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Am J Physiol Heart Circ Physiol 277: H1831-H1840, 1999;
0363-6135/99 $5.00
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Vol. 277, Issue 5, H1831-H1840, November 1999

Critical PO2 of skeletal muscle in vivo

Keith N. Richmond, Ross D. Shonat, Ronald M. Lynch, and Paul C. Johnson

Department of Physiology, University of Arizona Health Sciences Center, Tucson, Arizona 85724


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The main purpose of this study was to determine the interstitial oxygen tension at which aerobic metabolism becomes limited (critical PO2) in vivo in resting skeletal muscle. Using an intravital microscope system, we determined the interstitial oxygen tension at 20-µm-diameter tissue sites in rat spinotrapezius muscle from the phosphorescence lifetime decay of a metalloporphyrin probe during a 1-min stoppage of muscle blood flow. In paired experiments NADH fluorescence was measured at the same sites during flow stoppage. NADH fluorescence rose significantly above control when interstitial PO2 fell to 2.9 ± 0.5 mmHg (n = 13) and was not significantly different (2.4 ± 0.5 mmHg) when the two variables were first averaged for all sites and then compared. Similar values were obtained using the abrupt change in rate of PO2 decline as the criterion for critical PO2. With a similar protocol, we determined that NADH rose significantly at a tissue site centered 30 µm from a collecting venule when intravascular PO2 fell to 7.2 ± 1.5 mmHg. The values for critical interstitial and critical intravascular PO2 are well below those reported during free blood flow in this and in other muscle preparations, suggesting that oxygen delivery is regulated at levels well above the minimum required for oxidative metabolism. The extracellular critical PO2 found in this study is slightly greater than previously found in vitro, possibly due to differing local conditions rather than a difference in metabolic set point for the mitochondria.

metabolic hypothesis; reduced nicotinamide adenine dinucleotide; oxidative metabolism; oxygen delivery; in vivo microscopy; phosphorescence lifetime


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

OXYGEN DELIVERY to resting skeletal muscle in vivo is generally assumed to be at a level sufficient to sustain the oxidative requirements of the tissue. The oxygen tension required at the mitochondrial level to support oxidative metabolism (critical PO2) is <1 mmHg (15, 30, 43), whereas tissue oxygen tension reported in most skeletal muscles in vivo is 15-25 mmHg (3, 27, 42). In further support of this assumption, a number of studies have shown that oxygen consumption of resting skeletal muscle is independent of blood flow until flow is reduced by at least 50% below normal levels (21, 28, 35). Also, using endogenous NADH levels as an indicator of tissue metabolic state, we have found in the cat sartorius muscle that a reduction in blood flow of at least 50% was required to increase NADH fluorescence (23).

There are, however, reports that oxygen consumption in skeletal muscle decreases linearly with flow reduction below normal levels (9, 42, 45), and there is one report that oxygen consumption is flow limited even at normal flow rates (5). The latter findings could be explained if flow distribution in the muscle were markedly heterogeneous (25), if the diffusivity for oxygen in muscle were low, or if the critical PO2 were higher in vivo than reported for isolated cells or mitochondria. The last possibility has been suggested to explain how blood flow in resting skeletal muscle could be regulated by oxygen demand even with the tissue oxygen levels well above the values obtained for critical PO2 in vitro (8).

To resolve the question of critical PO2 in vivo, we studied a surgically exposed skeletal muscle preparation (the rat spinotrapezius) with intact circulation and innervation and used measurement techniques similar to those employed previously for in vitro determination of critical PO2 in single fibers from the same muscle (29). In the present study we briefly stopped blood flow in the muscle and measured the time required for mitochondrial metabolism at localized (20-µm diameter) tissue sites to become oxygen limited as signaled by a rise in endogenous NADH fluorescence. Separately, we used an oxygen-sensitive optical probe to measure the interstitial PO2 at the same sites in the muscle during flow stoppage. Combining data from the two studies enabled us to determine interstitial oxygen tension at the time NADH fluorescence rose. A sudden change in the rate of PO2 decrease during flow stoppage provided a separate assessment of critical PO2. Tissue sites selected for study were in the vicinity of postcapillary venules and because of their location should reflect the minimum tissue PO2 levels during free flow.

Using a similar protocol we determined the intravascular PO2 in a 20-µm venule at which NADH fluorescence at a tissue site centered 30 µm away began to rise. We also compared PO2 values for postcapillary venules and adjacent tissue regions during free flow conditions.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Muscle Preparation

Studies were performed on 18 fasted juvenile male Sprague-Dawley rats (75-100 g body wt) anesthetized with pentobarbital sodium (60 mg/kg, Nembutal, Abbott) administered by intraperitoneal injection. Supplemental anesthesia consisting of alpha -chloralose (2%) and urethan (10%) was infused continuously through a femoral venous cannula at the rate of 1.5 ml/h. The left femoral artery was cannulated for the measurement of systemic arterial pressure with a Statham pressure transducer (P23 Gb). A tracheal tube was inserted to maintain a patent airway. Animal use was in accord with the National Institutes of Health guidelines and approved by the University of Arizona Institutional Animal Care and Use Committee.

