Km Neelofar

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.

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.

Elucidating the impact of glucosylation on human serum albumin: A multi-technique approach

Early glycation products as well as advance glycation end products are involved in pathogenesis of diabetes. Most of studies carried out on AGEs and their possible role in assessing diabetes complications, whereas only a few were focused to highlight the role of Amadori products. In this study, an attempt has been made to investigate a structural and immunological characterizations of Amadori-albumin upon early glucosylation because albumin undergoes fast glycation under hyperglycaemic condition. Amadori-albumin formation was determined by NBT assay and Amadori adducts in glycated samples were confirmed by LC-MS. Structural alterations in Amadori-albumin were characterized by loss in fluorescence intensity, loss in secondary and tertiary structures, exposure of hydrophobic patches, shifting in Amide bands and increment in hydrodynamic radius. Further, presence to autoantibodies against Amadori-albumin in diabetes patients were confirmed by direct binding ELISA and inhibition ELISA. Immunological studies results showed that autoantibodies present in diabetic patients with and without chronic kidney disease (CKD) showed significant binding with Amadori-albumin in comparison to the native protein. Anti Amadori-albumin antibodies predominantly present in CKD patients compare to without CKD patients. Band shift assay results showed true interaction between Amadori-albumin and autoantibodies present in CKD patients. Glucosylation results showed structural alterations in Amadori-albumin and hence generation of neo-epitopes in HSA molecule. Such modifications rendering the protein highly immunogenic that may be recognized as foreign molecule by immune cells and induced autoantibodies in diabetic patients. These finding signify the role of Amadori-albumin in kidney dysfunction in diabetes and raised level of autoantibodies may be used as biomarker for progression of CKD.

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 β-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.

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.

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 Parkinsons 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.

Study of IL4-590C/T and IL6-174G/C Gene Polymorphisms in Type 2 Diabetic Patients With Chronic Kidney Disease in North Indian Population: GENE POLYMORPHISM IN DIABETIC PATIENTS

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.

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.

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.

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

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.