Noninvasive Monitoring of Severe Pulmonary Artery Hypertension in Atrial Septal Defect Patients: Role of Serum Bilirubin Combined with Uric Acid

Background : Atrial septal defect (ASD) patients commonly experience severe pulmonary arterial hypertension (SPAH), which is frequently associated with a poor prognosis. While serum bilirubin levels, indicative of liver function, are known predictors of right heart failure (RHF), their potential to differentiate SPAH in ASD patients is yet to be ascertained. The purpose of this study was to discover the potential correlations between serum bilirubin levels and ASD patients with SPAH. Methods : In this cross-sectional study, 102 ASD patients admitted from December 2019 to November 2020 were enrolled and divided into two cohorts: those with SPAH and those without. Blood tests were conducted to measure serum direct bilirubin (DBIL), total bilirubin (TBIL), alanine aminotransferase (ALT), aspartate aminotransferase (AST), uric acid (UA) and N-terminal pro B-type natriuretic peptide (NT-proBNP). Additionally, all participants underwent transthoracic echocardiography, and invasive hemodynamic data were gathered through right heart catheterization. Results : ASD patients with SPAH exhibited significantly elevated serum DBIL (5.2 ± 3.0 vs. 2.4 ± 1.5 µmol/L, p < 0.001) and TBIL (24.6 ± 20.7 vs. 10.1 ± 4.8 µmol/L, p < 0.001) levels in comparison to those without SPAH. However, ALT and AST levels remained comparable between the cohorts. Additionally, the SPAH cohort displayed higher serum UA (403.5 ± 131.6 vs. 317.8 ± 67.9 µmol/L, p < 0.001) and NT-proBNP levels. Serum DBIL levels, when analyzed independently of other variables, correlated with an increased risk of mean pulmonary arterial pressure (mPAP) in ASD patients ( β = 1.620, p = 0.010). A DBIL concentration of 2.15 mg/dL effectively differentiated ASD patients with SPAH from those without, with a sensitivity of 92.9% and a specificity of 51.4% (area under the curve [AUC]: 0.794, 95% confidence interval [CI]: 0.701–0.886, p < 0.001). Notably, the combination of DBIL and UA had a higher sensitivity of 92.9% and specificity of 71.6% (AUC: 0.874, 95% CI: 0.799–0.949, p < 0.001). Conclusions : Elevated serum DBIL and TBIL levels in ASD patients with SPAH were correlated with poor cardiac function and heightened pulmonary artery pressure. The combination of DBIL and UA has emerged as a strong noninvasive predictor for SPAH in ASD patients, presenting a potentially novel therapeutic biomarker.


Introduction
Atrial septal defect (ASD) is a frequently occurring congenital heart deficit.Between 6% and 35% of ASD patients develop pulmonary arterial hypertension (PAH), which can lead to increased mortality, diminished cardiac function, and atrial tachyarrhythmias [1][2][3][4][5][6].Severe pulmonary arterial hypertension (SPAH) in patients with ASD has a poor prognosis [7].Currently, therapeutic strategies for ASD with SPAH are controversial.Our previous study demonstrated that mean pulmonary arterial pressure (mPAP) was a simple but powerful predictor of the benefits of ASD closure in these patients, with an optimal cut-off value of 35 mmHg and an area under the curve (AUC) of 0.919 [5].
Nevertheless, in ASD patients with SPAH, closure may not always decrease pulmonary artery pressure due to factors like vessel remodeling and decreased vascular compliance in ASD patients with SPAH [6].Yong et al. [8] reported that after transcatheter ASD closure, most patients with severe PAH continue to have elevated pulmonary artery pressures, which might be due to irreversible vessel changes [7].Therefore, it is important to identify SPAH in ASD patients to choose the best timing for ASD occlusion.While right heart catheterization has long been the gold standard for determining SPAH, the procedure is invasive.There is a growing emphasis on biomarker studies to prompt an earlier initiation of more aggressive therapies.These biomarkers may be used for identifying potential SPAH in ASD patients.Bilirubin is a promising biomarker for assessing patients within this population.It is a metabolic byproduct of hemoglobin breakdown that also acts as an endogenous antioxidant molecule [9].Total bilirubin (TBIL) is well established prognostic factor in heart failure [10] and PAH [8].However, most prior PAH studies focused on patients with predominantly idiopathic or connective tissue diseaserelated cases.Few studies have focused solely on ASDassociated PAH, which has distinct pulmonary hemodynamics and pathophysiology.Since the role of bilirubin in ASD-SPAH remains unknown, this study was undertaken to investigate the role of serum bilirubin to assess its significance in ASD patients, especially in those with SPAH.

