Alok Raghav

Glycated serum albumin: A potential disease marker and an intermediate index of diabetes control

Glycation is a non-enzymatic spontaneous process in proteins which has remarkable impact on its physical and functional aspect. This alteration with addition of carbohydrate residue to human serum albumin leads to several pathological events such as diabetic nephropathy, neuropathy, retinopathy and cardiovascular complications. Human serum albumin is the major protein and is most susceptible to non-enzymatic glycation. Structural and biological properties of functional albumin alter due to the addition of reducing carbohydrate to free amino terminal residues vivo. These irreversible changes in functional albumin are stable which makes this modified albumin as new gold standard future diagnostic marker in diabetes associated complications. Glycated albumin can be used to determine the glycemic control due to short half life than erythrocytes which makes it an alternate reliable disease marker in diabetes. In this review, Human serum albumin glycation has been overviewed, stating concept of glycation and sites that are prone to this modifications. Impact of non-enzymatic addition of carbohydrate to albumins structural and biological properties has also been elaborated. Accurate measurements of glycated albumin with implications of new highly sensitive techniques have also been described briefly. Interestingly human serum albumin imposed glycation can serve as future tool not for diagnosing diabetes but also its potential in assessment of diabetes associated complications.

Nonenzymatic glycosylation of human serum albumin and its effect on antibodies profile in patients with diabetes mellitus

Background Albumin glycation and subsequent formation of advanced glycation end products (AGEs) correlate with diabetes and associated complications. Methods Human Serum Albumin (HSA) was modified with D-glucose for a 40 day period under sterile conditions at 37°C. Modified samples along with native HSA (unmodified) were analyzed for structural modifications by UV and fluorescence, FTIR, Liquid chromatography mass spectrometry (LCMS) and X–ray crystallography. New-Zealand white female rabbits immunized with AGEs, represent auto-antibodies formation as assessed by competitive and direct binding enzyme-linked immunosorbent assay (ELISA). Neo-epitopesagainst In-vitro formed AGEs were characterized in patients with diabetes mellitus type 2 (n = 50), type 1 (n = 50), gestational diabetes (n = 50) and type 2 with chronic kidney disease (CKD) with eGFR level 60–89 mL/min (n = 50) from serum direct binding ELISA. Results Glycated-HSA showed amarked increase in hyperchromicity of 65.82%,71.98%, 73.62% and 76.63% at λ280 nm along with anincreasein fluorescence intensity of 65.82%, 71.98%, 73.62% and 76.63% in glycated-HSA compared to native. FTIR results showed theshifting of Amide I peak from 1656 cm_1 to 1659 cm_1 and Amide II peak from 1554 cm_1 to 1564 cm_1 in glycated-HSA, with anew peak appearance of carbonyl group at 1737 cm-1. LCMS chromatogram of glycated-HSA showed thepresence of carboxymethyl lysine (CML) at 279.1 m/z. Immunological analysis showed high antibody titre>1:12,800 in theserum of rabbits immunized with glycated-HSA (modified with 400 mg/dL glucose) and inhibition of 84.65% at anantigen concentration of 20μg/mL. Maximum serum auto-antibody titre was found in T2DM (0.517±0.086), T1DM (0.108±0.092), GDM (0.611±0.041) and T2DM+CKD (0.096±0.25) patients immunized with glycated-HSA (modified with 400 mg/dL glucose). Conclusions Non-enzymatic glycosylation of HSA manifests immunological complications in diabetes mellitus due to change in its structure that enhances neo-epitopes generation.

