|
| HOME | SUBSCRIPTIONS | CURRENT ISSUE | PAST ISSUES | CARDIOSOURCE | SEARCH | HELP | FEEDBACK |
|
|
|
|||||||||
|
|||||||||||
|
J Am Coll Cardiol, 1998; 32:373-379 © 1998 by the American College of Cardiology Foundation |
a Department of Experimental Cardiology, Huntington Medical Research Institutes, Pasadena, California, USA
* Third Department of Internal Medicine, Showa University, School of Medicine, Tokyo, Japan
Manuscript received July 25, 1997; revised manuscript received April 2, 1998, accepted April 17, 1998.
Address for correspondence: Dr. Richard J. Bing, Huntington Medical Research Institutes, Department of Experimental Cardiology, 99 North El Molino Avenue, Pasadena, California 91101
 |
Abstract |
|---|
 Top Abstract MethodsResultsDiscussionReferences |
|---|
Background. Previously we reported that in experimental myocardial infarction (rabbits) NOx is released mainly by inflammatory cells (macrophages) in the myocardium 3 days after onset of ischemia. NOx is formed in heart muscle from NO; NO originates through the activity of the inducible form of nitric oxide synthase (iNOS).
Methods. Eight patients with acute anterior MI and an equal number of controls were studied. Coronary venous blood was obtained by coronary sinus catheterization; NOx concentrations in coronary sinus, in arterial and peripheral venous plasma were measured. Left ventricular end-diastolic pressure was determined. Measurements were carried out 24, 48 and 72 h after onset of symptoms. The type and location of coronary arterial lesions were determined by coronary angiography. Plasma NO3– was reduced to NO2– by nitrate reductase before determination of NO2– concentration by chemiluminescence.
Results. The results provided evidence that in patients with acute anterior MI, the myocardial production of nitrite and nitrate (NOx) was increased, as well as the coronary arterial–venous difference. Increased NOx production by the infarcted heart accounted for the increase of NOx concentration in arterial and the peripheral venous plasma. The peak elevation of NOx occurred on days 2 and 3 after onset of the symptoms, suggesting that NOx production was at least in part the result of production of NO by inflammatory cells (macrophages) in the heart.
Conclusions. The appearance of oxidative products of NO (NO2– and NO3–) in peripheral blood of patients with acute MI is the result of their increased release from infarcted heart during the inflammatory phase of myocardial ischemia. Further studies are needed to define the clinical value of these observations.
| ||||||||||||||||||||||
In contrast, the inducible form of NO synthase (iNOS) is mostly produced in macrophages activated by cytokines and endotoxin (6). It eliminates intracellular pathogens, damaging cells by inhibiting ATP production and oxidative phosphorylation and DNA synthesis (7). In infection, lipopolysaccharide released from bacterial walls, stimulates production of iNOS primarily in macrophages (8). The large amount of NO produced causes extensive vasodilation and hypotension. The iNOS is also activated in heart muscle under pathologic conditions, such as in heart failure, dilated cardiomyopathy and cardiac allograft rejection (9,10). It has been found that in infarcted rabbit heart muscle after ligation of a coronary artery, iNOS activity increases on the first postoperative day and persists for at least 14 days, declining to control levels 3 weeks after the onset of ischemia. iNOS was also localized in infiltrating macrophages of the human heart (11). Animals with myocardial ischemia showed consistent elevation of plasma nitrite (NO2–) and nitrate (NO3–) concentration 3 days after onset of myocardial infarction (12). Several findings suggest a causal relationship between NO production by the heart and elevated plasma levels of NOx, such as a relationship in time between NOx plasma concentration and NOx production and between coronary arteriovenous difference of NOx and myocardial iNOS activity.
