In vitro fermentation of nuts results in the formation of butyrate and c9,t11 conjugated linoleic acid as chemopreventive metabolites.

PURPOSE: The consumption of foods rich in dietary fiber and polyunsaturated fatty acids such as nuts can contribute to a healthy diet. Therefore, the formation of fermentation end-products which might exert chemopreventive effects regarding colon cancer was investigated after an in vitro simulated digestion and fermentation of nuts using human fecal microbiota. METHODS: Fermentation supernatants (FS) and pellets (FP) were obtained after an in vitro fermentation of hazelnuts, almonds, macadamia, pistachios and walnuts. Short-chain fatty acids (SCFA) and bile acids (BA) in FS as well as fatty acids in FP were analyzed via gas chromatography. Malondialdehyde (MDA) levels in FS were determined photometrically. RESULTS: Fermentation of nuts resulted in 1.9- to 2.8-fold higher concentrations of SCFA compared to the control and a shift of molar ratios toward butyrate production. In vitro fermentation resulted in the formation of vaccenic acid (C18:1t11, 32.1 ± 3.2 % FAME; fatty acid methyl ester) and conjugated linoleic acid (c9,t11 CLA, 2.4 ± 0.7 % FAME) exclusively in fermented walnut samples. Concentrations of secondary BA deoxycholic-/iso-deoxycholic acid (6.8-24.1-fold/4.9-10.9-fold, respectively) and levels of MDA (1.3-fold) were significantly reduced in fermented nut samples compared to the control. CONCLUSION: This is the first study that demonstrates the ability of the human fecal microbiota to convert polyunsaturated fatty acids from walnuts to c9,t11 CLA as a potential chemopreventive metabolite. In addition, the production of butyrate and reduction in potential carcinogens such as secondary BA and lipid peroxidation products might contribute to the protective effects of nuts regarding colon cancer development.

The phytochemical glaucarubinone promotes mitochondrial metabolism, reduces body fat, and extends lifespan of Caenorhabditis elegans.

Naturally occurring compounds that promote energy expenditure and delay aging in model organisms may be of significant interest, since these substances potentially provide pharmaceutical approaches to tackle obesity and promote healthy lifespan in humans. We aimed to test whether pharmaceutical concentrations of glaucarubinone, a cytotoxic and antimalarial quassinoid known from different species of the plant family Simaroubaceae, are capable of affecting metabolism and/or extending lifespan in a nematodal model organism for aging processes, the roundworm Caenorhabditis elegans. Adult C. elegans roundworms, maintained on agar plates, were fed with E. coli strain OP50 bacteria, and glaucarubinone was applied to the agar to test (i) whether it alters respiration rates and mitochondrial activity, (ii) whether it affects body fat content, and (iii) whether it may promote longevity by quantifying survival in the presence and absence of the compound. We have found that glaucarubinone induces oxygen consumption and reduces body fat content of C. elegans. Moreover and consistent with the concept of mitohormesis, glaucarubinone extends C. elegans lifespan when applied at a concentration of 1 or 10 nanomolar. Taken together, glaucarubinone is capable of reducing body fat and promoting longevity in C. elegans, tentatively suggesting that this compound may promote metabolic health and lifespan in mammals and possibly humans.

Differential effects of resveratrol and SRT1720 on lifespan of adult Caenorhabditis elegans.

Resveratrol and SRT1720 have been shown to act as sirtuin activators that may ameliorate type 2 diabetes and metabolic diseases in mice. Moreover, resveratrol extends lifespan in model organisms like C. elegans, N. FURZERI, and possibly D. melanogaster. The aim of the study was to test whether pharmacological concentrations of resveratrol and SRT1720 are capable of extending lifespan in a nematodal model organism for aging processes, the roundworm Caenorhabditis elegans. Several hundreds of adult C. ELEGANS roundworms were maintained on agar plates and fed E. COLI strain OP50 bacteria. Resveratrol (5 micromolar, 500 nanomolar) or SRT1720 (1 micromolar, 100 nanomolar) was applied to the agar to test whether they may promote longevity by quantifying survival in the presence and absence of the respective compounds. At a dose of 5 micromolar, which is pharmacologically relevant and 20 times lower than previously published concentrations, resveratrol significantly extends C. elegans lifespan by 3.6% (mean lifespan) and 3.4% (maximum lifespan). By unexpected contrast, SRT1720, which was previously proposed to be several hundred times more active than resveratrol, did not extend lifespan at none of the concentrations tested. Thus, in the model organisms C. elegans, resveratrol is capable of promoting longevity at a concentration that pharmacologically relevant and 20 times lower than previously published doses. The sirtuin activator SRT1720 did not extend lifespan, suggesting that in C. elegans, some relevant effects of resveratrol cannot be mimicked by SRT1720.

