Anti-glycating and anti-cytotoxic effect of silibinin on albumin at early glycation: A physiochemical study.

During persistent hyperglycaemia, albumin, one of the major blood proteins, can undergo fast glycation. It can be expected that timely inhibition of protein glycation might be add quality years to diabetic patients' life. Therefore, this study was designed to analyse the role of silibinin to reduced or delay amadori adduct formation at early glycation and its beneficial effect to improve the glycated albumin structure and conformation. We also analysed cytotoxic effect of amadori-albumin in the presence of silibinin on murine macrophage cell line RAW cells by MTT (3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide) assay. Formation of early glycated product (furosine) in all samples was confirmed by LCMS. Albumin incubated with glucose only showed presence of furosine like structure. Albumin treated with silibinin in the presence of glucose did not show such furosine like peak. This LCMS result showed the silibinin play a protective role in the formation of early glycated product. HMF contents were also reduced in the presence of silibinin, when albumin was incubated with increasing concentrations of silibinin (100 and 200 µM) in the presence of glucose. ANS binding fluorescence decrease by increasing silibinin concentrations with amadori-albumin. SDS-PAGE was also showed that no significant difference in the band mobility of albumin treated with silibinin as compared to native albumin. The secondary conformational alteration in amadori-albumin due to silibinin were confirmed by FTIR. This spectrum showed slight shift in amide I and Amide II band in albumin co-incubated with glucose and silibinin as compared to albumin incubated with glucose only. We further discussed about cytotoxic effect of amadori albumin and its prevention by silibinin. MTT assay results demonstrated that amadori-albumin showed cytotoxic effect on RAW cells but silibinin showed protective role and increased the cell viability. Moreover, the results showed that silibinin has anti-glycating potential and playing a role to prevent the formation of Amadori-albumin in-vitro. Silibinin possesses strong anti-glycating capacity and can improve albumin structure and function at early stage. It might be useful in delaying the progression of diabetes mellitus and its secondary complications at early stage.

A comparative analysis of fructosamine with other risk factors for kidney dysfunction in diabetic patients with or without chronic kidney disease.

BACKGROUND: Hyperglycemia is the driving force for the development of diabetic nephropathy leading to the end stage renal disease. It is well known that in hyperglycaemic condition, serum proteins become glycated through non-enzymatic glycation. With the other risk factors, serum fructosamine may be an important risk factor for kidney impairment. To assess coexistence of frequently documented risk factors of kidney dysfunction with serum fructosamine in diabetic patients with chronic kidney disease (CKD). METHODS: In this study, total 150 individuals, as control, type2 diabetic patients without complication and with CKD were included. Blood samples were collected from all the samples to estimate blood glucose, HbA1c, serum creatinine, fructosamine levels and lipid profile. Statistical analysis i.e. regression and correlation between serum fructosamine and other documented risk factors for diabetic CKD has been done. P < 0.001 was considered significant. RESULTS: Serum fructosamine, HbA1c, creatinine levels, cholesterol and LDL were increased significantly (P < 0.001) in diabetic patients with CKD compared to without complications. Systolic and diastolic blood pressure and BMI were also significantly higher in diabetic patients compared to control. Serum creatinine, total cholesterol and LDL showed a significant positive correlation but HDL showed a negative correlation with fructosamine in CKD diabetic patients. No significant correlation was found with any risk factors in diabetic patients without complications expect HbA1c. CONCLUSION: It is concluded that elevated serum fructosamine level is strongly associated with kidney dysfunction in diabetic patients. As there is a significant link between serum fructosamine and other risk factors for CKD diabetic patients.

A study on correlation between oxidative stress parameters and inflammatory markers in type 2 diabetic patients with kidney dysfunction in north Indian population.

