Resistance to O2 diffusion in anemic red muscle: roles of flux density and cell PO2.

Normal and anemic dog gracilis muscles were compared at equal O2 uptake rates (VO2) to locate the principal site of resistance to diffusive O2 transport. Anemia halved the hematocrit and the number of red blood cells per square millimeter of muscle cross section. Flow doubled in anemia, and flow times arterial O2 content, PO2 of effluent blood, and O2 extraction per red blood cell were approximately the same as control. Nevertheless, intracellular PO2 was significantly lower in anemia. At any instant the aggregate red blood cell surface area for O2 release was about half normal. Because the flux (VO2) was the same as control, the driving force for diffusion from red blood cell to myocyte should have doubled. An estimate of the total driving force from red blood cell to mitochondria was greater in anemia. This increase was much less than a factor of 2 because lower intracellular PO2 increases myoglobin-facilitated diffusion, thus decreasing resistance inside the myocyte. The role of myoglobin and the coupling of convective to diffusive transport are discussed. We conclude that the principal resistance to O2 diffusion lies outside the myocyte.

O2 transport and its interaction with metabolism; a systems view of aerobic capacity.

This commentary demonstrates that VO2max depends, in part, on diffusive O2 transport; exercise hyperemia is necessary but not sufficient. Experiments and new mathematical models place the principal site of resistance to O2 diffusion between the surface of a red cell and the sarcolemma. The large drop in PO2 over this short distance is caused by high flux density and absence of heme protein O2 carrier in this region. PO2 gradients within red myocytes are shallow at high VO2 because myoglobin acts as O2 carrier and PO2 buffer. At high VO2 cell PO2 is less than 5 torr, the myoglobin P50. Low cell PO2 relative to blood PO2 is essential to a) maintain the driving force on diffusion as capillary PO2 falls, and b) to increase myoglobin-facilitated diffusion and the overall O2 conductance. O2 per se does not limit mitochondrial ATP production under normal circumstances because the low O2 drive on electron transport is compensated by greater phosphorylation and redox drives. These metabolic adaptations support transcapillary diffusion by defending VO2 at the low cell PO2 required to extract O2 from blood. Thus aerobic capacity is a distributed property, dependent on the interaction of transport and metabolism as a system.

Interaction of blood flow, diffusive transport and cell metabolism in isovolemic anemia.

1) High blood flow can compensate for half-normal hematocrit, leaving the rate at which O2 is offered to the capillaries unchanged. Nevertheless, intracellular PO2 is lower in anemia, indicating impaired diffusive transport. 2) Anemia increases O2 flux per red cell and decreases functional capillary surface area. These changes increase flux density and the extracellular component of resistance to diffusive O2 transport, in accord with current theory (Federspiel and Popel, 1986; Groebe, 1990; Hellums, 1977). 3) Maintenance of diffusive flux in presence of anemia required a larger delta PO2 between Hb and Mb, and higher intracellular O2 conductance brought about by greater Mb-facilitated diffusion. Both compensations depend on lower PmbO2. 4) PmbO2 and creatine charge fall with increasing VO2 and ATP demand. These responses, as well as adaptive changes in redox help maintain VO2 in the presence of a lower O2 drive on electron transport. 5) Greater engagement of reserves of both transport and metabolism limits the range of aerobic performance in anemia. 6) The match between the transcapillary and mitochondrial O2 fluxes depends on interaction of transport and metabolism as a system.

Arteriovenous oxygen diffusion shunt is negligible in resting and working gracilis muscles.

Distribution of O2 within and among arterioles and venules was determined in dog and rat gracilis muscles with a cryospectrophotometric method. Saturation in 40-microns arterioles was not demonstrably different from saturation in the aorta even when flow was abnormally low. Arterioles greater than 40 microns ran parallel to venules. Measurements and a mathematical model indicate that diffusive shunting is negligible for typical separation distances between arterioles and venules. Most separation distances were greater than 30 microns. In some venule segments less than 15 microns from an arteriole, saturation within 10 microns of the wall facing the arteriole was higher than at other locations within the venule. However, saturation in the population of venules did not increase with venule diameter, and mean venular saturation was not different from saturation in effluent blood. We make the following conclusions: 1) a small arteriovenous diffusive O2 flux exists in postural muscles; 2) contribution of this flux to O2 mass balance is negligible; 3) O2 diffusivity of the arteriolar wall and surrounding tissue in vivo cannot be much higher than O2 diffusivity determined in vitro; and 4) effluent PO2 closely approximates mean end-capillary PO2.

