Eur J Appl Physiol (2016) 116:363–371 DOI 10.1007/s00421-015-3295-5
ORIGINAL ARTICLE
Acute l‑arginine supplementation has no effect on cardiovascular or thermoregulatory responses to rest, exercise, and recovery in the heat Christopher J. Tyler1 · Thomas R. M. Coffey1 · Gary J. Hodges2
Received: 22 June 2015 / Accepted: 5 November 2015 / Published online: 13 November 2015 © Springer-Verlag Berlin Heidelberg 2015
Abstract Purpose To investigate the effect of acute l-arginine (L-ARG) supplementation on cardiovascular and thermoregulatory responses to rest, exercise, and recovery in the heat. Methods Eight healthy men (age 27 ± 6 years; stature 176 ± 6 cm; body mass 76 ± 4 kg; maximal power output 237 ± 39 W) participated in a double-blind, crossover study, attending the laboratory for two experimental trials. On each occasion, participants consumed 500 ml of a blackcurrant-flavoured cordial beverage 30 min before completing a 90 min experiment in the heat (35 °C and 50 % rh). The experiment consisted of 30 min of seated rest, followed by 30 min submaximal cycling (60 % maximal power output) and 30 min passive seated recovery. On one visit the drink contained 10 g of dissolved L-ARG while on the other visit it did not. Results L-ARG supplementation increased plasma L-ARG concentrations (peak +223 ± 80 % after 60 min of the 90 min experiment); however, supplementation had no effect on rectal temperature, mean skin temperature, heart rate, arterial pressure, forearm skin vascular conductance, oxygen consumption or sweat loss at rest, during exercise, or during recovery in the heat (p > 0.05).
Communicated by Narihiko Kondo. * Christopher J. Tyler [email protected] 1
2
Department of Life Sciences, University of Roehampton, London SW15 4JD, England, UK Department of Kinesiology, Brock University, St. Catharines, Ontario, Canada
Conclusion Acute ingestion of 10 g L-ARG supplementation failed to elicit any changes in the cardiovascular or thermoregulatory responses to active or passive heat exposure in young, healthy males. Keywords Nitric oxide · Skin blood flow · Thermoregulation · Vasodilation · Heat loss Abbreviations ANOVA Analysis of variance CVC Cutaneous vascular conductance CVCmax Maximal cutaneous vascular conductance DAP Diastolic arterial pressure eNOS Endothelial nitric oxide synthase HR Heart rate l-ARG l-arginine MAP Mean arterial pressure nNOS Neuronal nitric oxide synthase NO Nitric oxide NO2 Nitrite NOS Nitric oxide synthase PLA Placebo trial rh Relative humidity SAP Systolic arterial pressure Trectal Rectal temperature Tskin Mean-weighted skin temperature VO2 Oxygen consumption Wmax Maximal power output
Introduction Increasing nitric oxide (NO) bioavailability can improve cardiovascular health (Adams et al. 1997; Clarkson et al. 1996; Rector et al. 1996) and enhance exercise performance
13
364
(Bailey et al. 2010a, b; Vanhatalo et al. 2011; Wylie et al. 2013). Much of the recent literature has focussed on increasing the bioavailability of NO using dietary nitrate supplementation and this has been shown to reduce the oxygen cost of submaximal exercise (Lansley et al. 2011; Vanhatalo et al. 2010; Kuennen et al. 2015), lower arterial pressure (Vanhatalo et al. 2010; Keen et al. 2015; Levitt et al. 2015), increase muscle blood flow and increase cutaneous vascular conductance (CVC) (Ferguson et al. 2013; Keen et al. 2015; Levitt et al. 2015). Dietary nitrate supplementation is one of the two pathways in which NO bioavailability can be elevated, the other is the nitric oxide synthase (NOS) -dependent l-arginine (L-ARG) pathway (Palmer et al. 1987). Beneficial physiological effects of L-ARG supplementation have regularly been observed in some clinical populations (Adams et al. 1997; Clarkson et al. 1996; Rector et al. 1996); however, the effect of L-ARG supplementation in healthy, non-diseased participants is equivocal with beneficial physiological and performance effects reported in some (Bailey et al. 2010b; BodeBoger