Heparitin Sulfate Research Articles

The hexosaminohexuronide of the bovine lens capsule

Two fractions of a purified glucosaminoglucuronide, one soluble, the other insoluble in water, were obtained from bovine lens capsules by extraction with 80% ethanol containing 5% KOH at room temperature and purified by repeated reprecipitation with 60% acetone. The two fractions represented about 80% of the total hexuronide of the lens capsule, and on the basis of their extinction coefficients in four different color reactions, paper chromatography of their hydrolyzate with Dowex 50-H +, and optical rotation, appeared to be identical or closely related to heparitin sulfate.

Acid mucopolysaccharides of human aorta: Part 1. Variations with maturation

Summary Acid mucopolysaccharide (MPS) contents in the intima and remaining outer wall of human aortas were studied to observe variations with age. Attempts were made to dissociate changes occurring with aging from those related to atherogenesis by the careful selection of vessels. MPS were isolated from 76 female aortas with and without lesions, and compared to 15 samples from Negro females, the only ones available essentially free of atherosclerotic disease. Aortas available in this population limits the study from extending into older age groups. Detailed MPS analyses were carried out on the lesion-free vessels. The total amount of MPS increases to around the third decade, with more marked changes of compounds occurring in the intima. Chondroitin sulfates C and B both increased in concentration in the intima and then decreased; chondroitin sulfate A progressively increased, while hyaluronic acid and heparitin sulfate showed a tendency to reach peak levels at an early age and then only gradually decreased. Other compounds were noted, but were not in sufficient amount to be clearly characterized. The importance of observing changes of each specific MPS compound and a careful selection of tissue to be studied are to be emphasized in these types of observations.

Enzymic degradation of ω-heparin (Whale heparin)

ω-Heparin isolated from whale organs was digested with crude heparinase from heparin-adapted flavobacteria and also with an eliminase separated from the crude enzyme. ω-Heparin was degraded, by the crude heparinase, to the three major monosaccharides, an α-keto acid, N-sulfated glucosamine, and N-, O-disulfated glucosamine. A minor products, N-acetylglucosamine and an N-acetylglucosamine-containing unsaturated oligosaccharide were also found in the digest. The eliminate digestion produced several unsaturated oligosaccharides. Two were indicated to contain unsaturated uronic acid and N-N-acetylglucosamine with and without O-sulfate. These products were similar to those found in the degradation products of α-heparin and heparitin sulfate with these enzymes. In addition, a study of the temperature optimum and the effects of inhibitors on the degradation of ω-heparin with the eliminase showed ω-heparin to be intermediary between α-heparin and heparin sulfate. Moreover, the unsaturated oligosaccharides produced and the changes in the reducing values after the action of these enzymes indicated that ω-heparin was not a mixture of α-heparin and heparin sulfate, but a complex polysaccharide consisting of an N-acetylglucosamine-containing polysaccharide to which α-heparin-type polyssacharides are attached.

Comparison of heparin specimens isolated from bovine, porcine, and whale organs

The heparin preparations isolated by the same procedure from lung and intestine of cattle, pig, and whale have been investigated to verify the difference of whale heparin from that of other terrestrial mammalia. The analytical data, electrophoretic mobilities, nuclear magnetic resonance (NMR) spectra, optical rotatory dispersion (ORD) spectra, sedimentation figures in ultracentrifugation, and molecular weight of these compounds confirmed that the terrestrial mammalia have α-heparin, and whale, a marine mammal, ω-heparin. Although the fine structure and location of Osulfate groups of ω-heparin have not yet been established, ω-heparin was proved again to be different from α-heparin and also heparitin sulfate. No organ specificity, as far as lung and intestine concerned, was found. Heparin samples obtained from lung and intestine of whale had much less sulfur (sulfate), but higher dextrorotation than those from cattle and pig. All of the sulfate groups of these compounds were found to be titratable. The whale heparin contained N-acetylglucosamine (approximately one-fourth of the total glucosamine) as in integral component of the compound. Whale heparin (ω-heparin) was eluted as two major fractions (1.25 M Fr. and 1.5 M Fr.) by ion-exchange (Dowex-1) chromatography, while the others (α-heparin) as mainly one fraction (2.0 M Fr.) with aqueous sodium chloride. But the two major fractions of ω-heparin were shown to be identical in chemical, physical, and biological properties except for minor differences of sulfur and acetyl contents. NMR-spectra of these compounds and heparitin sulfate indicated that all of these materials have α-linkages and glucosamine 6-sulfates. ORD-spectra of the heparins showed weak positive Cotton effects at 225 mμ, while chondroitin sulfates clear negative Cotton effects at 220 mμ.

