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Journal of Sports Sciences Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/rjsp20
Effect of sulphurous mineral water in haematological and biochemical markers of muscle damage after an endurance exercise in well-trained athletes a
a
b
a
a
Marisol Soria , Carlos González-Haro , Santiago Esteva , Jesús F. Escanero & José R. Pina a
Pharmacology and Physiology Department, School of Medicine, University of Zaragoza, Zaragoza, Spain b
Physiology and Immunology Department, School of Biology, University of Barcelona, Barcelona, Spain Published online: 06 Feb 2014.
To cite this article: Marisol Soria, Carlos González-Haro, Santiago Esteva, Jesús F. Escanero & José R. Pina (2014) Effect of sulphurous mineral water in haematological and biochemical markers of muscle damage after an endurance exercise in welltrained athletes, Journal of Sports Sciences, 32:10, 954-962, DOI: 10.1080/02640414.2013.868921 To link to this article: http://dx.doi.org/10.1080/02640414.2013.868921
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
Journal of Sports Sciences, 2014 Vol. 32, No. 10, 954–962, http://dx.doi.org/10.1080/02640414.2013.868921
Effect of sulphurous mineral water in haematological and biochemical markers of muscle damage after an endurance exercise in well-trained athletes
MARISOL SORIA1, CARLOS GONZÁLEZ-HARO1, SANTIAGO ESTEVA2, JESÚS F. ESCANERO1, & JOSÉ R. PINA1 1
Pharmacology and Physiology Department, School of Medicine, University of Zaragoza, Zaragoza, Spain and 2Physiology and Immunology Department, School of Biology, University of Barcelona, Barcelona, Spain
Downloaded by [University of Otago] at 02:46 03 October 2014
(Accepted 20 November 2013)
Abstract To investigate the effects of sulphurous mineral water (SMW) after a hydroponic treatment on muscle damage, antioxidant activity and peripheral blood changes induced by submaximal exercise. Thirty well-trained male triathletes were supplemented with SMW or placebo: 3 weeks of placebo, 30 days of wash out and 3 weeks of SMW. After both periods, participants ran for 2 h at 70% maximal aerobic speed. Antioxidant enzymes, lipid peroxidation, antioxidant capacity and blood cell markers were compared between placebo and SMW at pre-exercise (T0), immediately post-exercise (T1), 24 h post-exercise (T2) and 48 h post-exercise (T3). Total thiols decreased until T3 vs. T0 for both placebo and SMW; transient red blood cells, haemoglobin and haematocrit increased were shown at T1 vs. T0 and for leucocytes until T2 vs. T0, only for placebo group. Total thiols increased significantly in SMW vs. placebo at T0; Thiobarbituric acid reactive species was significantly higher at T0, T1, T2 and T3; catalase increased significantly at T1; creatine phosphokinase decreased significantly at T1, T2 and T3, although no significant differences were found at T0. Furthermore, red blood cells, haemoglobin and haematocrit were significantly higher and leucocytes were significantly lower at T0 and T1 in SMW group vs. placebo group. This study suggests that three weeks of SMW supplementation may protect from exercise-induced muscle damage. Keywords: hydroponic treatment, muscle damage, lipid peroxidation, ergogenic aid
Introduction Different authors have observed therapeutic and preventive benefits using sulphurous waters for a large variety of inflammatory diseases affecting the respiratory tract, skin, liver and skeletal muscle (Constantino, Lampa, & Nappi, 2006; Gupta & Nicol, 2004; Sukenik, Flusser, & Abu-Shakra, 1999). Furthermore, it has been related with pain reduction in osteoarthritis patients (Kovács et al., 2012). For the last few years, it has been documented that some sulphide (HS)-based therapies may play an important role in antioxidant strategies against oxidative damage associated with degenerative diseases (Casetta, Govoni, & Granieri, 2005). Although an excess of free radicals leads an oxidative stress state provoking negative biological disruptions for sport performance, certain level of free radicals are needed to maintain the cellular redox balance (Nikolaidis et al., 2008, 2012). Some authors have suggested that exercise increase oxygen uptake raising free radicals production such as superoxide and hydroxyl, highly reactive. These substances act against some
cell structures and produce lipid peroxidation, protein oxidation, deoxyribonucleic acid damage and muscle damage (Alessio, 1993). Furthermore, a decrement in antioxidant capacity of tissues and whole blood is produced (oxidative stress) (Alessio, 1993; Nikolaidis et al., 2008, 2012). In that point, it has been suggested that plasma thiobarbituric acid reactive species are related with lipid peroxidation of cell membranes elicited by peroxyl radicals (Alessio, 1993). Also, total thiols contribute significantly to the antioxidant capacity of plasma (Wayner, Burton, Ingold, Barclay, & Locke, 1987). Plasma protein-bound sulphhydryl groups are said to provide the first line of defence during the peroxyl radical attack (Inayama, Kumagai, Sakane, Saito, & Matsuda, 1996; Thomas, Poland, & Honzatko, 1995). It has been reported that plasma protein-bound sulphhydryl groups contributed significantly to the plasma antioxidant capacity after the exposure of human plasma to aqueous peroxyl radicals, and the sulphhydryl groups prevented plasma lipid peroxidation (Wayner et al., 1987).
