A direct microassay for serum retinol (vitamin A alcohol) by using size-exclusion high-pressure liquid chromatography with fluorescence detection

Abstract

Serum retinol (bound to plasma retinol-binding protein, RBP) can be determined by direct injection of as little as 20 μl of serum or plasma by using size-exclusion high-pressure liquid chromatography (SE-HPLC) with fluorescence detection. Toyo Soda TSK G-3000SW columns (0.75 × 7.5-cm guard column plus 0.75 × 30-cm analytical column) were eluted with 0.2 m NaCl/0.01 m phosphate buffer (pH 6.8) at 1 ml/min, with detection at 280 nm for protein elution. Fluorescence of the retinol-RBP complex was monitored with excitation at 334 nm (interference filter) and emission at 425 nm (long-pass filter). The retinol-RBP complex eluted as two peaks, the holo-RBP-transthyretin complex (apparent molecular weight 70,000) and holo-RBP (apparent molecular weight 9000). Identities of these peaks were established by immunodiffusion assay of the proteins and by extraction and analysis of retinol. Nonideal interactions with the column packing seem to be responsible for the low apparent molecular weight of holo-RBP. The first peak predominated when large volumes of serum (100 to 250 μl) were injected, and the second when small volumes (5 to 50 μl) were analyzed. The integrated area of the two fluorescence peaks due to retinol bound to RBP was proportional to the volume of a serum sample injected over the range 5 to 250 μl. Rat serum samples were analyzed for retinol by SE-HPLC and by conventional extraction and HPLC analysis; over the range 4 to 67 μg retinol/dl serum (0.14 to 2.34 μ m), a linear relationship was observed between the results of the two assay techniques ( n = 25, r = 0.93). Reproducibility was ±4% within-day, ±6% between days. At a signal:noise ratio of 5:1, the minimum amount of retinol quantitable was 1 ng, corresponding to 5 μg retinol/dl (0.17 μ m) for a 20-μl injection.