et al. 1998; Koppo et al. 2009; Schaefer et al. 2002), but not all (Bescos et al. 2009; Bode-Boger et al. 1998; Koppo et al. 2009; Schaefer et al. 2002), studies. Cutaneous blood flow is regulated by two branches of the sympathetic nervous system- the noradrenergic vasoconstrictor nerves and the cholinergic active vasodilator nerves (Kellogg et al. 1995). While resting in temperate ambient conditions and during dynamic exercise, the cutaneous vasculature is predominantly controlled by the noradrenergic vasoconstrictor system and endothelial NOS (eNOS) (Kellogg et al. 1999; McNamara et al. 2014); however, increases in body temperature lead to sympathetic cholinergic mediation of skin blood flow regulated by neuronal NOS (nNOS) (Kellogg et al. 1995, 1999). The cholinergic active vasodilator system accounts for 80–95 % of the increased skin blood flow observed during passive heat stress (Johnson and Kellogg 2010) and in situations where the magnitude of skin blood flow increases is naturally reduced (e.g., ageing), the skin blood flow response to passive heat stress has been shown to be augmented by intradermal L-ARG administration (Holowatz et al. 2006). Holowatz et al. (2006) suggested that L-ARG supplementation may enhance the vasodilatory response in older populations because of a reduced endogenous L-ARG concentrations, and therefore lower NO bioavailability, while younger individuals may have sufficient L-ARG concentrations to meet the vasodilatory stimulus provided by passive heating and so do not benefit from supplementation (Holowatz et al. 2006). Passive and active heating protocols both place the body under thermal stress and elevate cutaneous blood flow; however, the mechanisms which control this increase in peripheral blood flow differ. Previous investigations have
13
Eur J Appl Physiol (2016) 116:363–371
used passive heating protocols (Holowatz et al. 2006; Levitt et al. 2015) during which the increase in skin blood flow appears to be dependent on nNOS (Kellogg et al. 2009), whereas skin blood flow increases during exercise appear dependent on eNOS (McNamara et al. 2014). Recent data have shown that dietary nitrate supplementation increases rectal temperature without altering skin temperature during marching in hot conditions (Kuennen et al. 2015). No skin blood flow data were recorded and so the authors used the lack of effect on skin temperature to suggest that there was a lack of effect on skin blood flow. Unlike the nitrate-NO pathway (Govoni et al. 2008), the L-ARG-NO pathway is NOS-dependent (Moncada and Higgs 1993) and so when a greater strain is placed upon this system, such as during exercise and possibly during recovery from exercise in the heat, it seems prudent to suggest that L-ARG may offer a hyperaemia-induced thermoregulatory benefit to healthy individuals, but this is yet to be investigated. Recently, it has been reported that nitrate supplementation increases rectal temperature by ~11 % despite a ~6 % reduction in the oxygen cost of submaximal treadmill exercise in a hot environment (Kuennen et al. 2015) but effects of oral L-ARG supplementation on whole-body thermoregulatory and cardiovascular responses to active and passive hyperthermia are also unknown. An intervention which enhances heat dissipation and reduces thermal strain would be of interest to a range of researchers, athletes, and coaches and elevating NO bioavailability by systemic L-ARG supplementation may be such an intervention. The aim of this study was to investigate the physiological responses to an acute oral dose of L-ARG during rest, exercise and recovery in the heat, in recreationally active, healthy males. We hypothesised that acute L-ARG supplementation would offer no hyperaemiainduced thermoregulatory benefit to healthy individuals during passive heat exposure but that it might augment heat loss responses during active heat stress, when the cutaneous vasculature is predominantly controlled by eNOS rather than nNOS.
