Oxygen Transport to Tissue: Satellite Symposium of the 28th International Congress of Physiological Sciences, Budapest, Hungary, 1980

Silver

SESSION I

HETEROGENEITIES AND O2 TRANSPORT, MODEL AND EXPERIMENTAL

Outline

Chapter 1: TISSUE OXYGEN SUPPLY AND CRITICAL OXYGEN PRESSURE

Chapter 2: REDOX GRADIENTS IN OXYGEN DELIVERY TO TISSUE

Chapter 3: CHANNELS OF OXYGEN TRANSPORT FROM BLOOD TO MITOCHONDRIA

Chapter 4: OXYGEN TENSION PROFILES AND OXYGEN CONSUMPTION INHOMOGENEITIES IN THE ARTERIAL WALL: IMPLICATIONS FOR ATHEROGENESIS

Chapter 5: CORRELATION OF ELECTRICAL SIGNALS AND MITOCHONDRIAL REDOX STATE DURING SPREADING DEPRESSION

Chapter 6: RATE OF O2 CONSUMPTION AND REDOX STATE OF CYTOCHROME C (+C1) MEASURED IN LIVER IN SITU BY REFLECTANCE SPECTROPHOTOMETRY: EFFECT OF ETHANOL INGESTION

Chapter 7: HETEROGENEOUS OXYGEN TRANSPORT WITHIN MALIGNANT TUMORS

Chapter 8: EVALUATION OF ERG AS A MODEL OF CEREBRAL TISSUE PO2 CHANGES WITH ENVIRONMENTAL STRESS

Chapter 9: A THEORETICAL METHOD OF ANALYSIS OF THE DYNAMICS OF OXYGEN EXCHANGE IN THE MICROCIRCULATION OF THE INTESTINAL TRACT IN THE DOG THAT IS APPLICABLE IN THE SHOCK STATE

Chapter 10: QUANTITATIVE ANALYSIS OF REFLECTION SPECTRA BY SIMULATION EXPERIMENTS ON TISSUE

Chapter 11: EXPERIMENTAL AND MATHEMATICAL STUDIES OF OXYGEN TRANSPORT IN THE FETAL CEREBRAL MICROCIRCULATION

Chapter 12: EFFECT OF DIFFUSION HETEROGENEITY ON OXYGEN TENSION IN TISSUE

Chapter 13: INTRACELLULAR MICROHETEROGENEITY OF OXYGEN CONCENTRATIONS

Chapter 14: NON-INVASIVE PO2 MONITORING AT THE PALPEBRAL CONJUNCTIVA

Chapter 15: MICROREGIONAL OXYGEN TRANSMISSIBILITY FOLLOWING ISCHEMIA IN THE BRAIN CORTEX

Chapter 16: DISCUSSION AND SUMMARY SESSION I HETEROGENEITIES AND O2 TRANSPORT, MODEL AND EXPERIMENTAL

TISSUE OXYGEN SUPPLY AND CRITICAL OXYGEN PRESSURE

D.W. Lübbers,     Max-Planck-Institut für Systemphysiologie, Rheinlanddamm 201, 4600 Dortmund 1, FRG

Publisher Summary

This chapter discusses tissue oxygen supply and critical oxygen pressure. It is well known that for the whole animal as well as for the isolated organ, in a certain range, the O2 consumption, vO2, is independent of the O2 offered by the respired gas mixture or by the arterial blood. It is found that when oxygen is reduced below this range, a reaction threshold is reached and compensatory mechanisms are put into action to maintain the O2 consumption and, thus, the energy consumption at the same level. There is a point at which the compensatory mechanisms are exhausted. This state can be called critical threshold, critical state of oxygen supply, or simply critical oxygen supply. It means, in this state the oxygen supply limits the oxygen consumption.

Recently it has been questioned whether it is still sensible to use the term critical oxygen pressure as an essential parameter to describe tissue hypoxia or anoxia. In the following I like to show the usefulness but also the limitation of this expression. Since the expression was coined from physiological experiments I will begin to discuss these physiological results.

