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* Division of Life Sciences, Department of Cell Biology & Neurosciences, Rutgers University, New Brunswick/Piscataway, New Jersey 08854;
Robert C. Byrd Health Sciences Center, Department of Pharmacology & Toxicology, West Virginia University Medical School, Morgantown, West Virginia 26506;
Department of Adapted Physical Activity Studies, State University of Campinas—Campinas, Sao Paulo, Brazil 13083-970
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Abstract |
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Key Words: global myocardial ischemia • 2,6,8-trihydroxypurine • mechanical function
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Introduction |
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Acetaminophen is a phenol (4), and many phenols have antioxidant properties (5,6). Oxidative stress in tissues has been a topic of intense investigation both in the basic science laboratory and in clinical research in recent years (7,8). Taking basic discoveries in oxidative stress to a clinical application, i.e., translational research, is of significant contemporary interest (9–11). Increased use of acetaminophen by an aging population (12) justifies a more indepth exploration into the unknown mechanisms of action of this compound. Such has been an important objective of our recent investigations (13–15). In one of these studies, we found evidence of antiarrhythmic properties of acetaminophen (14). In a preliminary experiment (unpublished results), we found concentration-dependent positive inotropic actions of acetaminophen and modest, dose-dependent, coronary vasoconstriction. In the current article we report significant, anti-oxidant–mediated, cardioprotective actions of acetaminophen when it is delivered, upon reperfusion, to the postischemia myocardium.
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Materials and Methods |
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Upon completion of instrumentation, hearts were perfused in increments of approximately 2 ml/min at 3- to 4-min intervals until a control rate of 7 ml/min was reached. Flow was controlled at this rate throughout the experiment except during low-flow ischemia, when it was reduced to about 1 ml/min (Peri-Star pump, model 291, World Precision Instruments, Sarasota, FL). Hearts were allowed approximately 30 min post-instrumentation for monitored variables to achieve steady-state conditions. Monitored variables included: heart rate (HR, paced at spontaneous rate plus approximately 15%, cpm [cycles per minute], left ventricular developed pressure (LVDP, mm Hg; the difference between peak systolic and end–diastolic pressures in the left ventricle), ±dP/dtmax(mm Hg/sec, contractility), pressure rate product (PRP, LVDP x HR, mm Hg/cpm), coronary perfusate flowrate (CPF, ml/min), coronary perfusion pressure (CPP, mm Hg), and calculated coronary vascular resistance (CVR, mm Hg/ml/min).
Perfusate and Perfusion Modality.
Perfusate was a modified Krebs-Henseleit physiological buffer solution (vehicle for acetaminophen) containing (in mM): NaCl (128.0), KCl (4.7), MgSO4•7H20 (1.5), CaCl2 (2.5), KH2PO4 (1.2), NaHCO3 (24.9), glucose (10.0), pyruvate (2.0), and insulin (200 µU/ml). It was warmed to 38°C and equilibrated with 95% O2 and 5% CO2 (pH 7.40 ± 0.02). Aortic flow (antegrade coronary flow) was achieved retrogradely from a water-jacketed 1.5-liter reservoir, and was continuously monitored ultrasonically (model 2N423 flowprobe, model T101 flowmeter, Transonic Inc., Ithaca, NY).
LVDP was monitored isovolumetrically (model P231D, Gould-Statham, Oxnord, CA) and inflow (coronary arterial) and outflow (pulmonary arterial, i.e., coronary venous) perfusate samples were collected anaerobically for monitoring pH and perfusate gases (0.5–1.0 ml). Standard electrodes were used to measure partial pressures of oxygen (PO2, mm Hg), and carbon dioxide (PCO2, mm Hg), as well as pH, CO2 content, and base excess (model 248 blood gases/pH analyzer, Bayer Diagnostics, Norwood, MA) as previously reported from this (18–20) and other laboratories (21).
Experimental Protocols.
