A novel strategy to simultaneously enhance bioaccessible lipids and antioxidants in hetero/mixotrophic Chlorella vulgaris as functional ingredient.

Microalgae are a promising source of polyunsaturated fatty acids as well as bioactive antioxidant compounds such as carotenoids, phenolics and tocopherols. However, the accumulation of these biomolecules is often promoted by conflicting growth conditions. In this study, a phased bioprocessing strategy was developed to simultaneously enhance the lipid and antioxidant amounts by tailoring nitrogen content in the cultivation medium and applying light stress. This approach increased the overall contents of total fatty acids, carotenoids, phenolics, and α-tocopherol in Chlorella vulgaris by 2.2-, 2.2-, 1.5-, and 2.1-fold, respectively. Additionally, the bioaccessibility of the lipids and bioactives from the obtained biomasses improved after pulsed electric field (5 µs, 20 kV cm-1, 31.8 kJ kg-1sus) treatment (up to +12%) and high-pressure homogenization (100 MPa, 5-6 passes) (+41-76%). This work represents a step towards the generation of more efficient algae biorefineries, thus expanding the alternative resources available for essential nutrients.

Biochemical and Morphological Characterization of Heterotrophic Crypthecodinium cohnii and Chlorella vulgaris Cell Walls.

Microalgae are attractive for the food and cosmetic industries because of their nutrient composition. However, the bioaccessibility and extractability of nutrients in microalgae are limited by the rigid and indigestible cell wall. The goal of this study is to explore the cell wall polysaccharides (CWPSs) composition and morphology in heterotrophic Crypthecodinium cohnii and Chlorella vulgaris biomasses during growth. Our results showed that glucose was the major component of CWPSs and exopolysaccharides in C. cohnii. C. vulgaris CWPSs have a similar sugar profile in exponential and stationary phases, essentially composed of rhamnose and galactose. C. vulgaris cell wall thickness increased from 82 nm in the exponential phase to 114 nm in the stationary phase and consisted of two main layers. C. cohnii's cell wall was 133 nm thick and composed of several membranes surrounding thecal plates. Understanding of the microalgae cell wall helps developing a more efficient and targeted biorefinery approach.

Biochemical and Nutritional Evaluation of Chlorella and Auxenochlorella Biomasses Relevant for Food Application.

Microalgae are a source of potentially healthy and sustainable nutrients. However, the bioaccessibility of these nutrients remains uncertain. In this study, we analyzed the biomass composition of five commercial Chlorella and Auxenochlorella strains, and Chlorella vulgaris heterotrophically cultivated in our laboratory. Protein accounted for 65 ± 3% (w w-1) dry matter (DM) in all biomasses, except for the lab-grown C. vulgaris that contained 20% (w w-1) DM protein. The fatty acids content was comparable and ranged between 7 and 10% (w w-1) DM. Most of the biomasses had a ω6-polyunsaturated fatty acids (PUFAs)/ω3-PUFAs ratio <4, as recommended by nutritional experts. A recently published harmonized protocol for in vitro digestion was used to evaluate fatty acids and protein bioaccessibilities. Protein bioaccessibility ranged between 60 and 74% for commercial Chlorella and Auxenochlorella biomasses and was 43% for the lab-grown C. vulgaris. Fatty acids bioaccessibility was <7% in commercial biomasses and 19% in the lab-grown C. vulgaris. Taken together, the results show that microalgae are promising sources of bioaccessible protein. The limited fatty acids bioaccessibility indicates the need for alternative upstream and downstream production strategies.

Vaccenic acid and trans fatty acid isomers from partially hydrogenated oil both adversely affect LDL cholesterol: a double-blind, randomized controlled trial.

