Introduction

Interindividual tolerance to chronic hypoxaemia is highly varied and may determine survival in the clinical context. Specific mechanisms to explain this heterogeneous response to a sustained reduction in systemic oxygen availability remain unclear. Ascent to high altitude has been suggested as a paradigm for studying human responses to hypoxia in order to probe specific adaptive mechanisms pertinent to critical illness [1]. In combination with exercise, this can provide an effective method of manipulating the balance between oxygen supply and utilisation. In skeletal muscle this equilibrium of tissue oxygen metabolism governs exercise capacity, whilst amongst other organs it is one of the primary determinants of organ function.

The signal from near-infrared spectroscopy (NIRS) can be used to estimate skeletal muscle microcirculatory oxygenation, and principally reflects the venous haemoglobin oxygen status [2, 3]. Skeletal muscle oxygenation decreases during exercise and the magnitude of this response is dependent on exercise intensity [4–6]. This desaturation occurs despite an increase in systemic oxygen flux, local vasodilatation leading to enhanced regional blood flow, and increased tissue oxygen extraction. The sustained reduction in exercise capacity, measured by maximal oxygen consumption (VO2 max), at high altitude is a highly reproducible phenomenon despite adequate acclimatisation [7–10]. The precise mechanism of this persistent decline in exercise capacity remains unclear, but changes in oxygenation of the peripheral circulation detectable by NIRS may help elucidate the phenomenon and further our understanding of the adaptive mechanisms to chronic hypoxaemia in the clinical environment.

We hypothesised that, when measured at altitude, absolute values of skeletal muscle tissue oxygen saturation (StO2) measured by NIRS would be lower than those at sea level (SL) both whilst resting and during exercise. We also hypothesised that the difference in StO2 from rest to maximal exercise would remain unchanged and the rate of desaturation would increase at altitude. We therefore sought to quantify the change in muscle oxygenation using NIRS during exercise on ascent to high altitude.

Methods

Subject selection

Ethical approval for the present study was obtained from the University College London Committee on the Ethics of Non-NHS Human Research, and all participants gave written informed consent. The subjects were 24 healthy volunteers trekking to the base camp of Mount Everest (5,300 m) in spring 2007; six females and 18 males with a mean age of 35.2 years. This group consisted of two predetermined cohorts. All shared an identical ascent profile to 5,300 m; climbers (n = 14) then ascended from 5,300 m to various altitudes, reaching a maximum elevation of 8,848 m, whilst the base-camp team (n = 10) remained at 5,300 m for the duration of the study.

Study settings

Baseline measurements of exercising skeletal muscle StO2 were performed at SL (75 m) before departure to high altitude. Further measurements were taken at 3,500 m (days 4 to 6 of the expedition), on arrival at 5,300 m (5,300 m-a; days 15 to 17) and before departure from 5,300 m (5,300 m-b; days 69 to 71). Excessive exercise at altitude has been associated with an increased risk of acute mountain sickness [11]. Any subject suffering symptoms of acute mountain sickness at altitude was therefore not studied. Other exclusion criteria for cardiopulmonary exercise testing were based on the American Thoracic Society/American College of Chest Physicians guidelines for clinical exercise testing [12]; all subjects with an absolute or relative contraindication as defined by these guidelines were excluded from exercise.

Measurement of skeletal muscle oxygenation

Measurements were made using the InSpectra™ Tissue Spectrometer (Model 325; Hutchinson Technology Inc., Hutchinson, MN, USA) incorporating a 15 mm probe. The spectrometer calculates StO2 by applying algorithms to the NIRS signal reflected from tissue below the probe:

The spectrometer was connected to a laptop computer for the storage of data during exercise. The NIRS probe was placed on the skin of the dominant thigh over the lower third of the vastus lateralis muscle, 10 cm proximal to the knee joint. Once a signal had been confirmed on the spectrometer, the probe was attached firmly with Elastoplast tape. Measurements from the spectrometer were recorded during the predetermined exercise protocol and 60 seconds into the rest period that followed exercise. The InSpectra™ Tissue Spectrometer recorded StO2 every 3 seconds during standard data collection, and a time-point marker was entered into the computer software to ensure synchronisation of exercise and StO2 data.

