Imidazolone, a novel advanced glycation end product, is present at high levels in kidneys of rats with streptozotocin-induced diabetes.

We produced a monoclonal antibody to imidazolones A and B, novel advanced glycation end products formed from the reaction of 3-deoxyglucosone (3-DG) with the guanidino group of arginine. Liquid chromatography/mass spectrometry demonstrated that the formation of imidazolone A by incubating 3-DG with arginine is very rapid, reaching a maximum concentration within 24 h, but the formation of imidazolone B is very slow and low in quantity even after 2 weeks. Thus, at physiological conditions the formation of imidazolone A is dominant, while that of imidazolone B is negligible. Immunochemistry demonstrated that the imidazolone content in the kidneys of streptozotocin-induced diabetic rats was significantly higher than in the control rats. Serum levels of 3-DG in the diabetic rats were also significantly higher than in control rats. 3-DG attacks the arginine residues of the tissue proteins, producing imidazolone at high levels in the kidneys affected by diabetic nephropathy.

Immunohistochemical detection of imidazolone, a novel advanced glycation end product, in kidneys and aortas of diabetic patients.

To investigate the role of the Maillard reaction in the pathogenesis of diabetic complications, we produced several clones of monoclonal antibodies against advanced glycation end products (AGEs) by immunizing mice with AGE-modified keyhole limpet hemocyanin, and found that one clone (AG-1) of the anti-AGE antibodies reacted specifically with imidazolones A and B, novel AGEs. Thus, the imidazolones, which are the reaction products of the guanidino group of arginine with 3-deoxyglucosone (3-DG), a reactive intermediate of the Maillard reaction, were found to be common epitopes of AGE-modified proteins produced in vitro. We determined the erythrocyte levels of imidazolone in diabetic patients using ELISA with the monoclonal anti-imidazolone antibody. The imidazolone levels in the erythrocytes of diabetic patients were found to be significantly increased as compared with those of healthy subjects. Then we studied the localization of imidazolone in the kidneys and aortas obtained from diabetic patients by immunohistochemistry using the antibody. Specific imidazolone immunoreactivity was detected in nodular lesions and expanded mesangial matrix of glomeruli, and renal arteries in an advanced stage of diabetic nephropathy, as well as in atherosclerotic lesions of aortas. This study first demonstrates the localization of imidazolone in the characteristic lesions of diabetic nephropathy and atherosclerosis. These results, taken together with a recent demonstration of increased serum 3-DG levels in diabetes, strongly suggest that imidazolone produced by 3-DG may contribute to the progression of long-term diabetic complications such as nephropathy and atherosclerosis.

Amyloid beta 2-microglobulin is modified with imidazolone, a novel advanced glycation end product, in dialysis-related amyloidosis.

We have recently demonstrated by immunohistochemistry that amyloid beta 2-microglobulin (beta 2m) is modified with advanced glycation end products (AGEs) in dialysis-related amyloidosis (DRA). To further investigate the role of the Maillard reaction in the pathogenesis of DRA, we produced a monoclonal antibody to imidazolone, a novel AGE, and a reaction product of arginine and 3-deoxyglucosone (3-DG) which was accumulated in uremic serum. Then we determined the localization of imidazolone in the amyloid tissues by immunohistochemistry using the antibody. The connective tissues in carpal tunnel and ligamentum flavum were obtained from six patients with carpal tunnel syndrome and two patients with destructive spondyloarthropathy. Imidazolone was localized to all the beta 2m-positive amyloid deposits in these patients. Western blotting using the antibody demonstrated that beta 2m extracted from the synovium amyloid of hemodialysis patients was modified with imidazolone. Further, beta 2m isolated from the blood ultrafiltrate of hemodialyzed patients was also modified with imidazolone. In vitro incubation of beta 2m with 3-DG produced imidazolone-modified beta 2m. In conclusion, amyloid tissue beta2m is modified with imidazolone in patients with DRA. 3-DG accumulating in uremic serum may be involved in the modification of beta 2m with imidazolone.

Amyloid beta 2-microglobulin is modified with N epsilon-(carboxymethyl)lysine in dialysis-related amyloidosis.

