Prolonged absorption and susceptibility to enterohepatic circulation after oral administration of ergot alkaloids in ewes

Abstract

The objective of this study was to evaluate the pharmacokinetics profile of ergot alkaloids when administered to sheep orally. Although ergot alkaloids frequently contaminate animal feed, current understanding of their pharmacokinetics in animals cannot adequately predict toxicity. Blood samples were collected from ewes at 0.5, 1, 3, 5, and 12 h after oral exposure to 4 ergot alkaloids: ergocornine, ergocristine, ergocryptine, and ergosine, followed by serum analysis of these alkaloids using high performance liquid chromatography and tandem mass spectrometry. The alkaloids showed extended absorption time, in addition to clear signs of enterohepatic circulation. This pharmacokinetic profile suggests potential enhanced toxicity in animals with disorders related to secretion of bile acid. It may also explain the high susceptibility of sheep to ergot poisoning compared to other species. An extended sampling protocol (> 12 h) is necessary, however, to identify the pharmacokinetic properties of ergot alkaloids in ewes. In conclusion, ewes exposed to ergot alkaloids showed a prolonged absorption phase and enterohepatic circulation, which is in contrast with human ergot pharmacokinetics.

Introduction

Ergot alkaloids are fungal metabolites found in cereal and other crops. They are produced by a group of fungi of the genus Claviceps, most often by Claviceps purpura (1). The common occurrence in animal feed products at varying concentrations, depending on the feed type, source, and cultivar, has toxicological implications (2,3). In addition, ergot alkaloids vary in chemical structure and potency, although they all share a common ergoline ring (4).

Chronic ingestion of ergot alkaloids in animals is associated with severe vasoconstriction, which causes gangrene, decreased milk production, and abortion (5). Despite those significant clinical manifestations, there is still no definitive association between plasma concentrations of ergot alkaloids and concentrations ingested. As a result, reported allowable limits that are deemed safe in animal feed vary considerably among different countries. For example, grains destined for livestock consumption in the United States should contain < 0.3 ppm total ergot. In Europe and the United Kingdom, allowable levels are < 0.1 and 0.001 ppm, respectively. In Canada, the maximum allowable levels in cattle and swine feed are 2 to 3 ppm and 4 to 6 ppm, respectively (6).

It is essential to know the pharmacokinetic behavior and parameters of ergot alkaloids to better understand the clinical disease caused by ergot poisoning. Urine, feces, and to a lesser extent milk, are the primary routes for elimination of ergot alkaloids. Limited data are available on clearance rates for ergot alkaloids in livestock. The lack of sufficient quantities of reasonably priced pure ergot alkaloids needed to characterize the full pharmacokinetic profile of ergot alkaloids remains an obstacle to researchers (7). In a study examining the recovery of ergot alkaloids from lamb feces, recovery ranged from 7.35 to 26.1% (8).

The objective of this short communication was to evaluate the pharmacokinetics profile of the ergot alkaloids ergocornine, ergocristine, ergocryptine, and ergosine to determine their toxicity when administered to sheep orally.

Materials and methods

Animals

All protocols were approved by the Animal Care and Ethics Committee at the University of Saskatchewan. A total of 6 healthy adult ewes (3 y old) were used. Before the experiment, all animals were weighed and clinically examined, and body temperature and heart rate were recorded. Blood samples were collected from each animal and a complete blood (cell) count (CBC) was done to evaluate red and white blood cell counts, as well as platelet count and total plasma protein, to ensure all animals were healthy.

Experimental protocol

Ergot-containing sclerotia were collected, finely ground, and the concentrations of 4 alkaloids: ergocornine, ergocristine, ergocryptine, and ergosine, were determined using high performance liquid chromatography and tandem mass spectrometry (HPLC/MS) at Prairie Diagnostic Services, Saskatoon, Saskatchewan. Using a stomach tube, each ewe was orally fed ground sclerotia at a dose of 550 μg/kg body weight (BW) (total ergot), dissolved in 50 mL water. Calculated doses for each alkaloid are provided in Table I. Blood samples were collected before exposure and at 0.5, 1, 3, 5, and 12 h after each exposure. The serum was separated and stored at −20°C until analysis by HPLC/MS. Animals were euthanized after the last dose and tissues were collected and stored.

Table I.

Concentration of 4 ergot alkaloids determined within ground sclerotia using HPLC/MS. The total concentration of these alkaloids was used to formulate a single oral dose (550 μg/kg BW) that was administered to each sheep using a stomach tube.

