Departments of 1Radiology, 2Physiology and Biophysics, and 3Bioengineering, University of Washington Medical Center, Seattle 98195; 4Children's Hospital and Regional Medical Center, Seattle, 98105; and Departments of 5Pediatrics and 6Anesthesiology, University of Washington Medical Center, Seattle, Washington 98195
Submitted 9 June 2003 ; accepted in final form 18 September 2003
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ABSTRACT |
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P/O; oxidative phosphorylation; proton leak; optical spectroscopy
In the absence of proton leak, a maximum of 4.66 ATP would be generated for each O2 consumed (or P/O = 2.33) by the mitochondria (26, 33). However, substantial mitochondrial proton leak has been suggested from measurements showing that only 3457% of the total oxygen consumption in mammalian skeletal muscle is actually due to ATP turnover reactions (38, 39). These studies result in in vivo P/O values <1.5, which are similar to values from mitochondria isolated from liver tissue (14, 19). In contrast, direct measurements of ATPase and oxygen consumption rates in isolated mammalian cardiac and amphibian skeletal muscles find in situ P/O values between 2.0 and 2.4 (31, 37, 40). Thus direct measurements of P/O in situ indicate mitochondria may be more tightly coupled than either in vitro or proton leak measurements suggest. This discrepancy points to the need for measurements of mitochondrial coupling under physiological conditions.
In this report, we demonstrate a method to determine the in vivo P/O in mouse skeletal muscle by directly measuring both ATPase and oxygen consumption rates in vivo. Several groups have used a combination of near-infrared (NIRS) and magnetic resonance spectroscopies (MRS) to assess both tissue oxygenation and energetic state (4, 29, 41) as well as to compare NIRS and MRS measurements of oxygenation (48). The limitation to these analyses is the inability of the NIRS data to separate the signals for hemoglobin (Hb) and myoglobin (Mb) saturations, which precludes a quantitative measurement of intracellular PO2 and tissue oxygen consumption. Our procedure for measuring in vivo P/O builds on these well-established, noninvasive techniques of 31P-MRS and optical spectroscopy. We use an ischemic protocol to deplete the oxygen stores in the tissue, which allows us to measure the ATPase and oxygen consumption rates independently. The new advance permitting the measurement of local tissue oxygen consumption is the ability to separate Hb from Mb saturation in optical spectra (35, 42, 44). This measurement, combined with determination of ATP synthesis by 31P-MRS, yields the in vivo P/O and degree of mitochondrial coupling in intact skeletal muscle.
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METHODS |
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For the optical experiments, the hair was removed from the lower hindlimb with a commercial hair removal cream (Neet). The leg was secured between the fiber-optic bundles by fixing the ankle and foot in place so that light traveled through the leg just distal to the knee. During the optical experiments the animals breathed 100% O2 to maintain high Hb saturation of the blood. For the MRS experiments, the leg was secured in the same manner so that the entire volume of the hindlimb musculature was sampled. Ischemia was induced during both optical and MRS experiments by suspending a 0.5-kg weight from a 3.2-mm cord wrapped around the leg. After the final spectra were acquired, the hindlimb was removed under anesthesia and frozen between aluminum blocks at liquid N2 temperature. The animals were then euthanized with an overdose of anesthetic. The animals were kept warm with forced air throughout the experiments and tissue sampling to maintain the temperature of the leg at 37 ± 0.5°C.
Another group of animals (n = 6) was treated systemically with 3 mg 2,4-dinitrophenol (DNP)/kg body mass before the optical and MRS experiments. The mice were injected intraperitoneally with 1 mg DNP/ml saline solution. Preliminary experiments indicated that 20 min was sufficient for the effect of DNP to reach a steady state. Therefore, 20 min was allowed before the start of the experiment for the effect of the DNP to reach a steady state. Optical and MRS experiments were carried out as described below.
Optical spectroscopy. Optical transmission spectra were acquired with a 7-mm optical fiber bundle (no. K42347, Edmund Scientific) to carry illuminating light and a 2-mm fiber bundle for transmitted light. The fiber bundles were mounted with a fixed separation distance of 6 mm in a custom-made probe stand. Illumination from a constant-intensity quartz-tungsten-halogen white light source (model 66184, Oriel Instruments) was passed through a 1.0-in. water filter and electromechanical shutter (no. 76995, Oriel Instruments) before transmission to the optical probe to decrease tissue heating. Constant light intensity was insured by a photo-feedback system (no. 68850, Oriel Instruments). Spectra from 450 to 950 nm were acquired via a diffraction spectrograph (no. 100S, American Holographics) with a 512-pixel photodiode array (no. C4350, Hamamatsu) using a 200-ms exposure time. Spectral acquisition was gated to acquire data at 1-s intervals, and the data were converted into digital form with a 16-bit analog-to-digital converter (no. AT-MIO-16X, National Instruments).
