The effect of α-Tocopherol supplementation on training-induced elevation of S100B protein in sera of basketball players

Clinical Biochemistry 40 (2007) 900 – 906

The effect of α-Tocopherol supplementation on training-induced elevation of S100B protein in sera of basketball players Kleopatra H. Schulpis a , Marcos Moukas b , Theodore Parthimos b , Theodore Tsakiris b , Nickolaos Parthimos b , Stylianos Tsakiris b,⁎ b

a Institute of Child Health, Research Center, “Aghia Sophia” Children’s Hospital, GR-11527 Athens, Greece Department of Experimental Physiology, Medical School, University of Athens, PO Box 65257, GR-15401 Athens, Greece

Received 27 January 2007; received in revised form 4 April 2007; accepted 10 April 2007 Available online 27 April 2007

Abstract Objective: To investigate the effect of α-Tocopherol (α-T) supplementation on S100B elevated serum levels in basketball players' training. Design: Blood was obtained from 10 basketball players pre-exercise (group A), post-exercise (group B) and after 30 days on α-T (200 mg/24 h orally) supplementation pre- (group C) and post-training (group D). Blood samples were taken for the evaluation of total antioxidant status (TAS), α-T and catecholamines in plasma and S100B and muscle enzyme levels in serum. Methods: TAS, muscle enzymes: creatine kinase (CK), lactate dehydrogenase (LDH), and S100B protein levels were measured with commercial kits, whereas α-T and catecholamine levels with HPLC methods. Results: TAS was found higher in the groups with α-T addition (groups C and D) than in the other ones. On the contrary, CK, LDH and S100B were remarkably lower (116.8 + 9.5 U/L, 427 + 22 U/L, 0.18 + 0.04 μg/L, respectively) in group D than those in group B (286 + 12 U/L, 688 + 26 U/L, 0.28 + 0.06 μg/L, p b 0.001, respectively). S100B levels were negatively correlated with TAS (r = −0.64, p b 0.001) and positively with CK levels (r = 0.58, p b 0.001). Conclusions: α-T supplementation may reduce S100B increased release from muscle and nerves induced by training. S100B serum evaluation may be a useful biomarker for the effect of training on the participation of the neuromuscular system. © 2007 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved. Keywords: S100B; Oxidation; Free radicals; α-Tocopherol; Exercise

Introduction The term S100B refers to members of a multigenic family of calcium modulated mass (∼ 10,000 Da), that were firstly identified (on the basis of methods available at that time) as a protein fraction detectable in brain but not in nonneural extracts and called S100 because of their solubility in a 100% saturated solution with ammonium sulphate [1–3]. At present, at least 200 proteins have been identified as belonging to S100 protein family, the members of which are characterized by the presence of a pair of so-called EF hand (i.e., helix–loop–helix) calcium binding motifs [4].

⁎ Corresponding author. Fax: +302107462571. E-mail address: [email protected] (S. Tsakiris).

In particular, S100B, a homodimer of a subunit (β-subunit) that constitutes the bulk of the fraction originally isolated from brain extracts, was regarded for more than a decade as specific to the nervous system. A later study showed that the protein was not restricted to the nervous system [3,5,6]. Since then, the location of S100B has been extensively studied in mammalian tissues, including human tissues [5,6]. In the nervous system, the protein appears to be most abundant in glial cells, although its presence in neuronal subpopulations has also been reported [7,8]. In other nonneuronal tissues, adipose tissue constitutes a site of concentration for the protein comparable to the nervous tissue [9]. Physical exercise is characterized by an increase in oxygen consumption by the whole body and in particular by the muscle tissue. This increase in oxygen uptake is associated with a rise on the production of reactive oxygen species (ROS). The high

