Ana Maria Freire Tovar

Age-Related Changes in Populations of Aortic Glycosaminoglycans: Species With Low Affinity for Plasma Low-Density Lipoproteins, and Not Species With High Affinity, Are Preferentially Affected

Glycosaminoglycans were extracted from the intima and media layers of normal human thoracic aortas from donors of different ages. The arterial segments were devoid of macroscopically visible lesions obtained from patients who had no clinically evident cardiovascular disease. Total glycosaminoglycan content increases during the first 40 years of life. Changes in the content of hyaluronic acid and heparan sulfate are less noticeable. The content of chondroitin sulfate (mainly the 6-isomer) increases, whereas dermatan sulfate remains constant. Plasma LDL-affinity chromatography of dermatan sulfate+chondroitin 4/6-sulfate fractions allowed the separation of LDL high- and low-affinity glycosaminoglycan species. Remarkably, only glycosaminoglycan species with low affinity for plasma LDL increase with age in the disease-free areas of human thoracic aortas studied. These results suggest that age-related changes in glycosaminoglycan composition of the arterial wall do not contribute to increased deposition of plasma LDL. However, the alternative explanation that individuals with arterial glycosaminoglycans that avidly bind LDL would develop early and severe cardiovascular disease and would thus be excluded from our analysis cannot be ruled out.

Human venous and arterial glycosaminoglycans have similar affinity for plasma low-density lipoproteins.

We compared the glycosaminoglycan content of human venous and arterial walls. The most abundant glycosaminoglycan in human veins is dermatan sulfate whereas chondroitin 4/6-sulfate is preponderant in arteries. The concentrations of chondroitin 4/6-sulfate and heparan sulfate are approximately 4.8- and approximately 2.5-fold higher in arteries than in veins whereas dermatan sulfate contents are similar in the two types of blood vessels. Normal and varicose saphenous veins do not differ in their glycosaminoglycan contents. It is known that certain glycosaminoglycan species from the arterial wall, mainly high-molecular-weight fractions of dermatan sulfate+chondroitin 4/6-sulfate have greater affinity for plasma LDL. These types of glycosaminoglycans can be identified on a LDL-affinity column. We now demonstrated that a similar population of glycosaminoglycan also occurs in veins, although with a lower concentration than in the arteries due to less chondroitin 4/6-sulfate with affinity for LDL. The concentrations of dermatan sulfate species, which interact with LDL, are similar in arteries and veins. The presence of these glycosaminoglycans with affinity to plasma LDL in veins raises interesting questions concerning the role of these molecules in the pathogenesis of atherosclerosis. Possibly, the presence of these glycosaminoglycans in the vessel wall are not sufficient to cause retention of LDL and consequently endothelial dysfunction, but may require additional intrinsic factors and/or the hydrodynamic of the blood under the arterial pressure.

Sulfonation and anticoagulant activity of botryosphaeran from Botryosphaeria rhodina MAMB-05 grown on fructose

Botryosphaeran (EPS(FRU)), an exopolysaccharide of the beta-(1-->3,1-->6)-d-glucan type with 31% branching at C-6, is produced by the fungus Botryosphaeria rhodina MAMB-05 when grown on fructose as carbon source. Botryosphaeran was derivatized by sulfonation to induce anticoagulant activity. The effectiveness of the sulfonation reaction by chlorosulfonic acid in pyridine was monitored by the degree of substitution and FT-IR analysis of the sulfonated EPS(FRU) (once sulfonated, EPS(FRUSULF); and re-sulfonated, EPS(FRURESULF)). Activated partial thromboplastin time (APTT) and thrombin time (TT) tests of EPS(FRURESULF) indicated significant in vitro anticoagulant activity that was dose-dependent. EPS(FRU) did not inhibit any of the coagulation tests.

