Effect of fiber-bound polyphenols from highland barley on lipid oxidation products of cooked pork during in vitro gastrointestinal digestion.

BACKGROUND: The gastrointestinal (GI) tract is a major site of lipid oxidation, and the lipid oxidation products are related to an increased risk of various chronic diseases. In this study, the inhibition capacity of bound-polyphenol rich insoluble dietary fiber (BP-IDF) from highland barley (HB) to lipid oxidation was evaluated during simulated GI digestion. RESULTS: We found that the level of lipid hydroperoxides (LOOH) and aldehydes were significantly inhibited when highland barley bound-polyphenol rich insoluble dietary fiber (HBBP-IDF) co-digestion with cooked pork. The lipid oxidation products were more effectively scavenged during simulated gastric digestion, with inhibition of 77.4% for LOOH, 52.3% for malondialdehyde, 46.5% for 4-hydroxy-2-hexenal and 48.7% for 4-hydroxy-2-nonenel, respectively. The fiber-bound polyphenols are the principal scavengers of lipid oxidation products. CONCLUSION: These findings suggest that HBBP-IDF could be used as a functional ingredient able to scavenge lipid oxidation products across the GI tract. © 2023 Society of Chemical Industry.

4-hydroxy-2-alkenals in foods: a review on risk assessment, analytical methods, formation, occurrence, mitigation and future challenges.

Undoubtedly, significant advances were performed concerning 4-hydroxy-2-alkenals research on foods, and their formation by double oxidation of polyunsaturated fatty acids. But further studies are still needed, especially on their occurrence in foods enriched with n-3 and n-6 fatty acids, as well as in foods for infants and processed foods. Major factors concerning the formation of 4-hydroxy-2-alkenals were discussed, namely the influence of fatty acids composition, time/temperature, processing conditions, salt, among others. Regarding mitigation, the most effective strategies are adding phenolic extracts to foods matrices, as well as other antioxidants, such as vitamin E. Exposure assessment studies revealed 4-hydroxy-2-alkenals values that could not be considered a risk for human health. However, these toxic compounds remain unaltered after digestion and can easily reach the systemic circulation. Therefore, it is crucial to develop in vivo research, with the inclusion of the colon phase, as well as, cell membranes of the intestinal epithelium. In conclusion, according to our review it is possible to eliminate or effectively decrease 4-hydroxy-2-alkenals in foods using simple and economic practices.

Resveratrol attenuates 4-hydroxy-2-hexenal-induced oxidative stress in mouse cortical collecting duct cells.

Resveratrol (RSV) may provide numerous protective eff ects against chronic inflammatory diseases. Due to local hypoxia and hypertonicity, the renal medulla is subject to extreme oxidative stress, and aldehyde products formed during lipid peroxidation, such as 4-hydroxy-2-hexenal (HHE), might be responsible for tubular injury. This study aimed at investigating the eff ects of RSV on renal and its signaling mechanisms. While HHE treatment resulted in decreased expression of Sirt1, AQP2, and nuclear factor erythroid 2-related factor 2 (Nrf2), mouse cortical collecting duct cells (M1) cells treated with HHE exhibited increased activation of p38 MAPK, extracellular signal regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and increased expression of NOX4, p47(phox), Kelch ECH associating protein 1 (Keap1) and COX2. HHE treatment also induced NF-κB activation by promoting IκB-α degradation. Meanwhile, the observed increases in nuclear NF-κB, NOX4, p47(phox), and COX2 expression were attenuated by treatment with Bay 117082, N-acetyl-l-cysteine (NAC), or RSV. Our findings indicate that RSV inhibits the expression of inflammatory proteins and the production of reactive oxygen species in M1 cells by inhibiting NF-κB activation.

Effect of drying processes on the occurrence of lipid oxidation-derived 4-hydroxy-2-hexenal and 4-hydroxy-2-nonenal in Spanish mackerel (Scomberomorus niphonius).

