Human skeletal muscle intracellular oxygenation: the impact of ambient oxygen availability
1 Department of Medicine, University of California, San Diego, La Jolla, CA 90293-0623, USA
2 Norwegian University of Science and Technology, Faculty of Medicine NO-7489 Trondheim, Norway
3 NMR Laboratory, AFM and CEA, IFR 14, Institut de Myologie, Pitié-Salpêtrière University Hospital, Paris, France
Abstract
Intracellular oxygen (O2) availability and the impact of ambient hypoxia have far reaching ramifications in terms of cell signalling and homeostasis; however, in vivo cellular oxygenation has been an elusive variable to assess. Within skeletal muscle the extent to which myoglobin desaturates (deoxy-Mb) and the extent of this desaturation in relation to O2 availability provide an endogenous probe for intracellular O2 partial pressure (PiO2). By combining proton nuclear magnetic resonance spectroscopy (1H NMRS) at a high field strength (4 T), assessing a large muscle volume in a highly efficient coil, and extended signal averaging (30 min) we assessed the level of skeletal muscle deoxy-Mb in 10 healthy men (30 ± 4 years) at rest in both normoxia and hypoxia (10% O2). In normoxia there was an average deoxy-Mb signal of 9 ± 1%, which, when converted to PiO2 using an O2/Mb half-saturation (P50) of 3.2 mmHg, revealed an PiO2 of 34 ± 6 mmHg. In ambient hypoxia the deoxy-Mb signal rose to 13 ± 3% (PiO2= 23 ± 6 mmHg). However, intersubject variation in the defence of arterial oxygenation (SaO2) in hypoxia (SaO2 range: 86–67%) revealed a significant relationship between the changes in SaO2 and PiO2(r2= 0.5). These data are the first to document resting intracellular oxygenation in human skeletal muscle, highlighting the relatively high PiO2 values that contrast markedly with those previously recorded during exercise (2–5 mmHg). Additionally, the impact of ambient hypoxia on PiO2 and the relationship between changes in SaO2 and PiO2 stress the importance of the O2 cascade from air to cell that ultimately effects O2 availability and O2 sensing at the cellular level.
, 百拇医药
Introduction
Changes in intracellular oxygen availability have far reaching consequences likely involved in such diverse processes as angiogenesis (Richardson et al. 1999c; Wagner, 2001) and hypoxic pulmonary vasoconstriction (Wang et al. 2005; Wolin et al. 2005). However, data directly assessing intracellular oxygenation are sparse, especially in humans in vivo. Several very different methods have been employed in an attempt to assess local O2 availability including the use of PO2 electrodes (Hofer et al. 1992), O2 phosphorescence quenching (Rumsey et al. 1989), near infrared spectroscopy (Marcinek et al. 2003; Miura et al. 2004), and proton nuclear magnetic resonance spectroscopy (1H NMRS) of myoglobin (Mb) (Richardson et al. 1995b). Unlike the other methods which cause significant tissue damage or raise questions regarding the compartment of measurement, 1H NMRS of Mb is non-invasive and is clearly limited to muscle tissue, the only site in which this endogenous intracellular marker of oxygenation is found. Thus, this NMRS approach is ideally suited for in vivo human investigations.
, 百拇医药
The measurement of deoxy-Mb by NMRS to determine skeletal muscle oxygenation has been utilized in several studies (Richardson et al. 1995b, 1998a, 2001, 2002; Mole et al. 1999; Vanderthommen et al. 2003). However, until now the assessment of deoxy-Mb has been limited to exercise where, due to falling PiO2, there is sufficient signal visible even with a relatively low signal-to-noise ratio. Previously, the lack of a resting deoxy-Mb signal prior to muscular contraction or supra-systolic cuff occlusion has been interpreted as indicative of a relatively high PiO2 in resting skeletal muscle (Richardson et al. 1995b, 2001; Mole et al. 1999; Chung et al. 2005). Therefore, although it is known that muscle PiO2 falls to very low values of 2–5 mmHg during exercising (Mole et al. 1999; Richardson et al. 2001), the starting point for skeletal muscle oxygenation or resting PiO2 is, as of yet, unknown.
, http://www.100md.com
Hypoxia is both an important stimulus and a constant threat to the human body and its vital organs throughout life. Environmental changes such as exposure to high altitude reduce ambient O2 availability, while lung, vascular, and sleep disorders can result in hypoxia under normoxic conditions. It is known that hypoxia mediates adaptive changes in metabolism, O2 sensing and gene expression. However, although much research has examined the consequences of experimental hypoxic conditions, data documenting hypoxically mediated changes in cellular oxygenation in humans are sparse, if not non-existent.
, 百拇医药
Consequently, we performed 1H NMRS at a high field strength (4 Tesla (T)) with a large volume coil and extended signal averaging (30 min) to assess resting O2 saturation (deoxy-Mb) in muscles of the lower leg in both normoxia and hypoxia (10% O2). This study had three specific goals: (1) to measure the resting intracellular oxygenation level and calculate the PiO2 of human skeletal muscle in vivo, (2) to test the hypothesis that ambient hypoxia will significantly reduce the level of cellular oxygenation and hence lower PiO2 in human skeletal muscle, and (3) to assimilate these findings into the current understanding of skeletal muscle intracellular oxygenation and PiO2 during exercise.
, 百拇医药
Methods
Subjects
The protocol received unrestricted approval from the Pitié-Salpêtrière University Hospital Ethics Committee (Paris, France) and all procedures conformed to the standards set by the Declaration of Helsinki. The 8 male and 2 female subjects who volunteered to participate in the study gave written informed consent. Subjects were not recruited with regard to exercise habits and were therefore of varying activity levels. Mean age, height and body weight were 30 ± 4 years, 174 ± 3 cm, and 69 ± 3 kg, respectively.
, http://www.100md.com
Experimental protocol and set-up
The experimental protocol was focused on resting muscle oxygenation in the muscles of the lower leg under conditions of normoxia and hypoxia (10% O2). The studies were carried out in a 4 T, 46 cm internal bore, superconducting magnet (Magnex 4/60) interfaced to a Bruker Biospec NMR spectrometer. Before the experiments, all subjects were familiarized with the experimental set-up and were accustomed to lying supine in the magnet. The calf of the subject's dominant leg was carefully positioned inside a 17 cm inner diameter transversal electromagnetic (TEM) 1H transmit-and-receive volume coil. An air-cuff was wrapped above the knee of this leg and inflated to 240 mmHg for 10 min to achieve complete vascular occlusion and determine the maximum deoxy-Mb signal in the lower leg. Throughout the normoxic and hypoxic experiments subjects breathed through a low resistance two-way breathing valve (2700; Hans-Rudolph Inc., Kansas City, MO, USA) connected to either room air or a 150 l reservoir containing the hypoxic gas. Heart rate and arterial blood O2 saturation were monitored continuously using a NMR-compatible patient-monitoring device (Maglife, Bruker, Wissembourg, France) and arterial PO2 (PaO2 was calculated from blood O2 saturation using a standard O2 dissociation curve. The importance of remaining still throughout the experiments was explained to all subjects and there was good compliance.
, 百拇医药
1H NMRS of deoxy-Mb
Within the 4 T magnet the n– proton of the proximal histidine F8 of myoglobin in the deoxygenated state was selectively excited by an 0.8 ms Gaussian pulse (1024 accumulations, 256 complex points, spectral width 200 Hz, TR time 20 ms). The B0 field homogeneity was optimized with Fastmap, an automatic localized 1st and 2nd order shim procedure (Gruetter, 1993). The other necessary adjustments and the acquisition of reference images and spectra in resting conditions took about 20 min.
, 百拇医药
NMRS data processing
The deoxy-Mb spectra were processed with standard ParaVision and XWIN NMRS software. After a 100 Hz line-broadening exponential multiplication and Fourier transformation, zero and 1st order phases were applied to the first deoxy-Mb spectrum of each series and all others were processed using these same parameter settings. Automatic baseline correction (± 20 p.p.m.) was applied, and then the Mb peak of each spectrum was quantified by integration over the same p.p.m. range of that subject's deoxy-Mb signal (10 p.p.m.) attained at minutes 8–10 of ischaemia.
, http://www.100md.com
Calculation of PiO2
The plateaued deoxy-Mb signals obtained during the 8th and 10th minutes of cuff occlusion (240 mmHg) represent the complete deoxygenation of Mb (Wang et al. 1990). The fractional deoxy-Mb (Fdeoxy-Mb) was determined by normalizing the signal areas to the average signal obtained during the last minutes of cuff ischaemia. The conversion from Fdeoxy-Mb to PO2 values was then calculated from the oxygen-binding curve for Mb:
, 百拇医药
Where FMbO2 is the fraction of Mb that is oxygenated and P50 is the O2 pressure where 50% of the Mb binding sites are bound with O2. The temperature-dependent Mb half-saturation (P50) of 3.2 mmHg was used (Rossi-Fanelli & Antonini, 1958).
Calculation of diffusional conductance (DmO2) and mean capillary PO2 (PcapO2)
A Bohr integration technique was used to calculate an estimate of PcapO2 and DmO2 at rest. Mean capillary PO2 was calculated as previously described (Wagner, 1992) as the numerical average of the arterial PO2 values assessed by arterial Hb saturation and prior measures of femoral venous PO2 at rest (Agusti et al. 1994), computed equally spaced in time along the capillary as it traverses the muscle bed from arterial to venous end. This technique has been discussed in detail elsewhere (Roca et al. 1989; Hogan et al. 1991a) based on a proposal by Wagner (1988) with the assumption of a homogeneously perfused muscle. Briefly, the drop in PO2 along a capillary is calculated using Fick's law of diffusion (eqn (1)).