Distribution and composition of acid mucopolysaccharides in normal and atherosclerotic human aortas

The relationship between tissue concentration and composition of the acid mucopolysaccharides (acid MPS) of the human aorta and the stage of atherosclerosis and/or age has been examined. Acid MPS have been analysed in the intima, the media, and excised atherosclerotic lesions from aortas at different stages of atherosclerosis. The analyses comprised the determination of the total acid MPS; the acid MPS that are digestible by testicular hyaluronidase, i.e. hyaluronic acid (HA) plus chondroitin sulphate C (CSA-C); the hyaluronidase-resistant MPS, i.e. heparitin sulphate (HS) plus chondroitin sulphate B (CSA-B), and the four components separately. The concentrations of total acid MPS (per dry defatted weight) vary according to kind of tissue and lesions, but show little correlation with the stage of atherosclerosis. The concentrations of resistant acid MPS are highest in the fatty streaks and spots and lowest in the ulcerated lesions. The concentration of HA decreases with increasing stage of atherosclerosis and/or ageing; that of HS shows a maximum at stage I; that of CSA-C is maximal at II; no correlation was found between CSA-B concentration and stage of the disease. A decreased permeability of the arterial wall and a decreased fibrinolytic activity consequent upon these changes in MPS composition are seen as possible factors in the development of atherosclerotic plaques.

Separation of glycosaminoglycans on thin layers of silica gel

Mixtures of chondroitin sulfates A, B, and C, plus keratosulfate, heparitin sulfate, and hyaluronic acid have been resolved on thin layers of plain silica gel. Glycosaminoglycans in 1 μg amounts can be readily separated and visualized.

Methods for the separation of acid mucopolysaccharides of the human aorta

Various workers have obtained inconsistent and variable results in attempting to separate the four main components of acid mucopolysaccharides of the human aorta (hyaluronic acid, heparitin sulphate, chondroitin sulphuric acid B and chon-droitin sulphuric acid C) by precipitation with aqueous alcohol mixtures or chromatography on ion-exchange resins. Evidence is presented which suggests that these difficulties are due to physico-chemical interaction between the long-chain macromolecules. A method is described whereby aorta mucopolysaccharide composition can be reproducibly determined by a preliminary degradation of two components with hyaluronidase, separation of the products by gel filtration, and ion-exchange chromatography of the resultant fractions.

Acid mucopolysaccharides of amyloid tissue

The acid mucopolysaccharides (MPS) were isolated from different organs from patients with primary and secondary amyloidosis, and the MPS composition of these tissues was compared with those isolated from control individuals. Although each organ contains different amounts and types of MPS, amyloid tissue in general shows an increase in total MPS, primarily due to heparitin sulfate. In the myocardium involved in primary (familial) amyloidosis (only one sample available for study), the increase in MPS is apparently due to hyaluronic acid, in contrast to liver, spleen, kidney, and heart tissue involved in secondary amyloidosis. A slight increase in chondroitin sulfate B was also noted in certain secondary amyloid tissues. The amino acid composition of peptide material bound to heparitin sulfate was determined, and no abnormality was observed from the similar MPS-protein complex isolated from control tissue.

