INTRODUCTION

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Sepsis is a major cause of morbidity and mortality affecting an estimated 500,000 persons annually at a cost of $5-10 billion among hospital patients, in the United States alone (1, 2). The high mortality of patients with sepsis (20-40%) correlates with the extent and severity of systemic organ injury (3) and the development of altered oxygen metabolism (4, 5). Alterations of oxygen metabolism during critical illness have been well documented and are characterized by impaired tissue oxygen extraction and a "pathologic" dependency of oxygen consumption (O₂) on oxygen delivery (O₂) (4, 5).

The putative mechanism for altered oxygen metabolism during sepsis and other acute illnesses relates to insufficient tissue oxygen delivery (4, 5). However, previous investigations by ourselves (6) and others (9) suggest that altered oxygen metabolism during sepsis is not sufficiently explained by impaired oxygen delivery to the tissues. Instead, it is possible that, in addition to altered metabolism of glucose, proteins, and lipids (13), sepsis is associated with disruption of oxygen-consuming pathways. This notion is supported by clinical trials wherein augmentation of systemic oxygen delivery did not improve outcome in critically ill patients with established sepsis (14, 15). These observations led us to examine the possibility that sepsis-induced alterations of oxygen metabolism, such as O₂-O₂ alterations, may result from damage to oxygen- using intracellular organelles, the most significant being the mitochondria.

In animal models of sepsis, other investigators have shown that sepsis is associated with mitochondrial dysfunction (16) and ultrastructural evidence of mitochondrial injury (17, 18). However, these studies were not designed to prevent tissue hypoxia through resuscitative measures (e.g., fluid or blood transfusion) and did not monitor for changes in O₂-O₂ relationships that are characteristic of sepsis and other critical illnesses (4, 5). Thus, the relationship between mitochondrial injury and sepsis-induced alterations of oxygen metabolism has not been defined.

The present study was designed, therefore, to test the hypothesis that sepsis-induced alterations of oxygen metabolism correlate with the extent and severity of mitochondrial injury. To test this hypothesis, we used the in situ autoperfused feline ileum preparation to examine simultaneously ileal O₂-O₂ relationships and mitochondrial ultrastructure 2 h after intravenous endotoxin (LPS) administration and in time-matched control animals. In support of the hypothesis, the results indicated that LPS-induced O₂-O₂ alterations in the ileum correlated with the severity of mitochondrial injury. Although causality was not verified, the association between LPS- induced O₂-O₂ alterations and mitochondrial injury suggests that sepsis-induced alterations in oxygen metabolism may be the result of impaired intracellular oxygen use.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Animal Preparation

The preparation used for this study has been described previously (6- 8). Briefly, having been fasted overnight, random source male cats were anesthetized with intramuscular ketamine-HCl (25 mg/kg) and intravenous sodium pentobarbital (10 mg/kg). All animals had a cuffed endotracheal tube placed via tracheostomy and were mechanically ventilated. A carotid artery and a femoral artery and vein were then cannulated. The femoral artery cannula was connected to a pressure transducer linked to a chart recorder to enable continuous monitoring of arterial pressure (). All arterial and venous blood samples taken during the experiment were withdrawn into heparinized syringes and processed immediately for measurement of pH, PCO₂, and PO₂, using a blood gas analyzer (ABL3; Radiometer America, Westlake, OH), and for hemoglobin concentration and oxygen saturation and content using a co-oximeter (OSM3; Radiometer America) that was specifically calibrated for feline blood. Fluids lost owing to insensible water loss and blood sample acquisition were replenished by administering buffered isotonic saline via the femoral vein [5-10 (ml/kg)/h]. Finally, rectal temperature was monitored throughout the experiment and maintained at 39° C.

Surgical Procedure

After a midline abdominal incision, the spleen and omentum were carefully retracted, and a 40- to 50-g segment of ileum, with its blood and lymph supply intact, was isolated. After resecting the remainder of the small and large intestines, the superior mesenteric artery was cannulated and perfused via a connection made with the carotid artery cannula. The superior mesenteric vein was then cannulated (PE 240 tubing, 1.67-mm i.d.), and the effluent was collected in a reservoir. Using an extracorporeal circuit that was primed with heparinized, white cell-free blood from a donor cat, the venous effluent was reinfused via the femoral vein. Venous pressures were maintained at 0 mm Hg throughout the experiment. Finally, continuous measurement of superior mesenteric venous blood flow was provided through an inline flow probe with the meter connected to the chart recorder.

