Efficient in vitro digestion of lipids and proteins in bovine milk fat globule membrane ingredient (MFGMi) and whey-casein infant formula with added MFGMi.

The relative immaturity of the infant digestive system has the potential to affect the bioavailability of dietary lipids, proteins, and their digested products. We performed a lipidomic analysis of a commercial bovine milk fat globule membrane ingredient (MFGMi) and determined the profile of lipids and proteins in the bioaccessible fraction after in vitro digestion of both the ingredient and whey-casein-based infant formula without and with MFGMi. Test materials were digested using a static 2-phase in vitro model, with conditions simulating those in the infant gut. The extent of digestion and the bioaccessibility of various classes of neutral and polar lipids were monitored by measuring a wide targeted lipid profile using direct infusion-mass spectrometry. Digestion of abundant proteins in the ingredient and whey-casein infant formula containing the ingredient was determined by denaturing PAGE with imaging of Coomassie Brilliant Blue stained bands. Cholesterol esters, diacylglycerides, triacylglycerides, phosphatidylcholines, and phosphatidylethanolamines in MFGMi were hydrolyzed readily during in vitro digestion, which resulted in marked increases in the amounts of free fatty acids and lyso-phospholipids in the bioaccessible fraction. In contrast, sphingomyelins, ceramides, and gangliosides were largely resistant to simulated digestion. Proteins in MFGMi and the infant formulas also were hydrolyzed efficiently. The results suggest that neutral lipids, cholesterol esters, phospholipids, and proteins in MFGMi are digested efficiently during conditions that simulate the prandial lumen of the stomach and small intestine of infants. Also, supplementation of whey-casein-based infant formula with MFGMi did not appear to alter the profiles of lipids and proteins in the bioaccessible fraction after digestion.

Modulation of the oral glucocorticoid system during black raspberry mediated oral cancer chemoprevention.

Recent reports suggest that glucocorticoids (GCs), which can be synthesized in the oral mucosa, play an important role in cancer development. Therefore, the objectives of this study were to characterize the role of the oral GC system in oral cancer, and determine the effect of black raspberry (BRB) administration on GC modulation during oral cancer chemoprevention. We determined the expression of GC enzymes in various oral cancer cell lines, and investigated the role of the GC inactivating enzyme HSD11B2 on CAL27 oral cancer cells using siRNA mediated knockdown approaches. Using two in vivo models of oral carcinogenesis with 4-nitroquinoline 1-oxide carcinogen on C57Bl/6 mice and F344 rats, we determined the effect of BRB on GC modulation during head and neck squamous cell carcinoma chemoprevention. Our results demonstrate that HSD11B2, which inactivates cortisol to cortisone, is downregulated during oral carcinogenesis in clinical and experimental models. Knockdown of HSD11B2 in oral cancer cells promotes cellular proliferation, invasion and expression of angiogenic biomarkers EGFR and VEGFA. An ethanol extract of BRB increased HSD11B2 expression on oral cancer cells. Dietary administration of 5% BRB increased Hsd11b2 gene and protein expression and reduced the active GC, corticosterone, in cancer-induced mouse tongues. Our results demonstrate that the oral GC system is modulated during oral carcinogenesis, and BRB administration upregulates Hsd11b2 during oral cancer chemoprevention. In conclusion, our findings challenge the use of synthetic GCs in head and neck cancer, and support the use of natural product alternatives that potentially modulate GC metabolism in a manner that supports oral cancer chemoprevention.

Macrophage maturation from blood monocytes is altered in people with HIV, and is linked to serum lipid profiles and activation indices: A model for studying atherogenic mechanisms.

