5-hydroxymethylfurfural and Diabetes-Mellitus--Type-1

This study analyzes effect of glycation on ApoB-100 residues by D-ribose as D-ribosylated-glycated LDL might be responsible for the cause of diabetes mellitus because of its far higher antigenic ability. The binding characteristics of circulating auto-antibodies in type 1 and type 2 diabetes patients against native and modified LDL were assessed.. T1 Diabetes (n = 43), T2 diabetes patients (n = 100) were examined by direct binding ELISA as well as inhibition ELISA, were compared with healthy age-matched controls (n = 50).. High degree of specific binding was observed by 74.42% of T1 diabetes and 45.0% of T2 diabetes patient's sera toward glycated LDL, in comparison to its native analog. Competitive inhibition ELISA reiterates the direct binding results. Furthermore, ketoamine content, Hydroxymethylfurfural (HMF) content and carbonyl content were also estimated in patient's sera healthy subjects. The increase in total serum protein carbonyl levels in the diabetes patients was largely due to an increase in oxidative stress. The increase in ketoamine as well as HMF content inpatients sera than healthy subjects is an agreement of induced glycation reaction in patients than healthy subjects.. D-ribosylated-LDL has resulted in structural perturbation causing generation of neo-antigenic epitopes that are better antigens for antibodies in T1 and T2 diabetes patients.

Topics: Adult; Aged; Apolipoprotein B-100; Autoantibodies; Diabetes Mellitus, Type 1; Diabetes Mellitus, Type 2; Enzyme-Linked Immunosorbent Assay; Epitopes; Female; Furaldehyde; Glycation End Products, Advanced; Glycosylation; Humans; Lipoproteins, LDL; Male; Middle Aged; Protein Binding; Ribose

This study analyzes effect of glycation on proteins rich in lysine residues as hyperglycemia induced protein glycation has been mainly reported in diabetes mellitus at the intrachain lysine residues leading to the formation of Amadori modified proteins. We have studied the effect of glucose on poly-l-lysine (PLL), a homopolymer of lysine residues. Levels of Amadori products in the glycated PLL were evaluated by fructosamine assay and the presence of 5-hydroxymethylfurfural (HMF) in the glycated PLL was analyzed by thiobarbituric acid assay. Fluorescence and FT-IR spectroscopy were applied to characterize the modified PLL. Binding characteristics of experimentally induced antibodies against glycated PLL and the presence of antibodies against glycated PLL in the sera of diabetes patients was evaluated by solid phase enzyme immunoassays. The fructosamine assay showed significantly high yield of early glycation (Amadori) products in the glycated PLL, which was confirmed by increased yield of HMF from Amadori products of glycated PLL. Loss in fluorescence intensity and appearance of a new band corresponding to Amadori products were observed in FT-IR spectrum of the glycated PLL. Glycated PLL was found to be highly immunogenic in rabbits as compared to the native form. Serum antibodies from diabetes patients showed appreciably high recognition of the glycated PLL. The results conclusively show the glycation induced damage to the lysine molecules and specific recognition of Amadori-lysine residues by serum antibodies from diabetes patients. The glycated lysine residues may serve as a diagnostic biomarker for early glycation process in diabetes mellitus.

Topics: Adult; Animals; Antibodies; Diabetes Mellitus, Type 1; Diabetes Mellitus, Type 2; Female; Fructosamine; Furaldehyde; Glycosylation; Humans; Lysine; Male; Middle Aged; Polylysine; Rabbits

To evaluate the relative value of glycosylated serum proteins (GSPs) versus glycosylated hemoglobin (HbA1c) in assessing glycemic control in diabetes mellitus, we performed regular monitoring of GSPs and HbA1c in 30 subjects with insulin-dependent diabetes mellitus (IDDM) or non-insulin-dependent diabetes mellitus (NIDDM) who performed frequent self-glucose monitoring. Analysis of the relationship between patterns of glycemic control and GSPs and HbA1c demonstrated that subjects with IDDM and NIDDM appeared similar when the more traditional indicators of glycemic control such as mean blood glucose level (166.9 +/- 20.9 v 177.4 +/- 39.6 mg/dL) or HbA1c (83.57 +/- 12.8 v 80.24 +/- 15.7 mmol hydroxymethyl furfuraldehyde [HMF]/mol hemoglobin [Hgb]) were used. However, when GSP levels or the standard deviation of mean glucose levels (SDMG) were used to assess glycemic control, higher levels were found in subjects with IDDM (52 +/- 10.3 mg/g protein and 28.59 +/- 7.60 mg/dL) versus NIDDM (44.6 +/- 15.2 mg/g protein and 21.6 +/- 15.9 mg/dL). Using multivariate analysis, GSPs were predictive of SDMG (P = .046), whereas HbA1c added no significant further information (P = .27). Our results suggest that GSPs may be more sensitive than HbA1c assay to the greater fluctuations in blood glucose levels generally associated with IDDM.

