Copyright © 2017 John Libbey Eurotext. Downloaded by NYU LANGONE MED CTR SCH OF MED HEALTH SCIENCES LIBRARY on 30/06/2017.
doi:10.1684/mrh.2014.0372
Magnesium Research 2014; 27 (4): 155-64
ORIGINAL ARTICLE
Variations in serum magnesium and hormonal levels during incremental exercise 2, ˜ Marisol Soria1 , Carlos González-Haro1 , Miguel Angel Ansón1 , Carmen Inigo 2 1 Maria Luisa Calvo , Jesús Fernando Escanero 1 Pharmacology and Physiology Department School of Medicine, University of Zaragoza, Spain; 2 Clinical Biochemistry Service, Hospital Miguel Servet, Zaragoza, Spain
Correspondence: Jesús Fernando Escanero. Department of Pharmacology and Physiology. School of Medicine. ˜ University of Zaragoza, Spain. Postal address: C/ Domingo Miral, s/n. 50.009. Zaragoza, Espana.
Abstract. In this study, we examined the relationship between plasma magnesium levels and hormonal variations during an incremental exercise test until exhaustion in 27, well-trained, male endurance athletes. After a warm-up of 10 min at 2 W/kg, the test began at an initial workload of 2.5 W/kg and continued with increments of 0.5 W/kg every 10 min until exhaustion. Plasma magnesium, catecholamine, insulin, glucagon, parathyroid hormone (PTH), calcitonin, aldosterone and cortisol levels were determined at rest, at the end of each stage and three, five and seven minutes post-exercise. With the incremental exercise test, no variations in plasma magnesium levels were found, while plasma adrenaline, noradrenaline, PTH, glucagon and cortisol levels increased significantly. Over the course of the exercise, plasma levels of insulin decreased significantly, but those of calcitonin remained steady. During the recovery period, catecholamines and insulin returned to basal levels. These findings indicate that the magnesium status of euhydrated endurance athletes during incremental exercise testing may be the result of the interrelation between several hormonal variations. Key words: magnesium, incremental exercise, hormonal regulation
Some recent publications [1, 2] indicate important progress in the understanding of the molecular mechanisms of intestinal and renal absorption of magnesium (Mg), and it can be expected that there will be further clarification over the next few years. Magnesium homeostasis depends on the balance between intestinal absorption and renal excretion, with kidney tubules having primary control. At times of temporary Mg deficiency, the body depends on the availability of Mg in bone to keep serum levels constant [3]. Therefore, Mg homeostasis is dependent on three organs: the intestine, facilitating Mg uptake; the bone, the body’s Mg storage system; and the kidneys, which are responsible for Mg excretion. Hormonal regulation of the Mg balance began to be studied some time ago, but the process is
still not fully understood. To date, it has not even been determined at which molecular level these hormones operate. Many hormones have been reported to be involved in the Mg balance. Some of them, such as parathyroid hormone (PTH), calcitonin, glucagon, arginine vasopressin, insulin and aldosterone [4], act in the cortical thick ascending limb (cTAL) of the loop of Henle; these hormones increase the Mg reabsorption in this part of the nephron. The actions of these hormones are mediated by various pathways affecting luminal voltage and paracellular structure. In addition, others, such as PTH, calcitonin, glucagon, arginine vasopressin, insulin and aldosterone have been found to increase Mg reabsorption in the distal convoluted tubule in a similar way to their action in the cTAL [4].
155 ˜ To cite this article: Soria M, González-Haro C, Ansón MA, Inigo C, Calvo ML, Escanero JF. Variations in serum magnesium and hormonal levels during incremental exercise. Magnes Res 2014; 27(4): 155-64 doi:10.1684/mrh.2014.0372
M. SORIA, ET AL.
Copyright © 2017 John Libbey Eurotext. Downloaded by NYU LANGONE MED CTR SCH OF MED HEALTH SCIENCES LIBRARY on 30/06/2017.
