J Thorac Cardiovasc Surg 1994;107:482-0486
© 1994 Mosby, Inc.
Cardiac and Pulmonary Transplantation
From Oregon Health Sciences University, Division of Cardiopulmonary Surgery, Portland, Ore.
Received for publication March 12, 1993. Accepted for publication July 7, 1993. Address for reprints : Angelo A. Vlessin, MD, Phd, Oregon Health Sciences University, L223A, 3181 S.W. Sam Jackson Park Rd., Portland, OR 97201.
Purine efflux from transplanted human cardiac allografts was investigated as a potential biochemical correlate to graft preservation and eventual function. Coronary sinus effluent from 14 allografts was sampled at 1, 5, 10, 15, 20, and 25 minutes after reperfusion. The plasma fraction from each sample was analyzed for hypoxanthine, xanthine, urate, inosine, and adenosine by high-performance liquid chromatography. Total organ preservation time, aortic crossclamp and bypass times, and initial cardiac index off bypass were recorded. An inotropic score was calculated from the dosages of inotropic agents each recipient required immediately after transplantation. Inosine and adenosine were not detectable in the coronary sinus effluent at any time during reperfusion. Hypoxanthine concentration rose sevenfold (p < 0.001) 1 minute after reperfusion. Xanthine concentration peaked later at 5 minutes after reperfusion, a twofold increase (p < 0.02). As reperfusion continued, hypoxanthine and xanthine concentrations returned toward baseline levels. The rise in coronary sinus xanthine concentration provides evidence for hypoxanthine degradation by xanthine oxidase during the immediate reperfusion period. The extent of hypoxanthine efflux correlated with total graft ischemic time (p < 0.05), inotropic score (p < 0.005), and the time from crossclamp release to cessation of bypass (p < 0.01). Hypoxanthine efflux can be used as a sensitive and objective biochemical indicator of graft preservation and immediate function. (J THORAC CARDIOVASC SURG 1994;107:482-6)
Several different cardiac preservation solutions are currently in use throughout the world. All these solutions strive to accomplish one task, that is, to preserve cellular integrity during the storage period.
The most successful solutions are used cold and possess an ionic composition simulating that of the intracellular milieu. During the storage period, the donor myocardium remains completely anoxic. Without oxygen, mitochondrial functions halt and adenosine triphosphate (ATP) cannot be regenerated from adenosine diphosphate via oxidative phosphorylation. By equalizing the ionic distribution across the cell membrane, the preservation solution decreases the activity of the ATP-driven ion pumps in the plasma membrane. The high potassium concentration depolarizes the myocardium and stops ATP-depleting muscle contraction. Cooling has a profound impact on organ preservation by decreasing the basal metabolic rate and further diminishing the need for ATP. 1 Therefore, from a biochemical perspective, this organ-storage regimen minimizes cellular energy requirements and conserves myocardial ATP.
Unfortunately, current cardiac preservation solutions are not optimal and ATP is gradually lost during graft storage. ATP is degraded in a stepwise fashion to adenosine diphosphate, adenosine monophosphate, adenosine, inosine, and hypoxanthine. Hypoxanthine is further catabolized to xanthine and urate by xanthine dehydrogenase or oxidase, or both. 2 The degree of ATP degradation reflects the adequacy, as well as the duration, of preservation.
In contrast to liver and kidney transplantation methods, the lack of a long-term, cold preservation solution for myocardium currently requires prompt implantation of donor hearts. Postoperative assessment of donor heart function is often subjective and relies on indirect measurements of cardiac performance. The combination of a narrow range of brief ischemic times and lack of a sensitive and objective means of appraising preservation makes the clinical evaluation of new storage solutions and regimens difficult. With this in mind, our study objectives were to (1) characterize purine efflux from transplanted human cardiac allografts and (2) evaluate the utility of purine efflux as an indicator of organ preservation and eventual function.
