Characterization of dermatan sulfate and heparan sulfate in the urine of a patient with the Hunter syndrome

Glycosaminoglycan isolated from the urine of a patient with the Hunter syndrome was composed of heparan sulfate (59.9%), dermatan sulfate (30.6%) and chondroitin sulfate (9.5%), and was heterogeneous in molecular weight (1,500-10,000) and in sulfate content (0.35-2.05 moles/mole of hexosamine). About 60% of dermatan sulfate and 10% of heparan sulfate had molecular weight of 7,000 to 10,000, while about 10% of the former and 60% of the latter had those of 1,500 to 3,500. Sulfate contents of dermatan sulfate and heparan sulfate were inversely related to their molecular weights. Higher total- and N-sulfate contents were measured in smaller molecular-weight heparan sulfate, and higher acetyl content was in larger molecular-weight heparan sulfate. On the basis of the chemical properties of dermatan sulfate and heparan sulfate isolated in this experiment, their catabolic processes in the Hunter syndrome were discussed.     

Bioavailability of Desmin, a low molecular weight dermatan sulfate, affer subcutaneous administration to healthy volunteers

The bioavailability of two different s.c. doses of Desmin (a new low molecular weight dermatan sulfate) was evaluated in 12 healthy volunteers (6 men, 6 women aged 22–45 years) who were injected, on 3 separate days and with a wash-out period of at least 21 days between each administration, with 200 and 300 mg of Desmin by the s.c. route and 200 mg by the i.v. route. Immediately before injection and at various times thereafter (after 15 min and 30 min for i.v. only and after 1, 2, 3, 4, 6, 8, 12, and 24 h for both s.c. and i.v. dosing), blood samples were drawn to investigate bioavailability by measuring several coagulation parameters: activated partial thromboplastin time, thrombin time, inhibition of factor Xa, Heptest, and heparin cofactor II. Furthermore the local tolerance of the s.c. and i.v. injections were investigated. The s.c. administration of the two Desmin doses had a negligible effect on the activated partial thromboplastin time and a very small effect on the thrombin time, measured with human thrombin; in contrast, Heptest, heparin cofactor II, and anti-Xa activities increased, with a good drug bioavailability (more than 100%). The plasma effects of Desmin were dose dependent only when measured by Heptest, which also gave a greater response after the s.c. administrations. There were no symptoms of intolerance or pain at the injection site after single i.v. and s.c. Desmin administrations.     

Separation of dermatan sulfate from heparan sulfate in mucopolysaccharidosis urine by chromatography on Sephadex G-75.

Oligosaccharides of testicular hyaluronidase-degraded dermatan sulfate were separated from undegraded dermatan sulfate by chromatography on Sephadex G-75, but not by chromatography on Sephadex G-25. All but the smallest of these oligosaccharides were recovered in excellent yield following dialysis and precipitation with cetyl pyridinium chloride (CPC). G-75 chromatography of dialyzed, concentrated Hunter urine mucopolysaccharides precipitated with CPC resolved most of the large dermatan sulfate into a void volume related peak which was free of heparan sulfate. Decreasing amounts of dermatan sulfate oligosaccharides were eluted with sephadex-retarded polysaccharides, including small amounts which appeared with otherwise pure heparan sulfate.     

Fibrin moderates the catalytic action of heparin but not that of dermatan sulfate on thrombin inhibition in human plasma

Three recent studies have reported that fibrin in solution significantly inhibits the ability of heparin to catalyze the inhibition of thrombin by antithrombin III. In addition, heparin inhibits the release of fibrinopeptide A by clot-bound thrombin less effectively than it inhibits the release of fibrinopeptide A by thrombin in solution. We have also reported that dermatan sulfate, which catalyzes thrombin inhibition by heparin cofactor II, inhibits thrombus growth in rabbits more effectively than heparin. Because the results of these studies suggest that fibrin inhibits the reactivity of thrombin with antithrombin III-heparin but not with heparin cofactor II-dermatan sulfate, we compared the relative catalytic effects of heparin and dermatan sulfate on thrombin inhibition in plasma, both in the presence and absence of fibrin. We quantitated the rates of thrombin inhibition by antithrombin III and heparin cofactor II by specific enzyme-linked immunosorbent assays. When it was generated, fibrin was kept in solution by adding 2 mmol/L Gly-Pro-Arg-Pro to plasma. Fibrinogen-fibrin reduced the reactivity of thrombin with plasma antithrombin III, both in the presence of and in the absence of heparin. In contrast, the catalytic action of dermatan sulfate on thrombin inhibition by plasma heparin cofactor II was unimpaired by fibrinogen-fibrin. Based on the ability of dermatan sulfate to inhibit thrombus growth in rabbits, failure of fibrinogen-fibrin to moderate the catalytic action of dermatan sulfate may account for its greater antithrombotic effectiveness relative to that of heparin.     