Partial least squares analysis and calibration sets. Partial least squares (PLS) analysis was used to determine the Hb and Mb saturations from the optical spectra of the mouse leg. PLS analysis is an extension of linear regression that is useful for extracting information on specific spectral components from complex spectra (21). PLS analysis is able to separate signals emanating from Hb and Mb in complex optical spectra by using information from the entire range of wavelengths. The analysis generates weighting coefficients for each wavelength (560850 nm) that correspond to a known value for Hb or Mb saturation in a calibration set. In this study, two distinct PLS algorithms were applied to each spectrum, one to determine Mb saturation and one for Hb saturation. These weighting coefficients were then applied to each experimental spectrum to predict the unknown saturation of Hb or Mb. Before the PLS algorithm was applied, spectra were preprocessed by taking the second derivative with respect to wavelength to remove the effect of baseline offsets. For a more complete description of the PLS analysis, see Refs. 2, 21, 44.
For this analysis, the calibration spectra must approximate the spectra acquired from the tissue of interest. The construction of the calibration set for the PLS analysis is explained in detail in Marcinek et al. (35). Briefly, the spectra of oxy and deoxy Hb and Mb and oxidized and reduced cytochrome c (cyt c) in scattering media are collected. Oxy and deoxy spectra of each absorber are mathematically added in different proportions to generate composite spectra that span the range of saturation and redox states for each absorber. The concentration of each absorber is optimized such that that the composite spectra approximate the in vivo spectra.
Two calibration sets were used to test the sensitivity and predictability of the PLS method for determining Hb and Mb saturation (42). One calibration set was used to determine the PLS coefficients for Mb and Hb saturation. These coefficients were then used to predict the saturation of Mb and Hb from the second calibration set. The slopes of the predicted vs. known saturations of the second calibration set were 0.974 and 0.934 for Hb and Mb, respectively (35). A slope of unity corresponds to perfect agreement between the predicted vs. known in vitro saturations. The standard errors of the residuals for these regressions were 0.046 and 0.074 for Hb and Mb, respectively, which indicate prediction errors for Hb and Mb saturations from composite spectra of 4.6% and 7.4%, respectively (35).
31P-MRS. The magnetic resonance experiments were performed in a 4.7-T Bruker horizontal-bore magnet. The mouse leg was probed by using a three-turn solenoidal coil with an inner diameter of 8 mm and a length of 6 mm tuned to 31P resonance frequency (81.15 MHz). Static magnetic field (Bo) homogeneity was optimized by shimming using the proton peak from tissue water. Unfiltered phosphocreatine (PCr) line widths were 2030 Hz. A high signal-to-noise 31P-MRS spectrum was taken under fully relaxing conditions (128 acquisitions with a 16-s interpulse delay) at a spectral width of 3,500 Hz consisting of 1,024 data points. During the experimental procedure, spectra were acquired with a standard one-pulse sequence with a 1.2-s interpulse delay and a 55° flip angle. For each dynamic spectrum, 16 free induction decays were added to obtain higher signal-to-noise spectra with a resolution of 26 s (sum of acquisition time and time to write data). The fully relaxed and dynamic free induction decays were Fourier transformed, baseline corrected, line broadened with a 10-Hz exponential filter, and zero filled. Fully relaxed peak areas were calculated by integration of the processed spectra with the Omega software on a General Electric console. Peak areas relative to a standard spectrum were determined from the dynamic spectra with the Fit to Standard algorithm (25). Resting PCr concentrations were calculated with the PCr-to--ATP peak ratio from the fully relaxed spectra and the ATP concentrations measured by HPLC analysis of extracts from frozen hindlimbs (50). PCr concentrations throughout the dynamic experiments were determined by comparing the relative peak areas with the resting PCr concentrations. pH was determined from the chemical shift of Pi relative to PCr in each spectrum (47).
