Bojan Benko

Detection of autoantibodies against advanced glycation endproducts and AGE-immune complexes in serum of patients with diabetes mellitus

Advanced glycation of protein causes their immunogenicity. The evidence that advanced glycation endproducts (AGEs) have antigenic properties has led to a hypothesis that the AGE structure found in vivo may exert an autoimmune response. In the present study, we showed the sera of diabetic patients as well as of nondiabetic individuals to contain autoantibodies to epitopes of AGE structures. Contrary to what might be expected, we observed lower AGE antibody titers in diabetic subjects, and postulated that the antibodies against AGEs form immune complexes in vivo, hampering their determination. The existence of immune complexes containing AGE moiety was established by two independent criteria: (a) serum AGE-immune complexes (AGE-IC) were detected by enzyme-linked immunosorbent assay (ELISA) using an immunochemical bridge; and (b) soluble AGE-IC were precipitated from serum by polyethylene glycol and analyzed. We demonstrated the presence of circulating AGE-IC in sera, predominantly in the sera of diabetic subjects. We also found an inverse correlation between serum AGE level and AGE-IC (r=-0.8, P<0.000), indicating the serum level of AGEs to decline with an increasing presence of AGE-IC. The content of AGE in soluble immune complexes was significantly higher in diabetic patients than in control subjects (3.51+/-1.9 vs. 1.89+/-1.0 microgEq/ml (P<0.00004), and correlated inversely with free antibodies (r=-0.26, P<0.01). Interactions of AGE autoantibodies with AGE as a continuously produced antigen result in the formation of AGE-immune complexes that may play a role in the atherogenic processes.

Rat tissue collagen modified by advanced glycation: correlation with duration of diabetes and glycemic control.

Abstract Collagenous proteins are especially prone to nonenzymatic glycation, because they contain several dibasic amino acid residues with free amino groups, have a very slow turnover rate, and are exposed to ambient levels of glucose. The aim of this study was to determine the time-dependent course of advanced glycation process in diabetic rats in relation to glycemic control and duration of diabetes, compared to age-matched controls. Immunochemical assay with antibodies to advanced glycation end products (AGE) was first developed to qualitatively detect and quantify the AGE formed in rat tendon and aortic collagen. Individual collagen samples were extracted by extensive pepsin and collagenase digestion. The amount of AGE was measured by competitive ELISA and results were expressed as AGE U/mg collagen. Diabetic rats showed a significant increase in AGE content in aortic collagen at 20 weeks (n=6, 206.6 ± 16.7 U/mg collagen) compared with that measured at 4 and 12 weeks (n=6, 110 ± 12.8 U/mg collagen, and n=13, 184.9 ± 12.3 U/mg collagen at 4 and 12 weeks, respectively; p < 0.001 between 20 weeks and 4 weeks; p < 0.01 between 20 weeks and 12 weeks). The amount of AGE in tendon collagen of diabetic rats increased from 1.9 ± 0.38 U/mg at 4 weeks to 11.2 ± 6.1 U/mg collagen at 20 weeks, p < 0.001. Considerable disparity was observed in the intensity of glycation between aortic and tendon collagen. AGE-content per mg of aortic collagen was several-fold to that found in tendon collagen (p < 0.001). To investigate the effect of glycemic control on the advanced glycation process, total aortic AGE-collagen content was compared between untreated diabetic rats (D; n=13, 184.9 ± 12.3 U/mg) and diabetic rats treated for 12 weeks with insulin (DI; n=6, 133.9 ± 10.7 U/mg), or phlorizin (DP; n=6, 132.4 ± 8.9 U/mg), or by a combination of insulin/phlorizin (DIP; n=6, 124.3 ± 6.5 U/mg). In spite of therapy used, all groups of diabetic animals had a significantly higher aortic AGE-collagen content than those in the nondiabetic control group (C: n=8, 104.6 ± 14.9 U/mg) of the same age (D, DI, DP, DIP vs. C, p < 0.001). Comparison between the mean levels of glycated hemoglobin (D: 5.62 ± 0.38 % vs.C: 1.7 ± 0.05 %) and mean AGE levels in the studied group of animals yielded a very good exponential correlation (r = 0.89, p < 0.001). Glycation-derived late-stage collagen modification was detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and by immunoblotting confirmed to contain (an) AGE-structure(s). Our study provides strong immunochemical evidence of AGE formation in vivo during hyperglycemia, and of their temporal association with structural alterations of extracellular matrix proteins. The advanced glycation process is retarded and reduced in intensity, but not completely abolished, by glycemia regulation with, or independently of, insulin.

