Curry-Odorants and Their Metabolites Transfer into Human Milk and Urine.

SCOPE: The excretion of dietary odorants into urine and milk is evaluated and the impact of possible influencing factors determined. Furthermore, the metabolic relevance of conjugates for the excretion into milk is investigated. METHODS AND RESULTS: Lactating mothers (n = 20) are given a standardized curry dish and donated one milk and urine sample each before and 1, 2, 3, 4.5, 6, and 8 h after the intervention. The concentrations of nine target odorants in these samples are determined. A significant transition is observed for linalool into milk, as well as for linalool, cuminaldehyde, cinnamaldehyde, and eugenol into urine. Maximum concentrations are reached within 1 h after the intervention in the case of milk and within 2-3 h in the case of urine. In addition, the impact of glucuronidase treatment on odorant concentrations is evaluated in a sample subset of twelve mothers. Linalool, eugenol, and vanillin concentrations increased 3-77-fold in milk samples after treatment with ß-glucuronidase. CONCLUSION: The transfer profiles of odorants into milk and urine differ qualitatively, quantitatively, and in temporal aspects. More substances are transferred into urine and the transfer needs a longer period compared with milk. Phase II metabolites are transferred into urine and milk.

Identification of mozambioside roasting products and their bitter taste receptor activation.

Arabica coffee contains the bitter-tasting diterpene glycoside mozambioside, which degrades during coffee roasting, leading to yet unknown structurally related degradation products with possibly similar bitter-receptor-activating properties. The study aimed at the generation, isolation, and structure elucidation of individual pyrolysis products of mozambioside and characterization of bitter receptor activation by in vitro analysis in HEK 293T-Gα16gust44 cells. The new compounds 17-O-ß-d-glucosyl-11-hydroxycafestol-2-on, 11-O-ß-d-glucosyl-16-desoxycafestol-2-on, 11-O-ß-d-glucosyl-(S)-16-desoxy-17-oxocafestol-2-on, 11-O-ß-d-glucosyl-15,16-dehydrocafestol-2-on, and 11-O-ß-d-glucosyl-(R)-16-desoxy-17-oxocafestol-2-on were isolated from pyrolyzed mozambioside by HPLC and identified by NMR and UHPLC-ToF-MS. Roasting products 11-O-ß-d-glucosyl-(S)-16-desoxy-17-oxocafestol-2-on, 11-O-ß-d-glucosyl-15,16-dehydrocafestol-2-on, and 11-O-ß-d-glucosyl-(R)-16-desoxy-17-oxocafestol-2-on had lower bitter receptor activation thresholds compared to mozambioside. Molecular docking simulations revealed the binding modes of the compounds 11-O-ß-d-glucosyl-15,16-dehydrocafestol-2-on and 11-O-ß-d-glucosyl-(R)-16-desoxy-17-oxocafestol-2-on and their aglycone 11-hydroxycafestol-2-on in the two cognate receptors TAS2R43 and TAS2R46. The newly discovered roasting products 17-O-ß-d-glucosyl-11-hydroxycafestol-2-on, 11-O-ß-d-glucosyl-(S)-16-desoxy-17-oxocafestol-2-on, 11-O-ß-d-glucosyl-15,16-dehydrocafestol-2-on, and 11-O-ß-d-glucosyl-(R)-16-desoxy-17-oxocafestol-2-on were detected in authentic roast coffee brew by UHPLC-ToF-MS and could contribute to coffee's bitter taste impression.

Linalool, 1,8-Cineole, and Eugenol Transfer from a Curry Dish into Human Urine.

