Slippery Viscosity-Sensing Platform with Time Readout for the Detection of Hyaluronidase and Its Inhibitor.

Hyaluronidase (HAase) is a biomarker for cancer, and its detection is of great significance for early diagnosis. However, the requirement of sophisticated instruments, tedious operation procedures, and labeled molecules of conventional HAase biosensing methods hampers their widespread applications. Herein, we report a portable slippery viscosity-sensing platform with time readout for the first time and demonstrate HAase and tannic acid (TA, HAase inhibitor) detection as a model system. HAase specifically cleaves hyaluronic acid (HA) and decreases HA solution viscosity, thereby shortening the aqueous droplet's sliding time on a slippery surface. Thus, the HA solution viscosity alteration due to enzymatic hydrolysis is used to quantify the HAase concentration through the difference in the sliding time of the aqueous droplets on a slippery surface. The developed HAase sensing platform exhibits high sensitivity with a minimum detection limit of 0.23 U/mL and excellent specificity without the use of specialized instruments and labeled molecules. HAase detection in actual urine samples by a standard addition method is performed as well. Moreover, the quantitative detection of TA with an IC50 value of 37.68 ± 1.38 µg/mL is achieved. As an equipment-free, label-free, and high-portability sensing platform, this method holds promise in developing a user-friendly and inexpensive point-of-care testing (POCT) device for HAase detection, and its use can be extended to analyze other analytes with different stimuli-responsive polymers for great universality and expansibility in biosensing applications.

Blood and urine biomarkers associated with long-term respiratory dysfunction following neonatal hyperoxia exposure: Implications for prematurity and risk of SIDS.

Former preterm infants, many of whom required supplemental O2 support, exhibit sleep disordered breathing and attenuated ventilatory responses to acute hypoxia (HVR) beyond their NICU stay. There is an increasing awareness that early detection of biomarkers in biological fluids may be useful predictors/identifiers of short- and long-term morbidities. In the present study, we identified serotonin (5-HT), dopamine (DA) and hyaluronan (HA) as three potential biomarkers that may be increased by neonatal hyperoxia and tested whether they would be associated with an impaired HVR in a rat model of supplemental O2 exposure. Neonatal rats (postnatal age (P) 6 days, P6) exposed to hyperoxia (40% FIO2, 24 h/day between P1-P5 days of age) exhibited an attenuated early (1 min), but not the late (4-5 min) phase of the HVR compared to normoxia control rats; the attenuated early phase HVR was associated with increased levels of DA (urine and serum), 5-HT (platelet poor plasma only, PPP), and HA (serum only). At P21, both the early and late phases of the HVR were attenuated, but serum and urine levels of all 3 biomarkers were similar to age-matched control rats. These data indicate that changes in several serum and/or urine biomarkers (5-HT, DA, and HA) following short-term (days) neonatal hyperoxia can signify long-term (weeks) respiratory control dysfunction. Further studies are needed to determine whether early detection of similar biomarkers could be convenient predictors of increased risk of abnormalities in respiratory control including sleep disordered breathing in former preterm infants who had received prior supplemental O2 and who might also be at increased risk of SIDS.

Urinary hyaluronic acid: a versatile marker of bladder cancer.

PURPOSE: To evaluate the role of urinary hyaluronic acid (HA) as a diagnostic marker in urothelial carcinoma (UCC), squamous cell carcinoma (SCC), and adenocarcinoma (ADC) of urinary bladder and compare it with urine cytology. METHODS: HA was estimated in 170 subjects divided into three groups. Group I: UCC 88 patients, 28 with SCC and 12 with ADC; group II: 34 patients with benign bladder tumors; and group III: 10 healthy bladders. HA was estimated in urine and then readjusted to creatinine (HA/Cr) and protein (HA/Pr) in urine. Urine cytology was evaluated. RESULTS: The mean ± SD level HA was higher in UCC (589 ± 72), SCC (637 ± 45), and ADC (526 ± 30) as compared with benign (476 ± 92) and normal (277 ± 44) groups regardless the grade of tumor (p < 0.0001). A cutoff value of 490 ng/ml was calculated to detect malignancy with sensitivity of 98% and specificity of 66%. PPV, NPV, and ACC were 88.6%, 94.1%, and 90%, respectively. Urine cytology showed sensitivity of, specificity, PPV, NPV, and ACC of 52.6%, 90%, 90.45, 50%, and 65.5%, respectively. HA/Pr and HA/Cr, cutoff values for detection of malignancy were 84.9 and 9.6 but with less predictive values. Histopathological type was the only independent factor affecting level of HA on multivariate analysis, (p = 0.012, Exp (B) 14.98, 95% CI 1.8-121). CONCLUSION: Combination of urinary HA and urine cytology provides reliable marker of bladder cancer.

