HPLC-FLD测定不同脑区3种单胺神经递质及其代谢产物含量

目的 建立并优化测定脑组织中单胺类神经递质及其代谢产物含量的高效液相色谱荧光检测方法。方法 采用Inertsustain C₁₈色谱柱（250 mm×4.6 mm,5 μm）,流动相为甲醇和水,其中水相中每升含柠檬酸30 mmol、乙酸钠40 mmol、EDTA-2Na 0.2 mmol、辛烷磺酸钠0.4 mmol（pH=3.8）,梯度洗脱,流速为1.0 mL·min^-1,荧光检测波长λex=280 nm,λem=330 nm。结果 重酒石酸去甲肾上腺素、盐酸多巴胺、盐酸5-羟色胺、3-甲氧基-4-羟基苯乙胺、3,4-二羟基苯乙酸、高香草酸、5-羟吲哚乙酸7种成分在45 min内洗脱完全,分离效果良好;浓度0.9~400.0 μg·L^-1内线性关系良好,且相关系数>0.99,平均回收率>80%。结论 本方法简便、准确、分离度高、重复性好,适用于一般实验室脑组织中单胺类神经递质及其代谢产物的测定。     

HPLC-ECD法测定大鼠脑组织中单胺类神经递质的含量

目的:建立测定大鼠脑组织中的单胺类神经递质含量的方法。方法:采用高效液相色谱-电化学法,以2,5-二羟基苯甲酸为内标,测定大鼠大脑皮层、小脑、海马组织、下丘脑、嗅球中以去甲肾上腺素(NE)和5-羟色胺(5-HT)为代表的单胺类神经递质的含量。色谱柱为DIKMAC18,流动相为缓冲盐溶液(醋酸钠、庚烷磺酸钠、乙二胺四乙酸二钠和柠檬酸)-甲醇(梯度洗脱),流速为1.0 ml/min,工作电压为+0.7 V。结果:NE、5-HT的检测质量浓度线性范围分别为0.084⁴.2μg/ml(r=0.999 9)和0.0054.2μg/ml(r=0.999 9)和0.005⁰.25μg/ml(r=0.999 1),平均加样回收率分别为98.85%(RSD=2.89%,n=3)和101.5%(RSD=2.41%,n=3),日内(n=5)和日间(n=3)RSD均不大于3%;NE在小脑中的含量最低[(0.343±0.14)mg/g],在下丘脑中含量最高[(3.062±1.51)mg/g];5-HT在小脑中的含量最低[(0.059±0.04)mg/g],在大脑皮层中含量最高[(0.383±0.21)mg/g]。结论:本方法灵敏、简便、快速,可用于大鼠脑组织中NE、5-HT含量的测定。     

松果菊苷对脑缺血大鼠双侧脑组织中单胺类神经递质的影响

目的:研究对比大鼠脑缺血后缺血区和非缺血区单胺类神经递质及其代谢产物的变化,考察松果菊苷(ECH)对大鼠脑缺血后双侧脑组织中单胺类神经递质及其代谢产物的影响。方法:SD大鼠随机分为假手术对照组、模型组、ECH高、低剂量组(30,15 mg.kg-1.d-1)和川芎嗪组(CXQ,30 mg.kg-1.d-1)。各组大鼠腹腔注射相应的药物或生理盐水,qd,连续7 d。于给药d 3,进行大脑双侧脑组织纹状体埋置探针套管,末次给药1 h后,制作大鼠局灶性脑缺血模型(MCAO),造模后立刻进行微透析,将透析液注入高效液相-电化学检测器(HPLC-ECD),测定各组缺血后300 min内纹状体细胞外液中去甲肾上腺素(NE)、多巴胺(DA)、3,4二羟苯乙酸(DOPAC)、5-羟色胺(5-HT)和5-羟基吲哚乙酸(5-HIAA)的含量。结果:与假手术组相比,各模型组脑缺血后两侧脑组织细胞外液中5种物质水平均升高;与模型组比较,ECH高、低剂量组与CXQ组5种物质的峰值含量均有所降低。结论:大鼠脑缺血后,非缺血区单胺类神经递质及代谢产物升高,ECH对抗脑缺血损伤的作用可能与降低脑内单胺类神经递质的升高有关。     