Four experimental protocols (i–iv) were conducted (see details below). (i) This experiment was conducted to determine the efficacy of acetaminophen (against dysfunction/injury during reperfusion) when administered at the onset of reperfusion, postischemia. (ii) The second experiment was conducted to compare and contrast the efficacy of uric acid (2,6,8-trihydroxypurine, another phenol) with acetaminophen. This agent was also administered at the onset of reperfusion. (iii) A third experiment compared the effects of vehicle, acetaminophen, and uric acid on the production of blue light (chemiluminescence) during reperfusion (agents administered at the onset of reperfusion to identify their antioxidant/anti-chemiluminescent actions). (iv) Finally, in a fourth experiment, we measured the production of creatine kinase, a biochemical marker of tissue damage in myocardial ischemia and reperfusion. In this experiment efficacies of all three agents were compared.
Actions of Acetaminophen When Administered at Onset of Reperfusion.
The objective of this experiment was to determine whether administering acetaminophen at the onset of reperfusion, after a 20-min period of low-flow, global myocardial ischemia, would be efficacious in the injured, postischemia reperfused myocardium. The endpoints for determining efficacy were left ventricular and coronary circulatory functions. After each heart was instrumented and when monitored variables were in the steady state, a set of baseline (control) data were collected. Subsequently, hearts were submerged in warmed KHB (to maintain temperature during ischemia) and CPF was reduced to approximately 1 ml/min for 20 min. We have previously reported no aphysiological effects of submerging Langendorff-perfused guinea pig hearts in physiological salt solutions (20). As long as the hearts are adequately oxygenated, they continue to beat rhythmically and contract physiologically. Data, including samples of venous effluent perfusate, were collected only at 10 and 20 min of ischemia. Shortly before the period of ischemia either KHB or acetaminophen (0.35 mM, final concentration in the perfusate reservoir) was added to the perfusate reservoir. Timing and the dead space volume in the perfusion system were accurately calculated so the agent would begin arriving at the heart at the onset of reperfusion. After 20 min of ischemia, the heart chamber was drained (i.e., hearts were removed from submersion fluid) and CPF was restored to its pre-ischemia level. Monitored variables (e.g., developed pressure, coronary perfusion pressure), including samples of venous effluent, were collected at 1, 3, 6, 10, and 40 min of reperfusion.
Uric Acid and the Reperfused Myocardium.
Our main objective in this experiment was to compare another phenol, uric acid, with acetaminophen in the injured, reperfused myocardium. Uric acid is a circulating anti-oxidant in human plasma and has been of recent interest to one of the authors (KVD). This experiment was designed identically to that described above. The only difference was the replacement of acetaminophen with uric acid. In preliminary experiments we completed concentration/response studies to help identify an effective concentration of uric acid, i.e., one that could attenuate the production of peroxynitrite but that was not immediately toxic to the heart. We evaluated concentrations ranging from 0.1–0.4 mM. We used 0.18 mM because it was in the middle of this range and because it had no direct effects on the heart under baseline conditions. Data were collected at each of the eight time intervals described above.
Peroxynitrite and Acetaminophen Versus Uric Acid.
Three groups of hearts were studied; vehicle treated (n = 6), acetaminophen treated (n = 6), and uric acid treated (n = 6). Samples of coronary venous effluent perfusate (0.25–0.5 ml each) were collected in prechilled 1.5-ml capped vials at baseline, 10, and 20 min ischemia, and 1, 3, 6, 10, and 40 min reperfusion, i.e., eight samples from each heart. Samples were stored at -70°C until analysis. Chemiluminescence technology was used to assess the production of peroxynitrite as previously described (5, 6, 13).
Creatine Kinase in Vehicle-, Acetaminophen-, and Uric Acid-Treated Hearts.
Creatine kinase (CK) is a well-established marker of myocardial injury during ischemia and reperfusion (22–24). In this experiment we compared the effects of all three agents on the release of creatine kinase (n = 3–6 hearts per treatment group). Standard assays were used (product no. 47-10, Sigma Diagnostics, St. Louis, MO) and were based on the modified procedures of Nielsen and Ludvigsen (25) and Rosalki (26). Briefly, 1 ml of reconstituted reagent was added to 20 µl of sample in a 1 ml minimum cuvette. Each sample was incubated for 3 min at 30°C (±2°C) and processed spectrophotometrically at 340 nm (30-sec intervals for a total of 120 sec). Data are expressed in units per liter, where 1 unit of activity is defined as the amount of enzyme that produces 1.0 µmol/min of NADH.
