Loss Of Thiols Research Articles

Changes in the structural, aggregation behavior and gel properties of pork myofibrillar protein induced by theaflavins

This study explores the effect of different theaflavins (TFs) concentrations (0, 100, 300, 600 and 900 mg/L) on the structure, aggregation behavior and gelation properties of pork myofibrillar protein (MP). The protein structure and aggregation behavior were characterized by free sulfhydryl groups, surface hydrophobicity, fluorescence emission spectra, particle size and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The gel properties of samples were characterized by gel strength, cooking loss, microstructure and gel supernatant SDS-PAGE. The results showed a significant decrease in free thiol content with increasing TFs concentration, suggesting thiol-quinone covalent interaction between TFs and thiol group of MP. Intrinsic fluorescence spectroscopy confirmed a static quenching between TFs and MP. And TFs reduced the particle size of MP suspension and caused no protein aggregation bond in SDS-PAGE. For gel properties, TFs caused a decrease of gel strength from 96.77 g to 21.91 g and an increase in cooking loss from 40.34 % to 71.15 %. The bond of protein aggregates in gel supernatants SDS-PAGE revealed that some protein aggregates formed by disulfide bonding were not involve in gel formation with TFs addition. In conclusion, TFs cause thiol loss of MP and impaired MP gelling ability by interfering with disulfide bond formation during gelation.

The proteomic evidence on protein oxidation in pea protein concentrate-based low-moisture extrudates and its inhibition by antioxidants derived from plant extracts

This study aimed to assess the antioxidant activity of golden chlorella (GoC) and grape pomace (GrP) extracts both in vitro and in pea protein-based extrudates. We hypothesized that GoC/GrP would limit oxidation of proteins in the extrudates compared with commercial antioxidants. The results showed that GoC extract was effective in metal chelation and GrP extract possessed excellent radical scavenging activity and reducing power. Protein oxidation inevitably occurred after low-moisture extrusion in terms of elevated level of protein carbonyls and the gradual loss of thiols. LC-MS/MS revealed that the monoxidation and 4-hydroxynonenal adduction were the major oxidative modifications, and legumin was the most susceptible globulin for oxidation. The GoC/GrP extracts effectively retarded the oxidation progress in extrudates by lower intensity of oxidized peptides, whereas protein electrophoretic profiles remained unaffected. This study highlighted the great potential of GoC/GrP as natural antioxidants in plant-based foods.

Assessing the Impact of Pomegranate Peel Extract Active Packaging and High Hydrostatic Pressure Processing on Color and Oxidative Stability in Sliced Nitrate/Nitrite-Reduced Iberian Dry-Cured Loins

Our study aimed to assess the impact of active packaging with pomegranate peel extract (0.06 mg gallic acid eq./cm2) and/or high-pressure treatment (600 MPa, 7 min) on the instrumental color, lipid, and protein oxidation of Iberian dry loins formulated with reduced nitrate/nitrite levels (0, 37.5, and 150 mg/kg) during 100-day refrigerated storage (4 °C). CIE L*a*b* coordinates were measured, and malondialdehyde, carbonyls, and free thiol contents served as markers for lipid and protein oxidation. Active packaging lowered CIE L* (35.4 vs. 34.1) and a* (15.5 vs. 14.5) and increased yellowness (15.6 vs. 16.3) and hue (45.2 vs. 48.4), while pressurization increased CIE L* (33.1 vs. 36.3) and diminished a* values (16.1 vs. 13.9). Ongoing nitrate/nitrite amounts significantly influenced lipid peroxidation, protein carbonyl formation, and free thiol loss. Active packaging and high-pressure processing had varying effects on carbonyl and thiol contents. Neither pressurization nor active packaging impacted malondialdehyde formation. Pressurization enhanced the formation of 4-HNE (503 vs. 697 pg/g). Protein oxidation proved more sensitive to changes, with active packaging offering protection against protein carbonylation (15.4 vs. 14.7 nmol carbonyls/mg protein), while pressurization induced thiol loss (34.3 vs. 28.0 nmol Cys eq./mg protein). This comprehensive understanding provides essential insights for the meat industry, emphasizing the necessity for customized processing conditions to enhance color stability, lipid preservation, and protein integrity in dry-cured loin slices.