It is the purpose of this study to demonstrate that after occlusion of the anterior descending coronary artery in humans, NOx plasma levels are elevated and that this is caused by increased NOx production in the infarcted human heart muscle.
|
Methods |
|---|
|
TopAbstractMethodsResultsDiscussionReferences |
|---|
The study involved 16 patients, of these eight (6 men, 2 women), aged 48 to 80 years (mean 65.0 ± 3.3 years) with no evidence of acute myocardial infarction (AMI) served as control (Table 1). These patients were undergoing coronary arteriography using a Judkins catheter (6F) for evaluation of nonspecific chest pain and had no angiographic evidence of significant coronary artery stenosis (Table 1). All control patients were studied after an overnight fast and received 5,000 units of intravenous heparin at the initiation of the study. Antianginal and antihypertensive medications were discontinued at least 48 hours before this study. Routine cardiac catheterization was performed using a femoral approach and a biplane cineangio system (Toshiba). After a Cordis infinity micro pigtail catheter (6F) was inserted into the left ventricle, left ventricular end-diastolic pressure was recorded. Left ventriculography was performed and left ventricular ejection fraction was calculated (Table 1). A coronary sinus catheter (5F; Baxter) was placed in the coronary sinus (CS) through the left subclavian vein and the position was confirmed by injection of the contrast medium (iopamilone) and by typical position on fluoroscopy (13). Blood samples for analysis were drawn from the CS, femoral artery and femoral or brachial vein (henceforth referred to as the peripheral vein) before performing coronary angiography. A Cordis infinity Judkins type catheter (6F) was used for diagnostic coronary angiography.
|
30 min; 2) electrocardiographic ST-segment elevation of 0.1 mV in at least two leads in either anterior or lateral distribution; and 3) elevated creatine kinase MB isoenzymes within 24 h after the onset of symptoms. Excluded were patients with electrocardiographic or historic evidence of previous myocardial infarction, coronary artery bypass surgery or percutaneous transluminal coronary angioplasty. Tissue plasminogen activator or urokinase were not administered. Therapy included heparin and morphine when indicated. In most cases patients with AMI had sublingual nitrate once in the emergency room or catheterization laboratory before or after coronary angioplasty. Blood was drawn for determination of NOx >12 h after coronary angioplasty and the administration of nitroglycerine. Patients with AMI were subjected to cardiac catheterization within 6 h after onset of symptoms. Coronary angiography and angioplasty were performed using a standard femoral approach. All patients underwent direct coronary angioplasty (mean time from onset of chest pain to first inflation was 3.9 ± 0.6 h). The culprit artery was identified by coronary angiogram (Table 1). A significant coronary artery stenosis was defined as >75% luminal narrowing by visual estimate after intracoronary nitroglycerin (100 to 200 µg). The culprit arteries were revascularized using Scimed Bandit coronary angioplasty dilation catheter (2.5 to 3.5 mm by 20 mm, Boston Scientific) to Thrombolysis in Myocardial Infaction flow grade 3 until residual stenosis was <50% (14). Electrocardiographic ST-segment elevation decreased to normal levels in one patient (No. 2) after coronary angioplasty (Table 1). The remaining patients displayed continuous ST-segment elevation after coronary angioplasty. Sustained ventricular tachycardia, ventricular fibrillation or atrial fibrillation/flutter were not observed.
During hospitalization no patient had cardiogenic shock, reinfarction or infection. At time of discharge coronary angiography revealed that all culprit arteries had remained patent.
Measurement of plasma NO2– and NO3–. Blood was drawn in heparinized tubes for NO2– and NO3– determination, and immediately centrifuged at 1,200 g for 10 min for removal of the formed elements by plasma separation. The plasma samples were ultrafiltered using a micropartitioning device at 4,000 g for 1 h (Centrifree Micropartition System) to remove residual proteins. The deproteinized plasma samples were frozen at –20°C until analysis by chemiluminescence (15).