Reagents

Lipopolysaccharide (LPS) from Escherichia coli (serotype 0127:B8; Sigma, St. Louis, MO) was dissolved in buffered isotonic saline and used at a concentration of 1.0 mg/ml.

Experimental Protocol

On completion of the surgical procedures and after a ~ 30 to 40-min period over which the ileal preparation was allowed to stabilize, ileal samples were taken for later electron microscopy analysis (described below). After another ~ 15-min stabilization period, baseline measurements (Pa_O2, Pa_CO2, arterial pH, total Hb (hemoglobin), HbO₂ [oxyhemoglobin], Sa_O2, O₂ content, , and superior mesenteric venous blood flow) were obtained. Animals were then randomly assigned to two treatment groups: an experimental group (n = 11) that received intravenous LPS (3.0 mg/kg) as prepared above, and a time-matched control group (n = 5) that received intravenous buffered isotonic saline alone in a volume matching the LPS dose. All animals were observed and maintained over time, and evaluated experimentally at 2 h posttreatment.

In all groups, Pa_CO2 and Pa_O2 were kept within normal limits (6, 7) by adjusting the fraction of inspired oxygen (FI_O2) and minute ventilation as needed. In addition, a normal arterial pH was maintained by administering bicarbonate intravenously as needed. The Pa_O2-to-FI_O2 ratio was determined at baseline (time 0) and at hourly intervals thereafter, with the FI_O2 increased from that of room air (21% O₂) to 1.0 (100% O₂) for approximately 10 min. Additional donor blood was administered via the extracorporeal circuit, as necessary, to maintain above 90 mm Hg. All measurements taken at baseline were repeated after 2 h, and then ileal tissue samples needed for electron microscopy analysis (described below) were obtained. After the injection of colored microspheres (described below) to evaluate ileal perfusion at 2 h posttreatment and ~ 10 min for them to distribute, ileal O₂- O₂ determinations were made (described below). Measurements of ileal HbO₂ content were made in vivo every 15 min by reflectance spectrophotometry (described below). At the completion of the experiment, samples of ileum were taken for determination of ileal perfusion (described below), and then all animals were given a lethal intravenous injection of sodium pentobarbital.

Ileal Oxyhemoglobin Content Measurements

Ileal HbO₂ contents were measured in vivo at baseline and every 15 min by reflectance spectrophotometry. This technique, described by Jobsis and co-workers (19), has been used to detect changes in HbO₂ content in vivo (19, 20). A diode-array UV-visible spectrophotometer (model 8452A; Hewlett-Packard, Waldbronn, Germany) equipped with a remote reflectance fiberoptic probe (model RSA-HP-84F; Labsphere, North Sutton, NH) was used to detect relative changes in tissue HbO₂ levels. The relative HbO₂ content of the ileal tissues was determined from the difference in the absorbance of incident light (deuterium lamp, 190-820 nm) measured simultaneously at a wavelength of 580 nm and at a nearby, neutral (isosbestic) wavelength (570 nm) (19). Measurements made at 580 nm reflect changes in the relative HbO₂ content as well as changes in relative blood volume. Measurements made at 570 nm reflect changes in relative blood volume only, as this is an isosbestic wavelength with regard to changes in HbO₂ and is thus unaffected by oxygenation status (19).

Relative HbO₂ contents were expressed as a percentage of the maximal measurable range, established by making similar measurements with the animal breathing an FI_O2 of 1.0 at baseline (most oxygenated condition) and by excising the complete ileal segment at the end of the experiment (i.e., no further blood flow) and making measurements until tissue oxygen extraction had ended (least oxygenated condition). The latter condition was verified in several (but not all) experiments by ventilating the animal with an FI_O2 of 0 (100% N₂) and making measurements before the dropped below 60 mm Hg, yielding the same results. To standardize the results, the measurements made during no ileal blood flow were used when determining the least oxygenated condition of the measurable range.

Ileal Perfusion Determinations with Colored Microspheres

The technique of measuring tissue perfusion by the use of colored microspheres as described and validated by Kowallik and colleagues (21) was used. Briefly, at the end of the 2 h experimental period, monochromatic microspheres (yellow; 15 µm in diameter, approximately 1.0 × 10⁵/g of ileum) (Dye-Trak; Triton Technology, San Diego, CA) were injected into the superior mesenteric artery catheter supplying the ileal preparation and flushed with buffered isotonic saline. At the end of the experiment, multiple pieces of ileum (~ 2-3 g) were removed at random locations along the remaining intact ileal segment, and the mucosal and muscularis layers were separated from each other in all pieces and weighed.

