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Effects of Tumor Necrosis Factor Intestinal Structure and Metabolism

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J. J. B. VAN LANSCHOT, M.D., K. MEALY, M.D., and D. W. WILMORE, M.D.

Tumor necrosis factor (TNF), a polypeptide produced predominantly by activated macrophages, is an important mediator of sepsis. We analyzed the specific metabolic changes that occur in the gut following TNF administration. Following general anesthesia, hemodynamic and metabolic indices were measured serially in control dogs (n = 7) and animals receiving a continuous sublethal intravenous infusion of TNF (0.57.105 IU/kg/6 hours, n = 7). During TNF infusion mean arterial pressure gradually decreased despite fluid administration, which maintained wedge pressure and cardiac index, which were similar to control animals. While TNF significantly reduced intestinal blood flow to 12 ± 3 mL/min/kg compared to 28 ± 3 mL/min/kg (p < 0.01) in controls, intestinal oxygen consumption was maintained due to an increased extraction rate. Despite hypoperfusion the intestinal exchange of metabolic substrate (glucose, lactate, pyruvate, alanine, glutamine, glutamate, and ammonia) was comparable between the control and TNF-infused animals. However, when substrate carbon balance across the intestinal tract was calculated, it appeared that there was a limitation in fuel availability in the TNF animals. This may be due to competition for fuel between the gut and other major organs. Fuel limitation may jeopardize rapid cell proliferation and mucosal repair and with regional hypoperfusion these processes may account for the mucosal ulcerations observed at the termination of the study.

T

From the Laboratory for Surgical Metabolism and Nutrition, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts

Other investigations suggest that many of the responses that occur after infection are mediated, in part, by cytokines, and tumor necrosis factor (TNF) may be a major participant in this response.f8 With TNF administration, the hypothalamic-pituitary-adrenal axis is activated, protein metabolism is altered, and acute-phase changes occur.8"l Chronic TNF infusion causes an increase in liver mass as reflected by an increase in protein content and induces hypocellularity in the intestinal mucosa.'2 To further evaluate the effects of this cytokine on the gastrointestinal tract, we monitored exchange of substrate across the bowel during TNF infusion.

Materials and Methods

Study Design and Operative Procedure

HE CATABOLIC RESPONSE to critical illness is

characterized by a set of alterations in metabolism, including an increase in metabolic rate, an acceleration of skeletal muscle proteolysis and increased amino acid flux, negative nitrogen balance, and alterations of carbohydrate and fat metabolism. Previous studies suggest that the intestinal tract plays a central role in these metabolic alterations, particularly in the regulation of amino acid delivery from skeletal muscle to the liver.' 3 Supported by Trauma Grant 5 P50 GM36428-05. Dr. van Lanschot is the recipient of a fellowship from the Niels Stensen Foundation, Amsterdam, the Netherlands. Address reprint requests to Dr. J. J. B. van Lanschot, Laboratory for Surgical Metabolism and Nutrition, Brigham and Women's Hospital, Harvard Medical School, 75 Francis St., Boston, MA 02115. Accepted for publication March 5, 1990.

663

Fourteen mongrel dogs ofeither sex, weighing between 22 and 34 kg, were obtained from an animal farm at least 3 weeks before the study. The animals were housed in accordance with the recommendations of the Committee on Animal Care of the Harvard Medical School. To exclude the contribution of the spleen to the portal venous circulation, a splenectomy was performed in all dogs at least 2 weeks before the experiments. At that time, the splenectomized dogs were studied only if they satisfied strictly defined criteria that indicated full recovery.' Dogs were allocated randomly to a control group (n = 7) or to a group receiving TNF (n = 7). After an overnight fast, anesthesia was induced with pentobarbital sodium (25 to 30 mg/kg administered intravenously; Nembutal, Abbott Laboratories, Chicago, IL); during the experiment intravenous Nembutal injections (50 mg at a

