Monika Pischetsrieder

N ε-(Carboxymethyl)Lysine Adducts of Proteins Are Ligands for Receptor for Advanced Glycation End Products That Activate Cell Signaling Pathways and Modulate Gene Expression

Recent studies suggested that interruption of the interaction of advanced glycation end products (AGEs), with the signal-transducing receptor receptor for AGE (RAGE), by administration of the soluble, extracellular ligand-binding domain of RAGE, reversed vascular hyperpermeability and suppressed accelerated atherosclerosis in diabetic rodents. Since the precise molecular target of soluble RAGE in those settings was not elucidated, we tested the hypothesis that predominant specific AGEs within the tissues in disorders such as diabetes and renal failure,N ε-(carboxymethyl)lysine (CML) adducts, are ligands of RAGE. We demonstrate here that physiologically relevant CML modifications of proteins engage cellular RAGE, thereby activating key cell signaling pathways such as NF-κB and modulating gene expression. Thus, CML-RAGE interaction triggers processes intimately linked to accelerated vascular and inflammatory complications that typify disorders in which inflammation is an established component.

Receptor for Advanced Glycation End Products Mediates Inflammation and Enhanced Expression of Tissue Factor in Vasculature of Diabetic Apolipoprotein E–Null Mice

Abstract—Advanced glycation end products (AGEs) and their cell surface receptor, RAGE, have been implicated in the pathogenesis of diabetic complications. Here, we studied the role of RAGE and expression of its proinflammatory ligands, EN-RAGEs (S100/calgranulins), in inflammatory events mediating cellular activation in diabetic tissue. Apolipoprotein E– null mice were rendered diabetic with streptozotocin at 6 weeks of age. Compared with nondiabetic aortas and kidneys, diabetic aortas and kidneys displayed increased expression of RAGE, EN-RAGEs, and 2 key markers of vascular inflammation, vascular cell adhesion molecule (VCAM)-1 and tissue factor. Administration of soluble RAGE, the extracellular domain of the receptor, or vehicle to diabetic mice for 6 weeks suppressed levels of VCAM-1 and tissue factor in the aorta, in parallel with decreased expression of RAGE and EN-RAGEs. Diabetic kidney demonstrated increased numbers of EN-RAGE–expressing inflammatory cells infiltrating the glomerulus and enhanced mRNA for transforming growth factor-&bgr;, fibronectin, and &agr; 1 (IV) collagen. In mice treated with soluble RAGE, the numbers of infiltrating inflammatory cells and mRNA levels for these glomerular cytokines and components of extracellular matrix were decreased. These data suggest that activation of RAGE primes cells targeted for perturbation in diabetic tissues by the induction of proinflammatory mediators.

On-line haemodiafiltration versus haemodialysis: stable haematocrit with less erythropoietin and improvement of other relevant blood parameters.

Background: Controlled randomised studies to prove improved cardiovascular stability and improved anaemia management during on-line haemodiafiltration (oHDF) are scarce. Methods: 70 patients were treated with both haemodialysis (HD) and oHDF in a cross-over design during 2 × 24 weeks at a dialysis dose of eKt/V≧1.2. Patients randomised into group A started on HD and switched over to oHDF, whereas patients in group B began with oHDF and were treated with HD afterwards. Intradialytic morbid events (IME), such as symptomatic hypotension or muscle cramps, were noted in case of appearance. Blood parameters reflecting anaemic status, phosphate status, lipid metabolism, oxidative stress, and accumulation of advanced glycation end products were recorded either monthly or at the end of each study phase. Results: The mean incidence of IME was 0.15 IME per treatment, and there was no statistical difference between oHDF and HD. A higher haematocrit (oHDF 31.5% vs. HD 30.5%, p < 0.01) at a lower erythropoietin dose (oHDF 4,913 vs. HD 5,492 IU/week, p = 0.02) was found during oHDF, when the sequence of HD and oHDF had not been taken into account. For the study groups, the results were less distinct: in group A, a higher haematocrit (HD 30.4% vs. oHDF 32.0%, p < 0.01) at a comparable erythropoietin dose (HD 5,421 vs. oHDF 5,187 IU/week, ns) was observed during oHDF, whereas in group B an identical haematocrit (oHDF 30.8% vs. HD 30.7%, ns) was achieved at a reduced erythropoietin dose (oHDF 4,622 vs. HD 5,568 IU/week, p < 0.01). During oHDF, lower levels of free and protein-bound pentosidine and of serum phosphate were found. Conclusion: In contrast to other studies, no benefit regarding cardiovascular stability for oHDF was found, but oHDF could well offer a potential benefit regarding anaemia correction, inflammation, oxidative stress, lipid profiles, and calcium-phosphate product.

ISOLATION, PURIFICATION, AND CHARACTERIZATION OF AMADORIASE ISOENZYMES (FRUCTOSYL AMINE-OXYGEN OXIDOREDUCTASE EC 1.5.3) FROM ASPERGILLUS SP.