On the other hand, Irvine [5] remarked that plasma levels of several hormones increase with exercise, as part of the integrated response to stress. It seems that these increases generate responses that are beneficial in prolonged exercise. Although stress is a key issue in sport, we should take into account other hormonal changes, besides those caused by the regulatory stress hormones, including those that control the changes in plasma energy substrates (glucose and fatty acids), and others that control fluids and electrolytes. It may even be supposed that hormonal changes induced by stress mask other challenges. Further, it may be useful to assess hormonal status in sports for two reasons: one, to ascertain whether the physiological process of exercise is regulated more strongly by certain hormones; and second, because the hormonal control of some metabolites, such as Mg, which are difficult to explore under physiological conditions, might be demonstrated more clearly under the specific conditions of exercise. In relation to Mg and sport, two facts must be considered: 1. changes in serum Mg concentration caused by the exercise have been analysed in a previous study [6], and no variations were observed across the range of intensities tested, while osmolarity was maintained with ad libitum intake of water during the test; and 2. it has long been known [7] that Mg metabolism is primarily regulated by PTH, vitamin D3 and calcitonin, and is closely linked to the metabolism of calcium and potassium. This paper analyses the role of the variations in the aldosterone and mineral metabolismregulating hormones (PTH and calcitonin) in the regulation of Mg metabolism in euhydrated individuals during incremental exercise. The role of stress hormones and the hormones involved in energy metabolism were also analysed.
Methods and materials Participants The study participants were 27, well-trained, male, endurance athletes (19 triathletes - three of them elite-level- and eight road cyclists), aged 33.8 ± 6.7 years, with 9.3 ± 3.2 years of experience, and VO2 max of 60.2 ± 6.2 mL/kg/mn. Their morphological characteristics are summarised in
156
Table 1. Morphological characteristics of participants. Variable Body mass (kg) Height (m) BMI (kg/m2 ) Body fat (kg) Body fat (%) Lean body mass (kg) Lean body mass (%) BMC (kg) BMC (%) BMD (g/cm2 )
mean ± SD 74.7 ± 7.9 1.79 ± 0.05 23.3 ± 2.2 11.2 ± 6.4 15.0 ± 7.3 60.3 ± 4.6 81.3 ± 7.0 3.2 ± 0.3 4.3 ± 0.4 1.22 ± 0.08
BMI: body mass index; BMC: bone mineral content; BMD: bone mineral density.
table 1. All participants gave written, informed consent to participate in this study, and the study protocol was approved by the Ethics Committee of the University of Zaragoza. Body composition Dual-energy X-ray absorptiometry was performed using a Lunar Prodigy PrimoTM densitometer (General Electric, Madison, Wisconsin, USA) at two different X-ray energies (38 and 70 keV). Body mass, body fat, lean body mass, bone mineral content and bone mineral density were assessed for the whole body. Each scan took around seven minutes and the data were analysed using Lunar enCORE software (table 1). Study design In the present descriptive study, all participants underwent morning medical examinations that included the following routine measurements: spirometry, blood tests, and baseline ECG, and they also completed a health questionnaire. In addition, all participants were asked specifically about symptoms related to magnesium deficiency. Further, as described above, their body composition was measured using dual-energy X-ray absorptiometry. In the afternoon, participants performed an incremental, maximal exercise test. The inclusion criteria were; normal results for ECG function, blood screening and spirometry, and no symptoms of hypomagnesaemia such as neuromuscular hyperexcitability. One week later, participants performed a steady-state step test.
Copyright © 2017 John Libbey Eurotext. Downloaded by NYU LANGONE MED CTR SCH OF MED HEALTH SCIENCES LIBRARY on 30/06/2017.
Exercise intensity and plasma Mg and hormones levels
Training and nutritional conditions under which the athletes performed all tests were controlled: 2 h low intensity (∼50%VO2 max) training and a carbohydrate-rich diet (80%) over the 24 h prior to each test. Athletes did not participate in either strenuous training for the 72 h prior to the test, or in any competitions for one week prior to the test. Furthermore, during the three days prior to the steady-state step test, the magnesium intake in the diet was 454 mg/day, as determined by a nutritional survey.