BACKGROUND: Adverse effects of industrially produced trans fatty acids (iTFAs) on the risk of coronary artery disease are well documented in the scientific literature; however, effects of naturally occurring trans fatty acids (TFAs) from ruminant animals (rTFA), such as vaccenic acid (VA) and cis-9,trans-11 conjugated linoleic acid (c9,t11-CLA), are less clear. Although animal and cell studies suggest that VA and c9,t11-CLA may be hypocholesterolemic and antiatherogenic, epidemiologic data comparing rTFAs and iTFAs are inconsistent, and human intervention studies have been limited, underpowered, and not well controlled. OBJECTIVE: We determined the effects of VA, c9,t11-CLA, and iTFA, in the context of highly controlled diets (24 d each), on lipoprotein risk factors compared with a control diet. RESULTS: We conducted a double-blind, randomized, crossover feeding trial in 106 healthy adults [mean ± SD age: 47 ± 10.8 y; body mass index (in kg/m(2)): 28.5 ± 4.0; low-density lipoprotein (LDL) cholesterol: 3.24 ± 0.63 mmol/L]. Diets were designed to have stearic acid replaced with the following TFA isomers (percentage of energy): 0.1% mixed isomers of TFA (control), ∼3% VA, ∼3% iTFA, or 1% c9,t11-CLA. Total dietary fat (34% of energy) and other macronutrients were matched. Total cholesterol (TC), LDL cholesterol, triacylglycerol, lipoprotein(a), and apolipoprotein B were higher after VA than after iTFA; high-density lipoprotein (HDL) cholesterol and apolipoprotein AI also were higher after VA. Compared with control, VA and iTFA both increased TC, LDL cholesterol, ratio of TC to HDL cholesterol, and apolipoprotein B (2-6% change; P < 0.05); VA also increased HDL cholesterol, apolipoprotein AI, apolipoprotein B, and lipoprotein(a) (2-6% change; P < 0.05), whereas iTFA did not. c9,t11-CLA lowered triacylglycerol (P ≤ 0.01) and had no effect on other lipoprotein risk factors. CONCLUSIONS: With respect to risk of cardiovascular disease, these results are consistent with current nutrition labeling guidelines, with the requirement of VA, but not c9,t11-CLA, to be listed under TFA on the Nutrition Facts Panel. This trial was registered at clinicaltrials.gov as NCT00942656.

Structure- and dose-absorption relationships of coffee polyphenols.

Chlorogenic acids (CGAs) from coffee have biological effects related to human health. Thus, specific data on their bioavailability in the upper gastrointestinal tract are of high interest, since some molecules are absorbed here and so are not metabolized by colonic microflora. Up to now, no data on structure-absorption relationships for CGAs have been published, despite this being the most consumed group of polyphenols in the western diet. To address this gap, we performed ex vivo absorption experiments with pig jejunal mucosa using the Ussing chamber model (a model simulating the mucosa and its luminal/apical side). The main coffee polyphenols, caffeoylquinic acid (CQA), feruloylquinic acid (FQA), caffeic acid (CA), dicaffeoylquinic acid (diCQA), and D-(-)-quinic acid (QA), were incubated in individual experiments equivalent to gut lumen physiologically achievable concentrations (0.2-3.5 mM). Identification and quantification were performed with HPLC-diode array detection and HPLC-MS/MS. Additionally, the presence of ABC-efflux transporters was determined by Western blot analysis. The percentages of initially applied CGAs that were absorbed through the jejunal pig mucosa were, in increasing order: diCQA, trace; CQA, ≈ 1%; CA, ≈ 1.5%; FQA, ≈ 2%; and QA, ≈ 4%. No differences were observed within the CGA subgroups. Dose-absorption experiments with 5-CQA suggested a passive diffusion (nonsaturable absorption and a linear dose-flux relationship) and its secretion was affected by NaN3 , indicating an active efflux. The ABC-efflux transporters MDR 1 and MRP 2 were identified in pig jejunal mucosa for the first time. We conclude that active efflux plays a significant role in CGA bioavailability and, further, that the mechanism of CGA absorption in the jejunum is governed by their physicochemical properties.