The spinotrapezius muscle was isolated using a method originally described by Gray (13) and modified for these experiments as described previously (34). An incision was made in the skin overlying the left dorsal margin of the muscle, which was then isolated by blunt dissection from the underlying muscle layers and surrounding tissue, leaving the main arterial and venous vessels, as well as nerves, intact. Small vessels were sutured as necessary to prevent bleeding. Sutures sewn to the muscle margins were used to secure the muscle on the microscope stage. During the surgical procedure, a constant drip of Plasmalyte R solution (Travenol) was maintained. The solution, composed of (in meq/l) 140 sodium, 10 potassium, 5 calcium, 3 magnesium, 47 acetate, and 8 lactate, was adjusted to pH 7.4 and warmed to 37°C. On completion of the surgery, the animal was placed on its right side on a specially designed platform fitted to the microscope stage, and the exteriorized muscle was mounted on a Plexiglas plate at its in situ length and width. A quartz window at the center of the plate allowed transillumination of the tissue. The edges of the muscle were covered with moist gauze, and the entire muscle was covered with transparent polyvinyl film (Saran Wrap, Dow Corning), which provided an oxygen-impermeable barrier and prevented drying of the tissue. Animal body and muscle temperature were maintained at 37°C by electrically controlled heating coils lying beneath the platform.

Systemic Measurements

Arterial blood pressure, electrocardiogram, and heart rate were monitored continuously and recorded on a four-channel strip-chart recorder (model 2600, Gould-Brush). Arterial pH, PCO2, and PO2 were monitored periodically using a blood gas analyzer (ABL-330 Radiometer, Copenhagen, Denmark). Positive-pressure ventilation was used when necessary to maintain the appropriate physiological blood gas parameters.

In Vivo Microscope System

Because a detailed description of the microscope used in this study has been presented in previous reports (33, 39), only the salient characteristics of the system are presented below. The system has been used previously for in vivo measurements of NADH fluorescence (23, 24, 38) and oxygen tension (34) at localized (20-µm diameter) sites together with continuous video imaging and recording of the microscopic field of view. In the present study measurement of both NADH fluorescence and oxygen tension necessitated a manual change of filters and dichroic mirrors. However, the time required for these changes (15 s) precluded our making the two measurements simultaneously. Therefore, we monitored these two variables separately in paired studies and compared the time course of changes during the experimental procedure as described below. Matched Leitz objectives (magnification ×20, numerical aperature = 0.40) were used as condenser and objective in this application.

NADH Fluorescence Measurement

NADH fluorescence of the tissue was excited through the microscope condenser using the 366-nm line of a Hg arc lamp positioned beneath the microscope stage. The fluorescence emission at 450 nm was collected with the microscope objective, directed to a cooled photomultiplier tube, and detected using photon-counting techniques. In previous studies we found that the rise in the fluorescence signal during blood flow stoppage was quantitatively related to the increase in tissue NADH as determined by chemical analysis of tissue samples (39). The intensity of the excitation wavelength transmitted through the tissue was monitored with a second photomultiplier tube, and experiments in which this variable changed significantly during the occlusion period were not included in the data set for analysis. In this phase of the study data were collected at 1-s intervals.

Oxygen Measurement

Oxygen measurements were made using the method of Vanderkooi et al. (40) and applied to the microcirculation as described by Intaglietta et al. (14) and Shonat et al. (33). With the use of this technique, a metalloporphyrin compound was bound to albumin and injected into the circulation, where it distributed in the plasma and, in the preparation used in this study, also moved into the tissue spaces. When excited at the appropriate wavelength the compound produces a phosphorescence signal the decay time of which is a function of the oxygen tension in the area sampled. An average of 32 individual decay curves for each sampling point was log converted, and a best-fit estimate of decay time was obtained by linear regression. The calculated decay time was used to calculate the PO2 from the Stern-Volmer equation (40). If the linear coefficient of determination (R2) fell below 0.80, the measurement was rejected. This rejection criterion was employed previously as indicative of a low signal-to-noise ratio and/or an inhomogenous PO2 sampling region (33). The portion of the microscope system used for this measurement consisted of a strobe light source whose filtered output was directed through the microscope objective onto a tissue region ~100 µm in diameter. The phosphorescent signal was collected by the objective and passed through a dichroic mirror to a separate, red-sensitive photomultiplier tube. The oxygen probe was excited at 532 ± 23 nm, and emission was collected at >560 nm.