Intracellular PO2 in individual cardiac myocytes in dogs, cats, rabbits, ferrets, and rats.

Myoglobin (Mb) saturation in individual subepicardial myocytes was determined by cryospectroscopy in dogs, cats, ferrets, rabbits, and rats. Mb saturation within 800 microns of the epicardium is not affected by quick freezing or absorption of light by cytochromes. The PO2 in equilibrium with Mb (PMbO2) was calculated from the Mb oxydissociation curve. The minimum PMbO2 found among the 1,000 cells examined was 2.5 Torr, at least five times the critical PO2 for cytochrome turnover in myocardium. The maximum PMbO2 found was about one-half that in subepicardial venules, suggesting a large change in PO2 between capillaries and the cytosol. PMbO2 was the same in right and left ventricles and was unchanged by moderate hemodynamic stress. Median PMbO2 was remarkably uniform among species (range, 4.3-7.0 Torr in 20 animals), even though left ventricular work per minute varied approximately 200-fold, heart rate about fivefold, and arterial O2 content about twofold. Relatively uniform Mb saturation below venous PO2 should accelerate release of O2 from capillaries, promote Mb-facilitated O2 diffusion, and minimize diffusive O2 shunting.

Defining hypoxia: a systems view of VO2, glycolysis, energetics, and intracellular PO2.

The necessity for defining hypoxia as O2-limited energy flux rather than low partial pressure is explored from a systems perspective. Oxidative phosphorylation, the Krebs cycle, glycolysis, substrate supply, and cell energetics interact as subsystems; the set point is a match between ATP demand and aerobic ATP production. To this end the transport subsystem must match the transcapillary and mitochondrial O2 fluxes. High transcapillary O2 flux requires intracellular PO2 in the range 1-10 Torr. In this range the O2 drive on electron transport must be compensated by adaptive changes in the phosphorylation and redox drives. Thus the metabolic subsystem supports diffusive O2 transport by maintaining O2 flux at intracellular partial pressures required for O2 release from blood. Since responses to stress are distributed according to the state of the entire system, several simultaneous metabolic measurements, including intracellular PO2 (or a known direction of change in intracellular PO2) and the O2 dependence of a measurable function are required to judge the adequacy of O2 supply. ATP demand and aerobic capacity must also be evaluated, because the hypoxic threshold depends on the ratio of ATP demand to aerobic capacity. The application and limitation of commonly used criteria of hypoxia are discussed, and a more precise terminology is proposed.

Effect of tachycardia on intracellular PO2 and reserves of O2 transport in subendocardium of mouse left ventricle.

Intracellular PO2 (PmbO2) was determined by cryospectrophotometry in individual cardiac myocytes. The rate of progression of the freezing front was sufficient to trap the O2 distribution across the wall of the mouse left ventricle. The transmural PmbO2 distribution was uniform despite moderate tachycardia. Maximal heart rate produced a small but statistically significant transmural O2 gradient but no hypoxic myocytes in subendocardium. Reserves of diffusive as well as convective transport contribute to maintenance of aerobic metabolism during tachycardia.

Regulation of VO2 in red muscle: do current biochemical hypotheses fit in vivo data?

Observations used to test biochemical models of the regulation of O2 consumption (VO2) by cytosolic phosphate energy state must include a change in intracellular pH and/or a change in the adenine nucleotide or phosphate pools [Connett, R. J. Analysis of metabolic control: new insights using a scaled creatine kinase model. Am. J. Physiol. 254 (Regulatory Integrative Comp. Physiol. 23): R949-R959, 1988]. Data were collected over a wide range of energy turnover from canine muscles in situ. Intracellular PO2, glycolytic intermediates, adenine nucleotides, creatine, phosphocreatine (PCr), phosphate, and intracellular pH were determined for each muscle. PO2 was used to eliminate muscles in which VO2 could have been O2 limited (PO2 less than 0.5 Torr). This removed an important source of heterogeneity. Because adenine nucleotide and phosphate pools were constant relative to the creatine pool, discrimination among models depended solely on pH. The observed pH range from 7.2 to 5.9 did not permit separation of [PCr] from log[( ATP4-]/[ADP3-][H2PO4-]) (phosphorylation potential) as a regulatory parameter for VO2. However, [ADP] could be eliminated as an independent regulator. Because 90% of variability in VO2 was accounted for by phosphate energetics, an independent redox component must be small when intracellular PO2 greater than 0.5 Torr.