last 15 s of exercise, when at least two criteria recommended by BASES [8] were fulfilled. Blood lactate concentration ([La-]b) was measured with a Lactate-Pro analyser (Arkray, Kyoto, Japan), and blood samples were taken from every athlete at rest, at the end of each stage and 3, 5 and 7 minutes post-exercise. Finally, two urine samples were collected just before and immediately after the test to determine the level of hydration of the participants in terms of USG. Blood collection and processing
Incremental, maximal exercise test The exercise test was performed on an electromagnetically-braked, cycle ergometer (Cardioline, Milano, Italy). After 10 min of warmup at 100 W, the test began at a workload of 130 W, and then the load was increased by 30 W every 3 min until exhaustion. Throughout the test, VO2 and mechanical power output were measured in real time, breath-by-breath, using an Oxycon-Pro integrated indirect calorimetry system (Erich Jaeger, Höchberg, Germany). VO2 max was determined as the mean VO2 value during the last 15 s of the test, when at least two criteria recommended by the British Association of Sport and Exercise Sciences (BASES) [8] were fulfilled. Steady-state step test All participants carried out a second exercise test on the same cycle ergometer as in the first test. After a warm-up of 10 min at 2 W/kg, the test began at an initial workload of 2.5 W/kg and continued with increments of 0.5 W/kg every 10 min until exhaustion. This protocol has been used previously with amateur and elite road cyclists by San Millán et al. (2009) [9]. In order to start the test well hydrated, each participant drank 4 mL/kg of water 2 h before the exercise. During the exercise test, participants drank 0.842 ± 0.197 L of water (Mg content of 8.6 mg/L) and no significant differences in urine specific gravity (USG) were found comparing after with just before the test (1.014 ± 0.004 versus 1.014 ± 0.004 g/cm3 ) [10]. Throughout the test, VO2 and VCO2 were measured and averaged every 15 s using the Oxycon-Pro system, and heart rate was monitored and averaged every 5 s using a Polar RS800CX device (Polar, Finland). As before, VO2 PEAK was determined as the mean VO2 value during the
Venous blood samples were collected using a Teflon intravenous catheter (Baxter, Utrecht, Holland), inserted into an antecubital vein. Samples were collected in three types of tubes: 5 mL in a tube with 200 L of EDTA K anticoagulant (Vacutainer, Becton Dickinson, New Jersey, USA); 3 mL in a tube with lithium heparin containing reduced glutathione (1.2 mg/mL) to prevent catecholamine oxidation; and 3 mL in a tube without anticoagulant. Then, samples were centrifuged at 4◦ C for 15 minutes at 3,000 rpm to separate the plasma. The plasma was aliquoted into 1.8-mL Eppendorf tubes and stored at -80◦ C until further analysis. Plasma Mg analysis All Mg measurements were obtained by flame atomic absorption spectrophotometry using a Perkin–Elmer 3110 atomic absorption spectrometer (Perkin-Elmer, Uberlingen, Germany) equipped with an air–acetylene flame and an impact bead nebulizer. For Mg, we used a hollow cathode lamp operated at the intensities recommended by the manufacturer. Three different concentrations (2.0, 4.0 and 6.0 mg Mg/L) of Mg were used to calibrate standard curves, and absorbance was measured at 285.2 nm. To verify the assay accuracy and to monitor quality, two levels of BioRad Lyphochek controls were run at the beginning and end of each series, and after every 10 samples. Serum glucose and plasma ions Sodium and chloride were measured in serum using ion selective electrodes (ISEs) in a Hitachi 711 multitest analyser. We used SeronormTM (Nyegaard & Co., Oslo, Norway) as the calibrator, as recommended by the manufacturer. Glucose was determined using an enzymatic spectrophotometric method: glucose oxidase kit (Glucose, Roche Diagnostics, Rotkreuz, Switzerland).
157
M. SORIA, ET AL.
Copyright © 2017 John Libbey Eurotext. Downloaded by NYU LANGONE MED CTR SCH OF MED HEALTH SCIENCES LIBRARY on 30/06/2017.
