RESUMO
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.
Assuntos
Ácido Clorogênico/metabolismo , Café/metabolismo , Polifenóis/metabolismo , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Animais , Ácido Clorogênico/química , Coffea/química , Café/química , Esterificação , Fabaceae/química , Feminino , Humanos , Técnicas In Vitro , Absorção Intestinal , Mucosa Intestinal/metabolismo , Jejuno/metabolismo , Masculino , Estrutura Molecular , Proteína 2 Associada à Farmacorresistência Múltipla , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Polifenóis/química , Ácido Quínico/química , Ácido Quínico/metabolismo , Sus scrofaRESUMO
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.
Assuntos
Ácido Clorogênico/farmacocinética , Café/química , Intestino Delgado/efeitos dos fármacos , Absorção , Adulto , Disponibilidade Biológica , Ácido Clorogênico/administração & dosagem , Ácido Clorogênico/sangue , Ácido Clorogênico/urina , Cromatografia Líquida de Alta Pressão , Creatinina/urina , Estudos Cross-Over , Relação Dose-Resposta a Droga , Método Duplo-Cego , Feminino , Humanos , Ileostomia/métodos , Íleo/metabolismo , Intestino Delgado/metabolismo , Espectrometria de Massas em TandemRESUMO
A systematic investigation of the human metabolism of hydroxycinnamic acid conjugates was carried out. A set of 24 potential human metabolites of coffee polyphenols has been chemically prepared, and used as analytical standards for unequivocal identifications. These included glucuronide conjugates and sulfate esters of caffeic, ferulic, isoferulic, m-coumaric and p-coumaric acids as well as their dihydro derivatives. A particular focus has been made on caffeic and 3,4-dihydroxyphenylpropionic acid derivatives, especially the sulfate conjugates, for which regioselective preparation was particularly challenging, and have so far never been identified as human metabolites. Ten out of the 24 synthesized conjugates have been identified in human plasma and/or urine after coffee consumption. A number of these conjugates were synthesized, characterized and detected as hydroxycinnamic acid metabolites for the first time. This was the case of dihydroisoferulic acid 3'-O-glucuronide, caffeic acid 3'-sulfate, as well as the sulfate and glucuronide derivatives of 3,4-dihydroxyphenylpropionic acid.
Assuntos
Líquidos Corporais/metabolismo , Ácidos Cafeicos/sangue , Ácidos Cafeicos/urina , Café/metabolismo , Ácidos Cumáricos/sangue , Ácidos Cumáricos/urina , Comportamento de Ingestão de Líquido , Glucuronídeos/sangue , Glucuronídeos/urina , Ésteres do Ácido Sulfúrico/sangue , Ésteres do Ácido Sulfúrico/urina , Ácidos Cafeicos/química , Ácido Clorogênico/sangue , Ácido Clorogênico/urina , Cromatografia Líquida de Alta Pressão , Ácidos Cumáricos/química , Glucuronídeos/química , Humanos , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Ésteres do Ácido Sulfúrico/químicaRESUMO
Coffee and green tea are two of the most widely consumed hot beverages in the world. Their respective bioavailability has been studied separately, but absorption of their respective bioactive phenolics has not been compared. In a randomised cross-over design, nine healthy subjects drank instant coffee and green tea. Blood samples were collected over 12 h and at 24 h to assess return to baseline. After green tea consumption, (-)-epigallocatechin (EGC) was the major catechin, appearing rapidly in the plasma; (-)-EGC gallate (EGCg) and (-)-epicatechin (EC) were also present, but (-)-EC gallate and C were not detected. Dihydroferulic acid and dihydrocaffeic acid were the major metabolites that appeared after coffee consumption with a long time needed to reach maximum plasma concentration, suggesting metabolism and absorption in the colon. Other phenolic acid equivalents (caffeic acid (CA), ferulic acid (FA) and isoferulic acid (iFA)) were detected earlier, and they peaked at lower concentrations. Summations of the plasma area under the curves (AUC) for the measured metabolites showed 1.7-fold more coffee-derived phenolic acids than green tea-derived catechins (P = 0.0014). Furthermore, we found a significant correlation between coffee metabolites based on AUC. Inter-individual differences were observed, but individuals with a high level of CA also showed a correspondingly high level of FA. However, no such correlation was observed between the tea catechins and coffee phenolic acids. Correlation between AUC and maximum plasma concentration was also significant for CA, FA and iFA and for EGCg. This implies that the mechanisms of absorption for these two classes of compounds are different, and that a high absorber of phenolic acids is not necessarily a high absorber of catechins.