, http://www.100md.com
(1)
Where PiO2 is assessed by deoxy-Mb levels. DmO2 is an expression of all phenomena that facilitate O2 unloading at the muscle and is useful as a comparison among conditions and subject populations for gas exchange analyses (Hogan et al. 1990, 1991b).
Statistical analyses
Data were analysed using parametric statistics, following mathematical confirmation of normal distribution using a Shapiro-Wilk test. Specifically, the effect of hypoxia in terms of deoxy-Mb signal, PiO2, arterial O2 saturation and heart rate were assessed using paired t tests (Instat, San Diego, CA, USA). Linear regression analyses were also applied to these data to determine the strength and significance of the relationships between variables. Statistical significance was accepted at P 0.05. Data are presented as means ±S.E.M.
, 百拇医药
Results
Signal-to-noise ratio and cuff occlusion
The use of a large volume coil and increased field strength to 4 T significantly improved the signal-to-noise ratio in comparison to our previously published deoxy-Mb data employing a 7 cm surface coil (sample volume 100 ml) at 2 T (Richardson et al. 1995b, 1998, 2001). Specifically, as illustrated by two example spectra collected during minutes 8–10 of supra-systolic cuff occlusion of the leg (Fig. 1), the signal-to-noise ratio in the current study was 100 : 1 whereas our previous approach yielded a ratio of 5 : 1. With 2 min signal averaging, the deoxy-Mb at rest in normoxia was not clearly discernible prior to cuff inflation (Fig. 2, upper panel). Within the first 2 min of cuff ischaemia the deoxy-Mb peak developed from within the noise and plateaued in minutes 8–10. Again, with 2 min signal averaging, the deoxy-Mb signal was lost rapidly after cuff deflation. Due to the attainment of a plateau in the deoxy-Mb signal during cuff occlusion and our previous failure to enhance it even by superimposing exercise upon the supra-systolic (240 mmHg) blood pressure cuff occlusion (Richardson et al. 1999b), the data collected in the final minutes of cuff occlusion in each subject were considered to represent the maximal deoxy-Mb signal.
, http://www.100md.com
A comparison of the greater signal-to-noise ratio attained in the current 4 T system with a large tissue volume (the entire lower leg) (upper panel) in comparison to our previous data collected with a surface coil (sample volume 100 ml) at 2 T (lower panel). Both signals represent the final 2 min of supra-systolic cuff occlusion (8–10 min). au, arbitrary units.
Example spectra of the deoxy-Mb signal collected in the resting lower leg in normoxia (upper panel) and hypoxia (lower panel) in both cases contrasted with the signal attained during minutes 8–10 of supra-systolic cuff occlusion. The somewhat indefinable peak in normoxia contrasts with that attained in hypoxia as a consequence of the 2 min summation of these spectra, highlighting the need for extended averaging in normoxia to reveal a measurable signal.
, 百拇医药
Deoxy-Mb signal in normoxia and hypoxia
Although the improved signal-to-noise ratio set the stage for the novel assessment of human muscle PiO2 at rest, the average deoxy-Mb signal of less than 10% of the signal generated during cuff occlusion (Table 1) was still not clearly definable in normoxia with the 2 min signal averaging applied to the cuff data (Fig. 2, upper panel). In contrast, in hypoxia with an average deoxy-Mb signal of closer to 15% (Table 1) there was a clear signal with this 2 min averaging (Fig. 2, lower panel). Extended averaging (30 min) facilitated clear signal detection and quantification in both normoxia and hypoxia, but was clearly most important in normoxia (Fig. 3).
, http://www.100md.com
The deoxy-Mb signal in normoxia and hypoxia with 10, 20 and 30 min of signal averaging, highlighting the need in normoxia for extended averaging to define a clear peak.
Physiological response to hypoxia
As a group the 10 subjects responded to hypoxia in a typical fashion, with a fall in arterial O2 saturation, calculated arterial PO2, and a subsequent elevation in heart rate (Table 1). However, upon closer examination of the individual data there was a wide spectrum of responses that revealed some interesting relationships and internal consistency across variables, some measured completely independently. Presumably dependent upon the hypoxic ventilatory response, the subject's change in hypoxically induced arterial oxygenation and the subsequent change in arterial PO2 was quite varied and correlated significantly with the hypoxically induced change in PiO2(r2= 0.5) (Fig. 4). Again, presumably based ultimately upon the hypoxic ventilatory response, a relationship of similar strength was apparent between both of these variables and the change in heart rate from normoxia to hypoxia (r2= 0.6).
, 百拇医药
The significant relationship between the changes in arterial PO2 and intracellular PO2 induced by ambient hypoxia. Although the hypoxic gas breathed (10% O2) was uniform across all subjects, the response in terms of arterial and intracellular PO2 was not, presumably the consequence of variations in hypoxic ventilatory response. This relationship is supportive of the intuitive relationship between arterial PO2 and intracellular PO2, illustrating the importance of a diffusion gradient in the passive movement of O2 from air to cell.
, 百拇医药
Discussion
The unique assessment of intracellular oxygenation in the present study was facilitated by the performance of 1H NMRS of deoxy-Mb at a high field strength, the inclusion of a large muscle volume in a highly efficient coil, and the use of extended signal averaging. This research provides two novel insights into skeletal muscle oxygenation state. The first is the determination of deoxy-Mb levels at rest in normoxia, and the second is the effect of ambient hypoxia (10% O2) on muscle oxygenation. Specifically, it was determined that in normoxia Mb was 9 ± 1% deoxygenated and this increased to 13 ± 3% in hypoxia. In our view, any degree of Mb deoxygenation supports the role of Mb as a facilitator of O2 diffusion, and thus the observation that Mb is somewhat desaturated in normoxia and furthermore that Mb desaturation increases in hypoxia is consistent with Mb playing a significant role in O2 transport from blood to cell.
, 百拇医药
Not only does this study provide information about Mb desaturation and implications about the role of Mb itself, but by using the Mb/O2 dissociation curve it is possible to use this endogenous marker of skeletal muscle oxygenation to calculate the intracellular partial pressure of O2(PiO2). While recognizing the caveat of the as yet undetermined P50 for Mb in vivo, using a previously published value of 3.2 mmHg (Richardson et al. 1995b, 1999b, 2000) these data reveal a normoxic PiO2 of 34 mmHg, which fell to 23 mmHg upon exposure to hypoxia. The implications of these data are far reaching, from placing the previously documented exercising PiO2 values (2–5 mmHg) into context to developing a better understanding of PiO2 sensing as a consequence of hypoxia, that may play an essential role in regulating processes such as angiogenesis and metabolism in health and disease.
, 百拇医药
Deoxy-Mb signal detection at rest
Previously, the Mb peak has not been visible at rest while the peak was small or indistinguishable from the noise during progressive exercise from unweighted to 50–60% of maximum work rate (Richardson et al. 2001). In contrast, the current study achieved the assessment of resting muscle deoxy-Mb by focusing upon the fundamentals of the NMRS signal-to-noise ratio and the properties of Mb. Specifically, in contrast to most other studies performed at 1.5–2 T (Richardson et al. 1995b, 1998b, 2001; Mole et al. 1999; Ponganis et al. 2002; Chung et al. 2005) these studies were performed in a high field strength (4 T) magnet, thus improving signal-to-noise due to the effectively linear relationship between the NMR signal and field strength. Additionally, at 4 T the water suppression scheme is much simpler to implement due to the greater separation between Mb and the water peak, and therefore a shorter radio frequency pulse could be applied, minimizing the signal loss due to the fast Mb T2 decay. With this powerful improvement in hardware, the current pulse sequence was designed to maximize the available transverse magnetization. Also of importance was the use of a highly efficient coil that encompassed a large muscle volume, and data collection and signal averaging which were extended to 30 min. The NMR signal is linearly related to time, while noise increases to the square root of time, thus extended signal averaging provided a dramatic improvement in signal-to-noise ratio. The need to employ such long signal averaging was less in hypoxia because of the inherently greater signal (Figs 2 and 3); however, in several cases during normoxia this approach was essential to accurately quantify the deoxy-Mb peak.
, 百拇医药
This approach of evaluating deoxy-Mb and the subsequent capacity to routinely assess PiO2 non-invasively in skeletal muscle has the potential to greatly enhance our understanding of cell signalling (e.g. angiogenesis and the effects of sleep apnoea) and pathologies associated with skeletal muscle dysfunction that may be the consequence of extended periods of altered PiO2 (e.g. chronic obstructive lung disease, and congestive heart failure).
Exercising and resting PiO2 in context
, http://www.100md.com
The current consensus of 1H NMRS studies of deoxy-Mb to assess intracellular O2 availability would suggest that in skeletal muscle performing even moderate levels of work the intracellular PO2 is already low (5 mmHg) (Mole et al. 1999; Richardson et al. 2001). Although it is debated whether this fall occurs proportionately with exercise intensity (Mole et al. 1999) or achieves a nadir and plateaus beyond 50% of maximum effort (Richardson et al. 2001), the preponderance of data supports the latter profile (Richardson et al. 1995b, 2001; Vanderthommen et al. 2003). However, until the completion of this study the resting PiO2 was unknown due to inadequate signal-to-noise during resting data acquisition, and therefore the true magnitude of the intracellular oxygenation change with exercise was unclear. Now it is evident that the average fall in PiO2 from rest (34 mmHg) to moderately intense exercise in normoxia (3–5 mmHg) is relatively large, in the order of 30 mmHg. As calculated, muscle mean capillary PO2 remains 35–38 mmHg even at maximal exercise (Richardson et al. 1995b, 1999a) this newly documented fall in PiO2 reinforces the apparent importance of creating a steep O2 gradient from blood to working muscle cells, facilitating the high O2 flux rate required for exercise. However, based on these data, it is also clear that without a larger parallel increase in DO2 the high O2 flux rate necessary for maximal muscular work would be unattainable (see below).