Hormonal analysis Plasma catecholamine measurements. Plasma catecholamine concentrations were determined using high performance liquid chromatography (HPLC). Catecholamines were extracted by selective adsorption onto aluminium oxide (HPLC reagent Kit, Chromsystems, Gräfelfing, Germany) before the HPLC was run. Aluminium oxide was briefly shaken in extraction buffer (50 L) and 1 mL of plasma was added with 50 L of internal standard solution (600 pg dihydroxybenzylamine). The aluminium oxide was then washed three times, with a short period of centrifugation between washes. The catecholamines were extracted by brief shaking with 120 L of elution buffer and then centrifugation (final centrifugation) at 2,000 rpm for 1 min. Next, sample eluent was injected into the HPLC column (Resolve C18, 5-L spherical packing, Waters Corp., Milford, MA, USA) and eluted with a mobile phase. The flow rate was 1 mL/mn at 2,000 psi with a potential of 0.60 V. For each component of interest in the chromatogram, peak areas were determined by computer integration (Baseline 815, Waters Corp., Milford, MA, USA). Measurements of other hormones. The quantification of serum hormones was performed in automated analytical systems. Specifically, chemiluminescent immunoassays were used to measure levels of cortisol (Architec i2000sr, Abbott Laboratories, Abbott Park, IL, USA), calcitonin (Immulite 2000 Xpi, Siemens, New York, NY, USA), PTH (Unicel DxI 800 Access Immunoassay System, Beckman Coulter, Brea, CA, USA), and insulin (Architect ci8200, Abbott Laboratories). Aldosterone levels were determined by colorimetric direct enzyme-linked immunosorbent assay (EiAsy Way Aldosterone Kit; Diagnostic Biochem Canada Inc, Dorchester, Ontario, Canada) in an automated processing system. Serum glucagon was measured by radioimmunoassay (RIA) using a commercial glucagon assay (Euro Diagnostica AB, Malmö, Sweden). Analysis of USG
Urine samples were collected in 10-mL sterile containers and frozen at -80◦ C. Then, the USG was measured by photocolorimetry (Urisys 1800, Roche Diagnostics, Rotkreuz, Switzerland). This method has been found to be valid and reliable for the assessment of hydration status [11]. Partici-
158
pants were classified as euhydrated if their USG was ≤1.020; hypohydrated, for USG values from 1.020 to 1.029; and significantly hypohydrated, for USG≥1.030. These hydration-status groups were based on those most recently established by the American College of Sports Medicine [11]. Statistical analysis The data were expressed in SI units. The distribution of the data was examined using the Shapiro-Wilk’s test and analysed by one-way ANOVA and Pearson’s or Spearman’s correlation analysis. The statistical significance of differences for paired samples was tested using the non-parametric Wilcoxon test. The statistics were generated using IBM SPSS Statistics for Windows software. All values were expressed as means and standard deviations (SDs), and p values
doi:10.1684/mrh.2014.0372
Magnesium Research 2014; 27 (4): 155-64
ORIGINAL ARTICLE
Variations in serum magnesium and hormonal levels during incremental exercise 2, ˜ Marisol Soria1 , Carlos González-Haro1 , Miguel Angel Ansón1 , Carmen Inigo 2 1 Maria Luisa Calvo , Jesús Fernando Escanero 1 Pharmacology and Physiology Department School of Medicine, University of Zaragoza, Spain; 2 Clinical Biochemistry Service, Hospital Miguel Servet, Zaragoza, Spain
Correspondence: Jesús Fernando Escanero. Department of Pharmacology and Physiology. School of Medicine. ˜ University of Zaragoza, Spain. Postal address: C/ Domingo Miral, s/n. 50.009. Zaragoza, Espana.
Abstract. In this study, we examined the relationship between plasma magnesium levels and hormonal variations during an incremental exercise test until exhaustion in 27, well-trained, male endurance athletes. After a warm-up of 10 min at 2 W/kg, the test began at an initial workload of 2.5 W/kg and continued with increments of 0.5 W/kg every 10 min until exhaustion. Plasma magnesium, catecholamine, insulin, glucagon, parathyroid hormone (PTH), calcitonin, aldosterone and cortisol levels were determined at rest, at the end of each stage and three, five and seven minutes post-exercise. With the incremental exercise test, no variations in plasma magnesium levels were found, while plasma adrenaline, noradrenaline, PTH, glucagon and cortisol levels increased significantly. Over the course of the exercise, plasma levels of insulin decreased significantly, but those of calcitonin remained steady. During the recovery period, catecholamines and insulin returned to basal levels. These findings indicate that the magnesium status of euhydrated endurance athletes during incremental exercise testing may be the result of the interrelation between several hormonal variations. Key words: magnesium, incremental exercise, hormonal regulation
Some recent publications [1, 2] indicate important progress in the understanding of the molecular mechanisms of intestinal and renal absorption of magnesium (Mg), and it can be expected that there will be further clarification over the next few years. Magnesium homeostasis depends on the balance between intestinal absorption and renal excretion, with kidney tubules having primary control. At times of temporary Mg deficiency, the body depends on the availability of Mg in bone to keep serum levels constant [3]. Therefore, Mg homeostasis is dependent on three organs: the intestine, facilitating Mg uptake; the bone, the body’s Mg storage system; and the kidneys, which are responsible for Mg excretion. Hormonal regulation of the Mg balance began to be studied some time ago, but the process is
still not fully understood. To date, it has not even been determined at which molecular level these hormones operate. Many hormones have been reported to be involved in the Mg balance. Some of them, such as parathyroid hormone (PTH), calcitonin, glucagon, arginine vasopressin, insulin and aldosterone [4], act in the cortical thick ascending limb (cTAL) of the loop of Henle; these hormones increase the Mg reabsorption in this part of the nephron. The actions of these hormones are mediated by various pathways affecting luminal voltage and paracellular structure. In addition, others, such as PTH, calcitonin, glucagon, arginine vasopressin, insulin and aldosterone have been found to increase Mg reabsorption in the distal convoluted tubule in a similar way to their action in the cTAL [4].