LC-MS／MS法测定大鼠脑组织中单胺类神经递质及代谢产物

目的：建立LC—MS／MS法测定大鼠脑组织中5-羟色胺（5-HT）、去甲肾上腺素（NE）、多巴胺（DA）、5-羟吲哚乙酸（5-HIAA）、3-甲氧基-4-羟基苯乙二醇（MHPG）、3，4-双羟苯乙酸（DOPAC）、高香草酸（HVA）的含量。方法：采用Thermo C18色谱柱（4．6mm×250mm，5μm），柱温为室温；水（含0．05％的甲酸）-乙腈为流动相，流速为0．8mL·min^-1。ESI^＋多个反应监测（MRM）模式监测反应质荷比（m／z）：177．06→159．92（5-HT），170．00→152．00（NE），154．00→137．00（DA）；ESI^-多个反应监测（MRM）模式监测反应质荷比（m／z）：190．00→145．90（5-HIAA），263．00→165．00和263．00→150．00（MHPG），166．90→122．80（DOPAC），180．70→136．60（HVA）。结果：5-HT、NE和DA分别在4．45～4450ng·mL^-1、4．11～4110ng·mL^-1和4．10～4100ng·mL^-1浓度范围内线性良好（r≥0．999），最低检测浓度分别为4．45，4．11，4．10ng·mL^-1；5-HIAA、MHPG、DOPAC和HVA在12．5—1000ng·mL^-1浓度范围内线性良好（r≥0．998），最低检测浓度为12．5ng·mL^-1。日内和日间RSD均小于8．5％，重复性好。结论：本方法灵敏、快捷、准确、专一性强，可用于神经精神疾病神经递质的研究与分析。     

荧光-反相高效液相色谱法同时测定小鼠脑组织中3种单胺类神经递质

目的建立同时测定脑组织中3种单胺类神经递质含量的方法。方法反相高效液相色谱．荧光检测法。结果在色谱圈上去甲肾上腺素（NE）、多巴胺（DA）和5-羟色胺（5-HT）完全分离。NE、DA和5-HT在0．05μg·mL^-1～0．5μg·mL^-1浓度范围内都呈现良好的线性关系（r〉0．999）;NE、DA、5-HT最低检测限分别约为0．02／μg·mL^-1、0．02μg·mL^-1、0．01μg·mL^-1（S／N≥3）;3种单胺类神经递质高、中、低浓度的平均相对回收率在90．10％～106．41％之间;日内变异（RSD）均小于8％。结论本法简便、快速、准确,可用于脑组织中NE、DA、5-HT的含量测定。     

HPLC-荧光检测法测定美托洛尔的血药浓度

目的:建立以高效液相色谱-荧光检测法测定美托洛尔血药浓度的方法。方法:色谱柱为Symmetry C18,流动相为乙腈-水-三乙胺-磷酸(110:390:2.5:1.6),荧光检测波长为激发波长(Ex)=265nm、发射波长(Em)=298nm,流速为0.8mL.min-1,进样量为20μL。结果:美托洛尔血药浓度在2.0～100.0μg.L-1范围内线性关系良好(r=0.9996);低、中、高3种浓度的平均相对回收率分别为99.73%、98.21%、99.38%,日内RSD分别为2.89%、2.36%、1.32%,日间RSD分别为3.73%、3.03%、2.25%,最低检测限为1.0μg.L-1。结论:本方法精密、准确、特异性强、回收率高、操作简便,适用于临床血药浓度监测及药动学研究。     

高效液相色谱-电化学法测定大鼠纹状体细胞外液中单胺类神经递质含量

目的:建立应用常规高效液相色谱-电化学法(HPLC-ECD)测定大鼠脑微透析液中单胺类神经递质及其代谢产物。方法:将探针插入大鼠右侧纹状体,在清醒自由活动状态下,用Ringer氏液以1.5μL.min-1的速度灌流,每20 min收集l管透析液,将其注入HPLC-ECD仪,考察本方法的专属性、线性范围、精密度和准确度等,并对其中所含的NE、DA、DOPAC、HIAA、HVA和5-HT进行含量测定。结果:各递质在0.025～0.5 ng.μL-1范围内线性关系良好,方法加样回收完全,日间日内测定均RSD<3.8%。结论:本实验选用了高效液相色谱与电化学检测器连用的方法测定神经递质的含量,该方法简单准确、灵敏、特异性好。     

