Heparin Anti-Xa Activity, a Readily Available Unique Test to Quantify Apixaban, Rivaroxaban, Fondaparinux, and Danaparoid Levels.

BACKGROUND: Despite their usefulness in perioperative and acute care settings, factor-Xa inhibitor-specific assays are scarcely available, contrary to heparin anti-Xa assay. We assessed whether the heparin anti-Xa assay can (1) be used as a screening test to rule out apixaban, rivaroxaban, fondaparinux, and danaparoid levels that contraindicate invasive procedures according to current guidelines (>30 ng·mL-1, >30 ng·mL-1, >0.1 µg·mL-1, and >0.1 IU·mL-1, respectively), (2) quantify the anticoagulant level if found significant, that is, if it exceeded the abovementioned threshold. METHODS: In the derivation cohort then in the validation cohort, via receiver operating characteristics (ROC) curve analysis, we evaluated the ability of heparin anti-Xa assay to detect levels of factor-Xa inhibitors above or below the abovementioned safety thresholds recommended for an invasive procedure (screening test). Among samples with relevant levels of factor-Xa inhibitor, we determined the conversion factor linking the measured level and heparin anti-Xa activity in a derivation cohort. In a validation cohort, the estimated level of each factor-Xa inhibitor was thus inferred from heparin anti-Xa activity. The agreement between measured and estimated levels of factor-Xa inhibitors was assessed. RESULTS: Among 989 (355 patients) and 756 blood samples (420 patients) in the derivation and validation cohort, there was a strong linear relationship between heparin anti-Xa activities and factor-Xa inhibitors measured level (r = 0.99 [95% confidence interval {CI}, 0.99-0.99]). In the derivation cohort, heparin anti-Xa activity ≤0.2, ≤0.3, <0.1, <0.1 IU·mL-1 reliably ruled out a relevant level of apixaban, rivaroxaban, fondaparinux, and danaparoid, respectively (area under the ROC curve ≥0.99). In the validation cohort, these cutoffs yielded excellent classification accuracy (≥96%). If this screening test indicated relevant level of factor-Xa inhibitor, estimated and measured levels closely agreed (Lin's correlation coefficient close to its maximal value: 95% CI, 0.99-0.99). More than 96% of the estimated levels fell into the predefined range of acceptability (ie, 80%-120% of the measured level). CONCLUSIONS: A unique simple test already widely used to assay heparin was also useful for quantifying these 4 other anticoagulants. Both clinical and economic impacts of these findings should be assessed in a specific study.

Elevated Plasma Concentrations of Syndecan-1 Do Not Correlate With Increased Capillary Leakage of 20% Albumin.

BACKGROUND: Preclinical experiments show that an inflammatory reaction causes degradation of the endothelial glycocalyx layer and accelerated capillary leakage of albumin and fluid. The hypothesis in the present study was that elevated plasma concentrations of glycocalyx degradation products are associated with greater capillary leakage in humans. METHODS: This open clinical trial involved administration of an intravenous infusion of 20% albumin at 3 mL/kg over 30 minutes to 15 postburn patients who showed an activated inflammatory response. Blood samples and urine were collected for 300 minutes. The plasma concentrations of 2 biomarkers of glycocalyx degradation-syndecan-1 and heparan sulfate-were measured at 0, 60, and 300 minutes and compared to the capillary leakage of albumin and fluid obtained by mass balance calculations and population kinetic analysis. RESULTS: Patients were studied at 7 days (median) after a burn injury that covered 15% (maximum 48%) of the body surface area. The median plasma syndecan-1 concentration was 71 (25th-75th percentiles, 41-185) ng/mL. The 2 patients with highest values showed 2279 and 2395 ng/mL (normal 15 ng/mL). Heparan sulfate concentrations averaged 915 (673-1539) ng/mL. The infused amount of albumin was 57 (48-62) g, and 6.3 (5.1-7.7)% of that leaked from the plasma per hour.Linear correlation analysis of the relationship between the 10logarithm of the mean syndecan-1 and the albumin leakage showed a slope coefficient of -1.3 (95% confidence interval [CI], -3.6 to 1.0) and a correlation coefficient of -0.33 (P = .24). The kinetic analysis revealed that syndecan-1 served as a statistically significant covariate to the albumin leakage, but the relationship was inverse (power exponent -0.78, 95% CI, -1.50 to -0.05; P < .02). Heparan sulfate levels did not correlate with the capillary leakage of albumin or fluid in any of the analyses. CONCLUSIONS: A raised plasma concentration of syndecan-1 alone cannot be extrapolated to indicate increased capillary leakage of albumin and fluid.