Study Populations
This cross-sectional study included 102 patients with ASD admitted to Zhongshan Hospital, Fudan University, from December 2019 to November 2022.The diagnosis of PAH was based on right-heart catheterization values [11]: mPAP ≥20 mmHg, pulmonary arterial wedge pressure (PAWP) ≤15 mmHg, and pulmonary vascular resistance (PVR) >3 Wood units.Patients were categorized based on catheter mPAP as: normal (≤20 mmHg), mild (20-35 mmHg), moderate (35-45 mmHg), or severe PAH (>45 mmHg) [12].To identify patients with SPAH, these individuals were divided into two groups (ASD with SPAH and ASD without SPAH) according to a mPAP >45 mmHg.Exclusion criteria: (1) patients with patent foramen ovale, significant pulmonary stenosis, or Ebstein's anomaly; (2) patients with other identifiable causes for precapillary pulmonary hypertension, such as chronic thromboembolic pulmonary hypertension; (3) lung transplantation before ASD closure; (4) patients who have already been treated with specific PAH agents; (5) diseases resulting in hyperbilirubinemia, such as chronic hepatitis, malig- nant tumors, administration of hepatotoxic drugs, alcohol dependence, hemolysis and hematoma reabsorption, schistosomiasis, and biliary system diseases.A detailed medical history and abdominal ultrasonography were performed to exclude chronic liver disease.Urine samples were performed to screen for hemolysis or hematoma absorption.
Baseline characters and clinical data were collected from electronic medical records.TTE was performed using Vivid 7GE (G.E.Healthcare, Chicago, IL, USA) by an experienced sonographer, and echocardiographic parameters including left ventricular ejection fraction (LVEF), left atrial diameter (LAD), and left ventricular end-diastolic (LVED) were validated by two cardiology consultants.The diameter, type, and size of the defect and the anatomy of the ASD were examined by cardiology consultants.This study was performed in compliance with the Helsinki Declaration and was approved by the Ethics Committee.Written informed consent was obtained from all participants.

Blood Collection and Biochemical Measurements
The peripheral venous blood samples were obtained with the consent of the patients.Serum TBIL, direct bilirubin (DBIL), uric acid (UA), aspartate transaminase (ASL), alanine transaminase (ALT), and N-terminal pro B-type natriuretic peptide (NT-proBNP) were measured just before right heart catheterization at the Central Clinical Laboratory of our Hospital.The normal value of TBIL is 1.71-17.1 µmol/L, and DBIL 1.71-7.0µmol/L in our lab.

Right Heart Catheterization
Right heart catheterization was employed by cardiologists to evaluate PAH severity.A multipurpose catheter was inserted into the right atrium via the inferior vena cava and placed in the pulmonary veins across the ASD.A Swan-Ganz balloon-tipped catheter was placed in the pulmonary arteries through the right atrium and right ventricle.Hemodynamic parameters such as pulmonary arterial systolic pressure (PASP) and mPAP were then evaluated.

Statistical Analysis
Continuous variables with a normal distribution were presented as mean ± SD while non-normally distributed data were presented as median (25th-75th percentile).Categorical data were described as percentages.The Unpaired Student's t test and the χ 2 test were used to analyze the statistical differences between the two groups.Correlations between continuous data (for normally distributed data) were analyzed with Pearson correlation tests.Singlevariate and multi-variate linear regression analyses were performed to evaluate the degree of correlation between plasma biomarkers and clinical indicators.Sensitivity, specificity, as well as areas under the curve (AUC) were obtained using receiver operating characteristic (ROC) curve analysis.Results were considered statistically significant for p values < 0.05.All statistical analyses were performed using Stata 15.1 (StataCorp LP, College Station, TX, USA) or SPSS 26.0 (IBM Corp., Armonk, NY, USA).

Association of Serum Biomarkers and mPAP among ASD Patients
To explore the association between various parameters and PASP in ASD patients, linear regression analyses were performed (Table 3).Single-variable linear regression showed that ASD diameter, TBIL, DBIL, RVD, TAPSE, NT-proBNP and UA were significantly associated with increased mPAP among ASD patients with SPAH.Further multivariate linear regression analysis demonstrated that serum DBIL (β = 1.552, p = 0.015) independently cor-related with PASP, regardless of other variables including ASD diameter, RVD, TAPSE, TBIL, NT-proBNP, and UA.Similarly, UA levels (β = 0.030, p = 0.007) were independently associated with mPAP in ASD patients with SPAH, when controlled for other parameters such as ASD diameter, RVD, TAPSE, TBIL, NT-proBNP, and DBIL.