O2, is independent of the O2 offered by the respired gas mixture or by the arterial blood (see for example 19, 15, 4). When oxygen is reduced below this range a reaction threshold is reached and compensatory mechanisms are put into action to maintain the O2 consumption - and thus the energy consumption - at the same level. But there is a point at which the compensatory mechanisms are exhausted: This state can be called critical threshold or critical state of oxygen supply or simply critical oxygen supply. It means, in this state the oxygen supply limits the oxygen consumption. The situation of a critical O2 supply has been studied so extensively that it is impossible to review or even mention the main experimental work; instead of that, I shall discuss some examples to elucidate our problem. In the earlier experiments the different criteria for a sufficient oxygen supply that were applied, were: 1) oxygen consumption, 2) lactate balance, and 3) functional state. As later on measurements of tissue concentrations became possible, the tissue concentration of lactate, pyruvate and adenine nucleotides or a relationship such as the lactate/pyruvate ratio, the phosphate potential or the energy charge (see Siesjö, 1978) were used.

1) The O2 consumption criterion was used by Stainsby (1966). He measured the dependence of the O2 consumption of dog skeletal muscles (mm. gastrocnemius - plantaris) on the arterial PO2, PaO2. The critical situation of oxygen supply was produced by reducing PaO2. occurred during rest at a PaO2 of 8 kPa (60 mm Hg) and a PvO2 of 3.33 kPa (25 mm Hg) and during work at a PaO2 of 6.66 (50 mm Hg) and a PvO2 of 1.33 kPa (l0 mm Hg)• Although the O2 consumption during work was 8 times higher than during rest (40/ul O2/g. min as compared to 5/ul O2/g. min), the blood Po2 values during work were smaller. This difference can be explained by the increased number of perfused capillaries in the working muscle which reduce the supply area of a single capillary, and by the increased flow. The experiments demonstrated the strong influence of flow and capillary geometry.

2) The lactate balance criterion was used by o2 = 300/ul O2/g. min) it was increased to 1.87 kPa (14 mm Hg). These different transition points are in accordance with the changes of flow and tissue respiration.

3) The functional state criterion for O2 supply was used by Opitz and Schneider (1950) in their review and analysis of the oxygen supply of the brain. They found that the functional state can be at best correlated with the venous Po2 in the sinus sagittalis. The normal Pvo2 of 4.53 kPa (35mm Hg) can decrease to ca 3.73 kPa (28 mm Hg) without any detectable reaction but with a further decrease in Pvo2 blood flow increases to maintain the Pvo2 close to this level. Further reduction of Pvo2 shows first signs of changes in the ECG and in man higher mental functions are impaired. The critical oxygen supply is reached when the Pvo2 becomes smaller than 2.53 − 2.27 kPa (19 − 17 mm Hg). Under this condition man looses consciousness. The changes, however, are still reversible. They become irreversible when Pvo2 is lowered to 1.6 kPa (12 mm Hg) over a certain period of time.

The direct tissue measurements of lactate and adenine nucleotides corroborate these results (17). These examples show the complexity of our system but they also demonstrate that there is a definite state at which a critical O2 supply is reached. The occurrence of a critical O2 supply is influenced by many parameters but the venous Po2 - and not the venous O2 content - seems to be an important indicator of tissue oxygen supply. How can this be explained: It can be easily deduced from the physiological laws of oxygen supply, which concern 1) the O2 transport by blood 2) the O2 transport by diffusion and 3) the behavior of tissue oxygen consumption.

1) O2 transport by blood

The amount of oxygen which can be supplied to the tissue depends on a) the oxygen content of blood, Co2, and b) blood flow, B.

a) Oxygen content of blood. Under physiological conditions the main amount of oxygen is chemically bound to hemoglobin

Co2(chem) = 1.34. cHb. So2

CHb, concentration of hemoglobin in g/dl; So2, fractional oxygen saturation; 1.34, ml O2 per g hemoglobin.

and only a small amount of oxygen is physically dissolved

Co2(phys) = αp. Po2

αp, O2 solubility coefficient of plasma.

