Allopurinol
Prevents Intestinal Permeability Ischemia-Reperfusion Injury By W. Glaze Vaughan,
Jureta W. Horton,
Changes After
and Paula B. Walker
Dallas, Texas l Under normal conditions the intestinal mucosa is impermeable to potentially harmful materials from the intestinal lumen. Mucosal disruption promotes bacterial translocation, which is postulated to be a fuel source for sepsis and multiorgan failure. We have previously demonstrated that mesenteric ischemia-reperfusion (I/R) injury increases intestinal permeability (IP); however, the mechanism remains unclear. This study was designed to examine the hypothesis that changes in IP, after I/R injury, are mediated by xanthine oxidase-generated, oxygen-derived free radicals. Thirtythree Sprague-Dawley rats (weighing 300 to 400 g) were included in this study. Group 1 (n = 10) received enteral allopurinol, a xanthine oxidase inhibitor, IO mg/ kg daily for 1 week prior to mesenteric ischemia. Group 2 consisted of 11 untreated, ischemic animals. Groups 1 and 2 were subjected to superior mesenteric artery occlusion with interruption of collateral flow for 20 minutes to produce ischemic injury to the intestine. An additional 12 rats (group 3). served as nonischemic controls (sham). A loop of distal ileum was isolated and cannulated proximally and distally to allow luminal perfusion with warmed Ringer’s lactate at 1 mL/min. IP was determined in all groups by quantitatively measuring the plasma-to-luminal clearance of chromium (51Cr)-labeled ethylenediaminetetraacetate (EDTA) at baseline, during ischemia and 20,40, and 60 minutes after reperfusion. Complete ischemia produced significant increases in IP over baseline values in the untreated rats (group 2, baseline: 0.49 f 0.006, ischemia: 0.149 f 0.039) compared with sham rats (baseline: 0.41 f 0.006; ischemia: 0.047 f 0.009) or allopurinol-treated rats (baseline: 0.096 i: 0.020, ischemia: 0.073 f 0.012, P < .OOl). IP in allopurinol-treated rats remained unchanged 60 minutes postreperfusion (0.098 f 0.024) whereas 60 minutes of reperfusion produced significant increases in IP in untreated rats (0.196 f 0.049). We conclude that enteral allopurinol exerts a protective effect on the intestinal mucosa, preventing permeability changes in response to I/R injury. In addition, these data suggest that oxygen-derived free radicals contribute to l/R-mediated increases in IP. Copyright o 1992 by W.B. Saunders Company INDEX WORDS: Mesenteric ischemia; intestinal ity; ischemia-reperfusion injury; allopurinol.
permeabil-
From the Department of Surgery, The Universityof Texas Southwestem Medical Center, Dallas, TX. Supported in part by a research grant from the Southern Medical Association. Presented at the Jens G. Rosenkranz Resident Competition at the 43rd Annual Meeting of the Surgkal Section of the American Academy of Pediatrics, New Orleans, Louisiana, October 26-27, 1991. Address reprint requests to Jureta U? Horton, PhD, Department of Surgery, The University of Texas Southwestern Medical Center at Dallas, 5323 Harry Hines Blvd, Dallas, TX 75235-9031. Copyright o 1992 by WB. Saunders Company 0022-3468/92/2708-0007$03.00/0
