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l- carnitine protects plasma components against oxidative alterations
Nutrition 27 (2011) 693–699
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Basic nutritional investigation L-carnitine
protects plasma components against oxidative alterations
Joanna Kolodziejczyk Ph.D., Joanna Saluk-Juszczak Ph.D. *, Barbara Wachowicz Ph.D. Department of General Biochemistry, University of Lodz, Lodz, Poland
a r t i c l e i n f o
a b s t r a c t
Article history: Received 8 January 2010 Accepted 11 June 2010
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 mM) 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 mM 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. Ó 2011 Elsevier Inc. All rights reserved.
Keywords: L-Carnitine Oxidative stress Plasma proteins 3-Nitrotyrosine Carbonyl groups Thiols
Introduction Dietary supplements, antioxidants, and other nutrients play an important role in human body metabolism. It has been recognized that excess of some well-known nutrients may have potential hazardous effects; however, the molecular mechanisms of their biological activity are only partly understood. L-Carnitine (g-trimethylamine-b-hydroxybutyric acid) is widely used within the popular fitness media and athletic world, largely without scientific support. There is evidence for a beneficial effect of its supplementation in training, competition, and recovery from strenuous exercise and in regenerative athletics [1]. The function of L-carnitine in the transport of fatty acids into the mitochondrial matrix is well defined [2,3]. It may also
* Corresponding author. Tel./fax: þ48-42-635-4484. E-mail address: [email protected] (J. Saluk-Juszczak). 0899-9007/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.nut.2010.06.009
be involved in many biological processes. Recent data have indicate that L-carnitine also plays a role in the regulation of gene transcription, inhibits blood platelet aggregation, stimulates erythropoiesis, and acts as a radical scavenger [4]. Our previous study showed that carnitine modulates blood platelet function and metabolism [5]. Its effects on plasma components, blood coagulation, and fibrinolysis have not been studied. Oral L-carnitine supplementation is frequently reported to have beneficial effects on exercise capacity in clinical populations, but many experimental studies have been poorly controlled and results difficult to compare [6]. Thus, the explanation of the mechanisms of L-carnitine action is essential for the safety of its supplementation. The plasma components are continuously exposed to reactive oxygen species (ROS) and reactive nitrogen species (RNS) action. They may be injured as a result of reperfusion after ischemia and chronic ROS generation related to inflammatory processes [7–9]. In the pathogenesis of many diseases, the inflammatory
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processes, expression of proinflammatory mediators, and generation ROS and RNS are involved [10]. The overproduction of ROS in healthy individuals as an effect of variety of factors, including physical exercise, may also occur. During physical exercise the consumption of oxygen is increased and can result in an acute state of oxidative stress. Mitochondrial respiration and electron leakage from the electron transport chain (with subsequent production of the superoxide radical), prostanoid metabolism, the auto-oxidation of catecholamines, or oxidase activity (reduced nicotinamide adenine dinucleotide phosphate oxidase and xanthine oxidase) are the primary sources of increased ROS generation. Moreover, this initial increase in ROS during exercise may lead to an additional generation of prooxidants by phagocytic respiratory burst and amplified oxidative stress [11]. The plasma defense mechanisms against oxidative stress that result from an imbalance of pro-oxidants and antioxidants [12] and the function of exogenous antioxidants are very important for biological activities of human plasma components. Exercise in a general sense has always been associated with health, but strenuous exercise taken to the extreme initiates an immune and vascular proinflammatory situation. Under these conditions ROS and RNS are formed and peroxynitrite, a potent biological oxidant, is generated in the vessel wall [13]. Peroxynitrite is a highly reactive and strong oxidative species that has been implicated in the pathology of many diseases [14]. This reactive product from the rapid, diffusionally controlled reaction of nitric oxide with superoxide anion is capable of inducing oxidative/ nitrative changes in a wide variety of biomolecules [15]. Dietary supplements (including L-carnitine) are often used to foster exercise performance but may be associated with excessive body weight loss [16]. Therefore, our experiments were designed to investigate the role of L-carnitine (as a supplemented compound) in the protection of blood plasma proteins and lipids against oxidative/nitrative damage caused by peroxynitrite formed in vivo in the vessel wall during oxidative stress. We used in our experiments in vitro a very strong physiologic oxidant (peroxynitrite [ONOO]) that is formed in the vascular system and wanted to establish if L-carnitine can combat oxidative stress. Materials and methods Materials Human blood was obtained from healthy young (20–25 y) non-smoking men (n ¼ 10) who had fasted for at least 12 h. Blood was collected separately into a solution of citric acid, citrate, and dextrose (5:1, v/v) and centrifuged (3000 g, 15 min) to obtain plasma. Human plasma was preincubated (2 min, 37 C) with L-carnitine (final concentration 0.1–100 mM) and then the ONOO solution was added. For 3-nitrotyrosine (3-NT) immunodetection by a competition enzyme-linked immunosorbent assay (c-ELISA), streptavidin and biotinylated horseradish peroxidase complex/horseradish peroxidase polyclonal swine, anti-goat, mouse, rabbit immunoglobulins (Multi-Link, Dako, Denmark) were used. Anti–3-NT polyclonal antibodies were obtained from Abcam and o-phenylenediamine dihydrochloride (peroxidase substrate) was purchased from Sigma (St. Louis, MO, USA). All other reagents were of analytical grade and were provided by commercial suppliers.
