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Thermal stress and oxidant–antioxidant balance in experienced and novice winter swimmers
Journal of Thermal Biology 37 (2012) 595–601
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Thermal stress and oxidant–antioxidant balance in experienced and novice winter swimmers Celestyna Mila-Kierzenkowska a,n, Alina Woz´niak a, Tomasz Boraczyn´ski b, Micha" Szpinda c, Bartosz Woz´niak d, Alicja Jurecka e, Anna Szpinda c a
The Chair of Medical Biology, Collegium Medicum of Nicolaus Copernicus University, Kar!owicza 24, 85-094 Bydgoszcz, Poland ´zef Rusiecki Olsztyn University, Bydgoska 33, 10-243 Olsztyn, Poland Central Research Laboratory, Jo c Department of Normal Anatomy, Collegium Medicum of Nicolaus Copernicus University, Kar!owicza 24, 85-094 Bydgoszcz, Poland d Department of Neurosurgery, Stanislaw Staszic Specialist Hospital, Rydygiera 1, 64-920 Pi!a, Poland e Department of Orthopaedics and Musculoskeletal Traumatology, Collegium Medicum of Jagiellonian University, Os. Zlotej Jesieni 1, 31-826 Krakow, Poland b
a r t i c l e i n f o
abstract
Article history: Received 23 March 2012 Accepted 26 July 2012 Available online 4 August 2012
The adaptation of human organism to environmental stress plays an important role in maintaining good health. The exposure to both low and high ambient temperature may provoke thermal stress and such a condition potentially leads to the excessive production of reactive oxygen species, which may result in oxidative stress. The purpose of the study was to determine the effect of one session of swimming in ice-cold water and one hot sauna session (performed few months later) on oxidant– antioxidant balance in two groups of healthy volunteers: 21 experienced winter swimmers and 19 people who participated in winter swimming for the first time (novices). The activity of antioxidant enzymes: catalase, superoxide dismutase and glutathione peroxidase was measured in erythrocytes of studied persons. Moreover, in blood plasma and erythrocytes the concentration of lipid peroxidation products was estimated. No statistically significant differences in initial values of antioxidant enzymes activity and lipid peroxidation products level were revealed between experienced and novice winter swimmers. The crucial antioxidant enzyme that neutralizes reactive oxygen species generated as a result of thermal stress seems to be catalase, since statistically significant changes of CAT activity after sauna were observed. Increased TBARS level observed as a result of sauna bath proves that exposure of organism to high ambient temperature is a source of oxidative stress. However, such a stress was hardly noticed in regular winter swimmers. The regular baths in cold water combined with sauna probably lead to adaptive changes that protect the organism against harmful effects of thermal stress. & 2012 Elsevier Ltd. All rights reserved.
Keywords: Winter swimming Sauna Oxidative stress Reactive oxygen species Lipid peroxidation Adaptive response
1. Introduction The inevitable result of life in an oxygen-rich atmosphere is oxidative stress defined as a disturbance in oxidant–antioxidant systems in favor of the former (Davies, 2000). Although oxygen is essential for most life forms, it is also toxic due to generation of reactive oxygen species called ROS (Hermes-Lima and ZentenoSavin, 2002). ROS are potential toxic compounds produced continuously during the process of respiration, which represent a paradox in their biological function. They prevent diseases by assisting the immune system, mediating cell signaling and play a role in apoptosis, but on the other hand they can damage essential
Abbreviations: SOD, superoxide dismutase; CAT, catalase; GPx, glutathione peroxidase; TBARS, thiobarbituric acid reactive substance n Corresponding author. Tel.: þ48 52 585 3737; fax: þ 48 52 585 3742. E-mail addresses: [email protected] (C. Mila-Kierzenkowska), [email protected] (A. Woz´niak), [email protected] (T. Boraczyn´ski), [email protected] (M. Szpinda), [email protected] (B. Woz´niak), [email protected] (A. Jurecka), [email protected] (A. Szpinda). 0306-4565/$ - see front matter & 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jtherbio.2012.07.007
macromolecules, such as lipids, proteins and nucleic acids (Seifried et al., 2007). The first type of oxidative damage, caused by increased ROS generation, reported in the literature was lipid peroxidation (Davies, 2000). Extensive lipid peroxidation process is known to be related to ultimate disintegration of membranes and cell death (Avery, 2011). However, in organisms the enhancement in ROS formation is usually restricted by antioxidant mechanisms. Antioxidants are molecules that prevent uncontrolled formation of reactive oxygen species or inhibit their reactions with biological structures (Chaudiere and Ferrari-Iliou, 1999). The antioxidant system consists of antioxidant enzymes, like superoxide dismutase (SOD), catalase (CAT) glutathione peroxidase (GPx) as well as of a wide range of non-enzymatic compounds (Osorio et al., 2003). During the human lifetime the formation of ROS may be intensified by a range of different stress conditions (Avery, 2011), including the changes in environmental temperature. The cold exposure was proved to provoke the increased production of reactive oxygen species by changes in energy production (Blagojevic, 2007). An example of voluntary exposure of human to low environmental temperatures is winter swimming. Winter swimming refers to a
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regular swimming in cold water during winter season and is a common kind of habit in many countries. Immersion in cold water results in immediate and long-term physiologic alterations, consisting of metabolic, cardiovascular and hormonal changes (Kolletis and Kolletis, 2003) and is supposed to be a method of body hardening (Siems et al., 1994). Huttunen et al. (2004) presented that regular winter swimming improves well-being of patients who suffer from rheumatism, fibromyalgia and asthma. The winter swimmers usually combine the swimming in icecold water with a hot sauna bath. Sauna bathing, which is a special form of exposure to heat, is a popular form of wellness practiced in many countries. It is defined as short-term exposure to exceptionally high environmental temperatures (KukkonenHarjula and Kauppinen, 2006). Nowadays, innovative experiments investigating new therapeutic uses of sauna bathing have been performed throughout the world (Biro et al., 2003). The reactions of organism to sauna bathing are the expression of active thermoregulation and bear similarities with those of acute physical exercise (Ernst, 1989). After sauna, there occurs the sweating with loss of the body water and electrolytes, skin vasodilatation with an increase in heart rate and cardiac output, which results in decrease of blood pressure and hyperventilation (Kauppinen, 1997). A number of studies report the effect of sauna on hormonal changes in organism. Changes in the secretion of hormones induced by sauna are at times similar to changes provoked by any other stress situation. Exposure to heat for example induces the increase in noradrenalin level, but also the secretion of antidiuretic hormone and the rennin– angiotensin–aldosterone system is activated (Kukkonen-Harjula and Kauppinen, 1988). Moreover, sauna increases level of prolactin in blood plasma of women (Laatikainen et al., 1988). Sauna baths are also known to induce the changes in the lipid profile in blood serum. Pilch et al. (2010) demonstrated lowered concentration of total cholesterol and low-density lipoproteins (LDL) with concomitant increase of high-density lipoproteins (HDL) level in women after repeated sauna sessions. Though the sauna sessions cause various transient cardiovascular and hormonal changes, it is tolerated well by most healthy people (Hannuksela and Ellahham, 2001). Despite the well-known acute response to sauna, the long-term effects of regular sauna using are still speculative. The literature data postulate that both regular winter swimming and regular sauna visits may have a positive influence on human health by inducing some adaptive changes in organism. Adaptation response refers to the ability to better resist the damaging effects of some stress agents by prior pre-exposure to Table 1 Characteristics of the studied groups.
Number of participants Age (years) BMI (kg/m2) PWC170 (W/kg) VO2max (ml/min/kg)
Experienced winter swimmers
Novices
21 27.6 75.9 22.8 73.1 2.96 70.8 45.21 7 5.8
19 25.5 74.7 23.6 72.3 2.65 70.5 43.64 75.5
Values are expressed as mean 7 standard deviations (SD). Body Mass Index (BMI) was calculated using the formula weight/height2. PWC170 (Physical Work Capacity) test was performed on a cycle-ergometer Monark 828 E.
a low amount of this agent. The main physiological benefit of adaptive response is to protect the organism against stress damage caused by high doses of toxic agent (Crawford and Davies, 1994). Adaptive response to oxidative stress has been documented well for prokaryotic and eukaryotic cells (Crawford and Davies, 1994). Some authors postulate that organisms are able to adapt themselves to high exposure of ROS by increasing the expression of antioxidant enzymes and other forms of defense reactions against oxidative damage (Hermes-Lima and Zenteno-Savin, 2002). Blagojevic et al. (2011) showed that short-time exposure to cold leads to oxidative stress; however as it persists the organisms develop some adaptive changes toward reducing ROS formation and increasing the antioxidant action. The aim of this study was to determine the effect of exposure to low and high temperature on activity of antioxidant enzymes and concentration of lipid peroxidation products in blood of habitual and inexperienced winter swimmers.
