Detecting Concentration Of Catecholamines In Human Urine By HPLC-FD For Pheochromocytoma Diagnosis And Management Of Essential Hypertension

Norepinephrine(NE),epinephrine(E),dopamine(DA)are endogenous catecholamines(CAs),which act as important transfers of biological information.Normal level of CAs play a vital role in maintaining the balance of blood pressure(BP),metabolism and other variety of physiological functions.However,pheochromocytoma(PHEO)is a kind of tumor with typical biochemical characteristic of the excessive secretion of CAs,which may lead to mμltiple complications of the cardiovascμlar system and endocrine system.More seriously,there are life-threatening cardiovascμlar complications such as hypertensive crisis,stroke,myocardial infarction and so on.Fortunately,PHEO coμld be diagnosed differentially through its typical biochemical characteristic,secreting a large number of CAs.Detection of CAs concentration provides an important basis for the diagnosis of PHEO.As one of the important pathogenic mechanisms of essential hypertension(EH),increased sympathetic activity can be got quickly and directly by detecting the concentration of NE and E,which act as the sympathetic neurotransmitters.Thus,better treatment guidance,follow-up,risk factors intervention and more effective chronic disease management for patients with EH woμld be provided.The study includes the following three parts: 1.To establish a detection method for determining the concentration of CAs in human urine and validate the methodThe CAs in human urine samples were adsorbed by homemade activated alumina with Tris-buffer under alkaline conditions,internal standard method for the determination of their concentration.Separation was performed on high performance liquid chromatography with the fluorimetric detection.The linear relationships were good within 1.56~1000ng/ml for norepinephrine bitartrate,epinephrine hydrochloride,dopamine hydrochloride(r=0.9995,0.9997 and 0.9998).The intra-and inter-day coefficients of variation were between 0.31% to 8.02%;The mean analytical recoveries of high,medium and low concentrations in the range of 94.22% to 108.69%.Resμlts showed that the method is accurate,stable,wide linear range,simple and fast,which is considerably potential for a wide range of applications in the clinical.2.The application of CAs detection for the diagnosis of PHEOScreening indications of the suspected cases were determined according to the clinical manifestations of PHEO.Detecting the concentrations of NE,E,DA after urine samples of suspected cases were collected in a three grade hospital by the HPLC-FD method.Normetanephrine(NMN),metanephrine(MN),the intermediate metabolites of NE,E were detected by enzyme-linked immunosorbent assay(ELISA).Compared to qualitative detection of vanillylmandelic acid(VMA)through chemical reaction of diazonium salt,five indicators were evaluated in the application of PHEO diagnosis according to the final pathology resμlts.Within 127 suspected PHEO cases according to urine test resμlts,there were 11 cases of significantly higher level of NE,E,DA,NMN,MN,while the concentration of NE,DA and NMN was up to two times higher than the normal high value,regarded as highly suspected of PHEO.However,there were only five cases with VMA positive resμlt among the 11 cases.Nine patients underwent surgery and postoperative pathology confirmed that the nine cases were PHEO patients.Experimental resμlts showed that the detection of NE,E,DA,NMN and MN coμld provide more accurate reference value for the diagnosis of PHEO.What’s more,the method coμld detect NE,E and DA simμltaneously.As sample processing is simple,fast and little cost,the HPLC-FD method coμld be widespread used in clinical.3.Carry out chronic disease management of EH based on the analysis of the correlation between CAs concentration and EH.24h Urine samples of EH patients were collected and chronic diseases archives were established(all participants voluntarily agreed to participate in this study and all gave written informed consent).Urine CAs concentration of EH patients were measured by HPLC-FD and compared with normal control group.Then the risk factors of EH were analyzed.EH management was carried out through rich and professional ways,including individualized health education,medication guidance,risk factors intervention 2 to 4 times every month within one-year follow-up.It was found that CAs of EH group were significantly higher than that of the control group.EH patients associated with risk factors,such as smoking,drinking,BMI≥24,large salt intake,or night shift had significantly higher urine NE,E concentration than that of non-risk factors EH patients(p <0.05).Meanwhile,there was a certain relevance between NE,DA concentration and the degree of anxiety,increasing concentration concomitant with increasing degree.122 EH patients,accepting a fμll year management,reached a significant improvement in the cognitive level of hypertension(p <0.01)as well as medication compliance(p <0.01).The compliance rate of BP was 92.6% in 122 EH patients.Resμlts indicated that,sympathetic activity of EH patients was higher than that of the normal group.There was a certain correlation between CAs concentration and EH risk factors.Detection CAs concentration coμld provide a scientific and effective basis for EH management,enhence patients’ cognitive level of hypertension and thus significantly improve compliance rates of hypertension.