J. Anim Sci. 2008. 86:2761-2770. doi:10.2527/jas.2008-1025
© 2008 American Society of Animal Science
The effect of 7,8-methylenedioxylycoctonine-type diterpenoid alkaloids on the toxicity of methyllycaconitine in mice1
K. D. Welch2,
K. E. Panter,
D. R. Gardner,
B. T. Green,
J. A. Pfister,
D. Cook and
B. L. Stegelmeier
USDA-ARS, Poisonous Plant Research Laboratory, Logan, UT 84341
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Abstract
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Larkspur plants contain numerous norditerpenoid alkaloids, which include the 7,8-methylenedioxylycoctonine (MDL)-type alkaloids and the N-(methylsuccinimido)anthranoyllycoctonine (MSAL)-type alkaloids. The MSAL-type alkaloids are generally much more toxic (typically >20 times). Toxicity of many tall larkspurs, such as Delphinium barbeyi, has been attributed to its large concentration of MSAL-type alkaloids, including methyllycaconitine (MLA). However, the norditerpenoid alkaloids found in the greatest concentrations in most D. barbeyi populations are either deltaline or 14-O-acetyldictyocarpine (14-OAD), both less toxic MDL-type alkaloids. Although the individual toxicities of MLA, 14-OAD, and deltaline have been determined, the impact (additive or antagonistic) that large concentrations of deltaline or 14-OAD in the plant have on the toxicity of MLA is unknown. Consequently, the effect of MDL-type alkaloids on the toxicity of MLA was compared by using median lethal dose (LD50) and toxicokinetic profiles of the brainand muscle from mice receiving i.v. administration of these alkaloids, individually or in combination, at ratios of 1:1, 1:5, and 1:25 MLA to MDL-type alkaloids. The LD50 for MLA alone was 4.4 ± 0.7 mg/kg of BW, whereas the coadministration of MLA and deltaline at 1:1, 1:5, and 1:25 resulted in an LD50 of 2.7, 2.5, and 1.9 mg/kg of BW, respectively. Similarly, the coadministration of MLA and 14-OAD at 1:1, 1:5, and 1:25 resulted in an LD50 of 3.1, 2.2, and 1.5 mg/kg of BW, respectively. Coadministration of mixtures did not result in increased MLA bioavailability or alterations in clearance from the brain and muscle. Consequently, the increased toxicity of the mixtures was not a result of increased MLA bioavailability (based on the maximum concentrations observed) or alterations in MLA clearance from the brain and muscle, because these were unchanged. These results demonstrate that MDL-type alkaloids have an additive effect on MLA toxicity in mice and may also play a role in the overall toxicity of tall larkspur plants in cattle.
Key Words: Delphinium • diterpenoid alkaloid • larkspur • median lethal dose (LD50) • methyllycaconitine
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INTRODUCTION
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Larkspurs (Delphinium spp.) are one of the most serious toxic plant problems on foothill and mountain rangelands in the western United States (Pfister et al., 1999
). Total costs to the livestock industry have been estimated at millions of dollars annually (Nielsen et al., 1994). The toxicity of larkspur plants is due to more than 18 norditerpenoid alkaloids that occur as 1 of 2 types, the 7,8-methylenedioxylycoctonine (MDL)-type including deltaline and 14-O-acetyldictyocarpine (14-OAD), and the N-(methylsuccinimido)anthranoylly-coctonine (MSAL)-type, including methyllycaconitine (MLA; Pfister et al., 1999; Figure 1). Although the MSAL-type alkaloids are much more toxic (Manners et al., 1993, 1995), the MDL-type alkaloids are generally more abundant (Pfister et al., 1999; Gardner et al., 2002).