高热量饲料喂养大鼠对其脑中单胺类神经递质的影响

目的：研究高热量饲料诱发胰岛素抵抗（IR）动物模型大鼠脑内单胺类神经递质的变化方法：SD大鼠随机分为正常组和由高糖、高脂肪组成的高热量饲料喂养的模型组，模型组大鼠于高热量饲料喂养前一次性腹腔注射小剂量链脲霉素，自行配制高热量饲料，原材料由中国医学科学院实验动物研究所繁育厂提供。喂养18周，其问动态观察体重、胰岛素耐晕、糖耐量、糖异生及血脂，处死动物后称量腹腔脂肪，并测定肝脏组织FFA，TG及TC；在冰浴上迅速取其前脑，称重，加入适量的冰冷0．4MHCLO4溶液中，用超声波细胞粉碎机粉碎匀浆，离心（4℃，15，000g，10min），取上清，与一半体积的钾溶液混合，离心（4℃，15，000g，10min），取上清50μl进样HPLC-ECD分析，用反相高效液相色谱电化学检测法测定单胺类神经递质及其代谢产物（NE、DA、DOPAC、5-HT、5-HIAA）的含量。结果：给予高热量饲料6周可诱发实验动物产生以腹型肥胖、糖耐量异常、胰岛素耐量异常、糖异生能力增强、肝脏脂质沉积等为特征的氓动物模型，高热量饲料的加工方式影响氓动物模型的形成；给予高热量饲料大鼠模型组前脑中的单胺类神经递质及其代谢产物与正常对照组比NE、DA、DOPAC、5-HT、5-HIAA分别升高了8．5、1．5、1．7、1．8、2．1倍，其中除DA外均有统计学差异。结论：高热量饲料可诱发氓动物模型，且能使脑内单胺类神经递质及其代谢产物升高，但受饲料的加工方式的影响。     

无机汞对大鼠脑单胺类神经递质代谢的影响

用大鼠脑组织匀浆、游离脑细胞和脑细胞亚组分作实验材料，较全面地观察了氯化汞对单胺类神经递质含量及其合成、释放、摄取、降解等重要代谢环节的影响。结果表明，汞通过抑制脑线粒体外膜单胺氧化酶使得脑组织中５－羟色胺、多巴胺、去甲肾上腺素含量增高，５－羟吲哚乙酸含量下降，呈良好的剂量－效应关系；汞还明显改变了游离脑细胞对单胺类神经递质的释放和摄取能力。     

Interactions of methylenedioxymethamphetamine with monoamine transmitter release mechanisms in rat brain slices

Summary This study investigates the effects of methylenedioxymethamphetamine (MDMA) and amphetamine on monoamine release from rat superfused brain slices in both the presence and absence of vesicular stores of transmitter. MDMA caused the release of radioactivity from slices incubated with [³H]5-hydroxytryptamine, [³H]noradrenaline or [³H]dopamine with EC₅₀ values of 1.9 mol/l (95% confidence limits 1.5–2.3 mol/l), 4.5 mol/l (2.3–8.7 mol/l), and greater than 30 mol/l, respectively. In contrast, amphetamine (0.1–300 mol/l) was more effective in releasing radioactivity from slices incubated with [³H]dopamine than [³H]noradrenaline or [³H]5-hydroxytryptamine. When Ca²⁺ was excluded from the superfusion fluid, the MDMA induced release of radioactivity from slices incubated with [³H]dopamine was unaltered, but that from slices incubated with [³H]noradrenaline or [³H]5-hydroxytryptamine was enhanced. MDMA (10 mol/l) facilitated the stimulation-induced (5 Hz, 1 min) outflow of radioactivity from slices incubated with [³H]noradrenaline or [³H]5-hydroxytryptamine to 7.5-fold and 2.1-fold of control values, respectively, but had no effect on that from slices incubated with [³H]dopamine. Amphetamine (1 mol/l) increased the stimulation-induced outflow from slices incubated with [³H]noradrenaline, but not that from slices incubated with [³H]5-hydroxytryptamine or [³H]dopamine.Inhibition of monoamine oxidase by a 30-min incubation with pargyline (100 mol/l) enhanced the releasing action of MDMA on all three monoamines. Pargyline (100 mol/l) also enhanced the facilitation caused by MDMA, of the stimulation-induced outflow of radioactivity from slices incubated with [³H]noradrenaline, [³H]5-hydroxytryptamine or [³H]dopamine.In some experiments, slices were obtained from reserpinised rats (2.5 mg/kg s.c. 24 h prior) and pre-exposed for 30 min to the monoamine oxidase inhibitor pargyline (100 mol/l). Under these conditions, electrical stimulation evoked a small residual stimulation-induced outflow of radioactivity from slices incubated with [³H]noradrenaline, and failed to evoke an outflow of radioactivity from slices incubated with [³H]5-hydroxytryptamine or [³H]dopamine. However, a Ca²⁺-dependent stimulation-induced outflow of radioactivity was evoked in the presence of either MDMA (10 mol/l) or amphetamine (1 mol/l) from slices incubated with either [³H]dopamine or [³H]noradrenaline, but not from slices incubated with [³H]5-hydroxytryptamine. The stimulation-induced outflow of radioactivity from slices incubated with [³H]noradrenaline was enhanced in the presence of desipramine (1 mol/l), however this enhancement was less than that caused by 10 mol/l MDMA or 1 mol/l amphetamine. The Ca²⁺-dependent response to electrical stimulation in the presence of MDMA from slices incubated with [³H]noradrenaline was greatly reduced when rats were pretreated with a higher dose of reserpine (10 mg/kg s.c.).This study demonstrates that MDMA and amphetamine release radioactivity from brain slices incubated with [³H]noradrenaline, [³H]dopamine and [³H]5-hydroxytryptamine, but their order or potency as releasers of brain monoamines differs. The results also suggest that MDMA and amphetamine have significant effects on the exocytotic release of monoamines and may interact with both vesicular and cytoplasmic monoamines. Correspondence to J. J. Reid at the above address     