BACKGROUND: Research reports support the statement that oxidative stress and inflammation are well-known risk factors for chronic kidney disease (CKD) in patients with diabetes. This study was designed to ascertain the associated role of oxidative stress parameters and inflammatory markers in diabetes and related CKD among the north Indian population. METHODS: The study was divided into three groups as healthy subjects (group 1), patients with diabetes without complication (group 2), and with CKD (group 3). Serum levels of malondialdehyde (MDA) and nitric oxide (NO), superoxide dismutase (SOD), catalase (CAT), and glutathione reductase (GR) content were estimated in all individuals. Inflammatory cytokines interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α)-α were determined by enzyme-linked immuno-sorbent assay. RESULTS: MDA, protein carbonyl, and NO were significantly elevated in patients with type 2 diabetes as compared with healthy subjects (P ≤ 0.05). Total thiols content were found to be significantly decreased in patients with diabetes with CKD. The activity of antioxidant enzymes SOD, CAT, and GR showed a significant suppression in patients with type 2 diabetes with or without CKD as compared with healthy subjects. Nevertheless, the levels of proinflammatory cytokines IL-6 and TNF-α were significantly upregulated ( P ≤ 0.05) as compared with healthy subjects. CONCLUSION: Determination of antioxidant defense parameters and inflammatory markers contributes to understand the relationship between oxidative stress and inflammation on the development and prevention of chronic kidney disease in Indian patients with diabetes.

Inhibitory effect of silibinin on Amadori-albumin in diabetes mellitus: A multi-spectroscopic and biochemical approach.

Due to increased understanding of the damaging effects of glycation process, it is highly desirable to manage this process effectively either by prevention or by managing the consequences of glycation preferentially at early stage. The use of potential naturally occurring compounds as anti-glycating agents may provide an effective approach to control the development and progression of diabetic associated complications. In the present study, human serum albumin (albumin) was co-incubated with glucose and different concentrations of silibinin. Silibinin was demonstrated to possess anti-glycation activity. We found that silibinin inhibits glucoseinduced glycation at an early stage. We analyzed the effect of silibinin on albumin structure and its biochemical properties at early stage of glycation through various biophysical and biochemical techniques. Nitro blue tertazolium assay results showed that fructosamine formation was reduced in the presence of silibinin. UV-visible spectra results showed decrease in the absorbance with increasing concentrations of silibinin towards native albumin absorbance. Fluorescence results showed that the intensity was increased with increasing the silibinin concentrations as compared to Amadori-albumin. In addition, Far-UV CD spectra demonstrated some restoration of α-helicity when albumin was incubated with glucose in the presence of silibinin. Moreover, silibinin caused significant reduction in carbonyl contents with concomitant increase in free thiol, lysine and arginine residues. The anti-glycation activity of silibinin was concentration-dependent. From all the observations, we can conclude that silibinin might be acting as an obstacle in the binding of glucose with albumin and thus preventing the glycation induced changes in albumin. Silibinin may be effective in delaying glycation mediated pathologies in diabetic individuals.

Biophysical and immunological characterization of 2-dRib modified HSA and its implications in diabetes mellitus.

Glycoxidation of protein may lead to develop diabetes. In the present study, different concentrations of 2-deoxy d-ribose (2-dRib) were used to modify human serum albumin (HSA). Nitro Blue Tetrazolium (NBT) assay results showed that yield of the fructosamine content was directly proportional to the concentration of 2-dRib. UV and fluorescence spectroscopy results showed an increment in hyperchromicity and decrease in fluorescence intensity of 2-dRib modified HSA as compared to native HSA. Further secondary structural changes were confirmed by UV-circular dichroism (UV-CD) and Fourier transform infrared spectroscopy (FT-IR). To evaluate the immunogenicity of 2-dRib modified HSA, rabbits were immunized with native and 2-dRib modified HSA. Modified HSA sera showed high antibodies titre as compared to native HSA. Moreover, the binding affinity of native and modified HSA with diabetic patient's sera has been evaluated by direct binding ELISA. It was found that diabetic patient's sera showed high binding affinity with the modified HSA as compared to native HSA. On the basis of above findings, it can be concluded that 2-dRib is a potential glycating agent that can cause alteration in HSA structure and make HSA more immunogenic that might play a role in onset and progression of diabetes mellitus and its complications.

SLE autoantibodies are well recognized by peroxynitrite-modified-HSA: Its implications in the pathogenesis of SLE.

