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Segmental intestinal transplantation can be an adequate therapy for short bowel syndrome in growing dogs
Segmental Intestinal Transplantation Can Be an Adequate Therapy for Short Bowel Syndrome in Growing Dogs By M.C.J. Wolvekamp, E. Heineman, R.L. Marquet, MA.C. Meijssen, R.W.F. de Bruin, and J.C. Molenaar R o t t e r d a m a n d Maastricht, The Netherlands • This study was undertaken to investigate whether twostage segmental small bowel allotransplantation can maintain growth and development of young dogs (16 weeks, 5 to 6 kg) with surgically created short bowel syndrome {SBS). After near-total small bowel resection (group 1; n = 3), irreversible weight loss was noted. After a sham operation (group 2; n = 3), no growth disturbances were found. Major histocompatibility matched small bowel transplantation (SBT) with cyclosporine A as immunosuppressant, was performed in two stages (group 3; n = 7). During the first stage, one meter of jejunoileum from an adult donor was placed as a Roux loop. Four weeks later, the native small bowel was removed and replaced by the graft. Only one dog survived long-term; the dogs died from infectious complications. The addition of selective decontamination of the digestive tract and early gastrostomy feeding (group 4; n = 10) resulted in long-term survival in 60%. Follow-up at 4 months showed that their growth was about 20% compromised compared with that of the sham.operated animals. Functional analysis showed that electrolytes, urea, and D-xylose were normal, but there was an increase in the lactulose:mannitol ratio, fecal fat excretion, and postheparin diamine oxidase release. These results show that under the conditions described, segmental SBT functions sufficiently to treat SBS but does not maintain normal growth. Copyright © 1995 by W.B. Saunders Company INDEX WORDS: Segmental intestinal transplantation; short bowel syndrome; growing dogs; cyclosporine A immunosuppression; selective digestive tract decontamination.
MALL BOWEL transplantation (SBT) probably will become the preferred treatment for a variety of life-threatening gut-related diseases in early childhood. In transplant medicine, matching donor and recipient for major histocompatibility complex (MHC) antigens assists prolonged graft survival. 1,2 Thus, living relatives could donate a segment of bowel if segmental SBT could be proven to be a reasonable alternative to total parenteral nutrition (TPN). It is
S
From the Departments of Pediatric Surgery, General Surgery, and Internal Medicine II, Erasmus University Hospital, Rotterdam, The Netherlands, and the Department of Surgery, Academic Hospital, Maastricht, The Netherlands. Presented at the 41st Annual International Congress of the British Association of Paediatric Surgeons, Rotterdam, The Netherlands, June 29-July 1, 1994. Supported by the Sophia Foundation for Medical Research (project no. 120). Address reprint requests to E. Heineman, MD, PhD, Department of Surgery, Academic Hospital Maastricht, PO Box 5800, 6202 A Z Maastricht, The Netherlands. Copyright © 1995 by W.B. Saunders Company 0022-3468/95/3003-0005503. 00/0
396
still not clear whether segmental SBT functions sufficiently to maintain the growth and development of a child. In a previous study, we showed that adult dogs are able to survive long-term after transplantation with a segmental MHC-matched small bowel graft. 3 The present study was undertaken to investigate the value of MHC-matched two-stage segmental SB allotransplantation in the treatment of enterectomized puppies. We evaluated the functional capacity of a segmental graft, derived from an adult donor, by assessing the general condition of the animal, growth, serum urea and electrolytes, absorptive capacity of the intestine (D-xylose absorption and fecal fat excretion), mucosal integrity (lactulose:mannitol excretion ratio), and the adaptive response (postheparin diamine oxidase [DAO] release). MATERIALS AND METHODS
Animals Donors. Seventeen adult male and female Beagles, weighing 10 to 20 kg, were used. Their age range was 1 to 3 years. Recipients. Twenty-three healthy female Beagle puppies, 16 weeks of age and weighing 5 to 6 kg, were used. (All animals were from Harlan CPB, Zeist, The Netherlands.)
Ethics The experimental protocols adhered to the rules of "The Dutch Animal Experimentation Act" (1977) and the "Guidelines on the Protection of Experimental Animals" of the Council of the European Community (1986). Specific protocols were approved by the Committee on Animal Research of Erasmus University, Rotterdam.
MHC Matching Matching for antigens of the canine MHC DLA consisted of typing for class I and class II antigens, as described previously.3 Direct and indirect microeytotoxicity tests, based on a battery of approximately 60 alloantisera, were used to define the class I, serologically defined, antigens belonging to either DLA-A, DLA-B, or DLA-C. Typing for class II antigens (DLA-D) was performed with the unilateral mixed lymphocyte reaction, using a culture period of 6 days. The selection of donor-recipient pairs was based on a two-haplotype similarity.
