Hiroko Odani

Imidazolium crosslinks derived from reaction of lysine with glyoxal and methylglyoxal are increased in serum proteins of uremic patients: evidence for increased oxidative stress in uremia

Glyoxal (GO) and methylglyoxal (MGO) are reactive dicarbonyl compounds formed during autoxidation of both carbohydrates and lipids. They may react with lysine and arginine residues of proteins in Maillard or browning reactions, yielding advanced glycation or lipoxidation end products. Among these are the imidazolium crosslinks, N,N(‐di(N ϵ‐lysino))imidazolium (glyoxal‐lysine dimer, GOLD) and N,N(‐di(N ϵ‐lysino))‐4‐methyl‐imidazolium (methylglyoxal‐lysine dimer, MOLD). We have detected and measured GOLD and MOLD in human serum by electrospray ionization/mass spectrometry/mass spectrometry (ESI/MS/MS), using 15N4‐GOLD and 15N4‐MOLD as internal standards. In this report we show that levels of GOLD and MOLD are significantly elevated (3–4‐fold, P<0.01) in sera of non‐diabetic uremic patients, compared to age‐matched controls, and represent a major class of non‐enzymatic, Maillard reaction crosslinks in plasma proteins. These results provide strong evidence for increased non‐enzymatic crosslinking of tissue proteins by GO and MGO in uremia, implicating oxidative stress and resultant advanced glycation and lipoxidation reactions in tissue damage in uremia.

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.

Purification and complete amino acid sequence of novel β2-microglobulin

Abstract We have previously reported that novel β2-microglobulin (β2m) is a metabolite derived from β2m in ultrafiltrate of patients on long-term hemodialysis (LT-HD) [1]. Chromatofocusing showed the presence of at least two major novel β2ms with isoelectric points of 5.38 and 5.22. In the present study we purified one of major novel β2ms and determined the complete amino acid sequence. We demonstrate herein that the novel β2m has the same sequence as native β2m except for the 17th residue from the N-terminus which was identified as Asp instead of Asn in native β2m, suggesting a possible deamidation during LT-HD.

Improvement of chemical analysis of antibiotics XXIII : Identification of residual tetracyclines in bovine tissues by electrospray high-performance liquid chromatography-tandem mass spectrometry

To reliably identify the residual tetracycline antibiotics (TCs), oxytetracycline (OTC), tetracycline, chlortetracycline (CTC) and doxycycline (DC), in bovine tissues, we have established a confirmation method using electrospray ionization liquid chromatography-tandem mass spectrometry (ESI LC-MS-MS) with daughter ion scan. All TCs gave [M+H-NH3]+ and [M+H-NH3-H2O]+ as the product ions, except for DC when [M+H]+ was selected as the precursor ion. The combination of C18 cartridge clean-up and the present ESI LC-MS-MS method can reliably identify TCs fortified at a concentration of 0.1 ppm in bovine tissues, including liver, kidney and muscle, and has been successfully applied to the identification of residual OTC in bovine liver and residual CTC in bovine muscle samples previously found at concentrations of 0.58 ppm and 0.38 ppm by LC, respectively.

Separation of lac dye components by high-speed counter-current chromatography.

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.

Mass spectrometric study on the protein chemical modification of uremic patients in advanced Maillard reaction

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.

Relationship between dialysis induced hypotension and adenosine released by ischemic tissue

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.

Suppression of AGE Precursor Formation Following Unilateral Ureteral Obstruction in Mouse Kidneys by Transgenic Expression of α-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 α-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 α-dicarbonyl compounds under oxidative circumstances, but it was not sufficient to suppress acute renal fibrosis during 7 d of UUO by itself.

