Journal of Thermal Biology 88 (2020) 102492

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Journal of Thermal Biology

journal homepage: http://www.elsevier.com/locate/jtherbio

Influence of a high-temperature programme on serum, urinary and sweat

levels of selenium and zinc
J. Siquier-Coll a, *, I. Bartolom�e a, M. P�erez-Quintero a, D. Mun
~ oz b, M.C. Robles b,
a
M. Maynar-Marin ~o
a
Department of Physiology. School of Sport Sciences. University of Extremadura, Spain
b
Department of Physical Education and Sport, Sport Sciences Faculty, University of Extremadura, C�
aceres, Spain

A R T I C L E I N F O A B S T R A C T

Keywords: Introduction: The effect of hyperthermia on the antioxidant system in the human organism is well known.
Selenium Aim: The objective of this study was to observe the effects of heat on the concentration of Se and Zn, elements
Zinc related to antioxidant systems.
Heat acclimation
Methods: Twenty-nine subjects voluntarily participated in this study. They were divided into a control group (CG;
Serum
n ¼ 14) and an experimental group (EG; n ¼ 15). All of them underwent two incremental tests until exhaustion in
Sweat
Urine normothermia (22 � C, 20–40%RH) and hyperthermia (42 � C, 20–40%RH). EG experienced nine sessions of
repeated heat exposure at high temperatures (100 � C, 20%RH) for three weeks (HEHT). After the intervention,
the initial measurements were repeated. Urine and blood samples were collected before and after each test.
Additionally, sweat samples were collected after tests in hyperthermia.
Results: There were no significant changes in serum. An increase in the elimination of Zn and Se in EG was
observed in urine after HEHT (p < .05). The elimination of Zn by sweating decreased after HEHT in EG (p < .05).
Conclusions: Exposure to heat at high temperatures increases the urinary excretion of Se and Zn.

1. Introduction together with Zn, Se is considered an essential antioxidant element. The

It is known that physical exercise increases oxidative stress (Powers
et al., 2016). Physical activity and an increased body temperature
encourage a higher production of redox reactions (Gomes et al., 2011).
This phenomenon can be aggravated in hyperthermal situations due to
the physiological stress produced (McAnulty et al., 2005). According to
a study in animals, high temperatures increase this stress (Mills et al.,
1996), and it has been observed that hyperthermia produces oxidative
stress in humans as well (Quindry et al., 2013). However, these studies
are based on antioxidant markers, with scarce literature on selenium
(Se) and Zinc (Zn) in hot environments.
Se is an essential micronutrient for maintaining the functions of
selenoproteins and small selenium metabolites in the organism (Cheng
and Lei, 2016). Glutathione peroxidase (GPx), a powerful antioxidant
enzyme, stands out among the selenoproteins (Baltaci et al., 2016). This
enzyme catalyses the reduction of H2O2 produced by the action of su­
peroxide dismutase (SOD) to water (Lamprecht, 2014). Therefore,
recommended dietary allowance (RDA) has been established at 55
μg/day in adults (Cheng and Lei, 2016), and 200 μg/day in athletes
(Datta, 2019). However, the literature on the effect of exercise is limited,
and even more so in hyperthermic conditions.
Regarding Zinc, it is a multifunctional trace element (Maret, 2013),
that takes part in macronutrient metabolism and growth periods in
childhood and puberty (Rerksuppaphol and Rerksuppaphol, 2016). In
addition, it is linked to more than 300 enzymes, including lactate de­
hydrogenase and carbonic anhydrase, essential in metabolism during
exercise (Baranauskas et al., 2015). It stands out mainly for its antioxi­
dant function, forming part of Copper–Zinc superoxide dismutase
(Cu–Zn SOD) (Vincent et al., 2019). RDA is around 13.7 and 17.9
mg/day, and in athletes between 30 and 60 mg/day (Datta, 2019). Zn
losses through sweat from exercise and heat can lead to hypozincemia
(Wolinsky and Driskell, 2005). This phenomenon could contribute to an
increase in oxidative stress due to its deficiency and the exercise in the
heat.