155 ˜ To cite this article: Soria M, González-Haro C, Ansón MA, Inigo C, Calvo ML, Escanero JF. Variations in serum magnesium and hormonal levels during incremental exercise. Magnes Res 2014; 27(4): 155-64 doi:10.1684/mrh.2014.0372
M. SORIA, ET AL.
Copyright © 2017 John Libbey Eurotext. Downloaded by NYU LANGONE MED CTR SCH OF MED HEALTH SCIENCES LIBRARY on 30/06/2017.
On the other hand, Irvine [5] remarked that plasma levels of several hormones increase with exercise, as part of the integrated response to stress. It seems that these increases generate responses that are beneficial in prolonged exercise. Although stress is a key issue in sport, we should take into account other hormonal changes, besides those caused by the regulatory stress hormones, including those that control the changes in plasma energy substrates (glucose and fatty acids), and others that control fluids and electrolytes. It may even be supposed that hormonal changes induced by stress mask other challenges. Further, it may be useful to assess hormonal status in sports for two reasons: one, to ascertain whether the physiological process of exercise is regulated more strongly by certain hormones; and second, because the hormonal control of some metabolites, such as Mg, which are difficult to explore under physiological conditions, might be demonstrated more clearly under the specific conditions of exercise. In relation to Mg and sport, two facts must be considered: 1. changes in serum Mg concentration caused by the exercise have been analysed in a previous study [6], and no variations were observed across the range of intensities tested, while osmolarity was maintained with ad libitum intake of water during the test; and 2. it has long been known [7] that Mg metabolism is primarily regulated by PTH, vitamin D3 and calcitonin, and is closely linked to the metabolism of calcium and potassium. This paper analyses the role of the variations in the aldosterone and mineral metabolismregulating hormones (PTH and calcitonin) in the regulation of Mg metabolism in euhydrated individuals during incremental exercise. The role of stress hormones and the hormones involved in energy metabolism were also analysed.
Methods and materials Participants The study participants were 27, well-trained, male, endurance athletes (19 triathletes - three of them elite-level- and eight road cyclists), aged 33.8 ± 6.7 years, with 9.3 ± 3.2 years of experience, and VO2 max of 60.2 ± 6.2 mL/kg/mn. Their morphological characteristics are summarised in
156
Table 1. Morphological characteristics of participants. Variable Body mass (kg) Height (m) BMI (kg/m2 ) Body fat (kg) Body fat (%) Lean body mass (kg) Lean body mass (%) BMC (kg) BMC (%) BMD (g/cm2 )
mean ± SD 74.7 ± 7.9 1.79 ± 0.05 23.3 ± 2.2 11.2 ± 6.4 15.0 ± 7.3 60.3 ± 4.6 81.3 ± 7.0 3.2 ± 0.3 4.3 ± 0.4 1.22 ± 0.08
BMI: body mass index; BMC: bone mineral content; BMD: bone mineral density.
