Bioaccessibility and Caco-2 Cell Uptake of Iron Chlorophyllin Using A Biologically Relevant Digestion Model.

Iron deficiency remains a top nutrient deficiency worldwide. Iron chlorophyllin (IC), a compound structurally analogous to heme, utilizes the protoporphyrin ring of chlorophyll to bind iron. IC has previously been shown to deliver more iron to Caco-2 cells than FeSO4, the most common form prescribed for supplementation. However, previous test conditions used digestive conditions outside of those observed in humans. This study sought to assess IC bioaccessibility and Caco-2 cell uptake using physiologically relevant digestive solutions, pH, and incubation time, as compared to other iron sources (i.e. FeSO4, and hemoglobin (Hb)). Co-digestion with ascorbic acid (AA) and albumin was also investigated. Following gastric, duodenal, and jejunal digestion, IC-bound iron was less bioaccessible than iron delivered as FeSO4, and IC-bound iron was less bioaccessible than Hb-bound iron. IC-bound iron bioaccessibility was not affected by AA and was enhanced 2x with co-digested with a low dose of albumin. However, Caco-2 cell incubation with IC-containing digesta increased cell ferritin 2.5x more than FeSO4 alone, and less than Hb. IC with AA or with 400 mg albumin also increased cell ferritin more than IC alone, with the greatest increases observed following incubation of digesta containing IC + AA + 400 mg albumin. These results suggest IC can serve as an improved source of iron for supplementation as compared to FeSO4. These results also support further in vivo investigations of IC-based iron delivery in populations at risk of iron deficiency.

Re-remembering the influence of randomized ß-carotene on cognitive decline.

The wave of individuals impacted by dementia continues to rise rapidly as worldwide lifespan increases. Dietary strategies to slow cognitive decline and prolong time to clinical dementia remain understudied, but with potentially powerful public health consequences. Indeed, previously conducted large, randomized, placebo-controlled trials of micronutrients remain an under-leveraged resource to study changes in cognitive performance. As a motivating example, we highlight an ancillary report from the Physicians' Health Study, where subjects randomized to ß-carotene (a provitamin A carotenoid) had a more attenuated change in longitudinal global cognitive performance and verbal memory, as compared to subjects randomized to placebo. Despite mechanistic evidence from cell and animal studies supporting a vitamin A-mediated role in the biology associated with cognition, limited follow-up work has been conducted. We argue that dietary factors (including provitamin A) deserve a second look, leveraging multi-omic approaches, to elucidate how they may mitigate cognitive decline and dementia risk.

Carotenoids and Their Health Benefits as Derived via Their Interactions with Gut Microbiota.

Carotenoids have been related to a number of health benefits. Their dietary intake and circulating levels have been associated with a reduced incidence of obesity, diabetes, certain types of cancer, and even lower total mortality. Their potential interaction with the gut microbiota (GM) has been generally overlooked but may be of relevance, as carotenoids largely bypass absorption in the small intestine and are passed on to the colon, where they appear to be in part degraded into unknown metabolites. These may include apo-carotenoids that may have biological effects because of higher aqueous solubility and higher electrophilicity that could better target transcription factors, i.e., NF-κB, PPARγ, and RAR/RXRs. If absorbed in the colon, they could have both local and systemic effects. Certain microbes that may be supplemented were also reported to produce carotenoids in the colon. Although some bactericidal aspects of carotenoids have been shown in vitro, a few studies have also demonstrated a prebiotic-like effect, resulting in bacterial shifts with health-associated properties. Also, stimulation of IgA could play a role in this respect. Carotenoids may further contribute to mucosal and gut barrier health, such as stabilizing tight junctions. This review highlights potential gut-related health-beneficial effects of carotenoids and emphasizes the current research gaps regarding carotenoid-GM interactions.

Fat-soluble vitamin and phytochemical metabolites: Production, gastrointestinal absorption, and health effects.