2. Material and methods The study was performed on two groups of healthy volunteers, who reported the absence of any chronic disease and no use of any medications. The basic characteristic of the subjects is shown in Table 1. The first group consisted of 21 men who have been regular winter swimmers (WS-R). The winter swimming experience in this group ranged from 1 to 12 years, but most of them were beginners (mean experience amounted to 4.1 years). The activity of winter swimmers starts in the fall (usually at October) and ends in April, and they swim in the ice-cold water once a week during the whole season (it means about 18–22 sessions of winter swimming per season). The other group included 19 men who participated in winter swimming for the first time in their life and we named them novice winter swimmers (WS-N). Subjects from this group were selected from the people who had participated in sauna only occasionally. The experiment consisted of two parts—one sessions of winter swimming and one session of sauna (Table 2). The bath in ice-cold water was performed in the winter (in the middle of the winter swimming season) at the small river Wada˛g near Olsztyn (Poland). Winter swimming took place in the morning following the short warming up. The temperature of the water at the day of experiment was 0 1C, while the air temperature was 4 1C. All the subjects spent 3 min in ice-cold water staying all the time in contact with doctor who supervised the study. The part of the study including sauna took place in the summer few months after the end of winter swimming season. All subjects spent 30 min without any breaks in sauna heated to 85 1C with relative humidity of 40%. Before the blood collection, the studied persons were not allowed showering/cooling action. In the course of the experiment, the heart rate of all the participants was monitored by means of a Polar meter and the results were analyzed by PC software. The blood samples were taken from every studied person altogether six times: before the start of the experiment (control), 5 and 30 min after the swimming in ice-cold water as well as before and 5, and 30 min after hot sauna bath. The blood were taken from the cubital vein and put into the tube contained
Table 2 The scheme of the study design (all participants were subjected to the same sessions). Date of exposure
January 2010 July 2010
Type of exposure
Blood samples
Cold bath (winter swimming)
Sauna bath
One 3 min session –
– One 30 min session
Taken before, 5 min and 30 min after the exit from cold water Taken before, 5 min and 30 min after the exit from sauna
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K2EDTA. The research has obtained the agreement of the Bioethics Committee at Collegium Medicum in Bydgoszcz of the Nicolaus Copernicus University in Torun´ and all subjects had given written informed consent to participate in the study. The activity of SOD, CAT and GPx was measured in erythrocytes. Superoxide dismutase activity was determined according to Misra and Fridovich (1972) by a procedure based on SOD impeding the reaction of auto-oxidation of adrenaline to adrenochrome in an alkaline environment. SOD activity was expressed as U/gHb. Beers and Sizer (1952) method was used to measure the activity of catalase. This method is based on the detection at a wavelength of 240 nm of the decrease of hydrogen peroxide absorbance. CAT activity was expressed as 104 IU/gHb. Glutathione peroxidase activity was performed by Paglia and Valentine (1967). The method is based on the measurement of changes in absorbance caused by oxidation of reduced nicotinamide adenine dinucleotide phosphate (NADPH), measured at a wavelength of 340 nm. NADPH is a coenzyme of reduction of glutathione disulfide. The obtained oxided glutathione is a product of reaction catalyzed by glutathione peroxidase. Activity of GPx was expressed as U/gHb. The concentration of thiobarbituric acid reactive substances (TBARS) was assayed both in the erythrocytes and in blood plasma. TBARS level was determined according to Buege and Aust (1978) method in the Esterbauer and Cheeseman (1990) modification. The method involves creation of colored complex between lipid peroxidation products and thiobarbituric acid at the temperature of 100 1C in acidic environment. The maximum absorption of that complex occurs at a wavelength of 532 nm. The main product of lipid peroxidation that reacts with thiobarbituric acid is malondialdehyde (MDA) and therefore the level of TBARS in plasma was expressed as nmol of MDA/ml and in the erythrocytes as nmol of MDA/gHb. All the data were statistically analyzed with the use of ANOVA test. The differences on the level of p r0.05 were accepted as statistically significant. 3. Results In presented study, some changes in activity of catalase in studied subjects were revealed (Fig. 1). The exposure to cold water had no statistically significant impact on activity of this enzyme, however increasing tendency was observed both in regular winter
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swimmers and in novices. The changes in CAT activity as a consequence of hot sauna bath were more distinct and statistically significant. 5 min after sauna CAT activity in WS-R increased by about 20% (po0.05) and in WS-N by about 35% (po0.01) versus the control value (Fig. 1). 30 min after sauna it decreased by about 22% (po0.05) in WS-R and by about 26% (po0.01) in WS-N as compared to the activity 5 min after sauna and was comparable to the control value (Fig. 1). Considering SOD and GPx activity, there were no statistically significant changes after both cold bath and sauna (Table 3). There were also no differences in initial activity of investigated antioxidant enzymes between WS-R and WS-N (Table 3). We have also found no statistically significant differences in basal TBARS level both in blood plasma and in erythrocytes between the group of WS-R and WS-N (Table 4). Nonetheless, some changes in concentration of lipid peroxidation products were observed as result of exposure to low temperature. The decreasing tendencies of TBARS level in blood plasma and erythrocytes was observed 5 and 30 min after cold bath, both in WS-R and WS-N; however most of them were statistically insignificant (Table 4). The only statistically significant difference was revealed in TBARSerythrocytes level in of WS-R 5 min after the bath in cold water, and it was about 26% lower than before the start of the experiment (p o0.05). The relevant changes in TBARS concentration were found after sauna bath. In WS-N, the exposure to high ambient temperature caused significant increase of TBARSplasma concentration (Fig. 2). 5 min after sauna it was about 40% (p o0.001) and after 30 min about 33% (po0.001) higher as compared to the control value. Moreover, 5 min after sauna TBARSplasma level was 56% higher (p o0.001) in WS-N and 19% higher (p o0.05) in WS-R than 5 min after cold bath. 30 min after sauna level of this product of lipid peroxidation in WS-N was 37% higher (p o0.001), while in WS-R 34% higher (po0.01) than 30 min after bath in cold water. TBARS concentration in erythrocytes after sauna sessions was also higher then after winter swimming (Table 4). In WS-N 5 min after sauna TBARSerythrocytes level was about 58% higher (po0.01) and 30 min after sauna bath about 54% higher (p o0.01) than that at 5 and 30 min after winter swimming, respectively. In WS-R 5 min after sauna bath it was 31% higher than 5 min after cold bath (p o0.001) as well as 30 min after sauna it was 40% higher than 30 min after exposure to cold water (po0.001).
100 95 CAT 104 (IU/ gHb)
90
regular winter swimmers novice winter swimmers
85 80 75 70 aa
65
a
60 55 50 before winter swimming
5 min after 30 min after winter winter swimming swimming
before sauna
5 min after sauna
30 min after sauna
Statistically significant differences: - versus before the exposure: * p<0.05, ** p<0.01 - versus 5 min after sauna: a p<0.05, aa p<0.01 Fig. 1. Activity of catalase (CAT) before and after exposure to cold (winter swimming) and heat (sauna bath) in people regularly taking baths in freezing water (regular winter swimmers) and in those who practiced it once (novice winter swimmers).
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Table 3 The activity of antioxidant enzymes: catalase (CAT), glutathione peroxidase (GPx), and superoxide dismutase (SOD) after exposure to low and high ambient temperature in people regularly taking baths in freezing water (regular winter swimmers) and in those who practiced it for the first time (novice winter swimmers). Exposure to cold water (winter swimming) Before the exposure
Exposure to hot temperature (sauna bathing)
After 5 min
After 30 min
Before the exposure
After 5 min
After 30 min
Regular winter swimmers GPx (U/gHb) 10.97 4.8 SOD (U/gHb) 862.0 7 178.5
12.5 7 4.7 879.2 7 177.4
12.3 7 4.1 898.0 7 144.3
10.8 73.6 878.4 7182.7
11.4 74.5 856.8 791.9
11.27 3.8 897.1 7 94.3
Novice winter swimmers GPx (U/gHb) 11.7 7 5.3 SOD (U/gHb) 942.8 7 169.5
12.5 7 3.6 923.7 7 141.0
12.0 7 4.3 970.6 7 180.1
11.8 74.6 928.9 7177.3
8.8 74.9 872.0 7108.1
11.97 4.4 896.6 7 82.6
Values are expressed as means7 standard deviations (SD) of the means.
