IF1 ablation prevents ATP synthase oligomerization, enhances mitochondrial ATP turnover and promotes an adenosine-mediated pro-inflammatory phenotype.

ATPase Inhibitory Factor 1 (IF1) regulates the activity of mitochondrial ATP synthase. The expression of IF1 in differentiated human and mouse cells is highly variable. In intestinal cells, the overexpression of IF1 protects against colon inflammation. Herein, we have developed a conditional IF1-knockout mouse model in intestinal epithelium to investigate the role of IF1 in mitochondrial function and tissue homeostasis. The results show that IF1-ablated mice have increased ATP synthase/hydrolase activities, leading to profound mitochondrial dysfunction and a pro-inflammatory phenotype that impairs the permeability of the intestinal barrier compromising mouse survival upon inflammation. Deletion of IF1 prevents the formation of oligomeric assemblies of ATP synthase and alters cristae structure and the electron transport chain. Moreover, lack of IF1 promotes an intramitochondrial Ca2+ overload in vivo, minimizing the threshold to Ca2+-induced permeability transition (mPT). Removal of IF1 in cell lines also prevents the formation of oligomeric assemblies of ATP synthase, minimizing the threshold to Ca2+-induced mPT. Metabolomic analyses of mice serum and colon tissue highlight that IF1 ablation promotes the activation of de novo purine and salvage pathways. Mechanistically, lack of IF1 in cell lines increases ATP synthase/hydrolase activities and installs futile ATP hydrolysis in mitochondria, resulting in the activation of purine metabolism and in the accumulation of adenosine, both in culture medium and in mice serum. Adenosine, through ADORA2B receptors, promotes an autoimmune phenotype in mice, stressing the role of the IF1/ATP synthase axis in tissue immune responses. Overall, the results highlight that IF1 is required for ATP synthase oligomerization and that it acts as a brake to prevent ATP hydrolysis under in vivo phosphorylating conditions in intestinal cells.

The mitochondrial negative regulator MCJ modulates the interplay between microbiota and the host during ulcerative colitis.

Recent evidences indicate that mitochondrial genes and function are decreased in active ulcerative colitis (UC) patients, in particular, the activity of Complex I of the electron transport chain is heavily compromised. MCJ is a mitochondrial inner membrane protein identified as a natural inhibitor of respiratory chain Complex I. The induction of experimental colitis in MCJ-deficient mice leads to the upregulation of Timp3 expression resulting in the inhibition of TACE activity that likely inhibits Tnf and Tnfr1 shedding from the cell membrane in the colon. MCJ-deficient mice also show higher expression of Myd88 and Tlr9, proinflammatory genes and disease severity. Interestingly, the absence of MCJ resulted in distinct microbiota metabolism and composition, including a member of the gut community in UC patients, Ruminococcus gnavus. These changes provoked an effect on IgA levels. Gene expression analyses in UC patients showed decreased levels of MCJ and higher expression of TIMP3, suggesting a relevant role of mitochondrial genes and function among active UC. The MCJ deficiency disturbs the regulatory relationship between the host mitochondria and microbiota affecting disease severity. Our results indicate that mitochondria function may be an important factor in the pathogenesis. All together support the importance of MCJ regulation during UC.

A Foodomics Approach: CE-MS for Comparative Metabolomics of Colon Cancer Cells Treated with Dietary Polyphenols.

The potential of capillary electrophoresis-mass spectrometry (CE-MS) for metabolomics is demonstrated through the analysis of metabolites from human HT29 colon cancer cells treated and non-treated with dietary polyphenols. Prior to CE-MS analysis, four different metabolite purification strategies are investigated. Namely, the results obtained after methanol deproteinization, ultrafiltration, and two solid-phase extraction methods using C18 and polymer-based cartridges are described. These generic methods can have broad applications to analyze metabolites in a large variety of matrices and fields, including the new Foodomics area.

Metabolomics study of early metabolic changes in hepatic HepaRG cells in response to rosemary diterpenes exposure.