In this study, dry-cured Spanish mackerel (Scomberomorus niphonius, DCSM) was prepared via three different methods (hot-air drying, cold-air drying, and sun drying). The content of 4-hydroxy-2-hexenal (HHE) and 4-hydroxy-2-nonenal (HNE) derived from lipid oxidation in whole processes was investigated by HPLC-MS/MS. The changes in fatty acid composition were detected by GC-MS, and the degree of lipid oxidation was evaluated by the levels of acid values (AV), peroxide values (POV), and thiobarbituric acid-reactive substances (TBARS). The results showed that the drying process significantly accelerated lipid oxidation in DCSM. The contents of HHE and HNE were significantly increased after processing. The content of HHE was higher by 18.44-, 13.45-, and 16.32-folds compared with that of HNE after three different processes, respectively. The HHE and HNE contents fluctuated upward during the hot-air and cold-air drying process. However, the contents of HHE and HNE increased time-dependent during the sun drying process, with the highest values of 86.33 ± 10.54 and 5.29 ± 0.54 mg/kg fish among the three different processes. Besides, there was a significant positive correlation between HHE contents and n-3 fatty acids content in hot-air drying and sun drying processes (Pearson's r = .991/.996), and HNE occurrence was closely related to n-6 fatty acid content in sun drying process (Pearson's r = .989). Regression analysis indicated that the content of HHE and TOTOXTBA values in DCSM showed good linear relationships (R 2 value = .907), which suggested that the content of HHE could be used to estimate the oxidative deterioration of dry-cured fish products.

Fast and sensitive UHPLC-QqQ-MS/MS method for simultaneous determination of typical α,ß-unsaturated aldehydes and malondialdehyde in various vegetable oils and oil-based foods.

This study aimed to develop and evaluate a method for simultaneously analyzing malondialdehyde (MDA) and two typical toxic α,ß-unsaturated aldehydes, 4-hydroxy-2-hexenal (HHE), and 4-hydroxy-2-nonenal (HNE), using ultra-high-performance liquid chromatography with triple quadrupole mass spectrometry (UHPLC-QqQ-MS/MS). The method possessed widely linear range (10-1000 ng/mL). The limit of detection (LOD) and limit of quantitation (LOQ) of MDA, HHE, and HNE were 2.0 and 5.0 ng/g, 2.0 and 5.0 ng/g, and 0.1 and 0.3 ng/g, respectively; the recovery rates all fall into 95.56-104.22 %. The method was sufficiently precise (<5%), and not affected by the analysis matrix. Application to 17 food products revealed total MDA, HHE, and HNE contents were 0.11-3.56, 0.05-3.32, and 0.09-3.70 µg/g, respectively. It will be useful in future research on the influence of food composition and main substrate structure on the generation and distribution of these three aldehydes and the implementation of corresponding control methods.

Pro-oxidative activity of trout and bovine hemoglobin during digestion using a static in vitro gastrointestinal model.

The degradation of trout and bovine hemoglobin (Hb) and their pro-oxidant activities in washed cod muscle mince (WCM) were studied using simple pH-shifts to simulate gastrointestinal (GI) conditions (pH 7 â†’ 6 â†’ 3 â†’ 7), as well as full static in vitro GI digestion. Following gastric acidification to pH 6, metHb formation increased, especially for trout Hb. Subsequent acidification to pH 3 promoted Hb unfolding and partial or complete heme group-loss. During full GI digestion, polypeptide/peptide analyses revealed more extensive Hb-degradation in the gastric than duodenal phase, without any species-differences. When digesting WCM +/-Hb, both Hbs strongly promoted malondialdehyde (MDA), 4-hydroxy-2-hexenal (HHE), and 4-hydroxy-2-nonenal (HNE) formation, peaking at the end of the gastric phase. Trout-Hb stimulated MDA and HHE more than bovine Hb in the first gastric phase. Altogether, partially degraded Hb, and/or free hemin -both mammal and fish-derived- stimulated oxidation of PUFA-rich lipids under GI-conditions, especially gastric ones.