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LANSCHOT, MEALY, AND WILMORE

time) were repeated to maintain light anesthesia. After endotracheal intubation, urinary catheter and rectal temperature probes were inserted. The dogs were allowed to breathe spontaneously a mixture of approximately 60% oxygen and air. Under sterile conditions, silicone catheters were inserted into the carotid artery and the external jugular vein. The carotid artery catheter was attached to a standard mercury manometer. A flow-directed triple lumen pulmonary artery Swan-Ganz catheter was inserted via the external jugular vein. The distal opening was connected to a pressure amplifier (Model CM; Honeywell, Inc., Pleasantville, NY). Into the jugular vein catheter, 0.9% saline was infused at a rate of 4 mL/kg/hour. The TNF-infused dogs needed additional 0.9% saline administered to maintain hourly measured wedge pressure. The abdominal cavity then was entered through a small midline incision. A silicone catheter was inserted into a mesenteric vein of the proximal small bowel and directed into the portal vein; its tip was located in the porta hepatis by palpation. Next a mesenteric infusion catheter was inserted into a mesenteric vein of the distal small bowel and advanced approximately 2 cm. Both catheters were exteriorized via the wound. During this procedure the bowel was only minimally handled. A solution of para-aminohippurate (PAH) sodium in 0.9% saline (0.5 g PAH/100 mL 0.9% saline) was constantly infused into the mesenteric infusion catheter at a rate of 0.36 mL/minute. After 90 minutes steady-state dye concentrations were achieved'3 and baseline measurements were performed. Subsequently, in selected animals, TNF (0.57 X 105 IU/ kg body weight; Asahi Chemical Industry Inc., New York, NY) was infused peripherally during a 6-hour period. Measurements

Vital signs were monitored hourly. Every 2 hours arterial, mixed venous, and portal venous blood samples were taken for blood gas analysis and total body oxygen consumption was measured by closed circuit respirometry. Cardiac output was calculated indirectly by the Fick method. At 3-hour intervals, arterial blood samples were obtained for determination of hematologic indices and concentrations of blood urea nitrogen and stress hormones (adrenalcortical tropic hormone [ACTH], cortisol, insulin, glucagon, and epinephrine). Every 3 hours simultaneous arterial and portal venous samples were obtained to determine concentrations of PAH, potassium, and various metabolic substrates (including glucose, lactate, pyruvate, glutamine, glutamate, alanine, and ammonia). Regional blood flow and substrate flux were determined as previously described.3"3 Total carbon balance (expressed per liter of intestinal blood flow) was calculated as the sum

Ann. Surg. - December 1990

of all carbons taken up or released by the gut via the exchange of these same substrates. Intestinal oxygen consumption was calculated by the reversed Fick method. Oxygen extraction and ammonia production (expressed per liter ofintestinal blood flow) also were calculated. The intestinal redox state was estimated by calculating the portal venous lactate/pyruvate ratio.'4 Aerobic and anaerobic blood cultures were taken from the portal venous catheters at the beginning and at the end of the study. Urine was collected during the 6-hour period and further analyzed for electrolytes and nitrogen products. Urea production (expressed per kilogram of body weight) was calculated from the 6-hour urine value corrected for changes in total body water and blood urea nitrogen concentration. At the end of the study, all TNF-infused animals and five of seven control animals were killed by administering an overdose ofanesthetic agent, and the positions of catheters were verified. The ligament of Treitz was identified and a point 30 cm from the ligament identified. Two 10cm segments ofjejunum were excised distal to this point. The segments were cleaned of fat, opened, and rinsed with chilled 0.9% saline. The proximal segment was blotted dry and weighed; it was then dried for at least 16 hours at 90 C and reweighed. The distal segment was diced, homogenized, and stored at -70 C until measurement of DNA and protein content. The same procedure was performed on two 5-cm proximal colon segments located 10 cm from the ileocecal valve.