Recent work from this laboratory revealed that advanced glycation end product was localized to amyloid deposits in patients with dialysis-related amyloidosis by immunohistochemistry using a monoclonal antibody to advanced glycation end product. To elucidate the epitope of the antibody, N alpha-p-tosyl-L-lysine-methyl ester was incubated with glucose in vitro, and then a compound reactive to the antibody was purified from the incubate by buthanol extraction, XAD-2 column chromatography, and high-performance liquid chromatography while the reactivity was examined by enzyme linked immunosorbent assay. The purified compound was identified as N epsilon-(carboxymethyl)-N alpha-p-tosyl-L-lysine-methyl ester by using secondary ion mass spectrometry, and 1H- and 13C-nuclear magnetic resonance spectroscopy. The epitope of the antibody was identified as -CH2-NH-CH2-COOH by enzyme-linked immunosorbent assay of compounds with structures similar to N epsilon-(carboxymethyl)lysine. Immunochemical study using the antibody demonstrated the presence of N epsilon-(carboxymethyl)lysine in the beta 2-microglobulin dimer (molecular weight 23929) isolated from the synovium amyloid of a hemodialysis patient with dialysis-related amyloidosis. In conclusion, amyloid beta 2-microglobulin is modified with N epsilon-(carboxymethyl)lysine in dialysis-related amyloidosis.

Determination of bradykinin-(1-5) in inflammatory exudate by a new ELISA as a reliable indicator of bradykinin generation.

We have developed an ELISA for BK-(1-5) (Arg1-Pro2-Pro3-Gly4-Phe5). In rat carrageenin-induced pleurisy, in which a plasma exudation peak was observed 5 h after carrageenin, BK levels in the exudates were negligible (< 60 pg/rat). BK-(1-7) (des-Phe8-Arg9-BK) was detectable (900-400 pg/rat) over the entire course of the inflammation. However, a larger amount of BK-(1-5) was detectable in association with the increase in plasma exudation, showing a peak (8800 +/- 1200 pg/rat) 3 h after carrageenin. Bromelain (10 mg/kg, i.v.) and soy bean trypsin inhibitor (0.3 mg/rat, intra-pleural) significantly reduced BK-(1-5) levels (by 60-93%, 3, 7 and 19 h after carrageenin) and plasma exudation rates (by 61-74%, 3 and 7 h after carrageenin). Dexamethasone (0.3 mg/kg, i.p.) reduced BK-(1-5) levels (by 78%) and decreased plasma exudation (by 70%) 3 h after carrageenin. In nasal allergy patients, antigen challenge of nasal mucosa elevated BK-(1-5) levels and active kallikrein levels in nasal washes. These results verify that BK-(1-5) determined by ELISA is a good indicator for release of kinins in vivo.

Lack of contribution of circulatory kinin elevated by captopril to induce hypotension in normotensive and hypertensive rats.

Captopril (10 mg/kg, i.p.) increased the arterial bradykinin (BK) level (Art-BK) of non-treated Sprague-Dawley rats (SD), determined by an ELISA, from 10.8 +/- 3.2 pg/ml to 32.9 +/- 5.4 pg/ml significantly (p < 0.05, n = 6). Intravenous infusion of BK (100-3000 ng/kg/min) dose-dependently increased heart rate (HR) and decreased mean blood pressure (MBP), the former at lower doses than the latter, and the hypotensive response became significant at 3000 ng/kg/min. Art-BK determined during infusion of the lowest dose of BK (100 ng/kg/min) was 12 times the endogenous Art-BK after captopril administration. In spontaneously hypertensive rats, Wistar Kyoto rats, and deoxycorticosterone acetate-salt treated hypertensive rats, Art-BK (450-1280 pg/ml) determined during intravenous BK-infusion (1000-3000 ng/kg/min), which induced significant hypotension, was 20 to 100 times the endogenous Art-BK (4.5-64 pg/ml) with captopril treatment. These results suggest that the increased Art-BK due to inhibition of kinin degradation by captopril could not account for the hypotension due to this angiotensin converting enzyme inhibitor in normotensive and hypertensive rats.

