The effect of 4 weeks fixed and mixed intermittent hypoxic training (IHT) on respiratory metabolic and acid-base response of capillary blood during submaximal bicycle exercise in male elite taekwondo players

[Purpose] The purpose of our study was to determine the effectiveness of 4 weeks fixed and mixed intermittent hypoxic training (IHT) and its difference from exercise training at sea-level on exercise load, respiratory metabolic and acid-base response of capillary blood during 80% maximal heart rate (HRmax) bicycle exercise in male elite Taekwondo players. [Methods] Male elite Taekwondo players (n = 25 out of 33) were randomly assigned to training at sea-level (n = 8, control group), training at 16.5%O2 (2000 m) simulated hypoxic condition (n = 9, fixed IHT group), and training at 14.5%O2 (3000 m) up to 2 weeks and 16.5%O2 (2000 m) simulated hypoxic condition (n = 8, mixed IHT group) for 3 weeks. We compared their average exercise load, respiratory metabolic, and acid-base response of the capillary blood during 80% HRmax submaximal bicycle exercise before and after 4 weeks training. [Results] Fixed and mixed IHT groups showed positive improvement in respiratory metabolic and acid-base response of the capillary blood during 80% HRmax submaximal bicycle exercise after 4 weeks training. However, all dependent variables showed no significant difference between fixed IHT and mix IHT. [Conclusion] Results suggested that mixed and fixed IHT is effective in improving respiratory metabolic and acid-base response of capillary blood in male elite Taekwondo players. Thus, IHT could be a novel and effective method for improving exercise performance through respiratory metabolic and acid-base response.


INTRODUCTION
Since the 1968 Olympic Games were held in Mexico City at 2240 m high altitude, altitude/hypoxic training has been studied for exercise performance in elite athletes 29 . Currently, this training is commonly applied to athletes and coaches with consequent improvement in exercise performance in individual and team sports 1, 40 . Erythropoiesis and exercise performance at sea-level can be improved by 2 methods through the living and training at altitude and hypoxic environments 11, 40 . First, living high training high (LHTH) was designed as way of living and training at 1,500-4,000 m high altitude environments to enhance red blood cell (RBC) count, hemoglobin (Hb) concentration, maximal oxygen consumption (VO2max), and exercise performance at sea-level. In particular, LHTH is the norm for enhanced performance in countries with natural altitude environments, including Kenya and Ethiopia 29 . Second, living high training low (LHTL) involves simultaneous beneficial effects of altitude/hypoxic acclimation (e.g., increased RBC count and Hb concentration) and sea-level training (i.e., maintenance of training intensity). In addition, LHTL has shown efficacy for enhancing athletic performance and records, resulting in positive hematological, metabolic, and neuromuscular adaptations 2, 27 . Elite athletes use living high at 2,000-3,000 m and simultaneously training low below 1,500 m 35, 39 . However, LHTH and LHTL methods require a relatively long time (> 12 h/day and 2 weeks) in a hypoxic dose 41 . Also, LHTH is conducted in high altitude areas such as Chamonix in France, Albuquerque in United States, and Kunming in China; and LHTL is conducted with living at very expensive hypoxic accommodations and hotels 3, 22, 29 . Also, such domestic and foreign places and facilities are inadequate 27, 29 .
In recent years, intermittent hypoxic training (IHT), a new training method at altitude/hypoxic environment, is gaining popularity. IHT involves living at sea-level and training (< 4 h/day, 2-5 times/week, and 2-4 weeks) using relatively inexpensive altitude/hypoxic environment device such as hypoxic mask and hypoxic exercise room. IHT The effect of 4 weeks fixed and mixed intermittent hypoxic training (IHT) on respiratory metabolic and acid-base response of capillary blood during submaximal bicycle exercise in male elite taekwondo players [Purpose] The purpose of our study was to determine the effectiveness of 4 weeks fixed and mixed intermittent hypoxic training (IHT) and its difference from exercise training at sea-level on exercise load, respiratory metabolic and acid-base response of capillary blood during 80% maximal heart rate (HRmax) bicycle exercise in male elite Taekwondo players.
[Methods] Male elite Taekwondo players (n = 25 out of 33) were randomly assigned to training at sea-level (n = 8, control group), training at 16.5%O2 (2000 m) simulated hypoxic condition (n = 9, fixed IHT group), and training at 14.5%O2 (3000 m) up to 2 weeks and 16.5%O2 (2000 m) simulated hypoxic condition (n = 8, mixed IHT group) for 3 weeks. We compared their average exercise load, respiratory metabolic, and acid-base response of the capillary blood during 80% HRmax submaximal bicycle exercise before and after 4 weeks training.
[Results] Fixed and mixed IHT groups showed positive improvement in respiratory metabolic and acid-base response of the capillary blood during 80% HRmax submaximal bicycle exercise after 4 weeks training. However, all dependent variables showed no significant difference between fixed IHT and mix IHT.
[Conclusion] Results suggested that mixed and fixed IHT is effective in improving respiratory metabolic and acid-base response of capillary blood in male elite Taekwondo players. Thus, IHT could be a novel and effective method for improving exercise performance through respiratory metabolic and acid-base response.

