Protein-bound Carbonyls Research Articles

Antioxidant and Anti-Inflammatory Effects of Vanillic Acid in Human Plasma, Human Neutrophils, and Non-Cellular Models In Vitro

Vanillic acid (VA) is a dietary benzoic acid derivative, flavoring agent, and food stabilizer. In this study, the antioxidant and anti-inflammatory potential of VA was explored in vitro and ex vivo in human immune cells and non-cellular models. In neutrophils, VA significantly downregulated the fMLP-induced oxidative burst and the generation of reactive oxygen species (ROS); it also suppressed the release of pro-inflammatory cytokines (TNF-α, IL-8) and the tissue-remodeling enzyme elastase-2 (ELA-2) in cells stimulated with LPS and fMLP+cytochalasin B. Additionally, VA showed good biocompatibility with human neutrophils and peripheral blood mononuclear cells (PBMCs) across the tested concentrations of 1–50 µg/mL. Furthermore, VA at 1–5 μg/mL enhanced the non-enzymatic antioxidant capacity of human plasma (NEAC) and prevented oxidative and nitrative damage to plasma proteins by protecting tyrosine moieties and thiols from peroxynitrite. VA also inhibited lipid peroxidation and the formation of thiobarbituric acid-reactive substances (at 50 μg/mL) and protein-bound carbonyls (at 5–50 μg/mL) in peroxynitrite-treated plasma. In non-cellular tests, VA acted as a hypochlorous acid and hydrogen peroxide scavenger and inhibited non-enzymatic protein glycation, outperforming the references Trolox and aminoguanidine. Along with existing data from animal models and studies on polyphenol intake, these results might support the synergic role of VA in dietary protection against chronic diseases related to oxidative stress and inflammation.

Influence of processing of seitan, tempeh, and firm regular tofu on protein and lipid oxidation and Maillard reaction products formation

The impact of production and cooking on protein oxidation, lipid oxidation and Maillard reaction products (MRPs) generation was evaluated for firm regular tofu, tempeh, and seitan, three plant-based alternatives to meat products. Tofu showed higher content of protein-bound carbonyls (25.5 ± 5.4 nmol/mg protein) indicating higher degree of protein oxidation, while seitan exhibited the lowest content of MRPs like N(6)-carboxymethyllysine, as revealed by LC-MS/MS. Through determination of the peroxide value and thiobarbituric acid reactive substances as well as analysis of volatile oxidation compounds by GC–MS, an overall higher lipid oxidation level was found in tofu. Likewise, a mean 92.2 % depletion of naturally occurring antioxidant tocopherol, assessed by HPLC-DAD, was observed in tofu. Furthermore, acrylamide was detected in tofu at 196.0 ± 15.4 μg/kg after frying at 190 °C. This study, therefore, concluded that practices used in tempeh and seitan processing are promising strategies to limit oxidation and Maillard reactions in food.

Attenuation of albumin glycation and oxidative stress by minerals and vitamins: An in vitro perspective of dual-purpose therapy.

Nonenzymatic glycation of proteins is accelerated in the context of elevated blood sugar levels in diabetes. Vitamin and mineral deficiencies are strongly linked to the onset and progression of diabetes. The antiglycation ability of various water- and fat-soluble vitamins, along with trace minerals like molybdenum (Mo), manganese (Mn), magnesium (Mg), chromium, etc., have been screened using Bovine Serum Albumin (BSA) as in vitro model. BSA was incubated with methylglyoxal (MGO) at 37°C for 48h, along with minerals and vitamins separately, along with controls and aminoguanidine (AG) as a standard to compare the efficacy of the minerals and vitamins. Further, their effects on renal cells' (HEK-293) antioxidant potential were examined. Antiglycation potential is measured by monitoring protein glycation markers, structural and functional modifications. Some minerals, Mo, Mn, and Mg, demonstrated comparable inhibition of protein-bound carbonyl content and ß-amyloid aggregation at maximal physiological concentrations. Mo and Mg protected the thiol group and free amino acids and preserved the antioxidant potential. Vitamin E, D, B1 and B3 revealed significant glycation inhibition and improved antioxidant potential in HEK-293 cells as assessed by estimating lipid peroxidation, SOD and glyoxalase activity. These results emphasize the glycation inhibitory potential of vitamins and minerals, indicating the use of these micronutrients in the prospect of the therapeutic outlook for diabetes management.

