Co-Administration of Propionate or Protocatechuic Acid Does Not Affect DHA-Specific Transcriptional Effects on Lipid Metabolism in Cultured Hepatic Cells.

Long-chain n-3 polyunsaturated fatty acids (n-3 LC-PUFAs) are collectively recognized triglyceride-lowering agents, and their preventive action is likely mediated by changes in gene expression. However, as most studies employ fish oil, which contains a mixture of n-3 LC-PUFAs, the docosahexaenoic acid (DHA)-specific transcriptional effects on lipid metabolism are still unclear. The aim of the present study was to further elucidate the DHA-induced transcriptional effects on lipid metabolism in the liver, and to investigate the effects of co-administration with other bioactive compounds having effects on lipid metabolism. To this purpose, HepG2 cells were treated for 6 or 24 h with DHA, the short-chain fatty acid propionate (PRO), and protocatechuic acid (PCA), the main human metabolite of cyanidin-glucosides. Following supplementation, we mapped the global transcriptional changes. PRO and PCA alone had a very slight effect on the transcriptome; on the contrary, supplementation of DHA highly repressed the steroid and fatty acid biosynthesis pathways, this transcriptional modulation being not affected by co-supplementation. Our results confirm that DHA effect on lipid metabolism are mediated at least in part by modulation of the expression of specific genes. PRO and PCA could contribute to counteracting dyslipidemia through other mechanisms.

(Poly)phenolic Content and Profile and Antioxidant Capacity of Whole-Grain Cookies are Better Estimated by Simulated Digestion than Chemical Extraction.

It is widely recognized that the biological effects of phytochemicals cannot be attributed to the native compounds present in foods but rather to their metabolites endogenously released after intake. Bioavailability depends on bioaccessibility, which is the amount of the food constituent that is released from the matrix in the gastrointestinal tract. The use of chemical extraction to evaluate the content and profile of phytochemicals does not mirror the physiological situation in vivo, and their bioaccessibility should be considered while assessing their nutritional significance in human health. The current study was designed to compare the (poly)phenolic profile and content and antioxidant capacity of whole-grain (WG) cookies using chemical extraction and a more physiological approach based on simulated digestion. Three types of organic WG cookies (made with durum, Italian khorasan, or KAMUT® khorasan wheat) were considered, either fermented by Saccharomyces Cerevisiae or sourdough. Although the flour type and the fermentation process influenced the release of phytochemicals from the cookie matrix, in almost all samples, the simulated digestion appeared the most efficient procedure. Our results indicate that the use of chemical extraction for evaluation of the phytochemicals content and antioxidant capacity of food could lead to underestimation and underline the need for more physiological extraction methods.

Modulation of Adipocyte Differentiation and Proadipogenic Gene Expression by Sulforaphane, Genistein, and Docosahexaenoic Acid as a First Step to Counteract Obesity.

Obesity is characterized by excess body fat accumulation due to an increase in the size and number of differentiated mature adipocytes. Adipocyte differentiation is regulated by genetic and environmental factors, and its inhibition could represent a strategy for obesity prevention and treatment. The current study was designed with two aims: (i) to evaluate the changes in the expression of adipogenic markers (C/EBPα, PPARγ variant 1 and variant 2, and GLUT4) in 3T3-L1 murine preadipocytes at four stages of the differentiation process and (ii) to compare the effectiveness of sulforaphane, genistein, and docosahexaenoic acid in reducing lipid accumulation and modulating C/EBPα, PPARγ1, PPARγ2, and GLUT4 mRNA expression in mature adipocytes. All bioactive compounds were shown to suppress adipocyte differentiation, although with different effectiveness. These results set the stage for further studies considering natural food constituents as important agents in preventing or treating obesity.

Health benefits of ancient grains. Comparison among bread made with ancient, heritage and modern grain flours in human cultured cells.

Nowadays the higher nutritional value of whole grains compared to refined grains is recognized. In the last decade, there has been a renewed interest in the ancient wheat varieties for producing high-value food products with enhanced health benefits. This study compared two ancient grains, two heritage grains, and four modern grains grown in the same agronomic conditions considering not only their chemical characteristics, but also their biological effects. Whole grain flours were obtained and used to make bread. Bread was in vitro digested, the digesta were supplemented to HepG2 cells, and the biological effects of supplementation were evaluated. In addition, cells previously supplemented with the different digested bread types were then exposed to inflammatory agents to evidence possible protective effects of the pre-treatments. Despite the impossibility to discriminate bread made with different grains based on their chemical composition, results herein reported evidence that their supplementation to cultured cells exerts different effects, confirming the potential health benefits of ancient grains. This research represents an advancement for the evaluation of the apparent positive effects of ancient grains and the formulation of cereal-based products with added nutritional value.

