Dietary phenolics and their microbial metabolites are poor inhibitors of trimethylamine oxidation to trimethylamine N-oxide by hepatic flavin monooxygenase 3.

High circulating levels of trimethylamine N-oxide (TMAO) have been associated with cardiovascular disease risk. TMAO is formed through a microbiome-host pathway utilizing primarily dietary choline as a substrate. Specific gut microbiota transform choline into trimethylamine (TMA), and, when absorbed, host hepatic flavin-containing monooxygenase 3 (FMO3) oxidizes TMA into TMAO. Chlorogenic acid and its metabolites reduce microbial TMA production in vitro. However, little is known regarding the potential for chlorogenic acid and its bioavailable metabolites to inhibit the last step: hepatic conversion of TMA to TMAO. We developed a screening methodology to study FMO3-catalyzed production of TMAO from TMA. HepG2 cells were unable to oxidize TMA into TMAO due to their lack of FMO3 expression. Although Hepa-1 cells did express FMO3 when pretreated with TMA and NADPH, they lacked enzymatic activity to produce TMAO. Rat hepatic microsomes contained active FMO3. Optimal reaction conditions were: 50 µM TMA, 0.2 mM NADPH, and 33 µL microsomes/mL reaction. Methimazole (a known FMO3 competitive substrate) at 200 µM effectively reduced FMO3-catalyzed conversion of TMA to TMAO. However, bioavailable chlorogenic acid metabolites did not generally inhibit FMO3 at physiological (1 µM) nor supra-physiological (50 µM) doses. Thus, the effects of chlorogenic acid in regulating TMAO levels in vivo are unlikely to occur through direct FMO3 enzyme inhibition. Potential effects on FMO3 expression remain unknown. Intestinal inhibition of TMA production and/or absorption are thus likely their primary mechanisms of action.

Redox phospholipidomics analysis reveals specific oxidized phospholipids and regions in the diabetic mouse kidney.

While it is generally accepted that oxidative stress impacts the diabetic kidney and contributes to pathogenesis, there is a substantial lack of knowledge about the molecular entity and anatomic location of a variety of reactive species. Here we provide a novel "oxidative stress map" of the diabetic kidney - the first of its kind, and identify specific, oxidized and other reactive lipids and their location. We used the db/db mouse model and Desorption Electrospray Ionization (DESI) mass spectrometry combined with heatmap image analysis. We analyzed a comprehensive array of phospholipid peroxide species in normal (db/m) and diabetic (db/db) kidneys using DESI imaging. Oxilipidomics heatmaps of the kidneys were generated focusing on phospholipids and their potential peroxidized products. We identified those lipids that undergo peroxidation in diabetic nephropathy. Several phospholipid peroxides and their spatial distribution were identified that were specific to the diabetic kidney, with significant enrichment in oxygenated phosphatidylethanolamines (PE) and lysophosphatidylethanolamine. Beyond qualitative and semi-quantitative information about the targets, the approach also reveals the anatomic location and the extent of lipid peroxide signal propagation across the kidney. Our approach provides novel, in-depth information of the location and molecular entity of reactive lipids in an organ with a very heterogeneous landscape. Many of these reactive lipids have been previously linked to programmed cell death mechanisms. Thus, the findings may be relevant to understand what impact phospholipid peroxidation has on cell and mitochondria membrane integrity and redox lipid signaling in diabetic nephropathy.

Bioaccessibility, gut microbial metabolism and intestinal transport of phenolics from 100% Concord grape juice and whole grapes are similar in a simulated digestion and fecal fermentation model.

