3. RESULTADOS
3.1 CAPÍTULO 3 Evaluation of antioxidant capacity in plasma and saliva
3º artigo (submetido à Applied Physiology, Metabolism, and Nutrition em 23/05/2006, #06-32).
Evaluation of antioxidant capacity in plasma and saliva from young athletes by the use of differential pulse voltammetry
Paulo Guimarães Gandra, Armindo Antônio Alves, Lázaro Alessandro Soares Nunes, Lauro Tatsuo Kubota, Denise Vaz de Macedo
Abstract
Background: Low–molecular weight antioxidants play a major role protecting biological systems against reactive oxygen species and their antioxidant action can be referred as total antioxidant capacity (TAC). Objectives: Present the use of differential pulse voltammetry (DPV) for plasma and saliva TAC evaluation. Methods: Plasma and saliva TAC and urate concentrations, erythrocyte enzymatic antioxidants activities, plasma creatine kinase activity, white blood cells count, a
squat jump and a line drill tests were monitored in twelve young athletes at the 2nd
and 7th week of a training period. Training was carried out in 5 sessions/week, 3.5
h/d. Results: Saliva and plasma TAC correlated with each other. Moreover, these two parameters correlated significantly with their respective urate concentrations.
Physical fitness level was higher and plasma TAC was decreased in the 7th week
of training (787.82 ± 154.71 µmol Trolox equiv. L-1) compared to the 2nd week
(879.28 ± 197.92 µmol Trolox equiv. L-1). Conclusions: The electroanalytical
method presented here is a cheap, sensitive and simple method to measure the TAC of biological fluids and can be a very interesting tool to monitor the TAC of athletes’ body fluids during training and exercise. Our results also suggest that the decreased plasma antioxidant capacity is part of a normal process at the initial stage of a training cycle because was associated with a positive physical fitness adaptation without alterations in inflammatory and muscular damage markers. Key-words: antioxidant enzymes, CK, differential pulse voltammetry, performance, plasma, saliva, total antioxidant capacity, training, uric acid, total leucocytes.
Introduction
Skeletal muscle contractile activity is directly and indirectly implicated in increased production of reactive oxygen species (ROS) (Reid & Durhan 2002, Ji 1999). Muscle and blood antioxidant systems can undergo adaptations to avoid the deleterious actions of ROS such as lipid peroxidation, DNA damage and protein oxidation (Child et al. 1998, Brites et al. 1999, Ji 1999, Liu et al. 1999, Powers et
al. 1999, Smolka et al. 2000). When oxidants overcome the antioxidant defense
systems, an oxidative stress situation is established. Elevated levels of oxidative stress can be associated with overtraining, which in turn is associated with diminished athletic performance (Tiidus 1997, Mackinnon 2000). On the other hand, increased ROS levels can also result in positive effects in skeletal muscle like enhanced force production (Reid 2001) and positive adaptations resulting from redox-sensitive signaling pathways (Zhou et al. 2001, Andreucci et al. 2002, Catani
et al. 2004).
The antioxidant systems are composed of enzymatic and non-enzymatic antioxidants, and proteins (Ji 1999, Smolka et al. 2000, Reid & Durhan 2002). The non-enzymatic antioxidant system is mainly composed of low-molecular-weight antioxidants (LMWA). LMWA are found in body fluids and tissues at higher concentrations than enzyme antioxidants, playing an important role in antioxidant defense of biological systems. Blood transports and redistributes antioxidants to tissues and extracellular fluids. In this way, plasma LMWA can be affected by systemic metabolic alterations, like those resulting from exercise (Child et al. 1998).