A Randomized, Multicenter, Open-Label, Blinded End Point, Phase 2, Feasibility, Efficacy, and Safety Trial of Preoperative Microvascular Protection in Patients Undergoing Major Abdominal Surgery.

BACKGROUND: The endothelial glycocalyx, a carbohydrate-rich layer coating all endothelial surfaces, plays a fundamental role in the function of microcirculation. The primary aim of this study was to evaluate the feasibility of using dexamethasone and albumin to protect the endothelial glycocalyx in patients undergoing abdominal surgery. Secondary and exploratory outcomes included efficacy and safety. METHODS: We conducted a multicenter, open-label, blinded end point, phase 2, randomized trial. Patients undergoing colorectal, pancreas, or liver surgery were recruited and randomized to receive either intravenous dexamethasone (16 mg) and 20% albumin (100 mL) at induction of anesthesia, then 200 mL of 20% albumin with each subsequent 1000 mL of crystalloid administered (dexamethasone and albumin [Dex-Alb] group), or crystalloid fluid only with no dexamethasone (control group). Feasibility end points included patient recruitment and retention, consent rate, and successful study drug administration. The primary efficacy end point was the measurement of plasma syndecan-1 level on postoperative day (POD) 1, and secondary end points were heparan sulfate levels and inflammatory markers measured at 4 perioperative timepoints. Safety end points included errors in administration of the intervention, hyperglycemia, occurrence of postoperative complications, and patient retention. RESULTS: Seventy-two patients were randomized. All feasibility end points were achievable. There were no statistically significant differences observed in median (interquartile range) syndecan-1 levels on POD 1 (39 ng·mL-1 [20-97] in the Dex-Alb group versus 41 ng·mL-1 [19-84] in the control group; difference in medians -2.1, 95% confidence interval [CI], -13 to 8.6; P = .69). The Dex-Alb group had lower POD 1 heparan sulfate levels (319 ng·mL-1 [161-717] in the Dex-Alb group versus 1422 [670-2430] ng·mL-1 in the control group; difference in medians -1085, 95% CI, -1779 to -391) and C-reactive protein (CRP) levels on POD 1 (48 [29-77] mg·L-1 in the Dex-Alb group versus 85 mg·L-1 [49-133] in the control group; difference in medians -48, 95% CI, -75 to -21). Fewer patients had one or more postoperative complication in the Dex-Alb group than in the control group (6 [17%] vs 18 patients [50%]; odds ratio = 0.2, 95% CI, 0.06-0.6). CONCLUSIONS: Intravenous dexamethasone and albumin administration was feasible but did not reduce syndecan-1 on POD 1 in patients undergoing abdominal surgery. Given the clinically important CIs observed between the groups for heparan sulfate, CRP, and postoperative complications, a larger trial assessing the associations between dexamethasone and albumin administration and these outcomes is warranted.

Quality performance for indirect Xa inhibitor monitoring in patients using international external quality data.

Objectives Chromogenic anti-activated factor X (FXa) assays are currently the "gold standard" for monitoring indirect anticoagulants. However, anti-FXa has been shown to vary according to the choice of reagents. In the present study, the performance of anti-FXa measurement was evaluated in order to gain more insight into the clinical applications. Furthermore, the longitudinal coefficient of variation (CV) was studied to investigate whether there is improvement over time. Methods Laboratory tests results were evaluated for samples spiked with unfractionated heparin (UFH), low-molecular-weight-heparin (LMWH), fondaparinux and danaparoid sodium. External quality assessment (EQA) data from multiple years were used from more than 100 laboratories. Results Comparison of the results for all methods showed significant differences in measured values between the frequently used methods (ANOVA: p < 0.001). The largest differences were observed for LMWH and UFH measurements. These differences may be caused by differences in method composition, such as the addition of dextran sulphate. Substantial interlaboratory variation in anti-FXa monitoring was observed for all parameters, particularly at low concentrations. Our results showed that below 0.35 IU/mL, the CVs for UFH and LMWH increase dramatically and results below this limit should be used with caution. Conclusions Our study demonstrates that the choice of the anti-FXa method is particularly important for UFH and LMWH measurement. The variation in measurements may have an effect on clinical implications, such as therapeutic ranges. Furthermore, the longitudinal EQA data demonstrated a constant performance and, in at least 50% of the cases, improvement in the CV% of the anti-Xa results over time.

