Metabolic Changes And Their Roles In Adult Patients With Pulmonary Arterial Hypertension Associated With Congenital Heart Disease

Pulmonary arterial hypertension（PAH）is one of the clinical types of pulmonary hypertension（PH）.PAH associated with congenital heart disease（PAH-CHD）is a kind of pre-capillary PH,which refers to the elevated pulmonary arterial pressure caused by congenital heart disease（CHD）allowing prevalent left-to-right shunting.In China,CHD has become the first cause of PAH and the number of adult patients with PAH-CHD is increasing every year.Similar to idiopathic PAH（IPAH）,the prognosis of PAH-CHD is poor.Although targeted therapy improves the prognosis of patients with PAH-CHD to some extent,it is far from cure.The ultimate treatment for those patients with advanced PAH-CHD is heart-lung transplantation.Metabolic alterations including energy metabolism shifting are gradually considered to be the universal hallmarks from PAH models to patients.The metabolic characteristics of PAH-CHD patients have attracted much attention since they are different from IPAH patients.However,there were few metabonomic studies on the pure PAH-CHD cohort,which leads to the incomplete metabolic profile of PAH-CHD patients,and the correlations between metabolic alterations and clinical findings remain unclear.Hence,we conducted this metabolism-oriented study to solve these problems in three aspects:firstly a clinical retrospective analysis of adult PAH-CHD patients was performed to explore the relationship between the metabolic changes and PAH progress,and then the metabonomic analysis of plasma from adults patients with PAH-CHD and CHD and healthy subjects was performed to demonstrate the metabolic characteristics of PAH-CHD and search for potential biomarkers using ¹H-NMR based metabonomic platform,and in the third part we analyzed the metabolic differences between the patients with IPAH and PAH-CHD.PartⅠ:The retrospective analysis of adult patients with PAH-CHDObjective:By retrospectively analyzing the clinical data,we sought to explore the metabolic changes of adult PAH-CHD patients and the relationship of metabolic changes and PAH,and to analyze the relevant factors of right ventricular systolic dysfunction in adult PAH-CHD patients.Methods:According to the results from cardiac catheterization,adult CHD patients with PAH hospitalized from January 1,2018 to December 31,2020 were consecutively enrolled as the PAH-CHD group（127 cases）,while those without PAH were enrolled as the CHD group（154 cases）.The detailed clinical data of the two groups were collected.The relevant clinical data were analyzed to search for the potential blood biomarkers with diagnostic and prognostic value between the two groups.The factors associated with right ventricular systolic function were analyzed by univariate analysis and Logistic regression analysis.Results:（1）Compared with that of CHD group,the BMI of PAH-CHD group significantly decreased（P<0.01）,and the patients with prediabetes and hyperuricemia were more prevalent（all P<0.01）in PAH-CHD group as well as the active smokers（P<0.05）and patients with post-tricuspid defect（P<0.01）.The proportion of patients with cardiac function classⅢ-Ⅳand patients using digoxin and diuretics were higher in PAH-CHD group than in CHD group（all P<0.01）.（2）The parameters from transthoracic echocardiography（TTE）showed that the right ventricle size,the diameter of main pulmonary artery and the thickness of interventricular septum and right ventricular wall of PAH-CHD group were significantly greater than CHD group（all P<0.01）.In PAH-CHD group,the tricuspid valve regurgitation was more obvious（P<0.05）and the right ventricular systolic functional parameters（including FAC,TAPSE and systolic tricuspid annulus velocity）were significantly lower in PAH-CHD group when compared with CHD group（all P<0.01）.（3）Compared with CHD group,the PAP,PVR,RC time and RVSWI in PAH-CHD group were significantly increased while the Sv O2,Qp/Qs,RVCI,LVCI and Cp were significantly decreased（all P<0.01）.（4）Compared with CHD group,RBC,Hb,RDW-SD and RDW-CV in PAH-CHD group were significantly increased（all P<0.01）as well as the levels of pro BNP（P<0.01）,hs-c Tn T（P<0.01）,LDH（P<0.01）,HBDH（P<0.01）,Hb A1c（P<0.01）,TB（P<0.01）,DB（P<0.01）and TBA（P<0.05）.In PAH-CHD group,the level of TC（P<0.05）and HDL-C（P<0.01）decreased significantly while free fatty acid（FFA）and TG/HDL-C ratio increased significantly（all P<0.01）compared with CHD group.The activity of AST,γ-GT,ALP and ADA was significantly up-regulated in PAH-CHD group（all P<0.01）,while the activity of Che was down-regulated（P<0.05）.BUN,Cr and UA significantly increased（all P<0.01）while CO₂Cp decreased significantly（P<0.01）in PAH-CHD group.The levels of hs CRP,TSH and FT4 were significantly increased in PAH-CHD group（all P<0.01）.