Butyric acid increases transepithelial transport of ferulic acid through upregulation of the monocarboxylate transporters SLC16A1 (MCT1) and SLC16A3 (MCT4).

Ferulic acid is released by microbial hydrolysis in the colon, where butyric acid, a major by-product of fermentation, constitutes the main energy source for colonic enterocytes. We investigated how varying concentrations of this short chain fatty acid may influence the absorption of the phenolic acid. Chronic treatment of Caco-2 cells with butyric acid resulted in increased mRNA and protein abundance of the monocarboxylate transporters SLC16A1 (MCT1) and SLC16A3 (MCT4), previously proposed to facilitate ferulic acid absorption in addition to passive diffusion. Short term incubation with butyric acid only led to upregulation of MCT4 while both conditions increased transepithelial transport of ferulic acid in the apical to basolateral, but not basolateral to apical, direction. Chronic treatment also elevated intracellular concentrations of ferulic acid, which in turn gave rise to increased concentrations of ferulic acid metabolites. Immunofluorescence staining of cells revealed uniform distribution of MCT1 protein in the cell membrane, whereas MCT4 was only detected in the lateral plasma membrane sections of Caco-2 cells. We therefore propose that MCT1 may be acting as an uptake transporter and MCT4 as an efflux system across the basolateral membrane for ferulic acid, and that this process is stimulated by butyric acid.

Predicting phenolic acid absorption in Caco-2 cells: a theoretical permeability model and mechanistic study.

There is a considerable need to rationalize the membrane permeability and mechanism of transport for potential nutraceuticals. The aim of this investigation was to develop a theoretical permeability equation, based on a reported descriptive absorption model, enabling calculation of the transcellular component of absorption across Caco-2 monolayers. Published data for Caco-2 permeability of 30 drugs transported by the transcellular route were correlated with the descriptors 1-octanol/water distribution coefficient (log D, pH 7.4) and size, based on molecular mass. Nonlinear regression analysis was used to derive a set of model parameters a', ß', and b' with an integrated molecular mass function. The new theoretical transcellular permeability (TTP) model obtained a good fit of the published data (R² = 0.93) and predicted reasonably well (R² = 0.86) the experimental apparent permeability coefficient (P(app)) for nine non-training set compounds reportedly transported by the transcellular route. For the first time, the TTP model was used to predict the absorption characteristics of six phenolic acids, and this original investigation was supported by in vitro Caco-2 cell mechanistic studies, which suggested that deviation of the P(app) value from the predicted transcellular permeability (P(app)(trans)) may be attributed to involvement of active uptake, efflux transporters, or paracellular flux.

A novel combined analytical UV and MS approach for the quantification of oleuropein metabolites in human biological samples when authentic standards are not available.

The beneficial health effects of phytochemicals depend on their bioavailability and the form under which they reach systemic circulation, usually as phase II metabolites. The lack of authentic standards for these metabolites makes their quantification in biological samples challenging. A new analytical approach to get a more accurate quantification of oleuropein metabolites in biological samples after ingestion of olive leaf extract was proposed. This approach was based on the calculation of a response factor in QTOF MS for each metabolite, comparing their quantification in UV and MS using urine samples concentrated in the metabolites of interest. Glucuronide and sulfate conjugates of hydroxytyrosol and homovanillyl alcohol were more accurately quantified in plasma and urine and for the first time, oleuropein aglycone conjugates and their hydroxylated and hydrogenated derivatives were quantified after consumption of olive products. This approach could be extensible to the analysis of other phenolic metabolites when authentic standards are not available, opening a valuable method for bioavailability studies.

An oleuropein-based dietary supplement may improve joint functional capacity in older people with high knee joint pain: findings from a multicentre-RCT and post hoc analysis.

