Peritoneal Dialysis International, Vol. 17, pp 179-185 0896-8608/97 $300 + 00 Printed in Canada All rights reserved Copyright 1997 International Soeiety for Peritoneal Dialysis CHRONIC ADMINISTRATION OF IRON DEXTRAN INTO THE PERITONEAL CAVITY OF RATS Sang-Eun Park, Zbylut J. Twardowski, Harold L. Moore, Ramesh Khanna, and Karl D. Nolph Division of Nephrology, Department of Medicine, University of Missouri, Harry S. Truman Veterans Administration Hospital, Dalton Research Center, Columbia, Missouri, U.S.A..Objective: To determine the influence of chronic iron dextran administrations into the peritoneal cavity of rats on function and anatomy of the peritoneal membrane, as well as on erythropoiesis and serum iron..design: Prospective randomized animal study..setting: Animallaboratory..Animals: 36 Sprague-Dawley rats..interventions: The rats were divided into three groups (n = 12). The animals were given standard 1.5% Dianeal (control group) or 1.5% Dianeal containing iron dextran in a concentration of 2 mg/l [Iow-dose group (LDG)] or 10 mg/l [high-dose group (HDG)]..Main outcome measures: On the 8th day, at 3 months, and at 6 months a 2-hour peritoneal equilibration test (PET) and blood tests including hematocrit, serum iron, and total ironbinding capacity (TIBC) were done. After the final PET at 6 months, the peritoneal membrane was evaluated by gross inspection and by light microscopy..results: Hematocrit and serum iron levels increased only in the HDG and LDG. Peritoneal transport of small solutes decreased significantly in the HDG compared to baseline. All cases of the HDG group revealed peritoneal adhesions and fibrosis around the peritoneal catheter as well as massive iron deposits on the peritoneum. Similar but less pronounced changes were found in the LDG..Conclusions: These findings suggest an efficient absorption of iron from the peritoneal cavity of rats, however, dialysate iron dextran concentrations of 2 mg/l or greater are toxic to the peritoneal membrane. Therefore, future studies should be performed to determine the minimal effective and nontoxic iron dextran concentrations for intraperitoneal administration. KEY WORDS: Iron dextran; peritoneal fibrosis; iron toxicity; rats. A nemia is one of the most serious complications of end-stage renal disease that often cannot be corrected by dialysis. The use of recombinant human erythropoietin (rhuepo) to treat anemia in end Correspondence to: Z.J. Twardowski, Division of Nephrology, MA 436 Health Sciences Center, University of Missouri, Columbia, Missouri, 65212, U.S.A. Received 3 October 1996; accepted 18 January 1997. stage renal disease has been a major advance in the care of patients receiving hemodialysis or peritoneal dialysis (PD) (1). Such erythropoietin replacement therapy is frequently associated with the development of functional iron deficiency due to the massive transfer ofbone marrow iron stores to erythroid progenitor cells, so erythropoietin replacement therapy requires long-term maintenance therapy with iron, with fre quent monitoring for adequacy of iron stores (2-6). Compared to hemodialysis patients, PD patients are less prone to iron deficiency, however, they do require iron supplementation to compensate for losses of blood through the gastrointestinal tract, menstruation, and phlebotomy for laboratory tests. Available iron delivery routes are intravenous injection (7-9), intramuscular injection (10), and oral admin istration of iron (11,12). Intravenous administration is inconvenient for PD patients because of poor intravenous access. In addition, intravenous iron dextran infusion has been occasionally associated with serious reactions including hypotension, respiratory failure, and anaphylaxis (13,14). Intramuscular iron dextran injection has been suggested, but there have been some reports of development of sarcoma at the injection site after chronic intramuscular iron injection (15,16). Oral administration of iron may be limited by patient non-compliance, poor oral absorption, gastrointestinal side effects, and interactions with other oral medications (11). If the intraperitoneal route of iron delivery were available, it would be much more convenient for the PD patients. Our previous acute studies (17,18) with the 6-hour cycle showed that more than 70% of iron dextran injected into the peritoneal cavity ofrats is absorbed from the peritoneal cavity. There was no evidence of macroscopic change of the peritoneum or microscopic iron deposition on the peritoneum in the acute study. The goals of this chronic study were to determine the influence of chronic iron dextran administrations into the peritoneal cavity of rats on function and anatomy of the peritoneal membrane, serum iron, and hematocrit.