The tissue samples were digested in 4 M KOH at 72° C for 4 h, and the colored microspheres were then recovered from the tissues by filtering the mix with a polyester microfilter (pore size of 10 µm). The microfilter, along with the trapped microspheres, was carefully placed into a microcentrifuge tube, mixed with 600 µl of dimethylformamide, and centrifuged at 2,000 × g for 3 min. The extracted dye was measured spectrophotometrically (absorbance at 448 nm) and compared with a standard curve to determine its relative concentration. The concentration of dye correlates directly with blood flow over a clinically relevant range [0-1,000 (ml/min)/kg] (21). The blood flow to the mucosal and muscularis samples correlates with the dye concentration per kilogram of tissue and the measured blood flow [(ml/min)/kg] at the time of injection of the microspheres.

Tissue Processing for Electron Microscopy

Ileal tissue samples were obtained for electron microscopy processing and evaluation at baseline and again at 2 h posttreatment. Each time, ~ 1.0- to 1.5-cm pieces of ileum were taken from either end of the intact segment. Several cross-sectional rings (~ 2-3 mm wide) of ileal tissue were excised from the proximal ends of these pieces (i.e., away from the ligature), diced further along their longitudinal axes, and immediately submerged into isotonic fixative (4% paraformaldehyde, 2.5% glutaraldehyde, and 0.1 M sucrose in 0.1 M phosphate buffer, pH 7.4) for approximately 2 h at room temperature. The tissue was then minced such that longitudinal sections through the villi were easily obtained. Tissue pieces were then repeatedly rinsed in isotonic buffer (0.1 M sucrose in 0.1 M phosphate buffer, pH 7.4), postfixed in 1% osmium tetroxide in rinse buffer for 1 h at room temperature, rinsed again in rinse buffer, and stored overnight at 4° C. The next morning, the tissue pieces were allowed to come to room temperature, then dehydrated through an ascending series of ethanol solutions. After rinsing with propylene oxide, they were infiltrated with and embedded in Spurr medium which then polymerized overnight at 60° C. The next day, thin sections (~ 80-90 nm) were cut on a Reichart Ultracut E microtome, mounted on copper grids, stained with 2% uranyl acetate and Reynolds lead citrate, and then later examined using a Phillips CM-12 transmission electron microscope at 60 kV.

Mitochondrial Injury Determinations

Mitochondrial ultrastructure was evaluated by electron microscopy at three different locations within each ileal sample. Specifically, mitochondrial ultrastructure was analyzed within the villus tips, villus crypts, and the circumferential muscle layer. These sites were selected because of their relative susceptibilities to injury (e.g., the villus tips are more prone to various forms of injury than are other mucosal structures) and their unique morphological and functional characteristics (i.e., mucosal tissue has different characteristics than does smooth muscle). Mitochondrial ultrastructure was examined at three randomly selected and widely separated areas at each of these three locations on two different grids prepared from samples obtained at each time point. Examinations were made in a blinded fashion by two reviewers, and the severity of ultrastructural injury was quantified by determining a composite score (based on the scale of 0-5 as shown in Table 1) that represented all of the mitochondria visualized within the microscopy field. Thus, each reviewer had six total evaluations of mitochondrial injury made at each of the three locations (i.e., tips, crypts, and muscle) per time point. The results obtained by the two reviewers were pooled to yield a mean mitochondrial injury score at each of the three locations for each sample.

A strategy designed to grade consistently the severity of mitochondrial ultrastructural injury was established. It was derived from a scoring system based on the progressive stages of cellular injury, as described by Trump and colleagues (22). This staging system enumerates the characteristic progression of ultrastructural changes that occurs within cells in various models of inflammatory injury (e.g., ischemia- reperfusion). The association between the degree of mitochondrial injury and the defined stages of cellular injury (Table 1) was employed to quantify and standardize the severity of ultrastructural injury to the ileal mitochondria.