Methods Synthesis of ONOO and treatment of plasma with ONOO Peroxynitrite was synthesized according to the method described by Pryor et al. [17]. Freeze fractionation (70 C) of the peroxynitrite solution formed a yellow top layer, which was retained for further studies. The top layer typically contained 80 to 100 mM peroxynitrite, as determined spectrophotometrically at 302 nm in 0.1 M NaOH (l 302 nm ¼ 1679 M/cm). Human plasma samples were exposed to ONOO for 15 min at room temperature. Some experiments were also performed with decomposed ONOO, which was prepared by allowing the
ONOO to decompose at neutral pH (7.4) in 100 mM potassium phosphate buffer (60 min, room temperature). Estimation of carbonyl groups in plasma proteins by colorimetric method Plasma samples for carbonyl groups detection were precipitated on ice with cold trichloroacetic acid (final concentration 20%) and centrifuged for 5 min and then 1 mL of 10 mM 2,4-dinitrophenylhydrazine (DNPH) in 2 M HCl was added to obtain a 1-mg/mL solution of protein. To corresponding samples containing reagent blanks, 1 mL of 2 M HCl was inserted. The samples were placed in the dark for 1 h at room temperature and mixed every 10 min. Then they were precipitated with trichloroacetic acid to a final concentration of 10% and centrifuged for 5 min. Supernatants were removed and the sediment of proteins was washed with 10% trichloroacetic acid and then washed three times with 1 mL of an ethanol/ethyl acetate mixture (v/v, 1:1) to remove unbound DNPH. Samples were resuspended in 6 M guanidine hydrochloride (in 2 M HCl) for 15 min with vortex mixing. Carbonyl content was determined at 366 nm (3 ¼ 22 000 M/cm) [18]. Immunodetection of 3-NT by c-ELISA Detection of nitrotyrosine-containing proteins by a c-ELISA method in plasma was carried out according to a modified method, described by Khan et al. [19]. Wells of a 96-well microtiter dish were coated with 100 mL of nitro-fibrinogen (nitro-Fg; concentration 1 mg/mL and 10 mol of 3-NT/mol of protein) overnight at 4 C. The linearity of the c-ELISA method was confirmed by the construction of a standard curve ranging from 10 to 1000 nM nitro-Fg equivalents. Concentrations of nitrated proteins that inhibit anti-nitrotyrosine antibody binding were estimated from the standard curve and expressed as nitro-Fg equivalents. Determination of thiols A high-performance liquid chromatographic (HPLC) technique was used to analyze low-molecular-weight thiols (glutathione [GSH], cysteine, and homocysteine) from human plasma treated with L-carnitine and/or ONOO. HPLC analysis was performed with a Hewlett-Packard 1100 system (Hewlett-Packard, NJ, USA) according to the method of Glowacki et al. [20] and Bald et al. [21]. Free total thiol groups in plasma proteins with 5,50 -dithio-bis(2-nitro-benzoic acid) were determined [22]. Production of thiobarbituric acid-reactive substances in human plasma Incubation of plasma (control samples and samples treated with peroxynitrite and/or L-carnitine) was stopped by cooling the samples in an ice bath. Samples of plasma were transferred to an equal volume of 20% (v/v) cold trichloroacetic acid in 0.6 M HCl and centrifuged at 1200 g for 15 min. One volume of clear supernatant was mixed with 0.2 vol of 0.12 M thiobarbituric acid in 0.26 M Tris at pH 7.0 and immersed in a boiling-water bath for 15 min. Absorbance at 532 nm was measured and results were expressed as nanomoles of thiobarbituric-acid reactive substances per milliliter of plasma [23]. Statistical analysis To eliminate any uncertain data, the Q-Dixon test was performed. The statistical analysis was performed with one-way analysis of variance for repeated measurements. Statistically significant differences were also assessed by applying Student’s paired t test. P < 0.05 was considered statistically significant.
Results The results demonstrated that L-carnitine added to plasma at concentrations of 0.1 to 100 mM inhibits in vitro the oxidation of blood plasma proteins (measured as levels of carbonyl groups and protein thiols) caused by treatment of human plasma with 100 mM ONOO. In plasma samples without the added L-carnitine and treated with ONOO only, the level of carbonyl groups was assumed as 100%. We observed that the level of carbonyl groups in the presence of added L-carnitine (0.1–10 mM) was decreased by about 40% compared with plasma samples treated only with ONOO (P < 0.01; Fig. 1). L-Carnitine at concentration of 100 mM did not change peroxynitrite-induced carbonyl group formation (P > 0.05; Fig. 1). To determine whether added L-carnitine could protect tyrosine residues in plasma proteins against ONOO-induced nitration, we used a c-ELISA method. L-Carnitine (0.1–100 mM) effectively prevented peroxynitrite-induced nitration of tyrosine residues in plasma proteins (0.1–10 mM, P < 0.01; 100 mM, P < 0.001; Fig. 2). In plasma samples treated with ONOO only
plasma protein carbonyl groups [%]
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695
140 120 100
*
*
80
*
60 40 20 0
0
0.1
1
10
100
Concentrations of carnitine [µM] Fig. 1. Effects of L-carnitine (0.1–100 mM) added to plasma on carbonyl group formation (protein oxidation) induced by peroxynitrite (ONOO; 100 mM). Results are expressed as percentage of 100 mM ONOO-induced plasma carbonyl group formation. Results are representative of four independent experiments done in triplicate and are expressed as mean SD. * Effects were statistically significant according to Student’s paired t test: L-carnitine (0.1–10 mM) þ ONOO treated plasma versus ONOO treated plasma (P < 0.01).
(without added L-carnitine), about 0.25 to 0.30 mmol of 3-NT-Fg equivalent per milligram of plasma protein was formed and was assumed as 100%. L-Carnitine decreased the level of 3-NT by about 40% to 60% (Fig. 2). Incubation of plasma with peroxynitrite resulted in a decrease of protein thiols. The added L-carnitine (0.1–100 mM) had a protective effect on the lower level of SH groups in plasma proteins (P < 0.05; Fig. 3). The presence of added L-carnitine at concentrations of 1 to 100 mM also protected SH groups of GSH against the oxidation induced by peroxynitrite (P < 0.05; Fig. 4); the lowest concentration of L-carnitine (0.1 mM) had no antioxidative effect. The estimation of homocysteine and cysteine levels in plasma after ONOO treatment demonstrated that L-carnitine caused increases in homocysteine (P < 0.05; Fig. 5) and cysteine (P < 0.05; Fig. 6) levels. The effect of 100 mM L-carnitine on the cysteine level was statistically insignificant (P > 0.05; Fig. 6).
The L-carnitine (0.1–10 mM) added to plasma had inhibitory effects on the lipid peroxidation caused by peroxynitrite and was measured as the production of thiobarbituric acid-reactive substances (P < 0.02; Fig. 7). In plasma incubated with the highest concentration of L-carnitine (100 mM), its inhibitory effect was not observed (P > 0.05; Fig. 7). Discussion Overproduction of ROS/RNS under pathophysiologic conditions plays an important role in the pathogenesis of different diseases, including cardiovascular diseases and vasculopathies [24]. The rapid reaction between O2 and NO leads to the generation of the strong oxidative and nitrative factor, peroxynitrite (ONOO), that is responsible for many oxidative/nitrative alterations of biomolecules [25]. Because peroxynitrite is formed in the cardiovascular system in vivo, we used this nitrative and
120
3-nitrotyrosine [%]
100 80
*
*
*
60
*
40 20 0
0
0.1
1
10
100
Concentrations of carnitine [µM] Fig. 2. Effects of L-carnitine (0.1–100mM) added to plasma on nitration of tyrosine residues induced by peroxynitrite (ONOO; 100 mM) measured by competitive enzymelinked immunosorbent assay. Results are expressed as a percentage of that recorded for 100 mM ONOO-induced plasma 3-nitrotyrosine formation. Results are representative of three independent experiments done in triplicate and are expressed as mean SD. * Effects were statistically significant according to Student’s paired t test: L-carnitine (0.1–100 mM) þ ONOO-treated plasma versus ONOO-treated plasma (L-carnitine 0.1–10 mM, P < 0.01; 100 mM, P < 0.001).