（5）ROC curve analysis showed that there were six blood indexes with AUC>0.70including HBDH（AUC 0.848,P<0.01）,LDH（AUC 0.837,P<0.01）,pro BNP（AUC 0.813,P<0.01）,Hb（AUC 0.780,P<0.01）,RBC（AUC 0.727,P<0.01）and UA（AUC 0.719,P<0.01）,and the AUC for combination of these six blood indexes was 0.913.（6）The factors associated with right ventricular systolic dysfunction were age,RDW,Sa O₂,markers of heart failure and myocardial injury,Hb A1c,UA,remodeling of right ventricle and pulmonary artery,Cp and so on.The post-tricuspid defects were more common in patients with right ventricular systolic dysfunction（P<0.05）.Logistic regression showed that only post-tricuspid defect（OR=6.70,P<0.05）was the independent risk factor of right ventricular systolic dysfunction in adult patients with PAH-CHD.Conclusion:（1）PAH-CHD patients tend to complicate with metabolic disorders of glucose metabolism,lipid metabolism,purine metabolism and bilirubin metabolism.（2）The panel of RBC,Hb,pro BNP,UA,LDH and HBDH from peripheral venous blood assays could be used as the potential biomarkers to identify PAH-CHD from CHD patients.（3）PAH-CHD patients with post-tricuspid defects have the increased risk developing right ventricular systolic dysfunction.PartⅡ:Study on the characteristics of blood metabolites in adult PAH-CHD patients based on the ¹H-NMR metabonomic techniquesObjective:To investigate the metabolic characteristics of adult PAH-CHD patients and their roles in the pathogenesis of PAH and to search for novel potential biomarkers of PAH-CHD through detecting and quantifying the plasma metabolites of adult PAH-CHD patients and controls using untargeted ¹H-NMR based metabonomic platform combinated with correlation analysis between characteristic metabolites and clinical evaluation indexes of PAH.Methods:Adult patients with CHD were prospectively enrolled and divided into PAH-CHD group（PAH-CHD patients,21 cases）and CHD group（CHD patients without PAH,36 cases）.The patients of PAH-CHD group were further divided into two subgroups according to the defect location:pre-tricuspid defect subgroup（12 cases）and post-tricuspid defect subgroup（9 cases）.The patients of CHD group were further divided into two subgroups according to the m PAP levels:pre PAH subgroup（m PAP>20mm Hg and PVR<3WU）（11 cases）and m PAP≤20mm Hg subgroup（25 cases）.Twenty-three health controls were also enrolled in our study as HC group.The 1D ¹H-NMR plasma spectrums were acquired by using 850MHz NMR spectrometer（Bruker,Germany）following the plasma extracted from fasting venous blood of each subjects and sample preparation.The spectrums were preprocessed,metabolites were identified and the integral of each metabolite was extracted from each spectrum.Then,the integrals of metabolites were analyzed by multivariate,univariate analysis and pathway analysis between each two groups,by which the characteristic metabolites and metabolic pathways were screened out.The biological significance of metabolic alterations of PAH-CHD was translated by integrating the metabolic pathways involved with online database.ROC curve analysis and correlation analysis were carried out to identify the potential biomarkers and assess their roles in clinical evaluation of adult PAH-CHD patient.Results:（1）A total of 31 metabolites were identified from the typical 1D ¹H-NMR spectra.（2）Multivariate analysis showed the metabolic pattern of PAH-CHD group was different from that of CHD group or HC group and the metabolic pattern of CHD group was different from that of HC group.It also showed the metabolic pattern of PAH-CHD group was different from that of pre PAH subgroup and there was no metabolic pattern difference between the pre-tricuspid defect and post-tricuspid defect subgroup.（3）Combined with univariate analysis,the characteristic metabolites of PAH-CHD group（vs CHD group）were lipoprotein（i.e.LDL/VLDL）,choline,glucose,alanine,lactate and serine,and the characteristic metabolites of PAH-CHD group（vs HC group）were choline,methanol,alanine,glucose.Pathway analysis showed the characteristic metabolic pathways of PAH-CHD were glycine,serine and threonine metabolism,pyruvate metabolism,phenylalanine,tyrosine and tryptophan biosynthesis,phenylalanine metabolism when referred to CHD,and they were glycine,serine and threonine metabolism,pyruvate metabolism,phenylalanine,tyrosine and tryptophan biosynthesis,phenylalanine metabolism,histidine metabolism when referred to HC.