Dose-response plasma appearance of coffee chlorogenic and phenolic acids in adults.

SCOPE: Coffee contains phenolic compounds, mainly chlorogenic acids (CGAs). Even though coffee intake has been associated with some health benefits in epidemiological studies, the bioavailability of coffee phenolics is not fully understood. OBJECTIVE AND STUDY DESIGN: We performed a dose-response study measuring plasma bioavailability of phenolics after drinking three increasing, but still nutritionally relevant doses of instant pure soluble coffee. The study design was a one treatment (coffee) three-dose randomized cross-over design, with a washout period of 2 wks between visits. RESULTS: CGAs, phenolic acids, and late-appearing metabolites all increased with increasing ingested dose. Hence, the sum of area under the curve was significantly higher for the medium to low dose, and high to medium dose, by 2.23- and 2.38-fold, respectively. CGAs were not well absorbed in their intact form, regardless of the dose. CGA and phenolic acids appeared rapidly in plasma, indicating an early absorption in the gastrointestinal tract. Late-appearing metabolites were the most abundant, regardless of the dose. CONCLUSION: This study confirmed previous findings about coffee bioavailability but also showed that coffee phenolics appear in a positive dose-response manner in plasma when drank at nutritionally relevant doses.

Dose-response plasma appearance of green tea catechins in adults.

SCOPE: Tea is an infusion of the Camellia sinensis leaves. The most prevalent bioactive compounds in green tea are catechins (C), which are of great interest for their potential health-promoting effects. However, metabolism and bioavailability of C are not fully understood. METHODS AND RESULTS: This study investigates the human bioavailability (plasma appearance) of C after drinking three doses of infused green tea in a randomized cross-over design. The sum of area under the curve increased between the small (0.75% w/v, 180 mg total C) and medium (1.25%) dose of ingested green tea but not between the medium and the high (1.75%) dose. The overall pattern for the sum of C did not reflect the fate of individual C. While (-)-epigallocatechin and 4'-O-Me-epigallocatechin showed saturation in plasma between the medium and high green tea doses, (-)-epigallocatechin gallate and (-)-epicatechin did not "saturate" and increased proportionally with the ingested dose. Regardless of the dose, C appeared rapidly in plasma as monophasic curves, suggesting absorption in the small intestine and minimal entero-hepatic circulation. CONCLUSION: As a conclusion, when studying dose response of polyphenols and metabolites, one must look not only at the overall pattern of plasma appearance, but also at data specific for each metabolite.

Benefits of structured and free monoacylglycerols to deliver eicosapentaenoic (EPA) in a model of lipid malabsorption.

In the present study, we used a preclinical model of induced lipolytic enzyme insufficiency, and hypothesized that the use of monoacylglycerols (MAG) will enhance their bioavailability and delivery to the tissues. Experimental diets containing 20% lipids were fed to rats for 21 days with or without Orlistat. The control diet of fish oil (FO), a source of EPA and DHA, was tested against: structured (A) vanillin acetal of sn-2 MAG (Vanil + O) and (B) diacetyl derivative of sn-2 MAG (Acetyl + O) and (C) free MAG (MAG + O). FA profiles with an emphasis on EPA and DHA levels were determined in plasma, red blood cells (RBC), liver, spleen, brain and retina. We observed significant reduction of lipid absorption when rats co-consumed Orlistat. As expected, the FO groups with and without Orlistat showed the biggest difference. The Vanil + O, Acetyl + O and MAG + O groups, demonstrated higher levels of EPA (5.5 ± 1.9, 4.6 ± 1.6 and 5.6 ± 0.6, respectively) in RBC compared with FO + O diets (3.3 ± 0.2, 2.6 ± 0.2). Levels of EPA incorporation, in plasma, were similar to those obtained for RBC, and similar trends were observed for the collected tissues and even with DHA levels. These observations with two MAG derivatives providing the fatty acid esterified in the sn-2 position, show that these molecules are efficient vehicles of EPA in malabsorption conditions which is in line with our hypothesis. Free MAG, characterized as having exclusively sn-1(3) isomers of EPA, demonstrated better absorption efficiencies and accretion to tissues when compared to structured MAG. The study demonstrated that structured and free MAG can be used efficiently as an enteral vehicle to supply bioactive fatty acids such as EPA and DHA in lipid malabsorption where diminished lipolytic activity is the underlying cause.