OBJECTIVES: To investigate a 6-month intervention with an olive leaf extract (OLE) on knee functionality and biomarkers of bone/cartilage metabolism and inflammation. DESIGN: This randomized, double-blind, placebo-controlled, multi-centric trial included 124 subjects with knee pain or mobility issues. Subjects received twice a day one capsule of placebo or 125 mg OLE (Bonolive™, an OLE containing 50 mg of oleuropein) for 6 months. The co-primary endpoints were Knee injury and Osteoarthritis Outcome Score (KOOS) and serum Coll2-1NO2. The secondary endpoints were the subscales of the KOOS, knee pain VAS at rest and at walking, OARSI core set of performance-based tests and multiple inflammatory and bone or cartilage remodeling serum biomarkers and concentration of oleuropein's metabolites in urine. RESULTS: At 6 months, OLE group was not efficient on global KOOS score, changes of inflammatory and cartilage remodeling biomarkers compared to placebo. Post hoc analyses demonstrated a large and significant treatment effect of OLE in a sub-group of subjects with high walking pain at baseline (p = 0.03). This was observed at 6 months for the global KOOS score, and each different subscale and for pain at walking (p = 0.02). OLE treatment was well tolerated. CONCLUSION: OLE was not effective on joint discomfort excepted in a sub-group of subjects with high pain at treatment initiation. As oleuropein is well tolerated, OLE can be used to relieve knee joint pain and enhance mobility in subjects with articular pain.

Absorption and metabolism of chlorogenic acids in cultured gastric epithelial monolayers.

Gastric absorption of feruloylquinic acid and di-O-caffeoylquinic acid analogs has never been investigated despite their potential contribution to the proposed beneficial health effects leading to reduced risk of type 2 diabetes. Using a cultured gastric epithelial model, with an acidic apical pH, the relative permeability coefficients (P(app)) and metabolic fate of a series of chlorogenic acids (CGAs) were investigated. Mechanistic studies were performed in the apical to basal direction and demonstrated differential rates of absorption for different CGA subgroups. For the first time, we show intact absorption of feruloylquinic acids and caffeoylquinic acid lactones across the gastric epithelium (P(app) ∼ 0.2 cm/s). Transport seemed to be mainly by passive diffusion, because good linearity was observed over the incubation period and test concentrations, and we speculate that a potential carrier-mediated component may be involved in uptake of certain 4-acyl CGA isomers. In contrast, absorption of intact di-O-caffeoylquinic acids was rapid (P(app) ∼ 2-10 cm/s) but nonlinear with respect to time and concentration dependence, which was potentially limited by interaction with an efflux transporter and/or pH gradient dependence. For the first time, methylation is shown in gastric mucosa. Furthermore, isoferulic acid, dimethoxycinnamic acid, and ferulic acid were identified as novel gastric metabolites of CGA biotransformation. We propose that the stomach is the first location for the release of hydroxycinnamic acids, which could explain their early detection after coffee consumption.

Circulating Structurally Related (-)-Epicatechin Metabolite Species and Levels after Sustained Intake of a Cocoa Powder High in Polyphenols Are Comparable to Those Achieved after a Single Dose.

BACKGROUND: While the bioavailability of cocoa polyphenols, particularly of the monomer (-)-epicatechin, has been investigated after a single-dose intake, the effect of sustained cocoa consumption on the metabolic profile of the structurally related (-)-epicatechin metabolites (SREMs) has not been investigated. METHODS: A randomized, controlled crossover clinical trial in healthy young adults (18-40 year) was conducted to evaluate SREMs after consumption of a single-dose and after daily consumption of 1.3 g of polyphenol-rich cocoa powder for 28 days. The circulating SREMs were measured by ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). RESULTS: Twenty subjects (eleven males and nine females) were enrolled. The SREMs concentrations increased to 1741 ± 337 nM after a single-dose and to 1445 ± 270 nM after sustained supplementation. Sulfate conjugates showed higher levels in females (p < 0.05). The epicatechin-3'-glucuronide (E3'G) and epicatechin-3'-sulfate (E3'S) were the most abundant metabolites in all subjects. A high intra-individual correlation (r = 0.72, p < 0.001) between SREMs concentrations after single-dose and sustained supplementation was observed. The antioxidant capacity of plasma did not change in response to the intervention and was not correlated with any of the SREMs. CONCLUSION: The individual SREMs profile and concentrations after a 28-day supplementation are comparable to those after a single dose.

Acute dose-response effect of coffee-derived chlorogenic acids on the human vasculature in healthy volunteers: a randomized controlled trial.