Correspondence: Carlos González-Haro, Pharmacology and Physiology Department, School of Medicine, Zaragoza, Spain. E-mail: [email protected] © 2014 Taylor & Francis
Downloaded by [University of Otago] at 02:46 03 October 2014
Effects of sulphurous mineral water on muscular damage Some authors have suggested that the antioxidant status is related with the attenuation of free radical levels, reduction of oxidation damage in general and with muscle damage specifically (Nikolaidis et al., 2008, 2012). The semi-essential amino acid cysteine is the main source of the free sulphhydryl group of glutathione and limits its synthesis. Glutathione serves multiple functions in protecting tissues from oxidative damage and keeping the intracellular environment in the reduced state (Meister, 1983). The current information in scientific literature suggests that some substances increase the cysteine availability promoting glutathione synthesis such as N-acetyl cysteine (Zembron-Lacny, Slowinska-Lisowska, Szygula, & Witkowski, 2008), branched-chain amino acids (Coombes & McNaughton, 2000) or whey protein (Micke, Beeh, Schlaak, & Buhl, 2001). However, no studies have addressed the effects of sulphurous mineral water (SMW) in peripheral blood changes, muscle damage and oxidative response when exercise performed. Thus, the aim of the present study was to investigate the effects of SMW supplement on haematological parameters, biochemical markers of muscle damage and oxidative response in well-trained athletes after induced exercise.
Methods Participants Thirty well-trained male triathletes (age 27.3 ± 4.1 years, height 174.6 ± 7.2 cm, body mass 73.5 ± 4.5 kg, body mass index 24.2 ± 1.7 kg · m−2, years of experience 4.5 ± 1.7 years) participated in this study. All participants were informed verbally and in writing about the nature of the study, including all potential risks. Written informed consent was obtained prior to participation. Also, each participant completed a personal information sheet and a standard medical history form. The inclusion criteria were: age ranged between 18–31 years; participants free of any type of injury and under the same
A week
1
programme of training; participants were not allowed to intake any ergogenic dietary supplement during one month before the starting the study. This study was conducted according to the Helsinki Declaration for the Ethical Treatment of Human Subjects (last modified in 2004), and the institutional review board gave ethics approval.
Study design This study was conducted as a prospective, simpleblind, placebo-controlled trial using one group throughout the study. One week before of the beginning of the study, each participant carried out a maximal aerobic speed running field test. All triathletes underwent a first period of three weeks in which they drank 0.5 L · day−1 of placebo, this was followed over the next 30 days by a washing out period, and finally during three more weeks triathletes drank 0.5 L · day−1 of SMW (SMW, Laboratories Averroes, Spain). Both beverages were taken after the longest daily training when the participants were with highest hydration needs. At the end of each supplement period, a submaximal running field test was performed (Figure 1). Characteristics of SMW and placebo beverages are listed in Table I. Taste of placebo and SMW supplements was different but participants were not familiarised previously with SMW taste. Participants were instructed to continue their normal training programme during the study; however, a minimum of 48 h lay-off from exercise was required before and after each experimental trial. Prior to all trials, participants followed a high-carbohydrate diet (~75% carbohydrates, high glycemic index). Participants were instructed on how to use household measures to record their food intake, and a nutritionist was in charge of advising them and reviewing all completed food intake records. Furthermore, on the day before both running test, participants performed a 30 min continuous training at low intensity (~50% maximal oxygen uptake).
Washing out
P
SMW
3
2 T1
T0 1 2
3
955
T3
T2
MAS running test Submaximal running field test
Exercise
24 h
48 h
Figure 1. Study design. Notes: P, Placebo period; SMW, sulphurous mineral water period; T0, pre-exercise; T1, immediately post-exercise; T2, 24 h post-exercise; and T3, 48 h post-exercise.
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M. Soria et al. running field tests, which consisted of 2 h running at 70% maximal aerobic speed on field and flat terrain.
Table I. Characteristics of SMW and placebo (P) beverages.
Sodium Saccharin (mg) pH Conductivity (mcS · cm–1) Total hardness (CaCO3) (mg · L–1) Calcium (mg · L–1) Magnesium (mg · L–1) Hydrogen sulphide (mg · L–1) Sodium (mg · L–1) Bicarbonates (HCO3) (mg · L–1) Carbonates (CO3) (mg · L–1) Silicon dioxide (mg · L–1)
SMW
P
– 9.4 364 4.5 1.6 0.01 8.0 172 93 14 95
60.0 7.6 828 441.0 102.6 44.9 – – – – –
Downloaded by [University of Otago] at 02:46 03 October 2014
Exercise protocols Training volume performed by each participant was quantified weekly (in km per week) for the two treatment periods and for the washing out period. One week before starting the study, all participants carried out an incremental running field test until exhaustion to determine the maximal aerobic speed. A modification of maximal aerobic speed tests proposed by Léger and Boucher (1980) was conducted on a 400 m running synthetic track, with marker cones at every 50 m along the track. The first stage was set at speed of 9 km · h–1; thereafter, the speed increased by 1 km · h–1 every 2 min until the exhaustion. The change in speed was indicated by audio cues from the pre-recorded tape. The test was ceased when the participants falls 5 or more meters short of the designed marker, or when the participant feels they cannot continue the stage. Maximal aerobic speed was calculated as follows:
Blood sample collection Blood samples from each participant were collected in three separate tubes of 10 mL from the antecubital vein of the arm by venepuncture using standard and sterile procedures. Blood samples were collected into a serum separation tube, an EDTA tube and a heparinised tube from each individual. The serum tube was immediately centrifuged at 2,500 rpm for 10 min, and serum aliquots were stored at –80ºC until assayed. The whole blood from the EDTA tube was analysed for cell count. The anti-coagulated sample was used for determining superoxide dismutase and catalase activities in the haemolysate. Plasma volume changes were estimated by haematocrit variations. All results were corrected for changes in plasma volume. Four samples were collected for each submaximal running field test: pre-exercise (T0), immediately post-exercise (T1), 24 h postexercise (T2) and 48 h post-exercise (T3). Determination of plasma total thiols concentration Plasma total thiols were measured using the method of Ellman modified by Hu (1994), a spectrophotometric method based on the colorimetric reaction of sulphhydryls. The method depends on the capacity that plasma total thiols have to react with 5, 5′dithiobis-2-nitrobenzoic acid. The reaction produces a coloured complex that can be measured photometrically at 405 nm. Plasma values range from 400 to 600 µmol · L−1.
