Lucas Actis-Goretta

Bioavailability of bioactive food compounds: a challenging journey to bioefficacy

Bioavailability is a key step in ensuring bioefficacy of bioactive food compounds or oral drugs. Bioavailability is a complex process involving several different stages: liberation, absorption, distribution, metabolism and elimination phases (LADME). Bioactive food compounds, whether derived from various plant or animal sources, need to be bioavailable in order to exert any beneficial effects. Through a better understanding of the digestive fate of bioactive food compounds we can impact the promotion of health and improvement of performance. Many varying factors affect bioavailability, such as bioaccessibility, food matrix effect, transporters, molecular structures and metabolizing enzymes. Bioefficacy may be improved through enhanced bioavailability. Therefore, several technologies have been developed to improve the bioavailability of xenobiotics, including structural modifications, nanotechnology and colloidal systems. Due to the complex nature of food bioactive compounds and also to the different mechanisms of absorption of hydrophilic and lipophilic bioactive compounds, unravelling the bioavailability of food constituents is challenging. Among the food sources discussed during this review, coffee, tea, citrus fruit and fish oil were included as sources of food bioactive compounds (e.g. (poly)phenols and polyunsaturated fatty acids (PUFAs)) since they are examples of important ingredients for the food industry. Although there are many studies reporting on bioavailability and bioefficacy of these bioactive food components, understanding their interactions, metabolism and mechanism of action still requires extensive work. This review focuses on some of the major factors affecting the bioavailability of the aforementioned bioactive food compounds.

Intestinal absorption, metabolism, and excretion of (–)-epicatechin in healthy humans assessed by using an intestinal perfusion technique

BACKGROUND (-)-Epicatechin is a dietary flavonoid present in many foods that affects vascular function, but its action is limited by incomplete absorption, conjugation, and metabolism. Factors that influence this activity may be attributed to instability in the gastrointestinal lumen, low permeability across the intestinal wall, or active efflux from enterocytes and extensive conjugation. OBJECTIVE With the use of a multilumen perfusion catheter, we investigated the jejunal absorption, systemic availability, metabolism, and intestinal, biliary, and urinary excretion of (-)-epicatechin in humans. DESIGN In a single-center, randomized, open, controlled study in 8 healthy volunteers, 50 mg purified (-)-epicatechin was perfused into an isolated jejunal segment together with antipyrine as a marker for absorption. (-)-Epicatechin and conjugates were measured in intestinal perfusates, bile, plasma, and urine. RESULTS Forty-six percent of the dose was recovered in the perfusate either as unchanged (-)-epicatechin (22 mg) or conjugates (0.8 mg); with stability taken into account, this result indicates that ∼46% of the dose had apparently been absorbed. The conjugates were predominantly sulfates, which indicated conjugation by sulfotransferases followed by efflux from the enterocytes. In contrast, epicatechin glucuronides were dominant in plasma, bile, and urine. CONCLUSIONS Almost one-half of the (-)-epicatechin is apparently absorbed in the jejunum but with substantial interindividual differences in the extent of absorption. The data suggest that the nature and substitution position of (-)-epicatechin conjugation are major determinants of the metabolic fate in the body, influencing whether the compound is effluxed into the lumen or absorbed into the blood and subsequently excreted.

UPLC-MS/MS quantification of total hesperetin and hesperetin enantiomers in biological matrices.