Ileal O₂-O₂ Determinations

Superior mesenteric blood flow was sequentially decreased from baseline in a stepwise manner (i.e., ~ 1- to 2-ml/min decrements) to approximately 10% of baseline, using a variable resistance clamp in the arterial circuit. After each decrement in blood flow, at least 5 min was allowed for equilibration, and then systemic arterial and ileal venous blood samples were obtained and immediately analyzed for their oxygen contents. The measured superior mesenteric venous blood flow and oxygen contents were used to calculate ileal O₂ and O₂ according to the Fick equation. Ileal O₂-O₂ relationships were analyzed using least-squares regression to determine the critical thresholds for oxygen delivery (O₂c) and oxygen consumption (O₂c), as described previously (6). Finally, using the ratio of O₂ to O₂ at the minimal oxygen delivery (i.e., the lowest blood flow) and at the O₂c, the maximum oxygen extraction (O₂ERm) and critical oxygen extraction (O₂ERc) were determined, respectively.

Statistical Analyses

All data are expressed as means ± SEM. Relationships between O₂ and O₂ were determined using linear regression. The critical threshold for oxygen delivery (O₂c) was obtained using least-squares regression, as described previously (6). Values for O₂c, O₂c, O₂ERm, and O₂ERc were compared using a one-way analysis of variance (ANOVA) (23). Comparisons of hemodynamic and arterial blood parameters, relative HbO₂ contents and other spectrophotometric measurements, ileal perfusion distributions, and mitochondrial injury were made using a one-way ANOVA (treatment) with repeated measures (time). Post hoc analyses between group means at specific time points were performed using the Fisher test for least significant difference, which takes into consideration interactions due to multiple comparisons (23). Finally, relationships between mitochondrial injury and O₂c and O₂ERm were determined using linear regression and tested statistically by employing the Spearman correlation for bivariate analyses (23). Significance was based on a value of p  0.05.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Hemodynamic and Arterial Blood Parameters

At 2 h posttreatment, no significant differences were observed between the two groups with respect to either the measured arterial-venous pressure difference or the total blood flow across the ileal preparation (Table 2). In addition, the calculated vascular resistance in the ileum of the LPS-treated group was remarkably similar at 2 h to that of the control group. Although the LPS-treated animals required greater volume resuscitation relative to time-matched control animals (32 ± 3 versus 79 ± 8 ml; control versus LPS-treated animals, p < 0.01), arterial Hb concentrations and measured O₂ contents were not significantly different between the two groups and remained somewhat constant over time compared with baseline (Table 2). Furthermore, the arterial pH was well maintained over time compared with baseline and relatively consistent between the groups at 2 h (Table 2). However, the LPS-treated group required significantly greater amounts of administered bicarbonate to maintain the pH over time (10.8 ± 1.3 versus 17.3 ± 1.8 mEq; control versus LPS-treated animals, p < 0.01).

Ileal Oxyhemoglobin Contents

In vivo spectrophotometric measurements of relative HbO₂ contents changed little over 2 h, with control animals averaging nearly 97% and LPS-treated animals maintaining more than 92% of baseline values. Furthermore, there was no significant difference in the relative HbO₂ contents between the two groups at 2 h (Figure 1). The maximum absorbances for the control group (1.112 ± 0.097 and 1.098 ± 0.098 absorbance units [AU] at 580 and 570 nm, respectively) and the LPS-treated group (1.076 ± 0.033 and 1.061 ± 0.032 AU at 580 and 570 nm, respectively) were essentially the same for each group. Likewise, the minimum absorbances for the control group (0.800 ± 0.068 and 0.827 ± 0.068 AU at 580 and 570 nm, respectively) and the LPS-treated group (0.876 ± 0.037 and 0.902 ± 0.036 AU at 580 and 570 nm, respectively) were similar between the groups. Most importantly, the overall maximal measurable change in absorbance between the most and least oxygenated states was not significantly different between the two groups (0.042 ± 0.008 versus 0.041 ± 0.003 AU; control versus LPS) and was similar to that shown by other investigators (19). In addition, measurements made at 570 nm (which reflect relative changes in blood volume) were not significantly different at baseline (1.011 ± 0.055 versus 1.075 ± 0.035 AU; control versus LPS), and increased to 112.7 ± 1.6 and 106.0 ± 3.6% of baseline at 2 h in the control and LPS-treated groups, respectively.

View larger version (28K): [in this window] [in a new window]   Figure 1.   Relative oxyhemoglobin (HbO2) content in the ileal tissues, as a percentage of the maximal measurable range, in the control and LPS-treated groups 2 h after LPS administration compared with baseline (values are means ± SEM). Note that the HbO2 content remained relatively unaltered over time in both groups and indicated adequate O2 availability in the ileal blood through 2 h.