Modification of histones by the myeloperoxidase-derived oxidant hypochlorous acid (HOCl) alters their reactivity with vascular smooth muscle cells

In the nucleus, histones are essential in the packaging of DNA and the regulation of gene expression. These histones can also be released to the extracellular space by mechanisms such as necrosis and neutrophil extracellular trap (NET) formation. Histones are cytotoxic and cause sterile inflammation, and as a result, have been implicated in tissue damage in several pathologies, including atherosclerosis. Myeloperoxidase (MPO) is also present on NETs, which is catalytically active and able to produce hypochlorous acid (HOCl). This could modify histones and alter their extracellular reactivity. In this study, we compared the reactivity of histones with and without modification by HOCl with primary human coronary artery smooth muscle cells (HCASMCs). Histones induced a loss in viability and cell death primarily by apoptosis, which was attenuated on modification of the histones by HOCl. Exposure of HCASMCs to histones also resulted in the increased expression of the pro-inflammatory genes monocyte chemoattractant protein-1 (MCP-1), interleukin 6 (IL-6), and vascular cell adhesion molecule-1 (VCAM-1) and a decrease in intracellular thiols. In addition, there were changes in the expression of the stress related gene heme oxygenase-1 (HO-1). Modification of the histones with HOCl had no significant influence on changes in gene expression or thiol loss, in contrast to the cytotoxicity studies. Together, these studies provide new insight into the pathways by which histones could promote vascular dysfunction, which could be relevant to inflammatory diseases, such as atherosclerosis and sepsis, which are associated with elevated NET release and high circulating histones, respectively.

Oxidative preservative and sensory effects of Harpephyllum caffrum (wild plum) peel extracts in fresh ground beef patties

Indigenous, underutilized fruits-byproducts are potential sources of novel antioxidants for the food industry. The current study evaluated the effects of different levels of purified ethanolic extracts of Harpephyllum caffrum Bernh peel (HCP, 0, 250, 450 and 650 ppm) on shelf life and sensory attributes of fresh ground beef patties stored at 4 °C during 9 days in comparison with sodium metabisulfite (SMB, 450 ppm). Antioxidant type × day interactions influenced (P ≤ 0.05) shelf life parameters, including loss of colour, myoglobin and protein oxidation. The HCP extracts improved colour and protein (i.e., reduced thiol loss) stability during storage compared to the control. The HCP treatments had lower (P ≤ 0.05) TBARS on day 9 compared to the control. Also, HCP reduced (P ≤ 0.05) tenderness and juiciness, and increased undesirable flavours in beef patties compared to control and SMB. The study indicates that HCP is a potential alternative to SMB in improving oxidative shelf life of the beef patties, with additional studies recommended to reduce adverse effects on sensory quality.

Hybrid Sausages Using Pork and Cricket Flour: Texture and Oxidative Storage Stability.

This study aimed to study the functionalities of cricket flour (CF) and the effects of the addition of CF on the texture and oxidative stability of hybrid sausages made from lean pork and CF. Functional properties of CF, including protein solubility, water-holding capacity, and gelling capacity, were examined at different pHs, NaCl concentrations, and CF contents in laboratory tests. The protein solubility of CF was significantly affected by pH, being at its lowest at pH 5 (within the range 2-10), and the highest protein solubility toward NaCl concentrations was found at 1.0 M (at pH 6.8). A gel was formed when the CF content was ≥10%. A control sausage was made from lean pork, pork fat, salt, phosphate, and ice water. Three different hybrid sausages were formulated by adding CF at 1%, 2.5%, and 5.0% levels on top of the base (control) recipe. In comparison to control sausage, the textural properties of the CF sausages in terms of hardness, springiness, cohesiveness, chewiness, resilience, and fracturability decreased significantly, which corresponded to the rheological results of the raw sausage batter when heated at a higher temperature range (~45-80 °C). The addition of CF to the base recipe accelerated both lipid and protein oxidation during 14 days of storage, as indicated by the changes in TBARS and carbonyls and the loss of free thiols and tryptophan fluorescence intensity. These results suggest that the addition of CF, even at low levels (≤5%), had negative effects on the texture and oxidative stability of the hybrid sausages.