Rosemary diterpenes have demonstrated diverse biological activities, such as anti-cancer, antiinflammatory, as well as other beneficial effects against neurological and metabolic disorders. In particular, carnosic acid (CA), carnosol (CS) and rosmanol (RS) diterpenes have shown interesting results on anti-cancer activity. However, little is known about the toxic effects of rosemary diterpenes at the concentrations needed to exert their antiproliferative effect on cancer cells. In our study, CA, CS and RS exhibited a concentration-dependent effect on cell viability of two human colon cancer cell lines (HT-29 and HCT116) after 24 h exposure. HT-29 cell line was more resistant to the inhibitory effect of the three diterpenes than HCT116 cell line. Among the three diterpenes, RS exerted the strongest effect in both cell lines. To investigate the hepatotoxicity of CA, CS and RS, undifferentiated and differentiated HepaRG cells were exposed to increasing concentrations of the diterpenes (from 10 to 100 µM). Differentiated cells were found to be more resistant to the toxic activity of the three diterpenes than undifferentiated HepaRG, probably related to a higher detoxifying function of differentiated HepaRG cells compared with the undifferentiated cells. The metabolic profiles of differentiated HepaRG cells in response to CA, CS and RS were examined to determine biochemical alterations and deepen the study of the effects of rosemary phenolic diterpenes at molecular level. A multiplatform metabolomics study based on liquid- and gas-chromatography hyphenated to high resolution mass spectrometry revealed that rosemary diterpenes exerted different effects when HepaRG cells were treated with the same concentration of each diterpene. RS revealed a greater metabolome alteration followed by CS and CA, in agreement with their observed cytotoxicity. Metabolomics provided valuable information about early events in the metabolic profiles after the treatment with the investigated diterpenes from rosemary.

An Ultrahigh-Performance Liquid Chromatography-Time-of-Flight Mass Spectrometry Metabolomic Approach to Studying the Impact of Moderate Red-Wine Consumption on Urinary Metabolome.

Moderate red-wine consumption has been widely described to exert several benefits in human health. This is mainly due to its unique content of bioactive polyphenols, which suffer several modifications along their pass through the digestive system, including microbial transformation in the colon and phase-II metabolism, until they are finally excreted in urine and feces. To determine the impact of moderate wine consumption in the overall urinary metabolome of healthy volunteers ( n = 41), samples from a red-wine interventional study (250 mL/day, 28 days) were investigated. Urine (24 h) was collected before and after intervention and analyzed by an untargeted ultrahigh-performance liquid chromatography-time-of-flight mass spectrometry metabolomics approach. 94 compounds linked to wine consumption, including specific wine components (tartaric acid), microbial-derived phenolic metabolites (5-(dihydroxyphenyl)-γ-valerolactones and 4-hydroxyl-5-(phenyl)-valeric acids), and endogenous compounds were identified. Also, some relationships between parallel fecal and urinary metabolomes are discussed.

GC-MS based metabolomics of colon cancer cells using different extraction solvents.

The increasing incidence of colorectal cancer enforces the development of novel methodologies and protocols to deepen in the molecular mechanisms that govern disease pathophysiological events. The aim of this work is to deepen in the optimum metabolite extraction protocol from adherent mammalian cells of colon cancer for high throughput metabolomics using gas chromatography coupled to mass spectrometry (GC-MS). GC-MS results showed that metabolic information obtained from colon cancer cells was highly dependent on metabolite extraction selection, which at the same time is extremely influenced by the analytical platform. A further purpose of this investigation is to uncover an unexplored portion of HT-29 colon cancer cells metabolome, complementary to other already explored by CE-MS and LC-MS methods. At this respect, a total of 150 metabolites were identified in HT-29 colon cancer cells by GC-MS. The extraction protocol with acetonitrile-isopropanol-water was the most appropriate for fatty acids and related pathways analysis. Most of the metabolites involved in pathways of amino acids, glutathione, amino sugars and other polar metabolites were better extracted with acidified water, although water extraction showed the best overall reproducibility. Although pathways involving nitrogenous bases could be investigated using organic or aqueous extracts, a higher number of metabolites involved in these pathways were identified in the aqueous extracts. In addition, metabolite extraction protocol was observed to be crucial for the determination of potentially interesting clusters of metabolites.

The immunosuppressive effect of the tick protein, Salp15, is long-lasting and persists in a murine model of hematopoietic transplant.