The oxygen probe used to produce the phosphorescence signal was palladium meso-tetra[4-carboxyphenyl]porphine (Porphyrin Products, Logan, UT). Palladium porphyrine (240 mg) was added to 1 ml of DMSO (Sigma, St. Louis, MO) and warmed to 40-50°C to dissolve the probe. This solution was combined with 23 ml of physiological saline containing bovine serum albumin (60 mg/ml, fraction V; ICN Biochemicals) and buffered to a pH of 7.4 to provide a stock solution of 10 mg probe/ml. An amount of the stock solution sufficient to provide a concentration of 15 mg probe/kg body weight was injected as a bolus through the femoral venous cannula and allowed to equilibrate until sufficient phosphorescence signal was present in the tissue before the experimental protocol was begun. There was no apparent change in blood flow to the muscle with injection of the probe.

The phosphorescence signal was processed by custom software (33) to obtain on-line oxygen tension at 2-s intervals. Oxygen tension was displayed on a computer monitor and stored together with all collection parameters in an MS-DOS-based 386 personal computer (WJM, Tucson, AZ) for off-line analysis.

Experimental Protocol

Paired studies were performed in which either NADH fluorescence or oxygen tension at a site 20 µm in diameter was monitored during a 1-min occlusion of blood flow to the muscle. Muscle blood flow was interrupted by simultaneous occlusion of the veins and arteries at the proximal end of the muscle using a small metal bar (1-mm diameter) mounted on a micromanipulator (Narshige). This arrangement provided rapid occlusion and release with minimal tissue movement.

Critical tissue PO2 determination. The first series of experiments was designed to measure changes in mitochondrial NADH fluorescence and extracellular oxygen at a tissue site during blood flow stoppage. The center of the measuring site selected was 30 µm from the inner wall of a 20-µm (ID) venule. Criteria used for selecting a site for measurement were 1) a location near a postcapillary venule with a diameter of 20 µm (±5 µm); 2) stable blood flow, i.e., an absence of vasomotion; and 3) an absence of other venules or arterioles within 50 µm of the selected tissue site and venule.

One oxygen and one NADH measurement protocol was performed at each tissue site, with the order of the protocols determined randomly. The protocol for the oxygen measurement consisted of a 1-min control period, a 1-min occlusion period, and a 1-min postocclusion period. For the NADH fluorescence measurement this 3-min experimental period was preceded and followed by a 1-min period of oxygen measurement. This was done to verify that oxygen tension under free flow conditions was the same for the oxygen and NADH measurement protocols.

Intravascular PO2 during shift in tissue redox state. The second series of experiments was designed to compare oxygen tension in a 20-µm venule and NADH fluorescence at a 20-µm-diameter tissue site centered 30 µm from the inner wall of the venule during a 1-min occlusion. The criteria for tissue site and venule selection and the protocol were identical to those described above for the first experimental series.

Comparison of intravascular and tissue PO2. To determine the PO2 levels at intravascular and tissue sites similar to those in the protocol described above, the third series of experiments consisted of an oxygen measurement for 1 min at a 20-µm venule and for 1 min at a tissue site. Measurements were made during free flow conditions with a 1-min period between determinations at the two sites to allow for repositioning of the microscope stage. The order of the measurements was randomly determined.

Statistics

For the purpose of analysis, the NADH fluorescence data during the occlusion protocols were divided into four phases: the preocclusion control period, the period of rapid change during blood flow stoppage, the period of slow change during blood flow stoppage, and the postocclusion recovery period. The NADH fluorescence data for these periods were analyzed using linear regression (SigmaPlot, Jandel). The time at which NADH fluorescence began to increase during the occlusion was defined by regression analysis of the fluorescence signal during the preocclusion control period. These data were used to calculate a 95% confidence interval. When two consecutive data points rose above the 95% confidence interval (after the occlusion began), mitochondrial metabolism was assumed to be altered. The first of these time points was matched to the oxygen measurement at that specific time and used to determine the tissue or the venular PO2 at which tissue oxidative metabolism began to be limited.

Linear regression analysis was also performed on the oxygen tension data for the four periods described above. In addition, the second and third periods, which encompassed the duration of blood flow stoppage, were analyzed using a nonlinear, four-parameter curve-fitting procedure (SigmaPlot). The demarcation point between the two periods provided a separate estimate of critical PO2.

Comparisons between venular and tissue oxygen tension measurements during free flow conditions were made using unpaired Student t-tests (SigmaPlot). Significance was set at the P < 0.05 level. All data are expressed as means ± SE.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Tissue PO2 and NADH Fluorescence During Blood Flow Stoppage

Tissue PO2 and NADH fluorescence were monitored at 13 sites during successive 1-min occlusions. Figure 1 shows the results from successive NADH fluorescence and oxygen measurements at one tissue site. The PO2 during the preocclusion control period averaged 17.1 ± 0.5 mmHg.


