Toxicon 60 (2012) 791–796 Contents lists available at SciVerse ScienceDirect Toxicon journal homepage: www.elsevier.com/locate/toxicon Detection of monofluoroacetate in Palicourea and Amorimia species Stephen T. Lee a,*, Daniel Cook a, Franklin Riet-Correa b, James A. Pfister a, William R. Anderson c, Flavia G. Lima d, Dale R. Gardner a a Poisonous Plant Research Laboratory, Agricultural Research Service, United States Department of Agriculture, 1150 E. 1400 N., Logan, UT 84341, USA b Hospital Veterinario, CSTR, Universidade Federal de Campina Grande, Patos 58700-310, Paraíba, Brazil c University of Michigan Herbarium, Ann Arbor, MI 48108, USA d School of Veterinary Medicine, Federal University of Goiás, Goiânia 74001-970, Goiás, Brazil article info abstract Article history: Numerous plant species worldwide including Palicourea marcgravii and Tanaecium bila- Received 15 March 2012 biatum in Brazil cause sudden death and are known to contain monofluoroacetate (MFA). Received in revised form 29 May 2012 Other species in Brazil including some species traditionally assigned to Mascagnia but now Accepted 31 May 2012 properly called Amorimia species and other Palicourea species are reported to cause sudden Available online 12 June 2012 death in livestock and are suspected to contain MFA due to the similarity of clinical signs. In this study, an HPLC–APCI–MS method to detect and quantify MFA was developed and Keywords: was used to investigate plant material from field collections and/or herbarium specimens Monofluoroacetate Palicourea of Mascagnia, Amorimia, and Palicourea species suspected of causing sudden death. MFA Amorimia was detected in Amorimia amazonica, Amorimia camporum, Amorimia exotropica, Amorimia Mascagnia pubiflora, Amorimia rigida, and Amorimia septentrionalis as well as Palicourea aeneofusca. Sudden death syndrome MFA concentrations differ greatly between Palicourea species and Amorimia species, which may explain the incidence of poisoning and the amount of plant material required to cause sudden death between these taxa. Published by Elsevier Ltd. 1. Introduction recumbency leading to death. Numerous other plant species in Brazil including some species traditionally Numerous plant species worldwide cause sudden death assigned to Mascagnia, Pseudocalymma elegans, Fridericia syndrome in livestock; a number of these species are sus- japurensis (synonym Arrabidaea japurensis) and other Pal- pected or known to contain the toxic organofluorine icourea and Tanaecium species are reported to cause sudden compound monofluoroacetate (MFA; Twigg et al., 1996). death in livestock and are suspected to contain MFA due to For example, Dichapetalum cymosum native to southern the similarity of clinical signs; however, the presence of Africa (Marais, 1944); Acacia georginae, Oxylobium parvi- MFA has not been verified in these species (Tokarnia et al., florum, and Gastrolobium grandiflorum plants in Australia 1990, 2000, 2002; Vasconcelos et al., 2008a; Riet-Correa (Alpin et al., 1983); and Palicourea marcgravii (Oliveira, et al., 2009). 1963; Moraes-Moreau et al., 1995) and Tanaecium bilabia- Since the mid-part of the 20th century, poisoning of tum (synonym Arrabidaea bilabiata; Krebs et al., 1994)in livestock by species then assigned to Mascagnia or “tingui” Brazil contain MFA and cause sudden death syndrome. was reported throughout the northeast and southeast Clinical signs associated with sudden death are loss of regions of Brazil (Tokarnia et al., 1990, 2000, 2002). Five balance, ataxia, labored breathing, muscle tremors, and Mascagnia species, Mascagnia elegans, Mascagnia exo- tropica, Mascagnia pubiflora, Mascagnia rigida, and Mas- cagnia aff. rigida, are reported to cause sudden death in * Corresponding author. Tel.: þ1 435 752 2941; fax: þ1 435 753 5681. livestock (Tokarnia et al., 1990, 2000, 2002; Riet-Correa E-mail address: [email protected] (S.T. Lee). et al., 2009). M. rigida is one of the most important 0041-0101/$ – see front matter Published by Elsevier Ltd. http://dx.doi.org/10.1016/j.toxicon.2012.05.029 792 S.T. Lee et al. / Toxicon 60 (2012) 791–796 poisonous plants of Brazil because of its widespread Table 1 distribution throughout the nine states of northeastern Survey and detection of monofluoroacetate in herbarium specimens of Amorimia species and Mascagnia divaricata. Brazil and the southeastern region in the states of Minas Gerais and Espírito Santo (Tokarnia et al., 2000). Recent Taxa Specimens MFA MFA taxonomic research using morphological and molecular sampled detected concentration (%) studies of Mascagnia species led to the description of a new Amorimia amazonica 81<0.0007 < genus, Amorimia, to which four of the Mascagnia species Amorimia camporum 51 0.0007 fl Amorimia exotropica 7 2 0.02 (exotropica, pubi ora, rigida, and aff. rigida) suspected of Amorimia kariniana 10nf causing sudden death syndrome have been assigned Amorimia maritima 60nf (Anderson, 2006; Davis and Anderson, 2010). Amorimia Amorimia pubiflora 18 3 0.006 species are distinguished from Mascagnia species by leaf Amorimia rigida 8 2 0.002 Amorimia septentrionalis 6 1 0.002 glands on