Assuntos
Ácidos Cafeicos/farmacocinética , Camellia sinensis/química , Catequina/farmacocinética , Coffea/química , Café/química , Ácidos Cumáricos/farmacocinética , Chá/química , Adulto , Área Sob a Curva , Catequina/análogos & derivados , Estudos Cross-Over , Feminino , Humanos , Absorção Intestinal , Masculino , Fenóis/sangue , Fenóis/farmacocinéticaRESUMO
The intestinal absorption and metabolism of 385 micromol chlorogenic acids following a single intake of 200 mL of instant coffee by human volunteers with an ileostomy was investigated. HPLC-MS(3) analysis of 0-24h post-ingestion ileal effluent revealed the presence of 274+/-28 micromol of chlorogenic acids and their metabolites accounting for 71+/-7% of intake. Of the compounds recovered, 78% comprised parent compounds initially present in the coffee, and 22% were metabolites including free and sulfated caffeic and ferulic acids. Over a 24h period after ingestion of the coffee, excretion of chlorogenic acid metabolites in urine accounted for 8+/-1% of intake, the main compounds being ferulic acid-4-O-sulfate, caffeic acid-3-O-sulfate, isoferulic acid-3-O-glucuronide and dihydrocaffeic acid-3-O-sulfate. In contrast, after drinking a similar coffee, urinary excretion by humans with an intact colon corresponded to 29+/-4% of chlorogenic acid intake. This difference was due to the excretion of higher levels of dihydroferulic acid and feruloylglycine together with sulfate and glucuronide conjugates of dihydrocaffeic and dihydroferulic acids. This highlights the importance of colonic metabolism. Comparison of the data obtained in the current study with that of Stalmach et al. facilitated elucidation of the pathways involved in post-ingestion metabolism of chlorogenic acids and also helped distinguish between compounds absorbed in the small and the large intestine.
Assuntos
Ácido Clorogênico/farmacocinética , Café/química , Ileostomia , Adulto , Disponibilidade Biológica , Ácidos Cafeicos/química , Ácidos Cafeicos/farmacocinética , Ácidos Cafeicos/urina , Ácido Clorogênico/química , Ácido Clorogênico/urina , Cromatografia Líquida de Alta Pressão , Ácidos Cumáricos/química , Ácidos Cumáricos/farmacocinética , Ácidos Cumáricos/urina , Feminino , Humanos , Íleo/metabolismo , Absorção Intestinal , Masculino , Pessoa de Meia-Idade , Estrutura Molecular , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
Hydroxycinnamic acids are a class of phenolic antioxidants found widely in dietary plants. Their biotransformation in the human organism primarily involves Phase II conjugation reactions. In this study, activities of UDP-glucuronosyltransferases (UGTs) and sulfotransferases (SULTs) towards major dietary hydroxycinnamic acids (caffeic, dihydrocaffeic, dihydroferulic, ferulic and isoferulic acids) were investigated. Conjugate formation was evaluated using human liver and intestinal S9 homogenates, and in vitro characterization was carried out using recombinant human UGTs and SULTs. Analysis of the kinetics of hydroxycinnamic acid conjugation in human S9 homogenates revealed that intrinsic clearance (V(max)/K(m)) is much greater for sulfation than for glucuronidation. Assessment of activity using a panel of recombinant human SULTs showed that SULT1A1 is most active in the sulfation of caffeic, dihydrocaffeic and isoferulic acids, while SULT1E1 is most active in the sulfation of ferulic and dihydroferulic acids. Only isoferulic acid was significantly glucuronidated by human liver S9 homogenates, explained by the high activity of liver-specific UGT1A9. Studies on the kinetics of active SULTs and UGTs demonstrated a markedly lower K(m) for SULTs. To further corroborate our findings, we carried out an intervention study in healthy humans to determine the hydroxycinnamic acid conjugates in urine after consumption of hydroxycinnamate-rich coffee (200 ml). Analysis showed that sulfates are the main conjugates in urine, with the exception of isoferulic acid, which is mainly glucuronidated. These data suggest that sulfates are the predominant hydroxycinnamic acid conjugates in humans, and that SULT mediated sulfation is a major factor determining the bioavailability of hydroxycinnamic acids in vivo.
