Endocan levels in children with renal hypodysplasia.

AIMS: Severe reduction in nephron numbers that are characteristic of renal hypodysplasia (RHD) are one of the cause of childhood chronic kidney disease (CKD). Glomerular hyperfiltration, glomerular hypertrophy, progressive glomerular scarring, and interstitial fibrosis due to reduced nephron number are risk factors for CKD. In recent years, studies on specific markers for early diagnosis of renal failure and mortality have been carried out. The objectives of this study were to identify serum and urinary endocan levels that are expressed in glomerular endothelial cells and tubular epithelial cells in RHD. MATERIALS AND METHODS: 29 children with RHD were compared to 26 healthy controls in terms of serum and urinary endocan levels. RESULTS: The mean serum endocan level in the RHD group and the control group was 700.72 ± 323.19 and 426.86 ± 233.14 pg/mL, respectively. The mean serum endocan level was significantly higher (p = 0.003) in the RHD group. The mean urinary endocan level in the RHD group was 63.62 ± 92.46 pg/mL, and in the control group it was 80.26 ± 142.49 pg/mL. The mean urinary endocan level did not change between groups (p = 0.95). There was also a significant correlation between serum endocan level and uric acid level in the study group (r = 0.325, p = 0.028). CONCLUSION: To our knowledge, this was the first study that evaluated serum and urinary endocan levels in children with RHD. Although serum endocan level was found to be significantly higher in patients with RHD, further studies are needed to validate whether endocan could serve as a marker of poor renal prognosis in RHD.

Erythrocytic membrane anionic charge, sialic acid content, and their correlations with urinary glycosaminoglycans in preeclampsia and eclampsia.

Compared to healthy pregnant women, changes in erythrocytic membrane anionic charge (EAC) and urinary glycosaminoglycans (UGAGS) have been reported in African women with preeclampsia. A single previous study showed a decrease in erythrocytic membrane sialic acid (EMSA) in preeclampsia compared to healthy pregnancy; however, EMSA was not significantly different between women with preeclampsia and non-pregnant women. No study has focused on the relationships between EAC, EMSA, and UGAGS in preeclampsia and eclampsia compared to healthy pregnant and non-pregnant women of reproductive age. Moreover, the erythrocyte membrane contains sialoglycoproteins and proteoglycans involved in creating the negatively charged cell surface, disruption of which leads to erythrocyte aggregation seen in preeclampsia/eclampsia. However, the etiopathogenesis of preeclampsia and eclampsia remains unclear. Therefore, we evaluated the relationship between EAC, UGAGS, and EMSA in preeclampsia and eclampsia. Three groups of 30 women each were enrolled: Group A (non-pregnant women), Group B (healthy pregnant women without complications), and Group C (women with preeclampsia/eclampsia). EMSA was diminished under oxidative stress prevalent in eclampsia and preeclampsia which might have caused a decreased EAC. EAC was negatively correlated with UGAGS and positively correlated with EMSA (p < .001). EMSA was negatively correlated with UGAGS (p < .001). In conclusion, a loss of sialic acid from the erythrocyte membrane causes a significant decrease in the EAC which mirrors the decrease in the negative charge of the renal glomerular basement membrane and might lead to proteinuria and increased UGAGS excretion in preeclampsia and eclampsia.

Endocan as a marker of microvascular inflammation in kidney transplant recipients.