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nmol -SH protein groups/ml of plasma
696
500
*
*
*
*
ONOO
-
400
without with
300 200 100 0
0
0.1
1
10
100
Concentrations of carnitine [µM] Fig. 3. Effects of L-carnitine (0.1–100mM) added to plasma on total free thiols in the absence or presence of ONOO (100 mM). Results are expressed as nanomoles of thiols per milliliter of plasma. Results are representative of four independent experiments done in triplicate and are expressed as mean SD. * Effects were statistically significant according to Student’s paired t test: L-carnitine (0.1–100 mM) þ ONOO-treated plasma versus ONOO-treated plasma (P < 0.05). ONOO, peroxynitrite.
oxidative agent at the final concentration of 100 mM to establish a potential antioxidative action of L-carnitine added to human plasma, where L-carnitine and other endogenous antioxidants are present. In our previous studies we observed that peroxynitrite at this concentration induces the nitration of tyrosine residues in plasma proteins, formation of protein carbonyl groups, and thiol oxidation [26]. It has been estimated that the bolus addition of 250 mM ONOO is roughly equivalent to a steady-state level of 1 mM maintained for 7 min [27]. These concentrations could be readily formed in the blood vessel, where production rates of NO and superoxide radicals considerably increase [28,29]. L-Carnitine is biosynthesized from the amino acids lysine and methionine in the liver and kidney, but its significant amount derives from dietary intake, predominantly from food made from animals. It is required for the transport of long-chain fatty acids from the cytosol into the mitochondria, where they undergo
b-oxidation for the generation of metabolic energy. In the human body skeletal muscles are the main reservoir of L-carnitine. In blood plasma L-carnitine at concentrations of 41 mM (females) and 51 mM (males) is present [30]. Free L-carnitine constitutes about 80% of this amount; the rest (20%) includes short-chain acylcarnitines acetylcarnitine and propionyl-L-carnitine [31]. Oral or parenteral supplementation is effective under pathologic conditions, when the carnitine amount is lower, to maintain tissue and plasma levels of carnitine [32]. Plasma L-carnitine concentration positively correlates with its dietary intake. However, the determination of L-carnitine content in foodstuff is based on old and probably inadequate methodology [33]. Although the role of L-carnitine as an essential cofactor for mitochondrial transport and b-oxidation is well established, its other physiologic effects have recently been described. The biological activity of L-carnitine includes its antioxidative properties; however, the mechanism of its activity as a typical
nmol GSH groups/ml of plasma
8
* 6
*
ONOOwithout
*
with 4
2
0
0
0.1
1
10
100
Concentrations of carnitine [µM] Fig. 4. Effects of L-carnitine (0.1–100 mM) added to plasma on the level of plasma GSH measured by high-performance liquid chromatography (in the absence of ONOO or in the presence of 100 mM ONOO). The level of SH glutathione groups was measured by high-performance liquid chromatography. Results are expressed as nanomoles of GSH per milliliter of plasma, are representative of four independent experiments done in triplicate, and are expressed as mean SD. * Effects were statistically significant according to Student’s paired t test: L-carnitine (0.1–10 mM) treated plasma versus control (P < 0.05). The effect of 100 mM L-carnitine was not statistically significant. GSH, glutathione; ONOO, peroxynitrite.
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697
nmol HCSH/ml of plasma
12
*
10
*
ONOO-
*
* 8
without with
6 4 2 0 0
0.1
1
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Concentrations of carnitine [µM] Fig. 5. Effects of L-carnitine (0.1–100 mM) added to plasma on the level of HCSH, measured by high-performance liquid chromatography in plasma in the absence or presence of ONOO (100 mM). Results are expressed as nanomoles of HCSH per milliliter of plasma. Results are representative of four independent experiments done in triplicate and are expressed as mean SD. * Effects were statistically significant according to Student’s paired t test: L-carnitine (0.1–100 mM) þ ONOO-treated plasma versus ONOOtreated plasma (P < 0.05). HCSH, homocysteine; ONOO, peroxynitrite.
antioxidant is only partly recognized. A study by Derin et al. [34] suggested that the antioxidative action of L-carnitine might be a result of scavenging and inhibition of hydroxyl radical formation in the Fenton reaction system. Data from a clinical study support the hypothesis that carnitine favorably modulates oxidative stress, most likely by preventing membrane fatty acid peroxidation [35]. Arduini et al. [36] reported that the L and D forms of carnitine have similar non-enzymatic free radical scavenging activities [36]. According to Sayed-Ahmed et al. [37], only L-carnitine prevented the progression of atherosclerotic lesions. The protective effects of L-carnitine against damage to the heart caused by diabetes-induced alterations and additional ischemia and reperfusion in diabetic rats have been described by Schneider et al. [38]. The L-carnitine may be an important agent in the protection of myocardial alterations in diabetes with additional ischemia and reperfusion, because it stabilizes mitochondrial and cellular functions and acts through its antioxidative or radical scavenging potential [38].
These observations are very helpful in understanding the molecular basis of L-carnitine supplementation. Nevertheless, the available data are too general to provide L-carnitine as an antioxidative therapeutic agent in the clinical routine. For example, the effect of L-carnitine on blood plasma components is poorly understood. Our designed in vitro experiments for the first time have established the effect of L-carnitine as an antioxidant in the protection of plasma proteins and lipids against oxidative stress caused by ONOO. We studied the effects of L-carnitine on peroxynitrite-induced modifications of human plasma proteins and lipids in vitro, because the generation of ROS/RNS in the vascular system is responsible for the structural and functional changes of these plasma components [39–41]. The nitration of tyrosine residues, leading to 3-NT formation, is an established marker of ONOO generation and NO action [42,43]. Our previous studies demonstrated that peroxynitrite significantly increased carbonyl groups formation and induced 3-NT generation in blood plasma
6
nmol CSH/ml of plasma
ONOO-
* 4
*
without
*
with
2
0 0
0.1
1
10
100
Concentrations of carnitine [µM] Fig. 6. Effects of L-carnitine (0.1–100mM) added to plasma on the level of CSH in plasma in the absence or presence of ONOO (100 mM). Results are expressed as nanomoles of CSH per milliliter of plasma. Results are representative of four independent experiments done in triplicate and are expressed as mean SD. * Effects were statistically significant according to Student’s paired t test: L-carnitine (0.1–10 mM) þ ONOO-treated plasma versus ONOO-treated plasma (P < 0.05). The effect of 100 mM L-carnitine was not statistically significant. CSH, cysteine; ONOO, peroxynitrite.