The Influence of Butter and Oils on Oxidative Reactions during In Vitro Gastrointestinal Digestion of Meat and Fish.

Oxidative reactions during cooking and gastrointestinal digestion of meat and fish lead to the formation of various lipid- and protein oxidation products, some of which are toxic. In the present study, it was investigated how the addition of 3% butter or oils affect lipid- and protein oxidation during cooking and in vitro digestion of meat (chicken thigh, chicken breast, beef) and fish (mackerel, cod). These muscle foods were selected based on their differences in heme-Fe and PUFA contents, and n-6/n-3 PUFA ratio, and therefore varying potential to form oxidation products during digestion. Without additional fat, mackerel digests displayed the highest n-3 PUFA oxidation (4-hydroxy-2-hexenal, propanal, thiobarbituric reactive acid substances), and chicken digests the highest n-6 PUFA oxidation (4-hydroxy-2-nonenal, hexanal), whereas both lipid- and protein oxidation (protein carbonyl compounds) were low in cod and beef digests. Lipid oxidative reactions were generally not altered by the addition of butter to any muscle matrix, whereas the addition of fish oil and safflower oil in different ratios (3:0, 2:1, 1:2, 0:3) as n-3 PUFA and n-6 PUFA source respectively, stimulated oxidative reactions, especially during digestion of beef. Since beef was considered the muscle matrix with the highest potential to stimulate oxidation in the added fat substrate, in a second experiment, beef was cooked and digested with 3% butter or seven commercial vegetable oils (sunflower-, maize-, peanut-, rapeseed-, olive-, rice bran- or coconut oil), all labeled 'suitable for heating'. No relevant oxidative reactions were however observed during digestion of beef with any of these commercial vegetable oils.

The effect of heating on the formation of 4-hydroxy-2-hexenal and 4-hydroxy-2-nonenal in unsaturated vegetable oils: Evaluation of oxidation indicators.

The formation of 4-hydroxy-2-hexenal (HHE) and 4-hydroxy-2-nonenal (HNE) in vegetable oils and model oil systems were quantitatively assessed by RP-HPLC. Regardless of heating temperature, HHE was only detected in rapeseed and linseed oil, while HNE was detected in all tested oils. Intrinsic tocopherols suppressed HHE/HNE formation, but with similar inhibition rates (10.49-16.04%). Linear correlations were observed between HHE/HNE contents and corresponding n-3/6 fatty acid content in oils (R2 = 0.989/0.971). Model oil system revealed that HHE/HNE formation was closely related to methyl linolenate (MLN) and methyl linoleate (ML) contents. Low levels of ML (<0.5%) and MLN (<1.0%) did not lead to HHE/HNE formation. Therefore, HHE/HNE was classified as the characteristic aldehydes of n-3 and n-6 type oils, respectively. Heat map evaluation and regression analysis indicated HHE could predict the n-3 type oils oxidation, while HNE was a good indicator to estimate the oxidative deterioration of n-6 and n-9 type oils.

Long-Chain n-3 PUFA Content and n-6/n-3 PUFA Ratio in Mammal, Poultry, and Fish Muscles Largely Explain Differential Protein and Lipid Oxidation Profiles Following In Vitro Gastrointestinal Digestion.