* Corresponding author. Exercise Physiology Laboratory, School of Sport Sciences, University of Extremadura, University Avenue, 10003, C�
aceres, Extremadura,
Spain.
E-mail address: jsiquier@alumnos.unex.es (J. Siquier-Coll).

https://doi.org/10.1016/j.jtherbio.2019.102492
Received 13 October 2019; Received in revised form 28 November 2019; Accepted 23 December 2019
Available online 30 December 2019
0306-4565/© 2019 Elsevier Ltd. All rights reserved.
J. Siquier-Coll et al.
The increase in temperature produced by heat and exercise can affect
the concentrations of these trace elements. Therefore, the objective of
this research was to observe the acute effect of a maximum incremental
test until exhaustion in hyperthermic and normothermic conditions on
levels of Se and Zn in serum, sweat and urine before and after nine days
of exposure to high temperatures (HEHT).
2. Material and methods
This section and the sample were similar to the one used by Siquier-
Coll et al. (2019a).
2.1. Participants
Twenty-nine male university students voluntarily participated in this
study. Previously to the experimental period, all of them were informed
about the aim, characteristics and risks of the research. Before beginning
the experiments, all the participants provided their written consent and
accepted their voluntary participation. The subjects were divided into
an experimental group (EG) and a control group (CG). This work was
approved by the bioethics committee of the University of Extremadura
under the Helsinki Declaration ethic guidelines of 1975, updated at the
World Medical Assembly in Seoul 2008, for research involving human
subjects. The anthropometric and descriptive characteristics of the
participants are presented in Table 1.
2.2. Experimental protocol
The testing was carried out on two different days of measurements
separated by 48 h in order to ensure physical recovery. The order of the
tests was: day 1-normothermia trial until exhaustion (23 � 2 � C, 40–60%
RH); day2-hyperthermia trial until exhaustion (42 � 2 � C, 40–60%RH).
The participants were exposed to 15 min of heat (42 � 2 � C, 40–60%RH)
before starting the measurements on the second day. In order to control
the circadian rhythms, all the tests were performed from 9 a.m. to 2 p.m.
and at the same time for each participant.
The tests started with a blood extraction from the antecubital vein of
each participant and with the collection of a urine sample. Both samples
were obtained in fasting conditions. Then the participants had a similar
breakfast consisting of a 250 ml glucosaline drink which did not contain
any of the elements studied. One hour after the breakfast, every
participant performed an exercise test until exhaustion (described
below). The protocol of the tests was the same for both days of mea­
surements, but the first day the tests were performed in normothermic
conditions and the second day in a hyperthermic environment. Once
finished, another blood sample was drawn from each participant. The
first urination after the test was also obtained from each individual.
Once the first two tests were carried out, the sample was randomised,
dividing the participants into EG (n ¼ 15) and CG (n ¼ 14). Sweats
samples were collected at the end of the hyperthermia trials. In the
normothermia test there was not enough sweating to obtain a valid and
reliable sample for analysis. EG experienced nine sessions of heat
Table 1
Descriptive characteristics of the sample.
CONTROL (n ¼ 14) EXPERIMENTAL (n ¼ 15)
Age (years) 22.04 � 2.29 21.70 � 1.99
Height (cm) 172.91 � 3.76 176.65 � 7.17
Weight (kg) 70.67 � 5.69 74.47 � 11.28
BMI (kg/m2) 23.61 � 1.27 23.93 � 3.01
Fat mass (kg) 11.15 � 2.46 12.32 � 5.14
Fat mass (%) 15.66 � 2.36 16.05 � 4.56
Fat-free mass (kg) 60.06 � 4.33 62.49 � 7.43
Fat-free mass (%) 84.34 � 2.36 83.96 � 4.55
VO2 (ml/min/kg) 44.24 � 4.23 39.54 � 5.93
VO2 (L/min) 3.04 � 0.29 3.06 � 0.62
2
Journal of Thermal Biology 88 (2020) 102492
exposure at high temperatures in a sauna during three weeks. CG did not
experience any heat exposure or specific training plan. After that, all the
initial measurements (test in normothermic and hyperthermic condi­
tions) were again taken in both groups to check the possible changes
after exposure to heat. Fig. 1 shows the experimental design followed in
this study.
2.3. Health security protocol
Previously to the experimental period, all participants were exam­
ined by a physician in order to avoid any case of illness or contraindi­
cation to participating in the study. At this point the participants had to
comply with the inclusion criteria: be a healthy male, not have taken any
supplementation, medication or over-the-counter medication, drug or
alcohol in the previous four weeks, have a healthy lifestyle, not practice
more than 3 h of physical activity per week and not follow a specific
training plan.
Once the first fitness screening was completed, the cardiocirculatory
system of each participant was evaluated in resting conditions using an
electrocardiograph (Sanro BTL-08 SD ECG) and a tensiometer (visiomat;
comfort 20/40). Before the tests, the basal electrocardiograms were
analysed by a physician. Furthermore, heart activity was monitored in
real time in the tests by mean of an electrocardiograph [Mortara; (Ref
9293-029-60)] during the exercise and recuperation times. Core tem­
perature (Tc), measured in the buccal mucosa, and skin temperature
(Tsk), measured in the frontal region of the head in triplicate, were
monitored using an infrared thermometer [TAT 5000 “Exergen Tem­
poral Scanner” (Corp., USA)] at the beginning and end of the tests.
In order to avoid cases of breathing difficulties, two forced spirom­
etry tests were carried out before the exercise tests. A spirometer (Spi­
robank G) was used to measure respiratory capacity.