Management recommendations for grazing cattle on larkspur-containing ranges is based on concentration of MSAL-type alkaloids in the larkspur (Pfister et al., 2002; Ralphs et al., 2002). Delphinium barbeyi is a problematic species of tall larkspur plants because of its large concentration of MLA. However, the most abundant norditerpenoid alkaloids in most D. barbeyi populations are the less toxic MDL-type alkaloids, either deltaline or 14-OAD (Manners et al., 1993; Pfister et al., 1999; Gardner et al., 2002). The relative concentrations of these 2 alkaloids are location dependent, with deltaline being more abundant in some populations and 14-OAD predominating in others (Gardner et al., 2002). Although toxicities of MLA, 14-OAD, and deltaline have been determined individually (Manners et al., 1991; Panter et al., 2002), effects of the large concentration of deltaline or 14-OAD in these plants on toxicity of MLA are not known. To understand what contributions MDL-type alkaloids have on the overall toxicity of larkspurs, the effects of deltaline and 14-OAD on the toxicity of MLA were assessed in mice by comparing the lethality of i.v. administration of these alkaloids administered individually and in combination.
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MATERIALS AND METHODS
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All procedures were conducted under veterinary supervision and were approved by the Utah State University Animal Care and Use Committee.
Alkaloid Preparation
The larkspur alkaloids used in this study were extracted from D. barbeyi (Pelletier et al., 1981, 1989; Manners et al., 1991). Alkaloids, both individually purified alkaloids and a total alkaloid extract, were suspended in physiological buffered saline solution, and the pH was decreased with HCl to achieve solubility. Ammonium hydroxide was then added to the solutions to raise the pH to as close to physiological pH (5.5 to 7.0) as possible while still retaining solubility. Solutions were stored in sterile injection vials at 4°C until use. No adverse effects were seen after injections of solutions (0.05 to 0.2 mL) with lesser pH (5.5 to 7.0). The total alkaloid extract was analyzed by Fourier-transform infrared spectroscopy (Gardner et al., 1997) for measurement of MSAL-type and total alkaloids.
Animals
Median lethal dose (LD50) of each solution was determined in male Swiss Webster mice (Simonson Laboratories Inc., Gilroy, CA) weighing 23 ± 2 g. Between 0.05 and 0.2 mL of the purified alkaloid(s), in buffered saline, were injected via the tail vein. Mice were observed for clinical effects and mortality, and the LD50 of the solutions was determined by using a modified up-and-down method (Bruce, 1987). This method is preferred because fewer animals are required; however, it results in unbalanced numbers in each group. The LD50 values were calculated by using SAS Proc Probit in a logistic regression (SAS Inst. Inc., Cary, NC).
For toxicokinetic studies, deltaline was chosen to represent MLD-type alkaloids, and brain and muscle samples were analyzed. A total of 140 male Swiss Webster mice (Simonson Laboratories Inc.) weighing 24 ± 2 g were randomly divided into 4 treatment groups (MLA alone, 1:1, 1:5, or 1:25 ratio of MLA to deltaline) to obtain samples at 7 time points after injection (1, 2, 5, 10, 15, 30 and 60 min). Mice were dosed via the tail vein with 70% of the LD50 dose for each treatment, as determined in the LD50 studies. This dose has been shown to produce clinical signs but not be lethal (Stegelmeier et al., 2003). Brain and skeletal muscle from the biceps femoris from the pelvic limb were collected and frozen at –20°C until analyzed.
Sample Extractions
Samples (0.2 to 0.3 g) were prepared by CHCl3 extraction as described previously (Stegelmeier et al., 2003) and stored at –20°C until analysis by HPLC-mass spectrometry. This method of extraction has been shown to have approximately a 95% recovery rate and approximately 11% variation.
Sample and Standard Preparation
Samples were resuspended in 1.0 mL of methanol:20 mM ammonium acetate (50:50) and mixed for 1 min with a Vortex Genie (Scientific Industries Inc., Bohemic, NY), followed by filtration through a 0.2-µm nylon syringe filter into HPLC autosampler vials. Calibration standards of MLA and deltaline were prepared in ethanol from stock solutions (1.0 mg/mL) stored at –20°C. Diluted standards were prepared fresh daily to 1,000, 500, 250, 125, 62.5, and 31.2 ng/mL. Deltaline and MLA standards were injected as a mixture. Standards were analyzed after every 40 samples.
Mass Spectrometry Analysis
Analysis of MLA and deltaline in the samples was accomplished by using a Surveyor HPLC and autosampler system coupled to a ThermoFinnigan LCQ Advantage Max mass spectrometer (Thermo Finnigan, San Jose, CA), with modification to the methods described previously (Turek et al., 1995; Gardner et al., 1999; Stegelmeier et al., 2003). The instrument parameters were maximized for detection of the alkaloid reserpine by using the autotune feature. Samples (25 µL) were injected with a Surveyor autosampler onto a Betasil C18 HPLC column (100 x 2 mm, 5 µm, 100 A, Keystone Scientific, Bellefonte, PA). The column was eluted by using a gradient flow of methanol (A) to 20 mM ammonium acetate (B), starting with 65% A and linear gradient to 80% A from 0 to 6 min, 80% A from 6 to 7 min, and reequilibration at 65% A from 7 to 12 min. The flow rate was 0.3 mL/min. Retention times for MLA and deltaline under these conditions were approximately 3.7 and 5.3 min, respectively. Flow from the HPLC was connected directly to the electrospray source of the mass spectrometer. The mass spectrometer was operated in a full-scan MS-MS mode after fragmentation of selected parent ions of 683.3 (MH+, for MLA) and 508.3 m/z (MH+, for deltaline). Selected ion chromatograms for m/z = 665.3 and m/z = 476.2 were used for detection and quantification of MLA and deltaline, respectively.
Analysis and Statistics
Confidence (fiducial) intervals (95%) were calculated for LD50 values by using logistic regression. Statistical comparisons of toxicokinetic profiles between groups were performed by using ANOVA with a post hoc test of significance between individual groups. Differences were considered significant at P < 0.05. The alkaloid concentrations were plotted by using SigmaPlot for Windows (SPSS Inc., Richmond, CA). Nonlinear regression of the data was obtained by fitting the data to the 3-parameter equation, f = [a + (b x x)]/[1 + (c x x)], by using SigmaPlot for Windows (SPSS Inc.). Kinetic profiles were analyzed by using standard pharmacokinetic software (1998, PK Solutions 2.0 for Noncompartmental Pharmacokinetic Data Analysis, Summit Research Services, Montrose, CO). A curve-stripping procedure was used to determine the basic pharmacokinetic parameters of half-life and rate for the elimination phase of the MLA concentration curve. The following variables were determined: t1/2 = 0.693/Kelim, Cmax, Tmax, AUC. The t1/2 is the elimination half life, and Cmax and Tmax describe the concentration and time of maximal alkaloid concentrations. A trapezoidal method was used to determine the area under the curve (AUC) of a concentration versus time graph.
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RESULTS
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The acute toxicity of MLA was compared with the toxicity of MDL-type alkaloids administered individually versus their coadministration as mixtures with MLA having the following composition: 1:1, 1:5, and 1:25 MLA to MDL-type alkaloid. The LD50 for MLA alone was 4.4 ± 0.7 mg/kg of BW, whereas the LD50 for deltaline alone was 113.3 ± 6.4 mg/kg of BW. Even though deltaline was approximately 25 times less toxic than MLA, the coadministration of deltaline with MLA affected lethality (Figure 2). There was a dose-dependent increase (P < 0.05) in toxicity as the ratio of deltaline to MLA was increased from 1:1 to 1:5 to 1:25, with the respective LD50 values of 2.7 ± 0.3, 2.5 ± 0.2, and 1.9 ± 0.1 mg/kg. Similar results (P < 0.05) were obtained when 14-OAD was coadministered with MLA (Figure 2). Additionally, the coadministration of lycoctonine with MLA resulted in a similar dose-dependent increase in toxicity (Figure 2).
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Figure 2. The effect of coadministration of various 7,8-methylenedioxylycoctonine (MDL)-type alkaloids on the toxicity of methyllycaconitine (MLA). Data represent the median lethal dose (LD50) of MLA alone, MDL-type alkaloids alone, and MLA plus MDL-type alkaloids at ratios of 1:1, 1:5, and 1:25 MLA to MDL-type alkaloids. Results represent the mean ± SD of 24 to 86 mice per group; *P < 0.05 as compared with the MLA group. 14-OAD = 14-O-acetyldictyocarpine.
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Clinical signs were similar to those described by Stegelmeier et al. (2003). Within seconds of injection, mice were reluctant to move and they sat hunched with diffuse piloerection, resulting in a scruffy appearance. Within 1 min, mice developed muscle tremors and spastic jerky muscular convulsions. These jerky convulsions were followed by dyspnea, which caused the nose and toes to become cyanotic. Typically, animals died within 2 min of treatment. Animals that did not die seemed to have recovered and were completely normal within 20 min. There were no differences in the time to death among any of the treatment groups.
The effect of large MDL-type alkaloid concentrations on the toxicokinetic profile of MLA was determined by using deltaline as the model MDL-type alkaloid (Figures 3A and 3B). Elimination profiles of MLA in brain and muscle tissues were compared between mice administered MLA alone versus mice that received MLA and deltaline together to determine whether the increased toxicity of the mixtures could be attributed to changes in concentration of MLA in these tissues. In the brain, the coadministration of deltaline had no affect on the rate of elimination (P > 0.05) or elimination half life (P > 0.05) of MLA (Table 1). In the muscle, there was no effect on the rate of elimination (P > 0.05) of MLA, but there was a difference (P < 0.05) in the elimination half life of MLA, with this being greater (P < 0.05) in the 1:1 and 1:25 groups (Table 2). For both brain and muscle, the time to reach maximal MLA concentration (Tmax) did not differ (P > 0.05) between treatments. However, the maximal concentration of MLA obtained (Cmax) was reduced (P < 0.05) for both tissues by the addition of deltaline (Tables 1 and 2). Consequently, the total amount of MLA exposure to brain and muscle tissues, as depicted by AUC, was less (P < 0.05) in the deltaline coadministration groups. However, coadministration did not result in a dose-dependent change in Cmax or AUC in either tissue (P > 0.05).
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Figure 3. The effect of coadministration of deltaline on the toxicokinetics of methyllycaconitine (MLA). Data represent the tissue concentration of MLA from mice dosed with MLA alone and MLA plus deltaline at ratios of 1:1, 1:5, and 1:25 MLA to deltaline. Results represent the mean ± SD of the concentration of MLA in A) brain and B) muscle for 4 to 5 mice per group. The insets in the graphs are an expansion of the 1- to 10-min data points. The MLA and mixtures were dosed at 70% of their median lethal dose (LD50), as determined in Figure 2 .
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Table 1. Effect of coadministration of deltaline on the toxicokinetic profile of methyllycaconitine (MLA) in the brain1
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Table 2. The effect of coadministration of deltaline on the toxicokinetic profile of methyllycaconitine (MLA) in muscle1
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To assess the validity of the additive effect of MDL-type alkaloids on the toxicity of MLA, the toxicity of a total alkaloid extract from D. barbeyi was tested. The total alkaloid extract contained approximately a 1:5 ratio of MLA to MDL-type alkaloids (as determined by Fourier-transform infrared spectroscopy), with deltaline and 14-OAD being the predominant MDL-type alkaloids in the extract (Figure 4). The LD50 of the total alkaloid extract (2.0 ± 0.2 mg/kg of BW) was less (P < 0.05) than that of pure MLA and was very similar (P > 0.05) to that of the mixtures of MLA and either deltaline, 14-OAD, or lycoctonine at a 1:5 ratio (LD50: 2.5 ± 0.2, 2.2 ± 0.3, and 2.0 ± 0.2 mg/kg of BW, respectively; Figure 5).
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Figure 5. Comparison of the toxicity of a total alkaloid extract from Delphinium barbeyi versus the coadministration of purified alkaloids. The data represent the median lethal dose (LD50) of methyllycaconitine (MLA) alone, a total alkaloid extract, and MLA plus 7,8-methylenedioxylycoctonine (MDL)-type alkaloids at a 1:5 ratio. Results represent the mean ± SD of 25 to 86 mice per group; *P < 0.05 as compared with the MLA group; #P < 0.05 as compared with the total alkaloid extract group. 14-OAD = 14-O-acetyldictyocarpine.