SCOPE: Muscle food characteristics (fatty acid profile, heme-Fe, intrinsic antioxidants) that relate to the formation of (patho)physiological oxidation products during gastrointestinal digestion are investigated. METHODS AND RESULTS: Muscles (n = 33) from 18 mammal, poultry, and fish species, of which some are mixed with lard to standardize their fatty acid profile, are digested in vitro. Lipid oxidation is assessed by thiobarbituric reactive substances (TBARS), n-3 PUFA derivative 4-hydroxy-2-hexenal and propanal, n-6 PUFA derivative 4-hydroxy-2-nonenal and hexanal, and protein oxidation by carbonylation. Digests of n-3 PUFA-rich fish demonstrated the highest n-3 PUFA oxidation, whereas digests of various poultry and rabbit muscles showed highest n-6 PUFA oxidation, which correlated significantly with the n-6/n-3 PUFA ratio. Without lard addition, lipid oxidation is significantly higher in chicken and pork loin digests versus beef and deer digests, whereas the opposite occurred when these muscles are mixed with lard. Protein carbonylation correlates significantly with levels of TBARS and the sum of hydroxy-alkenals in digests. The n-6/n-3 PUFA ratio correlates well with the 4-hydroxy-2-nonenal/4-hydroxy-2-hexenal ratio in digests. CONCLUSIONS: Muscular fatty acid profiles largely explain type and extent of lipid and protein oxidation during gastrointestinal digestion. Red meat only stimulates oxidation when digested with specific fat sources.

Short-chain lipid peroxidation products form covalent adducts with pyruvate kinase and inhibit its activity in vitro and in breast cancer cells.

Pyruvate kinase catalyses the last step in glycolysis and has been suggested to contribute to the regulation of aerobic glycolysis in cancer cells. It can be inhibited by oxidation of cysteine residues in vitro and in vivo, which is relevant to the more pro-oxidant state in cancer and proliferating tissues. These conditions also favour lipid peroxidation and the formation of electrophilic fragmentation products, including short-chain aldehydes that can covalently modify proteins. However, as yet few studies have investigated their interactions with pyruvate kinase, so we investigated the effects of three different aldehydes, acrolein, malondialdehyde and 4-hydroxy-2(E)-hexenal (HHE), on the structure and activity of the enzyme. Analysis by LC-MS/MS showed unique modification profiles for each aldehyde, but Cys152, Cys423 and Cys474 were the residues most susceptible to electrophilic modification. Analysis of enzymatic activity under these conditions showed that acrolein was the strongest inhibitor, and at incubation times longer than 2 h, pathophysiological concentrations induced significant effects. Treatment of MCF-7 cells with the aldehydes caused similar losses of pyruvate kinase activity to those observed in vitro, and at lower concentrations than those required to cause cell death, with time and dose-dependent effects; acrolein adducts on Cys152 and Cys358 were detected. Cys358 and Cys474 are located at or near the allosteric or active sites, and formation of adducts on these residues probably contributes to loss of activity at low treatment concentrations. This study provides the first detailed analysis of the structure-activity relationship of C3 and C6 aldehydes with pyruvate kinase, and suggests that reactive short-chain aldehydes generated in diseases with an oxidative aetiology or from environmental exposure such as smoking could be involved in the metabolic alterations observed in cancer cells, through alteration of pyruvate kinase activity.

Effect of milk proteins and food-grade surfactants on oxidation of linseed oil-in-water emulsions during in vitro digestion.

Health benefits are associated with polyunsaturated fatty acids, but their sensitivity to oxidation may generate toxic oxidation species. The objective of this study was to compare the effect of milk proteins (casein, whey protein) and surfactants (Citrem, Tween 20) on the in vitro digestion and oxidation of linseed oil emulsions. The emulsion produced with Tween 20 resisted coalescence in the gastric phase and showed the highest concentrations of free fatty acids and reactive carbonyl compounds in the intestinal digestion phase. The Citrem-stabilized emulsion showed extensive coalescence in the gastric environment, which reduced lipolysis and the formation of advanced oxidation species. The protein-stabilized emulsions showed aggregation with some coalescence in the gastric phase, and casein provided better protection than whey protein against oxidation. This study suggests that the mechanism of emulsion destabilization in the gastric environment and the type of protein can modulate lipolysis and oxidation during in vitro digestion.

Simultaneous Analysis of Malondialdehyde, 4-Hydroxy-2-hexenal, and 4-Hydroxy-2-nonenal in Vegetable Oil by Reversed-Phase High-Performance Liquid Chromatography.

