title: A new flow cytometry method to measure oxidative status: the Peroxidation of Leukocytes Index Ratio (PLIR).
abstract: OBJECTIVE
  A complex relationship between immune system and metabolic pathway exists and can induce oxidative stress. The objective of this study was to design a new methodology allowing the measurement of oxidative status of leukocytes.
  
  
  RESULTS
  We developed a flow cytometry technique, based on C11-BODIPY 581/591 staining, to evaluate peroxidation in leukocytes. We defined the Peroxidation of Leukocytes Index Ratio (PLIR) as the ratio between the damage after AAPH-induced and PMA-induced peroxidation, using Trolox as standard antioxidant. Sensitivity of the method was assessed by correlating results with plasma antioxidant capacity (TRAP and FRAP), levels of endogenous antioxidants (uric acid and sulfhydryls) and markers of metabolic status (cholesterol, triglycerides, glucose and insulin). PLIR measures the ratio between the resistance to exogenous and endogenous ROS injury, independently from baseline level of oxidation, which was directly correlated with plasma cholesterol on lymphocytes (0.738, p=0.029), monocytes (0.691, p=0.047) and neutrophils (0.690, p=0.047). PLIR of lymphocytes was inversely correlated with uric acid (-0.810, p=0.009) and FRAP (-0.738, p=0.029) levels. On the other hand, PLIR of monocytes was directly correlated with the total scavenger antioxidant capacity attributable to nutritional antioxidants (0.738, p=0.029), calculated as the difference between TRAP and the contribution of uric acid and sulfhydryls to its value.
  
  
  CONCLUSIONS
  This study reports a feasible and reproducible new flow cytometry assay for assessing the leukocytes redox status. PLIR discriminates between reducing and scavenger activities and is able to appreciate the potentially dangerous effect of uric acid on innate immune response.
authors: Agricultural Research Council CRA, ex National Institute for Food and Nutrition Research INRAN, Rome, Italy. i.peluso@tiscali.it
authors: Ilaria
authors: I
authors: Peluso
authors: Gaspare
authors: G
authors: Adorno
authors: Anna
authors: A
authors: Raguzzini
authors: Lourdes
authors: L
authors: Urban
authors: Andrea
authors: A
authors: Ghiselli
authors: Mauro
authors: M
authors: Serafini
mesh_terms: D000328:Adult; D000578:Amidines / Q000494:pharmacology; D000975:Antioxidants / Q000378:metabolism*; D001372:Aza Compounds / Q000378:metabolism; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D002784:Cholesterol / Q000378:metabolism; D002839:Chromans / Q000378:metabolism; D005227:Fatty Acids / Q000378:metabolism; D005260:Female; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D007962:Leukocytes / Q000187:drug effects / Q000378:metabolism*; D008214:Lymphocytes / Q000187:drug effects / Q000378:metabolism; D008297:Male; D008875:Middle Aged; D009000:Monocytes / Q000187:drug effects / Q000378:metabolism; D009504:Neutrophils / Q000187:drug effects / Q000378:metabolism; D010084:Oxidation-Reduction / Q000187:drug effects; D018384:Oxidative Stress / Q000187:drug effects; D017382:Reactive Oxygen Species / Q000378:metabolism*; D015203:Reproducibility of Results; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
chemical_list: D000578:Amidines; D000975:Antioxidants; D001372:Aza Compounds; C463799:BODIPY581; D002839:Chromans; D005227:Fatty Acids; D017382:Reactive Oxygen Species; C046728:2,2'-azobis(2-amidinopropane); D002784:Cholesterol; D013755:Tetradecanoylphorbol Acetate; C010643:6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid