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ANIMAL PRODUCTION |
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* Rangeland Ecology and Management Department, University of Idaho, Moscow 83844-1135; and
and
ARS-USDA, Poisonous Plant Research Laboratory, Logan, UT 84341
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionImplicationsLiterature Cited |
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Key Words: Body Condition • Lupinus • Quinolizidine Alkaloids • Sheep • Toxic Plants
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionImplicationsLiterature Cited |
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). Range livestock adapt to and usually survive encounters with poisonous plants through learned foraging strategies to avoid these plants or by inherent mechanisms of detoxification (Provenza et al., 1992; Launchbaugh et al., 2001). Acquired preferences and aversions based on ingestive feedback allow herbivores to select plants and plant parts in inverse proportion to their toxicant content to decrease poisoning (Bernays et al., 1989; Pfister, 1999). Livestock also possess metabolic abilities to negate or decrease the toxic effects of plant toxins once they are ingested (Freeland and Janzen, 1974; McArthur et al., 1991; Smith, 1992; Launchbaugh, 1996).
Many animal and plant characteristics affect the ability of an animal to practice selective intake or to detoxify allelochemicals. An important, though little studied, factor that undoubtedly influences the detoxification of ingested phytotoxins is the nutritional status or body condition of a grazing animal. Well-fed animals may effectively process toxins from plants, whereas nutrient-deprived animals may be more likely to eat poisonous plants or they may be metabolically compromised in their ability to detoxify or excrete the toxins (Foley et al., 1995).
The objective of this experiment was to determine whether body condition and/or short-term supplementation of energy would affect the concentration and disposition (absorption and elimination) of quinolizidine alkaloids from silvery lupine (Lupinus argenteus) ingested by domestic sheep (Ovis aries). Primary emphasis was placed on anagyrine due to its importance in "crooked calf disease" (Panter et al., 1999).
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionImplicationsLiterature Cited |
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. Briefly, a 100-mg sample of ground lupine seed was combined with 5 mL of 1N HCl and 4 mL CHCl3 and placed in a mechanical shaker for 15 min, and then centrifuged to separate the aqueous and organic phases. The aqueous fraction was transferred to a clean tube and basified to a pH between 9 and 9.5 by drop-wise addition of concentrated NH4OH. The basified solution was extracted twice with CHCl3 (4 mL, then 2 mL). The combined CHCl3 fractions were filtered through anhydrous Na2SO4, dried under a stream of N2 at 50°C, and then reconstituted in 5 mL of CHCl3 for analysis. Two microliters of this final solution was injected into an HP 5890 GC equipped with a split/splitless injector, flame ionization detector, and a J&W DB-5 (30 m x 0.33 mm i.d.) capillary column (Agilent). Injector temperature was 250°C and operated in the split mode. Split vent flow rate was 60 mL/min and was purged after 1 min. Oven temperature was programmed as follows: 100°C for 1 min, 100 to 200°C at 50°C/min, and 200 to 320°C at 5°C/min. Alkaloid peaks, retention times, and peak areas were compared with two known standard plant samples: Lupinus caudatus (USDA accession No. 82-7), containing quinolizidine alkaloids, and L. formosus (USDA accession No. 87-3), containing piperidine alkaloids (Gardner and Panter, 1993, 1994). Peak identifications were based on GC/mass spectroscopy analysis (Thermo-Finnigan, Austin, TX) and comparison of retention times with those alkaloid peaks from the standard plant samples (Figure 1). The interassay variation of 85 replicates over the last 6 yr was less than 5% (T. Weirenga, ARS-USDA Poisonous Plant Research Lab, Logan, UT, unpublished data).