Experimental Groups The dogs were subdivided into four experimental groups: group 1 (n = 3), short bowel controls; group 2 (n = 3), sham-operated animals; group 3 (n = 7) MHC-matched segmental jejunoileal allografts; and group 4 (n = 10), MHC-matched segmental jejunoileal allografts + selective digestive tract decontamination Journal of Pediatric Surgery, Vo130,No 3 (March),1995:pp 396-401
SEGMENTAL INTESTINAL TRANSPLANTATION IN DOGS
397
After a midline incision, short bowel control animals had resection of the total small bowel, ie, from just distal to the ligament of Treitz to 5 cm proximal to the ileocecal valve. Thereafter, the bowel was reanastomosed end-to-end. The operative techniques of the sham operation and the two-stage segmental intestinal allotransplantation are described in detail elsewhere. 4 In summary, a sham operation is a control operation without interrupting the vascular, neural, or lymphatic supply. In these animals, a Roux-en-Y loop was created approximately 20 cm proximal to the ileocecal valve. This loop was used for histological and sonographic monitoring, as described previously. 4 During the first stage of a two-stage segmental allotransplantation (OK1), heterotopic SBT was performed, leaving the recipient's own bowel intact. The transplant (1 m) was harvested on a vascular pedicle, consisting of both superior mesenteric artery (with aortic cuff) and vein, from a fully MHCmatched donor. The graft was revascularized by anastomosis of the mesenteric vessels to the abdominal aorta and caval vein end-toside. The distal end of the graft was anastomosed as an isopersitaltic Roux-en-Y loop in continuity with the recipient's terminal ileum, approximately 5 cm proximal to the ileocecal valve. The proximal end was exteriorized as an open ileostomy. Four weeks later, the recipient's small gut was excised and the graft anastomosed in its place (OK2).
Assessment of the absorptive capacity. The integrity and the adaptive status of the small intestine were assessed in group 2 and in long-term group 4 survivors (Table 1). D-xylose absorption test. The dogs were fasted overnight, before the xylose challenge. D-xylose (0.5 g/kg) was dissolved in water and administered orally. Blood samples were collected before, and a half hour, 1, and 2 hours after the ingestion of xylose. A simplified assay procedure developed by Eberts et al6 was used to obtain the xylose blood level time curve. Fecal fat excretion. This was determined using a method described by Van de Kamer et al. 7 Lactulose:mannitol excretion test. The dogs were fasted overnight, and a specimen of urine (for baseline sugar concentrations) was taken immediately before the test. Four hundred milligrams of D-lactulose and 100 mg of D-mannitol (per kilogram) were dissolved in 2 mE of water. After oral administration, urine was collected for 5 hours. After 2 hours, the dogs were allowed to drink water freely. The urine samples were stored at -20°C until analysis. The lactulose and mannitol content was analysed using a gas liquid chromatographic method. 8 The results were expressed as the lactulose:mannitol concentration ratio measured in the urine pool 5 hours after ingestion. Postheparin DAO assessment. Food was withheld for the 12 hours preceding the test. Heparin (1,500 IU; Thromboliquine; Organon Technica BV, Boxtel, The Netherlands) dissolved in 5 mL of saline was injected intravenously. Blood samples were collected at 0, 15, 30, 60, and 120 minutes after the injection. The DAO concentration in plasma was determined as described by Romijn et al.9
Perioperative Treatment
Statistics
The perioperative management of groups 1, 2, and 3 consisted of "standard" care described previously.5 Groups 3 and 4 received 10 mg/kg/d of CsA in olive oil (Sandimmune; Sandoz, Basel, Switzerland) intramuscularly, from 1 day before surgery through the seventh postoperative day. From then on, CsA was administered orally, in capsules, at a dose of 20 mg/kg/d. In addition, group 4 animals received SDD treatment beginning 1 week before surgery and continuing throughout the experimental period. The SDD regimen consisted of 6.5 mg/kg/d of ciprofloxacin (Bayer Nederland BV, Mijdrecht, The Netherlands), 10 rag/ kg/d of polymyxin B (Pfizer Chemicals, New York, NY), and 60,000 U twice daily of nystatin (Labaz Sanofi-Winthrop, Maassluis, The Netherlands). Food was withheld for the 36 hours preceding surgery; then an energy-containing solution was administered orally (Extran; Nutricia, Zoetermeer, The Netherlands), supported with force feeding via the gastrostomy (Nutrison Pediatrics; Nutricia, Zoetermeer, The Netherlands) in the first postoperative week, after both the first-stage and second-stage operations. Thereafter, irradiated (0.9 megarad) dog food (Puppap; Hope Farms, Woerden, The Netherlands) was given, and water was freely available.
All data were expressed as mean _+ standard deviation. Differences in the tested parameters between groups (weight, fecal fat
(SDD) + early gastrostomy feeding. In groups 3 and 4, irnmunosuppression with cyclosporine A (CsA) was used.
Operative Procedure
Evaluation The general condition of the dogs and the appearance of the enterostomy, if present, were observed daily. Survival The animals were killed if their general condition deteriorated or if they lost more than 30% of their preoperative body weight. Weight. Weight was measured twice weekly. Blood analysis. For group 2 animals and group 4 in long-term survivors, routine blood chemistry data were recorded regularly. For animals receiving CsA, plasma trough levels were determined using a radioimmunoassay (CycloTrac SP-Serum/Plasma; Incstar Corp, Stillwater, MN).
Table 1. Experimental Design of Functional Assessment of the Graft Weeks After OK2 -6 -5 - 4 OK1 -2 0 OK2 1 2 3 4
TestRelatedto Specific Functional Componentof the Small Intestine Absorption
Permeability
Adaptation
Lactuiose:mannitol ratio Fecal fat excretion Fecal fat excretion Fecal fat excretion Fecal fat excretion Fecal fat excretion D-xylose absorption Lactulose:mannitol ratio
6 7 8 9 10 11 12 13 14
Postheparin DAO release Fecal fat excretion
Fecal fat excretion D-xylose absorption Lactulose: mannitol ratio Postheparin DAO release
Abbreviations: OK1, sham operation and heterotopic placement of allograft. OK2, removal of native small intestine and orthotopic placement of allograft.