Systemic lupus erythematosus (SLE) is an autoimmune disorder where the role of inflammatory processes in the etiopathogenesis is well documented. Despite extensive research, the trigger for initiation of the disease has not been identified. Peroxynitrite, a strong nitrating/oxidizing agent has been reported in SLE and other autoimmune diseases. In this study, human serum albumin (HSA) was exposed to peroxynitrite for 30min at 37°C. The structure of HSA was grossly perturbed when examined by various physico-chemical techniques. Peroxynitrite mediated nitration of HSA was confirmed by LCMS/MS. Furthermore, increase in hydrodynamic radius of peroxynitrite-modified-HSA suggests the attachment of nitro group(s). Aggregation in peroxynitrite-modified-HSA was evident in a TEM scan. Nitration, oxidation, cross linking, aggregation etc conferred immunogenicity on peroxynitrite-modified-HSA. High titre antibodies were elicited in rabbits immunized with peroxynitrite-modified-HSA. Induced antibodies were highly specific for peroxynitrite-modified-HSA but showed considerable binding with other nitrated molecules. Direct binding/inhibition ELISA carried out with autoantibodies in SLE sera showed preferential binding with peroxynitrite-modified-HSA. Anti-nDNA positive IgG from SLE sera showed preference for peroxynitrite-modified-HSA when subjected to immunoassay (direct binding and inhibition) and mobility shift assay. Our results reinforce the role of augmented inflammation in SLE progression.

Deciphering the enhanced inhibitory, disaggregating and cytoprotective potential of promethazine towards amyloid fibrillation.

Increasing evidence proposed that amyloid deposition by proteins play a crucial role in an array of neurotoxic and degenerative disorders like Parkinson's disease, systemic amyloidosis etc, that could be controlled by anti-aggregation methodologies which either inhibit or disaggregate such toxic aggregates. The present work targets the amyloid inhibiting and disaggregating potential of promethazine (PRM) against human insulin (HI) and human lysozyme (HL) fibrillogenesis. Biophysical techniques like Rayleigh scattering measurements (RLS), Thioflavin T (ThT) and 8-Anilinonaphthalene-1-sulfonic acid (ANS) fluorescence measurement, circular dichroism (CD) and dynamic light scattering (DLS) measurements illustrated the inhibitory action of PRM. The half maximal inhibitory concentration (IC50) of PRM for HI and HL was estimated to be 114.81±1.21µM and 186.20±1.03µM, respectively. Microscopic techniques revealed the absence of fibrillar structures when HI and HL was co-incubated with PRM. Cytoprotective behavior of PRM was investigated by cell based cytotoxicity assay performed on SH-SY5Y neuronal cell lines. The half maximal disaggregation concentration (DC50) was calculated as 21.37±0.89µM and 45.70±0.76µM, signifying that PRM is much potent to disaggregate pre formed fibrils rather than to inhibit fibrillation. Thus, PRM could be beneficial as therapeutic agent that can aid in the cure of amyloid related diseases.

Hyperglycemia induced reactive species trigger structural changes in human serum albumin of type 1 diabetic subjects.

Chronic oxidative stress fuels pathogenesis of a large set of diseases. Oxidative stress is the cause and consequence of numerous diseases including type 1 diabetes mellitus (T1DM), in which there is selective destruction of insulin producing pancreatic ß-cells. Studies have documented that hyperglycemia produces profound stress. In vivo production of numerous reactive oxygen, nitrogen, chlorine species and lipid/sugar oxidation products in T1DM patients may be the result of persistent hyperglycemia. Post-translational modifications by reactive species may create new antigenic epitopes and play a role in the development of autoimmune response. In this paper our main focus was to establish the effect of existing hyperglycemia induced oxido-nitrosative stress in T1DM patients on the integrity of human serum albumin. Raised nitric oxide, carbonyl, RBC hemolysis, lowered ferric reducing antioxidant power (FRAP), thiol and deformed RBC in T1DM are all highly suggestive of persistent oxido-nitrosative stress. Hyperglycemia induced generation of advanced glycation end products (AGEs) was established by LCMS. Chronic oxido-nitrosative stress can modify HSA in T1DM patients, producing immunologically active albumin. Therefore, it is speculated that the aberrant HSA may play a role in the initiation/progression of T1DM.

