Development and application of methods for extraction and LC/MS/MS analysis of sex steroids and conjugates from fish feces.

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Date
2015-03-24
Authors
Peters, Lisa E.
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Abstract
Non-lethal monitoring of animals using fecal steroid analysis is frequently employed to assess the health/reproductive status of individuals or populations, and may be applied to environmental monitoring of fish. Fecal steroid analysis requires both the non-polar parent and the polar glucuronide/sulfate conjugated forms to be considered. To address this challenge in fish, a reproductive steroid extraction method with HPLC/MS/MS analysis was first developed for Cortland’s in vitro bioassay medium. A 2-step liquid:liquid extraction method successfully captured parent sex steroids and their conjugates. This method was then applied to investigate routes of endocrine disruption in trout gonads exposed to environmentally relevant concentrations of polybrominated diphenyl ether (PBDE) flame retardants, and selected metabolites. Of the two classes, hydroxylated metabolites of PBDE-47 had the greatest effect on steroidogenesis in both male and female trout gonadal tissues. The extraction and LC/MS/MS analysis techniques were then applied to fish feces. The most effective matrix treatment for feces was a lipid removal agent, CleanasciteTM. It removed lipid and pigmented compounds left by acid washing, and prolonged column life without affecting hormone recoveries. Using paired plasma and feces samples from rainbow trout it was determined that fecal estrogens could predict plasma concentrations. Changes in plasma estrogens were typically not reflected in feces, which varied less, until the subsequent week. Fecal concentrations of E2-17 glucuronide best predicted plasma E2 concentrations in female rainbow trout, while the strongest relationship was between plasma E2-3 sulfate and fecal E2-17 glucuronide. Plasma clearance time and partitioning of estrogens into feces, urine and bile was then monitored in two experiments. The first involved in vivo administration of radiolabeled E2 into the blood of rainbow trout, and in the second radiolabelled E2 was introduced into the fish gut. Overall, E2 was cleared from plasma in 72 hours, and estrogens in feces can be a composite of hormones metabolized over > 4 days. Approximately 68% of E2-H3 injected into the gut entered enterohepatic circulation. Understanding hormone metabolism and clearance, and the roles of dietary uptake and enterophepatic circulation, will facilitate further development of fecal steroid analysis as a non-lethal assessment tool for monitoring fish reproduction.
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fish, steroids
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