Validation of a mass spectrometry method to quantify oak ellagitannins in wine samples

Detection and individual quantification of oak wood ellagitannins in oak barrel aged red wine samples are difficult mainly due to their low levels and the similarity between their structures. In this work, a quantification method using mass spectrometry has been developed and validated to quantify wine ellagitannins after sample fractionation with a previously reported method. The use of an internal standard is a requirement to correct mass signal variability. (-)-Gallocatechin, among the different tested compounds, was the only one that proved to be a suitable internal standard making possible the accurate and individual quantification of the main oak wood ellagitannins. The developed methodology has been used to detect and quantify these ellagitannins in different Spanish commercial wines, proving its usefulness.     

Comparison of the bioavailability of natural palm oil carotenoids and synthetic beta-carotene in humans

Palm oil carotenoids are a mixture of alpha- and beta-carotenes, which are used as food colorants. They may also be applied as a functional food ingredient because of the provitamin A activity of alpha- and beta-carotenes and their proposed beneficial roles in the prevention of chronic diseases. This paper discusses the results of an incomplete balanced crossover study with 69 healthy adult volunteers to compare palm oil carotenoids with synthetic beta-carotene in their efficacies to increase plasma levels of carotenoids. Four days of supplementation with natural palm oil carotenoids (7.6 mg/day of alpha-carotene, 11.9 mg/day of all-trans-beta-carotene, 7.5 mg/day of cis-beta-carotene) or synthetic beta-carotene (23.8 mg/day of all-trans-beta-carotene, 4.4 mg/day of cis-beta-carotene), added to a mixed meal, resulted in significant increases in plasma levels of the supplied carotenoids as compared to consumption of a low-carotenoid meal (i.e., 7.2-fold increase in alpha-carotene and 3.5-fold increase in all-trans-beta-carotene following palm oil carotenoids; 6.9-fold increase in all-trans beta-carotene following synthetic beta-carotene). As the carotenoid content differed between the treatments, the relative plasma responses were calculated per milligram of beta-carotene intake. These were similar for the two supplements, suggesting that the presence of alpha-carotene does not affect the bioavailability of beta-carotene from palm oil. It was concluded that 4 days of supplementation with palm oil carotenoids or synthetic beta-carotene improves the plasma beta-carotene status substantially, whereas alpha-carotene is additionally delivered by the palm oil supplement.     

A new process to develop a cocoa powder with higher flavonoid monomer content and enhanced bioavailability in healthy humans

Cocoa is a food rich in polyphenols, mainly the flavonoid procyanidins and flavan-3-ols. The improvement of the cardiovascular function in humans upon cocoa consumption has been specifically linked to the presence of flavan-3-ol derived metabolites in plasma, especially epicatechin glucuronide. In this context, a flavonoid-enriched cocoa-derived product could potentially exert stronger health benefits. The aim of the present study was to obtain a cocoa powder with a higher flavonoid content (mainly enriched in monomer compounds) and assess its flavonoid bioavailability in humans. For this purpose, an unfermented, nonroasted, and blanch-treated cocoa powder (A) was obtained. The powder contained four times more procyanidins than a conventional (B) cocoa powder. Powder A contained eight times more epicatechin and procyanidin B2 than powder B. Cocoa milk drinks were prepared with powder A (MDA) and B (MDB). The bioavailability of flavonoids in both drinks was assessed in a crossover intervention with healthy volunteers. The content of epicatechin glucuronide, the main metabolite detected in plasma, was five-fold higher upon consumption of MDA as compared with MDB. The urinary excretion of metabolites, mainly methyl epicatechin sulfate, was higher upon MDA consumption as compared with MDB, ranging from two- to 12-fold higher depending on the metabolite. These results, together with previous reports regarding the cardiovascular benefits linked to the presence of procyanidin metabolites in plasma, suggest that further clinical trials to validate the health benefits of a flavonoid-enriched cocoa powder are warranted.     

