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

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 \u3c 0.001), pre-myograph incubation temperature (39 vs. 21 °C, 1 h) had no effect (p \u3e 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 \u3e 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\u3c 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

Characterising the impact of ergot alkaloids on digestibility and growth performance of lambs

The negative impacts of ergot contamination of grain on the health of humans and animals were first documented during the fifth century AD. Although ergotism is now rare in humans, cleaning contaminated grain concentrates ergot bodies in screenings which are used as livestock feed. Ergot is found worldwide, with even low concentrations of alkaloids in the diet (<100 ppb total), reducing the growth efficiency of livestock. Extended periods of increased moisture and cold during flowering promote the development of ergot in cereal crops. Furthermore, the unpredictability of climate change may have detrimental impacts to important cereal crops, such as wheat, barley, and rye, favoring ergot production. Allowable limits for ergot in livestock feed are confusing as they may be determined by proportions of ergot bodies or by total levels of alkaloids, measurements that may differ widely in their estimation of toxicity. The proportion of individual alkaloids, including ergotamine, ergocristine, ergosine, ergocornine, and ergocryptine is extremely variable within ergot bodies and the relative toxicity of these alkaloids has yet to be determined. This raises concerns that current recommendations on safe levels of ergot in feeds may be unreliable. Furthermore, the total ergot alkaloid content is greatly dependent on the geographic region, harvest year, cereal species, variety and genotype and can vary greatly depending on the chosen analytical method. Considerable animal-to-animal variation in the ability of the liver to detoxify ergot alkaloids also exists and the impacts of factors, such as pelleting of feeds or use of binders to reduce bioavailability of alkaloids require study. Accordingly, unknowns greatly outnumber the knowns for cereal ergot and further study to help better define allowable limits for livestock would be welcome

MOLECULARLY IMPRINTED POLYMERS SYNTHESIZED AS ADSORBENTS FOR ERGOT ALKALOIDS: CHARACTERIZATION AND \u3cem\u3eIN VITRO\u3c/em\u3e AND \u3cem\u3eEX VIVO\u3c/em\u3e ASSESSMENT OF EFFECTS ON ERGOT ALKALOID BIOAVAILABILITY

Alkaloid toxicities negatively impact livestock health and production and are of serious economic concern to animal industries. To date, few strategies have been developed to evaluate alkaloid levels in feed or to counteract alkaloid toxicities. The present research evaluated the applicability of imprinting technology to synthesize polymers that have potential to interact with ergot alkaloids and therefore reduce their bioavailability in the GIT. The studies also evaluated applicability of synthesized polymers for use in the ruminal environment using an in vitro ruminal fermentation model, and for the ability to ameliorate vasoconstriction using ex vivo myographic evaluations. In the first experiment, styrene-based molecularly imprinted polymer (MIP) was synthesized using ergotamine as the imprinting template and evaluated for specificity of adsorption to various ergot alkaloids. Cross reactivity with related alkaloids exists due to similarities in structure and functional groups. Both polymers (MIP and NIP) showed strong adsorption intensity and no difference was observed for estimated maximum adsorption capacity between MIP and NIP. Morphologically, MIP was highly porous with greater surface area than NIP. Solid phase extraction indicated stronger adsorption of MIP than NIP to ergot alkaloids suggesting the potential for MIP as a sorbent material for solid phase extraction (SPE) columns used for sample clean-up prior to HPLC or LC-MS/MS analysis of complex samples. In Experiment 2, methacrylic acid-based polymers were synthesized with ergotamine as a template. Among the 4 alkaloids evaluated for selectivity, adsorption difference between MIP and NIP interacted with alkaloid concentration, although differences were generally consistent across concentrations. Imprinting did not affect lysergol and bromocriptine adsorption, but resulted in higher adsorption to methylergonovine. However, there was no difference between MIP and NIP for adsorption of ergotamine. Hydrophobic interactions and H-bonding were the primary interactive forces between polymers and alkaloid adsorbents. Morphologically, MIP had greater surface area and porosity implying a larger surface for adsorption. In addition to its application as SPE sorbent, this MIP was a suitable candidate for application as a feed adsorbent to reduce the bioavailability of certain alkaloid in the gut. In experiment 3, methacrylic acid-based polymers were evaluated for their effect on in vitro ruminal fermentation. There were no interactions between polymer type and inclusion level, and no differences between polymer types for cumulative gas production or rate of gas production. Total gas production and rate of gas production were unaffected by inclusion level. Polymers did not affect total or individual VFA concentrations, ammonia-N or methane concentration at any inclusion level. However, a logarithmic increase in polymer dose level decreased the pH linearly with maximum depression of 0.24 units. This study indicated that, within the range of expected use levels, polymers were essentially inert and would not be expected to affect ruminal fermentation. In experiment 4, ex vivo myographic bioassays were used to determine the impact of polymers on ergotamine bioavailability. Responses measured in the ex vivo myographic studies had similar trend as the responses generated from in vitro isothermal adsorption studies. Results of that study also showed that ex vivo myographic responses could be predicted from in vitro isothermal adsorption studies with more than 80% accuracy. These studies indicate that synthetic polymers are potentially effective adsorbents to mitigate ergot toxicity with little evidence of substantial differences between MIP and NIP

Biological Activity, Analytical Detection, and Degradation Assessment of the S-epimers of Ergot Alkaloids

