The Recovery and Characterization of Acid Glycosaminoglycans in Normal Human Urine: Influence of a Circadian Rhythm

Abstract

A method is described for the recovery of acid GAG (AGAG) as solid Ba salts from relatively small volumes (100+/-50 ml) of normal adult human urine. It is pointed out that (a) the amount recovered, (b) the molecular characteristics (i.e. molecular weight and charge density), (c) the ionic strength at which the AGAG are recovered and (d) the "critical electrolyte concentrations" of the AGAG-precipitating reagent (or anion exchanger) are inseparably linked, and that different fractions are obtained by variations in any parameter. Our product is defined as that fraction of heterodisperse material precipitated at room temperature by cetyl trimethylammonium bromide from urine diluted with 3 volumes of 0.05 M sodium acetate buffer pH 5.7, in the presence of asbestos powder. It is less degraded than other AGAG remaining in the urine supernatant. The product contained three electrophoretically separa0le components, characterized by susceptibility to pronase digestion, infrared and ultraviolet spectroscopy, hexosamine identification, tests for uronic and sialic acids, and the "critical electrolyte concentrations" of their Alcian blue complexes, as "chondroitin sulphate", "heparan sulphate" and acid glycoprotein. The method is simple, rapid and suita0le for use on multiple samples. The product is directly usable in electrophoretic analyses, etc. Using this method AGAG was recovered from the urine of nine males and five females, aged 25-43, collected during a 24-hr cycle, in six 4-hr aliquots. The ratios of "chondroitin sulphate" to "heparan sulphate" quantitated from the electrophoretic strip, showed a marked peak in every subject at 18-22 hrs.