People with HIV (PWH) are at increased risk for atherosclerotic cardiovascular disease (ASCVD). Proportions of vascular homing monocytes are enriched in PWH; however, little is known regarding monocyte-derived macrophages (MDMs) that may drive atherosclerosis in this population. We isolated PBMCs from people with and without HIV, and cultured these cells for 5 days in medium containing autologous serum to generate MDMs. Differential gene expression (DGE) analysis of MDMs from PWH identified broad alterations in innate immune signaling (IL-1ß, TLR expression, PPAR ßδ) and lipid processing (LXR/RXR, ACPP, SREBP1). Transcriptional changes aligned with the functional capabilities of these cells. Expression of activation markers and innate immune receptors (CD163, TLR4, and CD300e) was altered on MDMs from PWH, and these cells produced more TNFα, reactive oxygen species (ROS), and matrix metalloproteinases (MMPs) than did cells from people without HIV. MDMs from PWH also had greater lipid accumulation and uptake of oxidized LDL. PWH had increased serum levels of free fatty acids (FFAs) and ceramides, with enrichment of saturated FAs and a reduction in polyunsaturated FAs. Levels of lipid classes and species that are associated with CVD correlated with unique DGE signatures and altered metabolic pathway activation in MDMs from PWH. Here, we show that MDMs from PWH display a pro-atherogenic phenotype; they readily form foam cells, have altered transcriptional profiles, and produce mediators that likely contribute to accelerated ASCVD.

Uptake and metabolism of ß-apo-8'-carotenal, ß-apo-10'-carotenal, and ß-apo-13-carotenone in Caco-2 cells.

ß-Apocarotenoids are eccentric cleavage products of carotenoids formed by chemical and enzymatic oxidations. They occur in foods containing carotenoids and thus might be directly absorbed from the diet. However, there is limited information about their intestinal absorption. The present research examined the kinetics of uptake and metabolism of ß-apocarotenoids. Caco-2 cells were grown on 6-well plastic plates until a differentiated cell monolayer was achieved. ß-Apocarotenoids were prepared in Tween 40 micelles, delivered to differentiated cells in serum-free medium, and incubated at 37°C for up to 8 h. There was rapid uptake of ß-apo-8'-carotenal into cells, and ß-apo-8'-carotenal was largely converted to ß-apo-8'-carotenoic acid and a minor metabolite that we identified as 5,6-epoxy-ß-apo-8'-carotenol. There was also rapid uptake of ß-apo-10'-carotenal into cells, and ß-apo-10'-carotenal was converted into a major metabolite identified as 5,6-epoxy-ß-apo-10'-carotenol and a minor metabolite that is likely a dihydro-ß-apo-10'-carotenol. Finally, there was rapid cellular uptake of ß-apo-13-carotenone, and this compound was extensively degraded. These results suggest that dietary ß-apocarotenals are extensively metabolized in intestinal cells via pathways similar to the metabolism of retinal. Thus, they are likely not absorbed directly from the diet.

Substrate Specificity of Purified Recombinant Chicken ß-Carotene 9',10'-Oxygenase (BCO2).

Provitamin A carotenoids are oxidatively cleaved by ß-carotene 15,15'-dioxygenase (BCO1) at the central 15-15' double bond to form retinal (vitamin A aldehyde). Another carotenoid oxygenase, ß-carotene 9',10'-oxygenase (BCO2) catalyzes the oxidative cleavage of carotenoids at the 9'-10' bond to yield an ionone and an apo-10'-carotenoid. Previously published substrate specificity studies of BCO2 were conducted using crude lysates from bacteria or insect cells expressing recombinant BCO2. Our attempts to obtain active recombinant human BCO2 expressed in Escherichia coli were unsuccessful. We have expressed recombinant chicken BCO2 in the strain E. coli BL21-Gold (DE3) and purified the enzyme by cobalt ion affinity chromatography. Like BCO1, purified recombinant chicken BCO2 catalyzes the oxidative cleavage of the provitamin A carotenoids ß-carotene, α-carotene, and ß-cryptoxanthin. Its catalytic activity with ß-carotene as substrate is at least 10-fold lower than that of BCO1. In further contrast to BCO1, purified recombinant chicken BCO2 also catalyzes the oxidative cleavage of 9-cis-ß-carotene and the non-provitamin A carotenoids zeaxanthin and lutein, and is inactive with all-trans-lycopene and ß-apocarotenoids. Apo-10'-carotenoids were detected as enzymatic products by HPLC, and the identities were confirmed by LC-MS. Small amounts of 3-hydroxy-ß-apo-8'-carotenal were also consistently detected in BCO2-ß-cryptoxanthin reaction mixtures. With the exception of this activity with ß-cryptoxanthin, BCO2 cleaves specifically at the 9'-10' bond to produce apo-10'-carotenoids. BCO2 has been shown to function in preventing the excessive accumulation of carotenoids, and its broad substrate specificity is consistent with this.