, http://www.100md.com
Only one published manuscript has previously used 1H NMRS of deoxy-Mb to measure PiO2 at rest; however, that study was in the Mirounga angustirostris (elephant seal) and the deoxy-Mb signal was only visible during cycles of sleep apnoea when cellular oxygenation fell (Ponganis et al. 2002). Thus, although this comparative physiology study revealed the interesting observation that muscle PiO2 was reduced in this diving sea creature with an extended breath hold, it was limited by the lower field strength (1.5 T), a relatively small volume of muscle examined with a surface coil (13 cm), and a lack of any attempt to employ extended signal averaging, and thus true baseline muscle oxygenation measurements were not attained. Also, currently published only in abstract form, a 1H NMRS study performed in a muscle of the human hand reported a distinct Mb peak with 32 min of data acquisition at rest in normoxia (Amara et al. 2004). Other common methods of assessing tissue PO2 such as PO2 electrodes and phosphorescence quenching have revealed close agreement in animal preparations (Buerk et al. 1998), but both methods predominantly assess a complex milieu that is probably a combination of intracellular, interstitial, and vascular signals. With these approaches the oxygen tension reported in most skeletal muscles in vivo has been within the range of 14–25 mmHg (Whalen et al. 1973; Buerk et al. 1998; Richmond et al. 1999; Golub & Pittman, 2005). The current investigation, by virtue of Mb's restriction to skeletal muscle, was limited to an assessment of muscle intracellular oxygenation and revealed a 9 ± 1% Mb desaturation and a calculated PiO2 of 34 ± 6 mmHg in normoxia. Although somewhat higher than the previous tissue PO2 measurements described above in both method and result, these values certainly seem reasonable within the established O2 cascade from air to muscle (Fig. 5).
, 百拇医药
A comparison of the O2 cascade measured at rest in the current study and during maximal knee-extensor exercise with similar methodologies in a previous investigation (Richardson et al. 1995b).
It is generally accepted that exercise can evoke up to a 50-fold increase in (Rowell, 1993). However, based upon the following equation:
it has been debated whether this is primarily achieved by an increase in DO2 or the change in PO2 from blood to cell. Fuel was added to the controversy by the finding in several studies that from 50 to 60% of maximum work rate intracellular PO2 reaches a plateau which is then invariant with increasing work rate and increasing (Richardson et al. 1995b, 1998b, 2001). From these studies it was concluded that, when viewed in conjunction with our calculations of a mean capillary PO2 of 40 mmHg, there is very little benefit to be gained by a significant reduction in intracellular PO2 from moderate to high intensity exercise and therefore an increase in DO2 must account for the increasing muscle (Richardson et al. 2001). The current data reinforce this concept, remembering the likely 40- to 50-fold increase in skeletal muscle from rest to exercise and the calculated change in PO2 from blood to cell of 10 mmHg at rest to 35 mmHg (a 3.5-fold increase) at maximal exercise. It is clear that the major cause of this increased is an increase in DO2 and not a change in PO2, i.e.
, http://www.100md.com
It is also interesting to note that the calculated mean capillary PO2 in hypoxia was reduced from the normoxic value of 44 mmHg to 35 mmHg resulting in a very similar PO2 driving gradient from blood to cell in both normoxia and hypoxia (10 mmHg). This result is intuitively correct for, although muscle was not assessed in the current study, there is little evidence that metabolic rate is altered by acute hypoxia and therefore the driving gradient from blood to cell should be comparable in normoxia and hypoxia.
, 百拇医药
Defending oxygenation and hypoxic ventilatory response
When studying hypoxia it is important to remember that alterations in ambient O2 availability do not affect all subjects equally. Therefore, caution should be used when anticipating the result of a change in the fraction of inspired O2(FIO2) and the subsequent impact upon in vivo O2 availability. Theoretically, because of the O2 cascade from air to tissue, graded reductions in FIO2 should ultimately alter in vivo O2 availability all the way to the myocyte (Richardson et al. 1995b). However, on an individual basis, in hypoxia the defence of alveolar PO2 by an increase in alveolar ventilation can markedly influence this chain of events by limiting the impact of reduced ambient O2 availability in arterial blood. In fact, this hypoxic ventilatory response (HVR) varies widely between individuals, and has been used to distinguish between those who will thrive and those who will perish at high altitude (Bartsch et al. 2001). Although unanticipated, the importance of recognizing this phenomenon and its impact upon attempts to manipulate O2 availability became apparent in the current investigation, where 50% of the variance in PiO2 could be explained by the change in arterial PO2 (Fig. 4). Hence, the fall in skeletal muscle PiO2 was attenuated in those subjects with a brisk HVR, making teleological sense and providing perhaps the first evidence, through arterial O2 saturation, of the importance of human HVR in terms of cellular O2 homeostasis.
, 百拇医药
Despite an apparently strong HVR in some subjects, the ambient hypoxia of 10% O2 significantly reduced the average intracellular Mb saturation by 44% and calculated PiO2 by 33%. Although the complete ramifications of such a change within resting muscle cells are unknown (cell signalling and growth factor responses) it is clear that such a perturbation, although relatively large, still leaves the cells far above the suggested ‘critical PO2’ (between 0.1 and 0.5 mmHg) below which muscle metabolism is compromised (Chance & Quistorff, 1978; Wilson et al. 1979; Richmond et al. 1999). Indeed, as measured by metabolite concentration, there seems to be little impact of hypoxia on resting metabolism (Sahlin & Katz, 1989) or the kinetics from rest to exercise (Haseler et al. 2004); however, during even moderate intensity we have previously noted that muscle metabolism may be modulated by changes in O2 availability (Haseler et al. 1998). Taken together these data reinforce the concept that O2 availability and metabolism are more tightly coupled during exercise when PiO2 falls to low levels than at rest when there is a relative abundance of O2.
, 百拇医药
Differential effects of hypoxia on PiO2 at rest and exercise
It is interesting to note the large difference in impact upon PiO2, in absolute terms, between hypoxia at rest and during exercise. Specifically, in the current study a reduction in the ambient O2 to 10% resulted in an 11 mmHg change in PiO2 at rest (from 34 to 23 mmHg), whereas in previous investigations during exercise (using 12% O2) we have repeatedly seen closer to a 1 mmHg reduction (from 3 to 2 mmHg) (Richardson et al. 1995b, 1999b, 2002). The concept that reductions in intracellular oxygenation, caused by extracellular PO2 changes, are attenuated during exercise appears to be advantageous in terms of homeostasis under stress. This supposition is supported by data from Kindig et al. (2003) in single muscle fibres from a frog, which revealed that a PiO2 range of 23–56 mmHg at rest (achieved by bathing the cells in low, medium and high levels of extracellular PO2) fell to a smaller range of 3–23 mmHg during contraction. This phenomenon may occur as a result of the mitochondrial transition from a somewhat quiescent state during rest to an active more governing role, in terms of determining PiO2, during exercise. Therefore, these data support the theory that during a hypoxic challenge resting PiO2 is most likely the simple consequence of ambient hypoxia upon passive diffusion, while during exercise the large increase in metabolic rate and subsequent O2 consumption reduce PiO2 and facilitate O2 transport to a greater extent, somewhat staving off the effect of ambient hypoxia.
, http://www.100md.com
Limitations
It should be recognized that the calculation of PiO2 from the measured deoxy-Mb signal is markedly affected by the value of the yet to be determined P50 for Mb in vivo. The authors recommend that the reader recalculate the basic PiO2 data reported here with values within the range reported in the literature (1.5–5.5 mmHg; Wittenburg & Wittenberg, 1989) to truly appreciate the impact of this variable. However, the consistent use of a P50 of 3.2 mmHg in this and our other publications in this area allows these findings at rest to be placed in the context of our previous work during exercise. Again with regard to the P50, it should be recognized that due to the shape of the oxygen-binding curve for Mb there is likely to be some degree of deoxy-Mb signal apparent even at unphysiologically high PiO2 levels. For example, to achieve a < 1% deoxy-Mb signal using varying P50 values of 2.4, 3.2 and 5.5 mmHg one would have to have a PiO2 of 237, 317 and 545 mmHg, respectively. However, even in the unlikely scenario that the cells were in equilibrium with the highest physiologically available PO2 (arterial blood 100 mmHg), in each of these examples the level of deoxy-Mb signal would still be far below the deoxy-Mb level reported here in normoxia (2.5, 3 and 5% for each P50, respectively). This suggests that regardless of the P50 for Mb in vivo the current data do reveal a degree of true Mb desaturation.
, http://www.100md.com
References
Agusti AG, Roca J, Barbera JA, Casademont J, Rodriguez-Roisin R & Wagner PD (1994). Effect of sampling site on femoral venous blood gas values. J Appl Physiol 77, 2018–2022.
Amara CE, Marcinek DJ, Shankland EG, Kushmerick MJ & Conley KE (2004). Comparison of MR and optical spectroscopy to measure myoglobin desaturation in human FDI. Med Sci Sports Exerc 36, S321.
