Accepted Article
Acute effects of different degrees of ultra-endurance exercise on systemic inflammatory responses 1
Short title: Strenuous exercise and inflammation
Mario Comassi†, Edoardo Vitolo†, Lorenza Pratali‡, Serena Del Turco‡, Cinzia Dellanoce§,
†
Chiara Rossi†, Eleonora Santini†, Anna Solini†
Department of Clinical and Experimental Medicine University of Pisa; ‡Institute of Clinical Physiology, CNR, Pisa; §Institute of Clinical Physiology, CNR, Milan, Italy
Corresponding Author:
Anna Solini, MD, PhD Department of Clinical and Experimental Medicine University of Pisa Via Roma, 67 – I-56100 Pisa, Italy
Tel: +39 050993482
Fax: +39 050553235
E-mail: [email protected]
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/imj.12625 This article is protected by copyright. All rights reserved.
1
Accepted Article
Word count: 2470 Abstract
Background - Intense physical stress might promote inflammatory responses, while a regular physical exercise has positive influence. Little is known on the acute metabolic and inflammatory responses to different levels of strenuous exercise in trained athletes. Aim - To compare the short-term effect of two different ultra-endurance competitions on the
inflammatory profile in male triathletes. Methods - We studied 14 Ironman (IR) and 13 Half Ironman (HIR) before and after their own specific race. We assessed body composition and measured blood cells, lipids, iron metabolism and plasma levels of some acute-phase cytokines and inflammatory markers. Results - After the race, IR showed reduced total body water and free fat mass, not related with the duration of exercise, and increased white cells and platelets; HDL levels also increased. IR, but not HIR, showed reduced iron levels, increased ferritin and transferrin, reduced % saturated transferrin. HIR showed higher basal IL-6, TNF-α, IL-10, IL-1β than IR; however, the post-performance rise was greater in IR. Irisin increased only in HIR and osteocalcin decreased in IR. In the whole study group, ΔWBC was directly related with ΔMCP-1, and Δ ferritin was inversely related with Δ osteocalcin.
Conclusions - A single ultra-endurance competition induces an inflammatory response depending
on the duration of physical effort, with increased acute-phase cytokines, and an altered iron metabolism. Irisin, whose biological meaning is still uncertain, seems to be associated with acute variations of some metabolic parameters.
Keywords: physical exercise, ironman, iron metabolism, osteocalcin, irisin
This article is protected by copyright. All rights reserved.
2
Accepted Article
Introduction
A sedentary lifestyle is considered an independent risk factor for the development of atherosclerosis and coronary heart disease (CHD), and the most important scientific societies unequivocally advise an increase in physical activity in the prevention and treatment of CHD; however, mechanisms that underlie the effectiveness of exercise in preventing or treating CHD have not been completely established.1 In the attempt to better clarify this complex issue, the link between exercise and inflammation has recently received special attention.2 Acute bouts of exercise result in a transient
increase in acute-phase reactants and cytokines, related to the amount of exercise and muscle injury;3 on the other hand, regular activity has been repeatedly associated with a chronic antiinflammatory effect having a potential positive influence on the vasculature.4 Beside weight
reduction, likely the main determinant of the reduced cardiovascular risk exerted by physical activity, other beneficial effects of physical exercise include the modulation of the activation of specific cytokine cascades, the signaling pathways associated with insulin resistance and the activity and reactivity of the vascular endothelium;5 however, mechanisms underlying such effects are not fully clarified. A competition of Ironman (IR) triathletes is an ultra-endurance event in which athletes perform to
3.8km swim, 180km cycling and 42.197km running without interruption; competition on the middle distances is defined as the Half Ironman (HIR) race. This extreme sport activity requires a meticulous and demanding training, and exposes athletes to a strenuous physical stress able to radically influence their physiology, including pathologic structural remodeling of the heart and large arteries, and to cause significant changes in biochemical parameters.6 Chronic endurance training leads to improvements in glucose metabolism and lipoprotein profile; regarding changes that acutely occur immediately after strenuous exercise, a raise in markers of acute-phase inflammation has been reported,7 while little is known on the inflammatory molecules more strictly related to the development of atherosclerotic process and to an increased cardiovascular risk, like This article is protected by copyright. All rights reserved.