An overview of in vitro and in vivo glycation of albumin: a potential disease marker in diabetes mellitus.

Non-enzymatic glycation of macromolecules, especially proteins leading to their oxidation is increased in diabetes mellitus due to hyperglycaemia and play an important role in associated complications of the disease. Protein glycation mostly occurs in intra chain lysine residues resulting in the formation of early stage Amadori products which are finally converted to advance glycation end products (AGEs). This review deals with the structural studies of in vitro and in vivo glycated human serum albumin (HSA). The aim of this review is to explain the disturbance in secondary and tertiary structure of albumin upon glucosylation and the immunogenic potential of modified albumin. Amadori-albumin may have enough potential to provoke the immunoregulatry cells and generate autoantibodies in diabetic patients. Role of Amadori-albumin in the induction of autoantibodies in type2 diabetes especially in chronic kidney disease (CKD) patients has been discussed. This review also considers various studies that investigate the effects of glycation on the structural and immunological properties of HSA. The use of glycated albumin (GA) as a short to intermediate term marker for glycaemic control in diabetes is also focused.

Glycosylation Gap in Patients with Diabetes with Chronic Kidney Disease and Healthy Participants: A Comparative Study.

AIM: The aim of this study it to determine the level of glycosylation gap in patients with type 2 diabetes and its relation with kidney dysfunction. MATERIALS AND METHODS: In this study, 150 individuals were enrolled (aged 20-75 year) and divided into three groups. Group 1 included 50 nondiabetic individuals who served as control. Group 2 included 50 patients with type 2 diabetes without chronic kidney disease (CKD), and in Group 3, there were 50 patients with type 2 diabetes with CKD. Glycated hemoglobin (HbA1c) and fructosamine (FA) were measured in all groups to determine the glycosylation gap (GG), predicted HbA1c, and mean blood glucose (MBG). GG is defined as the difference between measured HbA1c and HbA1c predicted from FA based on the population regression of HbA1c on FA. The variables were compared by correlation analysis. RESULTS: Serum creatinine level was significantly high in patients with CKD (1.93 ± 0.99) as compared to patients with diabetes and control (0.891 ± 0.16; 0.912 ± 0.1), respectively. The study demonstrated a significant elevation in serum FA, measured HbA1c and predicted HbA1c, MBG in patients with diabetes with CKD as compared with those of without CKD, and controls. GG was found in healthy control (0.51 ± 0.78), patients with type 2 diabetes without CKD (0.62 ± 0.45), and patients with diabetes with CKD (1.0 ± 0.91), respectively. CONCLUSION: It is concluded that GG may be a useful clinical research tool for evaluating pathological source of variation in diabetes complications such as kidney disease.

Non-enzymatic glucosylation induced neo-epitopes on human serum albumin: A concentration based study.

Hyperglycaemia induced non enzymatic glycation is accelerated in diabetic patients and aggressively involved in diabetes progression. Human serum albumin (HSA) is the most abundant protein in blood circulation. In hyperglycaemia, it undergoes fast glycation and results in the impairment of structure. Our previous study has demonstrated structural alterations in Amadori-albumin modified with different glucose concentrations from physiological to pathophysiological range. Here, we focused on immunological characterization of Amadori-albumin. Immunogenicity of Amadori-albumin was analysed by direct binding and competitive ELISA. Amadori-albumin was found to be highly immunogenic (expect albumin modified with 5mM) and induced high titre antibodies depending upon the extent of modification. Very high titre antibodies were obtained with albumin modified with 75mM glucose as compared to native albumin. Anti-Amadori-albumin-IgG from rabbit sera exhibited increased recognition of Amadori-albumin than native albumin in competitive immunoassay. Alteration induced in albumin after glucosylation has made it highly immunogenic. Induced antibodies were quite specific for respective immunogens but showed cross-reaction with other Amadori/native proteins. It suggests that glucosylation has generated highly immunogenic epitopes on albumin. Formation of high molecular weight immune complex with retarded mobility further supports specificity of anti-Amadori-albumin-IgG towards Amadori-albumin. It may be concluded that due to early glycation, an array of modification occurred in HSA structure. Such gross structural changes might favour polymerization of most of the native epitopes into potent immunogenic neo-epitopes, but some original epitopes were still active and has contributed in the immunogenicity. It could be concluded that induction of anti-Amadori-albumin antibodies may be due to protection of glucose modified albumin from protiolytic breakdown. We assumed that this type of protein modifications might occur in diabetic patients in hyperglycaemic conditions that may be recognised as foreign molecules and can induce autoantibodies. Increased level of anti-Amadori-albumin autoantibodies may be used as a biomarker in disease diagnosis and its progression.

