In vitro and in vivo digestion of red cured cooked meat: oxidation, intestinal microbiota and fecal metabolites.

Mechanisms explaining epidemiological associations between red (processed) meat consumption and chronic disease risk are not yet elucidated, but may involve oxidative reactions, microbial composition alterations, inflammation and/or the formation of toxic bacterial metabolites. First, in vitro gastrointestinal digestion of 23 cooked beef-lard minces, to which varying doses of nitrite salt (range 0-40 g/kg) and sodium ascorbate (range 0-2 g/kg) were added, showed that nitrite salt decreased protein carbonylation up to 3-fold, and inhibited lipid oxidation, demonstrated by up to 4-fold lower levels of 'thiobarbituric acid reactive substances', 32-fold lower 4-hydroxynonenal, and 21-fold lower hexanal values. The use of ascorbate increased the antioxidant effect of low nitrite salt levels, whereas it slightly increased protein carbonylation at higher doses of nitrite salt. The addition of a low dose of ascorbate without nitrite salt slightly promoted oxidation during digestion, whereas higher doses had varying antioxidant effects. Second, 40 rats were fed a diet of cooked chicken- or beef-lard minces, either or not cured, for three weeks. Beef, compared to chicken, consumption increased lipid oxidation (2- to 4-fold) during digestion, and gut protein fermentation (cecal iso-butyrate, (iso-)valerate, and fecal indole, cresol), but oxidative stress and inflammation were generally not affected. Cured, compared to fresh, meat consumption significantly increased stomach protein carbonylation (+16%), colonic Ruminococcaceae (2.1-fold) and cecal propionate (+18%), whereas it decreased cecal butyrate (-25%), fecal phenol (-69%) and dimethyl disulfide (-61%) levels. Fecal acetaldehyde and diacetyl levels were increased in beef-fed rats by 2.8-fold and 5.9-fold respectively, and fecal carbon disulfide was 4-fold higher in rats consuming cured beef vs. fresh chicken. Given their known toxicity, the role of acetaldehyde and carbon disulfide in the relation between meat consumption and health should be investigated in future studies.

Untargeted Metabolomics Reveals Elevated L-Carnitine Metabolism in Pig and Rat Colon Tissue Following Red Versus White Meat Intake.

SCOPE: The consumption of red and processed meat, and not white meat, associates with the development of various Western diseases such as colorectal cancer and type 2 diabetes. This work aims at unraveling novel meat-associated mechanisms that are involved in disease development. METHODS AND RESULTS: A non-hypothesis driven strategy of untargeted metabolomics is applied to assess colon tissue from rats (fed a high dose of beef vs. white meat) and from pigs (fed red/processed meat vs. white meat), receiving a realistic human background diet. An increased carnitine metabolism is observed, which is reflected by higher levels of acylcarnitines and 3-dehydroxycarnitine (rats and pigs) and trimethylamine-N-oxide (rats). While 3-dehydroxycarnitine is higher in HT29 cells, incubated with colonic beef digests, acylcarnitine levels are reduced. This suggests an altered response from colon cancer cell line towards meat-induced oxidative stress. Moreover, metabolic differences between rat and pigs are observed in N-glycolylneuraminic acid incorporation, prostaglandin, and fatty acid synthesis. CONCLUSION: This study demonstrates elevated (acyl)carnitine metabolism in colon tissue of animals that follow a red meat-based diet, providing mechanistic insights that may aid in explaining the nutritional-physiological correlation between red/processed meat and Western diseases.

Commercial luncheon meat products and their in vitro gastrointestinal digests contain more protein carbonyl compounds but less lipid oxidation products compared to fresh pork.

Because of the large diversity in processed meat products and the potential involvement of oxidation processes in the association between red and processed meat consumption and chronic diseases, the concentration of oxidation products after gastrointestinal digestion of commercial luncheon meat products was investigated. A broad spectrum of meat products (n = 24), displaying large variation in macro- and micronutrient composition and processing procedures, was digested in vitro by simulating digestion fluids of the human gastrointestinal tract. Lipid and protein oxidation was assessed in the meat products before digestion and in the corresponding digests by measurement of free malondialdehyde, 4-hydroxy-2-nonenal, hexanal and protein carbonyl compounds. Compared to an unprocessed cooked pork mince, that was included as a reference in the digestion experiment, levels of lipid oxidation products were low in the digests of most meat products. Only the digests of Parma ham had slightly higher or comparable levels as the reference pork. In contrast, protein carbonyl compounds were comparable or up to 6 times higher in the processed meat products compared to the reference pork. Particularly raw-cooked and precooked-cooked meat products and corresponding digests had higher protein carbonyl levels, but also lower protein contents and higher fat to protein ratios. In conclusion, most luncheon meat products and corresponding digests contained lower amounts of free lipid oxidation products, but more protein carbonyl compounds compared to the reference pork.

Untargeted Metabolomics to Reveal Red versus White Meat-Associated Gut Metabolites in a Prudent and Western Dietary Context.

SCOPE: To improve understanding of the epidemiological link between red and processed meat consumption and chronic diseases, more insight into the formation of metabolites during meat digestion is warranted. METHODS AND RESULTS: Untargeted mass-spectrometry-based metabolomics is applied to explore the impact of red and processed meat consumption (compared to chicken), combined with a prudent or Western dietary pattern. A pig feeding study (n = 32), as a sentinel for humans, is conducted in a 2 × 2 factorial design for 4 weeks. The luminal content of the small intestine and colon are collected to determine their metabolic fingerprints. Seventy-six metabolites (38 in the small intestine, 32 in the colon, and 6 in both intestinal compartments) contributing to the distinct gut metabolic profiles of pigs fed either chicken or red and processed meat are (tentatively) identified. Consumption of red and processed meat results in higher levels of short- and medium-chain acylcarnitines and 3-dehydroxycarnitine, irrespective of dietary context, whereas long-chain acylcarnitines and monoacylglycerols are associated with the red and processed Western diet. CONCLUSION: The identification of red and processed meat-associated gut metabolites in this study contributes to the understanding of meat digestion in a complex but controlled dietary context and its potential health effects.