Exercise protocol

The subjects performed an incremental ramp test to the limit of tolerance using an electromagnetically braked cycle ergometer (Lode Corival; Lode, Groningen, the Netherlands) and a breath-by-breath cardiopulmonary exercise testing system (Metamax 3b; Cortex, Leipzig, Germany). A full calibration of the breath-by-breath system was performed before each test. Prior to the incremental exercise test, subjects warmed up with a low-intensity 30-minute constant work rate protocol. A ramp slope of 20 to 35 W/minute was chosen depending on the sex, age and physical fitness of the subjects in order to obtain a test duration of approximately 10 to 15 minutes [13]. The ramp slope was kept constant for all subjects throughout the study. Resting measurements were recorded for 3 minutes, followed by 3 minutes of unloaded cycling (ULC) and then the incremental ramped exercise protocol.

Measurement of peripheral oxygen saturation

Resting peripheral oxygen saturation (SpO2) was measured using a pulse oximeter (Onyx 9500; Nonin, Plymouth, MN, USA) on the subject's right index finger.

Analysis plan

VO2 max was calculated as the average oxygen consumption for the individual breaths taken in the final 20 seconds of the exercise test. The time for VO2 max reported was the middle time point of the 20-second time interval. Data were plotted on graphs of StO2 versus time and were visually assessed for quality and completeness. A number of NIRS plots were rejected from analysis on the grounds that there was little or no change in signal from rest throughout the exercise protocol. These plots represented nonphysiological data, and other authors have adopted a similar quality control technique [14].

The resting StO2 was calculated as the average of readings during a 30-second rest period before the exercise protocol started. Individual subject StO2 at each principle stage of the exercise protocol (end of 3 minutes ULC, VO2 max and 60 seconds after cessation of exercise) was calculated as the average of three recordings (spanning 9 seconds) around the designated time of occurrence. The mean (confidence interval) of these StO2 values is reported for each altitude along with differences between stage means (ΔStO2). The rate of change in StO2 (StO2 rate) between the end of ULC and VO2 max was calculated by dividing the difference in StO2 between stages by the time taken between stages.

Two-tailed paired t tests were used to assess the affect of altitude on StO2, ΔStO2 and StO2 rate. Comparison between groups containing different individuals was by unpaired t test. Correlation between the rate of decline in StO2 and SpO2 was by Pearson's product-moment coefficient, and that between StO2 and nonparametric data was with Spearman's rank correlation coefficient. P < 0.05 was taken to indicate statistical significance in all instances.

Results

Data collection and quality control

Data collection was incomplete due to a combination of technical difficulties and subjects failing to fulfil the inclusion criteria for exercise testing at altitude. All of the 24 subjects completed the exercise protocol at SL; missing the exercise protocol due to illness were two subjects at 3,500 m, one subject at 5,300 m-a and two subjects at 5,300 m-b. NIRS data were missing due to technical failure for four subjects at SL, for two subjects at 3,500 m and for three subjects at 5,300 m-a. Three subjects (all female) were completely removed from the analysis as part of the quality control screening. NIRS data meeting the predefined quality criteria were available for 18 subjects at SL, for 16 subjects at 3,500 m, for 16 subjects on arrival at 5,300 m and for 16 subjects on departure from 5,300 m. Only six subjects had complete data at each altitude time point, and their individual data can be seen in Figure 1. The data presented are all of the available data at each altitude time point.

