Imidazolium labelling permits the sensitive mass-spectrometric detection of N-glycosides directly from serum.

A novel imidazolium derivative (GITag) shows superior ionisation and consequently allows increased mass spectrometric detection capabilities of oligosaccharides and N-glycans. Here we demonstrate that human serum samples can be directly labelled by GITag on a MALDI target plate, abrogating prevalently required sample pretreatment or clean-up steps.

Association of a Marker of N-Acetylglucosamine With Progressive Multiple Sclerosis and Neurodegeneration.

Importance: N-glycan branching modulates cell surface receptor availability, and its deficiency in mice promotes inflammatory demyelination, reduced myelination, and neurodegeneration. N-acetylglucosamine (GlcNAc) is a rate-limiting substrate for N-glycan branching, but, to our knowledge, endogenous serum levels in patients with multiple sclerosis (MS) are unknown. Objective: To investigate a marker of endogenous serum GlcNAc levels in patients with MS. Design, Setting, and Participants: A cross-sectional discovery study and cross-sectional confirmatory study were conducted at 2 academic MS centers in the US and Germany. The discovery study recruited 54 patients with MS from an outpatient clinic as well as 66 healthy controls between April 20, 2010, and June 21, 2013. The confirmatory study recruited 180 patients with MS from screening visits at an academic MS study center between April 9, 2007, and February 29, 2016. Serum samples were analyzed from December 2, 2013, to March 2, 2015. Statistical analysis was performed from February 23, 2020, to March 18, 2021. Main Outcomes and Measures: Serum levels of GlcNAc plus its stereoisomers, termed N-acetylhexosamine (HexNAc), were assessed using targeted tandem mass spectroscopy. Secondary outcomes (confirmatory study) comprised imaging and clinical disease markers. Results: The discovery cohort included 66 healthy controls (38 women; mean [SD] age, 42 [20] years), 33 patients with relapsing-remitting MS (RRMS; 25 women; mean [SD] age, 50 [11] years), and 21 patients with progressive MS (PMS; 14 women; mean [SD] age, 55 [7] years). The confirmatory cohort included 125 patients with RRMS (83 women; mean [SD] age, 40 [9] years) and 55 patients with PMS (22 women; mean [SD] age, 49 [80] years). In the discovery cohort, the mean (SD) serum level of GlcNAc plus its stereoisomers (HexNAc) was 710 (174) nM in healthy controls and marginally reduced in patients with RRMS (mean [SD] level, 682 [173] nM; P = .04), whereas patients with PMS displayed markedly reduced levels compared with healthy controls (mean [SD] level, 548 [101] nM; P = 9.55 × 10-9) and patients with RRMS (P = 1.83 × 10-4). The difference between patients with RRMS (mean [SD] level, 709 [193] nM) and those with PMS (mean [SD] level, 405 [161] nM; P = 7.6 × 10-18) was confirmed in the independent confirmatory cohort. Lower HexNAc serum levels correlated with worse expanded disability status scale scores (ρ = -0.485; P = 4.73 × 10-12), lower thalamic volume (t = 1.7; P = .04), and thinner retinal nerve fiber layer (B = 0.012 [SE = 7.5 × 10-11]; P = .008). Low baseline serum HexNAc levels correlated with a greater percentage of brain volume loss at 18 months (t = 1.8; P = .04). Conclusions and Relevance: This study suggests that deficiency of GlcNAc plus its stereoisomers (HexNAc) may be a biomarker for PMS. Previous preclinical, human genetic, and ex vivo human mechanistic studies revealed that N-glycan branching and/or GlcNAc may reduce proinflammatory responses, promote myelin repair, and decrease neurodegeneration. Combined, the data suggest that GlcNAc deficiency may be associated with progressive disease and neurodegeneration in patients with MS.

JF1/B6F1 Ngly1-/- mouse as an isogenic animal model of NGLY1 deficiency.

