| Objective:To establish an HPLC-ECD method for quantitative determination of eleven compounds related to metabolism of bioamines in rat brain, to compare these eleven compounds in cortex and hippocampus, and to form CUMS induced depression model and study the influence of pathway AA-COX2/5-LO intervention on bioamines in the cortex of depression rat.Methods:1. The determination was conducted with the SHIMADZU HPLC system coupled with an amperometric detector ED723. Separations were performed on a Hypersil ODS2 column(250 mm×4.6 mm, 5.0 mm) at a flow rate of 1.0 m L/min. The mobile phase was composed of acetonitrile- buffer solution, which contained 25 mmol/L sodium acetate, 25 mmol/L citric acid, 0.01 mmol/L EDTA-2Na and 1.0 mmol/L OSA, adjusting p H to3.35 with acetic acid,(v/v, 10:90). The column temperature was set at30 °C and detector was set at 700 m V within output range of 10 mA. The injection volume was 20 mL.2. To compare these eleven compounds in cortex and hippocampus:determination of the concentration of Tyr, Trp, DA, NE, E, 5-HT, 3-MT,DOPAC, MHPG, HVA, 5-HIAA in the cortices and hippocampi from eight SD rats, using mean±SD for statistic description.3. To form CUMS induced depression model and intervene pathway AA-COX2/5-LO: 10 subjects were randomly selected from 90 male SD rats as control group. Six weeks CUMS administrate to the other 80 rats, 70 rats with significant differences in behavior indexes were selected,randomly divided into CUMS group, Sertraline 5 mg/kg group, Meloxicam3mg/kg group, Meloxicam 1 mg/kg group, Caffeic acid 30 mg/kg group,Caffeic acid 10 mg/kg group, Meloxicam 1 mg/kg+Caffeic acid 10 mg/kg group. The control group and CUMS group were lavaged with isovolumetric CMC-Na, others were treated with drugs lavage for 21 day.4. The influence of pathway AA-COX2/5-LO intervention on bioamines in the cortex of depression rat: every group underwent behavioral test after the 22 th day lavage and selected 5 male SD rats from each group. Determination of the concentration of Tyr, Trp, DA, NE, E,5-HT, 3-MT, DOPAC, MHPG, HVA, 5-HIAA in the cortices, using mean±SD for statistic description.Results:The eleven compounds of interest showed good linearity in a defined range with r2≥0.9984. The LOD were 0.04-1.6 ng and LOQ were 0.12-5 ng,respectively. The intra- and inter-day precision of all analytes in the three spiked levels were ≤8.83% and ≤9.17%, respectively. The relative recovery ranges from 94.12% to 108.07%, the extraction recovery between the range of 73.54%-99.90%.The eleven compounds in cortex and hippocampus from the eight rats were compared. The E and 3-MT are not detected in both of cortex and hippocampus. The concentrations of DA and HVA in hippocampus as well as 5-HT in cortex were all lower than LOQ. The concentration of Tyr in hippocampus was significantly higher than that in the cortex, whereas the level of DOPAC showed opposite change in these two brain regions.Meanwhile, it was observed that the levels of MHPG, NE, 5-HIAA and Trp failed to show any significant differences between the cortex and hippocampus.The eleven compounds in rats cortices from Control group, CUMS group, Sertraline 5 mg/kg group, Meloxicam 3 mg/kg group, Meloxicam 1mg/kg group, Caffeic acid 30 mg/kg group, Caffeic acid 10 mg/kg group,Meloxicam 1 mg/kg+Caffeic acid 10 mg/kg group were compared, and the data were analyzed with statistical method of One-Way ANOVA by SPSS17.0 software. Compared with Control group, the concentration of NE of CUMS group decreased significantly(P<0.05), indicating the CUMS induced depression models were successfully formed; the concentration of NE of Meloxicam 3 mg/kg group and Caffeic acid 30 mg/kg group weretrends to increase; the concentration of NE in big doses group of Meloxicam and Caffeic acid is obviously higher than the small doses group(P<0.05), showing the big doses of Meloxicam and Caffeic acid may be curative for depression. The concentration of HVA, DOPAC, Tyr in Meloxicam 1 mg/kg+Caffeic acid 10 mg/kg group is obviously higher than Meloxicam 1 mg/kg group or Caffeic acid 10 mg/kg group, showing the pathway AA-COX2/5-LO dual intervention was not superior to simplex intervention.Conclusions:The developed method is rapid, sensitive and reliable. It was successfully applied to quantitative determination of eleven compounds related to metabolism of bioamines in rat brain.The eleven compounds in cortex and hippocampus from the same rats were compared, which provided references for monitoring of its variations in the field of neuroscience.The CUMS induced depression models were successfully formed.Meloxicam 3 mg/kg and Caffeic acid 30 mg/kg had apparent influence on the concentration of bioamines with their precursor amino acids and metabolites. Moreover, pathway AA-COX2/5-LO dual intervention was not superior to simplex intervention. |
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