Content and synthesis of glycosaminoglycans in the developing lung.

The function of lung is fundamentally linked to the connective tissue composition of the alveolar interstitium. The composition and synthesis of one class of interstitial connective tissue components, the glycosaminoglycans (GAG), was determined in lung parenchyma of rabbits at different stages of development. Parenchymal GAG content ranged between 0.2 and 0.4% (wt/wt) of dry weight, with highest concentration in adult lung. There were significant changes in types of GAG present at different ages. Fetal lungs contained a relatively high proportion of chondroitin 4-sulfate while the GAG in lung parenchyma of older animals was predominantly dermatan sulfate, heparan sulfate, and heparin. Methods were developed for the study of rates of synthesis of GAG by incorporation of [1-14C]glucosamine into lung explants. The rate of synthesis of total GAG per cell increased with development to a maximum in lung from weanling rabbits and fell to low rates of synthesis in mature rabbits. Fetal rabbit lung parenchyma synthesized mostly hyaluronic acid and heparan sulfate, while in weanling rabbit parenchyma hyaluronic acid and chondroitin 4/6-sulfate synthesis was greatest. In mature animals, the rates of synthesis of all types of GAG were relatively low but there was a relatively greater emphasis on synthesis of dermatan sulfate and heparin. These results may have significance in changes in lung function during development and in effects on other connective tissue components.     

Glycosaminoglycans of the hemodialysis-associated carpal synovial amyloid and of amyloid-rich tissues and fibrils of heart, liver, and spleen

Significant amounts of glycosaminoglycans (GAGs) were found in amyloid fibril preparations. Using two-dimensional electrophoresis to fractionate GAG mixtures, we quantified and identified for the first time the GAGs of the fibrils from carpal synovium of patients with amyloid associated with chronic hemodialysis. The total GAG content was small, but the GAG distribution (high relative content of chondroitin sulfate and hyaluronic acid and lack of the other GAGs) was unique, unlike that for the other amyloid fibril preparations. The amyloid-rich heart, liver, and spleen tissues, as well as the fibrils isolated from these tissues of patients with systemic forms (primary amyloid and secondary amyloid) of amyloid disease, were also analyzed for GAGs. Fibrils from heart tissue of a patient with primary amyloidosis, now examined for the first time, contained four major GAGs (chondroitin sulfate, dermatan sulfate, hyaluronic acid, and heparan sulfate).     

A longitudinal evaluation of urinary glycosaminoglycan excretion in normoalbuminuric type 1 diabetic patients.

BACKGROUND: Previously, we found high urinary glycosaminoglycan (GAG) concentration, together with an altered electrophoretic pattern, in normoalbuminuric type 1 diabetic subjects with hemoglobin A(1c) (HbA(1c)) > or =8.0%. The purpose of this study was long-term evaluation of GAG excretion variations in these patients compared to those with HbA(1c) < 8.0% at baseline who maintained better metabolic control over time. METHODS: We enrolled 26 normotensive, normoalbuminuric type 1 diabetic patients and divided them into two groups according to mean HbA(1c) levels during the follow-up period. GAGs were isolated from 24-h urine samples on two separate occasions, at baseline and after a mean (+/-SD) follow-up of 6.8+/-1.1 years. RESULTS: All patients remained normoalbuminuric at follow-up, and had normal urinary alpha(1)-microglobulin levels. In patients with HbA(1c) <8.0%, total GAG levels and low sulfated chondroitin sulfate-proteoglycan/chondroitin sulfate ratio were almost unchanged during the follow-up period. In contrast, these increased in patients with HbA(1c) > or =8.0% and were significantly related to both HbA(1c) levels and the duration of poor glycemic control. CONCLUSIONS: Our results show a strong influence of hyperglycemic environment on GAG metabolism in diabetes and indicate that the distribution pattern of urinary GAGs, besides their total concentration, may be predictive of altered GAG metabolism in type 1 diabetes.     