Assuntos
Ácidos Cumáricos/metabolismo , Ácidos Cumáricos/farmacocinética , Glucuronosiltransferase/metabolismo , Sulfotransferases/metabolismo , Adulto , Biotransformação , Ácidos Cafeicos/metabolismo , Células Cultivadas , Cromatografia Líquida de Alta Pressão , Cinamatos/metabolismo , Ácidos Cumáricos/urina , Dieta , Feminino , Humanos , Cinética , Masculino , Sulfatos/metabolismo , Adulto JovemRESUMO
Chlorogenic acids (CGA) are antioxidants found in coffee. They are becoming of interest for their health-promoting effects, but bioavailability in humans is not well understood. We hypothesized that adding whole milk or sugar and nondairy creamer to instant coffee might modulate the bioavailability of coffee phenolics. Nine healthy participants were asked to randomly drink, in a crossover design, instant coffee (Coffee); instant coffee and 10% whole milk (Milk); or instant coffee, sugar, and nondairy creamer already premixed (Sugar/NDC). All 3 treatments provided the same amount of total CGA (332 mg). Blood was collected for 12 h after ingestion and plasma samples treated using a liquid-liquid extraction method that included a full enzymatic cleavage to hydrolyze all CGA and conjugates into phenolic acid equivalents. Hence, we focused our liquid chromatography-Electrospray ionization-tandem MS detection and quantification on caffeic acid (CA), ferulic acid (FA), and isoferulic acid (iFA) equivalents. Compared with a regular black instant coffee, the addition of milk did not significantly alter the area under the curve (AUC), maximum plasma concentration (C(max)), or the time needed to reach C(max) (T(max)). The C(max) of CA and iFA were significantly lower and the T(max) of FA and iFA significantly longer for the Sugar/NDC group than for the Coffee group. However, the AUC did not significantly differ. As a conclusion, adding whole milk did not alter the overall bioavailability of coffee phenolic acids, whereas sugar and nondairy creamer affected the T(max) and C(max) but not the appearance of coffee phenolics in plasma.
Assuntos
Café/química , Gorduras na Dieta/farmacologia , Sacarose Alimentar/farmacologia , Leite , Fenóis/farmacocinética , Adulto , Animais , Antioxidantes/farmacocinética , Área Sob a Curva , Disponibilidade Biológica , Ácidos Cafeicos/farmacocinética , Cromatografia Líquida de Alta Pressão , Cinamatos/farmacocinética , Ácidos Cumáricos/farmacocinética , Estudos Cross-Over , Feminino , Humanos , Masculino , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
Previous studies on coffee examined absorption of phenolic acids (PA) in the small intestine, but not the contribution of the colon to absorption. Nine healthy volunteers ingested instant soluble coffee ( approximately 335 mg total chlorogenic acids (CGAs)) in water. Blood samples were taken over 12 h, and at 24 h to assess return to baseline. Many previous studies, which used glucuronidase and sulfatase, measured only PA and did not rigorously assess CGAs. To improve this, plasma samples were analyzed after full hydrolysis by chlorogenate esterase, glucuronidase and sulfatase to release aglycone equivalents of PA followed by liquid-liquid extraction and ESI-LC-ESI-MS/MS detection. Ferulic, caffeic and isoferulic acid equivalents appeared rapidly in plasma, peaking at 1-2 h. Dihydrocaffeic and dihydroferulic acids appeared in plasma 6-8 h after ingestion (T(max=)8-12 h). Substantial variability in maximum plasma concentration and T(max) was also observed between individuals. This study confirms that the small intestine is a significant site for absorption of PA, but shows for the first time that the colon/microflora play the major role in absorption and metabolism of CGAs and PA from coffee.