Alkaloid	Concentration (ppb) Dry weight	Oral dose (μg/kg BW)
Ergocornine	216 500	26.4
Ergocristine	3 653 000	445.9
Ergocryptine	540 100	65.9
Ergosine	89 570	10.9
Total	4 499 170	549.1

Detection limit for each alkaloid was 1.25 parts per billion (ppb). HPLC/MS — High performance liquid chromatography and mass spectrometry.

Chemicals and reagents

Ergocornine, ergocristine, ergocryptine, and ergosine with purities of > 95.9% were purchased from Romer Labs (Washington, Missouri, USA). Methanol (HPLC grade), acetonitrile (HPLC grade), and formic acid (98%) were supplied by EMD Millipore (Oakville, Ontario). Water was purified in-house using a Barnstead Nanopure filtration system (> 16.8 MΩ-cm) (Fisher Scientific, Mississauga, Ontario). Ammonium acetate (10 mM, HPLC grade) was purchased from Fisher Scientific. Ammonium hydroxide (29%) was purchased from JT Baker (Radnor, Pennsylvania, USA). Hypersep Retain CX extraction cartridges (60 mg/3 mL) were purchased from Thermo Scientific (Fisher Scientific).

Sample processing and extraction

Sample extraction was carried out as described in a previous study, with modifications (9). Zen serum samples were thawed at room temperature prior to extraction. Serum was spiked with 5% formic acid in water at 0.2 mL/mL serum prior to extraction. Hypersep Retain CX extraction cartridges (Fisher Scientific) were pre-conditioned according to package insert, with 2 mL of methanol, followed by 2 mL of Barnstead water.

A total of 3 mL of serum was loaded onto cartridges and allowed to elute via gravity. Cartridges were rinsed with 2 mL of 5% formic acid in water, followed by 2 mL of methanol. Ergot alkaloids were eluted into clean test tubes with 1 mL of 97.5% methanol/2.5% ammonium hydroxide, 5 times. The eluent was evaporated under filtered air at room temperature. The residue was reconstituted in 85% acetonitrile/15% aqueous 10 mM ammonium acetate and transferred to an autosampler vial with insert for analysis.

Instrumental conditions

Serum quantification of ergot alkaloids was carried out as previously described by Krska et al (10). The HPLC/MS analysis of ergot alkaloid standards was carried out on a Sciex 4000 QTRAP in electrospray positive mode, attached to an Agilent 1200 high performance liquid chromatography (HPLC) system, equipped with an Agilent Zorbax Eclipse XDB-C18 narrow bore 2.1 × 150 mm, 5 μm HPLC column.

The mobile phase A was aqueous 10 mM ammonium acetate, and the mobile phase B was acetonitrile. The HPLC separation required a 21-min concentration gradient from 5% B to 80% B over 13 min and then returning to original settings for the remainder of run. The flow rate was 0.3 mL/min. The injection volume was 10 μL and the column temperature was maintained at 30°C. The auto-sampler system temperature was 4°C. Nitrogen gas was used as the nebulizer and collision gas. Chromatograms from multiple reaction monitoring (MRM) of singly protonated molecular ions [(M+H+)+] were used for quantitation of the ergot alkaloids. Detection limits were 0.1 ng/mL. The calibration range was 0.1, 0.25, 0.75, 1.25, 2.5, and 12.5 ng/mL.

Results

After receiving the oral dose of ergot alkaloids, all animals remained normal for the duration of the collection period. Results of blood sample analysis showed gradually increasing blood concentrations of ergot alkaloids during the 12-hour collection period. The trend was most pronounced with ergocristine, as shown in Figure 1.

Figure 1.

Blood concentrations of 4 ergot alkaloids (ergocornine, ergocristine, ergocryptine, and ergosine) versus time in hours. Ergot-exposed sheep received a single oral dose of 550 μg/kg BW total ergot dissolved in water, based on the levels of 4 ergot alkaloids determined previously. Data presented is mean ± SD and N = 6.

To further understand the behavior of each ergot alkaloid, logt-ransformed concentrations were recorded for each alkaloid, as shown in Figure 2. This figure shows that ergocristine had the highest concentration at the 12-hour time point (2.4 ng/mL), followed by ergosine (0.65 ng/mL), ergocryptine (0.52 ng/mL), and ergocornine (0.33 ng/mL).

Figure 2.