Table 4 The concentration of concentration of lipid peroxidation products—thiobarbituric acid reactive substances (TBARS) after exposure to low and high ambient temperature in blood plasma and erythrocytes of people regularly taking baths in freezing water (regular winter swimmers) and in those who practiced it for the first time (novice winter swimmers). Exposure to cold water (winter swimming)
Exposure to hot temperature (sauna bathing)
Before the exposure
After 5 min
After 30 min
Before the exposure
After 5 min
After 30 min
Regular winter swimmers TBARSplasma (10 1 nmolMDA/ml) TBARSerythrocytes [nmolMDA/gHb]
3.47 0.4 28.1 7 8.1
3.2 70.5 20.8 75.9n
2.9 70.4 22.9 77.9
3.4 7 0.3 27.9 7 6.7
3.87 0.5abbb 27.37 3.6aaabb
3.97 0.6aabb 32.17 7.2aaabbb
Novice winter swimmers TBARSerythrocytes (nmolMDA/gHb)
24.07 9.3
17.9 76.1
18.6 76.2
26.1 7 8.4
28.27 5.7aab
28.77 6.1aaabb
are expressed as means7 standard deviations (SD) of the means. Statistically significant differences: before exposure: np o0.05. 5 min after cold bath: ap o 0.05, aapo 0.01, aaap o 0.001. 30 min after cold bath: bp o0.05, bbp o0.01, bbbp o 0.001.
TBARS in plasma [10-1 nmol MDA/ml]
Values Versus Versus Versus
5.5 5
novice winter swimmers
4.5
aaa
4
bbb
3.5 3 2.5 2 before winter swimming
5 min after 30 min winter after winter swimming swimming
before sauna
5 min after 30 min sauna after sauna
Statistically significant differences: - versus before exposure: *** p<0.001 - versus 5 min after cold bath: aaa p<0.001 - versus 30 min after cold bath: bbb p< 0.001 Fig. 2. Concentration of thiobarbituric acid reactive substances (TBARS) before and after exposure to cold (winter swimming) and heat (sauna bath) in blood plasma of people who practiced taking baths in freezing water only once (novice winter swimmers).
Although we found no alterations in SOD and GPx activity in investigated groups during exposure to changed environmental conditions, we noticed some correlations between the antioxidant enzymes activity. In WS-R SOD and GPx activity was positively
correlated 30 min after bath in cold water (r ¼0.543, p o0.01), while CAT and GPx activity negatively correlated 30 min after sauna (r ¼ 0.461, po0.05). In WS-N the positive correlations were found between SOD and CAT activity (r ¼0.529) as well as
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between SOD and GPx activity (r ¼0.596) 30 min after bath in cold water, both on the level of statistical significance of p o0.05. 30 min after sauna positive correlation (r ¼0.74) was observed between the activity of SOD and GPx (p o0.01). Heart rate measured before winter swimming, during staying in the cold water and 3 min after the exit from the river is presented in Table 5. Similarly as in case of winter swimming heart rate was measured before the entry to sauna as well as during sauna bath and at recovery period (Table 6).
4. Discussion Winter swimming and sauna baths are the forms of recreation that refer to a thermal stress that may lead to disturbances in oxidant–antioxidant balance since the generation of reactive oxygen species is intensified under different stress conditions (Avery, 2011). In turn, repeated exposure to oxidative stress may induce some adaptive changes in organisms that help them to resist stress damage. Siems et al. (1999) demonstrated the improved antioxidative protection of organism as a result of adaptive response to repeated cold-induced oxidative stress. They observed higher initial GH level and higher activity of SOD and CAT, but not GPx in winter swimmers comparing with people who had never participated in winter swimming. In our previous studies, the improvement of antioxidant capacity as an effect of whole-body exposure to extremely low temperatures was also revealed in sportsmen subjected to intense physical exercise (Mila-Kierzenkowska et al., 2009). Increased baseline activity of key antioxidant enzymes as well as the changes in other elements of antioxidant defense system in preparation for a putative oxidative stress as a result of repeated cold action were also observed in warm-blooded animals, what seems to be an evolutionary adaptation of organisms exposed to low ambient temperatures (Blagojevic et al., 2011). The effect of baths in ice-cold water on total peroxyl radical trapping antioxidant capacity of plasma (TRAP) was previously examined by Dugue et al. (2005). The authors found slightly increased TRAP value in winter swimmers after cold exposure, nonetheless no long-term changes in basal values of TRAP could be observed. In presented paper we have found no differences in initial activity of antioxidant enzymes and no differences in basal level of lipid peroxidation products between regular winter swimmers and novices. Adaptation of human to
599
cold involves metabolic and insulative mechanism, but the time courses of development of individual mechanism to adaptation differ (Jansky, 2003). The variation in obtained results may be due to fact studied in this paper that winter swimmers possibly experienced winter swimming too short. In this paper we have found no statistically significant changes of antioxidant enzymes activity in erythrocytes because of short exposure to cold, however some increasing tendency in CAT, SOD and GPx activity was observed after swimming in cold water both in regular winter swimmers and in people who practiced winter swimming for the first time. Moreover, the positive correlation in activity of SOD and GPx in group of regular winter swimmers as well as the positive correlation in SOD and CAT, and SOD and GPx in novice winter swimmers were found 30 min after the cold bath. The increasing tendency in antioxidant enzymes activity and statistically significant correlations between studied antioxidant enzymes provide the evidence that after exposure to cold, intensified production of ROS occurs, as some other authors (Blagojevic, 2007; Blagojevic et al., 2011; Brenke et al., 1994; Siems et al., 1994; 1999) previously reported. Increased activity of SOD, CAT and GPx one hour after swimming in cold water was also observed in mouse liver (Akhalaya et al., 2006). The mechanisms related to generation of reactive oxygen species after cold exposure are multifold and difficult to determine, yet shivering thermoregulation is supposed to be one of the most important (Bleakley and Davison, 2010). During shivering, which is an involuntary rhythmic muscle contraction, the enhancement in heat production occurs thus the respiration and oxygen consumption increase which results in intensified ROS production. Nonetheless, non-shivering thermogenesis, which emerges in brown adipose tissue under the conditions of low ambient temperatures to generate the heat, may be also of great importance (Bleakley and Davison, 2010). The increased ROS formation after exposure to low temperatures may also ensue from the oxidation of molecules, including catecholamines. Catecholamines like dopamine, adrenaline and noradrenaline may be auto-oxidized and form superoxide anion, which is subsequently capable of oxidizing more the originating compound thus setting up a complex free-radical chain reaction (Kruk and K"adna, 1998). The increase of some catecholamines after whole-body exposure to cold water was observed by some authors (Lepp¨aluoto et al., 2008; Vybiral et al., 2000). Huttunen et al. (2001) postulated that attenuation of the catecholamine responses to cold water observed during winter swimming season may be an element of adaptation to
Table 5 Heart rate measured before, during and after exposure to cold water in form of winter swimming (WS) in people regularly taking baths in freezing water (regular winter swimmers) and in those who practiced it for the first time (novice winter swimmers). Before WS
Regular winter swimmers Heart rate (bpm) 7 SD 123.9 7 26.3 Novice winter swimmers Heart rate (bpm) 7 SD 133.5 7 34.6
30th second of WS
60th second of WS
90th second of WS
120th second of WS
150th second of WS
180th second of WS
3 min after WS
127.8 721.6
124.9 7 22.2
121.4 720.7
118.6 7 20.9
114.1 719.6
108.7 7 17.2
110.07 180
130.77 30.1
124.2 7 30.4
119.9 728.0
115.8 7 26.2
113.2 725.7
112.6 7 25.5
111.1 729.6
Table 6 Heart rate measured before, during and after exposure to heat (sauna) in people regularly taking baths in freezing water (regular winter swimmers) and in those who practiced it once (novice winter swimmers). Before sauna
Regular winter swimmers Heart rate (bpm) 7 SD 82.8 7 18.8 Novice winter swimmers Heart rate (bpm) 7SD 81.1 7 21.3
5th minute of sauna
10th minute of sauna
15th minute of sauna
20th minute of sauna
25th minute of sauna
30th minute of sauna
5 min after sauna
107.1 715.3
113.0 7 18.6
121.0 7 19.5
129.5 722.6