Dose-dependent absorption of chlorogenic acids in the small intestine assessed by coffee consumption in ileostomists.

SCOPE: Until now, the question of how the ingested doses of chlorogenic acids (CGA) from coffee influence their absorption and metabolism remains unresolved. To assess absorption in the small intestine, we performed a dose-response study with a randomized, double-blinded, crossover design with ileostomist subjects. METHODS AND RESULTS: After a polyphenol-free diet, the volunteers consumed, on three separate occasions, coffee with different total CGA contents (high 4525 µmol; medium 2219 µmol; low 1053 µmol). CGA concentrations in plasma, ileal effluent, and urine were subsequently determined by HPLC-DAD-ESI-MS and -ESI-MS/MS. The results show that the consumption of higher CGA concentrations leads to a faster ileal excretion. This corresponds to a renal excretion of 8.0 ± 4.9% (high), 12.1 ± 6.7% (medium), and 14.6 ± 6.8% (low) of total CGA and metabolites. Glucuronidation of CGA became slightly greater with increasing dose. After enzyme treatment, the area under the curve (AUC)(0-8h) for CGA metabolites in plasma was 4412 ± 751 nM × h(0-8) (-1) (high), 2394 ± 637 nM × h(0-8) (-1) (medium), 1782 ± 731 nM × h(0-8) (-1) (low), respectively. Additionally, we were able to identify new metabolites of CGA in urine and ileal fluid. CONCLUSION: We conclude that the consumption of high CGA concentrations via coffee might influence the gastrointestinal transit time and consequently affect CGA absorption and metabolism.

A state-of-the-art overview of the effect of metabolic conjugation on the biological activity of flavonoids.

Diets rich in flavonoids are associated with various positive health effects. Most in vitro research conducted to elucidate the modes of action of flavonoids uses flavonoid aglycones, but not their circulating conjugated metabolites. Conjugation alters the physico-chemical properties of flavonoids and it is widely assumed that this can affect their biological activity. This article gives a state-of-the-art overview of scientific literature reporting on the effect of metabolic conjugation on the biological activity of flavonoids. The biological activity of flavonoid aglycones is compared to that of their conjugates for a broad range of endpoints. Even though there is only limited literature available, it is shown that contrary to common belief, conjugation does not always decrease the biological activity of flavonoids. There are also endpoints which are unaffected by conjugation, and endpoints on which the conjugates have a higher or inverse activity when compared to the aglycone. The effects of conjugation can differ depending on the type and position of conjugation, the flavonoid concentration, the endpoint studied and the assay system used so that no general rules can be deducted. It is concluded that further studies on the effects of conjugation have to be done on a case-by-case basis, and characterization of the stability and metabolic fate of the flavonoids in the assay system under consideration is needed to avoid false positive or false negative outcomes.

Elucidation of (-)-epicatechin metabolites after ingestion of chocolate by healthy humans.