精神分裂症治疗前后单胺类神经递质分析及疗效评定

目的观察精神分裂症患者治疗前后血浆中单胺类神经递质含量变化及与疗效的相关性。方法采用放免法测定85例精神分裂症患者治疗前后血浆多巴胺(DA)、5-羟色胺(5-HT)、去甲肾上腺素(NE)和生长激素(GH)含量及简明精神病量表(BPRS)的减分率来评定疗效。结果治疗前后5-HT含量分别为(84.93±68.13)和(94.33±68.84)mmol/L,DA(34.14±10.24)和(32.89±9.63)mmol/L,NE(67.94±82.84)和(56.63±29.83)mmol/L,GH(3.69±10.02)和(2.28±4.20)mmol/L,前后对照均无统计学差异(P>0.05)。5-HT、DA、NE和GH含量变化与疗效无明显相关(P>0.05)。结论精神分裂症患者治疗前后血浆中单胺类神经递质未发现明显差异,与疗效是否相关需进一步细化精神症状。     

The activities of monoamine oxidase-A and-B,succinate dehydrogenase and acid phosphatase in the rat brain after hemitransection

Summary The activities of monoamine oxidase-A and-B were determined in four brain regions (limbic system, occipitotemporal cortex, hemispheres and striatum) of the rat 0, 3, 6, 9 and 14 days after hemitransection of the left side. No larger or consistent change in the activity of monoamine oxidase-A towards 5-hydroxytryptamine was found for the left (hemitransected) side with respect to the right side for any of the rats. The monoamine oxidase-B activity towards -phenethylamine increased in the left side striatum to a significant level by 3 days, and in the hemispheres and occipito-temporal cortex on the left side, with respect to the right side by 9 days, but no significant changes were found for the limbic system. A small decrease in the activity of succinate dehydrogenase was found in the striatum on the left side by 9 days after hemitransection, but no change in the activity of acid phosphatase was found in this brain region.     

高效液相色谱法测定大鼠脑组织中川陈皮素含量

目的建立大鼠脑组织中川陈皮素含量的高效液相色谱紫外检测法。方法脑组织匀浆液经乙腈蛋白沉淀,再采用乙酸乙酯和正己烷进行萃取,吸取有机相,N2气流吹干后,流动相复溶,离心,取上清液待测。采用Kro—masilC18柱（4．6mm×150mm,5μm）,流动相为甲醇-水（65：35,v／v）,流速0．8mL·min^-1,柱温为40℃,检测波长335nm,以尼莫地平为内标进样分析。结果川陈皮素、内标和内源性杂质分离良好,线性范围6．25～1200ng·g^-1（r=0．9992）,定量下限为6．25ng·g^-1,萃取回收率〉59％,方法回收率〉99％,批内、批间RSD均〈15％。结论本方法灵敏度高、专属性强,适合于大鼠脑组织中川陈皮素含量测定。     