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) and were offered to ewes being conditioned to LBC or ABC, respectively. The amount of diets offered was readjusted when lambs were weaned and the presentation of the feeds was changed as outlined later in this section (all amounts are reported on an as-fed basis). During wk 1 to 5, the low-nutrient diet was a chopped mixture containing 60% alfalfa hay (1-00-066; NRC, 1985) and 40% barley straw (1-00-498; NRC, 1985). The high nutrient diet comprised chopped alfalfa hay and 200 g of rolled corn (4-02-931; NRC, 1985) per ewe, per day. The diets were offered at 3% of BW for ewes in both groups. From wk 6 to 9, the diets were changed to a pelleted feed. The pelleted low-nutrient diet consisted 50% alfalfa and 50% barley straw, with no additional supplement, whereas the high-nutrient diet consisted of 100% alfalfa pellets and an additional 400 g of rolled corn per ewe, per day. The amount of pellets offered was gradually increased over 5 d from 1.50 to 2.25% BW for LBC ewes, and from 2.50 to 3.25% BW for ABC ewes. These amounts were divided into two daily allotments offered at 0700 and 1900, and all feed was consumed.
Body condition was scored on a 0-to-5 scale by tactile and visual estimation of body fat depositions (American Sheep Industry Association, 1997) by two trained technicians on a weekly basis during conditioning. Only one of these technicians was blind to the treatments. The mean body condition at the end of the conditioning period was 1 for LBC and 2.5 for ewes in ABC. The mean BW (±SD) of ewes in LBC at the beginning and at the end of the conditioning were 76.7 ± 6.2 and 62.8 ± 5.5 kg, respectively, whereas the weights for ABC were 83.9 ± 5.6 and 81.2 ± 6.2 kg. The average BW of ABC ewes was higher than that of LBC ewes before (P < 0.05) and after (P < 0.05) the conditioning period. However, LBC ewes lost a greater percentage of BW during conditioning than did ABC ewes (18.1 ± 4.3% vs. 3.2 ± 5.7% respectively; P < 0.05).
Two days before the experiment began, all ewes were fed 2.2% BW of a basal diet divided into two allotments each day at 0700 and 1900 to provide similar rumen fill and nutrient intake between groups. The basal diet consisted of a coarsely chopped mixture of grass and alfalfa hay that had 16% CP and 58% in vitro DM digestibility.
Ewes in ABC and LBC were assigned randomly to treatments with supplement (S) or without daily supplement (NS). Ewes assigned to the supplement treatments (five ewes in LBC and five in ABC) were offered a supplement at 0.4% BW that consisted of 50% soybean meal (5-04-610, NRC, 1985) and 50% corn meal (4-02-931, NRC, 1985). This supplement was intended to provide a supply of both protein and energy, and the level offered was fixed at 0.4% BW. The supplement was offered in two portions at 0800 and 2000 each day beginning the day of the experiment and continuing through the 60-h sampling period.
Experimental Procedure
The experimental measurements began after all ewes received a single oral dose of 8.5 g of ground silvery lupine seed and pods/kg of BW of at 1100. Lupine (ground to 1-mm particle size) was diluted in 5 L of lukewarm tap water and gavaged using a livestock pump system (The Magrath Co., McCook, NE). Blood samples (30 mL) were drawn via jugular vein puncture at 0, 0.5, 1, 2, 3, 4, 6, 8, 12, 18, 24, 36, 48, and 60 h after gavaging. Blood was allowed to clot for 1 h at room temperature and was then centrifuged for 30 min at 1,060 x g. Serum was immediately harvested and frozen in a standard chest freezer (–5 to –10°C).
