158 Ann Thorac Surg 45: , Feb Copyright by The Society of Thoracic Surgeons

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1 Value of Urinary Polyamines as Noninvasive Markers of Cardiac Allograft Rejection in the Dog Michel Carrier, M.D., Diane H. Russell, Ph.D., Thomas P. Davis, Ph.D., Robert W. Emery, M.D., and Jack G. Copeland, M.D. ABSTRACT A noninvasive marker of cardiac allograft rejection would be useful clinically. Lymphocyte proliferation and organ rejection may cause changes in urinary polyamine excretion. To test this hypothesis, cervical heterotopic heart transplantations were performed in a group of 6 nonimmunosuppressed dogs and in a group of 9 dogs treated with cyclosporine (N = 3) or cyclosporine and steroids (N = 6). A group (N = 3) having a sham operation was also studied. Serial biopsies of the transplanted hearts were performed. Urinary polyamine levels were measured daily by high-pressure liquid chromatography of urine specimens. Between 2 and 4 days after transplantation, the transplanted hearts of all animals without immunosuppression demonstrated histological rejection. An early increase in putrescine levels and in total urinary polyamine levels was observed in this group. In the treated groups, histological rejection appeared from the second to the eighth day after transplantation. Each episode of rejection occurred from 1 day to 4 days after a significant increase in urinary polyamine levels compared with the preoperative baseline level (p < 0.01). In contrast, polyamine excretion in 3 dogs after sham operations remained unchanged. Thus, urinary excretion of polyamines increases before the appearance of histological rejection; this suggests that changes in urinary polyamine levels may be a useful marker of cardiac allograft rejection. The diagnosis of cardiac allograft rejection is currently based on the results of serial endomyocardial biopsy and histological evaluation of the allograft [l, 21. This technique is used regularly during the early period after transplantation to monitor the immunological response of the recipient to the allograft. Despite excellent clinical results obtained with this method introduced by Caves and colleagues [3], many difficulties persist [4-61. A noninvasive method providing a daily evaluation of the recipient s immunological status would be extremely useful clinically. The measurement of polyamine excretion as a marker of the rejection process offers many of the advantages of an ideal noninvasive approach [7]. Intracellular poly- From the Departments of Cardiovascular and Thoracic Surgery and of Pharmacology, University of Arizona, Tucson, AZ. Presented at the Twenty-third Annual Meeting of The Society of Thoracic Surgeons, Toronto, Ont, Canada, Sept 21-23, Address reprint requests to Dr. Carrier, Montreal Heart Institute, 5000 E Belanger St, Montreal, Que, Canada H1T 1C8. amines are compoynds related to growth and differentiation of cells [S]. Their biosynthesis is intimately associated with the modulation of the regulatory process related to deoxyribonucleic acid and ribonucleic acid metabolism [9]. Polyamine accumulation occurs after cellular stimulation, and urinary levels reflect cellular proliferation or degeneration or both in a variety of pathological disorders [lo]. Putrescine and spermidine are the two major amines used as urinary markers of cellular metabolic activity. Putrescine is excreted as a function of the number of cells in the proliferative state, and spermidine is excreted during proliferation and is liberated from dead cells [8]. Based on observations made from human tissue culture and during lymphocyte transformation, there is a theoretical possibility that polyamines may serve as markers of lymphocyte proliferation and organ rejection [ll-131. As lymphocytes proliferate in response to antigenic stimulation, intracellular polyamines are released into the bloodstream and ultimately are filtered into the urine (Fig 1). Therefore, alterations in urinary polyamine levels may be a predictive factor of rejection by disclosing cellular proliferation in the immune system. To test this hypothesis, cervical heterotopic heart transplantations were performed in immunosuppressed and nonimmunosuppressed animals. Material and Methods Cervical heterotopic heart transplantation was performed in 15 dogs. The hearts of donor animals weighing 8 to 12 kg were excised and preserved with topical hypothermia. Simultaneously, in male recipients weighing 15 to 25 kg, the left carotid artery and jugular vein were exposed through a longitudinal neck incision. An end-to-side anastomosis was performed between the donor pulmonary artery and recipient jugular vein and between the donor aorta and recipient carotid