Article pubs.acs.org/JAFC
Accumulation, Persistence, and Effects of Indospicine Residues in Camels Fed Indigofera Plant Eddie T. T. Tan,†,§ Rafat Al Jassim,† A. Judy Cawdell-Smith,‡ Selina M. Ossedryver,⊥ Bruce R. D’Arcy,†,‡ and Mary T. Fletcher*,† †
Queensland Alliance for Agriculture and Food Innovation (QAAFI), The University of Queensland, Health and Food Sciences Precinct, Coopers Plains, QLD, Australia ‡ School of Agriculture and Food Sciences, Faculty of Science, The University of Queensland, Brisbane, QLD, Australia § Food Technology Programme, Faculty of Applied Sciences, Universiti Teknologi MARA, Shah Alam, Selangor, Malaysia ⊥ Department of Agriculture and Fisheries, Health and Food Sciences Precinct, Coopers Plains, QLD, Australia ABSTRACT: Indospicine (L-2-amino-6-amidinohexanoic acid) is a natural hepatotoxin found in all parts of some Indigofera plants such as Indigofera linnaei and Indigofera spicata. Several studies have documented a susceptibility to this hepatotoxin in different species of animals, including cattle, sheep, dogs, and rats, which are associated with mild to severe liver disease after prolonged ingestion. However, there is little published data on the effects of this hepatotoxin in camels, even though Indigofera plants are known to be palatable to camels in central Australia. The secondary poisoning of dogs after prolonged dietary exposure to residual indospicine in camel muscle has raised additional food safety concerns. In this study, a feeding experiment was conducted to investigate the in vivo accumulation, excretion, distribution, and histopathological effects of dietary indospicine on camels. Six young camels (2−4 years old), weighing 270−390 kg, were fed daily a roughage diet consisting of Rhodes grass hay and lucerne chaff, supplemented with Indigofera and steam-flaked barley. Indigofera (I. spicata) was offered at 597 mg DM/kg body weight (bw)/day, designed to deliver 337 μg indospicine/kg bw/day, and fed for a period of 32 days. Blood and muscle biopsies were collected over the period of the study. Concentrations of indospicine in the plasma and muscle biopsy samples were quantitated by validated ultraperformance liquid chromatography−tandem mass spectrometry (UPLC-MS/MS). The highest concentrations in plasma (1.01 mg/L) and muscle (2.63 mg/kg fresh weight (fw)) were found at necropsy (day 33). Other tissues were also collected at necropsy, and analysis showed ubiquitous distribution of indospicine, with the highest indospicine accumulation detected in the pancreas (4.86 ± 0.56 mg/kg fw) and liver (3.60 ± 1.34 mg/kg fw), followed by the muscle, heart, and kidney. Histopathological examination of liver tissue showed multiple small foci of predominantly mononuclear inflammatory cells. After cessation of Indigofera intake, indospicine present in plasma in the remaining three camels had a longer terminal elimination half-life (18.6 days) than muscle (15.9 days), and both demonstrated monoexponential decreases. KEYWORDS: indospicine, Indigofera, camel, residue, hepatotoxic, ultraperformance liquid chromatography−tandem mass spectrometry
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INTRODUCTION Leguminous Indigofera plants are not only widely distributed across rangeland regions of Australia1 but also widespread in subtropical and tropical areas throughout the world.2 Because the plants are rich in protein, highly palatable, and digestible, they are considered nutritious animal feed.3−5 However, the presence of naturally occurring indospicine (1, Figure 1) in some Indigofera species has limited their agricultural usage, whereas indospicine (1) levels in many other Indigofera spp. are still poorly defined.4,6−8 An analogue of arginine (2), indospicine (1) is a toxic amino acid and was first isolated from Indigofera spicata.9 Indospicine (1) is known to cause liver lesions in simple-stomached animals6,10−13 and in compartmental-stomached animals such as goats,14 sheep,15 and cattle16 after extended intake, with different degrees of hepatotoxic responses. Despite the hepatotoxicity of indospicine (1), the capability of ruminants to metabolize indospicine (1) remains ambiguous, with no clear clinical evidence of liver disease being perceived after the © XXXX American Chemical Society
consumption of indospicine-containing plants in some areas.17−19 It has been suggested that Australian feral camels as wild herbivores have foregut microorganisms with a better capacity to degrade plant toxins that intoxicate domesticated herbivore livestock.20 Even though Indigofera plants are a preferred food plant of camels,21 no evidence of indospicine (1) toxicity is reported in these animals, which perhaps suggests that camel foregut microorganisms can metabolize indospicine (1). However, questions have been raised again following the detection of indospicine (1)11,22 in camel meat associated with dog deaths after prolonged ingestion of a camel meat diet.11 This finding was unexpected and suggests that the camel gastrointestinal tract may not be capable of detoxifying Received: June 15, 2016 Revised: July 25, 2016 Accepted: July 30, 2016
