Reduced Protein Oxidation Research Articles

Effect of Protein Oxidation on Microbial, Free Amino Acids and Biogenic Amine Content in Chinese Dry Sausage

The objective of this study was to investigate the effect of protein oxidation on microbial, free amino acids (FAAs) and biogenic amines (BAs) in Chinese dry sausage during processing. Three different batches of Chinese dry sausages were examined: control batch, pro-oxidation batch and anti-oxidation batch. The experimental results revealed that anti-oxidation batch showed the lowest degree of protein oxidation compared with control batch, while the results of the pro-oxidation batch were just opposite. With the increasing protein oxidation, estimated by protein carbonyls and sulfhydryls, microbial content and free amino acid content increased, while BA content decreased. The protein carbonyls were positively correlated with total FAAs (P < 0.01) and total BA content (P < 0.05), while sulfhydryls content was negatively correlated with total FAA content (P < 0.01), total viable counts (P < 0.01), and total BA content (P < 0.01). In conclusion, reduced protein oxidation contributes to improved quality of Chinese dry sausage. This research provided a new strategy to improve the quality of meat product manufacture.

Diphenyl diselenide subcutaneous supplementation of dairy sheep: effects on oxidant and antioxidant status, inflammatory response and milk composition

Diphenyl diselenide ((PhSe)2) is a organoselenium compound with potent antioxidant properties. Therefore, the aim of the present study was to evaluate whether subcutaneous supplementation of (PhSe)2 in dairy sheep has positive effects on milk composition, as well as on the prevention of oxidative stress and exacerbated inflammatory response. For this, 16 primiparous recently calved sheep were divided into the following two groups, with eight animals in each: Group A, the control group; and Group B, the group subcutaneously supplemented with five doses of (PhSe)2 of 3.0µmol/kg each every 7 days. Blood samples from supplemented animals showed increased concentration of antioxidant enzymes (catalase, superoxide dismutase, glutathione peroxidase and glutathione-S-transferase), and reduced reactive oxygen species and lipid peroxidation, which prevented oxidative damage in the lactation period, as well as increased seric interleukin-10, an anti-inflammatory cytokine. In the sera, supplemented animals showed increased total antioxidant capacity and ferric-reducing ability of plasma compared with the control group. As a consequence, supplemented animals showed increased antioxidant variables, as well as reduced protein oxidation in milk samples. Moreover, milk from supplemented sheep showed a higher fat content, and lower total protein and lactose contents in some periods in the study, than did not-supplemented ewes. Seric concentrations of interleukin-1 were lower on Days 30 and 45 in supplemented animals, as well as the concentrations of tumour necrosis factor α in all periods, than were those in the control group, whereas the interleukin-10 concentrations were higher. Thus, dairy sheep supplementation of (PhSe)2 activated antioxidant and anti-inflammatory responses, and increased milk fat content. Moreover, this protocol increased the antioxidant and, consequently, reduced the oxidant concentration in milk, which is desirable for product quality.

Impact of Interfacial Composition on Lipid and Protein Co-Oxidation in Oil-in-Water Emulsions Containing Mixed Emulisifers.

The impact of interfacial composition on lipid and protein co-oxidation in oil-in-water emulsions containing a mixture of proteins and surfactants was investigated. The emulsions consisted of 5% v/v walnut oil, 0.5% w/v whey protein isolate (WPI), and 0 to 0.4% w/v Tween 20 (pH 3 and pH 7). The protein surface load, magnitude of the ξ-potential, and mean particle diameter of the emulsions decreased as the Tween 20 concentration was increased, indicating the whey proteins were displaced by this nonionic surfactant. The whey proteins were displaced from the lipid droplet surfaces more readily at pH 3 than at pH 7, which may have been due to differences in the conformation or interactions of the proteins at the droplet surfaces at different pH values. Emulsions stabilized by whey proteins alone had relatively low lipid oxidation rates when incubated in the dark at 45 °C for up to 8 days, as determined by measuring lipid hydroperoxides and 2-thiobarbituric acid-reactive substances (TBARS). Conversely, the whey proteins themselves were rapidly oxidized, as shown by carbonyl formation, intrinsic fluorescence, sulfhydryl group loss, and electrophoresis measurements. Displacement of whey proteins from the interface by Tween 20 reduced protein oxidation but promoted lipid oxidation. These results indicated that the adsorbed proteins were more prone to oxidation than the nonadsorbed proteins, and therefore, they could act as better antioxidants. Protein oxidation was faster, while lipid oxidation was slower at pH 3 than at pH 7, which was attributed to a higher antioxidant activity of whey proteins under acidic conditions. These results highlight the importance of interfacial composition and solution pH on the oxidative stability of emulsions containing mixed emulsifiers.

