Evaluation of vaccination against methyllycaconitine toxicity in mice.

The purpose of this study was to determine whether larkspur toxins conjugated to protein carriers would promote active immunity in mice. Mice were injected with several larkspur toxin-protein conjugates or adjuvant alone to determine whether the resulting immunological response altered animal susceptibility to methyllycaconitine, the major toxic larkspur alkaloid. Although vaccinations increased the calculated lethal dose 50% (LD50) for intravenous methyllycaconitine toxicity, overlapping confidence intervals did not provide evidence of differences between the vaccinated and control groups. In the lycoctonine conjugate (LYC)-vaccinated group, mouse survival was related (P = 0.001) to serum titers for methyllycaconitine doses up to 4.5 mg/kg of body weight. When mice withlow antibody titers were removed from the vaccinated groups in which titer was related to survival, the recalculated LD50 estimates were 20% greater than the LD50 of the control group. However, the 95% confidence intervals of the recalculated LD50 groups overlapped with the control groups. Overall, these results suggest that vaccination altered methyllycaconitine toxicity in mice and that vaccination may be useful in decreasing the effects of larkspur toxins in animals. Additional studies are warranted to continue development of potential larkspur vaccines for livestock.

Development of enzyme-linked immunosorbent assays for the hepatotoxic alkaloids riddelliine and riddelliine N-oxide.

Pyrrolizidine alkaloid-containing plants are widely distributed throughout the world and are particularly common in the genus Senecio. The structural types and concentrations of the alkaloids vary among plant species. In addition, within a species of plant, concentrations vary with environment and location. Many pyrrolizidine alkaloids are toxic and cause poisoning in livestock and in humans. Rapid, sensitive, and specific diagnostic techniques are needed to identify poisoned animals and to determine the particular plants and conditions under which livestock are likely to be poisoned. In this study, two competitive inhibition enzyme-linked immunosorbent assays for riddelliine, riddelliine N-oxide, and other closely related pyrrolizidine alkaloids were developed using polyclonal antibodies. One assay is class specific toward the free base forms of the pyrrolizidine alkaloids; the other assay showed cross-reactivity to both the free base and N-oxide forms of the alkaloids. The assay with the lowest limit of detection had an I(50) of 803.9 pg with a limit of detection of 47.5 pg for riddelliine. Spike and recovery studies for riddelliine in bovine blood ranged from 45 to 74%. The assay that showed cross-reactivity between the N-oxide and free base forms of the pyrrolizidine alkaloids allowed estimation of the total pyrrolizidine alkaloid content in Senecio riddellii in admixture with alfalfa. These findings suggest that these techniques will be excellent tools to diagnose poisoned animals and identify highly toxic plants.

Analysis of limonoid glucosides from citrus by electrospray ionization liquid chromatography-mass spectrometry.

An electrospray ionization liquid chromatography-mass spectrometry (ESI-LC-MS) method for the detection and quantitation of limonoid glucosides has been developed. Negative ions [M - H(+)](-) characteristic of six limonoid glucosides can be detected and quantified from selected ion monitoring chromatograms using carminic acid as an internal standard. The described method has been applied to the analysis of limonoid glucoside content in various liquid and solid Citrus spp. samples as well as complex mixtures of partially purified limonoid glucosides. Rapid and sensitive qualitative screening of samples for limonoid glucosides can also be accomplished with slight modifications of the method.

Development of enzyme-linked immunosorbent assays for toxic larkspur (Delphinium spp.) alkaloids.

Larkspur (Delphinium spp.) poisons thousands of cattle on western rangelands each year. Because poisoning does not cause specific lesions, and poisoned animals are rarely found before they die, definitively identifying poisoned animals is difficult. Additionally, toxin concentrations in larkspur plants vary with environment, plant, and location. Rapid, sensitive, and specific diagnostic techniques are needed to identify poisoned animals and to determine when and what plants are likely to poison livestock. In this study, three competitive inhibition enzyme-linked immunosorbent assays (CI-ELISA) for toxic larkspur alkaloids were developed. One assay is class-specific toward the N-(methylsuccinimido)anthranoyllycoctonine (MSAL) alkaloids, and two assays are specific for individual alkaloids. The assay with the lowest limit of detection had an I(50) of 191 pg with a limit of detection of 30.5 pg for methyllycaconitine. Spike and recovery studies using bovine blood and brain tissue ranged from 52 to 89%. These findings suggest that with additional development these techniques are likely to be excellent tools for diagnosing poisoned animals and identifying highly toxic plants.