Materials and methods Participants Nine healthy, recreationally active, non-heat acclimated, non-smoking, Caucasian males volunteered for the study; however, one of the participants experienced gastrointestinal discomfort following ingestion of the L-ARG and withdrew from the study. The mean (±standard deviation) age, stature, body mass and maximum power output in the heat (Wmax) of the 8 participants were 27 ± 6 years, 176 ± 6 cm, 76 ± 4 kg, and 237 ± 39 W, respectively. It
Eur J Appl Physiol (2016) 116:363–371
was calculated that a sample size of eight participants would provide sufficient statistical power (0.8; β = 0.20) to detect a difference in skin blood flow using an estimated effect size of d = 0.95 [the estimated effect size of 10 g intravenous L-ARG on skin blood flow was d = 1.40 (Giugliano et al. 1997); however, the bioavailability from oral dosages is ~70 % that of infusion (Bode-Boger et al. 1998) so the estimated effect size was reduced proportionately] and an alpha level of 0.05. None of the participants were users of dietary supplements or taking any medication. The participants were blinded to the main aim of the investigation but were informed of any associated risks and discomforts before giving their oral and written informed consent to participate. Once consent was given the participants completed a health screen questionnaire (American College of Sports Medicine Position Stand and American Heart Association 1998) and this screening procedure was repeated prior to each laboratory visit to assess the health status of the individual. Following the completion of the trials, each participant was provided with a post-participation information sheet, where full details of the study were provided and participants were asked for their consent to allow the use of their data in the final analysis. All 8 participants provided their consent at this stage. The study was approved by the University of Roehampton’s Ethical Advisory Committee. Pre‑trial (visit 1) Prior to the main trials, participants completed an incremental, cycle ergometer (Monark 874E, Monark, Sweden) test to determine Wmax (Kuipers et al. 1985) in a walk-in environmental chamber (Design Environmental, Wales, UK) set to 35 °C and 50 % relative humidity (rh). To determine Wmax, participants initially cycled for 5 min at 100 W after which the workload was increased by 50 W every 2.5 min until a heart rate of 160 b min−1 was reached. Upon attaining a heart rate of 160 b min−1 the work was increased by 21 W every 2.5 min until volitional exhaustion. The maximum workload was calculated using the following equation: Wmax = Wcom + ((t/150) × �W ) (Kuipers et al. 1985) where Wcom is the last workload completed, t is the time in seconds that the final, uncompleted, stage was sustained for and ΔW is the final load increment (21 W). Following the Wmax test, participants undertook a partial familiarisation where they sat for 15 min and cycled for 10 min to be familiarised with the ambient conditions and the power output required during the experimental trials (60 % Wmax). Participants abstained from alcohol, caffeine, strenuous exercise, and completed a food record for the 24 h period prior to the initial pre-test. They adopted the same diet and abstained from strenuous exercise, caffeine, and alcohol for 24 h prior to each subsequent trial.
365
Trials (visits 2 and 3) Following the preliminary test, participants visited the laboratory twice at the same time of the day (±1 h) separated by 7–9 days. Participants arrived at the laboratory ~45 min before the commencement of the trial and ~2 h postprandial. On arrival, nude body mass was recorded (Seca, Birmingham, UK), a rectal probe (Rectal 401, Varioham Eurosensor ltd, UK) was self-inserted ~10 cm past the anal sphincter for measurement of rectal temperature (Trectal), and a heart rate (HR) monitor (Polar, UK) was attached. Four skin thermistors (Varioham Eurosensor ltd, UK) were placed on the sternal notch, forearm, thigh, and calf, and connected to a digital reader (Thermistor Thermometer 5831, DigiTec Corp, USA) for the subsequent calculation of mean-weighted skin temperature (Tskin) (Ramanathan 1964). All thermistors were attached via a transparent dressing (Tagaderm, 3 M Health Care, USA) and waterproof tape (Transpore, 3 M Health Care, USA). A blood pressure cuff (Digital Blood Pressure Monitor UA-767, A&D Instruments LTD, Japan) was placed on the upper, right arm and a laser-Doppler local heater probe (PeriFlux 5000, Perimed AB, Stockholm, Sweden) was attached to the left forearm and maintained at 35 °C to clamp local skin temperature to environmental conditions. CVC was calculated as laser-Doppler