MAS ¼ Sf þ ðt=120Þ Plasma thiobarbituric acid reactive species assay where MAS: Maximal aerobic speed; Sf: Final speed last stage completed; t: time last stage not completed. Heart rate was recorded during the test, and maximal heart rate was measured at the end of the test by means of a heart rate monitor (XtrainerPlus, Polar, Finland). In addition, the maximal oxygen uptake was estimated with the equation proposed by Léger and Boucher (1980): VO2max mL kg 1 min1 ¼ 2:209 þ ð3:163MAS Þ þ 0:000525542MAS 3
The thiobarbituric acid reactive species assay is a wellestablished method for screening and monitoring lipid peroxidation. Thiobarbituric acid reactive species measurement of malonyldialdehyde concentration provides as an index of lipid peroxidation. A commercially available kit for this assay was used according to the manufacturer’s instructions (Cayman Chemical Co, Ann Arbor, MI). Malonyldialdehyde reacts with thiobarbituric acid to yield a malonyldialdehyde–thiobarbituric acid coloured reactant which has an absorption peak at 532 nm.
VO2max : Maximal oxygen uptake. Maximal aerobic speed running field test was performed in order to determine the individual velocity to be maintained during the subsequent submaximal
Superoxide dismutase analysis Superoxide dismutase activity in erythrocyte haemolysate was measured according to the method of Sun, Oberley, and Li (1988) by determining the inhibition of nitroblue tetrazolium reduction with xanthine/xanthine oxidase used as an O2+ generator.
Effects of sulphurous mineral water on muscular damage Activity was assessed in the ethanol phase of the lysate after 1.0 mL of ethanol/chloroform mixture (5/3, v/v) had been added to the same volume of the haemolysate and centrifuged. One unit of superoxide dismutase is defined as the amount of haemoglibine that inhibits the rate of nitroblue tetrazolium reduction by 50%. Results were expressed as units per gram of haemoglobin (U · g−1 Hb).
Downloaded by [University of Otago] at 02:46 03 October 2014
Catalase analysis Supernatant catalase activity was determined according to the Aebi method (Aebi, 1974) by monitoring the initial rate of disappearance of hydrogen peroxide (initial concentration, 10 mM) at 240 nm in a cuvette containing 10.5 mM hydrogen peroxide in 1 mL of 50 mM phosphate buffer (pH 7, 25°C), in a spectrophotometer. Results were expressed as the constant rate per second per gram of Hb (K · gr−1 Hb). Protein concentrations in plasma samples were measured according to Lowry method. Determination of plasma enzymes and haematological parameters Plasma creatine phosphokinase was measured using an auto-analyser (Model 747–400, Hitachi, Japan) and Boehringer Mannheim reagents. The catalytic concentration of the enzymes was expressed as U · L−1 at 37°C. Cell count; haematocrit and haemoglobin were determined in a Counter T660 (Coulter Electronics, Margercy, France). Haematocrit was used for applying corrections to the plasma parameters and the haemoglobin for estimating red blood cells enzymes concentrations. Plasma osmolarity determination One hundred mL of plasma was alloquated into two vessels for the duplicate assessment of plasma osmolarity by the freezing point-depression method (automatic cryoscopic osmometer, Gonotec Osmotat 030, Gonoter, Berlin, Germany). Statistical analyses Results were presented as mean ± SD. A paired student’s t-test was used to compare the effects of SMW and placebo on all the variables studied. An analysis of variance (ANOVA) with repeated measures was conducted to compare each parameter over time. When a significant effect of time was detected, paired t-tests and the false discovery rate multiple-comparisons procedure were used to determine at what time differences occurred specifically. Statistical significance was set at P < 0.05, and the analysis was performed using SPSS version 17.0 (SPSS, Chicago, IL, USA).
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Results Training and running tests Volume of training did not show significant differences between placebo, washing out and SMW periods for swimming (19.7 ± 2.3, 20.1 ± 3.0 and 21.2 ± 1.6 km), cycling (266.7 ± 11.5, 265.3 ± 25.1 and 276.7 ± 40.4 km) and running (40.7 ± 4.0, 42.1 ± 2.5 and 43.0 ± 3.6 km). The maximal physical and physiological response of participants at the maximal aerobic speed running field test was: maximal aerobic speed = 18.6 ± 1.3 km · h–1, maximal oxygen uptake = 64.6 ± 4.9 mL · kg–1 · min–1 and maximal heart rate = 190 ± 11 bpm. In addition, no significant differences for the 2 h running test intensity between placebo and SMW periods were shown, neither for % maximal aerobic speed (69.3 ± 1.2% vs. 70.5 ± 1.4%) nor for heart rate (148 ± 12 vs. 152 ± 15 bpm).