Physiology: does gut hormone PYY3-36 decrease food intake in rodents?

Batterham et al. report that the gut peptide hormone PYY3-36 decreases food intake and body-weight gain in rodents, a discovery that has been heralded as potentially offering a new therapy for obesity. However, we have been unable to replicate their results. Although the reasons for this discrepancy remain undetermined, an effective anti-obesity drug ultimately must produce its effects across a range of situations. The fact that the findings of Batterham et al. cannot easily be replicated calls into question the potential value of an anti-obesity approach that is based on administration of PYY3-36.

CYP4F2 repression and a modified alpha-tocopherol (vitamin E) metabolism are two independent consequences of ethanol toxicity in human hepatocytes.

The expression of CYP4F2, a form of cytochrome P-450 with proposed role in α-tocopherol and long-chain fatty acid metabolism, was explored in HepG2 and HepaRG human hepatocytes during ethanol toxicity. Cytotoxicity, ROS production, and JNK and ERK1/2 kinase signaling increased in a dose and time-dependent manner during ethanol treatments; CYP4F2 gene expression decreased, while other CYP4F forms, namely 4F11 and 12, increased along with 3A4 and 2E1 isoforms. α-Tocopherol antagonized the cytotoxicity and CYP4F2 gene repression effect of ethanol in HepG2 cells. Ethanol stimulated the tocopherol-ω-hydroxylase activity and the other steps of vitamin E metabolism, which points to a minor role of CYP4F2 in this metabolism of human hepatocytes. PPAR-γ and SREBP-1c followed the same expression pattern of CYP4F2 in response to ethanol and α-tocopherol treatments. Moreover, the pharmacological inhibition of PPAR-γ synergized with ethanol in decreasing CYP4F2 protein expression, which suggests a role of this nuclear receptor in CYP4F2 transcriptional regulation. In conclusion, ethanol toxicity modifies the CYP expression pattern of human hepatic cells impairing CYP4F2 transcription and protein expression. These changes were associated with a lowered expression of the fatty acid biosynthesis regulators PPAR-γ and SREBP-1c, and with an increased enzymatic catabolism of vitamin E. CYP4F2 gene repression and a sustained vitamin E metabolism appear to be independent effects of ethanol toxicity in human hepatocytes.

PYY3-36 as an anti-obesity drug target.

The neuropeptide Y (NPY)/peptide YY (PYY) system has been implicated in the physiology of obesity for several decades. More recently ignited enormous interest in PYY3-36, an endogenous Y2-receptor agonist, as a promising anti-obesity compound. Despite this interest, there have been remarkably few subsequent reports reproducing or extending the initial findings, while at the same time studies finding no anti-obesity effects have surfaced. Out of 41 different rodent studies conducted (in 16 independent labs worldwide), 33 (83%) were unable to reproduce the reported effects and obtained no change or sometimes increased food intake, despite use of the same experimental conditions (i.e. adaptation protocols, routes of drug administration and doses, rodent strains, diets, drug vendors, light cycles, room temperatures). Among studies by authors in the original study, procedural caveats are reported under which positive effects may be obtained. Currently, data speak against a sustained decrease in food intake, body fat, or body weight gain following PYY3-36 administration and make the previously suggested role of the hypothalamic melanocortin system unlikely as is the existence of PYY deficiency in human obesity. We review the studies that are in the public domain which support or challenge PYY3-36 as a potential anti-obesity target.