Endocan is a water-soluble proteoglycan exclusively secreted by vascular endothelium. Endocan levels may be elevated in kidney transplant recipients experiencing antibody-mediated rejection (ABMR), which is characterized by vascular inflammation in transplanted kidney. We evaluated the clinical relevance of endocan as markers of microvascular inflammation in patients who underwent kidney transplantation. Plasma and urinary endocan levels were measured in 203 kidney transplant recipients and were compared across different etiologies of allograft dysfunction and various pathologic scores. Both plasma and urinary endocan levels were significantly higher in patients with acute ABMR than those in patients with normal pathology, acute tubular necrosis (ATN), acute pyelonephritis, BK virus associated nephropathy (BKVN), and T-cell mediated rejection (TCMR). Patients with chronic active ABMR also exhibited significantly higher plasma and urinary endocan levels than patients with long-term graft survival. Scores of glomerulitis and peritubular capillaritis, which are typical features of microvascular inflammation, were significantly elevated in patients with higher plasma and/or urinary endocan levels. Furthermore, plasma and urinary endocan levels could effectively discriminate ABMR from ATN, BKVN, and TCMR. Finally, patients exhibiting high urinary and plasma endocan levels in acute ABMR group showed significantly worse renal survival. Altogether, plasma and urinary endocan levels may serve as potential markers of microvascular inflammation in kidney transplant recipients.

Significance of urinary glycosaminoglycans/proteoglycans in the evaluation of type 1 and type 2 diabetes complications.

Because of the high incidence of kidney disease in diabetic patients, the early diagnosis of renal impairment is a key point for intervention and management. Although urinary albumin excretion currently represents the accepted standard to assess both diabetic nephropathy and cardiovascular risk, it has some limitations as structural changes in the glomerular basement membrane may occur before the onset of microalbuminuria. It is therefore important to identify urinary markers that may provide greater sensitivity, earlier detection, and greater predictive power for diabetes complications. In this respect, urinary glycosaminoglycans/proteoglycans (GAGs/PGs) have been long associated with several kidney diseases as well as diabetic nephropathies as their levels increase more readily than albuminuria. In particular, heparan sulfate, a key component of the glomerular basement membrane responsible for its charge-dependent permeability, is excreted into urine at higher concentrations during the early kidney remodeling events caused by the altered glucose metabolism in diabetes. Over the past few years, also urinary trypsin inhibitor has been linked to a chronic inflammatory condition in both type 1 and 2 diabetes. The underlying mechanisms of such increase are not completely known since either a systemic inflammatory condition or a more localized early renal impairment could play a role. Nevertheless, the association with other inflammatory markers and a detailed urinary trypsin inhibitor structural characterization in diabetes remain to be elucidated. This review will discuss a great deal of information on the association between urinary GAGs/PGs and type 1 and 2 diabetes, with particular emphasis on renal involvement, and their potential as markers useful in screening, diagnosis and follow up to be associated with the current standard tests.

Plasma PP13 and urinary GAGs/PGs as early markers of pre-eclampsia.

PURPOSE: To evaluate at 11-13 weeks' gestation biochemical markers that may predict complications of pregnancy such as pre-eclampsia, proteinuria, and hypertension. METHODS: Analyses were performed on first-morning urine and plasma samples from first trimester pregnant women with increased risk of developing pre-eclampsia such as positive personal or family history of cardiovascular disease and diabetes mellitus. A total of 62 women were enrolled, 24 of them presented complications such as pre-eclampsia, proteinuria, and hypertension during pregnancy. The remaining 38 women had a physiological course of pregnancy and formed the reference group. Urine glycosaminoglycans/proteoglycans (GAGs/PGs) distribution was determined by electrophoresis on cellulose acetate strips. Urinary N-acetyl-ß-glucosaminidase was estimated kinetically. Plasma levels of placental protein 13 (PP13) were measured by enzyme-linked immunosorbent assay. RESULTS: No significant differences in total GAG excretion and N-acetyl-ß-glucosaminidase (NAG) concentration were observed between the two groups of pregnant women, whereas we detected increased relative content of total urinary trypsin inhibitor (UTI plus low-sulfated chondroitin sulfate) (p = 0.001) and reduced excretion of heparan sulfate (p = 0.007) and chondroitin sulfate (p = 0.011) in women presenting with pregnancy complications respect to controls. Plasma levels of PP13 were significantly reduced in the group of women who went on to develop complications compared with controls (p = 0.022). CONCLUSIONS: The reduced plasma levels of PP13 and the alteration of the relative content of urinary GAGs and PGs observed in our study could be a promising tool for the prediction of pre-eclampsia in an early stage of pregnancy.