MATERIALS AND METHODS ANIMAL TREATMENT Thirty-six male Sprague-Dawley rats weighing 300-350 g were divided into three groups (n = 12). Group 1 (control group) was given standard 1.5% Dianeal PD-2 (Baxter Health Care Inc., IL, U.S.A.). Group 2 (low-dose group) was given 1.5% Dianeal with 2 mg/l concentration of iron dextran. Group 3 (high-dose group) was given 1.5% Dianeal with 10 mg/l concentration of iron dextran. For the experimental, low-, and high-dose groups the iron in the form of iron dextran injection (InFeD, Schein Pharmaceutical, Florham Park, NJ, U.S.A.) was added to the dialysis solution. Iron dextran is available in 2-mL ampules containing 50 mg/ml elemental iron. The solution contains approximately 20% dextran with molecular weight 160 to 180 KD. For the low-dose group, 50 μg of elemental iron was added to 25 ml of dialysis solution (2 mg/l). For the high-dose group, 250 μg of elemental iron was added to 25 ml of dialysis solution (10 mg/l). The iron was delivered by infusion of the dialysis solution through an intraperitoneal catheter. CATHETER (19) IMPLANTATION The animals were placed supine on a 37 C heating pad in a large jar and anesthetized with methoxy flurane (Metofane, Mallinckrodt Veterinary, Mundelein, IL, U.S.A.). The anesthesia was main tained with a nose cone. The abdomen was shaved and disinfected with Amukin (Amuchina, Genova, Italy). The area was then covered with Tegaderm (3M Corporation, St. Paul, MN, U.S.A). An incision was made in the skin along the midline starting at the xiphoid process and extending caudally approximately 3 cm. A blunt dissection was then done on the midline through the abdominal wall approximately 0.75 to 1.5 cm below the xiphoid process. Once the cavity was penetrated, the tip of the catheter was advanced into the cavity and secured with a single stitch through the cuff material and the superficial muscle layer. The procedure was performed carefully to avoid bleeding and trauma to the peritoneum. The hole into the peritoneum was large enough to allow the catheter to pass through but small enough that the catheter fitted snugly. With the use of the 3 mm trocar, the other end of the catheter was tunneled under the skin to the point near the animal's scapula that had been shaved and disinfected. The trocar was then forced through the skin and the catheter pulled through the tunnel. When the second cuffwas next to the exit hole, the catheter was trimmed leaving approximately 1.5 to 2.0 cm exposed. The catheter was capped. The animals were allowed to wake up, placed again in their cages, and allowed free access to food and water. The animals were given buprenorphine subcutaneously for analgesia with the dose adjusted as needed. This analgesic therapy was continued for 72 hours post surgery. INTRAPERITONEAL DIALYSIS SOLUTION ADMINISTRATIONS Twenty-five ml of either the control or one of the experimental dialysis solutions was administered into the peritoneal cavity through the catheter each morning, 7 daysa-week, for 6 months. The first administration was done on the day of catheter implantation. The nonabsorbed dialysate was drained each morning before the next administration and cultured for bacterial growth. Every effort was made to avoid system contamination. TREATMENT OF PERITONITIS Diagnosis of peritonitis was based on culture results (more than 10 colonies on a blood agar plate). Upon diagnosis ofperitonitis, the animal was treated intraperitoneally with a loading dose of vancomycin (1 g/l) and gentamicin (1.7 mg/kg), followed with a maintenance dose of 30 mg/l and 8 mg/l, respectively, per exchange for one week. PERITONEAL MEMBRANE FUNCTION AND BLOOD TESTS On the eighth day, at 3 months, and at the last exchange (6 months) the animals were anesthetized, as outlined above, 2 hours after the instillation of the dialysis solution. After a 2-hour dwell, a 5 ml dialy sate sample was collected through the catheter and sent for chemistry and bacteriology analysis. Imme diately thereafter, a 2 ml sample of blood was taken by direct cardiac puncture for hematocrit, serum iron, and total iron-binding capacity (TIBC). In the case of peritonitis, the dialysate and blood samples were deferred until the membrane inflammation had subsided, that is, 2 days after an antibiotic had been discontinued. Peritoneal equilibration tests (PET) were performed by measuring glucose concentration in the fresh dialysis solution (DO) and dialysate after a 2-hour dwell (D). Glucose concentrations in the samples were determined by an autoanalyzer ASTRA-8 (Beckman Instruments, Brea, CA, U.S.A.). LABORATORY TESTS Serum iron, TIBC, and hematocrit were determined on blood samples obtained through cardiac