398
WOLVEKAMP ET AL
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RESULTS
Survival The mean survival time for the enterectomized dogs (group 1) was 11.3 days. The sham-operated animals (group 2) survived through the experimental period without complications. In group 3, only one of the seven dogs survived through the study period. One dog died of venous thrombosis (technical complication). Five of the remaining six dogs were killed because of postoperative complications. All had positive blood cultures of gut-derived bacteria, including hemolytic Escherichia coli and Staphylococcus aureus, in the early postoperative period. One dog died of multiorgan failure on day 10, and another died of transplant intussusception on day 14. One died of obstructive jaundice on day 48, and another because of pneumonia on day 53. In only one dog was rejection the cause of death (on day 24). In group 4, there were no episodes of bacteremia. Four dogs died, only one because of rejection (23 days after OK2). Of the other three, one died of arterial thrombosis, one because of general deterioration (on the 21st day) found to be caused by pulmonary artery thrombosis, and one of acute peritonitis (on day 34) caused by intussusception. The remaining six dogs
Fig 1. Weight curves after sham operation (group 2; n = 3) and twostage segmental allotransplantation (group 4; n = 6). ok1, time of sham operation or heterotopic placement of allograft; ok2, time of orthotopic placement of allograft.
had no postoperative complications and survived through the experimental period.
Weight Group 1 animals (SBS control) had rapid irreversible weight loss. Group 2 animals (sham-operated) had a normal growth pattern compared with nonoperated historical controls (data supplied by Harlan CPB). Throughout the study period, the growth pattern of group 4 animals was poorer than that of group 2 animals (Fig 1).
Blood Analysis There were no striking differences in routine blood chemistry values (including electrolytes, protein, urea, bilirubin, and transaminase) between groups 2 and 4 with the exception of serum ammonia levels. The ammonia levels were significantly higher in group 4 than in group 2, especially in the long-term survivors.
D-Xylose Absorption Test The D-xylose absorption test results of groups 2 and 4 are depicted in Fig 2A. Area-under-curve calculations showed that D-xylose absorption was similar in both groups at 4 and 12 weeks.
Fecal Fat Analysis From week 3 after the orthotopic positioning of the segmental graft, fecal fat excretion was significantly higher in group 4 compared with group 2 (Fig 2B).
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Functional evaluation after sham operation (group 2; n = 3) and two-stage segmental allotransplantation (group 4; n = 6).
Lactulose:Mannitol Absorption Test In the allotransplanted group, the mean lactulose: mannitol excretion ratio increased significantly, from 0.04 _+ 0.03 preoperatively to 0.08 + 0.06 at week 5 after OK2, and to 0.11 _+ 0.07 at week 13 (Fig 2C).
Postheparin DA 0 Assessment Six weeks after transplantation, postheparin DAO release was significantly higher in segmental graft animals than in the sham-operated dogs of the same age (Fig 2D). Fourteen weeks after orthotopic placement, postheparin DAO release did not differ significantly between the groups. DISCUSSION
The postoperative course of human SBT is often complicated by infectious episodes, which are associated with fungal and/or bacterial translocation. 1° After intestinal transplantation, ischemic injury, lymphatic and neural disruption, and altered motility and
systemic venous drainage increase the susceptibility to sepsis. 11,I2 Previous studies of adult dogs showed that DLAmatched segmental allotransplantation resulted in long-term survival without the need for SDD. 3 In the present study, we found that young recipients had an increased susceptibility to posttransplant infection associated with bacterial translocation of Enterobacteriaceae and S aureus--species that translocate more easily through a leaky intestine• The time of initiation of enteral feeding may influence the postoperative course positively, because immediate postoperative enteral feeding is known to decrease the translocation of bacteria, to reduce the hypermetabolic response to stress, and to enhance immunocompetence.13,14 A comparison of the survival in group 3 and group 4 suggests that SDD together with early enteral feeding led to a lower mortality rate, without infectious complications or bacterial translocation. The low rejection rate was a surprising finding because it has
400
WOLVEKAMP ET AL
been claimed that younger transplant recipients had a greater risk of graft rejection2; thus, MHC matching is promising for young recipients of a small bowel graft. Enterectomized dogs with short bowel syndrome that had a segmental jejunal allograft were able to overcome the symptoms of the surgically created syndrome. Although others 15 have shown that weight gain after segmental allografting is possible, no comparison with sham-operated control animals had been made. In the present study, long-term surviving dogs gained weight, but their growth was less than that of the sham-operated animals. The weight curve shows that after segmental allografting, weight was lost in the early postoperative phase, then stabilised, and finally increased at a normal rate but without catch-up. The dogs probably were undernourished in the early postoperative period. This could imply that if they had been given TPN, they might have grown normally. Routine blood analysis showed that group 4 animals were comparable to group 2 animals, indicating that a small bowel graft is able to maintain normal balance. The elevated serum ammonia levels in group 4 most probably were caused by caval shunting of the mesenteric vessels, a phenomenon already found in autotransplanted young dogs (unpublished data). D-xylose absorption was not impaired in the allotransplanted dogs. Although the D-xylose absorption test has been widely used to study the absorptive capacity of the small intestine, 16 its value may be limited because of variations in renal function and gastrointestinal transit time, which affect the excretion of orally administered xylose. 17 Moreover, subtle changes in intestinal permeability are not detected with D-xylose because it is actively absorbed. 18 Analysis of fecal fat excretion showed that this increased significantly from day 21 after transplantation. However, the values were still within the normal range. Because our control dogs did not receive CsA, which can impair fat absorption, ~9 we conclude that the transplanted segment of ileum maintained nearnormal fat absorption. The lactulose:mannitol absorption test is independent of the amount of urinary excretion. Lactulose, a disaccharide, is absorbed by a paracellular route, and