Enzyme additions as a tool to assess the potential bioavailability of organically bound nutrients

Potential enzyme activities in soil and water samples are measured by addition of an excess amount of suitable substrate and subsequent determination of product release. If the approach is reversed and an excess of enzyme is added, substrate availability becomes rate-limiting and the maximum release of product indicates the availability of a given substrate in a sample. This approach has been used in a range of studies using phosphatase enzyme additions to soil, manure and sediment extracts, soil suspensions, and lake and sea water (n = 41). Significantly fewer studies have used enzymes from the carbon, nitrogen and sulfur cycles (n = 14). In this review, the methodological aspects of enzyme additions are discussed using examples from studies in which enzymes from the phosphorus cycle were used. A meta-analysis performed for various soil extracts and water samples revealed that the majority of studies (75th percentile) indicate availability of organic phosphorus to enzymatic hydrolysis of up to 60%, with crude phytase preparations showing the lowest substrate specificity and greatest release of phosphorus. Compared to addition of enzymes from the phosphorus cycle, lower substrate degradation was generally achieved by addition of enzymes from the carbon, nitrogen and sulfur cycles to soil suspensions and soil organic matter extracts. Enzyme additions can be a valuable tool in process research, provided all the necessary controls are included and assay conditions optimized to ensure that the reaction reaches completion. Recommendations for the development of a standard protocol are made.     

Effect of simultaneous consumption of milk and coffee on chlorogenic acids' bioavailability in humans

Different studies have shown that milk may interact with polyphenols and affect their bioavailability in humans. The present study investigated the effect of the simultaneous consumption of coffee and milk on the urinary excretion of chlorogenic acids (CGA) and metabolites. Subjects were submitted to consumption of water, instant coffee (609 mmol of CGA) dissolved in water, and instant coffee dissolved in whole milk. Urine was collected for 24 h after consumption of each treatment for analysis of CGA and metabolites by HPLC/LC-MS. The amount of CGA and metabolites recovered after consumption of combined coffee-milk (40% ± 27%) was consistently lower in all subjects compared to that of coffee alone (68% ± 20%). Concluding, the simultaneous consumption of milk and coffee may impair the bioavailability of coffee CGA in humans.     

Soil fulvic acid properties as a means to assess the use of pig slurry amendment

Soil amendment with animal manures is a common practice for either increasing soil organic matter (SOM) and nutrient content or disposing of wastes from intensive animal industries. However, the application of organic amendments that are not sufficiently mature and stable may adversely affect soil properties, especially the content and quality of SOM pools. In this work, the effect of the consecutive annual additions of pig slurry (PS) at rates of 0 (control), 90 and 150 m³ ha⁻¹ per year over a 4-year period on the soil fulvic acid (FA) fraction of SOM was investigated in a field plot experiment conducted under semiarid conditions on a Calcic Luvisol in Toledo province, Spain. The FAs isolated from PS and control and amended soils were characterized for chemical, compositional, structural and functional properties by use of elemental and functional group analysis, and ultraviolet/visible, Fourier transform infrared (FT IR), fluorescence and electron spin resonance (ESR) spectroscopies. PS-FA was characterized by a prevalent aliphatic character, large contents of acidic functional groups, S- and N-containing groups and polysaccharide components, extended molecular heterogeneity, small organic free radical (OFR) contents and small degrees of aromatic ring polycondensation, polymerization and humification. With respect to the control soil FA, the PS-amended soil FAs were characterized by a smaller extraction yield, O and OFR contents and ratios of absorbances at 465 and 665 nm, and larger C, N, S, COOH and phenolic OH contents, C/N ratios and aliphaticity. Statistical analysis of experimental data showed that, with some exceptions, these effects generally increased with increasing cumulative amount of PS applied to soil over time. In conclusion, cumulative PS application to soil over time modifies the content and properties of the FA fraction of SOM. Thus, this material should not be considered as a mature organic amendment and should be treated appropriately before it is applied to soil, so as to increase the degree of humification and enhance its potential as a soil organic fertilizer.     

Metabolism of antioxidant and chemopreventive ellagitannins from strawberries, raspberries, walnuts, and oak-aged wine in humans: identification of biomarkers and individual variability

Ellagitannins (ETs) are dietary polyphenols, containing ellagic acid (EA) subunits, with antioxidant and cancer chemopreventive activities that might contribute to health benefits in humans. However, little is known about their metabolic fate. We investigate here the metabolism of different dietary ETs and EA derivatives in humans. Forty healthy volunteers were distributed in four groups. Each group consumed, in a single dose, a different ET-containing foodstuff, i.e., strawberries (250 g), red raspberries (225 g), walnuts (35 g), and oak-aged red wine (300 mL). After the intake, five urine fractions (F) were collected at 8 (F1), 16 (F2), 32 (F3), 40 (F4), and 56 (F5) h. Neither ETs nor EA were detected in urine after LC-MS/MS analysis. However, the microbial metabolite 3,8-dihydroxy-6H-dibenzo[b,d]pyran-6-one (urolithin B) conjugated with glucuronic acid was detected along the fractions F3-F5 in all of the subjects, independently of the consumed foodstuff. The mean percentage of metabolite excretion ranged from 2.8 (strawberries) to 16.6% (walnuts) regarding the ingested ETs. Considerable interindividual differences were noted, identifying "high and low metabolite excreters" in each group, which supported the involvement of the colonic microflora in ET metabolism. These results indicate that urolithin B (a previously described antiangiogenic and hyaluronidase inhibitor compound) is a biomarker of human exposure to dietary ETs and may be useful in intervention studies with ET-containing products. The antioxidant and anticarcinogenic effects of dietary ETs and EA should be considered in the gastrointestinal tract whereas the study of potential systemic activities should be focused on the bioavailable urolithin B derivatives.     

