Protein oxidation and in vitro digestibility of heat-treated fermented sausages: How do they change with the effect of lipid formulation during processing?

In the present work, it was aimed to evaluate the oxidative stress indicators and in vitro digestibility during the processing of heat-treated Turkish sausages (sucuk) manufactured using different lipid formulations. The utilization of olive oil in sausage formulations had considerable impacts on proximate composition, pH, and water activity. The increased olive oil content increased primary lipid oxidation products, whereas it decreased the secondary ones. The use of olive oil increased the total carbonyl content, while it decreased the α-aminoadipic semialdehyde concentration. In general, pepsin, trypsin, and α-chymotrypsin activities of the treatments were similar to each other. The heat treatment during the processing significantly increased most of the oxidation markers. Though strong correlations were recorded between specific oxidation markers, no relationship was detected between oxidation parameters and in vitro digestibility. The results indicated that the lipid formulation and processing operations had significant impact on chemical and functional properties of heat-treated fermented sausages, within the complex interrelationships between oxidation mechanisms. PRACTICAL APPLICATIONS: The present work pointed out the changes and correlations between specific oxidation markers and in vitro digestibility of heat-treated fermented sausages during the production procedure. Oxidation reactions that occur in both proteins and lipids could have drastic impact on overall quality; for this reason, it is of great importance to provide the data for lightening these impacts regarding the product types and production applications. Since heat-treated muscle foods are widely manufactured to meet industrial needs, the data obtained from this research would contribute to understand the effects of formulation and processing operations in the formation of oxidation products and change in digestibility, thereby to pioneer further research on this topic.

Intellectual Disability Associated With Pyridoxine-Responsive Epilepsies: The Need to Protect Cognitive Development.

Pyridoxine (vitamin B6)-responsive epilepsies are severe forms of epilepsy that manifest as seizures immediately after birth, sometimes in utero, sometimes months, or years after birth. Seizures may be treated efficiently by life-long supplementation with pyridoxine or its biologically active form, pyridoxal phosphate, but even so patients may become intellectually disabled, for which there currently is no effective treatment. The condition may be caused by mutations in several genes (TNSALP, PIGV, PIGL, PIGO, PNPO, PROSC, ALDH7A1, MOCS2, or ALDH4A1). Mutations in ALDH7A1, MOCS2, and ALDH4A1 entail build-up of reactive aldehydes (α-aminoadipic semialdehyde, γ-glutamic semialdehyde) that may react non-enzymatically with macromolecules of brain cells. Such reactions may alter the function of macromolecules, and they may produce "advanced glycation end products" (AGEs). AGEs trigger inflammation in the brain. This understanding points to aldehyde-quenching, anti-AGE, or anti-inflammatory therapies as possible strategies to protect cognitive development and prevent intellectual disability in affected children. Studies on how aldehydes traverse cell membranes and how they affect brain function could further the development of therapies for patients with pyridoxine-responsive epilepsies.

Analysis of Protein Oxidative Modifications in Follicular Fluid from Fertile Women: Natural Versus Stimulated Cycles.

Oxidative stress is associated with obstetric complications during ovarian hyperstimulation in women undergoing in vitro fertilization. The follicular fluid contains high levels of proteins, which are the main targets of free radicals. The aim of this work was to determine specific biomarkers of non-enzymatic oxidative modifications of proteins from follicular fluid in vivo, and the effect of ovarian stimulation with gonadotropins on these biomarkers. For this purpose, 27 fertile women underwent both a natural and a stimulated cycle. The biomarkers, glutamic semialdehyde (GSA), aminoadipic semialdehyde (AASA), Nε-(carboxymethyl)lysine (CML), and Nε-(carboxyethyl)lysine (CEL), were measured by gas-liquid chromatography coupled to mass spectrometry. Results showed that follicular fluid contained products of protein modifications by direct metal-catalyzed oxidation (GSA and AASA), glycoxidation (CML and CEL), and lipoxidation (CML). GSA was the most abundant biomarker (91.5%). The levels of CML amounted to 6% of the total lesions and were higher than AASA (1.3%) and CEL (1.2%). In the natural cycle, CEL was significantly lower (p < 0.05) than in the stimulated cycle, suggesting that natural cycles are more protected against protein glycoxidation. These findings are the basis for further research to elucidate the possible relevance of this follicular biomarker of advanced glycation end product in fertility programs.