3
Accepted Article
monocyte chemoattractant protein-1 (MCP-1) and high-sensitivity C reactive protein (hsCRP),8 or to the metabolic adaptation to physical exercise, like irisin, whose expression, enhanced during exercise, is able to stimulate browning of white fat tissue and uncoupling protein-1 (UCP-1) expression, with an improvement in glucose homeostasis.9 In these triathletes, information on the effects of acute bout of exercise on iron metabolism are also lacking, making the relationship between iron trafficking and pro-inflammatory activation of macrophages even more complex.10 Iron homeostasis may also regulate bone metabolism;11, 12 in this view, bone metabolism has been quite largely studied after acute exercise,13, 14 but few studies have evaluated the performance of bone formation markers in elite athletes, characterized by a higher bone turnover,15 linking them to iron metabolism. To contribute in clarifying this complex issue, we planned a study aimed at evaluating how a IR or a HIR race might affect the inflammatory profile in amateur athletes, paying special attention to cytokines involved in the modulation of acute
phase responses and bone remodeling.
Subject and methods
Participants - Study subjects were recruited on a volunteer basis among male participants of Elbaman®, an Ironman and Half Ironman triathlon competition which takes place in the Elba island, Italy, in the early Autumn. The protocol was approved by the Ethical Committee of Valle d’Aosta region (n. 65943; July 13, 2013) and all the volunteers signed an informed consent according to the Declaration of Helsinki. Exclusion criteria were the use of any medication, antioxidant or related supplements and febrile illness in the seven days preceding the study. The triathletes (all males, well-trained, non-smokers, free of acute and chronic illnesses) did not exercise for the 24 h preceding the race.
This article is protected by copyright. All rights reserved.
4
Accepted Article
Main outcome measurements - The day before the race, a brief medical history was collected in all
athletes; weight and height, blood pressure values, heart rate and saturation (by pulse-oximeter Tuff-Sat, Datex-Ohmeda, GE Healthcare Europe, Milan, Italy) were registered, and body composition was assessed by bioelectrical impedance analysis using a TANITA TBF-300A Body Composition Analyzer, on the basis of standardized mathematical formulae. The following parameters were measured: body mass index (BMI), fat mass (FM), fat free mass (FFM), total body water (TBW). A venous blood sample was drawn from an antecubital vein, collected into vacutainers and centrifuged at 1500rpm for 10 min at 4°C; a mobile testing laboratory was used at the race to ensure appropriate collection, separation, and storage of samples. The day of the race, the ambient temperature was 23.2°C. The mean duration of exercise was 12:48:21±1:14:18 hours for IR and 6:14:43±0:37:25 hours for HIR. Athletes were allowed to eat and drink ad libitum during the race, and nutrition was provided during the cycling and running courses by the organizers. At the end, they were placed in thermal blankets, and all tests were repeated within fifteen minutes; after that, athletes were fed. Biochemical parameters and a complete evaluation of iron metabolism were measured by standard methods; hsCRP was determined by nephelometric analysis. The acute phase cytokines TNF-α (an adipokine involved in systemic inflammation), IL-1β (involved in the proliferation, differentiation
and apoptosis of cells, and associated with septic shock, and wound healing), IL-6 (able to stimulate
acute phase protein synthesis, as well as the production of neutrophils in the bone marrow), and IL10 (showing prevailing anti-inflammatory effects), the chemotactic factor MCP-1, irisin and osteocalcin (a marker of bone mineral density) were assessed by ELISA according to the manufacturer's instructions (Quantikines kits, R&D Systems, Wiesbaden, Germany; intra-assay coefficients of variation were 3.1, 5.4, 7.4, 3.6, 4.9, 6.0 and 3.1 %, respectively). 40 athletes participated in the race; fourteen athletes competing in the IR distance and thirteen in the HIR distance completed the race, and were therefore selected for data collection.
This article is protected by copyright. All rights reserved.
