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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.

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.

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. 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. 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). These results suggest that the anti-IgA1 hinge peptide antibody plays a role in the inhibition of glomerular IgA deposition.

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.

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.

A new AGE, N ω -carboxymethylarginine (CMA), was found in acid-soluble skin collagen of a newborn bovine prepared in vitro glycation with 1 M glucose incubation at 37 °C for about 30 days. CMA production was increased with incubation time in parallel, and after 30-day incubation, the yield was 100 times higher than that of pentosidine. This result suggested the importance of CMA as a major AGE in collagen. We have detected and measured the CMA level in human serum proteins by electrospray ionization/liquid chromatography/mass spectrometry (ESI/LC/MS), using CMA standard concentration curve. In this study, we first show the existence of CMA in vivo, and its serum level is significantly elevated in diabetic serum proteins, compared to age-matched control serum proteins. These results provide the possibility of CMA as a new diagnostic marker of glycation in diabetes.

A new advanced glycation end product (AGE), N(omega)-carboxymethyl-arginine (CMA), was found in acid-soluble skin collagen of a newborn bovine prepared by in vitro glycation with 1 M glucose incubation at 37 degrees C for about 30 days [ 1 ]. CMA production was increased with incubation time in parallel, and after 30 days incubation the yield was 100 times higher than that of pentosidine [ 1 ]. This result suggested the importance of CMA as a major AGE in collagen. We have detected and measured the CMA level in human serum proteins by electrospray ionization/liquid chromatography/mass spectrometry (ESI/LC/MS), using CMA standard concentration curve. In this report, we first show the existence of CMA in vivo, and its serum level is significantly elevated in diabetic serum proteins, compared to age-matched control serum proteins. These results provide strong evidence that CMA is a new diagnostic marker of glycation in diabetes.

A reliable identification method for laccaic acids which are the main components in lac dye has been established using ESI LC/MS/MS with a volatile mobile phase containing acetylacetone. Addition of acetylacetone to the mobile phase enables us to separate laccaic acids without reduction of peak resolution. The volatile mobile phase, acetonitrile-0.05% aqueous TFA solution (1-4) containing 0.005 M acetylacetone, is applicable to directly interfaced ESI LC/MS/MS without clogging problems. In all ES tandem mass spectra of laccaic acids, [M-H-CO2]- and [M-H-2CO2]- appeared in considerable abundance, together with the weak ions of [M-H]-, [M-H-H2O]-, [M-H-CO2-H2O]-, and [M-H-2CO2-H2O]- when [M-H]- was selected as the precursor ion. Laccaic acids can be easily identified by these product ions. The method has been successfully applied to identification of laccaic acids in a jelly and in “Shi-kou,” which is a stick lac imported 1,200 years ago.

Nω-carboxymethylarginine (CMA) is a new acid-labile advanced glycation endproduct (AGE). We immunized rabbits with glycated collagen and succeeded in raising the antibody against CMA. The antibody specifically reacted against glycated collagen and CMA, but did not recognize non-glycated collagen or Ne-carboxymethyllysine (CML). The anti-CMA antibody may be applied to estimate CMA as the new diagnostic marker.

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.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.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 (p0.05). 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.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.

アミロイド骨関節症は長期透析患者にとって重篤な合併症である. 一方インターロイキンー1β (IL-1β) は炎症反応に重要な役割を果していると考えられており, 今回我々は, アミロイド骨関節症を有する血液透析 (HD) 患者の, 末梢血単球IL-1β産生能および末梢血単球細胞内IL-1β濃度について検討した. 対象は, 骨嚢胞性病変または手根管症候群 (CTS) を認め, 骨関節痛を有するHD患者 (A群) 12例, 骨嚢胞性病変, CTSを認めず, 骨関節痛のないHD患者 (B群) 22例, 健常人13例である. HD開始時の患者末梢血および健常人末梢血から, 単球分離用特殊プレートで単球を分離し, 培養液中に1×106 cell/mlとなるように調整, LPSを10μg/ml添加し48時間培養後, 培養上清のIL-1β濃度を酵素抗体法 (ELISA) にて測定した. さらに単球細胞内IL-1β濃度は, 単球を1×106 cell/mlとなるよう調整後, 直ちに凍結, 溶解を3回繰り返し, 遠沈後上清をELISAにて測定した. 結果は, 48時間培養上清のIL-1β濃度は, 健常人では5.01±3.34ng/mlであったのに対し, HD患者群では6.11±2.35ng/mlと有意差を認めなかった. A群, B群間にも有意差を認めなかった. 単球細胞内IL-1β濃度は, 健常人では, 49.58±47.90pg/mlであったのに対し, HD患者群では194.72±179.30pg/mlと有意に高値を示していた. またB群は130.02±125.30pg/mlであり, 健常人と有意差を認めなかったが, A群は251.80±202.90pg/mlと健常人より有意に高値であった. 以上より, アミロイド骨関節症を有する患者の末梢血単球が何らかの形で活性化されていて, このことが, 骨関節病変, 痛みの発現に関与している可能性もあり, 今後さらに検討が必要と考えられた.

