Eur J Nutr DOI 10.1007/s00394-014-0785-x

ORIGINAL CONTRIBUTION

Effect of polyphenol supplements on redox status of blood cells: a randomized controlled exercise training trial Lucrecia Carrera-Quintanar • Lorena Funes • Nestor Vicente-Salar • Cristina Blasco-Lafarga Antoni Pons • Vicente Micol • Enrique Roche



Received: 25 November 2013 / Accepted: 10 October 2014  Springer-Verlag Berlin Heidelberg 2014

Abstract Purpose The effect of endogenous antioxidants can be either an immediate response (relying on enzymatic activities) or a long-term adaptation (relying on gene modulation events), both susceptible to be modified by antioxidants from diet and supplementation. The aim of this work was to delve in these aspects in circulating white blood cells in a group of volunteers (n = 33, 20–22 years) performing eccentric exercises and consuming or not (n = 8) different polyphenolic antioxidants (Lippia citriodora extract-PLX n = 8, almond beverage n = 9 or a mixture of both n = 8) during 21 days.

Electronic supplementary material The online version of this article (doi:10.1007/s00394-014-0785-x) contains supplementary material, which is available to authorized users. L. Carrera-Quintanar  N. Vicente-Salar  E. Roche (&) Department of Applied Biology-Nutrition and Institute of Bioengineering, University Miguel Hernandez, Avda de la Universidad sn, 03202 Elche, Alicante, Spain e-mail: [email protected]

Methods We have designed a single-blind, parallelgroup, randomized controlled trial. Antioxidant enzyme activities, oxidative stress markers, and antioxidant gene expression were determined. Results Neutrophils and lymphocytes expressed high amounts of oxidative markers compared to plasma. Concerning enzymatic activities, increased superoxide dismutase levels were detected when certain supplements were consumed. However, catalase levels did not change. As for glutathione peroxidase levels, no differences were detected in lymphocytes, while neutrophils expressed increased levels in both placebo and PLX groups. Glutathione reductase activity was decreased in all groups, except in neutrophils of PLX group. At the level of gene expression, neither PLX nor the almond beverage interfered with the expression of genes coding for the corresponding enzymes. However, the combined intake of both supplements affected the expression of glutathione reductase and Cu–Zn and Mn-superoxide dismutases in neutrophils. Conclusions Altogether, these results suggest that blood cell types respond and adapt differently to exercise-induced oxidative damage.

L. Funes  V. Micol Institute of Molecular and Cellular Biology, University Miguel Hernandez, Elche, Alicante, Spain

Keywords Almond beverage  Lippia citriodora  Phenolic compounds  Weight lifting

C. Blasco-Lafarga Department of Physical Education and Sports, University of Valencia, Valencia, Spain

Introduction

A. Pons Department for Basic Biology and Health Sciences, University of Balearic Islands, Palma de Mallorca, Spain A. Pons  V. Micol  E. Roche CIBERobn (Fisiopatologı´a de la Obesidad y la Nutricio´n CB12/ 03/30038), Instituto de Salud Carlos III, Madrid, Spain

Strenuous or prolonged exercise increases reactive oxygen substances (ROS) and therefore oxidative stress in the skeletal muscle; thus, many athletes take antioxidant supplements to reduce this effect [1]. If left untreated or uncontrolled, muscle stress can cause overt structural and functional damage due to the repetitive contractile activity

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or contusions during vigorous exercise or contact sports, resulting in an inflammatory response [2, 3]. Neutrophils respond to this inflammation to remove cell debris and waste products, causing a respiratory burst [4]. This respiratory burst is essentially an oxidative reaction where superoxide radicals generated by NADPH oxidase are the initial step of a cascade of events which include additional oxidative reactions, i.e., myeloperoxidase [5]. On the other hand, lymphocytes, the second most abundant circulating white blood cell, undergo leukocytosis and lymphopenia after exhaustive aerobic exercise [6, 7], which subsequently compromises the immune response [8, 9]. Altogether, vigorous exercise causes oxidative muscle damage which is reflected in changes in circulating parameters. Therefore, vitamin antioxidant supplementation could minimize ROS production, theoretically providing a beneficial effect to the affected systems in order to improve performance and recovery. However, antioxidant use for physical performance and body redox status has been long debated [1, 10, 11]. Some studies indicate a positive effect on physical performance [12, 13] while others indicate the opposite [14, 15] or no effect [16–18]. The same type of contradictory results can be observed regarding modulation of exercise-induced oxidative stress [16, 19–24]. Although these discrepancies are due to the large variability in the experimental designs used in these studies, there are two main aspects which can be concluded from them. First, there is an immediate antioxidant response that depends on enzymatic activities, i.e., superoxide dismutase (SOD), catalase, glutathione peroxidase (GPX), and glutathione reductase (GRD). Second, there is a long-term adaptation process supported by the expression of antioxidant genes. Both events (response and adaptation) are modulated differently by antioxidants provided in the form of supplements. For example, large doses of vitamins C and E can work as excellent ROS scavengers, but eventually interfere with the induction of antioxidant enzyme gene expression, becoming an unfavorable long-term antioxidant adaptation. This is because ROS are instrumental transducers in the induction of endogenous genes coding for antioxidant defenses [25]. However, an interesting question that remains is whether other dietary antioxidants work in a similar manner as the antioxidant vitamins. In this regard, polyphenolic compounds represent a new group of substances that are still poorly characterized in sport performance and recovery. These compounds are present in seeds, fruits, and vegetables and exert a variety of effects including antioxidant and anti-inflammatory responses [26]. In this context, we have recently investigated the antioxidant capability of a Lippia citriodora extract (commercially called PLX, acronym coming from ‘‘PoLyphenol eXtract’’) on the oxidative status in plasma

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and blood cells of university students performing a 21-day aerobic training routine (3 days/week) [27–29]. In another study, we analysed the antioxidant effect of an almondbased isotonic energy drink enriched with vitamins C and E [7, 30, 31]. The antioxidant potential of this beverage resides in the polyphenolic compounds present in almonds [32] (46 mg/L hesperidin, 90 mg/L verbascoside, and 40 mg/L of other polyphenols) and the added vitamins (75 and 25 mg of vitamins C and E, respectively), although these were present in more discrete doses than in other studies in order to not impair endogenous adaptive responses [7]. Taking PLX and the almond beverage (AB) as paradigms of polyphenolic antioxidants, we aimed to study the basal antioxidant response and adaptation process in the most abundant population of circulating white blood cells (neutrophils and lymphocytes) in a group of university students performing a strenuous weightlifting training program and consuming the same diet, but with combinations of supplements (PLX, AB or a mixture of both: AB ? PLX).

