Use of flow cytometry and monochlorobimane to quantitate intracellular glutathione concentrations in feline leukocytes.
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.
Webb
Craig
C
Department of Clinical Sciences, Colorado State University, Ft. Collins, CO 80523, USA.
Bedwell
Cathy
C
Guth
Amanda
A
Avery
Paul
P
Dow
Steven
S
D000818:Animals; D002415:Cats / Q000097:blood*; D002845:Chromatography / Q000379:methods; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005033:Ethylmaleimide / Q000494:pharmacology; D016181:Feline Acquired Immunodeficiency Syndrome / Q000097:blood* / Q000821:virology; D005434:Flow Cytometry / Q000379:methods* / Q000662:veterinary; D005456:Fluorescent Dyes / Q000737:chemistry*; D005978:Glutathione / Q000097:blood*; D005982:Glutathione Transferase / Q000037:antagonists & inhibitors; D016089:Immunodeficiency Virus, Feline / Q000254:growth & development*; D007962:Leukocytes / Q000737:chemistry / Q000378:metabolism*; D018384:Oxidative Stress / Q000276:immunology; D011720:Pyrazoles / Q000737:chemistry*; D013047:Specific Pathogen-Free Organisms; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity
D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D011720:Pyrazoles; C059597:monochlorobimane; D005982:Glutathione Transferase; D005978:Glutathione; D005033:Ethylmaleimide