puncture on the eighth day, at 3 months, and at 6 months. Serum Fe and TIBC were determined by a colorimetric procedure using a commercial iron and total ironbindingcapacity kit (Sigma Chemical company, St. Louis, MO, U.S.A.). TISSUE SAMPLING After the final PET at 6 months, the abdomen was opened, the solution completely drained, and the peritoneal membrane was evaluated by gross inspection and by light microscopy after staining with Prussian blue. Tissue samples were taken from the small intestine, mesentery, and 3 regions of the abdomen, both lateral walls, and the diaphragm. The study was terminated before 6 months in animals with poorly-functioning or nonfunctioning catheters, but tissue samples were obtained for pathology at that time. The catheter was considered to be poorlyfunctioning if fluid could be infused into the peritoneal cavity, but could not be drained (one-way obstruction). In nonfunctioning catheters, the fluid could be neither infused nor drained (two-way obstruction). STATISTICAL ANALYSIS The results of this chronic study were analyzed by looking at the quantitative morphological changes in the peritoneum, including adhesions, fibrosis, and iron deposition. Regarding PET and laboratory data, simple t- tests for unpaired comparisons were performed by a Sigmastat program. One way ANOV A and the Student- Newman-Keuls method were also applied. Variables are shown as mean ± SD; p < 0.05 was considered statistically significant. RESULTS Table 1 shows hematocrit, serum iron, and TIBC at baseline, at 3 months, and at 6 months of study. The mean hematocrit was significantly higher than at baseline in the high-dose group at 3 months (47.9 ± 1.5 vs 43.0 ± 2.7; p < 0.05) and at 6 months (48.4 ± 1.8 vs 43.0 ± 2.7; p < 0.05). At 6 months, the mean hematocrits were significantly higher in the high-dose and low-dose groups compared to the control group (48.4 ± 1.8,46.4 ± 0.9, and 44.2 ± 1.4, respectively; p < 0.05). There was also a significant difference in hematocrit between the high-dose group and the low-dose group at 6 months (48.4 ± 1.8 vs 46.4 ± 0.9). Finally, at 6 months, the hematocrit was higher in the low-dose group compared to baseline (46.4 ± 0.9 vs 44.1 ± 2.3; p < 0.05). There were no significant differences in the mean serum iron among the three groups at baseline. Serum iron levels were significantly higher in the high-dose group at 3 and 6 months, compared to baseline and the control group. At 3 months, serum iron in the high-dose group was also significantly higher than in the low-dose group. In the lowdose group, serum iron was significantly higher than in the control group at 3 and 6 months. In the control At baseline, body weights in grams (mean ± SD) were 362 ± 21.7,332.7 ± 36.1, and 335.8 ± 21.5 in control, low-dose, and high-dose groups, respectively. The differences were not significant. Seven, eight, and three rats had functioning catheters at 3 months in the control, low-dose, and highdose groups, respectively. At 6 months, the number of functioning catheters dropped to five, six, and three in these groups, respectively. The animals with poorly functioning or nonfunctioning catheters were sacrificed and tissue samples taken for pathology. In the control group, five rats were sacrificed by the third month and two more by the sixth month; in the lowdose group, four rats were terminated by the third month and two mo re by the sixth month. In the highdose group, nine rats were sacrificed by the third month. All other animals completed 6 months of study.