Difference in acid mucopolysaccharides contents among aortas, carotid and cerebral arteries: A preliminary note

To examine the changes in acid mucopolysaccharide (AMPS) of a small sample of artery, we adapted the existing technique. The metachromatic bands of AMPS separated electrophoretically on cellulose acetate strips were dissolved in N,N-dimethylformamide and its colour density was measured spectrophotometrically. By use of this technique, we described as a preliminary study that the contents of hyaluronic acid and chondroitin sulfate were low in cerebral artery, and that the cerebral and internal carotid arteries contained less total acid mucopolysaccharides, particularly, chondroitin sulfates, and rather higher heparitin sulfate compared with the carotid sinus, common carotid artery and aorta.

Observations on the basophilia of amyloids.

Uptake of Alcian Blue by amyloids in various human tissues was usually negligible in paraffin sections stained at pH 2.6, but was considerable in sections stained at pH 5.7 in magnesium chloride. Amyloids took up toluidine blue at pH 5.7 and were then birefringent in polarised light. The “critical electrolyte concentratons” in magnesium chloride at pH 5.7 at which Alcian blue ceased to stain indicate that the polyanion(s) of amyloids contain both ester sulfate and non-ester sulfate ionic groups, presumably carboxyls. Other evidence supports this interpretation. The “critical electrolyte concentration” pattern is more compatible with the presence of heparitin sulphate than with any other sulphated polysaccharide. The presence of sialoprotein is not excluded. Masking of polyanions in amyloids suggests strongly that a polycation, probably a protein, is present. The behaviour of a series of polyanion-polycation complexes in a range of pH and salt concentrations has been studied. Congo red, an acid dye, stained polycations under the conditions used to stain amyloids. It is proposed that amyloids contain insoluble complexes formed by the interaction of protein with polyanion; in this case of a carbohydrate nature, and usually sulfated.

Modification of urinary mucopolysaccharide excretion in patients with Hurler's syndrome.

Several variants of Hurler's syndrome can be distinguished both genetically and biochemically. All these patients excrete excess acid mucopolysaccharides in their urine, but the ratio of chondroitin sulfate B to heparitin sulfate varies in different families. When the patients are in a basic condition and are maintained on a constant diet, the amount of 24 hour urinary acid mucopolysaccharides and the ratio of chondroitin sulfate B to heparitin sulfate show little daily fluctuation. Seven children with Hurler's syndrome were studied in their basic state and consecutively while receiving corticosteroids, thyroid, human growth hormone, hydroxychloroquine, and salicylates. It was demonstrated that administration of hydroxychloroquine increases urinary excretion of acid mucopolysaccharides, while prednisone tends to decrease the excretion of urinary acid mucopolysaccharides. Thyroid and corticosteroids produced marked changes in the ratio of chondroitin sulfate B to heparitin sulfate, especially in patients who excreted predominantly heparitin sulfate in their basic state. Salicylates and human growth hormone did not induce significant changes in the pattern of urinary excretion of acid mucopolysaccharides.

Heparin metabolism: isolation and characterization of uroheparin.

THE naturally occurring mucopolysaccharides (MPS) of mammals include the chondroitin sulphates, hyaluronic acid, heparitin sulphate, keratosulphate, chondroitin, and heparin. Most are located extracellularly, primarily in connective tissue. Only heparin is stored intracellularly, chiefly in mast cells and basophils. Little is known of its metabolism in humans, although rat tissues rapidly take it up after injection1. Although in dogs small amounts of circulating heparin bound to protein have been reported2, circulating extracellular heparin has not been demonstrated in humans and several attempts to detect heparin excretion in normal human urine have so far been unsuccessful.

