INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

One of the most important current concepts in the care of patients with respiratory failure is the recognition that mechanical ventilation can worsen or even cause lung injury. Current ventilator strategies to limit this injury include minimizing lung stretch and using high PEEP levels. This results in relative hypoventilation, and the "permissive" generation of a hypercapnic acidosis (HCA), and constitutes an improved clinical strategy in the management of patients with respiratory failure (1). It is supported by recent data which suggest that survival rates from acute respiratory distress syndrome (ARDS) may be rising (4). This permissive hypercapnia is "tolerated" often reluctantlyyet the possibility that increased CO₂ tension per se may play a role in organ protection has not been considered in the clinical context to date.

An increasing body of literature exists which suggests that respiratory, and metabolic, acidosis can exert protective effects on tissue injury in several different organ systems, including the central nervous system (5), the myocardium (6, 7), and the lung (8, 9). In our laboratory, we have demonstrated (8) that HCA protects against acute lung injury (ALI) independent of the ventilatory strategy employed. This has led to the suggestion that the beneficial effects of permissive hypercapnia in patients with ARDS may be due at least in part to HCA per se, in addition to potential benefits from reduced ventilator associated lung injury (VALI) (8). This finding further raises the possibility of a potential role for therapeutic hypercapnia in ALI (10).

The mechanism of these protective effects of HCA in ALI may be mediated in part via inhibition of XO based on the ability of HCA to inhibit the activity of the enzyme in vitro (8). The protective effect of hypercapnia and/or acidosis is not confined to the lung; in fact the evidence is better developed in other organ systems (5, 11). Acidosis may reduce organ cellular respiration and oxygen consumption, thereby reducing oxygen demand in the setting of reduced supply (15). For example, acidosis reversibly reduces cardiac contractility (12), thereby reducing organ oxygen demand to a sustainable level in the setting of acute organ ischemia. This constitutes a protective response. Furthermore, acidosis may protect against ongoing tissue production of additional organic acids (by a negative feedback loop), potentially providing a mechanism for cellular metabolic shutdown at times of nutrient shortage (16). However, our understanding of these potential protective effects of hypercapnia and/or acidosis, particularly in ALI, remains incomplete. Delineation of these mechanisms is important for two reasons. The first relates to the clinical potential that exists for the use of therapeutic hypercapnia (10). Second, buffering of HCA toward arbitrary pH goals is a controversial but common practice (17, 18). Aside from the controversy surrounding the systemic effects of this practice (19, 20), the pulmonary effects, in terms of ALI, have not been defined.

The current study therefore explored whether the mechanisms of lung protection in the presence of acidosis were primarily related to pH versus PCO₂. We hypothesized that a rank order of protection is conferred by elevated PCO₂ and/or lowered pH in acute lung injury. Specifically, we hypothesized that: (1) HCA would afford greater protection versus metabolic acidosis because of the ability of CO₂ to rapidly equilibrate across cell membranes and generate a more rapid intracellular acidosis (21, 22), and (2) that buffering HCA may attenuate or even abolish its protective effects in acute lung injury. We investigated, in addition, the effects of acidosis versus PCO₂ on the in vitro activity of exogenous xanthine oxidase (XO), hypothesizing that in vitro XO activity would be pH rather than PCO₂ dependent.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Male New Zealand White rabbits weighing 3.0 to 4.0 kg were used in all experiments. All experimental work conformed to the guidelines of the Canadian Council for Animal Care and was approved by the Animal Care Committee of The Toronto Hospital.

Experimental Outline

In this study, the experiments were organized into a series of ex vivo isolated lung experiments, and a series of uric acid assays. Series I examined the effects of: (1) control conditions (FI_CO2, 5%; pH, 7.4); (2) hypercapnic acidosis (HCA: FI_CO2, 25%; pH, 6.9); (3) metabolic acidosis (FI_CO2, 5%; pH, 6.9); (4) buffered hypercapnia (FI_CO2, 25%; pH, 7.4) on acute lung injury produced by warm ischemia-reperfusion. Series II examined the in vitro effects of: control, HCA, metabolic acidosis, and buffered hypercapnia on the activity of xanthine oxidase (XO). For this series, we measured the generation of uric acid from purine by addition of XO, in prepared perfusate.