THE IMPACT OF COMBINED EXERCISE TRAINING ON THE DEVELOPMENT OF CARDIOVASCULAR AND NEUROIMMUNE COMPLICATIONS INDUCED BY FRUCTOSE OVERLOAD IN HYPERTENSIVE RATS

The aim of the present study was to evaluate the impact of combined exercise training on the development of cardiovascular and neuroimmune complications induced by fructose overload in sedentary hypertensive (SH), SHR + fructose (HF) and SHR + fructose + training (Treadmill + ladder, 60 days, 40–60% of maximal capacity) (HFTC). The groups were divided into subgroups evaluated at 7, 15, 30 and 60 days (n=6/group/time). Fructose was offered in drinking water (10%). Metabolic, hemodynamic, autonomic, inflammatory, and oxidative stress parameters were evaluated. Regarding the metabolic profile, the HF group showed increase in white adipose tissue, reduction in insulin sensitivity (HF: 3.15±0.2 vs. H: 3.96±0,1 and HFTC: 4.32±0.2 %/min) and increased triglycerides (TG) when compared to H group at 60 days of protocol (HF:139±7 vs. H: 106±5 and HFTC:107±8 mg/dL); these changes were not observed in HFTC group. Fructose consumption (HF) induced a further increase in mean arterial pressure (MAP) at 30 and 60 days in SHR (HF‐30 days: 153±4 e HF‐60 days: 184±4 vs. H‐30 days: 141±3 e H‐60 days: 165±3 mmHg). Combined exercise training reduced MAP in 30 and 60 days of fructose overload in relation to the HF group (HFTC: 142±8 and 167±6 mmHg). There was a decrease on heart rate variability (VAR‐PI) in 7 days and 60 days in the HF group (10.5±1.3 and 40.81± 6.12 ms2) in relation to the H group (23±1.5 and 59.7±3.4 ms2). The HF group had a lower alpha index (spontaneous baroreflex) at 7 days compared to the H group (0.23±0.03 vs. 0.35±0.01 ms/mmHg), which was not observed in the HFTC group. There was an increase in IL‐6 and TNFα in cardiac tissue at 15, 30 and 60 days in the HF and HFTC groups compared to the H groups. The HFTC group presented higher IL‐10 values in the heart at 7 days compared to the HF group (28±1 vs. 16±1 pg/mg/prot). The HF group presented increase in cardiac lipoperoxidation at 30 and 60 days in relation to the H group. The HFTC group decreased lipoperoxidation compared to HF group at 60 days (1302±58 vs. 1956±215 cps/mg/prot) and there was also reduction in protein oxidation in this time. In addition, there was an increase in NADPH oxidase in the HF group at 60 days when compared to the H group and reduction in this parameter in the HFTC group in relation to the HF group at the same time (H: 0.18±0.02; HF: 0.46 ±0.04; HFTC 0.35 ±0.03 μm/mg/prot). The HFTC group increase glutathione redox balance in 7, 15, 30 and 60 days in relation to H and HF groups. These findings reinforce the role of the autonomic nervous system in the genesis of cardiometabolic dysfunctions and evidence an important role of combined exercise training in the prevention of these alterations.Support or Funding InformationFAPESP: 2015/11223‐6, CAPES‐PROSUP (1277269), UNINOVEThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Sanglifehrin A Has No Protective Effects in Non‐reperfused Myocardial Infarction: The Role of Mitochondria