Quantifying Hb and Mb. The frozen muscles of the hindlimb in the region of interest were separated from skin and bone on an iced aluminum block. The muscle tissue was pulverized at liquid N2 temperatures and separated into a portion for HPLC analysis and one for quantification of Hb and Mb with SDS-PAGE. Extracts of mouse muscle tissue were prepared for SDS-PAGE as described previously (35). The gels were stained with Coomassie blue and imaged, and the Mb and Hb bands of the standards and muscle extracts were quantified with NIH Image. Horse Mb and mouse Hb were used as standards and run on each gel. Quantification was repeated three times for each sample, and the mean Hb and Mb concentrations were used for the calculation of the oxygen consumption rates. Hb and Mb concentrations were measured for each animal.
Oxygen consumption. The oxygen consumption rate was determined from the rate of decline of tissue oxygen content according to
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Data analysis. All statistical analyses were conducted with GraphPad Prism v. 3.0a software for the Macintosh (GraphPad Software). ATPase and oxygen consumption rates were determined from the slopes of the least squares regression lines through the plots of PCr concentration ([PCr]) and total oxygen stores during ischemia, respectively. Two-tailed t-tests were used for comparisons between control and DNP-treated groups. Data are presented as means ± SE. Significance was defined as P < 0.05.
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RESULTS |
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Hb and Mb saturation in ischemia. Optical spectroscopy was used to measure the dynamics of Hb and Mb saturation during ischemia. Figure 2 presents a representative plot of the dynamics of Mb saturation for one animal during the ischemia and recovery protocol. Desaturation began soon after onset of ischemia and reached a nadir after 5 min, which represented 0% saturation. Full saturation (100%) was set by the peak achieved after reoxygenation during the hyperemic period of recovery (peak intensity at 7.5 min.). Mb saturation returned to the resting value in 45 min after the start of the recovery period and was 87 ± 3.5% (n = 5) of the hyperemic peak value. This disparity in the hyperemic and resting Mb saturation levels indicates that Mb was significantly desaturated from resting muscle. A similar process was used to set the saturation end points for Hb. Full saturation was set by using the value from resting muscle with the animal breathing 100% oxygen; complete desaturation was set as the value achieved after 5 min of ischemia. For both Hb and Mb, complete desaturation is apparent from the asymptote of the saturation curve after 5 min of ischemia.
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Oxygen consumption rate. Measurement of oxygen consumption involved determining the rate of deoxygenation of Hb and Mb during ischemia as shown in Fig. 3A. After a rapid, complete interruption of blood flow in the mouse hindlimb, Hb and Mb began to desaturate as the oxygen stores were depleted by the tissue oxygen consumption. This depletion rate measures the rate of oxygen consumption. The rate of tissue oxygen consumption was constant, as shown by the linear change in oxygen content, down to approximately half-saturation of Mb. During this early period of ischemia, oxygen stores in the tissue were sufficient for oxidative phosphorylation to meet the ATP demand of the muscle. This is clear from the constant PCr concentration in this animal over the first 90 s of ischemia (Fig. 3B).
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Figure 4 presents an example of the decrease in oxygen stores during ischemia for a control and a DNP-treated animal. The mouse hindlimb has a low concentration of Mb (30 ± 2.0 µmol/g tissue; n = 5) relative to the resting Hb concentration (55 ± 13 µmol/g tissue; n = 5). Thus 13.1 ± 1.1% of the tissue oxygen content at rest was bound to Mb compared with 78.8 ± 1.2% bound to Hb. The remaining 8.1 ± 1.2% was dissolved in the tissue. The solid regression lines in Fig. 4 show the range of oxygen content used to calculate resting oxygen consumption. The linear decrease in oxygen stores indicates that the oxygen uptake rate is constant over this range of intracellular PO2 values, 103 mmHg (Mb saturation of 8050% in Fig. 3) for the control and DNP-treated groups (35).