Consumption of diets rich in fruits and vegetables, which provide some fat-soluble vitamins and many phytochemicals, is associated with a lower risk of developing certain degenerative diseases. It is well accepted that not only the parent compounds, but also their derivatives formed upon enzymatic or nonenzymatic transformations, can produce protective biological effects. These derivatives can be formed during food storage, processing, or cooking. They can also be formed in the lumen of the upper digestive tract during digestion, or via metabolism by microbiota in the colon. This review compiles the known metabolites of fat-soluble vitamins and fat-soluble phytochemicals (FSV and FSP) that have been identified in food and in the human digestive tract, or could potentially be present based on the known reactivity of the parent compounds in normal or pathological conditions, or following surgical interventions of the digestive tract or consumption of xenobiotics known to impair lipid absorption. It also covers the very limited data available on the bioavailability (absorption, intestinal mucosa metabolism) and summarizes their effects on health. Notably, despite great interest in identifying bioactive derivatives of FSV and FSP, studying their absorption, and probing their putative health effects, much research remains to be conducted to understand and capitalize on the potential of these molecules to preserve health.

A mechanism for red coloration in vertebrates.

Red coloration is a salient feature of the natural world. Many vertebrates produce red color by converting dietary yellow carotenoids into red ketocarotenoids via an unknown mechanism. Here, we show that two enzymes, cytochrome P450 2J19 (CYP2J19) and 3-hydroxybutyrate dehydrogenase 1-like (BDH1L), are sufficient to catalyze this conversion. In birds, both enzymes are expressed at the sites of ketocarotenoid biosynthesis (feather follicles and red cone photoreceptors), and genetic evidence implicates these enzymes in yellow/red color variation in feathers. In fish, the homologs of CYP2J19 and BDH1L are required for ketocarotenoid production, and we show that these enzymes are sufficient to produce ketocarotenoids in cell culture and when ectopically expressed in fish skin. Finally, we demonstrate that the red-cone-enriched tetratricopeptide repeat protein 39B (TTC39B) enhances ketocarotenoid production when co-expressed with CYP2J19 and BDH1L. The discovery of this mechanism of ketocarotenoid biosynthesis has major implications for understanding the evolution of color diversity in vertebrates.

Dietary Energy Intake and Presence of Aberrant Crypt Foci Are Associated with Phospholipid, Purine, and Taurine Metabolite Abundances in C57BL/6N Mouse Colon.

SCOPE: Colon metabolomes associated with high-fat (H) versus energy-restricted (E) diets in early colorectal cancer (CRC) models have never been directly compared. The objectives of this study are to elucidate metabolites associated with diet, aberrant crypt foci (ACF), and diet:ACF interaction, using a lifetime murine model. METHODS AND RESULTS: Three-week-old mice consumed control (C), E, or H initiation diets for 18 weeks. ACF formation is initiated weeks 16-21 with azoxymethane injections, followed by progression diet crossover (to C, E, or H) through week 60. Colon extracts are analyzed using ultra-high-performance liquid chromatography-high resolution mass spectrometry (UHPLC-HRMS). Metabolites associated with diet, ACF, or diet:ACF are determined using regression models (FDR-adjusted p-value <0.05). No metabolites are significantly associated with initiation diets, but concentrations of acylcarnitines and phospholipids are associated with C, E, and H progression diets. Purines, taurine, and phospholipids are associated with ACF presence. No significant associations between metabolites and diet:ACF interaction are observed. CONCLUSIONS: These results suggest that recent, rather than early-life, diet is more closely associated with the colon metabolome, particularly lipid metabolism. Results from this study also provide candidate biomarkers of early CRC development and provide support for the importance of early diet on influencing pre-CRC risk.

A novel family of secreted insect proteins linked to plant gall development.

In an elaborate form of inter-species exploitation, many insects hijack plant development to induce novel plant organs called galls that provide the insect with a source of nutrition and a temporary home. Galls result from dramatic reprogramming of plant cell biology driven by insect molecules, but the roles of specific insect molecules in gall development have not yet been determined. Here, we study the aphid Hormaphis cornu, which makes distinctive "cone" galls on leaves of witch hazel Hamamelis virginiana. We found that derived genetic variants in the aphid gene determinant of gall color (dgc) are associated with strong downregulation of dgc transcription in aphid salivary glands, upregulation in galls of seven genes involved in anthocyanin synthesis, and deposition of two red anthocyanins in galls. We hypothesize that aphids inject DGC protein into galls and that this results in differential expression of a small number of plant genes. dgc is a member of a large, diverse family of novel predicted secreted proteins characterized by a pair of widely spaced cysteine-tyrosine-cysteine (CYC) residues, which we named BICYCLE proteins. bicycle genes are most strongly expressed in the salivary glands specifically of galling aphid generations, suggesting that they may regulate many aspects of gall development. bicycle genes have experienced unusually frequent diversifying selection, consistent with their potential role controlling gall development in a molecular arms race between aphids and their host plants.