Analysis of NO2– + NO3– required reduction of NO3– to NO2– with aspergillus nitrate reductase (Sigma Chemical). All samples were run in duplicate. Nitrate reductase 50 µl (0.2 U), FAD 5 µl (5 mmol/liter), NADPH 5 µl (6 mmol/liter) and phosphate buffer (1.2 mmol/liter) were added to 50 µl of deproteinized plasma to yield a final volume of 150 µl and subsequently incubated at 36°C for 1 h to allow for sufficient conversion of NO3– to NO2–. After incubation, 50 µl of the sample were injected into the reaction vessel. The NO3– content of the sample was calculated by subtracting the amount of NO2– in untreated samples from the amount of NO2– in the reduced samples (12). The sample containing nitrate reductase contains basal NO2– plus NO2– derived from reduction of NO3–.
Chemiluminescence was used for detection of NO2– after its reduction to NO. We used a reducing medium consisting of a ferrocene derivative dissolved in acetonitrile containing 1% perchloric acid (Sigma). In this solution, NO is generated from NO2– by a one-electron reduction pathway. In the reaction vessel, helium is used as the carrier gas of NO to the NO analyzer, at a rate of 35 ml/min. Reducing solutions (30 mg of 1,1-dimethylferrocene in 3 ml of acetonitrile, acidified with 50 µl of 70% perchloric acid) were freshly prepared and used within 2 h (15). Under these conditions, NO2– in the injected samples (50 µl) was reduced to NO and detected by chemiluminescence with a NO analyzer. Data were recorded automatically on a chart recorder (Soltec Corporation) as previously described (15).
NO2– and NO3– standard curves. Standard curves for NO2– (0 to 80 µmol/L) and NO3– (0 to 80 µmol/liter) were calculated by adding small aliquots (10 µl/50 µl sample) of sodium nitrite or sodium nitrate solution (in degassed ultrapure water) (12). A standard curve relating the luminescence produced by the added NO2– or NO3– was constructed, and the data were fitted to a straight line (linear regression) (12). All standard curves had r >0.96.
Calculation of NOx production equivalent. To estimate the production of NOx by the heart, the arterial–venous difference of NOx and coronary flow had to be known. NOx production by the heart was calculated as: Output of NOx from the heart (CSNOx x coronary flow) – Input of NOx into the heart (ArterialNOx x coronary flow) (16). Because coronary flow was not measured directly in these patients, data were used from patients with AMI, in which coronary flow was calculated from positron emission tomography imaging, using [13N]ammonia (17). Czernin et al. (17) established a regression correlation (r = 0.74) relating the rate pressure product (RPP) to myocardial blood flow as defined by the equation: y = 0.00009x – 0.07, where y = myocardial blood flow and x = RPP.
Using this equation we calculated mean coronary flow in our patients. The production equivalent of NOx by the heart could then be calculated. The term production equivalent was used in preference to production because coronary flow was not determined directly (12).
Statistical analysis. Two analyses were performed to evaluate the change in mean NOx levels over time. A two-way analysis of variance with repeated-measures was used to analyze the difference in mean NOx levels over time in the venous, arterial and coronary sinus plasma in patients with AMI. The repeated measures model used a general covariance matrix, allowing for unequal correlations over time. The design of this study was an unbalanced two-way repeated measures analysis of variance. All parameters (differences, means, p values) were estimated via maximum likelihood. A second analysis was performed comparing the mean NOx levels in the control patients to the patients with AMI at 24, 48 and 72 h based on between-group repeated measures variances for the peripheral venous, arterial and coronary sinus blood.
To compare the three sources of mean NOx levels to each other within each time period (control 24, 48 and 72 h), post-hoc t tests (based on the covariance structure of the analysis of variance model) were performed using the Tukey-Fisher least significant difference method. The within-subject variances were used to calculate the standard errors.