Anatomical determinants of O2 flux density at coronary capillaries.

Calculations indicate that the PO2 in plasma falls to zero approximately 3 microns from an erythrocyte at O2 consumption (VO2) characteristic of myocardium (Federspiel, W.A., and A. Popel, Microvasc. Res. 32: 164-189, 1986). We measured distances between individual red cells along capillaries in rat hearts rapidly frozen in situ. Cell spacing varied widely even in branches of the same capillary. Plasma gaps between red cells were divided into two populations, those less than 5 microns and those greater than 5 microns. Mean gap lengths were 2.1 and 16.5 microns, respectively. Although the number of long plasma gaps was underestimated, gaps greater than 5 microns accounted for one-third of observed capillary length. Frozen muscles were also viewed in cross section. Because the depth of penetration of light was approximately equal to 3 microns, counts of red cell-containing capillary profiles in cross section depend on cell spacing as well as on number of cell-containing flow paths. Counts varied markedly with arterial O2 partial pressure, indicating that the capillary surface area functional for O2 transport changes in response to stress. The adaptive role of change in O2 flux density (flux per area) is discussed in light of new knowledge of tissue O2 gradients.

Precapillary O2 loss and arteriovenous O2 diffusion shunt are below limit of detection in myocardium.

1. Mean intracellular PO2 is much lower than mean venous PO2 in subepicardium. 2. The drop in Hb saturation between aorta and terminal arterioles is within the 5% error of our method. 3. Arteriolar O2 has no effect on saturation in paired countercurrent venules in myocardium. 4. Saturation in coronary venules is independent of venule diameter and indistinguishable from saturation in macroscopic epicardial veins. 5. Since diffusive O2 shunting is negligible and PO2 is approximately linearly related to saturations over the observed range, mean coronary venous PO2 should closely approximate mean-end capillary PO2. 6. O2 mass transport from blood to tissue requires a steep PO2 gradient between the capillary and the surface of a tissue cell.

Intracellular PO2 in long axis of individual fibers in working dog gracilis muscle.

Dog gracilis muscles were frozen in situ during twitch contraction at 25-100% of aerobic capacity. O2 saturation of myoglobin (Mb) was determined from spectrophotometric measurements along individual fibers. Intracellular PO2 was calculated from the oxymyoglobin dissociation curve. At all work rates, long lengths of fibers were found in which saturation and PO2 were within the 4% error of measurement. During work at approximately 25% of aerobic capacity, Mb functioned at high saturation on the shallow slope of its dissociation curve. Consequently PO2 was poorly buffered. Although the range of saturation was small, PO2 varied up to 15 Torr along a fiber, and gradients up to 0.3 Torr/micron were observed. In contrast, at high O2 consumption (VO2), Mb functioned on the steep slope of its dissociation curve. Therefore gradients in intracellular PO2 along the axis of a myocyte were small (less than 0.05 Torr/micron) despite large gradients in Mb saturation (up to 0.5%/micron). Changes in intracellular PO2 over hundreds of microns did not reflect the large drop in intracapillary PO2 between arterioles and venules. Because intracellular PO2 is low and relatively uniform in the long axis of a fiber, the driving force for release of O2 from blood is dominated by intravascular PO2 in working red muscle.

How large is the drop in PO2 between cytosol and mitochondrion?

The subject of this brief review is the size of the local drops in PO2 around consuming mitochondria. We show that large drops (several Torr or more) are in conflict with the predictions of basic diffusion theory, when one uses accepted values for relevant parameters. In particular, oxygen diffusion coefficients must be reduced by at least a factor of 10 below measured values to reconcile Fick's law with large PO2 drops. Experimental evidence offered for large drops is often ambiguous because of system heterogeneities. In those cases where tractable models of heterogeneous systems can be developed, the experimental data are consistent with drops in PO2 on the order of a few hundredths of a Torr between cytosol and mitochondrion.

Minimum intracellular PO2 for maximum cytochrome turnover in red muscle in situ.