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ERUPTION of the intestinal mucosal barrier predisposes to extraluminal egress of potentially harmful bacteria and/or their toxins. Recently, attention has been focused on the role of the gastrointestinal tract as a source of pathogens that may cross a disrupted mucosal barrier initiating a septic process and perpetuating multiorgan failure.’ The exact mechanisms leading to mucosal injury, due to mesenteric ischemia-reperfusion (I/R), have not been fully elucidated, yet there is an increasing body of evidence to suggest a role for xanthine oxidase (X0)-derived reactive oxygen metabolites.2-6 This laboratory recently reported a model of transient intestinal ischemia that included occlusion of the superior mesenteric artery (SMA) and ligation of collateral arcades from the right colic artery and jejunal arteries; SMA occlusion with interruption of collateral flow for 20 minutes produced significant alterations in intestinal blood flow and mucosal permeability.’ In a previous study, using this same model of mesenteric ischemia, Megison et al8 showed an improved 7-day survival in those animals that had received prophylactic low-dose enteral allopurinol, suggesting a role for oxygen-derived free radicals in intestinal I/R injury. The purpose of this present study was to examine the mechanisms of I/R-mediated changes in mucosal permeability. More specifically, this study attempts to answer the question: Will allopurinol prevent the permeability changes observed after a period of transient, occlusive mesenteric ischemia followed by reperfusion? MATERIALS
AND METHODS
Adult male Sprague-Dawley rats (Dominion Laboratories, Omaha, NE) weighing 300 to 400 g were included for study. All animals were allowed food and water ad lib until 18 hours prior to the experiment; at this time solid food was removed but animals had free access to water until time of the experiment. Experimental animals (n = 10) received enteral allopurinol, a X0 inhibitor, 10 mg/kg daily for 7 days prior to mesenteric &hernia. A second group of 11 untreated, ischemic rats served as ischemic controls. An additional group, consisting of 12 rats, served as untreated, nonischemic controls (sham). All rats were anesthetized with methoxyilurane; ketamine hydrochloride, 20 mglkg, was administered intramuscularly to supplement inhalation anesthesia. This study was approved by the Institutional Review Board for Animal Research at The University of Texas Southwestern Medical Center. All animals were handled in accordance with the guidelines set
Journal of Pediatric Surgery,
Vol 27, No 8 (August), 1992:
pp 968-973
ALLOPURINOL PREVENTS INTESTINAL I/R INJURY
forth by the American Institute of Health.
Physiological Society and the National
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Table 1. Hemodynamic and Metabolic Responses to Treated and Untreated lschemia Baseline
Surgical Procedures Mean
All animals were allowed to breathe spontaneously. Cannulae were inserted into the left carotid artery to measure systemic arterial pressure (physiological recorder, model 81; Siemans Medical Systems Inc, Grand Prairie, TX) and into the external jugular vein for acquisition of blood samples and administration of drugs and fluid. Heparin (5,000 U/kg) was injected intravenously in all rats after surgical preparation. Body temperature was maintained at 38°C by use of a heating pad and a heating lamp. Animals underwent laparotomy and the SMA and collateral vessels were occluded as previously described9 (Fig 1). A loop of distal ileum (approximately 10 cm in length) was isolated and cannulae were placed both proximally and distally, via enterotomies, for continuous intraluminal perfusion. The viscera were covered with plastic wrap to minimize evaporative fluid and heat loss and to avoid alterations in mesenteric blood flow caused by hypothermia. In the sham group (n = 12), rats undenvent laparotomy with SMA and collateral vessel isolation without occlusion. All rats were studied at identical time periods regardless of group assignment.