After absorption in the gastrointestinal tract, (-)-epicatechin is extensively transformed into various conjugated metabolites. These metabolites, chemically different from the aglycone forms found in foods, are the compounds that reach the circulatory system and the target organs. Therefore, it is imperative to identify and quantify these circulating metabolites to investigate their roles in the biological effects associated with (-)-epicatechin intake. Using authentic synthetic standards of (-)-epicatechin sulfates, glucuronides, and O-methyl sulfates, a novel LC-MS/MS-MRM analytical methodology to quantify (-)-epicatechin metabolites in biological matrices was developed and validated. The optimized method was subsequently applied to the analysis of plasma and urine metabolites after consumption of dark chocolate, an (-)-epicatechin-rich food, by humans. (-)-Epicatechin-3'-ß-d-glucuronide (C(max) 290 ± 49 nM), (-)-epicatechin 3'-sulfate (C(max) 233 ± 60 nM), and 3'-O-methyl epicatechin sulfates substituted in the 4', 5, and 7 positions were the most relevant (-)-epicatechin metabolites in plasma. When plasmatic metabolites were divided into their substituent groups, it was revealed that (-)-epicatechin glucuronides, sulfates, and O-methyl sulfates represented 33 ± 4, 28 ± 5, and 33 ± 4% of total metabolites (AUC(0-24)(h)), respectively, after dark chocolate consumption. Similar metabolites were found in urine samples collected over 24h. The total urine excretion of (-)-epicatechin was 20 ± 2% of the amount ingested. In conclusion, we describe the entire metabolite profile and its degree of elimination after administration of (-)-epicatechin-containing food. These results will help us understand more precisely the mechanisms and the main metabolites involved in the beneficial physiological effects of flavanols.

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 ß-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.

Interaction of hesperetin glucuronide conjugates with human BCRP, MRP2 and MRP3 as detected in membrane vesicles of overexpressing baculovirus-infected Sf9 cells.

The citrus flavonoid hesperetin (4'-methoxy-3',5,7-trihydroxyflavanone) is the aglycone of hesperidin, the major flavonoid present in sweet oranges. Hesperetin 7-O-glucuronide (H7G) and hesperetin 3'-O-glucuronide (H3'G) are the two most abundant metabolites of hesperetin in vivo. In this study, their interaction with specific ABC transporters, believed to play a role in the disposition and bioavailability of hesperetin, was studied using Sf9 membranes from cells overexpressing human BCRP (ABCG2), MRP2 (ABCC2) and MRP3 (ABCC3). Both H7G and H3'G were tested for their potential to activate and inhibit ATPase activity, and to inhibit vesicular transport by these transporters. Both H7G and H3'G demonstrated interaction with all tested ABC transporters, especially with BCRP and MRP3. An interesting difference between H7G and H3'G was seen with respect to the interaction with BCRP: H7G stimulated the ATPase activity of BCRP up to 76% of the maximal effect generated by the reference activator sulfasalazine, with an EC(50) of 0.45 µM, suggesting that H7G is a high affinity substrate of BCRP, whereas H3'G did not stimulate BCRP ATPase activity. Only moderate inhibition of BCRP ATPase activity at high H3'G concentrations was observed. This study provides information on the potential of hesperetin glucuronide conjugates to act as specific ABC transporter substrates or inhibitors and indicates that regio-specific glucuronidation could affect the disposition of hesperetin.

Identification of novel circulating coffee metabolites in human plasma by liquid chromatography-mass spectrometry.

This study reports a liquid chromatography-mass spectrometry method for the detection of polyphenol-derived metabolites in human plasma without enzymatic treatment after coffee consumption. Separation of available standards was achieved by reversed-phase ultra performance liquid chromatography and detection was performed by high resolution mass spectrometry in negative electrospray ionization mode. This analytical method was then applied for the identification and relative quantification of circulating coffee metabolites. A total of 34 coffee metabolites (mainly reduced, sulfated and methylated forms of caffeic acid, coumaric acid, caffeoylquinic acid and caffeoylquinic acid lactone) were identified based on mass accuracy (<4 ppm for most metabolites), specific fragmentation pattern and co-chromatography (when standard available). Among them, 19 circulating coffee metabolites were identified for the first time in human plasma such as feruloylquinic acid lactone, sulfated and glucuronidated forms of feruloylquinic acid lactone and sulfated forms of coumaric acid. Phenolic acid derivatives such as dihydroferulic acid, dihydroferulic acid 4'-O-sulfate, caffeic acid 3'-O-sulfate, dimethoxycinnamic acid, dihydrocaffeic acid and coumaric acid O-sulfate appeared to be the main metabolites circulating in human plasma after coffee consumption. The described method is a sensitive and reliable approach for the identification of coffee metabolites in biological fluids. In future, this analytical method will give more confidence in compound identification to provide a more comprehensive assessment of coffee polyphenol bioavailability studies in humans.