flux divided by mean arterial pressure (MAP) and was standardised to %CVCmax. CVCmax was calculated as the mean of a 5 min plateau in laserDoppler flux observed during a bout of local skin heating to 43 °C (~35 min of heating). Upon arrival at the laboratory, participants consumed one of two drinks. Both drinks were made up of 200 ml blackcurrant cordial diluted in 300 ml of water- one contained 10 g of dissolved commercially available L-ARG powder (NOW Foods, USA) whereas the other did not (PLA). The drinks were prepared by an impartial technician and administered in a double-blind, crossover manner. A single, 10 g oral dose of L-ARG was selected based upon previous data suggesting that it is the largest tolerable dose and one that would definitely increase plasma L-ARG concentrations (Tang et al. 2011). The data were not unblinded until the completion of data analysis. Following the ingestion of the L-ARG or PLA beverage, participants rested in a seated position for 30 min in ambient, laboratory conditions before entering the environmental chamber (35 °C, 50 % rh) for a total duration of 90 min. During the 90 min, participants initially rested in a seated position for 30 min, then cycled in a seated position at 60 % Wmax (143 ± 22 W) for 30 min and finally undertook 30 min of passive, seated, recovery. The time-periods were chosen to maximise the L-ARG concentrations during the trial because previous data have shown that plasma L-ARG concentrations peak ~60 min after ingestion (i.e., after the
13
30 min rest in the heat) and remain elevated for ~120 min (i.e., the end of test) (Bode-Boger et al. 1998). Participants consumed 100 ml of water at the beginning of each 30 min section to reduce participant discomfort associated with perceptions of thirst. HR, Tskin and Trectal were recorded every 5 min during the 90 min trials, whereas oxygen consumption (VO2) was measured every 10 min using the Douglas bag method. CVC and arterial pressure were recorded at the start, end and at 5 min intervals of the rest and recovery periods but no measurements were taken during the exercise period due to issues with limb movement. MAP was calculated using the formula ((DAP × 2) + SAP)/3 (where DAP = diastolic arterial pressure and SAP = systolic arterial pressure). After the completion of each trial, participants towel-dried and recorded a dry post-exercise nude body mass (Seca 813, Seca Birmingham, UK; ±0.1 kg) from which sweat loss was calculated, taking into account the pre-trial body mass and the 100 ml of water consumed at the beginning of each 30 min section.
Eur J Appl Physiol (2016) 116:363–371
Plasma l-arginine concentration (µmol.L-1)
366
600 500 400 300 200 100 0
Pre
0
30 Time (min)
60
90
Fig. 1 Mean ± SD plasma l-arginine concentrations for l-arginine (dashed line, open squares) and placebo (solid line, filled circles) trials at rest and immediately before and after consecutive 30 min bouts of rest, submaximal exercise and recovery in the heat. 0–30 min = seated rest; 30–60 min = submaximal cycling exercise; 60–90 min = seated recovery. Main effect for trial, time and trial × time interaction (p
ORIGINAL ARTICLE
Acute l‑arginine supplementation has no effect on cardiovascular or thermoregulatory responses to rest, exercise, and recovery in the heat Christopher J. Tyler1 · Thomas R. M. Coffey1 · Gary J. Hodges2
Received: 22 June 2015 / Accepted: 5 November 2015 / Published online: 13 November 2015 © Springer-Verlag Berlin Heidelberg 2015
Abstract Purpose To investigate the effect of acute l-arginine (L-ARG) supplementation on cardiovascular and thermoregulatory responses to rest, exercise, and recovery in the heat. Methods Eight healthy men (age 27 ± 6 years; stature 176 ± 6 cm; body mass 76 ± 4 kg; maximal power output 237 ± 39 W) participated in a double-blind, crossover study, attending the laboratory for two experimental trials. On each occasion, participants consumed 500 ml of a blackcurrant-flavoured cordial beverage 30 min before completing a 90 min experiment in the heat (35 °C and 50 % rh). The experiment consisted of 30 min of seated rest, followed by 30 min submaximal cycling (60 % maximal power output) and 30 min passive seated recovery. On one visit the drink contained 10 g of dissolved L-ARG while on the other visit it did not. Results L-ARG supplementation increased plasma L-ARG concentrations (peak +223 ± 80 % after 60 min of the 90 min experiment); however, supplementation had no effect on rectal temperature, mean skin temperature, heart rate, arterial pressure, forearm skin vascular conductance, oxygen consumption or sweat loss at rest, during exercise, or during recovery in the heat (p > 0.05).