BACKGROUND: Epidemiological studies have reported lower risk of cardiovascular disease with moderate coffee consumption. In addition, emerging evidence indicates that consumption of coffee beverages enriched in chlorogenic acids (CGAs) may influence blood pressure and endothelial function, suggesting that the beneficial cardiovascular effect of coffee may relate to its CGA content. OBJECTIVES: We conducted a double-blind randomized crossover trial to test the effect of acute consumption of a decaffeinated green coffee extract (DGCE), rich in CGAs, on endothelial function in healthy subjects. METHODS: We compared 3 different doses of DGCE (302, 604, and 906 mg, respectively) with a placebo. Endothelial function was defined as the percentage change in the internal diameter of the brachial artery in response to flow-mediated dilation (%FMD). In addition, we followed the plasma concentration-time profiles of 25 systemic CGA metabolites over 24 h after DGCE consumption and we explored the relation between systemic concentrations of CGAs and the effect on %FMD. RESULTS: The DGCE formulations containing different amounts of CGAs resulted in dose-proportional increases in overall total polyphenol concentrations. The systemic appearance of total CGAs was biphasic, in agreement with previous results suggesting 2 sites of absorption in the gastrointestinal tract. Compared with the placebo group, a significant FMD increase (>1%) was observed 8.5, 10, and 24 h after consumption of 302 mg DGCE (∼156.4 mg CGAs). The differences with placebo observed in the other 2 groups were not statistically significant. Evaluation of the relation between phenolic exposure and %FMD showed a positive tendency toward a larger effect at higher concentrations and different behavior of CGA metabolites depending on the conjugated chemical position. CONCLUSIONS: We demonstrated an acute improvement in %FMD over time after ingestion of a DGCE, explained at least partly by the presence in the blood circulation of CGAs and their metabolites. This trial was registered at clinicaltrials.gov as NCT03520452.

Limitations of currently available in vitro oestrogenicity bioassays for effect-based testing of whole foods as the basis for decision making.

The idea that previously unknown hazards can be readily revealed in complex mixtures such as foods is a seductive one, giving rise to the hope that data from effect-based assays of food products collected in market surveys is of suitable quality to be the basis for data-driven decision-making. To study this, we undertook a comparative study of the oestrogenicity of blinded cereal samples, both in a number of external testing laboratories and in our own facility. The results clearly showed little variance in the activities of 9 samples when using a single method, but great differences between the activities from each method. Further exploration of these findings suggest that the oestrogenic activity is likely an inherent part of the natural food matrix which the varying sample preparation methods are able to release and extract to differing degrees. These issues indicate the current poor suitability of these types of datasets to be used as the basis for consumer advice or food decision-making. Data quality must be improved before such testing is used in practice.

Transport and metabolism of ferulic acid through the colonic epithelium.

Ferulic acid is an important antioxidant found in food, beverages, supplements, and herbal medicines. However, its mechanism of absorption in the colon has never been examined, even though this is its main site of in vivo absorption. Ferulic acid was efficiently transported as the free form through an in vitro model for the colonic epithelium consisting of cocultured Caco-2 and mucus-producing HT29-MTX cells, with only a small amount transported as feruloyl-glucuronide or sulfate, together with some free dihydroferulic acid. This pattern of metabolism and permeation was also seen with the use of rat everted ascending and descending colon sacs. In the cell model, free ferulic acid permeated by passive diffusion, as judged by the linearity of the uptake over time and nonsaturable concentration dependence. The permeation was independent of tight junctions but strongly linked to the hydrophobicity of the different phenolic acids tested, suggesting a transcellular rather than a paracellular transport. Using inhibitors, we showed that only a small proportion (<20%) of the free ferulic acid transport was carrier-mediated. The production of metabolites in the basal chamber was lowered by 3-[[3-[2-(7-chloroquinolin-2-yl)-vinyl]phenyl]-(2-dimethylcarbamoylethylsulfanyl)methylsulfanyl] propionic acid (MK571) and increased by cyclosporin A, implying an involvement of multidrug resistance protein and P-glycoprotein transporters in the efflux of metabolites, respectively to the serosal and luminal sides. These results show that the form of ferulic acid available to the blood after passage across the colonic barrier would be mainly the free form, together with only a small percentage of conjugated and reduced ferulic acid.

Effect of dihydrocaffeic acid on UV irradiation of human keratinocyte HaCaT cells.