The Metabolism and Potential Bioactivity of Chlorophyll and Metallo-chlorophyll Derivatives in the Gastrointestinal Tract.

Chlorophyll is the vivid chromophore which imparts the green color to plant leaves, and is consumed by humans through green vegetables. The basic porphyrin structure of chlorophyll binds magnesium in plants, but can bind different divalent metals (e.g., copper, zinc, iron) facilitated by food processing techniques and/or chemical synthesis. This review covers the known elements of chlorophyll and metallo-chlorophyll absorption, distribution, metabolism, excretion in vitro and in vivo. The review discusses what is understood about the ability of these novel metallo-chlorophyll derivatives to deliver essential metals. This review also detail chlorophyll and metallo-chlorophyll toxin binding properties which largely occur during digestion, focusing on toxins including dioxins, heterocyclic aromatic amines, polyaromatic hydrocarbons, and aflatoxin. Finally, the article highlights the gaps in the understanding of the metabolism and metal and toxin-binding bioactivity of this family of molecules.

Comparison of the carotenoid profiles of commonly consumed smear-ripened cheeses.

The objective of this study was to identify the carotenoids imparting the orange colour to the rind, and pale yellow color to the core, of selected smear-ripened cheeses. The cheeses investigated were Charloe, Ashbrook, Taleggio, and Limburger, and were sourced from artisanal markets. Samples of the rind and core were extracted using non-polar solvents, followed by saponification to hydrolyze triglycerides to remove fatty acids, and to release carotenoid esters. Extracts were tested using ultra-high pressure liquid chromatograph-diode array detector-high resolution mass spectrometry (UHPLC-DAD-MS and -MS/MS), and identities of α- and ß-carotene, lycopene, and ß-cryptoxanthin confirmed with authentic standards. ß-Carotene was the predominant species in both the rind and core, absorbing ~70% of the signal at 450 nm in all cheese extracts tested, as well as minor quantities of ß-cryptoxanthin and α-carotene. Carotenoids unique to the rind included lycopene as well as the rare bacterial carotenoids previously identified in bacterial isolates of cheeses (i.e. decaprenoxanthin, sarcinaxanthin, and echinenone). This is the first detailed characterisation of carotenoids extracted directly from smear-ripened cheeses, and reveals that smear-ripened cheese can contribute both provitamin A carotenoids as well as C50 carotenoids to the human diet.

Temperature and storage time increase provitamin A carotenoid concentrations and bioaccessibility in post-harvest carrots.

The aim was to enhance provitamin A carotenoid (proVA CAR) concentrations and bioaccessibility in carrots by manipulating post-harvest factors. To that end, we assessed the effects of Ultraviolet-C light, pulsed light, storage temperature, and storage duration. We also measured CAR bioaccessibility by using an in vitro model. Pulsed light, but not Ultraviolet-C, treatment increased proVA CAR concentrations in the cortex tissue (p < 0.05). Longer storage times and higher temperatures also increased concentrations (p < 0.05). The maximal increase induced by pulsed light was obtained after treatment with 20 kJ/m2 and 3-days of storage at 20 °C. However, the positive effect induced by pulsed light decreased considerably over the next seven days. ProVA CAR in carrots with the highest concentrations also proved to be more bioaccessible (p < 0.05). Thus, proVA CAR concentrations in stored carrots can be increased significantly through storage times and temperatures. Pulsed light can also significantly increase proVA CAR concentrations, but only temporarily.

Catechin-Rich Green Tea Extract and the Loss-of-TLR4 Signaling Differentially Alter the Hepatic Metabolome in Mice with Nonalcoholic Steatohepatitis.