133.8 7 24.3
140.3 7 24.0
109.37 22.9
95.4 717.5
102.8 7 20.4
108.8 7 22.9
116.9 722.1
124.8 7 22.7
132.1 7 25.3
106.97 19.2
600
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cold. Yet, further studies revealed that the changes in humoral status were rather due to adaptation to the research situation or refer to a seasonal variation (Hirvonen et al., 2002). Although the changes in activity of studied enzymes after exposure to cold are relatively small and statistically insignificant, considerable changes in CAT activity were observed after exposure of organism to heat. CAT activity statistically significantly increased 5 min and decreased 30 min after sauna bath. Relevant changes in SOD, CAT and GPx activities after heat stress were also observed in rats, but the results varied depending on the age and ¨ zturk ¨ ¨ usl ¨ u, ¨ 2004). Increased CAT stress duration (O and Gum activity 5 min after sauna must be due to increase in production of hydrogen peroxide (H2O2). Induction of oxidative stress and increased reactivity of ROS like H2O2 after exposure to heat was ¨ zturk ¨ ¨ usl ¨ u, ¨ 2004). previously reported in literature (O and Gum Catalase is the enzyme that destroys H2O2 by catalyzing its twoelectron dismutation into oxygen and water, but only at high concentration of H2O2 (Chaudiere and Ferrari-Iliou, 1999). Moreover, the rate of this is proportional to H2O2 concentration over a wide range of the latter and is among the highest enzymatic rates (Kirkman and Gaetani, 2006). Hydrogen peroxide may be also an inactivating agent of this enzyme and the mechanism of this progressive and transient inactivation involves the formation and interconversion of several redox intermediates (Vlastis et al., 2010). Hence such inactivation may be responsible for decrease in CAT activity observed in this study 30 min after sauna bath. On the other hand, decrease in CAT activity may be due to lowered hydrogen peroxide concentration as a consequence of increased catalase activity during first minutes after sauna. In normal conditions when H2O2 level is relatively low, it is essentially degraded in erythrocytes by GPx (Chaudiere and Ferrari-Iliou, 1999). It is possible that 30 min after sauna CAT activity decrease because the H2O2 is then neutralized by GPx, which may be confirmed by a negative correlation between CAT and GPx activity observed in group of winter swimmers 30 min after sauna bath. These changes reflect the highly dynamic nature of ROS formation and all the processes associated with it. The advantage of antioxidant enzymes over the non-enzymatic scavengers is that the enzyme may act very fast for they can be induced or inhibited according to actual requirement in cell immediately by endogenous effectors (Harris, 1992). It is worth to note that an important feature of antioxidant enzymes is also that they act in synergy to neutralize reactive oxygen species and such a synergistic interaction are reinforced by mutual protections (Chaudiere and Ferrari-Iliou, 1999). Presented in this paper correlation between studied antioxidant enzymes may be a confirmation of this statement. One of the most important effects of increased ROS formation observed in cells is lipid peroxidation. Although this process hardly ever contributes directly to killing, products of oxidized lipids like MDA or 4-hydroxynonenal (HNE) may themselves initiate further oxidative damage, which could prove fatal (Avery, 2011). Siems et al. (1999) proved that intensive short-term exposure to cold induces oxidative stress. In a group of winter swimmers the higher level HNE, which is one of the lipid peroxidation products was observed 15 min after the bathing in ice-cold water. One hour after the cold bath reduced level of glutathione (GH) and uric acid was also found, but HNE level was similar to the value before the cold exposure. The results obtained in this paper are at variance with those of Siems et al. (1999). After the swimming in cold water, we have observed a decreasing tendency in concentration of TBARS in plasma and especially in erythrocytes of both winter swimmers and novices. Moreover, in a group of winter swimmers statistically significant decrease in TBARS level in erythrocytes was noticed 5 min after cold bath as compared with control value. The decrease of these lipid peroxidation products may be a result of highly efficient antioxidant protection, including antioxidant enzymes and/or
non-enzymatic systems in the removal of harmful compounds under condition of cold stress. The lower total oxidative status (TOS) with concomitant higher total antioxidative status (TAS) after the exposure to extremely low temperatures was observed by Lubkowska et al. (2008). The other explanation of this lowering TBARS concentration may be the phenomena of peripheral hyperemia that occurs few minutes after the exposure to ice-cold water. This hyperemia results in better metabolism and faster elimination of harmful products of metabolism (Suszko, 2003). However, it cannot be excluded that intensified lipid peroxidation occurs in other tissues after the baths in ice-cold water. In neonatal chicks, after the exposure to low temperature, higher malondialdehyde level was observed in brain and in heart, but not in plasma, liver and muscle (Mujahid and Furuse, 2009). Osorio et al. (2003) also observed increased oxidative stress index calculated from the ratio TBARS/GSH and TBARS/Vitamin E in pregnant rats, yet such changes were not found in rats which were exercised. The authors imply that physical training allows a more efficient activation of antioxidant mechanism under thermal stress thus it may be a kind of adaptation of organism to cold exposure. The mechanisms responsible for cold adaptation in humans have been studied for years, but still are not fully understood (Vybiral et al., 2000). In presented paper no statistically significant differences in basal activity of antioxidant enzymes or in concentration of lipid peroxidation products were observed between regular and inexperienced winter swimmers. Nonetheless, the profile of changes in TBARS concentration in winter swimmers may suggest the existence of adaptive mechanisms protecting erythrocytes against cold stress damage. The reperfusion of oxygenated blood to ischemic organs, which take place after exposure to cold, is proved to be responsible for overproduction of reactive oxygen species. In such conditions ROS are formed mostly in mitochondrial respiration in the early phase of reperfusion and by activated phagocytes in early stage that is followed by induction of lipid peroxidation, protein degradation and DNA damage (Hermes-Lima and Zenteno-Savin, 2002). Lower level of lipid peroxidation after cold exposure in group of winter swimmers may testify their adaptation to ischemia and reperfusion as a result of repeated exposure to low ambient temperatures. Jansky´ et al. (1996) reported that repeated exposure of young sportsmen to cold water induced changes in regulation of thermal homeostasis. The metabolic response to cold was delayed and subjective shivering was attenuated. The decrease in metabolic rate thus the lesser oxygen consumption may be also responsible for decrease of lipid peroxidation in regular winter swimmers. In presented paper, the effect of exposure to the heat was also examined in the same groups of subjects. Higher TBARS level in blood plasma was found 5 and 30 min after visit in sauna as compared to control value, but only in novice group. In winter swimmers, the concentration of TBARS in blood plasma was only slightly increased (statistically insignificant). Those results clearly prove that heat stress is a reason of oxidative stress that is reflected by increased lipid peroxidation. However, oxidative stress after exposure to high temperature is observed mainly in novices and hardly in regular winter swimmers, that implies again the existence of adaptive response in people regularly exposed to thermal stress. Increased TBARS levels after heat stress were also observed ¨ zturk ¨ and Gum ¨ usl ¨ u, ¨ 2004). Masuda in animals like rats and cows (O et al. (2004) found lower level of 8-epi-prostaglandin F2a, which is a chemically stable product of lipid peroxidation, in blood of patients with coronary risk factor after the sauna therapy. The authors suggest that repeated sauna sessions may protect against oxidative stress, which leads to prevention of atherosclerosis. The adaptation of organism to heat stress in form of sauna bathing may be of great importance for human health, especially considering the fact that elevated temperatures during summer months are associated with excess in morbidity and mortality (McGeehin and Mirabelli, 2001).
