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United States Department of Agriculture

Agricultural Research Service

Title: Development of Enzyme-Linked Immunosorbent Assays for Isocupressic Acid and Serum Metabolites of Isocupressic Acid

Authors
item Lee, Stephen
item Gardner, Dale
item Garrossian, Massoud - FRONTIER SCIENTIFIC
item Panter, Kip
item Serreqi, Alessio - BIOSYNTECH CANADA, INC
item Schoch, Thomas
item Stegelmeier, Bryan

Submitted to: Journal of Agricultural and Food Chemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: March 10, 2003
Publication Date: April 25, 2003
Citation: LEE, S.T., GARDNER, D.R., GARROSSIAN, M., PANTER, K.E., SERREQI, A.N., SCHOCH, T.K., WIERENGA, T.L. DEVELOPMENT OF ENZYME-LINKED IMMUNOSORBENT ASSAYS FOR ISOCUPRESSIC ACID AND SERUM METABOLITES OF ISOCUPRESSIC ACID. JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY. 2003.

Interpretive Summary: The consumption of ponderosa pine, lodgepole pine, common juniper and monterey cypress causes abortions in pregnant cattle. Isocupressic acid is the abortifacient compound in these plants. However, isocupressic acid is rapidly metabolized to the serum metabolites; agathic acid, dihydroagathic acid and tetrahydroagathic acid. Rapid and sensitive diagnostic techniques are needed to identify poisoned animals, study toxicokinetics and to elucidate the mechanism of isocupressic acid-induced abortion in cattle. In this study four competitive inhibition enzyme-linked immunosorbent assays for isocupressic acid and its sera metabolites were developed. One assay was specific to isocupressic acid while the other three assays showed cross reactivity with agathic acid, dihydroagathic acid, tetrahydroagathic acid in addition to isocupressic acid. The assay specific for isocupressic acid had a limit of detection of 44.1 pg. The other assays which demonstrated cross reactivity with the isocupressic acid blood metabolites also had comparably low limits of detection. One assay was used to follow the absorption and elimination profile of isocupressic acid metabolites in both blood and urine after oral dosage of a cow with common juniper. The simple extraction-ELISA methods described in this paper demonstrate the potential of using these tools for the rapid screening of biological samples for the presence and levels of isocupressic acid and its blood and urine metabolites and will be beneficial in the diagnosis of animal poisonings and pharmacological studies.

Technical Abstract: The consumption of ponderosa pine (Pinus ponderosa), lodgepole pine (Pinus contorta), common juniper (Juniperus communis) and Monterey cypress (Cupressus macrocarpa) causes abortions in pregnant cattle. Recent studies have identified isocupressic acid as the primary abortifacient compound in these plants. In vitro and in vivo studies using rumen and blood have shown isocupressic acid rapidly metabolized to agathic acid dihydroagathic acid and tetrahydroagathic acid. Rapid and sensitive diagnostic techniques are needed to identify poisoned animals, study toxicokinetics, and to elucidate the mechanism of isocupressic acid-induced abortion in cattle. In this study four competitive inhibition enzyme-linked immunosorbent assays for isocupressic acid and its sera metabolites were developed using-polyclonal antibodies. One assay is specific to isocupressic acid while the other three assays show cross reactivity with agathic acid, dihydroagathic acid, tetrahydroagathic acid in addition to isocupressic acid. The assay specific for isocupressic acid had a limit of detection of 44.1 pg. The other assays which demonstrated cross reactivity with the isocupressic acid blood metabolites also had comparably low limits of detection. One assay was used to follow the absorption and elimination profile of isocupressic acid metabolites in both blood and urine after oral dosage of a cow with common juniper. The simple extraction ELISA methods described in this paper demonstrate the potential of using these tools for the rapid screening of biological samples for the presence and levels of isocupressic acid and its blood and urine metabolites and will be beneficial in the diagnosis of animal poisoning and pharmacological studies.

Last Modified: 4/18/2014
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