Background. Immunoglobulin A nephropathy (IgAN) is characterized by an aberrant structure of O-glycans in the IgA1hingeregion.Recently,under-galactosylatedIgA1has 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),patientswithotherformsofkidneydisease(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.

To elucidate the specific changes of pancreatic gamma-glutamyl-transpeptidase (gamma-GTP) associated with malignant transformation, some properties of gamma-GTP purified from pancreatic cancer were compared with those of gamma-GTPs from normal pancreas and other tissues. Four of five pancreatic cancer gamma-GTPs showed distinctly slower electrophoretic mobility than normal pancreatic enzymes. Isoelectric points of pancreatic cancer gamma-GTPs varied in each case, but they were all higher than those of normal pancreatic enzymes. This difference in isoelectric points of gamma-GTPs between cancerous tissue and normal tissue was reduced by neuraminidase treatment. Lectin affinity chromatography revealed two of five pancreatic cancer gamma-GTPs with a greater affinity to concanavalin A (Con A) than normal pancreas gamma-GTPs. Four of five pancreatic cancer gamma-GTPs had a greater affinity to Lens culinaris agglutinin (LCA) than normal pancreas gamma-GTPs. Normal pancreas gamma-GTPs had little affinity to Phaseolus vulgaris erythroagglutinating (E-PHA), but two of five pancreatic cancer gamma-GTPs had an apparent affinity to E-PHA and one of them had a slight affinity to E-PHA. These results indicate that the transformational changes of pancreatic cancer gamma-GTP are mainly induced in the sugar chains of the enzyme molecule, resulting in lower content of sialic acid and higher content of fucose and bisecting GlcNAc residue (the beta-N-acetylglucosamine residue linked at the C-4 of the beta-mannosyl residue of the trimannosyl core of the asparagine-linked sugar chain) as compared with the normal pancreatic enzyme.

Human serum immunoglobulin IgA1 is produced in bone marrow and interacts with specific cellular receptors that mediate biological events. In this study, we have analyzed the detailed glycoform structure of the human serum IgA1 Fc O-glycosylated hinge region by electrospray ionization liquid mass spectrometry. The IgA1 fragments containing the hinge glycopeptide were separated from 4 IgA nephropathy patient (IgAN) pooled sera, 10 non-IgAN pooled sera with other primary glomerulonephritides, and 5 healthy control subject pooled sera by trypsin treatment and Jacalin affinity chromatography. The molecular weights of IgA1 hinge glycopeptide were estimated using mass spectrometry, and 13 sialo and 8 asialo glycopeptide groups were identified. The results obtained clearly showed a decrease of GalNAc, Gal, and sialic acid in IgAN compared with non-IgAN and normal controls, and those strongly suggested the possibility that the decreased galactosylation and sialylation of the IgA1 hinge result in its glomerular deposition in IgAN.

The Maillard reaction, initiated by the nonenzymatic reaction of reducing sugar with protein, is proposed to play a significant role in protein aging and the complications of aging and diabetes. In this study, we detected and quantified some advanced glycation endproducts (AGEs) in human serum proteins of control and uremic patients by a highly selective and specific assay, electrospray ionization liquid chromatography-mass spectrometry-mass spectrometry (ESI-LC-MS-MS). From our results, levels of each AGEs in serum of uremic patients were significantly elevated, compared to age-matched controls. These results provide the evidence for increased modifications of proteins by Maillard reaction in uremia.

High-speed counter-current chromatography has been successfully applied to the separation of the lac dye components. A 25-mg quantity of the sample was separated using a two-phase solvent system composed of tert. -butyl methyl ether– n -butanol–acetonitrile–water (2:2:1:5). The fractions were analyzed by high-performance liquid chromatography and electrospray tandem mass spectrometry. The separation yielded 2.6 mg of 97.2% pure laccaic acid C, 9.5 mg of 98.1% pure laccaic acid A, 3.6 mg of 98.2% pure laccaic acid B, and 0.5 mg of a 95.0% pure anthraquinonedicarboxylic acid with a molecular mass of 360.

First, this investigation showed that plasma levels of inosine, hypoxanthine, and xanthine, which are metabolites of adenosine, rose sharply when blood pressure dropped suddenly along with symptoms during a hemodialysis session (sudden hypotension), but not when it decreased gradually with eventual symptoms (gradual hypotension). Because adenosine has an action to dilate vessels, this result indicates the possibility that the increased release of adenosine would be a cause of sudden hypotension. Second, it was found that the frequency of sudden hypotension decreases with the administration of caffeine, which is an adenosine-receptor antagonist, whereas the frequency of gradual hypotension did not change. This result supports the above-mentioned hypothesis that adenosine may well be a mediator of sudden hypotension, but not of gradual hypotension. Third, our investigation demonstrated no significant differences in plasma norepinephrine level, in plasma renin activity, or in mean blood pressure between the hemodialysis session in which caffeine was administered and the session in which a placebo was given. These findings suggest that the effect of caffeine administration to prevent sudden hypotension is not mediated by the stimulation of the sympathetic nervous system or activation of the renin-angiotensin system, but by the adenosine-receptor antagonism.