A
DOI: 10.1021/acs.jafc.6b02707 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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clearance from the muscle after the cessation of I. spicata intake. The camels were from the feral camel population in central Australia and were captured and sold by a commercial supplier. Animal protocols for this study were approved by the Animal Ethics Committee of University of Queensland, Queensland, Australia (AEC Approval No. SAFS/047/14/SLAI). All procedures performed in these studies involving camels were in accordance with the ethical standards of The University of Queensland. The camels were allowed to acclimatize to the yards and handling for 3 months before the commencement of the experiment, especially handling through the modified cattle crush facilities, to minimize any stress associated with blood and biopsy sample collections. The camels were fed 597 mg I. spicata DM/kg bw/ day to deliver 337 μg indospicine/kg bw/day for a period of 32 days. The I. spicata was fed with chaff to avoid refusals of feed by camels and was fed as two equal meals at 9:00 a.m. and 2:00 p.m. daily. I. spicata was introduced to the diet in increasing increments over the initial 3 days (33, 66, and 100% of daily dose, respectively) to reach the daily dose of 597 mg I. spicata DM/kg bw/day. The camels were allowed ad libitum intake of a roughage-based diet consisting of Rhodes grass hay (Chloris gayana) and Lucerne hay. Steam-flaked barley was introduced on day 20 and maintained until slaughter to maintain the body weight of the camels. In the morning of day 33, three camels (camels 1−3) were humanely euthanized using a captive bolt pistol and exsanguinated prior to necropsy. The remaining three camels (camels 4−6) were transferred to a Rhodes grass diet without incorporation of I. spicata until day 132 to determine the rate of excretion of indospicine (1). Blood, Biopsy, and Necropsy Sample Collections. Biopsy and blood sample collections were started 10 days prior to the commencement of the treatment phase to ensure that the camels had no prior exposure to indospicine (1). Muscle biopsies were obtained from the gluteal muscles. The hair over the biopsy site was trimmed and the surface of the skin scrubbed with alcohol and chlorhexidine. Local anesthetic (20 mg/L lignocaine hydrochloride, Ilium Lignocaine 20, Troy Laboratories Pty Ltd., Glendenning, Australia) was injected intradermally over the site of the biopsy. A 2−3 cm incision was made through the skin subcutaneous tissue and muscle sheath. A small piece of muscle (0.2−0.8 g) was removed using biopsy Acu.Punch (Acuderm Inc., Fort Lauderdale, FL, USA) and forceps, after which the incision was closed with a single suture. The wound was then sprayed with an antiseptic tincture, Chloromide (Troy Laboratories Pty Ltd.). The tissue sample was blotted dry and placed in a CryoTube vial (Sigma-Aldrich Pty Ltd., Castle Hill, Australia) and kept in an icebox for transportation to the Health and Food Sciences Precinct, Brisbane, where it was stored at −30 °C before indospicine (1) analysis. Biopsy samples were taken from all camels at weekly intervals until day 33. The biopsies were continued with the remaining three camels at 14-day intervals up to day 76, then on days 105 and 132. Biopsy sampling was alternated between the left and right rump of the camels throughout the experiment. Venous blood samples were collected from the jugular vein prior to the morning dosing, at weekly intervals until day 33 (camels 1−3) or day 76 for the remaining three camels. Further samples were collected on days 91, 105, and 132. Blood for hematological analysis was collected into EDTA anticoagulant (1 × 9 mL Vacuette) and lithium heparin for clinical biochemistry (2 × 6 mL BD Vacutainer) and indospicine (1) hepatotoxin assay (2 × 10 mL BD Vacutainer). The blood samples for indospicine (1) assay were stored on ice and transported to Health and Food Sciences Precinct, Brisbane. They were centrifuged at 4400 rpm for 10 min at 19 °C (Sigma 4K10), with plasma harvested and stored at −30 °C until analysis. The blood samples for hematology and clinical biochemistry analysis were transported to the Diagnostic Services-Clinical Pathology Laboratory at the School of Veterinary Science, The University of Queensland, Gatton Campus. Samples of liver, pancreas, heart, kidney, and rump muscle were collected at necropsy and stored at −30 °C until analyzed for indospicine. Separate tissue samples were collected into 10% neutral buffered formalin for histopathology. Indospicine Extraction: Tissue Samples. The indospicine (1) tissue analysis was undertaken according to the extraction procedure
Figure 1. Chemical structures of indospicine (1), an analogue of arginine (2), and D3-L-indospicine (3) utilized as an internal standard for UPLC-MS/MS analysis.