Chemical Analysis Whole blood concentration of PAH, glutamine, glutamate, alanine, and ammonia were measured as previously described.'5 Concentrations of stress hormones also were determined as previously described." Tissue DNA content was determined using a modification of the method of Burton'6 and protein content was measured using Lowry's technique.'7 Standard analytical methods were used for the measurement of other substrates, electrolytes, and hematologic indices. Data Analysis

Statistical calculations were performed on a personal computer (Tandy 1000 SX, Tandy Corp., Fort Worth, TX) using standard software packages (Ability Plus, Migent Inc., NV; Statgraphics, STSC Inc., Rockville, MD; SAS Software, SAS Institute, Cary, NC). Student's paired and unpaired t tests, repeated measures analysis of variance (ANOVA), and chi square test with Yates' correction were used when appropriate. Results are expressed as

665

EFFECTS OF TNF ON INTESTINAL STRUCTURE AND METABOLISM

Vol. 212 - No. 6

mean ± the standard error of the mean (SEM). Differences were considered significant when p < 0.05. Results

Systemic Response Anesthesia dose was similar in both groups of animals (control group: 41.1 ± 3.1 mg pentobarbital/kg body weight versus TNF group: 36.0 ± 1.7 mg/kg body weight). Body temperature increased during the 6 hours of study in both groups ofanimals and the increase was comparable (from 38.1 ± 0.2 C to 40.2 ± 0.1 C in the control group and from 38.7 ± 0.1 C to 40.5 ± 0.2 C in the TNF group). Both groups developed a tachypnea (from 16 ± 1 to 28 ± 6 breaths/minute in the control group and from 17 ± 2 to 46 ± 5 breaths/minute in the TNF group), which was significant by 4 hours and persisted at 6 hours; no differences were observed between the two groups.

While mean arterial pressure was stable throughout the 6-hour study in control dogs, blood pressure decreased to approximately 80 mmHg by 6 hours in the dogs receiving TNF (Fig. 1). Despite this decrease in mean arterial pressure, the TNF dogs developed a relative bradycardia; at 6 hours their heart rate was significantly lower in comparison to the controls. Cardiac index fell significantly during the first 2 hours of the study period in both groups (Fig. 1) and then stabilized. No significant difference occurred between the two groups. To maintain a constant wedge pressure, additional fluid was administered to all seven dogs receiving TNF; this ranged in volume from 400 to 1000 mL 0.9% saline/6 hours (mean = 779 ± 84 mL/6 hours). In both groups of animals the hematocrit decreased significantly during the 6 hours (from 44% ± 2% to 39% ± 2% in control animals and from 46% ± 2% to 40% ± 2% in TNF-treated animals; p < 0.01); no differences were observed between the two

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666

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LANSCHOT, MEALY,

AND WILMORE

Ann. Surg. - December 1990

TABLE 1. Arterial Substrate Concentrations and Base Excess (BE) Values in Control and TNF-infused Animals

Time (hours) Substrate Glucose

Control

[mg/mL]

TNF Control TNF

Lactate [mmol/L] Base Excess [mEq/L] Pyruvate

Control TNF Control TNF Control TNF Control TNF

[gmol/L]

Glutamine

[,gmol/L] [jmol/L]

Alanine

0

3

6

0.84 ± 0.04 0.84 ± 0.02 1.29 ± 0.28 1.11 ± 0.23 -4.5 ± 0.9 -2.9 ± 0.7 91 ± 19 67 ± 16 548 ± 24 643 ± 26 324 ± 40 310 ± 23

0.91 ± 0.04 0.76 ± 0.03 1.16 ± 0.18 1.71 ± 0.24 -3.5 ± 0.8 -5.5 ± 0.8 93 ± 14 110 ± 11 600 ± 31 616 ± 25 342 ± 58 398 ± 20

0.99 ± 0.07 0.70 ± 0.04 1.10 ± 0.23 2.20 ± 0.34 -3.3 ± 0.9 -8.0 ± 0.9 94 ± 17 129 ± 14 633 ± 53 691 ± 39 344 ± 51 605 ± 59

