Urinary Heparan Sulfate Research Articles

An At-Risk Population Screening Program for Mucopolysaccharidoses by Measuring Urinary Glycosaminoglycans in Taiwan.

Background: The mucopolysaccharidoses (MPSs) are a group of rare lysosomal storage disorders characterized by the accumulation of glycosaminoglycans (GAGs) and which eventually cause progressive damage to various tissues and organs. We developed a feasible MPS screening algorithm and established a cross-specialty collaboration platform between medical geneticists and other medical specialists based on at-risk criteria to allow for an earlier confirmative diagnosis of MPS. Methods: Children (<19 years of age) with clinical signs and symptoms compatible with MPS were prospectively enrolled from pediatric clinics between July 2013 and June 2018. Urine samples were collected for a non-specific total GAG analysis using the dimethylmethylene blue (DMB) spectrophotometric method, and the quantitation of three urinary GAGs (dermatan sulfate (DS), heparan sulfate (HS), and keratan sulfate (KS)) was performed by liquid chromatography/tandem mass spectrometry (LC-MS/MS). The subjects with elevated urinary GAG levels were recalled for leukocyte enzyme activity assay and genetic testing for confirmation. Results: Among 153 subjects enrolled in this study, 13 had a confirmative diagnosis of MPS (age range, 0.6 to 10.9 years—three with MPS I, four with MPS II, five with MPS IIIB, and one with MPS IVA). The major signs and symptoms with regards to different systems recorded by pediatricians at the time of the decision to test for MPS were the musculoskeletal system (55%), followed by the neurological system (45%) and coarse facial features (39%). For these 13 patients, the median age at the diagnosis of MPS was 2.9 years. The false negative rate of urinary DMB ratio using the dye-based method for these 13 patients was 31%, including one MPS I, two MPS IIIB, and one MPS IVA. However, there were no false negative results with urinary DS, HS and KS using the MS/MS-based method. Conclusions: We established an at-risk population screening program for MPS by measuring urinary GAG fractionation biomarkers using the LC-MS/MS method. The program included medical geneticists and other medical specialists to increase awareness and enable an early diagnosis by detecting MPS at the initial onset of clinical symptoms.

Long-term outcomes of enzyme replacement therapy for Taiwanese patients with Mucopolysaccharidosis I

Long-term outcomes of enzyme replacement therapy for Taiwanese patients with Mucopolysaccharidosis I

Development and validation of an LC-MS/MS Method for the quantitation of heparan sulfate in human urine.

Heparan sulfate is a linear polysaccharide and serves as an important biomarker to monitor patient response to therapies for MPS III disorder. It is challenging to analyze heparan sulfate intact owing to its complexity and heterogeneity. Therefore, a sensitive, robust and validated LC-MS/MS method is needed to support the clinical studies for the quantitation of heparan sulfate in biofluids under regulated settings. Presented in this work are the results of the development and validation of an LC-MS/MS method for the quantitation of heparan sulfate in human urine using selected high-abundant disaccharides as surrogates. During sample processing, a combination of analytical technologies have been employed, including rapid digestion, filtration, solid-phase extraction and chemical derivatization. The validated method is highly sensitive and is able to analyze heparan sulfate in urine samples from healthy donors. Disaccharide constitution analysis in urine samples from 25 healthy donors was performed using the assay and demonstrated the proof of concept of using selected disaccharides as a surrogate for validation and quantitation.

Prospective Evaluation of Chondroitin Sulfate, Heparan Sulfate and Hyaluronic Acid in Prostate Cancer.

