Boni R L, Simpson J T, Naritsin D B, Saito K, Markey S P
Section on Analytical Biochemistry, National Institute of Mental Health, Bethesda, MD 20892.
Biol Mass Spectrom. 1994 Jan;23(1):27-32. doi: 10.1002/bms.1200230106.
In a number of infectious and inflammatory diseases, stimulation of the immune system can lead to increased accumulation of tryptophan metabolites via induction of kynurenine pathway enzymes in extrahepatic tissues. We developed a liquid chromatographic/mass spectrometric (LC/MS) method suitable for tracing the disposition of 13C isotopomers of L-tryptophan and L-kynurenine in various cultured cell, tissue slice, and whole animal model systems used to investigate tryptophan flux through the kynurenine pathway. The method employs extractive derivatization of the analytes and their 2H internal standards with pentafluorobenzyl bromide in order to enhance the negative ion chemical ionization (NICI) mass spectrometric response. Normal-phase liquid chromatographic separation of derivatized analytes was optimized using a silica column with organic solvents, followed by particle beam transfer and NICI-MS. Standard curves were linear over the range 1-250 ng per sample. Particle beam and mass spectrometric operating parameters were optimized with direct flow injections of 1-(methylamino) anthraquinone, which is an ideal test compound for the evaluation of LC/NICI-MS. The developed method was used to quantify the conversion of (13C6)L-tryptophan to (13C6)L-kynurenine by human monocytes (THP-1) stimulated with interferon-gamma, lung and brain tissue slices obtained from gerbils immune-stimulated with pokeweed mitogen. The effect of whole body immune stimulation on the plasma levels of endogenous L-kynurenine in mice stimulated with interferon-gamma was also quantified.
在许多感染性和炎症性疾病中，免疫系统的刺激可通过诱导肝外组织中的犬尿氨酸途径酶，导致色氨酸代谢产物的积累增加。我们开发了一种液相色谱/质谱（LC/MS）方法，适用于追踪L-色氨酸和L-犬尿氨酸的13C同位素异构体在各种培养细胞、组织切片和全动物模型系统中的处置情况，这些模型系统用于研究色氨酸通过犬尿氨酸途径的通量。该方法采用五氟苄基溴对分析物及其2H内标进行萃取衍生化，以增强负离子化学电离（NICI）质谱响应。使用含有有机溶剂的硅胶柱对衍生化分析物进行正相液相色谱分离，并进行优化，随后进行粒子束转移和NICI-MS分析。标准曲线在每个样品1-250 ng的范围内呈线性。通过直接流动注射1-（甲氨基）蒽醌对粒子束和质谱操作参数进行了优化，1-（甲氨基）蒽醌是评估LC/NICI-MS的理想测试化合物。所开发的方法用于定量干扰素-γ刺激的人单核细胞（THP-1）、用商陆丝裂原免疫刺激的沙鼠获得的肺和脑组织切片中（13C6）L-色氨酸向（13C6）L-犬尿氨酸的转化。还定量了全身免疫刺激对干扰素-γ刺激的小鼠内源性L-犬尿氨酸血浆水平的影响。