Isolation of heparitin sulfate from skin of normal rats

Isolation of heparitin sulfate from skin of normal rats

Acid hydrolysis of acidic mucopolysaccharides

The hydrolysis of mucopolysaccharides with dilute hydrochloric acid was studied. Glycosidic bond cleavage of the β- D- and α- L-linked mucopolysaccharides occurred with unexpected facility and produced partially sulfated and N-acetylated disaccharides as well as higher oligosaccharides and smaller proportions of monosaccharides. These products were isolated by means of gel filtration with Sephadex G-25. The α- D-linked substances, heparin, N-acetylheparin, and heparitin sulfate, remained relatively resistant to hydrolysis under the conditions used although N-deacetylation occurred more readily with N-acetylheparin than with the other polysaccharides examined. Oligosaccharides of chondroitin 4- and 6-sulfate were found to undergo hydrolysis readily to N-acetylated and partially sulfated disaccharides.

Effects of preparations of heparin and heparitin sulfate on fibrinolysis

Some preparations of heparin and heparitin sulfate were found to be contaminated with protease inhibitors. By column chromatography two different inhibitors, both of an acid type, could be demonstrated. Inhibitor-free heparin preparations exerted an enhancing effect on fibrinolysis caused by plasminogen activators. Inhibitor-free heparitin sulfate had no enhancing effect. It is believed that these findings explain the discrepant earlier reports on the effects of heparin on fibrinolysis.

A new ion association-fractionation technique for identifying individual mucopolysaccharides and other polyanions in tissue sections.

A new histochemical ions association-fractionation technique has been developed for identifying individual mucopolysaccharides and other biological polyanions in tissue sections. This method is based on the differential precipitation of individual polyanions with quaternary ammonium salts in solutions of specific ionic concentration and the subsequent formation of red ion association complexes with the bound quaternary moieties. Hyaluronic acid, the chondroitin sulfates, heparitin sulfate, heparin and RNA were readily identified. Further differentiation was effected by incorporating acetone-dried powder extracts from Flarobacterium heparinum which has been adapted to chondroitin sulfate and heparin.

Separation and identification of acid polysaccharides by thin-layer chromatography.

Thin-layer chromatographic procedure are described for the separation and identificationn of a number of acid polysaccharides. With the present methods, chondroitin sulfates A and/or C, chondroitin sulfate B, keratosulfate, and heparitin sulfate and/or heparin may be distinguished among the normally occurring known sulfated APS. A number of polysaccharides, as yet unclassified, are also revealed by these procedures.

Structure of heparitin sulfate in tissues of the Hurler syndrome

Structure of heparitin sulfate in tissues of the Hurler syndrome

Electroretinography and Fundus Oculi Findings in Hurler's Disease and Allied Mucopolysaccharidoses

Introduction It has become apparent in recent years that Hurler's syndrome (gargoylism) is not a single entity but is one of a spectrum of genetically-determined biochemical disorders. A major advance in understanding and differentiating the condition occurred with the discovery by Dorfman and Lorencz8in 1957, and Meyer21in 1958, of increased mucopolysaccharides (MPS's) in the urine of patients with Hurler's disease. Meyer suggested that abnormally elevated amounts of mucopolysaccharides, chondroitin sulfate B, and heparitin sulfate, result from a genetic abnormality in the differentiation of the fibroblasts which produce these substances. There is an alternate theory of faulty binding of mucopolysaccharides to protein because of an abnormality in the latter.9 Detailed qualitative and quantitative examination of the urinary MPS's is necessary for accurate diagnosis and classification.18On clinical, biochemical, and genetic grounds, McKusick22has grouped the mucopolysaccharide diseases as follows: MPS 1.—The autosomal-recessive Hurler's syndrome

The Enzymatic Degradation of Heparin and Heparitin Sulfate: I. THE FRACTIONATION OF A CRUDE HEPARINASE FROM FLAVOBACTERIA

The Enzymatic Degradation of Heparin and Heparitin Sulfate: I. THE FRACTIONATION OF A CRUDE HEPARINASE FROM FLAVOBACTERIA