Comparison of variables over time for placebo period and SMW period SMW was well tolerated for all participants, and no discomforts were reported. With placebo, plasma total thiols concentration in T1 remained unchanged when compared to T0, although it showed a decrease (P < 0.05) at T2 and T3 respect to T0. In contrast, with SMW, plasma total thiols concentration was lower (P < 0.05) post-exercise (T1, T2 and T3) with respect to T0. Furthermore, there were no significant differences in plasma thiobarbituric acid reactive species, superoxide dismutase and catalase concentrations after exercise (T1, T2 and T3) with respect T0, in both SMW and placebo supplementations (Figure 2). Plasma creatine phosphokinase concentration with placebo was higher at all specific points of time measured post-exercise (T1, P < 0.05; T2 and T3, P < 0.001) with respect to T0; with SMW there was just a slight increase (P < 0.05) at T2 and T3 with respect to T0 (Figure 3). With placebo, there was a significantly transient increment of red blood cells, haemoglobin and haematocrit values at T1 respect to T0, which returned to T0 levels at T2 and T3. With SMW, these parameters did not change significantly. The leucocytes count was modified after both supplementations. With placebo, leucocytes count increased at T1 (P < 0.001), remained decreased at T2 (P < 0.05) and returned to T0 levels at T3. However, with SMW, there was just a slight increase (P < 0.05) at T1 and recovered the levels of T0 at T2 and T3 (Figure 2). Moreover, no significant differences were shown for mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular
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M. Soria et al.
600
16
–SH groups
32
TBARS
550 500 450
14
28
12
24
10
20
–1
350 300 250 200 150
(U.g–1 Hb)
(u mol-L )
–1
(u mol.L )
400 8
16
6
12
4
8
2
4
100 50 0 T1
T3
T0
T2
T0
T3
RBC
5.5 5.0 4.5 4.0 –1
(g.dL )
3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0
T1
T2
T3
T0
T1
Hct
T2
80
35
70
30
60 (fL)
25
–1
90
40
50 40
15
30
10
20
5
10
0
0 T3
T0
9 –1 (x10 .L )
MCHC
T0
T1
T2
T3
T1
14 13 12 11 10 9 8 7 6 5 4 3 2 1 0
T2
T3
T1
T2
T3
T2
T3
T2
T3
MCH
T0
T1
360
WBC
T3
Pasm
300 240
–1
42 39 36 33 30 27 24 21 18 15 12 9 6 3 0
T2
(mOsm.L )
T0
T1
38 36 34 32 30 29 26 24 22 20 18 16 14 12 10 8 6 4 2 0
T2 Hb
T0
T3
(Pg.cell )
100
45
T1
18 17 16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 0
MCV
50
20
–1
T1
6.0
RBC–CAT
55
(%)
T2
12 –1 (x10 .L )
–1
(u.g Hb)
32 30 28 26 24 22 20 18 16 14 12 10 8 6 4 2 0 T0
(g.dL )
Downloaded by [University of Otago] at 02:46 03 October 2014
0
0 T0
RBC–SOD
180 120 60 0
T0
Time
T1
T2 Time
T3
T0
T1 Time
Figure 2. Creatine phosphokinase comparisons between sulphurous mineral water vs. placebo periods for each condition with respect to T0. Notes: CK, creatine phosphokinase. **P < 0.001, differences between placebo vs. SMW (student’s t-test); †P < 0.05, differences with respect to T0 in sulphurous mineral water period (ANOVA with repeated measures); §P < 0.05 and ¶P < 0.001, differences with respect to T0 in placebo period (P < 0.05) (ANOVA with repeated measures).
haemoglobin concentration and plasma osmolarity after both supplementations. Comparison of the effects between SMW vs. placebo periods of supplementation At T0, a higher level (P < 0.001) of total thiols groups was found in SMW with respect to placebo. However, no significant differences between SMW and placebo were found at T1, T2 and T3. Plasma thiobarbituric acid reactive species concentrations were higher (P < 0.001) after SMW period with respect to placebo period at every specific point
(T0, T1, T2 and T3). Plasma superoxide dismutase concentration did not show any significant differences between SMW and placebo periods at any of the time points measured. Plasma catalase concentration was higher (P < 0.05) in SMW with respect to placebo at T1, although no significant differences were detected at T0, T2 and T3 (Figure 2). There did not exist significant differences in plasma creatine phosphokinase concentration at T0 between SMW and placebo, although it was lower (P < 0.001) at T1, T2 and T3 in SMW with respect to placebo (Figure 3). However, red blood cells count, haemoglobin and haematocrit concentration
CK (U-L–1)
Effects of sulphurous mineral water on muscular damage 2400 2200 2000 1800 1600 1400 1200 1000 800 600 400 200 0 T0
T2
T1 SMW
Downloaded by [University of Otago] at 02:46 03 October 2014
959
T3
P
Figure 3. Oxidative stress biomarkers and haematological variables comparisons of each treatment over time (pre-exercise (T0) and postexercise (T1, T2 and T3) and between P vs. sulphurous mineral water periods. Notes: All values are mean ± SD. –SH groups, plasma thiols; TBARS, thiobarbituric acids reactants; RBC–SOD, erythrocyte superoxide dismutase; RBC–CAT, erythrocyte catalase; red blood cells; Hb, haemoglobin; Hct, haematocrit; MCV, mean cell volume; MCH, mean cell haemoglobin; MCHC, mean cell haemoglobin concentration; WBC, leucocytes. *P < 0.05 and **P
Journal of Sports Sciences Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/rjsp20
Effect of sulphurous mineral water in haematological and biochemical markers of muscle damage after an endurance exercise in well-trained athletes a
a
b
a
a
Marisol Soria , Carlos González-Haro , Santiago Esteva , Jesús F. Escanero & José R. Pina a
Pharmacology and Physiology Department, School of Medicine, University of Zaragoza, Zaragoza, Spain b
Physiology and Immunology Department, School of Biology, University of Barcelona, Barcelona, Spain Published online: 06 Feb 2014.