Journal of Exercise Nutrition & Biochemistry
does not improve oxygen transporting capacity of the blood by increased erythropoiesis because of exposure to relatively short interval 1, 30 . However, IHT reportedly may enhance exercise performance by stimulating an increase in glycolysis enzyme activity, glucose transporting capacity, acid-base balance capacity, skeletal muscle mitochondrial density, capillary-to-fiber ratio, and fiber cross-sectional area via upregulation of hypoxia-inducible factor 1α (HIF-1α) 7, 19, 32, 34, 38 . Therefore, IHT has an advantage of eliciting an enhanced exercise performance by positive physiological change though additional hypoxic stimulation in athletes who cannot attend altitude/hypoxic training during the season 14 . Levine 20 and Wilber 40 reported that IHT is more efficient in increasing exercise performance by reducing fatigue; it should be performed after reducing the hypoxic stimulation or exercise intensity at second half of training before post-test.
Besides, on exercise at high altitude and hypoxic environments, PO2 of the arterial blood decreases and this leads to metabolic acidosis through increased ATP synthesis by anaerobic metabolic process, increase of hydrogen ion, and decrease of pH 10, 15, 21 . In high intensity exercise, skeletal muscle fatigue is caused due to increase of hydrogen ion by response of CO2 and HCO3 -, and damage to actin-myosin cross-bridge cycling by anaerobic glycolysis and accumulation of blood lactate, and lowering the sensitivity of calcium ion to troponin 15, 25, 26, 36 . Thus, it is important to review the pre and post changes in respiratory metabolic and acid-base response of the blood during submaximal exercise to explain the improved exercise performance. However, previous domestic and foreign studies on the effectiveness of IHT on acid-base response of the blood during submaximal exercise are inadequate.
Therefore, the purpose of our study was to determine the effectiveness of 4 weeks fixed and mixed IHT and its difference from exercise training at sea-level on exercise load, respiratory metabolic and acid-base response of the capillary blood during 80% HRmax bicycle exercise in male elite Taekwondo players.

Participants
Our study included 33 male elite Taekwondo players who did not participate in any exercise and training program at hypobaric hypoxia and normobaric hypoxia environments in the previous 6 months. The participants were non-smokers, and without history of musculoskeletal, cardiovascular, or pulmonary disease. The participants received information about the purpose, process and, possible adverse effects and provided written consent prior to the start of the study. Participants were randomly assigned to training at sea-level during 4 weeks (n=8, control group), training at 16.5%O2 (2000 m) simulated hypoxic condition during 4 weeks (n = 9, fixed IHT group), and training at 14.5%O2 (3000 m) up to 2 weeks and 16.5%O2 (2000 m) simulated hypoxic condition (n = 8, mixed IHT group) for 3 weeks. Twenty-five of the participant completed the study (>95% compliance) and were subjected to analyses. Data from the remaining 5 subjects were discarded due to medication, withdrawal, and noncompliance. There were no significant differences in physical characteristics among groups before training ( Table 1). All procedures were in accordance with the ethical standards of the responsible committee on human experimentation and with the Helsinki Declaration.

Experimental design
Twenty-five male elite Taekwondo players in control group (n = 8), fixed IHT group (n = 9), and mixed IHT group (n = 8) performed training session corresponding to 80% HRmax at each environment during 4 weeks. Training session consisted of treadmill (30 min) and bicycle (30 min) exercise at a total duration of 1 h, 3 days per week, for 4 weeks. The exercise intensity set to 80% HRmax can lead to physiological changes associated with exercise performance and improved time trial in exercise training at hypoxic condition 22 . HRmax was determined using the predicted HRmax formula (Male = 206 -0.69 age) 24 . We designed a study to measure the effectiveness of IHT group vs. control group. Therefore, at pre and post, we analyzed average exercise load, respiratory metabolic (minute ventilation; VE, oxygen consumption; VO2, carbon dioxide excretion; VCO2, respiratory exchange rate; RER, lactate blood level) and acid-base response of the capillary blood (oxygen saturation; SCO2, oxygen partial pressure; PO2, carbon dioxide partial pressure; PCO2, potential of hydrogen; pH, bicarbonate ion; HCO3 -) during 80% HRmax submaximal bicycle exercise (5, 10, 15, and 30 min).
A nitrogen generator (Separation & Filter Energy Technology Cooperation, Korea) was used to produce normobaric hypoxic environments. The various normobaric hypoxic conditions were simulated by introducing nitrogen into the environmental chamber (width 6.5 m × length 7.5 m × high 3 m), using a nitrogen generator with the capacity to simulate normobaric hypoxic conditions for altitudes of up to 6000 m. The temperature within the environmental chamber was maintained at 20 ± 2oC and the humidity was maintained at 60 ± 2% for all conditions.