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production of ROS may be responsible for a series of physiological and biochemical changes that occur during exercise. It has been reported that strenuous exercise produces a decrease in antioxidant levels and an increase in the marker of lipid peroxidation in target issues and blood lipoproteins [10]. The potential of dietary antioxidants as an endogenous defence to detoxify lipid peroxides produced during exercise has received increasing attention in recent years. Results of human studies on the effect of supplementation with antioxidant vitamin on lipid peroxidation or enzyme muscle damage are controversial. Some studies suggested favorable effects of antioxidant vitamin supplementation on these parameters after exercise [11], whereas others failed to demonstrate these effects [12,13]. Vitamin E supplementation was centered around its effect on performance. With the exception of exercise performed at a high altitude, other studies reported no effect on athletes' performance when on vitamin E supplementation. Since vitamin E has been found to protect cellular membranes from lipid peroxidation, studies have focused on the ability of vitamin E supplementation to reduce the increase in oxidative stress or on muscle damage caused by exercise [14,15]. Furthermore, brain tissue is especially prone to the deleterious effects of free radicals for various reasons, including modest antioxidant defences [16]. Activation of neuronal nitric oxide synthase and thus NO formation because of the high Ca2+ traffic across neuronal membranes [17] and high oxygen demand can lead to increased formation of free radicals [18]. High concentrations of the neurotransmitters dopamine, its precursors levodopa and noradrenaline, which are autoxizable molecules that react with oxygen to generate O2, are also present in the brain. Hydrogen peroxide and reactive quinones/semiquinones [19] and cell membranes enriched in polyunsaturated fatty acid side chains, which are especially sensitive to free radicals attack and therefore to lipid peroxidation [20], may also lead to free radical damage in brain tissue. Since free radical production is closely implicated with neural and muscle function, we aimed to investigate the potential role of antioxidant status on S100B serum concentrations in basketball players pre- and post-training, with and without vitamin E (α-Tocopherol, α-T) supplementation.

was recorded with a scale (Bilance Salus, Italy) to nearest 100 g. Blood pressure, both systolic and diastolic, was determined with an electronic (Nova-test) instrument, with which heart rate (bt/min) was simultaneously measured. The training units consisted in a general warm up and stretching (about 10 min), a technical–tactical part (about 30 min), a heavy training load part including training of counterattacks and simulated full or half-court basketball games (about 40 min), and finally a cool down phase (about 10 min) [22]. All basketball players routinely took part in this training program two or three times a week.

Subjects and methods

Evaluation of total antioxidant status TAS was measured in plasma of players before and after the game as previously reported by Miller et al. [23]. Plasma was frozen for up to 14 days before analysis. 2,2′-azino-di-(3Ethylbenzthiazoline sulphonate) (ABTS) was incubated with peroxidase (metmyoglobin) and H2O2 to produce the radical cation ABTS +, which had a relatively stable blue–green color measured spectrophotometrically at 600 nm. Antioxidants in the added sample cause suppression of the above color production to a degree proportional to their concentration. The assay range was 0–2.5 mmol/L. Samples with concentrations N 2.5 mmol/L were diluted with 0.9% NaCl and reassayed. The present method calculates both the radical scavenging effect and the effect on the rate of ABTS + oxidation (free radical production). Intra- and inter-assay variations were 3.4% and 3.9%, respectively.

The study was performed in accordance to the Helsinki Declaration as amended in 1983 and 1989, and approved by the Greek Ethics Committee. Subjects Ten (n = 10) male basketball players, all members of an adolescent champion team aged 18.5 ± 0.6 years, height 195 ± 5 cm, weight 74.0 ± 1.5 kg volunteered to participate in this study after signing the specific informed consent. Anthropometric measures were taken according to Lohman et al. [21]. In particular, standing height (cm) was measured with precision 0.1 cm using a stadiometer (SECA, model 220, Germany). Body weight (kg) with light indoor clothing, without shoes,

Methods The study was divided into two parts: in part A, the players were clinically examined and blood was drawn for laboratory tests before entering the warm up stage (group A) and at the end of forced training (group B). After the end of part A, the players were supplemented with α-T (200 mg/ 24 h/os) for 30 successive days (part B) [14]. During the time of supplementation, the players routinely continued to be trained as previously. After α-T supplementation, they were clinically re-evaluated and blood was drawn before (4–5 min) (group C) and at the end of the training (2–3 min) (group D) for re-determination of the same biochemical and enzyme parameters. Evaluation of lactate/pyruvate Duplicate 25 μL capillary blood samples were collected from the left thumb 2–3 min following the test for lactate and pyruvate evaluation. Additionally, 7.0 mL blood was obtained into heparinized tubes for erythrocyte membrane preparation and TAS evaluation. Lactate and pyruvate determinations: Samples for lactate and pyruvate estimations were centrifuged at 1000×g for 10 min and analyzed enzymatically. For the measurement of blood lactate and pyruvate concentrations, commercial available kits were utilized: Lactate Pap No 61192 Biomerieux and pyruvate Roche Pyr 124982. Coefficients of variations for these analyses were 2.2% and 2.0%, respectively.