N-Glycanase 1 (NGLY1) deficiency is a congenital disorder caused by mutations in the NGLY1 gene. Because systemic Ngly1-/- mice with a C57BL/6 (B6) background are embryonically lethal, studies on the mechanism of NGLY1 deficiency using mice have been problematic. In this study, B6-Ngly1-/+ mice were crossed with Japanese wild mice-originated Japanese fancy mouse 1 (JF1) mice to produce viable F2 Ngly1-/- mice from (JF1×B6)F1 Ngly1-/+ mice. Systemic Ngly1-/- mice with a JF1 mouse background were also embryonically lethal. Hybrid F1 Ngly1-/- (JF1/B6F1) mice, however, showed developmental delay and motor dysfunction, similar to that in human patients. JF1/B6F1 Ngly1-/- mice showed increased levels of plasma and urinary aspartylglycosamine, a potential biomarker for NGLY1 deficiency. JF1/B6F1 Ngly1-/- mice are a useful isogenic animal model for the preclinical testing of therapeutic options and understanding the precise pathogenic mechanisms responsible for NGLY1 deficiency.

Bariatric surgery decreases oxidative stress and protein glycosylation in patients with morbid obesity.

BACKGROUND: There is growing evidence that oxidative stress (OS) is a critical factor linking obesity with its associated comorbidities, such as cardiovascular diseases. AIM: To evaluate the degree of OS in people with morbid obesity and its relationship with glycoproteins, determined using 1H-NMR spectroscopy, before and after bariatric surgery (BS). METHODS: In this observational cohort study, plasma from 24 patients with BMI ≥ 40 kg/m2 (age: 21-65 years) was used to measure metabolites implicated in OS. We measured glycoprotein (GlycA, GlycB and GlycF) areas and shape factors (H/W = height/width). RESULTS: One year after BS, oxidized low-density lipoprotein had decreased by 49% (P < .0001), malondialdehyde by 32% (P = .0019) and lipoprotein (a) by 21% (P = .0039). The antioxidant enzymes paraoxonase-1 and catalase increased after BS (43%, P < .0001 and 54%, P = .0002, respectively). Superoxide dismutase-2 had fallen 1 year after BS (32%, P = .0052). After BS, both the glycoprotein areas and shape factors decreased by 20%-26%. These glycoproteins were significantly correlated with OS parameters. The plasma atherogenic index was 63% higher in obese individuals than 1 year after BS and correlated positively with glycoproteins. CONCLUSION: For the first time, we here demonstrate the relationship between OS parameters and glycoproteins in people with morbid obesity. So glycoproteins could therefore be a good indicator, together with the oxidative state to assess patient prognosis after BS.

GlycA, a novel marker for low grade inflammation, reflects gut microbiome diversity and is more accurate than high sensitive CRP in reflecting metabolomic profile.

INTRODUCTION: Gut microbiota is, along with adipose tissue, recognized as a source for many metabolic and inflammatory disturbances that may contribute to the individual's state of health. OBJECTIVES: We investigated in cross-sectional setting the feasibility of utilizing GlycA, a novel low grade inflammatory marker, and traditional low grade inflammatory marker, high sensitivity CRP (hsCRP), in reflecting serum metabolomics status and gut microbiome diversity. METHODS: Fasting serum samples of overweight/obese pregnant women (n = 335, gestational weeks: mean 13.8) were analysed for hsCRP by immunoassay, GlycA and metabolomics status by NMR metabolomics and faecal samples for gut microbiome diversity by metagenomics. The benefits of GlycA as a metabolic marker were investigated against hsCRP. RESULTS: The GlycA concentration correlated with more of the metabolomics markers (144 out of 157), than hsCRP (55 out of 157) (FDR < 0.05). The results remained essentially the same when potential confounding factors known to associate with GlycA and hsCRP levels were taken into account (P < 0.05). This was attributable to the detected correlations between GlycA and the constituents and concentrations of several sized VLDL-particles and branched chain amino acids, which were statistically non-significant with regard to hsCRP. GlycA, but not hsCRP, correlated inversely with gut microbiome diversity. CONCLUSION: GlycA is a superior marker than hsCRP in assessing the metabolomic profile and gut microbiome diversity. It is proposed that GlycA may act as a novel marker that reflects both the gut microbiome and adipose tissue originated metabolic aberrations; this proposal will need to be verified with regard to clinical outcomes. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov, NCT01922791, August 14, 2013.

Aspartylglycosamine is a biomarker for NGLY1-CDDG, a congenital disorder of deglycosylation.