Figure 1
figure 1

Individual changes in tissue oxygen saturation during exercise. Individual changes in tissue oxygen saturation (StO2) during exercise in the subjects with complete data at all altitude time points (n = 6). 5300 m-a, on arrival at 5,300 m (days 15 to 17); 5300 m-b, before departure from 5,300 m (days 69 to 71); ULC, unloaded cycling; VO2 max, maximal oxygen consumption; 60 sec-rest, 60 seconds after completion of exercise.

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Changes in absolute tissue oxygen saturation during exercise

The mean StO2 at rest, at the end of ULC, at VO2 max and 60 seconds after VO2 max are presented in Table 1 for each altitude along with the mean weight and resting SpO2. At SL, the mean StO2 increased by 6.7% during ULC, from a resting value of 74.4% (68.6 to 80.1) to 81.1% (73.8 to 88.3) (P = 0.001). The mean StO2 progressively declined during the incremental workload protocol to a nadir of 36.4% (28.5 to 44.3) at VO2 max (P < 0.0001), and then rose rapidly to a level above resting StO2 (82.3% (76.2 to 88.5); P < 0.0001) 60 seconds after exercise had ceased. At altitude the incremental exercise protocol resulted in a reproducible pattern of muscle desaturation. Figure 2 shows this pattern of change in mean StO2 at SL and during ascent to altitude. At 3,500 m and 5,300 m-b, the pattern was identical to SL but absolute values were approximately 15% lower at all stages of the exercise protocol. At 5,300 m-a, the resting StO2 was only 4.2% below that observed at SL (not significant) and the pattern of change was considerably flatter than other altitudes, such that the mean StO2 at VO2 max was 16.1% higher than at SL (P = 0.043). Figures 3 and 4 show typical NIRS plots (change in StO2 during exercise vs. time) from the same subject at SL and at 5,300 m-b, respectively.

Table 1 Weight, SpO2 and StO2 during the exercise protocol at different altitude time points
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Figure 2
figure 2

Tissue oxygen saturation at specific stages of the work protocol. Mean tissue oxygen saturation (StO2) at specific stages of the work protocol at sea level and different altitude time points. 5300(a), on arrival at 5,300 m (days 15 to 17); 5300(b), before departure from 5,300 m (days 69 to 71); Rest, resting; ULC, after 3 minutes of unloaded cycling; VO2 max, at maximal oxygen consumption; Rec, 60 seconds after exercise has ceased.

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Figure 3
figure 3

Near-infrared spectroscopy plot at sea level. Typical near-infrared spectroscopy plot from sea level (75 m). A, start of unloaded cycling; B, start of loaded cycling; C, maximum oxygen consumption.

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Figure 4
figure 4

Near-infrared spectroscopy plot at altitude. Typical near-infrared spectroscopy plot from 5,300 m-b (same subject as Figure 2). A, start of unloaded cycling; B, start of loaded cycling; C, maximum oxygen consumption.

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At 5,300 m-b there were no differences in the absolute StO2 values at any stage of exercise between climbers and the base-camp team, although climbers had a significantly higher SpO2 (86.6% vs. 79.2%, respectively; P = 0.005).

Tissue oxygen saturation differences between exercise stages

The differences between mean values at different StO2 stages of exercise at SL and at altitude are presented in Table 2. There was no difference in ΔStO2 between rest and VO2 max or between ULC and VO2 max at any altitude except 5,300 m-a, where the difference between rest and VO2 max was 17.5%, compared with 38.0% at SL (P = 0.018).

Table 2 Change in StO2 between specific stages of the exercise protocol at different altitude time points
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At 5,300 m-b there was a significant difference in the ΔStO2 value between climbers and the base-camp team from ULC to VO2 max; the base-camp team had a reduction of 62.9%, compared with only 41.0% in the climbers (P = 0.031).

Time-related tissue oxygen saturation gradient and subject rankings

StO2 gradients (StO2 rate values) between exercise stages at each altitude are presented in Table 3. StO2 rate was increased at 5,300 m-b compared with SL (P = 0.008) but not at the other altitude time points.