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Evaluation of muscle enzymes, catecholamine and α-T blood levels Serum lactate dehydrogenase (LDH) and creatine kinase (CK) were determined by an enzymatic method at 405 nm using commercial kits (Hyman Geselschaft fur Biochemie and Diagnostica, Taunustein, Germany). α-T was measured by reversed-phase HPLC method as described by Arnaud et al. [24]. A C18 Nova Pak column (Millipore, Frankfurt, Germany) and multi-wavelength UV detector were used to determine α-T. The linear concentration ranged from 0.18 to 91.8 μM between run coefficients of variation [25]. Plasma catecholamine, adrenaline (A), noradrenaline (NA) and dopamine (DA) levels were measured by a sensitive HPLC with reversed-phase ion-pair chromatography and electrochemical detection with an ESA Coulochem (Model 1100 A). The CV for A, NA and DA were 2.4, 2.6 and 2.3% respectively [26]. Evaluation of serum S100B concentrations Serum S100B protein was analyzed with the use of the immunoluminometric assay Sangtec 100 (Sangtec Medical AB, Bromma, Sweden) and fully automated luminescence analyzer (LIA-mat system). Sangtec 100 measures the β-subunit of S100 as defined by 3 monoclonal antibodies. The detection limit of the assay is 0.02 μg/L, while the measuring range is between 0.02 and 30 μg/L. The analysis represents the total amount of S100 and S100B in the sample as the assay is specific for the beta-chain. Intra- and inter-assay variations were 3.5 and 3.8%, respectively [27]. Statistical analysis Data were analyzed with ANOVA for repeated measurements. Pearson's test was utilized for the coefficient correlations evaluation. P valuesb 0.05 were considered statistically significant. Results As expected, blood pressure, both systolic and diastolic, as well as cardiac rate were statistically significantly increased post-exercise in both groups (group B and group D).

Table 1 Biochemical characteristics in basketball players (n = 10) pre- and post-training with or without α-Tocopherol (α-T) supplementation Group A Group B α-T supplementation (pre-training) (post-training) Group C Group D (pre-training) (post-training) Lactate (mM) 1.92 ± 0.22a Pyruvate (μM) 82.95 ± 2.27a Lactate/ 23.15 ± 2.25a Pyruvate

5.22 ± 0.63b 1.94 ± 0.20c 222.73 ± 4.15b 81.10 ± 1.98c 23.44 ± 2.32b 23.92 ± 2.30c

Values are expressed as mean ± SD. Lactate: a/b, a/d, b/c, c/d = p b 0.001; a/c, b/d = NS. Pyruvate: a/b, a/d, c/d, b/c = p b 0.001; a/c, b/d = NS. Lactate/Pyruvate: a/b, a/d, a/c, b/c, b/d, c/d = NS. NS = non-statistically significant.

5.80 ± 0.70d 235.00 ± 4.80d 24.68 ± 2.38d

Table 2 Catecholamines and α-Tocopherol (α-T) levels in the blood of basketball players pre- and post-training with or without α-T supplementation

α-T (μmol/L) DA (pmol/L) A (pmol/L) NA (nmol/L)

Group A (pre-training)

Group B (post-training)

α-T supplementation Group C (pre-training)

Group D (post-training)

23.3 ± 5.5a 55.0 ± 5.9a 230 ± 31a 1.53 ± 0.41a

22.5 ± 5.0b 140 ± 16b 875 ± 46b 3.2 ± 0.8b

32.7 ± 6.2c 60.0 ± 6.4c 220 ± 35c 1.45 ± 0.40c

30.5 ± 4.9d 148 ± 18d 890 ± 50d 3.9 ± 0.8d

α-T = α-Tocopherol; DA = dopamine; A = adrenaline; NA = Noradrenaline. Values are expressed as mean ± SD. α-T: a/c, a/d, b/c, b/d = p b 0.01; a/b, c/d = NS; DA: a/b, c/d, a/d, b/c = p b 0.01; a/c, b/d = NS. A: a/b, a/d, b/c, c/d = p b 0.01; a/c, b/d = NS; NA: a/b, a/d, b/c, c/d = p b 0.01; a/c, b/d = NS. NS = non-statistically significant.

As shown in Table 1, both lactate and pyruvate blood levels were remarkably increased post-training (group B and group D). In contrast, the ratios lactate/pyruvate were similar in all the studied groups. As presented in Table 2, α-T levels remained unaltered prevs. post-exercise, whereas catecholamine levels were remarkably increased post-training (group B, group D). As illustrated in Table 3, the muscle enzyme, CK and LDH, serum levels were statistically elevated post-training (group B, group D). Importantly, the enzyme levels were lower in the group with α-T addition (group D) than in group B at the same time of study. TAS levels were statistically decreased postexercise (group B and group D). Interestingly, TAS level was statistically higher in group C than those in all the other groups of study and significantly lowered in group D reaching that of group A. As presented in Table 4, statistically significant positive correlations were found between the muscle enzyme CK levels with S100B protein, whereas the protein negatively correlated with TAS in all studied groups. No correlations were found between S100B levels and lactate, pyruvate levels as well as catecholamine and α-T levels (data not shown). Table 3 Plasma total antioxidant status (TAS), serum muscle enzymes and S100B levels in the sera of basketball players pre- and post-forced training with or without αTocopherol (α-T) supplementation Group A Group B α-T supplementation (pre-training) (post-training) Group D Group C (pre-training) (post-training) CK (U/L) 86.0 ± 7.4a LDH (U/L) 387 ± 13a TAS (mmol/L) 1.47 ± 0.13a S100B (μg /L) 0.11 ± 0.03a

