Methylglyoxal-derivative advanced glycation endproducts: detection by competitive immunofluorometric assay and quantifying in serum and urine.

BACKGROUND: Advanced glycation endproducts (AGE) are a family of heterogeneous chemical structures formed on the host protein in the conditions of carbonyl or oxidative stress. Among AGE precursors, methylglyoxal (MG) is considered one of the key intermediates. METHODS: In the current study, we describe and evaluate a solid phase time-resolved fluoroimmunoassay (DELFIA) based on the competitive reaction between MG-AGE antibody and competitive antigen for detecting MG-adducts in serum and urine. The fluorometry assay was validated by comparison with previously established competitive ELISA. RESULTS: The concentration of MG-adducts assayed by competitive DELFIA in the sera of diabetic patients (DM, n=66) were higher in comparison to non-diabetic controls (C, n=28); DM median=294 mgEq/L (10th and 90th percentile 158-564) vs. C median=224 mgEq/L (10th and 90th percentile 124-290) (p=0.0022). In diabetic subjects, urinary excretion of MG-adducts exceeded the mean level measured in controls; DM median=36.0 mgEq/L (10th and 90th percentile 1-210) vs. C median=11.5 mgEq/L (10th and 90th percentile 1-49) p=0.0036. MG-adducts in urine were low and undetectable in 8 of 28 control subjects and 2 of 66 diabetics. The percentage recovery of MG-adduct added in the same concentration to control and diabetic pool sera showed under-recovery in the latter. Comparison of total AGE level and the amount of MG-adducts revealed MG-derivatives to account for 37% of the heterogeneous structure commonly termed AGE. CONCLUSIONS: The fluoroimmunoassay for MG-derivative AGE evaluated can be utilized on biomonitoring of MG-adducts in serum and its urinary excretion. The competitive DELFIA assay was found to be substantially more sensitive than the standard ELISA having a wider dynamic range, while sharing similar quenching attributes.

Advanced glycated human serum albumin as AGE-carrier protein in enzyme-linked immunosorbent assay.

Competitive ELISAs for advanced glycation end product (AGE) measurements are based on anti-AGE-antibody and in vitro prepared AGE-carrier competitive antigen. AGE-human serum albumin (HSA) prepared by non-enzymatic reaction between protein and glucose is often used as a competitive antigen. The aim of the study was to examine the impact of pH on AGE-HSA preparation. The sets of AGE-HSA analyzed by gel filtration chromatography, IEF and UV/VIS showed significant modifications of native albumin. A competitive ELISA was developed by using polyclonal-AGE-antibody and in vitro prepared AGE-HSA at pH 4.5-8.0. AGE-HSA preparations showed an impact on the ELISA ability to recognize AGE-immunoreactivity of serum proteins. The highest AGE-immunoreactivity was recorded with the antigen prepared at pH 4.5; detection limit declined by approximately 50% with antigens prepared at pH 6.5, 7.5 or 8.0. Study results confirmed the impact of pH on the glycation adduct formation, thus significantly modifying the results of competitive ELISA measurements.

Cut-off values for total serum immunoglobulin E between non-atopic and atopic children in north-west Croatia.

