Vecuronium bromide

Study objective: Human plasma L-arginine serves as a substrate pool for endothelial-derived nitric oxide (NO) synthase. In this pilot study, we tested the hypothesis that plasma L-arginine and other metabolites of the L-arginine NO pathway could correlate with postoperative pulmonary hypertension after cardiopulmonary bypass (CPB).

Design: Forty-two patients (median age, 0.5 years; range, 0.1 to 28 years) with atrial septal defect (n = 15), ventricular septal defect (n = 18), atrioventricular canal (n = 8), and aortopulmonary window (n = 1) were enrolled. The influence of patient age, preoperative pulmonary hypertension, duration of CPB, plasma L-arginine, guanosine 3', 5'-cyclic monophosphate (cGMP), and nitrate on postoperative pulmonary hypertension during the first 24 h after CPB was studied by logistic regression.

Results: Nineteen of 42 patients were found to have preoperative pulmonary hypertension. Thirteen of 42 patients showed persistent pulmonary hypertension after intraeardiac repair with a mean pulmonary artery pressure (PAP) of 38 mm Hg (range, 23 to 55 mm Hg) at 24 h after CPB. L-arginine concentrations in plasma were significantly lower 24 h after CPB than before: 52 [micro]mol/L (range, 18 to 95 [micro]mol/L) vs 79 [mciro]mol/L (range, 31 to 157 [micro]mol/L). Plasma cGMP levels were higher and plasma nitrate levels were lower immediately after weaning from CPB (p < 0.0033). On logistic regression analysis, only patient age (p = 0.02) and preoperative PAP (p = 0.01) were related to postoperative pulmonary hypertension.

Conclusion: Low plasma L-arginine does not relate to persistent pulmonary hypertension in patients with left-to-right shunt after CPB and intracardiac repair.

Key words: defects; hypertension; L-arginine; nitric oxide; pediatrics; pulmonary

Abbreviations: cGMP = guanosine 3', 5'-cyclic monophosphate; CPB = cardiopulmonary bypass; NO = nitric oxide; PAP = pulmonary artery pressure; Qp = pulmonary blood flow; Qs = systemic blood flow

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Pulmonary hypertension after intracardiac repair for children with left-to-right shunt contributes considerably to perioperative mortality after intracardiac repair. (1) Patients with increased pulmonary blood flow (Qp) show an impaired endothelium-dependent pulmonary artery relaxation. (2) Plasma L-arginine is a substrate and an important source for endothelial-derived nitric oxide (NO) synthase. (3) Guanosine 3', 5'-cyclic monophosphate (cGMP) acts as an intracellular second messenger of NO, and nitrate is the final end product of the L-arginine NO pathway.

Experimental (4) and clinical (5) data suggest a substrate deficiency for L-arginine related to left-to-right shunt. However, no studies are available that analyze the impact of low plasma L-arginine and metabolites of the L-arginine NO pathway for the development of postoperative pulmonary hypertension in children with left-to-right shunt after intracardiac repair. We therefore tested the hypothesis that plasma L-arginine, eGMP, and nitrate would relate to postoperative pulmonary hypertension in patients with left-to-right shunt after intraeardiae repair with cardiopulmonary bypass (CPB).

MATERIALS AND METHODS

Between January 2001 and June 2004, 42 consecutive children and young adults with congenital heart disease and left-to-right shunt were enrolled. These patients underwent cardiac catheterization for hemodynamic assessment prior to surgery for intracardiac repair.

Informed consent was obtained from the parents of each child and the patients > 7 years of age. The study protocol was approved by the Institutional Review Board of the University of Heidelberg Medical Center.

Patients

Clinical data of 42 patients (male, n = 23; female, n = 19) at a median age of 0.5 years (range, 0.1 to 28 years) are summarized on Table 1. Patients presented with atrial septum defect (n = 15), ventricular septal defect (n = 18), incomplete (n = 2) or complete (n = 6) atrioventricular canal (additional trisomy 21 in seven of eight patients), or aortopulmonary window (n = 1).

Hemodynamic Assessment

Cardiac catheterization was performed in all patients prior to surgical correction. Patients were sedated with phenobarbital and morphine. Oxygen consumption was measured using a metabolic analyzer (Deltatrac II; Datex Ohmeda; Duisburg, Germany). Qp and systemic blood flow (Qs) were calculated by means of the Fick equation. The Qp/Qs ratio and the resistance to pulmonary perfusion (pulmonary vascular resistance) were calculated according to standard formula. (6) Preoperative pulmonary hypertension was defined as mean pulmonary artery pressure (PAP) > 25 mm Hg as measured on cardiac catheterization. (7)

Surgical Management

Surgical management was standardized during this study. Intracardiac repair was performed through median sternotomy with standard CPB using bieaval cannulation, moderate hypothermia at 24 to 26[degrees]C, and antegrade extracellular cardioplegia.