ABSTRACTPurpose:The present study evaluates chondroitin sulfate (CS) and heparan sulfate (HS) in the urine and hyaluronic acid (HA) in the plasma of patients with prostate cancer before and after treatment compared to a control group.Materials and Methods:Plasma samples were used for HA dosage and urine for quantification of CS and HS from forty-four cancer patients and fourteen controls. Clinical, laboratory and radiological information were correlated with glycosaminoglycan quantification by statistical analysis.Results:Serum HA was significantly increased in cancer patients (39.68 ± 30.00 ng/ mL) compared to control group (15.04 ± 7.11 ng/mL; p=0.004) and was further increased in high-risk prostate cancer patients when compared to lower risk patients (p = 0.0214). Also, surgically treated individuals had a significant decrease in seric levels of heparan sulfate after surgical treatment, 31.05 ± 21.01 μg/mL (before surgery) and 23.14 ± 11.1 μg/mL (after surgery; p=0.029). There was no difference in the urinary CS and HS between prostate cancer patients and control group. Urinary CS in cancer patients was 27.32 ± 25.99 μg/mg creatinine while in the men unaffected by cancer it was 31.37 ± 28.37 μg/mg creatinine (p=0.4768). Urinary HS was 39.58 ± 32.81 μg/ mg creatinine and 35.29 ± 28.11 μg/mg creatinine, respectively, in cancer patients and control group (p=0.6252).Conclusions:Serum HA may be a useful biomarker for the diagnosis and prognosis of prostate cancer. However, urinary CS and HS did not altered in the present evaluation. Further studies are necessary to confirm these preliminary findings.

Butanolysis derivatization: improved sensitivity in LC-MS/MS quantitation of heparan sulfate in urine from mucopolysaccharidosis patients.

Heparan sulfate (HS) is a complex oligosaccharide that is a marker of a number of diseases, most notably several of the mucopolysaccharidoses (MPS). It is a very heterogeneous compound and its quantification at physiological concentrations in patient samples is challenging. Here, we demonstrate novel derivatization chemistry for depolymerization/desulfation and alkylation of HS based on butanolysis. The resultant alkylated disaccharides are quantifiable by LC-MS/MS. This new method is at least 70-fold more sensitive than a previously published methanolysis method. Disaccharide yield over time is compared for methanolysis, ethanolysis, and butanolysis. Maximum disaccharide concentration was observed after 2 h with butanolysis and 18 h with ethanolysis whereas a maximum was not reached over the 24 h of the experiment with methanolysis. The sensitivity of the new technique is illustrated by the quantification of HS in 5 μL urine samples from MPS patients and healthy controls. HS was quantifiable in all samples including controls. Disaccharide reaction products were further characterized using exact mass MS/MS.

Mental retardation in mucopolysaccharidoses correlates with high molecular weight urinary heparan sulphate derived glucosamine.

Mucopolysaccharidoses (MPS) are characterized by mental retardation constantly present in the severe forms of Hurler (MPS I), Hunter (MPS II) and Sanfilippo (MPS III) diseases. On the contrary, mental retardation is absent in Morquio (MPS IV) and Maroteaux-Lamy (MPS VI) diseases and absent or only minimal in the attenuated forms of MPS I, II and III. Considering that MPS patients affected by mental disease accumulate heparan sulfate (HS) due to specific enzymatic defects, we hypothesized a possible correlation between urinary HS-derived glucosamine (GlcN) accumulated in tissues and excreted in biological fluids and mental retardation. 83 healthy subjects were found to excrete HS in the form of fragments due to the activity of catabolic enzymes that are absent or impaired in MPS patients. On the contrary, urinary HS in 44 patients was observed to be composed of high molecular weight polymer and fragments of various lengths depending on MPS types. On this basis we correlated mental retardation with GlcN belonging to high and low molecular weight HS. We demonstrate a positive relationship between the accumulation of high molecular weight HS and mental retardation in MPS severe compared to attenuated forms. This is also supported by the consideration that accumulation of other GAGs different from HS, as in MPS IV and MPS VI, and low molecular weight HS fragments do not impact on central nervous system disease.