To obtain the relative HbO₂ content in vivo during the least oxygenated state, measurements were made in the setting of no ileal blood flow (as described in METHODS). These measurements were verified by comparing them with those made while ventilating the animals with an FI_O2 of 0, which yielded similar results. Namely, the difference in the absorbance measured at 570 nm and simultaneously at 580 nm was 0.027 ± 0.003 AU in the absence of blood flow and 0.025 ± 0.004 AU for an FI_O2 of 0. Correspondingly, this measurement was 0.027 ± 0.005 AU for controls and 0.026 ± 0.002 AU for the LPS-treated group. These measurements were consistent with those found by Jobsis and coworkers (19).

Ileal Perfusion Measurements

Measurements of the distribution of ileal blood flow to the mucosal and muscularis layers using colored microspheres demonstrated no significant difference over time from baseline or between the two groups at 2 h (Figure 2). Most importantly, the results showed that mucosal blood flow was maintained at 2 h, presenting no measurable evidence of shunting toward the muscularis layer after treatment with LPS.

View larger version (40K): [in this window] [in a new window]   Figure 2.   Blood flow to the mucosal and muscularis portions of the ileum, as determined from the distribution of colored microspheres, in the control and LPS-treated groups 2 h after LPS administration relative to baseline (values are means ± SEM). Note, in particular, that ileal blood flow and its distribution to the mucosa were maintained in the LPS-treated group compared with the control group and were relatively unchanged in both groups relative to baseline.

Ileal O₂-O₂ Relationships

As expected, on the basis of our previous experience with this model (7, 8), LPS treatment was associated with altered oxygen metabolism in the ileum after 2 h. Specifically, the LPS-treated group demonstrated a dramatic elevation in the ileal O₂c [25.1 ± 1.7 versus 18.3 ± 0.2 (ml/min)/kg; p < 0.01] and a significantly impaired ileal O₂ERm (60.2 ± 2.3 versus 74.1 ± 3.6%; p < 0.01) relative to time-matched control animals. Likewise, LPS treatment was associated with a significant decrease in the ileal O₂ERc (47.9 ± 3.9 versus 63.1 ± 4.6%; LPS-treated versus control animals, p < 0.01). In addition, although not significantly different, the ileal O₂c was slightly higher after LPS treatment compared with control animals (12.2 ± 0.6 versus 11.0 ± 0.7 (ml/min)/kg, respectively].

Mitochondrial Ultrastructure

LPS-treated animals demonstrated significant mitochondrial ultrastructural injury in the ileal mucosa relative to time-matched control animals (Figures 3 and 4). Although mitochondrial injury varied from mild to profoundly severe in the LPS-treated animals, on average, LPS treatment was associated with significant swelling of all intramitochondrial spaces, including the cristae (Figure 3). In the more severe instances, mitochondrial swelling was often accompanied by a disruption in membrane integrity as well. However, there were no significant differences in the relative presence or severity of mitochondrial injury between the ileal villus tips and crypts for either group. Thus, the results from these two locations were pooled for comparison of mitochondrial injury between the groups (Figure 4). In contrast to the findings in the mucosal layer, no significant change in mitochondrial ultrastructure was noted in the muscularis layer of the ileum over time or between groups. Finally, no evidence of mitochondrial injury was observed in any of the control ileal samples (Figures 3 and 4).

View larger version (171K): [in this window] [in a new window]   Figure 3.   Representative electron micrographs of ileal villus mitochondria from control (A) and LPS-treated (B-D) animals at 2 h posttreatment. Note that the mitochondria from the LPS-treated animals demonstrated a range of ultrastructural injury, from relatively mild to dramatically severe, consisting of swelling of all intramitochondrial spaces, particularly the cristae, and disruption of membrane integrity. Mitochondria from control animals were ultrastructurally normal in all respects. (Original magnification, ×55,000; staining, 2% uranyl acetate and Reynolds lead citrate.)

View larger version (16K): [in this window] [in a new window]   Figure 4.   Injury scores of ileal villus mitochondria for the control and LPS-treated groups 2 h after LPS administration relative to baseline (values are means ± SEM). Mitochondrial ultrastructural injury was dramatically evident and quite severe within 2 h of LPS treatment (*p < 0.001, compared with baseline and time-matched control group). In contrast, mitochondrial injury was virtually absent in control animals and changed little from baseline over time.

