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Assessing Hormonal Activity of Xenobiotics in Serum: The Role of HPLC and CALUX Assay

This study investigates the hormone-like effects of xenobiotic compounds, specifically 2,3,7,8-TCDD (Dioxin), found in blood serum. Utilizing the CALUX assay, which is sensitive to hormones and hormone-like substances, poses a challenge due to potential false positives from naturally occurring hormones such as estradiol. The methodology includes a two-step sample preparation process: first, removing interfering substances via solid phase extraction (SPE), followed by separating hormones using high-pressure liquid chromatography (HPLC). This approach aims to generate accurate results for environmental health assessments.

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Assessing Hormonal Activity of Xenobiotics in Serum: The Role of HPLC and CALUX Assay

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  1. Consider this:You want to determine the hormone-like effect of xenobiotic compounds found in blood serum 2,3,7,8-TCDD (”Dioxin”) Cl O Cl Cl O Cl Problem 1:Your serum contains a mixture of both hormones and xenobiotic compounds Xenobiotic substance Estradiol (Female sex hormone) Naturally occuring hormone Xenobiotic compound:A substance foreign to a living system. The job and the problem You want to do this by using a test (an assay) that responds to hormones and hormone-like substances (the CALUX assay) Problem 2:The test gives a false positive if hormones are present. Environmental Studies - Spring 2005 HPLC Lab (Environ.- & Occu. Med.)

  2. Sample preparation strategy Two steps are necessary: Removal of interfering substances from serum sample Removal of hormones from the clean serum sample Environmental Studies - Spring 2005 HPLC Lab (Environ.- & Occu. Med.)

  3. Serum Wash Elute Water Methanol Serum Waste Sample Waste TCDD Estradiol Removal of interfering substances from serum sample Solid phase extraction (SPE) This is called sample preparation To remove water, salts and proteins from serum What is left after this step is a sample of hormones and ”other things” Environmental Studies - Spring 2005 HPLC Lab (Environ.- & Occu. Med.)

  4. Imagine a tube filled with tiny particles like grains of sand – completely packed Imagine that we have a continous flow of liquid through the tube Sample with a mixture of TCDD and Estradiol Now we put a mixture of TCDD and Estradiol on top of the tube Flow The particles in the tube interacts with the mixture Sample Removal of hormones from the clean serum sample High pressure liquid chromatography (HPLC) Because of the flow the mixture moves down the tube Interaction is not equally strong for all components Environmental Studies - Spring 2005 HPLC Lab (Environ.- & Occu. Med.)

  5. Estradiol TCDD Sample with a mixture of TCDD and Estradiol Flow Removal of hormones from the clean serum sample High pressure liquid chromatography (HPLC) Imagine a tube filled with tiny particles like grains of sand – completely packed Imagine that we have a continous flow of liquid through the tube Now we put a mixture of TCDD and Estradiol on top of the tube Because of the flow the mixture moves down the tube The particles in the tube interacts with the mixture Interaction is not equally strong for all components Some components moves slower Separation complete Environmental Studies - Spring 2005 HPLC Lab (Environ.- & Occu. Med.)

  6. Thank you Environmental Studies - Spring 2005 HPLC Lab (Environ.- & Occu. Med.)

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