Heparin from bovine intestinal mucosa: Glycans with multiple sulfation patterns and anticoagulant effects

Pharmaceutical grade heparins from porcine intestine and bovine lung consist mainly of repeating tri-sulfated units, of the disaccharide →4-α-IdoA2S-1→4-α-GlcNS6S-1→. Heparin preparations from bovine intestine, in contrast, are more heterogeneous. Nuclear magnetic resonance (NMR) and disaccharide analysis after heparinase digestions show that heparin from bovine intestine contains α-glucosamine with significant substitutive variations: 64% are 6-O-sulfated and N -sulfated, as in porcine intestinal heparin while 36% are 6-desulfated. Desulfated α-iduronic acid units are contained in slightly lower proportions in bovine than in porcine heparin. NMR data also indicate N-, 3- and 6-trisulfated α-glucosamine (lower proportions) and α-GlcNS-1→4-α-GlcA and α-IdoA2S-1→4-α-GlcNAc (higher amounts) in bovine than in porcine heparin. Porcine and bovine heparins can be fractionated by anion exchange chromatography into three fractions containing different substitutions on the α-glucosamine units. Each individual fraction shows close disaccharide composition and anticoagulant activity, regardless of their origin (bovine or porcine intestine). However, these two heparins differ markedly in the proportions of the three fractions. Interestingly, fractions with the typical heparin disaccharides of porcine intestine are present in bovine intestinal heparin. These fractions contain high in vitro anticoagulant activity, reduced antithrombotic effect and high bleeding tendency. These observations indicate that the prediction of haemostatic effects of heparin preparations cannot rely exclusively on structural analysis and anticoagulant assays in vitro . Minor structural components may account for variations on in vivo effects. In conclusion, we suggest that pharmaceutical grade bovine intestinal heparin, even after purification procedures, is not an equivalent drug to porcine intestinal heparin.

Structural and functional analyses of bovine and porcine intestinal heparins confirm they are different drugs

Anticoagulant heparins are mostly obtained from porcine intestine. Occasionally they are also obtained from bovine intestine. Structural and functional analyses of pharmaceutical-grade heparins from these two sources using multiple methods such as NMR spectroscopy, in vitro and in vivo assays of the anticoagulant, antithrombotic and bleeding effects, complemented by fractionation on anion exchange chromatography, confirm they are different drugs. Although bovine heparin is more heterogeneous and less sulfated, heparins from both sources are overall made of a similar mixture of fractions, however with different proportions. Therefore, high-anticoagulant composites from bovine origin, similar to porcine counterparts, can be properly obtained.

Structural and haemostatic features of pharmaceutical heparins from different animal sources: challenges to define thresholds separating distinct drugs

Heparins extracted from different animal sources have been conventionally considered effective anticoagulant and antithrombotic agents despite of their pharmacological dissimilarities. We performed herein a systematic analysis on the physicochemical properties, disaccharide composition, in vitro anticoagulant potency and in vivo antithrombotic and bleeding effects of several batches of pharmaceutical grade heparins obtained from porcine intestine, bovine intestine and bovine lung. Each of these three heparin types unambiguously presented differences in their chemical structures, physicochemical properties and/or haemostatic effects. We also prepared derivatives of these heparins with similar molecular weight differing exclusively in their disaccharide composition. The derivatives from porcine intestinal and bovine lung heparins were structurally more similar with each other and hence presented close anticoagulant activities whereas the derivative from bovine intestinal heparin had a higher proportion of 6-desulfated α-glucosamine units and about half anticoagulant activity. Our findings reasonably indicate that pharmaceutical preparations of heparin from different animal sources constitute distinct drugs, thus requiring specific regulatory rules and therapeutic evaluations.

Heparan sulfates from arteries and veins differ in their antithrombin-mediated anticoagulant activity.

Because he moved to another city a few weeks after hospital discharge, a repeat TG assay was not possible. A telephone interview conducted 6 months later revealed that he did not experience any clinically obvious clotting abnormality again. In our patient, treatment with IVIG led to a normalization of hemostasis as confirmed by the TG assay, the disappearance of FV inhibitor, and the rise in FV activity. IVIGwas given on the basis of its reported efficacy in patients with acquired hemophilia A.However, a spontaneous recovery cannot be ruled out withcertainty.Theamountof thrombingenerated, the lagphase and the time to thrombin peak are influenced by FV, as FV is part of every step in the process of TG [4]. Platelet FV rather than plasma FV plays a significant role in the amplification phase of coagulation. FV inhibitor leads to an impaired initiation phase, and this in turn results in impaired platelet activation. On the other hand, FV on the surface of activated platelets becomes neutraliszed by FV inhibitor, which leads to a reduction in prothrombinase activity and thrombin burst. The amount of FV needed to produce an adequate thrombin burst is still unclear. In our patient, minimal changes in FV activity from 7.4% to 8.6% led to a considerable increase in thrombin formation. Higher concentrations of FV did not result in any further increase in ETP, but did result in further reductions in the lag phase and the time to peak. Al Dieri et al. [5] observed no severe impairment in ETP at FV levels of 2% in patients with congenital FV deficiency. However, they used much higher TF concentrations (15 pM) in their assay. Therefore, their data are hardly comparable to ours. Furthermore, a measurable amount of the coagulation factor in the presence of an acquired autologous inhibitor may not necessarily correlate with its hemostatic function. Studies in patients with acquired autoantibody hemophilia A have shown that the inactivation has a type II pattern, with some residual FVIII activity being identifiable even after incubation at high concentrations of antibody. The presence of the inhibitor rather than the measurable coagulation factor activity is thus important in assessing the hemostatic state. In conclusion, the TG assay could be useful for monitoring hemostatic changes in patients with FV deficiency due to an inhibitor. However, there are issues of standardization to be dealt with. Clinical correlation with TG cut-off values is also lacking. These issues have to be addressed before the assay can be used in clinical routine practice.