    Thus the total amount of oxygen

Co2(blood) = Co2(chem) + Co2(phys)

depends essentially on the hemoglobin concentration and the fractional O2 saturation. The fractional O2 saturation depends on the blood Po2. This dependence is described by the O2 dissociation curve.

b) Effect of flow. The O2 content of the arterial blood is offered and delivered to the tissue. In steady state the difference between the O2 content of arterial and venous blood, the AVDo2 times blood flow corresponds to the tissue respiration

    It is important to note that with constant tissue respiration the AVDo2 is a hyperbolic function: that means that small flow changes are very effective in offering more O2 or in reducing the O2 supply, whereas at high flow the same absolute change has practically no effect.

2) O2 transport by diffusion

The oxygen transport within the tissue is mainly performed by diffusion. The parameters, which govern the diffusion process can be easily seen from the diffusion equation for a simple layer

    D, diffusion coefficient; x, thickness of diffusion layer.

The O2 flux, Io2, depends a) on the oxygen conductivity (D.α) and b) on the Po2 gradient, Δ Po2/Δx.

a) In the product (D.α) D determines the speed with which the molecules travel - according to equation s−2 = 6 D.t, s−2 is the square of the mean distance which the molecule travels during time t - and α gives the number of molecules which actually travel. The oxygen conductivity (D.α) characterizes the individual property of the tissue; it increases with temperature as well as with content of water and lipids, but under normal physiological conditions its variation is only small.

b) The Po2 gradient is the important factor for the O2 transport. We should mention that fo£ diffusion of gases the oxygen pressure is the driving force and not the oxygen content. This is especially important for systems with varying values of α. The importance of the oxygen pressure for the O2 transport in the tissue explains why the critical oxygen supply could be correlated to the venous oxygen pressure and not to the venous oxygen content of the blood.

Two other important factors which influence the diffusion are c) the O2 consumption and d) the distances over which the oxygen has to be transported. The influence of these factors can be shown in a simple model consisting of a capillary which supplies oxygen to the surrounding cylindrical space (Krogh model (8))

with

c, capillary; t, tissue; z, cylinder.

This Krogh-Erlang equation shows that

c) the O2 consumption is linearly related to the oxygen pressure difference, Δ Po2, which is necessary to transport the oxygen into the tissue and that d) the geometry enters as approximately a squared function. This explains rhat in the resting muscle with a few open capillaries and consequently a large supply area a higher capillary Po2 is necessary to supply the tissue with oxygen than in the working muscle.

Fig. 1 shows the calculated Po2 decrease in skeletal muscle assuming according to Stainsby (1966) a radius of rz = 80 /um in resting and of rz = 18 /um in the working state. One sees that the Δ Po2 of 1.8 kPa (13.5 mm Hg) in the resting state is larger than the Δ Po2 of 0.35 kPa (2.6 mm Hg) in the working state. In spite of an 8 times smaller O2 consumption, the about 4 fold increase in radius (from 18 to 80/um) produces a Po2 decrease about 5 times larger in the resting than in the working muscle. Since in the resting state the total amount of oxygen which has to leave a single capillary is larger than that in the working state, the Po2 gradient in the neighborhood of the capillary is much steeper in the resting state than in the working state. This demonstrates directly how efficient the reduction of the radius of the tissue cylinder is in regard to the tissue oxygen transport.

Fig. 1 Calculated Po2 profile in resting and working skeletal muscle

3) Behavior of tissue oxygen consumption

The main consumer of oxygen is oxidative phosphorylation. The reactions involved are thoroughly discussed in other papers. For our point of view it is important to note that the mitochondria with their respiratory chains are perfect oxygen sinks. Under normal physiological conditions each molecule of oxygen which meets the mitochondria reacts with the cytochrome oxidase if ATP is needed so that the oxygen concentration becomes zero. This means that the total capillary Po2 is available for the oxygen transport.

With isolated mitochondria it has been shown (5, 2) that down to Po2 values of 0.0027 kPa (0.02 mm Hg) the respiratory rate can remain unchanged. This corresponds to an oxygen concentration of about 0.033/uM in the medium; in lipids the actual concentration may be somewhat larger because of the higher α. Below this Po2 value the O2 consumption decreases. With isolated mitochondria we could titrate the redox state of cytochrome aa3 by adding stepwise very small amounts of oxygen (21, 12). 100% oxidation was reached at Po2 values in the medium of ca. 0.008 kPa (0.06 ± 0.07 mm Hg; n = 20), a Po2 value hardly detectable by a Platinum electrode.