Absorption and Distribution Kinetics of the 13C-Labeled Tomato Carotenoid Phytoene in Healthy Adults.

BACKGROUND: Phytoene is a tomato carotenoid that may contribute to the apparent health benefits of tomato consumption. Although phytoene is a less prominent tomato carotenoid than lycopene, it is a major carotenoid in various human tissues. Phytoene distribution to plasma lipoproteins and tissues differs from lycopene, suggesting the kinetics of phytoene and lycopene differ. OBJECTIVE: The objective of this study was to characterize the kinetic parameters of phytoene absorption, distribution, and excretion in adults, to better understand why biodistribution of phytoene differs from lycopene. METHODS: Four adults (2 males, 2 females) maintained a controlled phytoene diet (1-5 mg/d) for 42 d. On day 14, each consumed 3.2 mg (13)C-phytoene, produced using tomato cell suspension culture technology. Blood samples were collected at 0, 1-15, 17, 21, and 24 h and 2, 3, 4, 7, 10, 14, 17, 21, and 28 d after (13)C-phytoene consumption. Plasma-unlabeled and plasma-labeled phytoene concentrations were determined using ultra-HPLC-quadrupole time-of-flight-mass spectrometry, and data were fit to a 7-compartment carotenoid kinetic model using WinSAAM 3.0.7 software. RESULTS: Subjects were compliant with a controlled phytoene diet, consuming a mean ± SE of 2.5 ± 0.6 mg/d, resulting in a plasma unlabeled phytoene concentration of 71 ± 14 nmol/L. A maximal plasma (13)C-phytoene concentration of 55.6 ± 5.9 nM was achieved 19.8 ± 9.2 h after consumption, and the plasma half-life was 2.3 ± 0.2 d. Compared with previous results for lycopene, phytoene bioavailability was nearly double at 58% ± 19%, the clearance rate from chylomicrons was slower, and the rates of deposition into and utilization by the slow turnover tissue compartment were nearly 3 times greater. CONCLUSIONS: Although only differing from lycopene by 4 double bonds, phytoene exhibits markedly different kinetic characteristics in human plasma, providing insight into metabolic processes contributing to phytoene enrichment in plasma and tissues compared with lycopene. This trial was registered at clinicaltrials.gov as NCT01692340.

The human enzyme that converts dietary provitamin A carotenoids to vitamin A is a dioxygenase.

ß-Carotene 15-15'-oxygenase (BCO1) catalyzes the oxidative cleavage of dietary provitamin A carotenoids to retinal (vitamin A aldehyde). Aldehydes readily exchange their carbonyl oxygen with water, making oxygen labeling experiments challenging. BCO1 has been thought to be a monooxygenase, incorporating oxygen from O2 and H2O into its cleavage products. This was based on a study that used conditions that favored oxygen exchange with water. We incubated purified recombinant human BCO1 and ß-carotene in either (16)O2-H2(18)O or (18)O2-H2(16)O medium for 15 min at 37 °C, and the relative amounts of (18)O-retinal and (16)O-retinal were measured by liquid chromatography-tandem mass spectrometry. At least 79% of the retinal produced by the reaction has the same oxygen isotope as the O2 gas used. Together with the data from (18)O-retinal-H2(16)O and (16)O-retinal-H2(18)O incubations to account for nonenzymatic oxygen exchange, our results show that BCO1 incorporates only oxygen from O2 into retinal. Thus, BCO1 is a dioxygenase.

A comparison of plasma and prostate lycopene in response to typical servings of tomato soup, sauce or juice in men before prostatectomy.