Ionic liquid based microextraction of targeted lipids from serum using UPLC-MS/MS with a chemometric approach: a pilot study

In the present study an ionic liquid based vortex assisted surfactant-enhanced emulsification microextraction (IL-VASEME) method followed by Plackett–Burman Design (PBD) and Central Composite Design (CCD) using liquid chromatography-electrospray mass spectrometry (LC-ESI-MS/MS) have been used for the determination of fatty acids, triglycerides and phospholipids in serum samples. The study helped in the detection of these lipid classes in a single run within 5 min. The extraction method parameters were statistically optimized by a design of experiment (DOE) approach to reduce the extraction time. The ionic liquid, 1-butyl-3-methylimidazolium hexafluorophosphate (C4MIMPF6) was used as an extraction solvent, while the surfactant, Triton X-100 was used as an emulsifying agent. The statistical method, PBD screened the most significant factors such as ionic liquid volume, surfactant strength and pH for optimizing the conditions for the separation of lipids. The screened factor values were based on the CCD design, which optimized the conditions like 45 μL of ionic liquid, 7.5 pH and 1.25% of surfactant strength for extraction of lipids. The limit of detection (LOD) and limit of quantification (LOQ) were 0.012–0.034 ng mL−1 and 0.046–0.114 ng mL−1 respectively. The recovery of lipids was in the range of 90.9–114%. The intraday and interday precision in the serum sample ranged between 1.42–4.48% and 3.75–10.8% respectively. This study reveals that the proposed IL-VASEME coupled with liquid chromatography tandem mass spectrometry method is more sensitive for the extraction and determination of lipids in a single step as compared to traditional methods.

Impact of glycation on structural and antioxidant function of human serum albumin: Relevance in diabetic complications

AIM Non-enzymatic glycation impairs the structural and functional characterstics of human serum albumin (HSA) native conformation. Prolonged hyperglycemia causes cross links formation in proteins that may contribute to progression of diabetic complications. METHODS HSA (20μM) was incubated with different concentration of d-glucose100, 200, 300 and 400mg/dl for a period of 40 days in phosphate buffer saline (20mM pH=7.4) under sterile conditions. Incubated samples were extensively dialyzed and structural changes were analyzed by far and near UV circular dichroism spectra measurement. Fructosamine assay with nitroblue tetrazolonium was performed to confer isomerisation between glucose and protein. Aggregations of the glycated product (AGEs) formed during reduction of nitrobluetetrazolium dye were evaluated by transmission electron microscopy. Crosslinks aggregates were investigated by in-situ Congo red binding assay. Red blood cells hemolysis test was performed to decipher the antioxidant activity of albumin samples. RESULTS Fructosamine content in glycated albumin demonstrates the non-enzymatic addition of glucose to HSA and confers the formation of monoformazone (marker of glycation). Significant changes were found in the glycated samples of HSA compared to native (unmodified) in far and near UV circular dichroism. Transmission electron microscopy, Congo red staining, showed the formation of crosslinks aggregated mass in glycated HSA. Glycation of albumin reduces the antioxidant capacity of native albumin confirmed by red blood cells hemolysis test. CONCLUSION The finding of present study brings new evidences on the detrimental alterations of on albumin vital functions after non-enzymatic glycation with glucose. These results may emphasize the albumin associated diabetic complications under glycemic range of diabetes mellitus.

Glycation, oxidation and glycoxidation of IgG: a biophysical, biochemical, immunological and hematological study

Glycation and oxidation induce structural alterations in the proteins in an interdependent manner with consequent pathological implications. The published literature presents wide range of modifications in conformational characteristics of proteins by glycation and oxidation; however, there is little data that could elaborate the cumulative effect of both the processes. This study has analysed the modifications in IgG by methylglyoxal (MG) (glycative stress), hydroxyl radical () (oxidative stress) and by their combined action i.e. treatment of MG glycated IgG (glycoxidation). It further addresses the implications of the altered structural integrity of IgG on its immunological characteristics and impact on haematological parameters in rabbits. Using circular dichroism, FTIR, SDS-PAGE analysis, thioflavin-T fluorescence assay, congo red absorbance analysis, dynamic light scattering, transmission electron microscopy, ELISA, blood cell counts and rectal temperature studies, we report that the glycoxidative modification caused maximum alteration in the IgG as compared to the glycatively and oxidatively modified protein. Far-UV CD results confirmed the highest decline in the beta-pleated sheet content of the protein by glycoxidation. The damage led to the reduced flexibility and enhanced electronic interactions in IgG as observed by near-UV CD. Modifications caused cross-linking and adduct formation in the serum protein. The electron micrograph confirmed amorphous aggregation in modified IgG. The modifications increased the hydrodynamic radius of IgG by allowing the attachment of and MG residues. The glycoxidatively modified IgG induced the maximum antibody titres that showed high specificity towards the altered IgG. The glycoxidation of IgG leads to activation of inflammatory pathways.

