INTRODUCTION

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UNDER AEROBIC CONDITIONS, venous CO₂ content (Cv_CO2) is higher than arterial CO₂ content (Ca_CO2). The PCO₂ gap (PCO₂) between mixed venous and arterial blood is normally 4-6 Torr (3). An increased venoarterial PCO₂ is observed during various forms of circulatory failure due to cardiogenic, obstructive, hypovolemic, or distributive shock (5). Several authors (1, 11, 13) have reported, in experimental studies, that, when systemic O₂ delivery (O₂) was reduced below its critical value (O_2 crit, the O₂ at which a decrease in O₂ uptake and an increase in lactate occur, defining dysoxia), a brisk increase in PCO₂ was observed. This was associated with a similar increase in arterial-to-venous pH difference (pH). These authors (1, 11, 13) suggested that such a brisk increase in PCO₂ (or pH) could be used as a reliable marker of tissue dysoxia because critical O_2 crit, calculated using the O₂ uptake (O₂)-to-O₂, lactate-to-O₂, or PCO₂-to-O₂ dual-regression analysis, gave the same result. Increase in venous PCO₂ (Pv_CO2) would represent increased tissue PCO₂ related to an anaerobic CO₂ production secondary to tissue dysoxia and buffering of excess H⁺ by HCO₃.

All studies that have addressed this issue used reduced blood flow to produce tissue dysoxia. However, for a given tissue CO₂ production and at steady state, a decrease in tissue blood flow mandates an increase in tissue PCO₂, regardless of the presence or absence of tissue dysoxia. Therefore, the presence of a decreasing flow acts as a confounding variable and results in difficulties in drawing any definitive conclusion on the meaning of increased PCO₂ in hypoxia. Moreover, to date, this issue has been addressed exclusively for the whole body and not at the organ level. It may be possible that regions behave differently from each other or from the body as a whole. The aim of this study was to evaluate the PCO₂ in a regional model of progressive tissue hypoxia produced by decreasing either flow or Ca_CO2.

	METHODS

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Animal preparation. This study was approved by the University of Alabama at Birmingham Institutional Animal Care and Use Committee. Dogs of either sex and mixed breed were used. All animals were initially anesthetized with intravenous pentobarbital sodium (30 mg/kg) and intubated with a cuffed endotracheal tube. Catheters were inserted into the pulmonary artery (via the internal jugular vein) and common carotid artery for continuous measurement of vascular pressures and blood sampling. Lamps suspended above the operating table were used to maintain core temperature near 37°C. Standard limb leads were used to obtain heart rate continuously by means of a cardiotachometer (type 9857 cardiotachometer coupler, Beckman Instruments, Schiller Park, IL).

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Experimental protocol. After all pressures and flows were stable for at least 30 min, the experiment began with a 30-min control period, during which measurements were obtained every 15 min. In the progressive ischemic hypoxia (IH) group, was then decreased every 15 min to produce values of ~60, 45, 40, 30, 20, 15, and 10 ml · kg¹ · min¹. In the hypoxic hypoxia (HH) group, was set at 60 mg · kg¹ · min¹ and limb O₂ was reduced by decreasing arterial PO₂ from 100 to ~15 Torr (i.e., Ca_O2 of 17 to 2 ml O₂/100 ml) in eight steps at 15-min intervals. A flow rate of 60 ml · kg¹ · min¹ was chosen for progessive hypoxia because it is within the range of resting blood flow to normal skeletal muscle and for the practical reason that a moderate flow was necessary to achieve the desired low PO₂ values using the membrane oxygenator. Pa_CO2, Pv_CO2, Ca_O2, Cv_O2, arterial pH (pH_a), and venous pH (pH_v) were determined every 15 min, 13 min after the change in hindlimb arterial flow or PO₂.

Statistics. Data were analyzed within and between groups using repeated-measures ANOVA and Newman-Keuls test. Paired and unpaired t-tests were used, as appropriate, for one-time comparisons. Statistical significance was accepted at P < 0.05 for all comparisons.

	RESULTS

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We wished to maintain systemic hemodynamics within normal range so we could examine the direct local effects of ischemia and hypoxia on the hindlimb without confounding baroreceptor or chemoreceptor influence. Systemic hemodynamics and O₂ parameters remained stable throughout the study without any between-group differences. Cardiac output averaged 136 ± 6 (SE) ml · kg¹ · min¹ for the 12 dogs. Pa_O2 was 82 ± 2 Torr, and Pa_CO2 was 34 ± 2 Torr. Mean arterial pressure was 128 ± 2 Torr and systemic O₂ was 6.67 ± 0.07 ml · kg¹ · min¹. Hematocrit was 39.0 ± 0.4%. These values are typical for paralyzed, pentobarbital sodium-anesthetized dogs.