Pharmacologic properties of an unfractionated heparin butyryl derivative with long-lasting effects.

This article reports on the pharmacologic properties of an O-acylated butyryl derivative (C4-UH) of unfractionated heparin (UH). In a purified system, the ability of C4-UH to catalyze the inhibition of thrombin and of factor Xa in the presence of antithrombin III was similar to that of UH. Addition of albumin (10 mg/ml) to the reagents reduced the antithrombin and antifactor Xa catalytic potency of C4-UH 68-fold and 20-fold, respectively, and did not alter those of UH. As judged from the prolongation of the activated partial thromboplastin time and the thrombin clotting time, the anticoagulant activities of C4-UH were two times weaker than those of UH. After calibration against UH, the antifactor Xa-specific and antithrombin-specific activities were two and 6.6 times lower, respectively. After bolus intravenous injection into rabbits, the apparent clearances of C4-UH were reduced 2.4 (antifactor Xa activity) and 3.2 times (antithrombin activity) in comparison with those of UH. This property accounted for the higher plasma concentrations generated during a constant infusion of the same dose. In the Wessler thromboplastin model, the minimum doses providing the maximum antithrombotic effect after bolus injection were equivalent for both compounds when expressed as antifactor Xa units; the duration of the antithrombotic effect of this derivative was prolonged, whereas the hemorrhagic potential was unaffected. This study opens a new concept for heparin derivatives having lower clearances and long-lasting effects. These properties could be linked to nonspecific binding of C4-UH to plasma proteins, thereby reducing the amount of free compound available to interact with antithrombin III.     

Increase of dermatan sulfate in a case of pulmonary fibrosis.

The quantity and composition of acid glycosaminoglycans (mucopolysaccharides) were compared between a fibrotic lung and normal lungs. The acid glycosaminoglycans were isolated by proteolysis, fractionation with ethanol and precipitation with cetylpyridinium chloride, and were identified by chromatography, electrophoresis and incubation with mucopolysaccharide-lyases. Evidence was obtained for the presence of hyaluronic acid, chondroitin sulfate A(C), dermatan sulfate and heparan sulfate. Quantitation of individual glycosaminoglycans by using specific mucopolysaccharide-lyases revealed that the quantity of dermatan sulfate in the fibrotic lung exceeded that in the normal lungs.     

An anticoagulant dermatan sulfate proteoglycan circulates in the pregnant woman and her fetus.

Investigation of the in vitro ability of plasma from pregnant women to inhibit exogenous thrombin (25 nM) demonstrated that heparin cofactor II inhibited more thrombin (3.0 +/- 0.7 nM, mean +/- SD) than plasma from women 3-5 d postpartum (1.9 +/- 0.5 nM) or plasma from nonpregnant adults (1.5 +/- 0.4 nM). Levels of heparin cofactor II were only slightly increased over normal in both pregnant and postpartum women and did not account for the observed increase in thrombin bound to heparin cofactor II. Assay of pregnancy plasma for dermatan sulfate anticoagulant activity demonstrated the presence of activity equivalent to 0.23 +/- 0.02 micrograms/ml of porcine mucosal dermatan sulfate. This activity could not be demonstrated in normal adult plasma or plasma from women on the contraceptive pill. The mass of dermatan sulfate in pregnancy and umbilical cord plasmas was increased over adult control plasma by 0.20 micrograms/ml (53%) and 0.29 micrograms/ml (76%), respectively. The glycosaminoglycan-containing fraction of plasma was isolated and an assay for anticoagulant dermatan sulfate confirmed its presence in both pregnancy and cord plasmas but minimal activity in adult plasma. Gel chromatography of isolated fractions from both pregnancy and cord plasmas revealed a polydisperse, active species with apparent Mr 150,000 D. Reductive elimination decreased the apparent Mr of the active species on gel chromatography to 31,000 D for cord and 21,000 D for pregnancy products. This confirmed the presence of an anticoagulant active dermatan sulfate proteoglycan circulating in the plasmas of pregnant women at term and fetuses at delivery.     