Materials and methods Trial design Male subjects were randomly assigned to one of the four parallel groups, in a 1:1:1:1 ratio, to consume placebo (PLB group), 1.2 g daily (1 capsule every 12 h: the first capsule at breakfast and the second after dinner) of Lippia extract (PLX group), 250 mL/day of the enriched functional AB (supplemented with 10 mg of vitamin E/100 mL and 30 mg of vitamin C/100 mL) (AB group), and 250 mL of AB plus 0.55 g of PLX (AB ? PLX group). Both drinks were taken at breakfast early in the morning. The design was a single-blind, parallel-group, randomized controlled trial conducted at Miguel Hernandez University of Elche (Spain). Participants Volunteers were selected from students of Sport Sciences and Physical Activity at the University Miguel Hernandez of Elche (Spain). Volunteers were regular practitioners of different rowing disciplines at the University and had participated in various national championships. Eligible participants were all male aged 20–22 years who met the inclusion criteria: to have commenced their training in the sport discipline, to perform weightlifting exercises during their regular training, to not present any chronic disorder, non-smokers, and to follow regular nutritional habits. Exclusion criteria included the consumption of

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antioxidants or any type of supplement (amino acids, vitamins, minerals, and vegetable extracts) at the moment of the study, consumption of anti-inflammatory drugs or to present muscle lesions. Interventions Volunteers were informed about the objectives and demands of the study and gave their written consent to participate. The protocol was in accordance with local legal requirements and the Helsinki Declaration for research on human beings and approved by the Ethical Committee of the corresponding Autonomic Governments. All subjects performed a supervised eccentric contraction-based resistance training routine for 60 min, 3 alternative days a week, during 3 weeks. The program is shown as Supplementary Material. Caloric expenditure was calculated taking into account three components: resting metabolic rate, thermic effect after meals, and physical activity expenditure. The resting metabolism was calculated according to Harris–Benedict equation. The thermal effect of food was estimated as the 8.5 % of the sum of resting metabolic rate plus physical activity expenditure. The physical activity expenditure was estimated from reference tables. Diet was designed using Dietsource software (Novartis, Barcelona, Spain) and adapted to strength exercises (55 % carbohydrates, 25 % lipids, and 20 % proteins). Daily energy intakes were 3,000 ± 95.4 kcal (training days) and 1,947 ± 44.5 kcal (resting days). Daily diet vitamin C intakes were 376.1 ± 17 mg (training days) and 113.7 ± 3 mg (resting days). Daily diet vitamin E intakes were 9.7 ± 0.6 mg (training days) and 4.8 ± 0.4 mg (resting days). The daily vitamin C and E intakes in the AB and AB ? PLX groups, due to its respective almond drink intake, were 451.1 ± 17 mg (training days) and 188.7 ± 3 mg (resting days) for vitamin C, and 34.7 ± 0.6 mg (training days) and 29.8 ± 0.4 mg (resting days) for vitamin E. Finally, vegetable servings were 4–5/ day. Subjects did not take any additional supplements other than the ones provided in this study. Participants were observed 2 days per week in order to supervise diet accomplishment. Capsules of Lippia citriodora extract (PLX, 600 mg/capsule containing 400 mg PLX and 200 mg excipients) and placebo (600 mg crystalline microcellulose/ capsule) were kindly provided by Monteloeder SL (Elche, Spain). Almond beverage supplemented with vitamins (AB) was kindly provided by Liquats Vegetals SL (Viladrau, Gerona, Spain). The drink was elaborated as indicated in [33] and packaged in white cartons observing only the expiration date. The Lippia extract and AB compositions have been extensively characterized in [27, 33, 34]. Anthropometry for body composition, fitness assessment by Burpee test, and blood sampling were performed at the beginning (day 1) and at the end of the intervention

(day 21). Anthropometry was performed according to International Society for Advancement of Kinanthropometry (ISAK) recommendations [35]. Age and anthropometric parameters of the volunteers participating in the study are shown in Table 1. Fitness assessment to control training progression throughout the experiment was analyzed using the Burpee test, which consists in four counts beginning in a standing position. The first count implies dropping in a squat position with the hands on the floor. Then, the legs are extended back in one quick motion to reach the front plank position (count 2). Count 3 consists in returning to the squat position in one quick movement. Finally, the subject returns to the upright standing position (count 4). Each individual was asked to perform the maximum number of repetitions (four counts = one repetition) during 1 min. The number of complete repetitions and cardiac frequency were measured. Three series of the complete test were performed with 2 min recovery in between to ensure muscular exhaustion and thus eccentric damage. Means for repeats and heart rate (HR) were calculated at the end of the session for each individual. Increase in the number of repetitions and changes in HR at the end of the study indicate an improved physical performance. The results of the Burpee test for the four groups are indicated in Table 1. Blood samples were obtained from the antecubital vein after overnight fasting (10–12 h from the last training session) in EDTA vacutainers at day 1 and 21, respectively. Lymphocytes, neutrophils, erythrocytes, and plasma were purified following an adaptation of the method described by Boyum [36]. Circulating glucose was determined by the glucose oxidase method coupled to the peroxidase reaction [37]. Circulating triglycerides were determined as previously shown [38]. Uric acid determination was according to Fossati et al. [39]. Creatinine was determined by Jaffe´ direct reaction [40]. Ferritin was determined using an enzyme-linked fluorescent assay (BioMerieux, Madrid) according to manufacturer’s instructions. Lactate was determined by a lactate oxidase/peroxidase coupled colorimetric reaction [41]. Serum Na? was determined by potentiometry using selective Spotlyte electrodes (Menarini, Badalona, Spain). Plasma proteins, such as creatine phosphokinase (CK), myoglobin (Mb), aspartate aminotransferase/serum glutamic oxaloacetic transaminase (AST/ GOT), alanine aminotransferase/serum glutamic pyruvic transaminase (ALT/GPT), and c-glutamyltranferase (GGT), were measured using automated standard laboratory procedures [42]. All antioxidant enzymatic activities were determined on a microplate reader (SPECTROstar Omega, BMG LabTech GmbH, Offenburg, Germany) at 37 C. SOD, catalase, GPX, and GRD were determined according to [43–46], respectively. Oxidative stress markers, protein carbonyl