Assuntos
Ácidos Cafeicos/sangue , Café/metabolismo , Colo/metabolismo , Ácidos Cumáricos/sangue , Intestino Delgado/metabolismo , Adulto , Ácido Clorogênico/sangue , Cromatografia Líquida de Alta Pressão , Feminino , Humanos , Masculino , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
Green tea containing 634 micromol of flavan-3-ols was ingested by human subjects with an ileostomy. Ileal fluid, plasma, and urine collected 0-24 h after ingestion were analysed by HPLC-MS. The ileal fluid contained 70% of the ingested flavan-3-ols in the form of parent compounds (33%) and 23 metabolites (37%). The main metabolites effluxed back into the lumen of the small intestine were O-linked sulphates and methyl-sulphates of (epi)catechin and (epi)gallocatechin. Thus, in subjects with a functioning colon substantial quantities of flavan-3-ols would pass from the small to the large intestine. Plasma contained 16 metabolites, principally methylated, sulphated, and glucuronidated conjugates of (epi)catechin and (epi)gallocatechin, exhibiting 101-256 nM peak plasma concentration and the time to reach peak plasma concentration ranging from 0.8 to 2.2 h. Plasma pharmacokinetic profiles were similar to those obtained with healthy subjects, indicating that flavan-3-ol absorption occurs in the small intestine. Ileostomists had earlier plasma time to reach peak plasma concentration values than subjects with an intact colon, indicating the absence of an ileal brake. Urine contained 18 metabolites of (epi)catechin and (epi)gallocatechin in amounts corresponding to 6.8+/-0.6% of total flavan-3-ol intake. However, excretion of (epi)catechin metabolites was equivalent to 27% of the ingested (-)-epicatechin and (+)-catechin.
Assuntos
Catequina/metabolismo , Flavonóis/metabolismo , Ileostomia , Absorção Intestinal , Chá/química , Adulto , Disponibilidade Biológica , Biotransformação , Catequina/análogos & derivados , Catequina/sangue , Catequina/química , Catequina/urina , Cromatografia Líquida de Alta Pressão , Feminino , Flavonóis/sangue , Flavonóis/química , Flavonóis/urina , Conteúdo Gastrointestinal/química , Glucuronídeos/sangue , Glucuronídeos/química , Glucuronídeos/metabolismo , Glucuronídeos/urina , Meia-Vida , Humanos , Masculino , Pessoa de Meia-Idade , Estrutura Molecular , Espectrometria de Massas em TandemRESUMO
A validated method was developed for the simultaneous determination of the hydroxycinnamates caffeic acid (CA), dihydrocaffeic acid (DHCA), ferulic acid (FA), dihydroferulic acid (DHFA), and isoferulic acid (IFA) in human plasma as metabolites derived from coffee consumption. The method includes a protein precipitation step prior to enzymatic hydrolysis of the conjugated metabolites (sulfate, glucuronide, and/or ester) back to their aglycone forms. After liquid-liquid extraction, the reconstituted extract was analysed by high-performance liquid chromatography coupled to negative electrospray ionisation tandem mass spectrometry. Calibration curves were constructed from spiked human plasma samples in the range of 0-4800 nM for each of the targeted analytes. Two internal standards, 3-(4-hydroxyphenyl)-propionic acid (500 nM) and 1,3-dicaffeoylquinic acid (200 nM), were spiked at the beginning of the sample preparation and before analysis, respectively. Good performance data were obtained with limits of detection and quantification of the five hydroxycinnamates ranging between 1-15 nM and 3-50 nM, respectively. Within and between-days precisions were respectively calculated between 8-18% and 8-30% (at 50 nM added initially), between 6-9% and 6-12% (at 200 nM), and between 5-9% and 5-9% (at 500 nM). Precision calculated from different analysts ranged from 18% to 44% (at 50 nM), from 8% to 16% (at 200 nM), and from 4% to 8% (at 500 nM). Using this method, we determined plasma levels in humans and measured the efficiency of deconjugation using our enzymatic cocktail.