Some methodologies quantify the total antioxidant capacity (TAC) of biological samples conferred by LMWA representing a very interesting way to analyze the non-enzymatic fraction of antioxidant systems (Cao & Prior 1998, Janaszewska & Bartosz 2002). Besides the practical advantage, the TAC considers the synergism between each antioxidant of the system, which is important because of their complex interactions (Cao & Prior 1998, Ghiselli et al. 2000). The several methods used to quantify TAC in fluids conferred by LMWA have different limitations. (Cao & Prior 1998, Janaszewska & Bartosz 2002, Gandra et al. 2004). Recently, Chevion et al. (1997) and Kohen et al. (2000) proposed cyclic voltammetry (CV) as a fast, simple and cheap analytical method to quantify the TAC of biological fluids and tissues.
Saliva may be preferable to plasma for evaluating the TAC of subjects exposed to oxidative stress since it can be collected by non invasive techniques and reflects changes in plasma (Meucci et al. 1998, Kohen et al. 2000). However only one study focused on saliva TAC after a physical exercise intervention (Atsumi et al. 1999). Thus, the influence of physical training on the TAC conferred by LMWA in plasma and saliva still remains to be elucidated, as well as the relationship between the antioxidant capacities of these body fluids during different exercise training programs.
Differential pulse voltammetry (DPV), like CV, is a commonly used method in analytical chemistry working under the same principle as CV. Both methods consider most LMWA to be reducing agents acting directly as ROS quenchers and thus the reducing power of a biological sample may reflect its antioxidant capacity
(Chevion et al. 1997, Kohen et al. 2000). However, DPV has well-established advantages over CV, such as good discrimination against background currents, better sensitivity and lower detection limits (Kissinger & Ridgway 1996), and may be a very interesting tool to monitor the antioxidant capacity of athletes’ body fluids during training and exercise (Gandra et al. 2004).
Our purpose was to use the differential pulse voltammetry method to measure plasma and saliva TAC and to investigate possible effects of exercise training intervention on these parameters in young basketball athletes in the beginning of their competitive season. The use of other metabolic markers to reflect the oxidative stress status and the physical fitness were simultaneously analyzed for further correlations.
Materials and Methods
Subjects
Twelve volunteer young male basketball players, with mean age of 18.5 ± 0.5 years old, 187.6 ± 7.8 cm high, 87.35 ± 13.12 kg, and 11.36 ± 4.58 % body fat, participated in this study. They were athletes from the same team and analysis were taken during the training period that preceded a competition season. Each athlete gave his informed consent after receiving information on the nature of the study. The study protocol was approved by the Research Ethics Committee of the Dentistry School at the State University of Campinas (No. 019/2004).
The subjects were analyzed for the first seven weeks of the one-year training period. Throughout the course of the study all subjects participated in physical and technical/tactical training 5 times per week in 2 or 3 sessions per day totalizing about 3.5 h of training per day. Two specific physical-capacity tests were performed to analyze whether training improved physical fitness.
Sample collection
Samplings were restricted to the 2nd (W
2) and 7th (W7) weeks of training. The
last training session prior to sampling was of lower intensity and duration (< 1 h of low-intensity exercise) to minimize interference of acute responses to physical exercise. After this last session, athletes were allowed 3 days of rest and then blood and saliva were sampled at 07:00 AM, after an overnight fast, and the physical capacity test was performed in the evening.
Blood samples
For total antioxidant capacity and biochemical determinations 10 mL of blood was collected from a superficial forearm vein by heparinized tubes and centrifuged at 1000 x g for 15 min (4ºC). Plasma aliquots (500μL) were obtained and stored frozen (-80ºC) until required for assay. The erythrocyte pellets were washed tree times with cold PBS (0.2 M phosphate buffer, pH 7.4, plus NaCl 0.9%) solution and hemolyzed (1:1, v/v) with cold water. Aliquots (500μL) were immediately frozen (-80ºC) until determination of catalase (CAT) and gluthatione
reductase (GR) activities. For the haematological analyses, 4 mL of blood was
sampled in K3EDTA.
Saliva samples
For total antioxidant capacity and urate determination, approximately 2mL of unstimulated whole saliva was collected in plastic tubes and stored frozen (-80ºC). Saliva was centrifuged at 1500 x g for 5 min (4ºC) and the TAC and urate concentration were assessed in saliva supernatant.