Red and processed meat consumption within two different dietary patterns: Effect on the colon microbial community and volatile metabolites in pigs.

Pigs were fed either red and processed meat or chicken meat within either a prudent or a Western dietary pattern for four weeks (2 × 2 full factorial design). The colon microbial community and volatile organic compounds were assessed (either quantified or based on their presence). Results show that Lactobacilli were characteristic for the chicken × prudent dietary pattern treatment and Paraprevotella for the red and processed meat × prudent dietary pattern treatment. Enterobacteriaceae and Desulfovibrio were characteristic for the chicken × Western dietary pattern treatment and Butyrivibrio for the red and processed meat × Western dietary pattern treatment. Campylobacter was characteristic for chicken consumption and Clostridium XIVa for red and processed meat, irrespective of the dietary pattern. Ethyl valerate and 1-methylthio-propane were observed more frequently in pigs fed red and processed meat compared to chicken meat. The prevalence of 3-methylbutanal was >80% for pigs receiving a Western dietary pattern, whereas for pigs fed a prudent dietary pattern the prevalence was <35%. The concentration of butanoic acid was significantly higher when the prudent dietary pattern was given, compared to the Western dietary pattern, but no differences for other short chain fatty acids or protein fermentation products were observed.

Background Diet Influences TMAO Concentrations Associated with Red Meat Intake without Influencing Apparent Hepatic TMAO-Related Activity in a Porcine Model.

Red meat has been associated with an increased cardiovascular disease (CVD) risk, possibly through gut microbial-derived trimethylamine-N-oxide (TMAO). However, previous reports are conflicting, and influences from the background diet may modulate the impact of meat consumption. This study investigated the effect of red and white meat intake combined with two different background diets on urinary TMAO concentration and its association with the colon microbiome in addition to apparent hepatic TMAO-related activity. For 4 weeks, 32 pigs were fed chicken or red and processed meat combined with a prudent or western background diet. 1H NMR-based metabolomics analysis was conducted on urine samples and hepatic mRNA expression of TMAO-related genes determined. Lower urinary TMAO concentrations were observed after intake of red and processed meat when consumed with a prudent compared to a western background diet. In addition, correlation analyses between urinary TMAO concentrations and relative abundance of colon bacterial groups suggested an association between TMAO and specific bacterial taxa. Diet did not affect the hepatic mRNA expression of genes related to TMAO formation. The results suggest that meat-induced TMAO formation is regulated by mechanisms other than alterations at the hepatic gene expression level, possibly involving modulations of the gut microbiota.

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.

Impact of Red versus White Meat Consumption in a Prudent or Western Dietary Pattern on the Oxidative Status in a Pig Model.

Human diets contain a complex mixture of antioxidants and pro-oxidants that contribute to the body's oxidative status. In this study, 32 pigs were fed chicken versus red and processed meat in the context of a prudent or Western dietary pattern for 4 weeks, to investigate their oxidative status. Lipid oxidation products (malondialdehyde, 4-hydroxy-2-nonenal, and hexanal) were higher in the chicken versus red and processed meat diets (1.7- to 8.3-fold) and subsequent in vitro (1.3- to 1.9-fold) and in vivo (1.4 to 3-fold) digests ( P < 0.001), which was presumably related to the higher polyunsaturated fatty acid content in chicken meat and/or the added antioxidants in processed meat. However, diet had only a marginal or no effect on the systemic oxidative status, as determined by plasma oxygen radical absorbance capacity, malondialdehyde, glutathione, and glutathione peroxidase activity in blood and organs, except for α-tocopherol, which was higher after the consumption of the chicken-Western diet. In conclusion, in contrast to the hypothesis, the consumption of chicken in comparison to that of the red and processed meat resulted in higher concentrations of lipid oxidation products in the pig intestinal contents; however, this was not reflected in the body's oxidative status.

Selective pharmacological inhibitors of HDAC6 reveal biochemical activity but functional tolerance in cancer models.

Our study investigates the biochemical and functional impact of selective histone deacetylase 6 (HDAC6) inhibitors, a promising class of novel therapeutics, in several cancer models. Selective HDAC6 inhibitors (Tubathian A, Tubastatin A, Tubacin and Ricolinostat) and a non-selective HDAC inhibitor (Vorinostat) were evaluated on cancer cell lines derived from multiple tumour types in both an in vitro and in vivo setting as potential cancer therapeutics. Selective HDAC6 inhibitors resulted in α-tubulin acetylation with no impact on histone acetylation but failed to show any anti-cancer properties. Only the use of high concentrations of selective HDAC6 inhibitors resulted in co-inhibition of other HDAC enzymes and consequently in reduced growth, migratory and/or invasive activity of cancer cells in vitro as well as in vivo. The specificity of HDAC6 inhibition was confirmed using a CRISPR/Cas9 knockout cell line. Our results suggest that selective HDAC6 inhibitors may fall short as potential single agent anti-cancer drugs and prove that many previous data regarding this promising class of compounds need to be interpreted with great care due to their use in high concentrations resulting in low selectivity and potential off-target effects.