group compared to baseline, serum iron was significantly lower at 3 months but was not significantly different at 6 months (Table 1). There was no significant difference in TIBC at baseline among the three groups. Although mean TIBC was increased in the low-dose group at 3 months, there was no significant difference among the three groups at 6 months (Table 1). Peritoneal transport properties were assessed by calculating the ratio of dialysate glucose after a 2hour dwell (D) to glucose concentration in dialysis solution (DO). The higher the ratio the lower the transport of small solutes. Baseline 2-hour DIDO ratios of glucose were 0.24, 0.26, 0.23 in the control, low-dose, and high-dose groups respectively. This is consistent with other PD studies in rats. There were no significant differences in glucose DIDO ratios at 2hour dwell among the groups at baseline. Mean DIDO ratios of glucose were significantly increased at 3 months compared to baseline in all groups (p < 0.05). Compared to the other groups, the DIDO ratio of glucose was higher in the high-dose group at 6 months (p < 0.05). At 6 months, DIDO glucose ratios after a 2-hour dwell in the high-dose, low-dose, and the control groups were 0.38 ± 0.10,0.33 ± 0.07, and 0.25 ± 0.12, respectively (Table 2). At 6 months, one case (20%) in the control group and the three cases (50%) in the low-dose group revealed adhesions of the peritoneum, while all cases (n = 3) of the high-dose group revealed adhesions. Fibrosis around the catheter was noted in one case (20% ) in the control group, two cases (33% ) in the lowdose group, and in all cases (100%) in the high-dose group. Compared to the control and low-dose groups, which didn't show any pigmentation of the peritoneum, two cases in the high-dose group showed dark brown pigmentation on the peritoneum of the mesentery and the abdominal wall by gross inspection (Table 3, Figure 1). Pericatheter fibrosis was found in all rats sacrificed before the end of the study and was the reason for catheter failure. At 6 months, Prussian blue staining of the peritoneum did not reveal any iron deposits in the control group. In the low-dose group at 6 months, there were numerous iron particles in the peritoneum of the abdominal wall and mesentery (Figure 2). In the high-dose group at 6 months, there were massive iron deposits in the peritoneum of the mesentery and abdominal wall (Figure 3). Dark spots, visible macroscopically as brown pigmentation on the peritoneal membrane, were produced by massive iron aggregates, as revealed microscopically after Prussian blue staining (Figure 4). Mild iron deposits were found in the high-dose group animals sacrificed at 1.5 months, but no such deposits were seen in the control and low-dose groups at 1.5 and 3 months. The incidence of peritonitis was 0.55Imonth/rat in the control group, 0.56Imonth/rat in the low-dose group and 0.52Imonth/rat in the high-dose group. There was no significant difference of incidence of peritonitis among the groups.