Serum alkaloid content was determined by GC after acid/base extraction similar to that for plant material (Gardner and Panter, 1993, 1994). Briefly, a 10-mL aliquot was basified with 10 drops of concentrated NH4OH, and then extracted twice with CHCl3 (4 mL both times) with mechanical shaking for 15 min. After shaking, the samples were centrifuged to separate the phases. The organic (CHCl3) phase was transferred to a clean tube and acidified with 5 mL of 1N HCl, mechanically mixed for 5 min, and then centrifuged to separate phases. The aqueous phase was transferred to a clean tube, basified with 1.1 mL of concentrated NH4OH, and then extracted twice with CHCl3 (4 mL, then 2 mL). The combined CHCl3 fractions were filtered through anhydrous Na2SO4, and then dried under a stream of N2 at 50°C and reconstituted in 200 µL of CHCl3 for analysis. Conditions for GC analysis were the same as those described for plant material; peak identification was confirmed by GC/mass spectroscopy. This method was validated in serum using caffeine as an internal standard and total and individual alkaloids were measured against a standard curve for anagyrine (S. T. Lee, ARS-USDA Poisonous Plant Research Lab, Logan, UT, unpublished data). Coefficients of variation for anagyrine in plant standards and in serum were 5.1 and 5.5%, respectively.
Two standard plant samples (USDA Accession No. 82-7 and 87-3) were used for quality control and comparison of alkaloid peak areas. These two standard plant samples were run with each batch of serum extractions to ensure adequacy of the extraction process.
Toxicokinetic Evaluation
Absorption and elimination profiles were analyzed using standard pharmacokinetic software (PK Solutions for Non-Compartmental Pharmacokinetic Data Analysis, Summit Research Services, Montrose, CO). A curve-stripping procedure was used to determine the basic pharmacokinetic parameters of half-life, rate and concentration intercept for each phase of the blood level curve. The following parameters were determined for total alkaloid and anagyrine: intercept: Cn = Coefficient of each exponential term; slope: s = –
n/2.303; rate constant: n = –2.303 s; half-life: t
= 0.693/n; Cmax = maximum observed concentration; Tmax = time point at Cmax; and AUC 
(area under the curve).
A trapezoidal method was used to determine the AUC of a concentration vs. time graph. The general equation describing the disposition of drugs in serum is given by the summation expression: C = 
Cn exp (–nt), where Cn and n are the zero-time intercepts and rate constants, respectively, for each exponential term. The corresponding triexponential function can be written as: C = Ae–
t + De–ßt + Ee–
t. In this expression, the intercepts are given letter designations indicating the three common drug disposition phases that are typically encountered after an oral dose: Absorption, Distribution, and Elimination.
Statistical Analyses
Four treatments resulted from combinations of body condition (LBC or ABC) and supplementation (S or NS): LBC-S, LBC-NS, ABC-S, and ABC-NS. Comparison of serum alkaloid levels in these groups was accomplished with a repeated-measures ANOVA, with time as the repeated measure. This initial analysis indicated that short-term supplementation had no effect on alkaloid disposition, and supplementation was removed from the statistical model.
Subsequently, the experiment was reanalyzed as a completely randomized design with two treatments. Variables analyzed were serum alkaloid concentration (µg/mL) over sampling time, biological half-life (h) of the alkaloids, and AUC for specific alkaloids. Mixed-model analysis of variance for correlated repeated measures (MIXED procedure using the ARI option for covariance structure; SAS Inst., Inc., Cary, NC) was performed to examine the effect of body condition on serum alkaloid concentrations. The AUC for total alkaloid and anagyrine in serum was analyzed separately. Because biological half-life of absorption or elimination did not have the time factor, these variables were analyzed using two-way analyses of variance to test the effects of body condition and supplementation (PROC GLM of SAS). For all analyses, multiple comparisons for main effects were performed to delineate differences using least squares means after significant (P < 0.05) F-tests.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionImplicationsLiterature Cited |
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). Three peaks in the chromatograms represented known alkaloids (anagyrine, 5,6-dehydrolupanine, and lupanine). Three other peaks represented alkaloids whose chemical structures were not elucidated (alkaloids D, E, and F, as designated in the USDA-ARS Poisonous Plant Research Laboratory records; K. E. Panter, unpublished results). The peaks representing alkaloids D, E, and F appeared in the chromatograms at about 10.9, 11.3 and 10.1 min, respectively (Figure 1). The plant material contained 1.64% total alkaloids (dry weight basis). The major alkaloids were anagyrine (28.8%), alkaloid E (28.3%), alkaloid D (16.0%), 5,6 dehydrolupanine (11.5%), and lupanine (8.2%). These five alkaloids accounted for about 93% of the total alkaloid content in the plant material.