286 ± 12b 688 ± 26b 0.92 ± 0.08b 0.28 ± 0.06b (+155%)

80.5 ± 6.0c 332 ± 15c 2.95 ± 0.10c 0.12 ± 0.02c

CK = creatine kinase; LDH = lactate dehydrogenase. Values are expressed as mean ± SD. CK: a/b, a/d, b/c, b/d, c/d = p b 0.01; a/c = NS. LDH: a/b, a/d, b/c, b/d, c/d = p b 0.01; a/c = NS. TAS: a/b, a/c, b/c, a/d, b/d, c/d = p b 0.01. S100B: a/b, a/d, b/c, b/d, c/d = p b 0.01; a/c = NS. NS = non-statistically significant.

116.8 ± 9.5d 427 ± 22d 2.10 ± 0.10d 0.18 ± 0.04d (+50%)

K.H. Schulpis et al. / Clinical Biochemistry 40 (2007) 900–906 Table 4 Coefficient correlations between total antioxidant status (TAS), creatine kinase (CK) and the protein S100B serum levels in the groups of study Groups TAS A B C D CK A B C D

S100B − 0.64 ⁎⁎ − 0.52** − 0.67** − 0.52** 0.58** 0.50 ⁎ 0.54** 0.48*

⁎ p b 0.01. ⁎⁎ p b 0.001.

As illustrated in Fig. 1, mean + SD S100B levels were remarkably increased post-training and significantly lowered with α-T supplementation at the same time of study. Discussion Anaerobic exercise has been associated with a substantial lactic acidosis both in blood and muscle [28] and also with a major increase in plasma catecholamine levels [29], as found in our players. Obviously, the remarkably increased blood pressure as well as the almost 2-fold higher cardiac rate, postexercise, could be implicated with the significant action of their sympathetic system.

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To examine acute oxidative stress in response to exercise, most researchers have assessed various stress markers in blood and urine. Few studies have examined oxidative stress in muscle tissue of humans in response to exercise [30,31]. In addition, most commonly increased are by-products of lipid peroxidation, but changes in the status of antioxidant compounds, such as glutathione, protein and DNA oxidative products and antioxidant enzymes activities as well as total antioxidant status have been used. These are all indirect measures of free radical action [11,28]. In this study, TAS was remarkably reduced in the players without α-T supplementation at the end of the training, as reported by other authors too [11,28,32]. In addition, TAS was significantly increased in the players with α-T addition before the beginning of the exercise. This finding is in agreement with that previously reported by other authors, as a result of the increase of the antioxidant action of the vitamin, by minimizing the basic (pre-exercise) free radical production for lipid peroxidation [11,32,33]. Interestingly, TAS was decreased in the players post-training (group D) possibly showing a tissue depletion of the other antioxidants [14,34], since α-T was measured unaltered in the plasma of our players. Additionally, TAS level in group D was found statistically significantly higher than those measured in the athletes pre- (group A) and posttraining (group B) without vitamin addition. The latter shows a remarkable amelioration of the players' total antioxidant capacity. Furthermore, the muscle enzyme, CK and LDH, levels were remarkably increased in both groups post-training, as expected [30]. It is important that CK and LDH serum levels were lower

Fig. 1. S100B blood concentrations in the players pre-training (group A), post-training (group B), pre-training with α-Tocopherol (α-T) supplementation (group C) and post-training with α-T addition (group D). Values are expressed as mean ± SD of three measurement calculations of the same sample for each case.