The structural alteration of O‐glycosylated hinge peptides in serum IgA1 before and after tonsillectomy in IgA nephropathy

The human IgA1 molecule has exceptional O-glycans in its hinge region. The fundamental structure of the Oglycans is the linkage between the a-anomeric carbon atom in N-acetylgalactosamine (GalNAc) and the hydroxy group of serine or threonine (GalNAc a1-O-Ser (Thr)). A wide structural variety of the sugar side-chains in glycoproteins is observed under physiological conditions. The phenomenon is called ‘the microheterogeneity of carbohydrates’. In several previous studies analysing the structural varieties of the O-glycans in the hinge region of serum IgA1 and the deposited IgA1 in glomeruli (glomerular IgA1) in IgA nephropathy (IgAN), it was found that the hinge glycopeptides in IgA1 were under-glycosylated. Recently, we investigated that the O-glycan structure of IgA1 molecules produced by tonsillar lymphocytes (tonsillar IgA1) were also under-glycosylated in IgAN patients. These findings suggested that the underglycosylated IgA1 might play a role in the cause of IgAN. Upper respiratory infections and tonsillitis often precede increases in haematuria and proteinuria in patients with IgAN. Béné et al. reported that tonsillectomy might be an efficient treatment for preventing the progression of IgAN. Recently, Hotta et al. reported that tonsillectomy combined with steroid pulse therapy (combination therapy) had a strong effect on IgAN. Therefore, tonsillectomy and combination therapy might have an influence upon the structural varieties of O-glycans of IgA1 in IgAN.

IgA1 produced by tonsillar lymphocytes is under‐O‐glycosylated in IgA nephropathy

Background: The human IgA1 has exceptional Oglycans in its hinge region. The fundamental structure of the O-glycans is the linkage between the a-anomeric carbon atom in N-acetylgalactosamine (GalNAc) and the hydroxy group of serine or threonine (GalNAc a1-O-Ser (Thr)). A wide structural variety of the sugar side chains in glycoproteins is observed under physiological conditions. The phenomenon is called ‘the microheterogeneity of carbohydrate’. In several previous studies analyzing the structural varieties of the O-glycans in the hinge region of serum IgA1 in IgA nephropathy (IgAN), it has been found that the hinge glycopeptides in IgA1 were under-glycosylated. Recently, we investigated the O-glycan structure of the deposited IgA1 in glomeruli that was obtained from 290 renal biopsy specimens of 278 IgAN patients. The results suggested that the O-glycan side chains in the deposited IgA1 were highly under-glycosylated. These findings suggested that the under-glycosylated IgA1 might play a role in the cause of IgAN. Upper respiratory infections and tonsillitis often precede increases in hematuria and proteinuria in patients with IgA nephropathy. Also, in some cases, tonsillectomy is an efficient treatment for preventing the progression of IgAN. Therefore, the mucosal immune system in the respiratory tract is considered to have a relation with the pathogenesis of IgAN. Aim: In this study, we investigated the O-glycan structure of IgA1 produced by tonsillar lymphocytes (tonsillar IgA1) to find the origin of the deposited IgA1 in the glomeruli of IgAN patients. Methods: Extracted tonsils were obtained from seven IgAN patients and five chronic tonsillitis patients as controls. Tonsillar lymphocytes separated from the extracted tonsils were cultured for 7 days. IgA in the culture medium was obtained by the anti-human IgA antibody coupled activated sepharose 4 B column, and IgA1 hinge glycopeptides was purified by trypsin digest, jacalin-agarose column and high performance liquid chromatography. The isolated hinge glycopeptides were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS). The varieties of the O-glycans in tonsillar IgA1 were determined from the molecular weights measured by MALDI-TOFMS. Results: No significant difference was found in the percentage of asialo-type O-glycans between the IgAN patients and controls. A significant increase in the percentage of asialo-agalacto-type O-glycans were found in the tonsillar IgA1 in four out of seven IgAN patients (57.1%) compared with controls. Conclusion: We present the first information about the precise structure of O-glycans in the IgA1 produced by tonsillar lymphocytes (tonsillar IgA1). In conclusion, we analyzed the tonsillar IgA1 to find the origin of the under-glycosylated IgA1 deposited in the glomeruli of IgAN patients. The under-glycosylation was found in the tonsillar IgA1 in some IgAN patients. These results indicated a possibility that the under-glycosylation tonsillar IgA1 has a relation to the pathogenesis of IgAN.