Determination of bound sulfur in serum by gas dialysis/high-performance liquid chromatography

A sensitive high-performance liquid chromatography (HPLC) method has been developed for the determination of bound sulfur in mammalian serum. In this work, bound sulfur is defined as divalent sulfur that is easily liberated as sulfide by reduction with dithiothreitol. Released sulfide was treated by flow gas dialysis and converted into a fluorescent derivative, thionine, through the reaction with p-phenylenediamine and ferric ion. Thionine was then determined by HPLC using fluorometric detection. The calibration curve for bound sulfur is linear in the range from 0.1 to 10 microM, and the sensitivity of this method allows detection of less than 10 nM bound sulfur (0.5-ml sample). Recoveries of low-molecular-weight and high-molecular-weight bound sulfur added to human serum at 1.0-5.0 microM levels averaged between 94 and 101%. The proposed method was applied to the determination of bound sulfur in human and various animal sera. The mean concentration in normal human serum was 1.16 +/- 0.09 microM for males (n = 5) and 1.07 +/- 0.18 microM for females (n = 5). The mean concentrations ranged from 1.55 to 6.18 microM in animal sera. The form of the sulfur bound in serum was also discussed.