BackgroundExcessive reactive oxygen species (ROS) production and mitochondrial permeability transition pore (mPTP) formation due to mitochondrial dysfunction is one of the hallmarks of heart failure. The mPTP plays a critical role in pathogenesis of myocardial infarction (MI)/ischemia‐reperfusion (IR). Cyclopilin D (CypD) activation leads to mPTP formation thereby stimulating excessive ROS production and subsequent protein oxidation. Inhibition of CypD has been linked to cardioprotection through inhibition of mPTP formation and therefore mitochondrial dysfunction. However, failure of recent CIRCUS clinical trials indicates the necessity to further study the efficacy of CypD inhibition during MI/IR injury.HypothesisInhibition of CypD by sanglifehrin A (SfA) protects the heart from reperfused but not non‐reperfused MI by preserving mitochondrial and cardiac function.MethodsMI was performed in female rats (200–250 g) by coronary artery ligation (CAL) for 30 min followed by 2‐ and 28‐ day post‐surgery with or without reperfusion. The animals were divided in the following groups: i) Sham (S, no MI); ii) Sham+SfA (SS); iii) MI (I, no reperfusion); iv) I+SfA (IS); v) MI+reperfusion (IR); and vi) IR+SfA (IRS). SfA (5 mg/kg, IV) was administered 25 min after the start of sham surgery or MI. Cardiac and left ventricular mitochondrial functions were analyzed at 2‐ and 28‐days post‐surgery.ResultsSfA significantly improved ejection fraction (EF%) after 2 (28±2 vs. 53±3) and 28 (35±3 vs. 44±2) days in reperfused hearts. SfA administration to permanent occluded hearts has no effect on EF% at 2 (36±4 vs. 40±4) and 28 (32±3 vs. 40±4) days. Left ventricular respiratory control index (RCI) was significantly reduced (7.5±0.5 vs. 5.6±0.3) in animals subjected to IR when compared to controls after 2 days but not after 28 days. Formation of the mPTP was the same in all groups at 2 and 28 days. An increase in ROS production was observed in permanent occluded hearts (10.0±0.8 vs 7.3±0.6) after the addition of 1 mM calcium, and SfA significantly reduced protein oxidation in reperfused hearts (16.2±1.4 vs 21.8±2.1) at 2 days. SfA significantly reduced ROS production, at 1 mM calcium, in all groups at 28 days although no differences were detected in protein oxidation levels. A 14% increase in respirasome content was observed in IR hearts at 2 days and a 13% in permanent occluded hearts at 28 days. Menstrual cycle affected post‐ischemic recovery, where cycles with high estrogen levels positively correlated (r2=0.48) with an increase in EF% in reperfused MI hearts.ConclusionCypD inhibition has cardioprotective effects in reperfused infarcted hearts but it has no beneficial effects in hearts with non‐reperfused MI. Hearts of female rats presumably develop compensatory mechanisms post‐MI that preserve mitochondrial function in response to oxidative stress.Support or Funding InformationSupported by the American Physiological Society Porter Physiology Development Fellowship and the National Institutes of Health (Grants SC1HL118669, SC1GM128210)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Lowering greening of cookies made from sunflower butter using acidic ingredients and effect on reducing capacity, tryptophan and protein oxidation

Lowering of greening formed from oxidized chlorogenic acid (CGA) and amino groups, and favoured at alkaline pH, was investigated using acidic ingredients (sour cream, buttermilk, yoghurt, and honey) in sunflower butter cookies. Cookies with maple syrup added were used as a positive control. Changes in greening intensity, greening reactants (total phenols, CGA, protein), antioxidant capacity, tryptophan and Schiff base fluorescence were measured. Percentage greening, pH and aw of cookies followed the same order: maple syrup > sour cream ≥ buttermilk > yoghurt > honey. pH was positively correlated with greening intensity (r = 0.77) and negatively correlated with CGA (r = −0.96). Total phenolic content, antioxidant capacity, tryptophan and Schiff bases were similar among cookies. The results suggest it is possible to decrease greening by minimizing storage time and using acidic ingredients. Minimal greening with acidic ingredients can extend the application of sunflower butter as a baking ingredient without loss of free radical-scavenging capacity, or higher protein oxidation.

Application of exogenous xyloglucan oligosaccharides affects molecular responses to salt stress in Arabidopsis thaliana seedlings .