No diseases were reported during the whole study.
2.4. Familiarisation period
Before the start of the experimental period, one week of prior
familiarisation was completed by all participants. During this week,
each participant visited the laboratory and became acquainted with the
physicians, the laboratory gear and tools and performed two submaxi­
mal tests on the cycloergometer (Ergoline 900; Bitz, Germany). Both
tests started at 50 W, increasing intensity by 25 W every two minutes
until reaching 75% of the estimated FCmax. Familiarisation tests were
performed in both normothermic (23 � 2 � C, 40–60%RH) and hyper­
thermic (42 � 2 � C, 40–60%RH) conditions, separated by 48 h.
During the tests, Heart Rate (HR) was measured with an ECG
[Mortara; (Ref 9293-029-60)] and respiratory variables were measured
using a gas analyser “Geratherm Respiratory GMBH [Ergostik (Ref
40.400; Corp Bad Kissinguen)]”.
2.5. Body composition
The anthropometric measurements were taken in the morning, in
fasting conditions, and at the same time for each participant. Body
height was measured using a wall stadiometer (Seca 220). Body weight,
fat-free mass and fat mass were measured by electric bioimpedance,
using a body composition analyser BF-350 (Tanita Corp. Japan).
2.6. Incremental exercise test until voluntary exhaustion
Each participant performed two maximal exercise tests in laboratory
conditions. The subjects performed a 50 W warm-up for 5 min. The first
test was carried out at room temperature, and the second one in a sauna
(Harvia C105S Logix Combi Control; 3–15 W; Finland). Both tests were
performed on the same cycloergometer, starting at an initial power of
50 W (W). Every two minutes, the power increased by 25 W until
voluntary exhaustion. The tests ended when the subject was unable to
J. Siquier-Coll et al.
Fig. 1. Experimental design and phases.
Fig. 1 describes the experimental design and the phases of this study. CG ¼ Control Group; EG ¼ Experimental Group; HEHT¼ Heat Exposure at high temperature.
sustain the power of the stage during more than 15 s or if the subject
reached exhaustion. During the trial, HR [Mortara; (Ref 9293-029-60)]
as well as respiratory variables [Geratherm Respiratory GMBH, Ergostik
(Ref 40.400; Corp Bad Kissinguen)] were recorded in real time. Sweat
Rate was calculated with the equation proposed by Murray (1996) to
calculate sweat loss after exercise and divided by the duration of the test
to calculate the sweat loss in each trial.
2.7. Heat exposure
The sessions consisted of five series of ten minutes in a sauna (Harvia
C105S Logix Combi Control; 3–15 W; Finland) at 100 � C (20%RH) with a
recovery of five minutes between series at ambient temperature (22 � C).
In order to control the circadian rhythms, EG performed the session in
the morning (from 9 a.m. to 14 p.m.) and at the same time for each
participant.
2.8. Sample collection
2.8.1. Serum samples
Two extractions of 5 mL of venous blood were drawn from the
antecubital vein of each participant using plastic syringes fitted with a
stainless-steel needle. The first samples were extracted before the exer­
cise test and the second ones, just after it. Once extracted, the samples
were collected in a metal-free polypropylene tube (previously washed
with diluted nitric acid).
Later, the blood samples were centrifuged at 2500 rpm for 10 min at
room temperature to isolate the serum. The serum was aliquoted into an
Eppendorf tube (previously washed with diluted nitric acid) and
conserved at 80 � C until biochemical analysis.
Haematocrit was obtained by centrifuging the whole blood into a
glass capillary containing heparin in a Microcen microfuge (Alresa,
Spain). Both haematocrits were used to correct the changes in plasma
volume by means of the Van Beaumont (1972) equations.
3
Journal of Thermal Biology 88 (2020) 102492
2.8.2. Urine samples
Urine samples were obtained from each participant before and after
the test, just after both blood extractions. The urine samples were
collected in polyethylene tubes previously washed with diluted nitric
acid and frozen at 80 � C until analysis. Before the analysis, the samples
were thawed at room temperature and homogenised by shaking.
2.8.3. Sweat samples
The sweat was collected at the end of hyperthermia tests. Prior to the
trials, the participants’ backs were washed following the guidelines of
Ely et al. (2011) in order to avoid sample contamination. The back of
each participant was rinsed with a liberal amount of MQ distilled water.
After that, an antibacterial liquid soap without perfume and without
content of the minerals studied was used. Once the back had been soa­
ped, the area was rinsed with abundant quantities of 18 MQ distilled
water. Additionally, just after the trials in hyperthermia, the sweat
samples were collected and aliquoted into an Eppendorf tube (previ­
ously washed with diluted nitric acid) and conserved at 80 � C until
biochemical analysis.
2.9. Serum, sweat and urinary trace element determination
2.9.1. Sample preparation
Zn and Se analyses were performed by inductively coupled plasma
mass spectrometry (ICP-MS) following the protocol used by (Maynar
et al., 2018a). To prepare the analysis, the organic matrix was decom­
posed by heating it for 10 h at 90 � C after the addition of 0.8 mL of HNO3
and 0.4 mL of H2O2 to 2 mL of serum, erythrocyte or urine samples. The
samples were then dried at 200 � C on a hot plate. Sample reconstitution
was carried out by adding 0.5 mL of nitric acid, 10 μL of indium (In) (10
mg/L) as the internal standard, and ultrapure water to complete 10 mL.
2.9.2. Standard and reference material preparation
Reagent blanks, element standards, and certified reference materials
J. Siquier-Coll et al. Journal of Thermal Biology 88 (2020) 102492