High affinity of a fucosylated chondroitin sulfate for plasma low density lipoprotein.

Factors that influence the binding of sulfated polysaccharides to plasma low density lipoprotein (LDL) were investigated. Among the naturally occurring polysaccharides tested, a fucosylated chondroitin sulfate from an echinoderm exhibited the strongest interaction with LDL. Defucosylation and desulfation totally abolished the interaction with LDL while reduction of carboxyl groups had little effect. These data indicate that the sulfated fucose branches are essential for binding of fucosylated chondroitin sulfate to LDL. In addition, there was a positive correlation between the binding to LDL and increasing length of the sulfated polysaccharide chains. The possibility of a practical use of this fucosylated chondroitin sulfate for the binding of LDL is discussed.

Lipid composition and catalytic properties of sarcoplasmic reticulum from normal and dystrophic chicken muscle

SummaryThe lipid composition of sarcoplasmic reticulum vesicles isolated from the pectoralis muscle of dystrophic chickens (line 413) and of the genetically related line of normal chickens (line 412) were studied. The total lipid content in the dystrophic preparation was increased by 12% when compared to the normal. The increase was restricted to the neutral lipids in the membrane, with a significant increase of 25% in the cholesterol and of 23% in the triacylglycerol contents. Identical phospholipid composition and content was found. Most of the fatty acid acyl chains from the phospholipids were similar, with an increase in the degree of saturation of phosphatidylcholine, phosphatidylserine and phosphatidylinositol. Similar profiles of the fatty acid acyl chains from triacylglycerol were found. The affinities of the ATPase enzyme for ATP and Ca2+ were similar in the normal and dystrophic preparations. The K0.5 for ATP in both preparations was 28 µM for the Mg2+-dependent activity, and 5–8 µM for the Ca2+-dependent activity which exhibited a secondary increase in the velocity of hydrolysis, in the millimolar ATP concentration range. The K0.5 for Ca2+ in both preparations was approximately 0.3 µM.

Systematic Analysis of Pharmaceutical Preparations of Chondroitin Sulfate Combined with Glucosamine

Glycosaminoglycans are carbohydrate-based compounds widely employed as nutraceuticals or prescribed drugs. Oral formulations of chondroitin sulfate combined with glucosamine sulfate have been increasingly used to treat the symptoms of osteoarthritis and osteoarthrosis. The chondroitin sulfate of these combinations can be obtained from shark or bovine cartilages and hence presents differences regarding the proportions of 4- and 6-sulfated N-acetyl β-d-galactosamine units. Herein, we proposed a systematic protocol to assess pharmaceutical batches of this combination drug. Chemical analyses on the amounts of chondroitin sulfate and glucosamine in the batches were in accordance with those declared by the manufacturers. Anion-exchange chromatography has proven more effective than electrophoresis to determine the type of chondroitin sulfate present in the combinations and to detect the presence of keratan sulfate, a common contaminant found in batches prepared with shark chondroitin sulfate. 1D NMR spectra revealed the presence of non-sulfated instead of sulfated glucosamine in the formulations and thus in disagreement with the claims declared on the label. Moreover, 1D and 2D NMR analyses allowed a precise determination on the chemical structures of the chondroitin sulfate present in the formulations. The set of analytical tools suggested here could be useful as guidelines to improve the quality of this medication.