Assuntos
Ácidos Cafeicos/sangue , Cromatografia Líquida/métodos , Cinamatos/sangue , Ácidos Cumáricos/sangue , Espectrometria de Massas em Tandem/métodos , Área Sob a Curva , Humanos , Hidrólise , Cinética , Limite de Detecção , Reprodutibilidade dos Testes , Espectrometria de Massas por Ionização por Electrospray/métodosRESUMO
This article reports the development and validation of a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the comprehensive quantification of flavan-3-ol and phenolic acid constituents of milk-based food products. Isotope dilution-based sample preparation consisted of protein precipitation using acidic methanol enriched with the stable isotope labelled internal standards and ultrafiltration to preserve overall polyphenol composition, but to eliminate milk proteins in order to comply with LC. Reversed-phase liquid chromatography was optimized to achieve separation of 22 analytes in 8 min in order to reduce suppression effects, achieve a wide dynamic range and, most importantly, to resolve isomeric compounds. Negative-ion electrospray mass spectrometric detection and fragmentation of analytes was optimized, final transitions were selected for maximized selectivity, reliable quantification and reduction of false positives. The quantitative performance of the method was validated, the main features include: (1) range of lower limits of detection 5-15 ng/mL for flavan-3-ols, 60-100 ng/mL for procyanidins, 1-60 ng/mL for other phenolic acids, (2) lower limits of quantification 15-45 ng/mL for flavan-3-ols, 200-300 ng/mL for procyanidins, 3-200 ng/mL for other phenolic acids, (3) averaged intra-day precision 9.5%, (4) calibrated range 60-300,000 ng/mL for flavan-3-ols, 900-900,000 ng/mL for procyanidins, 9-225,000 ng/mL for other phenolic acids, (5) averaged accuracy 99.5%. Applications for yoghurt and ice-cream products are given. The presented data suggest that this method will help to better characterize the polyphenol composition of milk-based food products for quality control, assessment of dietary intake and for polyphenol bioavailability/bioefficacy studies.
Assuntos
Cromatografia Líquida/métodos , Laticínios/análise , Flavonoides/análise , Hidroxibenzoatos/análise , Espectrometria de Massas em Tandem/métodos , Animais , Isótopos de Carbono , Cinamatos/análise , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
Human subjects drank coffee containing 412 mumol of chlorogenic acids, and plasma and urine were collected 0 to 24 h after ingestion and were analyzed by high-performance liquid chromatography-mass spectrometry. Within 1 h, some of the components in the coffee reached nanomole peak plasma concentrations (C(max)), whereas chlorogenic acid metabolites, including caffeic acid-3-O-sulfate and ferulic acid-4-O-sulfate and sulfates of 3- and 4-caffeoylquinic acid lactones, had higher C(max) values. The short time to reach C(max) (T(max)) indicates absorption of these compounds in the small intestine. In contrast, dihydroferulic acid, its 4-O-sulfate, and dihydrocaffeic acid-3-O-sulfate exhibited much higher C(max) values (145-385 nM) with T(max) values in excess of 4 h, indicating absorption in the large intestine and the probable involvement of catabolism by colonic bacteria. These three compounds, along with ferulic acid-4-O-sulfate and dihydroferulic acid-4-O-glucuronide, were also major components to be excreted in urine (8.4-37.1 mumol) after coffee intake. Feruloylglycine, which is not detected in plasma, was also a major urinary component (20.7 mumol excreted). Other compounds, not accumulating in plasma but excreted in smaller quantities, included the 3-O-sulfate and 3-O-glucuronide of isoferulic acid, dihydro(iso)ferulic acid-3-O-glucuronide, and dihydrocaffeic acid-3-O-glucuronide. Overall, the 119.9 mumol excretion of the chlorogenic acid metabolites corresponded to 29.1% of intake, indicating that as well as being subject to extensive metabolism, chlorogenic acids in coffee are well absorbed. Pathways for the formation of the various metabolites within the body are proposed. Urinary dihydrocaffeic acid-3-O-sulfate and feruloylglycine are potentially very sensitive biomarkers for the consumption of relatively small amounts of coffee.