Modification of beta 2m with advanced glycation end products as observed in dialysis-related amyloidosis by 3-DG accumulating in uremic serum.

beta 2microglobulin (beta 2m) isolated from the amyloid deposits in patients with dialysis-related amyloidosis (DRA) has been demonstrated to be modified with advanced glycation end products (AGEs). We demonstrated that AGE was localized to amyloid deposits in patients with DRA by immunohistochemistry using a monoclonal anti-AGE antibody. To clarify the mechanism of AGE modification of beta 2m-amyloid, we studied the effects of 3-deoxyglucosone (3-DG), a potent protein crosslinking the intermediate of the Maillard reaction, on the AGE modification of beta 2m, and quantified the serum levels of 3-DG in patients undergoing hemodialysis (HD) and continuous ambulatory peritoneal dialysis (CAPD), and undialyzed patients. The serum levels of 3-DG were markedly increased in the dialyzed and undialyzed uremic patients. Although the serum level of 3-DG decreased after HD with a mean reduction rate of 67%, it was still significantly higher than in normal serum. Incubation of beta 2m with 3-DG at 37 degrees C emitted fluorescence characteristic for AGE, and caused AGE modification and dimer formation of beta 2m as demonstrated by Western blotting using the same monoclonal anti-AGE antibody used for immunohistochemical demonstration of AGE in DRA. The AGE-modified dimer of beta 2m could be extracted from the amyloid tissue of a patient with DRA. 3-DG showed more intense and faster reactivity with beta 2m to form AGE and dimer as compared with glucose, and aminoguanidine suppressed the AGE and dimer formation of beta 2m by 3-DG. In conclusion, 3-DG accumulating in uremic serum may be involved in the AGE modification of beta 2m-amyloid.

Serum levels of 3-deoxyglucosone and tissue contents of advanced glycation end products are increased in streptozotocin-induced diabetic rats with nephropathy.

To investigate a role of the Maillard reaction in the pathogenesis of diabetic nephropathy, we measured serum levels of 3-deoxyglucosone (3-DG), a potent protein cross-linking intermediate of the Maillard reaction, and tissue contents of advanced glycation end products (AGEs) in streptozotocin (STZ)-induced diabetic rats. We quantified serum 3-DG using gas chromatography/mass spectrometry, and measured AGE contents in tissues using a competitive enzyme-linked immunosorbent assay with a monoclonal anti-AGE antibody. The STZ-induced diabetic rats showed nephropathy with proteinuria, hypoproteinemia, hyperlipidemia and reduced creatinine clearance. Serum levels of 3-DG in the STZ-induced diabetic rats (mean +/- 3.46 +/- 0.23 mumol/l) were significantly (p < 0.01) higher than those in control rats (1.23 +/- 0.13 mumol/l). AGE contents in the kidney and the lens obtained from the STZ-induced diabetic rats (398 +/- 45 and 816 +/- 200 arbitrary units, respectively) were also significantly (p < 0.01) higher than those in the control rats (122 +/- 10 and 299 +/- 50 arbitrary units, respectively). The results indicate that increased levels of serum 3-DG and renal tissue. AGEs may be related to the occurrence of diabetic nephropathy.

Optimal conditions for collection of blood samples for assay of alpha 2-macroglobulin-trypsin complex-like substance (MTLS).

We have developed an enzyme-linked immunosorbent assay (ELISA) for the specific quantification of alpha 2-macroglobulin-trypsin complex-like substance (MTLS). To exclude artifacts in measured values of MTLS, the conditions for collection of blood samples are critical. In the present study, we have determined the optimal conditions for blood collection and investigated the role of MTLS as a clinical tool for diagnosis in pancreatitis. Results obtained are as follows: (1) MTLS levels of all sera were more than 10-fold higher than the corresponding plasma; (2) MTLS levels of heparinized plasma were the lowest among plasma with three anticoagulants (sodium citrate, sodium EDTA and heparin); (3) some kinds of blood collection tubes containing heparin were not suitable for the sampling; (4) MTLS values of plasma obtained by blood collection tubes containing Trasylol and sodium EDTA were demonstrated more stable and lower than those obtained by heparin tubes; and (5) under these conditions, we can exclude elevation of MTLS values caused by inappropriate blood sampling and find the time course of the elevation reflecting clinical course of a patient with acute pancreatitis and a patient after endoscopic retrograde cholangiopancreatography (ERCP). The optimal conditions for collection of blood samples were as follows. Blood sampling should be performed by blood collection tubes containing Trasylol (50 microliters/ml blood) and sodium EDTA (1.5 mg/ml blood). The samples were immediately stored at 4 degrees C and centrifuged at 3,000 rpm for 15 min. The plasma were stored in plastic tubes at 4 degrees C until assayed.