Soil salinity is one of the most devastating problems which reduces crop production and increases desertification. New approaches to overcome the negative effect of salinity on plants include the use of plant biostimulants, such as Xyloglucan oligosaccharides (XGOs) derived from the breakdown of xyloglucans from plant cell walls. The present study aimed at verifying the influence of exogenous XGOs derived from Tamarindus indica L. cell walls, on Arabidopsis thaliana’s tolerance to salt stress by understanding the gene expression, enzymatic and metabolic changes resulting from its application. A. thaliana plants were grown in liquid media and after 15 days they were treated by a salt shock with 100 mM of sodium chloride, with or without XGOs at 0.1 mg L-1. Gene expression of four oxidative stress markers as well as catalase and peroxidase activities and content of glutathione, total carbonyl, polyphenolics and chlorophyll were quantified. Bioinformatic models were used to obtain the co-expression network of the four oxidative stress response gene markers from microarray data of Arabidopsis under salt stress. Results showed that in saline conditions, XGOs dramatically increased catalase gene expression and enzymatic activity, peroxidase activity, and chlorophyll a/b ratio, while reducing protein oxidation and total polyphenols. Thus, XGOs may act to counteract negative effects of oxidative stress under saline conditions.

Effect of plant extracts on lipid and protein oxidation of mackerel (Scomber scombrus) mince during frozen storage.

The effects of different plant extracts [green tea extract (GTE), grape seed extract (GSE), and pomegranate rind extract (PRE)] at a level of 100ppm equivalent phenolics and butylated hydroxytoluene (BHT) on the changes in quality of fish (Scomber scombrus) mince during 6months frozen storage at -18±1°C were investigated. During storage, significant oxidative reactions in both the lipids and proteins were observed with the increase in thiobarbituric acid reactive substances (TBARS) and carbonyls and decrease in sulphydryl groups and protein solubility. BHT and PRE effectively inhibited lipid oxidation as lower peroxide and TBARS values were observed. Moreover, antioxidants added to minced fish significantly reduced protein oxidation compared to control without any antioxidant. The minced fish containing PRE had lower carbonyl and higher sulphydryl contents, but no significant differences for carbonyl and sulphydry contents were observed among antioxidant sources. Protein solubility decreased with increase in storage period. The loss of protein solubility was higher in control samples than in antioxidant treated ones. Among antioxidant sources, PRE was an excellent antioxidant toward both lipid and protein oxidations. Therefore, it could be a potential source of natural antioxidants in minced fish during frozen storage.

Cholinergic Stimulation Improves Oxidative Stress and Inflammation in Experimental Myocardial Infarction

We previously reported that cholinergic stimulation with pyridostigmine (PY) induces anti-inflammatory cell recruitment soon after myocardial infarction (MI). In this study, we evaluated the anti-inflammatory effects of PY during the proliferative phase of cardiac repair by analyzing the infiltration of macrophages, Treg lymphocytes, oxidative stress and inflammatory cytokines. Wistar rats underwent control sham surgery or ligation of the left coronary artery and were randomly allocated to remain untreated (untreated infarcted group, I) or to receive PY (30 mg·kg(−1)·day(−1)) in the supplied water (infarcted treated group, I + PY). Blood pressure and heart rate variability were registered at day 5 post-MI. The animals were euthanized 7 days after thoracotomy, when the hearts were removed and processed for immunohistochemistry (CD68, CD206, FOXP3), cytokines (IL-1β, IL-6, IL-10, TNF-α) and oxidative stress (superoxide dismutase, catalase, glutathione peroxidase, lipidic and protein peroxidation). PY treatment increased parasympathetic modulation, M2 macrophages and the anti-oxidant enzyme activity but reduced protein oxidation (carbonyls) and the concentration of IL-1β, IL-6, TNF-α and IL-10. Cholinergic stimulation induces parasympathetic neuro-immune modulation and anti-inflammatory cell enrollment as well as prevents oxidative stress and cytokine production after MI.

The effects of modified atmosphere packaging and enzyme inhibitors on protein oxidation of tilapia muscle during iced storage

The objectives of this study were to investigate whether modified atmosphere packaging (MAP) and inhibitors of calpain, caspase-3, and cathepsins can reduce protein oxidation of tilapia muscle during iced storage. Parameters such as protein solubility, total carbonyl and sulfhydryl content as well as electrophoretic pattern of myofibrillar proteins from tilapia muscle were examined. We found that protein solubility, sulfhydryl group contents, and electrophoretic band intensities of myosin heavy chain and actin in MAP samples were significantly higher, whereas carbonyl content was significant lower than those in AP samples (P < 0.05). The extent of degradation in myosin heavy chain and actin in muscle tissue stored in AP was higher than that in MAP, suggesting that the latter was a better method to reduce protein oxidation during iced storage. After storage for 14 d with different enzyme inhibitors, carbonyl content, sulfhydryl content, and protein solubility in muscle samples were significantly affected by treatment (P < 0.05 for all three measures). The best preservation effect, similar to that of MAP, was achieved by the inclusion of the calpain inhibitor MDL-28170.

