Albumin from sera of rheumatoid arthritis patients share multiple biochemical, biophysical and immunological properties with in vitro generated glyco-nitro-oxidized-albumin.

The purpose of the present study is to explore the effects of endogenous stressors on structure and function of rheumatoid arthritis (RA) patients' albumin. In contrast to glycated-albumin or nitro-oxidized-albumin, high titre antibodies against glyco-nitro-oxidized-albumin were found in the sera of RA patients. Also, compared to the other two modified forms of albumin, glyco-nitro-oxidized-albumin showed highest percent inhibition. Albumin isolated from RA patients' sera displayed hyperchromicity and quenching of tyrosine and tryptophan fluorescence. Fluorescence spectroscopy studies also revealed the presence of dityrosine and advanced glycation end products in RA patient's albumin. RA patients' albumin showed weaker binding with 1-anilinonaphthalene-8-sulfonic acid dye. Secondary structure alterations were demonstrated by circular dichroism and Fourier transform infrared spectroscopy. Biochemical investigations revealed substantial decline in the availability of free side chains of amino acid residues; increased carbonyls and decreased sulfhydryls in RA patients' albumin. The functional impairment in RA patients' albumin was revealed by their low binding with bilirubin and cobalt. Liquid chromatography mass spectrometry analysis revealed the presence of Nε-(carboxymethyl) lysine and 3-nitrotyrosine in RA patients' albumin. The amyloidogenic aggregation of RA patients' albumin was confirmed by Congo red absorption and thioflavin-T fluorescence assays. The morphology of the aggregates was visualized under scanning and transmission electron microscope. From the above findings, we inferred that endogenous stress in RA patients have modified albumin and produce structural/functional abnormalities. Also, the presence of anti-glyco-nitro-oxidized-albumin antibodies along with other clinical features may be used as biomarker for the diagnosis and assessment of treatment responses in RA patients.Communicated by Ramaswamy H. Sarma.

Studies on the synergistic action of methylglyoxal and peroxynitrite on structure and function of human serum albumin.

Albumin, an important serum protein, is continuously exposed to various oxidizing/nitrating and glycating agents. Depending upon the nature/concentration of reactive species present, the protein may be glycated, oxidized/nitroxidized or glyco-nitro-oxidized. Peroxynitrite is a powerful nitroxidant and has been reported to damage a wide array of macromolecules. On the other hand, methylglyoxal is a very strong reactive dicarbonyl and a potent precursor for the formation of advanced glycation end products under pathological conditions. In certain pathological conditions albumin may be modified by peroxynitrite and methylglyoxal simultaneously. There is dearth of literature suggests that structural/conformational and functional alteration in albumin upon glycation and oxidation/nitroxidation, however the alterations produced by glyco-nitro-oxidation has not yet been explored. Therefore, in this study, simultaneous effect of glycation and nitroxidation on the structure and conformation, vis-a-vis function of albumin was explored. Glyco-nitro-oxidized albumin showed decreased free amino acid content together with decreased affinity of albumin towards cobalt. Molecular docking model and molecular dynamic simulations showed close interaction and formation of stable complexes between methylglyoxal, peroxynitrite and albumin. Formation of carboxymethyl lysine and 3-nitrotyrosine in glyco-nitro-oxidized albumin were confirmed by MALDI-TOF MS and UP-LC MS. Aggregate formation in glyco-nitro-oxidized albumin was visualized by transmission electron microscopy. On the basis of these results, it may be speculated that, albumin modified with endogenously generated methylglyoxal and peroxynitrite might be a driving factor in the progression of heightened inflammatory autoimmune responses. The work presents a ground to study the role of glyco-nitro-oxidized albumin in the pathogenesis and progression of various autoimmune diseases including rheumatoid arthritis. Communicated by Ramaswamy H. Sarma.

Impact of endogenous stress on albumin structure in systemic lupus erythematosus (SLE) patients.

Systemic lupus erythematosus (SLE) is an inflammatory, autoimmune disorder of unknown etiology. The inflammatory stress in SLE patients may modify macromolecules and produce structural/functional abnormalities. The present study is aimed at examining the consequences of stresses on the structure of albumin in SLE patients. Albumin was isolated from the sera of SLE/healthy subjects. Multiple physicochemical techniques were used to elucidate, structure of albumin. Advanced glycation end products in SLE patients' albumin were identified by the AGE specific fluorescence. Quenching of tryptophan, tyrosine fluorescence and surface protein hydrophobicity was observed in SLE patients' albumin. Protein-bound carbonyls were elevated while free thiol, lysine, arginine, and alpha helicity was found to be decreased in SLE albumin. Furthermore, changes in the secondary structure of SLE albumin were observed as shift in the position of amide I/II bands. Functionality of SLE albumin was also compromised as its cobalt-binding ability was substantially declined. Adduction of moieties was detected by dynamic light scattering (DLS) and confirmed by matrix assisted laser desorption/ionization. DLS, thioflavin T and transmission electron microscopy results confirmed aggregates in SLE patients' albumin. This study may be helpful in understanding the role of modified albumin in the cofounding pathologies associated with SLE.

Nitroxidized-Albumin Advanced Glycation End Product and Rheumatoid Arthritis.

Rheumatoid arthritis (RA) is an autoimmune disease whose major clinical consequence is inflammation of small joints and contiguous structures. Oxidative and nitrosative stress along with increased formation of advanced glycation end products (AGEs) play an important role in the disease process. Generation of reactive species during glycation of proteins further adds to the oxidative and nitrosative stress. Albumin, being the most abundant plasma protein, is frequently targeted by different oxidizing and nitrating agents, including peroxynitrite (OONO-) anion. Albumin is also targeted and modified by dicarbonyl metabolites (glyoxal and methylglyoxal) which are formed in oxidative and non-oxidative processes during the synthesis of AGEs. The endogenously formed OONO- and dicarbonyls may modify plasma albumin including those albumin that have travelled or migrated to synovial cells and caused nitration, oxidation, and glycation. These modifications may produce crosslinks, aggregate in albumin and confer immunogenicity. Simultaneous modification of albumin by OONO- and dicarbonyls may generate nitroxidized-AGE-albumin which may persist in circulation for a longer duration compared to native albumin. Nitroxidized-AGE-albumin level (or serum autoantibodies against nitroxidized- AGE-albumin) along with other pre-clinical features may help predict the likely onset of RA.

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.

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.