Four “amadoriase” enzyme fractions, which oxidatively degrade glycated low molecular weight amines and amino acids under formation of hydrogen peroxide and glucosone, were isolated from an Aspergillus sp. soil strain selected on fructosyl adamantanamine as sole carbon source. The enzymes were purified to homogeneity using a combination of ion exchange, hydroxyapatite, gel filtration, and Mono Q column chromatography. Molecular masses of amadoriase enzymes Ia, Ib, and Ic were 51 kDa, and 49 kDa for amadoriase II. Apparent kinetic constants for Nε-fructosyl Nα-t-butoxycarbonyl lysine and fructosyl adamantanamine were almost identical for enzymes Ia, Ib, and Ic, but corresponding values for enzyme II were significantly different. FAD was identified in all enzymes based on its typical absorption spectrum. N-terminal sequence was identical for enzymes Ia and Ib (Ala-Pro-Ser-Ile-Leu-Ser-Thr-Glu-Ser-Ser-Ile-Ile-Val-Ile-Gly-Ala-Gly-Thr-Trp-Gly-) and Ic except that the first 5 amino acids were truncated. The sequence of enzyme II was different (Ala-Val-Thr-Lys-Ser-Ser-Ser-Leu-Leu-Ile-Val-Gly-Ala-Gly-Thr-Trp-Gly-Thr-Ser-Thr-). All enzymes had the FAD cofactor-binding consensus sequence Gly-X-Gly-X-X-Gly within the N-terminal sequence. In summary, these data show the presence of two distinct amadoriase enzymes in the Aspergillus sp. soil strain selected on fructosyl adamantanamine and induced by fructosyl propylamine. In contrast to previous described enzymes, these novel amadoriase enzymes can deglycate both glycated amines and amino acids.

Glycation products in infant formulas: chemical, analytical and physiological aspects.

Infant formulas are milk-based products, which are adapted to the composition of human milk. To ensure microbiological safety and long shelf life, infant formulas usually undergo rigid heat treatment. As a consequence of the special composition and the heat regimen, infant formulas are more prone to thermally induced degradation reactions than regular milk products. Degradation reactions observed during milk processing comprise lactosylation yielding the Amadori product lactulosyllysine, the formation of advanced glycation end products (AGEs), and protein-free sugar degradation products, as well as protein or lipid oxidation. Several methods have been developed to estimate the heat impact applied during the manufacturing of infant formulas, including indirect methods such as fluorescence analysis as well as the analysis of defined reaction products. Most studies confirm a higher degree of damage in infant formulas compared to regular milk products. Differences between various types of infant formulas, such as liquid, powdered or hypoallergenic formulas depend on the analyzed markers and brands. A considerable portion of protein degradation products in infant formulas can be avoided when process parameters and the quality of the ingredients are carefully controlled. The nutritional consequences of thermal degradation products in infant formulas are largely unknown.

Two immunochemical assays to measure advanced glycation end-products in serum from dialysis patients.

Abstract Advanced glycation end-products are uremic toxins that accumulate in the serum and tissues of patients with chronic renal failure. Here, we established two enzyme-linked immunosorbent assays (ELISAs) for N ε-carboxymethyllysine and imidazolone to analyze advanced glycation end-products in human serum. Both ELISAs detected advanced glycation end-products bound to human serum albumin in a dose-dependent way. Whereas the formation of imida-zolone was independent of the presence of oxygen, concentrations of N ε-carboxymethyllysine epitopes increased 20-fold under oxidative conditions. The N ε-carboxymethyllysine ELISA showed a similar response to free, peptide-bound and protein-bound N ε-carboxymethyllysine, whereas the imidazolone antibody showed slightly higher affinity toward peptide-bound compared to protein-bound imidazolone. In human serum, linear dilution ranges from 1:10 to 1:40 (N ε-carboxymethyllysine ELISA) and from 1:2 to 1:8 (imidazolone ELISA) were found. The recovery of N ε-carboxymethyllysine from serum was 101±10% and 94±12%, respectively, and 93±15% and 97±12% for imidazolone. The coefficients of variation for intra-assay variability were 0.26–2.7% (N ε-carboxymethyllysine) and 0.1–2.4% (imidazolone), and 8.3–13.4% (N ε-carboxymethyllysine) and 7.8–12.5% (imidazolone) for inter-assay variability. In serum samples from hemodialysis patients (n=20) and controls (n=20), an approximately two-fold increase was detected in the patient group (p<0.001). The combination of the N ε-carboxymethyllysine and imidazolone ELISAs is a valuable tool to measure serum concentrations of advanced glycation end-products for clinical studies.

Identification and site-specific relative quantification of beta-lactoglobulin modifications in heated milk and dairy products.