Animal Response Rates of Alkaloid Absorption and Elimination
Basic toxicokinetic parameters were determined for total alkaloid and for anagyrine in the LBC and ABC groups (Table 1, Figure 2). Absorption, distribution, elimination, and peak height were evaluated as AUC using the trapezoidal rule to calculate AUC. There was a difference in AUC between LBC and ABC for total alkaloid (P = 0.003) and anagyrine (P = 0.001; Figure 2). This difference strongly suggests that overall disposition of lupine alkaloids, and especially the teratogen anagyrine, is altered in a relatively dramatic way based on body condition. Although further research is needed to evaluate the mechanism of this difference, these data suggest that animals in thin body condition are more vulnerable and respond differently than animals in higher body condition when ingesting lupine.
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for anagyrine and total alkaloid respectively. Biological half-life of absorption for total and individual alkaloids did not differ among treatments (P = 0.90), except for anagyrine, whereby the absorption half-life was significantly larger (P = 0.02) for LBC. However, some differences in elimination half-life were observed for other individual alkaloids (Table 1) and were significant for anagyrine, lupanine, and alkaloid E. Low body condition ewes experienced slower elimination of lupanine and alkaloid E than ABC ewes (P < 0.01), whereas the elimination of anagyrine was marginally affected by body condition (P < 0.06). These data would explain the increased serum alkaloid levels (calculated by AUC) noted in Figure 2 for both anagyrine and total alkaloids.
The dose of lupine used in this research resulted in a single total oral alkaloid dose of 139 mg/kg BW. This dose generated no clinical signs of toxicity at any time after dosing. Gardner and Panter (1993) also reported a lack of observed toxicosis when sheep were dosed with a single 150-mg/kg BW dose of alkaloids from L. argenteus containing 1.92% total alkaloids.
Blood samples from the sheep contained measurable levels of alkaloids beginning 0.5 h after dosing, and peaked by 2 to 3 h (Figure 3). Alkaloids could be detected in the blood stream for up to 48 h (Figure 2). Anagyrine fell below detection limits between 24 and 36 h after dosing. All alkaloids observed in the plant material, except alkaloid D, were detected in serum (Figure 1). Alkaloid F occurred at such low concentrations (<1.5 µg/mL) for a very short time (<3 h) that analyses were not performed. There was an effect of time on total alkaloid and anagyrine concentration (P < 0.001), but no interaction between time and body condition was observed. Lupanine, 5,6-dehydrolupanine and alkaloid E (data not shown) cleared from the body by 36, 48, and 18 h, respectively, and clearance of these alkaloids followed a pattern similar to that of anagyrine and total alkaloid as shown in Figure 3. These data clearly suggest that body condition is an important factor in disposition of lupine alkaloids.
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), and we propose these to be lupine alkaloid metabolites. These unknown peaks were also reported by Gardner and Panter (1993), and further research is needed to evaluate and identify these apparent lupine metabolites in the serum. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionImplicationsLiterature Cited |
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). Anagyrine is one of the key alkaloids of interest in this study as it relates to the incidence of lupine-induced "crooked calf disease" in the western United States (Panter et al., 1999). Understanding the disposition of this and other alkaloids from lupines in the mammalian system is important in providing key information to evaluate management strategies to reduce livestock losses. As range animals are introduced to summer pastures in varying body condition, understanding the relationship between body condition, nutritional status, and alkaloid absorption and elimination is important.