Body composition
All participants fasted overnight prior to measurement of body composition (i.e., height, weight, free fat mass, and body fat percentage). They wore lightweight clothing and were asked to remove any metal items. An X-SCAN PLUS (Jawon medical, Korea) was used to measure height and body composition.
Respiratory metabolic response Respiratory metabolic response measurements were performed at 5 min, 10 min, 20 min, and 30 min over the duration of the exercise protocol. VE, VO2, VCO2, and RER were measured with the Vmax-229 breath-bybreath auto metabolism analyzer (SensorMedics, USA), the Combi 75XLⅡ(Combi, Japan) and breathing valve in the facemask form. For the blood lactate level, 80 μL blood was collected in a capillary tube using the fingertip method, and the sample was analyzed using the YSI-1500 lactate analyzer (YSI Inc., USA).
Acid-base response of the capillary blood Acid-base response measurements were performed at 5 min, 10 min, 20 min, and 30 min over the duration of the exercise protocol. SCO2, PO2, PCO2, pH, and HCO3were measured by using the Gem Premier 3000 blood gas analyzer (Radiometer, USA). For the analysis of acid-base response, we collected blood samples by finger-tip method in the capillary vessel. Acid-base response of the capillary blood is similar to the results of the arterial blood and guarantee the reliability of results 36, 42 ; also, it is an easier sampling method, as compared to arterial blood 6 . We collected blood samples by finger-tip method after heating the capillary vessel to 43 o Celsius using electric pad and massage for influx of arterial blood, based on previous reports on heating the blood collection region and using stimulants such as irritant cream, glyceryl trinitrate paste, and nicotinate paste 8, 9, 18, 31 . Immediately after blood sampling, change of acid-base response was evaluated within 10 s of closing front and back of the collection tube to prevent reaction of blood sample with the gases in the atmosphere.

Statistical analysis
All data were presented as means ± standard deviations. Two-way repeated analysis of covariance (ANCO-VA) was used to determine the interaction and main effects between times and groups during exercise. Post-hoc test between times in each group, and paired t-test and post hoc test between groups at each time was for LSD (least significant difference) was conducted. All statistical analyses were performed using SPSS version 22.0 (IBM Corp., Armonk, NY) for Windows. The level of significance was set at p < .05.

Change in bicycle exercise load
Bicycle exercise load to submaximal exercise at pre and post was presented in Table 2. No significant interaction and main effect was detected, and all groups showed a similar increasing rate (control group = 11.9%, fixed IHT group = 11.4%, mixed IHT group = 11.8%) in bicycle exercise load.

Change in respiratory metabolic response
Respiratory metabolic response variables measured during submaximal exercise throughout the training were shown in Table 3-7. VE presented significant main effect within time and significant increase in control group at 10 min exercise. Overall, VE showed lower tendency of increase rate in fixed and mixed IHT groups, as compared with control group (Table 3). VO2 presented significant main effect within time at 10, 20, and 30 min exercise. Post-hoc analyses showed significantly increased VO2 in all groups at 10 and 30 min exercise and significant increase in control group and fixed IHT group at 20 min exercise  (Table 4). VCO2 presented significant interaction between condition and time at 5, 10, and 20 min exercise and significant main effect within time at 10, 20, and 30 min exercise.
The results of post-hoc were as follows. At 5 min exercise, fixed IHT group showed significantly lower value at post, as compared with control group. At 10 min exercise, VCO2 showed significant increase in control group and significantly higher value in control group, as compared with fixed and mixed IHT group at post. At 20 min exercise, VCO2 showed significant increase in control group and fixed IHT group, and significantly lower value in mixed IHT group, as compared with control group and fixed IHT group at post. At 30 min exercise, there was significant increase in control group and fixed IHT group (Table 5). RER presented significant main effect within time at all exercise times. By posthoc analyses, control group showed significant increase at 30 min exercise and fixed IHT group showed significant increase at 5 min exercise (Table 6). Blood lactate level  (Table 7).