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nmol TBARS/ml of plasma
2
*
ONOO-
* without
*
with
* 1
0 0
0.1
1
10
100
Concentrations of carnitine [µM] Fig. 7. Effects of carnitine (0.1–100 mM) added to plasma on the level of TBARS in human blood plasma in the absence or presence of ONOO (100 mM). Data represent mean SEM of five independent experiments done in triplicate. * Effects of L-carnitine at the concentration of 0.1–10 mM on the level of TBARS in blood plasma were statistically significant according to one-way analysis of variance (P < 0.02). The effect of 100 mM L-carnitine was not statistically significant (P > 0.05). ONOO, peroxynitrite; TBARS, thiobarbituric-acid reactive substances.
proteins. The different dietary antioxidants may protect plasma components against nitration and oxidation caused by peroxynitrite [44]. The results of this study indicate that L-carnitine possesses antioxidative and antinitrative properties and prevents the nitration of tyrosine residues in plasma proteins caused by ONOO. L-Carnitine (0.1–100 mM) effectively decreased the 3-NT and carbonyl group generation in plasma proteins, but the inhibitory effects were not dose-dependent. The oxidation of protein thiols to mixed disulfides is an early response to oxidative stress [45]. In our experiments 100 mM ONOO added to plasma resulted in the rapid decrease (>50%) of the level of total plasma protein thiols (Fig. 3), indicating that free thiols form disulfide bonds. In the presence of L-carnitine the level of SH groups was significantly higher. It indicates that Lcarnitine (0.1–100 mM) added to plasma protects against thiol oxidation (Fig. 3). Protein thiols act as a redox buffer; their concentration is much greater than those of low-molecularweight thiols [46]. The alteration of thiol/disulfide status in plasma seems to correspond to the level of plasma lowmolecular-weight thiols. Protein-bound thiols are highly unstable and can be present as free thiols, disulfides, and mixed disulfides when conjugated with GSH, cysteine, homocysteine, and g-glutamylcysteine. Plasma GSH level, in contrast to cell level, is very low and GSH becomes oxidized in response to peroxynitrite. GSH is a dominant ligand of protein thiols and their reactivity with GSH depends on thiol/disulfide redox states. The alteration of thiol/disulfide status in plasma seems also to correspond to the level of plasma homocysteine [46]. We observed that L-carnitine protects low-molecular-weight thiols (GSH, homocysteine, and cysteine) against oxidation induced by peroxynitrite (Figs. 4–6). L-Carnitine modulates blood platelet activation through antioxidant mechanisms and inhibition of arachidonic acid cascade [5,35]. Our results confirm the protective activity of L-carnitine against plasma lipid peroxidation. Augustyniak et al. [47] reported that L-carnitine also protects the lipid and protein parts of low-density lipoprotein particles against oxidative modifications [47]. The administration of L-carnitine to rats intoxicated with ethanol significantly protected lipids against oxidative modifications in blood serum and the liver [48].
The results of Volek et al. [49] provided indirect evidence of a positive effect of L-carnitine supplementation on the significant inhibition of free radical formation. However, another study in vivo showed that carnitine supplementation has little impact on exercise-induced oxidative stress biomarkers [50]. In conclusion, L-carnitine has antioxidative properties and protects plasma components against the toxic action of ROS and RNS. The beneficial effects of L-carnitine as a diet supplement seem to depend on its antioxidative activity. However, supplementation at very high doses (100 mM in our in vitro experiments) of L-carnitine, which is the most popular and favored supplement among athletes, seems to be less effective. Further study in vivo is required. References [1] Karlic H, Lohninger A. Supplementation of L-carnitine in athletes: does it make sense? Nutrition 2004;20:709–15. [2] Carter AL, Abney TO, Lapp DF. Biosynthesis and metabolism of carnitine. J Child Neurol 1995;10(suppl 2):S3–7. [3] Walter P, Schaffhauser AO. L-carnitine, a ‘vitamin-like substance’ for functional food. Ann Nutr Metab 2000;44:75–96. [4] Lohninger A, Pittner G, Pittner F. L-carnitine: new aspects of a known compoundda brief survey. Monatsh Chem 2005;136:1255–68. [5] Saluk-Juszczak J, Olas B, Wachowicz B, Glowacki R, Bald E. L-carnitine modulates blood platelet oxidative stress. Cell Biol Toxicol 2010;21:37–43. [6] Galloway SDR, Broad EM. Oral L-carnitine supplementation and exercise metabolism. Monatsh Chem 2005;136:1391–410. [7] Sorg J. Oxidative stress: a theoretical model or a biological reality? CR Biologies 2004;327:649–62. [8] Matsuoka H. Endothelial dysfunction associated with oxidative stress in human. Diabetes Res Clin Pract Suppl 2001;2:S65–72. [9] Griendling KK, FitzGerald GA. Oxidative stress and cardiovascular injury. Part I: Basic mechanisms and in vivo monitoring of ROS. Circulation 2003;108:1912–6. [10] Esmon CT. Crosstalk between inflammation and thrombosis. Maturitas 2004;47:305–14. [11] Jackson MJ. Exercise and oxygen radical production by muscle. In: Sen CK, Packer L, Hanninen O, editors. Handbook of oxidants and antioxidants in exercise. Amsterdam: Elsevier Science; 2000. p. 57–68. [12] Kinnunen S, Atalay M, Hyyppä S, Lehmuskero A, Hänninen O, Oksala N. Effects of prolonged exercise on oxidative stress and antioxidant defence in endurance horse. J Sports Sci Med 2005;4:415–21. [13] Pedersen BK, Hoffman-Goetz L. Exercise and the immune system: regulation, integration, and adaptation. Physiol Rev 2000;2:1055–81. [14] Mazzulli JR, Hodara R, Summer L, Ischiropoulos H. Oxidative stress and protein deposition diseases. Protein Rev 2007;24:123–33.
J. Kolodziejczyk et al. / Nutrition 27 (2011) 693–699 [15] Reiter CD, Teng RJ, Beckman JS. Reaction of superoxide and nitric with peroxynitrite. J Biol Chem 2000;275:32460–6. [16] Stefano GB, Prevo V, Cadet P, Dardik I. Vascular pulsations stimulating nitric oxide release during cyclic exercise may benefit health: a molecular approach. Int J Mol Med 2001;7:119–29. [17] Pryor WA, Cueto R, Jin X, Koppenol WH, Ngu-Schwemlein M, Squadrito Gl, et al. A practical method for preparing peroxynitrite solutions of low ionic strenght and free of hydrogen peroxide. Free Radic Biol Med 1995;1:75–83. [18] Levine RL, Williams JA, Stadtman ER, Shacter E. Carbonyl assays for determination of oxidatively modified proteins. Methods Enzymol 1994;233:346–57. [19] Khan J, Brennan DM, Bradley N, Gao B, Bruckdorfer R, Jacobs M. 3-Nitrotyrosine in the proteins of human plasma determined by an ELISA method. Biochem J 1998;330:795–801. [20] Glowacki R, Wójcik K, Bald E. Facile and sensitive method for the determination of mesna in plasma by high-performance liquid chromatography with ultraviolet detection. J Chromatogr 2001;914:29–35. [21] Bald E, Chwatko G, Glowacki R, Kusmierek K. Analysis of plasma thiols by high-performance liquid chromatography with ultraviolet detection. J Chromatogr 2004;1032:109–15. [22] Rice-Evans CA, Diplock AT, Symons MCR. Footprints of protein damage. In: Techniques in free radical research. Amsterdam: Elsevier; 1991, p 185–94. [23] Wachowicz B, Kustron J. Effect of cisplatin on lipid peroxidation in pig blood platelets. Cytobios 1992;70:41–7. [24] Madamanchi NR, Vendrov A, Runge MS. Oxidative stress and vascular disease. Arteroscler Thromb Vasc Biol 2005;25:29–38. [25] Dedon PC, Tannenbaum SR. Reactive nitrogen species in the chemical biology of inflammation. Arch Biochem Biophys 2004;423:12–22. [26] Nowak P, Saluk-Juszczak J, Olas B, Ko1odziejczyk J, Wachowicz B. The protective effects of selenoorganic compounds against peroxynitrite-induced changes in plasma proteins and lipids. Cell Mol Biol Lett 2006;11:1–11. [27] Beckmann JS, Chen J, Ischiropoulos H, Crow JP. Oxidative chemistry of peroxynitrite. Methods Enzymol 1994;233:229–40. [28] Bartosz G. Peroxynitrite: mediator of the toxic action of nitric oxide. Acta Biochim Pol 1996;43:645–59. [29] Soszynski M, Bartosz G. Effect of peroxynitrite on erythrocytes. Biochim Biophys Acta 1996;1291:107–14. [30] Ramsay RR, Gandour RD, van der Leij FR. Molecular enzymology of carnitine transfer and transport. Biochim Biophys Acta 2001;1546:21–43. [31] Cerretelli P, Marconi C. L-carnitine supplementation in humans. The effects on physical performance. Int J Sports Med 1990;11:1–14. [32] Brevetti G, Chiariello M, Ferulano G, Policicchio A, Nevola E, Rossini A, et al. Increases in walking distance in patients with peripheral vascular disease treated with L-carnitine: a double-blind, cross-over study. Circulation 1988;77:767–73. [33] Steiber A, Kerner J, Hoppel CL. Carnitine: a nutritional, biosynthetic and functional perspective. Mol Aspects Med 2004;25:455–73. [34] Derin N, Izgut-Uysal VN, Agac A, Aliciguzel Y, Demir N. L-carnitine protects gastric mucosa by decreasing ischemia reperfusion induced lipid peroxidation. J Physiol Pharmacol 2004;55:595–606.