indospicine (1) and that, if detoxification occurs, it is only partial, where a proportion of the ingested indospicine (1) finds its way into the animal tissues and accumulates there. Little is known about the toxicokinetics of indospicine (1), and its effect on camels is not clear. In central Australia, an estimated 300,000 feral camels roam regions in which Indigofera are known to be prevalent.23 Because there are more than 3000 camels used for human consumption and a further 2000 for pet food each year in Australia,23 concern has been raised about indospicine-contaminated feral camel meat entering both the human and pet food chains. The findings of the study described here are the first to provide insights into the bioaccumulation, distribution, and excretion of indospicine (1) and its histopathological effects on young camels fed a roughagebased diet containing I. spicata with a known indospicine (1) concentration. The quantitation of indospicine (1) was performed by adaptation of previously developed and validated ultraperformance liquid chromatography−tandem mass spectrometry (UPLC-MS/MS) coupled with stable isotope dilution assay (SIDA).22
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MATERIALS AND METHODS
Standards and Reagents. Synthesized indospicine (1) (standards, >99% pure) and D3-L-indospicine (3) (internal standard, >99% pure) used in UPLC-MS/MS analysis were provided by Prof. James De Voss and Dr. Robert Lang, The University of Queensland. Internal (1 mg/L) and external standard (0.005−2 mg/L) solutions for indospicine (1) were prepared in H2O with 0.1% heptafluorobutyric acid (HFBA). Acetonitrile was of high-performance liquid chromatography grade, and HFBA (99.5%) was of ion chromatography grade. All other reagents and chemicals used were of analytical grade. Plant Materials. Approximately 148 kg of fresh I. spicata was collected over a period of 4 months (January 23−May 19, 2014) from Yeerongpilly (27°31′31.3″ S; 153°00′43.7″ E), Queensland. The plant material was transported to the Health and Food Sciences Precinct, Brisbane, and air-dried to 49 kg, with an average moisture loss of 67%. This dried plant material was chaffed and stored frozen (−20 °C) until commencement of the feeding experiment. Dry matter (DM) was determined by heating to constant weight at 105 °C under an atmosphere of nitrogen using an automated LECO Thermogravimetric TGA 701 Analyzer (LECO Corp., St. Joseph, MI, USA). A composite sample was then taken, extracted, and analyzed for indospicine (1) concentration (565.1 ± 79.7 mg/kg DM (mean ± SD)).24 For plant identification and preservation purposes, a separate sample was pressed between absorbent papers and deposited at the Queensland Herbarium as voucher no. AQ751378. Animals and Treatments. Six young camels aged 2−4 years with an average initial weight of 316 ± 45.6 kg (270−387 kg) were used in this study to investigate the deposition of indospicine (1) and its B
DOI: 10.1021/acs.jafc.6b02707 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Table 1. Repeatability, Reproducibility, and Recovery of the UPLC-MS/MS Method for Indospicine Quantitation by Spiking Indospicine into Camel Plasma Samples (LOQ = 0.1 mg/L, LOD = 0.05 mg/L) quantitated concentration (mg/L) n
spike concentration (mg/L)
camel 1 camel 2 camel 3 camel 4 camel 5 camel 6
mean ± SD (mg/L)
RSD (%)
recovery (%)
intraday precision (repeatability)
6
2.5 5 50
2.06 5.51 46.28
2.95 4.45 44.92
2.38 4.18 47.31
2.77 5.33 60.87
2.36 4.67 54.07
2.66 5.16 50.42
2.53 ± 0.32 4.88 ± 0.53 50.64 ± 5.99
12.79 10.83 11.82
101.19 97.67 101.28
interday precision (reproducibility)
6
2.5 5 50
1.91 4.64 40.84
2.58 4.98 51.68
2.74 5.10 47.53
2.36 4.90 40.94
2.70 6.50 49.03
2.63 4.79 51.50
2.48 ± 0.31 5.15 ± 0.68 46.92 ± 4.92
12.56 13.18 10.49
99.47 103.03 93.84
that was developed in our laboratory and reported previously.22 Centrifugal filter units (Merck Millipore, Kilsyth, Australia) were prerinsed with reverse osmosis water (300 μL) and centrifuged twice (10000 rpm, 20 min), then inverted and centrifuged for 1 min at 1000 rpm. Camel biopsy and necropsy samples were thawed, finely chopped, weighed (0.2−0.8 g), and mixed with 0.1% HFBA in a ratio of 0.1 g/5 mL. The samples were then homogenized for 15 s using a Polytron T25 Basic homogenizer (Labtek, Brendale, Australia) for 15 s. The homogenates were chilled (4 °C) for 20 min and then centrifuged at 4500 rpm for 20 min at 18 °C. Supernatant (1.0 mL) was then transferred to a vial and spiked with the internal standard D3L-indospicine (3) and vortexed for 10 s. Spiked supernatant (450 μL) was transferred into a prerinsed centrifugal filter, which was centrifuged at 10000 rpm for 20 min, after which the filtrate was transferred to an insert for UPLC-MS/MS analysis. Indospicine Extraction: Plasma Samples. The indospicine (1) plasma extraction procedure was adapted from our previous study.22 Plasma samples were removed from storage and allowed to thaw at room temperature. Plasma (200 μL) was diluted (25×) with 0.1% HFBA in a 15 mL Falcon tube and vortexed for 10 s. An aliquot of the diluted plasma (1.0 mL) was spiked with 100 μL of the internal standard D3-L-indospicine (3) and vortexed for 10 s. A portion (450 μL) was transferred into prerinsed Amicon Ultra, 