Bartsch P, Grunig E, Hohenhaus E & Dehnert C (2001). Assessment of high altitude tolerance in healthy individuals. High Alt Med Biol 2, 287–296.
, http://www.100md.com
Buerk DG, Tsai AG, Intaglietta M & Johnson PC (1998). In vivo tissue pO2 measurements in hamster skinfold by recessed pO2 microelectrodes and phosphorescence quenching are in agreement. Microcirculation 5, 219–225.
Chance B & Quistorff B (1978). Study of tissue oxygen gradients by single and multiple indicators. Adv Exp Med Biol 94, 331–338.
Chung Y, Mole PA, Sailasuta N, Tran TK, Hurd R & Jue T (2005). Control of respiration and bioenergetics during muscle contraction. Am J Physiol Cell Physiol 288, C730–C738.
, http://www.100md.com
Golub AS & Pittman RN (2005). Erythrocyte-associated transients in PO2 revealed in capillaries of rat mesentery. Am J Physiol Heart Circ Physiol 288, H2735–H2743.
Gruetter R (1993). Automatic, localized adjustment of all first- and second-order Shim coils. Magn Reson Med 29, 804–811.
Haseler LJ, Kindig CA, Richardson RS & Hogan MC (2004). The role of oxygen in determining phosphocreatine onset kinetics in exercising humans. J Physiol 558, 985–992.
, http://www.100md.com
Haseler LJ, Richardson RS, Videen JS & Hogan JS (1998). Phosphocreatine hydrolysis during submaximal exercise: the effect of FIO2. J Appl Physiol 85, 1457–1470.
Hofer SOP, van der Kleij AJ & Bos KE (1992). Tissue oxygenation measurement: A directly applied Clark-type electrode in muscle tissue. In Advances in Experimental Medicine and Biology, ed. Erdmann W & Bruley DR, pp. 779–784. Plenum Press, New York.
Hogan MC, Bebout DE & Wagner PD (1991a). Effect of hemoglobin concentration on maximal O2 uptake in canine gastrocnemius muscle in situ. J Appl Physiol 70, 1105–1112.
, 百拇医药
Hogan MC, Bebout DE & Wagner PD (1991b). Effect of increased HbO2 affinity on VO2max at a constant O2 delivery in dog muscle in situ. J Appl Physiol 70, 2656–2662.
Hogan MC, Bebout DE, Wagner PD & West PD (1990). Maximal O2 uptake of in situ dog muscle during hypoxemia with constant perfusion. J Appl Physiol 69, 570–576.
Kindig CA, Howlett RA & Hogan MC (2003). Effect of extracellular PO2 on the fall in intracellular PO2 in contracting single myocytes. J Appl Physiol 94, 1964–1970.
, http://www.100md.com
Marcinek DJ, Ciesielski WA, Conley KE & Schenkman KA (2003). Oxygen regulation and limitation to cellular respiration in mouse skeletal muscle in vivo. Am J Physiol Heart Circ Physiol 285, H1900–H1908.
Miura H, McCully K, Nioka S & Chance B (2004). Relationship between muscle architectural features and oxygenation status determined by near infrared device. Eur J Appl Physiol 91, 273–278.
Mole P, Chung Y, Tran T, Sailasuta N, Hurd R & Jue T (1999). Myoglobin desaturation with exercise intensity in human gastrocnemius muscle. Am J Physiol 277, R173–R180.
, http://www.100md.com
Ponganis PJ, Kreutzer U, Sailasuta N, Knower T, Hurd R & Jue T (2002). Detection of myoglobin desaturation in Mirounga angustirostris during apnea. Am J Physiol Regul Integr Comp Physiol 282, R267–R272.
Richardson RS (1998). Oxygen transport: air to muscle cell. Med Sci Sports Exerc 30, 53–59.
Richardson RS (2000). Intracellular Po2 and bioenergetic measurements in skeletal muscle: the role of exercise paradigm. Am J Physiol Regul Integr Comp Physiol 278, R1111–R1113.
, 百拇医药
Richardson RS, Grassi B, Gavin TP, Haseler LJ, Tagore K, Roca J & Wagner PD (1999a). Evidence of O2 supply-dependent VO2 max in the exercise-trained human quadriceps. J Appl Physiol 86, 1048–1053.
Richardson RS, Knight DR, Poole DC, Kurdak SS, Hogan MC, Grassi B & Wagner PD (1995a). Determinants of maximal exercise VO2 during single leg knee-extensor exercise in humans. Am J Physiol 268, H1453–H1461.
Richardson RS, Leigh JS, Wagner PD & Noyszewski EA (1999b). Cellular PO2 as a determinant of maximal mitochondrial O2 consumption in trained human skeletal muscle. J Appl Physiol 87, 325–331.
, http://www.100md.com
Richardson RS, Mudaliar SRD, Mathieu-Costello O & Wagner PD (1998a). VEGF mRNA response to acute exercise in skeletal muscle is attenuated following chronic training. Med Sci Sports Exerc 30, S50.
Richardson RS, Newcomer SC & Noyszewski EA (2001). Skeletal muscle intracellular PO2 assessed by myoglobin desaturation: response to graded exercise. J Appl Physiol 91, 2679–2685.
Richardson RS, Noyszewski EA, Kendrick KF, Leigh JS & Wagner PD (1995b). Myoglobin O2 desaturation during exercise. Evidence of limited O2 transport. J Clin Invest 96, 1916–1926.
, 百拇医药
Richardson RS, Noyszewski EA, Leigh JS & Wagner PD (1998b). Lactate efflux from exercising human skeletal muscle: role of intracellular PO2. J Appl Physiol 85, 627–634.
Richardson RS, Noyszewski EA, Saltin B & Gonzalez-Alonso J (2002). Effect of mild carboxy-hemoglobin on exercising skeletal muscle: intravascular and intracellular evidence. Am J Physiol Regul Integr Comp Physiol 283, R1131–R1139.
Richardson RS, Wagner H, Mudaliar SR, Henry R, Noyszewski EA & Wagner PD (1999c). Human VEGF gene expression in skeletal muscle: effect of acute normoxic and hypoxic exercise. Am J Physiol 277, H2247–H2252.
, 百拇医药
Richmond KN, Shonat RD, Lynch RM & Johnson PC (1999). Critical PO2 of skeletal muscle in vivo. Am J Physiol 277, H1831–H1840.
Roca J, Hogan MC, Story D, Bebout DE, Haab P, Gonzalez R, Ueno O & Wagner PD (1989). Evidence for tissue diffusion limitation of VO2max in normal humans. J Appl Physiol 67, 291–299.
Rossi-Fanelli A & Antonini E (1958). Studies of the oxygen and carbon monoxide equilibria of human myoglobin. Arch Biochem Biophys 77, 478–492.
, 百拇医药
Rowell LB (1993). Human Cardiovascular Control. Oxford University Press Inc, New York.
Rumsey WL, Vanderkooi JM & Wilson DF (1989). Imaging of phosphorescence: a novel method for measuring oxygen distribution in perfused tissue. Science 241, 1649–1651.
Sahlin K & Katz A (1989). Hypoxaemia increases the accumulation of inosine monophosphate (IMP) in human skeletal muscle during submaximal exercise. Acta Physiol Scand 136, 199–203.
, 百拇医药
Vanderthommen M, Duteil S, Wary C, Raynaud JS, Leroy-Willig A, Crielaard JM & Carlier PG (2003). A comparison of voluntary and electrically induced contractions by interleaved 1H- and 31P-NMRS in humans. J Appl Physiol 94, 1012–1024.
Wagner PD (1988). An integrated view of the determinants of maximum oxygen uptake. In Oxygen Transfer from the Atmosphere to Tissues, ed. Gonzalez NC & Fedde MR, pp. 245–256. Plenum Publishing Corporation, New York.
, 百拇医药
Wagner PD (1992). Gas exchange and peripheral diffusion limitation. Med Sci Sports Exerc 24, 54–58.
Wagner PD (2000). New ideas on limitations to VO2max. Exerc Sport Sci Rev 28, 10–14.
Wagner PD (2001). Skeletal muscle angiogenesis. A possible role for hypoxia. Adv Exp Med Biol 502, 21–38.
Wang J, Noyszewski EA & Leigh JS (1990). In vivo MRS measurement of deoxymyoglobin in human forearms. Magn Reson Med 14, 562–567.
, 百拇医药
Wang J, Shimoda LA, Weigand L, Wang W, Sun D & Sylvester JT (2005). Acute hypoxia increases intracellular [Ca2+] in pulmonary arterial smooth muscle by enhancing capacitative Ca2+ entry. Am J Physiol Lung Cell Mol Physiol 288, L1059–L1069.
Whalen WJ, Nair P & Ganfield RA (1973). Measurements of oxygen tension in tissues with a micro oxygen electrode. Microvasc Res 5, 254–262.
Wilson DF, Erecinska M, Drown C & Silver IA (1979). The oxygen dependence of cellular energy metabolism. Arch Biochem Biophys 195, 485–493.
, 百拇医药
Wittenburg BA & Wittenberg JB (1989). Transport of oxygen in muscle. Annu Rev Physiol 51, 857–878.
