Increase in the peripheral blood methylglyoxal levels in 10% of hospitalized chronic schizophrenia patients.

Increasing evidence indicates that enhanced peripheral carbonyl stress markers exist in subtype of schizophrenia, although it may not be the primary cause. This study aimed to investigate whether plasma concentrations of methylglyoxal, 3-deoxy-glucosone, and glyoxal, which are reactive intermediates of protein metabolism in carbonyl stress, are changed in patients with schizophrenia and can function as potential biomarkers for schizophrenia with enhanced carbonyl stress. Plasma concentrations of these di-carbonyls were simultaneously estimated in 40 patients with schizophrenia and 40 healthy controls. As a result, no statistically significant differences were observed in mean plasma concentrations of three di-carbonyls between patients and controls. However, a remarkable increase in methylglyoxal concentrations was observed in four patients but not in controls. This increase was not found with regard to 3-deoxyglucosone and glyoxal both of patients and controls. Our correlation analysis showed that both the plasma methylglyoxal and glyoxal concentrations were significantly correlated with 3-deoxyglucosone concentrations in 40 patients and 40 controls. However, the plasma methylglyoxal concentrations did not show any significant correlation with the glyoxal concentrations in the patients or the controls. In four patients with extremely high methylglyoxal levels, the plasma methylglyoxal and glyoxal concentrations were not correlated to the 3-deoxyglucosone concentrations. Methylglyoxal is a physiological substrate of the glyoxalase system, and the accelerated accumulation of this compound lowers the glyoxalase I activity. These results suggested that this increase in four patients with high methylglyoxal levels may indicate the presence of a subtype of chronic schizophrenia that is associated with enhanced carbonyl stress.

Evaluation of the specific structures of IgA1 hinge glycopeptide in 30 IgA nephropathy patients by mass spectrometry.

BACKGROUND: Although many reports have described the abnormal structures of O-glycan in the IgA1 hinge region in IgA nephropathy (IgAN), the specific glycopeptide that can be used as a diagnostic biomarker for IgAN has not yet been identified. To pursue this diagnostic approach, we used mass spectrometric analysis to search for specific structures in IgA1 hinge glycopeptides in 30 IgAN patients contrasted with 30 healthy controls. METHOD: IgA1 hinge glycopeptides were individually purified from the sera of 30 biopsy-proven IgAN patients and 30 healthy controls. The structure of each glycopeptide was analyzed by ion trap mass spectrometry. Sugar conformations in each glycopeptide were estimated by collision-induced dissociation tandem mass spectrometry. Furthermore, to search for specific O-glycans in IgAN patients with a statistical significance, the identified hinge glycopeptides were analyzed by Fisher exact test. RESULT: A total of 57 hinge glycopeptides were identified from each of the 2 sample groups. Among the structures of the hinge glycopeptides identified, statistical significance was found for 6 glycopeptides (O-glycan compositions were 33-mer hinge core peptides + xGalNAc + yGal + zNeu5Ac; x:y:z = 5:3:3, 5:3:0, 5:2:1, 4:2:2, 3:1:1, 3:1:0) by Fisher exact test (p<0.05). In these 6 O-glycan compositions, 3 compositions (x:y:z = 4:2:2, 3:1:1, 3:1:0) were only observed in IgAN patients and were absent in the 30 controls. CONCLUSION: Statistically specific O-glycans were identified in 30 IgAN patients compared with 30 controls. These results open the possibility for preparation of lectin and/or antibodies binding to specific glycopeptides in IgAN.

Serum under-galactosylated IgA1 is increased in Japanese patients with IgA nephropathy.