Intestinal Permeability The cannulated ileal segment was perfused with warm lactated Ringer’s solution at the rate of 1 mL/min. SLCr-labeIed ethylenediaminetetraacetate (EDTA) (New England Nuclear, Boston, MA) was injected through the jugular catheter to achieve plasma counts of 25.000 counts/mL (approximately 150 FCi/kg body weight). After 30 minutes equilibration of the 51Cr-labeled EDTA, the luminal perfusate was collected for 20-minute intervals at designated experimental periods: at baseline, during ischemia (20 minutes of total SMA occlusion with collateral interruption), and 20. 40. and 60 minutes after reperfusion. One milliliter of blood was obtained at the end of each period, then replaced with an equivalent volume of warmed lactated Ringer’s solution. 51Crlabeled EDTA counts in plasma and in aliquots of perfusate (a 4-mL aliquot of each 20.minute perfusate collection) were measured in a gamma scintillation counter (model 5320; Packard Instruments Co, Legona Hills, CA). After completing the experiment. the perfused ileal loop was removed, rinsed with saline, and
arterial blood
lschemia
Aeperfusion
pressure (mm Hg)
Sham
101 t 5
89 + 5
102 2 6
Untreated
106 + 5
95 2 6
90 t 9
Treated
114+
12
106 k 10
90 f 4 228 + 10
Heart rate (beatsimin) 250 + 10
202 2 15
Untreated
240 t 9
158 k 9V
180 + lO*t
Treated
188 f 9
197 k 9
265 + 5*t
Sham
PH Sham
7.40 + 0.01
7.42 + 0.01
Untreated
7.37 zk 0.01
7.36 + 0.01
7.42 c 0.03
Treated
7.42 + 0.02
7.44 + 0.01
7.43 + 0.02
Sham
40 k 1
36 2 2
35 2 2
Untreated
42 -c 3
42 5 3
32 + 2t
Treated
40 + 1
35 r 2t
20 -t_2*t
Sham
80 i 3
a8 2 5
95 2 4
Untreated
a7 + 7
99 + 4
92 + 5
Treated
a7 + 3
103 ? 2t
105 2 4*t
7.41 t 0.01
PCOz (mm Hg)
POP(mm H9)
HCO, (MEqlL) Sham
26 +
24 -+ 1
22 t 1
Untreated
25 +
24 + 1
20 + 1*t
Treated
26 + 1
24 i 1
16 + 2”t
Sham
38 +
40 + 1
38 + 1
Untreated
40 2 1
40 ? 1
41 +2
Treated
42 t 1
44k2
45 + 2*
Hematocrit (%)
NOTE. All values are mean k SE. *Significant difference between groups at P < .05. tsignificant change from baseline within each group at P < .05
weighed. The bowel segment was then dried in a drying oven for 24 hours or until a constant weight was achieved. The bowel segment was then reweighed and total water content was calculated from wet and dry weight. After completing the experimental procedure, the animals were killed with an intravenous injection of nembutal. The plasma to luminal clearance of 51Cr-labeled EDTA was calculated according to the formula described by Nylander et al’“: cpm/mL (perfusate) X mL/min (perfusion rate) X 100 cpm/mL (plasma) x weight (g) where clearance (mL/min/lOO g tissue) is equal to gamma counts per minute (cpm) per milliliter perfusate times the luminal perfusion rate (mL/min) times 100 divided by cpm per milliliter of plasma times the wet weight of the ileal segment in grams.
Data Analysis All values are expressed as the mean t SEM. Clearance values were compared among untreated, allopurinol treated, and sham animals by using a two-way ANOVA. Further statistical evaluation using a multiple comparisons procedure was used if ANOVA showed differences among groups. Differences were considered significant at P I .05. RESULTS
Fig 1.
Experimental
model.
Table 1 summarizes the hemodynamic and metabolic responses to transient ischemia and reperfusion.
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VAUGHAN,
Mean arterial blood pressure tended to decrease after 20 minutes of ischemia, whether untreated or treated, but these changes were not statistically significant. Untreated ischemia was associated with a significant bradycardia, whereas heart rate was unchanged in allopurinol-treated or sham ischemic rats. Reperfusion in both untreated and treated rats was associated with a compensated metabolic acidosis as indicated by the unchanged arterial pH and decreases in arterial PCOZ and serum bicarbonate. Ischemia and reperfusion in untreated rats produced significant and persistent increases in plasmato-luminal clearance of Wr-labeled EDTA compared with sham ischemic control or allopurinoltreated rats (P I .OOl). It is important to note that the intraoperative preparation and manipulation of the intestine and arterial supply was the same for all animals. 51Cr-labeled EDTA clearance increased from 0.49 2 0.006 to 0.149 2 0.039 mL/min/lOO g of tissue during 20 minutes of untreated ischemia, representing a 67% increase in intestinal permeability over baseline values. During the initial 20 minutes of reperfusion in the untreated ischemic group, there was a 126% increase in clearance compared with baseline values. In contrast, animals pretreated with allopurinol had no increase in plasma-to-luminal clearance of Wr-labeled EDTA either during the ischemic period or at any time during reperfusion (Fig 2). The animals subjected to sham ischemia had no change in clearance of Wr-labeled EDTA throughout the experimental period. It was of interest that clearance in the allopurinol-treated rat, subjected to 20 minutes of intestinal ischemia, was not statistically different from that measured in the sham ischemia group, confirming that allopurinol pretreatment prevented ischemic as well as reperfusion-mediated changes in intestinal permeability.