Series I: Surgical Dissection

After premedication with intramuscular ketamine (85 mg/kg), anesthesia was induced intravenously with pentobarbital sodium (range, 15 to 25 mg/kg), and heparin (1,000 IU) was administered. The surgical preparation used in this study was similar to that previously reported (8, 23), with several modifications. Incremental boluses of pentobarbital (5 mg/kg) were administered as required. Briefly, a tracheotomy was performed and pancuronium bromide (1 mg intravenously) was administered after depth of anesthesia was confirmed by absence of response to paw clamp. The lungs were ventilated using a small animal ventilator (Model no. 683; Harvard Apparatus, South Natick, MA) with FI_CO2, 1.0; rate, 20/min; tidal volume, 4 ml/kg; and 2 cm H₂O positive end-expiratory pressure (PEEP). The carotid artery was cannulated for arterial pressure measurement, with additional pentobarbital administered for any elevation in baseline mean arterial pressure  10%. A sternotomy was performed, and the pulmonary artery and left atrium were cannulated. The lungs were then ventilated with 5% CO₂-95% O₂ and flushed with blood-free Krebs-Henseleit solution containing 3% bovine serum albumin, using a peristaltic pump (Model no. M312; Gilson, Veillier, France). The heart and lungs were excised from the chest and suspended by the tracheotomy tube from a counterbalance force-displacement transducer (Model no. 60-2995; Harvard Apparatus) connected to a chart recorder (Recordall Series 5000; Fisher Scientific, Nepean, ON, Canada) for continuous measurement of lung weight. The lungs were then perfused in a recirculating manner at a flow rate of 150 ml/min. The perfusate temperature was maintained at 37° C, and the total volume in the circuit was 500 ml. Pulmonary artery (Ppa) and left atrial (Pla) pressures were referenced to the height of the left atrium and measured continuously via T-piece stopcock connections in the tubing leading to each cannula. The pulmonary cannula ended in a single end lumen, whereas the left atrial cannula had side holes in addition to the end lumen, in order to prevent occlusion in the event of atrial collapse. The venous reservoir height was adjusted to maintain Pla at + 2 mm Hg. The left atrium was wrapped loosely with string and glued to maintain constant left atrial volume, preventing it from acting as a pressure capacitor during vascular occlusion, and thus causing distortion (i.e., underestimation) of pulmonary capillary hydrostatic pressure measurement. All gas mixtures were supplied premixed in cylinders (Praxair, Mississauga, ON, Canada).

Series I: Measurements

Perfusate gases. Samples of perfusate were taken from the reservoir and were measured for pH, PCO₂, and PO₂, using an ABL-300 blood gas analyzer (Radiometer, Copenhagen, Denmark).

Vascular and airway pressures. Ppa, Pla, and airway pressures were recorded from a standard monitor (Spacelabs Monitor Model no. 90303B; Spacelabs Medical Products Ltd., Mississauga ON, Canada), with hard copy tracings for determination of microvascular pressures recorded using a Gould 5-channel recorder (Gould 8-channel Recorder 2800; Gould Inc., Instruments Division, Cleveland, OH).

Pulmonary capillary hydrostatic pressure [Pcap]. To determine pulmonary capillary hydrostatic pressure (Pcap), mechanical ventilation was stopped and the lungs were inflated with continuous positive airway pressure (CPAP) of 3 mm Hg. The pulmonary arterial and left atrial cannulae were simultaneously occluded using the double occlusion technique (23, 24), and Pcap was calculated as the mean of the arterial and venous pressures at 3 s after occlusion.

Isogravimetric pressure. Isogravimetric pressure (Piso) is the maximum Pcap at which the lungs do not gain weight, indicating that the Starling forces are balanced (23, 24). Piso was determined by discontinuing flow through the lung and opening a shunt between arterial and venous tubing so that Ppa and Pla were identical and thus equal to Pcap. Pla was then altered in 1 mm Hg increments by alteration of the height of the venous reservoir, and the effect on lung weight was examined. Piso was defined as the highest Pcap at which the lung did not gain weight over a 4-min period.

Pulmonary capillary filtration coefficient. K_f,c was then calculated by a modification of the methods of Drake and colleagues (23, 24). When Pcap equals Piso, the Starling forces are balanced so that there is no net transfer of fluid across the pulmonary capillary membrane. When the Pcap is suddenly increased from Piso to Piso + 7 mm Hg, the other Starling forces initially remain unchanged so that pulmonary edema formation occurs at a rate of 7 × K_f,c. However, lung weight gain after the increase in Pcap is due to both intravascular volume expansion and the edema formation. The expansion is rapid and essentially complete within 3 min. Therefore, the rate of weight gain was recorded on semilog plot every minute from 3 to 10 min and extrapolated back to time = 0 by linear regression.