SCOPE: For most substances, there are several routes of excretion from the human body. This study focuses on urinary excretion of dietary odorants and compares the results with previously obtained results on excretion into milk. METHODS AND RESULTS: Lactating mothers (n = 18) are given a standardized curry dish and donate urine samples before and after the intervention. The odorants 1,8-cineole, linalool, cuminaldehyde, cinnamaldehyde, 4-hydroxy-2,5-dimethyl-3(2H)-furanone, sotolone, eugenol, vanillin, and γ-nonalactone are quantitatively analyzed. A significant transition of up to 6 µg g-1 creatinine into urine is observed for linalool, 1,8-cineole, and eugenol. Maximum concentrations are reached 1.5 h after the intervention for 1,8-cineole and eugenol as well as 2.5 h after the intervention for linalool. Comparison with previous results reveals that the excretion pattern of odorants into urine is divergent from the one into milk. In a second intervention study (n = 6), excretion of phase II metabolites into urine is studied using ß-glucuronidase treatment. Linalool and eugenol concentrations are 23 and 77 times higher after treatment than before treatment with ß-glucuronidase, respectively. CONCLUSION: The study demonstrates transition of linalool, 1,8-cineole, and eugenol from the diet into urine and excretion of glucuronides in the case of linalool, eugenol, and vanillin.

Consumption of Roasted Coffee Leads to Conjugated Metabolites of Atractyligenin in Human Plasma.

Roasted coffee contains atractyligenin-2-O-ß-d-glucoside and 3'-O-ß-d-glucosyl-2'-O-isovaleryl-2-O-ß-d-glucosylatractyligenin, which are ingested with the brew. Known metabolites are atractyligenin, atractyligenin-19-O-ß-d-glucuronide (M1), 2ß-hydroxy-15-oxoatractylan-4α-carboxy-19-O-ß-d-glucuronide (M2), and 2ß-hydroxy-15-oxoatractylan-4α-carboxylic acid-2-O-ß-d-glucuronide (M3), but the appearance and pharmacokinetic properties are unknown. Therefore, first time-resolved quantitative data of atractyligenin glycosides and their metabolites in plasma samples from a pilot human intervention study (n = 10) were acquired. None of the compounds were found in the control samples and before coffee consumption (t = 0 h). After coffee, neither of the atractyligenin glycosides appeared in the plasma, but the aglycone atractyligenin and the conjugated metabolite M1 reached an estimated cmax of 41.9 ± 12.5 and 25.1 ± 4.9 nM, respectively, after 1 h. M2 and M3 were not quantifiable until their concentration enormously increased ≥4 h after coffee consumption, reaching an estimated cmax of 2.5 ± 1.9 and 55.0 ± 57.7 nM at t = 10 h. The data suggest that metabolites of atractyligenin could be exploited to indicate coffee consumption.

Structure-Based Discovery of Mouse Trace Amine-Associated Receptor 5 Antagonists.

Trace amine-associated receptors (TAARs) were discovered in 2001 as new members of class A G protein-coupled receptors (GPCRs). With the only exception of TAAR1, TAAR members (TAAR2-9, also known as noncanonical olfactory receptors) were originally described exclusively in the olfactory epithelium and believed to mediate the innate perception of volatile amines. However, most noncanonical olfactory receptors are still orphan receptors. Given its recently discovered nonolfactory expression and therapeutic potential, TAAR5 has been the focus of deorphanization campaigns that led to the discovery of a few druglike antagonists. Here, we report four novel TAAR5 antagonists identified through high-throughput screening, which, along with the four ligands published in the literature, constituted our starting point to design a computational strategy for the identification of TAAR5 ligands. We developed a structure-based virtual screening protocol that allowed us to identify three new TAAR5 antagonists with a hit rate of 10%. Despite lacking an experimental structure, we accurately modeled the TAAR5 binding site by integrating comparative sequence- and structure-based analyses of serotonin receptors with homology modeling and side-chain optimization. In summary, we have identified seven new TAAR5 antagonists that could serve as lead candidates for the development of new treatments for depression, anxiety, and neurodegenerative diseases.

Sweetener System Intervention Shifted Neutrophils from Homeostasis to Priming.