SCOPE: Catechin-rich green tea extract (GTE) limits inflammation in nonalcoholic steatohepatitis (NASH) consistent with a Toll-like receptor 4 (TLR4)-dependent mechanism. It is hypothesized that GTE supplementation during NASH will shift the hepatic metabolome similar to that attributed to the loss-of-TLR4 signaling. METHODS AND RESULTS: Wild-type (WT) and loss-of-function TLR4-mutant (TLR4mut ) mice are fed a high-fat diet containing 0% or 2% GTE for 8 weeks prior to performing untargeted mass spectrometry-based metabolomics on liver tissue. The loss-of-TLR4 signaling and GTE shift the hepatic metabolome away from that of WT mice. However, relatively few metabolites are altered by GTE in WT mice to the same extent as the loss-of-TLR4 signaling in TLR4mut mice. GTE increases acetyl-coenzyme A precursors and spermidine to a greater extent than the loss-of-TLR4 signaling. Select metabolites associated with thiol metabolism are similarly affected by GTE and the loss-of-TLR4 signaling. Glycerophospholipid catabolites are decreased by GTE, but are unaffected in TLR4mut mice. Conversely, the loss-of-TLR4 signaling but not GTE increases several bile acid metabolites. CONCLUSION: GTE limitedly alters the hepatic metabolome consistent with a TLR4-dependent mechanism. This suggests that the anti-inflammatory activities of GTE and loss-of-TLR4 signaling that regulate hepatic metabolism to abrogate NASH are likely due to distinct mechanisms.

Vitamin A and D Absorption in Adults with Metabolic Syndrome versus Healthy Controls: A Pilot Study Utilizing Targeted and Untargeted LC-MS Lipidomics.

SCOPE: Persons with metabolic syndrome (MetS) absorb less vitamin E than healthy controls. It is hypothesized that absorption of fat-soluble vitamins (FSV) A and D2 would also decrease with MetS status and that trends would be reflected in lipidomic responses between groups. METHODS AND RESULTS: Following soymilk consumption (501 IU vitamin A, 119 IU vitamin D2 ), the triglyceride-rich lipoprotein fractions (TRL) from MetS and healthy subjects (n = 10 age- and gender-matched subjects/group) are assessed using LC-MS/MS. Absorption is calculated using area under the time-concentration curves (AUC) from samples collected at 0, 3, and 6 h post-ingestion. MetS subjects have ≈6.4-fold higher median vitamin A AUC (retinyl palmitate) versus healthy controls (P = 0.07). Vitamin D2 AUC is unaffected by MetS status (P = 0.48). Untargeted LC-MS lipidomics reveals six phospholipids and one cholesterol ester with concentrations correlating (r = 0.53-0.68; P < 0.001) with vitamin A concentration. CONCLUSIONS: The vitamin A-phospholipid association suggests increased hydrolysis by PLB, PLRP2, and/or PLA2 IB may be involved in the trend in higher vitamin A bioavailability in MetS subjects. Previously observed differences in circulating levels of these vitamins are likely not due to absorption. Alternate strategies should be investigated to improve FSV status in MetS.

COLMAR Lipids Web Server and Ultrahigh-Resolution Methods for Two-Dimensional Nuclear Magnetic Resonance- and Mass Spectrometry-Based Lipidomics.

Accurate identification of lipids in biological samples is a key step in lipidomics studies. Multidimensional nuclear magnetic resonance (NMR) spectroscopy is a powerful analytical tool for this purpose as it provides comprehensive structural information on lipid composition at atomic resolution. However, the interpretation of NMR spectra of complex lipid mixtures is currently hampered by limited spectral resolution and the absence of a customized lipid NMR database along with user-friendly spectral analysis tools. We introduce a new two-dimensional (2D) NMR metabolite database "COLMAR Lipids" that was specifically curated for hydrophobic metabolites presently containing 501 compounds with accurate experimental 2D 13C-1H heteronuclear single quantum coherence (HSQC) chemical shift data measured in CDCl3. A new module in the public COLMAR suite of NMR web servers was developed for the (semi)automated analysis of complex lipidomics mixtures (http://spin.ccic.osu.edu/index.php/colmarm/index2). To obtain 2D HSQC spectra with the necessary high spectral resolution along both 13C and 1H dimensions, nonuniform sampling in combination with pure shift spectroscopy was applied allowing the extraction of an abundance of unique cross-peaks belonging to hydrophobic compounds in complex lipidomics mixtures. As shown here, this information is critical for the unambiguous identification of underlying lipid molecules by means of the new COLMAR Lipids web server, also in combination with mass spectrometry, as is demonstrated for Caco-2 cell and lung tissue cell extracts.