331 - ProliNONOate Alters Fibrin Clot Formation Through Peroxynitrite Formation and Protein Oxidation

When a blood clot forms, the protein thrombin cleaves fibrinogen into fibrin that polymerizes into a branched network of fibers that forms the structure of the clot. The kinetics of fiber formation affect fiber diameter, fiber network density, rate of clot lysis and potentially clot stiffness. We previously showed that exposure to nitric oxide, an important regulator of blood flow, alters the characteristics of these fibrin clots. The addition of ProliNONOate to fibrinogen or thrombin together or separately lead to a decreased the rate of fiber formation, increased the time the clot took to gel, increased fiber diameters, and decreased clot density. We hypothesize the effects of nitric oxide on the fibrin clots are due to oxidation of the proteins due to the creation of peroxynitrite. Peroxynitrite is created when nitric oxide reacts with superoxide anion and is known to cause nitration of tyrosine resides and oxidation of methionine and cysteine residues. Therefore, we measured carbonyl formation and tyrosine nitration upon exposure to ProliNONOate. Here we show the addition of the peroxynitrite scavenging ferric porphyrin complex, Iron(III)-5,10,15,20-tetrakis94-sulonatophenyl) porphyrinato chloride (FeTPPS) to the samples immediately prior to the addition of proliNONOate reduces protein oxidation. The addition of FeTPPS reduced or eliminated protein carbonyl formation. Previously we reported the formation of 880 ± 480 mmol of carbonyls per mole of fibrinogen after the addition of ProliNONOate however, with the addition of FeTPPS no carbonyls were detected. Carbonyls detected on thrombin exposed to ProliNONOate were reduced from 390 ± 80 mmol/mol thrombin to 18 mmol/mole thrombin with the addition of FeTPPS. FeTPPS abrogated the changes to the rate of fibrin formation. When FeTPPS was added to fibrinogen before the addition of ProliNONOate there was no significate change in the rate of fiber formation. Without FeTPPS we previously reported a decreased rate of clot formation. Here we show the exposure of blood clot proteins fibrinogen and thrombin to NO donor ProliNONOate lead to peroxynitrite formation which caused the previously reported protein oxidation and altered fibrin fiber formation kinetics and thereby altered various fibrin clot properties.

Breads Fortified with Freeze-Dried Vegetables: Quality and Nutritional Attributes. Part II: Breads Not Containing Oil as an Ingredient.

The present article describes the second part of a study investigating the effect of adding vegetables on the nutritional, physico-chemical, and oxidative properties of wheat bread, and specifically focuses on bread that does not contain oil as an added ingredient. Wheat flour breads fortified with freeze-dried carrot, tomato, beetroot or broccoli were developed and assessed for their nutritional composition, antioxidant potential, oxidative stability, and storage properties. Using a simulated in vitro model, the study also examined the impact of vegetable addition on the oxidative stability of macronutrients during gastro-intestinal digestion. Adding vegetables improved the nutritional and functional attributes of the oil-free breads. However, they demonstrated a lower antioxidant potential compared to their oil-containing counterparts. Similarly, the textural and storage properties of the oil-free vegetable breads were poorer compared to the oil-containing breads. As expected, in the absence of oil the oil-free breads were associated with lower lipid oxidation both in their fresh form and during gastro-intestinal digestion. Adding vegetables reduced protein oxidation in the fresh oil-free breads but had no effect during gastro-intestinal digestion. The impact of vegetables on macronutrient oxidation in the oil-free breads during digestion appears to be vegetable-specific with broccoli exacerbating it and the others having no effect. Of the evaluated vegetables, beetroot showed the most promising nutritional and physico-chemical benefits when incorporated into bread that does not contain added oil.

Life-extending Dietary Restriction Reduces Oxidative Damage of Proteins in Grasshoppers but Does Not Alter Allocation of Ingested Nitrogen to Somatic Tissues.