A novel surface-enhanced Raman scattering-based ratiometric approach for detection of hyaluronidase in urine.

A ratiometric surface-enhanced Raman scattering (SERS) based method is described for the determination of the activity of hyaluronidase (HAase). Gold nanorods (AuNRs) were functionalized with 4-thiobenzonitrile (TBN) to act as the Raman reporter (TBN-AuNRs), and 4-thiophenylacetylene-functionalized gold-silver alloy nanoparticles (TPA-AuAgNPs) were used as the reference. Hyaluronic acid (HA) acts as the HAase recognition element. The TBN-modified AuNRs aggregate in the presence of HA due to the strong electrostatic interaction between the positively charged TBN-AuNRs and negatively charged HA. This strongly enhances the Raman signal of TBN at 2220 cm-1. However, HA has no significant effect on the dispersion of the modified AuAg NPs which are electroneutral. Hence, no change can be seen in the Raman intensity of TPA at 1974 cm-1. In the presence of HAase, HA is digested into smaller fragments. This results in good dispersion of the TBN-AuNRs and a weaker TBN Raman signal. Hence, the ratio of the Raman peaks at 1974 and 2220 cm-1 increases. Under the optimized conditions, the ratio changes in the 5-70 U mL-1 HAase activity range, and the detection limit is 1.7 U mL-1 (based on the 3σ rule). Moreover, this method has been successfully applied in the determination of the activity of HAase in artificial urine and it is expected to be a new method for the diagnosis of cancer, especially bladder cancer.

Urinary sulfated glycosaminoglycan insufficiency and chondroitin sulfate supplement in urolithiasis.

Familial members of urolithiasis have high risk for stone development. We observed the low sulfated glycosaminoglycan (GAG) excretion in urolithiasis patients and their descendants. In this study, we investigated urinary excretion of sulfated GAG, chondroitin sulfate (CS), heparan sulfate (HS) and hyaluronic acid (HA) in urolithiasis and their children, and explored the effect of CS and HA supplement in urolithic hyperoxaluric rats. The 24-hour urines were collected from urolithiasis patients (28) and their children (40), as well as healthy controls (45) and their children (33) to measure urinary sulfated GAG, CS, HS and HA excretion rate. Our result showed that urinary sulfated GAG and CS were diminished in both urolithiasis patients and their children, while decreased HS and increased HA were observed only in urolithiasis patients. Percentage of HS per sulfated GAG increased in both urolithiasis patients and their children. In hyperoxaluric rats induced by ethylene glycol and vitamin D, we found that CS supplement could prevent stone formation, while HA supplement had no effect on stone formation. Our study revealed that decreased urinary GAG and CS excretion are common in familial members of urolithiasis patients, and CS supplement might be beneficial in calcium oxalate urolithiasis prophylaxis for hyperoxaluric patients.

Hyaluronic acid and hyaluronidase as possible novel urine biomarkers for the diagnosis of prostate cancer.

