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Liquid-chromatographic assay for free and transthyretin-bound retinol-binding protein in serum from normal humans.

We quantified vitamin A-transporting retinol-binding protein (RBP) in serum or plasma by size-exclusion "high-performance" liquid chromatography, using a TSK 2000 column and fluorescent detection of the bound retinol. Serum or plasma samples filtered through a 0.20-microns (pore size) Millex filter were applied directly to the column. The pH 7.0 mobile phase contained sodium phosphate, disodium EDTA, and mercaptoethanol. Two peaks with RBP immunological activity were eluted: the smaller peak containing at least 86% of the vitamin A, which was identified as transthyretin-bound RBP; the larger peak containing a small amount (less than 14%) of a highly fluorescent vitamin A-containing protein, identified as free RBP. Both free and transthyretin-bound RBP can be quantified by this method.