MATERIALS AND METHODS
Sample and data collection
Fourteen donor hearts were harvested as previously described using 1 L cold (4° to 10° C) cardioplegia solution. 3 During implantation, a single-lumen catheter was placed into the coronary sinus under direct vision. The right atrium was closed around the catheter with a running Prolene suture (Ethicon, Inc., Somerville, N.J.). An arterial blood sample (2 ml) was drawn into a heparinized tube just before aorta crossclamp release (time 0). Coronary sinus samples (2 ml) were then drawn into heparinized tubes at 1, 5, 10, 15, 20, and 25 minutes after crossclamp release. All samples were immediately placed on ice and centrifuged. One ml of plasma was withdrawn and mixed into a separate tube containing 0.5 ml of 6% HClO4. Samples were stored at -70° C until assayed. Total ischemic time was defined as the time from donor heart removal to aorta crossclamp release. Total cardiopulmonary bypass time, total aorta crossclamp time, and time from aorta crossclamp release to cessation of cardiopulmonary bypass were recorded prospectively.
This study was approved by the Human Research Committee at our institution.
Intravenous inotropic support was initiated in the following manner to wean patients from cardiopulmonary bypass. Dopamine or dobutamine was used initially in all cases at dosages of 0 to 12 µg/kg · min. Isoproterenol was added (0 to 0.1 µg/kg · min) for heart rates less than 90 beats/min. Epinephrine administration was initiated only if cardiac output or blood pressure could not be maintained with maximal dosages of the previously noted inotropic agents. Inotropic scores were calculated from the dosages of inotropic agents required to wean each patient from cardiopulmonary bypass (Table I).
Coronary sinus plasma hypoxanthine and xanthine concentrations versus reperfusion time are illustrated in Fig. 1. Hypoxanthine concentration rose approximately sevenfold 1 minute after aortic crossclamp release. Concentrations then declined gradually, approaching prereperfusion levels after 20 minutes. Coronary sinus xanthine concentrations displayed a similar pattern, but the magnitude of change (twofold) was less dramatic. Although our assay method was capable of detecting adenosine and inosine, measurable quantities of these two purines were not present in any of the samples analyzed. Plasma urate concentrations remained unchanged during the reperfusion period.
ATP catabolism has become a hallmark of experimental myocardial ischemia. 1, 6, 7 Several animal studies have demonstrated strong correlations between degree of ischemic tissue injury, ATP loss, and subsequent organ function. 7-9 ATP degradation during ischemia leads to a concomitant rise in tissue purine catabolites. Therefore it is no surprise that the extent of purine efflux from the ischemic myocardium also correlates positively with the degree of injury and negatively with subsequent cardiac function. 10 Despite this simple scheme, the predominant purine that accumulates during the ischemic period is species-dependent. 10, 11 Important age-dependent differences have also been described. 7 To our knowledge, the pattern of purine efflux from ischemic human myocardium has, until now, never been described.
Hypoxanthine is the predominant purine catabolite that accumulates in human cardiac allografts before transplantation (Fig. 1). During reperfusion, appreciable quantities of xanthine were also released, signifying conversion of hypoxanthine to xanthine by xanthine dehydrogenase or oxidase, or both, within the myocardium. This efflux of xanthine substantiates the existence of xanthine dehydrogenase or oxidase, or both, in the human myocardium, which has been a subject of current controversy. 12, 13
Demonstrating a correlation between purine (hypoxanthine) efflux and ensuing graft function is a difficult task in the clinical setting. The methods used in animal models that accurately quantify cardiac function (e.g., intraventricular balloons to measure developed tension, compliance) cannot be used. Indirect indicators of immediate graft function must be relied on instead. In addition, human cardiac transplantation currently mandates prompt implantation of donor hearts. As a result, this study examines only a narrow window of ischemic times (1.8 to 4.4 hours) from which changes that undoubtedly relate to the length of organ storage are sought.
Despite these obstacles, the present study does show a positive correlation between coronary sinus plasma hypoxanthine concentration (at 1 minute of reperfusion) and the initial inotropic requirement and the ease of weaning the patient from cardiopulmonary bypass (i.e., time from aortic crossclamp release to cessation of bypass). A technical problem with the optimal procurement of one allograft led to an elevated coronary sinus hypoxanthine concentration despite a short total graft ischemic time. Exclusion of this allograft from the study group did yield a significant positive correlation between hypoxanthine efflux and length of organ storage (total ischemic time). Hence hypoxanthine efflux from ischemic myocardium can be exploited as a sensitive and objective index of graft preservation.
In summary, purine efflux from ischemic human myocardium was described for the first time. Additional new evidence is provided for the existence of xanthine dehydrogenase or oxidase, or both, in the human myocardium. The extent of hypoxanthine efflux correlates well with indirect measures of immediate graft function and may find utility as an objective biochemical indicator of graft preservation.
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