The goal of the study is to examine the possible use of HA (hyaluronic acid) and HAase (hyaluronidase) as novel urine biomarkers for the early diagnosis for prostate cancer (Pca). After a prostatic massage, the urine of 118 high-risk patients for Pca was collected, and the patients were submitted to ultrasound-guided transrectal biopsy. HA and HAase were detected and analyzed with Enzyme-Linked Immunosorbent Assay, and a statistical analysis of the urine levels of the two biomarkers according to the histology results was performed. HAase and HA were independently associated with Pca, and both HAase and HA showed significant predictive ability for prostate cancer. With an optimal cut-off point of 183.71 HAase had 70% sensitivity maintaining at the same time a 55.2% specificity, while the optimal cut-off point for HA was 50.13 with 65% sensitivity and 53.9% specificity. Patients with HAase more than 183.71 ng/ml had 3.67 times greater likelihood for prostate cancer and Patients with HA more than 50.13 ng/ml had 2.31 times greater likelihood for prostate cancer. The need of novel biomarkers that will improve the efficacy of PSA is urgent. HAase and HA showed significant predictive ability for prostate cancer and were independently associated with Pca, and greater levels were associated with greater odds for prostate cancer. To Our Knowledge, this is the first study referring to the detection of HAase and HA as potential urine biomarkers for the early diagnosis of Pca.

Minimal shedding of the glycocalyx layer during abdominal hysterectomy.

BACKGROUND: Surgery with and without hypervolaemia may cause shedding (breakdown) of the endothelial glycocalyx layer, but the severity of this problem is unclear. METHODS: In this preliminary report of a larger clinical trial, the plasma and urine concentrations of three biomarkers of glycocalyx shedding (syndecan-1, hyaluronic acid and heparan sulfate) were measured in seven patients before, during, and after open hysterectomy. The fluid therapy consisted of 25 ml/kg (approximately 2 l) of Ringer's lactate, which was infused over 30 min when the surgery started. The resulting plasma volume expansion at the end of the infusion was estimated from the haemodilution. RESULTS: The mean plasma concentration of syndecan-1 was 21.7 ng/ml before surgery and averaged 19.7 ng/ml during and after the surgery. The plasma concentration of hyaluronic acid decreased from 38.0 to 27.7 ng/ml (P < 0.05), while heparan sulfate increased from 3.4 to 5.5 µg/ml (P < 0.05). The urine concentrations of syndecan-1 decreased significantly, while they increased for hyaluronic acid and heparan sulfate. Despite the vigorous fluid load, the urine flow did not exceed 1 ml/min. CONCLUSIONS: No clear evidence was found for shedding of the endothelial glycocalyx layer when 2 l of Ringer's lactate was infused over 30 min during abdominal hysterectomy. Urine analyses yielded patterns of changes that differed from those in plasma. TRIAL REGISTRATION: ISRCTN81005631 . Registered May 17, 2016.

Classification using longitudinal trajectory of biomarker in the presence of detection limits.

Discriminant analysis is commonly used to evaluate the ability of candidate biomarkers to separate patients into pre-defined groups. Recent extension of discriminant analysis to longitudinal data enables us to improve the classification accuracy based on biomarker profiles rather than on a single biomarker measurement. However, the biomarker measurement is often limited by the sensitivity of the given assay, resulting in data that are censored at either the lower or the upper limit of detection. Inappropriate handling of censored data may affect the classification accuracy of biomarker and hinder the evaluation of its potential discrimination power. We develop a discriminant analysis method for censored longitudinal biomarker data based on mixed models and evaluate its performance by area under the receiver operation characteristic curve. Through the simulation study, we show that our method is better than the simple substitution methods in terms of parameter estimation and evaluating biomarker performance. Application to a biomarker study of patients with acute kidney injury demonstrates that our method may shed light on the potential clinical utility of biomarkers by taking into account both longitudinal trajectory and limit of detection issues.

Plasma and urinary glycosaminoglycans in the course of juvenile idiopathic arthritis.

OBJECTIVES: The aim of the study was to perform analyses of plasma and urinary glycosaminoglycan isolated from juvenile idiopathic arthritis (JIA). METHODS, RESULTS: Chondroitin/dermatan sulfate (CS/DS), heparan sulfate/heparin (HS/H) and hyaluronic acid (HA) were evaluated in samples obtained from JIA patients before and after treatment. Electrophoretic analysis of GAGs identified the presence of CS, DS and HS/H in plasma of healthy subjects and JIA patients. CS were the predominant plasma GAGs constituent in all investigated subject. The plasma CS level in untreated patients was significantly decreased. Therapy resulted in an increase in this glycan level. However, plasma CS concentration still remained higher than in controls. Increased levels of DS and HA in untreated JIA patients were recorded. Anti-inflammatory treatment led to normalization of these parameters concentrations. Plasma and urinary concentrations of HS/H were similar in all groups of individuals. Urinary CS/DS and HA were decreased only in untreated patients. CONCLUSIONS: The data presented indicate that changes in plasma and urinary glycosaminoglycan occur in the course of JIA. There are probably the expression of both local articular cartilage matrix and systemic changes in connective tissue remodeling.