Dietary restriction (DR) extends life span and reduces reproduction in most animals. The disposable soma hypothesis suggests that this longevity is the result of reduced investment in reproduction and increased nutrient allocation to the soma, permitting an increase in cellular maintenance. To investigate the role of nutrient allocation upon life-extending DR, tissue-specific nitrogen allocation was tracked in grasshoppers (Romalea microptera) upon a full or restricted (60% of full) diet. In addition, carbonyl (oxidized protein) assays addressed tissue maintenance. To develop a labeled diet on which grasshoppers could thrive, hydroponically grown Romaine lettuce was enriched with 15N. This allowed quantification of nitrogen allocation upon a normal or restricted diet. There was a 50% decrease in reproductive investment upon DR. At the same time, relative allocation of 15N to the ovary did not change. Most important, relative allocation was similar between restricted and full diet grasshoppers for somatic tissues (ie, mandibular and femur muscle, dried hemolymph, gut, and fat body). Carbonyl assays of muscles, hemolymph, and gut revealed an overall reduction in protein oxidation upon DR. These data suggest that DR does not alter nutrient allocation but does reduce protein oxidation, an observation that is inconsistent with the basic predictions of the disposable soma hypothesis.

Effect of liposome-encapsulated hemoglobin resuscitation on proteostasis in small intestinal epithelium after hemorrhagic shock.

Gut barrier dysfunction is the major trigger for multiorgan failure associated with hemorrhagic shock (HS). Although the molecular mediators responsible for this dysfunction are unclear, oxidative stress-induced disruption of proteostasis contributes to the gut pathology in HS. The objective of this study was to investigate whether resuscitation with nanoparticulate liposome-encapsulated hemoglobin (LEH) is able to restore the gut proteostatic mechanisms. Sprague-Dawley rats were recruited in four groups: control, HS, HS+LEH, and HS+saline. HS was induced by withdrawing 45% blood, and isovolemic LEH or saline was administered after 15 min of shock. The rats were euthanized at 6 h to collect plasma and ileum for measurement of the markers of oxidative stress, unfolded protein response (UPR), proteasome function, and autophagy. HS significantly increased the protein and lipid oxidation, trypsin-like proteasome activity, and plasma levels of IFNγ. These effects were prevented by LEH resuscitation. However, saline was not able to reduce protein oxidation and plasma IFNγ in hemorrhaged rats. Saline resuscitation also suppressed the markers of UPR and autophagy below the basal levels; the HS or LEH groups showed no effect on the UPR and autophagy. Histological analysis showed that LEH resuscitation significantly increased the villus height and thickness of the submucosal and muscularis layers compared with the HS and saline groups. Overall, the results showed that LEH resuscitation was effective in normalizing the indicators of proteostasis stress in ileal tissue. On the other hand, saline-resuscitated animals showed a decoupling of oxidative stress and cellular protective mechanisms.

Color attributes and oxidative stability of longissimus lumborum and psoas major muscles from Nellore bulls

The influence of muscle source on color stability of fresh beef from purebred Bos indicus cattle was investigated. Longissimus lumborum (LL) and psoas major (PM) muscles obtained from twelve (n=12) Nellore bull carcasses (24h post-mortem) were fabricated into 2.54-cm steaks, aerobically packaged, and stored at 4°C for nine days. Steaks were analyzed on day 0 for proximate composition and myoglobin concentration, whereas pH, instrumental color, metmyoglobin reducing activity (MRA), lipid oxidation, and protein oxidation were evaluated on days 0, 3, 6, and 9. LL steaks exhibited greater (P<0.05) redness, color stability, and MRA than PM counterparts. On the other hand, PM steaks demonstrated greater (P<0.05) myoglobin content, lipid oxidation, and protein oxidation than LL steaks. These results indicated the critical influence of muscle source on discoloration of fresh beef from Bos indicus animals and suggested the necessity to engineer muscle-specific strategies to improve color stability and marketability of beef from Bos indicus cattle.

