Nitro toxins were analyzed by both Fourier transform infrared spectroscopy spectroscopy (FT-IR) ( Schoch et al.,

1998) and spectrophotometric methods ( Matsumoto et al., 1961; Williams, 1981; Majak et al., 1992). Chemical analysis demonstrated the presence of indospicine in all samples of I. lespedezioides analyzed ( Table 1). The concentration ranged Obeticholic Acid mw from a low of 63 μg/g up to 1178 μg/g. In a previous analysis of I. lespedezioides, Aylward et al. (1987) reported an indospicine concentration of 0.02% (200 μg/g). Nitro toxins were detected only in the sample collected from Amajari. The FT-IR spectrum showed a weak signal at 1556 cm−1 indicative of 3-nitropropionic acid. The presence of nitro toxins was verified in the use of a colorimetric

assay ( Williams, this website 1981) in which a slightly pink solution was observed but the concentration was below the level of quantitation. To confirm the presence of nitro toxins the samples were analyzed using a third method reported by Matsumoto et al. (1961); only the sample from Amajairi was found to contain a detectable level of nitro toxin at a concentration of 2.5 mg/g as 3-nitropropionic acid equivalents. Majak et al. (1992) reported a slightly lower concentration at 1.5 mg/g 3-NPA in a sample of I. linnaei. I. linnaei and I. hendecaphylla also contain indospicine but it has not been shown that this toxin is responsible for the clinical syndrome. In Australia the disease in horses was treated and prevented with arginine or arginine containing substances ( Hooper et al., 1971), and it has been suggested that indospicine may competitively interfere with the incorporation of arginine into proteins due to inhibition of arginase activity and nitric oxide synthase ( Madsen and

Hegarty, 1970; Pass et al., 1996). The presence of indospicine in the three Indigofera species causing nervous signs in horses highly suggests that this amino acid is responsible for the clinical signs of the disease as suggested previously ( Hegarty and Pound, 1968; Hooper et al., 1971). However, the disease has not been reproduced dosing indospicine to experimental animals. Anitro toxin has also been suspected as a cause of the disease ( Majak et al., 1992), and similar conditions have been observed in other livestock ingesting nitro toxin-containing plants ( Shenk et al., 1976; oxyclozanide James et al., 1981), in possums and rats dosed with 3-nitropropionic acid ( Hamilton and Gould, 1987; Gregory et al., 2000), and in humans with moldy sugar cane poisoning which is considered a 3-nitropropionic acid toxicosis ( Liu et al., 1970; Hu, 1992). However, we found the nitro toxins to be either non-detectable or low compared to known nitro toxic plants such as some Astragalus species and would question if these levels would be toxic as Williams (1981) previously suggested and reported. In conclusion, I. lespedezioides causes nervous signs in horses in the state of Roraima.