(Seronorm, lot 0511545, Sero AS Billingstand, Norway) were prepared
identically and used for accuracy testing. Before the analysis, the com­
mercial control materials were diluted according to the manufacturer’s
recommendations.
2.9.3. Sample analysis
Digested solutions were assayed in an ICP-MS Nexion analyser model
300D (PerkinElmer, Inc., Shelton, CT, USA) equipped with a triple
quadrupole mass detector and a reaction cell/collision device that al­
lows operation in three modes: without reaction gas (STD); by kinetic
energy discrimination (KED) with helium as the collision gas; and in
reaction mode (DRC) with ammonia as the reaction gas. Both collision
and reaction gases such as plasmatic argon had a purity of 99.999% and
were supplied by Praxair (Madrid, Spain). Two mass flow controllers
regulated gas flows. The frequency of the generator was free-swinging
and worked at 40 Mhz. Three replicates were analysed per sample.
The sample quantifications were performed with indium (In) as the in­
ternal standard. The values of the standard materials of each element
(10 μg/L) used for quality controls were in agreement with intra and
inter-assay variation coefficients of less than 5%.
2.10. Statistical evaluation
Statistical analyses were carried out with SPSS 22.0 for Windows.
The results are expressed as the mean and standard deviation (x � sd).
The Kolmogorov–Smirnov test was applied to examine the distribution
of the variables, and Leven’s test was used to verify their homogeneity.
The difference between normothermia and hyperthermia, normo­
thermia before and after intervention, hyperthermia before and after
intervention and pre-post difference data were determined using the
Wilcoxon test for paired samples in both groups. The Mann-Whitney U
test was used to determine the differences between groups.
3. Results
Fig. 2 shows the values of haematocrit, sweat rate, skin temperature
(Tsk) and core temperature (Tc) before and after each trial. A significant
elevation in haematocrit can be observed after each test in both groups
(p < .01). EG experimented lower values pre-post-test in hyperthermia
in comparison to normothermia after HEHT (p < .05). In relation to
temperature, Tsk was significant higher after all trials in EG (p < .05),
but only in the hyperthermia tests in CG (p < .01). Previous to the first
trial, tsk was lower in EG than in CG (p < .01). Before HEHT, a higher
Tsk before and after the hyperthermia trial compared to the normo­
thermia test can be observed in EG (p < .01). The same thing happened
after HEHT in both groups (p < .01). Initial Tc was significantly lower in
EG than CG (p < .01). Both groups experimented a rise in Tc after the
hyperthermia trial previous to HEHT (p < .01) in comparison to Tc after
the normothermia test (p < .01). In the same way, after HEHT, Tc was
higher after the test in hyperthermia in both groups with respect to the
normothermia trial. Additionally, Tc before the trial was higher in hy­
perthermia than in normothermia (p < .01). Regarding the sweat rate,
there was an increase in sweating in the hyperthermia tests with respect
to the normothermia trials in EG (p < .01) and CG (p < .05).
The data related to the concentration of Se and Zn in serum with and
without correction (C) are presented in Table 2. Zn underwent changes
in normothermia before and after HETH (p < .05) in CG, and in hy­
perthermia after HEHT in EG (p < .05). The post-test value in hyper­
thermia after HEHT was significant higher in EG than in CG (p < .05). No
difference was found in Zn–C. With respect to Se, the values were lower
in CG than in EG at the beginning (p < .01), after normothermia (p <
.01), before and after hyperthermia trials (p < .05) previous to HETH,