Influence of roasting conditions on health-related compounds in different nuts.

Due to their health-beneficial ingredients the consumption of nuts can contribute to a healthy diet. The composition of hazelnuts, almonds, macadamia nuts, pistachios and walnuts regarding health-promoting and potentially harmful compounds was examined before and after roasting under different time and temperature conditions. Fatty acid compositions were not affected by roasting. Malondialdehyde increased with higher roasting temperatures (17-fold in walnuts). Levels of tocopherol isomers were reduced after roasting (α-T: 38%, ß-T: 40%, γ-T: 70%) and hydrophilic antioxidant capacity decreased significantly in hazelnuts (1.4-fold), macadamia nuts (1.7-fold) and walnuts (3.7-fold). Increasing roasting temperatures supported the formation of significant amounts of acrylamide only in almonds (1220 µg kg(-1)). In general, nuts roasted at low/middle temperatures (120-160°C) exhibited best sensory properties. Therefore, desired sensory quality along with a favourable healthy nut composition may be achieved by roasting over a low to medium temperature range.

Tocopherols are metabolized in HepG2 cells by side chain omega-oxidation and consecutive beta-oxidation.

The metabolism of tocopherols by omega- and beta-oxidation of the phytyl side chain has been inferred from the identification of the final products carboxyethyl-hydroxychromans (CEHC) and immediate precursors, alpha- and gamma-carboxymethylbutyl-hydroxychromans (CMBHCs). This hypothesis is here corroborated by the identification of a further alpha-tocopherol metabolite, alpha-carboxymethylhexyl-hydroxychroman (alpha-CMHHC), and evidence for the involvement of a P450-type cytochrome. HepG2 cells, when exposed to 100 microM all-rac-alpha-tocopherol, released alpha-CEHC, alpha-CMBHC, and alpha-CMHHC into the medium. The detection of those metabolites required pretreatment of the cells with alpha-tocopherol for 10 d. In contrast, analogous metabolites of gamma and delta-tocopherol were detectable without any preconditioning, while corresponding metabolites of RRR-alpha-tocopherol could not be detected at all. The formation of alpha-CEHC from all-rac-alpha-tocopherol was enhanced up to 5-fold by pretreatment of the HepG2 cells with rifampicin, known to induce CYP3A-type cytochromes with the capability of catalyzing omega-oxidation. In contrast, clofibrate did not reveal any effect. This observation suggests that a CYP3A-type cytochrome initiates tocopherol metabolism by omega-oxidation. It further reveals that inducible omega-oxidation is the rate-limiting step in tocopherol metabolism. It is discussed that competition of microsomal omega-oxidation with specific binding by the alpha-tocopherol transfer protein (alpha-TTP) determines the metabolic fate of the individual tocopherols.

Allium chemistry: synthesis, natural occurrence, biological activity, and chemistry of Se-alk(en)ylselenocysteines and their gamma-glutamyl derivatives and oxidation products.

Syntheses are reported for gamma-glutamyl Se-methylselenocysteine (Sa), selenolanthionine (16), Se-1-propenylselenocysteine (Gd), Se-2-methyl-2-propenyl-L-selenocysteine (6e), and Se-2-propynyl-L-selenocysteine (6f). Oxidation of 8a and Se-methylselenocysteine (Ga) gives methaneseleninic acid (24), characterized by X-ray crystallography, and dimethyl diselenide (25). Oxidation of Se-2-propenyl-L-selenocysteine (6c) gives allyl alcohol and 3-seleninoalanine (22). Compound 22 is also formed on oxidation of 16 and selenocystine (4). Oxidation of 6d gives 2-[(E,Z)-1-propenylseleno]propanal (36). These oxidations occur by way of selenoxides, detected by chromatographic and spectroscopic methods. The natural occurrence of many of the Se-alk(en)ylselenocysteines and their gamma-glutamyl derivatives and oxidation products is discussed. Three homologues of the potent cancer chemoprevention agents 6a and 6c, namely 6d-f, were evaluated for effects on cell growth, induction of apoptosis, and DNA-damaging activity using two murine mammary epithelial cell lines. Although each compound displays a unique profile of activity, none of these compounds (Gd-f) is likely to exceed the chemopreventive efficacy of selenocysteine Se-conjugates Ga and 6c.