Total antioxidant capacity (TAC)
Differential Pulse Voltammetry measurements followed the method for cyclic voltammetry described by Chevion et al. (1997) and Kohen et al. (2000). Plasma and saliva samples were diluted in PBS (1:1 v/v) to a final volume of 800 µL. The analyses were carried out in a potentiostat from Eco Chemic (Autolab PG-Stat30 model), Utrecht, Netherlands, connected to a microcomputer. A glassy carbon electrode (3 mm diameter) was used as working electrode, a saturated calomel electrode as reference and a platinum wire as auxiliary electrode in a 500 to 2000 µL cell. The applied potential ranged from -0.3 V to 1.0 V for plasma samples and from -0.3 V to 1.5 V for saliva samples. After each scan, the working electrode was polished manually with aqueous slurry of aluminum powder (Ø = 0.3 µm) on a damp smooth cloth to avoid protein aggregation on its surface. DPV analysis used modulation amplitudes of 75 mV, a scan rate of 9mV/s and pulse width of 0.180ms.
For amperometric methods, a sample reducing power is a result of two parameters: the potential at the anodic peak current (Ep), which is determined by the redox potential of the analyzed compounds, and the anodic peak current value (Ip), which is a direct function of the concentration of the compound (Kissinger &
Heineman 1983, Mabbott 1983)(Fig. 1a). However, for biological samples, different
antioxidant compounds with close Ep values can overlay the anodic peaks, resulting in only one peak. In these cases, the anodic peak area is a better parameter than the Ip value since alterations in the peak components can change the peak shape and the area, neither of which is expressed in the Ip value
(Chevion & Chevion 2000) (Fig. 1b).To determine values for area under the curve
(A), a base line was obtained from a straight line joining the two minimum points of the anodic peak by using the GPES software (General Purpose Electrochemical Software, version 4.9, Eco Chemie B.V., Utrecht, Netherlands) (Fig. 1a and 1b).
The ferric-reducing ability of plasma (FRAP) assay measures the increase in the absorbance at 593 nm resulting from the reduction of a ferric-tripyridyltriazine
(FeIII-TPTZ) complex to ferrous form (FeII), as a consequence of the presence of
antioxidants. The FRAP assay was based on Benzie and Strain (1996) and the
final results converted and expressed as µmol Trolox equivalent. L-1. This assay
was conducted in one plasma and one saliva sample, which were submitted also to a DPV analysis for TAC to establish a relationship between FRAP and DPV methodologies, thus expressing DPV results as Trolox equivalent.
Biochemical determinations
Creatine kinase (CK) activity and urate concentrations were determined spectrophotometrically with available commercial kits in an automated Autolab analyzer (Boehringer Mannheim).
Erythrocytes were diluted 1:20 in cold water for CAT and GR activity determination. CAT and GR activity were determined using a DU® Series 600
spectrophotometer (Beckman). Erythrocyte CAT activity was measured according to the method of Aebi (1984) and expressed as the rate constant of first-order reactions (k). GR activity was determined as described by Smith et al. (1988). Both erythrocyte enzyme activities were normalized to their hemoglobin concentration.
Haematological determinations
Total blood leukocyte count (WBC) and erythrocyte hemoglobin content
were determined by the hematological counter KX-21N Sysmex®.
Physical performance tests
The subjects executed 3 trials of a squat-jump test and 2 trials of a line-drill test with at least 3-min interval between trials. Only the best result for each test was considered.
The squat jump was performed from a semi-squat position without swinging the arms. The jump height was calculated by the flight time, determined by a contact mat attached to a microcomputer to register the time elapsed in which the
individual was not in contact with the mat. The estimated error of these measurements is 3% (Young et al. 1995).