Oxidation affects dye binding of myofibrillar proteins via alteration in net charges mediated by a reduction in isoelectric point

In order to study the effect of oxidation on the dye-binding behavior of myofibrillar proteins, selected dyes with different charges (positively charged Sarfarin O (SO), neutral bromophenol blue (BPB), and negatively charged Orange G (OG)) were incubated with myofibrils oxidized by the Fenton system with H2O2 (10 mM). Upon oxidation, loss of free thiols, formation of carbonyls, particle size, and hydrophobicity of myofibrillar proteins (MPs) increased. The absolute value of Zeta-potential increased by 14.48 % after oxidation, the myofibrillar proteins shifted to a more acidic isoelectric point (pI) upon oxidation. Oxidation decreased net positive charges of myofibrillar protein and the binding ability of MPs towards OG in the environment with pH less than pI and the affinity of MPs towards SO in the environment with pH more than pI were thus increased. Here we propose a hypothesis that oxidation-induced change in net charges is the driving force affecting the amount of protein-bound dye. This paper aims to examine the effect of oxidation on the net charges of myofibrillar proteins and to provide insight into the mechanism of oxidation-induced changes in protein-bound dyes.

Cysteine Sulfenylation of Protein Disulfide Isomerase Links Oxidative Stress to Thrombosis

Oxidative stress increases the risk of clinically significant thrombosis in the setting of inflammation, malignancy, infection, and dyslipidemia. Oxidants generated during oxidative stress are prothrombotic; however, the mechanisms by which oxidants induce thrombus formation is poorly understood. Protein disulfide isomerase (PDI) is a thiol isomerase that serves an important role in thrombus formation. It has a domain structure of a-b-b'-a', in which the aand a' domains are catalytic and the b and b' are substrate binding. A flexible x linker is between the b' and a'domains. PDI is uniquely sensitive to oxidative cysteine modification within the CGHC catalytic motif of its a and a'domains. Yet whether PDI cysteine modifications mediates thrombosis in an oxidative environment has not previously been studied. We hypothesized that oxidized PDI links oxidative stress to a prothrombotic phenotype through post-translational modification of proteins. We have previously shown that dyslipidemia promotes hydrogen peroxide generation from platelets and oxidizes proteins. Using hydrogen peroxide as a model oxidant, we found that oxidation of recombinant PDI promoted the loss of free thiols and inhibited reductase activity in a dose-dependent manner (IC50= 3.3 ± 0.12 µM). To understand how free thiols were lost, we used a benzothiazine-based nucleophilic probe, termed BTD, to label sulfenic acid. The transient cysteine sulfenic acid (S-OH) was detected by BTD labeling in wildtype PDI following exposure to hydrogen peroxide, but not in a PDI mutant in which all active site cysteines had been mutated to alanine. PDI exposure to oxidized lipoprotein particles (OxLDL) or 3-morpholinosydnonimine (SIN-1), a peroxynitrite donor, also promoted its sulfenylation. Using single cysteine mutants of PDI to identify the sulfenylation site, we found that Cys56 was preferentially sulfenylated as mutation of this cysteine prevented further labeling of the sulfenic acid probe. To assess whether interdomain interactions influenced sulfenylation, we evaluated BTD labeling in isolated fragments of PDI. Sulfenylation was identified in the a domain, consistent with Cys56 labeling, and was significantly enhanced in the ab fragment, implicating an interaction between the a and b domains in sulfenylation. No labeling was observed in an isolated b'xa' fragment. Further analysis of structural elements revealed that a conserved Arg120 within the a domain regulates Cys56 reactivity. Indeed, mutation of Arg120 to Ala prevented sulfenylation by peroxides compared to the wildtype control. Maleimide labeling showed that sulfenylated PDI is an intermediary that resolves into disulfided PDI. To determine whether this disulfided PDI promotes oxidase activity, we used a denatured and reduced RNAse that refolds when disulfides are transferred from oxidized PDI to the RNAse. PDI oxidized by hydrogen peroxide promoted RNAse refolding, whereas reduced PDI did not. A selective inhibitor of sulfenylation, arsenite, prevented RNAse refolding, indicating that hydrogen peroxide-induced oxidation of PDI proceeds through a sulfenic acid intermediate. Since PDI is secreted from cells, we evaluated whether activated cells release sulfenylated PDI using both human vein endothelial cells (HUVECs) and platelets. Sulfenylated PDI was increased in the cultured media of HUVECs stimulated with thrombin and in the releasate of platelets stimulated via PAR1 or glycoprotein VI. OxLDL exposure sensitizes platelets to activation by low doses of agonists. Inhibition of PDI using a neutralizing antibody inhibited augmentation of platelet aggregation by oxLDL, suggesting that oxidation of PDI by oxLDL promotes platelet activation. The ability of oxLDL to enhance the prothrombotic effect of PDI was tested in vivo in a murine model of laser-induced thrombus formation. Mice were infused with either control IgG or IgG directed at PDI, followed by the infusion of oxLDL. OxLDL significantly enhanced thrombus formation in this model. The effect of oxLDL was inhibited by anti-PDI IgG but not control IgG antibody or buffer, implicating PDI as an effector of oxLDL-augmented thrombus formation. In conclusion, our study demonstrates sulfenylation of PDI and shows that PDI sulfenylation retains PDI oxidase activity. Oxidation of PDI by oxidants such as oxLDL occurs in platelets and in endothelium and promotes thrombosis in the setting of oxidative stress.