Immunohistochemical detection of advanced glycation end products in dialysis-related amyloidosis.

beta 2-Microglobulin (beta 2m) isolated from the amyloid deposits in patients with dialysis-related amyloidosis (DRA) has been demonstrated to be modified with advanced glycation end products (AGE). We produced a monoclonal anti-AGE antibody which localized AGE to amyloid deposits in patients with DRA by immunohistochemistry. The connective tissues in the carpal tunnel were obtained from surgical specimens in six patients with DRA. AGE were localized to the beta 2m-positive amyloid deposits in these patients using the monoclonal anti-AGE antibody. AGE were also detected in infiltrating cells surrounding the amyloid deposits. The AGE-positive cells were identified as macrophages, since they showed positive staining with anti-CD68 antibody. In conclusion, AGE were demonstrated by immunohistochemical technique to be present in both beta 2m-positive amyloid deposits and surrounding macrophages in patients with DRA.

NH2-terminal sequence of novel pancreatic stone protein-related protein in human urine.

In a previous report, we detected the novel PSP-related protein in urine from a patient with diabetic nephropathy. This protein was different from PSP S1 and PSP S2-5 on the elution points of Mono S chromatography. To investigate the NH2-terminal sequence, we purified this protein by reverse phase chromatography and performed amino acid analysis. The results showed that the NH2-terminal sequence of this protein was one residue (arginine) longer than that of PSP S1.

Immunological characterization of pancreatic stone protein in human urine.

In order to study the mechanism and origin of urine pancreatic stone protein (PSP), PSP was analyzed in the urine and sera from healthy subjects, patients with renal disease, and intensive care patients by Mono S chromatography and Western blotting. The elution patterns could be classified into three types. In control urine, a single peak of immunoreactive PSP (peak I) was identified at the position of PSP-S2-5 (type A). In three of seven patients with renal disease, another peak of urine immunoreactive PSP (peak II) was recognized at the position slower than that corresponding to that of PSP-S1 (type B). In urine from one patient with diabetic nephropathy, a third peak of immunoreactive PSP (peak III) was eluted between peaks I and II (type C). In Western blotting, the bands in urine from patients with renal disease and of those in ICU mainly appeared at the positions of high-molecular-weight types of PSP and PSP-S2-5, respectively. These results suggest that the kidney can be another major source of urine PSP in addition to the pancreas.

Enzyme immunoassay and characterization of pancreatic stone proteins in human urine.

An enzyme immunoassay of pancreatic stone protein (PSP) in human urine was developed. Mean analytical recovery of pure PSP-S2-5 added to urine was 102.3% (SD 5.9%), and the precision of the assay was 2.0-2.7% within an assay and 2.5-2.9% between assays. In healthy volunteers (age 20-55 years), the mean value of the PSP concentration, expressed as ratios to urine creatinine, was 129 +/- 88 (mean +/- SD) micrograms/g without any differences for sex. Urine PSP correlated with urine N-acetylglucosaminidase (NAG) (r = 0.354). The molecular forms of immunoreactive PSP in urine were characterized by using cation exchange chromatography (Mono S), SDS-PAGE, N-terminal sequence, and enzyme immunoassay analysis. The urine PSP, eluted at the position corresponding to PSP-S2-5 on cation exchange chromatography, was converted to PSP-S1 by trypsin digestion. The difference in mobility on SDS-PAGE between urine PSP and PSP-S2-5 seems to be due to a glycosylated undecapeptide (N-terminal 1-11). The proposed method offers a sensitive, specific, and reproducible tool for laboratory analysis of human urine PSP levels.