Detection of a "nonaromatic" NIH shift during in vivo metabolism of the monoterpene carvone in humans

High-resolution gas chromatography in combination with mass spectrometry and high-resolution mass spectrometry was used to determine the positions and extent of labeling in the metabolites of carvone, namely in alpha,4-dimethyl-5-oxo-3-cyclohexene-1-acetic acid (dihydrocarvonic acid), alpha-methylene-4-methyl-5-oxo-3-cyclohexene-1-acetic acid (carvonic acid), and 5-(1,2-dihydroxy-1-methylethyl)-2-methyl-2-cyclohexen-1-one (uroterpenolone), after human ingestion of 9,9-dideutero- and 9-(13)C-carvone. Carvonic acid was formed by oxidation at the methyl carbon of the isopropenyl group of carvone, whereas dihydrocarvonic acid was formed by oxidation at the methylene position, most probably via carvone epoxide. A "nonaromatic" NIH shift must occur during the subsequent reactions yielding dihydrocarvonic acid. Additionally, dehydrogenation of dihydrocarvonic acid and hydrogenation of carvonic acid were observed, resulting in minor amounts of both acids owning a carboxy group of opposite origin. Uroterpenolone was found to be exclusively formed by oxidation at the methylene carbon of the isopropenyl group of carvone, and thus, most probably by hydrolysis of carvone epoxide.     

Oxidative metabolism of the soy isoflavones daidzein and genistein in humans in vitro and in vivo.

The soy isoflavones daidzein and genistein are found in high concentrations in human plasma and urine after soy consumption. However, in vitro and in vivo data regarding the oxidative metabolism of isoflavones in humans are scarce. Therefore, we have studied the oxidative metabolites of these compounds formed in human liver microsomes and excreted in urine of male and female humans ingesting soy products for 2 days. Human liver microsomes transformed the soy isoflavone daidzein to three monohydroxylated and three dihydroxylated metabolites according to GC/MS analysis. On the basis of a previous study with rat liver microsomes and with the help of reference substances, these metabolites were identified as 6,7,4'-trihydroxyisoflavone, 7,3',4'-trihydroxyisoflavone, 7,8,4'-trihydroxyisoflavone, 7,8,3',4'-tetrahydroxyisoflavone, 6,7,8,4'-tetrahydroxyisoflavone, and 6,7,3',4'-tetrahydroxyisoflavone. Significant amounts of the same metabolites except 6,7,8,4'-tetrahydroxyisoflavone were also found in urine of female and male volunteers after soy intake. Genistein was metabolized by human liver microsomes to six hydroxylation products. The main metabolites were the three aromatic monohydroxylated products 5,6,7,4'-tetrahydroxyisoflavone, 5,7,8,4'-tetrahydroxyisoflavone and 5,7,3',4'-tetrahydroxyisoflavone. The aliphatic monohydroxylated metabolite 2,5,7,4'-tetrahydroxyisoflavone and two aromatic dihydroxylated metabolites, 5,7,8,3',4'-pentahydroxyisoflavone and 5,6,7,3',4'-pentahydroxyisoflavone, were formed in trace amounts. The same hydroxylated genistein metabolites except the aliphatic hydroxylated one could also be detected in human urine samples. Methylated forms of the catechol metabolites, which were generated by incubations with catechol-O-methyltransferase in vitro could be detected only in trace amounts in the urine samples. This implies that this reaction does not play a major role in the biotransformation of the hydroxylated daidzein and genistein metabolites in vivo. Most of these oxidative metabolites are described as human in vivo metabolites for the first time. Their biological significance remains to be established.     

