Nineteen sheep were anesthetized with 30 mg/kg sodium pentobarbital, then intubated and mechanically ventilated (Dual Phase Control Respirator Pump Ventilator; Harvard Apparatus, South Natick, MA, USA) with a tidal volume of 15 ml/kg, a fraction of inspired oxygen (F_IO₂) of 0.21 and positive end-expiratory pressure adjusted to maintain O₂ arterial saturation at more than 90%. The respiratory rate was set to keep the end-tidal PCO₂ at 35 mmHg. Neuromuscular blockade was performed with intravenous pancuronium bromide (0.06 mg/kg). Additional pentobarbital boluses (1 mg/kg per hour) were administered as required.

Catheters were advanced through the left femoral vein to administer fluids and drugs, and through the left femoral artery to measure blood pressure and to obtain blood gases. A pulmonary artery catheter was inserted through right external jugular vein (Flow-directed thermodilution fiberoptic pulmonary artery catheter; Abbott Critical Care Systems, Mountain View, CA, USA).

An orogastric tube was inserted to allow drainage of gastric contents. A midline laparotomy and splenectomy were then performed. An electromagnetic flow probe was placed around the superior mesenteric artery to measure intestinal blood flow. A catheter was placed in the mesenteric vein through a small vein proximal to the gut to draw blood gases. A tonometer was inserted through a small ileotomy to measure intramucosal PCO₂. Lastly, after careful hemostasis, the abdominal wall incision was closed.

Arterial, systemic, pulmonary and central venous pressures were measured with corresponding transducers (Statham P23 AA; Statham, Halo Rey, Puerto Rico). Cardiac output was measured by thermodilution with 5 ml of saline solution (HP OmniCare Model 24 A 10; Hewlett Packard, Andover, MA, USA) at 0°C. An average of three measurements taken randomly during the respiratory cycle were considered and were normalized to body weight to yield Q. Intestinal blood flow was measured by the electromagnetic method (Spectramed Blood Flowmeter model SP 2202 B; Spectramed Inc., Oxnard, CA, USA) with in vitro calibrated transducers 5–7 mm in diameter (Blood Flowmeter Transducer; Spectramed Inc.). Occlusive zero was controlled before and after each experiment. Non-occlusive zero was corrected before each measurement. Superior mesenteric blood flow was normalized to gut weight (Q_intestinal).

Arterial, mixed venous and mesenteric venous partial pressure of oxygen (PO₂), PCO₂ and pH were measured with a blood gas analyzer (ABL 5; Radiometer, Copenhagen, Denmark), and hemoglobin and oxygen saturation were measured with a co-oximeter calibrated for sheep blood (OSM 3; Radiometer). Arterial, mixed venous and mesenteric venous contents (C_aO₂, C_vO₂ and C_vmO₂, respectively) were calculated as (Hb × 1.34 × O₂ saturation) + (PO₂ × 0.0031). Systemic and intestinal oxygen transport and oxygen consumption (DO₂, VO₂, DO_2i and VO_2i, respectively) were calculated as DO₂ = Q × C_aO₂; VO₂ = Q × (C_aO₂ - C_vO₂); DO_2i = Q_intestinal × C_aO₂, and VO_2i = Q_intestinal × (C_aO₂ - C_vmO₂).

Intramucosal PCO₂ was measured with a tonometer 15 (TRIP Sigmoid Catheter; Tonometrics, Inc., Worcester, MA, USA) filled with 2.5 ml of saline solution; 1.0 ml was discarded after an equilibration period of 30 min and PCO₂ was measured in the remaining 1.5 ml. Its value was corrected to the corresponding equilibration period and was used to calculate ΔPCO₂.

Mixed venous–arterial and mesenteric venous–arterial PCO₂ differences were also calculated. Arterial, mixed venous and mesenteric venous CO₂ contents (CCO₂) and their differences were calculated with Douglas's algorithm 16. Systemic and intestinal CO₂ production (VCO₂ and VCO_2i, respectively) were calculated as VCO₂ = Q × mixed venoarterial CCO₂, and VCO_2i = Q_intestinal × mesenteric venoarterial CCO₂. Global blood capacity for transporting CO₂ was evaluated as the ratio between venoarterial CCO₂ and PCO₂ differences (R_a-v). This index has been used to evaluate the amount of CO₂ transported by the blood in relation to the venoarterial gradient of PCO₂ 17.