（4）ROC curve analysis showed that choline（AUC 0.833）,glucose（AUC0.704）,alanine（AUC 0.745）,lactate（AUC 0.783）,serine（AUC 0.831）and their combination（AUC 0.938）could be used as potential biomarkers between PAH and CHD group.In addition,correlation analysis showed that choline,glucose,lactate and serine were positively correlated with PVR and m PAP（all P<0.05）while alanine was negatively correlated with them（all P<0.01）,and choline and lactate were positively correlated with pro BNP（all P<0.05）while alanine was negatively correlated with pro BNP（P<0.05）.Conclusion:（1）The characteristics of plasma metabolites in adult PAH-CHD patients were significantly different from CHD patients or healthy subjects,but the metabolic characteristics between PAH-CHD patients with pre-and post-tricuspid defects are not different.（2）Choline,glucose,alanine,lactate and serine significantly correlate with m PAP and PVR;choline and lactate also significantly correlate with pro BNP.These findings suggest that these metabolites may be the potential biomarkers of aldult PAH-CHD.（3）The metabolic alterations of adult PAH-CHD patients are characterized by the disorders of glucose,lipid and amino acid metabolism and the decreased mitochondrial aerobic oxidation function,which promotes the PAH progress.PartⅢ:Study on the differences of blood metabolic profiles between adult PAH-CHD and IPAH patients based on ¹H-NMR techniqueObjective:To investigate the metabolic characteristics of adult IPAH patients based on untargeted ¹H-NMR technique and to comparatively analyze the blood metabolic characteristics between adult PAH-CHD and IPAH patients.Methods:Twenty-one adult PAH-CHD patients（as PAH-CHD group）,12 adult IPAH patients（as IPAH group）and 23 healthy adults（as HC group）were enrolled in the study.Fasting venous blood samples were collected from enrolled patients and healthy subjects followed by plasma extraction and NMR samples preparation,then the 1D ¹H NMR spectra of each sample was carried out by 850MHz NMR spectrometer（Bruker,Germany）and preprocessed prior to assigning the metabolites and extracting their integrals.Multivariate analysis and univariate analysis were performed for IPAH group（vs HC）and PAH-CHD group（vs IPAH）followed by pathway analysis to screen out the characteristic metabolites and metabolic pathways.ROC curve analysis was used to evaluate the predictive ability of potential biomarkers from characteristic metabolites between groups.Results:（1）The metabolic profile of IPAH was different from that of HC.The characteristic metabolites of IPAH were lipoprotein,glucose,glutamine,alanine,acetylacetate and glycine and characteristic metabolic pathways involved were histidine metabolism,ketone synthesis and degradation,glycine,serine and threonine metabolism and pyruvate metabolism.（2）ROC curve analysis showed that each AUC of the 6 characteristic metabolites were all above 0.80,while 0.99for combination with all of them.（3）PAH-CHD represented significant differences in metabolic pattern compared with IPAH.The characteristic metabolites were lipoprotein,glucose,choline,methanol,glutamine and serine.The characteristic metabolic pathways were glycine,serine and threonine metabolism,biosynthesis of phenylalanine,tyrosine and tryptophan,phenylalanine metabolism,histidine metabolism and ketone body synthesis and degradation.（4）ROC curve analysis showed that lipoprotein（AUC 0.857）,glucose（AUC 0.885）,choline（AUC 0.933）,methanol（AUC 0.893）,glutamine（AUC 0.865）,serine（AUC 0.913）and their combinations（AUC 0.961）could be used as potential biomarkers to identify IPAH from PAH-CHD patients.Conclusion:（1）The metabolic disorders of adult IPAH patients are characterized by the decrease of glycolysis,increase of fatty acidβ-oxidation and ketone body synthesis.（2）The metablic characteristics of adult PAH-CHD patients are different from adult IPAH patients.They mainly represent increased lactate production,decreased fatty acidβ-oxidation,dysfunction of tricarboxylic acid cycle and increased ketone body decomposition when referred to adult IPAH patients.（3）The characteristic metabolites including lipoprotein,glucose,choline,methanol,glutamine and serine can be used as potential biomarkers to identify IPAH from PAH-CHD.