Circulating heparan sulfate chains and body weight contribute to anti-Xa levels in cancer patients using the prophylactic dose of enoxaparin.

Hospitalized cancer patients are at increased risk of thrombosis and prophylaxis with heparin is recommended. Heparanase is a protein capable of degrading heparan sulfate (HS) chains. The first objective of the study was to examine the effects of weight on anti-Xa levels in cancer patients treated with a fixed dose of enoxaparin as thromboprophylaxis. The second aim was to assess a potential correlation between plasma pre-treatment coagulation parameters and anti-Xa levels in an assumption that heparanase degradation activity towards heparins and HS chains could affect anti-Xa levels. Two blood samples (prior to and 3 h after drug injection) of 76 cancer patients with an indication for prophylaxis with enoxaparin (40 mg) were evaluated for coagulation markers. Sub-prophylactic levels of anti-Xa (< 0.2 U/ml) were found in 16/76 (21%) patients; in 13/76 (13%) patients the values were supra-prophylactic (> 0.5 U/ml). In the subgroup of patients weighing > 80 kg, 7/14 (50%) individuals had a sub-prophylactic level. Overall, anti-Xa levels appeared to correlate with patient's weight (r = - 0.48, p < 0.0001), pre-treatment partial thromboplastin time (PTT), D-dimer, HS, heparanase levels and procoagulant activity. We concluded that plasma anti-Xa levels correlated with patient's weight. A substantial portion of cancer patients receiving enoxaparin prophylaxis was undertreated. For patients > 80 kg, a weight-adjusted prophylactic dose of enoxaparin could be considered. Elevated enoxaparin anti-Xa levels correlated with pre-treatment parameters of coagulation system activation. High pre-treatment HS and elevated plasma anti-Xa levels may potentially serve as biomarkers for the identification of patients at increased thrombosis risk.

Hypervolemia does not cause degradation of the endothelial glycocalyx layer during open hysterectomy performed under sevoflurane or propofol anesthesia.

BACKGROUND: Fluid-induced hypervolemia may stimulate the release of natriuretic peptides and cause degradation (shedding) of the endothelial glycocalyx layer. Sevoflurane is believed to protect the glycocalyx, but the importance of using sevoflurane to prevent shedding during routine surgery is unclear. METHODS: The plasma concentrations of brain natriuretic peptide and two biomarkers of glycocalyx shedding, syndecan-1, and heparan sulfate, were measured in 26 patients randomized to receive general anesthesia with sevoflurane or propofol during open abdominal hysterectomy. The fluid therapy consisted of 25 mL/kg (approximately 2 L) of Ringer´s lactate over 30 minutes. Blood hemoglobin and plasma albumin were used to indicate plasma volume expansion and capillary leakage. RESULTS: The plasma concentrations of brain natriuretic peptide and shedding products showed low levels throughout the surgery (median brain natriuretic peptide, 21 ng/L; syndecan-1, 12.9 ng/mL; and heparan sulfate, 6.5 µg/mL), but the heparan sulfate concentration increased 2 hours post-operatively (to 17.3 µg/mL, P < .005). No differences were noted between the propofol and sevoflurane groups in any of the measured parameters. Albumin was apparently recruited to the bloodstream during the first 20 minutes, when the intravascular retention of infused fluid was almost 100%. The urine flow was <1 mL/min, despite the vigorous volume loading. CONCLUSIONS: No relevant elevations of brain natriuretic peptide or degradation products of the glycocalyx layer were observed when hypervolemia was induced during open abdominal hysterectomy performed with sevoflurane or propofol anesthesia. Plasma volume expansion from Ringer´s lactate was pronounced.