Synthesis of hepatic glycosaminoglycans in the early stages of galactosamine hepatitis: a rapid decline of heparan sulfate is followed by elevation of chondroitin sulfate and dermatan sulfate

Administration of a single dose of D-galactosamine to rats causes time-dependent, biphasic changes of total glycosaminoglycan synthesis in liver. A rapidly occurring inhibition is followed by a significantly enhanced (greater than 2 fold) production of 35S-labeled glycosaminoglycans in later stages of injury. Degree and duration of the inhibitory phase are dose-dependent; 50% inhibition is reached at 80 mg/kg and maximum inhibition (nearly 80%) at about 300 mg/kg body weight 2 h after injection of D-galactosamine. The hepatotoxin impairs preferentially the production of heparan sulfate, whereas that of chondroitin sulfate and dermatan sulfate is diminished only slightly and for a rather short period of time. The synthesis of the latter, however, is more stimulated than that of heparan sulfate in later stages of injury. The specific radioactivity of 35S-labeled 3'-phosphoadenosine-5'-phosphosulfate (PAPS) did not change significantly during the course of acute liver damage. Glycosaminoglycan synthesis in regenerating liver was nearly unaffected by D-galactosamine. Uridine at the dose applied partially reversed D-galactosamine-inhibited synthesis of proteoheparan sulfate. In accordance with the labeling studies the content of glucosamine-containing glycosaminoglycans in treated liver decreased, whereas that of galactosamine-containing glycosaminoglycans slightly increased, resulting in a nearly 50% reduction of the glucosamine/galactosamine ratio 5 h after administration of D-galactosamine. Ion exchange chromatographic studies of 35S-labeled specific types of glycosaminoglycans from normal and galactosamine-injured liver revealed only minor structural differences.     

Purification and structural elucidation of chondroitin sulfate/dermatan sulfate from Atlantic bluefin tuna (Thunnus thynnus) skins and their anticoagulant and ACE inhibitory activities

Chondroitin sulfate/dermatan sulfate (CS/DS) was extracted from Atlantic bluefin tuna (Thunnus thynnus) skin (SGAT) and was purified and characterized. SGAT was characterized by acetate cellulose electrophoresis, FTIR spectroscopy, ¹³C NMR spectroscopy and SAX-HPLC. According to the results obtained for specific chondroitinases (ABC and AC) and the SAX-HPLC separation of generated unsaturated repeating disaccharides, the polymer was found to contain a disaccharide monosulfated in positions 6 and 4 of GalNAc and disulfated disaccharides in different percentages. These results were confirmed by ¹³C NMR experiments. The average molecular mass was 24.07 kDa, as determined by PAGE analysis. SGAT was evaluated for its in vitro anticoagulant activity via activated partial thromboplastin time, thrombin time and prothrombin time tests. The polymer showed strong inhibitory activity against angiotensin I-converting enzyme (IC₅₀ = 0.25 mg mL⁻¹). Overall, the results suggest that this newly extracted CS/DS can be useful for pharmacological applications.

Chondroitin sulfate/dermatan sulfate (CS/DS) was extracted from Atlantic bluefin tuna (Thunnus thynnus) skin (SGAT) and was purified and characterized.     