BACKGROUND: NGLY1-CDDG is a congenital disorder of deglycosylation caused by a defective peptide:N-glycanase (PNG). To date, all but one of the reported patients have been diagnosed through whole-exome or whole-genome sequencing, as no biochemical marker was available to identify this disease in patients. Recently, a potential urinary biomarker was reported, but the data presented suggest that this marker may be excreted intermittently. METHODS: In this study, we performed untargeted direct-infusion high-resolution mass spectrometry metabolomics in seven dried blood spots (DBS) from four recently diagnosed NGLY1-CDDG patients, to test for small-molecule biomarkers, in order to identify a potential diagnostic marker. Results were compared to 125 DBS of healthy controls and to 238 DBS of patients with other diseases. RESULTS: We identified aspartylglycosamine as the only significantly increased compound with a median Z-score of 4.8 (range: 3.8-8.5) in DBS of NGLY1-CDDG patients, compared to a median Z-score of -0.1 (range: -2.1-4.0) in DBS of healthy controls and patients with other diseases. DISCUSSION: The increase of aspartylglycosamine can be explained by lack of function of PNG. PNG catalyzes the cleavage of the proximal N-acetylglucosamine residue of an N-glycan from the asparagine residue of a protein, a step in the degradation of misfolded glycoproteins. PNG deficiency results in a single N-acetylglucosamine residue left attached to the asparagine residue which results in free aspartylglycosamine when the glycoprotein is degraded. Thus, we here identified aspartylglycosamine as the first potential small-molecule biomarker in DBS for NGLY1-CDDG, making a biochemical diagnosis for NGLY1-CDDG potentially feasible.

Surface plasmon resonance biosensor for the accurate and sensitive quantification of O-GlcNAc based on cleavage by ß-D-N-acetylglucosaminidase.

Abnormal O-linked-N-acetylglucosamine (O-GlcNAc) concentrations have been associated with a variety of diseases (e.g., cancer, Alzheimer's disease, cardiovascular disease, etc.). However, O-GlcNAc detection is complicated, time-consuming and has poor specificity, therefore, the accurate detection of O-GlcNAc is difficult. In this study, an accurate and sensitive surface plasmon resonance (SPR) biosensor for O-GlcNAc detection that is based on ß-D-N-acetylglucosaminidase (OGA) and Au nanoparticles (AuNPs) was developed. In this strategy, AuNPs were used to amplify the SPR signal and improve the biosensor's sensitivity; OGA was used to cleave O-GlcNAc from O-GlcNAcylated biomolecules. The interaction between AuNPs labeled wheat germ agglutinin (AuNPs/WGA) and O-GlcNAcylated biomolecules on a modified Au film treated with and without OGA was recorded by SPR. The change of the SPR signal moves linearly with the amount of O-GlcNAc on the Au film and thus could be used for the detection of O-GlcNAc. By recording the difference of the SPR signals, this method can avoid disturbances from other sugars and nonspecific adsorption of AuNPs and thus enable the accurate detection of O-GlcNAc. The accurate detection range of O-GlcNAc was 4.65 × 10-12 to 4.65 × 10-7 M which was obtained by quantifying the amount of a standard O-GlcNAcylated peptide (O-GlcNAc-CREB), and the detection limit is 4.65 × 10-13 M. More importantly, the strategy was successfully used to detect O-GlcNAc in a real α-crystallin protein, cancer cell lysates and blood samples with satisfactory results. The study's results imply that this accurate and sensitive method has the potential to be applied in the early clinical diagnosis of O-GlcNAc-related diseases.

GlycA and hsCRP are independent and additive predictors of future cardiovascular events among patients undergoing angiography: The intermountain heart collaborative study.