The determination of serum and urinary endocan concentrations in patients with bladder cancer.

Background Endocan (endothelial cell-specific molecule-1) is a proteoglycan and plays an important role in angiogenesis and inflammation. The aim of this study was to evaluate of serum and urinary concentrations of endothelial cell-specific molecule-1 in bladder cancer. Methods The study included 50 bladder cancer patients, 50 with urinary tract infection and 51 healthy volunteers. Serum and urinary endothelial cell-specific molecule-1 concentrations were measured with enzyme linked immunosorbent assay. Results In bladder cancer group, serum and urinary endothelial cell-specific molecule-1 concentrations were significantly higher than in the healthy subjects ( P = 0.003 and P < 0.0001). Urinary endothelial cell-specific molecule-1 concentrations in cases with urinary tract infection were higher than in healthy volunteers ( P = 0.002). There were no significant differences between bladder cancer and urinary tract infection groups in terms of serum and urinary endothelial cell-specific molecule-1 concentrations. Urinary endothelial cell-specific molecule-1 concentrations were higher than those of corresponding serum endothelial cell-specific molecule-1 concentrations ( P < 0.0001 for bladder cancer and urinary tract infection groups, P = 0.002 for healthy subjects). In bladder cancer group, there was a positive correlation between serum endothelial cell-specific molecule-1 and urinary endothelial cell-specific molecule-1 concentrations ( r = 0.32, P = 0.002). For serum endothelial cell-specific molecule-1, sensitivity and specificity were 50%, and 77%, and for urinary endothelial cell-specific molecule-1, 62%, and 71%, respectively. Conclusion Serum and urinary endothelial cell-specific molecule-1 concentrations increase in bladder cancer. This parameter also increases in serum and urine of cases with urinary tract infection. That urinary endothelial cell-specific molecule-1 values were higher than serum endothelial cell-specific molecule-1 values in all groups may be attributed to direct exfoliation of epithelial cells in bladder to urine.

Impact of first trimester fasting glycemic levels on expression of proteoglycans in pregnancy.

AIM: The aim of this study was to assess the influence of glucose metabolism on the expression of glycosaminoglycans (GAGs) and proteoglycans (PGs) in pregnant women. MATERIAL AND METHODS: Seventy-six women in the first trimester of pregnancy (10-13 weeks) attending the Gynecologic and Obstetric Clinic, University of Sassari, were enrolled and gave early morning urine samples. Groups I, II and III included women with serum glucose values of 65-89 mg/dL, 90-99 mg/dL and 100-125 mg/dL, respectively. Urine GAGs/PGs distribution was determined by electrophoresis on cellulose acetate strips. Urinary N-Acetyl-ß-glucosaminidase was estimated kinetically. RESULTS: Analysis of urinary GAGs/PGs electrophoretic profiles showed a significant increase in heparan sulfate (HS) excretion (P = 0.017) as well as a reduced chondroitin sulfate (CS) excretion (P = 0.048) in the group II pregnant women compared with the group I, and higher values of the HS/CS ratio in groups II and III compared to group I. Furthermore, we observed a positive correlation among fasting blood glucose levels and the relative content of HS, the HS/CS and urinary trypsin inhibitor/CS ratios, and the N-Acetyl-ß-glucosaminidase levels. CONCLUSIONS: The assessment of risk factors for gestational diabetes mellitus should also take into account fasting blood glucose values of 90-99 mg/dL, as the findings of our study indicated an alteration in the metabolism of GAGs during the early stages of pregnancy.

Development of an indirect sandwich ELISA for detection of urinary antigen, using Legionella pneumophila PAL protein.