优化低分子肝素钠注射流程降低经皮冠状动脉介入治疗术后患者皮下出血的发生率

目的 探讨优化低分子肝素钠皮下注射流程对经皮冠状动脉介入治疗(percutaneous coronary intervention,PCI)术后患者皮下出血发生率的影响。 方法 将2015年10月-2017年6月应用低分子肝素钠皮下注射的PCI术后患者97例分为2组,选取2015年10月-2016年7月入院的41例患者为对照组,采用常规注射流程;选取2016年8月-2017年6月入院的56例患者为实验组,采用优化注射流程。比较2组患者的皮下出血情况。 结果 实验组患者的皮下出血程度轻于对照组(Z=-2.907,P=0.004)。 结论 优化低分子肝素钠皮下注射流程可降低患者皮下出血的发生率,保障低分子肝素钠抗血栓效果。     

Thin-layer gel filtration of urinary acid mucopolysaccharides in Hunter''s syndrome and in normal individuals

Acid mucopolysaccharides in urines in Hunter's syndrome and in normal urine were fractionated by column chromatography and an enzymatic method with chondroitinase ABC.

Molecular weight distributions of individual acid mucopolysaccharides were compared with each other by thin-layer gel filtration chromatography on Sephadex G-200, superfine.

Approximate molecular weight of dermatan sulfate in Hunter's urine estimated by thin-layer gel filtration was 12000. Dermatan sulfate isolated from mature rat skin (approx.M.W. 21000) was degraded with testicular hyaluronidase to a material, the molecular weight of which was nearly equal to that of dermatan sulfate in Hunter's urine.

Heparitin sulfate in Hunter's urine was monodisperse and low in molecular weight (approx. 5000). On the contrary, heparitin sulfate in normal urine was extremely polydisperse and heterogeneous. The molecular weight distribution of this material seemed to be much larger than that of other urinary acid mucopolysaccharides.     

Tissue distribution of the low molecular weight heparin, tinzaparin, following administration to rats by the oral route.

Heparins are antithrombotic drugs given by intravenous and subcutaneous routes. However, we have observed that heparins have antithrombotic activity in a rat model when administered orally despite low plasma levels, with low molecular weight heparins (LMWHs) being effective at lower single doses than unfractionated heparins (UFH). Since LMWHs may have other pharmaceutical uses and little is known regarding the pharmacokinetics of oral LMWHs, our objectives were to determine the distribution of the LMWH tinzaparin (Logiparin) following oral dosing. To study distribution at different doses, 0.025-15 mg/kg tinzaparin was given by stomach tube to rats. Gut and non-gut tissues were sampled 4 h later. In a time course study, plasma and tissue samples were collected at eight time points within 24 h after oral administration (60 mg/kg, 4 rats/time interval). Accumulated urine and faeces were collected over 4 and 24 h using metabolic cages. Gut tissue and washes, faeces, urine and non-gut tissue were extracted and analysed for heparin by agarose gel electrophoresis with toluidine blue staining. Activated partial thromboplastin time (APTT) and anti-Xa activity, by Heptest and chromogenic assay, estimated plasma tinzaparin concentrations. Stomach and lung tinzaparin concentrations demonstrated a dose-effect. Peak concentrations in tissue and washes of stomach, duodenum, jejunum, ileum and colon were at 6-30, 15-30, 30 min, 2 and 4 h, respectively. Amounts found at peak times in combined tissue and washes accounted for 46% and 0.5% in stomach (15 min) and colon (4 h), respectively. Tinzaparin was recovered from liver, lung, endothelial samples, and urine at 24 h, but not in faeces. Non-significant increases were seen in APTT and the Heptest, however, anti-Xa activity was significantly greater than control at all times examined, peaking at 2 h. No bleeding was observed. Results are consistent with oral absorption of tinzaparin with wide tissue distribution, likely on endothelium with little in plasma, as previously observed for UFH. Oral administration of LMWHs should be further studied.     

应用低分子肝素对梗死相关血管开通率及维持的影响

目的 评价溶栓前后应用低分子肝素（法安明）对梗死相关血管开通率及维持的影响。方法 将符合溶栓标准的８３例患者分成法安明组（４２例）和对照组（４１例），法安明组溶栓前给予法安明静注，并在溶栓后１２ｈ皮下注射，５０００Ｕ，每天２次，应用２周。对照组溶栓后１２ｈ皮下给予肝素钙７５００Ｕ，每天２次，应用２周。观察两组９０ｍｉｎ造影ＴＩＭＩ３级血流情况及住院期间心脏事件及出血发生率。结果 ９０ｍｉｎ造影梗死     

