Contractile Response of Bovine Lateral Saphenous Vein to Ergotamine Tartrate Exposed to Different Concentrations of Molecularly Imprinted Polymer

Abstract

Ergot alkaloids, in their active isomeric form, affect animal health and performance, and adsorbents are used to mitigate toxicities by reducing bioavailability. Adsorbents with high specificity (molecularly imprinted polymers: MIP) adsorb ergot alkaloids in vitro, but require evaluation for biological implications. Using ex vivo myography, synthetic polymers were evaluated for effects on the bioactivity of ergotamine tartrate (ETA). Polymers were first evaluated using isotherms. Lateral saphenous veins were collected from 17 steers for four independent studies: dose response of ETA, adsorbent dose response, validation of pre-myograph incubation conditions and MIP/ non-molecularly imprinted polymer (NIP) comparison. Norepinephrine normalized percent contractile response to increasing ETA exhibited a sigmoidal dose response (max: 88.47 and log of the effective molar concentration (EC₅₀) (-log [ETA]) of 6.66 ± 0.17 M). Although sample preparation time affected contractile response (p < 0.001), pre-myograph incubation temperature (39 vs. 21 °C, 1 h) had no effect (p > 0.05). Isothermal adsorption showed a maximum adsorption of 3.27E-008 moles·mg^-1 and affinity between 0.51 and 0.57 mg (R²: 0.83-0.92) for both polymers, with no significant difference between polymers (p > 0.05). No significant differences in maximum inhibitory (p = 0.96) and IC₅₀ responses (p = 0.163) between MIP and NIP were noticed. Normalized percent contraction could be predicted from the in vitro adsorption data (R² = 0.87, p < 0.01), for both polymers. These studies indicate that synthetic polymers are potentially effective adsorbents to mitigate ergot toxicity caused by ergot alkaloids, with little evidence of significant differences between MIP and NIP in aqueous media.

Keywords: ergot alkaloids; imprinted polymer; myograph.

Figure 1

Norepinephrine normalized contractile response curve of smooth muscle (lateral saphenous vein) to increasing concentrations of ETA. The points are means and the vertical bars show the SEM (n = 10). Percent contraction (NE normalized) = 88.5 + ((5.7 − 88.5)/(1 + 10^{6.664−intital [ETA]})) (Equation (3), Section 5.7).

Figure 2

Effect of incubation temperature and length of incubation in buffer containing ETA (7.813 × 10⁻⁷ M) on the contractile response of saphenous veins. Each data point was the mean of six replications of blood vessels ± SEM. Incubation time (60 min) reduced (p < 0.001) the contractile response and there was no effect (p > 0.05) of temperature or interaction between incubation time and temperature (p = 0.26). Different letters (a,b) above columns denote significant differences (p < 0.001) between incubation times.

Figure 3

The contractile response of lateral saphenous veins exposed to ETA treatment (7.813 × 10⁻⁷ M) that were filtered through polypropylene filtration columns fitted with quartz frits, at 39 °C. Each data point represents mean of six replications of blood vessels ± SEM. There was no significant difference (p = 0.8752) in response between the filtration and no filtration methods. Similar letters (a) above columns denote lack of significant difference (p > 0.05) between columns.

Figure 4

Ergotamine adsorption of polymers (molecularly imprinted polymers, MIP and non-molecularly imprinted polymer, NIP), in modified Krebs–Henseleit buffer media fitted to binding saturation curve with specific binding sites.

Figure 5

(a) Effect of increasing doses of MIP and NIP on the NE-normalized contractile response of saphenous veins, induced by ETA (7.813 × 10⁻⁷ M) in a 1 h, 39 °C incubation. (b) T-test analysis of area under the curve for comparison of MIP and NIP. Data are expressed as mean ± SEM (n = 12). Similar letters (a) above columns denote lack of significant difference (p > 0.05) between columns.

Figure 6

Regression plot between the measured and the predicted percent contractile response (NE-normalized) in the presence of increasing concentrations of (a) molecularly imprinted polymer and (b) non-molecularly imprinted polymer. Dotted lines represent 95% confidence intervals. The residuals (c) MIP and (d) NIP are shown on the right side of the plot.