Diagnostic Center for Population and Animal Health, College of Veterinary Medicine, Michigan State University, Lansing, MI 48910-8104, USA.
Toxicol Mech Methods. 2011 Mar;21(3):171-82. doi: 10.3109/15376516.2010.538753. Epub 2010 Dec 13.
1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is widely used as a neurotoxin in several models of Parkinson's disease in mice. MPTP is metabolized to 1-methyl-4-phenylpyridinium (MPP(+)), which is a mitochondrial toxicant of central dopamine (DA) neurons. There are species, strain, and age differences in sensitivity to MPTP. Simultaneous measurement of the MPTP active metabolite MPP(+) and dopamine (DA) in the brain would be helpful in mechanistic studies of this neurotoxin. The objective of this study was to develop a liquid chromatography-mass spectrometry (LC/MS) method for analysis of MPTP and MPP(+) in brain tissue and correlate these in the same sample with changes in DA measured via HPLC coupled with electrochemical detection. Twenty-five C57BL/6J7 8-week old female mice were used in the study. Mice were given a single subcutaneous injection of MPTP (20 mg/kg) and were sacrificed 1, 2, 4, or 8 h later. Zero time control mice received an injection of 0.9% normal saline (10 ml/kg) and were killed 1 h later. Brains were rapidly harvested and quickly frozen, and microdissected brain regions were placed in 0.1 M phosphate-citric acid buffer containing 20% methanol (pH 2.5). A new LC/MS method was successfully developed that utilized selected reaction monitoring (SRM) of MPP(+) m/z 170→127, 170→128, and 170→154 fragmentation for quantitation and area ratios (m/z 127)/(m/z 128) and (m/z 154)/(128) for identity confirmation. A similar SRM strategy from m/z 174 was unable to detect any significant levels of MPTP down to 0.4 ppb. According to this method, MPP(+) was detected in the nucleus accumbens (NA) and the striatum (ST), with the levels in the NA being 3-times higher than those in the ST. The advantage of this approach is that the tissue buffer used in this procedure allowed concurrent measurement of striatal DA, thus enabling direct correlation between accumulation of tissue MPP(+) and depletion of DA concentrations in discrete regions of the brain.
1-甲基-4-苯基-1,2,3,6-四氢吡啶（MPTP）被广泛用作几种小鼠帕金森病模型中的神经毒素。MPTP 代谢为 1-甲基-4-苯基吡啶鎓（MPP(+)），这是一种中多巴胺（DA）神经元的线粒体毒物。不同物种、品系和年龄对 MPTP 的敏感性存在差异。同时测量大脑中的 MPTP 活性代谢物 MPP(+)和多巴胺（DA）将有助于对这种神经毒素的机制研究。本研究的目的是开发一种液相色谱-质谱（LC/MS）方法，用于分析脑组织中的 MPTP 和 MPP(+)，并将其与通过 HPLC 与电化学检测相结合测量的 DA 变化相关联。在这项研究中使用了 25 只 8 周龄的 C57BL/6J7 雌性小鼠。小鼠接受单次皮下注射 MPTP（20mg/kg），1、2、4 或 8 小时后处死。零时间对照小鼠接受 0.9%生理盐水（10ml/kg）注射，1 小时后处死。大脑迅速采集并迅速冷冻，微切割的大脑区域置于含有 20%甲醇（pH2.5）的 0.1M 磷酸-柠檬酸缓冲液中。成功开发了一种新的 LC/MS 方法，该方法利用 MPP(+)m/z170→127、170→128 和 170→154 片段的选择反应监测（SRM）进行定量，并用质量比（m/z127）/（m/z128）和（m/z154）/（m/z128）进行身份确认。从 m/z174 获得的类似 SRM 策略无法检测到低至 0.4ppb 的任何显着水平的 MPTP。根据该方法，在伏隔核（NA）和纹状体（ST）中检测到 MPP(+)，NA 中的水平是 ST 中的 3 倍。这种方法的优点是，该程序中使用的组织缓冲液允许同时测量纹状体 DA，从而能够直接将组织 MPP(+)的积累与大脑不同区域 DA 浓度的耗竭相关联。