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To determine the maximum extent to which the MDL-type alkaloids increased the toxicity of MLA, the ratio of 14-OAD to MLA was increased to 1:50 and 1:100. The LD50 of the 1:50 mixture was 1.2 ± 0.1 and 0.8 ± 0.0 for the 1:100 mixture (Figure 6). All of the LD50 data for MLA and MLA plus 14-OAD were plotted, and a nonlinear regression curve was fitted to the data by using a 3-parameter equation, f = [a + (b x x)]/[1 + (c x x)] (Figure 6). Results from the nonlinear regression analysis indicated that the additive effect of 14-OAD on the toxicity of MLA had a maximum effect at approximately a 1:25 ratio of MLA to 14-OAD.
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Figure 6. Relationship between increasing ratios of 14-O-acetyldictyocarpine (14-OAD) to methyllycaconitine (MLA) and toxicity. The data represent the median lethal dose (LD50) of MLA plus deltaline at ratios of 1:0, 1:1, 1:5, 1:25, 1:50, and 1:100 methyllycaconitine (MLA) to deltaline. A nonlinear regression of the data was obtained by fitting the data to the 3-parameter equation, f = [a + (b x x)]/[1 + (c x x)].
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DISCUSSION
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Previous research has demonstrated that the physiological effects of MLA are attributable to its high affinity to nicotinic acetylcholine receptors (nAChR) in muscle and in the central and autonomic nervous systems (Benn and Jacyno, 1983; Stegelmeier et al., 1998). Methyllycaconitine has been shown to be a potent and selective competitive antagonist with nanomolar affinity at 
7-nAChR and with micromolar affinity at other nAChR (Ward et al., 1990; Alkondon et al., 1992; Lopez et al., 1998; Daly, 2005). In this regard, the binding affinity of larkspur alkaloids to nicotinic receptors has been linked to toxicity (Macallan et al., 1988; Dobelis et al., 1999). Although the MSAL-type alkaloids are much more toxic (typically >20 times; Manners et al., 1993, 1995), the MDL-type alkaloids are generally 1 to 5 times more abundant, with some populations of plants having 25 times more MDL-type alkaloids (Pfister et al., 1999; Gardner et al., 2002). Consequently, the focus of this study was to determine the influence of large concentrations of MDL-type alkaloids on the toxicity of larkspur plants.
In this study, results using the 2 most abundant MDL-type alkaloids, deltaline and 14-OAD, were essentially the same in that they both caused a dose-dependent increase in the toxicity when coadministered with MLA (Figure 2). The effect of these 2 alkaloids on the toxicity of MLA was additive, because their coadministration with MLA at 1:1, 1:5, 1:25 ratios resulted in decreases in the LD50 by approximately 25, 50, and 60%, respectively, compared with that of MLA alone. Solutions containing both MLA and a MDL-type alkaloid showed increased toxicity compared with MLA alone, suggesting that MDL-type alkaloids exacerbate MSAL toxicity and therefore play an important part in the toxicity of larkspur plants.
Results from lycoctonine coadministration with MLA indirectly addressed the question of competition for cholinergic receptors. Although lycoctonine is missing the anthranilic acid ester moiety on the C18 carbon on MLA, coadministration of lycoctonine with MLA resulted in a decrease in the LD50 compared with MLA alone, similar to deltaline and 14-OAD. This finding suggests there is no competition between the MSAL and MDL-type alkaloids for the same cholinergic receptors at these concentrations. If there were competition for the same binding site, the presence of a large concentration of the much less toxic MDL-type alkaloids would have resulted in an increase in the LD50 compared with that of MLA alone. In this regard, MLA is reported to be fairly selective for the 7-nAChR subunit (Davies et al., 1999), whereas the receptor subunit specificity of the MDL-type alkaloids is unknown. However, a study by Kukel and Jennings (1994) demonstrated that MDL-type alkaloids can displace -bungarotoxin (known to bind 7-nAChR) from rat and housefly neural membranes, suggesting that MDL-type alkaloids can compete for binding to the 7-nAChR subunit. Results from this study did not provide evidence that the MDL-type alkaloids compete with MLA for cholinergic receptor subunit binding at these concentrations.