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Concluding we state that both winter swimming and sauna affect the oxidant–antioxidant balance, however the changes are more distinct after sauna session. Among antioxidant enzymes, catalase seems to play the most important role in neutralizing reactive oxygen species that occurs as a result of thermal stress. In presented paper, higher level of lipid peroxidation products was observed after sauna than before the exposure to heat, but only in novice winter swimmers who rarely participated in sauna baths prior to the experiment. It may suggest that in winter swimmers who regularly practice swimming combined with hot sauna some adaptive changes exist and protect the organism against oxidative damage. References Akhalaya, M.Y., Platonow, A.G., Baizhumanov, A.A., 2006. Short-term cold exposure improves antioxidant status and general resistance of animals. Bull. Exp. Biol. Med. 14, 26–29. Avery, S.V., 2011. Molecular target of oxidative stress. Biochem. J. 434, 201–210. Beers, R.T., Sizer, I.W., 1952. A spectrophotometric method for measuring the breakdown of hydrogen peroxide by catalase. J. Biol. Chem. 195, 133–140. Biro, S., Masuda, A., Kihara, T., Thei, C., 2003. Clinical implication of thermal therapy in lifestyle-related diseases. Exp. Biol. Med. 228, 1245–1249. Blagojevic, D.P., 2007. Antioxidant systems in supporting environmental and programmed adaptation to low temperatures. CryoLetters 28, 137–150. Blagojevic, D.P., Grubor-Lajsic, G.N., Spasic, M.B., 2011. Cold defence responses: the role of oxidative stress. Front. Biosci. 1, 416–427. Bleakley, C.M., Davison, G.W., 2010. What is the biochemical and physiological rationale for using cold-water immersion in sports recovery. Br. J. Sports Med. 44, 179–187. Brenke, R., Siems, W., Maass, R., 1994. Fitness by cold stimulation of various intensity: effects on metabolism of purines and free radicals. Wien. Med. Wochenschr. 144, 66–68. Buege, J.A., Aust, S,D., 1978. Microsomal lipid peroxidation. In: Fleisher, S., Packer, I. (Eds.), Methods in Enzymology. Academic Press, New York. Chaudiere, J., Ferrari-Iliou, R., 1999. Intracellular antioxidants: from chemical to biochemical mechanisms. Food Chem. Toxicol. 37, 949–962. Crawford, D.R., Davies, J.A., 1994. Adaptive response and oxidative stress. Environ. Health Perspect. 102, 25–28. Davies, K.J.A., 2000. Oxidative stress, antioxidant defenses and damage removal, repair and replacement systems. Life 50, 279–289. Dugue, B., Smolander, J., Westerlund, T., Oksa, J., Nieminen, R., Moilanen, E., Mikkelsson, M., 2005. Acute and long-term effects of winter swimming and whole-body cryotherapy on plasma antioxidative capacity in healthy women. Scand. J. Clin. Lab. Invest. 65, 395–402. Ernst, E., 1989. Sauna—a hobby or for health. J. R. Soc. Med. 82, 639. Esterbauer, H., Cheeseman, K.H., 1990. Determination of aldehydic lipid peroxidation products: malondialdehyde and 4-hydroxynonenal. In: Packer, L., Glazer, A.N. (Eds.), Methods in Enzymology. Academic Press, New York. Hannuksela, M.L., Ellahham, S., 2001. Benefits and risks of sauna bathing. Am. J. Med. 2, 118–128. Harris, E.D., 1992. Regulation of antioxidant enzymes. FASEB J. 6, 2675–2683. Hermes-Lima, M., Zenteno-Savin, T., 2002. Animal response to drastic changes in oxygen availability and physiological oxidative stress. Comp. Biochem. Phys. C 133, 537–556. Hirvonen, J., Lindeman, S., Matti, J., Huttunen, P., 2002. Plasma catecholamines, serotonin and their metabolites and beta-endorphin of winter swimmers during one-winter. Possible correlations on psychological traits. Int. J. Circumopolar Health 61, 363–372. Huttunen, P., Kokko, L., Ylijukuri, V., 2004. Winter swimming improve general well-being. Int. J. Circumoplar Health 63, 140–144. Huttunen, P., Rintamaki, H., Hirvonen, J., 2001. Effects of regular winter swimming on the activity of the sympathoadrenal system before and after a single cold water immersion. Int. J. Circumpolar Health 60, 400–406.
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Contents lists available at SciVerse ScienceDirect
Journal of Thermal Biology journal homepage: www.elsevier.com/locate/jtherbio
Thermal stress and oxidant–antioxidant balance in experienced and novice winter swimmers Celestyna Mila-Kierzenkowska a,n, Alina Woz´niak a, Tomasz Boraczyn´ski b, Micha" Szpinda c, Bartosz Woz´niak d, Alicja Jurecka e, Anna Szpinda c a
The Chair of Medical Biology, Collegium Medicum of Nicolaus Copernicus University, Kar!owicza 24, 85-094 Bydgoszcz, Poland ´zef Rusiecki Olsztyn University, Bydgoska 33, 10-243 Olsztyn, Poland Central Research Laboratory, Jo c Department of Normal Anatomy, Collegium Medicum of Nicolaus Copernicus University, Kar!owicza 24, 85-094 Bydgoszcz, Poland d Department of Neurosurgery, Stanislaw Staszic Specialist Hospital, Rydygiera 1, 64-920 Pi!a, Poland e Department of Orthopaedics and Musculoskeletal Traumatology, Collegium Medicum of Jagiellonian University, Os. Zlotej Jesieni 1, 31-826 Krakow, Poland b
a r t i c l e i n f o
abstract
Article history: Received 23 March 2012 Accepted 26 July 2012 Available online 4 August 2012
The adaptation of human organism to environmental stress plays an important role in maintaining good health. The exposure to both low and high ambient temperature may provoke thermal stress and such a condition potentially leads to the excessive production of reactive oxygen species, which may result in oxidative stress. The purpose of the study was to determine the effect of one session of swimming in ice-cold water and one hot sauna session (performed few months later) on oxidant– antioxidant balance in two groups of healthy volunteers: 21 experienced winter swimmers and 19 people who participated in winter swimming for the first time (novices). The activity of antioxidant enzymes: catalase, superoxide dismutase and glutathione peroxidase was measured in erythrocytes of studied persons. Moreover, in blood plasma and erythrocytes the concentration of lipid peroxidation products was estimated. No statistically significant differences in initial values of antioxidant enzymes activity and lipid peroxidation products level were revealed between experienced and novice winter swimmers. The crucial antioxidant enzyme that neutralizes reactive oxygen species generated as a result of thermal stress seems to be catalase, since statistically significant changes of CAT activity after sauna were observed. Increased TBARS level observed as a result of sauna bath proves that exposure of organism to high ambient temperature is a source of oxidative stress. However, such a stress was hardly noticed in regular winter swimmers. The regular baths in cold water combined with sauna probably lead to adaptive changes that protect the organism against harmful effects of thermal stress. & 2012 Elsevier Ltd. All rights reserved.
Keywords: Winter swimming Sauna Oxidative stress Reactive oxygen species Lipid peroxidation Adaptive response
1. Introduction The inevitable result of life in an oxygen-rich atmosphere is oxidative stress defined as a disturbance in oxidant–antioxidant systems in favor of the former (Davies, 2000). Although oxygen is essential for most life forms, it is also toxic due to generation of reactive oxygen species called ROS (Hermes-Lima and ZentenoSavin, 2002). ROS are potential toxic compounds produced continuously during the process of respiration, which represent a paradox in their biological function. They prevent diseases by assisting the immune system, mediating cell signaling and play a role in apoptosis, but on the other hand they can damage essential
Abbreviations: SOD, superoxide dismutase; CAT, catalase; GPx, glutathione peroxidase; TBARS, thiobarbituric acid reactive substance n Corresponding author. Tel.: þ48 52 585 3737; fax: þ 48 52 585 3742. E-mail addresses: [email protected] (C. Mila-Kierzenkowska), [email protected] (A. Woz´niak), [email protected] (T. Boraczyn´ski), [email protected] (M. Szpinda), [email protected] (B. Woz´niak), [email protected] (A. Jurecka), [email protected] (A. Szpinda). 0306-4565/$ - see front matter & 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jtherbio.2012.07.007
macromolecules, such as lipids, proteins and nucleic acids (Seifried et al., 2007). The first type of oxidative damage, caused by increased ROS generation, reported in the literature was lipid peroxidation (Davies, 2000). Extensive lipid peroxidation process is known to be related to ultimate disintegration of membranes and cell death (Avery, 2011). However, in organisms the enhancement in ROS formation is usually restricted by antioxidant mechanisms. Antioxidants are molecules that prevent uncontrolled formation of reactive oxygen species or inhibit their reactions with biological structures (Chaudiere and Ferrari-Iliou, 1999). The antioxidant system consists of antioxidant enzymes, like superoxide dismutase (SOD), catalase (CAT) glutathione peroxidase (GPx) as well as of a wide range of non-enzymatic compounds (Osorio et al., 2003). During the human lifetime the formation of ROS may be intensified by a range of different stress conditions (Avery, 2011), including the changes in environmental temperature. The cold exposure was proved to provoke the increased production of reactive oxygen species by changes in energy production (Blagojevic, 2007). An example of voluntary exposure of human to low environmental temperatures is winter swimming. Winter swimming refers to a
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regular swimming in cold water during winter season and is a common kind of habit in many countries. Immersion in cold water results in immediate and long-term physiologic alterations, consisting of metabolic, cardiovascular and hormonal changes (Kolletis and Kolletis, 2003) and is supposed to be a method of body hardening (Siems et al., 1994). Huttunen et al. (2004) presented that regular winter swimming improves well-being of patients who suffer from rheumatism, fibromyalgia and asthma. The winter swimmers usually combine the swimming in icecold water with a hot sauna bath. Sauna bathing, which is a special form of exposure to heat, is a popular form of wellness practiced in many countries. It is defined as short-term exposure to exceptionally high environmental temperatures (KukkonenHarjula and Kauppinen, 2006). Nowadays, innovative experiments investigating new therapeutic uses of sauna bathing have been performed throughout the world (Biro et al., 2003). The reactions of organism to sauna bathing are the expression of active thermoregulation and bear similarities with those of acute physical exercise (Ernst, 1989). After sauna, there occurs the sweating with loss of the body water and electrolytes, skin vasodilatation with an increase in heart rate and cardiac output, which results in decrease of blood pressure and hyperventilation (Kauppinen, 1997). A number of studies report the effect of sauna on hormonal changes in organism. Changes in the secretion of hormones induced by sauna are at times similar to changes provoked by any other stress situation. Exposure to heat for example induces the increase in noradrenalin level, but also the secretion of antidiuretic hormone and the rennin– angiotensin–aldosterone system is activated (Kukkonen-Harjula and Kauppinen, 1988). Moreover, sauna increases level of prolactin in blood plasma of women (Laatikainen et al., 1988). Sauna baths are also known to induce the changes in the lipid profile in blood serum. Pilch et al. (2010) demonstrated lowered concentration of total cholesterol and low-density lipoproteins (LDL) with concomitant increase of high-density lipoproteins (HDL) level in women after repeated sauna sessions. Though the sauna sessions cause various transient cardiovascular and hormonal changes, it is tolerated well by most healthy people (Hannuksela and Ellahham, 2001). Despite the well-known acute response to sauna, the long-term effects of regular sauna using are still speculative. The literature data postulate that both regular winter swimming and regular sauna visits may have a positive influence on human health by inducing some adaptive changes in organism. Adaptation response refers to the ability to better resist the damaging effects of some stress agents by prior pre-exposure to Table 1 Characteristics of the studied groups.