Comparison of oxidation extent, structural characteristics, and oxidation sites of myofibrillar protein affected by hydroxyl radicals and lipid-oxidizing system

To compare the differences between direct protein oxidation (PO) and lipid-derived PO, the myofibrillar protein (MP) of obscure pufferfish was oxidatively modified by the hydroxyl radical oxidizing system (HOS) and the lipid-oxidizing system (LOS). The degree of oxidation, structural characteristics, and oxidation sites in MP were assessed. The results showed there was no significant thiol loss in LOS, compared with a 77.64% loss observed in case of the HOS. The secondary structure of MP was more vulnerable to HOS, but the tertiary structure was more susceptible to LOS. The cross-linking was largely attributed to the reversible disulfide links in HOS and the irreversible covalent linkages in LOS. Six amino acids and 10 specific oxidant products were identified in HOS. Only three amino acids and three specific oxidant products were identified in LOS. These findings may help deepen the understanding regarding the mechanism underlying PO in protein- and lipid-rich food materials.

Substitution of SERCA2 Cys674 aggravates cardiac fibrosis by promoting the transformation of cardiac fibroblasts to cardiac myofibroblasts

Sarcoplasmic/endoplasmic reticulum Ca2+ ATPase 2 (SERCA2) is vital to maintain intracellular calcium homeostasis, and its redox Cys674 (C674) is the key to regulating activity. Our goal was to investigate whether the redox state of SERCA2 C674 is critical for cardiac fibrosis and the mechanisms involved. Heterozygous SERCA2 C674S knock-in (SKI) mice, in which half of C674 was substituted by serine, were used to mimic the partial loss of the reactive C674 thiol in pathological conditions. In cardiac fibroblasts, the substitution of C674 thiol increased Ca2+ levels in cytoplasm and mitochondria, and intracellular ROS levels, and activated calcineurin/nuclear factor of activated T-lymphocytes (NFAT) pathway, increased the protein expression of profibrotic factors TGF beta 1 (TGF-β1), alpha smooth muscle actin, collagen I and collagen III, and promoted the transformation of cardiac fibroblasts to cardiac myofibroblasts, which could be reversed by calcineurin/NFAT inhibitor, SERCA2 agonist, or ROS scavenger. Activation of SERCA2 or scavenging ROS is beneficial to alleviate cardiac fibrosis caused by the substitution of C674. In conclusion, the partial loss of the reactive C674 thiol in the SERCA2 exacerbates cardiac fibrosis by activating the calcineurin/NFAT/TGF-β1 pathway to promote the transformation of cardiac fibroblasts to cardiac myofibroblasts, which highlights the importance of C674 redox state in maintaining the homeostasis of cardiac fibroblasts. SERCA2 is a potential therapeutic target for the treatment of cardiac fibrosis.

Abstract 12156: Loss of Thiol on C674 SERCA2 Leads to Ca2 + Mishandling With Hypertrophy in Right Ventricular Exposed to Hypoxia