A group of toxic aldehydes such as, malondialdehyde (MDA), 4-hydroxy-2-hexenal (HHE), and 4-hydroxy-2-nonenal (HNE) have been found in various vegetable oils and oil-based foods. Then simultaneous determination of them holds a great need in both the oil chemistry field and food field. In the present study, a simple and efficient analytical method was successfully developed for the simultaneous separation and detection of MDA, HHE, and HNE in vegetable oils by reversed-phase-high-performance liquid chromatography (RP-HPLC) coupled with photodiode array detector (PAD) at dual-channel detection mode. The effect of various experimental factors on the extraction performance, such as coextraction solvent system, butylated hydroxytoluene addition, and trichloroacetic acid addition were systematically investigated. Results showed that the linear ranges were 0.02-10.00 µg/mL for MDA, 0.02-4.00 µg/mL for HHE, and 0.03-4.00 µg/mL for HNE with the satisfactory correlation coefficient of >0.999 for all detected aldehydes. The limit of detection (LOD) and limit of quantification (LOQ) of MDA, HHE, and HNE were ∼0.021and 0.020 µg/mL, ∼0.009 and 0.020 µg/mL, and ∼0.014 and 0.030 µg/mL, respectively. Their recoveries were 99.64-102.18%, 102.34-104.61%, and 98.87-103.04% for rapeseed oil and 96.38-98.05%, 96.19-101.34%, and 96.86-99.04% for French fries, separately. Under the selected conditions, the developed methods was successfully applied to the simultaneous determination of MDA, HHE, and HNE in different tested vegetable oils. The results indicated that this method could be employed for the quality assessment of vegetable oils.

Chemistry and analysis of HNE and other prominent carbonyl-containing lipid oxidation compounds.

The process of lipid oxidation generates a diverse array of small aldehydes and carbonyl-containing compounds, which may occur in free form or esterified within phospholipids and cholesterol esters. These aldehydes mostly result from fragmentation of fatty acyl chains following radical oxidation, and the products can be subdivided into alkanals, alkenals (usually α,ß-unsaturated), γ-substituted alkenals and bis-aldehydes. Isolevuglandins are non-fragmented di-carbonyl compounds derived from H2-isoprostanes, and oxidation of the ω-3-fatty acid docosahexenoic acid yield analogous 22 carbon neuroketals. Non-radical oxidation by hypochlorous acid can generate α-chlorofatty aldehydes from plasmenyl phospholipids. Most of these compounds are reactive and have generally been considered as toxic products of a deleterious process. The reactivity is especially high for the α,ß-unsaturated alkenals, such as acrolein and crotonaldehyde, and for γ-substituted alkenals, of which 4-hydroxy-2-nonenal and 4-oxo-2-nonenal are best known. Nevertheless, in recent years several previously neglected aldehydes have been investigated and also found to have significant reactivity and biological effects; notable examples are 4-hydroxy-2-hexenal and 4-hydroxy-dodecadienal. This has led to substantial interest in the biological effects of all of these lipid oxidation products and their roles in disease, including proposals that HNE is a second messenger or signalling molecule. However, it is becoming clear that many of the effects elicited by these compounds relate to their propensity for forming adducts with nucleophilic groups on proteins, DNA and specific phospholipids. This emphasizes the need for good analytical methods, not just for free lipid oxidation products but also for the resulting adducts with biomolecules. The most informative methods are those utilizing HPLC separations and mass spectrometry, although analysis of the wide variety of possible adducts is very challenging. Nevertheless, evidence for the occurrence of lipid-derived aldehyde adducts in biological and clinical samples is building, and offers an exciting area of future research.

Relationship between 4-hydroxy-2-hexenal contents and commercial grade by organoleptic judgement in Japanese dried laver Porphyra spp.