注射低分子肝素钙后按压与否对皮下出血的影响

目的:探讨注射低分子肝素钙后是否应按压10 min对患者皮下出血及疼痛的影响.方法:采用自身对照法,将62例接受低分子肝素钙治疗的患者以自身肚脐为界,划分为右侧(试验侧)和左侧(对照侧).右侧试验侧注射后不按压,左侧对照侧注射后常规按压10～15 min,患者均使用相同的注射方法总共完成264例次(每人注射次数为3～7次)注射后,评价左右两侧皮下淤血程度及淤血出现的时间.结果:试验侧注射后的皮下出血程度低于对照侧(P＜0.05),注射后是否按压与皮下出血出现时间的早晚无统计学差异(P＞0.05).结论；注射低分子肝素钙后不按压比按压10 min以上可有效控制和减少患者皮下淤血的发生.     

Chemical structure of urinary dermatan sulfate excreted by a patient with the Hunter syndrome

The chemical structure of dermatan sulfate (DS) in the urine of a patient the Hunter syndrome was studied through the analysis of disaccharide units which were derived from the urinary DS by digestion with chondroitinase ABC and separated on a Dowex 1 column. The DS was basically composed of repeating disaccharide units of iduronyl N-acetylgalactosamine 4-sulfate. About 90% of the excess sulfate were linked to the iduronate residues as an additional sulfate group in the unit. N-Acetylgalactosamine 6-sulfate and N-acetylgalactosamine 4,6-disulfate residues were minor components. No non-sulfated disaccharide unit was detected in the digestion products. Only sulfoiduronate residue was found as the non-reducing terminal sugar of the DS molecule, consistent with the lack of iduronosulfate sulfatase in this disease.     

The use of low molecular weight heparins for postsurgical deep vein thrombosis prevention in orthopaedic patients

The prophylactic antithrombotic efficacy of a low molecular weight heparin was compared with a traditional unfractionated calcium heparin after orthopaedic surgery in 140 patients. Deep vein thromboses were detected in legs either by Doppler sonography or [125I]fibrinogen uptake tests in five (7.1%) and seven (10%) patients, respectively. The capacity of both drugs to prevent deep vein thrombosis was demonstrated. Compared with the control group, those who used low molecular weight heparin showed a significant increase of activated factor X inhibition and smaller increases in activated partial thromboplastin times. Tolerability of both drugs was good, with a low incidence of local side-effects.     

Characteristics of urinary glycosaminoglycans excreted by a patient with the Hurler-Scheie compound syndrome

Glycosaminoglycan isolated from urine of a patient with the Hurler-Scheie compound syndrome consisted of dermatan sulfate (60%), heparan sulfate (34%) and chondroitin sulfate (6%). About 60% of both dermatan and chondroitin sulfates had molecular weight 8,000-10,000, while 95% of the heparan sulfate had molecular weight less than 6,000. The total sulfate content of the glycosaminoglycans increased with decrease in molecular weight. N-sulfate content in the heparan sulfate, however, had no relation to molecular weight, and was 0.33 mole per mole of glucosamine on the average. About 70% of the heparan sulfate with the lowest molecular weight (1,500) were composed of three repeating disaccharide units of heparan sulfate and two acetyl, one N-sulfate and three O-sulfate groups linked to the units. The dermatan sulfate contained 1.0-1.2 moles of sulfate per mole of galactosamine. Of the excess sulfate 45-65% were bound to iduronate residues and the rest to C-6 of N-acetylgalactosamine 4-sulfate residues. Most of the dermatan sulfate (83.2-100%) had nonsulfated iduronic acid at the non-reducing end. This finding is consistent with the defect of iduronidase in this disease.     