Wolin MS, Ahmad M & Gupte SA (2005). Oxidant and redox signaling in vascular oxygen sensing mechanisms: basic concepts, current controversies, and potential importance of cytosolic NADPH. Am J Physiol Lung Cell Mol Physiol 289, L159–L173., http://www.100md.com(Russell S. Richardson, Sandrine Duteil, )
2 Norwegian University of Science and Technology, Faculty of Medicine NO-7489 Trondheim, Norway
3 NMR Laboratory, AFM and CEA, IFR 14, Institut de Myologie, Pitié-Salpêtrière University Hospital, Paris, France
Abstract
Intracellular oxygen (O2) availability and the impact of ambient hypoxia have far reaching ramifications in terms of cell signalling and homeostasis; however, in vivo cellular oxygenation has been an elusive variable to assess. Within skeletal muscle the extent to which myoglobin desaturates (deoxy-Mb) and the extent of this desaturation in relation to O2 availability provide an endogenous probe for intracellular O2 partial pressure (PiO2). By combining proton nuclear magnetic resonance spectroscopy (1H NMRS) at a high field strength (4 T), assessing a large muscle volume in a highly efficient coil, and extended signal averaging (30 min) we assessed the level of skeletal muscle deoxy-Mb in 10 healthy men (30 ± 4 years) at rest in both normoxia and hypoxia (10% O2). In normoxia there was an average deoxy-Mb signal of 9 ± 1%, which, when converted to PiO2 using an O2/Mb half-saturation (P50) of 3.2 mmHg, revealed an PiO2 of 34 ± 6 mmHg. In ambient hypoxia the deoxy-Mb signal rose to 13 ± 3% (PiO2= 23 ± 6 mmHg). However, intersubject variation in the defence of arterial oxygenation (SaO2) in hypoxia (SaO2 range: 86–67%) revealed a significant relationship between the changes in SaO2 and PiO2(r2= 0.5). These data are the first to document resting intracellular oxygenation in human skeletal muscle, highlighting the relatively high PiO2 values that contrast markedly with those previously recorded during exercise (2–5 mmHg). Additionally, the impact of ambient hypoxia on PiO2 and the relationship between changes in SaO2 and PiO2 stress the importance of the O2 cascade from air to cell that ultimately effects O2 availability and O2 sensing at the cellular level.
, 百拇医药
Introduction
Changes in intracellular oxygen availability have far reaching consequences likely involved in such diverse processes as angiogenesis (Richardson et al. 1999c; Wagner, 2001) and hypoxic pulmonary vasoconstriction (Wang et al. 2005; Wolin et al. 2005). However, data directly assessing intracellular oxygenation are sparse, especially in humans in vivo. Several very different methods have been employed in an attempt to assess local O2 availability including the use of PO2 electrodes (Hofer et al. 1992), O2 phosphorescence quenching (Rumsey et al. 1989), near infrared spectroscopy (Marcinek et al. 2003; Miura et al. 2004), and proton nuclear magnetic resonance spectroscopy (1H NMRS) of myoglobin (Mb) (Richardson et al. 1995b). Unlike the other methods which cause significant tissue damage or raise questions regarding the compartment of measurement, 1H NMRS of Mb is non-invasive and is clearly limited to muscle tissue, the only site in which this endogenous intracellular marker of oxygenation is found. Thus, this NMRS approach is ideally suited for in vivo human investigations.
, 百拇医药
The measurement of deoxy-Mb by NMRS to determine skeletal muscle oxygenation has been utilized in several studies (Richardson et al. 1995b, 1998a, 2001, 2002; Mole et al. 1999; Vanderthommen et al. 2003). However, until now the assessment of deoxy-Mb has been limited to exercise where, due to falling PiO2, there is sufficient signal visible even with a relatively low signal-to-noise ratio. Previously, the lack of a resting deoxy-Mb signal prior to muscular contraction or supra-systolic cuff occlusion has been interpreted as indicative of a relatively high PiO2 in resting skeletal muscle (Richardson et al. 1995b, 2001; Mole et al. 1999; Chung et al. 2005). Therefore, although it is known that muscle PiO2 falls to very low values of 2–5 mmHg during exercising (Mole et al. 1999; Richardson et al. 2001), the starting point for skeletal muscle oxygenation or resting PiO2 is, as of yet, unknown.
, http://www.100md.com
Hypoxia is both an important stimulus and a constant threat to the human body and its vital organs throughout life. Environmental changes such as exposure to high altitude reduce ambient O2 availability, while lung, vascular, and sleep disorders can result in hypoxia under normoxic conditions. It is known that hypoxia mediates adaptive changes in metabolism, O2 sensing and gene expression. However, although much research has examined the consequences of experimental hypoxic conditions, data documenting hypoxically mediated changes in cellular oxygenation in humans are sparse, if not non-existent.
, 百拇医药
Consequently, we performed 1H NMRS at a high field strength (4 Tesla (T)) with a large volume coil and extended signal averaging (30 min) to assess resting O2 saturation (deoxy-Mb) in muscles of the lower leg in both normoxia and hypoxia (10% O2). This study had three specific goals: (1) to measure the resting intracellular oxygenation level and calculate the PiO2 of human skeletal muscle in vivo, (2) to test the hypothesis that ambient hypoxia will significantly reduce the level of cellular oxygenation and hence lower PiO2 in human skeletal muscle, and (3) to assimilate these findings into the current understanding of skeletal muscle intracellular oxygenation and PiO2 during exercise.
, 百拇医药
Methods
Subjects
The protocol received unrestricted approval from the Pitié-Salpêtrière University Hospital Ethics Committee (Paris, France) and all procedures conformed to the standards set by the Declaration of Helsinki. The 8 male and 2 female subjects who volunteered to participate in the study gave written informed consent. Subjects were not recruited with regard to exercise habits and were therefore of varying activity levels. Mean age, height and body weight were 30 ± 4 years, 174 ± 3 cm, and 69 ± 3 kg, respectively.
, http://www.100md.com
Experimental protocol and set-up
The experimental protocol was focused on resting muscle oxygenation in the muscles of the lower leg under conditions of normoxia and hypoxia (10% O2). The studies were carried out in a 4 T, 46 cm internal bore, superconducting magnet (Magnex 4/60) interfaced to a Bruker Biospec NMR spectrometer. Before the experiments, all subjects were familiarized with the experimental set-up and were accustomed to lying supine in the magnet. The calf of the subject's dominant leg was carefully positioned inside a 17 cm inner diameter transversal electromagnetic (TEM) 1H transmit-and-receive volume coil. An air-cuff was wrapped above the knee of this leg and inflated to 240 mmHg for 10 min to achieve complete vascular occlusion and determine the maximum deoxy-Mb signal in the lower leg. Throughout the normoxic and hypoxic experiments subjects breathed through a low resistance two-way breathing valve (2700; Hans-Rudolph Inc., Kansas City, MO, USA) connected to either room air or a 150 l reservoir containing the hypoxic gas. Heart rate and arterial blood O2 saturation were monitored continuously using a NMR-compatible patient-monitoring device (Maglife, Bruker, Wissembourg, France) and arterial PO2 (PaO2 was calculated from blood O2 saturation using a standard O2 dissociation curve. The importance of remaining still throughout the experiments was explained to all subjects and there was good compliance.
, 百拇医药
1H NMRS of deoxy-Mb
Within the 4 T magnet the n– proton of the proximal histidine F8 of myoglobin in the deoxygenated state was selectively excited by an 0.8 ms Gaussian pulse (1024 accumulations, 256 complex points, spectral width 200 Hz, TR time 20 ms). The B0 field homogeneity was optimized with Fastmap, an automatic localized 1st and 2nd order shim procedure (Gruetter, 1993). The other necessary adjustments and the acquisition of reference images and spectra in resting conditions took about 20 min.
, 百拇医药
NMRS data processing
The deoxy-Mb spectra were processed with standard ParaVision and XWIN NMRS software. After a 100 Hz line-broadening exponential multiplication and Fourier transformation, zero and 1st order phases were applied to the first deoxy-Mb spectrum of each series and all others were processed using these same parameter settings. Automatic baseline correction (± 20 p.p.m.) was applied, and then the Mb peak of each spectrum was quantified by integration over the same p.p.m. range of that subject's deoxy-Mb signal (10 p.p.m.) attained at minutes 8–10 of ischaemia.
, http://www.100md.com
Calculation of PiO2
The plateaued deoxy-Mb signals obtained during the 8th and 10th minutes of cuff occlusion (240 mmHg) represent the complete deoxygenation of Mb (Wang et al. 1990). The fractional deoxy-Mb (Fdeoxy-Mb) was determined by normalizing the signal areas to the average signal obtained during the last minutes of cuff ischaemia. The conversion from Fdeoxy-Mb to PO2 values was then calculated from the oxygen-binding curve for Mb:
, 百拇医药
Where FMbO2 is the fraction of Mb that is oxygenated and P50 is the O2 pressure where 50% of the Mb binding sites are bound with O2. The temperature-dependent Mb half-saturation (P50) of 3.2 mmHg was used (Rossi-Fanelli & Antonini, 1958).
Calculation of diffusional conductance (DmO2) and mean capillary PO2 (PcapO2)
A Bohr integration technique was used to calculate an estimate of PcapO2 and DmO2 at rest. Mean capillary PO2 was calculated as previously described (Wagner, 1992) as the numerical average of the arterial PO2 values assessed by arterial Hb saturation and prior measures of femoral venous PO2 at rest (Agusti et al. 1994), computed equally spaced in time along the capillary as it traverses the muscle bed from arterial to venous end. This technique has been discussed in detail elsewhere (Roca et al. 1989; Hogan et al. 1991a) based on a proposal by Wagner (1988) with the assumption of a homogeneously perfused muscle. Briefly, the drop in PO2 along a capillary is calculated using Fick's law of diffusion (eqn (1)).