BACKGROUND: Immunoglobulin A nephropathy (IgAN) is characterized by an aberrant structure of O-glycans in the IgA1 hinge region. Recently, under-galactosylated IgA1 has been found to be increased in Caucasian IgAN patients. Thus, we examined this in Japanese IgAN patients. METHODS: An enzyme-linked immunosorbent assay of binding between Helix aspersa (HAA) and serum IgA was performed in Japanese IgAN patients and the HAA-IgA binding levels were compared among IgAN patients (n = 41), patients with other forms of kidney disease (OKD, n = 43) and healthy controls (n = 38). The clinicopathological severity of IgAN was then analysed between patients with high and low HAA-IgA binding levels. The levels were also compared in 11 patients before and after the combination of tonsillectomy and steroid pulse therapy. Furthermore, we examined the O-glycan structure of IgA1 hinge glycopeptides by mass spectrometry (MS). RESULTS: The HAA-IgA binding levels were significantly higher in IgAN patients compared with either healthy controls (P = 0.0025) or those with OKD (P = 0.016). To reflect the absolute level of under-galactosylated IgA, we multiplied the HAA-IgA binding level by the serum IgA concentration to produce an indicative value. The specificity and sensitivity of this value were 89% and 66%, respectively. MS showed that peak distribution of IgA1 hinge glycopeptides was shifted to smaller molecular weights in high HAA-IgA-binding IgAN patients. There was no correlation between the HAA-IgA binding level and either disease severity or the use of combination therapy. CONCLUSIONS: HAA-IgA binding is significantly increased in Japanese IgAN patients. This potential IgAN marker is not affected by disease severity or therapeutic intervention.

Protective role of anti-synthetic hinge peptide antibody for glomerular deposition of hypoglycosylated IgA1.

BACKGROUND: The KM mouse lacks endogenous genes for immunoglobulins and carries the entire human IgH locus and the IgLk transgene. Therefore, human IgA1 does not provoke a hetero-immune response. We had observed mesangial IgA deposits in KM mice given desialo-degalacto (DeS/DeGal) IgA1. METHODS: In this study, the mice were immunized with synthetic IgA1 hinge (glyco-)peptide before administration of DeS/DeGal IgA1, and the effects of the pre-immunization were evaluated. Mice were divided into sHP, 5GalNAc-sHP and non-immunization groups. In two pre-immunization groups, KLH-conjugated sHP or KLH-5GalNAc-sHP, which has five GalNAc residues, was subcutaneously given three times every 2 weeks. Two weeks after the final pre-immunization, DeS/DeGal IgA1 was administered daily for 5 weeks. Serial serum levels of anti-sHP and anti-IgA1 antibodies were evaluated by ELISA. On the day of the last administration of IgA1, renal biopsy was performed. RESULTS: Mesangial IgA deposits were observed in all non-immunized mice. In pre-immunized mice, IgA deposition was not detected in 6 of 13 sHP mice and 1 of 4 5GalNAc-sHP mice. The intensities of IgA deposits were significantly different between sHP groups and non-immunized (P = 0.003) groups. There was a significant inverse correlation between the intensities of IgA deposits and the anti-sHP antibody titers (P = 0.016). CONCLUSIONS: These results suggest that the anti-IgA1 hinge peptide antibody plays a role in the inhibition of glomerular IgA deposition.

Suppression of renal alpha-dicarbonyl compounds generated following ureteral obstruction by kidney-specific alpha-dicarbonyl/L-xylulose reductase.

Renal unilateral ureteral obstruction (UUO) causes acute generation of alpha-dicarbonyl stress substances, such as glyoxal, 3-deoxyglucosone, and methylglyoxal, in the kidneys. These alpha-dicarbonyl compounds are prone to form advanced glycation end products (AGEs) via the nonenzymatic Maillard reaction. Using transgenic (Tg) mice overexpressing a kidney-specific short-chain oxidoreductase, alpha-dicarbonyl/L-xylulose reductase (DCXR), we measured generation of alpha-dicarbonyls following UUO by means of electrospray ionization/liquid chromatography/mass spectrometry in their kidney extracts. The accumulation of 3-deoxyglucosone was significantly reduced in the kidneys of the mice Tg for DCXR compared to their wild-type littermates, demonstrating 4.91 +/- 2.04 vs. 6.45 +/- 1.85 ng/mg protein (P = 0.044) for the obstructed kidneys, and 3.68 +/- 1.95 vs. 5.20 +/- 1.39 ng/mg protein (P = 0.026) for the contralateral kidneys. Despite the reduction in accumulated alpha-dicarbonyls, collagen III content in kidneys of the Tg mice and their wild-type littermates showed no difference as monitored by in situ hybridization. Collectively, DCXR may function in the removal of renal alpha-dicarbonyl compounds under oxidative circumstances, but it is not sufficient to suppress acute renal fibrosis during 7 days UUO.