Number of participants Age (years) BMI (kg/m2) PWC170 (W/kg) VO2max (ml/min/kg)
Experienced winter swimmers
Novices
21 27.6 75.9 22.8 73.1 2.96 70.8 45.21 7 5.8
19 25.5 74.7 23.6 72.3 2.65 70.5 43.64 75.5
Values are expressed as mean 7 standard deviations (SD). Body Mass Index (BMI) was calculated using the formula weight/height2. PWC170 (Physical Work Capacity) test was performed on a cycle-ergometer Monark 828 E.
a low amount of this agent. The main physiological benefit of adaptive response is to protect the organism against stress damage caused by high doses of toxic agent (Crawford and Davies, 1994). Adaptive response to oxidative stress has been documented well for prokaryotic and eukaryotic cells (Crawford and Davies, 1994). Some authors postulate that organisms are able to adapt themselves to high exposure of ROS by increasing the expression of antioxidant enzymes and other forms of defense reactions against oxidative damage (Hermes-Lima and Zenteno-Savin, 2002). Blagojevic et al. (2011) showed that short-time exposure to cold leads to oxidative stress; however as it persists the organisms develop some adaptive changes toward reducing ROS formation and increasing the antioxidant action. The aim of this study was to determine the effect of exposure to low and high temperature on activity of antioxidant enzymes and concentration of lipid peroxidation products in blood of habitual and inexperienced winter swimmers.
2. Material and methods The study was performed on two groups of healthy volunteers, who reported the absence of any chronic disease and no use of any medications. The basic characteristic of the subjects is shown in Table 1. The first group consisted of 21 men who have been regular winter swimmers (WS-R). The winter swimming experience in this group ranged from 1 to 12 years, but most of them were beginners (mean experience amounted to 4.1 years). The activity of winter swimmers starts in the fall (usually at October) and ends in April, and they swim in the ice-cold water once a week during the whole season (it means about 18–22 sessions of winter swimming per season). The other group included 19 men who participated in winter swimming for the first time in their life and we named them novice winter swimmers (WS-N). Subjects from this group were selected from the people who had participated in sauna only occasionally. The experiment consisted of two parts—one sessions of winter swimming and one session of sauna (Table 2). The bath in ice-cold water was performed in the winter (in the middle of the winter swimming season) at the small river Wada˛g near Olsztyn (Poland). Winter swimming took place in the morning following the short warming up. The temperature of the water at the day of experiment was 0 1C, while the air temperature was 4 1C. All the subjects spent 3 min in ice-cold water staying all the time in contact with doctor who supervised the study. The part of the study including sauna took place in the summer few months after the end of winter swimming season. All subjects spent 30 min without any breaks in sauna heated to 85 1C with relative humidity of 40%. Before the blood collection, the studied persons were not allowed showering/cooling action. In the course of the experiment, the heart rate of all the participants was monitored by means of a Polar meter and the results were analyzed by PC software. The blood samples were taken from every studied person altogether six times: before the start of the experiment (control), 5 and 30 min after the swimming in ice-cold water as well as before and 5, and 30 min after hot sauna bath. The blood were taken from the cubital vein and put into the tube contained
Table 2 The scheme of the study design (all participants were subjected to the same sessions). Date of exposure
January 2010 July 2010
Type of exposure
Blood samples
Cold bath (winter swimming)
Sauna bath
One 3 min session –
– One 30 min session
Taken before, 5 min and 30 min after the exit from cold water Taken before, 5 min and 30 min after the exit from sauna
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K2EDTA. The research has obtained the agreement of the Bioethics Committee at Collegium Medicum in Bydgoszcz of the Nicolaus Copernicus University in Torun´ and all subjects had given written informed consent to participate in the study. The activity of SOD, CAT and GPx was measured in erythrocytes. Superoxide dismutase activity was determined according to Misra and Fridovich (1972) by a procedure based on SOD impeding the reaction of auto-oxidation of adrenaline to adrenochrome in an alkaline environment. SOD activity was expressed as U/gHb. Beers and Sizer (1952) method was used to measure the activity of catalase. This method is based on the detection at a wavelength of 240 nm of the decrease of hydrogen peroxide absorbance. CAT activity was expressed as 104 IU/gHb. Glutathione peroxidase activity was performed by Paglia and Valentine (1967). The method is based on the measurement of changes in absorbance caused by oxidation of reduced nicotinamide adenine dinucleotide phosphate (NADPH), measured at a wavelength of 340 nm. NADPH is a coenzyme of reduction of glutathione disulfide. The obtained oxided glutathione is a product of reaction catalyzed by glutathione peroxidase. Activity of GPx was expressed as U/gHb. The concentration of thiobarbituric acid reactive substances (TBARS) was assayed both in the erythrocytes and in blood plasma. TBARS level was determined according to Buege and Aust (1978) method in the Esterbauer and Cheeseman (1990) modification. The method involves creation of colored complex between lipid peroxidation products and thiobarbituric acid at the temperature of 100 1C in acidic environment. The maximum absorption of that complex occurs at a wavelength of 532 nm. The main product of lipid peroxidation that reacts with thiobarbituric acid is malondialdehyde (MDA) and therefore the level of TBARS in plasma was expressed as nmol of MDA/ml and in the erythrocytes as nmol of MDA/gHb. All the data were statistically analyzed with the use of ANOVA test. The differences on the level of p r0.05 were accepted as statistically significant. 3. Results In presented study, some changes in activity of catalase in studied subjects were revealed (Fig. 1). The exposure to cold water had no statistically significant impact on activity of this enzyme, however increasing tendency was observed both in regular winter
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swimmers and in novices. The changes in CAT activity as a consequence of hot sauna bath were more distinct and statistically significant. 5 min after sauna CAT activity in WS-R increased by about 20% (po0.05) and in WS-N by about 35% (po0.01) versus the control value (Fig. 1). 30 min after sauna it decreased by about 22% (po0.05) in WS-R and by about 26% (po0.01) in WS-N as compared to the activity 5 min after sauna and was comparable to the control value (Fig. 1). Considering SOD and GPx activity, there were no statistically significant changes after both cold bath and sauna (Table 3). There were also no differences in initial activity of investigated antioxidant enzymes between WS-R and WS-N (Table 3). We have also found no statistically significant differences in basal TBARS level both in blood plasma and in erythrocytes between the group of WS-R and WS-N (Table 4). Nonetheless, some changes in concentration of lipid peroxidation products were observed as result of exposure to low temperature. The decreasing tendencies of TBARS level in blood plasma and erythrocytes was observed 5 and 30 min after cold bath, both in WS-R and WS-N; however most of them were statistically insignificant (Table 4). The only statistically significant difference was revealed in TBARSerythrocytes level in of WS-R 5 min after the bath in cold water, and it was about 26% lower than before the start of the experiment (p o0.05). The relevant changes in TBARS concentration were found after sauna bath. In WS-N, the exposure to high ambient temperature caused significant increase of TBARSplasma concentration (Fig. 2). 5 min after sauna it was about 40% (p o0.001) and after 30 min about 33% (po0.001) higher as compared to the control value. Moreover, 5 min after sauna TBARSplasma level was 56% higher (p o0.001) in WS-N and 19% higher (p o0.05) in WS-R than 5 min after cold bath. 30 min after sauna level of this product of lipid peroxidation in WS-N was 37% higher (p o0.001), while in WS-R 34% higher (po0.01) than 30 min after bath in cold water. TBARS concentration in erythrocytes after sauna sessions was also higher then after winter swimming (Table 4). In WS-N 5 min after sauna TBARSerythrocytes level was about 58% higher (po0.01) and 30 min after sauna bath about 54% higher (p o0.01) than that at 5 and 30 min after winter swimming, respectively. In WS-R 5 min after sauna bath it was 31% higher than 5 min after cold bath (p o0.001) as well as 30 min after sauna it was 40% higher than 30 min after exposure to cold water (po0.001).