Discussion
This study demonstrates that elevated bilirubin levels (both DBIL and TBIL) are present in patients with both ASD and SPAH.Both DBIL and TBIL levels correlated with the cardiac function markers NT-proBNP, RVD, TAPSE, LVED, PASP and mPAP.Moreover, DBIL alone or in combination with UA can differentiate between ASD patients with SPAH and without SPAH with relatively high sensitivity and specificity.
Our study showed serum TBIL and DBIL were increased in ASD patients with SPAH, but without any concurrent increase in AST or ALT.The mechanisms causing elevated serum levels of TBIL and DBIL in ASD patients with SPAH are still unclear.One hypothesis suggests that for ASD patients, the elevated mPAP may increase the right atrial and ventricular load, which is expected to increase the risk of right heart failure (RHF).This RHF condition could manifest as liver dysfunction, potentially due to hepatic stasis and reduced perfusion.Changes in the right heart hemodynamic may be due to elevated central venous pressure (CVP) via the hepatic vein into the small hepatic veins [13].This impairment can further hinder the delivery of oxygen and nutrients to hepatocytes, resulting in the expansion of sinusoidal fenestrations [13].The resulting cholestatic and hypoxic hepatic injury caused by elevated CVP may contribute to an increase of bilirubin.In fact, DBIL has been identified as a critical marker of elevated CVP in other studies [10,14,15].Furthermore, other researchers have highlighted the positive correlation between serum bilirubin with right atrial pressure and the severity of tricuspid regurgitation [16].In our study, the damage to hepatocytes was limited, as indicated by the absence of statistical differences in ALT and AST between ASD with or without the SPAH.However, the liver biomarkers DBIL and TBIL were reflective of right heart function and were associated with the risk of SPAH in ASD patients.
Some basic researchers have sought to explore the relationship between bilirubin and PAH.Bilirubin is a tetrapyrrole pigment in blood that has both direct and indirect forms, and is known for its antioxidant and antiinflammatory activity [17][18][19].Physiological concentrations of bilirubin inhibit nuclear factor kappa-B (NF-κB) activation and inflammasome, contributing to its antiinflammatory effects [17].Thus, specific quantities of bilirubin may serve as a mitochondria-targeting agent for treating relevant diseases [20].Prior studies have shown that bilirubin reductase attenuates the effects of hypoxia on apoptosis in pulmonary artery smooth muscle cells by modulating the bilirubin-mediated (extracellular signalregulated kinase 1/2) ERK1/2 pathway [21].Furthermore, it has been shown that oxidative stress serves as a critical etiological factor in the progression of PAH [22][23][24].A study by Curjuric et al. [25] examined the influences of air pollution on adult lung disease, identifying a protective effect of bilirubin on lung function, emphasizing its potential preventive and curative significance.Therefore, increased bilirubin may have a protective effect on ASD patients with SPAH.
Our study demonstrated that the serum DBIL and TBIL levels can predict SPAH in ASD patients, with DBIL demonstrating a sensitivity of 92.9% and a specificity of 51.4%, while TBIL exhibited a sensitivity of 89.3% and a specificity of 62.2%.Echoing our observations, Xu et al. [26] showed that abnormally elevated DBIL was independently linked to all-cause mortality among idiopathic PAH patients, and treating PAH survivors significantly decreased serum DBIL levels.In contrast, almost no decrease in serum DBIL was found in non-survivors [27].In addition, hyper-bilirubinemia (TBIL) was associated with advanced RHF, which then markedly reduced survival in patients with PAH [24].Gong et al. [28] found that elevated serum bilirubin and reduced six-minute walk distance (6MWD) was identified as a predictor of adverse outcomes in patients with chronic thromboembolic pulmonary hypertension.While our study did not evaluate the prognostic implications for ASD patients with abnormal DBIL and TBIL levels, we observed that patients with SPAH presented more severe structural and hemodynamic symptoms compared to their non-SPAH counterparts.Therefore, our future research will focus on defining the role of DBIL and TBIL in predicting the prognosis of ASD patients with SPAH.
We observed that ASD patients with SPAH had significantly higher UA levels when compared to those without SPAH.Supporting this, Yan et al. [29] found that baseline hyperuricemia and high variability in serum UA were associated with higher 5-year mortality in patients with idiopathic PAH (IPAH).Savale et al. [30] suggests that UA levels can partly reflect the severity of PAH, with higher concentrations of UA promoting mild proliferation of pulmonary artery smooth muscle cells in patients with idiopathic PAH and in rat models.In our studies, DBIL levels combined with UA had a sensitivity (92.9%) and specificity (71.6%) to discriminated ASD individuals with SPAH.Therefore, UA combined with DBIL might better predict poor prognosis in these patients.

Limitations
Our study has several limitations.Firstly, 6MWD was not analyzed in regression analysis due to incomplete data, preventing us from supplementing the study with 6MWD results.Secondly, the organizational roles of DBIL and TBIL in ASD patients with SPAH have not been explored in our study.Attempts to solve this problem using both animal and cellular experiments are underway.Thirdly, long-term follow-up should be conducted to evaluate whether DBIL and TBIL could predict the prognosis of ASD patients suffering from SPAH.In conclusion, given our current sample size, it's challenging to rule out potential discrepancies.Further extensive, large-scale studies are needed to validate our findings.

Conclusions
Overall, we found that elevated serum DBIL and TBIL levels in ASD patients with SPAH are correlated with prognostic clinical markers.Utilizing DBIL combined with UA may serve as a safe, cost-effective, and powerful predictor of SPAH in patients with ASD, potentially introducing a novel therapeutic biomarker.