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  		Fig. 1.   Top: NADH fluorescence (open diamonds) and oxygen tension (open circles) measured in a tissue site during a period of flow stoppage. Solid lines are linear regression except during flow stoppage, where a four-parameter fit to oxygen tension data is also shown. Dotted line in control period before occlusion is 95% confidence limit for NADH fluorescence. Note that oxygen tension begins to fall immediately on flow stoppage, whereas NADH fluorescence does not increase until 13 s later. AU, arbitrary units. Bottom: regression coefficient (R2) of phosphorescence lifetime decay curve fit to a single exponential for individual oxygen measurements shown in top.

Occlusion of the supply arteries to the spinotrapezius muscle caused immediate flow stoppage verified visually as a rapid cessation of red blood cell movement. Concurrent with flow stoppage was a rapid decrease in the interstitial oxygen tension. Oxygen tension fell in the first 13 s of occlusion and showed little change for the remainder of the 60-s occlusion period. The PO2 change during this 13-s period appeared to be somewhat better described by the four-parameter, nonlinear curve than by linear regression. The nonlinear fit was used to identify the critical PO2 and not to predict the PO2 fall. NADH fluorescence intensity began to increase 13 s after flow stoppage and reached a plateau after 30 s. The initial rise in NADH fluorescence corresponded to a PO2 value of 2.1 mmHg, which was taken as the critical PO2. As is apparent in Fig. 1, the initial rise in NADH fluorescence also coincided with an abrupt change in the rate of fall of PO2. The sudden change may also reflect the point at which oxygen consumption became supply limited. Extrapolating the linear portions of the rapid and slow changes in PO2 to their intersection yielded a value of 1.6 mmHg in this experiment.

On release of the occlusion both the oxygen tension and NADH fluorescence rapidly returned to preocclusion control values. In some instances (not shown in Fig. 1) the oxygen tension rose above and NADH fluorescence intensity fell below preocclusion values before returning to control levels. The gradual decrease in NADH fluorescence intensity over the period of the experiment was due most likely to photobleaching and was seen in all experiments.

A plot of the mean values of PO2 for this series of experiments is shown in Fig. 2. The mean PO2 during the preocclusion control period was 15.0 ± 0.3 mmHg, and oxygen tension fell rapidly in the early phase of the occlusion. NADH fluorescence rose significantly at 9.9 ± 1.0 s after the beginning of flow stoppage. The average NADH fluorescence profiles are not shown for clarity. The mean interstitial critical PO2 was determined from the pooled data by calculating the oxygen tension at this time from the nonlinear curve. From Fig. 2 it can be seen that the calculated critical PO2 at this time point was 2.4 ± 0.5 mmHg. The corresponding value obtained using a linear regression of oxygen tension decline was essentially identical (2.4 ± 0.8 mmHg). The critical PO2 can also be calculated by taking the mean of the individual determinations in each experiment. For all 13 sites tested the mean critical PO2 calculated in this way was slightly, but not significantly, higher (2.9 ± 0.5 mmHg).


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  		Fig. 2.   Extracellular tissue oxygen tension in 13 postcapillary venular regions during flow stoppage. Solid lines are linear regression except during flow stoppage, where a four-parameter fit to oxygen tension data is also shown. Mean critical PO2 was defined by intersection of regression line for oxygen data and line representing average time after stop flow for NADH to rise above 95% confidence level.

As in the example shown in Fig. 1, the rise in NADH fluorescence appeared to coincide with the abrupt change in slope of the PO2 decline. The latter averaged 2.4 ± 0.4 mmHg when the individual data points were first averaged and 2.8 ± 0.5 mmHg when calculated from individual experiments and then averaged. After the inflection point in the PO2 trace there was an additional modest but significant decline, reaching 1.2 ± 0.1 mmHg at the end of the occlusion period.

Venular PO2 and Tissue NADH Fluorescence During Blood Flow Stoppage

The protocol for the second series of experiments was identical to the preceding except that oxygen tension was measured in a 20-µm venule centered 30 µm from the tissue site. Figure 3 is representative of the venular oxygen tension and tissue NADH fluorescence data obtained during a 1-min occlusion. The mean oxygen tension during the control period for the experiment shown in Fig. 3 was 19.8 ± 2.7 mmHg. On occlusion of the arteries and flow stoppage, the fall in venular oxygen tension was slower than in the tissue, and the change in slope at low PO2 was less abrupt. The pattern of NADH fluorescence change with occlusion is very similar to that seen in the previous series, but there was a longer delay after occlusion before fluorescence intensity increased (17 s). On release of the occlusion, NADH fluorescence intensity fell concurrently with a rise in oxygen tension. In the example shown in Fig. 3, the oxygen tension rose transiently above the preocclusion control level, whereas NADH fluorescence intensity fell slightly below preocclusion levels, but this "overshoot" of NADH and oxygen tension was not seen in all preparations.


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  		Fig. 3.   Oxygen tension (open circles) measured in a 20-µm venule and NADH fluorescence (open diamonds) measured from an adjacent tissue site during a 1-min period of flow stoppage. Solid lines are linear regression except during flow stoppage, where a four-parameter fit to oxygen tension data is also shown. Dotted line is 95% confidence limit for NADH fluorescence in control period before occlusion.