HORTON, AND WALKER
ATP Xanthine Dehydrogenase
t ADP
4 C
,’ H :
t AMP
I Xanthine Oxidase
Adenosine
:
I
t lnosine
\r Hypoianthine .
d
Xanthine
6
Uric Acid
\A 0;
OH;HzOz
1 REPERFUSION
1
Fig 3. Proposed mechanism for I/ R-mediated production of oxygenderived free radicals.
DISCUSSION
This study demonstrates that prophylactic administration of allopurinol protects against I/R-mediated changes in intestinal mucosal permeability and supports the hypothesis that oxygen radicals play a major role in the mucosal injury associated with I/R. A proposed mechanism for the protective effects of allopurinol includes X0 inhibition and prevention of toxic oxygen radical formation.ll During ischemia, adenosine triphosphate is degraded to hypoxanthine, and xanthine dehydrogenase is converted to X0. On reperfusion, X0 catalyzes the conversion of hypoxanthine to uric acid with release of the superoxide radical; subsequent reactions result in the formation of hydroxyl ions and hydrogen peroxide (Fig 3). These toxic metabolites have the capacity to cause systemic cellular damage, with resulting multiorgan dysfunction.12 As stated by Chiu et al13: It is, therefore, logical that every effort should be made to prevent this process from advancing to complete destruction of the normal protective barrier function, with the consequences to extraintestinal tissues which may spell disaster for the organism.
0.3
Clearance (ml/min/lWg) 0.2
Baseline
lschemia
20 min. -
40 min.
60 min.
Reprfuolon -
Fig 2. Effect of treated (alto) versus untreated (ischemia) I/R on intestinal mucosal clearance (permeability). *Significant change from baseline within each group at P c -05. tsignificant difference from sham controls at P -z .05.
Twenty minutes of complete intestinal ischemia in the rat was associated with significant hemodynamic and metabolic derangements. Reperfusion produced a metabolic acidosis that was corrected by an altered respiratory rate. It is likely that the postreperfusion acidosis was related to the accumulation of lactate during ischemia followed by a washout of the byproducts of anaerobic metabolism during reperfusion. In this present study, intestinal barrier dysfunction was evident from the increase in plasma-to-luminal clearance of Wr-labeled EDTA. It was of interest that animals pretreated with allopurinol had no significant
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PREVENTS INTESTINAL
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I/R INJURY
change in intestinal permeability despite the 99.6% decrease in intestinal blood flow previously demonstrated with our model of SMA occlusion.7 The protective effects of allopurinol would be expected to consist of postreperfusion inhibition of X0; therefore, it was of interest that allopurinol pretreatment prevented changes in intestinal permeability during ischemia. This allopurinol-mediated protection supports a previous study by Megison et a&l4 who reported significantly less histopathologic evidence of ischemic mucosal injury with allopurinol pretreatment. These data suggest that the protective effects of allopurinol may not be related solely to X0 inhibition; instead, allopurinol and its metabolite, oxypurinol, likely act as free radical scavengers independent of enzyme inhibition.15 Alternatively, allopurinol may preserve intestinal tissue adenine nucleotide content, increasing high energy phosphate availability to maintain cell membrane function.“j Under normal conditions, the intestinal mucosa is impermeable to macromolecules and provides a barrier against luminal bacteria and their toxins. Numerous studies have shown that several types of trauma, with and without hemorrhagic shock, alter intestinal permeability, promoting a movement of indigenous bacteria colonizing the gastrointestinal tract through the mucosa into mesenteric lymph nodes and systemic organs; this process has been termed bacterial translocation.17-20 The phenomena of bacterial translocation includes several components: hypoperfusion, bacterial overgrowth, impaired host immunity, and