GC/MS/MS detection of pyrrolic metabolites in animals poisoned with the pyrrolizidine alkaloid riddelliine.

Pyrrolic metabolites from pyrrolizidine alkaloids (PAs) were detected in liver and dried blood samples using a gas chromatography/tandem mass spectrometry (GC/MS/MS) selected product-ion-monitoring method. A calibration curve was constructed using a protein-metabolite conjugate spiked into dried bovine blood. These spiked samples served as a model for tissues from animals poisoned by the toxic metabolite of PAs. Tissue samples from pigs fed various amounts of the PA alkaloid riddelliine (from Senecio riddellii) were analyzed for pyrrolic metabolites, and the results were applied to the calibration curve to provide a measure of the degree of PA poisoning. Pyrrolic metabolites were detected in liver and blood samples of all poisoned animals at levels between 2 and 64 ppm. Although differences in metabolite levels could be discerned under the reported experimental conditions, the amount detected did not correlate with the dose of riddelliine given; and livers fixed with formalin gave greatly reduced recovery than those same livers either frozen or freeze dried.

Larkspur (Delphinium spp.) poisoning in livestock.

Larkspurs (Delphinium spp.) are toxic plants that contain numerous diterpenoid alkaloids which occur as one of two structural types: (1) lycotonine, and (2) 7,8-methylenedioxylycoctonine (MDL-type). Among the lycoctonine type alkaloids are three N-(methylsuccinimido) anthranoyllycoctonine (MSAL-type) alkaloids which appear to be most toxic: methyllycaconitine (MLA), 14-deacetylnudicauline (DAN), and nudicauline. An ester function at C-18 is an important structural requirement for toxicity. Intoxication results from neuromuscular paralysis, as nicotinic acetylcholine receptors in the muscle and brain are blocked by toxic alkaloids. Clinical signs include labored breathing, rapid and irregular heartbeat, muscular weakness, and collapse. Toxic alkaloid concentration generally declines in tall larkspurs with maturation, but alkaloid concentration varies over years and from plant to plant, and is of little use for predicting consumption by cattle. Knowledge of toxic alkaloid concentration is valuable for management purposes when cattle begin to eat larkspur. Cattle generally begin consuming tall larkspur after flowering racemes are elongated, and consumption increases as larkspur matures. Weather is also a major factor in cattle consumption, as cattle tend to eat more larkspur during or just after summer storms. Management options that may be useful for livestock producers include conditioning cattle to avoid larkspur (food aversion learning), grazing tall larkspur ranges before flowering (early grazing) and after seed shatter (late grazing), grazing sheep before cattle, herbicidal control of larkspur plants, and drug therapy for intoxicated animals. Some potentially fruitful research avenues include examining alkaloid chemistry in low and plains larkspurs, developing immunologic methods for analyzing larkspur alkaloids, developing drug therapy, and devising grazing regimes specifically for low and plains larkspur.

Pyrrolizidine alkaloid plants, metabolism and toxicity.

More than 350 PAs have been identified in over 6,000 plants in the Boraginaceae, Compositae, and Leguminosae families (Table 1). About half of the identified PAs are toxic and several have been shown to be carcinogenic in rodents. PA-containing plants have worldwide distribution, and they probably are the most common poisonous plants affecting livestock, wildlife, and humans. In many locations, PA-containing plants are introduced species that are considered invasive, noxious weeds. Both native and introduced PA-containing plants often infest open ranges and fields, replacing nutritious plants. Many are not palatable and livestock avoid eating them if other forages are available. However, as they invade fields or crops, plant parts or seeds can contaminate prepared feeds and grains which are then readily eaten by many animals. Human poisonings most often are a result of food contamination or when PA-containing plants areused for medicinal purposes. This is a review of current information on the diagnosis, pathogenesis, and molecular mechanisms of PA toxicity. Additional discussion includes current and future research objectives with an emphasis on the development of better diagnostics, pyrrole kinetics, and the effects of low dose PA exposure.