Introduction: Right ventricular (RV) dysfunction is the strongest predictor of mortality in pulmonary artery hypertension (PAH). Reactive oxygen species in heart failure cause irreversible oxidation of sarcoplasmic reticulum (SR) Ca 2+ -ATPase2 (SERCA2) C674, which results in SERCA2 dysfunction and intracellular Ca 2+ overload. However, the contribution of the loss of the thiol on C674 SERCA2 and RV failure in PAH remains unclear. Hypothesis: The loss of the thiol on C674 SERCA2 contributes to the RV dysfunction in PAH. Methods & Results: We employed the SERCA2 C674S heterozygote knock-in (SKI) mice, in which C674 is replaced by serine, mimics oxidative modification of SERCA2 by loss of the specific thiol. Wild-type mice (WT) and SKI were exposed to either normoxia (Nx) or chronic hypoxia (Hx) for four weeks. Hypoxia elevated RV systolic pressure (WT-Nx: 21.4mmHg vs. WT-Hx: 37.0mmHg, P<0.0001) and represented pulmonary vascular remodeling similarly both in WT and SKI. In histology, SKI-Hx showed the markedly increased Fulton's Index with higher levels of the mRNA expression of atrial natriuretic peptide and B-type natriuretic peptide than WT-Hx. In the exercise test, exercise duration was shorter and peak oxygen consumption was decreased in SKI-Hx than WT Hx. The mRNA levels of SERCA2 were not changed among the four groups. In Ca 2+ transient measurement of isolated myocytes with Fluo-4, SKI prolonged the time to 50% of cytosolic Ca 2+ extrusion (T50) and lower the peak Ca 2+ transient amplitude (F/F0) than WT under Nx. Prolonged T50 and lowered peak F/F0 were observed in WT-Hx, both of which additionally progressed in SKI-Hx. Conclusions: Hypoxia impaired SR Ca 2+ homeostasis in RV. SKI additionally impaired SR Ca 2+ homeostasis with the progression of RV hypertrophy. The loss of the thiol on C674 SERCA2 contributed to the pathogenesis of RV failure in PAH.

Improved effect of flaxseed gum on the weakened gelling properties of myofibrillar protein induced by catechin

The effect of flaxseed gum (FG) on the weakened gelling properties of myofibrillar proteins (MP) induced by catechin was investigated. Different levels of FG and catechin were incorporated into MP and the chemical changes of MP were studied; MP emulsions and gels with different levels of FG and catechin were prepared and their properties were studied. Catechin promoted the loss of thiol and the exposure of the hydrophobic groups of MP and increased the particle size and apparent viscosity of emulsions, resulting in a poor gel network. The incorporation of FG enhanced the gel strength, water holding capacity and dynamic rheological properties, which might be attributed to the formation of uniform and stabilized emulsions with high apparent viscosity and the enhanced disulfide cross-linking and hydrophobic interactions during heat-induced gelation. FG could be a potential approach in overcoming the deterioration of protein gels caused by catechin.

Formation of Lipid and Protein Oxidation Products during In Vitro Gastrointestinal Digestion of Dry-Cured Loins with Different Contents of Nitrate/Nitrite Added.

The effect of nitrate/nitrite (0, 37.5, 75, and 150 mg/kg) in the dry-cured loin formulation on the formation of lipid and protein oxidation products during in vitro digestion was evaluated. Dry-cured loins formulated with nitrate/nitrite resulted in significantly less lipid and protein oxidation than uncured loins before and after simulated digestion. Compared to loins added with 0 mg/kg nitrate/nitrite, dry-cured loins with 37.5, 75, and 150 mg/kg contained a significantly lower content of conjugated dienes, malondialdehyde, carbonyls, and non-heme iron, and higher amounts of nitrosylmioglobin and thiols. During in vitro digestion, the content of conjugated dienes, malondialdehyde, and carbonyls increased, while thiol content decreased, indicating the development of lipid and protein oxidative processes. At the end of the intestinal phase, the 75 mg/kg digests had a significantly higher content of conjugated dienes, while no differences were found among the other digests. During the in vitro intestinal phase (180 and 240 min), nitrate/nitrite curing resulted in significantly lower malondialdehyde concentrations in the 37.5, 75, and 150 mg/kg loin digests than in the uncured loin digests. No significant differences were observed at the end of the intestinal digestion phase between the cured loin digests. Digests of dried loins without nitrate/nitrite addition showed higher carbonyl contents than the nitrate/nitrite cured counterparts. The loss of thiols was significantly higher in loin digests without added nitrate/nitrite than in loin digests with different amounts of curing salts. The addition of 37.5 mg/kg nitrate/nitrite in the cured loin formulation prevents the formation of lipid peroxidation products and carbonyls from protein oxidation and thiol loss during digestion

Quantifying dynamic range in red blood cell energetics: Evidence of progressive energy failure during storage.