DISCUSSION Recombinant human erythropoietin (rhuepo ) has been associated with subjective and objective improvements in quality-of-life in patients with anemia of chronic renal failure (1). However, resistance to rhuepo therapy may occur due to insufficient iron stores. Although many patients treated with rhuepo take oral iron, patients continue to develop iron deficiency (20). Parenteral iron is used as an effective alternative for replacing iron stores to sustain erythropoiesis in hemodialysis patients treated with rhuepo (10). In PD patients, intravenous iron administrations are inconvenient; an intraperitoneal route of iron delivery would be much more convenient. Iron dextran (InFeD) contains ferric iron combined with dextran which has a molecular weight of 160 to 180 KD. It is commercially available and is widely used for dialysis patients, in spite of known anaphylactoid reactions to dextran (13,21). Hematocrit increased in those groups that were administered solutions containing iron dextran. Mean serum iron levels increased markedly at 3 and 6 months in the high-dose group and at 6 months in the low-dose group. While serum iron levels decreased significantly at 3 months in both the low-dose and control groups, this may be due to blood sampling by cardiac puncture. TIBC increased at 3 months in the low-dose group, but there was no significant difference in TIBC among the groups at 6 months. In this chronic study, these results suggest that the improved erythropoiesis observed with both the low and high-dose groups, when compared with the control group, was most likely due to an improvement in availability of iron induced by intraperitoneal iron administration. We investigated the effect of chronic intraperitoneal iron administration on the peritoneum using the peritoneal equilibration test in rats. The D/DO ratio increased significantly from baseline in all the groups at 3 months. Compared to the DIDO ratio of the control group at 6 months, which returned back to baseline level, the D/DO ratio of the high-dose group was still high at 6 months. An increased D/DO ratio of glucose means that the transport of glucose is reduced. This indicates a decrease in the effective peritoneal surface area or permeability. Based on the DIDO ratio, peritoneal function was poor in the highdose group compared to the control group at 6 months. These results indicate that chronic intraperitoneal administration of iron dextran affects the function of peritoneum. Gross inspection of the peritoneum showed striking alterations in both the low and high-dose groups. While only one case in the control group revealed adhesions of the peritoneum and fibrosis around the
catheter, all cases in the high-dose group showed adhesions and fibrosis around the catheter. Brown pigmentation, resulting from deposition of iron, was noted in the highdose group only. This means that chronic intraperitoneal administrations of a high dose of iron dextran can cause anatomical changes including adhesion, fibrosis, and iron deposition. Histological findings confirmed massive iron deposits on the peritoneum of the mesentery and on the parietal peritoneum, especially in the high-dose group. Chronic intravenous or intraperitoneal administration of iron dextran can damage some organs including the peritoneum. The main target organs of iron deposition are the liver, bone marrow, the spleen, and other tissues (22). Schwartz et al. studied a guinea pig model ofiron-overload toxicity induced by intraperitoneal iron dextran administration (23). They found marked iron deposition including iron particles and aggregates in the liver, heart, and bone marrow. Mild iron deposition was noted in our study in the high-dose group at 1.5 months and in the lowdose group at 6 months. The total amount of iron dextran administered was 9 mg for 6 months in the low-dose group and 11 mgfor 1.5 months in the highdose group. The total amo unt of iron dextran was 45 mg for 6 months in the high-dose group which revealed marked iron aggregates of iron particles. These facts support the hypothesis that the degree of iron deposition is directly proportional to the amount of iron dextran administered intraperitoneally. In this chronic study, more than half of the animals were terminated early due to poor catheter function. The major cause of poor catheter function was frequent peritonitis leading to peritoneal adhesions. The other possible cause of poor catheter function seemed to be due to the development of peritoneal adhesions