the abaxial surface, abaxially hairy petals, large Amorimia velutina 10nf gland-bearing bracts, straight erect styles, and fruit Mascagnia divaricata 80nf (samara) morphology (Anderson, 2006). The objective of this research was to develop a method to detect and quantify MFA in taxa suspected of causing tissues were sampled from the specimens. Numbers of sudden death in livestock. P. marcgravii was verified to specimens sampled for each taxon are shown in Table 1.A contain MFA as previously reported. Amorimia species, M. map showing the distribution of the Amorimia species is elegans (under its earlier synonym, Mascagnia divaricata), shown in Fig. 1. Voucher numbers of the surveyed taxa are and Palicourea aeneofusca suspected of causing sudden in Supplementary Table 1. death syndrome were investigated for the presence of MFA. Collections of A. septentrionalis were made in the state of Additionally, MFA concentrations were compared between Paraíba, Brazil at two locations (S709.470 W3719.060; P. marcgravii and other species suspected of causing sudden elevation 305 m; S712.240 W3715.110; elevation 749 m). death. Ten plants per location were separated into leaves, stems, and floral parts and seeds if available. Specimens were 2. Materials and methods verified as A. septentrionalis and voucher specimens were deposited at University of Michigan Herbarium and the 2.1. Plant material USDA-ARS Poisonous Plant Research Laboratory Herbarium (PPRL). Voucher numbers of these taxa are in Collections of P. marcgravii were made in the states of Supplementary Table 1. Further, one collection of Amorimia Goiás (S1628.820 W4921.400; elevation 800 m) and São sp. (M. aff. rigida) was made in a cultivated garden in Paulo, Brazil (S2157.120 W4727.800; elevation 628 m). Seropédica, in the state of Rio de Janeiro, courtesy of Carlos Mature leaves from 10 plants were collected from the Goiás Tokarnia; no voucher specimen is available for this location, while leaves from 10 plants were separated into collection. mature leaves and immature developing leaves at the São Paulo location. P. aeneofusca was collected 90 km inland from the coastal area of Paraíba (S657.510 W3542.920; 2.2. Extraction elevation 589 m). Mature leaves from 10 plants were har- fl vested. Identifications of Palicourea spp. were verified by Extraction of plant material for mono uoroactetate local botanists. Voucher specimens were deposited at the (MFA) analysis was accomplished by weighing 100 mg of USDA-ARS Poisonous Plant Research Laboratory herbarium ground plant material into a 13 mL screw top test tube fl (PPRL). Voucher numbers of P. aeneofusca are in equipped with Te on lined caps (Pierce, Rockford, IL, USA). Supplementary Table 1. Water (deionized, 5 mL) was added to each test tube and A number of samples from Malpighiaceae taxa were placed in a mechanical shaker for 30 min, then centrifuged provided courtesy of the University of Michigan Herbarium to separate the plant residue and water extract. The water (MICH): Amorimia amazonica (Mascagnia amazonica); extract was transferred to a clean 13 mL test tube. The plant Amorimia camporum, a new species described by Anderson residue was extracted once more with 5 mL water for (2006), specimens previously identified as M. pubiflora; 30 min. The water extracts were combined for a total of fi Amorimia exotropica (M. exotropica); Amorimia kariniana, 10 mL. A 1 mL aliquot of the water extracts was ltered fi a new species described by Anderson (2006), specimens through a lter made from a kimwipe and disposable previously identified as M. amazonica; Amorimia maritima pipette and transferred to a 1 mL autosample vial for (Mascagnia maritima); Amorimia pubiflora (M. pubiflora); analysis. P. marcgravii samples were initially diluted 1 to 5 Amorimia rigida (M. rigida); Amorimia septentrionalis, a new and then transferred to a 1 mL autosample vial for analysis. species described by Anderson (2006), specimens previ- Samples of Palicourea with MFA concentration values ously identified as M. rigida; Amorimia velutina, a new higher than the standard curve when diluted 1:5 v:v were species described by Anderson (2006), specimens previ- further diluted 1:20 v:v and re-analyzed. ously identified as M. rigida; and M. divaricata (M. elegans). These specimens from the University of Michigan 2.3. HPLC–APCI–MS Herbarium were examined for the presence of MFA. All specimens were identified by William Anderson at the The HPLC-APCI-MS method used in this study was University of Michigan Herbarium. Vegetative and floral based on a previously reported method (Noonan et al., S.T. Lee et al. / Toxicon 60 (2012) 791–796 793 Fig. 1. Map showing the distribution of Amorimia species, modified by W. R. Anderson from his map on the Malpighiaceae website (Anderson et al., 2006). 2007). In this study, samples were injected (20 mL) onto the analysis of MFA in plant samples (Noonan et al., 2007). a Poroshell 120 EC-C18 reversed phase column (50 Â 3mm The new method was specific for MFA and was linear over 2 i.d., 2.7 mm) (Agilent Technologies, Santa Clara, CA, USA) orders of magnitude (0.078 mg/mL–10.0 mg/mL MFA/H2O).