artery. Total time of ischemia did not exceed 45 minutes. Transcutaneous electrocardiographic leads were placed on the right atrium and ventricle. Plastic biopsy targets were sutured on the surface of the left ventricle away from major coronary vessels. The heart was placed in a cervical subcutaneous pocket. Needle myocardial biopsies were done through these defined areas. A group of 6 nonimmunosuppressed animals was monitored until arrest of allograft heart activity, at which time the animals were killed and the heterotopic hearts were excised and examined microscopically. A group of 9 animals was immunosuppressed with cyclosporine (20 mg per kilogram of body weight per day orally) (3 animals), or cyclosporine (20 mg/kg/day orally) with 158 Ann Thorac Surg 45: , Feb Copyright by The Society of Thoracic Surgeons

2 159 Carrier et al: Urinary Polyamines as Markers of Cardiac Allograft Rejection Ornithine Putrescine Spermidine Spermine 5At.l Liver Conjugation I I Kidney Excretion Urinary Polyamines Putrescine Acefylputrescine Acetylspermidine Acetylspermine Spermine Cadaverine Cellular Growth and Differentiation Fig 1. Intracellular biosynthesis and urinary excretion of polyamines. (ODC = ornithine decarboxylase; SAM = S-adenosylmethionine.) methylprednisolone (15 mg/kg intravenously) at the end of the operation and prednisone (tapering from 2 mg/kg/ day to 1 mg/kg/day orally) (6 dogs) until 14 days after transplantation. At 14 days, the immunosuppressive drugs were stopped, and the animals were killed a few days later after arrest of the allograft heart. No attempt was made to treat the acute rejection episodes, and cyclosporine doses were not modified according to the blood level. A third group of 3 animals had a sham cervical operation and was placed on the same protocol as the animals in the first group (no immunosuppression). Urine specimens for polyamine measurements were collected every morning in the first and third groups, and twice a day (every twelve hours) in the immunosuppressed group. Specimens were obtained by urethral catheterization or from spontaneous micturition, and were stored at - 80 C until assayed. Urinary polyamine levels were measured using high-pressure liquid chromatography [14]. Determination of putrescine, spermidine, N1-acetylspermidine, N8-acetylspermidine, cadaverine, and total polyamine content in the urine was performed. N-acetylputrescine does not appear in substantial quantities as an acetyl derivative in the urine of dogs and therefore was excluded from the analysis. Daily ECGs were recorded using the two myocardial leads, and QRS voltage was measured. Allograft biopsies were performed every day for the first group and every other day for the second group of animals. Biopsy specimens were stained with hematoxylin-eosin, trichrome, and methyl green-pyronine. Blood samples for cyclosporine trough levels were obtained every day in the treated group. The levels were determined by radioimmunoassay. This protocol was reviewed and approved by the University of Arizona Laboratory Animal Care Committee. All animals received humane care in compliance with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH Publication No , revised 1985). Statistical comparisons were made using the Student t test. Significance level was determined at a probability of less than Results are expressed as the mean k the standard error of the mean. Results Mean survival of the transplanted hearts was 8 k 1 days in the group without immunosuppression and 15.5 k 1 days in the immunosuppressed animals. Two dogs died of acute rejection before the end of the 14-day course of immunosuppressive therapy. Between 2 and 4 days after transplantation, all transplanted hearts in animals without immunosuppression demonstrated histological rejection characterized by perivascular and interstitial mononuclear cell infiltration [l]. An early increase in putrescine and total urinary polyamine levels was observed during the second to the sixth postoperative day in this group (Fig 2). The increased putrescine level was responsible for the increase in total urinary polyamine excretion during these periods. Histological rejection appeared from the second to the sixth postoperative day in animals immunosuppressed with cyclosporine alone (Fig 3) and from the fourth to the eighth day after transplantation in animals on a regimen of cyclosporine and prednisone (Fig 4). Rejection as determined by histological criteria progressed in both groups from the described mild form to an extremely severe form immediately before death of the allograft. High putrescine and total polyamine values were observed in the early posttransplantation period prior to the appearance of mild histological rejection in all