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ATP synthesis rates. PCr is constant during the deoxygenation phase (Fig. 3B). The PCr dynamics are indicative of the balance of ATP supply to demand via the creatine kinase reaction (7). The constancy of the PCr during the first 2 min of ischemia indicates that the rate of ATP synthesis was sufficient to meet the resting ATP demands of the muscle tissue until low Mb and Hb saturations were reached. At the point that intracellular PO2 begins to inhibit oxidative phosphorylation (low Mb saturation in Fig. 3A), PCr is consumed by the creatine kinase reaction to maintain the ATP supply in the muscle. This decline in PCr measures the cellular ATPase rate (regression line in Fig. 3B). We have found (13, 30) that the ATPase rate does not change with brief anoxia, indicating no change in cellular ATP demand in the presence of sufficient PCr to meet ATP needs. Figure 5 plots the mean pH values throughout ischemia for the control animals. There is a slight alkanalization (nonsignificant slope, n = 6; P = 0.45) of the tissue due to the consumption of protons by the creatine kinase reaction throughout the period of PCr consumption used to determine the ATPase rate. During this initial period there is no detectable increase in glycolytic ATP supply as indicated by the alkanalization of the tissue and the linear decrease in PCr concentration (7, 11). The significance of the lack of change in ATPase with anoxia and the absence of an increase in the glycolytic ATP supply is that the PCr decline measures not only the cellular ATP demand but also the mitochondrial ATP supply needed to meet that demand in the steady state in resting muscle (7). This cellular ATPase rate, and therefore the mitochondrial ATP supply, was not affected by systemic treatment with DNP despite a nearly twofold increase in respiration rate (Table 1; two-tailed t-tests, P = 0.02 for oxygen consumption and P = 0.74 for ATPase rate).
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P/O. In vivo P/O was determined by dividing the resting ATPase rate by the oxygen consumption rate measured during ischemia for each animal. Figure 6 compares the mean P/O values from the control and DNP-treated groups with the theoretical maximum for a muscle oxidizing palmitate. No significant difference is apparent between the theoretical P/O value and the measured value for mouse skeletal muscle in vivo (P = 0.51, 1-sample t-test). The similarity of the theoretical and measured P/O values indicates that only a small fraction of oxygen consumption is uncoupled from phosphorylation. However, systemic treatment with the mitochondrial uncoupler DNP significantly reduced the in vivo P/O of the resting skeletal muscle in the mouse hindlimb from control values [2.16 ± 0.24 vs. 1.37 ± 0.22 for control and 3 mg DNP/kg body mass (DNP3) groups, respectively; two-tailed t-test, P = 0.035]. This DNP treatment raised the fraction of oxygen consumption that did not lead to phosphorylation from 9% to >40%. Thus measurement of the P/O is sensitive to the application of an uncoupler. These results indicate that in resting mouse skeletal muscle there is little reduction of the in vivo P/O below the theoretical value for fully coupled mitochondria.
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DISCUSSION |
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Oxygen consumption (PLS). Oxygen consumption was determined in the intact mouse hindlimb by using a PLS analysis to separately quantify Hb and Mb saturations from in vivo optical spectra. The similar optical spectra of Hb and Mb make it difficult to differentiate the two with standard discrete wavelength analysis. However, the small differences at a given wavelength become large when examined across the full spectrum. PLS analysis takes advantage of these differences across the spectrum (560850 nm in this case) to distinguish the saturations of Hb and Mb from in vivo spectra (2, 3). These changes in saturation during ischemia can then be used to calculate the rate of oxygen consumption of the muscle. The rates of oxygen consumption determined with this technique were close to those reported for isolated mouse soleus and extensor digitorum longus (EDL) muscles (8.19.2 nmol O2·g1·s1), after adjusting to 37°C with a Q10 of 2 (15, 49).
The main potential source of error in our determination of oxygen consumption in vivo is our assumption of 100% Hb saturation at rest. We maximized Hb saturation at rest by having the mouse breathe 100% O2 during the optical experiments. If instead we assume that Hb is 80% saturated in the resting mouse hindlimb, our calculated resting oxygen consumption rate would decrease to 5.3 ± 0.4 nmol O2·g1·s1. This would yield an in vivo P/O of 2.6 ± 0.3, which is above the theoretical maximum for mitochondrial ATP synthesis (i.e., 2.33). Thus we conclude that Hb saturation at rest must be close to 100% in the resting mouse hindlimb while the animal is breathing 100% oxygen.
This optical method measures the total oxygen consumption of the tissues in the hindlimb including mitochondrial and nonmitochondrial sinks. Nonmitochondrial oxygen consumption has been reported to be 914% of the total resting oxygen consumption in rat skeletal muscle (18, 38). If 10% of the measured oxygen consumption in the mouse hindlimb is due to nonmitochondrial oxygen sinks, the mitochondrial P/O measured in this study increases to 2.40 ± 0.27 vs. 2.33 for the theoretical value. Thus a small nonmitochondrial oxygen uptake eliminates the apparent uncoupling.