Figure 4 shows the combined oxygen data for 12 venular sites. The mean PO2 during the preocclusion control period was 22.7 ± 0.3 mmHg. As in Fig. 3, the oxygen tension continued to decline through the entire occlusion period. Figure 4 also illustrates the slower return of venular oxygen tension to control values in comparison to the tissue sites. The oxygen tension at which NADH fluorescence increased after blood flow stoppage was determined as described for the previous experimental series using both linear and nonlinear fitting. The increase in NADH fluorescence intensity took place 16.3 ± 2.4 s after the beginning of the occlusion. The venular PO2 at this time, calculated using a linear fit to the fast component of the change in oxygen tension, was 8.3 ± 1.5 mmHg, whereas with the four-parameter nonlinear function the value was 7.7 ± 1.3 mmHg. The two values were not significantly different. The data were not amenable to separate determination of critical PO2 from comparison of the fast and slow rates of PO2 decline.


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  		Fig. 4.   Oxygen measurement in 12 20-µm postcapillary venules during flow stoppage. Solid lines are linear regression except during flow stoppage, where a four-parameter fit to oxygen tension data is also shown.

Venular PO2 Versus Tissue PO2

The third series of experiments was designed to compare, during free flow conditions, the oxygen tension at tissue and venular sites similar to those studied in the previous series. Whereas the oxygen measurements obtained during the control period of the preceding series provided some information on this point, a direct comparison between the PO2 at a venular site and at an adjacent tissue site during free flow conditions with a short time interval (60 s) between measurements was not possible in that series.

Figure 5 is an example of the data from one experiment and illustrates typical temporal variations at the two sites. The mean PO2 for the venule at this site was 20.8 ± 0.4 mmHg and ranged from 17.8 to 24.7 mmHg, whereas mean tissue PO2 was 17.0 ± 0.3 mmHg and ranged from 13.9 to 20.8 mmHg.


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  		Fig. 5.   Oxygen measurement from a 20-µm venule followed by extracellular tissue oxygen measurement at an adjacent tissue site during free flow conditions.

Figure 6 presents data from 17 paired measurements. As is apparent in this plot, tissue PO2 is closely related to venular PO2 at all sites, and a high degree of correlation (R2 = 0.82) was observed. Also, there was a difference of 3.0 ± 0.6 mmHg (range 0.5-8.2 mmHg) that appeared to be independent of the absolute level of PO2. At all but two sites, the venular PO2 was significantly higher than the adjacent tissue PO2 (P < 0.05). The mean venular PO2 was 17.7 ± 1.3 mmHg with a range of 5.5-27.5 mmHg. The mean tissue PO2 was 14.6 ± 1.4 mmHg and ranged from 3.3 to 24.2 mmHg. At no site was the tissue PO2 less than that determined in the preceding series to be critical for oxidative metabolism.


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  		Fig. 6.   Combined data for 17 determinations of oxygen tension in a 20-µm venule and interstitial PO2 at an adjacent tissue site as shown in Fig. 5. * Difference between venular and interstitial PO2 was not significant.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Principal Findings

The principal purpose of this study was to determine the critical PO2 of rat spinotrapezius muscle in vivo. We found that the interstitial fluid PO2 at which tissue oxidative metabolism was altered significantly was 2.4-2.9 mmHg. With the use of a similar measurement technique (comparison of PO2 and rise of NADH fluorescence) to determine the critical PO2 in vitro for single myocytes isolated from the same muscle, a mean extracellular critical PO2 value of 1.25 ± 0.22 mmHg (n = 6) was obtained (29). The difference, although small, is significant at the level of P < 0.05. For reasons that are discussed below this difference may be a result of experimental conditions in which the two measurements are made rather than a true difference between the in vivo and in vitro critical PO2.

Reliability of In Vivo Measurement and Sources of Error

Oxygen measurement. The phosphorescence lifetime technique has been used previously in this and other laboratories for determination of oxygen tension in the microcirculation (4, 14, 33, 34). In a previous report from our laboratory (33), we showed that this method produces reliable estimates of PO2 in blood and utilized the method to determine venular PO2 (34). Applications of the method to tissue oxygen determinations have been less common. However, in a recent study it has been shown that tissue PO2 values obtained with the phosphorescence technique are essentially identical to those obtained with the oxygen microelectrode (4). A necessary requirement for this application is that sufficient dye leak from the blood into the tissue to provide an adequate signal. Our criterion of an R2 value >0.80 for fitting the phosphorescence decay curve to a monoexponential process was intended to satisfy this requirement. Because the dye is bound to albumin, it presumably distributes in the interstitial fluid space but does not enter the muscle fibers to a significant degree (41). In addition, the probe must remain bound to albumin to give an accurate measure of PO2. This has not been tested directly, although it can be inferred from our results. Unbound probe has a much shorter lifetime, and this would lead to a large increase in calculated PO2, much higher than we see in vivo. Thus it is unlikely that the probe is dissociated from albumin in the interstitium.