The Glycosaminoglycans of Normal and Arthritic Cartilage

The cartilages from the hip joints of 13 normal and 15 osteoarthritic humans were analyzed for glycosaminoglycan content and distribution. The GAGs were separated by elution with CPC on a short cellulose column by the technique of Svejcar and Robertson after digestion of the tissue with pronase and papain. The eluates were identified by a variety of methods including determination of molar ratios, N-acetyl-hexosamine determinations after hyaluronidase treatment and thin-layer chromatography of unhydrolyzed and hydrolyzed GAGs.From the data obtained, it was demonstrated that cartilage from arthritic patients showed a significant increase in the concentration of chondroitin 4-sulfate and a significant decrease in keratan sulfate, with only slight changes in the total amount of GAG present. Calculations of the molar ratios showed variation in the sulfation with chondroitin 4-sulfate appearing in the "supersulfated" state in the arthritic cartilage.The data lead to speculation regarding the process of osteoarthritis, and it is concluded that the changes seen are more likely to represent an altered pattern of synthesis rather than selective degradation. Since the changes suggest a younger cartilage, a theory is advanced that the chondrocyte responds to the chronic stress of osteoarthritis by modulation to a chondroblastic phase.     

Increased excretion of urinary glycosaminoglycans in meningococcal septicemia and their relationship to proteinuria

OBJECTIVES: Meningococcal septic shock is a devastating illness associated with an increase in vascular permeability leading to hypovolemia and accumulation of plasma proteins and fluid in tissues. The capillary leak syndrome is often associated with widespread thrombosis in the skin, limbs, and digits. We postulated that the increase in vascular permeability and the intravascular thrombosis might be caused by an inflammation-induced loss of endothelial and basement membrane glycosaminoglycans (GAGs), which play a role in the permeability and thromboresistant properties of the microvasculature. DESIGN: Prospective, single-center observational study. SETTING: University-affiliated meningococcal research unit and pediatric intensive care unit. PATIENTS: Eighteen children requiring intensive care for meningococcal sepsis, 18 children with steroid-responsive nephrotic syndrome, and 18 healthy control children. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Serum concentrations and urine excretion of glycosaminoglycans were measured and related to changes in glomerular permeability to plasma proteins. The size-distribution and nature of glycosaminoglycans were defined by Polyacrylamide Gel Electrophoresis and specific enzyme digestion. Urinary excretion of heparan sulfate, chondroitin-4-sulfate, and chondroitin-6-sulfate were significantly increased in meningococcal disease (MD) relative to healthy controls and children with steroid-responsive nephrotic syndrome. The urinary GAGs in MD were of similar size to those in controls when analyzed after pronase digestion. However, analysis of proteoglycan size before proteolytic digestion showed the urinary GAGs in MD were of lower molecular weight and unattached to proteins. The fractional excretion of albumin and immunoglobulin G in MD increased with severity of disease. Patients with severe or fatal MD had albumin clearances overlapping those seen in steroid-responsive nephrotic syndrome. There was a significant correlation between proteinuria in MD and urinary excretion of heparan sulfate (r2 = 0.611, p < .0001), chondroitin-4-sulfate (r2 = 0.721, p < .0001), and chondroitin-6-sulfate (r2 = 0.395, p < .0001). CONCLUSIONS: The capillary leak in meningococcal disease is associated with increased plasma and urine concentrations of GAGs, which may be proteolytically cleaved from endothelial and basement membrane sites. The correlation between the severity of protein leakage and the urine excretion of GAGs suggests that loss of GAGs may be causally related to the increase in permeability to proteins.     

Enzymatic determination of urinary glycosaminoglycans from orthopedic patients

Crude glycosaminoglycan (GAG) fraction was directly precipitated with cetylpyridinium chloride without prior dialysis of urine of orthopedic patients. The crude GAG fraction was then fractionated with trichloroacetic acid (TCA). The TCA-insoluble peptide-bound GAG fraction thus obtained was treated with alkali to eliminate the peptide moiety for enzymatic analysis. The GAG compositions of this fraction and the TCA-soluble fraction were determined by digestion with mucopolysaccharidases (chondroitinase AC, chondroitinase B, chondroitinase C, heparitinase and Streptomyces hyaluronidase). When the amount of the crude GAG fraction was small, no significant amount of the TCA-insoluble peptide-bound GAG fraction was obtained. The GAG composition of this case was also determined by the same procedures after direct alkali-treatment of the crude GAG fraction. The data indicated that the proportion of the TCA-insoluble peptide-bound GAG fraction was very small. The alkali-treated TCA-insoluble peptide-bound GAG fraction contained a larger proportion of heparan sulfate than the TCA-soluble GAG fraction. It was clearly demonstrated that the patients with Werner's syndrome and mucopolysaccharidosis I-S (Scheie) excreted large amounts of hyaluronic acid and dermatan sulfate respectively, into urines. It was indicated in most cases that major urinary GAG were chondroitin 4-sulfate, chondroitin 6-sulfate plus chondroitin and heparan sulfate, while minor ones were dermatan sulfate and hyaluronic acid. In addition, the data suggested a wide range of the degree of desulfation or urinary GAG, and the presence of significant amounts of keratan sulfate plus acidic glycopeptides in the urinary GAG fractions. The present data provided more precise information on urinary GAG from orthopedic patients than those reported previously.     