One possible explanation for the results from this study is that enough nAChR are present in this strain of mice to ensure that there is no competition for the same receptor subunits at the concentrations of MLA and MDL-type alkaloids used in this study. However, as demonstrated in Figure 6, the additive effect of the MDL-type alkaloids to the toxicity of MLA-containing solutions was found to be saturated at approximately a 1:25 ratio of MLA to MDL-type alkaloid. These results suggest that specific receptors, or receptor subunits, are involved in this effect and that all the larkspur alkaloid-sensitive cholinergic receptors have been bound by alkaloids. Consequently, addition of more alkaloids would not increase the toxicity of the solution. Additionally, the toxicity of these 1:50 and 1:100 mixtures was similar to that of the 1:25 mixture, and definitely not less toxic, which suggests that even at a 1:100 ratio, there is no competition between MLA and MDL-type alkaloids for binding at the same subunits of the cholinergic receptor at these concentrations.
It could also be argued that differences in the affinity of MLA versus the MDL-type alkaloids for the 7-nAChR subunit could explain why the coadministration of large concentrations of MDL-type alkaloids did not compete with MLA for receptor binding. It is possible that the affinity of MLA is so much greater than that of the MDL-type alkaloids that MLA completely outcompetes the MDL-type alkaloids for binding to 7-nAChR subunits, and consequently, any effect the MDL-type alkaloids have is only additive to that of MLA. Additional research is needed to test these hypotheses and to identify the receptor(s) that the MDL-type alkaloids bind.
A potential explanation for the potentiating effect of MDL-type alkaloids on the toxicity of MLA could be due to an alteration in the toxicokinetic profile of MLA, such as a decrease in the clearance-excretion or metabolism of MLA. If the MDL-type alkaloids are eliminated from the body via the same pathway as that of MLA, then the presence of large concentrations of MDL-type alkaloids could saturate that elimination pathway, resulting in a delay in the elimination of MLA, which would effectively enhance its toxicity. However, in this study we found that large concentrations of deltaline had no effect on the kinetics of elimination of MLA in brain and muscle tissues, which indicates that the increased toxicity observed when deltaline was coadministered with MLA could be due to an additive effect of the deltaline at nAChR and not simply an alteration in the elimination of MLA.
One key aspect of this study was to demonstrate that the toxicity of a mixture of pure MLA and MDL-type alkaloids at a 1:5 ratio had toxicities similar to a solution of a total alkaloid extract from D. barbeyi that contained approximately 5 times as much of the MDL-type alkaloids (predominantly deltaline and 14-OAD) as MLA. The slightly lesser LD50 value for the total alkaloid extract could be explained by the small amount of 14-deactylnudicauline, another MSAL-type alkaloid found in the extract. These results suggest that the use of purified compounds gives a close approximation to the overall toxicity of the plant itself.
In conclusion, the results of this study confirm previous reports that MSAL-type alkaloids such as MLA cause greater toxicity than MDL-type alkaloids when administered i.v. and are likely the primary factors responsible for the toxicity of larkspur plants. However, in plants that contain large quantities of MDL-type alkaloids, these less toxic alkaloids appear to enhance the overall toxicity of the plant and should be considered when predicting the potential toxicity of larkspur populations. Consequently, when chemical analyses are performed on larkspur plants to assess their toxic potential, the concentrations of both the MSAL and MDL-type alkaloids should be determined, with more weight given to the MSAL-type alkaloids. Differential relative toxicities of alkaloids by species should also be considered because of differential bioavailability of alkaloids with oral dosing (monogastric vs. ruminant) and known species sensitivity (mice vs. relatively insensitive sheep vs. cattle). Consequently, similar experiments need to be performed in cattle to verify these results.
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Footnotes
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1 The authors thank Kendra Dewey, Ed Knoppel, and Scott Larsen for their expert technical support. 
2 Corresponding author: Kevin.Welch{at}ars.usda.gov
Received for publication March 10, 2008.
Accepted for publication May 28, 2008.
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Abstract
INTRODUCTION