A second consideration is whether the phosphorescence decay method consumes sufficient oxygen to significantly lower the tissue PO2. However, in a previous study (4), it was shown that the tissue PO2 reading obtained with the oxygen microelectrode did not change significantly during photoexcitation of the dye. Moreover, we note that in the present study, the time delay for the rise in NADH (when PO2 was not being measured) was the same as for the sudden change in rate of PO2 decrease determined with the optical method. If photoexcitation of the porphyrin dye significantly increased the rate of oxygen depletion in the tissue, we would have expected the sudden change in rate of fall of PO2 to precede the rise in NADH fluorescence.

NADH measurement. Tissue NADH fluorescence changes have been used previously to assess changes in the mitochondrial metabolic state both in vivo and in vitro as reviewed by Toth et al. (38, 39). This method was used in previous studies of tissue metabolic state in skeletal muscle (23, 38) in this laboratory, and the rise in fluorescence was shown to be in quantitative agreement with changes in tissue NADH levels (mitochondrial and cytoplasmic) as determined by chemical analysis of tissue samples (38, 39).

Sampling volume. By use of a pinhole aperture, the sampling area for both oxygen and NADH measurements was limited to a spot 20 µm in diameter referred to the tissue. However, the depth of the sampling site for NADH determined by transillumination was probably greater by a factor of 5 or 10 (39). As a consequence it is likely that the NADH measurement was obtained from a tissue region of ~20 µm diameter and >= 100 µm in depth. The muscle itself is ~200-µm thick, and the diameter of the muscle fibers is estimated to be 50-60 µm (29). Thus it is likely that more than one cell was present in the sampling volume. Because the oxygen tension was determined by epi-illumination, the sampling volume may have been more limited. It is also likely that there is some variation in the PO2 within its sampling volume. The signals obtained by both methods represent an average that would be weighted in favor of the tissue region best in focus within the sampling site (39).

The spinotrapezius muscle is of mixed fiber type with approximately equal numbers of slow-oxidative (SO), fast-oxidative-glycolytic (FOG), and fast-glycolytic (FG) fibers (37). Mitochondrial volume density for these fiber types ranges from 10% (SO) to 1% (FG) (10). This would lead to a disproportionate contribution of SO fibers to the NADH signal when such fibers are present in the sampling volume.

A possible source of error in the use of the NADH signal is the criterion for determination of the earliest rise in NADH fluorescence. Because our criterion was an elevation above the 95% confidence interval, it is possible that NADH may have begun to rise slightly earlier. In addition, the suitability of NADH as a rapid reporter of metabolic shift may be considered. Wilson et al. (43) have suggested that a change in redox state of cytochrome aa3 may precede a change of the NAD/NADH redox couple.

Error because of protocol. An additional limitation in determination of critical PO2 in this study is the use of separate rather than simultaneous measurements of PO2 and NADH fluorescence. This could lead to errors because of differences in initial tissue or venular PO2 at the time of occlusion, rate of fall of blood flow during occlusion, and changes in tissue conditions between consecutive runs. We minimized the first problem by determining that tissue PO2 was the same before PO2 and NADH protocols, the second by using a rapid occlusion and verifying visually the sudden stoppage of flow, and the third by randomly choosing the order in which the two measurements were made.

Error in measured critical PO2. As indicated above, one reason for the variability in the values obtained for critical PO2 may be the protocol itself. Because these errors may be random rather than systematic, the average value may be a more accurate assessment of critical PO2 than any single determination. A second source of error may be the heterogeneous distribution of the tissue fibers with their different mitochondrial densities and oxygen consumption. By making both NADH and PO2 measurements at the same site, the effect of these two factors on critical PO2 at that site should be minimized. A third and inescapable source of error is the heterogeneity of interstitial PO2 within the sampling volume. Because NADH fluorescence will rise as soon as critical PO2 is reached at any region within the sampling volume, the NADH signal may increase significantly, whereas the mean PO2 is still above the critical level. The heterogeneity of tissue PO2 in our data sample was minimized by our criterion of a regression coefficient (R2) >0.80 for acceptance of the monoexponential fit to the decay curve. Thus instances where heterogeneity was substantial were likely to have been eliminated. Finally, it should be noted that the critical PO2 value obtained from the rise in NADH signal is very close to that obtained from the abrupt shift in rate of PO2 decline. The latter would indicate exhaustion of the oxygen supply to the mitochondria.