Two types of dialysis induced hypotension apparently exist. One type presents as gradually decreasing blood pressure with eventual symptoms (gradual hypotension), whereas the other presents as abruptly and sharply decreasing blood pressure, along with symptoms (abrupt hypotension). In the current study, the authors found that the plasma hypoxanthine concentration during abrupt hypotension was significantly higher than before the hypotension occurred (20 min after saline was administered or at the beginning of dialysis), whereas comparison of the plasma hypoxanthine concentration during gradual hypotension and that before the hypotension occurred (20 min after saline was administered or at the beginning of dialysis) revealed no significant difference. The current results indicate that abnormally increased adenosine triphosphate (ATP) degradation associated with tissue ischemia occurred during abrupt hypotension but not during gradual hypotension. It can be speculated that the increased release of adenosine due to abnormally increased ATP degradation caused the abrupt hypotension. This conclusion seems reasonable given that adenosine directly decreases small vessel tone and inhibits prejunctional release of norepinephrine.

We have previously reported that novel beta 2-microglobulin (beta 2m) is a metabolite derived from beta 2m in ultrafiltrate of patients on long-term hemodialysis (LT-HD). Chromatofocusing showed the presence of at least two major novel beta 2m's with isoelectric points of 5.38 and 5.22. In the present study we purified one of major novel beta 2m's and determined the complete amino acid sequence. We demonstrate herein that the novel beta 2m has the same sequence as native beta 2m except for the 17th residue from the N-terminus which was identified as Asp instead of Asn in native beta 2m, suggesting a possible deamidation during LT-HD.

Background: The IgA1 molecule, which is predominantly deposited in glomeruli of IgA nephropathy, has the characteristic O-glycan side chain in its hinge region. Several lines of evidence have demonstrated that underglycosylated IgA1 acquires self-aggregation1 and binding affinities to the extra-cellular matrix,1,2 resulting in deposition in the glomeruli.3,4 However, there is little information concerning actual biological activities of the underglycosylated IgA1 deposited in glomeruli, which lead to mesangial cell proliferation and/or matrix expansion. Aim: To clarify the influence of O-glycan side chain in the hinge of IgA1 on its biological activity in mesangial cells, we performed a comprehensive gene expression profiling analysis of human cultured mesangial cells stimulated by enzymatically underglycosylated IgA1 using cDNA array. Methods: IgA1 was purified by affinity chromatography using anti IgA column followed by Jacalin column. Asialo/agalacto IgA1 was obtained by enzyme digestion using sialidase and β-galactosidase. Heat aggregated IgA1 was obtained by incubation at 63°C for 150 min. Cultured human mesangial cells were stimulated for 3 h by non-treated IgA1, heat aggregated IgA1 or asialo/agalacto IgA1, respectively, and total RNA was obtained. Only the enzyme stimulation was performed as a negative control for asialo/agalacto IgA1. After DNase treatment, isotope labelled probes were prepared by reverse transcription and hybridized to the Atlas Human 1.2 Array (CLONTECH, Palo Alto, CA, USA) according to the manufacturer’s protocol. A total of 1176 arrayed genes were quantitatively evaluated using BAS. Results: Expression profiles of 1176 genes were obtained. In all experiments, none of the three negative controls on the cDNA array was detected. The expression profiles of mesangial cells by each IgA1 stimulation resembled one another, but they were widely different from that of mononuclear cells. On the whole, 10% of the genes expressed in the mesangial cells were up-regulated by asialo/agalacto IgA1 stimulation compared to that by enzyme stimulation. The results suggested that under-O-glycosylated IgA1 has a biological function in human mesangial cells in vitro. In good agreement with previous studies,5 heat aggregated IgA1 stimulation up-regulated IL-6 and TNF-α expressions in mesangial cells compared to by non-treated IgA1. Asialo/agalacto IgA1 stimulation also up-regulated these molecules, suggesting its pathogenetic role in IgA nephropathy. However, since a similar tendency was revealed by enzyme stimulation alone, further study may be necessary to establish these observations. Conclusion. In the present study, we investigated the comprehensive expression profiles of mesangial cells stimulated by various types of IgA1 using cDNA array. This approach may serve as a useful database not only to evaluate the biological activities of under-O-glycosylated IgA1 but also to elucidate the pathogenesis of IgA nephropathy.