Neo-Epitopes Generated on Hydroxyl Radical Modified GlycatedIgG Have Role in Immunopathology of Diabetes Type 2

Glycoxidation plays a crucial role in diabetes and its associated complications. Among the glycoxidation agents, methylglyoxal (MG) is known to have very highglycationpotential witha concomitant generation of reactive oxygen species (ROS) during its synthesis and degradation. The presentstudy probes the MG and ROSinduced structural damage to immunoglobulin G (IgG) and alterations in its immunogenicity in diabetes type 2 patients (T2DM). Human IgG was first glycated with MG followed by hydroxyl radical (OH•) modification. Glycoxidation mediated effects on IgG were evaluated by various physicochemical techniques likeultraviolet (UV) and fluorescence spectroscopy, 8-anilinonaphthalene-1-sulfonic acid (ANS) binding studies, carbonyl andfree sulfhydryl groups assay, matrix assisted laser desorption ionization mass spectrometry-time of flight (MALDI-TOF), red blood cell (RBC) haemolysis assay, Congored (CR) staining analysis and scanning electron microscopy (SEM). The results revealed hyperchromicityin UV, advanced glycation end product (AGE)specific and ANS fluorescence, quenching in tyrosine and tryptophan fluorescence intensity,enhanced carbonyl content,reduction in free sulfhydryl groups,pronounced shift in m/z value of IgGand decrease in antioxidant activity in RBC induced haemolysis assayupon glycoxidation. SEM and CRstaining assay showed highly altered surface morphology in glycoxidised sample as compared to the native. Enzyme linked immunosorbent assay (ELISA) and band shift assay were performed to assess the changes in immunogenicity of IgG upon glyoxidation and its role in T2DM. The serum antibodies derived from T2DM patients demonstrated strong affinity towards OH• treated MG glycatedIgG (OH•-MG-IgG) when compared to native IgG (N-IgG) or IgGs treated with MG alone (MG-IgG) or OH• alone (OH•-IgG). This study shows the cumulating effect of OH• on the glycation potential of MG. The results point towards the modification of IgG in diabetes patients under the effect of glycoxidative stress, leading to the generation of neo-epitopes on theIgG molecule and rendering it immunogenic.

New insights into non-enzymatic glycation of human serum albumin biopolymer: A study to unveil its impaired structure and function

Albumin glycation and subsequent formation of advanced glycation end products (AGEs) correlate with diabetes and associated complications. Human Serum Albumin (HSA) was modified with d-glucose for a 40day period under sterile conditions at 37°C. Modified samples along with native HSA (unmodified) were analyzed for impairment in biochemical characteristics ((fructosamine, carbonyl, thiol, lysine, arginine, hydroxymethyl furfural [HMF] content), electrochemical (electrical conductance), simulated gastric fluid assay, simulated intestinal fluid assay, spectroscopic properties (UV, fluorescence), optical (surface contact angle) and fluid dynamics (viscosity and Stokes radius). Impairment in drug binding capacity of glycosylated-HSA was assessed with molecular docking experiment using metformin. UV-absorbance and fluorescence measurement were performed for drug bound glycated and native-HSA. Extensively modified HSA has been used to study its relevance in diabetes mellitus. Glucose modified-HSA resulted in AGEs formation. It suggests deleterious impairment in biochemical, electrochemical, spectroscopic, optical and fluidity properties of HSA at high concentrations of glucose. The results of the present study can be useful to understand the phenomenon of proteins damage in hyperglycemic conditions.