0.5 mL, 3K centrifugal filters and centrifuged (10000 rpm, 20 min). The filtrate was transferred to a limited volume insert for UPLC-MS/MS analysis. A validation of the indospicine (1) extraction from plasma was carried out by spiking three different indospicine (1) concentrations (2.5, 5.0, and 50.0 mg/L) in the blank camel plasma samples collected from camel 5 before commencement of treatment phase of the animal experiment. UPLC-MS/MS Analysis. Indospicine (1) quantitation was done according to the validated UPLC-MS/MS method.22 Indospicine (1) separation and detection were carried out using a Waters ACQUITY UPLC system liquid chromatograph (Waters, Rydalmere, Australia) and a Waters Micromass Quattro Premier triple-quadrupole mass spectrometer with an electrospray ionization (ESI) source, operated in the positive mode. Chromatographic separation was carried out at 30 °C on a 100 mm × 2.1 mm i.d., 1.7 μm, BEH C18 column (Waters) with 0.1% HFBA (v/v) in H2O (pH 2.15) (mobile phase A) and 0.1% HFBA in acetonitrile (mobile phase B). The flow rate was set at 0.2 mL/min with the following gradient: 0 min, 99% A; 4 min, 70% A; 7 min, 70% A; 8 min, 99% A; 10 min, 99% A. Indospicine (1) was quantitated utilizing selected reaction monitoring (SRM) with transition of m/z 174.2 → 111.0 (verified by transition of m/z 174.2 → 157.1) and D3-L-indospicine (3) with transition of m/z 177.1 → 114.0 (verified by transition of m/z 177.1 → 113.0). The capillary voltage was 2.79 kV; cone gas flow was 50 L/h; and desolvation gas flow was 600 L/h. The source and desolvation temperatures were set at 150 and 350 °C, respectively. The argon gas collision energies for indospicine (1) (15 and 12 eV) and D3-L-indospicine (3) (15 and 15 eV) were set with a cone voltage at 25 V. Hematological and Biochemical Analyses. Collected blood samples were mixed well, and hematological values of red blood cells (RBC), hemoglobin (Hgb), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean
corpuscular hemoglobin concentration (MCHC), red cell distribution width (RDW), white blood cells (WBC), neutrophils (NEU), lymphocytes (LYMPH), monocytes (MONO), eosinophils (EOSIN), and basophils (BASO) were measured by a Cell-Dyn 3700 Automated Haematology Analyzer (Abbott Diagnostic, North Ryde, Australia). Serum was separated from the clotted blood, and the biochemical parameters of the serum including albumin (ALB), alkaline phosphatase (ALP), aspartate aminotransferase (AST), total bilirubin (T BIL), calcium (Ca), cholesterol (CHOL), creatine kinase (CK), creatinine (CREAT), γ-glutamyl transferase (GGT), glucose (GLUC), magnesium (Mg), phosphate (PO4), total protein (TP), triglycerides (TRIG), urea (UREA), sodium (Na), potassium (K), chlorine (Cl), glutamate dehydrogenase (GLDH), bicarbonate (BICARB), and globulin (GLOB) were measured using an Olympus AU400 Clinical Chemistry Analyzer (Integrated Sciences, Chatswood, Australia). Packed cell volume (PCV) was measured after centrifugation, and fibrinogen was measured using a heat precipitation method.25 EDTA samples were also smeared and stained by using a Hematek Automated Slide Stainer (Bayer, Pymble, Australia) and manually differentiated through smear examination. Histopathological Examination. Specimens of heart, pancreas, liver spleen, kidney, and skeletal muscle from each of three camels were collected at necropsy, fixed in 10% neutral buffered formalin, and processed by routine methods. Hematoxylin and eosin stained sections were examined using light microscopy.
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RESULTS AND DISCUSSION Plasma Recovery. Repeatability and reproducibility of the modified method are reported in Table 1. Interday and intraday precisions were all >85%, with a recovery range between 97.67 and 103.03%. Diet and Body Weight Changes. Camel body weight was measured weekly before morning feeding and biopsy sampling from day 10 to day 27 in the necropsied camels and to day 132 in the remaining three animals. The weight was recorded to the nearest 0.5 kg using a load bar scale. The mean ± SD body weight (n = 6) was 325.8 ± 47.3 kg at the starting point of the study, and the average body weight measured a week before necropsy (day 33) was 300.4 ± 43.3 kg, with an average weight loss of 8%. All camels lost weight in the first 20 days (Figure 2) while having ad libitum intake of a basal diet (chaffed Rhodes grass (C. gayana)) supplemented with I. spicata, except camel 1. The situation was rectified by supplementing the low-quality Rhodes grass basal diet with Lucerne hay and steam-flaked barley at day 20 (to satisfy animal ethics conditions). However, the camels continued to lose weight until I. spicata was removed from the diet after day 32. This observation appears to be consistent with previous research, which found I. spicata could depress live weight gain.13 Despite the weight loss, the camels were grossly normal with no clear evidence of clinical illness as C
DOI: 10.1021/acs.jafc.6b02707 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Figure 3. Indospicine concentrations of plasma during the first 32 days of feeding period (n = 6) and following the cessation of I. spicata feeding (n = 3) experiment. Data points represent individual values of all young camels.