BACKGROUND: GlycA is an inflammatory marker that is raised in patients with cardiometabolic diseases and associated with cardiovascular (CV) events. We sought to determine if GlycA adds independent value to hsCRP for CV risk prediction. METHODS: Patients in the Intermountain Heart Collaborative Study who underwent coronary angiography and had plasma GlycA and hsCRP levels were studied (n = 2996). Patients were followed for 7.0 ±â€¯2.8 years. GlycA and hsCRP were moderately correlated (r = 0.46, P < .0001). GlycA and hsCRP concentrations were stratified into high and low categories by their median values. Multivariable cox hazard regression was utilized to determine the associations of GlycA quartiles, as well as high and low categories of GlycA and hsCRP, with major adverse cardiovascular events (MACE) defined as the composite of death, myocardial infarction (MI), heart failure (HF) hospitalization, and stroke. RESULTS: The highest GlycA quartile was associated with future MACE [HR: 1.43; 95% CI: 1.22-1.69; P < .0001]. Patients with high GlycA and high hsCRP had more diabetes, hyperlipidemia, hypertension, HF, renal failure and MI, but not coronary artery disease. High GlycA and hsCRP (H/H) versus low GlycA and hsCRP (L/L) was associated with MACE, death and HF hospitalization, but not MI or stroke. Combined MACE rates were 33.5%, 41.3%, 35.7% and 49.1% for L/L, L/H, H/L and H/H categories of GlycA/hsCRP, respectively (P-trend < .0001). The interaction between GlycA and hsCRP was significant for the outcome of death (P = .03). CONCLUSION: In this study, levels of GlycA and hsCRP were independent and additive markers of risk for MACE, death and HF hospitalization.

GlycA, a novel biomarker of systemic inflammation and cardiovascular disease risk.

BACKGROUND: GlycA is a novel spectroscopic marker of systemic inflammation with low intra-individual variability and other attributes favoring its clinical use in patients with chronic inflammatory and autoimmune diseases. GlycA is unique in its composite nature, reflecting both increased glycan complexity and circulating acute phase protein levels during local and systemic inflammation. Recent studies of GlycA from cross-sectional, observational and interventional studies have been highly informative, demonstrating that GlycA is elevated in acute and chronic inflammation, predicts death in healthy individuals and is associated with disease severity in patients with chronic inflammatory diseases such as rheumatoid arthritis, psoriasis and lupus. Moreover, following treatment with biological therapy in psoriasis, reduction in skin disease severity was accompanied by a decrease in GlycA levels and improvement in vascular inflammation. CONCLUSIONS: Collectively, these findings suggest GlycA is a marker that tracks systemic inflammation and subclinical vascular inflammation. However, larger prospective studies and randomized trials are necessary in order to assess the impact of novel therapies on GlycA in patients with chronic inflammatory conditions, which may be concomitant with cardiovascular benefits.

Novel Protein Glycan-Derived Markers of Systemic Inflammation and C-Reactive Protein in Relation to Glycemia, Insulin Resistance, and Insulin Secretion.

OBJECTIVE: N-acetylglucosamine/galactosamine (GlycA) and sialic acid (GlycB) moieties of glycosylated serum proteins are nonspecific measures of inflammation, but conclusive data on their relationship with insulin resistance or insulin secretion are missing. Therefore, we aimed to examine the relation of GlycA, GlycB, and C-reactive protein (CRP) to direct measures of insulin sensitivity (insulin sensitivity index [SI]) and insulin secretion (acute insulin response [AIR]). RESEARCH DESIGN AND METHODS: This study used cross-sectional analyses and included 1,225 participants with and without type 2 diabetes in the Insulin Resistance Atherosclerosis Study (IRAS). SI and AIR were measured using the frequently sampled intravenous glucose tolerance test, and GlycA and GlycB were measured using nuclear magnetic resonance spectroscopy. RESULTS: GlycA and GlycB had a strong correlation with CRP (r = 0.60 [P < 0.001] and r = 0.46 [P < 0.001], respectively). In a linear regression model with both GlycA and CRP as independent variables, GlycA (ß × 1 SD, -0.04 ± 0.02; P < 0.01) and CRP (-0.06 ± 0.02; P < 0.001) were independently associated with SI even after adjusting for demographics, smoking, physical activity, plasma glucose, and BMI. However, neither CRP nor GlycA had an independent relationship with AIR. CONCLUSIONS: GlycA may complement CRP in evaluating the relationship between inflammation, glucose tolerance, and insulin resistance.

Circulating N-Linked Glycoprotein Side-Chain Biomarker, Rosuvastatin Therapy, and Incident Cardiovascular Disease: An Analysis From the JUPITER Trial.