the amount excreted is a reliable index of mucosal leakiness. Mannitol, a monosaccharide, is absorbed by the transcellular route and reflects the mucosal surface area. The higher lactulose:mannitol ratio found in the allotransplanted dogs showed that subtle changes in intestinal permeability, not apparent with D-xylose, were present at both 29 and 85 days posttransplant. Andre et al 2° compared the lactulose:mannitol ratio and the chromium 51 EDTA test, another permeability test, directly in inflammatory bowel diseases, and concluded that the combination of these tests provided increased sensitivity and therefore might be useful to monitor the small bowel mucosal architecture in transplant recipients. DAO is an enzyme that is unique because its blood levels correlate positively with the integrity of the intestinal mucosa. 21 It is known to be released from the intestine into the blood upon stimulation by intravenously injected heparin. 2a In cases of intestinal mucosal damage, like small bowel atrophy, a lower plasma release of DAO was found. ;1,23 Rose et a124 have shown that postheparin serum DAO is not significantly affected by autotransplantation and systemic vascular anastomosis, suggesting that it may serve as a potential indicator of the condition of the transplanted intestine. In the present study, the area-under-curve values of the heparin stimulation curve of allotransplanted animals were significantly higher 43 days after orthotopic grafting. This was not found 99 days after graft placement. The temporary increase in intestinal DAO activity might be explained by the hypothesis that the segmental graft has an adaptive response to overcome the effects of short bowel syndrome. This is supported by results of Rokkas et a125 suggesting that DAO is an important regulator of intestinal mucosal growth. In conclusion, this study shows that a two-stage segmental allotransplantation of 1 m of small bowel combined with SDD and early gastrostomy feeding is able to prevent the symptoms of surgically created short bowel syndrome. Although it did maintain adequate nutritional status, it did not maintain a normal growth pattern.
REFERENCES 1. Karuppan SS, Lindholm A, Moller E: Fewer acute rejection episodes and improved outcome in kidney-transplanted patients with selection criteria based on crossmatching. Transplantation 53:666-673, 1992 2. Pirsch JD, D'Alessandro AM, Sollinger HW, et al: The effect of donor age, recipient age, and HLA match on immunologic graft survival in cadaver renal transplant recipients. Transplantation 53:55-59, 1992 3. Meijssen MAC, Heineman E, de Bruin RWF, et al: Long-
term survival of DLA-matched segmental intestinal allografts in dogs. Transplantation 56:1062-1066, 1993 4. Wolvekamp MCJ, Robben SGF, Vermey M, et al: Ultrasonography; A useful tool in the follow-up of small-bowel transplantation in growing dogs. Surg Res Comm 15:183-197, 1994 5. Meijsen MAC, Heineman E, de Bruin RWF, et al: Value of in vivo electrophysiological measurements to evaluate canine small bowel autotransplants. Gut 32:1329-1335, 1991 6. Eberts TJ, Sample RHB, Glick MR, et al: A simplified,
SEGMENTAL INTESTINAL TRANSPLANTATION IN DOGS
colorimetric micromethod for xylose in serum or urine, with phloroglucinol. Clin Chem 25:1440-1443, 1979 7. Van de Kamer JH, Ten Bokkel Huinink H, Weyers HA: Rapid method for the determination of fat in feces. J Biol Chem 177:347-355, 1949 8. Jansen G, Muskiet FAJ, Schierbeek H, et al: Capillary gaschromatographic profiling of urinary? plasma and erythrocyte sugars and polyols as their trimethylsilyl derivatives, preceded by a simple and rapid prepurification method. Clin Chem Acta 157:277293, 1986 9. Romijn JC, Verkoelen CF, Splinter TAW: Species-dependent differences of the biochemical properties of diamine oxidase. Int J Biochem 18:835-839, 1986 10. Tzakis AG, Todo S, Reyes J, et al: Clinical intestinal transplantation: Focus on complications. Transplant Proc 24:12381240, 1992 11. Browne BJ, Johnson CP, Edmiston CE, et al: Transplantation promotes bacterial overgrowth and translocation. J Surg Res 51:512-517, 1991 12. Grant D, Hurlbut D, Zhong R, et al: Intestinal permeability and bacterial translocation following transplantation in the rat. Transplantation 52:221-224, 1991 13. Alexander JW: Nutrition and infection. Arch Surg 121:966972, 1986 14. Moore EE, Jones TN: Benefits of immediate jejunostomy feeding after major abdominal trauma--A prospective, randomized study. J Trauma 26:874-881, 1986 15. Kimura K, Larosa CA, Blank MA, et al: Successful segmental intestinal transplantation in enterectomized pigs. Ann Surg 211:158-164, 1990 16. Quigley EMM, Thompson JS, Rose SG: The long-term function of canine jejunoileal autotransplants--Insights into allograft physiology. Transplant Proc 24:1105-1106, 1992
401
