One of the reasons that ergot alkaloid metabolism in ungulates has not been defined adequately is the lack of a robust and logistically favorable analytical method for determination of all the alkaloids and their metabolites. Currently, the primary methods for quantification of ergot alkaloids include HPLC (Craig et al., 1994; Jaussaud et al., 1998), ELISA (Hill and Agee, 1994), and  high performance liquid chromatography-mass spectrometry (HPLC-MS) (Yates et al., 1985; Lehner et al., 2004). The ELISA method is rapid but not specific for a given ergot alkaloid. In fact, Hill and Agee's (1994) data would support a bias toward simpler ergots, such as lysergic acid, over the more complex forms like ergovaline. As a result, use of the ELISA method is restricted primarily to total ergot alkaloid level determinations and thus is of limited use for aiding in the determination of mechanisms associated with alkaloid absorption and metabolism. However, this assay has been functioning well, based on all reports so far, as a diagnostic test for ergot alkaloid exposure.

The HPLC methods of Craig et al. (1994) and Jaussaud et al. (1998) are specific but suffer from a need for pure standards of each alkaloid of interest to establish identities of alkaloids by retention time. As such, these methods are limited in their use for discovery research concerning alkaloid metabolites. These methods, however, are excellent for determining profiles of known alkaloids in a number of matrices (e.g., animal serum, feces, and urine). The HPLC-MS methods (Yates et al., 1985; Lehner et al., 2004) offer the greatest opportunity for discovery work involving the full array of ergot alkaloids and their metabolites. As with HPLC methods that use fluorescence detection, analysis by HPLC-MS provides identification and quantification of ergot alkaloids with known chemical structures. However, a third dimension of information in the form of a mass spectrum also is acquired for each component in the sample mixture. Molecular mass can be derived from this MS data for confirmation of known alkaloids separated by HPLC. For novel ergot alkaloids and any corresponding metabolites as a result of biotransformation, a single HPLC-MS analysis can provide retention times, molecular mass, and empirical formulas from isotopic distribution information. Furthermore, tandem mass spectrometry (MS/MS), in which a molecular ion can be isolated and fragmented within the mass analyzer, can provide clues for elucidation of chemical structure of unknown ergot alkaloids and corresponding metabolites. The growing availability of HPLC-MS/MS will provide us with the ability to examine other ergot alkaloids and metabolites, allowing for a better understanding of toxicant fate in ungulates.

If our knowledge of the mechanisms for metabolism and the absorption/excretory routes for the ergot alkaloids is limited in ungulates, our knowledge of the kinetic rates of clearing is even more restricted. Little research has been conducted in ungulates, largely due to the lack of sufficient and/or affordably priced quantities of pure ergot alkaloids of interest available for large animal studies. Jaussaud et al. (1998) reported that the plasma clearance of ergovaline administered via an intravenous injection (17 mg/kg BW) to sheep was 0.02 L/min/kg BW. Similarly, Bony et al. (2001) injected geldings with 15 mg ergovaline/kg BW and observed a plasma clearance rate of 0.02 L/min/kg BW. However, because only plasma ergovaline concentrations were evaluated in these studies, it remains unclear as to whether ergovaline was sequestered in tissues, metabolized (e.g., to lysergic acid), or actually eliminated from the body. Fully understanding the metabolism, distribution, and clearance of these alkaloids in ungulates would facilitate the design of better protocols for improving animal tolerance to E+ tall fescue.

 

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