These low critical Po2 values measured by the Pt electrode in steady state could not be detected in kinetic measurements (20). Here the redox state of cytochrome aa3 changed at Po2 values in the range between 0.2 kPa −0.93 kPa (1.5 − 7 mm Hg). Whereas in steady state experiments a good reproducibility could be achieved, the same was not possible in kinetic experiments. This may have been caused by methodological artifacts:

1) Because of the finite response time of the Pt electrode the Po2 tracing of the electrode runs behind the true Po2 of the medium and thereby falsifies the true signal: the reading of the electrode is too high (and too late).

2) The observed kinetics depends not only on the kinetics of the respiratory chain but also on the response time of the electrode.

3) Furthermore, it cannot be excluded that the mitochondria of the cells have a fixed layer of medium which also would delay the electrode signal. The exact determination of the critical Po2, i.e. the deviation from linearity, which indicates the change in respiratory rate, is diffucult since the respiratory rate is not always sufficiently constant. For example, in a test (n = 180) only about 50% of all curves showed a normal statistical scatter of the respiratory rate (16). In all other cases systematic deviations of the respiratory rate occurred; often - but the opposite is also possible - the respiratory rate descreased slightly down to lower Po2 values, in this case the point of deviation is found to be different with a large Po2 range from that found with a small one: With large ranges the critical Po2 was found between 1.87 and 1.47 kPa (14-11 mm Hg) and with small ranges only between 0.4 and 0.13 kPa (3 − 1 mm Hg). Similar data (20, 21, 12,) were found with liver, kidney and ascites tumor cells and their corresponding mitochondria. The variation of the respiratory rate was somewhat substrate-dependent. This points to the fact that constant O2 consumption and the entrance of limitations at the same Po2 level can only be expected if the energy need and substrate supply remain²unchanged. That is obviously not always the case.

In general, then, our analysis suggests that in hypoxic tissue the region with normal oxygen supply is surrounded by a zone of hypoxia in which the o2 concentration limits the O2 consumption. Under this condition the critical O2 supply is determined by the critical capillary Po2 which is reached when in the periphery of the tissue the critical mitochondrial Po2 is reached.

As already mentioned from tissue experiments it has been determined that with decreasing O2 supply at first a reaction threshold is reached at which for example blood flow increases before a critical state of oxygen supply occurs. This leads to the important question of whether or not these reactions are caused by local critical hypoxia (Hypoxia hypothesis 15, 22).

We tried to answer this question experimentally. The Krogh-Erlang equation shows that local tissue Po2 mirrors the capillary Po2, oxygen conductivity, tissue respiration and geometry, i.e. the local balance between oxygen supply and oxygen consumption (10).

Fig. 2 shows for example the normal Po2 histogram of a brain (guinea pig, light barbiturate anesthesia) (9). As expected, the local Po2 varies considerably. It is interesting that 5% of all Po2 values are in the lowest class. In this class values very close to zero (and sometimes not distinguishable from zero) are often found, without any sign of hypoxia. With Po2 needle electrodes (3) it is sometimes difficult to ascertain the exact zero, but using membrane covered multiwire electrodes (7) it has been verified that these low Po2 values occur in normal tissue. The Po2 histogram also shows that many tissue Po2 values are much lower than the venous Po2 of 4.53 kPa (34 mm Hg). This points to the fact that the capillary network of the tissue is much more complicated than assumed in the Krogh model. It is known that capillaries have different lengths and consequently with the same pressure gradient they must have different flow velocities.

Fig. 2 Po2 histogram of guinea pig brain

Fig. 3 shows histograms of Po2 and of mean flow velocity from the surface of a beating cat heart (18). With air respiration the Po2 histogram of the heart muscle is shifted more to the right than that of the guinea pig brain. The histogram of mean velocities measured by H2 - pH2 clearance (13) shows large differences in mean flow velocities. This is understandable if one takes into account that the lengths of the capillaries in heart muscle vary between 100 and 800/um with the maximum fraction having a length of 400/um. These different capillaries will have different Po2 profiles and thus the venous Po2 is a mixture of the different capillary venous Po2 values.