Probability distributions of myoglobin (Mb) saturation and intracellular PO2 were determined with subcellular spatial resolution in dog gracilis muscles during steady-state twitch contraction at 5-100% of maximal rate of O2 consumption (VO2). Calculations (Clark, A., and P. A.A. Clark. Biophys. J. 48: 931-938, 1985) and measurements (Gayeski, T. E. J., and C. R. Honig. Adv. Exp. Med. Biol. 200: 487-494, 1986) indicate that the PO2 in equilibrium with Mb is virtually identical to the PO2 at cytochrome aa3. Median intracellular PO2 and PO2 in the lower tails of probability distributions were poorly correlated with VO2. The variability of cell PO2 was greatly diminished when median PO2 was less than the PO2 for half saturation of MB, since Mb acts as a PO2 buffer. The lower tails of PO2 distributions contained almost no anoxic loci even when median PO2 was less than 1 Torr. VO2 was well correlated with the concentration ratio of phosphocreatine to free creatine (PCr/Crf) over a wide range of PO2. PO2 greater than or equal to 0.5 Torr supported maximal VO2 and energy demand. We conclude that 1) the mechanism of action of cytochrome aa3 is the same in red muscle in vivo as in mitochondria in vitro, and 2) an upper bound on the apparent Michaelis constant for maximal VO2 of red muscle is approximately 0.06 Torr.

Comparison of intracellular PO2 and conditions for blood-tissue O2 transport in heart and working red skeletal muscle.

1. Neither anoxic nor hypoxic cells were found in epicardium of anaesthetized dogs, cats, rabbits and rats despite heterogeneity of flow (Wieringa et al., 1982) and haematocrit (Honig et al., in press) in the coronary capillary network. 2. Median PO2 in unstressed dog heart and cat heart are 4.8 and 5.2 torr, respectively. These values are close to the P50 of the oxymyoglobin dissociation curve, and well above PcritO2. 3. A dense, interconnected capillary network and high capillary haematocrit appear essential to achieve high O2 extraction at flows characteristic of maximally working myocardium. 4. Mb promotes O2 transport in myocardium by: a) maximizing the driving force for transcapillary diffusion, b) minimizing spatial variability in PmbO2, c) facilitating O2 diffusion in myocytes and, d) permitting close capillary packing without a diffusion shunt for O2. 5. The O2 conductance of the red cell-capillary system is a major determinant of O2 mass transfer in red muscle.

O2 gradients from sarcolemma to cell interior in red muscle at maximal VO2.

The intracellular distribution of O2 in cross sections of dog gracilis muscles was determined by myoglobin (Mb) cryospectrophotometry. The volume sampled by the photometer was approximately 30 micron3 and contained 1-2 mitochondria. Measurements could be made to within 3 micron of capillaries without interference from hemoglobin. Mb saturation was uniform at all loci examined when respiration was blocked with cyanide. During twitch contraction at maximum O2 consumption, saturations within a cell cross section varied by up to 20%. The corresponding difference in partial pressure of O2 (PO2) was 1.5 Torr. Circumferential O2 gradients parallel to and 5 micron from the sarcolemma were greatest near capillaries. They did not exceed 0.1 Torr/micron and were dissipated within 25 micron of the sarcolemma. Gradients perpendicular to the sarcolemma were less than 0.02 Torr/micron. Saturation was not significantly correlated with cell diameter. Minimum PO2 was seldom located at the center of the cell cross section. Differences in saturation between contiguous cells often exceeded 10%. The distribution of O2 within cells appeared to reflect both an intercellular O2 flux and and an O2 flux from adjacent capillaries. Data agree qualitatively and quantitatively with mathematical models that take account of the particulate nature of blood and facilitated diffusion by Mb.

Lactate efflux is unrelated to intracellular PO2 in a working red muscle in situ.

Blood flow, lactate extraction, and tissue lactate concentration were measured in an autoperfused pure red muscle (dog gracilis). Muscles were frozen in situ during steady-twitch contraction at frequencies of 1-8 Hz [10-100% of maximum O2 consumption (VO2max)]. Myoglobin saturation was determined spectrophotometrically with subcellular spatial resolution. Intracellular PO2 (Pto2) was calculated from the oxymyoglobin-dissociation curve. Tissue lactate was well correlated with VO2 but not with Pto2. Lactate efflux increased markedly above a threshold work rate near 50% VO2max. Efflux was neither linearly correlated with tissue lactate nor related to Pto2. Pto2 exceeded the minimum PO2 for maximal VO2 in each of 2,000 cells examined in muscles frozen at 1-6 Hz. A small population of anoxic cells was found in three muscles at 8 Hz, but lactate efflux from these muscles was not greater than from six other muscles at 8 Hz. Our conclusions are that 1) the concept of an anaerobic threshold does not apply to red muscle and 2) in absence of anoxia neither tissue lactate nor blood lactate can be used to impute muscle O2 availability or glycolytic rate. A mechanism by which the blood-tissue lactate gradient could support aerobic metabolism is discussed.