Effect of sevoflurane and propofol on tourniquet-induced endothelial damage: a pilot randomized controlled trial for knee-ligament surgery.

BACKGROUND: The glycocalyx layer is a key structure in the endothelium. Tourniquet-induced ischemic periods are used during orthopedic surgery, and the reactive oxygen species generated after ischemia-reperfusion may mediate the shedding of the glycocalyx. Here, we describe the effects of tourniquet-induced ischemia-reperfusion and compare the effects of sevoflurane and propofol on the release of endothelial biomarkers after ischemia-reperfusion in knee-ligament surgery. METHODS: This pilot, single-center, blinded, randomized, controlled trial included 16 healthy patients. After spinal anesthesia, hypnosis was achieved with sevoflurane or propofol according to randomization. During the perioperative period, five venous blood samples were collected for quantification of syndecan-1, heparan sulfate, and thrombomodulin from blood serum by using ELISA assays kits. Sample size calculation was performed to detect a 25% change in the mean concentration of syndecan-1 with an alpha of 0.05 and power of 80%. RESULTS: For our primary outcome, a two-way ANOVA with post-hoc Bonferroni correction analysis showed no differences in syndecan-1 concentrations between the sevoflurane and propofol groups at any time point. In the sevoflurane group, we noted an increase in syndecan-1 concentrations 90 min after tourniquet release in the sevoflurane group from 34.6 ± 24.4 ng/mL to 47.9 ± 29.8 ng/mL (Wilcoxon test, p < 0.01) that was not observed in patients randomized to the propofol group. The two-way ANOVA showed no intergroup differences in heparan sulfate and thrombomodulin levels. CONCLUSIONS: Superficial endothelial damage without alterations in the cell layer integrity was observed after tourniquet knee-ligament surgery. There was no elevation in serum endothelial biomarkers in the propofol group patients. Sevoflurane did not show the protective effect observed in in vitro and in vivo studies. TRIAL REGISTRATION: The trial was registered in www.clinicaltrials.gov (ref: NCT03772054, Registered 11 December 2018).

Characterization of Fluid Biomarkers Reveals Lysosome Dysfunction and Neurodegeneration in Neuronopathic MPS II Patients.

Mucopolysaccharidosis type II is a lysosomal storage disorder caused by a deficiency of iduronate-2-sulfatase (IDS) and characterized by the accumulation of the primary storage substrate, glycosaminoglycans (GAGs). Understanding central nervous system (CNS) pathophysiology in neuronopathic MPS II (nMPS II) has been hindered by the lack of CNS biomarkers. Characterization of fluid biomarkers has been largely focused on evaluating GAGs in cerebrospinal fluid (CSF) and the periphery; however, GAG levels alone do not accurately reflect the broad cellular dysfunction in the brains of MPS II patients. We utilized a preclinical mouse model of MPS II, treated with a brain penetrant form of IDS (ETV:IDS) to establish the relationship between markers of primary storage and downstream pathway biomarkers in the brain and CSF. We extended the characterization of pathway and neurodegeneration biomarkers to nMPS II patient samples. In addition to the accumulation of CSF GAGs, nMPS II patients show elevated levels of lysosomal lipids, neurofilament light chain, and other biomarkers of neuronal damage and degeneration. Furthermore, we find that these biomarkers of downstream pathology are tightly correlated with heparan sulfate. Exploration of the responsiveness of not only CSF GAGs but also pathway and disease-relevant biomarkers during drug development will be crucial for monitoring disease progression, and the development of effective therapies for nMPS II.

Correlation between acute degradation of the endothelial glycocalyx and microcirculation dysfunction during cardiopulmonary bypass in cardiac surgery.