Salp15, a salivary protein of Ixodes ticks, inhibits the activation of naïve CD4 T cells. Treatment with Salp15 results in the inhibition of early signaling events and the production of the autocrine growth factor, interleukin-2. The fate of the CD4 T cells activated in the presence of Salp15 or its long-term effects are, however, unknown. We now show that Salp15 binding to CD4 is persistent and induces a long-lasting immunomodulatory effect. The activity of Salp15 results in sustained diminished cross-antigenic antibody production even after interruption of the treatment with the protein. Transcriptionally, the salivary protein provokes an acute effect that includes known activation markers, such as Il2 or Cd44, and that fades over time. The long-term effects exerted by Salp15 do not involve the induction of either anergy traits nor increased populations of regulatory T cells. Similarly, the treatment with Salp15 does not result in B cell anergy or the generation of myeloid suppressor cells. However, Salp15 induces the increased expression of the ectoenzyme, CD73, in regulatory T cells and increased production of adenosine. Our study provides a profound characterization of the immunomodulatory activity of Salp15 and suggests that its long-term effects are due to the specific regulation of CD73.

Nano-liquid Chromatography-orbitrap MS-based Quantitative Proteomics Reveals Differences Between the Mechanisms of Action of Carnosic Acid and Carnosol in Colon Cancer Cells.

Carnosic acid (CA) and carnosol (CS) are two structurally related diterpenes present in rosemary herb (Rosmarinus officinalis). Although several studies have demonstrated that both diterpenes can scavenge free radicals and interfere in cellular processes such as cell proliferation, they may not necessarily exert the same effects at the molecular level. In this work, a shotgun proteomics study based on stable isotope dimethyl labeling (DML) and nano-liquid chromatography-tandem mass spectrometry (nano-LC-MS/MS) has been performed to identify the relative changes in proteins and to gain some light on the specific molecular targets and mechanisms of action of CA and CS in HT-29 colon cancer cells. Protein profiles revealed that CA and CS induce different Nrf2-mediated response. Furthermore, examination of our data revealed that each diterpene affects protein homeostasis by different mechanisms. CA treatment induces the expression of proteins involved in the unfolded protein response in a concentration dependent manner reflecting ER stress, whereas CS directly inhibits chymotrypsin-like activity of the 20S proteasome. In conclusion, the unbiased proteomics-wide method applied in the present study has demonstrated to be a powerful tool to reveal differences on the mechanisms of action of two related bioactive compounds in the same biological model.

Capillary Electrophoresis in Food and Foodomics.

Quality and safety assessment as well as the evaluation of other nutritional and functional properties of foods imply the use of robust, efficient, sensitive, and cost-effective analytical methodologies. Among analytical technologies used in the fields of food analysis and foodomics, capillary electrophoresis (CE) has generated great interest for the analyses of a large number of compounds due to its high separation efficiency, extremely small sample and reagent requirements, and rapid analysis. The introductory section of this chapter provides an overview of the recent applications of capillary electrophoresis (CE) in food analysis and foodomics. Relevant reviews and research articles on these topics are tabulated including papers published in the period 2011-2014. In addition, to illustrate the great capabilities of CE in foodomics the chapter describes the main experimental points to be taken into consideration for a metabolomic study of the antiproliferative effect of carnosic acid (a natural diterpene found in rosemary) against HT-29 human colon cancer cells.

Foodomics study on the effects of extracellular production of hydrogen peroxide by rosemary polyphenols on the anti-proliferative activity of rosemary polyphenols against HT-29 cells.

A number of studies have demonstrated a strong association between the antioxidant properties of rosemary polyphenols and their chemoprotective activity. However, the prooxidant effects of rosemary polyphenols have been rarely reported. In this work, a foodomics study is performed to investigate the in vitro autooxidation of carnosic acid (CA), carnosol (CS) and a polyphenol-enriched rosemary extract (SC-RE) in cell culture conditions. The results revealed that rosemary polyphenols autooxidation in culture medium generated H2 O2 at different rates. Generated H2 O2 levels by SC-RE and CA, but not CS, were correlated with intracellular reactive oxygen species (ROS) generation in HT-29 cells, and were partially involved in their anti-proliferative effect in this cell line. These compounds also induced different effects on glutathione metabolism. Results also indicated that high extracellular H2 O2 concentrations, resulting of using high (45 µg/mL) SC-RE concentration in culture media, exerted some artifactual effects related with cell cycle, but they did not influence the expression of relevant molecular biomarkers of stress.

Recent advances in the application of capillary electromigration methods for food analysis and Foodomics.