Enzymology of vertebrate carotenoid oxygenases.

Mammals and higher vertebrates including humans have only three members of the carotenoid cleavage dioxygenase family of enzymes. This review focuses on the two that function as carotenoid oxygenases. ß-Carotene 15,15'-dioxygenase (BCO1) catalyzes the oxidative cleavage of the central 15,15' carbon-carbon double of ß-carotene bond by addition of molecular oxygen. The product of the reaction is retinaldehyde (retinal or ß-apo-15-carotenal). Thus, BCO1 is the enzyme responsible for the conversion of provitamin A carotenoids to vitamin A. It also cleaves the 15,15' bond of ß-apocarotenals to yield retinal and of lycopene to yield apo-15-lycopenal. ß-Carotene 9',10'-dioxygenase (BCO2) catalyzes the cleavage of the 9,10 and 9',10' double bonds of a wider variety of carotenoids, including both provitamin A and non-provitamin A carotenoids, as well as the xanthophylls, lutein and zeaxanthin. Indeed, the enzyme shows a marked preference for utilization of these xanthophylls and other substrates with hydroxylated terminal rings. Studies of the phenotypes of BCO1 null, BCO2 null, and BCO1/2 double knockout mice and of humans with polymorphisms in the enzymes, has clarified the role of these enzymes in whole body carotenoid and vitamin A homeostasis. These studies also demonstrate the relationship between enzyme expression and whole body lipid and energy metabolism and oxidative stress. In addition, relationships between BCO1 and BCO2 and the development or risk of metabolic diseases, eye diseases and cancer have been observed. While the precise roles of the enzymes in the pathophysiology of most of these diseases is not presently clear, these gaps in knowledge provide fertile ground for rigorous future investigations. This article is part of a Special Issue entitled Carotenoids: Recent Advances in Cell and Molecular Biology edited by Johannes von Lintig and Loredana Quadro.

Green Tea Extract Treatment in Obese Mice with Nonalcoholic Steatohepatitis Restores the Hepatic Metabolome in Association with Limiting Endotoxemia-TLR4-NFκB-Mediated Inflammation.

SCOPE: Catechin-rich green tea extract (GTE) alleviates nonalcoholic steatohepatitis (NASH) by lowering endotoxin-TLR4 (Toll-like receptor-4)-NFκB (nuclear factor kappa-B) inflammation. This study aimed to define altered MS-metabolomic responses during high-fat (HF)-induced NASH that are restored by GTE utilizing livers from an earlier study in which GTE decreased endotoxin-TLR4-NFκB liver injury. METHODS AND RESULTS: Mice are fed a low-fat (LF) or HF diet for 12 weeks and then randomized to LF or HF diets containing 0% or 2% GTE for an additional 8 weeks. Global MS-based metabolomics and targeted metabolite profiling of catechins/catechin metabolites are evaluated. GTE in HF mice restores hepatic metabolites implicated in dyslipidemia insulin resistance, and inflammation. These include 122 metabolites: amino acids, lipids, nucleotides, vitamins, bile acids, flavonoids, xenobiotics, and carbohydrates. Hepatic amino acids, B-vitamins, and bile acids are inversely correlated with biomarkers of insulin resistance, liver injury, steatosis, and inflammation. Further, phosphatidylcholine metabolites are positively correlated with biomarkers of liver injury and NFκB inflammation. Thirteen catechin metabolites are identified in livers of GTE-treated mice, mostly as phase II conjugates of parental catechins or microbial-derived valerolactones. CONCLUSION: The defined anti-inflammatory/metabolic interactions advance an understanding of the mechanism by which GTE catechins protect against NFκB-mediated liver injury in NASH.

The Effects of Doxorubicin-based Chemotherapy and Omega-3 Supplementation on Mouse Brain Lipids.