Is cytotoxicity a determinant of the different in vitro and in vivo effects of bioactives?

BACKGROUND: Foodstuffs of both plant and animal origin contain a wide range of bioactive compounds. Although human intervention studies are mandatory to assess the health effects of bioactives, the in vitro approach is often used to select the most promising molecules to be studied in vivo. To avoid misleading results, concentration and chemical form, exposure time, and potential cytotoxicity of the tested bioactives should be carefully set prior to any other experiments. METHODS: In this study the possible cytotoxicity of different bioactives (docosahexaenoic acid, propionate, cyanidin-3-O-glucoside, protocatechuic acid), was investigated in HepG2 cells using different methods. Bioactives were supplemented to cells at different concentrations within the physiological range in human blood, alone or in combination, considering two different exposure times. RESULTS: Reported data clearly evidence that in vitro cytotoxicity is tightly related to the exposure time, and it varies among bioactives, which could exert a cytotoxic effect even at a concentration within the in vivo physiological blood concentration range. Furthermore, co-supplementation of different bioactives can increase the cytotoxic effect. CONCLUSIONS: Our results underline the importance of in vitro cytotoxicity screening that should be considered mandatory before performing studies aimed to evaluate the effect of bioactives on other cellular parameters. Although this study is far from the demonstration of a toxic effect of the tested bioactives when administered to humans, it represents a starting point for future research aimed at verifying the existence of a potential hazard due to the wide use of high doses of multiple bioactives.

Influence of elastic modulus of intraradicular posts on the fracture load of roots restored with full crowns / Influência do modulo de elasticidade de retentores intrarradiculares na carga para fratura de raízes restauradas com coroas totais

Objective: This study aimed to evaluate the fracture load and displacement of roots restored with posts of different elastic modulus. Material and method: Thirty-six replicas of epoxy resin mixed with glass microfibers were made from an endodontically-treated human premolar root prepared to a length of 12 mm with a custom drill, leaving the apical 4 mm unprepared. Replicas were randomly restored with (n = 12): FP-LM (fiber post with low elastic modulus- 50 GPa), FP-HM (fiber post with high elastic modulus - 67 GPa) and MP (metallic post - 208 GPa), using self-curing adhesive and dual resin cement. Cores were built up with composite resin and metallic crowns were cemented in all the roots with self-adhesive resin cement with self-curing mode. Specimens were subjected to a fracture load test (45° inclination/0.5 mm/min) and displacement was registered at 100 N. Result: One-way ANOVA showed that elastic modulus of the post did not affect the fracture load means (p = 0.203) (FP-LM: 237.4 ± 65.11 N; FP-HM: 236.7 ± 92.85 N; MP: 295.8 ± 108.7 N) but was statistically significant for the displacement (p < 0.00): Tukey's test showed that FP-LM displacement mean (0.81 ± 0.15 mm) was significantly higher than those for FP-HM (0.46 ± 0.26 mm; p = 0.00) and MP (0.62 ± 0.07 mm; p = 0.04). Conclusion: Posts with different elastic modulus exhibit similar fracture loads, but a lower displacement is achieved when fiber posts with a high elastic modulus and metallic posts are used.