BACKGROUND: The aim of this study was to determine cut-off values for total serum immunoglobulin E (IgE) between non-atopic and atopic children with respiratory symptoms. Children of 0-16 years of age were evaluated for respiratory symptoms of >4-week duration. METHODS: Children were divided into two groups: non-atopic children (n=3355) who were non-IgE-sensitized with undetectable allergen-specific IgE (<0.35 kIU(A)/L), and atopic children (n=4620) who were sensitized to > or =1 allergens (specific IgE > or =0.35 kIU(A)/L). Upper and lower centiles were determined and cut-off values calculated using receiver operating characteristic (ROC) analysis. RESULTS: Serum total IgE increased with age in both groups, although at a variable level and rate, and reached a plateau at 9 and 10 years in non-atopic and atopic children, respectively. Atopic children had on average 14-fold higher serum total IgE compared to non-atopic children. In both groups, the median was lower than the corresponding mean and the distribution skewness was always positive (group I, 0.87; group II, 0.91). In almost all age groups, the 95th percentile for non-atopic children corresponded to the calculated cut-off values, whereas the 10th percentile for atopic children corresponded to the respective cut-off values only until the age of 8 years, after which greater differences between the cut-off values and the 10th percentile were recorded. Cut-off values for total serum IgE in children up to 16 years were determined with diagnostic sensitivity, specificity and area under the ROC curve of 96%, 91% and 0.950, respectively. CONCLUSIONS: The 95th percentile for total IgE in non-atopic children and the 10th percentile in atopic children could be taken as cut-off values in children up to 8 years of age, after which significant percentile discrepancies between non-atopic and atopic children were recorded. Since atopic subjects show a more irregular centile distribution, cut-off values are best determined by ROC analysis.

Anti-IgE therapy with omalizumab in asthma and allergic rhinitis.

The pharmacology, efficacy, dosage, adverse effects, and economics of anti IgE (omalizumab) are discussed. Omalizumab is the generic name for the human/murine chimeric (recombinant humanized) monoclonal IgG antibody. Anti-IgE prevents IgE from attaching to effector cells, and thereby blunts IgE-mediated inflammatory responses. After subcutaneous administration its absorption is slow, reaching peak concentration in serum after an average of 7-8 days. At recommended doses, serum free IgE levels decrease within 1 hour following the first dose and are maintained between doses. Dose and dosing frequency are adjusted according to body mass and serum total IgE concentration before the start of treatment. Omalizumab administered subcutaneously is an effective treatment for add-on therapy in patients with poorly controlled, moderate-to-severe allergic asthma and allergic rhinitis (adults and adolescents > 12 years). It reduces the requirement for inhaled corticosteroids while protecting against disease exacerbation. Omalizumab is well tolerated, but the safety profile requires long-term assessment in adults as well as in children.

Thrombopoietin and interleukin-6 in children with pneumonia-associated thrombocytosis.

BACKGROUND: The aim of this study was to investigate why some, but not all, children develop thrombocytosis during the course of pneumonia. METHODS: The retrospective study included 40 healthy children and 75 children with pneumonia: 17 patients with platelet count within the reference values, i.e., platelet count 450 x 10(9)/L. Erythrocyte sedimentation rate, leukocyte and platelet counts, and concentrations of hemoglobin, C-reactive protein, interleukin-6 and thrombopoietin were determined in the blood of patients and control groups of children. RESULTS: Patients with thrombocytosis were slightly younger (3.0 +/- 1.8 years and median 2.5 years, respectively) than patients with normal platelet count (3.8 +/- 2.4 years and median 4 years, respectively). Additionally, according to clinical and radiological findings, pneumonia in children with thrombocytosis had a more severe and protracted course. Serum thrombopoietin concentrations were found to be 91.2 +/- 41.7 ng/L (range: 14.3-166.7 ng/L) in patients with normal platelet count (313 +/- 70 x 10(9)/L, range: 206-428 x 10(9)/L). In patients with thrombocytosis (581 +/- 131 x 10(9)/L, range: 450-830 x 10(9)/L) serum thrombopoietin ranged from 63.6 to 1115.9 ng/L (526.6 +/- 268.4 ng/L). In these patients both concentration of hemoglobin (114 +/- 12 g/L) and iron (4.3 +/- 1.3 micromol/L) significantly decreased as compared with the control group. CONCLUSIONS: Study results suggested the possible development of reactive thrombocytosis in children with pneumonia. As platelets are involved in inflammatory reaction, reactive thrombocytosis might be part of the mechanism of defense. Reactive thrombocytosis may develop as a sequel of either anemia or inflammatory reaction (or both).