Involvement of heparan sulfate in the renoprotective effects of imidapril, an angiotensin-converting enzyme inhibitor, in diabetic db/db mice

We investigated the renoprotective effects of imidapril hydrochloride ((-)-(4 S)-3-[(2 S)-2-[[(1 S)-1-ethoxycarbonyl-3-phenylpropyl] amino] propionyl]-1-methyl-2-oxoimidazolidine-4-carboxylic acid hydrochloride, imidapril), an angiotensin-converting enzyme inhibitor, in a diabetic animal model. We used BKS.Cg-+Leprdb/+Leprdb (db/db) mice, a genetic animal model of obese type 2 diabetes. Diabetic db/db mice suffered from glomerular hyperfiltration, albuminuria and hypoalbuminemia. Oral administration of 5 mg/kg/day of imidapril for 3 weeks suppressed renal hyperfiltration, reduced albuminuria and normalized hypoalbuminemia. Imidapril did not influence body weights, blood pressure or blood glucose concentrations in db/db mice. Urinary excretion of heparan sulfate (HS) in non-treated 11-week-old db/db mice was significantly lower than that in age-matched non-diabetic db/+m mice. HS is a component of HS proteoglycans, which are present in glomerular basement membranes and glycocalyx of cell surfaces. Reduced urinary HS excretion indicated glomerular HS loss in db/db mice. Imidapril increased urinary excretion of HS to concentrations observed in db/+m mice, indicating that imidapril prevented the loss of renal HS. These results suggest that imidapril ameliorates renal hyperfiltration and loss of renal contents of HS. Improvement of filtration function and maintenance of HS, which is an important structural component of glomeruli, may contribute to renoprotective effects of imidapril.

A Novel Mutation (c.200T&gt;C) in the NAGLU Gene of a Korean Patient with copolysaccharidosis IIIB

Mucopolysaccharidosis (MPS) IIIB is a lysosomal storage disorder (LSD) caused by abnormalities of the enzyme α-N-acetylglucosaminidase (NAGLU) that is required for degradation of heparan sulfate. The patient in this study was a 4-yr-old boy. He presented with normal height and weight, pectus carinatum, and multiple persistent Mongolian spots on his back. He had mild dysmorphic features with prominent speech developmental delays and, to a lesser extent, motor developmental delays. The cetylpyridinium chloride precipitation test revealed excessive mucopolysacchariduria (657.2 mg glycosaminoglycan/g creatinine; reference range, <175 mg glycosaminoglycan/g creatinine). Thin layer chromatography showed urinary heparan sulfate excretion. NAGLU enzyme activity was significantly decreased in leukocytes (not detected; reference range, 0.9-1.51 nmol/hr/mg protein) as well as in plasma (0.14 nmol/hr/mg protein; reference range, 22.3-60.9 nmol/hr/mg protein). PCR and direct sequencing analysis of the NAGLU gene showed that the patient was a compound heterozygote for 2 mutations: c.200T>C (p.L67P) and c.1444C>T (p.R482W). The c.200T>C mutation was a novel finding. This is the first report of a Korean patient with MPS IIIB who was confirmed by molecular genetic analyses and biochemical investigation.

Mild mental retardation and low levels of urinary heparan sulfate in a patient with the attenuated phenotype of mucopolysaccharidosis type IIIA

ObjectivesWe report the case of a 28-year-old female subject affected by the attenuated phenotype of mucopolysaccharidosis type IIIA characterized by moderate slowly evolving mental retardation in which the urinary content of heparan sulfate was demonstrated as being substantially low compared to that found in patients with the severe phenotype. Design and methodsThe specific evaluation of macromolecular heparan sulfate by electrophoresis and the determination of related glucosamine in the urine were performed. ResultsIn our patient, the urinary macromolecular heparan sulfate content (4.2μg/mg creatinine) was ~7.5-times higher than in healthy subjects (0.56μg/mg creatinine±0.9 SD) while it was ~28-times lower compared to the severe mucopolysaccharidosis IIIA group (117μg/mg creatinine±44.8 SD). Furthermore, the urinary glucosamine (86.4μg/mg creatinine) was ~2.4-times greater than in healthy subjects (36.0μg/mg creatinine±18.2 SD) but ~2.4-times lower than in severe subjects (208.1μg/mg creatinine±55.0 SD). ConclusionsThe above data could reflect the reduced heparan sulfate storage in her tissues and organs, and in particular in the brain, consequently explaining her moderate mental retardation. Furthermore, the clinical presentation of patients with an attenuated form of MPS III confirms the need for a specific evaluation of urinary GAGs in all young and adult subjects showing a not well-defined or not particularly severe mental retardation, along with an early MPS diagnosis. Such investigation should also be associated with a more specific characterization of heparan sulfate.

