A reversed phase HPLC method for the simultaneous determination of all monosaccharides contained in galactosaminoglycan isomers from human aorta proteoglycans.

Monosaccharides obtained by reduction and hydrolysis of galactosaminoglycan isomers, are entirely determined as their perbenzoyl derivatives by reversed phase HPLC, without removal of hexosamines prior to benzoylation. The method is suitable for the analysis of arterial proteoglycan constituent galactosaminoglycans, providing specific, precise and reproducible results. Moreover, synthesis and characterization of tri-O-benzoyl-1,6-L-anhydroidose and N-benzoyl-tetra-O-benzoyl-alpha- and -beta-D-galactosamine have been accomplished.

LDL interaction with proteoglycans isolated from human aortas with different atherosclerotic involvement.

BACKGROUND: Proteoglycan (PG)-LDL interaction is likely to be involved in lipid deposition in arterial wall. The relative content and the structural properties of different PG populations change in human aorta with atherosclerotic degeneration. Therefore, we extracted and separated these PGs from human aorta samples with increasing severity of atherosclerotic involvement and studied their interactions with human LDL. MATERIALS AND METHODS: PGs were extracted with 6 M urea, purified by ion-exchange chromatography and separated into two different populations (PGI and PGII) on the basis of hydrodynamic size and glycosaminoglycan composition. The interaction of both PGI and PGII with LDL was studied separately by precipitation assay. RESULTS: The ratio PGI/PGII decreased markedly with increasing severity of the disease. Both PGI and PGII formed insoluble complexes with LDL. However, the shape of saturation curves was markedly different. An excess of PGI from normal or intermediately affected aorta inhibited insoluble complex formation with LDL. On the contrary, an excess either of PGII or of PGI from severely affected aorta did not inhibit insoluble complex formation. In the case of PGII, the maximum of percentage cholesterol precipitated was higher when PGII from severely affected aorta was used. CONCLUSIONS: The different interactions of LDL with either PGI or PGII are likely to depend on the different structural properties of PGs. The decrease of PGI/PGII ratio following atherosclerotic degeneration could play an important role in lipid deposition in arterial wall.

[Study of the interaction between plasma proteoglycans and LDL by means of fluorescence spectroscopy]. / Studio dell'interazione tra proteoglicani plasmatici ed LDL mediante spettroscopia di fluorescenza.

The relevance of the interaction between LDL and PGs in the development of atherosclerotic processes is well known. However, the exact nature of the interaction and the consequent structural and/or conformational modifications of the lipoprotein remain to be clarified. It has been demonstrated that after this interaction the LDL particle is not recognized by specific cellular receptors and enters the scavenger pathway operating in different cell types. These effects have been shown by using aortic PGs, but PGs are also present in the plasma compartment and may interact constantly with LDL, taking part in the regulation of lipid metabolism. In order to assess the capability of plasma PGs to induce LDL modifications, we investigated their interactions by studying the changes in the organizational parameters of LDL by fluorescence spectroscopy. Plasma PGs were isolated by DEAE Sephacel ion exchange chromatography and Sephacryl S300 gel filtration in two different families: a low-charge PG and a high-charge PG. Human LDL was prepared from plasma of normolipemic donors by ultracentrifugal flotation between 1.025-1.045 g/ml. Steady-state anisotropy measures were obtained by analyzing the rotational diffusion rate of DPH after incubation of LDL with plasma PGs in a physiological ratio. In our experimental conditions, LDL incubation with plasma low-charge PG did not modify DPH fluorescence anisotropy, whereas LDL treatment with highly charged PGs induced a marked decrease of this parameter, suggesting a significant effect on LDL microviscosity. The data show that both the charge and the GAG composition of PGs appear to be critical factors in LDL-PG interaction.(ABSTRACT TRUNCATED AT 250 WORDS)

Structural and functional modifications of human aorta proteoglycans in atherosclerosis.

Proteoglycans (PGs) were extracted from minced normal human aorta intima and media and adjacent atherosclerotic plaques. Samples obtained from each individual artery which showed different degrees of atherosclerotic involvement were studied separately. Comparing normal and atherosclerotic areas from the same aorta, the hexuronic acid content was always lower in the atherosclerotic minces. Atherosclerotic samples always contained a higher percentage amount of chondroitinase AC resistant material. PGs were sequentially extracted with increasing guanidine hydrochloride (GuHCl) concentrations. 0.4 M GuHCl extracted about 13% of total PGs, containing mostly chondroitin sulphate (CS), whilst 4 M GuHCl extracted about 50% of total PGs, containing CS, dermatan sulphate (DS), heparan sulphate and hyaluronic acid. PGs from atherosclerotic minces showed a higher DS amount, based on electrophoretic glycosaminoglycan (GAG) analysis. PGs extracted with 4 M GuHCl were further characterized by gel-chromatography and by CsCl density gradient centrifugation. The relative content of PGs with highest hydrodynamic size appeared to be markedly reduced in all the atherosclerotic samples. LDL/GAGs and LDL/PGs interactions were studied by affinity chromatography. GAGs obtained by papain digestion of PGs extracted from atherosclerotic areas contained a glycosaminoglycuronan interacting more strongly with human LDL than GAGs from normal areas of the same artery. The complete elution of PGs required higher NaCl concentration than GAGs. Moreover, PGs from atherosclerotic samples showed higher affinity for LDL than PGs from normal areas of the same aorta.

[Post-heparin lipase activity in beta-thalassemia major: preliminary data]. / Attivita lipasica post-eparinica nella beta-thalassemia major: dati preliminari.

We studied serum lipids and post-heparin triglyceride lipase activities in 8 patients with Beta-Thalassaemia Major, under high transfusion programme and regular chelation therapy and in 8 control subjects. Total cholesterol and HDL-cholesterol were significantly lower in patients with Cooley's anaemia, whereas triglyceride levels did not differ in the two groups. Post-heparin triglyceride lipase activities were determined according with the method of Krauss et A1. using glyceryl-tri-(1-14C)oleate as substrate and NaCl to inactivate the extrahepatic lipase. These enzymatic activities (both hepatic and extrahepatic) resulted significantly lower in thalassaemic patients. We suppose that the decreased levels of these enzymatic activities could play a role in determining the decrease of HDL-cholesterol that we observed in our thalassaemic patients.