Tomato product consumption and estimated lycopene intake are hypothesised to reduce the risk of prostate cancer. To define the impact of typical servings of commercially available tomato products on resultant plasma and prostate lycopene concentrations, men scheduled to undergo prostatectomy (n 33) were randomised either to a lycopene-restricted control group ( < 5 mg lycopene/d) or to a tomato soup (2-2¾ cups prepared/d), tomato sauce (142-198 g/d or 5-7 ounces/d) or vegetable juice (325-488 ml/d or 11-16·5 fluid ounces/d) intervention providing 25-35 mg lycopene/d. Plasma and prostate carotenoid concentrations were measured by HPLC. Tomato soup, sauce and juice consumption significantly increased plasma lycopene concentration from 0·68 (sem 0·1) to 1·13 (sem 0·09) µmol/l (66 %), 0·48 (sem 0·09) to 0·82 (sem 0·12) µmol/l (71 %) and 0·49 (sem 0·12) to 0·78 (sem 0·1) µmol/l (59 %), respectively, while the controls consuming the lycopene-restricted diet showed a decline in plasma lycopene concentration from 0·55 (sem 0·60) to 0·42 (sem 0·07) µmol/l ( - 24 %). The end-of-study prostate lycopene concentration was 0·16 (sem 0·02) nmol/g in the controls, but was 3·5-, 3·6- and 2·2-fold higher in tomato soup (P= 0·001), sauce (P= 0·001) and juice (P= 0·165) consumers, respectively. Prostate lycopene concentration was moderately correlated with post-intervention plasma lycopene concentrations (r 0·60, P =0·001), indicating that additional factors have an impact on tissue concentrations. While the primary geometric lycopene isomer in tomato products was all-trans (80-90 %), plasma and prostate isomers were 47 and 80 % cis, respectively, demonstrating a shift towards cis accumulation. Consumption of typical servings of processed tomato products results in differing plasma and prostate lycopene concentrations. Factors including meal composition and genetics deserve further evaluation to determine their impacts on lycopene absorption and biodistribution.

Substrate specificity of purified recombinant human ß-carotene 15,15'-oxygenase (BCO1).

Humans cannot synthesize vitamin A and thus must obtain it from their diet. ß-Carotene 15,15'-oxygenase (BCO1) catalyzes the oxidative cleavage of provitamin A carotenoids at the central 15-15' double bond to yield retinal (vitamin A). In this work, we quantitatively describe the substrate specificity of purified recombinant human BCO1 in terms of catalytic efficiency values (kcat/Km). The full-length open reading frame of human BCO1 was cloned into the pET-28b expression vector with a C-terminal polyhistidine tag, and the protein was expressed in the Escherichia coli strain BL21-Gold(DE3). The enzyme was purified using cobalt ion affinity chromatography. The purified enzyme preparation catalyzed the oxidative cleavage of ß-carotene with a Vmax = 197.2 nmol retinal/mg BCO1 × h, Km = 17.2 µM and catalytic efficiency kcat/Km = 6098 M(-1) min(-1). The enzyme also catalyzed the oxidative cleavage of α-carotene, ß-cryptoxanthin, and ß-apo-8'-carotenal to yield retinal. The catalytic efficiency values of these substrates are lower than that of ß-carotene. Surprisingly, BCO1 catalyzed the oxidative cleavage of lycopene to yield acycloretinal with a catalytic efficiency similar to that of ß-carotene. The shorter ß-apocarotenals (ß-apo-10'-carotenal, ß-apo-12'-carotenal, ß-apo-14'-carotenal) do not show Michaelis-Menten behavior under the conditions tested. We did not detect any activity with lutein, zeaxanthin, and 9-cis-ß-carotene. Our results show that BCO1 favors full-length provitamin A carotenoids as substrates, with the notable exception of lycopene. Lycopene has previously been reported to be unreactive with BCO1, and our findings warrant a fresh look at acycloretinal and its alcohol and acid forms as metabolites of lycopene in future studies.

Bioactive compounds or metabolites from black raspberries modulate T lymphocyte proliferation, myeloid cell differentiation and Jak/STAT signaling.