BACKGROUND: Non-nutritive sweeteners (NNS) are part of personalized nutrition strategies supporting healthy glycemic control. In contrast, the consumption of non-nutritive sweeteners has been related to person-specific and microbiome-dependent glycemic impairments. Reports on the effects of NNS on our highly individual cellular immune system are sparse. The recent identification of taste receptor expression in a variety of immune cells, however, suggested their immune-modulatory relevance. METHODS: We studied the influence of a beverage-typical NNS system on the transcriptional profiling of sweetener-cognate taste receptors, selected cytokines and their receptors, and on Ca2+ signaling in isolated blood neutrophils. We determined plasma concentrations of saccharin, acesulfame-K, and cyclamate by HPLC-MS/MS, upon ingestion of a soft drink-typical sweetener surrogate. In an open-labeled, randomized intervention study, we determined pre- versus post-intervention transcript levels by RT-qPCR of sweetener-cognate taste receptors and immune factors. RESULTS: Here we show that the consumption of a food-typical sweetener system modulated the gene expression of cognate taste receptors and induced the transcriptional regulation signatures of early homeostasis- and late receptor/signaling- and inflammation-related genes in blood neutrophils, shifting their transcriptional profile from homeostasis to priming. Notably, sweeteners at postprandial plasma concentrations facilitated fMLF (N-formyl-Met-Leu-Phe)-induced Ca2+ signaling. CONCLUSIONS: Our results support the notion of sweeteners priming neutrophils to higher alertness towards their adequate stimuli.

Metabolites of dietary atractyligenin glucoside in coffee drinkers' urine.

Arabica roast coffee contains a substantial amount of water soluble atractyligenin-2-O-ß-d-glucoside, which is ingested by consumption of coffee brew. Metabolomics data suggest this coffee compound is excreted as glucuronides, but the structures of conjugates have not been elucidated so far. We collected coffee drinkers' urine and isolated four metabolites by MS-guided liquid chromatographic fractionation. The structures were investigated by nuclear magnetic resonance (NMR) and time-of-flight mass spectrometry (ToF-MS) and identified as atractyligenin-19-O-ß-d-glucuronide (M1), 2ß-hydroxy-15-oxoatractylan-4α-carboxy-19-O-ß-d-glucuronide (M2), and 2ß-hydroxy-15-oxoatractylan-4α-carboxylic acid-2-O-ß-d-glucuronide (M3). An unconjugated metabolite (M4) was confirmed as atractyligenin. We analyzed spot urines from n = 6 coffee drinking individuals and detected the metabolites M1, M2 and M4 in every sample, and M3 in four out of six samples, suggesting interindividual differences in metabolism.

Metabolites of Key Flavor Compound 2,3,5-Trimethylpyrazine in Human Urine.

Pyrazines are among the most important compound class conveying the odor impressions "roasty", "nutty", and "earthy". They are formed by the Maillard reaction and occur ubiquitously in heated foods. The excretion of metabolites of the key flavor odorant 2,3,5-trimethylpyrazine, abundant in the volatile fraction of roasted coffee, was investigated. Based on literature suggestions, putative phase 1 and phase 2 metabolites were synthesized, characterized by nuclear magnetic resonance and mass spectroscopy data and used as standards for targeted, quantitative analysis of coffee drinkers' urine using stable-isotope-dilution-ultrahigh-performance liquid chromatography tandem mass spectroscopy (SIDA-UHPLC-MS/MS). The analysis of spot urine samples from a coffee intervention study revealed 3,6-dimethylpyrazine-2-carboxylic acid, 3,5-dimethylpyrazine-2-carboxylic acid, and 5,6-dimethylpyrazine-2-carboxylic acid were quantitatively dominating metabolites. Only negligible traces of pyrazinemethanols (3,6-dimethyl-2-pyrazinemethanol and 3,5,6-trimethylpyrazine-2-ol), glucuronides ((3,6-dimethylpyrazine-2-yl-)methyl-O-ß-D-glucuronide and (3,5-dimethylpyrazine-2-yl-)methyl-O-ß-D-glucuronide), and sulfates ((3,6-dimethylpyrazine-2-yl-)methyl-sulfate and (3,5-dimethylpyrazine-2-yl-)methyl-sulfate) were detected.

An aqueous extract of the brown alga Eisenia bicyclis extends lifespan in a sex-specific manner by interfering with the Tor-FoxO axis.