Figure 2. Body weight changes in camels after continuous ingestion (days 1−32) and cessation (days 33−132) of I. spicata. Euthanasia and exsanguination of three camels (camels 1−3) were done on day 33.
average steady-state indospicine (1) plasma concentration of 0.96 ± 0.19 mg/L, where indospicine (1) absorbed within this period may be approximate to the amount of indospicine (1) eliminated. However, the overall indospicine (1) deposition in plasma within the steady-state period varied widely between individual young camels (0.63−1.36 mg/L). In addition, Figure 3 shows that the average indospicine (1) level remained relatively steady for over a week after cessation of I. spicata intake, which was possibly due to the longer mean retention time of plant materials in the foregut and intestine of camels (up to 4 days32), allowing continued absorption and accumulation of indospicine (1) in plasma during this period. This delayed decline of indospicine in plasma may also be due to redistribution of the indospicine from other compartments, such as the pancreas, liver, and muscle, back into the plasma (multicompartment model). After cessation of I. spicata feeding, indospicine (1) elimination from plasma with a monoexponential phase was observed. A fitted indospicine elimination curve was plotted from day 33 to day 132 using a statistical software (Genstat 6th ed.). The fitted curve with percentage variance of 95.1% and standard error of observations of 0.07 mg/L revealed that the indospicine (1) present in plasma has a terminal elimination half-life (t1/2) of 18.6 days (days 33−51.6) with an elimination rate constant of 0.037 day−1. A note of caution is due here, because nonlinear regression analysis shows the average indospicine (1) concentration data point of day 41 has a large standardized residual and has high statistical leverage on the maximal indospicine (1) concentration data point of day 33. Indospicine (1) in plasma was below the quantifiable level (3 months. Twenty-four hour toxicokinetics investigations to examine the disposition of indospicine (1) in feral young camels were not carried out to minimize the stress of frequent repeated bleeds on such large nondomesticated animals. Indospicine (1) progressively accumulated in muscle and attained higher concentrations than in plasma at the end of the I. spicata exposure. The concentrations of indospicine (1)
had been reported in other compartmental-stomached animals.15,26,27 Hematological and Biochemical Changes. Some isolated elevations of AST and GLDH levels were observed (camels 1, 4, and 6 had elevated AST and GLDH on days −10, 6, and 20, respectively), with no apparent correlation with the repeated daily indospicine (1) dose of 337 μg/kg bw for 32 days. Dogs, by comparison, are more susceptible to indospicine (1), and dietary exposure of a similar dose (mean daily dose rate of 450 μg indospicine/kg bw) for four consecutive days has been reported to raise the plasma ALT concentration with restricted liver lesions.28 The observed intermittent higher concentrations of AST and GLDH reflect hepatic injury; however, the random spikes were not persistent and therefore were most probably not due to indospicine (1). The cause of injury was not identified. All other clinical biochemistry and hematological values remained within the normal reference ranges29−31 throughout the feeding experiment with no clinical signs of illness. Distribution of Indospicine in the Plasma and Biopsy Tissues. A minimal number of young camels was used in this experiment in compliance with the reduction principle of 3Rs stipulated by the AEC of the University of Queensland. No indospicine (1) was detected in plasma or biopsied muscle of any of the experimental camels prior to the commencement of the feeding experiment. Continuous ingestion of indospicine (1) for a period of 32 days with an oral daily intake of I. spicata designed to supply 337 μg indospicine/kg bw, divided into two meals, morning and afternoon, resulted in a rapid rise of indospicine (1) in plasma (Figure 3) over the first 13 days of feeding, followed by a slower rate before an apparent steady state was reached. The concentrations of indospicine (1) during the rapid increase stage (days 1−20) and slower increase period (days 21−32) varied greatly among individual camels, but showed less variation following the cessation of Indigofera feeding (days 32−132). The maximum average indospicine (1) plasma concentration of the six camels was 1.01 ± 0.15 mg/L (0.83−1.16 mg/L, n = 6) at 33 days. The average indospicine (1) in plasma plot (Figure 3) suggested that the indospicine (1) may have risen to a plateau during days 20−41, with an D
DOI: 10.1021/acs.jafc.6b02707 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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than the limit of quantitation (0.1 mg/kg). Because Indigofera plants are known to have a seasonal abundance dependent on rainfall, camels in the central desert of Australia may have only periodic intake of Indigofera plants. This seasonality may further explain why 50% of the culled camel rump tissue samples collected in situ from Simpson Desert camels had low levels (0.1−3.7 mg/kg) of indospicine.22 However, the possibility of high indospicine (1) levels being present in camel muscle is greatly increased when the camels are harvested within the period when indospicine-producing Indigofera plants are readily accessible after the first summer rain. Figure 5 shows that plasma levels of indospicine (1) were positively correlated with the levels present in muscle with
increased for all collected biopsied tissues and maintained the same pattern of increase, reaching a maximum of 2.63 ± 0.28 mg/kg fw before the feeding of I. spicata was stopped. Differences in concentrations between animals were observed, but to a lower extent than those observed for plasma (Figures 3 and 4). Considering the camels in this study were allowed ad
Figure 4. Indospicine concentrations of muscle during the first 32 days of the feeding period (n = 6) and following the cessation of I. spicata feeding (n = 3) experiment. Data points represent individual values of all young camels. Figure 5. Comparison and correlation plot of indospicine concentrations (mean ± SD) between muscle and plasma samples over the period of the feeding experiment.