immunosuppressed animals. Rejection episodes occurred from 1 day to 4 days after total polyamine excretion increased to an average of 30 k 4.7 nmol/mg of creatinine in the nonimmunosuppressed group, 24.2 k 1.4 nmoy mg in the cyclosporine-treated group, and 53.8 k 9.4 nmoymg in the cyclosporine-prednisone-treated group. All these changes were significantly different ( p < 0.01) from preoperative baseline values for each of the groups (Fig 5). Three animals were followed before and after a sham cervical operation. They did not show any significant variation in polyamine excretion (Fig 6). Decreases in QRS voltage on the ECG during the postoperative course of all animals undergoing transplantation reflected the histological changes observed on biopsy specimens (Fig 7). Cyclosporine trough levels were extremely variable, but fell rapidly at the end of treatment periods (Fig 8). Comment Urinary excretion of polyamines increased prior to histological rejection in all animals receiving a transplant (see Figs 2-4). This increase in polyamine excretion is postulated to reflect lymphocyte proliferation and differentiation in response to antigenic stimulation. Lymphocyte transformation increases markedly within 24 hours of allograft transplantation in untreated animals, thereby reflecting early postimmunological reactivity against the graft [15]. An elevation of activated

3 ~ The Annals of Thoracic Surgery Vol 45 No 2 February 1988 I - N-6 *p< PUTRESCINE I TOTAL POLYAMINES REJECTION Fig 2. Urinary polyamine excretion before and after transplantation in animals without immunosuppression. The longitudinal bar represents histological rejection. At 4 days after transplantation, all animals demonstrated histological rejection, W I * I T N=3 PUTRESCINE TOTAL POLYAMINES *p<0.05 REJECTION Fig 3. Urinary polyamine excretion before and after transplantation in animals immunosuppressed with cyclosporine alone. The longitudinal bar represents histological rejection. All animals demonstrated histological rejection within 6 days after transplantation *.r N-6 PUTRESCINE - TOTAL POLYAMINES * = REJECTION I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I Fig 4. Urinary polyamine excretion before and after transplantation in animals immunosuppressed with cyclosporine and steroids. The longitudinal bar represents histological rejection. All animals demonstrated histological rejection within 8 days after transplantation.

4 161 Carrier et al: Urinary Polyamines as Markers of Cardiac Allograft Rejection W PUTRESCINE +p<0.05 TOTAL POLYAMINES TRANSPLANT B=PRE-REJECTION * r A B A B A B NO CYCLOSPORINE CYCLOSPORINE IMMUNOSUPPRESSION CN-3) AND CN-6) PREDNISONE CN-6) Fig 5. Polyamine peaks I day to 4 days before detection of histological signs of rejection. % 16- H z mh 62 ZE WI- 12- > 8- -la 0s a- : 4- C PUTRESCINE TOTAL POLYAMINES I I Fig 6. Urinary excretion of polyamines before and after sham cervical operat ions TREATED GROUP CN-9) NO IMMUNOSUPPRESSION CN-6). T T Fig 7. Changes in QRS voltage from ECG of animals undergoing transplantation.

5 162 The Annals of Thoracic Surgery Vol 45 No 2 February 1988 A W J 600- Wn 500- z\ =r %w N= J u 100- >- u 0 I Fig 8. Cyclosporine trough levels in the plasma of immunosuppressed animals. lymphocytes and lymphoblasts in the peripheral blood occurs prior to rejection [16, 171, and urinary polyamine excretion follows this pattern of cellular proliferation and differentiation. The immunosuppression used in the present study was ineffective in preventing histological acute rejection, and did not modify the baseline excretion of polyamines compared with the animals undergoing transplantation without immunosuppression. Cyclosporine was administered orally in this experiment, and absorption 'of the drug is known to vary markedly. Furthermore, toxic levels of the drug may have contributed to the gastrointestinal symptoms observed and to the decreased absorption [MI. The highest polyamine values were observed in the group with cyclosporine and steroids. Despite immunosuppressive therapy, an elevation in polyamine excretion was documented as early as the first 2 days after transplantation while histological rejection appeared a few days later. The peak value of polyamine excretion preceding histological rejection by 1 day to 4 days was higher in animals receiving cyclosporine and steroids than in dogs given cyclosporine alone or those with no immunosuppression. This might represent a steroid effect on metabolic activity and cellular turnover. The stable excretion of polyamines after the sham operations supports our hypothesis that polyamine elevation in the immediate posttransplantation period is related to lymphocyte reactivity against the graft, since the surgical trauma did