Change t 4 t 4 -

t

* * NS t NS t NS NS NS NS NS t

t, significant increase over time; 4, significant decrease over time; -, no change over time; *p < 0.05, tP < 0.01. groups. White blood cell count increased significantly in control dogs from 17.0 ± 1.9 X 109/L at the start to 30.1 ± 3.7 X 109/L after 6 hours (p < 0.01); in the TNF-treated dogs it decreased significantly from 14.4 ± 2.7 to 6.4 ± 1.5 X 109/L (p < 0.05). Arterial glucose concentration increased significantly in the control dogs, while it decreased in the TNF-treated dogs (Table 1). In control animals the arterial lactate concentration and base excess did not change, but lactate increased and base excess decreased in the TNF group such that significant differences were present between groups at 6 hours. At this time arterial pH was significantly lower in the TNF group as compared to controls (7.27 ± 0.03 versus 7.34 ± 0.02, p < 0.05). Arterial concentrations of pyruvate and glutamine were not significantly different for the two groups. In the TNF-treated dogs, the arterial alanine concentration increased with time but remained unchanged in controls. At 3 hours and at 6 hours, arterial concentrations of ACTH, cortisol, glucagon, and TABLE 2. Arterial Concentrations of Stress Hormones in Control and TNF-infused Animals

Time (hours) Hormones ACTH

[pg/mL]

Cortisol

[jg/dL] Insulin [IU/mL]

Glucagon [pg/mL]

Epinephrine [pg/mL] *

P < 0.05,

Control TNF Control TNF Control TNF Control TNF Control TNF

0

3

136 ± 21 128 ± 24 8.6 ± 0.8 8.5 ± 1.2 10.5 ± 1.9 8.7 ± 2.2 418 ± 64 395 ± 36 679 ± 252 408 ± 260

29 ± 6 366 ± 67t 5.3 ± 0.6 11.6 ± 1.3t 6.7 ± 1.2 6.0 ± 2.7 418 ± 55 700 ± 77* 295 ± 86 724 ± 176*

tP < 0.01 vs. controls.

epinephrine all were significantly greater in the TNF-infused animals (Table 2). No significant differences in urine volume and urinary electrolyte and nitrogen excretion were observed between control and TNF-treated animals (Table 3). Urea nitrogen generation also was similar in the two groups (90 ± 11 versus 102 ± 4 mg/kg/6 hours).

Hemodynamic and Metabolic Changes in the Gut While intestinal blood flow remained unchanged in control dogs, it decreased significantly in the animals receiving TNF and differences were observed between the two groups of animals at 3 and 6 hours (Fig. 2, p < 0.05). Intestinal oxygen consumption rose significantly with time (Fig. 2, p < 0.05), but no differences occurred between the two groups. Despite the decrease in intestinal blood flow with TNF infusion, the intestinal redox state (expressed as portal venous lactate/pyruvate ratio) was not significantly altered and tended to decrease with the TNF infusion (18.25 ± 2.06 initially versus 14.83 ± 0.48 at 6 hours). No difference in intestinal flux of metabolic substrates was observed between the two groups (Table 4). Intestinal oxygen extraction rate (expressed as the number of mil-

6

25 701 5.8 10.7 7.2 3.1

±8 ± 195t ± 1.3 ± 1.0* ± 0.9 ± 1.5* 471 ± 64 1489 ± 108t 640 ± 248 1751 ± 462*

TABLE 3. Urine Volume and Urinary Excretion of Different Substances in Control and TNF-infused Animals

Urine Measurements

Control

TNF

Urine volume [mL/kg/6 hrs] Sodium [meq/kg/6 hrs] Potassium [meq/kg/6 hrs] Total N [g/kg/6 hrs] Urea N [mg/kg/6 hrs] Creatinine [g/kg/6 hrs]

5.0 ± 1.3 0.43 ± 0.24 0.66 ± 0.13 0.13 ± 0.02 111 ± 14 0.008 ± 0.001

9.6 ± 2.3* 0.90 ± 0.31* 0.87 ± 0.13* 0.10 ± 0.01* 90 ± 2* 0.008 ± 0.001*

*

p > 0.05 vs. controls.