To evaluate the correlation between the commercial grade determined by organoleptic judgment panel and chemical substances in dried laver Porphyra spp., we analyzed the contents of free amino acids, 5'-nucleotides, total lipids, fatty acids, α-tocopherol, lipophilic pigments, and aldehydes in several grades of laver that had been classified by an organoleptic judgment panel. Compared with the lower-grade laver samples, the excellent-grade laver samples contained higher concentrations of free amino acids, 5'-nucleotides, total lipids, α-tocopherol, chlorophyll a, and ß-carotene and lower concentrations of aldehydes such as 4-hydroxy-2-hexenal (HHE), propanal, butanal, and 1-hexanal, which are formed during lipid peroxidation of n-3 or n-6 polyunsaturated fatty acids. In addition, the HHE content was strongly correlated with the propanal content in the analyzed laver (r(2)=0.9123). These results showed that the commercial grade assigned by an organoleptic judgment panel was correlated with chemical substances associated with color, taste, and the prevention of lipid oxidation.

Lipoxidation adducts with peptides and proteins: deleterious modifications or signaling mechanisms?

Protein lipoxidation refers to the modification by electrophilic lipid oxidation products to form covalent adducts, which for many years has been considered as a deleterious consequence of oxidative stress. Oxidized lipids or phospholipids containing carbonyl moieties react readily with lysine to form Schiff bases; alternatively, oxidation products containing α,ß-unsaturated moieties are susceptible to nucleophilic attack by cysteine, histidine or lysine residues to yield Michael adducts, overall corresponding to a large number of possible protein adducts. The most common detection methods for lipoxidized proteins take advantage of the presence of reactive carbonyl groups to add labels, or use antibodies. These methods have limitations in terms of specificity and identification of the modification site. The latter question is satisfactorily addressed by mass spectrometry, which enables the characterization of the adduct structure. This has allowed the identification of lipoxidized proteins in physiological and pathological situations. While in many cases lipoxidation interferes with protein function, causing inhibition of enzymatic activity and increased immunogenicity, there are a small number of cases where lipoxidation results in gain of function or activity. For certain proteins lipoxidation may represent a form of redox signaling, although more work is required to confirm the physiological relevance and mechanisms of such processes. This article is part of a Special Issue entitled: Posttranslational Protein modifications in biology and Medicine.

High-fat diet induces changes in adipose tissue trans-4-oxo-2-nonenal and trans-4-hydroxy-2-nonenal levels in a depot-specific manner.

Protein carbonylation is the covalent modification of proteins by α,ß-unsaturated aldehydes produced by nonenzymatic lipid peroxidation of polyunsaturated fatty acids. The most widely studied aldehyde product of lipid peroxidation, trans-4-hydroxy-2-nonenal (4-HNE), is associated with obesity-induced metabolic dysfunction and has demonstrated reactivity toward key proteins involved in cellular function. However, 4-HNE is only one of many lipid peroxidation products and the lipid aldehyde profile in adipose tissue has not been characterized. To further understand the role of oxidative stress in obesity-induced metabolic dysfunction, a novel LC-MS/MS method was developed to evaluate aldehyde products of lipid peroxidation and applied to the analysis of adipose tissue. 4-HNE and trans-4-oxo-2-nonenal (4-ONE) were the most abundant aldehydes present in adipose tissue. In high fat-fed C57Bl/6J and ob/ob mice the levels of lipid peroxidation products were increased 5- to 11-fold in epididymal adipose, unchanged in brown adipose, but decreased in subcutaneous adipose tissue. Epididymal adipose tissue of high fat-fed mice also exhibited increased levels of proteins modified by 4-HNE and 4-ONE, whereas subcutaneous adipose tissue levels of these modifications were decreased. High fat feeding of C57Bl/6J mice resulted in decreased expression of a number of genes linked to antioxidant biology selectively in epididymal adipose tissue. Moreover, TNFα treatment of 3T3-L1 adipocytes resulted in decreased expression of GSTA4, GPx4, and Prdx3 while upregulating the expression of SOD2. These results suggest that inflammatory cytokines selectively downregulate antioxidant gene expression in visceral adipose tissue, resulting in elevated lipid aldehydes and increased protein carbonylation.