Protein Oxidation as a Relevant Factor Determining Protein Status in Humans

INTRODUCTIONDefining optimal protein intake in various metabolic conditions like the newborn, aging and during exercise is highly relevant. Recently, it has been suggested that high protein intake in newborn could lead to excessive weight gain in later life (Kirchberg et al. 2014). Limited protein oxidation could be a relevant parameter directly related to this.OBJECTIVESTo monitor protein oxidation we developed a non‐invasive 13C‐protein‐derived breath test using naturally enriched milk protein (fractions). We validated and applied this technique, in a pilot study, under various metabolic conditions in healthy adult males.METHOD/DESIGNAfter baseline sampling, 30 g of naturally labeled 13C‐milk protein were consumed. Breath samples were taken every 10 min during 5,5 h and 13CO2 was measured by Isotope Ratio Mass Spectrometer. The following variables were used to calculate the amount of substrate oxidized: administered dose, 13C enrichment of substrate, molecular weight of substrate, number of carbon atoms in a substrate molecule, estimated CO2‐production of the subject based on body surface area.RESULTSPostprandial kinetics of oxidation of whey (rapidly digestible protein) and casein (slowly digestible protein) derived from our breath test were comparable to literature data regarding the kinetics of appearance of amino acids in blood (Boirie et al. 1997). Using this test we could demonstrate that 24 ± 3% (mean ± sd) of the milk protein dose was oxidized over 5,5 h which served as our control (100%). A decrease of 31% ± 18% in milk protein oxidation was observed after a 3 day protein restricted diet (~10 g/day) compared to a normal diet and 30 minutes of cycling before protein consumption reduced protein oxidation by 39% ± 37%. A protein restricted diet plus exercise reduced oxidation by 52% ± 19%.CONCLUSIONSProtein oxidation, which can be monitored in breath, is a significant factor in protein metabolism. With our technique we are able to characterize changes in overall protein oxidation under various metabolic conditions such as diet and exercise, which could be relevant for defining optimal protein intake under these conditions and can be extended to measurements during aging and in newborn.Support or Funding InformationThe project is financially supported by Hanze Nutrition BV

Biomarkers of Subclinical Atherosclerosis and Natural Products as Complementary Alternative Medicine.

Cardiovascular diseases (CVD) are considered the leading cause of morbidity and mortality from chronic diseases in the world. In addition, about 20% of first and recurrent acute myocardial infarctions (MI) are silent. In this context, subclinical atherosclerosis culminates in evident CVD, through the evolution of early risk factors such as hypercholesterolemia, hypertriglyceridemia and others. The main problem in CVD is related to the long-time between the start of the subclinical atherosclerosis and the manifestation of the disease. The identification of subjects at risk of such events is obviously substantial, since identification leads to implementation and compliance with effective preventive measures that reduce such risk. In this sense, this review demonstrates biomarkers as an alternative to early detection of subclinical atherosclerosis. One of the proposed biomarkers is the Ischemia-modified albumin (IMA), being considered a promising biochemical biomarker for atherosclerotic conditions. Another marker that is gaining strength and is associated with the IMA are the advanced oxidation protein products (AOPP), its measurement provides information on the level of exposure to potentially harmful changes to proteins and metabolic control. And last but not least we have nitric oxide as an early marker mainly related to endothelial dysfunction. In this review also is evidenced the use of the Campomanesia xanthocarpa, a plant native to southern region from Brazil extensively used as complementary and alternative medicine, and natural products to reduce protein oxidation and improve the availability of nitric oxide and consequently vascular function, reducing the risk for development of CVD.

Increased Mammalian Target of Rapamycin Signaling Contributes to the Accumulation of Protein Oxidative Damage in a Mouse Model of Down's Syndrome

Background: Neurodegenerative diseases are characterized by increased levels of oxidative stress and an altered mammalian target of rapamycin (mTOR)/autophagy axis; however, the mutual relationship between these two events is controversial. Previous studies in Down's syndrome (DS) and Alzheimer's disease (AD) suggested that the accumulation of protein oxidative damage results from the increased free radical production, mainly related to metabolic alterations, mitochondrial degeneration and amyloid-β deposition, and aberrant activity of protein degradative systems. Summary: This study analyzed mTOR signaling in Ts65Dn mice, a model of DS, at 6 and 12 months of age compared with euploid mice showing the early aberrant hyperphosphorylation of mTOR coupled with the reduction of autophagosome formation. Moreover, the evaluation of protein oxidation shows an increase in protein nitration and protein-bound 4-hydroxynonenal in 12-month-old Ts65Dn mice suggesting the potential involvement of altered autophagy in the buildup of protein oxidative damage. In addition, data obtained on cell culture support the protective role of autophagy in reducing protein oxidation. Key Messages: Overall, this study provides further evidence for the role of mTOR hyperactivation and reduced autophagy in the accumulation of protein oxidative damage during DS and AD pathologies.