Communicated by Narihiko Kondo. * Christopher J. Tyler [email protected] 1
2
Department of Life Sciences, University of Roehampton, London SW15 4JD, England, UK Department of Kinesiology, Brock University, St. Catharines, Ontario, Canada
Conclusion Acute ingestion of 10 g L-ARG supplementation failed to elicit any changes in the cardiovascular or thermoregulatory responses to active or passive heat exposure in young, healthy males. Keywords Nitric oxide · Skin blood flow · Thermoregulation · Vasodilation · Heat loss Abbreviations ANOVA Analysis of variance CVC Cutaneous vascular conductance CVCmax Maximal cutaneous vascular conductance DAP Diastolic arterial pressure eNOS Endothelial nitric oxide synthase HR Heart rate l-ARG l-arginine MAP Mean arterial pressure nNOS Neuronal nitric oxide synthase NO Nitric oxide NO2 Nitrite NOS Nitric oxide synthase PLA Placebo trial rh Relative humidity SAP Systolic arterial pressure Trectal Rectal temperature Tskin Mean-weighted skin temperature VO2 Oxygen consumption Wmax Maximal power output
Introduction Increasing nitric oxide (NO) bioavailability can improve cardiovascular health (Adams et al. 1997; Clarkson et al. 1996; Rector et al. 1996) and enhance exercise performance
13
364
(Bailey et al. 2010a, b; Vanhatalo et al. 2011; Wylie et al. 2013). Much of the recent literature has focussed on increasing the bioavailability of NO using dietary nitrate supplementation and this has been shown to reduce the oxygen cost of submaximal exercise (Lansley et al. 2011; Vanhatalo et al. 2010; Kuennen et al. 2015), lower arterial pressure (Vanhatalo et al. 2010; Keen et al. 2015; Levitt et al. 2015), increase muscle blood flow and increase cutaneous vascular conductance (CVC) (Ferguson et al. 2013; Keen et al. 2015; Levitt et al. 2015). Dietary nitrate supplementation is one of the two pathways in which NO bioavailability can be elevated, the other is the nitric oxide synthase (NOS) -dependent l-arginine (L-ARG) pathway (Palmer et al. 1987). Beneficial physiological effects of L-ARG supplementation have regularly been observed in some clinical populations (Adams et al. 1997; Clarkson et al. 1996; Rector et al. 1996); however, the effect of L-ARG supplementation in healthy, non-diseased participants is equivocal with beneficial physiological and performance effects reported in some (Bailey et al. 2010b; BodeBoger et al. 1998; Koppo et al. 2009; Schaefer et al. 2002), but not all (Bescos et al. 2009; Bode-Boger et al. 1998; Koppo et al. 2009; Schaefer et al. 2002), studies. Cutaneous blood flow is regulated by two branches of the sympathetic nervous system- the noradrenergic vasoconstrictor nerves and the cholinergic active vasodilator nerves (Kellogg et al. 1995). While resting in temperate ambient conditions and during dynamic exercise, the cutaneous vasculature is predominantly controlled by the noradrenergic vasoconstrictor system and endothelial NOS (eNOS) (Kellogg et al. 1999; McNamara et al. 2014); however, increases in body temperature lead to sympathetic cholinergic mediation of skin blood flow regulated by neuronal NOS (nNOS) (Kellogg et al. 1995, 1999). The cholinergic active vasodilator system accounts for 80–95 % of the increased skin blood flow observed during passive heat stress (Johnson and Kellogg 2010) and in situations where the magnitude of skin blood flow increases is naturally reduced (e.g., ageing), the skin blood flow response to passive heat stress has been shown to be augmented by intradermal L-ARG administration (Holowatz et al. 2006). Holowatz et al. (2006) suggested that L-ARG supplementation may enhance the vasodilatory response in older populations because of a reduced endogenous L-ARG concentrations, and therefore lower NO bioavailability, while younger individuals may have sufficient L-ARG concentrations to meet the vasodilatory stimulus provided by passive heating and so do not benefit from supplementation (Holowatz et al. 2006). Passive and active heating protocols both place the body under thermal stress and elevate cutaneous blood flow; however, the mechanisms which control this increase in peripheral blood flow differ. Previous investigations have
13
Eur J Appl Physiol (2016) 116:363–371