Suppression of AGE precursor formation following unilateral ureteral obstruction in mouse kidneys by transgenic expression of alpha-dicarbonyl/L-xylulose reductase.

Unilateral ureteral obstruction (UUO) of kidneys causes acute generation of carbonyl stress. By electrospray ionization/liquid chromatography/mass spectrometry (ESI/LC/MS) we measured the content of methyl glyoxal, glyoxal, and 3-deoxyglucosone in mouse kidney extracts following UUO. UUO resulted in elevation of these dicarbonyls in the obstructed kidneys. Furthermore, the accumulation of 3-deoxyglucosone was significantly reduced in the kidneys of mice transgenic for alpha-dicarbonyl/L-xylulose reductase (DCXR) as compared to their wild-type littermates, demonstrating 4.91+/-2.04 vs. 6.45+/-1.85 ng/mg protein (P=0.044) for the obstructed kidneys, and 3.68+/-1.95 vs. 5.20+/-1.39 ng/mg protein (P=0.026) for the contralateral kidneys. On the other hand, collagen III content in kidneys showed no difference as monitored by in situ hybridization. Collectively, DCXR may function in the removal of renal alpha-dicarbonyl compounds under oxidative circumstances, but it was not sufficient to suppress acute renal fibrosis during 7 d of UUO by itself.

Structural analyses of O-glycan sugar chains on IgA1 hinge region using SELDI-TOFMS with various lectins.

The aim of the study was to develop a simple and precise method for identifying glycosylation of the IgA hinge region using surface-enhanced laser desorption/ionization (SELDI)-TOFMS with a lectin-coupled ProteinChip array. Serum IgA was isolated using an anti-IgA antibody column. Following reduction, alkylation, and trypsin digestion, the IgA fragments were applied on the ProteinChip coupled with jacalin, peanut agglutinin (PNA), or Vilsa villosa lectin (VVL). The SELDI-TOFMS peaks corresponding to the fragments containing IgA1 hinge glycopeptides trapped by each lectin were compared. The jacalin-, PNA-, and VVL-immobilized ProteinChips detected 13, 4, and 2 peaks, respectively. One major peak was confirmed as a glycopeptide by MS/MS analysis. These results suggest that a lectin-immobilized ProteinChip assay can be used to simplify the procedures for the analyses of the O-glycans in IgA1 hinge. This method potentially makes it possible to identify a disease-specific glycoform by selecting the appropriate ligand-coupled ProteinChip array.

IgA1 molecules produced by tonsillar lymphocytes are under-O-glycosylated in IgA nephropathy.

BACKGROUND: Human serum immunoglobulin A1 (IgA1) has a unique mucine-like structure in its hinge region that contains O-glycans and proline-rich peptides. We previously reported the under-O-glycosylation of the hinge in serum IgA1 and deposited IgA1 in glomeruli (glomerular IgA1) in IgA nephropathy. The clinical development and exacerbation of IgA nephropathy frequently are preceded by episodes of upper respiratory tract infections. Therefore, tonsils, which represent the predominant immunocompetent tissue of the upper respiratory tract, may be related to the pathogenesis of IgA nephropathy. In this study, we investigated the O-glycan structure of IgA1 produced by tonsillar lymphocytes (tonsillar IgA1), suspecting that tonsillar IgA1 is one of the origins of glomerular IgA1 in patients with IgA nephropathy. METHODS: Extracted tonsils were obtained from 7 patients with IgA nephropathy and 5 patients with chronic tonsillitis as controls. Tonsillar lymphocytes separated from extracted tonsils were cultured for 7 days, and IgA1 in the culture medium was purified. The varieties of O-glycans in tonsillar IgA1 were determined from the molecular weights measured by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. RESULTS: A significant increase in the percentage of asialo-agalacto type O-glycans was found in tonsillar IgA1 in 4 of 7 patients with IgA nephropathy (57.1%) compared with controls. Between the IgA nephropathy and control groups, the difference was statistically significant (P = 0.047). CONCLUSION: This study provides precise information about the structure of O-glycans in tonsillar IgA1 in patients with IgA nephropathy. Our results suggest that tonsils produced the underglycosylated IgA1 molecules in patients with IgA nephropathy.