Though differing only slightly in their degrees of sulfation, heparin preparations from pig mucosa and those from beef mucosa have consistently different 13C- and 1H-NMR spectra, which provide useful fingerprints for distinguishing the two types of heparin. Integrated areas of NMR signals associated with minor, undersulfated sequences (assigned by comparison with mono-dimensional spectra of selectively desulfated heparins and by analysis of two-dimensional spectra of heparins prepared from pig and beef mucosa) permit quantitation of differences in sulfation patterns. Undersulfation of pig mucosal heparins at position 6 of the hexosamine units, determined by 13C-NMR and expressed as percent glucosamines nonsulfated at C6 referred to total glucosamines, is substantially lower for pig mucosal heparins than for beef mucosal heparins (16.9-21.7% vs 36.7-40.7%; average values: 18.6% vs 40.3%). By contrast, undersulfation at position 2 of the iduronic acid units, determined by 1H-NMR and expressed as percent nonsulfated iduronic acid referred to total (sulfated + nonsulfated) iduronic acid is significantly higher for pig mucosal preparations (9.6-13.5% vs 2.1-2.7%; average values: 12.7% vs 2.3%). Pig mucosal heparins also have a significantly higher content of 3-O-sulfated glucosamine units, which are markers for the active site of heparin for antithrombin-III.

As previously observed for model glycolipids, micellar aggregates of the ganglioside GM-1 can be disrupted by the formation of inclusion complexes with α-cyclodextrin (α-CD). Evidence for such disaggregation was obtained from narrowing and shifting of 1H- and 13C-NMR signals, and decreasing of 13C-NMR relaxation times (T1) upon addition of α-CD to aqueous (D2O) solutions of the ganglioside. As a result of the α-CD-induced disaggregation, GM-1 becomes permeable through 100 000 Da cut-off ultrafiltration membranes (which are virtually impermeable to normal GM-1 aggregates), and can be freed from phospholipid contaminants. β-Cyclodextrin (β-CD), which gives weaker complexes with GM-1, does not produce any significant disaggregation effects. Also, fully methylated β-cyclodextrin (Meβ-CD) and hydroxyethyl-β-cyclodextrin (HEβ-CD) were ineffective. A solid complex (which precipitates from solutions at α-CD/GM-1 molar ratios > 5) was obtained, and characterized by CP/MAS 13C-NMR spectroscopy and by DSC.

The binding of single-stranded polydeoxyribonucleotides to adenosine A1 and A2 receptors was investigated. Defibrotide, a natural substance with established anti-thrombotic and anti-ischaemic effects, displaced [3H]CHA (N6-cyclohexyl-adenosine) and [3H]NECA (5'-N-ethylcarboxamido-adenosine) concentration dependently, completely and competitively. Ki values of 371 +/- 68 and 688 +/- 115 micrograms/ml (mean +/- S.E.M. of 4-5 replications) were computed for adenosine A1 and A2 sites, respectively. Higher and lower molecular weight polydeoxyribonucleotides displayed comparable affinity, whereas a double-stranded polydeoxyribonucleotide and a polyanion with a negative charge comparable to that of defibrotide were inactive. Defibrotide did not affect the total number of binding sites in radioligand saturation experiments. Defibrotide relaxed the K(+)-contracted guinea-pig trachealis muscle (IC50 = 4001 micrograms/ml) about one-third as potently as the CHA-contracted preparation and as potently as the resting preparation. NECA, a mixed adenosine A1/A2 receptor agonist, behaved similarly. The effects were abolished by the adenosine A1/A2 receptor blocker 8-phenyltheophylline, but not by the selective A1 blocker, 1,3-dipropyl-8-(2-amino-4-chlorophenyl)-xanthine. These results demonstrate that defibrotide binds to adenosine receptors and triggers pharmacological responses comparable to those of a known agonist.

Pneumatically assisted electrospray was demonstrated to be a powerful ionization source for the analysis of oligosaccharides. A mass spectrometer was interfaced to an HPLC system, using this interface, to determine oligosaccharides from the enzymatic digestion of heparin separated on a reversed-phase column. To set up the technique, and particularly to clarify the ionization process, purified disaccharides, from enzymatic digestion of chondroitin sulphates, were measured. The use of a suitable counter ion in the mobile phase, tetrapropylammonium (TPA to optimize the HPLC separation, gave, with sulphated di- and oligosaccharides, adducts [M + nTPA - (n + m)H]m-, which were unexpectedly stable to fragmentation; molecular ions [M - (n + 1)H]n−, in the presence of the counter ion, were observed only with desulphated or monosulphated disaccharides. The stability of the adducts and the use of a deuterated ion-pair reagent permitted an exact evaluation of the molecular masses of disaccharides and oligosaccharides of unknown structure. Spectra obtained in the absence of the counter ion contained singly or multiply charged molecular ions and fragmentation ions mainly from loss of the sulphate groups; under these ionization conditions the exact mass determination and interpretation of the spectra were difficult. After removal of the counter ion, tandem mass spectra could be obtained with some interesting data for the characterization of these molecules. Complete spectral analyses were performed with amounts of samples of 50 μg but, using microbore columns, one twentieth of this amount may give good spectra.

Pneumatically assisted electrospray was demonstrated to be a powerful ionization source for the analysis of oligosaccharides. A mass spectrometer was interfaced to an HPLC system, using this interface, to determine oligosaccharides from the enzymatic digestion of heparin separated on a reversed-phase column. To set up the technique, and particularly to clarify the ionization process, purified disaccharides, from enzymatic digestion of chondroitin sulphates, were measured. The use of a suitable counter ion in the mobile phase, tetrapropylammonium (TPA), to optimize the HPLC separation, gave, with sulphated di- and oligosaccharides, adducts [M + nTPA - (n + m)H]m-, which were unexpectedly stable to fragmentation; molecular ions [M - (n + 1)H]n-, in the presence of the counter ion, were observed only with desulphated or monosulphated disaccharides. The stability of the adducts and the use of a deuterated ion-pair reagent permitted an exact evaluation of the molecular masses of disaccharides and oligosaccharides of unknown structure. Spectra obtained in the absence of the counter ion contained singly or multiply charged molecular ions and fragmentation ions mainly from loss of the sulphate groups; under these ionization conditions the exact mass determination and interpretation of the spectra were difficult. After removal of the counter ion, tandem mass spectra could be obtained with some interesting data for the characterization of these molecules. Complete spectral analyses were performed with amounts of samples of 50 mug but, using microbore columns, one twentieth of this amount may give good spectra.