To cite this article: Marisol Soria, Carlos González-Haro, Santiago Esteva, Jesús F. Escanero & José R. Pina (2014) Effect of sulphurous mineral water in haematological and biochemical markers of muscle damage after an endurance exercise in welltrained athletes, Journal of Sports Sciences, 32:10, 954-962, DOI: 10.1080/02640414.2013.868921 To link to this article: http://dx.doi.org/10.1080/02640414.2013.868921
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
Journal of Sports Sciences, 2014 Vol. 32, No. 10, 954–962, http://dx.doi.org/10.1080/02640414.2013.868921
Effect of sulphurous mineral water in haematological and biochemical markers of muscle damage after an endurance exercise in well-trained athletes
MARISOL SORIA1, CARLOS GONZÁLEZ-HARO1, SANTIAGO ESTEVA2, JESÚS F. ESCANERO1, & JOSÉ R. PINA1 1
Pharmacology and Physiology Department, School of Medicine, University of Zaragoza, Zaragoza, Spain and 2Physiology and Immunology Department, School of Biology, University of Barcelona, Barcelona, Spain
Downloaded by [University of Otago] at 02:46 03 October 2014
(Accepted 20 November 2013)
Abstract To investigate the effects of sulphurous mineral water (SMW) after a hydroponic treatment on muscle damage, antioxidant activity and peripheral blood changes induced by submaximal exercise. Thirty well-trained male triathletes were supplemented with SMW or placebo: 3 weeks of placebo, 30 days of wash out and 3 weeks of SMW. After both periods, participants ran for 2 h at 70% maximal aerobic speed. Antioxidant enzymes, lipid peroxidation, antioxidant capacity and blood cell markers were compared between placebo and SMW at pre-exercise (T0), immediately post-exercise (T1), 24 h post-exercise (T2) and 48 h post-exercise (T3). Total thiols decreased until T3 vs. T0 for both placebo and SMW; transient red blood cells, haemoglobin and haematocrit increased were shown at T1 vs. T0 and for leucocytes until T2 vs. T0, only for placebo group. Total thiols increased significantly in SMW vs. placebo at T0; Thiobarbituric acid reactive species was significantly higher at T0, T1, T2 and T3; catalase increased significantly at T1; creatine phosphokinase decreased significantly at T1, T2 and T3, although no significant differences were found at T0. Furthermore, red blood cells, haemoglobin and haematocrit were significantly higher and leucocytes were significantly lower at T0 and T1 in SMW group vs. placebo group. This study suggests that three weeks of SMW supplementation may protect from exercise-induced muscle damage. Keywords: hydroponic treatment, muscle damage, lipid peroxidation, ergogenic aid
Introduction Different authors have observed therapeutic and preventive benefits using sulphurous waters for a large variety of inflammatory diseases affecting the respiratory tract, skin, liver and skeletal muscle (Constantino, Lampa, & Nappi, 2006; Gupta & Nicol, 2004; Sukenik, Flusser, & Abu-Shakra, 1999). Furthermore, it has been related with pain reduction in osteoarthritis patients (Kovács et al., 2012). For the last few years, it has been documented that some sulphide (HS)-based therapies may play an important role in antioxidant strategies against oxidative damage associated with degenerative diseases (Casetta, Govoni, & Granieri, 2005). Although an excess of free radicals leads an oxidative stress state provoking negative biological disruptions for sport performance, certain level of free radicals are needed to maintain the cellular redox balance (Nikolaidis et al., 2008, 2012). Some authors have suggested that exercise increase oxygen uptake raising free radicals production such as superoxide and hydroxyl, highly reactive. These substances act against some
cell structures and produce lipid peroxidation, protein oxidation, deoxyribonucleic acid damage and muscle damage (Alessio, 1993). Furthermore, a decrement in antioxidant capacity of tissues and whole blood is produced (oxidative stress) (Alessio, 1993; Nikolaidis et al., 2008, 2012). In that point, it has been suggested that plasma thiobarbituric acid reactive species are related with lipid peroxidation of cell membranes elicited by peroxyl radicals (Alessio, 1993). Also, total thiols contribute significantly to the antioxidant capacity of plasma (Wayner, Burton, Ingold, Barclay, & Locke, 1987). Plasma protein-bound sulphhydryl groups are said to provide the first line of defence during the peroxyl radical attack (Inayama, Kumagai, Sakane, Saito, & Matsuda, 1996; Thomas, Poland, & Honzatko, 1995). It has been reported that plasma protein-bound sulphhydryl groups contributed significantly to the plasma antioxidant capacity after the exposure of human plasma to aqueous peroxyl radicals, and the sulphhydryl groups prevented plasma lipid peroxidation (Wayner et al., 1987).