Fine characterization of mitral valve glycosaminoglycans and their modification with degenerative disease.

BACKGROUND: The levels and fine structure of complex polysaccharides, glycosaminoglycans (GAGs), were determined in segments of the posterior mitral valve leaflet (MVL) taken from 15 patients affected by mitral regurgitation and degenerative disease and were compared with segments from 15 multiorgan donors. METHODS: MVL GAGs were analyzed by agarose gel electrophoresis, and by HPLC and fluorophore-assisted carbohydrate electrophoresis to evaluate disaccharide patterns after treatment with chondroitinase ABC. RESULTS: GAGs from the control group were composed of approximately 37% hyaluronic acid and 63% chondroitin sulfate/dermatan sulfate with a charge density of approximately 0.61. Chondroitin sulfate/dermatan sulfate polymers contained approximately 23% of the disaccharide sulfated in position 6 on N-acetyl-galactosamine, approximately 38% of the 4-sulfated disaccharide and approximately 2% of the non-sulfated disaccharide (with a 4-sulfated/6-sulfated ratio of 1.7). The total amount of GAGs was 0.66 microg/mg tissue. The total amount of GAGs in patients suffering from mitral regurgitation and degenerative disease was approximately 51.5% higher (although the difference was not significant, probably because of the low number of subjects enrolled in the study). However, significantly higher hyaluronic acid content (approx. +38%, p<0.05) and lower sulfated GAG content (approx. -21%, p<0.005) were demonstrated. As a consequence, the total charge density decreased by approximately 23% (p<0.005). This macro-modification of GAG composition was also followed by a micro-alteration of the structure of the sulfated polysaccharides, in particular with a significant decrease in the 4-sulfated disaccharide (and a parallel increase in hyaluronic acid content) with no modification of the percentage of the 6-sulfated and non-sulfated disaccharides (with a significant decrease in the 4-/6-sulfated ratio). CONCLUSIONS: We assume that changes in the relative amount and distribution of GAGs in posterior MVL in subjects suffering from mitral regurgitation and degenerative disease are consistent with a decrease in the tension to which these tissues are subjected and with an abnormal matrix microstructure capable of influencing the hydration and of conditioning the mechanical weakness of these pathological tissues.     

Effects of cryopreservation and deconstruction on the dermal glycosaminoglycan content of human skin.

Since the concept of a fabricated skin replacement was first proposed, it has been recognized that a permanent skin replacement must contain a functional complex structure consisting of epidermis integrated with dermis. Although a practical solution for the replacement of missing epidermis exists through the culture expansion of the autologous epidermis, a practical solution for permanently replacing missing dermis has not been achieved. While it is generally recognized that the insoluble matrix components--largely collagen and elastin--are essential, the role of other matrix components such as glycosaminoglycans (GAGs) and proteoglycans remains undefined. This article describes both the qualitative and quantitative GAG composition of fresh and cryopreserved human dermis. Through the use of 2 different colorimetric assays and cellulose acetate electrophoresis, we found the following: 1) the principal dermal GAGs are those of the heparin family; 2) dermatan sulfate is the second most predominant GAG component; 3) chondroitin-6-sulfate is found at concentrations of 2 orders of magnitude less than the heparins; and 4) hyaluronan and keratan sulfate were both found as only minor constituents. When the GAG composition of fresh skin was compared with that of cryopreserved skin, no significant differences were observed. This study also examined the time course of GAG leaching during the preparation of deconstructed human dermis, which is human dermis reduced to the native insoluble matrix components by exhaustive saline soaking. We found that GAG leaching was readily detectable even within the first day. Sixty percent of total GAG leaching occurred by day 7. These investigations establish a benchmark for the reproduction of GAGs in synthetic dermal constructs. Further, the results of the leaching study generate important considerations for short-term skin storage and long-term skin banking. Because GAG leaching commences immediately, appropriate precautions must be taken to minimize the potential functional compromise of cryopreserved human dermis.     