Dihydrocaffeic acid, a dietary constituent and a microbial metabolite of flavonoids, is an antioxidant, but few biological effects have been examined. After its production by microflora in the colon, dihydrocaffeic acid is absorbed and found in plasma as a combination of free and metabolized forms. Excess solar UV radiation provokes damage and initiates immune response and inflammation in skin, sometimes leading to cancer. Dihydrocaffeic acid reduced the cytotoxicity and pro-inflammatory cytokine production (interleukin-6 and -8) in HaCaT cells, a keratinocyte model, following UV radiation. The effect of dihydrocaffeic acid may result from a combination of direct radical scavenging of the reactive oxygen species formed or reinforcement of the antioxidant potential of the keratinocytes, as well as a direct interference with the pathway involved in cytokine stimulation. The minimum structure required for such an effect appears to consist of a propionate side chain attached to a catechol moiety, as indicated by the efficacy of caffeic acid, but not of the methyl and glucuronide conjugates of dihydrocaffeic acid. The data obtained suggest that dihydrocaffeic acid is a potential candidate for photo-protection by interfering with the events initiated after UV exposure in keratinocytes.

Infant digestion physiology and the relevance of in vitro biochemical models to test infant formula lipid digestion.

Lipids play an important role in the diet of preterm and term infants providing a key energy source and essential lipid components for development. While a lot is known about adult lipid digestion, our understanding of infant digestion physiology is still incomplete, the greatest gap being on the biochemistry of the small intestine, particularly the activity and relative importance of the various lipases active in the intestine. The literature has been reviewed to identify the characteristics of lipid digestion of preterm and term infants, but also to better understand the physiology of the infant gastrointestinal tract compared to adults that impacts the absorption of lipids. The main differences are a higher gastric pH, submicellar bile salt concentration, a far more important role of gastric lipases as well as differences at the level of the intestinal barrier. Importantly, the consequences of improper in vitro replication of gastric digestions conditions (pH and lipase specificity) are demonstrated using examples from the most recent of studies. It is true that some animal models could be adapted to study infant lipid digestion physiology, however the ethical relevance of such models is questionable, hence the development of accurate in vitro models is a must. In vitro models that combine up to date knowledge of digestion biochemistry with intestinal cells in culture are the best choice to replicate digestion and absorption in infant population, this would allow the adaptation of infant formula for a better digestion and absorption of dietary lipids by preterm and term infants.

Acute effects of chlorogenic acids on endothelial function and blood pressure in healthy men and women.

Coffee is a rich source of polyphenols, primarily chlorogenic acids (CGA). Certain polyphenols and polyphenol-rich foods and beverages have been shown to improve endothelial function and lower blood pressure (BP). The aim of the present study was to investigate the acute effect of two doses of CGA (5-CGA) on endothelial function and BP. In a cross-over study, 16 healthy men and women received: (i) 0 mg purified 5-CGA (control group); (ii) 450 mg purified 5-CGA; (iii) 900 mg purified 5-CGA; and (iv) 200 mg purified (-)-epicatechin (positive control) in random order one week apart. Peak and continuous mean (60 to 240 s post ischaemia) flow-mediated dilation (FMD) was measured at baseline, 1 h and 4 h. BP was measured at baseline and every 30 min to 4 h. Plasma CGA and epicatechin levels were significantly increased at both 1 h and 4 h post their respective treatments. Peak FMD was not significantly altered by either dose of 5-CGA or the epicatechin, relative to control (p > 0.05). Relative to control, effects on continuous mean FMD response following 450 mg 5-CGA and 900 mg of 5-CGA (0.47 ± 0.16%, p = 0.016 and 0.65 ± 0.16%, p < 0.001, respectively) at 1 h and (0.18 ± 0.17%, p = 0.99 and 0.44 ± 0.16%, p < 0.05, respectively) at 4 h. There was no significant effect of any of the treatments on BP. In conclusion, the present study has found no significant effect of 5-CGA, at 450 and 900 mg, on peak FMD response. However, there were significant improvements in mean post-ischaemic FMD response, particularly at the 1 h time point in this group of healthy individuals.

Absorption of dimethoxycinnamic acid derivatives in vitro and pharmacokinetic profile in human plasma following coffee consumption.