Phenolic rich 100% grape juice has been associated with many health benefits, but its place in dietary guidance is controversial relative to whole fruit. Direct comparisons of phenolic profiles and bioavailability between these food forms are needed. Phenolic bioaccessibility and metabolism from Concord (CG) and Niagara (NG) grapes and corresponding 100% juices were investigated using an in vitro digestion coupled with anaerobic gut fermentation model. Intestinal transport of resulting bioaccessible phenolics and microbial metabolites was estimated using a Caco-2 cell model. Total bioaccessible phenolics from both upper and lower digestion were similar (P > 0.05) between NG (400.9 ± 26.3 µmol per 100 g) and NGJ (349.5 ± 8.3 µmol per 100 g) and significantly different (P < 0.05) between CG (417.2 ± 24.4 µmol per 100 g) and CGJ (294.3 ± 45.4 µmol per 100 g) total cellular transport of phenolics was similar (P > 0.05) between whole grapes (89.4 ± 5.3 µmol per 100 g for CG, and 71.8 ± 2.4 µmol per 100 g for NG) and 100% juices (88.0 ± 5.6 µmol per 100 g for CGJ, and 85.3 ± 9.4 µmol per 100 g for NGJ). Differences were observed between the location of phenolic metabolism, bioaccessibility and subsequent cellular transport of individual phenolics between grapes and juice matrices. Specifically, greater amounts of phenolics were transported from grape juices than whole grapes from the upper tract. However, cumulative bioaccessibility and transport from upper and lower GI digestion/fermentation together indicates that the absorbable phenolics from 100% grape juice is similar to that of whole grapes, suggesting that phenolic-mediated health benefits from consumption of whole fruit and juice may be similar.

Bioaccessibility and intestinal cell uptake of carotenoids and chlorophylls differ in powdered spinach by the ingredient form as measured using in vitro gastrointestinal digestion and anaerobic fecal fermentation models.

Insights into food matrix factors impacting bioavailability of bioactive carotenoids and chlorophylls from fruits and vegetable ingredients are essential to understanding their ability to promote health. The stability and bioaccessibility of carotenoids and chlorophylls were assessed from dehydrated, spray-dried, freeze-dried and fresh spinach ingredient forms using in vitro models simulating upper gastrointestinal (GI) digestion and lower GI anaerobic fecal fermentation. Intestinal transport of bioaccessible bioactives from both upper and lower GI compartments was assessed using the Caco-2 human intestinal cell model. Differences in carotenoid and chlorophyll contents were observed between ingredient forms and these influenced bioaccessibility. Lower carotenoid and chlorophyll contents in spray dried spinach resulted in the lowest total bioaccessible content among all spinach treatments (5.8 ± 0.2 µmoles per g DW carotenoid and chlorophyll). The total bioaccessible content was statistically similar between freeze-dried (12.5 ± 0.6 µmoles per g DW), dehydrated (12.5 ± 3.2 µmoles per g DW), and fresh spinach (14.2 ± 1.2 µmoles per g DW). Post anaerobic fermentation, cellular accumulation of carotenoids was higher (17.57-19.52 vs. 5.11-8.56%), while that of chlorophylls was lower (3.05-5.27 vs. 5.25-6.44%), compared to those observed following upper GI digestion. Collectively, these data suggest that spinach forms created by various drying technologies deliver similar levels of bioaccessible spinach bioactives and that the lower GI tract may serve as a site for significant absorption fostered by interactions with gut microbial communities that liberate additional bioactives from the spinach matrix.

Prune supplementation for 12 months alters the gut microbiome in postmenopausal women.

Prunes have health benefits, particularly in postmenopausal women. It is likely that the gut microbiome mediates some of these effects, but its exact role remains to be elucidated. This study aims to characterize the effect of prune supplementation on the gut microbiome of postmenopausal women. The fecal microbiome of 143 postmenopausal women ages 55-75 who met the compliance criteria in a randomized controlled trial of a 12-month dietary intervention in one of three treatment groups - no prunes (n = 52), 50 g prunes per day (n = 54), or 100 g prunes per day (n = 37) - was characterized at baseline and at the 12-month endpoint using 16S rRNA gene sequencing and QIIME2. Additional outcomes included assessment of select urinary phenolic metabolites and inflammatory markers. After 12 months, microbiomes of women consuming 50 g prunes had decreased evenness in bacteria taxa (Pielou's Evenness, Kruskal-Wallis p = 0.026). Beta diversity comparisons indicated significant differences in microbiomes among prune treatments (Bray-Curtis PERMANOVA, p = 0.005), and the effect was different at each prune dose (p = 0.057). Prunes enriched some bacterial taxa such as the family Lachnospiraceae (LEfSe LDA = 4.5). Some taxa correlated with urinary phenolic metabolites and inflammatory markers. Blautia negatively correlated with total urinary phenolics (r = -0.25, p = 0.035) and Lachnospiraceae UCG-001 negatively correlated with plasma concentrations of IL-1ß (r = -0.29, p = 0.002). Differing gut microbiomes and correlation of some taxa with select phenolic metabolites and inflammatory markers, particularly Lachnospiraceae, after prune consumption suggest a potential mechanism mediating health effects. The microbiome differences at each dose may have implications for the use of prunes as a non-pharmacological whole food intervention for gut health.

