Preparation of lipid-free tissue extracts for chromatographic determination of thyroid hormones and metabolites |
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Authors: | Janice T Gordon Floy L Crutchfield Anthony S Jennings Mary B Dratman |
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Institution: | 1. The Medical College of Pennsylvania, Philadelphia, Pennsylvania 19129 U.S.A.;2. The Medical Research Service, Veterans Administration Hospital, University and Woodland Avenues, Philadelphia, Pennsylvania 19104 U.S.A.;3. The Hospital of the University of Pennsylvania, Philadelphia, Pennsylvania 19104 U.S.A. |
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Abstract: | We have developed a new method of preparing tissues for analysis of thyroid hormones and metabolites which eliminates troublesome lipids and proteins. Frozen tissue is homogenized by grinding with dry ice in a Waring blender, and the moist powder obtained is extracted with chloroform:methanol (2:1). In a modification of the Folch procedure for the total separation of lipids, the filtered extracts then are partitioned into polar and nonpolar layers by the addition of 0.05% aqueous calcium chloride. The upper phase contains the iodocompounds of interest as well as all salts and small polar molecules. The lipids remain dissolved in the lower phase after it is back-extracted with pure upper phase. The combined upper or aqueous methanol layers are lyophilized and the residue is taken up in methanol to yield a concentrated solution ready for analysis of iodocompounds. The greater clarity of the extract permits application of larger samples for two-dimensional paper chromatography than has been customary. For gradient analysis by reverse-phase ion-pair high-performance liquid chromatography (HPLC), the nitrogen-evaporated methanol extract is dissolved in the initial mobile phase, 0.1% H3PO4, for injection onto the column. Using these methods we have achieved the first reported separation of 125I-labeled tissue iodothyronine metabolites by HPLC. The new method of extraction is of general applicability to any biological sample which might be analyzed in thyroid hormone metabolism research. |
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Keywords: | Author to whom all correspondence should be addressed: Medical Research Service Veterans Administration Hospital University and Woodland Avenues Philadelphia Pa 19104 |
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