Assuntos
Bebidas , Cinamatos/sangue , Cinamatos/urina , Café/metabolismo , Ácidos Cumáricos/sangue , Ácidos Cumáricos/urina , Metabolômica , Biomarcadores/sangue , Biomarcadores/urina , Biotransformação , Ácidos Cafeicos/sangue , Ácidos Cafeicos/urina , Cromatografia Líquida de Alta Pressão , Cinamatos/farmacocinética , Ácidos Cumáricos/farmacocinética , Glucuronatos/sangue , Glucuronatos/urina , Humanos , Hidroxilação , Metabolômica/métodos , Espectrometria de Massas por Ionização por Electrospray , Sulfatos/sangue , Sulfatos/urinaRESUMO
The present article describes the development and validation of an ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for the comprehensive quantification of anthocyanin and flavonol constituents of milk-based food products. Protein precipitation by acidified methanol and ultrafiltration was utilized as sample preparation to preserve overall polyphenol composition but to eliminate milk proteins in order to comply with UPLC. Reversed-phase chromatography was optimized to achieve separation of 27 analytes in 10 min in order to reduce suppression effects, achieve a wide dynamic range, and most importantly, to resolve isomeric compounds. Positive-ion electrospray mass spectrometric detection and fragmentation of analytes was optimized, final transitions were selected for maximized selectivity, reliable quantification, and reduction of false positives. The quantitative performance of the method was validated, the main features include (1) range of lower limits of detection 0.3-30 ng/mL for glycosylated analytes, 10-300 ng/mL for aglycones, (2) lower limits of quantification 1-100 ng/mL for glycosylated analytes, 30-1,000 ng/mL for aglycones, (3) averaged intraday precision 9%, (4) calibrated range 2-180,000 ng/mL for glycosylated analytes, 60-600,000 ng/mL for aglycones, and (5) averaged accuracy 101%. Applications for yogurt and ice cream products are given. The presented data suggest that this method will help to better characterize the polyphenol composition of milk-based food products for quality control, for assessment of dietary intake, and for polyphenol bioavailability/bioefficacy studies.
Assuntos
Antocianinas/análise , Cromatografia Líquida , Laticínios/análise , Flavonóis/análise , Leite/química , Espectrometria de Massas em Tandem , Animais , Humanos , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
Two sodium-dependent vitamin C transporter isoforms (SVCT1 and SVCT2) were identified as ascorbic acid transporters, but their roles in skin have, as yet, not been elucidated. Here we analyze the expression and function of SVCTs in healthy human skin cells and skin tissues, and in UVB-induced cutaneous tissue injury. SVCT1 was primarily found in the epidermis expressed by keratinocytes, whereas SVCT2 expression was in the epidermis and dermis in keratinocytes, fibroblasts, and endothelial cells. Uptake experiments revealed that ascorbic acid affinity of SVCT1 was lower than SVCT2 (K(m)=75 muM and K(m)=44 muM, respectively), but maximal velocity was 9-times higher (36 nmol/min/well). In keratinocytes, SVCT1 was found to be responsible for vitamin C transport, although SVCT2 gene expression was higher. On UVB irradiation, SVCT1 mRNA expression in murine skin declined significantly in a time- and dose-dependent manner, whereas SVCT2 mRNA levels were unchanged. Furthermore, UVB irradiation of keratinocytes in vitro was accompanied by reduced ascorbic acid transport. In summary, these data indicate that the two vitamin C transporter isoforms fulfill specific functions in skin: SVCT1 is responsible for epidermal ascorbic acid supply, whereas SVCT2 mainly facilitates ascorbic acid transport in the dermal compartment. UVB-induced oxidative stress in mice resulted in depletion of SVCT1 mRNA levels and led to significantly decreased ascorbic acid uptake in keratinocytes, providing evidence on why ascorbic acid levels are decreased on UVB irradiation in vivo.