Bivariate analysis of the parameters of oxygen metabolism and the corresponding degree of mitochondrial injury determined at 2 h established significant and noteworthy correlations. Specifically, a positive correlation existed between the O₂c and mitochondrial injury (Figure 5); whereas a negative correlation existed between the O₂ERm and mitochondrial injury (Figure 6).

View larger version (16K): [in this window] [in a new window]   Figure 5.   The relationship between the critical oxygen delivery ( ·DO2c), as determined from individual O2- ·DO2 relationships, and the corresponding degree of mitochondrial injury in the control and LPS-treated animals, 2 h posttreatment. Note that there was a positive and significant correlation between the parameters.

View larger version (16K): [in this window] [in a new window]   Figure 6.   The relationship between the maximum oxygen extraction (O2ERm), calculated from measurements made during individual O2- ·DO2 determinations, and the corresponding degree of mitochondrial injury in the control and LPS-treated animals, 2 h posttreatment. Note that there was a negative and significant correlation between the parameters.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

The cause of organ dysfunction in sepsis is unknown. However, sepsis-induced disruption of oxygen metabolism is commonly observed and is believed to play a critical role in the pathogenesis of organ dysfunction (4, 5). The manifestations of sepsis-induced alterations of oxygen metabolism have been well documented both clinically (4, 5) and experimentally (7, 8). Namely, abnormal O₂-O₂ relationships in sepsis are characterized by impaired tissue oxygen extraction, elevated tissue O₂, and a dependency of O₂ on O₂ over a broad range of oxygen deliveries (4, 5). Moreover, the fact that O₂ is dependent on O₂ at higher than normal oxygen deliveries suggests that the tissues are anaerobic, a notion supported by the observation that tissue acidosis (11), hyperlactatemia (13), and abnormal O₂-O₂ relationships (7) coexist in sepsis.

Our experiments (6) and those of others (9) provide evidence that impaired tissue oxygen delivery may not be sufficient to explain altered oxygen metabolism during sepsis. In this regard, several studies (9) have demonstrated that tissue oxygen levels actually increase during sepsis. In addition, the intestinal mucosal layer has been shown to demonstrate persistent lactatemia and acidosis (signs of increased anaerobic respiration) despite increases in mucosal oxygen concentrations (11) and mucosal blood flow (12). Likewise, using the in situ autoperfused feline ileal preparation employed in this study, we have shown that microvascular injury, combined with conditions favoring tissue edema formation (i.e., the putative causes of impaired oxygen delivery to the tissues), does not result in altered O₂-O₂ relationships (6). Furthermore, in LPS-treated animals, the severity of microvascular injury in the ileum does not correlate with the development of ileal O₂-O₂ alterations (7). Taken together, these studies suggest that impaired oxygen use, rather than insufficient oxygen delivery, may be responsible for the O₂-O₂ alterations in sepsis.

On the basis of these observations, it follows that the principal oxygen-consuming organelles (e.g., mitochondria) may be injured in the tissues during sepsis or after LPS administration. Indeed, in a primate model of gram-negative sepsis, Simonson and coworkers have shown that mitochondrial morphology and function were impaired in skeletal muscle of the forearm (24). Moreover, using models of septic shock in which no attempt was made to maintain adequate tissue perfusion, others have demonstrated the presence of mitochondrial injury in various systemic organs (17, 18). By optimizing tissue perfusion (i.e., minimizing ischemia) and measuring parameters of tissue oxygen metabolism, the present study provides further insights into the pathogenesis of mitochondrial injury in the ileum during sepsis. The functional status of the ileum is of particular interest owing to its putative role in the perpetuation of a systemic inflammatory state during critical illness, as in sepsis (3).

In this study, we have demonstrated that time-dependent alterations in ileal oxygen metabolism correlate with the development of ileal mitochondrial ultrastructural injury. Two hours after LPS treatment, both O₂-O₂ alterations and mitochondrial injury (Figures 3 and 4) are present in the ileum. In keeping with our original hypothesis, highly significant correlations exist between variables of altered ileal oxygen metabolism and the severity of ileal mitochondrial injury (Figures 5 and 6). Importantly, LPS-induced alterations in ileal oxygen metabolism and mitochondrial ultrastructure occur despite unchanged relative ileal HbO₂ contents (Figure 1) and ileal mucosal blood flow distributions (Figure 2). Although these organ-specific indices of oxygen availability to the tissues cannot absolutely exclude the potential existence of microregional tissue hypoxia, the experiments were designed to carefully minimize and examine for the presence of tissue hypoxia and ischemia. Accepting the current technical limitations for measuring oxygen delivery and content in living tissue, the results of this study and of other investigations (10) provide compelling evidence suggesting that inadequate oxygen delivery to the tissues may not be the primary cause of altered oxygen metabolism in the ileum during sepsis.