Determination of urinary hexosamines for diagnosis of bladder pain syndrome

Bladder pain syndrome (BPS) is a chronic disease characterized by urgency, bladder pain, and frequency, and urinary glycosaminoglycans are thought to reflect bladder epithelial deficiency in BPS. Sensitive and specific evaluation of total urinary glycosaminoglycans may be useful for the clinical diagnosis of BPS and its treatment. A procedure for the simultaneous determination of glucosamine and galactosamine produced from urinary glycosaminoglycans has been performed in BPS patients and healthy subjects. The total content of urinary hexosamines in BPS patients significantly increased by ~130% with the increase in glucosamine greater than galactosamine. A significant increase in total hexosamines content and in particular in glucosamine belonging to urinary heparan sulfate was determined in BPS patients compared with controls. We propose HS and in particular its low-molecular mass fragments and glucosamine assay as useful markers for a biochemical diagnosis of BPS and for monitoring this syndrome.

Kidney post-transplant monitoring of urinary glycosaminoglycans/proteoglycans and monokine induced by IFN-γ (MIG)

Allograft rejection during the first year after renal transplantation can lead to persistent allograft dysfunction and reduced long-term graft survival. Thus, it is important to define early predictors of kidney damage, less invasive than allograft biopsy. Urinary glycosaminoglycan/proteoglycan concentration and distribution, N-acetyl-β-(D)-glucosaminidase (NAG), and monokine induced by IFN-γ (MIG) levels were evaluated in the immediate post-transplant and during a 1-year follow-up. We observed increased urinary levels of MIG, urinary trypsin inhibitor and its degradation products, the lack of urinary heparan sulfate excretion, and the decreased chondroitin sulfate relative content at day 1 post-transplant in most patients who developed complications in the postoperative period. Moreover, urinary MIG levels showed significant correlations with NAG, C-reactive protein, and GFR at day 1 post-transplant. The monitoring of glycosaminoglycan/proteoglycan urinary pattern and the levels of urine MIG could serve as useful markers for predicting possible complications of transplantation, unraveling an early inflammatory state, on whose basis the immunosuppressive therapy could be appropriately modified.

Efficient analysis of urinary glycosaminoglycans by LC-MS/MS in mucopolysaccharidoses type I, II and VI

Mucopolysaccharidoses (MPSs) are complex storage disorders caused by specific lysosomal enzyme deficiencies, resulting in the accumulation of glycosaminoglycans (GAGs) in urine, plasma, as well as in various tissues. We devised and validated a straightforward, but accurate and precise tandem mass spectrometry methodology coupled to high performance liquid chromatography (LC-MS/MS) for the quantification of GAGs in urine. The method is applicable to the investigation of patients with MPS I, II, and VI, by quantifying dermatan sulfate (DS) and heparan sulfate (HS) in urine. We analyzed urine samples from 28 MPS patients, aged 1 to 42 years, and 55 control subjects (41 days to 18 years old). Levels of DS and HS in urine from healthy controls of all ages were below the limit of quantification. The levels of DS and HS in urine from 6 treated patients with MPS I were lower than in 6 untreated patients in DS (0.7–45 vs 9.3–177 mg/mmol creat) and HS (0–123 mg/mmol creatinine vs 38–418 mg/mmol creatinine); similar results were obtained for 9 patients with MPS II and 7 patients with MPS VI. Analyses were performed on as little as 250 μL of urine. Methanolysis took 75 min per sample; the total analysis run time for each LC-MS/MS injection was 8 min. Results indicate that the method is applicable to a wide variety of situations in which high accuracy and precision are required, including the evaluation of the effectiveness of existing and emerging treatments.