Food has a decisive influence on our health, to the extent where even lifespan can be directly affected by it. In the present work, we have examined the effects of an aqueous extract of the marine brown alga Eisenia bicyclis in terms of its potential to extend lifespan. For this purpose, we used the fruit fly Drosophila melanogaster as a model. The experiments showed that small amounts of Eisenia extract can extend lifespan by up to 40%. This effect is not only related to the median but also to the maximum lifespan. Interestingly, this life-extending effect is sex-specific, i.e. it occurs exclusively in females. Even under stressful nutritional conditions such as a high sugar diet, this effect is detectable. Mechanistic studies showed that this life-prolonging effect depends on a functional Tor and a functional FoxO signaling pathway. It can be concluded that components of the Eisenia extract prolong lifespan by interacting with the Tor-FoxO axis. This study may serve to stimulate further investigations, which on the one hand show such a life-prolonging effect also in other organisms and on the other hand identify the substances responsible for this effect. Finally, it may also encourage the increased use of arame as a health-promoting food supplement.

Activation Spectra of Human Bitter Taste Receptors Stimulated with Cyclolinopeptides Corresponding to Fresh and Aged Linseed Oil.

Linseed oil is rich in unsaturated fatty acids, and its increased consumption could aid in health-promoting nutrition. However, rapid oxidation of linseed oil and concomitant development of bitterness impair consumers' acceptance. Previous research revealed that cyclolinopeptides, a group of cyclic peptides inherent to linseed oil, dominantly contribute to the observed bitterness. In the present study, fresh and stored linseed oil and flaxseed were analyzed for the presence of cyclolinopeptides using preparative high-performance liquid chromatography combined with mass spectrometry- and nuclear magnetic resonance-based identification and quantification. The purified compounds were tested for the activation of all 25 human bitter taste receptors of which only two responded exclusively to methionine-oxidized cyclolinopeptides. Of those, the methionine sulfoxide-containing cyclolinopeptide-4 elicited responses at relevant concentrations. We conclude that this compound is the main determinant of linseed oil's bitterness and propose strategies to reduce the development of bitterness.

Overlapping activation pattern of bitter taste receptors affect sensory adaptation and food perception.

The composition of menus and the sequence of foodstuffs consumed during a meal underlies elaborate rules. However, the molecular foundations for the observed taste- and pleasure-raising effects of complex menus are obscure. The molecular identification and characterization of taste receptors can help to gain insight into the complex interrelationships of food items and beverages during meals. In our study, we quantified important bitter compounds in chicory and chicory-based surrogate coffee and used them to identify responsive bitter taste receptors. The two receptors, TAS2R43 and TAS2R46, are exquisitely sensitive to lactucin, lactucopicrin, and 11ß,13-dihydrolactucin. Sensory testing demonstrated a profound influence of the sequence of consumption of chicory, surrogate coffee, and roasted coffee on the perceived bitterness by human volunteers. These findings pave the way for a molecular understanding of some of the mixture effects underlying empirical meal compositions.

Dietary Linalool is Transferred into the Milk of Nursing Mothers.

SCOPE: Breast milk is repeatedly postulated to shape the first aroma and taste impressions of infants and thus impact their flavor learning. The objective of this study is to assess the transition of aroma compounds from a customary curry dish into milk. METHODS AND RESULTS: The article prepares a standardized curry dish and administers the dish to nursing mothers (n = 18) in an intervention study. The participants donate one milk sample before and three samples after the intervention. Due to their olfactory or quantitative relevance in the curry dish, 1,8-cineole, linalool, cuminaldehyde, cinnamaldehyde, 4-hydroxy-2,5-dimethyl-3(2H)-furanone, sotolone, eugenol, vanillin, and γ-nonalactone are defined as target compounds, and their transition into milk is quantified by gas chromatography-mass spectrometry. A significant transition into the milk is observed for linalool, and its olfactory relevance in this respect is supported by calculated odor activity values. In contrast, no relevant levels are detected for the other eight target compounds. CONCLUSION: Ingestion of a customary curry dish can lead to an alteration of the milk aroma, which might be perceived by the infant during breastfeeding. The current study also demonstrates that the extent of aroma transfer differs between both substances and individuals.

