Production of oxidative products of nitric oxide in infarcted human heart

Patients and methods

All participants in the study were informed of the procedure and gave written consent. The protocol was approved by the local ethics committees and is in line with the recommendations of the Declaration of Helsinki.

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.

Patients with acute myocardial infarction (AMI) (6 men, 2 women), aged 46 to 81 years (mean 66.0 = 4.5 years) (Table 1)displayed evidence of infarction as documented by the following criteria: 1) chest pain lasting ≥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 NO_x>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 gfor 10 min for removal of the formed elements by plasma separation. The plasma samples were ultrafiltered using a micropartitioning device at 4,000 gfor 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 NO_xproduction equivalent

To estimate the production of NO_xby the heart, the arterial–venous difference of NO_xand coronary flow had to be known. NO_xproduction by the heart was calculated as: Output of NO_xfrom the heart (CS_NOx× coronary flow) − Input of NO_xinto the heart (Arterial_NOx× 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 NO_xby 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 NO_xlevels over time. A two-way analysis of variance with repeated-measures was used to analyze the difference in mean NO_xlevels 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 NO_xlevels 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 NO_xlevels to each other within each time period (control 24, 48 and 72 h), post-hoc ttests (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 NO_xlevel of the control group to the mean NO_xlevels at 24, 48 and 72 h. Post-hoc ttests (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.

Patients

Patients in this study represent a clinically homogenous population; none of them had cardiogenic shock or congestive heart failure, and all had anterior myocardial infarction with the left anterior descending coronary artery as the culprit vessel. Furthermore all AMI patients had higher left ventricular end-diastolic pressure and lower left ventricular ejection fraction when compared to controls. Heart rate and blood pressure did not differ (Table 1). Other factors such as smoking, diuretics or calcium antagonists did not influence either hemodynamics or NO_xplasma concentration in patients with AMI (Tables 1 and 2). The effect of angiotension-converting enzyme inhibitors could not be evaluated, because all but one patient was given angiotension-converting enzyme inhibitors.

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 I₂by accumulation of endogenous bradykinin. This demonstrates the potential ability to increase NO production through the kinin pathway (18).

Formation of NO_x

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 NO_x(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).

NO_xand myocardial infarction

The results of the present study provide evidence that in patients with anterior AMI at 48 h after onset of symptoms, NO_xproduction equivalent by the heart is increased together with its coronary arterial–venous difference (Tables 2 and 3). This accounts for the increase of NO_xin arterial and peripheral venous plasma (Table 2, Fig. 1). In experimental myocardial infarction activated macrophages are a major source of NO (26). An elevated NO_xproduction by the heart and increased concentrations of NO_xin 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 NO_xin peripheral plasma observed after myocardial infarction are the result of increased production of NO_xby macrophages in infarcted heart muscle, the possibility must be considered that other factors are involved. Part of plasma NO_xoriginates 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 NO_x. It is unlikely that coronary angioplasty influences the results as collections of plasma for NO_xdetermination were carried out ≥18 h after angioplasty. Another possible reason for the increase of NO_xin plasma is accumulation of nitrate anion if the production of NO_xexceeds its elimination within the half-life of NO_x(3.8 h) (16). Similarly, diminished clearance of NO_xby the kidney can lead to increased NO_xplasma 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 NO_xin 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 NO_xin CS plasma of patients after onset of acute myocardial infarction. It also suggests that in certain instances involving multiple factors NO_xin plasma may not be the exclusive reflection of iNOS in heart muscle.

Our observations of increased arteriocoronary sinus difference and of NO_xmyocardial production equivalent strongly point to the heart as a source of increased plasma levels of NO_xin 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 NO_xby 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 NO_xdeterminations in peripheral blood of patients with myocardial infarction can be drawn. More patients need to be studied and the various parameters responsible for increased NO_xproduction should be further defined. However, the evidence of a cardiac origin in the increase in plasma NO_xconcentration in patients with myocardial infarction appears convincing.