Submitted to: Journal of Agricultural and Food Chemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: January 2, 2002
Publication Date: March 16, 2002
Citation: Garrossian, M., Gardner, D.R., Panter, K.E., James, L.F. 2002. Preparation of tetrahydroagathic acid: a serum metabolite of isocupressic acid, a cattle abortifacient in ponderosa pine.. Journal of Agricultural and Food Chemistry. Interpretive Summary: Consumption of ponderosa pine needles causes late-term abortions in cattle and is a serious poisonous plant problem in foothill and mountain rangelands of the Western US. The toxic compound in ponderosa pine needles was identified as isocupressic acid. However, isocupressic is rapidly metabolized to several different compounds once cattle ingest it. The metabolism of isocupressic acid makes it difficult for discovering the biochemical mechanism of action of the abortion and for identification of chemical markers to diagnose pine needle abortion cases. In this paper chemical procedures are presented for the synthesis of three isocupressic acid metabolites and in one case the prepared compound (tetrahydroagathic acid) was fully characterized and used to prove that it was present in the serum of cattle that had ingested plant material containing isocupressic acid. The developed chemical procedures will be useful in preparing metabolites of isocupressic acid that can then be used in further studies on the abortion mechanism. It was also found that one of the metabolites, tetrahydroagathic acid, which remains in the serum longer than any other metabolite, maybe a useful chemical marker for diagnosing ponderosa pine needle-induced abortions. Tetrahydroagathic acid was the only metabolite of isocupressic found in the serum of five cows that had aborted several days after reportedly eating pine needles.
Technical Abstract: Isocupressic acid (1) was used to synthetically prepare a mixture of (8S, 13R,S)-labda-15,19-dioic acid (tetrahydroagathic acid) (5) via a two-step oxidation procedure followed by hydrogenation of the double bonds at C13 and C8. Reduction of the C8,17 double bond was sterospecific producing only the 8S isomer and confirmed by the nOe interaction between the resulting C17 and C20 methyls. The 13R and 13S isomers of 5 were separate and analyzed by HPLC/MS and (13S)-tetrahydroagathic acid was isolated and identified by comparison to a standard prepared by hydrogenation of naturally occurring (13S)-dihydroagathic acid (4). (13R,S)-dihydroagathic acid was prepared by selective sodium metal catalyzed hydrogenation of the C13,14 allylic double bond of agathic acid (3). The prepared compounds were then used as standards to confirm the presence of 4 and 5 and their respective 13R and 13S isomers in bovine serum samples. Tetrahydroagathic acid was shown to be the only metabolite detected in serum samples taken from a suspected cattle abortion case submitted for diagnosis and thus 5 could be a valuable diagnostic marker for pine needle-induced abortions.