Effect of oxygen consumption by measuring method on Po2 transients associated with the passage of erythrocytes in capillaries of rat mesentery

Amy G. Tsai, Pedro Cabrales, Paul C. Johnson, Marcos Intaglietta


Golub, Aleksander S., and Roland N. Pittman. Erythrocyte-associated transients in PO2 revealed in capillaries of rat mesentery. Am J Physiol Heart Circ Physiol 288: H2735–H2743, 2005. Mathematical models have predicted the existence of Po2 gradients between erythrocytes in capillaries in the usual case where plasma contributes substantial resistance to oxygen diffusion. According to theoretical predictions, these gradients could be detected as rapid Po2 fluctuations (erythrocyte-associated transients, EATs) along the capillary. However, verification of a model and correct choice of its parameters can be made only on the basis of direct experimental measurements. We used phosphorescence quenching microscopy to measure Po2 in 52 capillaries of rat mesentery to obtain plasma Po2 values 100 times/s at a given point along a capillary. A 532-nm laser generated 10-μs pulses of light, concentrated by a ×100 objective, onto a spot 0.9 μm in diameter. The presence of erythrocytes in the excitation region was detected on the basis of phosphorescence amplitude (PA), proportional to the amount of plasma encountered by the laser beam, and on the basis of the intensity of transmitted laser light (LT), detected by a photodiode placed under the capillary. The data revealed correlated waveforms in PA, LT, and Po2 in capillaries. The magnitude of the Po2 gradients between erythrocytes and plasma was correlated with average capillary Po2. EATs in Po2 were more readily detected in capillaries with relatively low oxygenation. The correlation coefficients between PA and Po2 for the half of the capillaries (n = 26) below the median Po2 (mean Po2 = 17 mmHg; R = −0.72) was higher than that for the other half (mean Po2 = 39 mmHg; R = −0.38). These results support the theoretical predictions of EATs and plasma Po2 gradients in capillaries.

To the Editor: Golub and Pittman report the measurement of Po2 gradients between red blood cells (RBCs) in mesenteric capillaries, indicating that capillary Po2 is ∼20 mmHg when measured at locations where RBCs are present, and that this drops to about 6–7 mmHg in the intra-RBC plasma space (2). This result suggests that the lower Po2 between RBCs in capillaries corresponds to tissue Po2 and that tissue oxygenation is maintained by transients in oxygen delivery supplied by RBCs as they pass through the capillaries.

This configuration is plausible when capillaries are the only suppliers of oxygen to the tissue; however, there is substantial evidence that arterioles contribute significantly to the tissue oxygen supply. Oxygen exit from arteriolar RBCs elevates tissue Po2 to the extent that capillary RBCs and tissue are in virtual equilibrium (3).

According to Golub and Pittman, RBCs travel surrounded by plasma with a low Po2 (6 mmHg) differing from the usual observation that tissue and capillary Po2 average ∼20 mmHg in microvascular preparations (2). When the mesentery is spread over an impermeable support, areas outside of the diffusion field of arterioles have low Po2s, a configuration unlikely to exist in situ.

With the consideration of the unusual oxygen distribution found in this study, it is possible that results may be due to a systematic error arising from oxygen consumption by the measuring method.

Golub and Pittman (2) state: “Oxygen consumption by the phosphorescence quenching method was tested using blood taken from the jugular catheter and placed in a microslide with a 40-μm path length. …”; “Excitation conditions were the same as the in vivo experiments, except that the blood sample in the microslide remained motionless. …”; and “Results show that in the Po2 interval of 60–25 mmHg, the Po2 decrease in stationary blood was 1.2 ± 0.6 mmHg/flash…”. This should be compared with the Po2 decrease of 0.02 mmHg/flash of Tsai et al. (5), when excitation was directed to water, which has 20 times less oxygen carrying capacity than blood, indicating a difference of 500× in oxygen consumption per flash between methods.

Using a method that lowers blood Po2 by 1.2 mmHg/flash to determine plasma Po2, which has a 10-fold lower oxygen-carrying capacity than blood (in the Po2 range of these experiments), results in oxygen measurements that are ∼12 mmHg lower than normal due to oxygen consumption by the method.