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Eur J Nutr Table 1 Age, anthropometric parameters, and results of the Burpee test in the four groups of volunteers participating in the study

Parameter (units)

PLX

PLB

n

8

8

AB ? PLX

AB 9

8

Age (years)

22 ± 2.3

21 ± 1.8

20 ± 0.2

20 ± 0.6

Height (cm)

181 ± 1.9

181 ± 3.5

177 ± 2.2

178 ± 1.9

Weight (kg)

79 ± 2.4

71 ± 4.1

75 ± 2.9

73 ± 2.2

BMI (kg/m2)

23 ± 0.7

21 ± 1.1

23 ± 0.9

23 ± 0.7

Fat mass (%)

12 ± 0.6

13 ± 0.6

12 ± 0.5

13 ± 1.2

Muscle mass (%)

45 ± 1.9

45 ± 0.9

45 ± 2.7

44 ± 1.7

Burpee test Repeats day 1 HR (bpm) day 1 Repeats day 21 HR (bpm) heart rate (beats per min)

HR (bpm) day 21

25.7 ± 0.9

28.2 ± 1.4

30.3 ± 1

30.6 ± 1.3

147.2 ± 4.6

168.7 ± 3.9

153.5 ± 5.2

166.5 ± 3.6

29.9 ± 0.9

30.2 ± 1.5

32.5 ± 1

164.1 ± 4.0

171.8 ± 3.6

derivatives, and malondialdehyde (MDA) were determined according to [47, 48]. Total RNA was isolated from lymphocytes and neutrophils using the Tripure extraction kit (Roche Diagnostics, Barcelona, Spain). RNA (1 lg) was reverse transcribed using 50 U of Expand reverse Transcriptase (Roche Diagnostics) and 20 pmol oligo-dT for 60 min at 37 C in a 20 lL final volume, according to manufacturer´s instructions. cDNA (0.5 lL) was amplified using the LightCycler FastStart DNA MasterPLUS SYBR Green 1 kit (Roche Diagnostics), performed at 95 C/10 s (denaturation), Tm/7 s (annealing) and 72 C/12 s (synthesis) for 40 cycles, using the following Tm and primers: •

Cu–Zn-SOD (Tm = 55 C): Forward: 50 -GCCAAAGGATGAAGAGAGGCAT G-30 Reverse: 50 -GCGGCCAATGATGCAATGGT-30



Mn-SOD (Tm = 59 C): Forward: 50 -GTGTCCAAGGCTCAGGTTGGGG-30 Reverse: 50 -GGAATAAGGCCTGTTGTTCCTTGCAG-30



Catalase (Tm = 60 C): Forward: 50 -TTTGGCTACTTTGAGGTCAC-30 Reverse: 50 -TCCCCATTTGCATTAACCAG-30



GPX (Tm = 58 C): Forward: 50 -GCCTGCAGCTGTGTAGTGCTGG-30 Reverse: 50 -GCTGGTTTTTCCTTTGGGTTTAGG TG-30



GRD (Tm = 65 C): Forward: 50 -CAAGGAAGAAAAGGTGGTTGGGA TC-30

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171 ± 3.4

32.3 ± 1.6 174.2 ± 3.1

Reverse: 50 -GTCAAAGTCTGCCTTCGTTGCTCC30 The relative quantification was performed using the 2 method [49], normalized to 36B4 rRNA as the non-variant control (Tm = 53 C): ðDDct Þ

Forward: 50 -CTTCCTGGAGGGTGTCCGCAAT-30 Reverse: 50 -GGGAAGGTGTAATCCGTCTCCACA-30

Outcomes The primary endpoint was to assess whether antioxidant supplementation can modulate immediate antioxidant response, by analyzing SOD, catalase, GPX, and GRD activities in volunteers. The secondary endpoint was to assess whether the same supplements can regulate longterm adaptation processes, relying on the expression of the corresponding genes in the same population. Likewise, since we needed to monitor the health status of each individual during the study and the efficacy of the antioxidant response, the study was complemented with determinations of circulating parameters and measurements of oxidative stress markers.

Sample size We considered a big (Cohen) effect size (d = 1), with a two-sided 5 % significance level and a power of 80 % for age, body mass index (BMI), fat mass, and muscle mass (n = 7 in each case per group, except in muscle mass where n = 10). Due to the limited size (n \ 10), onesample K–S test (Kolmogorov–Smirnov test) was performed in order to assess that each sample fits to a normal distribution. Statistical significance: p \ 0.05.