The glycosaminoglycans of the human artery and their changes in atherosclerosis.

The changes in levels of glycosaminoglycans (GAGs) of the intima and media of the human artery in atherosclerosis were determined by a recently introduced two-dimensional electrophoresis technique that permits direct measurments of each of these macromolecules. To identify the arterial GAGs, they were fractionated by chromatography on a DEAE-Sephadex A-25 column, and the resulting three fractions (hyaluronic acid [HA], heparan sulfate [HS], and the partially separated chondroitin sulfates B [CSB] and C [CSC]) were analyzed for their electrophoretic mobilities by this electrophoretic method, for their digestability by highly specific hydrolases (leech hyaluronidase, heparinase, and chondroitinases ABC and AC) and for their iduronic acid content. From these studies we concluded that normal and atherosclerotic human aortas contain CSB, CSC, HA, and HS. Further, we demonstrated that CSB is a hybrid consisting of approximately 40% CSA and 60% CSB and that CSC appears to be a polymer consisting essentially of glucuronic acid and N-acetylgalactosamine-6-sulfate. Classical CSA as well as chondroitin (CH) were not present in detectable amounts. In the relatively normal intima, the mean concentrations of the GAGs were found to be 4.7, 20.9, 1.3, and 5.1 mg/g of dry, defatted, decalcified tissue for CSB, CSC, HA, and HS, respectively. With the progression of atherosclerosis, there was a pronounced decrease in the total GAG content (from 32 to 18 mg) associated with a decrease in the CSC and HS levels but without a change in the HA concentrations. Of particular interest, however, was the increase in the CSB level. In the media whose total GAG content averaged approximately 20 mg, no significant changes in these GAG levels were noted with the progression of the disease except for that of CSC. These findings may be important in explaining the increased lipoprotein and collagen deposition in the diseased aorta.     

Elimination of glycosaminoglycans in duodenal peptic ulcer and problems of its pathogenesis

Changes in the excretion and composition of proteoglycans specific for duodenal ulcer were studied in 50 patients with duodenal ulcer, 30 patients with gastric ulcer, 30 patients with chronic endogenous gastroduodenitis and in 35 healthy persons. In all the examinees proteoglycans were isolated from daily urine, their carbohydrate components--glycosaminoglycans (GAG)--were separated and divided into fractions (keratan sulfate, hyaluronic acid, heparan sulfate, chondroitin sulfate-4, chondroitin sulfate-6, dermatan sulfate, and heparin) by column chromatography on unmodified cellulose. It has been established that only peptic ulcer is characterized by disorders in GAG excretion differing in the period of exacerbation and remission. Changes in the composition of proteoglycans excreted with urine resulted probably from a deficiency of chondroitin sulfate-6 in patients with chronic duodenal ulcer. The deficiency was more marked during exacerbation but did not disappear in the period of remission of duodenal ulcer either.     

Hypoxia differentially enhances the effects of transforming growth factor-beta isoforms on the synthesis and secretion of glycosaminoglycans by human lung fibroblasts

Interstitial lung diseases associated with hypoxia, such as lung fibrosis, are characterized by enhanced production of transforming growth factor-beta (TGF-beta) and increased deposition of extracellular matrix (ECM) molecules, including glycosaminoglycans (GAGs). In this study, we investigated the effect of hypoxia (3% O(2)) on TGF-beta-induced GAG synthesis by primary human pulmonary fibroblasts, established from lung biopsies. Total GAG synthesis was assessed by the incorporation of [(3)H]glucosamine into GAGs associated with the cell layer (cells and ECM) or secreted in the medium. GAGs were isolated and purified by gel filtration, fractionated by electrophoresis on cellulose acetate membranes, and characterized using GAG-degrading enzymes. GAG molecules identified in the cell layer and the medium were: hyaluronic acid, and chondroitin, dermatan, and heparan sulfates. All TGF-beta isoforms time dependently induced [(3)H]glucosamine incorporation into GAGs of the cell layer or the medium. Characterization of individual GAG molecules indicated that this was attributed to dermatan and heparan sulfates in the cell layer and to hyaluronic acid and chondroitin and dermatan sulfates in the medium. Hypoxia enhanced the effect of all TGF-beta isoforms, particularly that of TGF-beta3, on the secretion of hyaluronic acid and chondroitin and dermatan sulfates. In the cell layer, hypoxia stimulated only the effect of TGF-beta2-induced [(3)H]glucosamine incorporation into GAGs. Our data indicate that hypoxia differentially enhances the effect of TGF-beta isoforms on the secretion and deposition of GAGs and may hasten ECM remodeling associated with the pathogenesis of lung fibrosis.     