Effect of Different Warm-Up Durations on the Plasma Oxidative Stress Biomarkers Following Anaerobic Exercise in Amateur Handball Players

The aim of this study was to assess the effects of three warm-up durations on post-exercise oxidative stress biomarkers, as well as recovery from the Wingate test in 14 amateur handball players. These players completed the Wingate test three times: after 5 min, after 10 min, and then after 15 min of warm-up, spread over 2 weeks with an interval of 1 week between each session. At the end of each session of the Wingate test, blood samples were taken: at rest (PR), after warming up (PWU) and immediately at the end of the test (P0), then after 10-min (P10) and after 20-min (P20) of recovery. The measured parameters are the total antioxidant status (TAS), superoxide dismutase (SOD), glutathione reductase (GR), glutathione peroxidase (GPx), malondialdehyde (MDA), protein-bound-carbonyls (PCs) and thiobarbituric acid reactive substances (TBARSs). The main findings revealed that anaerobic exercise induces oxidative stress, as evidenced by changes in antioxidant status and significant increases in concentrations of the majority of oxidative stress indicators (p < 0.05). At P20, plasma GPx, SOD, GR, TBARSs, and PC contents, are lower and significantly different after a 15-min warm-up than after a 10-min or 5-min warm-up. ANOVA showed a significant ”warm-up × recovery” effect on plasma GPx, SOD, GR, TBARSs, and PC contents [F(8.104) = 3.61; p < 0.001; η2p = 0.22, F(8.104): from 1.88 to 19.97; η2p: from 0.19 to 0.61: 0.05 < p < 0.001], respectively. We concluded that a 15-min warm-up was more efficient than the other duration in reducing a free radical rise, emphasizing the importance of the warm-up length on plasma oxidative stress biomarkers.

Fast and slow skeletal myosin binding protein-C and aging

Aging is associated with skeletal muscle strength decline and cardiac diastolic dysfunction. The structural arrangements of the sarcomeric proteins, such as myosin binding protein-C (MyBP-C) are shown to be pivotal in the pathogenesis of diastolic dysfunction. Yet, the role of fast (fMyBP-C) and slow (sMyBP-C) skeletal muscle MyBP-C remains to be elucidated. Herein, we aimed to characterize MyBP-C and its paralogs in the fast tibialis anterior (TA) muscle from adult and old mice. Immunoreactivity preparations showed that the relative abundance of the fMyBP-C paralog was greater in the TA of both adult and old, but no differences were noted between groups. We further found that the expression level of cardiac myosin binding protein-C (cMyBP-C), an important modulator of cardiac output, was lowered by age. Standard SDS-PAGE along with Pro-Q Diamond phosphoprotein staining did not identify age-related changes in phosphorylated MyBP-C proteins from TA and cardiac muscles; however, it revealed that MyBP-C paralogs in fast skeletal and cardiac muscle were highly phosphorylated. Mass spectrometry further identified glycogen phosphorylase, desmin, actin, troponin T, and myosin regulatory light chain 2 as phosphorylated myofilament proteins in both ages. MyBP-C protein-bound carbonyls were determined using anti-DNP immunostaining and found the carbonyl level of fMyBP-C, sMyBP-C, and cMyBP-C to be similar between old and adult animals. In summary, our data showed some differences regarding the MyBP-C paralog expression and identified an age-related reduction of cMyBP-C expression. Future studies are needed to elucidate which are the age-driven post-translational modifications in the MyBP-C paralogs.