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The only rule considered to stop the study was related to a drastic decline in the number of valid observations necessary to obtain statistically significant results. In addition, a particular subject could be excluded from the study for the following reasons: protocol violations, muscle lesions, and refuse to follow the study due to personal reasons. Participants’ selection A total of 119 male students were screened for the study in the university. Participants were considered eligible if they met the above-mentioned inclusion criteria. The assignment to the distinct groups was carried out following a near homogeneity between groups in terms of age, body composition, and training routine (see Table 1). The assignment was performed by the research team. A single-blind study was performed, where the participants were unaware of the type of supplement they were receiving. The data obtained were collected, blinded, and statistically analyzed by two independent researchers that were not in contact with the volunteers. Statistical methods Statistical analysis was carried out using the SPSS-10 software for Windows. Results were expressed as the mean ± SEM, and the values with a p \ 0.05 were considered statistically significant. The effect of supplementation (PLX, AB or AB ? PLX) on the changes induced by the weightlifting routine was tested by a two-way ANOVA with the supplementation (S) and the 3 weeks of eccentric exercise (E) as factors. The sets of data in which there were significant effects were tested by the one-way ANOVA test.

Results The aim of this study was to assess the effect of different polyphenolic compounds as antioxidant supplements in athletes, analyzing the basal response and adaptation of the major antioxidant enzymes, i.e., SOD, catalase, GPX, and GRD in neutrophils and lymphocytes. To this end, an intervention study was performed with university volunteers performing regular training sessions of eccentric weight lifting during 3 weeks and either consuming a placebo or antioxidants in the form of supplements such as a Lippia citriodora extract (PLX), an almond beverage enriched in vitamins C and E (AB) or a mixture of both supplements (AB ? PLX). These compounds have been developed in our laboratories, and their effect on performance in extensive aerobic exercise has been previously described [7, 28–31]. The recruitment process began in

October 2007, and the intervention was carried out in February 2008. Participant flow is shown in Fig. 1. Neutrophil and lymphocyte oxidative damage during training period The first question in this research was to know the extent of oxidative damage produced during the 21-day training period by determining MDA and protein carbonyls as markers of oxidized lipids and proteins, respectively. The training program formed part of the pre-season routine of the volunteers that participated in the study and contained a high percentage of eccentric exercises that produced oxidative damage [50]. In this context, the cellular compartment (neutrophils and lymphocytes) presented higher values of oxidative stress markers than the plasmatic compartment (Table 2). In addition, there was no difference in MDA expression among the different groups of volunteers, except for a protective effect in the AB group (Table 2a). Similar high values were observed for protein carbonyls in the PLB, PLX, and AB groups. However, a modest but significant increase, according to the ANOVA test, was observed in AB ? PLX group (Table 2a). Regarding lymphocytes, the eccentric routine increased the MDA but not carbonyl levels in the PLB group (Table 2b). Nevertheless, a reduction in MDA levels and protein carbonyls were observed in the PLX group (Table 2b). In addition, the consumption of AB and AB ? PLX avoided the increase of MDA due to eccentric exercise (Table 2b). Nevertheless, high levels of oxidative alterations were detected in both cell types when compared with plasma (Table 2c), the medium that surrounds neutrophils and lymphocytes. Oxidative changes were detected in plasma due to exercise performance according to the ANOVA test, as indicated by increased MDA levels that were mitigated only in the group consuming PLX. Also, protein carbonyl levels were different in the AB ? PLX group compared to the rest of the groups. Concerning other circulating parameters obtained from the blood analysis, the following changes were observed in the placebo and experimental groups (Table 3). A slight but significant decrease in fasting glycaemia (around 4 %) was observed in all groups. Also, a significant increase (around 20 %) in circulating uric acid levels was observed in all groups. These small changes in glycaemia and uric acid were due to exercise performance according to the ANOVA test. In any case, the above-mentioned parameters presented normal values for healthy individuals. Regarding circulating proteins that could reflect the status of tissue integrity, there were no major changes in the different markers, except for Mb, which is a marker for

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R E C R U I T M E N T

A S S I G N M E N T S

M O N I T O R I N G

Selected for evaluation (n=119) Excluded (n= 79) •Did not meet the selection criteria

Randomized (n=40)

Assigned to the intervention: PLB group (n=10)

Assigned to the intervention: PLX® group (n=10)

Interrupted the intervention (n=2)

Interrupted the intervention (n=2)

Interrupted the intervention (n=1)

Interrupted the intervention (n=2)

Motive: voluntarily declined due to personal reasons (n=1) and muscle lesions (n=1)

Motive: muscle lesions (n=2)

Motive: protocol violations (n=1)

Motive: voluntarily declined due to personal reasons (n=1) and protocol violations (n=1)

Analysed (n=8)

Analysed (n=8)

A N A L Y S I S

Assigned to the intervention: AB group (n=10)

Analysed (n=9)

Assigned to the intervention: AB+PLX® group (n=10)

Analysed (n=8)

Fig. 1 Participant flow diagram

muscle and heart damage. Circulating Mb levels were not significantly changed in PLB, AB, or AB ? PLX groups. Only the PLX consuming group presented a *20 % decrease in circulating Mb, suggesting a protective effect of this supplement in muscle tissue. Finally, no significant changes in platelet counting were observed, except for the AB ? PLX group, where a modest *7 % increase was detected. Antioxidant response of neutrophils and lymphocytes to exercise-induced oxidative stress Antioxidant enzymes play an instrumental role in preventing oxidative damage by scavenging ROS. The first line of defense is SOD (both cytosolic and mitochondrial) which converts the superoxide anion (O2 ) produced in intracellular oxidative processes, mainly the mitochondria, to hydrogen peroxide (H2O2). The activity level of this enzyme was decreased in the neutrophils of the PLB group (Table 4a), although not in the lymphocytes (Table 4b).