Increased excretion of urinary glycosaminoglycans in a case of Rothmund Thomson syndrome.

The exercretion of urinary total glycosaminoglycans (GAG) in a case of Rothmund Thomson syndrome associated with osteosarcoma was increased about 2--3 times that of normal control. Since the excretion of urinary total GAG in two cases of osteosarcoma was within normal range, the increased excretion of total GAG might be on account of the metabolic disorder of GAG in Rothmund Thomson syndrome. The prominent fractions obtained by Dowex 1 column chromatography from this syndrome were 0.75 M Fr and 1.0 M Fr, in which the major GAG were indicated to be partially degraded forms of heparan sulfate and chondroitin sulfate, respectively.     

Neonatal screening for mucopolysaccharidoses by determination of glycosaminoglycans in the eluate of urine‐impregnated paper: preliminary results of an improved DMB‐based procedure

Background: The fact that mucopolysaccharidoses (MPSes) are now treatable, and that the earlier treatment is initiated the better, is an indication for neonatal screening. The most efficient approach seems likely to be a multi‐tier procedure in which screening for urinary glycosaminoglycan (GAG) is followed by enzyme determinations in heelprick blood of newborns screening positive. Hitherto the method of choice for the determination of GAG has been the measurement of absorbance by a complex of GAG and 1,9‐dimethylmethylene blue (DMB). Method: We evaluated a DMB method in which absorbance by DMB is measured following its addition to the eluate obtained from paper‐borne newborn urine samples and is normalized relative to urinary creatinine. Calibration is performed with chondroitin‐6‐sulfate (Ch‐6‐S). Results: The limits of detection and quantification of GAG were 1.98 and 5.94 mg/dl, respectively. The within‐run coefficients of variation (CVs) of the GAG/creatinine ratio for 25, 31, and 70 mg/dl solutions of Ch‐6‐S in urine were 21.8, 16.4, and 10.5%, respectively, and the corresponding between‐run CVs were 25.0, 13.5, and 10.1%. Recovery from the urine spiked with 31 mg Ch‐6‐S/dl was 94.8%. Accuracy was also acceptable for all other GAGs except hyaluronic acid. For neonatal screening, the diagnostic threshold was tentatively established as 800 mg GAG/g creatinine, the 95th centile of samples from 903 infants aged 3–28 days, but the value of the GAG/creatinine ratio was negatively correlated with age. Application of the new method to samples from older individuals with and without MPS achieved 100% sensitivity and specificity when used with an age‐dependent threshold taken from the literature on the original DMB method. Conclusion: If used in the first tier of a multi‐tier screening protocol, the proposed method would allow the detection of abnormal levels of all GAGs except hyaluronic acid. J. Clin. Lab. Anal. 24:149–153, 2010. © 2010 Wiley‐Liss, Inc.     

Dermatan sulfate activates nuclear factor-kappab and induces endothelial and circulating intercellular adhesion molecule-1

Proteoglycans (PGs) can influence cell behaviors through binding events mediated by their glycosaminoglycan (GAG) chains. This report demonstrates that chondroitin sulfate B, also known as dermatan sulfate (DS), a major GAG released during the inflammatory phase of wound repair, directly activates cells at the physiologic concentrations of DS found in wounds. Cultured human dermal microvascular endothelial cells exposed to DS responded with rapid nuclear translocation of nuclear factor-kappaB (NF-kappaB), increased expression of intercellular adhesion molecule-1 (ICAM-1) mRNA, and increased ICAM-1 cell surface protein. Heparan sulfate and chondroitin sulfates A and C had no effect on activation of NF-kappaB or induction of ICAM-1. Inhibition of NF-kappaB activation blocked the effect of DS. The increase in cell surface ICAM-1 did not involve TNF-alpha or IL-1 release by endothelial cells, but it was facilitated by autocrine factors whose release was initiated by DS. The ICAM-1-inductive activity of DS was confirmed in vivo. Injection of DS, but not heparin or other chondroitin sulfates, into mice greatly increased circulating levels of soluble ICAM. These data provide evidence that DS, but not other GAGs, initiates a previously unrecognized cell signaling event that can act during the response to injury.     