Dietary Piperine is Transferred into the Milk of Nursing Mothers.

INTRODUCTION: The diet of breastfeeding mothers could bring nurslings into contact with flavor compounds putatively contributing to early sensory programming of the infant. The study investigates whether tastants from a customary curry dish consumed by mothers are detectable in their milk afterwards and can be perceived by the infant. METHODS AND RESULTS: Sensory evaluation identifies pungency as the dominating taste impression of the curry dish. Its ingredients of chili, pepper, and ginger suggest the flavor compounds capsaicin, piperine, and 6-gingerol as analytical targets. Breastfeeding mothers are recruited for an intervention trial involving the consumption of the curry dish and subsequent collection of milk samples for flavor compound analysis. Targeted and untargeted mass spectrometric (MS)- investigations identify exclusively piperine as an intervention-derived compound in human milk. However, concentrations are below the human taste threshold. CONCLUSION: Piperine from pepper-containing foods transfers into the mother's milk within 1 h and is delivered to the nursling. Concentrations of 50 and 200 nM of piperine are 70-350 times below the human taste threshold, but TRPV1 (Transient Receptor Potential Vanilloid-1 ion channel) desensitization through frequent exposure to sub-taste-threshold concentrations could contribute to an increased tolerance at a later age.

Comparative assessment of purified saponins as permeabilization agents during respirometry.

Saponins are a diverse group of secondary plant metabolites, some of which display hemolytic toxicity due to plasma membrane permeabilization. This feature is employed in biological applications for transferring hydrophilic molecules through cell membranes. Widely used commercial saponins include digitonin and saponins from soap tree bark, both of which constitute complex mixtures of little definition. We assessed the permeabilization power of pure saponins towards cellular membranes in an effort to detect novel properties and to improve existing applications. In a respirometric assay, we characterized half-maximal permeabilization of the plasma membrane for different metabolites, of the mitochondrial outer membrane for cytochrome C and the full solubilization of mitochondrial inner membrane protein complexes. Beyond the complete list as repository for the field, we highlight several findings with direct applicability. First, we identified and validated α-chaconine as alternative permeabilization agent in respirometric assays of cultured cells and isolated synaptosomes, superior to digitonin in its tolerability for mitochondria. Second, we identified glycyrrhizic acid to form exceptionally small pores impermeable for adenosine diphosphate. Third, in a concentration dependent manner, tomatine proved to be able to selectively permeabilize the mitochondrial outer, but not inner membrane, allowing for novel states in which to determine cytochrome C oxidase activity. In summary, we provide a list of the permeabilization properties of 18 pure saponins. The identification of two saponins, namely tomatine and chaconine, with direct usability in improved or novel cell biological applications within this small subgroup demonstrates the tremendous potential for further functional screening of pure saponins.

Numerous Compounds Orchestrate Coffee's Bitterness.

Coffee is one of the most consumed hot beverages worldwide and is highly regarded because of its stimulating effect despite having a pronounced bitterness. Even though numerous bitter ingredients have been identified, the detailed molecular basis for coffee's bitterness is not well understood except for caffeine, which activates five human bitter taste receptors. We elucidated the contribution of other bitter coffee constituents in addition to caffeine with functional calcium imaging experiments using mammalian cells expressing the cDNAs of human bitter taste receptors, sensory experiments, and in silico modeling approaches. We identified two human bitter taste receptors, TAS2R43 and TAS2R46, that responded to the bitter substance mozambioside with much higher sensitivity than to caffeine. Further, the structurally related bitter substances bengalensol, cafestol, and kahweol also activated the same pair of bitter taste receptors much more potently than the prototypical coffee bitter substance caffeine. However, for kahweol, a potent but weak activator of TAS2R43 and TAS2R46, we observed an inhibitory effect when simultaneously applied together with mozambioside to TAS2R43 expressing cells. Molecular modeling experiments showed overlapping binding sites in the receptor's ligand binding cavity that suggest that the partial agonist kahweol might be useful to reduce the overall bitterness of coffee-containing beverages. Taken together, we found that the bitterness of coffee is determined by a complex interaction of multiple bitter compounds with several human bitter taste receptors.