Postrepair Evaluation and Monitoring

After weaning from CPB and warming, the BP in the pulmonary artery and aorta was measured in all patients. In those patients with persistent pulmonary hypertension defined as mean PAP > 25 mm Hg, (7) pulmonary arterial lines were placed for continuous pressure recording (n = 18).

Persistent pulmonary hypertension during the first 24 h after intracardiac repair was defined as mean PAP > 25 mm Hg as measured with pulmonary arterial lines or--in patients without pulmonary arterial lines--as an increase in right ventricular systolic pressure (> 30 mm Hg) as measured on continuous-wave Doppler echocardiography (tricuspid regurgitant velocity). Significant residual left-to-right shunt or right ventricular outflow tract obstruction were ruled out by echocardiography.

Treatment Modalities for Postoperative Pulmonary Hypertension

Patients with pulmonary hypertension after cardiac repair were placed on mechanical ventilation to keep the arterial pH > 7.45 and the Pa[O.sub.2] > 20 kPa. These patients were sedated with fentanyl and paralyzed with vecuronium bromide for at least 24 h. Inhaled NO was initiated whenever the mean pulmonary/systemic BP ratio was > 0.5. The presence of pulmonary hypertensive crises was defined as an acute rise in mean pulmonary/ systemic BP ratio > 0.7 followed by a decrease in systemic arterial BP and oxygen saturation. (8)

During the first 24 h after cardiac surgery, IV infusions for fluid replacement did not contain amino acid preparations. Oral nutrition was started only thereafter.

Blood Sampling

In all patients, blood samples were obtained from the systemic arterial line (radial artery) at following time points: (1) during surgery immediately before starting CPB; (2) during surgery immediately after weaning from CPB; and (3) at 3, 6, 12, and 24 h after weaning from CPB. Blood samples were collected in cooled tubes containing sodium ethylenediamine tetra-acetic acid (for cGMP and nitrate) or Li-heparin (1.5 IU/mL) for L-arginine. The tubes were immediately placed on ice and centrifuged at 2,000g (15 min, 4[degrees]C). The plasma was separated and stored at - 80[degrees]C.

Measurement of L-Arginine

For measurement of L-arginine, we adopted a high-performance liquid chromatography method. (9) Basic amino acids were extracted by using a cation-exchange cartridge and subjected to precolumn derivatization with o-phthaldialdeliyde. A short reversed-phase column was used, and great care was taken to separate the curve for L-hydroxy-arginine from the curve for L-arginine at the baseline of the chromatograln to exclude overlap between the two curves. An external standard with known concentration for L-arginine was used to calculate the L-arginine concentrations of samples.

The coefficients of variation for L-arginine in physiologic concentrations as a measure of the intra-assay and interassay imprecision were < 3.1% and < 6.7%, respectively. The overall recoveries for L-arginine were > 90%.

Measurement of cGMP was performed as previously described (9) using a radioimmunoassay kit purchased from Coulter Immunotech (Krefeld, Germany). For quantitative determination of nitrate in plasma, we performed a modified Griess reaction using an enzyme immunometric assay kit purchased from Assays Designs (Ann Arbor, MI).

Data Analysis

Because of the explorative hypothesis-generating nature of our study', a sample size of at least 40 patients was thought appropriate to note differences between study groups. Data are given as median (range). The Wilcoxon matched-pair, signed-rank test with adjustment of the significance level [alpha] using the Bonferroni method for multiple comparisons was used to test the hypothesis that plasma L-arginine, cGMP, and nitrate as measured before CPB would differ from values obtained after CPB. The level of statistical significance was calculated as p < 0.0033.

The influence of preoperative PAP, patient age, time for CPB, plasma L-arginine, cGMP, and nitrate as predictors for the presence or absence of postoperative pulmonary hypertension was examined by performing a logistic regression (SAS version 8.2; SAS Institute; Cary, NC). The model for logistic regression was fitted with the help of the SAS macro "glinnnix'" (SAS Institute). Independent variables were plasma L-arginine, eGMP and nitrate, preoperative PAP, time for CPB, and age of patient. In addition, the random effect of the patient itself on the development of postoperative pulmonary hypertension was taken into account as an intercept of the statistical analysis.