5
Accepted Article
Statistical analysis - Data are expressed as mean±SD. Group comparisons were performed using the
nonparametric Mann-Whitney U-test or the unpaired t-test, for variables with non-normal or normal distribution, respectively, and χ2 for categorical variables. Variations of the different parameters
after the race respect to baseline were evaluated by analysis of variance (ANOVA) for repeated observations, correcting with post-hoc Bonferroni test. Relationships between variables were assessed using Spearman’s correlation analysis and multiple linear regression analysis. The statistical significance was determined on a probability level of
Acute effects of different degrees of ultra-endurance exercise on systemic inflammatory responses 1
Short title: Strenuous exercise and inflammation
Mario Comassi†, Edoardo Vitolo†, Lorenza Pratali‡, Serena Del Turco‡, Cinzia Dellanoce§,
†
Chiara Rossi†, Eleonora Santini†, Anna Solini†
Department of Clinical and Experimental Medicine University of Pisa; ‡Institute of Clinical Physiology, CNR, Pisa; §Institute of Clinical Physiology, CNR, Milan, Italy
Corresponding Author:
Anna Solini, MD, PhD Department of Clinical and Experimental Medicine University of Pisa Via Roma, 67 – I-56100 Pisa, Italy
Tel: +39 050993482
Fax: +39 050553235
E-mail: [email protected]
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/imj.12625 This article is protected by copyright. All rights reserved.
1
Accepted Article
Word count: 2470 Abstract
Background - Intense physical stress might promote inflammatory responses, while a regular physical exercise has positive influence. Little is known on the acute metabolic and inflammatory responses to different levels of strenuous exercise in trained athletes. Aim - To compare the short-term effect of two different ultra-endurance competitions on the
inflammatory profile in male triathletes. Methods - We studied 14 Ironman (IR) and 13 Half Ironman (HIR) before and after their own specific race. We assessed body composition and measured blood cells, lipids, iron metabolism and plasma levels of some acute-phase cytokines and inflammatory markers. Results - After the race, IR showed reduced total body water and free fat mass, not related with the duration of exercise, and increased white cells and platelets; HDL levels also increased. IR, but not HIR, showed reduced iron levels, increased ferritin and transferrin, reduced % saturated transferrin. HIR showed higher basal IL-6, TNF-α, IL-10, IL-1β than IR; however, the post-performance rise was greater in IR. Irisin increased only in HIR and osteocalcin decreased in IR. In the whole study group, ΔWBC was directly related with ΔMCP-1, and Δ ferritin was inversely related with Δ osteocalcin.
Conclusions - A single ultra-endurance competition induces an inflammatory response depending
on the duration of physical effort, with increased acute-phase cytokines, and an altered iron metabolism. Irisin, whose biological meaning is still uncertain, seems to be associated with acute variations of some metabolic parameters.
Keywords: physical exercise, ironman, iron metabolism, osteocalcin, irisin
This article is protected by copyright. All rights reserved.
2
Accepted Article
Introduction
A sedentary lifestyle is considered an independent risk factor for the development of atherosclerosis and coronary heart disease (CHD), and the most important scientific societies unequivocally advise an increase in physical activity in the prevention and treatment of CHD; however, mechanisms that underlie the effectiveness of exercise in preventing or treating CHD have not been completely established.1 In the attempt to better clarify this complex issue, the link between exercise and inflammation has recently received special attention.2 Acute bouts of exercise result in a transient
increase in acute-phase reactants and cytokines, related to the amount of exercise and muscle injury;3 on the other hand, regular activity has been repeatedly associated with a chronic antiinflammatory effect having a potential positive influence on the vasculature.4 Beside weight
reduction, likely the main determinant of the reduced cardiovascular risk exerted by physical activity, other beneficial effects of physical exercise include the modulation of the activation of specific cytokine cascades, the signaling pathways associated with insulin resistance and the activity and reactivity of the vascular endothelium;5 however, mechanisms underlying such effects are not fully clarified. A competition of Ironman (IR) triathletes is an ultra-endurance event in which athletes perform to
3.8km swim, 180km cycling and 42.197km running without interruption; competition on the middle distances is defined as the Half Ironman (HIR) race. This extreme sport activity requires a meticulous and demanding training, and exposes athletes to a strenuous physical stress able to radically influence their physiology, including pathologic structural remodeling of the heart and large arteries, and to cause significant changes in biochemical parameters.6 Chronic endurance training leads to improvements in glucose metabolism and lipoprotein profile; regarding changes that acutely occur immediately after strenuous exercise, a raise in markers of acute-phase inflammation has been reported,7 while little is known on the inflammatory molecules more strictly related to the development of atherosclerotic process and to an increased cardiovascular risk, like This article is protected by copyright. All rights reserved.