The line drill is a common field test applied to basketball players, representing a high-intensity exercise. It consists of repeated maximal speed sprints, beginning from a standing position on the court baseline, to different lines at the following distances: 5.8 m, 14.3 m, 22.9 m and 28.7 m from baseline (Hoffman et al. 2000). As the subjects reached each line they sprinted back to the starting point and sprinted to the next line and so on, until they reached the final distance and returned back to the baseline completing 143.4 m of continuous repeated sprints. The total time of the test was registered.
Statistics
The results were expressed as mean ± standard deviation (SD). The results before and after training were compared by Student t-test for paired samples. Correlations were calculated by Pearson’s linear correlation using the statistical software GraphPad (GraphPad Software, Inc., San Diego, California, USA). Statistical significance was set at p < 0.05. The significant difference for the Ep values was set at 50 mV.
Results
Characterization of saliva anodic peak
Saliva anodic peaks components have not been described yet. Since urate is the major saliva LMWA, it was tested whether urate could be a component of
saliva anodic peaks. Thus, urate was added in different amounts to a saliva sample diluted in PBS, resulting in corresponding increases in the first anodic peak of the saliva (Fig. 1C). Additions of ascorbate were also performed resulting in the same patterns of increases in saliva first anodic peak as observed for urate. In the present study we also found the presence of a second anodic peak in saliva samples at a peak potential of approximately 1V (Fig 1D). Deproteinization did not affect saliva anodic peaks.
Effect of training on antioxidant status
Antioxidant capacities determined by DPV are expressed by anodic peak area and Ep values for plasma and saliva. However, the first anodic peak areas for plasma and saliva were treated as plasma and saliva TAC and converted to µmol of Trolox equivalent per liter. To obtain an expression to convert the area values to Trolox equivalent, a direct correlation between the TAC obtained by DPV, represented by the area of plasma and saliva first anodic peak, and FRAP in Trolox equivalent was performed. The following equations were used for plasma and saliva, respectively:
Plasma TAC = 441.85 * A1 / 2.07 e-7 µmol Trolox equivalent.L-1
Saliva TAC = 232.70 * A1 / 1.63 e-7 µmol Trolox equivalent.L-1,
were A1 is the first anodic peak area.
Plasma TAC was significantly decreased in W7, while only the second
anodic peak for plasma showed significant decrease in the peak potential (Ep2) in
50mV (Table 1). Saliva TAC and Ep1 values showed a tendency toward decrease in W7 (Table 1). Plasma TAC was significantly correlated with plasma urate
concentration in both W2 (r=0.9877, p<0.0001) and W7 (r=0.9643, p<0.0001).
Saliva TAC was significantly correlated with saliva urate concentration in W2
(r=0.6345, p<0.05) and W7 (r=0.6967, p<0.05). Plasma and saliva TAC were not
correlated in W2, but significant correlation emerged in W7 and also for the sum of
all the analysis (r=0.6225, p<0.05, and r=0.5871, p<0.01, respectively).
Effect of training on biochemical parameters
Blood WBC and plasma CK activity were not significantly different in W7
compared to W2 (Table 2). On the other hand, a significant decrease in plasma and
saliva urate concentrations occurred in W7. Plasma and saliva urate concentrations
were significantly correlated with each other only in W2 (r= 0.6067, p<0.05) and for
the sum of all the analyses (r=0.5267, p<0.01).
The activities of both erythrocyte antioxidant enzymes, CAT and GR, were not significantly affected by training.
Effect of training on performance parameters
The performance tests indicated a significant increase in squat-jump height (34.1 ± 5.5 cm in W2 versus 38.6 ± 5.7cm in W7, P < 0.0001) and a significant
Discussion
Analysis of the DPV tracing have shown the presence of two anodic peaks in hydrophilic extraction of human plasma and saliva, composed probably of the same LMWA described for cyclic voltammetry, since they are voltammetric methods based on the same principle. However, Ep values for plasma and saliva anodic peak currents reported here were lower than those described by Chevion et
al. (1997) and Kohen et al. (1992) due to use of a saturated calomel instead of
Ag/AgCl as reference electrode.