17. Behrens ME, Northrop C, Wraight P, et al: Evaluation of mannitol, lactulose and SlCr-labelled ethylenediaminetetraacetate as markers of intestinal permeability in man. Clin Sci 73:197-204, 1987 18. Nathavitharana KA, Lloyd DR, Raafat F, et al: UrinarY mannitol: Lactul0se excretion ratios and jejunal architecture. Arch Dis Child 63:1054-1059, 1988 19. Wassef R, Cohen Z, Nordgren S, et al: Cyclospori~e absorption in intestinal transplantation. Transplantation 39:496499, 1985 20. Andre C, Descos L, Minaire Y: Comparisons between lactulose-mannitol test and 51Cr-labelled ethylene diamine tetraacetate test in inflammatory bowel diseases. Hepato Gastroentero! 37:1i3-117, 1990 (suppl 2) 21. D'Agostino L, Ciacci C, Daniele B, et al: Postheparin plasma diamine oxidase in subjects with small bowel atrophy: Dig Dis Sci 32:313-317, 1987 22. Rokkas T, Vaja S, Taylor P, et al: Is the intestine the sole source of heparin-stimulated plasma diamine oxidase? Acute effects of jejunectomy, ileoectomy and total enterectomy. Diges~ tion 46:439-446, 1990 (suppl 2) 23. Luk GD, Bayless TM, Baylin SB: Plasma postheparin diamine oxidase. Sensitive provocative test for quantitating length of acute intestinal mucosa injury in the rat. J Clin Invest 71:13081 1315, 1983 : 24. Rose SG, Thompson JS, Spanta AD, et al: The effect of intestina! autotransplantation on serum diamine oxidase activity. ) Surg Res 50:223-227, 1991 25. Rokkas T, Vaja S, Murphy GM, et al: Aminoguanidine blocks intestinal diamine oxidase (DAO) activity and enhances the intestinal adaptive response to resection in the rat. Digestion 46:447-457, 1990 (suppl 2)
MALL BOWEL transplantation (SBT) probably will become the preferred treatment for a variety of life-threatening gut-related diseases in early childhood. In transplant medicine, matching donor and recipient for major histocompatibility complex (MHC) antigens assists prolonged graft survival. 1,2 Thus, living relatives could donate a segment of bowel if segmental SBT could be proven to be a reasonable alternative to total parenteral nutrition (TPN). It is
S
From the Departments of Pediatric Surgery, General Surgery, and Internal Medicine II, Erasmus University Hospital, Rotterdam, The Netherlands, and the Department of Surgery, Academic Hospital, Maastricht, The Netherlands. Presented at the 41st Annual International Congress of the British Association of Paediatric Surgeons, Rotterdam, The Netherlands, June 29-July 1, 1994. Supported by the Sophia Foundation for Medical Research (project no. 120). Address reprint requests to E. Heineman, MD, PhD, Department of Surgery, Academic Hospital Maastricht, PO Box 5800, 6202 A Z Maastricht, The Netherlands. Copyright © 1995 by W.B. Saunders Company 0022-3468/95/3003-0005503. 00/0
396
still not clear whether segmental SBT functions sufficiently to maintain the growth and development of a child. In a previous study, we showed that adult dogs are able to survive long-term after transplantation with a segmental MHC-matched small bowel graft. 3 The present study was undertaken to investigate the value of MHC-matched two-stage segmental SB allotransplantation in the treatment of enterectomized puppies. We evaluated the functional capacity of a segmental graft, derived from an adult donor, by assessing the general condition of the animal, growth, serum urea and electrolytes, absorptive capacity of the intestine (D-xylose absorption and fecal fat excretion), mucosal integrity (lactulose:mannitol excretion ratio), and the adaptive response (postheparin diamine oxidase [DAO] release). MATERIALS AND METHODS
Animals Donors. Seventeen adult male and female Beagles, weighing 10 to 20 kg, were used. Their age range was 1 to 3 years. Recipients. Twenty-three healthy female Beagle puppies, 16 weeks of age and weighing 5 to 6 kg, were used. (All animals were from Harlan CPB, Zeist, The Netherlands.)
Ethics The experimental protocols adhered to the rules of "The Dutch Animal Experimentation Act" (1977) and the "Guidelines on the Protection of Experimental Animals" of the Council of the European Community (1986). Specific protocols were approved by the Committee on Animal Research of Erasmus University, Rotterdam.
MHC Matching Matching for antigens of the canine MHC DLA consisted of typing for class I and class II antigens, as described previously.3 Direct and indirect microeytotoxicity tests, based on a battery of approximately 60 alloantisera, were used to define the class I, serologically defined, antigens belonging to either DLA-A, DLA-B, or DLA-C. Typing for class II antigens (DLA-D) was performed with the unilateral mixed lymphocyte reaction, using a culture period of 6 days. The selection of donor-recipient pairs was based on a two-haplotype similarity.
Experimental Groups The dogs were subdivided into four experimental groups: group 1 (n = 3), short bowel controls; group 2 (n = 3), sham-operated animals; group 3 (n = 7) MHC-matched segmental jejunoileal allografts; and group 4 (n = 10), MHC-matched segmental jejunoileal allografts + selective digestive tract decontamination Journal of Pediatric Surgery, Vo130,No 3 (March),1995:pp 396-401
SEGMENTAL INTESTINAL TRANSPLANTATION IN DOGS
397
After a midline incision, short bowel control animals had resection of the total small bowel, ie, from just distal to the ligament of Treitz to 5 cm proximal to the ileocecal valve. Thereafter, the bowel was reanastomosed end-to-end. The operative techniques of the sham operation and the two-stage segmental intestinal allotransplantation are described in detail elsewhere. 4 In summary, a sham operation is a control operation without interrupting the vascular, neural, or lymphatic supply. In these animals, a Roux-en-Y loop was created approximately 20 cm proximal to the ileocecal valve. This loop was used for histological and sonographic monitoring, as described previously. 4 During the first stage of a two-stage segmental allotransplantation (OK1), heterotopic SBT was performed, leaving the recipient's own bowel intact. The transplant (1 m) was harvested on a vascular pedicle, consisting of both superior mesenteric artery (with aortic cuff) and vein, from a fully MHCmatched donor. The graft was revascularized by anastomosis of the mesenteric vessels to the abdominal aorta and caval vein end-toside. The distal end of the graft was anastomosed as an isopersitaltic Roux-en-Y loop in continuity with the recipient's terminal ileum, approximately 5 cm proximal to the ileocecal valve. The proximal end was exteriorized as an open ileostomy. Four weeks later, the recipient's small gut was excised and the graft anastomosed in its place (OK2).