During storage, red blood cells (RBCs) undergo significant biochemical and morphologic changes, referred to collectively as the "storage lesion". It was hypothesized that these defects may arise from disrupted oxygen-based regulation of RBC energy metabolism, with resultant depowering of intrinsic antioxidant systems. As a function of storage duration, the dynamic range in RBC metabolic response to three models of biochemical oxidant stress (methylene blue, hypoxanthine/xanthine oxidase, and diamide) was assessed, comparing glycolytic flux by NMR and UHPLC-MS methodologies. Blood was processed/stored under standard conditions (AS-1 additive solution) with leukoreduction. Over a 6-week period, RBC metabolic and antioxidant status were assessed at baseline and following exposure to the three biochemical oxidant models. Comparison was made of glycolytic flux (1 H-NMR tracking of [2-13 C]-glucose and metabolomic phenotyping with [1,2,3-13 C3 ] glucose), reducing equivalent (NADPH/NADP+ ) recycling, and thiol-based (GSH/GSSG) antioxidant status. As a function of storage duration, we observed the following: (1) a reduction in baseline hexose monophosphate pathway (HMP) flux, the sole pathway responsible for the regeneration of the essential reducing equivalent NADPH; with (2) diminished stress-based dynamic range in both overall glycolytic as well as proportional HMP flux. In addition, progressive with storage duration, RBCs showed (3) constraint in reducing equivalent (NADPH) recycling capacity, (4) loss of thiol based (GSH) recycling capacity, and (5) dysregulation of metabolon assembly at the cytoplasmic domain of Band 3 membrane protein (cdB3). Blood storage disturbs normal RBC metabolic control, depowering antioxidant capacity and enhancing vulnerability to oxidative injury.

Protective effects of dietary carnosine during in-vitro digestion of pork differing in fat content and cooking conditions.

Muscle carnosine represents an important health advantage of meat. Ground pork samples with intrinsic or added carnosine; fat content; and cooked under low or high intensity as a 2×2×2 factorial were digested in-vitro. Changes in free carnosine and in markers of lipid (hexanal, 4-hydroxynonenal (4-HNE), malondialdehyde (MDA) and protein (protein-carbonyls, thiols) oxidation, and of advanced glycation end-products (AGEs) Nε -(carboxymethyl)lysine (CML) were determined in the saliva, gastric, and duodenal digests. During digestion, the different markers overall indicated increased oxidation and decreased free carnosine. Increasing pork carnosine level significantly reduced protein carbonyls, loss of thiols, and 4-HNE during in-vitro gastric digestion, irrespective of fat and cooking level of the meat. Increased carnosine also significantly reduced hexanal, MDA and CML up to the duodenum phase in moderately cooked lean pork. Besides substantiating the formation of AGEs during digestion, these results show a potentially important role of dietary carnosine occurring in the gastrointestinal tract. PRACTICAL APPLICATIONS: The ailments epidemiologically associated with red meat consumption could be counteracted by ingesting carnosine into meat. The health advantages of dietary carnosine, however, have never been demonstrated during digestion, a unique and complex oxidative environment compounded by the composition and cooking of the meat. The results obtained substantiated that AGEs formation occurred in-vitro in the GIT. They also showed that increased carnosine had an immediate health beneficial role during pork digestion in reducing the formation of different harmful molecules, including AGEs, modulated by the composition and cooking of the meat. However, in exerting this protective role in the GIT, the remaining free level of carnosine, gradually decreased during digestion. Carnosine, as an important meat compositional factor may, depending on the fat content and cooking conditions, change the image of meat from representing a health risk to a health benefit. Carnosine level may also explain discrepancies observed in the literature.