Targeted metabolomics of pellicle and saliva in children with different caries activity.

Pellicle is the initial proteinaceous layer that is formed almost instantaneously on all solid surfaces in the oral cavity. It is of essential relevance for any interactions and metabolism on the tooth surface. Up to now, there is no information on the metabolome of this structure. Accordingly, the present study aims to characterise the metabolomic profile of in-situ pellicle in children with different caries activity for the first time in comparison to saliva. Small molecules such as carbohydrates, amino acids, organic acids, and fatty acids, putatively involved in the formation of caries were quantified using mass spectrometry (MS)-based techniques, such as (stable isotope dilution analysis)-ultra-performance liquid chromatography-tandem MS and gas chromatography/electron ionisation-MS. Pellicle and corresponding saliva samples were collected from caries-active, caries-free and caries-rehabilitated 4- to 6-year-old children. The most abundant analytes in pellicle were acetic acid (1.2-10.5 nmol/cm2), propionic acid (0.1-8.5 nmol/cm2), glycine (0.7-3.5 nmol/cm2), serine (0.08-2.3 nmol/cm2), galactose (galactose + mannose; 0.035-0.078 nmol/cm2), lactose (0.002-0.086 nmol/cm2), glucose (0.018-0.953 nmol/cm2), palmitic acid (0.26-2.03 nmol/cm2), and stearic acid (0.34-1.81 nmol/cm2). Significant differences depending on caries activity were detected neither in saliva nor in the corresponding pellicle samples.

Activity and distribution pattern of enzymes in the in-situ pellicle of children.

OBJECTIVE: This study investigated, for the first time, pellicle enzymes with respect to their activity, distribution and fluorescence pattern in children with different caries experience. DESIGN: In-situ pellicles were collected from 41 children (aged 4-6 years) with different caries status; 17 of them were caries-free (dmf = 0), 12 had dental restorations but no current caries (dmf ≥ 2) and 12 had at least two carious lesions (dmf ≥ 2). Bovine enamel samples were fixed on individual upper jaw braces for pellicle formation. After 30 min of intraoral exposure, the pellicle and saliva samples were analysed for the activities of amylase, lysozyme, peroxidase and glucosyltransferase (GTF). The distribution of these enzymes, including GTF-isoforms B, C and D, and the pellicle ultrastructure were examined by gold-immunolabelling and transmission electron microscopy (TEM). Furthermore, interactions between pellicle enzymes and adherent bacteria were visualised using combined fluorescence and immunofluorescence labelling. RESULTS: There were no significant differences in the pellicle enzyme activities between the study groups. TEM analysis revealed the absence of GTF C and D in the pellicle of caries-active children. Amylase, peroxidase and GTF-isoforms showed a random distribution within the pellicle layer; lysozyme was found in the form of clusters. A similar ultrastructural pattern was observed for all subjects. Fluorescence labelling technique enabled visualisation of all enzymes, except for GTF B. CONCLUSION: Pellicle enzyme activities and ultrastructure are not associated with children's caries status. Further investigation is needed to assess the influence of individual GTF-isoforms on caries susceptibility in children.

Bioavailability and Biological Effects of 2- O-ß-d-Glucopyranosyl-carboxyatractyligenin from Green Coffee in Caenorhabditis elegans.