Statistical Analysis.
All data are presented as means plus or minus one SEM. Differences in group variances were identified using analysis of variance (ANOVA, repeated measures design). Differences in group means were identified using a priori tests including Fisher’s (least significant difference) and Tukey’s w-procedure. Significance was established at P < 0.05 in all cases.
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Results |
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)Coronary perfusion pressure was 38 ± 2 mm Hg under baseline, pre-ischemia control conditions in hearts in all three groups. Coronary perfusion pressure dropped to approximately 10–15 mm Hg during ischemia and did not differ significantly amongst the various treatment groups. Upon reperfusion, CPP in vehicle-treated hearts increased slowly but steadily, becoming significantly greater than that in acetaminophen-treated hearts by 40 min of reperfusion. Coronary perfusion pressure in acetaminophen-treated hearts returned to baseline levels in the first minute of reperfusion, and maintained this plateau throughout reperfusion (Fig. 2). Treatment with uric acid caused marked, statistically significant increments in CPP throughout the period of reperfusion. Changes in calculated CVR in the three groups paralleled changes in CPP and are summarized, with other indices of mechanical function, in Tables I and II.
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). This initial burst, and the subsequent production of blue light, was abolished during the 40 min of reperfusion in the presence of acetaminophen. During the first 3 min of reperfusion, uric acid also attenuated the production of blue light (when compared with vehicle-treated hearts). However, at 6–10 min, there were no significant differences between vehicle- and uric acid-treated hearts, and by 40 min chemiluminescence in the presence of uric acid was elevated significantly above that for vehicle.
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. For example, the t
(min) for both the production and abatement of blue light, in the presence of acetaminophen, exceeded the pre-determined detection time of the luminometer (20 min).
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Discussion |
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Uric acid is of physiologic importance to humans because it is the terminal oxidation product of purine metabolism (28). Human plasma contains uric acid at concentrations approaching 0.5 mM (29), it is a polyphenol, and such antioxidants are the circulatory system’s first line of defense against oxidant-induced tissue injury (11). Our initial objective was to compare equimolar concentrations of uric acid and acetaminophen. However, at concentrations approaching 0.35 mM marked cardiotoxicity (e.g., concentration-dependent negative inotropy) was evident with uric acid. We are well aware that by the laws of mass action, we supplied only about half the antioxidant concentration of uric acid as acetaminophen. Thus, comparing the two under the present circumstances might not be justified. However, as can be seen from the data, e.g., Figs. 1, 2, and 4
even 0.18 mM uric acid seemed to be cardiotoxic during our ischemia/reperfusion protocol. In the presence of peroxynitrite, uric acid is nitrated to an as-yet-unidentified uric acid derivative (30). This product might play a pivotal role in tissue pathophysiology, producing cardiovascular as well as non-cardiovascular actions. Recent epidemiologic studies reveal that elevated uric acid concentrations are an independent risk factor for cardiovascular mortality in the general human population (31).
The maintenance of coronary perfusion pressure and coronary vascular resistance by acetaminophen during reperfusion (relative to vehicle and uric acid) is also consistent with its antioxidant actions as seen in this and our earlier studies (13–15). Nor is the coronary perfusion pressure of approximately 40 mm Hg under baseline conditions aphysiological for this guinea pig heart preparation. For example, at a coronary perfusion pressure of 40 mm Hg and a controlled flow rate of 7 ml/min, these hearts are neither ischemic nor hypoxic. They have coronary flow reserves of at least 100% (in some cases it is 2- to 3-fold greater than baseline values), and normal oxygen delivery, extraction, and consumption for crystalloid-perfused Langendorff hearts (personal observations).
Peroxynitrite.
The burst of blue light seen in the early minutes of reperfusion in the presence of vehicle is consistent with our earlier observations. We have attributed the oxidation of luminol to the actions of peroxynitrite. However, ischemia/reperfusion alters the metabolism of purines in the myocardium and increases the activity of enzymes such as xanthine oxidase and nitric oxide synthase. Byproducts of this activity, e.g., superoxide and nitric oxide, are also known to oxidize luminol (5, 6, 32). Clearly, there are other nonidentified myocardial oxidants that can cause the production of luminol-dependent blue light. Thus, at the present, we are only able to conclude that peroxynitrite, generated by the combination of superoxide and nitric oxide, is possibly one of several potential targets of the antioxidant actions of acetaminophen. Still, in the presence of acetaminophen chemiluminescence was markedly attenuated (e.g., see Table III). The increased maximum slope, t rise, and t decline denote marked attenuation of the ability of peroxynitrite (other oxidants) to oxidize luminol to produce blue light. This is consistent with important cardioprotective properties of acetaminophen.