Bioactive phytochemicals from natural products, such as black raspberries (BRB; Rubus occidentalis), have direct anticancer properties on malignant cells in culture and in xenograft models. BRB components inhibit cancer progression in more complex rodent carcinogenesis models. Although mechanistic targets for BRB phytochemicals in cancer cells are beginning to emerge, the potential role in modulating host immune processes impacting cancer have not been systematically examined. We hypothesized that BRB contain compounds capable of eliciting potent immunomodulatory properties that impact cellular mediators relevant to chronic inflammation and tumor progression. We studied both an ethanol extract from black raspberries (BRB-E) containing a diverse mixture of phytochemicals and two abundant phytochemical metabolites of BRB produced upon ingestion (Cyanidin-3-Rutinoside, C3R; Quercitin-3-Rutinoside, Q3R). BRB-E inhibited proliferation, and viability of CD3/CD28 activated human CD4(+) and CD8(+) T lymphocytes. BRB-E also limited in vitro expansion of myeloid-derived suppressor cells (MDSC) and their suppressive capacity. Pre-treatment of immune cells with BRB-E attenuated IL-6-mediated phosphorylation of signal transducer and activator of transcription-3 (STAT3) and IL-2-induced STAT5 phosphorylation. In contrast, pre-treatment of immune cells with the C3R and Q3R metabolites inhibited MDSC expansion, IL-6-mediated STAT3 signaling, but not IL-2-induced STAT5 phosphorylation and were less potent inhibitors of T cell viability. Together these data indicate that BRB extracts and their physiologically relevant metabolites contain phytochemicals that affect immune processes relevant to carcinogenesis and immunotherapy. Furthermore, specific BRB components and their metabolites may be a source of lead compounds for drug development that exhibits targeted immunological outcomes or inhibition of specific STAT-regulated signaling pathways.

Xanthones in mangosteen juice are absorbed and partially conjugated by healthy adults.

The proposed health-promoting effects of the pericarp from mangosteen fruit have been attributed to a family of polyphenols referred to as xanthones. The purpose of this study was to determine the bioavailability of xanthones from 100% mangosteen juice in healthy adult participants (n = 10). Pericarp particles accounted for 1% of the mass and 99% of the xanthone concentration in the juice. The juice provided 5.3 ± 0.1 mmol/L total xanthones with α-mangostin, garcinones (C, D, and E), γ-mangostin, gartanins, and other identified xanthones accounting for 58, 2, 6, 4, and 5%, respectively. Participants ingested 60 mL mangosteen juice with a high-fat breakfast. Free and conjugated (glucuronidated/sulfated) xanthones were detected in serum and urine. There was marked variation in the AUC (762-4030 nmol/L × h), maximum concentration (113 ± 107 nmol/L), and time to maximum concentration (3.7 ± 2.4 h) for α-mangostin in sera during the 24-h collection. Similarly, xanthones in 24-h urine ranged from 0.9 to 11.1 µmol and accounted for 2.0 ± 0.3% (range 0.3-3.4%) of the ingested dose. There were no significant differences between female and male participants in mean pharmacokinetic values of α-mangostin in serum and urinary xanthones. Only 15.4 ± 0.7% of total xanthones in pericarp particles in the juice partitioned into mixed micelles during in vitro digestion. These results show that xanthones in mangosteen juice are absorbed when ingested along with a high-fat meal, although release of xanthones from pericarp particles during digestion may be limited.

Lipidome Alterations with Exercise Among People With and Without HIV: An Exploratory Study.