Urinary trehalase activity in chronic glomerulonephritis.

To determine the diagnostic role of urinary trehalase in chronic glomerular disease, urinary trehalase activity and other urinary markers such as N-acetyl-beta-D-glucosaminidase (NAG), alanine aminopeptidase (AAP), alkaline phosphatase (ALP), gamma-glutamyltranspeptidase (gamma-GTP), lactate dehydrogenase (LDH), lysozyme and beta 2-microglobulin (BMG) were measured in patients with chronic glomerulonephritis, nephrotic syndrome and chronic renal failure. Urinary trehalase activity was significantly increased in chronic glomerular disease, especially nephrotic syndrome, as compared with that in the healthy subjects. The highest incidence of elevated excretion was observed for trehalase with 52% in chronic glomerular disease, followed by NAG. Urinary trehalase activities in the patients were significantly correlated with the urinary levels of protein, NAG and AAP and total score of tubular damage, but not correlated with urinary levels of BMG or lysozyme. In patients with chronic glomerulonephritis and nephrotic syndrome, there was no significant difference in urinary trehalase activities between with and without hematuria. These results indicate that in some patients with chronic glomerular disease, there is tubular involvement as substantiated by elevation of the other urinary enzymes and BMG. Urinary trehalase is elevated more often in these types of disease than other markers of tubular damage.

Enzyme immunoassay and immunochemical characterization of pancreatic stone protein in human serum.

Monoclonal antibodies were raised against human pancreatic stone protein (PSP) and used for one-step enzyme immunoassay (EIA). PSP-S2-5 was employed as the standard in the assay. The assay's measurable range was 25-1,500 ng/ml and within run coefficient of variation was 3.7-6.4%. Analytical recovery of the assay was 101.5 +/- 5.65% (mean +/- SD). The results of experiments in which serum was fractionated by Mono S (cation exchange chromatography) suggested that most of immunoreactive material in human serum is PSP-S2-5. The EIA offers simple, rapid, and specific analysis of serum PSP level for clinical diagnosis.

Enzyme immunoassay for specific analysis of pancreatic stone proteins in human pancreatic juice.

In order to study the concentration of pancreatic stone protein (PSP) in human pancreatic juice, we investigated the influence of the insoluble form of PSP-S1 converted from PSP-S2-5 on PSP determination and the assay method for PSP-S1 precipitate after solubilizing PSP-S1. When bovine trypsin was added to pancreatic juice, PSP-S1 was converted from PSP-S2-5 and precipitated about 45-85% after 1 h. The precipitated PSP-S1 was dissolved in 0.1 M sodium acetate buffer, pH 4.0, and the concentration was measured by the enzyme immunoassay, with similar reactivity to PSP-S1 and PSP-S2-5. The proposed method can offer accurate and specific analysis of the PSP level in pancreatic juice. The results of the fractionation of pancreatic juice and duodenal juice on Mono S cation-exchange chromatography suggested that the major component of PSP was PSP-S2-5 in pancreatic juice and PSP-S1 in duodenal juice.

Enzyme immunoassay for urinary albumin at low concentration in diabetes mellitus.

Microalbuminuria, an increased excretion of urinary albumin undetectable by Albustix test strips, appears to predict the late development of diabetic nephropathy at a stage that albuminuria might be reduced by good metabolic control Once albuminuria is detected by Albustix, it indicates the likelihood of diabetic nephropathy. Microalbuminuria is also related to an increased prevalence of proliferative retinopathy, blindness, and peripheral neuropathy. It is therefore important to detect microalbuminuria by a sensitive, rapid and simple method. Microalbuminuria can be measured by radial immunodiffusion, immunoelectrophoresis, radioimmunoassay, enzyme immunoassay, latex-bead immunoagglutination, turbidimetric immunoassay and dye-binding. The disadvantages of radioimmunoassays are: short shelf life, isotope-related health and safety hazards, and the expense of equipment used to measure gamma-emitting isotopes. Our aim in this study was to develop a simple, rapid and sensitive one-step sandwich enzyme immunoassay using anti-human albumin monoclonal antibodies for screening microalbuminuria.