Figures 1 and 2 depict the changes seen in hindlimb O₂ and ER_O2 as O₂ was decreased by progressive IH or HH. In both groups, the O₂-to-O₂ graph describes the typical biphasic relationship. Mean O_2 crit was slightly higher in HH than in IH, but the difference was not statistically significant. ER_O2 at O_2 crit was significantly larger in IH than in HH (79 ± 2 and 66 ± 4%, respectively). Venous PO₂ at O_2 crit (Fig. 3) was not different between groups (23 ± 1 and 21 ± 2 Torr in IH and HH, respectively). For the lowest O₂ obtained, Pv_O2 was significantly higher in IH than in HH (15 ± 1 and 9 ± 2 Torr, respectively). Beyond O_2 crit, ER_O2 rose continuously and quite similarly in both groups, reaching a maximal extraction ratio of ~85-90%.

View larger version (17K): [in this window] [in a new window]   Fig. 1.   Hindlimb O2 uptake as a function of limb O2 delivery (O2) for ischemic hypoxia (IH; ) and hypoxic hypoxia (HH; ). There was no statistically significant difference at any O2. Critical O2 (O2 crit) was not different in IH and HH.

View larger version (16K): [in this window] [in a new window]   Fig. 2.   Hindlimb O2 extraction ratio as a function of limb O2 for IH () and HH (). There was no statistically significant difference at any O2, although O2 extraction at O2 crit was significantly larger in IH than in HH (79 ± 2 vs. 66 ± 4%, respectively).

View larger version (14K): [in this window] [in a new window]   Fig. 3.   Hindlimb venous PO2 (PvO2) as a function of limb O2 for IH () and HH (). * P < 0.05 vs. HH with ANOVA.

Figure 4 depicts the changes seen in hindlimb CO₂ as O₂ was decreased by progressive IH or HH. In both groups, the CO₂-to-O₂ graph describes a very similar biphasic relationship. The hindlimb respiratory exchange ratio (R) increased in both groups, with a trend to decrease by the end of the experiment in HH (Fig. 5).

View larger version (18K): [in this window] [in a new window]   Fig. 4.   Hindlimb CO2 production as a function of limb O2 for IH () and HH (). There was no statistically significant difference at any O2.

View larger version (15K): [in this window] [in a new window]   Fig. 5.   Hindlimb respiratory exchange ratio (R = CO2 production/O2 uptake) as a function of limb O2 for IH () and HH (). * P < 0.05 vs. HH with ANOVA.

PCO₂ significantly increased in IH but did not change in HH (Fig. 6). The increase in PCO₂ in IH occurred before reaching O_2 crit. At O_2 crit, PCO₂ approached 16 Torr. There was no evidence of changes in the slope of the PCO₂-to-O₂ relationship. pH increased significantly only in IH (Fig. 7).

View larger version (15K): [in this window] [in a new window]   Fig. 6.   Hindlimb PCO2 (outflow PCO2  inflow PCO2) as a function of limb O2 for IH () and HH (). * P < 0.05 vs. HH with ANOVA.

View larger version (17K): [in this window] [in a new window]   Fig. 7.   Hindlimb pH (inflow pH  outflow pH) as a function of limb O2 for IH () and HH (). * P < 0.05 vs. HH with ANOVA.

	DISCUSSION

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The main result of this study is that occurrence of an increased PCO₂ during ischemia is related to decreased blood flow and impaired CO₂ washout. Dysoxia per se is not sufficient to increase PCO₂. In presence of a constant flow, dysoxia with CO₂ generated from anaerobiosis does not promote PCO₂ widening.

Tissue dysoxia occurs when O₂ is inadequate to support O₂ demand (4, 8). O₂ represents the terminal electron acceptor for oxidative phosphorylation. In the absence of adequate O₂, the intermediates in the electron transport system are converted to their reduced states, and electron transport is compromised (4). In response to declines in cellular O₂, the tissues employ a series of responses to maintain a balance between ATP production (main cellular energy source) and cellular energy needs. The predominant mechanism is an increase in ER_O2 of capillary blood (ER_O2 = O₂/O₂). However, with severe decreases in O₂, compensatory increases in ER_O2 may not be sufficient to provide the mitochondria with the O₂ required to sustain aerobic metabolism. The cells must then use anaerobic sources of energy to produce ATP, resulting in the generation of lactate and H⁺ ions. In our study, we did not measure lactate production. However, information gained from the O₂-to-O₂ relationship clearly identifies the onset of tissue dysoxia in our hindlimb preparation. Maximal O₂ extraction was comparable in both groups, meaning that physiological responses to impaired O₂ delivery were similarly present in IH and HH. Also, O₂ fell to the same level in both groups by the end of the experiment (~1 ml O₂ · kg¹ · min¹), suggesting that a similar severity of dysoxia was reached. We can assume then that HH and IH were comparable in terms of dysoxia. Moreover, dysoxia started at very similar O_2 crit in IH and HH, excluding any possibility of an earlier O₂ debt accumulation in one group that was responsible for a larger CO₂ accumulation