OBJECTIVE: The association between the shedding of the endothelial glycocalyx (EG) and the pathogenesis of microcirculatory perfusion disturbances has been discussed in experimental studies. This discussion, however, has limited relevance in a clinical setting. We investigated EG shedding in patients undergoing cardiopulmonary bypass (CPB) and its association with alterations in microvascular perfusion. METHODS: The plasma levels of syndecan-1, heparan sulfate, and hyaluronan were used as markers of glycocalyx degradation. Microcirculatory parameters included perfused vessel density (PVD) and De Backer Scores. Sidestream dark field imaging (SDF) was applied to visualize sublingual microcirculation during the preoperative resting state (T0), after sternum splitting, after aortic clamping, 5 min before aortal declamping, 1 h after CPB (T4), 4 h after CPB, 24 h after CPB (T6), and 48 h after CPB. RESULTS: Thirty patients undergoing cardiac surgery were recruited. The plasma levels of glycocalyx degradation markers increased after CPB. This increase indicated severe glycocalyx shedding at T4 relative to that at T0. By T6, the plasma levels of glycocalyx degradation markers had decreased to baseline levels in a stepwise manner. PVD and the De Backer Scores decreased at T4 and recovered at T6. Glycocalyx marker concentrations were correlated with microvascular alterations during cardiac surgery. CONCLUSIONS: Glycocalyx components are closely related to microcirculation perfusion disorders. Damage to the glycocalyx during surgery with CPB may play a key role in microcirculation perfusion dysfunction.

Glycosaminoglycan Neutralization in Coagulation Control.

The glycosaminoglycans (GAGs) heparan sulfate, dermatan sulfate, and heparin are important anticoagulants that inhibit clot formation through interactions with antithrombin and heparin cofactor II. Unfractionated heparin, low-molecular-weight heparin, and heparin-derived drugs are often the main treatments used clinically to handle coagulatory disorders. A wide range of proteins have been reported to bind and neutralize these GAGs to promote clot formation. Such neutralizing proteins are involved in a variety of other physiological processes, including inflammation, transport, and signaling. It is clear that these interactions are important for the control of normal coagulation and influence the efficacy of heparin and heparin-based therapeutics. In addition to neutralization, the anticoagulant activities of GAGs may also be regulated through reduced synthesis or by degradation. In this review, we describe GAG neutralization, the proteins involved, and the molecular processes that contribute to the regulation of anticoagulant GAG activity.

Intravenous fluid resuscitation is associated with septic endothelial glycocalyx degradation.

BACKGROUND: Intravenous fluids, an essential component of sepsis resuscitation, may paradoxically worsen outcomes by exacerbating endothelial injury. Preclinical models suggest that fluid resuscitation degrades the endothelial glycocalyx, a heparan sulfate-enriched structure necessary for vascular homeostasis. We hypothesized that endothelial glycocalyx degradation is associated with the volume of intravenous fluids administered during early sepsis resuscitation. METHODS: We used mass spectrometry to measure plasma heparan sulfate (a highly sensitive and specific index of systemic endothelial glycocalyx degradation) after 6 h of intravenous fluids in 56 septic shock patients, at presentation and after 24 h of intravenous fluids in 100 sepsis patients, and in two groups of non-infected patients. We compared plasma heparan sulfate concentrations between sepsis and non-sepsis patients, as well as between sepsis survivors and sepsis non-survivors. We used multivariable linear regression to model the association between volume of intravenous fluids and changes in plasma heparan sulfate. RESULTS: Consistent with previous studies, median plasma heparan sulfate was elevated in septic shock patients (118 [IQR, 113-341] ng/ml 6 h after presentation) compared to non-infected controls (61 [45-79] ng/ml), as well as in a second cohort of sepsis patients (283 [155-584] ng/ml) at emergency department presentation) compared to controls (177 [144-262] ng/ml). In the larger sepsis cohort, heparan sulfate predicted in-hospital mortality. In both cohorts, multivariable linear regression adjusting for age and severity of illness demonstrated a significant association between volume of intravenous fluids administered during resuscitation and plasma heparan sulfate. In the second cohort, independent of disease severity and age, each 1 l of intravenous fluids administered was associated with a 200 ng/ml increase in circulating heparan sulfate (p = 0.006) at 24 h after enrollment. CONCLUSIONS: Glycocalyx degradation occurs in sepsis and septic shock and is associated with in-hospital mortality. The volume of intravenous fluids administered during sepsis resuscitation is independently associated with the degree of glycocalyx degradation. These findings suggest a potential mechanism by which intravenous fluid resuscitation strategies may induce iatrogenic endothelial injury.