Targeted analysis of Coffea arabica and Coffea canephora green coffees (total sample size n = 57) confirmed 2- O-ß-d-glucopyranosyl-carboxyatractyligenin (6) as the quantitatively dominating carboxyatractyligenin derivative. Its abundance in Arabicas (2425 ± 549 nmol/g, n = 48) exceeded that in Robustas (34 ± 12 nmol/g, n = 9) roughly by a factor of 70. Coffee processing involving heat (e.g., steam treatment and decaffeination) reduced concentrations of 6 and increased those of the decarboxylated derivative. The bioavailability of compound 6 in Caenorhabditis elegans was demonstrated by ultraperformance liquid chromatography-tandem mass spectrometry analysis of extracts prepared from nematode cultures incubated in a liquid medium containing 6. A toxicity assay performed to assess the impact of 6 in vivo showed a 20-fold higher median lethal dose (LD50 = 11.7 ± 1.2 mM) concentration compared to that of the known phytotoxic adenine-nucleotide transporters inhibitor carboxyatractyloside (2, LD50 = 0.61 ± 0.05 mM), whereas 1 mM 6 and 0.1 mM 2 were sufficient to decrease the survival of wild type C. elegans, already 10-20-fold lower doses reduced reproduction. Because the insulin/insulin-like growth factors signaling cascade (IIS) is a key regulator of life span and stress resistance, the impact of compound 6 on the survival of long-living daf-2 C. elegans was tested. As the susceptibility of these nematodes to 6 was as high as that in wild type, an impact on central metabolic processes independent of IIS was suggested. Analysis of the in vivo adenosine triphosphate (ATP) content of adult C. elegans revealed no changes after 1 and 24 h, but a 50% reduction after treatment with 1 mM 6 during the entire postembryonic development. These data speak for a developmental-stage-dependent modulation of the ATP pool by 6.

Synephrine as a Specific Marker for Orange Consumption.

To validate the suitability of synephrine, known to be a highly abundant alkaloid in oranges, as a dietary biomarker for orange consumption, a highly sensitive and robust stable isotope dilution analysis (SIDA) as well as an ECHO method, using the analyte itself as a pseudointernal standard injected into the analysis run to provide an "echo peak" of the analyte, was developed to quantitate synephrine by liquid chromatography-tandem mass spectrometry (LC-MS/MS) in citrus juices and human urine before and after the ingestion of orange juice. A citrus juice screening revealed high synephrine concentrations of 150-420 nmol/mL in orange (n = 11) and tangerine (n = 2) juices, whereas 20-100 times lower levels were found in juice from grapefruit (n = 14), lemon (n = 5), pomelo (n = 2), and lime (n = 4). Application of the SIDA to quantitate synephrine in sulfatase/glucuronidase-treated urine samples (n = 10) after orange juice consumption showed an increase of synephrine from trace levels (0.1 ± 0.1 nmol/mL) in the 2-day washout phase to a maximum concentration of 8.9 (±5.5) nmol/mL found 4 h after ingestion of orange juice. Whereas proline betaine was recently reported as a dietary biomarker indicating the ingestion of any citrus product and Chinese artichoke, synephrine can be used a reliable additional biomarker with high specificity for orange and tangerine.

High-Throughput Quantitation of Proline Betaine in Foods and Suitability as a Valid Biomarker for Citrus Consumption.

Proline betaine has been proposed as a candidate dietary biomarker for citrus intake. To validate its suitability as a dietary biomarker and to gain insight into the range of this per-methylated amino acid in foods and beverages, a quick and accurate stable isotope dilution assay was developed for quantitative high-throughput HILIC-MS/MS screening of proline betaine in foods and urine after solvent-mediated matrix precipitation. Quantitative analysis of a variety of foods confirmed substantial amounts of proline betaine in citrus juices (140-1100 mg/L) and revealed high abundance in tubers of the vegetable Stachys affinis, also known as Chinese artichocke (∼700 mg/kg). Seafood including clams, shrimp, and lobster contained limited amounts (1-95 mg/kg), whereas only traces were detected in fish, cuttlefish, fresh meat, dairy products, fresh vegetable (<3 mg/kg), coffee, tea, beer, and wine (<7 mg/L). The human excretion profiles of proline betaine in urine were comparable when common portions of orange juice or fried Stachys tubers were consumed. Neither mussels nor beer provided enough proline betaine to detect significant differences between morning urine samples collected before and after consumption. As Stachys is a rather rare vegetable and not part of peoples' daily diet, the data reported here will help to monitor the subject's compliance in future nutritional human studies on citrus products or the exclusion of citrus products in the wash-out phase of an intervention study. Moreover, proline betaine measurement can contribute to the establishment of a toolbox of valid dietary biomarkers reflecting wider aspects of diet to assess metabolic profiles as measures of dietary exposure and indicators of dietary patterns, dietary changes, or effectiveness of dietary interventions.