The Establishment Of Of Pheochromocytoma New Diagnostic Methods And The Treatment Of Heart Failure In Sgl-3, Sgl-1 Pharmacology Research

Background Pheochromocytoma is a rare tumor of adrenal medulla.If the tumor is not diagnosed and treated in time,it could lead to various cardiovascular or cerebrovascular diseases and even death.The imaging or biochemical tests now used for detecting pheochromocytoma have been proved to have some shortages,such as low sensitivity or specificity.Recently both metanephrine and normetanephrine,two O-methylated metabolites of catecholamine,were recognized internationally as the preferred monitoring indicators for pheochromocytoma.The objectives of this study are to establish the method of measuring metanephrine and normetanephrine in 24h urine using high performance liquid chromatography,to investigate its application and reliability,and to set up the diagnostic criteria and range of normal values.Moreover,the superiority of metanephrine and normetanephrine measurement in the diagnosis of the pheochromocytoma could be validated by comparing catecholamine levels in plasma and urine.Methods In the study,solid-phase extraction cartridge is used for the extraction of metanephrine and normetanephrine from the samples,and high performance liquid chromatography is used for their measurement.The variations of equipment,intra-assay variations,inter-assay variations and recoveries of metanephrine and normetanephrine were detected to examine the stability of the method.The measurement of metanephrines in 24h urine samples of 324 hypertensive non-pheochromocytoma patients(controls) and 7 verified pheochromocytoma patients(cases) were carried out by the method.The diagnostic criteria of metanephrine and normetanephrine were established by using receiver operating characteristic curves,and their range of normal values was set up by percentile rank method.The concentration of catecholamine in plasma and 24h urine of all patients and most controls were measured.Resultsâ‘ Peak value of both metanephrine and normetanephrine were detected within 10 minutes.â‘¡The variations of equipment,intra-assay variations,inter-assay variations and recoveries of metanephrine were 2.3%,5.5%,5.1%and 93.4% respectively,while the corresponding values of normetanephrine were 1.9%,4.8%,4.7% and 87.9%.â‘¢The average values of metanephrine and normetanephrine in controls were 152±114μg/24h and 294±267μg/24h,while the corresponding values for cases were 1426±1956μg/24h and 4376±2591μg/24h.â‘£The diagnostic criteria and 99% upper of normal values of metanephrine were 415μg/24h and 419μg/24h,while the relevant values for normetanephrine were 1464μg/24h and 1184μg/24h.⑤The sensitivity and specificity of metanephrine were 71.4%and 98.8%,and the corresponding figures for normetanephrine were 100%and 100%.The diagnostic sensitivity and specificity of plasma catecholamines were 71.4%and 79.0%,and the corresponding figures for 24h urine catecholamines were 100%and 82.4%.Conclusion A quick,reliable and accurate method of quantifying metanephrine and normetanephrine in 24h urine was established by using high performance liquid chromatographic assay,and the diagnostic criteria and range of normal values of these two indexes were set up first time in China.In comparison with the plasma and 24h urine catecholamines,the measurement of metanephrine and normetanephrene is now the best biochemical assay for detecting pheochromocytomas. BackgroundAlthough the treatment of congestive heart failure(CHF) has improved a lot during last decades of years,the morbidity and mortality of CHF are still at high levels.Therefore,it is still an important and necessary work to develop new anti-heart failure drugs.SGL-3 is a traditional Chinese medicine,and has been proved to be very effective in the treatment of CHF.One of the objectives of this study was to demonstrate the protective effects of SGL-3 through in vitro and in vivo experiments,and to find out its pharmacological mechanism.SGL-1 is a flavone compound which has been proved to be a powerful antioxidant.Since the excessive reactive oxygen species(ROS) plays an important role in the process of doxorubicin-induced cardiotoxicity,we tried to demonstrate the protective effects of SGL-1 against doxorubicin-induced apoptosis and injury in H9c2 cells.Methodsâ‘ SGL-3 in vitro study.In cultured rat neonatal cardiac myocytes,the inhibition of angiotensin-â…¡-induced(1μmol/L)cardiac myocyte hypertrophy by SGL-3(10μg/mL) was observed using[³H]leucine incorporation and phalloidine assay.â‘¡SGL-3 in vivo study.In vivo,twenty-weeks-old male Wistar rats were subjected to suprarenal abdominal aorta constriction(AAC) operation(n=48) or sham operation (n=12).Twenty weeks after surgery,heart failure was detected in AAC rats by using echocardiography,then the AAC rats were treated with either SGL-3(150 mg·kg^-1·d^-1, 500 mg·kg^-1·d^-1 and 1500 mg·kg^-1·d^-1,PO,n=12 in each group) or Captopril(270 mg·kg^-1·d^-1,PO) plus Metoprolol(1,800 mg·kg^-1·d^-1,PO)(n=12) for 18 weeks respectively.Cardiac hemodynamic and morphological parameters were obtained by using multi-functional physiology recorder and echocardiography.The pathologic changes were observed using hematoxylin and eosin staining,masson staining and sirius red staining.Moreover,the levels of plasma ANP and other indexes of renin-angiotensin-aldosterone system(RAAS) were also measured.â‘¢SGL-1 in vitro study.The 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT) staining,Hoechst33258 staining and lactate dehydrogenase(LDH) measurement were used to investigate the anti-apoptosis effect of SGL-1.The reactive oxygen species were observed using 2,7-dichlorofluorescein diacetate(DCFH-DA) probe,while the activities of malondialdehyde(MDA),superoxide dismutase(SOD) and glutathione peroxidase(GPx) were measured to investigate the antioxidative activity of SGL-1.Resultsâ‘ In cultured rat neonatal cardiac myocytes,SGL-3 significantly inhibited cardiac myocyte hypertrophy which was induced by angiotensin-â…¡,reduced[³H]leucine incorporation by 74%±26%and cell surface area by 104%±31%.â‘¡The in vivo results revealed that SGL-3 could improve cardiac systolic function more effectively(EF:51.8%±8.42%for AAC group,75.6%±7.65%for sham operation group, 69.3%±7.73%for SGL-3 treated group) than Captopril plus Metoprolol(EF: 56.3%±8.41%).Histological examination of cardiac structure showed that the extent of cardiac myocyte hypertrophy was restrained,and in compare with vehicle,myocardial fibrosis,circulating renin-aldosterone system and ANP secreting were remarkedly inhibited in AAC rats treated with SGL-3(p＜0.05).â‘¢In H9c2 cells,SGL-1 significantly relieved doxorubicin-induced apoptosis(82.8%vs. 48.8%) and cell injury(1.83 times compared with control vs.1.23 times),reduced the production of ROS and MDA(2.5 times vs.1.46 times),and raised the activities of SOD (58.9%vs.89.3%) and GPx(46.1%vs.87.3%).Moreover,we found that SGL-1 did not influence the anticancer activity of doxorubicin(30.9%vs.29.5%).Conclusionsâ‘ SGL-3 attenuated the cardiac myocyte hypertrophy both in vitro and in vivo and inhibited the process from hypertrophy to heart failure.The major mechanisms might be the improvement of cardiac functions and the inhibition of RAAS.â‘¡SGL-1 significantly reduced the apoptosis and injury induced by doxorubicin.These effects might be related to the scavenging of ROS and the improvement of oxidative stress in cardiac myocytes.