The response variable was chosen as "development of postoperative puhnonary hypertension at any time point after the operation." Missing values were handled by using the MI procedure of the SAS 8.2 software package: the MCMC (Markov Chain Monte Carlo) method for imputation of missing values was used. In order to avoid overfitting, (10) a logistic regression model with four covariates (L-arginine, cGMP, nitrate, and preoperative PAP) was fitted in an initial approach. In a second approach, a more complex logistic regression model (including age of patient and time for CPB) was fitted. Statistical significance was accepted at p < 0.0.5.

For assessing colinearity, the means of L-arginine, cGMP, and nitrate were calculated for each patient. The correlation between the covariates was examined by calculating the Kendall [tau] rank correlation coefficients and graphically with scatter plots.

RESULTS

Plasma L-Arginine, cGMP, and Nitrate After CPB

Plasma L-arginine was significantly lower at 3, 6, 12, and 24 h after weaning from CPB (p < 0.0033, Wilcoxon test; Fig 1). Plasma cGMP levels increased significantly only immediately after weaning from CPB (p < 0.0033, Wilcoxon test; Fig 2). Plasma nitrate levels were significantly lower only immediately after weaning from CPB (p < 0.0033, Wilcoxon test; Fig 3).

[FIGURES 1-3 OMITTED]

Impact of Plasma Levels of L-Arginine, cGMP, and Nitrate on Postoperative Pulmonary Hypertension

Thirteen of 42 patients were found to have postoperative pulmonary hypertension during the study period of 24 h after intracardiac repair (Table 1). In these patients, mean PAP was 30 mm Hg (range, 25 to 43 mm Hg) at 3 h after CPB and 38 into Hg (range, 23 to 55 mm Hg) at 24 h after CPB. Three patients required inhaled NO. Pulmonary hypertensive crises were not observed during the study period. None of the patients in this series received nitroglycerin, and only one patient was treated with sodium nitroprusside. Renal function as measured by urine output and serum creatinine was normal in all patients. In-hospital mortality of the patients studied in this series was zero.

On logistic regression, age of patient at intracardiac repair (p = 0.02), preoperative PAP (p = 0.01), and the random effect of the patient itself (intercept) (p = 0.007) were related to the development of postoperative pulmonary hypertension (Table 2). Time for CPB (p = 0.24), plasma L-arginine (p = 0.14), plasma eGMP (p = 0.09), and plasma nitrate (p = 0.28) were not related to the development of postoperative pulmonary hypertension. Both preoperative PAP (p < 0.0001) and patient age (p < 0.0005) showed a correlation with time for CPB on testing for colinearity (Kendall -r rank correlation coefficients).

DISCUSSION

One of the key questions to understand the pathophysiology of postoperative pulmonary hypertension is whether the ability of the body to endogenously synthesize NO is distorted by CPB and/or the underlying left-to-right shunt in these patients. Low plasma levels of L-arginine as measured in this series after CPB are consistent with previous findings by Barr and coworkers (5) in 26 infants. One might speculate that low plasma L-arginine could indicate a state of reduced substrate availability for NO synthase. It is well known that the proportion of L-arginine in human plasma represents only a small proportion of the total L-arginine pool. Castillo and coworkers (3) have shown that plasma-L-arginine is responsible for > 54% of ongoing NO synthesis in the healthy human. The half saturating L-arginine concentration (Michaelis constant (Km) = 2, 9 [micro]M) for endothelial NO synthase (11) suggests that this enzyme should be well saturated with substrate even at the reduced levels of plasma L-arginine that could be found in our patients after CPB, and this is consistent with the result of our statistical analysis that shows that the reduction of plasma L-arginine was not related to the development of postoperative pulmonary hypertension.

cGMP is catalyzed by NO synthase and acts as an intracellular second messenger to activate protein kinase G, which in turn leads to smooth-muscle cell relaxation. Plasma levels of cGMP peaked after CPB, which is consistent with previous findings. (8) High plasma cGMP levels as observed in this series immediately after weaning from CPB could result from cellular damage by extracorporeal circulation. The fact that plasma cGMP was not significantly different at 3, 6, 12, and 24 h after weaning from CPB argues against an increased activation of the L-arginine NO pathway during this period.