Vol. 212 - No. 6

EFFECTS OF TNF ON INTESTIP,1IAL STRUCTURE AND METABOLISM

intestinal flow 40rri/*Lkg 40 30-

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667

most pronounced in the distal ileum. Mesenteric and portal venous thrombosis were not present in these animals. None of the five control dogs that underwent autopsy showed comparable small bowel lesions. No gross pathologic lesions were found in the liver, kidney, and adrenal glands. Pulmonary atelectasis was observed frequently in both groups of animals. These lesions could be macroscopically reversed by overinflation of the lungs. Jejunal and colonic dry weight:wet weight ratios were similar in the TNF-infused and control groups (jejunal segment: 0.20 ± 0.01 versus 0.20 + 0.00; colonic segment: 0.20 ± 0.01 versus 0.19 ± 0.01). Jejunal and colonic DNA/ protein ratios also were unchanged after TNF infusion (jejunal segment: 0.065 ± 0.007 versus 0.060 ± 0.001; colonic segment: 0.034 ± 0.004 versus 0.041 ± 0.003), suggesting that no change in intestinal cellularity occurred during this 6-hour study. Before the commencement of the study, positive portal venous blood cultures were found in two of six control dogs and three of the seven TNF-infused dogs (chi square test, not significant [NS]). At the end of the study one of the six control dogs and three of the seven TNF-infused dogs had positive blood cultures (chi square test, NS).

I

0.6

Discussion

0.40.2

____.___-____ 2

3

TIvE (hour)

4 56

FIG. 2. Alterations of intestinal blood flow an(d intestinal oxygen consumption (V02) during 6 hours in control ani mals (solid lines) and in TNF-infused animals (dashed lines).

liliters of oxygen extracted per liter a)f blood circulating through the gut) and intestinal ammo nia production rate (expressed as micromoles of ammoni a released to 1 L of blood circulating through the gut) were significantly higher in the TNF-treated group as compare(d to controls (Table 5, p < 0.01 and p < 0.05, respectiNrely). Total carbon extraction rate (expressed as net mic,romoles of carbon extracted per liter of blood via the e) change of glucose, lactate, pyruvate, glutamine, glutamiate, and alanine), however, was not statistically differerit between the two groups. The potassium flux did not c,hange significantly during time in the two groups; nor iwas any significant difference in potassium flux detecte(d between the two groups (data not shown). Structural and Functional Aspects of tthe Gut Six of the seven TNF-infused anin ials had focal, submucosal small bowel infarctions at atutopsy, which were

The purpose of this study was to examine the metabolic effects of TNF infusion on the gastrointestinal tract and not the effects of shock and death. Others have demonstrated that there is major disruption of the intestinal mucosa associated with lethal doses of TNF administered by intraperitoneal bolus injection.9 In this study a sublethal infusion of TNF induced mild hemodynamic, metabolic, and endocrine changes similar to those previously reported from this laboratory in other canine studies.""8 Mean arterial blood pressure decreased from 124 to 76 mmHg in the TNF-infused animals and this was associated with a corresponding decrease in portal blood flow from 26 to 12 mL/min/kg (approximately 54%). In response to the decrease in intestinal perfusion, oxygen extraction increased, oxygen consumption normalized, and aerobic metabolism was preserved. This was confirmed by the intestinal production of lactate and pyruvate, which was similar in the TNF-infused animals to that of the control group (Table 4). These responses are at variance with the alterations in intestinal blood flow that occurred during controlled hemorrhagic hypotension in the dog.'9 When mean arterial pressure was reduced from 130 to 60 mmHg, by volume depletion portal blood flow decreased approximately 59% (from 309 to 127 mL/minute), changes that

VAN LANSCHOT, MEALY, AND WILMORE

668

Ann. Surg. - December 1990

TABLE 4. Intestinal Flux of Different Metabolic Substrates in Control and TNF-infused Animals

Time (hours) Substrate Glucose

[mg/kg/min] Lactate

[(rmol/L/kg/min]

Pyruvate

[umol/L/kg/min] Glutamine

[,umol/L/kg/min]

Glutamate [umol/L/kg/min] Alanine

[,gmol/L/kg/min]

Ammonia

[,ug/kg/min]