Influence of simulated food and oral processing on carotenoid and chlorophyll in vitro bioaccessibility among six spinach genotypes.

Increasing the density of micronutrients and phytochemicals in vegetable foods through plant breeding and processing is of value for consumers. However, the extent to which interactions between genetics and processing (G × P) can be leveraged for green leafy vegetables to improve the delivery of such compounds is unknown. Using spinach as a model, a three-phase in vitro digestion method with and without simulated oral processing (mastication) and coupling to a Caco-2 human intestinal cell culture model was used to determine whether bioaccessibility and intestinal uptake of carotenoids and chlorophylls can be modified from six spinach genotypes, fresh or processed as blanched, sterilized, and juiced products. Carotenoid and chlorophyll bioaccessibility varied significantly with the genotype (p < 0.001) and processing treatment (p < 0.001), with processing having a more profound influence on the bioaccessibility, decreasing micellarization of phytochemicals from juiced (25.8-29.3%), to fresh (19.5-27.9%), to blanched (14.9-20.5%), and sterilized spinach (10.4-13.0%). Oral mastication had a significant influence on the carotenoid bioaccessible content of sterilized spinach (0.3-0.5 µmoles per g DW) as compared to fresh spinach (0.1-0.3 µmoles per g DW), most likely due to the additive effect of thermal processing and mastication on facilitating digestive breakdown of the spinach matrix. Caco-2 accumulation of carotenoid and chlorophyll was modestly but significantly (<0.001) lower in fresh spinach (2.4%) compared to other treatment samples (3.7-4.8%). These results suggest that the genotype, processing treatment, and genotype × processing (G × P) interaction may affect carotenoid and chlorophyll bioaccessibility in spinach and that food processing remains a dominant factor in modulating the bioavailability of these phytochemicals.

Starch-phenolic complexes are built on physical CH-π interactions and can persist after hydrothermal treatments altering hydrodynamic radius and digestibility of model starch-based foods.

Leveraging phenolic complexation to optimize starch functionality and digestibility is restrained by the obscurity of their physicochemical nature and molecular basis. To define starch-phenolic complexes under hydrothermal treatments, maize amylopectin and potato starch were complexed with caffeic acid, ferulic acid and gallic acid. Starch hydrothermal stability and digestibility were measured by differential scanning calorimeter and Englyst's method, respectively. While monosaccharide compositions and glycosidic linkages were analyzed by GC-MS, hydrodynamic radius and proton magnetic resonance of gelatinized complexes were measured by dynamic light scattering and NMR respectively. Compared with native starches, starch-phenolic complexes were not chemically modified and had modestly lower estimated glycemic indexes and significantly lower gelatinization temperatures (p < 0.05). Starch-phenolic complexes also had significantly lower levels of phenolic proton intensities and hydrodynamic radii relative to the control starch-phenolic mixtures (p < 0.05). These results suggested that phenolics may complex with starch through non-covalent CH-π bonds along α-(1 → 4) glycosidic chains.

Modulating Phenolic Bioaccessibility and Glycemic Response of Starch-Based Foods in Wistar Rats by Physical Complexation between Starch and Phenolic Acid.

This study assessed the impact of caffeic and ferulic acid complexation with maize amylopectin or potato starch on glycemic parameters. In comparison to starch-phenolic mixtures, starch-phenolic complexes resulted in significant modification of phenolic bioaccessibility and cellular uptake (p < 0.05). In addition, glucose release from in vitro digestion of starch was modestly reduced in the complexes compared to native starch alone (21.2-26.8 versus 29.8-30.5 mM). Furthermore, intestinal glucose transport, assessed in Caco-2 cell monolayers, was not affected by the presence of complexes (82.4-124 versus 100% at 90 min). However, a reduced glycemic response was evident in a Wistar rat model, with significant reduction in 240 min of blood glucose area under the curve following oral administration of the potato starch-ferulic acid complex compared to native potato starch (26 170 ± 556 versus 28 951 ± 486 mg min dL-1; p < 0.001). These alterations were attributed to complexation-induced resistant starch formation and phenolic entrapment, providing an alternative mechanistic approach to modulate glycemic properties of starch-based foods.