Age-related comorbidities and physical function impairments in aging people with HIV (PWH) can be improved through exercise interventions. The mechanisms underlying these improvements, including lipidomic changes, are unknown. Sedentary adults (50-75 years old) with or without HIV participated in supervised endurance/resistance exercise for 24 weeks. Plasma lipid concentrations (∼1,200 lipid species from 13 lipid classes) at baseline and week 24 were measured by mass spectrometry. Given multiple comparisons, unadjusted and Benjamini-Hochberg corrected p values are reported. Analyses are considered exploratory. Twenty-five PWH and 24 controls had paired samples at baseline and week 24. The change in total triacylglycerol (TAG) concentrations after exercise intervention differed between groups (unadj-p = 0.006, adj-p = 0.078) with concentrations increasing among controls, but not among PWH. Changes in concentrations of TAG species composed of long-chain fatty acids differed between groups (unadj-p < 0.04) with increases among controls, but not among PWH. Changes in total diacylglycerol (DAG) concentration from baseline to week 24 differed between groups (unadj-p = 0.03, adj-p = 0.2) with an increase in PWH and a nonsignificant decrease in controls. Baseline to week 24 changes in DAGs composed of palmitic acid (16:0), palmitoleic acid (16:1), and stearic acid (18:0) differed by serostatus (unadj-p = 0.009-0.03; adj-p 0.10-0.12), with nonsignificant increases and decreases in concentrations in PWH and controls, respectively. Concentrations of individual lysophosphatidylcholine (LPC) and ceramide (CER) species also differed by HIV serostatus (unadj-p < = 0.05). Although exploratory, the effects of exercise on the lipidome may differ among people with and without HIV, potentially due to underlying alterations in lipid processing and fatty acid oxidation in PWH. Clinical Trials NCT02404792.

Assessment of dietary carotenoid intake and biologic measurement of exposure in humans.

Carotenoids are a diverse family of phytochemicals with over 1000 different carotenoids present in nature. A human diet containing a variety of plant foods typically includes approximately 50 different carotenoids, although six (α-carotene, ß-carotene, ß-cryptoxanthin, lycopene, lutein, and zeaxanthin) comprise over 90% of total carotenoid intake. Most carotenoids do not meet the definition of a nutrient, but several can be cleaved to form vitamin A and are important contributors to vitamin A nutriture and prevention of vitamin A deficiency. Large epidemiologic studies suggest that diets rich in total or specific carotenoids are associated with a reduced risk of several diseases including various types of cancer, cardiovascular disease, cognitive disorders, and age-related macular degeneration. However, accurate measurement of dietary intake is challenging and current methods of dietary assessment, including food frequency questionnaires, diet records and 24-h recalls, have strengths and limitations regarding estimating carotenoid intake. Additionally, carotenoid bioavailability from the diet is influenced by many variables including food processing and cooking, meal composition, and individual characteristics of the host including age, digestive efficiency, nutritional status and genetic polymorphisms. Carotenoids are deposited in many human tissues and can be measured using a variety of techniques including high performance liquid chromatography (HPLC) and mass spectrometry (MS). Continued research is necessary to improve dietary intake assessment and establish biologically relevant dose-response relationships in the context of individual variability to advance our understanding of diet, disease risk, and health promotion.

Erratum to "Phenolic profile, in vitro antimicrobial activity and antioxidant capacity of Vaccinium meridionale Swartz pomace" [Heliyon 6 (5) (May 2020) e03845].

[This corrects the article DOI: 10.1016/j.heliyon.2020.e03845.].

Phenolic profile, in vitro antimicrobial activity and antioxidant capacity of Vaccinium meridionale swartz pomace.

Berries of Vaccinium meridionale Swartz contain a variety of phytochemicals, which are believed to account for their bioactive properties. The potential of Vaccinium meridionale Swartz pomace as a source of bioactive compounds was investigated. The dietary fiber (DF) content was assessed by the AOAC method, phenolic compounds were characterized and quantified via HPLC-PDA and UPLC-QTOF-MS. The in vitro antibacterial activity was tested against Gram-positive and Gram-negative bacteria. The antioxidant properties were assessed by the ORAC and the ABTS assays. The DF content was 52.4 ± 3.7%, phenolic compounds comprised anthocyanins (ACNs) (747.6 ± 167.5 mg cyanidin-3-glucoside/100 g FW), hydroxycinammic acids (HCAs) (229.2 ± 68.4 mg chlorogenic acid equivalents/100 g FW), flavonols (335.0 ± 139.5 rutin equivalents/100 g FW), and procyanidins (PACs) (140.9 ± 33.3 mg cocoa procyanidin equivalents/100 g FW). Staphylococcus aureus was more sensitive than E. coli. The ORAC value was 250.0 ± 32.0 µmol TE/g fresh weight (FW). Results suggest that the residue from V. meridionale S. can be utilized to obtain valuable nutraceuticals for the development of functional foods.