Correspondence: Carlos González-Haro, Pharmacology and Physiology Department, School of Medicine, Zaragoza, Spain. E-mail: [email protected] © 2014 Taylor & Francis
Downloaded by [University of Otago] at 02:46 03 October 2014
Effects of sulphurous mineral water on muscular damage Some authors have suggested that the antioxidant status is related with the attenuation of free radical levels, reduction of oxidation damage in general and with muscle damage specifically (Nikolaidis et al., 2008, 2012). The semi-essential amino acid cysteine is the main source of the free sulphhydryl group of glutathione and limits its synthesis. Glutathione serves multiple functions in protecting tissues from oxidative damage and keeping the intracellular environment in the reduced state (Meister, 1983). The current information in scientific literature suggests that some substances increase the cysteine availability promoting glutathione synthesis such as N-acetyl cysteine (Zembron-Lacny, Slowinska-Lisowska, Szygula, & Witkowski, 2008), branched-chain amino acids (Coombes & McNaughton, 2000) or whey protein (Micke, Beeh, Schlaak, & Buhl, 2001). However, no studies have addressed the effects of sulphurous mineral water (SMW) in peripheral blood changes, muscle damage and oxidative response when exercise performed. Thus, the aim of the present study was to investigate the effects of SMW supplement on haematological parameters, biochemical markers of muscle damage and oxidative response in well-trained athletes after induced exercise.
Methods Participants Thirty well-trained male triathletes (age 27.3 ± 4.1 years, height 174.6 ± 7.2 cm, body mass 73.5 ± 4.5 kg, body mass index 24.2 ± 1.7 kg · m−2, years of experience 4.5 ± 1.7 years) participated in this study. All participants were informed verbally and in writing about the nature of the study, including all potential risks. Written informed consent was obtained prior to participation. Also, each participant completed a personal information sheet and a standard medical history form. The inclusion criteria were: age ranged between 18–31 years; participants free of any type of injury and under the same
A week
1
programme of training; participants were not allowed to intake any ergogenic dietary supplement during one month before the starting the study. This study was conducted according to the Helsinki Declaration for the Ethical Treatment of Human Subjects (last modified in 2004), and the institutional review board gave ethics approval.
Study design This study was conducted as a prospective, simpleblind, placebo-controlled trial using one group throughout the study. One week before of the beginning of the study, each participant carried out a maximal aerobic speed running field test. All triathletes underwent a first period of three weeks in which they drank 0.5 L · day−1 of placebo, this was followed over the next 30 days by a washing out period, and finally during three more weeks triathletes drank 0.5 L · day−1 of SMW (SMW, Laboratories Averroes, Spain). Both beverages were taken after the longest daily training when the participants were with highest hydration needs. At the end of each supplement period, a submaximal running field test was performed (Figure 1). Characteristics of SMW and placebo beverages are listed in Table I. Taste of placebo and SMW supplements was different but participants were not familiarised previously with SMW taste. Participants were instructed to continue their normal training programme during the study; however, a minimum of 48 h lay-off from exercise was required before and after each experimental trial. Prior to all trials, participants followed a high-carbohydrate diet (~75% carbohydrates, high glycemic index). Participants were instructed on how to use household measures to record their food intake, and a nutritionist was in charge of advising them and reviewing all completed food intake records. Furthermore, on the day before both running test, participants performed a 30 min continuous training at low intensity (~50% maximal oxygen uptake).
Washing out
P
SMW
3
2 T1
T0 1 2
3
955
T3
T2
MAS running test Submaximal running field test
Exercise
24 h
48 h
Figure 1. Study design. Notes: P, Placebo period; SMW, sulphurous mineral water period; T0, pre-exercise; T1, immediately post-exercise; T2, 24 h post-exercise; and T3, 48 h post-exercise.
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M. Soria et al. running field tests, which consisted of 2 h running at 70% maximal aerobic speed on field and flat terrain.
Table I. Characteristics of SMW and placebo (P) beverages.
Sodium Saccharin (mg) pH Conductivity (mcS · cm–1) Total hardness (CaCO3) (mg · L–1) Calcium (mg · L–1) Magnesium (mg · L–1) Hydrogen sulphide (mg · L–1) Sodium (mg · L–1) Bicarbonates (HCO3) (mg · L–1) Carbonates (CO3) (mg · L–1) Silicon dioxide (mg · L–1)
SMW
P
– 9.4 364 4.5 1.6 0.01 8.0 172 93 14 95
60.0 7.6 828 441.0 102.6 44.9 – – – – –
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Exercise protocols Training volume performed by each participant was quantified weekly (in km per week) for the two treatment periods and for the washing out period. One week before starting the study, all participants carried out an incremental running field test until exhaustion to determine the maximal aerobic speed. A modification of maximal aerobic speed tests proposed by Léger and Boucher (1980) was conducted on a 400 m running synthetic track, with marker cones at every 50 m along the track. The first stage was set at speed of 9 km · h–1; thereafter, the speed increased by 1 km · h–1 every 2 min until the exhaustion. The change in speed was indicated by audio cues from the pre-recorded tape. The test was ceased when the participants falls 5 or more meters short of the designed marker, or when the participant feels they cannot continue the stage. Maximal aerobic speed was calculated as follows:
Blood sample collection Blood samples from each participant were collected in three separate tubes of 10 mL from the antecubital vein of the arm by venepuncture using standard and sterile procedures. Blood samples were collected into a serum separation tube, an EDTA tube and a heparinised tube from each individual. The serum tube was immediately centrifuged at 2,500 rpm for 10 min, and serum aliquots were stored at –80ºC until assayed. The whole blood from the EDTA tube was analysed for cell count. The anti-coagulated sample was used for determining superoxide dismutase and catalase activities in the haemolysate. Plasma volume changes were estimated by haematocrit variations. All results were corrected for changes in plasma volume. Four samples were collected for each submaximal running field test: pre-exercise (T0), immediately post-exercise (T1), 24 h postexercise (T2) and 48 h post-exercise (T3). Determination of plasma total thiols concentration Plasma total thiols were measured using the method of Ellman modified by Hu (1994), a spectrophotometric method based on the colorimetric reaction of sulphhydryls. The method depends on the capacity that plasma total thiols have to react with 5, 5′dithiobis-2-nitrobenzoic acid. The reaction produces a coloured complex that can be measured photometrically at 405 nm. Plasma values range from 400 to 600 µmol · L−1.