SCOPE: This study reports the 24 h human plasma pharmacokinetics of 3,4-dimethoxycinnamic acid (dimethoxycinnamic acid) after consumption of coffee, and the membrane transport characteristics of certain dimethoxycinnamic acid derivatives, as present in coffee. METHODS AND RESULTS: Eight healthy human volunteers consumed a low-polyphenol diet for 24 h before drinking 400 mL of commercially available coffee. Plasma samples were collected over 24 h and analyzed by HPLC-MS(2) . Investigation of the mechanism of absorption and metabolism was performed using an intestinal Caco-2 cell model. For the first time, we show that dimethoxycinnamic acid appears in plasma as the free aglycone. The time to reach the C(max) value of approximately 0.5 µM was rapid, T(max) = 30 min, and showed an additional peak at 2-4 h for several subjects. In contrast, smaller amounts of dimethoxy-dihydrocinnamic acid (C(max) ∼ 0.1 µM) peaked between 8 and 12 h after coffee intake. In the cell model, dimethoxycinnamic acid was preferentially transported in the free form by passive diffusion, and a small amount of dimethoxycinnamoylquinic acid hydrolysis was observed. CONCLUSION: These findings show that dimethoxycinnamic acid, previously identified in plasma after coffee consumption, was rapidly absorbed in the free form most likely by passive diffusion in the upper gastrointestinal tract.

Characterization of hydroxycinnamic acid glucuronide and sulfate conjugates by HPLC-DAD-MS(2): enhancing chromatographic quantification and application in Caco-2 cell metabolism.

Hydroxycinnamic acids (HCAs) are emerging naturally occurring anti-inflammatory bioactive compounds. Determination of HCA metabolism has been restricted by the lack of authentic standards for the in vivo metabolites, and so the bioavailability of metabolites is often estimated post-hydrolysis. Recently a set of HCA conjugates were chemically synthesized allowing their detection in biological fluids in a very limited number of studies. However, authentic standards are not widely available and for many investigators accurate quantification of HCA conjugates remains a major analytical challenge. Consequently, we have characterized novel physicochemical properties of 14 authentic standards of HCA conjugates; the resulting data will permit for the first time the accurate quantification of HCA conjugates relative to the parent aglycone without the need for standards. MS operating conditions were optimized to achieve excellent sensitivity, and limits of detection by MS ranged from 3 to 15nM for 12 out of 14 conjugates and 30 to 50nM for the remaining. Intra-day and inter-day precision and accuracy was calculated at <±6% and <±10% respectively. For the first time response factors were determined by triple-quadrupole MS and spectroscopic detection methods, providing essential correction factors. Moreover, we present original analysis of UV-absorbance spectral shifts for HCA conjugates with regio-isomerization, which is advantageous for their differentiation. We demonstrate the usefulness of this method to assess the fate of hydroxycinnamic acids in the Caco-2 cell intestinal model and the impact of metabolism on HCA physicochemistry. For the first time, four HCA conjugates have been unequivocally identified as novel Caco-2 monoculture intestinal metabolites: ferulic acid-4-O-glucuronide, dihydroferulic acid-4-O-sulfate, caffeic acid-4-O-sulfate, and caffeic acid-3-O-sulfate. The characterization data presented here will significantly improve quantification and understanding of the bioavailability in vivo.

Investigation of the metabolic fate of dihydrocaffeic acid.

The antioxidant dihydrocaffeic acid is a dietary constituent and a microbial metabolite of flavonoids. Orally administered to rats, dihydrocaffeic acid was very rapidly absorbed most probably by the gastric or duodenal epithelium and excreted in urine as free and conjugated forms. LC-MS2 analysis of plasma and urine samples allowed confident identification of the dihydrocaffeic acid metabolites. The parent compound was glucuronidated, sulphated or methylated, on one of the hydroxyl groups present on its phenyl ring. All the dihydrocaffeic acid metabolites peaked in plasma within the first 30min following ingestion, suggesting a metabolism possibly by the gastric or duodenal cells and by the liver. Using in vitro and ex vivo models of the intestinal epithelium and the liver, the identity and source of the metabolites detected in vivo were examined. The data obtained suggest that, in rats, intestinal cells are more able to glucuronidate dihydrocaffeic acid, whereas liver favours sulphation. Moreover, glucuronidation, sulphation and methylation seem to be regio-selective, preferably on the 3-OH of dihydrocaffeic acid. The methyl conjugate, dihydroferulic acid, was shown to be oxidized into ferulic acid by intestinal and hepatic cells, which were also able to perform the reverse reaction, the reduction of ferulic acid into dihydroferulic acid. As a conclusion, the main form of dihydrocaffeic acid circulating in plasma after its ingestion is a mixture of different primary and secondary metabolites.

A systematic study of carboxylic acids in negative ion mode electrospray ionisation mass spectrometry providing a structural model for ion suppression.

We report a systematic investigation of the effects and structural requirements for ion suppression in negative ion mode electrospray ionisation mass spectrometry of a series of carboxylic acids and present a structural model rationalising ion suppression effects.