BACKGROUND: GlycA, a novel protein glycan biomarker of N-acetyl side chains of acute-phase proteins, was recently associated with incident cardiovascular disease (CVD) in healthy women. Whether GlycA predicts CVD events in the setting of statin therapy in men and women without CVD but with evidence of chronic inflammation is unknown. METHODS AND RESULTS: In the Justfication for the Use of Statins in Prevention: an Intervention Trial Evaluating Rosuvastatin (JUPITER) trial (NCT00239681), participants with low-density lipoprotein cholesterol <130 mg/dL and high-sensitivity C-reactive protein (hsCRP) ≥2 mg/L were randomized to rosuvastatin 20 mg/day or placebo. GlycA was quantified by nuclear magnetic resonance spectroscopy in 12 527 before randomization and 10 039 participants at 1 year. A total of 310 first primary CVD events occurred during maximum follow-up of 5.0 years (median, 1.9). GlycA changed minimally after 1 year on study treatment: 6.8% and 4.7% decrease in the rosuvastatin and placebo groups, respectively. Overall, baseline GlycA levels were associated with increased risk of CVD: multivariable-adjusted hazard ratio (HR) per SD increment, 1.20 (95% CI, 1.08-1.34; P=0.0006). After additionally adjusting for hsCRP, this was slightly attenuated (HR, 1.18; 95% CI, 1.04-1.35; P=0.01). On-treatment GlycA levels were also associated with CVD; corresponding multivariable-adjusted HRs per SD before and after additionally adjusting for hsCRP: 1.27 (95% CI, 1.13-1.42; P<0.0001) and 1.24 (95% CI, 1.07-1.44; P=0.004), respectively. Tests for heterogeneity by treatment arm were not significant (P for interaction, >0.20). CONCLUSION: In the JUPITER trial, increased levels of GlycA were associated with an increased risk of CVD events independent of traditional risk factors and hsCRP. CLINICAL TRIALS REGISTRATION: URL: http://www.clinicaltrials.gov. Unique identifier: NCT00239681.

Circulating N-Linked Glycoprotein Acetyls and Longitudinal Mortality Risk.

RATIONALE: Circulating glycoprotein N-acetyl glucosamine residues have recently been associated with incident cardiovascular disease and diabetes mellitus. OBJECTIVE: Using a plasma glycan biosignature (GlycA) to identify circulating N-acetyl glycan groups, we examined the longitudinal association between GlycA and mortality among initially healthy individuals. METHODS AND RESULTS: We quantified GlycA by 400 MHz (1)H nuclear magnetic resonance spectroscopy in 27,524 participants in the Women's Health Study (NCT00000479). The primary outcome was all-cause mortality. We replicated the findings in an independent cohort of 12,527 individuals in the Justification for the Use of statins in Prevention: an Intervention Trial Evaluating Rosuvastatin (JUPITER) trial (NCT00239681). We also undertook secondary examination of cardiovascular disease and cancer mortality in the Women's Health Study. In the Women's Health Study, during 524,515 person-years of follow-up (median, 20.5 years), there were 3523 deaths. Risk factor-adjusted multivariable Cox proportional hazard ratio (95% confidence interval) per SD increment in GlycA for all-cause mortality was significantly increased at 5 years (1.21 [1.06-1.40]) and during maximal follow-up (1.14 [1.09-1.16]). Similar risk for all-cause mortality was observed in the replication cohort (1.33 [1.21-1.45]). In the Women's Health Study, risk of cardiovascular disease mortality was increased at 5 years (1.43 [1.05-1.95]) and during maximal follow-up (1.15 [1.04-1.26]) and of cancer mortality at 5 years (1.23 [1.02-1.47]) and during maximal follow-up (1.08 [1.01-1.16]). Examination of correlations and mortality associations adjusted for high-sensitivity C-reactive protein, fibrinogen, and intercellular adhesion molecule-1, suggested that GlycA reflects summative risk related to multiple pathways of systemic inflammation. CONCLUSIONS: Among initially healthy individuals, elevated baseline circulating glycoprotein N-acetyl methyl groups were associated with longitudinal risk of all-cause, cardiovascular, and cancer mortality.

The glycosylation of AGP and its associations with the binding to methadone.