Effect of sodium ascorbate and sodium nitrite on protein and lipid oxidation in dry fermented sausages.

The effects of sodium nitrite and ascorbate on lipid and protein oxidation were studied during the ripening process of dry fermented sausages. Samples were taken at day 0, 2, 8, 14, 21 and 28 of ripening to assess lipid (malondialdehyde) and protein (carbonyls and sulfhydryl groups) oxidation. Sodium ascorbate and nitrite were separately able to reduce the formation of malondialdehyde. Their combined addition resulted in higher amounts of carbonyl compounds compared to their separate addition or the treatment without any of both compounds. Moreover, sodium nitrite limited the formation of γ-glutamic semialdehyde whereas sodium ascorbate showed a pro-oxidant effect. A loss of thiol groups was observed during ripening, which was not affected by the use of sodium ascorbate nor sodium nitrite. In conclusion, sodium nitrite and ascorbate affected protein and lipid oxidation in different manners. The possible pro-oxidant effect of their combined addition on carbonyl formation might influence the technological and sensory properties of these products.

Apple phenolics as inhibitors of the carbonylation pathway during in vitro metal-catalyzed oxidation of myofibrillar proteins.

The effect of apple phenolics on the oxidative damage caused to myofibrillar proteins by an in vitro metal-catalyzed oxidation system was investigated. Three pure phenolic compounds (chlorogenic acid, (-)-epicatechin and phloridzin) and an apple peel extract were added to myofibrillar proteins in three concentrations (50, 100 and 200µM), and a blank treatment was included as a control. All suspensions were subjected to Fe(3+)/H2O2 oxidation at 37°C during 10days, and protein oxidation was evaluated as carbonylation (α-amino adipic and γ-glutamic semialdehydes) and Schiff base cross-links. Significant inhibition by apple phenolics was found as compared to the control treatment, with (-)-epicatechin being the most efficient antioxidant and phloridzin showing the weakest antioxidant effect. The higher concentrations of apple extract showed effective antioxidant activity against protein oxidation in myofibrillar proteins, emphasizing the potential of apple by-products as natural inhibitors of protein oxidation in meat products.

Protein oxidation and proteolysis during storage and in vitro digestion of pork and beef patties.

The effect of protein oxidation on proteolysis during meat digestion was investigated following storage and subsequent in vitro digestion of beef and pork patties. Protein oxidation was evaluated as thiol oxidation, total carbonylation, and specific carbonylation (α-amino adipic and γ-glutamic semialdehyde). Furthermore, 4-hydroxyphenylalanine, a hydroxylation product of phenylalanine, was identified and quantified as a new protein oxidation marker. After 7days of chilled illuminated storage (4°C), significant oxidative modifications were quantified and the oxidative degradation was continued during in vitro digestion. The observed effects were more abundant in beef patties. Protein oxidation before digestion resulted in impaired proteolysis during digestion.

Sources of superoxide/H2O2 during mitochondrial proline oxidation.

p53 Inducible gene 6 (PIG6) encodes mitochondrial proline dehydrogenase (PRODH) and is up-regulated several fold upon p53 activation. Proline dehydrogenase is proposed to generate radicals that contribute to cancer cell apoptosis. However, there are at least 10 mitochondrial sites that can produce superoxide and/or H2O2, and it is unclear whether proline dehydrogenase generates these species directly, or instead drives production by other sites. Amongst six cancer cell lines, ZR75-30 human breast cancer cells had the highest basal proline dehydrogenase levels, and mitochondria isolated from ZR75-30 cells consumed oxygen and produced H2O2 with proline as sole substrate. Insects use proline oxidation to fuel flight, and mitochondria isolated from Drosophila melanogaster were even more active with proline as sole substrate than ZR75-30 mitochondria. Using mitochondria from these two models we identified the sites involved in formation of superoxide/H2O2 during proline oxidation. In mitochondria from Drosophila the main sites were respiratory complexes I and II. In mitochondria from ZR75-30 breast cancer cells the main sites were complex I and the oxoglutarate dehydrogenase complex. Even with combinations of substrates and respiratory chain inhibitors designed to minimize the contributions of other sites and maximize any superoxide/H2O2 production from proline dehydrogenase itself, there was no significant direct contribution of proline dehydrogenase to the observed H2O2 production. Thus proline oxidation by proline dehydrogenase drives superoxide/H2O2 production, but it does so mainly or exclusively by providing anaplerotic carbon for other mitochondrial dehydrogenases and not by producing superoxide/H2O2 directly.