Postoperative microcirculatory perfusion and endothelial glycocalyx shedding following cardiac surgery with cardiopulmonary bypass.

We investigated microcirculatory perfusion disturbances following cardiopulmonary bypass in the early postoperative period and whether the course of these disturbances mirrored restoration of endothelial glycocalyx integrity. We performed sublingual sidestream dark field imaging of the microcirculation during the first three postoperative days in patients who had undergone on-pump coronary artery bypass graft surgery. We calculated the perfused vessel density, proportion of perfused vessels and perfused boundary region. Plasma was obtained to measure heparan sulphate and syndecan-1 levels as glycocalyx shedding markers. We recruited 17 patients; the mean (SD) duration of non-pulsatile cardiopulmonary bypass was 103 (18) min, following which 491 (29) ml autologous blood was transfused through cell salvage. Cardiopulmonary bypass immediately decreased both microcirculatory perfused vessel density; 11 (3) vs. 16 (4) mm.mm-2 , p = 0.052 and the proportion of perfused vessels; 92 (5) vs. 69 (9) %, p < 0.0001. The proportion of perfused vessels did not increase after transfusion of autologous salvaged blood following cardiopulmonary bypass; 72 (7) %, p = 0.19 or during the first three postoperative days; 71 (5) %, p < 0.0001. The perfused boundary region increased after cardiopulmonary bypass; 2.2 (0.3) vs. 1.9 (0.3) µm, p = 0.037 and during the first three postoperative days; 2.4 (0.3) vs. 1.9 (0.3) µm, p = 0.003. Increased plasma heparan sulphate levels were inversely associated with the proportion of perfused vessels during cardiopulmonary bypass; R = -0.49, p = 0.02. Plasma syndecan-1 levels were inversely associated with the proportion of perfused vessels during the entire study period; R = -0.51, p < 0.0001. Our study shows that cardiopulmonary bypass-induced acute microcirculatory perfusion disturbances persist in the first three postoperative days, and are associated with prolonged endothelial glycocalyx shedding. This suggests prolonged impairment and delayed recovery of both microcirculatory perfusion and function after on-pump cardiac surgery.

Glycosaminoglycans analysis in blood and urine of patients with mucopolysaccharidosis.

To explore the correlation between glycosaminoglycan (GAG) levels and mucopolysaccharidosis (MPS) type, we have evaluated the GAG levels in blood of MPS II, III, IVA, and IVB and urine of MPS IVA, IVB, and VI by tandem mass spectrometry. Dermatan sulfate (DS), heparan sulfate (HS), keratan sulfate (KS; mono-sulfated KS, di-sulfated KS), and the ratio of di-sulfated KS in total KS were measured. Patients with untreated MPS II had higher levels of DS and HS in blood while untreated MPS III had higher levels of HS in blood than age-matched controls. Untreated MPS IVA had higher levels of KS in blood and urine than age-matched controls. The ratio of blood di-sulfated KS/total KS in untreated MPS IVA was constant and higher than that in controls for children up to 10 years of age. The ratio of urine di-sulfated KS/total KS in untreated MPS IVA was also higher than that in age-matched controls, but the ratio in untreated MPS IVB was lower than controls. ERT reduced blood DS and HS in MPS II, and urine KS in MPS IVA patients, although GAGs levels remained higher than the observed in age-matched controls. ERT did not change blood KS levels in MPS IVA. MPS VI under ERT still had an elevation of urine DS level compared to age-matched controls. There was a positive correlation between blood and urine KS in untreated MPS IVA patients but not in MPS IVA patients treated with ERT. Blood and urine KS levels were secondarily elevated in MPS II and VI, respectively. Overall, measurement of GAG levels in blood and urine is useful for diagnosis of MPS, while urine KS is not a useful biomarker for monitoring therapeutic efficacy in MPS IVA.

Incomplete biomarker response in mucopolysaccharidosis type I after successful hematopoietic cell transplantation.

