxidative action of hop cones, however, is poorly understood. The aim of our present study was to investigate in vitro changes in human blood platelets induced by peroxynitrite (ONOO−, the compound of particular importance for vascular thrombosis and inflammatory process) in the presence of hop cone extract (Humulus lupulus). The antioxidative action of the extract was also compared with the properties of a well-characterized antioxidative commercial monomeric polyphenol, resveratrol (3,4′,5-trihydroxystilbene) in a model system in vitro. Various biomarkers of oxidative/nitrative stress, such as carbonyl groups, 3-nitrotyrosine and thiobarbituric acid reactive substances (TBARS) were estimated. The 3-nitrotyrosine formation and carbonyl group generation was assessed by the use of a competition ELISA test and ELISA test, respectively. Tested plant extract (12.5–50 µg/ml), like resveratrol, significantly inhibited protein carbonylation and nitration in the blood platelets treated with ONOO− (0.1 mM). The extract from hop cones, like resveratrol, also caused a distinct reduction of platelet lipid peroxidation induced by ONOO−. The present results indicate that the hope cone extract has in vitro protective effects against ONOO−, such as induced oxidative/nitrative damage to the human platelet proteins and lipids. However, in comparative studies the extract was not found to be a more effective antioxidant than the solution of pure resveratrol.

Red cabbage anthocyanins may protect blood plasma proteins and lipids

The present in vitro study was designed to examine the antioxidative activity of red cabbage anthocyanins (ATH) in the protection of blood plasma proteins and lipids against damage induced by oxidative stress. Fresh leaves of red cabbage were extracted with a mixture of methanol/distilled water/0.01% HCl (MeOH/H2O/HCl, 50/50/1, v/v/w). Total ATH concentration [µM] was determined with cyanidin 3-glucoside as a standard. Phenolic profiles in the crude red cabbage extract were determined using the HPLC method. Plasma samples were exposed to 100 µM peroxynitrite (ONOO−) or 2 mM hydrogen peroxide (H2O2) in the presence/absence of ATH extract (5–15 µM); oxidative alterations were then assessed. Pre-incubation of plasma with ATH extract partly reduced oxidative stress in plasma proteins and lipids. Dose-dependent reduction of both ONOO− and H2O2-mediated plasma protein carbonylation was observed. ATH extract partly inhibited the nitrative action of ONOO−, and significantly decreased plasma lipid peroxidation caused by ONOO− or H2O2. Our results demonstrate that anthocyanins present in red cabbage have inhibitory effects on ONOO− and H2O2-induced oxidative stress in blood plasma components. We suggest that red cabbage ATH, as dietary antioxidants, should be considered as potentially usable nutraceuticals in the prevention of oxidative stress-related diseases.