Legionella pneumophila peptidoglycan-associated lipoprotein (PAL) protein is an extremely conserved antigen among Legionella species. In this study, rabbit and rat anti-PAL immunoglobulin G antibodies were produced by immunization with purified, recombinant PAL (r-PAL) protein of L. pneumophila serogroup 1 and used as capture and detection antibodies in the PAL antigen-based enzyme-linked immunosorbent assay (ELISA) to detect urinary PAL antigen. Urine samples were obtained from rats experimentally infected with L. pneumophila serogroup 1. The PAL antigen was measured in urine samples of 40 infected and 40 uninfected rats. After choosing the cut-off value of 0.192, the sensitivity and specificity of the PAL antigen-based ELISA were 87.5 and 97.5 %, respectively. The results obtained by PAL antigen base ELISA were compared with those obtained by Biotest. The PAL antigen was detected efficiently by both of the assays and all of the control human urine samples were negative by the ELISA test. The PAL antigen-based ELISA assay was relatively simple to perform, precise, highly sensitive and specific, and reproducible. Based on our data the PAL antigen-based ELISA described here is the first indirect sandwich ELISA for urinary antigen detection which could easily be applied for diagnosis of Legionnaires disease.

Major basic protein homolog (MBP2): a specific human eosinophil marker.

Human eosinophil granule major basic protein (MBP1) is an exceedingly basic (isoelectric point >11) 14-kDa protein, comprising the core of the secondary eosinophil granule. Recently, a less cationic homolog of MBP, termed MBPH or simply, MBP2, has been discovered. We prepared a panel of mAbs to MBP2 and used these Abs to localize and quantitate this molecule in leukocytes and biological fluids. Specific mAbs for MBP2 were selected using slot-blot analyses and used in a two-site immunoassay, Western blotting, and immunofluorescence microscopy. The sensitivity of the immunoassay was markedly improved by reduction and alkylation of MBP2. MBP1 is more abundant than MBP2 in lysates of eosinophils and their granules, as judged by immunoassay and Western blotting. By immunofluorescence, MBP1 is present in eosinophils, basophils, and a human mast cell line (HMC1), whereas MBP2 is only detected in eosinophils. Neither MBP1 nor MBP2 could be detected in any other peripheral blood leukocyte. MBP2 levels measured in plasma and serum were essentially identical. In contrast to past measurements for MBP1, MBP2 was not detected above normal levels in sera from pregnant donors. However, measurement of serum MBP2 discriminated patients with elevated eosinophils from normal subjects, and MBP2 was also detectable in other biological specimens, such as bronchoalveolar lavage, sputum, and stool. These results indicate that MBP2 is present only in eosinophils and that it may be a useful biomarker for eosinophil-associated diseases.

Urinary glycosaminoglycan composition in chronic glomerulonephritis.

BACKGROUND: Glycosaminoglycans (GAG) play an important role in regulating glomerular permeability, and a reduction in their plasmatic concentration or urinary loss has been implicated in the pathogenesis of diseases associated with increased albumin permeability. The purpose of this study was to evaluate GAG excretion in renal pathology by analyzing the composition of urinary GAG in antibody mediated glomerular injury, such as mesangial glomerulonephritis (IgAGN) and primitive membranous glomerulonephritis (MGN), to verify the effects of glomerular capillary wall lesion with and without mesangial cell injury. METHODS: Urinary GAG excretion was analyzed in 20 patients with IgAGN, 18 patients with MGN, and in 18 healthy subjects (controls). GAG were isolated from 24-hr urine using ion-exchange chromatography on DEAE-Sephacel, and the results are expressed as mg hexuronate/g creatinine (Cr). GAG composition was determined by cellulose acetate electrophoresis and expressed as relative percentages by densitometric scanning of Alcian Blue stained strips. RESULTS: We found total GAG levels significantly higher in the urine of patients with MGN in comparison with controls and patients with IgAGN. The electrophoretic pattern analysis demonstrated low sulfated chondroitin sulfate proteoglycan (LSC-PG) in all patients compared to 44% in controls (8/18), but also low sulfated chondroitin sulfate (LSC) in 18.4% of patients (7/38) and slow migrating LSC (SM-LSC) in 8% of patients (3/38), only in the MGN group. Moreover, in patients with MGN, the LSC-PG relative content was significantly higher when compared to that observed in controls. Finally, in IgAGN and MGN patients, a significant reduction in chondroitin sulfate (CS) relative content was observed. CONCLUSIONS: It seems likely that an increase in total GAG levels takes place when a reduction in renal function occurs, but the alteration of CS and herapan sulfate (HS) relative contents, and the presence of LSC-PG and free LSC also in the urine of IgAGN patients, allows us to suggest that the GAG distribution pattern becomes abnormal before an increase in total urine GAG excretion. In addition, the quali-quantitative determination of urinary GAG and GAGprotein complex could constitute an additional non-invasive marker to appraise the metabolism of renal connective tissue in some renal diseases.