Nitrate is the final end product of the L-arginine NO pathway and--as measured in the fasting state--may indeed reflect the actual ongoing NO synthesis. Conflicting data exist from previous studies concerning plasma nitrate levels in patients with left-to-right shunt after intracardiac repair: Duke and coworkers (12) reported no evidence of an increased activation of the L-arginine NO pathway with little changes in plasma nitrate after intracardiac repair. In children with left-to-right shunt and mild or severe pulmonary hypertension before intracardiac repair, Hiramatsu et al (13) also reported no change in plasma nitrate as measured immediately after weaning from CPB. In contrast, Seghaye et al (14) and Barr et al (5) reported a decrease in plasma nitrate at 12 h and 24 h after CPB in patients with left-to-right shunt and pulmonary hypertension. Finally Bando and coworkers (8) reported an increase in plasma nitrate and cGMP after CPB when comparing their data obtained in patients with high Qp and high pulmonary/pressure with patients with low Qp (eg, tetralogy of Fallot).

Plasma nitrate is influenced by several exogenous factors, such as treatment with NO donors (eg, nitroglycerin, sodium nitroprnsside, NO inhalation) or renal function. The controversies existing in the literature on plasma nitrate after intracardiac repair can be resolved when taking into account these factors: in our series, only 3 of 42 patients received inhaled NO, and only 1 patient received sodium nitroprusside. None of our patients was in renal failure. Duke et al (12) performed a subgroup analysis in 16 patients without renal impairment and without NO donors, and in these patients plasma nitrate actually decreased significantly after intracardiac repair, as in our and other (14) series, whereas patients receiving nitrate drugs showed a significant increase in plasma nitrate at 24 h after CPB. In the series presented by Bando et al, (8) 74% of their patients with pulmonary hypertension and high Qp received nitroglycerin after intracardiac repair, serving as explanation for the observed increase in plasma nitrate in their series. In addition, a mathematical correction was applied for the effect of hemodilution during CPB by these authors, (8) whereas such corrections were not attempted in the series of Duke et al, (12) Hiramatsu et al, (13) nor in our own series.

In summary, our findings indicate that endogenous synthesis of NO after CPB for left-to-right shunt is reduced only immediately after weaning from CPB, but shows little changes thereafter during the first 24 h after intracardiac repair for left-to-right shunt. In addition, there is no deficiency for L-arginine, the substrate for endothelial-derived NO synthase. Again, well-known factors such as age of patient at operation and the presence of preoperative pulmonary hypertension were the most important factors to predict pulmonary hypertension during the first 24 h after CPB.

The present study has several limitations. Tracer infusion studies using nonradioactive isotopes (eg, with L-[guanidino-(15) [N.sub.2]]arginine), which allow for a better characterization of the L-arginine turnover in humans, (3) were not performed. Our data do not argue against a trial analyzing the benefit of pharmacotherapy with L-arginine to prevent postoperative pulmonary hypertension. The amount of blood needed for the measurement of L-arginine and other metabolites by high-performance liquid chromatography in this series did not allow for additional measurements of other vasoactive factors in plasma. In this pilot study, we were therefore not able to measure other vasoactive factors that exert an effect on the balance of pulmonary vasodilatation/vasoconstriction after CPB (eg, endothelin-1). In addition, the effect of inhaled NO and sodium nitroprusside on the metabolites of the L-arginine NO pathway could not be analyzed since only few patients received such medications in our study.

CONCLUSION

In conclusion, we have shown that endogenous NO synthesis is not changed significantly after intracardiac repair in children with left-to-right shunt. The decrease in plasma L-arginine observed after CPB does not relate to postoperative pulmonary hypertension. Whether or not children undergoing CBP for left-to-fight shunt will benefit from a perioperative therapy with L-arginine remains to be established by future trials.

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* From the Department of Pediatric Cardiology (Drs. Gorenflo, Eitel, and Gross), Department of Cardiac Surgery (Drs. Ullmann and Hagl), Central Laboratory of the Department of Medicine (Dr. Fiehn), and the Department of Medical Biometry and Informatics (Dr. Dreyhaupt), University Medical Center, Heidelberg, Germany.

Drs. Gorenflo and Ullmann equally contributed to this work.

Drs. Gorenflo and Ullmann were supported by a grant from the German Heart Foundation (No. F02/02).

Manuscript received January 12, 2004; revision accepted November 1, 2004.

Reproduction of this article is prohibited without written permission from the American College of Chest Physicians (e-mail: permissions@chestnet.org).

Correspondence to: Matthias Gorenflo, MD, Department of Pediatric Cardiology, University Medical Center, INF 153, D-69120 Heidelberg, Germany; e-mail: Matthias_Gorenflo@med. uni-heidelberg.de

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