Abstract
A variety of procedures have been developed for determining the sulfate ester content of various biomolecules. Ion chromatography (IC), that is, quantitation of ionic substances by ion conductimetry after separation by anion-exchange chromatography, has been increasingly utilized for the determination of inorganic sulfate in clinical and environmental samples. We adopted suppressed-mode IC to the determination of lipid- or glycolipid-bound sulfate released by acid hydrolysis and found that it has the advantage of increased precision for wide concentration ranges (30 pmol to ~μmol) and lack of interference from other lipids. To minimize deterioration of the separation column, the lipophilic constituents in the acid hydrolysate were removed by a two-phase partition system of chloroform-methanol-water. The inorganic sulfate was quantitatively extracted into the aqueous phase by replacing water with an alkaline buffer.By this method, the concentration of sulfolipids was determined in the kidney of mammals with various body mass. Sulfolipids were more concentrated in the kidney of smaller animals, which have higher maximum urine concentrating activity per gram of body mass, supporting the hypothesis of the function of sulfolipids as an ion barrier on the luminal surface of renal tubules.
Highlights
A variety of procedures have been developed for determining the sulfate ester content of various biomolecules
We extended suppressed-mode Ion chromatography (IC) to the detemination of lipid- or glycolipid-bound sulfate after quantitative liberation from parent compounds
Sulfolipid sulfates have usually been liberated by acid hydrolysis or solvolysis [1]
Summary
Analytical grade chemicals from various suppliers were used throughout this work. High-purity water was prepared by a Puric water purification system (Organo). The several minor monosulfated glycolipids, including GlcCer I3-sulfate [20], SM3, and the isoglobo-series sulfoglycolipids [7, 8], were present in rat kidney and eluted simultaneously with SM4s in the monosulfated glycolipid fraction. Their concentrations were Ͻ1% of SM4s [7, 8, 20], except for SM3 in mouse kidney, which accounted for ϳ10% of SM4s [9]. Fractions 2, 4, and 5 containing SM4sϩSM3, HSO3Chol, and SB1a, respectively, were used for analysis
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