, http://www.100md.com
(1)
Where PiO2 is assessed by deoxy-Mb levels. DmO2 is an expression of all phenomena that facilitate O2 unloading at the muscle and is useful as a comparison among conditions and subject populations for gas exchange analyses (Hogan et al. 1990, 1991b).
Statistical analyses
Data were analysed using parametric statistics, following mathematical confirmation of normal distribution using a Shapiro-Wilk test. Specifically, the effect of hypoxia in terms of deoxy-Mb signal, PiO2, arterial O2 saturation and heart rate were assessed using paired t tests (Instat, San Diego, CA, USA). Linear regression analyses were also applied to these data to determine the strength and significance of the relationships between variables. Statistical significance was accepted at P 0.05. Data are presented as means ±S.E.M.
, 百拇医药
Results
Signal-to-noise ratio and cuff occlusion
The use of a large volume coil and increased field strength to 4 T significantly improved the signal-to-noise ratio in comparison to our previously published deoxy-Mb data employing a 7 cm surface coil (sample volume 100 ml) at 2 T (Richardson et al. 1995b, 1998, 2001). Specifically, as illustrated by two example spectra collected during minutes 8–10 of supra-systolic cuff occlusion of the leg (Fig. 1), the signal-to-noise ratio in the current study was 100 : 1 whereas our previous approach yielded a ratio of 5 : 1. With 2 min signal averaging, the deoxy-Mb at rest in normoxia was not clearly discernible prior to cuff inflation (Fig. 2, upper panel). Within the first 2 min of cuff ischaemia the deoxy-Mb peak developed from within the noise and plateaued in minutes 8–10. Again, with 2 min signal averaging, the deoxy-Mb signal was lost rapidly after cuff deflation. Due to the attainment of a plateau in the deoxy-Mb signal during cuff occlusion and our previous failure to enhance it even by superimposing exercise upon the supra-systolic (240 mmHg) blood pressure cuff occlusion (Richardson et al. 1999b), the data collected in the final minutes of cuff occlusion in each subject were considered to represent the maximal deoxy-Mb signal.
, http://www.100md.com
A comparison of the greater signal-to-noise ratio attained in the current 4 T system with a large tissue volume (the entire lower leg) (upper panel) in comparison to our previous data collected with a surface coil (sample volume 100 ml) at 2 T (lower panel). Both signals represent the final 2 min of supra-systolic cuff occlusion (8–10 min). au, arbitrary units.
Example spectra of the deoxy-Mb signal collected in the resting lower leg in normoxia (upper panel) and hypoxia (lower panel) in both cases contrasted with the signal attained during minutes 8–10 of supra-systolic cuff occlusion. The somewhat indefinable peak in normoxia contrasts with that attained in hypoxia as a consequence of the 2 min summation of these spectra, highlighting the need for extended averaging in normoxia to reveal a measurable signal.
, 百拇医药
Deoxy-Mb signal in normoxia and hypoxia
Although the improved signal-to-noise ratio set the stage for the novel assessment of human muscle PiO2 at rest, the average deoxy-Mb signal of less than 10% of the signal generated during cuff occlusion (Table 1) was still not clearly definable in normoxia with the 2 min signal averaging applied to the cuff data (Fig. 2, upper panel). In contrast, in hypoxia with an average deoxy-Mb signal of closer to 15% (Table 1) there was a clear signal with this 2 min averaging (Fig. 2, lower panel). Extended averaging (30 min) facilitated clear signal detection and quantification in both normoxia and hypoxia, but was clearly most important in normoxia (Fig. 3).
, http://www.100md.com
The deoxy-Mb signal in normoxia and hypoxia with 10, 20 and 30 min of signal averaging, highlighting the need in normoxia for extended averaging to define a clear peak.
Physiological response to hypoxia
As a group the 10 subjects responded to hypoxia in a typical fashion, with a fall in arterial O2 saturation, calculated arterial PO2, and a subsequent elevation in heart rate (Table 1). However, upon closer examination of the individual data there was a wide spectrum of responses that revealed some interesting relationships and internal consistency across variables, some measured completely independently. Presumably dependent upon the hypoxic ventilatory response, the subject's change in hypoxically induced arterial oxygenation and the subsequent change in arterial PO2 was quite varied and correlated significantly with the hypoxically induced change in PiO2(r2= 0.5) (Fig. 4). Again, presumably based ultimately upon the hypoxic ventilatory response, a relationship of similar strength was apparent between both of these variables and the change in heart rate from normoxia to hypoxia (r2= 0.6).
, 百拇医药
The significant relationship between the changes in arterial PO2 and intracellular PO2 induced by ambient hypoxia. Although the hypoxic gas breathed (10% O2) was uniform across all subjects, the response in terms of arterial and intracellular PO2 was not, presumably the consequence of variations in hypoxic ventilatory response. This relationship is supportive of the intuitive relationship between arterial PO2 and intracellular PO2, illustrating the importance of a diffusion gradient in the passive movement of O2 from air to cell.
, 百拇医药
Discussion
The unique assessment of intracellular oxygenation in the present study was facilitated by the performance of 1H NMRS of deoxy-Mb at a high field strength, the inclusion of a large muscle volume in a highly efficient coil, and the use of extended signal averaging. This research provides two novel insights into skeletal muscle oxygenation state. The first is the determination of deoxy-Mb levels at rest in normoxia, and the second is the effect of ambient hypoxia (10% O2) on muscle oxygenation. Specifically, it was determined that in normoxia Mb was 9 ± 1% deoxygenated and this increased to 13 ± 3% in hypoxia. In our view, any degree of Mb deoxygenation supports the role of Mb as a facilitator of O2 diffusion, and thus the observation that Mb is somewhat desaturated in normoxia and furthermore that Mb desaturation increases in hypoxia is consistent with Mb playing a significant role in O2 transport from blood to cell.
, 百拇医药
Not only does this study provide information about Mb desaturation and implications about the role of Mb itself, but by using the Mb/O2 dissociation curve it is possible to use this endogenous marker of skeletal muscle oxygenation to calculate the intracellular partial pressure of O2(PiO2). While recognizing the caveat of the as yet undetermined P50 for Mb in vivo, using a previously published value of 3.2 mmHg (Richardson et al. 1995b, 1999b, 2000) these data reveal a normoxic PiO2 of 34 mmHg, which fell to 23 mmHg upon exposure to hypoxia. The implications of these data are far reaching, from placing the previously documented exercising PiO2 values (2–5 mmHg) into context to developing a better understanding of PiO2 sensing as a consequence of hypoxia, that may play an essential role in regulating processes such as angiogenesis and metabolism in health and disease.
, 百拇医药
Deoxy-Mb signal detection at rest
Previously, the Mb peak has not been visible at rest while the peak was small or indistinguishable from the noise during progressive exercise from unweighted to 50–60% of maximum work rate (Richardson et al. 2001). In contrast, the current study achieved the assessment of resting muscle deoxy-Mb by focusing upon the fundamentals of the NMRS signal-to-noise ratio and the properties of Mb. Specifically, in contrast to most other studies performed at 1.5–2 T (Richardson et al. 1995b, 1998b, 2001; Mole et al. 1999; Ponganis et al. 2002; Chung et al. 2005) these studies were performed in a high field strength (4 T) magnet, thus improving signal-to-noise due to the effectively linear relationship between the NMR signal and field strength. Additionally, at 4 T the water suppression scheme is much simpler to implement due to the greater separation between Mb and the water peak, and therefore a shorter radio frequency pulse could be applied, minimizing the signal loss due to the fast Mb T2 decay. With this powerful improvement in hardware, the current pulse sequence was designed to maximize the available transverse magnetization. Also of importance was the use of a highly efficient coil that encompassed a large muscle volume, and data collection and signal averaging which were extended to 30 min. The NMR signal is linearly related to time, while noise increases to the square root of time, thus extended signal averaging provided a dramatic improvement in signal-to-noise ratio. The need to employ such long signal averaging was less in hypoxia because of the inherently greater signal (Figs 2 and 3); however, in several cases during normoxia this approach was essential to accurately quantify the deoxy-Mb peak.
, 百拇医药
This approach of evaluating deoxy-Mb and the subsequent capacity to routinely assess PiO2 non-invasively in skeletal muscle has the potential to greatly enhance our understanding of cell signalling (e.g. angiogenesis and the effects of sleep apnoea) and pathologies associated with skeletal muscle dysfunction that may be the consequence of extended periods of altered PiO2 (e.g. chronic obstructive lung disease, and congestive heart failure).
Exercising and resting PiO2 in context
, http://www.100md.com
The current consensus of 1H NMRS studies of deoxy-Mb to assess intracellular O2 availability would suggest that in skeletal muscle performing even moderate levels of work the intracellular PO2 is already low (5 mmHg) (Mole et al. 1999; Richardson et al. 2001). Although it is debated whether this fall occurs proportionately with exercise intensity (Mole et al. 1999) or achieves a nadir and plateaus beyond 50% of maximum effort (Richardson et al. 2001), the preponderance of data supports the latter profile (Richardson et al. 1995b, 2001; Vanderthommen et al. 2003). However, until the completion of this study the resting PiO2 was unknown due to inadequate signal-to-noise during resting data acquisition, and therefore the true magnitude of the intracellular oxygenation change with exercise was unclear. Now it is evident that the average fall in PiO2 from rest (34 mmHg) to moderately intense exercise in normoxia (3–5 mmHg) is relatively large, in the order of 30 mmHg. As calculated, muscle mean capillary PO2 remains 35–38 mmHg even at maximal exercise (Richardson et al. 1995b, 1999a) this newly documented fall in PiO2 reinforces the apparent importance of creating a steep O2 gradient from blood to working muscle cells, facilitating the high O2 flux rate required for exercise. However, based on these data, it is also clear that without a larger parallel increase in DO2 the high O2 flux rate necessary for maximal muscular work would be unattainable (see below).