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[35] Malaguarnera M, Vacante M, Avitabile T, Malaguarnera M, Cammalleri L, Motta M. L-Carnitine supplementation reduces oxidized LDL cholesterol in patients with diabetes. Am J Clin Nutr 2009;89:71–6. [36] Arduini A, Dottori S, Molajoni F, Kirk R, Martelli EA. Is the carnitine system part of the heart antioxidant network? In: Ferrari R, Dimauro S, Sherwood G, editors. L-carnitine and its role in medicine: from function to therapy. London: Academic Press; 1992. p. 169–81. [37] Sayed-Ahmed M, Khattab MM, Gad MZ, Mostafa N. L-carnitine prevents the progression of atherosclerotic lesions in hypercholesterolaemic rabbits. Pharmacol Res 2001;3:235–42. [38] Schneider R, Löster H, Aust W, Craatz S, Welt K, Fitzl G. Protective effects of L-carnitine on myocardium of experimentally diabetic rats with additional ischaemia and reperfusion. Monatsh Chem 2005;136:1467–81. [39] Davies MJ, Fu S, Wang H, Dean RT. Stable markers of oxidant damage to protein and their application in the study of human disease. Free Radic Biol Med 1999;11/12:1151–63. [40] Madamanchi NR, Vendrov A, Rune MS. Oxidative stress and vascular disease. Arterioscler Thromb Vasc Biol 2005;25:29–38. [41] Patel RP, McAndrew J, Sellak H, White R, Jo H, Freeman BA, DarleyUsmar VM. Biological aspects of reactive nitrogen species. Biochim Biophys Acta 2003;1411:385–400. [42] Reiter ChD, Teng R-J, Beckman JS. Superoxide reacts with nitric oxide to nitrate tyrosine at physiological pH via peroxynitrite. J Biol Chem 2000;42:32460–6. [43] Ischiropoulos H. Biological tyrosine nitration: a pathophysiological function of nitric oxide and reactive oxygen species. Arch Biochem Biophys 1998;1:1–11. [44] Olas B, Nowak P, Kolodziejczyk J, Ponczek M, Wachowicz B. Protective effects of resveratrol against oxidative/nitrative modifications of plasma proteins and lipids exposed to peroxynitrite. J Nutr Biochem 2006;17: 96–102. [45] Hansen JM, Choe H-S, Carney EW, Harris C. Differential antioxidant enzyme activities and glutathione content between rat and rabbit conceptuses. Free Radic Biol Med 2001;10:1078–88. [46] Schafer RQ, Buettner GR. Redox environment of the cell as viewed through the redox state of the glutathione disulfide/glutathione couple. Free Radic Biol Med 2001;11:1191–212. [47] Augustyniak A, Stankiewicz A, Skrzydlewska E. The influence of L-carnitine on oxidative modification of LDL in vitro. Toxicol Mech Methods 2008;18:455–62. [48] Augustyniak A, Skrzydlewska E. L-carnitine in the lipid and protein protection against ethanol-induced oxidative stress. Alcohol 2009;43: 217–23. [49] Volek JS, Kramer WJ, Rubin MR, Gomez AL, Ratamess NA, Gaynor P. L-carnitine L-tartrate supplementation favorably affects markers of recovery from exercise stress. Am J Physiol Endocrinol Metab 2002;282:E474–82. [50] Bloomer RJ, Smith WA. Oxidative stress in response to aerobic and anaerobic power testing: influence of exercise training and carnitine supplementation. Res Sports Med 2009;1:1–16.
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Basic nutritional investigation L-carnitine
protects plasma components against oxidative alterations
Joanna Kolodziejczyk Ph.D., Joanna Saluk-Juszczak Ph.D. *, Barbara Wachowicz Ph.D. Department of General Biochemistry, University of Lodz, Lodz, Poland
a r t i c l e i n f o
a b s t r a c t
Article history: Received 8 January 2010 Accepted 11 June 2010
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 mM) 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 mM 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. Ó 2011 Elsevier Inc. All rights reserved.