libitum access to drinking water and a previous study observed 20% higher values of plasma volume in hydrated camels than in dehydrated camels,33 a possible explanation for the greater fluctuation of indospicine observed in plasma may be because of the changes in plasma volumes after water intake. However, more research on this topic needs to be undertaken before the association between plasma volume and indospicine concentration is fully understood. These results permit extrapolation and provide further support for the hypothesis that higher indospicine (1) concentrations may be present in muscle when camels have dietary exposure to indospicine-containing plants for a prolonged period of time. Dietary exposure of plants with higher indospicine (1) levels and/or higher feeding quantities may also result in a much higher indospicine (1) level in muscle than seen in this study.34 The indospicine (1) in camel muscle was eliminated following first-order reactions upon cessation of the I. spicata diet. This suggests that camels have a potential indospicine (1) removal mechanism as has been proposed in horses.28 However, the long half-life of indospicine (1) resulted in it remaining in the muscle biopsy specimens with an average concentration of 0.23 ± 0.01 mg/kg fw (n = 3) up until the time of termination in this study (day 132). Figure 4 shows indospicine (1) persisted in muscle up to 3 months after cessation of I. spicata, with an elimination rate constant of 0.043 day−1 and a slightly shorter terminal elimination half-life (t1/2 = 15.9 days) than plasma established through a fitted indospicine (1) elimination curve with a percentage variance of 98% and standard error of observations of 0.125 mg/kg. The results of this study show that camels with muscle indospicine levels of 2.5 mg/kg, may need a month to clear the indospicine (1) levels to 33 days) may be needed for indospicine (1) to reach steady state in muscle compared to plasma (20 days). Feeding camels larger and/or more frequent quantities of I. spicata will result in steady states with higher indospicine levels in both plasma and muscle, but should not change the time to reach steady states.34 The higher indospicine concentration measured in muscle as compared to plasma is indicative of slower indospicine turnover from muscle, even though it has a shorter half-life than plasma. The lengthy time for elimination of indospicine (1) from camel muscle (Figure 5) increases the potential consumer exposure to E
DOI: 10.1021/acs.jafc.6b02707 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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concentration in the pancreas is less clear, but, intriguingly, this finding is consistent with high indospicine in the pancreas reported for the indospicine-intoxicated dogs.28,36 It is interesting to note that the highest level of arginine (2) is also observed in the rat pancreas at 30 min after intraperitoneal administration of arginine (2).37 We hypothesize that indospicine (1), which is an arginine (2) analogue and acts as an nitric oxide inhibitor,38,39 may inhibit protein synthesis, thereby inhibiting insulin secretion from the pancreas.40 Histopathological Findings. The histopathology of organs collected at necropsy is detailed in Table 3. Although extensive research on the effects of indospicine (1) on livers of different species of animals has been reported,26−28,41,42 there are no previously reported findings for camels. Histopathological examinations of the livers of the three camels necropsied at the end of 32-day feeding period showed mild histopathological changes despite blood biochemistry profiles throughout the feeding experiment being within the reference range. Multiple small, inflammatory foci, often accompanied by singlecell hepatocellular necrosis, were randomly distributed throughout the hepatic parenchyma. An incidental eosinophilic (parasitic) granuloma was also present in the liver of one animal. The liver lesions found in this feeding experiment should be interpreted with caution, due to the small number of animals used and the possibility that they had access to a variety of other plants, which may have caused the lesions. In addition, comparison should not be made between the findings reported for laboratory animals,6,27,43,44 which had wide differences in indospicine (1) levels and duration of indospicine (1) intake, and those found in this study, where the camels were fed a set concentration of indospicine (1) based on body weight. Moreover, differences in the susceptibility of foregut fermenters to such plant toxin may differ greatly from those of nonruminants, due to the role of microorganisms and the fermentation processes in the foregut. However, the results of this examination suggest that the duration of exposure and dose of indospicine (1) used in this subacute feeding experiment were not sufficient to reproduce the syndrome of complete liver failure observed in dogs.28 Changes in other tissues included a mild, multifocal, nonsuppurative (predominantly lymphocytic) interstitial myocardial infiltrate and, in one animal, mild, focal myositis. No lesions were observed in sections of pancreas, kidney, or spleen. Moderate numbers of tapeworms were found in the intestines of camels during post-mortem examination; however, no eggs of Monezia spp. were recovered from the fecal samples
indospicine (1) residues in camel meat. The short-term bioaccumulative nature of indospicine (1) in camel makes prediction through the modeling of blood and tissue sample analysis unreliable for determining the stages and effects of indospicine (1) exposure in camels. However, the correlation between plasma and muscle indospicine (1) concentrations determined in this study suggests that plasma analysis can potentially be used to estimate the concentration in muscle. This is limited by the apparent steady state that is attained in plasma, whereas indospicine (1) in tissue continues to accumulate. Distribution of Indospicine in Necropsy Samples. There is little information available concerning the accumulation of indospicine (1) in the internal organs of herbivores, including camels. Results from the investigation of indospicine (1) concentrations in the organs of three camels, at the termination of feeding I. spicata, indicate that the pancreas had the highest concentration of indospicine (1) (4.86 ± 0.56 mg/ kg fw) (Table 2). The liver was the second highest (3.60 ± 1.34 Table 2. Indospicine Concentrations of Necropsied Camel Tissues from the 32-Day Camel Feeding Experiment Incorporated with I. spicata indospicine concentrationa (mg/kg fw)
a
tissue
camel 1
camel 2
camel 3
pancreas liver muscleb heart kidney
5.32 5.05 2.16 2.13 1.31
5.02 3.33 2.28 2.20 1.55
4.24 2.42 1.88 1.97 1.07
mean ± SD 4.86 3.60 2.11 2.10 1.31
± ± ± ± ±
0.56 1.34 0.21 0.12 0.24
LOQ = 0.1 mg/kg fw, LOD = 0.05 mg/kg fw. bGluteal muscle.