A similar method was used to compare the mean NOx level of the control group to the mean NOx levels at 24, 48 and 72 h. Post-hoc t tests (based on the covariance structure of the analysis of variance model) using the Tukey-Fisher least significant difference method were used to compare these means. The total variance estimated from the analysis of variance model using a general covariance matrix was used to calculate these standard errors. All variances used to calculate the standard errors for the within-subject and between-subject comparisons were estimated from the repeated measures of the analysis of variance model using a general covariance matrix, which allowed for unequal correlations over time. A p value of 0.05 or less was considered statistically significant. Data processing was performed using the SAS (version 6.11) software package.
|
Results |
|---|
|
TopAbstractMethodsResultsDiscussionReferences |
|---|
Table 2 shows that after onset of symptoms the values for NOx in CS plasma are close to significance at 48 h (p < 0.07) as compared to arterial and peripheral venous NOx concentrations. At 24 h, CS NOx concentrations still exceed those in arterial and peripheral venous plasma (Table 2). Compared to the control group, all patients with AMI showed statistically significant increase in NOx concentrations in arterial, CS and peripheral venous plasma at 48 and 72 h after onset of symptoms (Fig. 1). These statistical differences were absent 24 h after onset of symptoms. In Figure 2 the NOx levels in arterial blood are plotted in each individual patient as a function of time from the presentation through the remainder of the study. The values of the control series are also shown. A general increase in arterial NOx concentrations in individual patients is noted. Changes in individual CS and peripheral venous plasma followed a similar trend (not shown).
|
|
|
The output of NOx from the heart is represented as the product of coronary flow and the NOx concentration in the CS plasma (12,16). Because coronary flow was not determined directly, it was calculated from the rate–pressure product as presented in Table 3, according to methods of Czernin et al. (17). From the coronary flow thus calculated the production equivalent of NOx by the heart was obtained (12). The cardiac NOx production equivalent is listed in Table 3. A multiple comparison test showed that at 48 h after onset of symptoms the NOx production equivalent was significantly greater than at control, again pointing to the heart as the source of NOx production (control 137 ± 37 nmol/100 g/min; AMI at 48 h = 472 ± 104 nmol/100 g/min; p < 0.05; Table 3).
|
|
Discussion |
|---|
|
TopAbstractMethodsResultsDiscussionReferences |
|---|
The angiotension-converting enzyme inhibitors may have influenced NO production as inhibition of angiotension-converting enzyme localized in the luminal site of the vascular endothelium results in increased synthesis of NO and prostaglandin I2 by accumulation of endogenous bradykinin. This demonstrates the potential ability to increase NO production through the kinin pathway (18).
Formation of NOx. It is well known that NO is produced in infarcted heart muscle primarily by the activity of iNOS (19). Activation of iNOS occurs in cardiomyocytes and during the inflammatory phase in activated macrophages (11). iNOS shows no requirement for Ca2+ and calmodulin, although calmodulin exists as a highly bound subunit (20). Activation of iNOS depends on the release of endogenous substances, including cytokines (tumor necrosis factor-alpha, interleukin-1 beta, interferon) (1). NO itself has an unpaired electron, and is physiologically and pharmacologically active (21). In contrast, as shown by Stuehr and Nathan (22), NO2– and NO3– are inactive, except at acidic pH when NO2– is converted into more reactive species. NO2– is formed by a reaction of NO with oxygen. The primary metabolite of NO is NO3– with <10% of the total appearing as NO2– (23); NO2– concentrations remain constant, whereas those of NO3– vary widely (12,16). The sum of NO2– and NO3– has been designated as NOx (12). The product of the reaction of NO with oxygen, superoxide and transition metals support additional nitrosative reactions (24). Hibbs et al. (8) showed that L-arginine is required for activation of macrophages to a bactericidal and tumoricidal state. Apparently NO is an intermediate in the oxidation of L-arginine to NO2– and NO3– and the citrulline pathway (25).
NOx and myocardial infarction. The results of the present study provide evidence that in patients with anterior AMI at 48 h after onset of symptoms, NOx production equivalent by the heart is increased together with its coronary arterial–venous difference (Tables 2 and 3). This accounts for the increase of NOx in arterial and peripheral venous plasma (Table 2, Fig. 1). In experimental myocardial infarction activated macrophages are a major source of NO (26). An elevated NOx production by the heart and increased concentrations of NOx in peripheral venous blood have been noted previously in rabbits 3 days after occlusion of a coronary artery (16). This may have been the result of formation of NO by activated macrophages (19). It is possible that both cardiomyocytes and inflammatory cells, primarily activated macrophages, are involved (11).