Glomerular expression of biglycan and decorin and urinary levels of decorin in primary glomerular disease.

AIMS: Recent studies have suggested that small leucine-rich proteoglycans (SLRP) of the extracellular matrix play a major role in modulating the activity of growth factors and in regulating the deposition of collagens. In this study, the expression of the SLRPs biglycan and decorin in the glomeruli of patients with primary glomerular disease (minimal change disease, IgA nephropathy, and membranous nephropathy) and urine immunoreactive levels examined. METHODS: Renal biopsy specimens were obtained from patients with minimal change disease, IgA nephropathy and membranous nephropathy. Immunohistochemical staining was performed on fresh-frozen samples using anti-biglycan and anti-decorin antibodies. Examination of urine proteoglycan excretion from a total of 26 patients and 8 normal volunteers was performed by indirect ELISA. RESULTS: In normal kidney samples, biglycan and decorin expression was found predominantly in the intrarenal arteries and tubulointerstitium, with only minimal expression in the glomeruli. Glomerular expression of these proteoglycans in glomerular disease was unchanged in all of the 4 patients examined with minimal change disease. In the case of IgA nephropathy or membranous nephropathy, some of the patients showed minimally increased immunostaining of either biglycan or decorin, but there were no signs of simultaneous upregulation of both proteoglycans. To further examine the changes in proteoglycan expression, ELISA was performed on urine samples. Urine biglycan levels were below detection levels, but high values of urine decorin immunoreactivity were found in the patients with glomerular disease. A significant negative correlation was found between urine decorin and creatinine clearance. CONCLUSION: These results suggest that distinct changes in the expression of the SLRPs biglycan and decorin may be seen in patients with primary glomerular disease. Moreover, the negative relationship between urine decorin levels and renal function supports the hypothesis that decorin may be involved in the pathophysiology of renal dysfunction in humans.

Urinary glycosaminoglycan and proteoglycan excretion in normoalbuminuric patients with type 1 diabetes mellitus.

BACKGROUND: Diabetic nephropathy may be related to an abnormal metabolism of glycosaminoglycans (GAG) in the glomerular basement membrane (GBM). The first manifestation of nephropathy is microalbuminuria, whose appearance indicates a loss of GBM selectivity. The present study evaluated whether GAG excretion becomes abnormal in parallel with microalbuminuria, and whether such abnormalities are also present in normoalbuminuric diabetic patients. METHODS: We measured urinary GAG excretion in 60 patients with type 1 (insulin-dependent) diabetes mellitus and in 22 healthy subjects. GAG were isolated from 24-h urine using ion-exchange chromatography on DEAE Sephacel. GAG composition was determined by cellulose acetate electrophoresis and expressed as percentages by densitometric scanning of Alcian Blue stained strips. RESULTS: On subgrouping for albuminuric status and glyco-metabolic control, we found high urinary GAG concentrations in all except the normoalbuminuric patients with good glyco-metabolic control. The urinary GAG electrophoretic pattern showed alterations in chondroitin sulphate (CS) and heparan sulphate (HS) relative contents. A higher frequency of low sulphated chondroitin sulphate-proteoglycan (LSC-PG) was observed in all patients, including those with normoalbuminuria and good glyco-metabolic control. CONCLUSIONS: This urinary pattern may be indicative of an abnormal GBM metabolism. Since GAG play an important role in GBM permeability, these anomalies might consequently represent a first step towards selective changes of GBM in type 1 diabetes mellitus.