Control TNF Control TNF Control TNF Control TNF Control TNF Control TNF Control TNF

0

3

6

1.25 ± 0.31 1.18 ± 0.49 1.40 ± 1.54 1.88 ± 1.65 0.45 ± 0.18 0.36 ± 0.32 0.94 ± 0.70 1.99 ± 0.52 -0.09 ± 0.03 -0.18 ± 0.07 -0.63 ± 0.21 -1.09 ± 0.49 -86 ± 21 -128 ± 39

1.22 ± 0.38 1.10 ± 0.29 1.82 ± 1.90 -0.55 ± 0.85 0.36 ± 0.15 -0.23 ± 0.09 2.33 ± 0.48 1.81 ± 0.39 -0.19 ± 0.06 -0.14 ± 0.04 -0.85 ± 0.59 -0.74 ± 0.26 -135 ± 16 -164 ± 44

1.33 ± 0.38 0.72 ± 0.13 -2.45 ± 1.14 -4.28 ± 1.65 0.06 ± 0.14 -0.50 ± 0.14 2.60 ± 0.97 1.48 ± 0.34 -0.22 ± 0.05 -0.14 ± 0.01 -0.55 ± 0.27 -0.88 ± 0.24 -160 ± 23 -126 ± 14

Group Effect NS NS NS NS NS

NS

NS

NS, not significant. - flux indicates release; + flux indicates uptake. Differences between

the groups (group effects) were analyzed by repeated measures ANOVA.

were similar to that observed in our animals. However, with hemorrhagic shock, intestinal oxygen consumption decreased from 19.0 to 12.7 mL/minute. While the hemodynamic changes were similar to those in our study, the changes in oxygen consumption were different for the TNF-infused animals, which maintained intestinal oxygen consumption between 25 to 30 mL/minute, a value similar to controls. It appears that these two quite varied stimuli result in different intestinal responses. It is possible that differences in anesthetic technique or a greater decrease in blood pressure (to a mean arterial pressure of 60 mmHg in the hemorrhagic shock model versus 76 in the TNF-infused dogs) could explain these differences. In contrast to dogs undergoing hemorrhagic shock, endotoxin administration to dogs resulted in a decrease in blood pressure, a decrease in portal blood flow, and an increase in total resistance of the splanchnic bed.20 At the same time there was an increase in intestinal weight and augmented sensitivity of small mesenteric veins to epinephrine. This observation formed the basis for the hy-

pothesis that splanchnic sequestration of plasma occurred during septic shock and that this loss ofcirculating volume contributed to the associated hypovolemia and hypotension associated with sepsis. Tumor necrosis factor is thought to mediate the responses to endotoxin,7-9 but similar changes in fluid sequestration were not observed in this study. The dry weight:wet weight ratio was similar in the TNF-infused and control animals, demonstrating that sequestration of fluid did not occur in the present study. In addition the arterial and portal venous hematocrit was monitored serially throughout the study, and no significant hemoconcentration occurred across the gut in any animals studied (data not shown). Thus it appears that the gut insult in this study was mild and compensatory adjustments were made to maintain bowel metabolism in the face of a decreasing portal blood flow. The alterations that occurred after the administration of endotoxin also have been observed with administration of greater doses of TNF resulting in lactic acidosis, hypotension, and death.9

TABLE 5. Intestinal Oxygen and Carbon Extraction Rate and Ammonia Production Rate

Time (hours)

Extraction or Production Rate

02-Extraction rate

[mL/L]

NH3-Production rate

[umol/L] C-Extraction rate

[gmol/L] NS, not significant.

Group

Group Studied

0

Control TNF Control TNF Control TNF

22.4 ± 3.4 21.0 ± 3.2 -192 ± 22 -263 ± 34 1972 ± 405 1725 ± 392

3 36.4 ± 63.1 ± -250 ± -609 ± 1587 ± 2690 ±

2.8 7.3 29 121 499

357

6

Effect

40.4 ± 2.9 60.7 ± 4.4 -336 ± 37 -643 ± 104 1725 ± 439 1353 ± 592

p < 0.01 p