Wet-to-dry weight. At the end of experiment, the lungs were dissected from the heart-lung block and wet weight was measured. The lungs were then placed in a drying chamber, and weighed on a daily basis. Dry weight was recorded as the plateau weight where sequential weighs demonstrated that maximal dehydration occurred.

Series I: Exclusion Criteria

Prior to randomization, baseline perfusion was commenced for 10 min with 95% O₂-5% CO₂. For all preparations, the following exclusion criteria were applied: air leak from preparation; appearance of gas bubbles or emboli in perfusate cannulae; weight gain > 2 g during baseline perfusion; weight gain > 3 g during estimation of baseline K_f,c; perfusate leakage > 0.3 ml/min from preparation during baseline perfusion; Ppa > 20 mm Hg; weight gain > 3 g during the second 15 min interval after baseline K_f,c; weight gain > 1 g during 5 min immediately after addition of purine. Preparations were discarded where any of these parameters were exceeded during the baseline stage.

Series I: Injury Induced by Warm Ischemia and Reperfusion

A model of warm ischemia-reperfusion was developed, modified from a pervious report (25). After baseline perfusion, and provided none of the exclusion criteria were met, the preparations were then randomized. Depending on group allocation, ventilation gas mixture was altered to either 5% CO₂, 75% O₂, 20% N₂ or to 25% CO₂, 75% O₂. The target pH and PCO₂ were achieved over a 30-min period. In the metabolic acidosis and buffered hypercapnia groups, the target pH was achieved by titration with hydrochloric acid and sodium hydroxide, respectively. Pilot studies confirmed that with this approach, the perfusate osmolarity was maintained at 290 ± 5 mOsm/L in all groups. After stabilization at target pH and PCO₂, purine (0.006 g) was added to replace physiologic sources, and reperfusion continued for an additional 10 min. Ventilation and then perfusion were then stopped, and lung inflation was maintained using CPAP 3 mm Hg with the same gas mixture and the temperature maintained at 37° C in a humidified chamber. After 45 min, mechanical ventilation was recommenced. Perfusion was then commenced at 15 ml/min and increased in a standardized fashion, increasing by 15 ml/min each minute for the first 10 min. The perfusion continued for an additional 30 min, at which stage final values for Ppa, Pcap, Piso, and K_f,c were measured. Wet:dry weight was then measured.

Series II: In Vitro Xanthine Oxidase Activity

To assess the effects of perfusate pH and PCO₂ on the enzymatic activity of exogenous xanthine oxidase, the generation of uric acid was measured in the same perfusate solution (Krebs-Henseleit solution containing 3% bovine serum albumin; temperature, 37° C; 500 ml per beaker) that was used in the perfused lung experiments. The perfusate preparations were allocated to four groups, (1) control (FI_CO2, 5%; pH, 7.4), (2) HCA (FI_CO2, 25%; pH, 6.9), (3) metabolic acidosis (FI_CO2, 5%; pH, 6.9), and (4) buffered hypercapnia (FI_CO2, 25%; pH, 7.4). The perfusate was bubbled with 5% CO₂, 75% O₂, 20% N₂ or with 25% CO₂, 75% O₂, as appropriate; osmolarity was maintained at 290 ± 5 mOsm/L; and the target pH and PCO₂ was achieved in the metabolic acidosis and buffered hypercapnia groups by means of titration of hydrochloric acid and sodium hydroxide, respectively. Upon stability of pH and PCO₂, purine (1.5 mM) was added, followed 2 min later by xanthine oxidase (0.006 U/ml). Oxypurinol (2.4 mM) was used to terminate the reaction (26) in samples taken at timed intervals, and the concentration of uric acid was assayed using an Olympus AU 800 (Olympus Corp of America, New Hyde Park, NY) clinical chemistry system, using a coupled uricase enzyme assay (27).

