Equine inflammatory disease is accompanied by a neutrophil activation resulting in the release of granulocytic enzyme myeloperoxidase (MPO). To measure MPO in horse plasma as marker of neutrophil activation, the authors purified equine neutrophil MPO and developed a specific enzyme immunoassay using 2 specific polyclonal antibodies obtained from rabbit (primary antibody) and guinea pig (secondary antibody). The sandwich complex "primary antibody-MPO-secondary antibody" was detected using a goat anti-guinea pig immunoglobulin antibody conjugated to alkaline phosphatase. The enzyme-linked immunosorbent assay (ELISA) showed good precision and accuracy, with intra- and interassay coefficients of variation below 10% for MPO concentrations ranging from 0.78 to 50 ng/ml. A stable plasma MPO value, unaffected by time elapsed between blood collection and centrifugation, was obtained with plasma from EDTA anticoagulated blood. The mean MPO value measured in 38 healthy horses was 181.80 +/- 64.74 ng/ml. In 20 horses suffering from obstruction of the large or small intestine, MPO concentrations measured at the time of arrival at the intensive care unit were significantly higher than mean normal value, ranging from 477.88 to 2,748.13 ng/ml. Work is in progress to apply this MPO ELISA technique to other biological fluids and other equine diseases.
Ginkgo biloba extract is known to be efficient in diseases associated with free radical generation. The purpose of this work was to study, under in vitro conditions, the action of Ginkgo biloba extract (Gbe) against superoxide anion (O2-.), which is directly or indirectly implicated in cell damage. Gbe appears to have both an O2-. scavenging effect and also a superoxide dismutase activity. Its antiradical effect was demonstrated by low temperature electron spin resonance and in a non-enzymatic system (phenazine methosulfate-NADH), and its enzymatic activity was shown by polarographic determination.
An original method called SIEFED (specific immunological extraction followed by enzymatic detection) was developed for the specific detection of the activity of equine myeloperoxidase (MPO). The method consists of the extraction of MPO from aqueous solutions by immobilized anti-MPO antibodies followed by washing (to eliminate proteins and interfering molecules) and measurement of MPO activity using a detection system containing a fluorogenic substrate, hydrogen peroxide, and nitrite as reaction enhancer. The SIEFED technique was applied to study active MPO in horse biological fluids and the effects of 2 polyphenolic molecules, curcumin and resveratrol, on MPO activity. The detection limit of the SIEFED was 0.23 mU/ml. The SIEFED exhibited good precision with intra-assay and interassay coefficients of variation below 10% and 20%, respectively, for MPO activities ranging from 0.25 to 6.4 mU/ml. The activity of MPO was generally higher than 1 mU/ml in the fluids collected from horses with inflammatory diseases. Curcumin and resveratrol exerted a dose-dependent inhibition on MPO activity and, as they were removed before the enzymatic detection of MPO, the results suggest a direct drug-nzyme interaction or an enzyme structure modification by the drug. The SIEFED is a new tool that would be useful for specific detection of active MPO in complex media and for selection of MPO activity modulators.
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