Fig. 2. Temperature and haematocrit before and after the incremental test until exhaustion and sweat rate in each trial.
Fig. 2 shows the external temperature (A), internal temperature (B) and haematocrit (C) before and after each test, and the sweat rate in each trial (D). CG ¼ Control
Group; EG ¼ Experimental Group; Tsk ¼ External temperature: Tc: Internal temperature; HEHT ¼ Heat exposure at high temperature; HEHT ¼ pre-normothermic
trial before HEHT; ANB HEHT ¼ post-normothermic trial before HEHT; PHB HEHT ¼ pre-hyperthermic trial before HEHT; AHB HEHT ¼ post-hyperthermic trial
before HEHT;PNA HEHT ¼ pre-normothermic trial after HEHT; ANA HEHT ¼ post-normothermic trial after HEHT; PHA HEHT ¼ pre-hyperthermic trial after HEHT;
AHA HEHT ¼ post-hyperthermic trial after HEHT; NB HEHT ¼ Normothermic test before HEHT; HB HEHT ¼ Hyperthermic test before HEHT; NA HEHT ¼
Normothermic test after HEHT; HA HEHT ¼ Hyperthermic trial after HEHT; *Pre-post differences of each test (p < .05); ** Intra-group pre-post differences of each
test (p < .01);¢¢ Difference with respect to the control group (p < .01); þþ before-before and after-after differences before HEHT (p < .01); ∞ Before- Intra-group
before and after-after differences after HEHT (p < .05); ∞∞ Intra-group before-before and after-after differences after HEHT (p < .01). ¢ Differences between group
(p < .05). # Intra-groups differences between NB HEHT -HB HEHT test and NA HEHT-HA HEHT trials.

4
J. Siquier-Coll et al. Journal of Thermal Biology 88 (2020) 102492

Table 2
Serum concentrations of Se and Zn before and after each trial. The values are presented without and with (C) concentration for possible haemoconcentration.
PRE-HEHT POST-HEHT

Normothermic (22 C)�

Hyperthermic (42 C)
�
Normothermic (22 � C) Hyperthermic (42 � C)

Before After Before After Before After Before After

Zn (μg/L) Control 741.31 � 793.82 � 781.14 � 852.23 � 810.04 � 853.94 � 798.43 � 794.47 � 82.11
92.43 145.89a 175.08 161.47 138.85 152.87a 125.88
Experimental 829.07 � 151 859.67 � 810.22 � 859.79 � 924.97 � 942.73 � 847.67 � 972.94 �
195.81 161.83 281.22 325.66 233.73 188.34 238.74b e
Zn–C (μg/ Control 741.31 � 734.45 � 781.14 � 798.27 � 810.04 � 806.29 � 798.43 � 763.85 �
L) 92.43 161.39 175.08 141.11 138.85 157.63 125.88 153.23
Experimental 829.07 � 151 788.77 � 810.22 � 770.66 � 924.97 � 873.87 � 847.67 � 875.07 �
181.14 161.83 269.1 325.66 222.03 188.34 235.09
Se (μg/L) Control 87.84 � 15.6 93.64 � 18.51a 91.95 � 12.21 94.04 � 13.54 90.97 � 10.42 93.56 � 18.21 88.5 � 18.42 93.23 � 17.39a
Experimental 114.24 � 119.28 � 114.6 � 119.38 � 100.37 � 106.32 � 12b d 93.99 � 105.43 � 8.35b c
15.82f 21.27f 16.88f 23.92f 12.31d e 8.79∞ e

Se–C (μg/ Control 87.84 � 15.6 86.42 � 19.32 91.95 � 12.21 88.23 � 12.72 90.97 � 10.42 88.78 � 19.84 88.5 � 18.42 88.99 � 18.05
L) Experimental 114.24 � 109.59 � 114.6 � 16.88 105.57 � 100.37 � 12.31 98.67 � 13.42c 93.99 � 8.79 94.57 � 9.22
15.82 19.75e 25.37
a
Pre-post differences of each test (p < .05).
b
Pre-post differences of each test (p < .01).
c
Differences after-after and before-before with respect to the test with the same thermal conditions (p < .05).
d
After-after and before-before differences with respect to the test with the same thermal conditions (p < .05).
e
Differences with respect to the same parameter of the control group (p < .05).
f
Differences with respect to the same parameter of the control group (p < .01).