Assuntos
Ácido Ascórbico/farmacologia , Transportadores de Ânions Orgânicos Dependentes de Sódio/metabolismo , Estresse Oxidativo/efeitos da radiação , Pele/efeitos dos fármacos , Pele/metabolismo , Simportadores/metabolismo , Raios Ultravioleta , Animais , Biópsia , Células Cultivadas , Regulação da Expressão Gênica , Humanos , Queratinócitos/efeitos dos fármacos , Queratinócitos/metabolismo , Cinética , Camundongos , Camundongos Endogâmicos C57BL , Transportadores de Ânions Orgânicos Dependentes de Sódio/genética , Isoformas de Proteínas/metabolismo , RNA Mensageiro/genética , Transportadores de Sódio Acoplados à Vitamina C , Simportadores/genéticaRESUMO
Dietary bioactive compounds (vitamin E, carotenoids, polyphenols, vitamin C, Se and Zn) have beneficial effects on skin health. The classical route of administration of active compounds is by topical application direct to the skin, and manufacturers have substantial experience of formulating ingredients in this field. However, the use of functional foods and oral supplements for improving skin condition is increasing. For oral consumption, some dietary components could have an indirect effect on the skin via, for example, secondary messengers. However, in the case of the dietary bioactive compounds considered here, we assume that they must pass down the gastrointestinal tract, cross the intestinal barrier, reach the blood circulation, and then be distributed to the different tissues of the body including the skin. The advantages of this route of administration are that the dietary bioactive compounds are metabolized and then presented to the entire tissue, potentially in an active form. Also, the blood continuously replenishes the skin with these bioactive compounds, which can then be distributed to all skin compartments (i.e. epidermis, dermis, subcutaneous fat and also to sebum). Where known, the distribution and mechanisms of transport of dietary bioactive compounds in skin are presented. Even for compounds that have been studied well in other organs, information on skin is relatively sparse. Gaps in knowledge are identified and suggestions made for future research.
Assuntos
Suplementos Nutricionais , Micronutrientes/farmacocinética , Pele/metabolismo , Administração Oral , Ácido Ascórbico/farmacocinética , Disponibilidade Biológica , Carotenoides/farmacocinética , Flavonoides/farmacocinética , Humanos , Fenóis/farmacocinética , Polifenóis , Selênio/farmacocinética , Vitamina E/farmacocinética , Zinco/farmacocinéticaRESUMO
Keratinocyte growth factor (KGF), a member of the fibroblast growth factor (FGF) family, is a specific mitogen for different types of epithelial cells and a potent survival factor for these cells under stress conditions. KGF expression increases strongly after injury to various tissues, including the skin and the intestine, and signaling via the KGF receptor was shown to be crucial for repair of skin wounds and for liver regeneration. Here we demonstrate an increased expression of KGF in chronic liver disease associated with fibrosis. The extent of KGF overexpression correlated strongly with the stage of fibrosis. As the cellular source of KGF we identified activated hepatic stellate cells (HSCs)/myofibroblasts. In contrast to the ligand, the KGF receptor, FGFR2-IIIb, was exclusively expressed by hepatocytes, but not by activated HSCs or other parenchymal or nonparenchymal liver cells. Based on the known effects of KGF on hepatocytes in vitro, our findings suggest that HSC/myofibroblast-derived KGF may enhance liver regeneration and/or hepatocyte survival in patients with chronic liver disease.