Lactate, sodium, potassium, chloride and serum total proteins were measured with an automatic analyzer every 60 min (Automatic Analyzer Hitachi 912; Boehringer Mannheim Corporation, Indianapolis, IN, USA). Anion gap was calculated as ([Na⁺] + [K⁺]) - ([Cl^-] + [HCO₃^-]). Anion gap was corrected for changes in plasma protein concentration 18.

Basal measurements were taken after a stabilization period longer than 30 min. Then animals were assigned to the following groups: (1) sham group (n = 6), consisting of sheep receiving 100 ml of saline in 10 min, followed by an infusion necessary to keep intestinal blood flow at basal levels; (2) normal blood flow group (n = 7), consisting of sheep receiving 5 μg/kg Escherichia coli lipopolysaccharide dissolved in 100 ml of saline in 10 min, and then saline infusion so as to maintain intestinal blood flow at basal levels; and (3) increased blood flow group (n = 6), consisting of sheep receiving 5 μg/kg Escherichia coli lipopolysaccharide dissolved in 100 ml of saline in 10 min, followed by saline infusion so as to increase intestinal blood flow by 50% from basal levels.

F_IO₂ was increased to 0.50 in endotoxemic sheep to avoid deep hypoxemia.

Measurements were performed at 30 min intervals for 120 min from the start of endotoxin administration.

At the end of the experiment, the animals were killed with an additional dose of pentobarbital and a KCl bolus. A catheter was inserted in the superior mesenteric artery and Indian ink was instilled through it. Dyed intestinal segments were dissected, washed and weighed for the calculation of gut indexes.

The local Animal Care Committee approved the study. Care of animals was in accordance with National Institute of Health guidelines.

Data were assessed for normality and expressed as means ± SD. Differences within groups were analyzed with a repeated-measures analysis of variance and Dunnett's multiple comparisons test to compare each time point with basal. One-time comparisons between groups were tested with a one-way analysis of variance and a Newman–Keuls multiple comparison test.

Sham, normal blood flow and increased blood flow groups received 10 ± 6, 24 ± 9 and 91 ± 38 ml/kg per hour, respectively, of normal saline solution (P < 0.05) to achieve resuscitation goals. Variations of intestinal blood flow from basal values, at the end of the experiment, were 8 ± 5%, – 1 ± 22% and 60 ± 22%, respectively (P < 0.05). As expected, the increased blood flow group had higher central venous and pulmonary wedge pressures, intestinal blood flow, cardiac output and systemic oxygen transport than the normal blood flow group. The increased blood flow group had also higher intestinal oxygen consumption (Table 1).

Metabolic acidosis developed in both groups with endotoxemia, but was greater in the increased blood flow group because of hyperchloremia and an increased anion gap (Table 2 and Fig. 1). These variables did not change in the sham group. Lactate levels remained stable in the three groups (Table 2).

ΔPCO₂ increased in the normal blood flow group and remained unchanged in the increased blood flow and sham groups (Fig. 2). Systemic and intestinal venoarterial PCO₂ differences were also higher in the normal blood flow group than in the others (Table 3). Systemic and intestinal R_a-v were lower in both endotoxemic groups.

We used a short-term infusion of endotoxin followed by saline expansion to induce a state of normodynamic shock, with preserved cardiac output and intestinal blood flow 1920. A state of normodynamic shock was chosen as a control group to avoid CO₂ accumulation caused by macrovascular hypoperfusion. We found that intramucosal acidosis and systemic metabolic acidosis occurred, in spite of stable systemic and gut oxygen transports and consumptions.