Sanfilippo B in an elderly female psychiatric patient: a rare but relevant diagnosis in presenile dementia

Sanfilippo B is a rare autosomal recessive mucopolysaccharidosis (MPS IIIB) caused by a deficiency of N-acetyl-alpha-D-glucosaminidase (NAGLU). A mild mentally retarded elderly female patient is described with a slowly progressive dementia who had given birth to a daughter who developed normally. Metabolic screening revealed an enhanced concentration of heparan sulfate in urine. Enzymatic assay demonstrated deficiency of N-acetyl-alpha-D-glucosaminidase. Mutations in the NAGLU gene were found. One mentally retarded and hospitalized elder brother was also found to have MPS IIIB, whereas a second brother, who had died earlier, is suspected to have had the same metabolic disorder. Prior to the development of dementia, both the patient and her brother showed autistic like features, signs of ideomotor apraxia and weakness in verbal comprehension. Screening for metabolic disorders, in particular MPSes, should always be considered in patients with a history of mental deficit and dementia or progressive functional decline.

Invited comment

Acta Psychiatrica ScandinavicaVolume 122, Issue 2 p. 166-166 Invited comment Ute Moog MD, PhD, Ute Moog MD, PhD Institute of Human Genetics, Heidelberg University, Im Neuenheimer Feld 366, 69120 Heidelberg, Germany E-mail: ute.moog@med.uni-heidelberg.deSearch for more papers by this author Ute Moog MD, PhD, Ute Moog MD, PhD Institute of Human Genetics, Heidelberg University, Im Neuenheimer Feld 366, 69120 Heidelberg, Germany E-mail: ute.moog@med.uni-heidelberg.deSearch for more papers by this author First published: 02 July 2010 https://doi.org/10.1111/j.1600-0447.2009.01520.xCitations: 4Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article.Citing Literature Volume122, Issue2August 2010Pages 166-166 RelatedInformation

Copper(II)-selective chelation improves function and antioxidant defences in cardiovascular tissues of rats as a model of diabetes: comparisons between triethylenetetramine and three less copper-selective transition-metal-targeted treatments

Treatment with the Cu(II)-selective chelator triethylenetetramine (TETA) improves cardiovascular disease in human patients, and cardiac and vascular/renal disease in rats used as a model of diabetes. Here we tested two hypotheses: first, that TETA elicits greater improvement in organ function than less Cu-selective transition-metal-targeted treatments; second, that the therapeutic actions of TETA are consistent with mediation through suppression of oxidative stress. Rats were made diabetic with streptozotocin (55 mg/kg, i. v.) and treated from 8 weeks after disease induction for the following 8 weeks with effective dosages of oral TETA, or one of three less Cu-selective transition-metal-targeted treatments: D-penicillamine, deferiprone or Zn acetate. Treatment effects were measured in ex vivo cardiac and aortic tissues, plasma and urine. Diabetes damaged both cardiac and renal/vascular function by impairing the ability of cardiac output to respond physiologically to rising afterload, and by significantly elevating the urinary albumin/creatinine ratio. Diabetes also lowered total antioxidant potential and heparan sulphate levels in cardiac and arterial tissues, and serum ferroxidase activity, whereas it elevated urinary heparan sulphate excretion. TETA treatment rectified or partially rectified all these defects, whereas the other three experimental treatments were ineffectual. By contrast, none of the four drug treatments lowered diabetes-mediated elevations of plasma glucose or lipid concentrations. TETA may limit the cardiac and renal/vascular damage inflicted by diabetes through its actions to reinforce antioxidant defence mechanisms, probably acting through selective chelation of 'loosely-bound'/chelatable Cu(II). It may also improve heparan sulphate homeostasis and bolster antioxidant defence by increasing vascular extracellular superoxide dismutase activity. Urinary albumin/creatinine ratio might prove useful for monitoring TETA treatment.