If, as these findings suggest, tissue hypoxia is not the proximal cause of mitochondrial injury and altered oxygen metabolism in sepsis, then what is? One possible explanation evokes reactive oxygen intermediates (ROIs) as a cause of impaired mitochondrial function during acute inflammatory injury. Namely, various ROIs are known to be produced in tissues in models of sepsis (25), including superoxide anion (25, 26) and nitric oxide (NO) (27, 28). These ROIs could cause mitochondrial dysfunction resulting in impaired oxygen use and consequent oxygen metabolism alterations. In this regard, ROIs can inhibit mitochondrial oxidative phosphorylation (29), in turn, leading to increased formation of further ROIs (30). Besides potentially causing further mitochondrial damage and/or dysfunction, these ROIs, combined with the impaired electron flow through the inhibited electron transport chain, would result in less efficient oxygen use. Thus, ROI-induced mitochondrial inhibition could explain why the ileum was less effective in using the oxygen available to it (i.e., decreased O₂ERm) in this model of sepsis. Regardless of the oxygen-consuming pathways functioning in this setting (e.g., oxidative phosphorylation, ROI formation), in the absence of a reduction in O₂ (as was the case for the LPS-treated group), a decrease in oxygen extraction by the tissues would result in an increase in the critical O₂ (as demonstrated in the present study). To the extent that mitochondrial impairment results in inefficient oxygen use and additional ROI production during sepsis, mitochondrial inhibition could explain why augmenting oxygen delivery often fails to improve outcome in patients with established sepsis (14, 15).

Nitric oxide is a ROI that may play a critical role in sepsis-induced alterations of oxygen metabolism. NO alone (31, 32) or in combination with superoxide (29, 30) (i.e., peroxynitrite, the reactive product of NO and superoxide [29, 30, 33]) is known to impair mitochondrial oxygen consumption through inhibition of oxidative phosphorylation. Indeed, NO overproduction, as is seen during sepsis (27, 28), is capable of causing cell death associated with profound mitochondrial injury (34). A potential association between LPS-induced NO dysregulation and mitochondrial injury is supported by observations in our laboratory. Specifically, the severity of ileal mitochondrial injury correlated with the immunoprevalence of inducible nitric oxide synthase and 3-nitrotyrosine (indicates nitrosylation of tyrosine residues on proteins due to peroxynitrite) staining in ileal tissues (35). Despite these observations, the role of NO and other ROIs in the pathogenesis of O₂-O₂ alterations and mitochondrial injury remains undefined.

In summary, this study is provocative in that the results demonstrate a strong statistical correlation between mitochondrial injury and altered oxygen metabolism during sepsis. Although regional measures of oxygen delivery and blood flow distribution were unchanged from baseline in the LPS-treated group, the existence of microregional blood flow heterogeneity (i.e., hypoxia) was difficult to exclude owing to the limitations of current techniques for in vivo tissue oxygen measurement. Nonetheless, the results of this study are significant in that, regardless of the cause, mitochondrial injury occurred in the ileum despite efforts to provide sufficient oxygen to the tissues. Moreover, to the extent that the observed association between mitochondrial injury and altered oxygen metabolism reflects a causal relationship, an answer to the paradox regarding oxygen metabolism during sepsis may be provided. Namely, signs of anaerobic metabolism (e.g., tissue acidosis) develop in the gut during sepsis despite increased tissue oxygen content (10, 11) and increased tissue oxygen delivery (12). These observations are consistent with the possibility that impaired ileal oxygen use results from mitochondrial injury during sepsis. Mitochondrial dysfunction may also explain the results of large, well-designed clinical trials demonstrating no benefit to increasing systemic oxygen delivery in patients with established sepsis (14, 15). However, although the present study showed a significant association between mitochondrial injury and oxygen metabolism abnormalities in sepsis, causality was not demonstrated, and the mechanisms responsible for these findings remain unclear. Future investigations into the causes of mitochondrial injury during sepsis may lead to the development of new approaches to the treatment of critically ill patients presenting with oxygen metabolism alterations.