Topics: Adult; Blood Glucose; Blood Glucose Self-Monitoring; Blood Proteins; Colorimetry; Diabetes Mellitus, Type 1; Diabetes Mellitus, Type 2; Furaldehyde; Glycated Hemoglobin; Glycated Serum Proteins; Glycoproteins; Humans; Middle Aged; Multivariate Analysis

We developed a simple isocratic high performance liquid chromatography (HPLC) method for the quantitative determination of 5-hydroxymethyl-2-furfuraldehyde (5-HMF) liberated by mild hydrolysis of small amounts of glycosyl proteins. The absorbance of hydrolysate components after HPLC separation was recorded at 280 nm. To detect substances possibly interfering with the 5-HMF peak we always recorded the ratio of the peak heights A280 nm/A254 nm which was a constant value of 4.4. For each sample the blank was obtained by reduction with NaBH4 before hydrolysis with oxalic acid 1 mol/l. The best NaBH4/protein ratio was found to be 4 mg/mg. With this method we measured the nonenzymatic glycosylation (glycation) as 5-HMF in samples with a protein concentration as low as 0.8 mg/ml. 5-HMF produced per milligram of protein was independent from protein concentration for a wide range (0.8-10 mg/ml). The mean coefficient of variation for within assay and between precision was 6.8 and 11.6%, respectively. The 5-HMF measured on plasma proteins from normal subjects (n = 7) was 0.16 +/- 0.04 nmol/mg. Protein from insulin-dependent diabetic patients was 0.31 +/- 0.07 nmol/mg. With this method we succeeded in detecting an excessive glycation of platelet membrane proteins in 13 type-I diabetic patients.

Topics: Blood Platelets; Chromatography, High Pressure Liquid; Diabetes Mellitus, Type 1; Furaldehyde; Glycoproteins; Humans; Hydrolysis; Microchemistry

Human platelet membrane proteins (PMP), incubated in vitro in the presence of various concentrations of glucose, undergo nonenzymatic glycation, as evidenced by incorporation of [3-3H]glucose radioactivity into the acid-precipitable fraction. The time course of the reaction is linear for the first hours, and the rate of glycation depends on the glucose concentration in the medium: at a glucose concentration of 80 mmol/L, up to 60 nmol of glucose is bound per milligram of PMP. The ketoaminic nature of the glucose/protein linkages was demonstrated by the finding of 5-hydroxymethylfurfuraldehyde by liquid-chromatographic analysis of acid hydrolysates of PMP. We analyzed PMP from 13 subjects with type I poorly controlled diabetes and from 10 nondiabetics. Nonenzymatic glycation, evaluated as nanomoles of the aldehyde per milligram of protein, was much greater in diabetic patients than in nondiabetics: 1.58 +/- 0.70 vs 0.37 +/- 0.18 (mean +/- SD).

Topics: Blood Platelets; Cell Fractionation; Chromatography, Liquid; Diabetes Mellitus, Type 1; Furaldehyde; Glucose; Humans; In Vitro Techniques; Membrane Proteins

In 18 control subjects and in 41 Type 1 (insulin-dependent) diabetic patients (13 with normal proteinuria, group A; 15 with microproteinuria, group B; and 13 with clinical proteinuria, group C), mean blood glucose, glycosylated haemoglobin and non-enzymatic glycosylated serum and urinary proteins, expressed as 5-hydroxymethylfurfural (5-HMF), were measured. In each group of diabetic patients, the levels of mean daily blood glucose, glycosylated haemoglobin and serum 5-HMF/mg protein were higher than in the control subjects. The urinary 5-HMF/mg proteinuria and the urinary/serum 5-HMF concentration ratio values were raised in group A and reduced in groups B and C. Moreover, they showed a negative correlation with 24-h urinary protein excretion in the control subjects and in each group of diabetic patients. The urinary 5-HMF/day in groups A, B and C was greater than in the control subjects. The urinary 5-HMF/day did not correlate with the mean daily blood glucose levels and, only in group A, did it correlate with serum 5-HMF and glycosylated haemoglobin. This suggests that, in this group, functional factors result in the increased renal elimination of 5-HMF and, therefore, of non-enzymatically glycosylated proteins. However, in the other groups of patients, this elimination depends on the degree of proteinuria.

Topics: Blood Glucose; Blood Proteins; Diabetes Mellitus, Type 1; Diabetic Nephropathies; Diabetic Retinopathy; Furaldehyde; Glycated Hemoglobin; Glycated Serum Proteins; Glycoproteins; Humans; Proteinuria

We describe here some useful modifications of the thiobarbituric acid (TBA) assay for measurement of nonenzymatic glucosylation of serum protein. The modified assay minimizes interference by glucose without a lengthy dialysis step, and does not require an independent blank determination. These modifications should make the TBA assay more convenient for evaluating glycemic control in diabetes. Serum protein is first precipitated with cold ethanol to remove endogenous glucose. The protein is then hydrolyzed in an oxalic acid solution to release glucose as hydroxymethylfurfural (HMF). The HMF is reacted with TBA to form a chromophore which is extracted into isobutanol for spectrophotometric analysis (lambda max = 435 nm). The absorbance at 435 nm is corrected by subtracting a blank reading at 500 nm, and the nmol HMF released is determined using a standard curve prepared with pure HMF. Normal values of this assay for both adults and children are 0.38 +/- 0.10 nmol HMF/mg serum protein (means +/- 2 SD). When the assay was applied to serum samples from a group of 39 Type I diabetic children more than 90% of the children exceeded the normal range of the assay.

Topics: Adolescent; Adult; Blood Proteins; Chemical Precipitation; Child; Diabetes Mellitus, Type 1; Furaldehyde; Humans; Hydrolysis; Methods; Reference Values; Spectrophotometry; Thiobarbiturates