Substitution of synthetic nitrates and antioxidants by spices, fruits and vegetables in Clean label Spanish chorizo

Natural extracts obtained from fruits and vegetables processing are important sources of phenolic compounds and nitrates, with excellent antioxidant and antimicrobial properties. The aim of this study was to elaborate a Clean label dry-cured meat product (Spanish “chorizo”) using Mediterranean Diet ingredients (Citrus, Acerola, Rosemary, Paprika, Garlic, Oregano, Lettuce + Arugula + Watercress, Spinach + Celery, Chard + Beet). For that, a self-life study for 150 days was carried out, when physical-chemical (colour, pH, aw, thiol loss, volatile compounds profile), microbiological, and organoleptic changes were determined. The combination of citrus extracts and leafy green vegetables halved the hexanal and nonanal content for 150 days. In addition, this change did not affect to the sensory properties of the product, which obtained the highest acceptance avoiding the oxidative damage (colour, volatile compounds release, thiol loss) and the microbiological growth. Nevertheless, rosemary extract incorporation altered sensory quality, unless it also avoided protein and lipid oxidation, as well as microbiological growth. Otherwise, Control sample elaborated with synthetic sources of nitrates and nitrites showed a lower sensory quality due to the increased hardness, protein oxidation, hexanal, and nonanal concentrations, related to lipid oxidation, and hence, to rancid flavour apparition.

Electrophilic reactivity of the Busulfan metabolite, EdAG, towards cellular thiols and inhibition of human thioredoxin-1

Busulfan is an alkylating agent used in chemotherapy conditioning regimens prior to hematopoietic stem cell transplantation (HSCT). However, its administration is associated with a great risk of adverse toxicities, which have been historically attributed to busulfan’s mechanism of non-specific DNA alkylation. A phase II generated metabolite of busulfan, EdAG (γ-glutamyldehydroalanylglycine), is a dehydroalanine analog of glutathione (GSH) with an electrophilic moiety, suggesting it may bind to proteins and disrupt biological function. However, EdAG’s reactions with common cellular thiols such as glutathione (GSH) and l-cysteine are understudied, along with possible inhibition of glutathionylation-dependent enzymes (with active site cysteine residues). We established a physiologically-relevant in vitro model to readily measure thiol loss over time. Using this model, we compared the apparent rates of thiol depletion in the presence of EdAG or arecoline, a toxic constituent of the areca (betel) nut and known GSH depletor. Simulated kinetic modeling revealed that the mean (±SE) alpha (α) second order rate constants describing GSH and l-cysteine depletion in the presence of EdAG were 0.00522 (0.00845) μM−1∙min−1 and 0.0207 (0.00721) μM−1∙min−1, respectively; in the presence of arecoline, the apparent rates of depletion were 0.0619 (0.009) μM−1∙min−1 and 0.2834 (0.0637) μM−1∙min−1 for GSH and l-cysteine, respectively. Under these experimental conditions, we conclude that EdAG was a weaker electrophile than arecoline. Arecoline and EdAG both depleted apparent l-cysteine concentrations to a much greater extent than GSH, approximately 4.58-fold and 3.97-fold change greater, respectively. EdAG modestly inhibited (∼20%) the human thioredoxin-1 (hTrx-1) catalyzed reduction of insulin with a mean IC50 of 93 μM [95% CI: 78.6–110 μM). In summary, EdAG’s ability to spontaneously react with endogenous thiols and inhibit hTrx-1 are potentially biochemically relevant in humans. These findings continue to support the growing concept that EdAG, an underrecognized phase II metabolite of busulfan, plays a role in untoward cellular toxicities during busulfan pharmacotherapy.

Pinpointing oxidative stress behind the white striping myopathy: depletion of antioxidant defenses, accretion of oxidized proteins and impaired proteostasis.

This study aimed to investigate the molecular mechanisms involved in the onset of the white striping (WS) myopathy with particular attention to the role of oxidative stress and protein oxidation in the loss of meat quality. It was found that WS-M (moderate degree; white stripes <1 mm thickness) and WS-S (severe degree; white stripes >1 mm thickness) breast presented higher pH, hardness, redness, lipid, and collagen content, and lower lightness than normal breast. Compared with the latter, WS-S had a more severe loss of protein thiols (70.7% less thiols than in N), reduced activity of antioxidant enzymes such as catalase (23 versus 40 U g-1 ), glutathione peroxidase (0.21 versus 0.54 U g-1 ), and superoxide dismutase (56 versus 73 U g-1 ), and consequently, had greater accretion of thiobarbituric acid reactive substances (0.64 versus 0.22 mg MDAkg-1 muscle), allysine (3.1 versus 1.9 nmol mg-1 protein) and Schiff base structures (645 versus 258 fluorescent units). The analysis of sarcoplasmic proteins revealed that muscles severely affected by the myopathy suffered a chronic impairment of physiological (upregulation of sarcoplasmic reticulum Ca2+ ATPase, sarcalumenin and calsequestrin-2) and metabolic processes (downregulation of pyruvate kinase, creatine kinase, and l-lactate dehydrogenase). The overexpression of ribonuclease / angiogenin inhibitor 1 and Kelch-like proteins in WS chicken breasts indicates altered protein turnover plausibly mediated by oxidative stress and accumulation of oxidized proteins. © 2020 Society of Chemical Industry.