Fig. 3 histogram of the beating heart muscle of the cat.

Consequently, the absolute value of the mixed venous Po2 is not related in a simple way to anoxic or hypoxic zones as assumed in tne Krogh model. Therefore, one needs very local methods such as the Po2 histogram to detect such changes. To answer our question we found that flow velocity changed despite no detectable anoxic Po2 values in tissue. We can therefore assume that at decreasing O2 supply in the tissue a signal is produced which has nothing to do with the critical state of oxygen supply which concerns the energy need. What kind of signal that may be - whether a single or several chemical reactions are involved - is an open question and needs further research. As a beginning two types of reactions shall be described:

1) Wilson et al. (1979) observed with cells of warm-blooded animals that starting at an O2 content of the medium of 20/uM, with decreasing O2 content the O2 consumption remained constant down to rather low O2 values (KM less than 1/uM), that however the redox state of cytochrome c, the ATp/ADP and NADH/NAD ratios changed over the total range. At constant respiratory rate this mechanism can produce a Po2 dependent signal for other reactions.

2) But it is also possible that the O2 consumption changes directly, being dependent on the Po2.

a) It has been found that the carotid body has an O2 dependent O2 consumption (1). Since this fact is important for the O2 sensing mechanism it may be due to the very special situation in this organ. From optical measurements a special cytochrome oxidase has been proposed (14).

b) The experimental results with the hemoglobin-free perfused liver which were obtained together with Kessler (Kessler et al. 1980) may have a more general meaning: It was found that the O2 consumption of the isolated liver was reduced to 2/3 if the Pa O2 decreases from 80 − 24 kPa (600 − 180 mm Hg). In this range the redox state of cytochrome aa3 remained unchanged. Only at a Pa o2 of 20 kPa (150 mm Hg) both the Po2 histogram and the amount of reduced cytochrome aa3 showed that tissue hypoxia occurred and became responsible for the further decrease in O2 consumption. It may be that here other oxidases - Kessler proposed the monoamino oxidase in the outer mitochondrial membrane - were responsible for such an O2 economizing effect. However, all oxidases with a suitable KM value could act - as M. Kessler put it - as signal oxidases.

In conclusion we think that the term critical oxygen supply is useful since it describes the threshold at which the O2 transport limits the rate of oxygen consumption of a tissue. In this state, within the tissue local Po2 values occur at which the mitochondrial O2 consumption becomes rate limiting in regard to a constant energy need: Thus we can define a critical oxygen transport pressure for the tissue and a critical oxygen pressure for the O2 consumption of the mitochondria (11). Since the capillary network is a complicated structure, the critical supply situation describes only a mean situation. This limits the usefulness of this expression and the applicability of the overall methods which are used to describe this state.

It is important to stress that far above the critical O2 supply situation the tissue possesses reactions which respond to higher oxygen pressures. This fact has been described by the term reaction threshold (15), but it is not necessary that for all reactions a real threshold exists.

References

1. Acker, H., Lübbers, D. W. The kinetics of local tissue Po2 decrease after perfusion stop within the carotid body of the cat in vivo and in vitro. Pflügers Arch. 1977; 369:135–140.

2. Bârzu, O., Satre, M. Determination of oxygen affinity of respiratory systems using oxyhemoglobin as oxygen donor. Analyt. Biochem. 1970; 36:428–433.

3. Baumgärtl, H., Lübbers, D.W. (in print): Microcoaxial needle sensor for polarographic measurement of local O2 pressure in the cellular range of living tissue. Its construction and properties. In: Handbook of POS. Eds. E. Gnaiger, H. Forster. Springer, Heidelberg

4. Bretschneider, W. H. Über den Mechanismus der hypoxischen Coronarerweiterung. In: Lochner W., Witzleb E., eds. Probleme der Coronardurchblutung. Berlin-Göttingen-Heidelberg: Springer; 1958:44–83.

5. Chance, B., Schoener, B., Schindler, F. The intracellular oxidation-reduction state. In: Dickens F., Neil E., eds. Oxygen in the Animal Organism. London-New York-Paris-Frankfurt: Pergamon Press;