and before normothermia test (p < .05) and post hyperthermia test (p <
Table 4
.05) after HETH. CG experimented a pre-post difference after the
Sweat levels of Zn and Se in the hyperthermic condition with and without
normothermia trial (p < .05) before HEHT and post hyperthermia test
correction (C) for sweat excreted during the incremental test until exhaustion.
after HEHT (p < .05). The concentration of Se in EG underwent a rise
CONTROL EXPERIMENTAL
after normothermia and hyperthermia trials after HEHT (p < .01).
Additionally, after HEHT, the pre-post data in the normothermia (p < PRE-HEHT POST- HEHT PRE- HEHT POST- HEHT
.01) test and the post-value in the hyperthermia trial (p < .05) were Zn (μg/ 1704.39 � 1756.82 � 1624.19 � 1097.73 �
lower than in the same parameter before HEHT. Se–C did not show L) 1348.86 1353.63 905.72 477.8
changes in CG. In EG, Se–C was significantly higher after the first trial in Zn–C 1488.21 � 1113.722 � 1492.11 � 738.14 �
(μg/h) 1907.99 910.85 1455.46 462.68*
normothermia with respect to CG (p < .05). A decrease can also be
Se (μg/L) 5.17 � 1.59 12.77 � 24.91 3.64 � 1.67þ 4.01 � 2.05þ
observed in Se–C post normothermia test after HEHT in comparison to Se–C 3.82 � 2.07 7.27 � 13.04 3.12 � 2.49 2.73 � 1.81
the same data before HEHT in EG (p < .05). (μg/h)
Table 3 presents the urine levels of Zn and Se. The concentration of
*Pre-post differences of each test (p < .05); **Pre-post differences of each test (p
Zn showed changes after the normothermia trials in CG (p < .05) and < .01); þDifferences with respect to the same parameter of the control group (p
post normothermia test after HEHT in EG (p < .05). No more alterations < .05).
were observed in Zn. Regarding Se, there were pre-post changes after the
first hyperthermia test in CG (p < .05) and in normothermia and hy­
4. Discussion
perthermia trials after HEHT in EG (p < .05). Furthermore, the con­
centration of Se post hyperthermia test was higher after HEHT with
Exercise results in an increase in body temperature (Tc) accompa­
respect to the same value before HEHT (p < .05).
nied by dehydration in the human body, being greater in hot conditions
The concentrations of Zn and Se in sweat are described in Table 4.
(Wingo, 2015). Increased Tc involves redistributing blood flow to pe­
The values are expressed without and with correction (C) for the sweat
ripheral areas to remove heat through the skin by evaporation (Gonza­
rate. Zn–C was significant lower after HEHT in EG (p < .05). A lower
lez-Alonso, 2012). This situation leads to an increase in sweating in hot
concentration of Se pre and post HEHT can be observed in EG in com­
environments, resulting in haemoconcentration, which would explain
parison to CG.
the higher levels of haematocrit after testing (Veltmeijer et al., 2017).

Table 3
Urine levels of Zn and Se before and after the incremental tests until exhaustion.
PRE-HEHT POST-HEHT

Normothermic (22 C)
�
Hyperthermic (22 C)
�
Normothermic (22 � C) Hyperthermic (22 � C)

Before After Before After Before After Before After

Zn (μg/ Control 459 � 261.62 441.98 � 381.5 � 541.24 � 550.26 � 607.44 � 492.71 � 558.91 � 325.1
L) 240.95a 414.02 361.88 369.99 354.68a 255.25
Experimental 549.91 � 606.67 � 427.8 � 416.45 � 532.03 � 659.04 � 542.02 � 626.25 �
390.68 300.15 295.31 245.13 282.14 289.2a 395.01 304.05b
Se (μg/ Control 22.35 � 12.62 23.83 � 10.02 15.76 � 11.1 25.8 � 9.9a 24.61 � 13.19 25 � 11.95 22.92 � 11.71 31.76 � 15.21
L) Experimental 24.78 � 12.16 25.81 � 13.21 22.11 � 10.6 21.08 � 8.46 26.05 � 13.64 31.56 � 13.43a 27.96 � 12.59 35.75 � 12.06a
b

a
Pre-post differences of each test (p < .05).
b
Differences after-after and before-before with respect to the test with the same thermal conditions (p < .05).