Data Analysis and Statistics

All data were entered into a standard spread sheet (Excel 7.0; Microsoft Corp., Redmond, WA) and exported for analysis using Sigmastat no. 2; Sandel Scientific, Corte Madera, CA). The data are summarized as means ± SEM. Statistical analysis utilized Kruskal-Wallis and Student-Newman-Keuls tests for nonparametric data, and ANOVA with Student-Newman-Keuls for parametric data. We considered differences significant when p < 0.05.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Series I: Injury Induced by Ischemia-Reperfusion

Baseline. All baseline variables were similar in the four groups (p = NS) (Table 1). Specifically, there was no significant difference in perfusate pH in the control versus buffered hypercapnia groups, or in the hypercapnic acidosis (HCA) versus metabolic acidosis groups. There was no significant difference in perfusate PCO₂ in the control versus metabolic acidosis groups, or in the HCA versus buffered hypercapnia groups. Baseline values of Pcap, Piso, K_f,c, Ppa, and Paw were comparable among the groups (Table 1 and Figures 1 and 5).

View larger version (15K): [in this window] [in a new window]   Figure 1.   Kf,c measured before and after IR injury. Results are mean ± SEM of the data. *p < 0.05 post-IR versus pre-IR; n = 10 per group.

View larger version (15K): [in this window] [in a new window]   Figure 5.   Peak airway pressure (Paw) at baseline, before IR, and after IR. Results are mean ± SEM of the data. *p < 0.05 in all groups over time; n = 10 per group.

Microvascular pressure measurements. There were no significant differences in Pcap among the groups or within each group before ischemia-reperfusion (IR) versus the following IR (p = NS) (Table 1).

Pulmonary capillary permeability measurements. There were no differences in baseline capillary permeability (K_f,c) between the groups (p = NS) (Figure 1). The final K_f,c was similar in the control and buffered hypercapnia groups, and was greater in those groups compared with both HCA and metabolic acidosis (p < 0.05) (Figure 1). Furthermore, the rank order of final K_f,c among the groups was: control  buffered hypercapnia > metabolic acidosis > hypercapnic acidosis (p < 0.05) (Figure 1). This indicates that permeability after reperfusion was worst (and comparable) in the control and buffered hypercapnia groups (indicating minimal protection), was least in the HCA group (indicating maximal protection), and was intermediate in the metabolic acidosis group (indicating intermediate protection).

Isogravimetric pressure measurements. There was a significant decrease in isogravimetric pressure (Piso) in all groups after versus before IR (p < 0.05) (Table 1). The magnitude of the decrease in Piso (Piso; Baseline Piso  Final Piso) was greatest in the buffered hypercapnia group compared with all other groups (p < 0.05) (Figure 2). Piso was comparable in control, hypercapnic acidosis, and metabolic acidosis groups (p = NS) (Figure 2).

View larger version (18K): [in this window] [in a new window]   Figure 2.   Reduction in isogravimetric pressure (Piso; Baseline Piso  Final Piso) after IR injury. Results are mean ± SEM of the data. *p < 0.05 versus other groups; n = 10 per group.

Wet:dry measurements. The wet:dry weights ratio was less in the HCA versus the control and buffered hypercapnia groups (p < 0.05) (Figure 3). The ratio was less with metabolic acidosis versus buffered hypercapnia (p < 0.05) (Figure 3), and was comparable between the control and buffered hypercapnia groups (p = NS) (Figure 3).

View larger version (26K): [in this window] [in a new window]   Figure 3.   Wet:dry weight ratios after IR injury. Results are mean ± SEM of the data. *p < 0.05; n = 10 per group.

Pulmonary artery pressure measurements. Baseline Ppa was similar in all groups (p = NS) (Table 1). There was a significant increase in Ppa in all groups after IR (p < 0.05) (Figure 4). Despite comparable injury (in terms of permeability and weight gain), the elevation in Ppa (Post-Reperfusion Ppa  Pre-Ischemia Ppa; Ppa) was significantly less in the buffered hypercapnia group compared with the control group (p < 0.05) (Figure 4). In fact, despite worse injury, the Ppa was similar in the buffered hypercapnia group compared with both the HCA and the metabolic acidosis groups (p = NS) (Figure 4).

View larger version (18K): [in this window] [in a new window]   Figure 4.   Elevation in pulmonary artery pressure (Ppa; postreperfusion Ppa  Preischemia Ppa) after IR injury. Results are mean ± SEM of the data. *p < 0.05 versus other groups; n = 10 per group.

Airway pressure measurements. There was a significant increase in Paw in all groups over time (p < 0.05) (Figure 5). However, there were no significant differences between the groups before or after IR (p = NS) (Figure 5).

