Concentration, activity and biochemical characterization of myeloperoxidase in fresh and post-thaw equine semen and their implication on freezability.

Myeloperoxidase (MPO) is a pro-oxidant enzyme associated with decreased motility in thawed equine semen. This study aimed to describe MPO concentration, activity and subunits in raw and thawed semen and to correlate these data with motilities in raw and thawed semen. Semen samples from five stallions were collected four times. Motilities were assessed in raw and thawed semen. MPO assays were performed in raw seminal plasma, raw sperm-rich pellet and thawed semen. Total and active MPO concentrations were, respectively, assayed by enzyme-linked immunosorbent assay and specific immunological extraction followed by enzymatic detection. MPO subunits present in semen were characterized by Western blot. Purified active MPO was added in saline solution and freezing extender to control its activity during freezing procedure. Differences between medians were determined using Kruskal-Wallis test, and correlations were determined using Spearman's test for nonparametric data. Active MPO concentration was low in seminal plasma and thawed semen, but high in pellet (p = 0.0058), as the opposite relation was observed for total MPO concentration (p < 0.0001). In seminal plasma and post-thaw semen, inactive 86-kDa MPO precursor was mainly observed. Purified MPO activity was decreased in the extender (p = 0.0286). MPO activity in pellet was highly correlated with thawed progressive motility (r = -0.5576, p = 0.0086). Inactive MPO precursor and unknown low molecular weight inactive MPO precursor subunits explain low MPO activity in semen. Major MPO activity was observed in pellet, and post-thaw loss of activity is partially explained by MPO inactivation in extender. Thawed semen motility was negatively correlated with MPO activity in pellet, becoming a potential freezability predictor.

Activation of equine neutrophils by phorbol myristate acetate or N-formyl-methionyl-leucyl-phenylalanine induces a different response in reactive oxygen species production and release of active myeloperoxidase.

Neutrophil (PMN) contribution to the acute inflammatory processes may lead to an excessive generation of reactive oxygen metabolites species (ROS) and secretion of granule enzymes. We compared the effects of either phorbol myristate acetate (PMA) or N-formyl-methionyl-leucyl-phenylalanine (fMLP) in combination with a pre-treatment by cytochalasin B (CB) on the production of ROS and the release of total and active myeloperoxidase (MPO) by isolated equine PMNs. The ROS production was assessed by lucigenin dependent chemiluminescence (CL) and ethylene release by alpha-keto-gamma-methylthiobutyric acid (KMB) oxidation. In the supernatant of activated PMNs, total equine MPO was measured by ELISA and active MPO by the SIEFED (Specific Immunologic Extraction Followed by Enzymatic Detection) technique that allows for the study of the interaction of a compound directly with the enzyme. The stimulation of PMNs with CB-fMLP only modestly increased the release of MPO, but more than 70% of released MPO was active. PMA stimulation markedly increased the production of ROS and release of MPO, but more than 95% of released MPO was inactive. When PMNs were pre-incubated with superoxide dismutase (SOD) prior to PMA activation, the lucigenin enhanced CL, which is linked to the superoxide anion (O2-) production, was much more decreased than KMB oxidation, linked to the hydroxyl-like radical production. The addition of SOD prior to the activation of PMNs by PMA also limited the loss of the activity of released MPO. These results confirm the key role of O2- generation in the ROS cascade in PMN and reveal its critical role on MPO inactivation.

In vivo administration of acepromazine or promethazine to horse decreases the reactive oxygen species production response of subsequently isolated neutrophils to stimulation with phorbol myristate acetate.

The previous experiments have shown that some phenothiazines have antioxidant and anti-inflammatory properties in vitro. In this study the inhibition of the production of reactive oxygen species (ROS) by neutrophils was studied in two groups of horses, which received a dose of 0.1 mg/kg of either acepromazine or promethazine intravenously. Blood samples were collected before (T0) and 0.5, 1, 3 and 5 h after drug administration. The chemiluminescence (CML) response of neutrophils was measured ex vivo in the presence of luminol for a period of 10 min and the maximum CML value (peak value) recorded. There was a significant inhibition of the ROS production in the acepromazine treated group (49% inhibition) at 5 h after administration and in the promethazine group (24% inhibition) at 3 h after administration (P < 0.05 vs. T0). These findings are of therapeutic relevance in the use of phenothiazines in equine patients with inflammatory diseases where neutrophil activation and ROS production are implicated.

Effects of unfractionated and fractionated heparins on myeloperoxidase activity and interactions with endothelial cells: possible effects on the pathophysiology of equine laminitis.

As heparins are sometimes used to prevent equine laminitis, the interactions between equine neutrophil myeloperoxidase (MPO), unfractionated (UFH) and fractionated low molecular weight (LMWH) heparins and digital endothelium have been investigated. The effects of the heparins on purified equine MPO activity were tested by immunocapture followed by enzymatic detection. Endothelium-MPO interactions were assessed by measuring total and active MPO uptake by arterial and venous digital endothelial cells in culture with or without the addition of heparins. A dose-dependent MPO inhibition by UFH and LMWH was seen, with the greatest reduction in MPO activity noted with the highest concentration of LMWH. The MPO capture was greater in arterial cells, but heparins better inhibited MPO capture in venous cells. The activity of cell-bound MPO was almost completely suppressed by the heparins, and no differences were observed between UFH and LMWH. The results confirm the anti-inflammatory properties of heparins and allow a better understanding of the potential role of MPO in laminitis.