disruption of the intestinal barrier. There is increasing clinical and experimental evidence suggesting that gut failure with subsequent bacterial translocation are initiating events in the development and perpetuation of multisystem organ failure syndrome.21~22Our study provides evidence that even a brief period of intestinal ischemia followed by reperfusion alters the permeability characteristics of the intestine, potentiating the migration of intestinal bacteria into the lymphatic and portal circulation. These data are consistent with previous reports of increased intestinal permeability after a single dose of endotoxin,23 after a major burn injury, 17,24and after severe hemor-
rhagic shock. 25 Our finding that allopurinol, in clinically acceptable doses, ablated the I/R-mediated increases in intestinal permeability confirmed a previous proposal by Baker et al that I/R injury, mediated by oxygen-derived free radicals, represents a significant portion of the total injury seen after intestinal hypoperfusion.17 That allopurinol pretreatment can prevent reperfusion injury after ischemia is not a new concept. Several studies have shown that allopurinol and free radical scavengers enhance the viability of skin flaps, improve organ preservation, limit cardiac infarct size after I/R, and improve survival after mesenteric ischemia.26,27 A significant finding in our present study is that the protective effect of allopurinol was achieved with low enteral doses that would not cause intolerable side effects. The clinical significance of this present study in adult rats remains speculative; however, our findings suggest that inhibitors of oxygen radical formation, such as allopurinol, may be useful as prophylactic agents to reduce reperfusion injury in multiple clinical settings. If a patient population at risk for an ischemic event could be identified, for example, patients with vascular disease and neonates prone to necrotizing enterocolitis, allopurinol could be studied for its protective effects, since it is innocuous and can be administered once a day. In conclusion, low-dose, enteral allopurinol prevented the I/R changes in intestinal permeability. Our results suggest that oxygen-derived free radicals are mediators in the pathogenesis of I/R injury, although a number of other factors such as cytokines, complement, and arachidonic acid metabolites may interact in a complex fashion, contributing to the resultant injury. Further investigations into the pathogenic mechanisms of mesenteric I/R injury are needed before effective therapies can be designed and implemented in the clinical setting.
ACKNOWLEDGMENT We would like to thank Nancy Bain for her excellent secretarial skills in the preparation of this manuscript.
REFERENCES 1. Saadia R, Schein M, Boffard KD: Gut barrier function and the surgeon. Br J Surg 77:487-492,199O 2. Granger DN, Parks DA: Contributions of ischemia and reperfusion to mucosal lesion formation. Am J Physiol 250:G749G753,1986 3. McCord JM: Oxygen-derived free radicals in postischemic tissue injury. N Engl J Med 312:159-163,1985 4. Parks DA, Bulkey B, Granger DN, et al: Ischemic injury in the cat small intestine: Role of superoxide radicals. Gastroenterology 82:9-15. 1982
5. Granger DN, Hollwarth ME, Parks DA: Ischemia-reperfusion injury: Role of oxygen-derived free radicals. Acta Physiol Stand 546147~63,1986(suppl) 6. Grisham MB, Hernandez LA, Granger DN: Xanthine oxidase and neutrophil infiltration in intestinal ischemia. Am J Physiol 251:G567-G574,1986 7. Horton JW: Alterations in intestinal permeability and blood flow in a new model of mesenteric ischemia. Circ Shock 36:134-139, 1992 8. Megison SM, Horton JW, Chao H, et al: High dose versus low