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in the players with α-T addition post-exercise (group D) than those determined in the blood of players without the supplementation of the vitamin at the same time of study (group B). This finding could be explained by the increase of αT concentrations in the blood of the players after supplementation [11]. Supplementation with α-T may protect the cells, e.g., muscle cells, from glutamate-induced “cell death” and by ameliorating lipid peroxidation injury during the exercise. The latter may be implicated with the decrease of CK and LDH levels in the serum of basketball players post-training, as we recently reported [35]. The primary mechanism of the protection by α-T is described in details by Cunha et al. [36]. S100A1 and S100B proteins are among the best characterized members of the S100 multigenic family of small, acidic proteins, containing the EF-hand Ca2+ binding motif [1,2,4]. As mentioned, S100 proteins, expressed exclusively in vertebrates, are proposed to be involved in a large number of cellular events where they act as event triggers or activators. S100B is mostly expressed in the nervous system, primarily in astrocytes, neurons, muscle and Schwann cells [6,37] as well as in adipose tissue [9]. In some cell types, S100B is secreted into the extracellular space by a yet unknown mechanism [38,39]. Its release is regulated by serotonin (5-HT) through 5-HT1A receptors [40]. Recently Scaccianoce et al. [41] found a relationship between stress and circulating levels of the protein. In this study, the strong positive relation found between the protein and CK levels shows that the observed remarkably increased levels of S100B in the athletes, post-training, may be the consequence of S100B release by the strong contraction of their muscles also leading to lipolysis, another source of S100B production, as mentioned above [6,9,37,41]. The latter is further supported not only by the strong relationship observed between S100B and muscle enzyme levels in the basketball players but also by a previous study on athletes of swimming race [42]. In addition, the statistically higher levels of the protein in the sera of players post-exercise (group B) may be also due to the longterm participation of the hypothalamus–hypophysis–adrenals axis during the eventful training [40,41]. Similarly, we cannot exclude the possibility of the presence of a psychogenic stress during this training which may additionally act in the S100B protein releasing in the blood stream, since most players are very anxious to succeed a great victory in favor of their team. This suggestion is supported by the previously reported elevation of S100B levels determined in the CSF of rat subjected to predator stress [40,43,44]. There is also evidence for catecholamine-dependent relationship between serum S100B and stress [45]. In this study, no relationship was found between catecholamine and S100B blood levels. This finding may be due to the evaluation of catecholamine levels in the blood and not in the CSF of the players [45]. Since stress is closely related to exercise and free radical production, we may take into account that free radicals might mediate S100B production. The latter may additionally induce antiinflammatory action not only on muscles against free radical injury but also in CNS [3,6]. These suggestions are reinforced by the negative relationship found between the protein and TAS

as well as by the positive relation observed between S100B and CK levels in basketball players. Furthermore, α-T has been shown to have anti-inflammatory effects both in vivo and in vitro. α-T therapy, especially in high doses [46] has been shown to decrease the release of proinflammatory cytokines (such as interleukin-β, interleukin 6 and tumor necrosis factor-α (TNF-α) and the chemokine interleukin-8) as well as to decrease adhesion of monocytes to endothelium. Consequently, α-T has been shown to decrease Creactive protein (CRP) levels in patients with related risk factors such as diabetes and smoking [46,47]. Importantly, Jain et al. [48] have shown that the vitamin increases glutathione (GSH) production, which plays a significant role in the cell antioxidant capacity. In this respect, α-T may not only reduce the “inflammatory effects” of free radicals on neural and muscle tissues during training but also increase the antioxidant capacity of the cells, resulting in a decrease of CK, LDH and S100B levels in the sera of basketball players. As mentioned above, the levels of the protein S100B were remarkably increased in the serum of the basketball players after the end of their strenuous exercise (+ 155%, group B). The observed significant lower level of S100B (+ 50%) in the players with α-T supplementation post-training (group D), as compared with that without the addition of the antioxidant, could be due to the action of the vitamin (Fig. 1). α-T may not only offer the antioxidant and anti-inflammatory protection via amelioration of lipid peroxidation and GSH increase [48], as shown by the higher TAS level and lower muscle enzyme levels in the players, but also protect the “cell death” from glutamate entry as previously reported [45,46]. Consequently, the lower S100B levels measured in the players with α-T addition post-training may be due to each one or all the above described beneficial effects of the lipid soluble vitamin. Conclusions (a) S100B levels were remarkably increased in basketball players post-training possibly due to their muscle contractions and lipolysis during the game without excluding the possibility of CNS contribution by S100B release in the blood stream induced by a predatory stress; (b) The inverse correlation found between total antioxidant status (TAS) and the protein S100B levels may be due to the anti-inflammatory action of S100B on the free radical injury of muscles produced during training; (c) The observed post-exercise significantly lower S100B levels in the players with α-T supplementation may be the consequence of the reduced lipid peroxidation by the lipid soluble vitamin and protection of the harmful action of free radicals on the neuronal and muscle tissues, as evidenced by the lower muscle enzyme levels measured in their sera; and (d) S100B serum evaluation may be a useful biomarker of the participation of the nervous and muscular system during training.

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