Oxidative phosphorylation results in the formation of CO₂ and water. When O₂ is progressively decreased below O_2 crit, this is followed by 1) a decrease in tissue O₂ and aerobic CO₂ production and 2) an increase in H⁺ concentration associated with tissue CO₂ production resulting from cellular buffering by bicarbonates. Total CO₂ production (CO₂) beyond O_2 crit is, therefore, the sum of decreased aerobic CO₂ production and increased anaerobic CO₂ production. CO₂ is related to O₂, i.e., CO₂ = R × O₂, with R being stable and principally affected by the fuel source used for aerobic metabolism (3, 10). Anaerobic sources of CO₂ may, however, increase R when O₂ is lowered beyond O_2 crit. This was observed by Cohen et al. (3) in hemorrhaged pigs; airway CO₂ production decreased during hemorrhage but less than O₂, and, consequently, R increased. Our results are consistent at the organ level; when flow and O₂ were progressively decreased (IH), CO₂ decreased, but R increased, suggesting some production of anaerobic CO₂. When flow was kept constant while Ca_O2 was decreased (HH), we observed a similar decrease in CO₂, with a trend for an increase in R. Whatever the increase in R, we must admit, however, that anaerobic sources of CO₂ are much less important than aerobic ones because CO₂ consistently and dramatically decreased when O₂ was lowered beyond O_2 crit. This occurred similarly in IH and HH, suggesting an absence of gross difference in CO₂ for these two forms of hypoxia.

Besides aerobic and anaerobic production of CO₂, two other factors affecting PCO₂ are CO₂ dissociation curve and tissue blood flow. The CO₂ dissociation curve is influenced by the saturation of hemoglobin with O₂, a phenomenon known as the Haldane effect (12). The lower the saturation of hemoglobin with O₂, the larger the CO₂ saturation of hemoglobin for a given PCO₂. This might account for a smaller PCO₂ in HH, a situation in which larger hemoglobin deoxygenation would increase the blood's ability to carry CO₂. The similar value of Pv_O2 at O_2 crit, when PCO₂ is already larger in IH than in HH, limits this explanation above O_2 crit. Below O_2 crit, the Haldane effect may contribute, however, in magnifying the difference in PCO₂ that was observed between HH and IH. This would explain why R tends to rapidly decrease by the end of the experiment in HH. CO₂ decreases more rapidly than O₂ because more CO₂ is transported by red blood cells.

For a given tissue CO₂ production, a lower blood flow must be associated with a higher Pv_CO2. In this study, there was a clear inverse linear relation between hindlimb Pv_CO2 and blood flow. Because PCO₂ did not increase in HH, despite comparable levels of tissue dysoxia, decreased blood flow appears to be another cause of the PCO₂ widening observed in the IH group. Increased Pv_CO2 was associated with a decrease in pH_v and a widening in pH in the IH group. pH_v remained almost constant in HH. These results suggest that Pv_CO2 was the primary determinant of pH_v in this model and that respiratory acidosis very likely accounts for expanding pH.

During hindlimb ischemia in this study, PCO₂ was ~16 Torr at the onset of tissue dysoxia. This value is similar to values found in experimental models of progressive hemorrhage or tamponnade (1, 13), in which whole body PCO₂ varied from 12.9 (13) to 14.9 Torr (11) at O_2 crit. However, in contrast to previous studies done in the whole animal (1, 11, 13), this value cannot be easily determined in our experiments by considering a brisk increase on the PCO₂-to-O₂ relationship and cannot provide a useful tool to determine O_2 crit. If PCO₂ is ~15 Torr or larger at the systemic or regional level, one may say that there is a great risk of dysoxia associated with a decrease in flow; if PCO₂ is <15 Torr, one may say nothing about the presence or absence of dysoxia. If we assume that a PCO₂ gradient of 5 Torr exists between the tissues and the venous blood, a PCO₂ of 15 Torr is compatible with the 20 Torr tissue-to-artery PCO₂ value that represents a situation at risk of dysoxia, as determined in a mathematical model by Schlichtig and Bowles (9).

In summary, in the isolated hindlimb model, lowering O₂ by decreasing flow results in an increased PCO₂, whereas lowering O₂ by decreasing blood oxygenation does not affect PCO₂. For the first time, we demonstrated that absence of increased PCO₂ does not preclude the presence of tissue dysoxia.