Objetivo: Este trabalho avaliou a carga para fratura e deslocamento de raízes restauradas com pinos de diferentes módulos de elasticidade. Material e método: Trinta e seis réplicas de microfibras de vidro embutidas em resina epóxi foram fabricadas a partir de uma raiz de um pré-molar tratado endodonticamente preparado em 12 mm de comprimento com brocas customizadas, deixando 4 mm apicais sem preparo. As raízes foram randomicamente restauradas com (n = 12): FP-LM (pino de fibra com um baixo módulo de elasticidade - 50 GPa), FP-HM (pino de fibra com um alto módulo de elasticidade - 67 GPa) e MP (pinos metálicos - 208 GPa), usando adesivo autopolimerizável e cimento resinoso dual. Núcleos foram confeccionados com resina composta e coroas metálicas foram cimentadas em todas as raízes com cimento resinoso autoadesivo com modo de polimerização químico. Os espécimes foram submetidos ao teste de fratura em 45° (inclinação de 45° / 0,5 mm/min) e o deslocamento foi registrado aos 100 N. Resultado: Anova 1 fator mostrou que o módulo de elasticidade dos retentores não afetou as médias de fratura (p = 0,203) (FP-LM: 237,4 ± 65,11 N; FP-HM: 236,7 ± 92,85 N; MP: 295,8 ± 108,7 N) mas foi estatisticamente significante para o deslocamento (p < 0,00): o teste de Tukey mostrou que a média de deslocamento do grupo FP-LM (0,81 ± 0,15 mm) foi significativamente maior do que o grupo FP-HM (0,46 ± 0,26 mm; p = 0,00) e MP (0,62 ± 0,07 mm; p = 0,47). Conclusão: Pinos com diferentes módulos de elasticidade mostram resistência similar, porém um menor deslocamento é obtido quando pinos de fibra com alto módulo de elasticidade (FP-HM/MP) são usados.

Evidence of a DHA Signature in the Lipidome and Metabolome of Human Hepatocytes.

Cell supplementation with bioactive molecules often causes a perturbation in the whole intracellular environment. Omics techniques can be applied for the assessment of this perturbation. In this study, the overall effect of docosahexaenoic acid (DHA) supplementation on cultured human hepatocyte lipidome and metabolome has been investigated using nuclear magnetic resonance (NMR) in combination with traditional techniques. The effect of two additional bioactives sharing with DHA the lipid-lowering effect-propionic acid (PRO) and protocatechuic acid (PCA)-has also been evaluated in the context of possible synergism. NMR analysis of the cell lipid extracts showed that DHA supplementation, alone or in combination with PCA or PRO, strongly altered the cell lipid profile. The perfect discrimination between cells receiving DHA (alone or in combination) and the other cells reinforced the idea of a global rearrangement of the lipid environment induced by DHA. Notably, gas chromatography and fluorimetric analyses confirmed the strong discrimination obtained by NMR. The DHA signature was evidenced not only in the cell lipidome, but also in the metabolome. Results reported herein indicate that NMR, combined with other techniques, represents a fundamental approach to studying the effect of bioactive supplementation, particularly in the case of molecules with a broad spectrum of mechanisms of action.

Ancient wheat and health: a legend or the reality? A review on KAMUT khorasan wheat.

After WWII, the industrialized agriculture selected modern varieties of Triticum turgidum spp. durum and spp. aestivum (durum wheat and common wheat) based on higher yields and technological characteristics. Nowadays, the use of whole ancient grains and pseudo cereals is considered nutritionally important. How ancient grains have positive effects is not entirely known, the fragmentation of the scientific knowledge being also related to the fact that ancient grains are not a homogeneous category. The KAMUT® trademark indicates a specific and ancient variety of grain (Triticum turgidum ssp. turanicum, commonly khorasan wheat), and guarantees certain attributes making studies sufficiently comparable. In this work, studies on KAMUT® khorasan wheat have been systematically reviewed, evidencing different aspects supporting its benefits. Although it is not possible to establish whether all ancient grains share these positive characteristics, in total or in part, this review provides further evidences supporting the consumption of ancient grains.

PUFA and oxidative stress. Differential modulation of the cell response by DHA.

Although an increased dietary intake of long-chain n-3 PUFA is considered an effective preventive strategy, a theoretical concern related to the possible increase of lipid peroxidation induced by a PUFA-rich diet still remains a problem. In this study, the effects of different PUFA (linoleic, α-linolenic, arachidonic, eicosapentaenoic and docosahexaenoic acid) on cytotoxicity, lipid oxidation, and modulation of antioxidant defenses were evaluated in HepG2 cells submitted to an oxidative stress (H2O2). Results clearly evidenced that all supplemented PUFA, but DHA, enhanced cell susceptibility to H2O2. Overall, our results underline that PUFA cannot be considered as a single category but as individual compounds, and research on mechanisms of action and preventive effects should deal with the individual fatty acids, particularly in the case of DHA.

