Subjects
The subjects for this study were eight junior national level competitive young cyclists in Norway. The group included one female and seven male athletes (age: 17 ± 1 year, height: 180 ± 5 cm, weight: 70.6 ± 7.8 kg). Subjects gave their informed consent after obtaining all the information they requested concerning this study. To avoid acclimatization in hypoxic condition, the subjects lived at sea level and trained under normoxic and simulated hypoxic conditions. It is reported that some competitive athletes have a serum ferritin level that is suggestive of reduced bone marrow stores [17, 18]. When such athletes attempt hypoxic training, they often do not thrive, and clearly do not increase erythrocyte volume or
O2max. Therefore, all the subjects took iron supplement during the study. The subjects took liquid iron supplementation during the study.
Training Protocol
The subjects were randomly assigned to two training groups. Group A (n = 4) trained under normoxic condition (NC; 21% O2) for 2 hours/day, 3 days/week for 3 weeks while Group B (n = 4) used the same training protocol under hypoxic condition (HC; 15% O2). After each training period, five weeks of self-training under usual field conditions was implemented as a washout period. After the self-training, training conditions were switched from NC to HC in Group A, from HC to NC in Group B (Fig. 1). The work intensity during training was set to be at lactate threshold (LT) under each NC and HC. During each training session, heart rate (HR) was recorded to monitor the subjects' exercise stress (Polar CIC, Port Washington, NY). Also, plasma lactate ([La]) was analyzed by an enzymatic method (KDK lactate pro analyzer, Japan) during each training. Arterial oxygen saturation was measured by pulseoximetry (MicrO2, Siemens, Germany).
Hypoxic condition
We simulated HC in the training room by replacing O2 gas in the room air with N2 gas. HT was performed in the room at ambient fractional O2 concentration of 15 % (corresponding to an altitude of approximately 2500 m). O2 concentration in the room was maintained by the system's feed back mechanism which activated a fan that brought in fresh air from the outside whenever the O2 concentration in the room was lower than the O2 set point in the system. CO2 gas in the room was also monitored and the CO2 built up in the room was reduced by using CO2 scrubber. Several air conditioning systems ensured that there were no changes in room temperature and humidity during the training session. The complete set up for simulating normobaric hypoxia in the room was evaluated and approved for use with humans by a Norwegian governmental safety organization.
Experimental protocol
Bicycle exercise test was performed at sea level within one week before and after each training period for evaluating training effects. Subjects had a brachial arterial line and then rested for one hour prior to exercise. After the resting blood samples were taken, subjects moved on to the stationary bike (Lode, Netherlands).
Measurements were carried out on two types of incremental bicycle exercise tests. Before the submaximal test (Submaximal), 50 W warming up was performed for 10–15 min. Submaximal was started just below LT (stage 1), which was determined prior to the test. The workload (~25 W) increased every 5 min to stage 5 for determination of
O2 at LT (
O2@LT). More than 15 min after the end of Submaximal,
O2max protocol (Vmax) was carried out to determine
O2max. Vmax test also started just below LT, and ~25 W was added every 30 sec until voluntary exhaustion. After each exercise protocol, subjects continued to cycle at 50 W in order to avoid the health risks due to sudden cessation of exercise.
Expired gas was collected into Douglas bags. Gas fractions from the Douglas bag were measured by mass spectrometer (Ametec process & analytic instruments Division, USA). [La] was analyzed from arterial blood samples immediately following each exercise stage. A heart rate monitor was fitted around the chest of each subject for the biotelemetry of the heart rate. Arterial oxygen saturation was measured by pulseoximetry. Muscle oxygenation of vastus lateralis (VL) was monitored using NIRcws. The muscle oxygenation was measured after each training period but not before each training period.
Hematology assessments
Plasma volume was measured by using the Evans blue dye indicator-dilution technique. After the subject rested in a supine position for 30 min, a baseline blood sample was drawn and a known quantity of Evans blue dye was injected. Venous blood was drawn 10, 20 and 30 min after injection of the dye. Samples were spun and measurements of absorbance were taken at 620 and 740 nm by spectrophotometry (UV-1202, Shimadzu, Japan). Hematocrit was measured by averaging five runs on a Sysmex K-1000 total blood analyzer (TOA Medical Electronics, Japan). Blood volume was estimated by dividing plasma volume using one minus hematocrit; appropriate corrections were used for trapped plasma and peripheral sampling. Total red cell volume was defined as blood volume minus plasma volume. Hb concentration was measured from a 3 ml sample of venous blood in an EDTA-vacutainer, and it was measured with the same analyzer as hematocrit. Ferritin was measured on plasma samples taken before and after each training period by an immuno turbidimetric method (Modular P, Roche, Swiss).
NIRcws
Oxygenation of the vastus lateralis muscle was measured by NIRcws (HEO-100, OMRON, Japan). The basic principle of this NIRcws device has been discussed in detail in previous studies [9, 11, 13]. The NIRcws probe contains a light source and an optical detector, with a distance of 3.0 cm between the light source and the detector. Thus, the depth of penetration is evaluated to be ~1.5 cm, as extensively discussed previously [14, 15]. A pair of two-wavelength light emitting diodes, with wavelength of 760 and 850 nm, were used as the light source. A silicon photodiode was used as the photodetector. In this study, oxygenated Hb and/or Mb changes (ΔOxy-Hb/Mb) and total Hb changes (ΔTotal-Hb) were calculated using the algorithm reported previously [13]. Also, deoxygenated Hb/Mb (Δ[Hb/Mb]), and muscle tissue deoxygenation (Δ[Deoxy]) were calculated by Δ[Total-Hb] - Δ[Oxy-Hb/Mb] and Δ[Hb/Mb] - Δ[Oxy-Hb/Mb], respectively.
In NIRcws measurements, the absolute O2 concentration or saturation determination is difficult because of unquantifiable biophysical quantities such as optical path length [12]. In muscle oxygenation measurement, a subctaneous fat layer greatly affects the detected light intensity [9]. As the fat layer thickness varies greatly in humans, optical densities of Δ[Total-Hb] and Δ[Oxy-Hb/Mb] cannot be compared between individuals. In this study, therefore, relative deoxygenation change at each stage during Submaximal (%d [Deoxy]) was normalized by the full deoxygenation during Vmax at each test. The %d [Deoxy] was evaluated during the last 30 sec of each stage. The muscle oxygenation recovery was taken from the maximal deoxygenation measured over the last few seconds of exercise and the minimum deoxygenation measured in the overshoot recovery. Half time reoxygenation (T1/2), the time to reach a value of half-maximal recovery, was determined after Submaximal. The probe was firmly attached to the skin overlying the lower one-third of VL muscle (~12 cm from the top of patella). No sliding was observed in any subjects.
Since Mb has similar absorption spectra to Hb, NIRS signal gives mixed information of both Hb and Mb. However, it is reported that Mb concentration is no greater than 25% [11] or 20% [16]. Therefore, we can conclude that the signals are derived mainly from Hb. The specific probe position was recorded as the distance from the top of the patella in the first experiment in December, and the probe was placed at exactly the same location for each test after December.
Statistics
The results are presented as mean value ± standard error (SE). The hematological assessments and measurements parameters during Vmax test were statistically analyzed with one-way ANOVA. The HR, [La] and %d [Deoxy] at Submaximal were compared by two-way ANOVA (time and training). The monitored parameters during each training and T1/2 after Submaximal were compared by paired t-test. Statistical significance was set at p < 0.05 for all comparisons.