BACKGROUND: Residual disease, primarily involving musculoskeletal tissue, is a common problem in patients with neuronopathic mucopolysaccharidosis type I (MPS I, Hurler or severe Hurler-Scheie phenotype) after a successful hematopoietic cell transplantation (HCT). The concentration of the GAG derived biomarkers heparan sulfate (HS) and dermatan sulfate (DS), may reflect residual disease and is used for monitoring biochemical response to therapies. This study investigates the response of HS and DS in blood and urine to HCT in MPS I patients. METHODS: In 143 blood- and urine samples of 17 neuronophatic MPS I patients, collected prior and post successful HCT, the concentration of the disaccharides derived after full enzymatic digestion of HS and DS were analyzed by multiplex liquid chromatography tandem-mass spectrometry (LC-MS/MS). RESULTS: Median follow up after HCT was 2.4years (range 0-11years). HCT led to a rapid decrease of both HS and DS. However, only 38% of the patients reached normal HS levels in blood and even less patients (6%) reached normal DS levels. In none of the patients normalization of HS or DS was observed in urine. CONCLUSIONS: Biomarker response after HCT is incomplete, which may reflect residual disease activity. Novel therapeutic strategies should aim for full metabolic correction to minimize clinical manifestations.

Glycosaminoglycan levels in dried blood spots of patients with mucopolysaccharidoses and mucolipidoses.

Mucopolysaccharidoses (MPSs) and mucolipidoses (ML) are groups of lysosomal storage disorders in which lysosomal hydrolases are deficient leading to accumulation of undegraded glycosaminoglycans (GAGs), throughout the body, subsequently resulting in progressive damage to multiple tissues and organs. Assays using tandem mass spectrometry (MS/MS) have been established to measure GAGs in serum or plasma from MPS and ML patients, but few studies were performed to determine whether these assays are sufficiently robust to measure GAG levels in dried blood spots (DBS) of patients with MPS and ML. MATERIAL AND METHODS: In this study, we evaluated GAG levels in DBS samples from 124 MPS and ML patients (MPS I=16; MPS II=21; MPS III=40; MPS IV=32; MPS VI=10; MPS VII=1; ML=4), and compared them with 115 age-matched controls. Disaccharides were produced from polymer GAGs by digestion with chondroitinase B, heparitinase, and keratanase II. Subsequently, dermatan sulfate (DS), heparan sulfate (HS-0S, HS-NS), and keratan sulfate (mono-sulfated KS, di-sulfated KS, and ratio of di-sulfated KS in total KS) were measured by MS/MS. RESULTS: Untreated patients with MPS I, II, VI, and ML had higher levels of DS compared to control samples. Untreated patients with MPS I, II, III, VI, and ML had higher levels of HS-0S; and untreated patients with MPS II, III and VI and ML had higher levels of HS-NS. Levels of KS were age dependent, so although levels of both mono-sulfated KS and di-sulfated KS were generally higher in patients, particularly for MPS II and MPS IV, age group numbers were not sufficient to determine significance of such changes. However, the ratio of di-sulfated KS in total KS was significantly higher in all MPS patients younger than 5years old, compared to age-matched controls. MPS I and VI patients treated with HSCT had normal levels of DS, and MPS I, VI, and VII treated with ERT or HSCT had normal levels of HS-0S and HS-NS, indicating that both treatments are effective in decreasing blood GAG levels. CONCLUSION: Measurement of GAG levels in DBS is useful for diagnosis and potentially for monitoring the therapeutic efficacy in MPS.

Newborn screening for mucopolysaccharidoses: a pilot study of measurement of glycosaminoglycans by tandem mass spectrometry.

BACKGROUND: Mucopolysaccharidoses (MPS) are a group of inborn errors of metabolism that are progressive and usually result in irreversible skeletal, visceral, and/or brain damage, highlighting a need for early diagnosis. METHODS: This pilot study analyzed 2862 dried blood spots (DBS) from newborns and 14 DBS from newborn patients with MPS (MPS I, n = 7; MPS II, n = 2; MPS III, n = 5). Disaccharides were produced from polymer GAGs by digestion with chondroitinase B, heparitinase, and keratanase II. Heparan sulfate (0S, NS), dermatan sulfate (DS) and mono- and di-sulfated KS were measured by liquid chromatography tandem mass spectrometry (LC-MS/MS). Median absolute deviation (MAD) was used to determine cutoffs to distinguish patients from controls. Cutoffs were defined as median + 7× MAD from general newborns. RESULTS: The cutoffs were as follows: HS-0S > 90 ng/mL; HS-NS > 23 ng/mL, DS > 88 ng/mL; mono-sulfated KS > 445 ng/mL; di-sulfated KS > 89 ng/mL and ratio di-KS in total KS > 32 %. All MPS I and II samples were above the cutoffs for HS-0S, HS-NS, and DS, and all MPS III samples were above cutoffs for HS-0S and HS-NS. The rate of false positives for MPS I and II was 0.03 % based on a combination of HS-0S, HS-NS, and DS, and for MPS III was 0.9 % based upon a combination of HS-0S and HS-NS. CONCLUSIONS: Combination of levels of two or more different GAGs improves separation of MPS patients from unaffected controls, indicating that GAG measurements are potentially valuable biomarkers for newborn screening for MPS.