Structural analysis and fatty acid-binding properties of two Croatian variants of human serum albumin.

BACKGROUND: The aim of the present work was to characterize the molecular defects of a slow-migrating (albumin Zagreb) and a fast-migrating (albumin Krapina) genetic variant of human serum albumin detected in heterozygous persons living in Croatia and to elucidate the fatty acid-binding properties of the two alloalbumins. METHODS: Purification and structural identification of the variants were performed by conventional protein chemistry methods, whereas types and amounts of albumin-bound, endogenous fatty acids were determined by gas chromatography. RESULTS: Protein sequencing established that albumin Zagreb is a proalbumin variant (-1Arg-->Gln), and that albumin Krapina is due to a mutation within the mature polypeptide chain (573Lys-->Glu). The gas chromatographic results showed that the fatty acid-binding properties of the proalbumin variant are normal, while the amino acid substitution in position 573 resulted in a general decrease of fatty acid binding. CONCLUSIONS: The structural defects of the first alloalbumins, detected by routine clinical electrophoresis among the Croatian population, were characterized. Albumin Zagreb is caused by a hot-spot mutation occurring in a CpG sequence in the albumin gene. It is commonly assumed that bisalbuminaemia has no direct clinical relevance. However, the present study suggests that naturally occurring mutations can affect the ligand-binding properties of human serum albumin.

A microassay for measurement of Fc function of human immunoglobulin preparations by using tetanus toxoid as antigen.

In order to minimize possible adverse reactions, the functional integrity of proteins in products derived from human plasma has to be unaffected by methods of preparation and storage conditions. Numerous biologically relevant functions of IgG, a major component of immunoglobulin for intravenous use preparations (IVIG), rely on the integrity of Fc fragments. Manufacturers are obliged to prove that Fc-mediated functions are maintained in IVIG preparations. The European Pharmacopoeia's monograph proposes a Rubella antigen-based test for Fc function of immunoglobulins. We present a modification of the proposed method achieved by using more convenient and readily available tetanus toxoid as an alternative antigen target and by adapting the procedure for the use on microtitre plates, thus greatly enhancing its feasibility and sample throughput. The test conditions were optimized so that batch-to-batch variability in tetanus antibody content did not influence the result. The precision of the test was within +/- 5%. By using this test, we compared Fc functionality of 9 commercial IVIG-7S preparations, which were prepared by using different virus inactivation/removal approaches. No significant differences between them have been found.

In vitro glycation of human immunoglobulin G.

BACKGROUND: Glucose can covalently bind to the proteins by nonenzymatic process often termed glycation. Glycation of IgG is of special interest due to its possible influence on the functionality of immunoglobulins and overall immunocompetence. METHODS: The glycation of IgG was studied using radioactive D-[U-14C]-glucose. RESULTS: The kinetics, concentration/temperature dependence and distribution of glycation binding sites of human IgG were studied under various conditions. (a) In average, 0.7 glucose molecules were found to be bound per IgG after 90 days of incubation at 37 degrees C with normal serum IgG and glucose concentrations, and 3.1 molecules after incubation with glucose in the concentration characteristic for diabetics. (b) Incubation of the same solutions for 270 days at 4-8 degrees C resulted in binding of 0.3 and 0.8 glucose molecules per IgG, respectively. (c) After 90 days at 37 degrees C in the presence of 0.56 mol/l of glucose, IgG was glycated with averagely 47 glucose molecules per IgG and with 9 after 270 days at 4-8 degrees C. (d) At very high glucose concentration (1.67 mol/l) in concentrated IgG solution (87 g/l), the molar ratio glucose/IgG reached 41 after incubation at 60 degrees C for 10 h. CONCLUSIONS: Glycation of IgG with glucose nearly resembled the first order reaction kinetics. No preferential glycation sites were found as bound glucose was equally distributed throughout F(ab)2 and Fc fragments as well as on the light and heavy immunoglobulin chains.