Correlation Study Of The Glucuronic Acid Pathway Metabolites In Plasma And Pulmonary Hypertension Based On The Mass Spectrometry Technology

Background:Idiopathic pulmonary hypertension（IPAH）is a rare disease which the incidence is the second only lower than that of thalassemia.The median survival period of patients is only 2.8 years.In the case of adequate treatment,the one-year mortality is still as high as15%.what is more,there is still no specific drug in clinic.Studies have found that pulmonary hypertension has a similar Warburg effect with tumor,that is,cells still tend to glycolysis to obtain energy even under aerobic conditions.This suggests that the disorder of glucose metabolism pathway plays an important role in the occurrence and development of pulmonary hypertension.The glucuronic acid pathway is one of the bypasses of glucose metabolism.Its product,Uracil diphosphate-glucuronic acid（UDP-Glc UA）is one of the components of glycosaminoglycan（GAG）,and GAG is one of the components of extracellular matrix.The extracellular matrix can be degraded by lysosomes in cells to finally produce D-glucuronic acid（D-Glc UA）.UDP-glucose-6-dehydrogenase（UGDH）is a key enzyme in the glucuronic acid pathway.Studies have found that UGDH can promote the migration of tumor cells and may be used as a new specific candidate biomarker of prostate cancer^[1].Due to the similar characteristics of pulmonary hypertension and tumor and the important role of metabolites of this pathway in the development of tumor,we have reason to believe that it also plays an important role in the development of pulmonary hypertension.Objective:Based on the mass spectrometry quantitative technology,the changes of metabolites among glucuronic acid pathway under rat pulmonary hypertension model induced by Monocrotaline were studied,and the metabolites with significant changes were screened out.The correlation between its concentration and hemodynamic indexes and vascular remodeling indexes of pulmonary hypertension was analyzed.It is proved that the index is still significantly increased in clinical research,and the change trend of its concentration in human body is preliminarily studied,so as to provide a basis for its later use as a diagnosis and treatment index of pulmonary hypertension.Methods:（1）To establish a mass spectrometry quantitative technology:establish an ultra performance liquid chromatography mass spectrometer method that can simultaneously,accurately and precisely quantify the concentrations of D-Glc UA and UDP-Glc UA in rat plasma and human plasma;（2）Animal experiment:the rat model of pulmonary hypertension was established to collect plasma samples and lung tissue samples.The concentrations of D-Glc UA and UDP-Glc UA in plasma samples were detected and statistically analyzed with those in control group to screen out metabolites with significant changes in concentration（P<0.05）.Pearson correlation coefficient in statistical software SAS 9.4was used for correlation analysis to study the correlation between differential metabolites and right ventricular systolic pressure,right heart index[RV/（LV+S）]and pulmonary artery remodeling[（adventitia diameter-intima diameter）/adventitia diameter];（3）Clinical study:plasma samples from patients with idiopathic pulmonary hypertension and healthy volunteers were collected to confirm that the differential metabolites were still significantly elevated in the patient’s plasma.Plasma samples of healthy volunteers at different time points within 3days were collected to study the concentration changes of the differential metabolites.Results:（1）The linearity of D-Glc UA and UDP-Glc UA in plasma is good in the concentration range of 50.000～1000.00 ng·m L^-1（r>0.99）.The established UPLC-MS/MS method can detect the concentrations of D-Glc UA and UDP-Glc UA in plasma simultaneously,accurately and accurately;（2）Compared with the control group,the hemodynamic indexes such as RVSP and[RV/（LV+S）]were significantly increased,and HE staining showed obvious pulmonary vascular remodeling;（3）Compared with the control group,the concentration of D-Glc UA in plasma of pulmonary hypertension rats was significantly higher（P<0.001）,which was a significant differential metabolite.UDP-Glc UA was not detected in the plasma of both groups.（4）There was a strong correlation between plasma D-Glc UA concentration and right ventricular systolic pressure（r=0.8094）;There was a strong correlation between plasma D-Glc UA concentration and right heart index[RV/（LV+S）]（r=0.8627）;There was a strong correlation between plasma D-Glc UA concentration and the degree of pulmonary artery remodeling（r=0.8871）;（5）Compared with the control group,The plasma concentration of D-Glc UA in patients with idiopathic pulmonary hypertension increased significantly（P<0.01）.UDP-Glc UA was not detected in the plasma of both groups,and the results were consistent with the animal experiment;（6）The plasma concentration of D-Glc UA in healthy volunteers was not disturbed by high fat diet and other factors,and there was no significant change in the monitoring time of 3 days（P>0.05）,which indicates the D-Glc UA was a relatively stable index.Conclusion:（1）The plasma concentration of D-Glc UA in monocrotaline induced pulmonary artery rats was significantly higher than that of the control group,and was strongly correlated with pulmonary artery hemodynamic indexes（right ventricular systolic pressure and right heart index）and the degree of pulmonary artery remodeling;（2）The plasma concentration of D-Glc UA in patients with idiopathic pulmonary hypertension was significantly higher than that in healthy volunteers,and its concentration was not disturbed by factors such as high-fat diet.It was a relatively stable index.