Bioaccessibility of the Bioactive Peptide Carnosine during in Vitro Digestion of Cured Beef Meat.

A bioactive compound is a food component that may have an impact on health. Its bioaccessibility, defined as the fraction released from the food matrix into the gastrointestinal tract during digestion, depends on compound stability, interactions with other food components, and supramolecular organization of food. In this study, the effect of pH on the bioaccessibility of the bioactive dipeptide carnosine was evaluated in two commercial samples of the Italian cured beef meat bresaola at two key points of digestion: before the gastric and after the duodenal phases. The digestion process was simulated using an in vitro static system, whereas capillary zone electrophoresis (CZE) and (1)H nuclear magnetic resonance (NMR) were used for quantitative analysis. The gap between the total carnosine content, measured by CZE, and its free diffusible fraction observable by NMR spectroscopy, was 11 and 19% for two independent bresaola products, where such percentages represent the fraction of carnosine not accessible for intestinal absorption because it was adsorbed to the food matrix dispersed in the digestion fluid.

The foodomics approach for the evaluation of protein bioaccessibility in processed meat upon in vitro digestion.

The present work describes a foodomics protocol coupling an in vitro static simulation of digestion to a combination of omics techniques, to grant an overview of the protein digestibility of a meat-based food, namely Bresaola. The proteolytic activity mediated by the digestive enzymes is evaluated through Bradford and SDS-PAGE assays, combined to NMR relaxometry and spectroscopy, to obtain information ranging from the microscopic to the molecular level, respectively. The simple proteomics tool adopted here points out that a clear increase of bioaccessible proteins occurs in the gastric phase, rapidly disappearing during the following duodenal digestion. However, SDS-PAGE and the Bradford assay cannot follow the fate of the digested proteins when the products are sized <5 kDa. Conversely, NMR spectroscopy is able to capture the overall molecular profile of small fragments and peptides, which are mainly formed during the duodenal phase, thus giving the kinetics of the whole digestion process. Time domain NMR relaxometry, finally, detects the swelling phenomenon occurring during the gastric phase, when the digestion fluid enters the meat matrix.

European consumers' perceived seriousness of their eating habits relative to other personal health risks.

OBJECTIVE: Poor eating habits are a key priority on the European public health agenda due to their large health and economic implications. Healthy eating interventions may be more effective if consumers perceive their eating habits as a more serious personal health risk. This study investigates European consumers' perceived seriousness of their eating habits, its determinants and relative importance among other potential personal health risks including weight, stress and pollution. METHOD: A quantitative survey was conducted during Spring 2011 among samples representative for age, gender and region in five European countries (n=3003). RESULTS: Participants were neutral towards the seriousness of their eating habits for personal health. Eating habits were ranked third after stress and weight. Gender, age, country, health motive, body mass index, and subjective health status were important determinants of the perceived seriousness of their eating habits, whereas perceived financial condition, smoking and education were insignificant. CONCLUSION: Eating habits were perceived more seriously by women, Italians, obese, and younger individuals with stronger health motives and fair subjective health status. Nevertheless, other health risks were often considered more important than eating habits. More or specific efforts are required to increase Europeans' awareness of the seriousness of their eating habits for personal health.

Sugar cane and sugar beet molasses, antioxidant-rich alternatives to refined sugar.

Molasses, the main byproduct of sugar production, is a well-known source of antioxidants. In this study sugar cane molasses (SCM) and sugar beet molasses (SBM) were investigated for their phenolic profile and in vitro antioxidant capacity and for their protective effect in human HepG2 cells submitted to oxidative stress. According to its higher phenolic concentration and antioxidant capacity in vitro, SCM exhibited an effective protection in cells, comparable to or even greater than that of α-tocopherol. Data herein reported emphasize the potential health effects of molasses and the possibility of using byproducts for their antioxidant activity. This is particularly important for consumers in developing countries, as it highlights the importance of consuming a low-price, yet very nutritious, commodity.

Pro- and anti-oxidant effects of polyunsaturated fatty acid supplementation in HepG2 cells.