In summary, the Po2 of RBCs in capillaries, plasma, and tissue are in virtual equilibrium. A significant decrease in capillary plasma Po2 measured by phosphorescence quenching that lowers the measured Po2 by 1.2 mmHg/flash in blood is the result of oxygen consumption by the method when applied to plasma measurements. This high oxygen consumption precludes measuring Po2 in the tissue, a reference point not reported in this study. Thus the presence of RBC-associated transients in Po2 in capillaries is a consequence of how the method is implemented by Golub and Pittman (2). This result is erroneous and leads them to conclusions contrary to data in the literature. As an example, they indicate that hemodilution jeopardizes oxygen transport, whereas experimental results show that 75% hemodilution increases oxygen release normalized to hematocrit by 50–450% depending on the plasma viscosity (1, 4).



EATs or Not EATs?

To the Editor: As we and others have pointed out previously (3, 4), oxygen consumption by the method remains a major issue in phosphorescence quenching microscopy. There are two obvious ways to cope with the problem: 1) reduce the density of the excitation energy by enlarging the excitation spot or 2) minimize the size of the light spot to near the diffraction limit, so that oxygen depletion caused by the excitation can be rapidly replenished by diffusion from the adjacent fluid (plasma, in the situation now considered). We have developed a novel measuring technique based on the second approach, using a small excitation spot with subsequent reduction of the potential oxygen consumption artifact. Unfortunately, details of the technique related to the oxygen consumption matter were omitted in the accepted version of our manuscript, due to space limitations.

In our system, an excitation light pulse creates a depression in Po2 due to photoactivated oxygen consumption. The estimated characteristic diffusion time, T, to replenish the oxygen inside the 0.9-μm diameter excitation volume of plasma is ∼80 μs (TR2/D, radius R = 0.45 × 10−4 cm, oxygen diffusion coefficient D = 2.4 × 10−5 cm2/s). In a hypothetical case, when all the oxygen is depleted from the excitation volume, the Po2 will be restored to its initial value within 200 μs, and the effect of consumption on the Po2 measurement will be negligible. Significant oxygen consumption by a light pulse could be recognized from an examination of the phosphorescence decay curve: the initial part of the curve would start from a slow exponential decay, moving to a much faster decay rate during the following 200 μs. The semilogarithmic plot of our typical decay curve (Fig. 3 of Ref. 1) does not exhibit this behavior. This supports the consistent finding of our in vitro test that consumption by the method is moderate (1.2 mmHg/flash) and that compensation by oxygen diffusion into the excitation region begins at the moment of the excitation flash.

Following the recommendation in their letter to compare our results with the low oxygen consumption achieved by Tsai et al. (4), we have reviewed their methodology. These authors estimated oxygen consumption in a sealed 75-mm-long glass capillary tube. The tube was masked so that only a 0.5-mm length was exposed to 45 min of pulsed illumination at 30 flashes/s. Immediately after the 45 min of illumination, the contents of the tube were mixed and the Po2 of the solution was measured. From the calculation presented in their paper, Tsai et al. apparently assumed that the oxygen consumption process involved the entire volume of the 75-mm-long container, although only the 0.5-mm segment of the probe solution was illuminated. For the 45-min (2,700 s) illumination period, the depression in Po2 could only propagate for a distance L ≈ 2.5 mm in each direction from the center of the tube (LMath); i.e., only about 1/15 of the entire volume. Thus, the real consumption in the affected region of this test was their reported value of 0.02 mmHg /flash × 15 ≈ 0.3 mmHg/flash.

In addition, the oxygen consumption by the method is roughly proportional to the Po2 in the illuminated section of the tube. After about the first 150 flashes (∼5 s), the region under the 0.5-mm-wide slit was almost depleted of oxygen, and the consumption rate was then limited by diffusion from the surrounding regions, thereby reducing the decrement in Po2 per flash.

Oxygen consumption in their test was also reduced by dissolving the albumin-bound phosphor in water, which did not contain organic molecules (except for the diluted probe itself) available as fuel for photo-oxidation. Therefore, the application of the results of this test to Po2 measurements in plasma or interstitial fluid, containing photo-oxidizable substrates in abundance (2), becomes problematic.

Thus low oxygen consumption due to the phosphorescence quenching method, reported by Tsai et al. (4), appears to underestimate the actual oxygen consumption by at least a factor of 15. The actual value was practically the same as the one we reported in 1998 (3) in a test employing a similar excitation system (EG&G FX-249 xenon flash lamp), where we warned against the use of high-frequency flash illumination to measure tissue Po2.

