Angelique Stalmach

Metabolite Profiling of Hydroxycinnamate Derivatives in Plasma and Urine after the Ingestion of Coffee by Humans: Identification of Biomarkers of Coffee Consumption

Human subjects drank coffee containing 412 μmol 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 (Cmax), 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 Cmax values. The short time to reach Cmax (Tmax) 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 Cmax values (145–385 nM) with Tmax 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 μmol) after coffee intake. Feruloylglycine, which is not detected in plasma, was also a major urinary component (20.7 μmol 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 μmol 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.

Green tea flavan-3-ols: colonic degradation and urinary excretion of catabolites by humans.

Following the ingestion of green tea, substantial quantities of flavan-3-ols pass from the small to the large intestine (Stalmach et al. Mol. Nutr. Food Res. 2009, 53, S44-S53; Mol. Nutr. Food Res. 2009, doi: 10.1002/mnfr.200900194). To investigate the fate of the flavan-3-ols entering the large intestine, where they are subjected to the action of the colonic microflora, (-)-epicatechin, (-)-epigallocatechin, and (-)-epigallocatechin-3-O-gallate were incubated in vitro with fecal slurries and the production of phenolic acid catabolites was determined by GC-MS. In addition, urinary excretion of phenolic catabolites was investigated over a 24 h period after ingestion of either green tea or water by healthy volunteers with a functioning colon. The green tea was also fed to ileostomists, and 0-24 h urinary excretion of phenolic acid catabolites was monitored. Pathways are proposed for the degradation of green tea flavan-3-ols in the colon and further catabolism of phenolic compounds passing into the circulatory system from the large intestine, prior to urinary excretion in quantities corresponding to ca. 40% of intake compared with ca. 8% absorption of flavan-3-ol methyl, glucuronide, and sulfate metabolites in the small intestine. The data obtained point to the importance of the colonic microflora in the overall bioavailability and potential bioactivity of dietary flavonoids.

Absorption, metabolism and excretion of Choladi green tea flavan-3-ols by humans

Ten healthy human subjects consumed 500 mL of Choladi green tea, containing 648 mumol of flavan-3-ols after which plasma and urine were collected over a 24 h period and analysed by HPLC-MS. Plasma contained a total of ten metabolites, in the form of O-methylated, sulphated and glucuronide conjugates of (epi)catechin and (epi)gallocatechin, with 29-126 nM peak plasma concentrations (C(max)) occurring 1.6-2.3 h after ingestion, indicative of absorption in the small intestine. Plasma also contained unmetabolised (-)-epigallocatechin-3-gallate and (-)-epicatechin-3-gallate with respective C(max) values of 55 and 25 nM. Urine excreted 0-24 h after consumption of green tea contained 15 metabolites of (epi)catechin and (epi)gallocatechin, but (-)-epigallocatechin-3-gallate and (-)-epicatechin-3-gallate were not detected. Overall flavan-3-ol metabolite excretion was equivalent to 8.1% of intake, however, urinary (epi)gallocatechin metabolites corresponded to 11.4% of (epi)gallocatechin ingestion while (epi)catechin metabolites were detected in amounts equivalent to 28.5% of (epi)catechin intake. These findings imply that (epi)catechins are highly bioavailable, being absorbed and excreted to a much greater extent than most other flavonoids. It is also evident that flavan-3-ol metabolites are rapidly turned over in the circulatory system and as a consequence C(max) values are not an accurate quantitative indicator of the extent to which absorption occurs.

Bioavailability of chlorogenic acids following acute ingestion of coffee by humans with an ileostomy

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.

Absorption, metabolism, and excretion of green tea flavan‐3‐ols in humans with an ileostomy

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.

Recent advances in capillary electrophoresis coupled to mass spectrometry for clinical proteomic applications

Proteome analysis using capillary electrophoresis coupled to mass spectrometry (CE‐MS) has been used in a number of clinical applications in the past years. The main focus of CE‐MS‐based studies has been on the investigation of urine, due to the stability of the urinary proteome, ease of collection, and also the low molecular weight range of the urinary proteome, mostly peptides below 30 kDa. The reproducibility of this approach has enabled analysis of over 20 000 samples in a comparable way, giving enormous statistical power to any additional study involving this methodological setup. In this article, we review the major technological issues associated with the application of CE‐MS in the routine investigation of the urinary proteome for clinical applications. We pinpoint recent developments that may have a chance to improve on the currently used approach, and highlight obstacles that need to be solved. In the second part of the article, we review the recent clinical applications, aiming to highlight relevant issues, and possible future routine applications in clinical diagnosis. In the end, we provide a short outlook, and indicate future developments to be expected, as well as problems that need to be solved to enable routine application of CE‐MS in a clinical setting.