Therapeutic Efficacy of Natural Product 'C-Phycocyanin' in Alleviating Streptozotocin-Induced Diabetes via the Inhibition of Glycation Reaction in Rats.

Diabetes is a long-term metabolic disorder characterized by persistently elevated blood sugar levels. Chronic hyperglycemia enhances glucose-protein interactions, leading to the formation of advanced glycation end products (AGEs), which form irreversible cross-links with a wide variety of macromolecules, and accumulate rapidly in the body tissues. Thus, the objective of this study was to assess the therapeutic properties of C-phycocyanin (C-PC) obtained from Plectonema species against oxidative stress, glycation, and type 2 diabetes mellitus (T2DM) in a streptozotocin (STZ)-induced diabetic Wistar rat. Forty-five days of C-PC administration decreased levels of triglycerides (TGs), blood glucose, glycosylated hemoglobin, (HbA1c), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), liver and kidney function indices, and raised body weight in diabetic rats. C-PC suppressed biochemical glycation markers, as well as serum carboxymethyllysine (CML) and fluorescent AGEs. Additionally, C-PC maintained the redox state by lowering lipid peroxidation and protein-bound carbonyl content (CC), enhancing the activity of high-density lipoprotein cholesterol (HDL-C) and renal antioxidant enzymes, and preserving retinal and renal histopathological characteristics. Thus, we infer that C-PC possesses antidiabetic and antiglycation effects in diabetic rats. C-PC may also act as an antidiabetic and antiglycation agent in vivo that may reduce the risk of secondary diabetic complications.

Oxidative Damage of Proteins Precedes Loss of Cholinergic Phenotype in the Septal Neurons of Olfactory Bulbectomized Mice.

The development of cholinergic deficit is considered an early sign of a number of pathological conditions, including Alzheimer's disease. Cholinergic dysfunction underlies cognitive decline associated with both normal aging and Alzheimer's disease. Here, we studied a possible mechanism of functional impairment of cholinergic neurons using an olfactory bulbectomy model. Male mice were subjected to olfactory bulbectomy or sham surgery. Three weeks after that they were trained in Morris water maze and then euthanized one month after surgery. The cholinergic indices as well as the indices of oxidative stress were studied using immunohistochemistry, western blot and ELISA. Gene expression was studied using RT-qPCR. The experimental treatment was followed by impaired learning of a standard spatial task in a water maze. This was associated with a decrease in the number of cells containing choline acetyltransferase (ChAT), in relation to total number of neurons in the medial septum and lower ChAT enzymatic activity in the hippocampus. However, the levels of mRNAs of ChAT, vesicular ACh transporter and acetylcholine esterase remained unchanged in bulbectomized mice compared to sham-operated animals. These alterations were preceded by the accumulation of protein-bound carbonyls, indicating oxidative damage of proteins, whereas oxidative damage of nucleic acids was not detected. We assume that in olfactory bulbectomy model, oxidative damage of proteins may cause cholinergic dysfunction rather than irreversible neuronal damage. These data indicate that cholinergic neurons of the basal forebrain are very sensitive to oxidative stress, which may be responsible for the appearance of early cognitive decline in Alzheimer's disease.

Nitrate alleviates ammonium toxicity in wheat (Triticum aestivum L.) by regulating tricarboxylic acid cycle and reducing rhizospheric acidification and oxidative damage