Nitrogen metabolism components as a tool to discriminate between organic and conventional citrus fruits

The aim of this work was to develop a method for authenticity control of organically grown orange fruits. Due to the different kinds of nitrogen fertilization of the soil in organically and conventionally managed farms, the study tried to verify the possibility to differentiate Navelina and Tarocco orange fruits obtained by these production systems through the detection of markers linked to nitrogen metabolism. In addition to the classic quality parameters, total nitrogen (N) and synephrine contents in juice and (15)N/(14)N isotope ratio (expressed as delta(15)N per thousand) in proteins of pulp and amino acids of juice were determined. The results obtained indicated that total N and synephrine contents were significantly higher in conventional fruits, whereas the delta(15)N per thousand values were higher in the organic ones. The new markers identified in this research by linear discriminant analysis of the data may constitute a useful tool to differentiate organic citrus fruits or juices from conventional ones.     

Nuclear magnetic resonance spectroscopic based studies of the metabolism of black tea polyphenols in humans

Epidemiological studies indicate that a high intake of flavonoids is associated with an improved health status. Tea is one of the most abundant sources of flavonoids in the human diet. The bioavailability and biotransformation of tea flavonoids are, however, not clearly understood. The aim of the present study was to investigate the metabolism of black tea via a nonspecific screening method. (1)H nuclear magnetic resonance (NMR) spectroscopy was used to obtain nonselective profiles of urine samples collected from three human volunteers before and after a single dose of black tea. The complex spectroscopic profiles were interpreted with the use of pattern recognition techniques. Hippuric acid was confirmed as the major urinary black tea metabolite. One previously unknown metabolite was detected and identified as 1,3-dihydroxyphenyl-2-O-sulfate (sulfate conjugate of pyrogallol) using HPLC directly coupled to mass spectrometry and (1)H NMR spectroscopy. This study shows that NMR-pattern recognition studies can be used for the discovery of unknown flavonoid metabolites in humans.     

Biofortification of rice with zinc: assessment of the relative bioavailability of zinc in a Caco-2 cell model and suckling rat pups

Staple foods, such as rice, can now be enriched in micronutrients through conventional breeding (i.e., biofortification) to enhance dietary intake of vulnerable populations. The objectives of this study were (1) to establish a rapid, high capacity Caco-2 cell model to determine the relative bioavailability of zinc (Zn) from samples of staple food breeding lines for potential use as a guideline for selection/breeding and (2) to determine the relative bioavailability of Zn from conventional rice varieties and one Zn-biofortified type. Polished or undermilled, parboiled rice samples were digested in vitro with pepsin and pH adjustment, and by pancreatic enzymes. Zn uptake from digested samples was measured in Caco-2 cells in culture. A previously validated rat pup model was also used to assess Zn absorption in vivo, using gastric intubation and (65)Zn labeling. Pups were killed after 6 h, and radioactivity in tissues and in small intestine perfusate and cecum-colon contents was used to measure Zn bioavailability. A biofortified rice variety contained substantially more Zn than conventional varieties, with no change in phytate content. Absorbed Zn (μg/g rice) was significantly higher from the new variety in both the in vitro Caco-2 cell model (2.1-fold) and the rat pup model (2.0-fold). Results from the two models were highly correlated, particularly for the polished samples. Biofortification of rice with Zn results in significantly increased Zn uptake in both models. Since results from the Caco-2 cell model correlated well with those from rat pups, this cell model is likely to predict results in human populations and can be used for screening purposes.     

In vitro metabolism of trans-permethrin and its major metabolites, PBalc and PBacid, in humans

To estimate the metabolic profile of trans-permethrin in humans, a comparison of the in vitro metabolism of trans-permethrin in humans and rats was conducted using hepatic microsomes, and cytochrome P450 and UDP-glucuronyltransferase isoforms, which catalyze the metabolism of 3-phenoxybenzyl alcohol (PBalc) and 3-phenoxybenzoic acid (PBacid), respectively. In humans and rats, the major metabolic reaction of trans-permethrin in microsomal incubations was the cleavage of ester linkage to give PBalc, followed by oxidation to 4'-OH-PBalc, 4'-OH-PBacid, and PBacid. As to 4'-hydroxylation of PBalc, several CYPs were able to catalyze the reaction, and CYP2E1 was identified as a predominant isoform. PBacid and its conjugates (glucuronide and glycine) are major urinary metabolites of trans-permethrin in mammals. PBacid is also a metabolite of several pyrethroids, and has been used as a biomarker of human exposure to pyrethroids. Our study indicated that there was no difference in glucuronyltransferase activity of PBacid between humans and rats, and that only UGT1A9 can catalyze the glucuronidation of PBacid among human UGTs. Some UGT1A9 variants are known to have poor glucuronidation activity. From these results, it was assumed that deficiency or polymorphism of UGT1A9 might affect the profile of PBacid and its conjugates in urine collected from persons exposed to trans-permethrin or other pyrethroids. These results are helpful for understanding the metabolism of trans-permethrin in humans and determining methods for quantification of target analytes for assessment of human exposure to trans-permethrin and other pyrethroids that give PBacid and its conjugates as urinary metabolites.     