Synthetic vs. Natural Hydroxytyrosol for Clean Label Lamb Burgers.

Clean labelling refers to consumers’ desire for manufacturers to be more transparent in the way their products are made and sourced. Natural antioxidants (spices, herbs, fruits, or vegetables) have been proven to offer the same functionality as their synthetic counterparts, with the advantage of being label friendly and process compatible, maintaining meat quality and reducing food waste. Lamb meat has the challenges to have an intense flavour and fat composition to test the effectiveness of some of these natural antioxidants like hydroxytyrosol (HXT). The current paper was designed to test both natural (HXTo) and synthetic (HXTs) antioxidants using four lamb patty batches: one Control (C) (which included sulphites); a reference (R) sample (14.6% carnosic acid and 6% carnosol from natural rosemary extracts, 200 ppm); a sample containing synthetic hydroxytyrosol (HXTs, 99% purity, 200 ppm); and a sample with added organic hydroxytyrosol (HXTo, sample 7% purity from olive tree leaves, 200 ppm). A shelf-life study was carried out for 6 days at 4 °C, testing proximal composition and mineral bioavailability, pH changes, colour (by CIELab), total antioxidant capacity (TAC by oxygen radical absorbance capacity (ORAC)), lipid and protein oxidation (thiobarbituric acid reactive substances (TBARs) and thiol loss, respectively), volatile compound profiles (by HPC-MS), sensory evaluation, and microbiological growth (as total vial count (TVC) and total coliform count (TCC)). Results revealed that lamb burgers with added HXTs had better-preserved raw lamb meat in the test conditions, with reduced colour losses, lipid oxidation, and release of volatile compounds, the half the microbiological growth (TVC) of the Control, the best TAC, and significantly increased (p < 0.05) minerals bioavailability, while maintaining sensory acceptability. In summary, natural antioxidants are an adequate strategy for lamb meat burgers. Regarding HXTo, obtained from olives, the synthetic analogue is even more effective in terms of preservative and antioxidant activity, and in maintaining the nutritional value, sensory characteristics, and safety of food products.

Impact of Copper Fungicide Use in Hop Production on the Total Metal Content and Stability of Wort and Dry-Hopped Beer

Transition metals, including copper, iron, and manganese, are known to catalyze the generation of reactive oxygen species (ROS) in beer leading to reduced product stability. Metals in beer are generally derived from raw ingredients. The present study aims to evaluate the impact of brewing and dry-hopping using hops treated with copper-based fungicides (CBFs) on the final transition metal content of model buffer solutions and pilot-scale systems of wort and beer. Copper levels in model wort and beer solutions were elevated (105.6% and 230.4% increase, respectively) when CBF-treated hops were used. In laboratory-prepared wort, elevated copper concentrations were not observed when CBF-treated hops were used for boiling. Dry hopping of beer using CBF-treated hops led to significant increases in total copper content (ca. 75 µg/kg vs. ca. 40–50 µg/kg in the control-hopped beer) when yeast was absent from the treated beer, but not when yeast was present. It was observed that manganese levels were significantly elevated in all hopped beers (ca. 495–550 µg/kg vs. 90–125 µg/kg in the unhopped control), regardless of hop treatment. A hop varietal thiol, 4-Mercapto-4-methylpentan-2-one, was spiked into treated beers, and the rate of oxidative loss was monitored during aging. Rates of thiol loss in treated beer samples did not differ across CBF treatments but were significantly lower in unhopped controls in the absence of yeast (p &lt; 0.0001) and correlated significantly with total manganese content of the beers (R2 = 0.4228, p = 0.0006). The rate of staling in hopped beers as measured by the rate of 1-hydroxyethyl radical generation did not differ among hop treatments, suggesting that excess copper content contributed from the hops does not negatively impact the oxidative stability of the beers. These findings suggest that brewers can use CBF-treated hops without any negative implications for the shelf stability of their beers and do not contraindicate the use of CBF in hops production when necessary.