ABSTRACT Ammonium (NH4 +) is one of the most important nutrients required by plants. However, a high concentration of NH4 + as the sole nitrogen source suppresses plant growth. Although nitrate (NO3 −) can alleviate NH4 + toxicity, the mechanisms underlying this ability have not been fully elucidated. In this study, wheat plants were cultivated in hydroponic solution with 7.5 mM NO3 − (control), 7.5 mM NH4 + (sole ammonium, SA) or 7.5 mM NH4 + plus 1.0 mM NO3 − (ammonium and nitrate, AN). The results showed that compared with the control, the SA treatment significantly decreased root growth, protein content and the concentrations of most intermediates and the activity of enzymes from the tricarboxylic acid (TCA) cycle. Moreover, increased the activity of plasma membrane H+-ATPase and the rate of H+ efflux along roots, caused solution acidification, and increased the activity of mitochondrial respiratory chain complexes I–IV and the contents of protein-bound carbonyls and malondialdehyde in roots. SA treatment induced ultrastructure disruption and reduced the viability of root cells. Compared with the SA treatment, the AN treatment increased root growth, protein content, the concentrations of most intermediates and the activity of enzymes from the TCA cycle. Furthermore, AN treatment decreased the rate of H+ efflux, retarded medium acidification, decreased protein carbonylation and lipid peroxidation in roots and relieved ultrastructure disruption and increased the viability of root cells. Taken together, these results indicate that NO3 –-dependent alleviation of NH4 + toxicity in wheat seedlings is closely associated with physiological processes that mediate TCA cycle, relieve rhizospheric acidification and decrease the production of ROS and oxidative damage.

Biochemical markers of oxidative stress in maize seedlings exposed to rose-grass aphids

We studied the influence of rose-grass aphid (Metopolophium dirhodum L.) infestation on the biochemical markers of oxidative stress in seedlings of two maize (Zea mays L.) varieties (relatively resistant Ambrozja and resistant Plomyk). We compared the generation of superoxide anion radicals O2-, level of hydrogen peroxide (H2O2), lipid peroxidation products (MDA) as well as markers of protein damage (protein-bound carbonyl groups). The studied parameters were measured at 24, 48, 72 and 96 h post-initial aphid infestation compared to the non-infested control seedlings. Our studies indicated that the rose-grass aphid feeding evoked oxidative stress in the maize seedlings. Investigated Z. mays cultivars exhibited excessive generation of superoxide anion radicals in response to insect treatments. Rose-grass aphid feeding increased the H2O2 level in maize tissues with similar levels observed at most periods post-infestation with M. dirhodum, also increased lipid peroxidation products with the maximal levels at 48 and 72 h for Ambrozja and 48, 72 and 96 h post-infestation for Plomyk varieties. Further at 48 and 72 h post-initial aphid infestation, there was an increase in protein bound carbonyl groups content in the maize seedlings after infestation with aphids.

Ezetimibe attenuates experimental diabetes and renal pathologies via targeting the advanced glycation, oxidative stress and AGE-RAGE signalling in rats

The current in-vivo study was premeditated to uncover the protective role of ezetimibe (EZ) against advanced glycation endproducts (AGEs)-related pathologies in experimental diabetes. Our results showed that EZ markedly improved the altered biochemical markers of diabetes mellitus (DM) (FBG, HbA1c, insulin, microalbumin, and creatinine) and cardiovascular disease (in-vivo lipid/lipoprotein level and hepatic HMG-CoA reductase activity) along with diminished plasma carboxymethyl-lysine (CML) and renal fluorescent AGEs level. Gene expression study revealed that EZ significantly down-regulated the renal AGEs-receptor (RAGE), nuclear factor-κB (NFκB-2), transforming growth factor-β (TGF-β1), and matrix metalloproteinase-2 (MMP-2) mRNA expression, however, the neuropilin-1 (NRP-1) mRNA expression was up-regulated. In addition, EZ also maintained the redox status via decreasing the lipid peroxidation and protein-bound carbonyl content (CC) and increasing the activity of high-density lipoprotein (HDL)-associated-paraoxonase-1 (PON-1) and renal antioxidant enzymes as well as also protected renal histopathological features. We conclude that EZ exhibits antidiabetic and reno-protective properties in diabetic rats.