31P-MRS. The rate of ATP synthesis of mitochondria is determined from the initial breakdown rate of PCr measured with 31P-MRS during ischemia. This use of ATPase to measure ATP supply is based on the requirement that the rate of ATP synthesis must match the ATP demand of the cell under steady-state conditions. Another method used to measure ATP synthesis rates in tissue in vivo is saturation transfer by 31P-MRS (8, 27, 28, 31, 40). This method uses the rate of phosphate exchange among Pi, PCr, and ATP to quantify mitochondrial ATP synthesis and yields P/O (2.12.4) similar to those reported here (31, 40). However, this technically sophisticated technique requires extensive controls to quantify the multisite exchange of phosphate, as well as the sources and sinks of ATP synthesis and use (9, 31). The method presented here simplifies the measurement of oxidative ATP synthesis by using PCr breakdown to quantify ATP demand during tissue anoxia.
P/O values. The in vivo P/O for mitochondrial ATP synthesis measured in this study, 2.16 ± 0.24, is close to the stoichiometric maximum of 2.33 for mitochondria oxidizing palmitate. The theoretical value for oxidation of palmitate is based on a net yield of 107 ATP from the consumption of 23 O2 molecules (P/O = 2.33), calculated assuming ATP-to-NADH and ATP-to-FADH2 ratios of 2.5 and 1.5, respectively (26, 33). The absence of a detectable glycolytic flux in the resting muscle indicates that the mitochondria are primarily oxidizing fatty acids. These calculations indicate that >90% of the total cellular oxygen consumption in mouse skeletal muscle at rest is due to ATP synthesis. Treatment with DNP reduced the in vivo P/O in the mouse hindlimb to 1.37 ± 0.22. Therefore, partial uncoupling of the mitochondria reduced the percentage of total cellular oxygen consumption resulting in ATP production to <60%.
An in vivo P/O of 2.16 is consistent with other direct measurements of ATPase and oxygen consumption rates in isolated cardiac and skeletal muscles obtained using independent techniques. Paul and Kushmerick (37) measured a P/O of 2 in stimulated, isolated frog sartorius, based on biochemical determinations of the change in phosphorylated compounds (PCr, ATP) and oximetry to measure oxygen uptake. Values of 2.12.4 for P/O were also found in isolated working rat heart preparations by using saturation transfer 31P-MRS to measure the rate of mitochondrial ATP synthesis (31, 40). These results also agree with direct measurements from mitochondria isolated from rat hearts, where the P/O was determined to be 2.4 (14).
Indirect calculation of mitochondrial coupling based on proton leak measurements in muscle tissue (38, 39) and hepatocytes (23, 36) yielded low P/O values of 1.5. These values are based on measurements of the contribution of proton leak to mitochondrial oxygen consumption after chemically blocking F1F0 ATPase activity. The chemical interventions necessary for the measurement of proton leak are a potential explanation of the difference between the in vivo P/O values measured directly in intact tissue and those calculated from measurements of proton leak. In studies in situ in the rat hindlimb, blocking the mitochondrial ATPase with oligomycin resulted in an increase in mitochondrial membrane potential (38, 39). To reverse this effect of oligomycin, electron transport was chemically inhibited to return the membrane potential to the preoligomycin levels. It is possible that these multiple chemical interventions led to changes in mitochondrial function either through direct effects on the mitochondria or alteration in the metabolic state of the cell that led to an overestimation of the contribution of proton leak to cellular respiration.
In summary, we describe a method that uses MRS and optical spectroscopy during ischemia to independently measure ATP and oxygen flux rates in vivo in skeletal muscle. These measured fluxes are then used to determine the mitochondrial P/O in skeletal muscle in vivo. This approach is applicable to a variety of physiological conditions, such as exercising or diseased muscle, and is only limited by the requirements that there is no blood flow to the tissue and that the muscle can be made anoxic. The in vivo P/O measured in the intact mouse hindlimb was not significantly different from the theoretical maximum, indicating that ATP synthesis is tightly coupled to mitochondrial oxygen consumption under these conditions.
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ACKNOWLEDGMENTS |
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GRANTS
This project was supported by National Institutes of Health Grants AR-45184, AR-36281, and AG-00057.
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FOOTNOTES |
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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. Section 1734 solely to indicate this fact.
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