used passive heating protocols (Holowatz et al. 2006; Levitt et al. 2015) during which the increase in skin blood flow appears to be dependent on nNOS (Kellogg et al. 2009), whereas skin blood flow increases during exercise appear dependent on eNOS (McNamara et al. 2014). Recent data have shown that dietary nitrate supplementation increases rectal temperature without altering skin temperature during marching in hot conditions (Kuennen et al. 2015). No skin blood flow data were recorded and so the authors used the lack of effect on skin temperature to suggest that there was a lack of effect on skin blood flow. Unlike the nitrate-NO pathway (Govoni et al. 2008), the L-ARG-NO pathway is NOS-dependent (Moncada and Higgs 1993) and so when a greater strain is placed upon this system, such as during exercise and possibly during recovery from exercise in the heat, it seems prudent to suggest that L-ARG may offer a hyperaemia-induced thermoregulatory benefit to healthy individuals, but this is yet to be investigated. Recently, it has been reported that nitrate supplementation increases rectal temperature by ~11 % despite a ~6 % reduction in the oxygen cost of submaximal treadmill exercise in a hot environment (Kuennen et al. 2015) but effects of oral L-ARG supplementation on whole-body thermoregulatory and cardiovascular responses to active and passive hyperthermia are also unknown. An intervention which enhances heat dissipation and reduces thermal strain would be of interest to a range of researchers, athletes, and coaches and elevating NO bioavailability by systemic L-ARG supplementation may be such an intervention. The aim of this study was to investigate the physiological responses to an acute oral dose of L-ARG during rest, exercise and recovery in the heat, in recreationally active, healthy males. We hypothesised that acute L-ARG supplementation would offer no hyperaemiainduced thermoregulatory benefit to healthy individuals during passive heat exposure but that it might augment heat loss responses during active heat stress, when the cutaneous vasculature is predominantly controlled by eNOS rather than nNOS.
Materials and methods Participants Nine healthy, recreationally active, non-heat acclimated, non-smoking, Caucasian males volunteered for the study; however, one of the participants experienced gastrointestinal discomfort following ingestion of the L-ARG and withdrew from the study. The mean (±standard deviation) age, stature, body mass and maximum power output in the heat (Wmax) of the 8 participants were 27 ± 6 years, 176 ± 6 cm, 76 ± 4 kg, and 237 ± 39 W, respectively. It
Eur J Appl Physiol (2016) 116:363–371
was calculated that a sample size of eight participants would provide sufficient statistical power (0.8; β = 0.20) to detect a difference in skin blood flow using an estimated effect size of d = 0.95 [the estimated effect size of 10 g intravenous L-ARG on skin blood flow was d = 1.40 (Giugliano et al. 1997); however, the bioavailability from oral dosages is ~70 % that of infusion (Bode-Boger et al. 1998) so the estimated effect size was reduced proportionately] and an alpha level of 0.05. None of the participants were users of dietary supplements or taking any medication. The participants were blinded to the main aim of the investigation but were informed of any associated risks and discomforts before giving their oral and written informed consent to participate. Once consent was given the participants completed a health screen questionnaire (American College of Sports Medicine Position Stand and American Heart Association 1998) and this screening procedure was repeated prior to each laboratory visit to assess the health status of the individual. Following the completion of the trials, each participant was provided with a post-participation information sheet, where full details of the study were provided and participants were asked for their consent to allow the use of their data in the final analysis. All 8 participants provided their consent at this stage. The study was approved by the University of Roehampton’s Ethical Advisory Committee. Pre‑trial (visit 1) Prior to the main trials, participants completed an incremental, cycle ergometer (Monark 874E, Monark, Sweden) test to determine Wmax (Kuipers et al. 1985) in a walk-in environmental chamber (Design Environmental, Wales, UK) set to 35 °C and 50 % relative humidity (rh). To determine Wmax, participants initially cycled for 5 min at 100 W after which the workload was increased by 50 W every 2.5 min until a heart rate of 160 b min−1 was reached. Upon attaining a heart rate of 160 b min−1 the work was increased by 21 W every 2.5 min until volitional exhaustion. The maximum workload was calculated using the following equation: Wmax = Wcom + ((t/150) × �W ) (Kuipers et al. 1985) where Wcom is the last workload completed, t is the time in seconds that the final, uncompleted, stage was sustained for and ΔW is the final load increment (21 W). Following the Wmax test, participants undertook a partial familiarisation where they sat for 15 min and cycled for 10 min to be familiarised with the ambient conditions and the power output required during the experimental trials (60 % Wmax). Participants abstained from alcohol, caffeine, strenuous exercise, and completed a food record for the 24 h period prior to the initial pre-test. They adopted the same diet and abstained from strenuous exercise, caffeine, and alcohol for 24 h prior to each subsequent trial.