Urinary glycosaminoglycan (uGAG) excretion in healthy pediatric and adolescent population.

OBJECTIVES: The influence of age and gender factor on the urinary excretion of total glycosaminoglycans (uGAGs) and their particular types: chondroitin/dermatan sulfates (CS/DSs), heparan sulfates (HSs) and hyaluronan (HA) was analyzed in healthy pediatric and adolescent population. DESIGN AND METHODS: Urine samples were collected from 95 healthy children. Sulfated GAGs excreted in the urine were quantitated using standardized dye-binding method, while the concentrations of HA were determined by immunoassay. RESULTS: Age-dependent decline in total uGAG excretion (r=-0.686; p<0.001), resulting from a decrease in particular GAG fractions i.e. CS/DS (r=-0.757; p<0.001), HS (r=-0.401; p<0.05) and HA (r=-0.638; p<0.001), was found in healthy subjects. The observed differences were not gender specific with the exception of HS, in which excretion declines with age in males (r=-0.501; p<0.05) and does not change in females. Changes in the distribution pattern of uGAG were also found. CS/DS were the predominant uGAG's fraction, representing from 55% to 76% of the total GAGs. Children up to 3 years excreted more GAGs than older subjects and with a higher proportion of CS/DS and less content of HS. Moreover, the relative contribution of HA was increased twofold in adolescents, aged 15-18, as compared to younger subjects. A negative correlation existed between uGAG excretion and body height, except for HS, for which this relationship was found only in males. CONCLUSIONS: Changes in urinary distribution pattern of particular GAG types during physiological human growth and development were found. Evaluation of urinary GAG screening procedures during pathological conditions should be based on the GAG/creatinine ratios with age and gender taken into account.

Evaluation of the metabolism of glycosaminoglycans in patients with interstitial cystis.

INTRODUCTION: Painful bladder syndrome/interstitial cystitis (PBS/IC) pathogenesis is not fully known, but evidence shows that glycosaminoglycans (GAG) of bladder urothelium can participate in its genesis. The loss of these compounds facilitates the contact of urine compounds with deeper portions of bladder wall triggering an inflammatory process. We investigated GAG in urine and tissue of PBS/IC and pure stress urinary incontinence (SUI) patients to better understand its metabolism. MATERIALS AND METHODS: Tissue and urine of 11 patients with PBS/IC according to NIDDK criteria were compared to 11 SUI patients. Tissue samples were analyzed by histological, immunohistochemistry and immunofluorescence methods. Statistical analysis were performed using t Student test and Anova, considering significant when p < 0.05. RESULTS: PBS/IC patients had lower concentration of GAG in urine when compared to SUI (respectively 0.45 ± 0.11 x 0.62 ± 0.13 mg/mg creatinine, p < 0.05). However, there was no reduction of the content of GAG in the urothelium of both groups. Immunofluorescence showed that PBS/IC patients had a stronger staining of TGF-beta, decorin (a proteoglycan of chondroitin/dermatan sulfate), fibronectin and hyaluronic acid. CONCLUSION: the results suggest that GAG may be related to the ongoing process of inflammation and remodeling of the dysfunctional urothelium that is present in the PBS/IC.

Sensitivity and specificity of urinary hyaluronic acid and hyaluronidase in detection of bladder transitional cell carcinoma.

