Oxidation of homovanillic acid as a selective assay for eosinophil peroxidase in eosinophil peroxidase-myeloperoxidase mixtures and its use in the detection of human eosinophil peroxidase deficiency.
Biochemical assays for peroxidase activity do not usually distinguish between different peroxidases. The guaiacol assay, for example, which is one of the most commonly used assays for peroxidase activity, is sensitive to both eosinophil peroxidase (EPO) and the peroxidase of neutrophils, i.e., myeloperoxidase (MPO), thus preventing distinction of the two peroxidases in mixed neutrophil-eosinophil populations. In this paper we describe a simple and sensitive method for selective assays of EPO in EPO-MPO mixtures or mixed populations of neutrophils and eosinophils. The method is based on the peroxidase-mediated oxidation of homovanillic acid (HVA) under appropriate assay conditions in which EPO is still very active in catalyzing the reaction whilst MPO-mediated HVA oxidation is almost undetectable. Optimal assay conditions were as follows: pH 10.5, 10 microM hydrogen peroxide, 0.8 mM HVA and an incubation time of 120 min at 37 degrees C. Under these conditions the assay permits EPO activities as low as 0.025 guaiacol U/ml to be measured even in the presence of 0.175 guaiacol U/ml of MPO. In mixed neutrophil-eosinophil cell suspensions the test permits the detection of as few as 5 X 10(3) eosinophils even in the presence of about 700 X 10(3) neutrophils (eosinophils: neutrophils ratio 1:140) with no appreciable interference by the latter cells. The method described here has been applied to studies of human EPO deficiency and proved to be successful in the identification of individuals with partial EPO deficiency, which is not feasible with non quantitative methods (for example, cytochemistry) or unselective biochemical assay of peroxidase activity.
Menegazzi
R
R
Istituto di Patologia Generale, Università di Trieste, Italy.
Zabucchi
G
G
Zuccato
P
P
Cramer
R
R
Piccinini
C
C
Patriarca
P
P
D047209:Eosinophil Peroxidase; D004804:Eosinophils / Q000201:enzymology*; D006719:Homovanillic Acid / Q000378:metabolism*; D006801:Humans; D009504:Neutrophils / Q000201:enzymology; D010084:Oxidation-Reduction; D009195:Peroxidase / Q000032:analysis* / Q000502:physiology; D010544:Peroxidases / Q000097:blood* / Q000172:deficiency
D047209:Eosinophil Peroxidase; D010544:Peroxidases; D009195:Peroxidase; D006719:Homovanillic Acid