This review work presents and discusses the main applications of capillary electromigration methods in food analysis and Foodomics. Papers that were published during the period February 2013-February 2015 are included following the previous review by Garcia-Cañas et al. (Electrophoresis, 2014, 35, 147-169). Analysis by CE of a large variety of food-related molecules with different chemical properties, including amino acids, hazardous amines, peptides, proteins, phenols, polyphenols, lipids, carbohydrates, DNAs, vitamins, toxins, contaminants, pesticides, residues, food additives, as well as small organic and inorganic compounds. This work includes recent results on food quality and safety, nutritional value, storage, bioactivity, as well as applications of CE for monitoring food processing. The use, among other CE developments, of microchips, CE-MS, and chiral CE in food analysis and Foodomics is also discussed.

Metabolomics of adherent mammalian cells by capillary electrophoresis-mass spectrometry: HT-29 cells as case study.

In this work, the optimization of an effective protocol for cell metabolomics is described with special emphasis in the sample preparation and subsequent analysis of intracellular metabolites from adherent mammalian cells by capillary electrophoresis-mass spectrometry. As case study, colon cancer HT-29 cells, a human cell model to investigate colon cancer, are employed. The feasibility of the whole method for cell metabolomics is demonstrated via a fast and sensitive profiling of the intracellular metabolites HT-29 cells by capillary electrophoresis-time-of-flight mass spectrometry (CE-TOF MS). The suitability of this methodology is further corroborated through the examination of the metabolic changes in the polyamines pathway produced in colon cancer HT-29 cells by difluoromethylornithine (DFMO), a known potent ornithine decarboxylase inhibitor. The selection of the optimum extraction conditions allowed a higher sample volume injection that led to an increase in CE-TOF MS sensitivity. Following a non-targeted metabolomics approach, 10 metabolites (namely, putrescine, ornithine, gamma-aminobutyric acid (GABA), oxidized and reduced glutathione, 5'-deoxy-5'-(methylthio)adenosine, N-acetylputrescine, cysteinyl-glycine, spermidine and an unknown compound) were found to be significantly altered by DFMO (p<0.05) in HT-29 cells. In addition to the effect of DFMO on polyamine metabolism, minor modifications of other metabolic pathways (e.g., related to intracellular thiol redox state) were observed.

Potential of prodendronic polyamines with modulated segmental charge density as novel coating for fast and efficient analysis of peptides and basic proteins by CE and CE-MS.

In this work, the suitability of a new polymer family has been investigated as capillary coatings for the analysis of peptides and basic proteins by CE. This polymer family has been designed to minimize or completely prevent protein-capillary wall interactions and to modify the EOF. These coating materials are linear polymeric chains bearing as side cationizable moiety a dentronic triamine derived from N,N,N',N'-tetraethyldiethylenetriamine (TEDETA), which is linked to the backbone through a spacer (unit labeled as TEDETAMA). Four different polymers have been prepared and evaluated: a homopolymer which comprised only of those cationizable repetitive units of TEDETAMA, and three copolymers that randomly incorporate TEDETAMA together with neutral hydrosoluble units of N-(2-hydroxypropyl) methacrylamide (HPMA) at different molar percentages (25:75, 50:50 and 75:25). It has been demonstrated that the composition of the copolymers influences the EOF and therefore the separation of the investigated biopolymers. Among the novel polymers studied, poly-(TEDETAMA-co-HPMA) 50:50 copolymer was successfully applied as coating material of the inner capillary surface in CE-UV and CE-MS, providing EOF reversing together with fast and efficient baseline separation of peptides and basic proteins. Finally, the feasibility of the polymer-coated capillary was shown through the analysis of lysozyme in a cheese sample.

Faecal metabolomic fingerprint after moderate consumption of red wine by healthy subjects.