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However, the presence of the different supplements seemed to significantly increase (PLX group) or maintain (ABand AB ? PLX-groups) SOD activity values in neutrophils (Table 4a). Regarding lymphocytes, SOD activity did not differ in the PLX–AB and AB ? PLX groups (Table 4b). After the SODs produce H2O2, it is generally detoxified by other antioxidant enzymes, mainly catalase or the GPX– GRD tandem. Catalase degrades H2O2, producing O2 and H2O. While catalase activity did not differ among the placebo and experimental groups (Table 4a, b), significant differences were observed in the GPX–GRD. These two antioxidants work together to remove hydrogen peroxide, relying on the reduction of glutathione from GSSG to GSH. Regarding neutrophils, there were no changes in GPX levels in AB and AB ? PLX groups; however, PLB- and PLX groups did present significant changes, due to exercise performance according to the ANOVA test (Table 4a). On the other hand, GRD tended to decrease in all groups except for the PLX consuming group where the

Eur J Nutr Table 2 Oxidative markers in neutrophils (a), lymphocytes (b), and plasma (c) due to supplement consumption (S), to exercise training (E) or to an interaction between both factors (S 9 E), according to ANOVA test

Marker

Group

Day 1

Day 21

S

18.3 ± 4.5

E

S9E

a) Neutrophils MDA (mmols/L)

Protein carbonyls (mmols/L)

PLB

19.8 ± 4.1

PLX

18.2 ± 1.2

20.0 ± 0.2

AB

20.7 ± 1.7

13.7 ± 0.8§

AB ? PLX

20.7 ± 2.0

17.7 ± 3.5 10.7 ± 1.3

PLB

11.3 ± 1.1

PLX

12.3 ± 1.3

12.0 ± 1.0

AB

11.3 ± 0.8

12.7 ± 0.3

AB ? PLX

11.7 ± 0.8

16.3 ± 0.8}

PLB

24.3 ± 2.8

35.3 ± 3.3}

27.3 ± 1.4

18.3 ± 1.9§,}

AB

24.6 ± 2.6

28.6 ± 3.2

AB ? PLX

27.0 ± 3.0

25.3 ± 2.8

PLB PLX

17.7 ± 1.3 17.3 ± 0.9

19.0 ± 0.9 12.3 ± 1.1§,}

*

*

b) Lymphocytes MDA (mmols/L)

PLX

Protein carbonyls (mmols/L)

Intracellular MDA and protein carbonyl concentrations were calculated assuming a value of 300 lL/109 cells [60] * Value is significantly different (p \ 0.05) according to ANOVA test, § Significant differences due to supplement consumption compared to placebo, } Significant differences due to exercise training



AB

15.0 ± 0.8

13.0 ± 0.7

AB ? PLX

14.3 ± 0.6

15.5 ± 0.6

PLB

161 ± 33

249 ± 29}

174 ± 9

91 ± 9§,}

* *

*

*

*

*

*

*

*

c) Plasma MDA (lmols/L)

PLX

Protein carbonyls (lmols/L)



}

* *

AB

175 ± 24

338 ± 5

AB ? PLX

198 ± 25

288 ± 9}

94 ± 3

98 ± 2

104 ± 2

101 ± 1

98 ± 1

99 ± 1

87 ± 1

100 ± 2}

PLB PLX AB 

AB ? PLX

activity values were maintained (Table 4a). Regarding lymphocytes, GPX did not present significant changes in the different groups during the experiment (Table 4b). Nevertheless, GRD activity was decreased in all groups due to exercise performance according to ANOVA test, being significant only in PLB, PLX, and AB ? PLX groups (Table 4b). Antioxidant adaptation of neutrophils and lymphocytes to exercise-induced oxidative stress Gene expression of the antioxidant enzymes was also analyzed by qRT-PCR in both neutrophils and lymphocytes. In the case of neutrophils, the PLB group presented at day 21 increased Mn-SOD gene expression levels (Fig. 2b) and decreased GRD (Fig. 2d), suggesting that exercise could be responsible for these changes. In addition, both SOD genes and GRD, but not GPX, were significantly lower at day 21 compared to day 1 only in the AB ? PLX group. Finally, the consumption of PLX or AB seemed to maintain at the end of the study, and the

* *

*

expression values were observed at day 1 for Cu–Zn-SOD, Mn-SOD, GPX, and GRD genes (Fig. 2a–d). Regarding lymphocytes, there were no significant differences in the expression levels of Cu–Zn-SOD, Mn-SOD, catalase, GPX, and GRD genes in any of the groups (not shown). Results observed in lymphocytes are reinforced by the data presented in Table 4c, indicating that erythrocytes, an anucleated cell where gene modulation events do not occur, present an almost similar pattern of enzyme activities for the GPX-GRD tandem in all groups of volunteers, with the exception of an increased GPX activity only in the PLX group.

Discussion Although one of the limitations of our study is the low number of participants, their homogeneity was very high in terms of gender, age, body composition, and training routine. In addition, participants were university students practicing different rowing disciplines at regional and

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123 139.6 ± 0.3

140.6 ± 0.4

22.1 ± 3.7 19.3 ± 0.9

AST/GOT (U/L)

ALT/GPT (U/L)