Table 3 StO2 decline between unloaded cycling and VO2 max during exercise at different altitude time points
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At 5,300 m-b there was a significant difference in StO2 rate between climbers and the base-camp team from ULC to VO2 max; the base-camp team had a desaturation rate of 0.127%/second, compared with only 0.086%/second in the climbers (P = 0.047).

Subjects were numerically ranked at each altitude based on their rate of StO2 decline from the end of ULC to VO2 max.

Rankings were compared at each altitude by Spearman's rank correlation coefficient. There was a significant correlation between the subject rank order at SL when compared with 3,500 m (correlation coefficient = 0.780, P = 0.002), compared with 5,300 m-a (correlation coefficient = 0.688, P = 0.007) and compared with 5,300 m-b (correlation coefficient = 0.41, P = 0.041).

Relationship between tissue oxygen saturation and peripheral oxygen saturation

There was no correlation between the resting SpO2 and the resting StO2 at any altitude. There was a correlation, however, between resting SpO2 and StO2 at VO2 max at 5,300 m-b (r = 0.557, P = 0.019). At 5,300 m-b there was also a negative correlation between the resting SpO2 and StO2 rate (r = -0.562, P = 0.023), such that those individuals with a lower resting SpO2 desaturated more rapidly during exercise.

Discussion

Summary of findings

The present study demonstrated in selected individuals that NIRS could be used to measure skeletal muscle StO2 in the vastus lateralis during exhaustive exercise and to generate reproducible results in the hypoxic environment encountered at high altitude. Three minutes of unloaded exercise at SL led to a small rise in StO2 that progressively declined as the exercise workload increased. StO2 reached a plateau shortly before VO2 max was achieved, and then, following cessation of exercise, rapidly recovered to a level above the resting value.

Figures 3 and 4 show typical examples of the change in StO2 during unloaded and loaded exercise at different altitudes. At 3,500 m, the absolute changes in StO2 were similar to those at SL but values were approximately 15% lower at rest and throughout exercise. On arrival at higher altitude (5,300 m-a), the resting StO2 was similar to that at SL and the changes during exercise (ΔStO2 values) were considerably less marked than those observed at SL, giving the appearance of a flattened pattern of StO2 (Figures 1 and 2). By the end of the expedition (5,300 m-b), days 69 to 71 at altitude, the pattern was similar to that observed at SL and at 3,500 m (that is, unchanged) except that the time-related ΔStO2 decline in StO2 (StO2 rate) during exercise was significantly more rapid than that at SL (P = 0.008).

At altitude, the maximum exercise capacity was reduced-leading to a reduction in VO2 max as has been previously documented [7–10, 15] - and the decline in VO2 max was proportional to elevation.

Study limitations

There were marked interindividual differences in absolute values at rest and during exercise (Table 1). One StO2 reason for these differences is related to the fact that near-infrared light emitted by and received by the spectrometer probe has an unknown pathlength, and therefore the absolute cannot be calculated [16]. Furthermore, significant StO2 heterogeneity in oxygenation has been demonstrated both within and between specific muscles so probe placement on the thigh is important and may have a significant effect upon results [17–19]. StO2 is therefore a value that is specific to the tissue beneath an individual probe. As a result of this, comparison of absolute StO2 values between individuals is of limited value. The relative changes in StO2 as a result of specific stimuli (ΔStO2), and the rate of change in the response (StO2 rate), however, may provide valuable insight into the dynamics of oxygen supply and demand in a subject.