Comparison of advanced glycation endproducts on haemoglobin (Hb-AGE) and haemoglobin A1c for the assessment of diabetic control

Glycation process in vivo results in two different products: early and advanced glycation endproducts (AGEs). The mechanism of early product formation has been well described, with HbA1c as the best-studied example. The finding that advanced glycation endproducts are also formed on haemoglobin suggests that HbA1c is a precursor for Hb-AGE formation. HbA1c has been well established as an important indicator for glycaemia monitoring, but the diagnostic role of Hb-AGE has not yet been clarified. A question is whether HbA1c and Hb-AGE are competitive or complementary parameters. In our study, Hb-AGE was quantified by the competitive ELISA technique using polyclonal anti-AGE-RNase antibodies to detect AGE immunoreactivities of proteins precipitated in red cell hemolysate. Results are expressed as AGE units/mg Hb. Hb-AGE was analysed in three groups of patients divided according to HbA1c values as follows: group I (n = 25) HbA1c < 7%, Hb-AGE = 6.93 (5.7-7.3) U/mg; group II (n = 25) HbA1c = 7-10%, Hb-AGE = 8.62 (7.7-10.2) U/mg; and group III (n = 25) HbA1c > 10%, Hb-AGE = 12.47 (10.8-13.9) U/mg (median (interquartile range)). A close relation between the amounts of red cell HbA1c and Hb-AGE was observed in all diabetic subjects (n = 75) r = 0.77, P < 0.001. Patients with HbA1c level > 8% were considered to be in poor glycaemic control and those with HbA1c < 8% in good control. In the well-controlled subgroup (n = 33), HbA1c and Hb-AGE were less tightly correlated (r = 0.37, P <0.001). However, in those patients with a higher level of HbA1c = 12.55 (8.9-13.3)% (n = 42), the related Hb-AGE was 11.5 (10.3-12.8) U/mg Hb, yielding a more significant correlation (r = 0.51, P < 0.001). The content of Hb-AGE did not correlate with age (r = 0.09), diabetes duration (r = 0.05) or severity of retinopathy and/or nephropathy. The observed difference may reflect a different kinetic rate of HbA1c production and subsequently the rate of Hb-AGE formation. The discrepancy in the correlation between HbA1c and Hb-AGE suggests that they are complementary rather than opposed parameters. The amount of haemoglobin-linked AGEs does not correlate with the presence or absence of retinopathy and/or nephropathy. It seems that Hb-AGE represents only the metabolic status, equally in the subjects with and without diabetic microangiopathy.

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

Abstract 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 0–16years 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 (<0.35kIUA/L), and atopic children (n=4620) who were sensitized to ≥1 allergens (specific IgE ≥0.35 kIUA/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 10years 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 8years, 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 16years 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 8years 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.

Bisalbuminemia in Two Croatian Families

BACKGROUND Bisalbuminemia is a dysproteinemia characterized by the occurrence of two albumin fractions on serum protein separation by electrophoresis on cellulose acetate sheets. Bisalbuminemia may occur as a hereditary trait or as analytical interference with some drugs, especially penicillin. METHODS Two patients with the finding of bisalbuminemia are presented. Both patients (patient 1 was a 4-1/2-month-old male infant, and patient 2 was a 15-year-old boy) were admitted for respiratory infection. RESULTS Bisalbuminemia was detected by serum protein electrophoresis and confirmed by isoelectric focusing in pH gradient gel (pH range 4.0-6.5). This finding was supported by simultaneous detection of abnormal albumin in the mother of patient 1, while the father had normal albumin. The abnormal fast albumin in both patients had an increased relative mobility of 1.08 when measured from the sample application position. CONCLUSIONS The results presented are the first description of albumin mutations in Croatia (that according to the CISMEL group could be classified as ZC/HZ), and present the first step in identification prior to determination of structural change and amino acid sequence in the albumin molecule.

Quality of electrophoresis and isoelectric focusing images compressed using JPEG and JPEG2000

Aim: High quality of all image elements must be maintained for the purpose of telepathology. The aim of this study was to compress the images of electrophoresis and isoelectric focusing samples using JPEG (Joint Photographic Expert Group) and JPEG2000 algorithms, and to assess the quality of the compressed images before and after electronic transmission. Methods: Scanograms of serum protein electrophoresis samples of a patient with Zagreb albumin and albumin samples for isoelectric focusing of a patient with Krapina albumin were selected for the study together with a photographed scanogram of isoelectric focusing. Each image was compressed at eight compression rates (from 3.00 bpp (bit per pixel) to 0.1 bpp) using JPEG and JPEG2000 compression algorithms. All images (N = 51), both compressed and uncompressed, we retransmitted by email for assessment to eight medical biochemists: six from Croatia, one from Italy and one from Denmark. Image quality was also assessed by objective measures, i.e. compared to the quality of PSNR (Peak-Signal-to-Noise-Ratio), SNR (Signal-to-Noise-Ratio), OQF (Optimized Quality Factor) and MSE (Mean Squared Error) images. Results: All images compressed using the JPEG2000 algorithm were subjectively rated as excellent. Contrarily, images compressed using JPEG at 0.1 bpp were rated as completely useless, those at 0.2 bpp as moderately blurred, and those at 0.3-3.00 as excellent. At JPEG compression at 0.3 bpp, PSNR and SNR values corresponded to PSNR and SNR values obtained by JPEG2000 compression at 0.1 bpp.