table 1. All participants gave written, informed consent to participate in this study, and the study protocol was approved by the Ethics Committee of the University of Zaragoza. Body composition Dual-energy X-ray absorptiometry was performed using a Lunar Prodigy PrimoTM densitometer (General Electric, Madison, Wisconsin, USA) at two different X-ray energies (38 and 70 keV). Body mass, body fat, lean body mass, bone mineral content and bone mineral density were assessed for the whole body. Each scan took around seven minutes and the data were analysed using Lunar enCORE software (table 1). Study design In the present descriptive study, all participants underwent morning medical examinations that included the following routine measurements: spirometry, blood tests, and baseline ECG, and they also completed a health questionnaire. In addition, all participants were asked specifically about symptoms related to magnesium deficiency. Further, as described above, their body composition was measured using dual-energy X-ray absorptiometry. In the afternoon, participants performed an incremental, maximal exercise test. The inclusion criteria were; normal results for ECG function, blood screening and spirometry, and no symptoms of hypomagnesaemia such as neuromuscular hyperexcitability. One week later, participants performed a steady-state step test.
Copyright © 2017 John Libbey Eurotext. Downloaded by NYU LANGONE MED CTR SCH OF MED HEALTH SCIENCES LIBRARY on 30/06/2017.
Exercise intensity and plasma Mg and hormones levels
Training and nutritional conditions under which the athletes performed all tests were controlled: 2 h low intensity (∼50%VO2 max) training and a carbohydrate-rich diet (80%) over the 24 h prior to each test. Athletes did not participate in either strenuous training for the 72 h prior to the test, or in any competitions for one week prior to the test. Furthermore, during the three days prior to the steady-state step test, the magnesium intake in the diet was 454 mg/day, as determined by a nutritional survey.
last 15 s of exercise, when at least two criteria recommended by BASES [8] were fulfilled. Blood lactate concentration ([La-]b) was measured with a Lactate-Pro analyser (Arkray, Kyoto, Japan), and blood samples were taken from every athlete at rest, at the end of each stage and 3, 5 and 7 minutes post-exercise. Finally, two urine samples were collected just before and immediately after the test to determine the level of hydration of the participants in terms of USG. Blood collection and processing
Incremental, maximal exercise test The exercise test was performed on an electromagnetically-braked, cycle ergometer (Cardioline, Milano, Italy). After 10 min of warmup at 100 W, the test began at a workload of 130 W, and then the load was increased by 30 W every 3 min until exhaustion. Throughout the test, VO2 and mechanical power output were measured in real time, breath-by-breath, using an Oxycon-Pro integrated indirect calorimetry system (Erich Jaeger, Höchberg, Germany). VO2 max was determined as the mean VO2 value during the last 15 s of the test, when at least two criteria recommended by the British Association of Sport and Exercise Sciences (BASES) [8] were fulfilled. Steady-state step test All participants carried out a second exercise test on the same cycle ergometer as in the first test. After a warm-up of 10 min at 2 W/kg, the test began at an initial workload of 2.5 W/kg and continued with increments of 0.5 W/kg every 10 min until exhaustion. This protocol has been used previously with amateur and elite road cyclists by San Millán et al. (2009) [9]. In order to start the test well hydrated, each participant drank 4 mL/kg of water 2 h before the exercise. During the exercise test, participants drank 0.842 ± 0.197 L of water (Mg content of 8.6 mg/L) and no significant differences in urine specific gravity (USG) were found comparing after with just before the test (1.014 ± 0.004 versus 1.014 ± 0.004 g/cm3 ) [10]. Throughout the test, VO2 and VCO2 were measured and averaged every 15 s using the Oxycon-Pro system, and heart rate was monitored and averaged every 5 s using a Polar RS800CX device (Polar, Finland). As before, VO2 PEAK was determined as the mean VO2 value during the
Venous blood samples were collected using a Teflon intravenous catheter (Baxter, Utrecht, Holland), inserted into an antecubital vein. Samples were collected in three types of tubes: 5 mL in a tube with 200 L of EDTA K anticoagulant (Vacutainer, Becton Dickinson, New Jersey, USA); 3 mL in a tube with lithium heparin containing reduced glutathione (1.2 mg/mL) to prevent catecholamine oxidation; and 3 mL in a tube without anticoagulant. Then, samples were centrifuged at 4◦ C for 15 minutes at 3,000 rpm to separate the plasma. The plasma was aliquoted into 1.8-mL Eppendorf tubes and stored at -80◦ C until further analysis. Plasma Mg analysis All Mg measurements were obtained by flame atomic absorption spectrophotometry using a Perkin–Elmer 3110 atomic absorption spectrometer (Perkin-Elmer, Uberlingen, Germany) equipped with an air–acetylene flame and an impact bead nebulizer. For Mg, we used a hollow cathode lamp operated at the intensities recommended by the manufacturer. Three different concentrations (2.0, 4.0 and 6.0 mg Mg/L) of Mg were used to calibrate standard curves, and absorbance was measured at 285.2 nm. To verify the assay accuracy and to monitor quality, two levels of BioRad Lyphochek controls were run at the beginning and end of each series, and after every 10 samples. Serum glucose and plasma ions Sodium and chloride were measured in serum using ion selective electrodes (ISEs) in a Hitachi 711 multitest analyser. We used SeronormTM (Nyegaard & Co., Oslo, Norway) as the calibrator, as recommended by the manufacturer. Glucose was determined using an enzymatic spectrophotometric method: glucose oxidase kit (Glucose, Roche Diagnostics, Rotkreuz, Switzerland).
