Webb Craig, Bedwell Cathy, Guth Amanda, Avery Paul, Dow Steven
Department of Clinical Sciences, Colorado State University, Ft. Collins, CO 80523, USA.
Vet Immunol Immunopathol. 2006 Aug 15;112(3-4):129-40. doi: 10.1016/j.vetimm.2006.02.009. Epub 2006 May 26.
Oxidative stress and abnormal glutathione metabolism is thought to play an important role in various diseases of cats. However, current assays for the reduced form of glutathione (GSH) are time-consuming and semi-quantitative and do not allow assessment of GSH concentrations in individual cell populations. Therefore, we developed a flow cytometric assay for rapid determination of intracellular GSH concentrations in feline blood leukocytes. The assay was based on the ability of the non-fluorescent substrate monochlorobimane (mBCl) to form fluorescent adducts with GSH in a reaction catalyzed by the enzyme glutathione-S-transferase. Using flow cytometry, we found that mBCl was sensitive and specific for intracellular detection of the reduced form of GSH in feline leukocytes. Intracellular GSH concentrations were also stable for at least 24h in EDTA preserved whole blood samples stored at 4 degrees C. Neutrophils and monocytes from normal cats had significantly higher intracellular concentrations of GSH than T cells and B cells. The effects of FIV infection on intracellular GSH concentrations in cats were assessed using flow cytometry. We found that neutrophils from FIV-infected cats had significantly increased GSH concentrations, whereas intracellular GSH concentrations were significantly decreased in CD4(+) and CD8(+) lymphocytes from FIV-infected cats, compared to age-matched control animals. We conclude that a flow cytometric assay based on mBCl may be used to accurately and rapidly assess the effects of various disease states and treatments on GSH concentration in cat leukocytes and to help assess intracellular oxidative stress.
氧化应激和异常的谷胱甘肽代谢被认为在猫的各种疾病中起重要作用。然而，目前用于检测还原型谷胱甘肽（GSH）的方法耗时且为半定量，无法评估单个细胞群体中的GSH浓度。因此，我们开发了一种流式细胞术检测方法，用于快速测定猫血液白细胞中的细胞内GSH浓度。该检测方法基于非荧光底物单氯双氢乙二胺（mBCl）在谷胱甘肽-S-转移酶催化的反应中与GSH形成荧光加合物的能力。使用流式细胞术，我们发现mBCl对猫白细胞中还原型GSH的细胞内检测敏感且特异。在4℃储存的EDTA保存的全血样本中，细胞内GSH浓度在至少24小时内也保持稳定。正常猫的中性粒细胞和单核细胞的细胞内GSH浓度显著高于T细胞和B细胞。使用流式细胞术评估了猫免疫缺陷病毒（FIV）感染对猫细胞内GSH浓度的影响。我们发现，与年龄匹配的对照动物相比，FIV感染猫的中性粒细胞的GSH浓度显著增加，而FIV感染猫的CD4(+)和CD8(+)淋巴细胞的细胞内GSH浓度显著降低。我们得出结论，基于mBCl的流式细胞术检测方法可用于准确、快速地评估各种疾病状态和治疗对猫白细胞中GSH浓度的影响，并有助于评估细胞内氧化应激。