Semi-log plots of concentrations of individual ergot alkaloids (ergocornine, ergocristine, ergocryptine, and ergosine) in blood versus time in hours. Ergot-exposed sheep received a single oral dose of 550 μg/kg BW total ergot dissolved in water, based on the levels of 4 ergot alkaloids determined previously. Data presented is mean ± SD and N = 6.

No elimination phase was observed within the time points selected, but a plateau of concentrations was achieved for ergocornine, ergocryptine, and ergosine. As seen in Figure 2, all alkaloids displayed a transient increase in concentrations at the half-hour time point. However, the slopes of change for 4 points, including the 1-, 3-, 5-, and 7-hour time points, were equal to 0.041, 0.143, 0.059, and 0.072 for ergocornine, ergocristine, ergocryptine, and ergosine, respectively.

Discussion

Ergot toxicosis or ergotism has been associated with devastating clinical effects to humans and livestock, which results in significant economic losses (4,5). Due to wet environmental conditions, field and storage mycotoxins have become more prevalent in western Canada over the past few years (11). For example, in 2013, almost 50%, 30%, and 25% of the wheat from Alberta, Saskatchewan, and Manitoba, respectively, was downgraded due to ergot (11).

Regulators worldwide often struggle to define safe limits of ergot alkaloids in feed. Canada has defined the maximum allowable feed levels of ergot alkaloids in cattle and swine as 2 to 3 and 4 to 6 ppm, respectively, whereas other regulators chose higher or lower levels (6). Although levels are often based on experimental trials that report adverse effects above the chosen safe levels, these “safe levels” often rely on measuring the total concentration of ergot alkaloids within feed regardless of the different mix that could be present.

Despite the similar chemical structure of these alkaloids, it is well-known that each has its unique biological activity and probable toxicity, which is determined by its unique pharmacokinetic and dynamic characteristics. Determining accurate safe levels of ergot alkaloids within feed, therefore, requires further studies to determine several important pharmacokinetic and dynamic parameters describing the behavior of each alkaloid. Such studies in livestock are extremely limited due to insufficient quantities of reasonably priced pure ergot alkaloids, as well as technical limitations in measuring them accurately within the serum and body fluids.

The rate and extent of absorption of each ergot alkaloid across the gastrointestinal tract is related to its unique physicochemical characteristics, which in turn determine its solubility, ionization, and partitioning between water and lipid phases. Most ergot alkaloids are weak bases and are therefore charged at low pH, making them more likely to be absorbed in the rumen and the small intestine than in the abomasum (12,13,14).

This study attempted to evaluate the pharmacokinetic profile of 4 ergot alkaloids (ergocryptine, ergocornine, ergocristine, and ergosine) in sheep after oral administration. Characterizing the full pharmacokinetic profile of the examined ergot alkaloids would require the determination of alkaloid concentration in urine and fecal samples, which is beyond the scope of this study. Conventionally, in pharmacokinetic studies, pure drugs are followed up to 5 half-lives to elucidate the full-elimination pharmacokinetic profile. An alkaloid mixture derived from ergot sclerotia was used in the current study instead of pure alkaloids primarily because of cost.

The design of sample collection in our experiment was based on the assumption that ergot alkaloids are weak bases and are therefore readily absorbed in the rumen (12–14). In addition, human pharmacokinetic trials of ergotamine reported a mean terminal half-life of 2 to 3 h and an initial rapid distribution phase, with a mean absorption half-life of 10.5 min (15). We, therefore, assumed that a 12-hour sampling regime would allow ergot alkaloids to reach terminal elimination and provide a full pharmacokinetic profile. Unexpectedly, however, we noticed a prolonged absorption phase that prevented all the examined alkaloids from reaching the elimination phase.

The prolonged absorption was likely substantiated by the enterohepatic circulation observed at 0.5 h in all ergot alkaloids examined. Enterohepatic circulation is known to have significant clinical implications. For example, pathologies that reduce bile flow, such as chronic hepatic or biliary disorders as well as gastrointestinal disorders, could significantly influence pharmacokinetics parameters (16). As such, these conditions could significantly increase area under the curve for the examined ergot derivatives, increasing the overall exposure and enhancing toxicity (17). Impairment of enterohepatic circulation can also increase or decrease half-life. Additional studies should be conducted to help determine the exact effect.

In conclusion, unlike humans, ewes have an extended absorption phase that is longer than 12 h and ergot alkaloids are susceptible to enterohepatic circulation. This warrants further investigation to optimize collection of samples over a longer period. A comprehensive elucidation of the pharmacokinetic profile is still critical in order to further understand the behavior of ergot alkaloids.