New Approaches to the Measurement of the Concentration
and Peroxidase Activity of Myeloperoxidase
in Human Blood Plasma

I. V. Gorudkoa,1, O. S. Tcherkalinab, A. V. Sokolovb, M. O. Pulinab, E. T. Zakharovab,
V. B. Vasilyev
b, S. N. Cherenkevicha, and O. M. Panasenkoc

a Department of Biophysics, Belarusian State University, pr. Nezavisimosti 4, Minsk, 220030 Belarus

b Institute for Experimental Medicine, Russian Academy of Medical Sciences,
ul. Akademika Pavlova 12, St. Petersburg, 197376 Russia

c Research Institute of Physico-Chemical Medicine, ul. Malaya Pirogovskaya 1a, Moscow, 119992 Russia

Received March 2, 2009; in final form, March 20, 2009

Abstract—A novel method for spectrophotometrical measurement of myeloperoxidase (MPO) activity in
plasma with o-dianisidine (DA) as a substrate is proposed. We have determined the optimal conditions, includ-
ing the pH and hydrogen peroxide concentration, under which MPO is the main contributor to DA oxidation in
plasma. Specific MPO inhibitors, salicylhydroxamic acid or 4-aminobenzoic acid hydrazide, are added to mea-
sure the activity of other heme-containing peroxidases (mainly hemoglobin and its derivatives) and subtract
their contribution from the total plasma peroxidase activity. Plasma MPO concentrations are quantified by a
new enzyme-linked immunosorbent assay (ELISA) developed by us and based on the use of antibodies raised
in rats and rabbits. The sensitivity of this ELISA is high: 0.2–250 ng/ml. A direct and significant (P < 0.0001)
correlation was observed between the MPO activities measured spectrophotometrically and the MPO level
determined by ELISA in blood samples from 38 healthy donors. The proposed approaches to MPO measure-
ment in plasma can be used to evaluate the enzyme activity and concentration, as well as the efficacy of mech-
anisms by which MPO is regulated under physiological conditions and against the background of various
inflammatory diseases.

Key words: myeloperoxidase, plasma peroxidase activity, blood plasma, o-dianisidine, hemoglobin, enzyme-
linked immunosorbent assay.

DOI: 10.1134/S1068162009050057

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