Creatine Kinase.
The concentrations of this important biochemical indicator of myocardial tissue damage varied within treatment groups, especially during ischemia and reperfusion. The most consistent result, however, was the marked, and statistically significant reduction in CK in the presence of acetaminophen during reperfusion. The expected effects of ischemia on CK were also observed, i.e., a significant increment during the 20-min period of restricted coronary perfusate flow (22). Importantly, CK concentrations did not vary significantly among the three treatment groups under baseline, control conditions, or during ischemia.
Others have reported changes in CK in isolated perfused hearts during ischemia/reperfusion (33–35), but there are no previous reports of the actions of acetaminophen on these changes. The significant reduction in CK in the presence of vehicle and acetaminophen in the early minutes of reperfusion are explained, in part, by washout. However, this trend was sustained in acetaminophen-treated hearts but not in the presence of either vehicle or uric acid. By the end of the period of reflow CK had returned to or above levels seen during baseline and ischemia conditions in the latter groups. Hence, washout cannot explain acetaminophen’s actions in the later minutes of reperfusion. For example, by 10 and 40 min CK levels in the acetaminophen-treated group were lower than their own baseline/ischemia values. Clearly, the comparative results with uric acid and vehicle strengthen the argument that acetaminophen’s cardioprotective properties are revealed at the biochemical, as well as the morphological and physiological (functional) levels (13, 14).
Time–Course and Therapeutic Doses of Acetaminophen.
Our current interest in acetaminophen began a few years ago when we administered it to hearts 20 min before the onset of ischemia and found it to be beneficial (13). Later, we administered acetaminophen midway through a 20-min period of ischemia and also demonstrated cardioprotection (15). In this experiment, acetaminophen was introduced to the hearts at the onset of reperfusion. Collectively, these three experiments reveal that acetaminophen has important cardioprotective actions under all three temporal conditions. From our perspective, acetaminophen appears to be most efficacious when administered before the onset of ischemia. Time and additional experiments by others are needed to confirm this opinion. Nonetheless, our combined results raise questions about the potential beneficial effects of administering acetaminophen chronically (12).
The concentration of acetaminophen used in these experiments [0.35 mM] yields circulating concentrations in the coronary venous effluent perfusate of approximately 50 µg/ml. Several hours after therapeutic dosing, plasma concentrations in humans are typically 10–20 µg/ml, although values as high as 50 µg/ml have been reported. Cytotoxicity is generally thought to occur at plasma concentrations 
300 µg/ml. Thus, 0.35 mM acetaminophen is greater than concentrations needed for therapeutic efficacy but is well below concentrations needed to produce hepato-/cytotoxicity. We have not conducted experiments using concentrations that would closely estimate therapeutic doses (e.g., 0.1–0.15 mM).
Summary and Conclusions.
Administering acetaminophen at the onset of reperfusion following a 20-min period of low-flow, global myocardial ischemia, provides cardioprotection during reperfusion. Biochemical and functional evidence to support this conclusion have been presented here. Uric acid, an alternative polyphenol, provides some degree of cardioprotection (biochemical) in the early minutes of reperfusion, but is markedly cardiotoxic in the later minutes of reperfusion. This is true of concentrations that, under basal physiological conditions, show little or no signs of toxicity in our Landendorff heart preparation. There is mounting evidence that acetaminophen has important cardiovascular actions other than those mediated directly on the myocardium (36, 37). Clearly, more work on the cytoprotective properties of acetaminophen in the mammalian myocardium and other mammalian organ systems is warranted.
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Footnotes |
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1 To whom requests for reprints and correspondence should be addressed at Cell Biology & Neuroscience, Rutgers University, 604 Allison Road, Piscataway, NJ 08854. E-mail: merrill{at}biology.rutgers.edu 
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