81.5 ± 8.0

179.1 ± 14.9

23.2 ± 1.8

23.4 ± 2.9

24.1 ± 2.3

29.1 ± 2.9

152.7 ± 12.8

19.5 ± 0.9

25.0 ± 3.6

24.5 ± 3.7

32.5 ± 2.6

81.5 ± 7.6

135.7 ± 0.6

9.5 ± 0.6

4.2 ± 0.1

0.92 ± 0.1

151.2 ± 7.1

81.7 ± 0.5 89.3 ± 11.3

2.3 ± 0.2

3.1 ± 0.2

213.1 ± 9.0

40.6 ± 0.6

46.3 ± 0.4

15.2 ± 0.2

5.2 ± 0.1

1

PLX group

180.3 ± 15.0

22.9 ± 1.9

22.9 ± 2.7

21.9 ± 2.6

26.5 ± 3.3**

78.2 ± 8.3

139.1 ± 0.2

8.9 ± 0.3

5.6 ± 0.5*

0.91 ± 0.1

153.9 ± 7.4

79.7 ± 0.4* 76.1 ± 6.3

2.3 ± 0.2

3.3 ± 0.4

219.6 ± 10.4

40.9 ± 0.8

45.3 ± 0.2

15.8 ± 0.6

5.4 ± 0.1

21

147.3 ± 13.7

18.8 ± 2.5

23.1 ± 2.2

23.4 ± 1.9

34.8 ± 1.5

83.5 ± 8.4

139.0 ± 0.1

8.6 ± 0.5

4.3 ± 0.3

0.97 ± 0.0

153.0 ± 7.4

82.5 ± 0.7 62.6 ± 5.5

2.1 ± 0.1

3.4 ± 0.1

205.8 ± 9.5

42.5 ± 0.5

46.3 ± 0.4

15.2 ± 0.1

5.6 ± 0.1

1

AB group

182.2 ± 18.7

21.2 ± 1.8

24.0 ± 1.7

19.9 ± 1.6

38.6 ± 3.4

81.9 ± 6.9

140.6 ± 0.3

9.2 ± 0.7

5.6 ± 0.2*

1.08 ± 0.0

149.3 ± 3.8

79.4 ± 1.1* 64.0 ± 9.1

2.6 ± 0.1

3.6 ± 0.3

209.7 ± 8.4

42.9 ± 0.4

46.1 ± 0.4

15.1 ± 0.1

5.4 ± 0.1

21

151.6 ± 14.3

19.5 ± 1.8

23.5 ± 2.8

18.4 ± 1.8

36.7 ± 1.8

94.5 ± 8.5

139.9 ± 0.2

8.5 ± 0.8

4.5 ± 0.1

92 ± 0.0

144.6 ± 4.8

81.8 ± 1.3 68.1 ± 9.3

2.4 ± 0.1

2.9 ± 0.1

211.2 ± 4.5

41.0 ± 0.5

43.9 ± 0.3

14.5 ± 0.2

5.1 ± 0.1

1

AB ? PLX group

180.9 ± 17.1

20.9 ± 2.3

23.9 ± 3.6

21.0 ± 1.3

33.3 ± 3.9

94.6 ± 8.9

140.0 ± 0.3

8.9 ± 0.6

5.9 ± 0.2*

1.09 ± 0.1

131.0 ± 4.5

80.0 ± 1.1* 64.1 ± 8.1

2.6 ± 0.1

3.0 ± 0.2

224.5 ± 8.5**

40.9 ± 0.5

44.6 ± 0.3

14.8 ± 0.1

5.2 ± 0.0

21

** Significant differences at p \ 0.05, due to supplement consumption according to ANOVA test

* Significant differences at p \ 0.05, due to exercise performance according to ANOVA test

AST/GOT aspartate aminotransferase/serum glutamic oxaloacetic transaminase, ALT/GPT alanine aminotransferase/serum glutamic pyruvic transaminase, CK creatin phosphokinase, GGT cglutamyltransferase, and RDW-SD red cell distribution width

143.2 ± 10.8

22.6 ± 3.0

GGT (U/L)

CK (U/L)

83.8 ± 7.1 28.7 ± 2.9

Ferritin (ng/mL)

Myoglobin (ng/mL)

Circulating proteins

Na (mEq/L)

9.1 ± 0.9

9.3 ± 0.7

Lactate (mg/dL)

5.1 ± 0.3*

4.3 ± 0.4

Uric acid (mg/dL)

0.94 ± 0.1

164.1 ± 4.8

0.93 ± 0.0

152.3 ± 5.8

78.4 ± 0.5* 64.9 ± 3.7

2.4 ± 0.2

3.4 ± 0.4

Creatine (mg/dL)

Total cholesterol (mg/dL)

Glucose (mg/dL) Triglycerides (mg/dL)

82.3 ± 0.6 61.1 ± 3.3

2.3 ± 0.2

Lymphocytes (103 cells/lL)

Circulating metabolites/elements

3.2 ± 0.1

Neutrophils (103 cells/lL)

194.9 ± 7.9

40.2 ± 0.9

217.0 ± 3.1

41.9 ± 0.6

Platelets (103 cells/lL)

RDW-SD (fL)

44.7 ± 0.5

45.0 ± 0.5

Hematocrit (%)

15.0 ± 0.9

5.2 ± 0.2

21

14.8 ± 0.6

5.3 ± 0.1

1

PLB group

Haemoglobin (g/dL)

Erythrocytes (106 cells/lL)

Hemogram

Day

Parameter (units)

Table 3 Haematological and serum parameters determined in the four experimental groups of volunteers

Eur J Nutr

Eur J Nutr Table 4 Antioxidant enzymatic activities in neutrophils (a), lymphocytes (b), and erythrocytes (c) due to supplement consumption (S), to exercise training (E) or to an interaction between both factors (S 9 E), according to ANOVA test Enzymatic activity

Group

Day 1

Day 21

S

E

S9E

a) Neutrophils SOD (pkat/109 cells)

32.6 ± 5.1

12.6 ± 1.8}

PLX

40.6 ± 4.7

52.4 ± 4.2§,}

AB

24.4 ± 2.9

32.5 ± 5.3

PLB 

Catalase (k5/ 109 cells)

9

GPX (nkat/10 cells)

GRD (nkat/109 cells)

* *

*

AB ? PLX

41.0 ± 3.3

33.1 ± 4.3

PLB

52.8 ± 10.2

35.1 ± 4.0

PLX AB

38.6 ± 6.0 42.3 ± 3.0

29.9 ± 4.1 20.2 ± 2.7

AB ? PLX

31.0 ± 3.1

29.4 ± 4.5

PLB

88.8 ± 9.8

206.4 ± 29.5}

*

PLX

99.7 ± 12.1

161.5 ± 5.4}

*

AB

149.1 ± 23.5

122.2 ± 12.8

AB ? PLX

138.6 ± 20.9

123.6 ± 11.0

PLB

1,400 ± 147.3

676.0 ± 148.6}

PLX

1,335 ± 244.6

1,407 ± 158.4

AB

1,645 ± 216.3

762.3 ± 138.8}

*

AB ? PLX

1,270 ± 199.1

780.2 ± 87.6}

*

53.7 ± 5.6



*

*

b) Lymphocytes SOD (pkat/109 cells)