Glycosaminoglycans from renal brush border membranes of rabbits

Brush border membranes were prepared from the rabbit kidney cortex by sucrose density gradient centrifugation and digested with pronase. A glycosaminoglycan fraction was isolated from the digest by ion-exchange chromatography. Electrophoretogram on cellulose acetate membrane (Separax) of the glycosaminoglycan fraction indicated the presence of two components, which were identified as chondroitin sulfate A/C and dermatan sulfate on the basis of their mobility in Separax electrophoresis and sensitivity to chondroitinase ABC. Neither hyaluronic acid nor heparan sulfate was detected. The results of high-performance liquid chromatography of the pyridylamino derivatives of unsaturated disaccharides in the digests with chondroitinase AC and ABC of the glycosaminoglycan fraction indicated that the ratio of chondroitin 4-sulfate, chondroitin 6-sulfate and dermatan sulfate was 5.5 : 1.0 : 9.1.     

Alterations in serum glycosaminoglycan profiles in Graves' patients.

BACKGROUND: Qualitative and quantitative analyses of glycosaminoglycans (GAGs) in serum obtained from Graves' disease (GD) patients without extrathyroidal complications were carried out to provide a clearer understanding of the role of these macromolecules in the disease pathogenesis. METHODS: GAGs were isolated from-SIMEL (Italian Society of Laboratory Medicine) Inter-associative Study Group on Diabetes Mellitus serum of 17 GD patients before treatment and after attainment of the euthyroid state, as well as from 20 healthy individuals. GAGs were quantified using the hexuronic acid assay and subjected to electrophoretic fractionation. RESULTS: Increased amounts of total GAGs were found in GD patients. Attainment of euthyroidism led to a decrease in, but not normalization of, total serum GAGs level. Electrophoretic analyses of GAGs before and after treatment identified the presence of chondroitin sulfate (CS), heparan sulfate/heparin (HS/H) and dermatan sulfate (DS) in serum from healthy subjects and GD patients. CS was the predominant serum GAG constituent in all subjects investigated. Enhanced CS levels in both GD patient groups were accompanied by increased structural heterogeneity of these compounds. Normalization of thyroid function did not change CS levels. DS levels in serum of untreated GD patients were elevated in comparison to healthy subjects. Anti-thyroid treatment led to a significant decrease in DS to levels below those in controls. DS in all serum samples investigated displayed a similar structure. HS/H levels in serum of untreated GD patients was seven-fold higher than in healthy subjects. In addition, HS/H in untreated GD patients were characterized by higher structural heterogeneity than those isolated from control subjects and euthyroid GD patients. Anti-thyroid therapy led to a decrease in HS/H concentrations towards normal values. CONCLUSIONS: Our results indicate that in the course of GD, the metabolism of particular types of GAGs is regulated by different mechanisms, including a hyperthyroid state and immunological abnormalities. Furthermore, qualitative and quantitative changes in serum GAGs seem to reflect GD-associated systemic changes in extracellular matrix properties.     

Evaluation of reliability for urine mucopolysaccharidosis screening by dimethylmethylene blue and Berry spot tests

