成年大鼠脑透析液和血清中褪黑素含量的测定

成年大鼠脑透析液和血清中褪黑素含量的测定

孟涛;郑志竑;叶婧;林玲

【摘要】目的：采用高效液相色谱（HPLC）‐荧光法和酶联免疫（ELISA）法测定成年大鼠脑微透析液及血清中的褪黑素（MLT）含量，比较2种检测方法的灵敏性、准确性及可靠性。方法将微透析引导管置入正常成年大鼠纹状体内1周后，采用脑微透析技术动态收集2：00，6：00，10：00，14：00，18：00和22：00的透析液，并于次日22：00行尾静脉取血；用HPLC‐荧光法和ELISA法分别测定透析液及血清样本中的MLT水平。结果HPLC‐荧光法测定MLT检出限为0．1 pg／mL ，在1．0～500 pg／mL之间呈线性关系，回归方程 y＝10446 x ＋8603．5（r＝0．9987，P＜ 0．001），日间、日内精密度均＜6％；ELISA 法检出限为5．92 pg／mL ，检测线性范围为12．35～1000 pg／mL。用HPLC‐荧光法测定2：00，6：00，10：00，14：00，18：00和22：00大鼠脑透析液中的MLT含量分别为（32．99 ± 2．22），（18．25 ± 2．27），（4．03 ± 1．70），（2．92 ± 1．08），（12．08 ± 1．77）和（21．83 ± 2．04）pg／m L ，具有明显的昼夜节律性，探针回收率为12％～13％；22：00血清中的MLT含量为（82．62 ± 8．09）pg／mL ，提取回收率＞90％，且血清与透析液中的MLT水平呈高度正相关（r＝0．987，P＜0．001）；用ELISA法测定血清中的MLT含量为（81．31±9．62）pg／mL ，且用2种方法测定血清中MLT差别无统计学意义（P＝0．115）。结论用HPLC‐荧光法测定大鼠脑微透析液和血清中的MLT 含量具有灵敏、快速、准确、重复性好等特点，且HPLC‐荧光法和ELISA法测定血清中的MLT具有较好的一致性。%Objective To determine brain dialysate and serum melatonin (MLT ) levels in adult rats by high‐performance liquid chromatography (HPLC) with fluorescence

detection and enzyme‐linked immunosorbent a ssay (ELISA) ,and compare the sensitivity ,accuracy and reliability of the two detection methods . Methods The guide tube was implanted into the striatum of healthy adult rats . One week af‐ter implantation ,dialysate samples were collected at

2 :00 ,6:00 ,10 :00 ,14:00 ,18 :00 ,22 :00 h in stria‐tum by brain microdialysis . Blood samples were collected from the rat tail vein at 22 :00 h on the follow‐ing day . HPLC with fluorescence detection and ELISA were applied to detect dialysate and serum MLT levels . Results The lowest concentration of MLT that can be detected by HPLC with fluorescence de‐tection was 0 .1 pg/mL . There was a regressive equation y=10 446 x+8 60

3 .5 (r=0 .998 7 ,P<0 .05) in linear range 1 .0~500 pg/mL of MLT

levels ,with an intra‐assay and inter‐assay of below 6% . The detection limit of MLT by ELISA was 5 .92 pg/mL ,and the detection range was 12 .35~1 000 pg/mL . The mean concentrations of dialysate MLT that showed a distinct fluctuation were (in pg/mL )

(32.99±2.22),(18.25±2.27),(4.03±1.70),(2.92±1.08),(12.08±1.77),(21.83±2.04 )at2:00, 6 :00 ,10:00 ,14:00 ,18:00 and 22 :00 h by HPLC with fluorescence detection . The recovery of the probes was from 12% to 13% . Besides ,the mean concentration of serum MLT was (82 .62 ± 8 .09) pg/mL by t his method . The extraction recovery of serum MLT was more than 90% . There was a distinct positive correlation between serum MLT and dialysate MLT levels at 22:00 pm ,and pearson’s correlation coeffi‐cient between the two was 0 .987 at the 0 .001 level (t wo‐tailed) .In addition ,the mean concentration of serum MLT was (81 .31 ± 9 .62) pg/mL by ELISA . There