Assuntos
Fatores de Crescimento de Fibroblastos/biossíntese , Cirrose Hepática/metabolismo , Hepatopatias/metabolismo , Fígado/citologia , Animais , Tetracloreto de Carbono/toxicidade , Células Cultivadas , Modelos Animais de Doenças , Fator 7 de Crescimento de Fibroblastos , Fibroblastos/metabolismo , Hepatócitos/metabolismo , Humanos , Imuno-Histoquímica , Fígado/metabolismo , Cirrose Hepática/induzido quimicamente , RNA Mensageiro/análise , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos , Receptores de Fatores de Crescimento de Fibroblastos/biossíntese , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Growth factors are polypeptides that stimulate the division of certain cell types at low concentrations. Fibroblast growth factor (FGF) 7 (FGF-7) and its homologue FGF-10 act specifically on various types of epithelial cells including keratinocytes of the skin, intestinal epithelial cells and hepatocytes. In addition, FGF-7 and FGF-10 have been shown to be more than growth factors: they can protect epithelial cells from damaging effects induced, for example, by radiation and oxidative stress. Therefore, they are currently in clinical trials for the treatment of oral mucositis, a severe side-effect of cancer therapy characterized by painful inflammation and ulceration of the oral epithelium. To gain insight into the mechanisms of FGF-7/FGF-10 action in epithelial cells, we searched for genes that are regulated by these growth factors. Indeed, we identified genes that help us to explain the mechanisms that underlie the effects of FGF-7. Most interestingly, several genes were identified that are likely to mediate the cytoprotective effect of FGF-7 for epithelial cells in vitro and possibly also in injured and diseased tissues in vivo.
Assuntos
Citoproteção/fisiologia , Fatores de Crescimento de Fibroblastos/metabolismo , Fatores de Crescimento de Fibroblastos/fisiologia , Regulação da Expressão Gênica , Divisão Celular/fisiologia , Epitélio/metabolismo , Humanos , Fígado/fisiologia , Cicatrização/fisiologiaRESUMO
Fibroblast growth factors (FGFs) regulate early development and organogenesis. In particular, a subfamily of FGFs is essential for the formation and differentiation of epithelial tissues and organs. Recent studies revealed a crucial role for these FGFs in repair of the skin, intestine and liver. In addition, the cytoprotective potential of FGFs suggests their use for the protection of epithelial cells under conditions of stress in vivo. Indeed, the first successful clinical trials using FGFs for the treatment of radiation- and chemotherapy-induced mucosal epithelial damage have been announced.
Assuntos
Citoproteção , Células Epiteliais/fisiologia , Cicatrização , Animais , Fator 10 de Crescimento de Fibroblastos , Fator 7 de Crescimento de Fibroblastos , Fatores de Crescimento de Fibroblastos/fisiologia , Camundongos , Camundongos Knockout , Morfogênese , Receptores de Fatores de Crescimento de Fibroblastos/fisiologia , Fenômenos Fisiológicos da Pele , Linfócitos T/fisiologiaRESUMO
Several growth factors have been suggested to play a crucial role in liver regeneration, but a functional proof is still missing. Since fibroblast growth factors are important for the initiation of mammalian liver development, we determined the roles of these mitogens in liver repair by targeted expression of a dominant-negative fibroblast growth factor receptor (FGFR) in hepatocytes of transgenic mice. The liver of young animals appeared histologically normal, and liver function was not obviously impaired. In aged transgenic mice, the frequency of fatty liver development was strongly increased compared to control animals. Following partial hepatectomy, transgenic mice showed markedly reduced hepatocyte proliferation because of an arrest in the late G(1) phase of the cell cycle. These data demonstrate a key role of FGFR signalling in repair after liver injury.