100 95 CAT 104 (IU/ gHb)
90
regular winter swimmers novice winter swimmers
85 80 75 70 aa
65
a
60 55 50 before winter swimming
5 min after 30 min after winter winter swimming swimming
before sauna
5 min after sauna
30 min after sauna
Statistically significant differences: - versus before the exposure: * p<0.05, ** p<0.01 - versus 5 min after sauna: a p<0.05, aa p<0.01 Fig. 1. Activity of catalase (CAT) before and after exposure to cold (winter swimming) and heat (sauna bath) in people regularly taking baths in freezing water (regular winter swimmers) and in those who practiced it once (novice winter swimmers).
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Table 3 The activity of antioxidant enzymes: catalase (CAT), glutathione peroxidase (GPx), and superoxide dismutase (SOD) after exposure to low and high ambient temperature in people regularly taking baths in freezing water (regular winter swimmers) and in those who practiced it for the first time (novice winter swimmers). Exposure to cold water (winter swimming) Before the exposure
Exposure to hot temperature (sauna bathing)
After 5 min
After 30 min
Before the exposure
After 5 min
After 30 min
Regular winter swimmers GPx (U/gHb) 10.97 4.8 SOD (U/gHb) 862.0 7 178.5
12.5 7 4.7 879.2 7 177.4
12.3 7 4.1 898.0 7 144.3
10.8 73.6 878.4 7182.7
11.4 74.5 856.8 791.9
11.27 3.8 897.1 7 94.3
Novice winter swimmers GPx (U/gHb) 11.7 7 5.3 SOD (U/gHb) 942.8 7 169.5
12.5 7 3.6 923.7 7 141.0
12.0 7 4.3 970.6 7 180.1
11.8 74.6 928.9 7177.3
8.8 74.9 872.0 7108.1
11.97 4.4 896.6 7 82.6
Values are expressed as means7 standard deviations (SD) of the means.
Table 4 The concentration of concentration of lipid peroxidation products—thiobarbituric acid reactive substances (TBARS) after exposure to low and high ambient temperature in blood plasma and erythrocytes of people regularly taking baths in freezing water (regular winter swimmers) and in those who practiced it for the first time (novice winter swimmers). Exposure to cold water (winter swimming)
Exposure to hot temperature (sauna bathing)
Before the exposure
After 5 min
After 30 min
Before the exposure
After 5 min
After 30 min
Regular winter swimmers TBARSplasma (10 1 nmolMDA/ml) TBARSerythrocytes [nmolMDA/gHb]
3.47 0.4 28.1 7 8.1
3.2 70.5 20.8 75.9n
2.9 70.4 22.9 77.9
3.4 7 0.3 27.9 7 6.7
3.87 0.5abbb 27.37 3.6aaabb
3.97 0.6aabb 32.17 7.2aaabbb
Novice winter swimmers TBARSerythrocytes (nmolMDA/gHb)
24.07 9.3
17.9 76.1
18.6 76.2
26.1 7 8.4
28.27 5.7aab
28.77 6.1aaabb
are expressed as means7 standard deviations (SD) of the means. Statistically significant differences: before exposure: np o0.05. 5 min after cold bath: ap o 0.05, aapo 0.01, aaap o 0.001. 30 min after cold bath: bp o0.05, bbp o0.01, bbbp o 0.001.
TBARS in plasma [10-1 nmol MDA/ml]
Values Versus Versus Versus
5.5 5
novice winter swimmers
4.5
aaa
4
bbb
3.5 3 2.5 2 before winter swimming
5 min after 30 min winter after winter swimming swimming
before sauna
5 min after 30 min sauna after sauna
Statistically significant differences: - versus before exposure: *** p<0.001 - versus 5 min after cold bath: aaa p<0.001 - versus 30 min after cold bath: bbb p< 0.001 Fig. 2. Concentration of thiobarbituric acid reactive substances (TBARS) before and after exposure to cold (winter swimming) and heat (sauna bath) in blood plasma of people who practiced taking baths in freezing water only once (novice winter swimmers).
Although we found no alterations in SOD and GPx activity in investigated groups during exposure to changed environmental conditions, we noticed some correlations between the antioxidant enzymes activity. In WS-R SOD and GPx activity was positively
correlated 30 min after bath in cold water (r ¼0.543, p o0.01), while CAT and GPx activity negatively correlated 30 min after sauna (r ¼ 0.461, po0.05). In WS-N the positive correlations were found between SOD and CAT activity (r ¼0.529) as well as
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between SOD and GPx activity (r ¼0.596) 30 min after bath in cold water, both on the level of statistical significance of p o0.05. 30 min after sauna positive correlation (r ¼0.74) was observed between the activity of SOD and GPx (p o0.01). Heart rate measured before winter swimming, during staying in the cold water and 3 min after the exit from the river is presented in Table 5. Similarly as in case of winter swimming heart rate was measured before the entry to sauna as well as during sauna bath and at recovery period (Table 6).
4. Discussion Winter swimming and sauna baths are the forms of recreation that refer to a thermal stress that may lead to disturbances in oxidant–antioxidant balance since the generation of reactive oxygen species is intensified under different stress conditions (Avery, 2011). In turn, repeated exposure to oxidative stress may induce some adaptive changes in organisms that help them to resist stress damage. Siems et al. (1999) demonstrated the improved antioxidative protection of organism as a result of adaptive response to repeated cold-induced oxidative stress. They observed higher initial GH level and higher activity of SOD and CAT, but not GPx in winter swimmers comparing with people who had never participated in winter swimming. In our previous studies, the improvement of antioxidant capacity as an effect of whole-body exposure to extremely low temperatures was also revealed in sportsmen subjected to intense physical exercise (Mila-Kierzenkowska et al., 2009). Increased baseline activity of key antioxidant enzymes as well as the changes in other elements of antioxidant defense system in preparation for a putative oxidative stress as a result of repeated cold action were also observed in warm-blooded animals, what seems to be an evolutionary adaptation of organisms exposed to low ambient temperatures (Blagojevic et al., 2011). The effect of baths in ice-cold water on total peroxyl radical trapping antioxidant capacity of plasma (TRAP) was previously examined by Dugue et al. (2005). The authors found slightly increased TRAP value in winter swimmers after cold exposure, nonetheless no long-term changes in basal values of TRAP could be observed. In presented paper we have found no differences in initial activity of antioxidant enzymes and no differences in basal level of lipid peroxidation products between regular winter swimmers and novices. Adaptation of human to
599
cold involves metabolic and insulative mechanism, but the time courses of development of individual mechanism to adaptation differ (Jansky, 2003). The variation in obtained results may be due to fact studied in this paper that winter swimmers possibly experienced winter swimming too short. In this paper we have found no statistically significant changes of antioxidant enzymes activity in erythrocytes because of short exposure to cold, however some increasing tendency in CAT, SOD and GPx activity was observed after swimming in cold water both in regular winter swimmers and in people who practiced winter swimming for the first time. Moreover, the positive correlation in activity of SOD and GPx in group of regular winter swimmers as well as the positive correlation in SOD and CAT, and SOD and GPx in novice winter swimmers were found 30 min after the cold bath. The increasing tendency in antioxidant enzymes activity and statistically significant correlations between studied antioxidant enzymes provide the evidence that after exposure to cold, intensified production of ROS occurs, as some other authors (Blagojevic, 2007; Blagojevic et al., 2011; Brenke et al., 1994; Siems et al., 1994; 1999) previously reported. Increased activity of SOD, CAT and GPx one hour after swimming in cold water was also observed in mouse liver (Akhalaya et al., 2006). The mechanisms related to generation of reactive oxygen species after cold exposure are multifold and difficult to determine, yet shivering thermoregulation is supposed to be one of the most important (Bleakley and Davison, 2010). During shivering, which is an involuntary rhythmic muscle contraction, the enhancement in heat production occurs thus the respiration and oxygen consumption increase which results in intensified ROS production. Nonetheless, non-shivering thermogenesis, which emerges in brown adipose tissue under the conditions of low ambient temperatures to generate the heat, may be also of great importance (Bleakley and Davison, 2010). The increased ROS formation after exposure to low temperatures may also ensue from the oxidation of molecules, including catecholamines. Catecholamines like dopamine, adrenaline and noradrenaline may be auto-oxidized and form superoxide anion, which is subsequently capable of oxidizing more the originating compound thus setting up a complex free-radical chain reaction (Kruk and K"adna, 1998). The increase of some catecholamines after whole-body exposure to cold water was observed by some authors (Lepp¨aluoto et al., 2008; Vybiral et al., 2000). Huttunen et al. (2001) postulated that attenuation of the catecholamine responses to cold water observed during winter swimming season may be an element of adaptation to
Table 5 Heart rate measured before, during and after exposure to cold water in form of winter swimming (WS) in people regularly taking baths in freezing water (regular winter swimmers) and in those who practiced it for the first time (novice winter swimmers). Before WS
Regular winter swimmers Heart rate (bpm) 7 SD 123.9 7 26.3 Novice winter swimmers Heart rate (bpm) 7 SD 133.5 7 34.6
30th second of WS
60th second of WS
90th second of WS