Uptake and metabolism of hydroxycinnamic acids (chlorogenic, caffeic, and ferulic acids) by HepG2 cells as a model of the human liver

Hydroxycinnamic acids are antioxidant polyphenols common in the human diet, although their potential health benefits depend on their bioavailability. To study the hepatic uptake and metabolism, human hepatoma HepG2 cells were incubated for 2 and 18 h with caffeic, ferulic, and chlorogenic acids. Moderate uptake of caffeic and ferulic acids was observed versus a low absorption of chlorogenic acid, where esterification of the caffeic acid moiety markedly reduced its absorption. Methylation was the preferential pathway for caffeic acid metabolism, along with glucuronidation and sulfation, while ferulic acid generated glucuronides as the only metabolites. Ferulic acid appeared to be more slowly taken up and metabolized by HepG2 cells than caffeic acid, with 73% and 64% of the free, nonmetabolized molecules detected in the culture medium after 18 h, respectively. In conclusion, hydroxycinnamic acids can be metabolized by the liver as suggested by the results obtained using HepG2 cells as a hepatic model system.     

NMR analytical approach to clarify the molecular mechanisms of the antioxidative and radical-scavenging activities of antioxidants in tea using 1,1-diphenyl-2-picrylhydrazyl

(+)-catechin, ethyl gallate, ascorbic acid, and alpha-tocopherol were reacted with 1,1-diphenyl-2-picrylhydrazyl (DPPH), and the reaction mixtures were subjected to 13C-nuclear magnetic resonance (NMR) analyses to clarify the molecular mechanisms of the antioxidative and radical-scavenging activities of each antioxidant. When ascorbic acid was reacted with DPPH, it was oxidized to dehydroascorbic acid by DPPH. When a mixture of ascorbic acid and (+)-catechin was reacted with DPPH, ascorbic acid scavenged DPPH radical faster than (+)-catechin. Ascorbic acid also scavenged DPPH radical faster than ethyl gallate and alpha-tocopherol. When (+)-catechin was reacted with DPPH, the B-ring of (+)-catechin changed to an o-quinone structure. However, it was reduced to (+)-catechin by ethyl gallate or alpha-tocopherol. alpha-Tocopherol and ethyl gallate had almost identical antioxidative activities. Therefore, the order of radical-scavenging ability (speed) suggested by our 13C NMR study was as follows: ascorbic acid > alpha-tocopherol = ethyl gallate > (+)-catechin.     

Reproduction and pre-dispersal survival of Iberian lynx in a subpopulation of the Doñana National Park

Little is known about the reproduction of the Iberian lynx (Lynx pardinus) even though it is the most endangered felid in the world. We studied during 9 years several reproductive parameters of the Iberian lynx in one of the subpopulations situated in Doñana National Park (south-western Spain), by means of radio-tracking, direct observations and photo-trapping. The potential breeding subpopulation was usually composed of 3 adult females, which bred 83% of the total possible 29 female-reproductive year. The minimum total number of cubs born during the study was 64 (7.7 ± 0.69 per year). There was no correlation between the number of cubs born or number of breeding females and population size of European rabbit (Oryctolagus cuniculus, the main prey of the lynx). All known births (n = 16) occurred in March except one in April and another in June. Mean litter size was 3.0 ± 0.16 (n = 16, range = 2-4). Sex-ratio (n = 59 cubs) was 1.03:1.00 (females:males). Nevertheless, the commonest picture was a female with 2 cubs older than 3 months. Altogether, at age of 3 months, 75% of cubs survived. Number of lynx alive at 10 months old and before dispersing was 69% and 57%, respectively. Sex did not affect survival for any age. Normally, it was not possible to know the causes of the death of cubs younger than 3 months. Survival at this age was not related with mother, mother age, or rabbit abundance. All females that bred were older than 3 years. The age of last reproduction was 9 years. For 3 females that were tracked during almost their complete reproductive life, the life time reproductive output was between 11 and 19 cubs. Iberian lynx reproductive parameters did not respond to wide changes in prey abundance during the study. Conservation plans considering the extraction of cubs with a low survival probability should be considered by managers, for instance, in translocation campaigns.