Evaluation of leukocyte arylsulphatase a, serum interleukin-6 and urinary heparan sulphate following tamoxifen therapy in breast cancer

Leukocyte arylsulphatase A (AS-A) was shown to be significantly high in newly-diagnosed breast cancer patients. Previous reports imply a connection between serum interleukin-6 (IL-6) and breast cancer, possibly through a modulation of enzymes involved in estrogen synthesis. Abnormal distribution of heparan sulphate proteoglycans (HSPGs) in malignant breast epithelial cells suggests that they play a key role in the regulation of cell growth. Estradiol is believed to be effective in modulating glycosaminoglycans (GAGs) and their depolymerizing enzymes. Therefore, in this study, attempts were made to evaluate the activity of leukocyte arylsulphatase A, serum interleukin-6, urinary GAGs and heparan sulphate (HS) in response to tamoxifen (TAM) therapy in mastectomised breast cancer patients. Thirty-four patients (aged 30–82 years) were administered TAM (20 mg twice daily). Blood and urine samples of each patient were collected three times (at the beginning, and in third and sixth month of TAM therapy), and biochemical parameters were measured. There was no difference between baseline leukocyte AS-A activity and that measured after three months. At the end of six months, enzyme activity was significantly higher than the former values ( p = 0.022), but within the reference intervals reported in the literature. Although this increase might imply a normalization, the duration of TAM therapy is not long enough to make a decision about either regression or aggravation of the disease. TAM did not have any effect on serum IL-6, urinary HS and GAG levels which may be due to insensitivity of these variables to TAM during the short period of therapy. Both urinary GAG and HS levels measured at sixth month exhibited a positive correlation with the baseline level of leukocyte AS-A ( p = 0.005 and 0.009, respectively), suggesting that positive responses to the drug might be seen in patients with low AS-A activity.

More Dengue, More Questions

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Urinary heparan sulfate proteoglycan excretion in black African women with pre-eclampsia.

The heparan sulfate proteoglycan of the glomerular basement membrane is considered to be mainly responsible for the charge selectivity of the glomerular basement membrane. Decreased heparan sulfate proteoglycan results in a decreased anionic charge of the glomerular basement membrane with increased heparan sulfate proteoglycan in the urine, and is believed to be responsible for the proteinuria in pre-eclampsia. To determine the urinary heparan and chondroitin sulfate proteoglycan levels in women with pre-eclampsia. Eighty-four patients were studied: 28 were normotensive pregnant, 28 were nonproteinuric hypertensive, and 28 were pre-eclamptic. Urine samples were obtained and urinary glycosaminoglycan concentrations were determined using the dimethyl-methylene blue assay. Plotting absorbance against the concentrations of heparan and chondroitin sulfate proteoglycans drew a standard curve. The concentration of heparan and chondroitin sulfate proteoglycans was read-off from the linear portion of the standard curve. The standard solutions contained 25, 50, 100 and 200 mg/l heparan or chondroitin sulfate. The Mann-Whitney U-test was used to detect differences between the three groups, and the Pearson's correlation coefficient was calculated for clinical data correlation of the pre-eclamptic group. Urinary excretion of heparan sulfate proteoglycan (123.1 +/- 22.1 mg/l) was significantly increased in the pre-eclamptic group compared with the normotensive pregnant group (60.5 +/- 5.1 mg/l; p < 0.0001) and the hypertensive nonproteinuric group (63. 2 +/- 3.7 mg/l; p < 0.0001). Urinary chondroitin sulfate proteoglycan excretion followed a similar pattern, being significantly increased in the pre-eclamptic group (88.86 +/- 9.79 mg/l) compared with the normotensive pregnant group (49.1 +/- 8.49 mg/l; p < 0.0001) and the hypertensive nonproteinuric group (43. 9 +/- 5.7 mg/l; p < 0.0001). A significant Pearson's correlation between 24-h urine output vs. 24-h protein excretion (r = 0.51; p < 0.001), and between loss of HSPG versus 24-h urinary protein excretion (r = 0.72; p < 0.0001) was obtained in the pre-eclamptic group. This study demonstrates a reduction of glomerular charge in pre-eclampsia. The strong correlation between the severity of proteinuria and the loss of charge supports the hypothesis that the loss of glomerular charge induces structural changes of the filtration barrier, and may be the mechanism responsible for the proteinuria in pre-eclampsia. Furthermore, the elevated levels of urinary proteoglycan (heparan and chondroitin sulfate proteoglycans) in this disorder show a loss into the urine rather than a neutralization of these macromolecules.