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dose enteral allopurinol for prophylaxis in mesenteric &hernia. Circ Shock 30:323-329,199O 9. Megison SM, Horton JW, Chao H, et al: A new model for intestinal ischemia in the rat. J Surg Res 49:168-173,199O 10. Nylander 0, Kvietys P, Granger DN: Effects of hydrochloric acid on duodenal and jejunal mucosal permeability in the rat. Am J Physiol257:G653-G660,1989 11. Granger DN: Role of xanthine oxidase and granulocytes in ischemia-reperfusion injury. Am J Physiol255:H1269-H1275, 1988 12. Carrico CJ, Meakins JL, Marshall JC, et al: Muhiple-organfailure syndrome. Arch Surg 121:196-208,1986 13. Chiu C-J, McArdle AH, Brown R, et al: Intestinal mucosal lesions in low-flow states. Arch Surg 101:478-483,197O 14. Megison SM, Horton JW, Chao H, et al: Prolonged survival and decreased mucosal injury after low-dose enteral allopurinol prophylaxis in mesenteric ischemia. J Pediatr Surg 25:917-921, 1990 15. Das DK, Engelman RM, Clement R, et al: Role of xanthine oxidase inhibitor as free radical scavenger: A novel mechanism of action of allopurinol and oxypurinol in myocardial salvage. Biothem Biophys Res Commun 148:314-319,1987 16. Allan G, Cambridge D, Lee-Tsang-Tan L, et al: The protective action of allopurinol in an experimental model of haemorrhagic shock and reperfusion. Br J Pharm 89:149-155,1986 17. Baker JW, Deitch EA, Berg RD. et al: Hemorrhagic shock induces bacterial translocation from the gut. J Trauma 28:896-906, 1988 18. Deitch EA: Intestinal permeability is increased in burn patients shortly after injury. Surgery 107:411-416, 1990
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19. Carter EA, Tompkins RG, Schiffrin E, et al: Cutaneous thermal injury alters macromolecular permeability of rat small intestine. Surgery 107:335-341,199O 20. Deitch EA, Winterton J, Li M, et al: The gut as a portal of entry for bacteremia. Role of protein malnutrition. Ann Surg 205:681-692,1987 21. Deitch EA: Potential role of gut failure and bacterial translocation as promoters and potentiators of the multiple organ failure syndrome, in Bihari DJ, Cerra FB (eds): Multiple Organ Failure. Fullerton, CA, Society of Critical Care Medicine, 1989, pp 297-325 22. Baue AE: Gastrointestinal tract. An active metabolic organ that can fail, in Baue AE (ed): Multiple Organ Failure, Patient Care and Prevention. St Louis, MO, Mosby Year Book, 1990, pp 364-372 23. O’Dwyer ST, Michie HR, Ziegler TR, et al: A single dose of endotoxin increases intestinal permeability in healthy humans. Arch Surg 123:1459-1464,198s 24. Zielger TR, Smith RJ, O’Dwyer ST, et al: Increased intestinal permeability associated with infection in burn patients. Arch Surg 123:1313-1319,1988 25. Deitch EA, Bridges W, Baker J, et al: Hemorrhagic shockinduced bacterial translocation is reduced by xanthine oxidase inhibition or inactivation. Surgery 104:191-198,1988 26. Parks DA, Bulkley GB, Granger DN: Role of oxygen free radicals in shock, ischemia, and organ preservation. Surgery 94:428-432,1983 27. Przyklenk K, Kloner RA: “Reperfusion injury” by oxygenderived free radicals? Circ Res 64:86-96, 1989
Discussion Louis Mar-won (Washington, DC): This is a welldesigned, carefully controlled study assessing the value of pretreatment with enteral allopurinol to obviate the changes in permeability associated with distal intestinal ischemia and reperfusion. These experiments demonstrate that oxygen radical generation associated with I/R contributes to the changes in intestinal permeability, and that these changes can be blocked by inhibition of X0 and the use of free radical scavengers. Although I/R has been suggested as an etiology of necrotizing enterocolitis, it is clearly not the entire story, yet this is still an important study since it is probable that local blood flow changes, combined with other factors, predispose the infant gut to necrotizing enterocolitis. The usefulness of this model is in the generation of toxic products associated with alterations in regional blood flow, and the demonstration that free radical generation contributes to changes in normal intestinal homeostasis. I have several questions. You propose that bacterial translocation from the lumen into the blood is facilitated by permeability changes caused by I/R, yet the mechanism used to study intestinal permeability measures movement from the blood into the lumen.