Assessment of the absorptive capacity. The integrity and the adaptive status of the small intestine were assessed in group 2 and in long-term group 4 survivors (Table 1). D-xylose absorption test. The dogs were fasted overnight, before the xylose challenge. D-xylose (0.5 g/kg) was dissolved in water and administered orally. Blood samples were collected before, and a half hour, 1, and 2 hours after the ingestion of xylose. A simplified assay procedure developed by Eberts et al6 was used to obtain the xylose blood level time curve. Fecal fat excretion. This was determined using a method described by Van de Kamer et al. 7 Lactulose:mannitol excretion test. The dogs were fasted overnight, and a specimen of urine (for baseline sugar concentrations) was taken immediately before the test. Four hundred milligrams of D-lactulose and 100 mg of D-mannitol (per kilogram) were dissolved in 2 mE of water. After oral administration, urine was collected for 5 hours. After 2 hours, the dogs were allowed to drink water freely. The urine samples were stored at -20°C until analysis. The lactulose and mannitol content was analysed using a gas liquid chromatographic method. 8 The results were expressed as the lactulose:mannitol concentration ratio measured in the urine pool 5 hours after ingestion. Postheparin DAO assessment. Food was withheld for the 12 hours preceding the test. Heparin (1,500 IU; Thromboliquine; Organon Technica BV, Boxtel, The Netherlands) dissolved in 5 mL of saline was injected intravenously. Blood samples were collected at 0, 15, 30, 60, and 120 minutes after the injection. The DAO concentration in plasma was determined as described by Romijn et al.9
Perioperative Treatment
Statistics
The perioperative management of groups 1, 2, and 3 consisted of "standard" care described previously.5 Groups 3 and 4 received 10 mg/kg/d of CsA in olive oil (Sandimmune; Sandoz, Basel, Switzerland) intramuscularly, from 1 day before surgery through the seventh postoperative day. From then on, CsA was administered orally, in capsules, at a dose of 20 mg/kg/d. In addition, group 4 animals received SDD treatment beginning 1 week before surgery and continuing throughout the experimental period. The SDD regimen consisted of 6.5 mg/kg/d of ciprofloxacin (Bayer Nederland BV, Mijdrecht, The Netherlands), 10 rag/ kg/d of polymyxin B (Pfizer Chemicals, New York, NY), and 60,000 U twice daily of nystatin (Labaz Sanofi-Winthrop, Maassluis, The Netherlands). Food was withheld for the 36 hours preceding surgery; then an energy-containing solution was administered orally (Extran; Nutricia, Zoetermeer, The Netherlands), supported with force feeding via the gastrostomy (Nutrison Pediatrics; Nutricia, Zoetermeer, The Netherlands) in the first postoperative week, after both the first-stage and second-stage operations. Thereafter, irradiated (0.9 megarad) dog food (Puppap; Hope Farms, Woerden, The Netherlands) was given, and water was freely available.
All data were expressed as mean _+ standard deviation. Differences in the tested parameters between groups (weight, fecal fat
(SDD) + early gastrostomy feeding. In groups 3 and 4, irnmunosuppression with cyclosporine A (CsA) was used.
Operative Procedure
Evaluation The general condition of the dogs and the appearance of the enterostomy, if present, were observed daily. Survival The animals were killed if their general condition deteriorated or if they lost more than 30% of their preoperative body weight. Weight. Weight was measured twice weekly. Blood analysis. For group 2 animals and group 4 in long-term survivors, routine blood chemistry data were recorded regularly. For animals receiving CsA, plasma trough levels were determined using a radioimmunoassay (CycloTrac SP-Serum/Plasma; Incstar Corp, Stillwater, MN).
Table 1. Experimental Design of Functional Assessment of the Graft Weeks After OK2 -6 -5 - 4 OK1 -2 0 OK2 1 2 3 4
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Permeability
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6 7 8 9 10 11 12 13 14
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Fecal fat excretion D-xylose absorption Lactulose: mannitol ratio Postheparin DAO release
Abbreviations: OK1, sham operation and heterotopic placement of allograft. OK2, removal of native small intestine and orthotopic placement of allograft.
398
WOLVEKAMP ET AL
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RESULTS
Survival The mean survival time for the enterectomized dogs (group 1) was 11.3 days. The sham-operated animals (group 2) survived through the experimental period without complications. In group 3, only one of the seven dogs survived through the study period. One dog died of venous thrombosis (technical complication). Five of the remaining six dogs were killed because of postoperative complications. All had positive blood cultures of gut-derived bacteria, including hemolytic Escherichia coli and Staphylococcus aureus, in the early postoperative period. One dog died of multiorgan failure on day 10, and another died of transplant intussusception on day 14. One died of obstructive jaundice on day 48, and another because of pneumonia on day 53. In only one dog was rejection the cause of death (on day 24). In group 4, there were no episodes of bacteremia. Four dogs died, only one because of rejection (23 days after OK2). Of the other three, one died of arterial thrombosis, one because of general deterioration (on the 21st day) found to be caused by pulmonary artery thrombosis, and one of acute peritonitis (on day 34) caused by intussusception. The remaining six dogs
Fig 1. Weight curves after sham operation (group 2; n = 3) and twostage segmental allotransplantation (group 4; n = 6). ok1, time of sham operation or heterotopic placement of allograft; ok2, time of orthotopic placement of allograft.
had no postoperative complications and survived through the experimental period.