Comparative assessment of phenolic bioaccessibility from 100% grape juice and whole grapes.

Juicing of grapes includes contact with phenolic rich seeds and skins that otherwise rely on maceration for phenolic release. To understand if 100% grape juice can provide a matrix with highly bioaccessible phenolics relative to whole fruit, differences in phenolic content and bioaccessibility from commonly consumed table, Concord (CG) and Niagara (NG) grapes and their 100% juices were compared. Phenolic contents in whole grapes and 100% juices were assayed by LC-MS prior to in vitro digestion to determine phenolic bioaccessibility. Phenolic compounds were concentrated in CG and NG seeds as flavan-3-ols (222.2-285.5 mg per 100 g fw). CG skins were rich in anthocyanins (201.4 mg per 100 g fw) and flavonols (15.5 mg per 100 g fw). Product form had a significant impact on content (p < 0.01), relative bioaccessibility, and absolute bioaccessibility (p < 0.01). CG had a higher total phenolic content (21.9-50.7 mg per 100 g fw) compared to CGJ (5.8 mg per 100 g fw), though NG (4.9-10.8 mg per 100 g fw) was similar in phenolic content to NGJ (9.4-10.8 mg per 100 g fw). Absolute bioaccessibility of total phenolics from CGJ (5.2 mg per 100 g fw) was similar to CG (2.6-9.6 mg per 100 g fw), while NGJ (5.1-5.7 mg per 100 g fw) had higher bioaccessible phenolic content than NG (0.8-1.1 mg per 100 g fw). Differences in bioaccessible content were driven by high relative bioaccessibility of anthocyanins in CGJ (86-135%) compared to CG (14-39%) as well as for flavan-3-ols and phenolic acids from CGJ/NGJ (48-101; 39-85%) compared to CG/NG (0-3; 9-67%). Comparisons between juices and table grapes followed similar trends. A greater fraction of skin and seed phenolics was extracted through juicing and made bioaccessible, making 100% grape juice and whole fruit similar in phenolic delivery to consumers.

Development of a genetic framework to improve the efficiency of bioactive delivery from blueberry.

In the present study, we applied a novel high-throughput in vitro gastrointestinal digestion model to phenotype bioaccessibility of phenolics in a diverse germplasm collection representing cultivated highbush blueberries. Results revealed significant (P < 0.05) differences between accessions, years, and accession by year interaction for relative and absolute bioaccessibility of flavonoids and phenolic acids. Broad sense heritability estimates revealed low to moderate inheritances of relative and absolute bioaccessibility, suggesting that besides environmental variables, genetics factors could control bioaccessibility of phenolics. Acylated anthocyanins had significantly higher relative bioaccessibility than non-acylated anthocyanins. Correlation analysis indicated that relative bioaccessibility did not show significant association with fruit quality or raw concentration of metabolites. The study also identified accessions that have high relative and absolute bioaccessibility values. Overall, combining the bioaccessibility of phenolics with genetic and genomic approaches will enable the identification of genotypes and genetic factors influencing these traits in blueberry.

Banana flour phenolics inhibit trans-epithelial glucose transport from wheat cakes in a coupled in vitro digestion/Caco-2 cell intestinal model.

Some fruit phenolics are reported to attenuate intestinal glucose transport through inhibitory action at the luminal brush border membrane. This effect may contribute, in part, to the ability of flavonoid-rich food to regulate glucose homeostasis of meals rich in available carbohydrates. For the first time, the potential of green banana flours to inhibit transepithelial glucose transport was investigated in the context of a model starchy meal (wheat cake) using a simulated digestion/Caco-2 human intestinal cell model. A 10% replacement of wheat flour with any of the four banana flours (native and extruded oven-dried and freeze-dried) resulted in cakes with significantly higher total phenolics (68-198 µg per 100 g, p < 0.05), especially using extruded banana flour (197-198 µg per 100 g), as measured by LC/MS. Banana cakes, especially those containing oven-dried and/or extruded banana flours, exhibited from 45.0 to 54.5% higher glucose transport inhibition than the control cake. Interestingly, the digesta of cakes made with freeze-dried and extruded banana flour presented a significantly higher phenolic content (1116 µM, p < 0.05) than the other digesta (745-791 µM), while the phenolic content in control digesta was only 548 µM. These results suggested that the amounts of quercetin and myricetin, even in traces, were critical determinants of glucose transport inhibition.