157
M. SORIA, ET AL.
Copyright © 2017 John Libbey Eurotext. Downloaded by NYU LANGONE MED CTR SCH OF MED HEALTH SCIENCES LIBRARY on 30/06/2017.
Hormonal analysis Plasma catecholamine measurements. Plasma catecholamine concentrations were determined using high performance liquid chromatography (HPLC). Catecholamines were extracted by selective adsorption onto aluminium oxide (HPLC reagent Kit, Chromsystems, Gräfelfing, Germany) before the HPLC was run. Aluminium oxide was briefly shaken in extraction buffer (50 L) and 1 mL of plasma was added with 50 L of internal standard solution (600 pg dihydroxybenzylamine). The aluminium oxide was then washed three times, with a short period of centrifugation between washes. The catecholamines were extracted by brief shaking with 120 L of elution buffer and then centrifugation (final centrifugation) at 2,000 rpm for 1 min. Next, sample eluent was injected into the HPLC column (Resolve C18, 5-L spherical packing, Waters Corp., Milford, MA, USA) and eluted with a mobile phase. The flow rate was 1 mL/mn at 2,000 psi with a potential of 0.60 V. For each component of interest in the chromatogram, peak areas were determined by computer integration (Baseline 815, Waters Corp., Milford, MA, USA). Measurements of other hormones. The quantification of serum hormones was performed in automated analytical systems. Specifically, chemiluminescent immunoassays were used to measure levels of cortisol (Architec i2000sr, Abbott Laboratories, Abbott Park, IL, USA), calcitonin (Immulite 2000 Xpi, Siemens, New York, NY, USA), PTH (Unicel DxI 800 Access Immunoassay System, Beckman Coulter, Brea, CA, USA), and insulin (Architect ci8200, Abbott Laboratories). Aldosterone levels were determined by colorimetric direct enzyme-linked immunosorbent assay (EiAsy Way Aldosterone Kit; Diagnostic Biochem Canada Inc, Dorchester, Ontario, Canada) in an automated processing system. Serum glucagon was measured by radioimmunoassay (RIA) using a commercial glucagon assay (Euro Diagnostica AB, Malmö, Sweden). Analysis of USG
Urine samples were collected in 10-mL sterile containers and frozen at -80◦ C. Then, the USG was measured by photocolorimetry (Urisys 1800, Roche Diagnostics, Rotkreuz, Switzerland). This method has been found to be valid and reliable for the assessment of hydration status [11]. Partici-
158
pants were classified as euhydrated if their USG was ≤1.020; hypohydrated, for USG values from 1.020 to 1.029; and significantly hypohydrated, for USG≥1.030. These hydration-status groups were based on those most recently established by the American College of Sports Medicine [11]. Statistical analysis The data were expressed in SI units. The distribution of the data was examined using the Shapiro-Wilk’s test and analysed by one-way ANOVA and Pearson’s or Spearman’s correlation analysis. The statistical significance of differences for paired samples was tested using the non-parametric Wilcoxon test. The statistics were generated using IBM SPSS Statistics for Windows software. All values were expressed as means and standard deviations (SDs), and p values
