Identification of protoxins and a microbial basis for red maple (Acer rubrum) toxicosis in equines

The leaves of Acer rubrum (red maple), especially when wilted in the fall, cause severe oxidative damage to equine erythrocytes, leading to potentially fatal methemoglobinemia and hemolytic anemia. Gallic acid and tannins from A. rubrum leaves have been implicated as the toxic compounds responsible for red maple toxicosis, but the mechanism of action and toxic principle(s) have not been elucidated to date. In order to investigate further how red maple toxicosis occurs, aqueous solutions of gallic acid, tannic acid, and ground dried A. rubrum leaves were incubated with contents of equine ileum, jejunum, cecum, colon, and liver, and then analyzed for the metabolite pyrogallol, as pyrogallol is a more potent oxidizing agent. Gallic acid was observed to be metabolized to pyrogallol maximally in equine ileum contents in the first 24 hr. Incubation of tannic acid and A. rubrum leaves, individually with ileum contents, produced gallic acid and, subsequently, pyrogallol. Ileum suspensions, when passed through a filter to exclude microbes but not enzymes, formed no pyrogallol, suggesting a microbial basis to the pathway. Bacteria isolated from ileum capable of pyrogallol formation were identified as Klebsiella pneumoniae and Enterobacter cloacae. Therefore, gallotannins and free gallic acid are present in A. rubrum leaves and can be metabolized by K. pneumoniae and E. cloacae found in the equine ileum to form pyrogallol either directly or through a gallic acid intermediate (gallotannins). Identification of these compounds and their physiological effects is necessary for the development of effective treatments for red maple toxicosis in equines.