mg/kg fw) followed by the heart muscle (2.10 ± 0.12 mg/kg fw), skeletal muscle (2.11 ± 0.21 mg/kg fw), and kidney (1.31 ± 0.24 mg/kg fw). It is not surprising that indospicine (1) concentrations are high in camel livers because this organ has a vital role to detoxify, filter, and expel toxins of all nature. However, it is interesting to find that indospicine (1) concentrations were even higher in the pancreas, with levels in this tissue being more than double the level present in muscle or heart tissues (Table 2). Higher indospicine (1) levels in the liver could be attributed to indospicine (1) passing through (and being filtered by) the liver before it reaches the general circulatory system for distribution to other sites.35 The reason for the high
Table 3. Histopathological Observations in Animals Receiving Repeated Dose of Indospicine Acquired from I. spicata tissue liver
muscle
heart
kidney pancreas spleen
camel 1 multiple small foci of predominantly mononuclear inflammatory cells with occasional neutrophils within the hepatic parenchyma: single, or occasionally two, necrotic hepatocytes within some foci focal areas of individual muscle fibers occasionally infiltrated by macrophages and lesser numbers of lymphocytes: often accompanied by lymphocytic infiltration within adjacent endomysim, in some cases with degeneration of muscle fiber mild, multifocal, nonsuppurative (predominantly lymphocytic) interstitial myocardial infiltrate no abnormalities detected no abnormalities detected no abnormalities detected
camel 2
camel 3
as camel 1
as camel 1; in addition, a single, moderately large, unencapsulated, eosinophilic granuloma
no abnormalities detected
a single focus on macrophages replaced a muscle fiber (in transverse section)
mild, multifocal, nonsuppurative (predominantly lymphocytic) perivascular and interstitial myocardial infiltrate no abnormalities detected no abnormalities detected no abnormalities detected
a single, small focus of lymphocytes within the myocardial interstitium
F
no abnormalities detected no abnormalities detected no abnormalities detected DOI: 10.1021/acs.jafc.6b02707 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Journal of Agricultural and Food Chemistry
(3) Hassen, A.; Rethman, N. F. G.; van Niekerk, W. A.; Tjelele, T. J. Influence of season/year and species on chemical composition and in vitro digestibility of five Indigofera accessions. Anim. Feed Sci. Technol. 2007, 136, 312−322. (4) Hassen, A.; Rethman, N. F. G.; Apostolides, Z.; Van Niekerk, W. A. Forage production and potential nutritive value of 24 shrubby Indigofera accessions under field conditions in South Africa. Trop. Grasslands 2008, 42, 96−103. (5) Hassen, A. Characterization and Evaluation of Indigofera Species as Potential Forage and Cover Crops for Semi-arid and Arid Ecosystems. Ph.D. thesis, University of Pretoria, Pretoria, South Africa, 2006. (6) Hegarty, M. P.; Pound, A. W. Indospicine, a hepatotoxic amino acid from Indigofera spicata: isolation, structure, and biological studies. Aust. J. Biol. Sci. 1970, 23, 831−842. (7) Wilson, P. G.; Rowe, R. A revision of the Indigofereae (Fabaceae) in Australia. 1. Indigastrum and the simple or unifoliolate species of Indigofera. Telopea 2004, 10, 651−682. (8) Hegarty, M.; Lowry, J.; Tangendjaja, B. Toxins in forages. In Forages in Southeast Asian and South Pacific Agriculture. Proceedings of an International Workshop Held at Cisarua, Indonesia, Aug 19−23, 1985; Blair, G. J., Ivory, D. A., Evans, T. R., Eds.; ACIAR: Canberra, Australia, 1986; pp 128−132. (9) Hegarty, M. P.; Pound, A. W. Indospicine, a new hepatotoxic amino acid from Indigofera spicata. Nature 1968, 217, 354−355. (10) Christie, G. S.; Madsen, N. P.; Hegarty, M. P. Acute biochemical changes in rat liver induced by the naturally-occurring amino acid indospicine. Biochem. Pharmacol. 1969, 18, 693−700. (11) FitzGerald, L. M.; Fletcher, M. T.; Paul, A. E. H.; Mansfield, C. S.; O’Hara, A. J. Hepatotoxicosis in dogs consuming a diet of camel meat contaminated with indospicine. Aust. Vet. J. 2011, 89, 95−100. (12) Ossedryver, S. M.; Baldwin, G. I.; Stone, B. M.; McKenzie, R. A.; van Eps, A. W.; Murray, S.; Fletcher, M. T. Indigofera spicata (creeping indigo) poisoning of three ponies. Aust. Vet. J. 2013, 91, 143−149. (13) Aylward, J. H.; Court, R. D.; Haydock, K. P.; Strickland, R. W.; Hegarty, M. P. Indigofera species with agronomic potential in the tropics. Rat toxicity studies. Aust. J. Agric. Res. 1987, 38, 177−186. (14) Young, M. P. Investigation of the Toxicity of Horsemeat Due to Contamination by Indospicine. Ph.D. thesis, University of Queensland, Brisbane, Australia, 1992. (15) Maskasame, C. Toxicity and Nutritional Value of Some Promising Pasture Legumes in Rats and Sheep. M.V.Sc. thesis, University of Queensland, Brisbane, Australia, 1984. (16) AZRI, Arid Zone Research Institute. Indospicine in beef. In Northern Territory Department of Primary Industries and Fisheries Technical Annual Report 1987−1988; NTDPIF: Alice Springs, Australia, 1989; p 39. (17) Bell, A. T.; Everist, S. L. Indiogofera enneaphylla: a plant toxic to horses (Birdsville disease). Aust. Vet. J. 1951, 27, 185−188. (18) Nath, K.; Malik, N.; Singh, O. Chemical composition and nutritive value of Indigofera enneaphylla and I. cordifolia as sheep feeds. Aust. J. Exp. Agric. 