Change in acid-base response of the capillary blood
Acid-base response variables measured during submaximal exercise throughout the training were shown in Table 8-12. All acid-base response variables (SCO2, PO2, PCO2, pH, HCO3 -) showed no significant interaction effect. SCO2 presented significant main effect within time at 20 and 30 min exercise. By post-hoc analyses, SCO2 showed significant decrease in control group at 20 and 30 min exercise (Table 8). PO2 presented significant main effect within time at 10, 20, and 30 min exercise. In addition, PO2 showed significant decrease in control group at 10 and 30 min exercise (Table 9). PCO2 presented significant main effect within time at 10 min exercise,  (Table 10). No significant interaction and main effect in pH was detected (Table 11). HCO3presented significant main effect within time at 10 min exercise. Post-hoc analyses indicated significant decrease in HCO3in control group at 10 min exercise (Table 12).

DISCUSSION
Exercise at high altitude and hypoxic environments, results in a decrease in PO2 of the arterial blood, which leads to metabolic acidosis through increase of ATP synthesis by anaerobic metabolic process, increase of hydrogen ion, and decrease of pH 10, 15, 21 . High intensity exercise causes skeletal muscle fatigue due to hydrogen ion increase by CO2 and HCO3response, damage to The results of our study indicated that bicycle exercise load to submaximal exercise showed a similar tendency toward increase in all groups (control group = 11.9%, fixed IHT group = 11.4%, mixed IHT group = 11.8%), with no difference between groups at pre and post. In respiratory metabolic response, fixed and mixed IHT groups presented significantly lower VCO2 at post, lower tendency for increase in VE and VO2, and higher tendency for decrease in blood lactate level, as compared with control group. In acid-base response of the blood, fixed and mixed IHT groups presented lower tendency for decrease in SCO2, PO2, and HCO3 -, as compared with control group. However, PCO2 and pH showed no difference between groups.
Generally, IHT increases glycolysis enzyme activity, glucose transporting capacity, mitochondria density, capillary density, cross section area of skeletal muscle, and activity of the motor unit by stimulating the neuromuscular system. These positive changes improve the effectiveness of oxygen utilization 12, 23, 32-34, 38 . Also, exercise performance is enhanced by improvement of inflow rate in oxygen to skeletal muscle tissue and oxygen utilization capacity in mitochondria 13, 28 . In our study, fixed and mixed IHT groups showed positive results in VE, VO2, VCO2, blood lactate level, PO2, SCO2, and HCO3 -, as compared with control group despite same increase rate on bicycle exercise load at 80% HRmax through training in all groups. Especially, positive changes of VO2 and SCO2 during exercise of the same load in our study were consistent with results of previous studies that IHT improves exercise economy such as oxygen utilization capacity in skeletal muscle tissue 12, 16, 28, 30 . It is likely that enhanced exercise economy through IHT improves PO2, blood lactate level, and HCO3by increased aerobic energy metabolic rate and consolidated tolerance and removal capacity to fatigue-causing substance during anaerobic energy metabolism 26, 28, 30 . Also, improvement of VE and HCO3in mixed and fixed IHT groups was possibly caused by inhibition of sympathetic nervous system, hyperactivity of parasympathetic nervous system, and adaptation of chemical receptors (carotid bodies and aortic bodies) in the central and peripheral nerve through continuous stimulation of hypoxic ventilatory response 4, 28 .
We verified the effect of IHT on respiratory metabolic and acid-base response of the blood as well as efficiency difference between mixed IHT (training at 16.5%O2-2000 m simulated hypoxic condition during 4 weeks) and fixed IHT (training at 14.5%O2-3000 m up to 2 weeks and 16.5%O2-2000 m simulated hypoxic condition for 3 weeks). Our study was based on previous reports that the effect of IHT is greatly influenced by hypoxic environment condition and training intensity, and IHT should be performed after reducing hypoxic stimulation or exercise intensity at second half of training before post-test to reduce fatigue. However, no significant difference in all dependent variables occurred between fixed IHT group and mixed IHT group. These results are possibly due to mixed IHT group training at the more severe hypoxic condition (fixed IHT group = 2000 m vs mixed IHT = 3000 m) up to 2 weeks and training under the same hypoxic condition from 3 weeks to 4 weeks. Thus, it is likely that IHT group did not perform training after reduced hypoxic conditions or exercise intensity at second half of training before post-test to reduce fatigue, as compared with the control group.

CONCLUSION
Our results suggested that mixed and fixed IHT is effective in improving respiratory metabolic and acid-base response of the capillary blood in male elite Taekwondo players. Thus, IHT can be considered a novel and effective method for improving exercise performance through cardiopulmonary and acid-base response. However, no significant difference in all dependent variables was detected between fixed IHT group and mixed IHT group.