Catalase (k5/109 cells)

PLB

34.4 ± 5.5

PLX

41.1 ± 3.1

59.2 ± 6.2

AB

59.3 ± 5.7

66.6 ± 7.9

AB ? PLX PLB

37.9 ± 4.8 51.0 ± 9.2

34.9 ± 4.4 22.8 ± 2.5

PLX

32.4 ± 4.9

27.5 ± 3.8

AB

48.5 ± 7.1

58.0 ± 4.4

AB ? PLX 9

GPX (nkat/10 cells)

PLB

GRD (nkat/10 cells)

64.2 ± 5.5 123.0 ± 18.0

PLX

68.4 ± 6.5

89.1 ± 9.8

AB

99.2 ± 12.4

71.6 ± 7.5

AB ? PLX 9

51.3 ± 0.2 122.8 ± 21.6

71.8 ± 9.1

58.6 ± 4.7

PLB

1,046 ± 185.7

332.5 ± 39.7}

*

PLX

1,029 ± 238.0

560.2 ± 65.8}

*

AB

512.1 ± 197.0

355.2 ± 44.9

AB ? PLX

479.3 ± 88.8

268.6 ± 38.6}

*

15.9 ± 2.0

10.2 ± 1.7}

*

PLX

26.2 ± 0.7

16.9 ± 1.2}

*

AB AB ? PLX

21.6 ± 1.8 25.4 ± 1.7

30.7 ± 2.4 24.7 ± 1.5 2.8 ± 0.3

c) Erythrocytes SOD (pkat/g Hb)

PLB 

Catalase (k5/g Hb)

GPX (nkat/g Hb)

PLB

3.0 ± 0.2

PLX

2.8 ± 0.1

2.9 ± 0.1

AB

2.7 ± 0.1

2.7 ± 0.2

AB ? PLX

2.8 ± 0.2

2.4 ± 0.1

PLB

29.7 ± 2.8

24.4 ± 1.4

PLX

28.0 ± 2.3

61.5 ± 14.4§,}

AB

21.0 ± 2.1

16.9 ± 1.1

AB ? PLX

20.6 ± 0.8

18.1 ± 1.0

*

*

*

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Eur J Nutr Table 4 continued Enzymatic activity

Group

GRD (nkat/g Hb)

PLB

Day 1

Day 21

S

E

56.4 ± 7.4

21.9 ± 3.6}

*

PLX

48.7 ± 7.8

28.7 ± 4.4}

*

AB AB ? PLX

63.1 ± 13.8 51.3 ± 8.0

15.3 ± 2.9} 18.9 ± 3.2}

* *



S9E

Significant differences (p \ 0.05) according to ANOVA (*), due to supplement consumption compared to PLB (§) and to exercise training (}) Hb haemoglobin

Fig. 2 Relative levels of neutrophil antioxidant enzyme mRNAs. Figure depicts the mRNA levels of the PLB group (white), PLX group (black), AB group (dashed) and AB ? PLX group (dotted) at the end of the experiment (day 21). The mRNA levels at the beginning of each experimental period (day 1) were arbitrarily referred to as 1. (*) Significant differences (p \ 0.05) with respect to day 1

2.5

7.0

A

B

*

6.0

2.0

5.0 1.5

4.0 3.0

1.0

2.0

*

0.5 0

1.0

*

0

Cu-Zn-SOD 2.0

Mn-SOD 1.5

C

D

1.5 1.0 1.0 0.5

*

*

0.5

0

0

GPX

national levels. Therefore, we can state that the results are applicable to young people with similar lifestyles, training, and eating habits. In Spain, young people represent around 15 % of the population In this context, a large variety of metabolites, peptides, and enzymes are involved in the antioxidant process in response to exercise. We have focused our interest in the modulation of activities and gene expression levels of the main antioxidant enzymes in neutrophils and lymphocytes of volunteers taking polyphenol-based antioxidant supplements (provided in the form of PLX, AB or a mixture of both) while performing eccentric exercises. The training routine followed in this study provoked oxidative damage in the lipid component (evidenced by MDA increases) of lymphocytes and plasma (Table 2b, c). The PLX consuming group presented a positive antioxidant potentiating effect, reducing oxidative damage in this particular cell type (Table 2b).

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GRD

The first consideration is that the consumption of the supplements does not cause apparently main alterations in participants, regarding circulating parameters. All significant changes were considered normal for healthy individuals. Nevertheless, modest but significant changes were observed for platelet number in the AB ? PLX group. Since platelets present certain growth factors, a plausible explanation to this observation is that the modest increase in platelet number with this supplement may favor connective tissue repair [51]; however, this needs to be confirmed with additional experiments. As in other studies [18, 25], we have considered that the antioxidant response to exercise has two components. The first consists in the antioxidant response of the cells in the moment of stress, while the second is the adaptation by genetic modulating processes when there is a persistent pro-oxidant situation. The latter case takes more time to come in effect, since it requires transcriptional and

