Identification of N(omega)-carboxymethylarginine, a new advanced glycation endproduct in serum proteins of diabetic patients: possibility of a new marker of aging and 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.

Mass spectrometry proves under-O-glycosylation of glomerular IgA1 in IgA nephropathy.

BACKGROUND: The IgA1 molecule, which is predominantly deposited in glomeruli in IgA nephropathy (IgAN), is a unique serum glycoprotein because it has O-glycan side chains in its hinge region. Our study was conducted to investigate the O-glycan structure in the glomerular IgA1 in IgAN. METHODS: The IgA1 was separated from 290 renal biopsy specimens of 278 IgAN patients and from four serum IgA1 samples (IgAN, 2; control, 2). The variety of O-glycan glycoform was determined by estimating the precise molecular weights of the IgA1 hinge glycopeptides using matrix-assisted laser desorption ionization time of flight mass spectrometry. RESULTS: The peak distribution of IgA1 hinge glycopeptides clearly shifted to lesser molecular weights in both glomerular and serum IgA1 in IgAN compared with the serum IgA1 of controls. In the five major peaks of IgA1 hinge glycopeptides in each sample, the numbers of carbohydrates composing O-glycans (GalNAc, Gal, and NANA) in the deposited and serum IgA1 in IgAN patients were significantly fewer than those in the serum IgA1 in the control groups. CONCLUSION: The O-glycan side chains in the hinge of the glomerular IgA1 were highly underglycosylated in IgAN. These results indicate that the decreased sialylation and galactosylation of the IgA1 hinge glycopeptides play a crucial role in its glomerular deposition in IgAN.

Direct evidence for decreased sialylation and galactosylation of human serum IgA1 Fc O-glycosylated hinge peptides in IgA nephropathy by mass spectrometry.

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.

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.

Increase in three alpha,beta-dicarbonyl compound levels in human uremic plasma: specific in vivo determination of intermediates in advanced Maillard reaction.

Methylglyoxal (MGO), glypxal (GO) and 3-deoxyglucosone (3-DG) are reactive alpha,beta-dicarbonyl intermediates in advanced Maillard reaction, which form advanced glycation and oxidation end products (AGEs) by reaction with both lysine and arginine residues in protein. We measured these three dicarbonyl compound levels in human plasma to estimate the relationship between accumulation of alpha, beta-dicarbonyl compounds and AGE formation reactions in uremia and diabetes in human plasma by a highly selective and specific assay, electrospray ionization liquid chromatography mass spectrometry (ESI/LC/MS). We show that 3-DG and MGO levels are significantly higher in uremia and diabetes compared with age-matched healthy controls. Only the GO level in uremic plasma is significantly higher compared to diabetes and healthy controls. In both diabetic and uremic patients, these dicarbonyl compounds promote AGE accumulation in vivo, and especially in uremic patients, increased accumulation of GO could result from accelerating oxidative stress.

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.

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(epsilon)-lysino))imidazolium (glyoxal-lysine dimer, GOLD) and N,N(-di(N(epsilon)-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.

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.

First evidence for accumulation of protein-bound and protein-free pyrraline in human uremic plasma by mass spectrometry.

Glucose-derived advanced glycation end products (AGEs) cross-link proteins and cause various biological tissue damage. One of them, pyrraline [epsilon-2-(formyl-5-hydroxymethyl-pyrrol-1-yl) -L-norleucine], has been demonstrated by utilizing antibody to accumulate in plasma and sclerosed matrix of diabetic individuals, suggesting responsibility for diabetic complications. To elucidate the involvement of pyrraline in uremia, we examined the pyrraline levels in patients with chronic renal failure by a mass spectrometric approach. Here we show that protein-free pyrraline as well as pyrraline with binding protein are significantly increased in non-diabetic uremic plasma compared to healthy subjects. Our results suggest that circulating pyrraline could be a substance contributing to complications in uremia.

Role of adenosine in dialysis-induced hypotension.

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.

Determination of a new immunosuppressant, mycophenolate mofetil, and its active metabolite, mycophenolic acid, in rat and human body fluids by high-performance liquid chromatography.

Mycophenolate mofetil, a new immunosuppressant, is a morpholinoethyl ester of mycophenolic acid. A new selective, sensitive and simple high-performance liquid chromatographic method was developed for the determination of mycophenolic acid and mycophenolate mofetil in biological samples. The preparation of samples was based on liquid-liquid extraction. The compounds were separated on a CN column using acetonitrile-0.01 M phosphate buffer (1:4, v/v) as the mobile phase. UV detection was used at wavelengths 215 and 304 nm. The detection limit was 5 ng per injection volume. This method enabled pharmacokinetic and pharmacodynamic studies in humans and rats.

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.

Purification and complete amino acid sequence of novel beta 2-microglobulin.

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.

Characterization of gamma-glutamyltranspeptidase from human pancreatic cancer.

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.

[Evaluation of CA 15-3 radioimmunoassay].

The CA 15-3 RIA kit (Centocor Co.) based on a solid-phase radioimmunoassay was evaluated both experimentally and clinically. The results of a basic study of the reproducibility, recovery and dilution were very satisfactory. Serum levels of CA 15-3 were determined in 211 patients with malignancies including 13 breast cancers, 81 patients with benign diseases and 13 controls. Elevated levels of this antigen (greater than 25 U/ml) were observed in 16.7% of esophageal cancers, 9.7% of gastric cancers, 7.9% of colon cancers, 10% of hepatocellular cancers, 10% of biliary tract cancers, 10% of pancreatic cancers, 33% of lung cancers, and 75% of recurrent breast cancers. Thus, in malignant diseases the positivity was low except for recurrent breast cancer. On the other hand, the false-positive rate in benign diseases was only 3.7% and the false-positive cases had a level of 40 U/ml or less. These results indicate that the serum levels of CA 15-3 may have diagnostic value in patients with recurrent breast cancer.

[CA 19-9 enzyme immunoassay].

The carbohydrate antigenic determinant (CA 19-9) EIA kit (FUJIREVIO INC) was examined both experimentally and clinically. In the basic study, the standard curve showed linearity from to 240 U/ml, and the intra-and interassay reproducibility was good. Serum levels of CA 19-9 were determined by EIA in 246 cancer patients and 180 patients with benign diseases, and CA 19-9 levels by EIA were compared with those by RIA in concurrent measurement. There was an excellent correlation between the two. The positive of CA 19-9 EIA was almost the same as that of CA 19-9 RIA in cancer patients. False-positive cases by CA 19-9 EIA were somewhat more numerous than those by CA 19-9 RIA in patients with benign diseases, especially liver diseases. However, CA 19-9 assay by EIA is simple and has an excellent reproducibility and accuracy, as does that by RIA. Thus, the CA 19-9 EIA is a very useful assay.