Chemical speciation influences comparative activity of selenium-enriched garlic and yeast in mammary cancer prevention.

A recent human intervention trial showed that daily supplementation with selenized yeast (Se-yeast) led to a decrease in the overall cancer morbidity and mortality by nearly 50%; past research has also demonstrated that selenized garlic (Se-garlic) is very effective in mammary cancer chemoprevention in the rat model. The goal of this study was to compare certain biological activities of Se-garlic and Se-yeast and to elucidate the differences based on the chemical forms of selenium found in these two natural products. Characterization of organic selenium compounds in yeast (1922 microg/g Se) and garlic (296 microg/g Se) was carried out by high-performance liquid chromatography with inductively coupled plasma mass spectrometry or with electrospray mass spectrometry. Analytical speciation studies showed that the bulk of the selenium in Se-garlic and Se-yeast is in the form of gamma-glutamyl-Se-methylselenocysteine (73%) and selenomethionine (85%), respectively. The above methodology has the sensitivity and capability to account for >90% of total selenium. In the rat feeding studies, supplementation of Se-garlic in the diet at different levels consistently caused a lower total tissue selenium accumulation when compared to Se-yeast. On the other hand, Se-garlic was significantly more effective in suppressing the development of premalignant lesions and the formation of adenocarcinomas in the mammary gland of carcinogen-treated rats. Given the present finding on the identity of selenomethionine and gamma-glutamyl-Se-methylselenocysteine as the major form of selenium in Se-yeast and Se-garlic, respectively, the metabolism of these two compounds is discussed in an attempt to elucidate how their disposition in tissues might account for the differences in cancer chemopreventive activity.

A combination of lipoic acid plus coenzyme Q10 induces PGC1α, a master switch of energy metabolism, improves stress response, and increases cellular glutathione levels in cultured C2C12 skeletal muscle cells.

Skeletal muscle function largely depend on intact energy metabolism, stress response, and antioxidant defense mechanisms. In this study, we tested the effect of a combined supplementation of α-lipoic acid (LA) plus coenzyme Q10 (Q10) on PPARγ-coactivator α (PGC1α) activity, expression of glutathione-related phase II enzymes and glutathione (GSH) levels in cultured C2C12 myotubes. Supplementation of myotubes with 250 µmol/L LA plus 100 µmol/L Q10 significantly increased nuclear levels of PGC1α, a master switch of energy metabolism and mitochondrial biogenesis. The increase of nuclear PGC1α was accompanied by an increase in PPARγ transactivation, a downstream target of PGC1α, and an increase in mitochondrial transcription factor A mRNA centrally involved in mitochondrial replication and transcription. Furthermore, supplementation of myotubes with LA plus Q10 resulted in an increase of genes encoding proteins involved in stress response, GSH synthesis, and its recycling. In LA-plus-Q10-treated myotubes a significant 4-fold increase in GSH was evident. This increase in GSH was accompanied by increased nuclear Nrf2 protein levels, partly regulating γGCS and GST gene expression. Present data suggest that the combined supplementation of skeletal muscle cells with LA plus Q10 may improve energy homeostasis, stress response, and antioxidant defense mechanisms.

A cell-based high-throughput assay system reveals modulation of oxidative and nonoxidative glucose metabolism due to commonly used organic solvents.