Keywords: L-Carnitine Oxidative stress Plasma proteins 3-Nitrotyrosine Carbonyl groups Thiols
Introduction Dietary supplements, antioxidants, and other nutrients play an important role in human body metabolism. It has been recognized that excess of some well-known nutrients may have potential hazardous effects; however, the molecular mechanisms of their biological activity are only partly understood. L-Carnitine (g-trimethylamine-b-hydroxybutyric acid) is widely used within the popular fitness media and athletic world, largely without scientific support. There is evidence for a beneficial effect of its supplementation in training, competition, and recovery from strenuous exercise and in regenerative athletics [1]. The function of L-carnitine in the transport of fatty acids into the mitochondrial matrix is well defined [2,3]. It may also
* Corresponding author. Tel./fax: þ48-42-635-4484. E-mail address: [email protected] (J. Saluk-Juszczak). 0899-9007/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.nut.2010.06.009
be involved in many biological processes. Recent data have indicate that L-carnitine also plays a role in the regulation of gene transcription, inhibits blood platelet aggregation, stimulates erythropoiesis, and acts as a radical scavenger [4]. Our previous study showed that carnitine modulates blood platelet function and metabolism [5]. Its effects on plasma components, blood coagulation, and fibrinolysis have not been studied. Oral L-carnitine supplementation is frequently reported to have beneficial effects on exercise capacity in clinical populations, but many experimental studies have been poorly controlled and results difficult to compare [6]. Thus, the explanation of the mechanisms of L-carnitine action is essential for the safety of its supplementation. The plasma components are continuously exposed to reactive oxygen species (ROS) and reactive nitrogen species (RNS) action. They may be injured as a result of reperfusion after ischemia and chronic ROS generation related to inflammatory processes [7–9]. In the pathogenesis of many diseases, the inflammatory
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processes, expression of proinflammatory mediators, and generation ROS and RNS are involved [10]. The overproduction of ROS in healthy individuals as an effect of variety of factors, including physical exercise, may also occur. During physical exercise the consumption of oxygen is increased and can result in an acute state of oxidative stress. Mitochondrial respiration and electron leakage from the electron transport chain (with subsequent production of the superoxide radical), prostanoid metabolism, the auto-oxidation of catecholamines, or oxidase activity (reduced nicotinamide adenine dinucleotide phosphate oxidase and xanthine oxidase) are the primary sources of increased ROS generation. Moreover, this initial increase in ROS during exercise may lead to an additional generation of prooxidants by phagocytic respiratory burst and amplified oxidative stress [11]. The plasma defense mechanisms against oxidative stress that result from an imbalance of pro-oxidants and antioxidants [12] and the function of exogenous antioxidants are very important for biological activities of human plasma components. Exercise in a general sense has always been associated with health, but strenuous exercise taken to the extreme initiates an immune and vascular proinflammatory situation. Under these conditions ROS and RNS are formed and peroxynitrite, a potent biological oxidant, is generated in the vessel wall [13]. Peroxynitrite is a highly reactive and strong oxidative species that has been implicated in the pathology of many diseases [14]. This reactive product from the rapid, diffusionally controlled reaction of nitric oxide with superoxide anion is capable of inducing oxidative/ nitrative changes in a wide variety of biomolecules [15]. Dietary supplements (including L-carnitine) are often used to foster exercise performance but may be associated with excessive body weight loss [16]. Therefore, our experiments were designed to investigate the role of L-carnitine (as a supplemented compound) in the protection of blood plasma proteins and lipids against oxidative/nitrative damage caused by peroxynitrite formed in vivo in the vessel wall during oxidative stress. We used in our experiments in vitro a very strong physiologic oxidant (peroxynitrite [ONOO]) that is formed in the vascular system and wanted to establish if L-carnitine can combat oxidative stress. Materials and methods Materials Human blood was obtained from healthy young (20–25 y) non-smoking men (n ¼ 10) who had fasted for at least 12 h. Blood was collected separately into a solution of citric acid, citrate, and dextrose (5:1, v/v) and centrifuged (3000 g, 15 min) to obtain plasma. Human plasma was preincubated (2 min, 37 C) with L-carnitine (final concentration 0.1–100 mM) and then the ONOO solution was added. For 3-nitrotyrosine (3-NT) immunodetection by a competition enzyme-linked immunosorbent assay (c-ELISA), streptavidin and biotinylated horseradish peroxidase complex/horseradish peroxidase polyclonal swine, anti-goat, mouse, rabbit immunoglobulins (Multi-Link, Dako, Denmark) were used. Anti–3-NT polyclonal antibodies were obtained from Abcam and o-phenylenediamine dihydrochloride (peroxidase substrate) was purchased from Sigma (St. Louis, MO, USA). All other reagents were of analytical grade and were provided by commercial suppliers.
Methods Synthesis of ONOO and treatment of plasma with ONOO Peroxynitrite was synthesized according to the method described by Pryor et al. [17]. Freeze fractionation (70 C) of the peroxynitrite solution formed a yellow top layer, which was retained for further studies. The top layer typically contained 80 to 100 mM peroxynitrite, as determined spectrophotometrically at 302 nm in 0.1 M NaOH (l 302 nm ¼ 1679 M/cm). Human plasma samples were exposed to ONOO for 15 min at room temperature. Some experiments were also performed with decomposed ONOO, which was prepared by allowing the
ONOO to decompose at neutral pH (7.4) in 100 mM potassium phosphate buffer (60 min, room temperature). Estimation of carbonyl groups in plasma proteins by colorimetric method Plasma samples for carbonyl groups detection were precipitated on ice with cold trichloroacetic acid (final concentration 20%) and centrifuged for 5 min and then 1 mL of 10 mM 2,4-dinitrophenylhydrazine (DNPH) in 2 M HCl was added to obtain a 1-mg/mL solution of protein. To corresponding samples containing reagent blanks, 1 mL of 2 M HCl was inserted. The samples were placed in the dark for 1 h at room temperature and mixed every 10 min. Then they were precipitated with trichloroacetic acid to a final concentration of 10% and centrifuged for 5 min. Supernatants were removed and the sediment of proteins was washed with 10% trichloroacetic acid and then washed three times with 1 mL of an ethanol/ethyl acetate mixture (v/v, 1:1) to remove unbound DNPH. Samples were resuspended in 6 M guanidine hydrochloride (in 2 M HCl) for 15 min with vortex mixing. Carbonyl content was determined at 366 nm (3 ¼ 22 000 M/cm) [18]. Immunodetection of 3-NT by c-ELISA Detection of nitrotyrosine-containing proteins by a c-ELISA method in plasma was carried out according to a modified method, described by Khan et al. [19]. Wells of a 96-well microtiter dish were coated with 100 mL of nitro-fibrinogen (nitro-Fg; concentration 1 mg/mL and 10 mol of 3-NT/mol of protein) overnight at 4 C. The linearity of the c-ELISA method was confirmed by the construction of a standard curve ranging from 10 to 1000 nM nitro-Fg equivalents. Concentrations of nitrated proteins that inhibit anti-nitrotyrosine antibody binding were estimated from the standard curve and expressed as nitro-Fg equivalents. Determination of thiols A high-performance liquid chromatographic (HPLC) technique was used to analyze low-molecular-weight thiols (glutathione [GSH], cysteine, and homocysteine) from human plasma treated with L-carnitine and/or ONOO. HPLC analysis was performed with a Hewlett-Packard 1100 system (Hewlett-Packard, NJ, USA) according to the method of Glowacki et al. [20] and Bald et al. [21]. Free total thiol groups in plasma proteins with 5,50 -dithio-bis(2-nitro-benzoic acid) were determined [22]. Production of thiobarbituric acid-reactive substances in human plasma Incubation of plasma (control samples and samples treated with peroxynitrite and/or L-carnitine) was stopped by cooling the samples in an ice bath. Samples of plasma were transferred to an equal volume of 20% (v/v) cold trichloroacetic acid in 0.6 M HCl and centrifuged at 1200 g for 15 min. One volume of clear supernatant was mixed with 0.2 vol of 0.12 M thiobarbituric acid in 0.26 M Tris at pH 7.0 and immersed in a boiling-water bath for 15 min. Absorbance at 532 nm was measured and results were expressed as nanomoles of thiobarbituric-acid reactive substances per milliliter of plasma [23]. Statistical analysis To eliminate any uncertain data, the Q-Dixon test was performed. The statistical analysis was performed with one-way analysis of variance for repeated measurements. Statistically significant differences were also assessed by applying Student’s paired t test. P < 0.05 was considered statistically significant.
