Rapid determination of monoamine neurotransmitters and their metabolites by high performance liquid chromatography
- VernacularTitle:高效液相色谱法快速检测单胺类神经递质及其代谢产物
- Author:
Limin LUN
;
Linjie CHE
;
Shiwei LI
- Publication Type:Journal Article
- From:
Chinese Journal of Tissue Engineering Research
2006;10(22):180-182
- CountryChina
- Language:Chinese
-
Abstract:
BACKGROUND: The determination of monoamine neurotransmitters and their metabolites is important in the experimental.research of Parkinson disease (PD) and experimental diagnosis of phaochromocytoma.OBJECTIVE: To develop a high performance liquid chromatography (HPLC) method for simultaneous and rapid determination of monoamine neurotransmitters and their metabolites which include epinephrine(E), norepinephrine(NE), dopamine(DA), 5'-hydroxytryptamine(5'-HT), 5-hydroxy3-indoleacetic acid(5-HIAA), homovanillic acid(HVA) and 3,4-dihydroxyphenylacetic acid(DOPAC).DESIGN: Case control observation and a randomized controlled animal experiment.SETTING: Department of Clinical Laboratory, Affiliated Hospital of Medical College, Qingdao University; Haiyang Branch, Affiliated Hospital of Medical College, Qingdao University.PARTICIPANTS: This experiment was carried out at Staff Room of Physiology, edical College, Qingdao University and Department of Clinical Laboratory, Affiliated Hospital of Medical College of Qingdao University from October to December 2003. ①Two patients with phaeochromocytoma were all confirmed by CT, MRI or pathologic section as well as physical sign. Twelve health volunteers were all university students and were excluded to suffer hypertension, endocrine system diseases and other diseases. ② Total 12 healthy female Wister rats weighed between 150 g to 180 g were chosen and randomly divided into control group and PD group with 6 rats in each.METHODS: The standard chromatograms of the standard solution of mixtures of seven substances (E, NE, DA, 5'-HT, 5-HIAA, HVA and DOPAC)were prepared and the precisions and recovery rates of the same samples were determined respectively. Urine samples of 24 hours obtained from 12 healthy volunteers and 2 patients with phaeochromocytoma were analyzed respectively by HPLC. The unilateral forebrain bundles in PD group were destroyed with 6-hydroxydopamine (6-OHDA) for monitoring release characteristics of monoamine neurotransmitters and their metabolites in healthy and marred caudate putamen nucleus striatum of normal and PD rats striatum.MAIN OUTCOME MEASURES: ①Determination of the precisions and recovery rates of monoamines. ②The contents of catecholamines in 24 hours urine of 12 health volunteers and 2 patients with phaeochromocytoma .③The release characteristics of monoamines and their metabolites in rats striatum. RESULTS: The results and analysis of catecholamines substances in 24hours urine were all obtained from 12 health volunteers and 2 patients with pheochromocytoma. There were no animals died in the experinment, and all of them were involved in the result analysis. ①There was a favorable linear relationship in the range of 2-1 000 μg/L for DA, E, NE and DOPAC, 10-1 000 μg/L for 5-HIAA, HVA and 5-HT for 30-1 000 μg/L with HPLC. The minimal detection limit of DA, E, NE and DOPAC was 2 μg/L, 5-HIAA 10 μg/L, HVA and 5-HT 30 μg/L. The coefficient of variation (CV) in the same batch was 3.0%-8.6%, and the CV in the different batchs was 3.8%-9.7%. The average rates of recovery of DA, NE, E and other 4 kinds of substances were 107.3%, 89.4%, 80.1% and 89.2%-95.7%, respectively.②The contents of CA in 24 hours urine of the 2 patients with phaeochromocytomas were higher than those of the 12 healthy volunteers. One of patient samples showed that E was mainly higher than that of control group. Other samples showed that NE was mainly higher than that of control group. Otherwise , E and NE were ten times higher than that of control group. ③The contents of DA and its metabolites such as DOPAC and HVA in intact sides of hemiparkinsonian rats had no significant changes ,while the contents of DA, DOPAC and HVA in lesion sides were significantly decreased compared with the controls (P ＜ 0.01 ).There were no significant changes in the contents of 5-HT and its metabolites 5-HIAA. ④The turnover rates of DA (DOPAC/DA and HVA/DA) in intact and lesion sides of hemiparkinsonian rats were all higher than those of the controls(P ＜ 0.05).CONCLUSION: The method we developed is suitable for the experimental research of normal and hemiparkinsonian rat models and for the diagnostic testing of phaeochromocytoma.