Series II: In Vitro Inhibition of Xanthine Oxidase (XO)

The target pH values were 7.4 in the control and buffered hypercapnia groups, and 6.9 in the HCA and metabolic acidosis groups. The target PCO₂ values were 34 mm Hg in the control and metabolic acidosis groups, and 110 mm Hg in the HCA and buffered hypercapnia groups. The concentration of uric acid produced at 1 and 2 h after addition of XO was comparable in the control versus buffered hypercapnia groups (p = NS) (Figure 6), and was comparable in the HCA versus metabolic acidosis groups (p = NS) (Figure 6). XO activity was less in the HCA and metabolic acidosis groups than in the control and buffered hypercapnia groups (p < 0.05) (Figure 6), indicating that the activity of XO is critically dependent on pH and is independent of PCO₂.

View larger version (14K): [in this window] [in a new window]   Figure 6.   Uric acid at specific time points after the in vitro addition of xanthine oxidase to purine under conditions described by each group allocation. Results are mean ± SEM of the data. *p < 0.05 ANOVA for both acidosis groups versus both normal pH groups; n = 3 per group.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Rationale for Hypercapnic Acidosis in Acute Lung Injury

Permissive hypercapnia has been discussed as a potentially beneficial strategy in ARDS (2, 28), and its use has been reported to improve clinical outcome as reported in a recent clinical trial (3). The current conceptual framework for instituting permissive hypercapnia emphasizes reduced lung stretch, with potentially less ventilator-induced lung injury (31), and, tolerance to the resultant elevated Pa_CO2 (17, 18, 28, 29), relegating the role of the elevated CO₂ to that of a bystander. This approach assumes that the hypercapnic acidosis generated reflects the underlying protective hypoventilatory strategy rather than having any direct therapeutic role per se. The possibility that hypercapnic acidosis may exert clinically important organ protection has received little attention to date (10). We have shown previously that hypercapnic acidosis is highly protective in both ischemia-reperfusion (IR) and free-radical-mediated acute lung injury (ALI) (8). The findings of the current study further confirm and expand upon our previous work (8), generating further insights into the mechanisms of the protective role of hypercapnic acidosis in organ injury.

Acidosis Is Protective in Acute Lung Injury

We have confirmed previous work documenting that metabolic (9) and hypercapnic (8) acidosis is protective in the setting of ALI. We have extended those findings, confirming that HCA is more protective than metabolic acidosis, in terms of microvascular permeability. This supports our first hypothesis suggesting that the elevated levels of PCO₂ in HCA result in the rapid inward diffusion of highly soluble CO₂ molecules, thus lowering the intracellular pH by subsequent dissociation. This occurs more rapidly than is possible by inward equilibration of relatively impermeant H⁺ ions associated with metabolic acidosis (22) and is consistent with the differences observed in protection observed with hypercarbic and normocarbic acidosis in other organ systems (7, 11). In the myocardium, for example, it has been demonstrated that intracellular acidosis is more rapidly augmented when the extracellular acidosis is due to hypercapnia as opposed to a metabolic source (21). This finding provides an important mechanistic insight and suggests that the protective effect of HCA is at least partly dependent on intracellular mechanisms.

Buffering of Hypercapnic Acidosis in ALI

Sodium bicarbonate is commonly used, albeit somewhat controversially (17, 20) in a wide variety of conditions, including permissive hypercapnia (17). In the current study, buffered hypercapnia was less protective than either hypercapnic or metabolic acidosis, and buffering hypercapnic acidosis to normal pH greatly attenuated the protective effect of HCA. In fact, buffering the hypercapnic acidosis was associated with the largest reduction in Piso of all the groups. It should be remembered, however, that although there was no significant difference between the control and buffered hypercapnia groups with regard to K_f,c, there was definitely no protective effect with buffered hypercapnia. We suggest that this represents a Hypercapnia Paradox, whereby hypercapnia in the setting of acidosis is highly protective, but when buffered to normal pH is not. In terms of mechanistic insights, this may suggest that it is the acidosis (whether extracellular or intracellular) rather than the carbon dioxide per se that is protective. Alternatively, it may be that extremely high levels of intracellular CO₂, as observed after administration of sodium bicarbonate (32) is harmful. The exact mechanisms of the deleterious effects of buffering HCA are unclear. One possible explanation involves the suggestion that hydrogen ions protect against IR lung injury via alterations of amiloride-sensitive Na-H exchange channels (9). This is supported by the clinical use of sodium bicarbonate therapy for tricyclic cardiac toxicity, which is based on altered sodium channel kinetics (33). In cardiac myocytes, sodium channel alterations associated with sodium bicarbonate administration are due to the alterations in extracellular pH and to changes in extracellular sodium concentration (33). Therefore, although not confirmed by the current series of experiments, we speculate that the deleterious effects of buffering HCA in this model might involve alterations in the functional opening status of microvascular ion channels, resulting in altered pulmonary fluid regulatory mechanisms. The functional characterization of such channels has been reviewed in depth elsewhere (34). Development and investigation of such a hypothesis would require extensive additional work, but it would provide critical clinically relevant insights.