Methadone remains the most common form of pharmacological therapy for opioid dependence; however, there is a lack of explanation for the reports of its relatively low success rate in achieving complete abstinence. One hypothesis is that in vivo binding of methadone to the plasma glycoprotein alpha-1-acid glycoprotein (AGP), to a degree dependent on the molecular structure, may render the drug inactive. This study sought to determine whether alterations present in the glycosylation pattern of AGP in patients undergoing various stages of methadone therapy (titration < two weeks, harm reduction < one year, long-term > one and a half years) could affect the affinity of the glycoprotein to bind methadone. The composition of AGP glycosylation was determined using high pH anion exchange chromatography (HPAEC) and intrinsic fluorescence analysed to determine the extent of binding to methadone. The monosaccharides galactose and N-acetyl-glucosamine were elevated in all methadone treatment groups indicating alterations in AGP glycosylation. AGP from all patients receiving methadone therapy exhibited a greater degree of binding than the normal population. This suggests that analysing the glycosylation of AGP in patients receiving methadone may aid in determining whether the therapy is likely to be effective.

Exploring leukocyte O-GlcNAcylation as a novel diagnostic tool for the earlier detection of type 2 diabetes mellitus.

CONTEXT: Because current tests available for the diagnosis of diabetes have shortcomings, a novel screening method for the earlier and more efficient detection of type 2 diabetes would be a significant clinical advance. OBJECTIVE: The hexosamine biosynthetic pathway usually acts as a fuel sensor, and its activation leads to O-linked ß-N-acetylglucosamine (O-GlcNAc) modification of target proteins (O-GlcNAcylation) in a glucose-responsive manner. O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) are responsible for O-GlcNAc addition and removal, respectively. Because higher hexosamine biosynthetic pathway flux is linked to insulin resistance/type 2 diabetes, we hypothesized that increased O-GlcNAcylation of leukocyte proteins can detect the onset of pre- and overt diabetes. DESIGN, SETTING AND PATIENTS: Seventy-four participants from Bellville and Stellenbosch (Western Cape, South Africa) were recruited and classified as normal, prediabetic, and diabetic individuals (American Diabetes Association criteria). MAIN OUTCOME MEASURES: Leukocytes isolated from study subjects were evaluated for O-GlcNAc, OGA, and O-GlcNAc transferase expression by flow cytometry and immunofluorescence microscopy. RESULTS: Flow cytometric analysis of leukocyte subtypes revealed increased O-GlcNAcylation in granulocytes vs. lymphocytes (P < 0.001). Diabetic individuals displayed higher leukocyte O-GlcNAcylation (P < 0.01), whereas granulocyte analysis showed an increase for prediabetic subjects (P < 0.01). However, OGA expression increased in leukocytes of diabetic subjects and is likely an adaptation to attenuate higher O-GlcNAcylation observed (P < 0.001). CONCLUSIONS: Together our data demonstrate that leukocyte (particularly granulocyte) O-GlcNAcylation could help detect pre- and overt diabetes and offer clinical value as unique markers for the earlier and more efficient detection of type 2 diabetes.

Human serum N-glycan profiles are age and sex dependent.

BACKGROUND: protein glycosylation varies with the physiological and pathological status of the cell. Consequently, analysis of protein-linked glycans has growing importance both in basic glycobiological research and as a potential tool for monitoring the physiological state in humans. DESIGN, SETTING AND PARTICIPANTS: a total of 265 healthy northern Chinese men and women were grouped by age and gender. The mean age in males and females was similar. OBJECTIVE: the study is aimed to evaluate the effects of the age and gender on the human serum N-glycans profiles in the clinical diagnose of ageing and disease. METHODS: the 265 human serum N-glycan profiles were obtained by DNA sequencer-assisted fluorophore-assisted carbohydrate electrophoresis. Comparison of N-glycan profiles was carried out among the different genders and age groups and the data were analysed with the GeneMapper software. RESULTS: age-related changes in the three N-glycan structures (NGA2F, NGA2FB and NA2F) were observed. Interestingly, fucosylation of N-glycans was significantly different (P < 0.0001) between men and women: more core-α-1,6-fucosylated glycans were detected in women, whereas more branching-α-1,3-fucosylated N-glycans were seen in men. CONCLUSIONS: the N-glycome profile in serum is gender and age dependent. This should be taken into consideration in the development of serum glycome markers.

Novel potential markers of nasopharyngeal carcinoma for diagnosis and therapy.