Weight Group 1 animals (SBS control) had rapid irreversible weight loss. Group 2 animals (sham-operated) had a normal growth pattern compared with nonoperated historical controls (data supplied by Harlan CPB). Throughout the study period, the growth pattern of group 4 animals was poorer than that of group 2 animals (Fig 1).
Blood Analysis There were no striking differences in routine blood chemistry values (including electrolytes, protein, urea, bilirubin, and transaminase) between groups 2 and 4 with the exception of serum ammonia levels. The ammonia levels were significantly higher in group 4 than in group 2, especially in the long-term survivors.
D-Xylose Absorption Test The D-xylose absorption test results of groups 2 and 4 are depicted in Fig 2A. Area-under-curve calculations showed that D-xylose absorption was similar in both groups at 4 and 12 weeks.
Fecal Fat Analysis From week 3 after the orthotopic positioning of the segmental graft, fecal fat excretion was significantly higher in group 4 compared with group 2 (Fig 2B).
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Lactulose:Mannitol Absorption Test In the allotransplanted group, the mean lactulose: mannitol excretion ratio increased significantly, from 0.04 _+ 0.03 preoperatively to 0.08 + 0.06 at week 5 after OK2, and to 0.11 _+ 0.07 at week 13 (Fig 2C).
Postheparin DA 0 Assessment Six weeks after transplantation, postheparin DAO release was significantly higher in segmental graft animals than in the sham-operated dogs of the same age (Fig 2D). Fourteen weeks after orthotopic placement, postheparin DAO release did not differ significantly between the groups. DISCUSSION
The postoperative course of human SBT is often complicated by infectious episodes, which are associated with fungal and/or bacterial translocation. 1° After intestinal transplantation, ischemic injury, lymphatic and neural disruption, and altered motility and
systemic venous drainage increase the susceptibility to sepsis. 11,I2 Previous studies of adult dogs showed that DLAmatched segmental allotransplantation resulted in long-term survival without the need for SDD. 3 In the present study, we found that young recipients had an increased susceptibility to posttransplant infection associated with bacterial translocation of Enterobacteriaceae and S aureus--species that translocate more easily through a leaky intestine• The time of initiation of enteral feeding may influence the postoperative course positively, because immediate postoperative enteral feeding is known to decrease the translocation of bacteria, to reduce the hypermetabolic response to stress, and to enhance immunocompetence.13,14 A comparison of the survival in group 3 and group 4 suggests that SDD together with early enteral feeding led to a lower mortality rate, without infectious complications or bacterial translocation. The low rejection rate was a surprising finding because it has
400
WOLVEKAMP ET AL
been claimed that younger transplant recipients had a greater risk of graft rejection2; thus, MHC matching is promising for young recipients of a small bowel graft. Enterectomized dogs with short bowel syndrome that had a segmental jejunal allograft were able to overcome the symptoms of the surgically created syndrome. Although others 15 have shown that weight gain after segmental allografting is possible, no comparison with sham-operated control animals had been made. In the present study, long-term surviving dogs gained weight, but their growth was less than that of the sham-operated animals. The weight curve shows that after segmental allografting, weight was lost in the early postoperative phase, then stabilised, and finally increased at a normal rate but without catch-up. The dogs probably were undernourished in the early postoperative period. This could imply that if they had been given TPN, they might have grown normally. Routine blood analysis showed that group 4 animals were comparable to group 2 animals, indicating that a small bowel graft is able to maintain normal balance. The elevated serum ammonia levels in group 4 most probably were caused by caval shunting of the mesenteric vessels, a phenomenon already found in autotransplanted young dogs (unpublished data). D-xylose absorption was not impaired in the allotransplanted dogs. Although the D-xylose absorption test has been widely used to study the absorptive capacity of the small intestine, 16 its value may be limited because of variations in renal function and gastrointestinal transit time, which affect the excretion of orally administered xylose. 17 Moreover, subtle changes in intestinal permeability are not detected with D-xylose because it is actively absorbed. 18 Analysis of fecal fat excretion showed that this increased significantly from day 21 after transplantation. However, the values were still within the normal range. Because our control dogs did not receive CsA, which can impair fat absorption, ~9 we conclude that the transplanted segment of ileum maintained nearnormal fat absorption. The lactulose:mannitol absorption test is independent of the amount of urinary excretion. Lactulose, a disaccharide, is absorbed by a paracellular route, and
the amount excreted is a reliable index of mucosal leakiness. Mannitol, a monosaccharide, is absorbed by the transcellular route and reflects the mucosal surface area. The higher lactulose:mannitol ratio found in the allotransplanted dogs showed that subtle changes in intestinal permeability, not apparent with D-xylose, were present at both 29 and 85 days posttransplant. Andre et al 2° compared the lactulose:mannitol ratio and the chromium 51 EDTA test, another permeability test, directly in inflammatory bowel diseases, and concluded that the combination of these tests provided increased sensitivity and therefore might be useful to monitor the small bowel mucosal architecture in transplant recipients. DAO is an enzyme that is unique because its blood levels correlate positively with the integrity of the intestinal mucosa. 21 It is known to be released from the intestine into the blood upon stimulation by intravenously injected heparin. 2a In cases of intestinal mucosal damage, like small bowel atrophy, a lower plasma release of DAO was found. ;1,23 Rose et a124 have shown that postheparin serum DAO is not significantly affected by autotransplantation and systemic vascular anastomosis, suggesting that it may serve as a potential indicator of the condition of the transplanted intestine. In the present study, the area-under-curve values of the heparin stimulation curve of allotransplanted animals were significantly higher 43 days after orthotopic grafting. This was not found 99 days after graft placement. The temporary increase in intestinal DAO activity might be explained by the hypothesis that the segmental graft has an adaptive response to overcome the effects of short bowel syndrome. This is supported by results of Rokkas et a125 suggesting that DAO is an important regulator of intestinal mucosal growth. In conclusion, this study shows that a two-stage segmental allotransplantation of 1 m of small bowel combined with SDD and early gastrostomy feeding is able to prevent the symptoms of surgically created short bowel syndrome. Although it did maintain adequate nutritional status, it did not maintain a normal growth pattern.