Pulmonary Vascular Tone

Buffered hypercapnia preparations had lower Ppa than the other groups, both before and after IR injury. Furthermore, the magnitude of the increase in Ppa (i.e., Ppa) was least in the buffered hypercapnia group, despite the severe injury incurred in that group. This confirms previous work demonstrating that hypercapnia per se (as opposed to hypercapnic acidosis) is a potent pulmonary vasodilator (35). Furthermore, acidosis regardless of etiology acts as a pulmonary vasoconstrictor. HCA results in pulmonary vasoconstriction, demonstrating that pulmonary vascular tone is more sensitive to pH than to PCO₂. These findings suggest that the direct effects of pH and PCO₂ are independent.

Role of Xanthine Oxidase

Our studies of the in vitro activity of xanthine oxidase, a pivotal enzyme in ischemia-reperfusion injury, demonstrate that its activity is critically pH-dependent, with acidosis markedly reducing enzyme activity. This confirms our previous findings (8) and lends further weight to the contention that it is the acidosis rather than the hypercapnia per se that is protective in the setting of acute lung injury. The differential effect of HCA versus metabolic acidosis in an ex vivo model of ALI compared with their identical effect on in vitro xanthine oxidase activity may be explained by the ability of HCA to more rapidly establish an intracellular acidosis. This in turn implies a more prominent role for intracellular subtypes of xanthine oxidase (36) or flavoenzymes (37) in IR injury compared with the extracellular free radical sources.

Limitations of the Study

Extrapolation of our results to the clinical situation must be restricted by the inherent limitations of an isolated buffer- perfused model. The perfused lung is denervated, isolated from the systemic circulation, and perfused with a blood-free perfusate. The particular concerns relate to the lack of pulmonary-systemic interactions in the context of organ injury and the potential systemic effects of profound hypercapnia. A further limitation of the current study is the lack of a definite mechanism to explain the observed effects. The pH-dependent nature of both organ protection in lung IR injury and XO activity has been demonstrated, and it is known that XO plays a pivotal role in tissue injury. However, we still do not have definitive proof that the protection afforded by acidosis was in fact mediated via inhibition of endogenous (intracellular or extracellular) XO. Furthermore, the effect of buffered metabolic acidosis on ALI, i.e., generating a metabolic acidosis and then buffering it to normal pH, was not assessed. This was considered less relevant to the clinical strategy that currently pertains in the management of ALI, i.e., permissive hypercapnic acidosis, and the possible utility of therapeutic hypercapnia but may have relevance in injury to other organ systems, e.g., brain or myocardial ischemia, and deserves exploration (10).

Clinical Significance

There are several clinical implications of importance arising from this study. First, although acidosis is clearly both a natural response to, and a marker of, tissue dysoxia, and a predictor of adverse outcome, it is not necessarily harmful per se. Second, HCA is more protective than metabolic acidosis in the setting of ALI. Third, the protective effects of HCA are abolished if the pH is buffered towards normal. This suggests that in the absence of correcting the primary problem, buffering of HCA may be detrimental where ALI related to IR may be a concern. The use of bicarbonate to correct acidosis, e.g., during cardiac arrest, has become increasingly controversial (20), and it has been removed from routine use in cardiac arrest algorithms (38). The results of the current study support this concern.

We conclude that in the current model of IR-induced acute lung injury: (1) hypercapnic and metabolic acidosis are protective, (2) hypercapnic acidosis is most protective, (3) buffering of hypercapnic acidosis attenuates its protective effects, (4) buffered hypercapnia is a potent pulmonary vasodilator in the context of ALI, (5) metabolic and hypercapnic acidosis inhibit in vitro XO activity to a similar degree, and (6) because metabolic and hypercapnic acidosis inhibit in vitro XO activity to a similar degree, the differential effects on ALI cannot be explained solely on the basis of extracellular XO inhibition.