During milk processing, proteins can be severely modified by oxidation, condensation, and Maillard reaction, leading to changes in their nutritional and technological properties. In this study, major modifications of beta-lactoglobulin, formed during the heating and processing of milk, were screened by mass spectrometry. For this purpose, beta-lactoglobulin was isolated from the milk samples by gel electrophoresis and analyzed by matrix-assisted laser desorption/ionization mass spectrometry after in-gel digestion with endoproteinase AspN. In heated milk, lactulosyllysine was detected at lysine 47 and 138 or 141 as well as methionine sulfoxide at methionine 7, 24, and 145. All these modifications increased gradually when raw milk was heated for 20, 40, and 60 min at 120 degrees C. The major modifications were also relatively quantified in dairy products, such as raw, high-temperature, ultra-high-temperature, sterilized, and condensed milk as well as infant formulas. The highest contents of lactulosyllysine at Lys47 were detected in powdered infant formulas, whereas lactulosyllysine at Lys138/141 was predominant in condensed milk samples. Methionine sulfoxide at Met7 and Met24 showed a trend toward higher modification rates in more severely processed products.

Analysis of protein glycation products by MALDI-TOF/MS.

Abstract: Matrix‐assisted laser desorption ionization‐mass spectrometry with time‐of‐flight detection (MALDI‐TOF/MS) is a promising tool to analyze advanced glycation end product (AGE)‐modified proteins. The combination of soft ionization (MALDI) with time‐of‐flight mass detection allows analysis of peptides and proteins of a molecular mass up to 300 kDa with minimal sample workup. Because the direct structural analysis of intact AGE proteins is not possible due to the formation of broad and poorly resolved peaks, peptide mapping was introduced into the analysis of AGE proteins by MALDI‐TOF/MS, allowing site‐specific analysis of defined AGEs. When methylglyoxal‐modified lysozyme was subjected to MALDI‐TOF/MS peptide mapping, methylimidazolone and argpyrimidine attached to the arginine residue and carboxyethyl (CEL) bound to the lysine were detected on peptideaa1‐7 (KVFGRCE). In contrast, only one methylimidazolone was found on peptideaa8‐35 (LAAAMKRHGLDNYRGYSLGNWVCAAKFE) and peptideaa120‐129 (VQAWIRGCRL), respectively. The analysis of AGE protein, which had been incubated with glucose, revealed the presence of an Amadori product and a carboxymethyl residue (CML) on peptideaa1‐7 and peptideaa8‐35, as well as an imidazolone A on peptideaa120‐129. Furthermore, the early Maillard reaction of lysozyme, which had been glycated by seven different sugars, was monitored by MALDI‐TOF/MS peptide mapping. Finally, this approach was successfully applied for site‐ and product‐specific relative quantification of AGEs. For example, kinetics of CML and Amadori product formation on peptideaa1‐7, as well as imidazolone A formation on peptideaa120‐129, were determined.

Analysis of Protein Glycation Products by Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry

The term protein glycation summarizes non-enzymatic reactions between amino groups of proteins and sugars or sugar degradation products, leading to early glycation products (intact sugar attached) and advanced glycation end-products (AGEs). Protein glycation is involved in the progression of several diseases, such as diabetes, uremia, and atherosclerosis. However, qualitative and quantitative analysis of in vitro or in vivo glycated proteins is still a challenging task. The introduction of matrix-assisted laser desorption ionization time-of-flight technique (MALDI-TOF) changed mass spectrometry (MS) into a valuable tool for biomedical analysis, because the soft ionization procedure allows the measurement of proteins up to 100 kDa. In the last few years, MALDI-TOF-MS was applied to the investigation of glycation processes: the analyses of plasma proteins from diabetic or uremic patients allowed a precise determination of the average number of sugar residues attached to serum albumin or immunoglobulins of each patient. Thus, a more individualized diagnosis of each patient was achieved by MALDI-TOF-MS than by other diagnostic tools. In a similar way, the glycation rate of hemoglobin, isolated from diabetic blood and of beta-2-microglobulin isolated from amyloid plaques from uremic patients was determined. The application of MALDI-TOF-MS for in vitro studies revealed important new insights into glycation mechanisms. Whereas the measurement of the intact proteins allows the determination of the average glycation rate, peptide mapping prior to MALDI-TOF-MS can reveal the exact structures of the glycation products and the glycation site. Furthermore, when the unmodified peptide is used as internal standard, MALDI-TOF-MS can also be used for reliable, site specific relative quantification of defined glycation products.

Development and validation of an enzyme-linked immunosorbent assay (ELISA) for quantification of lysozyme in cheese.

A competitive enzyme-linked immunosorbent assay (ELISA) was developed to quantify the amount of the preservative and potential allergen lysozyme in cheese using a commercially available monoclonal antibody against hen egg white lysozyme. The limit of detection for lysozyme in a cheese matrix amounted to 2.73 ng/mL, and the working range comprises 3.125-800 ng/mL. Intra- and interassay coefficients of variation were lower than 12%. Neither cross-reactivity with alpha-lactalbumin and human lysozyme nor unspecific interference with matrix components was observed. The recovery of lysozyme-spiked cheese ranged from 87.4 to 93.6% at four concentrations (50, 100, 200, and 400 mg/kg). The ELISA method was also compared to a high-performance liquid chromatography (HPLC) method, confirming the reliability and accuracy of the ELISA. A total of 21 commercially available cheese samples produced with and without lysozyme were analyzed with ELISA as well as HPLC. Both methods showed good agreement with a correlation index of R2=0.990.