Although it is likely from the evidence presented here that the elevated levels of NOx in peripheral plasma observed after myocardial infarction are the result of increased production of NOx by macrophages in infarcted heart muscle, the possibility must be considered that other factors are involved. Part of plasma NOx originates as a consequence of the activity of the endothelial form of NO synthase. Evidence for this is the finding of Akiyama et al. (12) that S-methylisothiourea, which primarily inhibits iNOS, only partially lowers plasma levels of NOx. It is unlikely that coronary angioplasty influences the results as collections of plasma for NOx determination were carried out 18 h after angioplasty. Another possible reason for the increase of NOx in plasma is accumulation of nitrate anion if the production of NOx exceeds its elimination within the half-life of NOx (3.8 h) (16). Similarly, diminished clearance of NOx by the kidney can lead to increased NOx plasma levels. This may be the case in severe congestive heart failure as reported by Winlaw et al. (27). However, none of the patients in this series had signs of congestive heart failure.
Another possible explanation for increased appearance of NOx in CS plasma is enhanced NO production originating in coronary vascular endothelium beginning 24 h after reperfusion. Kim et al. (28) have shown that brief ischemic episodes are responsible for enhanced coronary artery endothelial function. This effect is delayed in onset and prolonged in duration. This may be one of the factors responsible for the increased concentration of NOx in CS plasma of patients after onset of acute myocardial infarction. It also suggests that in certain instances involving multiple factors NOx in plasma may not be the exclusive reflection of iNOS in heart muscle.
Our observations of increased arteriocoronary sinus difference and of NOx myocardial production equivalent strongly point to the heart as a source of increased plasma levels of NOx in myocardial infarction.
Nitric oxide and cardiac disorders. Cardiac diseases other than myocardial infarction are accompanied by changes in plasma concentration of NO3– (29,30). Winlaw et al. (27) reported that plasma NO3– increased significantly in patients with chronic congestive heart failure; however, because only peripheral plasma concentrations were measured, decrease in renal function may have been partially responsible. Haywood et al. (31) found increased expression of inducible form of nitric oxide synthase messenger RNA in the hearts of patients with chronic congestive heart failure. Langrehr et al. (29) demonstrated in allografted rats a significant increase in NO2–/NO3– levels in peripheral blood before critical signs of rejection were noted. Treatment with FK-506, an inhibitor of transplant rejection, abolished this increase. Winlaw et al. (32) found in rats an eightfold increased excretion of urinary NO2– in untreated allograft rejection. Akiyama et al. (12) also reported reduction of plasma levels of NOx by S-methylisothiourea, a selective inhibitor of iNOS in experimental myocardial infarction in rabbits.
Conclusions. From data presented in this report, no definite conclusions on future applications of NOx determinations in peripheral blood of patients with myocardial infarction can be drawn. More patients need to be studied and the various parameters responsible for increased NOx production should be further defined. However, the evidence of a cardiac origin in the increase in plasma NOx concentration in patients with myocardial infarction appears convincing.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
|
References |
|---|
|
TopAbstractMethodsResultsDiscussionReferences |
|---|
This article has been cited by other articles:
|
|
 |
|