The depth of tissue beneath the spectrometer probe that near-infrared light can penetrate is directly related to the distance between the illumination and detection fibres (optodes) of the device. The banana-shaped light beam has a maximum depth of approximately one-half the distance between the optodes [20]. The InSpectra™ Tissue Spectrometer was fitted with a 15 mm probe, which means the maximum depth of penetration was approximately 7.5 mm. For some individuals this may not represent muscle tissue oxygenation but overlying adipose tissue or skin. This factor may further increase the interindividual variation in the NIRS signal. Most of the NIRS plots that were rejected as a result of minimal change in oxygenation throughout exercise were from females; this has been previously noted as a substantial confounding factor during skeletal muscle NIRS studies [21]. Other authors also report needing to remove subjects from the final analysis as a result of poor signal response due to excessive subcutaneous adipose tissue [14]. Validated objective criteria for rejection of nonphysiological NIRS data do not exist. Subjective removal of data may therefore confound results; however, data removal was performed without the knowledge of the subject or altitude identification.

As the NIRS light beam must pass through skin during both emission and reflection, skin blood flow and oxygenation will contribute to the overall NIRS signal [22, 23]. Ambient laboratory temperature may therefore have exerted an effect on results by inducing vasoconstriction within the skin at low temperature. All studies were performed in purpose-built insulated temporary laboratories where the average temperature throughout the 3-month expedition was 24.1°C at SL, 19.6°C at 3,500 m and 21.5°C at 5,300 m. Diurnal temperature variation may have resulted in temperatures considerably lower than these mean values for those tests performed early in the morning at altitude. The preliminary warm-up exercise protocol, however, should have provided sufficient stimulus to negate the effect of cold-induced vasoconstriction.

Interpretation of results

Although a rarely regarded tissue in the critically ill patient, early reduction in skeletal muscle StO2 detected by NIRS has been shown to herald poor outcome by identifying patients at risk of infectious complications or multiple organ failure [24]. Rather than a quantitative measurement of tissue oxygenation, however, the StO2 value derived from a NIRS signal reflects the localised equilibrium of oxygen delivery and utilisation [25] and closely follows regional venous oxygenation [14, 26]. Furthermore, correlation between changes in the continuous-wave NIRS signal and that of 31P magnetic resonance spectroscopy [25] and of 1H nuclear magnetic resonance [27] suggests that NIRS can be used effectively in the evaluation of localised muscle oxidation. As a measure of oxygen saturation, StO2 could be regarded as a surrogate marker for tissue oxygen content, therefore indicating crude alterations in tissue oxygen extraction. Previous work has demonstrated that acute exposure to hypoxia results in a greater degree of skeletal muscle deoxygenation during exercise when compared with normoxia [28].

The change in absolute StO2 observed in the present study at SL has been previously reported by other authors at SL; typically, there is an initial increase in oxygenation followed by a decline until the minimum plateau value at VO2 max [4, 14, 29]. The commonly observed plateau of StO2 shortly before VO2 max at SL (Figure 3) tended not to be as prominent at 5,300 m-b (Figure 4), although this was difficult to quantify. The significance of this plateau has been suggested as representing the limit of tissue oxygen extraction [14, 28]. The relationship between maximal oxygen extraction and VO2 max may therefore be altered after prolonged exposure to hypoxia and requires more detailed investigation. Similar values for ΔStO2 at SL, 3,500 m and 5,300 m-b suggest that the overall balance between oxygen delivery and utilisation at these altitude time points were similar. Oxygen delivery exceeded utilisation during ULC, resulting in an increase in StO2 - but as the work rate increased the situation was reversed, until the peri-VO2 max plateau when oxygen extraction was maximal and exhaustion was imminent.