related to the high concentration of intraperitoneal iron dextran. In conclusion, this study suggests that iron dextran is efficiently absorbed from the peritoneal cavity; dose-related development of adhesions and peritoneal iron deposits indicate that the concentration of iron in the peritoneal cavity should be lower than 2 mg/l. Future studies are needed to determine the optimal intraperitoneal iron dextran concentration: high enough to deliver a sufficient amount ofiron, yet low enough to avoid peritoneal toxicity. ACKNOWLEDGMENT Supported in part by a grant fromamgen, Inc. (EPO 94-033). REFERENCES 1. Eschbach JW, Egrie JC, Downing MR, Browne JK, Adamson JW. Correction of anemia of end-stage renal disease with recombinant human erythropoietin: results of a combined phase I and II clinical trial. N Engl J Med 1987; 316:73-8. 2. Van Wyck DB, Stivelman JC, Ruiz J, Kirlin LF, Katz MA, Ogden DA. Iron status in patients receiving erythropoietin for dialysis-associated anemia. Kidney Int 1989; 35:712-6. 3. Anastassiades EG, HowarthD, HowarthJ,etal. Monitoring ofiron requirements in renal patients on erythropoietin. Nephrol Dial Transplant 1993; 8:846-53. 4. Macdougall IC, Cavill I, Hulme B, et al. Detection of functional iron deficiency during erythropoietin treatment: a new approach. Br Med J 1992; 304:225-6. 5. Sunder-Plassmann G, Horl WH. Iron metabolism and iron substitution during erythropoietin therapy. Clinical Investigator 1994; 72(Suppl 6):Sll-15. 6. York S. Current perspectives: iron management during therapy with recombinant human erythropoietin. ANNA J 1993; 20:645-50. 7. Burns DL, Mascioli EA, Bistrian BR. Parenteral iron dextran therapy: a review. Nutrition 1995; 11:163-8. 8. RosenlofK, Kivivuori SM, Gronhagen-Riska C, Teppo AM, Slimes MA. Iron availability is transiently improved by intravenous iron medication in patients on chronic hemodialysis. Clin Nephrol 1995; 43:249-55. 9. Fishbane S, Frei GL, Maesaka J. Reduction in recombinant human erythropoietin doses by the use of chronic intravenous iron supplementation. Am J Kidney Dis 1995; 26:41-6. 10. Suh H, Wadhwa NK. Iron dextran treatment in peritoneal dialysis patients on erythropoietin. In: Khanna R, Nolph KD, Prowant BF, Twardowski ZJ, Oreopoulos DG, eds. Advances in peritoneal dialysis. Toronto: Peritoneal Dialysis Bulletin 1992; 8:464-6. 11. Wingard RL, Parker RA, Ismail N, Hakim RM. Efficacy of oral iron therapy in patients receiving recombinant human erythropoietin. Am J Kidney Dis 1995; 25:433-9. 12. Raja R, Bloom E, Johnson R. Comparative effects of erythropoietin with oral iron in peritoneal dialysis and hemodialysis patients. In: Khanna R, Nolph KD, Prowant BF, Twardowski ZJ, Oreopoulos DG, eds. Advances in peritoneal dialysis. Toronto: Peritoneal Dialysis Publications 1993; 9:177-80. 13. Michelson E. Anaphylactoid reaction to dextran. N Engl J Med 1968; 278:552. 14. Hamstra RD, Block MH, Schocket AL. Intravenous iron dextran in clinical medicine. JAMA 1980; 243:1726-31. 15. MacKinnon AE, Bancewicz J. Sarcoma after injection of intramuscular iron. Br Med J 1973; 2:277-9. 16. Grasso P. Sarcoma after intramuscular iron injection. Br Med J 1973; 2:667. 17. Suzuki K, Twardowski ZJ, Nolph KD, Khanna R, Moore HL. Absorption of iron from the peritoneal cavity ofrats. In: Khanna R, ed. Advances in peritoneal dialysis. Toronto: Peritoneal Dialysis Publications 1994; 10:42-3. 18. Suzuki K, Twardowski ZJ, Nolph KD, Khanna R, Moore HL. Absorption of iron dextran from the peritoneal cavity ofrats. In: Khanna R, ed. Advances in
peritoneal dialysis. Toronto: Peritoneal Dialysis Publications 1995; 11:57-9. 19. Moore HL. A chronic catheter model for PD exchanges in the ambulatory rat.perit Dial Int 1992; 12(Suppl1): S141. 20. Domoto DT, Martin KJ. Failure of CAPD patients to respond to an oral iron absorption test. In: Khanna R, Nolph KD, Prowant BF, Twardowski ZJ, Oreopoulos DG, eds. Advances in peritoneal dialysis. Toronto: Peritoneal Dialysis Bulletin 1992; 8:102-4. 21. Fleming LW, Stewart WK, Parratt D. Dextran anti bodies, complement conversion and circulating immune complexes after intravenous iron dextran therapy in dialysed patients. Nephrol Dial Transplant 1992; 7:35-9. 22. Carthew P,EdwardsRE, SmithAG, DormanB, Francis JE. Rapid induction of hepatic fibrosis in the gerbil after the parenteral administration of iron dextran complex. Hepatology 1991; 13:534-9. 23. Schwartz KA, Fisher J, Adams ET. Morphological investigations of the guinea pig model ofiron overload. Toxicol Pathol1993; 21:311-20.