REFERENCES 1. Karuppan SS, Lindholm A, Moller E: Fewer acute rejection episodes and improved outcome in kidney-transplanted patients with selection criteria based on crossmatching. Transplantation 53:666-673, 1992 2. Pirsch JD, D'Alessandro AM, Sollinger HW, et al: The effect of donor age, recipient age, and HLA match on immunologic graft survival in cadaver renal transplant recipients. Transplantation 53:55-59, 1992 3. Meijssen MAC, Heineman E, de Bruin RWF, et al: Long-
term survival of DLA-matched segmental intestinal allografts in dogs. Transplantation 56:1062-1066, 1993 4. Wolvekamp MCJ, Robben SGF, Vermey M, et al: Ultrasonography; A useful tool in the follow-up of small-bowel transplantation in growing dogs. Surg Res Comm 15:183-197, 1994 5. Meijsen MAC, Heineman E, de Bruin RWF, et al: Value of in vivo electrophysiological measurements to evaluate canine small bowel autotransplants. Gut 32:1329-1335, 1991 6. Eberts TJ, Sample RHB, Glick MR, et al: A simplified,
SEGMENTAL INTESTINAL TRANSPLANTATION IN DOGS
colorimetric micromethod for xylose in serum or urine, with phloroglucinol. Clin Chem 25:1440-1443, 1979 7. Van de Kamer JH, Ten Bokkel Huinink H, Weyers HA: Rapid method for the determination of fat in feces. J Biol Chem 177:347-355, 1949 8. Jansen G, Muskiet FAJ, Schierbeek H, et al: Capillary gaschromatographic profiling of urinary? plasma and erythrocyte sugars and polyols as their trimethylsilyl derivatives, preceded by a simple and rapid prepurification method. Clin Chem Acta 157:277293, 1986 9. Romijn JC, Verkoelen CF, Splinter TAW: Species-dependent differences of the biochemical properties of diamine oxidase. Int J Biochem 18:835-839, 1986 10. Tzakis AG, Todo S, Reyes J, et al: Clinical intestinal transplantation: Focus on complications. Transplant Proc 24:12381240, 1992 11. Browne BJ, Johnson CP, Edmiston CE, et al: Transplantation promotes bacterial overgrowth and translocation. J Surg Res 51:512-517, 1991 12. Grant D, Hurlbut D, Zhong R, et al: Intestinal permeability and bacterial translocation following transplantation in the rat. Transplantation 52:221-224, 1991 13. Alexander JW: Nutrition and infection. Arch Surg 121:966972, 1986 14. Moore EE, Jones TN: Benefits of immediate jejunostomy feeding after major abdominal trauma--A prospective, randomized study. J Trauma 26:874-881, 1986 15. Kimura K, Larosa CA, Blank MA, et al: Successful segmental intestinal transplantation in enterectomized pigs. Ann Surg 211:158-164, 1990 16. Quigley EMM, Thompson JS, Rose SG: The long-term function of canine jejunoileal autotransplants--Insights into allograft physiology. Transplant Proc 24:1105-1106, 1992
401
17. Behrens ME, Northrop C, Wraight P, et al: Evaluation of mannitol, lactulose and SlCr-labelled ethylenediaminetetraacetate as markers of intestinal permeability in man. Clin Sci 73:197-204, 1987 18. Nathavitharana KA, Lloyd DR, Raafat F, et al: UrinarY mannitol: Lactul0se excretion ratios and jejunal architecture. Arch Dis Child 63:1054-1059, 1988 19. Wassef R, Cohen Z, Nordgren S, et al: Cyclospori~e absorption in intestinal transplantation. Transplantation 39:496499, 1985 20. Andre C, Descos L, Minaire Y: Comparisons between lactulose-mannitol test and 51Cr-labelled ethylene diamine tetraacetate test in inflammatory bowel diseases. Hepato Gastroentero! 37:1i3-117, 1990 (suppl 2) 21. D'Agostino L, Ciacci C, Daniele B, et al: Postheparin plasma diamine oxidase in subjects with small bowel atrophy: Dig Dis Sci 32:313-317, 1987 22. Rokkas T, Vaja S, Taylor P, et al: Is the intestine the sole source of heparin-stimulated plasma diamine oxidase? Acute effects of jejunectomy, ileoectomy and total enterectomy. Diges~ tion 46:439-446, 1990 (suppl 2) 23. Luk GD, Bayless TM, Baylin SB: Plasma postheparin diamine oxidase. Sensitive provocative test for quantitating length of acute intestinal mucosa injury in the rat. J Clin Invest 71:13081 1315, 1983 : 24. Rose SG, Thompson JS, Spanta AD, et al: The effect of intestina! autotransplantation on serum diamine oxidase activity. ) Surg Res 50:223-227, 1991 25. Rokkas T, Vaja S, Murphy GM, et al: Aminoguanidine blocks intestinal diamine oxidase (DAO) activity and enhances the intestinal adaptive response to resection in the rat. Digestion 46:447-457, 1990 (suppl 2)