Određivanje oksidacije proteina u hrani za ribe

This research aimed to develop a reliable, easy-to-perform and cheap method for measuring protein oxidation in complex samples such as aquaculture feed within various protein sources. For that purpose modified 2,4-dinitrophenylhydrazine (DNPH)-based method for quantification of protein carbonyls was employed whilst the modification of the method consisted of using different solutions for the extraction (distilled water and different concentrations of KCl and NaCl solutions), time of protein extraction (after homogenization and over the night) and concentration of trichloroacetic acid (10 and 25% TCA) for protein precipitation. Extraction during the night, higher TCA concentration and the use of 0.5 M KCl extraction solution resulted in the highest protein amount measured by the Lowry method and 280 nm protein estimation. On the other hand, the lowest protein yield was obtained by using distilled water for the extraction. Furthermore, the lowest amount of protein carbonyls was in the case when extraction was performed with distilled water (DW), while the highest content of protein carbonyls was reached with 0.15 M KCl and 0.5 M KCl extraction solutions. It was observed that the amount of proteinbound carbonyls compounds was increasing during storage under accelerated conditions and, in comparison to the original method, the modified method for measuring protein oxidation resulted in a higher amount of carbonyls during all points of storage.

Glycation of soy proteins leads to a range of fractions with various supramolecular assemblies and surface activities

Dry and subsequent wet heating were used to glycate soy proteins with dextran or glucose, followed by fractionation based on size and solubility. Dry heating led to protein glycation (formation of furosine, Nε-(carboxymethyl)-l-lysine, Nε-(carboxyethyl)-l-lysine, and protein-bound carbonyls) and aggregation (increased particle size); while subsequent wet heating induced partial unfolding and de-aggregation. The measurable free amino group content of soy proteins changed from 0.77 to 0.14, then to 0.62 mmol/g upon dry and subsequent wet heating; this non-monotonic evolution is probably due to protein structural changes, and shows that this content should be interpreted with caution as a glycation marker. After both heating steps, the smaller-sized water-soluble fractions showed higher surface activity than the larger insoluble ones, and dextran conjugates exhibited a higher surface activity than their glucose counterparts. We thereby achieved a comprehensive understanding of the properties of various fractions in plant protein fractions, which is essential when targeting applications.

Effect of d-glucose on the chemical characteristics and irradiation off-odor performance in porcine meat emulsion system

In present study, using gamma-ray irradiation at dose of 0 kGy and 3 kGy, the effect of D-glucose on off-odor performance in porcine muscle protein emulsions was monitored by determining lipid oxidation, protein oxidation events, profile of free amino acids and volatile compounds. Results showed that sarcoplasmic emulsion produced more off-odor substances than myofibrillar emulsion after irradiation, including sulfides, benzene derivatives and benzaldehyde. 3 kGy irradiation significantly increased TBARS and protein-bound carbonyls amount in sarcoplasmic emulsion (P < 0.05). Furthermore, the protein-bound carbonyls and Schiff base formation in sarcoplasmic emulsion exhibited a glucose-responsive increase following irradiation (P < 0.05) rather than in myofibrillar emulsion. Multivariate analysis further proposed that formation of benzaldehyde during irradiation required the cooperation of lipid peroxidation interfaced with sarcoplasmic components and glucose even facilitated such process. However, unlike sarcoplasmic emulsion, myofibrillar proteolysis in presence of glucose was less impaired upon irradiation probably owing to a more moderate protein carbonylation.

Evaluation of main post-translational modifications occurring in naturally generated peptides during the ripening of Spanish dry-cured ham.