Comparison With Literature Findings on Critical PO2

Isolated mitochondria. The Michaelis constant (Km) of cytochrome oxidase reported for isolated mitochondria from heart (22, 36) and skeletal muscle (7) ranges from 0.05 to 0.5 mmHg. It has been suggested that Km values obtained in isolated mitochondria or isolated cells may be significantly different from those extant in vivo due to the absence of regulatory mechanisms normally present in vivo. Tamura et al. (36) reported that the apparent Km of cytochrome aa3 for oxygen (determined optically) was higher in the isolated perfused rat heart than in mitochondria isolated from the heart (6 vs. 0.3 mmHg). However, Gayeski et al. (12), using myoglobin saturation to determine the intracellular PO2 in a quick-frozen dog gracilis muscle, concluded that the Km for cytochrome aa3 was not higher in vivo than reported in isolated mitochondria, and maximal oxygen consumption could be maintained at intracellular oxygen tensions >0.5 mmHg. Although it is noted that PO2 estimates from frozen tissue sections may be affected by changes in myoglobin saturation during the freezing process (6), this finding suggests that critical PO2 in vivo does not differ from that in vitro.

Isolated cells. It would be expected that the Km of suspensions of cells, utilizing extracellular PO2 measurements, would be higher than those from isolated mitochondria (20) due to the additional diffusion barrier between the mitochondria and the extracellular space. Wilson et al. (43) reported the Km of cytochrome oxidase for cardiac cells in suspension was ~1 µM (0.7 mmHg). In the study by Richmond et al. (29) of isolated myocytes of rat spinotrapezius muscle, the critical PO2 determined from the rise in NADH fluorescence was slightly higher (1.25 ± 0.2 mmHg) and is not unexpected, because critical PO2 reports the first detectable evidence of oxygen lack. Rumsey et al. (32) calculated that the drop in oxygen concentration from the plasma membrane to mitochondria of cardiac myocytes should range from 0.27 to 2.11 µM (0.18-1.4 mmHg), depending on the metabolic rate and the distribution of individual mitochondria within the myocyte, specifically, their distance from the cell wall. The extracellular critical PO2 value obtained from isolated rat spinotrapezius muscle cells is consistent with these calculations (29). As indicated above, it is possible that because of PO2 gradients in the interstitium, our in vivo estimate for extracellular critical PO2 may overstate the actual value. If this were not the case, our data would be consistent with only a small effect of in vivo conditions, of the order of 1 mmHg, on critical PO2. Our data from the present study therefore do not support the hypothesis that mitochondrial oxidative metabolism in vivo becomes limited at substantially higher intracellular oxygen tensions than those found in vitro.

Comparison of Interstitial Critical PO2 With Tissue PO2

The mean oxygen tension in the interstitium measured at perivenular sites in this preparation during free flow conditions (Fig. 6) was 14.6 ± 1.4 mmHg (range 3.3-24.2 mmHg). Other investigators have employed oxygen microelectrodes to measure oxygen tension (3, 19, 27) in muscle but did not determine whether the electrode tip was within the cell or in the interstitial space during recording. These investigators reported mean tissue oxygen tensions well above our estimated critical PO2 and also with a wide range of values. Lash and Bohlen (19) reported a midcapillary tissue PO2 in resting rat spinotrapezius muscle of 27.8 ± 13.7 (SE) mmHg using a suffusing solution equilibrated with 10% O2-5% CO2-85% N2. Boegehold and Johnson (3) found a mean tissue PO2 of 22.8 ± 3.3 (SE) mmHg and a range of 4-40 mmHg in the region of venous capillaries in cat sartorius muscle using a suffusion solution of 5% CO2-95% N2. However, the suffusate PO2 at the entrance to the muscle reservoir averaged 9.7 mmHg, and 20 µm above the muscle surface was 23.5 ± 1.5 (SE) mmHg. In rat cremaster muscle Prewitt and Johnson (27) reported a tissue PO2 in the region of venous capillaries with an N2 equilibrated solution of 19 ± 3.4 (SE) mmHg and a range of 2.9-41 mmHg, but when a 10% O2 suffusing solution was used, the mean rose only slightly to 20 ± 1.7 (SE) mmHg, whereas the range decreased (14-24 mmHg).

The studies described above with the Whalen oxygen microelectrode necessitated the use of suffusing solution equilibrated with N2 or an N2-O2 gas mixture over the muscle. Even without oxygen in the gas mixture it is evident from the report by Boegehold and Johnson (3) that oxygen diffused into the flowing suffusate, perhaps from the atmosphere and sites within the muscle itself. Because our preparation was covered with an O2-impermeable film (Saran Wrap), the values obtained may be closer to those found in unexposed tissue. In addition, our measurements were made in the vicinity of venules, where values may be lower than those in the vicinity of the capillary network.

