Evaluation of a direct fluorometric method for determination of serum retinol.

We evaluated the usefulness of a fluorometric method for determining serum retinol (Futterman et al., Invest Ophthalmol Vis Sci 1975;14:125-30) in which fluorescence (excitation 335 nm; emission 460 nm) of retinol is directly measured in unextracted, diluted serum. Using serum from 466 individual donors, we compared values so obtained with those by a "high-performance" liquid-chromatographic method. The correlation coefficient (r) was 0.74. When we compared fluorometric retinol values with retinol-binding protein values for the 466 samples, r was 0.71. About 1% of the 466 samples had markedly higher values by fluorometry than by chromatography, the result of positive interferences. For two serum pools, we obtained CVs of 1.58% (n = 57) and 1.79% (n = 57) in long-term precision studies lasting 60 days. Although the fluorometric method of Futterman et al. has not been widely adopted, we find that it is simple to perform and that results compare favorably with the chromatographic method in precision and accuracy. It is unique among commonly used serum retinol methods in that the serum need not be extracted with organic solvents.

Stability of vitamin A in frozen sera.

We compared values for vitamin A measured in fresh sera with values obtained after storage at -20 degrees C for five to eight years. Loss of vitamin A from long-stored sera during the extraction step was eliminated by adding ascorbic acid to the extraction solvent, ethanol. Retinol-binding protein was also measured in the stored sera. Not only did vitamin A values remain stable during the years of storage, but also the correlation between concentrations of vitamin A and retinol-binding protein in the stored sera was typical of that observed with fresh sera.