, http://www.100md.com
Only one published manuscript has previously used 1H NMRS of deoxy-Mb to measure PiO2 at rest; however, that study was in the Mirounga angustirostris (elephant seal) and the deoxy-Mb signal was only visible during cycles of sleep apnoea when cellular oxygenation fell (Ponganis et al. 2002). Thus, although this comparative physiology study revealed the interesting observation that muscle PiO2 was reduced in this diving sea creature with an extended breath hold, it was limited by the lower field strength (1.5 T), a relatively small volume of muscle examined with a surface coil (13 cm), and a lack of any attempt to employ extended signal averaging, and thus true baseline muscle oxygenation measurements were not attained. Also, currently published only in abstract form, a 1H NMRS study performed in a muscle of the human hand reported a distinct Mb peak with 32 min of data acquisition at rest in normoxia (Amara et al. 2004). Other common methods of assessing tissue PO2 such as PO2 electrodes and phosphorescence quenching have revealed close agreement in animal preparations (Buerk et al. 1998), but both methods predominantly assess a complex milieu that is probably a combination of intracellular, interstitial, and vascular signals. With these approaches the oxygen tension reported in most skeletal muscles in vivo has been within the range of 14–25 mmHg (Whalen et al. 1973; Buerk et al. 1998; Richmond et al. 1999; Golub & Pittman, 2005). The current investigation, by virtue of Mb's restriction to skeletal muscle, was limited to an assessment of muscle intracellular oxygenation and revealed a 9 ± 1% Mb desaturation and a calculated PiO2 of 34 ± 6 mmHg in normoxia. Although somewhat higher than the previous tissue PO2 measurements described above in both method and result, these values certainly seem reasonable within the established O2 cascade from air to muscle (Fig. 5).
, 百拇医药
A comparison of the O2 cascade measured at rest in the current study and during maximal knee-extensor exercise with similar methodologies in a previous investigation (Richardson et al. 1995b).
It is generally accepted that exercise can evoke up to a 50-fold increase in (Rowell, 1993). However, based upon the following equation:
it has been debated whether this is primarily achieved by an increase in DO2 or the change in PO2 from blood to cell. Fuel was added to the controversy by the finding in several studies that from 50 to 60% of maximum work rate intracellular PO2 reaches a plateau which is then invariant with increasing work rate and increasing (Richardson et al. 1995b, 1998b, 2001). From these studies it was concluded that, when viewed in conjunction with our calculations of a mean capillary PO2 of 40 mmHg, there is very little benefit to be gained by a significant reduction in intracellular PO2 from moderate to high intensity exercise and therefore an increase in DO2 must account for the increasing muscle (Richardson et al. 2001). The current data reinforce this concept, remembering the likely 40- to 50-fold increase in skeletal muscle from rest to exercise and the calculated change in PO2 from blood to cell of 10 mmHg at rest to 35 mmHg (a 3.5-fold increase) at maximal exercise. It is clear that the major cause of this increased is an increase in DO2 and not a change in PO2, i.e.
, http://www.100md.com
It is also interesting to note that the calculated mean capillary PO2 in hypoxia was reduced from the normoxic value of 44 mmHg to 35 mmHg resulting in a very similar PO2 driving gradient from blood to cell in both normoxia and hypoxia (10 mmHg). This result is intuitively correct for, although muscle was not assessed in the current study, there is little evidence that metabolic rate is altered by acute hypoxia and therefore the driving gradient from blood to cell should be comparable in normoxia and hypoxia.
, 百拇医药
Defending oxygenation and hypoxic ventilatory response
When studying hypoxia it is important to remember that alterations in ambient O2 availability do not affect all subjects equally. Therefore, caution should be used when anticipating the result of a change in the fraction of inspired O2(FIO2) and the subsequent impact upon in vivo O2 availability. Theoretically, because of the O2 cascade from air to tissue, graded reductions in FIO2 should ultimately alter in vivo O2 availability all the way to the myocyte (Richardson et al. 1995b). However, on an individual basis, in hypoxia the defence of alveolar PO2 by an increase in alveolar ventilation can markedly influence this chain of events by limiting the impact of reduced ambient O2 availability in arterial blood. In fact, this hypoxic ventilatory response (HVR) varies widely between individuals, and has been used to distinguish between those who will thrive and those who will perish at high altitude (Bartsch et al. 2001). Although unanticipated, the importance of recognizing this phenomenon and its impact upon attempts to manipulate O2 availability became apparent in the current investigation, where 50% of the variance in PiO2 could be explained by the change in arterial PO2 (Fig. 4). Hence, the fall in skeletal muscle PiO2 was attenuated in those subjects with a brisk HVR, making teleological sense and providing perhaps the first evidence, through arterial O2 saturation, of the importance of human HVR in terms of cellular O2 homeostasis.
, 百拇医药
Despite an apparently strong HVR in some subjects, the ambient hypoxia of 10% O2 significantly reduced the average intracellular Mb saturation by 44% and calculated PiO2 by 33%. Although the complete ramifications of such a change within resting muscle cells are unknown (cell signalling and growth factor responses) it is clear that such a perturbation, although relatively large, still leaves the cells far above the suggested ‘critical PO2’ (between 0.1 and 0.5 mmHg) below which muscle metabolism is compromised (Chance & Quistorff, 1978; Wilson et al. 1979; Richmond et al. 1999). Indeed, as measured by metabolite concentration, there seems to be little impact of hypoxia on resting metabolism (Sahlin & Katz, 1989) or the kinetics from rest to exercise (Haseler et al. 2004); however, during even moderate intensity we have previously noted that muscle metabolism may be modulated by changes in O2 availability (Haseler et al. 1998). Taken together these data reinforce the concept that O2 availability and metabolism are more tightly coupled during exercise when PiO2 falls to low levels than at rest when there is a relative abundance of O2.
, 百拇医药
Differential effects of hypoxia on PiO2 at rest and exercise
It is interesting to note the large difference in impact upon PiO2, in absolute terms, between hypoxia at rest and during exercise. Specifically, in the current study a reduction in the ambient O2 to 10% resulted in an 11 mmHg change in PiO2 at rest (from 34 to 23 mmHg), whereas in previous investigations during exercise (using 12% O2) we have repeatedly seen closer to a 1 mmHg reduction (from 3 to 2 mmHg) (Richardson et al. 1995b, 1999b, 2002). The concept that reductions in intracellular oxygenation, caused by extracellular PO2 changes, are attenuated during exercise appears to be advantageous in terms of homeostasis under stress. This supposition is supported by data from Kindig et al. (2003) in single muscle fibres from a frog, which revealed that a PiO2 range of 23–56 mmHg at rest (achieved by bathing the cells in low, medium and high levels of extracellular PO2) fell to a smaller range of 3–23 mmHg during contraction. This phenomenon may occur as a result of the mitochondrial transition from a somewhat quiescent state during rest to an active more governing role, in terms of determining PiO2, during exercise. Therefore, these data support the theory that during a hypoxic challenge resting PiO2 is most likely the simple consequence of ambient hypoxia upon passive diffusion, while during exercise the large increase in metabolic rate and subsequent O2 consumption reduce PiO2 and facilitate O2 transport to a greater extent, somewhat staving off the effect of ambient hypoxia.
, http://www.100md.com
Limitations
It should be recognized that the calculation of PiO2 from the measured deoxy-Mb signal is markedly affected by the value of the yet to be determined P50 for Mb in vivo. The authors recommend that the reader recalculate the basic PiO2 data reported here with values within the range reported in the literature (1.5–5.5 mmHg; Wittenburg & Wittenberg, 1989) to truly appreciate the impact of this variable. However, the consistent use of a P50 of 3.2 mmHg in this and our other publications in this area allows these findings at rest to be placed in the context of our previous work during exercise. Again with regard to the P50, it should be recognized that due to the shape of the oxygen-binding curve for Mb there is likely to be some degree of deoxy-Mb signal apparent even at unphysiologically high PiO2 levels. For example, to achieve a < 1% deoxy-Mb signal using varying P50 values of 2.4, 3.2 and 5.5 mmHg one would have to have a PiO2 of 237, 317 and 545 mmHg, respectively. However, even in the unlikely scenario that the cells were in equilibrium with the highest physiologically available PO2 (arterial blood 100 mmHg), in each of these examples the level of deoxy-Mb signal would still be far below the deoxy-Mb level reported here in normoxia (2.5, 3 and 5% for each P50, respectively). This suggests that regardless of the P50 for Mb in vivo the current data do reveal a degree of true Mb desaturation.
, http://www.100md.com
References
Agusti AG, Roca J, Barbera JA, Casademont J, Rodriguez-Roisin R & Wagner PD (1994). Effect of sampling site on femoral venous blood gas values. J Appl Physiol 77, 2018–2022.
Amara CE, Marcinek DJ, Shankland EG, Kushmerick MJ & Conley KE (2004). Comparison of MR and optical spectroscopy to measure myoglobin desaturation in human FDI. Med Sci Sports Exerc 36, S321.
