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Title:   Horse-killing toxins identified in maple leaves

Issue: For centuries, horses have been known to suffer from hemolytic anemia, methemoglobinemia and death after eating red maple leaves. Recently, horses have been observed suffering these symptoms after ingesting leaves from other trees, most notably the sugar maple. It is important to horse owners in the Eastern US to find out what compounds are causing this problem and which species of trees carry these toxins.

Furthermore, the horse is similar in one way to the large subpopulation of humans which is susceptible to favism. Both groups lack sufficient glucose-6-phosphate dehydrogenase activity to protect red blood cells from oxidation when presented with an oxidizing toxin (fava bean alkaloids in human favism and red maple toxins in horses). Symptoms are similar (methemoglobinemia and anemia) and we believe that discoveries of protective strategies for the horses may be lead to new ways to prevent the human syndrome.

Response: Maple leaves were extracted with water and methanol. These extracts were then separated into defined fractions by thin layer chromatography. Test tubes containing red blood cells from horses were used to detect the presence of toxins in these fractions. Once fractions were found that could harm blood cells in a test tube in exactly the same way that eating maple leaves harms the blood of live horses, a gas chromatograph/mass spectrometer was used to identify the compounds responsible.

We found that gallic acid causes methemoglobinemia and is plentiful in both water and methanol extracts of red maple, sugar maple and silver maple, and in the extract fractions from these species that oxidize blood cells. Furthermore, 2,3-dihydro-3,5-dihydroxy-6-methoxy-4H-pyran-4-one, (a potential co-oxidant) is found in the water extract and probably amplifies the oxidizing power of the gallic acid. This would also explain why water extracts were more effective than methanol extracts in oxidizing hemoglobin to methemoglobin

A third, as yet unidentified, factor was discovered that causes a little oxidation, but a great deal of hemolysis.

Impact: We now expect not only red maple, but also sugar maple and silver maple leaves to poison horses. Similar analysis of Norway maple leaves indicates that poisoning due to the ingestion of leaves from this ornamental tree is much less likely.