Detection of oxidized and glycated proteins in clinical samples using mass spectrometry--a user's perspective.

BACKGROUND: Proteins in human tissues and body fluids continually undergo spontaneous oxidation and glycation reactions forming low levels of oxidation and glycation adduct residues. Proteolysis of oxidised and glycated proteins releases oxidised and glycated amino acids which, if they cannot be repaired, are excreted in urine. SCOPE OF REVIEW: In this review we give a brief background to the classification, formation and processing of oxidised and glycated proteins in the clinical setting. We then describe the application of stable isotopic dilution analysis liquid chromatography-tandem mass spectrometry (LC-MS/MS) for measurement of oxidative and glycation damage to proteins in clinical studies, sources of error in pre-analytic processing, corroboration with other techniques - including how this may be improved - and a systems approach to protein damage analysis for improved surety of analyte estimations. MAJOR CONCLUSIONS: Stable isotopic dilution analysis LC-MS/MS provides a robust reference method for measurement of protein oxidation and glycation adducts. Optimised pre-analytic processing of samples and LC-MS/MS analysis procedures are required to achieve this. GENERAL SIGNIFICANCE: Quantitative measurement of protein oxidation and glycation adducts provides information on level of exposure to potentially damaging protein modifications, protein inactivation in ageing and disease, metabolic control, protein turnover, renal function and other aspects of body function. Reliable and clinically assessable analysis is required for translation of measurement to clinical diagnostic use. Stable isotopic dilution analysis LC-MS/MS provides a "gold standard" approach and reference methodology to which other higher throughput methods such as immunoassay and indirect methods are preferably corroborated by researchers and those commercialising diagnostic kits and reagents. This article is part of a Special Issue entitled Current methods to study reactive oxygen species - pros and cons and biophysics of membrane proteins. Guest Editor: Christine Winterbourn.

Protein and lipid oxidative damage and complex I content are lower in the brain of budgerigar and canaries than in mice. Relation to aging rate.

What are the mechanisms determining the rate of animal aging? Of the two major classes of endothermic animals, bird species are strikingly long-lived compared to mammals of similar body size and metabolic rate. Thus, they are ideal models to identify longevity-related characteristics not linked to body size or low metabolic rates. Since oxidative stress seems to be related to the basic aging process, we measured specific markers of different kinds of oxidative damage to proteins, like glutamic and aminoadipic semialdehydes (GSA and AASA, specific protein carbonyls), Nɛ-(carboxyethyl)lysine (CEL), Nɛ-(carboxymethyl)lysine (CML), and Nɛ-(malondialdehyde)lysine (MDAL), as well as mitochondrial Complex I content and amino acid and membrane fatty acyl composition, in the brain of short-lived mice (maximum life span [MLSP] 3.5 years) compared with those of long-lived budgerigar 'parakeets' (MLSP, 21 years) and canaries (MLSP, 24 years). The brains of both bird species had significantly lower levels of compounds formed as a result of oxidative (GSA and AASA), glycoxidative (CEL and CML), and lipoxidative (CML and MDAL) protein modifications, as well as a lower levels of mitochondrial complex I protein. Although it is known that fatty acid unsaturation is lower in many tissues of long-lived compared to short-lived mammals, this is not true in the particular case of brain. In agreement with this, we also found that the brain tissue of bugerigars and canaries contains no fewer double bonds than that of mice. Amino acid composition analyses revealed that bird proteins have a significantly lower content of His, Leu and Phe, as well as, interestingly, of methionine, whereas Asp, Glu, Ala, Val, and Lys contents were higher than in the mammals. These results, together with those previously described in other tissues of pigeons (MLSP, 35 years) compared to rats (MLSP, 4 years), indicate that oxidative damage to proteins, lipids and mitochondrial DNA are lower in birds (very long-lived species) than in short-lived mammals of similar body size. The lower degree of oxidative modification of bird brain proteins was not due to decreases in the target amino acids (lysine for CEL, CML, MDAL, and AASA; and arg and pro for GSA), since these were present in bird brain proteins at higher or similar levels than in those of mice. These results are consistent with the possibility that decreases in oxidative protein modification are caused at least in part by the low rate of mitochondrial oxygen radical generation in these birds, as in all long-lived homeothermic vertebrates investigated so far.