Faecal metabolome contains information on the metabolites found in the intestine, from which knowledge about the metabolic function of the gut microbiota can be obtained. Changes in the metabolomic profile of faeces reflect, among others, changes in the composition and activity of the intestinal microorganisms. In an effort to improve our understanding of the biological effects that phenolic compounds (including red wine polyphenols) exert at the gut level, in this foodomic study we have undertaken a metabolome characterization of human faeces after moderate consumption of red wine by healthy subjects for 4 weeks. Namely, a nontargeted metabolomic approach based on the use of UHPLC-TOF MS was developed to achieve the maximum metabolite information on 82 human faecal samples. After data processing and statistical analysis, 37 metabolites were related to wine intake, from which 20 could be tentatively or completely identified, including the following: (A) wine compounds, (B) microbial-derived metabolites of wine polyphenols, and (C) endogenous metabolites and/or others derived from other nutrient pathways. After wine consumption, faecal metabolome was fortified in flavan-3-ols metabolites. Also, of relevance was the down regulation of xanthine and bilirubin-derived metabolites such as urobilinogen and stercobilin after moderate wine consumption. As far as we know, this is the first study of the faecal metabolome after wine intake.

Comprehensive foodomics study on the mechanisms operating at various molecular levels in cancer cells in response to individual rosemary polyphenols.

In this work, the contribution of carnosic acid (CA) and carnosol (CS), two major compounds present in rosemary, against colon cancer HT-29 cells proliferation is investigated using a comprehensive Foodomics approach. The Foodomics study reveals that CA induces transcriptional activation of genes that encode detoxifying enzymes and altered the expression of genes linked to transport and biosynthesis of terpenoids in the colon cancer cell line. Functional analysis highlighted the activation of the ROS metabolism and alteration of several genes involved in pathways describing oxidative degradation of relevant endogenous metabolites, providing new evidence about the transcriptional change induced by CA in HT-29 cells. Metabolomics analysis showed that the treatment with CA affected the intracellular levels of glutathione. Elevated levels of GSH provided additional evidence to transcriptomic results regarding chemopreventive response of cells to CA treatment. Moreover, the Foodomics approach was useful to establish the links between decreased levels of N-acetylputrescine and its degradation pathway at the gene level. The findings from this work and the predictions based on microarray data will help explore novel metabolic processes and potential signaling pathways to further elucidate the effect of CA in colon cancer cells.

Impact of glutathione-enriched inactive dry yeast preparations on the stability of terpenes during model wine aging.

The impact of the addition of glutathione-enriched Inactive dry yeast preparations (g-IDYs) on the stability of some typical wine terpenes (linalool, α-terpineol, ß-citronellol, and nerol) stored under accelerated oxidative conditions was evaluated in model wines. Additionally, the effects of a second type of IDY preparation with a different claim (fermentative nutrient) and the sole addition of commercial glutathione into the model wines were also assessed. Model wines were spiked with the low molecular weight fraction (<3 kDa permeate) isolated from the IDYs, avoiding the interaction of aroma compounds with other yeast components. An exhaustive chemical characterization of both IDY permeates was carried out by using targeted and nontargeted metabolomics approaches using CE-MS and FT-ICR-MS analytical platforms. The findings suggest that the addition of <3 kDa permeate isolated from any of the IDYs employed decreases the loss of typical wine terpenes in model wines submitted to accelerated aging conditions. The g-IDY preparation did indeed release reduced GSH into the model wines, although this compound did not seem exclusively related to the protective effect on some aroma compounds determined in both model wines. The presence of other sulfur-containing compounds from yeast origin in g-IDY, and also the presence of small yeast peptides, such as methionine/tryptophan/tyrosine-containing tripeptide in both types of IDYs, seemed to be related to the antioxidant activity determined in the two permeates and to the minor loss of some terpenes in the model wines spiked with them.

Capillary electrophoresis-mass spectrometry for Peptide analysis: target-based approaches and proteomics/peptidomics strategies.

In this chapter, the potential of capillary electrophoresis-mass spectrometry (CE-MS) for peptide analysis is demonstrated by the presentation of two different strategies typically followed in analysis of these biomolecules by CE-MS. The first one is a target-based approach and it is used to detect a toxic oligopeptide in a complex matrix. Namely, CE-MS using an ion trap MS analyzer is applied to detect and quantify γ-glutamyl-S-ethenyl-cysteine (GEC) bioactive dipeptide in a legume plant. The second one is a shotgun-like methodology used for proteomic analysis. Particularly, CE-MS using a time-of-flight MS analyzer is employed to investigate the substantial equivalence between a genetically modified (GM) variety of soybean and its conventional isogenic counterpart. These generic methods have broad applications for the analysis of peptides in a large variety of matrices, including applications in the area of proteomics and peptidomics.