PURPOSE: To the assess sensitivity and specificity of urinary levels of hyaluronic acid (HA) and hyaluronidase (HAase) as an individual or a combined test to diagnose bladder transitional cell carcinoma (TCC). MATERIALS AND METHODS: One hundred and ninety-four urine specimens were collected from individuals between July 2007 and March 2008. The urinary level of hyaluronic acid (HA) was measured by Enzyme-linked immunosorbent assay. Thereafter, the urinary levels of HA and HAase were normalized to urinary creatinine level and expressed as ng/mg and µ/mg. RESULTS: Eighty percent of patients with bladder cancer had urinary HA level < 500 ng/mg, and 90% of controls showed HA level < 500 ng/mg (P < .001). The mean urinary levels of HA in controls did not vary significantly (P < .05), whereas they significantly increased (2.5 to 6.5 folds) in all grades of TCC. More than 80% of patients with grades 2 and 3 TCC had urinary HAase level < 10 µ/mg and over 80% of controls showed HAase level < 10 µ/mg (P < .05). Hyaluronidase levels increased in patients with grades 2 and 3 bladder TCC. CONCLUSION: Measurement of urinary levels of HA and HAase (with 89% sensitivity and 83% specificity) appears to be a highly accurate and non-invasive method for detecting bladder TCC and evaluating its grade.

Evaluation of the metabolism of glycosaminoglycans in patients with interstitial cystis

Introduction: Painful bladder syndrome/interstitial cystitis (PBS/IC) pathogenesis is not fully known, but evidence shows that glycosaminoglycans (GAG) of bladder urothelium can participate in its genesis. The loss of these compounds facilitates the contact of urine compounds with deeper portions of bladder wall triggering an inflammatory process. We investigated GAG in urine and tissue of PBS/IC and pure stress urinary incontinence (SUI) patients to better understand its metabolism. Materials and Methods: Tissue and urine of 11 patients with PBS/IC according to NIDDK criteria were compared to 11 SUI patients. Tissue samples were analyzed by histological, immunohistochemistry and immunofluorescence methods. Statistical analysis were performed using t Student test and Anova, considering significant when p < 0.05. Results: PBS/IC patients had lower concentration of GAG in urine when compared to SUI (respectively 0.45 ± 0.11 x 0.62 ± 0.13 mg/mg creatinine, p < 0.05). However, there was no reduction of the content of GAG in the urothelium of both groups. Immunofluorescence showed that PBS/IC patients had a stronger staining of TGF-beta, decorin (a proteoglycan of chondroitin/dermatan sulfate), fibronectin and hyaluronic acid. Conclusion: the results suggest that GAG may be related to the ongoing process of inflammation and remodeling of the dysfunctional urothelium that is present in the PBS/IC. .

In vitro studies on the role of glycosaminoglycans in crystallization intensity during infectious urinary stones formation.

Proteus mirabilis cause urinary tract infections which are recurrent and can lead to formation of urinary calculi. Both bacterial and the host factors are involved in the development of urolithiasis. To determine the impact of glycosaminoglycans (GAGs) in the formation of P. mirabilis-induced urinary stones, we investigated the in vitro crystallization, aggregation and adhesion of crystals in the presence of GAGs naturally appearing in urine. Crystallization experiments were performed in synthetic urine infected with P. mirabilis in the presence of: hyaluronic acid (HA), heparan sulfate (HS), chondroitin sulfate A, B and C (ChSA, ChSB, ChSC). The intensity of crystallization and aggregation were established by counting particles and phase-contrast microscopy. To analyze the adhesion of crystals, we used normal urothelium and (45)Ca isotope-labeled crystals. In the presence of ChSC, both the size of the crystals formed and their number were higher compared with the control. GAGs increased crystals adhesion to the cells, but only for ChSA this effect was significant. Chondroitin sulfates, which accelerate the first stages of infection-induced stones formation, may play an important role in the pathogenesis of infectious urolithiasis.

Critical parameters for the analysis of anionic oligosaccharides by desorption electrospray ionization mass spectrometry.