3
Accepted Article
monocyte chemoattractant protein-1 (MCP-1) and high-sensitivity C reactive protein (hsCRP),8 or to the metabolic adaptation to physical exercise, like irisin, whose expression, enhanced during exercise, is able to stimulate browning of white fat tissue and uncoupling protein-1 (UCP-1) expression, with an improvement in glucose homeostasis.9 In these triathletes, information on the effects of acute bout of exercise on iron metabolism are also lacking, making the relationship between iron trafficking and pro-inflammatory activation of macrophages even more complex.10 Iron homeostasis may also regulate bone metabolism;11, 12 in this view, bone metabolism has been quite largely studied after acute exercise,13, 14 but few studies have evaluated the performance of bone formation markers in elite athletes, characterized by a higher bone turnover,15 linking them to iron metabolism. To contribute in clarifying this complex issue, we planned a study aimed at evaluating how a IR or a HIR race might affect the inflammatory profile in amateur athletes, paying special attention to cytokines involved in the modulation of acute
phase responses and bone remodeling.
Subject and methods
Participants - Study subjects were recruited on a volunteer basis among male participants of Elbaman®, an Ironman and Half Ironman triathlon competition which takes place in the Elba island, Italy, in the early Autumn. The protocol was approved by the Ethical Committee of Valle d’Aosta region (n. 65943; July 13, 2013) and all the volunteers signed an informed consent according to the Declaration of Helsinki. Exclusion criteria were the use of any medication, antioxidant or related supplements and febrile illness in the seven days preceding the study. The triathletes (all males, well-trained, non-smokers, free of acute and chronic illnesses) did not exercise for the 24 h preceding the race.
This article is protected by copyright. All rights reserved.
4
Accepted Article
Main outcome measurements - The day before the race, a brief medical history was collected in all
athletes; weight and height, blood pressure values, heart rate and saturation (by pulse-oximeter Tuff-Sat, Datex-Ohmeda, GE Healthcare Europe, Milan, Italy) were registered, and body composition was assessed by bioelectrical impedance analysis using a TANITA TBF-300A Body Composition Analyzer, on the basis of standardized mathematical formulae. The following parameters were measured: body mass index (BMI), fat mass (FM), fat free mass (FFM), total body water (TBW). A venous blood sample was drawn from an antecubital vein, collected into vacutainers and centrifuged at 1500rpm for 10 min at 4°C; a mobile testing laboratory was used at the race to ensure appropriate collection, separation, and storage of samples. The day of the race, the ambient temperature was 23.2°C. The mean duration of exercise was 12:48:21±1:14:18 hours for IR and 6:14:43±0:37:25 hours for HIR. Athletes were allowed to eat and drink ad libitum during the race, and nutrition was provided during the cycling and running courses by the organizers. At the end, they were placed in thermal blankets, and all tests were repeated within fifteen minutes; after that, athletes were fed. Biochemical parameters and a complete evaluation of iron metabolism were measured by standard methods; hsCRP was determined by nephelometric analysis. The acute phase cytokines TNF-α (an adipokine involved in systemic inflammation), IL-1β (involved in the proliferation, differentiation
and apoptosis of cells, and associated with septic shock, and wound healing), IL-6 (able to stimulate
acute phase protein synthesis, as well as the production of neutrophils in the bone marrow), and IL10 (showing prevailing anti-inflammatory effects), the chemotactic factor MCP-1, irisin and osteocalcin (a marker of bone mineral density) were assessed by ELISA according to the manufacturer's instructions (Quantikines kits, R&D Systems, Wiesbaden, Germany; intra-assay coefficients of variation were 3.1, 5.4, 7.4, 3.6, 4.9, 6.0 and 3.1 %, respectively). 40 athletes participated in the race; fourteen athletes competing in the IR distance and thirteen in the HIR distance completed the race, and were therefore selected for data collection.
This article is protected by copyright. All rights reserved.
5
Accepted Article
Statistical analysis - Data are expressed as mean±SD. Group comparisons were performed using the
nonparametric Mann-Whitney U-test or the unpaired t-test, for variables with non-normal or normal distribution, respectively, and χ2 for categorical variables. Variations of the different parameters
after the race respect to baseline were evaluated by analysis of variance (ANOVA) for repeated observations, correcting with post-hoc Bonferroni test. Relationships between variables were assessed using Spearman’s correlation analysis and multiple linear regression analysis. The statistical significance was determined on a probability level of