MAS ¼ Sf þ ðt=120Þ Plasma thiobarbituric acid reactive species assay where MAS: Maximal aerobic speed; Sf: Final speed last stage completed; t: time last stage not completed. Heart rate was recorded during the test, and maximal heart rate was measured at the end of the test by means of a heart rate monitor (XtrainerPlus, Polar, Finland). In addition, the maximal oxygen uptake was estimated with the equation proposed by Léger and Boucher (1980): VO2max mL kg 1 min1 ¼ 2:209 þ ð3:163MAS Þ þ 0:000525542MAS 3
The thiobarbituric acid reactive species assay is a wellestablished method for screening and monitoring lipid peroxidation. Thiobarbituric acid reactive species measurement of malonyldialdehyde concentration provides as an index of lipid peroxidation. A commercially available kit for this assay was used according to the manufacturer’s instructions (Cayman Chemical Co, Ann Arbor, MI). Malonyldialdehyde reacts with thiobarbituric acid to yield a malonyldialdehyde–thiobarbituric acid coloured reactant which has an absorption peak at 532 nm.
VO2max : Maximal oxygen uptake. Maximal aerobic speed running field test was performed in order to determine the individual velocity to be maintained during the subsequent submaximal
Superoxide dismutase analysis Superoxide dismutase activity in erythrocyte haemolysate was measured according to the method of Sun, Oberley, and Li (1988) by determining the inhibition of nitroblue tetrazolium reduction with xanthine/xanthine oxidase used as an O2+ generator.
Effects of sulphurous mineral water on muscular damage Activity was assessed in the ethanol phase of the lysate after 1.0 mL of ethanol/chloroform mixture (5/3, v/v) had been added to the same volume of the haemolysate and centrifuged. One unit of superoxide dismutase is defined as the amount of haemoglibine that inhibits the rate of nitroblue tetrazolium reduction by 50%. Results were expressed as units per gram of haemoglobin (U · g−1 Hb).
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Catalase analysis Supernatant catalase activity was determined according to the Aebi method (Aebi, 1974) by monitoring the initial rate of disappearance of hydrogen peroxide (initial concentration, 10 mM) at 240 nm in a cuvette containing 10.5 mM hydrogen peroxide in 1 mL of 50 mM phosphate buffer (pH 7, 25°C), in a spectrophotometer. Results were expressed as the constant rate per second per gram of Hb (K · gr−1 Hb). Protein concentrations in plasma samples were measured according to Lowry method. Determination of plasma enzymes and haematological parameters Plasma creatine phosphokinase was measured using an auto-analyser (Model 747–400, Hitachi, Japan) and Boehringer Mannheim reagents. The catalytic concentration of the enzymes was expressed as U · L−1 at 37°C. Cell count; haematocrit and haemoglobin were determined in a Counter T660 (Coulter Electronics, Margercy, France). Haematocrit was used for applying corrections to the plasma parameters and the haemoglobin for estimating red blood cells enzymes concentrations. Plasma osmolarity determination One hundred mL of plasma was alloquated into two vessels for the duplicate assessment of plasma osmolarity by the freezing point-depression method (automatic cryoscopic osmometer, Gonotec Osmotat 030, Gonoter, Berlin, Germany). Statistical analyses Results were presented as mean ± SD. A paired student’s t-test was used to compare the effects of SMW and placebo on all the variables studied. An analysis of variance (ANOVA) with repeated measures was conducted to compare each parameter over time. When a significant effect of time was detected, paired t-tests and the false discovery rate multiple-comparisons procedure were used to determine at what time differences occurred specifically. Statistical significance was set at P < 0.05, and the analysis was performed using SPSS version 17.0 (SPSS, Chicago, IL, USA).
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Results Training and running tests Volume of training did not show significant differences between placebo, washing out and SMW periods for swimming (19.7 ± 2.3, 20.1 ± 3.0 and 21.2 ± 1.6 km), cycling (266.7 ± 11.5, 265.3 ± 25.1 and 276.7 ± 40.4 km) and running (40.7 ± 4.0, 42.1 ± 2.5 and 43.0 ± 3.6 km). The maximal physical and physiological response of participants at the maximal aerobic speed running field test was: maximal aerobic speed = 18.6 ± 1.3 km · h–1, maximal oxygen uptake = 64.6 ± 4.9 mL · kg–1 · min–1 and maximal heart rate = 190 ± 11 bpm. In addition, no significant differences for the 2 h running test intensity between placebo and SMW periods were shown, neither for % maximal aerobic speed (69.3 ± 1.2% vs. 70.5 ± 1.4%) nor for heart rate (148 ± 12 vs. 152 ± 15 bpm).