Differences in rates of alkaloid absorption and elimination strongly suggest that overall disposition of lupine alkaloids, and especially the teratogen, anagyrine, is altered in a relatively dramatic way based on body condition. Although further research is needed to evaluate the mechanism of this difference, these data suggest that animals in thin body condition maintain higher levels of serum alkaloids for a greater duration than do animals in higher condition when ingesting lupine. Therefore, fetal exposure to these alkaloids may be altered because of maternal body condition. Furthermore, it has been observed that thin cows grazing lupine rangelands consume greater amounts of lupine than cows in higher body condition (Lopez-Ortiz, 2002).
These data suggest that physiological differences, including body condition, influence lupine alkaloid disposition in the mammalian system. These differences may occur during absorption, distribution, biotransformation, or elimination of the alkaloids (Rozman and Klaassen, 1995). Each of these processes can be affected by multiple factors varying among individuals, including physiological state, body composition, and diet composition.
Differences expected between LBC and ABC animals include a smaller size and mass of splanchnic organs, primarily the liver (Noziere et al., 1999). If animals in low body condition lose liver mass, this may decrease detoxification capacity for animals with low condition scores. It is uncertain, but doubtful, that a decreased liver detoxification capacity in LBC ewes explains the differences observed in this study. Most of the quinolizidine alkaloids contained in Lupinus caudatus do not undergo major biotransformation in sheep, cattle, or goats (Gardner and Panter 1993, 1994). Similarly, Petterson et al. (1994) recovered two quinolizidine alkaloids largely unchanged in the urine of humans within 72 h of dosing.
Low body condition can also lead to a decrease in muscle mass and fatty deposits (Birnie et al., 2000). A reduction of these tissues may have decreased the mass for alkaloid dissolution in the body of LBC ewes. In this case, the toxicants would be more likely to bind to plasma proteins, and one could expect higher circulating levels in the blood stream (DeBethizy and Hayes, 1989; Smith, 1992). Rozman and Klaassen (1995) contended that the concentration of a toxicant in blood depends largely on its volume of distribution, indicating that body water volume could influence the measured toxin concentrations and may have been a factor in the higher blood concentration of some alkaloids in these animals. It is unlikely however, that body water volume affected this study because body water increases as body fat decreases (Scholz et al., 1990; Zahn et al., 1991), and thinner ewes had higher serum concentrations of alkaloids than did fatter ewes.
Foley et al. (1995) proposed that toxin breakdown during detoxification requires energy and substrates for biotransformation and elimination. Thus, if biotransformation of lupine alkaloids takes place in the animals, high alkaloid concentration in blood might indicate a decreased energy supply for detoxification or elimination in LBC animals. Smith (1992) suggested that plant compounds undergoing phase II biotransformations are conjugated with co-substrates of detoxification to carry and eliminate them. Those substrates can be deficient in animal tissues, thus reducing the capacity to detoxify by decreasing elimination. However, detoxification of lupine alkaloids may consist of simply eliminating the original alkaloids from the system. Meeker and Kilgore (1991) found substantial amounts of anagyrine and lupanine in milk after dosing lactating goats with lupine.
There were unidentified compounds in the GC profiles of blood serum after dosing that did not match with either the plant profile or the ewe’s serum before dosage, possibly indicating postabsorptive metabolism of lupine alkaloids. Gardner and Panter (1993) observed these compounds in their blood profiles as well. These unidentified compounds could be metabolites or conjugated alkaloids, and their identification will require further work.
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Footnotes |
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2 Correspondence: 1150 E. 1400 N. (phone: 435-752-2941; e-mail: kpanter{at}cc.usu.edu).
Received for publication July 29, 2002. Accepted for publication May 28, 2004.
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This article has been cited by other articles:
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  S. T. Lee, K. E. Panter, J. A. Pfister, D. R. Gardner, and K. D. Welch The effect of body condition on serum concentrations of two teratogenic alkaloids (anagyrine and ammodendrine) from lupines (Lupinus species) that cause crooked calf disease J Anim Sci, October 1, 2008; 86(10): 2771 - 2778. [Abstract] [Full Text] [PDF]
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