Kehr J, Dechent P, Kato T, Ogren S O
Department of Neuroscience, Karolinska Institute, Stockholm, Sweden.
J Neurosci Methods. 1998 Sep 1;83(2):143-50. doi: 10.1016/s0165-0270(98)00074-0.
Microbore column liquid chromatography with post-column immobilized enzyme reactor (IMER) and electrochemical detection on redox polymer coated electrodes was used for detection of acetylcholine (ACh) and choline (Ch) in microdialysis samples. The sensitivity of the coated electrodes, decreased gradually by about 10%/day, with highest reduction of 30% within the first 16 h of use. A number of choline derivatives were tested as possible internal standards, of those acetylethylhomocholine (AEHCh) and butyrylcholine (BCh) were found the most suitable candidates since they both provided high enzymatic conversion in the IMER. Physostigmine produced a negative peak, possibly reflecting oxidation of eseroline--a decarbamoylated product of reversible reaction of physostigmine with immobilized acetylcholine esterase. The probes, implanted in the ventral hippocampi of awake rats were perfused at a flow-rate of 1.25 microl/min with Ringer solution containing 10 microM physostigmine or with artificial cerebrospinal fluid only. The concentrations of ACh in 10-microl samples at basal conditions were between 0.9-2.5 nM, whereas in the presence of physostigmine the ACh levels raised to 41-48 nM. Physostigmine concentration was reduced to 8.8 microM, indicating its in vivo delivery of about 12%. The coefficients of variation were reduced from 7.4% for external standard method after every sixth sample to 5.8% and 5.9% for internal standardization with AEHCh and BCh, respectively. The latter method shortened the total analysis time by about 15%, thus being especially suitable for continuous long-lasting off-line or on-line monitoring. Additionally, other endogenous cholines such as butyrylcholine or synthetic choline derivatives could be detected by the present method.
采用带有柱后固定化酶反应器（IMER）的微径柱液相色谱法以及氧化还原聚合物涂层电极上的电化学检测法，对微透析样品中的乙酰胆碱（ACh）和胆碱（Ch）进行检测。涂层电极的灵敏度以每天约10%的速度逐渐下降，在使用的前16小时内下降幅度最大可达30%。测试了多种胆碱衍生物作为可能的内标物，其中乙酰乙基高胆碱（AEHCh）和丁酰胆碱（BCh）被发现是最合适的候选物，因为它们在IMER中都能实现高效的酶促转化。毒扁豆碱产生一个负峰，可能反映了毒扁豆碱与固定化乙酰胆碱酯酶可逆反应的脱氨甲酰化产物依色林的氧化。将探针植入清醒大鼠的腹侧海马体，以1.25微升/分钟的流速用含有10微摩尔毒扁豆碱的林格溶液或仅用人造脑脊液进行灌注。在基础条件下，10微升样品中ACh的浓度在0.9 - 2.5纳摩尔之间，而在有毒扁豆碱存在的情况下，ACh水平升高至41 - 48纳摩尔。毒扁豆碱浓度降至8.8微摩尔，表明其体内递送率约为12%。变异系数从每隔六个样品采用外标法时的7.4%，分别降至采用AEHCh和BCh进行内标法时的5.8%和5.9%。后一种方法将总分析时间缩短了约15%，因此特别适合连续长时间的离线或在线监测。此外，本方法还可检测其他内源性胆碱，如丁酰胆碱或合成胆碱衍生物。