Glycation of clinically relevant chickpea allergen attenuates its allergic immune response in Balb/c mice

Glycation of food allergens may alter their immunological behaviour. We sought to investigate the impact of glycation on the allergenicity of a food protein. Herein, a chickpea protein (≈26kDa) was purified and characterized as lectin. Further, glycation of this purified protein was carried out. Thereafter, allergic behaviour of this glycated protein was compared with its native form, using various allergic parameters in Balb/c mice. The reduced allergenicity of glycated protein was observed as lesser allergic phenotypes, reduced serum immunoglobulins and allergic mediators, lower mast cells and eosinophil counts, lower protein expressions of Th2 cytokines and associated transcription factors. In addition, more Th1 and less Th2 cytokine production in exposed splenocyte, were evident in the glycated protein treated mice as compared to its native protein treatment. Thus, glycation of the chickpea allergen attenuated the sensitizing potential and allergic responses in Balb/c mice significantly and could also be clinically beneficial.

Financial burden of diabetic foot ulcers to world: a progressive topic to discuss always:

Diabetic foot complications are the most common occurring problems throughout the globe, resulting in devastating economic crises for the patients, families and society. Diabetic foot ulcers (DFUs) have a neuropathic origin with a progressive prevalence rate in developing countries compared with developed countries among diabetes mellitus patients. Diabetic patients that are of greatest risk of ulcers may easily be diagnosed with foot examination. Economic burden may be carefully examined. The budget costing must include both the clinical and social impact of the patients.

Glycemic and non-glycemic targets in younger and older North Indian subjects with type 2 diabetes in a Tertiary care hospital: A 10 year's retrospective data analysis

BACKGROUND Although optimizing glycemic and non-glycemic targets reduced micro- and macro-vascular complications in type 2 diabetes, multiple barriers hinder turning evidence into practice. Mounting evidence suggests that those with onset of disease in early or mid-adult life, compared with those with onset at an older age, may have a more severe disease course and worse glycemic control. AIMS & OBJECTIVE We tested the hypothesis that those diagnosed at younger age would have worse glycemic control, even after adjustment for duration of diabetes, higher BMI and other known risk factors for worse glycemic control. MATERIALS & METHODS A cross-sectional analysis of 560 type 2 diabetic subjects from North Indian populace in the year 1999-2012 who reported to endocrine clinic was performed. Sixty patients did not report in the successive year and final data analyses were done in 500 patients attending clinic regularly over a period of 10 years for evaluation of glycemic and non-glycemic targets. They were followed up at 3 monthly intervals with all patients undergoing anthropometric measurement (BMI (weight in kg/height in m(2)), diet and lifestyle advice by a diabetic educator and consultation by endocrinologist. Fasting and postprandial plasma glucose, A1c (3 monthly), besides evaluation of SMBG that was performed in 50% of these patients regularly. Fasting lipids, S. creatinine and microalbuminuria were assessed annually and blood pressure recoding was done at each visit. The treatment was modified as per the investigation reports. We classified age at diabetes diagnosis as younger (<60 years) vs older (≥60 years). The primary outcome of interest was HbA1c ≥9%. Secondary outcomes were HbA1c ≥8% to <9% and HbA1c ≥7% to <8%. RESULTS After adjustment for sex, duration of diabetes, hyperglycemic medications, BMI, co-morbid conditions, age <60 years at diagnosis remains significantly associated with greater odds of HbA1c ≥9% [OR 0.95(0.84-1.07)], HbA1c ≥8% to <9% [OR 1.04(0.93-1.15)] and HbA1c ≥7% to <8% [OR 1.05(0.85-1.17)] for female sex. Seventy two (72.7%) of patients <60 years achieved BP <140/90mmHg (p<0.001) as compared to 62.3% of patients ≥60 years who achieved BP <150/90mmHg (p<0.001) and LDL-cholesterol <100mg/dl in 33.7% patients and 39.1% respectively (p<0.002). CONCLUSION Younger age (<60 years) at type 2 diabetes diagnosis is significantly associated with worse subsequent glycemic control and lipid control, as younger patients at diagnosis have fewer competing co-morbidities and complications. As patient-centeredness is a priority in type 2 diabetes care, safe, aggressive and individualized treatment could benefit this higher-risk group.