OBJECTIVE: To search for markers of nasopharyngeal carcinoma (NPC) for diagnosis. DESIGN AND METHODS: Using gas chromatography and mass spectrometry, we evaluated 51 serum metabolites in 49 NPC, 37 throat cancer patients and 40 healthy controls. High metabolites were selected and confirmed in NPC tissues. Sensitivity and specificity were appraised for 53 NPC diagnoses. RESULTS: Metabolic profiling revealed that kynurenine, N-acetylglucosaminylamine, N-acetylglucosamine and hydroxyphenylpyruvate increased in NPC patient sera. Their sensitivity and specificity were respectively 79% and 71%, 78% and 69%, 83% and 68%, 84% and 73% for NPC diagnosis. These increases were confirmed in NPC cells. Four metabolites gradually increased from stage I to stage III. After radiotherapy, four metabolites decreased gradually, and tended to a normal level, and were associated with rate of tumor reduction. CONCLUSION: The results reveal that kynurenine, N-acetylglucosaminylamine, N-acetylglucosamine and hydroxyphenylpyruvate are potentially markers of NPC for diagnosis and therapy.

Eryptosis in hereditary spherocytosis and thalassemia: role of glycoconjugates.

The present work is aimed to study the mechanism of faster erythrocyte clearance in hereditary spherocytosis (HS), a heterogeneous disorders characterized by alterations in the proteins of the red cell membrane skeleton along with different kinds of thalassemia. The maximum exposure of phosphatidylserine (PS) is found in HS compared to those in both α- and ß-thalassemia. Interestingly, in HS more PS exposed cells were found in younger erythrocytes compared to normal and the thalassemics where aged cells showed higher loss of PS asymmetry. Loss of sialic acid and GlcNAc bearing glycoconjugates, presumably the glycophorins, was also found upon aging. The loss of PS asymmetry together with the cell surface glycoproteins mediated by membrane vesiculation, seemed to play key role in early clearance of erythrocytes from circulation following a mechanism similar to HbEß-thalassemia.

Increased O-linked beta-N-acetylglucosamine levels on proteins improves survival, reduces inflammation and organ damage 24 hours after trauma-hemorrhage in rats.

OBJECTIVE: To evaluate the effects of O-linked beta-N-acetylglucosamine (O-GlcNAc) levels on survival, inflammation, and organ damage 24 hrs after trauma-hemorrhage. We have previously shown that increasing protein O-GlcNAc levels by different mechanisms reduced inflammatory responses and improved organ function 2 hrs after trauma-hemorrhage. DESIGN: Prospective, randomized, controlled study. SETTING: Animal research laboratory. SUBJECTS: Male, adult Sprague-Dawley rats. INTERVENTIONS: Overnight fasted animals were subjected to either sham surgery or trauma-hemorrhage and during the resuscitation phase received glucosamine (270 mg/kg) to increase O-GlcNAc synthesis or O-(2-acetamido-2-deoxy-D-glucopyranosylidene) amino N-phenyl carbamate (PUGNAc, 7 mg/kg) to inhibit O-GlcNAc removal, or mannitol as control. MEASUREMENTS AND MAIN RESULTS: Survival was followed up for 24 hrs. Surviving rats were euthanized and inflammatory responses, and end organ injuries were assessed. Both glucosamine and PUGNAc increased 24-hr survival compared with controls (control: 53%, GN: 85%, PUGNAc: 86%, log-rank test, p < .05). PUGNAc attenuated the trauma-hemorrhage-induced increase in serum interleukin-6 (sham surgery: 8 +/- 6, control: 181 +/- 36, PUGNAc: 42 +/- 22 pg/mL, p < .05), alanine transaminase (sham surgery: 95 +/- 14, control: 297 +/- 56, PUGNAc: 126 +/- 21 IU, p < .05), aspartate transaminase (sham surgery: 536 +/- 110, control: 1661 +/- 215, PUGNAc: 897 +/- 155 IU, p < .05), and lactate dehydrogenase (sham surgery: 160 +/- 18, control: 1499 +/- 311, PUGNAc: 357 +/- 99 IU, p < .05); however, glucosamine had no effect on these serum parameters. Furthermore, PUGNAc but not glucosamine maintained O-GlcNAc levels in liver and lung and significantly attenuated the NF-kappaB DNA activation in the liver. In the liver and heart, increased inducible nitric oxide synthase expression was also attenuated in the PUGNAc-treated group. CONCLUSIONS: These results demonstrate that increasing O-GlcNAc with either glucosamine or PUGNAc improved 24-hr survival after trauma-hemorrhage. However, only PUGNAc treatment attenuated significantly the subsequent tissue injury and inflammatory responses, suggesting that inhibition of O-GlcNAc removal may represent a new therapeutic approach for the treatment of hypovolemic shock.