丙咪嗪单用及与尼莫地平合用对小鼠脑内单胺递质含量的影响

目的：通过测定小鼠脑内单胺递质含量变化探讨丙咪嗪（Ｉｍｉ）单用及与尼莫地平（Ｎｉｍ）合用与单胺递质的关系。方法：采用高效液相色谱－电化学法测定脑内单胺递质及其代谢物的含量。结果：单用及合用组多巴胺含量均增加,去甲肾上腺素和５－羟色胺含量单用组明显增高,且以７ｄ合用组变化明显。结论：７ｄ组Ｉｍｉ与Ｎｉｍ合用对单胺递质水平有一定影响。     

尼莫地平对小鼠脑内单胺递质含量的影响

目的：探讨尼莫地平增强学习记忆的作用机制。方法：采用高效液相色谱－电化学法测定脑内单胺递质及其代谢物的含量。结果：尼莫地平组去甲肾上腺素（NE）,多巴胺（DA）,5－羟色胺（5－HT）及它们的主要代谢产物高香草酸（HVA）,3,4－双羟苯乙酸（DOPAC）含量增加,结论：尼莫地平增强学习记忆的作用可能与单胺递质有关。     

Interactions of tricyclic antidepressant drugs with human and rat monoamine oxidase type B

Summary The effect of tricyclic antidepressant drugs on the deamination of phenylethylamine and benzylamine by monoamine oxidase (MAO) type B was investigated in vitro in human brain cortex, human platelet, and rat brain preparations. These drugs inhibited MAO activity as expected; however, an atypical biphasic response was observed with the tertiary amine tricyclic, clomipramine, and, to a somewhat lesser extent, with two other tertiary amine tricyclics, imipramine and amitriptyline, when benzylamine was used as the substrate in human tissue preparations. This atypical biphasic pattern was not found when we used the secondary amine antidepressant drugs, desipramine, desmethylclomipramine, or fluoxetine, or used phenylethylamine as the substrate, or used rat rather than human brain tissue. For the tricyclics exhibiting normal inhibition patterns, the same rank order of inhibition was observed with benzylamine as a substrate in all three types of tissue; however with phenylethylamine, differences in inhibition were found between rat and human tissues. These tricyclic-MAO interactional data suggest that secondary and tertiary amine tricyclics interact differently with human MAO type B, that rat and human MAO type B are not functionally identical, and also support other data that phenylethylamine and benzylamine are deaminated by different mechanisms. Send offprint requests to A. A. Reid at the above address     

Inhibition of rat brain monoamine oxidase by formamidines and related compounds

The efficacy of the pesticide chlordimeform or N′-(4-chloro-o-tolyl)-N,N′-dimethylform-amidine, six chlordimeform metabolites, and eleven related compounds as inhibitors of the oxidative deamination of radiocarbon-labelled biogenic amines by rat brain monoamine oxidase was examined. The I₅₀ value for chlordimeform with tyramine as substrate was 6.0 × 10^?5M. Inhibition following prolonged pre-incubation of chlordimeform with monoamine oxidase increased with time. and this was attributed, at least in part, to the formation of the more potent monoamine oxidase inhibitor 4′-chloro-o-formotoluidide, a known chlordimeform metabolite and degradation product. 4′-Chloro-o-formotoluidide was the most potent monoamine oxidase inhibitor examined yielding I₅₀ values of 2.6 × 10^?6m. 1.5 × 10^?6M and 3.2 × 10^?6M, with tyramine, dopamine, and serotonin, respectively, as substrates. The N-demethyl (demethylchlordimeform) and N-didemethyl chlordimeform metabolites gave I₅₀ values with tyramine of 3.3 × 10^?5M and 7.5 × 10^?5M, respectively. Three additional metabolites, 4-chloro-o-toluidine, 5-chloroanthranilic acid, and N-formyl-5-chloroanthranilic acid, were weak inhibitors with I₅₀ values of 1 × 10^?4m or higher. The other formamidine compounds also inhibited the oxidative deamination of tyramine; I₅₀ values ranged from 9.3 × 10^?5m to 7.5 × 10^?6m. Lineweaver-Burk plots revealed that chlordimeform, demethylchlordimeform, and 4′-chloro-o-formotoluidide were competitive inhibitors of the oxidative deamination of β-phenylethylamine, tyramine, dopamine, tryptamine, and serotonin. Inhibition was reversible since activity was restored by washing.