Bartsch P, Grunig E, Hohenhaus E & Dehnert C (2001). Assessment of high altitude tolerance in healthy individuals. High Alt Med Biol 2, 287–296.
, http://www.100md.com
Buerk DG, Tsai AG, Intaglietta M & Johnson PC (1998). In vivo tissue pO2 measurements in hamster skinfold by recessed pO2 microelectrodes and phosphorescence quenching are in agreement. Microcirculation 5, 219–225.
Chance B & Quistorff B (1978). Study of tissue oxygen gradients by single and multiple indicators. Adv Exp Med Biol 94, 331–338.
Chung Y, Mole PA, Sailasuta N, Tran TK, Hurd R & Jue T (2005). Control of respiration and bioenergetics during muscle contraction. Am J Physiol Cell Physiol 288, C730–C738.
, http://www.100md.com
Golub AS & Pittman RN (2005). Erythrocyte-associated transients in PO2 revealed in capillaries of rat mesentery. Am J Physiol Heart Circ Physiol 288, H2735–H2743.
Gruetter R (1993). Automatic, localized adjustment of all first- and second-order Shim coils. Magn Reson Med 29, 804–811.
Haseler LJ, Kindig CA, Richardson RS & Hogan MC (2004). The role of oxygen in determining phosphocreatine onset kinetics in exercising humans. J Physiol 558, 985–992.
, http://www.100md.com
Haseler LJ, Richardson RS, Videen JS & Hogan JS (1998). Phosphocreatine hydrolysis during submaximal exercise: the effect of FIO2. J Appl Physiol 85, 1457–1470.
Hofer SOP, van der Kleij AJ & Bos KE (1992). Tissue oxygenation measurement: A directly applied Clark-type electrode in muscle tissue. In Advances in Experimental Medicine and Biology, ed. Erdmann W & Bruley DR, pp. 779–784. Plenum Press, New York.
Hogan MC, Bebout DE & Wagner PD (1991a). Effect of hemoglobin concentration on maximal O2 uptake in canine gastrocnemius muscle in situ. J Appl Physiol 70, 1105–1112.
, 百拇医药
Hogan MC, Bebout DE & Wagner PD (1991b). Effect of increased HbO2 affinity on VO2max at a constant O2 delivery in dog muscle in situ. J Appl Physiol 70, 2656–2662.
Hogan MC, Bebout DE, Wagner PD & West PD (1990). Maximal O2 uptake of in situ dog muscle during hypoxemia with constant perfusion. J Appl Physiol 69, 570–576.
Kindig CA, Howlett RA & Hogan MC (2003). Effect of extracellular PO2 on the fall in intracellular PO2 in contracting single myocytes. J Appl Physiol 94, 1964–1970.
, http://www.100md.com
Marcinek DJ, Ciesielski WA, Conley KE & Schenkman KA (2003). Oxygen regulation and limitation to cellular respiration in mouse skeletal muscle in vivo. Am J Physiol Heart Circ Physiol 285, H1900–H1908.
Miura H, McCully K, Nioka S & Chance B (2004). Relationship between muscle architectural features and oxygenation status determined by near infrared device. Eur J Appl Physiol 91, 273–278.
Mole P, Chung Y, Tran T, Sailasuta N, Hurd R & Jue T (1999). Myoglobin desaturation with exercise intensity in human gastrocnemius muscle. Am J Physiol 277, R173–R180.
, http://www.100md.com
Ponganis PJ, Kreutzer U, Sailasuta N, Knower T, Hurd R & Jue T (2002). Detection of myoglobin desaturation in Mirounga angustirostris during apnea. Am J Physiol Regul Integr Comp Physiol 282, R267–R272.
Richardson RS (1998). Oxygen transport: air to muscle cell. Med Sci Sports Exerc 30, 53–59.
Richardson RS (2000). Intracellular Po2 and bioenergetic measurements in skeletal muscle: the role of exercise paradigm. Am J Physiol Regul Integr Comp Physiol 278, R1111–R1113.
, 百拇医药
Richardson RS, Grassi B, Gavin TP, Haseler LJ, Tagore K, Roca J & Wagner PD (1999a). Evidence of O2 supply-dependent VO2 max in the exercise-trained human quadriceps. J Appl Physiol 86, 1048–1053.
Richardson RS, Knight DR, Poole DC, Kurdak SS, Hogan MC, Grassi B & Wagner PD (1995a). Determinants of maximal exercise VO2 during single leg knee-extensor exercise in humans. Am J Physiol 268, H1453–H1461.
Richardson RS, Leigh JS, Wagner PD & Noyszewski EA (1999b). Cellular PO2 as a determinant of maximal mitochondrial O2 consumption in trained human skeletal muscle. J Appl Physiol 87, 325–331.
, http://www.100md.com
Richardson RS, Mudaliar SRD, Mathieu-Costello O & Wagner PD (1998a). VEGF mRNA response to acute exercise in skeletal muscle is attenuated following chronic training. Med Sci Sports Exerc 30, S50.
Richardson RS, Newcomer SC & Noyszewski EA (2001). Skeletal muscle intracellular PO2 assessed by myoglobin desaturation: response to graded exercise. J Appl Physiol 91, 2679–2685.
Richardson RS, Noyszewski EA, Kendrick KF, Leigh JS & Wagner PD (1995b). Myoglobin O2 desaturation during exercise. Evidence of limited O2 transport. J Clin Invest 96, 1916–1926.
, 百拇医药
Richardson RS, Noyszewski EA, Leigh JS & Wagner PD (1998b). Lactate efflux from exercising human skeletal muscle: role of intracellular PO2. J Appl Physiol 85, 627–634.
Richardson RS, Noyszewski EA, Saltin B & Gonzalez-Alonso J (2002). Effect of mild carboxy-hemoglobin on exercising skeletal muscle: intravascular and intracellular evidence. Am J Physiol Regul Integr Comp Physiol 283, R1131–R1139.
Richardson RS, Wagner H, Mudaliar SR, Henry R, Noyszewski EA & Wagner PD (1999c). Human VEGF gene expression in skeletal muscle: effect of acute normoxic and hypoxic exercise. Am J Physiol 277, H2247–H2252.
, 百拇医药
Richmond KN, Shonat RD, Lynch RM & Johnson PC (1999). Critical PO2 of skeletal muscle in vivo. Am J Physiol 277, H1831–H1840.
Roca J, Hogan MC, Story D, Bebout DE, Haab P, Gonzalez R, Ueno O & Wagner PD (1989). Evidence for tissue diffusion limitation of VO2max in normal humans. J Appl Physiol 67, 291–299.
Rossi-Fanelli A & Antonini E (1958). Studies of the oxygen and carbon monoxide equilibria of human myoglobin. Arch Biochem Biophys 77, 478–492.
, 百拇医药
Rowell LB (1993). Human Cardiovascular Control. Oxford University Press Inc, New York.
Rumsey WL, Vanderkooi JM & Wilson DF (1989). Imaging of phosphorescence: a novel method for measuring oxygen distribution in perfused tissue. Science 241, 1649–1651.
Sahlin K & Katz A (1989). Hypoxaemia increases the accumulation of inosine monophosphate (IMP) in human skeletal muscle during submaximal exercise. Acta Physiol Scand 136, 199–203.
, 百拇医药
Vanderthommen M, Duteil S, Wary C, Raynaud JS, Leroy-Willig A, Crielaard JM & Carlier PG (2003). A comparison of voluntary and electrically induced contractions by interleaved 1H- and 31P-NMRS in humans. J Appl Physiol 94, 1012–1024.
Wagner PD (1988). An integrated view of the determinants of maximum oxygen uptake. In Oxygen Transfer from the Atmosphere to Tissues, ed. Gonzalez NC & Fedde MR, pp. 245–256. Plenum Publishing Corporation, New York.
, 百拇医药
Wagner PD (1992). Gas exchange and peripheral diffusion limitation. Med Sci Sports Exerc 24, 54–58.
Wagner PD (2000). New ideas on limitations to VO2max. Exerc Sport Sci Rev 28, 10–14.
Wagner PD (2001). Skeletal muscle angiogenesis. A possible role for hypoxia. Adv Exp Med Biol 502, 21–38.
Wang J, Noyszewski EA & Leigh JS (1990). In vivo MRS measurement of deoxymyoglobin in human forearms. Magn Reson Med 14, 562–567.
, 百拇医药
Wang J, Shimoda LA, Weigand L, Wang W, Sun D & Sylvester JT (2005). Acute hypoxia increases intracellular [Ca2+] in pulmonary arterial smooth muscle by enhancing capacitative Ca2+ entry. Am J Physiol Lung Cell Mol Physiol 288, L1059–L1069.
Whalen WJ, Nair P & Ganfield RA (1973). Measurements of oxygen tension in tissues with a micro oxygen electrode. Microvasc Res 5, 254–262.
Wilson DF, Erecinska M, Drown C & Silver IA (1979). The oxygen dependence of cellular energy metabolism. Arch Biochem Biophys 195, 485–493.
, 百拇医药
Wittenburg BA & Wittenberg JB (1989). Transport of oxygen in muscle. Annu Rev Physiol 51, 857–878.
Wolin MS, Ahmad M & Gupte SA (2005). Oxidant and redox signaling in vascular oxygen sensing mechanisms: basic concepts, current controversies, and potential importance of cytosolic NADPH. Am J Physiol Lung Cell Mol Physiol 289, L159–L173., http://www.100md.com(Russell S. Richardson, Sandrine Duteil, )