Peptidyl post-translational modifications (PTMs) could influence the final quality of processed meat. In this study, the peptide oxidative phenomena in Spanish dry-cured ham (Biceps femoris muscle) was evaluated at different ripening times (9, 12, 15, 18 and 24months of processing) evidencing interactions amongst the lipid and protein oxidation, major peptidyl PTMs and the release of free amino acids (FAAs). Results showed that 12months of processing enabled the most abundant protein-bound carbonyls, while TBARS value was significantly favored (p<0.001) by ripening. However, FAAs were still intensively generated during overall ripening. Peptidomics and chemometrics further revealed that proteolysis mostly hampered the oxidized peptides rather than the deamidated ones during ripening. Myosin light chain (MYL1 and MYL3) showed high oxidative susceptibility owing to peptidyl methionine and proline oxidation as well as acetaldehyde adduct formation on lysine or histidine residues.

CHEMICAL AND MICROBIOLOGICAL CHARACTERISTICS OF HOMOGENISED RICOTTA CHEESE PRODUCED FROM BUFFALO WHEY

To extend the shelf life of buffalo Ricotta cheese, a process was assessed that included a second heat treatment followed by homogenisation and hot packaging. The microbiological and chemical characteristics as well as the oxidation degree of the product were determined over storage for 21 days using total antioxidant activity, redox potential, malondialdehyde content, and protein-bound carbonyl content. Homogenised buffalo Ricotta cheese has a longer shelf life than traditional Ricotta cheese, although the process could be optimised to reduce the total bacterial load during storage. No significant oxidative damage occurred during storage. This innovative process could promote the market expansion of Ricotta cheese.

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.

B procyanidins of Annona crassiflora fruit peel inhibited glycation, lipid peroxidation and protein-bound carbonyls, with protective effects on glycated catalase

Advanced glycation end-products (AGEs) have been reported as results of increased oxidative stress. Consequently, the search for new antioxidant and anti-glycating agents is under intense investigation. Plant-derived procyanidins have previously demonstrated anti-glycation properties. Thus, this study aimed to isolate procyanidins from Annona crassiflora fruit peel, a species from the Brazilian Savanna, and investigate their antioxidant and anti-glycation effects. Free radical scavenging and quenching properties, formation of reactive oxygen species (ROS), AGEs, protein carbonyl and thiol groups, lipid peroxidation, crosslinked AGEs, as well as glycated catalase activity, were analyzed. In addition, in silico assessment of absorption, distribution, metabolism, excretion and toxicity was carried out. The procyanidins-enriched fraction, named here as F7, showed high antioxidant and anti-glycation capacities, with inhibitory activities against lipid peroxidation, and AGEs and ROS formation. In addition, there were reductions in AGEs-induced crosslinks and protein carbonyls and protective effects against oxidation of thiol groups and glycated-catalase. ADMET predictions of F7 showed favorable absorption and distribution, with no hepatotoxicity or mutagenicity. Together, our results support the anti-glycation activities of the procyanidins-enriched fraction from A. crassiflora, and suggest that these effects are triggered, at least in part, by scavenging free radical and dicarbonyls intermediates.

Applicability of FTIR-ATR Method to Measure Carbonyls in Blood Plasma after Physical and Mental Stress.

Introduction Oxidative stress is a state of imbalance between the production of reactive oxygen species and antioxidant defenses. It results in the oxidation of all cellular elements and, to a large extent, proteins, causing inter alia the formation of carbonyl groups in their structures. The study focused on assessment of changes in the plasma protein-bound carbonyls in police horses after combat training and after rest and the applicability of infrared spectroscopy with a Fourier transform, utilizing the attenuated total reflectance (FTIR-ATR) in detecting plasma protein oxidation. Methods We evaluated the influence of both the different concentrations of hydrogen peroxide and combat training on protein carbonylation in horse blood plasma. The oxidation of plasma proteins was assessed using a spectrophotometric method based on the carbonyl groups derivatization with 2,4-dinitrophenylhydrazine (DNPH). The measured values were correlated with the carbonyl groups concentrations determined by means of the FTIR-ATR method. Results The linear correlation between the DNPH and FTIR-ATR methods was shown. The concentration of plasma protein-bound carbonyls significantly deceased in police horses after one-day rest when compared to the values measured directly after the combat training (a drop by 23%, p<0.05 and 29%, p<0.01 measured by DNPH and FTIR-ATR methods, respectively). These results were consistent with the proteins phosphorylation analysis. ConclusionThe FTIR-ATR method may be applied to measure the level of plasma proteins peroxidation.