PUFA are bioactive nutrients thought to be effective in the prevention of many chronic diseases. PUFA susceptibility to free radical oxidation represents the other side of the coin, and the role of PUFA as pro- or anti-oxidants is still an unanswered question. In this study we supplemented HepG2 cells with different PUFA, and observed different effects on cytotoxicity, oxidation and modulation of antioxidant defenses. These were not simply related to the length of carbon chain, or to the number and position of double bonds. ARA supply evidenced the induction of oxidative damage, while DHA supplemented cells appeared richer in antioxidant defenses. To our knowledge, our study is the first evidencing the different pro- or anti-oxidant effect of different fatty acids when supplemented to cells. Overall, this points out the importance of not generalizing dietary recommendations considering PUFA as one category, but to extend them to the individual fatty acids.

Phytosterol supplementation reduces metabolic activity and slows cell growth in cultured rat cardiomyocytes.

Besides being cholesterol-lowering agents, phytosterols (PS) can inhibit the growth and development of tumours. The anti-neoplastic activity is accounted for by PS incorporation into cell membranes, resulting in the interference of membrane functionality. The similarity between the PS cholesterol-lowering and anti-neoplastic effective doses deserves attention on the possible adverse effects even in non-neoplastic cells. To date, few studies have addressed the clarification of this important issue. In the present study, we supplemented primary, non-neoplastic neonatal rat cardiomyocytes with two different PS concentrations (3 or 6 µg/ml), both within the range of human plasma concentration. Cardiac cells were chosen as an experimental model since the heart has been reported as the target organ for subchronic toxicity of PS. Following supplementation, a dose-dependent incorporation of PS and a decrease in cholesterol content were clearly evidenced. PS did not induce apoptosis but caused a reduction in metabolic activity (measured as 3-(4,5-dimethyldiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) conversion) and a slowing down of cell growth. The lower MTT conversion and the similar lactate dehydrogenase release could suggest that PS more efficiently target mitochondria than plasma membrane integrity. The replacement of cholesterol by PS could also have caused the observed slowing down of cell growth and the reduction in metabolic activity, which could rely on the PS increase, cholesterol decrease, or both. The present study is the first report on the effect of PS in cardiac cells, and although it is difficult to translate the obtained results to the health of heart tissue, it raises concerns about the safety of long-term exposure to physiologically relevant PS concentrations.

Role of cereal type and processing in whole grain in vivo protection from oxidative stress.

The reduced risk of chronic diseases related to whole grain consumption is in part attributed to their high antioxidant content. Many studies have been performed on the in vitro antioxidant capacity of cereals, but in vivo studies are necessary. We have evaluated and compared the effect of whole grain durum wheat bread and whole grain Kamut khorasan bread on the oxidative status in rats. Two different bread-making processes were used for whole grain Kamut khorasan, sourdough and baker's yeast. After 7 weeks on the experimental diets rats were divided into two subgroups, one receiving an oxidative stress by doxorubicin injection. Our results evidenced both wheat durum and Kamut khorasan as good sources of antioxidants, and a lower oxidative state in rats fed the cereal-based diets. Furthermore, Kamut khorasan bread fed animals had a better response to stress than wheat durum fed, especially when a sourdough bread was supplied. Although further studies are needed, data herein reported suggest whole grains, particularly whole ancient grains, as a safe and convenient way of increasing antioxidant protection.

NMR comparison of in vitro digestion of Parmigiano Reggiano cheese aged 15 and 30 months.

The content of essential amino acids is an important aspect for determining the nutritional value of food proteins, but their digestibility is also a key property, deeply affected by food processing. The production of Parmigiano Reggiano cheese is closely related to the nutritional quality of the final product; in particular the high digestibility of its proteins is claimed to be proportional to cheese aging. Two different kinds of Parmigiano Reggiano, young (aged 15 months) and old (aged 30 months), were separately digested using an in vitro system that simulates digestive processes in the mouth, stomach and small intestine. Samples were collected at different stages of digestion and the process of protein hydrolysis was explored and compared by low-field (LF) and high-resolution (HR) NMR, together with other biochemical methods. HR-NMR allowed to simultaneously observe the quantity of free amino acids, peptides and proteins, also giving for these latter qualitative information about their dimension. LF-NMR, instead, gave the possibility to observe digestion with no treatments whatsoever, thus representing a technique suitable for on-line measurements. The results pointed out that cheeses with different aging times, although starting from distinct initial compositions, conclude digestion in a similar way, in terms of free amino acids and small organic compounds, but evolve with different kinetics of hydrolysis and peptide formation, discriminating the young from the old cheese.