Eur J Nutr

translational events, but lasts at least until the situation is normalized. Throughout the experimental design, we have stated differences between modification of enzymatic activities just after the acute administration of food or supplements, or adaptation due to modulation of gene expression leading to a new protein scenario. The first event is immediate, occurs between the first 1–4 h (depending on metabolite pharmacokinetics) following administration, and is due to the direct effect of the metabolites on the enzymes. We have reported that verbascoside, the main component of lemon verbena extract (PLX), shows a short term activation effect on blood cells GRD which may be explained by the observed in vitro activation of the enzyme by verbascoside [28]. In contrast, the modulation of gene expression by metabolites is a much slower process and depends on the synthesis/degradation rates of mRNA and proteins. Experiments oriented to determine how fast target proteins are expected to change in response to a sudden block in miRNA synthesis in mammalian cells estimate that protein levels would have recovery times ranging from 24 to 140 h, depending on their mean half-life [52, 53]. It is generally accepted that impaired adaptation to oxidative stress, reflected in low levels of antioxidant enzymes, results in a poor response. This has been extensively studied in protocols that administered large doses of vitamins C and E [1, 18, 25]. Nevertheless, this assumption is more complex with other antioxidants, such as polyphenols, as these structures present a high degree of variability and likely multitargeted cell functions [54–58]. Theoretically, an adequate antioxidant supplement should reinforce the endogenous response by, for example, enhancing antioxidant enzyme activities, while not interfering with the intracellular adaptation that depends on gene expression processes. Nevertheless, both aspects need to be analyzed in each particular situation since the modulation of the antioxidant response and adaptation depend on many variables that include the type of exercise, intensity, frequency, recovery time, diet, and supplement dose. Moreover, the different cell types and body systems respond and adapt differently to a specific oxidative insult. In the present study, it is difficult to define a clear pattern. Nevertheless, polyphenolic antioxidants seem to act similarly to antioxidant vitamins, where excess dose can interfere with the adaptation response (assessed by gene expression analysis). However, our study also indicates that the effect depends on the cell type analyzed and may differ considerably. The first observation is that neutrophils and lymphocytes present a different adaptation pattern for the same exercise routine and supplementation. In particular, the expression of the analyzed genes was similar in the lymphocytes of all the control and experimental groups. This possibly indicates that the changes on enzyme activity

may be modulated by the influence of exercise, as it is the case of GRD in all groups. Interestingly, in erythrocytes, a cell that does not present acute gene modulation events in its mature phenotype (mean half-life = 120 days), the pattern observed for GPX–GRD activities was very similar to that observed in lymphocytes. Therefore, these results suggest that lymphocytes and erythrocytes do not present an antioxidant adaptation response regarding these particular genes, thus basing the antioxidant defense in the enzymatic activities. Conversely to lymphocytes, neutrophils presented some antioxidant responses (dependent on enzymatic activities) in parallel with antioxidant adaptations (dependent on gene expression levels). For instance, a decreased GRD enzymatic activity and gene expression were observed in PLBand AB ? PLX groups. Nevertheless, this parallelism was not observed in the rest of the cases for GPX and GRD, suggesting that the supplements taken together do not seem to allow a proper adaptation. This may possibly be due to an excess of antioxidant intake (Table 4a; Fig. 2) that interferes with antioxidant response, as observed in studies administering high levels of antioxidant vitamins [1, 18, 25]. On the other hand, the results obtained with SOD activity and gene expression are difficult to interpret, due to the impossibility to distinguish between the cytosolic (Cu– Zn-SOD) and the mitochondrial (Mn-SOD) forms at the level of enzymatic activities. This study agrees with previous reports, that antioxidant excess can impair the adaptation response (i.e., see the effect of AB ? PLX on Cu–Zn-SOD, Mn-SOD genes as well as in GRD gene). Nevertheless, impaired adaptation of intracellular antioxidant defenses (monitored by alteration of gene expression events) has been well documented after administration of megadoses of vitamins C and E [59]. On the other hand, fewer evidences have been reported in the case of administration of high doses of polyphenols and non-vitamin antioxidants. These include carotenoids (known antioxidants), isoflavones (due to the estrogen-like activity), and epigallocatechin-3-gallate (causing hepatotoxicity) [59]. Therefore, our report suggests that high doses of polyphenols seem to interfere in a different manner in the antioxidant gene induction, replacing but not avoiding the antioxidant defense system that is exerted mainly by the exogenous compounds. Finally, it must be noted that catalase levels did not change at any level or in any cell type regardless of the group analyzed. This suggests that catalase acts constitutively and is possibly the principal pathway for ROS detoxification in this particular exercise routine. In addition, supplement consumption does not interfere with the training exercise routine since the same results in the Burpee test can be observed between PLB group and the rest of groups (Table 1). In any case, we can speculate that

123

Eur J Nutr

this antioxidant response and adaptation could also be involved in tissue recovery events. However, the antioxidant response is complex, and it is necessary to perform additional experiments in order to study alternative antioxidant pathways and other oxidative alterations. In conclusion, the precise dose of polyphenols in the form of dietary supplements remains to be elucidated, regarding the large variety of chemical structures exhibited by these compounds and the candidate functions, aside from antioxidant protection, that can be exerted in the different body systems. Our results support the general idea that free radicals are instrumental as intracellular transducers in order to elicit an antioxidant adaptation by means of gene modulation events. High doses of polyphenols seem to interfere in a different manner in the antioxidant gene induction depending on the cell type studied, as well as most likely the exercise routine performed. In addition, the antioxidant response (depending on the enzymatic activities present in the cell) and adaptation process (depending on gene expression events) in sport performance and recovery are complex mechanisms that do not necessarily go hand in hand. Acknowledgments We thank Jose Maria Adsuar for technical assistance in blood analysis. This work was supported by grants from Spanish Science Ministry AGL2007-62806/ALI to AP, AGL200760778 and AGL 2011-29857-C03-03 to VM and PROMETEO/2012/ 007 from Generalitat Valenciana to VM and ER. ER is recipient of Instituto de Salud Carlos III-FEDER (PS09/01093) and Fundacion Salud 2000-Merck Serono grants. AP, VM and ER are members of the ‘‘Centro de Investigacio´n Biome´dica en Red de Fisiopatologı´a de la Obesidad y Nutricio´n’’ CIBERobn (CB12/03/30038). LC-Q and LF were recipients of CONACYT-Mexico (ref 197139) and FPI (Spanish Science Ministry) fellowships, respectively. Conflict of interest Authors declare that there are no conflicts of interest.

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Effect of polyphenol supplements on redox status of blood cells: a randomized controlled exercise training trial.

The effect of endogenous antioxidants can be either an immediate response (relying on enzymatic activities) or a long-term adaptation (relying on gene...
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