5
J. Siquier-Coll et al.
On the other hand, this process produces an increase in redox reactions,
increasing serum cytokines and circulating leukocytes (Baltaci et al.,
2016). Thus, antioxidant defence mechanisms are put in place. Quindry
et al. (2013) indicated that moderate intensity exercise in the heat (33
�
C) produces a blood response of oxidative stress not observed with
neutral (20 � C) and cold (7 � C) temperatures. Simultaneously, research
conducted on athletes studied the acute effect of a treadmill exercise for
45 min (75–80% of maximum oxygen consumption) at room tempera­
ture (10–12 � C, 40–55% humidity) and in heat stress (30–32 � C, 75–78%
humidity). Heat exercise produced a marked increase in cellular dam­
age, oxidative stress, and anti-inflammatory and antioxidant response in
post-exercise plasma (Sureda et al., 2015). In a recent study, heat
acclimation (42 � C; 18% RH) and two tests were performed under the
same conditions to see the effect on oxidative stress. It was concluded
that ten days of heat acclimation increase oxidative stress levels (Kaldur
et al., 2014). In our study, we found a haemoconcentration after effort in
both normothermia and hyperthermia. This meant that if we did not
apply corrections for blood volume there would be increases in the el­
ements studied. But when the values were corrected for haemoconcen­
tration, no significant changes were found in serum concentrations of Se
and Zn after the acute effect of exercise and after HEHT. For a better
discussion, the results of each metal will be discussed separately.
Regarding selenium, it appears from the above studies that the
changes in selenium are due to diet alterations (National Academy of
Sciences, 2006), and there is scant literature on the effect of exercise on
this element. Thus, serum Se values are in accordance with established
plasma levels (Baltaci et al., 2016). Most research on post-exercise levels
reported a decrease in plasma/serum (Emre et al., 2004; Eskici et al.,
2016; Margaritis et al., 2005). This fall may be due to an increase in the
antioxidant response. Rodas et al. (2000) suggested that the decrease in
serum post-exercise may be associated with the transfer of lactate to the
blood from the muscle. Doker et al. (2014) reported higher levels of
serum Se after acute exercise in amateur swimmers, but not in controls.
In this sense, a recent study found an increase of Se serum after exercise
in well trained athletes. Nonetheless, the mentioned investigations did
not correct the values for haemoconcentration (Soria et al., 2015). In
heat stress, Wang et al. (2012) found a depletion of Se after intense
basketball exercise, but did not correct for haemoconcentration, which
is possibly why the values they obtained returned to baseline after 2 h. In
a recent study by our group, on the acute effect in Se, there was a serum
post-exercise decrease in Se, but their significance decreased after
correction for haemoconcentration (Siquier-Coll et al., 2019b), as in this
study. In another recent study where the correction for haemoconcen­
tration was performed, serum changes occurred in the group of athletes
and not in the control group, suggesting that continued physical activity
produces an antioxidant response (Maynar et al., 2018b). In addition, no
changes in urinary Se were observed in the above-mentioned study.
Similarly, Singh et al. (1991) found no significant differences after five
days of exercise and psychological stress. In contrast, Rodriguez et al.
(1995) reported a decrease in urinary excretion of Se with exercise.
Similarly, a recent study in female athletes also observed such a deple­
tion (Eskici et al., 2016). Conversely, recent research reported a rise in
urine Se after mountain ultra-marathon race (Jablan et al., 2017). We
did not observe changes in the acute effect, either in normothermia or in
hyperthermia (Siquier-Coll et al., 2019b). However, this study shows
how there was an increase in urine in CG after the test in hyperthermia
and in both thermal conditions after HEHT in EG. It seems that, as in
other trace elements reported above (Siquier- Coll et al., 2019a), expo­
sure to high temperatures stimulates urinary elimination of Se. Tang
et al. (2016) observed in workers how the excretion levels of Se in sweat
were higher at higher temperatures. A study reported that after 16 days
of heat acclimation (37.8 � C) Se was not affected (Consolazio et al.,
1964), as found in this study.
Serum Zn levels are within the levels set by Lu et al. (2015) for the
same technique. It has been suggested that long-term activities decrease
Zn in plasma/serum, while short, high-intensity activities increase levels
6
Journal of Thermal Biology 88 (2020) 102492
of Zn in plasma/serum (van Rij et al., 1986). This is due to the redis­
tribution that occurs in Zn’s homeostasis during the exercise, where
there is a redistribution from erythrocytes to plasma (Lukaski et al.,
1984). Singh et al. (1991) found lower plasma levels of Zn after five days
of training, which may be due to the high lipid peroxidation and anti­
oxidant response to it. Simultaneously, a review concluded that athletes