120th second of WS
150th second of WS
180th second of WS
3 min after WS
127.8 721.6
124.9 7 22.2
121.4 720.7
118.6 7 20.9
114.1 719.6
108.7 7 17.2
110.07 180
130.77 30.1
124.2 7 30.4
119.9 728.0
115.8 7 26.2
113.2 725.7
112.6 7 25.5
111.1 729.6
Table 6 Heart rate measured before, during and after exposure to heat (sauna) in people regularly taking baths in freezing water (regular winter swimmers) and in those who practiced it once (novice winter swimmers). Before sauna
Regular winter swimmers Heart rate (bpm) 7 SD 82.8 7 18.8 Novice winter swimmers Heart rate (bpm) 7SD 81.1 7 21.3
5th minute of sauna
10th minute of sauna
15th minute of sauna
20th minute of sauna
25th minute of sauna
30th minute of sauna
5 min after sauna
107.1 715.3
113.0 7 18.6
121.0 7 19.5
129.5 722.6
133.8 7 24.3
140.3 7 24.0
109.37 22.9
95.4 717.5
102.8 7 20.4
108.8 7 22.9
116.9 722.1
124.8 7 22.7
132.1 7 25.3
106.97 19.2
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cold. Yet, further studies revealed that the changes in humoral status were rather due to adaptation to the research situation or refer to a seasonal variation (Hirvonen et al., 2002). Although the changes in activity of studied enzymes after exposure to cold are relatively small and statistically insignificant, considerable changes in CAT activity were observed after exposure of organism to heat. CAT activity statistically significantly increased 5 min and decreased 30 min after sauna bath. Relevant changes in SOD, CAT and GPx activities after heat stress were also observed in rats, but the results varied depending on the age and ¨ zturk ¨ ¨ usl ¨ u, ¨ 2004). Increased CAT stress duration (O and Gum activity 5 min after sauna must be due to increase in production of hydrogen peroxide (H2O2). Induction of oxidative stress and increased reactivity of ROS like H2O2 after exposure to heat was ¨ zturk ¨ ¨ usl ¨ u, ¨ 2004). previously reported in literature (O and Gum Catalase is the enzyme that destroys H2O2 by catalyzing its twoelectron dismutation into oxygen and water, but only at high concentration of H2O2 (Chaudiere and Ferrari-Iliou, 1999). Moreover, the rate of this is proportional to H2O2 concentration over a wide range of the latter and is among the highest enzymatic rates (Kirkman and Gaetani, 2006). Hydrogen peroxide may be also an inactivating agent of this enzyme and the mechanism of this progressive and transient inactivation involves the formation and interconversion of several redox intermediates (Vlastis et al., 2010). Hence such inactivation may be responsible for decrease in CAT activity observed in this study 30 min after sauna bath. On the other hand, decrease in CAT activity may be due to lowered hydrogen peroxide concentration as a consequence of increased catalase activity during first minutes after sauna. In normal conditions when H2O2 level is relatively low, it is essentially degraded in erythrocytes by GPx (Chaudiere and Ferrari-Iliou, 1999). It is possible that 30 min after sauna CAT activity decrease because the H2O2 is then neutralized by GPx, which may be confirmed by a negative correlation between CAT and GPx activity observed in group of winter swimmers 30 min after sauna bath. These changes reflect the highly dynamic nature of ROS formation and all the processes associated with it. The advantage of antioxidant enzymes over the non-enzymatic scavengers is that the enzyme may act very fast for they can be induced or inhibited according to actual requirement in cell immediately by endogenous effectors (Harris, 1992). It is worth to note that an important feature of antioxidant enzymes is also that they act in synergy to neutralize reactive oxygen species and such a synergistic interaction are reinforced by mutual protections (Chaudiere and Ferrari-Iliou, 1999). Presented in this paper correlation between studied antioxidant enzymes may be a confirmation of this statement. One of the most important effects of increased ROS formation observed in cells is lipid peroxidation. Although this process hardly ever contributes directly to killing, products of oxidized lipids like MDA or 4-hydroxynonenal (HNE) may themselves initiate further oxidative damage, which could prove fatal (Avery, 2011). Siems et al. (1999) proved that intensive short-term exposure to cold induces oxidative stress. In a group of winter swimmers the higher level HNE, which is one of the lipid peroxidation products was observed 15 min after the bathing in ice-cold water. One hour after the cold bath reduced level of glutathione (GH) and uric acid was also found, but HNE level was similar to the value before the cold exposure. The results obtained in this paper are at variance with those of Siems et al. (1999). After the swimming in cold water, we have observed a decreasing tendency in concentration of TBARS in plasma and especially in erythrocytes of both winter swimmers and novices. Moreover, in a group of winter swimmers statistically significant decrease in TBARS level in erythrocytes was noticed 5 min after cold bath as compared with control value. The decrease of these lipid peroxidation products may be a result of highly efficient antioxidant protection, including antioxidant enzymes and/or
non-enzymatic systems in the removal of harmful compounds under condition of cold stress. The lower total oxidative status (TOS) with concomitant higher total antioxidative status (TAS) after the exposure to extremely low temperatures was observed by Lubkowska et al. (2008). The other explanation of this lowering TBARS concentration may be the phenomena of peripheral hyperemia that occurs few minutes after the exposure to ice-cold water. This hyperemia results in better metabolism and faster elimination of harmful products of metabolism (Suszko, 2003). However, it cannot be excluded that intensified lipid peroxidation occurs in other tissues after the baths in ice-cold water. In neonatal chicks, after the exposure to low temperature, higher malondialdehyde level was observed in brain and in heart, but not in plasma, liver and muscle (Mujahid and Furuse, 2009). Osorio et al. (2003) also observed increased oxidative stress index calculated from the ratio TBARS/GSH and TBARS/Vitamin E in pregnant rats, yet such changes were not found in rats which were exercised. The authors imply that physical training allows a more efficient activation of antioxidant mechanism under thermal stress thus it may be a kind of adaptation of organism to cold exposure. The mechanisms responsible for cold adaptation in humans have been studied for years, but still are not fully understood (Vybiral et al., 2000). In presented paper no statistically significant differences in basal activity of antioxidant enzymes or in concentration of lipid peroxidation products were observed between regular and inexperienced winter swimmers. Nonetheless, the profile of changes in TBARS concentration in winter swimmers may suggest the existence of adaptive mechanisms protecting erythrocytes against cold stress damage. The reperfusion of oxygenated blood to ischemic organs, which take place after exposure to cold, is proved to be responsible for overproduction of reactive oxygen species. In such conditions ROS are formed mostly in mitochondrial respiration in the early phase of reperfusion and by activated phagocytes in early stage that is followed by induction of lipid peroxidation, protein degradation and DNA damage (Hermes-Lima and Zenteno-Savin, 2002). Lower level of lipid peroxidation after cold exposure in group of winter swimmers may testify their adaptation to ischemia and reperfusion as a result of repeated exposure to low ambient temperatures. Jansky´ et al. (1996) reported that repeated exposure of young sportsmen to cold water induced changes in regulation of thermal homeostasis. The metabolic response to cold was delayed and subjective shivering was attenuated. The decrease in metabolic rate thus the lesser oxygen consumption may be also responsible for decrease of lipid peroxidation in regular winter swimmers. In presented paper, the effect of exposure to the heat was also examined in the same groups of subjects. Higher TBARS level in blood plasma was found 5 and 30 min after visit in sauna as compared to control value, but only in novice group. In winter swimmers, the concentration of TBARS in blood plasma was only slightly increased (statistically insignificant). Those results clearly prove that heat stress is a reason of oxidative stress that is reflected by increased lipid peroxidation. However, oxidative stress after exposure to high temperature is observed mainly in novices and hardly in regular winter swimmers, that implies again the existence of adaptive response in people regularly exposed to thermal stress. Increased TBARS levels after heat stress were also observed ¨ zturk ¨ and Gum ¨ usl ¨ u, ¨ 2004). Masuda in animals like rats and cows (O et al. (2004) found lower level of 8-epi-prostaglandin F2a, which is a chemically stable product of lipid peroxidation, in blood of patients with coronary risk factor after the sauna therapy. The authors suggest that repeated sauna sessions may protect against oxidative stress, which leads to prevention of atherosclerosis. The adaptation of organism to heat stress in form of sauna bathing may be of great importance for human health, especially considering the fact that elevated temperatures during summer months are associated with excess in morbidity and mortality (McGeehin and Mirabelli, 2001).
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