A 96-well format screening system was generated to quantify changes in nonoxidative glucose metabolism and oxidative pyruvate metabolism. D-Glucose uptake from the supernatant media was quantified by the glucose oxidase method, and L-lactate production of cells was quantified by the lactate dehydrogenase method applied on supernatant media. Mitochondrial membrane potential was quantified using tetramethylrhodamine methyl ester (TMRM) fluorescence, and reactive oxygen species (ROS) formation was determined by quantification of dihydrodichlorofluorescein fluorescence. Adenosine triphosphate (ATP) content of myocytes was determined using the luciferin reaction, and cellular respiration was quantified using commercially available, precoated microtiter plates. These six assays were used to determine the putative influence of organic solvents, namely dimethyl sulfoxide (DMSO), ethanol, methanol, and N-methylpyrrolidone (NMP) at concentrations of 0.01, 0.1, 1.0, and 5.0% (vol/vol), respectively, on glucose and pyruvate metabolism after 4 and 24 hours. In summary, all solvents induced significant changes in regard to one or several of the parameters evaluated, affecting cellular glucose uptake, glycolysis, mitochondrial metabolism, or oxidative phosphorylation. Accordingly, this comprehensive HTS evaluation should enable researchers to choose specific organic solvents on a rational basis to avoid nonspecific effects in cultured cells and tissue culture based experimental setups.

Vitamin E analogues as inducers of apoptosis: structure-function relation.

Recent results show that alpha-tocopheryl succinate (alpha-TOS) is a proapoptotic agent with antineoplastic activity. As modifications of the vitamin E (VE) molecule may affect its apoptogenic activity, we tested a number of newly synthesised VE analogues using malignant cell lines. Analogues of alpha-TOS with lower number of methyl substitutions on the aromatic ring were less active than alpha-TOS. Replacement of the succinyl group with a maleyl group greatly enhanced the activity, while it was lower for the glutaryl esters. Methylation of the free succinyl carboxyl group on alpha-TOS and delta-TOS completely prevented the apoptogenic activity of the parent compounds. Both Trolox and its succinylated derivative were inactive. alpha-tocotrienol (alpha-T3 H) failed to induce apoptosis, while gamma-T3 H was apoptogenic, and more so when succinylated. Shortening the aliphatic side chain of gamma-T3 by one isoprenyl unit increased its activity. Neither phytyl nor oleyl succinate caused apoptosis. These findings show that modifications of different functional moieties of the VE molecule can enhance apoptogenic activity. It is hoped that these observations will lead to the synthesis of analogues with even higher apoptogenic and, consequently, antineoplastic efficacy.

Selenium speciation in enriched and natural samples by HPLC-ICP-MS and HPLC-ESI-MS with perfluorinated carboxylic acid ion-pairing agents.

Selenium-enriched plants, such as hyperaccumulative phytoremediation plants (Astragalus praleongus, 517 micrograms g-1 Se, and Brassica juncea, 138 micrograms g-1 Se in dry sample), yeast (1200, 1922 and 2100, micrograms g-1 Se in dry sample), ramp (Allium tricoccum, 48, 77, 230, 252, 405 and 524 micrograms g-1 Se in dry sample), onion (Allium cepa, 96 and 140 micrograms g-1 Se in dry sample) and garlic (Allium sativum, 68, 112, 135, 296, 1355 micrograms g-1 Se in dry sample) were analyzed by HPLC-ICP-MS for their selenium content and speciation after hot water and enzymatic extractions. Reference samples with natural selenium levels, such as onion and garlic controls, cooking garlic powder, baking yeast powder and a commercial garlic supplement were also analyzed. Selected samples were also examined by HPLC-electrospray ionization (ESI)-MS. HPLC was mostly carried out with 0.1% heptafluorobutanoic acid (HFBA) as ion-pairing agent in 1 + 99 v/v methanol-water solution, but 0.1% trifluoroacetic acid (TFA) in 1 + 99 v/v methanol-water solution was also utilized to permit chromatography for compounds that did not elute with HFBA. More than 75% of the total eluting selenium compounds, based upon element specific detection, were identified from retention time data and standard spiking experiments, and between 60 and 85% of compounds were identified by MS, with up to 25% of the total eluting molecular selenium species being unidentified as yet. Limits of quantification (LOQ, defined as the concentration giving an S/N of 10) for HPLC-ICP-MS were in the range 2-50 ng mL-1 Se in the injected extracts for the selenium-enriched samples and 2-10 ng mL-1 Se for the natural selenium level samples. LOQ values for HPLC-ESI-MS were ca. 100 times higher than those measured by HPLC-ICP-MS.