Bioavailability of C-Linked Dihydrochalcone and Flavanone Glucosides in Humans Following Ingestion of Unfermented and Fermented Rooibos Teas

High-performance liquid chromatography-mass spectrometry (HPLC-MS(n)) detected aspalathin and nothofagin, C-glycosides of apigenin and luteolin, and four eriodictyol-C-glycoside isomers in unfermented and fermented rooibos teas. The fermented drink contained 10-fold higher levels of aspalathin and nothofagin and a 4-fold lower eriodictyol-C-glycoside content than the fermented tea. The total flavonoid contents in 500 mL servings of the teas were 84 (fermented) and 159 mumol (unfermented). Following the ingestion of 500 mL of the teas by 10 volunteers, 0-24 h urine and plasma samples were collected for analysis. HPLC-MS(n) identified eight metabolites in urine. These were O-linked methyl, sulfate, and glucuronide metabolites of aspalathin and an eriodictyol-O-sulfate. The main compound excreted was an O-methyl-aspalathin-O-glucuronide (229 nmol) following ingestion of the unfermented drink and eriodictyol-O-sulfate (68 nmol) after ingestion of the fermented beverage. The overall metabolite levels excreted were 82 and 352 nmol, accounting for 0.09 and 0.22% of the flavonoids in the fermented and unfermented drinks, respectively. Most of the aspalathin metabolites were excreted within 5 h of tea consumption, suggesting absorption in the small intestine. Urinary excretion of the eriodictyol-O-sulfate occurred mainly during the 5-12 h collection period, indicative of absorption in the large intestine. Despite exhaustive searches, no flavonoid metabolites were detected in plasma.

Gastrointestinal stability and bioavailability of (poly)phenolic compounds following ingestion of Concord grape juice by humans

The in vitro gastrointestinal stability of (poly)phenolic compounds in Concord grape juice was compared with recoveries in ileal fluid after the ingestion of the juice by ileostomists. Recoveries in ileal fluid indicated that 67% of hydroxycinnamate tartarate esters, and smaller percentages of the intake of other (poly)phenolic compounds, pass from the small intestine to the colon. The juice was also ingested by healthy subjects with an intact functioning colon. Peak plasma concentrations (C(max) ) ranged from 1.0 nmol/L for petunidin-3-O-glucoside to 355 nmol/L for dihydrocoumaric acid. Urinary excretion, as an indicator of bioavailability, varied from 0.26% for total anthocyanins to 24% for metabolites of hydroxycinnamate tartarate esters. The C(max) times of the anthocyanins indicated that their low level absorption occurred in the small intestine in contrast to hydroxycinnamate metabolites which were absorbed in both the small and the large intestine where the colonic microflora appeared responsible for hydrogenation of the hydroxycinnamate side chain. The bioavailability of the complex mixture of (poly)phenolic compounds in Concord grape juice, was very similar to that observed in previous studies when compounds were either fed individually or as major components in products containing a restricted spectrum of (poly)phenolic compounds.

The Antioxidant and Chlorogenic Acid Profiles of Whole Coffee Fruits Are Influenced by the Extraction Procedures

Commercial whole coffee fruit extracts and powder samples were analyzed for chlorogenic acids (CGA), caffeine and antioxidant activities. CGA and caffeine were characterized by LC-MS(n) and HPLC accordingly, and quantified by UV absorbance. ORAC, HORAC, NORAC, SORAC and SOAC (antioxidant capacities) were assessed. Three caffeoylquinic acids, three feruloylquinic acids, three dicaffeoylquinic acids, one p-coumaroylquinic acid, two caffeoylferuloylquinic acids and three putative chlorogenic lactones were quantified, along with a methyl ester of 5-caffeoylquinic acid (detected in one sample, the first such report in any coffee material). Multistep whole coffee fruit extracts displayed higher CGA content than single-step extracts, freeze-dried, or air-dried whole raw fruits. Caffeine in multistep extracts was lower than in the single-step extracts and powders. Antioxidant activity in whole coffee fruit extracts was up to 25-fold higher than in powders dependent upon the radical. Total antioxidant activity of samples displayed strong correlation to CGA content.

Bioavailability of coffee chlorogenic acids and green tea flavan-3-ols.

This paper reviews recent human studies on the bioavailability of chlorogenic acids in coffee and green tea flavan-3-ols in which the identification of metabolites, catabolites and parent compounds in plasma, urine and ileal fluid was based on mass spectrometric methodology. Both the chlorogenic acids and the flavan-3-ols are absorbed in the small intestine and appear in the circulatory system predominantly as glucuronide, sulfate and methylated metabolites. Even when absorption occurs in the small intestine, feeding studies with ileostomists reveal that substantial amounts of the parent compounds and some of their metabolites appear in ileal fluid indicating that in volunteers with a functioning colon these compounds will pass to the large intestine where they are subjected to the action of the colonic microflora. A diversity of colonic-derived catabolites are absorbed into the bloodstream and pass through the body prior to excretion in urine. There is growing evidence that these compounds, which were little investigated until recently, are produced in quantity in the colon and form a key part of the bioavailability equation of flavonoids and related compounds that occur in fruits, vegetables and beverages. Recent evidence indicates that some colon-derived phenolic acids have in vitro anti-inflammatory activity.