Effect of peroxynitrite on human serum albumin: a multi technique approach.

In this study, human serum albumin (HSA), the most abundant protein of blood plasma, was modified with varying concentrations of peroxynitrite. The peroxynitrite-induced changes in HSA was monitored by spectroscopy, SDS-PAGE, 1-anilinonaphthalene-8-sulfonic acid (ANS), thermal denaturation studies, and matrix-assisted laser desorption/inonization-time of flight mass spectrometry (MALDI-TOF MS). Aggregate formation was studied by thioflavin T binding and scanning electron microscopy (SEM). The results indicated formation of 3-nitrotyrosine, 6-nitrotryptophan, dityrosine, and carbonyls in modified samples and showed retarded mobility in SDS-polyacrylamide gel. Reduction in α-helicity and surface protein hydrophobicity confirmed the secondary and tertiary structure alterations in peroxynitrite-modified-HSA. Also, attachment of nitro group and increase in melting temperature was observed in modified sample. Furthermore, significant enhancement in the fluorescence intensity of ThT upon binding with peroxynitrite-modified-HSA and images under scanning electron microscope are suggestive of protein aggregation. It is, therefore, speculated that HSA modified by endogenously formed peroxynitrite might act as a trigger for nitration/aggregation and suggested the role of peroxynitrite-modified-HSA in SLE.

Study of IL4-590C/T and IL6-174G/C Gene Polymorphisms in Type 2 Diabetic Patients With Chronic Kidney Disease in North Indian Population.

To explore the associations between potential functional promoter polymorphisms in pro-inflammatory and anti-inflammatory (IL-4(-590C/T) and IL-6(-174G/C) cytokine genes, and kidney dysfunction in North Indian type 2 diabetic subjects with chronic kidney disease. A total of 150 subjects aged 25-75 year were included in this study. The glomerular filtration rate (GFR) and serum creatinine were estimated. PCR was performed to analyse genotype distribution in IL-4 (-590T/C) and IL-6 (-174G/C) among healthy, type 2 diabetic patients with or without CKD. The genotype distributions were determined by Hardy-Weinberg equilibrium. CKD patients showed lower GFR (59.36 ± 1.33 ml/min/1.73 m2 ) and higher serum creatinine (1.93 ± 0.99% mg) level in comparison to diabetic patients without CKD and healthy subjects. Genotypic distribution of the different genotypes among the study groups in IL-4 gene was genotype CC = 30, TC = 12, and TT = 8 in CKD patients. In type 2 diabetic patients without CKD, genotype distribution was CC = 38, TC = 10, and TT = 2. In healthy subjects, distribution of genotype was CC = 35, TC = 14, and TT = 1. The distribution of different genotype among the study groups for IL-6 gene was GG = 27, GC = 20, and CC = 3 in healthy subjects; GG = 28, GC = 19, and CC = 3 in diabetic patients without CKD and GG = 38, GC = 11, and CC = 1 in diabetic patients with CKD. There was no significant difference in the distribution of genotype frequencies between healthy subjects and diabetic patients without CKD but a significant difference was found in diabetic patients with CKD. The functional promoter polymorphisms IL4-590C/T and IL6-174G/C, which affect the IL-4 and IL-6 levels in north Indian subjects, were associated with kidney dysfunction and CKD. J. Cell. Biochem. 118: 1803-1809, 2017. © 2016 Wiley Periodicals, Inc.