Small proteoglycans in human diabetic nephropathy: discrepancy between glomerular expression and protein accumulation of decorin, biglycan, lumican, and fibromodulin.

Small leucine-rich proteoglycans (SLRPs), for example, decorin, biglycan, fibromodulin, and lumican, are extracellular matrix organizers and binding partners of TGF-b. Decorin is also involved in growth control and angiogenesis. Hence, these proteoglycans are likely of importance in the pathogenesis of diabetic glomerulosclerosis. In normal kidney, SLRPs were preferentially expressed in the tubulointerstitium. Weak expression occurred in the mesangial matrix. Biglycan was expressed by glomerular endothelial cells and, together with fibromodulin, by distal tubular cells and in collecting ducts. In all stages of diabetic nephropathy, there was a marked up-regulation of the proteoglycans in tubulointerstitium and glomeruli. Decorin and lumican became expressed in tubuli. However, in glomeruli, overexpression was not mirrored by local proteoglycan accumulation except in advanced nephropathy. In severe glomerulosclerosis, increased decorin concentrations were found in plasma and urine, and urinary TGF-b/decorin complexes could be demonstrated indirectly. The failure to detect an increased glomerular proteoglycan quantity during the development of nephropathy could be explained by assuming that they are secreted into the mesangial matrix, but cleared via the vasculature or the urinary tract, in part as complexes with TGF-b. They could thereby counteract the vicious circle being characterized by increased TGF-b production and increased matrix deposition in diabetic nephropathy.

Small proteoglycans of normal adult human kidney: distinct expression patterns of decorin, biglycan, fibromodulin, and lumican.

BACKGROUND: Among the members of the small leucine-rich proteoglycan family, decorin, biglycan, and fibromodulin have been proposed to be potent modulators of transforming growth factor-beta (TGF-beta) activity, thereby playing an important role in the pathogenesis of fibrotic kidney diseases. Furthermore, decorin expression influences the expression of p21WAF1/CIP1, which has been related to kidney hypertrophy and hyperplasia. However, none of the members of this proteoglycan family have been investigated in normal adult human kidney cortex, thus making it impossible to correlate disease-mediated alterations of their expression with the normal situation in vivo. METHODS: The chondroitin/dermatan sulfate proteoglycans, decorin and biglycan, and the keratan sulfate proteoglycans, fibromodulin and lumican, were investigated in normal human adult renal cortex by immunohistochemistry on the light and electron microscopic level and by in situ hybridization. Northern blot and reverse transcription-polymerase chain reaction (RT-PCR) methods were used to get an estimate of their expression in isolated glomeruli. Decorin excretion with the urine was measured by Western blotting. RESULTS: Two bands of decorin and a single band of biglycan mRNA were identified in Northern blots of isolated glomeruli. Amplification by RT-PCR was required to detect the signals for fibromodulin and lumican. All four proteoglycans were preferentially expressed in the renal interstitium with accumulations around tubules. Weak expression was found in the mesangial matrix. Biglycan was expressed by glomerular endothelial cells and, together with fibromodulin, was synthesized and deposited in distal tubular cells and collecting ducts. Immunogold labeling indicated the presence of the proteoglycans in the glomerular basement membrane, which was interpreted as a result of glomerular filtration. Indirect evidence suggested tubular reuptake of decorin after glomerular filtration. CONCLUSION: The data indicate that the different cells of the adult human kidney are characterized by a distinct expression pattern of the four small proteoglycans. It is suggested that these proteoglycans may have distinct pathophysiological roles depending upon whether they are expressed by mesangial cells, endothelial cells, epithelial cells, or cells of the tubulointerstitium.