Identification of the botanical origin of commercial pine nuts responsible for dysgeusia by gas-liquid chromatography analysis of Fatty Acid profile.

Over the last 10 years, complaints were increasingly reported from consumers that experienced dysgeusia following the consumption of pine nuts. In the present study, pine nuts samples (N = 16) from consumers that reported dysgeusia have been analyzed to identify the botanical origin of critical pine nuts samples. The fatty acid composition of the samples was performed, and diagnostic index values were used to identify the botanical origin of the samples. Pinus armandii nuts were identified in all the samples pure or in mixture with P. koraiensis nuts. P. armandii is not reported as edible pine nuts by the Food and Agriculture Organization (FAO). This study confirmed that consumption of P. armandii nuts may lead to dysgeusia. Based on the present study and previous work, we advise import companies to trade pine nuts from traditionally recognized species such as P. pinea, P. sibirica, P. koraiensis, or P. gerardiana.

Flavanols from green tea and phenolic acids from coffee: critical quantitative evaluation of the pharmacokinetic data in humans after consumption of single doses of beverages.

Coffee contains a complex mixture of chlorogenic acids, which are mainly ferulic and caffeic acids ester-linked to quinic acid. Green tea contains flavanols, mainly (-)-epigallocatechin gallate (EGCG), (-)-epigallocatechin (EGC) and (-)-epicatechin (EC). For healthy humans, we identified seven studies on green tea in liquid form and five on coffee beverage reporting single-dose plasma pharmacokinetics. Weighted averages, based on the number of subjects, and elimination of outliers, allowed estimation of some pharmacokinetic parameters. After consumption of an "average" cup of green tea containing 112 mg of (-)-epigallocatechin gallate, 51 mg of EGC and 15 mg of EC in 200 mL, the predicted C(max) values (total free and sulfate/glucuronide conjugates) in plasma are 125, 181 and 76 nM, respectively, together with 94 nM methyl-EGC and 51 nM methyl-EC (standard deviation <20%). After consumption of an "average" cup of coffee (160 mg total chlorogenic acids (0.46 mmol)/200 mL), predicted C(max) values of caffeic, ferulic, isoferulic, dihydrocaffeic and dihydroferulic acids are 114, 96, 50, 384 and 594 nM, respectively (too few studies to calculate standard deviation). Most studies report a very low amount of intact chlorogenic acids in plasma, with one exception. More studies on absorption of chlorogenic acids from coffee are required, including dose-response studies.

Interaction of hydroxycinnamic acids and their conjugates with organic anion transporters and ATP-binding cassette transporters.