Why not study the movement from the lumen to the blood directly, or bacterial translocation after I/R itself? Does the plasma to luminal clearance of a macromolecule such as labeled EDTA correlate directly with movement of bacteria from the lumen to the blood? Since intestinal absorption stops but secretion continues in a number of pathological conditions, most notably intestinal obstruction, changes in permeability in one direction may not necessarily occur in the other. Why was there a significant difference in baseline EDTA clearance between the allopurinol-treated group and the sham ischemia group? These data suggest that pretreatment with allopurinol causes changes in permeability. While you noted no difference during ischemia between the allopurinol-treated group and the sham group, did a significant change of permeability occur during reperfusion in these groups? There were no statistical analysis provided in the manuscript between the allopurinol-treated group and the sham group during reperfusion. Would you please discuss in greater detail the
ALLOPURINOL
PREVENTS INTESTINAL
I/R INJURY
choice of the route of administration of allopurinol, the dosage, and the time course of pretreatment. Do you propose a direct protective effect on the mucosa, or is the allopurinol absorbed systemically? Can a shorter pretreatment course produce similar results? And finally, what was the time interval between the last allopurinol dose and the ischemia? Kathy Anderson (Washington, DC): Did you see any lesions in the gut? And, if so, what were they? We don’t usually see these babies until they have actually had their insult. Did you have a group, or would you think it would be a good idea to have a group where you produce ischemia first, and then treat with allopurinol to see if you can diminish the injury? W. Glaze Vaughan (response): Dr Marmon, with regard to your question concerning the movement of macromolecules and bacteria across the gut barrier, we measured plasma to gut lumen clearance of 51Cr-labeled EDTA, we did not examine bacterial translocation from the gut lumen to peripheral organs and plasma. While our model allowed us to examine permeability changes in the gastrointestinal tract that occurred secondary to ischemia and reperfusion, the data provide no information with regard to the subsequent translocation of bacteria. Other investigators have used radiolabeled albumin and a variety of other substances to define alterations in the permeability characteristics of the gastrointestinal tract after the ischemia and reperfusion related either to hemorrhagic shock or burn trauma. However, several studies have suggested that SICr-labeled EDTA is a more suitable compound to evaluate intestinal mucosal integrity. With regard to the differences in baseline permeability in those animals treated with enteral allopurino1 for 5 days before the ischemic episode, we have no information regarding the effects of allopurinol on permeability characteristics of the bowel. We had changed vendors for the purchase of rats during this part of the study, and these data may represent
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differences in permeability from animal to animal and, thus, intragroup variability. At present, we are studying groups of animals with long-term enteral allopurinol administration in the absence of ischemic insult to determine the specific effects of allopurinol on bowel permeability. With regard to the route of allopurinol administration, we chose the enteral route for administration because: (1) this is easily accomplished in the rat model; and (2) parenteral allopurinol is not available at the present time. In this regard, Burroughs has a phase III trial in progress using parenteral allopurinol; but to date, information regarding efficacy and safety has not been made available. We chose the dose of 10 mglkg, which has been shown to have no side effects and yet accomplishes X0 inhibition; thus, we could prevent free radical formation without super normal allopurinol doses such as those previously described in the literature (50 to 100 mg/kg body weight). With regard to the time interval over which allopurinol was administered, we selected to administer the drug once a day for 5 days. This was based on previous work in our laboratory showing that we could accomplish X0 inhibition over this time period. We also know that maximal plasma concentration occurs approximately 1% hours after oral administration, and the primary metabolite of allopurinol (oxypurinol) has a circulating time of approximately 18 hours. Therefore, we achieved X0 inhibition for 20 to 24 hours. Dr Anderson, we did not evaluate the lesions histologically. However, in previous studies from our laboratory, histopathologic studies showed significant mucosal changes after 20 minutes of SMA occlusion, including disruptive changes in the villus tips along the length of the villi as well as flattening, shortening, and necrosis of the villus tips. In response to your second question, we have not examined the efficacy of allopurinol administration after the ischemic injury; however, those studies are in progress at the present time.