Azaftig, a urinary proteoglycan from a cachectic cancer patient, causes profound weight loss in mice.

Cachexia is a complex medical condition characterized by significant weight loss associated with decreased body fat and protein; the condition may present itself with or without anorexia. We have isolated and partially characterized a proteoglycan (azaftig) from the urine of cancer and AIDS patients experiencing weight loss. When given to mice, the purified azaftig resulted in a significant decrease in body weight and body fat without any effect on appetite. The results of these studies show that azaftig may be one of the many factors participating in the emergence of the cachectic state.

Isolation, characterization, and distribution of a 24-kDa proteoglycan in the urine of cachectic cancer and AIDS patients.

Substantial weight loss in individuals with AIDS or cancer is associated with a poor prognosis and increased mortality. We have isolated and partially characterized a proteoglycan (named azaftig) from the urine of a cancer patient experiencing weight loss. Furthermore, we have raised a polyclonal antibody to azaftig in rabbits and developed a procedure to measure the level of this proteoglycan in urine by Western blot. We report the presence of azaftig in the urine of cancer and AIDS patients experiencing weight loss, but not in the control or weight-stable subjects. The azaftig-like immunoreactivity was present in 69.2% (9/13) of patients with weight loss, but only in 27.0% (3/11) of weight-stable cancer or AIDS patients and none of the control subjects (n = 8).

Quantitation of proteoglycans as glycosaminoglycans in biological fluids using an alcian blue dot blot analysis.

A method for quantitation of intact proteoglycans as GAGs in biological fluids (blood plasma, synovial fluid) or 4 M guanidine extracts of tissues has been published previously (S. Björnsson, Anal. Biochem. 210, 282-291, 1993). The method is based on the specific interaction between sulfated polymers and the tetravalent cationic dye Alcian blue at pH 1.5 in 0.4 M guanidine-HCl and in the presence of 0.25% Triton. The absorbance assay has a measuring range of 1-20 microgram of glycosaminoglycan (GAG) which is not sensitive enough to measure the low contents of proteoglycans in blood plasma, urine, or wound fluid. A dot blot assay is now described in which the Alcian blue-GAG complexes are collected on a polyvinylidene fluoride membrane, by filtration in a dot blot apparatus, and the stain is quantitated as reflectance by scanning and densitometry. The assay requires 10 microliter of sample and has a measuring range of 10-800 ng of GAG, corresponding to a concentration of 1-80 mg/liter, suitable for proteoglycans in biological fluids. The procedures for chemistry, scanning, densitometry, and curve fitting were each evaluated separately. The error contributed by chemistry accounted for a minor portion of the imprecision. The imprecision contributed by scanning was the most important source of within-run and between-run imprecision, and was caused by inequalities of the charge-coupled device along the scanning arm. Unexpectedly, curve fitting was also a major source of total imprecision in dot blot quantitation and differed with the type of equation used. The between-run imprecision calculated as CV (SD/mean . 100) was 13.0% at 8 mg/liter. The response of the assay was identical for six different commercial preparations of GAGs (chondroitin-4-sulfate, chondroitin-6-sulfate, dermatan sulfate, keratan sulfate, heparan sulfate, and heparin) despite differences in degree of sulfation known to exist. There was no positive or negative interference by blood plasma, apart from a slight negative interference on the quantitation of heparan sulfate. Analysis of 319 paired blood plasma and urine specimens from hospitalized patients showed a variation of plasma GAGs of 0.1-17.6 and urine-GAGs of 0.0-45.6 mg/liter. There was no correlation between plasma and urine GAG concentrations.