Sulfated oligosaccharides derived from glycosaminoglycans (GAGs) are fragile compounds, highly polar and anionic. We report here on the rare but successful application of desorption electrospray ionization (DESI) - LTQ-Orbitrap mass spectrometry (MS) to the high-resolution analysis of anionic and sulfated oligosaccharides derived from the GAGs hyaluronic acid and heparin. For that purpose, key parameters affecting DESI performance, comprising the geometric parameters of the DESI source, the probed surface and the spraying conditions, applied spray voltage, flow rates and solvent composition were investigated. Under suitable conditions, the DESI technique allows the preservation of the structural integrity of such fragile compounds. DESI enabled the sensitive detection of anionic hyaluronic acid and heparin oligosaccharides with a limit of detection (LOD) down to 5 fmol (≈10 pg) for the hyaluronic acid decasaccharide. Detection of hyaluronic acid oligosaccharides in urine sample was also successfully achieved with LOD values inferior to the ng range. Multistage tandem mass spectrometry (MS(n) ) through the combination of the DESI source with a hybrid linear ion trap-orbitrap mass spectrometer allowed the discrimination of isomeric sulfated oligosaccharides and the sequence determination of a hyaluronic acid decasaccharide. These results open promising ways in glycomic and glycobiology fields where structure-activity relationships of bioactive carbohydrates are currently questioned.

Hyaluronic acid fillers on the horizon: roundtable discussion.

In this roundtable discussion, the physicochemical properties and potential clinical applications of two new ranges of hyaluronic acid fillers are reviewed. These fillers display enhanced tissue integration after implantation due to novel manufacturing processes, and one of the ranges is customized for specific clinical applications by variation of filler gel calibration and cross-linking.

Current and emerging bladder cancer urinary biomarkers.

Bladder cancer continues to be one of the most common malignancies. Those who have been already diagnosed are at high risk for recurrence, especially if the pathology demonstrates high-grade disease. Diagnosis and surveillance is reliant on invasive evaluation with cystoscopy. Urinary cytology has been used to aid in diagnosis, but its use is limited. Other assays have been developed that may aid in clinical decision making. The ultimate goal will be the development of a highly sensitive and specific urinary marker for bladder cancer. This would provide a noninvasive means of diagnosing the disease and limit the number of unnecessary cystoscopies. This article will review the currently available urinary bladder cancer markers. It will also review new and investigational urinary markers that have shown promise for future clinical use.

Testing for urinary hyaluronate improves detection and grading of transitional cell carcinoma.

OBJECTIVE: The purpose of this study is to establish a method for the diagnosis and grading of transitional cell carcinoma (TCC), which is responsible for 90% of bladder tumors, using a recently developed ultrasensitive assay for the measurement of hyaluronan (HA). MATERIALS AND METHODS: Urine samples were collected prior to surgery (cystoscopy, transurethral resection for bladder cancer (TURBT), and cystectomy) in 350 patients. After the procedure, pathologic examination revealed that 160 patients had TCC. HA was measured directly in the urine by a noncompetitive enzyme-linked immunosorbent assay (ELISA)-like fluorometric assay. Using the receiver operator characteristic curve (ROC), t-test, Dunn test, Kruskal-Wallis test, and Mann-Whitney test, we evaluated the differences between groups (those with TCC vs. those without TCC). RESULTS: By analyzing the ROC curve, we chose a urinary HA cutoff value of 13.0 µg/l for indicating risk of TCC. Using the value this of 13.0 µg/l, we found that this test had an overall sensitivity of 82.3% and an overall specificity of 81.2%. The positive predictive value of this assay was 78.9%, the negative predictive negative value was 84.2%, and the predictive accuracy was 81.7%. Logistic regression analysis revealed that every 1 µg/l increase in HA increased a patient's likelihood of having TCC by 3.9%. The sensitivity of this test to detect superficial tumors was 76.6%, whereas its sensitivity for detecting invasive tumors was 94.6%. The urinary HA excretion of patients with TCC, classified according to the TNM staging system and the World Health Organization (WHO) grading system, were compared, and a significant difference was observed between the HA levels of patients with superficial tumors compared with invasive tumors (P = 0.005) as well as between patients with low- vs. high-grade carcinomas (P < 0.001). Patients with urinary HA levels >35 µg/l had a 4.63 times increased risk of having an aggressive, invasive, high grade tumor (P = 0.005). CONCLUSIONS: Our results support the postulate that urinary HA may be used as a tumor marker to aid in the diagnosis and grading of TCC. Additionally, more invasive tumors produce and release more HA in urine than superficial tumors, thus higher HA levels indicate more aggressive disease.

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.