Urinary heparan sulfate proteoglycan excretion in black African women with pre-eclampsia

Background. The heparan sulfate proteoglycan of the glomerular basement membrane is considered to be mainly responsible for the charge selectivity of the glomerular basement membrane. Decreased heparan sulfate proteoglycan results in a decreased anionic charge of the glomerular basement membrane with increased heparan sulfate proteoglycan in the urine, and is believed to be responsible for the proteinuria in pre-eclampsia. Aim. To determine the urinary heparan and chondroitin sulfate proteoglycan levels in women with pre-eclampsia. Materials and methods. Eighty-four patients were studied: 28 were normotensive pregnant, 28 were nonproteinuric hypertensive, and 28 were pre-eclamptic. Urine samples were obtained and urinary glycosaminoglycan concentrations were determined using the dimethyl-methylene blue assay. Plotting absorbance against the concentrations of heparan and chondroitin sulfate proteoglycans drew a standard curve. The concentration of heparan and chondroitin sulfate proteoglycans was read-off from the linear portion of the standard curve. The standard solutions contained 25, 50, 100 and 200 mg/l heparan or chondroitin sulfate. The Mann–Whitney U-test was used to detect differences between the three groups, and the Pearson's correlation coefficient was calculated for clinical data correlation of the pre-eclamptic group. Results. Urinary excretion of heparan sulfate proteoglycan (123.1 ± 22.1 mg/l) was significantly increased in the pre-eclamptic group compared with the normotensive pregnant group (60.5 ± 5.1 mg/l; p < 0.0001) and the hypertensive nonproteinuric group (63. 2 ± 3.7 mg/l; p < 0.0001). Urinary chondroitin sulfate proteoglycan excretion followed a similar pattern, being significantly increased in the pre-eclamptic group (88.86 ± 9.79 mg/l) compared with the normotensive pregnant group (49.1 ± 8.49 mg/l; p < 0.0001) and the hypertensive nonproteinuric group (43. 9 ± 5.7 mg/l; p < 0.0001). A significant Pearson's correlation between 24-h urine output vs. 24-h protein excretion (r = 0.51; p < 0.001), and between loss of HSPG versus 24-h urinary protein excretion (r = 0.72; p < 0.0001) was obtained in the pre-eclamptic group. Conclusion. This study demonstrates a reduction of glomerular charge in pre-eclampsia. The strong correlation between the severity of proteinuria and the loss of charge supports the hypothesis that the loss of glomerular charge induces structural changes of the filtration barrier, and may be the mechanism responsible for the proteinuria in pre-eclampsia. Furthermore, the elevated levels of urinary proteoglycan (heparan and chondroitin sulfate proteoglycans) in this disorder show a loss into the urine rather than a neutralization of these macromolecules.

The effect of aminoguanidine on urinary heparan sulphate levels in experimental diabetes.

In the present study, we investigated whether the ameliorating effect of aminoguanidine on diabetes-related proteinuria and nephropathy is associated with glomerular basement membrane heparan sulphate contents. STZ-induced diabetic rats developed proteinuria (at the tenth week: diabetic rats, 713 +/- 418 mg protein per millimole creatinine; control rats, <30) and increased urinary heparan sulphate excretion (diabetic rats, 1,400 +/- 83 microg/mmol creatinine; control rats, 41 +/- 13; p < 0.001), suggesting loss of glomerular basement membrane charge. Aminoguanidine treatment of diabetic rats diminished urinary heparan sulphate levels (196 +/- 52), suggesting high incorporation of heparan sulphate-associated charge into glomerular basement membrane. Aminoguanidine administration to diabetic rats also relatively improved proteinuria (456 +/- 255). It is concluded that aminoguanidine treatment has a relative beneficial effect by restoring the diabetes-induced change in renal basement membrane heparan sulphate levels.