Depression and Smoking Augment the Production of Circulating Autoantibodies against Glycated HSA in Rheumatoid Arthritis Patients

Background: Depression and smoking contribute to the prognosis of autoimmune rheumatoid arthritis (RA). Advanced glycation end products (AGEs) are also detected in RA patients. This study correlates RA in patients with various levels of depression and a history of smoking through the detection of antibodies against AGEs of proteins. Methods: Sixty RA subjects were selected and divided into 4 groups based on their levels of depression and smoking habits. The division was as follows: group I consisted of RA patients classified as depressed (RA-D); group II consisted of RA patients with a history of smoking (RA-S); group III consisted of RA patients suffering from depression who were also smokers (RA-DS); and group IV consisted of patients with RA alone (RA-A), i.e., not depressed and non-smokers. In vitro human serum albumin (HSA) was modified by glucose, and the modifications were studied by biochemical and biophysical techniques. Glycated (G)-HSA was used as an antigen, and autoantibodies against G-HSA (G-HSA-Abs) were screened in serum samples of different groups of RA subjects. Oxidative stress levels in all patients and healthy individuals were analyzed by protein-bound carbonyl content estimations. Results: Significant biochemical and biophysical changes were detected in G-HSA when compared to native (N)-HSA. All patients and control subjects were screened for circulating G-HSA-Abs and N-HSA-Abs. From the cohort of different samples, serum autoantibodies from RA-DS showed a high recognition of G-HSA-Abs titres compared to RA-D or RA-S. RA-A exhibited the least binding of circulating G-HSA-Abs of all the groups. The oxidative stress marker, the carbonyl content also exhibited highest levels in RA-DS, followed by RA-D and RA-S. Band shift assay showed the highest titres of immunoglobulin G in the serum samples of RA-DS. Conclusions: Smoking and concomitant depression in RA subjects may lead to enhanced oxidative stress levels responsible for the gradual formation and/or exposing of cryptic epitopes on HSA that induce the production of G-HSA-Abs. Hence, we postulate that by reducing depression and corresponding oxidative stress, it may be possible to control or limit the severity of the precipitation of RA disease activity and improve prognosis.

Aged garlic has more potent antiglycation and antioxidant properties compared to fresh garlic extract in vitro

Protein glycation involves formation of early (Amadori) and late advanced glycation endproducts (AGEs) together with free radicals via autoxidation of glucose and Amadori products. Glycation and increased free radical activity underlie the pathogenesis of diabetic complications. This study investigated whether aged garlic has more potent antiglycation and antioxidant properties compared to fresh garlic extract in vitro in a cell-free system. Proteins were glycated by incubation with sugars (glucose, methylglyoxal or ribose) ±5–15 mg/mL of aged and fresh garlic extracts. Advanced glycation endproducts were measured using SDS-PAGE gels and by ELISA whereas Amadori products were assessed by the fructosamine method. Colorimetric methods were used to assess antioxidant activity, free radical scavenging capacity, protein-bound carbonyl groups, thiol groups and metal chelation activities in addition to phenolic, total flavonoid and flavonol content of aged and fresh garlic extracts. Aged garlic inhibited AGEs by 56.4% compared to 33.5% for an equivalent concentration of fresh garlic extract. Similarly, aged garlic had a higher total phenolic content (129 ± 1.8 mg/g) compared to fresh garlic extract (56 ± 1.2 mg/g). Aged garlic has more potent antiglycation and antioxidant properties compared to fresh garlic extract and is more suitable for use in future in vivo studies.