Comparison of variables over time for placebo period and SMW period SMW was well tolerated for all participants, and no discomforts were reported. With placebo, plasma total thiols concentration in T1 remained unchanged when compared to T0, although it showed a decrease (P < 0.05) at T2 and T3 respect to T0. In contrast, with SMW, plasma total thiols concentration was lower (P < 0.05) post-exercise (T1, T2 and T3) with respect to T0. Furthermore, there were no significant differences in plasma thiobarbituric acid reactive species, superoxide dismutase and catalase concentrations after exercise (T1, T2 and T3) with respect T0, in both SMW and placebo supplementations (Figure 2). Plasma creatine phosphokinase concentration with placebo was higher at all specific points of time measured post-exercise (T1, P < 0.05; T2 and T3, P < 0.001) with respect to T0; with SMW there was just a slight increase (P < 0.05) at T2 and T3 with respect to T0 (Figure 3). With placebo, there was a significantly transient increment of red blood cells, haemoglobin and haematocrit values at T1 respect to T0, which returned to T0 levels at T2 and T3. With SMW, these parameters did not change significantly. The leucocytes count was modified after both supplementations. With placebo, leucocytes count increased at T1 (P < 0.001), remained decreased at T2 (P < 0.05) and returned to T0 levels at T3. However, with SMW, there was just a slight increase (P < 0.05) at T1 and recovered the levels of T0 at T2 and T3 (Figure 2). Moreover, no significant differences were shown for mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular
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600
16
–SH groups
32
TBARS
550 500 450
14
28
12
24
10
20
–1
350 300 250 200 150
(U.g–1 Hb)
(u mol-L )
–1
(u mol.L )
400 8
16
6
12
4
8
2
4
100 50 0 T1
T3
T0
T2
T0
T3
RBC
5.5 5.0 4.5 4.0 –1
(g.dL )
3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0
T1
T2
T3
T0
T1
Hct
T2
80
35
70
30
60 (fL)
25
–1
90
40
50 40
15
30
10
20
5
10
0
0 T3
T0
9 –1 (x10 .L )
MCHC
T0
T1
T2
T3
T1
14 13 12 11 10 9 8 7 6 5 4 3 2 1 0
T2
T3
T1
T2
T3
T2
T3
T2
T3
MCH
T0
T1
360
WBC
T3
Pasm
300 240
–1
42 39 36 33 30 27 24 21 18 15 12 9 6 3 0
T2
(mOsm.L )
T0
T1
38 36 34 32 30 29 26 24 22 20 18 16 14 12 10 8 6 4 2 0
T2 Hb
T0
T3
(Pg.cell )
100
45
T1
18 17 16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 0
MCV
50
20
–1
T1
6.0
RBC–CAT
55
(%)
T2
12 –1 (x10 .L )
–1
(u.g Hb)
32 30 28 26 24 22 20 18 16 14 12 10 8 6 4 2 0 T0
(g.dL )
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0
0 T0
RBC–SOD
180 120 60 0
T0
Time
T1
T2 Time
T3
T0
T1 Time
Figure 2. Creatine phosphokinase comparisons between sulphurous mineral water vs. placebo periods for each condition with respect to T0. Notes: CK, creatine phosphokinase. **P < 0.001, differences between placebo vs. SMW (student’s t-test); †P < 0.05, differences with respect to T0 in sulphurous mineral water period (ANOVA with repeated measures); §P < 0.05 and ¶P < 0.001, differences with respect to T0 in placebo period (P < 0.05) (ANOVA with repeated measures).
haemoglobin concentration and plasma osmolarity after both supplementations. Comparison of the effects between SMW vs. placebo periods of supplementation At T0, a higher level (P < 0.001) of total thiols groups was found in SMW with respect to placebo. However, no significant differences between SMW and placebo were found at T1, T2 and T3. Plasma thiobarbituric acid reactive species concentrations were higher (P < 0.001) after SMW period with respect to placebo period at every specific point
(T0, T1, T2 and T3). Plasma superoxide dismutase concentration did not show any significant differences between SMW and placebo periods at any of the time points measured. Plasma catalase concentration was higher (P < 0.05) in SMW with respect to placebo at T1, although no significant differences were detected at T0, T2 and T3 (Figure 2). There did not exist significant differences in plasma creatine phosphokinase concentration at T0 between SMW and placebo, although it was lower (P < 0.001) at T1, T2 and T3 in SMW with respect to placebo (Figure 3). However, red blood cells count, haemoglobin and haematocrit concentration
CK (U-L–1)
Effects of sulphurous mineral water on muscular damage 2400 2200 2000 1800 1600 1400 1200 1000 800 600 400 200 0 T0
T2
T1 SMW
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T3
P
Figure 3. Oxidative stress biomarkers and haematological variables comparisons of each treatment over time (pre-exercise (T0) and postexercise (T1, T2 and T3) and between P vs. sulphurous mineral water periods. Notes: All values are mean ± SD. –SH groups, plasma thiols; TBARS, thiobarbituric acids reactants; RBC–SOD, erythrocyte superoxide dismutase; RBC–CAT, erythrocyte catalase; red blood cells; Hb, haemoglobin; Hct, haematocrit; MCV, mean cell volume; MCH, mean cell haemoglobin; MCHC, mean cell haemoglobin concentration; WBC, leucocytes. *P < 0.05 and **P