Valuable markers for contrast-induced nephropathy in patients undergoing cardiac catheterization.

BACKGROUND: Contrast-induced nephropathy (CIN) frequently complicates cardiac catheterization, so the objectives of present study were to investigate the usefulness of cystatin C before catheterization and establish a cut-off level for CIN, and to examine the changes in cystatin C and several other markers in patients with and without CIN. METHODS AND RESULTS: Prospective study of consecutive 87 patients who underwent elective catheterization: moderate renal disease defined as glomerular filtration rate 30-59 ml . min(-1) .1.73 mm(-2); cystatin C and creatinine (Cr), urinary liver-type fatty acid-binding protein (L-FABP), alpha1, beta2 microglobulins, N-acetyl-beta-D-glucosaminidase, and microalbumin were measured immediately before, and 1, 2, and 3 days after catheterization. CIN occurred in 18 patients and receiver-operating characteristic analysis showed a higher area-under-the-curve for cystatin C compared with serum Cr (0.933 vs 0.832 p=0.012). At a cut-off level of >1.2 mg/L, cystatin C before catheterization exhibited 94.7% (95% confidence interval: 0.851-1.015) sensitivity and 84.8% specificity for detecting CIN. Cystatin C levels were higher in CIN patients than in those without CIN, even before catheterization (cystatin C: 1.08+/-0.22 vs 1.36+/-0.28 mg/L, p=0.007). Urinary L-FABP was increased on days 1 and 2 in patients with moderate renal disease. CONCLUSION: Cystatin C was useful for predicting the occurrence of CIN. Urinary L-FABP was the only marker of transient renotubular damage.

Bilateral pulvinar signal intensity decrease on T2-weighted images in patients with aspartylglucosaminuria.

BACKGROUND: Aspartylglucosaminuria (AGU) is an autosomal recessive lysosomal disease caused by deficiency of aspartylglucosaminidase. A thalamic T2 signal intensity decrease is associated with lysosomal diseases. PURPOSE: To investigate thalamic signal intensity in AGU by performing a retrospective review of brain magnetic resonance (MR) imaging studies of AGU patients. MATERIAL AND METHODS: A total of 25 MR examinations were available for 11 patients aged between 3 and 32 years (four patients underwent bone marrow transplantation). Of these, 13 examinations were performed after bone marrow transplantation. Five patients had from two to six examinations, and six patients had one examination each. In every patient, the diagnosis of AGU was confirmed by blood and urine tests. Eighteen examinations were performed with a 1.0T imager including dual spin-echo T2 and proton density (PD) axial and coronal images, and 10 examinations also included T1-weighted images. Seven examinations were performed with a 1.5T imager including turbo spin-echo axial and coronal T2-weighted images and axial fluid-attenuated inversion recovery (FLAIR) images; three examinations included T1-weighted three-dimensional magnetization-prepared rapid acquisition gradient-echo (3D MPRAGE) images. The signal intensity of the thalamus and pulvinar in every sequence was compared to that of the putamina. RESULTS: In AGU, thalamic alterations were first detectable on T2-weighted images (25 examinations in 11 patients) from the age of 3 years 6 months, showing decreased signal intensity in 21 of 24 examinations. T1-weighted images (13 examinations) showed slightly increased thalamic signal intensity in five out of seven examinations from the age of 7 years, and PD images (19 examinations) showed decreased signal intensity from the age of 16 years (three examinations). The pulvinar showed decreased signal intensity on spin-echo T2-weighted images for 14 of 18 examinations or on FLAIR sequences for seven of seven examinations from the age of 6 years and 6 months, both in patients with and without bone marrow transplantation, but no pulvinar alterations were observable on T1 and PD images. CONCLUSION: In AGU, the thalamus is affected. Pulvinar changes are visible only on T2-weighted images, and these may be the first changes reported in the group of lysosomal diseases.