Ascidian dermatan sulfates attenuate metastasis,inflammation and thrombosis by inhibition of P‐selectin

See also Zacharski LR. Controlling cancer growth from within the blood coagulation mechanism. This issue, pp 1804–6. Summary. Background: Cancer‐associated thrombosis and enduring inflammation are strongly associated with cancer progression and metastasis. Heparin is the mostly clinically used anticoagulant/antithrombotic drug, and has recently been shown to exhibit antimetastatic and anti‐inflammatory activities that are linked to inhibition of P‐selectin and/or L‐selectin. P‐selectin‐mediated platelet–tumor cell and tumor cell–endothelium interactions facilitate the initial steps of metastasis. Objectives and Methods: The aim of the present study was to determine the capacity of dermatan sulfates to inhibit P‐selectin and to test their potential to affect thrombosis, inflammation and metastasis in respective experimental mouse models. Results: Two dermatan sulfates isolated from the ascidians Styela plicata and Phallusia nigra, composed of the same disaccharide core structure (IdoA2‐GalNAc)_n, but sulfated at carbon 4 or 6 of the GalNAc, respectively, have opposed heparin cofactor II (HCII) activities and are potent inhibitors of P‐selectin. The ascidian dermatan sulfates effectively attenuated metastasis of both MC‐38 colon carcinoma and B16‐BL6 melanoma cells and the infiltration of inflammatory cells in a thioglycollate peritonitis mouse model. Moreover, both glycosaminoglycans reduced thrombus size in an FeCl₃‐induced arterial thrombosis model, irrespective of their HCII activities. The analysis of arterial thrombi demonstrated markedly reduced platelet deposition after dermatan sulfate treatment, suggesting that the glycosaminoglycan inhibited P‐selectin and thereby the binding of activated platelets during thrombus formation. Conclusions: Collectively, these findings provide evidence that specific inhibition of P‐selectin represents a potential therapeutic target in thrombosis, inflammation and metastasis, and that ascidian dermatan sulfates may serve as antiselectin agents.     

Gentamicin concentrations in human subcutaneous tissue.

Wound infections frequently originate from the subcutaneous tissue. The effect of gentamicin in subcutaneous tissue has, however, normally been evaluated from concentrations in blood or wound fluid. The aim of the present study was to investigate the pharmacokinetic properties of gentamicin in human subcutaneous adipose tissue by a microdialysis technique. Seven healthy young volunteers each had four microdialysis probes placed in the fat (subcutaneous) layer of the abdominal skin. After the administration of a 240-mg gentamicin intravenous bolus, consecutive measurements of the drug concentrations in serum and subcutaneous interstitial fluid were obtained simultaneously for 6 h. The tissue gentamicin concentration peaked after 10 to 30 min. The peak concentration in the tissue was 6.7 +/- 2.0 mg.liter-1 (standard deviation), equivalent to 39.1% of the peak concentration in serum. The area under the concentration-versus-time curve for the first 6 h in the tissue was 1,281 +/- 390.0) mg.min liter-1, equivalent to 59.7% of the area under the concentration-versus-time curve in serum. It is concluded that the microdialysis technique can be used to make dynamic and quantitative measurements of the gentamicin concentration in human subcutaneous tissue. In this adipose tissue, the peak concentrations of gentamicin were approximately seven times the MIC for Pseudomonas aeruginosa and 33 times the MIC for Staphylococcus aureus after the administration of an intravenous bolus of 240 mg, indicating the presence of sufficient concentrations in the adipose tissue to be effective against common bacteria.     

Hepatic uptake of a modified low molecular weight heparin in rats.

Fractionated and unfractionated heparins are widely used as antithrombotic agents. Because of their heterogeneous composition, it is difficult to study the pharmacokinetics of these drugs. We now report on a new method for labeling low molecular weight heparins with 131I by binding tyramine to the anhydromannose end of the molecules. We examined the pharmacokinetics of the compound by intravenous injection of 131I-tyramine-heparin into Wistar rats. About 18% of the activity was found in the liver, whereas 33% was detected in urine. Biological activity in terms of Factor Xa inhibition was measurable. Since evidence from cell culture experiments implies that reticuloendothelial cell system receptors might be involved in heparin metabolism, maleylated BSA, a substance known to block scavenger receptors, was injected before the radiolabeled heparin compound. The liver uptake was reduced from 17.4 to 4.8%. Injection of unfractionated heparin before tracer application caused a considerable increase in urine excretion of the tracer substance. To our knowledge, this is the first report that liver uptake of heparins is linked to scavenger receptor mediated mechanisms in vivo. This interaction of heparins with scavenger receptors might play an important role in the biology of the vessel wall.