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.

Plasma syndecan-1 and heparan sulfate correlate with microvascular glycocalyx degradation in hemorrhaged rats after different resuscitation fluids.

The endothelial glycocalyx plays an essential role in many physiological functions and is damaged after hemorrhage. Fluid resuscitation may further change the glycocalyx after an initial hemorrhage-induced degradation. Plasma levels of syndecan-1 and heparan sulfate have been used as indirect markers for glycocalyx degradation, but the extent to which these measures are representative of the events in the microcirculation is unknown. Using hemorrhage and a wide range of resuscitation fluids, we studied quantitatively the relationship between plasma biomarkers and changes in microvascular parameters, including glycocalyx thickness. Rats were bled 40% of total blood volume and resuscitated with seven different fluids (fresh whole blood, blood products, and crystalloids). Intravital microscopy was used to estimate glycocalyx thickness in >270 postcapillary venules from 58 cremaster preparations in 9 animal groups; other microvascular parameters were measured using noninvasive techniques. Systemic physiological parameters and blood chemistry were simultaneously collected. Changes in glycocalyx thickness were negatively correlated with changes in plasma levels of syndecan-1 (r = -0.937) and heparan sulfate (r = -0.864). Changes in microvascular permeability were positively correlated with changes in both plasma biomarkers (r = 0.8, P < 0.05). Syndecan-1 and heparan sulfate were also positively correlated (r = 0.7, P < 0.05). Except for diameter and permeability, changes in local microcirculatory parameters (red blood cell velocity, blood flow, and wall shear rate) did not correlate with plasma biomarkers or glycocalyx thickness changes. This work provides a quantitative framework supporting plasma syndecan-1 and heparan sulfate as valuable clinical biomarkers of glycocalyx shedding that may be useful in guiding resuscitation strategies following hemorrhage.

The circulating glycosaminoglycan signature of respiratory failure in critically ill adults.

Systemic inflammatory illnesses (such as sepsis) are marked by degradation of the endothelial glycocalyx, a layer of glycosaminoglycans (including heparan sulfate, chondroitin sulfate, and hyaluronic acid) lining the vascular lumen. We hypothesized that different pathophysiologic insults would produce characteristic patterns of released glycocalyx fragments. We collected plasma from healthy donors as well as from subjects with respiratory failure due to altered mental status (intoxication, ischemic brain injury), indirect lung injury (non-pulmonary sepsis, pancreatitis), or direct lung injury (aspiration, pneumonia). Mass spectrometry was employed to determine the quantity and sulfation patterns of circulating glycosaminoglycans. We found that circulating heparan sulfate fragments were significantly (23-fold) elevated in patients with indirect lung injury, while circulating hyaluronic acid concentrations were elevated (32-fold) in patients with direct lung injury. N-Sulfation and tri-sulfation of heparan disaccharides were significantly increased in patients with indirect lung injury. Chondroitin disaccharide sulfation was suppressed in all groups with respiratory failure. Plasma heparan sulfate concentrations directly correlated with intensive care unit length of stay. Serial plasma measurements performed in select patients revealed that circulating highly sulfated heparan fragments persisted for greater than 3 days after the onset of respiratory failure. Our findings demonstrate that circulating glycosaminoglycans are elevated in patterns characteristic of the etiology of respiratory failure and may serve as diagnostic and/or prognostic biomarkers of critical illness.

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.