Comparison of Critical Interstitial PO2 With Venular PO2

The studies shown in Figs. 3-6 were designed to better understand the relation between PO2 at tissue sites and that at an adjacent venule. As shown in Fig. 4, during flow stoppage, NADH fluorescence intensity at the tissue site increased when venular PO2 fell to 7.7 ± 0.5 mmHg. From the data in Fig. 2 we assume that interstitial PO2 at the tissue site at this time is 2.4-2.9 mmHg, resulting in a venule-to-tissue PO2 difference of 4.8-5.3 mmHg. For comparison we have calculated the venular PO2 that would be predicted from the Krogh equation (18), assuming the venule was the sole source of oxygen and 2.4 mmHg was the critical PO2 in the interstitial fluid at the tissue site. The Krogh diffusion coefficient (K) used for this calculation was based on a recent measurement of the diffusivity and solubility of oxygen at 37°C by Bentley et al. (1) (K = 9.54 × 10-10 ml O2 · cm-1 · s-1 · mmHg-1). The PO2 difference between the inner wall of a 20-µm vessel and a tissue site centered 30 µm away as obtained from the Krogh equation was 5.9 mmHg, which is only slightly higher than the experimental estimate of 4.8-5.3 mmHg.

During free flow (Fig. 6), the tissue-to-venule PO2 gradient is less than during flow stoppage and appears to be relatively constant (3.0 ± 0.6 mmHg), although in two instances there was no significant difference between tissue and venular PO2 levels. In conjunction with the predictions from the Krogh equation, this finding suggests that the postcapillary venules provide part of the oxygen requirements of surrounding tissue. During free flow it is likely that adjacent capillaries also provide a significant amount of oxygen to the tissue site. During blood flow stoppage the latter source may have been more rapidly depleted, allowing the venules to become a more important source of oxygen.

It has been noted in other studies that PO2 levels gradually increase from the smaller to the larger venules (14, 33), possibly due to shunting effects of capillaries with higher flow rates. The present study indicates that PO2 in postcapillary venules is generally higher than surrounding tissue, but further study would be required to determine whether such a relationship holds in other parts of the venular network.

The mean venular oxygen tension in the paired venular/tissue PO2 series was 17.7 mmHg (Fig. 6), whereas that during the control period of the occlusion study (Fig. 3) was 23.1 mmHg. These two values are in the range obtained in 11- to 31-µm venules in a previous study in this preparation (34). The values are consistent with the end-capillary oxygen tension (20-25 mmHg) estimated for cardiac and red skeletal muscle (44).

Implications for Blood Flow Regulation

The metabolic hypothesis of blood flow regulation as described by Berne (2) proposes that blood flow is maintained at adequate levels through a feedback mechanism responsive to changes in oxidative metabolism in the parenchymal tissue. This regulation may come about through a partial shift to anaerobic metabolism and formation of vasodilator metabolites by the parenchyma when oxygen demand increases or blood flow decreases (11, 17, 23, 31, 38). In resting muscle this hypothesis would imply that there are normally present tissue sites where oxygen tension is below critical PO2, which our studies indicate would average 2.4-2.9 mmHg in the interstitium.

Tissue oxygen levels in resting muscle are generally much higher than this value. To reconcile these observations with the metabolic hypothesis, it has been suggested by Rosenthal et al. (30) and Jobsis et al. (16) that the critical PO2 in vivo is different from that measured in vitro. As indicated above our results do not support this postulate. In addition, during free flow tissue PO2 at all sites tested was above the critical level. Because there are occasional tissue sites that are near critical PO2, it is possible that feedback from such sites could play a role when blood flow falls. According to our findings in cat sartorius muscle (23), a 50% reduction in flow for at least 30 s would be required, using a rise in NADH as the criterion for shift in redox state.

In conclusion, this study shows that interstitial critical PO2 in resting rat spinotrapezius muscle is 2.4-2.9 mmHg. This value is slightly but significantly higher than that determined previously in isolated cells from the same muscle. The difference may be due to PO2 variations within the sampling volume in the in vivo measurement rather than a difference in critical PO2 for the mitochondria in vivo and in vitro. This critical PO2 is well below that found during free flow in tissue regions where PO2 is expected to be low, namely, in the vicinity of postcapillary venules. These findings do not support a role for metabolic feedback mechanisms in blood flow regulation in resting skeletal muscle. This study also shows that a small PO2 gradient exists between intravascular PO2 in collecting venules and that in tissue sites 30 µm away. This gradient increases during flow stoppage and approaches that predicted from the Krogh equation.


    ACKNOWLEDGEMENTS

The authors gratefully acknowledge the skilled technical support of Bethany Skovan, Vivian Wu, and Harry W. Schechner.


    FOOTNOTES

This study was supported by National Heart Lung and Blood Institute Grants HL-07249, HL-15390, and HL-17421.

Current address of K. N. Richmond: Dept. of Physiology and Biophysics, Univ. of Washington, Seattle, WA 98195; current address of R. D. Shonat: Dept. of Biomedical Engineering, Worcester Polytechnic Institute, Worcester, MA 01609.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: P. C. Johnson, Dept. of Bioengineering, M0412, Engineering Bldg Unit 1, Univ. of California, San Diego, La Jolla, CA 92093-0412.

Received 12 March 1999; accepted in final form 15 June 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Heart Circ Physiol 277(5):H1831-H1840
0002-9513/99 $5.00 Copyright © 1999 the American Physiological Society


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