Results The results demonstrated that L-carnitine added to plasma at concentrations of 0.1 to 100 mM inhibits in vitro the oxidation of blood plasma proteins (measured as levels of carbonyl groups and protein thiols) caused by treatment of human plasma with 100 mM ONOO. In plasma samples without the added L-carnitine and treated with ONOO only, the level of carbonyl groups was assumed as 100%. We observed that the level of carbonyl groups in the presence of added L-carnitine (0.1–10 mM) was decreased by about 40% compared with plasma samples treated only with ONOO (P < 0.01; Fig. 1). L-Carnitine at concentration of 100 mM did not change peroxynitrite-induced carbonyl group formation (P > 0.05; Fig. 1). To determine whether added L-carnitine could protect tyrosine residues in plasma proteins against ONOO-induced nitration, we used a c-ELISA method. L-Carnitine (0.1–100 mM) effectively prevented peroxynitrite-induced nitration of tyrosine residues in plasma proteins (0.1–10 mM, P < 0.01; 100 mM, P < 0.001; Fig. 2). In plasma samples treated with ONOO only
plasma protein carbonyl groups [%]
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140 120 100
*
*
80
*
60 40 20 0
0
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Concentrations of carnitine [µM] Fig. 1. Effects of L-carnitine (0.1–100 mM) added to plasma on carbonyl group formation (protein oxidation) induced by peroxynitrite (ONOO; 100 mM). Results are expressed as percentage of 100 mM ONOO-induced plasma carbonyl group formation. Results are representative of four independent experiments done in triplicate and are expressed as mean SD. * Effects were statistically significant according to Student’s paired t test: L-carnitine (0.1–10 mM) þ ONOO treated plasma versus ONOO treated plasma (P < 0.01).
(without added L-carnitine), about 0.25 to 0.30 mmol of 3-NT-Fg equivalent per milligram of plasma protein was formed and was assumed as 100%. L-Carnitine decreased the level of 3-NT by about 40% to 60% (Fig. 2). Incubation of plasma with peroxynitrite resulted in a decrease of protein thiols. The added L-carnitine (0.1–100 mM) had a protective effect on the lower level of SH groups in plasma proteins (P < 0.05; Fig. 3). The presence of added L-carnitine at concentrations of 1 to 100 mM also protected SH groups of GSH against the oxidation induced by peroxynitrite (P < 0.05; Fig. 4); the lowest concentration of L-carnitine (0.1 mM) had no antioxidative effect. The estimation of homocysteine and cysteine levels in plasma after ONOO treatment demonstrated that L-carnitine caused increases in homocysteine (P < 0.05; Fig. 5) and cysteine (P < 0.05; Fig. 6) levels. The effect of 100 mM L-carnitine on the cysteine level was statistically insignificant (P > 0.05; Fig. 6).
The L-carnitine (0.1–10 mM) added to plasma had inhibitory effects on the lipid peroxidation caused by peroxynitrite and was measured as the production of thiobarbituric acid-reactive substances (P < 0.02; Fig. 7). In plasma incubated with the highest concentration of L-carnitine (100 mM), its inhibitory effect was not observed (P > 0.05; Fig. 7). Discussion Overproduction of ROS/RNS under pathophysiologic conditions plays an important role in the pathogenesis of different diseases, including cardiovascular diseases and vasculopathies [24]. The rapid reaction between O2 and NO leads to the generation of the strong oxidative and nitrative factor, peroxynitrite (ONOO), that is responsible for many oxidative/nitrative alterations of biomolecules [25]. Because peroxynitrite is formed in the cardiovascular system in vivo, we used this nitrative and
120
3-nitrotyrosine [%]
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*
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40 20 0
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Concentrations of carnitine [µM] Fig. 2. Effects of L-carnitine (0.1–100mM) added to plasma on nitration of tyrosine residues induced by peroxynitrite (ONOO; 100 mM) measured by competitive enzymelinked immunosorbent assay. Results are expressed as a percentage of that recorded for 100 mM ONOO-induced plasma 3-nitrotyrosine formation. Results are representative of three independent experiments done in triplicate and are expressed as mean SD. * Effects were statistically significant according to Student’s paired t test: L-carnitine (0.1–100 mM) þ ONOO-treated plasma versus ONOO-treated plasma (L-carnitine 0.1–10 mM, P < 0.01; 100 mM, P < 0.001).
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nmol -SH protein groups/ml of plasma
696
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*
*
ONOO
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300 200 100 0
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Concentrations of carnitine [µM] Fig. 3. Effects of L-carnitine (0.1–100mM) added to plasma on total free thiols in the absence or presence of ONOO (100 mM). Results are expressed as nanomoles of thiols per milliliter of plasma. Results are representative of four independent experiments done in triplicate and are expressed as mean SD. * Effects were statistically significant according to Student’s paired t test: L-carnitine (0.1–100 mM) þ ONOO-treated plasma versus ONOO-treated plasma (P < 0.05). ONOO, peroxynitrite.
oxidative agent at the final concentration of 100 mM to establish a potential antioxidative action of L-carnitine added to human plasma, where L-carnitine and other endogenous antioxidants are present. In our previous studies we observed that peroxynitrite at this concentration induces the nitration of tyrosine residues in plasma proteins, formation of protein carbonyl groups, and thiol oxidation [26]. It has been estimated that the bolus addition of 250 mM ONOO is roughly equivalent to a steady-state level of 1 mM maintained for 7 min [27]. These concentrations could be readily formed in the blood vessel, where production rates of NO and superoxide radicals considerably increase [28,29]. L-Carnitine is biosynthesized from the amino acids lysine and methionine in the liver and kidney, but its significant amount derives from dietary intake, predominantly from food made from animals. It is required for the transport of long-chain fatty acids from the cytosol into the mitochondria, where they undergo
b-oxidation for the generation of metabolic energy. In the human body skeletal muscles are the main reservoir of L-carnitine. In blood plasma L-carnitine at concentrations of 41 mM (females) and 51 mM (males) is present [30]. Free L-carnitine constitutes about 80% of this amount; the rest (20%) includes short-chain acylcarnitines acetylcarnitine and propionyl-L-carnitine [31]. Oral or parenteral supplementation is effective under pathologic conditions, when the carnitine amount is lower, to maintain tissue and plasma levels of carnitine [32]. Plasma L-carnitine concentration positively correlates with its dietary intake. However, the determination of L-carnitine content in foodstuff is based on old and probably inadequate methodology [33]. Although the role of L-carnitine as an essential cofactor for mitochondrial transport and b-oxidation is well established, its other physiologic effects have recently been described. The biological activity of L-carnitine includes its antioxidative properties; however, the mechanism of its activity as a typical
nmol GSH groups/ml of plasma
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Concentrations of carnitine [µM] Fig. 4. Effects of L-carnitine (0.1–100 mM) added to plasma on the level of plasma GSH measured by high-performance liquid chromatography (in the absence of ONOO or in the presence of 100 mM ONOO). The level of SH glutathione groups was measured by high-performance liquid chromatography. Results are expressed as nanomoles of GSH per milliliter of plasma, are representative of four independent experiments done in triplicate, and are expressed as mean SD. * Effects were statistically significant according to Student’s paired t test: L-carnitine (0.1–10 mM) treated plasma versus control (P < 0.05). The effect of 100 mM L-carnitine was not statistically significant. GSH, glutathione; ONOO, peroxynitrite.