  S. W. Rabkin and M. Y. C. Tsang The action of nitric oxide to enhance cell survival in chick cardiomyocytes is mediated through a cGMP and ERK1/2 pathway while p38 mitogen-activated protein kinase-dependent pathways do not alter cell death Exp Physiol, July 1, 2008; 93(7): 834 - 842. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 The TRIUMPH Investigators Effect of Tilarginine Acetate in Patients With Acute Myocardial Infarction and Cardiogenic Shock: The TRIUMPH Randomized Controlled Trial JAMA, April 18, 2007; 297(15): 1657 - 1666. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 I. Andreadou, E. K. Iliodromitis, E. Mikros, M. Constantinou, A. Agalias, P. Magiatis, A. L. Skaltsounis, E. Kamber, A. Tsantili-Kakoulidou, and D. T. Kremastinos The Olive Constituent Oleuropein Exhibits Anti-Ischemic, Antioxidative, and Hypolipidemic Effects in Anesthetized Rabbits J. Nutr., August 1, 2006; 136(8): 2213 - 2219. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Antoniades, D. Tousoulis, C. Vasiliadou, C. Pitsavos, C. Chrysochoou, D. Panagiotakos, C. Tentolouris, K. Marinou, N. Koumallos, and C. Stefanadis Genetic Polymorphism on Endothelial Nitric Oxide Synthase Affects Endothelial Activation and Inflammatory Response During the Acute Phase of Myocardial Infarction J. Am. Coll. Cardiol., September 20, 2005; 46(6): 1101 - 1109. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Bertuglia, A. Giusti, and P. Del Soldato Antioxidant activity of nitro derivative of aspirin against ischemia-reperfusion in hamster cheek pouch microcirculation Am J Physiol Gastrointest Liver Physiol, March 1, 2004; 286(3): G437 - G443. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. H. Han, E. Qamirani, A. G. Nelson, D. R. Hyduke, G. Chaudhuri, L. Kuo, and J. C. Liao Regulation of nitric oxide consumption by hypoxic red blood cells PNAS, October 14, 2003; 100(21): 12504 - 12509. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. W. Cohen, D. S. Park, S. E. Woodman, T. M. Williams, M. Chandra, J. Shirani, A. Pereira de Souza, R. N. Kitsis, R. G. Russell, L. M. Weiss, et al. Caveolin-1 null mice develop cardiac hypertrophy with hyperactivation of p42/44 MAP kinase in cardiac fibroblasts Am J Physiol Cell Physiol, February 1, 2003; 284(2): C457 - C474. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Saito, F. Hu, L. Tayara, L. Fahas, H. Shennib, and A. Giaid Inhibition of NOS II prevents cardiac dysfunction in myocardial infarction and congestive heart failure Am J Physiol Heart Circ Physiol, July 1, 2002; 283(1): H339 - H345. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. M. Stevens, M. S. Jahania, J. E. Stivers, R. M. Mentzer Jr, and R. D. Lasley Effects of in vivo myocardial ischemia and reperfusion on interstitial nitric oxide metabolites Ann. Thorac. Surg., April 1, 2002; 73(4): 1261 - 1266. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 V. Stangl, G. Baumann, K. Stangl, and S. B Felix Negative inotropic mediators released from the heart after myocardial ischaemia-reperfusion Cardiovasc Res, January 1, 2002; 53(1): 12 - 30. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Zhang, J. W. Bissing, L. Xu, A. J. Ryan, S. M. Martin, F. J. Miller Jr, K. C. Kregel, G. R. Buettner, and R. E. Kerber Nitric oxide synthase inhibitors decrease coronary sinus-free radical concentration and ameliorate myocardial stunning in an ischemia-reperfusion model J. Am. Coll. Cardiol., August 1, 2001; 38(2): 546 - 554. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Saito, M. P. Pelletier, H. Shennib, and A. Giaid Nitric oxide system in needle-induced transmyocardial revascularization Ann. Thorac. Surg., July 1, 2001; 72(1): 129 - 136. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. J Bing Myocardial ischemia and infarction: growth of ideas Cardiovasc Res, July 1, 2001; 51(1): 13 - 20. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. A. Recchia, T. R. Vogel, and T. H. Hintze NO metabolites accumulate in erythrocytes in proportion to carbon dioxide and bicarbonate concentration Am J Physiol Heart Circ Physiol, August 1, 2000; 279(2): H852 - H856. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. J Bing, T. Yamamoto, M. Yamamoto, R. Kakar, and A. Cohen New look at myocardial infarction: toward a better aspirin Cardiovasc Res, July 1, 1999; 43(1): 25 - 31. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | SUBSCRIPTIONS | CURRENT ISSUE | PAST ISSUES | CARDIOSOURCE | SEARCH | HELP | FEEDBACK |