The relatively high resting StO2 and reduced ΔStO2 between rest and VO2 max observed at 5,300 m-a (Figures 1 and 2) is not in keeping with the findings at 3,500 m and 5,300 m-b. Identical equipment, protocols and investigators were used on arrival at and departure from 5,300 m, so technical failure or inaccuracy seems unlikely. Limited ability to extract oxygen could account for the reduced ΔStO2 value; however, the reason for this on arrival at 5,300 m is unclear. One hypothesis is that the small gain in weight observed from 3,500 m to 5,300 m may represent a degree of tissue oedema that could increase oxygen diffusion limitation in muscle tissue. The usual response of ascent to altitude is generally weight loss [30]; the gain in weight observed, or certainly the lack of loss, could therefore be tissue fluid accumulation, frequently observed in those affected detrimentally by high altitude [31]. The rate of decline in StO2 during loaded exercise (end of ULC to VO2 max), as presented in Table 3, is significantly more rapid at 5,300 m-b than at SL. This more rapid rate of decline was not seen on arrival at the same altitude 50 days previous to the departure measurements despite the small rise in mean at 5,300 m-b (P = 0.002). Weight loss at altitude SpO2 results in a greater proportion of lean tissue mass than fat mass [30], which leads to a reduction in the skeletal muscle fibre cross-sectional area [32]. In the group investigated in the present study, mean weight loss during the expedition was 5.8 kg in 72 days (P < 0.001).

At 5,300 m-b, those subjects with a lower resting SpO2 showed a more rapid rate of StO2 desaturation during exercise. One explanation for this could be that individuals with a low at rest had reduced systemic oxygen SpO2 availability due either to a blunted hypoxic ventilatory response or to an increased arterial-alveolar oxygen partial pressure difference. Following acclimatisation to high altitude, however, cardiac output remains unchanged from SL values for a given work load throughout exercise [9, 33], and the arterial oxygen content remains above SL values [34, 35] even at maximal exercise [36]. This implies that systemic oxygen delivery remains similar to that experienced at SL under conditions similar to those experienced by the subjects in this study. More distal mechanisms in the oxygen cascade - such as alterations in regional and/or microcirculatory blood flow, changes in the affinity of oxygen to haemoglobin or diffusion limitation within skeletal muscle tissue - may therefore be responsible for the observed findings.

At the end of the expedition (5,300 m-b) the base-camp team who remained at 5,300 m after ascent were found to have a significantly greater ΔStO2 and StO2 rate of decline during exercise than the climbers who ascended above 5,300 m. In the climbing cohort, the exposure to severe hypoxia at altitudes up to 8,848 m is likely to have induced a greater rise in haemoglobin than the base-camp team, thus increasing arterial oxygen content and preventing such a precipitous fall in skeletal muscle oxygenation during exercise. Alternatively, exposure to such hypoxic conditions may trigger a process of hypoxic preconditioning that affords a degree of protection following descent to lower heights.

In the clinical setting, abnormal heterogeneous blood flow in the sublingual microcirculation of critically ill patients [37–39] may account for the imbalance between oxygen delivery and consumption in this pathological state [40]. Reduced sublingual microcirculatory blood flow has been observed in climbers ascending to high altitude [41]. If abnormal blood flow also exists in skeletal muscle microvasculature, one could postulate that it may lead to a reduction in tissue oxygen delivery and hence in StO2.

Conclusion

NIRS is a useful tool for studying thigh skeletal muscle oxygenation during exercise but is limited by the depth of beam penetration in some individuals. The pattern of absolute change in exercising muscle StO2 on exposure to altitude is similar to that at SL and, despite the reduction in exercise capacity, demonstrates a similar reduction in StO2 from rest to maximal exertion. The rate of desaturation is more rapid after prolonged exposure to altitude (69 to 71 days), and at this altitude time point a lower resting SpO2 was associated with a more rapid rate of StO2 decline during exercise. Exposure to extreme hypoxia above 5,300 m appears to have a protective effect in reducing the degree of muscle desaturation during exercise on return to 5,300 m.

These findings suggest that mechanisms within the peripheral circulation or tissues govern local tissue oxygen flux and utilisation. Alterations at the distal portion of the oxygen cascade may be an important component of adaptation or maladaptation to chronic hypoxia secondary to high altitude exposure and disease. The heterogeneity of individual responses to chronic hypoxia may therefore be explained by changes in the peripheral circulation rather than the systemic circulation in both scenarios.