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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.

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.

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.

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.