chondroitin-sulfates and acetic-anhydride

Heparin, NAcHep, DS, and CS were labeled with deuterium by N-reacetylating, with the deuterated acetic anhydride (CD3CO)2O, GAGs previously N-deacetylated (by hydrazinolysis) to the desired extent. Degrees of deuteration of the present preparations, as determined by 2H- and 1H-NMR were 15%, 51%, 49%, and 79% for heparin, NAcHep, DS, and CS, respectively. The NMR analysis (including the 13C spectra) of the labeled products indicated that deuterium labeling did not involve any substantial modification of the GAG structures. Also NMR signals associated with specific sequences of heparin for antithrombin and of DS for heparin cofactor II were essentially the same in the unlabeled and in the deuterated GAGs. The substantial retention of the original structure was confirmed by data on the degree of sulfation (by conductimetry) and on the electrophoretic mobility in acid buffer. On the other hand, HPLC/SEC data indicated some depolymerization of heparin and DS in the N-deacetylation step of the labeling reactions. HPLC/MS spectrometry permitted a clear identification of disaccharide and tetrasaccharide fragments obtained from deuterated GAGs by enzymic (heparinase, chondroitinase ABC) or chemical depolymerization (deaminative cleavage, Smith degradation), opening new prospects for studies of human pharmacokinetics, with differentiation of exogenous from endogenous GAGs.

Topics: Acetic Anhydrides; Acetylation; Animals; Carbohydrate Sequence; Cattle; Chondroitin Sulfates; Chromatography, Gel; Dermatan Sulfate; Deuterium; Glycosaminoglycans; Heparin; Isotope Labeling; Magnetic Resonance Spectroscopy; Mass Spectrometry; Molecular Sequence Data; Molecular Weight; Sharks

Isolated glycosaminoglycans (GAGs) were quantified biochemically in the cerebella of 6-day-old rats. 14C-Labeled hyaluronic acid (HA) and chondroitin-4-sulfate (C-4-S), added prior to isolation of GAGs from tissue, served as internal standards to allow correction for unknown losses during the purification procedure and exact quantification of GAGs in the intact tissue. Three main constituents--HA, chondroitin sulfate (CS), and heparan sulfate (HS)--were found at concentrations of 1.82, 1.52, and 0.76 micrograms/mg protein amounting to 44%, 37%, and 19% of the total GAG fraction, respectively. Incorporation of [3H]glucosamine precursor into GAGs was higher for HS (56% of incorporated precursor) and lower for HA (29%) and CS (15%). The specific activities of individual GAGs were 64.7 nCi/micrograms for HS, 14.2 for HA, and 8.3 for CS.

Topics: Acetic Anhydrides; Animals; Cerebellum; Chondroitin Sulfates; Glucosamine; Glycosaminoglycans; Heparitin Sulfate; Hyaluronic Acid; Rats; Time Factors