Hyperglycemia induced structural and functional changes in human serum albumin of diabetic patients: a physico-chemical study.

Structural and functional changes in albumin are of particular interest as numerous studies in vivo have reported a strong involvement of glycated-HSA in the development and progression of chronic diabetic complications. Non-enzymatic addition of glucose molecules to a protein induces structural changes in it. These changes depend on the degree of glycation. In this study, conformational changes in glycated-HSA and its antioxidant capacity were evaluated. HSA was purified from diabetic patients with/without CKD and healthy subjects. Glycation induced an increase in the molecular mass of HSA as determined by mass spectroscopy. Further secondary and tertiary structural changes were observed by UV, circular dichroism (CD) spectroscopy, Fourier transform infrared spectroscopy (FTIR), tryptophan and 1-anilinonaphthalene-8-sulfonic acid (ANS) fluorescence. The mean α-helix content was found to be 59.46% for normal HSA and it was reduced down to 45.63% in HSA isolated from diabetic patients without CKD and to 37.48% in CKD-HSA. FTIR analysis showed Amide I and Amide II band shifting in HSA of diabetic patients without and with CKD. These findings indicate the secondary structure changes in glycated HSA. The tertiary structure is also affected by in vivo glycation as confirmed by intrinsic fluorescence and ANS fluorescence results. Consequently, these structural changes associated with glycation provoked a reduction in the free thiol group and a strong increment of protein carbonyl contents and the fructosamine level in glycated HSA. Antioxidant activity was evaluated by a RBC hemolysis test. The result indicates that the free radical scavenging capacities of HSA were decreased in diabetic patients with or without CKD. Our study revealed that structural and functional features of glycated HSA, isolated from diabetic patients with and without CKD were significantly different from the HSA isolated from non-diabetic subjects. Moreover these changes were more prominent in HSA isolated from diabetic patients with CKD. These findings suggest that active sites of HSA may not be available under extensive glycation, leading to the impairment of its important functions. Thus glycated HSA may be involved in the pathogenesis of diabetes and its complications such as chronic kidney disease.

Amadori albumin in diabetic nephropathy.

Nonenzymatic glycation of macromolecules in diabetes mellitus (DM) is accelerated due to persistent hyperglycemia. Reducing sugar such as glucose reacts non enzymatically with free €-amino groups of proteins through series of reactions forming Schiff bases. These bases are converted into Amadori product and further into AGEs. Non enzymatic glycation has the potential to alter the biological, structural and functional properties of macromolecules both in vitro and in vivo. Studies have suggested that amadori as well as AGEs are involved in the micro-macro vascular complications in DM, but most studies have focused on the role of AGEs in vascular complications of diabetes. Recently putative AGE-induced patho-physiology has shifted attention from the possible role of amadori-modified proteins, the predominant form of the glycated proteins in the development of the diabetic complications. Human serum albumin (HSA), the most abundant protein in circulation contains 59 lysine and 23 arginine residues that could, in theory be involved in glycation. Albumin has dual nature, first as a marker of intermediate glycation and second as a causative agent of the damage of tissues. Among the blood proteins, hemoglobin and albumin are the most common proteins that are glycated. HSA with a shorter half life than RBC, appears to be an alternative marker of glycemic control as it can indicate blood glucose status over a short period (2-3 weeks) and being unaffected by RBCs life span and variant haemoglobin, anemia etc which however, affect HbA1c. On the other hand, Amadori albumin may accumulate in the body tissues of the diabetic patients and participate in secondary complications. Amadori-albumin has potential role in diabetic glomerulosclerosis due to long term hyperglycaemia and plays an important role in the pathogenesis of diabetic nephropathy. This review is an approach to compile both the nature of glycated albumin as a damaging agent of tissues and as an intermediate diagnostic marker and its potential role in diabetic nephropathy.