Hesperidin (hesperetin-7-O-rutinoside), a flavonoid affecting vascular function, is abundant in citrus fruits and derived products such as juices. After oral administration, hesperidin is hydrolyzed by the colonic microbiota producing hesperetin-7-O-glucoside, the glucoside group is further cleaved and the resulting hesperetin is absorbed and metabolized. Flavanones have a chiral carbon generating (R)- and (S)-enantiomers, with potentially different biological activities. A rapid UPLC-MS/MS method for the analysis of (R)- and (S)-hesperetin enantiomers in human plasma and urine was developed and validated. Biological matrices were incubated with β-glucuronidase/sulfatase, and hesperetin was isolated by solid-phase extraction using 96-well plate mixed-mode cartridges having reversed-phase and anion-exchange functionalities. Racemic hesperetin was analyzed with a UPLC HSS T3 reversed phase column and hesperetin enantiomers with a HPLC Chiralpak IA-3 column using H(2)O with 0.1% CHOOH as solvent A and acetonitrile with 0.1% CHOOH as solvent B. The method was linear between 50 and 5000nM for racemic hesperetin in plasma and between 25 and 2500nM for (S)- and (R)-hesperetin in plasma. Linearity was achieved between 100 and 10,000nM for racemic hesperetin in urine and between 50 and 5000nM for (S)- and (R)-hesperetin in urine. Values of repeatability and intermediate reproducibility for racemic hesperetin and enantiomers in plasma and urine were below 15% of deviation in general, and maximum 20% for the lowest concentrations. In addition, the method was applied for the quantification of total hesperetin and of hesperetin enantiomers in human plasma and urine samples, obtained after oral ingestion of purified hesperetin-7-O-glucoside. In conclusion, the developed and validated method was sensitive, accurate and precise for the quantification of enantiomers of hesperetin in biological fluids.

Quantification of phenolic acids and their methylates, glucuronides, sulfates and lactones metabolites in human plasma by LC–MS/MS after oral ingestion of soluble coffee

Chlorogenic acids and derivatives like phenolic acids are potentially bioactive phenolics, which are commonly found in many foods. Once absorbed, chlorogenic and phenolic acids are highly metabolized by the intestine and the liver, producing glucuronidated and/or sulphated compounds. These metabolites were analyzed in human plasma using a validated liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) method. After protein precipitation, phenolic acids and their metabolites were extracted by using ethanol and chromatographic separation was achieved by reversed-phase using an Acquity UPLC BEH C18 column combined with a gradient elution system using 1% acetic acid aqueous solution and 1% acetic acid with 100% acetonitrile. The method was able to quantify 56 different compounds including 24 phenolic acids, 4 lactones, 15 sulfates and 13 glucuronides metabolites between 5 and 1000nM in plasma for most of them, except for m-dihydrocoumaric acid, 5-ferulloylquinic-glucuronide, 4-methoxycinnamic acid, 3-phenylpropionic acid, 3-(4-methoxyphenyl)propionic acid (25 to 1000nM) and p-dihydrocoumaric acid (50-1000nM). Values of repeatability and intermediate reproducibility were below 15% of deviation in general, and maximum 20% for the lowest concentrations. The validated method was successfully applied to quantify phenolic acids and their metabolites in plasma obtained after oral ingestion of soluble coffee. In conclusion, the developed and validated method is proved to be very sensitive, accurate and precise for the quantification of these possible dietary phenols.

Epicatechin B-Ring Conjugates: First Enantioselective Synthesis and Evidence for Their Occurrence in Human Biological Fluids

Herein, the first enantioselective total synthesis of a number of biologically relevant (-)-epicatechin conjugates is described. The success of this synthesis relied on (i) optimized conditions for the stereospecific cyclization step leading to the catechin C ring; on (ii) efficient conjugation reactions; and on (iii) optimized deprotection sequences. These standard compounds have been subsequently used to elucidate for the first time the pattern of (-)-epicatechin conjugates present in four different human biological fluids following (-)-epicatechin absorption.

The effect of glucuronidation on isoflavone induced estrogen receptor (ER)α and ERβ mediated coregulator interactions.