365
Trials (visits 2 and 3) Following the preliminary test, participants visited the laboratory twice at the same time of the day (±1 h) separated by 7–9 days. Participants arrived at the laboratory ~45 min before the commencement of the trial and ~2 h postprandial. On arrival, nude body mass was recorded (Seca, Birmingham, UK), a rectal probe (Rectal 401, Varioham Eurosensor ltd, UK) was self-inserted ~10 cm past the anal sphincter for measurement of rectal temperature (Trectal), and a heart rate (HR) monitor (Polar, UK) was attached. Four skin thermistors (Varioham Eurosensor ltd, UK) were placed on the sternal notch, forearm, thigh, and calf, and connected to a digital reader (Thermistor Thermometer 5831, DigiTec Corp, USA) for the subsequent calculation of mean-weighted skin temperature (Tskin) (Ramanathan 1964). All thermistors were attached via a transparent dressing (Tagaderm, 3 M Health Care, USA) and waterproof tape (Transpore, 3 M Health Care, USA). A blood pressure cuff (Digital Blood Pressure Monitor UA-767, A&D Instruments LTD, Japan) was placed on the upper, right arm and a laser-Doppler local heater probe (PeriFlux 5000, Perimed AB, Stockholm, Sweden) was attached to the left forearm and maintained at 35 °C to clamp local skin temperature to environmental conditions. CVC was calculated as laser-Doppler flux divided by mean arterial pressure (MAP) and was standardised to %CVCmax. CVCmax was calculated as the mean of a 5 min plateau in laserDoppler flux observed during a bout of local skin heating to 43 °C (~35 min of heating). Upon arrival at the laboratory, participants consumed one of two drinks. Both drinks were made up of 200 ml blackcurrant cordial diluted in 300 ml of water- one contained 10 g of dissolved commercially available L-ARG powder (NOW Foods, USA) whereas the other did not (PLA). The drinks were prepared by an impartial technician and administered in a double-blind, crossover manner. A single, 10 g oral dose of L-ARG was selected based upon previous data suggesting that it is the largest tolerable dose and one that would definitely increase plasma L-ARG concentrations (Tang et al. 2011). The data were not unblinded until the completion of data analysis. Following the ingestion of the L-ARG or PLA beverage, participants rested in a seated position for 30 min in ambient, laboratory conditions before entering the environmental chamber (35 °C, 50 % rh) for a total duration of 90 min. During the 90 min, participants initially rested in a seated position for 30 min, then cycled in a seated position at 60 % Wmax (143 ± 22 W) for 30 min and finally undertook 30 min of passive, seated, recovery. The time-periods were chosen to maximise the L-ARG concentrations during the trial because previous data have shown that plasma L-ARG concentrations peak ~60 min after ingestion (i.e., after the
13
30 min rest in the heat) and remain elevated for ~120 min (i.e., the end of test) (Bode-Boger et al. 1998). Participants consumed 100 ml of water at the beginning of each 30 min section to reduce participant discomfort associated with perceptions of thirst. HR, Tskin and Trectal were recorded every 5 min during the 90 min trials, whereas oxygen consumption (VO2) was measured every 10 min using the Douglas bag method. CVC and arterial pressure were recorded at the start, end and at 5 min intervals of the rest and recovery periods but no measurements were taken during the exercise period due to issues with limb movement. MAP was calculated using the formula ((DAP × 2) + SAP)/3 (where DAP = diastolic arterial pressure and SAP = systolic arterial pressure). After the completion of each trial, participants towel-dried and recorded a dry post-exercise nude body mass (Seca 813, Seca Birmingham, UK; ±0.1 kg) from which sweat loss was calculated, taking into account the pre-trial body mass and the 100 ml of water consumed at the beginning of each 30 min section.
Eur J Appl Physiol (2016) 116:363–371
Plasma l-arginine concentration (µmol.L-1)
366
600 500 400 300 200 100 0
Pre
0
30 Time (min)
60
90
Fig. 1 Mean ± SD plasma l-arginine concentrations for l-arginine (dashed line, open squares) and placebo (solid line, filled circles) trials at rest and immediately before and after consecutive 30 min bouts of rest, submaximal exercise and recovery in the heat. 0–30 min = seated rest; 30–60 min = submaximal cycling exercise; 60–90 min = seated recovery. Main effect for trial, time and trial × time interaction (p