1971, 11, 178−185. (19) Stehlé, H. Survey of Forage Crops in the Caribbean; Caribbean Commission Central Secretariat: Port of Spain, 1956. (20) Fowler, M. Plant poisoning in free-living wild animals: a review. J. Wildl. Dis. 1983, 19, 34−43. (21) Dörges, B.; Heucke, J.; Dance, R. The palatability of central Australian plant species to camels. Technote 116; www.nt.gov.au/d/ Content/File/p/Technote/TN116.pdf (accessed May 30, 2016). (22) Tan, E. T. T.; Fletcher, M. T.; Yong, K. W. L.; D’Arcy, B. R.; Al Jassim, R. Determination of hepatotoxic indospicine in Australian camel meat by ultra-performance liquid chromatography−tandem mass spectrometry. J. Agric. Food Chem. 2014, 62, 1974−1979. (23) McGregor, M.; Hart, Q.; Bubb, A.; Davies, R. Managing the impacts of feral camels across remote Australia: final report of the Australia feral camel management project; http://www.nintione.com. au/publication/afcmp-0017 (accessed June 14, 2014). (24) Tan, E. T. T.; Materne, C.; Silcock, R.; D’Arcy, B. R.; Al Jassim, R.; Fletcher, M. T. Seasonal and species variation of the hepatotoxin
collected from the three camels (including a duplicate of each). It is likely that the tapeworms found in the intestines were not fully matured to the point of egg release. Residue Risk. Results of this experiment provide solid evidence of indospicine (1) contamination in camel meat arising from feeding on indospicine (1)-rich Indigofera plants. The fact that all camels in this study maintained good health and did not show any clinical signs of adverse effects suggests that camels are less susceptible to indospicine (1) intoxication than other species of animals such as dogs. However, the potential for secondary food poisoning in other species, including humans, caused by ingestion of indospicine (1) residue in camel tissue with resultant hepatotoxicosis is of concern. Indospicine (1) residue in meat is a known cause of secondary food poisoning to dogs,11,28 but its effects on humans are unclear, and caution must be taken when camel meat harvested from areas where Indigofera plants are prevalent is consumed. The findings of this study have a number of important implications for future practices of harvesting feral camels from central Australia. The determination, in this study, of the half-life of indospicine (1) in muscle suggests that feral camels captured from central Australia should be maintained on a diet that is free of indospicine-containing Indigofera plants, for a period of at least a month (or more) before slaughtering, to ensure that the concentration of indospicine (1) in muscle is greatly reduced. Further risk analysis based on both the actual prevalence of indospicine residues in camels at slaughter and the toxicity of indospicine needs to be undertaken to provide a reliable withholding period.
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AUTHOR INFORMATION
Corresponding Author
*(M.T.F.) Phone: +61 7 3276 6089. Fax: +61 7 3216 6565. Email: Mary.fl
[email protected]. Funding
This study was partly supported by the Academic Training Scheme for Institutions of Higher Education (SLAI) Scholarship (provided by the Malaysian government and Universiti Teknologi MARA) and Top-up Assistance Program Scholarship (TUAP) (provided by The University of Queensland). Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We offer special thanks to Dianne Zischke for her daily management of the young camels, and we are grateful to Titilayo Falade, Ken Yong, and Benigni Temba, who have helped in collections of Indigofera plant material. We also thank Cindy Giles, Dennis Webber, Michael Gravel, Brian Burren, and Cameron Kath (Department of Agriculture and Fisheries, Queensland, Australia) for providing advice on UPLC-MS/MS, hematological, biochemical, and dry matter analyses. We gratefully acknowledge the staff in the Centre for Advanced Animal Science (CAAS), who have provided substantial assistance during the necropsy of camels.
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REFERENCES
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DOI: 10.1021/acs.jafc.6b02707 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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