BACKGROUND: The mucopolysaccharidosis (MPS) are a group of inherited metabolic disorders resulting from the deficiency of the enzyme responsible for intralysosomal catabolism of glycosaminoglycans (GAGs). GAGs are progressively accumulated in multiple tissues and released into the corporal fluids. The first laboratory approximation to MPS diagnosis is the identification of an increased urinary GAG excretion. For this, several semiquantitative and quantitative methods have been developed. The aim of this retrospective statistical study was to evaluate the reliability of MPS urine screening for the semiquantitative Berry spot test (BST) and the quantitative dimethylmethylene blue test (DMB). METHODS: The 24-h-urine samples (n = 246) were tested through BST, DMB, and for GAG excretion pattern by one-dimensional electrophoresis or thin layer chromatography. RESULTS: the 204 samples that demonstrated a normal GAG excretion pattern were considered as non-MPS samples. Forty-two samples presented an abnormal GAG excretion pattern. Enzyme analysis was available for 31 out of 42 patients (31/42), confirming that all were affected by MPS. Urinary GAG concentrations of MPS patients by DMB were increased 1.04- to 7.1-folds, compared to age-related normal levels. The sensitivity was 100% for DMB and 93.6% for BST. DMB demonstrated a specificity of 74.5%, while BST a specificity of 53.9%. The specificity of MPS screening increased to 84.3%, considering conjunctly DMB and BST. CONCLUSION: The DMB is a sensitive method, however, inclusion of BST could increase the specificity of MPS urine screening.     

Glycosaminoglycans of skin and urine in pseudoxanthoma elasticum: evidence for chondroitin 6-sulfate alteration

Abnormally elevated hyaluronic acid and dermatan sulfate were isolated from lesional skin of a patient with severe pseudoxanthoma elasticum. These glycosaminoglycans (estimated as uronic acid) surpassed the normal controls by 33.6 and 4.8 magnitudes, respectively. Urine chondroitin 6-sulfate of the same patient moved faster by electrophoresis on cellulose acetate than its counterpart isolated from urine of another four patients or from the normal controls. Hyaluronic acid and chondroitin 6-sulfate exceeded their counterparts in normal urine by 2- to 10- and 4- to 18-folds, respectively. These increments correlated with the pseudoxanthoma elasticum severity in three of the five patients studied. The data showed: the first conclusive evidence that dermatan sulfate increased in lesional skin of a patient, with severe pseudoxanthoma elasticum, who also had considerably augmented HA, alteration of the same patient's urine chondroitin 6-sulfate, and diversified urine glycosaminoglycans in pseudoxanthoma elasticum.     

Bench-to-bedside review: The role of glycosaminoglycans in respiratory disease

The extracellular matrix (ECM) plays a significant role in the mechanical behaviour of the lung parenchyma. The ECM is composed of a three-dimensional fibre mesh that is filled with various macromolecules, among which are the glycosaminoglycans (GAGs). GAGs are long, linear and highly charged heterogeneous polysaccharides that are composed of a variable number of repeating disaccharide units. There are two main types of GAGs: nonsulphated GAG (hyaluronic acid) and sulphated GAGs (heparan sulphate and heparin, chondroitin sulphate, dermatan sulphate, and keratan sulphate). With the exception of hyaluronic acid, GAGs are usually covalently attached to a protein core, forming an overall structure that is referred to as proteoglycan. In the lungs, GAGs are distributed in the interstitium, in the sub-epithelial tissue and bronchial walls, and in airway secretions. GAGs have important functions in lung ECM: they regulate hydration and water homeostasis; they maintain structure and function; they modulate the inflammatory response; and they influence tissue repair and remodelling. Given the great diversity of GAG structures and the evidence that GAGs may have a protective effect against injury in various respiratory diseases, an understanding of changes in GAG expression that occur in disease may lead to opportunities to develop innovative and selective therapies in the future.     

Galactose 6-sulfate sulfatase activity in Morquio syndrome

We have prepared a new substrate (o-beta-D-sulfo-galactosyl-(1-4)-beta-D-6-sulfo-2-acetamido-2-deoxyglucosyl- (1-4)-D-[1-3H]galactitol), from shark cartilage keratan sulfate, for the assay of galactose 6-sulfate sulfatase activity. Using this substrate, we found there was a striking deficiency of galactose 6-sulfate sulfatase activity, in addition to the known deficiency of N-acetylgalactosamine 6-sulfate sulfatase, in the cultured skin fibroblasts of patients with Morquio syndrome. Our results could be explained by the hypothesis that accumulation of keratan sulfate and chondroitin 6-sulfate in Morquio syndrome is due to a deficiency of galactose 6-sulfate sulfatase and N-acetylgalactosamine 6-sulfate sulfatase activity, which are necessary for the degradation of these two mucopolysaccharides.