Ergotism has an extensive history dating back centuries. The fungus Claviceps purpurea produces ergot sclerotia that contain secondary metabolites called ergot alkaloids. Ergot alkaloids cause toxic effects after consumption of ergot contaminated food and feed. Ergot contaminated food and feed currently poses a health risk to humans and animals. There are six common ergot alkaloids produced by the fungus that exist in two configurations. The two configurations are the C-8-R-isomer (R-epimer) and the C-8-S-isomer (S-epimer), the latter of which has been studied to a lesser extent. The S-epimers have been considered inactive in terms of biological activity compared to the R-epimers. The R-epimers can elicit their toxic effects by binding to receptors in vascular smooth muscle cells. In this dissertation, the aim was to further understand the S-epimers of the six ergot alkaloids common to Claviceps purpurea through biological, analytical, and degradation assessments. An arterial tissue bath technique was used to assess the bioactivity of selected S-epimers. Following the exposure of arteries to the S-epimers, vascular constriction was observed. Exposure of increasing concentrations of the S-epimers to the arteries resulted in an increase in the contractile response. In a following study, arterial contraction due to the S-epimers over time was assessed and a sustained vascular contraction was observed. The sustained vascular contraction of the S-epimer was different than the corresponding R-epimer, which may be due to differences in vascular contraction mechanisms. This is the first time that the S-epimers of ergot alkaloids have demonstrated bioactivity, contrary to historical belief. To support the vasoconstriction observed in the previous studies, a novel approach using an in silico method to assess ergot structure activity relationships was used, specifically to observe the S-epimer-vascular receptor relationship. The S-epimers bound to vascular receptors with relatively high affinity and strong molecular interactions, which has not been documented previously. Since the S-epimers demonstrated bioactivity and can bind to vascular receptors, it was important to assess the concentrations of the S-epimers in natural ergot contaminated samples. A newly validated analytical method to detect and quantify R and S-epimer concentrations using high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) was used. This new analytical method can be implemented in laboratories worldwide for the sensitive and accurate detection and quantification of ergot epimers. It was observed that the S-epimers of the six common ergot alkaloids constitute approximately 35% of the total ergot alkaloid concentration in the samples assessed. When the natural ergot contaminated samples were exposed to various temperatures over time, the R and S-epimer concentrations varied. Therefore, ergot contaminated samples should be analyzed prior to their use for food or feed to obtain the most accurate concentration results. The high concentration of the S-epimers and the lack of concentration stability warranted an investigation into the degradation of the S-epimers associated with their potential bioactivity which can lead to toxic effects. Some studies assessing degradation methods of ergot alkaloids only quantify or focus on the R-epimers and not the S-epimers. Through using degradation methods of ammonia and ultra-violet (UV) light, based on practicality, we observed that the R and S-epimer concentrations behave differently, following the degradation methods. Ammonia significantly reduced the concentration of total ergot alkaloids, while demonstrated varied effects on the concentrations of the R and S-epimers. The use of ammonia for the degradation of ergot alkaloids may be a practical means for livestock to consume ergot contaminated feed without any adverse effects. The use of UV light did not significantly alter the concentrations of the total ergot alkaloids; however, it decreased the concentration of the S-epimer, ergotaminine. Since the effects of the degradation methods on the R and S-epimer concentrations may vary, it is important to assess both configurations following degradation assessments. Overall, this research has furthered our understanding of the S-epimers of ergot alkaloids. Continuous research on the S-epimers of ergot alkaloids is needed to further understand the S-epimers in terms of their bioactivity, stability, and degradation. It was important to investigate the S-epimers since they were historically deemed as non-bioactive and have not been thoroughly researched, compared to the R-epimers. The results of this dissertation have demonstrated, for the first time, the bioactivity of the S-epimers and the use of a novel in silico method to assess how the S-epimers may contribute to the adverse effects associated with ergot alkaloids. In addition, high concentrations of S-epimers were observed within ergot contaminated samples and the use of ammonia as a method to degrade ergot epimers may be practical within the agriculture industry. Guidelines for the concentrations of ergot alkaloids in food and feed have been set worldwide; however, some guidelines only include the R-epimers and not the S-epimers. The results of this dissertation encourage the inclusion of the S-epimers into guidelines globally for both food and feed for the protection of human and animal health. The observation that the S-epimers are bioactive, have high concentrations in contaminated matrices, and may contribute to the toxic effects following ergot alkaloid consumption, will ultimately improve food and feed safety guidelines for both human and livestock populations

Investigation of the Ergopeptide Epimerization Process

Ergopeptides, like ergocornine and a-ergocryptine, exist in an S- and in an R-configuration. Kinetic experiments imply that certain configurations are preferred depending on the solvent. The experimental methods are explained in this article. Furthermore, computational methods are used to understand this configurational preference. Standard quantum chemical methods can predict the favored configurations by using minimum energy calculations on the potential energy landscape. However, the explicit role of the solvent is not revealed by this type of methods. In order to better understand its influence, classical mechanical molecular simulations are applied. It appears from our research that “folding” the ergopeptide molecules into an intermediate state (between the S- and the R-configuration) is mechanically hindered for the preferred configurations

Investigation of the Ergopeptide Epimerization Process

Ergopeptides, like ergocornine and a-ergocryptine, exist in an S- and in an R-configuration. Kinetic experiments imply that certain configurations are preferred depending on the solvent. The experimental methods are explained in this article. Furthermore, computational methods are used to understand this configurational preference. Standard quantum chemical methods can predict the favored configurations by using minimum energy calculations on the potential energy landscape. However, the explicit role of the solvent is not revealed by this type of methods. In order to better understand its influence, classical mechanical molecular simulations are applied. It appears from our research that “folding” the ergopeptide molecules into an intermediate state (between the S- and the R-configuration) is mechanically hindered for the preferred configurations