Non-prenylated isoflavone aglycones are known to have phyto-estrogenic properties and act as agonistic ligands on ERα and ERβ due to their structural resemblance to 17β-estradiol (E2). Genistein and daidzein are the two main dietary isoflavones; upon uptake they are extensively metabolized and exist nearly exclusively as their conjugated forms in biological fluids. Little is known about the effect of conjugation on the intrinsic estrogenic activities of these isoflavones. To characterize and compare the intrinsic estrogenic activities of genistein and daidzein, and their respective 7-O-glucuronide metabolites a cell-free assay system was employed that determines the ligand-induced changes in ERα- and ERβ-ligand binding domain (LBD) interactions with 154 different binding motifs derived from 66 different nuclear receptor coregulators. The glucuronides were 8 to 4400 times less potent than their respective aglycones to modulate ERα-LBD and ERβ-LBD-coregulator interactions. Glucuronidation changed the preferential activation of genistein from ERβ-LBD to ERα-LBD and further increased the slightly preferential activation of daidzein for ERα-LBD. The tested isoflavone compounds were less potent than E2 (around 5 to 1580 times for the aglycones) but modulated the LBD-coregulator interactions in a manner similar to E2. Our results show that genistein and daidzein remain agonistic ligands of ERα-LBD and ERβ-LBD in their conjugated form with a higher relative preference for ERα-LBD than the corresponding aglycones. This shift in receptor preference is of special interest as the preferential activation of ERβ is considered one of the possible modes of action underlying the supposed beneficial instead of adverse health effects of isoflavones.

The role of liquid chromatography and flow injection analyses coupled to isotope ratio mass spectrometry for studying human in vivo glucose metabolism.

Under most physiological conditions, glucose, or carbohydrate (CHO), homeostasis is tightly regulated. In order to mechanistically appraise the origin of circulating glucose (e.g. via either gluconeogenesis, glycogenolysis or oral glucose intake), and its regulation and oxidation, the use of stable isotope tracers is now a well-accepted analytical technique. Methodologically, liquid chromatography coupled to isotope ratio mass spectrometry (LC/IRMS) can replace gas chromatography coupled to combustion-isotope ratio mass spectrometry (GC/C/IRMS) for carrying out compound-specific (13)C isotopic analysis. The LC/IRMS approach is well suited for studying glucose metabolism, since the plasma glucose concentration is relatively high and the glucose can readily undergo chromatography in an aqueous mobile phase. Herewith, we report two main methodological approaches in a single instrument: (1) the ability to measure the isotopic enrichment of plasma glucose to assess the efficacy of CHO-based treatment (cocoa-enriched) during cycling exercise with healthy subjects, and (2) the capacity to carry out bulk isotopic analysis of labeled solutions, which is generally performed with an elemental analyzer coupled to IRMS. For plasma samples measured by LC/IRMS the data show a isotopic precision SD(δ(13)C) and SD(APE) of 0.7 ‰ and 0.001, respectively, with δ(13)C and APE values of -25.48 ‰ and 0.06, respectively, being generated before and after tracer administration. For bulk isotopic measurements, the data show that the presence of organic compounds in the blank slightly affects the δ(13)C values. Despite some analytical limitations, we clearly demonstrate the usefulness of the LC/IRMS especially when (13)C-glucose is required during whole-body human nutritional studies.

Modulation of (-)-epicatechin metabolism by coadministration with other polyphenols in caco-2 cell model

Widely consumed beverages such as red wine, tea, and cocoa-derived products are a great source of flavanols. Epidemiologic and interventional studies suggest that cocoa flavanols such as (–)-epicatechin may reduce the risk of cardiovascular diseases. The interaction of (–)-epicatechin with food components including other polyphenols could modify its absorption, metabolism, and finally its bioactivity. In the present study we investigate (–)-epicatechin absorption and metabolism when coexposed with other polyphenols in the intestinal absorptive Caco-2 cell model. Depending on the type of polyphenols coadministered, the total amount of 3′-O-methyl-epicatechin and 3′-O-sulfate-epicatechin conjugates found both in apical and basal compartments ranged from 19 to 801 nM and from 6 to 432 nM, respectively. The coincubation of (–)-epicatechin with flavanols, chlorogenic acid, and umbelliferone resulted in similar amounts of 3′-O-methyl-epicatechin effluxed into the apical compartment relative to control. Coincubation with isorhamnetin, kaempferol, diosmetin, nevadensin, chrysin, equol, genistein, and hesperitin promoted the transport of 3′-O-methyl-epicatechin toward the basolateral side and decreased the apical efflux. Quercetin and luteolin considerably inhibited the appearance of this (–)-epicatechin conjugate both in the apical and basolateral compartments. In conclusion, we could demonstrate that the efflux of (–)-epicatechin conjugates to the apical or basal compartments of Caco-2 cells is modulated by certain classes of polyphenols and their amount. Ingesting (–)-epicatechin with specific polyphenols could be a strategy to increase the bioavailability of (–)-epicatechin and to modulate its metabolic profile.