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Concentrations of carnitine [µM] Fig. 5. Effects of L-carnitine (0.1–100 mM) added to plasma on the level of HCSH, measured by high-performance liquid chromatography in plasma in the absence or presence of ONOO (100 mM). Results are expressed as nanomoles of HCSH per milliliter of plasma. Results are representative of four independent experiments done in triplicate and are expressed as mean SD. * Effects were statistically significant according to Student’s paired t test: L-carnitine (0.1–100 mM) þ ONOO-treated plasma versus ONOOtreated plasma (P < 0.05). HCSH, homocysteine; ONOO, peroxynitrite.
antioxidant is only partly recognized. A study by Derin et al. [34] suggested that the antioxidative action of L-carnitine might be a result of scavenging and inhibition of hydroxyl radical formation in the Fenton reaction system. Data from a clinical study support the hypothesis that carnitine favorably modulates oxidative stress, most likely by preventing membrane fatty acid peroxidation [35]. Arduini et al. [36] reported that the L and D forms of carnitine have similar non-enzymatic free radical scavenging activities [36]. According to Sayed-Ahmed et al. [37], only L-carnitine prevented the progression of atherosclerotic lesions. The protective effects of L-carnitine against damage to the heart caused by diabetes-induced alterations and additional ischemia and reperfusion in diabetic rats have been described by Schneider et al. [38]. The L-carnitine may be an important agent in the protection of myocardial alterations in diabetes with additional ischemia and reperfusion, because it stabilizes mitochondrial and cellular functions and acts through its antioxidative or radical scavenging potential [38].
These observations are very helpful in understanding the molecular basis of L-carnitine supplementation. Nevertheless, the available data are too general to provide L-carnitine as an antioxidative therapeutic agent in the clinical routine. For example, the effect of L-carnitine on blood plasma components is poorly understood. Our designed in vitro experiments for the first time have established the effect of L-carnitine as an antioxidant in the protection of plasma proteins and lipids against oxidative stress caused by ONOO. We studied the effects of L-carnitine on peroxynitrite-induced modifications of human plasma proteins and lipids in vitro, because the generation of ROS/RNS in the vascular system is responsible for the structural and functional changes of these plasma components [39–41]. The nitration of tyrosine residues, leading to 3-NT formation, is an established marker of ONOO generation and NO action [42,43]. Our previous studies demonstrated that peroxynitrite significantly increased carbonyl groups formation and induced 3-NT generation in blood plasma
6
nmol CSH/ml of plasma
ONOO-
* 4
*
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*
with
2
0 0
0.1
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Concentrations of carnitine [µM] Fig. 6. Effects of L-carnitine (0.1–100mM) added to plasma on the level of CSH in plasma in the absence or presence of ONOO (100 mM). Results are expressed as nanomoles of CSH per milliliter of plasma. Results are representative of four independent experiments done in triplicate and are expressed as mean SD. * Effects were statistically significant according to Student’s paired t test: L-carnitine (0.1–10 mM) þ ONOO-treated plasma versus ONOO-treated plasma (P < 0.05). The effect of 100 mM L-carnitine was not statistically significant. CSH, cysteine; ONOO, peroxynitrite.
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nmol TBARS/ml of plasma
2
*
ONOO-
* without
*
with
* 1
0 0
0.1
1
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Concentrations of carnitine [µM] Fig. 7. Effects of carnitine (0.1–100 mM) added to plasma on the level of TBARS in human blood plasma in the absence or presence of ONOO (100 mM). Data represent mean SEM of five independent experiments done in triplicate. * Effects of L-carnitine at the concentration of 0.1–10 mM on the level of TBARS in blood plasma were statistically significant according to one-way analysis of variance (P < 0.02). The effect of 100 mM L-carnitine was not statistically significant (P > 0.05). ONOO, peroxynitrite; TBARS, thiobarbituric-acid reactive substances.
proteins. The different dietary antioxidants may protect plasma components against nitration and oxidation caused by peroxynitrite [44]. The results of this study indicate that L-carnitine possesses antioxidative and antinitrative properties and prevents the nitration of tyrosine residues in plasma proteins caused by ONOO. L-Carnitine (0.1–100 mM) effectively decreased the 3-NT and carbonyl group generation in plasma proteins, but the inhibitory effects were not dose-dependent. The oxidation of protein thiols to mixed disulfides is an early response to oxidative stress [45]. In our experiments 100 mM ONOO added to plasma resulted in the rapid decrease (>50%) of the level of total plasma protein thiols (Fig. 3), indicating that free thiols form disulfide bonds. In the presence of L-carnitine the level of SH groups was significantly higher. It indicates that Lcarnitine (0.1–100 mM) added to plasma protects against thiol oxidation (Fig. 3). Protein thiols act as a redox buffer; their concentration is much greater than those of low-molecularweight thiols [46]. The alteration of thiol/disulfide status in plasma seems to correspond to the level of plasma lowmolecular-weight thiols. Protein-bound thiols are highly unstable and can be present as free thiols, disulfides, and mixed disulfides when conjugated with GSH, cysteine, homocysteine, and g-glutamylcysteine. Plasma GSH level, in contrast to cell level, is very low and GSH becomes oxidized in response to peroxynitrite. GSH is a dominant ligand of protein thiols and their reactivity with GSH depends on thiol/disulfide redox states. The alteration of thiol/disulfide status in plasma seems also to correspond to the level of plasma homocysteine [46]. We observed that L-carnitine protects low-molecular-weight thiols (GSH, homocysteine, and cysteine) against oxidation induced by peroxynitrite (Figs. 4–6). L-Carnitine modulates blood platelet activation through antioxidant mechanisms and inhibition of arachidonic acid cascade [5,35]. Our results confirm the protective activity of L-carnitine against plasma lipid peroxidation. Augustyniak et al. [47] reported that L-carnitine also protects the lipid and protein parts of low-density lipoprotein particles against oxidative modifications [47]. The administration of L-carnitine to rats intoxicated with ethanol significantly protected lipids against oxidative modifications in blood serum and the liver [48].
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