had lower plasma levels of Zn due to exercise despite high intake (Chu
et al., 2018). Thus, Maynar et al. (2018b) found changes in Zn con­
centrations after the acute post-exercise effect in athletes, but not in
control subjects. Two studies reported an elevation of Zn levels in serum
in well-athletes trained athletes (Soria et al., 2016), and in elite swim­
mers, amateur swimmers and sedentary controls (Doker et al., 2014).
However, these researches did not correct values for haemoconcentra­
tion. The participants in this study had average levels of VO2max so,
since they are not athletes, this could be the explanation of why no
changes were observed. Besides, Chu et al. (2016) emphasised in their
review that in cyclists there are no changes due to the lower participa­
tion of muscle groups, this being another possible reason why no sig­
nificant changes were found in post-exercise serum. Concerning heat, it
has been emphasised that exposure to extreme temperatures can result
in stimulation with subsequent changes in the metabolism of Zn (Ohno
et al., 1990). In a study of basketball players, no plasma changes were
observed after 5 days of intense heat exercise (Wang et al., 2012).
Conversely, Uhari et al. (1983) obtained a decrease in serum Zn on the
4th day of heat exposure. No changes in serum Zn–C (corrected for
haemoconcentration) were observed in this study after acute exercise or
after HEHT.
Related to excretion pathways, urine is a minority route of Zn
excretion, with values between 0.4 and 0.7 mg/day (National Academy
of Sciences, 2006). In our previous published study on the acute effect,
no changes in this metal were observed in erythrocytes or serum
(Siquier-Coll et al., 2019b). The results in the bibliography found are
disparate, while in some studies the concentration of urinary Zn in­
creases (Anderson et al., 1984; Granell, 2014; Kaya, 2008), in others
there are no changes, perhaps because the test has to be of maximum
intensity to elicit changes. Maynar et al. (2018b) found no post-exercise
changes in controls, but did in athletes. In this research, post-exercise
elimination increased after testing in normothermia after HEHT in
both groups and in EG the Zn concentration was higher after the
post-HEHT hyperthermia test compared to the homologous test before
HEHT, but there were no changes in the previous acute effect as shown
in our recently published study (Siquier-Coll et al., 2019b). The urinary
elimination of Zn in heat during 16 days of heat exposure and exercise
was in a range of 0.57–0.75 mg/day (Consolazio et al., 1964). Heat
exercise can increase the excretion of Zn by urine due to an increase in
the catabolism of muscle proteins caused by the rise in the load of amino
acids filtered by the kidneys (Coates et al., 2010). Thus, a possible in­
crease in the catabolism of muscle proteins may have affected the in­
crease in urinary Zn in EG after HEHT. Regarding the other excretion
pathway, sweat, Aruoma et al. (1988) reported concentrations of Zn in
sweat on the back of 7.3–8.8 mol/l. It seems that increases in sweat Zn
occur, as in urine, through muscle damage (C� ordova and Navas, 1998).
On the other hand, other authors report that the excretion of Zn de­
creases with exercise time (DeRuisseau et al., 2002; Montain et al., 2007;
Tipton et al., 1993). One study observed concentrations of Zn in sweat in
winter (25 � C) and in summer (35 � C) without finding significant
changes (Hoshi et al., 2002). On the contrary, Tang et al. (2016) found
higher concentrations of Zn at higher temperatures in workers on 8 h
shifts. In relation to repeated exposure to heat, Consolazio et al. (1964)
observed excretion values of 1.83 mg/h, decreasing to 0.29.-0.32
mg/hour after acclimation at 37.8 � C. However, recent studies have not
found significant changes in acclimatised subjects (Chinevere et al.,
2008; Ely et al., 2013). In this study, as in our previous study (Siquier-
Coll et al., 2019a), there were lower levels of Zn excreted by sweat after
HEHT, which are countered by increased urinary excretion.
J. Siquier-Coll et al.
5. Conclusions
From all the above-mentioned data, we can conclude that the acute
performance of maximum physical activity until exhaustion does not
produce significant changes in serum or urinary concentrations either in
normothermia or hyperthermia. However, repeated exposure to heat at
high temperatures results in increased urinary elimination of Se and Zn,
which in the case of Zn is accompanied by a decrease in its sweat
excretion. It is necessary to monitor the concentrations of both elements
when carrying out intense activities at high temperatures as they could
cause a serum decrease in these important elements.
CRediT authorship contribution statement
J. Siquier-Coll: Investigation, Writing - original draft, Writing - re­
view & editing, Formal analysis. I. Bartolome �: Investigation, Formal
analysis. M. Pe
�rez-Quintero: Investigation. D. Mun ~ oz: Validation. M.
C. Robles: Visualization. M. Maynar-Marin ~ o: Conceptualization,
Methodology, Writing - review & editing.
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