Effects of environmental temperature and dietary manganese on egg production performance, egg quality, and some plasma biochemical traits of broiler breeders.

An experiment was conducted to investigate the effects of environmental temperature and dietary Mn on egg production performance, egg quality, and some plasma biochemical traits of broiler breeders. A completely randomized factorial design involved 2 environmental temperatures (a normal temperature, 21 ± 1°C, and a high temperature, 32 ± 1°C) × 3 dietary Mn treatments (a Mn-unsupplemented corn–soybean meal basal diet or the basal diet supplemented with 120 mg of Mn/kg of diet as either MnSO4·H2O or manganese proteinate). There were 6 treatments with 6 replicates (4 birds per replicate). High temperature decreased egg weight (P < 0.0001), laying rate (P < 0.0001), egg yield (P < 0.0001), feed intake (P < 0.0001), egg:feed ratio (P < 0.0001), eggshell strength (P < 0.05) and thickness (P < 0.0001), plasma triiodothyronine level (P < 0.05), and alkaline phosphatase activity (P < 0.04) whereas it increased rectal temperature (P < 0.0001); plasma malondialdehyde level (P < 0.02); and activities (P < 0.002) of lactic dehydrogenase, aspartate aminotransferase, and creatine kinase. Broiler breeders fed the diets supplemented with Mn regardless of source had greater (P < 0.05) eggshell strength and lower (P ≤ 0.05) plasma triiodothyronine level and protein carbonyl content than those fed the control diet. The broiler breeders fed the diet supplemented with the organic Mn had greater (P < 0.01) eggshell thickness than those fed the control diet. There were interactions (P < 0.05) between environmental temperature and dietary Mn in laying rate, egg yield, feed intake, and egg:feed ratio. Under normal temperature, dietary Mn did not affect the above 4 parameters; however, under high temperature, broiler breeders fed the diet supplemented with the organic Mn showed greater (P < 0.03) improvements in these 4 parameters than those fed the control diet. The results from this study indicated that high temperature significantly impaired egg production performance and eggshell quality and induced lipid peroxidation and tissue damage whereas dietary supplementation of either organic or inorganic Mn improved eggshell strength and thermotolerance and reduced protein oxidation and that the organic Mn could alleviate the negative effect of high temperature on egg production performance of broiler breeders at the period of 32 to 45 wk of age.

Reducing protein oxidation in low-flow electrospray enables deeper investigation of proteoforms by top down proteomics

Enabling the implementation of top down proteomic techniques within clinical workflows requires a dramatic increase in sensitivity. It has been previously demonstrated that electrospray ionization (ESI) becomes more efficient with decreasing volumetric flow rates at the emitter. Therefore, narrow inner diameter (I.D.) columns used in front-end chromatographic separations yield increased sensitivity. However, the smaller cross-sectional area of a narrow I.D. column places a larger fraction of the eluent in fluid communication with the electrode within the high voltage union that facilitates electrospray ionization (ESI), leading to increased oxidation of solution-phase proteins. Oxidation of proteins alters their chemical state of the protein, complicates data analysis, and reduces the depth of proteome coverage attained in a typical top-down proteomics experiment. Excessive protein oxidation results in poor deconvolution and exact mass calculations from MS1 spectra, interferes with peak isolation for MS/MS fragmentation, and effectively reduces sensitivity by splitting ion current. All of these factors deteriorate top down mass spectral data quality, an effect that becomes more pronounced as column diameter decreases. Artificial protein oxidation can also mislead investigations of in vivo protein oxidation. All of these effects are accentuated in comparison to bottom up proteomics due to the increased probability of having oxidizable residues within a particular species with increasing mass. Herein, we describe a configuration (which we term “Low Protein Oxidation (LPOx)”) for proteomics experiments created by re-arranging liquid chromatography (LC) plumbing and present its application to artificial protein oxidation and show a marked improvement in detection sensitivity. Using a standard mixture of five intact proteins, we demonstrate that the LPOx configuration reduces protein oxidation up to 90% using 50μm I.D. columns when compared to a conventional LC plumbing configuration with 50μm I.D. column. As a proof-of-concept study, at least 11 distinct proteoforms of serum Apolipoprotein A1 were detected with the LPOx configuration. This innovative LC configuration can be applied to the top down identification and characterization of proteoforms obscured by abundant artificial protein oxidation at low flowrates, all while using reduced amounts of valuable protein samples.