not affect polyamine excretion. The decrease in QRS voltage on the ECG corroborated the histological findings of early rejection. The voltage decreased rapidly after transplantation in the animals without immunosuppression and more slowly in the treated animals. This study demonstrates that urinary polyamine excretion increases before the histological appearance of mild rejection in both immunosuppressed and nonimmunosuppressed animals following heterotopic heart transplantation. The high polyamine excretion level prior to rejection is postulated to represent increased cellular metabolic activity of the immune system preceding allograft rejection. Polyamine excretion is also influenced by the immunosuppressive protocol used, and steroids may be at least partly responsible for the variations in polyamine changes prior to rejection. Increases in polyamine excretion were observed in all animals undergoing transplantation, whereas no changes occurred in the group having sham operations. This suggests that urinary polyamine assay may be a useful adjunct in the noninvasive approach to the monitoring of allograft rejection. This approach deserves further animal and clinical studies to determine its potential value as a marker for human allograft rejection and as a determinant of cellular metabolic activity of the immune system. References 1. Billingham ME: Diagnosis of cardiac rejection by endomyocardial biopsy. J Heart Transplant 1:25, Copeland JG, Emery RW, Levinson MM, et al: Cyclosporine: an immunosuppressive panacea? J Thorac Cardiovasc Surg 91:26, Caves PK, Stinson EB, Billingham ME, et al: Diagnosis of human cardiac allograft rejection by serial cardiac biopsy. J Thorac Cardiovasc Surg 66:461, Novitzky D, Rose AG, Cooper DKC, Reichart B: Histopathologic changes at the site of endomyocardial biopsy: potential for confusion with acute rejection. J Heart Transplant 5:79, Sibley RK, Olivari MT, Bolman RM, Ring WS: Endomyocardial biopsy in the cardiac allograft recipient: a review of 570 biopsies. Ann Surg 203:177, Haverich A, Scott WC, Dawkins KD, et al: Asymmetric pattern of rejection following orthotopic cardiac transplantation in primates. J Heart Transplant 3:280, Womble JR, Larson DF, Copeland JG, Russell DH: Urinary polyamine levels are markers of altered T lymphocyte proliferationfloss and rejection in heart transplant patients. Transplant Proc , Russell DH, Dune BGM Polyamines as biochemical markers of normal and malignant growth. Russell DH, Dune BGM (eds): Progress in Cancer Research Therapy. New York, Raven, 1978, Vol. 8, pp 1-13

6 163 Carrier et al: Urinary Polyamines as Markers of Cardiac Allograft Rejection 9. Williams-Ashman HG, Canellakis ZN: Polyamines in mammalian biology and medicine. Perspect Biol Med 22421, Dune BGM, Salmon SE, Russell DH: Polyamines as markers of responses and disease activity in cancer chemotherapy. Cancer Res 37214, Menashe M, Faber J, Bachrach U: Formation of N- acetylputrescine and N1-acetylspermidine in cultured human lymphocytes. Biochem J 188:263, Morton LJ, Graziano KD, Mardiney MR, Russell DH: Specific increases in polyamines in mixed lymphocyte reactions. In Russell DH (ed): Polyamines in Normal and Neoplastic Growth. New York, Raven, 1973, pp Kay JE, Lindsay VJ: Polyamine synthesis during lymphocyte activation. Exp Cell Res 77428, Seiler N, Knodgen B: Determination of polyamines and related compounds by reversed-phase high-performance liquid chromatography. J Chromatogr 339:45, Tennenbaum JI, St-Pierre RL, Vasko JS: Early detection of canine heart allograft rejection. Arch Surg 99:753, Ertel W, Reichenspurner H, Hammer C, et al: Immunologic monitoring in dogs after allogenic heterotopic heart transplantation. J Heart Transplant 3268, Ertel W, Reichenspumer H, Lersch C, et al: Cytoimmunologic monitoring in acute rejection and viral, bacterial or fungal infection following transplantation. J Heart Transplant 4:390, Painvin GA, Heimbecker RO, Keown PA, et al: The side effects of cyclosporin A clinical and histologic finding following 31 canine heterotopic cardiac allotransplantations. Curr Surg Mar/Apr:120, 1983 Notice from the American Board of Thoracic Surgery The Part I (written) examination will be held at the Hiatt- Regency, DallasRort Worth Airport, Dallas, TX, in February, The closing date for registration is August 1, To be admissible for the Part I1 (oral) examination, a candidate must have successfully completed the Part I (writ ten) examination. A candidate applying for admission to the certifying examination must fulfill all the requirements of the Board in force at the time the application is received. Please address all communications to the American Board of Thoracic Surgery, One American Plaza, Suite 803, Evanston, IL