Black raspberry components inhibit proliferation, induce apoptosis, and modulate gene expression in rat esophageal epithelial cells.

We have shown that a diet containing freeze-dried black raspberries (BRB) inhibits the development of chemically induced cancer in the rat esophagus. To provide insights into possible mechanisms by which BRB inhibit esophageal carcinogenesis, we evaluated an ethanol (EtOH) extract of BRB, and two component anthocyanins (cyanidin-3-O-glucoside and cyanidin-3-O-rutinoside) in BRB, for their effects on growth, apoptosis, and gene expression in rat esophageal epithelial cell lines. The EtOH extract and both anthocyanins selectively caused significant growth inhibition and induction of apoptosis in a highly tumorigenic cell line (RE-149 DHD) but not in a weakly tumorigenic line (RE-149). The uptake of anthocyanins from the EtOH extract into RE-149 DHD cells far exceeded their uptake into RE-149 cells, which may have accounted for the selective effects of the extract on growth and apoptosis of RE-149 DHD cells. The growth inhibitory and proapoptotic effects were enhanced by the daily addition of the EtOH extract and the anthocyanins to the medium. Interestingly, the EtOH extract did not alter cyclooxygenase-2 (COX-2) and nitric oxide synthase (i-NOS) expression in RE-149 DHD cells, whereas both anthocyanins downregulated the expressions of these genes. This differential effect may have been related to the relative amounts of anthocyanins in the extract vs. when they were added individually to the medium. We conclude that the selective effects of the EtOH extract on growth and apoptosis of highly tumorigenic rat esophageal epithelial cells in vitro may be due to preferential uptake and retention of its component anthocyanins, and this may also be responsible for the greater inhibitory effects of freeze-dried whole berries on tumor cells in vivo.

Dietary Black Raspberries Impact the Colonic Microbiome and Phytochemical Metabolites in Mice.

SCOPE: Black raspberries (BRB) are a rich source of bioactive phytochemicals, including anthocyanins and ellagitannins. These phytochemicals are poorly absorbed and may be transformed by gut microbiota into various metabolites that may impact the colonic mucosa or upon absorption have systemic bioactivity. The objective of this study is to define the impact of a BRB-containing diet on the colon microbiome in mice and quantify the phytochemical metabolites in the colon contents and circulation. METHODS AND RESULTS: Male mice were fed 10% w/w freeze-dried BRB powder for 6 weeks. The colonic microbiota was evaluated by 16S rRNA gene sequencing. Anthocyanin and ellagitannin metabolites, protocatechuic acid, and urolithins were analyzed by HPLC-MS/MS. The BRB diet impacted colon mucosal microbial composition with a more robust effect observed on the luminal microflora. BRB-derived protocatechuic acid and urolithins were quantified in the colon, luminal contents, plasma, liver, and prostate with protocatechuic acid present in higher concentrations compared to urolithins. CONCLUSION: This study highlights the complex interactions between dietary phytochemicals, the host microbiome, and metabolism. It is demonstrated that microbially produced phytochemical metabolites are present in the colon and systemic circulation where they may exert biological activity.

Altered Lipidome Composition Is Related to Markers of Monocyte and Immune Activation in Antiretroviral Therapy Treated Human Immunodeficiency Virus (HIV) Infection and in Uninfected Persons.