The reason for increased intestinal ΔPCO₂ in sepsis remains controversial 21. It might reflect hypoperfusion, but has also been found in normodynamic states 22. Vallet and colleagues studied endotoxemic dogs with low blood flow, resuscitated with dextran. Gut flow was increased and oxygen transport normalized, but oxygen uptake and mucosal PO₂ and pH remained low, results that were ascribed to flow redistribution from mucosal to serosal layers 13. Conversely, Revelly and colleagues described flow redistribution from serosa to mucosa induced by endotoxin 23. VanderMeer and colleagues found that intramucosal acidosis developed despite preserved blood flow and tissue PO₂ in endotoxemic pigs, attributed to changes in energetic metabolism 24. Thus, the concept of 'cytopathic hypoxia' was introduced 6.

However, cytopathic hypoxia and increased anaerobic CO₂ production might not be the sole explanation for the increase in ΔPCO₂. Vallet and colleagues 25 and Dubin and colleagues 26 recently showed that hypoperfusion is a key factor in the development of venous and tissue hypercarbia. In addition, Tugtekin and colleagues showed an association between increased ΔPCO₂ and diminished villi microcirculation 7.

This body of information suggests that intramucosal acidosis in sepsis is due mainly to microcirculatory alterations, even though cardiac output and regional flows might remain unchanged. Disturbed energetic metabolism might be present in sepsis, but it does not explain intramucosal acidosis. However, it might be a reasonable explanation for the development of systemic metabolic acidosis in our experiments. Increased anion-gap metabolic acidosis appeared despite preserved oxygen metabolism. As described previously, metabolic acidosis was not explained by elevations of lactate but by increases in unmeasured anions whose source and identification are still unknown 2728.

Increased blood flow by volume expansion prevented ΔPCO₂ elevation. PCO₂ gradients, venoarterial and tissue-arterial PCO₂ differences are the result of interactions between CO₂ production, blood capacity to transport CO₂ and blood flow to tissues. We and others have previously shown that ΔPCO₂ fails to reflect tissue hypoxia when blood flow is preserved 252629. Our results suggest that intramucosal acidosis is related mainly to local hypoperfusion, because the only difference between our groups, in terms of PCO₂ difference determinants, was the level of blood flow. We can speculate that volume expansion might improve microcirculation and, subsequently, CO₂ clearance. However, intramucosal acidosis might be corrected by the inhibition of inducible nitric oxide synthase and without microcirculatory recruitment 30. Improvement of cellular metabolism and/or redistribution of blood flow from the mucosa to other layers have been proposed as underlying mechanisms. We cannot exclude the possibility that increases in blood flow might decrease tissue hypoxia and anaerobically generated CO₂. Intestinal VO₂ increased after elevation of O₂ transport in the increased blood flow group, suggesting unmet needs in the normal blood flow group. Flow might have been inadequate in the face of increased metabolic requirements caused by endotoxemia 31.

Despite this apparent dependence on intestinal oxygen supply, CO₂ production remained stable. Possible reasons are error propagation in the VO₂ and VCO₂ calculations, or an increase in VO₂ due to non-metabolic processes, such as the production of inflammatory reactants and reactive oxygen species 32.

Other investigators have reported that volume expansion could not correct intramucosal acidosis, in both clinical and experimental settings 111314. Differences in the level of attained blood flow, timing of expansion or the type of injury might account for these findings opposite to ours.

Potential limitations of our study are related to the errors of saline tonometry, such as inadequate equilibration time, deadspace effect and underestimation of PCO₂ by blood gas analyzers 3334.

Metabolic acidosis was a prominent finding in our study. Expansion with large volumes of saline predictably produced hyperchloremic metabolic acidosis 35. In addition, metabolic acidosis arose as a result of unmeasured anions. Previous research has shown that during streptococcal infusion in pigs, metabolic acidosis decreased, but did not disappear, when oxygen transport was supported with dextran and red blood cells 36.

The reason for augmented unmeasured anions in the increased blood flow group is unclear. Possible causes are washout of tissue acids by high blood flow, or an impairment of oxygenation caused by tissue edema. Nevertheless, Gow and colleagues have shown that oxygen extraction is already altered in septic animals, so increased diffusion distances would not be relevant 37.

In addition, hyperchloremic acidosis might induce an inflammatory response, cellular dysfunction and apoptosis, and increased mortality in experimental septic shock 38394041. In this way, a deleterious effect of acidosis on cellular function with the subsequent production of unknown anions might be operative.