Plasma first anodic peak is determined mostly by urate and ascorbate due to their high concentration in human plasma (Chevion et al. 1997, Kohen et al. 2000). Urate has been reported as the most important LMWA of human saliva (Meucci et al. 1998), contributing to 70% of the total antioxidant capacity of this fluid (Mooe et al. 1998). Addition of urate and ascorbate to saliva samples increased saliva first anodic peak (Fig. 1c), confirming that this peak can be composed by these antioxidants. Saliva urate has been suggested as originating from plasma urate based primarily on the correlation of urate concentrations between plasma and saliva (Kondakova et al., 1999). Our data demonstrated significant correlation between saliva total antioxidant capacity and saliva urate concentration, a result also found by other TAC methods (Meucci et al. 1998).
The second anodic peak for plasma can be composed of lipoic acid, or be a result of hemolysis during plasma separation, being comprised of NADPH from red blood cells (Kohen et al. 2000). The decreased value of the second anodic peak
Ep for plasma (Ep2) may be a consequence of altered sample antioxidant content. Besides urate and ascorbate, saliva does not contain other LMWA in large
amounts[42] and the removal of proteins did not interfere in saliva second anodic
peak. Maybe cells from mouth lining, neutrophils or even bacteria present in saliva could be responsible for saliva second anodic peak. The nature of saliva second anodic peak still has to be elucidated.
Physical training and acute sessions of physical exercise can induce different kinds of alterations in plasma LMWA concentrations. Some works using different methodologies have reported that acute bouts of exercise increase plasma TAC, an effect associated with or accompanied by urate concentration increases (Child et al., 1998, Liu et al. 1999, Maxwell et al. 1993). Brites et al. (1999) described a total antioxidant capacity of plasma 25% higher for soccer players than for sedentary individuals. Conversely, Sharman et al. (2004) and
Balakrishnan & Anuradha (1998) observed a decreased plasma total antioxidant
capacity for subjects under different physical training programs. Different results for TAC during physical exercise and physical training may be a consequence of different populations studied, of different methodologies to determine TAC, or of different physical training models and moments of samplings.
The plasma CK activity and circulatory WBC that were used here as muscular alterations and inflammatory markers respectively were not changed by training, supporting that at the moment of sample collection the subjects were not under effect from acute exercise. Therefore, the significant decrease in plasma
to a decreased urate concentration, since these parameters were strongly correlated with each other. As a positive physical fitness adaptation was also detected, represented by improvements in the physical performance tests, such decrease in TAC is posited as a normal process at the initial phase of a training program.
However, saliva antioxidant capacity was not changed, although saliva total antioxidant capacity tended to be decreased in W7. Decrease in saliva total
antioxidant capacity was reported in children after 1 h of physical exercise (Atsumi
et al. 1999). The unchanged saliva antioxidant capacity observed here might reflect
a low oxidative stress level resulting from the initial phase of physical training that was not sufficiently intense or long to change saliva TAC. The unchanged erythrocyte antioxidant-enzyme activities supports this interpretation, although a tendency toward increased CAT activity was found, probably through a direct action of ROS on the enzyme structure (Tauler et al. 1999).
The results presented here reinforce the monitoring of some biomarkers as a powerful methodology for evaluating individual physical stress threshold in athletes during a competitive season. Such evaluations, however, require frequent and successive blood samplings to have practical importance. Saliva collection is a less invasive sampling method for assessing antioxidant status in athletes during a competitive period, permitting more studies about metabolic responses from athletes of different modalities, which could contribute for prophylactic actions. In this sense, differential pulse voltammetry is proposed as a cheap, sensitive, simple and reliable method to measure total antioxidant capacity of biological fluids, such
as plasma and saliva. Acknowledgements
The authors thank the athletes who participated in this study. This study was supported by the Brazilian agencies FAPESP (03/09923-2P) and CNPq. P.G.Gandra and L.A.S. Nunes received grants from the Brazilian agency CAPES.
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