Identification of epicatechin as one of the key bioactive constituents of polyphenol-enriched extracts that demonstrate an anti-allergic effect in a murine model of food allergy

Polyphenols are naturally derived bioactive compounds with numerous reported health benefits. We have previously reported on the beneficial effect of a polyphenol-enriched apple extract in a murine model of food allergy. The objectives of the present study were to elucidate the class of bioactive polyphenols that exhibit a beneficial anti-allergic effect and to assess whether the protective effect matches the in vivo bioavailable metabolite concentrations. Female BALB/c mice were sensitised to ovalbumin (OVA) following the protocol of a well-established murine model of food allergy. They were fed diets containing polyphenol-enriched extracts or purified epicatechin for 8 d after the last sensitisation. The sensitised mice were orally challenged with OVA after the intervention. The allergy symptoms, in addition to allergen-specific serum Ig concentrations and gene expression profiles in the intestine, of the control and treated mice were compared. Plasma samples were collected to compare the concentrations of bioavailable epicatechin metabolites in the treatment groups. Polyphenol-enriched fruit extracts containing epicatechin exhibited a significant anti-allergic effect in vivo. This effect was unambiguously attributed to epicatechin, as oral administration of this purified polyphenol to sensitised mice by inclusion in their diet modulated allergy symptoms in a dose-dependent manner. Immune parameters were also affected by the administration of epicatechin. Bioavailability measurements in plasma indicated that the attenuation of allergy symptoms could be due to the higher concentrations of bioavailable epicatechin metabolites. In conclusion, epicatechin is a key bioactive polyphenol that has the ability to modulate allergy outcomes in sensitised mice.

The effect of acute dark chocolate consumption on carbohydrate metabolism and performance during rest and exercise

Consumption of cocoa-enriched dark chocolate (DC) has been shown to alter glucose and insulin concentration during rest and exercise compared with cocoa-depleted control (CON). However, the impact of DC consumption on exercise metabolism and performance is uncertain. Therefore, we investigated carbohydrate metabolism via stable isotope tracer techniques during exercise after subjects ingested either DC or CON. Sixteen overnight-fasted male cyclists performed a single-blinded, randomized, crossover design trial, after consuming either DC or CON at 2 h prior to 2.5 h of steady-state (SS) exercise (∼45% peak oxygen uptake). This was followed by an ∼15-min time-trial (TT) and 60 min of recovery. [6,6-(2)H2]Glucose and [U-(13)C]glucose were infused during SS to assess glucose rate of appearance (Ra) and disappearance (Rd). After DC consumption, plasma (-)-glucose and insulin concentrations were significantly (p < 0.001) elevated throughout vs. CON. During SS, there was no difference in [6,6-(2)H2]glucose Ra between treatments, but towards the end of SS (last 60 min) there was a ∼16% decrease in Rd in DC vs. CON (p < 0.05). Accordingly, after DC there was an ∼18% significant decrease in plasma glucose oxidation (trial effect; p = 0.032), and an ∼15% increase in tracer-derived muscle glycogen utilization (p = 0.045) late during SS exercise. The higher blood glucose concentrations during exercise and recovery after DC consumption coincided with high concentrations of epicatechin and (or) theobromine. In summary, DC consumption altered muscle carbohydrate partitioning, between muscle glucose uptake and glycogen oxidation, but did not effect cycling TT performance.