Chemotherapy-induced cognitive impairment affects ~30% of breast cancer survivors, but the effects on how chemotherapy impacts brain lipids, and how omega-3 polyunsaturated fatty acid supplementation may confer protection, is unknown. Ovariectomized mice were randomized to two rounds of injections of doxorubicin + cyclophosphamide or vehicle after consuming a diet supplemented with 2% or 0% EPA+DHA, and sacrificed 4, 7, and 14 days after the last injection (study 1, n = 120) or sacrificed 10 days after the last injection (study 2, n = 40). Study 1 whole brain samples were extracted and analyzed by UHPLC-MS/MS to quantify specialized pro-resolving mediators (SPMs). Lipidomics analyses were performed on hippocampal extracts from study 2 to determine changes in the brain lipidome. Study 1 results: only resolvin D1 was present in all samples, but no differences in concentration were observed (P > 0.05). Study 2 results: chemotherapy was positively correlated with omega-9 fatty acids, and EPA+DHA supplementation helped to maintain levels of plasmalogens. No statistically significant chemotherapy*diet effect was observed. Results demonstrate a limited role of SPMs in the brain post-chemotherapy, but a significant alteration of hippocampal lipids previously associated with other models of cognitive impairment (i.e., Alzheimer's and Parkinson's disease).

The Effect of an Iron Supplement on Lycopene Metabolism and Absorption During Digestion in Healthy Humans.

SCOPE: To investigate the formation and absorption of lycopene (LYC) metabolites in the human upper gastrointestinal lumen, in the absence and presence of iron. METHODS: Healthy males (n = 7) consumed test meals that deliver ≈22 mg LYC + ≈0.3 mg apo-lycopenals from oleoresin without (-FeSO4 ) and with ferrous sulfate (160 mg, +FeSO4 ). Subjects were intubated with a naso-gastric/naso-duodenal tube. Digesta, blood plasma, and the triglyceride-rich lipoprotein (TRL) fractions of plasma were analyzed using LC-MS/MS, to measure LYC and apo-lycopenoids. RESULTS: Digesta LYC concentrations increased with time (p = 1.2 × 10-7 ), decrease with time × iron (p = 1.1 × 10-5 ), and remain ≈200× higher than apo-lycopenals/lycopenone. Digesta apo-8'-, -10'-, -12'-, -14'-, -15-lycopenal, and apo-13-lycopenone concentrations increased with time (p < 0.01), apo-12'-, -14'-, -15-lycopenal, apo-13-lycopenone increase with iron (p < 0.05), and time × iron decrease apo-8'-, -10'-, -12'-, -14'-, -15-lycopenal, apo-13-lycopenone concentrations (p < 0.01). A 1.9-fold decrease in LYC TRL area-under-the-time-concentration-curve is observed after the test meal +FeSO4 versus the test meal -FeSO4 (p = 0.02). Apo-lycopenals were detected in later TRL fractions, and no apo-lycopenols or apo-lycopenoic acids were observed in any samples. CONCLUSIONS: FeSO4 reduces LYC absorption. Apo-lycopenals appear to be absorbed from foods, and not made in significant quantities during digestion.

Novel Processing Technologies as Compared to Thermal Treatment on the Bioaccessibility and Caco-2 Cell Uptake of Carotenoids from Tomato and Kale-Based Juices.

This research aimed to measure the impact of novel food processing techniques, i.e., pulsed electric field (PEF) and ohmic heating (OH), on carotenoid bioaccessibility and Caco-2 cell uptake from tomato juice and high-pressure processing (HPP) and PEF on the same attributes from kale-based juices, as compared with raw (nonprocessed) and conventional thermally treated (TT) juices. Lycopene, ß-carotene, and lutein were quantitated in juices and the micelle fraction using high-performance liquid chromatography (HPLC)-diode array detection and in Caco-2 cells using HPLC-tandem mass spectrometry. Tomato juice results were as follows: PEF increased lycopene bioaccessibility (1.5 ± 0.39%) by 150% (P = 0.01) but reduced ß-carotene bioaccessibility (28 ± 6.2%) by 44% (P = 0.02), relative to raw juice. All processing methods increased lutein uptake. Kale-based juice results were as follows: TT and PEF degraded ß-carotene and lutein in the juice. No difference in bioaccessibility or cell uptake was observed. Total delivery, i.e., the summation of bioaccessibility and cell uptake, of lycopene, ß-carotene, and lutein was independent of type of processing. Taken together, PEF and OH enhanced total lycopene and lutein delivery from tomato juice to Caco-2 cells as well as TT, and may produce a more desirable product due to other factors (i.e., conservation of heat-labile micronutrients, fresher organoleptic profile). HPP best conserved the carotenoid content and color of kale-based juice and merits further consideration.