SCOPE: Hydroxycinnamic acids are abundant antioxidants in our diet. In humans, hydroxycinnamic acids are metabolized to form sulfates and glucuronides, with the majority recovered in urine. METHODS AND RESULTS: We assessed the potential roles of organic anion transporters (OATs) and ATP-binding cassette (ABC) transporters in the renal uptake and efflux of hydroxycinnamic acid conjugates. Uptake studies using OAT1 (SLC22A6)-, OAT2 (SLC22A7)-, and OAT3 (SLC22A8)-expressing 293H embryonic kidney cells showed that OAT1 and OAT3, but not OAT2, accepted hydroxycinnamic acid conjugates as substrates. OAT1 and OAT3 mediated the basolateral uptake of hydroxycinnamic acid sulfates and glucuronide conjugates, respectively. Hydroxycinnamic acid sulfates are substrates of OAT4 and were capable of trans-stimulating 5-carboxyfluorescein uptake mediated by OAT4. On the other hand, hydroxycinnamic acid conjugates are not substrates for the ABC transporters, multidrug resistance protein 2 (MRP2/ABCC2) or breast cancer resistance protein (BCRP/ABCG2), demonstrated by the inability to alter ATPase activity. Cis-inhibition studies with OATs and MRPs revealed that hydroxycinnamic acid conjugates have limited impact on the transport of model substrates significantly at physiological concentrations. CONCLUSION: Concerted action of OAT1, OAT3, and OAT4 is involved in the elimination of hydroxycinnamic acid conjugates into urine, whereas MRP2 and breast cancer resistance protein are not involved in the disposition of these conjugates.

Effect of trans fatty acid isomers from ruminant sources on risk factors of cardiovascular disease: study design and rationale.

Substantial evidence clearly demonstrates the deleterious effects of industrially-produced trans fatty acids (TFA); however, data are lacking from large, well controlled human feeding studies that directly compare the effects of industrially-produced and naturally-occurring TFA. The purpose of the current study is to determine whether consumption of TFA derived from different sources differentially affect risk factors of cardiovascular disease (CVD). The study was a randomized, crossover design, controlled-feeding intervention designed to compare the effects of the following diet treatments on risk factors of CVD: low TFA diet (base diet, 34% energy from fat; 0.1% energy from TFA), base diet with vaccenic acid (3.0% energy), base diet with mixed isomers of TFA from partially hydrogenated vegetable oil (3.0% energy), and base diet with cis-9, trans-11 CLA (1.0% energy). The added energy from TFA replaced energy from stearic acid. Participants were required to be between the ages of 25 and 65 years, have a body mass index between 20 and 38 kg/m(2), total cholesterol <280 mg/dl, fasting triacylglycerol <300 mg/dl, fasting glucose <126 mg/dl, and blood pressure <160/100 mm Hg (controlled with certain medications). Of the 116 participants who were randomized, a total of 95 completed the intervention. Results from this study will be important in determining whether ruminant TFA and industrially produced TFA differentially affect markers of cardiovascular risk, in the context of a highly controlled feeding study.

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.

Plasma pharmacokinetics of catechin metabolite 4'-O-Me-EGC in healthy humans.

BACKGROUND: Tea is an infusion of the leaves of the Camellia sinensis plant and is the most widely consumed beverage in the world after water. Green tea contains significant amounts of polyphenol catechins and represents a promising dietary component to maintain health and well-being. Epidemiological studies indicate that polyphenol intake may have potential health benefits, such as, reducing the incidence of coronary heart disease, diabetes and cancer. While bioavailability of green tea bioactives is fairly well understood, some gaps still remain to be filled, especially the identification and quantification of conjugated metabolites in plasma, such as, sulphated, glucuronidated or methylated compounds. AIM OF THE STUDY: In the present study, we aimed to quantify the appearance of green tea catechins in plasma with particular emphasis on their methylated forms. RESULTS: After feeding 400 mL of green tea, 1.25% infusion to 9 healthy subjects, we found significant amounts of EC, EGC and EGCg in plasma as expected. EGC was the most bioavailable catechin, and its methylated form (4'-O-Me-EGC) was also present in quantifiable amounts. Its kinetics followed that of its parent compound. However, the relative amount of the methylated form of EGC was lower than that of the parent compound, an important aspect which, in the literature, has been controversial so far. The quantitative results presented in our study were confirmed by co-chromatography and accurate mass analysis of the respective standards. We show that the relative abundance of 4'-O-Me-EGC is ~40% compared to the parent EGC. CONCLUSION: 4'-O-Me-EGC is an important metabolite derived from catechin metabolism. Its presence in significant amounts should not be overlooked when assessing human bioavailability of green tea.