A decrement in Po2 of 0.3 mmHg/flash is not significant for measurements in flowing blood, where, for local excitation in a microvessel, each volume of plasma receives only one flash. However, when applied to motionless interstitial fluid, flash illumination at 30 flashes/s may lead to an accumulated depression in Po2 of ∼9 mmHg. This offers an alternative explanation of the unexpectedly large Po2 drop across the arteriolar wall, reported by Tsai et al., who concluded that the large transmural Po2 was due to a high metabolic rate of the vascular wall [141–323 times higher than in surrounding tissue (4)].

In our in vitro test (1), we used whole blood to simulate the oxygen consumption in a capillary, but measurements were made in plasma, because the albumin-bound probe was distributed only in plasma. We made Po2 measurements using individual decay curves because the high signal-to-noise ratio (SNR) in our system obviates the need for averaging multiple curves to provide an acceptable SNR. The time between flashes (10 ms) was too short to mobilize oxygen from red blood cells (RBC), whose half-time for oxygen release is 180–240 ms (5). The arbitrary conversion of our 1.2 mmHg/flash into 12 mmHg/flash, made in the letter of Tsai et al., was based on an incorrect assumption that RBC deoxygenation occurred more rapidly than this in our test. Furthermore, 1.2 mmHg/flash is a maximal value obtained in stationary plasma, because in flowing blood the depletion is reduced by rapid inflow of oxygen into the depleted volume from adjacent plasma.

The statement in Tsai et al.’s letter that the Po2 “…drops to about 6–7 mmHg in the intra-RBC plasma space” is based on our Fig. 5 (Ref. 1). Unfortunately, the authors appear to have overlooked Fig. 6 and the text that follows it (Ref. 1), where we noted that approximately one-third of the capillaries had distinct EATs. In this group of obviously hypoxic capillaries with low hematocrit, the connection among Po2, LT, and PA is readily apparent. The clarity of these time courses is the reason they were selected for the illustration of the connection among the three signals and the association of these fluctuations with the passage of RBCs. However, in the majority of the capillaries we measured, especially those having high average Po2, fluctuations were smaller and their connection with RBCs was revealed by using correlation coefficients between measured parameters and the coefficient of variation in Po2. All of this information is presented in our study (Ref. 1, p. H2740–H2743).

The hypothesis that the excitation light pulse depresses the plasma Po2 between RBCs, but does not do this in the plasma adjacent to an RBC (Po2 peaks), is internally inconsistent. Possibly, this premise was based on the belief that oxygen can dissociate from oxyhemoglobin on a time scale of tens of microseconds. However, the implicit suggestion that hemoglobin-bound oxygen in an RBC can be involved in the compensation of oxygen depleted by a flash is incorrect. The half-time of oxygen release from an RBC is three orders of magnitude longer than the time course of a typical decay curve (5). The absence of deep Po2 fluctuations in capillaries with high Po2 (when oxygen consumption should be even higher) also does not support their hypothesis.

In summary and based on statements in their letter, it appears that Tsai et al. have misunderstood the principle of Po2 measurements using a submicron excitation spot. Their hypothesis that the depression in Po2 in the plasma between RBCs is caused by oxygen consumption is not consistent with high Po2 in the plasma associated with RBCs and the absence of deep fluctuations in Po2 for capillaries with high average Po2. A systematic error, by definition, can shift the baseline Po2, but it cannot create Po2 variations in the plasma. As pointed out above, the report by Tsai et al. (4) of low oxygen consumption by the method contains a serious flaw, which can explain their finding of an apparently large metabolic rate of the vascular wall.

The conclusion of Tsai et al. in their letter that “…the Po2s of RBCs in capillaries, plasma, and tissue are in virtual equilibrium” is at odds with the preponderance of evidence accumulated over the last 30 years. If Po2 were in equilibrium among RBCs, plasma, and the surrounding tissue, there would be no net oxygen flow from RBC to plasma, across the capillary wall to parenchymal cells, and finally to the mito-chondria. This picture of virtual equilibrium is contrary to our current understanding of microcirculatory oxygen transport. We continue to embrace the conventional notion that oxygen diffuses from RBC to plasma and then to surrounding tissue, driven by Po2 differences between RBCs and mitochondria. EATs are simply a manifestation of this Po2 gradient that contradicts the idea of a virtual Po2 equilibrium, declared by Tsai et al. The first detection of EATs reported in our study is in good agreement with many theoretical predictions cited therein (1).


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