Background: HIV infection and antiretroviral therapy (ART) have both been linked to dyslipidemia and increased cardiovascular disease (CVD) risk. Alterations in the composition of saturated (SaFA), monounsaturated (MUFA), and polyunsaturated (PUFA) fatty acids are related to inflammation and CVD progression in HIV-uninfected (HIV-) populations. The relationships among the lipidome and markers of monocyte and immune activation in HIV-infected (HIV+) individuals are not well understood. Methods: Concentrations of serum lipids and their fatty acid composition were measured by direct infusion-tandem mass spectrometry in samples from 20 ART-treated HIV+ individuals and 20 HIV- individuals. Results: HIV+ individuals had increased levels of free fatty acids (FFAs) with enrichment of SaFAs, including palmitic acid (16:0) and stearic acid (18:0), and these levels were directly associated with markers of monocyte (CD40, HLA-DR, TLR4, CD36) and serum inflammation (LBP, CRP). PUFA levels were reduced significantly in HIV+ individuals, and many individual PUFA species levels were inversely related to markers of monocyte activation, such as tissue factor, TLR4, CD69, and SR-A. Also in HIV+ individuals, the composition of lysophosphatidylcholine (LPC) was enriched for SaFAs; LPC species containing SaFAs were directly associated with IL-6 levels and monocyte activation. We similarly observed direct relationships between levels of SaFAs and inflammation in HIV uninfected individuals. Further, SaFA exposure altered monocyte subset phenotypes and inflammatory cytokine production in vitro. Conclusions: The lipidome is altered in ART-treated HIV infection, and may contribute to inflammation and CVD progression. Detailed lipidomic analyses may better assess CVD risk in both HIV+ and HIV- individuals than does traditional lipid profiling.

Optimization of extraction methods for quantification of microcystin-LR and microcystin-RR in fish, vegetable, and soil matrices using UPLC-MS/MS.

Human-driven environmental change has increased the occurrence of harmful cyanobacteria blooms in aquatic ecosystems. Concomitantly, exposure to microcystin (MC), a cyanobacterial toxin that can accumulate in animals, edible plants, and agricultural soils, has become a growing public health concern. For accurate estimation of health risks and timely monitoring, availability of reliable detection methods is imperative. Nonetheless, quantitative analysis of MCs in many types of biological and environmental samples has proven challenging because matrix interferences can hinder sample preparation and extraction procedures, leading to poor MC recovery. Herein, controlled experiments were conducted to enhance the use of ultra-performance liquid-chromatography tandem-mass spectrometry (UPLC-MS/MS) to recover MC-LR and MC-RR at a range of concentrations in seafood (fish), vegetables (lettuce), and environmental (soil) matrices. Although these experiments offer insight into detailed technical aspects of the MC homogenization and extraction process (i.e., sonication duration and centrifugation speed during homogenization; elution solvent to use during the final extraction), they centered on identifying the best (1) solvent system to use during homogenization (2-3 tested per matrix) and (2) single-phase extraction (SPE) column type (3 tested) to use for the final extraction. The best procedure consisted of the following, regardless of sample type: centrifugation speed = 4200 × g; elution volume = 8 mL; elution solvent = 80% methanol; and SPE column type = hydrophilic-lipophilic balance (HLB), with carbon also being satisfactory for fish. For sonication, 2 min, 5 min, and 10 min were optimal for fish, lettuce, and soil matrices, respectively. Using the recommended HLB column, the solvent systems that led to the highest recovery of MCs were methanol:water:butanol for fish, methanol:water for lettuce, and EDTA-Na4P2O7 for soils. Given that the recommended procedures resulted in average MC-LR and MC-RR recoveries that ranged 93 to 98%, their adoption for the preparation of samples with complex matrices before UPLC-MS/MS analysis is encouraged.

High-Pressure Processing of Broccoli Sprouts: Influence on Bioactivation of Glucosinolates to Isothiocyanates.

Effects of high-pressure processing (HPP, 100-600 MPa for 3 min at 30 °C) on the glucosinolate content, conversion to isothiocyanates, and color changes during storage in fresh broccoli sprouts were investigated. A mild heat treatment (60 °C) and boiling (100 °C) were used as positive and negative controls, respectively. Glucosinolates were quantified using liquid chromatography-mass spectrometry, and isothiocyanates were quantified using high-performance liquid chromatography-photodiode array detection. A formation of isothiocyanates was observed in all high-pressure-treated sprouts. The highest degree of conversion (85%) was observed after the 600 MPa treatment. Increased isothiocyanate formation at 400-600 MPa suggests an inactivation of the epithiospecifier protein. During storage, color changed from green to brownish, reflected by increasing a* values and decreasing L* values. This effect was less pronounced for sprouts treated at 100 and 600 MPa, indicating an influence on the responsible enzymes. In summary, HPP had no negative effects on the glucosinolate-myrosinase system in broccoli sprouts.