Effect of dietary polyphenols on K562 leukemia cells: a Foodomics approach.

In this work, a global Foodomics strategy has been applied to study the antiproliferative effect of dietary polyphenols from rosemary on two human leukemia lines, one showing a drug-sensitive phenotype (K562), and another exhibiting a drug-resistant phenotype (K562/R). To this aim, whole-transcriptome microarray together with an MS-based nontargeted analytical approach (via CE-TOF MS and UPLC-TOF MS) have been employed to carry out transcriptomics and metabolomics analyses, respectively. Functional enrichment analysis was done using ingenuity pathway analysis (IPA) software as a previous step for a reliable interpretation of transcriptomic and metabolomic profiles. Rosemary polyphenols altered the expression of approximately 1% of the genes covered by the whole transcriptome microarray in both leukemia cell lines. Overall, differences in the transcriptional induction of a number of genes encoding phase II detoxifying and antioxidant genes, as well as differences in the metabolic profiles observed in the two leukemia cell lines suggest that rosemary polyphenols may exert a differential chemopreventive effect in leukemia cells with different phenotypes. IPA predictions on transcription factor analysis highlighted inhibition of Myc transcription factor function by rosemary polyphenols, which may explain the observed antiproliferative effect of rosemary extract in the leukemia cells. Metabolomics analysis suggested that rosemary polyphenols affected differently the intracellular levels of some metabolites in two leukemia cell sublines. Integration of data obtained from transcriptomics and metabolomics platforms was attempted by overlaying datasets on canonical (defined) metabolic pathways using IPA software. This strategy enabled the identification of several differentially expressed genes in the metabolic pathways modulated by rosemary polyphenols providing more evidences on the effect of these compounds.

CE/LC-MS multiplatform for broad metabolomic analysis of dietary polyphenols effect on colon cancer cells proliferation.

In this study, an analytical multiplatform is presented to carry out a broad metabolomic study on the anti-proliferative effect of dietary polyphenols on human colon cancer cells. CE, RP/UPLC, and HILIC/UPLC all coupled to TOF MS were combined to achieve a global metabolomic examination of the effect of dietary polyphenols on HT29 colon cancer cells. By the use of a nontargeted metabolomic approach, metabolites showing significant different expression after the polyphenols treatment were identified in colon cancer cells. It was demonstrated that this multianalytical platform provided extensive metabolic information and coverage due to its complementary nature. Differences observed in metabolic profiles from CE-TOF MS, RP/UPLC-TOF MS, and HILIC/UPLC-TOF MS can be mainly assigned to their different separation mechanisms without discarding the influence of the different tools used for data processing. Changes in glutathione metabolism with an enhanced reduced glutathione/oxidized glutathione (GSH/GSSG) ratio were detected in polyphenols-treated cells. Moreover, significant alterations in polyamines content with important implications in cancer proliferation were observed after the treatment with polyphenols. These results from metabolomics can explain the chemopreventive effect of the tested dietary polyphenols on colon cancer and may be of importance for future prevention and/or treatment of this disease.

Global Foodomics strategy to investigate the health benefits of dietary constituents.

A global methodology, called Foodomics, which allows carrying out a comprehensive evaluation of the health benefits of food ingredients is presented in this work. The new methodology is based on the combination of several analytical platforms and data processing for Transcriptomics, Proteomics and Metabolomics studies, allowing the determination of changes induced by food ingredients at molecular level. Both, the whole methodological development and its potential are presented through the investigation of a case study following a hypothesis-free strategy. Namely, the chemopreventive effect of polyphenols from rosemary was examined on the total gene, protein and metabolite expression in human HT29 colon cancer cells. Conclusions on the bioactivity of polyphenols against colon cancer cells based on the results from each single platform (Transcriptomics, Proteomics or Metabolomics) are compared with the conclusions based on the integration of the whole results from the three platforms, corroborating the interest of using a global integrative strategy as Foodomics. To our knowledge, although many papers and reviews have been published on this topic, this is the first time that Transcriptomics, Proteomics and Metabolomics platforms are put together to study the health benefits from dietary ingredients against colon cancer cells at gene, protein and metabolite level. Advantages, drawbacks and current challenges of this global analytical strategy are discussed in this work. The results from our study provide new insights on the biological mechanisms involved in the cancer risk reduction properties of dietary constituents.